Although autophagy is generally protective, uncontrolled or excessive activation of autophagy can be detrimental. However, it is often difficult to distinguish death by autophagy from death with autophagy, and whether autophagy contributes to death in cardiomyocytes (CMs) is still controversial. Excessive activation of autophagy induces a morphologically and biochemically defined form of cell death termed autosis. Whether autosis is involved in tissue injury induced under pathologically relevant conditions is poorly understood. In the present study, myocardial ischemia/reperfusion (I/R) induced autosis in CMs, as evidenced by cell death with numerous vacuoles and perinuclear spaces, and depleted intracellular membranes. Autosis was observed frequently after 6 hours of reperfusion, accompanied by upregulation of Rubicon, attenuation of autophagic flux, and marked accumulation of autophagosomes. Genetic downregulation of Rubicon inhibited autosis and reduced I/R injury, whereas stimulation of autosis during the late phase of I/R with Tat–Beclin 1 exacerbated injury. Suppression of autosis by ouabain, a cardiac glycoside, in humanized Na⁺,K⁺-ATPase–knockin mice reduced I/R injury. Taken together, these results demonstrate that autosis is significantly involved in I/R injury in the heart and triggered by dysregulated accumulation of autophagosomes due to upregulation of Rubicon.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease associated with cardiomyopathy. DMD cardiomyopathy is characterized by abnormal intracellular Ca ²⁺ homeostasis and mitochondrial dysfunction. We used dystrophin and utrophin double-knockout ( mdx:utrn ^–/– ) mice in a sarcolipin (SLN) heterozygous-knockout ( sln ^+/– ) background to examine the effect of SLN reduction on mitochondrial function in the dystrophic myocardium. Germline reduction of SLN expression in mdx:utrn ^–/– mice improved cardiac sarco/endoplasmic reticulum (SR) Ca ²⁺ cycling, reduced cardiac fibrosis, and improved cardiac function. At the cellular level, reducing SLN expression prevented mitochondrial Ca ²⁺ overload, reduced mitochondrial membrane potential loss, and improved mitochondrial function. Transmission electron microscopy of myocardial tissues and proteomic analysis of mitochondria-associated membranes showed that reducing SLN expression improved mitochondrial structure and SR-mitochondria interactions in dystrophic cardiomyocytes. These findings indicate that SLN upregulation plays a substantial role in the pathogenesis of cardiomyopathy and that reducing SLN expression has clinical implications in the treatment of DMD cardiomyopathy.

Background/Objectives: Cardiomyopathy (CM) is a leading cause of morbidity and mortality in Duchenne muscular dystrophy (DMD) patients. Ferroptosis, an iron-dependent form of cell death characterized by lipid peroxidation, is implicated in various cardiovascular diseases. However, the role of ferroptosis in DMD-CM remains unexplored. Methods: Here, we used dystrophin and utrophin double-knockout (mdx:utr^−/−) mice as a model that exhibits cardiac pathological phenotypes similar to those seen in DMD patients to investigate the potential role of ferroptosis. Results: We observed an increased level of iron deposition and lipid peroxidation in the hearts of mdx:utr^−/− mice. Live/Dead viability assays revealed that mdx:utr^−/− cardiomyocytes exhibited greater susceptibility to ferroptosis than WT cardiomyocytes both at baseline and upon exposure to ferroptosis inducers. We also used mdx:utr^−/− mice with a heterozygous sarcolipin (SLN) knockout background (sln^+/−) to investigate the effect of SLN reduction on ferroptosis susceptibility in DMD-CM. Notably, ferroptosis was significantly suppressed in cardiomyocytes from mdx:utr^−/−:sln^+/− mice (p < 0.01). Western blot analysis confirmed the upregulation of transferrin receptor 1 (TfR1) and 15-lipoxygenase-1 (15LOX1), along with the downregulation of heme oxygenase-1 (HMOX-1) and ferroptosis suppressor protein 1 (FSP1) in mdx:utr^−/− hearts, while glutathione peroxidase 4 (GPX4) levels remained unchanged. A similar pattern of alterations in ferroptosis-related biomarkers was observed in human heart samples from DMD patients compared to healthy controls. Conclusions: Our results provide direct evidence that ferroptosis contributes to the pathology of DMD-CM and suggest that reducing SLN expression and inhibiting ferroptosis may represent potential therapeutic strategies for this condition.