Chronic overloading of the rat heart induces a cascade of adaptational events which compensate for the increase in work. Two of these have been extensively described: a qualitative event with an isomyosin change leading to an improved efficiency and a quantitative event resulting in cardiac hypertrophy. By means of immunofluorescence, we investigated if elements of the cytoskeleton, i.e. microtubules and intermediate filaments, could be triggers for these adaptational mechanisms. Studies of overloaded heart were performed in young rats with aortic stenosis or adult rats with aortic insufficiency. Cardiac myocytes were isolated and labelled by immunofluorescence with antibodies raised against V1 or V3 isomyosin, desmin or tubulin. The aim of the work was to visualize: (1) when and where the shift in the expression of isomyosins occurs within the myocytes; (2) the eventual changes in the pattern of intermediate filaments of desmin and/or of microtubules during the adaptation of myocytes to overload. We observed: (1) that the shift from the high (V1) to low (V3) ATPase isomyosin occurred in a population of myocytes soon after stenosis; (2) that changes in the pattern of microtubules occurred soon after induction of hypertrophy; (3) no changes in the distribution or intensity of the staining of desmin.
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.
Rat cardiac ventricular myosins were obtained from fetuses, from young, adult, and old normal animals, from hypophysectomised adults and from rats submitted to a chronic mechanical overload of the heart. The different proteins were compared by electrophoresis in non-denaturing conditions and by competitive enzymelinked immunosorbent assay (EL.ISA). For the latter purpose, antibodies specific of the rat cardiac V3 isomyosin were used. It was found that the V3 isomyosin of fetuses is indistinguishable from that of adult hypertrophied heart. This result strongly suggests that adaptation to chronic increase in cardiac work is mediated through the synthesis of the fetal form of myosin. Double immunolabeling of isolated myocytes with antibodies specific of the rat V1 or the rat V3 isomyosins showed that this isoenzymic redistribution occurs throughout the whole length of the myocytes, without any preferential localization of the newly synthesized V3 isoform. The existence of such a mechanism of adaptation was also searched for in human hearts. Competitive ELISA performed with antibodies against human cardiac heavy meromyosin indicated no differences in apparent affinities with the myosins of two "control" subjects and of three patients with noticeable cardiac hypertrophy.
Ventricular hypertrophy following chronic overload results from the hypertrophy of cardiomyocytes and the hyperplasia of non muscle cells in the heart. The mechanisms by which hemodynamic stress induces these cellular adaptations are unknown. However, an increasing body of evidence seems to favor local paracrine and/or autocrine mechanisms rather than circulating factors. Locally, it is well established that cardiac endothelium regulates myocardial contraction by the release of nitric oxide, prostacyclin, endothelin and other still unidentified factors. Similarly, endothelial factors seem involved in myocyte growth alteration, especially those occurring around coronary vessels, at the early stage of hemodynamic stresses. This involves factors such as endothelin and angiotensin II that are known to induce changes in both cardiac myocyte and non muscle cell growth. On the other hand, an autocrine function of myocardial cells has also been proposed. Mechanical stress provokes the release of cardiomyocyte-derived growth factors, including Fibroblast Growth Factors, that may induce angiogenesis and proliferation and survival of many cell types. Thus, in the heart, cardiomyocytes and non muscle cells sense acute mechanical stretch and exert paracrine and/or autocrine regulation of their growth.
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.
Chronic increases in haemodynamic load modify the expression of cardiac genes, leading to cardiac hypertrophy and a new phenotype. As an example, changes in the expression of the genes encoding the main contractile proteins, the isomyosin heavy chains, have been associated with modifications of the physiological properties of cardiac muscle. The cellular and molecular mechanisms which either do or do not initiate and maintain these changes in cardiac genomic expression remain to be elucidated. Using in situ hybridization we show that mRNAs encoding a cellular form of fibronectin (c-FN), a protein of the basal membrane which is not or poorly expressed in adult rat heart, are reexpressed as a result of severe hypertension with a similar time course than the beta-heavy chain of myosin (beta-MHC), also mostly expressed in fetal heart. The accumulation of the c-FN mRNAs was found in the wall of coronary arteries whilst that of the beta-MHC mRNAs occurred in the myocytes at the border zone of these arteries. Thus a high pressure in the arteries could be the trigger inducing the synthesis of factors which could, through a gradient, modulate the phenotype of both the smooth muscle cells of the media and the cardiocytes. Besides, using a model of cultured adult rat cardiocytes, we show that the differential expression of the MHC isoforms is dependent on the beta-adrenergic stimulation but that the regulation depends on the stage of development of the cells and differs for the alpha and beta MHC. These 2 complementary approaches for identifying the molecular mechanisms that control cardiac muscle growth should help for understanding cardiac adaptation triggered by haemodynamic overload, such as arterial hypertension as well as cardiac failure.
