Ann. Rev. PhysioL 1979. 41:539-52 Copyright @ 1979 by Annual Reviews Inc. All rights reserved

MOLECULAR ASPECTS

OF CARDIAC HYPERTROPHY

Radovan Zak and Murray Rabinowitz

Cardiology Section of the Department of Medicine and the Department of Biochemistry, University of Chicago and the Franklin McLean Memorial Research Institute [operated by the University of Chicago for the U.S. Research and Development Administration under Contract E(11-1}-69] Chicago, Illinois 60637

INTRODUCTION

+1236

The response of cardiac growth to physiological changes that alter the hemodynamic load is a biological problem of general interest. In the early phase of work overload, cardiac growth is an adaptive response that allows the individual to survive. When the overload is prolonged, however, changes in the organization of muscle cells occur, with consequent dimin­ished contractile function and eventual heart failure. The nature of the pathological lesions as well as the transition or distinction between physio­logical and pathological growth are the points of interest. Another interest­ing problem concerns the mechanisms by which the synthesis of cellular components is regulated and adjusted to physiological demands. The extent of ATP utilization serves in some way as a signal stimulating gene activity.

Many of the reaction steps involved in the translation of genetic informa­tion into the amino acid sequence of polypeptide chains have already been elucidated in studies of rather simple biological systems, such as cell-free preparations, bacteria, or cells in culture. Much less is known about the regulatory mechanisms involved in the switching on and off of individual genes during cell differentiation and growth. Studies of gene activity in higher organisms, where several types of cells are present within the same organ, are much more difficult. The heart, for example, contains at least eight distinct cell types, the nonmuscle cells outnumbering myocytes three to one. Even muscle-specific proteins, such as myosin, cannot be considered

539 0066-4278/79/0301-0539$01.00

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an unequivocal cellular marker, since myosin isozymes are found in a great variety of cells. It is thus obvious that the study of molecular events in developing cardiac hypertrophy must proceed in the direction of analysis of products of specific genes. Any advancement in this area of heart physi­ology is contingent upon our current knowledge of molecular biology. For this reason. we present in this article an overview of mechanisms regulating gene activity and discuss the most recent information on the molecular aspects of cardiac hypertrophy published subsequent to reviews by Meerson (51) and us (57).

CONTROL OF TRANSCRIPTION

The first level of control that is almost certainly implicated in the activation of synthetic processes in hemodynamically overloaded heart is the tran­scription of messenger RNA. The information available so far about regula­tion of genetic activity in eukaryotes is still fragmentary; nevertheless, it is apparent that the regulatory mechanisms in higher organisms are much more complex than those operating in prokaryotes.

Two characteristics of DNA organization in higher organisms reflect the complexity of the mechanisms regulating genetic expression. First, DNA is intimately associated with chromosomal proteins believed to maintain the DNA in a largely repressed condition. The DNA in eukaryotic chromo­somes exists in a form of chromatin composed of DNA closely associated with two classes of proteins: the highly basic histones and the mildly acidic nonhistones. The DNA is arranged in small, repeating nucleoprotein units along chromatin fibers.

Second, the amount of DNA in the haploid nuclei of higher organisms, in contrast to prokaryotes, vastly exceeds the number of genes needed. The genomes of higher cells contain segments of repeated nucleotide sequences distributed throughout the length of the DNA fiber. The repeated sequences have been postulated as being important in the regulation of transcription of structural genes (see 21). Different classes of repeated sequences are presumed to interact with cellular regulators, perhaps mediated through receptors and chromosomal proteins, to activate specific groups of struc­tural genes. The mechanism of structural-gene activation is still unknown, but RNA or protein products of repeated sequences have been postulated to result in a cascade of activity, so that a small number of molecules are capable of activating large numbers of structural genes. Of great interest are recent observations that many eukaryotic genes contain sequences that are not produced by the mature messenger RNA (31). The gene is thus a mosaic of expressed sequences (exons) in a matrix of silent regions (introns). The regulatory function of the silent sequences is still a mystery.

