Thomas R. Shannon

Ca2+/Calmodulin–Dependent Protein Kinase Modulates Cardiac Ryanodine Receptor Phosphorylation and Sarcoplasmic Reticulum Ca2+ Leak in Heart Failure

Abnormal release of Ca from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction and arrhythmogenesis in heart failure (HF). We previously demonstrated decreased Ca transient amplitude and SR Ca load associated with increased Na/Ca exchanger expression and enhanced diastolic SR Ca leak in an arrhythmogenic rabbit model of nonischemic HF. Here we assessed expression and phosphorylation status of key Ca handling proteins and measured SR Ca leak in control and HF rabbit myocytes. With HF, expression of RyR2 and FK-506 binding protein 12.6 (FKBP12.6) were reduced, whereas inositol trisphosphate receptor (type 2) and Ca/calmodulin–dependent protein kinase II (CaMKII) expression were increased 50% to 100%. The RyR2 complex included more CaMKII (which was more activated) but less calmodulin, FKBP12.6, and phosphatases 1 and 2A. The RyR2 was more highly phosphorylated by both protein kinase A (PKA) and CaMKII. Total phospholamban phosphorylation was unaltered, although it was reduced at the PKA site and increased at the CaMKII site. SR Ca leak in intact HF myocytes (which is higher than in control) was reduced by inhibition of CaMKII but was unaltered by PKA inhibition. CaMKII inhibition also increased SR Ca content in HF myocytes. Our results suggest that CaMKII-dependent phosphorylation of RyR2 is involved in enhanced SR diastolic Ca leak and reduced SR Ca load in HF, and may thus contribute to arrhythmias and contractile dysfunction in HF.

Elevated Sarcoplasmic Reticulum Ca2+ Leak in Intact Ventricular Myocytes From Rabbits in Heart Failure

Altered sarcoplasmic reticulum (SR) Ca2+-ATPase and Na+-Ca2+ exchange (NCX) function have been implicated in depressing SR Ca2+ content and contractile function in heart failure (HF). Enhanced diastolic ryanodine receptor (RyR) leak could also lower SR Ca2+ load in HF, but direct cellular measurements are lacking. In this study, we measure SR Ca2+ leak directly in intact isolated rabbit ventricular myocytes from a well-developed nonischemic HF model. Abrupt block of SR Ca2+ leak by tetracaine shifts Ca2+ from the cytosol to SR. The tetracaine-induced decline in [Ca2+]i and increase total SR Ca2+ load ([Ca2+]SRT) directly indicate the SR Ca2+ leak (before tetracaine). Diastolic SR Ca2+ leak increases with [Ca2+]SRT, and for any [Ca2+]SRT is greater in HF versus control. Mathematical modeling was used to compare the relative impact of alterations in SR Ca2+ leak, SR Ca2+-ATPase, and Na+-Ca2+ exchange on SR Ca2+ load in HF. We conclude that increased diastolic SR Ca2+ leak in HF may contribute to reductions in SR Ca2+ content, but changes in NCX in this HF model have more impact on [Ca2+]SRT.

β-Adrenergic Enhancement of Sarcoplasmic Reticulum Calcium Leak in Cardiac Myocytes Is Mediated by Calcium/Calmodulin-Dependent Protein Kinase

Enhanced cardiac diastolic Ca leak from the sarcoplasmic reticulum (SR) ryanodine receptor may reduce SR Ca content and contribute to arrhythmogenesis. We tested whether β-adrenergic receptor (β-AR) agonists increased SR Ca leak in intact rabbit ventricular myocytes and whether this depends on protein kinase A or Ca/calmodulin-dependent protein kinase II (CaMKII) activity. SR Ca leak was assessed by acute block of the ryanodine receptor by tetracaine and assessment of the consequent shift of Ca from cytosol to SR (measured at various SR Ca loads induced by varying frequency). Cytosolic [Ca] ([Ca]i) and SR Ca load ([Ca]SRT) were assessed using fluo-4. β-AR activation by isoproterenol dramatically increased SR Ca leak. However, this effect was not inhibited by blocking protein kinase A by H-89, despite the expected reversal of the isoproterenol-induced enhancement of Ca transient amplitude and [Ca]i decline rate. In contrast, inhibitors of CaMKII, KN-93, or autocamtide-2–related inhibitory peptide II or β-AR blockade reversed the isoproterenol-induced enhancement of SR Ca leak, and CaMKII inhibition could even reduce leak below control levels. Forskolin, which bypasses the β-AR in activating adenylate cyclase and protein kinase A, did not increase SR Ca leak, despite robust enhancement of Ca transient amplitude and [Ca]i decline rate. The results suggest that β-AR stimulation enhances diastolic SR Ca leak in a manner that is (1) CaMKII dependent, (2) not protein kinase A dependent, and 3) not dependent on bulk [Ca]i.

