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Oxidants have been suggested to enhance contractile function in unfatigued muscle. In this study we aimed to determine the effect of oxidants on "chemically skinned" diaphragm muscle fibre bundles. The sarcoplasmic reticulum and contractile proteins were exposed to superoxide anions (O2-) and hydrogen peroxide (H2O2) under controlled conditions. Application of O2-initially increased maximum Ca2+ -activated force but subsequently reduced maximum Ca2+ -activated force without altering myofilament Ca2+ sensitivity. Unlike myocardium, caffeine-induced Ca2+ release from the sarcoplasmic reticulum was also inhibited by O2- exposure in diaphragm fibre bundles. Application of H2O2 also increased maximum Ca2+ -activated force but had additional effects on resting tension (which increased to 25 % of the control maximum Ca2+ -activated force). H2O2 was without effect on myofilament Ca2+ sensitivity or caffeine-induced Ca2+ release from the sarcoplasmic reticulum. These data demonstrate that oxidants can potentiate contractile force in the diaphragm through a direct action on the contractile proteins. The potentiation of force is not sustained, however, and under these conditions the detrimental effects of O2- on Ca2+ release from the sarcoplasmic reticulum combined with the effects of oxidants on the contractile proteins will ultimately compromise excitation-contraction coupling in the diaphragm. Experimental Physiology (2001) 86.2, 161-168.

This study aimed to determine structural alterations occurring in cardiac myofilaments after exogenous application of oxidants and the effects of oxidants on contractile protein function in a rabbit coronary artery ligation model of heart failure. Myocardial "stiffness" was higher in the ligated animals (Lig) than sham-operated controls (Sh, 4.9+/-1.5 versus 1.6+/-0.8 mN.mm-1). Superoxide anion (O2-) exposure decreased active stiffness in both groups, whereas hypochlorous acid (HOCl) had no effect in Lig but increased stiffness in Sh. Resting stiffness was higher in Lig than Sh (0.6+/-0.2 versus 0.2+/-0.1 mN.mm-1), remaining unchanged after O2- exposure but increasing after HOCl in both groups. The frequency at minimum stiffness was lower in Lig than Sh (0.9+/-0.2 versus 1. 7+/-0.6 Hz) and was reduced in both groups after oxidant exposure. Myofilament calcium sensitivity (pCa50) was not altered by O2- in Sh but increased in Lig (pCa50 increased from 5.41+/-0.05 to 5.56+/-0. 06). Protease contamination in the xanthine oxidase used to generate O2- did not affect myofilament ultrastructure at the concentrations used here. These data demonstrate that contractile proteins from "failed" myocardium have a similar response to exogenously applied oxidants as controls and that application of protease-contaminated xanthine oxidase system does not degrade the contractile protein structure.

Oxygen-derived free radicals (FRs) and other reactive oxygen species (ROS) have been implicated in the deleterious aspects of myocardial infarction, neutrophil infiltration and post-ischaemic reperfusion. We studied their actions on the main intracellular organelles of Ca-compartmentation and force production (the sarcoplasmic reticulum (SR) and myofilaments) in rat heart preparations by using two forms of chemical 'skinning'. We recorded Ca(2+)-activated isometric tension or, in saponin-treated trabeculae where SR function is maintained, either tension alone or tension and [Ca2+] transients evoked by caffeine. A single, brief application of xanthine/xanthine oxidase (generating superoxide; O2-) rapidly and irreversibly inhibits Ca(2+)-activated force with a dose- and time-dependent action. The kinetics of residual force production are slowed. Rigor induction (by ATP withdrawal) before and during exposure to .O2- prevents this action, suggesting the .O2(-)-sensitive site is occluded in rigor. Myofilament Ca-sensitivity and SR function were unaffected by .O2- or physiologically relevant [H2O2] (< 10 microM). Briefly applying 10-50 microM hypochlorous acid (HOCl) increased Ca-sensitivity and resting tension, but reduced Ca-activated force, in a manner consistent with 'rigor-like' crossbridges being involved. HOCl also provoked spontaneous Ca-release but reduced net SR Ca-uptake. Electron microscopy reveals that the myofilament lattice suffers a characteristic disruption by HOCl but not by .O2-. We conclude that FRs and ROS associated with myocyte dysfunction, reperfusion and inflammation could contribute to post-ischaemic myocardial dysfunction.

Neutrophil activation occurs after myocardial infarction/ischaemia. They produce the reactive oxygen species (ROS) hypochlorous acid (HOCl) and hydrogen peroxide (H2O2) which could contribute to contractile dysfunction upon reperfusion. The myofilaments of 'skinned' rat cardiac muscle were exposed to ROS in various states of activation. Isometric force was measured at controlled degrees of activation. A single application of 10 microM HOCl for 1 min increased log [Ca2+] for half-maximal activation (log K1/2) from 5.23 to 5.32, initial maximum Ca-activated force (FCa, max) was reduced by 18.8 +/- 5.8% and resting tension increased to 15.4 +/- 8.0% of FCa, max. At 50 microM, a 1-min exposure to HOCl produced a greater increase in Ca-sensitivity (log K1/2 increased from 5.23 to 5.47), a greater reduction in FCa, max (falling by 42.3 +/- 23.2%) and a greater increase in resting tension (to 25 +/- 10.7% of FCa, max). The nature of the resting tension rise was examined by reducing pH before and during exposure to HOCl; the results are consistent with 'rigor-like' cross-bridges being involved. H2O2 was without effect on the myofilaments at physiologically relevant (< 10 microM) concentrations. These results suggest that ROS associated with inflammation could contribute to post-ischaemic myocardial dysfunction.