Abstract-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 CaMKIIdependent 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. Key Words: ryanodine receptor Ⅲ CaMKII Ⅲ phosphorylation Ⅲ heart failure Ⅲ arrhythmia C ontractile dysfunction in HF is caused by diminished sarcoplasmic reticulum (SR) Ca load that could arise from enhanced activity of Na/Ca exchange (NCX), reduced SR Ca ATPase (SERCA) function, and increased diastolic SR Ca leak via ryanodine receptors (RyR), 1-5 all of which we have demonstrated to occur in our arrhythmogenic rabbit model of nonischemic HF. [1][2][3] HF is also associated with a nearly 50% incidence of sudden cardiac death from ventricular tachycardia (VT) that degenerates to ventricular fibrillation (VF). 6 In 3D cardiac mapping studies in our HF rabbit model, we showed that spontaneously occurring VT initiates by nonreentrant mechanisms 7 associated with delayed afterdepolarizations. 2 These arise from spontaneous SR Ca release that activates a transient inward current (I ti ) carried primarily by NCX. 2 Thus abnormal SR Ca release via RyR may contribute to both contractile dysfunction and arrhythmogenesis.The cardiac RyR (RyR2) is the center of a large macromolecular protein complex that directly or indirectly interacts with RyR2 and modulates its function. The complex includes FK506 binding protein 12.6 (FKBP12.6), calmodulin (CaM), protein kinase A (PKA), Ca/CaM-dependent protein kinase (CaMKII), protein phosphatases PP1 and PP2A, mAKAP, and other associated proteins such as spinophilin, calsequestrin, and sorcin. 8 It was shown 9 in HF that PKA mediates RyR2 hyperphosphorylation at the RyR2-Ser2809 site (th...

We have developed a detailed mathematical model for Ca2+ handling and ionic currents in the rabbit ventricular myocyte. The objective was to develop a model that: 1), accurately reflects Ca-dependent Ca release; 2), uses realistic parameters, particularly those that concern Ca transport from the cytosol; 3), comes to steady state; 4), simulates basic excitation-contraction coupling phenomena; and 5), runs on a normal desktop computer. The model includes the following novel features: 1), the addition of a subsarcolemmal compartment to the other two commonly formulated cytosolic compartments (junctional and bulk) because ion channels in the membrane sense ion concentrations that differ from bulk; 2), the use of realistic cytosolic Ca buffering parameters; 3), a reversible sarcoplasmic reticulum (SR) Ca pump; 4), a scheme for Na-Ca exchange transport that is [Na]i dependent and allosterically regulated by [Ca]i; and 5), a practical model of SR Ca release including both inactivation/adaptation and SR Ca load dependence. The data describe normal electrical activity and Ca handling characteristics of the cardiac myocyte and the SR Ca load dependence of these processes. The model includes a realistic balance of Ca removal mechanisms (e.g., SR Ca pump versus Na-Ca exchange), and the phenomena of rest decay and frequency-dependent inotropy. A particular emphasis is placed upon reproducing the nonlinear dependence of gain and fractional SR Ca release upon SR Ca load. We conclude that this model is more robust than many previously existing models and reproduces many experimental results using parameters based largely on experimental measurements in myocytes.

Abstract-Increased diastolic SR Ca 2ϩ leak (J leak ) could depress contractility in heart failure, but there are conflicting reports regarding the J leak magnitude even in normal, intact myocytes. We have developed a novel approach to measure SR Ca 2ϩ leak in intact, isolated ventricular myocytes. After stimulation, myocytes were exposed to 0 Na ϩ , 0 Ca 2ϩ solution Ϯ1 mmol/L tetracaine (to block resting leak). Figure 1A). This rise increases both passive J leak and Ca 2ϩ pump-mediated J pumpR . Two schools of thought describe diastolic SR Ca 2ϩ flux as either (1) a strict forward pump versus leak balance (J pumpF ϭJ leak without appreciable J pumpR , Figure 1B) 1,2,6 or (2) approaching a thermodynamic equilibrium where J pumpF ϭJ pumpR (with little J leak , Figure 1C) ] i can approach a thermodynamic limit of 7000. 8,10 In this case ( Figure 1C), most of the diastolic J pumpF is balanced by J pumpR , which rises with [Ca 2ϩ ] SR during [Ca 2ϩ ] i decline (with little J leak required). 8 -12 Note that J pumpR cannot exceed J pumpF in an intact cell, but as it approaches J pumpF , it could create a steady-state balance where there is almost no net pump flux (J pump ), just enough to be counterbalanced by a very small J leak . This would require very little ATP consumption to retain diastolic [Ca 2ϩ ] SRT but would make [Ca 2ϩ ] SRT quite sensitive to energetic state (eg, ⌬G ATP ).

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.

