Sarcolemmal distribution of ICa and INCX and Ca2+ autoregulation in mouse ventricular myocytes.
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ABSTRACT:
SUBMITTER: Gadeberg HC
PROVIDER: S-EPMC5538864 | biostudies-other | 2017 Jul
REPOSITORIES: biostudies-other
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Project description:BACKGROUND AND PURPOSE The Ca(2+) paradox is an important phenomenon associated with Ca(2+) overload-mediated cellular injury in myocardium. The present study was undertaken to elucidate molecular and cellular mechanisms for the development of the Ca(2+) paradox. EXPERIMENTAL APPROACH Fluorescence imaging was performed on fluo-3 loaded quiescent mouse ventricular myocytes using confocal laser scanning microscope. KEY RESULTS The Ca(2+) paradox was readily evoked by restoration of the extracellular Ca(2+) following 10-20?min of nominally Ca(2+)-free superfusion. The Ca(2+) paradox was significantly reduced by blockers of transient receptor potential canonical (TRPC) channels (2-aminoethoxydiphenyl borate, Gd(3+), La(3+)) and anti-TRPC1 antibody. The sarcoplasmic reticulum (SR) Ca(2+) content, assessed by caffeine application, gradually declined during Ca(2+)-free superfusion, which was further accelerated by metabolic inhibition. Block of SR Ca(2+) leak by tetracaine prevented Ca(2+) paradox. The Na(+) /Ca(2+) exchange (NCX) blocker KB-R7943 significantly inhibited Ca(2+) paradox when applied throughout superfusion period, but had little effect when added for a period of 3?min before and during Ca(2+) restoration. The SR Ca(2+) content was better preserved during Ca(2+) depletion by KB-R7943. Immunocytochemistry confirmed the expression of TRPC1, in addition to TRPC3 and TRPC4, in mouse ventricular myocytes. CONCLUSIONS AND IMPLICATIONS These results provide evidence that (i) the Ca(2+) paradox is primarily mediated by Ca(2+) entry through TRPC (probably TRPC1) channels that are presumably activated by SR Ca(2+) depletion; and (ii) reverse mode NCX contributes little to the Ca(2+) paradox, whereas inhibition of NCX during Ca(2+) depletion improves SR Ca(2+) loading, and is associated with reduced incidence of Ca(2+) paradox in mouse ventricular myocytes.
| S-EPMC3010579 | biostudies-literature
Project description:Aging is associated with diminished cardiac function in males. Cardiac excitation-contraction coupling in ventricular myocytes involves Ca influx via the Ca current (ICa) and Ca release from the sarcoplasmic reticulum, which occur predominantly at t-tubules. Caveolin-3 regulates t-tubular ICa, partly through protein kinase A (PKA), and both ICa and caveolin-3 decrease with age. We therefore investigated ICa and t-tubule structure and function in cardiomyocytes from male wild-type (WT) and caveolin-3-overexpressing (Cav-3OE) mice at 3 and 24 months of age. In WT cardiomyocytes, t-tubular ICa-density was reduced by ~50% with age while surface ICa density was unchanged. Although regulation by PKA was unaffected by age, inhibition of caveolin-3-binding reduced t-tubular ICa at 3 months, but not at 24 months. While Cav-3OE increased cardiac caveolin-3 protein expression ~2.5-fold at both ages, the age-dependent reduction in caveolin-3 (WT ~35%) was preserved in transgenic mice. Overexpression of caveolin-3 reduced t-tubular ICa density at 3 months but prevented further ICa loss with age. Measurement of Ca release at the t-tubules revealed that the triggering of local Ca release by t-tubular ICa was unaffected by age. In conclusion, the data suggest that the reduction in ICa density with age is associated with the loss of a caveolin-3-dependent mechanism that augments t-tubular ICa density.
