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.

The quantity and the electrophoretic characteristics of desmin were analyzed in a familial skeletal muscle disorder, characterized by the intra-sarcoplasmic accumulation of an electron-dense granulo-filamentous material facing the Z-lines and reacting strongly with polyclonal anti-desmin antibodies. The analysis was performed on biopsies from the deltoid muscles of 4 patients, members of 2 families. In the 4 biopsies, an increase in the relative amount of desmin compared to that of actin or insoluble proteins (3 fold) and in the number of isovariants (6 instead of 3) was observed. The isovariants of desmin were similar to those described in Purkinje fibres of the heart as a phosphorylated form of the protein [(1987) Eur. J. Cell Biol. 44, 68-78]. Therefore, post-translational events could affect both the polymerization and the amount of desmin filaments in this autosomal dominant familial myopathy.

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)

Annexins are characterized by Ca2+-dependent binding to phospholipids. Annexin II mainly participates in cell-cell adhesion and signal transduction, whereas annexins V and VI also seem to regulate intracellular calcium cycling. Their abundance and localization were determined in left ventricle (LV) and right ventricle (RV) from hypertensive guinea pigs, during the transition from compensatory hypertrophy to heart failure. Immunoblot analysis of annexins II, V, and VI revealed an increased accumulation (2.6-, 1.45-, and 2.3-fold, respectively) in LV from hypertensive guinea pigs and no modification in RV. Immunofluorescent labeling of annexins II, V, and VI; of Na+-K+-ATPase; and of sarcomeric α-actinin showed that in control LV and RV, 1) annexin II is present in nonmuscle cells; 2) annexins V and VI are mainly observed in the sarcolemma and intercalated disks of myocytes; 3) annexins II, V, and VI strongly label endothelial cells and adventitia of coronary arteries; and 4) annexin VI is present in the media. At the onset of heart failure, the most striking changes are the increased protein accumulation in LV and the very strong labeling of annexins II, V, and VI in interstitial tissue, suggesting a role in fibrosis development and cardiac remodeling.

The effect of catecholamines on adult myocardial protein synthetic activity was studied by use of an experimental model of isolated adult rat cardiac myocytes maintained in culture for 1-6 days. During this period, the majority of myocytes retained their rod-shaped morphology, but the cell number decreased progressively (50% of the initial density after 2 days in culture). Between day 1 and day 3 in culture, the specific synthetic activities of total proteins and of electrophoretically purified myosin heavy chain and actin ([14C] phenylalanine incorporation into protein, in disintegrations per minute per microgram protein) decreased (-19%,-32%, and -73%, respectively). Addition of isoproterenol or norepinephrine (10 nM) from the onset of the culture for 3 days increased the specific activity of both total and noncontractile proteins (greater than 20%) but had no effect on the specific activity of myosin heavy chain and actin when compared with 3-day cultured control cells. beta-Adrenergic receptors are specifically required to mediate this increase in total protein synthesis. This finding was demonstrated by the inhibitory effects of propranolol; neither prazosin nor yohimbine showed any effect. The pattern of synthesized protein during adrenergic stimulation was qualitatively evaluated by use of [35S]methionine incorporation and gel electrophoresis. The general pattern of labeled proteins did not differ significantly from that of control cells; this occurrence suggests that isoproterenol harmoniously stimulates the synthesis of noncontractile proteins. These findings demonstrate that low doses of beta-adrenergic agonists have an anabolic effect on adult cardiac quiescent myocytes that do not affect the major contractile proteins. Regulation of myofibrillar protein synthesis may be more dependent on myocyte contractile activity.

