Mapping of a novel gene for familial hypertrophic cardiomyopathy to chromosome 11.

Familial hypertrophic cardiomyopathy (FHC) is a cardiac disorder transmitted as an autosomal dominant trait. FHC has been shown to be genetically heterogeneous with less than 50% of published pedigrees being associated with mutations in the beta myosin heavy chain (beta-MHC) gene on chromosome 14q11-q12. A second locus has recently been reported on chromosome 1. We examined the segregation of microsatellite markers in a French pedigree for which the disease is not linked to beta-MHC gene. We found significant linkage of the disease locus to several (CA)n repeats located on chromosome 11 (lod scores between +3.3 and +4.98). The data suggest the localization of the novel FHC gene in a region spanning 17 centiMorgans.

Apoptosis in pressure overload-induced heart hypertrophy in the rat.

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'-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.

Familial hypertrophic cardiomyopathy. Microsatellite haplotyping and identification of a hot spot for mutations in the beta-myosin heavy chain gene.

Familial hypertrophic cardiomyopathy (FHC) is a clinically and genetically heterogeneous disease. The first identified disease gene, located on chromosome 14q11-q12, encodes the beta-myosin heavy chain. We have performed linkage analysis of two French FHC pedigrees, 720 and 730, with two microsatellite markers located in the beta-myosin heavy chain gene (MYO I and MYO II) and with four highly informative markers, recently mapped to chromosome 14q11-q12. Significant linkage was found with MYO I and MYO II in pedigree 720, but results were not conclusive for pedigree 730. Haplotype analysis of the six markers allowed identification of affected individuals and of some unaffected subjects carrying the disease gene. Two novel missense mutations were identified in exon 13 by direct sequencing, 403Arg-->Leu and 403Arg-->Trp in families 720 and 730, respectively. The 403Arg-->Leu mutation was associated with incomplete penetrance, a high incidence of sudden deaths and severe cardiac events, whereas the consequences of the 403Arg-->Trp mutation appeared less severe. Haplotyping of polymorphic markers in close linkage to the beta-myosin heavy chain gene can, thus, provide rapid analysis of non informative pedigrees and rapid detection of carrier status. Our results also indicate that codon 403 of the beta-myosin heavy chain gene is a hot spot for mutations causing FHC.

Altered sarcoplasmic reticulum Ca2(+)-ATPase gene expression in the human ventricle during end-stage heart failure.

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 end-stage 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 less than 0.01 and -47%, P less than 0.05, respectively). The LV ratio of Ca2(+)-ATPase mRNA to 18S RNA positively correlated with cardiac index (P less than 0.02). The RV ratio correlated negatively with systolic, diastolic and mean pulmonary arterial pressures (P less than 0.02, P less than 0.02, and P less than 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.

Effects of chronic growth hormone hypersecretion on intrinsic contractility, energetics, isomyosin pattern, and myosin adenosine triphosphatase activity of rat left ventricle.

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 less than 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 less than 0.001). Increased curvature of the force-velocity relationship (+64%, P less than 0.05 vs. controls) indicated that the muscles contracted more economically, suggesting the involvement of V3 myosin. Total calcium- and actin-activated 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.

(Ca2+ + Mg2+)-dependent ATPase mRNA from smooth muscle sarcoplasmic reticulum differs from that in cardiac and fast skeletal muscles.

We have investigated some characteristics of the sarcoplasmic reticulum (Ca2+ + Mg2+)-dependent ATPase (Ca2+-ATPase) mRNA from smooth muscle using specific cDNA probes isolated from a rat heart cDNA library. RNA blot analysis has shown that the Ca2+-ATPase mRNA expressed in smooth muscle is identical in size to the cardiac mRNA but differs from that of fast skeletal muscle. S1 nuclease mapping has moreover shown that the cardiac and smooth muscle isoforms possess different 3'-end sequences. These results indicate that a distinct sarcoplasmic reticulum Ca2+-ATPase mRNA is present in smooth muscle.

Effect of amiodarone on myosin isoenzymic distribution in rat ventricular myocardium.

