We propose that prolonged CMV causes diaphragm disuse, which, in turn, leads to activation of the ALP through oxidative stress and the induction of the FOXO1 transcription factor.

Our data suggest that mitochondrial dysfunction lies at the nexus between oxidative stress and the impaired diaphragmatic contractility that develops during MV. Energy substrate oversupply relative to demand, resulting from diaphragmatic inactivity during MV, could play an important role in this process.

Although absence of the cytoskeletal protein dystrophin leads to dilated cardiomyopathy in humans, the functional role of dystrophin in cardiac muscle remains undefined. We have addressed the hypothesis that dystrophin could help protect the heart against injury and contractile dysfunction induced by mechanical stress. In normal and dystrophin‐deficient (mdx) mice, cardiac mechanical stress was first manipulated ex vivo in a perfused working heart preparation. Despite an afterload level in the normal physiologic range, ex vivo perfused mdx hearts developed severe contractile dysfunction and nonischemic tissue damage, as is shown by excessive LDH release without a rise in coronary lactate. Injury to dystrophin‐deficient hearts was significantly correlated with cardiac work, and reducing the afterload level improved contractility and prevented injury in mdx hearts studied ex vivo. The response to mechanical stress in vivo was also assessed by using the vital dye Evans blue, which penetrates into cardiomyocytes with a disrupted sarcolemma. In the mdx group only, cardiomyocyte injury was increased markedly by acute elevations of mechanical stress induced by isoproterenol or brief aortic occlusion. Strikingly accelerated mortality and cardiac necrosis were also observed in the mdx group subjected to chronically increased cardiac mechanical stress via subtotal aortic constriction. Taken together, our results provide the first direct evidence that dystrophin serves to protect cardiomyocytes from mechanical stress and workload‐induced damage. Accordingly, reducing cardiac work in patients with dystrophin deficiency could be beneficial not only in treating established cardiomyopathy, but also in preventing the onset of cardiac disease.

Myofiber necrosis and fibrosis are hallmarks of Duchenne muscular dystrophy (DMD), leading to lethal weakness of the diaphragm. Macrophages (MPs) are required for successful muscle regeneration, but the role of inflammatory monocyte (MO)-derived MPs in either promoting or mitigating DMD is unclear. We show that DMD (mdx) mouse diaphragms exhibit greatly increased expression of CCR2 and its chemokine ligands, along with inflammatory (Ly6Chigh) MO recruitment and accumulation of CD11bhigh MO-derived MPs. Loss-of-function of CCR2 preferentially reduced this CD11bhigh MP population by impeding the release of Ly6Chigh MOs from the bone marrow but not the splenic reservoir. CCR2 deficiency also helped restore the MP polarization balance by preventing excessive skewing of MPs toward a proinflammatory phenotype. These effects were linked to amelioration of histopathological features and increased muscle strength in the diaphragm. Chronic inhibition of CCR2 signaling by mutated CCL2 secreted from implanted mesenchymal stem cells resulted in similar improvements. These data uncover a previously unrecognized role of inflammatory MOs in DMD pathogenesis and indicate that CCR2 inhibition could offer a novel strategy for DMD management.

Nitric oxide (NO), a free radical that is negatively inotropic in the heart and skeletal muscle, is produced in large amounts during sepsis by an NO synthase inducible (iNOS) by LPS and/or cytokines. The aim of this study was to examine iNOS induction in the rat diaphragm after Escherichia Coli LPS inoculation (1.6 mg/kg i.p.), and its involvement in diaphragmatic contractile dysfunction. Inducible NOS protein and activity could be detected in the diaphragm as early as 6 h after LPS inoculation. 6 and 12 h after LPS, iNOS was expressed in inflammatory cells infiltrating the perivascular spaces of the diaphragm, whereas 12 and 24 h after LPS it was expressed in skeletal muscle fibers. Inducible NOS was also expressed in the left ventricular myocardium, whereas no expression was observed in the abdominal, intercostal, and peripheral skeletal muscles. Diaphragmatic force was significantly decreased 12 and 24 h after LPS. This decrease was prevented by inhibition of iNOS induction by dexamethasone or by inhibition of iNOS activity by N G -methyl-L -arginine. We conclude that iNOS was induced in the diaphragm after E. Coli LPS inoculation in rats, being involved in the decreased muscular force. ( J. Clin. Invest. 1996. 98:1550-1559.)

