Effects of reducing agents and oxidants on excitation‐contraction coupling in skeletal muscle fibres of rat and toad.

Abstract

1. The mechanically skinned fibre technique was used to examine the role of oxidationreduction in the control of Ca2+ release and contraction in rat and toad skeletal muscle fibres under physiological conditions of myoplasmic [Mg2+] and [ATP] and sarcoplasmic reticulum (SR) Ca2+ load. 2. None of the reducing agents, dithiothreitol (DIT, 10 mm), glutathione (GSH, 10 mM) or cysteine (1 and 5 mM), had any detectable effect on the peak force, duration or the total number of depolarization-induced responses that could be elicited in skinned fibres from either toad or rat muscle, except for a slight alteration in one case (GSH on the duration of the response in rat fibres) caused by an effect of the agent of the Ca2+ sensitivity of the contractile apparatus.3. Application of the reactive disulphide, 2,2'-dithiodipyridine (DTDP, 100 /SM), a potent oxidizing agent, never induced any measurable force response or noticeable depletion of SR Ca2P in any fibre under the conditions used. When all Ca2+ uptake was prevented, DTDP treatment of rat fibres was found to cause a 2-to 3-fold increase in the low rate of Ca2+ 'leak' from the SR. DTDP treatment also increased the responsiveness of toad muscle fibres to 1 or 2 mm caffeine. These effects could be largely reversed by treatment with DTT. These results indicate that oxidation of the Ca2+ release channel does not cause substantial channel opening under physiological conditions. 4. Depolarization-induced force responses in both rat and toad fibres were rapidly abolished in the presence of DTDP (10 or 100 /M), in a manner favoured by inactivation of the voltage sensors. The relatively impermeant oxidant, 5,5'-dithionitrobenzoic acid (DTNB, 100 /M), had an effect very similar to DTDP if applied intracellularly, but unlike DTDP, had little or no effect if applied extracellularly (at 5 mM) before skinning. Depolarization-induced responses could be restored by treatment with DTT (10 mM). Intracellular application of the sulfhydryl-alkylating agent, N-ethylmaleimide (NEM, 100 ,M), had effects very similar to DTDP and DTNB. 5. These results are not consistent with the proposal that excitation-contraction coupling in skeletal muscle primarily involves the oxidative linkage of the voltage sensors to the Ca2+ release channels, but do show that oxidation of an intracellularly accessible site can interfere with the coupling, in a process made more sensitive by voltage sensor inactivation.In vertebrate skeletal muscle, it is not known exactly how the the normal physiological level of 1 mm (approximately 0 1-

A change of heart: oxidative stress in governing muscle function?

“…In spite of suggestions that SH oxidation of tropomyosin and/or actin in combination with myosin light chain1 (MLC1) (Hertelendi et al 2008) are potential mediators of oxidative myocardial contractile depression, it is currently difficult to clearly state that oxidation of myofilament proteins causes cardiac dysfunction, since studies have reported equivocal effects in different muscle types and even within a single muscle type. The role of oxidation of myofilament proteins in cardiac dysfunction in the heart is currently confused, as decreased Ca 2+ sensitivity has been reported (Posterino and Lamb 1996;Lamb and Posterino 2003;Hertelendi et al 2008) while others reported no changes or even increases in Ca 2+ sensitivity (MacFarlane and Miller 1992;MacFarlane and Miller 1994). Interestingly, slow twitch muscle differed from fast twitch muscle in relation to the addition of cysteine oxidants during muscle contraction (Lamb et al 1995;Lamb and Posterino 2003), suggesting that oxidative responses may be dependent on positions and differences between cysteines in fast and slow skeletal muscle.…”

“…While recent evidence indicates a role for oxidative and nitrosative regulation of the Ca 2+ -sensitive proteome in excitation-contraction (EC) coupling, only phosphorylation is the best-established therapeutic target (Haycock et al 1996;Posterino and Lamb 1996;Gao et al 1996;Disatnik et al 1998;Duncan et al 2005;Dai et al 2007;Ullrich et al 2009;Canton et al 2014).…”

“…Curved arrow depicts the steeper end-systolic pressurevolume relationship in heart failure with preserved ejection fraction compared with heart failure with reduced ejection fraction Striated muscle is subjected to oxidative stress and evidence now suggests that oxidative stress also modulates contractile function in cardiac and skeletal muscle. Redox modifications of proteins are known to result in functionally important changes in protein structure, stability, interactivity, and activity (Haycock et al 1996;Posterino and Lamb 1996;Gao et al 1996;Disatnik et al 1998;Duncan et al 2005;Dai et al 2007;Sanchez et al 2008;Ullrich et al 2009;Canton et al 2014). However, due to the limited data available and the fact that most studies have focused mainly on post-translational modifications of single purified contractile proteins, our understanding of the redox-dependent mechanisms that control contraction-relaxation in the intact heart remains poor.…”

Depolarization-induced contraction and SR function in mechanically skinned muscle fibers from dystrophic mdx mice

“…Substituting K-HDTA for the release solution caused sustained tension, and the area under the tension curve was used as an approximate indicator of SR Ca 2ϩ content (29). After 2 min of equilibration in the release solution and complete SR Ca 2ϩ depletion, the SR was partially reloaded with Ca 2ϩ by incubation in the load solution for defined intervals (10, 20, 30, and 60 s).…”

“…Fibers then underwent a repeated depolarization (Na-HDTA) with a 30-s repriming period in K-HDTA between each DICR until they reached the point of rundown (DICR Ͻ50% of initial). The viability of SR CRC function was assessed by stimulating SR Ca 2ϩ release with a low-[Mg 2ϩ ] solution (K-HDTA with 0.1 mM free Mg 2ϩ ) (29). At the conclusion of the DICR measurement, P o was determined.…”