Abstract-Enhanced

Materials and MethodsExperiments were conducted in accordance with the Guide for the Care and Use of Experimental Animals at Loyola University Medical Center and conformed to the Guide for the Care and Use of Laboratory Animals published by NIH (publication No. 85-23, revised 1985). Ventricular myocytes were isolated from New Zealand White rabbits (Myrtle's Rabbitry, Inc, Thompson Station, Tenn) in which HF was induced by combined aortic insufficiency and stenosis 5,6 and diastolic SR Ca 2ϩ leak was measured 14 (see online data supplement available at http://www.circresaha.org). Briefly, cells were stimulated to steady state at different frequencies to vary load. Diastolic [Ca 2ϩ ] i was measured in 0 Na ϩ , 0 Ca 2ϩ normal Tyrode'sϮtetracaine followed by caffeine to measure RyRdependent changes in [Ca 2ϩ ] SRT ( Figure 1A). Results and DiscussionRabbit HF myocytes in this model have significantly reduced Ca 2ϩ transients, [Ca 2ϩ ] SRT , and blunted force-frequency relationship. 5,6 Cells used in this study had similar characteristics ( Figure 1B) Figure 1C). 14 The tetracaine-dependent shifts at each [Ca 2ϩ ] SRT were converted to SR Ca 2ϩ leak rates. 14 The leak- [Ca 2ϩ ] SRT relationship also shifted leftward in the HF (PϽ0.05) versus control data ( Figure 1D) Figure 2D). In our rabbit HF model, 5,6 enhanced NCX function appears dominant, whereas different balances of enhanced NCX and reduced SR Ca 2ϩ -ATPase could cause similar [Ca 2ϩ ] SRT reductions in other cases (where leak has not been assessed). 2,3,7 NCX changes seem most influential on [Ca 2ϩ ] SRT for the ranges in Figure 2, but further experimental tests may be required to further validate this. In addition, one would expect a diastolic SR Ca 2ϩ leak to be more influential at low heart rate (due to the longer diastolic interval), but in HF twitch depression is more apparent at higher heart rate versus low. In conclusion, SR Ca 2ϩ leak increases in HF, but it may be less influential than altered SR Ca 2ϩ -ATPase and NCX in causing the [Ca 2ϩ ] SRT reduction in HF, which reduces systolic function.

The cardiac sarcolemmal Na-Ca exchanger (NCX) is allosterically regulated by [Ca]i such that when [Ca]i is low, NCX current (INCX) deactivates. In this study, we used membrane potential (Em) and INCX 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 INCX (Ca efflux) and to decrease outward INCX. On the other hand, Ca activation would increase INCX in both directions. Thus, we attributed [Ca]i-dependent increases in outward INCX to allosteric regulation. Ca activation of INCX 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 (Δ680–685). 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 (KmCaAct = 125 ± 16 nM SEM ≈ resting [Ca]i). This, along with the observation that no delay was observed between measured [Ca]i and activation of INCX under our conditions, suggests that beat to beat changes in NCX function can occur in vivo. These changes in the INCX 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 KmCaAct from other species. Model predictions for Ca activation, on which our estimates of KmCaAct are based, confirm that Ca activation strongly influences outward INCX, explaining why it increases rather than declines with increasing [Ca]i.

Ca 2؉ signals, produced by Ca 2؉ release from cellular stores, switch metabolic responses inside cells. In muscle, Ca 2؉ sparks locally exhibit the rapid start and termination of the cell-wide signal. By imaging Ca 2؉ inside the store using shifted excitation and emission ratioing of fluorescence, a surprising observation was made: Depletion during sparks or voltage-induced cell-wide release occurs too late, continuing to progress even after the Ca 2؉ release channels have closed. This finding indicates that Ca 2؉ is released from a ''proximate'' compartment functionally in between store lumen and cytosol. The presence of a proximate compartment also explains a paradoxical surge in intrastore Ca 2؉ , which was recorded upon stimulation of prolonged, cell-wide Ca 2؉ release. An intrastore surge upon induction of Ca 2؉ release was first reported in subcellular store fractions, where its source was traced to the store buffer, calsequestrin. The present results update the evolving concept, largely due to N. Ikemoto and C. Kang, of calsequestrin as a dynamic store. Given the strategic location and reduction of dimensionality of Ca 2؉ -adsorbing linear polymers of calsequestrin, they could deliver Ca 2؉ to the open release channels more efficiently than the luminal store solution, thus constituting the proximate compartment. When store depletion becomes widespread, the polymers would collapse to increase store [Ca 2؉ ] and sustain the concentration gradient that drives release flux.calcium signaling ͉ calcium sparks ͉ excitation-contraction coupling ͉ sarcoplasmic reticulum ͉ skeletal muscle R apid changes in intracellular cytosolic [Ca 2ϩ ] are required for signaling functions in many cell types (1). These changes are achieved via Ca 2ϩ release through channels, ryanodine receptors (RyRs), which must open and close quickly. To increase its speed, gating of RyRs relies on effects of the permeant ion, including channel opening by elevated cytosolic [Ca 2ϩ ] (2). In muscle, the desirable fast kinetic features are already present in its elementary signaling events, Ca 2ϩ sparks (3), which involve the nearly simultaneous opening (4) of a number of channels (5), followed by their synchronized closing (4). Thus, this gating does not follow the usual Markovian rules for channels that evolve independently but requires timekeeping and synchronization (6). In cardiac muscle, depletion of sarcoplasmic reticulum (SR) Ca 2ϩ is the likely timer of channel closing, and the substantial depletion that follows the cardiac beat (7) was imaged as ''blinks'' associated with Ca 2ϩ sparks (8). The sensor that translates depletion into channel closing appears to be the main intra-SR buffer, calsequestrin (CSQ) (9).By contrast, in skeletal muscle, depletion associated with a twitch is only 8-15% (10). This low rate of depletion reflects a SR with larger terminal cisternae containing higher concentrations of a CSQ of greater binding capacity, thus constituting a much greater calcium reservoir. Despite the greater store, sparks of skeletal mus...