Ca Release Flux Research Articles

Increasing SERCA function promotes initiation of calcium sparks and breakup of calcium waves.

Increasing sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump activity enhances sarcoplasmic reticulum calcium (Ca) load, which increases both ryanodine receptor opening and driving force of Ca release flux. Both of these effects promote Ca spark formation and wave propagation. However, increasing SERCA activity also accelerates local cytosolic Ca decay as the wave front travels to the next cluster, which limits wave propagation. As a result, increasing SERCA pump activity has a biphasic effect on the propensity of arrhythmogenic Ca waves, but a monotonic effect to increase Ca spark frequency and amplitude. Waves of sarcoplasmic reticulum (SR) calcium (Ca) release can cause arrhythmogenic afterdepolarizations in cardiac myocytes. Ca waves propagate when Ca sparks at one Ca release unit (CRU) recruit new Ca sparks in neighbouring CRUs. Under normal conditions, Ca sparks are too small to recruit neighbouring Ca sparks where Ca sensitivity is also low. However, under pathological conditions such as a Ca overload or ryanodine receptor (RyR) sensitization, Ca sparks can be larger and propagate more readily as macro-sparks or full Ca waves. Increasing SERCA pump activity promotes SR Ca load, which promotes RyR opening and increases driving force of the Ca release flux from SR to cytosol, promoting Ca waves. However, high sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) activity can also decrease local cytosolic [Ca] as it approaches the next CRU, thereby reducing wave appearance and propagation. In this study, we use a physiologically detailed model of subcellular Ca cycling and experiments in phospholamban-knockout mice, to show how Ca waves are initiated and propagate and how different conditions contribute to the generation and propagation of Ca waves. We show that reducing diffusive coupling between Ca sparks by increasing SERCA activity prevents Ca waves by reducing [Ca] at the next CRU, as do Ca buffers, low intra-SR Ca diffusion and distance between CRUs. Increasing SR Ca uptake rate has a biphasic effect on Ca wave propagation; initially it enhances Ca spark probability and amplitude and CRU coupling, thereby promoting arrhythmogenic Ca wave propagation, but at higher levels SR Ca uptake can abort those arrhythmogenic Ca waves.

Dyadic Remodeling as a Cause of Impaired E-C Coupling in Early Stages of Myocardial Injury

In failing myocytes, impaired E-C coupling is often associated with loss of t-tubules and appearance of orphaned calcium release units. Impairment of communication between Ca-channels of sarcolemma and of sarcoplasmic reticulum (SR) has been observed in non-failing stages of cardiac hypertrophy. We therefore studied the relationship between Ca-signaling and ultrastructure of dyadic junctions in early stages of myocardial injury. Myocardial injury (IMI) was induced by a single dose of isoproterenol (150 mg/kg, i.p.) in adult male rats. On day 15, isolated myocytes were studied using laser scanning fluorescence confocal microscopy: Ca-spikes were activated by whole-cell patch-clamp and t-tubules were visualized by di-8-ANEPPS. Ultrastructure of dyadic junctions was evaluated from electron-microscopic images. Our results show that remodeling of IMI myocytes transpired as increased proportion of longitudinal elements of tubular system as well as increased proportion of dyads with defective contact between the membranes of sarcolemma and SR; however, there was no actual loss of t-tubules and ultrastructure of t-tubules in dyads of IMI myocytes was similar to control (CTR). In parallel, Ca-spike activation was slower and less synchronized in IMI. In both CTR and IMI, distribution of latencies of Ca-spikes and the time profile of Ca-release flux measured from whole cells consisted of two components. The fraction of the slow component of calcium release in IMI was close to the fraction of dyads with defective contact between the membranes of sarcolemma and SR. We conclude that overload of cardiac myocytes of the working part of injured myocardium leads to damage of tubulo-reticular junctions of dyadic microdomains, which results in impairment of local calcium signaling. Support: APVV-0721-10, VEGA 2/047/14 and VEGA 2/0095/15

Long-Lasting Sparks: Multi-Metastability and Release Competition in the Calcium Release Unit Network.

