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• W4295736708 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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 (iMSBmal1-/-) 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 iMSBmal1-/- 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. Editor's evaluation Riley et al. provide a fundamental study that advances our understanding of how muscle biology is regulated by the circadian clock. The authors use compelling methodology to reveal a novel molecular mechanism for circadian regulation of the muscle giant protein titin via the splicing factor RBM20. The work will be of broad interest to muscle and circadian biologists, with implications for muscle-related disorders. https://doi.org/10.7554/eLife.76478.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Circadian rhythms are intrinsically directed patterns seen in behavior and biology with an ~24 hr periodicity (Bass and Takahashi, 2010). Underlying circadian rhythms is a transcriptional–translational feedback loop referred to as the molecular clock. The molecular clock exists in virtually every cell in the body and functions to support homeostasis through regulating a daily pattern of gene expression (Merrow et al., 2005; Yoo et al., 2004). BMAL1 is a PAS/basic helix–loop–helix (bHLH) transcription factor that acts as the only nonredundant component of the core clock mechanism (Bunger et al., 2000; Hogenesch et al., 1998). Beyond its role in cellular time keeping, BMAL1 and its partner, CLOCK, have been described as transcriptional regulators of both rhythmic and nonrhythmic genes important for cell-type-specific functions (Bass and Takahashi, 2010; Bozek et al., 2009; Miller et al., 2007). The most well-studied aspect of these functions, particularly in the context of skeletal muscle, is the circadian clock regulation of carbohydrate and lipid metabolism (Reinke and Asher, 2019). Our lab has provided evidence suggesting a more expansive role for the skeletal muscle clock through maintenance of skeletal muscle structure and function (McCarthy et al., 2007; Andrews et al., 2010; Schroder et al., 2015). The first evidence for this role came from microarray analyses showing expression of the myogenic regulatory factor Myod1 as well as genes encoding contractile proteins are circadian in the hindlimb muscles of C57BL/6J mice (McCarthy et al., 2007). The molecular clock’s proposed role in maintaining muscle function was supported with whole-muscle and single-fiber mechanics data from the Bmal1 knockout mice (Andrews et al., 2010). Additionally, electron microscopy studies demonstrated sarcomere irregularities consistent with the diminished force output findings. However, these experiments were performed in systemic Bmal1 KO mice that exhibit significant behavioral abnormalities, thus, distinguishing the role of skeletal muscle molecular clock from activity patterns requires further study using more refined approaches and animal models. Historically, sarcomeres have been studied as a two-filament system; however, a third filament comprised of the giant protein titin is now broadly recognized as having a critical role in both the structure and contractile function of sarcomeres (Freundt and Linke, 2019). Titin is the largest known protein and spans the length of the half-sarcomere, from Z-line to M-line, functioning as the structural backbone for the sarcomere and aiding in both active and passive force generation (Freundt and Linke, 2019; Brynnel et al., 2018; Mijailovich et al., 2019). The I-band region of titin connects the thick filament to the thin filament and acts as a viscoelastic spring. This region is primarily composed of immunoglobulin-like (Ig) repeats and the PEVK region. Titin undergoes a remarkable amount of splicing within this region with inclusion or exclusion of these repeats yielding longer or shorter titin proteins, respectively (Guo et al., 2010; Guo et al., 2012; Li et al., 2013). Maintenance of titin splicing, and thus the isoform composition, is needed to