Subcellular heterogeneity of ryanodine receptor properties in ventricular myocytes with low T-tubule density

Rationale: In ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI). Objective: To characterize RyR functional properties in relation to TT proximity, at baseline and after MI. Methods: Myocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca2+ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca2+ release, F>F50 within 20 ms) or their absence (delayed areas). Spontaneous Ca2+ release events during diastole, Ca2+ sparks, reflecting RyR activity and properties, were subsequently assigned to either category. Results: In CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na+/Ca2+ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca2+ influx via Ca2+ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca2+ content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca2+ removal by NCX at the membrane was significantly lower in MI. Conclusion: TT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca2+ loss and raise SR Ca2+ content, but may promote Ca2+ waves

Changes in ryanodine receptor (RyR) function and synchronization of Ca2+ release from the sarcoplasmic reticulum (SR) of cardiomyocytes in ischemic cardiomyopathy

SUMMARY Heart failure (HF) remains an important health problem and economic burden in the current society (1). Improvements in acute myocardial infarction (MI) treatment, better drug therapy and secondary prevention lead to more patients surviving the initial cardiac damage and developing HF at a later timepoint. The focus is now on the underlying mechanisms of evolution to HF. There has been significantevidence that disturbance of the intracellular Ca2+ homeostasis in cardiomyocytes plays an important role in the pathophysiology of HF. A hallmark feature of HF pathogenesis is impaired sarcoplasmic reticulum (SR) function, with reduced SR Ca2+ load. In addition to defective SR Ca2+ resequestration caused by decreased sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity, several lines of evidence revealed the existence of a diastolic Ca2+ leak mediated by defective regulation of ryanodine receptors (RyRs). The overall aim of this thesis was to investigate the roleof changes in RyR function in regional contractile dysfunction in ischemic cardiomyopathy (ICM). We investigated the effect of proximity of T-tubules (TTs) on intrinsic RyR function. In cardiomyocytes isolated from healthy control pigs, we found the existence of a functional link between Ca2+ spark duration and TT proximity. Our results shown that the Na+/Ca2+ exchanger (NCX) plays an important role modulating diastolic Ca2+ release. NCX in the TTs was able to shorten the duration of spontaneous diastolic Ca2+ release through RyR. In a pig model of ischemic cardiomyopathy, this regulatory mechanism was lost after cellular remodeling, and a reduction of TTs and NCX prolonged spark duration close to themembrane, which might increase the propensity for generation of spontaneous Ca2+ waves. We next evaluated regional cellular remodelingand Ca2+ handling in the area remote to a moderate size MI in a pig model of ischemic cardiomyopathy without overt HF. We compared these findings to our earlier data on changes in the MI adjacent area. Our data indicate that, opposite to loss of TTs, dyssynchrony and slowing of Ca2+ release in the area adjacent to the MI, the area remote to the MI shows maintained TT density and even increased efficiency of excitation-contraction coupling. While RyR function and spark-mediated Ca2+ leak was potentiated in both substrates, differences in spark frequency suggest differences in the underlying mechanisms. Different triggers for remodeling -ischemia vs. stretch- are probably important, and different signaling pathways are expected to be involved in remodeling in the different areas of the heart. In the last part of our study, we investigated whether RyR function and Ca2+ handling could be modulated during post-MI remodeling. In a mouse model of ischemic cardiomyopathy, we investigated theeffect of voluntary exercise early after MI on post-MI remodeling. Early voluntary exercise training after MI restored cell contraction to normal values, predominantly because of changes in myofilament Ca2+ response, and had a beneficial effect on diastolic Ca2+ handling. However, the beneficial effect was not a complete reversal of remodeling as hypertrophy and loss of repolarizing K+ currents were not affected. Secondly, we investigated the effect of cardio-specific FK506-binding protein (12.6 kDa, FKBP12.6) overexpression on RyR function in a transgenic mouse model,with and without MI. At baseline, FKPB12.6 overexpression could effectively reduce RyR open probability (Po) and fractional Ca2+ release from the SR, with maintained [Ca2+]i transient (and contraction) by an increased SR Ca2+ content. However, reduction of RyR Po by this approach appeared insufficient to prevent left ventricular hypertrophy and reduce post-MI remodeling, which is even exaggerated in transgenic mice.To conclude, this work supports a role for TTs in modulation of RyR function in healthy cardiomyocytes. During remodeling in ICM, RyR function can be modulated because of alterations in TT density and/or because of intrinsic changes in RyR, depending on the location in the heart and the nature of the stimulus for remodeling. RyR function can be modulated by different therapeutic approaches. However, the exact role of such an approach is still undetermined and it is important to remember that post-MI remodeling includes more than only changes in RyR function.status: publishe

Dyssynchrony of Ca2+ release from the sarcoplasmic reticulum as subcellular mechanism of cardiac contractile dysfunction

