The new HNO donor, 1-nitrosocyclohexyl acetate, increases contractile force in normal and β-adrenergically desensitized ventricular myocytes

Contractile dysfunction and diminished response to β-adrenergic agonists are characteristics for failing hearts. Chemically donated nitroxyl (HNO) improves contractility in failing hearts and thus may have therapeutic potential. Yet, there is a need for pharmacologically suitable donors. In this study we tested whether the pure and long acting HNO donor, 1-nitrosocyclohexyl acetate (NCA), affects contractile force in normal and pathological ventricular myocytes (VMs) as well as in isolated hearts. VMs were isolated from mice either subjected to isoprenaline-infusion (ISO; 30 μg/g per day) or to vehicle (0.9% NaCl) for 5 days. Sarcomere shortening and Ca²⁺ transients were simultaneously measured using the IonOptix system. Force of contraction of isolated hearts was measured by a Langendorff-perfusion system. NCA increased peak sarcomere shortening by + 40-200% in a concentration-dependent manner (EC₅₀ ∼55 μM). Efficacy and potency did not differ between normal and chronic ISO VMs, despite the fact that the latter displayed a markedly diminished inotropic response to acute β-adrenergic stimulation with ISO (1 μM). NCA (60 μM) increased peak sarcomere shortening and Ca²⁺ transient amplitude by ∼200% and ∼120%, respectively, suggesting effects on both myofilament Ca²⁺ sensitivity and sarcoplasmic reticulum (SR) Ca²⁺ cycling. Importantly, NCA did not affect diastolic Ca²⁺ or SR Ca²⁺ content, as assessed by rapid caffeine application. NCA (45 μM) increased force of contraction by 30% in isolated hearts. In conclusion, NCA increased contractile force in normal and β-adrenergically desensitized VMs as well as in isolated mouse hearts. This profile warrants further investigations of this HNO donor in the context of heart failure.     

Mitochondrial Ca influx and efflux rates in guinea pig cardiac mitochondria:Low and high affinity effects of cyclosporine A

Ca²⁺ plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca²⁺ is controlled primarily by the mitochondrial Ca²⁺ uniporter and the mitochondrial Na⁺/Ca²⁺ exchanger, influencing NADH production through Ca²⁺-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca²⁺-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca²⁺ release. Here we selectively measure Ca²⁺ influx rate through the mitochondrial Ca²⁺ uniporter and Ca²⁺ efflux rates through Na⁺-dependent and Na⁺-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na⁺/Ca²⁺ exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ_m loss, Ca²⁺ release, NADH oxidation, swelling) of high extramitochondrial Ca²⁺ additions, allowing mitochondria to tolerate total mitochondrial Ca²⁺ loads of > 400 nmol/mg protein. For Ca²⁺ pulses up to 15 μM, Na⁺-independent Ca²⁺ efflux through the permeability transition pore accounted for ~ 5% of the total Ca²⁺ efflux rate compared to that mediated by the mitochondrial Na⁺/Ca²⁺ exchanger (in 5 mM Na⁺). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na⁺/Ca²⁺ exchanger-mediated Ca²⁺ efflux at higher concentrations (IC₅₀ = 2 μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~ 40% at 10 μM cyclosporine A, while having no effect on the mitochondrial Ca²⁺ uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca²⁺ load in cardiomyocytes, potentially explaining the paradoxical toxic effects of cyclosporine A at high concentrations. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Cardiodepressive effect elicited by the essential oil of Alpinia speciosa is related to L-type Ca current blockade

