Intracellular dyssynchrony of diastolic cytosolic [Ca²âº] decay in ventricular cardiomyocytes in cardiac remodeling and human heart failure.

RATIONALE: Synchronized release of Ca²âº into the cytosol during each cardiac cycle determines cardiomyocyte contraction. OBJECTIVE: We investigated synchrony of cytosolic [Ca²âº] decay during diastole and the impact of cardiac remodeling. METHODS AND RESULTS: Local cytosolic [Ca²âº] transients (1-µm intervals) were recorded in murine, porcine, and human ventricular single cardiomyocytes. We identified intracellular regions of slow (slowCaR) and fast (fastCaR) [Ca²âº] decay based on the local time constants of decay (TAUlocal). The SD of TAUlocal as a measure of dyssynchrony was not related to the amplitude or the timing of local Ca²âº release. Stimulation of sarcoplasmic reticulum Ca²âº ATPase with forskolin or istaroxime accelerated and its inhibition with cyclopiazonic acid slowed TAUlocal significantly more in slowCaR, thus altering the relationship between SD of TAUlocal and global [Ca²âº] decay (TAUglobal). Na⁺/Ca²âº exchanger inhibitor SEA0400 prolonged TAUlocal similarly in slowCaR and fastCaR. FastCaR were associated with increased mitochondrial density and were more sensitive to the mitochondrial Ca²âº uniporter blocker Ru360. Variation in TAUlocal was higher in pig and human cardiomyocytes and higher with increased stimulation frequency (2 Hz). TAUlocal correlated with local sarcomere relengthening. In mice with myocardial hypertrophy after transverse aortic constriction, in pigs with chronic myocardial ischemia, and in end-stage human heart failure, variation in TAUlocal was increased and related to cardiomyocyte hypertrophy and increased mitochondrial density. CONCLUSIONS: In cardiomyocytes, cytosolic [Ca²âº] decay is regulated locally and related to local sarcomere relengthening. Dyssynchronous intracellular [Ca²âº] decay in cardiac remodeling and end-stage heart failure suggests a novel mechanism of cellular contractile dysfunction.

FKBP12.6 overexpression does not protect against remodelling after myocardial infarction.

Reducing the open probability of the ryanodine receptor (RyR) has been proposed to have beneficial effects in heart failure. We investigated whether conditional FKBP12.6 overexpression at the time of myocardial infarction (MI) could improve cardiac remodelling and cell Ca(2+) handling. Wild-type (WT) mice and mice overexpressing FKBP12.6 (Tg) were studied on average 7.5 ± 0.2 weeks after MI and compared with sham-operated mice for in vivo, myocyte function and remodelling. At baseline, unloaded cell shortening in Tg was not different from WT. The [Ca(2+)](i) transient amplitude was similar, but sarcoplasmic reticulum (SR) Ca(2+) content was larger in Tg, suggesting reduced fractional release. Spontaneous spark frequency was similar despite the increased SR Ca(2+) content, consistent with a reduced RyR channel open probability in Tg. After MI, left ventricular dilatation and myocyte hypertrophy were present in both groups, but more pronounced in Tg. Cell shortening amplitude was unchanged with MI in WT, but increased with MI in Tg. The amplitude of the [Ca(2+)](i) transient was not affected by MI in either genotype, but time to peak was increased; this was most pronounced in Tg. The SR Ca(2+) content and Na(+)- Ca(2+) exchanger function were not affected by MI. Spontaneous spark frequency was increased significantly after MI in Tg, and larger than in WT (at 4 Hz, 2.6 ± 0.4 sparks (100 µm)(-1) s(-1) in Tg MI versus 1.6 ± 0.2 sparks (100 µm)(-1) s(-1) in WT MI; P < 0.05). We conclude that FKPB12.6 overexpression can effectively reduce RyR open probability with maintained cardiomyocyte contraction. However, this approach appears insufficient to prevent and reduce post-MI remodelling, indicating that additional pathways may need to be targeted.

