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

Abstract

Aims: The mitochondrial dysfunction in our lamb model of congenital heart disease with increased pulmonary blood flow (PBF) (Shunt) is associated with disrupted carnitine metabolism. Our recent studies have also shown that asymmetric dimethylarginine (ADMA) levels are increased in Shunt lambs and ADMA increases the nitration of mitochondrial proteins in lamb pulmonary arterial endothelial cells (PAEC) in a nitric oxide synthase (NOS)-dependent manner. Thus, we determined whether there was a mechanistic link between endothelial nitric oxide synthase (eNOS), ADMA, and the disruption of carnitine homeostasis in PAEC. Results: Exposure of PAEC to ADMA induced the redistribution of eNOS to the mitochondria, resulting in an increase in carnitine acetyl transferase (CrAT) nitration and decreased CrAT activity. The resulting increase in acyl-carnitine levels resulted in mitochondrial dysfunction and the disruption of mitochondrial bioenergetics. Since the addition of l-arginine prevented these pathologic changes, we examined the effect of l-arginine supplementation on carnitine homeostasis, mitochondrial function, and nitric oxide (NO) signaling in Shunt lambs. We found that the treatment of Shunt lambs with l-arginine prevented the ADMA-mediated mitochondrial redistribution of eNOS, the nitration-mediated inhibition of CrAT, and maintained carnitine homeostasis. In turn, adenosine-5¢-triphosphate levels and eNOS/heat shock protein 90 interactions were preserved, and this decreased NOS uncoupling and enhanced NO generation. Innovation: Our data link alterations in cellular l-arginine metabolism with the disruption of mitochondrial bioenergetics and implicate altered carnitine homeostasis as a key player in this process. Conclusion: l-arginine supplementation may be a useful therapy to prevent the mitochondrial dysfunction involved in the pulmonary vascular alterations secondary to increased PBF.

Endothelin-1 Induces a Glycolytic Switch in Pulmonary Arterial Endothelial Cells via the Mitochondrial Translocation of Endothelial Nitric Oxide Synthase

“…This is in agreement with our recent studies in which we showed that asymmetric ADMA, an endogenous NOS uncoupler, induces eNOS mitochondrial redistribution and the disruption of mitochondrial bioenergetics through the nitration of carnitine acetyl transferase and the attenuation of carnitine homeostasis (16). However, ADMA induces eNOS mitochondrial translocation via the nitration-mediated activation of Akt1 and increased phosphorylation of eNOS at S617 and S1,177 (19).…”

“…Furthermore, this correlates with an increase in lactate levels (14), suggesting that a glycolytic switch may also be induced in these lambs. Interestingly, these lambs also have decreased ATP levels (16), suggesting that the glycolytic switch is unable to maintain ATP demand. We did not observe a decrease in ATP in PAECs exposed to ET-1, nor have reductions in ATP been observed in PAECs isolated from patients with idiopathic pulmonary arterial hypertension (33).…”

“…Carnitine homeostasis is critical for maintaining normal mitochondrial function, and we have shown that its disruption is associated with alterations in mitochondrial bioenergetics (16). Using the Seahorse XF24 Analyzer (North Billerica, MA), we examined the effect of ET-1 on mitochondrial bioenergetics in PAECs.…”

“…Our recent studies indicate that asymmetric dimethylarginine (ADMA) disrupts mitochondrial bioenergetics in PAECs due to eNOS uncoupling secondary to a redistribution of eNOS from the plasma membrane to the mitochondria (16). Thus, we next evaluated the effect of ET-1 on eNOS uncoupling.…”

Asymmetric Dimethylarginine Induces Endothelial Nitric-oxide Synthase Mitochondrial Redistribution through the Nitration-mediated Activation of Akt1*

“…It is possible that this sequence could interact with the positively charged eNOS mitochondrial translocation loop located at aa 627-631 (RRKRK) (14). Furthermore, we have recently shown in an animal model of pulmonary hypertension associated with increased pulmonary blood flow that increases in ADMA correlates with increased activities of CHIP and Hsp70 (47) and increased translocation of eNOS to the mitochondrion (48). However, further studies will be required to determine whether Hsp70 and/or CHIP play a role in regulating eNOS translocation to the mitochondrion.…”