Cardiotoxicity of environmental contaminant tributyltin involves myocyte oxidative stress and abnormal Ca2+ handling.

Tributyltin (TBT) is an organotin environmental pollutant widely used as an agricultural and wood biocide and in antifouling paints. Countries began restricting TBT use in the 2000s, but their use continues in some agroindustrial processes. We studied the acute effect of TBT on cardiac function by analyzing myocardial contractility and Ca2+ handling. Cardiac contractility was evaluated in isolated papillary muscle and whole heart upon TBT exposure. Isolated ventricular myocytes were used to measure calcium (Ca2+) transients, sarcoplasmic reticulum (SR) Ca2+ content and SR Ca2+ leak (as Ca2+ sparks). Reactive oxygen species (ROS), as superoxide anion (O2•-) was detected at intracellular and mitochondrial myocardium. TBT depressed cardiac contractility and relaxation in papillary muscle and intact whole heart. TBT increased cytosolic, mitochondrial ROS production and decreased mitochondrial membrane potential. In isolated cardiomyocytes TBT decreased both Ca2+ transients and SR Ca2+ content and increased diastolic SR Ca2+ leak. Decay of twitch and caffeine-induced Ca2+ transients were slowed by the presence of TBT. Dantrolene prevented and Tiron limited the reduction in SR Ca2+ content and transients. The environmental contaminant TBT causes cardiotoxicity within minutes, and may be considered hazardous to the mammalian heart. TBT acutely induced a negative inotropic effect in isolated papillary muscle and whole heart, increased arrhythmogenic SR Ca2+ leak leading to reduced SR Ca2+ content and reduced Ca2+ transients. TBT-induced myocardial ROS production, may destabilize the SR Ca2+ release channel RyR2 and reduce SR Ca2+ pump activity as key factors in the TBT-induced negative inotropic and lusitropic effects.

A novel computational model of mouse myocyte electrophysiology to assess the synergy between Na+ loading and CaMKII.

Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) hyperactivity in heart failure causes intracellular Na(+) ([Na(+)]i) loading (at least in part by enhancing the late Na(+) current). This [Na(+)]i gain promotes intracellular Ca(2+) ([Ca(2+)]i) overload by altering the equilibrium of the Na(+)-Ca(2+) exchanger to impair forward-mode (Ca(2+) extrusion), and favour reverse-mode (Ca(2+) influx) exchange. In turn, this Ca(2+) overload would be expected to further activate CaMKII and thereby form a pathological positive feedback loop of ever-increasing CaMKII activity, [Na(+)]i, and [Ca(2+)]i. We developed an ionic model of the mouse ventricular myocyte to interrogate this potentially arrhythmogenic positive feedback in both control conditions and when CaMKIIδC is overexpressed as in genetically engineered mice. In control conditions, simulation of increased [Na(+)]i causes the expected increases in [Ca(2+)]i, CaMKII activity, and target phosphorylation, which degenerate into unstable Ca(2+) handling and electrophysiology at high [Na(+)]i gain. Notably, clamping CaMKII activity to basal levels ameliorates but does not completely offset this outcome, suggesting that the increase in [Ca(2+)]i per se plays an important role. The effect of this CaMKII-Na(+)-Ca(2+)-CaMKII feedback is more striking in CaMKIIδC overexpression, where high [Na(+)]i causes delayed afterdepolarizations, which can be prevented by imposing low [Na(+)]i, or clamping CaMKII phosphorylation of L-type Ca(2+) channels, ryanodine receptors and phospholamban to basal levels. In this setting, Na(+) loading fuels a vicious loop whereby increased CaMKII activation perturbs Ca(2+) and membrane potential homeostasis. High [Na(+)]i is also required to produce instability when CaMKII is further activated by increased Ca(2+) loading due to ß-adrenergic activation. Our results support recent experimental findings of a synergistic interaction between perturbed Na(+) fluxes and CaMKII, and suggest that pharmacological inhibition of intracellular Na(+) loading can contribute to normalizing Ca(2+) and membrane potential dynamics in heart failure.

