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.     

Effects of KT-362, a sarcolemmal and intracellular calcium antagonist, on calcium transients of cultured neonatal rat ventricular cells: A comparison with gallopamil and ryanodine

Summary We evaluated the effects of KT-362 (5-[3-([2-(3,4-dimethoxyphenyl)-ethyl]amino)-1-oxopropyl]-2,3,4,5-tetrahydro-1,5-benzothiazepine fumarate), a putative intracellular calcium antagonist, on the intracellular free calcium concentration ([Ca²⁺]_i) of cultured neonatal rat ventricular cells using microfluorometry of fura-2. The effects were compared with those of gallopamil (D-600), a sarcolemmal calcium channel antagonist, and ryanodine, a modulator of sarcoplasmic reticulum (SR) function. KT-362 decreased both systolic [Ca²⁺]_i (sCa) and diastolic [Ca²⁺]_i (dCa) in cell aggregates, in a concentration (1, 3, 10, and 30 M) and stimulation frequency (0.2, 0.5, and 1.0 Hz) dependent manner. The time to peak of the Ca²⁺ transient was significantly prolonged by KT-362 at a concentration of 30 M, while the half-life of the Ca²⁺ transient was prolonged at concentrations of 10 M. Gallopamil (1 M) decreased both sCa and dCa in a frequency (0.2, 0.5, and 1.0 Hz) dependent fashion, as was the case for KT-362, but did not change the time course of Ca²⁺ transients. Ryanodine (10 M) prolonged the time to peak and half-life of the Ca²⁺ transient, as was also the case for KT-362, while the effect of ryanodine on dCa differed from that of KT-362. Finally, the effect of KT-362 on Ca²⁺ transients could be mimicked by simultaneous application of gallopamil and ryanodine. These results suggest that KT-362 is a novel compound that exerts depressant effects on both sarcolemmal Ca²⁺ channels, and perhaps Ca²⁺ release channels of the sarcoplasmic reticulum, in cultured neonatal rat ventricular cells.     

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.     

Changes of intra-mitochondrial Ca in adult ventricular cardiomyocytes examined using a novel fluorescent Ca indicator targeted to mitochondria

In this study a Ca²⁺ sensitive protein was targeted to the mitochondria of adult rabbit ventricular cardiomyocytes using an adenovirus transfection technique. The probe (Mitycam) was a Ca²⁺-sensitive inverse pericam fused to subunit VIII of human cytochrome c oxidase. Mitycam expression pattern and Ca²⁺ sensitivity was characterized in HeLa cells and isolated adult rabbit cardiomyocytes. Cardiomyocytes expressing Mitycam were voltage-clamped and depolarized at regular intervals to elicit a Ca²⁺ transient. Cytoplasmic (Fura-2) and mitochondrial Ca²⁺ (Mitycam) fluorescence were measured simultaneously under a range of cellular Ca²⁺ loads. After 48 h post-adenoviral transfection, Mitycam expression showed a characteristic localization pattern in HeLa cells and cardiomyocytes. The Ca²⁺ sensitive component of Mitycam fluorescence was 12% of total fluorescence in HeLa cells with a K_d of  220 nM. In cardiomyocytes, basal and beat-to-beat changes in Mitycam fluorescence were detected on initiation of a train of depolarizations. Time to peak of the mitochondrial Ca²⁺ transient was slower, but the rate of decay was faster than the cytoplasmic signal. During spontaneous Ca²⁺ release the relative amplitude and the time course of the mitochondrial and cytoplasmic signals were comparable. Inhibition of mitochondrial respiration decreased the mitochondrial transient amplitude by  65% and increased the time to 50% decay, whilst cytosolic Ca²⁺ transients were unchanged. The mitochondrial Ca²⁺ uniporter (mCU) inhibitor Ru360 prevented both the basal and transient components of the rise in mitochondrial Ca²⁺. The mitochondrial-targeted Ca²⁺ probe indicates sustained and transient phases of mitochondrial Ca²⁺ signal, which are dependent on cytoplasmic Ca²⁺ levels and require a functional mCU.     

