Diversity Supplement: Mechanistic Assessment of Blood Flow Restricted Training for an ACL Injury

Vitamin D deficiency is also known to promote muscle pain and weakness, but little is known about the underlying mechanisms driving vitamin D’s integral role in skeletal muscle health. Vitamin D is a hormone that binds to the vitamin D [nuclear] receptor (VDR), and it has broad biological function primarily by forming a heterodimer with retinoid X receptor (RXR) and binding to the vitamin D response element (VDRE) to affect transcription of hundreds of target genes. The best known and characterized vitamin D functions are related to its role in maintaining calcium absorption and overall bone health, but it has roles in immunology, cancer, metabolic diseases, muscle health and others. Recent work has started to elucidate vitamin D’s integral role in molecular pathways in affecting tissue fibrosis and mitochondrial metabolism in muscle. Aim 1: Determine whether dietary vitamin D intake or circulating vitamin D concentrations affect vitamin D receptor (VDR) expression in muscle and/or localization with the pro-fibrotic SMAD3 transcriptional regulator in adolescents and young adults approximately 2 weeks after an ACL tear. Hypothesis: We hypothesize that lower vitamin D intake and circulating concentrations of 25- (OH)D will result in lower expression of VDR, less colocalization of SMAD3 and VDR, and greater muscle fibrosis in response to ACL injury and reconstruction surgery. ACL injury causes muscle impairment and fibrosis, which a co-investigators’ prior work shows is potentiated by TGF-β mediated pathways (Peck 2019). Evidence from liver, kidney, and intestine studies in mice show that vitamin D activated VDR binds to SMAD3 in the nucleus (TGF-β downstream target) and inhibits TGF-β–SMAD signal transduction without activation of classical VDRE-mediated transcription (Ding 2013, Ito 2013, Tao 2014). Consequently, VDR inhibits mRNA expression of the key fibrogenic targets, e.g. Acta2 and Col1a1 (Ding 2013) and tissue fibrosis (Ding 2013, Ito 2013, and Tao 2014). Vitamin D status associates with VDR expression in human muscle in a dose-dependent manner (Pojednic 2015). Co-investigators on the parent grant have characterized SMAD3 protein and muscle fibrosis markers in humans with ACL injury previously (Fry 2016, Peck 2019). Fibrosis markers are already being analyzed on the parent grant. Aim 2: Determine whether dietary vitamin D intake or status associates with loss of type- 2a muscle cross-sectional area or oxidative capacity in adolescents and young adults following an ACL tear. Hypothesis: We hypothesize that lower vitamin D intake and circulating concentrations of 25- (OH)D will result in lower type 2a fiber cross-sectional area, lower expression of SDH, and less mitochondrial capacity. The PI on the parent grant published findings showing that type 2a muscle cross-sectional area was reduced in patients having an ACL tear before surgery (Noehren 2016). Similarly, studies in older adults have demonstrated that supplementation with vitamin D increases type 2a fiber cross-sectional area (Sørensen 1979 and Ceglia 2013), and VDR expression, especially in type 2 fibers (Ceglia 2013). Work in C2C12 mouse myoblast cells showed that vitamin D induced a 1.8-fold increase individual myotube cross-sectional area (Girgis 2014). Additionally, in vitro muscle cell models show that knockout of VDR reduces mitochondrial respiration (Ashcroft 2020), whereas provision with biologically active vitamin D promotes mitochondrial respiration (Ryan 2016). Prior studies from our lab and others show an increase in succinate dehydrogenase (SDH) when muscle cells are exposed to vitamin D (Schnell 2019, Ryan 2016). Here we propose to determine whether insufficient dietary vitamin D intake or status exacerbates expected decrements in type 2a fiber cross sectional area associated with ACL tear. Furthermore, we will examine whether vitamin D intake/status or VDR expression associates with SDH expression or mitochondrial respiration.