ß-blocker timolol prevents arrhythmogenic Ca²âº release and normalizes Ca²âº and Zn²âº dyshomeostasis in hyperglycemic rat heart.

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 ß-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 and diabetic cardiac function: focus on recent in vitro and in vivo studies.

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.

Vitamin E in oxidant stress-related cardiovascular pathologies: focus on experimental studies.

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.

Intracellular free zinc during cardiac excitation-contraction cycle: calcium and redox dependencies.

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.

Ryanodine receptor: a new therapeutic target to control diabetic cardiomyopathy.

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.

Cardiac cellular actions of quebrachidine, an indole alkaloid isolated from Rauwolfia viridis Roem et Schult

INTRODUCTION: the search for new drugs with safer therapeutic profiles in Cardiology is still a need and natural products, particularly from plants, constitute an excellent source of new compounds. OBJECTIVE: to study the cardiac cellular actions of quebrachidine an indole alkaloid, extracted from the roots of Rauwolfia viridis R et S, known as Quebrachidine, which is structurally related to the antiarrhythmics ajmaline and prajmaline. METHODS: several complementary experimental approaches to evaluate the effects of quebrachidine on the electrophysiological and contractile properties of cardiac tissues and cells were used. RESULTS: quebrachidine increased the ventricular fibrillation threshold in anaesthetized rabbits. It decreased the maximum rate of depolarization and increased the duration of the ventricular action potential in different species. These actions were accompanied by a positive inotropic effect over a broad concentration range and were consistent with the increase in Ca2+ currents recorded in single ventricular cardiomyocytes. CONCLUSIONS: the present results demonstrate that quebrachidine keeps the antiarrhythmic profile of ajmaline and prajmaline but also demonstrates a net positive inotropic action on cardiac tissues predictive of better therapeutic safety margin. Our results suggest that ajmalan-like molecular structures could provide a sound basis for the search of effective antiarrhythmics with positive inotropic effect

INTRODUCCIÓN: la búsqueda de nuevos fármacos con perfiles terapéuticos más seguros en cardiología, es aun una necesidad y los productos naturales, particularmente de plantas, constituyen una fuente excelente de nuevos compuestos. OBJETIVOS: estudiar las acciones celulares cardíacas de la quebrachidina, un alcaloide indólico extraído de las raíces de Rauwolfia viridis R et S, el cual está estructuralmente relacionado con los antiarrítmicos ajmalina y prajmalina. MÉTODOS: se utilizaron diferentes modelos experimentales complementarios para evaluar los efectos de la quebrachidina sobre las propiedades electrofisiológicas y contráctiles de tejidos y células cardíacas. RESULTADOS: la quebrachidina incrementó el umbral para la fibrilación ventricular en conejos anestesiados. Este alcaloide redujo la velocidad máxima de despolarización y aumentó la duración del potencial de acción ventricular de diferentes especies. Estas acciones estuvieron acompañadas de un efecto inotrópico positivo en un amplio rango de concentraciones y asociadas a un incremento en las corrientes de Ca2+ en cardiomiocitos ventriculares aislados. CONCLUSIONES: estos resultados demuestran que la quebrachidina conserva el perfil antiarrítmico de la ajmalina y la prajmalina pero muestra un efecto inotrópico positivo neto en tejidos cardíacos lo cual predice un mejor margen de seguridad terapéutico. Los resultados sugieren que las estructuras moleculares con núcleo ajmalano pueden constituir una base firme para la búsqueda de antiarrítmicos con efecto inotrópico positivo

Cardiac cellular actions of quebrachidine, an indole alkaloid isolated from Rauwolfia viridis Roem et Schult

INTRODUCTION: the search for new drugs with safer therapeutic profiles in Cardiology is still a need and natural products, particularly from plants, constitute an excellent source of new compounds. OBJECTIVE: to study the cardiac cellular actions of quebrachidine an indole alkaloid, extracted from the roots of Rauwolfia viridis R et S, known as Quebrachidine, which is structurally related to the antiarrhythmics ajmaline and prajmaline. METHODS: several complementary experimental approaches to evaluate the effects of quebrachidine on the electrophysiological and contractile properties of cardiac tissues and cells were used. RESULTS: quebrachidine increased the ventricular fibrillation threshold in anaesthetized rabbits. It decreased the maximum rate of depolarization and increased the duration of the ventricular action potential in different species. These actions were accompanied by a positive inotropic effect over a broad concentration range and were consistent with the increase in Ca2+ currents recorded in single ventricular cardiomyocytes. CONCLUSIONS: the present results demonstrate that quebrachidine keeps the antiarrhythmic profile of ajmaline and prajmaline but also demonstrates a net positive inotropic action on cardiac tissues predictive of better therapeutic safety margin. Our results suggest that ajmalan-like molecular structures could provide a sound basis for the search of effective antiarrhythmics with positive inotropic effect(AU)

