CMYA5 establishes cardiac dyad architecture and positioning

Cardiac excitation-contraction coupling requires dyads, the nanoscopic microdomains formed adjacent to Z-lines by apposition of transverse tubules and junctional sarcoplasmic reticulum. Disruption of dyad architecture and function are common features of diseased cardiomyocytes. However, little is known about the mechanisms that modulate dyad organization during cardiac development, homeostasis, and disease. Here, we use proximity proteomics in intact, living hearts to identify proteins enriched near dyads. Among these proteins is CMYA5, an under-studied striated muscle protein that co-localizes with Z-lines, junctional sarcoplasmic reticulum proteins, and transverse tubules in mature cardiomyocytes. During cardiac development, CMYA5 positioning adjacent to Z-lines precedes junctional sarcoplasmic reticulum positioning or transverse tubule formation. CMYA5 ablation disrupts dyad architecture, dyad positioning at Z-lines, and junctional sarcoplasmic reticulum Ca2+ release, leading to cardiac dysfunction and inability to tolerate pressure overload. These data provide mechanistic insights into cardiomyopathy pathogenesis by demonstrating that CMYA5 anchors junctional sarcoplasmic reticulum to Z-lines, establishes dyad architecture, and regulates dyad Ca2+ release.

I think the MS is interesting and although the English is awkward in places it is easy to read and suitable for increasing knowledge in general cardiac physiology. Perhaps a bit more modesty for the apparently strong claims of the role of the protein might make the MS more accurate without detracting from the central message?
Considering the title, the introduction tells the reader very little about the background knowledge of what CMYA5 is and what it is known to do. Quite a bit of work has already been done of this protein including demonstration of effects on jSR structure and cardiac function, so it is disingenuous to say (l84) "little studied" in the context of the present paper. The demonstration of cardiac mitochondrial defects is also notable. (Perhaps even more intriguing are the demonstrations of both schizophrena association and brain defects related to this gene product -doesn't this imply a function beyond tt-SR assembly?). I would also point out that ASPH has also been designated as TRIM76.
The gene symbol CMYA5, arose from involvement of myospryn in cardiomyopathy and was originally hypothetical but a link to cardiomyopathy was suggested by co-expression of myospryn with known cardiomyopathy genes (Walker 2001). This hypothesis gained empirical support from the association of a myospryn cSNP haplotype with left ventricular wall thickness and diastolic dysfunction in patients (Nakagami et al. 2007). Such background should be included.
This protein has been implicated as an anchoring protein that may serve to localize A-kinase (among other possible binding functions) so this raises the question as to whether the (moderate) changes seen in the KO model are related to this function. However, there are no controls for this possible effect/role. In this context, if beta signalling or calcineurin is affected is compromised, is it really any surprise that the failing hard is impacted and how does this support the central hypothesis?
The authors state "We found that CMYA5 is required to position jSR adjacent to Z-lines, an early and essential step in dyad assembly" which would imply that in the KO no dyads should form but in fact they do (see Fig. 5D).
The proteomic approach is powerful, but it is quite unclear why the authors focus on CMY5 when many other protein that affect Ca signalling are associated with the gene product. I am struck by the fact that the highest ranked protein was phospolambam… This raises serious concerns as to causation in these experiments as many higher ranked protein are known to affect many aspect of EC coupling. 398: Who the authors say "GCaMP6f-junctin16and srCES29 were kind gifts from Dr. Heping Cheng, Peking University" when Cheng is a co-author? 388: "Aortic banding was performed on male mice between 25 and 30 g, using a modification of our previously described protocol35" The citation does not describe their protocol but is a general review of techniques by other authors .   Fig 1c. "Expression of BirA* dyadic biosensors in myocardium. Heart sections were stained for the myc epitope tag. The majority of cardiomyocytes were immunoreactive". Without quantification this is an overstatement of the result which show ~50% stained. Fig 1E: Where are the MW markers? It is clear that the biotinylation hits many proteins and there are clear differences between triadin and junctin. Why is this ignored? I would like to have seen an overlay of the ASPH expression or at least have the putative band corresponding to the mw of ASPH indicated.
