Muscle calcium stress cleaves junctophilin1, unleashing a gene regulatory program predicted to correct glucose dysregulation
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Junctophilin has been traditionally known as a structural anchor to keep excitation-contraction proteins in place for healthy contractile function of skeletal muscle. Here the authors provide an interesting and important role in skeletal muscle for Junctophilin, where it translocates to the nuclei and influences gene transcription. The authors provide convincing evidence for a novel role of junctophilin beyond its structural role as a regulator of gene expression.
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Abstract
Calcium ion movements between cellular stores and the cytosol govern muscle contraction, the most energy-consuming function in mammals, which confers skeletal myofibers a pivotal role in glycemia regulation. Chronic myoplasmic calcium elevation (“calcium stress”), found in malignant hyperthermia-susceptible (MHS) patients and multiple myopathies, has been suggested to underlie the progression from hyperglycemia to insulin resistance. What drives such progression remains elusive. We find that muscle cells derived from MHS patients have increased content of an activated fragment of GSK3β — a specialized kinase that inhibits glycogen synthase, impairing glucose utilization and delineating a path to hyperglycemia. We also find decreased content of junctophilin1, an essential structural protein that colocalizes in the couplon with the voltage-sensing Ca V 1.1, the calcium channel RyR1 and calpain1, accompanied by an increase in a 44 kDa junctophilin1 fragment (JPh44) that moves into nuclei. We trace these changes to activated proteolysis by calpain1, secondary to increased myoplasmic calcium. We demonstrate that a JPh44-like construct induces transcriptional changes predictive of increased glucose utilization in myoblasts, including less transcription and translation of GSK3β and decreased transcription of proteins that reduce utilization of glucose. These effects reveal a stress-adaptive response, mediated by the novel regulator of transcription JPh44.
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Author Response
Reviewer #1 (Public Review):
Junctophilin is mostly known as a structural anchor to keep excitation-contraction (E-C) proteins in place for healthy contractile function of skeletal muscle. Here the authors provide a new interesting role in skeletal muscle for Junctophilin (44 kD segment, JPh44), where it translocates to the nuclei and influences gene transcription. Also, the authors have shown that Calpain 1 can digest junctophilin to generate the 44 kDa segment. The field of skeletal muscle generally knows little about how E-C coupling proteins have dual role and influence gene regulation that subsequently may alter the muscle function and metabolism. This part of the manuscript is solid, informative, and novel. The authors use advanced imaging and genetic manipulations of junctophilin etc to support their …
Author Response
Reviewer #1 (Public Review):
Junctophilin is mostly known as a structural anchor to keep excitation-contraction (E-C) proteins in place for healthy contractile function of skeletal muscle. Here the authors provide a new interesting role in skeletal muscle for Junctophilin (44 kD segment, JPh44), where it translocates to the nuclei and influences gene transcription. Also, the authors have shown that Calpain 1 can digest junctophilin to generate the 44 kDa segment. The field of skeletal muscle generally knows little about how E-C coupling proteins have dual role and influence gene regulation that subsequently may alter the muscle function and metabolism. This part of the manuscript is solid, informative, and novel. The authors use advanced imaging and genetic manipulations of junctophilin etc to support their hypothesis. The authors then also aim to link this mechanism to hyperglycemia in individuals susceptible for malignant hyperthermia as they have elevated levels of the 44kDa segment. However, the power of the analyses are low and the included data comparisons complicates the possibility to interpret the results and its relevance. Nevertheless, the data supporting the novel dual role of junctophilin would likely be appreciated and gain attention to the muscle field.
Thanks for your constructive reading. We agreed (in our answer to Item 1) to your concern regarding power of the tests. To improve it we would need many more individual patients (which, after the pandemic peaks, are starting to be recruited again, although at a pace of no more than 2 per month). We are committed to updating the present report as soon as we obtain, say, 20 more MHS and MHN patients –a minimum to impact power of the tests. In any case, we claim that power is not an acute concern, as this communication deals mainly with positive results, where significance is of the essence.
We have established significance in most of the observations communicated here; in the few cases where p is marginal, significance is inferred by correlations.
