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Diastolic dysfunction and delayed ventricular repolarization are typically observed in the elderly, but whether these defects are intimately associated with the progressive manifestation of the aging myopathy remains to be determined. In this regard, aging in experimental animals is coupled with increased late Na+ current (INa,L) in cardiomyocytes, raising the possibility that INa,L conditions the modality of electrical recovery and myocardial relaxation of the aged heart. For this purpose, aging male and female wild-type (WT) C57Bl/6 mice were studied together with genetically engineered mice with phosphomimetic (gain of function, GoF) or ablated (loss of function, LoF) mutations of the sodium channel Nav1.5 at Ser571 associated with, respectively, increased and stabilized INa,L. At ∼18 mo of age, WT mice developed prolonged duration of the QT interval of the electrocardiogram and impaired diastolic left ventricular (LV) filling, defects that were reversed by INa,L inhibition. Prolonged repolarization and impaired LV filling occurred prematurely in adult (∼5 mo) GoF mutant mice, whereas these alterations were largely attenuated in aging LoF mutant animals. Ca2+ transient decay and kinetics of myocyte shortening/relengthening were delayed in aged (∼24 mo) WT myocytes, with respect to adult cells. In contrast, delayed Ca2+ transients and contractile dynamics occurred at adult stage in GoF myocytes and further deteriorated in old age. Conversely, myocyte mechanics were minimally affected in aging LoF cells. Collectively, these results document that Nav1.5 phosphorylation at Ser571 and the late Na+ current modulate the modality of myocyte relaxation, constituting the mechanism linking delayed ventricular repolarization and diastolic dysfunction. NEW & NOTEWORTHY: We have investigated the impact of the late Na current (INa,L) on cardiac and myocyte function with aging by using genetically engineered animals with enhanced or stabilized INa,L, due to phosphomimetic or phosphoablated mutations of Nav1.5. Our findings support the notion that phosphorylation of Nav1.5 at Ser571 prolongs myocardial repolarization and impairs diastolic function, contributing to the manifestations of the aging myopathy. [ABSTRACT FROM AUTHOR]

We previously showed that the transaminase inhibitor, aminooxyacetic acid, reduced respiration energized at complex II (succinate dehydrogenase, SDH) in mitochondria isolated from mouse hindlimb muscle. The effect required a reduction in membrane potential with resultant accumulation of oxaloacetate (OAA), a potent inhibitor of SDH. To specifically assess the effect of the mitochondrial transaminase, glutamic oxaloacetic transaminase (GOT2) on complex II respiration, and to determine the effect in intact cells as well as isolated mitochondria, we performed respiratory and metabolic studies in wildtype (WT) and CRISPR-generated GOT2 knockdown (KD) C2C12 myocytes. Intact cell respiration by GOT2KD cells versus WT was reduced by adding carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) to lower potential. In mitochondria of C2C12 KD cells, respiration at low potential generated by 1 µM FCCP and energized at complex II by 10 mM succinate + 0.5 mM glutamate (but not by complex I substrates) was reduced versus WT mitochondria. Although we could not detect OAA, metabolite data suggested that OAA inhibition of SDH may have contributed to the FCCP effect. C2C12 mitochondria differed from skeletal muscle mitochondria in that the effect of FCCP on complex II respiration was not evident with ADP addition. We also observed that C2C12 cells, unlike skeletal muscle, expressed glutamate dehydrogenase, which competes with GOT2 for glutamate metabolism. In summary, GOT2 KD reduced C2C12 respiration in intact cells at low potential. From differential substrate effects, this occurred largely at complex II. Moreover, C2C12 versus muscle mitochondria differ in complex II sensitivity to ADP and differ markedly in expression of glutamate dehydrogenase. NEW & NOTEWORTHY: Impairment of the mitochondrial transaminase, GOT2, reduces complex II (succinate dehydrogenase, SDH)-energized respiration in C2C12 myocytes. This occurs only at low inner membrane potential and is consistent with inhibition of SDH. Incidentally, we observed that C2C12 mitochondria compared with muscle tissue mitochondria differ in sensitivity of complex II respiration to ADP and in the expression of glutamate dehydrogenase. [ABSTRACT FROM AUTHOR]

Pulmonary hypertension (PH) is a condition in which remodeling of the pulmonary vasculature leads to hypertrophy of the muscular vascular wall and extension of muscle into nonmuscular arteries. These pathological changes are predominantly due to the abnormal proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), enhanced cellular functions that have been linked to increases in the cell membrane protein aquaporin 1 (AQP1). However, the mechanisms underlying the increased AQP1 abundance have not been fully elucidated. Here we present data that establishes a novel interaction between AQP1 and the proteolytic enzyme caspase-3. In silico analysis of the AQP1 protein reveals two caspase-3 cleavage sites on its C-terminal tail, proximal to known ubiquitin sites. Using biotin proximity ligase techniques, we establish that AQP1 and caspase-3 interact in both human embryonic kidney (HEK) 293A cells and rat PASMCs. Furthermore, we demonstrate that AQP1 levels increase and decrease with enhanced caspase-3 activity and inhibition, respectively. Ultimately, further work characterizing this interaction could provide the foundation for novel PH therapeutics. NEW & NOTEWORTHY: Pulmonary arterial smooth muscle cells (PASMCs) are integral to pulmonary vascular remodeling, a characteristic of pulmonary arterial hypertension (PAH). PASMCs isolated from robust animal models of disease demonstrate enhanced proliferation and migration, pathological functions associated with increased abundance of the membrane protein aquaporin 1 (AQP1). We present evidence of a novel interaction between the proteolytic enzyme caspase-3 and AQP1, which may control AQP1 abundance. These data suggest a potential new target for novel PAH therapies. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium channels (VGSCs) are macromolecular assemblies composed of a number of proteins regulating channel conductance and properties. VGSCs generate Naþ current (INa) in myocytes and play fundamental roles in excitability and impulse conduction in the heart. Moreover, VGSCs condition mechanical properties of the myocardium, a process that appears to involve the late component of INa. Variants in the gene SCN1B, encoding the VGSC α1- and α1B-subunits, result in inherited neurological disorders and cardiac arrhythmias. But the precise contributions of b1/b1B-subunits and VGSC integrity to the overall function of the adult heart remain to be clarified. For this purpose, adult mice with cardiac-restricted, inducible deletion of Scn1b (conditional knockout, cKO) were studied. Myocytes from cKO mice had increased densities of fast (þ20%)- and slow (+140%)-inactivating components of INa, with respect to control cells. By echocardiography and invasive hemodynamics, systolic function was preserved in cKO mice, but diastolic properties and ventricular compliance were compromised, with respect to control animals. Importantly, inhibition of late INa with GS967 normalized left ventricular filling pattern and isovolumic relaxation time in cKO mice. At the cellular level, cKO myocytes presented delayed kinetics of Ca2+ transients and cell mechanics, defects that were corrected by inhibition of INa. Collectively, these results document that VGSC b1/b1B-subunits modulate electrical and mechanical function of the heart by regulating, at least in part, Na+ influx in cardiomyocytes. [ABSTRACT FROM AUTHOR]

