Oxidant stress derails the cardiac connexon connection

Connexin 43 (Cx43) is the major protein component of gap junctions that electrically couple cardiomyocytes at the intercalated disc. Oxidant stress, reduced Cx43 expression, and altered subcellular localization are present in many forms of structural heart disease. These changes in Cx43 lead to alterations in electrical conduction in the ventricle and predispose to lethal cardiac arrhythmias. In their study in this issue of the JCI, Smyth et al. tested the hypothesis that oxidant stress perturbs connexon forward trafficking along microtubules to gap junctions (see the related article beginning on page 266). Failing human ventricular myocardium exhibited a reduction in Cx43 and the microtubule-capping protein EB1 at intercalated discs. Oxidant stress in the adult mouse heart reduced N-cadherin, EB1, and Cx43 colocalization. In HeLa cells and neonatal mouse ventricular myocytes, peroxide exposure displaced EB1 from the plus ends of microtubules and altered microtubule dynamics. Mutational disruption of the EB1-tubulin interaction mimicked the effects of oxidant stress, including a reduction in surface Cx43 expression. These data provide important new molecular insights into the regulation of Cx43 at gap junctions and may identify targets for preservation of cellular coupling in the diseased heart. Rapid propagation of electrical impulses in excitable tissue is essential to processes as diverse as cognition, movement, and the genesis of the heartbeat. Central to rapid conduction in the heart and other organs are gap junctions. Gap junctions are low-resistance conduits between cells, comprised of proteins called connexins. In the heart, connexins are key mediators of electrical conduction and are thus central to excitation and contraction. Connexins hexamerize to form connexons or hemichannels in the membranes of apposing cells that dock head-to-head to form intact gap junction channels (Figure ​(Figure1). 1). Open in a separate windowFigure 1Structure of the cardiac intercalated disc.(A) A schematic of the intercalated disc under normal conditions shows normal forward trafficking along microtubules of Cx43-containing connexons to the adherens junction for incorporation into gap junction plaques between cardiomyocytes. Connexins mediate electrical conduction between cells and are thus central to cardiomyocyte excitation and contraction. (B) In this issue of the JCI, Smyth et al. (9) show that in the setting of oxidative stress, the microtubule-capping protein EB1 dissociates from the microtubule plus end, impeding connexon trafficking to the adherens junction and reducing the generation of gap junction channels. This results in cellular uncoupling and slowed electrical conduction in the ventricle and may predispose to lethal cardiac arrhythmias. Adapted with permission from Cell (10) and ref. 9. The major connexin of working ventricular myocardium is connexin 43 (Cx43). Cx43 is richly endowed with protein interaction domains and sites of phosphorylation that contribute to regulation of the functional expression of gap junction channels. The carboxyl terminus contains a PDZ-binding domain, multiple consensus serine and tyrosine phosphorylation sites, and binding sites for tubulins. Post-translational modifications and protein-protein interactions are thought to be important for proper formation and localization of clusters of gap junctions into so-called “plaques,” although Cx43 with a truncated carboxyl terminus forms working gap junction channels (1).     

Electrical interaction between cardiomyocyte sheets separated by non‐cardiomyocyte sheets in heterogeneous tissues

Electrical coupling between cardiomyocytes is important in synchronous beating and normal heart functions. Cardiomyocytes are also electrically coupled to non‐cardiomyocytes. The electrical interactions between cardiomyocytes and non‐cardiomyocytes, or those between separated cardiomyocytes, are important for normal heart function because abnormalities of the coupling and variation of the cell population induce pathological heart functions and arrhythmias. In this study the three‐dimensional time course of the electrical interaction between two rat neonatal cardiomyocyte sheets separated by non‐cardiomyocyte sheets was analysed by a multiple‐electrode extracellular recording system. The two cardiomyocyte sheets separated by a single‐ or double‐layered mouse fibroblast NIH3T3 cell sheet coupled electrically at 113 ± 28 or 287 ± 87 min after layering, respectively. The time course of the electrical coupling, when the single‐layer NIH3T3 cell sheet was inserted, is similar to that of a layered cardiomyocyte sheet. Immunocytological analysis and dye transfer assay suggested the formation of gap junctions at heterocellular junctions of cardiomyocytes and NIH3T3 cells. On the other hand, when a double‐layered NIH3T3 cell sheet was inserted, an incomplete electrical coupling of two cardiomyocyte sheets, including a conduction delay, was observed. The electrical coupling of cardiomyocyte sheets was completely blocked (conduction block) by insertion of a triple‐layered NIH3T3 cell sheet, a communication‐defective HeLa cell sheet or a Ca²⁺‐antagonist LaCl₃‐treated cell sheet. These electrophysiological analyses of heterogeneously stacked cell sheets might provide insights into complex electrical conduction systems that resemble those of native or damaged heart and transplanted tissues. Copyright © 2009 John Wiley & Sons, Ltd.     

