Rac1 activation induces tumour necrosis factor‐α expression and cardiac dysfunction in endotoxemia

Induction of tumour necrosis factor‐α (TNF‐α) expression leads to myocardial depression during sepsis. However, the underlying molecular mechanisms are not fully understood. The aim of this study was to investigate the role of Rac1 in TNF‐α expression and cardiac dysfunction during endotoxemia and to determine the involvement of phosphoinositide‐3 kinase (PI3K) in lipopolysaccharide (LPS)‐induced Rac1 activation. Our results showed that LPS‐induced Rac1 activation and TNF‐α expression in cultured neonatal mouse cardiomyocytes. The response was inhibited in Rac1 deficient cardiomyocytes or by a dominant‐negative Rac1 (Rac1N17). To determine whether PI3K regulates Rac1 activation, cardiomyocytes were treated with LY294002, a PI3K selective inhibitor. Treatment with LY294002 decreased Rac1 activity as well as TNF‐α expression stimulated by LPS. Furthermore, inhibition of PI3K and Rac1 activity decreased LPS‐induced superoxide generation which was associated with a significant reduction in ERK1/2 phosphorylation. To investigate the role of Rac1 in myocardial depression during endotoxemia in vivo, wild‐type and cardiomyocyte‐specific Rac1 deficient mice were treated with LPS (2 mg/kg, i.p.). Deficiency in Rac1 significantly decreased myocardial TNF‐α expression and improved cardiac function during endotoxemia. We conclude that PI3K‐mediated Rac1 activation is required for induction of TNF‐α expression in cardiomyocytes and cardiac dysfunction during endotoxemia. The effect of Rac1 on TNF‐α expression seems to be mediated by increased NADPH oxidase activity and ERK1/2 phosphorylation.     

Functional abnormalities in iPSC‐derived cardiomyocytes generated from CPVT1 and CPVT2 patients carrying ryanodine or calsequestrin mutations

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia characterized by syncope and sudden death occurring during exercise or acute emotion. CPVT is caused by abnormal intracellular Ca²⁺ handling resulting from mutations in the RyR2 or CASQ2 genes. Because CASQ2 and RyR2 are involved in different aspects of the excitation‐contraction coupling process, we hypothesized that these mutations are associated with different functional and intracellular Ca²⁺ abnormalities. To test the hypothesis we generated induced Pluripotent Stem Cell‐derived cardiomyocytes (iPSC‐CM) from CPVT1 and CPVT2 patients carrying the RyR2^R420Q and CASQ2^D307H mutations, respectively, and investigated in CPVT1 and CPVT2 iPSC‐CM (compared to control): (i) The ultrastructural features; (ii) the effects of isoproterenol, caffeine and ryanodine on the [Ca²⁺]_i transient characteristics. Our major findings were: (i) Ultrastructurally, CASQ2 and RyR2 mutated cardiomyocytes were less developed than control cardiomyocytes. (ii) While in control iPSC‐CM isoproterenol caused positive inotropic and lusitropic effects, in the mutated cardiomyocytes isoproterenol was either ineffective, caused arrhythmias, or markedly increased diastolic [Ca²⁺]_i. Importantly, positive inotropic and lusitropic effects were not induced in mutated cardiomyocytes. (iii) The effects of caffeine and ryanodine in mutated cardiomyocytes differed from control cardiomyocytes. Our results show that iPSC‐CM are useful for investigating the similarities/differences in the pathophysiological consequences of RyR2 versus CASQ2 mutations underlying CPVT1 and CPVT2 syndromes.     

Protective effects of Araloside C against myocardial ischaemia/reperfusion injury: potential involvement of heat shock protein 90

The present study was designed to investigate whether Araloside C, one of the major triterpenoid compounds isolated from Aralia elata known to be cardioprotective, can improve heart function following ischaemia/reperfusion (I/R) injury and elucidate its underlying mechanisms. We observed that Araloside C concentration‐dependently improved cardiac function and depressed oxidative stress induced by I/R. Similar protection was confirmed in isolated cardiomyocytes characterized by maintaining Ca²⁺ transients and cell shortening against I/R. Moreover, the potential targets of Araloside C were predicted using the DDI‐CPI server and Discovery Studio software. Molecular docking analysis revealed that Araloside C could be stably docked into the ATP/ADP‐binding domain of the heat shock protein 90 (Hsp90) protein via the formation of hydrogen bonds. The binding affinity of Hsp90 to Araloside C was detected using nanopore optical interferometry and yielded KD values of 29 μM. Araloside C also up‐regulated the expression levels of Hsp90 and improved cell viability in hypoxia/reoxygenation‐treated H9c2 cardiomyocytes, whereas the addition of 17‐AAG, a pharmacologic inhibitor of Hsp90, attenuated Araloside C‐induced cardioprotective effect. These findings reveal that Araloside C can efficiently attenuate myocardial I/R injury by reducing I/R‐induced oxidative stress and [Ca²⁺]_i overload, which was possibly related to its binding to the Hsp90 protein.     