Notch receptor signaling is active during cardiac development and silenced in myocytes after birth. Conversely, outward K+ Kv currents progressively appear in postnatal myocytes leading to shortening of the action potential (AP) and acquisition of the mature electrical phenotype. In the present study, we tested the possibility that Notch signaling modulates the electrical behavior of cardiomyocytes by interfering with Kv currents. For this purpose, the effects of Notch receptor activity on electrophysiological properties of myocytes were evaluated using transgenic mice with inducible expression of the Notch1 intracellular domain (NICD), the functional fragment of the activated Notch receptor, and in neonatal myocytes after inhibition of the Notch transduction pathway. By patch clamp, NICD-overexpressing cells presented prolonged AP duration and reduced upstroke amplitude, properties that were coupled with reduced rapidly activating Kv and fast Na+ currents, compared with cells obtained from wild-type mice. In cultured neonatal myocytes, inhibition of the proteolitic release of NICD with a γ-secretase antagonist increased transcript levels of the Kv channel-interacting proteins 2 (KChIP2) and enhanced the density of Kv currents. Collectively, these results indicate that Notch signaling represents an important regulator of the electrophysiological behavior of developing and adult myocytes by repressing, at least in part, repolarizing Kv currents. [ABSTRACT FROM AUTHOR]

Diabetes and other metabolic conditions characterized by elevated blood glucose constitute important risk factors for cardiovascular disease. Hyperglycemia targets myocardial cells rendering ineffective mechanical properties of the heart, but cellular alterations dictating the progressive deterioration of cardiac function with metabolic disorders remain to be clarified. In the current study, we examined the effects of hyperglycemia on cardiac function and myocyte physiology by employing mice with high blood glucose induced by administration of streptozotocin, a compound toxic to insulin-producing β-cells. We found that hyperglycemia initially delayed the electrical recovery of the heart, whereas cardiac function became defective only after ~2 mo with this condition and gradually worsened with time. Prolonged hyperglycemia was associated with increased chamber dilation, thinning of the left ventricle (LV), and myocyte loss. Cardiomyocytes from hyperglycemic mice exhibited defective Ca2+ transients before the appearance of LV systolic defects. Alterations in Ca2+ transients involved enhanced spontaneous Ca2+ releases from the sarcoplasmic reticulum (SR), reduced cytoplasmic Ca2+ clearance, and declined SR Ca2+ load. These defects have important consequences on myocyte contraction, relaxation, and mechanisms of rate adaptation. Collectively, our data indicate that hyperglycemia alters intracellular Ca2+ homeostasis in cardiomyocytes, hindering contractile activity and contributing to the manifestation of the diabetic cardiomyopathy. [ABSTRACT FROM AUTHOR]

We have investigated cardiac myocytes derived from human-induced pluripotent stem cells (iPSC-CMs) from two normal control and two family members expressing a mutant cardiac troponin T (cTnTR173W) linked to dilated cardiomyopathy (DCM). cTnT is a regulatory protein of the sarcomeric thin filament. The loss of this basic charge, which is strategically located to control tension, has consequences leading to progressive DCM. iPSC-CMs serve as a valuable platform for understanding clinically relevant mutations in sarcomeric proteins; however, there are important questions to be addressed with regard to myocyte adaptation that we model here by plating iPSCCMs on softer substrates (100 kPa) to create a more physiologic environment during recovery and maturation of iPSC-CMs after thawing from cryopreservation. During the first week of culture of the iPSC-CMs, we have determined structural and functional characteristics as well as actin assembly dynamics. Shortening, actin content, and actin assembly dynamics were depressed in CMs from the severely affected mutant at 1 wk of culture, but by 2 wk differences were less apparent. Sarcomeric troponin and myosin isoform composition were fetal/neonatal. Furthermore, the troponin complex, reconstituted with wild-type cTnT or recombinant cTnT-R173W, depressed the entry of cross-bridges into the force-generating state, which can be reversed by the myosin activator omecamtiv mecarbil. Therapeutic doses of this drug increased both contractility and the content of F-actin in the mutant iPSC-CMs. Collectively, our data suggest the use of a myosin activation reagent to restore function within patientspecific iPSC-CMs may aid in understanding and treating this familial DCM. [ABSTRACT FROM AUTHOR]