| S-EPMC5946816 | biostudies-other
Project description:KEY POINTS:Small-conductance Ca2+ -activated K+ (SK) channels expressed in ventricular myocytes are dormant in health, yet become functional in cardiac disease. SK channels are voltage independent and their gating is controlled by intracellular [Ca2+ ] in a biphasic manner. Submicromolar [Ca2+ ] activates the channel via constitutively-bound calmodulin, whereas higher [Ca2+ ] exerts inhibitory effect during depolarization. Using a rat model of cardiac hypertrophy induced by thoracic aortic banding, we found that functional upregulation of SK2 channels in hypertrophic rat ventricular cardiomyocytes is driven by protein kinase A (PKA) phosphorylation. Using site-directed mutagenesis, we identified serine-465 as the site conferring PKA-dependent effects on SK2 channel function. PKA phosphorylation attenuates ISK rectification by reducing the Ca2+ /voltage-dependent inhibition of SK channels without changing their sensitivity to activating submicromolar [Ca2+ ]i . This mechanism underlies the functional recruitment of SK channels not only in cardiac disease, but also in normal physiology, contributing to repolarization under conditions of enhanced adrenergic drive. ABSTRACT:Small-conductance Ca2+ -activated K+ (SK) channels expressed in ventricular myocytes (VMs) are dormant in health, yet become functional in cardiac disease. We aimed to test the hypothesis that post-translational modification of SK channels under conditions accompanied by enhanced adrenergic drive plays a central role in disease-related activation of the channels. We investigated this phenomenon using a rat model of hypertrophy induced by thoracic aortic banding (TAB). Western blot analysis using anti-pan-serine/threonine antibodies demonstrated enhanced phosphorylation of immunoprecipitated SK2 channels in VMs from TAB rats vs. Shams, which was reversible by incubation of the VMs with PKA inhibitor H89 (1 ?mol L-1 ). Patch clamped VMs under basal conditions from TABs but not Shams exhibited outward current sensitive to the specific SK inhibitor apamin (100 nmol L-1 ), which was eliminated by inhibition of PKA (1 ?mol L-1 ). Beta-adrenergic stimulation (isoproterenol, 100 nmol L-1 ) evoked ISK in VMs from Shams, resulting in shortening of action potentials in VMs and ex vivo optically mapped Sham hearts. Using adenoviral gene transfer, wild-type and mutant SK2 channels were overexpressed in adult rat VMs, revealing serine-465 as the site that elicits PKA-dependent phosphorylation effects on SK2 channel function. Concurrent confocal Ca2+ imaging experiments established that PKA phosphorylation lessens rectification of ISK via reduction Ca2+ /voltage-dependent inhibition of the channels at high [Ca2+ ] without affecting their sensitivity to activation by Ca2+ in the submicromolar range. In conclusion, upregulation of SK channels in diseased VMs is mediated by hyperadrenergic drive in cardiac hypertrophy, with functional effects on the channel conferred by PKA-dependent phosphorylation at serine-465.
| S-EPMC7496687 | biostudies-literature
Project description:The sarcoplasmic reticulum (SR) in ventricular myocytes contains releasable Ca(2+) for activating cellular contraction. Recent measurements of intra-SR (luminal) Ca(2+) suggest a high diffusive Ca(2+)-mobility constant (D(CaSR)). This could help spatially to unify SR Ca(2+)-content ([Ca(2+)](SRT)) and standardize Ca(2+)-release throughout the cell. But measurements of localized depletions of luminal Ca(2+) (Ca(2+)-blinks), associated with local Ca(2+)-release (Ca(2+)-sparks), suggest D(CaSR) may actually be low. Here we describe a novel method for measuring D(CaSR). Using a cytoplasmic Ca(2+)-fluorophore, we estimate regional [Ca(2+)](SRT) from localized, caffeine-induced SR Ca(2+)-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 microm(2)/s, up to tenfold lower than previous estimates. Ignoring background SR Ca(2+)-leakage in our measurement protocol produces an artifactually high D(CaSR) (>40 microm(2)/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 Ca(2+)-signaling within sarcomeres during excitation-contraction coupling. Low D(CaSR) also implies that [Ca(2+)](SRT) may readily become spatially nonuniform, particularly under pathological conditions of spatially nonuniform Ca(2+)-release. Local control of luminal Ca(2+), imposed by low D(CaSR), may complement the well-established local control of SR Ca(2+)-release by Ca(2+)-channel/ryanodine receptor couplons.
