Halvor K. Mørk

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) Ca2+ release throughout the cell. We examined whether progressive changes in T‐tubule organization and Ca2+ 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 Ca2+ transients (fluo‐4 AM, 1 Hz) showed that regions of delayed Ca2+ release occurred at these gaps. Three‐week CHF cells exhibited an increased number of delayed release regions, and increased overall dyssynchrony of Ca2+ release. A common pattern of Ca2+ 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 Ca2+ release which may contribute to the slowing of SR Ca2+ release in this condition.

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

Alterations in trans‐sarcolemmal and sarcoplasmic reticulum (SR) Ca2+ 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 Ca2+‐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. Ca2+ transients were similarly altered. Reduction in Ca2+ transient magnitude resulted from complete loss of SR Ca2+ release between 4‐ and 7‐week KO, due to loss of a small remaining pool of SERCA2. Declining SR Ca2+ release was partly offset by increased L‐type Ca2+ current, which was facilitated by AP prolongation in 7‐week KO. Ca2+ entry via reverse‐mode Na+–Ca2+ exchange (NCX) was also enhanced. Up‐regulation of NCX and plasma membrane Ca2+‐ATPase increased Ca2+ extrusion rates in 4‐week KO. Diastolic dysfunction in 7‐week KO resulted from further SERCA2 loss, but also impaired NCX‐mediated Ca2+ extrusion following Na+ accumulation. Reduced Na+‐K+‐ATPase activity contributed to the Na+ gain. Normalizing [Na+] by dialysis increased the Ca2+ 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 Ca2+ extrusion mechanisms can maintain near‐normal diastolic function in the absence of SERCA2.

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.

Slow Ca2 + sparks de-synchronize Ca2 + release in failing cardiomyocytes: Evidence for altered configuration of Ca2 + 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.

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.

5-HT4-elicited positive inotropic response is mediated by cAMP and regulated by PDE3 in failing rat and human cardiac ventricles

The left ventricle in failing hearts becomes sensitive to 5‐HT parallelled by appearance of functional Gs‐coupled 5‐HT4 receptors. Here, we have explored the regulatory functions of phosphodiesterases in the 5‐HT4 receptor‐mediated functional effects in ventricular muscle from failing rat and human heart.

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.

Cyclic AMP‐dependent inotropic effects are differentially regulated by muscarinic Gi‐dependent constitutive inhibition of adenylyl cyclase in failing rat ventricle

BACKGROUND AND PURPOSE β‐Adrenoceptor (β‐AR)‐mediated inotropic effects are attenuated and Gi proteins are up‐regulated in heart failure (HF). Muscarinic receptors constitutively inhibit cAMP formation in normal rat cardiomyocytes. We determined whether constitutive activity of muscarinic receptors to inhibit adenylyl cyclase (AC) increases in HF and if so, whether it modifies the reduced β‐AR‐ or emergent 5‐HT4‐mediated cAMP‐dependent inotropic effects.

SERCA2 Knockout Mice Exhibit Impaired Control of Ca2+ Current but not Ventricular Arrhythmias

Impaired Ca2+ handling by the sarcoplasmic reticulum (SR) and Ca2+-dependent arrhythmias are hallmark features of human heart failure. We investigated the control of L-type Ca2+ current (ICa, L) when SR function is reduced and the consequences for arrhythmogenesis. Experiments were performed on cardiomyocytes isolated from conditional SERCA2 KO mice (KO) which had developed heart failure 7 weeks following gene disruption. SERCA2flox/flox (FF) mice served as controls. SR Ca2+ content was reduced to 4% (P<0.05) of FF values in KO cardiomyocytes, and SR Ca2+ release did not occur on a beat-to-beat basis. Marked up-regulation of the L-type Ca2+ channel in KO (α1C subunit= 178% FF, α2/δ1= 147% FF, P<0.05) was accompanied by a 40% increase in peak ICa, L (P<0.05). Loss of SR function resulted in slower Ca2+ current inactivation, prolonged duration of current activation, and loss of frequency-dependent facilitation. The larger magnitude and prolonged ICa,L in KO resulted in AP prolongation, which was not observed in the presence of nifedipine or upon removal of extracellular Ca2+. AP prolongation was associated with prolonged QT intervals corrected for heart rate in KO mice compared to FF (5.63 ms vs 4.90 ms, P<0.05). While AP prolongation is expected to be arrhythmogenic, incidence of early after-depolarizations in KO cardiomyocytes was not increased (FF= 2/13 cells, KO= 0/10 cells, P=NS). Telemetric ECG surveillance during pharmacological stress also revealed a similar incidence of ventricular arrhythmias in FF and KO mice. In conclusion, loss of SR function results in greater L-type Ca2+ entry, loss of Ca2+-dependent inactivation, and prolonged APs and QT interval. While such alterations would be expected to pro-arrhythmic in larger species, the relatively brief AP in failing mice may preclude occurrence of early after-depolarizations.

Myocardial infarction in mice induces hypertrophy and heart failure without reduced contraction of viable myocytes

were subjected to either pressure-overload (ascending aorticconstriction to induce left ventricular hypertension and pathological hypertrophy) or the sham operation for 1 week. Exercise-induced cardiac hypertrophy had a favourable effect on cardiac function in the pressure-overload model, attenuated pathological growth and inhibited fibrosis. Furthermore, markers of pathological hypertrophy were significantly lower in aortic-banded hearts from exercise trained mice than untrained mice. Conclusions: Exercise-induced cardiac hypertrophy provided sustained protection in a setting of pressure overload via beneficial effects on cardiac growth, fibrosis and molecular markers of pathological hypertrophy. Examination of the “preconditioning” capacity of regular physical activity/physiological hypertrophy and the molecular mechanisms responsible for this phenomenon could provide new strategies to protect the heart form pathological insults.