P2X4 receptors (P2X4Rs) are ligand-gated ion channels capable of conducting cations such as Na+. Endogenous cardiac P2X4R can mediate ATP-activated current in adult murine cardiomyocytes. In the present study, we tested the hypothesis that cardiac P2X receptors can induce Na+ entry and modulate Na+ handling. We further determined whether P2X receptor-induced stimulation of the Na+/Ca2+ exchanger (NCX) has a role in modulating the cardiac contractile state. Changes in Na+-K+-ATPase current (Ip) and NCX current (INCX) after agonist stimulation were measured in ventricular myocytes of P2X4 transgenic mice using whole cell patch-clamp techniques. The agonist 2-methylthio-ATP (2-meSATP) increased peak Ip from a basal level of 0.52 ± 0.02 to 0.58 ± 0.03 pA/pF. 2-meSATP also increased the Ca2+ entry mode of INCX (0.55 ± 0.09 pA/pF under control conditions vs. 0.82 ± 0.14 pA/pF with 2-meSATP) at a membrane potential of +50 mV. 2-meSATP shifted the reversal potential of INCX from -14 ± 2.3 to -25 ± 4.1 mV, causing an estimated intracellular Na+ concentration increase of 1.28 ± 0.42 mM. These experimental results were closely mimicked by mathematical simulations based on previously established models. KBR7943 or a structurally different agent preferentially opposing the Ca2+ entry mode of NCX, YM-244769, could inhibit the 2-meSATP-induced increase in cell shortening in transgenic myocytes. Thus, the Ca2+ entry mode of INCX participates in P2X agonist-stimulated contractions. In ventricular myocytes from wild-type mice, the P2X agonist could increase INCX, and KB-R7943 was able to inhibit the contractile effect of endogenous P2X4Rs, indicating a physiological role of these receptors in wild-type cells. The data demonstrate a novel Na+ entry pathway through ligand-gated P2X4Rs in cardiomyocytes. [ABSTRACT FROM AUTHOR]

Pulmonary hypertension (PH) results in pressure overload of the right ventricle (RV) of the heart, initiating pathological RV remodeling and ultimately leading to right heart failure. Substantial research indicates that signaling through the MAPK superfamily mediates pathological cardiac remodeling. These considerations led us to test the hypothesis that the regulatory protein MAPKKK-2 (MEKK2) contributes to RV hypertrophy in hypoxiainduced PH. Transgenic mice with global knockout of MEKK2 (MEKK2-/- mice) and age-matched wild-type (WT) mice were exposed to chronic hypobaric hypoxia (10% O2, 6 wk) and compared with animals under normoxia. Exposure to chronic hypoxia induced PH in WT and MEKK2-/- mice. In response to PH, WT mice showed RV hypertrophy, demonstrated as increased ratio of RV weight to body weight, increased RV wall thickness at diastole, and increased cardiac myocyte size compared with normoxic control animals. In contrast, each of these measures of RV hypertrophy seen in WT mice after chronic hypoxia was attenuated in MEKK2-/- mice. Furthermore, chronic hypoxia elicited altered programs of hypertrophic and inflammatory gene expression consistent with pathological RV remodeling in WT mice; MEKK2 deletion selectively inhibited inflammatory gene expression compared with WT mice. The actions of MEKK2 were mediated in part through regulation of the abundance and phosphorylation of its effector, ERK5. In conclusion, signaling by MEKK2 contributes to RV hypertrophy and altered myocardial inflammatory gene expression in response to hypoxiainduced PH. Therapies targeting MEKK2 may protect the myocardium from hypertrophy and pathological remodeling in human PH. [ABSTRACT FROM AUTHOR]

β-Adrenergic receptor (β-AR) stimulation increases extracellular ubiquitin (UB) levels, and extracellular UB inhibitsβ-AR-stimulated apoptosis in adult cardiac myocytes. This study investigates the role of exogenous UB in chronic β-AR-stimulated myocardial remodeling. L-Isoproterenol (ISO; 400 βg-kg-1.h-1) was infused in mice in the presence or absence of UB (1 μg.g-1.h-1). Left ventricular (LV) structural and functional remodeling was studied 7 days after infusion. UB infusion enhanced serum UB levels. In most parts, UB alone had no effect on morphometric or functional parameters. Heart weight-to-body weight ratios were increased to a similar extent in the ISO and UB+ ISO groups. Echocardiographic analyses showed increased percent fractional shortening, ejection fraction, and LV circumferential stress and fiber-shortening velocity in the ISO group. These parameters were significantly lower in UB +ISO vs. ISO. Isovolumic contraction and relaxation times and ejection time were significantly lower in ISO vs. UB + ISO. The increase in the number of TUNEL-positive myocytes and fibrosis was significantly higher in ISO vs. UB + ISO. Activation of Akt was higher, whereas activation of GSK-3β and JNKs was lower in UB β ISO vs ISO. Expression of MMP-2, MMP-9, and TIMP-2 was higher in UB β ISO vs ISO. In isolated cardiac fibroblasts, UB enhanced expression of MMP-2 and TIMP-2 in the presence of ISO. Neutralizing UB antibodies negated the effects of UB on MMP-2 expression, whereas recombinant UB enhanced MMP-2 expression. UB activated Akt, and inhibition of Akt inhibited UB + ISO-mediated increases in MMP-2 expression. Thus, exogenous UB plays an important role in β-AR-stimulated myocardial remodeling with effects on LV function, fibrosis, and myocyte apoptosis. [ABSTRACT FROM AUTHOR]

The heart is comprised of a syncytium of cardiac myocytes (CM) and surrounding nonmyocytes, the majority of which are cardiac fibroblasts (CF). CM and CF are highly interspersed in the myocardium with one CM being surrounded by one or more CF. Bidirectional cross talk between CM and CF plays important roles in determining cardiac mechanical and electrical function in both normal and diseased hearts. Genetically engineered animal models and in vitro studies have provided evidence that CM and CF can regulate each other's function. Their cross talk contributes to structural and electrical remodeling in both atria and ventricles and appears to be involved in the pathogenesis of various heart diseases that lead to heart failure and arrhythmia disorders. Mechanisms of CM-CF cross talk, which are not yet fully understood, include release of paracrine factors, direct cell-cell interactions via gap junctions and potentially adherens junctions and nanotubes, and cell interactions with the extracellular matrix. In this article, we provide an overview of the existing multiscale experimental and computational approaches for the investigation of cross talk between CM and CF and review recent progress in our understanding of the functional consequences and underlying mechanisms. Targeting cross talk between CM and CF could potentially be used therapeutically for the modulation of the cardiac remodeling response in the diseased heart and may lead to new strategies for the treatment of heart failure or rhythm disturbances. [ABSTRACT FROM AUTHOR]

