SR Ca Release Research Articles

‘Eventless’ InsP3‐dependent SR‐Ca2+ release affecting atrial Ca2+ sparks

Augmented inositol 1,4,5-trisphosphate receptor (InsP3R) function has been linked to a variety of cardiac pathologies, including cardiac arrhythmia. The contribution of inositol 1,4,5-trisphosphate-induced Ca(2+) release (IP3ICR) in excitation-contraction coupling (ECC) under physiological conditions, as well as under cellular remodelling, remains controversial. Here we test the hypothesis that local IP3ICR directly affects ryanodine receptor (RyR) function and subsequent Ca(2+)-induced Ca(2+) release in atrial myocytes. IP3ICR was evoked by UV-flash photolysis of caged InsP3 under whole-cell configuration of the voltage-clamp technique in atrial myocytes isolated from C57/BL6 mice. Photolytic release of InsP3 was accompanied by a significant increase in the Ca(2+) release event frequency (4.14 ± 0.72 vs. 6.20 ± 0.76 events (100 μm)(-1) s(-1)). These individual photolytically triggered Ca(2+) release events were identified as Ca(2+) sparks, which originated from RyR openings. This was verified by Ca(2+) spark analysis and pharmacological separation between RyR and InsP3R-dependent sarcoplasmic reticulum (SR)-Ca(2+) release (2-aminoethoxydiphenyl borate, xestospongin C, tetracaine). Significant SR-Ca(2+) flux but eventless SR-Ca(2+) release through InsP3R were characterized using SR-Ca(2+) leak/SR-Ca(2+) load measurements. These results strongly support the idea that IP3ICR can effectively modulate RyR openings and Ca(2+) spark probability. We conclude that eventless and highly efficient InsP3-dependent SR-Ca(2+) flux is the main mechanism of functional cross-talk between InsP3Rs and RyRs, which may be an important factor in the modulation of ECC sensitivity.

Altered calsequestrin glycan processing is common to diverse models of canine heart failure

Calsequestrin-2 (CSQ2) is a resident glycoprotein of junctional sarcoplasmic reticulum that functions in the regulation of SR Ca(2+) release. CSQ2 is biosynthesized in rough ER around cardiomyocyte nuclei and then traffics transversely across SR subcompartments. During biosynthesis, CSQ2 undergoes N-linked glycosylation and phosphorylation by protein kinase CK2. In mammalian heart, CSQ2 molecules subsequently undergo extensive mannose trimming by ER mannosidase(s), a posttranslational process that often regulates protein breakdown. We analyzed the intact purified CSQ2 from mongrel canine heart tissue by electrospray mass spectrometry. The average molecular mass of CSQ2 in normal mongrel dogs was 46,306 ± 41 Da, corresponding to glycan trimming of 3-5 mannoses, depending upon the phosphate content. We tested whether CSQ2 glycan structures would be altered in heart tissue from mongrel dogs induced into heart failure (HF) by two very different experimental treatments, rapid ventricular pacing or repeated coronary microembolizations. Similarly dramatic changes in mannose trimming were found in both types of induced HF, despite the different cardiomyopathies producing the failure. Unique to all samples analyzed from HF dog hearts, 20-40 % of all CSQ2 contained glycans that had minimal mannose trimming (Man9,8). Analyses of tissue samples showed decreases in CSQ2 protein levels per unit levels of mRNA for tachypaced heart tissue, also indicative of altered turnover. Quantitative immunofluorescence microscopy of frozen tissue sections suggested that no changes in CSQ2 levels occurred across the width of the cell. We conclude that altered processing of CSQ2 may be an adaptive response to the myocardium under stresses that are capable of inducing heart failure.

Oxidation of Ryanodine Receptor following Ischemia/Reperfusion Increases the Propensity of Ca Waves during Beta-Adrenergic Receptor Stimulation

