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The mechanisms that stimulate fetal heart growth during anemia are unknown. To examine the hypothesis that adrenal hormones contribute to this process, we determined the effects of adrenalectomy (Adx) on heart growth and the activation of cardiac mitogen-activated protein kinases (MAPKs) in the presence and absence of fetal anemia. To identify mechanisms contributing to the initiation of cardiac growth, the duration of anemia was limited to a period shorter than that previously described to result in increased cardiac mass. Four groups of fetal sheep were studied (Adx-Anemic, Adx-Control, Intact-Anemic, Intact-Control). Anemia was created by daily controlled hemorrhage for 5 days; hearts were collected for analysis at 133 d gestation (term 145 d). Cardiomyocyte morphometry, immunohistochemistry for Ki-67 (proliferation marker), and Western blotting for protein levels of MAPKs and proliferating cell nuclear antigen (PCNA) were performed. Blood pressure, heart rate, heart weight-to-body weight ratio, and cardiomyocyte length and width remained similar among groups throughout the study. PCNA levels in the Adx-Anemic group were twice as high as in any other group (both ventricles, p < 0.05). Levels of phosphorylated extracellular signal-regulated kinase (ERK) were ~60% higher in the Intact-Anemic and Adx-Anemic groups, compared with the Intact-Control and Adx-Control groups (p < 0.02). These results suggest that adrenal hormones may attenuate fetal cardiomyocyte proliferation in response to anemia (as evidenced by the increased PCNA in Adx-Anemic fetuses) and that phosphorylation of myocardial ERK results from fetal anemia, irrespective of the status of the fetal adrenal gland. Key words: MAPK, hyperplasia, cortisol, sheep, fetus, heart, cardiomyocyte. Les mecanismes qui stimulent la croissance foetale durant l'anemie sont inconnus. Pour verifier l'hypothese que les hormones surrenaliennes participent a ce processus, nous avons determine les effets d'une surrenalectomie (Adx) sur la croissance cardiaque et l'activation des proteines kinases activees par un mitogene cardiaque (MAPK), en presence et en l'absence d'anemie foetale. Pour identifier les mecanismes participant au declenchement de la croissance cardiaque, nous avons limite; davantage la periode anemique par rapport a celle decrite anteerieurement en vue d'augmenter la masse cardiaque. Nous avons examine; 4 groupes de foetus de brebis (Adx aneemique, Adx temoin, Intact anemique, Intact teemoin). Nous avons cree l'anemie en induisant une hemorragie controlee toes les jours pendant 5 jours, pubis nous avons prelate les course au jour 133 de la gestation (terme : 145 jours) pour les analyser. Nous avons realise une analyse morphometrique des cardiomyocytes, et eu recours a l'immunohistochimie pour evaluer l'antigene Ki-67 (marqueur de proliferation) et au transfert Western pour mesurer les taux proteiques des MAPK et l'antigene nucleaire de proliferation cellulaire (PCNA). La pression arterielle, la frequence cardiaque, le rapport poids-cardiaque a poids corporel, ainsi que la longueur et la largeur des cardiomyocytes sont demeures constants chez tous les groupes tout au long de l'etude. Les taux de PCNA chez le groupe Adx anemique ont ete 2 fois plus eleves que ceux des autres groupes (les 2 ventricules, p < 0,05). Les taux de la proteine ERK phosphorylee ont ete environ 60 % plus eleves chez les groupes Intact anemique et Adx aneemique que chez les groupes Intact temoin et Adx temoin (p < 0,02). Ces resultats donnent a penser que les hormones surrenaliennes pourraient attenuer la proliferation des cardiomyocytes foetaux en reponse a l'anemie (comme demontre par l'augmentation de PCNA dans les foetus Adx anemiques) et que la phosphorylation de la proteine ERK myocardique resulte de l'anemie fotale, peu importe l'etat de glande surrenale fotale. Mots-cles: MAPK, hyperplasie, cortisol, brebis, foetus, coeur, cardiomyocyte. [Traduit par la Redaction], Introduction To provide adequate nutrient delivery and meet the demand of increased systemic blood flow to support fetal tissue growth, fetal heart size and cardiac output increase as gestation advances. [...]

Existing models of electrical activity in myocardial tissue ate unable to easily capture the effects of ephaptic coupling. Homogenized models do not account for cellular geometry, while detailed spatial models are too complicated to simulate in three dimensions. Here we propose a unique model that accurately captures the geometric effects while being computationally efficient. We use this model to provide an initial study of the effects of changes in extracellular geometry, gap junctional coupling, and sodium ion channel distribution on propagation velocity in a single 1D strand of cells. In agreement with previous studies, we find that ephaptic coupling increases propagation velocity at low gap junctional conductivity while it decreases propagation at higher conductivities. We also find that conduction velocity is relatively insensitive to gap junctional coupling when sodium ion channels are located entirely on the cell ends and cleft space is small. The numerical efficiency of this model, verified by comparison with more detailed simulations, allows a thorough study in parameter variation and shows that cellular structure and geometry has a nontrivial impact on propagation velocity. This model can be relatively easily extended to higher dimensions while maintaining numerical efficiency and incorporating ephaptic effects through modeling of complex, irregular cellular geometry. cardiac electrophysiology | extracellular conductivity | microdomain effects doi/ 10.1073/pnas.1010154107

Unknown molecular responses to sarcomere protein gene mutations account for pathologic remodeling in hypertrophic cardiomyopathy (HCM), producing myocyte growth and increased cardiac fibrosis. To determine if hypertrophic signals activated myocyte enhancer factor-2 (Mef2), we studied mice carrying the HCM mutation, myosin heavy-chain Arg403Gln, ([MHC.sup.403/+]) and an Mef2-dependent [beta]-galactosidase reporter transgene. In young, prehypertrophic [MHC.sup.403/+] mice the reporter was not activated. In hypertrophic hearts, activation of the Mef2-dependent reporter was remarkably heterogeneous and was observed consistently in myocytes that bordered fibrotic foci with necrotic cells, [MHC.sup.403/+] myocytes with Mef2-dependent reporter activation reexpressed the fetal myosin isoform ([beta]MHC), a molecular marker of hypertrophy, although [MHC.sup.403/+] myocytes with or without [beta]MHC expression were comparably enlarged over WT myocytes. To consider Mef2 roles in severe HCM, we studied homozygous [MHC.sup.403/403] mice, which have accelerated remodeling, widespread myocyte necrosis, and neonatal lethality. Levels of Phosphorylated class II histone deacetylases that activate Mef2 were substantially increased in [MHC.sup.403/403] hearts, but Mef2-dependent reporter activation was patchy. Sequential analyses showed myocytes increased Mef2-dependent reporter activity before death. Our data dissociate myocyte hypertrophy, a consistent response in HCM, from heterogeneous Mef2 activation and reexpression of a fetal gene program. The temporal and spatial relationship of Mef2-dependent gene activation with myocyte necrosis and fibrosis in [MHC.sup.403/+] and [MHC.sup.403/403] hearts defines Mef2 activation as a molecular signature of stressed HCM myocytes that are poised to die. fibrosis | hypertrophy | sarcomere protein gene mutation doi/ 10.1073/pnas.1012826107

ROS, such as [H.sub.2][O.sub.2], are a component of pathological conditions in many organ systems and have been reported to be elevated in cardiac pathophysiology. The experiments presented here test the hypothesis that [H.sub.2][O.sub.2] induces alterations in cardiac myofilament function by the posttranslational modification of sarcomeric proteins indirectly through PKC signaling. In vitro assessment of actomyosin [Mg.sup.2+]-ATPase activity of myofibrillar fractions showed blunted relative ATP consumption in the relaxed state (pCa 8.0) in response to treatment with 0.5 mM [H.sub.2][O.sub.2] before myofilament isolation. The effect was attributable to downstream 'redox signaling,' inasmuch as the direct application of [H.sub.2][O.sub.2] to isolated myofibrils did not alter [Mg.sup.2+]-ATPase activity. [Ca.sup.2+]-ATPase activity, which was used as a measure of myofibrillar myosin function, was unaffected by [H.sub.2][O.sub.2]. Functional experiments using rat cardiac trabeculae treated with 0.5 or 5 mM H202 followed by detergent extraction of membranes demonstrated increased [Ca.sup.2+] sensitivity of force production, a faster rate of force redevelopment, and (for 5 mM) decreased maximum tension. Biochemical analysis of myocardial samples treated with 0.5 mM [H.sub.2][O.sub.2] demonstrated increased phosphorylation of two sarcomeric proteins: cardiac troponin I and myosin-binding protein-C. These changes were eliminated by a general PKC inhibitor. However, [H.sub.2][O.sub.2] and the general PKC activator PMA induced different phosphorylation patterns in cardiomyocytes in which PKC-[delta] was elevated by viral infection. These data provide evidence that PKC-dependent redox signaling affects the function of cardiac myofilaments and indicate modification of specific proteins through this signaling mechanism. force-pCa relationship; ATPase activity; phosphorylation; cardiac troponin I; myosin-binding protein C doi: 10.1152/ajpheart.00050.2010.

Insulin resistance (IR) is a condition where different organs are refractory to insulin stimulation of glucose uptake. ANG II has been suggested to be involved in the development of IR in the heart. The precise mechanism by which this occurs is still unknown. Here we have used dynamic fluorescent imaging techniques to show that ANG II inhibits insulin production of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] in cardiac myocytes. Fluorophore (Venus)-conjugated cAMP-dependent protein kinase-pleckstrin homology domain, which specifically binds to PI(3,4,5)P3, was transfected in neonatal rat cardiac myocytes. Insulin induced a robust increase in the fluorescence intensity at the cell surface, which was diminished by application of ANG II. The inhibitory action of ANG II was antagonized by RNH-6270 (an angiotensin type 1 receptor antagonist) but not by PD-122370 (an angiotensin type 2 receptor antagonist). BAPTA-AM ([Ca.sup.2+] chelator) largely attenuated the ANG II effect, whereas K-252b (PKC inhibitor) did not. Furthermore, an elevation of intracellular [Ca.sup.2+] induced by ionomycin mimicked the ANG II effect. Therefore, it is suggested that ANG II antagonizes insulin-mediated production of PI(3,4,5)P3 via a [Ca.sup.2+]-dependent but PKC-independent pathway in cardiac myocytes. protein kinase C; angiotensin type 1 receptor; heart; adenosine 3',5'cyclic monophosphate-dependent protein kinase; real-time imaging; fluorophore doi: 10.1152/ajpheart.00220.2009.