To bridge the gap between two-dimensional cell culture and tissue, various three-dimensional (3-D) cell culture approaches have been developed for the investigation of cardiac myocytes (CMs) and cardiac fibroblasts (CFs). However, several limitations still exist. This study was designed to develop a cardiac 3-D culture model with a scaffold-free technology that can easily and inexpensively generate large numbers of microtissues with cellular distribution and functional behavior similar to cardiac tissue. Using micromolded nonadhesive agarose hydrogels containing 822 concave recesses (800 μm deep × 400 μm wide), we demonstrated that neonatal rat ventricular CMs and CFs alone or in combination self-assembled into viable (Live/Dead stain) spherical-shaped microtissues. Importantly, when seeded simultaneously or sequentially, CMs and CFs self-sorted to be interspersed, reminiscent of their myocardial distribution, as shown by cell typespecific CellTracker or antibody labeling. Microelectrode recordings and optical mapping revealed characteristic triangular action potentials (APs) with a resting membrane potential of -66 ± 7 mV (n = 4) in spontaneously contracting CM microtissues. Under pacing, optically mapped AP duration at 90% repolarization and conduction velocity were 100 ± 30 ms and 18.0 ± 1.9 cm/s, respectively (n = 5 each). The presence of CFs led to a twofold AP prolongation in heterogenous microtissues (CM-to-CF ratio of 1:1). Importantly, Ba2+-sensitive inward rectifier K+ currents and Ca2+-handling proteins, including sarco(endo)plasmic reticulum Ca2+-ATPase 2a, were detected in CM-containing microtissues. Furthermore, cell type-specific adenoviral gene transfer was achieved, with no impact on microtissue formation or cell viability. In conclusion, we developed a novel scaffold-free cardiac 3-D culture model with several advancements for the investigation of CM and CF function and crossregulation. [ABSTRACT FROM AUTHOR]

Calmodulin- dependent protein kinase II (CaMKII) has been proposed to be a therapeutic target for heart failure (HF). However, the cardiac effect of chronic CaMKII inhibition in HF has not been well understood. We have tested alterations of Ca2+ handling, excitation-contraction coupling, and in vivo β-adrenergic regulation in pressureoverload HF mice with CaMKIIδ knockout (KO). HF was produced in wild-type (WT) and KO mice 1 wk after severe thoracic aortic banding (sTAB) with a continuous left ventricle (LV) dilation and reduction of ejection fraction for up to 3 wk postbanding. Cardiac hypertrophy was similar between WT HF and KO HF mice. However, KO HF mice manifested exacerbation of diastolic function and reduction in cardiac reserve to β-adrenergic stimulation. Compared with WT HF, L-type calcium channel current (ICa) density in KO HF LV was decreased without changes in ICa activation and inactivation kinetics, whereas ICa recovery from inactivation was accelerated and Ca2+-dependent ICa facilitation, a positive staircase blunted in WT HF, was recovered. However, ICa response to isoproterenol was reduced. KO HF myocytes manifested dramatic decrease in sarcoplasmic reticulum (SR) Ca2+ leak and slowed cytostolic Ca2+ concentration decline. Sarcomere shortening was increased, but relaxation was slowed. In addition, an increase in myofilament sensitivity to Ca2+ and the slow skeletal muscle troponin I-to-cardiac troponin I ratio and interstitial fibrosis and a decrease in Na/Ca exchange function and myocyte apoptosis were observed in KO HF LV. CaMKIIδ KO cannot suppress severe pressure-overload-induced HF. Although cellular contractility is improved, it reduces in vivo cardiac reserve to β-adrenergic regulation and deteriorates diastolic function. Our findings challenge the strategy of CaMKII inhibition in HF. [ABSTRACT FROM AUTHOR]

Studies concerning the physiological significance of Ca2+ sparks often depend on the detection and measurement of large populations of events in noisy microscopy images. Automated detection methods have been developed to quickly and objectively distinguish potential sparks from noise artifacts. However, previously described algorithms are not suited to the reliable detection of sparks in images where the local baseline fluorescence and noise properties can vary significantly, and risk introducing additional bias when applied to such data sets. Here, we describe a new, conceptually straightforward approach to spark detection in linescans that addresses this issue by combining variance stabilization with local baseline subtraction. We also show that in addition to greatly increasing the range of images in which sparks can be automatically detected, the use of a more accurate noise model enables our algorithm to achieve similar detection sensitivities with fewer false positives than previous approaches when applied both to synthetic and experimental data sets. We propose, therefore, that it might be a useful tool for improving the reliability and objectivity of spark analysis in general, and describe how it might be further optimized for specific applications. [ABSTRACT FROM AUTHOR]

Doxorubicin (Dox) is a potent antitumor agent used in cancer treatment. Unfortunately, Dox is myotoxic and results in significant reductions in skeletal muscle mass and function. Complete knowledge of the mechanism(s) by which Dox induces toxicity in skeletal muscle is incomplete, but it is established that Dox-induced toxicity is associated with increased generation of reactive oxygen species and oxidative damage within muscle fibers. Since muscular exercise promotes the expression of numerous cytoprotective proteins (e.g., antioxidant enzymes, heat shock protein 72), we hypothesized that muscular exercise will attenuate Dox-induced damage in exercise-trained muscle fibers. To test this postulate, Sprague-Dawley rats were randomly assigned to the following groups: sedentary, exercise, sedentary with Dox, or exercise with Dox. Our results show increased oxidative stress and activation of cellular proteases (calpain and caspase-3) in skeletal muscle of animals treated with Dox. Importantly, our findings reveal that exercise can prevent the Dox-induced oxidative damage and protease activation in the trained muscle. This exercise-induced protection against Dox-induced toxicity may be due, at least in part, to an exercise-induced increase in muscle levels of antioxidant enzymes and heat shock protein 72. Together, these novel results demonstrate that muscular exercise is a useful countermeasure that can protect skeletal muscle against Dox treatment-induced oxidative stress and protease activation in skeletal muscles. [ABSTRACT FROM AUTHOR]

