Contractility In Ventricular Myocytes Research Articles

Irx5 and transient outward K+ currents contribute to transmural contractile heterogeneities in the mouse ventricle.

Previous studies have established that transmural gradients of the fast transient outward K+ current (Ito,f) correlate with regional differences in action potential (AP) profile and excitation-contraction coupling (ECC) with high Ito,f expression in the epimyocardium (EPI) being associated with short APs and low contractility and vice versa. Herein, we investigated the effects of altering the Ito,f gradients on transmural contractile properties using mice lacking Irx5 (Irx5-KO) or lacking Kcnd2 (KV4.2-KO) or both. Irx5-KO mice exhibited decreased global LV contractility in association with elevated Ito,f, as well as reduced cell shortening and Ca2+ transient amplitudes in cardiomyocytes isolated from the endomyocardium (ENDO) but not in cardiomyocytes from the EPI. Transcriptional profiling revealed that the primary effect of Irx5 ablation on ECC-related genes was to increase Ito,f gene expression (i.e., Kcnd2 and Kcnip2) in the ENDO, but not the EPI. By contrast, KV4.2-KO mice showed selective increases in cell shortening and Ca2+ transients in isolated EPI cardiomyocytes, leading to enhanced ventricular contractility and mice lacking both Irx5 and Kcnd2 displayed elevated ventricular contractility, comparable to KV4.2-KO mice, demonstrating a dominant role of Irx5-dependent modulation of Ito,f in the regulation of contractility. Our findings show that the transmural electromechanical heterogeneities in the healthy ventricles depend on the Irx5-dependent Ito,f gradients. These observations provide a useful framework for assessing the molecular mechanisms underlying the alterations in contractile heterogeneity seen in the diseased heart.NEW & NOTEWORTHY Irx5 is a vital transcription factor that establishes the transmural heterogeneity of ventricular myocyte contractility, thereby ensuring proper contractile function in the healthy heart. Regional differences in excitation-contraction coupling in the ventricular myocardium are primarily mediated through the inverse relationship between Irx5 and the fast transient outward K+ current (Ito,f) across the ventricular wall.

Ophiopogonin D alleviates diabetic myocardial injuries by regulating mitochondrial dynamics

Ethnopharmacological relevanceOphiopogonin D (OP-D) is a steroidal saponin extracted from Ophiopogon japonicus (Thunb.) Ker Gawl. (Liliaceae), that has been traditionally used to treat cough, sputum, and thirst in some Asian countries. Recently, various pharmacological roles of OP-D have been identified, including anti-inflammatory, cardioprotective, and anti-cancer effects. However, whether OP-D can prevent diabetic myocardial injury remains unknown. Aim of the studyIn this study, we aimed to observe the effects of OP-D on the diabetic myocardium. Materials and methodsLeptin receptor-deficient db/db mice were used as an animal model for type 2 diabetes. The effects of OP-D on blood glucose, blood lipids, myocardial ultrastructure, and mitochondrial function in mice were observed after four weeks of intragastric administration. Palmitic acid was used to stimulate cardiomyocytes to establish a myocardial lipotoxicity model. Cell apoptosis, mitochondrial morphology, and function were observed. ResultsBlood glucose and blood lipid levels were significantly increased in db/db mice, accompanied by myocardial mitochondrial injury and dysfunction. OP-D treatment reduced blood lipid levels in db/db mice and relieved mitochondrial injury and dysfunction. OP-D inhibited palmitic acid induced-mitochondrial fission and dysfunction, reduced endogenous apoptosis, and improved cell survival rate in H9C2 cardiomyocytes. Both in vivo and in vitro models showed increased phosphorylation of DRP1 at Ser-616, reduced phosphorylation of DRP1 at Ser-637, and reduced expression of fusion proteins MFN1/2 and OPA1. Meanwhile, immunofluorescence co-localization analysis revealed that palmitic acid stimulated the translocation of DRP1 protein from the cytoplasm to the mitochondria in H9C2 cardiomyocytes. The imbalance of mitochondrial dynamics, protein expression, and translocation of DRP1 were effectively reversed by OP-D treatment. In isolated mice ventricular myocytes, palmitic acid enhanced cytoplasmic Ca2+ levels and suppressed contractility in ventricular myocytes, accompanied by activation of calcineurin, a key regulator of DRP1 dephosphorylation at Ser-637. OP-D reversed the changes caused by palmitic acid. ConclusionsOur findings indicate that OP-D intervention could alleviate lipid accumulation and mitochondrial injury in diabetic mouse hearts and palmitic acid-stimulated cardiomyocytes. The cardioprotective effect of OP-D may be mediated by the regulation of mitochondrial dynamics.