This study was undertaken to elucidate the effect of the essential oil from Alpinia speciosa (EOAs) on cardiac contractility and the underlying mechanisms. The essential oil was obtained from Alpinia speciosa leaves and flowers and the oil was analyzed by GC-MS method. Chemical analysis revealed the presence of at least 18 components. Terpinen-4-ol and 1,8-cineole corresponded to 38% and 18% of the crude oil, respectively. The experiments were conducted on spontaneously-beating right atria and on electrically stimulated left atria isolated from adult rats. The effect of EOAs on the isometric contractions and cardiac frequency in vitro was examined. EOAs decreased rat left atrial force of contraction with an EC₅₀ of 292.2 ± 75.7 μg/ml. Nifedipine, a well known L-type Ca²⁺ blocker, inhibited in a concentration-dependent manner left atrial force of contraction with an EC₅₀ of 12.1 ± 3.5 μg/ml. Sinus rhythm was diminished by EOAs with an EC₅₀ of 595.4 ± 56.2 μg/ml. Whole-cell L-type Ca²⁺ currents were recorded by using the patch-clamp technique. EOAs at 25 μg/ml decreased I_Ca,L by 32.6 ± 9.2% and at 250 μg/ml it decreased by 89.3 ± 7.4%. Thus, inhibition of L-type Ca²⁺ channels is involved in the cardiodepressive effect elicited by the essential oil of Alpinia speciosa in rat heart.     

The dynamics of mitochondrial Ca fluxes

We have investigated the kinetics of mitochondrial Ca²⁺ influx and efflux and their dependence on cytosolic [Ca²⁺] and [Na⁺] using low-Ca²⁺-affinity aequorin. The rate of Ca²⁺ release from mitochondria increased linearly with mitochondrial [Ca²⁺] ([Ca²⁺]_M). Na⁺-dependent Ca²⁺ release was predominant al low [Ca²⁺]_M but saturated at [Ca²⁺]_M around 400 μM, while Na⁺-independent Ca²⁺ release was very slow at [Ca²⁺]_M below 200 μM, and then increased at higher [Ca²⁺]_M, perhaps through the opening of a new pathway. Half-maximal activation of Na⁺-dependent Ca²⁺ release occurred at 5-10 mM [Na⁺], within the physiological range of cytosolic [Na⁺]. Ca²⁺ entry rates were comparable in size to Ca²⁺ exit rates at cytosolic [Ca²⁺] ([Ca²⁺]_c) below 7 μM, but the rate of uptake was dramatically accelerated at higher [Ca²⁺]_c. As a consequence, the presence of [Na⁺] considerably reduced the rate of [Ca²⁺]_M increase at [Ca²⁺]_c below 7 μM, but its effect was hardly appreciable at 10 μM [Ca²⁺]_c. Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca²⁺]_M transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca²⁺ buffering, and comparison of our results with data on total mitochondrial Ca²⁺ fluxes indicate that the mitochondrial Ca²⁺ bound/Ca²⁺ free ratio is around 10- to 100-fold for most of the observed [Ca²⁺]_M range and suggest that massive phosphate precipitation can only occur when [Ca²⁺]_M reaches the millimolar range.     

Effects of endogenous cannabinoid anandamide on excitation–contraction coupling in rat ventricular myocytes

A role for anandamide (N-arachidonoyl ethanolamide; AEA), a major endocannabinoid, in the cardiovascular system in various pathological conditions has been reported in earlier reports. In the present study, the effects of AEA on contractility, Ca²⁺ signaling, and action potential (AP) characteristics were investigated in rat ventricular myocytes. Video edge detection was used to measure myocyte shortening. Intracellular Ca²⁺ was measured in cells loaded with the fluorescent indicator fura-2 AM. AEA (1 μM) caused a significant decrease in the amplitudes of electrically evoked myocyte shortening and Ca²⁺ transients. However, the amplitudes of caffeine-evoked Ca²⁺ transients and the rate of recovery of electrically evoked Ca²⁺ transients following caffeine application were not altered. Biochemical studies in sarcoplasmic reticulum (SR) vesicles from rat ventricles indicated that AEA affected Ca²⁺-uptake and Ca²⁺-ATPase activity in a biphasic manner. [³H]-ryanodine binding and passive Ca²⁺ release from SR vesicles were not altered by 10 μM AEA. Whole-cell patch-clamp technique was employed to investigate the effect of AEA on the characteristics of APs. AEA (1 μM) significantly decreased the duration of AP. The effect of AEA on myocyte shortening and AP characteristics was not altered in the presence of pertussis toxin (PTX, 2 μg/ml for 4 h), AM251 and SR141716 (cannabinoid type 1 receptor antagonists; 0.3 μM) or AM630 and SR 144528 (cannabinoid type 2 receptor antagonists; 0.3 μM). The results suggest that AEA depresses ventricular myocyte contractility by decreasing the action potential duration (APD) in a manner independent of CB1 and CB2 receptors.     

