Morphine preconditioning confers cardioprotection in doxorubicin-induced failing rat hearts via ERK/GSK-3ß pathway independent of PI3K/Akt.

Preconditioning against myocardial ischemia-reperfusion (I/R) injury can be suppressed in some pathological conditions. This study was designed to investigate whether morphine preconditioning (MPC) exerts cardioprotection in doxorubicin (DOX)-induced heart failure in rats and the mechanisms involved. Phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt), extracellular signal-regulated kinase (ERK) and glycogen synthase kinase (GSK)-3ß pathways were examined. Normal and DOX-induced failing rat hearts were subjected to I/R injury using a Langendorff perfusion system with or without MPC or ischemic preconditioning (IPC). The PI3K inhibitor (wortmannin) or ERK inhibitor (PD98059) was infused before MPC. In normal hearts, both MPC and IPC significantly reduced infarct size and the rise in lactate dehydrogenase (LDH) level caused by I/R injury. Pretreatment with wortmannin or PD98059 abrogated the protective effects of MPC and suppressed the phosphorylation of Akt, ERK and GSK-3ß. In failing rat hearts, however, MPC retained its cardioprotection while IPC did not. This protective effect was abolished by PD98059 but not wortmannin. MPC increased the level of p-ERK rather than p-Akt. The phosphorylation of GSK-3ß induced by MPC was reversed by PD98059 only. IPC did not elevate the expression of p-ERK, p-Akt and p-GSK-3ß in failing rat hearts. We conclude that MPC is cardioprotective in rats with DOX-induced heart failure while IPC is not. The effect of MPC appears to be mediated via the ERK/GSK-3ß pathway independent of PI3K/Akt.

The function of calcineurin and ERK1/2 signal in the antihypertrophic effects of kappa-opioid receptor stimulation on myocardial hypertrophy induced by isoprenaline.

The aim of the present study, performed in an in vitro model of cardiac hypertrophy, was to examine the possible function of calcineurin and ERK1/2 in the inhibitory effects of kappa-opioid receptor stimulation on Ca2+ transients and myocardial hypertrophy induced by beta1-adrenoceptor stimulation. We determined the effects of trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamid methanesulfonate salt (U50,488H), a selective kappa-opioid receptor agonist, on the enhancement of spontaneous Ca2+ transients and the induction of hypertrophy by isoprenaline, a beta-adrenoceptor agonist, in cultured neonatal ventricular myocytes. Total protein content, [3H]leucine incorporation and cell size were used as indices of hypertrophy; calcineurin activity and phospho-ERK1/2 level were determined by immunoblotting. Isoprenaline (10 micromol x L(-1)) increased all the three indices of hypertrophy, Ca2+ transients, calcineurin activity and the level of phospho-ERK1/2. The effects of isoprenaline were abolished by 1 micromol x L(-1) U50,488H in the absence but not in the presence of nor-binaltorphimine, a kappa-opioid receptor antagonist. The inhibitory effects of U50,488H were reproduced by cyclosporine-A, an inhibitor of calcineurin, U0126, the inhibitor of ERK1/2 and verapamil, a L-type Ca2+ channel antagonist. In addition, suppression of calcineurin activity by cyclosporine-A was associated with modest suppression of ERK1/2 phosphorylation. Meanwhile, suppression of ERK1/2 phosphorylation by U0126 was associated with modest suppression of calcineurin activity. In conclusion, the inhibitory effects of kappa-opioid receptor stimulation involved calcineurin and ERK1/2, and the two signaling pathways showed interaction in the mechanism of antihypertrophic effects afforded by kappa-opioid receptor stimulation.

Ionic mechanisms underlying cardiac toxicity of the organochloride solvent trichloromethane.

