Susceptibility of isolated myofibrils to in vitro glutathionylation: Potential relevance to muscle functions.
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 alpha-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 alpha-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 alpha-cardiac actin in human heart. Due to the critical location of alpha-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.
Passarelli
Chiara
C
Children's Hospital and Research Institute "Bambino Gesù", Rome, Italy.
Di Venere
Almerinda
A
Piroddi
Nicoletta
N
Pastore
Anna
A
Scellini
Beatrice
B
Tesi
Chiara
C
Petrini
Stefania
S
Sale
Patrizio
P
Bertini
Enrico
E
Poggesi
Corrado
C
Piemonte
Fiorella
F
D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D005978:Glutathione / Q000378:metabolism*; D006321:Heart / Q000502:physiology; D006801:Humans; D018613:Microscopy, Confocal; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D009210:Myofibrils / Q000187:drug effects / Q000378:metabolism*; D018384:Oxidative Stress; D046188:Protein Modification, Translational / Q000187:drug effects; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000494:pharmacology
D000199:Actins; D017382:Reactive Oxygen Species; D005978:Glutathione