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Abstract: Objective: Previous studies have questioned whether there is an improved cardiac function after high-altitude training. Accordingly, the present study was designed specifically to test whether this apparent blunted response of the whole heart to training can be accounted for by altered mechanical properties at the cellular level. Methods: Adult rats were trained for 5 weeks under normoxic (N, NT for sedentary and trained animals, respectively) or hypobaric hypoxic (H, HT) conditions. Cardiac morphology and function were evaluated by echocardiography. Calcium Ca2+ sensitivity of the contractile machinery was estimated in skinned cardiomyocytes isolated from the left ventricular (LV) sub-epicardium (Epi) and sub-endocardium (Endo) at short and long sarcomere lengths (SL). Results: Cardiac remodelling was harmonious (increase in wall thickness with chamber dilatation) in NT rats and disharmonious (hypertrophy without chamber dilatation) in HT rats. Contrary to NT rats, HT rats did not exhibit enhancement in global cardiac performance evaluated by echocardiography. Stretch- dependent Ca2+ sensitization of the myofilaments (cellular index of the Frank-Starling mechanism) increased from Epi to Endo in N rats. Training in normoxic conditions further increased this stretch-dependent Ca2+ sensitization. Chronic hypoxia did not significantly affect myofibrilar Ca2+ sensitivity. In contrast, high-altitude training decreased Ca2+ sensitivity of the myofilaments at both SL, mostly in Endo cells, resulting in a loss of the transmural gradient of the stretch-dependent Ca2+ sensitization. Expression of myosin heavy chain isoforms was affected both by training and chronic hypoxia but did not correlate with mechanical data. Conclusions: Training at sea level increased the transmural gradient of stretch-dependent Ca2+ sensitization of the myofilaments, accounting for an improved Frank-Starling mechanism. High-altitude training depressed myofilament response to Ca2+, especially in the Endo layer. This led to a reduction in this transmural gradient that may contribute to the lack of improvement in LV function via the Frank-Starling mechanism. [Copyright &y& Elsevier]

Summary 1. The effects of the mitogen-activated protein kinase (MAPK) inhibitors PD 98059 and U 0126, useful tools to investigate MAPK involvement in intracellular signal transduction pathways, were assessed on cardiomyocytes. 2. In rat freshly isolated ventricular myocytes, under current-clamp conditions, PD 98059 (40 µmol/L) shortened the action potential. Under whole-cell patch-clamp, this compound slowly induced a fast activating sustained outward K+ current that was sensitive to 1 mmol/L Ba2+ , 100 µmol/L Gd3+ , 3 mmol/L 4-aminopyridine and 100 µmol/L tetracain. The PD 98059-induced current was prevented by 40 µmol/L AACOCF3 , a cytosolic phospholipase A2 inhibitor. 3. U 0126 (1 µmol/L), a recently developed highly potent p42/44 MAPK inhibitor, did not alter K+ currents. 4. PD 98059, but not U 0126, increased arachidonic acid content, probably as a consequence of its reported cyclo-oxygenase inhibitory effect. 5. These observations indicate that PD 98059 activates a TREK-1 like current. Thus, this MAPK inhibitor has to be used with caution because alterations in cell metabolism can be secondary to changes in electrophysiological behaviour. [ABSTRACT FROM AUTHOR]

The L-type Ca2+ current (ICa-L) plays a key role in the cardiac excitation-contraction (E-C) coupling. Thus, it is a major target for many transmitters and hormones modulating cardiac function and, therefore, for pharmacological drugs to regulate inotropy. Ca2+ (and other) ion currents are commonly studied in animal tissues for practical reasons. Investigations in human cardiomyocytes started extensively only ten years ago with the development of patch-clamp techniques, enzymatic cell dissociation procedures, and surgical techniques. These studies have already provided valuable information concerning the nature, biophysics, pharmacology and regulation of human cardiac ionic currents in normal and diseased tissues. Interesting advances have been made to understand the role of ICa-L in the development of chronic atrial fibrillation (AF). Alterations of single channel activity and regulation of macroscopic ICa-L have also been found in heart failure (HF), although some of the data are divergent and puzzling. The T-type Ca2+ current (ICa-T) has never been recorded in human cardiomyocytes. After a rapid overview of the basic properties of human cardiac Ca2+ currents, we focus on selected aspects of pathophysiology that are still unsolved. [ABSTRACT FROM AUTHOR]

Evaluates sarcoplasmic reticulum calcium release mechanisms to further explore the determinants of altered calcium signaling in skeletal muscle from rats with chronic myocardial overload. Examination of global calcium transients in single fibers in whole extensor digitorum longus muscle preparations; Discussion on the alteration of the biochemical properties of the skeletal muscle macromolecular calcium release complex during heart failure; Conclusion.