Cardiac contractile function depends on coordinated electrical activation throughout the heart. Dyssynchronous electrical activation of the ventricles has been shown to contribute to contractile dysfunction in heart failure, and resynchronization therapy has emerged as a therapeutic concept. At the cellular level, coupling of membrane excitation to myofilament contraction is facilitated by highly organized intracellular structures which coordinate Ca(2+) release. The cytosolic [Ca(2+)] transient triggered by depolarization-induced Ca(2+) influx is the result of a gradable and robust high gain process, Ca(2+)-induced Ca(2+) release (CICR), which integrates subcellular localized Ca(2+) release events. Lack of synchronization of these localized release events can contribute to contractile dysfunction in myocardial hypertrophy and heart failure. Different underlying mechanisms relate to functional and structural changes in sarcolemmal Ca(2+) channels, the sarcoplasmic Ca(2+) release channel or ryanodine receptor, RyR, their intracellular arrangement in close proximity in couplons and the loss of t-tubules. Dyssynchrony at the subcellular level translates in a reduction of the overall gain of CICR at the cellular level and forms an important determinant of myocyte contractility in heart failure

Effect of sarcolemmal fluxes on spark frequency and duration.

<p>A. Effect of NCX block by 5 mM nickel on spark frequency and duration in early (left) and delayed (right) release areas in CTRL cardiomyocytes (N_pigs = 3, n_cells = 7). B. Effect of I_CaL block by 50 µM cadmium on spark frequency and duration in early (left) and delayed (right) release areas in CTRL cardiomyocytes (N_pigs = 2, n_cells = 9). * denotes P<0.05.</p

NCX in myocytes from the area adjacent to MI.

<p>A. NCX function was measured as the tail current upon repolarization to −70 mV (arrow) after a step to +10 mV in CTRL and MI. B. Averaged NCX current density in CTRL (N_pigs = 9, n_cells = 30) and MI (N_pigs = 7, n_cells = 19), with [Ca²⁺]_i measured simultaneously. * denotes P<0.05.</p

Ca²⁺ removal by NCX during caffeine application.

<p>A. Example of current and [Ca²⁺]_i transient recording obtained during the last conditioning pulse from -70 to +10 mV and caffeine application (left). The decay of the current was fit by a 1- or 2-exponential according to the goodness of fit (R²>0.95) and with the 2 amplitudes being negative. The right panel is a typical example of a 2-exponential decay in a CTRL myocyte. B. Example of monophasic I_NCX decay (left) and mean data on incidence of biphasic decay (middle). The percentage of cells better fit by a biphasic exponential was significantly higher in CTRL than in MI (CTRL, N_pigs = 4, n_cells = 17; MI, N_pigs = 4, n_cells = 12, P<0.05). In addition, Tau of fast component of I_NCX decay tended to be faster in CTRL than in MI (right, CTRL, N_pigs = 4, n_cells = 8, <i>vs</i>. MI, N_pigs = 2, n_cells = 4, P = NS). * denotes P<0.05.</p

Effect of proximity of TTs to RyR on spontaneous Ca²⁺ sparks.

<p>A. Typical example of a line scan image during and after 1 Hz stimulation. After loading the SR with conditioning pulses from −70 to +10 mV at 1 Hz, stimulation was stopped and 15 seconds of diastole were recorded for Ca²⁺ sparks. Sparks were assigned to early (blue) and delayed (red) release areas corresponding to their position on the scan line. B. Spark frequency and morphology in early vs. delayed release areas in CTRL pigs (N_pigs = 12, n_cells = 41). * denotes P<0.05.</p

Modulation of Ca²⁺ sparks with TT loss during culture.

<p>A. Representative confocal images of TT staining with di-8-ANEPPS (left) and RyR staining (right) after 48 hours of culture. B. Representative line scan image of Ca²⁺ spark protocol in CULT. The type of release areas is marked in blue and red for early and delayed release areas respectively. C. Comparison of spark duration in early (left) and delayed (right) release areas in CTRL (N_pigs = 7, n_cells = 29) vs. CULT (N_pigs = 7, n_cells = 14). D. Fraction of cells showing long-lasting sparks (>47.6 ms) in CTRL (17/35 cells with long sparks) and CULT (10/15 cells with long sparks). E. NCX current density in CTRL (N_pigs = 4, n_cells = 14) and CULT (N_pigs = 4, n_cells = 7). * denotes P<0.05.</p

Effect of remodeling in the area adjacent to MI on Ca²⁺ sparks.

<p>A. Representative line scan image of Ca²⁺ spark recording in MI. The types of release areas are marked in blue and red for early and delayed release areas respectively. The artifact region (marked by –) was excluded for analysis. B. Whole-cell spark frequency and morphology in CTRL (N_pigs = 12, n_cells = 41) and MI (N_pigs = 7, n_cells = 33). C. Spark mass, calculated by amplitude*width*duration, and spark-mediated Ca²⁺ leak, calculated by spark frequency*spark mass, in CTRL (N_pigs = 12, n_cells = 41) and MI (N_pigs = 7, n_cells = 33). D. SR Ca²⁺ content as µmoles/L accessible cytosol, in CTRL (N_pigs = 4, n_cells = 15) and MI (N_pigs = 4, n_cells = 12). * denotes P<0.05.</p