P1593VEGFR1-PLC signaling is implicated in normal spontaneous firing of cardiac pacemaker cells

Abstract Spontaneous firing of sinoatrial node cells (SANC) is regulated by sarcoplasmic reticulum (SR) generated local subsarcolemmal calcium releases (LCRs). LCRs appear during diastolic depolarization (DD) and activate an inward sodium-calcium exchange current to accelerate the DD rate and thus spontaneous SANC firing. Vascular endothelial growth factor (VEGF) receptors VEGFR1 and VEGFR2 activate PLC, and suppression of VEGFR-PLC signaling decreases calcium transients and contractility in ventricular myocytes. We tested the idea that VEGFR-PLC signaling may contribute to normal spontaneous beating of SANC. We observed that expression of VEGFR1 (assessed by RT-qPCR) in rabbit sinoatrial node was comparable to that of β1-adrenergic receptors, but less than that in ventricle. The pan VEGFR1/2/3 inhibitor PTK787/ZK222584 (10 μmol/L) in a time-dependent manner: (1) suppressed spontaneous SANC beating rate by ∼50% (perforated patch-clamp technique); (2) markedly decreased the LCR size and number per each spontaneous cycle (confocal microscopy, calcium indicator Fluo-3) and prolonged the LCR period (the interval between action potential-induced calcium transient and occurrence of subsequent LCR). The PTK787/ZK222584-induced increase in the LCR period (from 348.2±30.3 to 619.9±103.4 msec; P<0.05) predicted the concomitant increase in the spontaneous cycle length (from 405.6±31.1 to 702.1±105.1 msec; P<0.05), suggesting that calcium cycling could be a major target of VEGFR-dependent regulation of SANC firing. All effects of PTK787/ZK222584 were reversible upon washout. To elucidate whether signaling of VEGFR1 or VEGFR2 regulated spontaneous SANC firing, we employed a selective VEGFR2 inhibitor ZM-323881 (5 μmol/L), which suppressed spontaneous beating rate in only 2 of 10 SANC. These results indicate that VEGFR1, but not VEGFR2, is likely a key receptor that modulates automaticity in majority of SANC. To clarify downstream targets from VEGFR1 we employed PLC inhibitor U-73122, which decreased the LCR size, number and prolonged the LCR period. The inactive analog U-73343 was without effect. Because LCRs are critically dependent upon the SR calcium load, supplied by L-type calcium current (ICa,L), we examined effects of U-73122 on ICa,L. U-73122, but not U-73343, markedly suppressed ICa,L amplitude by ∼50%, leading to a decrease in the calcium influx and, as a result, to decrease in the LCR parameters, prolongation of the LCR period and spontaneous SANC cycle length. Thus, basal VEGFR1 signaling activates PLC, which modulates intracellular SR calcium cycling and LCR characteristics in SANC. We conclude that VEGFR1-PLC is a novel mechanism involved in the regulation of normal automaticity of cardiac pacemaker cells. Acknowledgement/Funding Intramural Research Program, National Institute on Aging, NIH

Contractile heterogeneity in ventricular myocardium.

The transmural heterogeneity of the contractility in ventricular muscle has not been well-studied. Here, we investigated the calcium transient and sarcomere contraction/relaxation in the endocardial (Endo) and epicardial (Epi) myocytes. Endo and Epi myocytes were isolated from C57/BL6 mice by Langendorff perfusion. Ca2+ transient and sarcomere contraction/relaxation were recorded simultaneously at different stimulation frequencies using a dual excitation fluorescence photomultiplier system. We found that the Endo myocytes have higher baseline diastolic calcium, significantly larger calcium transient and stronger sarcomere shortening than Epi myocytes. However, both the rising and decline phases for calcium transient and sarcomere shortening were slower in Endo than in Epi myocytes. When simulation frequency was increased from 1 to 3 Hz, a greater percent increase in the diastole calcium level, Ca2+ transient and sarcomere shortening amplitude has been observed in the Endo myocytes. Accordingly, the frequency-dependent acceleration in the decay rate of calcium transient and sarcomere relaxation was more profound in the Endo than in Epi myocytes. Western blot analysis showed that CaMKII activity was significantly higher in Epi than in Endo myocardium before stimulation. However, this transmural heterogeneity was reversed by rapid pacing. CaMKII inhibition by KN93 diminished the frequency-dependent alterations of Ca2+ transient and sarcomere contraction. Our results suggest that the contractility of ventricular myocytes is heterogeneous. The Endo-myocardium is the major force generating layer in the heart, both at slow and fast heart rate, and the transmural heterogeneity of CaMKII activation plays an important role in the frequency-dependent alterations.

