the effects of 20-hete infarction in metabolic syndrome rats

The Effects of 20-HETE Antagonism on Myocardial

Infarction in Metabolic Syndrome Rats

Corinna Lozano

Science Research ProgramPawling High School

Introduction

Myocardial Infarctions (MI):

HEART ATTACK

● Blocked Coronary Artery● Induced in JCR/SD Rats● Loss of blood flow● Loss of oxygen

https://en.wikipedia.org/wiki/Myocardial_infarction

Introduction

Ischemia: an inadequate blood supply to an organ or part of the body especially the heart muscles (19)

● Damage to the heart● Cell death● Caused by heart attacks

https://www.slideshare.net/AliAlbarkaawi1/surgical-treatment-of-myocardial-ischemia

Introduction

● < 2 million people/year die from MI (26)

● 30% survival rate following MI (26)

● Metabolic Syndrome pateints less likely to survive

https://medicalxpress.com/news/2018-10-brain-cancer-survival-elderly.html

IntroductionMetabolic Syndrome: cluster of biochemical and physiological abnormalities

Associated with the development of cardiovascular disease and type 2 diabetes (16)

https://www.bioticsresearch.com/content/omega-3s-metabolic-syndrome

Side effects of include :

● High blood sugar ● Increased pressure ● Abnormal cholesterol or

triglyceride levels

Introduction

● JCR rats used as Metabolic Syndrome model○ Contain CP Gene○ James C. Russel (JCR)

● SD rats represent normal health or control group

Image created by Corinna Lozano

Russell, J. C., & Proctor, S. D. (2006, November 15). Small animal models of cardiovascular disease: tools for the study of the roles of metabolic syndrome, dyslipidemia, and atherosclerosis. Retrieved from https://www.sciencedirect.com/science/article/pii/S1054880706001463

Introduction● Metabolic syndrome linked

to 20-HETE (13)○ Symptoms of syndrome

similar to 20-HETE function

● 20-HETE levels are elevated in Metabolic Syndrome rats (JCR)

Molecular mechanisms and cell signaling of 20-hydroxyeicosatetraenoic acid in vascular pathophysiology. (n.d.). Retrieved from https://www.bioscience.org/2016/v21/af/4465/fulltext.php?bframe=figures.htm

Introduction

● Eicosanoid metabolite of arachidonic acid ● 20-HETE impairs Cell Growth● Involved in vascularization

○ Regulation vascular tone○ Blood flow to specific organs○ Sodium/fluid transport in kidney○ Vascular pathway remodeling○ Associated with development of

stroke and heart attacks

20-HETE~20-Hydroxyeicosatetraenoic acid (14)

https://www.semanticscholar.org/paper/Vascular-actions-of-20-HETE.-Hoopes-Garcia/357fffd0f2b959fb53318e5dde53f2e077648013/figure/2

Introduction


Introduction20-SOLA

20-SOLA

20-HETE Antagonist

Associated with

increased Blood Flow

Normalizes Blood

Pressure in Male JCR

rats

20-HETE levels

decreased

Water SolubleMore

Potent injected

instead of dissolved

Dissolved into Bloodstream


● Decreases 20-HETE levels to healthy level

● Mechanism unknown

● 20-SOLA is the easiest 20-HETE antagonist to administer

Introduction

● AMPK-activate protein kinase enzyme ● Highly conserved ● Involved in homeostasis of cell energy● Activates glucose and fatty acid uptake

*Activated by elevated AMP/ATP ratio due to cellular and environmental stressors

http://mcr.aacrjournals.org/content/13/7/1059

AMPK (19)

Introduction

○ ATP levels are low AMPK promotes ATP production ○ Protects cells from stress

■ Depletion of ATP ■ Energy sensing

*MECHANISMS UNKNOWN*

● Three phosphorylation sites○ Activated- Serine 485,Serine 491○ Inactivated- Threonine 172

P-AMPKalpha1 & AMPKalpha1&alpha2

https://medicinalherbals.net/list-ampk-activators-benefits-ampk-activation/

P-AMPKalpha and P-AMPKalpha1/alpha2 (19)

Introduction

Introduction: Ampk antibody


Literature Review

Ampk kinase is an energy sensing enzyme in which shows when cellular energy is low

D.Grahame et al 2012

20-SOLA is water soluble in which could be administered by I.V. for easy application

Victor Garcia et al 2017ndey V, Garcia V, Gilani A, Mishra P, Zhang FF, Paudyal MP, Falck JR, Nasjletti A, Wang WH, Schwartzman ML. The blood pressure-lowering effect of 20-HETE blockade in Cyp4a14(-/-) mice is associated with natriuresis.J Pharmacol Exp Ther. 2017;363:412–418. doi: 10.1124/jpet.117.243618

Literature Review

20-HETE levels significantly elevated in Metabolic Syndrome rats due to poor condition of their heart

Rocic et al 2017

https://www.semanticscholar.org/paper/Vascular-actions-of-20-HETE.-Hoopes-Garcia/357fffd0f2b959fb53318e5dde53f2e077648013/figure/1

Literature Review

Relationship between myocardial ischemic injury and major modes of cell death such as:

● Oncosis ● Apoptosis

University of Texas Health Sciences et al 2015

https://www.researchgate.net/figure/Androgen-driven-20-HETE-dependent-hypertension-in-mice-Mice-are-treated-with-either_fig1_236615072

Gap in research

● Lack of knowledge about 20-HETE and its involvement in heart attacks and ischemia

