Sequential mitochondrial remodeling in cardiomyocytes drives diastolic dysfunction and transition to HFrEF in anthracycline-induced cardiotoxicity

Abstract Aims Doxorubicin (DOX) is a highly effective chemotherapeutic agent whose clinical use is limited by cumulative cardiotoxicity. The subcellular origins of early cardiac injury remain unclear but cardiomyocyte (CM) mitochondrial dysfunction is implicated. However, vulnerability of specific CM mitochondrial subpopulations is unknown. Building on our previous work linking the postnatal maturation of crest-associated subsarcolemmal mitochondria (SSM) at the CM surface to diastolic function, we investigated the spatial and temporal susceptibility of SSM during DOX exposure and their contribution to early diastolic dysfunction. Methods and results Adult male mice received chronic DOX treatment (5 mg/kg/week for 5 weeks) to mimic cumulative clinical exposure. Cardiac function was monitored longitudinally, during treatment and after protocol completion, using echocardiography-Doppler imaging, and global longitudinal strain (GLS). Subcellular mitochondrial remodeling was assessed using atomic force microscopy (AFM) and transmission electron microscopy (TEM). A tamoxifen-inducible, CM-specific Ephrin-B1 knockout model was used to probe the functional role of SSM in DOX-induced injury. DOX induced a progressive and selective loss of crest/SSM at the CM surface very early within 3 days of exposure, while the architecture of interfibrillar mitochondria IFM remained preserved. This early SSM depletion paralleled impaired myocardial relaxation reflected by a prolonged isovolumic relaxation time, along with reduced GLS, all preceding changes in left ventricular ejection fraction or detectable IFM abnormalities. Notably, in mice lacking Ephrin-B1, and therefore mature crest/SSM, DOX exposure triggered an unusually rapid onset of systolic dysfunction, highlighting the cardioprotective role of these surface mitochondrial populations. Conclusions Crest/SSM at the CM surface are the earliest selective mitochondrial targets of DOX, and their loss precedes IFM remodeling. This spatial-temporal hierarchy reveals a compartment-specific functional distinction, with SSM supporting diastolic performance and IFM sustaining systolic contraction. Hence, preserving SSM emerges as a promising early target to prevent progression of anthracycline cardiotoxicity toward systolic failure. Clinically, our findings also support early diastolic monitoring as a sensitive approach for detecting anthracycline cardiotoxicity.