maintain muscle function. This study utilized inducible, skeletal muscle-specific Bmal1 (iMSBmal1-/-) knockout mice to further examine the role of the skeletal muscle molecular clock in regulating titin splicing. Importantly, these mice display muscle weakness defined by ex vivo measures of maximum isometric tetanic force (i.e., specific force) (Schroder et al., 2015). We show that loss of Bmal1 specifically in adult skeletal muscle alters splicing in titin’s spring region, resulting in sarcomere length disruption. Consistent with changes in splicing, we identify exon-specific protein changes in titin using a methodological approach for analyzing titin isoforms using liquid chromatography–mass spectrometry (LC-MS). Mechanistically, the skeletal muscle molecular clock regulates Rbm20, the gene encoding an RNA-binding protein known to regulate titin splicing. Overexpressing RBM20 in iMSBmal1-/- skeletal muscle is sufficient to restore titin isoform expression. Use of a chronic jet-lag model with wildtype mice confirms that circadian disruption is sufficient to change Rbm20 expression. Our findings establish the role of the skeletal muscle molecular clock in regulating Rbm20 expression and titin splicing and provide a mechanism by which circadian dysregulation might alter skeletal muscle physiology. Results Titin splicing is disrupted following Bmal1 knockout in adult skeletal muscle Since titin has been implicated in regulating sarcomere length and plays a prominent role in striated muscle force generation, we asked whether titin protein changes following Bmal1 knockout in skeletal muscle. Using sodium dodecyl sulfate-vertical agarose gel electrophoresis (SDS-VAGE) to analyze gross size changes in titin protein of iMSBmal1-/- tibialis anterior (TA) muscle, we found a shift in titin isoform composition (p<0.01; Figure 1A and B) with no change in total titin expression. The shift in titin isoforms changes the predominant form from a short isoform to a mixture of both short and long titin proteins. Figure 1 Download asset Open asset Skeletal muscle Bmal1 knockout results in altered splicing of titin’s spring region. (A) Sodium dodecyl sulfate-vertical agarose gel electrophoresis (SDS-VAGE) was performed to measure titin protein isoform in iMSBmal1+/+ and iMSBmal1-/- tibialis anterior muscles. (B) Quantification of titin isoforms in these muscles shows a significant shift in titin isoform from a predominantly short isoform to a mix of long and short isoforms in iMSBmal1-/- muscles but not iMSBmal1+/+ muscles (N = 8/group). (C) Percent spliced in (PSI) and ∆PSI of titin exons expressed in iMSBmal1+/+ and iMSBmal1-/- skeletal muscle. Exons within the proximal Ig domain were included more often in iMSBmal1-/- muscle compared to iMSBmal1+/+ muscle (N = 3/group). (D) Average PSI across three splicing events in the proximal Ig domain of tibialis anterior muscle show increased exon inclusion in iMSBmal1-/- mice compared to iMSBmal1+/+ controls (N=3/group). Each event is highlighted in Figure 2C. (E) Liquid chromatography–mass spectrometry (LC-MS)-quantified peptide abundance mapping onto exons identified using RNAseq confirms changes in titin splicing are translated to titin protein (N=3/group). §No peptides were detected that mapped to exons 52–69 in iMSBmal1+/+ muscle. Data plotted as mean ± SEM. Statistical significance determined by Student's t-test. *p<0.05, **p<0.01, ***p<0.0001. Figure 1—source data 1 Sodium dodecyl sulfate-vertical agarose gel electrophoresis (SDS-VAGE) gels used for quantifying titin isoform ratios in iMSBmal1+/+ and iMSBmal1-/- tibialis anterior (TA) muscle. https://cdn.elifesciences.org/articles/76478/elife-76478-fig1-data1-v2.zip Download elife-76478-fig1-data1-v2.zip Figure 1—source data 2 Titin peptide-level data used for quantifying domain-level changes to titin splicing. Data are provided for each individual and listed with the exon coding for the peptide. https://cdn.elifesciences.org/articles/76478/elife-76478-fig1-data2-v2.zip Download elife-76478-fig1-data2-v2.zip Titin mRNA