Reviewer #2 (Public Review):
Skeletal muscle is the main regulator of glycemia in mammals and a major puzzle in the field of diabetes is the mechanism by which skeletal muscle (as well as other tissues) become insensitive to insulin or decrease glucose intake. the authors had proposed in a previous publication that high intracellular calcium, by means of calpain activation, could cleave and decrease the availability of GLUT4 glucose transporters. In this manuscript, the authors identify two additional targets of calpain activation. One of them is GSK3β, a specialized kinase that when cleaved, inhibits glycogen synthase and impairs glucose utilization. The second target is junctophilin 1, a protein involved in the structure of the complex responsible for E-C coupling in skeletal muscle. The authors succeeded in showing that a fragment of junctophilin1 (JPh44) moves from the triad to other cytosolic regions including the nuclei and they show changes in gene expression under these conditions, some of them linked to glucose metabolism.
Overall, the manuscript shows a novel and audacious approach with a careful treatment of the data (that was not always easy nor obvious) that allow sensible conclusions and definitively constitutes a step forward in this field.
Thanks for the generous report.
Reviewer #3 (Public Review):
First, we express utmost gratitude for your critical work on our manuscript. Your concerns made us perform additional experiments and validations, eventually forcing us to abandon a couple of erroneous notions and therefore improving our understanding and interpretations. Because your concerns were already in the “Essentials” list assembled by the Editor, our responses here will mostly refer to our earlier answers to the items in that list.
- Figure 1 A and B show a western blot of proteins isolated from muscles of MHN and MHS individuals decorated with two different antibodies directed against JPH1. According to the manufacturer, antibody A is directed against the JPH1 protein sequence encompassing amino acids 387 to 512 while antibody B is directed against a no better specified C-terminal region of JPH1. Surprisingly, antibody B appears not to detect the full-length protein in lysates from human muscles, but recognizes only the 44 kDa fragment of JPH1. However, to the best of the reviewer's knowledge, antibody B has been reported by other laboratories to recognize the full-length JPH1 protein.
The reason for the failure of ab B to recognize the full human protein may be that it was raised against a murine immunogen (this interpretation was communicated to us by G.D. Lamb, who co-authored the 2013 paper by Murphy et al. where the failure was noted). It recognizes both JPh1 and JPh44 of murine muscle in our hands.
Thus, is not obvious why here this antibody should recognize only the shorter fragment.
We agree entirely. In spite of the difficulties in interpretation, the recognition of human JPh44 by the ab is, however, a fact, repeatedly demonstrated in the present study, which can be used to advantage.
In addition, in MHS individuals there is no direct correlation between reduction in the content of the full-length JPH1 protein and appearance of the 44 kDa JPH1fragment, since, as also reported by the authors, no significant difference between MHN and MHS can be observed concerning the amount of the 44 kDa JPH1.
Tentative interpretations of the lack of correlation have been presented in the response to Item 14, above.
Based on the data presented, it is very difficult to accept that antibody A and B have specific selectivity for JPH1 and the 44 kDa fragment of JPH1.
Indeed, we now acknowledge that Ab A reacts equally with JPh1 and the 44 kDa fragment (and provide quantitative evidence for it in Supplement 1 to Fig. 8). We also provide conclusive evidence of the specificity of ab B (e.g., Supplement 2 to Fig. 1).
- In Figure 2B staining of a nucleus is shown only with antibody B against the 44 kDa JPH1 fragment, while no nucleus stained with antibody A is shown in Fig 2A. Images should all be at the same level of magnification and nuclear staining of nuclei with antibody A should be reported. In Figure 2Db labeling of JPH1 covers both the nucleus and the cytoplasm, does it mean that JPH1 also goes to the nucleus? One would rather think that background immunofluorescence may provide a confounding staining and authors should be more cautious in interpreting these data.
These items are fully covered in our response to Item 16.
Images in 2D and 2E refer to primary myotubes derived from patients. The authors show that RyR1 signals co-localizes with full-length JPH1, but not with the 44 kDa fragment, recognized by antibody B. How do the authors establish myotube differentiation?
Myotubes are studied 5-10 days after switching cells to differentiation medium, which is DMEM-F12 supplemented with 2.5% horse serum, as explained in Figueroa et al 2019. Cells with more than 3 nuclei were considered myotubes. Myotubes with similar degree of maturation (number of nuclei) were selected for experimental comparisons.
- Figure 3 A-C. The authors show images of a full-length JPH1 tagged with GFP at the N-terminus and FLAG at the C- terminus. In Figure 3Ad and Cd the Flag signal is all over the cytoplasm and the nuclei: since these are normal mouse cells and fibers, it is surprising that the FLAG signal is in the nuclei with an intensity of signal higher than in patient's muscle.