Cardiac FKBP12.6 overexpression protects against triggered ventricular tachycardia in pressure overloaded mouse hearts.

Alterations in RyR2 function have been proposed as a major pathophysiological mechanism of arrhythmias and heart failure (HF). Cardiac FKBP12.6 overexpression protects against myocardial infarction-induced HF and catecholamine-promoted ventricular arrhythmias. We tested the hypothesis that FKBP12.6 overexpression protects against maladaptive LVH and triggered ventricular arrhythmias following transverse aorta constriction (TAC) in the mouse. The TAC-associated mortality rate was significantly lower in male transgenic (DT) than in Ctr mice (p < 0.05). TAC-associated maladaptive hypertrophy was blunted in DT mice especially 1 month post-TAC and their SERCA2a/PLB ratio remained unchanged 1 and 2 months post-TAC. Two months after TAC, trains of 30 stimuli (burst pacing) performed following isoproterenol injection (0.2 mg/kg, ip), induced VT in 50% of the TAC-Ctr and in none of the TAC-DT mice (p = 0.022). The increase in myocyte shortening and Ca(2+) spark frequency observed in sham-operated Ctr mice in response to 50 nM isoproterenol was reduced in DT mice, and abolished in TAC-DT mice. NCX1 function was reduced in Sham-DT and TAC-DT compared with Sham-Ctr and TAC-Ctr mice, respectively (p < 0.05 for the 2 comparisons). In mice killed after isoproterenol injection and burst pacing, RyR2 S2814 phosphorylation was decreased by 50% in TAC-DT versus TAC-Ctr mice (p < 0.05), with no change in RyR2 S2808 and PLB S16 and T17 phosphorylation. Cardiac FKBP12.6 overexpression in the mouse blunts pressure overload-induced maladaptive LV remodelling and protects against catecholamine-promoted burst pacing-induced ventricular tachycardia by decreasing cardiac sensitivity to adrenergic stress and RyR2 S2814 phosphorylation, and decreasing NCX1 activity.

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). Ca(2+) transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca(2+) release, F>F(50) within 20 ms) or their absence (delayed areas). Spontaneous Ca(2+) release events during diastole, Ca(2+) 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(+)/Ca(2+) exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca(2+) influx via Ca(2+) channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca(2+) 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; Ca(2+) 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 Ca(2+) loss and raise SR Ca(2+) content, but may promote Ca(2+) waves.

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.

Early exercise training after myocardial infarction prevents contractile but not electrical remodelling or hypertrophy.

AIMS: Exercise started early after myocardial infarction (MI) improves in vivo cardiac function and myofilament responsiveness to Ca(2+). We investigated whether this represents partial or complete reversal of cellular remodelling. METHODS AND RESULTS: Mice with MI following left coronary ligation were given free access to a running wheel (MI(EXE), N = 22) or housed sedentary (MI(SED), N = 18) for 8 weeks and compared with sedentary sham-operated animals (SHAM, N = 11). Myocytes were enzymatically isolated from the non-infarcted left ventricle. Myocytes in MI were significantly longer and even more so with exercise (165 +/- 3 microm in MI(EXE) vs. 148 +/- 3 microm in MI(SED) and 136 +/- 2 microm in SHAM; P < 0.05, mean +/- SEM); cell width was not different. Contraction was measured during electrical field stimulation at 1, 2, and 4 Hz. Unloaded cell shortening was significantly reduced in MI(SED) (at 1 Hz, L/L(0)=4.4 +/- 0.3% vs. 6.7 +/- 0.4% in SHAM; P < 0.05, also at 2 and 4 Hz). Exercise restored cell shortening to SHAM values (MI(EXE), L/L(0)=6.4 +/- 0.5%). Membrane currents and [Ca(2+)](i) were measured via whole-cell patch clamping, with Fluo-3 as Ca(2+) indicator, all at 30 degrees C. Ca(2+) transient amplitude, I(CaL) and sarcoplasmic reticulum Ca(2+) content were not different between the three groups. Diastolic Ca(2+) levels at 4 Hz were significantly elevated in MI(SED) only, with a trend to increased spontaneous Ca(2+) release events (sparks). Action potential duration was increased and transient outward K(+) currents significantly reduced after MI; this was unaffected by exercise. CONCLUSIONS: Early voluntary exercise training after MI restores cell contraction to normal values predominantly because of changes in the myofilament Ca(2+) response and has a beneficial effect on diastolic Ca(2+) handling. However, the beneficial effect is not a complete reversal of remodelling as hypertrophy and loss of repolarizing K(+) currents are not affected.

