Intracellular Ca- and PKC-dependent upregulation of T-type Ca channels in LPC-stimulated cardiomyocytes

Lysophosphatidylcholine (LPC) accumulation in intracellular and/or interstitial space in cardiomyocytes may underlie as a mechanism for tachycardia and various arrhythmias during cardiac ischemia, which is usually accompanied by elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). The present study was therefore designed to investigate possible mechanisms responsible for [Ca²⁺]_i elevation by LPC focusing on T-type Ca²⁺ channel current (I_Ca.T). LPC as well as phorbol 12-myristate 13-acetate (PMA) significantly accelerated the beating rates of neonatal rat cardiomyocytes. Augmentation of I_Ca.T by LPC was dependent on the intracellular Ca²⁺ concentration: an increase of I_Ca.T was significantly larger in high [Ca²⁺]_i condition (pCa = 7) than those in low [Ca²⁺]_i condition (pCa = 11). In heterologous expression system by use of human cardiac Ca_V3.1 and Ca_V3.2 channels expressed in HEK293 cells, LPC augmented Ca_V3.2 channel current (I_Cav3.2) in a concentration-dependent manner but not Ca_V3.1 channel current (I_Cav3.1). Augmentation of I_Cav3.2 by LPC was highly [Ca²⁺]_i dependent: I_Cav3.2 was unchanged when pCa was 11 but was markedly increased when [Ca²⁺]_i was higher than 10⁻¹⁰ M (pCa ≤ 10) by LPC application (10-50 μM). A specific inhibitor of protein kinase Cα (Ro-32-0432) attenuated the increase of I_Cav3.2 by LPC. LPC stimulates I_Ca.T in a [Ca²⁺]_i-dependent manner via PKCα activation, which may play a role in triggering arrhythmias in pathophysiological conditions of the heart.     

Sarcoplasmic Reticulum Function in Murine Ventricular Myocytes Overexpressing SR CaATPase

To examine the effects of the overexpression of sarcoplasmic reticulum (SR) CaATPase on function of the SR and Ca²⁺homeostasis, we measured [Ca²⁺]_itransients (fluo-3), and L-type Ca²⁺currents (I_Ca,L), Na/Ca exchanger currents (I_Na/Ca), and SR Ca²⁺content with voltage clamp in ventricular myocytes isolated from wild type (WT) mice and transgenic (SRTG) mice. The amplitude of [Ca²⁺]_itransients was insignificantly increased in SRTG myocytes, while the diastolic [Ca²⁺]_itended to be lower. The initial and terminal declines of [Ca²⁺]_itransients were significantly accelerated in SRTG myocytes, implying a functional upregulation of the SR CaATPase. We examined the functional contribution of only the SR CaATPase to the initial and the terminal phase of the decline of [Ca²⁺]_i, by abruptly inhibiting Na/Ca exchange with a rapid switcher device. The rate of [Ca²⁺] decline mediated by the SR CaATPase was increased by 40% in SRTG compared with WT myocytes. The function of the L-type Ca²⁺channel was unchanged in SRTG myocytes, while INa/Ca density was slightly (10%) decreased. Measured SR Ca²⁺content was significantly increased by 29% in SRTG myocytes. Thus, overexpression of SR CaATPase markedly accelerates the decline of [Ca²⁺]_itransients, and induces an increase in SR Ca²⁺content, with some downregulation of the Na/Ca exchanger.     

H(2)O(2)-induced left ventricular dysfunction in isolated working rat hearts is independent of calcium accumulation