We investigated the hypothesis that diaphragm compliance was abnormal in cardiomyopathic Syrian hamsters (CSH), an experimental model of myopathy. The passive elastic properties of isolated diaphragm muscles were analyzed at both the muscle and sarcomere levels. We used the following passive exponential relationship between stress (sigma) and strain (epsilon): sigma = (Eo/beta) (ebetaepsilon - 1), where Eo is the initial elastic modulus and beta is the stiffness constant. Immunocytochemistry procedures were used to analyze the distribution of two key elastic components of muscle, extracellular collagen and intracellular titin elastic components, as well as the extracellular matrix glycoprotein laminin. Muscle and sarcomere values of beta were nearly twofold lower in CSH (8.7 +/- 1.9 and 8.3 +/- 1.4, respectively) than in control animals (19.7 +/- 1.7 and 16.8 +/- 2.1, respectively) (P < 0.01 for each). Compared with controls, Eo was higher in CSH. Sarcomere slack length was significantly longer in CSH than in control animals (2.1 +/- 0.1 vs. 1.9 +/- 0.1 micrometer, P< 0.05). The surface area of collagen I was significantly larger in CSH (17.4 +/- 1.8%) than in control animals (12.4 +/- 0.7%, P < 0.05). There was no change in the distribution of titin or laminin labelings between the groups. These results demonstrate increased diaphragm compliance in cardiomyopathic hamsters. The increase in CSH diaphragm compliance was observed despite an increase in the surface area of collagen and was not associated with an abnormal distribution of titin or laminin.

During muscle development, an isozymic transition of the glycolytic enzyme enolase occurs from the embryonic and ubiquitous alphaalpha-isoform to the muscle-specific betabeta-isoform. Here, we demonstrate a stimulatory role of thyroid hormones on these two enolase genes during rat development in hindlimb muscles and an inhibitory effect on the muscle-specific enolase gene in cardiac muscle. In hindlimb muscles the ubiquitous alpha-transcript level is diminished by hypothyroidism, starting at birth. On the contrary, the more abundant muscle-specific beta-transcript is insensitive to hypothyroidism before establishment of the functional diversification of fibers and is greatly decreased thereafter. Our data support the hypothesis of a role of thyroid hormones in coordinating the expressions of contractile proteins and metabolic enzymes during muscle development. The subcellular localization of isoenolases, established here, is not modified by hypothyroidism. Our results underline the specificity of action of thyroid hormones, which modulate differentially two isozymes in the same muscle and regulate, in opposite directions, the expression of the same gene in two different muscles.

To examine whether the two components of the voltage-activated outward K+ current, an initially rapidly inactivating component (Ito,1) and a slowly inactivating sustained component (Isus), in human atrial myocytes are distinct currents differentially regulated, we studied their behavior during serum-induced growth of cultured myocytes. Currents were recorded in whole cell patch clamped myocytes. After 1 wk of culture (day 8), membrane capacitance was twice the value in freshly dissociated myocytes (178.7 +/- 23 vs. 83.1 +/- 5.5 pF; P < 0.001). Ito,1 density did not differ from that in freshly dissociated myocytes (at +40 mV: 4.38 +/- 0.8 vs. 3.71 +/- 0.6 pA/pF), whereas that of Isus was markedly increased (at +40 mV: 9.76 +/- 2 vs. 2.21 +/- 0.29 pA/pF; P < 0.001). After inactivation of Ito,1 by a prepulse, sustained depolarization elicited in cultured myocytes an Isus with a density of 10.22 +/- 1.18 pA/pF and an apparent tail current reversal potential of -73.5 +/- 3.2 mV, indicating high K+ selectivity. Isus was highly sensitive to 4-aminopyridine (55.4 +/- 4.4% inhibition in 50 microM) and to D-600 (with a concentration inhibiting 50% of maximal response of 34.2 x 10(-6) M). Addition of 5-10 nM staurosporine at day 3 prevented cell growth and reduced Ito,1 density but not the increase in Isus density, which was inhibited by 10 microM staurosporine. Our results indicate that Ito,1 and Isus are regulated independently during in vitro myocyte growth in human atrial myocytes and that the increase in Isus density is not mediated by a protein kinase C-dependent pathway.