Beta-Adrenergic receptor (beta-AR) stimulation generates the main positive inotropic response in the heart. However, after ischemia/reperfusion (I/R), beta-AR stimulation can generate arrhythmias. I/R also increased intracellular reactive oxygen species (ROS) production. We investigated whether ryanodine receptor (RyR) oxidation by ROS contributes to the transition from positive inotropic to arrythmogenic effect in ventricular myocytes after I/R. Measurements of contractile and electrical activity from ex vivo rabbit hearts revealed that global I/R produces severe tachi-arrhythmias. Ventricular myocytes isolated from ischemic hearts were characterized by increased both SR Ca leak rates and fractional SR Ca release compared to cells from non-ischemic hearts. Furthermore, myocytes from ischemic hearts showed increased ROS production, decreased level of free thiols in RyRs (RyR oxidation), and increased level of oxidized glutathione (GSSG). Pretreatment of myocytes from ischemic hearts with the reducing agent mercaptopropionylglycine attenuated the oxidization of free thiols in RyRs and normalized systolic SR Ca release and diastolic Ca leak. In myocytes from ischemic hearts, isoproterenol (ISO; 10 nM) led to the occurrence of spontaneous Ca waves, whereas in cells from non-ischemic hearts the same dose of ISO only caused a positive inotropic effect. Treatment of myocytes from non-ischemic hearts with H2O2 (0.1 mM) increased SR Ca leak and fractional SR Ca release to similar levels observed in myocytes from ischemic hearts. Moreover, application of ISO (10 nM) to myocytes from non-ischemic hearts pretreated with H2O2 increased the propensity of Ca waves. These results indicate that augmentation of ROS production following I/R causes RyR oxidation. This post-translational modification of the RyR plays a critical role in the transition from positive inotropic to arrhythmogenic effect during beta-AR stimulation.

Excision of the Cacnb2 Gene in Mice Results in Augmented SR-Ca Release and Impaired Cardiac Function In Vivo

In mammalian cardiac myocytes, L-type Ca channels comprise at least the pore-forming Cav1.2α1 and an auxiliary Cavs2 subunit. Since Cavs2 null mice die at embryonic day 10.5, we used our recently described inducible Cavs2-KO mouse line. The patch clamp technique was applied to investigate basic properties of L-type Ca channels as well as its s-adrenergic responsiveness. We employed video-imaging for global Ca transients and ultrafast 2D confocal imaging for studying Ca sparks. High resolution transthoracic echocardiography allowed us to characterise cardiac function and morphology in vivo. Despite the absence of the Cavs2-subunit, the L-type Ca current's voltage dependence was unchanged but its amplitude was reduced by 25%. Isoproterenol stimulation resulted in a comparable ICa increase in myocytes from both, mygliol and tamoxifen injected mice. Unexpectedly, global Ca transients displayed an increased steady-state amplitude and the SR-Ca content was significantly augmented as was fractional Ca release. Analysis of Ca sparks revealed a higher amplitude while their frequency was down by more than 50%. When investigating the heart in vivo we found indications of dilated cardiomyopathy concomitant with a decreased fractional shortening and ejection fraction. Echocardiographic studies in Doppler mode indicated massive alterations in the E/A wave ratio. From these data we conclude that in adult mice the impairments resulting from Cavs2-KO went well beyond solely altering L-type Ca channel function to largely modulating the SR-Ca release process and causing afflicted cardiac functions in vivo.This work was supported by the DFG.

Analysis of Calcium (Ca) Transients during Excitation-Contraction Coupling in Frog Twitch Muscle Fibers

Singly-dissected fibers were micro-injected with furaptra, a rapidly-responding Ca indicator. Resting fluorescence (FR) and action-potential evoked changes (ΔF) were measured at 16 oC; ΔF/FR was scaled to units of ΔfCaD, the change in fraction of furaptra in the Ca-bound form. Measured ΔfCaD was compared with ΔfCaD simulated with a kinetic model of the underlying myoplasmic Ca movements. During the period 30-200 ms after an action potential, simulated ΔfCaD decayed toward baseline more slowly than measured ΔfCaD. If ΔfCaD was simulated with a modified model that incorporated competition between Mg and Ca for occupancy of the regulatory sites on troponin (assumed dissociation constant of Mg's reaction with the regulatory sites, 2.2 mM; assumed myoplasmic free [Mg], 1 mM), good agreement with measured ΔfCaD was observed. The results support the conclusion that Mg, at physiological levels, competes with Ca for occupancy of the regulatory sites, as indicated in some experiments from fragmented preparations, including tension-pCa measurements in skinned fibers and biochemical studies of isolated troponin molecules. ΔfCaD in frog was also compared with our previous measurements and simulations of ΔfCaD in mouse fibers (reviewed in J. Gen. Physiol., 2012). In frog, the SR Ca release flux elicited by an action potential appears to be the sum of two components. The time course of the first component is similar to that of the entire flux waveform in both fast-and slow-twitch mouse fibers, while that of the second is several-fold slower; the fractional release amounts are ∼0.85 (first component) and ∼0.15 (second component). An anatomical basis for two release components in frog is the presence of both junctional and para-junctional Ca release channels, whereas the mouse fibers have only junctional channels (Felder and Franzini-Armstrong, 2002). Supported by NIH (GM 086167)