is well known to undergo significant number of splicing events to generate its many isoforms (Guo et al., 2010). To gain insight into the changes in titin isoform following loss of Bmal1 in adult skeletal muscle, we performed RNA sequencing to identify exon-specific changes in iMSBmal1-/- muscle (Guo et al., 2012; Schafer et al., 2015; Maatz et al., 2014). iMSBmal1-/- TA muscle showed increased percent sliced in (PSI) of exons 52–88 of the titin transcript compared to iMSBmal1+/+ control samples (Figure 1C). These exons code for a portion of titin’s proximal Ig segment, one of titin’s extensible segments within its spring region. Though the ∆PSI across this splicing event was stable, the variability in PSI across this event made statistical analysis difficult. To remedy this, we split this large splicing event into three, smaller splicing events (exons 52–69, 70–79, and 80–88) based on the similarity of PSI values within each sample (Figure 1C). In each of these smaller events, inclusion of exons was significantly greater in iMSBmal1-/- muscle compared to iMSBmal1+/+ muscle (Figure 1D; p<0.05). Thus, Bmal1 knockout in skeletal muscle altered splicing of titin’s I band localized spring region. We next asked whether this difference in Ttn mRNA splicing resulted in detectable differences in titin protein. To date, no paper has provided side-by-side comparisons of RNA splicing and exon inclusion of titin protein. To address this gap in the field, we homogenized skeletal muscle from iMSBmal1+/+ and iMSBmal1-/- mice using a previously published method for analyzing MYBPC isoforms (O’Leary et al., 2019). Proteins were digested with trypsin, and peptide abundances were quantified by label-free LC-MS analyses. A total of 1218 peptides corresponding to the Ensembl ENSMUST00000099981.10 titin transcript were identified in the LC-MS analysis of which 1009 met our minimum threshold for quantification and were used to calculate exon-specific peptide abundance. These peptides corresponded to 225 of the 363 known exons in the Ttn gene (Figure 1—source data 2). We then analyzed differential expression of peptides and identified that those representing exons 52–69 were not detected in iMSBmal1+/+ muscle and only identified in the iMSBmal1-/- muscle samples. The abundance of peptides from the translation of exons 70–79 and 80–88 were 18 and 16% greater in the iMSBmal1-/- compared to the iMSBmal1+/+ muscle samples, respectively (p<0.05; Figure 1E). Notably, the only other region that displayed significant differences at the protein level is encoded by exons 112–217. This region is lowly expressed in both iMSBmal1+/+ and iMSBmal1-/- muscle, thus the difference in our measurement may be a result of low read numbers across this region in the RNAseq data. These results demonstrate that the difference in Ttn mRNA splicing encoding exons within the proximal Ig region in the iMSBmal1-/- muscle is translated to titin protein. Our findings from these experiments also provide a unique analytical reference for the titin research community. iMSBmal1-/- muscle displays irregular sarcomere lengths We have previously reported that the global Bmal1 knockout mouse displays sarcomere abnormalities in skeletal muscle (Andrews et al., 2010); however, these sarcomeric changes could be a result of a loss of skeletal muscle Bmal1, a loss of behavioral rhythms, and/or other systemic interactions. Since titin acts as a sarcomeric ruler and our observed titin splicing change is present in titin protein, we measured sarcomere length in iMSBmal1-/- skeletal muscle. To determine whether iMSBmal1-/- skeletal muscle presents with abnormal sarcomere morphology to accompany the weakness, we longitudinally cryosectioned TA muscles and measured sarcomere lengths as the distance between peak fluorescence following α-actinin immunostaining (Figure 2A). Using this approach, mean sarcomere length did not significantly change following skeletal muscle-specific loss of Bmal1 (p=0.86; Figure 2B). However, sarcomere length homogeneity is a feature of healthy