We recently demonstrated early metabolic alterations in the dystrophin-deficient mdx heart that precede overt cardiomyopathy and may represent an early ''subclinical'' signature of a defective nitric oxide (NO)/cGMP pathway. In this study, we used genetic and pharmacological approaches to test the hypothesis that enhancing cGMP, downstream of NO formation, improves the contractile function, energy metabolism, and sarcolemmal integrity of the mdx heart. We first generated mdx mice overexpressing, in a cardiomyocyte-specific manner, guanylyl cyclase (GC) (mdx/GC ؉/0 ). When perfused ex vivo in the working mode, 12-and 20-week-old hearts maintained their contractile performance, as opposed to the severe deterioration observed in age-matched mdx hearts, which also displayed two to three times more lactate dehydrogenase release than mdx/GC ؉/0 . At the metabolic level, mdx/GC ؉/0 displayed a pattern of substrate selection for energy production that was similar to that of their mdx counterparts, but levels of citric acid cycle intermediates were significantly higher (36 ؎ 8%), suggesting improved mitochondrial function. Finally, the ability of dystrophin-deficient hearts to resist sarcolemmal damage induced in vivo by increasing the cardiac workload acutely with isoproterenol was enhanced by the presence of the transgene and even more so by inhibiting cGMP breakdown using the phosphodiesterase inhibitor sildenafil (44.4 ؎ 1.0% reduction in cardiomyocyte damage). Overall, these findings demonstrate that enhancing cGMP signaling, specifically downstream and independent of NO formation, in the dystrophin-deficient heart improves contractile performance, myocardial metabolic status, and sarcolemmal integrity and thus constitutes a potential clinical avenue for the treatment of the dystrophin-related cardiomyopathies.cardiomyopathy ͉ isotopes ͉ perfusion ͉ energy metabolism ͉ nitric oxide

Cystic fibrosis (CF) patients often have reduced mass and strength of skeletal muscles, including the diaphragm, the primary muscle of respiration. Here we show that lack of the CF transmembrane conductance regulator (CFTR) plays an intrinsic role in skeletal muscle atrophy and dysfunction. In normal murine and human skeletal muscle, CFTR is expressed and co-localized with sarcoplasmic reticulum-associated proteins. CFTR–deficient myotubes exhibit augmented levels of intracellular calcium after KCl-induced depolarization, and exposure to an inflammatory milieu induces excessive NF-kB translocation and cytokine/chemokine gene upregulation. To determine the effects of an inflammatory environment in vivo, sustained pulmonary infection with Pseudomonas aeruginosa was produced, and under these conditions diaphragmatic force-generating capacity is selectively reduced in Cftr −/− mice. This is associated with exaggerated pro-inflammatory cytokine expression as well as upregulation of the E3 ubiquitin ligases (MuRF1 and atrogin-1) involved in muscle atrophy. We conclude that an intrinsic alteration of function is linked to the absence of CFTR from skeletal muscle, leading to dysregulated calcium homeostasis, augmented inflammatory/atrophic gene expression signatures, and increased diaphragmatic weakness during pulmonary infection. These findings reveal a previously unrecognized role for CFTR in skeletal muscle function that may have major implications for the pathogenesis of cachexia and respiratory muscle pump failure in CF patients.

The availability of genetically modified mice requires the development of methods to assess heart function and metabolism in the intact beating organ. With the use of radioactive substrates and ex vivo perfusion of the mouse heart in the working mode, previous studies have documented glucose and fatty acid oxidation pathways. This study was aimed at characterizing the metabolism of other potentially important exogenous carbohydrate sources, namely, lactate and pyruvate. This was achieved by using (13)C-labeling methods. The mouse heart perfusion setup and buffer composition were optimized to reproduce conditions close to the in vivo milieu in terms of workload, cardiac functions, and substrate-hormone supply to the heart (11 mM glucose, 0.8 nM insulin, 50 microM carnitine, 1.5 mM lactate, 0.2 mM pyruvate, 5 nM epinephrine, 0.7 mM oleate, and 3% albumin). The use of three differentially (13)C-labeled carbohydrates and a (13)C-labeled long-chain fatty acid allowed the quantitative assessment of the metabolic origin and fate of tissue pyruvate as well as the relative contribution of substrates feeding acetyl-CoA (pyruvate and fatty acids) and oxaloacetate (pyruvate) for mitochondrial citrate synthesis. Beyond concurring with the notion that the mouse heart preferentially uses fatty acids for energy production (63.5 +/- 3.9%) and regulates its fuel selection according to the Randle cycle, our study reports for the first time in the mouse heart the following findings. First, exogenous lactate is the major carbohydrate contributing to pyruvate formation (42.0 +/- 2.3%). Second, lactate and pyruvate are constantly being taken up and released by the heart, supporting the concept of compartmentation of lactate and glucose metabolism. Finally, mitochondrial anaplerotic pyruvate carboxylation and citrate efflux represent 4.9 +/- 1.8 and 0.8 +/- 0.1%, respectively, of the citric acid cycle flux and are modulated by substrate supply. The described (13)C-labeling strategy combined with an experimental setup that enables continuous monitoring of physiological parameters offers a unique model to clarify the link between metabolic alterations, cardiac dysfunction, and disease development.