A decrease in the myocardial level of the mRNA encoding the Ca2+-ATPase of the sarcoplasmic reticulum (SR) has been recently reported during experimental cardiac hypertrophy and failure. To determine if such a deficit occurs in human endstage heart failure, we compared the SR Ca2+-ATPase mRNA levels in left (LV) and right ventricular (RV) specimens from 13 patients undergoing cardiac transplantation (6 idiopathic dilated cardiomyopathies; 4 coronary artery diseases with myocardial infarctions; 3 diverse etiologies) with control heart samples using a rat cardiac SR Ca2+-ATPase cDNA probe. We observed a marked decrease in the mRNA for the Ca2+-ATPase relative to both the 18S ribosomal RNA and the myosin heavy chain mRNA in LV specimens of patients with heart failure compared to controls (-48%, P < 0.01 and -47%, P < 0.05, respectively). The LV ratio of Ca2+-ATPase mRNA to 18S RNA positively correlated with cardiac index (P < 0.02).The RV ratio correlated negatively with systolic, diastolic and mean pulmonary arterial pressures (P < 0.02, P < 0.02, and P < 0.01, respectively). We suggest that a decrease of the SR Ca2+-ATPase mRNA in the myocardium plays an important role in alterations of Ca2+ movements and myocardial relaxation reported during human end-stage heart failure. (J. Clin. Invest. 1990. 85:305-309.) gene expression * heart failurehuman left ventricle -myosin heavy chain mRNA * sarcoplasmic reticulum Ca2+-ATPase mRNA

We studied the effect of chronic mechanical overloading on the isoenzyme composition of rat cardiac myosin in several experimental models: aortic stenosis (AS), aortic incompetence (AI), aortocaval fistula (ACF), overload of the non-infarcted area after left coronary ligation (INF), and overload of the spontaneously hypertensive rats (SHR). Samples of the left and right ventricles were isolated from these hearts, and myosins were analyzed by electrophoresis in non-dissociating conditions. The myosin isoenzymes were called V1, V2, and V3 in order of decreasing mobility, according to the nomenclature of Hoh et al. Controls of the Wistar and Wistar Kyoto (WKY) strains were almost exclusively V1, A slow age-dependent shift toward V3 was observed in the left ventricles of adult Wistar rats, which at 30 weeks of age (body weight 600 g) contained approximately 15% of this form. In all models of cardiac hypertrophy, an isoenzymic redistribution was observed with a significant increase in V3. The level of V3 was statistically correlated with the degree of hypertrophy in the AS, (n = 11, r - 0.6, P less than 0.05), the AI (n = 14, 4 = 0.88, P less than 0.001), and the AS + AI(n = 14, 4 = 0.69, P less than 0.01) but not in the ACF (n = 16, r = 0.46). The isoenzymic changes could account for the decreases in both myosin ATPase activity and cardiac contractility described previously in our laboratory and by others. They also demonstrate that changes in myosin isoenzymes represent a general response of the rat heart, to chronic mechanical overloading.

Pressure overload induces cardiac growth in the rat, which implies the hypertrophy of cardiac muscle cells and proliferation of nonmuscle cells. The cardiac cell loss observed in parallel has generally been attributed to necrosis. Using an in situ assay, we demonstrated a phase of apoptosis or programmed cell death during the first 7 d after pressure overload with a peak at day 4 while cardiac growth continued for over 30 d. The increase in apoptosis was confirmed by quantification of 180-1500-bp DNA oligonucleosomes with agarose gel electrophoresis and in situ labeling via 3 Ј -terminal deoxynucleotidyl transferase assay. While some apoptosis was observed in the basal state in nonmuscle cells, pressure overload induced apoptosis mainly in cardiomyocytes. These data suggest that cardiac hypertrophy is initiated by a wave of apoptosis of cardiomyocytes. Thus, apoptosis may be involved in the pathogenesis of heart remodeling. ( J. Clin. Invest. 1996. 97:2891-2897.) Key words: apoptosis • heart • hypertrophy • aortic stenosis • pressure overload

