Expression of the sarcomeric actin isogenes in the rat heart with development and senescence.

Sarcomeric actin genes, alpha-cardiac and alpha-skeletal, are coexpressed in neonatal rodent hearts and are regulated in response to hormonal and hemodynamic stimuli; however, their precise developmental pattern of expression has not been determined, and it is unknown whether they are coexpressed during senescence. We have, therefore, investigated the accumulation of sarcomeric actin transcripts in rat heart during fetal and postnatal development and with senescence by two different techniques: primer extension analysis with an oligonucleotide common to both sarcomeric actins and RNA hybridization with specific cardiac alpha-actin cRNA probes. We found that at 17-19 days in utero both isogenes are coexpressed and alpha-skeletal actin mRNAs represent 28.0 +/- 0.8% of the sarcomeric actin mRNA total. Skeletal actin mRNAs increase to 40% of the total 1 week after birth (NS, p = 0.15), remain constant for 3 weeks, and decrease to less than 20% of the total in ventricles and atria of 1-month-old rats. The alpha-skeletal actin transcripts further decline to less than 5% of the total at 2 months of age and do not reaccumulate in senescent animals. There was no significant difference between male and female rat ventricles. By comparison with the known accumulations of alpha- and beta-myosin heavy chain mRNAs, our results demonstrate that whatever the developmental stage the kinetics of expression for the sarcomeric myosin and actin multigene families are independent.

Switches in cardiac muscle gene expression as a result of pressure and volume overload.

In the mammalian heart, the expression of genes encoding proteins responsible for contraction, relaxation, and endocrine function changes in hypertrophy resulting from hemodynamic overload. Different mechanisms are involved in this mechanogenic transduction, including 1) differential expression of myosin and actin multigene families, which may account for the decreased velocity of contractile element shortening in hypertrophied heart, 2) nonactivation of the sarcoplasmic reticulum Ca(2+)-ATPase gene, which may explain the increased duration of isometric relaxation, and finally 3) activation in the ventricle of the atrial natriuretic factor gene that is responsible in part for the high plasma levels of this peptide. It is increasingly apparent that these changes are independently regulated, but little is known about the mechanisms underlying this regulation. Preliminary results indicate that it is now possible to analyze the early time course or transcription for each gene after the imposition of hemodynamic overload. This should significantly enhance our understanding of the regulatory mechanisms involved in the phenoconversions of the hemodynamically overloaded heart.

Ca(2+)-ATPase and function of sarcoplasmic reticulum during cardiac hypertrophy.

The properties of the calcium pump system of sarcoplasmic reticulum (SR) were studied in a series of 34 rats subjected to cardiac overload and 19 sham-operated animals. Total homogenates of left ventricle were analyzed by measuring the oxalate-supported Ca2+ uptake rate, the steady-state level of the phosphorylated intermediate of Ca(2+)-adenosine triphosphatase (Ca(2+)-ATPase) (EP), and the amount of Ca(2+)-ATPase mRNA. All three parameters decreased gradually as a function of the relative left ventricular weight increase. The calcium-sensitivity curves showed that the velocity of Ca2+ transport in SR from the hypertrophied heart is diminished at low as well as optimal Ca2+ concentrations, with the dissociation constant (Kd) value for Ca2+ unchanged from that of the control preparation. Taken together with the results presented in our recent publication (De la Bastie, Levitsky, Mercadier, Marotte, Wisnewsky, Brovkovivh, Schwartz, and Lompré, Circ. Res. 66: 554-564, 1990), these data strongly indicate that differences in the Ca2+ pump activities of SR from normal and hypertrophied rat hearts are due to quantitative rather than qualitative changes of the Ca(2+)-ATPase protein.

Skeletal actin mRNA increases in the human heart during ontogenic development and is the major isoform of control and failing adult hearts.