A knock-in mouse model of N-terminal R420W mutation of cardiac ryanodine receptor exhibits arrhythmogenesis with abnormal calcium dynamics in cardiomyocytes

Cardiac ryanodine receptor gene (RyR2) mutations cause fatal arrhythmogenic diseases such as catecholaminergic polymorphic ventricular tachycardia and arrhythmogenic right ventricular cardiomyopathy. The N-terminal region of RyR2 is one of the hot spots for mutations. In this study, we investigated cardiac phenotypes of a knock-in mouse model carrying R420W mutation of RyR2. The N-terminal R420W mutation has already been found in juvenile sudden death cadavers of unrelated families. The depolarization-induced Ca²⁺ transient amplitude was significantly lower in cardiomyocytes from RyR2^R420W/R420W mice compared with wild-type mice. The time to peak of the Ca²⁺ transient was significantly increased in RyR2^R420W/R420W mice. Furthermore, the prolonged decay time from the peak of the Ca²⁺ transient was detected in RyR2^R420W/R420W mice. ECG telemetry revealed that various types of arrhythmias were induced in RyR2^R420W/R420W mice in response to administration of caffeine and adrenaline. The mutant mice showed high occurrences of arrhythmias in response to heart stimulants compared with wild-type mice. These findings suggest that R420W mutation impairs depolarization-induced Ca²⁺ oscillation in cardiomyocytes, which possibly results in sudden death due to stress-induced arrhythmias.     

Role of myocardial viscoelasticity in disturbances of electrical and mechanical activity in calcium overloaded cardiomyocytes: mathematical modeling

Cardiomyocyte Ca²⁺ overload is closely linked to cardiac arrhythmias. We have earlier shown in a mathematical model that myocardium mechanical activity may contribute to rhythm disturbances induced by Ca²⁺ overload in cardiomyocytes with reduced Na⁺-K⁺ pump work (Sulman et al., 2008). The same model is used here to address possible contribution of the passive mechanical properties of cardiac muscle (i.e. myocardial viscous and elastic properties) to the arrhythmogenesis. In a series of contractions at regular pacing rate of 75 beats/min a model with higher viscosity demonstrated essentially earlier appearance of extrasystoles due to a faster cardiomyocyte Ca²⁺ loading up to a level triggering spontaneous Ca²⁺ releases from the sarcoplasmic reticulum. The model predicts that myocardial elasticity also may affect arrhythmogenesis in cardiomyocytes overloaded with Ca²⁺. Contribution of the mechanical properties of the myocardial tissue to the arrhythmia has been analyzed for wide ranges of both viscosity and elasticity coefficients. The results suggest that myocardial viscoelastic properties may be a factor affecting Ca²⁺ handling in cardiomyocytes and contributing to cardiac mechano-electric feedback in arrhythmogenesis.     

Distinctive electrophysiological characteristics of right ventricular out‐flow tract cardiomyocytes

Ventricular arrhythmias commonly originate from the right ventricular out‐flow tract (RVOT). However, the electrophysiological characteristics and Ca²⁺ homoeostasis of RVOT cardiomyocytes remain unclear. Whole‐cell patch clamp and indo‐1 fluorometric ratio techniques were used to investigate action potentials, Ca²⁺ homoeostasis and ionic currents in isolated cardiomyocytes from the rabbit RVOT and right ventricular apex (RVA). Conventional microelectrodes were used to record the electrical activity before and after (KN‐93, a Ca²⁺/calmodulin‐dependent kinase II inhibitor, or ranolazine, a late sodium current inhibitor) treatment in RVOT and RVA tissue preparations under electrical pacing and ouabain (Na⁺/K⁺ ATPase inhibitor) administration. In contrast to RVA cardiomyocytes, RVOT cardiomyocytes were characterized by longer action potential duration measured at 90% and 50% repolarization, larger Ca²⁺ transients, higher Ca²⁺ stores, higher late Na⁺ and transient outward K⁺ currents, but smaller delayed rectifier K⁺, L‐type Ca²⁺ currents and Na⁺‐Ca²⁺ exchanger currents. RVOT cardiomyocytes showed significantly more pacing‐induced delayed afterdepolarizations (22% versus 0%, P < 0.05) and ouabain‐induced ventricular arrhythmias (94% versus 61%, P < 0.05) than RVA cardiomyocytes. Consistently, it took longer time (9 ± 1 versus 4 ± 1 min., P < 0.05) to eliminate ouabain‐induced ventricular arrhythmias after application of KN‐93 (but not ranolazine) in the RVOT in comparison with the RVA. These results indicate that RVOT cardiomyocytes have distinct electrophysiological characteristics with longer AP duration and greater Ca²⁺ content, which could contribute to the high RVOT arrhythmogenic activity.     

