Disturbed Fatty Acid Oxidation Endoplasmic ReticulumStress and Apoptosis in Left Ventricle of Patients WithType 2 DiabetesMarko Ljubkovic1 Melanie Gressette2 Cristijan Bulat3 Marija Cavar1 Darija Bakovic14 Damir Fabijanic4

Ivica Grkovic5 Christophe Lemaire26 and Jasna Marinovic1

Diabetes 2019681924ndash1933 | httpsdoiorg102337db19-0423

Chronic heart failure is a common complication inpatients with type 2 diabetes mellitus (T2DM) T2DMis associated with disturbed metabolism of fat whichcan result in excessive accumulation of lipids in car-diac muscle In the current study we assessed mito-chondrial oxidation of carbohydrates and fatty acidslipid accumulation endoplasmic reticulum (ER) stressand apoptosis in diabetic left ventricle Left ventricularmyocardium from 37 patients (a group of patients withdiabetes and a group of patients without diabetes[ejection fraction gt50]) undergoing coronary arterybypass graft surgery was obtained by subepicardialneedle biopsy The group with diabetes had a signifi-cantly decreased rate of mitochondrial respirationfueled by palmitoyl-carnitine that correlated with bloodglucose dysregulation while there was no difference inoxidation of pyruvate Diabetic myocardium also hadsignificantly decreased activity of hydroxyacyl-CoA de-hydrogenase (HADHA) and accumulated more lipiddroplets and ceramide Also markers of ER stressresponse (GRP78 and CHOP) and apoptosis (cleavedcaspase-3) were elevated in diabetic myocardiumThese results show that even in the absence of con-tractile failure diabetic heart exhibits a decreasedmitochondrial capacity for b-oxidation increasedaccumulation of intracellular lipids ER stress andgreater degree of apoptosis Lower efficiency of mi-tochondrial fatty acid oxidation may represent a poten-tial target in combating negative effects of diabetes on theheart

Chronic heart failure is a common complication in patientswith type 2 diabetes mellitus (T2DM) (12) Being a com-plex metabolic disorder T2DM is associated with acceler-ated atherosclerosis and coronary artery disease (CAD)leading to an increase in incidence of myocardial infarc-tion heart failure and ischemic heart diseasendashassociateddeath (1) Nevertheless cardiac dysfunction in T2DM canoccur even in the absence of CAD as a part of diabeticcardiomyopathy Each 1 increase in HbA1c was previ-ously shown to result in 8 greater risk of heart failureindependent of other risk factors (3) Various mechanismshave been suggested as potential culprits of myocardialdamage in diabetes ranging from maladaptive inflamma-tory response and endocrine disorder to altered myocar-dial metabolism and utilization of energetic substrates (4)

T2DM also leads to altered metabolism of fat Thisresults in increased lipolysis elevated levels of circulatingfatty acids and their enhanced uptake by peripheraltissues including the heart (4) Consequently diabeticmyocardium ldquoburnsrdquo more fatty acids than nondiabetic(5) However it is not clear whether this is a physiologicalmetabolic response to excess of fatty acid substrate andintracellular deficit of glucose (6) or it can be detrimentalfor the heart (7)

Data from patients with diabetes are very sparse andare mostly obtained by MRI and positron emission to-mography (PET) measurements of cardiac contractile per-formance lipid deposition and energy substrate uptake(8) There are currently two studies that have directly

1Department of Physiology University of Split School of Medicine Split Croatia2Signalisation et Physiopathologie Cardiovasculaire INSERM UMR-S 1180 Uni-versiteacute Paris-Sud Universiteacute Paris-Saclay Chacirctenay-Malabry France3Department of Cardiac Surgery University Hospital Split Split Croatia4Department of Cardiology University Hospital Split Split Croatia5Department of Anatomy University of Split School of Medicine Split Croatia6Universiteacute Versailles St-Quentin Versailles France

Corresponding author Jasna Marinovic jasnamarinovicmefsthr

Received 25 April 2019 and accepted 29 July 2019

Clinical trial reg no NCT03179137 clinicaltrialsgov

This article contains Supplementary Data online at httpdiabetesdiabetesjournalsorglookupsuppldoi102337db19-0423-DC1

copy 2019 by the American Diabetes Association Readers may use this article aslong as the work is properly cited the use is educational and not for profit and thework is not altered More information is available at httpwwwdiabetesjournalsorgcontentlicense

1924 Diabetes Volume 68 October 2019

METABOLISM

investigated mitochondrial function in human diabeticatrial tissue both showing that diabetes is associatedwith mitochondrial dysfunction (910) However todate there are no studies investigating these processesin human ventricular muscle part of the myocardium moststricken by the diabetes complications Also atrial myo-cardium differs significantly from ventricular in mitochon-drial respiratory capacity (11) activity of enzymes involvedin aerobic metabolism (12) and response to noxious stim-uli leading to chronic heart failure (13)

In the current study we aimed to fill the gap betweenfindings obtained in animal models of the disease func-tional data recorded in imaging studies and reports fromhuman right atrium by directly assessing the mitochon-drial capacity for oxidation of carbohydrates and fattyacids in the samples of left ventricle obtained frompatients undergoing coronary artery bypass grafting(CABG) surgery Additionally we assessed some of theintracellular pathways that are linked to mitochondrialfunction and cellular energy balance and which can directlyresult in loss of functional myocytes such as endoplasmicreticulum (ER) stress and apoptosis

RESEARCH DESIGN AND METHODS

Study DesignThirty-seven hemodynamically stable CAD patients sched-uled for elective CABG surgery at the University HospitalSplit were included Emergency patients patients with leftventricular (LV) ejection fraction (LVEF) 50 andpatients with type 1 diabetes concomitant valve replace-ment and severe renal hepatic or pulmonary disease wereexcluded

The included patients fell into one of the two groupcategories nondiabetic (non-DM) group and diabetic (DM)group based on clinical diagnosis of T2DM the use ofdiabetes medication and measurements of fasting plasmaglucose (7 mmolL) or glycosylated hemoglobin(HbA1c 65) Pre intra- and postsurgical procedureswere performed according to the standard clinical routinesof the Department of Cardiac Surgery University HospitalSplit The study complies with the Declaration of Helsinkiand was approved by the ethics committees of the Uni-versity Hospital Split (2181-147-01) and University ofSplit School of Medicine (2181-198-03-04) All patientsgave written informed consent to participate in the studybefore being enrolled This was a single-center trial regis-tered as an observational study at httpwwwclinicaltrialsgov under identification no NCT03179137

Left Ventricular BiopsiesDuring the CABG procedure performed without the useof cardiopulmonary bypass and cardioplegia (ldquooff-pumprdquo)one to two cylinder-shaped biopsies (153 1 mm dimen-sions) were taken from anteroseptal part of the LV aspreviously described (14) Tissue was cut into severalpieces that were either transferred to the laboratory within15 min in ice-cold storage solution (in mmolL 277

CaK2EGTA 723 K2EGTA 656 MgCl2 57 Na2ATP15 phosphocreatine 20 imidazole 20 taurine 05 dithio-threitol and 50 K-methanesulfonate pH 71 at 0degC) andused for mitochondrial respiration measurements or im-mediately snap-frozen in liquid nitrogen and storedat 280degC for later analyses (enzyme activity expressionanalyses histological staining)

ChemicalsAll chemicals used for this study unless otherwise notedwere purchased from Sigma-Aldrich

Mitochondrial RespirationMyocardial biopsy samples were first dissected permea-bilized with saponin (50 mgmL) and incubated in thereaction vessel filled with a respiratory buffer (in mmolL277 CaK2EGTA 723 K2EGTA 138 MgCl2 3 K2HPO420 imidazole 20 taurine 05 DTT 90 K-methanesulfonate10 Na-methanesulfonate and 02 BSA pH 71 at30degC) where mitochondrial respiration was evaluatedwith a Clark-type electrode (Oxygraph Hansatech Instru-ments) (1415) Fatty acid oxidation was assessed usingpalmitoyl-carnitine as substrate (40 mmolL in the pres-ence of 5 mmolL malate) (Fig 1A) and carbohydrateoxidation by providing mitochondria with pyruvate(10 mmolL with addition of 5 mmolL malate) (Fig 1B)The tissue oxygen consumption rate (in pmol O2smgwet tissue weight) a proxy of mitochondrial respirationwas sequentially recorded in the presence of substratesonly upon addition of a saturating amount of ADP(25 mmolL) and trifluorocarbonylcyanide phenylhy-drazone (FCCP) (1 mmolL) an uncoupler of mitochon-drial oxygen consumption from ATP synthesis Ambientoxygen was maintained 210 mmolL to avoid its diffu-sion limitation in the fibers (Fig 1A and B)

RNA Isolation and Quantitative RT-PCRQuantitative RT-PCR of total ventricular RNA was per-formed as previously described (16) mRNA levels for alltarget genes (PPARa [peroxisome proliferatorndashactivatedreceptor a] FATCD36 [fatty acid translocasecluster ofdifferentiation 36] and CPT1 [carnitine palmitoyltrans-ferase 1]) were normalized to Ywhaz and PolR2A levelsFor detection of mature miRNA cDNA was prepared ina reverse transcription reaction using miScript HiSpecBuffer from the miScript II RT Kit (QIAGEN) Real-timePCR was performed using miRNA-specific miScript PrimerAssay (forward primer) and the miScript SYBR Green PCRKit PCR primers were obtained from Eurofins Genomics(Ebersberg Germany) and are listed in SupplementaryTable 1

Biochemical AssaysThe activity of citrate synthase and pyruvate dehydroge-nase (PDH) in homogenized LV tissue was assessed usingcommercial kits (CS0720 Sigma-Aldrich and AAMT008-1KIT Merck Millipore respectively) as previously de-scribed (14) Activity of hydroxyacyl-CoA dehydrogenase

diabetesdiabetesjournalsorg Ljubkovic and Associates 1925

(HADHA) in myocardial homogenate was assessed spec-trophotometrically (at 340 nm [30degC]) by monitoring con-version of acetoacetyl-CoA to hydroxybutyryl-CoA

Western BlottingFor probing the blots of tissue protein the followingprimary antibodies were used anti-VLCAD (very-long-chainacyl-CoA dehydrogenase) (sc-376239 Santa Cruz Biotech-nology) MitoProfile PDH WB Antibody Cocktail (ab110416Abcam) total OXPHOS human antibody cocktail (againstrepresentative subunits of electron transfer chain com-plexes ab110411 MitoSciences) anti-GRP78 (glucose-regulated protein 78) (3183 Cell Signaling Technology)anti-CHOP (sc-793 Santa Cruz Biotechnology) and antindashcaspase-3 (sc-7148 Santa Cruz Biotechnology) Chemilumi-nescent substrate was Luminata Forte (Merck Millipore) andblots were imaged using the ChemiDoc imaging system (Bio-Rad) Actin served as a loading control

Quantification of Intramyocardial Neutral LipidsIntramyocardial detection of neutral lipids was per-formed using oil red O (ORO) (Sigma-Aldrich) stainingBright-field images of 340 magnification were cap-tured and at least 10 frames per biopsy were used foranalysis using ImageJ software The extent of OROstaining is expressed as the pixel number over tissuearea

Ceramide DetectionIntramyocardial ceramide levels were assessed by immu-nostaining of 10-mm cryosections of the ventricular bi-opsies Primary anti-ceramide antibody was obtained fromSigma-Aldrich (CloneMID 15B4 product no C8104 1300dilution) Secondary antibody was goat anti-mouse AlexaFluor 488 (ab150121 Abcam) used at 1500 dilutionFluorescence detection was done using 340 objectiveusing an AxioVision microscope (ZEISS)

Figure 1mdashMitochondria from the left ventricle of patients with diabetes have reduced capacity for fatty acid oxidation A Representativetrace of O2 consumption recording in permeabilized LV fibers (tissue) in the presence of palmitoyl-carnitine and malate (PcM) followingaddition of ADP and the uncoupling agent FCCP The arrow indicates reoxygenation (ReOx) of the experimental vessel to prevent O2 diffusionlimitation in the tissue B Representative trace of O2 consumption recording in the presence of pyruvate and malate (PM) and uponsubsequent addition of ADP and FCCP C Mean values of O2 consumption rates (OCR) from palmitoyl-carnitine protocol described in AD Mean values of O2 consumption rates from pyruvate protocol described in B E Correlation between blood levels of HbA1c andmitochondrial respiration driven by palmitoyl-carnitine and malate following addition of ADP F Correlation between blood levels of HbA1c

and mitochondrial respiration driven by pyruvate and malate following addition of ADP Data in bar graphs are mean 6 SEM n = 18 patients innon-DM group and 13 in DM group ns nonsignificant P 005 vs non-DM group

1926 Metabolic Disturbances in Diabetic Left Ventricle Diabetes Volume 68 October 2019

TUNEL AssayTUNEL assay was performed in frozen myocardial biopsytissue slices using a commercial kit (ApopTag Fluoresceinin Situ Apoptosis Detection Kit Merck) Apoptotic cellswere quantified as percentage of FITC-stained nuclei intotal number of cells Image analysis was performed usingAdobe Photoshop CS6 (Adobe Systems)

Statistical AnalysisFisher exact test was used for comparison of categoricalparameters For metrical parameters normality of distri-bution was checked using the DrsquoAgostino-Pearson test andin case of normal distribution unpaired Student t test wasconducted for comparison Otherwise a nonparametricanalysis was performed (Mann-Whitney test) Data inTable 1 are presented as means 6 SD Data in figuresare presented as means 6 SEM Correlation analysis wasperformed using GraphPad Prism 6 software with a two-sided P value 005 considered significant

Data and Resource AvailabilityThe data sets generated andor analyzed during the cur-rent study are available from the corresponding author

upon reasonable request No applicable resources weregenerated or analyzed during the current study

RESULTS

Patient CharacteristicsMain characteristics of the two patient groups are shownin Table 1 Compared with the non-DM group patientswith diabetes had slightly lower LVEF as well as increasedEuropean System for Cardiac Operative Risk Evaluation(EuroSCORE) II and HbA1c parameters As expected thetwo groups also differed in antidiabetes medication ther-apy All patients successfully underwent the CABG surgerywith no complications evidently related to the LV biopsyprocedure

Decreased Mitochondrial Respiration Driven by FattyAcid in Diabetic MyocardiumWhen mitochondria were provided with palmitoyl-carnitine(in the presence of ADP) respiration was significantlylower in LV myocardium from DM patients compared withthe non-DM group (Fig 1C) On the other hand ADP-supported mitochondrial oxidation of pyruvate was notdifferent between the non-DM and DM groups (Fig 1D)

Table 1mdashClinical characteristics and demographics of patients enrolled in the study

Patients without diabetes (n = 21) Patients with diabetes (n = 16) P

Female sex n () 3 (14) 4 (25) 044

Age (years) 63 6 9 66 6 9 038

EuroSCORE II () 17 6 08 26 6 18 001

Clinical characteristicsHypertension n () 13 (62) 11 (69) 072BMI (kgm2) 284 6 28 297 6 70 053Fasting plasma glucose (mgdL) 1047 6 199 1951 6 752 0001HbA1c ( [mmolmol]) 57 6 05 [39 6 6] 73 6 11 [56 6 12] 0001HDL (mmolL) 11 6 04 12 6 03 07LDL (mmolL) 2 6 07 25 6 1 022TG (mmolL) 19 6 13 17 6 07 087

EchocardiographyLVEF () 673 6 79 602 6 69 001LV relative wall thickness 036 6 01 040 6 01 045LA diameter (mm) 43 6 07 42 6 07 087MV E velocity (cms) 813 6 23 781 6 24 079MV A velocity (cms) 979 6 20 987 6 23 094MV E-to-A ratio 086 6 031 079 6 012 055MV DT (ms) 2237 6 610 2513 6 315 036

Medications n ()Acetylsalicylic acid 17 (81) 13 (81) 100Clopidogrel 13 (62) 9 (56) 074b-Blocker 17 (81) 15 (94) 036ACE inhibitorARB 9 (43) 9 (56) 051Statin 16 (76) 10 (63) 048Nitrate 4 (19) 1 (6) 036Diuretic 7 (33) 10 (63) 01Calcium channel blocker 2 (10) 3 (19) 063Amiodarone 3 (14) 4 (25) 063Insulin 0 (0) 5 (31) 001Oral hypoglycemic agent 0 (0) 11 (69) 0001

