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Author(s): Ziqing Liu [1, 2]; Li Wang [1, 2]; Joshua D. Welch [3]; Hong Ma [1, 2]; Yang Zhou [1, 2]; Haley Ruth Vaseghi [1, 2]; Shuo Yu [1, 2]; [...]

Inhibition of cGMP-specific phosphodiesterase 5 (PDE5) ameliorates pathological cardiac remodeling and has been gaining attention as a potential therapy for heart failure. Despite promising results in males, the efficacy of the PDE5 inhibitor sildenafil in female cardiac pathologies has not been determined and might be affected by estrogen levels, given the hormone's involvement in cGMP synthesis. Here, we determined that the heart-protective effect of sildenafil in female mice depends on the presence of estrogen via a mechanism that involves myocyte eNOS-dependent cGMP synthesis and the cGMP-dependent protein kinase Ia (PKGIα). Sildenafil treatment failed to exert antiremodeling properties in female pathological hearts from Gαq- overexpressing or pressure-overloaded mice after ovary removal; however, estrogen replacement restored the effectiveness of sildenafil in these animals. In females, sildenafil-elicited myocardial PKG activity required estrogen, which stimulated tonic cardiomyocyte cGMP synthesis via an eNOS/ soluble guanylate cyclase pathway. In contrast, eNOS activation, cGMP synthesis, and sildenafil efficacy were not estrogen dependent in male hearts. Estrogen and sildenafil had no impact on pressure-overloaded hearts from animals expressing dysfunctional PKGIα, indicating that PKGIα mediates antiremodeling effects. These results support the importance of sex differences in the use of PDE5 inhibitors for treating heart disease and the critical role of estrogen status when these agents are used in females., Introduction Heart disease is the major cause of death in women as well as in men in developed countries. However, there are sex-specific clinical characteristics, some of which may be [...]

Glucose uptake and glycolysis are increased in the heart during ischemia, and this metabolic alteration constitutes an important contributing factor towards ischemic injury. Therefore, it is important to understand glucose uptake regulation in the ischemic heart. There are primarily 2 glucose transporters controlling glucose uptake into cardiac myocytes: GLUT1 and GLUT4. In the non-ischemic heart, insulin stimulates GLUT4 translocation to the sarcolemmal membrane, while both GLUT1 and GLUT4 translocation can occur following AMPK stimulation. Using a newly developed technique involving [³H]2-deoxyglucose, we measured glucose uptake in H9c2 ventricular myoblasts, and demonstrated that while insulin has no detectable effect on glucose uptake, phenformin-induced AMPK activation increases glucose uptake 2.5-fold. Furthermore, insulin treatment produced no discernible effect on either Akt serine 473 phosphorylation or AMPKα threonine 172 phosphorylation, while treatment with phenformin results in an increase in AMPKα threonine 172 phosphorylation, and a decrease in Akt serine 473 phosphorylation. Visualization of a dsRed-GLUT4 fusion construct in H9c2 cells by laser confocal microscopy showed that unlike insulin, AMPK activation did not redistribute GLUT4 to the sarcolemmal membrane, suggesting that AMPK may regulate glucose uptake via another glucose transporter. These studies suggest that AMPK is a major regulator of glucose uptake in cardiac myocytes. Key words: AMPK, glucose uptake, GLUT4, glycolysis, metabolism, cardiac myocyte, 2-deoxyglucose. La captation de glucose et laglycolyse sont accrues dans le cceur lors de l'ischemie, et cette modification metabolique constitue un facteur contributif important vers le dommage ischemique. En consequence, il est important de comprendre la regulation de la captation du glucose dans le cceur ischemique. Il existe 2 transporteurs de glucose qui controlent la captation de glucose dans les myocytes cardiaques, GLUT1 et GLUT4. Dans le ccur non ischemique, l'insuline stimule la translocation de GLUT4 vers la membrane du sarcolemme, alors que la translocation de GLUT1 et GLUT4 peut survenir a la suite de la stimulation de l'AMPK. Nous avons mesure la captation de glucose dans les myoblastes ventriculaires H9c2 a l'aide d'une nouvelle technique impliquant le [³H]2-deoxyglucose, et nous avons demontre que, alors que l'insuline n'exerce pas d'effet detectable sur la captation de glucose, l'activation de l'AMPK par la phenformine accroit la captation de glucose de 2.5 fois. De plus, le traitement a l'insuline ne produisait par d'effet detectable sur la phosphorylation d'Akt sur la serine 473 ou de l'AMPKα sur la threonine 172, alors que le traitement a la phenformine resultait en une augmentation de la phosphorylation de l'AMPKα sur la threonine 172, et en une diminution de la phosphorylation d'Akt sur la serine 473. L'observation en microscopie confocale au laser d'une construction impliquant une fusion dsRed-GLUT4 chez les cellules H92c a montre que contrairement a l'insuline, l'activation de l'AMPK ne produit pas de redistribution de GLUT4 a la membrane du sarcolemme, suggerant que l'AMPK peut reguler la captation de glucose par l'intermediaire d'un autre transporteur de glucose. Ces etudes suggerent que l'AMPK est un regulateur cle de la captation de glucose dans les myocytes cardiaques. [Traduit par la Redaction] Mots-cles: AMPK, captation de glucose, GLUT4, glycolyse, metabolisme, myocytes cardiaques, 2-deoxyglucose., Introduction 5'-AMP-activated protein kinase (AMPK) is a serine/threonine kinase that acts as a 'cellular fuel gauge,' owing to its ability to increase energy producing pathways, and to inhibit energy consuming [...]

Human mutations in or variants of TBX20 are associated with congenital heart disease, cardiomyopathy, and arrhythmias. To investigate whether cardiac disease in patients with these conditions results from an embryonic or ongoing requirement for Tbx20 in myocardium, we ablated Tbx20 specifically in adult cardiomyocytes in mice. This ablation resulted in the onset of severe cardiomyopathy accompanied by arrhythmias, with death ensuing within 1 to 2 weeks of Tbx20 ablation. Accounting for this dramatic phenotype, we identified molecular signatures that posit Tbx20 as a central integrator of a genetic program that maintains cardiomyocyte function in the adult heart. Expression of a number of genes encoding critical transcription factors, ion channels, and cytoskeletal/myofibrillar proteins was downregulated consequent to loss of Tbx20. Genome-wide ChIP analysis of Tbx20-binding regions in the adult heart revealed that many of these genes were direct downstream targets of Tbx20 and uncovered a previously undescribed DNA-binding site for Tbx20. Bioinformatics and in vivo functional analyses revealed a cohort of transcription factors that, working with Tbx20, integrated multiple environmental signals to maintain ion channel gene expression in the adult heart. Our data provide insight into the mechanisms by which mutations in TBX20 cause adult heart disease in humans., Introduction Coordinated expression of transcription factors in adult heart regulates gene expression programs responsible for cardiomyocyte function. As many of these factors are also essential for cardiac development, it is [...]

Myocardial infarction has been the leading cause of morbidity and mortality in developed countries over the past few decades. The transplantation of cardiomyocytes offers a potential method of treatment. However, cardiomyocytes are in high demand and their supply is extremely limited. Embryonic stem cells (ESCs), which have been isolated from the inner cell mass of blastocysts, can self-renew and are pluripotent, meaning they have the ability to develop into any type of cell, including cardiomyocytes. This suggests that ESCs could be a good source of genuine cardiomyocytes for future therapeutic purposes. However, problems with the yield and purity of ESC-derived cardiomyocytes, among other hurdles for the therapeutic application of ESC-derived cardiomyocytes (e.g., potential immunorejection and tumor formation problems), need to be overcome before these cells can be used effectively for cell replacement therapy. ESC-derived cardiomyocytes consist of nodal, atrial, and ventricular cardiomyocytes. Specifically, for treatment of myocardial infarction, transplantation of a sufficient quantity of ventricular cardiomyocytes, rather than nodal or atrial cardiomyocytes, is preferred. Hence, it is important to find ways of increasing the yield and purity of specific types of cardiomyocytes. Atrial and ventricular cardiomyocytes have differential expression of genes (transcription factors, structural proteins, ion channels, etc.) and are functionally distinct. This paper presents a thorough review of differential gene expression in atrial and ventricular myocytes, their expression throughout development, and their regulation. An understanding of the molecular and functional differences between atrial and ventricular myocytes allows discussion of potential strategies for preferentially directing ESCs to differentiate into chamber-specific cells, or for fine tuning the ESC-derived cardiomyocytes into specific electrical and contractile phenotypes resembling chamber-specific cells. atrial cardiomyocytes; ventricular cardiomyocytes; gene regulations; therapeutic application doi: 10.1152/ajpcell.00402.2009.

Previous studies have suggested that inhibition of the mammalian target of rapamycin (mTOR) by rapamycin suppresses myocardial hypertrophy. However, the role of mTOR in the progression of cardiac dysfunction in pathological hypertrophy has not been fully defined. Interestingly, recent reports indicate that the inflammatory response, which plays an important role in the development of heart failure, is enhanced by rapamycin under certain conditions. Our aim in this study was to determine the influence of mTOR on pathological hypertrophy and to assess whether cardiac mTOR regulates the inflammatory response. We generated transgenic mice with cardiac-specific overexpression of wild-type mTOR (mTOR-Tg). mTOR-Tg mice were protected against cardiac dysfunction following left ventricular pressure overload induced by transverse aortic constriction (TAC) (P < 0.01) and had significantly less interstitial fibrosis compared with littermate controls (WT) at 4 wk post-TAC (P < 0.01). In contrast, TAC caused cardiac dysfunction in WT. At 1 wk post-TAC, the proinflammatory cytokines interleukin (IL)-1[beta] and IL-6 were significantly increased in WT mice but not in mTOR-Tg mice. To further characterize the effects of mTOR activation, we exposed HL-1 cardiomyocytes transfected with mTOR to lipopolysaccharide (LPS). mTOR overexpression suppressed LPS-induced secretion of IL-6 (P < 0.001), and the mTOR inhibitors rapamycin and PP242 abolished this inhibitory effect of mTOR. In addition, mTOR overexpression reduced NF-[kappa]B-regulated transcription in HL-1 cells. These data suggest that mTOR mitigates adverse outcomes of pressure overload and that this cardioprotective effect of mTOR is mediated by regulation of the inflammatory reaction. cardiac hypertrophy; heart failure; inflammation; transgenic mice; mammalian target of rapamycin doi: 10.1152/ajpcell.00338.2010.

