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Gene Expression in Cardiac Hypertrophy Kenneth R. Boheler and Ketty Schwartz

Cardiac hypertrophy due to hemodynamic overload is a harbinger of

morbidity and mortality in humans. The development of hypertrophy

involves both quantitative and qualitative changes in gene expression

that are thought to produce an enlarged organ more capable of meeting

its new functional requirements. The genes are normal, but the way in

which they are regulated is modified. Analysis of these changes and the

mechanisms involved are essential if we are to understand the role that

hypertrophy plays in the pathogenesis of heart failure. (Trends Cardiovasc Med 1992;2:176-182)

Cardiac hypertmphy due to chronic hemo-

dynamic overload is the common end

result of most cardiac disorders and a

consistent feature of cardiac failure.

Cardiac myocytes are terminally differ-

entiated cells that in utero divide but

shortly after birth become nonprolifera-

tive (although recent data suggest that

some myocyte hyperplasia is present in

the adult and aging rat myocardium

[Anversa et al. 19911). Not only the size

but also the phenotype of the nondivid-

ing myocytes changes with chronic hemo-

dynamic overload. The changes are qual-

itative (phenotypic conversions charac-

terized by isoform switches) and quanti-

tative (characterized by modulation of

individual gene expressions), and in some

instances lead to the reexpression of a

phenotype analogous to that seen in the

fetal or senescent heart (reviewed in

Lomprk et al. [1991]). The ensemble of

these pressure-overload-induced changes,

termed mechanogenic transduction, pro-

duces an enlarged organ considered to

be better adapted to the new functional

demands. An important feature of this mechanogenic transduction is that it

Kenneth R. Boheler and Ketty Schwartz are at INSERM U127, H6pital Lariboisitie, 75010 Paris, France.

does not involve genetic defects: the

genes are normal, but the way in which

they are regulated is modified. Cardiac

hypertrophy is thus due to enhanced

growth of a fixed population of myocytes

with some altered expressions of normal

genes. This review describes the molecu-

lar phenotype of the hypertrophied heart,

with particular emphasis on the homol-

ogy with development and aging and on

the regulatorial levels that control gene

expressions in the heart.

l Qualitative Changes in Gene Expression

Qualitative changes in gene expression

are due to differential expression of

multigene families (Figure 1). The first

identified and most thoroughly studied

example of isoform switching involves

the myosin heavy-chain family (reviewed

in Lornpre et al. [1991]). The thick filament of the cardiac sarcomere can contain two possible myosin heavy- chain isoforms, a-MHC and P-MHC. An effect of a permanent hemodynamic overload is activation of the P-MHC gene and a repression of the a-MHC one. This

change occurs in all tissues and species tested so far, but the potential for an

increase in P-MHC depends upon the

176 01992, Elsevier Science Publishing Co., lOSO-1738/92/$5.00 TCM Vol. 2, No. 5, 1992

Fibronectin (fetal)

Collagen

(Types I, 111,

Creatine Kinase BB

Creatine Kinase MB Lactate Dehydrogenase

P-Myosin Heavy Chain

Atrial Light Chain 1

(M-LDH)

Skeletal a-actin

Smooth muscle actin Troponin T (fetal isoform)

fi Tropomyosin

Myocytes

ti

a3 NalK ATPase

Figure 1. Qualitative changes characterized by isofonn switching that occur during early to say whether these fetal genes hemodynamic overload-induced cardiac hypertrophy. share common regulatory mechanisms.

initial phenotype: it is high in rat ventricles and in human atria, which normally contain -90% a-MHC, and it is small in human ventricles, which contain mainly P-MHC. The change from a-MHC to P-MHC (or isomyosin Vl, the aa homodimer, to V3, the pp homodimer) results in a slower rate of ATP cycling by myosin and a lower velocity of contraction in the hypertrophied fiber. The result is an improved economy of force development that is usually considered adaptive. Be- cause P-MHC is predominant in rat fetal ventricles, the idea was developed that reactivation of a fetal program occurs with hemodynamic overloading. P-MHC also increases with aging (O’Neill et al. 1991), consistent with the idea that the continuous loss of myocytes that occurs in aged ventricles generates a greater workload on the remaining myocytes, which may serve as a chronic mechanical stimulus.

