Peptidomimetic Targeting of Cavß2 Overcomes Dysregulation of the L-Type Calcium Channel Density and Recovers Cardiac Function.

BACKGROUND: L-type calcium channels (LTCCs) play important roles in regulating cardiomyocyte physiology, which is governed by appropriate LTCC trafficking to and density at the cell surface. Factors influencing the expression, half-life, subcellular trafficking, and gating of LTCCs are therefore critically involved in conditions of cardiac physiology and disease. METHODS: Yeast 2-hybrid screenings, biochemical and molecular evaluations, protein interaction assays, fluorescence microscopy, structural molecular modeling, and functional studies were used to investigate the molecular mechanisms through which the LTCC Cavß2 chaperone regulates channel density at the plasma membrane. RESULTS: On the basis of our previous results, we found a direct linear correlation between the total amount of the LTCC pore-forming Cavα1.2 and the Akt-dependent phosphorylation status of Cavß2 both in a mouse model of diabetic cardiac disease and in 6 diabetic and 7 nondiabetic cardiomyopathy patients with aortic stenosis undergoing aortic valve replacement. Mechanistically, we demonstrate that a conformational change in Cavß2 triggered by Akt phosphorylation increases LTCC density at the cardiac plasma membrane, and thus the inward calcium current, through a complex pathway involving reduction of Cavα1.2 retrograde trafficking and protein degradation through the prevention of dynamin-mediated LTCC endocytosis; promotion of Cavα1.2 anterograde trafficking by blocking Kir/Gem-dependent sequestration of Cavß2, thus facilitating the chaperoning of Cavα1.2; and promotion of Cavα1.2 transcription by the prevention of Kir/Gem-mediated shuttling of Cavß2 to the nucleus, where it limits the transcription of Cavα1.2 through recruitment of the heterochromatin protein 1γ epigenetic repressor to the Cacna1c promoter. On the basis of this mechanism, we developed a novel mimetic peptide that, through targeting of Cavß2, corrects LTCC life-cycle alterations, facilitating the proper function of cardiac cells. Delivery of mimetic peptide into a mouse model of diabetic cardiac disease associated with LTCC abnormalities restored impaired calcium balance and recovered cardiac function. CONCLUSIONS: We have uncovered novel mechanisms modulating LTCC trafficking and life cycle and provide proof of concept for the use of Cavß2 mimetic peptide as a novel therapeutic tool for the improvement of cardiac conditions correlated with alterations in LTCC levels and function.

Modulation of sympathetic activity and heart rate variability by ivabradine.

AIMS: Bradycardic agents are currently used in the treatment of angina and heart failure; direct information on their effects on cardiac sympathetic nerve activity (SNA) may be relevant to their chronic use. The present study evaluates the effect of pacemaker inhibition on SNA; direct nerve recordings and indirect autonomic indexes are compared. METHODS AND RESULTS: Experiments were performed in 18 anaesthetized rats. SNA (direct nerve recording) and heart rate variability (HRV) indexes were evaluated in parallel. All parameters were recorded 10 min before to 60 min after administration of the If blocker ivabradine (IVA; 2 mg/kg, i.v.; n = 8) or vehicle (VEH; n = 5). IVA-induced RR interval (RR) prolongation (at 60 min +15.0 ± 7.1%, P < 0.01) was associated with decreased diastolic arterial pressure (DAP; -17.3 ± 8.4%, P < 0.05) and increased SNA (+51.1 ± 12.3%, P < 0.05). These effects were accompanied by increased RR variance (RRσ(2)), which showed strong positive correlation with RR. Frequency-domain HRV indexes (in normalized units) were unchanged by IVA. After baroreceptor reflexes had been eliminated by sino-aortic denervation (n = 5), similar IVA-induced RR prolongation (at 60 min +14.3 ± 5.9%, NS vs. intact) was associated with a larger DAP reduction (-30.9 ± 4.1%, P < 0.05 vs. intact), but failed to affect SNA. CONCLUSIONS: (i) IVA-induced bradycardia was associated with increased SNA, resulting from baroreceptor unloading; if this applied to chronic IVA use in humans, it would be of relevance for therapeutic use of the drug. (ii) Whenever mean HR is concomitantly changed, time-domain HRV indexes should not be unequivocally interpreted in terms of autonomic balance.