Intrinsic muscle clock is necessary for musculoskeletal health

Abstract

Key points: The endogenous molecular clock in skeletal muscle is necessary for maintenance of phenotype and function. Loss of Bmal1 solely from adult skeletal muscle (iMSBmal1(-/-) ) results in reductions in specific tension, increased oxidative fibre type and increased muscle fibrosis with no change in feeding or activity. Disruption of the molecular clock in adult skeletal muscle is sufficient to induce changes in skeletal muscle similar to those seen in the Bmal1 knockout mouse (Bmal1(-/-) ), a model of advanced ageing. iMSBmal1(-/-) mice develop increased bone calcification and decreased joint collagen, which in combination with the functional changes in skeletal muscle results in altered gait. This study uncovers a fundamental role for the skeletal muscle clock in musculoskeletal homeostasis with potential implications for ageing.

Abstract: Disruption of circadian rhythms in humans and rodents has implicated a fundamental role for circadian rhythms in ageing and the development of many chronic diseases including diabetes, cardiovascular disease, depression and cancer. The molecular clock mechanism underlies circadian rhythms and is defined by a transcription-translation feedback loop with Bmal1 encoding a core molecular clock transcription factor. Germline Bmal1 knockout (Bmal1 KO) mice have a shortened lifespan, show features of advanced ageing and exhibit significant weakness with decreased maximum specific tension at the whole muscle and single fibre levels. We tested the role of the molecular clock in adult skeletal muscle by generating mice that allow for the inducible skeletal muscle-specific deletion of Bmal1 (iMSBmal1). Here we show that disruption of the molecular clock, specifically in adult skeletal muscle, is associated with a muscle phenotype including reductions in specific tension, increased oxidative fibre type, and increased muscle fibrosis similar to that seen in the Bmal1 KO mouse. Remarkably, the phenotype observed in the iMSBmal1(-/-) mice was not limited to changes in muscle. Similar to the germline Bmal1 KO mice, we observed significant bone and cartilage changes throughout the body suggesting a role for the skeletal muscle molecular clock in both the skeletal muscle niche and the systemic milieu. This emerging area of circadian rhythms and the molecular clock in skeletal muscle holds the potential to provide significant insight into intrinsic mechanisms of the maintenance of muscle quality and function as well as identifying a novel crosstalk between skeletal muscle, cartilage and bone.

Figure 1. Core molecular clock gene expression is disrupted with loss of Bmal1 in skeletal muscle

A heat map from median normalized microarray data of mRNAs expressed in a circadian manner from pooled samples (n = 4) of the iMSBmal1 ^+/+ and iMSBmal1 ^−/− GTN collected from the circadian time course is shown. Bright yellow represents mRNA expression that is increased and blue represents mRNA expression that is decreased compared to median levels. Note the significant disruption of circadian expression patterns in the GTN of the iMSBmal1 ^−/− mice.

Figure 2. Real time PCR confirms disruption in the expression of several core molecular clock and clock controlled genes

A, The circadian expression profiles of Bmal1, Per2, Rora, Reverbα, Clock, Cry1, Dbp, and Myod1 mRNA expression every 4h over 24h. The histogram below each timecourse graph provides summary data of the amplitude results obtained with JTK_cycle for each mRNA. The histograms for average fold change provide summary data over the 24h for level of expression as mean ± SEM with significant differences (P < 0.05) designated by an asterisk (^∗). n = 4/time point; ^∗ P < 0.05. B, The relative intensity from microarray expression data generated from pooled samples (n = 4) at each time point (6 time points) of the iMSBmal1 ^+/+ and iMSBmal ^−/− GTN collected from the circadian time course. Array data for Pgc1α, Sox6, Mef2a, and Six1 in the GTN of iMSBmal1 ^+/+ and iMSBmal1 ^−/− mice are shown; ^∗ P < 0.05.

