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.     

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.     

Calcium-sensitizing inotropic agents in the treatment of heart failure: A critical view

Summary Interventions that augment the contractile state of the heart are associated with, or caused by, alterations in Ca²⁺ exchange in heart muscles. New inotropic agents have been developed that increase the sensitivity of the myofilaments to Ca²⁺. To examine the effect of calcium-sensitizing agents on force development, we measured systolic and diastolic intracellular Ca²⁺ concentration ([Ca²⁺]_i) and constructed [Ca²⁺]_i-force relationships in normal (n=6) and myopathic human hearts (n=10). Using the bioluminescent calcium indicator aequorin, we found that the diastolic [Ca²⁺]_i was 225±52 nM in normal muscles, whereas in myopathic muscles diastolic [Ca²⁺]_i was significantly higher at 361±68 nM. Calcium-sensitizing agents that shift the [Ca²⁺]_i-force relationship toward lower [Ca²⁺]_i increase the diastolic force of myopathic hearts significantly more than in normal human hearts. This leads us to the conclusion that inotropic agents that increase the sensitivity of the myofilaments to Ca²⁺ further impair relaxation in myopathic hearts, resulting in a reduced contractile reserve and diminished actice force production.     

Mitochondrial free calcium regulation during sarcoplasmic reticulum calcium release in rat cardiac myocytes

Cardiac mitochondria can take up Ca²⁺, competing with Ca²⁺ transporters like the sarcoplasmic reticulum (SR) Ca²⁺-ATPase. Rapid mitochondrial [Ca²⁺] transients have been reported to be synchronized with normal cytosolic [Ca²⁺]_i transients. However, most intra-mitochondrial free [Ca²⁺] ([Ca²⁺]_mito) measurements have been uncalibrated, and potentially contaminated by non-mitochondrial signals. Here we measured calibrated [Ca²⁺]_mito in single rat myocytes using the ratiometric Ca²⁺ indicator fura-2 AM and plasmalemmal permeabilization by saponin (to eliminate cytosolic fura-2). The steady-state [Ca²⁺]_mito dependence on [Ca²⁺]_i (with 5 mM EGTA) was sigmoid with [Ca²⁺]_mito < [Ca²⁺]_i for [Ca²⁺]_i below 475 nM. With low [EGTA] (50 μM) and 150 nM [Ca²⁺]_i (± 15 mM Na⁺) cyclical spontaneous SR Ca²⁺ release occurred (5–15/min). Changes in [Ca²⁺]_mito during individual [Ca²⁺]_i transients were small ( 2–10 nM/beat), but integrated gradually to steady-state. Inhibition SR Ca²⁺ handling by thapsigargin, 2 mM tetracaine or 10 mM caffeine all stopped the progressive rise in [Ca²⁺]_mito and spontaneous Ca²⁺ transients (confirming that SR Ca²⁺ releases caused the [Ca²⁺]_mito rise). Confocal imaging of local [Ca²⁺]_mito (using rhod-2) showed that [Ca²⁺]_mito rose rapidly with a delay after SR Ca²⁺ release (with amplitude up to 10 nM), but declined much more slowly than [Ca²⁺]_i (time constant 2.8 ± 0.7 s vs. 0.19 ± 0.06 s). Total Ca²⁺ uptake for larger [Ca²⁺]_mito transients was  0.5 μmol/L cytosol (assuming 100:1 mitochondrial Ca²⁺ buffering), consistent with prior indirect estimates from [Ca²⁺]_i measurements, and corresponds to  1% of the SR Ca²⁺ uptake during a normal Ca²⁺ transient. Thus small phasic [Ca²⁺]_mito transients and gradually integrating [Ca²⁺]_mito signals occur during repeating [Ca²⁺]_i transients.     

