Voltage-clamped Cardiomyocytes Research Articles

Assessment of Ca2+ Sensitivity of SK Channels in Rat Ventricular Cardiomyocytes using Intrinsic CA2+ Cycling Machinery

Small conductance Ca2+ activated K+ (SK) channels are thought to play significant roles in ventricular arrhythmias. It has been hypothesized that increase in Ca2+ sensitivity is the main underlying cause of functional upregulation of SKs in diseased hearts. We developed the protocol to assess Ca2+ sensitivity using simultaneous recordings of currents with intracellular Ca2+ in voltage clamped cardiomyocytes. By converting Rhod-2 fluorescence into [Ca2+]i we estimated Ca2+ sensitivity of rat SK2 channels overexpressed in cells using Adenovirus gene transfer. We obtained apparent affinity of rSK2 ∼ 500 nM which is in consistence with published values. To test whether SK channels from diseased hearts are more sensitive to Ca2+ we used rat model of cardiac hypertrophy induced by thoracic aortic banding (TAB). In contrast to Shams, myocytes from TAB hearts exhibited significant prolongation of APD upon application of SK inhibitor UCL-1684 (1 μM). Voltage clamp experiments showed SK inhibitor-sensitive current with amplitude ∼1 pA/pF. Simultaneous measurements of caffeine-induced ISK (10 mM) and [Ca]i at −30 mV before and after application of UCL-1684 demonstrated that sensitivity of SKs in TAB myocytes is low (Kd ∼900 nM). This result suggests that sensitivity to [Ca2+]i in hypertrophy is rather reduced than increased. To explore potential mechanisms responsible for changes in SK sensitivity to [Ca2+]i we treated cultured rat myocytes overexpressing SK2 with Phenylephrine (100 μM) and Propranolol (1 μM). We found that SK2 Ca2+ dependence in Phe/Pro was shifted to the right (Kd ∼850 nM), which was preventable by coexpressing CRNK, an inhibitory peptide for ROS and Ca2+-dependent tyrosine kinase Pyk2. Western blot analysis demonstrated that Pyk2 levels were upregulated in TABs. These data implicate Pyk2 as a novel negative regulator of SK Ca2+ sensitivity in diseased hearts.

Novel Calmodulin Mutation (CALM3-A103V) Associated with CPVT Syndrome Activates Arrhythmogenic Ca Waves and Sparks

Introduction: Calmodulin (CaM) is an essential Ca2+ signaling protein in a wide range of biological processes. In the dyadic cleft CaM binds to RyR2, Cav1.2 and CaMKII. CaM mutations are associated with an autosomal dominant syndrome of sudden death that can present with clinical features of catecholaminergic polymorphic ventricular tachycardia (CPVT) or long QT syndrome (LQTS). CPVT-linked CaM mutations activate ryanodine receptor (RyR2) Ca release channels; whereas LQTS-CaMs have no effects on RyR2 channels but prolong the action potential by impairing L-type Ca current (LTCC) inactivation. Here we report a novel CaM mutation – A103V – in CALM3, in a 10 year old female with episodes of exertion-induced syncope and normal QT interval consistent with CPVT. Her mother is mutation positive and also had a positive exercise stress test.Methods: CaM-A103V Ca binding affinity was measured by spectrofluorometry. Ca handling and LTCC were tested in mouse ventricular myocytes dialyzed with WT or the mutant CaM and compared to a published CPVT CaM mutant (N54I).Results: A103V modestly lowered CaM Ca binding affinity (3-fold reduction vs WT-CaM). In permeabilized myocytes, A103V-CaMs at a physiological intracellular concentration (100nM) promoted significantly higher spontaneous Ca wave and spark activity, a typical cellular phenotype of CPVT. Even a 1:4 mixture of A103V-CaM:WT-CaM activated Ca waves, demonstrating functional dominance. In voltage-clamped cardiomyocytes dialyzed with WT or mutant CaM, CaM-A103V had significantly less effects on LTCC inactivation compared to published CaM mutants that cause LQTS.Conclusion: A103V CaM shares functional characteristics with the established CPVT-CaM N54I. A small proportion of A103V-CaM is sufficient to evoke arrhythmogenic Ca disturbances, which explains the autosomal dominant inheritance.