Sex differences in repolarization and slow delayed rectifier potassium current and their regulation by sympathetic stimulation in rabbits

Slow delayed rectifier potassium current (IKs) is important in action potential (AP) repolarization and repolarization reserve. We tested the hypothesis that there are sex-specific differences in IKs, AP, and their regulation by β-adrenergic receptors (β-AR's) using whole-cell patch-clamp. AP duration (APD90) was significantly longer in control female (F) than in control male (M) myocytes. Isoproterenol (ISO, 500 nM) shortened APD90 comparably in M and F, and was largely reversed by β1-AR blocker CGP 20712A (CGP, 300 nM). Inhibition of IKs with chromanol 293B (10 μM) resulted in less APD prolongation in F at baseline (3.0 vs 8.9 %, p < 0.05 vs M) and even in the presence of ISO (5.4 vs 20.9 %, p < 0.05). This suggests that much of the ISO-induced APD abbreviation in F is independent of IKs. In F, baseline IKs was 42 % less and was more weakly activated by ISO (19 vs 68 % in M, p < 0.01). ISO enhancement of IKs was comparably attenuated by CGP in M and F. After ovariectomy, IKs in F had greater enhancement by ISO (72 %), now comparable to control M. After orchiectomy, IKs in M was only slightly enhanced by ISO (23 %), comparable to control F. Pretreatment with thapsigargin (to block SR Ca release) had bigger impact on ISO-induced APD shortening in F than that in M (p < 0.01). In conclusion, we found that there are sex differences in IKs, AP, and their regulation by β-AR's that are modulated by sex hormones, suggesting the potential for sex-specific antiarrhythmic therapy.

Size‐dependent heterogeneity of contractile Ca2+ sensitization in rat arterial smooth muscle

Each segment along arterial vessels adapts to different circumstances, including blood pressure and sympathetic innervation. PKC and Rho-associated kinase (ROCK) Ca(2+)-sensitizing pathways leading to myosin phosphatase inhibition are critically involved in α(1)-adrenoceptor-mediated vascular smooth muscle contraction in distinctive time-dependent manners. We tested whether the amplitude and time course of each pathway varies dynamically between arterial segments. Using pharmacological approaches, we determined the time-dependent roles of Ca(2+) release, Ca(2+) influx, PKC and ROCK in α(1)-agonist-induced contraction and phosphorylation of key proteins in denuded rat small mesenteric artery, midsized caudal artery and thoracic aorta. SR Ca(2+) release and voltage-dependent Ca(2+) influx were essential for the initial rising and late sustained phases, respectively, of phenylephrine-induced contraction, regardless of arterial size. In small mesenteric arteries, α(1A)-subtype-specific antagonists and inhibitors of PKC, but not ROCK, markedly reduced the initial and late phases of contraction in a non-additive manner and suppressed phosphorylation of myosin light chain (MLC) and CPI-17, but not myosin targeting subunit of myosin light chain phosphatase (MYPT1). In aorta, an α(1D)-specific antagonist reduced both the initial and late phases of contraction with a significant decrease in MLC but not CPI-17 or MYPT1 phosphorylation. ROCK inhibitors, but not PKC inhibitors, suppressed the sustained phase of contraction with a decrease in MLC and MYPT1 phosphorylation in the aorta. The effect of ROCK inhibitors was additive with the α(1D)-antagonist. The results for midsized arteries were intermediate. Thus, the PKC-CPI-17 Ca(2+)-sensitizing pathway, which is dependent on PKC subtype and a Ca(2+)-handling mechanism, and is downstream of α(1A) receptors, plays a major role in α(1)-agonist-induced contraction of small resistance arteries in the splanchnic vascular beds. The effect of PKC and ROCK increases and decreases, respectively, with decreasing arterial size.

