The specific inhibition of the cardiac electrogenic sodium/bicarbonate cotransporter leads to cardiac hypertrophy.

Two alkalinizing mechanisms coexist in cardiac myocytes to maintain intracellular pH: sodium/bicarbonate cotransporter (electroneutral isoform NBCn1 and electrogenic isoform NBCe1) and sodium/proton exchanger (NHE1). Dysfunction of these transporters has previously been reported to be responsible for the development of cardiovascular diseases. The aim of this study was to evaluate the contribution of the downregulation of the NBCe1 to the development of cardiac hypertrophy. To specifically reduce NBCe1 expression, we cloned shRNA into a cardiotropic adeno-associated vector (AAV9-shNBCe1). After 28 days of being injected with AAV9-shNBCe1, the expression and the activity of NBCe1 in the rat heart were reduced. Strikingly, downregulation of NBCe1 causes significant hypertrophic heart growth, lengthening of the action potential in isolated myocytes, an increase in the duration of the QT interval and an increase in the frequency of Ca2+ waves without any significant changes in Ca2+ transients. An increased compensatory expression of NBCn1 and NHE1 was also observed. We conclude that reduction of NBCe1 is sufficient to induce cardiac hypertrophy and modify the electrical features of the rat heart.

Positive inotropic and negative lusitropic effect of angiotensin II: intracellular mechanisms and second messengers.

In the cat ventricle angiotensin II exerts a positive inotropic effect produced by an increase in intracellular calcium associated with a prolongation of relaxation. The signaling cascades involved in these effects as well as the subcellular mechanisms of the negative lusitropic effect are still not clearly defined. The present study was directed to investigate these issues in cat papillary muscles and isolated myocytes. The functional suppression of the sarcoplasmic reticulum (SR) with either 0.5 microm ryanodine or 0.5 microm ryanodine plus 1 microm thapsigargin or the preincubation of the myocytes with the specific inhibitor of the inositol 1,4,5-triphosphate (IP3) receptors [diphenylborinic acid, ethanolamine ester (2-APB), 5-50 microm] did not prevent the positive inotropic effect and the increment in Ca2+ transient produced by 1 microm angiotensin II. In contrast, protein kinase C (PKC) inhibitors, chelerythrine (20 microm) and calphostin C (1 microm) completely inhibited both, the angiotensin II-induced increase in L-type calcium current and positive inotropic effect. The prolongation of half relaxation time produced by 0.5 microm angiotensin II [207+/-15.4 msec (control) to 235+/-19.98 msec (angiotensin II), P<0.05] was completely blunted by PKC inhibition. This antirelaxant effect, which was independent of intracellular pH changes, was associated with a prolongation of the action potential duration and was preserved after either the inhibition of the SR and the SR Ca2+ ATPase (ryanodine plus thapsigargin) or of the reverse mode of the Na+/Ca2+ exchanger (KB-R7943, 5 microm). We conclude that in feline myocardium the positive inotropic and negative lusitropic effects of angiotensin II are both entirely mediated by PKC without any significant participation of the IP3 limb of the phosphatidylinositol/phospholipase C cascade. The results suggest that the antirelaxant effect of angiotensin II might be determined by the decrease in Ca2+ efflux through the Na+/Ca2+ exchanger produced by the angiotensin II-induced prolongation of the action potential duration.

Glucagon-like peptide-1 increases cAMP but fails to augment contraction in adult rat cardiac myocytes.

The gut hormone, glucagon-like peptide-1 (GLP-1), which is secreted in nanomolar amounts in response to nutrients in the intestinal lumen, exerts cAMP/protein kinase A-mediated insulinotropic actions in target endocrine tissues, but its actions in heart cells are unknown. GLP-1 (10 nmol/L) increased intracellular cAMP (from 5.7+/-0.5 to 13.1+/-0.12 pmol/mg protein) in rat cardiac myocytes. The effects of cAMP-doubling concentrations of both GLP-1 and isoproterenol (ISO, 10 nmol/L) on contraction amplitude, intracellular Ca(2+) transient (CaT), and pH(i) in indo-1 and seminaphthorhodafluor (SNARF)-1 loaded myocytes were compared. Whereas ISO caused a characteristic increase (above baseline) in contraction amplitude (160+/-34%) and CaT (70+/-5%), GLP-1 induced a significant decrease in contraction amplitude (-27+/-5%) with no change in the CaT after 20 minutes. Neither pertussis toxin treatment nor exposure to the cGMP-stimulated phosphodiesterase (PDE2) inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine or the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine nor the phosphatase inhibitors okadaic acid or calyculin A unmasked an ISO-mimicking response of GLP-1. In SNARF-1-loaded myocytes, however, both ISO and GLP-1 caused an intracellular acidosis (DeltapH(i) -0.09+/-0.02 and -0.08+/-0.03, respectively). The specific GLP-1 antagonist exendin 9-39 and the cAMP inhibitory analog Rp-8CPT-cAMPS inhibited both the GLP-1-induced intracellular acidosis and the negative contractile effect. We conclude that in contrast to beta-adrenergic signaling, GLP-1 increases cAMP but fails to augment contraction, suggesting the existence of functionally distinct adenylyl cyclase/cAMP/protein kinase A compartments, possibly determined by unique receptor signaling microdomains that are not controlled by pertussis toxin-sensitive G proteins or by enhanced local PDE or phosphatase activation. Furthermore, GLP-1 elicits a cAMP-dependent modest negative inotropic effect produced by a decrease in myofilament-Ca(2+) responsiveness probably resulting from intracellular acidification.