Making and using calcium-selective mini- and microelectrodes.

Detection and measurement of intracellular calcium concentration ([Ca(2+)](i)) have relied on various methods, the popularity of which depends on their ease of use and applicability to different cell types. Historically, Ca(2+)-selective electrodes have been used concomitantly with absorption indicators such as arsenazo-III, but their interest has been eclipsed by the introduction of a large number of fluorescent calcium probes with calcium sensitivities varying from the nanomolar to the micromolar range such as fura-2, indo-1, fluo-4, and many others. In this chapter, we emphasize the utility of Ca(2+)-selective electrodes and show that their use is complementary to use of fluorescent indicators; indeed, each method has advantages and disadvantages. We first describe the preparation and application of Ca(2+)-selective minielectrodes based on the Ca(2+) ligand ETH 129 (Schefer et al., 1986) that have a larger dynamic range and faster response time than most commercially available calcium electrodes. The second part of the chapter is dedicated to ETH 129-based Ca(2+)-selective microelectrodes (MEs), and their application in the determination of [Ca(2+)](i) in cardiac cells. Since numerous reviews and books have been dedicated to the theoretical aspects of ion-selective ME principles and technology, this chapter is not intended for investigators who have no experience with MEs.

Calcium and cardiomyopathies.

Regulation of Calcium (Ca) cycling by the sarcoplasmic reticulum (SR) underlies the control of cardiac contraction during excitation-contraction (E-C) coupling. Moreover, alterations in E-C coupling occurring in cardiac hypertrophy and heart failure are characterized by abnormal Ca-cycling through the SR network. A large body of evidence points to the central role of: a) SERCA and its regulator phospholamban (PLN) in the modulation of cardiac relaxation; b) calsequestrin in the regulation of SR Ca-load; and c) the ryanodine receptor (RyR) Ca-channel in the control of SR Ca-release. The levels or activity of these key Ca-handling proteins are altered in cardiomyopathies, and these changes have been linked to the deteriorated cardiac function and remodeling. Furthermore, genetic variants in these SR Ca-cycling proteins have been identified, which may predispose to heart failure or fatal arrhythmias. This chapter concentrates on the pivotal role of SR Ca-cycling proteins in health and disease with specific emphasis on their recently reported genetic modifiers.

Confocal imaging of CICR events from isolated and immobilized SR vesicles.

The Ca2+ concentration inside the sarcoplasmic reticulum ([Ca2+]SR) is a difficult parameter to measure in ventricular cardiac myocytes. Interference from Ca2+-sensitive dye loading into cellular compartments other than the SR interferes with free Ca2+ measurement. In addition, the composition of the cytosol surrounding the SR in intact cells cannot be easily controlled. We have developed a method to measure localized [Ca2+]SR in immobilized membrane vesicles during rapid solution switches. Ca2+ uptake and release in rat SR membrane vesicles was monitored using confocal microscopy. Vesicles were immobilized on a coverslip using an agarose matrix. Perfusion with a Ca2+-containing solution supplemented with ATP initiated SR Ca2+ uptake, causing a rise in intravesicular fluorescence in vesicles containing the low-affinity Ca2+ indicator fluo-5N. Perfusion with caffeine caused SR Ca2+ release and a decrease in intravesicular flourescence. Although caffeine-dependent release was readily visible with extravesicular Ca2+-green, Ca2+ which leaked from the SR was detected only indirectly as eventless release. We conclude that SR Ca2+ uptake and release can be selectively measured in functional SR vesicles using a confocal microscope. Caffeine-dependent release is directly measurable though SR Ca2+ leak can only be inferred as subresolution events, presumably because channels in separate vesicles were not close enough to result in concerted Ca2+-induced Ca2+ release.