striated muscle, and we noted that sarcomere length was significantly more variable in iMSBmal1-/- muscle compared to iMSBmal1+/+ muscle (F = 22.12, p<0.05; Figure 2B). Figure 2 Download asset Open asset Sarcomere lengths are variable in iMSBmal1-/- muscle. Representative images from (A) iMSBmal1+/+ and (B) iMSBmal1-/- tibialis anterior muscles. Muscles were longitudinally cryosectioned and stained with a primary antibody against α-actinin-2. (C) Sarcomere lengths are significantly more variable as based on an F-test in iMSBmal1-/- muscles compared to iMSBmal1+/+ controls. N = 4/group. #Significant difference in variance (p<0.05). Titin isoform shift accounts for sarcomere length irregularity in iMSBmal1-/- muscle After defining the region of titin mRNA that changed in iMSBmal1-/- muscle, we asked whether splicing in this region would be sufficient to account for the increased sarcomere length variability. To test this, we performed immunohistochemistry using epitope-specific antibodies against titin’s Z1Z2 and N2A domains because these epitopes flank the region with increased exon inclusion, the proximal Ig segment of titin. The positional difference between the two domains provides a proxy for proximal Ig domain length (Figure 3A). Representative images of iMSBmal1+/+ and iMSBmal1-/- muscle can be seen in Figure 3B and C, respectively. The significant difference in sarcomere length variability persisted as compared through an F-test of group variance though the sarcomere lengths themselves were longer than in our previous measurements. iMSBmal1+/+ muscle had sarcomere lengths of 2.12 ± 0.01 µm while iMSBmal1-/- muscle had sarcomere lengths of 2.28 ± 0.02 µm (F = 3.62, p<0.001; Figure 3D). Once we determined that sarcomere length variability was maintained, we asked whether proximal Ig domain length was significantly altered following Bmal1 knockout in adult skeletal muscle. iMSBmal1-/- had significantly longer proximal Ig domain lengths (229.5 ± 1.357 nm) than the proximal Ig domain from iMSBmal1+/+ samples (182.4 ± 1.388 nm; p<0.0001; Figure 3E). This difference in lengths accounts for the difference we see in sarcomere lengths in these samples. However, the variation around the mean for proximal Ig domain length was not significantly different (F = 1.338, p=0.12). These changes confirm that the proximal Ig domain length in adult skeletal muscle is correlated with a corresponding change in sarcomere length. Figure 3 Download asset Open asset The change in titin’s spring length in iMSBmal1-/- muscle accounts for sarcomere length variability. (A) Proximal Ig length was determined as the positional difference between the Z1Z2 domain of titin and the N2A domain of titin. These antibodies flank the proximal Ig domain. Created with BioRender.com. Representative images of immunohistochemistry using titin-epitope-specific and α-actinin-2 antibodies for (B) iMSBmal1+/+ and (C) iMSBmal1-/- skeletal muscle. Green: N2A titin; red: Z1Z2 titin; blue: α-actinin-2. (D) Sarcomere length variability is significantly lower in iMSBmal1+/+ compared to iMSBmal1-/- skeletal muscle. (E) A significantly longer proximal Ig length is correlated with this change in sarcomere length variability. N = 250–350 sarcomeres/three biological replicates per group. Data plotted as mean ± SEM. Statistical significance determined by Student's t-test. ***p<0.001, ****p<0.0001. # Significant difference in variance determined by F-test (p<0.05). Changes in titin I-band splicing are directly linked to changes in sarcomere length While the changes to sarcomere length of iMSBmal1-/- TA muscle are associated with titin splicing and length, this model does not causally link titin splicing within the proximal Ig region to sarcomeric structure. Thus, we thought it necessary to manipulate titin splicing without the influence of the clock. To directly test whether the region of titin that is altered in iMSBmal1-/- muscle could contribute to changes in sarcomere length independent of clock changes, we used U7 snRNPs to direct titin splicing from