Quantitative measurement of Ca²(+) in the sarcoplasmic reticulum lumen of mammalian skeletal muscle

Skeletal muscle stores Ca²⁺ in the sarcoplasmic reticulum (SR) and releases it to initiate contraction, but the concentration of luminal Ca²⁺ in the SR ([Ca²⁺]_SR) and the amount that is released by physiological or pharmacological stimulation has been difficult to measure. Here we present a novel, yet simple and direct, method that provides the first quantitative estimates of static content and dynamic changes in [Ca²⁺]_SR in mammalian skeletal muscle, to our knowledge. The method uses fluo-5N loaded into the SR of single, mammalian skeletal muscle cells (murine flexor digitorum brevis myofibers) and confocal imaging to detect and calibrate the signals. Using this method, we have determined that [Ca²⁺]_{SR, free} is 390 μM. 4-Chloro-m-cresol, an activator of the skeletal muscle ryanodine receptor, reduces [Ca²⁺]_{SR, free} to ∼8 μM, when values are corrected for background fluorescence from cytoplasmic pools of dye. Prolonged electrical stimulation (10 s) at 50 Hz releases 88% of the SR Ca²⁺ content, whereas stimulation at 1 Hz (10 s) releases only 20%. Our results lay the foundation for molecular modeling of the dynamics of luminal SR Ca²⁺ and for future studies of the role of SR Ca²⁺ in healthy and diseased mammalian muscle.     

Ca2+-mobility in the sarcoplasmic reticulum of ventricular myocytes is low

The sarcoplasmic reticulum (SR) in ventricular myocytes contains releasable Ca²⁺ for activating cellular contraction. Recent measurements of intra-SR (luminal) Ca²⁺ suggest a high diffusive Ca²⁺-mobility constant (D_CaSR). This could help spatially to unify SR Ca²⁺-content ([Ca²⁺]_SRT) and standardize Ca²⁺-release throughout the cell. But measurements of localized depletions of luminal Ca²⁺ (Ca²⁺-blinks), associated with local Ca²⁺-release (Ca²⁺-sparks), suggest D_CaSR may actually be low. Here we describe a novel method for measuring D_CaSR. Using a cytoplasmic Ca²⁺-fluorophore, we estimate regional [Ca²⁺]_SRT from localized, caffeine-induced SR Ca²⁺-release. Caffeine microperfusion of one end of a guinea pig or rat myocyte diffusively empties the whole SR at a rate indicating D_CaSR is 8-9 μm²/s, up to tenfold lower than previous estimates. Ignoring background SR Ca²⁺-leakage in our measurement protocol produces an artifactually high D_CaSR (>40 μm²/s), which may also explain the previous high values. Diffusion-reaction modeling suggests that a low D_CaSR would be sufficient to support local SR Ca²⁺-signaling within sarcomeres during excitation-contraction coupling. Low D_CaSR also implies that [Ca²⁺]_SRT may readily become spatially nonuniform, particularly under pathological conditions of spatially nonuniform Ca²⁺-release. Local control of luminal Ca²⁺, imposed by low D_CaSR, may complement the well-established local control of SR Ca²⁺-release by Ca²⁺-channel/ryanodine receptor couplons.     

