This study investigates the role of mitochondrial DNA (mtDNA) that escapes from autophagy in the pathogenesis of heart failure. The authors found that mtDNA that escapes autophagy can trigger inflammatory responses through Toll-like receptor 9 (TLR9) in cardiomyocytes, leading to myocarditis and dilated cardiomyopathy. Cardiac-specific deletion of lysosomal deoxyribonuclease II (DNase II) in mice resulted in increased mortality, severe myocarditis, and dilated cardiomyopathy after pressure overload. Early in the disease process, DNase II-deficient hearts showed infiltration of inflammatory cells and increased mRNA expression of inflammatory cytokines, with mtDNA accumulation in autolysosomes. Administration of inhibitory oligodeoxynucleotides against TLR9 or ablation of Tlr9 attenuated the development of cardiomyopathy in DNase II-deficient mice. Additionally, Tlr9-ablation improved pressure overload-induced cardiac dysfunction and inflammation even in wild-type Dnase2a alleles. These findings provide new insights into the mechanisms of chronic inflammation in failing hearts.This study investigates the role of mitochondrial DNA (mtDNA) that escapes from autophagy in the pathogenesis of heart failure. The authors found that mtDNA that escapes autophagy can trigger inflammatory responses through Toll-like receptor 9 (TLR9) in cardiomyocytes, leading to myocarditis and dilated cardiomyopathy. Cardiac-specific deletion of lysosomal deoxyribonuclease II (DNase II) in mice resulted in increased mortality, severe myocarditis, and dilated cardiomyopathy after pressure overload. Early in the disease process, DNase II-deficient hearts showed infiltration of inflammatory cells and increased mRNA expression of inflammatory cytokines, with mtDNA accumulation in autolysosomes. Administration of inhibitory oligodeoxynucleotides against TLR9 or ablation of Tlr9 attenuated the development of cardiomyopathy in DNase II-deficient mice. Additionally, Tlr9-ablation improved pressure overload-induced cardiac dysfunction and inflammation even in wild-type Dnase2a alleles. These findings provide new insights into the mechanisms of chronic inflammation in failing hearts.