Flecainide ameliorates arrhythmogenicity through NCX flux in Andersen-Tawil syndrome-iPS cell-derived cardiomyocytes

Andersen-Tawil syndrome (ATS) is a rare inherited channelopathy. The cardiac phenotype in ATS is typified by a prominent U wave and ventricular arrhythmia. An effective treatment for this disease remains to be established. We reprogrammed somatic cells from three ATS patients to generate induced pluripotent stem cells (iPSCs). Multi-electrode arrays (MEAs) were used to record extracellular electrograms of iPSC-derived cardiomyocytes, revealing strong arrhythmic events in the ATS-iPSC-derived cardiomyocytes. Ca²⁺ imaging of cells loaded with the Ca²⁺ indicator Fluo-4 enabled us to examine intracellular Ca²⁺ handling properties, and we found a significantly higher incidence of irregular Ca²⁺ release in the ATS-iPSC-derived cardiomyocytes than in control-iPSC-derived cardiomyocytes. Drug testing using ATS-iPSC-derived cardiomyocytes further revealed that antiarrhythmic agent, flecainide, but not the sodium channel blocker, pilsicainide, significantly suppressed these irregular Ca²⁺ release and arrhythmic events, suggesting that flecainide's effect in these cardiac cells was not via sodium channels blocking. A reverse-mode Na^+/Ca²⁺exchanger (NCX) inhibitor, KB-R7943, was also found to suppress the irregular Ca²⁺ release, and whole-cell voltage clamping of isolated guinea-pig cardiac ventricular myocytes confirmed that flecainide could directly affect the NCX current (I_NCX). ATS-iPSC-derived cardiomyocytes recapitulate abnormal electrophysiological phenotypes and flecainide suppresses the arrhythmic events through the modulation of I_NCX.     

Roles of I(f) and intracellular Ca2+ release in spontaneous activity of ventricular cardiomyocytes during murine embryonic development

In recent years, the contribution of I(f), an important pacemaker current, and intracellular Ca²⁺ release (ICR) from sarcoplasmic reticulum to pacemaking and arrhythmia has been intensively studied. However, their functional roles in embryonic heart remain uncertain. Using patch clamp, Ca²⁺ imaging, and RT‐PCR, we found that I(f) regulated the firing rate in early and late stage embryonic ventricular cells, as ivabradine (30 µM), a specific blocker of I(f), slowed down action potential (AP) frequency. This inhibitory effect was even stronger in late stage cells, though I(f) was down‐regulated. In contrast to I(f), ICR was found to be indispensable for the occurrence of APs in ventricular cells of different stages, because abolishment of ICR with ryanodine and 2‐aminoethoxydiphenyl borate (2‐APB), specific blockers of ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs), completely abolished APs. In addition, we noticed that RyR‐ and IP3R‐mediated ICR coexisted in early‐stage ventricular cells and RyRs functionally dominated. While at late stage RyRs, but not IP3Rs, mediated ICR. In both early and late stage ventricular cells, Na‐Ca exchanger current (I_Na/Ca) mediated ICR‐triggered depolarization of membrane potential and resulted in the initiation of APs. We also observed that different from I(f), which presented as the substantial component of the earlier diastolic depolarization current, application of ryanodine, and/or 2‐APB slowed the late phase of diastolic depolarization. Thus, we conclude that in murine embryonic ventricular cells I(f) regulates firing rate, while RyRs and IP3Rs (early stage) or RyRs (late stage)‐mediated ICR determines the occurrence of APs. J. Cell. Biochem. 114: 1852–1862, 2013. © 2013 Wiley Periodicals, Inc.     

The effect of Sirt1 deficiency on Ca2+ and Na+ regulation in mouse ventricular myocytes