Calcium (Ca) sparks are elementary events of biological Ca signaling. A normal Ca spark has a brief duration in the range of 10 to 100 ms, but long-lasting sparks with durations of several hundred milliseconds to seconds are also widely observed. Experiments have shown that the transition from normal to long-lasting sparks can occur when ryanodine receptor (RyR) open probability is either increased or decreased. Here, we demonstrate theoretically and computationally that long-lasting sparks emerge as a collective dynamical behavior of the network of diffusively coupled Ca release units (CRUs). We show that normal sparks occur when the CRU network is monostable and excitable, while long-lasting sparks occur when the network dynamics possesses multiple metastable attractors, each attractor corresponding to a different spatial firing pattern of sparks. We further highlight the mechanisms and conditions that produce long-lasting sparks, demonstrating the existence of an optimal range of RyR open probability favoring long-lasting sparks. We find that when CRU firings are sparse and sarcoplasmic reticulum (SR) Ca load is high, increasing RyR open probability promotes long-lasting sparks by potentiating Ca-induced Ca release (CICR). In contrast, when CICR is already strong enough to produce frequent firings, decreasing RyR open probability counter-intuitively promotes long-lasting sparks by decreasing spark frequency. The decrease in spark frequency promotes intra-SR Ca diffusion from neighboring non-firing CRUs to the firing CRUs, which helps to maintain the local SR Ca concentration of the firing CRUs above a critical level to sustain firing. In this setting, decreasing RyR open probability further suppresses long-lasting sparks by weakening CICR. Since a long-lasting spark terminates via the Kramers’ escape process over a potential barrier, its duration exhibits an exponential distribution determined by the barrier height and noise strength, which is modulated differently by different ways of altering the Ca release flux strength.

Urocortin 2 Protects Against Pacing-Induced Alternans via Phosphorylation of Phospholamban in Cardiac Myocytes from Normal and Failing Hearts

Cardiac alternans is a high risk indicator for cardiac arrhythmias, stroke and sudden cardiac death. The cardioactive peptide Urocortin 2 (Ucn2) exhibits beneficial effects in normal and failing hearts, and elicits PKA-dependent positive inotropic and lusitropic effects in normal myocytes. Thus, we investigated if Ucn2 protects against pacing-induced alternans and elucidated the underlying mechanism.Experiments were performed on single rabbit atrial and ventricular myocytes from normal and failing hearts. Chronic heart failure was induced by combined pressure and volume overload. Ca alternans was induced by incrementally increasing the pacing frequency until stable Ca alternans occurred at room temperature. Global Ca transients were measured with the fluorescent Ca indicators Fluo-4 or Indo-1 and monitored simultaneously with mechanical alternans (sarcomere length). In some experiments, cytosolic Ca alternans and intra-SR Ca alternans were simultaneously recorded with the Ca indicators Rhod-2 and Fluo-5N, respectively.The average alternans ratio (AR = 1-(small-amplitude/large-amplitude)) in atrial myocytes was 0.79, and in normal and failing ventricular myocytes the ARs were 0.69 and 0.64, respectively. Ucn2 (100 nM) completely abolished Ca and mechanical alternans (within 2-3 min) in atrial and ventricular myocytes from normal and failing hearts. An increased sarcoplasmic reticulum (SR) Ca content, together with an enhanced SR Ca release flux, suggested that Ucn2 normalized alternans through effects on SR Ca ATPase (SERCA). Ucn2 increased significantly the level of cyclic adenosine monophosphate (cAMP) in normal cells (∼12-fold), and enhanced phosphorylation of phospholamban (PLB) at Ser16 in normal myocytes (∼10-fold) and to a lesser extent (∼5-fold) in failing myocytes. These data demonstrate that Ucn2 rescues alternans presumably via increased SERCA activity in atrial and ventricular myocytes and thus protects normal and failing hearts from proarrhythmic alternans.