Trichloromethane (chloroform) is widely used for industrial chemical synthesis and also as an organic solvent in laboratories or ingredient of pesticides. Sudden death resulted from cardiac arrhythmias has been reported in clinic with acute trichloromethane intoxication. The present study was designed to investigate ionic mechanisms underlying arrhythmogenic effect (cardiac toxicity) of trichloromethane in isolated rat hearts and ventricular myocytes and HEK 293 cells stably expressing human Nav1.5, HCN2, or hERG channel using conventional electrophysiological approaches. It was found that trichloromethane (5mM) induced bradycardia and atrial-ventricular conduction blockade or ventricular fibrillation, and inhibited cardiac contractile function in isolated rat hearts. It shortened action potential duration (APD) in isolated rat ventricular myocytes, and increased the threshold current for triggering action potential, but had no effect on the inward rectifier K(+) current I(K1). However, trichloromethane significantly inhibited the L-type calcium current I(Ca.L) and the transient outward potassium current I(to) in a concentration-dependent manner (IC(50)s: 1.01 and 2.4mM, respectively). In HEK 293 cells stably expressing cardiac ion channel genes, trichloromethane reduced hNav1.5, HCN2, and hERG currents with IC(50)s of 8.2, 3.3, and 4.0mM, respectively. These results demonstrate for the first time that trichloromethane can induce bradycardia or ventricular fibrillation, and the arrhythmogenic effect of trichloromethane is related to the inhibition of multiple ionic currents including I(Ca.L), I(to), I(Na), HCN2, and hERG channels.

Intracerebroventricular administration of morphine confers remote cardioprotection--role of opioid receptors and calmodulin.

The current study aimed to delineate the mechanism of remote preconditioning by intracerebroventricular morphine (RMPC) against myocardial ischemia-reperfusion injury. Male Sprague-Dawley rats were given an intracerebroventricular morphine injection before myocardial ischemia and reperfusion injury. Ischemia-reperfusion injury was achieved by 30min of left coronary artery occlusion followed by 120min of reperfusion. The effects of remote preconditioning by intracerebroventricular morphine preconditioning were also determined upon selective blockade of the δ, κ or µ-opioid receptors, or calmodulin (CaM). The infarct size, as a percentage of the area at risk, was determined by 2,3,5-triphenyltetrazolium staining. Remote preconditioning by intracerebroventricular morphine reduced infarct size in the ischemic/reperfused myocardium, and the effect was abolished by the selective blockade of any one of the three δ, κ and µ opioid receptors or CaM. Furthermore, remote preconditioning by intracerebroventricular morphine increased the expression of CaM in the hippocampus and the plasma level of calcitonin gene-related peptide (CGRP). The results of the present study provide evidence that the cardioprotection of remote preconditioning by intracerebroventricular morphine involves not only all three types of opioid receptors in the central nervous system, but also CaM, which releases CGRP, one of the mediators of remote preconditioning.

Polyol pathway impairs the function of SERCA and RyR in ischemic-reperfused rat hearts by increasing oxidative modifications of these proteins.

A number of studies have shown that the polyol pathway, consisting of aldose reductase (AR) and sorbitol dehydrogenase (SDH), contributes to ischemia-reperfusion (I/R)-induced myocardial infarction due to depletion of ATP. In this report we show that the polyol pathway in I/R heart also contributes to the impairment of sacro/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and ryanodine receptor (RyR), two key players in Ca(2+) signaling that regulate cardiac contraction. Rat hearts were isolated and retrogradely perfused with either Krebs' buffer containing 1 microM AR inhibitor, zopolrestat, or 200 nM SDH inhibitor, CP-170,711, and challenged by 30 min of regional ischemia and 45 min of reperfusion. We found that post-ischemic contractile function of the isolated perfused hearts was improved by pharmacological inhibition of the polyol pathway. I/R-induced contractile dysfunction is most likely due to impairment in Ca(2+) signaling and the activities of SERCA and RyR. All these abnormalities were significantly ameliorated by treatment with ARI or SDI. We showed that the polyol pathway activities increase the level of peroxynitrite, which enhances the tyrosine nitration of SERCA and irreversibly modifies it to form SERCAC674-SO(3)H. This leads to reduced level of S-glutathiolated SERCA, contributing to its inactivation. The polyol pathway activities also deplete the level of GSH, leading to decreased active RyR, the S-glutathiolated RyR. Thus, in I/R heart, inhibition of polyol pathway improved the function of SERCA and RyR by protecting them from irreversible oxidation.

Protein kinase C mediates the effects of delta-opioid receptor stimulation on survival and apoptosis in neonatal cardiomyocytes cultured in serum-deprived condition.

