Signaling In Ventricular Myocytes Research Articles

β-N-Methylamino-L-Alanine (BMAA) Modulates the Sympathetic Regulation and Homeostasis of Polyamines.

The neurotoxin β-N-methylamino-L-alanine (BMAA) is a non-proteinogenic amino acid produced by cyanobacteria. Non-neuronal toxicity of BMAA is poorly studied with a reported increase in reactive oxygen species and a decrease in the antioxidant capacity of liver, kidney, and colorectal adenocarcinoma cells. The aim of this research is to study the toxicity of BMAA (0.1-1 mM) on mitochondria and submitochondrial particles with ATPase activity, on the semicarbazide-sensitive amino oxidases (SSAOs) activity of rat liver, and on an in vitro model containing functionally active excitable tissues-regularly contracting heart muscle preparation with a preserved autonomic innervation. For the first time the BMAA-dependent inhibition of SSAO activity, the elimination of the positive inotropic effect of adrenergic innervation, and the direct and reversible inhibition of adrenaline signaling in ventricular myocytes with 1 mM BMAA were observed. Additionally, it is confirmed that 1 mM BMAA can activate mitochondrial ATPase indirectly. It is concluded that a higher dose of BMAA may influence multiple physiological and pathological processes as it slows down the degradation of biogenic amines, downregulates the sympathetic neuromediation, and embarrasses the cell signaling of adrenergic receptors.

Protein tyrosine phosphatase 1B is a mediator of cyclic ADP ribose-induced Ca2+ signaling in ventricular myocytes

Cyclic ADP-ribose (cADPR) releases Ca2+ from ryanodine receptor (RyR)-sensitive calcium pools in various cell types. In cardiac myocytes, the physiological levels of cADPR transiently increase the amplitude and frequency of Ca2+ (that is, a rapid increase and decrease of calcium within one second) during the cardiac action potential. In this study, we demonstrated that cADPR levels higher than physiological levels induce a slow and gradual increase in the resting intracellular Ca2+ ([Ca2+]i) level over 10 min by inhibiting the sarcoendoplasmic reticulum Ca2+ ATPase (SERCA). Higher cADPR levels mediate the tyrosine-dephosphorylation of α-actin by protein tyrosine phosphatase 1B (PTP1B) present in the endoplasmic reticulum. The tyrosine dephosphorylation of α-actin dissociates phospholamban, the key regulator of SERCA, from α-actin and results in SERCA inhibition. The disruption of the integrity of α-actin by cytochalasin B and the inhibition of α-actin tyrosine dephosphorylation by a PTP1B inhibitor block cADPR-mediated Ca2+ increase. Our results suggest that levels of cADPR that are relatively higher than normal physiological levels modify calcium homeostasis through the dephosphorylation of α-actin by PTB1B and the subsequent inhibition of SERCA in cardiac myocytes.

Enhanced nucleoplasmic Ca2 + signaling in ventricular myocytes from young hypertensive rats

Arterial hypertension causes left ventricular (LV) myocyte hypertrophy. Alterations in nuclear Ca2+ may be involved in regulation of histone acetylation, transcription and hypertrophy. Regulation of nuclear Ca2+ in hypertension, however, is unknown. Therefore, we elucidated cellular mechanisms underlying nuclear Ca2+ regulation in LV myocytes from hypertensive versus normotensive rats and evaluated possible consequences for Ca2+-dependent regulation of histone acetylation. LV myocytes and myocyte nuclei were isolated from young spontaneously hypertensive rats (SHR) shortly after development of hypertension. Normotensive Wistar-Kyoto rats (WKY) served as controls. Cytoplasmic and nucleoplasmic Ca2+ transients (CaTs) were imaged simultaneously using linescan confocal microscopy and Fluo-4. LV myocytes and nuclei from SHR exhibited hypertrophy. Cytoplasmic and nucleoplasmic CaTs were increased in SHR. The increase in nucleoplasmic Ca2+, however, exceeded the increase in cytoplasmic Ca2+, indicating enhanced nuclear Ca2+ signaling in SHR. Ca2+ load of sarcoplasmic reticulum and perinuclear Ca2+ stores was also increased in SHR, while fractional release from both stores remained unchanged. Intranuclear Ca2+ propagation was accelerated in SHR, associated with preserved density of nuclear envelope invaginations and elevated nuclear expression of nucleoporins and SR-Ca2+-ATPase, SERCA2a. Nuclear Ca2+/calmodulin-dependent protein kinase II delta (CaMKIIδ) expression was elevated and histone deacetylases exhibited redistribution from nucleus to cytosol associated with increased histone acetylation in SHR. Thus, in early hypertension, there is remodeling of nuclear Ca2+ handling resulting in enhanced nuclear Ca2+ signaling. Enhanced nuclear Ca2+ signaling, in turn, increases nuclear localization and activity of CaMKIIδ driving nuclear export of histone deacetylases and increased histone acetylation.