Reactive oxygen species (ROS) and intracellular Ca²⁺ overload play key roles in myocardial ischemia-reperfusion (IR) injury but the relationships among ROS, Ca²⁺ overload and LV mechanical dysfunction remain unclear. We tested the hypothesis that H₂O₂ impairs LV function by causing Ca²⁺ overload by increasing late sodium current (I_Na), similar to Sea Anemone Toxin II (ATX-II). Diastolic and systolic Ca²⁺ concentrations (d[Ca²⁺]_i and s[Ca²⁺]_i) were measured by indo-1 fluorescence simultaneously with LV work in isolated working rat hearts. H₂O₂ (100 μM, 30 min) increased d[Ca²⁺]_i and s[Ca²⁺]_i. LV work increased transiently then declined to 32% of baseline before recovering to 70%. ATX-II (12 nM, 30 min) caused greater increases in d[Ca²⁺]_i and s[Ca²⁺]_i. LV work increased transiently before declining gradually to 17%. Ouabain (80 μM) exerted similar effects to ATX-II. Late I_Na inhibitors, lidocaine (10 μM) or R56865 (2 μM), reduced effects of ATX-II on [Ca²⁺]_i and LV function, but did not alter effects of H₂O₂. The antioxidant, N-(2-mercaptopropionyl)glycine (MPG, 1 mM) prevented H₂O₂-induced LV dysfunction, but did not alter [Ca²⁺]_i. Paradoxically, further increases in [Ca²⁺]_i by ATX-II or ouabain, given 10 min after H₂O₂, improved function. The failure of late I_Na inhibitors to prevent H₂O₂-induced LV dysfunction, and the ability of MPG to prevent H₂O₂-induced LV dysfunction independent of changes in [Ca²⁺]_i indicate that impaired contractility is not due to Ca²⁺ overload. The ability of further increases in [Ca²⁺]_i to reverse H₂O₂-induced LV dysfunction suggests that Ca²⁺ desensitization is the predominant mechanism of ROS-induced contractile dysfunction.     

Activation of Connexin-43 Hemichannels Can Elevate [Ca]iand [Na]iin Rabbit Ventricular Myocytes During Metabolic Inhibition

ATP depletion due to ischemia or metabolic inhibition (MI) causes Na⁺and Ca²⁺accumulation in myocytes, which may be in part due to opening of connexin-43 hemichannels. Halothane (H) has been shown to reduce conductance of connexin-43 hemichannels and to protect the heart against ischemic injury. We therefore investigated the effect of halothane on [Ca²⁺]_iand [Na⁺]_iin myocytes during MI. Isolated rabbit left ventricular myocytes were loaded with 4μ m fluo-3 AM for 30 min, or with 5 μ m sodium green AM for 60 min at 37°C. After washing, the myocytes were exposed to: (1) Normal HEPES solution; (2) MI solution (2 m NaCN, 20 m 2-deoxy- -glucose and 0-glucose); or (3) MI+H (0.95 m , 4.7 m ) for 60 min. Propidium iodide (PI, 25 μ m) was added to all samples before data acquisition. The fluorescence intensity was measured by flow cytometry with 488 nm excitation and 530 nm emission for fluo-3 or sodium green, and 670 nm for PI. The [Ca²⁺]_iand [Na⁺]_iwere then calculated by calibration. In some experiments, the effect of 10 μ m tetrodotoxin (TTX) and 20 μ m nifedipine (NIF) were studied. Metabolic inhibition for 60 min caused a significant increase in [Ca²⁺]_iand [Na⁺]_iin myocytes when compared to controls, which was significantly reduced by halothane in a dose-dependent fashion. In the presence of TTX and NIF, halothane also significantly reduced the rise in the [Ca²⁺]_iand [Na⁺]_iin myocytes subjected to MI. 1-heptanol, another gap junction blocker, had similar effects. Thus, halothane reduced [Ca²⁺]_iand [Na⁺]_ioverload produced by MI in myocytes. This effect is not solely due to block of voltage-gated Na⁺and Ca²⁺channels, and is likely mediated by inhibiting the opening of connexin-43 hemichannels.     

Diastolic transient inward current in long QT syndrome type 3 is caused by Ca overload and inhibited by ranolazine

Long QT syndrome variant 3 (LQT-3) is a channelopathy in which mutations in SCN5A, the gene coding for the primary heart Na⁺ channel alpha subunit, disrupt inactivation to elevate the risk of mutation carriers for arrhythmias that are thought to be calcium (Ca²⁺)-dependent. Spontaneous arrhythmogenic diastolic activity has been reported in myocytes isolated from mice harboring the well-characterized ΔKPQ LQT-3 mutation but the link to altered Ca²⁺ cycling related to mutant Na⁺ channel activity has not previously been demonstrated. Here we have investigated the relationship between elevated sarcoplasmic reticulum (SR) Ca²⁺ load and induction of spontaneous diastolic inward current (I_TI) in myocytes expressing ΔKPQ Na⁺ channels, and tested the sensitivity of both to the antianginal compound ranolazine. We combined whole-cell patch clamp measurements, imaging of intracellular Ca²⁺, and measurement of SR Ca²⁺ content using a caffeine dump methodology. We compared the Ca²⁺ content of ΔKPQ^+/− myocytes displaying I_TI to those without spontaneous diastolic activity and found that I_TI induction correlates with higher sarcoplasmic reticulum (SR) Ca²⁺. Both spontaneous diastolic I_TI and underlying Ca²⁺ waves are inhibited by ranolazine at concentrations that preferentially target I_NaL during prolonged depolarization. Furthermore, ranolazine I_TI inhibition is accompanied by a small but significant decrease in SR Ca²⁺ content. Our results provide the first direct evidence that induction of diastolic transient inward current (I_TI) in ΔKPQ^+/− myocytes occurs under conditions of elevated SR Ca²⁺ load.     