Muscular dystrophies are often associated with significant cardiac disease that can be the prominent feature associated with gene mutations in sarcoglycan. Cardiac cell death is a main feature of cardiomyopathy in sarcoglycan deficiency and may arise as a cardiomyocyte intrinsic process that remains unclear. Deficiency of [delta]-sarcoglycan ([delta]-SG) induces disruption of the dystrophin-associated glycoprotein complex, a known cause of membrane instability that may explain cardiomyocytes cytosolic [Ca.sup.2+] increase. In this study we assessed the hypothesis that cytosolic [Ca.sup.2+] increase triggers cardiomyocyte death through mitochondrial [Ca.sup.2+] overload and dysfunction in the [delta]-SG-deficient CHF147 hamster. We showed that virtually all isolated CHF147 ventricular myocytes exhibited elevated cytosolic and mitochondrial [Ca.sup.2+] levels by the use of the Fura-2 and Rhod-2 fluorescent probes. Observation of living cells with Mito-Tracker red lead to the conclusion that ~15% of isolated CHF147 cardiomyocytes had disorganized mitochondria. Transmission electron microscope imaging showed mitochondrial swelling associated with crest and membrane disruption. Analysis of the mitochondrial permeability transition pore (MPTP) activity using calcein revealed that mitochondria of CHF147 ventricular cells were twofold leakier than wild types, whereas reactive oxygen species production was unchanged. Bax, Bcl-2, and LC3 expression analysis by Western blot indicated that the intrinsic apoptosis and the cell death associated to autophagy pathways were not significantly activated in CHF147 hearts. Our results lead to conclusion that cardiomyocytes death in [delta]-SG-deficient animals is an intrinsic phenomenon, likely related to [Ca.sup.2+]-induced necrosis. In this process [Ca.sup.2+] overload-induced MPTP activation and mitochondrial disorganization may have an important role. calcium; mitochondria; cardiac muscle; sarcolemmopathy doi: 10.1152/ajpcell.00545.2009.

We demonstrate here a cardiac tissue-engineering strategy addressing multicellular organization, integration into host myocardium, and directional cues to reconstruct the functional architecture of heart muscle. Microtemplating is used to shape poly(2-hydroxyethyl methacrylate-co-methacrylic acid) hydrogel into a tissue-engineering scaffold with architectures driving heart tissue integration. The construct contains parallel channels to organize cardiomyocyte bundles, supported by micrometer-sized, spherical, interconnected pores that enhance angiogenesis while reducing scarring. Surface-modified scaffolds were seeded with human ES cell-derived cardiomyocytes and cultured in vitro. Cardiomyocytes survived and proliferated for 2 wk in scaffolds, reaching adult heart densities. Cardiac implantation of acellular scaffolds with pore diameters of 30-40 [micro]m showed angiogenesis and reduced fibrotic response, coinciding with a shift in macrophage phenotype toward the M2 state. This work establishes a foundation for spatially controlled cardiac tissue engineering by providing discrete compartments for cardiomyocytes and stroma in a scaffold that enhances vascularization and integration while controlling the inflammatory response. angiogenesis | cardiomyocyte | human embryonic stem cell | hydrogel doi/ 10.1073/pnas.1006442107

We present a multiscale model and an adaptive numerical scheme for simulating cardiac action potential propagation along a linear strand of heart muscle cells. This model couples macroscale partial differential equations posed over the tissue to microscale equations posed over discrete cellular geometry. The microscopic equations are used only near action potential wave fronts, and the macroscopic equations are used everywhere else. We study the effects of gap-junctional and ephaptic coupling on conduction in the multiscale model and its fully macroscale and fully microscale analogues. Our simulations reveal that the adaptive multiscale model accurately reproduces the action potential wave forms and wave speeds of the fully microscale model. They also demonstrate that, at low gap-junctional conductivities, the accuracy of fully macroscale simulations is sensitive to numerical grid spacing. Moreover, adaptive multiscale simulations capture the effect of ephaptic coupling, whereas fully macroscale simulations do not. We propose two ways of generalizing our multiscale model to higher dimensions, and we argue that such generalizations may be necessary to obtain accurate three-dimensional simulations of cardiac conduction in certain pathophysiological parameter regimes. cardiac electrophysiology | gap junction | electric-field mechanism | mathematical model doi/ 10.1073/pnas.1008443107

Endoplasmic reticulum (ER) stress (ERS) is involved in various cardiovascular diseases. Our previous study verified that ERS took part in the development of cardiac hypertrophy; however, its mechanism is still unclear. This study aimed to investigate the roles of the calcineurin (CAN) signal pathway in hypertrophy induced by the ERS inductor thapsigargin (TG) in neonatal cardiomyocytes from Sprague-Dawley rats. Investigation of ER chaperone expression, ER staining, and calreticulin immunoflourescence were used to detect the ERS response, mRNA expression of atrial natriuretic peptide and brain natriuretic peptide, total protein synthesis rate, and cell surface area were used to evaluate cardiac hypertrophy induced by TG. TG induced a significant ERS response along with hypertrophy in a dose-and time-dependent manner in cardiomyocytes, which was verified by treatment with tunicamycin, another ERS inducer. Furthermore, TG induced a significant elevation of the intracellular [Ca.sup.2+] level, CaN activation, and myocyte enhancer factor 2c (MEF2c) expression in a dose- and time-dependent manner in cardiomyocytes. Cyclosporine A, a CaN inhibitor, markedly suppressed MEF2c nuclear translocation and inhibited TG-induced hypertrophy. These results demonstrate that ERS induces cardiac hypertrophy and that the CaN-MEF2c pathway is involved in ERS-induced hypertrophy in cardiomyocytes. thapsigargin doi: 10.1152/ajpheart.00980.2009.

Cardiac c-[kit.sup.+] cells are generally believed to be the major population of stem/progenitor cells in the heart and can be used as a cell source for cardiomyoplasty; however, the cellular electrophysiological properties are not understood in this type of cells. The present study was designed to investigate functional ion channels in undifferentiated mouse cardiac c-[kit.sup.+] cells using approaches of whole cell patch voltage clamp, RT-PCR, and cell proliferation assay. It was found that three types of ionic currents were present in mouse cardiac c-[kit.sup.+] cells, including a delayed rectifier [K.sup.+] current ([IK.sub.DR]) inhibited by 4-aminopyridine (4-AP), an inward rectifier [K.sup.+] current ([I.sub.Kir]) decreased by [Ba.sup.2+], and a volume-sensitive chloride current ([I.sub.Cl.vol]) inhibited by 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB). RT-PCR revealed that the corresponding ion channel genes, Kv1.1, Kv1.2, and Kv1.6 (for [IK.sub.DR]), Kir.1.1, Kir2.1, and Kir2.2 (likely responsible for [I.sub.Kir), and Clcn3 (for [I.sub.Cl.vol]), were significant in mouse cardiac c-[kit.sup.+] cells. The inhibition of [I.sub.Cl.vol] with NPPB and niflumic acid, but not [IK.sub.DR] with 4-AP and tetraethylammonium, reduced cell proliferation and accumulated the cell progression at [G.sub.0]/[G.sub.1] phase in mouse cardiac c-[kit.sup.+] cells. Our results demonstrate that three types of functional ion channel currents (i.e., [IK.sub.DR], [I.sub.Kir], and [I.sub.Cl.vol]) are present in mouse cardiac c-[kit.sup.+] cells, and [I.sub.Cl.vol] participates in regulating cell proliferation. mouse cardiac stem/progenitor cells; delayed rectifier potassium current; inward rectifier potassium current; volume-sensitive chloride current doi:10.1152/ajpcell.00207.2009.

Most lysosomal storage diseases (LSDs) are life-threatening genetic diseases. The pathogenesis of these diseases is poorly understood. Induced pluripotent stem (iPS) cell technology offers new opportunities for both mechanistic studies and development of stem cellbased therapies. Here we report the generation of disease-specific iPS cells from mouse models of Fabry disease, globoid cell leukodystrophy (GLD), and mucopolysaccharidosis VII (MPSVII). These mouse model-derived iPS cells showed defects in disease-specific enzyme activities and significant accumulation of substrates for these enzymes. In the lineage-directed differentiation studies, Fabry-iPS and GLD-iPS cells were efficiently differentiated into disease-relevant cell types, such as cardiomyocytes and neural stem cells, which might be useful in mechanistic and therapeutic studies. Notably, MPSVII-iPS cells demonstrated a markedly impaired ability to form embryoid bodies (EBs) in vitro. MPSVII-EBs exibited elevated levels of hyaluronan and its receptor CD44, and markedly reduced expression levels of E-cadherin and cell-proliferating marker. Partial correction of enzyme deficiency in MSPVII-iPS cells led to improved EB formation and reversal of aberrant protein expression. These data indicate a potential mechanism for the partial lethality of MPSVII mice in utero, and suggest a possible abnormality of embryonic development in MPSVII patients. Thus, our study demonstrates the unique promise of iPS cells for studying the pathogenesis and treatment of LSDs. cardiomyocyte/embryoid body/neural stem cell

Patients with primary (AL) cardiac amyloidosis suffer from progressive cardiomyopathy with a median survival of less than 8 months and a 5-year survival of amyloid | cell death | heart failure | TAB1 | reactive oxygen species doi/10.1073/pnas.0912263107

Atrial fibrillation (AF) is a common arrhythmia that increases the risk of stroke and heart failure. Here, we have shown that mast cells, key mediators of allergic and immune responses, are critically involved in AF pathogenesis in stressed mouse hearts. Pressure overload induced mast cell infiltration and fibrosis in the atrium and enhanced AF susceptibility following atrial burst stimulation. Both atrial fibrosis and AF inducibility were attenuated by stabilization of mast cells with cromolyn and by BM reconstitution from mast cell-deficient WBB6F1-[Kit.sup.[W/W-v]] mice. When cocultured with cardiac myocytes or fibroblasts, BM-derived mouse mast cells increased platelet-derived growth factor A (PDGF-A) synthesis and promoted cell proliferation and collagen expression in cardiac fibroblasts. These changes were abolished by treatment with a neutralizing antibody specific for PDGF α-receptor (PDGFR-α). Consistent with these data, upregulation of atrial Pdgfa expression in pressure-overloaded hearts was suppressed by BM reconstitution from WBB6F1-[Kit.sup.[W/W-v]] mice. Furthermore, injection of the neutralizing PDGFR-α-specific antibody attenuated atrial fibrosis and AF inducibility in pressure-overloaded hearts, whereas administration of homodimer of PDGF-A (PDGF-AA) promoted atrial fibrosis and enhanced AF susceptibility in normal hearts. Our results suggest a crucial role for mast cells in AF and highlight a potential application of controlling the mast cell/PDGF-A axis to achieve upstream prevention of AF in stressed hearts., Introduction Atrial fibrillation (AF) is a supraventricular arrhythmia that is characterized by rapid and fibrillatory atrial activation with an irregular ventricular response. AF remains the most common arrhythmia encountered in [...]

Cardiac pathology, such as myocardial infarction (MI), activates intracellular proteases that often trigger programmed cell death and contribute to maladaptive changes in myocardial structure and function. To test whether inhibition of calpain, a [Ca.sup.2+]-dependent cysteine protease, would prevent these changes, we used a mouse MI model. Calpeptin, an aldehydic inhibitor of calpain, was intravenously administered at 0.5 mg/kg body wt before MI induction and then at the same dose subcutaneously once per day. Both calpeptin-treated (n = 6) and untreated (n = 6) MI mice were used to study changes in myocardial structure and function after 4 days of MI, where end-diastolic volume (EDV) and left ventricular ejection fraction (EF) were measured by echocardiography. Calpain activation and programmed cell death were measured by immunohistochemistry, Western blotting, and TdT-mediated dUTP nick-end labeling (TUNEL). In MI mice, calpeptin treatment resulted in a significant improvement in EF [EF decreased from 67 [+ or -] 2% pre-MI to 30 [+ or -] 4% with MI only vs. 41 [+ or -] 2% with MI + calpeptin] and attenuated the increase in EDV [EDV increased from 42 [+ or -] 2 [micro]l pre-MI to 73 [+ or -] 4 [micro]l with MI only vs. 55 [+ or -] 4 [micro]l with MI + calpeptin]. Furthermore, calpeptin treatment resulted in marked reduction in calpain- and caspase-3-associated changes and TUNEL staining. These studies indicate that calpain contributes to MI-induced alterations in myocardial structure and function and that it could be a potential therapeutic target in treating MI patients. cardiomyocytes; cell death doi: 10.1152/ajpheart.00338.2009.

