P87Regulation of myocyte function by gch1 overexpression mediated increase in nitric oxide

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Abstract

Purpose

By causing uncoupling of nitric oxide synthase (NOS) activity, myocardial depletion of the NOS co-factor BH4 has been associated with adverse LV remodelling and impaired relaxation in animal models of cardiac disease. Oral supplementation of BH4 has been shown to be beneficial under these conditions; however, whether this is due to a genuine increase in myocardial BH4 availability or to a systemic antioxidant effect of BH4 remains to be established. Here, we evaluated the effect of increasing myocardial BH4 availability by transgenic overexpression of GTP cyclohydrolase-1 (GCH) under the control of the MHC promoter (GCH-Tg) on cardiomyocyte function.

Methods & Results

There was no difference in body weight, cardiac mass or myocyte size between genotypes. As expected, BH4 and total biopterin concentrations (HPLC) in myocytes from GCH-Tg were significantly increased compared with controls. These increases resulted in a 2-fold increase in myocardial NOS activity, which was mostly accounted for by the neuronal NOs (nNOS) isoform. The speed of relaxation and the rate of decay of the [Ca2+]i transient were faster in mGCH-Tg myocytes and isolated hearts. These findings were associated with a reduction in total PLB and an increase in the PLB-Ser16 phosphorylated fraction. nNOS inhibition with SMTC (100nM) abolished the difference in the rate of relaxation and PLB-Ser16 phosphorylated fraction between genotypes. The Ca2+ load in the sarcoplasmic reticulum (SR) did not differ between genotypes, nor did myocyte contraction (3 Hz, 35C) or the amplitude of the [Ca2+]i transient (Fura-2, 3 Hz, 35C), despite a reduction in Ca2+ current density in the mGCH-Tg myocytes, which was reversed by specific nNOS inhibition with SMTC (100nM). Fractional release of Ca2+ from the SR was increased in mGCH-Tg myocytes.

Conclusions

Myocardial BH4 content regulates cardiac function through a nNOS-mediated effect on intracellular Ca2+ fluxes. The BH4-mediated increase in NOS-derived NO improves relaxation (by increasing the PLB phosphorylated fraction) and decreases ICa . However, contractility and [Ca2+]i transient amplitude are maintained in mGCH Tg myocytes by an increase in the fractional release of Ca2+ from the SR suggesting that nNOS-derived NO may increase the gain of EC coupling. Together, these findings indicate that strategies aimed at increasing myocardial BH4 availability may hold promise in the treatment of heart failure in the presence or absence of NOS uncoupling, suggesting that this therapeutic approach may have wider than expected applicability.

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