Glutathionylation of the β1 Subunit Prevents the E1Na3 to E2P Forward Reaction in the Na+, K+ ATPase

Exposure of voltage clamped cardiomyocytes to receptor-coupled oxidant signalling has been associated with a decrease in electrogenic Na+, K+ pump current activity vattributed to glutathionylation of the pump's β1 subunit. The mechanism by which glutathionylation, a reversible oxidative modification, affects turnover is not understood. We investigated the effect of β1 subunit glutathionylation on the E1Na3 → E2P forward reaction using the fluorescent probe anthryl-ouabain (AO) that binds to E2P species.We induced glutathionylation in Na+, K+ ATPase enriched pig kidney membrane fragments by exposure to the chemical oxidant peroxynitrite. The Na+, K+ ATPase was stabilised in the E1Na3 poise by incubation in 100 mM NaCl and 20 mM Histidine. Glutathionylation of the β1 subunit was confirmed by immunoblotting techniques. MgATP was added to initiate the E1Na3 → E2P conversion and the E2P species was detected by the binding of AO. Addition of MgATP induced an increase in AO fluorescence indicative of a shift to E2P. The increase in fluorescence was blocked by the exposure to peroxynitrite. When glutathionylation was reversed by exposure to glutaredoxin 1, confirmed by the immublotting technique, the shift to E2P was restored as indicated by AO fluorescence.In an independent series of experiments we utilised baseline glutathionylation of a fraction of the Na+, K+ ATPase that is detectable without exposure to oxidants. We followed the E1Na3 → E2P conversion with RH421 fluorescence. As expected, addition of MgATP markedly increased fluorescence. We added 2.5 mM dithiothreitol (DTT) to rapidly reverse glutathionylation. This further increased RH421 fluorescence indictating that DTT made additional Na+, K+ ATPase available to undergo the E1Na3 → E2P conversion.We conclude that glutathionylation of the β1 subunit blocks the forward E1Na3 → E2P reaction.

Direct CaMKII Activation by Endogenous Nitric Oxide Modulates Calcium Spark Frequency in Cardiomyocytes

During β-adrenergic stimulation of cardiomyocytes, phosphorylation of the ryanodine receptors (RyRs) by protein kinases may contribute their increased open probability. We previously reported that β-adrenergic stimulation (β-ARS) with isoproterenol (Iso) increases the Ca2+ spark frequency by about 4-fold in quiescent, voltage-clamped cardiomyocytes. Kinase inhibitors (KN-93, AIP, H89) and nitric oxide synthase (NOS) inhibitors (L-NAME, AAAN) indicated that CaMKII and NOS were involved in the change of spark frequency.Here we show a new mechanism where CaMKII is activated by nitric oxide (NO). using confocal NO imaging with DAF-2 we observed a significant (∼19%) raise in endogenous NO production matching the time-course of the increase in spark frequency upon β-ARS. In contrast, this increase in NO was not observed in the presence of L-NAME, confirming that the DAF-2 fluorescence was reflecting NO and not ROS. In parallel experiments, bypassing the classical β-ARS pathway by application of a NO donor, increased the frequency of sparks to the same extent as during β-ARS with Iso. This increase induced by NO was blocked by the CaMKII inhibitor AIP, suggesting that NO first activated CaMKII, which subsequently phosphorylated the RyRs. Biochemical in-vitro CaMKII activity assays confirmed direct activation by NO, in a Ca2+ independent manner. CaMKII has been reported to be activated by ROS. However, control experiments revealed that ROS were not involved in the examined pathway. Neither incubation of the cells with SOD mimetics or ROS scavengers (Mn-TBAP, TIRON) prevented the increase in spark frequency upon Iso application.Based on these findings we conclude that endogenously produced NO activates CaMKII, contributing to the regulation of RyR Ca2+ sensitivity and Ca2+ spark frequency upon β-adrenergic stimulation of cardiomyocytes. Supported by SNF

Paradoxical Loss of Sarcoplasmic Reticulum Calcium Release Refractoriness Caused by Overexpressing Calsequestrin in Cardiac Muscle

L-type Ca current (ICa) triggers Carelease from the sarcoplasmic reticulum (SR) by activating RyR2 Ca release channels. Here we probe the role of the SR protein calsequestrin in the regulation of SR Ca2+ release. Voltage-clamped cardiomyocytes loaded with Fluo-4 were stimulated with 1 Hz train (S1 stimuli). Ca2+ release was activated with ICa tail currents to maintain constant trigger. Application of premature extra stimuli (S2) at successively shorter S1-S2 coupling interval resulted in significant depression of Ca2+ release during S2 stimuli in wild-type myocytes. In calsequestrin knockout myocytes (Casq2 KO), the amplitudes of S2 and S1 were the same even at shortest S1-S2 intervals (Figure). This result suggests that calsequestrin is the primary regulator of release refractoriness. To test this hypothesis further, we next used myocytes from calsequestrin overexpressing mice (Casq2 OverX). Casq2 OverX cardiomyocytes exhibit significantly depressed Ca release during S1 transients despite increased SR Ca2+ content. Surprisingly, Ca release amplitudes during the premature S2 stimulus exceeded S1 at short S1-S2 intervals (Figure). Thus, either too little or too much calsequestrin causes loss of SR Ca release refractoriness. Supported by NIH-RO1-HL88635.View Large Image | View Hi-Res Image | Download PowerPoint Slide

