Skeletal Muscle NADPH Oxidase Is Increased and Triggers Stretch-Induced Damage in the mdx Mouse

Recent studies have shown that oxidative stress contributes to the pathogenesis of muscle damage in dystrophic (mdx) mice. In this study we have investigated the role of NADPH oxidase as a source of the oxidative stress in these mice. The NADPH oxidase subunits gp91phox, p67phox and rac 1 were increased 2–3 fold in tibilais anterior muscles from mdx mice compared to wild type. Importantly, this increase occurred in 19 day old mice, before the onset of muscle necrosis and inflammation, suggesting that NADPH oxidase is an important source of oxidative stress in mdx muscle. In muscles from 9 week old mdx mice, gp91phox and p67phox were increased 3–4 fold and NADPH oxidase superoxide production was 2 times greater than wild type. In single fibers from mdx muscle NADPH oxidase subunits were all located on or near the sarcolemma, except for p67phox,which was expressed in the cytosol. Pharmacological inhibition of NADPH oxidase significantly reduced the intracellular Ca2+ rise following stretched contractions in mdx single fibers, and also attenuated the loss of muscle force. These results suggest that NADPH oxidase is a major source of reactive oxygen species in dystrophic muscle and its enhanced activity has a stimulatory effect on stretch-induced Ca2+ entry, a key mechanism for muscle damage and functional impairment

Functional cyclophilin D moderates platelet adhesion, but enhances the lytic resistance of fibrin

In the course of thrombosis, platelets are exposed to a variety of activating stimuli classified as ‘strong’ (e.g. thrombin and collagen) or ‘mild’ (e.g. ADP). In response, activated platelets adhere to injured vasculature, aggregate, and stabilise the three-dimensional fibrin scaffold of the expanding thrombus. Since ‘strong’ stimuli also induce opening of the mitochondrial permeability transition pore (MPTP) in platelets, the MPTP-enhancer Cyclophilin D (CypD) has been suggested as a critical pharmacological target to influence thrombosis. However, it is poorly understood what role CypD plays in the platelet response to ‘mild’ stimuli which act independently of MPTP. Furthermore, it is unknown how CypD influences platelet-driven clot stabilisation against enzymatic breakdown (fibrinolysis). Here we show that treatment of human platelets with Cyclosporine A (a cyclophilin-inhibitor) boosts ADP-induced adhesion and aggregation, while genetic ablation of CypD in murine platelets enhances adhesion but not aggregation. We also report that platelets lacking CypD preserve their integrity in a fibrin environment, and lose their ability to render clots resistant against fibrinolysis. Our results indicate that CypD has opposing haemostatic roles depending on the stimulus and stage of platelet activation, warranting a careful design of any antithrombotic strategy targeting CypD

Oxidation of myofibrillar proteins causes contractile dysfunction in human heart failure

Background: Previous studies in microembolized pig hearts (Eur. Heart. J. 27, 875-881, 2006) demonstrated that augmented intracellular accumulation of reactive oxygen species causes oxidative modifications at the level of the contractile machinery. This study was aimed at investigating the role of myofibrillar protein (MP) oxidation\nitrosylation and the relationship between MP oxidation and contractile impairment in human failing myocardium (NYHA class IV). Methods and Results: As compared to samples from non-failing donor hearts (NF-group, n = 15), left ventricular biopsies from explanted failing hearts (NYHA class IV, HF-group, n = 33) displayed a 2.3 \ub1 0.29- and 2.6 \ub1 0.53-fold increase in actin and tropomyosin (Tm) carbonylation respectively, and a 2.2 \ub1 0.47-fold higher level of high-molecular-mass complexes of Tm due to disulphide cross-bridge formation. MP were also modified by reactive nitrogen species. The extent of S-nitrosylation was 1.3 \ub1 0.15-fold higher in the HF-group. Interesting, actin and Tm carbonylation along with Tm oxidation significantly correlated with both loss of viability (r2 = 0.646, P = 6.16E-12; r2 = 0.453, P = 1.58E-07; r2 = 0.221, P = 0.001, respectively), as indicated by plasma TnI levels and contractile impairment (r2 = 0.599, P = 6.88E-112; r2 = 0.457, P = 1.37E-07; r2 = 0.646, P = 6.16E-12, respectively), as shown by reduced left ventricular ejection fraction (LVEF). Conclusion: This study demonstrates that HF-related oxidative and nitrosative stresses induce covalent changes of MP and that these MP changes play a relevant role in contractile impairment as suggested by the inverse correlation between MP oxidation and LVEF

