Dual Effect of Phosphate Transport on Mitochondrial Ca2+ Dynamics

The large inner membrane electrochemical driving force and restricted volume of the matrix confer unique constraints on mitochondrial ion transport. Cation uptake along with anion and water movement induces swelling if not compensated by other processes. For mitochondrial Ca²⁺ uptake, these include activation of countertransporters (Na⁺/Ca²⁺ exchanger and Na⁺/H⁺ exchanger) coupled to the proton gradient, ultimately maintained by the proton pumps of the respiratory chain, and Ca²⁺ binding to matrix buffers. Inorganic phosphate (P_i) is known to affect both the Ca²⁺ uptake rate and the buffering reaction, but the role of anion transport in determining mitochondrial Ca²⁺ dynamics is poorly understood. Here we simultaneously monitor extra- and intra-mitochondrial Ca²⁺ and mitochondrial membrane potential (ΔΨ_m) to examine the effects of anion transport on mitochondrial Ca²⁺ flux and buffering in P_i-depleted guinea pig cardiac mitochondria. Mitochondrial Ca²⁺ uptake proceeded slowly in the absence of P_i but matrix free Ca²⁺ ([Ca²⁺]_mito) still rose to ∼50 μm. P_i (0.001–1 mm) accelerated Ca²⁺ uptake but decreased [Ca²⁺]_mito by almost 50% while restoring ΔΨ_m. P_i-dependent effects on Ca²⁺ were blocked by inhibiting the phosphate carrier. Mitochondrial Ca²⁺ uptake rate was also increased by vanadate (V_i), acetate, ATP, or a non-hydrolyzable ATP analog (AMP-PNP), with differential effects on matrix Ca²⁺ buffering and ΔΨ_m recovery. Interestingly, ATP or AMP-PNP prevented the effects of P_i on Ca²⁺ uptake. The results show that anion transport imposes an upper limit on mitochondrial Ca²⁺ uptake and modifies the [Ca²⁺]_mito response in a complex manner.     

Intracellular calcium movements during relaxation and recovery of superfast muscle fibers of the toadfish swimbladder

The mating call of the Atlantic toadfish is generated by bursts of high-frequency twitches of the superfast twitch fibers that surround the swimbladder. At 16°C, a calling period can last several hours, with individual 80–100-Hz calls lasting ∼500 ms interleaved with silent periods (intercall intervals) lasting ∼10 s. To understand the intracellular movements of Ca²⁺ during the intercall intervals, superfast fibers were microinjected with fluo-4, a high-affinity fluorescent Ca²⁺ indicator, and stimulated by trains of 40 action potentials at 83 Hz, which mimics fiber activity during calling. The fluo-4 fluorescence signal was measured during and after the stimulus trains; the signal was also simulated with a kinetic model of the underlying myoplasmic Ca²⁺ movements, including the binding and transport of Ca²⁺ by the sarcoplasmic reticulum (SR) Ca²⁺ pumps. The estimated total amount of Ca²⁺ released from the SR during a first stimulus train is ∼6.5 mM (concentration referred to the myoplasmic water volume). At 40 ms after cessation of stimulation, the myoplasmic free Ca²⁺ concentration ([Ca²⁺]) is below the threshold for force generation (∼3 µM), yet the estimated concentration of released Ca²⁺ remaining in the myoplasm (Δ[Ca_M]) is large, ∼5 mM, with ∼80% bound to parvalbumin. At 10 s after stimulation, [Ca²⁺] is ∼90 nM (three times the assumed resting level) and Δ[Ca_M] is ∼1.3 mM, with 97% bound to parvalbumin. Ca²⁺ movements during the intercall interval thus appear to be strongly influenced by (a) the accumulation of Ca²⁺ on parvalbumin and (b) the slow rate of Ca²⁺ pumping that ensues when parvalbumin lowers [Ca²⁺] near the resting level. With repetitive stimulus trains initiated at 10-s intervals, Ca²⁺ release and pumping come quickly into balance as a result of the stability (negative feedback) supplied by the increased rate of Ca²⁺ pumping at higher [Ca²⁺].     