D-Enantiomers Take a Close Look at the Functioning of a Cardiac Cationic L-Amino Acid Transporter

Cationic amino acid transporters are highly selective for L-enantiomers such as L-arginine (L-Arg). Because of this stereoselectivity, little is known about the interaction of these transporters with D-isomers. To study whether these compounds can provide information on the molecular mechanism of transport, inward currents activated by L-Arg with low apparent affinity were measured in whole-cell voltage-clamped cardiomyocytes as a function of extracellular L-Arg and D-Arg concentrations. D-Arg inhibited L-Arg currents in a membrane-potential ( V M)-dependent competitive manner, indicating the presence of D-Arg binding sites in the carrier. Analysis of these steady-state currents showed that L- and D-Arg binding reactions dissipate a similar small fraction of the membrane electric field. Since D-Arg is not transported, these results suggest that enantiomer recognition occurs at conformational transitions that initiate amino acid translocation. The V M dependence of maximal current levels suggests that inward currents arise from the slow outward movement of negative charges in the unliganded transporter. Translocation of the L-Arg-bound complex, on the other hand, appears to be electroneutral. D-Arg-dependent transient charge movements, also detected in these cells, displayed a V M-dependent charge distribution and kinetics that are consistent with amino acid binding in an ion well in a shallow, water-filled extracellular binding pocket.

Pressure Puff Induced Calcium Signals in Voltage Clamped Cardiomyocytes

ICa-gated release of Ca2+ from the SR is the dominant mechanism mediating cardiac E-C coupling. On the other hand, in the absence of Ca2+ entry, supplemental Ca2+ release may be activated mechanically, either from the SR secondary to nitrosylation of RyRs or from mitochondria as a direct effect of puff-induced shear force. Here we have probed the puff-induced Ca2+ release in voltage-clamped cardiomyocytes, where the Ca2+-indicator rhod-2 was targeted to mitochondria by: a) staining with the AM-form of the dye for 45 minutes, b) incubating without dye for 24-30 hours and c) dialyzing with dye-free internal solution through the voltage-clamp pipette for 20 minutes before initiating measurements. In such cells, which remained responsive for periods as long as 2 hours, we measured relatively slow (∼1s) puff-induced deceases in fluorescence suggestive of mitochondrial Ca2+ release as previously found in non-dialyzed and permeabilized cells (Belmonte and Morad, 2008, J Physiol 586:1376). To clarify the [Ca2+]i- signaling under these conditions, our experimental paradigm included activation of ICa both at the beginning and end of a 2 second priming interval where the cell was exposed to control solution, 10 mM Caffeine or zero Na+ (and high K+). The caffeine-induced Ca2+ signal was biphasic with internal solution containing 0.2 or 14mM EGTA, generating INaCa only in 0.2 but not in 14mM EGTA during the rapid initial rise of Ca2+, suggesting that the maintained component of the Ca2+ signal arises from a confined and most likely mitochondrial space, not detected by NCX. We conclude that patch clamped Rhod-2 loaded myocytes that were washed overnight and dialyzed for 1-2 hour with 14mM EGTA produced reliable mitochondrial Ca2+ signals supporting the finding that the PF-triggered Ca2+-transients are caused by mitochondrial Ca2+ release.

Ca2+ Wave Development in Ventricular Cardiomyocytes from Mice with Inducible Knockout of SERCA2

