Skeletal Muscle Dysfunction in Experimental Pulmonary Hypertension Kosmas Kosmas1,2, Zoe Michael2,3, Fotios Spyropoulos1,2, Jeffrey Widrick2,4, Ravi Jasuja2, Aimilia Papathanasiou1,2, Helen Christou 1,21Department of Pediatric Newborn Medicine, Brigham and Women’s Hospital, Boston, MA 02115, USA 2Harvard Medical School, Boston, MA 02215, USA 3Department of Pediatrics, Boston Children’s Hospital, Boston, MA 02215, USA 4Department of Genetics, Boston Children’s Hospital, Boston, MA 02215,

Abstract

BackgroundPulmonary arterial hypertension (PAH), is a progressive disease that seriously compromises quality of life due to dyspnea and fatigue on exertion that worsens over time such that patients are ultimately immobile. Although hemodynamic compromise is the main contributor to exercise intolerance in PAH, accumulating evidence supports that intrinsic skeletal muscle dysfunction also occurs and may contribute to this symptomatology. We previously showed that treatment with the carbonic anhydrase inhibitor Acetazolamide (ACTZ) ameliorates the hemodynamic components of experimental PAH but its effects on skeletal muscle function and exercise tolerance are unknown. Our objective is to characterize the mechanisms of skeletal muscle dysfunction in a preclinical model of severe PAH and evaluate the effect of ACTZ treatment.HypothesesWe hypothesized that reduced exercise endurance in experimental PAH is associated with increased proportion of fast glycolytic (Type II) fibers to slow oxidative (Type I) fibers, and sarcomeric dysfunction in skeletal muscles and that treatment with ACTZ, alters muscle fiber specificity, improves skeletal muscle contractile properties and exercise tolerance.MethodsWe used the adult rat Sprague Dawley sugen/hypoxia (Su/Hx) model of severe pulmonary hypertension and performed hemodynamic and exercise tolerance studies (treadmill). We evaluated the abundance of Type I and Type II fiber specific markers and sarcomeric organization in muscles from Su/Hx and ACTZ treated rats and measured twitch and tetanic forces, contraction and relaxation times in diaphragm, soleus and extensor digitorum longus (EDL). We used the rat myoblast cell line (L6) to evaluate the molecular mechanisms of muscle fiber switch using immunofluorescence, RNA and protein expression studies.ResultsWe found that diaphragm and soleus muscle from the adult rat sugen/hypoxia (Su/Hx) model of severe PAH have increased abundance of type II fiber markers and thick sarcomeric aggregates and these changes are accompanied by decreased twitch and tetanic forces, contraction and half‐relaxation time and exercise endurance despite absence of muscle atrophy. Acetazolamide (ACTZ), treatment restored the above derangements. In vitro rat myotube differentiation was associated with a predominance of type II fiber markers in conjunction with increased abundance of the transcription factor FoxO1. This pattern of increased abundance of FoxO1 and increased type II markers was also seen in Su/Hx soleus muscles and was reversed in response to ACTZ treatment.ConclusionsWe conclude that a shift in skeletal muscle fiber type specification may underlie skeletal muscle dysfunction in experimental PAH and that treatment with ACTZ may ameliorate skeletal muscle dysfunction through an inhibitory effect on FoxO1‐mediated fiber type specification.