Mitochondrial calcium overload is a key determinant in heart failure

Calcium (Ca2+) released from the sarcoplasmic reticulum (SR) is crucial for excitation-contraction (E-C) coupling. Mitochondria, the major source of energy, in the form of ATP, required for cardiac contractility, are closely interconnected with the SR, and Ca2+ is essential for optimal function of these organelles. However, Ca2+ accumulation can impair mitochondrial function, leading to reduced ATP production and increased release of reactive oxygen species (ROS). Oxidative stress contributes to heart failure (HF), but whether mitochondrial Ca2+ plays a mechanistic role in HF remains unresolved. Here, we show for the first time, to our knowledge, that diastolic SR Ca2+ leak causes mitochondrial Ca2+ overload and dysfunction in a murine model of postmyocardial infarction HF. There are two forms of Ca2+ release channels on cardiac SR: type 2 ryanodine receptors (RyR2s) and type 2 inositol 1,4,5-trisphosphate receptors (IP3R2s). Using murine models harboring RyR2 mutations that either cause or inhibit SR Ca2+ leak, we found that leaky RyR2 channels result in mitochondrial Ca2+ overload, dysmorphology, and malfunction. In contrast, cardiac-specific deletion of IP3R2 had no major effect on mitochondrial fitness in HF. Moreover, genetic enhancement of mitochondrial antioxidant activity improved mitochondrial function and reduced posttranslational modifications of RyR2 macromolecular complex. Our data demonstrate that leaky RyR2, but not IP3R2, channels cause mitochondrial Ca2+ overload and dysfunction in HF

Genetically enhancing mitochondrial antioxidant activity improves muscle function in aging

Age-related skeletal muscle dysfunction is a leading cause of morbidity that affects up to half the population aged 80 or greater. Here we tested the effects of increased mitochondrial antioxidant activity on age-dependent skeletal muscle dysfunction using transgenic mice with targeted overexpression of the human catalase gene to mitochondria (MCat mice). Aged MCat mice exhibited improved voluntary exercise, increased skeletal muscle specific force and tetanic Ca(2+) transients, decreased intracellular Ca(2+) leak and increased sarcoplasmic reticulum (SR) Ca(2+) load compared with age-matched wild type (WT) littermates. Furthermore, ryanodine receptor 1 (the sarcoplasmic reticulum Ca(2+) release channel required for skeletal muscle contraction; RyR1) from aged MCat mice was less oxidized, depleted of the channel stabilizing subunit, calstabin1, and displayed increased single channel open probability (Po). Overall, these data indicate a direct role for mitochondrial free radi! cals in promoting the pathological intracellular Ca(2+) leak that underlies age-dependent loss of skeletal muscle function. This study harbors implications for the development of novel therapeutic strategies, including mitochondria-targeted antioxidants for treatment of mitochondrial myopathies and other healthspan-limiting disorders

Mitochondrial oxidative stress promotes atrial fibrillation

Oxidative stress has been suggested to play a role in the pathogenesis of atrial fibrillation (AF). Indeed, the prevalence of AF increases with age as does oxidative stress. However, the mechanisms linking redox state to AF are not well understood. In this study we identify a link between oxidative stress and aberrant intracellular Ca(2+) release via the type 2 ryanodine receptor (RyR2) that promotes AF. We show that RyR2 are oxidized in the atria of patients with chronic AF compared with individuals in sinus rhythm. To dissect the molecular mechanism linking RyR2 oxidation to AF we used two murine models harboring RyR2 mutations that cause intracellular Ca(2+) leak. Mice with intracellular Ca(2+) leak exhibited increased atrial RyR2 oxidation, mitochondrial dysfunction, reactive oxygen species (ROS) production and AF susceptibility. Both genetic inhibition of mitochondrial ROS production and pharmacological treatment of RyR2 leakage prevented AF. Collectively, our results indicate that alterations of RyR2 and mitochondrial ROS generation form a vicious cycle in the development of AF. Targeting this previously unrecognized mechanism could be useful in developing effective interventions to prevent and treat AF