Abstract 23: Disruption of TRAF2-TAK1-NF-κB Signaling Axis Triggers K-48 Linked Poly-Ubiquitylation of RIP1 and Necrotic Cell Death in Doxorubicin Cardiotoxicity

The anthracycline doxorubicin (Dox) is a highly effective anti-tumour agent, however, its use is limited by its severe cardiotoxic effects that manifests as heart failure. The decline in cardiac performance induced by doxorubicin remains poorly defined. A critical survival role for the canonical IKKβ -mTOR-NF-κB signaling pathway has been demonstrated in ventricular myocytes. In this report, we demonstrate that, Dox impairs IKKβ-mTOR- NF-κB signaling in ventricular myocytes accompanied by mitochondrial perturbations including mPTP, loss of mitochondrial membrane potential and ROS production. IKKβ- NF-κB signaling involves TRAF 2 mediated ligation of K63- ubiquitin chains to RIP1 (Receptor Interacting Protein 1) which serves as scaffold for recruitment of ubiquitylated Tak1 complexes and phosphorylation-dependent activation of IKKβ -NF-kB signaling. Interestingly, ventricular myocytes treated with dox demonstrated reduction in expression levels of TRAF2 and TAK1, in vivo and in vitro. This was accompanied by a decline in K63- and concomitant increase in K-48 linked polyubiquitination on RIP1, impaired NF-kB activation and necrotic cell death of cardiac myocytes. Interestingly, inhibiting the kinase activity of RIP1 with Necrostatin-1, (Nec1) suppressed necrotic cell injury induced by dox but not NF-kB activation. Concordant with these findings was a marked increase in necrotic cell death in cardiac myocytes defective for IKKB signaling or MEF cells deficient for p65 treated with dox. Notably, mitochondrial perturbations, including PT-pore opening , ROS production, calcium uptake, LDH, Tn(T) and HMGB-1 release and necrotic cell injury induced by dox were completely abrogated by restoring NF-kB signaling in cardiac myocytes or Nec-1. Herein, we provide novel evidence that K-48 linked poly ubiquitylation of RIP1 provides a functional switch that impairs NF-kB activation and signals necrosis in cells treated with dox. Interventions that modulate NF-kB activity may prove beneficial in mitigating the cardiotoxic effects of dox.

Tako-tsubo cardiomyopathy: How to understand possible pathophysiological mechanism and the role of 123I-MIBG imaging

Tako-tsubo cardiomyopathy (TCM) is an increasingly recognized clinical syndrome characterized by acute reversible apical ventricular dysfunction, commonly preceded by exposure to severe physical or emotional stress. In this review, we give a short overview on clinical presentation and treatment of TCM and discuss the possible pathophysiological mechanisms of TCM and the role of various non-invasive imaging modalities in TCM with a focus on the potential role of 123I-meta-iodobenzylguanidine (MIBG) scintigraphy. Currently, the dominating hypothesis on the pathophysiology of TCM postulates that high levels of the neurotransmitter epinephrine may trigger a change in intracellular signaling in ventricular myocytes. More specific, epinephrine stimulates G-protein coupled β2 adenoreceptors (β2AR) which are located on ventricular myocytes. Normal levels of this neurotransmitter predominantly stimulate the intracellular G-protein, and induce a positive inotropic effect. However, with significant increasing levels of epinephrine, the predominance of stimulation is shifted from G-stimulating to the G-inhibitor protein coupling, which leads to a negative inotropic effect. Interestingly, this negative inotropic effect is the largest in the apical myocardium where the β2AR:β1AR ratio is the highest within the heart. Echocardiography and ventriculography are essential to diagnose TCM, but new imaging tools are promising to diagnose TCM and to evaluate therapeutic efficacy. Cardiovascular magnetic resonance can be used to differentiate TCM from other myocardial diseases, such as myocarditis. 123I-meta-iodobenzylguanidine (123I-MIBG) scintigraphy can be used to assess ventricular adrenergic activity and may guide optimization of individual (pharmacological) therapy. These new insights into the possible pathophysiological mechanisms and novel diagnostic imaging modalities can be used as starting point for the development of international guidelines of TCM which may increase the awareness, and optimize the treatment of TCM.

