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Nicotine, a key constituent of tobacco/electronic cigarettes causes cardiovascular injury and mortality. Nicotine is known to induce oxidative stress and mitochondrial dysfunction in cardiomyocytes leading to cell death. However, the underlying mechanisms remain unclear. Pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) is a member of metal-dependent protein phosphatase (PPM) family and is known to dephosphorylate several AGC family kinases and thereby regulate a diverse set of cellular functions including cell growth, survival, and death. Our lab has previously demonstrated that PHLPP1 removal reduced cardiomyocyte death and cardiac dysfunction following injury. Here, we present a novel finding that nicotine exposure significantly increased PHLPP1 protein expression in the adolescent rodent heart. Building upon our in vivo finding, we determined the mechanism of PHLPP1 expression in cardiomyocytes. Nicotine significantly increased PHLPP1 protein expression without altering PHLPP2 in cardiomyocytes. In cardiomyocytes, nicotine significantly increased NADPH oxidase 4 (NOX4), which coincided with increased reactive oxygen species (ROS) and increased cardiomyocyte apoptosis which were dependent on PHLPP1 expression. PHLPP1 expression was both necessary and sufficient for nicotine induced mitochondrial dysfunction. Mechanistically, nicotine activated extracellular signal-regulated protein kinases (ERK1/2) and subsequent eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) to increase PHLPP1 protein expression. Inhibition of protein synthesis with cycloheximide (CHX) and 4EGI-1 abolished nicotine induced PHLPP1 protein expression. Moreover, inhibition of ERK1/2 activity by U0126 significantly blocked nicotine induced PHLPP1 expression. Overall, this study reveals a novel mechanism by which nicotine regulates PHLPP1 expression through ERK-4E-BP1 signaling axis to drive cardiomyocyte injury. [Display omitted] • Nicotine increases PHLPP1 expression in cardiomyocytes leading to increased ROS, mitochondrial dysfunction, and death. • Overexpression of PHLPP1 alone increased ROS and mitochondrial dysfunction in cardiomyocytes. • Removal of PHLPP1 protected cardiomyocytes from nicotine induced oxidative stress, mitochondrial dysfunction, and damage. • Nicotine increased PHLPP1 through ERK/4E-BP1 signaling axis. [ABSTRACT FROM AUTHOR]

• The imaging protocol visualises intracellular calcium signals and their sources. • It correlates calcium spark TIRF images with DNA-PAINT images of RyR channels. • It is a powerful method for studying nanoscale structure-function relationship. Nanometre-scale cellular information obtained through super-resolution microscopies are often unaccompanied by functional information, particularly transient and diffusible signals through which life is orchestrated in the nano-micrometre spatial scale. We describe a correlative imaging protocol which allows the ubiquitous intracellular second messenger, calcium (Ca2+), to be directly visualised against nanoscale patterns of the ryanodine receptor (RyR) Ca2+ channels which give rise to these Ca2+ signals in wildtype primary cells. This was achieved by combining total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals, with the subsequent DNA-PAINT imaging of the RyRs. We report a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets and demonstrate how such data can be used to visually identify the ensembles of Ca2+ channels that are locally activated during the genesis of cytoplasmic Ca2+ signals. [ABSTRACT FROM AUTHOR]

Cardiovascular diseases are leading causes that threaten people's life. To investigate cells that are involved in disease development and tissue repair, various technologies have been introduced. Among these technologies, lineage tracing is a powerful tool to track the fate of cells in vivo , providing deep insights into cellular behavior and plasticity. In cardiac diseases, newly formed cardiomyocytes and endothelial cells are found from proliferation of local cells, while fibroblasts and macrophages are originated from diverse cell sources. Similarly, in response to vascular injury, various sources of cells including media smooth muscle cells, endothelium, resident progenitors and bone marrow cells are involved in lesion formation and/or vessel regeneration. In summary, current review summarizes the development of lineage tracing techniques and their utilizations in investigating roles of different cell types in cardiovascular diseases. [ABSTRACT FROM AUTHOR]

To develop an adult guinea pig model of lipotoxicity and explore the underlying mechanisms associated with changes in the expression of the delayed rectifier potassium current (I K). Lipotoxicity may represent a common link among metabolic disorders and a higher vulnerability to arrhythmias. Whole-cell patch clamp, and palmitic acid (PA, a potent inducer of lipotoxicity), were used to assess mechanisms of short-term (∼50 days) high-fat diet (HFD) feeding on atrial electrophysiology in guinea pig hearts and myocytes. HFD fed guinea pigs were significantly heavier, displayed hypertriglyceridemia and hypercholesterolemia; but no signs of hyperglycemia or inflammation compared to low-fat diet fed controls. Increasing cardiac PA levels, resulted in shortened atrial action potential duration, and increased I K density. Inhibition of phosphoinositide 3-kinase (PI3K) prevented increases in I K due to PA. Acute (≥1hr) exposure of atrial myocytes to exogenous PA (1 mM) increased the density of the rapid delayed rectifier potassium current I Kr , while it was decreased with the unsaturated oleic acid (OA, 1 mM). Serine-threonine protein phosphatase-2 (PP2A) inhibition with cantharidin reversed the effect of OA on I Kr. Our data provide evidence of a novel lipotoxic guinea pig model with signs of vulnerability to arrhythmias. Inhibition of PA/PI3K/ I K and/or activation of the OA/PP2A/ I Kr pathways may be therapeutically beneficial for lipotoxic arrhythmias. • Short-term high-fat diet feeding selectively induced lipotoxicity in guinea pig. • Lipotoxic hearts showed shortened atrial action potential and increased IK density. • PI3K inhibition prevented increases in IK due to palmitic acid. • PP2A inhibition reversed decreases in the rapid IK current due to oleic acid. • Lipotoxicity is a key mechanism in obesity-related arrhythmias. [ABSTRACT FROM AUTHOR]

Modulation of endothelial nitric oxide synthase (eNOS) activation is recognized as a main trigger of the cardioprotective effects of exercise training on heart vulnerability to ischemia-reperfusion (IR). However, this enzyme is expressed both in coronary endothelial cells and cardiomyocytes and the contribution of each one to such cardioprotection has never been challenged. The aim of this study was to investigate the role of eNOS from the cardiomyocytes vs. the endothelium in the exercise cardioprotection. Male Wistar rats were assigned to a chronic aerobic training (Ex) (vs. sedentary group; Sed) and we investigated the role of eNOS in the effects of exercise on sensitivity to IR or anoxia-reoxygenation (A/R) at whole heart, isolated cardiomyocytes and left coronary artery (LCA) levels. We observed that exercise increased eNOS activation (Ser1177 phosphorylation) and protein S-nitrosylation in whole heart but not at cardiomyocyte level, suggesting the specific target of endothelial cells by exercise. Consistently, in isolated cardiomyocytes submitted to the A/R procedure, exercise reduced cell death and improved cells contractility, but independently of the eNOS pathway. Next, to evaluate the contribution of endothelial cells in exercise cardioprotection, LCA were isolated before and after an IR procedure performed on Langendorff hearts. Exercise improved basal relaxation sensitivity to acetylcholine and markedly reduced the alteration of endothelium-dependent coronary relaxation induced by IR. Furthermore, inactivation of coronary endothelial cells activity just before IR, obtained with a bolus of Triton X-100, totally suppressed cardioprotective effects of exercise on both left ventricular functional recovery after IR and infarct size, whereas no effect of Triton X-100 was observed in Sed group. In conclusion, these results show that coronary endothelial cells rather than cardiomyocytes play a key role in the eNOS-dependent cardioprotection of exercise. [ABSTRACT FROM AUTHOR]

Muscle satellite cells can proliferate and differentiate into myocytes; this biological function has important implications for muscle development, aging, repair, and wasting diseases. Established analytical methods, including western blotting, PCR, and immunohistochemistry, has been used to characterize the stages of satellite cells growth and development but there is currently a lack of methods to simultaneously monitor cell proliferation and identify changes in cell properties (i.e., expression of myogenic regulatory factors) for each individual cell during the progression to become myocytes. Image-based flow cytometry has the capability to simultaneously obtain morphometric data, monitor cell division, and detect expression of multiple proteins of interest. In this article we demonstrate the use of image-based flow cytometry and cell-trace dye to monitor satellite cells proliferation and to identify different stages of satellite cell differentiation. [ABSTRACT FROM AUTHOR]