Influence of Infrasound Exposure on the Whole L-type Calcium Currents in Rat Ventricular Myocytes

This study was designed to examine the effect of infrasound exposure (5 Hz at 130 dB) on whole-cell L-type Ca²⁺ currents (WLCC) in rat ventricular myocytes and the underlying mechanism(s) involved. Thirty-two adult Sprague-Dawley rats were randomly assigned to infrasound exposure and control groups. [Ca²⁺]_i, WLCC, mRNA expression of the a_1c subunit of L-type Ca²⁺ channels (LCC), and SERCA2 protein were examined on day 1, 7, and 14 after initiation of infrasound exposure. Fluo-3/AM fluorescence and the laser scanning confocal microscope techniques were used to measure [Ca²⁺]_i in freshly isolated ventricular myocytes. The Ca²⁺ fluorescence intensity (FI), denoting [Ca²⁺]_i in cardiomyocytes, was significantly elevated in a time-dependent manner in the exposure groups. There was a significant increase in WLCC in the 1-day group and a further significant increase in the 7- and 14-day groups. LCC mRNA expression measured by RT-PCR revealed a significant rise in the 1-day group and a significant additional rise in the 7- and 14-day groups compared with control group. SERCA2 expression was significantly upregulated in the 1-day group followed by an overt decrease in the 7- and 14-day groups. Prolonged exposure of infrasound altered WLCC in rat cardiomyocytes by shifting the steady-state inactivation curves to the right (more depolarized direction) without altering the slope and biophysical properties of I _Ca,L. Taken together, our data suggest that changes in [Ca²⁺]_I levels as well as expression of LCC and SERCA2 may contribute to the infrasound exposure-elicited cardiac response. Zhaohui Pei and Zhiqiang Zhuang contributed equally to this work.     

Enhanced L-type calcium currents in cardiomyocytes from transgenic rats overexpressing SERCA2a

BACKGROUND:

Previous research reported that transgenic rats overexpressing the sarco(endo)plasmic reticulum Ca²⁺-ATPase SERCA2a exhibit improved contractile function of the myocardium. Furthermore, impaired Ca²⁺ uptake and reduced relaxation rates in rats with diabetic cardiomyopathy were partially rescued by transgenic expression of SERCA2a in the heart.

OBJECTIVE:

To explore whether enhanced Ca²⁺ cycling in the cardiomyocytes of SERCA2a transgenic rats is associated with changes in L-type Ca²⁺ (I_Ca-L) currents.

METHODS:

The patch-clamp technique was used to measure whole-cell currents in cardiomyocytes from transgenic rats overexpressing SERCA2a and from wild-type (nontransgenic) animals.

RESULTS:

The amplitudes of I_Ca-L currents at depolarizing pulses ranging from −45 mV to 0 mV (350 ms duration, 1 Hz) were significantly higher in cardiomyocytes of SERCA2a transgenic rats than in nontransgenic rats (1985±48 pA [n=32] versus 1612±55 pA [n=28], respectively). The inactivation kinetics of I_Ca-L showed subtle differences with increased tau fast and tau slow decay constants in cardiomyocytes of SERCA2a transgenic animals. Beta-adrenergic stimulation with 50 nM isoproterenol reduced tau fast and tau slow decay constants in cardiomyocytes of transgenic rats to values that were not significantly different from those in normal cardiomyocytes. Furthermore, isoproterenol enhanced I_Ca-L currents 3.2-fold and 2.3-fold in cardiomyocytes with and without the SERCA2a transgene, respectively, and this effect was abolished by buffering intracellular Ca²⁺ with BAPTA.

