Negative chronotropism, positive inotropism and lusitropism of 3,5-di-t-butyl-4-hydroxyanisole (DTBHA) on rat heart preparations occur through reduction of RyR2 Ca2+ leak

Abstract

3,5-Di-t-butyl-4-hydroxyanisole (DTBHA) is considered as an activator of the skeletal muscle sarcoplasmic reticulum (SR) Ca²⁺-uptake, endowed with antioxidant and L-type Ca²⁺ channel blocking activities. In this study we assessed the cardiac effects of DTBHA on Langendorff perfused rat hearts, isolated rat atria and rat cardiac SR membrane vesicles, as well as on several SERCA isoforms of membrane preparations. Moreover, in order to clarify its molecular mechanism of action Ca²⁺ imaging experiments were carried out on HEK293 cells transiently transfected with RyR2 channel. Docking of DTBHA at the rat RyR2 protein was investigated in silico. In Langendorff perfused rat hearts, DTBHA significantly increased, in a concentration-dependent manner, left ventricular pressure and diastole duration, while reducing heart rate and the time-constant of isovolumic relaxation, leaving unaltered coronary perfusion pressure. At the maximum concentration tested (30 µM), it significantly prolonged PQ interval, but left the corrected QT intervals unaffected. In spontaneously beating atria, DTBHA decreased sinus rate in a concentration-dependent manner. DTBHA, at concentrations higher than 10 µM, increased Ca²⁺ uptake in cardiac SR without affecting Ca²⁺-dependent ATPase activity assayed on several SERCA isoforms. Moreover, DTBHA antagonized thapsigargin-stimulated Ca²⁺ leak in cardiac SR and reduced caffeine-induced, RyR2-activated Ca²⁺ release in RyR2 expressing HEK293 cells. Using computational approaches, DTBHA showed a good affinity outline into binding sites of RyR2 protein. In conclusion, DTBHA behaved like a negative chronotropic, a positive inotropic and a lusitropic agent on rat heart preparations and improved cardiac SR Ca²⁺ uptake by lowering SR Ca²⁺ leak.

Keywords: Antioxidant phenol derivative; Cardiac sarcoplasmic reticulum membrane vesicles; Isolated rat atria; Langendorff isolated rat heart; RyR2 channel.