Effects of prasugrel on membrane potential and contractile activity of rat ventricular myocytes.

Though prasugrel is one of the important P2Y12 inhibitors currently in use for antiplatelet therapy, its potential effects on contractility and electrical activity of ventricular myocytes have not yet been investigated. Hence this study was designed to study the impact of prasugrel on contractile function and membrane potential of isolated ventricular myocytes. Freshly isolated rat ventricular myocytes were used in this study. Myocyte contraction was measured during electrical stimulation of cardiomyocytes and the action potential (AP) recordings were obtained with current clamp mode of the patch-clamp amplifier. AP duration and fractional shortening of ventricular myocytes did not show any change with the administration of 1μM of prasugrel. However, remarkable depolarization of resting membrane potential followed by apparent fibrillation episodes was detected in the cardiomyocytes. Similar events were observed in the contractile activity of myocytes during field stimulation. Also, a higher concentration of prasugrel (10μM) elicited repeated fibrillations, which disappeared after washout or nitric oxide synthase (NOS) inhibition with L-NAME. In contrast, the same concentration of ticagrelor, another P2Y12 inhibitor did not induce fibrillation events though it decreased the contractility of ventricular myocytes significantly. The perfusion of ventricular myocytes with L-NAME did not alter the negative inotropic effect of ticagrelor. Prasugrel, a widely used antithrombotic agent, may induce depolarization in the membrane potential of myocytes as well as fibrillation via NO mediated pathway.

Calcitriol (1,25-dihydroxyvitamin D3) increases L-type calcium current via protein kinase A signaling and modulates calcium cycling and contractility in isolated mouse ventricular myocytes

Calcitriol, the bioactive metabolite of vitamin D, exerts its effects through interaction with the nuclear vitamin D receptor (VDR) to induce genomic responses. Calcitriol may also induce rapid responses via plasma membrane-associated VDR, involving the activation of second messengers and modulation of voltage-dependent channels. VDR is expressed in cardiomyocytes, but the molecular and cellular mechanisms involved in the rapid responses of calcitriol in the heart are poorly understood. The aim of the present study was to analyze the rapid nongenomic effect of calcitriol on L-type calcium channels, intracellular Ca2+ ([Ca2+]i) transients, and cell contractility in ventricular myocytes. We used the whole-cell patch-clamp technique to record L-type calcium current (ICaL) and confocal microscopy to study global [Ca2+]i transients evoked by electrical stimulation and cell shortening in adult mouse ventricular myocytes treated with vehicle or with calcitriol. In some experiments, ICaL was recorded using the perforated patch-clamp technique. Calcitriol treatment of cardiomyocytes induced a concentration-dependent increase in ICaL density (Half maximal effective concentration (EC50) = 0.23 nM) and a significant increase in peak [Ca2+]i transients and cell contraction. The effect of calcitriol on ICaL was prevented by pretreatment of cardiomyocytes with the protein kinase A (PKA) inhibitor KT-5720 but not with the β-adrenergic blocker propranolol. The effect of calcitriol on ICaL was absent in myocytes isolated from VDR knockout mice. Calcitriol induces a rapid response in mouse ventricular myocytes that involves a VDR-PKA-dependent increase in ICaL density, enhancing [Ca2+]i transients and contraction.