This study addressed the hypothesis that cardiac Sirtuin 1 (Sirt1) deficiency alters cardiomyocyte Ca²⁺ and Na⁺ regulation, leading to cardiac dysfunction and arrhythmogenesis. We used mice with cardiac‐specific Sirt1 knockout (Sirt1^?/?). Sirt1^flox/flox mice were served as control. Sirt1^?/? mice showed impaired cardiac ejection fraction with increased ventricular spontaneous activity and burst firing compared with those in control mice. The arrhythmic events were suppressed by KN93 and ranolazine. Reduction in Ca²⁺ transient amplitudes and sarcoplasmic reticulum (SR) Ca²⁺ stores, and increased SR Ca²⁺ leak were shown in the Sirt1^?/? mice. Electrophysiological measurements were performed using patch‐clamp method. While L‐type Ca²⁺ current (I_{Ca, L}) was smaller in Sirt1^?/? myocytes, reverse‐mode Na⁺/Ca²⁺ exchanger (NCX) current was larger compared with those in control myocytes. Late Na⁺ current (I_{Na, L}) was enhanced in the Sirt1^?/? mice, alongside with elevated cytosolic Na⁺ level. Increased cytosolic and mitochondrial reactive oxygen species (ROS) were shown in Sirt1^?/? mice. Sirt1^?/? cardiomyocytes showed down‐regulation of L‐type Ca²⁺ channel α1c subunit (Cav1.2) and sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 2a (SERCA2a), but up‐regulation of Ca²⁺/calmodulin‐dependent protein kinase II and NCX. In conclusions, these findings suggest that deficiency of Sirt1 impairs the regulation of intracellular Ca²⁺ and Na⁺ in cardiomyocytes, thereby provoking cardiac dysfunction and arrhythmogenesis.     

Taxol,a microtubule stabilizer,prevents ischemic ventricular arrhythmias in rats

Microtubule integrity is important in cardio‐protection, and microtubule disruption has been implicated in the response to ischemia in cardiac myocytes. However, the effects of Taxol, a common microtubule stabilizer, are still unknown in ischemic ventricular arrhythmias. The arrhythmia model was established in isolated rat hearts by regional ischemia, and myocardial infarction model by ischemia/reperfusion. Microtubule structure was immunohistochemically measured. The potential mechanisms were studied by measuring reactive oxygen species (ROS), activities of oxidative enzymes, intracellular calcium concentration ([Ca²⁺]_i) and Ca²⁺ transients by using fluorometric determination, spectrophotometric assays and Fura‐2‐AM and Fluo‐3‐AM, respectively. The expression and activity of sarcoplasmic reticulum Ca²⁺‐ATPase (SERCA2a) was also examined using real‐time polymerase chain reaction, Western blot and pyruvate/Nicotinamide adenine dinucleotide‐coupled reaction. Our data showed that Taxol (0.1, 0.3 and 1 μM) effectively reduced the number of ventricular premature beats and the incidence and duration of ventricular tachycardia. The infarct size was also significantly reduced by Taxol (1 μM). At the same time, Taxol preserved the microtubule structure, increased the activity of mitochondrial electron transport chain complexes I and III, reduced ROS levels, decreased the rise in [Ca²⁺]_i and preserved the amplitude and decay times of Ca²⁺ transients during ischemia. In addition, SERCA2a activity was preserved by Taxol during ischemia. In summary, Taxol prevents ischemic ventricular arrhythmias likely through ameliorating abnormal calcium homeostasis and decreasing the level of ROS. This study presents evidence that Taxol may be a potential novel therapy for ischemic ventricular arrhythmias.     

Heart-specific Deletion of CnB1 Reveals Multiple Mechanisms Whereby Calcineurin Regulates Cardiac Growth and Function

Calcineurin is a protein phosphatase that is uniquely regulated by sustained increases in intracellular Ca²⁺ following signal transduction events. Calcineurin controls cellular proliferation, differentiation, apoptosis, and inducible gene expression following stress and neuroendocrine stimulation. In the adult heart, calcineurin regulates hypertrophic growth of cardiomyocytes in response to pathologic insults that are associated with altered Ca²⁺ handling. Here we determined that calcineurin signaling is directly linked to the proper control of cardiac contractility, rhythm, and the expression of Ca²⁺-handling genes in the heart. Our approach involved a cardiomyocyte-specific deletion using a CnB1-LoxP-targeted allele in mice and three different cardiac-expressing Cre alleles/transgenes. Deletion of calcineurin with the Nkx2.5-Cre knock-in allele resulted in lethality at 1 day after birth due to altered right ventricular morphogenesis, reduced ventricular trabeculation, septal defects, and valvular overgrowth. Slightly later deletion of calcineurin with the α-myosin heavy chain Cre transgene resulted in lethality in early mid adulthood that was characterized by substantial reductions in cardiac contractility, severe arrhythmia, and reduced myocyte content in the heart. Young calcineurin heart-deleted mice died suddenly after pressure overload stimulation or neuroendocrine agonist infusion, and telemetric monitoring of older mice showed arrhythmia leading to sudden death. Mechanistically, loss of calcineurin reduced expression of key Ca²⁺-handling genes that likely lead to arrhythmia and reduced contractility. Loss of calcineurin also directly impacted cellular proliferation in the postnatal developing heart. These results reveal multiple mechanisms whereby calcineurin regulates cardiac development and myocyte contractility.     