Expression of the two sarcomeric actins, alpha-skeletal and alpha-cardiac, is regulated in the rodent heart in response to developmental, hormonal, and hemodynamic stimuli. Little is known in man, except that both isogenes were found to be coexpressed in three adult ventricles. In this report, we investigated the isoactin mRNA composition in ventricles from 21 control patients (4 fetal, 5 juvenile, 12 adult) and from 15 patients undergoing cardiac transplantation (5 idiopathic dilated cardiomyopathies, 5 ischemic myopathies with myocardial infarcts, 5 diverse etiologies) by two different and complementary techniques: RNA dot blot analysis with specific cDNA probes, and primer extensions with an oligonucleotide common to alpha-cardiac and alpha-skeletal actins. In the case of dot blot analysis, quantification of each isoform was performed by using as standards RNA transcripts obtained from cloned human alpha-actin sequences, and the total amount of sarcomeric actin mRNA was evaluated as a function of total poly(A+)RNA. We found that both isogenes are always coexpressed, and that the isoactin pattern changes during development. In utero and in neonatal hearts, alpha-skeletal actin mRNA represents less than or equal to 20% of sarcomeric actins, it increases to 48 +/- 6% during the first decade after birth and becomes the predominant isoform of adult hearts (60.4 +/- 8.5%). The 15 adult failing hearts exhibited the same isoactin pattern as the control ones (62.84 +/- 11.06%), and there was no difference in expression between patients with dilated cardiomyopathy or ischemic heart disease. These observations demonstrate that cardiac development in man, in contrast to rodent heart, is characterized by an up-regulation of the skeletal actin gene, the expression of which does not change in hypertrophied and failing hearts, and suggest that the actin and myosin heavy chain families are independently regulated in human heart.

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

The cardiac ventricular myosin heavy chain phenotype is developmentally and hormonally regulated, but less is known concerning the actin phenotype. In this study, the levels of accumulation of alpha-skeletal and alpha-cardiac actin mRNAs were investigated in rat and human ventricles by primer extension assays. In rat, the two iso-mRNAs are present in approximately equal amounts from birth until 15 days of age and the cardiac form is predominant in adult and senescent hearts. Hypothyroid development has no effect, at least during the first two weeks of age. In man, the two isoactins are co-expressed to similar ratios in one control heart and in one failing heart. It therefore appears that myosin heavy chain and actin multigene families are both expressed in a species specific fashion but are independently regulated within a species. Preliminary results from nuclear run-on assays are presented that indicate differences in the level of transcription of the alpha-actin and beta-myosin heavy chain isogenes in the rat heart.

Function of the sarcoplasmic reticulum and expression of its Ca2(+)-ATPase gene in pressure overload-induced cardiac hypertrophy in the rat.

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)

Atrial natriuretic factor gene expression in rat ventricle during experimental hypertension.

Activation of atrial natriuretic factor (ANF) gene expression has been reported in the rat ventricle in several models of hemodynamic overload, including hypertension. However, nothing is known about the potential trigger(s) and the time course of this activation during the development of hypertension. We measured aortic blood pressure, left ventricular hypertrophy (LVH), and left ventricular ANF mRNA concentration (LV ANF mRNA) in a first group of rats (study A) killed at 5 and 18 h and 2, 4, 6, 9, 15, and 30 days after suprarenal coarctation of the abdominal aorta. Coarctation induced a progressive rise in aortic blood pressure and left ventricular mass. We observed a biphasic accumulation of ANF mRNA in the left ventricle with a peak at day 4 averaging 20 times the control value long before stable hypertension and hypertrophy were achieved, followed by a decrease until day 9. This decrease was followed by a new rise, which stabilized around 10 times the control value seen during stable hypertension and hypertrophy. In a second group of rats killed at days 4 and 30 (study B), we determined, in addition to the previous parameters, left ventricular end-diastolic pressure (LVEDP), plasma renin (PRC), and plasma ANF concentrations. LVEDP and PRC were markedly increased at day 4, but at day 30, during stable hypertension and hypertrophy, these parameters returned to control values, whereas plasma ANF was increased. Using immunocytochemistry, we looked in a third group of rats (study C) for the presence of the immunoreactive peptide at days 4 and 30.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and expression of the rat heart sarcoplasmic reticulum Ca2+-ATPase mRNA.

