The skeletal muscle circadian clock regulates titin splicing through RBM20
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Evaluation Summary:
This manuscript will be of broad interest to the field of muscle biology, muscle physiology, exercise physiology, metabolism and circadian rhythms. This manuscript identifies a new molecular pathway that connects circadian rhythms to muscle structure and function through titin isoform switching.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Circadian rhythms are maintained by a cell-autonomous, transcriptional–translational feedback loop known as the molecular clock. While previous research suggests a role of the molecular clock in regulating skeletal muscle structure and function, no mechanisms have connected the molecular clock to sarcomere filaments. Utilizing inducible, skeletal muscle specific, Bmal1 knockout (iMS Bmal1 -/- ) mice, we showed that knocking out skeletal muscle clock function alters titin isoform expression using RNAseq, liquid chromatography–mass spectrometry, and sodium dodecyl sulfate-vertical agarose gel electrophoresis. This alteration in titin’s spring length resulted in sarcomere length heterogeneity. We demonstrate the direct link between altered titin splicing and sarcomere length in vitro using U7 snRNPs that truncate the region of titin altered in iMS Bmal1 -/- muscle. We identified a mechanism whereby the skeletal muscle clock regulates titin isoform expression through transcriptional regulation of Rbm20 , a potent splicing regulator of titin. Lastly, we used an environmental model of circadian rhythm disruption and identified significant downregulation of Rbm20 expression. Our findings demonstrate the importance of the skeletal muscle circadian clock in maintaining titin isoform through regulation of RBM20 expression. Because circadian rhythm disruption is a feature of many chronic diseases, our results highlight a novel pathway that could be targeted to maintain skeletal muscle structure and function in a range of pathologies.
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Author Response
Reviewer #1 (Public Review):
The manuscript by Dr Riley and colleagues reports a novel link between molecular clock operative in skeletal muscle and titin mRNA, encoding for essential regulator of sarcomere length and muscular strength. Surprisingly, this clock-mediated regulation of titin occurs at the level of splicing, as demonstrated by SDS-VAGE analyses of skeletal muscle from muscle-specific Bmal1KO mice compared to Bmal1wt counterpart. Concomitant with switch of predominant isoform of titin, skeletal muscle of muscle specific Bmal1KO mice exhibited irregular sarcomere length. Moreover, the authors show that this shift of titin splice is causal for such sarcomere length irregularity and for altered sarcomere length in muscle from the mice with compromised clock function. Importantly, the authors provide …
Author Response
Reviewer #1 (Public Review):
The manuscript by Dr Riley and colleagues reports a novel link between molecular clock operative in skeletal muscle and titin mRNA, encoding for essential regulator of sarcomere length and muscular strength. Surprisingly, this clock-mediated regulation of titin occurs at the level of splicing, as demonstrated by SDS-VAGE analyses of skeletal muscle from muscle-specific Bmal1KO mice compared to Bmal1wt counterpart. Concomitant with switch of predominant isoform of titin, skeletal muscle of muscle specific Bmal1KO mice exhibited irregular sarcomere length. Moreover, the authors show that this shift of titin splice is causal for such sarcomere length irregularity and for altered sarcomere length in muscle from the mice with compromised clock function. Importantly, the authors provide compelling evidence that Rbm20, encoding for RNA-binding protein that mediates splicing of titin, is cooperatively regulated by Bmal1-Clock heterodimer and MyoD, via enhancer element in intron 1 of Rbm20, thus identifying Rbm20 as a novel direct clock-regulated gene in the skeletal muscle. Strikingly, rescue of Rbm20 in muscle specific Bmal1KO animals' results in rescue of titin splicing pattern and protein size, suggesting that Rbm20 mediates the regulatory effect of Bmal1 on titin splicing and represents a mechanistic link between the clock and regulator of sarcomere length and regularity.
We thank reviewer 1 for the very kind comments. We agree that the circadian regulation of titin in any capacity is surprising. We are excited about the implications of our work for cardiac muscle and its therapeutic potential in human skeletal muscle.
