Muscular dystrophies (MDs) are a heterogeneous group of diseases that share a common end-point represented by muscular wasting. MDs are caused by mutations in a variety of genes encoding for different molecules, including extracellular matrix, transmembrane and membrane-associated proteins, cytoplasmic enzymes and nuclear proteins. However, it is still to be elucidated how genetic mutations can affect the molecular mechanisms underlying the contractile impairment occurring in these complex pathologies. The intracellular accumulation of reactive oxygen species (ROS) is widely accepted to play a key role in contractile derangements occurring in the different forms of MDs. However, scarce information is available concerning both the most relevant sources of ROS and their major molecular targets. This review focuses on (i) the sources of ROS, with a special emphasis on monoamine oxidase, a mitochondrial enzyme, and (ii) the targets of ROS, highlighting the relevance of the oxidative modification of myofilament proteins.
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.
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.
?-Lactalbumin (LA) forms with oleic acid (OA) a complex which has been reported to induce the selective death of tumor cells. However, the mechanism by which this complex kills a wide range of tumor cell lines is as yet largely unknown. The difficulty in rationalizing the cytotoxic effects of the LA/OA complex can be due to the fact that the molecular aspects of the interaction between the protein and the fatty acid are still poorly understood, in particular regarding the oligomeric state of the protein and the actual molar ratio of OA over protein in the complex. Here, the effect of LA addition to an OA aqueous solution has been examined by dynamic light scattering measurements and transmission electron microscopy. Upon protein addition, the aggregation state of the rather insoluble OA is dramatically changed, and more water-soluble and smaller aggregates of the fatty acid are formed. A mixture of LA and an excess of OA forms a high molecular weight complex that can be isolated by size-exclusion chromatography and that displays cellular toxicity toward Jurkat cells. On the basis of gel filtration data, cross-linking experiments with glutaraldehyde, and OA titration, we evaluated that the isolated LA/OA complex is given by 4-5 protein molecules that bind 68-85 OA molecules. The protein in the complex adopts a molten globule-like conformation, and it interacts with the fatty acid mostly through its ?-helical domain, as indicated by circular dichroism measurements and limited proteolysis experiments. Overall, we interpret our and previous data as indicating that the cellular toxicity of a LA/OA complex is due to the effect of a protein moiety in significantly enhancing the water solubility of the cytotoxic OA and, therefore, that the protein/OA complex can serve mainly as a carrier of the toxic fatty acid in a physiological milieu.
Several studies documented the key role of oxidative stress and abnormal production of reactive oxygen species (ROS) in the pathophysiology of muscular dystrophies (MDs). The sources of ROS, however, are still controversial as well as their major molecular targets. This study investigated whether ROS produced in mitochondria by monoamine oxidase (MAO) contributes to MD pathogenesis. Pargyline, an MAO inhibitor, reduced ROS accumulation along with a beneficial effect on the dystrophic phenotype of Col6a1(-/-) mice, a model of Bethlem myopathy and Ullrich congenital MD, and mdx mice, a model of Duchenne MD. Based on our previous observations on oxidative damage of myofibrillar proteins in heart failure, we hypothesized that MAO-dependent ROS might impair contractile function in dystrophic muscles. Indeed, oxidation of myofibrillar proteins, as probed by formation of disulphide cross-bridges in tropomyosin, was detected in both Col6a1(-/-) and mdx muscles. Notably, pargyline significantly reduced myofiber apoptosis and ameliorated muscle strength in Col6a1(-/-) mice. This study demonstrates a novel and determinant role of MAO in MDs, adding evidence of the pivotal role of mitochondria and suggesting a therapeutic potential for MAO inhibition.
Our objective was to address the balance of inducible nitric oxide (NO) synthase (iNOS) and arginase and their contribution to contractile dysfunction in heart failure (HF). Excessive NO formation is thought to contribute to contractile dysfunction; in macrophages, increased iNOS expression is associated with increased arginase expression, which competes with iNOS for arginine. With substrate limitation, iNOS may become uncoupled and produce reactive oxygen species (ROS). In rabbits, HF was induced by left ventricular (LV) pacing (400 beats/min) for 3 wk. iNOS mRNA [quantitative real-time PCR (qRT-PCR)] and protein expression (confocal microscopy) were detected, and arginase II expression was quantified with Western blot; serum arginine and myocardial nitrite and nitrate concentrations were determined by chemiluminescence, and protein S-nitrosylation with Western blot. Superoxide anions were quantified with dihydroethidine staining. HF rabbits had increased LV end-diastolic diameter [20.0 + or - 0.5 (SE) vs. 17.2 + or - 0.3 mm in sham] and decreased systolic fractional shortening (11.1 + or - 1.4 vs. 30.6 + or - 0.7% in sham; both P < 0.05). Myocardial iNOS mRNA and protein expression were increased, however, not associated with increased myocardial nitrite or nitrate concentrations or protein S-nitrosylation. The serum arginine concentration was decreased (124.3 + or - 5.6 vs. 155.4 + or - 12.0 micromol/l in sham; P < 0.05) at a time when cardiac arginase II expression was increased (0.06 + or - 0.01 vs. 0.02 + or - 0.01 arbitrary units in sham; P < 0.05). Inhibition of iNOS with 1400W attenuated superoxide anion formation and contractile dysfunction in failing hearts. Concomitant increases in iNOS and arginase expression result in unchanged NO species and protein S-nitrosylation; with substrate limitation, uncoupled iNOS produces superoxide anions and contributes to contractile dysfunction.
The complexes formed by partially folded human and bovine alpha-lactalbumin with oleic acid (OA) have been reported to display selective apoptotic activity against tumor cells. These complexes were named human (HAMLET) or bovine (BAMLET) alpha-lactalbumin made lethal to tumor cells. Here, we analyzed the OA complexes formed by fragments of bovine alpha-lactalbumin obtained by limited proteolysis of the protein. Specifically, the fragments investigated were 53-103 and the two-chain fragment species 1-40/53-123 and 1-40/104-123, these last being the N-terminal fragment 1-40 covalently linked via disulfide bridges to the C-terminal fragment 53-123 or 104-123. The OA complexes were obtained by mixing the fatty acid and the fragments in solution (10-fold and 15-fold molar excess of OA over protein fragment) or by chromatography of the fragments loaded onto an OA-conditioned anion exchange column and salt-induced elution of the OA complexes. Upon binding to OA, all fragments acquire an enhanced content of alpha-helical secondary structure. All OA complexes of the fragment species showed apoptotic activity for Jurkat tumor cells comparable to that displayed by the OA complex of the intact protein. We conclude that the entire sequence of the protein is not required to form an apoptotic OA complex, and we suggest that the apoptotic activity of a protein-OA complex does not imply specific binding of the protein.
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