Reviewer #2 (Remarks to the Author): These authors use an AAV-based targeted proteome biosensor to identify by mass spectrometry new proteins associated to dyads in the P1 mouse heart. CMYA5 was a protein highly enriched in virus-transduced cardiomyocytes. CMYA5 co-localizes with RYR2 in cardiomyocytes. Using PLA, the authors determine that RYR2 and CMYA5 are in close proximity, showing a striated pattern throughout cardiomyocytes. These data suggest that CMYA5 is a dyadic protein associated with RYR2. Generation of null mice by Crispr-Cas9 gene edition reveals that mutants show reduced systolic function and ventricular dilatation, indicating that CMYA5 is essential for heart function.
Dyads are critical for E-C coupling and examination of T tubules system shows that they are disrupted. T tubule disruption is a cell autonomous phenotype occurring in the absence of cardiac dysfunction, as AAV-mediated mosaic ablation of CMYA5 in a low number of cardiomyocytes indicates. TEM reveals changes in T tubules shape and dyad architecture. The authors then study the interrelationships between T-tubules, jSR, and Z-lines in wild type and Cmya5KO adult atria and P7 and foetal ventricular cardiomyocytes. Their results show that dyads are built on scaffolding provided by sarcomeres so that CMYA5 localizes to Z-lines, and subsequently tethers jSR adjacent to these structures. T-tubules subsequently form and co-localize with jSR, yielding organized, properly positioned dyads. Then, they assay CMYA5MD9 (C-terminal region) and FSD2, using AAV vectors, for their ability to rescue Cmya5KO hearts, without success. The authors claim that the N-terminal region of CMYA5 (aa 1-2730) is essential for its localization at Z-lines.
Visualization of Ca2+ dynamics in contracting wild type and Cmya5KO cardiomyocytes indicated that CMYA5 is required to coordinate E-C coupling and regulate RYR2 activity. Finally, these authors use a pressure overload (TAC) model to establish that CMYA5 stabilizes dyad structure and function to biomechanical stress. Overall, the paper is very complete and has very carefully performed and elegant experiments.
One suggestion to complement the negative results with CMYA5MD9 C-terminal region is to test if, as predicted, the N-terminal region of CMYA5 (aa 1-2730) is essential for its localization at Z-lines.
Reviewer #3 (Remarks to the Author): Review on Lu et al. "CMYA5 establishes cardiac dyad architecture and positioning" The authors applied an innovative proximity proteomics approach in intact, living hearts to identify proteins enriched near dyads. Proteins being biotinylated in close proximity to the known transmembrane jSR proteins junctin and triadin 1 were enriched by streptavidin and identified by mass spectrometry in comparison to a control setup. Results are visualized in Figure 1C and compiled in Supplemental data 1. Data were uploaded to PRIDE database and thus will be publicly available.
The analysis looks sound. However, the authors do not comment at all the proteins identified beside CMYA5. Phospholamban (rank 1) or Striated muscle-specific serine/threonine-protein kinase (rank 2) seem to be much more interesting candidates, because CMYA5 was found only at rank 448. If the authors have used proximity proteomics only to confirm that the already -based on literature dataselected candidate CMYA5 is part of the dyad, the results part should be rewritten in this way. In the current version of the first section of the results, the selection of the candidate on which the experiments of the whole manuscript concentrates, seems to be artificial. At least the authors should explain, why no other candidates have been taken into consideration, but that other proteins might be important in maintenance of the dyad architecture and cell integrity.
No information is provided on protein sample preparation and the number of replicates being analysed.
No information at all is provided on the LC-MS/MS method used and therefore sensitivity of the analysis cannot be evaluated. l. 458 "oC" is missing l. 546 "mass spectrometry" should be used instead of "mass spectroscopy"

Point-to-point Responses Reviewer #1 (Remarks to the Author):
I think the MS is interesting and although the English is awkward in places it is easy to read and suitable for increasing knowledge in general cardiac physiology. Perhaps a bit more modesty for the apparently strong claims of the role of the protein might make the MS more accurate without detracting from the central message?
A: We thank you for the overall positive comments. In the revised manuscript, we moderated or conditioned several of the statements as recommended by the reviewer.
Considering the title, the introduction tells the reader very little about the background knowledge of what CMYA5 is and what it is known to do. Quite a bit of work has already been done of this protein including demonstration of effects on jSR structure and cardiac function, so it is disingenuous to say (l84) "little studied" in the context of the present paper.
