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.     

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.     

Intracellular [Na<Superscript>+</Superscript>] modulates synergy between Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger and L-type Ca<Superscript>2+</Superscript> current in cardiac excitation–contraction coupling during action potentials

Excitation–contraction coupling (ECC) in cardiac myocytes involves triggering of Ca²⁺ release from the sarcoplasmic reticulum (SR) by L-type Ca channels, whose activity is strongly influenced by action potential (AP) profile. The contribution of Ca²⁺ entry via the Na⁺/Ca²⁺ exchanger (NCX) to trigger SR Ca²⁺ release during ECC in response to an AP remains uncertain. To isolate the contribution of NCX to SR Ca²⁺ release, independent of effects on SR Ca²⁺ load, Ca²⁺ release was determined by recording Ca²⁺ spikes using confocal microscopy on patch-clamped rat ventricular myocytes with [Ca²⁺]_i fixed at 150 nmol/L. In response to AP clamps, normalized Ca²⁺ spike amplitudes (ΔF/F ₀) increased sigmoidally and doubled as [Na⁺]_i was elevated from 0 to 20 mmol/L with an EC₅₀ of ~10 mmol/L. This [Na⁺]_i-dependence was independent of I _Na as well as SR Ca²⁺ load, which was unchanged under our experimental conditions. However, NCX inhibition using either KB-R7943 or XIP reduced ΔF/F ₀ amplitude in myocytes with 20 mmol/L [Na⁺]_i, but not with 5 mmol/L [Na⁺]_i. SR Ca²⁺ release was complete before the membrane repolarized to −15 mV, indicating Ca²⁺ entry into the dyad (not reduced extrusion) underlies [Na⁺]_i-dependent enhancement of ECC. Because I _Ca,L inhibition with 50 mmol/L Cd²⁺ abolished Ca²⁺ spikes, our results demonstrate that during cardiac APs, NCX enhances SR Ca²⁺ release by synergistically increasing the efficiency of I _Ca,L-mediated ECC.     

Ranolazine improves diastolic dysfunction in isolated myocardium from failing human hearts--role of late sodium current and intracellular ion accumulation

The goal of this study was to test the hypothesis that the novel anti-ischemic drug ranolazine, which is known to inhibit late I_Na, could reduce intracellular [Na⁺]_i and diastolic [Ca²⁺]_i overload and improve diastolic function. Contractile dysfunction in human heart failure (HF) is associated with increased [Na⁺]_i and elevated diastolic [Ca²⁺]_i. Increased Na⁺ influx through voltage-gated Na⁺ channels (late I_Na) has been suggested to contribute to elevated [Na⁺]_i in HF. In isometrically contracting ventricular muscle strips from end-stage failing human hearts, ranolazine (10 µmol/L) did not exert negative inotropic effects on twitch force amplitude. However, ranolazine significantly reduced frequency-dependent increase in diastolic tension (i.e., diastolic dysfunction) by ~ 30% without significantly affecting sarcoplasmic reticulum (SR) Ca²⁺ loading. To investigate the mechanism of action of this beneficial effect of ranolazine on diastolic tension, Anemonia sulcata toxin II (ATX-II, 40 nmol/L) was used to increase intracellular Na⁺ loading in ventricular rabbit myocytes. ATX-II caused a significant rise in [Na⁺]_i typically seen in heart failure via increased late I_Na. In parallel, ATX-II significantly increased diastolic [Ca²⁺]_i. In the presence of ranolazine the increases in late I_Na, as well as [Na⁺]_i and diastolic [Ca²⁺]_i were significantly blunted at all stimulation rates without significantly decreasing Ca²⁺ transient amplitudes or SR Ca²⁺ content. In summary, ranolazine reduced the frequency-dependent increase in diastolic tension without having negative inotropic effects on contractility of muscles from end-stage failing human hearts. Moreover, in rabbit myocytes the increases in late I_Na, [Na⁺]_i and [Ca²⁺]_i caused by ATX-II, were significantly blunted by ranolazine. These results suggest that ranolazine may be of therapeutic benefit in conditions of diastolic dysfunction due to elevated [Na⁺]_i and diastolic [Ca²⁺]_i.     

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.     

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.     