Ca2+ Overload and Sarcoplasmic Reticulum Instability in tric-a Null Skeletal Muscle

The sarcoplasmic reticulum (SR) of skeletal muscle contains K⁺, Cl⁻, and H⁺ channels may facilitate charge neutralization during Ca²⁺ release. Our recent studies have identified trimeric intracellular cation (TRIC) channels on SR as an essential counter-ion permeability pathway associated with rapid Ca²⁺ release from intracellular stores. Skeletal muscle contains TRIC-A and TRIC-B isoforms as predominant and minor components, respectively. Here we test the physiological function of TRIC-A in skeletal muscle. Biochemical assay revealed abundant expression of TRIC-A relative to the skeletal muscle ryanodine receptor with a molar ratio of TRIC-A/ryanodine receptor ∼5:1. Electron microscopy with the tric-a^−/− skeletal muscle showed Ca²⁺ overload inside the SR with frequent formation of Ca²⁺ deposits compared with the wild type muscle. This elevated SR Ca²⁺ pool in the tric-a^−/− muscle could be released by caffeine, whereas the elemental Ca²⁺ release events, e.g. osmotic stress-induced Ca²⁺ spark activities, were significantly reduced likely reflecting compromised counter-ion movement across the SR. Ex vivo physiological test identified the appearance of “alternan” behavior with isolated tric-a^−/− skeletal muscle, i.e. transient and drastic increase in contractile force appeared within the decreasing force profile during repetitive fatigue stimulation. Inhibition of SR/endoplasmic reticulum Ca^{2+ ATPase} function could lead to aggravation of the stress-induced alternans in the tric-a^−/− muscle. Our data suggests that absence of TRIC-A may lead to Ca²⁺ overload in SR, which in combination with the reduced counter-ion movement may lead to instability of Ca²⁺ movement across the SR membrane. The observed alternan behavior with the tric-a^−/− muscle may reflect a skeletal muscle version of store overload-induced Ca²⁺ release that has been reported in the cardiac muscle under stress conditions.     

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.     

Ca2+ Modulation of sarcoplasmic reticulum Ca2+ release in rat skeletal muscle fibers

Ca²⁺ transients and the rate of Ca²⁺ release (dCa_REL/dt) from the sarcoplasmic reticulum (SR) in voltage-clamped, fast-twitch skeletal muscle fibers from the rat were studied with the double Vaseline gap technique and using mag-fura-2 and fura-2 as Ca²⁺ indicators. Single pulse experiments with different returning potentials showed that Ca²⁺ removal from the myoplasm is voltage independent. Thus, the myoplasmic Ca²⁺ removal (dCa_REM/dt) was studied by fitting the decaying phase of the Ca²⁺ transient (Melzer, Ríos & Schneider, 1986) and dCa_REL/dt was calculated as the difference between dCa/dt and dCa_REM/dt. The fast Ca²⁺ release decayed as a consequence of Ca²⁺ inactivation of Ca²⁺ release. Double pulse experiments showed inactivation of the fast Ca²⁺ release depending on the prepulse duration. At constant interpulse interval, long prepulses (200 msec) induced greater inactivation of the fast Ca²⁺ release than shorter depolarizations (20 msec). The correlation (r) between the myoplasmic [Ca²⁺]_i and the inhibited amount of Ca²⁺ release was 0.98. The [Ca²⁺]_i for 50% inactivation of dCa_REL/dt was 0.25 m, and the minimum number of sites occupied by Ca²⁺ to inactivate the Ca²⁺ release channel was 3.0. These data support Ca²⁺ binding and inactivation of SR Ca²⁺ release.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association (USA). Part of this work was developed in Dr. Stefani's laboratory at Baylor College of Medicine.     

Sarcoplasmic Reticulum K+ (TRIC) Channel Does Not Carry Essential Countercurrent during Ca2+ Release

The charge translocation associated with sarcoplasmic reticulum (SR) Ca²⁺ efflux is compensated for by a simultaneous SR K⁺ influx. This influx is essential because, with no countercurrent, the SR membrane potential (V_m) would quickly (<1 ms) reach the Ca²⁺ equilibrium potential and SR Ca²⁺ release would cease. The SR K⁺ trimeric intracellular cation (TRIC) channel has been proposed to carry the essential countercurrent. However, the ryanodine receptor (RyR) itself also carries a substantial K⁺ countercurrent during release. To better define the physiological role of the SR K⁺ channel, we compared SR Ca²⁺ transport in saponin-permeabilized cardiomyocytes before and after limiting SR K⁺ channel function. Specifically, we reduced SR K⁺ channel conduction 35 and 88% by replacing cytosolic K⁺ for Na⁺ or Cs⁺ (respectively), changes that have little effect on RyR function. Calcium sparks, SR Ca²⁺ reloading, and caffeine-evoked Ca²⁺ release amplitude (and rate) were unaffected by these ionic changes. Our results show that countercurrent carried by SR K⁺ (TRIC) channels is not required to support SR Ca²⁺ release (or uptake). Because K⁺ enters the SR through RyRs during release, the SR K⁺ (TRIC) channel most likely is needed to restore trans-SR K⁺ balance after RyRs close, assuring SR V_m stays near 0 mV.     