Calcium cycling proteins and heart failure: mechanisms and therapeutics

Ca²⁺-dependent signaling is highly regulated in cardiomyocytes and determines the force of cardiac muscle contraction. Ca²⁺ cycling refers to the release and reuptake of intracellular Ca²⁺ that drives muscle contraction and relaxation. In failing hearts, Ca²⁺ cycling is profoundly altered, resulting in impaired contractility and fatal cardiac arrhythmias. The key defects in Ca²⁺ cycling occur at the level of the sarcoplasmic reticulum (SR), a Ca²⁺ storage organelle in muscle. Defects in the regulation of Ca²⁺ cycling proteins including the ryanodine receptor 2, cardiac (RyR2)/Ca²⁺ release channel macromolecular complexes and the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 2a (SERCA2a)/phospholamban complex contribute to heart failure. RyR2s are oxidized, nitrosylated, and PKA hyperphosphorylated, resulting in “leaky” channels in failing hearts. These leaky RyR2s contribute to depletion of Ca²⁺ from the SR, and the leaking Ca²⁺ depolarizes cardiomyocytes and triggers fatal arrhythmias. SERCA2a is downregulated and phospholamban is hypophosphorylated in failing hearts, resulting in impaired SR Ca²⁺ reuptake that conspires with leaky RyR2 to deplete SR Ca²⁺. Two new therapeutic strategies for heart failure (HF) are now being tested in clinical trials: (a) fixing the leak in RyR2 channels with a novel class of Ca²⁺-release channel stabilizers called Rycals and (b) increasing expression of SERCA2a to improve SR Ca²⁺ reuptake with viral-mediated gene therapy. There are many potential opportunities for additional mechanism-based therapeutics involving the machinery that regulates Ca²⁺ cycling in the heart.     

S100A1 DNA-based Inotropic Therapy Protects Against Proarrhythmogenic Ryanodine Receptor 2 Dysfunction

Restoring expression levels of the EF-hand calcium (Ca²⁺) sensor protein S100A1 has emerged as a key factor in reconstituting normal Ca²⁺ handling in failing myocardium. Improved sarcoplasmic reticulum (SR) function with enhanced Ca²⁺ resequestration appears critical for S100A1''s cyclic adenosine monophosphate-independent inotropic effects but raises concerns about potential diastolic SR Ca²⁺ leakage that might trigger fatal arrhythmias. This study shows for the first time a diminished interaction between S100A1 and ryanodine receptors (RyR2s) in experimental HF. Restoring this link in failing cardiomyocytes, engineered heart tissue and mouse hearts, respectively, by means of adenoviral and adeno-associated viral S100A1 cDNA delivery normalizes diastolic RyR2 function and protects against Ca²⁺- and β-adrenergic receptor-triggered proarrhythmogenic SR Ca²⁺ leakage in vitro and in vivo. S100A1 inhibits diastolic SR Ca²⁺ leakage despite aberrant RyR2 phosphorylation via protein kinase A and calmodulin-dependent kinase II and stoichiometry with accessory modulators such as calmodulin, FKBP12.6 or sorcin. Our findings demonstrate that S100A1 is a regulator of diastolic RyR2 activity and beneficially modulates diastolic RyR2 dysfunction. S100A1 interaction with the RyR2 is sufficient to protect against basal and catecholamine-triggered arrhythmic SR Ca²⁺ leak in HF, combining antiarrhythmic potency with chronic inotropic actions.     