Potentiation of fractional sarcoplasmic reticulum calcium release by total and free intra-sarcoplasmic reticulum calcium concentration.

Our aim was to measure the influence of sarcoplasmic reticulum (SR) calcium content ([Ca](SRT)) and free SR [Ca] ([Ca](SR)) on the fraction of SR calcium released during voltage clamp steps in isolated rabbit ventricular myocytes. [Ca](SRT), as measured by caffeine application, was progressively increased by conditioning pulses. Sodium was absent in both the intracellular and in the extracellular solutions to block sodium/calcium exchange. Total cytosolic calcium flux during the transient was inferred from I(Ca), [Ca](SRT), [Ca](i), and cellular buffering characteristics. Fluxes via the calcium current (I(Ca)), the SR calcium pump, and passive leak from the SR were evaluated to determine SR calcium release flux (J(rel)). Excitation-contraction (EC) coupling was characterized with respect to both gain (integral J(rel)/integral I(Ca)) and fractional SR calcium release. Both parameters were virtually zero for a small, but measurable [Ca](SRT). Gain and fractional SR calcium release increased steeply and nonlinearly with both [Ca](SRT) and [Ca](SR). We conclude that potentiation of EC coupling can be correlated with both [Ca](SRT) and [Ca](SR). While fractional SR calcium release was not linearly dependent upon [Ca](SR), intra-SR calcium may play a crucial role in regulating the SR calcium release process.

Quantitative Assessment of the SR Ca2+ Leak-Load Relationship

Abstract— Increased diastolic SR Ca2+ leak (Jleak) could depress contractility in heart failure, but there are conflicting reports regarding the Jleak magnitude even in normal, intact myocytes. We have developed a novel approach to measure SR Ca2+ leak in intact, isolated ventricular myocytes. After stimulation, myocytes were exposed to 0 Na+, 0 Ca2+ solution ±1 mmol/L tetracaine (to block resting leak). Total cell [Ca2+] does not change under these conditions with Na+-Ca2+ exchange inhibited. Resting [Ca2+]i declined 25% after tetracaine addition (126±6 versus 94±6 nmol/L;P <0.05). At the same time, SR [Ca2+] ([Ca2+]SRT) increased 20% (93±8 versus 108±6 &mgr;mol/L). From this Ca2+ shift, we calculate Jleak to be 12 &mgr;mol/L per second or 30% of the SR diastolic efflux. The remaining 70% is SR pump unidirectional reverse flux (backflux). The sum of these Ca2+ effluxes is counterbalanced by unidirectional forward Ca2+ pump flux. Jleak also increased nonlinearly with [Ca2+]SRT with a steeper increase at higher load. We conclude that Jleak is 4 to 15 &mgr;mol/L cytosol per second at physiological [Ca2+]SRT. The data suggest that the leak is steeply [Ca2+]SRT-dependent, perhaps because of increased [Ca2+]i sensitivity of the ryanodine receptor at higher [Ca2+]SRT. Key factors that determine [Ca2+]SRT in intact ventricular myocytes include (1) the thermodynamically limited Ca2+ gradient that the SR can develop (which depends on forward flux and backflux through the SR Ca2+ ATPase) and (2) diastolic SR Ca2+ leak (ryanodine receptor mediated).