Arrhythmogenic Calmodulin Mutations Affect the Activation and Termination of Cardiac Ryanodine Receptor-mediated Ca2+ Release

The intracellular Ca²⁺ sensor calmodulin (CaM) regulates the cardiac Ca²⁺ release channel/ryanodine receptor 2 (RyR2), and mutations in CaM cause arrhythmias such as catecholaminergic polymorphic ventricular tachycardia (CPVT) and long QT syndrome. Here, we investigated the effect of CaM mutations causing CPVT (N53I), long QT syndrome (D95V and D129G), or both (CaM N97S) on RyR2-mediated Ca²⁺ release. All mutations increased Ca²⁺ release and rendered RyR2 more susceptible to store overload-induced Ca²⁺ release (SOICR) by lowering the threshold of store Ca²⁺ content at which SOICR occurred and the threshold at which SOICR terminated. To obtain mechanistic insights, we investigated the Ca²⁺ binding of the N- and C-terminal domains (N- and C-domain) of CaM in the presence of a peptide corresponding to the CaM-binding domain of RyR2. The N53I mutation decreased the affinity of Ca²⁺ binding to the N-domain of CaM, relative to CaM WT, but did not affect the C-domain. Conversely, mutations N97S, D95V, and D129G had little or no effect on Ca²⁺ binding to the N-domain but markedly decreased the affinity of the C-domain for Ca²⁺. These results suggest that mutations D95V, N97S, and D129G alter the interaction between CaM and the CaMBD and thus RyR2 regulation. Because the N53I mutation minimally affected Ca²⁺ binding to the C-domain, it must cause aberrant regulation via a different mechanism. These results support aberrant RyR2 regulation as the disease mechanism for CPVT associated with CaM mutations and shows that CaM mutations not associated with CPVT can also affect RyR2. A model for the CaM-RyR2 interaction, where the Ca²⁺-saturated C-domain is constitutively bound to RyR2 and the N-domain senses increases in Ca²⁺ concentration, is proposed.     

Identification of loss-of-function RyR2 mutations associated with idiopathic ventricular fibrillation and sudden death

Mutations in cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most CPVT RyR2 mutations characterized are gain-of-function (GOF), indicating enhanced RyR2 function as a major cause of CPVT. Loss-of-function (LOF) RyR2 mutations have also been identified and are linked to a distinct entity of cardiac arrhythmia termed RyR2 Ca²⁺ release deficiency syndrome (CRDS). Exercise stress testing (EST) is routinely used to diagnose CPVT, but it is ineffective for CRDS. There is currently no effective diagnostic tool for CRDS in humans. An alternative strategy to assess the risk for CRDS is to directly determine the functional impact of the associated RyR2 mutations. To this end, we have functionally screened 18 RyR2 mutations that are associated with idiopathic ventricular fibrillation (IVF) or sudden death. We found two additional RyR2 LOF mutations E4146K and G4935R. The E4146K mutation markedly suppressed caffeine activation of RyR2 and abolished store overload induced Ca²⁺ release (SOICR) in human embryonic kidney 293 (HEK293) cells. E4146K also severely reduced cytosolic Ca²⁺ activation and abolished luminal Ca²⁺ activation of single RyR2 channels. The G4935R mutation completely abolished caffeine activation of and [³H]ryanodine binding to RyR2. Co-expression studies showed that the G4935R mutation exerted dominant negative impact on the RyR2 wildtype (WT) channel. Interestingly, the RyR2-G4935R mutant carrier had a negative EST, and the E4146K carrier had a family history of sudden death during sleep, which are different from phenotypes of typical CPVT. Thus, our data further support the link between RyR2 LOF and a new entity of cardiac arrhythmias distinct from CPVT.     