The aims of the present study were to determine whether Delta opioid receptor (delta-OR) stimulation improved the survival of cardiomyocytes cultured in serum-deprived conditions, which impaired their growth. [D-Ala2, D-Leu5]-enkephalin (DADLE), a selective delta-OR agonist, at a concentration range of 0.1 micromol/L to 10 micromol/L for 48 h increased the viability of the cardiomyocyte under serum deprivation conditions. DADLE (0.1 micromol/L) also decreased the early cell apoptosis rate and the expression of Caspase-3. The effects of 0.1 micromol/L DADLE were abolished by 10 micromol x L(-1) naltrindole, a selective delta-OR antagonist, or by blockade of protein kinase C (PKC) with its blockers, 10 micromol x L(-1) GF109203X or 1 micromol/L staurosporine. Furthermore, 0.1 micromol x L(-1) DADLE increased the expression of PKC, an effect abrogated by 10 micromol x L(-1) naltrindole. The observations indicate that delta-OR stimulation improves the viability and reduces the apoptosis via PKC pathway in neonatal cardiomyocytes cultured in serum deprived conditions.

Effects of remifentanil on intracellular Ca(2+) and its transients induced by electrical stimulation and caffeine in rat ventricular myocytes.

BACKGROUND: Preconditioning with remifentanil confers cardioprotection. Since Ca(2+) overload is a precipitating factor of injury, we determined the effects of remefentanil on intracellular Ca(2+) ([Ca(2+)](i)) and its transients induced by electrical stimulation and caffeine, which reflects Ca(2+) handling by Ca(2+) handling proteins, in rat ventricular myocytes. METHODS: Freshly isolated adult male Sprague-Dawley rat myocytes were loaded with Fura-2/AM and [Ca](i) was determined by spectrofluorometry. Remifentanil at 0.1 - 1000 microg/L was administered. Ten minutes after administration, either 0.2 Hz electrical stimulation was applied or 10 mmol/L caffeine was added. The [Ca(2+)](i), and the amplitude, time resting and 50% decay (t(50)) of both transients induced by electrical stimulation (E [Ca(2+)](i)) and caffeine (C [Ca(2+)](i)) were determined. RESULTS: Remifentanil (0.1 - 1000.0 microg/L) decreased the [Ca(2+)](i) in a dose-dependent manner. It also decreased the amplitude of both transients dose-dependently. Furthermore, it increased the time to peak and t(50) of both transients dose-dependently. CONCLUSION: Remifentanil reduced the [Ca(2+)](i) and suppressed the transients induced by electrical stimulation and caffeine in rat ventricular myocytes.

Testosterone-augmented contractile responses to alpha1- and beta1-adrenoceptor stimulation are associated with increased activities of RyR, SERCA, and NCX in the heart.

We hypothesized that testosterone at physiological levels enhances cardiac contractile responses to stimulation of both alpha(1)- and beta(1)-adrenoceptors by increasing Ca(2+) release from the sarcoplasmic reticulum (SR) and speedier removal of Ca(2+) from cytosol via Ca(2+)-regulatory proteins. We first determined the left ventricular developed pressure, velocity of contraction and relaxation, and heart rate in perfused hearts isolated from control rats, orchiectomized rats, and orchiectomized rats without and with testosterone replacement (200 microg/100 g body wt) in the presence of norepinephrine (10(-7) M), the alpha(1)-adrenoceptor agonist phenylephrine (10(-6) M), or the nonselective beta-adrenoceptor agonist isoprenaline (10(-7) M) in the presence of 5 x 10(-7) M ICI-118,551, a beta(2)-adrenoceptor antagonist. Next, we determined the amplitudes of intracellular Ca(2+) concentration transients induced by electrical stimulation or caffeine, which represent, respectively, Ca(2+) release via the ryanodine receptor (RyR) or releasable Ca(2+) in the SR, in ventricular myocytes isolated from the three groups of rats. We also measured (45)Ca(2+) release via the RyR. We then determined the time to 50% decay of both transients, which represents, respectively, Ca(2+) reuptake by sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and removal via the sarcolemmal Na(+)/Ca(2+) exchanger (NCX). We correlated Ca(2+) removal from the cytosol with activities of SERCA and its regulator phospholamban as well as NCX. The results showed that testosterone at physiological levels enhanced positive inotropic and lusitropic responses to stimulation of alpha(1)- and beta(1)-adrenoceptors via the androgen receptor. The increased contractility and speedier relaxation were associated with increased Ca(2+) release via the RyR and faster Ca(2+) removal out of the cytosol via SERCA and NCX.

