Establishment of New Skeletal Muscle Cell Lines with Fluorescence Reporters for Inflammation, and Their Utility in Drug Screening

Background: Muscle wasting (MW) accompanied by systemic inflammation contributes to poor prognosis in critical illnesses, including burn injury (BI). Studies on aging have identified sarcopenia and inflammatory traits as crucial determinants of health in seniors. Despite the acknowledged significance of inflammatory responses in skeletal muscles during diseases and aging, a versatile cellular reporting system representing skeletal muscle inflammatory response is lacking. In this study, we established cell lines expressing enhanced green fluorescent protein (EGFP) under the control of NF-κB or STAT3 promoters, major transcription factors regulating inflammatory cytokine expression such as IL-1β and TNF-α. To assess their utility, we conducted drug screenings to mitigate inflammatory responses induced by oxidative stress and burn-derived serum stress.
Methods: Transgenes encoding EGFP under NF-κB or STAT3 promoters were transfected into the C2C12 mouse myoblast cell line, along with ROSA26-CRISPR/Cas9 for gene integration at the Safe Harbor Site of the genome. Stable cell lines were obtained through cycles of G418 drug selection, fluorescence-activated cell sorting (FACS), and subcloning. EGFP expression was evaluated in response to oxidative stress (0.5mM H2O2) or burn-derived 10% serum (from day3 burn rats, 30% BSA) for 24 hours. Drug efficacy testing involved trehalose (autophagy/mitophagy modulator) and humanin (anti-inflammatory peptide, Alzheimer’s disease drug candidate).
Results: Both oxidative stress and burn-serum-induced stress increased EGFP expression by 1.4 to 2.6-fold in both NF-κB and STAT3 cell lines, demonstrating the versatility of our cell lines in measuring inflammatory responses. Trehalose and humanin effectively suppressed the inflammatory response.
Conclusions: Our novel muscle cell lines serve as a promising reporter system for assessing skeletal muscle inflammatory responses to stresses. Compared to conventional methods like RT-PCR, ELISA, and Western Blotting, the fluorescence-based reporter system offers simpler and quicker assessments of drug efficacy. The positive outcomes with trehalose and humanin suggest a potential connection between autophagy/mitophagy, skeletal muscle inflammation, and Alzheimer’s disease within skeletal muscle biology.