Stretch-Dependent Regulation of Calcium Signaling in Heart - Who are the Key Players?

An acute physiologic stretch of a cardiomyocyte triggers an increase in the production of reactive oxygen species (ROS) by the membrane-localized enzyme complex NADPH oxidase 2 (Nox2, X-ROS signaling). The ROS act locally to sensitize nearby Ca2+ release channels in the sarcoplasmic reticulum, the ryanodine receptors type 2 (RyR2), resulting in a brief increase in the frequency of calcium sparks. During sustained, cyclical stretch the rate of ROS production remains elevated and is graded by both the degree and frequency of cyclic stretch. The elevated ROS production results in a sustained increase of calcium spark rate, thus coupling the mechanical load on the heart cell to its calcium signaling sensitivity. However, an increase in RyR2 sensitivity alone is insufficient to explain a persistent increase in Ca2+ sparks, implicating additional stretch-dependent players in the sustained elevation of calcium signaling sensitivity with increased mechanical stress. We initially investigated three potential players based on their previous links to mechanical stress and modulation by ROS signaling. Here we report on the contribution of nitric oxide (NO), Ca2+/Calmodulin-dependent kinase II (CaMKII), and mechano-sensitive channels (MSC) on stretch-dependent regulation of calcium signaling in ventricular myocytes. We find that both NO and CaMKII have little to no effect on the rapid acute increase in the Ca2+ spark rate with stretch (≤10s), but each contribute significantly to maintaining elevated calcium signaling sensitivity with prolonged cyclic stretch (≥1min). We also explore the role of ROS signaling in the stretch-dependent activation of these signaling pathways, and how this affects both diastolic calcium sparks as well as systolic calcium transients and contractility in electrically paced myocytes.

Subcellular Origin and Tissue-Wide Synchronization of Abnormal Ca Release in the Genesis of Ca-Dependent Atrial Arrhythmia

Introduction. Abnormal diastolic Ca release (DCR) from the sarcoplasmic reticulum has been implicated in both ventricular as well as atrial fibrillation (AF). Atrial cells lack the extensive T-tubule network that facilitates Ca signaling in ventricular myocytes. How the distinct structural organization of atrial cells affects the genesis of AF is currently unknown.Methods and Results. We used rapid 2D confocal imaging to map Ca changes in atrial single myocytes and tissue preparations derived from CASQ2 knock-out (CASQ2.KO) mice manifesting Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). Isolated CASQ2.KO atrial cells showed frequent local and cell-wide DCR events when exposed to isoproterenol. DCR predominantly originated nearly simultaneously at several “eager” sites localized at cell periphery. The distribution of latencies to local DCR events in CASQ2.KO cells displayed a left-ward shift consistent with abbreviated RyR2 refractoriness associated with CPVT. This abbreviated RyR2 refractoriness translated into highly synchronous spontaneous Ca oscillations across the intact atrial tissue. Conditional overexpression of SERCA2a in CASQ.KO mice exacerbated proarrhythmic Ca oscillations by increasing the proportion of interior eager release sites and enhancing DCR synchronicity in both isolated atrial cells and atrial tissue preparations.Conclusions. Our results suggest that aberrant DCR involves a certain set of eager Ca release sites that 1) only in part coincide with sites involved in normal EC coupling at cell periphery and 2) act as pacemakers for spontaneous Ca oscillations present in atrial cells. Due to abbreviated release site refractoriness, DCR is synchronized between neighboring cells thereby leading to tissue-wide Ca oscillations that may form basis for AF. SERCA2a overexpression exacerbates arrhythmic Ca oscillations by further enhancing DCR synchronicity.