Extraction of Sub-microscopic Ca Fluxes from Blurred and Noisy Fluorescent Indicator Images with a Detailed Model Fitting Approach

The release of Ca from intracellular stores is key to cardiac muscle function; however, the molecular control of intracellular Ca release remains unclear. Depletion of the intracellular Ca store (sarcoplasmic reticulum, SR) may play an important role, but the ability to measure local SR Ca with fluorescent Ca indicators is limited by the microscope optical resolution and properties of the indicator. This leads to an uncertain degree of spatio-temporal blurring, which is not easily corrected (by deconvolution methods) due to the low signal-to-noise ratio of the recorded signals. In this study, a 3D computer model was constructed to calculate local Ca fluxes and consequent dye signals, which were then blurred by a measured microscope point spread function. Parameter fitting was employed to adjust a release basis function until the model output fitted recorded (2D) Ca spark data. This ‘forward method’ allowed us to obtain estimates of the time-course of Ca release flux and depletion within the sub-microscopic local SR associated with a number of Ca sparks. While variability in focal position relative to Ca spark sites causes more out-of-focus events to have smaller calculated fluxes (and less SR depletion), the average SR depletion was to 20±10% (s.d.) of the resting level. This focus problem implies that the actual SR depletion is likely to be larger and the five largest depletions analyzed were to 8±6% of the resting level. This profound depletion limits SR release flux during a Ca spark, which peaked at 8±3 pA and declined with a half time of 7±2 ms. By comparison, RyR open probability declined more slowly, suggesting release termination is dominated by neither SR Ca depletion nor intrinsic RyR gating, but results from an interaction of these processes.

Effects of Redox Environment on Calcium Alternans in Isolated Rabbit Cardiomyocytes

Cardiac alternans is a multifactorial phenomenon linked to cardiac arrythmias. At the cellular level cardiac alternans is defined by beat-to-beat alternations in contraction amplitude (mechanical alternans), action potential duration (electrical or action potential duration alternans) and Ca transient amplitude (Ca alternans) at constant stimulation frequency. The aim of this project was to characterize the effect of changes in the cellular redox environment on Ca alternans in cardiac myocytes. Single myocytes (from New Zealand White rabbits) were isolated enzymatically by retrograde Langendorff perfusion. Ca alternans were induced by incrementally increasing the pacing frequency (electrical field stimulation) until stable Ca alternans occurred. The frequency at which stable Ca alternans were observed varied from cell to cell and ranged from >1 to 2.5 Hz at room temperature. Global cytosolic Ca transients were measured with Indo-1. In some experiments, cytosolic Ca alternans and intra-SR Ca alternans were simultaneously measured with the fluorescent Ca indicators Rhod-2 and Fluo-5N, respectively. Confocal microscopy was used to measure Ca sparks with Fluo-4.Reducing agents dithiothreitol and reduced glutathione partially abolished Ca and mechanical alternans by restoring diastolic Ca and Ca transient amplitudes. A decreased sarcoplasmic reticulum (SR) Ca release flux but not Ca content, together with a decreased Ca spark frequency, suggest that reducing agents normalized alternans through effects on the SR Ca release channel (ryanodine receptor type-2). Addition of a membrane permeant superoxide dismutase mimetic, Tempol, had little effect on Ca alternans, suggesting the possible role of dithiotreitol directly acting on the ryanodine receptor. These data highlight that the redox state of the cell may be important in the generation of Ca and mechanical alternans during oxidative stress.

Analysis of Calcium (Ca) Transients during Excitation-Contraction Coupling in Frog Twitch Muscle Fibers

Singly-dissected fibers were micro-injected with furaptra, a rapidly-responding Ca indicator. Resting fluorescence (FR) and action-potential evoked changes (ΔF) were measured at 16 oC; ΔF/FR was scaled to units of ΔfCaD, the change in fraction of furaptra in the Ca-bound form. Measured ΔfCaD was compared with ΔfCaD simulated with a kinetic model of the underlying myoplasmic Ca movements. During the period 30-200 ms after an action potential, simulated ΔfCaD decayed toward baseline more slowly than measured ΔfCaD. If ΔfCaD was simulated with a modified model that incorporated competition between Mg and Ca for occupancy of the regulatory sites on troponin (assumed dissociation constant of Mg's reaction with the regulatory sites, 2.2 mM; assumed myoplasmic free [Mg], 1 mM), good agreement with measured ΔfCaD was observed. The results support the conclusion that Mg, at physiological levels, competes with Ca for occupancy of the regulatory sites, as indicated in some experiments from fragmented preparations, including tension-pCa measurements in skinned fibers and biochemical studies of isolated troponin molecules. ΔfCaD in frog was also compared with our previous measurements and simulations of ΔfCaD in mouse fibers (reviewed in J. Gen. Physiol., 2012). In frog, the SR Ca release flux elicited by an action potential appears to be the sum of two components. The time course of the first component is similar to that of the entire flux waveform in both fast-and slow-twitch mouse fibers, while that of the second is several-fold slower; the fractional release amounts are ∼0.85 (first component) and ∼0.15 (second component). An anatomical basis for two release components in frog is the presence of both junctional and para-junctional Ca release channels, whereas the mouse fibers have only junctional channels (Felder and Franzini-Armstrong, 2002). Supported by NIH (GM 086167)