Data are means6 SD unless otherwise indicated ARB angiotensin II receptor blocker LA left atrial MV mitral valve MV DT mitral valvedeceleration time P 005

diabetesdiabetesjournalsorg Ljubkovic and Associates 1927

Administration of FCCP accelerated the respiration in bothgroups to the same extent suggesting that maximal elec-tron transfer chain capacity is comparable between themThe rate of mitochondrial respiration driven by palmitoyl-carnitine was negatively correlated with blood levels ofHbA1c (r

2 = 021) Such correlation was not present forpyruvate-fueled mitochondrial respiration

Decreased b-Oxidation in Diabetic MyocardiumCitrate synthase activity a marker of mitochondrialcontent in the tissue was not different between thetwo groups (Fig 2A) Activity of HADHA (responsiblefor the second and third step of b-oxidation) was de-creased in the DM group (Fig 2B) revealing a reducedcapacity for b-oxidation in diabetic hearts Moreoverexpression of VLCAD an enzyme catalyzing the firststep of b-oxidation was significantly reduced in DMmyocardium (Fig 2C) There was no difference in mRNAexpression of other key fatty acid metabolism factorsPPARa FATCD36 and CPT1 (Fig 2D) Also levels ofmiR-33a miR-33b and miR-208a (miRNAs implicated infatty acid metabolism [1718]) were not different be-tween the groups

Activity and expression of the main subunits of PDHwere unaltered in the DM group compared with thenon-DM group (Fig 3A and B) In addition expressionof the representative components of five mitochondrialrespiratory complexes (OXPHOS IndashV) was also not differ-ent between the non-DM and DM groups (Fig 3C) thussupporting respirometry data which suggest that there isno difference in total mitochondrial capacity for oxidativephosphorylation

Increased Accumulation of Fat in Diabetic MyocardiumMyocardial staining using fat-soluble ORO dye revealedincreased accumulation of intracellular lipid droplets inDM groupmyocardium (Fig 4A and B) The staining extentwas positively correlated with the ratio of pyruvate- topalmitoyl-driven mitochondrial respiration (Fig 4C) in-dicating that decreased mitochondrial ability to oxidizepalmitoyl relative to pyruvate is associated with increasedaccumulation of triglycerides (TGs) in cardiomyocytesAlso anti-ceramide immunofluorescence staining revealedincreased levels of ceramide in diabetic myocardium (Fig4D and E)

ER Stress and Apoptosis in Diabetic MyocardiumDiabetic myocardium exhibited significantly increased ex-pression of GRP78 and CHOP indicating activation of theER stress response (Fig 5A and B) This was associatedwith increased levels of cleaved caspase-3 and a tendencytoward increased percentage of TUNEL-positive cells (P =009) (Fig 5C and D) pointing to increased apoptosis indiabetic myocardium

DISCUSSION

In the current study we found that the hearts ofpatients with type 2 diabetes even in the absence ofcontractile failure display 1) a decreased mitochondrialcapacity for fatty acidndashfueled respiration and un-changed mitochondrial oxidative capacity for carbohy-drates 2) a reduced expressionactivity of b-oxidationenzymes 3) an increased accumulation of intracellu-lar TGs and ceramide and 4) increased ER stress andapoptosis

Figure 2mdashMitochondrial b-oxidation is reduced in diabetic myocardium A Activity of citrate synthase indicator of mitochondrial contentin non-DM group (n = 18) and DM group (n = 13) B Activity of HADHA in non-DM and DM groups C Expression of VLCAD in non-DM andDM groups D Expression levels of mRNA coding for key factors involved in cardiac metabolism of fatty acids (PPARa CD36 and CPT1)in non-DM and DM myocardia E Expression levels of miR-33a -33b and -208a which target genes involved in b-oxidation and insulinsignaling in non-DM and DM myocardia Data in bar graphs are mean 6 SEM P 005 vs non-DM group

1928 Metabolic Disturbances in Diabetic Left Ventricle Diabetes Volume 68 October 2019

Mitochondrial Capacity for Substrate Oxidation inDiabetic LV MyocardiumDue to insulin resistance in T2DM there is a greaterextent of lipolysis in adipose tissue with increased fattyacid delivery to the myocardium (5) This is coupled withreduced insulin-stimulated entry of glucose into thecardiac myocytes (via GLUT4) (5) resulting in aug-mented myocardial reliance on fatty acid uptake andmetabolism for production of ATP (19) Evidence forsuch cardiac metabolic impact of T2DM was mostlyobtained in PET studies investigating substrate utiliza-tion at the level of whole heart (8) and were thusinfluenced by many variables including plasma concen-tration of substrates

Despite valuable PET data on overall cardiac substrateutilization it still remains unclear whether T2DM affectsintrinsic function of human ventricular mitochondria Inthe current study cellular and mitochondrial influx ofsubstrates was controlled by permeabilization of the tissueand providing it with fixed amounts of metabolitesthereby minimizing acute effects of plasma insulin andsubstrate availability on mitochondrial respiration Alsowith use of palmitoyl-carnitine some of the rate-limitingsteps in fatty acid utilization (sarcolemmal uptake byFATCD36 and mitochondrial translocation by CPT1)were bypassed By virtue of using pyruvate mitochondrialoxidation of carbohydrates independent of the insulin-mediated GLUT4 uptake was tested

Mitochondrial respiration driven by palmitoyl-carnitinewas decreased in diabetic myocardium while the oxidationof pyruvate was unaffected Patients with higher levelsof HbA1c exhibited greater impairment of mitochondrial

palmitoyl-carnitine oxidation pointing toward a relationshipbetween severity of insulin resistancechronic glycemialevels and mitochondrial dysfunction Indeed loss-of-insulin signaling in the heart by the selective cardiomyo-cyte deletion of insulin receptors in CIRKO mice inducedsignificant mitochondrial dysfunction with reduced mito-chondrial capacity for oxidation of substrates (20) Alsoincreased palmitate load was shown to induce mitochon-drial and cellular damage whereby rat cardiomyocytesincubated with high palmitate concentrations displayeddiminished ability to oxidize fatty acids and intracellularsteatosis (21) Considering that global insulin resistancecauses elevated levels of circulating fatty acids (includingpalmitate) it is possible that their elevated load leads tocardiomyocytesrsquo impairment of fatty acid metabolizationin mitochondria This combined with impairment ofmyocardial insulin signaling (which in itself was shownto induce mitochondrial dysfunction [20]) could furtheraugment the intracellular accumulation of palmitate andstart the cardiotoxic vicious cycle

Alterations in mitochondrial fatty acid oxidation werepreviously reported in atrial tissue from patients withT2DM (910) and in animal models of the disease(202223) Also data from a recent PET study investigat-ing LV substrate metabolism in patients with diabetesagree with our findings by showing that despite increasedabsolute rates of fatty acid oxidation diabetic heart oxi-dizes relatively less of the extracted fatty acids since theincrease in fatty acid esterification was proportionatelyhigher than the increase in oxidation (24) Thereforefindings from our and previous studies suggest that de-spite globally increased fatty acid usage by the diabetic

Figure 3mdashPDH and expression of mitochondrial respiratory chain complexes are unaltered in diabetic left ventricle A Activity of PDH the keyregulating enzyme of carbohydrate oxidation in non-DM and DM groups B Left panel image of representative blot probed with antibodiesagainst main subunits of PDH Right panel mean values of quantified chemiluminescence normalized to non-DM group C Left panel image ofrepresentative blot probed with antibodies aimed at characteristic subunit of each of the mitochondrial respiratory chain complexes (CI to V) Rightpanel mean values of quantified chemiluminescence normalized to non-DM group Data in bar graphs are mean6 SEM mOD mean optical density

diabetesdiabetesjournalsorg Ljubkovic and Associates 1929

hearts (due to increased fatty acid delivery and reducedglucose uptake) the mitochondrial machinery in diabeticmyocardium actually has less capacity for processing fattyacids (the maximum capacity is not likely to be achieved inthe in vivo setting but can result in reported lower percentof fatty acid oxidation relative to their extracted amount)(24) As a result accumulation of fatty acids upstreamfrom the bottleneck in their utilization pathway could leadto their excessive incorporation in various types of lipidsand consequent lipotoxicity (2526)

The finding of the unchanged expression of OXPHOSsubunits in DM group as well as the unchanged rate ofmaximal respiration driven by pyruvate (both ADP andFCCP stimulated) suggests that the observed reduction inmitochondrial oxidation of palmitoyl is not a result ofdefects in mitochondrial respiratory chain activity Rather

these findings suggest the main disruption leading to thedecreased capacity for fatty acid oxidation might be atthe level of b-oxidation Indeed measurement of theexpression and activity of VLCAD and HADHA respec-tively revealed that these b-oxidation steps are down-regulated in diabetic myocardium Along this lineproteomic analysis of insulin resistant myocardium fromCIRKO mice revealed significantly altered expressionof mitochondrial proteins with downregulation ofb-oxidation enzymes (20)

Accumulation of Lipids in Diabetic MyocardiumMeasurements of intracellular neutral lipids revealed a signif-icantly increased amount of lipid droplets inside LV myocar-dium of patients with diabetes This correlated with thedegree of impairment of mitochondrial palmitoyl-carnitine

Figure 4mdashAugmented accumulation of lipid droplets and ceramide in diabetic LV myocardium A Representative images of non-DM and DMleft ventricles stained with ORO and visualized by light microscopy B Quantified values of ORO staining C Correlation between ratioof mitochondrial respiration driven by pyruvate-malate (Pyr) to respiration driven by palmitoyl-carnitine-malate (Palm) (described in Fig 1)and ORO staining D Representative images of non-DM and DM left ventricles probed with anti-ceramide primary antibody (upper panels)and negative controls in which primary antibody was omitted (lower panels) in LV tissue slices obtained from patients without diabetes andpatients with diabetes Green Alexa Fluor 488 fluorescence indicates ceramide staining while blue fluorescence indicates nuclei stained withDAPI E Quantified values of ceramide staining in non-DM and DM myocardia Data in bar graphs are mean6 SEM P 005 vs non-DMgroup

1930 Metabolic Disturbances in Diabetic Left Ventricle Diabetes Volume 68 October 2019

oxidation suggesting that the observed mitochondrial dys-function might be responsible for the enhanced fataccumulation These results are in agreement with earlierdocumentation of an increased lipid content in humandiabetic myocardium based on MRI methodology (25)and on direct histological visualization (however thesewere diabetic hearts in end-stage contractile failure) (27)Accumulation of neutral fat was previously shown toresult in deterioration in cardiac function For exampledeficiency of adipose TG lipase (ATGL) an enzyme re-sponsible for degradation of lipid droplets induces mas-sive cardiac TG deposition and fatal cardiomyopathy (28)

Ceramide was found to be the critical cardiotoxin ina rodent model of lipotoxic cardiomyopathy as reductionin myocardial ceramide resulted in improved cardiac func-tion and survival (29) Moreover obese diabetic rats(Zucker diabetic fatty rats) were previously shown toexhibit cardiac dysfunction associated with excessivemyocardial storage of TG and ceramide (30) Despite theconvincing results on ceramide accumulation obtained inanimal models of diabetes and obesity the contentof ceramide was found to be unaltered in human atrialmyocardium obtained from patients with T2DM (31)However we also detected increased anti-ceramide stain-ing in diabetic myocardium with the most likely explana-tion for this discrepancy being the fact that we used LVmyocardium in contrast to the right atrial appendagesused before (31)

ER Stress and Apoptosis in Diabetic MyocardiumER stress is marked by an excess accumulation of mis-folded proteins that can be triggered by intracellularbuildup of saturated fatty acids (32) ER stress results

in activation of cellular prosurvival response called theldquounfolded protein responserdquo aiming to restore the normalfunction of the ER by increasing expression of proteinchaperones such as GRP78 as well as by decreasing globalprotein synthesis and ldquounloadingrdquo the ER However if theunfolded protein response does not succeed in restorationof normal ER function due to severechronic ER stress thecell turns on its self-destruction program by inducing theproapoptotic transcription factor CHOP (33) In the cur-rent study we detected an upregulation of GRP78 andinduction of CHOP in diabetic myocardium demonstrat-ing a greater degree of ER stress in human diabetic heartsThese data are also in agreement with the results obtainedin animal models of T2DM (34) Interestingly induction ofER stress by tunicamycin can also induce profound mito-chondrial remodeling including decreased ability to pro-cess fatty acids (16) Whether ER stress is the initiator orworks concomitantly with other disturbances (eg fattyacid overload of cardiomyocytes) leading to the downwardspiral of adverse mitochondrial remodeling in human di-abetic myocardium remains to be investigated

Lastly we demonstrate an augmented rate of apoptoticcell death in the DM group compared with the non-DMgroup Our findings are in line with earlier studies ondiabetes and cell death in human myocardium (35)

Limitations of the Current StudySince both patient groups suffered from CAD requiringsurgical revascularization we were not able to studyfactors influencing the diabetic myocardium in absence ofpotentially confounding CAD Also although there was noobvious difference in severity of CAD between DM andnon-DM patients we cannot assess possible differences in

Figure 5mdashER stress and apoptosis are increased in left ventricle of patients with type 2 diabetes A Image from representative blotsprobed against ER stress factors GRP78 and CHOP and cleaved caspase-3 (an indicator of apoptosis) in non-DM and DM groupsB Mean values of quantified chemiluminescence normalized to non-DM group C Representative colocalization images of non-DM andDM myocardia stained with DAPI (blue) or FITC (green [apoptotic nuclei]) D Average percentages of TUNEL-positive nuclei in non-DMand DM left ventricles Data in bar graphs are mean 6 SEM P 005 vs non-DM group

diabetesdiabetesjournalsorg Ljubkovic and Associates 1931

microvascular function between the two groups The con-centration of glucose and lipids in the blood was deter-mined in a fasted state prior to the surgery As we did notassess blood levels of insulin free fatty acid or glucose atthe time of the biopsy procedure we cannot assess theirpossible acute effects However by using permeabilizedmyocardial tissue and fixed concentrations of substrate formitochondrial functional assay we believe that acuteeffects of insulin and substrate availability on our resultswere minimized Finally due to the small amount ofbiopsy sample we could not perform all of the measure-ments in tissues from all of the patients

ConclusionOur study shows that LV myocardium of patients withT2DM has significantly altered mitochondrial functionwith decreased capacity to oxidize long-chain fatty acidsthrough reduced activity and expression of b-oxidationenzymes This is associated with excessive accumulationof fat in cardiac cells in form of intracellular lipid dropletsand ceramide and is paralleled with ER stress and in-creased apoptosis Therefore based on our and previousdata it seems that insulin resistance with abundantfatty acid loading of cardiomyocytes and ER stress leadsto decreased ability of mitochondria to handle largeamounts of fatty acids being delivered to the cardiomyo-cyte As a result although total fatty acid usage in diabetichearts is increased it is actually insufficient to clear allintracellular fatty acids This may lead to cardiac steatosisand lipotoxicity extending to a loss of cardiac myocytesby apoptosis Therefore besides targeting insulin resis-tance as the primary aim of antidiabetes therapy an-other target for treating diabetic myocardium might befurther increasing its mitochondrial capacity for fattyacid oxidation

Acknowledgments The authors thank medical personnel of Departmentof Cardiac Surgery at University Hospital Split especially Slavica Kotromanovic forhelp with collection of patient data and biopsy samples as well as Lucija Frankovicfor technical assistanceFunding This work was supported by Croatian Science Foundation grants6153 to ML and 3718 to DB and by the French grant ANR-10-LABX-33 to CLand MG as members of the Laboratory of Excellence LERMITDuality of Interest No potential conflicts of interest relevant to this articlewere reportedAuthor Contributions ML and JM designed the study researcheddata and wrote the manuscript ML MG CB MC DB DF IG CL andJM reviewed and edited the manuscript MG CB MC DB DF and IGresearched data CL planned the experiments and researched data ML and JMare the guarantors of this work and as such had full access to all the data in thestudy and take responsibility for the integrity of the data and the accuracy of thedata analysis

References1 Fox CS Coady S Sorlie PD et al Trends in cardiovascular complications ofdiabetes JAMA 20042922495ndash24992 Benjamin EJ Blaha MJ Chiuve SE et al American Heart AssociationStatistics Committee and Stroke Statistics Subcommittee Heart disease andstroke statistics-2017 update a report from the American Heart Association

