Temporal cardiomyocyte-specific TRF2 loss drives TERRA activation and systolic heart failure

Background Heart failure (HF) is the leading cause of mortality worldwide and is strongly associated with age-related myocardial degeneration. In patients with heart failure, shortened telomeres and reduced levels of telomeric repeat-binding factor 2 (TRF2) have been reported. We have previously demonstrated that HF induction in mice results in telomere attrition. Previous research on HF and telomere loss focused on either systemic TRF2 loss, or multigenerational studies of telomerase-deficient mice. which precludes discrimination between direct cardiomyocyte (CM) effects and systemic consequences in haematopoietic, immune or vascular compartments. Purpose We sought to determine whether primary telomere deprotection specific to CMs is sufficient to impair cardiac function, via targeted loss of the shelterin component TRF2, and to explore whether associated increases in telomeric repeat-containing RNA (TERRA), known to accumulate at the outer mitochondrial membrane, contribute to myocardial dysfunction. Methods TRF2 protein was confirmed by Western blot, and Immunofluorescence. Cardiac function was evaluated by echocardiography. Gene expression analysis was performed using RT-qPCR with the TaqMan system primers. For cells, TRF2 KD was done via TRF2 siRNA. Results In vitro, TRF2 knockdown (KD) in mouse cardiac fibroblasts, showed a significant increase in TERRA expression from Chromosome 18 (1.7-fold, p = 0.0039) and its sub-telomeric region (1.6-fold, p = 0.0448) compared to lipofectamine control. To investigate further we generated CM-specific inducible Cre driver mouse with Terf2 deletion (Terf2fl/fl; Myh6-MerCreMer, Cre heterozygous as experimental, and Cre negative as control mice). Terf2 deletion was induced by tamoxifen (TAM) administration (30 mg/kg, i.p., day 1-3) in mice aged 5 and 10 weeks, the study was terminated at week 8. Both experimental and control groups received TAM to control for Cre-independent effects. Echocardiographic assessment revealed that experimental mice exhibited a significant increase in heart-to-body weight ratio (p = 0.0031), reduced ejection fraction (p = 0.0444) compared to controls. No sex-specific differences were detected. At transcript level, CM TRF2 loss was associated with increased expression of other shelterin transcripts compared to control: Telomeric Repeat-Binding Factor 1 (1.5-fold), TERF1-interacting nuclear factor 2 (1.4-fold), and Protection of Telomeres 1b (1.5-fold). Conclusion Cardiomyocyte-specific TRF2 loss and consequent telomeric dysfunction are sufficient to induce systolic impairment, thus supporting the role of telomere loss in myocardial dysfunction rather than a secondary consequence of HF. These findings establish a direct mechanistic link between TRF2 loss, TERRA upregulation and myocardial dysfunction, and position telomere integrity as an upstream determinant and potential therapeutic target in age-related HF progression.