Plasma membrane Ca2+-ATPase isoform 4 antagonizes cardiac hypertrophy in association with calcineurin inhibition in rodents

How Ca²⁺-dependent signaling effectors are regulated in cardiomyocytes, given the extreme cytoplasmic Ca²⁺ concentration changes that underlie contraction, remains unknown. Cardiomyocyte plasma membrane Ca²⁺-ATPase (PMCA) extrudes Ca²⁺ but has little effect on excitation-contraction coupling, suggesting its potential role in controlling Ca²⁺-dependent signaling effectors such as calcineurin. We generated cardiac-specific inducible PMCA4b transgenic mice that displayed normal global Ca²⁺ transient and cellular contraction levels and reduced cardiac hypertrophy following transverse aortic constriction (TAC) or phenylephrine/Ang II infusion, but showed no reduction in exercise-induced hypertrophy. Transgenic mice were protected from decompensation and fibrosis following long-term TAC. The PMCA4b transgene reduced the hypertrophic augmentation associated with transient receptor potential canonical 3 channel overexpression, but not that associated with activated calcineurin. Furthermore, Pmca4 gene–targeted mice showed increased cardiac hypertrophy and heart failure events after TAC. Physical associations between PMCA4b and calcineurin were enhanced by TAC and by agonist stimulation of cultured neonatal cardiomyocytes. PMCA4b reduced calcineurin nuclear factor of activated T cell–luciferase activity after TAC and in cultured neonatal cardiomyocytes after agonist stimulation. PMCA4b overexpression inhibited cultured cardiomyocyte hypertrophy following agonist stimulation, but much less so in a Ca²⁺ pumping–deficient PMCA4b mutant. Thus, Pmca4b likely reduces the local Ca²⁺ signals involved in reactive cardiomyocyte hypertrophy via calcineurin regulation.     

β1-adrenoceptor stimulation promotes LPS-induced cardiomyocyte apoptosis through activating PKA and enhancing CaMKII and IκBα phosphorylation

IntroductionCaspase activation and cardiomyocyte apoptosis have been implicated in lipopolysaccharide (LPS)-induced cardiac contractile dysfunction. We have recently demonstrated that β1-adrenoceptor (AR) activation by endogenous norepinephrine contributes to cardiomyocyte apoptosis in endotoxemic mice. Here, we further investigated the molecular mechanisms for the enhancing effect of β₁-AR activation on LPS-induced cardiomyocyte apoptosis.MethodsThe adult mouse ventricular myocytes were exposed to LPS, dobutamine, protein kinase A (PKA) inhibitor or/and nifedipine, an L-type Ca²⁺ channel blocker. Male BALB/c mice were treated with LPS or/ and β₁-AR antagonist, atenolol. Cardiomyocyte apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assay and apoptosis-associated molecules were detected.ResultsLPS induced apoptosis in adult mouse ventricular myocytes, dobutamine (DOB), a β₁-AR agonist, promoted apoptosis, caspase-8, 9 and 3 activation and increased cytosolic Ca²⁺ concentration in LPS-challenged cardiomyocytes. DOB also up-regulated TNF-α expression, decreased Bcl-2 levels, promoted Bax translocation to mitochondria, mitochondrial membrane potential loss and cytochrome c release as well as IκBα, p38 MAPK, JNK and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) phosphorylation in LPS-treated cardiomyocytes. PKA inhibitor abolished the effects of DOB on caspase-9 activation, Bcl-2 levels as well as JNK and p38 MAPK phosphorylation, but not on IκBα phosphorylation, TNF-α expression and caspase-8 activation in LPS-stimulated cardiomyocytes. Pretreatment with nifedipine not only significantly blocked the enhancing effects of DOB on LPS-induced elevation in cytosolic Ca²⁺ concentration and CaMKII phosphorylation in cardiomyocytes, but also partly reversed the effects of DOB on caspase-9 and caspase-3/7 activities in LPS-treated cardiomyocytes. Furthermore, atenolol suppressed TNF-α expression, JNK, p38 MAPK and CaMKII phosphorylation, increased Bcl-2 expression, and inhibited cytochrome c release and cardiomyocyte apoptosis in the myocardium of endotoxemic mice.Conclusionsβ₁-AR activation promotes LPS-induced apoptosis through activating PKA, increasing CaMKII phosphorylation as well as enhancing IκBα phosphorylation and TNF-α expression in cardiomyocytes.     

PLCδ1 Protein Rescues Ischemia-reperfused Heart by the Regulation of Calcium Homeostasis