Predictions of microRNA-mRNA interactions typically rely on bioinformatic algorithms, but these algorithms only suggest the possibility of microRNA binding and may miss important interactions as well as falsely predict others. We developed an affinity purification approach to empirically identify microRNAs associated with the 3' UTR of the mRNA encoding Hand2, a transcription factor essential for cardiac development. In addition to miR-1, a known regulator of Hand2 expression, we determined that the Hand2 3'UTR also associated with miR-133a, a microRNA cotranscribed with miR-1 in cardiac and muscle cells. Using a sequential binding assay, we showed that miR-1 and miR-133a could occupy the Hand2 3'UTR concurrently, miR-133a inhibited Hand2 expression in tissue culture models, and miR-133a double knockout mice had elevated levels of Hand2 mRNA and protein. We conclude that Hand2 is regulated by miR-133a in addition to miR-1. The affinity purification assay should be generally applicable for identifying other microRNA-mRNA interactions. heart | cardiomyocytes | C2C12 | MS2 doi/ 10.1073/pnas.1013162107

A study was conducted to evaluate the capacity of patient-specific induced pluripotent stem-cell models from a family affected by long-QT syndrome to give rise to functional cardiomyocytes that recapitulate the electrophysiological characteristics of the disorder. Results indicated that in such cases the patient-derived cells recapitulated the electrophysiological characteristics of the disorder.

A zebrafish heart can fully regenerate after amputation of up to 20% of its ventricle. During this process, newly formed coronary blood vessels revascularize the regenerating tissue. The formation of coronary blood vessels during zebrafish heart regeneration likely recapitulates embryonic coronary, vessel development, which involves the activation and proliferation of the epicardium, followed by an epithelial-to-mesenchymal transition. The molecular and cellular mechanisms underlying these processes are not well understood. We examined the role of PDGF signaling in explant-derived primary cultured epicardial cells in vitro and in regenerating zebrafish hearts in vivo. We observed that mural and mesenchymal cell markers, including pdgfr[beta], are up-regulated in the regenerating hearts. Using a primary culture of epicardial cells derived from heart explants, we found that PDGF signaling is essential for epicardial cell proliferation. PDGF also induces stress fibers and loss of cell-cell contacts of epicardial cells in explant culture. This effect is mediated by Rho-associated protein kinase. Inhibition of PDGF signaling in vivo impairs epicardial cell proliferation, expression of mesenchymal and mural cell markers, and coronary blood vessel formation. Our data suggest that PDGF signaling plays important roles in epicardial function and coronary vessel formation during heart regeneration in zebrafish. epicardium | mesenchyrnal cells | mural cells | zebrafish heart regeneration doi/ 10.1073/pnas.0915016107

IL-18 and the extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN) stimulate the expression of proinflammatory cytokines and MMPs and are elevated in myocardial hypertrophy, remodeling, and failure. Here, we report several novel findings in primary cardiomyocytes treated with IL-18. First, IL-18 activated multiple transcription factors, including NF-[kappa]B (p50 and p65), activator protein (AP)-1 (cFos, cJun, and JunD), GATA, CCAAT/enhancer-binding protein, myocyte-specific enhancer-binding factor, interferon regulatory factor-1, p53, and specific protein (Sp)-1. Second, IL-18 induced EMMPRIN expression via myeloid differentiation primary response gene 88/IL-1 receptor-associated kinase/TNF receptor-associated factor-6/JNK-dependent Sp1 activation. Third, IL-18 induced a number of MMP genes, particularly MMP-9, at a rapid rate as well as tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-3 at a slower rate. Finally, the IL-18 induction of MMP-9 was mediated in part via EMMPRIN and through JNK- and ERK-dependent AP-1 activation and p38 MAPK-dependent NF-[kappa]B activation. These results suggest that the elevated expression of IL-18 during myocardial injury and inflammation may favor EMMPRIN and MMP induction and extracellular matrix degradation. Therefore, targeting IL-18 or its signaling pathways may be of potential therapeutic benefit in adverse remodeling. myocardial remodeling; extracellular matrix; extracellular matrix metalloproteinase inducer; matrix metalloproteinase; tissue inhibitor of metalloproteinase; c-Jun N[H.sub.2] terminal kinase; specific protein-1; activator protein-1; nuclear factor-[kappa]B doi: 10.1152/ajpheart.00451.2010.

The production of 20-hydroxyeicosatetraenoic acid (20-HETE) is increased during ischemia-reperfusion, and inhibition of 20-HETE production has been shown to reduce infarct size caused by ischemia. This study was aimed to discover the molecular mechanism underlying the action of 20-HETE in cardiac myocytes. The effect of 20-HETE on L-type [Ca.sup.2+] currents ([I.sub.Ca,L]) was examined in rat isolated cardiomyocytes by patch-clamp recording in the whole cell mode. Superfusion of cardiomyocytes with 20-HETE (10-100 nM) resulted in a concentration-dependent increase in [I.sub.Ca,L], and this action of 20-HETE was attenuated by a specific NADPH oxidase inhibitor, gp91ds-tat (5 [micro]M), or a superoxide scavenger, polyethylene glycol-superoxide dismutase (25 U/ml), suggesting that NADPH-oxidase-derived superoxide is involved in the stimulatory action of 20-HETE on [I.sub.Ca,L]. Treatment of cardiomyocytes with 20-HETE (100 nM) increased both NADPH oxidase activity and superoxide production by approximately twofold. To study the molecular mechanism mediating the 20-HETE-induced increase in NADPH oxidase activity, PKC activity was measured in cardiomyocytes. Incubation of the cells with 20-HETE (100 nM) significantly increased PKC activity, and pretreatment of cardiomyocytes with a selective PKC inhibitor, GF-109203 (1 [micro]M), attenuated the 20-HETE-induced increases in [I.sub.Ca,L] and in NADPH oxidase activity. In summary, 20-HETE stimulates NADPH oxidase-derived superoxide production, which activates L-type [Ca.sup.2+] channels via a PKC-dependent mechanism in cardiomyocytes. 20-HETE and 20-HETE-producing enzymes could be novel targets for the treatment of cardiac ischemic diseases. 20-hydroxyeicosatetraenoic acid; L-type calcium channel; protein kinase C; cardiac myocytes; reactive oxygen species doi: 10.1152/ajpheart.00067.2010.

Sulfonylurea receptor-containing ATP-sensitive potassium ([K.sub.ATP]) channels have been implicated in cardioprotection, but the cell type and constitution of channels responsible for this protection have not been clear. Mice deleted for the first nucleotide binding region of sulfonylurea receptor 2 (SUR2) are referred to as SUR2 null since they lack full-length SUR2 and glibenclamide-responsive [K.sub.ATP] channels in cardiac, skeletal, and smooth muscle. As previously reported, SUR2 null mice develop electrocardiographic changes of ST segment elevation that were shown to correlate with coronary artery vasospasm. Here we restored expression of the cardiomyocyte SUR2-[K.sub.ATP] channel in SUR2 null mice by generating transgenic mice with ventricular cardiomyocyte-restricted expression of SUR2A. Introduction of the cardiomyocyte SUR2A transgene into the SUR2 null background restored functional cardiac [K.sub.ATP] channels. Hearts isolated from rescued mice, referred to as MLC2A, had significantly reduced infarct size (27 [+ or -] 3% of area at risk) compared with SUR2 null mice (36 [+ or -] 3% of area at risk). Compared with SUR2 null hearts, MLC2A hearts exhibited significantly improved cardiac function during the postischemia reperfusion period primarily because of preservation of low diastolic pressures. Additionally, restoration of cardiac SUR2-[K.sub.ATP] channels significantly reduced the degree and frequency of ST segment elevation episodes in MLC2A mice. Therefore, cardioprotective mechanisms both dependent and independent of SUR2-[K.sub.ATP] channels contribute to cardiac function. ATP-sensitive potassium channel; SUR2; SUR2A; ischemia; vasospasm doi: 10.1152/ajpheart.00084.2010.

Both mechanical and humoral triggers have been put forward to explain the hypertrophic response of the challenged cardiomyocyte. The aim of the present study was to investigate whether cyclic equibiaxial stretch is a direct stimulus for isolated adult rabbit cardiomyocytes to develop hypertrophy and to explore the potential involvement of the autocrine/ paracrine factors ANG II, transforming growth factor (TGF)-[[beta].sub.1], and IGF-I in this process. Isolated cardiomyocytes were exposed to 10% cyclic equibiaxial stretch (1 Hz) for up to 48 h or treated with ANG II (100 nM), TGF-[[beta].sub.1] (5 ng/ml), IGF-I (100 ng/ml), ANG II type 1 ([AT.sub.1]) receptor blockers, or conditioned medium of stretched fibroblasts. Cyclic stretch significantly increased cell surface area (+3.1%), protein synthesis (+ 21%), and brain natriuretic peptide (BNP) mRNA expression (6-fold) in cardiomyocytes. TGF-[[beta].sub.1] expression increased (+42%) transiently at 4 h, whereas cardiomyocyte IGF-I expression was not detectable under all experimental conditions. The [AT.sub.1] receptor blockers candesartan and irbesartan (100 nM) did not prevent the stretch-induced hypertrophic response. Direct exposure to ANG II, TGF-[[beta].sub.1], or IGF-I did not enhance cardiomyocyte BNP expression. In cardiac fibroblasts, stretch elicited a significant approximately twofold increase in TGF-[[beta].sub.1] and IGF-I expression. Conditioned medium of stretched fibroblasts increased BNP expression in cardiomyocytes (~2-fold, P = 0.07). This study clearly indicates that cyclic stretch is a strong, direct trigger to induce hypertrophy in fully differentiated rabbit cardiomyocytes. The present findings do not support the notion that stretch-mediated hypertrophy of adult rabbit cardiomyocytes involves autocrine/paracrine actions of ANG II, TGF-[[beta].sub.1], or IGF-I. fibroblasts; transforming growth factor-[[beta].sub.1]; angiotensin II; insulin-like growth factor-I; brain natriuretic peptide doi: 10.1152/ajpheart.00822.2009.

