Susceptibility of isolated myofibrils to in vitro glutathionylation: Potential relevance to muscle functions

Abstract

In this study we investigated the molecular mechanism of glutathionylation on isolated human cardiac myofibrils using several pro-glutathionylating agents. Total glutathionylated proteins appeared significantly enhanced with all the pro-oxidants used. The increase was completely reversed by the addition of a reducing agent, demonstrating that glutathione binding occurs by a disulfide and that the process is reversible. A sensitive target of glutathionylation was a-actin, showing a different reactivity to the several pro-glutathionylating agents by ELISA. Noteworthy, myosin although highly sensitive to the in vitro glutathionylation does not represent the primary glutathionylation target in isolated myofibrils. Light scattering measurements of the glutathionylated a-actin showed a slower polymerisation compared to the non-glutathionylated protein and force development was depressed after glutathionylation, when the myofibrils were mounted in a force recording apparatus. Interestingly, confocal laser scanning microscopy of cardiac cryosections indicated, for the first time, the constitutive glutathionylation of acardiac actin in human heart. Due to the critical location of a-actin in the contractile machinery and to its susceptibility to the oxidative modifications, glutathionylation may represent a mechanism for modulating sarcomere assembly and muscle functionality under patho-physiological conditions in vivo. V C 2009 Wiley-Liss, Inc.

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

“…Several other sarcomeric proteins including myosin, actin and tropomyosin also contain a variety of cysteines that are modified by ROS in specific pathophysiological states (Aksenov et al 2001;Eaton et al 2002;Fratelli et al 2002;Canton et al 2004Canton et al , 2006Fiaschi et al 2006;Passarelli et al 2010). However, it has not been fully elucidated which cysteines are involved and how their function is altered.…”

Redox regulation of cytoskeletal dynamics during differentiation and de-differentiation

“…Already 1947, Bailey and Perry noted that the thiol state of actin does not affect actomyosin formation [69]. Although post-translational redox modifications of actin, especially S-glutathionylation, do not alter the binding efficiency of myosin, ATPase activity of actomyosin and thereby muscle functionality were significantly deceased via inhibition of disassembly of the inactive ADPbound complex [70,71]. Fiaschi et al demonstrated the involvement of Cys374 glutathionylation in this process by the expression of a Cys374Arg mutant, however, here actin S-glutathionylation seems to promote the disassembly of this complex [72].…”

Regulation of protein function by S-nitrosation and S-glutathionylation: processes and targets in cardiovascular pathophysiology

“…α-Actin is also particularly sensitive to binding of glutathione, as shown in isolated cardiac and skeletal myofibrils under conditions of oxidative stress (Passarelli et al, 2010). S-glutathionylation of cardiac α-actin occurs non-enzymatically, via spontaneous oxidation of a Cys residue to a cysteinyl-sulfenic acid intermediary (DalleDonne et al, 2003;Johansson and Lundberg, 2007).…”