Myocardial Ca²⁺ overload induced by ischemia/reperfusion (I/R) is a major element of myocardial dysfunction in heart failure. Phospholipase C (PLC) plays important roles in the regulation of the phosphoinositol pathway and Ca²⁺ homeostasis in various types of cells. Here, we investigated the protective role of PLCδ1 against myocardial I/R injury through the regulation of Ca²⁺ homeostasis. To investigate its role, PLCδ1 was fused to Hph1, a cell-permeable protein transduction domain (PTD), and treated into rat neonatal cardiomyocytes and rat hearts under respective hypoxia-reoxygenation (H/R) and ischemia-reperfusion conditions. Treatment with Hph1-PLCδ1 significantly inhibited intracellular Ca²⁺ overload, reactive oxygen species generation, mitochondrial permeability transition pore opening, and mitochondrial membrane potential elevation in H/R neonatal cardiomyocytes, resulting in the inhibition of apoptosis. Intravenous injections of Hph1-PLCδ1 in rats with I/R-injured myocardium caused significant reductions in infarct size and apoptosis and also improved systolic and diastolic cardiac functioning. Furthermore, a small ions profile obtained using time-of-flight secondary ion mass spectrometry showed that treatment with Hph1-PLCδ1 leads to significant recovery of calcium-related ions toward normal levels in I/R-injured myocardium. These results suggest that Hph1-PLCδ1 may manifest as a promising cardioprotective drug due to its inhibition of the mitochondrial apoptotic pathway in cells suffering from I/R injury.     

Stable fetal cardiomyocyte grafts in the hearts of dystrophic mice and dogs.

This report documents the formation of stable fetal cardiomyocyte grafts in the myocardium of dystrophic dogs. Preliminary experiments established that the dystrophin gene product could be used to follow the fate of engrafted cardiomyocytes in dystrophic mdx mice. Importantly, ultrastructural analyses revealed the presence of intercalated discs consisting of fascia adherens, desmosomes, and gap junctions at the donor-host cardiomyocyte border. To determine if isolated cardiomyocytes could form stable intracardiac grafts in a larger species, preparations of dissociated fetal canine cardiomyocytes were delivered into the hearts of adult CXMD (canine X-linked muscular dystrophy) dogs. CXMD dogs, like Duchenne muscular dystrophy patients and mdx mice, fail to express dystrophin in both cardiac and skeletal muscle. Engrafted fetal cardiomyocytes, identified by virtue of dystrophin immunoreactivity, were observed to be tightly juxtaposed with host cardiomyocytes as long as 10 wk after engraftment, the latest date analyzed. Confocal laser scanning microscopy revealed the presence of connexin43, a major constituent of the gap junction, at the donor-host cardiomyocyte border. The presence of intracardiac grafts was not associated with arrhythmogenesis in the CXMD model. These results indicate that fetal cardiomyocyte grafting can successfully augment cardiomyocyte number in larger animals.     

Pre-transplantation of Bone Marrow Mesenchymal Stem Cells Amplifies the Therapeutic Effect of Ultrasound-Targeted Microbubble Destruction–Mediated Localized Combined Gene Therapy in Post–Myocardial Infarction Heart Failure Rats

Although stem cell transplantation and single-gene therapy have been intensively discussed separately as treatments for myocardial infarction (MI) hearts and have exhibited ideal therapeutic efficiency in animal models, clinical trials turned out to be disappointing. Here, we deliver sarcoplasmic reticulum Ca²⁺-ATPase 2a (SERCA2a) and connexin 43 (Cx43) genes simultaneously via an ultrasound-targeted microbubble destruction (UTMD) approach to chronic MI hearts that have been pre-treated with bone marrow mesenchymal stem cells (BMSCs) to amplify cardiac repair. First, biotinylated microbubbles (BMBs) were fabricated, and biotinylated recombinant adenoviruses carrying the SERCA2a or Cx43 gene were conjugated to the surface of self-assembled BMBs to form SERCA2a-BMBs, Cx43-BMBs or dual gene-loaded BMBs. Then, the general characteristics of these bubbles, including particle size, concentration, contrast signal and gene loading capacity, were examined. Second, a rat myocardial infarction model was created by ligating the left anterior descending coronary artery and injecting BMSCs into the infarct and border zones. Four weeks later, co-delivery of SERCA2a and Cx43 genes to the infarcted heart were delivered together to the infarcted heart using the UTMD approach. Cardiac mechano-electrical function was determined 4 wk after gene transfection, and the infarcted hearts were collected for myocardial infarct size measurement and detection of expression of SERCA2a, Cx43 and cardiac-specific markers. Finally, to validate the role of BMSC transplantation, MI rats transplanted or not with BMSCs were transfected with SERCA2a and Cx43, and the cardiac mechano-electrical function of these two groups of rats was recorded and compared. General characteristics of the self-assembled gene-loaded BMBs were qualified, and the gene loading rate was satisfactory. The self-assembled gene-loaded BMBs were in microscale and exhibit satisfactory dual-gene loading capacity. High transfection efficiency was achieved under ultrasound irradiation in vitro. In addition, rats in which SERCA2a and Cx43 were overexpressed simultaneously had the best contractile function and electrical stability among all experimental groups. Immunofluorescence assay revealed that the levels of SERCA2a and/or Cx43 proteins were significantly elevated, especially in the border zone. Moreover, compared with rats that did not receive BMSCs, rats pre-treated with BMSCs have better mechano-electrical function after transfection with SERCA2a and Cx43. Collectively, we report a promising cardiac repair strategy for post-MI hearts that exploits the providential advantages of stem cell therapy and UTMD-mediated localized co-delivery of specific genes.     