Adenylyl cyclase (AC) type 5 (AC5) and AC type 6 (AC6) are the two major AC isoforms in the heart. Cardiac overexpression of AC6 has been shown to be protective in response to several interventions. In this investigation, we examined the effects of chronic pressure overload in AC6 transgenic (TG) mice. In the absence of any stress, AC6 TG mice exhibited enhanced contractile function compared with their wild-type (WT) littermates, i.e., increased (P < 0.05) left ventricular (LV) ejection fraction (EF) (75 [+ or -] 0.9 vs. 71 [+ or -] 0.5%) and LV dP/dt (7,850 [+ or -] 526 vs. 6,374 [+ or -] 315 mmHg/s). Forskolin (25 [micro] x [kg.sup.-1] x [min.sup.-1] for 5 min) increased LVEF more (P < 0.05) in AC6 TG mice (14.8 [+ or -] 1.0%) than in WT mice (7.7 [+ or -] 1.0%). Also, isoproterenol (0.04 [micro]g x [kg.sup.-1] x [min.sup.-1] for 5 min) increased LVEF more (P < 0.05) in AC6 TG mice (18.0 [+ or -] 1.2%) than in WT mice (11.6 [+ or -] 2.1%). Pressure overload, induced by 4 wk of transverse aortic constriction (TAC), increased the LV weight-to-body weight ratio and myocyte cross-sectional area similarly in both groups, but reduced LVEF more in AC6 TG mice (22%) compared with WT mice (9%), despite the higher starting level of LVEF in AC6 TG mice. LV systolic wall stress increased more in AC6 TG mice than in WT mice, which could be responsible for the reduced LVEF in AC6 TG mice with chronic pressure overload. In addition, LV dP/dt was no longer elevated in AC6 TG mice after TAC compared with WT mice. LV end-diastolic diameter was also greater (P < 0.05) in AC6 TG mice (3.8 [+ or -] 0.07 mm) than in WT mice (3.6 [+ or -] 0.05 mm) after TAC. Thus, in contrast to other interventions previously reported to be salutary with cardiac AC6 overpression, the response to chronic pressure overload was not; actually, AC6 TG mice fared worse than WT mice. The mechanism may be due to the increased LV systolic wall stress in AC6 TG mice with chronic pressure overload. cardiac function; transverse aortic constriction; hypertrophy; apoptosis; transgenic doi: 10.1152/ajpheart.00148.2010.

In experimental animal and cell culture models, activation of peroxisome proliferator-activated receptor (PPAR) [gamma] in heart has been shown to have beneficial effects on cardiac function and cardiomyocyte physiology. The goal of this study was to identify the signaling pathway by which PPAR[gamma] activation protects cardiomyocytes from the deleterious effects of hypertrophic stimuli. In primary cardiomyocyte cultures, we found that genetic or pharmacological activation of PPAR[gamma] protected cells from cardiac hypertrophy induced by [alpha]-adrenergic stimulation. Examination of gene expression in these cells revealed a surprising increase in the expression of adiponectin in cardiomyocytes and secretion of the high-molecular-weight form of the hormone into media. Using RNAi to block PPAR[gamma]-induced adiponectin production or adiponectin receptor gene expression, we found that the PPAR[gamma]-mediated antihypertrophic effect required cardiomyocyte-produced adiponectin, as well as an intact adiponectin signaling pathway. Furthermore, mice expressing constitutive-active PPAR[gamma] and cardiomyocyte specific adiponectin expression were protected from high-fat diet-induced cardiac hypertrophy and remodeling. These findings demonstrate that functional adiponectin hormone can be produced from the heart and raise the possibility that beneficial effects of PPAR[gamma] activation in heart could be due in part to local production of adiponectin that acts on cardiomyocytes in an autocrine manner. peroxisome proliferator-activated receptor [gamma]; adiponectin; thiazolidinediones; regulation of gene expression; cardiac hypertrophy; type 2 diabetes doi: 10.1152/ajpheart.01032.2009.

We have previously shown that the inhibition of myocardial nitric oxide (NO) and peroxynitrite-matrix metalloproteinase (MMP) signaling by early preconditioning (PC) is involved in its cardioprotective effect. Therefore, in the present study, we investigated the role of NO and peroxynitrite-MMP signaling in the development of late PC. PC was performed by five consecutive cycles of 4-min coronary occlusion and 4-min reperfusion in anesthetized rats in vivo. Twenty-four hours later, hearts were subjected to a 30-min coronary occlusion followed by 180-min reperfusion to measure infarct size. In separate experiments, heart tissue was sampled to measure biochemical parameters before and 3, 6, 12, or 24 h after the PC protocol, respectively. Late PC decreased infarct size, increased cardiac inducible NO synthase (iNOS) activity and gene expression, and decreased SOD activity at 24 h significantly compared with sham-operated controls. Late PC increased cardiac superoxide levels significantly at 24 h; however, it did not change cardiac NO levels. Cardiac peroxynitrite levels were significantly decreased. Downstream cellular targets of peroxynitrite, MMP-2 and MMP-9 activities were decreased in the late PC group at 24 h compared with the sham-operated group. To verify if PC-induced inhibition of MMPs had a causative role in the reduction of infarct size, in separate experiments, we measured infarct size after the pharmacological inhibition of MMPs by ilomastat and found a significant reduction of infarct size compared with the vehicle-treated group. In conclusion, this is the first demonstration that the inhibition of cardiac peroxynitrite-MMP signaling contributes to cardioprotection by late PC and that pharmacological inhibition of MMPs is able to reduce infarct size in vivo. Furthermore, increased expression of iNOS may play a role in the development of late PC; however, increased iNOS activity does not lead to increased NO production in late PC. nitric oxide; peroxynitrite; inducible nitric oxide synthase; nitric oxide synthase; superoxide; nitrotyrosine; superoxide dismutase; ilomastat; tissue inhibitors of metalloproteinase; xanthine oxidoreductase; NADPH oxidase; gene expression doi: 10.1152/ajpheart.00052.2010.

Cardiac remodeling during heart failure development induces a significant increase in the activity of the L-type [Ca.sup.2+] channel (Cavl.2). However, the effects of enhanced Cavl.2 activity on myocyte excitation-contraction (E-C) coupling, cardiac contractility, and its regulation by the [beta]-adrenergic system are not clear. To recapitulate the increased Cavl.2 activity, a double transgenic (DTG) mouse model overexpressing the Cav[beta]2a subunit in a cardiac-specific and inducible manner was established. We studied cardiac (in vivo) and myocyte (in vitro) contractility at baseline and upon [beta]-adrenergic stimulation. E-C coupling efficiency was evaluated in isolated myocytes as well. The following results were found: l) in DTG myocytes, L-type [Ca.sup.2+] current ([I.sub.Ca,L]) density, myocyte fractional shortening (FS), peak [Ca.sup.2+] transients, and sarcoplasmic reticulum (SR) [Ca.sup.2+] content (caffeine-induced [Ca.sup.2+] transient peak) were significantly increased (by 100.8%, 48.8%, 49.8%, and 46.8%, respectively); and 2) cardiac contractility evaluated with echocardiography [ejection fraction (EF) and (FS)] and invasive intra-left ventricular pressure (maximum dP/dt and -dP/dt) measurements were significantly greater in DTG mice than in control mice. However, 1) the cardiac contractility (EF, FS, dP/dt, and -dP/dt)-enhancing effect of the [beta]-adrenergic agonist isoproterenol (2 [micro]g/g body wt ip) was significantly reduced in DTG mice, which could be attributed to the loss of [beta]-adrenergic stimulation on contraction, [Ca.sup.2+] transients, [I.sub.Ca,L], and SR [Ca.sup.2+] content in DTG myocytes; and 2) E-C couplng efficiency was significantly lower in DTG myocytes. In conclusion, increasing Cavl.2 activity by promoting its high-activity mode enhances cardiac contractility but decreases E-C coupling efficiency and the adrenergic reserve of the heart. L-type calcium channel; [[beta].sub.2a], subunit; ventricular myocyte; [beta]-adrenergic response doi: 10.1152/ajpheart.00265.2010.