Ghrelin-induced growth hormone release from goldfish pituitary cells involves voltage-sensitive calcium channels

Ghrelin (GRL) is a stimulator of growth hormone (GH) release in many organisms, including goldfish. As a first study to examine the signalling mechanisms mediating GRL action on GH release in goldfish, we tested the hypothesis that GLR induces GH release from goldfish pituitary cells by enhancing Ca²⁺ entry through L-type voltage-sensitive Ca²⁺ channels (LVSCCs) using perifusion GH release and fura-2/AM Ca²⁺-imaging experiments. Goldfish (g)GRL₁₉ at 1 nM elicited reversible and repeatable GH responses from dispersed goldfish mixed pituitary cultures. However, the lack of a dose-response relationship in sequential treatments with decreasing concentrations of gGRL₁₉ (ranging from 10 to 0.01 nM) implicated rapid desensitization of the GH response. Sequential applications of gGRL₁₉ (1 nM) and salmon GnRH (100 nM), a known Ca²⁺-dependent stimulator of GH release, increased intracellular free Ca²⁺ levels ([Ca²⁺]_i) from the same identified somatotropes, suggesting co-expression of GRL and GnRH receptors on single cells. In contrast, 1 nM gGRL₁₉ failed to elicit GH release and elevation in [Ca²⁺]_i when the cells are incubated with nominally Ca²⁺-free media. When GH release and [Ca²⁺]_i increases were already stimulated by the LVSCC agonist Bay K8644 (10 μM), addition of 1 nM gGRL₁₉ did not further elevate these responses. Finally, the LVSCC inhibitors nifedipine (1 μM) and verapamil (1 μM) abolished 1 nM gGRL₁₉-induced GH release responses while nifedipine eliminated gGRL₁₉-induced [Ca²⁺]_i increase. Taken together, the results of this study provide evidence that entry of extracellular Ca²⁺ through LVSCCs is a key component of the GRL signalling pathway leading to GH release in the goldfish pituitary.     

Involvement of Src in L-type Ca channel depression induced by macrophage migration inhibitory factor in atrial myocytes

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that controls inflammatory processes, and inflammation is known to play an important role in the pathogenesis of atrial fibrillation (AF). The present study sought to investigate whether MIF expression is responsible for the changes in L-type Ca²⁺ currents (I_Ca,L) seen in AF. Whole-cell voltage-clamp recordings and biochemical assays were used to study the regulation and expression of I_Ca,L in human atrial myocytes and in HL-1 cells. Basal I_Ca,L was reduced in AF compared to sinus rhythm (SR) controls, mRNA and protein levels of the pore-forming α_1C subunit of L-type Ca²⁺ channel (LCC α_1C) were also decreased, while MIF expression levels were increased in AF. Levels of Src and activated Src (p-Src Y⁴¹⁶) were higher in AF than in SR. Treatment of atrial myocytes from a patient with SR with human recombinant MIF (rMIF) (40 nM, 1 h) was found to depress I_Ca,L amplitudes, while mouse rMIF (20 or 40 nM, 24 h) suppressed peak I_Ca,L in HL-1 cells by  69% and  83% in a concentration-dependent manner. Mouse rMIF impaired the time-dependent recovery from inactivation of I_Ca,L and down-regulated LCC α_1C subunit levels. The depression of I_Ca,L and decrease of LCC protein levels induced by rMIF were prevented by the Src inhibitors genistein and PP1. These results implicate MIF in the electrical remodeling that accompanies AF, probably by decreasing I_Ca,L amplitudes through impairment of channel function, down-regulation of LCC α_1C subunit levels, and the activation of c-Src kinases in atrial myocytes.     

H2O2-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.     

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.     

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.     

Differences in intracellular calcium homeostasis between atrial and ventricular myocytes