Opening of mitochondrial permeability transition pore in cardiomyocytes: Is ferutinin a suitable tool for its assessment?

Mitochondrial permeability transition pore (mPTP) opening is a critical event leading to cell injury during myocardial ischemia–reperfusion but having a reliable cellular model to study the effect of drugs targeting mPTP is an unmet need. This study evaluated whether the Ca²⁺ electrogenic ionophore ferutinin is a relevant tool to induce mPTP in cardiomyocytes. mPTP opening was monitored using the calcein/cobalt fluorescence technique in adult cardiomyocytes isolated from wild-type and cyclophylin D (CypD) knock-out mice. Concomitantly, the effect of ferutinin was assessed in isolated myocardial mitochondria. Our results confirmed the Ca²⁺ ionophoric effect of ferutinin in isolated mitochondria and cardiomyocytes. Ferutinin induced all the hallmarks of mPTP opening in cells (loss of calcein, of mitochondrial potential and cell death), but none of them could be inhibited by CypD deletion or cyclosporine A, indicating that mPTP opening was not the major contributor to the effect of ferutinin. This was confirmed in isolated mitochondria where ferutinin acts by different mechanisms dependent and independent of the mitochondrial membrane potential. At low ferutinin/mitochondria concentration ratio, ferutinin displays protonophoric-like properties, lowering the mitochondrial membrane potential and limiting oxidative phosphorylation without mitochondrial swelling. At high ferutinin/mitochondria ratio, ferutinin induced a sudden Ca²⁺ independent mitochondrial swelling, which is only partially inhibited by cyclosporine A. Together, these result show that ferutinin is not a suitable tool to investigate CypD-dependent mPTP opening in isolated cardiomyocytes because it possesses other mitochondrial properties such as swelling induction and mitochondrial uncoupling properties which impede its utilization.     

RNAi targeting ryanodine receptor 2 protects rat cardiomyocytes from injury caused by simulated ischemia-reperfusion

The effects of a small interfering RNA targeting ryanodine receptor 2 (si-Ryr2) on cardiomyocytes injury following a simulated ischemia-reperfusion (I/R) were investigated. Pretreated with si-Ryr2 or ryanodine, primary cultures of neonatal rat cardiomyocytes were subjected to a protocol of simulated I/R. Compared with control, the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and the generation of reactive oxygen species (ROS) was significantly augmented after I/R. Concomitant with these, cell injury assessed by Annexin V/PI staing, mitochondria membrane potential (ΔΨm) and the leakage of lactic dehydrogenase (LDH) and creatine phosphokinase (CPK) were aggravated. Si-Ryr2 treatment reduced [Ca²⁺]_i and ROS generation and protected the cardiomyocytes from subsequent I/R injury, as evidenced by stable ΔΨm and decreased Annexin V⁺ PI^- staing and enzymes release. Moreover, si-Ryr2 exerted more effective protection on I/R injury compared to ryanodine. The present study demonstrated for the first time that in neonatal cardiomyocytes, si-Ryr2 reduces cell death associated with attenuating [Ca²⁺]_i and ROS production. Furthermore, we attempt to speculate that si-Ryr2 excel ryanodine in Ryr2 function research of cardioprotection.     

