Abstract Body: Background: Innate immune activation is critical for cardiac hypertrophy; however, its upstream regulation is less well understood. Coiled-coil-helix-coiled-coil-helix domain 4 (CHCHD4) is an essential carrier for the import of various mitochondrial proteins including TP53-regulated inhibitor of apoptosis 1 (TRIAP1). CHCHD4 was recently identified as a dysregulated gene in patients with dilated cardiomyopathy and was separately found to mediate innate immune signaling in skeletal muscle. Therefore, we hypothesized that CHCHD4 may play a role in the development of cardiac hypertrophy by regulating innate immunity. Methods: Genetically modified C57BL/6 mice overexpressing CHCHD4 (CHCHD4 Tg) or haploinsufficient in CHCHD4 (CHCHD4^+/-) were utilized. Mice were injected either with low dose (2 mg/kg) or high dose (30 mg/kg) isoproterenol (ISO) daily for up to 3 wk to induce hypertrophy. Results: CHCHD4^+/- mice developed cardiac hypertrophy (HW/BW (mg/g): 3.62 ± 0.15 CHCHD4^+/- vs 2.70 ± 0.13 wild-type (WT) or 2.55 ± 0.16 CHCHD4 Tg, p<0.0001) when allowed to age to 28 wk and showed reduced left ventricular ejection fraction (EF) at 1 year when compared with wild-type or CHCHD4 Tg mice (EF (%): 50.94 ± 0.94 CHCHD4^+/- vs 58.33 ± 0.78 wild-type or 57.88 ± 1.32 CHCHD4 Tg, p<0.0001). After low dose ISO treatment, CHCHD4^+/- mice had reduced EF at 2 wk compared to WT or CHCHD4 Tg mice (EF (%): 44.60 ± 1.74 CHCHD4^+/- vs 54.28 ± 0.86 wild-type, p<0.0001; or vs 52.76 ± 1.33 CHCHD4 Tg, p=0.0006). After high dose ISO treatment, WT mice developed cardiac hypertrophy by 1 wk and impaired EF by 3 wk, while CHCHD4 Tg mice remained normal. ISO treatment caused reduction of CHCHD4 levels in both the 24 hr and 1 wk states. This reduction was associated with decreased TRIAP1 and activation of the cGAS-STING-NFkB pathway in wild-type, but not CHCHD4 Tg mice. Further, plasma mitochondrial DNA (mtDNA) was 2.3 times higher in WT mice treated with high dose ISO for 1 wk compared to WT control (n=8-11/group, p=0.011), while that of CHCHD4 Tg mice was not significantly changed by ISO treatment. Conclusions: Our findings suggest that CHCHD4 may be a key upstream regulator of innate immune activation mediating cardiac hypertrophy and that cell-free mtDNA in blood could be a potential biomarker of pathogenesis.