Cytoplasmic Ca2+ does not inhibit the cardiac muscle sarcoplasmic reticulum ryanodine receptor Ca2+ channel,although Ca2+-induced Ca2+ inactivation of Ca2+ release is observed in native vesicles

Single channel properties of cardiac and fast-twitch skeletal muscle sarcoplasmic reticulum (SR) release channels were compared in a planar bilayer by fusing SR membranes in a Cs⁺-conducting medium. We found that the pharmacology, Cs⁺ conductance and selectivity to monovalent and divalent cations of the two channels were similar. The cardiac SR channel exhibited multiple kinetic states. The open and closed lifetimes were not altered from a range of 10^–7 to 10^–3 M Ca²⁺, but the proportion of closed and open states shifted to shorter closings and openings, respectively.However, while the single channel activity of the skeletal SR channel was activated and inactivated by micromolar and millimolar Ca²⁺, respectively, the cardiac SR channel remained activated in the presence of high [Ca²⁺]. In correlation to these studies, [³H]ryanodine binding by the receptors of the two channel receptors was inhibited by high [Ca²⁺] in skeletal but not in cardiac membranes in the presence of adenine nucleotides. There is, however, a minor inhibition of [³H]ryanodine binding of cardiac SR at millimolar Ca²⁺ in the absence of adenine nucleotides.When Ca²⁺-induced Ca²⁺ release was examined from preloaded native SR vesicles, the release rates followed a normal biphasic curve, with Ca²⁺-induced inactivation at high [Ca²⁺] for both cardiac and skeletal SR. Our data suggest that the molecular basis of regulation of the SR Ca²⁺ release channel in cardiac and skeletal muscle is different, and that the cardiac SR channel isoform lacks a Ca²⁺-inactivated site.This work was supported by research grants from the National Institutes of Health HL13870 and AR38970, and the Texas Affiliate of the American Heart Association, 91A-188. M. Fill was the recipient of an NIH fellowship AR01834.     

Regulation of Ca2+ release from sarcoplasmic reticulum in skeletal muscles

Ca²⁺ release from skeletal sarcoplasmic reticulum (SR) could be regulated by at least three mechanisms: 1) Ca²⁺, 2) calmodulin, and 3) Ca²⁺/calmodulin-dependent phosphorylation. Bell-shaped Ca²⁺-dependence, of Ca²⁺ release from both actively- and passively-loaded SR vesicles suggest that opening and closing of the Ca²⁺ release channel could be regulated by [Ca²⁺ _o] . The time- and concentration-dependent inhibition of Ca ²⁺ release from skeletal SR by calmodulin was also studied using passively-Ca²⁺ loaded SR vesicles. Up to 50% of Ca ²⁺ release was inhibited by calmodulin (0.01–0.5 µM); this inhibition required 5–15 min preincubation time. The hypothesis that Ca²⁺/calmodulin-dependent phosphorylation of a 60 kDa protein regulates Ca²⁺ release from skeletal SR was tested by stopped-flow fluorometry using passively-Ca²⁺-loaded SR vesicles. Approximately 80% of the initial rates of Ca²⁺-induced Ca²⁺ release was inhibited by the phosphorylation within 2 min of incubation of the SR with Mg·ATP and calmodulin. We identified two types of 60 kDa phosphoproteins in the rabbit skeletal SR, which was distinguished by solubility of the protein in CHAPS. The CHAPS-soluble 60 kDa phosphoprotein was purified by column chromatography on DEAE-Sephacel, heparin-agarose, and hydroxylapatite. Analyses of the purified protein indicate that the CHAPS-soluble 60 kDa protein is an isoform of phosphoglucomutase (PGM). cDNAs encoding isoforms of PGM were cloned and sequenced using synthetic oligonucleotides. Two types of PGM isoforms (Type I and Type 11) were identified. The translated amino acid sequences show that Type II isoform is SR-form. Our results are significant in terms of understanding evidence of an association of glycolytic and glycogenolytic enzymes with SR and a role in the regulation of SR functions. (Mol Cell Biochem 114: 105-108, 1992)     

Assembly of a Tyr122 Hydrophobic Cluster in Sarcoplasmic Reticulum Ca2+-ATPase Synchronizes Ca2+ Affinity Reduction and Release with Phosphoenzyme Isomerization