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CARDIAC HYPERTROPHY 541

The exact means by which cellular metabolites interact with the chromo­somes to alter transcription is not known, but important information is being obtained from studies of the mechanism of action of anabolic hor­mones (45). It has been demonstrated in several models that the action of steroid honnones involves initial binding of the hormone to a receptor molecule in the cytoplasm of target cells. Subsequently, the hormone-recep­tor complex migrates into the nucleus, where it becomes associated with chromatin. Interaction of the receptor with the nuclear site results in activa­tion of RNA polymerase and in subsequent cellular response.

The actual process that leads to an opening of the repressed gene for transcription is still obscure. It is generally believed, however, that some uncoiling or loosening of DNA fibers within the chromatin is necessary for transcription to occur. How this loosening takes place is not known, al­though it is recognized that histones act as blocking agents and inhibitors of RNA synthesis from DNA templates (68). The relative constancy among diverse cell types and among different physiological states nevertheless makes it unlikely that histones are specific regulators of gene expression. The acidic nonhistones of chromatin are more likely candidates for the regulatory role. They are highly heterogeneous in molecular weight, show tissue specificity, bind preferentially to homologous DNA, and stimulate the transcription by RNA polymerase of isolated DNA or chromatin. It is of interest that these activities of acidic proteins can be altered by phospho­rylation. The extent of nuclear nonhistone protein phosphorylation could be related to the transcriptional activity of various tissues (13), including hearts of hyperthyroid rats (44). The elevated synthesis of RNA in hearts of animals treated with thyroid hormone seems to correlate with the activity of cyclic-AMP-dependent nuclear protein kinase.

Kun et al (43) have suggested another covalent modification of chromatin proteins that might be involved in regulation. They postulate that ADP­ribose, which can be liberated enzymatically from chromatin-bound poly­(ADP-ribose), may form a Schiff base with the E-amino group of lysine residues of proteins, and may consequently alter DNA transcription. It is of particular interest that polyamines, which are present in elevated amounts in growing organs, have been shown to serve as a trap for alde­hydes and might thus prevent chromatin modification by the degradation product of poly(ADP-ribose).

Besides changes in template activity, control of transcription could also involve modulation of the activity of RNA polymerase. Several types of RNA polymerase have been separated (see 7). The most abundant are the nucleolar polymerase. which synthesizes ribosome-like RNA (polymerase I), and the polymerase associated with chromatin (polymerase II), which is involved in the synthesis of RNA having a base composition similar to

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that of DNA, presumably messenger RNA. Activities of these two types are distinguished by preferential inhibition of polymerase II by a by-cyclic octapeptide, a-amanitin. Other enzymatic activities have been reported, such as a-amanitin-insensitive polymerase III, which is present in the nu­cleoplasm.

Studies of transcriptional control in heart are hampered by tissue heterogeneity, since nearly two thirds of the total nuclei in heart belong to nonmuscle cells (see 81). The time sequence and the intensity of response to a growth stimulus, such as hemodynamic overload, vary according to the cell type.

One of the best-documented and most striking changes in the enlarging myocardium is an increased synthesis of RNA, which can be demonstrated by labeling experiments several hours after imposition of a work-overload (57). The activity of DNA-dependent RNA polymerase in nuclei isolated from unfractionated hypertrophic myocardium rises rapidly after aortic constriction, and a peak value is reached on the second postoperative day (see 57). The main increase occurs in the activity of ribosomal RNA poly­merase. This change is preceded by increased template activity of cardiac chromatin (27).

Techniques have been developed recently for the separation of nuclei of muscle and of nonmuscle cells (18, 20). In hypertrophied heart the activity of polymerases II and III increased in nuclei of both cell populations while the activity of polymerase I, which showed the greatest change, occurred only in the muscle-cell nuclei. Assays of polymerase activity in the presence of ammonium sulfate of high molarity provide a measure of total RNA polymerase activity that is independent of chromatin activity. Molar am­monium sulfate removes most of the chromosomal proteins, allowing the endogenous DNA template to be fully utilized. Using this technique, it has been concluded that the observed changes in developing hypertrophy repre­sent an increase either in the activity or in the amount of RNA polymerase. Of interest, however, is the observation that the elevation of polymerase activity lags behind the increase in RNA synthesis. Changes in chromatin activity may account for the early rise in RNA synthesis, while an increase in the activity or amount of polymerase takes place later during the develop­ment of hypertrophy (20).