Enhancement of contraction and L-type Ca2+ current by murrayafoline-A via protein kinase C in rat ventricular myocytes

We previously reported that murrayafoline-A (1-methoxy-3-methyl-9H-carbazole, Mu-A) increases the contractility of ventricular myocytes, in part, via enhancing Ca2+ influx through L-type Ca2+ channels, and that it increases the Ca2+ transients by activation of protein kinase C (PKC). In the present study, we further examined the cellular mechanisms for the enhancement of contractility and L-type Ca2+ current (ICa,L) by Mu-A. Cell shortening and ICa,L were measured in rat ventricular myocytes using a video edge detection method and perforated patch-clamp technique, respectively. We found that the positive inotropic effect of Mu-A was not affected by pre-exposure to the β-adrenoceptor antagonist propranolol, the protein kinase A (PKA) inhibitors KT5720 or H-89, or the phospholipase C inhibitor U73122. Interestingly, the Mu-A-mediated increases in cell shortening and in the rate of contraction were completely suppressed by pre-treatment with the PKC inhibitor GF109203X. The stimulatory effect of Mu-A on ICa,L was not altered by inhibition of PKA (KT5720), G-protein coupled receptors (suramin), or α1-adrenoceptor (prazosin). However, pre-exposure to the PKC inhibitor, GF109203X or chelerythrine, abolished the Mu-A-induced increase in ICa,L. Pre-exposure to the Ca2+-calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 slightly reduced the stimulatory effects on contraction and ICa,L by Mu-A. Phosphorylation of PKC was enhanced by Mu-A in ventricular myocytes. These data suggest that Mu-A increases contraction and ICa,L via PKC in rat ventricular myocytes, and that the PKC-mediated responses in the presence of Mu-A may be partly mediated by CaMKII.

Protein kinaseC and Ca(2+) -calmodulin-dependent protein kinaseII mediate the enlarged reverse INCX induced by ouabain-increased late sodium current in rabbit ventricular myocytes.

What is the central question of this study? What are the effects of protein kinaseC (PKC) and Ca(2+) -calmodulin-dependent protein kinaseII (CaMKII) on late sodium current (INaL ), reverse Na(+) -Ca(2+) exchange current (reverse INCX ) or intracellular Ca(2+) levels changed by ouabain? What is the main finding and its importance? Ouabain, even at low concentrations (0.5-8.0μm), can increase INaL and reverse INCX , and these effects may contribute to the effect of the glycoside to increase Ca(2+) transients and contractility. Both PKC and CaMKII activities may mediate or modulate these processes. It has been reported that the cardiac glycoside ouabain can increase the late sodium current (INaL ), as well as the diastolic intracellular calcium concentration and contractile shortening. Whether an increase of INaL participates in a pathway that can mediate the positive inotropic response to ouabain is unknown. We therefore determined the effects of ouabain on INaL , reverse Na(+) -Ca(2+) exchange current (reverse INCX ), intracellular Ca(2+) ([Ca(2+) ]i ) levels and contractile shortening in rabbit isolated ventricular myocytes. Ouabain (0.1-8μm) markedly increased INaL and reverse INCX in a concentration-dependent manner, with significant effects at concentrations as low as 0.5 and 1μm. These effects of ouabain were suppressed by the INaL inhibitors TTX and ranolazine, the protein kinaseC inhibitor bisindolylmaleimide and the Ca(2+) -calmodulin-dependent protein kinaseII inhibitor KN-93. The enhancement by 0.5μm ouabain of ventricular myocyte contractility and intracellular Ca(2+) transients was suppressed by 2.0μm TTX. We conclude that ouabain, even at low concentrations (0.5-8.0μm), can increase INaL and reverse INCX , and these effects may contribute to the effect of the glycoside to increase Ca(2+) transients and contractility. Both protein kinaseC and Ca(2+) -calmodulin-dependent protein kinaseII activities may mediate or modulate these processes.

GW25-e0061 Up-regulation of neuronal nitric oxide synthase modulates myofilament Ca2+ sensitivity and controls left ventricular myocyte contractility in hypertensive rats

GW25-e0061 Up-regulation of neuronal nitric oxide synthase modulates myofilament Ca2+ sensitivity and controls left ventricular myocyte contractility in hypertensive rats

Effects of endogenous cannabinoid anandamide on excitation–contraction coupling in rat ventricular myocytes