Reviewer #2 (Public Review):
In this work the authors investigated whether deleting the BMAL1 gene, an integral component of the cellular clock that drives the circadian rhythms of cells, affects the giant protein titin. They report that deleting BMAL1 in skeletal muscle alters the splicing of titin and that this might underlie an increase in sarcomere length dispersion. They show that the effect is through the titin splicing factor RBM20. This work has high novelty and has the potential to add to our understanding of muscle physiology. It is unclear whether splicing of skeletal muscle titin indeed undergoes a circadian rhythm. This could be easily checked using protein gels or RNA seq in muscle samples collected at different times of the day.
We appreciate the question and recognize that our original manuscript did not clearly outline that the circadian clock regulates both rhythmic and non-rhythmic gene expression. In this study, the target of the muscle clock is expression of Rbm20 mRNA which is not a rhythmically expressed gene in muscle. This has now been addressed in the manuscript.
Based on the estimated titin turnover and incorporation rates of titin (Cadar et al., 2014), we do not believe that skeletal muscle titin splicing undergoes a circadian rhythm. However, we believe our data highlights the growing recognition of the molecular clock in regulating non-rhythmic processes. We have added data from a chronic phase advance model of circadian disruption with wildtype mice and identify that disrupted circadian rhythms are sufficient to change Rbm20 expression in skeletal muscle (Figure 5).
This work would be more convincing if the sarcomere length dispersion was investigated in greater detail. Showing this in one muscle type only (TA), in muscles fixed at one length only, and not showing sarcomere length dispersion in the rescue experiment of Figure 6, is rather limited.
We agree that our analysis of sarcomere length dispersion across joint angles would be interesting but we think it is beyond the scope of this study. As noted above, the premise of this study emerged from our early work in which we found that skeletal muscle from 2 different genetic mouse models of circadian disruption, Bmal1 KO mice as well as the Clock mutant mice, exhibit decreased maximum specific force with significant disruptions to sarcomere structure (Andrews et al., PNAS, 107 (44) 19090-19095 2010). The primary focus of this study was to address the mechanistic link between the muscle circadian clock, its transcriptional targets with a focus on sarcomere structure and our first clue was with the expression of titin isoforms. We included analysis of sarcomere length as an outcome measure because it is a fundamental feature of skeletal muscle, it has links to mechanical function and it is a structure that can be modified by titin spliceforms.
A small increase in sarcomere length variation as suggested in Figure 2 is unlikely to have a great functional consequence. If it were, how can muscles that express naturally long titin isoforms (soleus, EDL, diaphragm, etc), function well?
We did not intend to suggest that we see an increase in sarcomere length in Figure 2 and have clarified the figure and text accordingly. The change we see is related to the variability of sarcomere length; we do not see any change in the average sarcomere length. The topic of titin spliceform specialization and the contribution to sarcomere structure and function across different muscle groups (soleus vs. EDL vs. Diaphragm) is a really interesting question but beyond the scope of this study.
Reviewer #3 (Public Review):
This manuscript is using an inducible and skeletal muscle specific Bmal1 knockout mouse model (iMSBmal1-/-) that was published previously by the same group. In this study, they utilized the same mouse model and further investigated the effect of a core molecular clock gene Bmal1 on isoform switching of a giant sarcomeric protein titin and sarcomere length change resulted from titin isoform switching. Lance A. Riley et al found that iMSBmal1-/- mouse TA muscle expressed more longer titin due to additional exon inclusion of Ttn mRNA compared to iMSBmal+/+ mice. They observed that sarcomere length did not significantly change but more variable in iMSBmal1-/- muscle compared to iMSBmal+/+ muscle. In addition, they identified significant exon inclusion in the proximal Ig region, so they measured the proximal Ig length domain and confirmed that proximal Ig domain was significantly longer in iMSBmal1-/- muscle. Subsequently, they experimentally generated a shorter titin in C2C12 myotubes and observed that the shorter titin led to the shorter sarcomere length. Since RBM20 is a major regulator of Ttn splicing, they determined RBM20 expression level, and found that RBM20 expression was significantly lower in iMSBmal1-/- muscle. The reduced RBM20 expression was regulated by the molecular clock controlled transcriptional factor MyoD1. By performing a rescue experiment in vivo, the authors found that rescue of RBM20 in iMSBmal1-/- TA muscle restored titin isoform expression, however, they did not measure whether sarcomere length was restored. These data provide new information that the molecular cascades in the circadian clock mechanism regulate RBM20 expression and downstream titin isoform switching and sarcomere length change. Although the conclusion of this manuscript is mostly supported by the data, some aspects of experimental design and data analysis need be clarified and extended.