Very little is known about factors that affect the activity of RNA poly­merase. It is of interest, however, that polyamines have the ability in vitro to stimulate nuclear RNA polymerase (11) and labeling of proteins with amino acids (30). The polyamine levels are elevated early in the develop­ment of cardiac hypertrophy (10, 62), and the activity of ornithin decar­boxylase, a key enzyme in the biosynthetic pathway of spermine and spermidine, has been found to be elevated two hours after left ventricular pressure-overload (40, 47). .

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CARDIAC HYPERTROPHY 543

A rigorous demonstration of preferential synthesis of messenger RNA in cardiac hypertrophy is still elusive. Analysis of newly synthesized, pulse­labeled RNA by sucrose density-gradient centrifugation has revealed equal incorporation of label into ribosomal RNA and tRNA, with no evidence of the presence of messenger RNA with high specific radioactivity (57). These results are supported by the demonstration that the rates of tracer incorporation into RNA containing or lacking poly(A) segments are the same (74). Very early after aortic constriction, however, the activity of polymerase II is increased to a moderately greater extent than polymerase I (36). Also, Meerson et al (80) have recently reported that the proportion of heavy polysomes increases in developing hypertrophy. A conclusive answer concerning changes of messenger RNA concentration in hypertro­phy, however, still awaits the development of techniques that allow quanti­tation of specific mRNAs. Messenger RNA for myosin heavy chain has been isolated from embryonic chick muscle (e.g. 63) but not yet from mammalian heart, in part because of technical difficulties in obtaining intact heavy polysomes from differentiated heart muscle. When perfected, the use of cDNAs to measure the concentration of specific mRNA sequences may become extremely valuable in assaying the contributions of transcriptional and translational controls to the increase in protein synthesis during devel­oping hypertrophy.

Another level at which gene expression may be regulated involves pro­cesses that alter the activity of mRNA (post-transcriptional control) (e.g. 8). Three regulatory sites can be envisioned: (a) transport of mRNA from the nucleus to the cytoplasm; (b) post-transcriptional processing of the mRNA molecule, such as methylation of the 5' end of RNA or addition of poly(A) to the 3' end; or (c) modulation of functional levels ofmRNA. The importance of post-transcriptional control, at least during the development of skeletal muscle, is indicated by the demonstration that the mRNA se­quence for myosin heavy chain is present in the myoblast prior to active myosin synthesis in association with RNP particles (9, 34). At this time, only one study of possible post-transcriptional control in myocardium is available. In perfused hearts of reserpine-treated rabbits, the administration of norepinephrine resulted in enhanced activity of cytoplasmic Mn2+­stimulated polyadenylate activity (12).

CONTROL OF TRANSLATION

The translation of mRNA is a highly complex process, and consequently a large variety of regulatory mechanisms are operative, including: (a) modulation of the interaction of ribosomes with mRNA and aminoacyl­tRNA, which requires several protein factors; (b) changes in the activity of peptidyl transferase and in the extent of tRNA charging with available

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amino acids, including the activity of aminoacyl-tRNA synthetase and the availability of energy; and (c) modification of the functional state of riboso­mal subunits.

Information concerning translational control in cardiac hypertrophy is only fragmentary. In skeletal muscle, aminoacyl-tRNA synthetase activity has been found to fluctuate according to the rates of protein synthesis (55). In developing cardiac hypertrophy, in contrast, the activity of synthetase remains unaltered (28, 29). It is possible, however, that certain amino acids might be limiting since the enzymatic activities of synthetase with respect to different amino acids vary considerably (29).