Intrathecal morphine preconditioning induces cardioprotection via activation of delta, kappa, and mu opioid receptors in rats.

BACKGROUND: Small doses of intrathecal morphine provide cardioprotection similar to that conferred by IV morphine. However, the extent of intrathecal morphine preconditioning (IT-MPC) relative to that resulting from ischemic preconditioning (IPC) is unknown. Further, it is uncertain whether IT-MPC is mediated by opioid receptor dependent pathways. In this study, we compared the extent of cardioprotection conferred by IT-MPC with IPC and investigated the role of opioid receptors in this effect. METHODS: Eighty anesthetized, open-chest, male Sprague-Dawley rats were assigned to 1 of 13 groups (n = 6-7) after successful intrathecal catheter placement. Rats in the IPC group were subjected to three 5-min cycles of myocardial ischemia (induced by occlusion of the left main coronary artery) interspersed with 5 min of reperfusion. After IPC, myocardial ischemia and reperfusion injury was induced by 30 min of left main coronary artery occlusion followed by 2 h of reperfusion. In the IT-MPC groups, the rats were given 3 consecutive 5 min intrathecal morphine infusions (0.03, 0.3, 3.0, or 30.0 microg/kg, respectively) interspersed with 5 min infusion-free periods, before myocardial ischemia reperfusion injury. In 2 other groups either 300microg/kg of IV morphine or 10 microL of intrathecal normal saline were given. The selective delta, kappa, and mu opioid receptor antagonists naltrindole, nor-binaltorphimine, and D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), respectively, were given to groups of animals receiving IT-MPC to evaluate the relative role of the specific opioid receptor subtypes in IT-MPC preconditioning. Myocardial infarct size (IS), as a percentage of the area at risk (AAR), was determined by 2,3,5-triphenyltetrazolium staining. RESULTS: Intrathecal morphine 0.3 to 30 microg/kg reduced myocardial IS compared with intrathecal normal saline control animals. The IS/AAR were 33% +/- 10% (0.3 microg/kg), 29% +/- 10% (3 microg/kg) and 29% +/- 16% (30 mug/kg), versus 53% +/- 8% for the control group (P < 0.01). The reduction in IS/AAR with IT-MPC was similar to that produced by IV morphine (33% +/- 6%, P = 0.84) and IPC (22% +/- 4%, P = 0.41). Myocardial preconditioning due to IT-MPC was attenuated by co-administration of any one of the opioid receptor antagonists (IT-MPC + naltrindole 50% +/- 9%, IT-MPC + nor binaltorphimine 43% +/- 6%, IT-MPC + CTOP 53% +/- 9%, P = 0.14). CONCLUSIONS: IT-MPC produced comparable cardioprotection to myocardial IPC and IV morphine. Myocardial preconditioning from intrathecal morphine seems to involve delta, kappa, and mu opioid receptors.

kappa-Opioid receptor stimulation inhibits augmentation of Ca(2+) transient and hypertrophy induced by isoprenaline in neonatal rat ventricular myocytes - Role of CaMKIIdelta(B).

