Nrf3-Mediated Mitochondrial Superoxide Promotes Cardiomyocyte Apoptosis and Impairs Cardiac Functions by Suppressing Pitx2.

Abstract

Background: Myocardial infarction (MI) elicits mitochondria reactive oxygen species (ROS) production and cardiomyocyte (CM) apoptosis. Nrf3 (nuclear factor erythroid 2-related factor 3) has an established role in regulating redox signaling and tissue homeostasis. Here, we aimed to evaluate the role and mechanism of Nrf3 in injury-induced pathological cardiac remodeling.

Methods: Global (Nrf3-KO) and CM-specific (Nrf3^△CM) Nrf3 knockout mice were subjected to MI or ischemia/reperfusion injury, followed by functional and histopathological analysis. Primary neonatal mouse and rat ventricular myocytes and CMs derived from human induced pluripotent stem cells were used to evaluate the impact of Nrf3 on CM apoptosis and mitochondrial ROS production. Chromatin immunoprecipitation sequencing and immunoprecipitation-mass spectrometry analysis were used to uncover potential targets of Nrf3. MitoParaquat administration and CM-specific adeno-associated virus vectors were used to further confirm the in vivo relevance of the identified signal pathways.

Results: Nrf3 was expressed mainly in CMs in healthy human hearts, and an increased level of Nrf3 was observed in CMs within the border zone of infarcted human hearts and murine cardiac tissues after MI. Both global and CM-specific Nrf3 knockout significantly decreased injury-induced mitochondrial ROS production, CM apoptosis, and pathological cardiac remodeling, consequently improving cardiac functions. In addition, cardiac-specific Nrf3 overexpression reversed the ameliorative cardiac phenotypes observed in Nrf3-KO mice. Functional studies showed that Nrf3 promoted neonatal mouse ventricular myocyte, neonatal rat ventricular myocyte, and CMs derived from human induced pluripotent stem cell apoptosis by increasing mitochondrial ROS production. Critically, augmenting mitochondrial ROS with MitoParaquat blunted the beneficial effects of Nrf3 deletion on cardiac function and remodeling. Mechanistically, a redox regulator Pitx2 (paired-like homeodomain transcription factor 2) was identified as one of the main target genes of Nrf3. Specifically, Nrf3 binds to Pitx2 promoter, where it increases DNA methylation through recruiting heterogeneous nuclear ribonucleoprotein K and DNA-methyltransferase 1 complex, thereby inhibiting Pitx2 expression. CM-specific knockdown of Pitx2 blunted the beneficial effects of Nrf3 deletion on cardiac function and remodeling, and cardiac-specific Pitx2 overexpression attenuated MI-induced mitochondrial ROS production and CM apoptosis, as well as preserved cardiac functions after MI.

Conclusions: Nrf3 promotes injury-induced CM apoptosis and deteriorates cardiac functions by increasing mitochondrial ROS production through suppressing Pitx2 expression. Targeting the Nrf3-Pitx2-mitochondrial ROS signal axis may therefore represent a novel therapeutic approach for MI treatment.