Mitochondrial Ca2+-Handling in Fast Skeletal Muscle Fibers from Wild Type and Calsequestrin-Null Mice

Mitochondrial calcium handling and its relation with calcium released from sarcoplasmic reticulum (SR) in muscle tissue are subject of lively debate. In this study we aimed to clarify how the SR determines mitochondrial calcium handling using dCASQ-null mice which lack both isoforms of the major Ca²⁺-binding protein inside SR, calsequestrin. Mitochondrial free Ca²⁺-concentration ([Ca²⁺]_mito) was determined by means of a genetically targeted ratiometric FRET-based probe. Electron microscopy revealed a highly significant increase in intermyofibrillar mitochondria (+55%) and augmented coupling (+12%) between Ca²⁺ release units of the SR and mitochondria in dCASQ-null vs. WT fibers. Significant differences in the baseline [Ca²⁺]_mito were observed between quiescent WT and dCASQ-null fibers, but not in the resting cytosolic Ca²⁺ concentration. The rise in [Ca²⁺]_mito during electrical stimulation occurred in 20−30 ms, while the decline during and after stimulation was governed by 4 rate constants of approximately 40, 1.6, 0.2 and 0.03 s⁻¹. Accordingly, frequency-dependent increase in [Ca²⁺]_mito occurred during sustained contractions. In dCASQ-null fibers the increases in [Ca²⁺]_mito were less pronounced than in WT fibers and even lower when extracellular calcium was removed. The amplitude and duration of [Ca²⁺]_mito transients were increased by inhibition of mitochondrial Na⁺/Ca²⁺ exchanger (mNCX). These results provide direct evidence for fast Ca²⁺ accumulation inside the mitochondria, involvement of the mNCX in mitochondrial Ca²⁺-handling and a dependence of mitochondrial Ca²⁺-handling on intracellular (SR) and external Ca²⁺ stores in fast skeletal muscle fibers. dCASQ-null mice represent a model for malignant hyperthermia. The differences in structure and in mitochondrial function observed relative to WT may represent compensatory mechanisms for the disease-related reduction of calcium storage capacity of the SR and/or SR Ca²⁺-leakage.     

Myocardial mechanical dysfunction and calcium overload following rewarming from experimental hypothermia in vivo

Rewarming patients from accidental hypothermia are regularly complicated with cardiovascular instability ranging from minor depression of cardiac output to fatal circulatory collapse also termed “rewarming shock”. Since altered Ca²⁺ handling may play a role in hypothermia-induced heart failure, we studied changes in Ca²⁺ homeostasis in in situ hearts following hypothermia and rewarming. A rat model designed for studies of the intact heart in a non-arrested state during hypothermia and rewarming was used. Rats were core cooled to 15 °C, maintained at 15 °C for 4 h and thereafter rewarmed. As time-matched controls, one group of animals was kept at 37 °C for 5 h. Total intracellular myocardial Ca²⁺ content ([Ca²⁺]_i) was measured using ⁴⁵Ca²⁺. Following rewarming we found a significant reduction of stroke volume and cardiac output compared to prehypothermic control values as well as to time-matched controls. Likewise, we found that hypothermia and rewarming resulted in a more than six-fold increase in [Ca²⁺]_i to 3.01 ± 0.43 μmol/g dry weight compared to 0.44 ± 0.05 μmol/g dry weight in normothemia control. These findings indicate that hypothermia-induced alterations in the Ca²⁺-handling result in Ca²⁺ overload during hypothermia, which may contribute to myocardial failure during and after rewarming.     

Kinetics of the Ca, H, and Mg Interaction with the Ion-Binding Sites of the SR Ca-ATPase

Electrochromic styryl dyes were used to investigate mutually antagonistic effects of Ca²⁺ and H⁺ on binding of the other ion in the E₁ and P-E₂ states of the SR Ca-ATPase. On the cytoplasmic side of the protein in the absence of Mg²⁺ a strictly competitive binding sequence, H₂E₁?HE₁?E₁?CaE₁?Ca₂E₁, was found with two Ca²⁺ ions bound cooperatively. The apparent equilibrium dissociation constants were in the order of K_1/2(2 Ca) = 34 nM, K_1/2(H) = 1 nM and K_1/2(H₂) = 1.32 μM. Up to 2 Mg²⁺ ions were also able to enter the binding sites electrogenically and to compete with the transported substrate ions (K_1/2(Mg) = 165 μM, K_1/2(Mg₂) = 7.4 mM). In the P-E₂ state, with binding sites facing the lumen of the sarcoplasmatic reticulum, the measured concentration dependence of Ca²⁺ and H⁺ binding could be described satisfactorily only with a branched reaction scheme in which a mixed state, P-E₂CaH, exists. From numerical simulations, equilibrium dissociation constants could be determined for Ca²⁺ (0.4 mM and 25 mM) and H⁺ (2 μM and 10 μM). These simulations reproduced all observed antagonistic concentration dependences. The comparison of the dielectric ion binding in the E₁ and P-E₂ conformations indicates that the transition between both conformations is accompanied by a shift of their (dielectric) position.     