CONCLUSIONS:

These findings indicate that enhanced Ca²⁺ cycling in the hearts of SERCA2a transgenic rats, both under normal conditions and during beta-adrenergic stimulation, involves changes in I_Ca-L currents. Modified I_Ca-L kinetics may contribute, to some extent, to the improved contractile function of the myocardium of transgenic rats.     

Role of mitochondrial dysfunction in cardiac glycoside toxicity

Cardiac glycosides, which inhibit the plasma membrane Na⁺ pump, are one of the four categories of drug recommended for routine use to treat heart failure, yet their therapeutic window is limited by toxic effects. Elevated cytoplasmic Na⁺ ([Na⁺]_i) compromises mitochondrial energetics and redox balance by blunting mitochondrial Ca²⁺ ([Ca²⁺]_m) accumulation, and this impairment can be prevented by enhancing [Ca²⁺]_m. Here, we investigate whether this effect underlies the toxicity and arrhythmogenic effects of cardiac glycosides and if these effects can be prevented by suppressing mitochondrial Ca²⁺ efflux, via inhibition of the mitochondrial Na⁺/Ca²⁺ exchanger (mNCE). In isolated cardiomyocytes, ouabain elevated [Na⁺]_i in a dose-dependent way, blunted [Ca²⁺]_m accumulation, decreased the NADH/NAD + redox potential, and increased reactive oxygen species (ROS). Concomitant treatment with the mNCE inhibitor CGP-37157 ameliorated these effects. CGP-37157 also attenuated ouabain-induced cellular Ca²⁺ overload and prevented delayed afterdepolarizations (DADs). In isolated perfused hearts, ouabain's positive effects on contractility and respiration were markedly potentiated by CGP-37157, as were those mediated by β-adrenergic stimulation. Furthermore, CGP-37157 inhibited the arrhythmogenic effects of ouabain in both isolated perfused hearts and in vivo. The findings reveal the mechanism behind cardiac glycoside toxicity and show that improving mitochondrial Ca²⁺ retention by mNCE inhibition can mitigate these effects, particularly with respect to the suppression of Ca²⁺-triggered arrhythmias, while enhancing positive inotropic actions. These results suggest a novel strategy for the treatment of heart failure.     

Effects of nitric oxide donors on vascular smooth muscles depend on a type of vascular smooth-muscle preactivation

The abilities of such therapeutic nitrovasodilators as sodium nitroprusside (SNP) and glyceryl trinitrate (GTN) to dilate vascular smooth muscles (VSM) and affect intracellular calcium concentration level ([Ca²⁺]_i) in a rat tail artery were tested under different types of preactivation. To shed light on mechanisms underlying possible differences in the action of these two nitric oxide (NO) donors, simultaneous measurements of [Ca²⁺]_i and contractile force were done. All vascular rings were precontracted either using a high-K⁺-Krebs solution or phenylephrine (PE). It was shown that the effect of both NO donors strongly depended on a type of VSM preactivation. The EC₅₀ for GTN under K⁺ stimulation of VSM comprised (2.48±1.6)×10⁻⁵ M, whereas the mean EC₅₀ under PE stimulation was (3.05±2.3)×10⁻⁴ M (p<0.05, n=9). The EC₅₀ for SNP under K⁺ stimulation of VSM comprised (1.09±0.47)×10⁻⁷ M, whereas the EC₅₀ under PE stimulation was (8.01±2.4)×10⁻⁶ M (p<0.05, n=9). GTN demonstrated a significant discrepancy in the magnitude of changes in [Ca²⁺]_i and related VSM relaxant responses, indicating the ability of GTN to relax VSM in the absence of a proportional decrease in [Ca²⁺]_i. The main peculiarity of SNP action under K⁺ stimulation as compared to PE stimulation was the transient decrease in [Ca²⁺]_i while relaxation was sustained. Therefore, both NO donors demonstrated their ability to produce vasorelaxation as a result of an alteration in myofilament calcium sensitivity. These data clearly indicate that the sensitivity of VSM to NO donors is higher under K⁺ depolarization than that seen under PE stimulation, indicating that Ca²⁺ entry through voltage-operated calcium channels is more sensitive to NO as compared to calcium mobilization by means of Ca²⁺ entry through receptor-operated calcium channels or intracellular Ca²⁺ release, or both.     