The role that Ca²⁺ plays in ventricular excitation contraction coupling is well defined and much is known about the marked differences in the spatiotemporal properties of the systolic Ca²⁺ transient between atrial and ventricular myocytes. However, to date there has been no systematic appraisal of the Ca²⁺ homeostatic mechanisms employed by atrial cells and how these compare to the ventricle. In the present study we sought to determine the fractional contributions made to the systolic Ca²⁺ transient and the decay of [Ca²⁺]_i by the sarcoplasmic reticulum and sarcolemmal mechanisms. Experiments were performed on single myocytes isolated from the atria and ventricles of the rat. Intracellular Ca²⁺ concentration, membrane currents, SR Ca²⁺ content and cellular Ca²⁺ buffering capacity were measured at 23 °C. Atrial cells had smaller systolic Ca²⁺ transients (251 ± 39 vs. 376 ± 41 nmol.L^− 1) that decayed more rapidly (7.4 ± 0.6 vs. 5.45 ± 0.3 s^− 1). This was due primarily to an increased rate of SR mediated Ca²⁺ uptake (k_SR, 6.88 ± 0.6 vs. 4.57 ± 0.3 s^− 1). SR Ca²⁺ content was 289% greater and Ca²⁺ buffering capacity was increased ∼ 3-fold in atrial cells (B_max 371.9 ± 32.4 vs. 121.8 ± 8 μmol.L^− 1, all differences P < 0.05). The fractional release of Ca²⁺ from the SR was greater in atrial cells, although the gain of excitation contraction coupling was the same in both cell types. In summary our data demonstrate fundamental differences in Ca²⁺ homeostasis between atrial and ventricular cells and we speculate that the increased SR Ca²⁺ content may be significant in determining the increased prevalence of arrhythmias in the atria.     

Endothelin-induced changes in intracellular calcium concentration in rat vascular smooth muscle cells: Effects of extracellular calcium and atrial natriuretic factor

Summary The purpose of this study was to examine whether different mechanisms might underlie the changes in intracellular calcium concentration ([Ca²⁺]_i) stimulated by high and low concentrations of endothelin, and whether atrial natriuretic factor (ANF) has an inhibitory effect on endothelin-induced [Ca²⁺]_i changes in cultured rat vascular smooth muscle cells (VSMCs). In calcium-replete buffer, cultured monolayers of rat VSMCs superfused with endothelin at a high concentration (10 nM) exhibited a marked transient rise in [Ca²⁺]_i, followed by a sustained elevation, whereas a low concentration of endothelin (0.1 nM) induced a sustained monophasic elevation. When calcium-free buffer was used, 10 nM endothelin induced a transient rise in [Ca²⁺]_i of lesser amplitude, whereas 0.1 nM endothelin did not produce a significant rise. Pretreatment of VSMCs with ANF and cosuperfusion with endothelin failed to inhibit either transient or sustained endothelin-induced changes in [Ca²⁺]_i in calcium-replete buffer.     

Letm1, the mitochondrial Ca2+/H+ antiporter,is essential for normal glucose metabolism and alters brain function in Wolf–Hirschhorn syndrome