Modulation of Intracellular Calcium Waves and Triggered Activities by Mitochondrial Ca Flux in Mouse Cardiomyocytes

Recent studies have suggested that mitochondria may play important roles in the Ca²⁺ homeostasis of cardiac myocytes. However, it is still unclear if mitochondrial Ca²⁺ flux can regulate the generation of Ca²⁺ waves (CaWs) and triggered activities in cardiac myocytes. In the present study, intracellular/cytosolic Ca²⁺ (Ca_i ²⁺) was imaged in Fluo-4-AM loaded mouse ventricular myocytes. Spontaneous sarcoplasmic reticulum (SR) Ca²⁺ release and CaWs were induced in the presence of high (4 mM) external Ca²⁺ (Ca_o ²⁺). The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) reversibly raised basal Ca_i ²⁺ levels even after depletion of SR Ca²⁺ in the absence of Ca_o ²⁺ , suggesting Ca²⁺ release from mitochondria. FCCP at 0.01 - 0.1 µM partially depolarized the mitochondrial membrane potential (Δψ _m) and increased the frequency and amplitude of CaWs in a dose-dependent manner. Simultaneous recording of cell membrane potentials showed the augmentation of delayed afterdepolarization amplitudes and frequencies, and induction of triggered action potentials. The effect of FCCP on CaWs was mimicked by antimycin A (an electron transport chain inhibitor disrupting Δψ _m) or Ru360 (a mitochondrial Ca²⁺ uniporter inhibitor), but not by oligomycin (an ATP synthase inhibitor) or iodoacetic acid (a glycolytic inhibitor), excluding the contribution of intracellular ATP levels. The effects of FCCP on CaWs were counteracted by the mitochondrial permeability transition pore blocker cyclosporine A, or the mitochondrial Ca²⁺ uniporter activator kaempferol. Our results suggest that mitochondrial Ca²⁺ release and uptake exquisitely control the local Ca²⁺ level in the micro-domain near SR ryanodine receptors and play an important role in regulation of intracellular CaWs and arrhythmogenesis.     

Allosterically coupled calcium and magnesium binding sites are unmasked by ryanodine receptor chimeras

We studied cation regulation of wild-type ryanodine receptor type 1 (_WTRyR1), type 3 (_WTRyR3), and RyR3/RyR1 chimeras (Ch) expressed in 1B5 dyspedic myotubes. Using [³H]ryanodine binding to sarcoplasmic reticulum (SR) membranes, Ca²⁺ titrations with _WTRyR3 and three chimeras show biphasic activation that is allosterically coupled to an attenuated inhibition relative to _WTRyR1. Chimeras show biphasic Mg²⁺ inhibition profiles at 3 and 10 μM Ca²⁺, no observable inhibition at 20 μM Ca²⁺ and monophasic inhibition at 100 μM Ca²⁺. Ca²⁺ imaging of intact myotubes expressing Ch-4 exhibit caffeine-induced Ca²⁺ transients with inhibition kinetics that are significantly slower than those expressing _WTRyR1 or _WTRyR3. Four new aspects of RyR regulation are evident: (1) high affinity (H) activation and low affinity (L) inhibition sites are allosterically coupled, (2) Ca²⁺ facilitates removal of the inherent Mg²⁺ block, (3) _WTRyR3 exhibits reduced cooperativity between H activation sites when compared to _WTRyR1, and (4) uncoupling of these sites in Ch-4 results in decreased rates of inactivation of caffeine-induced Ca²⁺ transients.     