Adult zebrafish heart as a model for human heart? An electrophysiological study

The zebrafish has recently emerged as an excellent model for studies of heart development and regeneration. The physiology of the zebrafish heart has been suggested to resemble that of the human heart in many aspects, whereas, in contrast to mammals, the zebrafish has a remarkable ability to regenerate after heart injury. Thus, zebrafish have been proposed as a cost-effective model for genetic and pharmacological screens of factors affecting heart function and repair. However, realizing the full potential of the zebrafish heart as a model will require a better understanding of the electrophysiology of the adult zebrafish myocardium. Here, we characterize action potentials (APs) from intact adult atria and ventricles and find that the overall shape of zebrafish APs is similar to that of humans. We show that zebrafish, like most mammals, display functional acetylcholine-activated K⁺ channels in the atrium, but not in the ventricle. Furthermore, the zebrafish AP upstroke is dominated by Na⁺ channels, L-type Ca²⁺ channels contribute to the plateau phase and I_Kr channels are involved in repolarization. However, despite these similarities between zebrafish and mammalian electrophysiology, we also identified important differences. In particular, zebrafish display a robust T-type Ca²⁺ current in both atrial and ventricular cardiomyocytes. Interestingly, in most mammals T-type Ca²⁺ channels are only expressed in the developing heart or under pathophysiological conditions, indicating that adult zebrafish cardiomyocytes display a more immature phenotype.     

Hyperpolarization induced by K+ channel openers inhibits Ca2+ influx and Ca2+ release in coronary artery

The vasodilating mechanisms of the K⁺ channel openers—cromakalim, pinacidil, nicorandil, KRN2391, and Ki4032—were examined by measurement of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) using the fura-2 method in canine or porcine coronary arterial smooth muscle. The five K⁺ channel openers all produced a reduction of [Ca²⁺]_i in 5 and 30 mM KCl physiological salt solution (PSS), the effects of which were antagonized by tetrabutylammonium (TBA) or glibenclamide, but failed to affect [Ca²⁺]_i in 45 and 90 mM MCl-PSS. Cromakalim and Ki4032 only partially inhibited the 30 mM KCl-induced contractures, whereas pinacidil, nicorandil, and KRN2391 nearly abolished contractions produced by high KCl-PSS. The increased [Ca²⁺]_i and force produced by a thromboxane A₂ analogue, U46619, were inhibited by K⁺ channel openers and verapamil. In the absence of extracellular Ca²⁺, U46619 induced a transient increase in [Ca²⁺]_i with a contraction, which is effectively inhibited by cromakalim and Ki4032. Their inhibitory effects were blocked by TBA and counteracted by 20 mM KCl-induced depolarization. Cromakalim and Ki4032 did not affect caffeine-induced Ca²⁺ release. Cromakalim reduced U46619-induced IP₃ production and TBA blocked this inhibitory effect. Thus, cromakalim and Ki4032 are more specific K⁺ channel openers than pinacidil, nicorandil, and KRN2391. The vasodilation related with a reduction of [Ca²⁺]_i produced by K⁺ channel openers is due to the hyperpolarization of the plasma membrane resulting in not only the closure of voltage-dependent Ca²⁺ channels but also inhibition of the production of IP₃ and Ca²⁺ release from intracellular stores related to stimulation of the thromboxane A₂ receptor.     

Fibronectin increases the force production of mouse papillary muscles via α5β1 integrin