Effects of High [BAPTA] Inside Mouse Muscle Fibers Reveal a Role of Calcium in the Termination of Voltage-Operated Calcium Release from the SR

Striated muscles have a termination mechanism that causes the flux of Ca release from the SR, whether activated by an action potential or a voltage pulse, to rapidly cease after an early peak. This mechanism is viewed as a fundamental property that insures stability and “gradedness” in the control of Ca signals. To probe this termination, the evolution of Ca release flux was derived from cytosolic Ca transients elicited by SR-depleting depolarizations of long duration, in voltage-clamped cells of mouse FDB muscle. In the presence of 5 mM of the Ca buffer BAPTA, the release flux underwent major changes compared with a 10 mM [EGTA] “reference” situation (studied by Royer, J Physiol 2008). Ca release reached an early peak and then decayed to a sustained phase that was higher and briefer than in reference, often including a second rise or “hump”. In BAPTA, measurable release only lasted 100 ms or less. Its time integral -which measures the SR Ca content releasable by depolarization_was on average 1.4 mM (n=12 cells), compared with 2.1 mM for 18 cells in reference. An increase in flux with conserved releasable content indicates that BAPTA promotes flux and hastens emptying of the SR without greatly changing its storage properties (including lumenal [Ca2+]). A greater Ca flux driven by a similar [Ca2+] gradient requires greater and/or more sustained channel openness. These observations suggest the presence of a release channel-inhibiting mechanism (CDI) mediated by binding of cytosolic Ca2+ to open or closed channels, a mechanism more susceptible to interference by BAPTA than the slower-reacting EGTA. Work funded by NIAMS/NIH and an MDA grant to Dr. Jingsong Zhou, who we thank for continued support.

Sodium Current-Induced Release of Calcium from the Sarcoplasmic Reticulum in Rabbit Ventricular Myocytes

The hypothesis that Na current (INa) can induce release of Ca from the sarcoplasmic reticulum (SR) by activating reverse Na-Ca exchange (NCX) has been debated since 1990. We tested this hypothesis with epi-fluorescence imaging of adult rabbit ventricular myocytes loaded with the Ca indicator fluo-4. Ca release was triggered with an action potential clamp with and without an initial voltage ramp from −80 to −40 mV, for a duration of 1.5s. We confirmed that this protocol selectively blocked INa without altering Ca influx through L-type Ca channels (LCCs) and SR Ca load. With 0 mM Na in the pipette (to reduce intracellular Na), inactivating INa reduced SR Ca release flux by 27%±4% (n=9). With 5 mM Na in the pipette, the Ca release upon inactivation of INa was reduced by 33%±5% (n=4). We suggest that increased activation of reverse NCX by increased intracellular Na concentration mainly produced by INa explains these findings. These conclusions are in agreement with studies on normal and NCX knockout mice, which show that INa affects SR Ca release only in normal, but not in NCX knockout mice. In similar experiments, we applied 100 nM TTX to selectively block brain isoforms of Na channels. In the presence of TTX, the SR Ca release flux was reduced by 35%±3% (n=6). This effect of INa on Ca release can be explained by early reverse NCX, activated by TTX sensitive INa, which could prime the dyadic cleft with Ca. Furthermore, the results can be explained if INa activation of NCX, and subsequent priming of the dyadic cleft with Ca, increases the coupling fidelity between LCCs and ryanodine receptors within a couplon. Thus the presence of INa increases the likelihood that couplons are activated.