Apoptosis occurs in atrial cardiomyocytes in mitral and tricuspid valve disease. The purpose of this study was to examine the respective roles of the mitochondrial and tumor necrosis factor-α receptor associated death domain (TRADD)-mediated death receptor pathways for apoptosis in the atrial cardiomyocytes of heart failure patients due to severe mitral and moderate-to-severe tricuspid regurgitation. This study comprised eighteen patients (7 patients with persistent atrial fibrillation and 11 in sinus rhythm). Atrial appendage tissues were obtained during surgery. Three purchased normal human left atrial tissues served as normal controls. Moderately-to-severely myolytic cardiomyocytes comprised 59.7 ± 22.1% of the cardiomyocytes in the right atria and 52.4 ± 12.9% of the cardiomyocytes in the left atria of mitral and tricuspid regurgitation patients with atrial fibrillation group and comprised 58.4 ± 24.8% of the cardiomyocytes in the right atria of mitral and tricuspid regurgitation patients with sinus rhythm. In contrast, no myolysis was observed in the normal human adult left atrial tissue samples. Immunohistochemical analysis showed expression of cleaved caspase-9, an effector of the mitochondrial pathways, in the majority of right atrial cardiomyocytes (87.3 ± 10.0%) of mitral and tricuspid regurgitation patients with sinus rhythm, and right atrial cardiomyocytes (90.6 ± 31.4%) and left atrial cardiomyocytes (70.7 ± 22.0%) of mitral and tricuspid regurgitation patients with atrial fibrillation. In contrast, only 5.7% of cardiomyocytes of the normal left atrial tissues showed strongly positive expression of cleaved caspase-9. Of note, none of the atrial cardiomyocytes in right atrial tissue in sinus rhythm and in the fibrillating right and left atria of mitral and tricuspid regurgitation patients, and in the normal human adult left atrial tissue samples showed cleaved caspase-8 expression, which is a downstream effector of TRADD of the death receptor pathway. Immunoblotting of atrial extracts showed that there was enhanced expression of cytosolic cytochrome c, an effector of the mitochondrial pathways, but no expression of membrane TRADD and cytosolic caspase-8 in the right atrial tissue of mitral and tricuspid regurgitation patients with sinus rhythm, and right atrial and left atrial tissues of mitral and tricuspid regurgitation patients with atrial fibrillation. Taken together, this study showed that mitochondrial pathway for apoptosis was activated in the right atria in sinus rhythm and in the left and right atria in atrial fibrillation of heart failure patients due to mitral and tricuspid regurgitation, and this mitochondrial pathway activation may contribute to atrial contractile dysfunction and enlargement in this clinical setting. [ABSTRACT FROM AUTHOR]

Skeletal muscle growth and development is controlled by nutritional (amino acids, AA) as well as hormonal factors (insulin-like growth factor, IGF-I); however, how its interaction modulates muscle mass in fish is not clearly elucidated. The purpose of this study was to analyze the development of gilthead sea bream cultured myocytes to describe the effects of AA and IGF-I on proliferating cell nuclear antigen (PCNA) and myogenic regulatory factors (MRFs) expression, as well as on the transduction pathways involved in its signaling (TOR/AKT). Our results showed that AA and IGF-I separately increased the number of PCNA-positive cells and, together produced a synergistic effect. Furthermore, AA and IGF-I, combined or separately, increased significantly Myogenin protein expression, whereas MyoD was not affected. These results indicate a role for these factors in myocyte proliferation and differentiation. At the mRNA level, AA significantly enhanced PCNA expression, but no effects were observed on the expression of the MRFs or AKT2 and FOXO3 upon treatment. Nonetheless, we demonstrated for the first time in gilthead sea bream that AA significantly increased the gene expression of TOR and its downstream effectors 4EBP1 and 70S6K, with IGF-I having a supporting role on 4EBP1 up-regulation. Moreover, AA and IGF-I also activated TOR and AKT by phosphorylation, respectively, being this activation decreased by specific inhibitors. In summary, the present study demonstrates the importance of TOR signaling on the stimulatory role of AA and IGF-I in gilthead sea bream myogenesis and contributes to better understand the potential regulation of muscle growth and development in fish. [ABSTRACT FROM AUTHOR]

Abstract: Recent studies have placed an increasing amount of emphasis on the cardiovascular system and understanding how the heart and its vasculature can be regenerated following pathological stresses, such as hypertension and myocardial infarction. The remodeling process involves the permanent cellular constituents of the heart including myocytes, fibroblasts, endothelial cells, pericytes, smooth muscle cells and stem cells. It also includes transient cell populations, such as immune cells (e.g. lymphocytes, mast cells and macrophages) and circulating stem cells. Following injury, there are dramatic shifts in the various cardiac cell populations that can affect cell–cell and cell–extracellular matrix interactions and cardiac function. Cardiac fibroblasts are a key component in normal heart function, as well as during the remodeling process through dynamic cell–cell interactions and synthesis and degradation of the extracellular matrix. Fibroblasts dynamically interact with the various cardiac cell populations through mechanical, chemical (autocrine and/or paracrine) and electrophysiological means to alter gene and protein expression, cellular processes and ultimately cardiac function. Better understanding these cell–cell and cell–extracellular matrix interactions and their biological consequences should provide novel therapeutic targets for the treatment of heart disease. In this review we discuss the nature of these interactions and the importance of these interactions in maintaining normal heart function, as well as their role in the cardiac remodeling process. This article is part of a Special Issue entitled "Myocyte-Fibroblast Signalling in Myocardium." [Copyright &y& Elsevier]

Abstract: Voltage gated sodium channels (NaVs) are essential to propagate neuronal and cardiac electrical impulses. While the cardiac Na+ current (INa) is often all attributed to the cardiac isoform, NaV1.5, some evidence suggests that other Na+ channel isoforms are also expressed in the heart ventricle. One way to distinguish Na+ channels is by their sensitivity to tetrodotoxin (TTX); various “non-cardiac-type” Na+ channels are relatively sensitive to TTX (denoted tNaV channels) compared to NaV1.5 channels. tNaV channels have been detected in hearts with various pathological conditions such as hypertrophy, infarction and ischemia, where they might enhance the late Na+ current (INaL) thereby prolonging the action potential under such conditions (resulting in a prolonged QT interval on the EKG). The principal aim of this article is to evaluate the extent to which non-cardiac isotypes contribute to INaL under normal physiological conditions. INaL was measured in acutely dissociated dog cardiomyocytes using the patch-clamp technique. Our results indicate that 44% on average of the late INa current is due to non-cardiac NaVs. Previous studies indicated that the overexpression of non-cardiac NaV channels is responsible for the prolonged duration of the cardiac action potential (and, thereby, a prolonged QT interval) under pathophysiological conditions associated with various heart diseases. Our finding indicates that non-cardiac NaV channels are strong contributors to INaL under physiological conditions thereby suggesting that these channels are also major determinants of the duration of the cardiac action potential even in healthy hearts. Interestingly, these results may explain the observations of cardiac arrhythmias associated with prolonged QT intervals in people with inherited neuronal and musculoskeletal diseases involving mutations that enhance the current from non-cardiac-type NaVs, a connection which apparently was never made before. [Copyright &y& Elsevier]

Abstract: The development of local control theories in cardiac excitation–contraction coupling solved a major problem in the calcium-induced calcium release (CICR) hypothesis. Local control explained how regeneration, inherent in the CICR mechanism, might be limited spatially to enable graded Ca release (and force production). The key lies in the stochastic recruitment of individual calcium release units (couplons or CRUs) where adjacent CRUs are partially uncoupled by the distance between them. In the CRU, individual groups of sarcoplasmic reticulum calcium release channels (RyRs) are very close to the surface membrane where calcium influx, controlled by membrane depolarization, leads to high local Ca levels that enable a high speed response from RyRs that have a very low probability to opening at resting Ca levels. However, calcium diffusion from an activated CRU results in adjacent CRUs being exposed to much lower levels of Ca and probability of activation. This effectively uncouples the CRUs and limits overall regenerative gain to enable stability without compromising sensitivity. Nevertheless, it is still unclear how the CRU terminates its release of calcium on the physiological timescale, and possible mechanisms (and problems) are briefly reviewed. We suggest that modulation in RyR gating may serve to control average SR Ca levels to regulate other metabolic functions of the sarco(endo)plasmic reticulum beyond regulating contractility. This article is part of a special issue entitled "Local Signaling in Myocytes." [Copyright &y& Elsevier]

Abstract: The transverse (t-) tubule network is an important site for Ca influx and release during excitation-contraction coupling in cardiac ventricular myocytes; however, its role in Ca extrusion is less clear. The present study was designed to investigate the relative contributions of Ca extrusion pathways across the t-tubule and surface membranes. Ventricular myocytes were isolated from the hearts of adult male Wistar rats and detubulated using formamide. Intracellular Ca was monitored using fluo-3 and confocal microscopy. Caffeine (20mmol/L) was used to induce SR Ca release; carboxyeosin (20μmol/L) and nickel (10mmol/L) were used to inhibit the sarcolemmal Ca ATPase and Na/Ca exchanger (NCX) respectively. Carboxyeosin decreased the rate constant of decay of the caffeine-induced Ca transient in control cells, but had no effect in detubulated cells, suggesting that Ca extrusion via the Ca ATPase occurs only across the t-tubule membrane. However nickel decreased the rate constant of the caffeine-induced Ca transient in control and detubulated cells, although its effect was greater in control cells, suggesting that Ca extrusion via NCX occurs across the surface and t-tubule membranes. The PKA inhibitor H-89 (10μmol/L) was used to investigate the role of basal PKA activity in Ca extrusion; H-89 appeared to have no effect on Ca extrusion via the Ca ATPase, but reduced Ca extrusion via NCX at the t-tubules but not the surface membrane. Thus it appears that Ca extrusion via the sarcolemmal Ca ATPase occurs only at the t-tubules, and is not regulated by basal PKA activity, while Ca extrusion via NCX occurs across both the surface and t-tubule membranes, but predominantly across the t-tubule membrane due, in part, to localised stimulation of NCX by PKA at the t-tubules. This may be important in heart disease, in which changes in t-tubule structure and protein phosphorylation occur. [ABSTRACT FROM AUTHOR]