Ca2+ Scraps: Local Depletions of Free [Ca2+] in Cardiac Sarcoplasmic Reticulum During Contractions Leave Substantial Ca2+ Reserve

Abstract— Free [Ca2+] inside the sarcoplasmic reticulum ([Ca2+]SR) is difficult to measure yet critically important in controlling many cellular systems. In cardiac myocytes, [Ca2+]SR regulates cardiac contractility. We directly measure [Ca2+]SR in intact cardiac myocytes dynamically and quantitatively during beats, with high spatial resolution. Diastolic [Ca2+]SR (1 to 1.5 mmol/L) is only partially depleted (24% to 63%) during contraction. There is little temporal delay in the decline in [Ca2+]SR at release junctions and between junctions, indicating rapid internal diffusion. The incomplete local Ca2+ release shows that the inherently positive feedback of Ca2+-induced Ca2+ release terminates, despite a large residual driving force. These findings place stringent novel constraints on how excitation-contraction coupling works in heart and also reveal a Ca2+ store reserve that could in principle be a therapeutic target to enhance cardiac function in heart failure.

Assessment of intra-SR free [Ca] and buffering in rat heart

To measure the free intrasarcoplasmic reticulum [Ca] ([Ca]SR) in isolated rat cardiac microsomes, ventricular tissue was homogenized in the presence of the low-affinity Ca indicator furaptra. Stepwise increases in cuvette [Ca] ([Ca]c) in the presence of ATP caused progressive increases in steady-state intravesicular fluorescence ratio to a maximum (Rmax). Steady-state [Ca]SR/[Ca]c was approximately 7000. Therefore the resting [Ca]SR may approach 700 microM in the rat cardiac myocyte at [Ca]c = 100 nM. The sarcoplasmic reticulum (SR) Ca pump requires a free energy of deltaG approximately 44 kJ x mol(-1) to generate this [Ca] gradient (e.g., approximately 74% of deltaG(ATP)). Total SR 45Ca uptake was also measured in digitonin-permeabilized myocytes as a function of [Ca]c in the absence of precipitating ions. The steady-state SR Ca content at 100 nM [Ca]c was approximately 400 micromol/liter cytosolic volume. Used together, these data allowed evaluation of the in situ SR Ca-buffering properties. The SR Ca-binding site concentration was approximately 14 mM, and Kd(Ca) approximately 0.638 mM [Ca]SR.

Effects of FK-506 on Contraction and Ca2+ Transients in Rat Cardiac Myocytes

FK-506 binding protein (FKBP) has been reported to be closely associated with the ryanodine receptor in skeletal and cardiac muscle and to modulate sarcoplasmic reticulum (SR) Ca2+ release channel gating in isolated channels. FK-506 can inhibit the activity of FKBP, thereby reversing its effects on SR Ca2+ release. We investigated the function of FKBP during normal contractions and Ca2+ transients in intact rat ventricular myocytes loaded with fluorescent Ca2+ indicators. FK-506 significantly increased steady state twitch Ca2+ transients and contraction amplitudes even under conditions in which the SR Ca2+ load and Ca2+ current were unaltered, suggesting that FK-506 increases the fraction of SR Ca2+ released during excitation-contraction (E-C) coupling. Action potentials were somewhat prolonged, consistent with the larger Ca2+ transients causing greater inward Na(+)-Ca2+ exchange current. FK-506 did not affect SR Ca2+ uptake but modestly decreased Ca2+ extrusion via Na(+)-Ca2+ exchange in intact cells (although no effect on Na(+)-Ca2+ exchange was seen in sarcolemmal vesicles). In most cells, FK-506 caused an increase in SR Ca2+ content during steady state stimulation, as assessed by caffeine-induced contractures. This was probably due to the inhibition of Ca2+ efflux via Na(+)-Ca2+ exchange. FK-506 also accelerated the rest decay of SR Ca2+ content and increased the frequency of resting Ca2+ sparks about fourfold. The increase in frequency of these basic Ca2+ release events was not associated with changes in the amplitude or duration of the Ca2+ sparks. We conclude that FK-506 increases the fraction of SR Ca2+ released during normal twitches and enhances the rate of SR Ca2+ release during rest. FK-506 also inhibits Na(+)-Ca2+ exchange, although this effect may be indirect. These effects are consistent with an important SR-stabilizing effect of FKBP in intact rat ventricular myocytes.

