(714.9) Inhibition of Anaplerosis Prevented Mitochondrial Dysfunction and Vascular Remodeling in Pulmonary Hypertension

Poster Board Number: E73

Mathews Valuparampil Varghese (University of Arizona), Joel James (University of Arizona), Maki Niihori (University of Arizona), Olga Rafikova (University of Arizona), Ruslan Rafikov (University of Arizona)

Introduction: Pulmonary hypertension (PH) is a deadly disease of the lung vasculature. Recent studies reported mitochondrial dysfunction as a significant contributor to PH pathobiology, the prominent type of dysfunction is still unclear. Importantly, partial mitochondrial functioning is necessary to provide the metabolic intermediates for the proliferative growth of vascular cells in PH. Recently, we reported that in PH, the mitochondrial anabolic branch of metabolism - anaplerosis plays a critical role in the upregulation of the tricarboxylic acid cycle in pulmonary vascular cells with mitochondrial dysfunction.

Hypothesis: We hypothesize that the inhibition of both anaplerosis targets - pyruvate carboxylase (PC) and glutaminolysis - glutamate dehydrogenase 1 (GLUD1) in the early stages of PH could effectively attenuate mitochondrial dysfunction and vascular remodeling.

Methods: PH was induced in female Sprague Dawley rats by subcutaneous injection of sugen (50 mg/kg)/hypoxia (10% O2). The treatment groups received sugen/hypoxia (Su/Hx) and two-weeks intraperitoneal injection of inhibitors (PC inhibitor, phenylacetic acid (PAA), 20 mg/kg/every other day and R162, an inhibitor of GLUD1 (30mg/kg/daily).

Results: Hemodynamic analysis of five weeks of Su/Hx rats showed a progressive PH phenotype with increased right ventricular (RV) systolic pressure (111.08 ± 6.87 mmHg). Inhibiting anaplerosis at early stages of PH (2 weeks Su/Hx), decreased the RV hypertrophy (0.5 ± 0.02 to 0.42 ± 0.02, plt;0.05), RV systolic pressure (71.88 ± 3.59 mmHg to 54.14 ± 2.48 mmHg, plt;0.05), and Max dP/dt (3863.89 ± 180.27 mmHg/s to 2890.63 ± 154.52 mmHg/s, plt;0.01). Su/Hx group showed an increased glycolytic flux with hexokinase-I upregulation (plt;0.05), leading to an upregulation of anaplerosis. This was followed by the impairment in mitochondrial function with a reduction of pyruvate dehydrogenase (PDH) expression (plt;0.001)  and activation of mitochondrial glycerol-3-phosphate-dehydrogenase (GPD2) (plt;0.001), an important mediator of the glycerophosphate shuttle. The increased glycolysis also fluxed into the pentose phosphate pathway (PPP) through myo-inositol oxygenase (MIOX) (plt;0.001), a novel source of reactive oxygen species (ROS) generation in PH. These metabolic alterations lead to the upregulation of proliferative signaling pathways Akt (plt;0.01), STAT3 (plt;0.001), and P38 (plt;0.01). Importantly, the inhibition of the anaplerotic pathways by combination (PAA+R162) treatment in the Su/Hx PH model significantly resolved the activation of PPP, restored mitochondrial metabolism, and prevented the proliferative signaling leading to lung vascular remodeling.

Conclusion: The results suggest that inhibition of the anaplerotic pathway effectively attenuated mitochondrial dysfunction and vascular remodeling in Su/HX animals. Therefore, our findings indicate that simultaneous targeting of both PC and glutamine-mediated anaplerosis is a promising therapeutic target for the resolution of vascular remodeling in PH.

This work was supported by NIH grants R01HL133085 (OR), R01HL151447 (RR), and R01HL132918 (RR) and the AHA postdoctoral fellowship 831538 (MVV) and 834220 (JJ).