Abstract 19146: Disruption of TRAF2-TAK1-IKKB Signaling Triggers Mitochondrial Perturbations and Necrotic Cell Death in Doxorubicin Cardiotoxicity

The anthracycline doxorubicin (dox) is a highly effective anti-tumour agent, however, its use is limited by its severe cardiotoxic effects that manifests as heart failure. The decline in cardiac performance induced by doxorubicin remains poorly defined. A critical survival role for the canonical IKKβ -NF-κB signaling pathway has been demonstrated in ventricular myocytes. In this report, we demonstrate that, dox impairs IKKβ- NF-κB signaling in ventricular myocytes accompanied by mitochondrial perturbations including mPTP, loss of membrane potential and ROS production. IKKβ- NF-κB signaling involves TRAF 2 mediated ligation of K63 ubiquitin chains to RIP1 (Receptor Interacting Protein 1) which serves as scaffold for recruitment of ubiquitylated Tak1 complex and phosphorylation-dependent activation of IKKβ -NF-kB signaling. Interestingly, ventricular myocytes treated with dox demonstrated reduction in expression levels of TRAF2 and TAK1. This was accompanied by a decline in K63- ubiquitylation chains and concomitant increase in K-48 polyubiquitination of RIP1, impaired NF-kB activation and necrotic cell death. Interestingly, inhibiting the kinase activity of RIP1 with Necrostatin-1, (Nec1) suppressed necrotic cell injury induced by dox but not NF-kB activation. Concordant with these findings was a marked increase in necrotic cell death in cardiac myocytes defective for IKKB signaling or MEF cells deficient for p65 treated with dox. Notably, mitochondrial perturbations, including PT-pore opening , ROS production, calcium uptake, LDH, Tn(T) and HMGB-1 release and necrotic cell injury induced by dox were completely abrogated by restoring IKKB-NF-kB signaling in cardiac myocytes or Nec-1. Herein, we provide novel evidence that K-48 ubiquitylation of RIP1 by doxorubicin provides a functional switch that signals necrotic cell death in cells defective for IKKβ-NF-kB signaling. Hence, interventions that modulate IKKB- NF-kB activation may prove beneficial in mitigating the cardiotoxic effects of dox.

Functional Cross-Talk Between Aldosterone and Angiotensin-(1-7) in Ventricular Myocytes

High serum levels of aldosterone have been linked to the development of cardiac disease. In contrast, angiotensin (Ang)-(1-7) was extensively shown to possess cardioprotective effects, including the attenuation of cardiac dysfunction induced by excessive mineralocorticoid activation in vivo, suggesting possible interactions between these 2 molecules. Here, we investigated whether there is cross-talk between aldosterone and Ang-(1-7) and its functional consequences for calcium (Ca(2+)) signaling in ventricular myocytes. Short-term effects of aldosterone on Ca(2+) transient were assessed in Fluo-4/AM-loaded myocytes. Confocal images showed that Ang-(1-7) had no effect on Ca(2+) transient parameters, whereas aldosterone increased the magnitude of the Ca(2+) transient. Quite unexpectedly, addition of Ang-(1-7) to aldosterone-treated myocytes further enhanced the amplitude of the Ca(2+) transient suggesting a synergistic effect of these molecules. Aldosterone action on Ca(2+) transient amplitude was mediated by protein kinase A, and was related to an increase in Ca(2+) current (I(Ca)) density. Both changes were not altered by Ang-(1-7). When cardiomyocytes were exposed to aldosterone, increased Ca(2+) spark rate was measured. Ang-(1-7) prevented this change. In addition, a NO synthase inhibitor restored the effect of aldosterone on Ca(2+) spark rate in Ang-(1-7)-treated myocytes and attenuated the synergistic effect of these 2 molecules on Ca(2+) transient. These results indicate that NO plays an important role in this cross-talk. Our results bring new perspectives in the understanding of how 2 prominent molecules with supposedly antagonist cardiac actions cross-talk to synergistically amplify Ca(2+) signals in cardiomyocytes.

Influence of pH on Ca2+ current and its control of electrical and Ca2+ signaling in ventricular myocytes

