Rationale: Diaphragmatic function is a major determinant of the ability to successfully wean patients from mechanical ventilation (MV). Paradoxically, MV itself results in a rapid loss of diaphragmatic strength in animals. However, very little is known about the time course or mechanistic basis for such a phenomenon in humans. Objectives: To determine in a prospective fashion the time course for development of diaphragmatic weakness during MV; and the relationship between MV duration and diaphragmatic injury or atrophy, and the status of candidate cellular pathways implicated in these phenomena. Methods: Airway occlusion pressure (TwPtr) generated by the diaphragm during phrenic nerve stimulation was measured in short-term (0.5 h; n = 6) and long-term (>5 d; n = 6) MV groups. Diaphragmatic biopsies obtained during thoracic surgery (MV for 2–3 h; n = 10) and from brain-dead organ donors (MV for 24–249 h; n = 15) were analyzed for ultrastructural injury, atrophy, and expression of proteolysis-related proteins (ubiquitin, nuclear factor-κB, and calpains). Measurements and Main Results: TwPtr decreased progressively during MV, with a mean reduction of 32 ± 6% after 6 days. Longer periods of MV were associated with significantly greater ultrastructural fiber injury (26.2 ± 4.8 vs. 4.7 ± 0.6% area), decreased cross-sectional area of muscle fibers (1,904 ± 220 vs. 3,100 ± 329 μm²), an increase of ubiquitinated proteins (+19%), higher expression of p65 nuclear factor-κB (+77%), and greater levels of the calcium-activated proteases calpain-1, -2, and -3 (+104%, +432%, and +266%, respectively) in the diaphragm. Conclusions: Diaphragmatic weakness, injury, and atrophy occur rapidly in critically ill patients during MV, and are significantly correlated with the duration of ventilator support.

Increased calcium levels in dystrophic muscle have damaging consequences. In this report, Bellinger et al. show that nitrosylation of the ryanodine receptor calcium channel, leading to calcium leak through the channel, is an underlying cause of increased calcium levels in the muscle of dystrophic mdx mice. Treatment of the mice with a compound that inhibits calcium leak increases their muscle function and physical activity, pointing to a potential new treatment for muscular dystrophy ( pages 243–244 ). Duchenne muscular dystrophy is characterized by progressive muscle weakness and early death resulting from dystrophin deficiency. Loss of dystrophin results in disruption of a large dystrophin glycoprotein complex, leading to pathological calcium (Ca2+)-dependent signals that damage muscle cells1,2,3,4,5. We have identified a structural and functional defect in the ryanodine receptor (RyR1), a sarcoplasmic reticulum Ca2+ release channel, in the mdx mouse model of muscular dystrophy that contributes to altered Ca2+ homeostasis in dystrophic muscles. RyR1 isolated from mdx skeletal muscle showed an age-dependent increase in S-nitrosylation coincident with dystrophic changes in the muscle. RyR1 S-nitrosylation depleted the channel complex of FKBP12 (also known as calstabin-1, for calcium channel stabilizing binding protein), resulting in 'leaky' channels. Preventing calstabin-1 depletion from RyR1 with S107, a compound that binds the RyR1 channel and enhances the binding affinity of calstabin-1 to the nitrosylated channel, inhibited sarcoplasmic reticulum Ca2+ leak, reduced biochemical and histological evidence of muscle damage, improved muscle function and increased exercise performance in mdx mice. On the basis of these findings, we propose that sarcoplasmic reticulum Ca2+ leak via RyR1 due to S-nitrosylation of the channel and calstabin-1 depletion contributes to muscle weakness in muscular dystrophy, and that preventing the RyR1-mediated sarcoplasmic reticulum Ca2+ leak may provide a new therapeutic approach.

During exercise, defects in calcium (Ca ²⁺ ) release have been proposed to impair muscle function. Here, we show that during exercise in mice and humans, the major Ca ²⁺ release channel required for excitation–contraction coupling (ECC) in skeletal muscle, the ryanodine receptor (RyR1), is progressively PKA-hyperphosphorylated, S -nitrosylated, and depleted of the phosphodiesterase PDE4D3 and the RyR1 stabilizing subunit calstabin1 (FKBP12), resulting in “leaky” channels that cause decreased exercise tolerance in mice. Mice with skeletal muscle-specific calstabin1 deletion or PDE4D deficiency exhibited significantly impaired exercise capacity. A small molecule (S107) that prevents depletion of calstabin1 from the RyR1 complex improved force generation and exercise capacity, reduced Ca ²⁺ -dependent neutral protease calpain activity and plasma creatine kinase levels. Taken together, these data suggest a possible mechanism by which Ca ²⁺ leak via calstabin1-depleted RyR1 channels leads to defective Ca ²⁺ signaling, muscle damage, and impaired exercise capacity.

Background—: Under physiological conditions, Ca ²⁺ transfer from the endoplasmic reticulum (ER) to mitochondria might occur at least in part at contact points between the 2 organelles and involves the VDAC1/Grp75/IP3R1 complex. Accumulation of Ca ²⁺ into the mitochondrial matrix may activate the mitochondrial chaperone cyclophilin D (CypD) and trigger permeability transition pore opening, whose role in ischemia/reperfusion injury is well recognized. We questioned here whether the transfer of Ca ²⁺ from ER to mitochondria might play a role in cardiomyocyte death after hypoxia-reoxygenation. Methods and Results—: We report that CypD interacts with the VDAC1/Grp75/IP3R1 complex in cardiomyocytes. Genetic or pharmacological inhibition of CypD in both H9c2 cardiomyoblasts and adult cardiomyocytes decreased the Ca ²⁺ transfer from ER to mitochondria through IP3R under normoxic conditions. During hypoxia-reoxygenation, the interaction between CypD and the IP3R1 Ca ²⁺ channeling complex increased concomitantly with mitochondrial Ca ²⁺ content. Inhibition of either CypD, IP3R1, or Grp75 decreased protein interaction within the complex, attenuated mitochondrial Ca ²⁺ overload, and protected cells from hypoxia-reoxygenation. Genetic or pharmacological inhibition of CypD provided a similar effect in adult mice cardiomyocytes. Disruption of ER-mitochondria interaction via the downregulation of Mfn2 similarly reduced the interaction between CypD and the IP3R1 complex and protected against hypoxia-reoxygenation injury. Conclusions—: Our data (1) point to a new role of CypD at the ER-mitochondria interface and (2) suggest that decreasing ER-mitochondria interaction at reperfusion can protect cardiomyocytes against lethal reperfusion injury through the reduction of mitochondrial Ca ²⁺ overload via the CypD/VDAC1/Grp75/IP3R1 complex.