Pierre Coutu

Calcium-Handling Abnormalities Underlying Atrial Arrhythmogenesis and Contractile Dysfunction in Dogs With Congestive Heart Failure

Background—Congestive heart failure (CHF) is a common cause of atrial fibrillation. Focal sources of unknown mechanism have been described in CHF-related atrial fibrillation. The authors hypothesized that abnormal calcium (Ca2+) handling contributes to the CHF-related atrial arrhythmogenic substrate. Methods and Results—CHF was induced in dogs by ventricular tachypacing (240 bpm ×2 weeks). Cellular Ca2+-handling properties and expression/phosphorylation status of key Ca2+ handling and myofilament proteins were assessed in control and CHF atria. CHF decreased cell shortening but increased left atrial diastolic intracellular Ca2+ concentration ([Ca2+]i), [Ca2+]i transient amplitude, and sarcoplasmic reticulum (SR) Ca2+ load (caffeine-induced [Ca2+]i release). SR Ca2+ overload was associated with spontaneous Ca2+ transient events and triggered ectopic activity, which was suppressed by the inhibition of SR Ca2+ release (ryanodine) or Na+/Ca2+ exchange. Mechanisms underlying abnormal SR Ca2+ handling were then studied. CHF increased atrial action potential duration and action potential voltage clamp showed that CHF-like action potentials enhance Ca2+i loading. CHF increased calmodulin-dependent protein kinase II phosphorylation of phospholamban by 120%, potentially enhancing SR Ca2+ uptake by reducing phospholamban inhibition of SR Ca2+ ATPase, but it did not affect phosphorylation of SR Ca2+-release channels (RyR2). Total RyR2 and calsequestrin (main SR Ca2+-binding protein) expression were significantly reduced, by 65% and 15%, potentially contributing to SR dysfunction. CHF decreased expression of total and protein kinase A–phosphorylated myosin-binding protein C (a key contractile filament regulator) by 27% and 74%, potentially accounting for decreased contractility despite increased Ca2+ transients. Complex phosphorylation changes were explained by enhanced calmodulin-dependent protein kinase II&dgr; expression and function and type-1 protein-phosphatase activity but downregulated regulatory protein kinase A subunits. Conclusions—CHF causes profound changes in Ca2+-handling and -regulatory proteins that produce atrial fibrillation–promoting atrial cardiomyocyte Ca2+-handling abnormalities, arrhythmogenic triggered activity, and contractile dysfunction.

Mechanisms Underlying Rate-Dependent Remodeling of Transient Outward Potassium Current in Canine Ventricular Myocytes

Transient outward K+ current (Ito) downregulation following sustained tachycardia in vivo is usually attributed to tachycardiomyopathy. This study assessed potential direct rate regulation of cardiac Ito and underlying mechanisms. Cultured adult canine left ventricular cardiomyocytes (37°C) were paced continuously at 1 or 3 Hz for 24 hours. Ito was recorded with whole-cell patch clamp. The 3-Hz pacing reduced Ito by 44% (P<0.01). Kv4.3 mRNA and protein expression were significantly reduced (by ≈30% and ≈40%, respectively) in 3-Hz paced cells relative to 1-Hz cells, but KChIP2 expression was unchanged. Prevention of Ca2+ loading with nimodipine or calmodulin inhibition with W-7, A-7, or W-13 eliminated 3-Hz pacing-induced Ito downregulation, whereas downregulation was preserved in the presence of valsartan. Inhibition of Ca2+/calmodulin-dependent protein kinase (CaMK)II with KN93, or calcineurin with cyclosporin A, also prevented Ito downregulation. CaMKII-mediated phospholamban phosphorylation at threonine 17 was increased in 3-Hz paced cells, compatible with enhanced CaMKII activity, with functional significance suggested by acceleration of the Ca2+i transient decay time constant (Indo 1-acetoxymethyl ester microfluorescence). Total phospholamban expression was unchanged, as was expression of Na+/Ca2+ exchange and sarcoplasmic reticulum Ca2+-ATPase proteins. Nuclear localization of the calcineurin-regulated nuclear factor of activated T cells (NFAT)c3 was increased in 3-Hz paced cells compared to 1-Hz (immunohistochemistry, immunoblot). INCA-6 inhibition of NFAT prevented Ito reduction in 3-Hz paced cells. Calcineurin activity increased after 6 hours of 3-Hz pacing. CaMKII inhibition prevented calcineurin activation and NFATc3 nuclear translocation with 3-Hz pacing. We conclude that tachycardia downregulates Ito expression, with the Ca2+/calmodulin-dependent CaMKII and calcineurin/NFAT systems playing key Ca2+-sensing and signal-transducing roles in rate-dependent Ito control.