Defective cardiac mechanical activity in diabetes results from alterations in intracellular Ca(2+) handling, in part, due to increased oxidative stress. Beta-blockers demonstrate marked beneficial effects in heart dysfunction with scavenging free radicals and/or acting as an antioxidant. The aim of this study was to address how β-blocker timolol-treatment of diabetic rats exerts cardioprotection. Timolol-treatment (12-week), one-week following diabetes induction, prevented diabetes-induced depressed left ventricular basal contractile activity, prolonged cellular electrical activity, and attenuated the increase in isolated-cardiomyocyte size without hyperglycemic effect. Both in vivo and in vitro timolol-treatment of diabetic cardiomyocytes prevented the altered kinetic parameters of Ca(2+) transients and reduced Ca(2+) loading of sarcoplasmic reticulum (SR), basal intracellular free Ca(2+) and Zn(2+) ([Ca(2+)]i and [Zn(2+)]i), and spatio-temporal properties of the Ca(2+) sparks, significantly. Timolol also antagonized hyperphosphorylation of cardiac ryanodine receptor (RyR2), and significantly restored depleted protein levels of both RyR2 and calstabin2. Western blot analysis demonstrated that timolol-treatment also significantly normalized depressed levels of some [Ca(2+)]i-handling regulators, such as Na(+)/Ca(2+) exchanger (NCX) and phospho-phospholamban (pPLN) to PLN ratio. Incubation of diabetic cardiomyocytes with 4-mM glutathione exerted similar beneficial effects on RyR2-macromolecular complex and basal levels of both [Ca(2+)]i and [Zn(2+)]i, increased intracellular Zn(2+) hyperphosphorylated RyR2 in a concentration-dependent manner. Timolol also led to a balanced oxidant/antioxidant level in both heart and circulation and prevented altered cellular redox state of the heart. We thus report, for the first time, that the preventing effect of timolol, directly targeting heart, seems to be associated with a normalization of macromolecular complex of RyR2 and some Ca(2+) handling regulators, and prevention of Ca(2+) leak, and thereby normalization of both [Ca(2+)]i and [Zn(2+)]i homeostasis in diabetic rat heart, at least in part by controlling the cellular redox status of hyperglycemic cardiomyocytes.

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.

Diabetes mellitus is a major risk factor for cardiovascular complications. Intracellular Ca(2+) release plays an important role in the regulation of muscle contraction. Sarcoplasmic reticulum Ca(2+) release is controlled by dedicated molecular machinery, composed of a complex of cardiac ryanodine receptors (RyR2s). Acquired and genetic defects in this complex result in a spectrum of abnormal Ca(2+) release phenotypes in heart. Cardiovascular dysfunction is a leading cause for mortality of diabetic individuals due, in part, to a specific cardiomyopathy, and to altered vascular reactivity. Cardiovascular complications result from multiple parameters, including glucotoxicity, lipotoxicity, fibrosis, and mitochondrial uncoupling. In diabetic subjects, oxidative stress arises from an imbalance between production of reactive oxygen and nitrogen species and capability of the system to readily detoxify reactive intermediates. To date, the etiology underlying diabetes-induced reductions in myocyte and cardiac contractility remains incompletely understood. However, numerous studies, including work from our laboratory, suggest that these defects stem in part from perturbation in intracellular Ca(2+) cycling. Since the RyR2s are one of the well-characterized redox-sensitive ion channels in heart, this article summarizes recent findings on redox regulation of cardiac Ca(2+) transport systems and discusses contributions of redox regulation to pathological cardiac function in diabetes.