Ventricular phosphodiesterase-5 expression is increased in patients with advanced heart failure and contributes to adverse ventricular remodeling after myocardial infarction in mice.

BACKGROUND: Ventricular expression of phosphodiesterase-5 (PDE5), an enzyme responsible for cGMP catabolism, is increased in human right ventricular hypertrophy, but its role in left ventricular (LV) failure remains incompletely understood. We therefore measured LV PDE5 expression in patients with advanced systolic heart failure and characterized LV remodeling after myocardial infarction in transgenic mice with cardiomyocyte-specific overexpression of PDE5 (PDE5-TG). METHODS AND RESULTS: Immunoblot and immunohistochemistry techniques revealed that PDE5 expression was greater in explanted LVs from patients with dilated and ischemic cardiomyopathy than in control hearts. To evaluate the impact of increased ventricular PDE5 levels on cardiac function, PDE5-TG mice were generated. Confocal and immunoelectron microscopy revealed increased PDE5 expression in cardiomyocytes, predominantly localized to Z-bands. At baseline, myocardial cGMP levels, cell shortening, and calcium handling in isolated cardiomyocytes and LV hemodynamic measurements were similar in PDE5-TG and wild-type littermates. Ten days after myocardial infarction, LV cGMP levels had increased to a greater extent in wild-type mice than in PDE5-TG mice (P<0.05). Ten weeks after myocardial infarction, LV end-systolic and end-diastolic volumes were larger in PDE5-TG than in wild-type mice (57+/-5 versus 39+/-4 and 65+/-6 versus 48+/-4 muL, respectively; P<0.01 for both). LV systolic dysfunction and diastolic dysfunction were more marked in PDE5-TG than in wild-type mice, associated with enhanced hypertrophy and reduced contractile function in isolated cardiomyocytes from remote myocardium. CONCLUSIONS: Increased PDE5 expression predisposes mice to adverse LV remodeling after myocardial infarction. Increased myocardial PDE5 expression in patients with advanced cardiomyopathy may contribute to the development of heart failure and represents an important therapeutic target.

Remodeling of T-tubules and reduced synchrony of Ca2+ release in myocytes from chronically ischemic myocardium.

In ventricular cardiac myocytes, T-tubule density is an important determinant of the synchrony of sarcoplasmic reticulum (SR) Ca2+ release and could be involved in the reduced SR Ca2+ release in ischemic cardiomyopathy. We therefore investigated T-tubule density and properties of SR Ca2+ release in pigs, 6 weeks after inducing severe stenosis of the circumflex coronary artery (91+/-3%, N=13) with myocardial infarction (8.8+/-2.0% of total left ventricular mass). Severe dysfunction in the infarct and adjacent myocardium was documented by magnetic resonance and Doppler myocardial velocity imaging. Myocytes isolated from the adjacent myocardium were compared with myocytes from the same region in weight-matched control pigs. T-tubule density quantified from the di-8-ANEPPS (di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate) sarcolemmal staining was decreased by 27+/-7% (P<0.05). Synchrony of SR Ca2+ release (confocal line scan images during whole-cell voltage clamp) was reduced in myocardium myocytes. Delayed release (ie, half-maximal [Ca2+]i occurring later than 20 ms) occurred at 35.5+/-6.4% of the scan line in myocardial infarction versus 22.7+/-2.5% in control pigs (P<0.05), prolonging the time to peak of the line-averaged [Ca2+]i transient (121+/-9 versus 102+/-5 ms in control pigs, P<0.05). Delayed release colocalized with regions of T-tubule rarefaction and could not be suppressed by activation of protein kinase A. The whole-cell averaged [Ca2+]i transient amplitude was reduced, whereas L-type Ca2+ current density was unchanged and SR content was increased, indicating a reduction in the gain of Ca2+-induced Ca2+ release. In conclusion, reduced T-tubule density during ischemic remodeling is associated with reduced synchrony of Ca2+ release and reduced efficiency of coupling Ca2+ influx to Ca2+ release.