Mitochondrial metabolism, respiration, and ATP production necessitate ion transport across the inner mitochondrial membrane. Leucine zipper-EF-hand containing transmembrane protein 1 (Letm1), one of the genes deleted in Wolf–Hirschhorn syndrome, encodes a putative mitochondrial Ca²⁺/H⁺ antiporter. Cellular Letm1 knockdown reduced Ca²⁺_mito uptake, H⁺_mito extrusion and impaired mitochondrial ATP generation capacity. Homozygous deletion of Letm1 in mice resulted in embryonic lethality before day 6.5 of embryogenesis and ∼50% of the heterozygotes died before day 13.5 of embryogenesis. The surviving heterozygous mice exhibited altered glucose metabolism, impaired control of brain ATP levels, and increased seizure activity. We conclude that loss of Letm1 contributes to the pathology of Wolf–Hirschhorn syndrome in humans and may contribute to seizure phenotypes by reducing glucose oxidation and other specific metabolic alterations.Mitochondria are major effectors and regulators of intracellular [Ca²⁺]. Calcium-mediated signal transduction across the inner mitochondrial membrane (IMM) links increased metabolic demand to ATP production rate because Ca²⁺ regulates key metabolic enzymes, metabolite transporters, and the F₁F₀ H⁺-ATPase (1, 2). Excessive Ca²⁺ accumulation reduces mitochondrial membrane potential (ΔΨ_mito), impairs ATP production, precipitates phosphate, and triggers cell death. Ca²⁺ extrusion across the IMM represents a significant energy cost at normal ΔΨ_mito (∼−180 mV) and thus mitochondrial Ca²⁺ (Ca²⁺_mito) signaling is tightly controlled under physiological conditions (3–5).The mitochondrial Ca²⁺ uniporter (MCU), a highly Ca²⁺-selective channel (6), dominates fast Ca²⁺_mito uptake when [Ca²⁺]_cyto is high, significantly buffers extramitochondrial Ca²⁺, and depolarizes ΔΨ_mito (7, 8). Because the MCU’s V_max is orders of magnitude higher than that of Ca²⁺_mito exchange mechanisms, repetitive high Ca²⁺_cyto elevations trigger Ca²⁺_mito overload and can lead to the mitochondrial permeability transition (9, 10). Free [Ca²⁺]_mito in intact cells usually fluctuates below the micromolar range, suggesting that Ca²⁺ exchangers are crucial for maintaining Ca²⁺_mito homeostasis at low Ca²⁺_cyto levels to preserve mitochondrial homeostasis and bioenergetics. The Na⁺/Ca²⁺ exchanger, Ca²⁺/H⁺ antiporter, and potentially others, control Ca²⁺ across the IMM. Mitochondrial transporters and channels are rapidly being identified (11).We previously performed a genome-wide RNAi screen in Drosophila S2 cells that identified Letm1 (leucine zipper-EF-hand containing transmembrane protein 1) as a mitochondrial Ca²⁺/H⁺ antiporter (12). Letm1 is an evolutionarily conserved, ubiquitously expressed IMM protein. Heterozygous deletion of a chromosomal region containing Letm1 is associated with Wolf–Hirschhorn syndrome (WHS), a disease characterized by cranio-facial defects, growth and mental retardation, muscle hypotonia, congenital heart defects, and seizures (13). In the current study, we generated Letm1-deficient mice and found that Letm1 knockdown reduced Ca²⁺_mito uptake at low [Ca²⁺]_cyto, impaired mitochondrial ATP generation capacity, disrupted early embryonic development, altered glucose metabolism, and increased susceptibility to seizures. These results are consistent with the WHS seizure phenotype in humans and show that Letm1 alters mitochondrial energetic performance and metabolic pathways.     