● Ampk largely not understood

● Treatment for people who have undergone an MI/MS is not well established

https://www.nature.com/collections/vzwgqhtnrc

Hypothesis

20-SOLA will decrease ischemic damage in both JCR and SD rats but will be more prevalent in the

JCR rats

MethodologyMyocardial Infarct (MI) protocol:

30 minute occlusion of the left anterior descending coronary artery (LAD); After 30 minutes, blood flow is

reestablished and the left ventricle is reperfused

After 48 hours, 7 days, or 8 weeks, hearts are separated into IZ and NZ for following

experiments

Histology

Protein expression/localization - Western blot - Immunohistochemistry (IHC)

20-HETE levels - LC/MS/MS

LAD

Occluder

Normal zone(NZ) Collateral-

dependent zone(CZ)

LAD

LAD

LAD


Methodology

20-SOLA treatment: 20-HETE antagonist

● I.V. placed into SD and JCR rats ● Used to combat 20-HETE levels● MEASURED AT 48 HOURS,

1 WEEK AND 8 WEEK● Single treatment ● Cross sectional analysis of heart

Pandey V, Garcia V, Gilani A, Mishra P, Zhang FF, Paudyal MP, Falck JR, Nasjletti A, Wang WH, Schwartzman ML. The blood pressure-lowering effect of 20-HETE blockade in Cyp4a14(-/-) mice is associated with natriuresis.J Pharmacol Exp Ther. 2017;363:412–418. doi: 10.1124/jpet.117.243618

Pan

dey

et a

l. 20

17

Methodology

Western Blot Make gels

https://www.creative-diagnostics.com/The-Basis-of-Western-Blot.htm

Isolate protein

Transfer gel onto membrane

Add secondary antibody

BCA Protein Assay

Load and run gels

Analyze antibodies

Scan blot on odyssey scanner

Add primary antibody(AMPK)

ImageJ program

Compare blot samples

. Methodology


48 hours post MI

Effect of 20-SOLA on MI size at 48 hours post reperfusion

Results

p(Thr172)AMPK

JCR MI JCR MI+

20-SOLA

JCR MI JCR MI+

20-SOLA

JCR MI JCR MI+

20-SOLA

JCR control

SD MI SD MI+

20-SOLA

SD MI SD MI+

20-SOLA

SD MI SD MI+

20-SOLA

SD control

48 hours 7 days 8 weeks 48 hours 7 days 8 weeks

* p<0.05 vs. control# p<0.05 vs. SD

fold

incr

ease

/con

trol

4

3

2

1

*

*

*

* #

#

*

*

* *

$ p<0.05 vs. MI

$

* $ #

#

## $ $ $

Total AMPK

Discussion

● 20-HETE levels were ~2 times greater in JCR vs. SD rats at baseline before surgery

● 20-HETE levels increased during the ischemic period in both SD and JCR rats○ Increase was more significant in JCR (~4-fold vs. NZ)

than in SD (~3-fold vs. NZ) rats● 20-HETE levels were somewhat increased in the NZs of SD

and JCR rats○ Increase did not reach statistical significance○ Has effect of ischemia on remote myocardium

https://www.health.harvard.edu/heart-health/a-closer-look-at-heart-disease-risk

https://sera.sg/en/index.php/lessons/adult-cpraed-protocol/ccad-blog-illustration_recognising-heart-attack/

Discussion● 20-HETE elevated so role was assessed in

myocardial infarction using 20-SOLA (antagonist)

● Infarct expansion (the white on the tissue images, represents how great the heart attack was throughout the heart) ○ Resulted in MIs ~50% of the LV in JCR and

~25% of the LV in SD animals

● Data suggests 20-HETE antagonists able to decrease further damage to the myocardium

● 20-SOLA treatment had opposite effect in SD rats

https://mtkenyatimes.co.ke/heart-attack/

https://www.k9sforwarriors.org/blog/heart-heroes-one-mom-hundreds-veterans

Conclusion● 20-HETE levels were significantly elevated in response

to myocardial ischemia within 5 minutes of MI in both normal (SD) and MetS (JCR) rats

● Ischemic damage is greater in MetS (JCR) rats rather than normal (SD) rats

○ Supports hypothesis

http://www.mbaycih.com/metabolic-syndrome/

https://www.heart.org/en/health-topics/heart-attack/warning-signs-of-a-heart-attack

Conclusion● Determined that 20-HETE plays a causative role in collateral

cellular growth (CCG)

● AMPK was more prevalent in JCR rats after 20-SOLA treatment○ SD rats had higher AMPK levels without treatment

● 20-SOLA was found to lower 20-HETE levels in both samples○ Significantly more in JCR MI rats

● 20-SOLA aided the decrease in ischemia for both rat groups○ More significantly in JCR rats

https://www.ibeat.com/2017/01/06/whats-difference-heart-failure-heart-attack/

Future Research● Establish a more effective treatment for

MI in Metabolic Syndrome rats○ Relevant to the epidemic of

cardiovascular diseases plaguing populations globally

● Investigations involving:○ Human participants○ Role of AMPK on other

mechanisms● 20-SOLA increase effectiveness

https://www.medicinenet.com/coronary_artery_disease_screening_tests_cad/article.htm

https://www.medscape.com/viewarticle/843031

Acknowledgements● I would like to thank the following people for always

guiding and encouraging my interest in pursuing this research ○ Dr. Petra Rocic○ Science research peers○ Gillian Rinaldo ○ My family and friends

References

The Effects of 20-HETE Antagonism on Myocardial

Infraction on Metabolic Syndrome Rats

Corinna Lozano

Science Research Program

the effects of 20-hete infarction in metabolic syndrome rats

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