Ankyrin-B reduction enhances Ca spark-mediated SR Ca release promoting cardiac myocyte arrhythmic activity

Ankyrin-B (AnkB) loss-of-function may cause ventricular arrhythmias and sudden cardiac death in humans. Cardiac myocytes from AnkB heterozygous mice (AnkB(+/-)) show reduced expression and altered localization of Na/Ca exchanger (NCX) and Na/K-ATPase (NKA), key players in regulating [Na](i) and [Ca](i). Here we investigate how AnkB reduction affects cardiac [Na](i), [Ca](i) and SR Ca release. We found reduced NCX and NKA transport function but unaltered [Na](i) and diastolic [Ca](i) in myocytes from AnkB(+/-) vs. wild-type (WT) mice. Ca transients, SR Ca content and fractional SR Ca release were larger in AnkB(+/-) myocytes. The frequency of spontaneous, diastolic Ca sparks (CaSpF) was significantly higher in intact myocytes from AnkB(+/-) vs. WT myocytes (with and without isoproterenol), even when normalized for SR Ca load. However, total ryanodine receptor (RyR)-mediated SR Ca leak (tetracaine-sensitive) was not different between groups. Thus, in AnkB(+/-) mice SR Ca leak is biased towards more Ca sparks (vs. smaller release events), suggesting more coordinated openings of RyRs in a cluster. This is due to local cytosolic RyR regulation, rather than intrinsic RyR differences, since CaSpF was similar in saponin-permeabilized myocytes from WT and AnkB(+/-) mice. The more coordinated RyRs openings resulted in an increased propensity of pro-arrhythmic Ca waves in AnkB(+/-) myocytes. In conclusion, AnkB reduction alters cardiac Na and Ca transport and enhances the coupled RyR openings, resulting in more frequent Ca sparks and waves although the total SR Ca leak is unaffected. This could enhance the propensity for triggered arrhythmias in AnkB(+/-) mice.

Synchronization of Ca+2 Release in Multicellular Cardiac Preparations

Delayed afterdepolarizations (DADs) are frequently observed under conditions that increase cellular and SR Ca+2. The high Ca+2 can cause spontaneous SR Ca+2 release and oscillations in the membrane potential. It is these oscillations that evoke spontaneous action potentials in the myocytes, leading to arrhythmias. Since myocytes are electrically coupled, any depolarization that arises in one myocyte would be dampened due to dissipation of the current in the neighboring myocytes. It is believed that some synchronization mechanism exists that synchronizes DADs and action potentials in neighboring cells. In our current study, we are interested in examining whether there is a synchronization mechanism for the generation of triggered activity in multicellular cardiac preparations. Trabeculae isolated from rat hearts were mounted on a modified confocal microscope that was equipped with a force transducer. Trabeculae were loaded with Rhod-2 AM, and were washed with buffer containing BDM and blebbistatin to stop contraction and allow for Ca+2 imaging. Combination of 0.5 mM caffeine and a range of concentrations of isoproterenol (100–300 nM) were added to the perfusion to induce DADs. Line scans were acquired and fluorescence emitted were expressed as ΔF/F0. The addition of caffeine and isoproterenol show an increased incidence of DADs, with more DADs occuring as the concentration of isoproternol was increased. The amplitude of the DADs, in comparison to the stimulated twitch, was also increased with increasing isoproternol concentration. In similar experiments without BDM/blebbistatin, examining the force tracings, the DADs occured in a synchronous manner in neighboring myocytes eventually leading to contractions that are similar to those that were paced. The line scan images also show similar findings, where the Ca+2 transient amplitudes were analyzed. Collectively, these results suggest that intracellular, diastolic SR Ca release is can become synchronized in a multicellular cardiac preparation.

How Does Stochastic Ryanodine Receptor-Mediated Ca Leak Fail to Initiate a Ca Spark?

Spontaneous calcium (Ca) sparks are initiated by single ryanodine receptor (RyR) opening. Once one RyR channel opens, it elevates local [Ca] in the cleft space ([Ca]Cleft), which opens other RyR channels in the same Ca release unit (CaRU) via Ca-induced Ca-release. Experiments by Zima et al. (J. Physiol. 588:4743–4757, 2010) demonstrate that spontaneous Ca sparks occur only when intrasarcoplasmic-reticulum (SR) [Ca] ([Ca]SR) is above a threshold level, but that RyR-mediated SR Ca leak exists without Ca sparks well below this threshold [Ca]SR. We examine here how single RyR opening at lower [Ca]SR can fail to recruit Ca sparks at a CaRU, while still contributing to SR Ca leak. We assess this using a physiologically detailed mathematical model of junctional SR Ca release in which RyR gating is regulated by [Ca]SR and [Ca]Cleft. We find that several factors contribute to the failure of Ca sparks as [Ca]SR declines: 1), lower [Ca]SR reduces driving force and thus limits local [Ca]Cleft achieved and the rate of rise during RyR opening; 2), low [Ca]SR limits RyR open time (τO), which further reduces local [Ca]Cleft attained; 3), low τO and fast [Ca]Cleft dissipation after RyR closure shorten the opportunity for neighboring RyR activation; 4), at low [Ca]SR, the RyR exhibits reduced [Ca]Cleft sensitivity. We conclude that all of these factors conspire to reduce the probability of Ca sparks as [Ca]SR declines, despite continued RyR-mediated Ca leak. In addition, these same factors explain the much lower efficacy of L-type Ca channel opening to trigger local SR Ca release at low [Ca]SR during excitation-contraction coupling. Conversely, all of these factors are fundamentally important for increasing the propensity for pro-arrhythmic Ca sparks and waves in cardiac myocytes at high [Ca]SR.