The mechanism whereby events in and around the catalytic site/head of Ca²⁺-ATPase effect Ca²⁺ release to the lumen from the transmembrane helices remains elusive. We developed a method to determine deoccluded bound Ca²⁺ by taking advantage of its rapid occlusion upon formation of E1PCa₂ and of stabilization afforded by a high concentration of Ca²⁺. The assay is applicable to minute amounts of Ca²⁺-ATPase expressed in COS-1 cells. It was validated by measuring the Ca²⁺ binding properties of unphosphorylated Ca²⁺-ATPase. The method was then applied to the isomerization of the phosphorylated intermediate associated with the Ca²⁺ release process E1PCa₂ → E2PCa₂ → E2P + 2Ca²⁺. In the wild type, Ca²⁺ release occurs concomitantly with EP isomerization fitting with rate-limiting isomerization (E1PCa₂ → E2PCa₂) followed by very rapid Ca²⁺ release. In contrast, with alanine mutants of Leu¹¹⁹ and Tyr¹²² on the cytoplasmic part of the second transmembrane helix (M2) and Ile¹⁷⁹ on the A domain, Ca²⁺ release in 10 μm Ca²⁺ lags EP isomerization, indicating the presence of a transient E2P state with bound Ca²⁺. The results suggest that these residues function in Ca²⁺ affinity reduction in E2P, likely via a structural rearrangement at the cytoplasmic part of M2 and a resulting association with the A and P domains, therefore leading to Ca²⁺ release.     

Ca2+-induced Ca2+ Release Phenomena in Mammalian Sympathetic Neurons Are Critically Dependent on the Rate of Rise of Trigger Ca2+

The role of ryanodine-sensitive intracellular Ca²⁺ stores present in nonmuscular cells is not yet completely understood. Here we examine the physiological parameters determining the dynamics of caffeine-induced Ca²⁺ release in individual fura-2–loaded sympathetic neurons. Two ryanodine-sensitive release components were distinguished: an early, transient release (TR) and a delayed, persistent release (PR). The TR component shows refractoriness, depends on the filling status of the store, and requires caffeine concentrations ≥10 mM. Furthermore, it is selectively suppressed by tetracaine and intracellular BAPTA, which interfere with Ca²⁺-mediated feedback loops, suggesting that it constitutes a Ca²⁺-induced Ca²⁺-release phenomenon. The dynamics of release is markedly affected when Sr²⁺ substitutes for Ca²⁺, indicating that Sr²⁺ release may operate with lower feedback gain than Ca²⁺ release. Our data indicate that when the initial release occurs at an adequately fast rate, Ca²⁺ triggers further release, producing a regenerative response, which is interrupted by depletion of releasable Ca²⁺ and Ca²⁺-dependent inactivation. A compartmentalized linear diffusion model can reproduce caffeine responses: When the Ca²⁺ reservoir is full, the rapid initial Ca²⁺ rise determines a faster occupation of the ryanodine receptor Ca²⁺ activation site giving rise to a regenerative release. With the store only partially loaded, the slower initial Ca²⁺ rise allows the inactivating site of the release channel to become occupied nearly as quickly as the activating site, thereby suppressing the initial fast release. The PR component is less dependent on the store''s Ca²⁺ content. This study suggests that transmembrane Ca²⁺ influx in rat sympathetic neurons does not evoke widespread amplification by CICR because of its inability to raise [Ca²⁺] near the Ca²⁺ release channels sufficiently fast to overcome their Ca²⁺-dependent inactivation. Conversely, caffeine-induced Ca²⁺ release can undergo considerable amplification especially when Ca²⁺ stores are full. We propose that the primary function of ryanodine-sensitive stores in neurons and perhaps in other nonmuscular cells, is to emphasize subcellular Ca²⁺ gradients resulting from agonist-induced intracellular release. The amplification gain is dependent both on the agonist concentration and on the filling status of intracellular Ca²⁺ stores.     

Modulation of the Frequency of Spontaneous Sarcoplasmic Reticulum Ca2+ Release Events (Ca2+ Sparks) by Myoplasmic [Mg2+] in Frog Skeletal Muscle

The modulation by internal free [Mg²⁺] of spontaneous calcium release events (Ca²⁺ “sparks”) from the sarcoplasmic reticulum (SR) was studied in depolarized notched frog skeletal muscle fibers using a laser scanning confocal microscope in line-scan mode (x vs. t). Over the range of [Mg²⁺] from 0.13 to 1.86 mM, decreasing the [Mg²⁺] induced an increase in the frequency of calcium release events in proportion to [Mg²⁺]^−1.6. The change of event frequency was not due to changes in [Mg-ATP] or [ATP]. Analysis of individual SR calcium release event properties showed that the variation in event frequency induced by the change of [Mg²⁺] was not accompanied by any changes in the spatiotemporal spread (i.e., spatial half width or temporal half duration) of Ca²⁺ sparks. The increase in event frequency also had no effect on the distribution of event amplitudes. Finally, the rise time of calcium sparks was independent of the [Mg²⁺], indicating that the open time of the SR channel or channels underlying spontaneous calcium release events was not altered by [Mg²⁺] over the range tested. These results suggest that in resting skeletal fibers, [Mg²⁺] modulates the SR calcium release channel opening frequency by modifying the average closed time of the channel without altering the open time. A kinetic reaction scheme consistent with our results and those of bilayer and SR vesicle experiments indicates that physiological levels of resting Mg²⁺ may inhibit channel opening by occupying the site for calcium activation of the SR calcium release channel.     

