Sulfhydryl Redox State Affects Susceptibility to Ischemia and Sarcoplasmic Reticulum Ca²⁺ Release in Rat Heart

Implications for Ischemic Preconditioning

From the Scuola Superiore St Anna (R.Z.), Dipartimento di Scienze dell'Uomo e dell'Ambiente (G.Y., P.G., G.R., S.R.-T.), and Dipartimento di Cardiologia (M.M.), University of Pisa, Italy.

Correspondence to Riccardo Zucchi, MD, Scuola Superiore St Anna, via Carducci 40, I-56100 Pisa, Italy. E-mail rzucchi{at}bm.med.unipi.it

Abstract—We investigated the effect of sulfhydryl and disulfide reagents on ischemic preconditioning and on sarcoplasmic reticulum Ca²⁺ release. Isolated working rat hearts were subjected to ischemic preconditioning (three 3-minute periods of global ischemia) or to control aerobic perfusion, which was followed by 30 minutes of global ischemia and 120 minutes of retrograde reperfusion. Necrosis was evaluated on the basis of lactate dehydrogenase release and triphenyltetrazolium chloride staining. In parallel experiments, sarcoplasmic reticulum Ca²⁺ release and [³H]-ryanodine binding were determined before the sustained ischemia. Ischemic preconditioning was associated with protection versus ischemic injury, decreased Ca²⁺ release and reduced [³H]-ryanodine binding. The disulfide reducing agent dithiothreitol (1 mmol/L) removed the protection provided by ischemic preconditioning, if added to the perfusion buffer either before or after the preconditioning procedure. In preconditioned hearts, dithiothreitol increased sarcoplasmic reticulum Ca²⁺ release and ryanodine binding, whereas in control hearts it had no effect on either tissue injury or sarcoplasmic reticulum function. Perfusion of control hearts with the sulfhydryl blocking agents 4,4'-dithiodipyridine (25 µmol/L) and N-ethylmaleimide (16 µmol/L) increased the resistance to ischemia and reduced sarcoplasmic reticulum Ca²⁺ release and [³H]-ryanodine binding. These effects were not additive with those induced by preconditioning. Sulfhydryl and disulfide reagents produced similar effects on Ca²⁺ release and [³H]-ryanodine binding if added in vitro to preparations obtained from control and preconditioned hearts. We conclude that ischemic preconditioning is associated with the oxidation of sulfhydryl groups involved in the modulation of sarcoplasmic reticulum Ca²⁺ release.

Key Words: sarcoplasmic reticulum • Ca²⁺ • ischemia • ryanodine receptor • thiol

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