Store-Operated Ca2+ Entry (SOCE) Contributes to Normal Skeletal Muscle Contractility in young but not in aged skeletal muscle

Muscle atrophy alone is insufficient to explain the significant decline in contractile force of skeletal muscle during normal aging. One contributing factor to decreased contractile force in aging skeletal muscle could be compromised excitation-contraction (E-C) coupling, without sufficient available Ca(2+) to allow for repetitive muscle contractility, skeletal muscles naturally become weaker. Using biophysical approaches, we previously showed that store-operated Ca(2+) entry (SOCE) is compromised in aged skeletal muscle but not in young ones. While important, a missing component from previous studies is whether or not SOCE function correlates with contractile function during aging. Here we test the contribution of extracellular Ca(2+) to contractile function of skeletal muscle during aging. First, we demonstrate graded coupling between SR Ca(2+) release channel-mediated Ca(2+) release and activation of SOCE. Inhibition of SOCE produced significant reduction of contractile force in young skeletal muscle, particularly at high frequency stimulation, and such effects were completely absent in aged skeletal muscle. Our data indicate that SOCE contributes to the normal physiological contractile response of young healthy skeletal muscle and that defective extracellular Ca(2+) entry through SOCE contributes to the reduced contractile force characteristic of aged skeletal muscle.

Ec Coupling is Preserved in Ncx Knockout Mice Exposed to Metabolic Inhibitors

To determine whether ablating the cardiac sodium-calcium exchanger (NCX) prevents couplon loss during metabolic inhibition (MI), we recorded Ca transients or resting Ca spark frequency (CaSpF) in ventricular myocytes isolated from WT and ventricular-specific NCX knockout (KO) mice. In field stimulated myocytes loaded with 10 µM Fluo-3 AM, application of the mitochondrial and glycolytic inhibitors FCCP (50 nM) and 2-DG (10 mM) reduced Ca transients by 54.2 ± 3.3% in WT but only by 19.0 ± 6.0% in KO (p <0.05). Similarly, MI suppressed action potential evoked Ca sparks in patch clamped WT myocytes loaded with 1 mM fluo-3 salt by 60.0 ± 8.2% (p <0.05), but in KO by only 33.0 ± 3.4%. The reduction in ICa during MI was similar for both WT and KO and SR Ca stores remained at control levels in both cell types during MI. In patch clamped myocytes loaded with 1 mM K5Fluo-3 and held at −75 mV, MI reduced resting CaSpF in WT by 92.4 ± 6.7%, (p=0.008) but had no effect on CaSpF in KO Abolishing Na and Ca gradients by using the same “internal” solution for both the bath and pipette (current clamp mode, VRest = 0 mV), eliminated the KO's resistance to MI, causing CaSpF to decline to a similar extent in both WT and KO (59.0 ± 6.2% and 60.7 ± 10.9%, respectively). This finding suggests that elevated diadic cleft Ca, a major characteristic of KO myocytes (Pott et al, Biophys J, 92: 1431, 2007; Neco et al. Biophys J, 99: 755, 2010), maintains effective triggering of SR Ca release during MI despite reduced ICa.

Increased Levels of Micrornas miR-1 and miR-133 in Failing Heart Underlie Dissociation of Phosphatase Activity from RyR2 Complex Resulting in Enhanced RyR2 CaMKII-Dependent Phosphorylation and Cardiac Arrhythmias

