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Introduction: Sinoatrial node (SAN), the leading pacemaker region, generates electrical impulses that propagate throughout the heart. SAN dysfunction with bradyarrhythmia is well documented in heart failure (HF). However, the underlying mechanisms are not entirely understood. Mitochondria are critical to cellular processes that determine life or death of the cell. Therefore, proper communication with the mitochondria is essential for normal cellular function. We tested the hypothesis that alterations in the mitochondria-sarcoplasmic reticulum (SR) connectomics underlie SAN dysfunction in HF. Methods: We took advantage of a mouse model of pressure-overload induced HF by transverse aortic constriction (TAC) and a SAN-specific CRISPR/Cas9 mediated gene silencing of mitofusin-2 ( Mfn2 ). Results: TAC mice exhibited impaired cardiac function with HF, cardiac fibrosis, and profound SAN dysfunction. Ultrastructural imaging using electron microscope (EM) tomography revealed abnormal mitochondrial structure with increased SR-mitochondria distance. The expression of the SR-mitochondria tether GTPase protein Mfn-2 was significantly down-regulated and showed reduced colocalization with ryanodine receptor (RyR2) in HF SAN cells. Indeed, SAN-specific gene silencing of Mfn-2 led to alterations in the mitochondrial-SR microdomains[MFN1] and SAN dysfunction. Finally, disruption in the mitochondrial-SR microdomains resulted in abnormal mitochondrial Ca 2+ handling, alterations in localized PKA activity, and impaired mitochondrial function in HF SAN cells. Conclusion: The study provides insight on the role of mitochondrial-SR microdomains in SAN automaticity and into possible therapeutic targets for SAN dysfunction in HF patients.