The disruption of mitochondrial dynamics has been identified in cardiovascular diseases, including pulmonary hypertension (PH), ischemia-reperfusion injury, heart failure, and cardiomyopathy. Mitofusin 2 (Mfn2) is abundantly expressed in heart and pulmonary vasculature cells at the outer mitochondrial membrane to modulate fusion. Previously, we have reported reduced levels of Mfn2 and fragmented mitochondria in pulmonary arterial endothelial cells (PAECs) isolated from a sheep model of PH induced by pulmonary over-circulation and restoring Mfn2 normalized mitochondrial function. In this study, we assessed the effect of increased expression of Mfn2 on mitochondrial metabolism, bioenergetics, reactive oxygen species production, and mitochondrial membrane potential in control PAECs. Using an adenoviral expression system to overexpress Mfn2 in PAECs and utilizing ¹³C labeled substrates, we assessed the levels of TCA cycle metabolites. We identified increased pyruvate and lactate production in cells, revealing a glycolytic phenotype (Warburg phenotype). Mfn2 overexpression decreased the mitochondrial ATP production rate, increased the rate of glycolytic ATP production, and disrupted mitochondrial bioenergetics. The increase in glycolysis was linked to increased hypoxia-inducible factor 1α (HIF-1α) protein levels, elevated mitochondrial reactive oxygen species (mt-ROS), and decreased mitochondrial membrane potential. Our data suggest that disrupting the mitochondrial fusion/fission balance to favor hyperfusion leads to a metabolic shift that promotes aerobic glycolysis. Thus, therapies designed to increase mitochondrial fusion should be approached with caution.

It is well-established that mitochondrial dysfunction plays a critical role in the development of pulmonary hypertension (PH). However, the molecular mechanisms and how the individual electron transport complexes (ETC) may be affected are poorly understood. In this study, we identified decreased ETC Complex I activity and assembly and linked these changes to disrupted mitochondrial bioenergetics in pulmonary arterial endothelial cells (PAECs) isolated from a lamb model of PH with increased pulmonary blood flow (Shunt). These derangements were associated with decreased mitochondrial activity of the protein tyrosine kinase, pp60^Src. Treating Control PAECs with either the Src family kinase inhibitor, PP2, or the siRNA-mediated knockdown of pp60^Src was able to recapitulate the adverse effects on ETC Complex I activity and assembly and mitochondrial bioenergetics. Conversely, restoring pp60^Src activity in lamb PH PAECs re-established ETC Complex I activity, improved ETC Complex I assembly and enhanced mitochondrial bioenergetics. Phosphoprotein enrichment followed by two-dimensional gel electrophoresis and tandem mass spectrometry was used to identify three ETC Complex I subunits (NDUFS1, NDUFAF5, and NDUFV2) as pp60^Src substrates. Finally, we demonstrated that the pY levels of NDUFS1, NDUFAF5, and NDUFV2 are decreased in lamb PH PAECs. Enhancing mitochondrial pp60^Src activity could be a therapeutic strategy to reverse PH-related mitochondrial dysfunction.

Metabolic dysfunction-associated steatotic liver disease (MASLD) and steatohepatitis (MASH) are leading causes of cirrhosis and hepatocellular carcinoma. Defects in autophagy contribute to the development of MASLD, however, the role of the Unc-51-like autophagy-activating kinase 1 (ULK1) in the pathophysiology of MASLD remains unclear. Herein, we show that ULK1, a serine/threonine kinase and core autophagy protein, is significantly repressed in human MASH livers, and that hepatocyte-specific loss of ULK1, unexpectedly, promotes hepatic steatosis and progression to liver fibrosis, without affecting basal autophagy flux. Phospho-proteomics identified the transcriptional coactivator NCOA3 as a downstream phospho-target of ULK1. Mechanistically, ULK1 phosphorylates NCOA3 to repress its transcriptional activity and restrain the CREB/CBP-mediated de novo lipogenic program. Accordingly, a phosphorylation-deficient NCOA3 mutant drives CREB/CBP-mediated lipogenesis, whereas genetic or pharmacological NCOA3 inhibition prevents steatosis, hepatic inflammation, and profibrotic signaling. Hence, ULK1-mediated NCOA3 phosphorylation is a fundamental and druggable checkpoint against the entire MASLD spectrum.