Gene transfer of connexin43 into skeletal muscle

Cellular cardiomyoplasty using skeletal myoblasts may be beneficial for infarct repair. One drawback to skeletal muscle cells is their lack of gap junction expression after differentiation, thus preventing electrical coupling to host cardiomyocytes. We sought to overexpress the gap junction protein connexin43 (Cx43) in differentiated skeletal myotubes, using retroviral, adenoviral, and plasmid-mediated gene transfer. All strategies resulted in overexpression of Cx43 in cultured myotubes, but expression of Cx43 from constitutive viral promoters caused significant death upon differentiation. Dye transfer studies showed that surviving myotubes contained functional gap junctions, however. Retrovirally transfected myoblasts did not express Cx43 after grafting into the heart, possibly due to promoter silencing. Adenovirally transfected myoblasts expressed abundant Cx43 after forming myotubes in cardiac grafts, but grafts showed signs of injury at 1 week and had died by 2 weeks. Interestingly, transfection of already differentiated myotubes with adenoviral Cx43 was nontoxic, implying a window of vulnerability during differentiation. To test this hypothesis, Cx43 was expressed from the muscle creatine kinase (MCK) promoter, which is active only after myocyte differentiation. The MCK promoter resulted in high levels of Cx43 expression in differentiated myotubes but did not cause cell death during differentiation. MCK-Cx43-transfected myoblasts formed viable cardiac grafts and, in some cases, Cx43-expressing myotubes were in close apposition to host cardiomyocytes, possibly allowing electrical coupling. Thus, high levels of Cx43 during skeletal muscle differentiation cause cell death. When, however, expression of Cx43 is delayed until after differentiation, using the MCK promoter, myotubes are viable and express gap junction proteins after grafting in the heart. This strategy may permit electrical coupling of skeletal and cardiac muscle for cardiac repair.     

Phosphodiesterase 4B in the cardiac L-type Ca²⁺ channel complex regulates Ca²⁺ current and protects against ventricular arrhythmias in mice

β-Adrenergic receptors (β-ARs) enhance cardiac contractility by increasing cAMP levels and activating PKA. PKA increases Ca²⁺-induced Ca²⁺ release via phosphorylation of L-type Ca²⁺ channels (LTCCs) and ryanodine receptor 2. Multiple cyclic nucleotide phosphodiesterases (PDEs) regulate local cAMP concentration in cardiomyocytes, with PDE4 being predominant for the control of β-AR–dependent cAMP signals. Three genes encoding PDE4 are expressed in mouse heart: Pde4a, Pde4b, and Pde4d. Here we show that both PDE4B and PDE4D are tethered to the LTCC in the mouse heart but that β-AR stimulation of the L-type Ca²⁺ current (I_Ca,L) is increased only in Pde4b^–/– mice. A fraction of PDE4B colocalized with the LTCC along T-tubules in the mouse heart. Under β-AR stimulation, Ca²⁺ transients, cell contraction, and spontaneous Ca²⁺ release events were increased in Pde4b^–/– and Pde4d^–/– myocytes compared with those in WT myocytes. In vivo, after intraperitoneal injection of isoprenaline, catheter-mediated burst pacing triggered ventricular tachycardia in Pde4b^–/– mice but not in WT mice. These results identify PDE4B in the Ca_V1.2 complex as a critical regulator of I_Ca,L during β-AR stimulation and suggest that distinct PDE4 subtypes are important for normal regulation of Ca²⁺-induced Ca²⁺ release in cardiomyocytes.     

Angiotensin 1‐7 modulates electrophysiological characteristics and calcium homoeostasis in pulmonary veins cardiomyocytes via MAS/PI3K/eNOS signalling pathway

Background

Atrial fibrillation (AF) is the most common sustained arrhythmia, and pulmonary veins (PVs) play a critical role in triggering AF. Angiotensin (Ang)‐(1‐7) regulates calcium (Ca²⁺) homoeostasis and also plays a critical role in cardiovascular pathophysiology. However, the role of Ang‐(1‐7) in PV arrhythmogenesis remains unclear.

Materials and methods

Conventional microelectrodes, whole‐cell patch‐clamp and the fluo‐3 fluorimetric ratio technique were used to record ionic currents and intracellular Ca²⁺ in isolated rabbit PV preparations and in single isolated PV cardiomyocytes, before and after administration of Ang‐(1‐7).

Results

Ang (1‐7) concentration dependently (0.1, 1, 10 and 100 nmol/L) decreased PV spontaneous electrical activity. Ang‐(1‐7) (100 nmol/L) decreased the late sodium (Na⁺), L‐type Ca²⁺ and Na⁺‐Ca²⁺ exchanger currents, but did not affect the voltage‐dependent Na⁺ current in PV cardiomyocytes. In addition, Ang‐(1‐7) decreased intracellular Ca²⁺ transient and sarcoplasmic reticulum Ca²⁺ content in PV cardiomyocytes. A779 (a Mas receptor blocker, 3 μmol/L), L‐NAME (a NO synthesis inhibitor, 100 μmol/L) or wortmannin (a specific PI3K inhibitor, 10 nmol/L) attenuated the effects of Ang‐(1‐7) (100 nmol/L) on PV spontaneous electric activity.