INTRODUCCIÓN: la búsqueda de nuevos fármacos con perfiles terapéuticos más seguros en cardiología, es aun una necesidad y los productos naturales, particularmente de plantas, constituyen una fuente excelente de nuevos compuestos. OBJETIVOS: estudiar las acciones celulares cardíacas de la quebrachidina, un alcaloide indólico extraído de las raíces de Rauwolfia viridis R et S, el cual está estructuralmente relacionado con los antiarrítmicos ajmalina y prajmalina. MÉTODOS: se utilizaron diferentes modelos experimentales complementarios para evaluar los efectos de la quebrachidina sobre las propiedades electrofisiológicas y contráctiles de tejidos y células cardíacas. RESULTADOS: la quebrachidina incrementó el umbral para la fibrilación ventricular en conejos anestesiados. Este alcaloide redujo la velocidad máxima de despolarización y aumentó la duración del potencial de acción ventricular de diferentes especies. Estas acciones estuvieron acompañadas de un efecto inotrópico positivo en un amplio rango de concentraciones y asociadas a un incremento en las corrientes de Ca2+ en cardiomiocitos ventriculares aislados. CONCLUSIONES: estos resultados demuestran que la quebrachidina conserva el perfil antiarrítmico de la ajmalina y la prajmalina pero muestra un efecto inotrópico positivo neto en tejidos cardíacos lo cual predice un mejor margen de seguridad terapéutico. Los resultados sugieren que las estructuras moleculares con núcleo ajmalano pueden constituir una base firme para la búsqueda de antiarrítmicos con efecto inotrópico positivo(AU)

Ahnak1 modulates L-type Ca(2+) channel inactivation of rodent cardiomyocytes.

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.

Protective role of antioxidants in diabetes-induced cardiac dysfunction.

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.

Could early ischemic arrhythmia triggered by purinergic activation of the transient receptor potential channels be prevented by creatine?

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 Cß 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.

Transient receptor potential: a large family of new channels of which several are involved in cardiac arrhythmia.

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.

ATP/UTP activate cation-permeable channels with TRPC3/7 properties in rat cardiomyocytes.

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.

Effects of K-201 on the calcium pump and calcium release channel of rat skeletal muscle.

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.

[Transient receptor potential, TRP channels: a new family of channels broadly expressed]. / Les canaux TRP (transient receptor potential): une nouvelle famille de canaux à expression variée.

Transient receptor potential, TRP channels are a new superfamily of functionally versatile non-selective cation channels present from yeast to mammals. On the basis of their structural homology, TRP channels are subdivided in 7 groups : TRPC 1-7 Canonical, TRPV 1-6 Vanilloid, TRPM 1-8 Melastatin, TRPP 1-3 Polycystin, TRPML Mucolipin, TRPA Ankyrin and TRPN (NO mechanotransducer potential C), the latter not expressed in mammals. Their cloning and heterologous expression allowed to demonstrating that these channels are generally weakly voltage-dependent. They are activated by various ligands involving a signal transduction cascade as well as directly by multiple compounds, heat and pH. TRP channels are found in a broad range of cell types. TRP channels are essential in allowing animals to sense the outside world and cells to sense their local environment. Following mutations or anomalous behaviour, these channels have a major role in several human diseases.

Sex-related effects on diabetes-induced alterations in calcium release in the rat heart.

The present study was designed to determine whether the properties of local Ca(2+) release and its related regulatory mechanisms might provide insight into the role of sex differences in heart functions of control and streptozotocin-induced diabetic adult rats. Left ventricular developed pressure, the rates of pressure development and decay (+/-dP/dt), basal intracellular Ca(2+) level ([Ca(2+)](i)), and spatiotemporal parameters of [Ca(2+)](i) transients were found to be similar in male and female control rats. However, spatiotemporal parameters of Ca(2+) sparks in cardiomyocytes isolated from control females were significantly larger and slower than those in control males. Diabetes reduced left ventricular developed pressure to a lower extent in females than in males, and the diabetes-induced depressions in both +dP/dt and -dP/dt were less in females than in males. Diabetes elicited a smaller reduction in the amplitude of [Ca(2+)](i) transients in females than in males, a smaller reduction in sarcoplasmic reticulum-Ca(2+) load, and less increase in basal [Ca(2+)](i). Similarly, the elementary Ca(2+) events and their control proteins were clearly different in both sexes, and these differences were more marked in diabetes. Diabetes-induced depression of the Ca(2+) spark amplitude was significantly less in females than in matched males. Levels of cardiac ryanodine receptors (RyR2) and FK506-binding protein 12.6 in control females were significantly higher than those shown in control males. Diabetes induced less RyR2 phosphorylation and FK506-binding protein 12.6 unbinding in females. Moreover, total and free sulfhydryl groups were significantly less reduced, and PKC levels were less increased, in diabetic females than in diabetic males. The present data related to local Ca(2+) release and its related proteins describe some of the mechanisms that may underlie sex-related differences accounting for females to have less frequent development of cardiac diseases.

Alterations in the calcium homeostasis of skeletal muscle from postmyocardial infarcted 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.