We have previously reported that 1) inhibition of cyclooxygenase-2 and PGE2 production reduces hypertrophy after myocardial infarction in mice and 2) PGE2 acting through its EP4 receptor causes hypertrophy of neonatal ventricular myocytes (NVM5) via ERK1/2. It is known that EP4 couples to adenylate cyclase, cAMP, and PKA. The present study was designed to determine interactions between the cAMP-PKA pathway and ERKI/2 and to further characterize events downstream of ERK1/2. We hypothesized that PKA and the small GTPase Rap are upstream of ERKI/2 and that 90-kDa ribosomal S6 kinase (p90RSK) is activated downstream. Treatment of NVMs with PGE2 activated Rap, and this activation was inhibited in part by an EP4 antagonist and PKA inhibition. Transfection of a dominant negative mutant of Rap reduced PGE2 activation of ERK1/2. PGE2 activation of p90RSK was also dependent on EP4, PKA, and Rap. We also tested the involvement of Rap, ERK1/2, and p90RSK in PGE2 regulation of gene expression. PGE2 stimulation of brain natriuretic peptide promoter activity was blocked by either ERK1/2 inhibition or a dominant negative mutation of p90RSK. PGE2 stimulation of c-Fos was dependent on EP4, PKA, ERK1/2, and p90RSK, whereas only the latter two kinases were involved in PGE2 regulation of early growth response-1. Finally, we tested the involvement of EP4-dependent signaling in the NVM growth response and found that the overexpression of EP4 increased NVM cell size. We conclude that EP4-dependent signaling in NVMs in part involves PKA, Rap, ERK1/2, and p90RSK and results in the increased expression of brain natriuretic peptide and c-Fos. [ABSTRACT FROM AUTHOR]

We investigated how exercise training superimposed on chronic hypertension impacted left ventricular remodeling. Cardiomyocyte hypertrophy, apoptosis, and proliferation in hearts from female spontaneously hypertensive rats (SHRs) were examined. Four-month-old SHR animals were placed into a sedentary group (SHR-SED; n = 18) or a treadmill running group (SHR-TRD, 20 m/min, 1 h/day, 5 days/wk, 12 wk; n = 18). Age-matched, sedentary Wistar Kyoto (WKY) rats were controls (n = 18). Heart weight was greater in SHR-TRD vs. both WKY (P < 0.01) and SHR-SED (P < 0.05). Morphometricderived left ventricular anterior, posterior, and septal wall thickness were increased in SHR-SED relative to WKY and augmented in SHR-TRD. Cardiomyocyte surface area, length, and width were increased in SHR-SED relative to WKY and further increased in SHR-TRD. Calcineurin abundance was increased in SHR-SED vs. WKY (P < 0.001) and attenuated in SHR-TRD relative to SHR-SED (P < 0.05). Protein abundance and mRNA of Akt was not different among groups. The rate of apoptosis was increased in SHR-SED relative to WKY and mitigated in SHR-TRD. The abundance of Ki-67+ cells across groups was not statistically different across groups. The abundance of cardiac progenitor cells (c-Kit+ cells) was increased in SHR-TRD relative to WKY. These data suggest that exercise training superimposed on hypertension augmented cardiomyocyte hypertrophy, despite attenuating calcineurin abundance. Exercise training also mitigated apoptosis in hypertension and showed a tendency to enhance the abundance of cardiac progenitor cells, resulting in a more favorable cardiomyocyte number in the exercise-trained hypertensive heart. [ABSTRACT FROM AUTHOR]

The goal of this investigation was to determine the distribution of myocardial apoptosis in myocytes and nonmyocytes in primates and patients with heart failure (HF). Almost all clinical cardiologists and cardiovascular investigators believe that myocyte apoptosis is considered to be a cardinal sign of HF and a major factor in its pathogenesis. However, with the knowledge that 75% of the number of cells in the heart are nonmyocytes, it is important to determine whether the apoptosis in HF is occurring in myocytes or in nonmyocytes. We studied both a nonhuman primate model of chronic HF, induced by rapid pacing 2-6 mo after myocardial infarction (Ml), and biopsies from patients with ischemic cardiomyopathy. Dual labeling with a cardiac muscle marker was used to discriminate apoptosis in myocytes versus nonmyocytes. Left ventricular ejection fraction decreased following MI (from 78% to 60%) and further with HF (35%, P < 0.05). As expected, total apoptosis was increased in the myocardium following recovery from MI (0.62 cells/mm2) and increased further with the development of HF (1.91 cells/mm2). Surprisingly, the majority of apoptotic cells in MI and MI + HF, and in both the adjacent and remote areas, were nonmyocytes. This was also observed in myocardial biopsies from patients with ischemic cardiomyopathy. We found that macrophages contributed the largest fraction of apoptotic nonmyocytes (41% vs. 18% neutrophils, 16% fibroblast, and 25% endothelial and other cells). Although HF in the failing human and monkey heart is characterized by significant apoptosis, in contrast to current concepts, the apoptosis in nonmyocytes was eight- to ninefold greater than in myocytes. [ABSTRACT FROM AUTHOR]

Banerjee I, Fuseler JW, Intwala AR, Baudino TA. IL-6 loss causes ventricular dysfunction, fibrosis, reduced capillary density, and dramatically alters the cell populations of the developing and adult heart. Am J Physiol Heart Circ Physiol 296: H1694-H1704, 2009. First published February 20, 2009; doi: 10.1 l52/ajpheart.00908.200.-Interleukin-6 (IL-6) is a pleiotropic cytokine responsible for many different processes including the regulation of cell growth, apoptosis, differentiation, and survival in various cell types and organs, including the heart. Recent studies have indicated that IL-6 is a critical component in the cell-cell communication between myocytes and cardiac fibroblasts. In this study, we examined the effects of IL-6 deficiency on the cardiac cell populations, cardiac function, and interactions between the cells of the heart, specifically cardiac fibroblasts and myocytes. To examine the effects of IL-6 loss on cardiac function, we used the IL-6[sup-/-] mouse. IL-6 deficiency caused severe cardiac dilatation, increased accumulation of interstitial collagen, and altered expression of the adhesion protein periostin. In addition, flow cytometric analyses demonstrated dramatic alterations in the cardiac cell populations of IL-6[sup-/-] mice compared with wild-type littermates. We observed a marked increase in the cardiac fibroblast population in IL-6[sup-/-] mice, whereas a concomitant decrease was observed in the other cardiac cell populations examined. Moreover, we observed increased cell proliferation and apoptosis in the developing IL-6[sup-/-] heart. Additionally, we observed a significant decrease in the capillary density of IL-6[sup-/-] hearts. To elucidate the role of IL-6 in the interactions between cardiac fibroblasts and myocytes, we performed in vitro studies and demonstrated that IL-6 deficiency attenuated the activation of the STAT3 pathway and VEGF production. Taken together, these data demonstrate that a loss of IL-6 causes cardiac dysfunction by shifting the cardiac cell populations, altering the extracellular matrix, and disrupting critical cell-cell interactions. [ABSTRACT FROM AUTHOR]