Increased propensity of ventricular myocytes to arrhythmogenic spontaneous SR Ca release and afterdepolarizations in heart failure (HF) has been linked to abnormally high activity of RyR2. Growing evidence supports hyperphosphorylation of RyR2 at the CaMKII site S-2814 as a potential mechanism for altered RyR2 function. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation remain poorly understood. MicroRNAs are small noncoding RNAs that regulate protein expression by interfering with mRNAs of target genes. We recently reported that 2-fold overexpession of microRNA miR-1 enhances CaMKII-dependent RyR2 phosphorylation by disrupting protein phosphatase 2A scaffolding to the RyR2, resulting in increased activity of the channel and Ca-dependent afterdepolarizations in myocytes. In the present study, we used a canine model of nonischemic HF to test the hypothesis that the HF-related alterations in RyR2 phosphorylation levels are caused by a decrease in phosphatase activity localized to RyR2 due to enhanced expression of two most abundant muscle-specific microRNAs miR-1 and miR-133. qRT-PCR studies revealed that the levels of miR-1 and miR-133 were significantly increased in HF myocytes compared to controls (2 and 1.6 fold accordingly). Western blotting showed that PP2A regulatory (b56alpha) and catalytic subunits, specific targets of miR-1 and miR-133 validated by luciferase-reporter assay, were decreased in HF cells. Analysis using phospho-specific antibodies confirmed that RyR2 phosphorylation at Ser-2814 was significantly increased in HF myocytes compared to controls. CaMKII inhibitory peptide reduced the frequency of spontaneous Ca waves in paced current-clamped HF myocytes to low control values. These finding suggest that altered levels of major muscle-specific microRNAs contribute to abnormal RyR2 function in HF by depressing localized phosphatase activity to the channel, thus leading to excessive phosphorylation of RyR2s.

Ca efflux via the sarcolemmal Ca ATPase occurs only in the t-tubules of rat ventricular myocytes

The transverse (t-) tubule network is an important site for Ca influx and release during excitation-contraction coupling in cardiac ventricular myocytes; however, its role in Ca extrusion is less clear. The present study was designed to investigate the relative contributions of Ca extrusion pathways across the t-tubule and surface membranes. Ventricular myocytes were isolated from the hearts of adult male Wistar rats and detubulated using formamide. Intracellular Ca was monitored using fluo-3 and confocal microscopy. Caffeine (20mmol/L) was used to induce SR Ca release; carboxyeosin (20μmol/L) and nickel (10mmol/L) were used to inhibit the sarcolemmal Ca ATPase and Na/Ca exchanger (NCX) respectively. Carboxyeosin decreased the rate constant of decay of the caffeine-induced Ca transient in control cells, but had no effect in detubulated cells, suggesting that Ca extrusion via the Ca ATPase occurs only across the t-tubule membrane. However nickel decreased the rate constant of the caffeine-induced Ca transient in control and detubulated cells, although its effect was greater in control cells, suggesting that Ca extrusion via NCX occurs across the surface and t-tubule membranes. The PKA inhibitor H-89 (10μmol/L) was used to investigate the role of basal PKA activity in Ca extrusion; H-89 appeared to have no effect on Ca extrusion via the Ca ATPase, but reduced Ca extrusion via NCX at the t-tubules but not the surface membrane. Thus it appears that Ca extrusion via the sarcolemmal Ca ATPase occurs only at the t-tubules, and is not regulated by basal PKA activity, while Ca extrusion via NCX occurs across both the surface and t-tubule membranes, but predominantly across the t-tubule membrane due, in part, to localised stimulation of NCX by PKA at the t-tubules. This may be important in heart disease, in which changes in t-tubule structure and protein phosphorylation occur.

Na/K-ATpase α2-Subunit Preferentially Modulates Ca Transients and SR Ca Release in Cardiac Myocytes

Na/K-ATPase (NKA) is essential in regulating [Na]i, and thus cardiac myocyte Ca and contractility via Na/Ca exchange. Different NKAα-subunit isoforms are present in heart and may differ functionally, depending on differential membrane localization. In smooth muscle and astrocytes, NKA-α2 is located at the junctions with endo(sarco)plasmic reticulum, where they could regulate local Na, and indirectly junctional cleft [Ca]. In contrast, NKA-α1 is ubiquitously distributed and may regulate bulk [Na]i. It is controversial whether this model holds for cardiac myocytes. We measured the effect of selective NKA-α2/NKA-α1 inhibition with low concentrations of ouabain on [Na]i, Ca transients and fractional SR Ca release in cardiac myocytes from wild-type (WT) mice and transgenic mice in which NKA-α1 is ouabain-sensitive and NKA-α2 is ouabain-resistant (SWAP mice), respectively. Different [ouabain] (5 and 0.1 μM, respectively) were used to attain a similar level of total NKA inhibition in WT and SWAP mice. Ouabain increased Ca transients in WT (F/F0=1.84±0.13 vs. 1.54±0.12 under control conditions) but not in SWAP mice (1.49±0.15 vs. 1.46±0.12), despite a similar and modest increase in [Na]i (≤2 mM). Ca transient increase in WT mice was mediated specifically by NKA-α2 inhibition, because 5 μM ouabain had no effect in transgenic mice where both NKA-α1 and NKA-α2 are ouabain-resistant. Ouabain also significantly increased the fractional SR Ca release in WT mice (from 23±2% to 28±2%) but not SWAP mice (23±3% with and without ouabain). Dual-color immunofluorescence measurements coupled with spatial cross-correlation analysis revealed that NKA-α2 is co-localized to a significant extent with both Na/Ca exchanger (43% co-localization) and ryanodine receptors (43% co-localization) in cardiac myocytes. In conclusion, our data indicate that NKA-α2 has a more prominent role (vs. NKA-α1) in modulating Ca transients and SR Ca release in cardiac myocytes.

