BACKGROUNDIn hypertension with cardiac hypertrophy, the specific contributions to increased production of the cardiac natriuretic peptides (NP) atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) by load and the hypertrophic process are not known. In the present work we determine ANF and BNP synthesis and secretion in the aortic-banded rat treated with dosage schedules of the ACE inhibitor ramipril that result in the prevention or regression of both hypertension and hypertrophy (high dosage) or in the prevention or regression of hypertrophy alone with persistent hypertension (low dosage). Myosin heavy chain (MHC) isoform switch was studied as an indicator of ventricular cardiocyte hypertrophy as well as the levels of collagen III mRNA as a measure of changes in extracellular matrix.METHODS AND RESULTSRamipril was administered for 6 weeks just after suprarenal aortic banding, or rats were banded for 6 weeks, after which ramipril was administered during the following 6 weeks. Banding caused an increase in blood pressure, left ventricular weight-to-body weight ratio, plasma and ventricular NP, ventricular NP mRNA, collagen III, and beta-MHC mRNA. Ramipril at 1 mg/kg normalized all these parameters while ramipril at 10 micrograms/kg normalized left ventricular weight-to-body weight ratio but not blood pressure. Plasma and ventricular NP content and mRNA levels were partially normalized by ramipril (10 micrograms/kg). Ramipril (10 micrograms/kg) prevented increased collagen III mRNA levels but did not affect beta-MHC mRNA levels.CONCLUSIONS(1) NP production and secretion in aortic-banded rats are independently related to increased blood pressure and hypertrophy. (2) A load-dependent component is more important than a load-independent component in regulating left ventricular NP production. (3) ANF production is more sensitive than BNP production to the load-independent component. (4) Low-dose ramipril treatment reverses hypertrophy and the increased collagen III expression but does not reverse the increased beta-MHC isoform expression, suggesting that these are independently regulated processes. (5) Aortic banding and ACE inhibition do not affect atrial NP production and content.

Adult rat ventricular myocytes assume after 2 weeks in culture a flattened spread morphology and a loss in organized myofibrils. This sequence of phenotypic changes is accompanied by the reexpression of the fetal gene program. Although different signal transduction pathways were recently shown to be involved in cell growth and differentiation, not much is known about tyrosine kinase activation and cardiac myocyte differentiation. We investigated whether the tyrosine kinase signal transduction pathway is involved in the dedifferentiation of adult rat ventricular myocytes in long-term culture using a specific inhibitor of tyrosine phosphorylation, genistein. For this experiment, adult rat ventricular myocytes were cultured as previously described and incubated in culture medium containing different concentrations of genistein (10-250 microM). After 24 hr of incubation and in a concentration-dependent manner genistein prevented cell spreading. However, at high concentration, cells detached from the plates (10% to 100 microM and 95% at 250 microM). The effect of genistein on adult rat ventricular myocyte phenotype in culture was investigated by examining the expression of total actins and alpha-smooth muscle actin and alpha-sarcomeric actin in cells after 6 days of incubation with and without genistein. Myofibrillar proteins were extracted and separated by gel electrophoresis. Expression of alpha-smooth muscle actin and alpha-sarcomeric actin was determined by Western blotting using specific antibodies. While there was an increase in the amount of total actins and no change in the amount of alpha-sarcomeric actin in the cells exposed to genistein, the amount of alpha-smooth muscle actin decreased with increasing concentrations of genistein reaching undetectable levels at 100 microM. These results demonstrate that genistein inhibits cell spreading and the reexpression of alpha-smooth muscle actin in adult rat ventricular myocytes in culture in a dose-dependent manner, therefore, inhibiting the process of dedifferentiation.

We examined the relationship between cardiac hypertrophy, myosin heavy chain (MHC) isoform expression, and production of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) before and after the development of DOCA-salt hypertension. DOCA-salt rats exhibited significant left ventricular hypertrophy at the prehypertensive stage (1 week of treatment), without MHC isoform switch or change in natriuretic peptide gene expression. In the hypertensive stage (5 weeks of treatment), pronounced left ventricular hypertrophy was observed, and this was characterized by an increase in beta-MHC protein, resulting in a switch from 90% alpha-MHC to 51% alpha-MHC and 49% beta-MHC. ANF and BNP mRNA levels and peptide content were significantly increased at this stage. Unexpectedly, the MHC isoform switch was evident in the nonhypertrophied right ventricle to the same degree as in the left ventricle. Natriuretic peptide production was also increased in the right ventricle at 5 weeks of treatment, but to a lesser degree than in the left ventricle. In contrast, in the hypertrophied left atrium there was no MHC isoform switch, while ANF and BNP mRNA levels were augmented. Plasma ANF was significantly increased in the prehypertensive stage; this was accompanied by a partial depletion of atrial ANF stores. Plasma BNP was increased only in the hypertensive stage, reflecting an increase in ventricular BNP synthesis and secretion. These results suggest that I) cardiac hypertrophy, MHC isoform expression, and stimulation of natriuretic peptide production are processes that may be dissociated from each other; 2) increases in plasma ANF without a concomitant increase in plasma BNP reflect atrial hemodynamic overload, while increases in both ANF and BNP in plasma are associated with ventricular hypertrophy; and 3) there exist differences in the storage, secretion, and processing patterns of ANF and BNP in the atria.