Ca sparks are elementary Ca-re- lease events from intracellular Ca stores that are observed in virtually all types of muscle. Typically, Ca sparks are measured in the line-scan mode with confocal laser-scanning microscopes, yielding two-dimensional images (distance vs. time). The manual analysis of these images is time consuming and prone to errors as well as investigator bias. Therefore, we developed SparkMaster, an automated analysis program that allows rapid and reliable spark analysis. The underlying analysis algorithm is adapted from the threshold-based standard method of spark analysis developed by Cheng et al. (Biophys J 76: 606-617, 1999) and is implemented here in the freely available image-processing software ImageJ. SparkMaster offers a graphical user interface through which all analysis parameters and output options are selected. The analysis includes general image parameters (number of detected sparks, spark frequency) and individual spark parameters (amplitude, full width at half-maximum amplitude, full duration at half-maximum amplitude, full width, full duration, time-to peak, maximum steepness of spark upstroke, time constant of spark decay). We validated the algorithm using images with synthetic sparks embedded into backgrounds with different signal-to-noise ratios to determine an analysis criteria at which a high sensitivity is combined with a low frequency of false-positive detections. Finally, we applied SparkMaster to analyze experimental data of sparks measured in intact and permeabilized ventricular cardiomyocytes, permeabilized mammalian skeletal muscle, and intact smooth muscle cells. We found that SparkMaster provides a reliable, easy to use, and fast way of analyzing Ca sparks in a wide variety of experimental conditions. [ABSTRACT FROM AUTHOR]

Both angiotensin receptor antagonists and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been shown to attenuate cardiomyocyte hypertrophy after myocardial infarction. Whether combination treatment may be superior to either drug alone on cardiomyocyte hypertrophy remains unclear. After ligation of the left anterior descending artery, rats were randomized to both, one, or neither of the angiotensin receptor antagonists olmesartan (0.01, 0.1, 1, and 2 mg·kg-1·day-1) and HMG-CoA reductase inhibitor pravastatin (5 mg·kg-1·day-1) for 4 wk. Each drug, when given alone, decreased cardiomyocyte sizes isolated by enzymatic dissociation at the border zone when compared with vehicles. However, compared with either drug alone, combined olmesartan and pravastatin prevent cardiomyocyte hypertrophy to a larger extent, which was further confirmed by downregulation of the left ventricular atrial natriuretic peptide mRNA. The myocardial endothelin-1 levels at the border zone were 6.5-fold higher (P <0.0001) in the vehicle group compared with the sham group, which can be inhibited after pravastatin administration. Combination treatment significantly attenuated cardiomyocyte hypertrophy in a dose-dependent manner, although tissue endothelin-1 levels remained stable in combination groups of different olmesartan doses. Measurements of the arrhythmic score mirrored those of cardiomyocyte hypertrophy. Dual therapy with pravastatin and olmesartan, which produced an additive reduction in cardiomyocyte hypertrophy and cardiac fibrosis after myocardial infarction through different mechanisms, decreases the propensity of the heart to arrhythmogenesis. Pravastatin administration provided favorable ventricular remodeling, probably through decreased tissue endothelin-I level. In contrast, olmesartan-related attenuated cardiomyocyte hypertrophy is independent of endothelin-1 pathway. [ABSTRACT FROM AUTHOR]

The role of caveolae, membrane microenvironments enriched in signaling molecules, in myocardial ischemia is poorly defined. In the current study, we used cardiac myocytes prepared from adult rats to test the hypothesis that opioid receptors (OR), which are capable of producing cardiac protection in vivo, promote cardiac protection in cardiac myocytes in a caveolae-dependent manner. We determined protein expression and localization of δ-OR (DOR) using coimmunohistochemistry, caveolar fractionation, and immunoprecipitations. DOR colocalized in fractions with caveolin-3 (Cav-3), a structural component of caveolae in muscle cells, and could be immunoprecipitated by a Cav-3 antibody. Immunohistochemistry confirmed plasma membrane colocalization of DOR with Cav-3. Cardiac myocytes were subjected to simulated ischemia (2 h) or an ischemic preconditioning (IPC) protocol (10 min ischemia, 30 min recovery, 2 h ischemia) in the presence and absence of methyl-β-cyclodextrin (MβCD, 2 mM), which binds cholesterol and disrupts caveolae. We also assessed the cardiac protective effects of SNC-121 (SNC), a selective DOR agonist, on cardiac myocytes with or without MβCD and MβCD preloaded with cholesterol. Ischemia, simulated by mineral oil layering to inhibit gas exchange, promoted cardiac myocyte cell death (trypan blue staining), a response blunted by SNC (37 ± 3 vs. 59 ± 3% dead cells in the presence and absence of 1 μM SNC, respectively, P < 0.01) or by use of the IPC protocol (35 ± 4 vs. 62 ± 3% dead cells, P < 0.01). MI3CD treatment, which disrupted caveolae (as detected by electron microscopy), fully attenuated the protective effects of IPC or SNC, resulting in cell death comparable to that of the ischemic group. By contrast, SNC-induced protection was not abrogated in cells incubated with cholesterol-saturated M[3CD, which maintained caveolae structure and function. These findings suggest a key role for caveolae, perhaps through enrichment of signaling molecules, in contributing to protection of cardiac myocytes from ischemic damage. [ABSTRACT FROM AUTHOR]