F-ATPase of Drosophila melanogaster Forms 53-Picosiemen (53-pS) Channels Responsible for Mitochondrial Ca2+-induced Ca2+ Release

Mitochondria of Drosophila melanogaster undergo Ca²⁺-induced Ca²⁺ release through a putative channel (mCrC) that has several regulatory features of the permeability transition pore (PTP). The PTP is an inner membrane channel that forms from F-ATPase, possessing a conductance of 500 picosiemens (pS) in mammals and of 300 pS in yeast. In contrast to the PTP, the mCrC of Drosophila is not permeable to sucrose and appears to be selective for Ca²⁺ and H⁺. We show (i) that like the PTP, the mCrC is affected by the sense of rotation of F-ATPase, by Bz-423, and by Mg²⁺/ADP; (ii) that expression of human cyclophilin D in mitochondria of Drosophila S₂R⁺ cells sensitizes the mCrC to Ca²⁺ but does not increase its apparent size; and (iii) that purified dimers of D. melanogaster F-ATPase reconstituted into lipid bilayers form 53-pS channels activated by Ca²⁺ and thiol oxidants and inhibited by Mg²⁺/γ-imino ATP. These findings indicate that the mCrC is the PTP of D. melanogaster and that the signature conductance of F-ATPase channels depends on unique structural features that may underscore specific roles in different species.     

Ca2+/H+ exchange,lumenal Ca2+ release and Ca2+/ATP coupling ratios in the sarcoplasmic reticulum ATPase

The Ca²⁺ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca²⁺ in intracellular membrane bound compartments, thereby lowering cytosolic Ca²⁺ to induce relaxation. The stored Ca²⁺ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca²⁺, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca²⁺. We review here biochemical and biophysical evidence demonstrating that release of bound Ca²⁺ into the lumen of SR requires Ca²⁺/H⁺ exchange at the low affinity Ca²⁺ sites. Rise of lumenal Ca²⁺ above its dissociation constant from low affinity sites, or reduction of the H⁺ concentration by high pH, prevent Ca²⁺/H⁺ exchange. Under these conditions Ca²⁺ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca²⁺pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.     

Understanding Ca2+ alternans

JGP study reveals that insufficient reuptake of calcium into the sarcoplasmic reticulum underlies arrhythmogenic variations in cardiac calcium transients.

