Altered carnitine homeostasis is associated with decreased mitochondrial function and altered nitric oxide signaling in lambs with pulmonary hypertension

Abstract

SM. Altered carnitine homeostasis is associated with decreased mitochondrial function and altered nitric oxide signaling in lambs with pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 294: L46-L56, 2008. First published November 16, 2007 doi:10.1152/ajplung.00247.2007.-Utilizing aortopulmonary vascular graft placement in the fetal lamb, we have developed a model (shunt) of pulmonary hypertension that mimics congenital heart disease with increased pulmonary blood flow. Our previous studies have identified a progressive development of endothelial dysfunction in shunt lambs that is dependent, at least in part, on decreased nitric oxide (NO) signaling. The purpose of this study was to evaluate the possible role of a disruption in carnitine metabolism in shunt lambs and to determine the effect on NO signaling. Our data indicate that at 2 wk of age, shunt lambs have significantly reduced expression (P Ͻ 0.05) of the key enzymes in carnitine metabolism: carnitine palmitoyltransferases 1 and 2 as well as carnitine acetyltransferase (CrAT). In addition, we found that CrAT activity was inhibited due to increased nitration. Furthermore, free carnitine levels were significantly decreased whereas acylcarnitine levels were significantly higher in shunt lambs (P Ͻ 0.05). We also found that alterations in carnitine metabolism resulted in mitochondrial dysfunction, since shunt lambs had significantly decreased pyruvate, increased lactate, and a reduced pyruvate/lactate ratio. In pulmonary arterial endothelial cells cultured from juvenile lambs, we found that mild uncoupling of the mitochondria led to a decrease in cellular ATP levels and a reduction in both endothelial NO synthase-heat shock protein 90 (eNOS-HSP90) interactions and NO signaling. Similarly, in shunt lambs we found a loss of eNOS-HSP90 interactions that correlated with a progressive decrease in NO signaling. Our data suggest that mitochondrial dysfunction may play a role in the development of endothelial dysfunction and pulmonary hypertension and increased pulmonary blood flow. carnitine metabolism; oxidative stress THE DEVELOPMENT OF PULMONARY HYPERTENSION and its associated increased vascular reactivity are common accompaniments of congenital heart disease with increased pulmonary blood flow (3). Endothelial dysfunction is thought to be an early hallmark of pulmonary hypertension (4). There is increasing histological and physiological evidence that endothelial injury and the resulting aberration in the balance of its regulatory mechanisms play an important role in the development of pulmonary hypertension (4). Children with pulmonary hypertension have evidence of endothelial dysfunction as indicated by impaired endothelium-dependent relaxation in early disease and decreased endothelial nitric oxide synthase (eNOS) protein levels in late disease (11,23). However, data delineating the role of endotheliumderived NO in the disease have been less clear. For example, we (5) and others (72) have shown that pulmonary expression of NOS can be paradoxically inc...

Disruption of Endothelial Cell Mitochondrial Bioenergetics in Lambs with Increased Pulmonary Blood Flow

“…Our recent studies have shown that increased CrAT nitration and decreased CrAT activity correlates with a disruption in carnitine homeostasis and NO signaling in Shunt lambs (42). To determine whether l-arginine supplementation could preserve carnitine homeostasis and mitochondrial function in Shunt lambs, we examined the effect of l-arginine supplementation on the levels of total protein nitration, CrAT activity, and CrAT nitration in control and Shunt lambs exposed to l-arginine (500 mg/kg/day) or vehicle for 3 weeks.…”

“…Our previous studies demonstrated that increased levels of ADMA in Shunt lambs (47) correlated with a disruption in carnitine homeostasis and a loss of carnitine acetyl transferase (CrAT) activity (42). Carnitine homeostasis is of critical importance for maintaining oxidative phosphorylation (41).…”

“…Pulmonary mitochondrial function is attenuated in our lamb model of a congenital heart defect with increased pulmonary blood flow (PBF) (Shunt), and this correlates with a disruption of carnitine metabolism (42). One of the major correlations with the disrupted carnitine homeostasis was a reduction in CrAT activity associated with increased nitration (42). However, the mechanism by which CrAT becomes nitrated was not elucidated.…”

“…The principal enzyme affected has been identified as carnitine acetyltransferase (CrAT), which catalyzes a reversible equilibrium reaction between acyl-CoA and CoA, and acylcarnitine and carnitine (59). Pulmonary mitochondrial function is attenuated in our lamb model of a congenital heart defect with increased pulmonary blood flow (PBF) (Shunt), and this correlates with a disruption of carnitine metabolism (42). One of the major correlations with the disrupted carnitine homeostasis was a reduction in CrAT activity associated with increased nitration (42).…”

Nitration of Tyrosine 247 Inhibits Protein Kinase G-1α Activity by Attenuating Cyclic Guanosine Monophosphate Binding

“…Western Blot Analysis-Cells were prepared as previously described (21,22). Similarly, peripheral lung tissue from the control lambs and the lambs with pulmonary hypertension secondary to increased pulmonary blood flow (shunt) was prepared as described earlier (1).…”