Enterostatin, a pentapeptide cleaved from procolipase, suppresses fat intake after peripheral and central administration. Chronic treatment of rats with enterostatin decreases body weight and body fat. The effect was greater than could be accounted by the reduction in food intake alone. Hence, we have investigated the effect of enterostatin on energy metabolism. Male Sprague-Dawley rats adapted to a high-fat diet were implanted with lateral cerebral ventricular or amygdala cannulas. The metabolic effects were determined by indirect calorimetry. After habituation to the test cages, fasted rats were injected with either saline vehicle or enterostatin given either intraperitoneally (100 nmol) or intracerebroventricularly (1 nmol) or into specific brain regions [amygdala (0.01 nmol) or paraventricular nucleus (PVN) (0.1 nmol)]. Respiratory quotient (RQ) and energy expenditure were monitored over 2 h. Intraperitoneal enterostatin reduced RQ (saline: 0.81 ± 0.02 vs. enterostatin: 0.76 ± 0.01) and increased energy expenditure by 44%. Intracerebroventricular enterostatin increased the energy expenditure without any effects on RQ, whereas PVN enterostatin increased metabolic rate, while preventing the increase in RQ observed in the control animals. In contrast, neither RQ nor energy expenditure was altered after enterostatin was injected into the amygdala. Enterostatin activated AMP-activated protein kinase in primary cultures of human myocytes in a dose- and time-dependent manner and increased the rate of fatty acid β-oxidation. These findings suggest that enterostatin regulates energy expenditure and substrate partitioning through both peripheral and central effects. [ABSTRACT FROM AUTHOR]

The ability of gene transfer with the poreforming subunit of the human maxi-K channel (hSlo) to ameliorate the decline in erectile capacity commensurate with 12-24 wk of streptozotocin (STZ)-diabetes was examined in 181 Fischer-344 rats. A 2-mo period of STZ-diabetes was induced before gene transfer, and erectile capacity was evaluated by measuring the intracavernous pressure response (ICP) to cavernous nerve (CN) stimulation (ranging from 0.5 to 10 mA). In the first series of experiments, ANOVA revealed increased CN-stimulated ICP responses at 1 and 2 mo postinjection of 100 µg pcDNA-hSlo compared with control values. A second series of experiments further examined the dose dependence and duration of gene transfer. The ICP response to submaximal (0.5 mA) and maximal (10 mA) nerve stimulation was evaluated 3 or 4 mo postinjection of a single dose of pcDNA-hSlo ranging from 10 to 1,000 µg. ANOVA again revealed that hSlo overexpression was associated with increased CN-stimulated ICP responses compared with responses in corresponding control animals. Histological studies revealed no immune response to the presence of hSlo. PCR analysis documented that expression of both plasmid and transcript were largely confined to the corporal tissue. In the third series of pharmacological experiments, hSlo gene transfer in vivo was associated with iberiotoxin-sensitive relaxation responses to sodium nitroprusside in corporal tissue strips in vitro. The latter data indicate that gene transfer produces functional maxi-K channels that participate in the modulation of corporal smooth muscle cell tone. Taken together, these observations suggest a fundamental diabetes-related change in corporal myocyte maxi-K channel regulation, expression, or function that may be corrected by expression of recombinant hSlo. [ABSTRACT FROM AUTHOR]

In our studies with spontaneously hypertensive (SHR), Wistar-Kyoto (WKY), and Wistar rats, we observed normotensive WKY rats with cardiac hypertrophy determined by a greater left ventricular (LV) mass (LVM)-to-body weight (BW) ratio (LVM/BW) than that of normotensive Wistar rats. Thus we compared the following parameters in SHR, WKY, and Wistar rats: LVM/BW, cell capacitance as index of total surface area of the myocytes, length, width, and cross-sectional area of cardiac myocytes, LV collagen volume fraction, and myocardial stiffness. The LVM/BW of WKY (2.41 ± 0.03 mg/g, n = 41) was intermediate between SHR (2.82 ± 0.04 mg/g, n = 47) and Wistar rats (1.98 ± 0.04 mg/g, n = 28). A positive correlation between blood pressure and LVM was found in SHR, whereas no such relationship was observed in WKY or Wistar rats. Cell capacitance and cross-sectional area were not significantly different in SHR and WKY rats; these values were significantly higher than those of Wistar rats. The cell length was smaller but the width was similar in WKY compared with SHR. Papillary muscles isolated from the LV of WKY and SHR were stiffer than those from Wistar rats. Consistently, a greater level of myocardial fibrosis was detected in WKY and SHR compared with Wistar rats. These findings demonstrate blood pressure-independent cardiac hypertrophy in normotensive WKY rats. [ABSTRACT FROM AUTHOR]

We have developed a model of Ca2+ handling in ferret ventricular myocytes. This model includes a novel L-type Ca2+ channel, detailed intracellular Ca2+ movements, and graded Ca2+-induced Ca2+ release (CICR). The model successfully reproduces data from voltage-clamp experiments, including voltage- and time-dependent changes in intracellular Ca2+ concentration ([Ca2+]i), L-type Ca2+ channel current (ICaL) inactivation and recovery kinetics, and Ca2+ sparks. The development of graded CICR is critically dependent on spatial heterogeneity and the physical arrangement of calcium channels in opposition to ryanodine-sensitive release channels. The model contains spatially distinct subsystems representing the subsarcolemmal regions where the junctional sarcoplasmic reticulum (SR) abuts the T-tubular membrane and where the L-type Ca2+ channels and SR ryanodine receptors (RyRs) are localized. There are eight different types of subsystems in our model, with between one and eight L-type Ca2+ channels distributed binomially. This model exhibits graded CICR and provides a quantitative description of Ca2+ dynamics not requiring Monte-Carlo simulations. Activation of RyRs and release of Ca2+ from the SR depend critically on Ca2+ entry through L-type Ca2+ channels. In turn, Ca2+ channel inactivation is critically dependent on the release of stored intracellular Ca2+. Inactivation of ICaL depends on both transmembrane voltage and local [Ca2+]i near the channel, which results in distinctive inactivation properties. The molecular mechanisms underlying many ICaL gating properties are unclear, but [Ca2+]i dynamics clearly play a fundamental role. [ABSTRACT FROM AUTHOR]