Adult rat ventricular cardiocytes, when cocultured with epicardial mesothelial cells (EMC), demonstrate remarkable plasticity of phenotype accompanied by a significant increase in cardiocyte contractile protein content, suggesting that a factor with growth-promoting properties may take part in EMC-adult rat ventricular cardiocyte interactions. Endothelin (ET) has been shown to induce cell hypertrophy, including enhancement of expression of muscle-specific genes. We investigated the ability of EMC to synthesize and release ET. By light microscopy, specific immunostaining, with either ET-1 or Big ET-1 antibodies, was visualized in EMC as a fine punctate distributed throughout the cytoplasm. Reverse phase-high performance liquid chromatography (HPLC) of epicardial mesothelial cells conditioned medium showed several peaks of immunoreactive ET. The major peak eluted with the same retention time as that of ET-1. By Northern blot analysis, a specific 2.3-kilobase (kb) mRNA species was detected by hybridization to a cDNA insert encoding for rat prepro-ET-1. ET accumulated in the culture medium in a time-dependent manner, whereas cell content remained comparatively low. Angiotensin II (AII) dose-dependently stimulated release of immunoreactive ET into the culture medium.

Adult rat ventricular myocytes undergo a well-documented sequence of phenotypic changes during adaptation to primary culture. However, we observed that coculture of myocytes with a specific subset of nonmyocyte cardiac cells could slow and even reverse the process of adaptation. These nonmyocyte cells were isolated and identified by immunohistochemical and ultrastructural criteria as being of epicardial mesothelial origin. When added to long-term primary cultures of adult ventricular myocytes, epicardial mesothelial cells appeared to induce myofibrillar arrays that were more organized than those seen in noncocultured myocytes; these changes that occurred were concurrent with the appearance of large amplitude contractions and multicellular synchronous beating that was facilitated by gap junctions between myocytes and epicardial mesothelial cells. The changes in morphology and function were accompanied by a marked increase in beta-myosin heavy chain isoform transcription in cocultured myocytes, a return to the ratio of cardiac to skeletal alpha-actin expected in adult rat myocardium, and a much reduced expression of smooth muscle alpha-actin. These changes in myocyte phenotype and function appeared to require epicardial cell-myocyte contact, or close apposition, because media conditioned by epicardial mesothelial cells alone or in coculture had no effect. Thus, these rapid and reversible changes in myocyte ultrastructure, function, and gene expression may provide a useful in vitro model with which to study the mechanism responsible for regulating the plasticity of ventricular myocyte phenotype and the role of specific cell-cell interactions.

It has long been assumed that the primary influences regulating cardiac contractility are the extent of mechanical loading of muscle fibers and the activity of the autonomic nervous system. However, the vasoactive peptide endothelin, initially found in vascular endothelium, is among the most potent positively inotropic agents yet described in mammalian myocardium. In isolated adult rat ventricular cells, endothelin's action was slow in onset but very long lasting with an EC50 of 50 pM that approximates the reported KD of the peptide for its receptor in rat heart. When the calcium activity of the buffer superfusing isolated single fura-2-loaded myocytes paced at 1.5 Hz was varied from 0.1 to 0.9 mM [Ca2+]o, 100 pM endothelin increased contractile amplitude with no significant change in diastolic or systolic [Ca2+]i, thus appearing to sensitize the myofilaments to intracellular calcium. Pertussis toxin, or prior exposure to a beta-adrenergic agonist, reduced or abolished the increase in myocyte contractility induced by endothelin. This novel and potent pharmacologic action of endothelin points to the potential importance of local, paracrine factors, perhaps derived from microvascular endothelium or endocardium, in the control of the contractile function of the heart.

The effect of endothelin-1 on the phosphoinositide pathway was studied in slices of atria and mesenteric artery from normotensive and hypertensive (DOCA-salt) rats. Endothelin-1 induced a dose-dependent increase in inositol monophosphate production in both groups, but the reactivity of the phosphoinositide pathway was enhanced in DOCA-salt hypertensive rats. Endothelin-1 was more potent than noradrenaline and activation by these two agonists combined was additive. The phosphoinositide activation induced by endothelin-1 was independent of extracellular calcium, and was not prevented by nifedipine treatment or by removing calcium from the incubation medium. Endothelin-1 is a potent direct activator of the phosphoinositide pathway in cardiovascular tissues, and this effect is potentiated in DOCA-salt hypertension.