We previously demonstrated that the O-linked [beta]-N-acetylglucosamine (O-GlcNAc) posttranslational modification confers cardioprotection at least partially through mitochondrial-dependent mechanisms, but it remained unclear if O-GlcNAc signaling interfered with other mechanisms of cell death. Because ischemia/hypoxia causes endoplasmic reticulum (ER) stress, we ascertained whether O-GlcNAc signaling could attenuate ER stress-induced cell death per se. Before induction of ER stress (with tunicamycin or brefeldin A), we adenovirally overexpressed O-GlcNAc transferase (AdOGT) or pharmacologically inhibited O-GlcNAcase [via O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate] to augment O-GlcNAc levels or adenovirally overexpressed O-GlcNAcase to reduce O-GlcNAc levels. AdOGT significantly (P < 0.05) attenuated the activation of the maladaptive arm of the unfolded protein response [according to C/EBP homologous protein (CHOP) activation] and cardiomyocyte death (reflected by percent propidium iodide positivity). Moreover, pharmacological inhibition of O-GlcNAcase significantly (P < 0.05) mitigated ER stress-induced CHOP activation and cardiac myocyte death. Interestingly, overexpression of GCA did not alter ER stress markers but exacerbated brefeldin A-induced cardiomyocyte death. We conclude that enhanced O-GlcNAc signaling represents a partially proadaptive response to reduce ER stress-induced cell death. These results provide new insights into a possible interaction between O-GlcNAc signaling and ER stress and may partially explain a mechanism of O-GlcNAc-mediated cardioprotection. O-linked [beta]-N-acetylglucosamine; endoplasmic reticulum doi: 10.1152/ajpheart.00553.2009.

A number of type 1 receptor cytokine family members protect the heart from acute and chronic oxidative stress. This protection involves activation of two intracellular signaling cascades: the reperfusion injury salvage kinase (RISK) pathway, which entails activation of phosphatidylinositol 3-kinase (PI3-kinase) and ERK1/2, and JAK-STAT signaling, which involves activation of transcription factor signal transducer and activator of transcription 3 (STAT3). Obligatory for activation of both RISK and STAT3 by nearly all of these cytokines are the kinases JAK1 and JAK2. Yet surprisingly little is known about how JAK1 and JAK2 are regulated in the heart or how they couple to PI3-kinase activation. Although the JAKs are linked to antioxidative stress programs in the heart, we recently reported that these kinases are inhibited by oxidative stress in cardiac myocytes. In contrast, others have reported that cardiac JAK2 is activated by acute oxidative stress by an undefined process. Here we summarize recent insights into the regulation of JAK1 and JAK2. Besides oxidative stress, inhibitory regulation involves phosphorylation, nitration, and intramolecular restraints. Stimulatory regulation involves phosphorylation and adaptor proteins. The net effect of stress on JAK activity in the heart likely represents the sum of both inhibitory and stimulatory processes, along with their dynamic interaction. Thus the regulation of JAKs in the heart, once touted as the paragon of simplicity, is proving rather complicated indeed, requiring a second look. It is our contention that a better understanding of the regulation of this kinase family that is implicated in cardiac protection could translate into effective therapeutic strategies for preventing myocardial damage or repairing the injured heart. Janus kinase; oxidative stress; redox; cardiac remodeling; cytokine; cardiac myocyte doi: 10.1152/ajpheart.00032.2009.

An analysis of the clonality of cardiac progenitor cells (CPCs) and myocyte turnover in vivo requires genetic tagging of the undifferentiated cells so that the clonal marker of individual mother cells is traced in the specialized progeny. CPC niches in the atria and apex of the mouse heart were infected with a lentivirus carrying EGFP, and the destiny of the tagged cells was determined 1-5 months later. A common integration site was identified in isolated CPCs, cardiomyocytes, endothelial cells (ECs), and fibroblasts, documenting CPC self-renewal and multipotentiality and the clonal origin of the differentiated cell populations. Subsequently, the degree of EGFP-lentiviral infection of CPCs was evaluated 2-4 days after injection, and the number of myocytes expressing the reporter gene was measured 6 months later. A BrdU pulse-chasing protocol was also introduced as an additional assay for the analysis of myocyte turnover. Over a period of 6 months, each EGFP-positive CPC divided approximately eight times generating 230 cardiomyocytes; this value was consistent with the number of newly formed cells labeled by BrdU. To determine whether, human CPCs (hCPCs) are self-renewing and multipotent, these cells were transduced with the EGFP-lentivirus and injected after acute myocardial infarction in immunosuppressed rats. hCPCs, myocytes, ECs, and fibroblasts collected from the regenerated myocardium showed common viral integration sites in the human genome. Thus, our results indicate that the adult heart contains a pool of resident stem cells that regulate cardiac homeostasis and repair.

Ongoing investigation of the electrophysiology and pathophysiology of the human atria requires an accurate representation of the membrane dynamics of the human atrial myocyte. However, existing models of the human atrial myocyte action potential do not accurately reproduce experimental observations with respect to the kinetics of key repolarizing currents or rate dependence of the action potential and fail to properly enforce charge conservation, an essential characteristic in any model of the cardiac membrane. In addition, recent advances in experimental methods have resulted in new data regarding the kinetics of repolarizing currents in the human atria. The goal of this study was to develop a new model of the human atrial action potential, based on the Nygren et al. model of the human atrial myocyte and newly available experimental data, that ensures an accurate representation of repolarization processes and reproduction of action potential rate dependence and enforces charge conservation. Specifically, the transient outward [K.sup.+] current ([I.sub.t]) and ultrarapid rectifier [K.sup.+] current ([I.sub.Kur]) were newly formulated. The inwardly recitifying [K.sup.+] current ([I.sub.KI]) was also reanalyzed and implemented appropriately. Simulations of the human atrial myocyte action potential with this new model demonstrated that early repolarization is dependent on the relative conductances of [I.sub.t] and [I.sub.Kur], whereas densities of both [I.sub.Kur] and [I.sub.K1] underlie later repolarization. In addition, this model reproduces experimental measurements of rate dependence of [I.sub.t], [I.sub.Kur], and action potential duration. This new model constitutes an improved representation of excitability and repolarization reserve in the human atrial myocyte and, therefore, provides a useful computational tool for future studies involving the human atrium in both health and disease. ionic model; repolarization; potassium current doi:10.1152/ajpheart.00411.2009

Success of human myocardial tissue engineering for cardiac repair has been limited by adverse effects of scaffold materials, necrosis at the tissue core, and poor survival after transplantation due to ischemic injury. Here, we report the development of scaffold-free prevascularized human heart tissue that survives in vivo transplantation and integrates with the host coronary circulation. Human embryonic stem cells (hESCs) were differentiated to cardiomyocytes by using activin A and BMP-4 and then placed into suspension on a rotating orbital shaker to create human cardiac tissue patches. Optimization of patch culture medium significantly increased cardiomyocyte viability in patch centers. These patches, composed only of enriched cardiomyocytes, did not survive to form significant grafts after implantation in vivo. To test the hypothesis that ischemic injury after transplantation would be attenuated by accelerated angiogenesis, we created 'second-generation,' pre-vascularized, and entirely human patches from cardiomyocytes, endothelial cells (both human umbilical vein and hESC-derived endothelial cells), and fibroblasts. Functionally, vascularized patches actively contracted, could be electrically paced, and exhibited passive mechanics more similar to myocardium than patches comprising only cardiomyocytes. Implantation of these patches resulted in 10-fold larger cell grafts compared with patches composed only of cardiomyocytes. Moreover, the preformed human microvessels anastomosed with the rat host coronary circulation and delivered blood to the grafts. Thus, inclusion of vascular and stromal elements enhanced the in vitro performance of engineered human myocardium and markedly improved viability after transplantation. These studies demonstrate the importance of including vascular and stromal elements when designing human tissues for regenerative therapies. angiogenesis | human embryonic stem cells | tissue engineering myocardial infarction | cardiomyocyte

Regulation of sodium flux across the cell membrane plays a vital role in the generation of action potentials and regulation of membrane excitability in cells such as cardiomyocytes and neurons. Alteration of sodium channel function has been implicated in diseases such as epilepsy, long QT syndrome, and heart failure. However, single cell imaging of sodium dynamics has been limited due to the narrow selection of fluorescent sodium indicators available to researchers. Here we report, the detection of spatially defined sodium activity during action potentials. Fluorescent nanosensors that measure sodium in real-time, are reversible and are completely selective over other cations such as potassium that were used to image sodium. The use of the nanosensors in vitro was validated by determining drug-induced activation in heterologous cells transfected with the voltage-gated sodium channel [Na.sub.v] 1.7. Spatial information of sodium concentrations during action potentials will provide insight at the cellular level on the role of sodium and how slight changes in sodium channel function can affect the entirety of an action potential. cellular imaging | ion channels | sodium sparks

In mammalian adult cardiomyocytes, sarcoplasmic reticulum (SR) [Ca.sup.2+]-ATPase (SERCA) plays a major role in controlling the decline of cytosolic free [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) in comparison with sarcolemmal [Na.sup.+]/[Ca.sup.2+] exchanger (NCX). However, the functional importance of SERCA and NCX in cytosolic [Ca.sup.2+] removal during early cardiomyogenesis is still debated. In this study, the functional contributions of [Ca.sup.2+] transporters to [[[Ca.sup.2+]].sub.i] decline in mouse embryonic stem cell-derived cardiomyocytes (mESCMs), a suitable model for investigation of early cardiogenesis, at various differentiation stages were investigated. We estimated that even at early differentiation stages of mESCMs, SERCA was responsible for ~76% of total [Ca.sup.2+] removal, while NCX was responsible for ~21%. The contributions of SERCA and NCX to cytosolic [Ca.sup.2+] clearance were increased to -88% and decreased to 10%, respectively, at the late differentiation stage. Dynamical analysis of the transient decay phases in normal and [Na.sup.+]-free solutions suggests that the contribution of NCX to [[[Ca.sup.2+]].sub.i] decline is more apparent in the terminal slow decay phase than that in the initial fast phase. When SR function was suppressed in type 2 ryanodine receptor-null mESCMs or with ryanodine receptor and SERCA inhibitors (ryanodine and thapsigargin), NCX acted as the main pathway for [[[Ca.sup.2+]].sub.i] decline. We conclude that the rapid [[[Ca.sup.2+]].sub.i] decline is mainly achieved by the SR uptake even at the early differentiation stage of mESCMs, while NCX acts as the main [Ca.sup.2+] remover when SR function is suppressed. These findings suggest a critical role of SR in the regulation of [[[Ca.sup.2+]].sub.i] homeostasis even in differentiating cardiomyocytes. sarcoplasmic reticulum; calcium transporters; sarcoplasmic reticulum [Ca.sup.2+]-ATPase; sodium/calcium exchanger