We aimed to further define the pathway mediating the inhibitory effects of kappa-opioid receptor stimulation on Ca(2+) transients and hypertrophic responses to beta(1)-adrenoceptor stimulation. We determined the effects of trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamid methanesulfonate salt (U50,488H), a selective kappa-opioid receptor agonist, on the enhancement of spontaneous Ca(2+) transients and the induction of hypertrophy by isoprenaline, a beta-adrenoceptor agonist, in cultured neonatal ventricular myocytes. The results were compared with those found with KN93, a selective Ca(2+)/calmodulin-dependent kinase (CaMKII) inhibitor, propranolol, a beta-adrenoceptor antagonist, and verapamil, a L-type Ca(2+) channel antagonist. Hypertrophy of cardiomyocytes was characterized by increases in (i) total protein content; (ii) cell size; and (iii) [(3)H]leucine incorporation. 10 micromol/l isoprenaline increased all three parameters. We also determined the expression of nuclear CaMKIIdelta in response to U50,488H in the presence or absence of isoprenaline. To determine whether the effects of U50,488H were receptor-mediated, its effects were also measured following blockade of the kappa-opioid receptor with nor-binaltorphimine. kappa-Opioid receptor stimulation suppressed the stimulatory effect of isoprenaline on Ca(2+) transients and cardiac hypertrophy, as did KN93, propranalol and verapamil. U50,488H also suppressed the expression of nuclear CaMKIIdelta(B) in the presence, but not in the absence of isoprenaline. These results suggest that the inhibitory effect of kappa-opioid receptor stimulation on beta(1)-adrenoceptor stimulation may also involve CaMKIIdelta.

Polyol pathway mediates iron-induced oxidative injury in ischemic-reperfused rat heart.

Recent studies have shown that the polyol pathway is involved in ischemia-reperfusion (I/R)-induced myocardial infarction, but the mechanism is unclear. We previously found that lack of aldose reductase (AR), the first enzyme of the polyol pathway, attenuated the increase in transferrin (Tf) level in I/R brain, suggesting that AR contributes to iron-catalyzed free radical-induced damage. We therefore investigated if this mechanism occurs in I/R hearts. We found that inhibition of AR or sorbitol dehydrogenase (SDH), the second enzyme of the polyol pathway, both attenuated the I/R-mediated increases in HIF-1alpha, Tf, TfR, and intracellular iron content and reduced the I/R-induced infarct area of the heart. Further, administration of niacin, which replenishes NAD+, the cofactor for SDH, also normalized TfR and HIF-1alpha levels in I/R hearts. These results suggest that during I/R polyol pathway activity increases the cytosolic NADH/NAD+ ratio. This activates HIF-1alpha that induces the expression of TfR, which in turn increases Tf uptake and iron accumulation and exacerbates oxidative damage that increases the lipid peroxidation. This was confirmed by the fact that administration of the iron chelator deferoxamine attenuated the I/R-induced myocardial infarction.

2-hydroxyoleic acid affects cardiomyocyte [Ca2+]i transient and contractility in a region-dependent manner.

Monounsaturated fatty acids such as oleic acid are cardioprotective, modify the physicochemical properties of cardiomyocyte membranes, and affect the electrical stability of these cells by regulating the conductance of ion channels. We have designed a nonhydrolysable oleic acid derivative, 2-hydroxyoleic acid (2-OHOA), which regulates membrane lipid structure and cell signaling, resulting in beneficial cardiovascular effects. We previously demonstrated that 2-OHOA induces PKA activation and PKCalpha translocation to the membrane; both pathways are thought to regulate transient outward K(+) current (I(to)) depending on the stimulus and the species used. This study was designed to investigate the effect of 2-OHOA on isolated cardiomyocytes. We examined the dose- and time-dependent effect of 2-OHOA on cytosolic Ca(2+) concentration ([Ca(2+)](i)) transient and contraction of myocytes isolated from different parts of the rat ventricular myocardium. Although this drug had no effect on [Ca(2+)](i) transient and cell shortening in myocytes isolated from the septum, it increased (up to 95%) [Ca(2+)](i) transient and cell shortening in subpopulations of myocytes from the right and left ventricles. The pattern of the effects of 2-OHOA was similar to that observed following the application of the I(to) blocker 4-aminopyridine, suggesting that the drug may act on this channel. Unlike the effect of 2-OHOA on [Ca(2+)](i) transient and cell shortening, PKCalpha translocation to membranes was not region specific. Thus 2-OHOA-induced effects on [Ca(2+)](i) transients and cell shortening are likely related to reductions in I(to) function, but PKCalpha translocation does not seem to play a role. The present results indicate that 2-OHOA selectively increases myocyte inotropic responsiveness, which could underlie its beneficial cardiovascular effects.

delta-Opioid receptor stimulation enhances the growth of neonatal rat ventricular myocytes via the extracellular signal-regulated kinase pathway.