Tonic Regulation of Excitation–Contraction Coupling by Basal Protein Kinase C Activity in Isolated Cardiac Myocytes

A high-speed imaging technique was used to investigate the effects of inhibitors and activators of protein kinase C (PKC) on the [Ca²⁺]_i transients and contraction of fura-2 loaded rat ventricular cardiac myocytes. The amplitude of the [Ca²⁺]_i transient was reduced following treatment with 100 nm phorbol 12,13-dibutyrate (PDBu), whereas the PKC inhibitors staurosporine (0.5μm) and calphostin C (10μm) increased [Ca²⁺]_i transient amplitude, elevated basal [Ca²⁺]_i and slowed the decay of the [Ca²⁺]_i transient. These changes were paralleled by similar alterations in the rate and extent of cell shortening. The activity of nitrendipine-sensitive Ca²⁺ channels was monitored indirectly as the rate of Mn²⁺ quench of cytosolic fura-2 in electrically-paced cells. PDBu reduced Mn²⁺ influx by six-fold, whereas staurosporine and calphostin C increased the influx rate by eight-fold and seven-fold over basal quench, respectively. The caffeine releasable Ca²⁺ pool was reduced in the presence of PDBu and increased transiently in presence of staurosporine. The effects of PKC activation and inhibition on sarcoplasmic reticulum Ca²⁺ content may be secondary to alterations of sarcolemmal Ca²⁺ influx. However, the PKC inhibitors also decreased the rate of sarcoplasmic reticulum Ca²⁺ uptake in permeabilized myocytes, suggesting that a direct effect of PKC on the sarcoplasmic reticulum may contribute to the prolongation of the [Ca²⁺]_i transient under these conditions. The present work demonstrates that basal PKC activity has a potent depressant effect, mediated primarily through inhibition of sarcolemmal Ca²⁺ influx, which may play a key role in setting the basal tone of cardiac muscle.     

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.     

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.     

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.     

Different features of Ca2+ oscillations in differentiated and undifferentiated hepatocyte doublets

Cytosolic free Ca²⁺ ([Ca²⁺]_i) oscillations are postulated to play a critical role in cellular proliferation. By using doublets of normal rats (NR) and those 18 hours after two-thirds hepatectomy (PHR), we investigated cytosolic free Ca²⁺ ([Ca²⁺]_i) responses in liver regeneration. Normal rat hepatocyte doublets that retain their bile canaliculi are polarized and well differentiated. PHR doublets, which also retain their bile canaliculi, were characterized as undifferentiated by (1) decreased canalicular secretion of fluorescein-isothiocyanate-labeled glycocholate; (2) increased labeling index of hepatocytes in BrdU staining (∼30%); and (3) impaired transfer of fluorescent dye injected into one cell of the pair to the other. Addition of phenylephrine to NR and PHR doublets in the presence of extracellular Ca²⁺ resulted in [Ca²⁺]_i oscillations or a nonoscillatory-sustained increase in [Ca²⁺]_i, followed by a gradual return to the baseline. Extracellular Ca²⁺ was not required for [Ca²⁺]_i, oscillations but was necessary for a sustained increase in [Ca²⁺]_i. Simultaneous addition of prazosin, al-receptor blocker, to doublets immediately abolished these [Ca²⁺]_i, responses. The [Ca²⁺]_i level in each of the adjacent cells was synchronous in sustained increase in [Ca²⁺]_i, but asynchronous in [Ca²⁺]_i oscillations. As the phenylephrine concentration was increased (1 to 100 μmol/L), oscillations were replaced by a sustained increase in [Ca²⁺]_i in NR doublets. In contrast, in PHR doublets, oscillations remained, whereas the frequency of oscillations increased in a dose-dependent manner. These results indicate that the mechanisms of phenylephrineevoked [Ca²⁺]_i responses are different in differentiated and undifferentiated doublets and that the frequency modulation of [Ca²⁺]_i oscillations may be involved in the intracellular signal transduction in the cellular proliferation process during liver regeneration.     

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.     