Obestatin, a newly discovered peptide encoded by the ghrelin gene, induces the expression of genes regulating pancreatic [beta]-cell differentiation, insulin biosynthesis, and glucose metabolism. It also activates antiapoptotic signaling pathways such as phosphoinositide 3-kinase (PI3K) and ERKI/2 in pancreatic [beta]-cells and human islets. Since these kinases have been shown to protect against myocardial injury, we sought to investigate whether obestatin would exert cardioprotective effects. Both isolated perfused rat heart and cultured cardiomyocyte models of ischemia-reperfusion (I/R) were used to measure infarct size and cell apoptosis as end points of injury. The presence of specific obestatin receptors on cardiac cells as well as the signaling pathways underlying the obestatin effect were also studied. In the isolated heart, the addition of rat obestatin-(1-23) before ischemia reduced infarct size and contractile dysfunction in a concentration-dependent manner, whereas obestatin(23-1), a synthetic analog with an inverse aminoacid sequence, was ineffective. The cardioprotective effect of obestatin-(1-23) was observed at concentrations of 10-50 nmol/l and was abolished by inhibiting PI3K or PKC by the addition of wortmannin (100 nmol/1) or chelerythrine, (5 [micro]mol/1), respectively. In rat H9c2 cardiac cells or isolated ventricular myocytes subjected to I/R, 50 nmol/1 obestatin(1-23) reduced cardiomyocyte apoptosis and reduced caspase-3 activation; the antiapoptotic effect was blocked by the inhibition of PKC, PI3K, or ERK1/2 pathways. In keeping with these functional findings, radioreceptor binding results revealed the presence of specific high-affinity obestatin-binding sites, mainly localized on membranes of the ventricular myocardium and cardiomyocytes. Our data suggest that, by acting on specific receptors, obestatin-(l-23) activates PI3K, PKC-[epsilon], PKC-[delta], and ERK1/2 signaling and protects cardiac cells against myocardial injury and apoptosis induced by I/R. ischemia-reperfusion; myocardial infarction; obestatin receptors doi: 10.1152/ajpheart.00800.2009.

Cardiac diseases persistently increase the contractility demands of cardiac myocytes, which require activation of the sympathetic nervous system and subsequent increases in myocyte [Ca.sup.2+] transients. Persistent exposure to sympathetic and/or [Ca.sup.2+] stress is associated with myocyte death. This study examined the respective roles of persistent [beta]-adrenergic receptor ([beta]-AR) agonist exposure and high [Ca.sup.2+] concentration in myocyte death. Ventricular myocytes (VMs) were isolated from transgenic (TG) mice with cardiac-specific and inducible expression of the [[beta].sub.2a]-subunit of the L-type [Ca.sup.2+] channel (LTCC). VMs were cultured, and the rate of myocyte death was measured in the presence of isoproterenol (ISO), other modulators of [Ca.sup.2+] handling and the [beta]-adrenergic system, and inhibitors of caspases and reactive oxygen species generation. The rate of myocyte death was greater in TG vs. wild-type myocytes and accelerated by ISO in both groups, although ISO did not increase LTCC current ([I.sub.Ca-L]) in TG-VMs. Nifedipine, an LTCC antagonist, only partially prevented myocyte death. These results suggest both LTCC-dependent and -independent mechanisms in ISO induced myocyte death. ISO increased the contractility of wild type and TG-VMs by enhancing sarcoplasmic reticulum function and inhibiting sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase, [Na.sup.+]/[Ca.sup.2+] exchanger, and CaMKII partially protected myocyte from death induced by both [Ca.sup.2+] and ISO. Caspase and reactive oxygen species inhibitors did not, but [[beta].sub.2]-AR activation did, reduce myocyte death induced by enhanced [I.sub.Ca-L] and ISO stimulation. Our results suggest that catecholamines induce myocyte necrosis primarily through [[beta].sub.1]-AR-mediated increases in [I.sub.Ca-L], but other mechanisms are also involved in rodents. [Ca.sup.2+] signaling; [[beta].sub.2]-adrenergic receptor; mouse heart; necrosis; [[beta].sub.2a]-subunit doi: 10.1152/ajpheart.00392.2010.

Diabetes mellitus (DM) is closely related to cardiovascular morbidity and mortality, but the specific molecular basis linking DM with increased vulnerability to cardiovascular injury remains incompletely understood. Methylglyoxal (MG), a precursor to advanced glycation end products (AGEs), is increased in diabetic patient plasma, but its role in diabetic cardiovascular complications is unclear. Thioredoxin (Trx), a cytoprotective molecule with antiapoptotic function, has been demonstrated to be vulnerable to glycative inhibition, hut whether Trx is glycatively inhibited by MG, thus contributing to increased cardiac injury, has never been investigated. Cultured H9c2 cardiomyocytes were treated with MG (200 [micro]M) for 6 days. The following were determined pre- and post-simulated ischemia-reperfusion (SI-R: 8 h of hypoxia followed by 3 h of reoxygenation): cardiomyocyte death/ apoptosis, Trx expression and activity, AGE formation, Trx-apoptosis-regulating kinase-1 (Trx-ASK1) complex formation, and p38 mitogen-activated protein kinase (MAPK) phosphorylation and activity. Compared with vehicle, MG significantly increased SI-R-induced cardiomyocyte LDH release and apoptosis (P < 0.01). Prior to SI-R, Trx activity was reduced in MG-treated ceils, but Trx expression was increased moderately. Moreover, Trx-ASK1 complex formation was reduced, and both p38 MAPK activity and phosphorylation were increased. To investigate the effects of MG on Trx directly, recombinant human Trx (hTrx) was incubated with MG in vitro. Compared with vehicle, MG incubation markedly increased CML formation (a glycation footprint) and inhibited Trx activity. Finally, glycation inhibitor aminoguanidine administration during MG treatment of cultured cells reduced AGE formation, increased Trx activity, restored Trx-ASK1 interaction, and reduced p38 MAPK phosphorylation and activity, caspase-3 activation, and LDH release (P < 0.01). We demonstrated for the first time that methylglyoxal sensitized cultured cardiomyocytes to SI-R injury by posttranslational modification of Trx via glycation. Therapeutic interventions scavenging AGE precursors may attenuate ischemic-reperfusion injury in hyperglycemic state diseases such as diabetes. hypoxia and reoxygenation; apoptosis doi: 10.1152/ajpendo.00215.2010.

Studies in various types of cells find that, on average, each mitochondrion becomes involved in a fusion event every 15 min, depending on the cell type. As most contact events do not result in mitochondrial fusion, it is expected that properties of the individual mitochondrion determine the likelihood of a fusion event. However, apart from membrane potential, the properties that influence the likelihood of entering a fusion event are not known. Here, we tag and track individual mitochondria in H9c2, INS1, and primary [beta]-cells and determine the biophysical properties that increase the likelihood of a fusion event. We found that the probability for fusion is independent of contact duration and organelle dimensions, but it is influenced by organelle motility. Furthermore, the history of a previous fusion event of the individual mitochondrion influenced both the likelihood for a subsequent fusion event, as well as the site on the mitochondrion at which the fusion occurred. These observations unravel the specific properties that distinguish mitochondria that will enter fusion events from the ones that will not. Altogether, these properties may help to elucidate the molecular mechanisms that regulate fusion at the level of the single mitochondrion. mitochondrial fusion and fisson; mitochondrial movement; H9c2 cells; photoactivatable green fluorescent protein doi: 10.1152/ajpcell.00427.2009.

The vertebrate heart is formed from diverse embryonic territories, including the first and second heart fields. The second heart field (SHF) gives rise to the right ventricle and outflow tract, yet its evolutionary origins are unclear. We found that heart progenitor ceils of the simple chordate Ciona intestinalis also generate precursors of the atrial siphon muscles (ASMs). These precursors express Islet and Tbx1/10, evocative of the splanchnic mesoderm that produces the lower jaw muscles and SHF of vertebrates. Evidence is presented that the transcription factor COE is a critical determinant of ASM fate. We propose that the last common ancestor of tunicates and vertebrates possessed multipotent cardiopharyngeal muscle precursors, and that their reallocation might have contributed to the emergence of the SHF. 10.l126/science.1190181

Nuclear factors of activated T cells (NFATs) are [Ca.sup.2+]-sensitive transcription factors that have been implicated in hypertrophy, heart failure (HF), and arrhythmias. Cytosolic NFAT is activated by dephosphorylation by the [Ca.sup.2+]-sensitive phosphatase calcineurin, resulting in translocation to the nucleus, which is opposed by kinase activity, rephosphorylation, and nuclear export. Four different NFAT isoforms are expressed in the heart. The activation and regulation of NFAT in adult cardiac myocytes, which may depend on the NFAT isoform and cell type, are not fully understood. This study compared basal localization, import, and export of NFATcl and NFATc3 in adult atrial and ventricular myocytes to identify isoform-and tissue-specific regulatory mechanisms of NFAT activation under physiological conditions and in HF. NFAT-green fluorescent protein fusion proteins and NFAT immunocytochemistry were used to analyze NFAT regulation in adult cat and rabbit myocytes. NFATcl displayed basal nuclear localization in atrial and ventricular myocytes, an effect that was attenuated by reducing intracellular [Ca.sup.2+] concentration and inhibiting calcineurin, and enhanced by the inhibition of nuclear export. In contrast, NFATc3 was localized to the cytoplasm but could be driven to the nucleus by angiotensin II and endothelin-1 stimulation in atrial, but not ventricular, cells. Inhibition of nuclear export (by leptomycin B) facilitated nuclear localization in both cell types. Ventricular myocytes from HF rabbits showed increased basal nuclear localization of endogenous NFATc3 and reduced responsiveness of NFAT translocation to phenylephrine stimulation. In control myocytes, [Ca.sup.2+] overload, leading to spontaneous [Ca.sup.2+] waves, induced substantial translocation of NFATc3 to the nucleus. We conclude that the activation of NFAT in adult cardiomyocytes is isoform and tissue specific and is tightly controlled by nuclear export. NFAT is activated in myocytes from HF animals and may be secondary to [Ca.sup.2+] overload. nuclear factor of activated T cells; intracellular [Ca.sup.2+] concentration; calcineurin; nuclear translocation doi: 10.1152/ajpheart.01072.2009.