the 5′ splice site of titin exon 51 to the 3′ splice site of titin exon 89, resulting in shorter titin protein using an in vitro C2C12 myotube model. To visualize sarcomere length, we used a stable cell line in which α-actinin is labeled with GFP (eGFP-ACTN2-C2C12). These labeled myocytes were transfected with the titin-specific U7 snRNPs and plated on micropatterned gelatin hydrogels to support maturation of the myotube culture and sarcomere formation and alignment (Denes et al., 2019; Bettadapur et al., 2016). At day 10 of differentiation, eGFP-ACTN2-C2C12 myotubes were fixed then stained with a titin N2A epitope-specific antibody similarly to the tissue samples from iMSBmal1-/- muscle (Figure 4A). Representative images of control eGFP-ACTN2-C2C12 myotubes and Ttn-U7 transfected eGFP-ACTN2-C2C12 myotubes can be seen in Figure 4B and C, respectively. While control myotubes had an average sarcomere length of 2.756 ± 0.0495 µm, Ttn-U7 transfected myotubes had significantly shorter sarcomeres with an average sarcomere length of 2.387 ± 0.0347 µm (p<0.0001, n = 100–200 sarcomeres/two biological replicates; Figure 4D). Because ACTN2 is a key component of the Z-line, the distance between the N2A-epitope and eGFP-ACTN2 was used to calculate proximal Ig length in this experiment. Proximal Ig domain lengths were also significantly different. Ttn-U7 transfected myotubes had a proximal Ig length of 285.1 ± 3.131 nm compared to 448.0 ± 3.368 nm of control myotubes (p<0.0001; Figure 4E). These results confirm that altering the length of titin’s proximal Ig domain causes a change in sarcomere length in skeletal muscle myotubes. Figure 4 Download asset Open asset Directly shortening titin’s proximal Ig domain length results in shorter sarcomeres in vitro. (A) U7 snRNPs were designed to induce splicing or the proximal Ig domain with dysregulated splicing in iMSBmal1-/- muscle. Created with BioRender.com. Representative images of immunocytochemistry using a titin-epitope-specific antibody and stably expressing eGFP-ACTN2 C2C12 myotubes that were transfected with an (B) empty vector control or (C) Ttn-U7 splicing factors. Green: α-actinin-2; red: N2A titin. (D) Sarcomere lengths were significantly shorter in myotubes transfected Ttn-U7 vectors compared to empty vector controls. (E) Ttn-U7 vector transfection resulted in significantly shorter proximal Ig domain lengths in eGFP-ACTN2-C2C12 myotubes. N = 100-200 sarcomeres/two biological replicates per group. Data plotted as mean ± SEM. Statistical significance determined by Student's t-test. ****p<0.0001. Rbm20 is targeted by the skeletal muscle molecular clock Having established that iMSBmal1-/- skeletal muscle has an altered titin isoform composition resulting in sarcomere length heterogeneity, we looked to define the mechanism linking the skeletal muscle molecular clock to titin splicing. RBM20 is a well-characterized splicing factor known to target titin pre-mRNA (Guo et al., 2012; Li et al., 2013; Maatz et al., 2014). RBM20 is an RNA-binding protein that acts as a splicing repressor such that when present at high levels it binds along titin pre-mRNA preventing normal intronic splicing. When bound by many RBM20 proteins, titin pre-mRNA undergoes large exon-skipping events whereby the resultant, processed mRNA is shorter than if RBM20 was not present. We tested whether RBM20 levels change following loss of skeletal muscle Bmal1. Rbm20 mRNA expression was reduced by 34% in iMSBmal1-/- muscle compared to iMSBmal1+/+ muscle (p<0.05; Figure 5A). This reduced expression was also present at the protein level where RBM20 protein content decreased by 46% compared to iMSBmal1+/+ muscle (p<0.05; Figure 5B). Figure 5 Download asset Open asset Rbm20 is targeted by the positive limb of the skeletal muscle molecular clock. (A) Rbm20 mRNA is decreased by 34% in iMSBmal1-/- muscle compared to iMSBmal1+/+ muscle (N = 6–8/group). (B) iMSBmal1-/- muscle shows a 57% reduction in RBM20 protein levels compared to iMSBmal1+/+ control muscle (N = 4/group). (C) BMAL1, CLOCK, and MYOD1 