Induced Overexpression of Na+/Ca2+ Exchanger Does Not Aggravate Myocardial Dysfunction Induced by Transverse Aortic Constriction

BackgroundAlterations in expression and activity of cardiac Na⁺/Ca²⁺ exchanger (NCX1) have been implicated in the pathogenesis of heart failure.Methods and ResultsUsing transgenic mice in which expression of rat NCX1 was induced at 5 weeks of age, we performed transverse aortic constriction (TAC) at 8 weeks and examined cardiac and myocyte function at 15–18 weeks after TAC (age 23–26 weeks). TAC induced left ventricular (LV) and myocyte hypertrophy and increased myocardial fibrosis in both wild-type (WT) and NCX1-overexpressed mice. NCX1 and phosphorylated ryanodine receptor expression was increased by TAC, whereas sarco(endo)plasmic reticulum Ca²⁺-ATPase levels were decreased by TAC. Action potential duration was prolonged by TAC, but to a greater extent in NCX1 myocytes. Na⁺/Ca²⁺ exchange current was similar between WT-TAC and WT-sham myocytes, but was higher in NCX1-TAC myocytes. Both myocyte contraction and [Ca²⁺]_i transient amplitudes were reduced in WT-TAC myocytes, but restored to WT-sham levels in NCX1-TAC myocytes. Despite improvement in single myocyte contractility and Ca²⁺ dynamics, induced NCX1 overexpression in TAC animals did not ameliorate LV hypertrophy, increase ejection fraction, or enhance inotropic (maximal first derivative of LV pressure rise, +dP/dt) responses to isoproterenol.ConclusionsIn pressure-overload hypertrophy, induced overexpression of NCX1 corrected myocyte contractile and [Ca²⁺]_i transient abnormalities but did not aggravate or improve myocardial dysfunction.     

Platelet Cytosolic free Calcium Before and After Antihypertensive Treatment in Perinephritis Hypertension of the Rabbit

Cytosolic free calcium concentration ([Ca²⁺]_i) in platelets has been reported to be elevated in human essential hypertension, to be positively correlated with blood pressure and to decrease with blood pressure reduction. However, some groups have been unable to confirm these findings in either humans or hypertensive rats. We have examined the relationship between platelet [Ca²⁺]_i and blood pressure in the perinephritis model of hypertension in the rabbit. In addition, the effects of both acute and chronic treatment with verapamil or prazosin were studied. Mean arterial pressure, heart rate and platelet [Ca²⁺]_i were measured before and after treatment. Platelet [Ca²⁺]_i was measured by the Quin 2 fluorescence technique. Platelet [Ca²⁺]_i was similar for the normotensive and hypertensive rabbits, and no correlation between platelet [Ca²⁺]_i and blood pressure was observed. None of the antihypertensive treatments produced a lowering of platelet [Ca²⁺]_i. Therefore we conclude that platelet [Ca²⁺]_i is unlikely to be a universally useful index of [Ca²⁺]_i in vascular smooth muscle of resistance vessels.     

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.     

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.     

Recruitment of individually (all-or-none) responding cells, rather than amplitude enhancement, is the single-cell mechanism subserving the dose-responsive activation of intracellular calcium second messenger signaling by the human lutinizing-hormone receptor

We have investigated at the single-cell level how the human LH receptor mediates a dose-responsive increase in intracellular free calcium-ion concentrations ([Ca²⁺]_i). In human embryonic kidney cells (293 cells) stably transfected with the full-length human LH receptor cDNA. Intact dimeric LH, but not LH β- or α-subunits, evoked specific [Ca²⁺]_i signals. High-resolution fluorescence (fura-2) video-microscopy demonstrated cell-to-cell variability in [Ca²⁺]_i signaling responses in individual cells, viz., an all-or-none spike (9%), spike-and-plateau (25%), or plateau (52%) types of temporal signal. Oscillatory [Ca²⁺]_i responses were observed in 12–14% of LH-stimulated cells unrelated to LH concentration. The LH dose-response originated by higher concentrations of LH recruiting more individually responding cells (rather than altering [Ca²⁺]_i signal amplitude), and eliciting a [Ca²⁺]_i rise more rapidly, i.e., at reduced latency. Cobalt did not abolish the LH-stimulated [Ca²⁺]_i spike-and-lateau response, but decreased the percentage of cells with a plateau pattern. Quench experiments demonstrated influx of Mn²⁺ following the [Ca²⁺]_i spike, thus directly documenting divalent cation inflow during the plateau phase. Adenylyl-cyclase activation with forskolin or treatment with a cAMP analog failed to elicit the biphasic [Ca²⁺]_i resoonse, and pertussis toxin (PTX) did not alter LH-stimulated [Ca²⁺]_i signaling. However, overnight preincubation with LH reduced the percentage of [Ca²⁺]_i-responding cells following re-exposure to LH to 5.7% (vs 72% in control), suggesting LH-induced desensitization of the LH-receptor directed [Ca²⁺]_i signal. In summary, the present studies of human LH receptor signal transduction at the single-cell level show that increasing concentrations of LH achieve a dose-dependent intracellular Ca²⁺ signaling response by recruiting an increasing number of [Ca²⁺]_i-responding cells, while concomitantly decreasing the temporal latency of the biphasic [Ca²⁺]_i signal without altering the amplitude of its spike phase. Prolonged exposure to LH appears to desensitize the LH receptor-driven [Ca²⁺]_i signal.     