Modulation of L-type Ca2+ current (ICa,L) by H+ ions in cardiac myocytes is controversial, with widely discrepant responses reported. The pH sensitivity of ICa,L was investigated (whole cell voltage clamp) while measuring intracellular Ca2+ (Ca2+i) or pHi (epifluorescence microscopy) in rabbit and guinea pig ventricular myocytes. Selectively reducing extracellular or intracellular pH (pHo 6.5 and pHi 6.7) had opposite effects on ICa,L gating, shifting the steady-state activation and inactivation curves to the right and left, respectively, along the voltage axis. At low pHo, this decreased ICa,L, whereas at low pHi, it increased ICa,L at clamp potentials negative to 0 mV, although the current decreased at more positive potentials. When Ca2+i was buffered with BAPTA, the stimulatory effect of low pHi was even more marked, with essentially no inhibition. We conclude that extracellular H+ ions inhibit whereas intracellular H+ ions can stimulate ICa,L. Low pHi and pHo effects on ICa,L were additive, tending to cancel when appropriately combined. They persisted after inhibition of calmodulin kinase II (with KN-93). Effects are consistent with H+ ion screening of fixed negative charge at the sarcolemma, with additional channel block by H+o and Ca2+i. Action potential duration (APD) was also strongly H+ sensitive, being shortened by low pHo, but lengthened by low pHi, caused mainly by H+-induced changes in late Ca2+ entry through the L-type Ca2+ channel. Kinetic analyses of pH-sensitive channel gating, when combined with whole cell modeling, successfully predicted the APD changes, plus many of the accompanying changes in Ca2+ signaling. We conclude that the pHi-versus-pHo control of ICa,L will exert a major influence on electrical and Ca2+-dependent signaling during acid–base disturbances in the heart.

Inositol 1,4,5-trisphosphate supports the arrhythmogenic action of endothelin-1 on ventricular cardiac myocytes

Although ventricular cardiomyocytes express inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptors, it is unclear how these Ca2+ channels contribute to the effects of Gq-coupled agonists. Endothelin-1 augmented the amplitude of pacing-evoked Ca2+ signals (positive inotropy), and caused an increasing frequency of spontaneous diastolic Ca2+-release transients. Both effects of endothelin-1 were blocked by an antagonist of phospholipase C, suggesting that Ins(1,4,5)P3 and/or diacylglycerol production was necessary. The endothelin-1-mediated spontaneous Ca2+ transients were abolished by application of 2-aminoethoxydiphenyl borate (2-APB), an antagonist of Ins(1,4,5)P3 receptors. Incubation of electrically-paced ventricular myocytes with a membrane-permeant Ins(1,4,5)P3 ester provoked the occurrence of spontaneous diastolic Ca2+ transients with the same characteristics and sensitivity to 2-APB as the events stimulated by endothelin-1. In addition to evoking spontaneous Ca2+ transients, stimulation of ventricular myocytes with the Ins(1,4,5)P3 ester caused a positive inotropic effect. The effects of endothelin-1 were compared with two other stimuli, isoproterenol and digoxin, which are known to induce inotropy and spontaneous Ca2+ transients by overloading intracellular Ca2+ stores. The events evoked by isoproterenol and digoxin were dissimilar from those triggered by endothelin-1 in several ways. We propose that Ins(1,4,5)P3 receptors support the development of both inotropy and spontaneous pro-arrhythmic Ca2+ signals in ventricular myocytes stimulated with a Gq-coupled agonist.

Tyrosine kinases inhibitors block Fas-mediated deleterious effects in normoxic and hypoxic ventricular myocytes

Experimental evidences suggest an important role for Fas receptor activation in a wide range of myocardial pathologies, which are associated with a variety of arrhythmias and mechanical dysfunction. Our recent studies have shown that Fas-mediated arrhythmias and mechanical disturbances of ventricular myocytes can be accounted for by activation of the phospholipase C–1,4,5-inositol triphosphate–intracellular calcium release (PLC–1,4,5–IP 3–[Ca 2+] i) cascade, which is responsible for attenuating the transient outward current ( I to) and augmenting the L-type Ca 2+ current ( I Ca,L). We have also shown that whereas ventricular myocytes are resistant to Fas-mediated apoptosis, hypoxia predisposes myocytes to apoptosis induced by Fas activation by shifting the balance between pro-apoptotic and anti-apoptotic proteins towards the former. Since protein tyrosine phosphorylation has been shown to be involved in Fas signaling, we have hypothesized that inhibiting tyrosine kinases will attenuate Fas-mediated effects in ventricular myocytes in normoxic and hypoxic conditions. Therefore, we tested the effect of the tyrosine kinases inhibitors, genistein (50 μmol/l) and herbimycin A (50 μg/ml), on the abovementioned Fas-mediated effects in cultured neonatal rat ventricular myocytes (NRVM) and in freshly isolated adult murine ventricular myocytes. Fas receptor was activated by incubating NRVM with recombinant Fas ligand (rFasL, 50 ng/ml) and enhancing antibody (1 μg/ml), or by incubation of murine ventricular myocytes with the Fas-activating antibody Jo2 (10 μg/ml). The major findings were that genistein prevented Fas-mediated increase in 1,4,5-IP 3 production in NRVM (quantified by ion-exchange chromatography): 216 ± 41 counts per minute (cpm) in control, 605 ± 184 cpm in FasL-treated cardiomyocytes and 137 ± 51 cpm in rFasL + genistein-treated cultures. Accordingly, genistein or herbimycin A abolished the diastolic [Ca 2+] i-rise (as measured by fura-2 fluorescence) and arrhythmogenic activity in both rat and murine ventricular myocytes, and the Fas-mediated changes in I to and I Ca,L in murine ventricular myocytes. Importantly, genistein attenuated Fas-mediated apoptosis in hypoxic (22 h in 1% O 2) NRVM; the apoptotic ratios as measured by DAPI fluorescence assay were: 4.6 ± 1.0% in control, 12.5 ± 3.0% in rFasL and 7.3 ± 1.6% in rFasL + genistein-treated NRVM. This prevention of apoptosis by tyrosine kinases blockade was accompanied by inhibition of hypoxia-induced increased Fas expression and decreased expression of the anti-apoptotic protein xIAP. In conclusion, our findings indicate that tyrosine phosphorylation is involved in Fas signaling in ventricular myocytes, and can, therefore, serve as a novel potential target for attenuating Fas-mediated dysfunction in normoxic and hypoxic myocardium.

