Background: Circadian clocks are operative in all light-sensitive organisms, allowing an adaptation to the external world in anticipation of daily environmental changes. Over the last decades, substantial efforts in the field have been undertaken to unravel the tight connection between the circadian clock and most aspects of physiology. Majority of the studies have been conducted in rodent models, with molecular studies of the peripheral oscillators in humans lagging behind due to obvious difficulties in sample collection in human individuals in a repetitive manner. Since the skeletal muscle plays a central role in the regulation of whole-body metabolism, we aimed at characterizing circadian oscillators operative in primary human skeletal myotubes (hSKM) in vitro, and investigate their roles in regulating gene transcription, myokine secretion, insulin response and lipid metabolism. Moreover, the approaches established for the study of the circadian clockwork impact on the skeletal muscle cells have been extended to additional model of primary smooth muscle cells (SMCs) isolated from porcine arteries, allowing to assess the circadian rhythm in normal and pathological SMCs.
Methods: Human skeletal muscle biopsies for the in vitro part were obtained from Gluteus maximus and Rectus abdominus during planned surgery, whereas the Vastus lateralis was sampled for the in vivo part under standardized protocol, with the consent of donors. Human primary skeletal myoblasts differentiated from isolated satellite cells were cultured and differentiated into myotubes. We established experimental system for long-term bioluminescence recording in hSKM, employing delivery of the Bmal1-luciferase (Bmal1-luc) and Per2-luciferase (Per2-luc) circadian reporters by lentiviral transduction. Furthermore, we have developed settings allowing to disrupt the circadian clock in adult skeletal muscle cells by transfecting siRNA targeting CLOCK. Next, using an experimental approach combining long-term bioluminescence recording and outflow medium collection in cultured human primary cells, we assessed the basal secretion of a large panel of myokines in a circadian manner, in the presence or absence of a functional clock. RNA-seq was conducted on hSKM transfected either with siControl or siCLOCK, and harvested every 2 h during 48 h following in vitro synchronization, and on human skeletal muscle biopsies taken every 4 h during 24 h. Glucose uptake by hSKM was measured by the incorporation of 2-deoxy-[3H]-D-glucose before and after insulin stimulation. Finally, using circadian lipidomics analyses, we investigated temporal lipid profiles over 24 h in human skeletal muscle in vivo, and in hSKM cultured in vitro. Furthermore, these experimental settings were applied to primary
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porcine spindle and rhomboid SMCs. SMCs were cultured and subjected to transduction with Bmal1-luc or Per2-Bmal1-luc reporters for circadian bioluminescence recording. qRT-PCR for core clock and target genes were performed on samples synchronized by forskolin, and harvested every 4 h between 12 h and 36 h following in vitro synchronization. Treatment with PDGF-BB or FGF-2 of spindle SMCs was applied to induce the phenotypical switch toward rhomboid-like SMCs, with subsequent synchronization and monitoring of the circadian bioluminescence.
Results: Bioluminescence reporter assays revealed that hSKM, synchronized in vitro with forskolin pulse, exhibit a self-sustained circadian rhythm with a period length of 25.29 ± 0.13 h (Bmal1-luc) and 25.20 ± 0.19 h (Per2-luc). By transfecting primary hSKM with siRNA targeting CLOCK, efficient downregulation of CLOCK mRNA expression by at least 80% and protein by 74% has been observed, leading to significant dampening of the Bmal1-luc bioluminescent reporter circadian amplitude.
Molecular oscillators operative in hSKM have been further characterized by measuring endogenous core clock transcript expression around-the-clock using quantitative real-time polymerase chain reaction (qRT-PCR). Moreover, we demonstrated that the basal secretion of IL-6, IL-8 and MCP-1 by synchronized hSKM exhibited a circadian profile. Importantly, the secretion of IL-6 and several additional myokines was strongly downregulated upon siCLOCK-mediated clock disruption. Of note, glucose uptake by hSKM was reduced in the absence of functional cellular clocks, upon basal conditions and in response to insulin, suggesting an essential role for the human skeletal muscle clock in regulating insulin sensitivity. Furthermore, we demonstrated that circadian clock has an impact on muscle lipid metabolism, with about 20% of lipid metabolites exhibiting rhythmic profiles in human muscle biopsies collected around-the-clock in vivo, and in cultured hSKM, synchronized in vitro. These oscillations were strongly attenuated upon siRNA-mediated clock disruption in the primary myotubes. Additionally, our study provides the first large-scale circadian transcriptome analysis in human skeletal muscle, conducted by high throughput RNA sequencing (RNA-seq) following similar design in vivo and in vitro, allowing to distinguish cell-autonomous and systemic effects of the circadian clock on the muscle transcription. Importantly, genes involved in myokine secretion, insulin response and lipid metabolism have been identified, providing candidates to explain our previous observations. Of note, mRNA expression of core clock genes was in coherence with peak levels of lipid accumulation both in vivo and in vitro, and temporal lipid profiles correlated with transcript profiles of genes implicated in their biosynthesis. Regarding the SMC part, significantly longer period length of Per2-luc reporter oscillations
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has been observed in rhomboid SMCs, and PDGF-BB- and FGF-2-treated SMCs synchronized in vitro by forskolin pulse, as compared to the control counterparts. Endogenous gene expression revealed an upregulation of almost all core clock gene tested, along with a phase shift in rhomboid SMCs compared to spindle.
Conclusions: We provide for the first time an evidence that primary hSKM possess a high-amplitude cell-autonomous circadian clocks. Taken together, our data suggest an essential role for endogenous human skeletal muscle oscillators in regulating glucose uptake by the skeletal muscle, myokine secretion, and lipid metabolism. Our model for studying circadian rhythm in the primary human cells synchronized in vitro is of the particular importance, as it allows to dissect the cell-autonomous effects of the local clocks on transcript and lipid oscillations in hSKM, and to separate the cell-autonomous effects from the central ones, driven by diurnal cycles of rest/activity and food intake.
Indeed, we demonstrated that the muscle circadian clock bears important functional outputs in the absence of external synchronizers. Moreover, the experimental approach established in this work has been implemented for studying circadian clocks operative in cultured human pancreatic islets and porcine SMCs, and it can be further adapted to various cells types, primary cells or cell lines. This would allow for examining the molecular makeup of peripheral oscillators, and their impact on the organ transcription and function under physiological or pathophysiological conditions.
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