Phospholamban is required for CaMKII-dependent recovery of Ca transients and SR Ca reuptake during acidosis in cardiac myocytes.

Initially during acidosis, Ca transient amplitude (Delta[Ca]i) and the rate constant of [Ca]i decline (k(Ca)) are decreased, but later during acidosis Delta[Ca]i and k(Ca) partially recover. This recovery in rat myocytes could be inhibited by KN-93 suggesting that CaMKII-dependent protein phosphorylation (and enhanced SR Ca uptake) may be responsible. To test whether phospholamban (PLB) is required for the Delta[Ca]i and k(Ca) recovery during acidosis, we used isolated myocytes from PLB knockout (PLB-KO) vs. wild-type (WT) mice. [Ca]i was measured using fluo-3. During the initial phase of acidosis (1-4 min), Delta[Ca]i decreased in WT myocytes (n = 8) from 1.75 +/- 0.19 to 1.10 +/- 0.13 DeltaF/F0 (P < 0.05) and k(Ca) decreased from 3.20 +/- 0.22 to 2.38 +/- 0.18 s(-1) (P < 0.05). Later during acidosis (6-12 min), Delta[Ca]i partially recovered to 1.41 +/- 0.18 DeltaF/F0 and k(Ca) to 2.78 +/- 0.22 s(-1) (i.e. both recovered by approximately 50%). CaMKII inhibition using KN-93 completely prevented this recovery of Delta[Ca]i and k(Ca) during late acidosis in WT myocytes. In PLB-KO myocytes (n = 11) Delta[Ca]i decreased during early acidosis from 2.92 +/- 0.31 to 1.33 +/- 0.17 DeltaF/F0 (P < 0.05) and k(Ca) decreased from 10.45 +/- 0.56 to 7.58 +/- 0.68 s(-1) (P < 0.05). However, Delta[Ca]i did not recover during late acidosis and k(Ca) decreased even more (6.59 +/- 0.65 s(-1)). Parallel results were seen for contractile parameters. We conclude that PLB is crucial to the recovery of Delta[Ca]i and k(Ca) during acidosis. Moreover, PLB phosphorylation by CaMKII plays an important role in limiting the decline in Ca transients (and contraction) during acidosis.

Na/Ca exchange and Na/K-ATPase function are equally concentrated in transverse tubules of rat ventricular myocytes.

Formamide-induced detubulation of rat ventricular myocytes was used to investigate the functional distribution of the Na/Ca exchanger (NCX) and Na/K-ATPase between the t-tubules and external sarcolemma. Detubulation resulted in a 32% decrease in cell capacitance, whereas cell volume was unchanged. Thus, the surface-to-volume ratio was used to assess the success of detubulation. NCX current (I(NCX)) and Na/K pump current (I(pump)) were recorded using whole-cell patch clamp, as Cd-sensitive and K-activated currents, respectively. Both inward and outward I(NCX) density was significantly reduced by approximately 40% in detubulated cells. I(NCX) density at 0 mV decreased from 0.19 +/- 0.03 to 0.10 +/- 0.03 pA/pF upon detubulation. I(pump) density was also lower in detubulated myocytes over the range of voltages (-50 to +100 mV) and internal [Na] ([Na](i)) investigated (7-22 mM). At [Na](i) = 10 mM and -20 mV, I(pump) density was reduced by 39% in detubulated myocytes (0.28 +/- 0.02 vs. 0.17 +/- 0.03 pA/pF), but the apparent K(m) for [Na](i) was unchanged (16.9 +/- 0.4 vs. 17.0 +/- 0.3 mM). These results indicate that although thet-tubules represent only approximately 32% of the total sarcolemma, they contribute approximately 60% to the total I(NCX) and I(pump). Thus, the functional density of NCX and Na/K pump in the t-tubules is 3-3.5-fold higher than in the external sarcolemma.