[published correction appears in Circulation 2017135e646 published correctionappears in Circulation 2017136e196] Circulation 2017135e146ndashe6033 Stratton IM Adler AI Neil HA et al Association of glycaemia with mac-rovascular and microvascular complications of type 2 diabetes (UKPDS 35)prospective observational study BMJ 2000321405ndash4124 Jia G Hill MA Sowers JR Diabetic cardiomyopathy an update of mech-anisms contributing to this clinical entity Circ Res 2018122624ndash6385 Chong CR Clarke K Levelt E Metabolic remodeling in diabetic cardiomy-opathy Cardiovasc Res 2017113422ndash4306 Fillmore N Mori J Lopaschuk GD Mitochondrial fatty acid oxidation al-terations in heart failure ischaemic heart disease and diabetic cardiomyopathyBr J Pharmacol 20141712080ndash20907 Rector RS Payne RM Ibdah JA Mitochondrial trifunctional protein defectsclinical implications and therapeutic approaches Adv Drug Deliv Rev 2008601488ndash14968 Peterson LR Herrero P Schechtman KB et al Effect of obesity and insulinresistance on myocardial substrate metabolism and efficiency in young womenCirculation 20041092191ndash21969 Anderson EJ Kypson AP Rodriguez E Anderson CA Lehr EJ Neufer PDSubstrate-specific derangements in mitochondrial metabolism and redox balancein the atrium of the type 2 diabetic human heart J Am Coll Cardiol 2009541891ndash189810 Montaigne D Marechal X Coisne A et al Myocardial contractile dysfunctionis associated with impaired mitochondrial function and dynamics in type 2 diabeticbut not in obese patients Circulation 2014130554ndash56411 Lemieux H Semsroth S Antretter H Houmlfer D Gnaiger E Mitochondrialrespiratory control and early defects of oxidative phosphorylation in the failinghuman heart Int J Biochem Cell Biol 2011431729ndash173812 Bass A Stejskalovaacute M Ostaacutedal B Samaacutenek M Differences between atrialand ventricular energy-supplying enzymes in five mammalian species Physiol Res1993421ndash613 Cardin S Pelletier P Libby E et al Marked differences between atrial andventricular gene-expression remodeling in dogs with experimental heart failure JMol Cell Cardiol 200845821ndash83114 Cavar M Ljubkovic M Bulat C et al Trimetazidine does not alter metabolicsubstrate oxidation in cardiac mitochondria of target patient population Br JPharmacol 20161731529ndash154015 Kuznetsov AV Veksler V Gellerich FN Saks V Margreiter R Kunz WSAnalysis of mitochondrial function in situ in permeabilized muscle fibers tissuesand cells Nat Protoc 20083965ndash97616 Prola A Nichtova Z Pires Da Silva J et al Endoplasmic reticulum stressinduces cardiac dysfunction through architectural modifications and alteration ofmitochondrial function in cardiomyocytes Cardiovasc Res 2019115328ndash34217 Gerin I Clerbaux LA Haumont O et al Expression of miR-33 from an SREBP2intron inhibits cholesterol export and fatty acid oxidation J Biol Chem 201028533652ndash3366118 Blumensatt M Fahlbusch P Hilgers R et al Secretory products fromepicardial adipose tissue from patients with type 2 diabetes impair mitochondrialb-oxidation in cardiomyocytes via activation of the cardiac renin-angiotensinsystem and induction of miR-208a Basic Res Cardiol 2017112219 Carley AN Severson DL Fatty acid metabolism is enhanced in type 2 diabetichearts Biochim Biophys Acta 20051734112ndash12620 Boudina S Bugger H Sena S et al Contribution of impaired myocardialinsulin signaling to mitochondrial dysfunction and oxidative stress in the heartCirculation 20091191272ndash128321 Hickson-Bick DL Buja LM McMillin JB Palmitate-mediated alterations inthe fatty acid metabolism of rat neonatal cardiac myocytes J Mol Cell Cardiol200032511ndash51922 Beaudoin MS Perry CG Arkell AM et al Impairments in mitochondrialpalmitoyl-CoA respiratory kinetics that precede development of diabetic cardio-myopathy are prevented by resveratrol in ZDF rats J Physiol 20145922519ndash2533

1932 Metabolic Disturbances in Diabetic Left Ventricle Diabetes Volume 68 October 2019

TUNEL AssayTUNEL assay was performed in frozen myocardial biopsytissue slices using a commercial kit (ApopTag Fluoresceinin Situ Apoptosis Detection Kit Merck) Apoptotic cellswere quantified as percentage of FITC-stained nuclei intotal number of cells Image analysis was performed usingAdobe Photoshop CS6 (Adobe Systems)

Statistical AnalysisFisher exact test was used for comparison of categoricalparameters For metrical parameters normality of distri-bution was checked using the DrsquoAgostino-Pearson test andin case of normal distribution unpaired Student t test wasconducted for comparison Otherwise a nonparametricanalysis was performed (Mann-Whitney test) Data inTable 1 are presented as means 6 SD Data in figuresare presented as means 6 SEM Correlation analysis wasperformed using GraphPad Prism 6 software with a two-sided P value 005 considered significant

Data and Resource AvailabilityThe data sets generated andor analyzed during the cur-rent study are available from the corresponding author

upon reasonable request No applicable resources weregenerated or analyzed during the current study

RESULTS

Patient CharacteristicsMain characteristics of the two patient groups are shownin Table 1 Compared with the non-DM group patientswith diabetes had slightly lower LVEF as well as increasedEuropean System for Cardiac Operative Risk Evaluation(EuroSCORE) II and HbA1c parameters As expected thetwo groups also differed in antidiabetes medication ther-apy All patients successfully underwent the CABG surgerywith no complications evidently related to the LV biopsyprocedure

Decreased Mitochondrial Respiration Driven by FattyAcid in Diabetic MyocardiumWhen mitochondria were provided with palmitoyl-carnitine(in the presence of ADP) respiration was significantlylower in LV myocardium from DM patients compared withthe non-DM group (Fig 1C) On the other hand ADP-supported mitochondrial oxidation of pyruvate was notdifferent between the non-DM and DM groups (Fig 1D)

Table 1mdashClinical characteristics and demographics of patients enrolled in the study

Patients without diabetes (n = 21) Patients with diabetes (n = 16) P

Female sex n () 3 (14) 4 (25) 044

Age (years) 63 6 9 66 6 9 038

EuroSCORE II () 17 6 08 26 6 18 001

Clinical characteristicsHypertension n () 13 (62) 11 (69) 072BMI (kgm2) 284 6 28 297 6 70 053Fasting plasma glucose (mgdL) 1047 6 199 1951 6 752 0001HbA1c ( [mmolmol]) 57 6 05 [39 6 6] 73 6 11 [56 6 12] 0001HDL (mmolL) 11 6 04 12 6 03 07LDL (mmolL) 2 6 07 25 6 1 022TG (mmolL) 19 6 13 17 6 07 087

EchocardiographyLVEF () 673 6 79 602 6 69 001LV relative wall thickness 036 6 01 040 6 01 045LA diameter (mm) 43 6 07 42 6 07 087MV E velocity (cms) 813 6 23 781 6 24 079MV A velocity (cms) 979 6 20 987 6 23 094MV E-to-A ratio 086 6 031 079 6 012 055MV DT (ms) 2237 6 610 2513 6 315 036

Medications n ()Acetylsalicylic acid 17 (81) 13 (81) 100Clopidogrel 13 (62) 9 (56) 074b-Blocker 17 (81) 15 (94) 036ACE inhibitorARB 9 (43) 9 (56) 051Statin 16 (76) 10 (63) 048Nitrate 4 (19) 1 (6) 036Diuretic 7 (33) 10 (63) 01Calcium channel blocker 2 (10) 3 (19) 063Amiodarone 3 (14) 4 (25) 063Insulin 0 (0) 5 (31) 001Oral hypoglycemic agent 0 (0) 11 (69) 0001

Data are means6 SD unless otherwise indicated ARB angiotensin II receptor blocker LA left atrial MV mitral valve MV DT mitral valvedeceleration time P 005

diabetesdiabetesjournalsorg Ljubkovic and Associates 1927

Administration of FCCP accelerated the respiration in bothgroups to the same extent suggesting that maximal elec-tron transfer chain capacity is comparable between themThe rate of mitochondrial respiration driven by palmitoyl-carnitine was negatively correlated with blood levels ofHbA1c (r

2 = 021) Such correlation was not present forpyruvate-fueled mitochondrial respiration

Decreased b-Oxidation in Diabetic MyocardiumCitrate synthase activity a marker of mitochondrialcontent in the tissue was not different between thetwo groups (Fig 2A) Activity of HADHA (responsiblefor the second and third step of b-oxidation) was de-creased in the DM group (Fig 2B) revealing a reducedcapacity for b-oxidation in diabetic hearts Moreoverexpression of VLCAD an enzyme catalyzing the firststep of b-oxidation was significantly reduced in DMmyocardium (Fig 2C) There was no difference in mRNAexpression of other key fatty acid metabolism factorsPPARa FATCD36 and CPT1 (Fig 2D) Also levels ofmiR-33a miR-33b and miR-208a (miRNAs implicated infatty acid metabolism [1718]) were not different be-tween the groups

Activity and expression of the main subunits of PDHwere unaltered in the DM group compared with thenon-DM group (Fig 3A and B) In addition expressionof the representative components of five mitochondrialrespiratory complexes (OXPHOS IndashV) was also not differ-ent between the non-DM and DM groups (Fig 3C) thussupporting respirometry data which suggest that there isno difference in total mitochondrial capacity for oxidativephosphorylation

Increased Accumulation of Fat in Diabetic MyocardiumMyocardial staining using fat-soluble ORO dye revealedincreased accumulation of intracellular lipid droplets inDM groupmyocardium (Fig 4A and B) The staining extentwas positively correlated with the ratio of pyruvate- topalmitoyl-driven mitochondrial respiration (Fig 4C) in-dicating that decreased mitochondrial ability to oxidizepalmitoyl relative to pyruvate is associated with increasedaccumulation of triglycerides (TGs) in cardiomyocytesAlso anti-ceramide immunofluorescence staining revealedincreased levels of ceramide in diabetic myocardium (Fig4D and E)

ER Stress and Apoptosis in Diabetic MyocardiumDiabetic myocardium exhibited significantly increased ex-pression of GRP78 and CHOP indicating activation of theER stress response (Fig 5A and B) This was associatedwith increased levels of cleaved caspase-3 and a tendencytoward increased percentage of TUNEL-positive cells (P =009) (Fig 5C and D) pointing to increased apoptosis indiabetic myocardium

DISCUSSION

In the current study we found that the hearts ofpatients with type 2 diabetes even in the absence ofcontractile failure display 1) a decreased mitochondrialcapacity for fatty acidndashfueled respiration and un-changed mitochondrial oxidative capacity for carbohy-drates 2) a reduced expressionactivity of b-oxidationenzymes 3) an increased accumulation of intracellu-lar TGs and ceramide and 4) increased ER stress andapoptosis

Figure 2mdashMitochondrial b-oxidation is reduced in diabetic myocardium A Activity of citrate synthase indicator of mitochondrial contentin non-DM group (n = 18) and DM group (n = 13) B Activity of HADHA in non-DM and DM groups C Expression of VLCAD in non-DM andDM groups D Expression levels of mRNA coding for key factors involved in cardiac metabolism of fatty acids (PPARa CD36 and CPT1)in non-DM and DM myocardia E Expression levels of miR-33a -33b and -208a which target genes involved in b-oxidation and insulinsignaling in non-DM and DM myocardia Data in bar graphs are mean 6 SEM P 005 vs non-DM group

1928 Metabolic Disturbances in Diabetic Left Ventricle Diabetes Volume 68 October 2019

Mitochondrial Capacity for Substrate Oxidation inDiabetic LV MyocardiumDue to insulin resistance in T2DM there is a greaterextent of lipolysis in adipose tissue with increased fattyacid delivery to the myocardium (5) This is coupled withreduced insulin-stimulated entry of glucose into thecardiac myocytes (via GLUT4) (5) resulting in aug-mented myocardial reliance on fatty acid uptake andmetabolism for production of ATP (19) Evidence forsuch cardiac metabolic impact of T2DM was mostlyobtained in PET studies investigating substrate utiliza-tion at the level of whole heart (8) and were thusinfluenced by many variables including plasma concen-tration of substrates

Despite valuable PET data on overall cardiac substrateutilization it still remains unclear whether T2DM affectsintrinsic function of human ventricular mitochondria Inthe current study cellular and mitochondrial influx ofsubstrates was controlled by permeabilization of the tissueand providing it with fixed amounts of metabolitesthereby minimizing acute effects of plasma insulin andsubstrate availability on mitochondrial respiration Alsowith use of palmitoyl-carnitine some of the rate-limitingsteps in fatty acid utilization (sarcolemmal uptake byFATCD36 and mitochondrial translocation by CPT1)were bypassed By virtue of using pyruvate mitochondrialoxidation of carbohydrates independent of the insulin-mediated GLUT4 uptake was tested

Mitochondrial respiration driven by palmitoyl-carnitinewas decreased in diabetic myocardium while the oxidationof pyruvate was unaffected Patients with higher levelsof HbA1c exhibited greater impairment of mitochondrial

palmitoyl-carnitine oxidation pointing toward a relationshipbetween severity of insulin resistancechronic glycemialevels and mitochondrial dysfunction Indeed loss-of-insulin signaling in the heart by the selective cardiomyo-cyte deletion of insulin receptors in CIRKO mice inducedsignificant mitochondrial dysfunction with reduced mito-chondrial capacity for oxidation of substrates (20) Alsoincreased palmitate load was shown to induce mitochon-drial and cellular damage whereby rat cardiomyocytesincubated with high palmitate concentrations displayeddiminished ability to oxidize fatty acids and intracellularsteatosis (21) Considering that global insulin resistancecauses elevated levels of circulating fatty acids (includingpalmitate) it is possible that their elevated load leads tocardiomyocytesrsquo impairment of fatty acid metabolizationin mitochondria This combined with impairment ofmyocardial insulin signaling (which in itself was shownto induce mitochondrial dysfunction [20]) could furtheraugment the intracellular accumulation of palmitate andstart the cardiotoxic vicious cycle

Alterations in mitochondrial fatty acid oxidation werepreviously reported in atrial tissue from patients withT2DM (910) and in animal models of the disease(202223) Also data from a recent PET study investigat-ing LV substrate metabolism in patients with diabetesagree with our findings by showing that despite increasedabsolute rates of fatty acid oxidation diabetic heart oxi-dizes relatively less of the extracted fatty acids since theincrease in fatty acid esterification was proportionatelyhigher than the increase in oxidation (24) Thereforefindings from our and previous studies suggest that de-spite globally increased fatty acid usage by the diabetic

Figure 3mdashPDH and expression of mitochondrial respiratory chain complexes are unaltered in diabetic left ventricle A Activity of PDH the keyregulating enzyme of carbohydrate oxidation in non-DM and DM groups B Left panel image of representative blot probed with antibodiesagainst main subunits of PDH Right panel mean values of quantified chemiluminescence normalized to non-DM group C Left panel image ofrepresentative blot probed with antibodies aimed at characteristic subunit of each of the mitochondrial respiratory chain complexes (CI to V) Rightpanel mean values of quantified chemiluminescence normalized to non-DM group Data in bar graphs are mean6 SEM mOD mean optical density

diabetesdiabetesjournalsorg Ljubkovic and Associates 1929

hearts (due to increased fatty acid delivery and reducedglucose uptake) the mitochondrial machinery in diabeticmyocardium actually has less capacity for processing fattyacids (the maximum capacity is not likely to be achieved inthe in vivo setting but can result in reported lower percentof fatty acid oxidation relative to their extracted amount)(24) As a result accumulation of fatty acids upstreamfrom the bottleneck in their utilization pathway could leadto their excessive incorporation in various types of lipidsand consequent lipotoxicity (2526)

The finding of the unchanged expression of OXPHOSsubunits in DM group as well as the unchanged rate ofmaximal respiration driven by pyruvate (both ADP andFCCP stimulated) suggests that the observed reduction inmitochondrial oxidation of palmitoyl is not a result ofdefects in mitochondrial respiratory chain activity Rather

these findings suggest the main disruption leading to thedecreased capacity for fatty acid oxidation might be atthe level of b-oxidation Indeed measurement of theexpression and activity of VLCAD and HADHA respec-tively revealed that these b-oxidation steps are down-regulated in diabetic myocardium Along this lineproteomic analysis of insulin resistant myocardium fromCIRKO mice revealed significantly altered expressionof mitochondrial proteins with downregulation ofb-oxidation enzymes (20)