Sarcoplasmic reticulum Ca2+-ATPase cDNA clones have been isolated from an adult rat heart cDNA library and the nucleotide sequence of the Ca2+-ATPase mRNA determined. The sequence has an open reading frame of 997 codons. It is identical to a cDNA isolated from a rat stomach cDNA library and 90% isologous to the rabbit and human slow/cardiac cDNAs. Nuclease S1 mapping analysis indicates that this sequence corresponds to the main Ca2+-ATPase mRNA present in heart and in slow skeletal muscle and that it is expressed in various proportions in smooth and non-muscle tissues, together with another isoform which differs from the cardiac form in the sequence of its 3'-end.

Different sensitivity to trypsin of the human platelet plasma and intracellular membrane Ca2+ pumps.

The Ca2+ pumps associated with human platelet plasma and intracellular membranes have been further characterized by their sensitivity to trypsin. (a) Tryptic degradation of the Ca2+-ATPases has been followed by immunoblotting. It resulted in fragmentation into peptides of 80, 55, 35, and 24 kDa for both enzymes. Subcomplete hydrolysis obtained with a ratio of trypsin/membrane protein of 0.05-0.1 for the two Ca2+ pumps resulted in the total disappearance of the 100-, 80-, and 35-kDa fragments. However, maximum degradation was reached within 1 min for the intracellular enzyme but needed 5 min of incubation for the plasma membrane enzyme. (b) This effect of trypsin has been correlated with its effect on both the Ca2+-ATPase activities. The plasma membrane enzyme showed a maximum inhibition of 50-60% which was obtained using a trypsin/protein ratio of 0.1 and 5 min of incubation. A much higher trypsin sensitivity was observed for the intracellular enzyme because the maximum inhibition reached 80% after only 1 min of incubation. (c) Finally, the two Ca2+ transport systems studied showed different trypsin reactivities; the Ca2+ uptake by the plasma membrane vesicles was inhibited by 20-25%, and this maximum inhibition was observed after 5 min of incubation with trypsin. In contrast, the Ca2+ transport associated with the intracellular membrane vesicles was difficult to detect after trypsin treatment. Taken together, the results show that the two Ca2+ pumps can be distinguished by their trypsin sensitivity.

(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.

Messenger RNA content and complexity in normal and overloaded rat heart.

Cardiac overload was studied in rats by abdominal aortic constriction, which increased the left ventricular weight by 59% after 12 days. During the transition, which precedes the compensatory hypertrophy, both total RNA and poly(A)-containing messenger RNA increased. The concentration of these polynucleotides peaked by day 4 after constriction, rising from 1.27 +/- 0.3 to 1.88 +/- 0.2 mg g-1 fresh weight for total RNA, and from 38 +/- 24 to 62 +/- 12 micrograms g-1 fresh weight for poly(A)-containing RNA, and returned to normal by day 12. However, the total amount per ventricle of both RNAs remained high. Poly(A)-containing RNA prepared from normal heart was hybridized to its cDNA copy. These results were expressed as the percentage of hybridization vs. the log 10 of the product of the poly(A)-containing RNA concentration and the time (Rot), and in computer analysis were described by division into three different frequency components. In normal hearts, the Rot 1/2 values of these components were, respectively, 3.98 X 10(-3), 0.338 and 21.380 mol.s.1(-1), which correspond to 2-3, 240 and 12,200 different sequences that were copied 22,000-33,000, 310 and 5 times, respectively. Four and 30 days after banding there was a harmonious enhancement of the number of the copies without any change in the number of different sequences, and the three different hybridization curves were superimposed. In conclusion, cardiac overload raises the poly(A)-containing RNA concentration, probably by stimulating transcription, but no major changes occur in any of the frequency classes.

