The relative contributions of cardiomyocytes versus endothelial cells to cardiac BH4 production; studies in novel cardiomyocyte- and endothelial-specific GTPCH I-deficient mice

Abstract

BH4 is a critical cofactor for the NOS enzymes and is essential for NOS coupling and NO production. NO modulates cardiac function, although its exact role remains unclear. Recent advances have shown that both eNOS and nNOS are present in cardiomyocytes, yet evidence suggests that BH4 synthesis in myocytes is suboptimal. Currently, little is known about cardiac BH4, yet this information is essential to understand the roles of NOSs/BH4 in the heart. Using 2 novel floxed mouse models of cell-specific GTPCH I deficiency, the rate-limiting enzyme in de novo BH4 biosynthesis, we aimed to elucidate the relative contributions of myocytes and endothelial cells to cardiac BH4 levels. PCR confirmed cardiomyocyte- and endothelial-specific GCH1 excision in GCHfl/flMHCcre and GCHfl/flTie2cre mice, respectively, concomitant with depleted GTPCH1 protein and BH4 production in these cells. Whereas BH4 and total biopterins were significantly reduced in GCHfl/flTie2cre hearts compared to GCHfl/fl (0.55 ± 0.17 versus 1.88 ± 0.28 pmol BH4/mg protein, respectively), levels remained unchanged in GCHfl/flMHCcre hearts, indicating that myocytes are not a major contributor of cardiac biopterins. Furthermore, BH4 levels in isolated wild-type heart endothelial cells were >4-fold higher than in isolated cardiomyocytes (2.7 ± 0.97 versus 0.53 ± 0.23 mol/mg protein). However, despite low BH4 levels in wild-type cardiomyocytes, reducing this further in GCHfl/flMHCcre mice significantly increased heart:body weight. These studies demonstrate that endothelial cells, not cardiomyocytes, are a major source of cardiac biopterins. However, depleting BH4 specifically in myocytes elicits cardiac morphometric changes. Future studies will explore the impact of BH4 deficiency in these cell types on cardiovascular diseases.