1. The aims of the present study were to determine whether delta-opioid receptor stimulation enhanced proliferation of and to investigate the role of the extracellular signal-regulated kinase (ERK) pathway in ventricular myocytes from neonatal rats. 2. At concentratins ranging from 10 nmol/L to 10 micromol/L, [D-Ala2,D-Leu5]enkephalin (DADLE) concentration-dependently promoted myocardial growth and DNA synthesis and altered the cytoskeleton. 3. At 1 micromol/L, DADLE also increased the expression and phosphorylation of ERK. 4. These effects of 1 micromol/L DADLE were abolished by 10 micromol/L naltrindole, a selective delta-opioid receptor antagonist, 10 nmol/L U0126, a selective ERK antagonist, 1 micromol/L staurosporine, an inhibitor of protein kinase (PK) C, and 100 micromol/L Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt hydrate (Rp-cAMPS), an inhibitor of PKA. 5. In conclusion, delta-opioid receptor stimulation enhances the proliferation and development of the ventricular myocytes of neonatal rats. The ERK pathway and related signalling mechanisms, namely PKC and PKA, are involved.

Involvement of voltage-gated K+ and Na+ channels in gastric epithelial cell migration.

Epithelial cell migration plays an important role in gastrointestinal mucosal repair. We previously reported that multiple functional ion channels, including a Ba(2+)-sensitive K(+) inward rectifier K(ir)1.2, 4-aminopyridine (4-AP)-sensitive voltage-gated K(+) channels K(v)1.1, K(v)1.6 and K(v)2.1, and a nifedipine-sensitive, tetrodotoxin (TTX)-insensitive voltage-gated Na(+) channel Na(v)1.5 were expressed in a non-transformed rat gastric epithelial cell line (RGM-1). In the present study, we further investigated whether these ion channels are involved in the modulation of gastric epithelial cell migration. Cell migration was determined by monolayer wound healing assay. Results showed that blockade of K(v) with 4-AP or Na(v)1.5 with nifedipine inhibited RGM-1 cell migration in the absence or presence of epidermal growth factor (EGF), which effectively stimulated RGM-1 cell migration. Moreover, high concentration of TTX mimicked the action of nifedipine, suggesting that the action of nifedipine was mediated through specific blockade of Na(v)1.5. In contrast, inhibition of K(ir)1.2 with Ba(2+), either in basal or EGF-stimulated condition, had no effect on RGM-1 cell migration. In conclusion, the present study demonstrates for the first time that voltage-gated K(+) and Na(+) channels are involved in the modulation of gastric epithelial cell migration.

Cardioprotection by the female sex hormone -- interaction with the beta1-adrenoceptor and its signaling pathways.

Estrogen is a steroid and the predominant female sex hormone in the body. Ovariectomised (OVX) adult female rats exhibit greater myocardial injury compared to the sham rats following ischemic insult in the presence of beta-adrenoceptor stimulation. Estrogen replacement restores the response of OVX female rats to ischemic/beta-adrenoceptor stimulation to that of normal female rats, providing evidence for a cardioprotective role of estrogen during ischemic insult. The protective effect is due to down-regulation of the beta(1)-adrenoceptor. There is also evidence that estrogen suppresses the expression and activity of protein kinase A (PKA), a second messenger of the G(s) protein/adenylyl cyclase/cAMP/PKA pathway which ultimately influences contractile function. There is also preliminary evidence that estrogen may suppress the activity of Ca(2+)/calmodulin kinase II deltac isoform (CaMKII-deltac), another downstream second messenger of the beta(1)-adrenoceptor pathway, which is involved in PKA-independent cell apoptosis. Acute administration of estrogen at physiological level could inhibit myocardial beta(1)-adrenoceptor and attenuate Ca(2+) influx independent of the estrogen receptor. In addition, brain studies also show estrogen inhibits the activities activated by the beta-adrenoceptor in brain regions responsible for the regulation of arterial blood pressure. Thus, it can be appreciated that the interaction between estrogen and the beta(1)-adrenoceptor and its signaling pathways is a complex one. Estrogen plays an important role not only in reproduction but also in other regulatory functions such as cardioprotection.