Figure 3. Maintenance of muscle function is disrupted in the iMSBmal1^−/− and Bmal1 KO mice

A, grip strength measured in the iMSBmal1^+/+ (n = 8) and iMSBmal1^−/− (n = 10) at 55–60 weeks post‐treatment was reduced in the iMSBmal1^−/−. B, specific tension measured in the EDL of the iMSBmal1^+/+ and iMSBmal1^−/− mice at 20 weeks post‐treatment was also decreased in the iMSBmal1^−/− mice (n = 3). C, specific tension measured in the EDL of the iMSBmal1^+/+ and iMSBmal1^−/− mice at 58 weeks post‐treatment was also decreased in the iMSBmal1^−/− mice (n = 5). Three additional iMSBmal1^−/− muscles ripped mid‐belly during force measures indicating changes in biological material properties in these muscles. D, grip strength measured in the WT (n = 9) and Bmal1 KO (n = 5) littermates at 25–40 weeks of age was reduced in the Bmal1 KO mice. E, specific tension measured in the EDL of WT (n = 5) and Bmal1 KO (n = 6) mice at 20–22 weeks of age was also decreased in the Bmal1 KO mice. F, peak specific force at 300 Hz measured in the EDL was significantly reduced in both the iMSBmal1^−/− (58 weeks post‐treatment; n = 5) and Bmal1 KO (20–22 weeks post‐treatment; n = 6) mice but was unaffected in the cardiac‐specific Bmal1 KO (iCSΔBmal1^−/−; 65 weeks post‐treatment; n = 3). Summary values are presented as means ± SEM with significant differences (P < 0.05) designated by an asterisk (*).

Figure 4. A shift toward an oxidative fibre type is observed in the skeletal muscles of both the iMSBmal1^−/− and Bmal1 KO mice

A, representative images of fibre type staining (58 weeks post‐treatment) using fibre type‐specific myosin heavy chain antibodies to distinguish type I (pink), type IIA (green), type IIB (red) and type IIX (black) fibres in the TA muscle of the iMSBmal1^+/+ and iMSBmal1^−/− mice is shown. A shift toward a more oxidative fibre type is seen in the summary of the fibre type data at 58 weeks post‐treatment in the iMSBmal1^−/− (n = 9) when compared to iMSBmal1^+/+ (n = 4) mice. B, representative images of SDH staining (58 weeks post‐treatment) in the TA of the iMSBmal1^+/+ and iMSBmal1^−/− mice is shown. A trend toward more oxidative fibres in the iMSBmal1^−/− mice with SDH staining is seen in the summary data (iMSBmal1^+/+, 4; iMSBmal1^−/−, 9). C, myosin heavy chain gels revealed increased type IIA myosin in the plantaris of (58 weeks post‐treatment) iMSBmal1^−/− mice (n = 3). D, representative image of fibre type staining using fibre type‐specific myosin heavy chain antibodies to distinguish type I (pink), type IIA (green), type IIB (red), and type IIX (black) fibres (22 weeks) and summary data in the TA muscle of the WT and Bmal1 KO mice are shown. A shift toward a more oxidative fibre type is observed in the Bmal1 KO muscle (n = 4). E, representative images of SDH staining in the TA of the WT and Bmal1 KO mice is shown. An increase in SDH staining is seen in the summary data for WT and Bmal1 KO mice (n = 4). F, myosin heavy chain gels revealed increased type IIA myosin in the plantaris of the Bmal1 KO mice (22 weeks) (n = 3). Summary values are presented as means ± SEM with significant differences (P < 0.05) designated by an asterisk (*).

Figure 5. The iMSBmal1^−/− and Bmal1 KO mice exhibit differential effects on fibre area and centrally nucleated fibres

A, representative images of H&E stained TA muscle from iMSBmal1^+/+ (n = 4) and iMSBmal1^−/− (n = 5) mice at 58 weeks post‐treatment is shown. No differences in fibre size or the number of centrally nucleated fibres were observed as can be seen in the summary data. B, representative images of H&E stained TA muscle from WT and Bmal1 KO (n = 4) at 20–22 weeks of age. Fibre size was decreased and the number of centrally nucleated fibres was increased as can be seen in the summary data. Summary values are presented as means ± SEM with significant differences (P < 0.05) designated by an asterisk (*).