Coupled Ca2+/H+ transport by cytoplasmic buffers regulates local Ca2+ and H+ ion signaling

Ca²⁺ signaling regulates cell function. This is subject to modulation by H⁺ ions that are universal end-products of metabolism. Due to slow diffusion and common buffers, changes in cytoplasmic [Ca²⁺] ([Ca²⁺]_i) or [H⁺] ([H⁺]_i) can become compartmentalized, leading potentially to complex spatial Ca²⁺/H⁺ coupling. This was studied by fluorescence imaging of cardiac myocytes. An increase in [H⁺]_i, produced by superfusion of acetate (salt of membrane-permeant weak acid), evoked a [Ca²⁺]_i rise, independent of sarcolemmal Ca²⁺ influx or release from mitochondria, sarcoplasmic reticulum, or acidic stores. Photolytic H⁺ uncaging from 2-nitrobenzaldehyde also raised [Ca²⁺]_i, and the yield was reduced following inhibition of glycolysis or mitochondrial respiration. H⁺ uncaging into buffer mixtures in vitro demonstrated that Ca²⁺ unloading from proteins, histidyl dipeptides (HDPs; e.g., carnosine), and ATP can underlie the H⁺-evoked [Ca²⁺]_i rise. Raising [H⁺]_i tonically at one end of a myocyte evoked a local [Ca²⁺]_i rise in the acidic microdomain, which did not dissipate. The result is consistent with uphill Ca²⁺ transport into the acidic zone via Ca²⁺/H⁺ exchange on diffusible HDPs and ATP molecules, energized by the [H⁺]_i gradient. Ca²⁺ recruitment to a localized acid microdomain was greatly reduced during intracellular Mg²⁺ overload or by ATP depletion, maneuvers that reduce the Ca²⁺-carrying capacity of HDPs. Cytoplasmic HDPs and ATP underlie spatial Ca²⁺/H⁺ coupling in the cardiac myocyte by providing ion exchange and transport on common buffer sites. Given the abundance of cellular HDPs and ATP, spatial Ca²⁺/H⁺ coupling is likely to be of general importance in cell signaling.Most cells are exquisitely responsive to calcium (Ca²⁺) (1) and hydrogen (H⁺) ions (i.e., pH) (2). In cardiac myocytes, Ca²⁺ ions trigger contraction and control growth and development (3), whereas H⁺ ions, which are generated or consumed metabolically, are potent modulators of essentially all biological processes (4). By acting on Ca²⁺-handling proteins directly or via other molecules, H⁺ ions exert both inhibitory and excitatory effects on Ca²⁺ signaling. For example, in the ventricular myocyte, H⁺ ions can reduce Ca²⁺ release from sarcoplasmic reticulum (SR) stores, through inhibition of the SR Ca²⁺ ATPase (SERCA) pump and ryanodine receptor (RyR) Ca²⁺ channels (5, 6). In contrast, H⁺ ions can enhance SR Ca²⁺ release by stimulating sarcolemmal Na⁺/H⁺ exchange (NHE), which raises intracellular [Na⁺] and reduces the driving force for Ca²⁺ extrusion on Na⁺/Ca²⁺ exchange (NCX), leading to cellular retention of Ca²⁺ (7, 8). Ca²⁺ signaling is thus subservient to pH.Cytoplasmic Ca²⁺ and H⁺ ions bind avidly to buffer molecules, such that <1% of all Ca²⁺ ions and <0.001% of all H⁺ ions are free. Some of these buffers bind H⁺ and Ca²⁺ ions competitively, and this has been proposed to be one mechanism underlying cytoplasmic Ca²⁺/H⁺ coupling (9). Reversible binding to buffers greatly reduces the effective mobility of Ca²⁺ and H⁺ ions in cytoplasm (10, 11) and can allow for highly compartmentalized ionic microdomains, and hence a spatially heterogeneous regulation of cell function. In cardiac myocytes under resting (diastolic) conditions, the cytoplasm-averaged concentration of free [Ca²⁺] ([Ca²⁺]_i) and [H⁺] ([H⁺]_i) ions is kept near 10⁻⁷ M by membrane transporter proteins. Thus, [H⁺]_i is regulated by the balance of flux among acid-extruding and acid-loading transporter proteins at the sarcolemma [e.g., NHE and Cl⁻/OH⁻ (CHE) exchangers, respectively] (4). Similarly, the activity of SERCA and NCX proteins returns [Ca²⁺]_i to its diastolic level after evoked signaling events (3, 12). Despite these regulatory mechanisms, cytoplasmic gradients of [H⁺]_i and [Ca²⁺]_i do occur in myocytes and are an important part of their physiology. Gradients arise from local differences in transmembrane fluxes that alter [H⁺]_i or [Ca²⁺]_i. For example, spatial [H⁺]_i gradients are produced when NHE transporters, expressed mainly at the intercalated disk region, are activated (4, 13) or when membrane-permeant weak acids, such as CO₂, are presented locally (14). Similarly, release of Ca²⁺ through a cluster of RyR channels in the SR produces [Ca²⁺]_i nonuniformity in the form of Ca²⁺ sparks (15). Given the propensity of cytoplasm to develop ionic gradients, it is important to understand their underlying mechanism and functional role.The present work demonstrates a distinct form of spatial interaction between Ca²⁺ and H⁺ ions. We show that cytoplasmic [H⁺] gradients can produce stable [Ca²⁺]_i gradients, and vice versa, and that this interaction is mediated by low-molecular-weight (mobile) buffers with affinity for both ions. We demonstrate that the diffusive counterflux of H⁺ and Ca²⁺ bound to these buffers comprises a cytoplasmic Ca²⁺/H⁺ exchanger. This acts like a “pump” without a membrane, which can, for instance, recruit Ca²⁺ to acidic cellular microdomains. Cytoplasmic Ca²⁺/H⁺ exchange adds a spatial paradigm to our understanding of Ca²⁺ and H⁺ ion signaling.     

Effects of unipolar stimulation on voltage and calcium distributions in the isolated rabbit heart

