Human skeletal myotubes display a cell-autonomous circadian clock implicated in basal myokine secretion

Abstract

ObjectiveCircadian clocks are functional in all light-sensitive organisms, allowing an adaptation to the external world in anticipation of daily environmental changes. In view of the potential role of the skeletal muscle clock in the regulation of glucose metabolism, we aimed to characterize circadian rhythms in primary human skeletal myotubes and investigate their roles in myokine secretion.MethodsWe established a system for long-term bioluminescence recording in differentiated human myotubes, employing lentivector gene delivery of the Bmal1-luciferase and Per2-luciferase core clock reporters. Furthermore, we disrupted the circadian clock in skeletal muscle cells by transfecting siRNA targeting CLOCK. Next, we assessed the basal secretion of a large panel of myokines in a circadian manner in the presence or absence of a functional clock.ResultsBioluminescence reporter assays revealed that human skeletal myotubes, synchronized in vitro, exhibit a self-sustained circadian rhythm, which was further confirmed by endogenous core clock transcript expression. Moreover, we demonstrate that the basal secretion of IL-6, IL-8 and MCP-1 by synchronized skeletal myotubes has a circadian profile. Importantly, the secretion of IL-6 and several additional myokines was strongly downregulated upon siClock-mediated clock disruption.ConclusionsOur study provides for the first time evidence that primary human skeletal myotubes possess a high-amplitude cell-autonomous circadian clock, which could be attenuated. Furthermore, this oscillator plays an important role in the regulation of basal myokine secretion by skeletal myotubes.

Lipidomics reveals diurnal lipid oscillations in human skeletal muscle persisting in cellular myotubes cultured in vitro

“…The first peak of Bmal1-luc reporter activity was observed about 8 h after forskolin synchronization ( Fig. 3 A, Inset), and as previously demonstrated for endogenous BMAL1 in human primary myotubes (27). The Bmal1-luc reporter activity peak recorded 8 h after synchronization ( Fig.…”

“…In vivo, lipid levels peaked in the early morning hours, near the usual wake time, supporting a major role for the muscle clock in anticipating sleep-to-wake transition as has been proposed previously (44). To directly test the effect of cell-autonomous oscillators on lipid metabolic changes, we subsequently disrupted the endogenous clock, operative in human skeletal myotubes (27). Clock disruption had a strong impact on lipid metabolite oscillations.…”

“…Recent studies conducted in other types of isolated human primary cells have demonstrated that peripheral clocks are functional in vitro, as shown for skin fibroblasts (39), thyrocytes (40), and pancreatic islets (41-43). Moreover, cell-autonomous oscillators of human primary skeletal myotubes have been recently assessed (25)(26)(27). Remarkably, we observed that lipid oscillations persisted in human primary myotubes synchronized in vitro, with a comparable percentage of oscillating lipid species (18.6%) (Fig.…”

“…However, whether lipids in peripheral tissues oscillate in a cellautonomous manner has not yet been resolved. As the existence of self-sustained circadian oscillators operative in human primary skeletal myotubes established from muscle biopsies, cultured and synchronized in vitro, has been recently demonstrated (25)(26)(27), we sought to address this question. In this study, we explored the regulation of lipid metabolite oscillations in human skeletal muscle in vivo and in vitro, focusing on lipids involved in membrane organization (phospholipids and sphingolipids) and those involved in energy metabolism, such as triglycerides.…”

“…S3). To monitor core clock properties in synchronized human primary myotubes, circadian bioluminescence was continuously recorded for several days, using a lentiviral Bmal1-luc reporter (27). Bmal1-luc reporter profiles recorded from all 10 myotube cultures demonstrated a high degree of alignment (Fig.…”