A role for anandamide (N-arachidonoyl ethanolamide; AEA), a major endocannabinoid, in the cardiovascular system in various pathological conditions has been reported in earlier reports. In the present study, the effects of AEA on contractility, Ca2+ signaling, and action potential (AP) characteristics were investigated in rat ventricular myocytes. Video edge detection was used to measure myocyte shortening. Intracellular Ca2+ was measured in cells loaded with the fluorescent indicator fura-2 AM. AEA (1μM) caused a significant decrease in the amplitudes of electrically evoked myocyte shortening and Ca2+ transients. However, the amplitudes of caffeine-evoked Ca2+ transients and the rate of recovery of electrically evoked Ca2+ transients following caffeine application were not altered. Biochemical studies in sarcoplasmic reticulum (SR) vesicles from rat ventricles indicated that AEA affected Ca2+-uptake and Ca2+-ATPase activity in a biphasic manner. [3H]-ryanodine binding and passive Ca2+ release from SR vesicles were not altered by 10μM AEA. Whole-cell patch-clamp technique was employed to investigate the effect of AEA on the characteristics of APs. AEA (1μM) significantly decreased the duration of AP. The effect of AEA on myocyte shortening and AP characteristics was not altered in the presence of pertussis toxin (PTX, 2μg/ml for 4h), AM251 and SR141716 (cannabinoid type 1 receptor antagonists; 0.3μM) or AM630 and SR 144528 (cannabinoid type 2 receptor antagonists; 0.3μM). The results suggest that AEA depresses ventricular myocyte contractility by decreasing the action potential duration (APD) in a manner independent of CB1 and CB2 receptors.

Novel insights into the development of chagasic cardiomyopathy: Role of PI3Kinase/NO axis

BackgroundChagas' disease is one of the leading causes of heart failure in Latin American countries. Despite its great social impact, there is no direct evidence in the literature explaining the development of heart failure in Chagas' disease. Therefore, the main objective of the study was to investigate the development of the Chagas' disease towards its chronic phase and correlate with modifications in the cellular electrophysiological characteristics of the infected heart. Methods and resultsUsing a murine model of Chagas' disease, we confirmed and extended previous findings of altered electrocardiogram and echocardiogram in this cardiomyopathy. The observed changes in the electrocardiogram were correlated with the prolonged action potential and reduced transient outward potassium current density. Reduced heart function was associated with remodeling of intracellular calcium handling, altered extracellular matrix content, and to a set of proteins involved in the control of cellular contractility in ventricular myocytes. Furthermore, disruption of calcium homeostasis was partially due to activation of the PI3Kinase/nitric oxide signaling pathway. Finally, we propose a causal link between the inflammatory mediators and heart remodeling during chagasic cardiomyopathy. ConclusionAltogether our results demonstrate that heart failure in Chagas' disease may occur due to electrical and mechanical remodeling of cardiac myocytes, and suggest that AKT/PI3K/NO axis could be an important pharmacological target to improve the disease outcome.

PKC-ε mediates multiple endothelin-1 actions on systolic Ca2+ and contractility in ventricular myocytes

Endothelin-1 (ET-1) induces positive inotropy (enhanced contractility) in cardiac muscle, but establishing underlying cellular mechanisms has been controversial in part because of a growing number of signaling pathways and end effectors targeted by ET-1. Here we present evidence that ET-1 induces positive inotropism in ventricular tissue by increasing both systolic Ca2+ and myofilament Ca2+ sensitivity. To examine the roles of PKC-δ and PKC-ε in these acute responses to ET-1, kinase inactive dominant negative PKC (dn-PKC) constructs were expressed in adult rat ventricular myocytes. Yellow fluorescent protein (YFP) was fused to dn-PKC constructs to visualize expression and localization of dn-PKC in living myocytes. Due to an alanine to glutamate mutation in the pseudosubstrate site, dn-PKCs constitutively translocated to anchoring sites and were unaffected by agonist or phorbol ester treatment. Dn-PKC-δ-YFP mainly distributed at Z-lines and at intercalated disks in adult myocytes, whereas dn-PKC-ε-YFP stained the surface sarcolemma, T-tubules/Z-lines and perinuclear region. Myocytes expressing dn-PKC-δ-YFP showed normal systolic Ca2+ and contractile responses to ET-1. In contrast, the entire ensemble of ET-1 responses was blocked in myocytes expressing dn-PKC-ε-YFP including increased Ca2+ transients, enhanced myofilament Ca2+ sensitivity, and positive inotropy. This report provides direct evidence that PKC-ε is activated early and robustly following ET-1 stimulation and thus mediates multiple intracellular changes underlying the acute actions of ET-1 on myocardium.