R4496C RyR2 mutation impairs atrial and ventricular contractility

Ryanodine receptor (RyR2) is the major Ca²⁺ channel of the cardiac sarcoplasmic reticulum (SR) and plays a crucial role in the generation of myocardial force. Changes in RyR2 gating properties and resulting increases in its open probability (P_o) are associated with Ca²⁺ leakage from the SR and arrhythmias; however, the effects of RyR2 dysfunction on myocardial contractility are unknown. Here, we investigated the possibility that a RyR2 mutation associated with catecholaminergic polymorphic ventricular tachycardia, R4496C, affects the contractile function of atrial and ventricular myocardium. We measured isometric twitch tension in left ventricular and atrial trabeculae from wild-type mice and heterozygous transgenic mice carrying the R4496C RyR2 mutation and found that twitch force was comparable under baseline conditions (30°C, 2 mM [Ca²⁺]_o, 1 Hz). However, the positive inotropic responses to high stimulation frequency, 0.1 µM isoproterenol, and 5 mM [Ca²⁺]_o were decreased in R4496C trabeculae, as was post-rest potentiation. We investigated the mechanisms underlying inotropic insufficiency in R4496C muscles in single ventricular myocytes. Under baseline conditions, the amplitude of the Ca²⁺ transient was normal, despite the reduced SR Ca²⁺ content. Under inotropic challenge, however, R4496C myocytes were unable to boost the amplitude of Ca²⁺ transients because they are incapable of properly increasing the amount of Ca²⁺ stored in the SR because of a larger SR Ca²⁺ leakage. Recovery of force in response to premature stimuli was faster in R4496C myocardium, despite the unchanged rates of recovery of L-type Ca²⁺ channel current (I_Ca-L) and SR Ca²⁺ content in single myocytes. A faster recovery from inactivation of the mutant R4496C channels could explain this behavior. In conclusion, changes in RyR2 channel gating associated with the R4496C mutation could be directly responsible for the alterations in both ventricular and atrial contractility. The increased RyR2 P_o and fractional Ca²⁺ release from the SR induced by the R4496C mutation preserves baseline contractility despite a slight decrease in SR Ca²⁺ content, but cannot compensate for the inability to increase SR Ca²⁺ content during inotropic challenge.     

Mitochondrial PKC‐ε deficiency promotes I/R‐mediated myocardial injury via GSK3β‐dependent mitochondrial permeability transition pore opening

Mitochondrial fission is critically involved in cardiomyocyte apoptosis, which has been considered as one of the leading causes of ischaemia/reperfusion (I/R)‐induced myocardial injury. In our previous works, we demonstrate that aldehyde dehydrogenase‐2 (ALDH2) deficiency aggravates cardiomyocyte apoptosis and cardiac dysfunction. The aim of this study was to elucidate whether ALDH2 deficiency promotes mitochondrial injury and cardiomyocyte death in response to I/R stress and the underlying mechanism. I/R injury was induced by aortic cross‐clamping for 45 min. followed by unclamping for 24 hrs in ALDH2 knockout (ALDH2^?/?) and wild‐type (WT) mice. Then myocardial infarct size, cell apoptosis and cardiac function were examined. The protein kinase C (PKC) isoform expressions and their mitochondrial translocation, the activity of dynamin‐related protein 1 (Drp1), caspase9 and caspase3 were determined by Western blot. The effects of N‐acetylcysteine (NAC) or PKC‐δ shRNA treatment on glycogen synthase kinase‐3β (GSK‐3β) activity and mitochondrial permeability transition pore (mPTP) opening were also detected. The results showed that ALDH2^?/? mice exhibited increased myocardial infarct size and cardiomyocyte apoptosis, enhanced levels of cleaved caspase9, caspase3 and phosphorylated Drp1. Mitochondrial PKC‐ε translocation was lower in ALDH2^?/? mice than in WT mice, and PKC‐δ was the opposite. Further data showed that mitochondrial PKC isoform ratio was regulated by cellular reactive oxygen species (ROS) level, which could be reversed by NAC pre‐treatment under I/R injury. In addition, PKC‐ε inhibition caused activation of caspase9, caspase3 and Drp1Ser⁶¹⁶ in response to I/R stress. Importantly, expression of phosphorylated GSK‐3β (inactive form) was lower in ALDH2^?/? mice than in WT mice, and both were increased by NAC pre‐treatment. I/R‐induced mitochondrial translocation of GSK‐3β was inhibited by PKC‐δ shRNA or NAC pre‐treatment. In addition, mitochondrial membrane potential (?Ψ_m) was reduced in ALDH2^?/? mice after I/R, which was partly reversed by the GSK‐3β inhibitor (SB216763) or PKC‐δ shRNA. Collectively, our data provide the evidence that abnormal PKC‐ε/PKC‐δ ratio promotes the activation of Drp1 signalling, caspase cascades and GSK‐3β‐dependent mPTP opening, which results in mitochondrial injury‐triggered cardiomyocyte apoptosis and myocardial dysfuction in ALDH2^?/? mice following I/R stress.     