Differential alteration of cardiotonic effects of EMD 57033 and β-adrenoceptor agonists in volume-overload rabbit ventricular myocytes

Background: We investigated the effects of EMD 57033, a prototype Ca²⁺ sensitizer, and β-adrenoceptor agonists in ventricular myocytes isolated from the volume-overload (V-O) heart failure model of the rabbit. Methods and Results: V-O cardiac hypertrophy was induced in rabbits by the formation of an arterio-venous shunt between the carotid artery and jugular vein 12 to 15 weeks after the operation. Ventricular myocytes were enzymically isolated from normal and V-O rabbit hearts. The myocyte was loaded with a fluorescence Ca²⁺ dye, indo-1, and Ca²⁺ transients, and cell lengths were measured simultaneously. V-O myocytes were significantly larger than control myocytes. Duration of Ca²⁺ transients and cell shortening was significantly longer in the V-O myocytes than in control myocytes. Effects of cardiotonic interventions, including EMD 57033, isoproterenol, and dobutamine, on Ca²⁺ transients and cell shortening in V-O myocytes were compared with those in control rabbit myocytes. Isoproterenol and dobutamine increased the systolic cell shortening and peak Ca²⁺ transients and abbreviated the duration of cell shortening and Ca²⁺ transients. These responses were markedly attenuated in V-O myocytes. By contrast, the response of cell shortening to EMD 57033 was unaltered, and the Ca²⁺ sensitizing effect of EMD 57033 was rather enhanced in V-O myocytes. Conclusion: Our results indicate that the effectiveness of Ca²⁺ sensitizers is maintained in the V-O rabbit hypertrophy and heart failure model in contrast to the blunted response to β-adrenoceptor agonists, which provides an insight on therapeutic strategy with Ca²⁺ sensitizers for the treatment of contractile dysfunction in congestive heart failure.     

Nisoldipine blocks the increase of intracellular free calcium-ion concentration associated with elevated sodium–lithium countertransport activity in erythrocytes in patients with NIDDM

To understand the mechanism by which elevated sodium–lithium countertransport activity (SLC) associates with increased intracellular free calcium-ion concentration ([Ca²⁺]_i), we investigated the relationship between SLC and the effects of the extracellular Ca²⁺ concentration ([Ca²⁺]_o) and a Ca²⁺-channel blocker, nisoldipine, on [Ca²⁺]_i in erythrocytes from 48 patients with non-insulin-dependent (Type 2) diabetes mellitus (NIDDM). There was a significant correlation between SLC and [Ca²⁺]_i. Nisoldipine in the incubation medium significantly decreased [Ca²⁺]_i, and there was a significant positive correlation between SLC and the degree of [Ca²⁺]_i decrease. When the [Ca²⁺]_o was elevated, [Ca²⁺]_i was significantly increased, but nisoldipine almost completely suppressed this increase of [Ca²⁺]_i. There was a significant positive correlation between SLC and the degree of the suppression. These data suggest that elevated SLC correlates with increased [Ca²⁺]_i, and that the increased [Ca²⁺]_i might be due to the increased Ca²⁺ influx through a dihydropyridine-sensitive Ca²⁺ pathway. © 1997 by John Wiley & Sons, Ltd.     

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.     

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.