Contractility of ventricular myocytes is well preserved despite altered mechanisms of Ca2+ transport and a changing pattern of mRNA in aged type 2 Zucker diabetic fatty rat heart

There has been a spectacular rise in the global prevalence of type 2 diabetes mellitus and cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. The objective of the study was to investigate ventricular myocyte shortening, intracellular Ca(2+) signalling and expression of genes encoding cardiac muscle proteins in the aged Zucker diabetic fatty (ZDF) rat. There was a fourfold elevation in non-fasting blood glucose in ZDF rats (478.43 ± 29.22 mg/dl) compared to controls (108.22 ± 2.52 mg/dl). Amplitude of shortening, time to peak (TPK) and time to half (THALF) relaxation of shortening were unaltered in ZDF myocytes compared to age-matched controls. Amplitude and THALF decay of the Ca(2+) transient were unaltered; however, TPK Ca(2+) transient was prolonged in ZDF myocytes (70.0 ± 3.2 ms) compared to controls (58.4 ± 2.3 ms). Amplitude of the L-type Ca(2+) current was reduced across a wide range of test potentials (-30 to +40 mV) in ZDF myocytes compared to controls. Sarcoplasmic reticulum Ca(2+) content was unaltered in ZDF myocytes compared to controls. Expression of genes encoding cardiac muscle proteins, membrane Ca(2+) channels, and cell membrane ion transport and intracellular Ca(2+) transport proteins were variously altered. Myh6, Tnnt2, Cacna2d3, Slc9a1, and Atp2a2 were downregulated while Myl2, Cacna1g, Cacna1h, and Atp2a1 were upregulated in ZDF ventricle compared to controls. The results of this study have demonstrated that preserved ventricular myocyte shortening is associated with altered mechanisms of Ca(2+) transport and a changing pattern of genes encoding a variety of Ca(2+) signalling and cardiac muscle proteins in aged ZDF rat.

Possible Roles of Cannabinoid Receptors in Cardiac Myocytes

Anandamide is an endogenous ligand of G-protein coupled cannabinoid CB1 and CB2 receptors, which has been shown to have complex effects on the cardiovascular system culminating in a hypotensive response and a reduction in cardiac contractility. The aims of this study were to investigate electrophysiologically the effects of anandamide on cardiac ventricular action potential (AP) parameters and accompanying cell contractions, and to explore the involvement, if any, of CB1 and CB2 receptors. Guinea pig ventricular myocytes were stimulated to fire APs by a 2 ms depolarising current pulse applied via an intracellular microelectrode at a frequency of 1 Hz using an Axoclamp 2 (Axon Instruments) amplifier (bridge mode). Myocyte contraction was measured using an edge detection system. Anandamide (1, 3, 10 µM) caused a concentration-dependent reduction in AP duration (APD) at 90, 50 and 20 % repolarisation, which was accompanied by a reduction in the amplitude of myocyte contraction. At a concentration of 10 µM, anandamide reduced APD 90 by 38 ± 8 % (n = 7, p < 0.001). In addition, 10 µM R-(+)-methanandamide (a non-hydrolysable analogue of anandamide) similarly reduced APD and amplitude of contraction. In the presence of the CB2 receptor antagonist AM 630, but not the CB1 receptor antagonist AM 281, the magnitudes of these reductions in response to 10 µM anandamide were partially but significantly reduced. This study confirms that anandamide causes a reduction in ventricular myocyte contractility and may modulate ventricular cell Ca2+ handling. Since these effects appear only to be partially inhibited by CB2 receptor blockade, it is likely that anandamide has actions which are independent of CB1 and CB2 receptors.

Hydrogen sulfide interacts with nitric oxide in the heart: possible involvement of nitroxyl

The present study aims to investigate the interaction between nitric oxide (NO) and hydrogen sulfide (H(2)S), the two important gaseous mediators in rat hearts. Intracellular calcium in isolated cardiomyocytes was measured with a spectrofluorometric method using Fura-2. Myocyte contractility was measured with a video edge system. NaHS (50 µM, an H(2)S donor) had no significant effect on the resting calcium level, electrically induced (EI) calcium transients, and cell contractility in ventricular myocytes. Stimulating endogenous NO production with l-arginine or exogenous application of NO donors [sodium nitroprusside (SNP) and 2-(N,N-diethylamino)-diazenolate-2-oxide] decreased myocyte twitch amplitudes accompanied by slower velocities of both cell contraction and relaxation. Surprisingly, NaHS reversed the negative inotropic and lusitropic effects of the above three NO-increasing agents. In addition, the mixture of SNP + NaHS increased, whereas SNP alone decreased, the resting calcium level and the amplitudes of EI calcium transients. Angeli's salt, a nitroxyl anion (HNO) donor, mimicked the effect of SNP + NaHS on calcium handling and myocyte contractility. Three thiols, N-acetyl-cysteine, l-cysteine, and glutathione, abolished the effects of HNO and SNP + NaHS on myocyte contraction. Neither Rp-cAMP [a protein kinase A (PKA) inhibitor] nor Rp-cGMP [a protein kinase G (PKG) inhibitor] affected the effects of SNP + NaHS, suggesting a cAMP/PKA- or cGMP/PKG-independent mechanism. H(2)S may interact with NO to form a thiol sensitive molecule (probably HNO) which produces positive inotropic and lusitropic effects. Our findings may shed light on the interaction of NO and H(2)S and provide new clues to treat cardiovascular diseases.