Beta-Adrenoceptor Stimulation Reveals Ca2+ Waves and Sarcoplasmic Reticulum Ca2+ Depletion in Left Ventricular Cardiomyocytes from Post-Infarction Rats with and without Heart Failure

Abnormal cellular Ca²⁺ handling contributes to both contractile dysfunction and arrhythmias in heart failure. Reduced Ca²⁺ transient amplitude due to decreased sarcoplasmic reticulum Ca²⁺ content is a common finding in heart failure models. However, heart failure models also show increased propensity for diastolic Ca²⁺ release events which occur when sarcoplasmic reticulum Ca²⁺ content exceeds a certain threshold level. Such Ca²⁺ release events can initiate arrhythmias. In this study we aimed to investigate if both of these aspects of altered Ca²⁺ homeostasis could be found in left ventricular cardiomyocytes from rats with different states of cardiac function six weeks after myocardial infarction when compared to sham-operated controls. Video edge-detection, whole-cell Ca²⁺ imaging and confocal line-scan imaging were used to investigate cardiomyocyte contractile properties, Ca²⁺ transients and Ca²⁺ waves. In baseline conditions, i.e. without beta-adrenoceptor stimulation, cardiomyocytes from rats with large myocardial infarction, but without heart failure, did not differ from sham-operated animals in any of these aspects of cellular function. However, when exposed to beta-adrenoceptor stimulation, cardiomyocytes from both non-failing and failing rat hearts showed decreased sarcoplasmic reticulum Ca²⁺ content, decreased Ca²⁺ transient amplitude, and increased frequency of Ca²⁺ waves. These results are in line with a decreased threshold for diastolic Ca²⁺ release established by other studies. In the present study, factors that might contribute to a lower threshold for diastolic Ca²⁺ release were increased THR286 phosphorylation of Ca²⁺/calmodulin-dependent protein kinase II and increased protein phosphatase 1 abundance. In conclusion, this study demonstrates both decreased sarcoplasmic reticulum Ca²⁺ content and increased propensity for diastolic Ca²⁺ release events in ventricular cardiomyocytes from rats with heart failure after myocardial infarction, and that these phenomena are also found in rats with large myocardial infarctions without heart failure development. Importantly, beta-adrenoceptor stimulation is necessary to reveal these perturbations in Ca²⁺ handling after a myocardial infarction.     

Insulin inhibits β-adrenergic action in ischemic/reperfused heart: a novel mechanism of insulin in cardioprotection

Objective Sympathetic overactivity is closely connected with cell injury and contractile dysfunction during myocardial ischemia/reperfusion (MI/R). Insulin exerts protection for the I/R heart and the underlying mechanisms remain unclear. This study aimed to investigate the ability of insulin to modulate β-adrenergic actions on myocardial contraction and post-ischemic injury in acute MI/R and the underlying mechanism. Methods Isolated hearts from adult SD rats were subjected to MI/R (30 min/2 h) and treated with isoproterenol (ISO) or/and insulin. Myocardial contraction, cardiomyocyte apoptosis, myocardial injury and infarction were assessed. In a separate study, isolated ventricular myocytes were subjected to simulated I/R (15/30 min) and myocyte shortening and intracellular Ca²⁺ transient in response to ISO during reperfusion were assessed with presence or absence of insulin. Results In isolated I/R hearts, insulin largely reversed the ISO-associated contractile functional impairment at 2 h after MI/R, inhibiting ISO-induced declines in heart rate and left ventricular systolic pressure by 34.0% and 23.0% and preventing ISO-induced elevation in left ventricular end-diastolic pressure by 28.7% respectively (all P < 0.05). In addition, ISO alone resulted in enlarged infarct size, elevated CK and LDH activity and increased apoptotic index in I/R hearts compared with vehicle, which were inhibited by treatment of insulin (all P < 0.05). Interestingly, in SI/R cardiomyocytes, insulin alone at 10⁻⁷ mol/l increased cell contraction whereas attenuated the positive inotropic response to ISO (10⁻⁹ mol/l) during R as evidenced by a 18.7% reduction in peak twitch amplitude and a 23.9% reduction in calcium transient amplitude (both P < 0.05). Moreover, insulin blunted ISO-mediated increase in PKA activity, enhanced the PKA-dependent phosphorylation of phospholamban (PLB), resulting in increased sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a) activity. Conclusion Insulin attenuated the contractile response to β-AR stimulation and suppressed ISO-elicited cardiac dysfunction and cell injury in MI/R. The inhibitory effect of insulin on the β-adrenergic action involved the inhibition of PKA-mediated Ca²⁺ transient and promotion of post-ischemic Ca²⁺ handling.     