Strengths:
This paper links the circadian rhythms to skeletal muscle structure and function through a new molecular cascade: the core clock component Bmal1-transcription factor MyoD1-RBM20 expression-titin isoform switching-sarcomere length change.
Utilization of muscle specific bmal1 knockout mice could rule out the confounding factors from the molecular clock in other cell types
The authors performed the RNA sequencing and label free LC-MS analyses to determine the exon inclusion and exclusion through a side-by-side comparison which is a new approach to identify individual alternative spliced exons via both mRNA level and protein level.
We agree that the side-by-side analysis from RNAseq and LC-MS data are novel and provides a foundation for others wanting to study both titin mRNA and protein. In this version, we have expanded this work to include samples from our Rbm20 rescue model (Figure 6). Similarly, to our approach in the muscle specific Bmal1 knockout model, these results confirm our RNA-seq results and indicate that LC-MS is a suitable method to measure titin protein isoform. We note that while more work is needed to confirm the broad utility of the LC-MC approach, it may be a suitable alternative to RNA-seq for measuring region-specific, and possibly exon-specific, changes in titin isoform expression.
Weaknesses:
Both RBM20 expression and titin isoform expression varies in different skeletal muscles. The authors only detected their expression in TA muscle. It is not clear why the authors only chose TA muscle.
The reviewer, like Reviewer 2, raises a good point about muscle specificity as this is a significant challenge for research in the field of skeletal muscle. As we noted above, our primary focus was on the TA because our goal was to study the molecular links between the muscle circadian clock and titin expression with inclusion of analysis of a structural outcome, sarcomere length variability. This muscle is well suited for the combination of approaches employed. We recognize the limits of using a single muscle, but we note that the we used ChIPseq data that provided the initial clues that CLOCK and BMAL1 bind to a site within intron 1 of the Rbm20 gene came from gastrocnemius and not TA muscle samples . Our targeted ChIP-PCR confirms that CLOCK and BMAL1 bind to the same intron 1 location from TA muscle samples. In addition, we have included data from quadriceps and TA muscles in our chronic jet lag model in which we use an environmental manipulation to disrupt the muscle clocks. We believe that the edits to the text and inclusion of this data strengthen and extends our findings to other muscles through circadian disruption and not only a genetic knockout model.
The sarcomere length data are self-contradictory. The authors stated that sarcomere length was not significantly changed in muscle specific KO mice in Line 149, however, in Line 163, the measurements showed significantly longer in muscle specific KO muscle. The significance is also indicated in Figures 2C and 3B.
We apologize for the miscommunication. The significance indicated in Figure 2C refers to the significant difference in variability of sarcomere length and not a significant difference in sarcomere length. The difference in Figure 3B is to indicate a slightly longer but significantly different from control sarcomere length, but also a significant difference in sarcomere length variability. To make this difference clear, we have changed the symbol for significantly different variability from * to # in both Figures 2C and 3B. We hope this clarifies our findings.
Manipulating titin size using U7 snRNPs linking to the changes in sarcomere length and overexpressing RBM20 to switch titin size are the concepts that have been proved. These data do not directly support the impact of muscle specific Bmal1 KO on ttn splicing and RBM20 expression
We agree that the use of U7 snRNPs does not directly support the impact of muscle specific Bmal1 KO on titin splicing and RBM20 expression; however, that was not the goal of this set of experiments. Several papers have recently indicated titin’s role as a sarcomeric ruler (Tonino 2017, Brynnel 2018), but none of them have investigated the proximal Ig domain that we identified as regulated by the circadian clock disruption. Because of this, we thought it necessary to show this region specifically contributes to sarcomere length using our cell culture model. Further, we think this point strengthens our study as it suggests that in the absence of a clock effect, altering the proximal Ig domain of titin directly alters sarcomere length adding to the growing evidence base that titin acts as a sarcomeric ruler. We have edited the text of the results and the discussion to clarify this point.
There is no evidence to show if interrupted circadian rhythms in mice change RBM20 expression and ttn splicing, which is critical to validate the concept that circadian rhythms are linked to Ttn splicing through RBM20.