Ca²⁺ alternans (Ca-Alts) are beat-to-beat changes in the amplitude of the Ca²⁺ transients evoked in cardiomyocytes, which can lead to arrhythmias and sudden cardiac death. Ca-Alts can be induced by an elevated heart rate (tachycardia) or metabolic impairments such as ischemia or hypothermia, but the molecular mechanisms underlying the phenomenon are unclear. In this issue of JGP, Millet et al. reveal that Ca-Alts arise when SERCA pumps are unable to fully replenish Ca²⁺ levels in the SR (1).Jose Millet, Yuriana Aguilar-Sanchez, Ariel L. Escobar (left to right), and colleagues investigate the mechanisms underlying arrhythmogenic Ca-Alts. The FLOM technique shows how these beat-to-beat changes in Ca²⁺ transients can be induced in intact hearts by increased heart rate and local reductions in temperature produced by a cold finger. The researchers find that Ca-Alts result from insufficient replenishment of SR Ca²⁺ levels by SERCA pumps.“Ca-Alts are very arrhythmogenic,” says Ariel L. Escobar, a professor at the University of California, Merced. “If you develop these alternans, you have a very high chance of suffering ventricular fibrillation.”Yet the mechanisms underlying Ca-Alts remain unclear. Though they appear to involve changes in the amount of Ca²⁺ released from the SR (2,3,4), Ca-Alts could be triggered by variations in the duration of action potentials (APD-Alts) that stimulate calcium-induced calcium release, an incomplete recovery of the ryanodine receptor that releases Ca²⁺ from the SR, or incomplete refilling of the SR by SERCA ATPases.To investigate the phenomenon in more detail, Escobar and colleagues, including co-first authors Jose Millet and Yuriana Aguilar-Sanchez, developed a new technique called fluorescence local field optical mapping (FLOM), which uses optical conduits containing >70,000 optical fibers to map the fluorescence of calcium-sensitive or potentiometric dyes in the epicardium of intact mouse hearts. “This approach allows us to study the spatiotemporal dynamics of calcium and membrane potential changes in a functional heart,” Escobar explains.FLOM imaging confirmed that Ca-Alts can be induced by increased heart rate and/or global reductions in temperature, two conditions that also induce APD-Alts. More crucially, however, Escobar and colleagues used a small, crescent-shaped cold finger to show that local reductions in tissue temperature also induce Ca-Alts but do not cause APD-Alts, demonstrating that the two phenomena can be uncoupled and that Ca-Alts are not driven by changes in action potential duration.Because the crescent-shaped cold finger creates a temperature gradient within the epicardium, Escobar and colleagues were able to carefully analyze the temperature dependence of Ca²⁺ dynamics. The relaxation of Ca²⁺ transients becomes gradually slower at lower temperatures, and a thermodynamic analysis of this process suggested that it involves not only active mechanisms—such as the ATPases that pump Ca²⁺ into the SR—but also passive mechanisms such as diffusion and binding to cytosolic buffers.In contrast, the relatively steep temperature dependence of Ca-Alts indicated that they exclusively depend on an active process like SERCA-mediated Ca²⁺ reuptake into the SR. Indeed, Escobar and colleagues found that the Q₁₀ temperature coefficient of Ca-Alts is remarkably similar to the Q₁₀ of SERCA-mediated Ca²⁺ transport in vitro.To confirm the importance of Ca²⁺ reuptake in Ca-Alts, Escobar and colleagues treated hearts with the SERCA inhibitor Thapsigargin. Partial blockade of SERCA-mediated reuptake enhanced the level of Ca-Alts, the researchers found, indicating that Ca-Alts are induced when SERCA pumps fail to fully replenish SR Ca²⁺ stores between heart beats. This could occur when the heart is beating particularly fast or when the metabolic activity of cardiomyocytes is impaired by, for example, low temperatures.Escobar’s team is now developing a needle-shaped optical conduit that can be used to probe any layer within the ventricular wall. “We hope to measure Ca-Alts in each layer, including the endocardium where SERCA levels are lower and Ca-Alts tend to be initiated,” Escobar says.     

Mitochondrial Free Ca2+ Levels and Their Effects on Energy Metabolism in Drosophila Motor Nerve Terminals

Mitochondrial Ca²⁺ uptake exerts dual effects on mitochondria. Ca²⁺ accumulation in the mitochondrial matrix dissipates membrane potential (_ΔΨ_m), but Ca²⁺ binding of the intramitochondrial enzymes accelerates oxidative phosphorylation, leading to mitochondrial hyperpolarization. The levels of matrix free Ca²⁺ ([Ca²⁺]_m) that trigger these metabolic responses in mitochondria in nerve terminals have not been determined. Here, we estimated [Ca²⁺]_m in motor neuron terminals of Drosophila larvae using two methods: the relative responses of two chemical Ca²⁺ indicators with a 20-fold difference in Ca²⁺ affinity (rhod-FF and rhod-5N), and the response of a low-affinity, genetically encoded ratiometric Ca²⁺ indicator (D4cpv) calibrated against known Ca²⁺ levels. Matrix pH (pH_m) and _ΔΨ_m were monitored using ratiometric pericam and tetramethylrhodamine ethyl ester probe, respectively, to determine when mitochondrial energy metabolism was elevated. At rest, [Ca²⁺]_m was 0.22 ± 0.04 μM, but it rose to ∼26 μM (24.3 ± 3.4 μM with rhod-FF/rhod-5N and 27.0 ± 2.6 μM with D4cpv) when the axon fired close to its endogenous frequency for only 2 s. This elevation in [Ca²⁺]_m coincided with a rapid elevation in pH_m and was followed by an after-stimulus _ΔΨ_m hyperpolarization. However, pH_m decreased and no _ΔΨ_m hyperpolarization was observed in response to lower levels of [Ca²⁺]_m, up to 13.1 μM. These data indicate that surprisingly high levels of [Ca²⁺]_m are required to stimulate presynaptic mitochondrial energy metabolism.     