Accumulation of Lipids in Diabetic MyocardiumMeasurements of intracellular neutral lipids revealed a signif-icantly increased amount of lipid droplets inside LV myocar-dium of patients with diabetes This correlated with thedegree of impairment of mitochondrial palmitoyl-carnitine

Figure 4mdashAugmented accumulation of lipid droplets and ceramide in diabetic LV myocardium A Representative images of non-DM and DMleft ventricles stained with ORO and visualized by light microscopy B Quantified values of ORO staining C Correlation between ratioof mitochondrial respiration driven by pyruvate-malate (Pyr) to respiration driven by palmitoyl-carnitine-malate (Palm) (described in Fig 1)and ORO staining D Representative images of non-DM and DM left ventricles probed with anti-ceramide primary antibody (upper panels)and negative controls in which primary antibody was omitted (lower panels) in LV tissue slices obtained from patients without diabetes andpatients with diabetes Green Alexa Fluor 488 fluorescence indicates ceramide staining while blue fluorescence indicates nuclei stained withDAPI E Quantified values of ceramide staining in non-DM and DM myocardia Data in bar graphs are mean6 SEM P 005 vs non-DMgroup

1930 Metabolic Disturbances in Diabetic Left Ventricle Diabetes Volume 68 October 2019

oxidation suggesting that the observed mitochondrial dys-function might be responsible for the enhanced fataccumulation These results are in agreement with earlierdocumentation of an increased lipid content in humandiabetic myocardium based on MRI methodology (25)and on direct histological visualization (however thesewere diabetic hearts in end-stage contractile failure) (27)Accumulation of neutral fat was previously shown toresult in deterioration in cardiac function For exampledeficiency of adipose TG lipase (ATGL) an enzyme re-sponsible for degradation of lipid droplets induces mas-sive cardiac TG deposition and fatal cardiomyopathy (28)

Ceramide was found to be the critical cardiotoxin ina rodent model of lipotoxic cardiomyopathy as reductionin myocardial ceramide resulted in improved cardiac func-tion and survival (29) Moreover obese diabetic rats(Zucker diabetic fatty rats) were previously shown toexhibit cardiac dysfunction associated with excessivemyocardial storage of TG and ceramide (30) Despite theconvincing results on ceramide accumulation obtained inanimal models of diabetes and obesity the contentof ceramide was found to be unaltered in human atrialmyocardium obtained from patients with T2DM (31)However we also detected increased anti-ceramide stain-ing in diabetic myocardium with the most likely explana-tion for this discrepancy being the fact that we used LVmyocardium in contrast to the right atrial appendagesused before (31)

ER Stress and Apoptosis in Diabetic MyocardiumER stress is marked by an excess accumulation of mis-folded proteins that can be triggered by intracellularbuildup of saturated fatty acids (32) ER stress results

in activation of cellular prosurvival response called theldquounfolded protein responserdquo aiming to restore the normalfunction of the ER by increasing expression of proteinchaperones such as GRP78 as well as by decreasing globalprotein synthesis and ldquounloadingrdquo the ER However if theunfolded protein response does not succeed in restorationof normal ER function due to severechronic ER stress thecell turns on its self-destruction program by inducing theproapoptotic transcription factor CHOP (33) In the cur-rent study we detected an upregulation of GRP78 andinduction of CHOP in diabetic myocardium demonstrat-ing a greater degree of ER stress in human diabetic heartsThese data are also in agreement with the results obtainedin animal models of T2DM (34) Interestingly induction ofER stress by tunicamycin can also induce profound mito-chondrial remodeling including decreased ability to pro-cess fatty acids (16) Whether ER stress is the initiator orworks concomitantly with other disturbances (eg fattyacid overload of cardiomyocytes) leading to the downwardspiral of adverse mitochondrial remodeling in human di-abetic myocardium remains to be investigated

Lastly we demonstrate an augmented rate of apoptoticcell death in the DM group compared with the non-DMgroup Our findings are in line with earlier studies ondiabetes and cell death in human myocardium (35)

Limitations of the Current StudySince both patient groups suffered from CAD requiringsurgical revascularization we were not able to studyfactors influencing the diabetic myocardium in absence ofpotentially confounding CAD Also although there was noobvious difference in severity of CAD between DM andnon-DM patients we cannot assess possible differences in

Figure 5mdashER stress and apoptosis are increased in left ventricle of patients with type 2 diabetes A Image from representative blotsprobed against ER stress factors GRP78 and CHOP and cleaved caspase-3 (an indicator of apoptosis) in non-DM and DM groupsB Mean values of quantified chemiluminescence normalized to non-DM group C Representative colocalization images of non-DM andDM myocardia stained with DAPI (blue) or FITC (green [apoptotic nuclei]) D Average percentages of TUNEL-positive nuclei in non-DMand DM left ventricles Data in bar graphs are mean 6 SEM P 005 vs non-DM group

diabetesdiabetesjournalsorg Ljubkovic and Associates 1931

microvascular function between the two groups The con-centration of glucose and lipids in the blood was deter-mined in a fasted state prior to the surgery As we did notassess blood levels of insulin free fatty acid or glucose atthe time of the biopsy procedure we cannot assess theirpossible acute effects However by using permeabilizedmyocardial tissue and fixed concentrations of substrate formitochondrial functional assay we believe that acuteeffects of insulin and substrate availability on our resultswere minimized Finally due to the small amount ofbiopsy sample we could not perform all of the measure-ments in tissues from all of the patients

ConclusionOur study shows that LV myocardium of patients withT2DM has significantly altered mitochondrial functionwith decreased capacity to oxidize long-chain fatty acidsthrough reduced activity and expression of b-oxidationenzymes This is associated with excessive accumulationof fat in cardiac cells in form of intracellular lipid dropletsand ceramide and is paralleled with ER stress and in-creased apoptosis Therefore based on our and previousdata it seems that insulin resistance with abundantfatty acid loading of cardiomyocytes and ER stress leadsto decreased ability of mitochondria to handle largeamounts of fatty acids being delivered to the cardiomyo-cyte As a result although total fatty acid usage in diabetichearts is increased it is actually insufficient to clear allintracellular fatty acids This may lead to cardiac steatosisand lipotoxicity extending to a loss of cardiac myocytesby apoptosis Therefore besides targeting insulin resis-tance as the primary aim of antidiabetes therapy an-other target for treating diabetic myocardium might befurther increasing its mitochondrial capacity for fattyacid oxidation

Acknowledgments The authors thank medical personnel of Departmentof Cardiac Surgery at University Hospital Split especially Slavica Kotromanovic forhelp with collection of patient data and biopsy samples as well as Lucija Frankovicfor technical assistanceFunding This work was supported by Croatian Science Foundation grants6153 to ML and 3718 to DB and by the French grant ANR-10-LABX-33 to CLand MG as members of the Laboratory of Excellence LERMITDuality of Interest No potential conflicts of interest relevant to this articlewere reportedAuthor Contributions ML and JM designed the study researcheddata and wrote the manuscript ML MG CB MC DB DF IG CL andJM reviewed and edited the manuscript MG CB MC DB DF and IGresearched data CL planned the experiments and researched data ML and JMare the guarantors of this work and as such had full access to all the data in thestudy and take responsibility for the integrity of the data and the accuracy of thedata analysis

References1 Fox CS Coady S Sorlie PD et al Trends in cardiovascular complications ofdiabetes JAMA 20042922495ndash24992 Benjamin EJ Blaha MJ Chiuve SE et al American Heart AssociationStatistics Committee and Stroke Statistics Subcommittee Heart disease andstroke statistics-2017 update a report from the American Heart Association

[published correction appears in Circulation 2017135e646 published correctionappears in Circulation 2017136e196] Circulation 2017135e146ndashe6033 Stratton IM Adler AI Neil HA et al Association of glycaemia with mac-rovascular and microvascular complications of type 2 diabetes (UKPDS 35)prospective observational study BMJ 2000321405ndash4124 Jia G Hill MA Sowers JR Diabetic cardiomyopathy an update of mech-anisms contributing to this clinical entity Circ Res 2018122624ndash6385 Chong CR Clarke K Levelt E Metabolic remodeling in diabetic cardiomy-opathy Cardiovasc Res 2017113422ndash4306 Fillmore N Mori J Lopaschuk GD Mitochondrial fatty acid oxidation al-terations in heart failure ischaemic heart disease and diabetic cardiomyopathyBr J Pharmacol 20141712080ndash20907 Rector RS Payne RM Ibdah JA Mitochondrial trifunctional protein defectsclinical implications and therapeutic approaches Adv Drug Deliv Rev 2008601488ndash14968 Peterson LR Herrero P Schechtman KB et al Effect of obesity and insulinresistance on myocardial substrate metabolism and efficiency in young womenCirculation 20041092191ndash21969 Anderson EJ Kypson AP Rodriguez E Anderson CA Lehr EJ Neufer PDSubstrate-specific derangements in mitochondrial metabolism and redox balancein the atrium of the type 2 diabetic human heart J Am Coll Cardiol 2009541891ndash189810 Montaigne D Marechal X Coisne A et al Myocardial contractile dysfunctionis associated with impaired mitochondrial function and dynamics in type 2 diabeticbut not in obese patients Circulation 2014130554ndash56411 Lemieux H Semsroth S Antretter H Houmlfer D Gnaiger E Mitochondrialrespiratory control and early defects of oxidative phosphorylation in the failinghuman heart Int J Biochem Cell Biol 2011431729ndash173812 Bass A Stejskalovaacute M Ostaacutedal B Samaacutenek M Differences between atrialand ventricular energy-supplying enzymes in five mammalian species Physiol Res1993421ndash613 Cardin S Pelletier P Libby E et al Marked differences between atrial andventricular gene-expression remodeling in dogs with experimental heart failure JMol Cell Cardiol 200845821ndash83114 Cavar M Ljubkovic M Bulat C et al Trimetazidine does not alter metabolicsubstrate oxidation in cardiac mitochondria of target patient population Br JPharmacol 20161731529ndash154015 Kuznetsov AV Veksler V Gellerich FN Saks V Margreiter R Kunz WSAnalysis of mitochondrial function in situ in permeabilized muscle fibers tissuesand cells Nat Protoc 20083965ndash97616 Prola A Nichtova Z Pires Da Silva J et al Endoplasmic reticulum stressinduces cardiac dysfunction through architectural modifications and alteration ofmitochondrial function in cardiomyocytes Cardiovasc Res 2019115328ndash34217 Gerin I Clerbaux LA Haumont O et al Expression of miR-33 from an SREBP2intron inhibits cholesterol export and fatty acid oxidation J Biol Chem 201028533652ndash3366118 Blumensatt M Fahlbusch P Hilgers R et al Secretory products fromepicardial adipose tissue from patients with type 2 diabetes impair mitochondrialb-oxidation in cardiomyocytes via activation of the cardiac renin-angiotensinsystem and induction of miR-208a Basic Res Cardiol 2017112219 Carley AN Severson DL Fatty acid metabolism is enhanced in type 2 diabetichearts Biochim Biophys Acta 20051734112ndash12620 Boudina S Bugger H Sena S et al Contribution of impaired myocardialinsulin signaling to mitochondrial dysfunction and oxidative stress in the heartCirculation 20091191272ndash128321 Hickson-Bick DL Buja LM McMillin JB Palmitate-mediated alterations inthe fatty acid metabolism of rat neonatal cardiac myocytes J Mol Cell Cardiol200032511ndash51922 Beaudoin MS Perry CG Arkell AM et al Impairments in mitochondrialpalmitoyl-CoA respiratory kinetics that precede development of diabetic cardio-myopathy are prevented by resveratrol in ZDF rats J Physiol 20145922519ndash2533

1932 Metabolic Disturbances in Diabetic Left Ventricle Diabetes Volume 68 October 2019

Administration of FCCP accelerated the respiration in bothgroups to the same extent suggesting that maximal elec-tron transfer chain capacity is comparable between themThe rate of mitochondrial respiration driven by palmitoyl-carnitine was negatively correlated with blood levels ofHbA1c (r

2 = 021) Such correlation was not present forpyruvate-fueled mitochondrial respiration

Decreased b-Oxidation in Diabetic MyocardiumCitrate synthase activity a marker of mitochondrialcontent in the tissue was not different between thetwo groups (Fig 2A) Activity of HADHA (responsiblefor the second and third step of b-oxidation) was de-creased in the DM group (Fig 2B) revealing a reducedcapacity for b-oxidation in diabetic hearts Moreoverexpression of VLCAD an enzyme catalyzing the firststep of b-oxidation was significantly reduced in DMmyocardium (Fig 2C) There was no difference in mRNAexpression of other key fatty acid metabolism factorsPPARa FATCD36 and CPT1 (Fig 2D) Also levels ofmiR-33a miR-33b and miR-208a (miRNAs implicated infatty acid metabolism [1718]) were not different be-tween the groups

Activity and expression of the main subunits of PDHwere unaltered in the DM group compared with thenon-DM group (Fig 3A and B) In addition expressionof the representative components of five mitochondrialrespiratory complexes (OXPHOS IndashV) was also not differ-ent between the non-DM and DM groups (Fig 3C) thussupporting respirometry data which suggest that there isno difference in total mitochondrial capacity for oxidativephosphorylation

Increased Accumulation of Fat in Diabetic MyocardiumMyocardial staining using fat-soluble ORO dye revealedincreased accumulation of intracellular lipid droplets inDM groupmyocardium (Fig 4A and B) The staining extentwas positively correlated with the ratio of pyruvate- topalmitoyl-driven mitochondrial respiration (Fig 4C) in-dicating that decreased mitochondrial ability to oxidizepalmitoyl relative to pyruvate is associated with increasedaccumulation of triglycerides (TGs) in cardiomyocytesAlso anti-ceramide immunofluorescence staining revealedincreased levels of ceramide in diabetic myocardium (Fig4D and E)

ER Stress and Apoptosis in Diabetic MyocardiumDiabetic myocardium exhibited significantly increased ex-pression of GRP78 and CHOP indicating activation of theER stress response (Fig 5A and B) This was associatedwith increased levels of cleaved caspase-3 and a tendencytoward increased percentage of TUNEL-positive cells (P =009) (Fig 5C and D) pointing to increased apoptosis indiabetic myocardium

DISCUSSION

In the current study we found that the hearts ofpatients with type 2 diabetes even in the absence ofcontractile failure display 1) a decreased mitochondrialcapacity for fatty acidndashfueled respiration and un-changed mitochondrial oxidative capacity for carbohy-drates 2) a reduced expressionactivity of b-oxidationenzymes 3) an increased accumulation of intracellu-lar TGs and ceramide and 4) increased ER stress andapoptosis

Figure 2mdashMitochondrial b-oxidation is reduced in diabetic myocardium A Activity of citrate synthase indicator of mitochondrial contentin non-DM group (n = 18) and DM group (n = 13) B Activity of HADHA in non-DM and DM groups C Expression of VLCAD in non-DM andDM groups D Expression levels of mRNA coding for key factors involved in cardiac metabolism of fatty acids (PPARa CD36 and CPT1)in non-DM and DM myocardia E Expression levels of miR-33a -33b and -208a which target genes involved in b-oxidation and insulinsignaling in non-DM and DM myocardia Data in bar graphs are mean 6 SEM P 005 vs non-DM group

1928 Metabolic Disturbances in Diabetic Left Ventricle Diabetes Volume 68 October 2019

Mitochondrial Capacity for Substrate Oxidation inDiabetic LV MyocardiumDue to insulin resistance in T2DM there is a greaterextent of lipolysis in adipose tissue with increased fattyacid delivery to the myocardium (5) This is coupled withreduced insulin-stimulated entry of glucose into thecardiac myocytes (via GLUT4) (5) resulting in aug-mented myocardial reliance on fatty acid uptake andmetabolism for production of ATP (19) Evidence forsuch cardiac metabolic impact of T2DM was mostlyobtained in PET studies investigating substrate utiliza-tion at the level of whole heart (8) and were thusinfluenced by many variables including plasma concen-tration of substrates