Actin-stimulated myosin ATPase activity and crossbridge cycling generate force and lead to contraction. The activation may involve two different sarco- merit isoforms of actin, a-cardiac and a-skeletal, and pressure overload in- duces changes in their expressions. The

a-skeletal isoactin gene in rats is transitorily upregulated and, since it is also active in utero, represents the second example of a fetal program reactivation by hemodynamic overload (Schwartz et al. 1986; Izumo et al. 1988). a-Skeletal actin is not increased in the senescent myocardium (Carrier et al. 1992), and thus the analogy between compensated hypertrophy and aging is apt for P-MHC and less so with a-skeletal actin. In humans, in contrast to rodent heart, a-skeletal actin is upregulated during development and is the major isofonn of adult control, hypertrophied, and failing hearts (Boheler et al. 1991). Another actin isoform, a-smooth, normally expressed in vascular smooth muscles, is the first muscular isoactin expressed during cardiac myogenesis. It was recently found to be reactivated permanently with pressure-overload hypertrophy, and the high levels of smooth muscle actin mRNAs in the hypertrophied myocardium strongly suggest that this isoform is present in the myocytes (Black et al. 1991). The functional and structural effects of reinducing the two fetal actin isogenes, a-skeletal and vascular smooth, are unknown, and it is too

The expression of other multigene families controlling cardiac contraction is also modified in cardiac hypertrophy, and as more probes become available, the number will probably increase in the near future. The two other types of myosin subunits, myosin light chains 1 and 2, both exist as two isofotms, the atria1 and ventricular isotypes. The atria1 isoform ALCl increases in several forms of ventricular hypertrophy, whereas the ventricular types VLC 1 and VLC2 appear in pressure-overloaded human atria (Cum- mins 1982). The physiologic significance of such transitions is unclear, except that the transition of ALC2 to VIC2 in the atria may account for the increased calcium sensitivity of skinned atria1 fibers from patients with dilated cardiomyopathy, and may indirectly reduce the myosin ATPase activity and maximum velocity of shortening of the fiber, resulting in an improved economy for atria1 contraction (Wankerl et al. 1990). There is also evidence of qualitative changes in some other contractile proteins of the thin filament. The mRNA encoding the p isoform of tropomyosin, present during fetal and early postnatal life in rats, is transitorily activated at the onset of pressure-overload hypertrophy (Izumo et al. 1988). One subunit of the troponin complex, TnT, exists as two or more

TCM Vol. 2, No. 5, I992 01992, Elsevier Science Publishing Co., 1050-1738/92/$5.00 177

cardiac isoforms, which most probably, and in contrast to all other proteins previously mentioned, are produced by alternative splicing of a single gene. The fetal isotype is not upregulated with pressure overload in rats (Saggin et al. 1988), at least in the compensated stage, whereas in humans there appears to be a partial reactivation of fetal cardiac TnT expression in failing hearts (Anderson et al. 1991). If confirmed, this would be the first molecular basis of a thin filament dysregulation in heart failure and would partially provide an explanation to the unanswered question of why the myofi- brillar ATPase activities of human failing hearts are lower than in controls in spite of the same l3-MHC content.

The isofotm switches produced in response to increased afterload are not limited to components of the contractile apparatus. There are also changes in tissue content of isoenzymes that control intermediary metabolism. Increases of fetal type isoenzymes (BB+MB) of creatine kinase and of the M-LDH isoform of lactate dehydrogenase are believed to produce an increased glycolytic capacity that could be a favorable adaptation ensuring that ATP resynthesis via the creatine kinase reaction remains (Ing- wall et al. 198.Q although it should also be noted that this creatine kinase isofonn switch was not observed in hypertensive rats (Fontanet et al. 1991). There are also changes in membrane proteins, which, as for the contractile proteins, depend on the animal species. A twofold increase in the accumulation of the isomRNA encoding the a3 isoform of Na+/K+-ATPase (the cardiac glycoside receptor) was found in rat heart hypertrophy, but it is un- likely that this increase totally accounts for the altered properties of the enzyme (Charlemagne et al., unpublished data, 1992). In the failing human heart, Na+/K+- ATPase isoform expression is not altered and [sH]ouabain binding is nearly normal, suggesting that adaptation to digi- talization in the failing human heart results from other mechanisms (Allen et al. 1992).

