β3-Adrenergic receptor overexpression in cardiomyocytes preconditions mitochondria to withstand ischemia–reperfusion injury

β3-Adrenergic receptor (β3AR) agonists have been shown to protect against ischemia–reperfusion injury (IRI). Since β3ARs are present both in cardiomyocytes and in endothelial cells, the cellular compartment responsible for this protection has remained unknown. Using transgenic mice constitutively expressing the human β3AR (hβ3AR) in cardiomyocytes or in the endothelium on a genetic background of null endogenous β3AR expression, we show that only cardiomyocyte expression protects against IRI (45 min ischemia followed by reperfusion over 24 h). Infarct size was also limited after ischemia–reperfusion in mice with cardiomyocyte hβ3AR overexpression on top of endogenous β3AR expression. hβ3AR overexpression in these mice reduced IRI-induced cardiac fibrosis and improved long-term left ventricular systolic function. Cardiomyocyte-specific β3AR overexpression resulted in a baseline remodeling of the mitochondrial network, characterized by upregulated mitochondrial biogenesis and a downregulation of mitochondrial quality control (mitophagy), resulting in elevated numbers of small mitochondria with a depressed capacity for the generation of reactive oxygen species but improved capacity for ATP generation. These processes precondition cardiomyocyte mitochondria to be more resistant to IRI. Upon reperfusion, hearts with hβ3AR overexpression display a restoration in the mitochondrial quality control and a rapid activation of antioxidant responses. Strong protection against IRI was also observed in mice infected with an adeno-associated virus (AAV) encoding hβ3AR under a cardiomyocyte-specific promoter. These results confirm the translational potential of increased cardiomyocyte β3AR expression, achieved either naturally through exercise or artificially through gene therapy approaches, to precondition the cardiomyocyte mitochondrial network to withstand future insults.