Despite valuable PET data on overall cardiac substrateutilization it still remains unclear whether T2DM affectsintrinsic function of human ventricular mitochondria Inthe current study cellular and mitochondrial influx ofsubstrates was controlled by permeabilization of the tissueand providing it with fixed amounts of metabolitesthereby minimizing acute effects of plasma insulin andsubstrate availability on mitochondrial respiration Alsowith use of palmitoyl-carnitine some of the rate-limitingsteps in fatty acid utilization (sarcolemmal uptake byFATCD36 and mitochondrial translocation by CPT1)were bypassed By virtue of using pyruvate mitochondrialoxidation of carbohydrates independent of the insulin-mediated GLUT4 uptake was tested

Mitochondrial respiration driven by palmitoyl-carnitinewas decreased in diabetic myocardium while the oxidationof pyruvate was unaffected Patients with higher levelsof HbA1c exhibited greater impairment of mitochondrial

palmitoyl-carnitine oxidation pointing toward a relationshipbetween severity of insulin resistancechronic glycemialevels and mitochondrial dysfunction Indeed loss-of-insulin signaling in the heart by the selective cardiomyo-cyte deletion of insulin receptors in CIRKO mice inducedsignificant mitochondrial dysfunction with reduced mito-chondrial capacity for oxidation of substrates (20) Alsoincreased palmitate load was shown to induce mitochon-drial and cellular damage whereby rat cardiomyocytesincubated with high palmitate concentrations displayeddiminished ability to oxidize fatty acids and intracellularsteatosis (21) Considering that global insulin resistancecauses elevated levels of circulating fatty acids (includingpalmitate) it is possible that their elevated load leads tocardiomyocytesrsquo impairment of fatty acid metabolizationin mitochondria This combined with impairment ofmyocardial insulin signaling (which in itself was shownto induce mitochondrial dysfunction [20]) could furtheraugment the intracellular accumulation of palmitate andstart the cardiotoxic vicious cycle

Alterations in mitochondrial fatty acid oxidation werepreviously reported in atrial tissue from patients withT2DM (910) and in animal models of the disease(202223) Also data from a recent PET study investigat-ing LV substrate metabolism in patients with diabetesagree with our findings by showing that despite increasedabsolute rates of fatty acid oxidation diabetic heart oxi-dizes relatively less of the extracted fatty acids since theincrease in fatty acid esterification was proportionatelyhigher than the increase in oxidation (24) Thereforefindings from our and previous studies suggest that de-spite globally increased fatty acid usage by the diabetic

Figure 3mdashPDH and expression of mitochondrial respiratory chain complexes are unaltered in diabetic left ventricle A Activity of PDH the keyregulating enzyme of carbohydrate oxidation in non-DM and DM groups B Left panel image of representative blot probed with antibodiesagainst main subunits of PDH Right panel mean values of quantified chemiluminescence normalized to non-DM group C Left panel image ofrepresentative blot probed with antibodies aimed at characteristic subunit of each of the mitochondrial respiratory chain complexes (CI to V) Rightpanel mean values of quantified chemiluminescence normalized to non-DM group Data in bar graphs are mean6 SEM mOD mean optical density

diabetesdiabetesjournalsorg Ljubkovic and Associates 1929

hearts (due to increased fatty acid delivery and reducedglucose uptake) the mitochondrial machinery in diabeticmyocardium actually has less capacity for processing fattyacids (the maximum capacity is not likely to be achieved inthe in vivo setting but can result in reported lower percentof fatty acid oxidation relative to their extracted amount)(24) As a result accumulation of fatty acids upstreamfrom the bottleneck in their utilization pathway could leadto their excessive incorporation in various types of lipidsand consequent lipotoxicity (2526)

The finding of the unchanged expression of OXPHOSsubunits in DM group as well as the unchanged rate ofmaximal respiration driven by pyruvate (both ADP andFCCP stimulated) suggests that the observed reduction inmitochondrial oxidation of palmitoyl is not a result ofdefects in mitochondrial respiratory chain activity Rather

these findings suggest the main disruption leading to thedecreased capacity for fatty acid oxidation might be atthe level of b-oxidation Indeed measurement of theexpression and activity of VLCAD and HADHA respec-tively revealed that these b-oxidation steps are down-regulated in diabetic myocardium Along this lineproteomic analysis of insulin resistant myocardium fromCIRKO mice revealed significantly altered expressionof mitochondrial proteins with downregulation ofb-oxidation enzymes (20)

Accumulation of Lipids in Diabetic MyocardiumMeasurements of intracellular neutral lipids revealed a signif-icantly increased amount of lipid droplets inside LV myocar-dium of patients with diabetes This correlated with thedegree of impairment of mitochondrial palmitoyl-carnitine

Figure 4mdashAugmented accumulation of lipid droplets and ceramide in diabetic LV myocardium A Representative images of non-DM and DMleft ventricles stained with ORO and visualized by light microscopy B Quantified values of ORO staining C Correlation between ratioof mitochondrial respiration driven by pyruvate-malate (Pyr) to respiration driven by palmitoyl-carnitine-malate (Palm) (described in Fig 1)and ORO staining D Representative images of non-DM and DM left ventricles probed with anti-ceramide primary antibody (upper panels)and negative controls in which primary antibody was omitted (lower panels) in LV tissue slices obtained from patients without diabetes andpatients with diabetes Green Alexa Fluor 488 fluorescence indicates ceramide staining while blue fluorescence indicates nuclei stained withDAPI E Quantified values of ceramide staining in non-DM and DM myocardia Data in bar graphs are mean6 SEM P 005 vs non-DMgroup

1930 Metabolic Disturbances in Diabetic Left Ventricle Diabetes Volume 68 October 2019

oxidation suggesting that the observed mitochondrial dys-function might be responsible for the enhanced fataccumulation These results are in agreement with earlierdocumentation of an increased lipid content in humandiabetic myocardium based on MRI methodology (25)and on direct histological visualization (however thesewere diabetic hearts in end-stage contractile failure) (27)Accumulation of neutral fat was previously shown toresult in deterioration in cardiac function For exampledeficiency of adipose TG lipase (ATGL) an enzyme re-sponsible for degradation of lipid droplets induces mas-sive cardiac TG deposition and fatal cardiomyopathy (28)

Ceramide was found to be the critical cardiotoxin ina rodent model of lipotoxic cardiomyopathy as reductionin myocardial ceramide resulted in improved cardiac func-tion and survival (29) Moreover obese diabetic rats(Zucker diabetic fatty rats) were previously shown toexhibit cardiac dysfunction associated with excessivemyocardial storage of TG and ceramide (30) Despite theconvincing results on ceramide accumulation obtained inanimal models of diabetes and obesity the contentof ceramide was found to be unaltered in human atrialmyocardium obtained from patients with T2DM (31)However we also detected increased anti-ceramide stain-ing in diabetic myocardium with the most likely explana-tion for this discrepancy being the fact that we used LVmyocardium in contrast to the right atrial appendagesused before (31)

ER Stress and Apoptosis in Diabetic MyocardiumER stress is marked by an excess accumulation of mis-folded proteins that can be triggered by intracellularbuildup of saturated fatty acids (32) ER stress results

in activation of cellular prosurvival response called theldquounfolded protein responserdquo aiming to restore the normalfunction of the ER by increasing expression of proteinchaperones such as GRP78 as well as by decreasing globalprotein synthesis and ldquounloadingrdquo the ER However if theunfolded protein response does not succeed in restorationof normal ER function due to severechronic ER stress thecell turns on its self-destruction program by inducing theproapoptotic transcription factor CHOP (33) In the cur-rent study we detected an upregulation of GRP78 andinduction of CHOP in diabetic myocardium demonstrat-ing a greater degree of ER stress in human diabetic heartsThese data are also in agreement with the results obtainedin animal models of T2DM (34) Interestingly induction ofER stress by tunicamycin can also induce profound mito-chondrial remodeling including decreased ability to pro-cess fatty acids (16) Whether ER stress is the initiator orworks concomitantly with other disturbances (eg fattyacid overload of cardiomyocytes) leading to the downwardspiral of adverse mitochondrial remodeling in human di-abetic myocardium remains to be investigated

Lastly we demonstrate an augmented rate of apoptoticcell death in the DM group compared with the non-DMgroup Our findings are in line with earlier studies ondiabetes and cell death in human myocardium (35)

Limitations of the Current StudySince both patient groups suffered from CAD requiringsurgical revascularization we were not able to studyfactors influencing the diabetic myocardium in absence ofpotentially confounding CAD Also although there was noobvious difference in severity of CAD between DM andnon-DM patients we cannot assess possible differences in

Figure 5mdashER stress and apoptosis are increased in left ventricle of patients with type 2 diabetes A Image from representative blotsprobed against ER stress factors GRP78 and CHOP and cleaved caspase-3 (an indicator of apoptosis) in non-DM and DM groupsB Mean values of quantified chemiluminescence normalized to non-DM group C Representative colocalization images of non-DM andDM myocardia stained with DAPI (blue) or FITC (green [apoptotic nuclei]) D Average percentages of TUNEL-positive nuclei in non-DMand DM left ventricles Data in bar graphs are mean 6 SEM P 005 vs non-DM group

diabetesdiabetesjournalsorg Ljubkovic and Associates 1931

microvascular function between the two groups The con-centration of glucose and lipids in the blood was deter-mined in a fasted state prior to the surgery As we did notassess blood levels of insulin free fatty acid or glucose atthe time of the biopsy procedure we cannot assess theirpossible acute effects However by using permeabilizedmyocardial tissue and fixed concentrations of substrate formitochondrial functional assay we believe that acuteeffects of insulin and substrate availability on our resultswere minimized Finally due to the small amount ofbiopsy sample we could not perform all of the measure-ments in tissues from all of the patients

ConclusionOur study shows that LV myocardium of patients withT2DM has significantly altered mitochondrial functionwith decreased capacity to oxidize long-chain fatty acidsthrough reduced activity and expression of b-oxidationenzymes This is associated with excessive accumulationof fat in cardiac cells in form of intracellular lipid dropletsand ceramide and is paralleled with ER stress and in-creased apoptosis Therefore based on our and previousdata it seems that insulin resistance with abundantfatty acid loading of cardiomyocytes and ER stress leadsto decreased ability of mitochondria to handle largeamounts of fatty acids being delivered to the cardiomyo-cyte As a result although total fatty acid usage in diabetichearts is increased it is actually insufficient to clear allintracellular fatty acids This may lead to cardiac steatosisand lipotoxicity extending to a loss of cardiac myocytesby apoptosis Therefore besides targeting insulin resis-tance as the primary aim of antidiabetes therapy an-other target for treating diabetic myocardium might befurther increasing its mitochondrial capacity for fattyacid oxidation

Acknowledgments The authors thank medical personnel of Departmentof Cardiac Surgery at University Hospital Split especially Slavica Kotromanovic forhelp with collection of patient data and biopsy samples as well as Lucija Frankovicfor technical assistanceFunding This work was supported by Croatian Science Foundation grants6153 to ML and 3718 to DB and by the French grant ANR-10-LABX-33 to CLand MG as members of the Laboratory of Excellence LERMITDuality of Interest No potential conflicts of interest relevant to this articlewere reportedAuthor Contributions ML and JM designed the study researcheddata and wrote the manuscript ML MG CB MC DB DF IG CL andJM reviewed and edited the manuscript MG CB MC DB DF and IGresearched data CL planned the experiments and researched data ML and JMare the guarantors of this work and as such had full access to all the data in thestudy and take responsibility for the integrity of the data and the accuracy of thedata analysis

References1 Fox CS Coady S Sorlie PD et al Trends in cardiovascular complications ofdiabetes JAMA 20042922495ndash24992 Benjamin EJ Blaha MJ Chiuve SE et al American Heart AssociationStatistics Committee and Stroke Statistics Subcommittee Heart disease andstroke statistics-2017 update a report from the American Heart Association

[published correction appears in Circulation 2017135e646 published correctionappears in Circulation 2017136e196] Circulation 2017135e146ndashe6033 Stratton IM Adler AI Neil HA et al Association of glycaemia with mac-rovascular and microvascular complications of type 2 diabetes (UKPDS 35)prospective observational study BMJ 2000321405ndash4124 Jia G Hill MA Sowers JR Diabetic cardiomyopathy an update of mech-anisms contributing to this clinical entity Circ Res 2018122624ndash6385 Chong CR Clarke K Levelt E Metabolic remodeling in diabetic cardiomy-opathy Cardiovasc Res 2017113422ndash4306 Fillmore N Mori J Lopaschuk GD Mitochondrial fatty acid oxidation al-terations in heart failure ischaemic heart disease and diabetic cardiomyopathyBr J Pharmacol 20141712080ndash20907 Rector RS Payne RM Ibdah JA Mitochondrial trifunctional protein defectsclinical implications and therapeutic approaches Adv Drug Deliv Rev 2008601488ndash14968 Peterson LR Herrero P Schechtman KB et al Effect of obesity and insulinresistance on myocardial substrate metabolism and efficiency in young womenCirculation 20041092191ndash21969 Anderson EJ Kypson AP Rodriguez E Anderson CA Lehr EJ Neufer PDSubstrate-specific derangements in mitochondrial metabolism and redox balancein the atrium of the type 2 diabetic human heart J Am Coll Cardiol 2009541891ndash189810 Montaigne D Marechal X Coisne A et al Myocardial contractile dysfunctionis associated with impaired mitochondrial function and dynamics in type 2 diabeticbut not in obese patients Circulation 2014130554ndash56411 Lemieux H Semsroth S Antretter H Houmlfer D Gnaiger E Mitochondrialrespiratory control and early defects of oxidative phosphorylation in the failinghuman heart Int J Biochem Cell Biol 2011431729ndash173812 Bass A Stejskalovaacute M Ostaacutedal B Samaacutenek M Differences between atrialand ventricular energy-supplying enzymes in five mammalian species Physiol Res1993421ndash613 Cardin S Pelletier P Libby E et al Marked differences between atrial andventricular gene-expression remodeling in dogs with experimental heart failure JMol Cell Cardiol 200845821ndash83114 Cavar M Ljubkovic M Bulat C et al Trimetazidine does not alter metabolicsubstrate oxidation in cardiac mitochondria of target patient population Br JPharmacol 20161731529ndash154015 Kuznetsov AV Veksler V Gellerich FN Saks V Margreiter R Kunz WSAnalysis of mitochondrial function in situ in permeabilized muscle fibers tissuesand cells Nat Protoc 20083965ndash97616 Prola A Nichtova Z Pires Da Silva J et al Endoplasmic reticulum stressinduces cardiac dysfunction through architectural modifications and alteration ofmitochondrial function in cardiomyocytes Cardiovasc Res 2019115328ndash34217 Gerin I Clerbaux LA Haumont O et al Expression of miR-33 from an SREBP2intron inhibits cholesterol export and fatty acid oxidation J Biol Chem 201028533652ndash3366118 Blumensatt M Fahlbusch P Hilgers R et al Secretory products fromepicardial adipose tissue from patients with type 2 diabetes impair mitochondrialb-oxidation in cardiomyocytes via activation of the cardiac renin-angiotensinsystem and induction of miR-208a Basic Res Cardiol 2017112219 Carley AN Severson DL Fatty acid metabolism is enhanced in type 2 diabetichearts Biochim Biophys Acta 20051734112ndash12620 Boudina S Bugger H Sena S et al Contribution of impaired myocardialinsulin signaling to mitochondrial dysfunction and oxidative stress in the heartCirculation 20091191272ndash128321 Hickson-Bick DL Buja LM McMillin JB Palmitate-mediated alterations inthe fatty acid metabolism of rat neonatal cardiac myocytes J Mol Cell Cardiol200032511ndash51922 Beaudoin MS Perry CG Arkell AM et al Impairments in mitochondrialpalmitoyl-CoA respiratory kinetics that precede development of diabetic cardio-myopathy are prevented by resveratrol in ZDF rats J Physiol 20145922519ndash2533

1932 Metabolic Disturbances in Diabetic Left Ventricle Diabetes Volume 68 October 2019

Mitochondrial Capacity for Substrate Oxidation inDiabetic LV MyocardiumDue to insulin resistance in T2DM there is a greaterextent of lipolysis in adipose tissue with increased fattyacid delivery to the myocardium (5) This is coupled withreduced insulin-stimulated entry of glucose into thecardiac myocytes (via GLUT4) (5) resulting in aug-mented myocardial reliance on fatty acid uptake andmetabolism for production of ATP (19) Evidence forsuch cardiac metabolic impact of T2DM was mostlyobtained in PET studies investigating substrate utiliza-tion at the level of whole heart (8) and were thusinfluenced by many variables including plasma concen-tration of substrates