The extracellular matrix (ECM) protein-integrin-cytoskeleton axis plays a central role as a mechanotransducing protein assemblage in many cell types. However, how the process of mechanotransduction and the mechanically generated signals arising from this axis affect myofilament function in cardiac muscle are not completely understood. We hypothesize that ECM proteins can regulate cardiac function through integrin binding, and thereby alter the intracellular calcium concentration ([Ca²⁺]_i) and/or modulate myofilament activation processes. Force measurements made in mouse papillary muscle demonstrated that in the presence of the soluble form of the ECM protein, fibronectin (FN), active force was increased significantly by 40% at 1 Hz, 54% at 2 Hz, 35% at 5 Hz and 16% at 9 Hz stimulation frequencies. Furthermore, increased active force in the presence of FN was associated with 12-33% increase in [Ca²⁺]_i and 20-50% increase in active force per unit Ca²⁺. A function blocking antibody for α5 integrin prevented the effects of the FN on the changes in force and [Ca²⁺]_i, whereas a function blocking α3 integrin antibody did not reverse the effects of FN. The effects of FN were reversed by an L-type Ca²⁺ channel blocker, verapamil or PKA inhibitor. Freshly isolated cardiomyocytes exhibited a 39% increase in contraction force and a 36% increase in L-type Ca²⁺ current in the presence of FN. Fibers treated with FN showed a significant increase in the phosphorylation of phospholamban; however, the phosphorylation of troponin I was unchanged. These results demonstrate that FN acts via α5β1 integrin to increase force production in myocardium and that this effect is partly mediated by increases in [Ca²⁺]_i and Ca²⁺ sensitivity, PKA activation and phosphorylation of phospholamban.     

L-type Ca channel facilitation mediated by H2O2-induced activation of CaMKII in rat ventricular myocytes

The Ca²⁺-dependent facilitation (CDF) of L-type Ca²⁺ channels, a major mechanism for force-frequency relationship of cardiac contraction, is mediated by Ca²⁺/CaM-dependent kinase II (CaMKII). Recently, CaMKII was shown to be activated by methionine oxidation. We investigated whether oxidation-dependent CaMKII activation is involved in the regulation of L-type Ca²⁺ currents (I_Ca,L) by H₂O₂ and whether Ca²⁺ is required in this process. Using patch clamp, I_Ca,L was measured in rat ventricular myocytes. H₂O₂ induced an increase in I_Ca,L amplitude and slowed inactivation of I_Ca,L. This oxidation-dependent facilitation (ODF) of I_Ca,L was abolished by a CaMKII blocker KN-93, but not by its inactive analog KN-92, indicating that CaMKII is involved in ODF. ODF was not affected by replacement of external Ca²⁺ with Ba²⁺ or presence of EGTA in the internal solutions. However, ODF was abolished by adding BAPTA to the internal solution or by depleting sarcoplasmic reticulum (SR) Ca²⁺ stores using caffeine and thapsigargin. Alkaline phosphatase, β-iminoadenosine 5′-triphosphate (AMP-PNP), an autophosphorylation inhibitor autocamtide-2-related inhibitory peptide (AIP), or a catalytic domain blocker (CaM-KIINtide) did not affect ODF. In conclusion, oxidation-dependent facilitation of L-type Ca²⁺ channels is mediated by oxidation-dependent CaMKII activation, in which local Ca²⁺ increases induced by SR Ca²⁺ release is required.     

Oscillations in cytoplasmic free calcium concentration in human pancreatic islets from subjects with normal and impaired glucose tolerance

Summary Plasma insulin levels in healthy subjects oscillate and non-insulin-dependent diabetic patients display an irregular pattern of such oscillations. Since an increase in cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) in the pancreatic beta cell is the major stimulus for insulin release, this study was undertaken to investigate the dynamics of electrical activity, [Ca²⁺]_i-changes and insulin release, in stimulated islets from subjects of varying glucose tolerance. In four patients it was possible to investigate more than one of these three parameters. Stimulation of pancreatic islets with glucose and tolbutamide sometimes resulted in the appearance of oscillations in [Ca²⁺]_i, lasting 2–3 min. Such oscillations were observed even in some islets from patients with impaired glucose tolerance. In one islet from a diabetic patient there was no response to glucose, whereas that islet displayed [Ca²⁺]_i-oscillations in response to tolbutamide, suggesting that sulphonylurea treatment can mimic the complex pattern of glucose-induced [Ca²⁺]_i-oscillations. We also, for the first time, made patch-clamp recordings of membrane currents in beta-cells in situ in the islet. Stimulation with glucose and tolbutamide resulted in depolarization and appearance of action potentials. The islet preparations responded to stimulation with a number of different secretagogues with release of insulin. The present study shows that human islets can respond to stimulation with glucose and sulphonylurea with oscillations in [Ca²⁺]_i, which is the signal probably underlying the oscillations in plasma insulin levels observed in healthy subjects. Interestingly, even subjects with impaired glucose tolerance had islets that responded with oscillations in [Ca²⁺]_i upon glucose stimulation, although it is not known to what extent the response of these islets was representative of most islets in these patients.Abbreviations [Ca²⁺]_i Cytoplasmic free Ca²⁺ - NIDDM non-insulin-dependent diabetes mellitus - DMSO dimethylsulphoxide - PC pancreatic cancer     