Figure 6. The iMSBmal1^−/− and Bmal1 KO mice exhibit differential effects on satellite cell number

A, fibrosis was increased in the iMSBmal1^−/− (n = 5) compared to iMSBmal1^+/+ (n = 4) muscle as seen by increased interstitial staining using WGA. B, fibrosis was increased in the Bmal1 KO (n = 5) TA muscle compared to WT (n = 4) as seen by increased interstitial staining using WGA. C, representative Pax7 staining (red) of the TA muscle from 58 week post‐treatment iMSBmal1^+/+ and iMSBmal1^−/− mice is shown. Sections were counterstained with DAPI (blue) and laminin (green), and Pax7⁺/DAPI⁺ nuclei were quantified (white arrows). Pax7⁺/DAPI⁺ nuclei per muscle fibre were not different in the iMSBmal1^−/− mice (n = 6) compared to iMSBmal1^+/+ (n = 3). D, representative PAX7 staining (red) of the tibialis anterior muscle from 22‐week‐old WT and Bmal1 KO mice is shown. Sections were counterstained with DAPI (blue) and laminin (green), and PAX7⁺/DAPI⁺ nuclei were quantified (white arrows). PAX7⁺/DAPI⁺ nuclei per muscle fibre were reduced in the Bmal1 KO mice (n = 8) compared to WT (n = 7). Summary values are presented as mean ± SEM with significant differences (P < 0.05) between iMSBmal1^+/+ and iMSBmal1^−/− mice designated by an asterisk (*).

Figure 7. mRNA expression of genes involved in fibrosis and Wnt and Tgfβ signalling are differentially regulated in the GTN of the iMSBmal1^−/− mice 5 weeks post‐treatment

A, microarray data from the GTN of the iMSBmal1^−/− mice revealed differential expression of extracellular matrix genes. B, microarray data from the GTN of the iMSBmal1^−/− mice revealed differential expression of fibroblast associated genes. C, microarray data from the GTN of the iMSBmal1^−/− mice revealed differential expression of the genes for secreted proteins which have been demonstrated to impact the skeletal system.

Figure 8. Disruption of the molecular clock specifically in adult skeletal muscle is sufficient for disruption of systemic skeletal homeostasis and gait, similar to that observed in the Bmal1 KO

A, representative images of iMSBmal1 ^+/+ and iMSBmal1 ^−/− (58 weeks post‐treatment). Notice the smaller body mass as well as effects on coat condition and tail/ear cartilage. B, arthropathy similar to that observed in the germline Bmal1 KO is present in the iMSBmal1 ^−/− mice. Representative images of iMSBmal1 ^+/+ and iMSBmal1 ^−/− (58 weeks post‐treatment) lower hindlimb and foot stained with Alcian Blue (cartilage) and Alizarin Red which stains calcium (bone). Arrows mark differences in bone showing a thickening of the distal tibia (arrow 1), and calcification of the calcaneal tendon (arrow 2). Representative frames taken from high speed video of iMSBmal1 ^+/+ and iMSBmal1 ^−/− (58 weeks post‐treatment) at toe‐off during treadmill walking showing the marked reduction of range of motion in the ankle and MTP joints in the skeletal muscle‐specific knockout mice. The gait of the knockout mice was noticeably disrupted with more of a hopping behaviour at a speed at which the iMSBmal1 ^+/+ mice walked comfortably. In addition, gait analysis revealed decreased range of motion (ROM) in the iMSBmal1 ^−/− mice. C, representative images of iMSBmal1 ^+/+ and iMSBmal1 ^−/− (58 weeks post‐treatment) ribcage stained with Alcian Blue and Alizarin Red demonstrated increased calcification of the ribcage similar to that observed in the Bmal1 KO mouse. D, representative images of WT and Bmal1 KO (20–22 weeks) lower hindlimb and foot stained with Alcian Blue and Alizarin Red. Arrows mark differences in bone showing a thickening of the distal tibia (arrow 1), and calcification of the calcaneal tendon (arrow 2). Representative frames taken from high speed video of WT and Bmal1 KO (20–22 weeks) at toe‐off during treadmill walking showed the marked reduction of range of motion in the ankle and MTP joints in the global knockout mouse. E, representative images of WT and Bmal1 KO (20–22 weeks) ribcage stained with Alcian Blue and Alizarin Red demonstrated increased calcification of the ribcage.