Abstract: The t-tubule network is central to excitation–contraction coupling in mammalian cardiac ventricular myocytes, with recent studies showing that the majority of Ca influx via the L-type Ca current (I Ca) occurs across the t-tubule membrane. The present study investigated whether tonic phosphorylation of the L-type Ca channel is different at the t-tubule and surface membranes, and if this could account for the high density of I Ca at the t-tubules. Ventricular myocytes were isolated from male Wistar rats and detubulated using formamide. I Ca was recorded using the whole cell patch clamp technique, and Ca transients were recorded using fluo-3 in conjunction with confocal microscopy. The protein kinase A (PKA) inhibitor H-89 (10μmol/L) and the CaMKII inhibitor KN-93 (5μmol/L) decreased the amplitude of I Ca in intact cells but had no effect on I Ca amplitude in detubulated cells. These inhibitors also decreased the amplitude of the Ca transient in intact cells but not in detubulated cells. Antibody staining for phosphorylated L-type Ca channel showed significantly higher phosphorylation at the t-tubules than at the surface membrane in intact cells. Thus it appears that tonic phosphorylation of the L-type Ca channel maintains the amplitude of I Ca and occurs predominantly at the t-tubules. This may have important implications in heart disease, in which changes of phosphorylation and t-tubule density have been reported. [Copyright &y& Elsevier]

Abstract: Using deconvolved confocal microscopy of fluorescently labeled markers for z-disks, t-tubules and ryanodine receptors, we have examined sarcomere organization in cardiac myocytes from rat, rabbit and human. We show that sarcomeres exhibit dislocations in registration and occasionally more complex helicoidal topology. This organization was present at both slack (∼1.8 μm) and long sarcomere lengths (∼2.2 μm). Misregistrations in z-disks persisted over 15–20 sarcomere lengths and appeared to arise primarily from variations in fiber direction; particularly as myofibrils pass around nuclei. In addition, myofibrils twist along the cell length. T-tubules generally follow the sarcomere z-disks although additional elements bridging adjacent myofibrils and along the length of the myofibril are present to varying degrees in all cells. Ryanodine receptors (the sarcoplasmic reticulum Ca2+ release channel) are generally located within 250 nm of the local plane containing t-tubules and z-disks, but a small fraction (∼2%) is found on longitudinal elements of the t-system between z-disks. The results are discussed with respect to the possible role(s) of such complex z-disk organization and z-disk dislocations in the maintenance of cell structure and sarcomere assembly. In addition, the non-planar organization of z-disks may be important in the propagation of local Ca2+ waves which may have a useful role in helping maintain the uniformity of sarcomere activation in the presence of t-tubule remodeling. [Copyright &y& Elsevier]

Abstract: Biglycan, a proteoglycan component of extracellular matrix, has been suspected to contribute to the development of atherosclerosis, but overexpression of biglycan in transgenic mice has been shown to induce cardioprotective genes including nitric oxide (NO) synthases in the heart. Therefore, here we hypothesized if exogenous administration of biglycan exerts cytoprotection. Primary cardiomyocytes from neonatal rats were subjected to 150 min hypoxia and 2 h reoxygenation. Mortality of cardiomyocytes was dose-dependently attenuated by pretreatment with 1–100 nM biglycan. Biglycan enhanced eNOS mRNA and protein, and significantly increased NO content of cardiomyocytes. The NO synthase inhibitor l-nitro-arginine-methyl-ester significantly attenuated the cytoprotective effect of biglycan. This is the first demonstration that biglycan leads to cytoprotection against hypoxia/reoxygenation injury, and that this phenomenon is partially mediated by an NO-dependent mechanism. [Copyright &y& Elsevier]

Abstract: Voltage-gated sodium channels are composed of pore-forming α- and auxiliary β-subunits and are responsible for the rapid depolarization of cardiac action potentials. Recent evidence indicates that neuronal tetrodotoxin (TTX) sensitive sodium channel α-subunits are expressed in the heart in addition to the predominant cardiac TTX-resistant Nav1.5 sodium channel α-subunit. These TTX-sensitive isoforms are preferentially localized in the transverse tubules of rodents. Since neonatal cardiomyocytes have yet to develop transverse tubules, we determined the complement of sodium channel subunits expressed in these cells. Neonatal rat ventricular cardiomyocytes were stained with antibodies specific for individual isoforms of sodium channel α- and β-subunits. α-actinin, a component of the z-line, was used as an intracellular marker of sarcomere boundaries. TTX-sensitive sodium channel α-subunit isoforms Nav1.1, Nav1.2, Nav1.3, Nav1.4 and Nav1.6 were detected in neonatal rat heart but at levels reduced compared to the predominant cardiac α-subunit isoform, Nav1.5. Each of the β-subunit isoforms (β1–β4) was also expressed in neonatal cardiac cells. In contrast to adult cardiomyocytes, the α-subunits are distributed in punctate clusters across the membrane surface of neonatal cardiomyocytes; no isoform-specific subcellular localization is observed. Voltage clamp recordings in the absence and presence of 20 nM TTX provided functional evidence for the presence of TTX-sensitive sodium current in neonatal ventricular myocardium which represents between 20 and 30% of the current, depending on membrane potential and experimental conditions. Thus, as in the adult heart, a range of sodium channel α-subunits are expressed in neonatal myocytes in addition to the predominant TTX-resistant Nav1.5 α-subunit and they contribute to the total sodium current. [Copyright &y& Elsevier]

Abstract: The role that Ca2+ plays in ventricular excitation contraction coupling is well defined and much is known about the marked differences in the spatiotemporal properties of the systolic Ca2+ transient between atrial and ventricular myocytes. However, to date there has been no systematic appraisal of the Ca2+ homeostatic mechanisms employed by atrial cells and how these compare to the ventricle. In the present study we sought to determine the fractional contributions made to the systolic Ca2+ transient and the decay of [Ca2+]i by the sarcoplasmic reticulum and sarcolemmal mechanisms. Experiments were performed on single myocytes isolated from the atria and ventricles of the rat. Intracellular Ca2+ concentration, membrane currents, SR Ca2+ content and cellular Ca2+ buffering capacity were measured at 23 °C. Atrial cells had smaller systolic Ca2+ transients (251±39 vs. 376±41 nmol.L−1) that decayed more rapidly (7.4±0.6 vs. 5.45±0.3 s−1). This was due primarily to an increased rate of SR mediated Ca2+ uptake (k SR, 6.88±0.6 vs. 4.57±0.3 s−1). SR Ca2+ content was 289% greater and Ca2+ buffering capacity was increased ∼3-fold in atrial cells (B max 371.9±32.4 vs. 121.8±8 μmol.L−1, all differences P <0.05). The fractional release of Ca2+ from the SR was greater in atrial cells, although the gain of excitation contraction coupling was the same in both cell types. In summary our data demonstrate fundamental differences in Ca2+ homeostasis between atrial and ventricular cells and we speculate that the increased SR Ca2+ content may be significant in determining the increased prevalence of arrhythmias in the atria. [Copyright &y& Elsevier]

Abstract: Chronic ventricular pressure overload states, such as hypertension, and elevated levels of neurohormones (norepinephrine, angiotensin II, endothelin-1) initiate cardiac hypertrophy and dysfunction and share the property of being able to bind to Gq-coupled 7-transmembrane receptors. The goal of the current study was to determine the role of endogenous cardiac myocyte Gq signaling and its role in cardiac hypertrophy and dysfunction during high blood pressure (BP). We induced renal artery stenosis for 8 weeks in control mice and mice expressing a peptide inhibitor of Gq signaling (GqI) using a 2 kidney, 1 clip renal artery stenosis model. 8 weeks following chronic high BP, control mice had cardiac hypertrophy and depressed function. Inhibition of cardiomyocyte Gq signaling did not reverse cardiac hypertrophy but attenuated increases in a profile of cardiac profibrotic genes and genes associated with remodeling. Inhibition of Gq signaling also attenuated the loss of cardiac function. We determined that Gq signaling downstream of angiotensin II receptor stimulation negatively impacted β-adrenergic receptor (AR) responses and inhibition of Gq signaling was sufficient to restore βAR-mediated responses. Therefore, in this study we found that Gq signaling negatively impacts cardiac function during high BP. Specifically, we found that inhibition of AT1-Gq signaling augmented βAR mediated effects in a renal artery stenosis model of hypertension. These observations may underlie additional, beneficial effects of angiotensinogen converting enzyme (ACE) inhibitors and angiotensin receptor antagonists observed during times of hemodynamic stress. [Copyright &y& Elsevier]

