Sarcoplasmic Reticulum Calcium Release Is Required for Arrhythmogenesis in the Mouse.

Dysfunctional sarcoplasmic reticulum Ca2+ handling is commonly observed in heart failure, and thought to contribute to arrhythmogenesis through several mechanisms. Some time ago we developed a cardiomyocyte-specific inducible SERCA2 knockout mouse, which is remarkable in the degree to which major adaptations to sarcolemmal Ca2+ entry and efflux overcome the deficit in SR reuptake to permit relatively normal contractile function. Conventionally, those adaptations would also be expected to dramatically increase arrhythmia susceptibility. However, that susceptibility has never been tested, and it is possible that the very rapid repolarization of the murine action potential (AP) allows for large changes in sarcolemmal Ca2+ transport without substantially disrupting electrophysiologic stability. We investigated this hypothesis through telemetric ECG recording in the SERCA2-KO mouse, and patch-clamp electrophysiology, Ca2+ imaging, and mathematical modeling of isolated SERCA2-KO myocytes. While the SERCA2-KO animals exhibit major (and unique) electrophysiologic adaptations at both the organ and cell levels, they remain resistant to arrhythmia. A marked increase in peak L-type calcium (I CaL) current and slowed I CaL decay elicited pronounced prolongation of initial repolarization, but faster late repolarization normalizes overall AP duration. Early afterdepolarizations were seldom observed in KO animals, and those that were observed exhibited a mechanism intermediate between murine and large mammal dynamical properties. As expected, spontaneous SR Ca2+ sparks and waves were virtually absent. Together these findings suggest that intact SR Ca2+ handling is an absolute requirement for triggered arrhythmia in the mouse, and that in its absence, dramatic changes to the major inward currents can be resisted by the substantial K+ current reserve, even at end-stage disease.

Slow Ca²âº sparks de-synchronize Ca²âº release in failing cardiomyocytes: evidence for altered configuration of Ca²âº release units?

In heart failure, cardiomyocytes exhibit slowing of the rising phase of the Ca(2+) transient which contributes to the impaired contractility observed in this condition. We investigated whether alterations in ryanodine receptor function promote slowing of Ca(2+) release in a murine model of congestive heart failure (CHF). Myocardial infarction was induced by left coronary artery ligation. When chronic CHF had developed (10 weeks post-infarction), cardiomyocytes were isolated from viable regions of the septum. Septal myocytes from SHAM-operated mice served as controls. Ca(2+) transients rose markedly slower in CHF than SHAM myocytes with longer time to peak (CHF=152 ± 12% of SHAM, P<0.05). The rise time of Ca(2+) sparks was also increased in CHF (SHAM=9.6 ± 0.6 ms, CHF=13.2 ± 0.7 ms, P<0.05), due to a sub-population of sparks (≈20%) with markedly slowed kinetics. Regions of the cell associated with these slow spontaneous sparks also exhibited slowed Ca(2+) release during the action potential. Thus, greater variability in spark kinetics in CHF promoted less uniform Ca(2+) release across the cell. Dyssynchronous Ca(2+) transients in CHF additionally resulted from T-tubule disorganization, as indicated by fast Fourier transforms, but slow sparks were not associated with orphaned ryanodine receptors. Rather, mathematical modeling suggested that slow sparks could result from an altered composition of Ca(2+) release units, including a reduction in ryanodine receptor density and/or distribution of ryanodine receptors into sub-clusters. In conclusion, our findings indicate that slowed, dyssynchronous Ca(2+) transients in CHF result from alterations in Ca(2+) sparks, consistent with rearrangement of ryanodine receptors within Ca(2+) release units.

Control of Ca2+ release by action potential configuration in normal and failing murine cardiomyocytes.