Increased Ca Sparks and Waves Frequency and Unchanged [Na]i in Hearts from Ankyrin-B Heterozygous Mice

The interplay between Na/Ca exchanger (NCX) and Na/K-ATPase (NKA) is essential in regulating cardiac [Na]i, [Ca]i and contractility. The membrane targeting and stability of both NCX and NKA require direct interaction with cytoskeletal protein ankyrin B (AnkB). Humans with AnkB loss-of-function mutations and AnkB heterozygous mice (AnkB+/-) display a complex cardiac phenotype that includes ventricular arrhythmias and sudden death. Cardiac myocytes from AnkB+/- mice show reduced NCX and NKA expression at the T-tubules, larger cellular and SR Ca load and increased frequency of delayed afterdepolarizations (DADs). How the coordinated loss of NCX and NKA affects SR Ca release and [Na]i is poorly understood. We measured Ca sparks and waves in myocytes from AnkB+/- and wild-type (WT) mice with and without 1 μM isoproterenol. Under control conditions, Ca transients, SR Ca load and Ca sparks frequency (CaSpF), even normalized to SR Ca content, were higher in AnkB+/- mice at all frequencies. With isoproterenol, Ca transient amplitude and SR Ca content were similar in myocytes from WT and AnkB+/- mice, but CaSpF was still elevated in AnkB+/- mice. Ca waves occurred preponderantly in AnkB+/- mice. In some cases, Ca waves and sparks evolved into synchronous after-Ca transients that are indicative of DADs. This data suggest that SR Ca release is enhanced in AnkB+/- mice. We measured [Na]i to determine whether the increased CaSpF in AnkB+/- mice is due to higher [Na]i, which may elevate diastolic Ca. [Na]i was slightly, but not significantly, lower in AnkB+/- vs. WT mice (11.4±1.1 vs. 13.2±1.7 mM in WT). Isoproterenol lowered [Na]i by a comparable amount in both AnkB+/- and WT mice (to 8.1±0.9 mM and 11.1±1.5 mM). Thus, altered [Na]i does not explain the increased Ca sparks frequency in AnkB+/- mice.

Refractoriness of Ryanodine Receptors During Calcium Alternans in Rabbit Atrial Myocytes

Electro-mechanical and Ca alternans is a known pro-arrhythmic factor. At the cellular level Ca alternans appears as cytosolic Ca transients of alternating amplitude at regular beating frequency. Direct intra-sarcoplasmic reticulum (SR) [Ca] measurements with the low affinity Ca indicator fluo-5N entrapped in the SR revealed that alternans in diastolic SR content are not a prerequisite for cytosolic Ca alternans, and thus SR Ca content is not the sole determinant of alternans. The goal of this study was to determine whether alternans of the kinetics of recovery from inactivation of ryanodine receptors and refractoriness of release represent a key factor underlying cytosolic Ca alternans. Alternans was induced by electrical pacing (1.6 to 2.5 Hz). After Ca alternans was established, pacing was stopped and the occurrence of spontaneous Ca waves and Ca sparks was quantified. The time interval from cessation of stimulation to the appearance of the first Ca waves was significantly shorter and the frequency of Ca sparks was higher after the small Ca transient compared to the large transient. Application of 0.1 mM caffeine or 10 μM isoproterenol rescued Ca alternans and shortened the rest interval until appearance of Ca waves. Photolysis of caged Ca (DM-nitrophen) to produce photolytically triggered Ca release (PTCR) from the SR was used to probe the refractoriness of SR Ca release during alternans. During the decay phase of the Ca transient PTCR was significantly less during the large Ca transient. During the rising phase of the Ca transient PTCR was greater during the large Ca transient, and was capable of inducing a phase reversal of Ca alternans. We conclude that alternating ryanodine receptor inactivation recovery intervals, together with alternations in SR Ca load, represent key determinants of Ca alternans. (VMS and CL contributed equally).