Expression of inducible nitric oxide synthase depresses beta-adrenergic-stimulated calcium release from the sarcoplasmic reticulum in intact ventricular myocytes.

BACKGROUND: beta-adrenergic hyporesponsiveness in many cardiomyopathies is linked to expression of inducible nitric oxide synthase (iNOS) and increased production of NO. The purpose of this study was to examine whether iNOS expression alters the function of the sarcoplasmic reticulum (SR) Ca(2+) release channel (ryanodine receptor, RyR) during beta-adrenergic stimulation. METHODS AND RESULTS: Expression of iNOS was induced by lipopolysaccharide (LPS) injection (10 mg/kg) 6 hours before rat myocyte isolation. Confocal microscopy (fluo-3) was used to measure Ca(2+) spark frequency (CaSpF, reflecting resting RyR openings) and Ca(2+) transients. CaSpF was greatly increased by the adenylate cyclase activator forskolin (100 nmol/L) in normal myocytes (iNOS not expressed), but this effect was suppressed (by 77%) in LPS myocytes (iNOS expressed). When NO production by iNOS was inhibited by aminoguanidine (1 mmol/L), there was a further increase in the forskolin-induced CaSpF in LPS myocytes (to levels similar to the forskolin-stimulated CaSpF in normal myocytes). This effect was also seen in myocytes isolated from a failing human heart. There was no effect of aminoguanidine on forskolin-stimulated CaSpF in normal myocytes. ODQ (10 micromol/L), an inhibitor of NO stimulation of guanylate cyclase, did not restore the forskolin-induced rise in CaSpF in LPS myocytes. Aminoguanidine also increased twitch Ca(2+) transient amplitude in LPS myocytes after forskolin application (independent of changes in SR Ca(2+) load). CONCLUSIONS: iNOS/NO depresses beta-adrenergic-stimulated RyR function through a cGMP-independent pathway (eg, NO- and/or peroxynitrite-dependent redox modification). This mechanism limits beta-adrenergic responsiveness and may be an important signaling pathway in cardiomyopathies, including human heart failure.

LabHEART: an interactive computer model of rabbit ventricular myocyte ion channels and Ca transport.

An interactive computer program, LabHEART, was developed to simulate the action potential (AP), ionic currents, and Ca handling mechanisms in a rabbit ventricular myocyte. User-oriented, its design allows switching between voltage and current clamp and easy on-line manipulation of key parameters to change the original formulation. The model reproduces normal rabbit ventricular myocyte currents, Ca transients, and APs. We also changed parameters to simulate data from heart failure (HF) myocytes, including reduced transient outward (I(to)) and inward rectifying K currents (I(K1)), enhanced Na/Ca exchange expression, and reduced sarcoplasmic reticulum Ca-ATPase function, but unaltered Ca current density. These changes caused reduced Ca transient amplitude and increased AP duration (especially at lower frequency) as observed experimentally. The model shows that the increased Na/Ca exchange current (I(NaCa)) in HF lowers the intracellular [Ca] threshold for a triggered AP from 800 to 540 nM. Similarly, the decrease in I(K1) reduces the threshold to 600 nM. Changes in I(to) have no effect. Combining enhanced Na/Ca exchange with reduced I(K1) (as in HF) lowers the threshold to trigger an AP to 380 nM. These changes reproduce experimental results in HF, where the contributions of different factors are not readily distinguishable. We conclude that the triggered APs that contribute to nonreentrant ventricular tachycardia in HF are due approximately equally (and nearly additively) to alterations in I(NaCa) and I(K1). A free copy of this software can be obtained at http://www.meddean.luc.edu/lumen/DeptWebs/physio/bers.html.

Positive and negative effects of nitric oxide on Ca(2+) sparks: influence of beta-adrenergic stimulation.