Cardiomyocytes from failing hearts exhibit spatially nonuniform or dyssynchronous sarcoplasmic reticulum (SR) Ca(2+) release. We investigated the contribution of action potential (AP) prolongation in mice with congestive heart failure (CHF) after myocardial infarction. AP recordings from CHF and control myocytes were included in a computational model of the dyad, which predicted more dyssynchronous ryanodine receptor opening during stimulation with the CHF AP. This prediction was confirmed in cardiomyocyte experiments, when cells were alternately stimulated by control and CHF AP voltage-clamp waveforms. However, when a train of like APs was used as the voltage stimulus, the control and CHF AP produced a similar Ca(2+) release pattern. In this steady-state condition, greater integrated Ca(2+) entry during the CHF AP lead to increased SR Ca(2+) content. A resulting increase in ryanodine receptor sensitivity synchronized SR Ca(2+) release in the mathematical model, thus offsetting the desynchronizing effects of reduced driving force for Ca(2+) entry. A modest nondyssynchronous prolongation of Ca(2+) release was nevertheless observed during the steady-state CHF AP, which contributed to increased time-to-peak measurements for Ca(2+) transients in failing cells. Thus, dyssynchronous Ca(2+) release in failing mouse myocytes does not result from electrical remodeling, but rather other alterations such as T-tubule reorganization.

Tamoxifen administration routes and dosage for inducible Cre-mediated gene disruption in mouse hearts.

Tissue-specific and time-dependent control of in vivo gene disruption may be achieved using conditional knockout strategies in transgenic mice. Fusion of mutant estrogen receptor ligand-binding domains to Cre recombinase (Cre-ER(T), MerCreMer) combined with cardiac-directed gene expression has been used to generate several cardiac-specific tamoxifen-inducible Cre-expressing mouse lines. Such mice have successfully been used to generate Cre-loxP-mediated gene disruption in an inducible manner in the myocardium in vivo. However, information is sparse regarding the tamoxifen dosage, the time course of gene disruption and whether different administration routes differ in efficiency in obtaining gene disruption in the myocardium. We have evaluated these parameters in Serca2 ( flox/flox ) Tg(alphaMHC-MerCreMer) transgenic mice (SERCA2 KO). Serca2 mRNA transcript abundance was used as a sensitive indicator of Cre-loxP-dependent gene disruption in the myocardium. We found that 2 i.p. injections of tamoxifen in oil (1 mg/day, approximate total dose 80 mg/kg) was sufficient for efficient gene disruption with maximal reduction of Serca2 mRNA as early as 4 days after tamoxifen induction. Moreover, a simple protocol using tamoxifen-supplemented non-pelleted dry feed p.o. was comparable to i.p. injections in inducing gene disruption. These improvements may significantly improve animal welfare and reduce the workload in the production of cardiac conditional knockout mice.

Sodium accumulation promotes diastolic dysfunction in end-stage heart failure following Serca2 knockout.

Alterations in trans-sarcolemmal and sarcoplasmic reticulum (SR) Ca(2+) fluxes may contribute to impaired cardiomyocyte contraction and relaxation in heart failure. We investigated the mechanisms underlying heart failure progression in mice with conditional, cardiomyocyte-specific excision of the SR Ca(2+)-ATPase (SERCA) gene. At 4 weeks following gene deletion (4-week KO) cardiac function remained near normal values. However, end-stage heart failure developed by 7 weeks (7-week KO) as systolic and diastolic performance declined. Contractions in isolated myocytes were reduced between 4- and 7-week KO, and relaxation was slowed. Ca(2+) transients were similarly altered. Reduction in Ca(2+) transient magnitude resulted from complete loss of SR Ca(2+) release between 4- and 7-week KO, due to loss of a small remaining pool of SERCA2. Declining SR Ca(2+) release was partly offset by increased L-type Ca(2+) current, which was facilitated by AP prolongation in 7-week KO. Ca(2+) entry via reverse-mode Na(+)-Ca(2+) exchange (NCX) was also enhanced. Up-regulation of NCX and plasma membrane Ca(2+)-ATPase increased Ca(2+) extrusion rates in 4-week KO. Diastolic dysfunction in 7-week KO resulted from further SERCA2 loss, but also impaired NCX-mediated Ca(2+) extrusion following Na(+) accumulation. Reduced Na(+)-K(+)-ATPase activity contributed to the Na(+) gain. Normalizing [Na(+)] by dialysis increased the Ca(2+) decline rate in 7-week KO beyond 4-week values. Thus, while SERCA2 loss promotes both systolic and diastolic dysfunction, Na(+) accumulation additionally impairs relaxation in this model. Our observations indicate that if cytosolic Na(+) gain is prevented, up-regulated Ca(2+) extrusion mechanisms can maintain near-normal diastolic function in the absence of SERCA2.

Slowing of cardiomyocyte Ca2+ release and contraction during heart failure progression in postinfarction mice.