Testosterone and cardioprotection against myocardial ischemia.

Male gender is a risk factor for cardiovascular diseases. Testosterone being the main male sex hormone is therefore believed to be responsible for the deleterious effect of the male. However, there are recent studies showing that testosterone level is lower in patients with ischemic heart diseases, and testosterone treatment alleviates the symptoms. Earlier studies showed that functional androgen receptors are present in the heart and that testosterone acts directly at the myocardium. There is increasing evidence to suggest testosterone confers cardioprotection by direct action on the myocardium. Here, we review the recent literature on association between testosterone and myocardial ischemia in males, and the signal transduction mechanisms that mediate the action of testosterone in the heart. The studies reviewed in this article provide evidence that testosterone may confer protection via a varieties of mechanisms, which may be both genomic and non-genomic. Further studies are warranted to further delineate the integration of signaling mechanisms and to explore the possibility of using testosterone in the aging male population with ischemic heart diseases.

Chronic intermittent hypoxia alters Ca2+ handling in rat cardiomyocytes by augmented Na+/Ca2+ exchange and ryanodine receptor activities in ischemia-reperfusion.

This study examined Ca(2+) handling mechanisms involved in cardioprotection induced by chronic intermittent hypoxia (CIH) against ischemia-reperfusion (I/R) injury. Adult male Sprague-Dawley rats were exposed to 10% inspired O(2) continuously for 6 h daily from 3, 7, and 14 days. In isolated perfused hearts subjected to I/R, CIH-induced cardioprotection was most significant in the 7-day group with less infarct size and lactate dehydrogenase release, compared with the normoxic group. The I/R-induced alterations in diastolic Ca(2+) level, amplitude, time-to-peak, and the decay time of both electrically and caffeine-induced Ca(2+) transients measured by spectrofluorometry in isolated ventricular myocytes of the 7-day CIH group were less than that of the normoxic group, suggesting an involvement of altered Ca(2+) handling of the sarcoplasmic reticulum (SR) and sarcolemma. We further determined the protein expression and activity of (45)Ca(2+) flux of SR-Ca(2+)-ATPase, ryanodine receptor (RyR) and sarcolemmal Na(+)/Ca(2+) exchange (NCX) in ventricular myocytes from the CIH and normoxic groups before and during I/R. There were no changes in expression levels of the Ca(2+)-handling proteins but significant increases in the RyR and NCX activities were remarkable during I/R in the CIH but not the normoxic group. The augmented RyR and NCX activities were abolished, respectively, by PKA inhibitor (0.5 microM KT5720 or 0.5 microM PKI(14-22)) and PKC inhibitor (5 microM chelerythrine chloride or 0.2 microM calphostin C) but not by Ca(2+)/calmodulin-dependent protein kinase II inhibitor KN-93 (1 microM). Thus, CIH confers cardioprotection against I/R injury in rat cardiomyocytes by altered Ca(2+) handling with augmented RyR and NCX activities via protein kinase activation.

Role of reverse mode Na+/Ca2+ exchanger in the cardioprotection of metabolic inhibition preconditioning in rat ventricular myocytes.

This study determined the role of the reverse mode Na(+)/Ca(2+) exchanger (NCX) in cardioprotection of metabolic inhibition preconditioning in isolated ventricular myocyctes. Activity of the reverse mode NCX was assessed by changes of [Ca(2+)](i) upon withdrawal of extracellular Na(+). [Ca(2+)](i) was measured by spectrofluorometry, using Fura-2 as Ca(2+) indicator. The amplitude of contraction and exclusion of trypan blue by myocytes served as indices of contractile function and viability, respectively. Firstly, NCX activity significantly decreased during simulated reperfusion after severe metabolic inhibition (index ischaemia) in myocytes subjected to metabolic inhibition preconditioning. This inhibitory effect on NCX activity correlated with the enhancing effect of metabolic inhibition preconditioning on cell viability following ischaemic insult. Treatment myocytes with E4031, an activator of reverse mode NCX, during index ischaemia and reperfusion attenuated the enhancing effects of metabolic inhibition preconditioning on cell contraction and viability. Secondly, NCX activity was significantly higher at the end of metabolic inhibition preconditioning. More importantly, E4031 pretreatment mimicked the beneficial effects of metabolic inhibition preconditioning in myocytes and ischaemic preconditioning in the isolated perfused heart, respectively, and these effects were abolished by KB-R7943, an inhibitor of reverse mode NCX. The results indicate that increased reverse mode NCX activity during preconditioning triggered cardioprotection, and reduced reverse mode NCX activity during reperfusion after index ischaemia conferred cardioprotection.