Nitric oxide (NO) can have a positive or negative effect on cardiac contractility and the ryanodine receptor (RyR). This dual effect has been explained as being dependent on the concentration of NO. We find that cellular RyR response to NO is also dependent on the degree of beta-adrenergic stimulation, and thus the state of protein kinase A activation. Ca(2+) spark frequency (CaSpF) in rat ventricular myocytes was used as an index of resting RyR activity. CaSpF response to beta-adrenergic stimulation was used as an index of protein kinase A activation. High concentration of isoproterenol, a beta-adrenergic agonist, caused a large increase in CaSpF; addition of NO (spermine NONOate, 300 microM) then caused a decrease in CaSpF. Low concentration of isoproterenol produced only a slight increase in CaSpF, but the same NO concentration now caused a large increase in CaSpF. A dual effect was also observed in twitch. Thus the net direction of the effects of NO on RyR activity and Ca(2+) transients (directly or by alteration of sarcoplasmic reticulum Ca(2+) load) can be reversed, depending on the ambient level of beta-adrenergic activation.

A cardiac dihydropyridine receptor II-III loop peptide inhibits resting Ca(2+) sparks in ferret ventricular myocytes.

1. We studied the effect of a peptide (Ac-10C) on cardiac ryanodine receptor (RyR) opening. This decapeptide (KKERKLARTA) is a fragment of the cardiac dihydropyridine receptor (DHPR) from the cytosolic loop between the second and third transmembrane domains (II-III loop). Studies were carried out in ferret ventricular myocytes by simultaneously applying ruptured-patch voltage clamp and line-scan confocal microscopy with fluo-3 to measure intracellular [Ca(2+)] ([Ca(2+)](i)) and Ca(2+) sparks. 2. Inclusion of Ac-10C in the dialysing pipette solution inhibited resting Ca(2+) spark frequency (due to diastolic RyR openings) by > 50 %. This occurred without changing sarcoplasmic reticulum (SR) Ca(2+) content, which was measured via the caffeine-induced Ca(2+) transient amplitude and the caffeine-induced Na(+)-Ca(2+) exchange current (I(NCX)) integral. Ac-10C also reduced slightly the size of Ca(2+) sparks. 3. Ac-10C did not alter either resting [Ca(2+)](i) (assessed by indo-1 fluorescence) or DHPR gating (measured as L-type Ca(2+) current). 4. The SR Ca(2+) fractional release was depressed by Ac-10C at relatively low SR Ca(2+) content, but not at higher SR Ca(2+) content. 5. A control scrambled peptide (Ac-10CS) did not alter any of the measured parameters (notably Ca(2+) spark frequency or SR Ca(2+) fractional release). Thus, the Ac-10C effects may be sequence or charge distribution specific. 6. Our results suggest an inhibitory regulation of RyRs at rest via the cardiac DHPR II-III loop N-terminus region. The mechanism of the effect and whether this interaction is important in cardiac excitation-contraction coupling (E-C coupling) per se, requires further investigation.

Arrhythmogenesis and contractile dysfunction in heart failure: Roles of sodium-calcium exchange, inward rectifier potassium current, and residual beta-adrenergic responsiveness.

Ventricular arrhythmias and contractile dysfunction are the main causes of death in human heart failure (HF). In a rabbit HF model reproducing these same aspects of human HF, we demonstrate that a 2-fold functional upregulation of Na(+)-Ca(2+) exchange (NaCaX) unloads sarcoplasmic reticulum (SR) Ca(2+) stores, reducing Ca(2+) transients and contractile function. Whereas beta-adrenergic receptors (beta-ARs) are progressively downregulated in HF, residual beta-AR responsiveness at this critical HF stage allows SR Ca(2+) load to increase, causing spontaneous SR Ca(2+) release and transient inward current carried by NaCaX. A given Ca(2+) release produces greater arrhythmogenic inward current in HF (as a result of NaCaX upregulation), and approximately 50% less Ca(2+) release is required to trigger an action potential in HF. The inward rectifier potassium current (I(K1)) is reduced by 49% in HF, and this allows greater depolarization for a given NaCaX current. Partially blocking I(K1) in control cells with barium mimics the greater depolarization for a given current injection seen in HF. Thus, we present data to support a novel paradigm in which changes in NaCaX and I(K1), and residual beta-AR responsiveness, conspire to greatly increase the propensity for triggered arrhythmias in HF. In addition, NaCaX upregulation appears to be a critical link between contractile dysfunction and arrhythmogenesis.