Rats were given amiodarone (50 mg X kg-1 X day-1, orally) for 4 weeks and the distribution of ventricular isomyosins, a sensitive index of the effects of thyroid hormones on cardiac tissue, was analyzed. Amiodarone treatment induced a marked increase in both T4 and rT3 and tended to decrease T3 serum levels. At the pharmacologically active dosage we used, the drug induced a moderate redistribution of ventricular isomyosins in favour of V, at the expense of V1. Our results do not support the hypothesis that the major mechanism of action of amiodarone is mediated through hypothyroid-like effects.

[Left ventricular accumulation of messenger ribonucleic acid coding for the natriuretic atrial factor in various experimental models of cardiac hypertrophy in rats]. / Accumulation ventriculaire gauche de l'acide ribonucléique messager codant pour le facteur atrial natriurétique dans divers modèles expérimentaux d'hypertrophie cardiaque chez le rat.

Cardiac hypertrophy secondary to chronic hemodynamic overload is associated with an increase in the ventricular concentration of the messenger ribonucleic acid (mRNA) coding for the atrial natriuretic factor (ANF). We have compared, in male Wistar rats (10 week old, 200-220 g), using dot blot hybridization and a specific oligonucleotide probe, the left ventricular concentration of ANF mRNA (LV ANF mRNA) in 4 models of chronic hemodynamic overload inducing various patterns of left ventricular hypertrophy (LVH): a model of volume overload, the aortocaval fistula (ACF, n = 15); a model of pressure overload, coarctation of the abdominal aorta (CoA, n = 13) and 2 models of mixed overload, aortic regurgitation (AR, n = 7) and myocardial infarction (INF, n = 18). A month after surgery, LVH was 49 p. 100 for AR, 41 p. 100 for Co A and 21 p. 100 for ACF. Instead of a severe infarction, LVH was 6 p. 100 in INF demonstrating a marked hypertrophy of the non infarcted myocardium. For each model, LV ANF mRNA was compared to that in a corresponding group of sham-operated control rats and expressed as the percentage of ANF mRNA concentration in the pooled atria of the controls. In the 4 control groups LV ANF mRNA was 1 +/- 0.5 p. 100 that in the corresponding atria and the sham-operated animals were thus pooled in a single group (n = 19). In the 4 models of LVH, LV ANF mRNA markedly increased as compared to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Antithetical accumulation of myosin heavy chain but not alpha-actin mRNA isoforms during early stages of pressure-overload-induced rat cardiac hypertrophy.

Myocardial response to a hemodynamic overload involves changes in the expression of isogenes encoding myosin heavy chain (MHC) and actin: beta-MHC/alpha-MHC and skeletal/cardiac alpha-actin mRNA isoform ratios are increased. It is not known whether these changes are due to increased accumulations of the two neosynthesized transcripts, beta-MHC and skeletal alpha-actin, or whether the mRNA isoforms normally present, alpha-MHC and cardiac alpha-actin, are concomitantly decreased. To answer these questions, using dot-blot hybridizations, primer extension, and exonuclease VII mapping assays, we have analyzed the content of sarcomeric MHC and actin mRNAs in the poly(A+) RNA in left ventricles of 23-24-day-old rats 18 and 24 hours after a pressure overload induced by stenosis of the thoracic aorta. The results showed a 1.9-fold increase in poly(A+) RNA after the stenosis. Skeletal/cardiac alpha-actin mRNA isoforms were already increased fivefold (from 0.19 to 0.99) at 18 hours, and this was exclusively due to a 5.5-fold increase in skeletal alpha-actin mRNA. At 24 hours, this ratio was increased ninefold (from 0.14 to 1.22), and this was due to a 4.3-fold increase in the level of skeletal alpha-actin mRNAs (p < 0.001) and a 1.9-fold decrease of cardiac alpha-actin mRNA (p < 0.001), restoring the same proportion of sarcomeric actin mRNA in sham-operated and operated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of thyroid hormone on the accumulation of mRNA for skeletal and cardiac alpha-actin in hearts from normal and hypophysectomized rats.