Characterization of ionic currents and electrophysiological properties of goldfish somatotropes in primary culture

Growth hormone release in goldfish is partly dependent on voltage-sensitive Ca²⁺ channels but somatotrope electrophysiological events affecting such channel activities have not been elucidated in this system. The electrophysiological properties of goldfish somatotropes in primary culture were studied using the whole-cell and amphotericin B-perforated patch-clamp techniques. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) of identified somatotropes was measured using Fura-2/AM dye. Goldfish somatotropes had an average resting membrane potential of −78.4 ± 4.6 mV and membrane input resistance of 6.2 ± 0.2 GΩ. Voltage steps from a holding potential of −90 mV elicited a non-inactivating outward current and transient inward currents at potentials more positive than 0 and −30 mV, respectively. Isolated current recordings indicate the presence of 4-aminopyridine- and tetraethylammonium (TEA)-sensitive K⁺, tetrodotoxin (TTX)-sensitive Na⁺, and nifedipine (L-type)- and ω-conotoxin GVIA (N-type)-sensitive Ca²⁺ channels. Goldfish somatotropes rarely fire action potentials (APs) spontaneously, but single APs can be induced at the start of a depolarizing current step; this single AP was abolished by TTX and significantly reduced by nifedipine and ω-conotoxin GVIA. TEA increased AP duration and triggered repetitive AP firing resulting in an increase in [Ca²⁺]_i, whereas TTX, nifedipine and ω-conotoxin GVIA inhibited TEA-induced [Ca²⁺]_i pulses. These results indicate that in goldfish somatotropes, TEA-sensitive K⁺ channels regulate excitability while TTX-sensitive Na⁺ channels together with N- and L-type Ca channels mediates the depolarization phase of APs. Opening of voltage-sensitive Ca²⁺ channels during AP firing leads to increases in [Ca²⁺]_i.     

Effects of new intravascular contrast agents on [Ca2+]i transients and contraction in cultured ventricular myocytes

Summary We examined the effects of four kinds of intravascular contrast agents (amidtrizoic acid, iohexol, iopamidol, and ioxaglic acid) on [Ca²⁺]_i transients (indo-1 fluorescence) and cell contraction (video motion analyzer), using cultured chick embryo ventricular myocytes. Exposure of ventricular myocytes to amidtrizoic acid (a conventional contrast agent) reduced the [Ca²⁺]_i transients and the sensitivity of the contractile elements to [Ca²⁺]_i. Ioxaglic acid (a low osmotic contrast agent) also reduced the [Ca²⁺]_i transients, but did not significantly change the sensitivity of the contractile elements to [Ca²⁺]_i. Neither iohexol nor iopamidol (nonionic contrast agents) reduced the [Ca²⁺]_i transients, but both significantly decreased the sensitivity of the contractile elements to [Ca²⁺]_i. A marked negative inotropic effect of amidtrizoic acid was caused by both calcium binding and hypertonicity. The less marked depression of contractility produced by ioxaglic acid is possibly the result of calcium binding, but is not caused by hypertonicity. The negative inotropism produced by nonionic contrast agents (iohexol and iopamidol) was due to hypertonicity, but not due to alterations in the [Ca²⁺]_i transients.Exposure of ventricular myocytes to nonionic contrast agents (iohexol and iopamidol) slowed decay in the [Ca²⁺]_i transients with increased end-diastolic [Ca²⁺]_i. After washing out the nonionic contrast agents, these parameters returned to control levels. On the other hand, exposure to amidtrizoic acid decreased end-diastolic [Ca²⁺]_i without changing decay time in the [Ca²⁺]_i transients. After washing out amidtrizoic acid, there was a prolongation of half decay time in [Ca²⁺]_i transients with a significant increase in end-diastolic [Ca²⁺]_i and cell position. Diastolic dysfunction just after washout of amidtrizoic acid was possibly caused by an increase in [Na⁺]_i due to sodium influx during exposure to the contrast agent.     

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.     

Spontaneous and agonist-induced calcium oscillations in single human nonfunctioning adenoma cells

The effects of gonadotropin-releasing hormone (GnRH) and GnRH-associated peptide (GAP) on cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in 20 human nonfunctioning pituitary adenomas. We divided these tumors into three classes according to their response pattern to hypothalamic peptides. In type I adenomas (8 out of 20 adenomas), GnRH and GAP mobilized intracellular calcium ions stored in a thapsigargin (TG)-sensitive store. For the same concentration of agonist, two distinct patterns of GnRH-GAP-induced Ca²⁺ mobilization were observed (1) sinusoidal oscillations, and (2) monophasic transient. The latter is followed by a protein kinase C (PKC)-dependent increase in calcium influx through L-type channels. In type II adenomas (7 out of 20 adenomas), GnRH and GAP only stimulate calcium influx through dihydropyridine-sensitive Ca²⁺ channels by a PKC-dependent mechanism. TG (1 μM) did not affect [Ca²⁺]_i in these cells, suggesting that they do not possess TG-sensitive Ca²⁺ pools. All the effects of GnRH and GAP were blocked by an inhibitor of phospholipase C (PLC), suggesting that they were owing to the activation of the phosphoinositide turnover. Type I and type II adenoma cells showed spontaneous Ca²⁺ oscillations that were blocked by dihydropyridines and inhibition of PKC activity. GnRH and GAP had no effect on the [Ca²⁺]_i of type III adenoma cells that were also characterized by a low resting [Ca²⁺]_i and by the absence of spontaneous Ca²⁺ fluctuations. K⁺-induced depolarization provoked a reduced Ca²⁺ influx, whereas TG had no effect on the [Ca²⁺]_i of type III adenoma cells. The variety of [Ca²⁺]_i response patterns makes these cells a good cell model for studying calcium homeostasis in pituitary cells.     