Despite valuable PET data on overall cardiac substrateutilization it still remains unclear whether T2DM affectsintrinsic function of human ventricular mitochondria Inthe current study cellular and mitochondrial influx ofsubstrates was controlled by permeabilization of the tissueand providing it with fixed amounts of metabolitesthereby minimizing acute effects of plasma insulin andsubstrate availability on mitochondrial respiration Alsowith use of palmitoyl-carnitine some of the rate-limitingsteps in fatty acid utilization (sarcolemmal uptake byFATCD36 and mitochondrial translocation by CPT1)were bypassed By virtue of using pyruvate mitochondrialoxidation of carbohydrates independent of the insulin-mediated GLUT4 uptake was tested

Mitochondrial respiration driven by palmitoyl-carnitinewas decreased in diabetic myocardium while the oxidationof pyruvate was unaffected Patients with higher levelsof HbA1c exhibited greater impairment of mitochondrial

palmitoyl-carnitine oxidation pointing toward a relationshipbetween severity of insulin resistancechronic glycemialevels and mitochondrial dysfunction Indeed loss-of-insulin signaling in the heart by the selective cardiomyo-cyte deletion of insulin receptors in CIRKO mice inducedsignificant mitochondrial dysfunction with reduced mito-chondrial capacity for oxidation of substrates (20) Alsoincreased palmitate load was shown to induce mitochon-drial and cellular damage whereby rat cardiomyocytesincubated with high palmitate concentrations displayeddiminished ability to oxidize fatty acids and intracellularsteatosis (21) Considering that global insulin resistancecauses elevated levels of circulating fatty acids (includingpalmitate) it is possible that their elevated load leads tocardiomyocytesrsquo impairment of fatty acid metabolizationin mitochondria This combined with impairment ofmyocardial insulin signaling (which in itself was shownto induce mitochondrial dysfunction [20]) could furtheraugment the intracellular accumulation of palmitate andstart the cardiotoxic vicious cycle

Alterations in mitochondrial fatty acid oxidation werepreviously reported in atrial tissue from patients withT2DM (910) and in animal models of the disease(202223) Also data from a recent PET study investigat-ing LV substrate metabolism in patients with diabetesagree with our findings by showing that despite increasedabsolute rates of fatty acid oxidation diabetic heart oxi-dizes relatively less of the extracted fatty acids since theincrease in fatty acid esterification was proportionatelyhigher than the increase in oxidation (24) Thereforefindings from our and previous studies suggest that de-spite globally increased fatty acid usage by the diabetic

Figure 3mdashPDH and expression of mitochondrial respiratory chain complexes are unaltered in diabetic left ventricle A Activity of PDH the keyregulating enzyme of carbohydrate oxidation in non-DM and DM groups B Left panel image of representative blot probed with antibodiesagainst main subunits of PDH Right panel mean values of quantified chemiluminescence normalized to non-DM group C Left panel image ofrepresentative blot probed with antibodies aimed at characteristic subunit of each of the mitochondrial respiratory chain complexes (CI to V) Rightpanel mean values of quantified chemiluminescence normalized to non-DM group Data in bar graphs are mean6 SEM mOD mean optical density

diabetesdiabetesjournalsorg Ljubkovic and Associates 1929

hearts (due to increased fatty acid delivery and reducedglucose uptake) the mitochondrial machinery in diabeticmyocardium actually has less capacity for processing fattyacids (the maximum capacity is not likely to be achieved inthe in vivo setting but can result in reported lower percentof fatty acid oxidation relative to their extracted amount)(24) As a result accumulation of fatty acids upstreamfrom the bottleneck in their utilization pathway could leadto their excessive incorporation in various types of lipidsand consequent lipotoxicity (2526)

The finding of the unchanged expression of OXPHOSsubunits in DM group as well as the unchanged rate ofmaximal respiration driven by pyruvate (both ADP andFCCP stimulated) suggests that the observed reduction inmitochondrial oxidation of palmitoyl is not a result ofdefects in mitochondrial respiratory chain activity Rather

these findings suggest the main disruption leading to thedecreased capacity for fatty acid oxidation might be atthe level of b-oxidation Indeed measurement of theexpression and activity of VLCAD and HADHA respec-tively revealed that these b-oxidation steps are down-regulated in diabetic myocardium Along this lineproteomic analysis of insulin resistant myocardium fromCIRKO mice revealed significantly altered expressionof mitochondrial proteins with downregulation ofb-oxidation enzymes (20)

Accumulation of Lipids in Diabetic MyocardiumMeasurements of intracellular neutral lipids revealed a signif-icantly increased amount of lipid droplets inside LV myocar-dium of patients with diabetes This correlated with thedegree of impairment of mitochondrial palmitoyl-carnitine

Figure 4mdashAugmented accumulation of lipid droplets and ceramide in diabetic LV myocardium A Representative images of non-DM and DMleft ventricles stained with ORO and visualized by light microscopy B Quantified values of ORO staining C Correlation between ratioof mitochondrial respiration driven by pyruvate-malate (Pyr) to respiration driven by palmitoyl-carnitine-malate (Palm) (described in Fig 1)and ORO staining D Representative images of non-DM and DM left ventricles probed with anti-ceramide primary antibody (upper panels)and negative controls in which primary antibody was omitted (lower panels) in LV tissue slices obtained from patients without diabetes andpatients with diabetes Green Alexa Fluor 488 fluorescence indicates ceramide staining while blue fluorescence indicates nuclei stained withDAPI E Quantified values of ceramide staining in non-DM and DM myocardia Data in bar graphs are mean6 SEM P 005 vs non-DMgroup

1930 Metabolic Disturbances in Diabetic Left Ventricle Diabetes Volume 68 October 2019

oxidation suggesting that the observed mitochondrial dys-function might be responsible for the enhanced fataccumulation These results are in agreement with earlierdocumentation of an increased lipid content in humandiabetic myocardium based on MRI methodology (25)and on direct histological visualization (however thesewere diabetic hearts in end-stage contractile failure) (27)Accumulation of neutral fat was previously shown toresult in deterioration in cardiac function For exampledeficiency of adipose TG lipase (ATGL) an enzyme re-sponsible for degradation of lipid droplets induces mas-sive cardiac TG deposition and fatal cardiomyopathy (28)

Ceramide was found to be the critical cardiotoxin ina rodent model of lipotoxic cardiomyopathy as reductionin myocardial ceramide resulted in improved cardiac func-tion and survival (29) Moreover obese diabetic rats(Zucker diabetic fatty rats) were previously shown toexhibit cardiac dysfunction associated with excessivemyocardial storage of TG and ceramide (30) Despite theconvincing results on ceramide accumulation obtained inanimal models of diabetes and obesity the contentof ceramide was found to be unaltered in human atrialmyocardium obtained from patients with T2DM (31)However we also detected increased anti-ceramide stain-ing in diabetic myocardium with the most likely explana-tion for this discrepancy being the fact that we used LVmyocardium in contrast to the right atrial appendagesused before (31)

ER Stress and Apoptosis in Diabetic MyocardiumER stress is marked by an excess accumulation of mis-folded proteins that can be triggered by intracellularbuildup of saturated fatty acids (32) ER stress results

in activation of cellular prosurvival response called theldquounfolded protein responserdquo aiming to restore the normalfunction of the ER by increasing expression of proteinchaperones such as GRP78 as well as by decreasing globalprotein synthesis and ldquounloadingrdquo the ER However if theunfolded protein response does not succeed in restorationof normal ER function due to severechronic ER stress thecell turns on its self-destruction program by inducing theproapoptotic transcription factor CHOP (33) In the cur-rent study we detected an upregulation of GRP78 andinduction of CHOP in diabetic myocardium demonstrat-ing a greater degree of ER stress in human diabetic heartsThese data are also in agreement with the results obtainedin animal models of T2DM (34) Interestingly induction ofER stress by tunicamycin can also induce profound mito-chondrial remodeling including decreased ability to pro-cess fatty acids (16) Whether ER stress is the initiator orworks concomitantly with other disturbances (eg fattyacid overload of cardiomyocytes) leading to the downwardspiral of adverse mitochondrial remodeling in human di-abetic myocardium remains to be investigated

Lastly we demonstrate an augmented rate of apoptoticcell death in the DM group compared with the non-DMgroup Our findings are in line with earlier studies ondiabetes and cell death in human myocardium (35)

Limitations of the Current StudySince both patient groups suffered from CAD requiringsurgical revascularization we were not able to studyfactors influencing the diabetic myocardium in absence ofpotentially confounding CAD Also although there was noobvious difference in severity of CAD between DM andnon-DM patients we cannot assess possible differences in

Figure 5mdashER stress and apoptosis are increased in left ventricle of patients with type 2 diabetes A Image from representative blotsprobed against ER stress factors GRP78 and CHOP and cleaved caspase-3 (an indicator of apoptosis) in non-DM and DM groupsB Mean values of quantified chemiluminescence normalized to non-DM group C Representative colocalization images of non-DM andDM myocardia stained with DAPI (blue) or FITC (green [apoptotic nuclei]) D Average percentages of TUNEL-positive nuclei in non-DMand DM left ventricles Data in bar graphs are mean 6 SEM P 005 vs non-DM group

diabetesdiabetesjournalsorg Ljubkovic and Associates 1931

microvascular function between the two groups The con-centration of glucose and lipids in the blood was deter-mined in a fasted state prior to the surgery As we did notassess blood levels of insulin free fatty acid or glucose atthe time of the biopsy procedure we cannot assess theirpossible acute effects However by using permeabilizedmyocardial tissue and fixed concentrations of substrate formitochondrial functional assay we believe that acuteeffects of insulin and substrate availability on our resultswere minimized Finally due to the small amount ofbiopsy sample we could not perform all of the measure-ments in tissues from all of the patients

ConclusionOur study shows that LV myocardium of patients withT2DM has significantly altered mitochondrial functionwith decreased capacity to oxidize long-chain fatty acidsthrough reduced activity and expression of b-oxidationenzymes This is associated with excessive accumulationof fat in cardiac cells in form of intracellular lipid dropletsand ceramide and is paralleled with ER stress and in-creased apoptosis Therefore based on our and previousdata it seems that insulin resistance with abundantfatty acid loading of cardiomyocytes and ER stress leadsto decreased ability of mitochondria to handle largeamounts of fatty acids being delivered to the cardiomyo-cyte As a result although total fatty acid usage in diabetichearts is increased it is actually insufficient to clear allintracellular fatty acids This may lead to cardiac steatosisand lipotoxicity extending to a loss of cardiac myocytesby apoptosis Therefore besides targeting insulin resis-tance as the primary aim of antidiabetes therapy an-other target for treating diabetic myocardium might befurther increasing its mitochondrial capacity for fattyacid oxidation

Acknowledgments The authors thank medical personnel of Departmentof Cardiac Surgery at University Hospital Split especially Slavica Kotromanovic forhelp with collection of patient data and biopsy samples as well as Lucija Frankovicfor technical assistanceFunding This work was supported by Croatian Science Foundation grants6153 to ML and 3718 to DB and by the French grant ANR-10-LABX-33 to CLand MG as members of the Laboratory of Excellence LERMITDuality of Interest No potential conflicts of interest relevant to this articlewere reportedAuthor Contributions ML and JM designed the study researcheddata and wrote the manuscript ML MG CB MC DB DF IG CL andJM reviewed and edited the manuscript MG CB MC DB DF and IGresearched data CL planned the experiments and researched data ML and JMare the guarantors of this work and as such had full access to all the data in thestudy and take responsibility for the integrity of the data and the accuracy of thedata analysis

References1 Fox CS Coady S Sorlie PD et al Trends in cardiovascular complications ofdiabetes JAMA 20042922495ndash24992 Benjamin EJ Blaha MJ Chiuve SE et al American Heart AssociationStatistics Committee and Stroke Statistics Subcommittee Heart disease andstroke statistics-2017 update a report from the American Heart Association

[published correction appears in Circulation 2017135e646 published correctionappears in Circulation 2017136e196] Circulation 2017135e146ndashe6033 Stratton IM Adler AI Neil HA et al Association of glycaemia with mac-rovascular and microvascular complications of type 2 diabetes (UKPDS 35)prospective observational study BMJ 2000321405ndash4124 Jia G Hill MA Sowers JR Diabetic cardiomyopathy an update of mech-anisms contributing to this clinical entity Circ Res 2018122624ndash6385 Chong CR Clarke K Levelt E Metabolic remodeling in diabetic cardiomy-opathy Cardiovasc Res 2017113422ndash4306 Fillmore N Mori J Lopaschuk GD Mitochondrial fatty acid oxidation al-terations in heart failure ischaemic heart disease and diabetic cardiomyopathyBr J Pharmacol 20141712080ndash20907 Rector RS Payne RM Ibdah JA Mitochondrial trifunctional protein defectsclinical implications and therapeutic approaches Adv Drug Deliv Rev 2008601488ndash14968 Peterson LR Herrero P Schechtman KB et al Effect of obesity and insulinresistance on myocardial substrate metabolism and efficiency in young womenCirculation 20041092191ndash21969 Anderson EJ Kypson AP Rodriguez E Anderson CA Lehr EJ Neufer PDSubstrate-specific derangements in mitochondrial metabolism and redox balancein the atrium of the type 2 diabetic human heart J Am Coll Cardiol 2009541891ndash189810 Montaigne D Marechal X Coisne A et al Myocardial contractile dysfunctionis associated with impaired mitochondrial function and dynamics in type 2 diabeticbut not in obese patients Circulation 2014130554ndash56411 Lemieux H Semsroth S Antretter H Houmlfer D Gnaiger E Mitochondrialrespiratory control and early defects of oxidative phosphorylation in the failinghuman heart Int J Biochem Cell Biol 2011431729ndash173812 Bass A Stejskalovaacute M Ostaacutedal B Samaacutenek M Differences between atrialand ventricular energy-supplying enzymes in five mammalian species Physiol Res1993421ndash613 Cardin S Pelletier P Libby E et al Marked differences between atrial andventricular gene-expression remodeling in dogs with experimental heart failure JMol Cell Cardiol 200845821ndash83114 Cavar M Ljubkovic M Bulat C et al Trimetazidine does not alter metabolicsubstrate oxidation in cardiac mitochondria of target patient population Br JPharmacol 20161731529ndash154015 Kuznetsov AV Veksler V Gellerich FN Saks V Margreiter R Kunz WSAnalysis of mitochondrial function in situ in permeabilized muscle fibers tissuesand cells Nat Protoc 20083965ndash97616 Prola A Nichtova Z Pires Da Silva J et al Endoplasmic reticulum stressinduces cardiac dysfunction through architectural modifications and alteration ofmitochondrial function in cardiomyocytes Cardiovasc Res 2019115328ndash34217 Gerin I Clerbaux LA Haumont O et al Expression of miR-33 from an SREBP2intron inhibits cholesterol export and fatty acid oxidation J Biol Chem 201028533652ndash3366118 Blumensatt M Fahlbusch P Hilgers R et al Secretory products fromepicardial adipose tissue from patients with type 2 diabetes impair mitochondrialb-oxidation in cardiomyocytes via activation of the cardiac renin-angiotensinsystem and induction of miR-208a Basic Res Cardiol 2017112219 Carley AN Severson DL Fatty acid metabolism is enhanced in type 2 diabetichearts Biochim Biophys Acta 20051734112ndash12620 Boudina S Bugger H Sena S et al Contribution of impaired myocardialinsulin signaling to mitochondrial dysfunction and oxidative stress in the heartCirculation 20091191272ndash128321 Hickson-Bick DL Buja LM McMillin JB Palmitate-mediated alterations inthe fatty acid metabolism of rat neonatal cardiac myocytes J Mol Cell Cardiol200032511ndash51922 Beaudoin MS Perry CG Arkell AM et al Impairments in mitochondrialpalmitoyl-CoA respiratory kinetics that precede development of diabetic cardio-myopathy are prevented by resveratrol in ZDF rats J Physiol 20145922519ndash2533

1932 Metabolic Disturbances in Diabetic Left Ventricle Diabetes Volume 68 October 2019

hearts (due to increased fatty acid delivery and reducedglucose uptake) the mitochondrial machinery in diabeticmyocardium actually has less capacity for processing fattyacids (the maximum capacity is not likely to be achieved inthe in vivo setting but can result in reported lower percentof fatty acid oxidation relative to their extracted amount)(24) As a result accumulation of fatty acids upstreamfrom the bottleneck in their utilization pathway could leadto their excessive incorporation in various types of lipidsand consequent lipotoxicity (2526)