Cardiac [Na.sup.+]-[K.sup.+]-ATPase (NKA) regulates intracellular [Na.sup.+], which in turn affects intracellular [Ca.sup.2+] and contractility via the [Na.sup.+]/[Ca.sup.2+] exchanger. Extracellular [K.sup.+] concentration ([[K.sup.+]]) is a central regulator of NKA activity. Phospholemman (PLM) has recently been recognized as a critical regulator of NKA in the heart. PLM reduces the intracellular [Na.sup.+] affinity of NKA, an effect relieved by PLM phosphorylation. Here we tested whether the NKA [[alpha].sub.1]-vs. [[alpha].sub.2]-isoforms have different external [K.sup.+] sensitivity and whether PLM and PKA activation affects the NKA affinity for [K.sup.+] in mouse cardiac myocytes. We measured the external [[K.sup.+]] dependence of the pump current generated by the ouabain resistant NKA isoform in myocytes from wild-type (WT) mice (i.e., current due to NKA-[[alpha].sub.1]) and mice in which the NKA isoforms have swapped ouabain affinities ([[alpha].sub.1] is ouabain sensitive and [[alpha].sub.2] is ouabain resistant) to assess current due to NKA-[[alpha].sub.2]. We found that NKA-[[alpha].sub.1] has a higher affinity for external [K.sup.+] than NKA-[[alpha].sub.2] [half-maximal pump activation ([K.sub.0.5]) = 1.5 [+ or -] 0.1 vs. 2.9 [+ or -] 0.3 mM]. The apparent external [K.sup.+] affinity of NKA was significantly lower in myocytes from WT vs. PLM-knockout mice ([K.sub.0.5] = 2.0 [+ or -] 0.2 vs. 1.05 [+ or -] 0.08 mM). However, PKA activation by isoproterenol (1 [micro]M) did not alter the [K.sub.0.5] of NKA for external [K.sup.+] in WT myocytes. We conclude that 1) NKA-[[alpha].sub.1] has higher affinity for [K.sup.+] than NKA-[[alpha].sub.2] in cardiac myocytes, 2) PLM decreases the apparent external [K.sup.+] affinity of NKA, and 3) phosphorylation of PLM at the cytosolic domain does not alter apparent extracellular [K.sup.+] affinity of NKA. voltage-clamp; phosphorylation

L-type [Ca.sup.2+] currents conducted by [Ca.sub.v] 1.2 channels initiate excitation-contraction coupling in cardiac myocytes. Intracellular [Mg.sup.2+] ([Mg.sub.i]) inhibits the ionic current of [Ca.sub.v] l.2 channels. Because [Mg.sub.i] is altered in ischemia and heart failure, its regulation of [Ca.sub.v] 1.2 channels is important in understanding cardiac pathophysiology. Here, we studied the effects of [Mg.sub.i] on voltage-dependent inactivation (VDI) of [Ca.sub.v] l.2 channels using [Na.sup.+] as permeant ion to eliminate the effects of permeant divalent cations that engage the [Ca.sup.2+]-dependent inactivation process. We confirmed that increased [Mg.sub.i] reduces peak ionic currents and increases VDI of [Ca.sub.v] 1.2 channels in ventricular myocytes and in transfected cells when measured with [Na.sup.+] as permeant ion. The increased rate and extent of VDI caused by increased [Mg.sub.i] were substantially reduced by mutations of a cation-binding residue in the proximal C-terminal EF-hand, consistent with the conclusion that both reduction of peak currents and enhancement of VDI result from the binding of [Mg.sub.i] to the EF-hand ([K.sub.D] [approximately equal to] 0.9 mM) near the resting level of Mgi in ventricular myocytes. VDI was more rapid for L-type [Ca.sup.2+] currents in ventricular myocytes than for [Ca.sub.v] 1.2 channels in transfected cells. Coexpression of [Ca.sub.v] [[beta].sub.2b] subunits and formation of an autoinhibitory complex of truncated [Ca.sub.v] 1.2 channels with noncovalently bound distal C-terminal domain (DCT) both increased VDI in transfected cells, indicating that the subunit structure of the [Ca.sub.v] 1.2 channel greatly influences its VDI. The effects of noncovalently bound DCT on peak current amplitude and VDI required Mgi binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue. Our results demonstrate cooperative regulation of peak current amplitude and VDI of [Ca.sub.v] 1.2 channels by [Mg.sub.i], the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism.

Cardiomyocytes (CMs) are nonregenerative. Self-renewable pluripotent human embryonic stem cells (hESCs) can differentiate into CMs for cell-based therapies. We recently reported that [Ca.sup.2+] handling, crucial to excitation-contraction coupling of hESC-derived CMs (hESC-CMs), is functional but immature. Such immature properties as smaller cytosolic [Ca.sup.2+] transient amplitudes, slower kinetics, and reduced [Ca.sup.2+] content of sarcoplasmic reticulum (SR) can be attributed to the differential developmental expression profiles of specific [Ca.sup.2+] handling and regulatory proteins in hESC-CMs and their adult counterparts. In particular, calsequestrin (CSQ), the most abundant, high-capacity but low-affinity, [Ca.sup.2+]-binding protein in the SR that is anchored to the ryanodine receptor, is robustly expressed in adult CMs but completely absent in hESC-CMs. Here we hypothesized that gene transfer of CSQ in hESC-CMs suffices to induce functional improvement of SR. Transduction of hESC-CMs by the recombinant adenovirus Ad-CMV-CSQ-IRES-GFP (Ad-CSQ) significantly increased the transient amplitude, upstroke velocity, and transient decay compared with the control Ad-CMV-GFP (Ad-GFP) and Ad-CMV-CSQ[DELTA]-IRES-GFP (Ad-CSQ[DELTA], which mediated the expression of a nonfunctional, truncated version of CSQ) groups. Ad-CSQ increased the SR [Ca.sup.2+] content but did not alter L-type [Ca.sup.2+] current. Pharmacologically, untransduced wild-type, Ad-GFP-, Ad-CSQ[DELTA]-, and Ad-CSQ-transduced hESC-CMs behaved similarly. Whereas ryanodine significantly reduced the [Ca.sup.2+] transient amplitude and slowed the upstroke, thapsigargin slowed the decay. Neither triadin nor junctin was affected. We conclude that CSQ expression in hESC-CMs facilitates [Ca.sup.2+] handling maturation. Our results shed insights into the suitability of hESC-CMs for therapies and as certain heart disease models for drug screening. calcium transients; ryanodine receptor; adenovirus

Heart muscle excitation--contraction (E-C) coupling is governed by [Ca.sup.2+] release units (CRUs) whereby [Ca.sup.2+] influx via L-type [Ca.sup.2+] channels (Cav1.2) triggers [Ca.sup.2+] release from juxtaposed [Ca.sup.2+] release channels (RyR2) located in junctional sarcoplasmic reticulum (jSR). Although studies suggest that the jSR protein triadin anchors cardiac calsequestrin (Casq2) to RyR2, its contribution to E-C coupling remains unclear. Here, we identify the role of triadin using mice with ablation of the Trdn gene ([Trdn.sup.-/-]). The structure and protein composition of the cardiac CRU is significantly altered in [Trdn.sup.-/-] hearts, jSR proteins (RyR2, Casq2, junctin, and junctophilin 1 and 2) are significantly reduced in [Trdn.sup.-/-] hearts, whereas Cavl.2 and SERCA2a remain unchanged. Electron microscopy shows fragmentation and an overall 50% reduction in the contacts between jSR and T-tubules, Immunolabeling experiments show reduced colocalization of Cav1.2 with RyR2 and substantial Casq2 labeling outside of the jSR in [Trdn.sup.-/-] myocytes. CRU function is impaired in [Trdn.sup.-/-] myocytes, with reduced SR [Ca.sup.2+] release and impaired negative feedback of SR [Ca.sup.2+] release on Cav1.2 [Ca.sup.2+] currents ([l.sub.ca]). Uninhibited [Ca.sup.2+] influx via [I.sub.Ca] likely contributes to [Ca.sup.2+] overload and results in spontaneous SR [Ca.sup.2+] releases upon [beta]-adrenergic receptor stimulation with isoproterenol in [Trdn.sup.-/-] myocytes, and ventricular arrhythmias in [Trdn.sup.-/-] mice. We conclude that triadin is critically important for maintaining the structural and functional integrity of the cardiac CRU; triadin Ioss and the resulting alterations in CRU structure and protein composition impairs E-C coupling and renders hearts susceptible to ventricular arrhythmias. cardiac muscle | sarcoplasmic reticulum | calsequestrin | Cav1.2 | RyR2

Nonsteroidal anti-inflammatory drugs selective for inhibition of COX-2 increase heart failure and elevate blood pressure. The COX-2 gene was floxed and crossed into merCremer mice under the [alpha]-myosin heavy-chain promoter. Tamoxifen induced selective deletion of COX-2 in cardiomyocytes depressed cardiac output, and resulted in weight loss, diminished exercise tolerance, and enhanced susceptibility to induced arrhythmogenesis. The cardiac dysfunction subsequent to pressure overload recovered progressively in the knockouts coincident with increasing cardiomyocyte hypertrophy and interstitial and perivascular fibrosis. Inhibition of COX-2 in cardiomyocytes may contribute to heart failure in patients receiving nonsteroidal anti-inflammatory drugs specific for inhibition of COX-2. arrhythmia | heart failure | knockout | NSAIDs | fibrosis