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.     

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.     

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.     

Intracellular Levels of Na+ and TTX-sensitive Na+ Channel Current in Diabetic Rat Ventricular Cardiomyocytes

Intracellular Na⁺ ([Na⁺] _i ) is an important modulator of excitation–contraction coupling via regulating Ca²⁺ efflux/influx, and no investigation has been so far performed in diabetic rat heart. Here, we examined whether any change of [Na⁺] _i in paced cardiomyocytes could contribute to functional alterations during diabetes. Slowing down in depolarization phase of the action potential, small but significant decrease in its amplitude with a slight depolarized resting membrane potential was traced in live cardiomyocytes from diabetic rat, being parallel with a decreased TTX-sensitive Na⁺ channel current (I _Na) density. We recorded either [Na⁺] _i or [Ca²⁺] _i by using a fluorescent Na⁺ indicator (SBFI-AM or Na-Green) or a Ca²⁺ indicator (Fura 2-AM) in freshly isolated cardiomyocytes. We examined both [Na⁺] _i and [Ca²⁺] _i at rest, and also [Na⁺] _i during pacing with electrical field stimulation in a range of 0.2–2.0 Hz stimulation frequency. In order to test the possible contribution of Na⁺/H⁺ exchanger (NHE) to [Na⁺] _i , we examined the free cytoplasmic [H⁺] _i changes from time course of [H⁺] _i recovery in cardiomyocytes loaded with SNARF1-AM by using ammonium prepulse method. Our data showed that [Na⁺] _i in resting cells from either diabetic or control group was not significantly different, whereas the increase in [Na⁺] _i was significantly smaller in paced diabetic cardiomyocytes compared to that of the controls. However, resting [Ca²⁺] _i in diabetic cardiomyocytes was significantly higher than that of the controls. Here, a lower basal pH _i in diabetics compared with the controls correlates also with a slightly higher but not significantly different NHE activity and consequently a similar Na⁺ loading rate at resting state with a leftward shift in pH sensitivity of NHE-dependent H⁺-flux. NHE protein level remained unchanged, while protein levels of Na⁺/K⁺ ATPase and Na⁺/Ca²⁺ exchanger were decreased in the diabetic cardiomyocytes. Taken together, the present data indicate that depressed I _Na plays an important role in altered electrical activity with less Na⁺ influx during contraction, and an increased [Ca²⁺] _i load in these cells seems to be independent of [Na⁺] _i . The data with insulin treatment suggest further a recent balance between Na⁺ influx and efflux proteins associated with the [Na⁺] _i , particularly during diabetes.     

Experimental diabetes enhances Ca2+ mobilization and glutamate exocytosis in cerebral synaptosomes from mice

The present study was conducted to investigate the effects of the diabetic condition on the Ca²⁺ mobilization and glutamate release in cerebral nerve terminals (synaptosomes). Diabetes was induced in male mice by intraperitoneal injection of streptozotocin. Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and glutamate release in synaptosomes were determined using fura-2 and enzyme-linked fluorometric assay, respectively. Diabetes significantly enhanced the ability of the depolarizing agents K⁺ and 4-aminopyridine (4-AP) to increase [Ca²⁺]_i. In addition, diabetes significantly enhanced K⁺- and 4-AP-evoked Ca²⁺-dependent glutamate release. The pretreatment of synaptosomes with a combination of ω-agatoxin IVA (a P-type Ca²⁺ channel blocker) and ω-conotoxin GVIA (an N-type Ca²⁺ channel blocker) inhibited K⁺- or 4-AP-induced increases in [Ca²⁺]_i and Ca²⁺-dependent glutamate release in synaptosomes from the control and diabetic mice to a similar extent, respectively. These results indicate that diabetes enhances a K⁺- or 4-AP-evoked Ca²⁺-dependent glutamate release by increasing [Ca²⁺]_i via stimulation of Ca²⁺ entry through both P- and N-type Ca²⁺ channels.     