The finding of the unchanged expression of OXPHOSsubunits in DM group as well as the unchanged rate ofmaximal respiration driven by pyruvate (both ADP andFCCP stimulated) suggests that the observed reduction inmitochondrial oxidation of palmitoyl is not a result ofdefects in mitochondrial respiratory chain activity Rather

these findings suggest the main disruption leading to thedecreased capacity for fatty acid oxidation might be atthe level of b-oxidation Indeed measurement of theexpression and activity of VLCAD and HADHA respec-tively revealed that these b-oxidation steps are down-regulated in diabetic myocardium Along this lineproteomic analysis of insulin resistant myocardium fromCIRKO mice revealed significantly altered expressionof mitochondrial proteins with downregulation ofb-oxidation enzymes (20)

Accumulation of Lipids in Diabetic MyocardiumMeasurements of intracellular neutral lipids revealed a signif-icantly increased amount of lipid droplets inside LV myocar-dium of patients with diabetes This correlated with thedegree of impairment of mitochondrial palmitoyl-carnitine

Figure 4mdashAugmented accumulation of lipid droplets and ceramide in diabetic LV myocardium A Representative images of non-DM and DMleft ventricles stained with ORO and visualized by light microscopy B Quantified values of ORO staining C Correlation between ratioof mitochondrial respiration driven by pyruvate-malate (Pyr) to respiration driven by palmitoyl-carnitine-malate (Palm) (described in Fig 1)and ORO staining D Representative images of non-DM and DM left ventricles probed with anti-ceramide primary antibody (upper panels)and negative controls in which primary antibody was omitted (lower panels) in LV tissue slices obtained from patients without diabetes andpatients with diabetes Green Alexa Fluor 488 fluorescence indicates ceramide staining while blue fluorescence indicates nuclei stained withDAPI E Quantified values of ceramide staining in non-DM and DM myocardia Data in bar graphs are mean6 SEM P 005 vs non-DMgroup

1930 Metabolic Disturbances in Diabetic Left Ventricle Diabetes Volume 68 October 2019

oxidation suggesting that the observed mitochondrial dys-function might be responsible for the enhanced fataccumulation These results are in agreement with earlierdocumentation of an increased lipid content in humandiabetic myocardium based on MRI methodology (25)and on direct histological visualization (however thesewere diabetic hearts in end-stage contractile failure) (27)Accumulation of neutral fat was previously shown toresult in deterioration in cardiac function For exampledeficiency of adipose TG lipase (ATGL) an enzyme re-sponsible for degradation of lipid droplets induces mas-sive cardiac TG deposition and fatal cardiomyopathy (28)

Ceramide was found to be the critical cardiotoxin ina rodent model of lipotoxic cardiomyopathy as reductionin myocardial ceramide resulted in improved cardiac func-tion and survival (29) Moreover obese diabetic rats(Zucker diabetic fatty rats) were previously shown toexhibit cardiac dysfunction associated with excessivemyocardial storage of TG and ceramide (30) Despite theconvincing results on ceramide accumulation obtained inanimal models of diabetes and obesity the contentof ceramide was found to be unaltered in human atrialmyocardium obtained from patients with T2DM (31)However we also detected increased anti-ceramide stain-ing in diabetic myocardium with the most likely explana-tion for this discrepancy being the fact that we used LVmyocardium in contrast to the right atrial appendagesused before (31)

ER Stress and Apoptosis in Diabetic MyocardiumER stress is marked by an excess accumulation of mis-folded proteins that can be triggered by intracellularbuildup of saturated fatty acids (32) ER stress results

in activation of cellular prosurvival response called theldquounfolded protein responserdquo aiming to restore the normalfunction of the ER by increasing expression of proteinchaperones such as GRP78 as well as by decreasing globalprotein synthesis and ldquounloadingrdquo the ER However if theunfolded protein response does not succeed in restorationof normal ER function due to severechronic ER stress thecell turns on its self-destruction program by inducing theproapoptotic transcription factor CHOP (33) In the cur-rent study we detected an upregulation of GRP78 andinduction of CHOP in diabetic myocardium demonstrat-ing a greater degree of ER stress in human diabetic heartsThese data are also in agreement with the results obtainedin animal models of T2DM (34) Interestingly induction ofER stress by tunicamycin can also induce profound mito-chondrial remodeling including decreased ability to pro-cess fatty acids (16) Whether ER stress is the initiator orworks concomitantly with other disturbances (eg fattyacid overload of cardiomyocytes) leading to the downwardspiral of adverse mitochondrial remodeling in human di-abetic myocardium remains to be investigated

Lastly we demonstrate an augmented rate of apoptoticcell death in the DM group compared with the non-DMgroup Our findings are in line with earlier studies ondiabetes and cell death in human myocardium (35)

Limitations of the Current StudySince both patient groups suffered from CAD requiringsurgical revascularization we were not able to studyfactors influencing the diabetic myocardium in absence ofpotentially confounding CAD Also although there was noobvious difference in severity of CAD between DM andnon-DM patients we cannot assess possible differences in

Figure 5mdashER stress and apoptosis are increased in left ventricle of patients with type 2 diabetes A Image from representative blotsprobed against ER stress factors GRP78 and CHOP and cleaved caspase-3 (an indicator of apoptosis) in non-DM and DM groupsB Mean values of quantified chemiluminescence normalized to non-DM group C Representative colocalization images of non-DM andDM myocardia stained with DAPI (blue) or FITC (green [apoptotic nuclei]) D Average percentages of TUNEL-positive nuclei in non-DMand DM left ventricles Data in bar graphs are mean 6 SEM P 005 vs non-DM group

diabetesdiabetesjournalsorg Ljubkovic and Associates 1931

microvascular function between the two groups The con-centration of glucose and lipids in the blood was deter-mined in a fasted state prior to the surgery As we did notassess blood levels of insulin free fatty acid or glucose atthe time of the biopsy procedure we cannot assess theirpossible acute effects However by using permeabilizedmyocardial tissue and fixed concentrations of substrate formitochondrial functional assay we believe that acuteeffects of insulin and substrate availability on our resultswere minimized Finally due to the small amount ofbiopsy sample we could not perform all of the measure-ments in tissues from all of the patients

ConclusionOur study shows that LV myocardium of patients withT2DM has significantly altered mitochondrial functionwith decreased capacity to oxidize long-chain fatty acidsthrough reduced activity and expression of b-oxidationenzymes This is associated with excessive accumulationof fat in cardiac cells in form of intracellular lipid dropletsand ceramide and is paralleled with ER stress and in-creased apoptosis Therefore based on our and previousdata it seems that insulin resistance with abundantfatty acid loading of cardiomyocytes and ER stress leadsto decreased ability of mitochondria to handle largeamounts of fatty acids being delivered to the cardiomyo-cyte As a result although total fatty acid usage in diabetichearts is increased it is actually insufficient to clear allintracellular fatty acids This may lead to cardiac steatosisand lipotoxicity extending to a loss of cardiac myocytesby apoptosis Therefore besides targeting insulin resis-tance as the primary aim of antidiabetes therapy an-other target for treating diabetic myocardium might befurther increasing its mitochondrial capacity for fattyacid oxidation

Acknowledgments The authors thank medical personnel of Departmentof Cardiac Surgery at University Hospital Split especially Slavica Kotromanovic forhelp with collection of patient data and biopsy samples as well as Lucija Frankovicfor technical assistanceFunding This work was supported by Croatian Science Foundation grants6153 to ML and 3718 to DB and by the French grant ANR-10-LABX-33 to CLand MG as members of the Laboratory of Excellence LERMITDuality of Interest No potential conflicts of interest relevant to this articlewere reportedAuthor Contributions ML and JM designed the study researcheddata and wrote the manuscript ML MG CB MC DB DF IG CL andJM reviewed and edited the manuscript MG CB MC DB DF and IGresearched data CL planned the experiments and researched data ML and JMare the guarantors of this work and as such had full access to all the data in thestudy and take responsibility for the integrity of the data and the accuracy of thedata analysis

References1 Fox CS Coady S Sorlie PD et al Trends in cardiovascular complications ofdiabetes JAMA 20042922495ndash24992 Benjamin EJ Blaha MJ Chiuve SE et al American Heart AssociationStatistics Committee and Stroke Statistics Subcommittee Heart disease andstroke statistics-2017 update a report from the American Heart Association

[published correction appears in Circulation 2017135e646 published correctionappears in Circulation 2017136e196] Circulation 2017135e146ndashe6033 Stratton IM Adler AI Neil HA et al Association of glycaemia with mac-rovascular and microvascular complications of type 2 diabetes (UKPDS 35)prospective observational study BMJ 2000321405ndash4124 Jia G Hill MA Sowers JR Diabetic cardiomyopathy an update of mech-anisms contributing to this clinical entity Circ Res 2018122624ndash6385 Chong CR Clarke K Levelt E Metabolic remodeling in diabetic cardiomy-opathy Cardiovasc Res 2017113422ndash4306 Fillmore N Mori J Lopaschuk GD Mitochondrial fatty acid oxidation al-terations in heart failure ischaemic heart disease and diabetic cardiomyopathyBr J Pharmacol 20141712080ndash20907 Rector RS Payne RM Ibdah JA Mitochondrial trifunctional protein defectsclinical implications and therapeutic approaches Adv Drug Deliv Rev 2008601488ndash14968 Peterson LR Herrero P Schechtman KB et al Effect of obesity and insulinresistance on myocardial substrate metabolism and efficiency in young womenCirculation 20041092191ndash21969 Anderson EJ Kypson AP Rodriguez E Anderson CA Lehr EJ Neufer PDSubstrate-specific derangements in mitochondrial metabolism and redox balancein the atrium of the type 2 diabetic human heart J Am Coll Cardiol 2009541891ndash189810 Montaigne D Marechal X Coisne A et al Myocardial contractile dysfunctionis associated with impaired mitochondrial function and dynamics in type 2 diabeticbut not in obese patients Circulation 2014130554ndash56411 Lemieux H Semsroth S Antretter H Houmlfer D Gnaiger E Mitochondrialrespiratory control and early defects of oxidative phosphorylation in the failinghuman heart Int J Biochem Cell Biol 2011431729ndash173812 Bass A Stejskalovaacute M Ostaacutedal B Samaacutenek M Differences between atrialand ventricular energy-supplying enzymes in five mammalian species Physiol Res1993421ndash613 Cardin S Pelletier P Libby E et al Marked differences between atrial andventricular gene-expression remodeling in dogs with experimental heart failure JMol Cell Cardiol 200845821ndash83114 Cavar M Ljubkovic M Bulat C et al Trimetazidine does not alter metabolicsubstrate oxidation in cardiac mitochondria of target patient population Br JPharmacol 20161731529ndash154015 Kuznetsov AV Veksler V Gellerich FN Saks V Margreiter R Kunz WSAnalysis of mitochondrial function in situ in permeabilized muscle fibers tissuesand cells Nat Protoc 20083965ndash97616 Prola A Nichtova Z Pires Da Silva J et al Endoplasmic reticulum stressinduces cardiac dysfunction through architectural modifications and alteration ofmitochondrial function in cardiomyocytes Cardiovasc Res 2019115328ndash34217 Gerin I Clerbaux LA Haumont O et al Expression of miR-33 from an SREBP2intron inhibits cholesterol export and fatty acid oxidation J Biol Chem 201028533652ndash3366118 Blumensatt M Fahlbusch P Hilgers R et al Secretory products fromepicardial adipose tissue from patients with type 2 diabetes impair mitochondrialb-oxidation in cardiomyocytes via activation of the cardiac renin-angiotensinsystem and induction of miR-208a Basic Res Cardiol 2017112219 Carley AN Severson DL Fatty acid metabolism is enhanced in type 2 diabetichearts Biochim Biophys Acta 20051734112ndash12620 Boudina S Bugger H Sena S et al Contribution of impaired myocardialinsulin signaling to mitochondrial dysfunction and oxidative stress in the heartCirculation 20091191272ndash128321 Hickson-Bick DL Buja LM McMillin JB Palmitate-mediated alterations inthe fatty acid metabolism of rat neonatal cardiac myocytes J Mol Cell Cardiol200032511ndash51922 Beaudoin MS Perry CG Arkell AM et al Impairments in mitochondrialpalmitoyl-CoA respiratory kinetics that precede development of diabetic cardio-myopathy are prevented by resveratrol in ZDF rats J Physiol 20145922519ndash2533

1932 Metabolic Disturbances in Diabetic Left Ventricle Diabetes Volume 68 October 2019

oxidation suggesting that the observed mitochondrial dys-function might be responsible for the enhanced fataccumulation These results are in agreement with earlierdocumentation of an increased lipid content in humandiabetic myocardium based on MRI methodology (25)and on direct histological visualization (however thesewere diabetic hearts in end-stage contractile failure) (27)Accumulation of neutral fat was previously shown toresult in deterioration in cardiac function For exampledeficiency of adipose TG lipase (ATGL) an enzyme re-sponsible for degradation of lipid droplets induces mas-sive cardiac TG deposition and fatal cardiomyopathy (28)

Ceramide was found to be the critical cardiotoxin ina rodent model of lipotoxic cardiomyopathy as reductionin myocardial ceramide resulted in improved cardiac func-tion and survival (29) Moreover obese diabetic rats(Zucker diabetic fatty rats) were previously shown toexhibit cardiac dysfunction associated with excessivemyocardial storage of TG and ceramide (30) Despite theconvincing results on ceramide accumulation obtained inanimal models of diabetes and obesity the contentof ceramide was found to be unaltered in human atrialmyocardium obtained from patients with T2DM (31)However we also detected increased anti-ceramide stain-ing in diabetic myocardium with the most likely explana-tion for this discrepancy being the fact that we used LVmyocardium in contrast to the right atrial appendagesused before (31)

ER Stress and Apoptosis in Diabetic MyocardiumER stress is marked by an excess accumulation of mis-folded proteins that can be triggered by intracellularbuildup of saturated fatty acids (32) ER stress results

in activation of cellular prosurvival response called theldquounfolded protein responserdquo aiming to restore the normalfunction of the ER by increasing expression of proteinchaperones such as GRP78 as well as by decreasing globalprotein synthesis and ldquounloadingrdquo the ER However if theunfolded protein response does not succeed in restorationof normal ER function due to severechronic ER stress thecell turns on its self-destruction program by inducing theproapoptotic transcription factor CHOP (33) In the cur-rent study we detected an upregulation of GRP78 andinduction of CHOP in diabetic myocardium demonstrat-ing a greater degree of ER stress in human diabetic heartsThese data are also in agreement with the results obtainedin animal models of T2DM (34) Interestingly induction ofER stress by tunicamycin can also induce profound mito-chondrial remodeling including decreased ability to pro-cess fatty acids (16) Whether ER stress is the initiator orworks concomitantly with other disturbances (eg fattyacid overload of cardiomyocytes) leading to the downwardspiral of adverse mitochondrial remodeling in human di-abetic myocardium remains to be investigated

Lastly we demonstrate an augmented rate of apoptoticcell death in the DM group compared with the non-DMgroup Our findings are in line with earlier studies ondiabetes and cell death in human myocardium (35)

Limitations of the Current StudySince both patient groups suffered from CAD requiringsurgical revascularization we were not able to studyfactors influencing the diabetic myocardium in absence ofpotentially confounding CAD Also although there was noobvious difference in severity of CAD between DM andnon-DM patients we cannot assess possible differences in

Figure 5mdashER stress and apoptosis are increased in left ventricle of patients with type 2 diabetes A Image from representative blotsprobed against ER stress factors GRP78 and CHOP and cleaved caspase-3 (an indicator of apoptosis) in non-DM and DM groupsB Mean values of quantified chemiluminescence normalized to non-DM group C Representative colocalization images of non-DM andDM myocardia stained with DAPI (blue) or FITC (green [apoptotic nuclei]) D Average percentages of TUNEL-positive nuclei in non-DMand DM left ventricles Data in bar graphs are mean 6 SEM P 005 vs non-DM group

diabetesdiabetesjournalsorg Ljubkovic and Associates 1931

microvascular function between the two groups The con-centration of glucose and lipids in the blood was deter-mined in a fasted state prior to the surgery As we did notassess blood levels of insulin free fatty acid or glucose atthe time of the biopsy procedure we cannot assess theirpossible acute effects However by using permeabilizedmyocardial tissue and fixed concentrations of substrate formitochondrial functional assay we believe that acuteeffects of insulin and substrate availability on our resultswere minimized Finally due to the small amount ofbiopsy sample we could not perform all of the measure-ments in tissues from all of the patients

