Skeletal Muscle Mitochondrial Abnormalities and the Metabolic Syndrome in Pulmonary Arterial Hypertension

AIM 1:

To determine whether MS is associated with intramuscular lipid accumulation and impaired skeletal muscle metabolism and perfusion in human PAH.

Rationale: MS and IR are highly prevalent amongst PAH patients even in the absence of obesity and diabetes. There are several lines of evidence in the literature that IR develops with the accumulation of fatty-acid metabolites within insulin-responsive tissues, especially intramyocellular lipid deposition within skeletal muscles. Although the mechanism accounting for lipid accumulation remains elusive, a reduction in lipid oxidation as a result of reduction in mitochondrial density has been proposed. The objectives of Aim 1 are 1) to confirm that PAH patients have increased intramuscular lipid accumulation; 2) to determine whether intramuscular lipid accumulation is associated with impaired skeletal muscle metabolism; 3) to demonstrate that these abnormalities correlate with MS and IR and skeletal muscle function amongst PAH patients.

Experimental approaches: The proposed experiments will be performed on PAH patients (n=10-20) vs. 10 healthy but sedentary subjects matched for age, gender, height and weight (definition based on current recommendations), excluding patients with clinically relevant conditions (e.g. diabetes). These individuals are continuously identified through our systematic plasma biobanking process at the time of right heart catheterization (CER#20735), in which roughly 40% of PAH patients with no obesity/diabetes have MS. In addition to routinely performed analyses: A) blood sample will be drawn for Apolipoprotein A1, Apolipoprotein B, glycated hemoglobin, fasting blood glucose, insulin, adiponectin and leptin. B) MR imaging will be used to assess fat infiltration within the quadriceps muscle, liver and heart (see appendix for details). C) Volitional and non volitional strength and endurance of the dominant quadriceps and VO2peak on cycle ergometer will be assessed, as previously described. D) Percutaneous biopsy specimens of the vastus lateralis muscle of the nondominant leg will be taken. Part of the specimen (≈100mg) will be used for immunohistochemistry fiber typing (ethanol modified technique), capillarisation (quantitative IF using CD31-antibody) and intramyocellular lipid accumulation (Oil red O staining, which stains only the most hydrophobic and neutral lipids, as the investigators previously described. The extracellular flux analyzer Seahorse XF24 will be used on the remaining tissues for real time measurements of oxygen consumption and extracellular acidification rates (glycolysis). To ensure that physical inactivity is not responsible for skeletal muscle lipid accumulation, subjects' daily life physical activities will be objectively quantified during one week using a physical activity monitor (SenseWear® armband).

Interpretation: This multimodality approach will provide comprehensive information to confirm: 1) PAH patients exhibit significant increases in quadriceps muscle lipid accumulation compared to controls; 2) lipid accumulation is increased within the skeletal muscle of PAH patients with MS compared to PAH without MS despite similar levels of physical activity; 3) Lipid accumulation is associated with a reduction in lipid oxidation in vivo; 4) MS/IR and quadriceps muscle function correlate with muscle lipid accumulation/glucose oxidative phosphorylation capacity.