Alpha-myosin heavy chain isoform and atrial size in patients with various types of mitral valve dysfunction: a quantitative study.

The cardiac myosin phenotype, an important determinant of myocardial contractility, is modified by chronic increases in hemodynamic load. To quantify the proportion of atrial alpha-myosin heavy chain in various types of left atrial overload and to assess the possible relation between this proportion and atrial size, 34 patients were studied, 4 with Wolff-Parkinson-White syndrome, 29 with various types of mitral valve dysfunction and 1 with an atrial septal defect. Four normal autopsy hearts were also studied. The proportion of alpha-myosin heavy chain among total (alpha plus beta) myosin heavy chains was determined in each atrial sample, using an enzyme-linked immunosorbent assay. The size of the left atrium was assessed by one- and two-dimensional echocardiography. Alpha-myosin heavy chain was the main isoform present in the normal atria (85.5 +/- 9% of total myosin heavy chains). Patients with pure tight mitral stenosis (n = 9), mitral stenosis plus mild regurgitation (n = 8) and severe mitral regurgitation (n = 8), who had a higher indexed left atrial transverse diameter than those with Wolff-Parkinson-White syndrome (33 +/- 6, 39 +/- 10 and 46 +/- 5 versus 19.5 +/- 2 mm/m2, p less than 0.01, p less than 0.001 and p less than 0.001, respectively), also demonstrated a much smaller percent of alpha-myosin heavy chain content (28 +/- 20, 23.5 +/- 13 and 12 +/- 10 versus 58 +/- 18%, p less than 0.01, p less than 0.01 and p less than 0.001, respectively).(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.

Alpha-skeletal muscle actin mRNA's accumulate in hypertrophied adult rat hearts.

Cardiac hypertrophy due to a chronic hemodynamic overload is accompanied by isoformic changes of two proteins of the thick filament of the sarcomere, myosin, and creatine phosphokinase. We have looked for isoactin changes, using deoxyribonucleic acid probes complementary to alpha-skeletal and alpha-cardiac actin messenger ribonucleic acids. Three groups of rats were studied at various days after application of a pressure overload (2-4 days, n = 13, 8-15 days, n = 5, and 30-40 days, n = 7) and were compared to control animals (n = 11). Whereas alpha-skeletal actin messenger ribonucleic acids were hardly detectable in the normal hearts (0.6 +/- 0.16%), they accumulated significantly in the first 4 days after the aortic stenosis (4.6 +/- 3.1%, p less than 0.001 vs. controls) and then slowly declined (8-15 days, 3.2 +/- 1.7% and 30-40 days, 1.6 +/- 0.6%, p less than 0.05 and NS vs. controls). This figure is similar to that observed in 8-day-old rats (2.27 +/- 0.3%, p less than 0.01 vs. controls). We conclude that, in rat myocardium, the expression of messenger ribonucleic acids encoding the sarcomeric actins is altered at the onset of a pressure overload hypertrophy. Although the physiological significance of isoactin changes is unknown, our results show that the thin filament participates as well as the thick filament in the response of cardiac muscle to new functional requirements.

Physiological adaptation of the heart to pathological overloading.

Chronic overloading of the rat heart induces a cascade of adaptational events that compensate for the increase in work. At the myocardial level there are two types of adaptational mechanisms: qualitative, represented by the isomyosin changes leading to an improved efficiency; and quantitative, the hypertrophy. We present new approaches exploring possible adaptational changes at other levels within the myocardial cell. Studies of heart overload were performed either in young rats with experimental aortic stenosis or in humans with chronic compensatory hypertrophy. By means of double immunofluorescence labeling of isolated myocytes with anti-V1 and anti-V3 myosin immunoglobulins, we showed that the shift from high- to low-ATPase isomyosins occurs rapidly after aortic stenosis (2-3 days). Cardiac myocytes were shown to be poor in tubulin but a microtubule pattern was clearly visualized by an immunofluorescence approach. Their role in the onset of adaptational processes after aortic stenosis in not yet clear. On the other hand, we showed that in humans, contrary to small rodents, the adaptational process at the isomyosin level is very small or nonexistent.