Ca2+ waves develop when Ca2+ is spontaneously released from the sarcoplasmic reticulum (SR). Ca2+ then diffuses through the cytosol and triggers further release from neighboring ryanodine receptors. SERCA2 has been proposed to affect Ca2+ wave development in ventricular cardiomyocytes in two ways: 1) By its role as regulator of SR Ca2+ content. 2) By its influence on cytosolic Ca2+ in a propagating Ca2+ wave. We have studied the effect of an isolated reduction of SERCA2 abundance on Ca2+ wave development. Knockout of the Serca2 gene in cardiomyocytes was induced by a single i.p. injection of tamoxifen in Serca2flox/flox Tg(αMHC-MerCreMer) mice. Serca2flox/flox mice served as controls. Experiments were performed on ventricular cardiomyocytes with a 53% reduction in SERCA2 protein expression without any changes in expression of the L-type Ca2+ channel, Na+-Ca2+-exchanger or plasma membrane Ca2+ ATPase. In field stimulated cells SERCA2 mediated rate of Ca2+ reuptake was reduced by 42%. Basic characteristics of excitation-contraction-coupling were as expected with a 16% reduction in Ca2+ transient amplitude, SR Ca2+ content reduced by 16% and peak Ca2+ current increased by 40%. When SR Ca2+ content was increased by 10mM external Ca2+, only 27% of voltage clamped cardiomyocytes from knockout mice developed Ca2+ waves compared to 50% of control cells. Confocal imaging showed that Ca2+ waves in knockout mice propagated at 16% lower velocity, possibly due to a 12% reduction in Ca2+ wave amplitude. We conclude that decreased SERCA2 abundance reduces the overall propensity for Ca2+ wave development, and decreases Ca2+ wave velocity.

Quantitative Fluorescence Measurement of Cardiac Na+/Ca2+ Exchanger Inhibition by Kinetic Analysis in Stably Transfected HEK293 Cells

We developed a method to quantitatively evaluate the potency of Na+/Ca2+ exchanger (NCX) inhibitors with fluorescence microscopy in NCX1-transfected HEK 293 cells. The reverse mode and forward mode NCX activities were measured as the ascending slope of the early phase increase in cytoplasmic Ca2+ concentration after change to low Na+ extracellular solution and the descending rate (inverse of the exponential time constant) on return to normal solution, respectively. Both modes of NCX were inhibited by SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline) and KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate), and the concentration-inhibition relationships for both inhibitors were in good agreement with those previously reported in voltage clamped cardiomyocytes.

Soluble substances released from postischemic reperfused rat hearts reduce calcium transient and contractility by blocking the l-type calcium channel

OBJECTIVESThis study was designed to investigate the effects of cardiodepressant substances released from postischemic myocardial tissue on myocardial calcium-regulating pathways.BACKGROUNDWe have recently reported that new cardiodepressant substances are released from isolated hearts during reperfusion after myocardial ischemia.METHODSAfter 10 min of global ischemia, isolated rat hearts were reperfused, and the coronary effluent was collected for 30 s. We tested the effects of the postischemic coronary effluent on cell contraction, Ca2+transients and Ca2+currents of isolated rat cardiomyocytes by applying fluorescence microscopy and the whole-cell, voltage-clamp technique. Changes in intracellular phosphorylation mechanisms were studied by measuring tissue concentrations of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), as well as activities of cAMP-dependent protein kinase (cAMP-dPK) and protein kinase C (PKC).RESULTSThe postischemic coronary effluent, diluted with experimental buffer, caused a concentration-dependent reduction of cell shortening and Ca2+transient in the field-stimulated isolated cardiomyocytes of rats, as well as a reduction in peak L-type Ca2+current in voltage-clamped cardiomyocytes. The current reduction resulted from reduced maximal conductance—not from changes in voltage- and time-dependent gating of the L-type Ca2+channel. The postischemic coronary effluent modified neither the tissue concentrations of cAMP or cGMP nor the activities of cAMP-dPK and PKC. However, the effluent completely eliminated the activation of glycogen phosphorylase after beta-adrenergic stimulation.CONCLUSIONSNegative inotropic substances released from isolated postischemic hearts reduce Ca2+transient and cell contraction through cAMP-independent and cGMP-independent blockage of L-type Ca2+channels.

Electrophysiological effects of bimoclomol in canine ventricular myocytes.