Skeletal alpha-actin gene products are coexpressed with cardiac alpha-actins in cardiac tissue of adult humans, cows, and pigs; in prenatal rats; and during hypertrophy due either to increased hemodynamic load or the administration of alpha-adrenergic agonists. Because there is preferential synthesis of the beta-myosin heavy chain in each case, it has been suggested that the synthesis of skeletal alpha-actin in cardiac tissue is linked to that of beta-myosin heavy chain. To test this hypothesis, thyroid hormone, which causes cardiac hypertrophy with preferential synthesis of alpha-myosin heavy chain, was administered to normal and hypophysectomized rats. Animals were sacrificed from 2 to 24 hr after the injection of either 1 or 5 micrograms of hormone per 10 g of body weight. The relative amount of mRNA for skeletal and cardiac alpha-actin was measured by using the technique of primer extension. Thyroid hormone caused a rapid increase in the amount of skeletal alpha-actin mRNA relative to controls, more than 7 times in hearts from normal animals and 15 times in hearts from hypophysectomized animals. A small increase in cardiac alpha-actin mRNA also occurred. The rapid increase in transcripts for skeletal alpha-actin under conditions where the isoform of myosin heavy chain that is being synthesized is primarily alpha demonstrates independent patterns of activation of the actin and myosin heavy chain multigene families during cardiac growth in mammals.

Cardiac expressions of alpha- and beta-myosin heavy chains and sarcomeric alpha-actins are regulated through transcriptional mechanisms. Results from nuclear run-on assays in isolated rat cardiac nuclei.

In the heart, mRNA accumulations for sarcomeric actins and myosin heavy chains (MHC) are subject to diverse regulatorial processes. To study cardiac contractile protein transcriptional regulations, an in vitro transcription system using nonenzymatically isolated rat cardiac nuclei was characterized. Transcription was shown to be rapid and continuous during the first 20 min of incubation and 5.4-fold less than that seen from comparably isolated hepatocyte nuclei. Neither RNase nor DNase activities were detectable. Direct transcriptional analyses of the alpha- and beta-MHC and cardiac and skeletal alpha-actin genes from cardiac nuclei were performed. In 23-24-day-old rats, significant levels of transcription were seen for alpha-MHC and for the sarcomeric alpha-actins. beta-MHC was just detectable, and no positive signals were ever seen for fibronectin. We then compared the perecentages of MHC and sarcomeric alpha-actin expressions determined from 1) the transcriptional assays and 2) total isolated RNA (alpha-MHC: 90.1 +/- 4.8% (transcription), 93.0 +/- 4.7% (accumulation); beta-MHC: 9.9 +/- 4.8%, 7.0 +/- 4.7%; cardiac alpha-actin: 84.0 +/- 2.5%, 84.9 +/- 2.5%; skeletal alpha-actin: 16.1 +/- 2.5%, 15.0 +/- 2.5%). The results support the conclusion that the primary mechanisms controlling the accumulations of these gene products are transcriptional. Additionally, we show that an anti-sense mRNA showing strong homology or identity with the 5' end of the beta-MHC gene is transcribed in cardiac nuclei but not in hepatocyte nuclei.

Regulation of myosin heavy chain and actin isogenes during cardiac growth and hypertrophy.

Expression of myosin heavy chain (MHC) and actin multigene families changes in mammals during cardiac growth and hypertrophy, but whether or not there is a common regulatory pathway is unclear. To address this question, we have looked at the alpha- and beta-MHC, and at the alpha-skeletal and alpha-cardiac actin (alpha-skel act and alpha-card act) isomRNA transitions during development and senescence, both in rat and human hearts. Since the precise amounts of each isoactin mRNA were not precisely known in the above situations, we first analyzed the time- course of accumulations of the two sarcomeric transcripts by primer extension assays, which allow an umambiguous quantification of the ratios of the two actin transcripts. In rats, both isogenes are expressed in-utero. alpha-skel act represents 40% of the total one week after birth, remains constant for 3 weeks, decreases to less than 5% at two months and does not re-accumulate thereafter. In humans, in contrast, alpha-skel act represents < 20% in-utero and in neonates, increases to 48% during the first decade after birth and becomes the predominant isoform of adult hearts. In rats beta-MHC mRNAs accumulate at birth, become undetectable at 3 weeks and reaccumulate to as much as 80% during senescence, and in humans beta-MHC mRNAs predominate throughout all developmental stages. These data show that in both species, the multigene families encoding the major contractile proteins are not coordinately regulated during development and aging.(ABSTRACT TRUNCATED AT 250 WORDS)

[Accumulation of messenger RNA of the atrial natriuretic factor in the rat left ventricle at the compensatory hypertrophy stage in pressure overload]. / Accumulation de l'acide ribonucléique messager du facteur atrial natriurétique dans le ventricule gauche du rat à la phase d'hypertrophie compenséee d'une sucharge de pression.