What about the nonmyocyte compart- ments? Many studies have shown that fibrillar collagen and fibronectin accumulate within the interstitium and the adventitia of the intramyocardial arteries together with a medial thickening of these vessels. This remodeling is thought to be the main cause of abnormalities in

myocardial stiffness and in vasomotor reactivity of intramyocardial resistance vessels (reviewed in Weber and Brilla [ 199 11). It is now clear that this compart- ment undergoes profound molecular changes; the distribution and density of collagen types I and III change transiently in experimental hypertrophy, and that of type IV changes permanently. The fetal forms of cellular fibronectin produced by alternative splicing of a single gene encoding multiple fibronectin isotypes accumulate in the wall of coro- nary arteries and in focal areas of the myocardium early after rat aortic sten- osis (Samuel et al. 1991). It is hypothe- sized that these fetal variants influence proliferation and migration of nonmus- cular cells as well as adhesion of myocytes of the extracellular matrix proteins that play an active role in cardiac remodeling.

From the above observations, it is therefore clear that when isoform switches occur, the form normally expressed in fetal life is either transitorily or permanently reinduced by hemodynamic overload, and that reprogramming of cardiac gene expression is a common feature of all cardiac cell populations, including striated myocytes, smooth muscle cells, and fibroblasts.

l Quantitative Changes in Gene Expression

The central feature of myocyte hy- pet-trophy is an increased protein content per cell, but this global increase may also involve a selective induction, repression, or nonactivation of any one gene (Figure 2).

The first set of genes induced by pressure overload includes several transcription factors or proto-oncogenes, repre- senting what is now called the immediate early gene program. These proto- oncogenes are all transiently upregulated, but the kinetics vary with the experimental model. In the classic model of suprarenal aortic constriction that is usually considered to produce a rather moderate hemodynamic overload, accumulation of c-fis and c-myc transcripts are detected 30 min to 2 h after constriction, peak at 8 h, and return to baseline by 48 h after surgery (Komuro et al. 1988; Izumo et al. 1988). In the model of thoracic aortic constriction that produces an acute pressure over-

load, c-fos and c-myc expression as well as that of other proto-oncogenes (c-Jon, junJ3, and nur77), peaks as early as 15 min after constriction and returns to basal values within 2-3 h (Rockman et al. 1991). Fos is localized in myocytes, whereas Myc is expressed primarily in nonmuscle cells, showing a cellular segregation of these proto-oncogene products (Snoeckx et al. 1991). Their role in the development of cardiac hypertrophy is completely unknown although poten- tially critical. Heterodimers of Fos/Jun or homodimers of Jun act cooperatively to induce flexure at consensus AP- 1 sites in transcriptional promotor regions for a number of genes and may react with other factors to regulate differential gene expression (Kerppola and Curran 1991). As for Myc, it belongs to a larger family of proteins (helix-loophelix) that has been shown to be important in skeletal muscle differentiation (Tapscott and Wein- traub 1991). In heart, its role is entirely unclear, but there is evidence suggesting that it may be important early in cardiac development or cell division. Indeed, transgenic mice expressing c-myc transcripts undergo a proliferative response early on, which ends during postnatal development (Jackson et al. 1990). One of the challenges in the field of cardiac hypertrophy is thus to determine whether the immediate early gene program is necessary for or only coincidental with the activation of other cardiac genes.

Other ubiquitous genes involved in tissue response to stress and tissular growth are selectively upregulated after pressure overload. There is a transient and early expression of three heat-shock proteins (HSP70, HSP68, and HSP58) that could confer some degree of protection to the actively growing myocytes (Delcayre et al. 1988). There is also evidence for production of trophic sub- stances and other transcription factors within the heart. Experimental aortic banding increases the accumulation of mRNAs encoding transforming growth factor pl (TGFPl), insulinlike growth factor-I, and early growth response factor 1 (Egr-1), a serum-inducible zinc finger protein. Basic fibroblast growth factor (bFGF) immunoreactivity also increases markedly in the hypertrophied myocytes, and it is worth noting that basic and acidic FGF exert antithetical and re- ciprocal effects on skeletal a-actin transcription that is only observed in cardiac

178 01992, Elsevier Science Publishing Co., 1050-1738/92/$5.00 TCM Vol. 2, No. 5, 1992

TroDhic Trigeers Adrenergic agonists Angiotensin II Endothelin

TGFf.3

bFGF, aFGF

echanical M Stretch Load

Atrial

rRNA

SERCA2a 1

Phospholamban 4

Natriuretic Factor ‘?