Calcium has been proposed as a primary influence on cell death during ischemic episodes in myocardial cells. One component of calcium entry into a cell is resting calcium influx. This basal movement of calcium is blocked by 100 µmol/L gadolinium chloride (Gd[Cl.sub.3]) in cardiac myocytes. Therefore, Gd[Cl.sub.3] should be cardioprotective under anoxic conditions. To test this, cardiac myocytes isolated from adult male rats were subjected to anoxia (100% [N.sub.2]) in the presence or absence of 100 µmol/L Gd[Cl.sub.3] in one of 2 ways. In the first method, cells were suspended in media and rendered anoxic for 0, 30, and 60 min, after which cell morphology and viability were scored. After 60 min of anoxia, rod-shaped cells accounted for 46% ± 4% of total cells (viability 81%); 10 min of reoxygenation markedly reduced rod-shaped cells to 27% (viability 72%). Gd[Cl.sub.3] in the medium did not protect the cells (anoxic rods 49%, reoxygenated rods 30%, viability 77%). In the second method, cells were attached to a laminin substrate, rendered anoxic, and then videotaped for up to 6 h. In this system, cells maintained their shape for some time after the onset of anoxia, and then began to 'die' (i.e., to take on either a rigor form or hypercontracted form) at a measurable rate. Time to onset of 'death' ([t.sub.0]), time to 50% and 100% 'death' ([t.sub.50] and [t.sub.100]), and rate of 'death' were used to measure anoxic damage. Without Gd[Cl.sub.3], cells on average began to die 115 ± 32 min after the onset of anoxia ([t.sub.0]); they died at an average rate of 0.046 cells/min. [t.sub.50] was achieved in 149 ± 42 min, [t.sub.100] in 183 ± 54 min. Addition of 100 µmol/L Gd[Cl.sub.3] did not affect any of these parameters. We concluded that Gd[Cl.sub.3] was not cardioprotective for anoxic myocytes and that cell damage generated by anoxia could not be attributed to resting calcium influx. Key words: heart, myocyte, calcium, anoxia. Le calcium aurait une influence imporante sur la mort cellulaire durant des episodes d'ischemie dans les cellules myocardiques. Une composante de l'entree de calcium dans une cellule est le courant calcique entrant au repos. Ce mouvement basal de calcium est bloque par 100 [micron]mol/L de chlorure de gadolinium (Gd[Cl.sub.3]) dans les myocytes cardiaques. Par consequent, le Gd[Cl.sub.3] devrait avoir un effet cardioprotecteur dans des conditions anoxiques. Pour verifier cette hypothese, on a soumis des cardiomyocytes isoles de rats males adultes a une anoxie (100% [N.sub.2]) en presence ou en l'absence de 100 [micron]mol/L de Gd[Cl.sub.3], en utilisant deux methodes. Dans la premiere methode, les cellules ont ete suspendues dans des solutions et rendues anoxiques pendant 0, 30, et 60 min; la morphologie et la viabilite des cellules ont ensuite ete notees. Apres 60 min d'anoxie, les cellules en batonnet ont represente 46% [+ o -] 4% de la totalite des cellules (viabilite 81%); une reoxygenation pendant 10 min a reduit de facon marquee le nombre de cellules en batonnet a 27% (viabilite 72%). La presence de Gd[Cl.sub.3] dans la solution n'a pas protege les cellules (batonnets anoxiques 49%, batonnets reox. 30%, viabilite 77%). Dans la seconde methode, les cellules ont ete fixees sur un substrat de laminine, rendues anoxiques, puis leur comportement a ete enregistre sur bande video pendant une periode allant jusqu'a 6 h. Dans ce systeme, les cellules ont maintenu leur forme quelque temps apres le debut de l'anoxie, puis ont commence a << mourir >> (c.-a-d. a prendre une forme soit rigide, soit hypercontractee) a un taux mesurable. Le temps vers le debut de la << mort >> [t.sub.0], le temps vers une << mortalite >> de 50% et de 100% ([t.sub.50] et [t.sub.100]) et le taux de << mortalite >> ont ete utilises pour mesurer la lesion anoxique. Sans Gd[Cl.sub.3], les cellules en moyenne ont commence a << mourir >> 115 [+ o -] 32 min apres le declenchement de l'anoxie ([t.sub.0]); les cellules sont << mortes >> a un taux moyen de 0,046 cellules/min. [t.sub.50] a ete atteint en 149 [+ o -] 42 min, [t.sub.100] en 183 [+ o -] 54 min. L'ajout de 100 [micron]mol/L de Gd[Cl.sub.3] n'a influe sur aucun de ces parametres. Nous avons conclu que le Gd[Cl.sub.3] n'a pas eu d'effet cardioprotecteur sur les myocytes anoxiques et que l'alteration des cellules causee par l'anoxie ne pouvait etre attribue e a un courant entant de calcium au repos. Mots-cles : coeur, myocyte, calcium, anoxie. [Traduit par la Redaction], Introduction Loss of control of calcium pathways is one of the features of reoxygenation after myocardial ischemia in mammals. In the cardiac myocyte, voltage-gated calcium channels, stretch-activated channels, and background [...]

T-tubular invaginations of the sarcolemma of ventricular cardiomyocytes contain junctional structures functionally coupling L-type calcium channels to the sarcoplasmic reticulum calcium-release channels (the ryanodine receptors), and therefore their configuration controls the gain of calcium-induced calcium release (CICR). Studies primarily in rodent myocardium have shown the importance of T-tubular structures for calcium transient kinetics and have linked T-tubule disruption to delayed CICR. However, there is disagreement as to the nature of T-tubule changes in human heart failure. We studied isolated ventricular myocytes from patients with ischemic heart disease, idiopathic dilated cardiomyopathy, and hypertrophic obstructive cardiomyopathy and determined T-tubule structure with either the fluorescent membrane dye di-8-ANNEPs or the scanning ion conductance microscope (SICM). The SICM uses a scanning pipette to produce a topographic representation of the surface of the live cell by a non-optical method. We have also compared ventricular myocytes from a rat model of chronic heart failure after myocardial infarction. T-tubule loss, shown by both ANNEPs staining and SICM imaging, was pronounced in human myocytes from all etiologies of disease. SICM imaging showed additional changes in surface structure, with flattening and loss of Z-groove definition common to all etiologies. Rat myocytes from the chronic heart failure model also showed both T-tubule and Z-groove loss, as well as increased spark frequency and greater spark amplitude. This study confirms the loss of T-tubules as part of the phenotypic change in the failing human myocyte, but it also shows that this is part of a wider spectrum of alterations in surface morphology. calcium handling | heart failure | morphology | T-tubule

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.

Adult progenitor cell transplantation has been proposed for the treatment of heart failure, but the mechanisms effecting functional improvements remain unknown. The aim of this study was to test the hypothesis that, in failing hearts treated with cell transplantation, the mechanical properties and excitation-contraction coupling of recipient cardiomyocytes are altered. Adult rats underwent coronary artery ligation, leading to myocardial infarction and chronic heart failure. After 3 wk, they received intramyocardial injections of either [10.sup.7] green fluorescence protein (GFP)-positive bone marrow mononuclear cells or 5 x [10.sup.6] GFP-positive skeletal myoblasts. Four weeks after injection, both cell types increased ejection fraction and reduced cardiomyocyte size. The contractility of isolated GFP-negative cardiomyocytes was monitored by sarcomere shortening assessment, [Ca.sup.2+] handling by indo-1 and fluo-4 fluorescence, and electrophysiology by patch-clamping techniques. Injection of either bone marrow cells or skeletal myoblasts normalized the impaired contractile performance and the prolonged time to peak of the [Ca.sup.2+] transient observed in failing cardiomyocytes. The smaller and slower L-type [Ca.sup.2+] current observed in heart failure normalized after skeletal myoblast, but not bone marrow cell, transplantation. Measurement of [Ca.sup.2+] sparks suggested a normalization of sarcoplasmic reticulum [Ca.sup.2+] leak after skeletal myoblast transplantation. The increased [Ca.sup.2+] wave frequency observed in failing myocytes was reduced by either bone marrow cells or skeletal myoblasts. In conclusion, the morphology, contractile performance, and excitation-contraction coupling of individual recipient cardiomyocytes are altered in failing hearts treated with adult progenitor cell transplantation. heart failure; cell therapy; excitation-contraction coupling; cell electrophysiology; paracrine mechanims

We have recently demonstrated that tissue inhibitor of metalloproteinase-3 (TIMP-3) decreases neonatal cardiomyocyte proliferation (Hammoud L, Xiang F, Lu X, Brunner F, Leco K, Feng Q. Cardiovasc Res 75: 359-368, 2007). The aim of the present study was to delineate a pathway through which TIMP-3 exerts its antiproliferative effect. Experiments were conducted on neonatal cardiomyocyte cultures and heart tissues isolated from wild-type (WT) and [TIMP-3.sup.-/-] mice. Deficiency in TIMP-3 decreased p27 expression and increased cardiomyocyte proliferation in cardiomyocytes and neonatal hearts. A TIMP-3/epidermal growth factor (EGF) receptor (EGFR)/c-Jun N[H.sub.2]-terminal kinase (JNK)/SP-1/p27 pathway was investigated. JNK phosphorylation and EGFR protein levels were increased in [TIMP-3.sup.-/-] cardiomyocytes and heart tissues. Treatment with recombinant TIMP-3 decreased JNK phosphorylation and EGFR expression/ phosphorylation. Inhibition of JNK activity using SP-600125 decreased SP-1 phosphorylation, increased p27 expression, and decreased cardiomyocyte proliferation. Furthermore, treatment with the EGFR specific inhibitor PD-168393 or the EGF-neutralizing antibody decreased cardiomyocyte proliferation as well as phosphorylation of JNK and SP-1 in both WT and [TIMP-3.sup.-/-] cardiomyocytes. We conclude that TIMP-3 inhibits neonatal mouse cardiomyocyte proliferation by upregulating p27 expression. The effects of TIMP-3 are mediated via inhibition of EGFR expression/phosphorylation, and decreases in JNK and SP-1 signaling. epidermal growth factor receptor; signal transduction; c-Jun N[H.sub.2]-terminal kinase

Massive amounts of nucleotides are released during ischemia in the cardiovascular system. Although the effect of the purine nucleotide ATP has been intensively studied in myocardial infarction, the cardioprotective role of the pyrimidine nucleotide UTP is still unclear, especially in the cardiovascular system. The purpose of our study was to elucidate the protective effects of UTP receptor activation and describe the downstream cascade for the cardioprotective effect. Cultured cardiomyocytes and left anterior descending (LAD)-ligated rat hearts were pretreated with UTP and exposed to hypoxiaischemia. In vitro experiments revealed that UTP reduced cardiomyocyte death induced by hypoxia, an effect that was diminished by suramin. UTP caused several effects that could trigger a cardioprotective response: a transient increase of [[[Ca.sup.2+]].sub.i], an effect that was abolished by PPADS or RB2; phosphorylation of the kinases ERK and Akt, which was abolished by U0126 and LY294002, respectively; and reduced mitochondrial calcium elevation after hypoxia. In vivo experiments revealed that UTP maintained ATP levels, improved mitochondrial activity, and reduced infarct size. In conclusion, UTP administrated before ischemia reduced infarct size and improved myocardial function. Reduction of mitochondrial calcium overload can partially explain the protective effect of UTP after hypoxic-ischemic injury. Key words: calcium, cardioprotection, cardiac cell culture, heart, hypoxia, ischemia, nucleotides, preconditioning, pyrimidines. D'enormes quantites de nucleotides sont liberes dans le systeme vasculaire au cours de l'ischemie. L'effet des nucleotides puriques (ATP) durant l'infarctus du myocarde a fait l'objet de nombreuses etudes; toutefois, le role cardioprotecteur des nucleotides pyrimidiques (UTP) est encore peu connu dans le systeme cardiovasculaire. La presente etude a eu pour but d'identifier les effets protecteurs de l'activation du recepteur UTP et de decrire la cascade en aval de l'effet cardioprotecteur. Des cardiomyocytes en culture et des cours de rats presentant une ligature de l'artere descendante anterieure gauche (ADG) ont ete pretraites avec de l'UTP et exposes 'une hypoxie-ischemie. Des experiences in vitro ont revele que l'UTP a diminue le taux de mortalite des cardiomyocytes induite par l'hypoxie, un effet qui a ete attenue par la suramine. L'UTP a provoque les effets suivants, lesquels pourraient declencher une reponse cardioprotectrice : une augmentation transitoire de la concentration de [[[Ca.sup.2+]].sub.i], effet qui a ete supprime par le PPADS ou le R132; la phosphorylation des kinases ERK et Akt, qui a ete supprimee respectivement par l'U0126 et le LY294002; une diminution de l'elevation de calcium mitochondrial apres l'hypoxie. Des experiences in vivo ont revele que l'UTP a maintenu les taux d'ATP, augmente l'activite mitochondriale et diminue la taille de l'infarctus. En conclusion, l'UTP administre avant l'ischemie a reduit la taille de l'infarctus et ameliore la fonction myocardique. La reduction de la surcharge calcique mitochondriale pourrait expliquer en partie l'effet protecteur de l'UTP apres une lesion d'hypoxie-ischemie. Mots-cles : calcium, cardioprotection, culture de cellules cardiaques, cour, hypoxie, ischemie, nucleotides, preconditionnement, pyrimidines., [Traduit par la Redaction] Introduction Despite intensive research and progress in medicine, myocardial infarction and heart failure remain the leading causes of death in the Western world. Myocyte loss, both [...]