kappa-opioid receptor stimulation inhibits cardiac hypertrophy induced by beta1-adrenoceptor stimulation in the rat.

To test the hypothesis that kappa-opioid receptor stimulation inhibits cardiac hypertrophy induced by beta1-adrenoceptor stimulation, we determined the effects of trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methanesulfonate salt (U50,488H), a selective kappa-opioid receptor agonist, on cardiac hypertrophy induced by isoprenaline, a selective beta-adrenoceptor agonist, in neonatal ventricular myocytes upon blockade of beta2-adrenoceptor. Hypertrophy of cardiomyocytes was determined by increases in (i) total protein content; (ii) [3H]leucine incorporation; and iii) cell size. 10 micromol/l isoprenaline increased all three parameters. The effects were abolished by 2 micromol/l propranolol, a beta-adrenergic receptor antagonist, or 300 nmol/l CGP20712A, a beta1-adrenoceptor antagonist, but not by 100 nmol/l ICI118,551, a beta2-adrenoceptor antagonist. The effects were also abolished by Rp-cAMPs 100 micromol/l, a protein kinase A inhibitor and not by pertussis toxin 5 mg/l. The effects of isoprenaline in the presence or absence of ICI118,551 were also abolished by 1 micromol/l U50,488H. The inhibitory effects of U50,488H were abolished by 1 micromol/l nor-binaltorphimine, a selective kappa-opioid receptor antagonist. U50,488H also abolished the increases in the amplitude and frequency of the spontaneous intracellular Ca2+ transient induced by 10 micromol/l isoprenaline in the presence or absence of ICI118,551, an effect also abolished by nor-binaltorphimine. In conclusion the results show that kappa-opioid receptor stimulation abolished both the cardiac hypertrophy and enhanced amplitude and frequency of the spontaneous intracellular Ca2+ transient induced by beta1-adrenoceptor stimulation.

Altered Ca(2+) handling by ryanodine receptor and Na(+)-Ca(2+) exchange in the heart from ovariectomized rats: role of protein kinase A.

Our previous study has demonstrated that ovariectomy (Ovx) significantly increased the left ventricular developed pressure (LVDP) and the maximal rate of developed pressure over time (+/-dP/dt(max)) in the isolated perfused rat heart and the effects were reversed by female sex hormone replacement. In the present investigation, we studied the effects of Ovx for 6 wk on Ca(2+) homeostasis that determines the contractile function. Particular emphasis was given to Ca(2+) handling by ryanodine receptor (RyR) and Na(+)-Ca(2+) exchange (NCX). (45)Ca(2+) fluxes via the RyR, NCX, and Ca(2+)-ATPase (SERCA) were compared with their expression in myocytes from Ovx rats with and without estrogen replacement. Furthermore, we correlated the handling of Ca(2+) by these Ca(2+) handling proteins with the overall Ca(2+) homeostasis by determining the Ca(2+) transients induced by electrical stimulation and caffeine, which reveals the dynamic changes of cytosolic Ca(2+) concentration ([Ca(2+)](i)) in the heart. In addition, we determined the expression and contribution of protein kinase A (PKA) to the regulation of the aforementioned Ca(2+) handling proteins in Ovx rats. It was found that after Ovx there were 1) increased Ca(2+) fluxes via RyR and NCX, which were reversed not only by estrogen replacement, but more importantly by blockade of PKA; 2) an increased expression of PKA; and 3) no increase in expression of NCX and SERCA. We suggest that hyperactivities of RyR and NCX are a result of upregulation of PKA. The increased release of Ca(2+) through RyR and removal of Ca(2+) by NCX are believed to be responsible for the greater contractility and faster relaxation after Ovx.