T Muscarinic

Receptors 1

/&y

PI-Adrenergic

Receptors L

Myocytes

Heat-Shock Proteins

HSP 70 1‘

HSP 68 t

HSP 58 t

Figure 2. Quantitative changes during hemodynamic overload-induced cardiac hypertrophy, including those in the immediate early gene program (in italics) and other more long-term changes in gene expression. A nonexhaustive list of some potential mechanical and trophic triggers is also presented.

myocytes (reviewed in Schneider and Parker [1991] and Chien et al. [1991]). Proof is still required to determine whether these factors (or other members of the vast family of growth factors) play a direct role in vivo, but it is now clear that cardiac muscle cells in vitro are targets for regulation by peptide growth factors, and an autocrine or paracrine model of myocardial hypertrophy is strongly sug- gested by these findings.

The ventricular expression of atria1 natriuretic factor (ANF) in response to a number of diverse hypertrophic stimuli including pressure and volume overload is one of the best characterized examples of selective long-term induction. During embryonic development, the ANF gene is expressed in both the ventricles and the atria, but shortly after birth its expression is largely limited to the atria. With cardiac hypertrophy, this gene is reex- pressed in the ventricles of mice, rats, and humans, indicating that this induction is a generalized response to pressure overload (reviewed in Mercadier and Michel [1990]). Since this peptide hormone plays a significant role in regulating blood pressure and natriu-

resis, its reinduction may contribute to a reduction in pre- and afterload and thus normalize the working conditions of the cardiac pump. From the point of view of gene expression, this induction repre- sented the third example of a reactivated fetal program.

In contrast, failure to upregulate the expression of individual genes can lead to a relative decrease of the level of gene products in the hypertrophied myocardium. The Ca2+ATPuse of the sarcoplasmic reticulum is a product of a multigene family and, in ventricular and atria1 tissue, a single isoform predominates, the slow skeletal/cardiac form SERCAZa. With pressure overload, there is neither an induction of a new isofonn nor an apparent activation of this gene. The result is a net decrease in its transcripts in rats, rabbits and humans, paralleled by a decrease in its protein levels in rat and rabbit (Komuro et al. 1989; Nagai et al. 1989; de la Bastie et al. 1990; Mercad- ier et al. 1990). The mRNA decrease occurs relative to total rRNA content and total MHC RNA, indicating a potential imbalance between the contractile and relaxation properties of the heart, and is

probably implicated in delayed rates of Ca2+ transients and myocardial relaxation (see Gwathmey et al. [ 19913. Thus, it appears that, in hemodynamically overloaded hearts, contraction is regulated via isoform switches and relaxation via quantitative modulation of a single isoform. mRNA expressions of phospholamban, one of the modulators of the activity of SERCA2a, decrease in parallel to that of SERCA2a, which may play a role in the differential sensitivity of hypertrophied and normal hearts to p- adrenergic action (Nagai et al. 1989). Other examples of apparently nonacti- vated genes include the Na channel (see Chien et al. [1991]) and the P1-adrenergic and muscarinic receptors. For the receptors, it is not yet known whether this is due to nonactivation of the corre- sponding genes or to other types of mechanisms, since the results have come mostly from binding studies (reviewed in Bristow et al. [1990] and Delcayre and Swynghedauw [1991]).