Enhanced muscle shortening and impaired Ca2+ channel function in an acute septic myopathy model

Myopathies in critically ill patients are increasingly documented. Various animal models of chronic sepsis have been employed to investigate reduced membrane excitability or altered isometric contractility of skeletal muscle. In contrast, immediate changes occurring during acute sepsis are significantly under-characterised; L-type Ca(2+) channel function or isotonic shortening are examples. We recorded slowly activating L-type Ca(2+) currents (I (Ca)) in voltage-clamped single intact mouse skeletal muscle fibres and tested the effects of acute challenge with serum fractions from critical illness myopathy patients (CIM). Using a high-speed camera system, we simultaneously recorded unloaded fibre shortening during isotonic contractions with unprecedented temporal resolution (approximately 1,600 frames/s). Time courses of fibre lengths and shortening velocity were determined from automated imaging algorithms. CIM fractions acutely induced depression of I (Ca) amplitudes with no shifts in I (Ca)-V-relations. Voltage-dependent inactivation was unaltered and I (Ca) activation and inactivation kinetics were prolonged compared to controls. Unexpectedly, maximum unloaded speed of shortening was slightly faster following CIM serum applications, suggesting a direct action of CIM serum on weak-binding-state cross-bridges. Our results are compatible with a model where CIM serum might acutely reduce a fraction of functional L-type Ca(2+) channels and could account for reduced SR Ca(2+) release and force production in CIM patients. Acute increase in isotonic shortening velocity might be an early diagnostic feature suitable for testing in clinical studies. The acute challenge model is also robust against atrophy or fibre type changes that ordinarily would have to be considered in chronic sepsis models.

Myostatin represses physiological hypertrophy of the heart and excitation–contraction coupling

Although myostatin negatively regulates skeletal muscle growth, its function in heart is virtually unknown. Herein we demonstrate that it inhibits basal and IGF-stimulated proliferation and differentiation and also modulates cardiac excitation-contraction (EC) coupling. Loss of myostatin induced eccentric hypertrophy and enhanced cardiac responsiveness to beta-adrenergic stimulation in vivo. This was due to myostatin null ventricular myocytes having larger [Ca(2+)](i) transients and contractions and responding more strongly to beta-adrenergic stimulation than wild-type cells. Enhanced cardiac output and beta-adrenergic responsiveness of myostatin null mice was therefore due to increased SR Ca(2+) release during EC coupling and to physiological hypertrophy, but not to enhanced myofilament function as determined by simultaneous measurement of force and ATPase activity. Our studies support the novel concept that myostatin is a repressor of physiological cardiac muscle growth and function. Thus, the controlled inhibition of myostatin action could potentially help repair damaged cardiac muscle by inducing physiological hypertrophy.

Simultaneous recording of intramembrane charge movement components and calcium release in wild‐type and S100A1−/− muscle fibres

In the preceding paper, we reported that flexor digitorum brevis (FDB) muscle fibres from S100A1 knock-out (KO) mice exhibit a selective suppression of the delayed, steeply voltage-dependent component of intra-membrane charge movement current termed Q(gamma). Here, we use 50 microm of the Ca(2+) indicator fluo-4 in the whole cell patch clamp pipette, in addition to 20 mM EGTA and other constituents included for the charge movement studies, and calculate the SR Ca(2+) release flux from the fluo-4 signals during voltage clamp depolarizations. Ca(2+) release flux is decreased in amplitude by the same fraction at all voltages in fibres from S100A1 KO mice compared to fibres from wild-type (WT) littermates, but unchanged in time course at each pulse membrane potential. There is a strong correlation between the time course and magnitude of release flux and the development of Q(gamma). The decreased Ca(2+) release in KO fibres is likely to account for the suppression of Q(gamma) in these fibres. Consistent with this interpretation, 4-chloro-m-cresol (4-CMC; 100 microm) increases the rate of Ca(2+) release and restores Q(gamma) at intermediate depolarizations in fibres from KO mice, but does not increase Ca(2+) release or restore Q(gamma) at large depolarizations. Our findings are consistent with similar activation kinetics for SR Ca(2+) channels in both WT and KO fibres, but decreased Ca(2+) release in the KO fibres possibly due to shorter SR channel open times. The decreased Ca(2+) release at each voltage is insufficient to activate Q(gamma) in fibres lacking S100A1.