Hypothermia improves ventricular myocyte contractility under conditions of normal perfusion and after an interval of ischemia

Aim Recent investigations have reported improved myocardial function during hypothermia following resuscitation from cardiac arrest. The effects of hypothermia on myocyte contractility were investigated under conditions of normal perfusion and after a 10 min interval of ischemia. Methods Ventricular myocytes were obtained from 10 male Sprague–Dawley rats weighing 400 ± 50 g. The myocytes were randomized to be perfused at: 37 °C, 34 °C, 32 °C, or 30 °C. A subsequent set of myocytes was subjected to 10 min of ischemia at 37 °C, prior to being randomized to reperfusion at: 37 °C, 34 °C, 32 °C or 30 °C. Myocyte contractility was expressed as length-shortening percentage. Intracellular Ca 2+ transients were assessed in a separate group of myocytes preloaded with Fura-2/AM. Sensitivity to Ca 2+ was tested by increasing perfusate Ca 2+ content, i.e. 0.5 mM, 1 mM and 2 mM. Results During normal perfusion and following reperfusion after 10 min of ischemia, myocyte contractility increased at 34 °C compared to 37 °C ( P < 0.01). When the perfusion temperature was decreased to 32 °C and 30 °C, contractility further increased ( P < 0.001). Intracellular Ca 2+ transients were greater during perfusion at 34 °C compared to those at 37 °C ( P < 0.001) and further increased at 30 °C ( P < 0.001). Increases in extracellular Ca 2+ concentration from 0.5 mM to 2 mM resulted in greater myocyte contractility during perfusion at 30 °C compared to that observed at 37 °C ( P < 0.001). Effects of hypothermia on intracellular Ca 2+ transients and sensitivity to Ca 2+ persisted after ischemia. Conclusions Hypothermia improved myocyte contractility, intracellular Ca 2+ transients and sensitivity to Ca 2+ under conditions of normal perfusion and following reperfusion after 10 min of ischemia.

Expression of active p21-activated kinase-1 induces Ca2+ flux modification with altered regulatory protein phosphorylation in cardiac myocytes

p21-Activated kinase-1 (Pak1) is a serine-threonine kinase that associates with and activates protein phosphatase 2A in adult ventricular myocytes and, thereby, induces increased Ca2+ sensitivity of skinned-fiber tension development mediated by dephosphorylation of myofilament proteins (Ke Y, Wang L, Pyle WG, de Tombe PP, Solaro RJ. Circ Res 94: 194-200, 2004). We test the hypothesis that activation of Pak1 also moderates cardiac contractility through regulation of intracellular Ca2+ fluxes. We found no difference in field-stimulated intracellular Ca2+ concentration ([Ca2+]i) transient amplitude and extent of cell shortening between myocytes expressing constitutively active Pak1 (CA-Pak1) and controls expressing LacZ; however, time to peak shortening was significantly faster and rate of [Ca2+]i decay and time of relengthening were slower. Neither caffeine-releasable sarcoplasmic reticulum (SR) Ca2+ content nor fractional release was different in CA-Pak1 myocytes compared with controls. Isoproterenol application revealed a significantly blunted increase in [Ca2+]i transient amplitude, as well as a slowed rate of [Ca2+]i decay, increased SR Ca2+ content, and increased cell shortening, in CA-Pak1 myocytes. We found no significant change in phospholamban phosphorylation at Ser16 or Thr17 in CA-Pak1 myocytes. Analysis of cardiac troponin I revealed a significant reduction in phosphorylated species that are primarily attributable to Ser(23/24) in CA-Pak1 myocytes. Nonstimulated, spontaneous SR Ca2+ release sparks were significantly smaller in amplitude in CA-Pak1 than LacZ myocytes. Propagation of spontaneous Ca2+ waves resulting from SR Ca2+ overload was significantly slower in CA-Pak1 myocytes. Our data indicate that CA-Pak1 expression has significant effects on ventricular myocyte contractility through altered myofilament Ca2+ sensitivity and modification of the [Ca2+]i transient.

