Does NMJ Degradation Initiate the Fibrotic Phenotypic Switch In Skeletal Muscles Post-denervation via a TGFβ-1 Dependent Mechanism?
Jennifer Uong, BȘ Čǻňđ.1, Justin P. Chan, BA1, Henry Hoang, BȘ Čǻňđ.1, Winnie A. Pǻlįșpįș, MD2, Ranjan Gupta, MD2.
1University of California, Irvine, Irvine, CA, USA, 2University of California, Irvine, Orange, CA, USA.
PURPOSE: Traumatic peripheral nerve injuries often result in the permanent loss of muscle function. One reason for the failure of recovery is the end-stage process of muscle fibrosis whereby an increase in extracellular matrix (ECM) interferes with the functional properties of muscles. It has been widely observed that denervation results in the abnormal accumulation of ECM. However, little is known about the cellular and molecular mechanisms of this fibrosis in denervated skeletal muscles in comparison to other tissues. One particular cellular signaling cascade of interest is the transforming growth factor beta 1 (TGFβ-1) as TGFβ-1 and its downstream effectors are widely accepted as being critically important to the development of fibrosis. In addition to TGFβ-1’s role in the fibrotic process, it is also known to play a critical role in synaptogenesis. As neuromuscular junction (NMJ) degradation and fibrosis both occur following denervation, we sought to establish if there is a temporal correlation between these two phenomena. With TGFβ-1’s ubiquitous involvement in both synaptogenesis and fibrosis, it is our hypothesis that NMJ degradation initiates the phenotypic change of skeletal muscle tissue to fibrotic tissue via a TGFβ-1 dependent mechanism.
METHODS: A denervation model was created in 6 week old C57BL/6 mice by excising 10 mm segments from the right sciatic nerve and suturing the proximal nerve to the gluteal muscle with 9-0 suture so as to prevent regeneration. Denervated gastrocnemius muscles were then harvested at multiple time points for analysis by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot for levels of TGFβ-1 and possible downstream targets. Passive muscle stiffness measurements were also performed by subjecting isolated denervated muscles to ramp stretches and then measuring resistance to stretch. Muscle collagen content was measured using a hydroxyproline assay and visualized with Masson’s trichrome staining.
RESULTS: qRT-PCR was used to confirm the transcriptional changes associated with the switch towards a fibrotic phenotype. One week after injury, expression of TGFβ-1 in denervated gastrocnemius muscles was significantly increased compared to muscle from the contralateral side. Connective tissue remodeling proteins including matrix metalloproteinases (mmp-3) and scleraxis (scx) were also significantly upregulated. Genes involved in inflammation (TNFα) and collagen deposition (Col1A1 and Col1A2) were not significantly changed at this time point (Figure 1). Gene expression levels in muscle samples one month after denervation showed a similar trend (Figure 2).
CONCLUSIONS: Our preliminary data suggests that TGFβ-1 signaling plays a key role in the early stages of the phenotypic switch towards muscle fibrosis after nerve injury. There is a temporal correlation between NMJ degradation and upregulation of the pro-fibrotic genes post-denervation. This is an important first step in establishing this correlation prior to determining causality and supports further investigation in this critically important area.
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