Allosteric regulation of Na/Ca exchange current by cytosolic Ca in intact cardiac myocytes.

The cardiac sarcolemmal Na-Ca exchanger (NCX) is allosterically regulated by [Ca](i) such that when [Ca](i) is low, NCX current (I(NCX)) deactivates. In this study, we used membrane potential (E(m)) and I(NCX) to control Ca entry into and Ca efflux from intact cardiac myocytes to investigate whether this allosteric regulation (Ca activation) occurs with [Ca](i) in the physiological range. In the absence of Ca activation, the electrochemical effect of increasing [Ca](i) would be to increase inward I(NCX) (Ca efflux) and to decrease outward I(NCX). On the other hand, Ca activation would increase I(NCX) in both directions. Thus, we attributed [Ca](i)-dependent increases in outward I(NCX) to allosteric regulation. Ca activation of I(NCX) was observed in ferret myocytes but not in wild-type mouse myocytes, suggesting that Ca regulation of NCX may be species dependent. We also studied transgenic mouse myocytes overexpressing either normal canine NCX or this same canine NCX lacking Ca regulation (Delta680-685). Animals with the normal canine NCX transgene showed Ca activation, whereas animals with the mutant transgene did not, confirming the role of this region in the process. In native ferret cells and in mice with expressed canine NCX, allosteric regulation by Ca occurs under physiological conditions (K(mCaAct) = 125 +/- 16 nM SEM approximately resting [Ca](i)). This, along with the observation that no delay was observed between measured [Ca](i) and activation of I(NCX) under our conditions, suggests that beat to beat changes in NCX function can occur in vivo. These changes in the I(NCX) activation state may influence SR Ca load and resting [Ca](i), helping to fine tune Ca influx and efflux from cells under both normal and pathophysiological conditions. Our failure to observe Ca activation in mouse myocytes may be due to either the extent of Ca regulation or to a difference in K(mCaAct) from other species. Model predictions for Ca activation, on which our estimates of K(mCaAct) are based, confirm that Ca activation strongly influences outward I(NCX), explaining why it increases rather than declines with increasing [Ca](i).

Phospholamban decreases the energetic efficiency of the sarcoplasmic reticulum Ca pump.

We tested the hypothesis that increased Sarcoplasmic reticulum (SR) Ca content ([Ca](SRT)) in phospholamban knockout mice (PLB-KO) is because of increased SR Ca pump efficiency defined by the steady-state SR [Ca] gradient. The time course of thapsigargin-sensitive ATP-dependent (45)Ca influx into and efflux out of cardiac SR vesicles from PLB-KO and wild-type (WT) mice was measured at 100 nm free [Ca]. We found that PLB decreased the initial SR Ca uptake rate (0.13 versus 0.31 nmol/mg/s) and decreased steady-state (45)Ca content (0.9 versus 4.1 nmol/mg protein). Furthermore, at similar total SR [Ca], the pump-mediated Ca efflux rate was higher in WT (0.065 versus 0.037 nmol/mg/s). The pump-independent leak rate constant (k(leak)) was also measured at 100 nm free [Ca]. The results indicate that k(leak) was < 1% of pump-mediated backflux and was not different among nonpentameric mutant PLB (PLB-C41F), WT pentameric PLB (same expression level), and PLB-KO. Therefore differences in passive SR Ca leak cannot be the cause of the higher thapsigargin-sensitive Ca efflux from the WT membranes. We conclude that the decreased total SR [Ca] in WT mice is caused by decreased SR Ca influx rate, an increased Ca-pump backflux, and unaltered leak. Based upon both thermodynamic and kinetic analysis, we conclude that PLB decreases the energetic efficiency of the SR Ca pump.