Relaxant effects of Schisandra chinensis and its major lignans on agonists-induced contraction in guinea pig ileum

In this study, the herbal extracts of Schisandra chinensis were demonstrated to inhibit the contractions induced by acetylcholine (ACh) and serotonin (5-HT) in guinea pig ileum, and the 95% ethanol extract was more effective than the aqueous extract. Analysis with High Performance Liquid Chromatography (HPLC) indicated that schisandrin, schisandrol B, schisandrin A and schisandrin B were the major lignans of Schisandra chinensis, and the ethanol extract contained higher amount of these lignans than the aqueous extract. All four lignans inhibited the contractile responses to ACh, with EC₂₀ values ranging from 2.2 ± 0.4 μM (schisandrin A) to 13.2 ± 4.7 μM (schisandrin). The effectiveness of these compounds in relaxing the 5-HT-induced contraction was observed with a similar magnitude. Receptor binding assay indicated that Schisandra lignans did not show significant antagonistic effect on muscarinic M3 receptor. In Ca²⁺-free preparations primed with ACh or KCl, schisandrin A (50 μM) attenuated the contractile responses to cumulative addition of CaCl₂ by 37%. In addition, schisandrin A also concentration-dependently inhibited ACh-induced contractions in Ca²⁺-free buffer. This study demonstrates that Schisandra chinensis exhibited relaxant effects on agonist-induced contraction in guinea pig ileum, with schisandrin, schisandrol B, schisandrin A and schisandrin B being the major active ingredients. The antispasmodic action of schisandrin A involved inhibitions on both Ca²⁺ influx through L-type Ca²⁺ channels and intracellular Ca²⁺ mobilization, rather than specific antagonism of cholinergic muscarinic receptors.     

Mitochondrial Na/Ca exchanger, a new target for neuroprotection in rat hippocampal slices

We tested here the hypothesis that the pharmacological modulation of the mitochondrial Na⁺/Ca²⁺ exchanger (mNCX) could be a new neuroprotective strategy to rescue stressed vulnerable neurons from death. We used rat hippocampal slices incubated with veratridine to cause neuronal death through a mechanism involving Na⁺ and Ca²⁺ overload. CGP37157 (CGP), an inhibitor of the mNCX, rescued veratridine vulnerable neurons from death, showing an EC₅₀ of 5 μM. This neuroprotection was associated to mitigation of veratridine-elicited overproduction of free radicals and to inhibition of the p38 MAPK-linked apoptotic pathway. These results suggest that the mNCX could become a new target to develop compounds with potential therapeutic neuroprotective actions in neurodegenerative diseases.     

Store-operated Ca entry and depolarization explain the anomalous behaviour of myometrial SR: Effects of SERCA inhibition on electrical activity,Ca and force

In the myometrium SR Ca²⁺ depletion promotes an increase in force but unlike several other smooth muscles, there is no Ca²⁺ sparks-STOCs coupling mechanism to explain this. Given the importance of the control of contractility for successful parturition, we have examined, in pregnant rat myometrium, the effects of SR Ca²⁺-ATPase (SERCA) inhibition on the temporal relationship between action potentials, Ca²⁺ transients and force. Simultaneous recording of electrical activity, calcium and force showed that SERCA inhibition, by cyclopiazonic acid (CPA 20 μM), caused time-dependent changes in excitability, most noticeably depolarization and elevations of baseline [Ca²⁺]_i and force. At the onset of these changes there was a prolongation of the bursts of action potentials and a corresponding series of Ca²⁺ spikes, which increased the amplitude and duration of contractions. As the rise of baseline Ca²⁺ and depolarization continued a point was reached when electrical and Ca²⁺ spikes and phasic contractions ceased, and a maintained, tonic force and Ca²⁺ was produced. Lanthanum, a non-selective blocker of store-operated Ca²⁺ entry, but not the L-type Ca²⁺ channel blocker nifedipine (1–10 μM), could abolish the maintained force and calcium. Application of the agonist, carbachol, produced similar effects to CPA, i.e. depolarization, elevation of force and calcium. A brief, high concentration of carbachol, to cause SR Ca²⁺ depletion without eliciting receptor-operated channel opening, also produced these results. The data obtained suggest that in pregnant rats SR Ca²⁺ release is coupled to marked Ca²⁺ entry, via store operated Ca²⁺ channels, leading to depolarization and enhanced electrical and mechanical activity.     