Abstract: Mutations in the human ether-a-go-go-related gene (hERG) cause type 2 long QT syndrome. In this study, we investigated the pathogenic mechanism of the hERG splice site mutation 2398+1G>C and the genotype–phenotype relationship of mutation carriers in three unrelated kindreds with long QT syndrome. The effect of 2398+1G>C on mRNA splicing was studied by analysis of RNA isolated from lymphocytes of index patients and using minigenes expressed in HEK293 cells and neonatal rat ventricular myocytes. RT-PCR analysis revealed that the 2398+1G>C mutation disrupted the normal splicing and activated a cryptic splice donor site in intron 9, leading to the inclusion of 54 nt of the intron 9 sequence in hERG mRNA. The cryptic splicing resulted in an in-frame insertion of 18 amino acids in the middle of the cyclic nucleotide binding domain. In patch clamp experiments the splice mutant did not generate hERG current. Western blot and immunostaining studies showed that the mutant expressed an immature form of hERG protein that failed to reach the plasma membrane. Coexpression of the mutant and wild-type channels led to a dominant negative suppression of wild-type channel function by intracellular retention of heteromeric channels. Our results demonstrate that 2398+1G>C activates a cryptic site and generates a full-length hERG protein with an insertion of 18 amino acids, which leads to a trafficking defect of the mutant channel. [Copyright &y& Elsevier]

Abstract: Cardiac adaptation to aerobic exercise training includes improved cardiomyocyte contractility and calcium handling. Our objective was to determine whether cytosolic calcium/calmodulin-dependent kinase II and its downstream targets are modulated by exercise training. A six-week aerobic interval training program by treadmill running increased maximal oxygen uptake by 35% in adult mice, whereupon left ventricular cardiomyocyte function was studied and myocardial tissue samples were used for biochemical analysis. Cardiomyocytes from trained mice had enhanced contractility and faster relaxation rates, which coincided with larger amplitude and faster decay of the calcium transient, but not increased peak systolic calcium levels. These changes were associated with reduced phospholamban expression relative to sarcoplasmic reticulum calcium ATPase and constitutively increased phosphorylation of phospholamban at the threonine 17, but not at the serine 16 site. Calcium/calmodulin-dependent kinase IIδ phosphorylation was increased at threonine 287, indicating activation. To investigate the physiological role of calcium/calmodulin-dependent kinase IIδ phosphorylation, this kinase was blocked specifically by autocamtide-2 related inhibitory peptide II. This maneuver completely abolished training-induced improvements of cardiomyocyte contractility and calcium handling and blunted, but did not completely abolish the training-induced increase in Ca2+ sensitivity. Also, inhibition of calcium/calmodulin-dependent kinase II reduced the greater frequency-dependent acceleration of relaxation that was observed after aerobic interval training. These observations indicate that calcium/calmodulin-dependent kinase IIδ contributes significantly to the functional adaptation of the cardiomyocyte to regular exercise training. [Copyright &y& Elsevier]

Abstract: It is established that prolonged hypoxia leads to activation of KATP channels and action potential (AP) shortening, but the mechanisms behind the early phase of metabolic stress remain controversial. Under normal conditions IK1 channels are constitutively active while KATP channels are closed. Therefore, early changes in IK1 may underlie early AP shortening. This hypothesis was tested using transgenic mice with suppressed IK1 (AAA-TG). In isolated AAA-TG hearts AP shortening was delayed by ∼24 s compared to WT hearts. In WT ventricular myocytes, blocking oxidative phosphorylation with 1 mM cyanide (CN; 28 °C) led to a 29% decrease in APD90 within ∼3–5 min. The effect of CN was reversed by application of 100 μM Ba2+, a selective blocker of IK1, but not by 10 μM glybenclamide, a selective blocker of KATP channels. Accordingly, voltage-clamp experiments revealed that both CN and true hypoxia lead to early activation of IK1. In AAA-TG myocytes, neither CN nor glybenclamide or Ba2+ had any effect on AP. Further experiments showed that buffering of intracellular Ca2+ with 20 mM BAPTA prevented IK1 activation by CN, although CN still caused a 54% increase in IK1 in a Ca2+-free bath solution. Importantly, both (i) 20 μM ruthenium red, a selective inhibitor of SR Ca2+-release, and (ii) depleting SR by application of 10 μM ryanodine+1 mM caffeine, abolished the activation of IK1 by CN. The above data strongly argue that in the mouse heart IK1, not KATP, channels are responsible for the early AP shortening during hypoxia. [Copyright &y& Elsevier]

Abstract: Apelin, the ligand for the angiotensin receptor like-1, has been implicated in the pathogenesis of atrial fibrillation and heart failure. However, it is unknown if apelin has direct effects on cardiomyocyte contractility and electrophysiology. APJ-like immunoreactivity was localized to T-tubules and intercalated disc area in isolated adult rat ventricular myocytes. Apelin (1nM) significantly increased sarcomere shortening in normal as well as failing cardiomyocytes. The transient increase in shortening was not accompanied by increased [Ca2+] transient amplitude. Apelin significantly activated the sarcolemmal Na+/H+ exchanger (NHE) and increased intracellular pH. Moreover, apelin (10nM) increased conduction velocity in monolayers of cultured neonatal rat cardiac myocytes. Our results demonstrate for the first time that apelin has direct effects on the propagation of action potential and contractility in cardiomyocytes. One of the mechanisms involved in the inotropic effect may be an increased myofilament sensitivity to Ca2+ as apelin enhanced the activity of NHE with consequent intracellular alkalinization. [Copyright &y& Elsevier]

Abstract: There is an association between obesity and heart failure associated with LV dysfunction. Adiponectin is an adipocyte-derived hormone that is downregulated in obesity. Here, we examined the role of adiponectin in cardiac remodeling after myocardial infarction with loss- and gain-of-function genetic manipulations in an experimental model. Myocardial infarction was created in adiponectin-deficient (APN-KO) and wild-type (WT) mice by the permanent ligation of the left anterior descending (LAD) artery. For some experiments, adenoviral vectors expressing adiponectin or β-galactosidase were delivered systemically. Cardiac structure and function were assessed by echocardiographic and Millar catheter measurements. Myocardial capillary density was assessed by staining with anti-CD31 antibody. Myocyte apoptotic activity was determined by TUNEL-staining. Myocardial interstitial fibrosis was evaluated by Masson''s trichrome staining. APN-KO mice showed exacerbated left ventricular (LV) dilation, myocyte hypertrophy and contractile dysfunction compared with WT mice at 4 weeks after LAD ligation. Impaired LV function in APN-KO mice was coupled to myocyte hypertrophy, increased apoptotic activity and interstitial fibrosis in the remote zone, and reduced capillary density in the infarct border zone. No difference in infarct size was observed between WT and APN-KO mice. Administration of adenovirus-mediated adiponectin in WT mice resulted in decreased LV dilatation and improved LV function that was associated with increased capillary density in the infarct border zone and decreased myocyte hypertrophy, diminished myocardial apoptosis and decreased interstitial fibrosis in the remote zone. These data suggest that adiponectin protects against the development of systolic dysfunction after myocardial infarction through its abilities to suppress cardiac hypertrophy and interstitial fibrosis, and protect against myocyte and capillary loss. [Copyright &y& Elsevier]

Abstract: Myocardial gene therapy continues to show promise as a tool for investigation and treatment of cardiac disease. Progress toward clinical approval has been slowed by limited in vivo delivery methods. We investigated the problem in a porcine model, with an objective of developing a method for high efficiency, homogeneous myocardial gene transfer that could be used in large mammals, and ultimately in humans. Eighty-one piglets underwent coronary catheterization for delivery of viral vectors into the left anterior descending artery and/or the great cardiac vein. The animals were followed for 5 or 28 days, and then transgene efficiency was quantified from histological samples. The baseline protocol included treatment with VEGF, nitroglycerin, and adenosine followed by adenovirus infusion into the LAD. Gene transfer efficiency varied with choice of viral vector, with use of VEGF, adenosine, or nitroglycerin, and with calcium concentration. The best results were obtained by manipulation of physical parameters. Simultaneous infusion of adenovirus through both left anterior descending artery and great cardiac vein resulted in gene transfer to 78±6% of myocytes in a larger target area. This method was well tolerated by the animals. We demonstrate targeted, homogeneous, high efficiency gene transfer using a method that should be transferable for eventual human usage. [Copyright &y& Elsevier]

Abstract: 5-Hydroxytryptamine (5-HT) is proarrhythmic in atrial cells from patients in sinus rhythm (SR) via activation of 5-HT4 receptors, but its effects in atrial cells from patients with atrial fibrillation (AF) are unknown. The whole-cell perforated patch-clamp technique was used to record L-type Ca2+ current (I CaL), action potential duration (APD) and arrhythmic activity at 37°C in enzymatically isolated atrial cells obtained from patients undergoing cardiac surgery, in SR or with chronic AF. In the AF group, 5-HT (10μM) produced an increase in I CaL of 115±21% above control (n =10 cells, 6 patients) that was significantly smaller than that in the SR group (232±33%; p <0.05; n =27 cells, 12 patients). Subsequent co-application of isoproterenol (1μM) caused a further increase in I CaL in the AF group (by 256±94%) that was greater than that in the SR group (22±6%; p <0.05). The APD at 50% repolarisation (APD50) was prolonged by 14±3ms by 5-HT in the AF group (n =37 cells, 14 patients). This was less than that in the SR group (27±4ms; p <0.05; n =58 cells, 24 patients). Arrhythmic activity in response to 5-HT was observed in 22% of cells in the SR group, but none was observed in the AF group (p <0.05). Atrial fibrillation was associated with reduced effects of 5-HT, but not of isoproterenol, on I CaL in human atrial cells. This reduced effect on I CaL was associated with a reduced APD50 and arrhythmic activity with 5-HT. Thus, the potentially arrhythmogenic influence of 5-HT may be suppressed in AF-remodelled human atrium. [Copyright &y& Elsevier]