Concentration-dependent effects of bimoclomol, a novel heat shock protein (HSP) coinducer, were studied on the parameters of action potential and transmembrane ionic currents in enzymatically dispersed canine ventricular cardiomyocytes using conventional microelectrode and whole cell voltage clamp techniques. Bimoclomol (10-100 microM) decreased the maximum velocity of depolarization (Vmax) and amplitude of action potentials in a concentration-dependent manner. These effects were fully reversible after a 5-min period of washout in drug-free medium. Action potential duration measured at 50% or 90% level of repolarization (APD-50 and APD-90, respectively) was markedly shortened by bimoclomol. Both APD-50 and APD-90 were decreased, but the reduction in APD-50 was more pronounced. The APD-shortening effect of bimoclomol was significantly reduced in the presence of 20 nM charybdotoxin (inhibitor of the Ca-dependent K current) or 0.5 mM anthracene-9-carboxylic acid (inhibitor of the Ca-dependent Cl current) or 1 microM glibenclamide (inhibitor of the ATP-sensitive K current). In the presence of anthracene-9-carboxylic acid, APD-90 was lengthened by bimoclomol. The APD-shortening effect of bimoclomol was also partially antagonized by chelation of intracellular Ca2+ by application of the cell permeant form of BAPTA, or when using 10 mM EGTA-containing patch pipettes to record action potentials. The Vmax-depressant effect of bimoclomol was not affected by charybdotoxin, anthracene-9-carboxylic acid, glibenclamide, or BAPTA load. In voltage clamped cardiomyocytes bimoclomol (100 microM) had no effect on the amplitude of I(Ca), but decreased significantly the inactivation time constant of I(Ca) (from 19.8+/-1.6 ms to 16.8+/-1.2 ms at 0 mV). Bimoclomol also decreased significantly the amplitude of I(K1) (from -20.5+/-1.1 pA/pF to -16.6+/-0.8 pA/pF at -135 mV), causing reduction in slope of the negative branch of the I-V curve. At positive potentials, however, bimoclomol increased outward current. The bimoclomol-induced current, therefore, was studied in the presence of BaCl2, when I(K1) current was blocked. The bimoclomol-induced current had a reversal potential close to -90 mV. Bimoclomol (100 microM) had no effect on the amplitude or kinetic properties of the transient outward K current (I(to)) and the delayed rectifier K current (I(K)). It is concluded that bimoclomol exerts both Ca-independent (inhibition of I(Na) and I(K1), activation of the ATP-sensitive K current) and Ca-dependent effects (mediated by Ca-activated Cl and probably K currents) in canine ventricular myocytes.

Reversal of the ATP-liganded State of ATP-sensitive K+ Channels by Adenylate Kinase Activity

The mechanism that promotes transition from the ATP- to the ADP-liganded state of ATP-sensitive K+ (KATP) channels and consequent channel opening in a cytosolic environment of high ATP concentration has yet to be understood. A mechanism examined here that could reverse the ATP-inhibited state is based on the action of adenylate kinase to catalyze phosphoryl transfer between ATP and AMP, resulting in transformation of ATP into ADP. In membrane patches excised from guinea pig cardiomyocytes, AMP alone did not affect channel behavior but increased the open probability of ATP-inhibited KATP channels. This required MgCl2 and a hydrolyzable form of ATP and was prevented by P1,P5-di-adenosine-5'-pentaphosphate, an inhibitor of adenylate kinase. The single channel amplitude and kinetics of channel openings induced by the ADP-generating substrates of adenylate kinase, AMP and MgATP, were indistinguishable from the biophysical properties of the KATP channel exhibited after addition of MgADP. In whole cell voltage-clamped cardiomyocytes, introduction of exogenous adenylate kinase along with millimolar MgATP and AMP induced a K+ current that was suppressed by a sulfonylurea blocker of KATP channels. Enriched sarcolemmal membrane preparations were found to possess ATP.AMP phosphotransferase activity with properties attributable to an extramitochondrial isoform of adenylate kinase. These results indicate that adenylate kinase is a naturally occurring component of sarcolemmal membranes that could provide dynamic governance of KATP channel opening through its phosphoryl transfer catalytic action in the microenvironment of the channel.

A dual-pipette technique that permits rapid internal dialysis and membrane potential measurement in voltage-clamped cardiomyocytes

Guinea pig ventricular myocytes were voltage-clamped and dialysed using two glass patch pipettes (P1, P2) with tip openings of around 2 microns. A substantial improvement in the efficacy of dialysis from P2 was achieved by the application of positive pressure (15-30 cm H2O) to P2, and similar negative pressure to P1. Evidence of enhanced dialysis was obtained by measuring the effect on Ca channel current of P2 dialysates containing Ca, cAMP, GTP [gamma-S], trypsin, or the catalytic subunit of protein kinase A. Times to maximum response were 3-5 times shorter than those calculated or observed by others using a single-pipette method. The speeding-up was verified in comparative experiments with 100 microM GTP [gamma-S] dialysates; maximum stimulation of ICa occurred after 1.3-1.8 min with the dual-pipette method, versus 8.2 min with a single pipette. Other advantages of the dual-pipette method include the option of following a control dialysis from P1 with a test dialysis from P2, and the measurement of actual membrane potential. The disadvantages are that the rate of success is lower than with single-pipette experiments, and that smaller cardiomyocytes are difficult subjects.