Inhibition of mitochondrial calcium uptake rather than efflux impedes calcium release by inositol-1,4,5-trisphosphate-sensitive receptors

Mitochondria modulate cellular Ca²⁺ signals by accumulating the ion via a uniporter and releasing it via Na⁺- or H⁺-exchange. In smooth muscle, inhibition of mitochondrial Ca²⁺ uptake inhibits Ca²⁺ release from the sarcoplasmic reticulum (SR) via inositol-1,4,5-trisphosphate-sensitive receptors (IP₃R). At least two mechanisms may explain this effect. First, localised uptake of Ca²⁺ by mitochondria may prevent negative feedback by cytosolic Ca²⁺ on IP₃R activity, or secondly localised provision of Ca²⁺ by mitochondrial efflux may maintain IP₃R function or SR Ca²⁺ content. To distinguish between these possibilities the role of mitochondrial Ca²⁺ efflux on IP₃R function was examined. IP₃ was liberated in freshly isolated single colonic smooth muscle cells and mitochondrial Na⁺–Ca²⁺ exchanger inhibited with CGP-37157 (10 μM). Mitochondria accumulated Ca²⁺ during IP₃-evoked [Ca²⁺]_c rises and released the ion back to the cytosol (within 15 s) when mitochondrial Ca²⁺ efflux was active. When mitochondrial Ca²⁺ efflux was inhibited by CGP-37157, an extensive and sustained loading of mitochondria with Ca²⁺ occurred after IP₃-evoked Ca²⁺ release. IP₃-evoked [Ca²⁺]_c rises were initially unaffected, then only slowly inhibited by CGP-37157. IP₃R activity was required for inhibition to occur; incubation with CGP-37157 for the same duration without IP₃ release did not inhibit IP₃R. CGP-37157 directly inhibited voltage-gated Ca²⁺ channel activity, however SR Ca²⁺ content was unaltered by the drug. Thus, the gradual decline of IP₃R function that followed mitochondrial Na⁺–Ca²⁺ exchanger inhibition resulted from a gradual overload of mitochondria with Ca²⁺, leading to a reduced capacity for Ca²⁺ uptake. Localised uptake of Ca²⁺ by mitochondria, rather than mitochondrial Ca²⁺ efflux, appears critical for maintaining IP₃R activity.     

Sarcoplasmic reticulum Ca2+-pump density is higher in distal than in proximal segments of porcine left coronary artery

Densities of sarcoplasmic reticulum (SR) Ca²⁺-pump were compared in proximal and distal segments of pig left coronary artery using two biochemical methods: acylphosphate formation and immunoreactivity in Western blots, and a functional assay based on contraction to SR Ca²⁺-pump inhibitors. In the microsomes prepared from smooth muscle, the level of the 115 kDa SR Ca²⁺-pump acylphosphate was 7.1 ± 0.3 -fold greater in distal than in proximal segments. Similarly in Western blots using these microsomes, the reactivity of the 115 kDa band to an anti-SR Ca²⁺-pump antibody was 5.3 ± 0.8 -fold greater in distal than in proximal segments. Endothelium free coronary artery rings contracted to the SR Ca²⁺-pump inhibitors Cyclopiazonic acid (CPA, EC₅₀ = 0.19 ± 0.06 M) and thapsigargin (EC₅₀ = 0.0095 ± 0.0035 M). With 10 M CPA, the force of contraction per tissue wet weight was 4.2 ± 0.5-fold greater in distal than in proximal rings, and with 1 M thapsigargin it was 4.0 ± 1.0 -fold greater. The contractions produced by 60 mM KCl were used as a control. In contrast to the CPA and thapsigargin, the force per mg tissue weight produced by 60 mM KCl did not differ significantly between the proximal and distal segments. Thus, the results from the two biochemical methods and those from the contractility data were all consistent with the smooth muscle in the distal segments of the coronary artery containing a higher density of the SR Ca²⁺-pump than the proximal segments.Abbreviations CPA cyclopiazonic acid - DTT dithiothreitol - SR sarcoplasmic reticulum     