Background  The effect of electric stimulation on the polarization of cardiac tissue (virtual electrode effect) is well known; the corresponding response of intracellular calcium concentration ([Ca²⁺] _i ) and its dependence on coupling interval between conditioning stimulus (S1) and test stimulus (S2) has yet to be elucidated. Objective  Because uncovering the transmembrane potential (V _m)–[Ca²⁺] _i relationship during an electric shock is imperative for understanding arrhythmia induction and defibrillation, we aimed to study simultaneous V _m and [Ca²⁺] _i responses to strong unipolar stimulation. Methods  We used a dual-camera optical system to image concurrently V _m and [Ca²⁺] _i responses to unipolar stimulation (20 ms ± 20 mA) in Langendorff-perfused rabbit hearts. RH-237 and Rhod-2 fluorescent dyes were used to measure V _m and [Ca²⁺] _i , respectively. The S1–S2 interval ranged from 10 to 170 ms to examine stimulation during the action potential. Results  The [Ca²⁺] _i deflections were less pronounced than changes in V _m for all S1–S2 intervals. For cathodal stimulation, [Ca²⁺] _i at the central virtual cathode region increased with prolongation of S1–S2 interval. For anodal stimulation, [Ca²⁺] _i at the central virtual anode area decreased with shortening of the S1–S2 interval. At very short S1–S2 intervals (10–20 ms), when S2 polarization was superimposed on the S1 action potential upstroke, the [Ca²⁺] _i distribution did not follow V _m and produced a more complex pattern. After S2 termination [Ca²⁺] _i exhibited three outcomes in a manner similar to V _m: non-propagating response, break stimulation, and make stimulation. Conclusions  Changes in the [Ca²⁺] _i distribution correlate with the behavior of the V _m distribution for S1–S2 coupling intervals longer than 20 ms; at shorter intervals S2 creates more heterogeneous [Ca²⁺] _i distribution in comparison with V _m. Stimulation in diastole and at very short coupling intervals caused V _m–[Ca²⁺] _i uncoupling at the regions of positive polarization (virtual cathode). Returned for 1. Revision: 22 January 2008 1. Revision received: 13 May 2008 Returned for 2. Revision: 20 June 2008 2. Revision received: 2 July 2008     

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.     

Pancreatic beta-cells from obese-hyperglycemic mice are characterized by excessive firing of cytoplasmic Ca2+ transients

Pancreatic β-cells from obese-hyperglycemic (ob/ob) mice are widely used for studying the mechanisms of insulin release, including its regulation by the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). In this study, we compared changes of [Ca²⁺]_i in single β-cells isolated from ob/ob mice with those from lean mice using dual-wavelength microfluorometry and the indicator fura-2. There were no differences in the frequency, amplitude, and half-width of the slow oscillations induced by glucose. Most β-cells from the obese mice responded to 10 mM caffeine with transformation of the oscillations into sustained elevation of [Ca²⁺]_i, a process counteracted by ryanodine. The β-cells from the obese mice were characterized by ample generation of [Ca²⁺]_i transients, which increased in number in the presence of glucagon. The transients became less frequent when leptin was added at a concentration as low as 1 nM. It is suggested that the excessive firing of [Ca²⁺]_i transients in the ob/ob mice is owing to the absence of leptin and is mediated by activation of the phospholipase C signaling pathway.     

Acute hypoxia activates store-operated Ca2+ entry and increases intracellular Ca2+ concentration in rat distal pulmonary venous smooth muscle cells

Rationale

Exposure to acute hypoxia causes vasoconstriction in both pulmonary arteries (PA) and pulmonary veins (PV). The mechanisms on the arterial side have been studied extensively. However, bare attention has been paid to the venous side.

Objectives

To investigate if acute hypoxia caused the increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i), and Ca²⁺ influx through store-operated calcium channels (SOCC) in pulmonary venous smooth muscle cells (PVSMCs).

Methods

Fluorescent microscopy and fura-2 were used to measure effects of 4% O₂ on [Ca²⁺]_i and store-operated Ca²⁺ entry (SOCE) in isolated rat distal PVSMCs.

Measurements and main results

In PVSMCs perfused with Ca²⁺-free Krebs Ringer bicarbonate solution (KRBS) containing cyclopiazonic acid to deplete Ca²⁺ stores in the sarcoplasmic reticulum (SR) and nifedipine to prevent Ca²⁺ entry through L-type voltage-depended Ca²⁺ channels (VDCC), hypoxia markedly enhanced both the increase in [Ca²⁺]_i caused by restoration of extracellular [Ca²⁺] and the rate at which extracellular Mn²⁺ quenched fura-2 fluorescence. Moreover, the increased [Ca²⁺]_i in PVSMCs perfused with normal salt solution was completely blocked by SOCC antagonists SKF-96365 and NiCl₂ at concentrations that SOCE >85% was inhibited but [Ca²⁺]_i responses to 60 mM KCl were not altered. On the contrary, L-type VDCC antagonist nifedipine inhibited increase in [Ca²⁺]_i to hypoxia by only 50% at concentrations that completely blocked responses to KCl. The increased [Ca²⁺]_i caused by hypoxia was completely abolished by perfusion with Ca²⁺-free KRBS.