Mitochondrial Injury and Protection in Ischemic Pre- and Postconditioning

Mitochondrial damage is a determining factor in causing loss of cardiomyocyte function and viability, yet a mild degree of mitochondrial dysfunction appears to underlie cardioprotection against injury caused by postischemic reperfusion. This review is focused on two major mechanisms of mitochondrial dysfunction, namely, oxidative stress and opening of the mitochondrial permeability transition pore. The formation of reactive oxygen species in mitochondria will be analyzed with regard to factors controlling mitochondrial permeability transition pore opening. Finally, these mitochondrial processes are analyzed with respect to cardioprotection afforded by ischemic pre- and postconditioning

Oxidation of myofibrillar proteins in human heart failure

OBJECTIVES: We investigated the incidence and contribution of the oxidation/nitrosylation of tropomyosin and actin to the contractile impairment and cardiomyocyte injury occurring in human end-stage heart failure (HF) as compared with nonfailing donor hearts. BACKGROUND: Although there is growing evidence that augmented intracellular accumulation of reactive oxygen/nitrogen species may play a key role in causing contractile dysfunction, there is a dearth of data regarding their contractile protein targets in human HF. METHODS: In left ventricular (LV) biopsies from explanted failing hearts (New York Heart Association functional class IV; HF group) and nonfailing donor hearts (NF group), carbonylation of actin and tropomyosin, disulphide cross-bridge (DCB) formation, and S-nitrosylation in tropomyosin were assessed, along with plasma troponin I and LV ejection fraction (LVEF). RESULTS: The LV biopsies from the HF group had 2.14 \ub1 0.23-fold and 2.31 \ub1 0.46-fold greater levels in actin and tropomyosin carbonylation, respectively, and 1.77 \ub1 0.45-fold greater levels of high-molecular-weight complexes of tropomyosin due to DCB formation, compared with the NF group. Tropomyosin also underwent S-nitrosylation that was 1.3 \ub1 0.15-fold higher in the HF group. Notably, actin and tropomyosin carbonylation was significantly correlated with both loss of viability indicated by plasma troponin I and contractile impairment as shown by reduced LVEF. CONCLUSIONS: This study demonstrated that oxidative/nitrosylative changes of actin and tropomyosin are largely increased in human failing hearts. Because these changes are inversely correlated to LVEF, actin and tropomyosin oxidation are likely to contribute to the contractile impairment evident in end-stage HF

Monoamine oxidase inhibition prevents mitochondrial dysfunction and apoptosis in myoblasts from patients with collagen VI myopathies

AbstractAlthough mitochondrial dysfunction and oxidative stress have been proposed to play a crucial role in several types of muscular dystrophy (MD), whether a causal link between these two alterations exists remains an open question. We have documented that mitochondrial dysfunction through opening of the permeability transition pore plays a key role in myoblasts from patients as well as in mouse models of MD, and that oxidative stress caused by monoamine oxidases (MAO) is involved in myofiber damage. In the present study we have tested whether MAO-dependent oxidative stress is a causal determinant of mitochondrial dysfunction and apoptosis in myoblasts from patients affected by collagen VI myopathies. We find that upon incubation with hydrogen peroxide or the MAO substrate tyramine myoblasts from patients upregulate MAO-B expression and display a significant rise in reactive oxygen species (ROS) levels, with concomitant mitochondrial depolarization. MAO inhibition by pargyline significantly reduced both ROS accumulation and mitochondrial dysfunction, and normalized the increased incidence of apoptosis in myoblasts from patients. Thus, MAO-dependent oxidative stress is causally related to mitochondrial dysfunction and cell death in myoblasts from patients affected by collagen VI myopathies, and inhibition of MAO should be explored as a potential treatment for these diseases