Store-operated Ca2+ Entry in Malignant Hyperthermia-susceptible Human Skeletal Muscle

In malignant hyperthermia (MH), mutations in RyR1 underlie direct activation of the channel by volatile anesthetics, leading to muscle contracture and a life-threatening increase in core body temperature. The aim of the present study was to establish whether the associated depletion of sarcoplasmic reticulum (SR) Ca²⁺ triggers sarcolemmal Ca²⁺ influx via store-operated Ca²⁺ entry (SOCE). Samples of vastus medialis muscle were obtained from patients undergoing assessment for MH susceptibility using the in vitro contracture test. Single fibers were mechanically skinned, and confocal microscopy was used to detect changes in [Ca²⁺] either within the resealed t-system ([Ca²⁺]_t-sys) or within the cytosol. In normal fibers, halothane (0.5 mm) failed to initiate SR Ca²⁺ release or Ca²⁺_t-sys depletion. However, in MH-susceptible (MHS) fibers, halothane induced both SR Ca²⁺ release and Ca²⁺_t-sys depletion, consistent with SOCE. In some MHS fibers, halothane-induced SR Ca²⁺ release took the form of a propagated wave, which was temporally coupled to a wave of Ca²⁺_t-sys depletion. SOCE was potently inhibited by “extracellular” application of a STIM1 antibody trapped within the t-system but not when the antibody was denatured by heating. In conclusion, (i) in human MHS muscle, SR Ca²⁺ depletion induced by a level of volatile anesthetic within the clinical range is sufficient to induce SOCE, which is tightly coupled to SR Ca²⁺ release; (ii) sarcolemmal STIM1 has an important role in regulating SOCE; and (iii) sustained SOCE from an effectively infinite extracellular Ca²⁺ pool may contribute to the maintained rise in cytosolic [Ca²⁺] that underlies MH.     

By Regulating Mitochondrial Ca2+-Uptake UCP2 Modulates Intracellular Ca2+

Introduction

The possible role of UCP2 in modulating mitochondrial Ca²⁺-uptake (mCa²⁺-uptake) via the mitochondrial calcium uniporter (MCU) is highly controversial.

Methods

Thus, we analyzed mCa²⁺-uptake in isolated cardiac mitochondria, MCU single-channel activity in cardiac mitoplasts, dual Ca²⁺-transients from mitochondrial ((Ca²⁺)m) and intracellular compartment ((Ca²⁺)c) in the whole-cell configuration in cardiomyocytes of wild-type (WT) and UCP2^-/- mice.

Results

Isolated mitochondria showed a Ru360 sensitive mCa²⁺-uptake, which was significantly decreased in UCP2^-/- (229.4±30.8 FU vs. 146.3±23.4 FU, P<0.05). Single-channel registrations confirmed a Ru360 sensitive voltage-gated Ca²⁺-channel in mitoplasts, i.e. mCa1, showing a reduced single-channel activity in UCP2^-/- (Po,total: 0.34±0.05% vs. 0.07±0.01%, P<0.05). In UCP2^-/- cardiomyocytes (Ca²⁺)m was decreased (0.050±0.009 FU vs. 0.021±0.005 FU, P<0.05) while (Ca²⁺)c was unchanged (0.032±0.002 FU vs. 0.028±0.004 FU, P>0.05) and transsarcolemmal Ca²⁺-influx was inhibited suggesting a possible compensatory mechanism. Additionally, we observed an inhibitory effect of ATP on mCa²⁺-uptake in WT mitoplasts and (Ca²⁺)m of cardiomyocytes leading to an increase of (Ca²⁺)c while no ATP dependent effect was observed in UCP2^-/-.

Conclusion

Our results indicate regulatory effects of UCP2 on mCa²⁺-uptake. Furthermore, we propose, that previously described inhibitory effects on MCU by ATP may be mediated via UCP2 resulting in changes of excitation contraction coupling.     

Contributions of Ca2+-Independent Thin Filament Activation to Cardiac Muscle Function

Although Ca²⁺ is the principal regulator of contraction in striated muscle, in vitro evidence suggests that some actin-myosin interaction is still possible even in its absence. Whether this Ca²⁺-independent activation (CIA) occurs under physiological conditions remains unclear, as does its potential impact on the function of intact cardiac muscle. The purpose of this study was to investigate CIA using computational analysis. We added a structurally motivated representation of this phenomenon to an existing myofilament model, which allowed predictions of CIA-dependent muscle behavior. We found that a certain amount of CIA was essential for the model to reproduce reported effects of nonfunctional troponin C on myofilament force generation. Consequently, those data enabled estimation of ΔG_CIA, the energy barrier for activating a thin filament regulatory unit in the absence of Ca²⁺. Using this estimate of ΔG_CIA as a point of reference (∼7 kJ mol⁻¹), we examined its impact on various aspects of muscle function through additional simulations. CIA decreased the Hill coefficient of steady-state force while increasing myofilament Ca²⁺ sensitivity. At the same time, CIA had minimal effect on the rate of force redevelopment after slack/restretch. Simulations of twitch tension show that the presence of CIA increases peak tension while profoundly delaying relaxation. We tested the model’s ability to represent perturbations to the Ca²⁺ regulatory mechanism by analyzing twitch records measured in transgenic mice expressing a cardiac troponin I mutation (R145G). The effects of the mutation on twitch dynamics were fully reproduced by a single parameter change, namely lowering ΔG_CIA by 2.3 kJ mol⁻¹ relative to its wild-type value. Our analyses suggest that CIA is present in cardiac muscle under normal conditions and that its modulation by gene mutations or other factors can alter both systolic and diastolic function.     