ChIP-seq data were used to identify potential regulatory regions for Rbm20. A significant binding peak was found within intron 1 of this gene. (D) C57BL/6J mice subjected to repeated phase advances (CPA) show a significant reduction in Rbm20 mRNA expression in the quadriceps muscle (N = 4/group). (E) C57BL/6J mice subjected to CPA show a nonsignificant reduction in Rbm20 expression in the tibialis anterior muscle (N = 3/group). (F) ChIP-PCR confirms binding of each protein of the positive limb of the molecular clock at this site (N = 3/group). (G) Dual-luciferase activity shows significant activation of E-Rbm20-Luc with overexpression of the positive limb of the molecular clock (N = 3/group). Data plotted as mean ± SEM. Statistics performed using Student's t-test or one-way ANOVA with Tukey's post-hoc test. *p<0.05, **p<0.01, ****p<0.0001, ‡ Group effect p<0.05. Figure 5—source data 1 RBM20 expression is decreased in iMSBmal1-/- muscle compared to iMSBmal1+/+ muscle. Western blot of alternating lanes of iMSBmal1+/+ and iMSBmal1-/- muscle lysates probed with anti-RBM20 antibody (top). Western blot of alternating lanes of iMSBmal1+/+ and iMSBmal1-/- muscle lysates probed with anti-γ-tubulin antibody (bottom). https://cdn.elifesciences.org/articles/76478/elife-76478-fig5-data1-v2.zip Download elife-76478-fig5-data1-v2.zip We next used an environmental model to chronically disrupt the expression of the muscle clock to ask whether this was sufficient to change Rbm20 expression in muscle. C57BL/6J mice were subjected to 8 weeks of repeated phase advances, a well-established model of circadian disruption most commonly referred to as chronic jet lag. Prior studies using this model demonstrated significant disruption of circadian clock gene expression in skeletal muscle (Wolff et al., 2013). For this analysis, we included collections at two time points so that we could capture potential changes during either the rest (CT30) or active (CT42) parts of the day. In Figure 5D, we found that Rbm20 mRNA did exhibit a significant difference in expression in the quadriceps muscles under normal light:dark conditions. In contrast, muscles from the CPA mice exhibit depressed expression of Rbm20, mostly notably at CT30, during the subjective rest phase for the mice. The results for the TA muscle from the same mice follow a similar trend with reduced Rbm20 expression at both time points in the CPA group, but this change was not significant (p=0.09). The observation that Rbm20 mRNA expression was differentially expressed in the rest vs. active period of the quadriceps muscles was not expected but is consistent with the muscle clock regulating Rbm20 expression. In summary, these data indicate that Rbm20 expression is regulated downstream of the skeletal muscle circadian clock; this regulation is evident in two different muscles, and it provides further evidence that altering the muscle clock, either genetically or physiologically, leads to altered Rbm20 expression. Since skeletal muscle Bmal1 knockout and physiological disruption of circadian rhythms reduced Rbm20 expression, we asked whether Rbm20 is a direct transcriptional target of the core clock factors in skeletal muscle. Our first approach was to interrogate our skeletal muscle ChIP-seq data sets for BMAL1 and CLOCK as these proteins comprise the positive limb of the skeletal muscle circadian clock (Gabriel et al., 2021). We also looked into our skeletal muscle ChIP-seq data set for MYOD1 as we have previously shown with Tcap that the clock factors can function synergistically with MYOD1 to transcriptionally regulate gene expression (Hodge et al., 2019; Cao et al., 2010). As seen in Figure 5C, we detected significant binding of BMAL1, CLOCK, and MYOD1 within a common region of intron 1 of Rbm20. Analysis of this region identified a putative regulatory element as defined by the third phase of the ENCODE project (Moore et al., 2020). To confirm binding of these proteins, we performed targeted ChIP-PCR using primers flanking this 88 bp site. We identified significant