Can the authors supply images of entire myotubes, possibly captured in different Z planes? How can they distinguish between the cleaved and uncleaved JPH1 signals, especially in mouse myofibers, where calpain is supposed not to be so active as in MHS muscle fibers?
Answer fully provided to Items 16b and 17 in Essentials list.
- If the 44 kDa JPH1 fragment contains a transmembrane domain, it is difficult to understand the dual sarcoplasmic reticulum and nuclear localization. To justify this the authors, in the Discussion session, mention a hypothetical vesicular transport of the 44 kDa JPH1 fragment by vesicles. Traffic of proteins to the nucleus usually occurs through the nuclear pores and does not require vesicles. Even if diffusion from the SR membrane to the nuclear envelope occurs, the protein should remain in the compartment of the membrane envelope. There is no established evidence to support such an unusual movement inside the cells.
In agreement with the criticism, we have removed the speculation from the Discussion.
- In Figure 5, the authors show the effect of Calpain1 on the full-length and 44 kDa JPH1 fragment in muscles from MHS patients. Can the authors repeat the same analysis on recombinant JPH1 tagged with GFP and FLAG?
We agree that confirmatory evidence of the calpain effect on dual-tagged recombinant JPh1 would be desirable. However, we think an in-depth study is required to follow up on the number of JPh1 fragments generated by calpain (or by different calpain isoforms) and their positions, similar to the detailed study of JPh2 fragmentation Wang et al. in 2021 (5).
Can the authors provide images from MHN muscle fibers stained with JPH1 and Calpain1.
We complied with the request.
- In Figure 6, the authors show images of MHS derived myotubes transfected with FLAG Calpain1 and compare the distribution of endogenous JPH1 and RYR1 in two cells, one expressing FLAG Calpain1 (cell1) and one not expressing the recombinant protein. They state that cell1 shows a strong signal of JPH1 in the nucleus, while this is not observed in cell2. Nevertheless, it is not clear where the nucleus is located within cell2 since the distribution of JPH1 is homogeneous across the cell. Can the authors show a different cell?
In agreement, we now show a comparison between cultures with and without transfection in Supplement 1 to Fig. 6.
- In Figure 7, panels Bb and Db: nuclei appear to stain positive for JPH1. It is not clear why in panels Ac, Bc they show a RYR1 staining while in panels Cc and Dc they show N-myc staining. The differential localization to nuclei appears rather poor also in these panels.
We have entirely removed from the manuscript the description of experiments of exposure to extracellular calpain, including Fig. 7 and three associated tables.
- The strong nuclear staining in Figure 8, panels C and D is very different from the staining observed in Fig. 2 and Fig. 3. Transfection should not change the ratio between nuclear and cytoplasmic distribution.
Transfection is an intrusive procedure, which requires production and trafficking of an exogenous protein. This protein, furthermore, is an artificial construct (in this case, a “stand-in”, which adds to the native protein and therefore is akin to overexpression). For the above reasons, we believe that differences in intensity of nuclear staining may obey to multiple causes and should not be especially concerning.
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eLife assessment
Junctophilin has been traditionally known as a structural anchor to keep excitation-contraction proteins in place for healthy contractile function of skeletal muscle. Here the authors provide an interesting and important role in skeletal muscle for Junctophilin, where it translocates to the nuclei and influences gene transcription. The authors provide convincing evidence for a novel role of junctophilin beyond its structural role as a regulator of gene expression.
-
Reviewer #1 (Public Review):
Junctophilin is mostly known as a structural anchor to keep excitation-contraction (E-C) proteins in place for healthy contractile function of skeletal muscle. Here the authors provide a new interesting role in skeletal muscle for Junctophilin (44 kD segment, JPh44), where it translocates to the nuclei and influences gene transcription. Also, the authors have shown that Calpain 1 can digest junctophilin to generate the 44 kDa segment. The field of skeletal muscle generally knows little about how E-C coupling proteins have dual role and influence gene regulation that subsequently may alter the muscle function and metabolism. This part of the manuscript is solid, informative, and novel. The authors use advanced imaging and genetic manipulations of junctophilin etc to support their hypothesis. The authors then …
Reviewer #1 (Public Review):
Junctophilin is mostly known as a structural anchor to keep excitation-contraction (E-C) proteins in place for healthy contractile function of skeletal muscle. Here the authors provide a new interesting role in skeletal muscle for Junctophilin (44 kD segment, JPh44), where it translocates to the nuclei and influences gene transcription. Also, the authors have shown that Calpain 1 can digest junctophilin to generate the 44 kDa segment. The field of skeletal muscle generally knows little about how E-C coupling proteins have dual role and influence gene regulation that subsequently may alter the muscle function and metabolism. This part of the manuscript is solid, informative, and novel. The authors use advanced imaging and genetic manipulations of junctophilin etc to support their hypothesis. The authors then also aim to link this mechanism to hyperglycemia in individuals susceptible for malignant hyperthermia as they have elevated levels of the 44kDa segment. However, the power of the analyses are low and the included data comparisons complicates the possibility to interpret the results and its relevance. Nevertheless, the data supporting the novel dual role of junctophilin would likely be appreciated and gain attention to the muscle field.