We recognize this concern and have performed a new study in which we used a model of chronic jet lag in normal adult C57BL6 mice as a model to disrupt the muscle clock (Wolff, Duncan and Esser, JAP 2013). This new data has been added in Figure 5 and shows that by altering the lights on: lights off schedule every 4 days for 8 weeks, mimicking repeated jet lag, we disrupt Rbm20 expression in TA and gastrocnemius muscle (note, this is new data for both the muscle and clock fields). Concomitant with changes in clock gene expression we reported in 2013, we found that mRNA expression of Rbm20 is altered as well. These findings confirm that normal muscle clock disruption is sufficient to alter expression of Rbm20.
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Evaluation Summary:
This manuscript will be of broad interest to the field of muscle biology, muscle physiology, exercise physiology, metabolism and circadian rhythms. This manuscript identifies a new molecular pathway that connects circadian rhythms to muscle structure and function through titin isoform switching.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The manuscript by Dr Riley and colleagues reports a novel link between molecular clock operative in skeletal muscle and titin mRNA, encoding for essential regulator of sarcomere length and muscular strength. Surprisingly, this clock-mediated regulation of titin occurs at the level of splicing, as demonstrated by SDS-VAGE analyses of skeletal muscle from muscle-specific Bmal1KO mice compared to Bmal1wt counterpart. Concomitant with switch of predominant isoform of titin, skeletal muscle of muscle specific Bmal1KO mice exhibited irregular sarcomere length. Moreover, the authors show that this shift of titin splice is causal for such sarcomere length irregularity and for altered sarcomere length in muscle from the mice with compromised clock function. Importantly, the authors provide compelling evidence that …
Reviewer #1 (Public Review):
The manuscript by Dr Riley and colleagues reports a novel link between molecular clock operative in skeletal muscle and titin mRNA, encoding for essential regulator of sarcomere length and muscular strength. Surprisingly, this clock-mediated regulation of titin occurs at the level of splicing, as demonstrated by SDS-VAGE analyses of skeletal muscle from muscle-specific Bmal1KO mice compared to Bmal1wt counterpart. Concomitant with switch of predominant isoform of titin, skeletal muscle of muscle specific Bmal1KO mice exhibited irregular sarcomere length. Moreover, the authors show that this shift of titin splice is causal for such sarcomere length irregularity and for altered sarcomere length in muscle from the mice with compromised clock function. Importantly, the authors provide compelling evidence that Rbm20, encoding for RNA-binding protein that mediates splicing of titin, is cooperatively regulated by Bmal1-Clock heterodimer and MyoD, via enhancer element in intron 1 of Rbm20, thus identifying Rbm20 as a novel direct clock-regulated gene in the skeletal muscle. Strikingly, rescue of Rbm20 in muscle specific Bmal1KO animals' results in rescue of titin splicing pattern and protein size, suggesting that Rbm20 mediates the regulatory effect of Bmal1 on titin splicing and represents a mechanistic link between the clock and regulator of sarcomere length and regularity.
-
Reviewer #2 (Public Review):
In this work the authors investigated whether deleting the BMAL1 gene, an integral component of the cellular clock that drives the circadian rhythms of cells, affects the giant protein titin. They report that deleting BMAL1 in skeletal muscle alters the splicing of titin and that this might underlie an increase in sarcomere length dispersion. They show that the effect is through the titin splicing factor RBM20. This work has high novelty and has the potential to add to our understanding of muscle physiology.
It is unclear whether splicing of skeletal muscle titin indeed undergoes a circadian rhythm. This could be easily checked using protein gels or RNA seq in muscle samples collected at different times of the day.
This work would be more convincing if the sarcomere length dispersion was investigated in …
Reviewer #2 (Public Review):
In this work the authors investigated whether deleting the BMAL1 gene, an integral component of the cellular clock that drives the circadian rhythms of cells, affects the giant protein titin. They report that deleting BMAL1 in skeletal muscle alters the splicing of titin and that this might underlie an increase in sarcomere length dispersion. They show that the effect is through the titin splicing factor RBM20. This work has high novelty and has the potential to add to our understanding of muscle physiology.
It is unclear whether splicing of skeletal muscle titin indeed undergoes a circadian rhythm. This could be easily checked using protein gels or RNA seq in muscle samples collected at different times of the day.