Messenger RNA content and complexity in normal and overloaded rat heart: a preliminary report.

Cardiac overload is associated with two different changes in gene expression: an increase in overall protein synthesis leading to hypertrophy, and at least two isoenzymic redistributions affecting myosin and creatine kinase. This preliminary report was an attempt to study the cardiac genome in this model. Total RNA (ribonucleic acid) was prepared by a combination of the guanidine-ethanol and phenol procedures. A DNA (deoxyribonucleic acid)-, t(transfer)-RNA-free preparation containing non-degraded 28 and 18 S rRNA and 3% poly A+-m (messenger) RNA was obtained. The poly A+mRNA isolated on an oligo (dT) cellulose column had an average nucleotide length of 1300 to 1800 and its c (complementary) DNA was 600 to 1400 base pairs. The first analysis of the kinetics of hybridation revealed a complex pattern which could be described by division into three components. Northern blot and dot blot using a recombinant cDNA plasmid complementary to myosin heavy chain or to actin mRNA (a gift from A. Weydert and M. Buckingham) indicated the presence of these two mRNA, with the former being frequently degraded. After aortic stenosis, both the content and the concentration of total RNA and poly A+mRNA increased in parallel and peaked by the 4th day at +35%. This is the first suggestion that the control of gene expression is transcriptional in this model.

Left ventricular isomyosins in normal and hypertrophied rat and human hearts.

Regulation of rat cardiac contractility by changes in the expression of a particular form of myosin (V1-V3) has been demonstrated with a pressure overload. Previous reports of the effect of a volume overload have been controversial. Therefore, we measured the isomyosin composition and mechanical function in the same papillary muscles from rat hearts subjected to a chronic volume overload (aortic insufficiency, AI). A marked change in isomyosin composition from V1 to V3 occurred. Contractility, as assessed by shortening velocity Vmax, was also significantly decreased, and this decrease was correlated with the isomyosin transformation. The changes in isomyosin composition and speed of contraction with AI are thus similar to changes induced by aortic stenosis. Little experimental evidence exists for involvement of such changes in the regulation of human cardiac contractility. Using immunoglobulins highly specific for V1 and V3 in autopsy samples we have observed that the human left ventricle is mostly composed of a V3 isoform (HV3) and that small amounts (1 to 15%) of a V1 type (HV1) are present in foetal and some adult hearts. This HV1 is absent from the left ventricles of patients with valvular disease, assessed at the time of valve replacement (N = 30, samples provided by Dr P. Menasché). Myosin Ca2+-stimulated ATPase activities were not significantly different between normal and hypertrophied hearts. These data demonstrate the heterogeneity of human ventricular myosin, which is composed of V1 and V3 isomyosins, as in other mammalian species. Isomyosin shifts from V1 to V3 are possible in man, but they are quantitatively small and without noticeable influence on overall ATPase activities.

[Characterization of total RNA, messenger poly(A)+ RNA and homologous complementary DNA in experimental cardiac hypertrophy]. / Caractérisation de l'ARN total, de l'ARN messager poly (A)+ et de l'ADN complémentaire homologue dans l'hypertrophie cardiaque expérimentale.

Two biological modifications are observed during adaptation of cardiac tissue to work overload: an increase in total protein synthesis and a redistribution of myosin isoenzymes. These modifications suggest that changes in DNA transcription are involved in the early response of cardiac tissue to overload. Results are reported in this paper that show a parallel increase in total and polyadenylated RNA content and concentration in cardiac overloaded tissue in the rat. The characterization of cardiac poly (A)+ mRNA by mRNA X cDNA hybridization and the identification of specific mRNAs with recombinant plasmid cDNAs are given in this report as preliminary results.