Differences in Ca(2+)-handling and sarcoplasmic reticulum Ca(2+)-content in isolated rat and rabbit myocardium.

We made novel measurements of the influence of rest intervals and stimulation frequency on twitch contractions and on sarcoplasmic reticulum (SR) Ca(2+)-content (using rapid cooling contractures, RCCs) in isolated ventricular muscle strips from rat and rabbit hearts at a physiological temperature of 37 degrees C. In addition, the frequency-dependent relative contribution of SR Ca(2+)-uptake and Na(+)/Ca(2+)-exchange for cytosolic Ca(2+)-removal was assessed by paired RCCs. With increasing rest intervals (1-240 s) post-rest twitch force and RCC amplitude decreased monotonically in rabbit myocardium (after 240 s by 45+/-10% and 61+/-11%, respectively P<0. 05, n=14). In contrast, rat myocardium (n=11) exhibited a parallel increase in post-rest twitch force (by 67+/-16% at 240 s P<0.05) and RCC amplitude (by 20+/-14%P<0.05). In rabbit myocardium (n=11), increasing stimulation frequency from 0.25 to 3 Hz increased twitch force by 295+/-50% (P<0.05) and RCC amplitude by 305+/-80% (P<0.05). In contrast, in rat myocardium (n=6), twitch force declined by 43+/-7% (P<0.05), while RCC amplitude decreased only insignificantly (by 16+/-7%). The SR Ca(2+)-uptake relative to Na(+)/Ca(2+)-exchange (based on paired RCCs) increased progressively with frequency in rabbit, but not in rat myocardium (;66+/-2% at all frequencies). We conclude that increased SR Ca(2+)-load contributes to the positive force-frequency relationship in rabbits and post-rest potentiation of twitch force in rats. Decreased SR Ca(2+)-load contributes to post-rest decay of twitch force in rabbits, but may play only a minor role in the negative force-frequency relationship in rats. SR Ca(2+)-release channel refractoriness may contribute importantly to the negative force-frequency relationship in rat and recovery from refractoriness may contribute to post-rest potentiation.

Regulatory role of phospholamban in the efficiency of cardiac sarcoplasmic reticulum Ca2+ transport.

Phospholamban is an inhibitor of the sarcoplasmic reticulum Ca(2+) transport apparent affinity for Ca(2+) in cardiac muscle. This inhibitory effect of phospholamban can be relieved through its phosphorylation or ablation. To better characterize the regulatory mechanism of phospholamban, we examined the initial rates of Ca(2+)-uptake and Ca(2+)-ATPase activity under identical conditions, using sarcoplasmic reticulum-enriched preparations from phospholamban-deficient and wild-type hearts. The apparent coupling ratio, calculated by dividing the initial rates of Ca(2+) transport by ATP hydrolysis, appeared to increase with increasing [Ca(2+)] in wild-type hearts. However, in the phospholamban-deficient hearts, this ratio was constant, and it was similar to the value obtained at high [Ca(2+)] in wild-type hearts. Phosphorylation of phospholamban by the catalytic subunit of protein kinase A in wild-type sarcoplasmic reticulum also resulted in a constant value of the apparent ratio of Ca(2+) transported per ATP hydrolyzed, which was similar to that present in phospholamban-deficient hearts. Thus, the inhibitory effects of dephosphorylated phospholamban involve decreases in the apparent affinity of sarcoplasmic reticulum Ca(2+) transport for Ca(2+) and the efficiency of this transport system at low [Ca(2+)], both leading to prolonged relaxation in myocytes.

Sarcoplasmic reticulum Ca(2+) release causes myocyte depolarization. Underlying mechanism and threshold for triggered action potentials.