The atria produce several peptides that have natriuretic and vasoactive properties, collectively called atrial natriuretic factor. All these peptides share a single messenger ribonucleic acid, the amount of which greatly increases in the rat left ventricle when the latter is submitted to chronic volume overload. Using the molecular hybridization technique and a desoxyribonucleic acid probe complementary to the atrial natriuretic factor messenger ribonucleic acid, we now report that a very important increase in the amount of this messenger ribonucleic acid is also observed in rat ventricle at at the compensatory stage of a pressure overload induced cardiac hypertrophy. This result suggests that the pressure overload hypertrophied rat ventricle also has the potential to itself regulate it's loading conditions via the regulation of extracellular fluid volume and vascular resistance.

Cloning of OL1, a putative olfactory receptor and its expression in the developing rat heart.

By using a strategy based on nucleotide sequence homology, we have cloned an intronless DNA encoding a new putative member of G protein-coupled receptors. The deduced amino acid sequence of the rat OL1 receptor, together with its expression at high levels in a small subset of cells in the olfactory neuroepithelium indicate that OL1 is related to the recently discovered olfactory multigene family. PCR and in situ hybridization analyses showed the OL1 transcripts to be not only expressed in the olfactory epithelium, but also in the heart. This unexpected cardiac expression was developmentally regulated, being maximal at early postnatal stages and hardly detectable at adult stages. Moreover, this observation was not restricted to OL1 since it was extended to other putative olfactory receptors. Although its functional significance remains unknown, this transient cardiac expression suggests that receptors belonging to the olfactory superfamily, could be not only involved in odor coding, but also in cardiac morphogenesis. Another olfactory-specific gene transcript encoding PTP NE-3, a recently cloned receptor-type protein-tyrosine phosphatase, could also be detected in heart. The very low levels of expression observed in rat embryo and at early postnatal stages as compared to adult stages, suggest that protein-tyrosine phosphatases, as well as protein-tyrosine kinases, may play a role in the control of cardiac cell growth and morphogenetic processes.

Use of antibodies against dodecylsulfate-denatured heavy meromyosins to probe structural differences between muscular myosin isoenzymes.

Heavy meromyosin, a tryptic myosin fragment, was purified from rabbit fast twitch muscles and rat cardiac ventricles. Both types of heavy meromyosin were denatured by sodium dodecylsulfate and used to immunize guinea-pigs after chromatography on Sephadex G-10 to remove excess dodecylsulfate. Micro-complement fixation analysis showed that the antisera were specific to a denatured configuration of heavy meromyosin and myosin, and hardly recognized the native proteins. Cross-reactions performed with both rabbit skeletal and rat cardiac antisera indicated that the antigenic structures of denatured myosins varied according both to species (man, rabbit, rat or mouse), and to muscle-type (red skeletal slow twitch, while skeletal fast twitch, cardiac atria or cardiac ventricles). Denatured heavy meromyosin chromatography on Sephadex G-200 in the presence of 0.1% sodium dodecylsulfate enabled separation of several polypeptides groups. Of these, a polypeptide of Mr 29000 was the most reactive and exhibited the same immunological specificities as the whole myosin molecule. The use of antibodies against denatured heavy meromyosin in conjunction with micro-complement fixation therefore provides a discriminant means, not only for estimating the structural relationship between several myosin isoenzymes, but also for localizing constant and variable regions in the heavy chains of these isoenzymes.

Comparisons of rat cardiac myosins at fetal stages in young animals and in hypothyroid adults.