| S-EPMC2479570 | biostudies-literature
Project description:The fluo family of indicators is frequently used in studying Ca(2+) physiology; however, choosing which fluo indicator to use is not obvious. Indicator properties are typically determined in well-defined aqueous solutions. Inside cells, however, the properties can change markedly. We have characterized each of three fluo variants (fluo-2MA, fluo-3 and fluo-4) in two forms-the acetoxymethyl (AM) ester and the K(+) salt. We loaded indicators into rat ventricular myocytes and used confocal microscopy to monitor depolarization-induced fluorescence changes and fractional shortening. Myocytes loaded with the indicator AM esters showed significantly different Ca(2+) transients and fractional shortening kinetics. Loading the K(+) salts via whole-cell patch-pipette eliminated differences between fluo-3 and fluo-4, but not fluo-2MA. Cells loaded with different indicator AM esters showed different staining patterns-suggesting differential loading into organelles. Ca(2+) dissociation constants (K(d,Ca)), measured in protein-rich buffers mimicking the cytosol were significantly higher than values determined in simple buffers. This increase in K(d,Ca) (decrease in Ca(2+) affinity) was greatest for fluo-3 and fluo-4, and least for fluo-2MA. We conclude that the structurally-similar fluo variants differ with respect to cellular loading, subcellular compartmentalization, and intracellular Ca(2+) affinity. Therefore, judicious choice of fluo indicator and loading procedure is advisable when designing experiments.
| S-EPMC3393790 | biostudies-literature
Project description:Zebrafish (Danio rerio) have become a popular model in cardiovascular research mainly due to identification of a large number of mutants with structural defects. In recent years, cardiomyopathies and other diseases influencing contractility of the heart have been studied in zebrafish mutants. However, little is known about the regulation of contractility of the zebrafish heart on a tissue level. The aim of the present study was to elucidate the role of trans-sarcolemmal Ca(2+)-flux and sarcoplasmic reticulum Ca(2+)-release in zebrafish myocardium. Using isometric force measurements of fresh heart slices, we characterised the effects of changes of the extracellular Ca(2+)-concentration, trans-sarcolemmal Ca(2+)-flux via L-type Ca(2+)-channels and Na(+)-Ca(2+)-exchanger, and Ca(2+)-release from the sarcoplasmic reticulum as well as beating frequency and ?-adrenergic stimulation on contractility of adult zebrafish myocardium. We found an overall negative force-frequency relationship (FFR). Inhibition of L-type Ca(2+)-channels by verapamil (1 ?M) decreased force of contraction to 22 ± 7% compared to baseline (n=4, p<0.05). Ni(2+) was the only substance to prolong relaxation (5 mM, time after peak to 50% relaxation: 73 ± 3 ms vs. 101 ± 8 ms, n=5, p<0.05). Surprisingly though, inhibition of the sarcoplasmic Ca(2+)-release decreased force development to 54 ± 3% in ventricular (n=13, p<0.05) and to 52 ± 8% in atrial myocardium (n=5, p<0.05) suggesting a substantial role of SR Ca(2+)-release in force generation. In line with this finding, we observed significant post pause potentiation after pauses of 5 s (169 ± 7% force compared to baseline, n=8, p<0.05) and 10 s (198 ± 9% force compared to baseline, n=5, p<0.05) and mildly positive lusitropy after ?-adrenergic stimulation. In conclusion, force development in adult zebrafish ventricular myocardium requires not only trans-sarcolemmal Ca2+-flux, but also intact sarcoplasmic reticulum Ca(2+)-cycling. In contrast to mammals, FFR is strongly negative in the zebrafish heart. These aspects need to be considered when using zebrafish to model human diseases of myocardial contractility.