Hypoxia-inducible factor (HIF)-l[alpha] is a key regulator of anaerobic energy metabolism. We asked the following question: Does the breakdown of microtubular structures influence glycolysis in hypoxic cardiomyocytes by regulating HIF-l[alpha] Neonatal rat cardiomyocytes were cultured under hypoxic conditions, while microtubule-stabilizing (pacfitaxel) and -depolymerizing (colchicine) agents were used to change microtubular structure. Models of high microtubule-associated protein 4 (MAP4) expression and RNA interference of microtubulin expression were established. Microtubular structural changes and intracellular HIF-l[alpha] protein distribution were observed with laser confocal scanning microscopy. Content of key glycolytic enzymes, viability, and energy content of cardiomyocytes were determined by colorimetry and high-performance liquid chromatography. HIF-l[alpha] protein content and mRNA expression were determined by Western blotting and real-time PCR, respectively. Low doses of microtubule-stabilizing agent (10 [micro]mol/1 paclitaxel) and enhanced expression of MAP4 stabilized the reticular microtubular structures in hypoxic cardiomyocytes, increased the content of key glycolytic enzymes, ameliorated energy supply and enhanced cell viability, and upregulated HIF-1[alpha] protein expression and endonuclear aggregation. In contrast, the microtubule-depolymerizing agent (10 [micro]mol/1 colchicine) or reduced microtubulin expression had adverse affects on the same parameters, in particular, HIF-l[alpha] protein content and endonuclear aggregation. We conclude that microtubular structural changes influence glycolysis in the early stages of hypoxia in cardiomyocytes by regulating HIF-1[alpha] content. Stabilizing microtubular structures increases endonuclear and total HIF-l[alpha] expression, content of key glycolytic enzymes, and energy supply. These findings provide potential therapeutic targets for ameliorating cell energy metabolism during early myocardial hypoxia. pacfitaxel; colchicine; microtubule-associated protein 4; interference RNA; PCR; immunofluorescence; glycolytic enzymes; ATP; ADP; pyruvate kinase; hexokinase; phosphofructokinase; lactate; [alpha]-microtubulin doi: 10.1152/ajpheart.01039.2009.

Mutations in human ether-a-go-go-related gene 1 (hERG) are linked to long QT syndrome type 2 (LQT2). hERG encodes the pore-forming [alpha]-subunits that coassemble to form rapidly activating delayed rectifier [K.sup.+] current in the heart. LQT2-1inked missense mutations have been extensively studied in noncardiac heterologous expression systems, where biogenic (protein trafficking) and biophysical (gating and permeation) abnormalities have been postulated to underlie the loss-of-function phenotype associated with LQT2 channels. Little is known about the properties of LQT2-1inked hERG channel proteins in native cardiomyocyte systems. In this study, we expressed wild-type (WT) hERG and three LQT2-1inked mutations in neonatal mouse cardiomyocytes and studied their electrophysiological and biochemical properties. Compared with WT hERG channels, the LQT2 missense mutations G601S and N470D hERG exhibited altered protein trafficking and underwent pharmacological correction, and N470D hERG channels gated at more negative voltages. The [DELTA]Y475 hERG deletion mutation trafficked similar to WT hERG channels, gated at more negative voltages, and had rapid deactivation kinetics, and these properties were confirmed in both neonatal mouse cardiomyocyte and human embryonic kidney (HEK)-293 cell expression systems. Differences between the cardiomyocytes and HEK-293 cell expression systems were that hERG current densities were reduced 10-fold and deactivation kinetics were accelerated 1.5- to 2-fold in neonatal mouse cardiomyocytes. An important finding of this work is that pharmacological correction of trafficking-deficient LQT2 mutations, as a potential innovative approach to therapy, is possible in native cardiac tissue. human ether-a-go-go-related gene I; long QT syndrome 2; protein trafficking; E4031 doi: 10.1152/ajpheart.01236.2009.

The female sex has been associated with improved myocardial salvage after ischemia and reperfusion (I/R). Estrogen, specifically 17[beta]-estradiol, has been demonstrated to mediate this phenomenon by limiting cardiomyocyte apoptosis. We sought to quantitatively assess the effect of sex, ovarian hormone loss, and I/R on myocardial Bax, Bcl-2, and apoptosis repressor with caspase recruitment domain (ARC) expression. Male (n = 48), female (n = 26), and oophorectomized female (n = 20) rabbits underwent 30 rain of regional ischemia and 3 h of reperfusion. The myocardial area at risk and infarct size were determined using a double-staining technique and planimetry. In situ oligo ligation was used to assess apoptotic cell death. Western blot analysis was used to determine proapoptotic (Bax) and antiapoptotic (Bcl-2 and ARC) protein levels in all three ischemic groups and, additionally, in three nonischemic groups. Infarct size (43.7 [+ or -] 3.2%) and apoptotic cell death (0.51 [+ or -] 0.10%) were significantly attenuated in females compared with males (56.4 [+ or -] 1.6%, P < 0.01, and 4.29 [+ or -] 0.95%, P < 0.01) and oophorectomized females (55.7 [+ or -] 3.4%, P < 0.05, and 4.36 [+ or -] 0.51%, P < 0.01). Females expressed significantly higher baseline ARC levels (3.62 [+ or -] 0.29) compared with males (1.78 [+ or -] 0.18, P < 0.01) and oophorectomized females (1.08 [+ or -] 0.26, P < 0.01). Males expressed a significantly higher baseline Bax-to-Bcl-2 ratio (4.32 [+ or -] 0.99) compared with females (0.65 [+ or -] 0.13, P < 0.01) and oophorectomized females (0.42 [+ or -] 0.10, P < 0.01). I/R significantly reduced Bax-to-Bcl-2 ratios in males. In all other groups, ARC levels and Bax-to-Bcl-2 ratios did not significantly change. These results support the conclusion that in females, endogenous estrogen limits I/R-induced cardiomyocyte apoptosis by producing a baseline antiapoptotic profile, which is associated with estrogen-dependent high constitutive myocardial ARC expression. 17[beta]-estradiol: cardiomyocyte apoptosis: apoptosis repressor with caspase recruitment domain: Bcl-2: Bax doi: 10.1152/ajpheart.01021.2009.

Potassium ([K.sup.+]) channels in the inner mitochondrial membrane influence cell function and survival. Increasing evidence indicates that multiple signaling pathways and pharmacological actions converge on mitochondrial ATP-sensitive [K.sup.+] ([mitoK.sub.ATP]) channels and PKC to confer cytoprotection against necrotic and apoptotic cell injury. However, the molecular structure of [mitoK.sub.ATP] channels remains unresolved, and the mitochondrial phosphoprotein(s) that mediate cytoprotection by PKC remain to be determined. As mice deficient in the main sarcolemmal gap junction protein connexin 43 (Cx43) lack this cytoprotection, we set out to investigate a possible link among mitochondrial Cx43, [mitoK.sub.ATP] channel function, and PKC activation. By patch-clamping the inner membrane of subsarcolemmal murine cardiac mitochondria, we found that genetic Cx43 deficiency, pharmacological connexin inhibition by carbenoxolone, and Cx43 blockade by the mimetic peptide [.sup.43]GAP27 each substantially reduced diazoxide-mediated stimulation of [mitoK.sub.ATP] channels. Suppression of mitochondrial Cx43 inhibited [mitoK.sub.ATP] channel activation by PKC. [MitoK.sub.ATP] channels of interfibrillar mitochondria, which do not contain any detectable Cx43, were insensitive to both PKC activation and diazoxide, further demonstrating the role of Cx43 in [mitoK.sub.ATP] channel stimulation and the compartmentation of mitochondria in cell signaling. Our results define a role for mitochondrial Cx43 in protecting cardiac cells from death and provide a link between cytoprotective stimuli and [mitoK.sub.ATP] channel opening, making Cx43 an attractive therapeutic target for protection against cell injury., Introduction Ischemic injury can result in cell death and irreversible loss of function in a variety of biological systems (1), (2). Understanding of the intracellular signaling mechanisms by which cells [...]

Previous studies have shown that oscillations of the metabolism can occur in cardiomyocytes under conditions simulating ischemia/reperfusion. It is not known whether they can also occur during real ischemia with near-anoxic oxygen tension. Here, using oxygen clamp in on-chip picochambers, we exposed single resting cardiomyocytes to near anoxia (p[O.sub.2] < 0.1 mm Hg). We show that at near anoxia, the mitochondrial membrane potential ([DELTA][PSI]) was kept by the [F.sub.1][F.sub.0]-ATPase reversal, using glycolytic adenosine triphosphate (ATP). In many cells, activation of current through sarcolemmal [K.sub.ATP] channels ([I.sub.KATP]) started after a delay with one or several oscillations (frequency of 0.044 [+ or -] 0.002 Hz). These oscillations were time correlated with oscillations of [DELTA][PSI]. Metabolic oscillations at near anoxia are driven by glycolysis because (a) they were inhibited when glycolysis was blocked, (b) they persisted in cells treated with cytoplasmic reactive oxygen species scavengers, and (c) the highest rate of ATP synthesis during an oscillation cycle was associated with the generation of reducing equivalents. Glycolytic oscillations could be initiated upon rapid, but not slow, transition to near anoxia, indicating that the speed of ATP/ADP ratio drop is a determinant of their occurrence. At enhanced oxidative stress, the rate of ATP consumption was increased as indicated by rapid IKATP activation with large-scale oscillations. These results show that metabolic oscillations occur in cardiomyocytes at near anoxia and are driven by glycolysis and modulated by mitochondria through the rate of ATP hydrolysis, which, in turn, can be accelerated by oxidative stress. doi/10.1085/jgp.200910332

