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Zinc exists in biological systems as bound and histochemically reactive free Zn(2+) in the nanomolar range. Zinc is required as either structural or catalytic component for a large number of enzymes. It also modulates current passage through many ion channels. Here, we reinvestigated the effects of extracellular and intracellular Zn(2+) on the L-type Ca(2+) current (I (CaL)) and its modulation by β-adrenergic stimulation in rat ventricular cardiomyocytes. In the absence of Ca(2+) ions, Zn(2+) could permeate through the L-type channel at much lower concentrations and at a more positive voltage range, but with a lower permeability than Ca(2+). In the presence of Ca(2+), extracellular Zn(2+) demonstrated strong bimodal inhibitory effects on the I (CaL), with half-inhibition occurring around 30 nM, i.e., in the range of concentrations found in the plasma. Intracellular Zn(2+) also significantly inhibited the I (CaL) with a half-inhibitory effect at 12.7 nM. Moreover, β-adrenergic stimulation was markedly reduced by intracellular Zn(2+) at even lower concentrations (<1 nM) as a consequence of Zn(2+)-induced inhibition of the adenylyl cyclase. All these effects appeared independent of redox variations and were not affected by dithiothreitol. Thus, both basal intracellular and extracellular Zn(2+) modulate transmembrane Ca(2+) movements and their regulation by β-adrenergic stimulation. Considering that, in many pathological situations, including diabetes, the extracellular Zn(2+) concentration is reduced and the intracellular one is increased, our results help to explain both Ca(2+) overload and marked reduction in the β-adrenergic stimulation in these diseases.

Resveratrol, a natural phytoalexin found in wine has the potential to impact a variety of human diseases. Resveratrol like other polyphenols activates many of the same intracellular pathways as those activated by caloric restriction. It can quench reactive oxidative species, ROS and induce eNOS and iNOS expression. Resveratrol also can activate SIRT1, a NAD⁺-dependent deacetylase, that leads an improved in mitochondrial function, and then this procedure turns to activate the transcription factor Nrf2 that coordinates expression of key antioxidant mechanisms by binding to the antioxidant response elements. Resveratrol provides cardioprotection by triggering preconditioning and inducing autophagy. It also presents chemical similarities with estrogen and was reported to activate both nuclear and extranuclear estrogen receptors. Resveratrol treatment alleviated diabetes-induced cardiovascular system disorders via different endogeneous signaling pathways including oxidative stress/antioxidant defense system, glucose/insulin metabolism, overexpression of iNOS/nitrotyrosine, and preconditioning. Resveratrol treatment significantly reduced the blood glucose level in STZ-treated type 1 diabetic animals through insulin-dependent and insulin-independent pathways. Resveratrol triggers some of the similar intracellular insulin signalling components in myocardium such as eNOS, AKT through the AMPK pathway, and plays an essential role in Glut-4 translocation and glucose uptake in STZ-induced diabetic myocardium. However, resveratrol can exhibit hormetic action expressing health benefits at lower doses whereas being detrimental at higher doses. It might also exert antidiabetic effects by activating SIRT1 directly in the brain. This review includes a summary of the role of resveratrol and diabetic cardiac function including a brief discussion on in vitro and in vivo studies as well as our original observations in diabetic rats.

Ahnak1, a giant 700 kDa protein, has been implicated in Ca(2+) signalling in various cells. Previous work suggested that the interaction between ahnak1 and Cavbeta(2) subunit plays a role in L-type Ca(2+) current (I (CaL)) regulation. Here, we performed structure-function studies with the most C-terminal domain of ahnak1 (188 amino acids) containing a PxxP consensus motif (designated as 188-PSTP) using ventricular cardiomyocytes isolated from rats, wild-type (WT) mice and ahnak1-deficient mice. In vitro binding studies revealed that 188-PSTP conferred high-affinity binding to Cavbeta(2) (K (d) approximately 60 nM). Replacement of proline residues by alanines (188-ASTA) decreased Cavbeta(2) affinity about 20-fold. Both 188-PSTP and 188-ASTA were functional in ahnak1-expressing rat and mouse cardiomyocytes during whole-cell patch clamp. Upon intracellular application, they increased the net Ca(2+) influx by enhancing I (CaL) density and/or increasing I (CaL) inactivation time course without altering voltage dependency. Specifically, 188-ASTA, which failed to affect I (CaL) density, markedly slowed I (CaL) inactivation resulting in a 50-70% increase in transported Ca(2+) during a 0 mV depolarising pulse. Both ahnak1 fragments also slowed current inactivation with Ba(2+) as charge carrier. By contrast, neither 188-PSTP nor 188-ASTA affected any I (CaL) characteristics in ahnak1-deficient mouse cardiomyocytes. Our results indicate that the presence of endogenous ahnak1 is required for tuning the voltage-dependent component of I (CaL) inactivation by ahnak1 fragments. We suggest that ahnak1 modulates the accessibility of molecular determinants in Cavbeta(2) and/or scaffolds selectively different beta-subunit isoforms in the heart.