-
Reviewer #2 (Public Review):
Skeletal muscle is the main regulator of glycemia in mammals and a major puzzle in the field of diabetes is the mechanism by which skeletal muscle (as well as other tissues) become insensitive to insulin or decrease glucose intake. the authors had proposed in a previous publication that high intracellular calcium, by means of calpain activation, could cleave and decrease the availability of GLUT4 glucose transporters. In this manuscript, the authors identify two additional targets of calpain activation. One of them is GSK3β, a specialized kinase that when cleaved, inhibits glycogen synthase and impairs glucose utilization. The second target is junctophilin 1, a protein involved in the structure of the complex responsible for E-C coupling in skeletal muscle. The authors succeeded in showing that a fragment of …
Reviewer #2 (Public Review):
Skeletal muscle is the main regulator of glycemia in mammals and a major puzzle in the field of diabetes is the mechanism by which skeletal muscle (as well as other tissues) become insensitive to insulin or decrease glucose intake. the authors had proposed in a previous publication that high intracellular calcium, by means of calpain activation, could cleave and decrease the availability of GLUT4 glucose transporters. In this manuscript, the authors identify two additional targets of calpain activation. One of them is GSK3β, a specialized kinase that when cleaved, inhibits glycogen synthase and impairs glucose utilization. The second target is junctophilin 1, a protein involved in the structure of the complex responsible for E-C coupling in skeletal muscle. The authors succeeded in showing that a fragment of junctophilin1 (JPh44) moves from the triad to other cytosolic regions including the nuclei and they show changes in gene expression under these conditions, some of them linked to glucose metabolism.
Overall, the manuscript shows a novel and audacious approach with a careful treatment of the data (that was not always easy nor obvious) that allow sensible conclusions and definitively constitutes a step forward in this field.
-
Reviewer #3 (Public Review):
The manuscript reports two separate lines of evidence whereby in individuals with Malignant Hyperthermia susceptibility, the increased cytosolic calcium levels caused by leaky RYR1 mutant channels boost Calpain1 activity resulting in the activation of two different pathways, where one results into impaired glucose metabolism, while the other is expected to stimulate glucose utilization by skeletal muscles.
In the first set of data, the authors report evidence that muscles fibers of MHS patients contain increased levels of the 40kDa activated form of GSK3ß, which is generated by Calpain1-mediated cleavage of 47kDa full length GSK3ß protein. The activation of GSKß activity is associated to impaired glucose utilization by skeletal muscle and fits well with previous data on alterations of glucose storage in MHS …
Reviewer #3 (Public Review):
The manuscript reports two separate lines of evidence whereby in individuals with Malignant Hyperthermia susceptibility, the increased cytosolic calcium levels caused by leaky RYR1 mutant channels boost Calpain1 activity resulting in the activation of two different pathways, where one results into impaired glucose metabolism, while the other is expected to stimulate glucose utilization by skeletal muscles.
In the first set of data, the authors report evidence that muscles fibers of MHS patients contain increased levels of the 40kDa activated form of GSK3ß, which is generated by Calpain1-mediated cleavage of 47kDa full length GSK3ß protein. The activation of GSKß activity is associated to impaired glucose utilization by skeletal muscle and fits well with previous data on alterations of glucose storage in MHS patients reported by the same authors in a previous paper (Tamminemi et al., 2020).
In the second set of data, the authors report evidence indicating that skeletal muscles from individuals with MHS present reduced levels of JPH1 in the presence of a 44 kDa fragment of JPH1 (JPH44) that corresponds to the C-terminal region of JPH1, a cleavage again generated by the calcium-induced activation of Calpain1 proteolytic activity. They then go on to present data indicating that the JPH44 fragment, although expected to contain the transmembrane segment of JPH1, migrates to the nucleus where it activates the transcription of genes correlated with increased glucose metabolism, an activity that would oppose the effect of GSK3ß activation. These data on JPH44 show some analogy with the reported calcium-induced cleavage of JPH2 in cardiomyocytes, where a fragment of JPH2 translocate to the nucleus, where it activates a protective program to counteract cardiac stress conditions (Guo et al., 2018).
