Mitochondrial DNA integrity and antioxidant determinants of resilience to anthracycline-induced cardiotoxicity

Background

Anthracycline cardiotoxicity affects up to one third of patients at long-term follow-up, yet current risk stratification tools cannot predict individual susceptibility. Some patients develop severe heart failure whilst others tolerate high exposures without dysfunction, suggesting that resilience mechanisms, rather than cumulative dose, determine individual response.

Purpose

To identify molecular determinants distinguishing resilient from susceptible phenotypes using patient-derived cardiomyocytes representing clinical extremes.

Methods

We recruited patients from opposite ends of the cardiotoxicity spectrum: three resilient patients (RES) tolerated high doxorubicin doses (490±46 mg/m²) without cardiac dysfunction for over 30 years, whilst two cardiotoxic patients (TOX) developed severe heart failure requiring mechanical circulatory support after lower exposures (303±85 mg/m²). Patient-derived induced pluripotent stem cells were differentiated into cardiomyocytes and assembled into three-dimensional dynamic engineered heart tissues (dyn-EHT). We performed chronic low-dose doxorubicin exposure (four 48-hour cycles at 0.1 µmol/L), mitochondrial DNA sequencing, bioenergetic profiling, and antioxidant analysis.

Results

After chronic doxorubicin exposure, TOX-dyn-EHT exhibited progressive tissue dilatation (11.4±0.3 versus 7.9±0.3 mm in RES, p<0.001), increased fibrosis, and severe contractile dysfunction with a 56% decline in contractile force in TOX, whilst RES maintained contractile function (p=0.007). Metabolically, TOX cells exhibited 51% decline in ATP-linked respiration whilst RES maintained function (3% decline, p<0.001) and 31% reduction in glycolytic capacity versus 13% in RES (p=0.007). Adaptive uncoupling increased 98% in RES (p<0.001) versus 31% in TOX (p=0.048).

Mechanistically, mitochondrial DNA sequencing revealed two key findings replicated across independent differentiations. First, total indel burden was elevated in TOX versus RES cardiomyocytes (5.28 versus 3.43 per 1,000 reads, p=0.0026) and patient-matched fibroblasts (p=0.0499), with similar elevation observed in independent validation cohort. Second, TOX cardiomyocytes exhibited insertion-biased mutagenesis (76% versus 54% insertions, p<0.001), a cardiomyocyte-specific pattern, representing the first demonstration of cell-type-specific mutagenic signatures in anthracycline cardiotoxicity. Additionally, RES cardiomyocytes exhibited elevated antioxidant protein levels, most notably 3.14-fold higher glutathione S-transferase activity (0.597 versus 0.190 nmol/min/µg protein, p=0.011).

Conclusions

Patient iPSC-derived 3D models recapitulated both phenotypes, with RES maintaining function whilst TOX exhibited progressive failure. Pre-existing mitochondrial genome integrity and antioxidant capacity distinguish resilient from susceptible phenotypes, providing constitutional biomarkers for pre-treatment risk stratification and personalized cardioprotection.Figure 1Feed forwar model of AIC