The EF-hand Ca2+ Binding Domain Is Not Required for Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor

Activation of the cardiac ryanodine receptor (RyR2) by elevating cytosolic Ca²⁺ is a central step in the process of Ca²⁺-induced Ca²⁺ release, but the molecular basis of RyR2 activation by cytosolic Ca²⁺ is poorly defined. It has been proposed recently that the putative Ca²⁺ binding domain encompassing a pair of EF-hand motifs (EF1 and EF2) in the skeletal muscle ryanodine receptor (RyR1) functions as a Ca²⁺ sensor that regulates the gating of RyR1. Although the role of the EF-hand domain in RyR1 function has been studied extensively, little is known about the functional significance of the corresponding EF-hand domain in RyR2. Here we investigate the effect of mutations in the EF-hand motifs on the Ca²⁺ activation of RyR2. We found that mutations in the EF-hand motifs or deletion of the entire EF-hand domain did not affect the Ca²⁺-dependent activation of [³H]ryanodine binding or the cytosolic Ca²⁺ activation of RyR2. On the other hand, deletion of the EF-hand domain markedly suppressed the luminal Ca²⁺ activation of RyR2 and spontaneous Ca²⁺ release in HEK293 cells during store Ca²⁺ overload or store overload-induced Ca²⁺ release (SOICR). Furthermore, mutations in the EF2 motif, but not EF1 motif, of RyR2 raised the threshold for SOICR termination, whereas deletion of the EF-hand domain of RyR2 increased both the activation and termination thresholds for SOICR. These results indicate that, although the EF-hand domain is not required for RyR2 activation by cytosolic Ca²⁺, it plays an important role in luminal Ca²⁺ activation and SOICR.     

Role of LOX‐1 in monocyte adhesion‐triggered redox,Akt/eNOS and Ca2+ signaling pathways in endothelial cells

This study was conducted to examine the role of lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1) in monocyte adhesion‐induced redox‐sensitive, Akt/eNOS and Ca²⁺ signaling pathways in endothelial cells (ECs). LOX‐1 was blocked by an antibody‐neutralizing LOX‐1 TS92 or small interfering RNA. In cultured human aortic ECs, monocyte adhesion activated Rac1 and p47^phox, and increased NADPH oxidase activity and reactive oxygen species (ROS) generation within 30 min and NF‐κB phosphorylation within 1 h, resulting in redox‐sensitive gene expression. Akt and eNOS phosphorylation was induced 15 min after adding monocytes and returned to control level after 30 min, whereas NO production was not altered by monocyte adhesion. Blockade of LOX‐1 blunted the monocyte adhesion‐triggered redox‐sensitive signaling pathway and Akt/eNOS phosphorylation in ECs. Both endothelial intracellular Ca²⁺ mobilization and Ca²⁺ influx caused by monocyte attachment were markedly attenuated by pretreatment of ECs with TS92. This suggests that LOX‐1 is involved in redox‐sensitive, Akt/eNOS and Ca²⁺ signaling pathways in monocyte adhesion to ECs independent of oxidized low‐density lipoprotein (ox‐LDL). Furthermore, blockade of Ca²⁺ inhibited monocyte adhesion‐triggered Rac1 and p47^phox activation and ROS generation in ECs, whereas Ca²⁺ signaling was suppressed by blockade of NADPH oxidase and ROS generation. Finally, TS92 blocked the monocyte adhesion to ECs stimulated with or without tumor necrosis factor‐α or ox‐LDL. We provide evidence that LOX‐1 plays a role in redox‐sensitive, Akt/eNOS and Ca²⁺ signaling pathways in monocyte adhesion to ECs independent of the ox‐LDL–LOX‐1 axis. J. Cell. Physiol. 220: 706–715, 2009. © 2009 Wiley‐Liss, Inc.     

Acid‐sensing ion channel 1a is involved in ischaemia/reperfusion induced kidney injury by increasing renal epithelia cell apoptosis

Acidic microenvironment is commonly observed in ischaemic tissue. In the kidney, extracellular pH dropped from 7.4 to 6.5 within 10 minutes initiation of ischaemia. Acid‐sensing ion channels (ASICs) can be activated by pH drops from 7.4 to 7.0 or lower and permeates to Ca²⁺entrance. Thus, activation of ASIC1a can mediate the intracellular Ca²⁺ accumulation and play crucial roles in apoptosis of cells. However, the role of ASICs in renal ischaemic injury is unclear. The aim of the present study was to test the hypothesis that ischaemia increases renal epithelia cell apoptosis through ASIC1a‐mediated calcium entry. The results show that ASIC1a distributed in the proximal tubule with higher level in the renal tubule ischaemic injury both in vivo and in vitro. In vivo, Injection of ASIC1a inhibitor PcTx‐1 previous to ischaemia/reperfusion (I/R) operation attenuated renal ischaemic injury. In vitro, HK‐2 cells were pre‐treated with PcTx‐1 before hypoxia, the intracellular concentration of Ca²⁺, mitochondrial transmembrane potential (?ψm) and apoptosis was measured. Blocking ASIC1a attenuated I/R induced Ca²⁺ overflow, loss of ?ψm and apoptosis in HK‐2 cells. The results revealed that ASIC1a localized in the proximal tubular and contributed to I/R induced kidney injury. Consequently, targeting the ASIC1a may prove to be a novel strategy for AKI patients.