The synchronization of coupled oscillators plays an important role in many biological systems, including the heart. In heart diseases, cardiac myocytes can exhibit abnormal electrical oscillations, such as early afterdepolarizations (EADs), which are associated with lethal arrhythmias. A key unanswered question is how cellular EADs partially synchronize in tissue, as is required for them to propagate. Here, we present evidence, from computational simulations and experiments in isolated myocytes, that irregular EAD behavior is dynamical chaos. We then show in electrically homogeneous tissue models that chaotic EADs synchronize globally when the tissue is smaller than a critical size. However, when the tissue exceeds the critical size, electrotonic coupling can no longer globally synchronize EADs, resulting in regions of partial synchronization that shift in time and space. These regional partially synchronized EADs then form premature ventricular complexes that propagate into recovered tissue without EADs. This process creates multiple hat propagate 'shifting' foci resembling polymorphic ventricular tachycardia. Shifting foci encountering shifting repolarization gradients can also develop localized wave breaks leading to reentry and fibrillation. As predicted by the theory, rabbit hearts exposed to oxidative stress ([H.sub.2][O.sub.2]) exhibited multiple shifting foci causing polymorphic tachycardia and fibrillation. This mechanism explains how collective cellular behavior integrates at the tissue scale to generate lethal cardiac arrhythmias over a wide range of heart rates. chaos | foci | reentry | fibrillation

Computer modeling of cardiac propagation suggests that curvature of muscle fibers modulates conduction velocity (CV). The effect could be involved in arrhythmogenesis by altering the dynamics of reentrant wavefronts or by causing propagation block. To verify the existence of this effect experimentally, we measured CV in anisotropic neonatal rat ventricular myocyte monolayers. The orientation of the cells was directed by scratches machined into plastic coverslips. Each substrate contained a region in which scratch radius of curvature varied from 0.25 to 1.0 cm. The CV anisotropy ratio (longitudinal CV/transverse CV in straight fiber regions) was 2.3 [+ or -] 0.3 (n : 38). We initiated wavefronts transverse to fibers with the fibers either curving toward or away from the wavefronts. Action potentials were recorded using a potentiometric dye and a video camera. Propagation was faster (p = 0.0003) when fibers curved toward wavefronts than when fibers curved in the opposite direction. The mean CV difference was 0.38 [+ or -] 0.44 cm/s (n = 24), which is 3.5% of nominal straight fiber transverse CV (11.0 [+ or -] 3.2 cm/s). The effect was also present (p = 0.07) when pacing was slowed from 350 to 500 ms (n = 6). In a control group (n = 8) with uncurved fibers, CV was the same in both directions (p = NS). We conclude that fiber curvature is a factor in modulating cardiac propagation. Index Terms--Anisotropy, arrhythmia, cardiomyocytes, electro-physiology, optical mapping.

Although granulocyte colony-stimulating factor (G-CSF) reportedly plays a cardioprotective role in several models of cardiac injury, clinical use of this drug in cardiac patients has been controversial. Here, we tested, in vivo and in vitro, the effect of G-CSF on cardiac mitochondria, which play a key role in determining cardiac cellular fate and function. Mild stimulation of C57/BL6 mice with doxorubicin (Dox) did not induce cardiac apoptosis or fibrosis but did induce damage to mitochondrial organization of the myocardium as observed through an electron microscope. Cardiac catheterization and echocardiography revealed that Dox did not alter cardiac systolic function or left ventricular size but did reduce diastolic function, an early sign of cardiac damage. Treatment with G-CSF attenuated significantly the damage to mitochondrial organization and rescued diastolic function. In an in vitro model for rat neonatal cardiomyocytes, a subapoptotic dose of Dox induced severe mitochondrial damage, including marked swelling of the cardiac mitochondria and/or decreased mitochondrial membrane potential. These mitochondrial changes were completely blocked by pretreatment with G-CSF. In addition, G-CSF dramatically improved ATP generation, which rescued Dox-impaired mitochondrial electron transport and oxygen consumption mainly through complex IV. These findings clearly indicate that G-CSF protects cardiac mitochondria, which are key organelles in the determination of cardiac cellular fate, in the early phase of cardiac injury. doxorubicin; mitochondria; cardiac diastolic function; oxygen consumption

Altered myocardial [Ca.sup.2+] and [Na.sup.+] handling in congestive heart failure (CHF) may be expected to decrease the tolerance to ischemia by augmenting reperfusion [Ca.sup.2+] overload. The aim of the present study was to investigate tolerance to hypoxia-reoxygenation by measuring enzyme release, cell death, ATP level, and cell [Ca.sup.2+] and [Na.sup.+] in cardiomyocytes from failing rat hearts. CHF was induced in Wistar rats by ligation of the left coronary artery during isoflurane anesthesia, after which cardiac failure developed within 6 wk. Isolated cardiomyocytes were cultured for 24 h and subsequently exposed to 4 h of hypoxia and 2 h of reoxygenation. Cell damage was measured as lactate dehydrogenase (LD) release, cell death as propidium iodide uptake, and ATP by firefly luciferase assay. Cell [Ca.sup.2+] and [Na.sup.+] were determined with radioactive isotopes, and free intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) with fluo-3 AM. CHF cells showed less increase in LD release and cell death after hypoxia-reoxygenation and had less relative reduction in ATP level after hypoxia than sham cells. CHF cells accumulated less [Na.sup.+] than sham cells during hypoxia (117 vs. 267 nmol/mg protein). CHF cells maintained much lower [[[Ca.sup.2+]].sub.i] than sham cells during hypoxia (423 vs. 1,766 arbitrary units at 4 h of hypoxia), and exchangeable [Ca.sup.2+] increased much less in CHF than in sham cells (1.4 vs. 6.7 nmol/mg protein) after 120 rain of reoxygenation. Ranolazine, an inhibitor of late [Na.sup.+] current, significantly attenuated both the increase in exchangeable [Ca.sup.2+] and the increase in LD release in sham cells after reoxygenation. This supports the suggestion that differences in [Na.sup.+] accumulation during hypoxia cause the observed differences in [Ca.sup.2+] accumulation during reoxygenation. Tolerance to hypoxia and reoxygenation was surprisingly higher in CHF than in sham cardiomyocytes, probably explained by lower hypoxia-mediated [Na.sup.+] accumulation and subsequent lower [Ca.sup.2+] accumulation in CHF after reoxygenation. calcium; congestive heart failure; sodium

Downregulation of the sarcoplasmic reticulum calcium ATPase (SERCA2) is associated with diastolic dysfunction in the failing heart. Elevated plasma endothelin-1 (ET) levels are correlated with congestive heart failure suggesting that ET may play a pathophysiological role. We have investigated the ability of ET to regulate SERCA2 gene expression in isolated adult rat ventricular myocytes. We find that ET enhances net protein synthesis by ~40% but significantly downregulates SERCA2 mRNA expression, time dependently, by ~30-50%, and the expression of SERCA2 protein by ~ 50%. In myoyctes, ET binds to ETA receptor that couples to [G.sub.q] and [G.sub.i] proteins. Inhibition of [G.sub.q]-PLC-induced phosphoinositide (PI) hydrolysis with U73122 (1 [micro]M) or inhibition of [G.sub.i] protein with pertussis toxin (PTX) abolishes the ability of ET to downregulate SERCA2 mRNA gene expression. Further investigation suggests that ET coupling to PTX-sensitive [G.sub.i] with consequent lowering of cAMP is required for downregulation of SERCA2 mRNA levels. Increasing intracellular cAMP quantity using cAMP-specific PDE inhibitor Ro20-1724 or cAMP analog dibutyryl-cAMP reverses ET-induced downregulation of SERCA2 mRNA levels. The data indicate that, in adult myocytes, ET downregulates SERCA2 mRNA and protein levels, and the effect requires cross-talk between [G.sub.q] and PTX-sensitive [G.sub.i] pathways. hypertrophy; sarcoplasmic reticulum calcium ATPase; [G.sub.i]; [G.sub.q]

Phospholamban has been suggested to be a key regulator of cardiac sarcoplasmic reticulum (SR) Ca cycling and contractility and a potential therapeutic target in restoring the depressed Ca cycling in failing hearts. Our understanding of the function of phospholamban stems primarily from studies in genetically altered mouse models. To evaluate the significance of this protein in larger mammalian species, which exhibit Ca cycling properties similar to humans, we overexpressed phospholamban in adult rabbit cardiomyocytes. Adenoviral-mediated gene transfer, at high multiplicities of infection, resulted in an insignificant 1.22-fold overexpression of phospholamban. There were no effects on twitch Ca-transient amplitude or decay under basal or isoproterenol-stimulated conditions. Furthermore, the SR Ca load and Na/Ca exchanger function were not altered. These apparent differences between phospholamban overexpression in rabbit compared with previous findings in the mouse may be due to a significantly higher (l.5-fold) endogenous phospholamban-to-sarco(endo)plasmic reticulum Ca-ATPase (SERCA) 2a ratio and potential functional saturation of SERCA2a by phospholamban in rabbit cardiomyocytes. The findings suggest that important species-dependent differences in phospholamban regulation of SERCA2a occur. In larger mammals, a higher fraction of SERCA2a pumps are regulated by phospholamban, and this may influence therapeutic strategies to enhance cardiac contractility and functional cardiac reserve. calcium; adenoviral; contractility

Self-renewable, pluripotent human embryonic stem cells (hESCs) can be differentiated into cardiomyocytes (CMs), providing an unlimited source of cells for transplantation therapies. However, unlike certain cell lineages such as hematopoietic cells, CMs lack specific surface markers for convenient identification, physical separation, and enrichment. Identification by immunostaining of cardiac-specific proteins such as troponin requires permeabilization, which renders the cells unviable and nonrecoverable. Ectopic expression of a reporter protein under the transcriptional control of a heart-specific promoter for identifying hESC-derived CMs (hESC-CMs) is useful for research but complicates potential clinical applications. The practical detection and removal of undifferentiated hESCs in a graft, which may lead to tumors, is also critical. Here, we demonstrate a nondestructive, label-free optical method based on Raman scattering to interrogate the intrinsic biochemical signatures of individual hESCs and their cardiac derivatives, allowing cells to be identified and classified. By combination of the Raman spectroscopic data with multivariate statistical analysis, our results indicate that hESCs, human fetal left ventricular CMs, and hESC-CMs can be identified by their intrinsic biochemical characteristics with an accuracy of 96%, 98%, and 66%, respectively. The present study lays the groundwork for developing a systematic and automated method for the noninvasive and label-free sorting of (i) high-quality hESCs for expansion and (ii) ex vivo CMs (derived from embryonic or adult stem cells) for cell-based heart therapies.