| S-EPMC4418605 | biostudies-literature
Project description:AimsSpontaneous Ca2+ waves in cardiomyocytes are potentially arrhythmogenic. A powerful controller of Ca2+ waves is the cytoplasmic H+ concentration ([H+]i), which fluctuates spatially and temporally in conditions such as myocardial ischaemia/reperfusion. H+-control of Ca2+ waves is poorly understood. We have therefore investigated how [H+]i co-ordinates their initiation and frequency.Methods and resultsSpontaneous Ca2+ waves were imaged (fluo-3) in rat isolated ventricular myocytes, subjected to modest Ca2+-overload. Whole-cell intracellular acidosis (induced by acetate-superfusion) stimulated wave frequency. Pharmacologically blocking sarcolemmal Na+/H+ exchange (NHE1) prevented this stimulation, unveiling inhibition by H+. Acidosis also increased Ca2+ wave velocity. Restricting acidosis to one end of a myocyte, using a microfluidic device, inhibited Ca2+ waves in the acidic zone (consistent with ryanodine receptor inhibition), but stimulated wave emergence elsewhere in the cell. This remote stimulation was absent when NHE1 was selectively inhibited in the acidic zone. Remote stimulation depended on a locally evoked, NHE1-driven rise of [Na+]i that spread rapidly downstream.ConclusionAcidosis influences Ca2+ waves via inhibitory Hi+ and stimulatory Nai+ signals (the latter facilitating intracellular Ca2+-loading through modulation of sarcolemmal Na+/Ca2+ exchange activity). During spatial [H+]i-heterogeneity, Hi+-inhibition dominates in acidic regions, while rapid Nai+ diffusion stimulates waves in downstream, non-acidic regions. Local acidosis thus simultaneously inhibits and stimulates arrhythmogenic Ca2+-signalling in the same myocyte. If the principle of remote H+-stimulation of Ca2+ waves also applies in multicellular myocardium, it raises the possibility of electrical disturbances being driven remotely by adjacent ischaemic areas, which are known to be intensely acidic.
| S-EPMC5852542 | biostudies-literature
Project description:In ventricular myocytes, the physiological function of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum (ER/SR) Ca(2+) sensor, is unclear with respect to its cellular localization, its Ca(2+)-dependent mobilization, and its action on Ca(2+) signaling. Confocal microscopy was used to measure Ca(2+) signaling and to track the cellular movement of STIM1 with mCherry and immunofluorescence in freshly isolated adult rat ventricular myocytes and those in short-term primary culture. We found that endogenous STIM1 was expressed at low but measureable levels along the Z-disk, in a pattern of puncta and linear segments consistent with the STIM1 localizing to the junctional SR (jSR). Depleting SR Ca(2+) using thapsigargin (2-10 µM) changed neither the STIM1 distribution pattern nor its mobilization rate, evaluated by diffusion coefficient measurements using fluorescence recovery after photobleaching. Two-dimensional blue native polyacrylamide gel electrophoresis and coimmunoprecipitation showed that STIM1 in the heart exists mainly as a large protein complex, possibly a multimer, which is not altered by SR Ca(2+) depletion. Additionally, we found no store-operated Ca(2+) entry in control or STIM1 overexpressing ventricular myocytes. Nevertheless, STIM1 overexpressing cells show increased SR Ca(2+) content and increased SR Ca(2+) leak. These changes in Ca(2+) signaling in the SR appear to be due to STIM1 binding to phospholamban and thereby indirectly activating SERCA2a (Sarco/endoplasmic reticulum Ca(2+) ATPase). We conclude that STIM1 binding to phospholamban contributes to the regulation of SERCA2a activity in the steady state and rate of SR Ca(2+) leak and that these actions are independent of store-operated Ca(2+) entry, a process that is absent in normal heart cells.