All of the above data show that alterations in gene expressions in the hypertrophied and failing hearts are selective. Evaluation of these alterations in human hearts has been limited in the past by the availability of cardiac tissue, but quantification of gene expressions in endomyocardial biopsy specimens by polymerase chain reaction amplifications will cer-

TCM Vol. 2, No. 5, 1992 01992, Elsevier Science Publishing Co., IOSO-1738/92/$5.00 179

tainly soon advance our knowledge in this field (Feldman et al. 1991).

l Regulatory Levels

Since protein expressions depend upon both transcriptional and translational processes, differentiation between these events is necessary if we are to understand the regulatory levels that control gene expressions. The difficulties in studying these processes in vivo caused most early studies to be conducted in vitro, mostly through the use of beating or arrested perfused hearts exposed to various perfusion pressures. Increased aortic pressure per se is sufficient to acceler- ate protein synthesis in heart via acceleration of both peptide chain initia- tion and elongation, indicating increases in the translational capacity of preex- isting ribosomes (reviewed in Morgan and Baker [ 19911). There is also enhanced protein synthesis through pretranslational events due to an increase in ribosome content resulting from enhanced RNA polymerase I activity and rRNA synthesis. Direct evidence of pretranslational regulation of individual genes has come from in vivo studies in rats and rabbits, where the synthesis of P-MHC and its mRNA levels changed in parallel after pressure overload (Izumo et al. 1987; Nagai et al. 1987). Several promising in vivo approaches toward the elucidation of mechanisms that control gene expression in response to hemodynamic overload have been recently developed. We have succeeded in isolating transcriptionally active nuclei from hearts of neonatal and adult rats, and found that the transcript accumulations for a- and P-MHC and cardiac and skeletal a-actins correspond directly to the relative transcriptional activity for each respective gene in 23-day-old ani- mals (Boheler et al. 1992). Based on these results, we now speculate that the changes in P-MHC expression seen in vivo with overload will be regulated primarily through transcriptionally mediated mechanisms, but further proof is needed. Another approach is direct injection of DNA into the myocardium (reviewed in Ieinwand and Leiden [1991]). Unlike most cells, cardiac myocytes share with skeletal fibers the potential of tak- ing up and expressing foreign DNA. Since reporter plasmids containing rat a-myosin heavy-chain promoter regions

seem to be regulated after injection as the endogenous MHC gene, the model of gene transfer should prove useful in studying cardiac gene regulations in normal and pathologic conditions. Fi- nally the major difficulties of aortic constriction in transgenic mice (hemodynamic analysis and surgery) were both recently and successfully resolved, which will enable the delineation of the hemody- mimic responsive elements for a given gene (Rockman et al. 1991). The first data already suggest that distinct sets of regulatory sequences mediate atria1 specific and inducible expression of ANF during in vivo hypertrophy.

How is it possible for the cell to have a differential response and an induction of a large pool of RNAs, yet a specific regulation of a subset of RNAs? One in vivo example of how selective induc- tions or nonactivations may occur involves G protein expression. G protein subunits form complexes that couple the functions of receptors (for example, adrenergic) to that of effecters (for instance, adenylate cyclase or phosphol- ipase C) and whose modulation may induce significant functional changes in myocardial cells (reviewed in Neer and Clapham [ 19921). With isoproterenol infusions into rats, the concentrations of the inhibitory subunits Gi,* and Gia_s transcripts increase while those of the stimulator-y subunit G,, remain largely unchanged (Eschenhagen et al. 1991). Recent results from in vitro transcription assays indicate that the gene for

Gia-2 is transcriptionally activated to levels that correspond well with its accumulated mRNA; however, the gene for G,, is not activated (Muller et al., unpublished data, 1992). The difference in this activation probably resides in the promoter regions for these two genes, where Gicr.z unlike G,, contains a CAMP responsive element that is responsive to the P-adrenergic stimulation.

l Potential Triggers

The use of in vitro models and cell culture systems has led to the greatest insights into the possible triggers in the development of cardiac hypertrophy. Although they cannot mimic exactly the conditions of the intact heart, they are very useful in describing individual pathways by which a change in hemodynamic load is transduced into a change

in cardiac mass and cardiac phenotype. These systems will not be described here in detail because they have been reviewed at length elsewhere (Cooper et al. 1989; Simpson et al. 1989; Mor- gan and Baker 1991; Chien et al. 1991; Schneider et al. 1991). They include cultured myocytes isolated from neo- nate and adult hearts, isolated papillary muscles, skinned fibers, and beating or arrested isolated perfused hearts subjected to various pressure and volume-loading conditions. In vivo studies have also been extensively used, including chronic or acute administration of adrenergic agonists or antagonists, thyroxine, angiotensin II, converting enzyme inhibitors, and the unloading of papillary muscles. Very schematically, two types of triggers have been found: mechanical triggers via stretch, load, cell deformation, and contraction; and trophic triggers such as peptide- derived growth factors (including fibroblast growth factors, transforming growth factors, angiotensin IX, and endothelin), thyroxine, adrenocorticoids, insulin, growth hormone, or adrenorecep- tor activation.