Directed differentiation of embryonic stem cells indicates that mesodermal lineages in the mammalian heart (cardiac, endothelial, and smooth muscle cells) develop from a common, multipotent cardiovascular precursor. To isolate and characterize the lineage potential of a resident pool of cardiovascular progenitor cells (CPcs), we developed BAC transgenic mice in which enhanced green fluorescent protein (EGFP) is placed under control of the c-kit locus (c-[kit.sup.BAC]-EGFP mice). Discrete c-kit-[EGFP.sup.+] cells were observed at different stages of differentiation in embryonic hearts, increasing in number to a maximum at about postnatal day (PN) 2; thereafter, [EGFP.sup.+] cells declined and were rarely observed in the adult heart. [EGFP.sup.+] cells purified from PN 0-5 hearts were [nestin.sup.+] and expanded in culture; 67% of cells were fluorescent after 9 days. Purified cells differentiated into endothelial, cardiac, and smooth muscle cells, and differentiation could be directed by specific growth factors. CPc-derived cardiac myocytes displayed rhythmic beating and action potentials characteristic of multiple cardiac cell types, similar to ES cell-derived cardiomyocytes. Single-cell dilution studies confirmed the potential of individual CPcs to form all 3 cardiovascular lineages. In adult hearts, cryoablation resulted in c-kit-[EGFP.sup.+] expression, peaking 7 days postcryolesion. Expression occurred in endothelial and smooth muscle cells in the revascularizing infarct, and in terminally differentiated cardiomyocytes in the border zone surrounding the infarct. Thus, c-kit expression marks CPc in the neonatal heart that are capable of directed differentiation in vitro; however, c-kit expression in cardiomyocytes in the adult heart after injury does not identify cardiac myogenesis. bacterial artificial chromosome | progenitor cell | stem cell

Different vertebrate species have different cardiac regeneration rates: high in teleost fish, moderate in urodele amphibians, and almost negligible in mammals. Regeneration may occur through stem and progenitor cell differentiation or via dedifferentiation with residual cardiomyocytes reentering the cell cycle. In this review, we will examine the ability of zebrafish and newts to respond to cardiac damage with de novo cardiogenesis, whereas rodents and humans respond with a marked fibrogenic response and virtually no cardiomyocyte regeneration. Concerted strategies are needed to overcome this evolutionarily imposed barrier and optimize cardiac regeneration in mammals.

Bacterial endotoxin lipopolysaccharide (LPS) is responsible for the multiorgan dysfunction that characterizes septic shock and is causal in the myocardial depression that is a common feature of endotoxemia in patients. In this setting the myocardial dysfunction appears to be due, in part, to the production of proinflammatory cytokines. A line of evidence also indicates that LPS stimulates autophagy in cardiomyocytes. However, the signal transduction pathway leading to autophagy and its role in the heart are incompletely characterized. In this work, we wished to determine the effect of LPS on autophagy and the physiological significance of the autophagic response. Autophagy was monitored morphologically and biochemically in HL-1 cardiomyocytes, neonatal rat cardiomyocytes, and transgenic mouse hearts after the administration of bacterial LPS or TNF-[alpha]. We observed that autophagy was increased after exposure to LPS or TNF-[alpha], which is induced by LPS. The inhibition of TNF-[alpha] production by AG126 significantly reduced the accumulation of autophagosomes both in cell culture and in vivo. The inhibition of p38 MAPK or nitric oxide synthase by pharmacological inhibitors also reduced autophagy. Nitric oxide or H202 induced autophagy in cardiomyocytes, whereas Nacetyl-cysteine, a potent antioxidant, suppressed autophagy. LPS resulted in increased reactive oxygen species (ROS) production and decreased total glutathione. To test the hypothesis that autophagy might serve as a damage control mechanism to limit further ROS production, we induced autophagy with rapamycin before LPS exposure. The activation of autophagy by rapamycin suppressed LPSmediated ROS production and protected cells against LPS toxicity. These findings support the notion that autophagy is a cytoprotective response to LPS-induced cardiomyocyte injury; additional studies are needed to determine the therapeutic implications. lipopolysaccharide; HL-1 cardiac myocyte; green fluorescent proteinmicrotubule-associated protein light chain 3; oxidative stress

We have previously demonstrated that in a rat model of trauma-hemorrhage (T-H), glucosamine administration during resuscitation improved cardiac function, reduced circulating levels of inflammatory cytokines, and increased tissue levels of O-linked N-acetylglucosamine (O-GlcNAc) on proteins. The mechanism(s) by which glucosamine mediated its protective effect were not determined; therefore, the goal of this study was to test the hypothesis that glucosamine treatment attenuated the activation of the nuclear factor-[kappa]B (NF-[kappa]B) signaling pathway in the heart via an increase in protein O-GlcNAc levels. Fasted male rats were subjected to T-H by bleeding to a mean arterial blood pressure of 40 mmHg for 90 min followed by resuscitation. Glucosamine treatment during resuscitation significantly attenuated the T-H-induced increase in cardiac levels of TNF-[alpha] and IL-6 mRNA, I[kappa]B-[alpha] phosphorylation, NF-[kappa]B, NF-[kappa]B DNA binding activity, ICAM-1, and MPO activity. LPS (2 [micro]g/ml) increased the levels of I[kappa]B-[alpha] phosphorylation, TNF-[alpha], ICAM-1, and NF-[kappa]B in primary cultured cardiomyocytes, which was significantly attenuated by glucosamine treatment and overexpression of O-GlcNAc transferase; both interventions also significantly increased O-GlcNAc levels. In contrast, the transfection of neonatal rat ventricular myocytes with OGT small-interfering RNA decreased O-GlcNAc transferase and O-GlcNAc levels and enhanced the LPS-induced increase in I[kappa]B-[alpha] phosphorylation. Glucosamine treatment of macrophage cell line RAW 264.7 also increased O-GIcNAc levels and attenuated the LPS-induced activation of NF-[kappa]B. These results demonstrate that the modulation of O-GlcNAc levels alters the response of cardiomyocytes to the activation of the NF-[kappa]B pathway, which may contribute to the glucosamine-mediated improvement in cardiac function following hemorrhagic shock. nuclear factor-[kappa]B; cytokines; neonatal rat ventricular myocytes; O-linked N-acetylglucosamine

Malhotra A, Vashistha H, Yadav VS, Dube MG, Kalra SP, Abdellatif M, Meggs LG. Inhibition of p66ShcA redox activity in cardiac muscle cells attenuates hyperglycemia-induced oxidative stress and apoptosis. Am J Physiol Heart Circ Physiol 296: H380-H388, 2009. First published December 5, 2008; doi:10.1152/ajpheart.00225.2008. Apoptotic myocyte cell death, diastolic dysfunction, and progressive deterioration in left ventricular pump function characterize the clinical course of diabetic cardiomyopathy. A key question concerns the mechanism(s) by which hyperglycemia (HG) transmits danger signals in cardiac muscle cells. The growth factor adapter protein p66ShcA is a genetic determinant of longevity, which controls mitochondrial metabolism and cellular responses to oxidative stress. Here we demonstrate that interventions which attenuate or prevent HG-induced phosphorylation at critical position 36 Ser residue (phospho-Ser36) inhibit the redox function of p66ShcA and promote the survival phenotype. Adult rat ventricular myocytes obtained by enzymatic dissociation were transduced with mutant-36 p66ShcA (mu-36) dominant-negative expression vector and plated in serum-free media containing 5 or 25 mM glucose. At HG, adult rat ventricular myocytes exhibit a marked increase in reactive oxygen species production, upregulation of phospho-Ser36, collapse of mitochondrial transmembrane potential, and increased formation of p66ShcAJcytochrome-c complexes. These indexes of oxidative stress were accompanied by a 40% increase in apoptosis and the upregulation of cleaved caspase-3 and the apoptosis-related proteins p53 and Bax. To test whether p66ShcA functions as a redox-sensitive molecular switch in vivo, we examined the hearts of male Akita diabetic nonobese (C57BL/6J) mice. Western blot analysis detected the upregulation of phospho-Ser36, the translocation of p66ShcA to mitochondria, and the formation of p66ShcA/cytochrome-c complexes. Conversely, the correction of HG by recombinant adeno-associated viral delivery of leptin reversed these alterations. We conclude that p66ShcA is a molecular switch whose redox function is turned on by phospho-Ser36 and turned off by interventions that prevent this modification. diabetes mellitus; reactive oxygen species

Numerous cAMP-elevating agents regulate events required for efficient migration of arterial vascular smooth muscle cells (VSMCs). Interestingly, when the impact of cAMP-elevating agents on individual migration-related events is studied, these agents have been shown to have distinct, and sometimes unexpected, effects. For example, although cAMP-elevating agents inhibit overall migration, they promote VSMC adhesion to extracellular matrix proteins and the formation of membrane extensions, which are both events that are essential for and promote migration. Herein, we extend previous observations that identified phosphodiesterase-4D3 (PDE4D3) as an integral component of a PKA/A kinase-anchoring protein (AKAP) complex in cultured/hypertrophied rat cardiac myocytes to the case for nonhypertrophied cardiac myocytes. Moreover, we show that while rat aortic VSMCs also express PDE4D3, this protein is not detected in PKA/ AKAP complexes isolated from these cells. In contrast, we show that another PDE4D splice variant expressed in arterial vascular myocytes, namely, PDE4D8, integrates into PKA/AKAP-based signaling complexes in VSMCs. Consistent with the idea that a PDE4D8/PKA/ AKAP complex regulates specific VSMC functions, PKA and PDFAD8 were each recruited to leading-edge structures in migrating VSMCs, and inhibition of PDE4D8 recruitment to pseudopodia of migrating cells caused localized changes in actin dynamics. Our data are presented in the context that cardiac myocytes and arterial VSMCs may use distinct PDFAD variants to regulate selected pools of targeted PKA activity and that disruption of this complex may allow selective regulation of cAMP-dependent events between these two cardiovascular cell types. migration; vascular smooth muscle cell

