SERCA-1 conformational change exerted by the Ca2+-channel blocker diltiazem affects mammalian skeletal muscle function

Abstract

In skeletal muscle (SM), inward Ca²⁺-currents have no apparent role in excitation-contraction coupling (e-c coupling), however the Ca²⁺-channel blocker can affect twitch and tetanic muscle in mammalian SM. Experiments were conducted to study how diltiazem (DLZ) facilitates e-c coupling and inhibits contraction. 1) In complete Extensor Digitorum Longus (EDL) muscle and single intact fibres, 0.03 mM DLZ causes twitch potentiation and decreases force during tetanic activity, with increased fatigue. 2) In split open fibres isolated from EDL fibres, DLZ inhibits sarcoplasmic reticulum (SR) Ca²⁺-loading in a dose-dependent manner and has a potentiating effect on caffeine-induced SR Ca²⁺-release. 3) In isolated light SR (LSR) vesicles, SERCA1 hydrolytic activity is not affected by DLZ up to 0.2 mM. However, ATP-dependent Ca²⁺-uptake was inhibited in a dose-dependent manner at a concentration where e-c coupling is changed. 4) The passive Ca²⁺-efflux from LSR was reduced by half with 0.03 mM diltiazem, indicating that SR leaking does not account for the decreased Ca²⁺-uptake. 5) The denaturation profile of the SERCA Ca²⁺-binding domain has lower thermal stability in the presence of DLZ in a concentration-dependent manner, having no effect on the nucleotide-binding domain. We conclude that the effect of DLZ on SM is exerted by crossing the sarcolemma and interacting directly with the SERCA Ca²⁺-binding domain, affecting SR Ca²⁺-loading during relaxation, which has a consequence on SM contractility. Diltiazem effect on SM could be utilized as a tool to understand SM e-c coupling and muscle fatigue.