The benzothiazepine derivative K-201 has been suggested as a potential therapeutic agent due to its antiarrhythmogenic action. To understand how the drug alters calcium release from the sarcoplasmic reticulum (SR), we investigated its effects on the SR calcium channel and calcium pump by single channel electrophysiology, whole-cell confocal microscopy, and ATPase activity measurements on control and post-myocardial infarcted (PMI) rat skeletal muscle. In bilayers, K-201 induced two subconductance states corresponding to approximately 24% (S(1)) and approximately 13% (S(2)) of the maximum conductance. Dependence of event frequency and of time spent in S(1) and S(2) on the drug concentration was biphasic both in control and in PMI rats, with a maximum at 50 microM. At this concentration, the channel spends 26 +/- 4% and 24 +/- 4%, respectively, of the total time in these subconductance states at positive potentials, while no subconductances are observed at negative potentials. K-201 altered the frequency of elementary calcium release events: spark frequency decreased from 0.039 +/- 0.001 to 0.023 +/- 0.001 s(-1) sarcomere(-1), while the frequency of embers increased from 0.011 +/- 0.001 to 0.023 +/- 0.001 s(-1) sarcomere(-1). Embers with different amplitude levels were observed after the addition of the drug. K-201 inhibited the Ca(2+) ATPase characterized by IC(50,contr) = 119 +/- 21 muM and n (Hill,contr) = 1.84 +/- 0.48 for control and IC(50,PMI) = 122 +/- 18 microM and n (Hill,PMI) = 1.97 +/- 0.24 for PMI animals. These results suggest that although K-201 would increase the appearance of subconductance states, the overall calcium release is reduced by the drug. In addition, the effect of K-201 is identical on calcium release channels from control and PMI rats.

In chronic heart failure, skeletal muscles develop a weakness that is not associated to an impaired circulatory function but rather to alterations in the skeletal muscle fibers themselves. To understand these changes, the steps in excitation-contraction coupling of rats that underwent a left anterior coronary artery occlusion were studied. About 24 weeks after the myocardial infarction, neither the total amount nor the voltage dependence of intramembrane charge were altered. In contrast, calcium release from the sarcoplasmic reticulum was considerably suppressed, and its voltage dependence shifted toward more positive voltages. Elementary calcium-release events showed altered morphology as the relative proportion of embers increased. Calcium sparks were smaller in amplitude and had larger time-to-peak values. Isolated ryanodine receptors (RyR) displayed an unusual rectification with increased single-channel conductance at positive (cis vs trans) voltages. In addition, the bell-shaped calcium dependence of channel activity was broader, with a slight shift of activation to lower and a larger shift in inactivation to higher calcium concentrations. These data indicate that the number of channels that open during a calcium-release event is decreased and that RyR function is altered; thus, calcium-release is suppressed after a myocardial infarction. These observations give an explanation for the impaired skeletal muscle function in these animals.

We investigated the activation of mitogen-activated protein kinases (MAPKs) pathways by purinergic stimulation in cardiac myocytes from adult rat hearts. ATPgammaS increased the phosphorylation (activation) of the extracellular signal regulated kinase 1 and 2 (ERK1/2) and p38 MAPK. ERK1/2 and p38 MAPK activation was differential, ERK1/2 being rapid and transient while that of p38 MAPK slow and sustained. Using selective inhibitors, activation of ERK1/2 was shown to involve protein kinase C and MEK1/2 while that of p38 MAPK was regulated by both protein kinase C and protein kinase A. Furthermore, we show that purinergic stimulation induces the phosphorylation of the MAPK downstream target, mitogen- and stress-activated protein kinase 1 (MSK1), in cardiac myocytes. The time course of MSK1 phosphorylation closely follows that of ERK activation. Inhibitors of the ERK and p38 MAPK pathways were tested on the phosphorylation of MSK1 at two different time points. The results suggest that ERKs initiate the response but both ERKs and p38 MAPK are required for the maintenance of the complete phosphorylation of MSK1. The temporal relationship of MSK1 phosphorylation and cPLA2 translocation induced by purinergic stimulation, taken together with previous findings, is an indication that cPLA2 may be a downstream target of MSK1.