Na+/H+ exchange and its inhibition in cardiac ischemia and reperfusion

Summary The characterization of various ion transport systems has led to a better understanding of the effects, which seem to take part in the impairment of ischemic and reperfused cardiac tissue. This review discusses the role of the Na⁺/H⁺ exchange system in the pathophysiology of ischemia and reperfusion and the beneficial effects of its inhibition.At the onset of ischemia intracellular pH (pH_i) decreases due to anaerobic metabolism and ATP hydrolysis, leading to an activation of Na⁺/H⁺ exchange. This in turn increases intracellular Na⁺ (Na⁺ _i) and activates Na⁺/K⁺ ATPase, with a consecutive increase of energy consumption. Since cellular Na⁺ and Ca⁺⁺ transport are coupled by the Na⁺/Ca⁺⁺ exchange system, which depends on the Na⁺ gradient, the high Na⁺ _i leads to increased intracellular Ca⁺⁺ (Ca⁺⁺ _i). After a certain period, Na⁺/H⁺ exchange is inactivated by a decrease of extracellular pH.In case of reperfusion the acid extracellular fluid is washed out, which reactivates Na⁺/H⁺ exchange, leading to an unfavourably fast restoration of pH_i and a second time to Na⁺ and Ca⁺⁺ _i overflow.High Ca⁺⁺ _i is assumed to be one of the main reasons for ischemic and reperfusion injury, like arrhythmias, myocardial contracture, stunning and necrosis.It seems that the inhibition of Na⁺/H⁺ exchange can interrupt this process at an early phase and prevent or delay the consequences of ischemia and reperfusion as demonstrated by numerous investigators.     

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.     

Involvement of protein kinase C and intracellular Ca in goldfish brain somatostatin-28 inhibitory action on growth hormone release in goldfish

Goldfish brain somatostatin-28 (gbSS-28) is present in brain and pituitary tissues of goldfish. We assessed whether gbSS-28 targets Ca²⁺ and/or protein kinase C (PKC)-dependent signaling cascades in inhibiting growth hormone (GH) release. gbSS-28 decreased basal GH release from primary cultures of dispersed goldfish pituitary cells and intracellular free calcium levels ([Ca²⁺]_i) in goldfish somatotropes. gbSS-28 partially reduced [Ca²⁺]_i and GH responses induced by two endogeneous gonadotropin-releasing hormones (GnRHs), salmon (s)GnRH and chicken (c)GnRH-II. Furthermore, gbSS-28 reduced GH increases and abolished [Ca²⁺]_i elevations elicited by two PKC activators, tetradecanoyl 4β-phorbol-13-acetate and dioctanyl glycerol. The PKC inhibitors Gö6976 and Bis II abolished [Ca²⁺]_i responses to PKC activators, but only attenuated GnRH-induced increases in [Ca²⁺]_i and did not alter basal [Ca²⁺]_i. In cells pretreated with Bis II, gbSS-28 further reduced basal [Ca²⁺]_i. Our results suggest that gbSS-28 inhibits GnRH-induced GH release in part by attenuating PKC-mediated GnRH [Ca²⁺]_i signals. gbSS-28 reduces basal GH release also via reduction in [Ca²⁺]_i but PKC is not involved in this regard.     

Activated human platelet products induce proarrhythmic effects in ventricular myocytes

Sudden cardiac death remains one of the most prevalent modes of death and is mainly caused by ventricular fibrillation (VF) in the setting of acute ischemia resulting from coronary thrombi. Animal experiments have shown that platelet activation may increase susceptibility of ischemic myocardium to VF, but the mechanism is unknown. In the present study, we evaluated the effects of activated blood platelet products (ABPPs) on electrophysiological properties and intracellular Ca²⁺ (Ca²⁺_i) homeostasis. Platelets were collected from healthy volunteers. After activation, their secreted ABPPs were added to superfusion solutions. Rabbit ventricular myocytes were freshly isolated, and membrane potentials and Ca²⁺_i were recorded using patch-clamp methodology and indo-1 fluorescence measurements, respectively. ABPPs prolonged action potential duration and induced early and delayed afterdepolarizations. ABPPs increased L-type Ca²⁺ current (I_Ca,L) density, but left densities of sodium current, inward rectifier K⁺ current, transient outward K⁺ current, and rapid component of the delayed rectifier K⁺ current unchanged. ABPPs did not affect kinetics or (in)activation properties of membrane currents. ABPPs increased systolic Ca²⁺_i, Ca²⁺_i transient amplitude, and sarcoplasmic reticulum Ca²⁺ content. ABPPs did not affect the Na⁺− Ca²⁺ exchange current (I_NCX) in Ca²⁺-buffered conditions. Products secreted from activated human platelets induce changes in I_Ca,L and Ca²⁺_i, which result in action potential prolongation and the occurrence of early and delayed afterdepolarizations in rabbit myocytes. These changes may trigger and support reentrant arrhythmias in ischemia models of coronary thrombosis.