Deterioration of cardiac contractility during congestive heart failure (CHF) is believed to involve decreased function of individual cardiomyocytes and may include reductions in contraction magnitude and/or kinetics. We examined the progression of in vivo and in vitro alterations in contractile function in CHF mice and investigated underlying alterations in Ca(2+) homeostasis. Following induction of myocardial infarction (MI), mice with CHF were examined at early (1 wk post-MI) and chronic (10 wk post-MI) stages of disease development. Sham-operated mice served as controls. Global and local left ventricle function were assessed by echocardiography in sedated animals ( approximately 2% isoflurane). Excitation-contraction coupling was examined in cardiomyocytes isolated from the viable septum. CHF progression between 1 and 10 wk post-MI resulted in increased mortality, development of hypertrophy, and deterioration of global left ventricular function. Local function in the noninfarcted myocardium also declined, as posterior wall shortening velocity was reduced in chronic CHF (1.2 +/- 0.1 vs. 1.9 +/- 0.2 cm/s in sham). Parallel alterations occurred in isolated cardiomyocytes since contraction and Ca(2+) transient time to peak values were prolonged in chronic CHF (115 +/- 6 and 158 +/- 11% sham values, respectively). Surprisingly, contraction and Ca(2+) transient magnitudes in CHF were larger than sham values at both time points, resulting from increased sarcoplasmic reticulum Ca(2+) content and greater Ca(2+) influx via L-type channels. We conclude that, in mice with CHF following myocardial infarction, declining myocardial function involves slowing of cardiomyocyte contraction without reduction in contraction magnitude. Corresponding alterations in Ca(2+) transients suggest that slowing of Ca(2+) release is a critical mediator of CHF progression.

Cardiomyocytes from postinfarction failing rat hearts have improved ischemia tolerance.

Altered myocardial Ca(2+) and Na(+) handling in congestive heart failure (CHF) may be expected to decrease the tolerance to ischemia by augmenting reperfusion Ca(2+) overload. The aim of the present study was to investigate tolerance to hypoxia-reoxygenation by measuring enzyme release, cell death, ATP level, and cell Ca(2+) and Na(+) in cardiomyocytes from failing rat hearts. CHF was induced in Wistar rats by ligation of the left coronary artery during isoflurane anesthesia, after which cardiac failure developed within 6 wk. Isolated cardiomyocytes were cultured for 24 h and subsequently exposed to 4 h of hypoxia and 2 h of reoxygenation. Cell damage was measured as lactate dehydrogenase (LD) release, cell death as propidium iodide uptake, and ATP by firefly luciferase assay. Cell Ca(2+) and Na(+) were determined with radioactive isotopes, and free intracellular Ca(2+) concentration ([Ca(2+)](i)) with fluo-3 AM. CHF cells showed less increase in LD release and cell death after hypoxia-reoxygenation and had less relative reduction in ATP level after hypoxia than sham cells. CHF cells accumulated less Na(+) than sham cells during hypoxia (117 vs. 267 nmol/mg protein). CHF cells maintained much lower [Ca(2+)](i) than sham cells during hypoxia (423 vs. 1,766 arbitrary units at 4 h of hypoxia), and exchangeable Ca(2+) increased much less in CHF than in sham cells (1.4 vs. 6.7 nmol/mg protein) after 120 min of reoxygenation. Ranolazine, an inhibitor of late Na(+) current, significantly attenuated both the increase in exchangeable Ca(2+) and the increase in LD release in sham cells after reoxygenation. This supports the suggestion that differences in Na(+) accumulation during hypoxia cause the observed differences in Ca(2+) accumulation during reoxygenation. Tolerance to hypoxia and reoxygenation was surprisingly higher in CHF than in sham cardiomyocytes, probably explained by lower hypoxia-mediated Na(+) accumulation and subsequent lower Ca(2+) accumulation in CHF after reoxygenation.

Increased cardiomyocyte function and Ca2+ transients in mice during early congestive heart failure.