Conclusions

These results suggest that acute hypoxia enhances SOCE via activating SOCCs, leading to increased [Ca²⁺]_i in distal PVSMCs.KEYWORDS : Calcium signaling, pulmonary venous smooth muscle (PVSM), store-operated Ca²⁺ entry (SOCE), intracellular Ca²⁺ concentration ([Ca²⁺]_i)     

Adenosine 5'-triphosphate (ATP) receptors induce intracellular calcium changes in mouse leydig cells

Cytoplasmic calcium ([Ca²⁺]_i) changes evoked by adenosine 5¹-triphosphate (ATP) were recorded in cultured individual Leydig cells within 10–18 h after cell dispersion. [Ca²⁺]_i was monitored using Fura-2AM loaded cells with a digital ratio imaging system. Five micromolars ATP induced biphasic [Ca²⁺]_i responses in most cells (94%,n=100), characterized by a fast increase from a basal level (126±5 nMSE,n=60 cells) to a peak (5–7 times above basal levels) within seconds, followed by a slow decrease toward a plateau level (2–3 times above basal) within 5 min. The peak phase of the [Ca²⁺]_i response increased with ATP concentrations (1–100 μM ATP) in a dose-dependent manner with an IC₅₀ of 5.9±1.2 μM, and it desensitized in a reversible manner with repeated application of 5 μM ATP at <5-min intervals. The [Ca²⁺]_i peak response was dependent on Ca²⁺ release from an intracellular pool, whereas the plateau phase was dependent on extracellular [Ca²⁺]. ATP did not appear to induce formation of nonspecific membrane pores, since stimulation for 10 min with ATP (10–100 μM) in the presence of extracellular Lucifer yellow (LY) (5 mg/mL) did not result in dye loading of the cells. [Ca²⁺]_i transients were elicited by other adenosine nucleotides with an order of potencies (ATP>Adenosine diphosphate [ADP]>Adenosine> Adenosine monophosphate [AMP]) that was compatible with the expression of P₂ receptors. [Ca²⁺]_i responses were suppressed by the purinergic P₂ receptor antagonist, suramin. These results provide functional evidence for the expression of purinergic P₂ receptors in Leydig cells.     

Distinct modulation of myocardial performance,energy metabolism,and [Ca2+]i transients by positive inotropic drugs in normal and severely failing hamster hearts

Summary The present study compared the effects of amrinone, dobutamine, dibutyryl cAMP, digoxin, and isoproterenol on mechanical performance, the high energy phosphate metabolites, and the [Ca²⁺]_i transients in normal and cardiomyopathic hamster hearts with severe heart failure. In normal hearts dobutamine, dibutyryl cAMP, and isoproterenol increased left ventricular developed pressure, while amrinone and digoxin did not. However, the amplitude of [Ca²⁺]_i transients was augmented with all drugs. Diastolic [Ca²⁺]_i level was increased with dobutamine and lowered with dibutyryl cAMP and isoproterenol. In cardiomyopathic hearts with severe heart failure, left ventricular developed pressure, the amplitude of [Ca²⁺]_i transients, the phosphorylation potential, and [cAMP]_i were significantly depressed and left ventricular end-diastolic pressure and diastolic [Ca²⁺]_i were significantly elevated when compared with normal hearts. Amrinone, dibutyryl cAMP, and isoproterenol improved mechanical performance while increasing [cAMP]_i and the amplitude of [Ca²⁺]_i transients, and decreasing diastolic [Ca²⁺]_i. On the other hand, with dobutamine and digoxin diastolic [Ca²⁺]_i was further increased and mechanical performance deteriorated with digoxin. Thus, distinct differences exist in modulation of mechanical performance, high-energy phosphate metabolism, and [Ca²⁺]_i transients by positive inotropic drugs between normal and cardiomyopathic hearts with severe heart failure.Supported in part by the George D. Smith Foundation and NIH grant AA 07413-01. Peter Buser is a recipient of a Career Development Grant (SCORE # 32-29340,90) from the Swiss National Science Foundation.