Conclusion

Ang‐(1‐7) regulates PV electrophysiological characteristics and Ca²⁺ homoeostasis via Mas/PI3K/eNOS signalling pathway.     

The heme oxygenase inducer hemin protects against cardiac dysfunction and ventricular fibrillation in ischaemic/reperfused rat hearts: role of connexin 43

Cardiac expression of cytoprotective gene heme oxygenase‐1 (HO‐1) is modulated by ischaemia and reperfusion (I/R). We therefore hypothesized that pretreatment with hemin, an inductor of HO‐1, would precondition the heart against post‐ischaemic dysfunction and ventricular fibrillation (VF). Male Wistar rats were given either hemin or HO enzyme inhibitor zinc protoporphyrin IX (ZnPP IX). Isolated hearts were subjected to 30?min global ischaemia followed by 120?min of reperfusion or were aerobically perfused in a time‐matched non‐ischaemic protocol. Control animals received no pretreatment. Compared to non‐perfused controls, pretreatment with hemin increased HO‐1 mRNA 13‐fold (p<0.001) and HO‐1 protein 3.5‐fold (p?0.001), improved post‐ischaemic aortic flow, coronary flow, LVDP and ?Dp/dt (p<0.01) and decreased LVEDP (p<0.001) and the incidence of VF (p = 0.001). The improved post‐ischaemic cardiac function and reduction of VF were accompanied by a higher total connexin 43 (Cx43) level compared to non‐pretreated and ZnPP IX pretreated hearts, and accumulation of non‐phosphorylated gap junction protein Cx43 in intercalated discs and lateral plasma membrane of cardiomyocytes. Cardioprotection by HO‐1 appeared to be independent of cGMP. Administration of ZnPP IX had no effect on cardiac function or VF. Our results show that pharmacological modulation of HO‐1 pathway may provide a new therapeutic approach to protect the heart against post‐ischaemic dysfunction and I/R‐induced VF possibly by a Cx43 dependent mechanism.     

Effect of Na+ reduction and monensin on ion content and contractile response in normoxic and ischaemic reperfused rat hearts

Summary— The possibility was explored whether the functional properties of Na+/Ca²⁺ exchange are altered after ischaemia, thereby contributing to the elevated intracellular (i) Ca²⁺ levels in ischaemic reperfused hearts. The intracellular Na+, K+ and Ca²⁺ contents in rat Langendorff heart preparations were determined by atomic absorption spectrometry under normoxic conditions, after ischaemia (30 min) and after ischaemia (30 min) plus reperfusion (30 min). In addition, the influence of modulating the Na+ gradient (Na+_o/Na+_i) across the sarcolemma was studied with respect to cardiac contractility and intracellular ion content. This was done by either decreasing extracellular (o) Na+ or by increasing Na+_i with monensin, both in normoxic and reperfused hearts. Both Na+_o reduction and monensin led to an increase in contractility and coronary flow, an effect which was nearly abolished in reperfused hearts. Under normoxic conditions the intracellular ion contents amounted to Na+ = 12.4 ± 0.4, K+ = 99.0 ± 3.1 and Ca²⁺ = 0.64 ± 0.02 mmol/kg cell (means ± SEM, n = 7). In normoxic hearts, lowering Na+_o reduced and monensin increased Na+_i, thereby both leading to a decrease in Na+ gradient; no effect on total Ca²⁺_i content was observed. Na+_i increased twofold after ischaemia as compared to the normoxic situation, an effect which was aggravated (4 fold increase) in reperfused hearts. The opposite effects were observed for K+_i with a 25% decrease after ischaemia and a 40% decrease in reperfused hearts. Only after ischaemia plus reperfusion was Ca²⁺_i increased (6 fold). In reperfused hearts, lowering Na+_o again reduced and monensin increased Na+_i, whereas a further rise in Ca²⁺_i was now observed depending on the Na+ gradient across the sarcolemma: the larger the drop in Na+ gradient, the more pronounced the increase in Ca²⁺_i in the reperfused heart. We conclude that the Ca²⁺_i increase in reperfused hearts can be modulated by changing the Na+ gradient across the sarcolemma. This suggests that inhibited or reversed Na+/Ca²⁺ exchange is predominantly responsible for the rise in Ca²⁺_i in ischaemic hearts that are subjected to reperfusion.     