Overexpression of diacylglycerol kinase zeta inhibits endothelin-1-induced decreases in Ca2+ transients and cell shortening in mouse ventricular myocytes

Endothelin-1 (ET-1) is released in various cardiovascular disorders including congestive heart failure, and may modulate significantly the disease process by its potent action on vascular and cardiac muscle cell function and gene regulation. In adult mouse ventricular cardiomyocytes loaded with indo-1, ET-1 induced a sustained negative inotropic effect (NIE) in association with decreases in Ca²⁺ transients. The ET-1-induced effects on Ca²⁺ transients and cell shortening were abolished in diacylglycerol (DAG) kinase ζ-overexpressing mouse ventricular myocytes. A nonselective protein kinase C (PKC) inhibitor, GF109203X, inhibited the ET-1-induced decreases in Ca²⁺ transients and cell shortening in concentration-dependent manners, whereas a selective Ca²⁺-dependent PKC inhibitor, Gö6976, did not affect the ET-1-induced effects. A phospholipase Cβ inhibitor, U73122, and an inhibitor of phospholipase D, C₂-ceramide, partially, but significantly, attenuated the ET-1-induced effects. Derivatives of the respective inhibitors with no specific effects, U73343 and dihydro-C₂-ceramide, did not affect the ET-1-induced effects. Taken together, these results indicate that activation of a Ca²⁺-independent PKC isozyme by 1,2-DAG, which is generated by phospholipase Cβ and phospholipase D activation and inactivated by phosphorylation via DAG kinase, is responsible for the ET-1-induced decreases in Ca²⁺ transients and cell shortening in mouse ventricular cardiomyocytes.     

L-type Ca current in ventricular cardiomyocytes

L-type Ca²⁺ channels are mediators of Ca²⁺ influx and the regulatory events accompanying it and are pivotal in the function and dysfunction of ventricular cardiac myocytes. L-type Ca²⁺ channels are located in sarcolemma, including the T-tubules facing the sarcoplasmic reticulum junction, and are activated by membrane depolarization, but intracellular Ca²⁺-dependent inactivation limits Ca²⁺ influx during action potential. I_CaL is important in heart function because it triggers excitation-contraction coupling, modulates action potential shape and is involved in cardiac arrhythmia. L-type Ca²⁺ channels are multi-subunit complexes that interact with several molecules involved in their regulations, notably by β-adrenergic signaling. The present review highlights some of the recent findings on L-type Ca²⁺ channel function, regulation, and alteration in acquired pathologies such as cardiac hypertrophy, heart failure and diabetic cardiomyopathy, as well as in inherited arrhythmic cardiac diseases such as Timothy and Brugada syndromes.     

SR Ca store refill—a key factor in cardiac pacemaking

This study presents a theoretical analysis of the role of store Ca²⁺ uptake on sinoatrial node (SAN) cell pacemaking. Two mechanisms have been shown to be involved in SAN pacemaking, these being: 1) the membrane oscillator model where rhythm generation is based on the interaction of voltage-dependent membrane ion channels and, 2) the store oscillator model where cyclical release of Ca²⁺ from intracellular Ca²⁺ stores depolarizes the membrane through activation of the sodium-calcium exchanger (NCX). The relative roles of these oscillators in generation and modulation of pacemaker rate have been vigorously debated and have many consequences. The main new outcomes of our study are: 1) uptake of Ca²⁺ by intracellular Ca²⁺ stores increases the maximum diastolic potential (MDP) by reducing the cytosolic Ca²⁺ concentration [Ca²⁺]_c and hence decreasing the NCX current; 2) this hyperpolarization enhances recruitment of key pacemaker currents (e.g. the hyperpolarization-activated HCN current (I_f) and T-type Ca²⁺ current (I_T-Ca)); 3) the resultant enhanced Ca²⁺ entry during the pacemaker depolarization increases [Ca²⁺]_c causing advancement of the store Ca²⁺ release cycle and increased NCX current. In overview, the novel feature of our study is an investigation of the role of store Ca²⁺ uptake on SAN pacemaking. This occurs during the early diastolic period and causes enhanced I_f, I_T-Ca and store release (and hence I_NCX) during the later diastolic period. There is thus a symbiotic interaction between the two pacemaker “clocks” over the entire diastolic period, this providing robust and highly malleable SAN pacemaking. Accounting for store Ca²⁺ uptake also provides insight into hitherto unexplained SAN behaviour, as we exemplify for the sinus bradycardia exhibited in catecholaminergic polymorphic ventricular tachycardia (CPVT).     