Abstract: In the mammalian heart, the circadian protein Clock regulates glucose and fatty acid metabolism. In this study, we determined some of the factors that regulate Clock expression and subcellular distribution in myocytes. Using immunochemistry and biochemical subcellular fractionation, we have shown that Clock localizes to the Z-disk of the myofilaments. Increasing calcium and cross-bridge cycling with 10μM phenylephrine for 48h resulted in a threefold increase in Clock and a translocation of the protein to the nucleus. When myofilament cross-bridge cycling was inhibited with 10μM verapamil or 7.5mM butanedione monoxime for 48h, both significantly reduced the presence of Clock in the nucleus and cytoskeleton. These results suggest that the expression and subcellular distribution of Clock can be altered by changes in cross-bridge cycling, a major source of energy expenditure in myocytes. We suggest that the circadian Clock protein may help coordinate the sensing of energy expenditure with energy supply. [Copyright &y& Elsevier]

Abstract: Treatment of metastatic breast cancer with doxorubicin (Doxo) in combination with trastuzumab, an antibody targeting the ErbB2 receptor, results in an increased incidence of heart failure. Doxo therapy induces reactive oxygen species (ROS) and alterations of calcium homeostasis. Therefore, we hypothesized that neuregulin-1 beta (NRG), a ligand of the cardiac ErbB receptors, reduces Doxo-induced alterations of EC coupling by triggering antioxidant mechanisms. Adult rat ventricular cardiomyocytes (ARVM) were isolated and treated for 18–48 h. SERCA protein was analyzed by Western blot, EC coupling parameters by fura-2 and video edge detection, gene expression by RT-PCR, and ROS by DCF-fluorescence microscopy. At clinically relevant doses Doxo reduced cardiomyocytes contractility, SERCA protein and SR calcium content. NRG, similarly as the antioxidant N-acetylcystein (NAC), did not affect EC coupling alone, but protected against Doxo-induced damage. NRG and Doxo showed an opposite modulation of glutathione reductase gene expression. NRG, similarly as NAC, reduced peroxide- or Doxo-induced oxidative stress. Specific inhibitors showed, that the antioxidant action of NRG depended on signaling via the ErbB2 receptor and on the Akt- and not on the MAPK-pathway. Therefore, NRG attenuates Doxo-induced alterations of EC coupling and reduces oxidative stress in ARVM. Inhibition of the ErbB2/NRG signaling pathway by trastuzumab in patients concomitantly treated with Doxo might prevent beneficial effects of NRG in the myocardium. [Copyright &y& Elsevier]

Abstract: Objective: We have recently identified an activation site on (Na+ +K+)-ATPase and found that binding of antibody SSA412 to this specific site of the enzyme markedly augments (Na+ +K+)-ATPase catalytic activity. Demonstration of whether activation of (Na+ +K+)-ATPase affects heart function in animal in vivo was the object of this investigation. Methods: Male wild-type CD-1 mouse and specific antibody SSA412 were used for the study. A pressure–volume micromanometer-conductance catheter in anesthetized mouse assessed in vivo cardiac functions. Results: Specific antibody SSA412 infusion in mouse shifted pressure–volume loop leftward with increased stroke volume and enhanced end-systolic elastance. Global systolic parameters such as ejection fraction and cardiac output, and load independent contractile parameters including dP/dt max/IP, PMX/EDV, Ees, and PRSW, were all increased without any effect on relaxation following administration of SSA412. Cardiac preload indexed by EDV and afterload by ESP did not alter, suggesting that SSA412-enhanced myocardial performance is a direct cardiac effect caused by the activation of (Na+ +K+)-ATPase. Conclusion: Our study provides the first in vivo physiological evidence to demonstrate that activation of (Na+ +K+)-ATPase induces significant positive inotropic effect in intact animal heart. The finding may lead to new therapeutic strategies for the treatment of heart failure. [Copyright &y& Elsevier]

Abstract: Complement activation augments myocardial cell injury and apoptosis during ischemia/reperfusion (I/R), whereas complement system inhibition with C1 inhibitor (C1INH), a serine protease inhibitor, exerts markedly cardioprotective effects. Our recent data demonstrate that C1INH prevents vascular endothelial cell apoptosis and a “modified” form of the reactive center loop-cleaved, inactive C1INH (iC1INH) plays an anti-inflammatory role in endotoxin shock. The aim of this study was to determine whether C1INH protects against myocardial cell injury via an anti-apoptotic activity or anti-inflammatory effect. In a rat model of acute myocardial infarction (AMI) induced by I/R, administration of C1INH protected against cardiomyocytic apoptosis via normalization of ratio of the Bcl-2/Bax expression in the myocardial infarct area. C1INH improved parameters of cardiac function and hemodynamics and reduced myocardial infarct size (MIS). In addition, myocardial and blood myeloperoxidase (MPO) activity, a marker of neutrophil infiltration, was decreased by treatment of C1INH. In cultured H9c2 rat cardiomyocytic cells, C1INH blocked hypoxia/reoxygenation-induced apoptosis in the absence of sera associated with inhibition of cytochrome c translocation and suppression of caspase-3 activation. The proportion of Bcl-2/Bax expression induced by hypoxia/reoxygenation was reversed by C1INH. Importantly, iC1INH also revealed these similar effects, indicating that C1INH has a direct anti-apoptotic activity. Therefore, these studies support the hypothesis that C1INH, in addition to inhibition of activation of the complement and contact systems, improves outcome in I/R-mediated myocardial cell injury via an anti-apoptotic activity independent of serine protease inhibitory activity. [Copyright &y& Elsevier]

Abstract: In cardiac ventricular myocytes, Na current is generated mainly by the cardiac NaV1.5 isoform, but the presence of “neuronal” Na channel isoforms in the heart has been demonstrated recently. In this study, we quantified the density and sub-cellular distribution of cardiac and neuronal channel isoforms in rat ventricular myocytes. I Na was recorded using the patch clamp technique in control and detubulated myocytes. Detubulation reduced cell capacitance (by ∼29%) but maximum conductance was not altered (1.94±0.15, 14 control vs 1.98±0.19nS/pF, 17 detubulated myocytes). The kinetic properties of I Na were similar in both cell types suggesting good voltage control of surface and t-tubule membranes. We calculated Na channel densities assuming the sub-cellular current localization we recently provided (neuronal isoform: ∼11% of total sarcolemmal current, ∼3% of cell surface, and ∼31% of t-tubule current). Single channel conductances were assumed to be 2.2 and 2.5pS for the cardiac and neuronal isoforms, respectively, after accounting for the use of low Na concentration. We calculated that the density of the cardiac Na channel isoform is relatively constant (in channels/μm2: ∼11 in total sarcolemma, ∼13 at the cell surface, ∼10 at the t-tubules). In contrast, neuronal Na channel isoforms are concentrated at the t-tubules (in channels/μm2: ∼1 in total sarcolemma, ∼0.3 at the cell surface, ∼2.5 at the t-tubules). We conclude that, in contrast to skeletal muscle in which Na channel density is higher at the cell surface than the t-tubules, in ventricular cardiac myocytes the sub-cellular distribution of Na channel density is relatively homogeneous (∼13 channels/μm2). [Copyright &y& Elsevier]

Abstract: Atrial fibrillation (AF) causes myocyte stress by inducing structural changes, predominantly myolysis, which is related to the progression of AF. As heat shock proteins (Hsp) protect against cellular stress, their efficacy in preventing myolysis was investigated in a tachy-paced cell model for AF and in patients with AF. HL-1 atrial myocytes were subjected to tachy-pacing, which induced myolysis. Hsp overexpression was accomplished by a mild heat shock or by the drug geranylgeranylacetone (GGA). Hsp-gene-transfection studies were carried out to investigate roles of individual Hsp. In left and/or right atrial appendages from patients with paroxysmal (n =14), persistent (n =17) AF and controls (n =13) in sinus rhythm (SR), Hsp levels (Westerns) and localization (confocal microscopy) were determined. Heat shock and GGA administered prior to tachy-pacing resulted in almost complete protection against tachy-pacing-induced myolysis. Overexpression of Hsp27, but not of Hsp70, also provided complete protection against pacing-induced myolysis. In patients with paroxysmal AF, Hsp27 expression was significantly increased compared to SR and persistent AF. No changes in Hsp40, Hsc70, Hsp70 and Hsp90 expression levels were observed. Hsp27 levels correlated inversely with the duration of paroxysmal and persistent AF and the extent of myolysis. Furthermore, Hsp27 was localized on myofibrils in tachy-paced HL-1 myocytes and in human cardiomyocytes. These data demonstrate that upregulation of Hsp, especially Hsp27, protects tachy-paced atrial myocytes from myolysis. Therefore, the observed elevated Hsp27 expression in patients with paroxysmal AF might serve to protect myocytes from myolysis and limit the progression to persistent AF. Pharmacological induction of Hsp, with drugs such as GGA, may represent a novel therapeutic approach in AF. [Copyright &y& Elsevier]