Excessive activation of calmodulin kinase II (CaMKII) causes arrhythmias and heart failure, but the cellular mechanisms for CaMKII-targeted proteins causing disordered cell membrane excitability and myocardial dysfunction remain uncertain. Failing human cardiomyocytes exhibit increased CaMKII and voltage-gated [Ca.sup.2+] channel ([Ca.sub.v]1.2) activity, and enhanced expression of a specific [Ca.sub.v]1.2 [beta]-subunit protein isoform ([[beta].sub.2a]). We recently identified [Ca.sub.v]1.2 [[beta].sub.2a] residues critical for CaMKII phosphorylation (Thr 498) and binding (Leu 493), suggesting the hypothesis that these amino acids are crucial for cardiomyopathic consequences of CaMKII signaling. Here we show WT [[beta].sub.2a] expression causes cellular [Ca.sup.2+] overload, arrhythmia-triggering cell membrane potential oscillations called early afterdepolarizations (EADs), and premature death in paced adult rabbit ventricular myocytes. Prevention of intracellular [Ca.sup.2+] release by ryanodine or global cellular CaMKII inhibition reduced EADs and improved cell survival to control levels in WT [[beta].sub.2a]-expressing ventricular myocytes. In contrast, expression of [[beta].sub.2a] T498A or L493A mutants mimicked the protective effects of ryanodine or global cellular CaMKII inhibition by reducing [Ca.sup.2+] entry through [Ca.sub.v]1.2 and inhibiting EADs. Furthermore, [Ca.sub.v]1.2 currents recorded from cells overexpressing CaMKII phosphorylation- or binding-incompetent [[beta].sub.2a] subunits were incapable of entering a CaMKII-dependent high-activity gating mode (mode 2), indicating that [[beta].sub.2a] Thr 498 and Leu 493 are required for [Ca.sub.v]1.2 activation by CaMKII in native cells. These data show that CaMKII binding and phosphorylation sites on [[beta].sub.2a] are concise but pivotal components of a molecular and biophysical and mechanism for EADs and impaired survival in adult cardiomyocytes. arrhythmias | calcium | calcium channel | calmodulin kinase | cardiac myocytes doi/ 10.1073/pnas.0913760107

Adiponectin (APN) has traditionally been viewed as an adipocyte-specific endocrine molecule with cardioprotective effects. Recent studies suggest that APN is also expressed in cardiomyocytes. However, biological significances of this locally produced APN remain completely unknown. The aim of this study was to investigate the pathological and pharmacological significance of cardiac-derived APN in cardiomyocyte pathology. Adult cardiomyocytes from wild-type littermates (WT) or gene-deficient mice were pretreated with vehicle (V) or rosiglitazone (RSG) for 6 h followed by simulated ischemia-reperfusion (SI/R, 3 h/12 h). Compared with WT cardiomyocytes, myocytes from APN knockout (APN-KO) mice sustained greater SI/R injury, evidenced by greater oxidative/nitrative stress, caspase-3 activity, and lactate dehydrogenase (LDH) release (P < 0.05). Myocytes from adiponectin receptor i knockdown (AdipoR1-KD) or AdipoR1-KD/ AdipoR2-KO mice had slightly increased SI/R injury, but the difference was not statistically significant. RSG significantly (P < 0.01) increased APN mRNA and protein expression, upregulated AdipoR1/ AdipoR2 expression, reduced SI/R-induced apoptosis, and decreased LDH release in WT cardiomyocytes. However, the anti-oxidative/ anti-nitrative and cell protective effects of RSG were completely lost in APN-KO cardiomyocytes (P > 0.05 vs. vehicle group), although a comparable degree of AdipoR1/AdipoR2 upregulation was observed. The upregulatory effect of RSG on APN mRNA and protein expression was significantly potentiated in AdipoR1-KD/AdipoR2-KO cardiomyocytes. However, the cellular protective effects of RSG were significantly blunted, although not completely lost, in these cells. These results demonstrated that cardiomyocyte APN is biologically active in protecting cells against SI/R injury. Moreover, this locally produced APN achieves its protective effect primarily through paracrine/autocrine activation of APN receptors. myocardial ischemia; diabetes; cytokines; oxidative stress doi:10.1152/ajpendo.00663.2009.

The [Na.sup.+]/[Ca.sup.2+] exchanger (NCX) is proposed to be an important protein in the regulation of [Ca.sup.2+] movements in the heart. This [Ca.sup.2+] regulatory action is thought to modulate contractile activity in the heart under normal physiological conditions and may contribute to the Ca2 + overload that occurs during ischemic reperfusion challenge. To evaluate these hypotheses, adult rat cardiomyocytes were exposed to an adenovirus that codes for short hairpin RNA (shRNA) targeting NCX gene expression through RNA interference. An adenovirus transcribing a short RNA with a scrambled nucleotide sequence was compared with the NCX-shRNA nucleotide sequence and used as a control. Freshly isolated rat cardiomyocytes were infected with virus for 48 h before examination. Cardiomyocytes maintained their characteristic morphological appearance during this short time period after isolation. NCX expression was inhibited by up to ~60% by the shRNA treatment as determined by Western blot analysis. The depletion in NCX protein was accompanied by a significant depression of NCX activity in shRNA-treated cells. [Ca.sup.2+] homeostasis was unaltered in the shRNA-treated cells upon electrical stimulation compared with control cells. However, when cardiomyocytes were exposed to a simulated ischemic solution, NCX-depleted cells were significantly protected from the rise in cytoplasmic [Ca.sup.2+] and damage that was detected in control cells during ischemia and reperfusion. Our data support the role for NCX in ischemic injury to the heart and demonstrate the usefulness of altering gene expression with an adenoviral-delivery system of shRNA in adult cardiomyocytes. short hairpin ribonucleic acid doi: 10.1152/ajpheart.00932.2009

Wang X, McLennan SV, Allen T J, Tsoutsman T, Semsarian C, Twigg SM. Adverse effects of high glucose and free fatty acid on cardiomyocytes are mediated by connective tissue growth factor. Am J Physiol Cell Physiol 297: C1490-C1500, 2009. First published July 22, 2009; doi: 10.1152/ajpcell.00049.2009.--Diabetic cardiomyopathy is characterized by interstitial fibrosis and cardiomyocyte hypertrophy and apoptosis. Also known as CCN2, connective tissue growth factor (CTGF) is implicated in the fibrosis; however, whether it contributes to cardiomyocytes changes and adverse effects of high glucose and lipids on these cells remains unknown. Hearts from streptozotocin-induced diabetic rats had elevated CTGF and changes of pathological myocardial hypertrophy, fibrosis, and cardiomyocyte apoptosis. Rat H9c2 cardiomyocytes were then treated with recombinant human (rh)CTGF, high glucose, or the saturated free fatty acid palmitate. Each reagent induced cell hypertrophy, as indicated by the ratio of total protein to cell number, cell size, and gene expression of cardiac hypertrophy marker genes atrial natriuretic peptide (ANP), and [alpha]-skeletal actin. Each treatment also caused apoptosis measured by increased caspase3/7 activity, apoptotic cells by transferase-mediated dUTP nick end labeling (TUNEL) assay, and lower viable cell number. Further studies showed CTGF mRNA was rapidly induced by high glucose and palmitate in H9c2 cells and in mouse neonatal cardiomyocyte primary cultures, small interfering RNA against CTGF blocked the high glucose and palmitate induction of hypertrophy and apoptosis. In addition, these CTGF effects were through the tyrosine kinase A (TrkA) receptor with tyrosine kinase activity, which has previously been implicated in CTGF signaling: TrkA was phosphorylated by CTGF, and a specific TrkA blocker abrogated CTGF-induced effects on hypertrophy and apoptosis. For the first time in any system, fatty acid is newly identified as a regulator of CTGF, and this work implicates autocrine CTGF as a mediator of adverse effects of high glucose and fatty acids in cardiomyocytes. diabetic cardiomyopathy; apoptosis; hypertrophy doi: 10.1152/ajpcell.00049.2009

Studies have shown significant cardiovascular effects of exogenous apelin administration, including the potent activation of cardiac contraction. However, the role of the endogenous apelin-APJ pathway is less clear. To study the loss of endogenous apelin-APJ signaling, we generated mice lacking either the ligand (apelin) or the receptor (APJ). Apelin-deficient mice were viable, fertile, and showed normal development. In contrast, APJ-deficient mice were not born in the expected Mendelian ratio, and many showed cardiovascular developmental defects. Under basal conditions, both apelin and APJ null mice that survived to adulthood manifested modest decrements in contractile function. However, with exercise stress both mutant lines demonstrated consistent and striking decreases in exercise capacity. To explain these findings, we explored the role of autocrine signaling in vitro using field stimulation of isolated left ventricular cardiomyocytes lacking either apelin or APJ. Both groups manifested less sarcomeric shortening and impaired velocity of contraction and relaxation with no difference in calcium transient. Taken together, these results demonstrate that endogenous apelin-APJ signaling plays a modest role in maintaining basal cardiac function in adult mice with a more substantive role during conditions of stress. In addition, an autocrine pathway seems to exist in myocardial cells, the ablation of which reduces cellular contraction without change in calcium transient. Finally, differences in the developmental phenotype between apelin and APJ null mice suggest the possibility of undiscovered APJ ligands or ligand-independent effects of APJ. pressure-volume hemodynamics; cardiomyocyte doi: 10.1152/ajpheart.00686.2009.