Initiation and elongation processes have been evaluated mostly by anala­ysis of distribution of ribosomal classes. The proportion of polysomes, monoribosomes, and ribosomal subunits depends on the state and activity of the protein-synthesizing machinery. For example, a decreased rate of protein synthesis, coupled with a decreased proportion of polysomes and increased fraction of subunits, indicates decreased initiation. By such analy­ses, the action of anabolic hormones has been shown to enhance the initia­tion of polypeptide formation in skeletal muscle (70). Similarly, in perfused heart, increased pressure-load is accompanied by accelerated peptide-chain initiation (35).

Initiation factors appear to be involved in the selection of mRNA for translation. Thus a subfraction of initiation factor IF3 has been obtained that is required for translation of myosin mRNA in cell-free system (33). In addition, a new class of RNA called tcRNA (for translational control) has been isolated from IF3 (41). The tcRNA appears to be capable of discriminating against the translation of heterologous mRNA (e.g. globin synthesis by the reticulocyte-ceIl-free system is inhibited by muscle-derived tcRNA).

PROTEIN TURNOVER AND CARDIAC HYPERTROPHY

It has been established during the last several years that all intracellular proteins turn over with half-lives varying from a few minutes to several days or weeks (60, 82). Since turnover involves continuous degradation and resynthesis, the rate at which the level of a given protein changes is deter­mined by the balance of rates of synthesis and degradation. For example, in developing hypertrophy the rate of synthesis is the sum of two processes: (a) protein synthesis balanced by protein degradation in the turnover pro­cess, and (b) synthesis of protein in excess of degradation, which leads to cardiac enlargement. Similarly, in regression of hypertrophy, the rate of degradation includes degradation balanced by resynthesis in the turnover process, plus degradation in excess of resynthesis. In both cases, the transi-

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CARDIAC HYPERTROPHY 545

tion to the new steady state may be accomplished while the rate of protein turnover either remains unchanged or is increased or decreased compared to the steady state in the heart before the imposition of hemodynamic overload.

Studies of protein turnover in heart, as well as in other organs, are frustrated by technical problems, mostly related to the absence of precur­sors that would allow pulse-labeling of intracellular proteins. Amino acids are recycled, and the extent of recycling depends on the physiological state of the animal. Moreover, equilibria between various compartments of pre­cursor amino acids are very complex, and as a result the specific radioac­tivity of the immediate protein precursor, the aminoacyl-tRNA, differs from that of free amino acid in the intracellular compartment (46, 48). Available studies of protein synthesis in hypertrophic heart do not allow unequivocal interpretation. However, published data based on analyses of the specific radio-activities of intracellular free amino acids, rather than on measurements of aminoacyl-tRNA, indicate a rather modest increase in the rate of amino acid incorporation into total protein (24) and into myosin (52, 67) of the overloaded heart in vivo. Similarly, acute pressure-overload in the perfused heart leads to elevated labeling of myocardial proteins (37). The increased amino acid incorporation is unrelated to enhanced coronary flow, as was shown using the elegant perfusion systems of Schreiber et al (64).

Measurements of protein degradation are subject to similar technical difficulties. Use of a nonreutilizatable precursor of heme, however, indicates that degradation of cytochrome c decreases considerably during the first 24 hours of pressure-overload (58). In contrast, the rate of myosin degradation, which was calculated by comparing the amount of myosin in the enlarged heart with the increased rate of its synthesis, was found to increase in developing hypertrophy (52).

In regard to the activities of proteases involved in cardiac hypertrophy, only cathepsin D has been studied. The data are contradictory. Some stud­ies indicate no correlation between cathepsin D activity and the change in protein breakdown, either during development or regression of pressure­induced hypertrophy (79), or in hormone-induced hypertrophy (78). In one study, however, an increased latency of cathepsin D was found within the first two days of pressure-overload (37).

