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.     

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.     

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.     

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.     

Crosstalk between membrane potential and cytosolic Ca2+ concentration in beta cells from Sur1 −/− mice

Aims/hypothesis Islets or beta cells from Sur1^–/– mice were used to determine whether changes in plasma membrane potential (V_m) remain coupled to changes in cytosolic Ca²⁺ ([Ca²⁺]_i) in the absence of K_ATP channels and thus provide a triggering signal for insulin secretion. The study also sought to elucidate whether [Ca²⁺]_i influences oscillations in V_m in sur1^–/– beta cells.Methods Plasma membrane potential and ion currents were measured with microelectrodes and the patch–clamp technique. [Ca²⁺]_i was monitored with the fluorescent dye fura-2. Insulin secretion from isolated islets was determined by static incubations.Results Membrane depolarisation of Sur1^–/– islets by arginine or increased extracellular K⁺, elevated [Ca²⁺]_i and augmented insulin secretion. Oligomycin completely abolished glucose-stimulated insulin release from Sur1^–/– islets. Oscillations in V_m were influenced by [Ca²⁺]_i as follows: (1) elevation of extracellular Ca²⁺ lengthened phases of membrane hyperpolarisation; (2) simulating a burst of action potentials induced a Ca²⁺-dependent outward current that was augmented by increased Ca²⁺ influx through L-type Ca²⁺ channels; (3) Ca²⁺ depletion of intracellular stores by cyclopiazonic acid increased the burst frequency in Sur1^–/– islets, elevating [Ca²⁺]_i and insulin secretion; (4) store depletion activated a Ca²⁺ influx that was not inhibitable by the L-type Ca²⁺ channel blocker D600.Conclusions/interpretation Although V_m is largely uncoupled from glucose metabolism in the absence of K_ATP channels, increased electrical activity leads to elevations of [Ca²⁺]_i that are sufficient to stimulate insulin secretion. In Sur1^–/– beta cells, [Ca²⁺]_i exerts feedback mechanisms on V_m by activating a hyperpolarising outward current and by depolarising V_m via store-operated ion channels.     

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.     

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.     

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.     

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     

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 calcium ion response to glucose in beta-cells of calbindin-D28k nullmutant mice and in betaHC13 cells overexpressing calbindin-D28k

This article describes studies on the glucose-induced responses of intracellular Ca²⁺ concentration ([Ca²⁺]_i), insulin release, and redistribution of calbindin-D_28k, a calcium-binding regulatory protein, in β-cells of pancreatic islets of calbindin-D_28k knockout (KO) and wild-type mice (C57BL6) as well as in βHC-13 control cells and βHC-13 CaBP40 cells (β-cell line overexpressing calbindin-D_28k). Upon increasing the glucose concentration from 2.8 to 30 mM, islets of KO mice showed a significantly greater increase in [Ca²⁺]_i (mean increase in [Ca²⁺]_i, i.e., Δ[Ca²⁺], was 296 nM) compared with wild-type mice (Δ[Ca²⁺]_i=97 nM). βHC-13 CaBP40 cells showed little change in [Ca²⁺]_i upon elevation of glucose from 5.5 to 32.7 mM, whereas βHC-13 control cells exhibited significant increases in [Ca²⁺]_i (Δ[Ca²⁺]_i=510 nM). Similarly, upon addition of 30 mM glucose, the rate of insulin release increased from 25.2 (basal rate) to 145.2 pg/mL/min in βHC-13 control cells, whereas in βHC-13 CaBP40 cells the rate of insulin release was only 27.5 pg/mL/min in high glucose. Thus, levels of calbindin-D_28k in β-cells affect both [Ca²⁺]_i and insulin secretion in response to glucose. The three-dimensional reconstruct of confocal immunofluorescent images showed that glucose caused redistribution of calbindin-D_28k resulting in co-localization in the region of L-type voltage-dependent calcium channels (VDCC). This colocalization may be an important regulatory function concerning Ca²⁺ influx via L-type VDCC and exocytosis of insulin granules.     

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.     

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.     

Effect of stimulation and veratrine on total cellular calcium in rat and guinea-pig ventricular myocytes