The collection of gene expression data from human heart biopsies is important for understanding the cellular mechanisms of arrhythmias and diseases such as cardiac hypertrophy and heart failure. Many clinical and basic research laboratories conduct gene expression analysis using RNA from whole cardiac biopsies. This allows for the analysis of global changes in gene expression in areas of the heart, while eliminating the need for more complex and technically difficult single-cell isolation procedures (such as flow cytometry, laser capture microdissection, etc.) that require expensive equipment and specialized training. The abundance of fibroblasts and other cell types in whole biopsies, however, can complicate gene expression analysis and the interpretation of results. Therefore, we have designed a technique to quickly and easily purify cardiac myocytes from whole cardiac biopsies for RNA extraction. Human heart tissue samples were collected, and our purification method was compared with the standard nonpurification method. Cell imaging using acridine orange staining of the purified sample demonstrated that >98% of total RNA was contained within identifiable cardiac myocytes. Real-time RT-PCR was performed comparing nonpurified and purified samples for the expression of troponin T (myocyte marker), vimentin (fibroblast marker), and a-smooth muscle actin (smooth muscle marker). Troponin T expression was significantly increased, and vimentin and n-smooth muscle actin were significantly decreased in the purified sample (n = 8; P < 0.05). Extracted RNA was analyzed during each step of the purification, and no significant degradation occurred. These results demonstrate that this isolation method yields a more purified cardiac myocyte RNA sample suitable for downstream applications, such as real-time RTPCR, and allows for more accurate gene expression changes in cardiac myocytes from heart biopsies. real-time reverse transcriptase-polymerase chain reaction; vimentin; troponin T, smooth muscle actin; cardiomyocytes

Adiponectin (APN) exerts its metabolic regulation largely through AMP-dependent protein kinase (AMPK). However, the role of AMPK in APN's antiapoptotic effect in ischemic-reperfused (I/R) adult cardiomyocytes remains incompletely understood. The present study was designed to determine the involvement of AMPK in the antiapoptotic signaling of APN. Cardiomyocytes from adult male mice overexpressing a dominant-negative [[alpha].sub.2]-subunit of AMPK (AMPK-DN) or wild-type (WT) littermates were subjected to simulated I/R (SI/R) and pretreated with 2 [micro]g/ml globular domain of APN (gAPN) or vehicle. SI/R-induced cardiomyocyte apoptosis was modestly increased in AMPK-DN cardiomyocytes (P < 0.05). Treatment with gAPN significantly reduced SI/R-induced apoptosis in WT cardiomyocytes as well as in AMPK-DN cardiomyocytes, indicating that the antiapoptotic effect of gAPN is partially AMPK independent. Furthermore, gAPN-induced endothelial nitric oxide synthase (eNOS) phosphorylation was significantly reduced in AMPK-DN cardiomyocytes, suggesting that the APN-eNOS signaling axis is impaired in AMPK-DN cardiomyocytes. Additional experiments demonstrated that treatment of AMPK-DN cardiomyocytes with gAPN reduced SI/R-induced NADPH oxidase overexpression, decreased superoxide generation, and blocked peroxynitrite formation to the same extent as that observed in WT cardiomyocytes. Collectively, our present study demonstrated that although the metabolic and eNOS activation effect of APN is largely mediated by AMPK, the superoxide-suppressing effect of APN is not mediated by AMPK, and this AMPK-independent antioxidant property of APN increased nitric oxide bioavailability and exerted significant antiapoptotic effect. apoptosis; oxidative stress; nitric oxide; adipocytokine

Cardiomyocytes derived from human embryonic stem (ES) cells are a potential source for cell-based therapy for heart diseases. We studied the effect of bone morphogenetic protein (BMP)-4 in the presence of fetal bovine serum (FBS) on cardiac induction from human H1 ES cells during embryoid body (EB) development. Suspension culture for 4 days with 20% FBS produced the best results for the differentiation of early mesoderm and cardiomyocytes. The addition of Noggin reduced the incidence of beating EBs from 23.6% to 5.3%, which indicated the involvement of BMP signaling in the spontaneous cardiac differentiation. In this condition, treatment with 12.5-25 ng/ml BMP-4 during the 4-day suspension optimally promoted the cardiomyocyte differentiation. The incidence of beating EBs at 25 ng/ml BMP-4 reached 95.8% on day 6 of expansion and then plateaued until day 20. In real-time PCR analysis, the cardiac development-related genes MESP1 and Nkx2.5 were upregulated in the EB outgrowths by 25 ng/ml BMP-4. The activation of BMP signaling in EBs was confirmed by the increase in the phosphorylation of Smadl/5/8 and by the nuclear localization of phospho-Smadl/5/8 and Smad4. The addition of 150 ng/ml Noggin considerably decreased the incidence of beating EBs and Nkx2.5 expression, and Noggin alone increased Nestin expression and neural differentiation in EB outgrowths. The cardiomyocytes induced by 25 ng/ml BMP-4 showed proper cell biological characteristics and a course of differentiation as judged from isoproterenol administration, gene expression, protein assay, immunoreactivity, and subcellular structures. No remarkable change in the extent of apoptosis and proliferation in the cardiomyocytes was observed by BMP-4 treatment. These findings showed that BMP-4 in combination with FBS at the appropriate time and concentrations significantly promotes cardiomyocyte induction from human ES cells. fetal bovine serum; early mesoderm; Noggin; cardiomyogenesis; differentiation

It has been difficult to establish whether we are limited to the heart muscle cells we are born with or if cardiomyocytes are generated also later in life. We have taken advantage of the integration of carbon-14, generated by nuclear bomb tests during the Cold War, into DNA to establish the age of cardiomyocytes in humans. We report that cardiomyocytes renew, with a gradual decrease from 1% turning over annually at the age of 25 to 0.45% at the age of 75. Fewer than 50% of cardiomyocytes are exchanged during a normal life span. The capacity to generate cardiomyocytes in the adult human heart suggests that it may be rational to work toward the development of therapeutic strategies aimed at stimulating this process in cardiac pathologies.

We examined the effects of 48 h bilateral nephrectomy on plasma and cardiac tissue expression of angiotensin-(1-12) [ANG-(1-12)], ANG I, and ANG II in adult Wistar-Kyoto rats to evaluate functional changes induced by removing renal renin. The goal was to expand the evidence of ANG-(1-12) being an alternate renin-independent, angiotensin-forming substrate. Nephrectomy yielded divergent effects on circulating and cardiac angiotensins. Significant decreases in plasma ANG-(1-12), ANG I, and ANG II levels postnephrectomy accompanied increases in cardiac ANG-(1-12), ANG I, and ANG II concentrations compared with controls. Plasma ANG-(1-12) decreased 34% following nephrectomy, which accompanied 78 and 66% decreases in plasma ANG I and ANG II, respectively (P < 0.05 vs. controls). Contrastingly, cardiac ANG-(1-12) in anephric rats averaged 276 [+ or -] 24 fmol/mg compared with 144 [+ or -] 20 fmol/mg in controls (P < 0.005). Cardiac ANG I and ANG II values were 300 [+ or -] 15 and 62 [+ or -] 7 fmol/mg, respectively, in anephric rats compared with 172 [+ or -] 8 fmol/mg for ANG I and 42 [+ or -] 4 fmol/mg for ANG II in controls (P < 0.001). Quantitative immunofluorescence revealed significant increases in average grayscale density for cardiac tissue angiotensinogen, ANG I, ANG II, and [AT.sub.1] receptors of WKY rats postnephrectomy. Faint staining of cardiac renin, unchanged by nephrectomy, was associated with an 80% decrease in cardiac renin mRNA. These changes were accompanied by significant increases in [p47.sup.phox], Rac1, and Nox4 isoform expression. In conclusion, ANG-(1-12) may be a functional precursor for angiotensin peptide formation in the absence of circulating renin. renin; angiotensinogen; angiotensin II; blood pressure

Binary cardiac transgenic (Tg) overexpression of P2[X.sub.4] receptors (P2[X.sub.4]R) improved the survival of the cardiomyopathic calsequestrin (CSQ) mice. Here we studied the mechanism of rescue using binary P2[X.sub.4]R/CSQ Tg and CSQ Tg mice as models. Cellular and intact heart properties were determined by simultaneous sarcomere shortening (SS) and [Ca.sup.2+] transients in vitro and echocardiography in vivo. Similar to a delay in death, binary mice exhibited a slowed heart failure progression with a greater left ventricular (LV) fractional shortening (FS) and thickness and a concomitant lesser degree of LV dilatation in both systole and diastole at 8 or 12 wk. By 16 wk, binary hearts showed similarly depressed FS and thinned out LV and equal enlargement of LV as did 12-wk-old CSQ hearts. Binary cardiac myocytes showed higher peak basal cell shortening (CS) and SS as well as greater basal rates of shortening and relaxation than did the CSQ myocytes at either 8 or 12 wk. Similar data were obtained in comparing the [Ca.sup.2+] transient. At 16 wk, binary myocytes were like the 12-wk-old CSQ myocytes with equally depressed CS, SS, and [Ca.sup.2+] transient. CSQ myocytes were longer than myocytes from wild-type and binary mice at 12 wk of age. At 16 wk, the binary myocyte length increased to that of the 12-wk-old CSQ myocyte, parallel to LV dilatation. The data suggest a unique mechanism, which involves a reversal of cardiac myocyte dysfunction and a delay in heart failure progression. It represents an example of targeting the abnormal failing myocyte in treating heart failure. contractility; heart failure; hypertrophy

Deterioration of cardiac contractility during congestive heart failure (CHF) is believed to involve decreased function of individual cardiomyocytes and may include reductions in contraction magnitude and/or kinetics. We examined the progression of in vivo and in vitro alterations in contractile function in CHF mice and investigated underlying alterations in [Ca.sup.2+] homeostasis. Following induction of myocardial infarction (MI), mice with CHF were examined at early (1 wk post-MI) and chronic (10 wk post-MI) stages of disease development. Sham-operated mice served as controls. Global and local left ventricle function were assessed by echocardiography in sedated animals (~2% isoflurane). Excitation-contraction coupling was examined in cardiomyocytes isolated from the viable septum. CHF progression between 1 and 10 wk post-MI resulted in increased mortality, development of hypertrophy, and deterioration of global left ventricular function. Local function in the noninfarcted myocardium also declined, as posterior wall shortening velocity was reduced in chronic CHF (1.2 [+ or -] 0.1 vs. 1.9 [+ or -] 0.2 cm/s in sham). Parallel alterations occurred in isolated cardiomyocytes since contraction and [Ca.sup.2+] transient time to peak values were prolonged in chronic CHF (115 [+ or -] 6 and 158 [+ or -] 11% sham values, respectively). Surprisingly, contraction and [Ca.sup.2+] transient magnitudes in CHF were larger than sham values at both time points, resulting from increased sarcoplasmic reticulum [Ca.sup.2+] content and greater [Ca.sup.2+] influx via L-type channels. We conclude that, in mice with CHF following myocardial infarction, declining myocardial function involves slowing of cardiomyocyte contraction without reduction in contraction magnitude. Corresponding alterations in [Ca.sup.2+] transients suggest that slowing of [Ca.sup.2+] release is a critical mediator of CHF progression. excitation-contraction coupling; cardiac contraction; calcium; cardiomyocytes