Abstract: Electrical remodeling of the diseased heart contributes to contractile dysfunction and arrhythmias, and is characterized by down-regulation of K+ channels that control action potential morphology. We have recently shown that remodeling of K+ channels underlying the transient outward current (Ito) involves a shift in cell redox balance that is reflected by a depletion of the endogenous redox buffer, glutathione (GSH). This study used a pharmacological model to further examine the role of redox-mediated mechanisms in regulating cardiac K+ currents. Inhibition of major redox pathways was elicited in normal rats by daily injections of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of thioredoxin and glutathione reductases, and buthionine sulfoximine (BSO), a blocker of GSH synthesis. Fluorescence microscopy studies showed that [GSH] in isolated ventricular myocytes was decreased ~50% from control after 3 days of BCNU/BSO treatment (P <0.05), consistent with a shift in cell redox state. In voltage-clamp experiments, maximum Ito density was decreased 33% from control in left ventricular myocytes from BCNU/BSO-treated rats (P <0.05), while the inward rectifier and steady state outward currents were not significantly altered. Decreased Ito density correlated with significant decreases in Kv4.2 mRNA and proteins levels of Kv4.2 and Kv1.4. Down-regulation of Ito in myocytes from BCNU/BSO rats was reversed in vitro by exogenous GSH or N-acetylcysteine, a GSH precursor and antioxidant. Ito density and [GSH] were also up-regulated by receptor tyrosine kinase activation with insulin or a tyrosine phosphatase inhibitor. The effect of these activators on Ito was blocked by inhibitors of PI 3-kinase, MEK and p38 MAP kinases. These data suggest that expression of cardiac Ito channels is regulated by endogenous oxidoreductase systems and that receptor tyrosine kinase signaling functionally impacts K+ channel remodeling through its control of cell redox state. [Copyright &y& Elsevier]

Abstract: Acetaldehyde, the major metabolite of ethanol, which is far more toxic and reactive than ethanol, may be responsible for alcohol-induced cardiac damage. This study was designed to examine the impact of facilitated acetaldehyde metabolism using transfection of human aldehyde dehydrogenase-2 (ALDH2) transgene on acetaldehyde- and ethanol-induced cell injury. Fetal human cardiac myocytes were transfected with ALDH2, the efficacy of which was verified by flow cytometry, Western blot and ALDH2 activity assays. Generation of reactive oxygen species (ROS) was detected using 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA). Apoptosis was evaluated by 4′,6′-diamidino-2′-phenylindoladihydrochloride (DAPI) fluorescence microscopy, quantitative DNA fragmentation ELISA and caspase 3 activity. Acetaldehyde and ethanol elicited overt ROS generation and apoptosis in human cardiac myocytes following 24–48 h of incubation. Immunostaining revealed activation of the MAP kinase cascades ERK1/2, SAPK/JNK and p38 MAP kinase in acetaldehyde-treated myocytes. Interestingly, ALDH2 transgene significantly attenuated acetaldehyde-induced ROS generation, apoptosis and phosphorylation of ERK1/2 and SAPK/JNK. Time-dependent response (0–12 h) revealed ROS accumulation and activation of MAP kinases prior to acetaldehyde-induced apoptosis. In addition, acetaldehyde-induced ROS generation and apoptosis were antagonized by non-enzymatic antioxidants. Our results suggested that ALDH2 transgene overexpression may effectively alleviate acetaldehyde-elicited cell injury through an ERK1/2 and SPAK/JNK-dependent mechanism. Our data are consistent with the notion of acetaldehyde as a contributor to alcoholic cardiomyopathy and implicate the therapeutic potential of ALDH2 enzyme in alcoholic complications. [Copyright &y& Elsevier]

Abstract: Prostaglandin F2α (PGF2α) plays an important role in pathologic cardiac growth. After testing several immune cytokines, we found that interferon-γ (IFN-γ) inhibited responsiveness of adult myocytes to PGF2α. The present study was designed to test the hypothesis that IFN-γ inhibits cardiac hypertrophy induced by PGF2α. Incubation of cultured adult rat cardiac myocytes with PGF2α caused cell spreading, which was inhibited by IFN-γ. The inhibitory effect was not affected by nitric oxide (NO) synthase inhibitors. In addition, administration of fluprostenol, a more selective agonist at the PGF2α receptor, induced cardiac hypertrophy in rats. Chronic treatment with IFN-γ inhibited this myocardial growth, and the inhibitory effect of IFN-γ was not accompanied by an increase in myocardial NO synthase gene expression. Further, abdominal aortic constriction resulted in a substantial increase in heart, ventricular and left ventricular weights to BW ratio that was significantly attenuated by treatment with IFN-γ. The results demonstrate that IFN-γ inhibits the in vitro and in vivo effects of PGF2α on cardiac hypertrophy, and that the mechanism of action is likely independent of NO production. IFN-γ also attenuated cardiac hypertrophy induced by pressure overload, suggesting that PGF2α plays a role in the pathogeneses of this severe type of cardiac hypertrophy. [Copyright &y& Elsevier]

Abstract: S100A6 (calcyclin), a member of the S100 family of EF-hand Ca2+ binding proteins, has been implicated in the regulation of cell growth and proliferation. We have previously shown that S100B, another member of the S100 family, is induced postinfarction and limits the hypertrophic response of surviving cardiac myocytes. We presently report that S100A6 expression is also increased in the periinfarct zone of rat heart postinfarction and in cultured neonatal rat myocytes by treatment with several trophic agents, including platelet-derived growth factor (PDGF), the α1-adrenergic agonist phenylephrine (PE), and angiotensin II (AII). Cotransfection of S100A6 in cultured neonatal rat cardiac myocytes inhibits induction of the cardiac fetal gene promoters skeletal α-actin (skACT) and β-myosin heavy chain (β-MHC) by PDGF, PE, AII, and the prostaglandin F2α (PGF2α), induction of the S100B promoter by PE, and induction of the α-MHC promoter by triiodothyronine (T3). By contrast, S100B cotransfection selectively inhibited only PE induction of skACT and β-MHC promoters. Fluorescence microscopy demonstrated overlapping intracellular distribution of S100B and S100A6 in transfected myocytes and in postinfarct myocardium but heterodimerization of the two proteins could not be detected by co-immunoprecipitation. We conclude that S100A6 may function as a global negative modulator of differentiated cardiac gene expression comparable to its putative role in cell cycle progression of dividing cells. [Copyright &y& Elsevier]

Following diazoxide (DZ) induced hypoinsulinemia, cardiac luminal lipoprotein lipase (LPL) increases [Cardiovasc. Res. 3 (2003) 788]. To identify circulating mediators that maintain high LPL in vivo, DZ hearts were perfused for 1 h in the presence or absence of glucose, triglyceride (TG), palmitic acid or palmitoyl lysophosphatidylcholine (PLPC). Only PLPC maintained high luminal LPL in DZ hearts, likely through enzyme recruitment from the cardiomyocyte. PLPC perfusion activated whole heart protein kinase C (PKC) ε. As calphostin pretreatment blocked PLPC induced PKC activation, and increases in luminal LPL activity, PKC activation is essential for the effects of PLPC. Incubation of myocytes with PLPC had no effects on either surface or intracellular LPL or PKC suggesting that PKC activation occurs in cells other than the myocyte or that metabolism of PLPC is required for its downstream effects. Since exposure of endothelial cells to PLPC activated PKC, whole heart PKC activation likely occurred in these cells. Incubation of myocytes with LPA, a phospholipase D (PLD) mediated breakdown metabolite of PLPC, significantly enhanced basal and heparin-releasable myocyte LPL activity, an effect that was duplicated by co-incubation of control myocytes with exogenous PLD and PLPC. Our data suggest that at least in the whole heart, the LPL augmenting property of PLPC likely requires endothelial PKC activation, formation of LPA, and mobilization of enzyme from the myocyte to the coronary lumen. [Copyright &y& Elsevier]

Despite many studies into the pathophysiology of cardiac ischemia–reperfusion injury, a number of key details are as yet undisclosed. These include the timing and magnitude of the changes in both Na+/H+ exchange (NHE-1) and Na+-HCO3--cotransport (NBC) transport rates. We fluorimetrically measured Hi+ fluxes (JNHE-1 and JNBC) and Nai+ fluxes in single contracting rabbit ventricular myocytes subjected to metabolic inhibition, pseudo-ischemia (i.e. metabolic inhibition and extracellular acidosis of 6.4), and pseudo-reperfusion. Metabolic inhibition and pseudo-ischemia inhibited NHE-1 by 43 ± 3.1% and 91 ± 3.6%, and NBC by 66 ± 5.4% and 100%, respectively. Inhibition was due to both an acidic shift of the pHi at which NHE-1 and NBC become quiescent (set-point pHi) and a reduction of the steepness of the pHi-Hi+ flux profiles. NHE-1 and NBC did not contribute to Nai+ loading during metabolic inhibition (Nai+ 18 ± 1.7 mM) or pseudo-ischemia (Nai+ 21 ± 1.7 mM), because pHi acidified less than set-point pHi''s. Upon pseudo-reperfusion NBC recovered to 54 ± 7.3% but NHE-1 to 193 ± 11% of aerobic control flux, and set-point pHi''s returned to near neutral values. Metabolic inhibition and reperfusion caused an acid load of 18 ± 3.2 mM H+ 94% of which were extruded by the hyperactive NHE-1. At pseudo-reperfusion Nai+ rose sharply to 31 ± 5.8 mM within 1.5 min and that coincided with hypercontracture. Cariporide not only prevented the Nai+ transient, but also inhibited pHi recovery and prevented hypercontracture. Our results are consistent with the view that NHE-1 is active during metabolic inhibition if, like in whole hearts, pHi is driven more acidic than NHE-1 set-point pHi. Furthermore, either an acidic pHi or absence of additional Nai+ loading during reperfusion, or both, limit ischemia–reperfusion injury. [Copyright &y& Elsevier]