Crosstalk between L-type Ca2+ channels and the sarcoplasmic reticulum: alterations during cardiac remodelling.

In the cardiac dyad, sarcolemmal L-type Ca(2+) channels (LCCs) and sarcoplasmic reticulum (SR) Ca(2+) release channels (RyR) are structurally in close proximity. This organization provides for an efficient functional coupling, tuning SR Ca(2+) release for optimal contraction of the myocyte. Given that LCC are regulated by the prevailing [Ca(2+)], this structural organization is the setting for feedback mechanisms and crosstalk. A defective coupling of Ca(2+) influx via LCC to activation of RyR has been implicated in reduced SR Ca(2+) release in heart failure. Both functional changes in LCC properties and structural re-organization of LCC in T-tubules could be involved. LCC are regulated by cytosolic Ca(2+), and crosstalk with SR Ca(2+) handling occurs on a long-term basis, i.e. during steady-state changes in heart rate, on an intermediate-term basis, i.e. on a beat-to-beat basis during sudden rate changes, and on a very short- or immediate-term basis, i.e. during a single heartbeat. We review the properties and consequences of these different feedback mechanisms and the changes in heart failure and cardiac hypertrophy that have thus far been studied.

Early exercise training normalizes myofilament function and attenuates left ventricular pump dysfunction in mice with a large myocardial infarction.

The extent and mechanism of the cardiac benefit of early exercise training following myocardial infarction (MI) is incompletely understood, but may involve blunting of abnormalities in Ca(2+)-handling and myofilament function. Consequently, we investigated the effects of 8-weeks of voluntary exercise, started early after a large MI, on left ventricular (LV) remodeling and dysfunction in the mouse. Exercise had no effect on survival, MI size or LV dimensions, but improved LV fractional shortening from 8+/-1 to 12+/-1%, and LVdP/dt(P30) from 5295+/-207 to 5794+/-207 mm Hg/s (both P<0.05), and reduced pulmonary congestion. These global effects of exercise were associated with normalization of the MI-induced increase in myofilament Ca(2+)-sensitivity (DeltapCa(50)=0.037). This effect of exercise was PKA-mediated and likely because of improved beta(1)-adrenergic signaling, as suggested by the increased beta(1)-adrenoceptor protein (48%) and cAMP levels (36%; all P<0.05). Exercise prevented the MI-induced decreased maximum force generating capacity of skinned cardiomyocytes (F(max) increased from 14.3+/-0.7 to 18.3+/-0.8 kN/m(2) P<0.05), which was associated with enhanced shortening of unloaded intact cardiomyocytes (from 4.1+/-0.3 to 7.0+/-0.6%; P<0.05). Furthermore, exercise reduced diastolic Ca(2+)-concentrations (by approximately 30%, P<0.05) despite the unchanged SERCA2a and PLB expression and PLB phosphorylation status. Importantly, exercise had no effect on Ca(2+)-transient amplitude, indicating that the improved LV and cardiomyocyte shortening were principally because of improved myofilament function. In conclusion, early exercise in mice after a large MI has no effect on LV remodeling, but attenuates global LV dysfunction. The latter can be explained by the exercise-induced improvement of myofilament function.