New Insight Into Cardiomyocyte Ca Signaling Obtained By Fast Confocal Imaging

With ultra-fast 1D-(x-t) and 2D-(x-y-t) confocal microscopy (Zeiss LSM 5 Live) we studied the spatio-temporal properties of Ca sparks and Ca transients. Ca sparks which originated from subsarcolemmal sarcoplasmic reticulum (SR) release sites in atrial myocytes were elongated in the longitudinal direction of the cell. Ca sparks corresponding to Ca release from non-junctional SR in atrial myocytes and junctional SR in ventricular myocytes were variable spatially with some events being symmetrical and others asymmetrical. Anisotropic sparks occurred in transverse as well as longitudinal direction. Ca sparks originating from non-junctional SR and recorded in line-scan (x-t) mode at 40,000 lines/s revealed a step-like appearance in space (time-dependent step-like increase of width from the point of origin) and amplitude during the activation phase of the spark. These steps in space and amplitude may represent the sequential opening of individual ryanodine receptor (RyR) channels in a release cluster and support the notion that sparks represent Ca release from a group of RyRs. Mathematical analysis of global Ca transients recorded from field-stimulated ventricular myocytes at high temporal resolution allowed separation of Ca entry from Ca release flux. After the electrical stimulus a latency period of 2.5 ms was required to activate sarcolemmal Ca channels. SR Ca release was initiated with an additional delay of 3.0 ms. Maximal Ca release flux was observed 3.9 ms after initiation of Ca release when all release sites became active. Ca entry flux was ten times smaller than Ca release flux. When SR Ca release was eliminated after treatment with thapsigargin and caffeine, identical Ca entry flux was observed as in control conditions. In summary, ultra-fast confocal imaging allows investigation of Ca signals with time resolution similar to patch clamp technique, however in a much less invasive fashion.

Partial Inhibition of Sarcoplasmic Reticulum Ca Release Evokes Long-Lasting Ca Release Events in Ventricular Myocytes: Role of Luminal Ca in Termination of Ca Release

In cardiac myocytes, local sarcoplasmic reticulum (SR) Ca depletion during Ca sparks is believed to play an important role in the termination of SR Ca release. We tested whether decreasing the rate of SR Ca depletion by partially inhibiting SR Ca release channels (ryanodine receptors) delays Ca spark termination. In permeabilized cat ventricular myocytes, 0.7 mM tetracaine caused almost complete Ca spark inhibition followed by a recovery significantly below control level. The recovery was associated with increased SR Ca load and increased Ca spark duration. Additionally, SR Ca release events lasting several hundred milliseconds occurred consistently. These events had a significantly lower initial Ca release flux followed by a stable plateau, indicating delayed release termination and maintained SR Ca load. Increasing SR Ca load (without inhibiting SR Ca release rate) or decreasing SR Ca release rate (without increasing SR Ca load) both induced only a small increase in spark duration. These results show that the combination of decreased release flux and increased SR Ca load has synergistic effects and exerts major changes on the termination of Ca release events. Long-lasting Ca release events may originate from highly interconnected release junctions where Ca diffusion from neighboring sites partially compensates Ca depletion, thereby delaying SR Ca-dependent termination. Eventually, these events terminate by luminal Ca-independent mechanisms, such as inactivation, adaptation, or stochastic attrition.

A novel, rapid and reversible method to measure Ca buffering and time-course of total sarcoplasmic reticulum Ca content in cardiac ventricular myocytes.

This paper outlines a simple method of estimating both the Ca-buffering properties of the cytoplasm and the time-course of changes of sarcoplasmic reticulum (s.r.) Ca concentration during systole. The experiments were performed on voltage-clamped ferret single ventricular myocytes loaded with the free acid of fluo-3 through a patch pipette. The application of caffeine (10 mM) resulted in a Na-Ca exchange current and a transient increase of the free intracellular Ca concentration ([Ca2+]i). The time-course of change of total Ca in the cell was obtained by integrating the current and this was compared with the measurements of [Ca2+]i to obtain a buffering curve. This could be fit with a maximum capacity for the intrinsic buffers of 114+/-18 micromol l-1 and Kd of 0.59+/-0.17 microM (n=8). During the systolic rise of [Ca2+]i, the measured changes of [Ca2+]i and the buffering curve were used to calculate the magnitude and time-course of the change of total cytoplasmic Ca and thence of both s.r. Ca content and Ca release flux. This method provides a simple and reversible mechanism to measure Ca buffering and the time-course of both total cytoplasmic and s.r. Ca.

Perchlorate enhances transmission in skeletal muscle excitation-contraction coupling.