Restoration of diabetes-induced abnormal local Ca2+ release in cardiomyocytes by angiotensin II receptor blockade.

Stimulation of local renin-angiotensin system and increased levels of oxidants characterize the diabetic heart. Downregulation of ANG II type 1 receptors (AT(1)) and enhancement in PKC activity in the heart point out the role of AT(1) blockers in diabetes. The purpose of this study was to evaluate a potential role of an AT(1) blocker, candesartan, on abnormal Ca(2+) release mechanisms and its relationship with PKC in the cardiomyocytes from streptozotocin-induced diabetic rats. Cardiomyocytes were isolated enzymatically and then incubated with either candesartan or a nonspecific PKC inhibitor bisindolylmaleimide I (BIM) for 6-8 h at 37 degrees C. Both candesartan and BIM applied on diabetic cardiomyocytes significantly restored the altered kinetic parameters of Ca(2+) transients, as well as depressed Ca(2+) loading of sarcoplasmic reticulum, basal Ca(2+) level, and spatiotemporal properties of the Ca(2+) sparks. In addition, candesartan and BIM significantly antagonized the hyperphosphorylation of cardiac ryanodine receptor (RyR2) and restored the depleted protein levels of both RyR2 and FK506 binding protein 12.6 (FKBP12.6). Furthermore, candesartan and BIM also reduced the increased PKC levels and oxidized protein thiol level in membrane fraction of diabetic rat cardiomyocytes. Taken together, these data demonstrate that AT(1) receptor blockade protects cardiomyocytes from development of cellular alterations typically associated with Ca(2+) release mechanisms in diabetes mellitus. Prevention of these alterations by candesartan may present a useful pharmacological strategy for the treatment of diabetic cardiomyopathy.

Role of T-type Ca2+ channels in the heart.

After the first demonstration 30 years ago that Ca2+ could permeate through two different channels, the occurrence and role of T-type Ca2+ current, ICaT have been the matter of hundreds of publications, including the two 1985' reports in various cardiac tissues and species. Except for its specific biophysical characteristics, ICaT is no longer so easily distinguished from the L-type Ca2+ current, ICaL, since it is also sensitive to multiple compounds and various neuromediators including the beta-adrenergic agonists. Changes in ICaT occur during development, so that while it is recorded in all embryonic and neonatal cells investigated, ICaT has been reported in adult ventricular cells of only few species in control. However, under various pathological conditions, ICaT is often recorded at some phases of remodelling at least in some localized area and one or more of the three channel proteins, Cav3.1-3.3 are clearly re-expressed under the influence of IGF-1, endothelin, and angiotensin II. ICaT contributes to the control of electrical activity including pacemaker and arrhythmia. Furthermore ICaT, and its low-depolarisation window current, participate in Ca2+ entry, so that ICaT has been involved in the release of Ca2+ from internal stores, the Ca2+-induced Ca2+ release mechanism, although at much lower level than ICaL. ICaT contributes also to Ca2+-dependent hormonal secretion. This review further emphasizes the difficulties encountered in analysing this current.

Functional remodeling in post-myocardial infarcted rats: focus on beta-adrenoceptor subtypes.

Cellular electrophysiological remodeling of the infarcted heart may lead to the deterioration of cardiac function and/or to arrhythmias. The present study was designed to characterize the functional expression of the hyperpolarization-activated current (I(f)) and its modulation by beta(1)-, beta(2)- and beta(3)-adrenoceptor (AR) subtypes, in patch-clamped ventricular myocytes isolated from the heart of post-myocardial infarcted (PMI) rats and sham-operated control (SHAM) rats. Maximum specific conductance of I(f) was significantly higher in left ventricular myocytes (LVM) from PMI rats compared to right ventricular myocytes from PMI rats as well as LVM and RVM from SHAM rats. All other basic properties of I(f) were similar. beta(1)AR stimulation with noradrenaline caused a rightward shift of V(H) in LVM from PMI rats which was significantly smaller (52.2%) than in LVM from SHAM rats. Incubation with pertussis toxin (PTX) largely restored the effect of beta(1)AR in PMI cells (86.6% vs. SHAM cells), but did not affect beta(1)AR response in SHAM cells. beta(2)AR response was significantly and equally increased by PTX-pretreatment (by 94% in SHAM and 87% in PMI cells). Conversely, beta(3)AR stimulation by the selective agonist SR 58611A caused a leftward shift of the activation curve which was significantly larger in PMI cells than in SHAM cells (P<0.01). beta(3)AR response was blunted by PTX-pretreatment, by incubation with N(G)-monomethyl-l-arginine acetate or by the selective beta(3)AR antagonist SR 59230A 1 microM. In conclusion, I(f) is significantly overexpressed in LVM from PMI rat hearts. In these cells, I(f) modulation by beta(1)AR is significantly depressed while beta(3)AR modulation is markedly enhanced, probably reflecting the increased activity of PTX-sensitive G(i) proteins in PMI cells.