The use of confocal microscopy has shown that the nucleus plays an important role in excitation-contraction and excitation-secretion coupling of several excitable and nonexcitable cardiovascular cells. It has shown that the nuclear membranes, like the sarcolemmal membrane, possess ionic transporters as well as G protein-coupled receptors (GPCRs), which play a major role in modulating both cytosolic and nuclear ionic homeostasis and nuclear signalling. During spontaneous contraction of heart cells, the increase in cytosolic [Ca.sup.2+] was immediately followed by a transient increase in nuclear [Ca.sup.2+]. The nuclear [Ca.sup.2+] rise during excitation-contraction and excitation-secretion coupling was both dependent and independent of changes in cytosolic [Ca.sup.2+]. Nuclear membrane GPCRs, such as those of angiotensin II, neuropeptide Y, and ET-1, were functional and contributed to modulation of nuclear ionic homeostasis via direct and (or) indirect modulation of nuclear membrane ionic transporters such as channels, pumps, and exchangers. The signalling of nuclear membrane GPCRs may also contribute to modulation of gene expression, which may regulate proliferation and remodelling of cells and, indeed, life and death. Direct or indirect targeting of nuclear membrane ionic transporters and GPCRs may constitute a new target for drug action. Key words: nucleus, nuclear membranes, GPCR, calcium, receptors, apoptosis, Ang II, ET-1, NPY. L'utilisation de la microscopie confocale a permis de reveler que le noyau joue un role important dans le couplage excitation--contraction et excitation--secretion de plusieurs cellules cardiovasculaires excitables et non excitables. On a montre que, comme la membrane sarcolemmique, les membranes nucleaires possedent des transporteurs ioniques et des recepteurs couples aux proteines G (RCPG), qui jouent un role majeur dans la modulation de l'homeostasie ionique cytosolique et nucleaire ainsi que dans la signalisation nucleaire. Durant la contraction spontanee des cellules cardiaques, l'augmentation de [Ca.sup.2+] cytosolique a ete immediatement suivie d'une augmentation transitoire de [Ca.sup.2+] nucleaire. L'elevation du [Ca.sup.2+] nucleaire durant le couplage excitation-contraction/secretion a ete a la fois dependante et independante des modifications du [Ca.sup.2+] cytosolique. Les RCPG des membranes nucleaires, comme ceux de l'Ang II, de NPY et de l'ET-1, ont ete fonctionnels et participent a la modulation de l'homeostasie ionique nucleaire par une modulation directe et/ou indirecte des transporteurs ioniques des membranes nucleaires, tels que les canaux, les pompes et les echangeurs. La signalisation des RCPG des membranes nucleaires pourraient aussi participer a la modulation de l'expression genique, qui pourrait reguler la vie et la mort ainsi que la proliferation et le remodelage des cellules. Le ciblage direct ou indirect des transporteurs ioniques et des RCPG des membranes nucleaires pourrait constituer une nouvelle cible pour l'action medicamenteuse. Mots-cles : noyau, membranes nucleaires, RCPG, calcium, transporteurs, apoptose, Ang II, ET-1, NPY. [Traduit par la Redaction], Introduction To date, much of the focus on the cardiovascular system has been on the regulation, characterization, and modulation of the transsarcolemmal influx of ions and their pharmacologic modulation through [...]

Mesenchymal stem cells (MSCs) have the capacity to differentiate into osteoblasts, chondrocytes, adipocytes, myocytes, and cardiomyocytes. Several established methods are presently available for in vitro isolation of MSCs from bone marrow. However, the duration necessary to culture them can be a major handicap to cell-based therapies needed for such urgent cardiovascular conditions as acute myocardial infarction and acute hindlimb ischemia. The best timing of cardiomyocyte differentiation induction after MCS isolation and expansion is still an unresolved issue. Our goal was to investigate the possibility of obtaining functional cardiomyocytes from rat MSC within a shorter time period. We examined MSCs' colony-forming capacity, CD90 and CD34 immunoreactivity during the 14 days of culturing. Cardiomyocyte differentiation was induced by 5-azacytidine. Immunohistochemic staining, together with intracellular [Ca.sup.2+] measurement experiments, revealed that MSCs do not differentiate into any specific cell lineage but show the characteristics of MSCs on both the 9th and 14th days of the culture. To check the potential for differentiation into cardiomyocytes, experiments with caffeine application and depolarization with KCl were performed. The cells possessed some of the specific biochemical features of contracting cells, with slightly higher capacities on the 14th day. Cells from 9th and 14th days of the culture that were treated with 5-azacytidine had a higher expression of cardiac-specific markers such as troponin I, α-sarcomeric actin, and MEF2D compared with the control groups. This study illustrates that it is possible to get functional cardiomyocytes from in vitro MSC culture in a shorter time period than previously achieved. This reduction in time may provide emergency cases with access to cell-based therapies that may have previously been unavailable. Key words: mesenchymal stem cells, cardiomyocytes, differentiation, rat, in vitro, gene expression. Les cellules souches mesenchymateuses (CSM) ont la capacite de se differencier en osteoblastes, chondrocytes, adipocytes, myocytes et cardiomyocytes. Plusieurs methodes eprouvees permettent d'isoler in vitro les CSM de la moelle osseuse. Toutefois, la duree requise pour leur culture pourrait etre un desavantage majeur pour la therapie cellulaire d'urgences cardiovasculaires comme l'infarctus du myocarde et l'ischemie des membres inferieurs. Le meilleur moment de l'induction de la differenciation des cardiomyocytes apres l'isolement et l'expansion des CSM n'a toujours pas ete determine. Nous avons etudie la possibilite d'obtenir des cardiomyocytes fonctionnels de CSM de rats sur une courte periode de temps. Nous avons examine la capacite de formation de colonies des CSM et l'immunoreactivite a CD90 et CD34 durant les 14 jours de culture. La differenciation en cardiomyocytes a ete induite par la 5-azacytidine. La coloration immuno-histochimique ainsi que des mesures du [Ca.sup.2+] intracellulaire ont revele que les CSM ne se differencient pas en une lignee cellulaire specifique, mais montrent les caracteristiques des CSM lors des 9e et 14' jours de culture. Pour verifier le potentiel de differenciation en cardiomyocytes, nous avons fait des experiences avec application de cafeine et depolarisation au KCl. Les cellules possedaient certaines des caracteristiques biochimiques specifiques des cellules pouvant se contracter, avec des capacites legerement superieures le quatorzieme jour. Les cellules des 9e et 14e jours de culture traitees avec la 5-azacytidine ont montre une plus grande expression de marqueurs specifiques du cour, tels que la troponine I, l'actine α-sarcomerique et MEF2D, comparativement a celles des groupes temoins. Cette etude montre qu'il est maintenant possible d'obtenir des cardiomyocytes fonctionnels d'une culture in vitro de CSM dans une courte periode de temps. Cette reduction de temps pourrait fournir aux cas d'urgence un acces a des therapies cellulaires jusqu'alors inaccessibles. Mots-cles : cellules souches mesenchymateuses, cardiomyocytes, differenciation, rat, in vitro, expression genique. [Traduit par la Redaction], Introduction Stem cell research has gained tremendous interest in the past two decades because it provides opportunities to develop new strategies for debilitating diseases. Stem, or progenitor, cells have the [...]

Recent studies support the existence of a common progenitor for the cardiac and endothelial cell lineages, but the underlying transcriptional networks responsible for specification of these cell fates remain unclear. Here we demonstrated that Ets-related protein 71 (Etsrp71), a newly discovered ETS family transcription factor, was a novel downstream target of the homeodomain protein, Nkx2-5. Using genetic mouse models and molecular biological techniques, we demonstrated that Nkx2-5 binds to an evolutionarily conserved Nkx2-5 response element in the Etsrp71 promoter and induces the Etsrp71 gene expression in vitro and in vivo. Etsrp71 was transiently expressed in the endocardium/endothelium of the developing embryo (E7.75-E9.5) and was extinguished during the latter stages of development. Using a gene disruption strategy, we found that Etsrp71 mutant embryos lacked endocardial/endothelial lineages and were nonviable. Moreover, using transgenic technologies and transcriptional and chromatin immunoprecipitation (ChIP) assays, we further established that Tie2 is a direct downstream target of Etsrp71. Collectively, our results uncover a novel functional role for Nkx2-5 and define a transcriptional network that specifies an endocardial/endothelial fate in the developing heart and embryo. cardiac progenitor cells | endocardium | Etsrp71 | Tie2 | cardiac development

We analyzed the effect of conditional, [alpha]MHC-dependent genetic [beta]-catenin depletion and stabilization on cardiac remodeling following experimental infarct. [beta]-Catenin depletion significantly improved 4-week survival and left ventricular (LV) function (fractional shortening: [CT.sup.[delta]ex3-6]: 24 [+ or -] 1.9%; [beta]-[cat.sup.[delta]ex3-6]; 30.2 [+ or -] 1.6%, P < 0.001). [beta]-Catenin stabilization had opposite effects. No significant changes in adult cardiomyocyte survival or hypertrophy were observed in either transgenic line. Associated with the functional improvement, LV scar cellularity was altered: [beta]-catenin-depleted mice showed a marked subendocardial and subepicardial layer of small [cTnT.sup.pos] cardiomyocytes associated with increased expression of cardiac lineage markers Tbx5 and GATA4. Using a Cre-dependent lacZ reporter gene, we identified a noncardiomyocyte cell population affected by [alpha]MHC-driven gene recombination localized to these tissue compartments at baseline. These cells were found to be cardiac progenitor cells since they coexpressed markers of proliferation (Ki67) and the cardiomyocyte lineage ([alpha]MHC, GATA4, Tbx5) but not cardiac Troponin T (cTnT). The cell population overlaps in part with both the previously described c-[kit.sup.pos] and stem cell antigen-1 [(Sca-1).sup.pos] precursor cell population but not with the Islet-[1.sup.pos] precursor cell pool. An in vitro coculture assay of highly enriched (>95%) Sca-[1.sup.pos] cardiac precursor cells from [beta]-catenin-depleted mice compared to cells isolated from control littermate demonstrated increased differentiation toward [alpha]-[actin.sup.pos] and [cTnT.sup.pos] cardiomyocytes after 10 days ([CT.sup.[delta]ex3-6]: 38.0 [+ or -] 1.0% [alpha]-[actin.sup.pos] [beta]-[cat.sup.[delta]ex3-6]: 49.9 [+ or -] 2.4% [alpha]-[actin.sup.pos], P < 0.001). We conclude that [beta]-catenin depletion attenuates postinfarct LV remodeling in part through increased differentiation of [GATA4.sup.pos]/Sca-[1.sup.pos] resident cardiac progenitor cells.

Diabetic cardiomyopathy is characterized by reduced cardiac contractility independent of vascular disease. A contributor to contractile dysfunction in the diabetic heart is impaired sarcoplasmic reticulum function with reduced sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase (SERCA2a) pump activity, leading to disturbed intracellular calcium handling. It is currently unclear whether increasing SERCA2a activity in hearts with existing diabetic cardiomyopathy could still improve calcium flux and contractile performance. To test this hypothesis, we generated a cardiac-specific tetracycline-inducible double transgenic mouse, which allows for doxycycline (DOX)-based inducible SERCA2a expression in which DOX exposure turns on SERCA2a expression. Isolated cardiomyocytes and Langendorff perfused hearts from streptozotocin-induced diabetic mice were studied. Our results show that total SERCA2a protein levels were decreased in the diabetic mice by 60% compared with control. SERCA2a increased above control values in the diabetic mice after DOX. Dysfunctional contractility in the diabetic cardiomyocyte was restored to normal by induction of SERCA2a expression. Calcium transients from diabetic cardiomyocytes showed a delayed rate of diastolic calcium decay of 66%, which was reverted toward normal after SERCA2a expression induced by DOX. Global cardiac function assessed in the diabetic perfused heart showed diminished left ventricular pressure, rate of contraction, and relaxation. These parameters were returned to control values by SERCA2a expression. In conclusion, we have used mice allowing for inducible expression of SERCA2a and could demonstrate that increased expression of SERCA2a leads to improved cardiac function in mice with an already established diabetic cardiomyopathy in absence of detrimental effects. transgenic mice; doxycycline; cardiac myocytes