Electrophoretic mobility of sarcoplasmic reticulum vesicles is determined by amino acids of A + P + N domains of Ca-ATPase

Establishing the origin of electrophoretic mobility of sarcoplasmic reticulum (SR) vesicles is the primary goal of this work. It was found that the electrophoretic mobility originates from ionizable amino acids of cytoplasmic domains of the Ca²⁺-ATPase, the calcium pump of SR. The mobility was measured at pH 4.0, 4.7, 5.0, 6.0, 7.5, and 9.0 in the region of ionic strength from 0.05 to 0.2 M. Mobility measurements were supplemented by studies of SR vesicles by photoelectron microscopy. The median diameter of SR vesicles was 260 nm. Ca²⁺-ATPases were not resolved. The mobility data were standardized by interpolation to a reference ionic strength of 0.1 M. The mobility of the SR vesicles is determined by the charge of the Ca²⁺-ATPase. It is due to the ionizable amino acids selected from the amino acid sequence of SERCA1a Ca²⁺-ATPase. The pH dependence of charge residing in various domains of Ca²⁺-ATPase was computed using pKa values in free water. The charge correlated with measured mobility. It was shown that a linear relationship exists between the mobility of the SR vesicles, μ, and the total computed charge, Q, on three cytoplasmic domains of Ca²⁺-ATPase: A, P, and N. It is given by μ = α + β Q where the fitted values β = (0.043 ± 0.002) × 10⁻⁸ m² V⁻¹ s⁻¹ e⁻¹ and α = (0.16 ± 0.02) × 10⁻⁸ m² V⁻¹ s⁻¹. Since β and α values do not change from pH 4 to pH 9, one concludes that the hydrodynamic friction of the cytoplasmic domains of SR is independent of their charge.     

9-Phenanthrol and flufenamic acid inhibit calcium oscillations in HL-1 mouse cardiomyocytes

It is well established that intracellular calcium ([Ca²⁺]_i) controls the inotropic state of the myocardium, and evidence mounts that a “Ca²⁺ clock” controls the chronotropic state of the heart. Recent findings describe a calcium-activated nonselective cation channel (NSC_Ca) in various cardiac preparations sharing hallmark characteristics of the transient receptor potential melastatin 4 (TRPM4). TRPM4 is functionally expressed throughout the heart and has been implicated as a NSC_Ca that mediates membrane depolarization. However, the functional significance of TRPM4 in regards to Ca²⁺ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. Here, we show by Fura2 Ca-imaging that pharmacological inhibition of TRPM4 in HL-1 mouse cardiac myocytes by 9-phenanthrol (10 μM) and flufenamic acid (10 and 100 μM) decreases Ca²⁺ oscillations followed by an overall increase in [Ca²⁺]_i. The latter occurs also in HL-1 cells in Ca²⁺-free solution and after depletion of sarcoplasmic reticulum Ca²⁺ with thapsigargin (10 μM). These pharmacologic agents also depolarize HL-1 cell mitochondrial membrane potential. Furthermore, by on-cell voltage clamp we show that 9-phenanthrol reversibly inhibits membrane current; by fluorescence immunohistochemistry we demonstrate that HL-1 cells display punctate surface labeling with TRPM4 antibody; and by immunoblotting using this antibody we show these cells express a 130–150 kDa protein, as expected for TRPM4. We conclude that 9-phenanthrol inhibits TRPM4 ion channels in HL-1 cells, which in turn decreases Ca²⁺ oscillations followed by a compensatory increase in [Ca²⁺]_i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.