End-stage heart failure is believed to involve depressed cardiomyocyte contractility and Ca2+ transients. However, the time course of these alterations is poorly understood. We examined alterations in myocyte excitation-contraction coupling in a mouse model of early congestive heart failure (CHF) following myocardial infarction. One week after myocardial infarction was induced by ligation of the left coronary artery, CHF mice were selected based on established criteria (increased left atrial diameter, increased lung weight). Sham-operated animals (SHAM) served as controls. Echocardiographic measurements showed decreased global function in early CHF relative to SHAM, but increased local function in viable regions of the myocardium which deteriorated with time. Cardiomyocytes isolated from the non-infarcted septum also exhibited larger contractions in early CHF than SHAM (CHF=219.6+/-15.3% of SHAM values, P<0.05; 1 Hz field stimulation), and relaxation was more rapid (time to 50% relaxation=82.9+/-5.5% of SHAM values, P<0.05). Ca2+ transients (fluo-4 AM) were larger and decayed more rapidly in CHF than SHAM during both field stimulation (1 Hz) and voltage-clamp steps. Sarcoplasmic reticulum (SR) Ca2+ content was increased. Western blots showed that while SR Ca2+ ATPase (SERCA) expression was unaltered in CHF, phospholamban (PLB) was downregulated (60+/-11% of SHAM values, P<0.05). Thus, an increased SERCA/PLB ratio in CHF may promote SR Ca2+ re-uptake. Additionally, peak L-type Ca2+ current and Na+/Ca2+ exchanger expression were increased in CHF, suggesting increased sarcolemmal Ca2+ flux. Thus, in early CHF, alterations in Ca2+ homeostasis improve cardiomyocyte contractility which may compensate for loss of function in the infarction area.

Contribution of the Na+/Ca2+ exchanger to rapid Ca2+ release in cardiomyocytes.

Trigger Ca(2+) is considered to be the Ca(2+) current through the L-type Ca(2+) channel (LTCC) that causes release of Ca(2+) from the sarcoplasmic reticulum. However, cell contraction also occurs in the absence of the LTCC current (I(Ca)). In this article, we investigate the contribution of the Na(+)/Ca(2+) exchanger (NCX) to the trigger Ca(2+). Experimental data from rat cardiomyocytes using confocal microscopy indicating that inhibition of reverse mode Na(+)/Ca(2+) exchange delays the Ca(2+) transient by 3-4 ms served as a basis for the mathematical model. A detailed computational model of the dyadic cleft (fuzzy space) is presented where the diffusion of both Na(+) and Ca(2+) is taken into account. Ionic channels are included at discrete locations, making it possible to study the effect of channel position and colocalization. The simulations indicate that if a Na(+) channel is present in the fuzzy space, the NCX is able to bring enough Ca(2+) into the cell to affect the timing of release. However, this critically depends on channel placement and local diffusion properties. With fuzzy space diffusion in the order of four orders of magnitude lower than in water, triggering through LTCC alone was up to 5 ms slower than with the presence of a Na(+) channel and NCX.

T-tubule disorganization and reduced synchrony of Ca2+ release in murine cardiomyocytes following myocardial infarction.

In cardiac myocytes, initiation of excitation-contraction coupling is highly localized near the T-tubule network. Myocytes with a dense T-tubule network exhibit rapid and homogeneous sarcoplasmic reticulum (SR) Ca(2+) release throughout the cell. We examined whether progressive changes in T-tubule organization and Ca(2+) release synchrony occur in a murine model of congestive heart failure (CHF). Myocardial infarction (MI) was induced by ligation of the left coronary artery, and CHF was diagnosed by echocardiography (left atrial diameter >2.0 mm). CHF mice were killed at 1 or 3 weeks following MI (1-week CHF, 3-week CHF) and cardiomyocytes were isolated from viable regions of the septum, excluding the MI border zone. Septal myocytes from SHAM-operated mice served as controls. T-tubules were visualized by confocal microscopy in cells stained with di-8-ANEPPS. SHAM cells exhibited a regular striated T-tubule pattern. However, 1-week CHF cells showed slightly disorganized T-tubule structure, and more profound disorganization occurred in 3-week CHF with irregular gaps between adjacent T-tubules. Line-scan images of Ca(2+) transients (fluo-4 AM, 1 Hz) showed that regions of delayed Ca(2+) release occurred at these gaps. Three-week CHF cells exhibited an increased number of delayed release regions, and increased overall dyssynchrony of Ca(2+) release. A common pattern of Ca(2+) release in 3-week CHF was maintained between consecutive transients, and was not altered by forskolin application. Thus, progressive T-tubule disorganization during CHF promotes dyssynchrony of SR Ca(2+) release which may contribute to the slowing of SR Ca(2+) release in this condition.