Essential Role of the RIα Subunit of cAMP-Dependent Protein Kinase in Regulating Cardiac Contractility and Heart Failure Development.

BACKGROUND: The heart expresses 2 main subtypes of cAMP-dependent protein kinase (PKA; type I and II) that differ in their regulatory subunits, RIα and RIIα. Embryonic lethality of RIα knockout mice limits the current understanding of type I PKA function in the myocardium. The objective of this study was to test the role of RIα in adult heart contractility and pathological remodeling. METHODS: We measured PKA subunit expression in human heart and developed a conditional mouse model with cardiomyocyte-specific knockout of RIα (RIα-icKO). Myocardial structure and function were evaluated by echocardiography, histology, and ECG and in Langendorff-perfused hearts. PKA activity and cAMP levels were determined by immunoassay, and phosphorylation of PKA targets was assessed by Western blot. L-type Ca2+ current (ICa,L), sarcomere shortening, Ca2+ transients, Ca2+ sparks and waves, and subcellular cAMP were recorded in isolated ventricular myocytes (VMs). RESULTS: RIα protein was decreased by 50% in failing human heart with ischemic cardiomyopathy and by 75% in the ventricles and in VMs from RIα-icKO mice but not in atria or sinoatrial node. Basal PKA activity was increased ≈3-fold in RIα-icKO VMs. In young RIα-icKO mice, left ventricular ejection fraction was increased and the negative inotropic effect of propranolol was prevented, whereas heart rate and the negative chronotropic effect of propranolol were not modified. Phosphorylation of phospholamban, ryanodine receptor, troponin I, and cardiac myosin-binding protein C at PKA sites was increased in propranolol-treated RIα-icKO mice. Hearts from RIα-icKO mice were hypercontractile, associated with increased ICa,L, and [Ca2+]i transients and sarcomere shortening in VMs. These effects were suppressed by the PKA inhibitor, H89. Global cAMP content was decreased in RIα-icKO hearts, whereas local cAMP at the phospholamban/sarcoplasmic reticulum Ca2+ ATPase complex was unchanged in RIα-icKO VMs. RIα-icKO VMs had an increased frequency of Ca2+ sparks and proarrhythmic Ca2+ waves, and RIα-icKO mice had an increased susceptibility to ventricular tachycardia. On aging, RIα-icKO mice showed progressive contractile dysfunction, cardiac hypertrophy, and fibrosis, culminating in congestive heart failure with reduced ejection fraction that caused 50% mortality at 1 year. CONCLUSIONS: These results identify RIα as a key negative regulator of cardiac contractile function, arrhythmia, and pathological remodeling.

Dual effect of cardiac FKBP12.6 overexpression on excitation-contraction coupling and the incidence of ventricular arrhythmia depending on its expression level.

FKBP12.6, a binding protein to the immunosuppressant FK506, which also binds the ryanodine receptor (RyR2) in the heart, has been proposed to regulate RyR2 function and to have antiarrhythmic properties. However, the level of FKBP12.6 expression in normal hearts remains elusive and some controversies still persist regarding its effects, both in basal conditions and during ß-adrenergic stimulation. We quantified FKBP12.6 in the left ventricles (LV) of WT (wild-type) mice and in two novel transgenic models expressing distinct levels of FKBP12.6, using a custom-made specific anti-FKBP12.6 antibody and a recombinant protein. FKBP12.6 level in WT LV was very low (0.16 ± 0.02 nmol/g of LV), indicating that <15% RyR2 monomers are bound to the protein. Mice with 14.1 ± 0.2 nmol of FKBP12.6 per g of LV (TG1) had mild cardiac hypertrophy and normal function and were protected against epinephrine/caffeine-evoked arrhythmias. The ventricular myocytes showed higher [Ca2+]i transient amplitudes than WT myocytes and normal SR-Ca2+ load, while fewer myocytes showed Ca2+ sparks. TG1 cardiomyocytes responded to 50 nM Isoproterenol increasing these [Ca2+]i parameters and producing RyR2-Ser2808 phosphorylation. Mice with more than twice the TG1 FKBP12.6 value (TG2) showed marked cardiac hypertrophy with calcineurin activation and more arrhythmias than WT mice during ß-adrenergic stimulation, challenging the protective potential of high FKBP12.6. RyR2R420Q CPVT mice overexpressing FKBP12.6 showed fewer proarrhythmic events and decreased incidence and duration of stress-induced bidirectional ventricular tachycardia. Our study, therefore, quantifies for the first time endogenous FKBP12.6 in the mouse heart, questioning its physiological relevance, at least at rest due its low level. By contrast, our work demonstrates that with caution FKBP12.6 remains an interesting target for the development of new antiarrhythmic therapies.

HIV-Tat induces a decrease in IKr and IKsvia reduction in phosphatidylinositol-(4,5)-bisphosphate availability.

Patients with HIV present with a higher prevalence of QT prolongation, of which molecular bases are still not clear. Among HIV proteins, Tat serves as a transactivator that stimulates viral genes expression and is required for efficient HIV replication. Tat is actively secreted into the blood by infected T-cells and affects organs such as the heart. Tat has been shown to alter cardiac repolarization in animal models but how this is mediated and whether this is also the case in human cells is unknown. In the present study, we show that Tat transfection in heterologous expression systems led to a decrease in hERG (underlying cardiac IKr) and human KCNE1-KCNQ1 (underlying cardiac IKs) currents and to an acceleration of their deactivation. This is consistent with a decrease in available phosphatidylinositol-(4,5)-bisphosphate (PIP2). A mutant Tat, unable to bind PIP2, did not reproduce the observed effects. In addition, WT-Tat had no effect on a mutant KCNQ1 which is PIP2-insensitive, further confirming the hypothesis. Twenty-four-hour incubation of human induced pluripotent stem cells-derived cardiomyocytes with Wild-type Tat reduced IKr and accelerated its deactivation. Concordantly, this Tat incubation led to a prolongation of the action potential (AP) duration. Events of AP alternans were also recorded in the presence of Tat, and were exacerbated at a low pacing cycle length. Altogether, these data obtained on human K+ channels both in heterologous expression systems and in human cardiomyocytes suggest that Tat sequesters PIP2, leading to a reduction of IKr and IKs, and provide a molecular mechanism for QT prolongation in HIV-infected patients.