Angiotensin II and cardiac excitation-contraction coupling: questions and controversies.

Angiotensin II (AngII) is a circulating peptide that produces a positive inotropic effect in the heart in several species, including humans. The subcellular mechanisms involved in producing this effect have been the focus of numerous studies; however, the results of these studies have generated considerable controversy. Although part of the controversy might arise from species and developmental differences, conflicting results have also been reported in the same species. To further complicate the understanding of the cardiac actions of AngII, the binding of the peptide to its transmembrane G-protein-coupled receptors has been shown to activate signalling cascades that involve numerous second messengers. Among these, inositol 1,4,5-triphosphate (IP3) and protein kinase C (PKC) have been shown to have the potential to modulate either one or both of the two basic mechanisms known to increase contractility: (i) an increase in the intracellular Ca2+ concentration ([Ca2+]i); or (ii) an increase in myofilament responsiveness to Ca2+. The aim of this review is to examine the effect of AngII on the fundamental components of cardiac excitation-contraction coupling: calcium currents, Na+/Ca2+ exchange, sarcoplasmic reticulum (SR)-CaZ+ release, calcium transients and contractile proteins. An answer to the following question is sought: Is the positive inotropic effect of AngII due to an increase in [Ca2+]i, to an increase in myofilament responsiveness to Ca2+, or to both?

Activation of distinct cAMP-dependent and cGMP-dependent pathways by nitric oxide in cardiac myocytes.

Nitric oxide (NO) donors were recently shown to produce biphasic contractile effects in cardiac tissue, with augmentation at low NO levels and depression at high NO levels. We examined the subcellular mechanisms involved in the opposing effects of NO on cardiac contraction and investigated whether NO modulates contraction exclusively via guanylyl cyclase (GC) activation or whether some contribution occurs via cGMP/PKG-independent mechanisms, in indo 1-loaded adult cardiac myocytes. Whereas a high concentration of the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 100 micromol/L) significantly attenuated contraction amplitude by 24.4+/-4.5% (without changing the Ca2+ transient or total cAMP), a low concentration of SNAP (1 micromol/L) significantly increased contraction amplitude (38+/-10%), Ca2+ transient (26+/-10%), and cAMP levels (from 6.2 to 8.5 pmol/mg of protein). The negative contractile response of 100 micromol/L SNAP was completely abolished in the presence of the specific blocker of PKG KT 5823 (1 micromol/L); the positive contractile response of 1 micromol/L SNAP persisted, despite the presence of the selective inhibitor of GC 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 micromol/L) alone, but was completely abolished in the presence of ODQ plus the specific inhibitory cAMP analog Rp-8-CPT-cAMPS (100 micromol/L), as well as by the NO scavenger oxyhemoglobin. Parallel experiments in cell suspensions showed significant increases in adenylyl cyclase (AC) activity at low concentrations (0.1 to 1 micromol/L) of SNAP (AC, 18% to 20% above basal activity). We conclude that NO can regulate both AC and GC in cardiac myocytes. High levels of NO induce large increases in cGMP and a negative inotropic effect mediated by a PKG-dependent reduction in myofilament responsiveness to Ca2+. Low levels of NO increase cAMP, at least in part, by a novel cGMP-independent activation of AC and induce a positive contractile response.

Dissociation between positive inotropic and alkalinizing effects of angiotensin II in feline myocardium.

The present study examines the intracellular pH (pHi) dependence of angiotensin (ANG) II-induced positive inotropic effect in cat papillary muscles contracting isometrically (0.2 Hz, 30 degrees C). Muscles were loaded with the fluorescent dye 2'-7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester for simultaneous measurement of pHi and contractility. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer (n = 4), there was a temporal dissociation between the positive inotropic and the alkalinizing effects of ANG II (0.5 microM). The positive inotropic effect of ANG II peaked at 9.7 +/- 0.8 min (240 +/- 57% above control) without significant changes in pHi. The increase in pHi became significant (0.05 +/- 0.01 pH units) only after 16 min of exposure to the drug, when the positive inotropic effect of ANG II was already fading. In HCO3- buffer (n = 7), the ANG II-induced positive inotropic effect occurred without significant pHi changes. In the presence of 5 microM ethyl isopropyl amiloride (EIPA, to specifically inhibit the Na+/H+ exchanger), the alkalinizing effect of ANG II was changed to a significant decrease in pHi, despite which ANG II still increased contractility by 87 +/- 16% (n = 6). The results indicate that in HEPES buffer only a fraction of the ANG II-induced positive inotropic effect can be attributed to a pHi change, whereas in a physiological CO2-HCO3- medium the positive inotropic effect of ANG II is independent of pHi changes. Furthermore, an ANG II-induced increase in myocardial contractility was observed even when ANG II administration elicited a decrease in pHi, as occurred after Na+/H+ exchanger blockade. The results show that in feline myocardium, the increase in contractility evoked by ANG II in a physiological CO2-HCO3- medium is not due to an increase in Ca2+ myofilament sensitivity secondary to an increase in myocardial pHi.