Leaky ryanodine receptors contribute to diaphragmatic weakness during mechanical ventilation

Ventilator-induced diaphragmatic dysfunction (VIDD) refers to the diaphragm muscle weakness that occurs following prolonged controlled mechanical ventilation (MV). The presence of VIDD impedes recovery from respiratory failure. However, the pathophysiological mechanisms accounting for VIDD are still not fully understood. Here, we show in human subjects and a mouse model of VIDD that MV is associated with rapid remodeling of the sarcoplasmic reticulum (SR) Ca2+ release channel/ryanodine receptor (RyR1) in the diaphragm. The RyR1 macromolecular complex was oxidized, S-nitrosylated, Ser-2844 phosphorylated, and depleted of the stabilizing subunit calstabin1, following MV. These posttranslational modifications of RyR1 were mediated by both oxidative stress mediated by MV and stimulation of adrenergic signaling resulting from the anesthesia. We demonstrate in the murine model that such abnormal resting SR Ca2+ leak resulted in reduced contractile function and muscle fiber atrophy for longer duration of MV. Treatment with β-adrenergic antagonists or with S107, a small molecule drug that stabilizes the RyR1–calstabin1 interaction, prevented VIDD. Diaphragmatic dysfunction is common in MV patients and is a major cause of failure to wean patients from ventilator support. This study provides the first evidence to our knowledge of RyR1 alterations as a proximal mechanism underlying VIDD (i.e., loss of function, muscle atrophy) and identifies RyR1 as a potential target for therapeutic intervention

Aberrant mitochondrial dynamics contributes to diaphragmatic weakness induced by mechanical ventilation.

In critical care patients, the “”temporary inactivity of the diaphragm caused by mechanical ventilation (MV) triggers a series of events leading to diaphragmatic dysfunction and atrophy, commonly known as ventilator-induced diaphragm dysfunction (VIDD). While mitochondrial dysfunction related to oxidative stress is recognized as a crucial factor in VIDD, the exact molecular mechanism remains poorly understood. In this study, we observe that 6 h of MV triggers aberrant mitochondrial dynamics, resulting in a reduction in mitochondrial size and interaction, associated with increased expression of dynamin-related protein 1 (DRP1). This effect can be prevented by P110, a molecule that inhibits the recruitment of DRP1 to the mitochondrial membrane. Furthermore, isolated mitochondria from the diaphragms of ventilated patients exhibited increased production of reactive oxygen species (ROS). These mitochondrial changes were associated with the rapid oxidation of type 1 ryanodine receptor (RyR1) and a decrease in the stabilizing subunit calstabin 1. Subsequently, we observed that the sarcoplasmic reticulum (SR) in the ventilated diaphragms showed increased calcium leakage and reduced contractile function. Importantly, the mitochondrial fission inhibitor P110 effectively prevented all of these alterations. Taken together, the results of our study illustrate that MV leads, in the diaphragm, to both mitochondrial fragmentation and dysfunction, linked to the up-/down-regulation of 320 proteins, as assessed through global comprehensive quantitative proteomics analysis, primarily associated with mitochondrial function. These outcomes underscore the significance of developing compounds aimed at modulating the balance between mitochondrial fission and fusion as potential interventions to mitigate VIDD in human patients

Leaky ryanodine receptors in β-sarcoglycan deficient mice: a potential common defect in muscular dystrophy