Sodium ions have been reported to alter the permeation properties of L- and N-type Ca2+ channels. Here in frog atrial cardiomyocytes under whole-cell patch-clamp conditions, we have examined the effects of lowering the external Na+ concentration on the amplitude of T-type Ca2+ current, ICaT, and on the relief of its steady-state inactivation by large depolarizing prepulses, ICaT facilitation. A partial reduction in Na+ ion concentration did not significantly alter ICaT amplitude elicited at -50 mV. However, after a large depolarization, low- Na+ solutions enhanced the relief of inactivation and induced ICaT facilitation. This facilitation occurred independently of the divalent charge carrier, high intracellular Ca2+ buffering or the intracellular Na+ content. Its effects were additional to the beta-adrenergic effects mediated by a decrease of Gi/o-protein inhibitory tone. In Ca2+-free solution the very large T-type current, then carried by Na+ ions, showed only a weak relief of inactivation. In conclusion, ICaT facilitation--which, as previously reported, is modulated by the transient voltage-dependent relief of Gi-protein inhibitory tone--is further enhanced in a low-Na+ solution. In Ca2+-free solution, relief of inactivation due to re-openings dependent on the divalent charge carrier is improbable. It thus appears that for a short while after a large depolarization, external Na+ compete with Ca2+ ions on permeation-controlling sites, so as to modulate channel re-openings and thus the amplitude of voltage-facilitated ICaT independently of the control exerted by the inhibitory G-protein.

Understanding transmembrane signalling process is one of the major challenge of the decade. In most tissues, since Fisher and Krebs's discovery in the 1950's, protein phosphorylation has been widely recognized as a key event of this cellular function. Indeed, binding of hormones or neurotransmitters to specific membrane receptors leads to the generation of cytosoluble second messengers which in turn activate a specific protein kinase. Numerous protein kinases have been so far identified and roughly classified into two groups, namely serine/threonine and tyrosine kinases on the basis of the target acid although some more recently discovered kinases like MEK (or MAP kinase kinase) phosphorylate both serine and tyrosine residues. Protein kinase C is a serine/threonine kinase that was first described by Takai et al. [1] as a Ca- and phospholipid-dependent protein kinase. Later on, Kuo et al. [2] found that PKC was expressed in most tissues including the heart. The field of investigation became more complicated when it was found that the kinase is not a single molecular entity and that several isoforms exist. At present, 12 PKC isoforms and other PKC-related kinases [3] were identified in mammalian tissues. These are classified into three groups. (1) the Ca-activated alpha-, beta-, and gamma-PKCs which display a Ca-binding site (C2); (2) the Ca-insensitive delta-, epsilon-, theta-, eta-, and mu-PKCs. The kinases that belong to both of these groups display two cysteine-rich domains (C1) which bind phorbol esters (for recent review on PKC structure, see [4]). (3) The third group was named atypical PKCs and include zeta, lambda, and tau-PKCs that lack both the C2 and one cysteine-rich domain. Consequently, these isoforms are Ca-insensitive and cannot be activated by phorbol esters [5]. In the heart, evidence that multiple PKC isoforms exist was first provided by Kosaka et at. [6] who identified by chromatography at least two PKC-related isoenzymes. Numerous studies were thus devoted to the biochemical characterization of these isoenzymes (see [7] for review on cardiac PKCs) as well as to the identification of their substrates. This overview aims at updating the present knowledge on the expression, activation and functions of PKC isoforms in cardiac cells.

The mechanism of enhancement of the L-type Ca current by a P2-purinergic agonist adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) was studied by recording single channel activity from cell-attached patches on rat isolated ventricular cells using patch pipettes containing 110 mM Ba2+. The application of ATP gamma S to the patch membrane through the pipette solution did not affect single channel activity. The addition of ATP gamma S to the bath containing a depolarizing solution was ineffective due to the voltage dependence of the purinergic stimulation. Bath application of ATP gamma S (100 microM) to control 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid (HEPES) solution increased the amplitude of ensemble average currents both by decreasing the probability of a blank sweep occurring and by increasing the number of openings per non-blank sweep. The single channel conductance (17 pS) was not changed by ATP gamma S. Both activation and inactivation curves were shifted towards hyperpolarized potentials by about 10 mV under P2-purinergic stimulation. Since ATP gamma S increased channel activity when applied via the bath, it must be supposed that a diffusible messenger is involved.