| S-EPMC4553828 | biostudies-literature
Project description:Key pointsPrevailing dogma holds that activation of the β-adrenergic receptor/cAMP/protein kinase A signalling pathway leads to enhanced L-type CaV 1.2 channel activity, resulting in increased Ca2+ influx into ventricular myocytes and a positive inotropic response. However, the full mechanistic and molecular details underlying this phenomenon are incompletely understood. CaV 1.2 channel clusters decorate T-tubule sarcolemmas of ventricular myocytes. Within clusters, nanometer proximity between channels permits Ca2+ -dependent co-operative gating behaviour mediated by physical interactions between adjacent channel C-terminal tails. We report that stimulation of cardiomyocytes with isoproterenol, evokes dynamic, protein kinase A-dependent augmentation of CaV 1.2 channel abundance along cardiomyocyte T-tubules, resulting in the appearance of channel 'super-clusters', and enhanced channel co-operativity that amplifies Ca2+ influx. On the basis of these data, we suggest a new model in which a sub-sarcolemmal pool of pre-synthesized CaV 1.2 channels resides in cardiomyocytes and can be mobilized to the membrane in times of high haemodynamic or metabolic demand, to tune excitation-contraction coupling.AbstractVoltage-dependent L-type CaV 1.2 channels play an indispensable role in cardiac excitation-contraction coupling. Activation of the β-adrenergic receptor (βAR)/cAMP/protein kinase A (PKA) signalling pathway leads to enhanced CaV 1.2 activity, resulting in increased Ca2+ influx into ventricular myocytes and a positive inotropic response. CaV 1.2 channels exhibit a clustered distribution along the T-tubule sarcolemma of ventricular myocytes where nanometer proximity between channels permits Ca2+ -dependent co-operative gating behaviour mediated by dynamic, physical, allosteric interactions between adjacent channel C-terminal tails. This amplifies Ca2+ influx and augments myocyte Ca2+ transient and contraction amplitudes. We investigated whether βAR signalling could alter CaV 1.2 channel clustering to facilitate co-operative channel interactions and elevate Ca2+ influx in ventricular myocytes. Bimolecular fluorescence complementation experiments reveal that the βAR agonist, isoproterenol (ISO), promotes enhanced CaV 1.2-CaV 1.2 physical interactions. Super-resolution nanoscopy and dynamic channel tracking indicate that these interactions are expedited by enhanced spatial proximity between channels, resulting in the appearance of CaV 1.2 'super-clusters' along the z-lines of ISO-stimulated cardiomyocytes. The mechanism that leads to super-cluster formation involves rapid, dynamic augmentation of sarcolemmal CaV 1.2 channel abundance after ISO application. Optical and electrophysiological single channel recordings confirm that these newly inserted channels are functional and contribute to overt co-operative gating behaviour of CaV 1.2 channels in ISO stimulated myocytes. The results of the present study reveal a new facet of βAR-mediated regulation of CaV 1.2 channels in the heart and support the novel concept that a pre-synthesized pool of sub-sarcolemmal CaV 1.2 channel-containing vesicles/endosomes resides in cardiomyocytes and can be mobilized to the sarcolemma to tune excitation-contraction coupling to meet metabolic and/or haemodynamic demands.
| S-EPMC6462464 | biostudies-literature
Project description:We conducted super-resolution light microscopy (LM) imaging of the distribution of ryanodine receptors (RyRs) and caveolin-3 (CAV3) in mouse ventricular myocytes. Quantitative analysis of data at the surface sarcolemma showed that 4.8% of RyR labeling colocalized with CAV3 whereas 3.5% of CAV3 was in areas with RyR labeling. These values increased to 9.2 and 9.0%, respectively, in the interior of myocytes where CAV3 was widely expressed in the t-system but reduced in regions associated with junctional couplings. Electron microscopic (EM) tomography independently showed only few couplings with caveolae and little evidence for caveolar shapes on the t-system. Unexpectedly, both super-resolution LM and three-dimensional EM data (including serial block-face scanning EM) revealed significant increases in local t-system diameters in many regions associated with junctions. We suggest that this regional specialization helps reduce ionic accumulation and depletion in t-system lumen during excitation-contraction coupling to ensure effective local Ca²? release. Our data demonstrate that super-resolution LM and volume EM techniques complementarily enhance information on subcellular structure at the nanoscale.
| S-EPMC3672889 | biostudies-literature