CONSEQUENCES OF CARDIAC HYPERTROPHY­CHANGES IN HEART STRUCTURE AND FUNCTION Cell Proliferation Activation of biosynthetic processes in myocardium that has been subjected to a sustained hemodynamic overload eventually results in cardiac enlarge­ment. The most recent studies of the cellular features of cardiac growth,

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reviewed by Rumyantsev (61), are consistent with previous observations. DNA synthesis and the consequent mitotic activity of cardiac myocytes continuously decline after birth and eventually cease altogether, at about 3-4 weeks of age in the rat. However, not every nuclear division results in cell proliferation since cells with more than one nucleus increase in number after birth (32, 39). The decline in mitotic activity is correlated in time with the loss of activities both of DNA polymerase (14,17,23) and of thymidine kinase (14). The measurements of enzymatic activities reported so far, however, have only a limited value since no distinction has been made between the nuclei of muscle and of nonmuscle cells. The control of DNA synthesis is quite different in the two cell populations. While the mitotic activity declines with age in both cell types, the rate of decline is much smaller in nonmuscle cells and, in contrast to myocytes, the repression of DNA synthesis is readily reversed by a variety of growth stimuli (see 81). Recently, however a procedure has been developed that allows separation of the nuclei of muscle and nonmuscle cells (18); the problems of regulation of DNA synthesis can thus be reexamined in a more rigorous way.

Very little is know about the factors regulating DNA replication. ADP­ribosylation of chromosomal proteins has been suggested in studies of a variety of differentiating cells as one possible regulatory factor. For exam­ple, in mixed nuclei isolated from heart, Claycomb (16) has detected an increase in the activity of poly(ADP-ribose) synthetase, with cell differentia­tion that seems inversely related to the rate of DNA synthesis. Unfortu­nately, these results are difficult to interpret quantitatively, since the concentration of NAD+ used in the assay system was much lower than the Km determined for this enzyme in rat tissues (25). Appearance of functional adrenergic innervation was also implicated in the control of DNA replica­tion, with noradrenaline and cyclic AMP as the chemical mediators (15).

In animal studies the imposition of work-overload produces a cellular response that depends on age and on the state of DNA synthesis. Overload­ing in the neonatal period results in an increased labeling with 3H-thymidine of nuclei in both muscle and nonmuscle cells (22, 75). In contrast, similar interventions in the adult 'result in labeling of the nuclei of connective cells only (6, 22).

Radioautographic procedures for measurements of cell proliferation have been supplemented recently with procedures for estimating changes in the cellular volume during cardiac growth. Radioautographic studies have sev­eral limitations: DNA synthesis does not necessarily indicate either cell division (see the discussion above of increased number of nuclei per cell) or nuclear division (e.g. endoreplication of DNA resulting in nuclei having more than two sets of chromosomes might contribute to nuclear labeling). Estimates of the total number of cells prior to and after growth stimulus

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CARDIAC HYPERTROPHY 547

thus may be a more direct approach. Unfortunately, the available proce­dures are not entirely satisfactory. However several promising new tech­niques have recently been applied to this century-old problem. By the use of the point-counting technique, the volume of heart occupied by myocytes can be determined in histological sections (56). Three different methods have been used recently to calculate the total number of muscle cells in the myocardium. In the first method, the two dimensions of myocytes have been measured after their separation by enzyme treatment (42). In the second, the cell diameter has been measured in histological sections and the volume calculated from the assumed length-width ratio (59). In the third method the cell width has been determined stereologically and the cell length defined in units of sarcomeres (69). The main advantage of the third method is its independence of the contractile state of myofibrils.

The application of the first method (42) to cardiac hypertrophy corrobo­rates the radioautographic evidence that in adult rat myocardial growth can be explained by enlargement of existing myocytes.

Recently studies of human autopsy material lends support to previous observations indicating essential differences between primates and other experimental animals. The appearance of polyploid nuclei is prominent in primates; the age of sequence of DNA endoreplication has been delineated in detail, and the times of onset thus indicated are 7 and 12 years of age in normally growing left and right ventricles, respectively (1). Moreover, the cytometric study of Astori et al (3), using sterological techniques, lends support to Linzbach's classical observation (see 81) that there is a critical weight during progressing cardiac enlargment at which cellular hypertro­phy is supplemented by addition of new muscle cells. The analysis of the frequency distribution of cell diameters and lengths is consistent with lon­gitudinal splitting of a certain cellular population.