Adenosine triphosphate (ATP) is released during neural stimulation and cardiac hypoxia and several mechanisms of its action have been reported in different tissues. ATP stimulates P1 and P2 purinergic receptors; it also activates receptor-operated channels and increases membrane permeability to small ions. In single rat ventricular cells under whole-cell patch-clamp, a stepwise application of ATP in the micromolar range affects the resting potential and membrane currents through an entirely novel mechanism of action which involves several steps. Extracellular ATP induces an inward current and depolarization of the cell, leading to automaticity. The inward current is non-specific for cations, its reversal potential is around -5 mV. The conductance change evoked by ATP is suppressed by 4,4-diisothiocyanostilbene 2,2-disulphonic acid (DIDS) and low-chloride media and is prolonged by adding intracellular bicarbonate. These effects are specific for ATP in the presence of magnesium and are not evoked by a non-hydrolysable analogue of ATP or in the presence of vanadate. Other nucleotides are ineffective. We propose that ATP hydrolysis activates the chloride/bicarbonate (Cl-/HCO3-) exchanger. The induced local acidification could then increase intracellular free calcium and as a consequence, increases the sarcolemmal conductance. Thus, a sudden release of ATP in pathological conditions would induce a depolarization which could generate ventricular arrhythmias.

Ca current (ICa) was measured by whole-cell voltage clamp in single cells isolated from frog ventricle, in which the Na current was inhibited by tetrodotoxin (0.3 microM) and K currents were blocked by substituting K with 120 mM intracellular and 20 mM extracellular Cs. The influence of stimulation by ATP (0.1-100 microM) was assessed in the presence of propranolol (1 microM) or pindolol (0.1 microM), prazozin (0.1 microM) and atropine (10 microM). ATP, in the micromolar range, had two types of effect. Like other P1-purinoagonists, it antagonized the increase in ICa elicited by beta-adrenostimulation. When added alone, 1 microM ATP could increase ICa up to twofold. An increase in ICa was also observed even after it had been maximally enhanced by intracellularly applied cAMP (50 microM). Voltage dependence and kinetics of ICa were not affected. These effects were considered to be related to P2-purinoceptor activation. At higher ATP concentrations the increase in ICa was less; at 100 microM, ATP reduced ICa. The ATP-induced increase in ICa was prevented by internal perfusion of the cells with GDP [beta-S] or neomycin, respectively, to block signal transduction to phospholipase C or its phosphodiesterase activity on the polyphosphoinositides. We conclude that P2-purinoceptor stimulation increases the Ca current in frog ventricular cells by a pathway that might involve phosphoinositide turnover.

In the absence of creatine phosphate, MgATP produced relaxation of rigor tension in chemically-skinned right papillary muscles of the rat, the half maximal effect being obtained at 1.8 mM MgATP. In the presence of 12 mM creatine phosphate and 250 microM ADP, a decrease in MgATP concentration even to 10(-9) M never induced rigor tension. At a very low MgATP concentration (10(-6) M), the half maximal relaxing effect was obtained with 2 mM creatine phosphate, a value close to the Km of isolated MM-creatine kinase for this substrate, or with 14 microM MgADP, a value 5 times lower than the reported Km. An exogenous MgATP regenerating system (phosphoenol pyruvate + pyruvate kinase) was not able to fully relax the fibres. When MM-creatine kinase was inhibited by fluorodinitrobenzene, the dependency of rigor tension on MgATP became the same as it was without creatine phosphate. After washing out the fluorodinitrobenzene the addition of exogenous MM-creatine kinase for half an hour fully relaxed rigor tension; moreover, this effect persisted even after prolonged washout. These results show that endogenous MM-creatine kinase is able to ensure maximal efficiency of myosin ATPase by producing a localized high MgATP/MgADP ratio; they also suggest the existence of rapidly exchangeable binding sites for MM-creatine kinase in cardiac myofibrils.

The effects of cardiac glycosides (1 microM ouabain, 50 microM dihydrooubain, 1 microM strophanthidin) on Ca current (ICa) were investigated on Cs-loaded single frog ventricular cells using the whole-cell patch-clamp technique (9). Cardiac glycosides exert both inhibitory and stimulatory effects on ICa in 20 Cs Ringer solution, but have only a stimulatory effect in 0 Cs, when the Na,K pump is blocked. The inhibitory response seems related to the inhibition of the Na,K pump by glycosides. The stimulatory effect on ICa may contribute to the positive inotropic effect of cardiac glycosides.