We used a heterotopic tranSp1anted working heart model to probe the collaborative role of bone marrow-derived progenitor cells (BPCs) and stromal cell-derived factor (SDF)-1[alpha] in attenuating tissue remodeling in recipient and tranSp1anted hearts. BPCs from male transgenic rats expressing green fluorescent protein ([GFP.sup.+] BPCs, 2 x [10.sup.6] cells) were injected intravenously into myeloablated female rats. One month later, heterotopic heart tranSp1antation was performed. The left anterior descending coronary artery (LAD) of the recipient heart was occluded permanently. Mesenchymal stem cells (MSCs; 2 x [10.sup.6] cells) with a null gene (null group) or overexpressing SDF-1[alpha] (SDF-1[alpha] group) were injected intramyocardially in the LAD perfusion region of both recipient and tranSp1anted hearts. Recipient and tranSp1anted hearts (n = l0 hearts/group) were harvested 21 days later for analysis. The survival of tranSp1anted hearts was assessed daily by palpation in additional animals (n = 7). Five days after LAD occlusion, subpopulations of [GFP.sup.+] BPCs in the circulation were significantly higher in the SDF-1[alpha] group. Y chromosome, 5-bromo-2'deoxyuridine, Ki67-positive nuclei, newly formed vessels, and [GFP.sup.+] cells significantly increased in tranSp1anted hearts of the SDF-1[alpha] group at 21 days after the injection of MSCs overexpressing SDF-1[alpha], whereas fewer TUNEL-positive nuclei were found. The survival of tranSp1anted hearts was also markedly increased in the SDF-1 a group (P < 0.05). Supplementation of endogenous cytokines released from the ischemic myocardium with exogenous MSCs overexpressing SDF-1[alpha] significantly increased BPC homing to acutely ischemic recipient and progressively ischemic tranSp1anted hearts. BPC recruitment resulted in the regeneration of new cardiomyocytes and blood vessels and extended survival of the tranSp1anted hearts. stromal cell-derived factor-1[alpha]; heterotopic heart tranSp1antation; stem cells; cell migration

Myocardial [Ca.sup.2+]/calmodulin-dependent protein kinase II (CaMKII) inhibition improves cardiac function following myocardial infarction (MI), but the CaMKII-dependent pathways that participate in myocardial stress responses are incompletely understood. To address this issue, we sought to determine the transcriptional consequences of myocardial CaMKII inhibition after MI. We performed gene expression profiling in mouse hearts with cardiomyocyte-delimited transgenic expression of either a CaMKII inhibitory peptide (AC3-I) or a scrambled control peptide (AC3-C) following MI. Of the 8,600 mRNAs examined, 156 were substantially modulated by MI, and nearly half of these showed markedly altered responses to MI with CaMKII inhibition. CaMKII inhibition substantially reduced the MI-triggered upregulation of a constellation of proinflammatory genes. We studied 1 of these proinflammatory genes, complement factor B (Cfb), in detail, because complement proteins secreted by cells other than cardiomyocytes can induce sarcolemmal injury during MI. CFB protein expression in cardiomyocytes was triggered by CaMKII activation of the NF-[kappa]B pathway during both MI and exposure to bacterial endotoxin. CaMKII inhibition suppressed NF-[kappa]B activity in vitro and in vivo and reduced Cfb expression and sarcolemmal injury. The Cfb-/- mice were partially protected from the adverse consequences of MI. Our findings demonstrate what we believe is a novel target for CaMKII in myocardial injury and suggest that CaMKII is broadly important for the genetic effects of MI in cardiomyocytes., Introduction The multifunctional [Ca.sup.2+]/calmodulin-dependent protein kinase II (CaMKII) is activated by increased intracellular [Ca.sup.2+] (1) and enhanced oxidant stress (2), both prominent features of myocardial disease. CaMKII inhibition protects against [...]

Based on extensive preclinical data, glycogen synthase kinase-3 (GSK-3) has been proposed to be a viable drug target for a wide variety of disease states, ranging from diabetes to bipolar disorder. Since these new drugs, which will be more powerful GSK-3 inhibitors than lithium, may potentially be given to women of childbearing potential, and since it has controversially been suggested that lithium therapy might be linked to congenital cardiac defects, we asked whether GSK-3 family members are required for normal heart development in mice. We report that terminal cardiomyocyte differentiation was substantially blunted in Gsk3b-/- embryoid bodies. While GSK-3[alpha]--deficient mice were born without a cardiac phenotype, no live-born Gsk3b-/- pups were recovered. The Gsk3b-/- embryos had a double outlet RV, ventricular septal defects, and hypertrophic myopathy, with near obliteration of the ventricular cavities. The hypertrophic myopathy was caused by cardiomyocyte hyperproliferation without hypertrophy and was associated with increased expression and nuclear localization of three regulators of proliferation--GATA4, cyclin D1, and c-Myc. These studies, which we believe are the first in mammals to examine the role of GSK-3[alpha] and GSK-3[beta] in the heart using loss-of-function approaches, implicate GSK-3[beta] as a central regulator of embryonic cardiomyocyte proliferation and differentiation, as well as of outflow tract development. Although controversy over the teratogenic effects of lithium remains, our studies suggest that caution should be exercised in the use of newer, more potent drugs targeting GSK-3 in women of childbearing age., Introduction The factors regulating proliferation of cardiomyocytes during development have been the focus of many investigations in the past decade. These studies have identified several growth factors, acting in a [...]

The Notch receptor mediates cell fate decision in multiple organs. In the current work we tested the hypothesis that Nkx2.5 is a target gene of Notch1 and raised the possibility that Notch1 regulates myocyte commitment in the adult heart. Cardiac progenitor cells (CPCs) in the niches express Notch1 receptor, and the supporting cells exhibit the Notch ligand Jagged1. The nuclear translocation of Notch1 intracellular domain (N1lCD) up-regulates Nkx2.5 in CPCs and promotes the formation of cycling myocytes in vitro. N1lCD and RBP-Jk form a protein complex, which in turn binds to the Nkx2.5 promoter initiating transcription and myocyte differentiation. In contrast, transcription factors of vascular cells are down-regulated by Jagged1 activation of the Notch1 pathway. Importantly, inhibition of Notch1 in infarcted mice impairs the commitment of resident CPCs to the myocyte lineage opposing cardiomyogenesis. These observations indicate that Notch1 favors the early specification of CPCs to the myocyte phenotype but maintains the newly formed cells in a highly proliferative state. Dividing Nkx2.5-positive myocytes correspond to transit amplifying cells, which condition the replicative capacity of the heart. In conclusion, Notch1 may have critical implications in the control of heart homeostasis and its adaptation to pathologic states. cardiac regeneration | myocardial infarction

To determine whether the expression of cardiac genes changes in a graded manner or by on/off switching when cardiac myocytes change genetic programs in living animals, we have studied two indicator genes that change their expression oppositely in mouse binucleate ventricular cardiomyocytes during development and in response to cardiac hypertrophy. One is a single-copy transgene controlled by an [alpha]-myosin heavy chain (aMHC) promoter and coding for CFP. The other is the endogenous [beta]-myosin heavy chain (bMHC) gene modified to code for a YFP-bMHC fusion protein. Using high-resolution confocal microscopy, we determined the expression of the two indicator genes in individual cardiomyocytes perinatally and after inducing cardiac hypertrophy by transverse aortic constriction. Our results provide strong evidence that the cardiac genes respond by switching their expression in an on/off rather than graded manner, and that responding genes within a single cell and within the two nuclei of cardiomyocytes do not necessarily switch concordantly. gene switching | myosin heavy chain

Membrane-associated guanylate kinase proteins (MAGUKs) are important determinants of localization and organization of ion channels into specific plasma membrane domains. However, their exact role in channel function and cardiac excitability is not known. We examined the effect of synapse-associated protein 97 (SAP97), a MAGUK abundantly expressed in the heart, on the function and localization of Kv1.5 subunits in cardiac myocytes. Recombinant SAP97 or Kv1.5 subunits tagged with green fluorescent protein (GFP) were over-expressed in rat neonatal cardiac myocytes and in Chinese hamster ovary (CHO) cells from adenoviral or plasmidic vectors. Immunocytochemistry, fluorescence recovery after photobleaching, and patchclamp techniques were used to study the effects of SAP97 on the localization, mobility, and function of Kv1.5 subunits. Adenovirus-mediated SAP97 overexpression in cardiac myocytes resulted in the clustering of endogenous Kv1.5 subunits at myocyte-myocyte contacts and an increase in both the maintained component of the outward [K.sup.+] current, [I.sub.Kur] (5.64 [+ or -] 0.57 pA/pF in SAP97 myocytes vs. 3.23 [+ or -] 0.43 pA/pF in controls) and the number of 4-aminopyridine-sensitive potassium channels in cell-attached membrane patches. In live myocytes, GFP-Kv1.5 subunits were mobile and organized in clusters at the basal plasma membrane, whereas SAP97 overexpression reduced their mobility. In CHO cells, Kvl.5 channels were diffusely distributed throughout the cell body and freely mobile. When coexpressed with SAP97, Kv subunits were organized in plaquelike clusters and poorly mobile. In conclusion, SAP97 regulates the [K.sup.+] current in cardiac myocytes by retaining and immobilizing Kv1.5 subunits in the plasma membrane. This new regulatory mechanism may contribute to the targeting of Kv channels in cardiac myocytes. potassium channel; membrane-associated guanylate kinase proteins; fluorescence recovery after photobleaching