Aims: Zinc exists in biological systems as bound and histochemically reactive free Zn(2+). It is an essential structural constituent of many proteins, including enzymes from cellular signalling pathways, in which it functions as a signalling molecule. In cardiomyocytes at rest, Zn(2+) concentration is in the nanomolar range. Very little is known about precise mechanisms controlling the intracellular distribution of Zn(2+) and its variations during cardiac function., Methods and Results: Live-cell detection of intracellular Zn(2+) has become feasible through the recent development of Zn(2+)-sensitive and -selective fluorophores able to distinguish Zn(2+) from Ca(2+). Here, in freshly isolated rat cardiomyocytes, we investigated the rapid changes in Zn(2+) homeostasis using the Zn(2+)-specific fluorescent dye, FluoZin-3, in comparison to Ca(2+)-dependent fluo-3 fluorescence. Zn(2+) sparks and Zn(2+) transients, in quiescent and electrically stimulated cardiomyocytes, respectively, were visualized in a similar manner to known rapid Ca(2+) changes. Both Zn(2+) sparks and Zn(2+) transients required Ca(2+) entry. Inhibiting the sarcoplasmic reticulum Ca(2+) release or increasing the Ca(2+) load in a low-Na(+) solution suppressed or increased Zn(2+) movements, respectively. Mitochondrial inhibitors slightly reduced both Zn(2+) sparks and Zn(2+) transients. Oxidation by H₂O₂ facilitated and acidic pH inhibited the Ca(2+)-dependent Zn(2+) release., Conclusion: It is proposed that Zn(2+) release during the cardiac cycle results mostly from intracellular free Ca(2+) increase, triggering production of reactive oxygen species that induce changes in metal-binding properties of metallothioneins and other redox-active proteins, aside from ionic exchange on these proteins.

The scope of this review is to summarize the important roles of vitamin E family members as protective agents in cardiovascular pathologies of different types of disease states and particularly in diabetes, including some of our research results, to illustrate how this recent knowledge is helping to better understand the roles of the vitamin E family in biology, in animals and humans specifically. Cardiovascular disease, a general name for a wide variety of diseases, disorders and conditions, is caused by disorders of the heart and blood vessels. Cardiovascular disease is the world's largest killer, claiming 17.1 million lives a year. Cardiovascular complications result from multiple parameters including glucotoxicity, lipotoxicity, fibrosis. Obesity and diabetes mellitus are also often linked to cardiovascular disease. In fact, cardiovascular disease is the most life-threatening of the diabetic complications and diabetics are 2- to 4-fold more likely to die of cardiovascular-related causes than non-diabetics. In order to prevent the tendency of cardiovascular disease, primary prevention is needed by modifying risk factors. Several recent studies, besides earlier ones, have reported beneficial effects of therapy with antioxidant agents, including trace elements, vitamins (E and/or C), other antioxidants, against the cardiovascular dysfunction. Hence, the use of peroxisome proliferator activated receptor-α (PPARα) agonists to reduce fatty acid oxidation, of trace elements such as selenium as antioxidant and other antioxidants such as vitamins E and C, contributes to the prevention of these dysfunctions. Moreover, therapy with antioxidants and the above vitamins to prevent or delay the onset and development of cardiovascular complications in diabetic patients and animal models has been investigated although these studies showed inconsistent results.

Despite its degradation by ectonucleotidases, a low ATP concentration is present in the interstitial space; moreover, its level can markedly increase during various physiopathological conditions. ATP and uridine 5'-triphosphate (UTP) releases correlate with the occurrence of ventricular premature beats and ventricular tachycardia. ATP facilitates several voltage-dependent ionic currents including the L-type Ca(2+) current. More recently, ATP and UTP were also shown to induce a poor voltage-dependent, long-lasting current carried by the heterotetrameric transient receptor potential (TRP) channels TRPC3/7. ATP effects result from its binding to metabotropic P2Y2 receptors that lead to diacylglycerol formation and activation of phospholipase Cβ and inositol-1,4,5-triphosphate production. ATP also favours TRPM4 activation by increasing Ca(2+) release from the sarcoplasmic reticulum. Indeed, TRPM4 current properties match those of the Ca(2+)-activated, nonselective cationic current supporting the delayed afterdepolarizations observed under conditions of Ca(2+) overload. In the present article, it was hypothesized that creatine, at a relatively high concentration, would serve as a buffer for the sudden release of ATP and UTP during the early phase of ischemia in association with previously described arrhythmic events. The potential preventive effect of creatine was tested by analyzing its ability to antagonize the arrhythmia that occurred on inducing a coronary ligature in rats that were or were not preinjected with creatine. Electrocardiogram recordings of creatine-injected rats clearly demonstrated that both ventricular premature beats and, particularly, ventricular tachycardia markedly decreased. The effect of creatine was even more striking in early deaths. However, an injection of beta-guanidinopropionate, a creatine analogue with 1000-fold lower kinetics, had no significant protective effect.

