Abstract 14: Identification of a Major Role for Cytochrome B5 Reductase 3 in Cardiomyocyte Metabolism and Function

Abstract

Cytochrome B 5 Reductase 3 (Cyb5R3) also known as methemoglobin reductase regulates redox signaling in erythrocytes and endothelial cells by maintaining heme iron in the reduced (Fe 2+ ) state. Knowing the importance of highly regulated redox signaling in other hematopoietic and somatic lineages, we conducted a pharmacological inhibition study using a CyB5R3 inhibitor in mice. Inhibition resulted in dilated cardiomyopathy (DCM) after 2 weeks. To determine the potential for human relevance, a high frequency point mutation (T117S) in African American populations was studied and served as a model to understand the impact of mutated CyB5R3 in human heart failure. We found T117S individuals associated with accelerated time to first acute cardiac events and time to death. With this evidence and the unknown function of CyB5R3 in cardiomyocytes, we created the first CyB5R3, cardiomyocyte specific inducible knockout (i-cKO) (Myh6-Cre ERT2 - flox/flox) and we observed >50% lethality 15 days post-last tamoxifen injection. These mice recapitulated a DCM phenotype similar to the pharmacological study. Hemodynamic measurements showed increased left and right ventricular stroke volumes and decreased ejection fractions. Histology displayed myocardial inflammation and early stage fibrosis while electron microscopy revealed myofibril dystrophy. With these results, we hypothesized CyB5R3 i-cKO mice develop impaired metabolism and bioenergetics due to loss of CyB5R3 mediated heme reduction. I-cKO animals had smaller mitochondrial size, a 30% loss of total ATP and a rise in lactate production, indicating glycolytic shift from oxidative phosphorylation. RNAseq analysis showed decreased transcription in mitochondrial complexes I, II and IV and decreased complex IV activity. Since oxygen is the final electron acceptor for complex IV, we hypothesized the loss of CyB5R3 impaired oxygen delivery to the mitochondria. Therefore, a “psuedohypoxic state” was created, which was supported via Hypoxyprobe staining, by labeling cardiomyocytes specifically with low pO 2 . Collectively, the results provide an important breakthrough in cardiomyocyte biology by identifying Cyb5R3 as the first heme iron reductase critical for regulating cardiac metabolism.