ConclusionOur study shows that LV myocardium of patients withT2DM has significantly altered mitochondrial functionwith decreased capacity to oxidize long-chain fatty acidsthrough reduced activity and expression of b-oxidationenzymes This is associated with excessive accumulationof fat in cardiac cells in form of intracellular lipid dropletsand ceramide and is paralleled with ER stress and in-creased apoptosis Therefore based on our and previousdata it seems that insulin resistance with abundantfatty acid loading of cardiomyocytes and ER stress leadsto decreased ability of mitochondria to handle largeamounts of fatty acids being delivered to the cardiomyo-cyte As a result although total fatty acid usage in diabetichearts is increased it is actually insufficient to clear allintracellular fatty acids This may lead to cardiac steatosisand lipotoxicity extending to a loss of cardiac myocytesby apoptosis Therefore besides targeting insulin resis-tance as the primary aim of antidiabetes therapy an-other target for treating diabetic myocardium might befurther increasing its mitochondrial capacity for fattyacid oxidation

Acknowledgments The authors thank medical personnel of Departmentof Cardiac Surgery at University Hospital Split especially Slavica Kotromanovic forhelp with collection of patient data and biopsy samples as well as Lucija Frankovicfor technical assistanceFunding This work was supported by Croatian Science Foundation grants6153 to ML and 3718 to DB and by the French grant ANR-10-LABX-33 to CLand MG as members of the Laboratory of Excellence LERMITDuality of Interest No potential conflicts of interest relevant to this articlewere reportedAuthor Contributions ML and JM designed the study researcheddata and wrote the manuscript ML MG CB MC DB DF IG CL andJM reviewed and edited the manuscript MG CB MC DB DF and IGresearched data CL planned the experiments and researched data ML and JMare the guarantors of this work and as such had full access to all the data in thestudy and take responsibility for the integrity of the data and the accuracy of thedata analysis

References1 Fox CS Coady S Sorlie PD et al Trends in cardiovascular complications ofdiabetes JAMA 20042922495ndash24992 Benjamin EJ Blaha MJ Chiuve SE et al American Heart AssociationStatistics Committee and Stroke Statistics Subcommittee Heart disease andstroke statistics-2017 update a report from the American Heart Association

[published correction appears in Circulation 2017135e646 published correctionappears in Circulation 2017136e196] Circulation 2017135e146ndashe6033 Stratton IM Adler AI Neil HA et al Association of glycaemia with mac-rovascular and microvascular complications of type 2 diabetes (UKPDS 35)prospective observational study BMJ 2000321405ndash4124 Jia G Hill MA Sowers JR Diabetic cardiomyopathy an update of mech-anisms contributing to this clinical entity Circ Res 2018122624ndash6385 Chong CR Clarke K Levelt E Metabolic remodeling in diabetic cardiomy-opathy Cardiovasc Res 2017113422ndash4306 Fillmore N Mori J Lopaschuk GD Mitochondrial fatty acid oxidation al-terations in heart failure ischaemic heart disease and diabetic cardiomyopathyBr J Pharmacol 20141712080ndash20907 Rector RS Payne RM Ibdah JA Mitochondrial trifunctional protein defectsclinical implications and therapeutic approaches Adv Drug Deliv Rev 2008601488ndash14968 Peterson LR Herrero P Schechtman KB et al Effect of obesity and insulinresistance on myocardial substrate metabolism and efficiency in young womenCirculation 20041092191ndash21969 Anderson EJ Kypson AP Rodriguez E Anderson CA Lehr EJ Neufer PDSubstrate-specific derangements in mitochondrial metabolism and redox balancein the atrium of the type 2 diabetic human heart J Am Coll Cardiol 2009541891ndash189810 Montaigne D Marechal X Coisne A et al Myocardial contractile dysfunctionis associated with impaired mitochondrial function and dynamics in type 2 diabeticbut not in obese patients Circulation 2014130554ndash56411 Lemieux H Semsroth S Antretter H Houmlfer D Gnaiger E Mitochondrialrespiratory control and early defects of oxidative phosphorylation in the failinghuman heart Int J Biochem Cell Biol 2011431729ndash173812 Bass A Stejskalovaacute M Ostaacutedal B Samaacutenek M Differences between atrialand ventricular energy-supplying enzymes in five mammalian species Physiol Res1993421ndash613 Cardin S Pelletier P Libby E et al Marked differences between atrial andventricular gene-expression remodeling in dogs with experimental heart failure JMol Cell Cardiol 200845821ndash83114 Cavar M Ljubkovic M Bulat C et al Trimetazidine does not alter metabolicsubstrate oxidation in cardiac mitochondria of target patient population Br JPharmacol 20161731529ndash154015 Kuznetsov AV Veksler V Gellerich FN Saks V Margreiter R Kunz WSAnalysis of mitochondrial function in situ in permeabilized muscle fibers tissuesand cells Nat Protoc 20083965ndash97616 Prola A Nichtova Z Pires Da Silva J et al Endoplasmic reticulum stressinduces cardiac dysfunction through architectural modifications and alteration ofmitochondrial function in cardiomyocytes Cardiovasc Res 2019115328ndash34217 Gerin I Clerbaux LA Haumont O et al Expression of miR-33 from an SREBP2intron inhibits cholesterol export and fatty acid oxidation J Biol Chem 201028533652ndash3366118 Blumensatt M Fahlbusch P Hilgers R et al Secretory products fromepicardial adipose tissue from patients with type 2 diabetes impair mitochondrialb-oxidation in cardiomyocytes via activation of the cardiac renin-angiotensinsystem and induction of miR-208a Basic Res Cardiol 2017112219 Carley AN Severson DL Fatty acid metabolism is enhanced in type 2 diabetichearts Biochim Biophys Acta 20051734112ndash12620 Boudina S Bugger H Sena S et al Contribution of impaired myocardialinsulin signaling to mitochondrial dysfunction and oxidative stress in the heartCirculation 20091191272ndash128321 Hickson-Bick DL Buja LM McMillin JB Palmitate-mediated alterations inthe fatty acid metabolism of rat neonatal cardiac myocytes J Mol Cell Cardiol200032511ndash51922 Beaudoin MS Perry CG Arkell AM et al Impairments in mitochondrialpalmitoyl-CoA respiratory kinetics that precede development of diabetic cardio-myopathy are prevented by resveratrol in ZDF rats J Physiol 20145922519ndash2533

1932 Metabolic Disturbances in Diabetic Left Ventricle Diabetes Volume 68 October 2019

microvascular function between the two groups The con-centration of glucose and lipids in the blood was deter-mined in a fasted state prior to the surgery As we did notassess blood levels of insulin free fatty acid or glucose atthe time of the biopsy procedure we cannot assess theirpossible acute effects However by using permeabilizedmyocardial tissue and fixed concentrations of substrate formitochondrial functional assay we believe that acuteeffects of insulin and substrate availability on our resultswere minimized Finally due to the small amount ofbiopsy sample we could not perform all of the measure-ments in tissues from all of the patients

ConclusionOur study shows that LV myocardium of patients withT2DM has significantly altered mitochondrial functionwith decreased capacity to oxidize long-chain fatty acidsthrough reduced activity and expression of b-oxidationenzymes This is associated with excessive accumulationof fat in cardiac cells in form of intracellular lipid dropletsand ceramide and is paralleled with ER stress and in-creased apoptosis Therefore based on our and previousdata it seems that insulin resistance with abundantfatty acid loading of cardiomyocytes and ER stress leadsto decreased ability of mitochondria to handle largeamounts of fatty acids being delivered to the cardiomyo-cyte As a result although total fatty acid usage in diabetichearts is increased it is actually insufficient to clear allintracellular fatty acids This may lead to cardiac steatosisand lipotoxicity extending to a loss of cardiac myocytesby apoptosis Therefore besides targeting insulin resis-tance as the primary aim of antidiabetes therapy an-other target for treating diabetic myocardium might befurther increasing its mitochondrial capacity for fattyacid oxidation

Acknowledgments The authors thank medical personnel of Departmentof Cardiac Surgery at University Hospital Split especially Slavica Kotromanovic forhelp with collection of patient data and biopsy samples as well as Lucija Frankovicfor technical assistanceFunding This work was supported by Croatian Science Foundation grants6153 to ML and 3718 to DB and by the French grant ANR-10-LABX-33 to CLand MG as members of the Laboratory of Excellence LERMITDuality of Interest No potential conflicts of interest relevant to this articlewere reportedAuthor Contributions ML and JM designed the study researcheddata and wrote the manuscript ML MG CB MC DB DF IG CL andJM reviewed and edited the manuscript MG CB MC DB DF and IGresearched data CL planned the experiments and researched data ML and JMare the guarantors of this work and as such had full access to all the data in thestudy and take responsibility for the integrity of the data and the accuracy of thedata analysis

References1 Fox CS Coady S Sorlie PD et al Trends in cardiovascular complications ofdiabetes JAMA 20042922495ndash24992 Benjamin EJ Blaha MJ Chiuve SE et al American Heart AssociationStatistics Committee and Stroke Statistics Subcommittee Heart disease andstroke statistics-2017 update a report from the American Heart Association

[published correction appears in Circulation 2017135e646 published correctionappears in Circulation 2017136e196] Circulation 2017135e146ndashe6033 Stratton IM Adler AI Neil HA et al Association of glycaemia with mac-rovascular and microvascular complications of type 2 diabetes (UKPDS 35)prospective observational study BMJ 2000321405ndash4124 Jia G Hill MA Sowers JR Diabetic cardiomyopathy an update of mech-anisms contributing to this clinical entity Circ Res 2018122624ndash6385 Chong CR Clarke K Levelt E Metabolic remodeling in diabetic cardiomy-opathy Cardiovasc Res 2017113422ndash4306 Fillmore N Mori J Lopaschuk GD Mitochondrial fatty acid oxidation al-terations in heart failure ischaemic heart disease and diabetic cardiomyopathyBr J Pharmacol 20141712080ndash20907 Rector RS Payne RM Ibdah JA Mitochondrial trifunctional protein defectsclinical implications and therapeutic approaches Adv Drug Deliv Rev 2008601488ndash14968 Peterson LR Herrero P Schechtman KB et al Effect of obesity and insulinresistance on myocardial substrate metabolism and efficiency in young womenCirculation 20041092191ndash21969 Anderson EJ Kypson AP Rodriguez E Anderson CA Lehr EJ Neufer PDSubstrate-specific derangements in mitochondrial metabolism and redox balancein the atrium of the type 2 diabetic human heart J Am Coll Cardiol 2009541891ndash189810 Montaigne D Marechal X Coisne A et al Myocardial contractile dysfunctionis associated with impaired mitochondrial function and dynamics in type 2 diabeticbut not in obese patients Circulation 2014130554ndash56411 Lemieux H Semsroth S Antretter H Houmlfer D Gnaiger E Mitochondrialrespiratory control and early defects of oxidative phosphorylation in the failinghuman heart Int J Biochem Cell Biol 2011431729ndash173812 Bass A Stejskalovaacute M Ostaacutedal B Samaacutenek M Differences between atrialand ventricular energy-supplying enzymes in five mammalian species Physiol Res1993421ndash613 Cardin S Pelletier P Libby E et al Marked differences between atrial andventricular gene-expression remodeling in dogs with experimental heart failure JMol Cell Cardiol 200845821ndash83114 Cavar M Ljubkovic M Bulat C et al Trimetazidine does not alter metabolicsubstrate oxidation in cardiac mitochondria of target patient population Br JPharmacol 20161731529ndash154015 Kuznetsov AV Veksler V Gellerich FN Saks V Margreiter R Kunz WSAnalysis of mitochondrial function in situ in permeabilized muscle fibers tissuesand cells Nat Protoc 20083965ndash97616 Prola A Nichtova Z Pires Da Silva J et al Endoplasmic reticulum stressinduces cardiac dysfunction through architectural modifications and alteration ofmitochondrial function in cardiomyocytes Cardiovasc Res 2019115328ndash34217 Gerin I Clerbaux LA Haumont O et al Expression of miR-33 from an SREBP2intron inhibits cholesterol export and fatty acid oxidation J Biol Chem 201028533652ndash3366118 Blumensatt M Fahlbusch P Hilgers R et al Secretory products fromepicardial adipose tissue from patients with type 2 diabetes impair mitochondrialb-oxidation in cardiomyocytes via activation of the cardiac renin-angiotensinsystem and induction of miR-208a Basic Res Cardiol 2017112219 Carley AN Severson DL Fatty acid metabolism is enhanced in type 2 diabetichearts Biochim Biophys Acta 20051734112ndash12620 Boudina S Bugger H Sena S et al Contribution of impaired myocardialinsulin signaling to mitochondrial dysfunction and oxidative stress in the heartCirculation 20091191272ndash128321 Hickson-Bick DL Buja LM McMillin JB Palmitate-mediated alterations inthe fatty acid metabolism of rat neonatal cardiac myocytes J Mol Cell Cardiol200032511ndash51922 Beaudoin MS Perry CG Arkell AM et al Impairments in mitochondrialpalmitoyl-CoA respiratory kinetics that precede development of diabetic cardio-myopathy are prevented by resveratrol in ZDF rats J Physiol 20145922519ndash2533

1932 Metabolic Disturbances in Diabetic Left Ventricle Diabetes Volume 68 October 2019

23 Kuo TH Moore KH Giacomelli F Wiener J Defective oxidative metabolism ofheart mitochondria from genetically diabetic mice Diabetes 198332781ndash78724 Peterson LR Saeed IM McGill JB et al Sex and type 2 diabetes obesity-independent effects on left ventricular substrate metabolism and relaxation inhumans Obesity (Silver Spring) 201220802ndash81025 Rijzewijk LJ van der Meer RW Smit JW et al Myocardial steatosis is anindependent predictor of diastolic dysfunction in type 2 diabetes mellitus J Am CollCardiol 2008521793ndash179926 Scheuermann-Freestone M Madsen PL Manners D et al Abnormal cardiacand skeletal muscle energy metabolism in patients with type 2 diabetes Cir-culation 20031073040ndash304627 Sharma S Adrogue JV Golfman L et al Intramyocardial lipid accumulation inthe failing human heart resembles the lipotoxic rat heart FASEB J 2004181692ndash170028 Hirano K Ikeda Y Zaima N Sakata Y Matsumiya G Triglyceride depositcardiomyovasculopathy N Engl J Med 20083592396ndash239829 Park TS Hu Y Noh HL et al Ceramide is a cardiotoxin in lipotoxic car-diomyopathy J Lipid Res 2008492101ndash2112

30 Zhou YT Grayburn P Karim A et al Lipotoxic heart disease in obese ratsimplications for human obesity Proc Natl Acad Sci U S A 2000971784ndash178931 Baranowski M Blachnio-Zabielska A Hirnle T et al Myocardium of type2 diabetic and obese patients is characterized by alterations in sphingolipid metabolicenzymes but not by accumulation of ceramide J Lipid Res 20105174ndash8032 Park M Sabetski A Kwan Chan Y Turdi S Sweeney G Palmitate induces ERstress and autophagy in H9c2 cells implications for apoptosis and adiponectinresistance J Cell Physiol 2015230630ndash63933 Yang L Zhao D Ren J Yang J Endoplasmic reticulum stress and protein qualitycontrol in diabetic cardiomyopathy Biochim Biophys Acta 20151852209ndash21834 Miki T Miura T Hotta H et al Endoplasmic reticulum stress in diabetic heartsabolishes erythropoietin-induced myocardial protection by impairment of phospho-glycogen synthase kinase-3beta-mediated suppression of mitochondrial perme-ability transition Diabetes 2009582863ndash287235 Chowdhry MF Vohra HA Galintildeanes M Diabetes increases apoptosis andnecrosis in both ischemic and nonischemic human myocardium role of caspasesand poly-adenosine diphosphate-ribose polymerase J Thorac Cardiovasc Surg2007134124ndash131 131e1ndash3

diabetesdiabetesjournalsorg Ljubkovic and Associates 1933