Calcium release channel RyR2 regulates insulin release and glucose homeostasis

The type 2 ryanodine receptor (RyR2) is a Ca²⁺ release channel on the endoplasmic reticulum (ER) of several types of cells, including cardiomyocytes and pancreatic β cells. In cardiomyocytes, RyR2-dependent Ca²⁺ release is critical for excitation-contraction coupling; however, a functional role for RyR2 in β cell insulin secretion and diabetes mellitus remains controversial. Here, we took advantage of rare RyR2 mutations that were identified in patients with a genetic form of exercise-induced sudden death (catecholaminergic polymorphic ventricular tachycardia [CPVT]). As these mutations result in a “leaky” RyR2 channel, we exploited them to assess RyR2 channel function in β cell dynamics. We discovered that CPVT patients with mutant leaky RyR2 present with glucose intolerance, which was heretofore unappreciated. In mice, transgenic expression of CPVT-associated RyR2 resulted in impaired glucose homeostasis, and an in-depth evaluation of pancreatic islets and β cells from these animals revealed intracellular Ca²⁺ leak via oxidized and nitrosylated RyR2 channels, activated ER stress response, mitochondrial dysfunction, and decreased fuel-stimulated insulin release. Additionally, we verified the effects of the pharmacological inhibition of intracellular Ca²⁺ leak in CPVT-associated RyR2-expressing mice, in human islets from diabetic patients, and in an established murine model of type 2 diabetes mellitus. Taken together, our data indicate that RyR2 channels play a crucial role in the regulation of insulin secretion and glucose homeostasis.     

Endothelium‐dependent hyperpolarizations: Past beliefs and present facts

Endothelium‐dependent relaxations are attributed to the release of various factors, such as nitric oxide, carbon monoxide, reactive oxygen species, adenosine, peptides and arachidonic acid metabolites derived from the cyclooxygenases, lipoxygenases, and cytochrome P450 monooxygenases pathways. The hyperpolarization of the smooth muscle cell can contribute to or be an integral part of the mechanisms underlying the relaxations elicited by virtually all these endothelial mediators. These endothelium‐derived factors can activate different families of K⁺ channels of the vascular smooth muscle. Other events associated with the hyperpolarization of both the endothelial and the vascular smooth muscle cells (endothelium‐derived hyperpolarizing factor (EDHF)‐mediated responses) contribute also to endothelium‐dependent relaxations. These responses involve an increase in the intracellular Ca²⁺ concentration of the endothelial cells followed by the opening of Ca²⁺‐activated K⁺ channels of small and intermediate conductance and the subsequent hyperpolarization of these cells. Then, the endothelium‐dependent hyperpolarization of the underlying smooth muscle cells can be evoked by direct electrical coupling through myoendothelial junctions and/or the accumulation of K⁺ ions in the intercellular space between the two cell types. These various mechanisms are not necessarily mutually exclusive and, depending on the vascular bed and the experimental conditions, can occur simultaneously or sequentially, or also may act synergistically.     

Afterdepolarizations and triggered activity as a mechanism for clinical arrhythmias

Afterdepolarizations cause triggered arrhythmias. One kind occurs after repolarization is complete, delayed afterdepolarizations (DADs). Another occurs as an interruption in repolarization, early afterdepolarizations (EADs). Afterdepolarizations initiate arrhythmias when they depolarize membrane potential to threshold potential for triggering action potentials. DADs usually occur mostly when Ca²⁺ in the sarcoplasmic reticulum (SR) is elevated. The SR leaks some of the Ca²⁺ into the myoplasm through Ca²⁺ release channels controlled by ryanodine receptors (RyR2) during diastole. The Na⁺‐Ca²⁺ exchanger extrudes elevated diastolic Ca²⁺ from the cell in exchange for Na⁺ (1 Ca²⁺ for 3 Na⁺) generating inward current causing DADs. DAD amplitude increases with decreasing cycle length, causing triggered activity during an increase in heart rate or during programmed electrical stimulation (PES). Coupling interval of the first triggered impulse is directly related to initiating cycle length. EADs are associated with an increased action potential duration (APD) causing long QT (LQT). EADs are caused by net inward currents (I_CaL, I_NCX) as a consequence. Hundreds of mutations can cause congenital LQT by altering repolarizing ion channels. Acquired LQT results from drug interaction with repolarizing ion channels. EAD‐triggered ventricular tachycardia is polymorphic and called “torsade de pointes.” Effects of PES on EAD‐triggered activity is related to effects of cycle length on APD. Shortening cycle length prevents EADs by accelerating repolarization. Typical PES protocols inhibit formation of EADs which can be therapeutic.