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The reduction in Ca2+ concentration during diastole and relaxation occurs differently in normal hearts and in hypertrophied hearts secondary to pressure overload. We have studied some possible molecular mechanisms underlying these differences by examining the function of the sarcoplasmic reticulum and the expression of the gene encoding its Ca2(+)-ATPase in rat hearts with mild and severe compensatory hypertrophy induced by abdominal aortic constriction. Twelve sham-operated rats and 31 operated rats were studied 1 month after surgery. Eighteen animals exhibited mild hypertrophy (left ventricular wt/body wt less than 2.6) and 13 animals severe hypertrophy (left ventricular wt/body wt greater than 2.6). During hypertrophy we observed a decline in the function of the sarcoplasmic reticulum as assessed by the oxalate-stimulated Ca2+ uptake of homogenates of the left ventricle. Values decreased from 12.1 +/- 1.2 nmol Ca2+/mg protein/min in sham-operated rats to 9.1 +/- 1.5 and 6.7 +/- 1.1 in rats with mild and severe hypertrophy, respectively (p less than 0.001 and p less than 0.001, respectively, vs. shams). This decrease was accompanied by a parallel reduction in the number of functionally active CA2(+)-ATPase molecules, as determined by the level of Ca2(+)-dependent phosphorylated intermediate: 58.8 +/- 7.4 and 48.1 +/- 13.5 pmol P/mg protein in mild and severe hypertrophy, respectively, compared with 69.7 +/- 8.2 in shams (p less than 0.05 and p less than 0.01, respectively, vs. shams). Using S1 nuclease mapping, we observed that the Ca2(+)-ATPase messenger RNA (mRNA) from sham-operated and hypertrophied hearts was identical. Finally, the relative level of expression of the Ca2(+)-ATPase gene was studied by dot blot analysis at both the mRNA and protein levels using complementary DNA clones and a monoclonal antibody specific to the sarcoplasmic reticulum Ca2(+)-ATPase. In mild hypertrophy, the concentrations of Ca2(+)-ATPase mRNA and protein in the left ventricle were unchanged when compared with shams (mRNA, 93.8 +/- 10.6% vs. sham, NS; protein, 105.5 +/- 14% vs. sham, NS). in severe hypertrophy, the concentration of Ca2(+)-ATPase mRNA decreased to 68.7 +/- 12.9% and that of protein to 80.1 +/- 15.5% (p less than 0.001 and p less than 0.05, respectively), whereas the total amount of mRNA and enzyme per left ventricle was either unchanged or slightly increased. The slow velocity of relaxation of severely hypertrophied heart can be at least partially explained by the absence of an increase in the expression of the Ca2(+)-ATPase gene and by the relative diminution in the density of the Ca2+ pumps.(ABSTRACT TRUNCATED AT 400 WORDS)

We studied papillary muscle mechanics and energetics, myosin phenotype, and ATPase activities in left ventricles from rats bearing a growth hormone (GH)-secreting tumor. 18 wk after tumor induction, animals exhibited a dramatic increase in body weight (+101% vs. controls) but no change in the ventricular weight/body weight ratio. The maximum isometric force of papillary muscles normalized per cross-sectional area rose markedly (+42%, P < 0.05 vs. controls), whereas the maximum unloaded shortening velocity did not change. This was observed despite a marked isomyosin shift towards V3 (32±5% vs. 8±2% in controls, P < 0.001). Increased curvature of the force-velocity relationship (+64%, P < 0.05 vs. controls) indicated that the muscles contracted more economically, suggesting the involvement of V3 myosin. Total calcium-and actinactivated myosin ATPase activities assayed on quickly frozen left ventricular sections were similar in tumor-bearing rats and in controls. After alkaline preincubation, these activities only decreased in tumor-bearing rats, demonstrating that V3 enzymatic sites were involved in total ATPase activity. These data demonstrate that chronic GH hypersecretion in the rat leads to a unique pattern of myocardial adaptation which allows the muscle to improve its contractile performance and economy simultaneously, thanks to myosin phenoconversion and an increase in the number of active enzymatic sites. (J. Clin. Invest.

We have analyzed the transition between isoforms of the glycolytic enzyme enolase (2-phospho-D-glycerate hydrolyase; EC 4.2.1.11) in rat heart during normal and pathological growth. A striking fall in embryonic alpha-enolase gene expression occurs during cardiac development, mostly controlled at pretranslational steps. In fetal and neonatal hearts, muscle-specific beta-enolase gene expression is a minor contributor to total enolase. Control mechanisms of beta-enolase gene expression must include posttranscriptional steps. Aortic stenosis induces a rapid and drastic decrease in beta-enolase transcript level in cardiomyocytes, followed by the fall in beta-subunit level. In contrast, alpha-enolase transcript level is not significantly altered, although the corresponding subunit level increases in nonmuscle cells. We conclude that, like fetal heart, hypertrophic heart is characterized by a high ratio of alpha- to beta-enolase subunit concentrations. This study indicates that the decrease in beta-enolase gene expression may be linked to beneficial energetic changes in contractile properties occurring during cardiac hypertrophy.

In striated muscle, chronic increases in workload result in changes in myosin phenotype. The aim of this study was to determine whether such changes occur in the diaphragm of patients with severe chronic obstructive pulmonary disease, a situation characterized by a chronic increase in respiratory load and lung volume. Diaphragm biopsies were obtained from 22 patients who underwent thoracic surgery. Myosin was characterized with electrophoresis in nondenaturing conditions, SDS-glycerol PAGE, and Western blotting with monoclonal antibodies specific for slow and fast myosin heavy chain (MHC) isoforms. Flow volume curves, total lung capacity, and functional residual capacity were measured before surgery in 20 patients. We found that the human diaphragm is composed of at least four myosin isoforms, one slow and three fast, resulting from the combination of three MHC species. Chronic overload was associated with an increase in the slow β-MHC species at the expense of the fast species (β-MHC, 78.2 ± 4.6 and 50.0 ± 6.5% in emphysematous and control patients, respectively; P < 0.005). Linear correlations were found between β-MHC percentage and forced expiratory volume in 1 s ( r = −0.52; P < 0.02), total lung capacity ( r = 0.44; P < 0.05), and functional residual capacity ( r = 0.65; P < 0.003). The human adult diaphragm is composed of a balanced proportion of slow and fast myosin isoforms. In patients with chronic obstructive pulmonary disease, the proportion of fast myosins decreases, whereas that of slow myosin increases. This increase appears to be closely related to lung hyperinflation and may reflect an adaptation of the diaphragm to the new functional requirements.