Developmental changes in beta2-adrenergic receptor signaling in ventricular myocytes: the role of Gi proteins and caveolae microdomains.

Cardiomyocyte beta2-adrenergic receptors (beta-ARs) provide a source of inotropic support and influence the evolution of heart failure. Recent studies identify distinct mechanisms for beta2-AR actions in neonatal and adult rat cardiomyocytes. This study examines whether ontogenic changes in cardiac beta2-AR actions can be attributed to altered Gi expression or changes in the spatial organization of the beta2-AR complex in membrane subdomains (caveolae). We show that beta2-ARs increase cAMP, calcium, and contractile amplitude in a pertussis toxin (PTX)-insensitive manner in neonatal cardiomyocytes. This is not caused by lack of Gi; Galphai expression is higher in neonatal cardiomyocytes than in those of adult rats. beta2-ARs provide inotropic support without detectably increasing cAMP, in adult cardiomyocytes. This cannot be attributed to dual coupling of beta2-ARs to Gs and Gi, because beta2-ARs do not promote cAMP accumulation in PTX-pretreated adult cardiomyocytes. Spatial segregation of beta2-ARs, Galphas/Galphai, and adenylyl cyclase to distinct membrane subdomains also is not a factor, because all of these proteins copurify in caveolin-3-enriched vesicles isolated from adult cardiomyocytes. However, these studies demonstrate that enzyme-based protocols routinely used to isolate ventricular cardiomyocytes lead to proteolysis of beta-ARs. The functional consequences of this limited beta-AR proteolysis is uncertain, because truncated beta1-ARs promote cAMP accumulation and truncated beta2-ARs provide inotropic support in adult cardiomyocytes. Collectively, these studies indicate that components of the beta2-AR signaling complex compartmentalize to restricted membrane subdomains in adult rat cardiomyocytes. Neither compartmentalization nor changes in Gi expression fully explain the ontogenic changes in beta2-AR responsiveness in the rat ventricle.

Ca(2+) signaling in cardiac myocytes overexpressing the alpha(1) subunit of L-type Ca(2+) channel.