Rat cardiac ventricular myosins were obtained from fetuses, from young normal animals, and from hypophysectomized adults. The purified proteins were compared by several techniques: (i) electrophoresis in non-denaturing conditions (pyrophosphate buffer), (ii) one- and two-dimensional analysis after proteolytic cleavage, (iii) immunological blotting after electrophoretic purification, and (iv) competitive enzyme-linked immunosorbent assay. Antibodies specific to each of the two major isoenzymes of adult rat heart (V1 and V3 according to the terminology of Hoh et al. (Hoh, J. F. Y., McGrath, P. A., and Hale, P. T. (1978) J. Mol. Cell. Cardiol. 10, 1053-1076) were used for the immunological studies. The heavy chains of the ventricular myosin isoenzymes of fetuses (V3F) were indistinguishable from those of the V3H isoenzyme present in hypophysectomized adults; both proteins differed from the V1 isoform of young animals. The light chains of V3F, V3H, and V1 were the same, except that V3F contained in addition a small amount of the embryonic light chain (Whalen, R. G., and Sell, S. M. (1980) Nature 286, 731-733). These results strongly suggest that adaptation of the adult rat heart to the hormonal deficiencies of hypophysectomy is mediated by the synthesis of the same myosin heavy chain form which is predominant in fetal hearts.

Myocardial adaptation to creatine deficiency in rats fed with beta-guanidinopropionic acid, a creatine analogue.

A creatine analogue, beta-guanidinopropionic acid (GPA), was fed to 12 young rats for several weeks. Another 12 animals were kept in the same conditions and age matched. Six pairs of animals were used to measure some energetic and mechanical parameters of the isovolumic perfused heart and to measure the accumulation of the phosphorylated form of GPA (GPAP) by 31P-nuclear magnetic resonance (NMR) spectroscopy. As a result of GPAP accumulation, phosphocreatine and ATP content decreased by 90 and 40%, respectively, and mechanical performance was impaired. Six other pairs of animals were used to assess the mechanical performances of Triton X-100-skinned fibers and the myosin isoenzyme distribution. It was found that the maximal force, Ca and ATP sensitivities, and myofibrillar creatine kinase efficacy of creatine-depleted hearts were similar to control values. There was, however, a decrease in the rate of cross-bridge cycling, and the isoenzymic expression of myosin was changed from the fast myosin V1 to the slower forms V2 and V3. In all animals, hypertrophy was observed in both right and left ventricles. We conclude that rat hearts subjected to a slow and persistent decrease in creatine and phosphocreatine respond with both quantitative and qualitative changes. These alterations, which most probably lead to an improvement in the economy of cardiac contraction, are nonetheless not sufficient to maintain maximal force.

Atrial natriuretic factor messenger ribonucleic acid and peptide in the human heart during ontogenic development.

We have investigated the level of expression of the atrial natriuretic factor (ANF) gene in the human heart during ontogenic development by determining the concentrations of ANF messenger ribonucleic acid (ANF mRNA), of immunoreactive ANF (IR ANF) and of receptor reactive ANF (RR ANF), in myocardial samples of the various heart chambers. We found the level was high and almost identical in the left and right ventricles in utero. It gradually decreased during ontogenic development to reach the low adult levels, with a more rapid decrease in the right than in the left ventricle after birth. In the atria, ANF gene expression was high as early as the 13th week of gestation, was higher in the right than in the left atrium, and appeared little affected by ontogenic development.

Differential effect of thyroxine on atrial and ventricular isomyosins in rats.

Two myosin heavy chains (MHCs), alpha and beta, which exhibit different levels of ATPase activity related to the different velocities of muscle shortening, are differentially expressed in rat cardiac ventricles, depending on the developmental stage and the thyroid status of the animals. In contrast, no changes have been reported concerning the expression of atrial MHCs in the same physiological and pathological conditions. We have now performed studies with sensitive techniques to test the hypothesis that the expression of alpha- and beta-MHCs can also be modulated in the rat atria, although at a low level. Atrial and ventricular isomyosin patterns of various groups of rats were examined by two-dimensional peptide mapping, immunofluorescence with specific anti-alpha- and anti-beta-MHC immunoglobulins, and electrophoresis under nondenaturing conditions. Normal ontogenic development of the atria is characterized by the disappearance of a small amount of beta-MHC, present at 19 days in utero. At 3 wk of age, atria and ventricles both contain only alpha-MHC. Severe hypothyroidism, produced either by methylthiouracil (MTU) treatment of pregnant females and of their litters or by hypophysectomy of adult animals, did not significantly deinduce atrial alpha-MHC but was characterized by a significant although slight accumulation of beta-MHC (less than 5% of total myosin). This latter effect was abolished by L-thyroxine restoration. It is concluded that alpha- and beta-MHC are developmentally and hormonally regulated both in atria and ventricles, although the extent of regulation is very different for the two tissues.