Gender-related differences in ventricular myocyte repolarization in the guinea pig

Abstract. It is well established that gender-differences exist in cardiac electrophysiology and these are thought to contribute to the increased risk of women, compared to men, for the potentially lethal ventricular arrhythmia, torsades de pointes. Data from animal models with abbreviated estrus cycles suggest that androgens may play a protective role in males. However, the role of female sex hormones in gender-differences in cardiac electrophysiology is less clear. This report describes gender differences in ventricular electrophysiology, investigated using the guinea pig heart. Ionic currents and action potentials were compared between ventricular myocytes isolated from male guinea pig hearts and those from females on the day of estrus (day 0) and 4 days post-estrus (day 4). The density of inward rectifier K⁺ current (I_K1) at –120 mV was significantly greater in male myocytes than in female myocytes either at day 0 or day 4. The peak L-type Ca²⁺ current (I_Ca) at +10 mV was also significantly larger in male myocytes than in day 0 and day 4 female myocytes. Moreover, I_Ca differed significantly between day 0 and day 4 female myocytes, strongly suggesting that I_Ca density varies around the estrus cycle. Delayed rectifier (I_K) tail currents were significantly different between male and female day 4 myocytes. Action potential duration (at 90% repolarization; APD₉₀) was significantly shorter in male myocytes than in female myocytes at day 0, but not at day 4, broadly consistent with the combined differences in I_K and I_Ca between the three groups. Taken together, our data are consistent with the contribution of multiple factors, rather than a single hormone, to gender differences in ventricular repolarization. Since female guinea pigs possess a conventional estrus cycle, our data suggest that this species may be well suited to elucidating the modulatory influence of ovarian steroids on ventricular repolarization and arrhythmic risk. Our findings suggest that further work examining the basis to gender differences in ventricular repolarization in the guinea pig is warranted.     

Ca2+ release-activated Ca2+ channel blockade as a potential tool in antipancreatitis therapy

Alcohol-related acute pancreatitis can be mediated by a combination of alcohol and fatty acids (fatty acid ethyl esters) and is initiated by a sustained elevation of the Ca²⁺ concentration inside pancreatic acinar cells ([Ca²⁺]_i), due to excessive release of Ca²⁺ stored inside the cells followed by Ca²⁺ entry from the interstitial fluid. The sustained [Ca²⁺]_i elevation activates intracellular digestive proenzymes resulting in necrosis and inflammation. We tested the hypothesis that pharmacological blockade of store-operated or Ca²⁺ release-activated Ca²⁺ channels (CRAC) would prevent sustained elevation of [Ca²⁺]_i and therefore protease activation and necrosis. In isolated mouse pancreatic acinar cells, CRAC channels were activated by blocking Ca²⁺ ATPase pumps in the endoplasmic reticulum with thapsigargin in the absence of external Ca²⁺. Ca²⁺ entry then occurred upon admission of Ca²⁺ to the extracellular solution. The CRAC channel blocker developed by GlaxoSmithKline, GSK-7975A, inhibited store-operated Ca²⁺ entry in a concentration-dependent manner within the range of 1 to 50 μM (IC₅₀ = 3.4 μM), but had little or no effect on the physiological Ca²⁺ spiking evoked by acetylcholine or cholecystokinin. Palmitoleic acid ethyl ester (100 μM), an important mediator of alcohol-related pancreatitis, evoked a sustained elevation of [Ca²⁺]_i, which was markedly reduced by CRAC blockade. Importantly, the palmitoleic acid ethyl ester-induced trypsin and protease activity as well as necrosis were almost abolished by blocking CRAC channels. There is currently no specific treatment of pancreatitis, but our data show that pharmacological CRAC blockade is highly effective against toxic [Ca²⁺]_i elevation, necrosis, and trypsin/protease activity and therefore has potential to effectively treat pancreatitis.