Disruption of the sarcolemma-associated dystrophin-glycoprotein complex underlies multiple forms of muscular dystrophy, including Duchenne muscular dystrophy and sarcoglycanopathies. A hallmark of these disorders is muscle weakness. In a murine model of Duchenne muscular dystrophy, mdx mice, cysteine-nitrosylation of the calcium release channel/ryanodine receptor type 1 (RyR1) on the skeletal muscle sarcoplasmic reticulum causes depletion of the stabilizing subunit calstabin1 (FKBP12) from the RyR1 macromolecular complex. This results in a sarcoplasmic reticular calcium leak via defective RyR1 channels. This pathological intracellular calcium leak contributes to reduced calcium release and decreased muscle force production. It is unknown whether RyR1 dysfunction occurs also in other muscular dystrophies. To test this we used a murine model of Limb-Girdle muscular dystrophy, deficient in β-sarcoglycan (Sgcb−/−). Skeletal muscle RyR1 from Sgcb−/− deficient mice were oxidized, nitrosylated, and depleted of the stabilizing subunit calstabin1, which was associated with increased open probability of the RyR1 channels. Sgcb−/− deficient mice exhibited decreased muscle specific force and calcium transients, and displayed reduced exercise capacity. Treating Sgcb−/− mice with the RyR stabilizing compound S107 improved muscle specific force, calcium transients, and exercise capacity. We have previously reported similar findings in mdx mice, a murine model of Duchenne muscular dystrophy. Our data suggest that leaky RyR1 channels may underlie multiple forms of muscular dystrophy linked to mutations in genes encoding components of the dystrophin-glycoprotein complex. A common underlying abnormality in calcium handling indicates that pharmacological targeting of dysfunctional RyR1 could be a novel therapeutic approach to improve muscle function in Limb-Girdle and Duchenne muscular dystrophies

Acute RyR1 Ca2+ leak enhances NADH-linked mitochondrial respiratory capacity

Sustained ryanodine receptor (RyR) Ca2+ leak is associated with pathological conditions such as heart failure or skeletal muscle weakness. We report that a single session of sprint interval training (SIT), but not of moderate intensity continuous training (MICT), triggers RyR1 protein oxidation and nitrosylation leading to calstabin1 dissociation in healthy human muscle and in in vitro SIT models (simulated SIT or S-SIT). This is accompanied by decreased sarcoplasmic reticulum Ca2+ content, increased levels of mitochondrial oxidative phosphorylation proteins, supercomplex formation and enhanced NADH-linked mitochondrial respiratory capacity. Mechanistically, (S-)SIT increases mitochondrial Ca2+ uptake in mouse myotubes and muscle fibres, and decreases pyruvate dehydrogenase phosphorylation in human muscle and mouse myotubes. Countering Ca2+ leak or preventing mitochondrial Ca2+ uptake blunts S-SIT-induced adaptations, a result supported by proteomic analyses. Here we show that triggering acute transient Ca2+ leak through RyR1 in healthy muscle may contribute to the multiple health promoting benefits of exercise

PKA phosphorylation activates the calcium release channel (ryanodine receptor) in skeletal muscle: defective regulation in heart failure

The type 1 ryanodine receptor (RyR1) on the sarcoplasmic reticulum (SR) is the major calcium (Ca2+) release channel required for skeletal muscle excitation–contraction (EC) coupling. RyR1 function is modulated by proteins that bind to its large cytoplasmic scaffold domain, including the FK506 binding protein (FKBP12) and PKA. PKA is activated during sympathetic nervous system (SNS) stimulation. We show that PKA phosphorylation of RyR1 at Ser2843 activates the channel by releasing FKBP12. When FKB12 is bound to RyR1, it inhibits the channel by stabilizing its closed state. RyR1 in skeletal muscle from animals with heart failure (HF), a chronic hyperadrenergic state, were PKA hyperphosphorylated, depleted of FKBP12, and exhibited increased activity, suggesting that the channels are “leaky.” RyR1 PKA hyperphosphorylation correlated with impaired SR Ca2+ release and early fatigue in HF skeletal muscle. These findings identify a novel mechanism that regulates RyR1 function via PKA phosphorylation in response to SNS stimulation. PKA hyperphosphorylation of RyR1 may contribute to impaired skeletal muscle function in HF, suggesting that a generalized EC coupling myopathy may play a role in HF