What are the intracellular mechanisms coupled to these initiating sig- nals? The data are largely circumstan- tial but highly suggestive of the involvement of protein-kinase-C- and/ or protein-kinase-A-dependent pathways and of ionic messengers like sodium and calcium. The best evidence, however, comes from in vitro studies and activation of protein kinase C (PKC). Stimulation of PKC by phorbol ester addition (phorbol myristate acetate) is capable of partially mimicking the a,-agonist-mediated induction of the immediate early gene program (c-fos, c-jun, Egr-1) and of skeletal a-actin, P-MHC, and myosin light-chain-2 gene expressions in cultured neonatal cells even in the presence of cycloheximide, a protein synthesis blocker (Simpson et al. 1989; Dunnmon et al. 1990). Further- more, transcription of the P-MHC isogene and other reporter plasmid constructs containing AP-1 recognition sites is preferentially stimulated by l3-PKC in cardiac neonatal myocytes (Kariya et al. 1991). Phorbol-ester- induced hypertrophic growth of neonatal cardiomyocytes apparently involves an increase in ribosomal DNA transcription (Al10 et al. 1991), and


stretch-activated induction of the early response gene c-fos is suppressed by protein kinase C inhibitors and induced with phorbol esters (Komuro et al. 1991). From these results has developed the hypothesis that activation of PKC may lead to phosphorylation of a transcription factor that can bind to the regulatory regions of accessible genes and the specific activation of responsive genes. Direct evidence in vivo is, however, lacking.

JM: 1991. Myocyte mitotic division in the

aging mammalian rat heart. Circ Res

69:1159-1164.

Black FM, Packer SE, Parker TG, et al.: 1991. The vascular smooth muscle a-actin gene is reactivated during cardiac hypertrophy pro- voked by load. J Clin Invest 88:1581-1588.

Boheler KR, Carrier L, de la Bastie D, et al.: 199 1. Skeletal actin mRNA increases in the human heart during ontogenic development and is the major isoform of control and failing adult hearts. J Clin Invest

88:323-330.

l Perspectives

As we have seen, cardiac hypertrophy due to chronic hemodynamic overload is a complicated process. At the molecular level, one of the primary goals of the future will be the complete analysis of the mechanisms regulating individual gene expressions in vivo. Several possible mechanisms have been described, but direct proof and their roles in vivo are still lacking. With the availability of cardiomyocyte AT-l tumor cell lineage (Delcarpio et al. 1991), it should be possible to differentiate further the mechanisms involved in gene regulation with cardiac hypertrophy, but the use of in vivo models and transgenic mice will be of paramount importance. They will provide the means to compare changes in gene expressions directly with physiologic functions, leading to a more complete understanding of which phenotypic changes have beneficial versus detrimental effects. This must be the ultimate goal if we are to elucidate the pathogenesis and treatment of heart failure.

Boheler KR, Chassagne C, Martin X, Wisnewsky C, Schwartz K: 1992. Cardiac expressions of a- and 8-myosin heavy chains and sarcomeric a-actins are regulated through transcriptional mechanisms. J Biol Chem 267:12,979-12,985.

Bristow MR, Hershberger RE, Port JD, et al.: 1990. P-Adrenergic pathways in nonfailing and failing human ventricular myocardium. Circulation 82(Suppl 1):112-125.

Carrier L, Boheler KR, Chassagne C, et al.: 1992. Expression of the sarcomeric actin isogenes in the rat heart with development and senescence. Circ Res 70:999-1005.

Chien KR, Knowlton KU, Zhu H, Chien S:

1991. Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response. FASEB J 5:3037- 3046.

Cooper G lV Kent RL, Mann DL: 1989. Load induction of cardiac hypertrophy. J Mol Cell Cardiol21 (Suppl5): 1 l-30.

Cummins P: 1982. Transitions in human atria1 and ventricular myosin light-chain

isoenzymes in response to cardiac pressure overload induced hypertrophy. Biochem J 205:195-204.

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