binding of BMAL1 (p<0.01), CLOCK (p<0.001), and MYOD1 (p<0.001; Figure 5F). To test whether this site served as an enhancer for Rbm20 transcription in muscle cells, we generated luciferase reporter constructs with either the Rbm20 proximal promoter alone (Rbm20-Luc) or the ~400 bp enhancer region from intron 1 cloned upstream of the Rbm20 promoter (E-Rbm20-Luc). We found that overexpression of the clock factors in E-Rbm20-Luc transfected myotubes resulted in a 255-fold increase in luciferase activity compared to empty vector control transfected myotubes (n = 3, p<0.0001, Figure 5G). Importantly, this increase is 9.8-fold higher than the increase seen in myotubes transfected with Rbm20-P-Luc, which lacks the enhancer element (p<0.0001). These data have identified a novel circadian clock-sensitive enhancer in the Rbm20 gene and support a model in which the skeletal muscle molecular clock modulates Rbm20 transcription through this intronic enhancer. RBM20 overexpression in iMSBmal1-/- muscle restores titin isoform expression Since Rbm20 expression is regulated by the skeletal muscle molecular clock, we tested whether overexpression of RBM20 expression, using direct muscle injection of Rbm20-AAV, in the iMSBmal1-/- muscle would be sufficient to restore titin splicing and protein uniformity (Figure 6A). Consistent with our prior measures, RBM20 protein levels decreased by 38% in iMSBmal1-/- muscle compared to iMSBmal1+/+ muscle injected with GFP-AAV (p<0.05). Injection of RBM20-AAV into iMSBmal1-/- muscle increased Rbm20 gene expression by 2.8-fold (padj<0.05) and RBM20 protein expression almost 2-fold above iMSBmal1+/+-GFP-AAV levels (p<0.05; Figure 6B). We performed SDS-VAGE with these muscles to determine titin isoform distribution. Consistent with earlier work, loss of muscle Bmal1 resulted in a significant increase in the long form of titin (p<0.01; Figure 6C). AAV expression of RBM20 in the iMSBmal1-/- muscle was sufficient to significantly reduce the amount of the long titin isoform to levels closer to wildtype (p<0.05; Figure 6C). These findings demonstrate that the decrease in RBM20 expression downstream of loss of muscle Bmal1 contributes significantly to titin isoform heterogeneity in skeletal muscle. Figure 6 with 1 supplement see all Download asset Open asset Rescuing RBM20 expression in iMSBmal1-/- muscle restores titin spring length and sarcomere length variability. (A) Protocol for conditionally knocking out skeletal muscle Bmal1 and overexpressing RBM20. (B) Transduction of AAV-RBM20 in iMSBmal1-/- muscle increases RBM20 91% over iMSBmal1+/+ AAV-GFP muscle and threefold over iMSBmal1-/- AAV-GFP muscle (N = 4/group). Molecular weight markers (MW) correspond to 150 kDa in RBM20 and HA blots and 50 kDa in tubulin blots. (C) Titin isoform expression is partially restored in iMSBmal1-/- AAV-RBM20 muscle (N = 4/group). (D) Percent spliced in (PSI) of every exon of Ttn between iMSBmal1-/--GFP and iMSBmal1-/--RBM20 muscle (N = 3/group). (E) Liquid chromatography–mass spectrometry (LC-MS)-quantified peptide abundance mapping onto exons identified using RNAseq confirms changes in titin splicing are translated to titin protein (N=3/group). Data plotted as mean ± SEM. Statistics performed using Student's t-test or one-way ANOVA with Tukey's post-hoc test. *p<0.05; **p<0.01. Figure 6—source data 1 RBM20 and HA protein expression are increased in iMSBmal1-/-AAV-RBM20 muscle lysates compared to iMSBmal1-/- AAV-GFP and iMSBmal1+/+ AAV-GFP. Western blot of HA protein expression across groups (top-left). Western blot of γ-tubulin expression across groups (top-right). Western blot of RBM20 expression across groups (middle left). Western blot of γ-tubulin expression across groups (middle right). Rescue of RBM20 expression results in titin isoform ratio similar to isof" @default.
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• W4295736708 title "Author response: The skeletal muscle circadian clock regulates titin splicing through RBM20" @default.
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