Machado–Joseph disease (MJD) is a neurodegenerative disorder, caused by the expansion of the (CAG)n tract in the MJD gene. This encodes the protein ataxin-3, of unknown function. The mouse Mjd gene has a structure similar to that of its human counterpart and it also contains a TATA-less promoter. Its 5′ flanking region contains conserved putative binding regions for transcription factors Sp1, USF, Arnt, Max, E47, and MyoD. Upon differentiation of P19 cells, the Mjd gene promoter is preferentially activated in endodermal and mesodermal derivatives, including cardiac and skeletal myocytes; and less so in neuronal precursors. Mouse ataxin-3 is ubiquitously expressed during embryonic development and in the adult, with strong expression in regions of the CNS affected in MJD. It is particularly abundant in all types of muscle and in ciliated epithelial cells, suggesting that it may be associated with the cytoskeleton and may have an important function in cell structure and/or motility. [Copyright &y& Elsevier]

Streptozotocin (STZ) has long been used to induce experimental diabetes mellitus to study diabetic complications such as diabetic cardiomyopathy. However, direct impact of STZ on cardiac function is unknown. This study was designed to evaluate the cardiac contractile effect of STZ in isolated adult rat ventricular myocytes. Contractile properties were assessed with an IonOptix MyoCam system including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90), and maximal velocities of shortening/relengthening (± dL/dt). Intracellular Ca2+ handling was evaluated with the fluorescent dye fura-2. Acute exposure of STZ (10-9–10-5 M) depressed PS, prolonged TR90, and decreased electrically stimulated intracellular Ca2+ rise in a concentration-dependent manner. TPS, ± dL/dt, resting intracellular Ca2+ level, and intracellular Ca2+ clearing rate were unaffected. The STZ-induced mechanical alterations were alleviated by the antioxidant vitamin C (100 μM) and the p38 MAP kinase inhibitor SB203580 (1 μM). 2′,7′-Dichlorofluorescein diacetate staining revealed enhanced production of reactive oxygen species following STZ treatment, which was prevented by either vitamin C or SB203580. Collectively, our data provided convincing evidence that the tool drug for experimental diabetes STZ may itself cause deleterious cardiac contractile dysfunction via an oxidative stress and p38 MAP kinase-dependent mechanism. Thus, caution should be taken when assessing diabetic heart complications using STZ-induced diabetic models. [Copyright &y& Elsevier]

Integrins are a family of cell-surface receptors that link the extracellular matrix (ECM) to the cellular cytoskeleton. The goal of this study was to determine the importance of the integrin β1 subunit in regulating cardiac L-type Ca2+ channel function. Neonatal rat ventricular myocytes were cultured on collagen membranes and infected with adenovirus expressing either the human β1A integrin (Adβ1A) or a chimeric protein consisting of the cytoplasmic tail domain of the β1A integrin and the extracellular/transmembrane domain of the interleukin-2 receptor (AdTAC-β1). Expression of the free β1 integrin tail (TAC-β1), but not the full-length β1A integrin, altered cell morphology and disrupted normal cell adhesion. When compared with myocytes infected with control virus, neither Adβ1A nor AdTAC-β1 infection produced any significant change in the current vs. voltage relationship of the whole-cell Ca2+ current (ICa) or the kinetics of ICa decay. Expression of TAC-β1, but not β1A, induced a negative shift in the Ca2+ channel steady-state inactivation curve. Application of the β-adrenergic receptor agonist isoproterenol produced over a 90% increase in ICa in control cells, but caused only an 18% increase in myocytes overexpressing the full-length β1A integrin. In addition, β-adrenergic stimulation resulted in a 5–10-fold increase in intracellular cAMP levels in control cells, but produced no significant response in Adβ1A-infected cells. In contrast, expression of TAC-β1 was associated with an augmentation in the Ca2+ channel response to isoproterenol (160% increase) and the Ca2+ channel agonist BayK8644. Thus, integrin/ECM interactions may be critical in the regulation of ICa [Copyright &y& Elsevier]

It is well established that β-adrenoceptor stimulation activates PKA and α1-adrenoceptor stimulation activates PKC. In normal ventricular myocytes, acute activation of α1-adrenoceptors inhibits β-adrenoceptor stimulated L-type Ca current (ICa-L) and direct activation of &epsiv;PKC leads to ICa-L inhibition. Because increased PKC activity has been observed chronically in in vivo setting such as failing human heart, we hypothesized that chronic in vivo activation of &epsiv;PKC alters ICa-L and its response to adrenergic stimulation. Therefore, we investigated the interaction between β- and α1-adrenoceptors vis-a&grave;-vis ICa-L in myocytes from transgenic mice (TG) with cardiac specific constitutive activation of &epsiv;PKC (&epsiv;PKC agonist). Whole-cell ICa-L was recorded from &epsiv;PKC agonist TG mice and age-matched non-TG (NTG) littermates under: (1) basal condition, (2) β-adrenoceptor agonist, isoproterenol (ISO), and (3) ISO plus α1-adrenoceptor agonist, methoxamine. The present results are the first to demonstrate that chronic in vivo activation of &epsiv;PKC leads to reduced basal ICa-L density. β-adrenoceptor activation of ICa-L is blunted in &epsiv;PKC agonist TG mice. α-adrenoceptor cross-talk with β-adrenoceptor signaling pathways vis-a&grave;-vis L-type Ca channels is impaired in &epsiv;PKC agonist TG mice. The diminished response to ISO and methoxamine suggests a protective feedback regulatory mechanism in &epsiv;PKC agonist TG mice and could be vital in the settings of excessive release of catecholamines during heart failure. [Copyright &y& Elsevier]

The hypothesis tested was that reoxygenation-induced contracture of myocardial cells, a form of reperfusion injury, can be due to a rigor-type mechanism. Isolated adult cardiomyocytes were exposed to 30- or 60-min anoxia (pH 6.4) and reoxygenation (pH 7.4). In cardiomyocytes, cytosolic Ca2+ and cell length, and in isolated rat hearts left ventricular end-diastolic pressure (LVEDP) were measured. During reoxygenation, cardiomyocytes developed contracture. When energy recovery was slowed down, less Ca2+ overload was required for contracture: (1) after 30-min anoxia Ca20 (cytosolic Ca2+ concentration in cells with 20% cell length reduction) was 1.42 ± 0.11 μmol/l; (2) after 30-min anoxia with partial mitochondrial inhibition during reoxygenation (NaCN, 0.1 mmol/l) Ca20 was reduced to 0.69 ± 0.05 μmol/l; (3) after 60-min anoxia Ca20 was reduced to 0.78 ± 0.05 μmol/l and (4) when energy recovery was accelerated (succinate, 0.2 mmol/l), Ca20 rose to 1.35 ± 0.05 μmol/l. In isolated hearts, the reperfusion-induced rise in LVEDP was modulated by the same interventions. Slow recovery of energy production favors reoxygenation-induced contracture in cardiomyocytes and hearts. This shows that rigor contracture contributes to reoxygenation-induced cell injury. [Copyright &y& Elsevier]

Reactive cardiomyocyte hypertrophy after myocardial infarction (MI) is an important risk factor for arrhythmias. Endothelin (ET)-1 has been implicated in the development of cardiac hypertrophy. We investigated the effect of pravastatin on ventricular hypertrophy during remodeling after MI and whether the attenuated hypertrophic effect was via reduced regional ET-1 expression. After ligation of the anterior descending artery, male Wistar rats were randomized to either vehicle, pravastatin, mevalonate, or a combination of the two drugs for 4 weeks. Sham operation served as controls. Pravastatin decreased cardiomyocyte sizes isolated by enzymatic dissociation at the border zone. The myocardial ET-1 levels at the border zone were 6.3-fold higher (P < 0.0001) in the vehicle group compared with sham group. The increased regional ET-1 levels can be inhibited after pravastatin administration. Immunohistochemical analysis confirmed the localization of ET-1 mainly in the cardiomyocytes. This was paralleled by a 9.8 ± 2.3-fold upregulation of preproET-1 mRNA assessed by real-time quantitative reverse transcription-polymerase chain reaction in the vehicle-treated rats, which reduced after administering pravastatin. Cardiomyocyte sizes at the border zone correlated positively with regional ET-1 levels (P = 0.001). Arrhythmic scores during programmed stimulation were significantly higher in the vehicle group than in the pravastatin-treated group (3.0 ± 1.3 vs. 1.3 ± 1.0, P < 0.0001). In contrast, pravastatin-induced effects were reversed by the addition of mevalonate, implicating 3-hydroxy-3-methyglutaryl-CoA reductase as the relevant target. The results of the present study suggest that the pravastatin administration after infarction can reduce the inducibility of ventricular arrhythmias as a result of attenuated cardiomyocyte hypertrophy probably through decreased tissue ET-1 level, which is linked to mevalonate metabolism. [Copyright &y& Elsevier]