Figure 1 A and B show a western blot of proteins isolated from muscles of MHN and MHS individuals decorated with two different antibodies directed against JPH1. According to the manufacturer, antibody A is directed against the JPH1 protein sequence encompassing amino acids 387 to 512 while antibody B is directed against a no better specified C-terminal region of JPH1. Surprisingly, antibody B appears not to detect the full-length protein in lysates from human muscles, but recognizes only the 44 kDa fragment of JPH1. However, to the best of the reviewer's knowledge, antibody B has been reported by other laboratories to recognize the full-length JPH1 protein.
Thus, is not obvious why here this antibody should recognize only the shorter fragment. In addition, in MHS individuals there is no direct correlation between reduction in the content of the full-length JPH1 protein and appearance of the 44 kDa JPH1fragment, since, as also reported by the authors, no significant difference between MHN and MHS can be observed concerning the amount of the 44 kDa JPH1.
Based on the data presented, it is very difficult to accept that antibody A and B have specific selectivity for JPH1 and the 44 kDa fragment of JPH1.In Figure 2B staining of a nucleus is shown only with antibody B against the 44 kDa JPH1 fragment, while no nucleus stained with antibody A is shown in Fig 2A. Images should all be at the same level of magnification and nuclear staining of nuclei with antibody A should be reported.
In Figure 2Db labeling of JPH1 covers both the nucleus and the cytoplasm, does it mean that JPH1 also goes to the nucleus? One would rather think that background immunofluorescence may provide a confounding staining and authors should be more cautious in interpreting these data.
Images in 2D and 2E refer to primary myotubes derived from patients. The authors show that RyR1 signals co-localizes with full-length JPH1, but not with the 44 kDa fragment, recognized by antibody B. How do the authors establish myotube differentiation?
3. Figure 3 A-C. The authors show images of a full-length JPH1 tagged with GFP at the N-terminus and FLAG at the C-terminus. In Figure 3Ad and Cd the Flag signal is all over the cytoplasm and the nuclei: since these are normal mouse cells and fibers, it is surprising that the FLAG signal is in the nuclei with an intensity of signal higher than in patient's muscle.
Can the authors supply images of entire myotubes, possibly captured in different Z planes? How can they distinguish between the cleaved and uncleaved JPH1 signals, especially in mouse myofibers, where calpain is supposed not to be so active as in MHS muscle fibers?4. If the 44 kDa JPH1 fragment contains a transmembrane domain, it is difficult to understand the dual sarcoplasmic reticulum and nuclear localization. To justify this the authors, in the Discussion session, mention a hypothetical vesicular transport of the 44 kDa JPH1 fragment by vesicles. Traffic of proteins to the nucleus usually occurs through the nuclear pores and does not require vesicles. Even if diffusion from the SR membrane to the nuclear envelope occurs, the protein should remain in the compartment of the membrane envelope. There is no established evidence to support such an unusual movement inside the cells.
5. In Figure 5, the authors show the effect of Calpain1 on the full-length and 44 kDa JPH1 fragment in muscles from MHS patients. Can the authors repeat the same analysis on recombinant JPH1 tagged with GFP and FLAG? Can the authors provide images from MHN muscle fibers stained with JPH1 and Calpain1.
6. In Figure 6, the authors show images of MHS derived myotubes transfected with FLAG Calpain1 and compare the distribution of endogenous JPH1 and RYR1 in two cells, one expressing FLAG Calpain1 (cell1) and one not expressing the recombinant protein. They state that cell1 shows a strong signal of JPH1 in the nucleus, while this is not observed in cell2. Nevertheless, it is not clear where the nucleus is located within cell2 since the distribution of JPH1 is homogeneous across the cell. Can the authors show a different cell?
7. In Figure 7, panels Bb and Db: nuclei appear to stain positive for JPH1. It is not clear why in panels Ac, Bc they show a RYR1 staining while in panels Cc and Dc they show N-myc staining. The differential localization to nuclei appears rather poor also in these panels.
8. The strong nuclear staining in Figure 8, panels C and D is very different from the staining observed in Fig. 2 and Fig. 3. Transfection should not change the ratio between nuclear and cytoplasmic distribution.
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