Mitochondrial membrane potential (DeltaΨm) is severely compromised in the myocardium after ischemia-reperfusion and triggers apoptotic events leading to cell demise. This study tests the hypothesis that mitochondrial ATP-sensitive K+ (mitoKATP) channel activation prevents the collapse of DeltaΨm in myocytes during anoxia-reoxygenation (A-R) and is responsible for cell protection via inhibition of apoptosis. After 3-h anoxia and 2-h reoxygenation, the cultured myocytes underwent extensive damage, as evidenced by decreased cell viability, compromised membrane permeability, increased apoptosis, and decreased ATP concentration. Mitochondria in A-R myocytes were swollen and fuzzy as shown after staining with Mito Tracker Orange CMTMRos and in an electron microscope and exhibited a collapsed DeltaΨm, as monitored by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Cytochrome c was released from mitochondria into the cytosol as demonstrated by cytochrome c immunostaining. Activation of mitoKATP channel with diazoxide (100 micromol/l) resulted in a significant protection against mitochondrial damage, ATP depletion, cytochrome c loss, and stabilized DeltaΨm. This protection was blocked by 5-hydroxydecanoate (500 μmol/l), a mitoKATP channel-selective inhibitor, but not by HMR-1098 (30 micromol/l), a putative sarcolemmal KATP channel-selective inhibitor. Dissipation of DeltaΨm also leads to opening of mitochondrial permeability transition pore, which was prevented by cyclosporin A. The data support the hypothesis that A-R disrupts DeltaΨm and induces apoptosis, which are prevented by the activation of the mitoKATP channel. This further emphasizes the therapeutic significance of mitoKATP channel agonists in the prevention of ischemia-reperfusion cell injury. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium channels (VGSCs) are macromolecular assemblies composed of a number of proteins regulating channel conductance and properties. VGSCs generate Na + current ( I ) in myocytes and play fundamental roles in excitability and impulse conduction in the heart. Moreover, VGSCs condition mechanical properties of the myocardium, a process that appears to involve the late component of Na ) in myocytes and play fundamental roles in excitability and impulse conduction in the heart. Moreover, VGSCs condition mechanical properties of the myocardium, a process that appears to involve the late component of I Na (conditional knockout, cKO) were studied. Myocytes from cKO mice had increased densities of fast (+20%)- and slow (+140%)-inactivating components of SCN1B , encoding the VGSC β1- and β1B-subunits, result in inherited neurological disorders and cardiac arrhythmias. But the precise contributions of β1/β1B-subunits and VGSC integrity to the overall function of the adult heart remain to be clarified. For this purpose, adult mice with cardiac-restricted, inducible deletion of Scn1b with GS967 normalized left ventricular filling pattern and isovolumic relaxation time in cKO mice. At the cellular level, cKO myocytes presented delayed kinetics of Ca I Na , with respect to control cells. By echocardiography and invasive hemodynamics, systolic function was preserved in cKO mice, but diastolic properties and ventricular compliance were compromised, with respect to control animals. Importantly, inhibition of late I Na with GS967 normalized left ventricular filling pattern and isovolumic relaxation time in cKO mice. At the cellular level, cKO myocytes presented delayed kinetics of Ca 2+ transients and cell mechanics, defects that were corrected by inhibition of I Na . Collectively, these results document that VGSC β1/β1B-subunits modulate electrical and mechanical function of the heart by regulating, at least in part, Na + influx in cardiomyocytes. NEW & NOTEWORTHY We have investigated the consequences of deletion of Scn1b , the gene encoding voltage-gated sodium channel β1-subunits, on myocyte and cardiac function. Our findings support the notion that Scn1b expression controls properties of Na + influx and Ca 2+ cycling in cardiomyocytes affecting the modality of cell contraction and relaxation. These effects at the cellular level condition electrical recovery and diastolic function in vivo, substantiating the multifunctional role of β1-subunits in the physiology of the heart.

P2X4 receptors (P2X4Rs) are ligand-gated ion channels capable of conducting cations such as Na(+). Endogenous cardiac P2X4R can mediate ATP-activated current in adult murine cardiomyocytes. In the present study, we tested the hypothesis that cardiac P2X receptors can induce Na(+) entry and modulate Na(+) handling. We further determined whether P2X receptor-induced stimulation of the Na(+)/Ca(2+) exchanger (NCX) has a role in modulating the cardiac contractile state. Changes in Na(+)-K(+)-ATPase current (Ip) and NCX current (INCX) after agonist stimulation were measured in ventricular myocytes of P2X4 transgenic mice using whole cell patch-clamp techniques. The agonist 2-methylthio-ATP (2-meSATP) increased peak Ip from a basal level of 0.52 ± 0.02 to 0.58 ± 0.03 pA/pF. 2-meSATP also increased the Ca(2+) entry mode of INCX (0.55 ± 0.09 pA/pF under control conditions vs. 0.82 ± 0.14 pA/pF with 2-meSATP) at a membrane potential of +50 mV. 2-meSATP shifted the reversal potential of INCX from -14 ± 2.3 to -25 ± 4.1 mV, causing an estimated intracellular Na(+) concentration increase of 1.28 ± 0.42 mM. These experimental results were closely mimicked by mathematical simulations based on previously established models. KB-R7943 or a structurally different agent preferentially opposing the Ca(2+) entry mode of NCX, YM-244769, could inhibit the 2-meSATP-induced increase in cell shortening in transgenic myocytes. Thus, the Ca(2+) entry mode of INCX participates in P2X agonist-stimulated contractions. In ventricular myocytes from wild-type mice, the P2X agonist could increase INCX, and KB-R7943 was able to inhibit the contractile effect of endogenous P2X4Rs, indicating a physiological role of these receptors in wild-type cells. The data demonstrate a novel Na(+) entry pathway through ligand-gated P2X4Rs in cardiomyocytes., (Copyright © 2014 the American Physiological Society.)