Changes in Cardiac Ultrastructure Recent data (2,3,54) are consistent with previous observations (see 58) that the volume of mitochondria relative to cell volume increases within the first day of pressure-overload and progressively decreases later on. The relation­ships between plasma membrane and cell volume, as well as between sar­cotubular membrane area and myofibrillar volume, remain constant in left ventricular hypertrophy caused by aortic constriction (53) and by adminis­tration of thyroxine (53).

Changes in Myosin ATPase

Correlation of depressed contractility following hemodynamic overload with changes in enzymatic properties of cardiac myosin continues to be actively investigated. Despite considerable improvement in the purity of

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isolated myofibrillar and myosin preparations, recent results, reviewed by Swynghedauw et al (71), are still contradictory. The major disagreement concerns the Ca2+ -stimulated and (K+)-EDT A-stimulated ATPase of puri­fied myosin, which was found to increase in dogs three weeks after pulmo­nary constriction by one group of investigators (76), but to decrease when another group used the rabbit as the experimental animal (65, 72). Changes in right ventricular (RV) systolic pressure and increases in cardiac weight were found to be comparable by both groups. In chronically overloaded myocardium, most investigators agree that myosin ATPase is reduced (50, 71, 76, 77).

From our current knowledge of myosin structure and function, we can postulate that the alteration in ATPase activity of myosin isolated from hypertrophic heart may be related to changes in either the molecular species or relative amounts of individual myosin light chains, to the synthesis of another type of myosin heavy chain, or to changes in the reactivity of sulfhydryl residues, SH, or SHz.

No change in the relative content oflight chains has been found in myosin when its ATPase activity was reduced (38, 72), while their content was altered in myosin with elavated ATPase (49, 76). Thyrotoxicosis, however, was found to result in elevated ATPase activity with no change in light­chain content (4). The role of sulfhydryl residues in the regulation of the ATPase activity of myosin has been studied by Alpert et al (65, 72). Analy­sis of ATPase activity after chemical modification of myosin gave results consistent with conformational change in the vicinity of the fast-reacting SH, group of myosin isolated from hypertrophic heart. Since the most likely explanation for altered conformation of the SH 1 moiety is an amino acid substitution, the results were interpreted as evidence for the appearance of a new cardiac isozyme of myosin.

Studies of myosin isolated from hearts of thyrotoxic animals (4, 5, 26) lend further support to the belief that synthesis of a new species of myosin heavy chains may occur in enlarged heart. These results agree with the conclusion of Alpert (65, 72) that the region near SH1 residues is important for the observed effects of thyroxine. Moreover, the electrophoreograms of cyanobromide (CNBr) digests indicate a substitution of methionine residues in thyrotoxic animals (26), substantiating the previous claim of altered primary structure of myosin produced by thyroxine treatment (73).

CONCLUSION

Despite continuous interest in cardiac hypertrophy, our knowledge of its molecular aspects is still elementary. Recently, however, several advance­ments of particular interest have been made: (a) Nuclei of muscle and

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CARDIAC HYPERTROPHY 549

nonmuscle cells have been separated, allowing for the first time the study of nuclear activity in specified cells (18). (b) Cardiac growth induced by pressure-overload (72) or by hormone treatment (26) has been shown to lead to myosin of altered ATPase, and strong evidence suggests that new species of myosin molecules thus appear. (c) The basis for assessment of protein synthesis and degradation has been established (46, 48). (d) Meth­ods are being developed to supplement radioautography in evaluating cell proliferation (42, 59, 69). (e) In spontaneously hypertensive rats it has been shown that blood pressure might not be the sole factor responsible for cardiac enlargement, but that hypertrophy can be the result of genetic cardiovascular abnormality (19, 66). (j) A hypothesis relating the extent of energy utilization to the nuclear activity via NAD+ metabolism has been proposed, which allows for experimental verification (43).

ACKNOWLEDGMENTS

This work was supported in part by U.S. Public Health Service Grants HL09172, HL04442, HL16637, and I-PI7-HLI7648 (Specialized Center of Research in Ischemic Heart Disease), and by grants from the National Heart and Lung Institute, from the Muscular Dystrophy Association of America, from the Chicago and Illinois Heart Association, and from the Louis Block Fund of The University of Chicago.

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