This work would be more convincing if the sarcomere length dispersion was investigated in greater detail. Showing this in one muscle type only (TA), in muscles fixed at one length only, and not showing sarcomere length dispersion in the rescue experiment of Figure 6, is rather limited.
A small increase in sarcomere length variation as suggested by Figure 2 is unlikely to have a great functional consequence. If it were, how can muscles that express naturally long titin isoforms (soleus, EDL, diaphragm, etc), function well?
-
Reviewer #3 (Public Review):
This manuscript is using an inducible and skeletal muscle specific Bmal1 knockout mouse model (iMSBmal1-/-) that was published previously by the same group. In this study, they utilized the same mouse model and further investigated the effect of a core molecular clock gene Bmal1 on isoform switching of a giant sarcomeric protein titin and sarcomere length change resulted from titin isoform switching. Lance A. Riley et al found that iMSBmal1-/- mouse TA muscle expressed more longer titin due to additional exon inclusion of Ttn mRNA compared to iMSBmal+/+ mice. They observed that sarcomere length did not significantly change but more variable in iMSBmal1-/- muscle compared to iMSBmal+/+ muscle. In addition, they identified significant exon inclusion in the proximal Ig region, so they measured the proximal Ig …
Reviewer #3 (Public Review):
This manuscript is using an inducible and skeletal muscle specific Bmal1 knockout mouse model (iMSBmal1-/-) that was published previously by the same group. In this study, they utilized the same mouse model and further investigated the effect of a core molecular clock gene Bmal1 on isoform switching of a giant sarcomeric protein titin and sarcomere length change resulted from titin isoform switching. Lance A. Riley et al found that iMSBmal1-/- mouse TA muscle expressed more longer titin due to additional exon inclusion of Ttn mRNA compared to iMSBmal+/+ mice. They observed that sarcomere length did not significantly change but more variable in iMSBmal1-/- muscle compared to iMSBmal+/+ muscle. In addition, they identified significant exon inclusion in the proximal Ig region, so they measured the proximal Ig length domain and confirmed that proximal Ig domain was significantly longer in iMSBmal1-/- muscle. Subsequently, they experimentally generated a shorter titin in C2C12 myotubes and observed that the shorter titin led to the shorter sarcomere length. Since RBM20 is a major regulator of Ttn splicing, they determined RBM20 expression level, and found that RBM20 expression was significantly lower in iMSBmal1-/- muscle. The reduced RBM20 expression was regulated by the molecular clock controlled transcriptional factor MyoD1. By performing a rescue experiment in vivo, the authors found that rescue of RBM20 in iMSBmal1-/- TA muscle restored titin isoform expression, however, they did not measure whether sarcomere length was restored. These data provide new information that the molecular cascades in the circadian clock mechanism regulate RBM20 expression and downstream titin isoform switching and sarcomere length change. Although the conclusion of this manuscript is mostly supported by the data, some aspects of experimental design and data analysis need be clarified and extended.
Strengths:
This paper links the circadian rhythms to skeletal muscle structure and function through a new molecular cascade: the core clock component Bmal1-transcription factor MyoD1-RBM20 expression-titin isoform switching-sarcomere length change.
Utilization of muscle specific bmal1 knockout mice could rule out the confounding factors from the molecular clock in other cell types
The authors performed the RNA sequencing and label free LC-MS analyses to determine the exon inclusion and exclusion through a side-by-side comparison which is a new approach to identify individual alternative spliced exons via both mRNA level and protein level.
Weaknesses:
Both RBM20 expression and titin isoform expression varies in different skeletal muscles. The authors only detected their expression in TA muscle. It is not clear why the authors only chose TA muscle.
The sarcomere length data are self-contradictory. The authors stated that sarcomere length was not significantly changed in muscle specific KO mice in Line 149, however, in Line 163, the measurements showed significantly longer in muscle specific KO muscle. The significance is also indicated in Figures 2C and 3B.
Manipulating titin size using U7 snRNPs linking to the changes in sarcomere length and overexpressing RBM20 to switch titin size are the concepts that have been proved. These data do not directly support the impact of muscle specific Bmal1 KO on ttn splicing and RBM20 expression
There is no evidence to show if interrupted circadian rhythms in mice change RBM20 expression and ttn splicing, which is critical to validate the concept that circadian rhythms are linked to Ttn splicing through RBM20.
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