Abstract 2492: Clenbuterol, Unlike Other B 2 -Adrenoceptor Agonists, Predominantly Activates the G i Protein in Isolated Rat Ventricular Myocytes

Clenbuterol (CLEN), a β 2 -adrenoceptor (AR) agonist, has been employed in combination with other pharmacological agents and LVADs to treat patients with severe heart failure (HF). Chronic administration of CLEN increases expression of Ca regulatory proteins in isolated cardiac myocytes and improves their contractility. However, the acute effects of CLEN on myocyte contractility compared to other β 2 -AR agonists and the signalling pathway of CLEN remain undefined. Here we compared the acute effects of CLEN to two other β 2 -AR agonists, fenoterol (FEN) and salbutamol (SAL). Isolated rat ventricular myocytes were superfused with β 2 AR agonists and sarcomere shortening was measured. CLEN produced a negative inotropism (30 μM: 65.3 ± 9.7% of control, n=9; p&lt;0.05; 100 μM: 37.9 ± 8.0%, n=9; p&lt;0.01) whereas FEN showed a positive inotropic response (30 μM: 184.8 ± 19.1% of control, n=9; p&lt;0.01; 100 μM: 198.1 ± 20.5%, n=9; p&lt;0.01). SAL had no significant effect. Selective β 1 AR blockade with 300 nM CGP 20712A did not affect CLEN’s action on sarcomere shortening but significantly reduced the contractile response to FEN and SAL (p&lt;0.05). Additional blockade of β 2 AR-Gs with 50 nM ICI 118,551 unveiled a negative inotropic response to FEN (100 μM: 61.5 ± 7.3% of control, n=7; p&lt;0.001) and SAL (100 μM: 67.4 ± 3.6% of control, n=7; p&lt;0.01) and did not alter the response to CLEN. Incubation with 2 μg/ml pertussis toxin (PTX) almost abolished the negative inotropic effects of CLEN: 30 μM (PTX: 95.1 ± 4.9%, n=6 vs control: 73.9 ± 5.5%, n=7; p&lt;0.05) and 100 μM (PTX: 96.2 ± 9.6%, n=6 vs control: 21.7 ± 5.5%, n=7; p&lt;0.001), suggesting the involvement of the inhibitory guanine nucleotide binding protein (G i ). In addition, overexpression of G i by adenoviral (AdV) transfection for 48 hrs induced a stronger negative inotropic effect upon application of CLEN compared with control cultured cells (AdV-Gi: clen 30 μM: 36.8 ± 8.3% of control, n=9; p&lt;0.01; 100 μM: 19.7 ± 6.5%, n=9; p&lt;0.01). In summary, CLEN significantly depresses contractility of ventricular myocytes, an effect not seen with FEN or SAL. CLEN predominantly acts through G i and the consequent downstream signaling pathways activation may explain the beneficial effects observed during administration of CLEN in patients treated with LVADs.

Clenbuterol affects rat ventricular myocyte contractility via an inhibitory G protein-mediated pathway

Clenbuterol, a β2-adrenoceptor (AR) agonist, has been employed in combination with other pharmacological agents and LVADs to treat patients with severe heart failure (HF). In experimental HF chronic clenbuterol (CLEN) administration induces cardiac hypertrophy and increases expression of Ca regulatory proteins resulting in improved contractility of isolated cardiac myocytes. However, the signaling pathway of CLEN remains undefined. The aims of this study were to assess the acute effects of CLEN on contractility in normal cardiomyocytes and use this to clarify its signaling pathways.

A56. PKC-ε mediates multiple endothelin-1 actions on systolic calcium and contractility in ventricular myocytes

Endothelin-1 (ET-1) induces positive inotropy (enhanced contractility) in cardiac muscle, but establishing underlying cellular mechanisms has been controversial in part because of a growing number of signaling pathways and end effectors targeted by ET-1. Here we present evidence that ET-1 induces positive inotropism in ventricular tissue by increasing both systolic Ca2+ and myofilament Ca2+ sensitivity. ET-1 also promotes twitch broadening, in contradistinction to β-adrenergic stimulation which narrows the cardiac twitch.