Cardiac Transgenic and Gene Transfer Strategies Converge to Support an Important Role for Troponin I in Regulating Relaxation in Cardiac Myocytes

Elucidating the relative roles of cardiac troponin I (cTnI) and phospholamban (PLN) in &bgr;-adrenergic–mediated hastening of cardiac relaxation has been challenging and controversial. To test the hypothesis that &bgr;-adrenergic phosphorylation of cTnI has a prominent role in accelerating cardiac myocyte relaxation performance we used transgenic (Tg) mice bearing near complete replacement of native cTnI with a &bgr;-adrenergic phospho-mimetic of cTnI whereby tandem serine codons 23/24 were converted to aspartic acids (cTnI S23/24D). Adult cardiac myocytes were isolated and contractility determined at physiological temperature under unloaded and loaded conditions using micro-carbon fibers. At baseline, cTnI S23/24D myocytes had significantly faster relaxation times relative to controls, and isoproterenol stimulation (Iso) had only a small effect to further speed relaxation in cTnI S23/24D myocytes (delta Iso: 7.2 ms) relative to the maximum Iso effect (31.2 ms) in control. The Ca2+ transient decay rate was similarly accelerated by Iso in Tg and nontransgenic (Ntg) myocytes. Gene transfer of cTnI S23/24D to myocytes in primary culture showed comparable findings. Gene transfer of cTnI with both serines 23/24 converted to alanines (cTnI S23/24A), or gene transfer of slow skeletal TnI, both of which lack PKA phosphorylation sites, significantly blunted Iso-mediated enhanced relaxation compared with controls. Gene transfer of wild-type cTnI had no effect on relaxation. These findings support a key role of cTnI in myocyte relaxation and highlight a direct contribution of the myofilaments in modulating the dynamics of myocardial performance.

Optimal range for parvalbumin as relaxing agent in adult cardiac myocytes: gene transfer and mathematical modeling.

Parvalbumin (PV) has recently been shown to increase the relaxation rate when expressed in intact isolated cardiac myocytes via adenovirus gene transfer. We report here a combined experimental and mathematical modeling approach to determine the dose-response and the sarcomere length (SL) shortening-frequency relationship of PV in adult rat cardiac myocytes in primary culture. The dose-response was obtained experimentally by observing the PV-transduced myocytes at different time points after gene transfer. Calcium transients and unloaded mechanical contractions were measured. The results were as follows. At low estimated [PV] (approximately 0.01 mM), contractile parameters were unchanged; at intermediate [PV], relaxation rate of the mechanical contraction and the decay rate of the calcium transient increased with little effects on amplitude; and at high [PV] (approximately 0.1 mM), relaxation rate was further increased, but the amplitudes of the mechanical contraction and the calcium transient were diminished when compared with control myocytes. The SL shortening-frequency relationship exhibited a biphasic response to increasing stimulus frequency in controls (decrease in amplitude and re-lengthening time from 0.2 to 1.0 Hz followed by an increase in these parameters from 2.0 to 4.0 Hz). The effect of PV was to flatten this frequency response. This flattening effect was partly explained by a reduction in the variation in fractional binding of PV to calcium during beats at high frequency. In conclusion, experimental results and mathematical modeling indicate that there is an optimal PV range for which relaxation rate is increased with little effect on contractile amplitude and that PV effectiveness decreases as the stimulus frequency increases.

Targeting Diastolic Dysfunction by Genetic Engineering of Calcium Handling Proteins

Diastolic heart failure (HF) is associated with significant morbidity and mortality, and is a growing medical problem in this country. Diastolic dysfunction is defined as an abnormality in myocardial relaxation that impairs filling during diastole and contributes to the clinical syndrome of HF. Effective clinical strategies to treat diastolic dysfunction are limited. This article focuses on the potential application of parvalbumin--a fast skeletal muscle calcium buffer--for remediation of slow relaxation in the failing heart.

Cardiac-directed parvalbumin transgene expression in mice shows marked heart rate dependence of delayed Ca2+ buffering action

Relaxation abnormalities are prevalent in heart failure and contribute to clinical outcomes. Disruption of Ca2+ homeostasis in heart failure delays relaxation by prolonging the intracellular Ca2+ transient. We sought to speed cardiac relaxation in vivo by cardiac-directed transgene expression of parvalbumin (Parv), a cytosolic Ca2+ buffer normally expressed in fast-twitch skeletal muscle. A key feature of Parvs function resides in its Ca2+/Mg2+ binding affinities that account for delayed Ca2+ buffering in response to the intracellular Ca2+ transient. Cardiac Parv expression decreased sarcoplasmic reticulum Ca2+ content without otherwise altering intracellular Ca2+ homeostasis. At high physiological mouse heart rates in vivo, Parv modestly accelerated relaxation without affecting cardiac morphology or systolic function. Ex vivo pacing of the isolated heart revealed a marked heart rate dependence of Parvs delayed Ca2+ buffering effects on myocardial performance. As the pacing frequency was lowered (7 to 2.5 Hz), the relaxation rates increased in Parv hearts. However, as pacing rates approached the dynamic range in humans, Parv hearts demonstrated decreased contractility, consistent with Parv buffering systolic Ca2+. Mathematical modeling and in vitro studies provide the underlying mechanism responsible for the frequency-dependent fractional Ca2+ buffering action of Parv. Future studies directed toward refining the dose and frequency-response relationships of Parv in the heart or engineering novel Parv-based Ca2+ buffers with modified Mg2+ and Ca2+ affinities to limit systolic Ca2+ buffering may hold promise for the development of new therapies to remediate relaxation abnormalities in heart failure.