Spontaneous sarcoplasmic reticulum (SR) Ca(2+) release causes delayed afterdepolarizations (DADs) via Ca(2+)-induced transient inward currents (I:(ti)). However, no quantitative data exists regarding (1) Ca(2+) dependence of DADs, (2) Ca(2+) required to depolarize the cell to threshold and trigger an action potential (AP), or (3) relative contributions of Ca(2+)-activated currents to DADs. To address these points, we evoked SR Ca(2+) release by rapid application of caffeine in indo 1-AM-loaded rabbit ventricular myocytes and measured caffeine-induced DADs (cDADs) with whole-cell current clamp. The SR Ca(2+) load of the myocyte was varied by different AP frequencies. The cDAD amplitude doubled for every 88+/-8 nmol/L of Delta[Ca(2+)](i) (simple exponential), and the Delta[Ca(2+)](i) threshold of 424+/-58 nmol/L was sufficient to trigger an AP. Blocking Na(+)-Ca(2+) exchange current (I(Na/Ca)) by removal of [Na](o) and [Ca(2+)](o) (or with 5 mmol/L Ni(2+)) reduced cDADs by >90%, for the same Delta[Ca(2+)](i). In contrast, blockade of Ca(2+)-activated Cl(-) current (I(Cl(Ca))) with 50 micromol/L niflumate did not significantly alter cDADs. We conclude that DADs are almost entirely due to I(Na/Ca), not I(Cl(Ca)) or Ca(2+)-activated nonselective cation current. To trigger an AP requires 30 to 40 micromol/L cytosolic Ca(2+) or a [Ca(2+)](i) transient of 424 nmol/L. Current injection, simulating I(ti)s with different time courses, revealed that faster I:(ti)s require less charge for AP triggering. Given that spontaneous SR Ca(2+) release occurs in waves, which are slower than cDADs or fast I(ti)s, the true Delta[Ca(2+)](i) threshold for AP activation may be approximately 3-fold higher in normal myocytes. This provides a safety margin against arrhythmia in normal ventricular myocytes.

Transmission of information from cardiac dihydropyridine receptor to ryanodine receptor: evidence from BayK 8644 effects on resting Ca(2+) sparks.

Coupling between L-type Ca(2+) channels (dihydropyridine receptors, DHPRs) and ryanodine receptors (RyRs) plays a pivotal role in excitation-contraction (E-C) coupling in cardiac myocytes, and Ca(2+) influx is generally accepted as the trigger of sarcoplasmic reticulum (SR) Ca(2+) release. The L-type Ca(2+) channel agonist BayK 8644 (BayK) has also been reported to alter RyR gating via a functional linkage between DHPR and RyR, independent of Ca(2+) influx. Here, the effect of rapid BayK application on resting RyR gating in intact ferret ventricular myocytes was measured as Ca(2+) spark frequency (CaSpF) by confocal microscopy and fluo 3. BayK increased resting CaSpF by 401+/-15% within 10 seconds in Ca(2+)-free solution, and depolarization had no additional effect. The effect of BayK on CaSpF was dose-dependent, but even 50 nmol/L BayK induced a rapid 245+/-12% increase in CaSpF. Nifedipine (5 micromol/L) had no effect by itself on CaSpF, but it abolished the BayK effect (presumably by competitive inhibition at the DHPR). The nondihydropyridine Ca(2+) channel agonist FPL-64176 (1 micromol/L) did not alter CaSpF (despite rapid and potent enhancement of Ca(2+) current, I(Ca)). In striking contrast to the very rapid and depolarization-independent effect of BayK on CaSpF, BayK increased I(Ca) only slowly (tau=18 seconds), and the effect was greatly accelerated by depolarization. We conclude that in ferret ventricular myocytes, BayK effects on I(Ca) and CaSpF both require drug binding to the DHPR, but postreceptor pathways may diverge in transmission to the gating of the L-type Ca(2+) channel and RyR.