Voltage-gated L-type Ca(2+) channels (LCCs) provide Ca(2+) ingress into cardiac myocytes and play a key role in intracellular Ca(2+) homeostasis and excitation-contraction coupling. We investigated the effects of a constitutive increase of LCC density on Ca(2+) signaling in ventricular myocytes from 4-month-old transgenic (Tg) mice overexpressing the alpha(1) subunit of LCC in the heart. At this age, cells were somewhat hypertrophic as reflected by a 20% increase in cell capacitance relative to those from nontransgenic (Ntg) littermates. Whole cell I(Ca) density in Tg myocytes was elevated by 48% at 0 mV compared with the Ntg group. Single-channel analysis detected an increase in LCC density with similar conductance and gating properties. Although the overexpressed LCCs triggered an augmented SR Ca(2+) release, the "gain" function of EC coupling was uncompromised, and SR Ca(2+) content, diastolic cytosolic Ca(2+), and unitary properties of Ca(2+) sparks were unchanged. Importantly, the enhanced I(Ca) entry and SR Ca(2+) release were associated with an upregulation of the Na(+)-Ca(2+) exchange activity (indexed by the half decay time of caffeine-elicited Ca(2+) transient) by 27% and SR Ca(2+) recycling by approximately 35%. Western analysis detected a 53% increase in the Na(+)-Ca(2+) exchanger expression but no change in the abundance of ryanodine receptor (RyR), SERCA2, and phospholamban. Analysis of I(Ca) kinetics suggested that SR Ca(2+) release-dependent inactivation of LCCs remains intact in Tg cells. Thus, in spite of the modest cardiac hypertrophy, the overexpressed LCCs form functional coupling with RyRs, preserving both orthograde and retrograde Ca(2+) signaling between LCCs and RyRs. These results also suggest that a modest but sustained increase in Ca(2+) influx triggers a coordinated remodeling of Ca(2+) handling to maintain Ca(2+) homeostasis.

Characterization of G-protein Signaling in Ventricular Myocytes From the Adult Mouse Heart: Differences From the Rat

We have developed a high yield technique for isolating ventricular myocytes from adult mouse hearts. This collagenase-trypsin procedure yields 3–6×106cells/heart. The cells are rod-shaped, roughly 20μM ×100 μM and Ca++tolerant, with viability of 65–80%. Binding studies with [125I]ICYP demonstrate the presence of β -adrenergic receptors at a density of 83 fmol/mg membrane protein. Assessment of the effects of the β1-specific antagonist CGP 20712A on [125I]ICYP binding and on isoproterenol (ISO)-sensitive adenylyl cyclase activity indicates that 67% of the receptors are β1and 33% are β2, compared to 16–20%β2in rat myocytes. Mouse myocytes respond to isoproterenol to produce cyclic AMP with an EC50≈110±20 n M. A functional Gipathway is demonstrated by inhibition of ISO-stimulated cyclic AMP accumulation by endothelin, carbachol and ATP and by sensitivity of this inhibition to pertussis toxin. As assessed by inositol phosphate production, endothelin and ATP stimulate the activity of the Gq-phospholipase C pathway, whereas carbachol, PGF2 αand α1-adrenergic receptor agonists show no significant effect. The inability of α1-adrenergic receptor agonists to induce phosphoinositide hydrolysis in mouse myocytes differs from a several fold α1-adrenergic activation that occurs in rat. Biochemical and pharmacological profiles, as well as the need for modifications in experimental design, indicate that mouse myocytes differ substantially from rat cardiac myocytes.

Outside-in Signalling of Fibronectin Stimulates Cardiomyocyte Hypertrophy in Cultured Neonatal Rat Ventricular Myocytes

Cardiac hypertrophy involves the accumulation of extracellular matrix proteins, such as fibronectin, leading to increasing myocardial stiffness, ventricular dysfunction and heart failure. To better understand the possible role of extracellular matrix-evoked intracellular signalling in ventricular myocytes, we investigated the effect of fibronectin on myocyte hypertrophic responses using cell culture models. Cell size in myocytes cultured on fibronectin-coated dishes was three times larger than that grown on non-coated dishes. However, the number of cells on fibronectin-coated dishes was not changed throughout the experiment. Protein synthesis was significantly increased by fibronectin, as were synthesis of atrial and brain natriuretic peptides. Fibronectin also elicited actin reorganization, co-localization of β 1 integrin and vinculin, formation of focal adhesions and tyrosine phosphorylation of focal adhesion kinase in myocytes. These fibronectin-mediated effects were inhibited in a dose-dependent manner by GRGDSP, a competitive antagonist of the fibronectin receptors; GRGDSP had no effect on cell number or viability. Blocking antibody for β 1 and β 3 integrin significantly suppressed fibronectin-induced secretion of natriuretic peptides. Myocyte hypertrophy was observed in myocyte-nonmyocyte co-culture that reflects more closely the myocyte environment in vivo. GRGDSP may also suppress the myocyte hypertrophic response in the co-culture. These findings demonstrate that the interaction of fibronectin and RGD-dependent integrins is involved in the hypertrophic responses of myocyte in vitro, and suggest that extracellular matrix proteins such as fibronectin are not merely passive adhesive molecules but are active participants in processes leading to myocyte hypertrophy.