A piperidinyl amide compound enhances Ca2+ signaling in cardiomyocytes by increasing activity of Ca2+ pump.

Abstract

In adult cardiomyocytes, the type 2a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) plays a vital role in intracellular Ca²⁺ regulation. Reduced SERCA2a function has been associated with decreased myocardial contraction and cardiac output in several heart diseases. Consequently, increasing SERCA2a activity is a high-priority target for treating cardiac pathologies associated with abnormal Ca²⁺ homeostasis. In our previous SERCA ATPase-based screening study, we identified several small molecules as potential activators of SERCA2a function, including Compound 9, a piperidinyl amide. With porcine cardiac sarcoplasmic reticulum (SR) preparations, we confirmed activation of both SERCA2a ATPase and Ca²⁺-uptake activities. In the current study, we analyzed the effect of Compound 9 on SERCA2a activity on intracellular Ca²⁺ dynamics in ventricular myocytes. Using FRET with human SERCA2a overexpressed in mammalian cells, we confirm that Compound 9 binds and alters SERCA structural dynamics independent of peptide regulators, including phospholamban (PLB). Confocal microscopy and in-cell Ca²⁺ imaging revealed that Compound 9 enhanced Ca²⁺ dynamics in mouse ventricular myocytes. Compound 9 (10 μM) increased the action potential-induced Ca²⁺ transients by 65% and SR Ca²⁺ load by 29%. Moreover, Compound 9 increased Ca²⁺ dynamics during adrenergic receptor stimulation and in PLB knockout cardiomyocytes, suggesting the stimulatory effect of Compound 9 is PLB independent. Overall, Compound 9 displays characteristics that can be beneficial to enhance cardiac intracellular Ca²⁺ dynamics by increasing SERCA2a function.