In the last two decades, remarkable advances have permitted a better understanding of the modulation of sympathetic tone and reactivity at the sympathetic nerve and at the effector cell levels. In man, several indirect approaches have permitted to suggest the possibility of increased sympathetic nerve activity and reactivity in an important subgroup of essential hypertensive patients. The demonstration of significant correlations between circulating levels of sympathetic transmitters and various parameters of cardiovascular functions supports the hypothesis of a participation of the sympathetic system in the maintenance of an elevated blood pressure in those patients. Moreover, several experimental evidences have indicated that the sensitivity of cardiovascular effector cells may be altered in hypertensive patients. The blunted beta receptor responsiveness and the normal or enhanced alpha receptor responsiveness which were observed suggest the possibility of an imbalance between adrenergic receptor functions in hypertension, which may explain the preferential alpha 1 modulation of blood pressure through changes in peripheral resistance in hypertensive patients. Such an abnormality could contribute to the development of cardiac and vascular wall hypertrophy during the evolution of hypertension. These studies therefore suggest that a variety of sympathetic dysfunctions could play a role in the development, maintenance and evolution of human essential hypertension.

The finding of elevated circulating catecholamine levels in experimental and human hypertension suggests an active sympathoadrenal participation in the pathogenesis of hypertension. In deoxycorticosterone acetate (DOCA)-salt hypertensive rats and in spontaneously hypertensive rats (SHR) the sympathoadrenal reactivity was found to be potentiated in response to various stimuli suggesting alterations in baroreflex functions or in local modulatory mechanisms. Several studies have suggested an attenuation of the alpha 2-presynaptic or local inhibitory mechanism and a potentiation of the beta 2-facilitatory presynaptic mechanism in the peripheral sympathetic system, thus possibly explaining the potentiated sympathoadrenal reactivity in those hypertensive animals. At postsynaptic adrenergic sites, beta-adrenoceptor numbers were reported to be decreased, whereas alpha 1-adrenoceptor numbers were unchanged in the cardiovascular system of DOCA hypertensive rats, thus favoring a dominance of alpha 1-postsynaptic responses in those animals. In support of this concept, the production of inositol monophosphate, used as an index of inositol triphosphate production, was found to be markedly enhanced following norepinephrine-induced alpha 1-stimulation in atria and ventricles as well as in mesenteric and femoral arteries of DOCA-salt hypertensive rats thus suggesting an increased reactivity of the second messenger system linked to alpha 1-adrenoceptors. Since similar abnormalities were also observed in SHR and in human hypertension, it thus appears that an imbalance between alpha- and beta-postsynaptic receptors may exist in various forms of hypertension. These studies therefore suggest the existence of multiple abnormalities in pre- and post-synaptic adrenergic mechanisms in experimental and human hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of the present study was to investigate alpha 1-adrenergic receptors in the heart as well as the activity and the sensitivity of the phosphoinositide pathway on tissue slices of atria, ventricles, and femoral artery of hypertensive rats treated for 4 weeks with deoxycorticosterone acetate (DOCA) and 1% saline. DOCA-salt hypertensive rats were characterized by an increased sympathoadrenal tone, as suggested by increased norepinephrine and epinephrine plasma levels. The basal activity of the phosphoinositide pathway, estimated by measuring the accumulation of inositol monophosphate in the presence of an excess of lithium, was found to be greater in atria than in ventricles and femoral artery in both normotensive and DOCA-salt hypertensive rats, but it was twofold greater in atria and ventricles of DOCA-salt hypertensive rats compared with normotensive rats. Following stimulation by norepinephrine, the production of inositol monophosphate was greater in atria and femoral artery than in ventricles in both groups. However, in DOCA-salt hypertensive rats, the production of inositol monophosphate was markedly enhanced, being about twofold greater in atria and femoral artery and about three times greater in ventricles than in tissues of normotensive rats. These differences between DOCA-salt hypertensive and normotensive animals do not appear to be associated with a difference in alpha 1-adrenergic receptor number or affinity since cardiac alpha 1-adrenergic receptor number was unchanged in hypertensive rats and the binding affinity to the receptor was significantly decreased in hypertensive rats compared with normotensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The sensitivity of the phosphatidylinositol (PI) pathway was evaluated in slices of atria (A), ventricles (V), and mesenteric artery (MA) in normotensive (NT) and DOCA-salt hypertensive (DOCA-HT) rats. During norepinephrine (NE) activation, the PI reactivity was two to three times greater in A, V, and MA of HT rats compared to NT rats. The long-term (2 weeks) administration of dietary lithium (Li) reduced the activation of PI by NE in left A and right V but caused no changes in MA of HT rats. The Li-treated hypertensive rats were also characterized by a lower systolic blood pressure and a lower ratio of ventricular weight/body weight. Plasma epinephrine (E) levels that were higher in HT rats were normalized in DOCA-HT + Li-treated rats, while the NE levels remained elevated in the DOCA-HT + Li group.