The effects of the anion perchlorate (present extracellularly at 8 mM) were studied on functional skeletal muscle fibers from Rana pipiens, voltage-clamped in a Vaseline gap chamber. Established methods were used to monitor intramembranous charge movement and flux of Ca release from the sarcoplasmic reticulum (SR) during pulse depolarization. Saponin permeabilization of the end portions of the fiber segment (Irving, M., J. Maylie, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:1-41) substantially reduced the amount of charge moving during conventional control pulses, thus minimizing a technical error that plagued our previous studies. Perchlorate prolonged the ON time course of charge movement, especially at low and intermediate voltages. The OFFs were also made slower, the time constant increasing twofold. The hump kinetic component was exaggerated by ClO4- or was made to appear in fibers that did not have it in reference conditions. ClO4- had essentially no kinetic ON effects at high voltages (> or = 10 mV). ClO4- changed the voltage distribution of mobile charge. In single Boltzmann fits, the midpoint potential V was shifted -20 mV and the steepness parameter K was reduced by 4.7 mV (or 1.78-fold), but the maximum charge was unchanged (n = 9). Total Ca content in the SR, estimated using the method of Schneider et al. (Schneider, M. F., B. J. Simon, and G. Szucs. 1987. Journal of Physiology. 392:167-192) for correcting for depletion, stayed constant over tens of minutes in reference conditions but decayed in ClO4- at an average rate of 0.3 mumol/liter myoplasmic water per s. ClO4- changed the kinetics of release flux, reducing the fractional inactivation of release after the peak. ClO4- shifted the voltage dependence of Ca release flux. In particular, the threshold voltage for Ca release was shifted by about -20 mV, and the activation of the steady component of release flux was shifted by > 20 mV in the negative direction. The shift of release activation was greater than that of mobile charge. Thus the threshold charge, defined as the minimum charge moved for eliciting a detectable Ca transient, was reduced from 6 nC/microF (0.55, n = 7) to 3.4 (0.53). The average of the paired differences was 2.8 (0.33, P < 0.01). The effects of ClO4- were then studied in fibers in modified functional situations. Depletion of Ca in the SR, achieved by high frequency pulsing in the presence of intracellular BAPTA and EGTA, simplified but did not eliminate the effects of ClO4-.(ABSTRACT TRUNCATED AT 400 WORDS)

The relationship between Q gamma and Ca release from the sarcoplasmic reticulum in skeletal muscle.

Asymmetric membrane currents and fluxes of Ca2+ release were determined in skeletal muscle fibers voltage clamped in a Vaseline-gap chamber. The conditioning pulse protocol 1 for suppressing Ca2+ release and the "hump" component of charge movement current (I gamma), described in the first paper of this series, was applied at different test pulse voltages. The amplitude of the current suppressed during the ON transient reached a maximum at slightly suprathreshold test voltages (-50 to -40 mV) and decayed at higher voltages. The component of charge movement current suppressed by 20 microM tetracaine also went through a maximum at low pulse voltages. This anomalous voltage dependence is thus a property of I gamma, defined by either the conditioning protocol or the tetracaine effect. A negative (inward-going) phase was often observed in the asymmetric current during the ON of depolarizing pulses. This inward phase was shown to be an intramembranous charge movement based on (a) its presence in the records of total membrane current, (b) its voltage dependence, with a maximum at slightly suprathreshold voltages, (c) its association with a "hump" in the asymmetric current, (d) its inhibition by interventions that reduce the "hump", (e) equality of ON and OFF areas in the records of asymmetric current presenting this inward phase, and (f) its kinetic relationship with the time derivative of Ca release flux. The nonmonotonic voltage dependence of the amplitude of the hump and the possibility of an inward phase of intramembranous charge movement are used as the main criteria in the quantitative testing of a specific model. According to this model, released Ca2+ binds to negatively charged sites on the myoplasmic face of the voltage sensor and increases the local transmembrane potential, thus driving additional charge movement (the hump). This model successfully predicts the anomalous voltage dependence and all the kinetic properties of I gamma described in the previous papers. It also accounts for the inward phase in total asymmetric current and in the current suppressed by protocol 1. According to this model, I gamma accompanies activating transitions at the same set of voltage sensors as I beta. Therefore it should open additional release channels, which in turn should cause more I gamma, providing a positive feedback mechanism in the regulation of calcium release.