Spontaneous Ca waves in ventricular myocytes from failing hearts depend on Ca2+-calmodulin-dependent protein kinase II

Increased cardiac ryanodine receptor (RyR)-dependent diastolic SR Ca leak is present in heart failure and in conditions when adrenergic tone is high. Increasing Ca leak from the SR could result in spontaneous Ca wave (SCaW) formation. SCaWs activate the inward Na/Ca exchanger (NCX) current causing a delayed afterdepolarization (DAD), potentially leading to arrhythmia. Here we examine SCaWs in ventricular myocytes isolated from failing and healthy rabbit hearts. Myocytes from healthy hearts did not exhibit SCaWs under baseline conditions versus 43% of those exposed to isoproterenol (ISO). This ISO-induced increase in activity was reversed by inhibition of Ca-calmodulin-dependent protein kinase II (CaMKII) by KN93. Inhibition of cAMP-dependent protein kinase (PKA) by H89 had no observed effect. Of myocytes treated with forskolin 50% showed SCaW activity, attributable to a large increase in SR Ca load ([Ca](SRT)) versus control. At similar [Ca](SRT) (121muM) myocytes treated with ISO plus KN93 had significantly fewer SCaWs versus those treated with ISO or ISO plus H89 (0.2+/-0.28 vs. 1.1+/-0.28 and 1.29+/-0.39 SCaWs cell(-)(1), respectively). In myocytes isolated from failing hearts ISO induced an increase in the percentage of cells generating SCaWs vs. baseline (74% vs. 11%) with no increase in [Ca](SRT). Inhibiting CaMKII reversed this effect (14%). At similar [Ca](SRT) (71microM) myocytes treated with ISO or ISO plus H89 had significantly more SCaWs per cell vs. untreated (2.5+/-0.5; 1.6+/-0.7 vs. 0.36+/-0.3, respectively). Treatment with ISO plus KN93 completely abolished this effect. The evidence suggests the ISO-dependent increase in SCaW activity in both healthy and failing myocytes is CaMKII-dependent, implicating CaMKII in arrhythmogenesis.

Ca flux, contractility, and excitation-contraction coupling in hypertrophic rat ventricular myocytes.

Left ventricular hypertrophy (approximately 40%) was induced in rats by banding of the abdominal aorta. After 16 wk, ventricular homogenates were prepared for biochemical measurements and ventricular myocytes were isolated for functional studies. In myocytes, the effects of banding on intracellular Ca handling, contraction, and excitation-contraction (E-C) coupling were determined using indo 1 fluorescence and whole cell voltage clamp. After steady-state field or voltage-clamp stimulation to load the sarcoplasmic reticulum (SR), SR Ca content assessed by caffeine-induced Ca transients was the same in sham and banded groups. Despite this, cell shortening amplitudes were significantly depressed in the banded group, suggesting altered contractile properties. In banded rats, the SR Ca-adenosinetriphosphatase (Ca-ATPase) mRNA level was reduced, as was homogenate thapsigargin-sensitive SR Ca-ATPase, but cytosolic free Ca concentration ([Ca]i) decline attributed to SR Ca-ATPase activity in intact cells was not slowed. Banding also reduced Na/Ca exchange mRNA level but did not affect either Na-dependent sarcolemmal 45Ca transport in homogenate or the rate of [Ca]i decline in intact cells attributed to Na/Ca exchange (during caffeine-induced contractures). Banding also did not change the rate of [Ca]i decline mediated by the combined function of the mitochondrial Ca uptake and sarcolemmal Ca-ATPase in intact cells. Ca current (ICa) density and voltage dependence were the same in sham and banded groups. Ryanodine receptor mRNA, protein content, and ryanodine affinity were also unchanged in the banded group. At 1 mM extracellular Ca concentration ([Ca]o), banding did not affect E-C coupling efficacy in intact cells under voltage clamp (i.e., same contraction for given ICa and SR Ca load). However, when [Ca]o was reduced to 0.5 mM, the efficacy of E-C coupling was greatly depressed in the banded group (even though ICa and SR Ca content were matched). In summary, unloaded myocyte contraction was depressed in these hypertrophic hearts, but Ca transport was little altered, at 1 mM [Ca]o. However, reduction of [Ca]o to 0.5 mM appears to unmask a depressed fractional SR Ca release in response to a given ICa trigger and SR Ca load.