Summary In rat cardiac myocytes, calcium efflux by Na⁺/Ca²⁺-exchange is expected only during ventricular systole following initial action potential repolarization. In contrast, in guinea-pigs, calcium influx via Na⁺/Ca²⁺-exchange is expected only during the initial portion of the action potential. Thus electrical stimulation is expected to result in reduced intracellular calcium ([Ca²⁺]_i) in rat and an increase in guinea pig. We tested this hypothesis by measuring total cellular calcium ([Ca]_tot) using⁴⁵Ca following stimulation of isolated rat and guinea-pig ventricular myocytes. Many studies have also emphasized that the rate and the direction of Na⁺/Ca²⁺-exchange across the sarcolemma are in part dependent on the magnitude of the transsarcolemmal sodium gradient. Thus, increasing intracellular sodium ([Na⁺]_i) is expected to result in an increased [Ca²⁺]_i. This hypothesis was also tested by measuring [Ca]_tot following veratrine administration. Enzymatically isolated rat and guinea-pig ventricular myocytes were divided into two groups; non-stimulated and stimulated (1 Hz). The concentration-dependent effects of veratrine (1,10,100 g/ml) on [Ca]_tot were determined in both these groups. In the absence of veratrine, non-stimulated rat myocytes had a significantly higher [Ca]_tot than did stimulated ones. Non-stimulated guinea-pig myocytes had a significantly lower [Ca]_tot when compared with stimulated ones Veratrine increased [Ca]_tot in both species in a concentration-dependent fashion. In addition, following veratrine the difference between [Ca]_tot in non-stimulated and stimulated rat myocytes was no longer significant. These results support those of others who have demonstrated that stimulation is associated with a gain of cellular calcium in both rabbit and guinea-pig ventricle and a calcium loss in rat ventricle. In addition, the increase in [Ca]_tot following veratrine was similar to the results of others that have shown an increase in [Ca²⁺]_i following exposure to veratrum alkaloids. Thus, we have demonstrated, using a technique limited to determination of [Ca]_tot, that this parameter may reflect changes produced by alteration of Na⁺/Ca²⁺-exchange.     

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.     

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.     

Theoretical investigation of action potential duration dependence on extracellular Ca in human cardiomyocytes

Reduction in [Ca²⁺]_o prolongs the AP in ventricular cardiomyocytes and the QT_c interval in patients. Although this phenomenon is relevant to arrhythmogenesis in the clinical setting, its mechanisms are counterintuitive and incompletely understood. To evaluate in silico the mechanisms of APD modulation by [Ca²⁺]_o in human cardiomyocytes. We implemented the Ten Tusscher-Noble-Noble-Panfilov model of the human ventricular myocyte and modified the formulations of the rapidly and slowly activating delayed rectifier K⁺ currents (I_Kr and I_Ks) and L-type Ca²⁺ current (I_CaL) to incorporate their known sensitivity to intra- or extracellular Ca²⁺. Simulations were run with the original and modified models at variable [Ca²⁺]_o in the clinically relevant 1 to 3 mM range. The original model responds with APD shortening to decrease in [Ca²⁺]_o, i.e. opposite to the experimental observations. Incorporation of Ca²⁺ dependency of K⁺ currents cannot reproduce the inverse relation between APD and [Ca²⁺]_o. Only when I_CaL inactivation process was modified, by enhancing its dependency on Ca²⁺, simulations predict APD prolongation at lower [Ca²⁺]_o. Although Ca²⁺-dependent I_CaL inactivation is the primary mechanism, secondary changes in electrogenic Ca²⁺ transport (by Na⁺/Ca²⁺ exchanger and plasmalemmal Ca²⁺-ATPase) contribute to the reversal of APD dependency on [Ca²⁺]_o. This theoretical investigation points to Ca²⁺-dependent inactivation of I_CaL as a mechanism primarily responsible for the dependency of APD on [Ca²⁺]_o. The modifications implemented here make the model more suitable to analyze repolarization mechanisms when Ca²⁺ levels are altered.     

Intracellular calcium activates TRPM2 and its alternative spliced isoforms

Melastatin-related transient receptor potential channel 2 (TRPM2) is a Ca²⁺-permeable, nonselective cation channel that is involved in oxidative stress-induced cell death and inflammation processes. Although TRPM2 can be activated by ADP-ribose (ADPR) in vitro, it was unknown how TRPM2 is gated in vivo. Moreover, several alternative spliced isoforms of TRPM2 identified recently are insensitive to ADPR, and their gating mechanisms remain unclear. Here, we report that intracellular Ca²⁺ ([Ca²⁺]_i) can activate TRPM2 as well as its spliced isoforms. We demonstrate that TRPM2 mutants with disrupted ADPR-binding sites can be activated readily by [Ca²⁺]_i, indicating that [Ca²⁺]_i gating of TRPM2 is independent of ADPR. The mechanism by which [Ca²⁺]_i activates TRPM2 is via a calmodulin (CaM)-binding domain in the N terminus of TRPM2. Whereas Ca²⁺-mediated TRPM2 activation is independent of ADPR and ADPR-binding sites, both [Ca²⁺]_i and the CaM-binding motif are required for ADPR-mediated TRPM2 gating. Importantly, we demonstrate that intracellular Ca²⁺ release activates both recombinant and endogenous TRPM2 in intact cells. Moreover, receptor activation-induced Ca²⁺ release is capable of activating TRPM2. These results indicate that [Ca²⁺]_i is a key activator of TRPM2 and the only known activator of the spliced isoforms of TRPM2. Our findings suggest that [Ca²⁺]_i-mediated activation of TRPM2 and its alternative spliced isoforms may represent a major gating mechanism in vivo, therefore conferring important physiological and pathological functions of TRPM2 and its spliced isoforms in response to elevation of [Ca²⁺]_i.