Aging is a progressive decline of body function, during which many tissues accumulate few cells with high levels of deleted mitochondrial DNA (mtDNA), leading to a defect of mitochondrial functions. Whether this mosaic mitochondrial deficiency contributes to organ dysfunction is unknown. To investigate this, we generated mice with an accelerated accumulation of mtDNA deletions in the myocardium, by expressing a dominant-negative mutant mitochondrial helicase. These animals accumulated few randomly distributed cardiomyocytes with compromised mitochondrial function, which led to spontaneous ventricular premature contractions and AV blocks at 18 months. These symptoms were not caused by a general mitochondrial dysfunction in the entire myocardium, and were not observed in mice at 12 months with significantly lower numbers of dysfunctional cells. Therefore, our results suggest that the disposition to arrhythmia typically found in the aged human heart might be due to the random accumulation of mtDNA deletions and the subsequent mosaic respiratory chain deficiency. Display omitted •Cardiomyocytes steadily accumulate mitochondrial DNA deletions with aging•This leads to the development of a tissue mosaic of mitochondrial deficiency•Impaired mitochondrial function in few cells promotes cardiac arrhythmia Baris et al. test the idea of mosaic respiratory chain deficiency contributing to aging-related human cardiac disease. Using a genetic mouse model of accelerated accumulation of mtDNA deletions in the heart, they show that a few cardiomyocytes with mitochondrial DNA deletions progressively accumulate during aging, leading to cardiac arrhythmias.