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.

Cardiac dysfunction occurs during type 1 and type 2 diabetes and results from multiple parameters including glucotoxicity, lipotoxicity, fibrosis and mitochondrial uncoupling. Oxidative stress arises from an imbalance between the production of ROS and the biological system's ability to readily detoxify the reactive intermediates. It is involved in the etiology of diabetes-induced downregulation of heart function. Several studies have reported beneficial effects of a therapy with antioxidant agents, including trace elements and other antioxidants, against the cardiovascular system consequences of diabetes. Antioxidants act through one of three mechanisms to prevent oxidant-induced cell damages. They can reduce the generation of ROS, scavenge ROS, or interfere with ROS-induced alterations. Modulating mitochondrial activity is an important possibility to control ROS production. Hence, the use of PPARalpha agonist to reduce fatty acid oxidation and of trace elements such as zinc and selenium as antioxidants, and physical exercise to induce mitochondrial adaptation, contribute to the prevention of diabetes-induced cardiac dysfunction. The paradigm that inhibiting the overproduction of superoxides and peroxides would prevent cardiac dysfunction in diabetes has been difficult to verify using conventional antioxidants like vitamin E. That led to use of catalytic antioxidants such as SOD/CAT mimetics. Moreover, increases in ROS trigger a cascade of pathological events, including activation of MMPs, PPARs and protein O-GlcNAcation. Multiple tools have been developed to counteract these alterations. Hence, well-tuned, balanced and responsive antioxidant defense systems are vital for proper prevention against diabetic damage. This review aims to summarize our present knowledge on various strategies to control oxidative stress and antagonize cardiac dysfunction during diabetes.

The transient receptor potential (TRP) family of ion channels comprises more than 50 cation-permeable channels expressed throughout the animal kingdom. TRPs can be grouped into 7 main subfamilies according to structural homology: the TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), TRPA (ankyrin), and TRPN (NO mechanopotential). During the past 20 years, the cloning and characterization after reexpression of most members of these cation channels have led to a plethora of data and more recently to some understanding of their roles in various cells and tissues. Specifically in the heart, TRPs are known to be involved in various diseases, including hypertrophy, heart failure, and arrhythmia. The later part of this review focuses on the potential contribution of TRPs to cardiac rhythm and their potential proarrhythmic effects. Furthermore, several neurotransmitters that activate the formation of diacylglycerol could modulate cardiac rhythm or, like ATP, induce arrhythmia.

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.

Extracellular purines and pyrimidines have major effects on cardiac rhythm and contraction. ATP/UTP are released during various physiopathological conditions, such as ischemia, and despite degradation by ectonucleotidases, their interstitial concentrations can markedly increase, a fact that is clearly associated with arrhythmia. In the present whole cell patch-clamp analysis on ventricular cardiomyocytes isolated from various mammalian species, ATP and UTP elicited a sustained, nonselective cationic current, I(ATP). UDP was ineffective, whereas 2'(3')-O-(4-benzoylbenzoyl)-ATP was active, suggesting that P2Y(2) receptors are involved. I(ATP) resulted from the binding of ATP(4-) to P2Y(2) purinoceptors. I(ATP) was maintained after ATP removal in the presence of guanosine 5'-[gamma-thio]triphosphate and was inhibited by U-73122, a PLC inhibitor. Single-channel openings are rather infrequent under basal conditions. ATP markedly increased opening probability, an effect prevented by U-73122. Two main conductance levels of 14 and 23 pS were easily distinguished. Similarly, in fura-2-loaded cardiomyocytes, Mn(2+) quenching and Ba(2+) influx were significant only in the presence of ATP or UTP. Adult rat ventricular cardiomyocytes expressed transient receptor potential channel TRPC1, -3, -4, and -7 mRNA and the TRPC3 and TRPC7 proteins that coimmunoprecipitated. Finally, the anti-TRPC3 antibody added to the patch pipette solution inhibited I(ATP). In conclusion, activation of P2Y(2) receptors, via a G protein and stimulation of PLCbeta, induces the opening of heteromeric TRPC3/7 channels, leading to a sustained, nonspecific cationic current. Such a depolarizing current could induce cell automaticity and trigger the arrhythmic events during an early infarct when ATP/UTP release occurs. These results emphasize a new, potentially deleterious role of TRPC channel activation.

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.