Calcium Handling Machinery and Sarcomere Assembly are Impaired Through Multipronged Mechanisms in Cancer Cytokine‐Induced Cachexia

Abstract Background Cachexia is a severe form of muscle wasting disorder particularly observed in patients with advanced cancer. The absence of effective strategies to ameliorate cachexia indicates our poor understanding of the mechanisms of cachexia. By employing system‐wide approaches, we investigated molecular mechanisms underlying cancer secreted pro‐inflammatory cytokine‐induced cachexia (CIC). Methods As cellular model systems, we employed mouse satellite stem cell‐derived primary muscle cells, mouse C2C12 myoblast progenitor cell‐derived myotubes, and neonatal rat cardiomyocytes. We induced CIC by incubating striated muscle cells with pro‐inflammatory cytokines TNF‐α and IFN‐γ. To understand the physiological effects of CIC, we probed the contractile properties of muscle cells following electrical stimulation and measured intracellular calcium transients. Effects of CIC on sarcomere organization were monitored by confocal microscopy. Large‐scale quantitative proteomics and RNA sequencing assays enabled us to examine molecular mechanisms underlying CIC. Using chromatin immunoprecipitation experiments, chromatin signalling and modulation of epigenetic marks on muscle‐specific genes were investigated. Results Here, we observed a drastic loss of striated muscle cell contraction in CIC, primarily, due to acutely disorganized sarcomere structures and impeded calcium handling process. In calcium transients, the extent of calcium (Ca 2+ ) release, as indicated by the calcium amplitude during the excitation–contraction coupling (ECC) process, was reduced (19.6 ± 2.35% in control to 8.6 ± 1.52% in CIC, p = 4.8 * 10 −11 ). Kinetics of calcium transients, i.e., the Ca 2+ release rate (26 ± 0.5 ms in control to 29 ± 5.1 ms in CIC, median p = 0.014), and calcium re‐uptake rate (137 ± 13 ms in control to 185 ± 24 ms in CIC, p = 0.032) were both prolonged. Proteomic analysis showed altered proteostasis in CIC, particularly related to sarcomere and sarcoplasmic reticulum (SR). Transcriptomic analysis unravelled upstream deregulation of global transcriptional events for sarcomeric and SR genes. Mechanistically, chromatin loading of transcriptionally active RNA Polymerase II on muscle‐specific genes, including Myh1 and Atp2a1 , was impeded. This was due to diminished transcriptionally active epigenetic marks H3K4 trimethylation on Myh1 and Atp2a1 , resulted in lower transcriptional activity of these muscle‐specific genes in CIC and ultimately reduced MyHC‐IId molecular motor protein and SERCA1 protein levels. Conclusions Our top‐down approach elucidated that the altered transcriptional mechanism and proteomic state perturbed functionally related machinery responsible for calcium handling and sarcomere organization in CIC. Knowledge of the underlying cause of muscle mass loss and compromised muscle function is key for developing therapeutic solutions to ameliorate cachectic conditions.