Abstract Body: Introduction: Aging-associated cardiac fibrosis drives diastolic dysfunction, yet the upstream mechanisms triggering cardiac fibroblast (CF) dysfunctions during cardiac aging remain unknown. Gata4, a transcription factor critical for cardiac homeostasis, declines progressively with age in both human and mouse hearts, with CFs showing the greatest reduction among all cardiac cell types. Whether age-dependent Gata4 loss drives CF dysfunctions remains unknown.
Methods: CF-specific (Tcf21-MerCreMer;Gata4^fl/fl) and cardiomyocyte-specific (αMHC-MerCreMer;Gata4^fl/fl) knockout (KO) mice were generated. Mitochondrial function was assessed by Seahorse XF respirometry and MitoTracker imaging; senescence was quantified by SA-β-gal, p16, and p21 over 10 days post-deletion. Single-nucleus RNA-seq (snRNA-seq) compared Gata4 KO and control mouse hearts. Human cardiac transcriptomic data (ages 20–70) were analyzed for correlation.
Results: CF-specific but not cardiomyocyte-specific Gata4 deletion induced robust senescence and fibrosis (p<0.001). snRNA-seq revealed that Gata4 KO CFs further showed coordinated downregulation of mitochondrial genes and upregulation of senescence-associated genes. Gata4 KO CFs displayed hyperfused mitochondrial morphology and significantly impaired basal respiration, maximal respiration, and ATP production (p<0.01). Critically, mitochondrial dysfunction was established at day 2 post-deletion when SA-β-gal staining remained negative; senescent cells emerged at day 3 and accumulated through day 10 (p<0.05), establishing temporal causality consistent with mitochondrial dysfunction-associated senescence (MiDAS). Gata4 KO CFs also displayed markedly increased SASP factor secretion including IL-6, CCL2, and TGF-β1, driving paracrine fibrogenic activation in neighboring cells. In human hearts, Gata4 strongly correlated with mitochondrial genes (r=0.93–0.98) and inversely correlated with senescence and fibrotic markers (r=−0.79 to −0.98). Importantly, Gata4 overexpression suppressed stress-induced senescence from >40% to <10% SA-β-gal+ cells (p<0.001).
Conclusion: In conclusion, age-dependent Gata4 decline drives MiDAS in CFs via mitochondrial gene suppression, establishing a conserved Gata4-mitochondria-senescence axis that sustains cardiac fibrosis. Restoring Gata4 identifies it as a novel upstream therapeutic target for age-associated cardiac fibrosis and diastolic dysfunction.