Voltage-Induced Ca2+ Release in Postganglionic Sympathetic Neurons in Adult Mice

Recent studies have provided evidence that depolarization in the absence of extracellular Ca²⁺ can trigger Ca²⁺ release from internal stores in a variety of neuron subtypes. Here we examine whether postganglionic sympathetic neurons are able to mobilize Ca²⁺ from intracellular stores in response to depolarization, independent of Ca²⁺ influx. We measured changes in cytosolic ΔF/F₀ in individual fluo-4 –loaded sympathetic ganglion neurons in response to maintained K⁺ depolarization in the presence (2 mM) and absence of extracellular Ca²⁺ ([Ca²⁺]_e). Progressive elevations in extracellular [K⁺]_e caused increasing membrane depolarizations that were of similar magnitude in 0 and 2 mM [Ca²⁺]_e. Peak amplitude of ΔF/F₀ transients in 2 mM [Ca²⁺]_e increased in a linear fashion as the membrane become more depolarized. Peak elevations of ΔF/F₀ in 0 mM [Ca²⁺]_e were ~5–10% of those evoked at the same membrane potential in 2 mM [Ca²⁺]_e and exhibited an inverse U-shaped dependence on voltage. Both the rise and decay of ΔF/F₀ transients in 0 mM [Ca²⁺]_e were slower than those of ΔF/F₀ transients evoked in 2 mM [Ca²⁺]_e. Rises in ΔF/F₀ evoked by high [K⁺]_e in the absence of extracellular Ca²⁺ were blocked by thapsigargin, an inhibitor of endoplasmic reticulum Ca²⁺ ATPase, or the inositol 1,4,5-triphosphate (IP₃) receptor antagonists 2-aminoethoxydiphenyl borate and xestospongin C, but not by extracellular Cd²⁺, the dihydropyridine antagonist nifedipine, or by ryanodine at concentrations that caused depletion of ryanodine-sensitive Ca²⁺ stores. These results support the notion that postganglionic sympathetic neurons possess the ability to release Ca²⁺ from IP₃-sensitive internal stores in response to membrane depolarization, independent of Ca²⁺ influx.     

Transport of Ca2+ from Sarcoplasmic Reticulum to Mitochondria in Rat Ventricular Myocytes

Studies with electron microscopy have shown that sarcoplasmic reticulum (SR) andmitochondria locate close to each other in cardiac muscle cells. We investigated the hypothesis thatthis proximity results in a transient exposure of mitochondrial Ca²⁺ uniporter (CaUP) to highconcentrations of Ca²⁺ following Ca²⁺ release from the SR and thus an influx of Ca²⁺into mitochondria. Single ventricular myocytes of rat were skinned by exposing them to aphysiological solution containing saponin (0.2 mg/ml). Cytosolic Ca²⁺ concentration ([Ca²⁺]_c)and mitochondrial Ca²⁺ concentration ([Ca²⁺]_m) were measured with fura-2 and rhod2,respectively. Application of caffeine (10 mM) induced a concomitant increase in[Ca²⁺]_c and [Ca²⁺]_m.Ruthenium red, at concentrations that block CaUP but not SR release, diminished thecaffeine-induced increase in [Ca²⁺]_m but not[Ca²⁺]_c. In the presence of 1 mM BAPTA, a Ca²⁺ chelator,the caffeine-induced increase in [Ca²⁺]_m was reduced substantially less than [Ca²⁺]_c. Moreover,inhibition of SR Ca²⁺ pump with two different concentrations of thapsigargin caused anincrease in [Ca²⁺]_m, which was related to the rate of [Ca²⁺]_c increase. Finally, electronmicroscopy showed that sites of junctions between SR and T tubules from which Ca²⁺ is released,or Ca²⁺ release units, CRUs, are preferentially located in close proximity to mitochondria.The distance between individual SR Ca²⁺ release channels (feet or ryanodine receptors) isvery short, ranging between approximately 37 and 270 nm. These results are consistent withthe idea that there is a preferential coupling of Ca²⁺ transport from SR to mitochondria incardiac muscle cells, because of their structural proximity.