The subcellular distribution of ATP-sensitive potassium (KATP) channel subunits in rat-isolated ventricular myocytes was investigated using a panel of subunit-specific antisera. Kir6.1 subunits were associated predominantly with myofibril structures and were co-localized with the mitochondrial marker MitoFluor red (correlation coefficient (cc) = 0.63 ± 0.05). Anti-Kir6.1 antibodies specifically recognized a polypeptide of 48 kDa in mitochondrial membrane fractions consistent with the presence of Kir6.1 subunits in this organelle. Both Kir6.2 and SUR2A subunits were distributed universally over the sarcolemma. Lower-intensity antibody-associated immunofluorescence was detected intracellularly, which was correlated with the distribution of MitoFluor red in both cases (cc, Kir6.2, 0.56 ± 0.05; SUR2A, 0.61 ± 0.06). A polypeptide of 40 kDa was recognized by anti-Kir6.2-subunit antibodies in western blots of both microsomal and mitochondrial membrane fractions consistent with the presence of this subunit in the sarcolemma and mitochondria. Similarly, SUR2A and SUR2B subunits were detected in western blots of microsomal membrane proteins consistent with a sarcolemmal localization for these polypeptides. SUR2B subunits were shown in confocal microscopy to co-localize strongly with t-tubules (cc, 0.81 ± 0.05). Together, the results indicate that Kir6.2 and SUR2A subunits predominate in the sarcolemma of ventricular myocytes consistent with a Kir6.2/SUR2A-subunit combination in the sarcolemmal KATPchannel. Kir6.1, Kir6.2 and SUR2A subunits were demonstrated in mitochondria. Combinations of these subunits would not explain the reported pharmacology of the mitochondrial KATP channel (Mol Pharmacol 59 (2001) 225) suggesting the possibility of further unidentified components of this channel. [Copyright &y& Elsevier]

A. I. U ndrovinas, V. A. M altsev, J., W. K yle, N. Silvermanand H. N. S abbah. Gating of the Late Na+ Channel in Normal and Failing Human Myocardium. Journal of Molecular and Cellular Cardiology (2002) 34, 1477–1489. We previously reported an ultraslow inactivating late Na+ current (INaL) in left ventricular cardiomyocytes (VC) isolated from normal (NVC) and failing (FVC) human hearts. This current could play a role in heart failure-induced repolarization abnormalities. To identify properties of NaCh contributing to INaL, we examined early and late openings in cell-attached patches of HEK293 cells expressing human cardiac NaCh α-subunit (α-HEK) and in VC of one normal and three failing human hearts. Two types of the late NaCh openings underlay INaL in all three preparations: scattered late (SLO) and bursts (BO). Amplitude analysis revealed that slope conductance for both SLO and BO was the same compared to the main level of early openings (EO) in both VC (21 vs 22.7 pS, NVC; 22.7 vs 22.6 pS, FVC) and α-HEK (23.2 vs 23 pS), respectively. Analysis of SLO latencies revealed voltage-independent ultraslow inactivation in all preparations with tendency to be slower in FVC compared to NCV. EO and SLO render one open voltage-independent state (τ∼0.4 ms) for NVC and FVC. One open (voltage-dependent) and two closed states (one voltage-dependent and another voltage-independent) were found in BO of both specimens. Burst duration tend to be longer in FVC (∼50 ms) than in NVC (∼30 ms). In FVC we found both modes SLO and BO at membrane potential of −10 mV that is attribute for take-off voltages (from −18 to −2 mV) for early afterdepolarizations (EAD''s) in FVC. In conclusions, we found a novel gating mode SLO that manifest slow (hundreds of ms), voltage-independent inactivation in both NVC and FVC. We were unable to reliably demonstrate any differences in the properties of the late NaCh in failing vs a normal human heart. Accordingly, the late current appears to be generated by a single population of channels in normal and failing human ventricular myocardium. Both SLO and BO could be implicated in EADs in HF. [Copyright &y& Elsevier]

Hypoxia–reoxygenation (H-R) is associated with alterations in oxidant–antioxidant balance and l-arginine–nitric oxide system. Tocopherols decrease the activity of reactive oxygen species (ROS) and yet are not beneficial in clinical trials. It has been proposed that mixed tocopherols as found in nature may be more tissue protective than α-tocopherol alone found in commercial preparations. We compared the effect of a mixed tocopherol preparation with that of α-tocopherol alone on superoxide dismutase (SOD) activity and iNOS expression in cultured myocytes exposed to H-R. Myocytes from Sprague–Dawley rat hearts were subjected to hypoxia for 24 h followed by reoxygenation for 3 h H-R. Parallel groups of myocytes were pretreated with α-tocopherol alone or a mixed-tocopherol preparation (containing α-, γ-, and δ-tocopherols) (50 μM) for 30 min. H-R resulted in myocyte injury (determined by LDH release), a decrease in SOD activity and an upregulation of iNOS expression/activity. Both tocopherol preparations attenuated cell injury and markedly decreased the effects of H-R on SOD activity and iNOS expression/activity (all P < 0.05 vs H-R group, n = 5). However, mixed-tocopherol preparation was much superior to α-tocopherol in terms of myocyte protection from the adverse effect of H-R (P < 0.05). Lack of efficacy of commercial tocopherol preparations in clinical trials may reflect absence of γ- and δ-tocopherols. [Copyright &y& Elsevier]

Transplantation of neonatal cardiomyocytes is a novel approach for the treatment of heart failure and myocardial infarction, but quantitative information on long-term cell survival and development is limited. Male donor cardiomyocytes were isolated from neonatal Fischer 344 rats (1–2 days), purified, and injected into the left ventricular wall of female syngeneic adult rats. One hour to 12 weeks later, genomic DNA was isolated from recipient hearts. The amount of male DNA per sample was determined by quantitative real-time TaqMan PCR of the male-specific Sry gene. Transplanted cell survival was 57±9% at 0–1 h, 24±6% at 24 h, 28±11% at 7 days, 27±3% at 14 days, 23±8% at 4 weeks and 15±3% at 12 weeks. The caspase inhibitor AcYVADcmk failed to improve transplanted cell survival at 24 h, suggesting that apoptosis did not play a major role in cell loss. Histology revealed that transplanted cells became more elongated over time, developed cross-striations, and that their nuclei increased in size. However, at 12 weeks, transplanted cells and their nuclei were still smaller than those of host myocardium. We established a quantitative survival profile for neonatal cardiomyocytes transplanted into normal adult myocardium. There was significant loss of cells within 24 h, but 15% of transplanted cells survived 12 weeks. Those cells that did survive underwent differentiation and developed visible sarcomeres, suggesting a potential contribution toward ventricular function. [ABSTRACT FROM AUTHOR]

Abstract: Calcium-induced calcium release (CICR) is an inherently regenerative process due to the Ca2+-dependent gating of ryanodine receptors (RyRs) in the sarco/endoplasmic reticulum (SR) and is critical for cardiac excitation–contraction coupling. This process is seen as Ca2+ sparks, which reflect the concerted gating of groups of RyRs in the dyad, a specialised junctional signalling domain between the SR and surface membrane. However, the mechanism(s) responsible for the termination of regenerative CICR during the evolution of Ca2+ sparks remain uncertain. Rat cardiac RyR gating was recorded at physiological Ca2+, Mg2+ and ATP levels and incorporated into a 3D model of the cardiac dyad which reproduced the time-course of Ca2+ sparks, Ca2+ blinks and Ca2+ spark restitution. Model CICR termination was robust, relatively insensitive to the number of dyadic RyRs and automatic. This emergent behaviour arose from the rapid development and dissolution of nanoscopic Ca2+ gradients within the dyad. These simulations show that CICR does not require intrinsic inactivation or SR calcium sensing mechanisms for stability and cessation of regeneration that arises from local control at the molecular scale via a process we call ‘induction decay’. [Copyright &y& Elsevier]

Varicella Zoster Virus (VZV) antigen has been reported to be present in the majority of temporal artery biopsies with implications for antiviral treatment in patients with giant cell arteritis. Using immunohistochemistry with VZV antibodies we found reactivity present in diverse myocyte types (smooth, skeletal and cardiac), diverse arteries (including temporal, coronary, and vertebral) and diverse clinical settings. This phenomenon is likely due to shared epitopes between VZV proteins and muscle elements and not due to actual VZV infection. We conclude that VZV immunohistochemistry should be used with caution for screening of VZV infection in the setting of temporal artery biopsies. [ABSTRACT FROM AUTHOR]