We added a paragraph to the introduction to briefly review what is known about this protein. The protein has been the subject of several studies and multiple interacting proteins have been reported. However, its function in muscle cells, particularly cardiomyocytes, has not been well studied, for example using genetic knockout. While we were preparing the initial submission, the first in vivo functional study was reported (Tsoupri et al, 2021). This study characterized the CMYA5 knockout mouse and reported cardiac dysfunction and abnormal ultrastructural features. One EM image showed abnormal T-tubules and jSR. However, this manuscript did not address the effect on dyad development or function, nor did it assess the dramatic effect of pressure overload on the cardiac phenotype. We included the paragraph below in the revised introduction: CMYA5 (cardiomyopathy-associated protein5 ), also known as myospryn, is an under-studied ~450 kDa member of the tripartite motif-containing super-family (TRIM) that is selectively expressed in cardiac and skeletal muscle.11,12 TRIM proteins contain four protein-protein binding domains (RING, BBox1, BBox2, and coiled-coiled) in a conserved order and generally function as part of large protein complexes.13 Based on co-expression of CMYA5 with known cardiomyopathy genes, it was initially hypothetically linked to cardiomyopathy.14 This link gained empirical support from the association of a CMYA5 coding single nucleotide polymorphism with left ventricular wall thickness and diastolic dysfunction.15 CMYA5 was previously reported to interact with multiple muscle proteins, including RYR2,12 the Z-line protein ACTN2,11 desmin,16 titin,17 and protein kinase A (PKA).18 However, little has been reported about the in vivo function of CMYA5. A recent study published while this manuscript was in preparation demonstrated that CMYA5 knockout causes cardiac dysfunction and mis-localization of RYR2.19 However the effect of CMYA5 knockout on dyad formation, structure, and function was not investigated in detail.
The demonstration of cardiac mitochondrial defects is also notable. (Perhaps even more intriguing are the demonstrations of both schizophrena association and brain defects related to this gene product -doesn't this imply a function beyond tt-SR assembly?). I would also point out that ASPH has also been designated as TRIM76.
We checked mitochondrial alignment by in situ heart imaging with Mito-tracker Red but did not find any difference between WT and KO hearts. Further we examined the morphology of mitochondria by EM and found the mitochondria were normal. We are uncertain why our results diverge from those of Tsoupri et al. with respect to cardiac mitochondria. These data are included in the revised manuscript, Supplementary Fig. 5.
The gene symbol CMYA5, arose from involvement of myospryn in cardiomyopathy and was originally hypothetical but a link to cardiomyopathy was suggested by co-expression of myospryn with known cardiomyopathy genes (Walker 2001). This hypothesis gained empirical support from the association of a myospryn cSNP haplotype with left ventricular wall thickness and diastolic dysfunction in patients (Nakagami et al. 2007). Such background should be included.
We revised the introduction to include these points. Please see paragraph quoted from Introduction above.
This protein has been implicated as an anchoring protein that may serve to localize A-kinase (among other possible binding functions) so this raises the question as to whether the (moderate) changes seen in the KO model are related to this function. However, there are no controls for this possible effect/role. In this context, if beta signalling or calcineurin is affected is compromised, is it really any surprise that the failing hard is impacted and how does this support the central hypothesis?
We checked PKA localization did not find any mislocation in isolated Cmya5 KO cardiomyocytes. We also looked at potential activation of NFAT3 by calcineurin, by measuring the extent of its nuclear localization. We did not detect a significant difference in NFAT3 nuclear localization in CMYA5 KO heart. These data are in revised Supplementary Fig.  5.

What about interactions with titin (Hackman et al. 2002)?
CMYA5 was previously reported to interact with the C-terminus of Titin, which is located at the M-line of sarcomeres. However, in cardiomyocytes we found that CMYA5 is localized near the Z-line, not the M-line. Furthermore, we validated that titin localization was normal in Cmya5 KO by immunostaining in isolated CMs. These data are included in revised Supplementary Fig. 5.
The authors state "We found that CMYA5 is required to position jSR adjacent to Z-lines, an early and essential step in dyad assembly" which would imply that in the KO no dyads should form but in fact they do (see Fig. 5D).
Although dyads are present, they are highly distorted and much of the jSR loses its positioning adjacent to Z-lines. We stated this more precisely in the revised text: "We found that CMYA5 is required to efficiently position jSR adjacent to Z-lines…". In other sections we state that CMYA5 regulates normal dyad assembly.
The proteomic approach is powerful, but it is quite unclear why the authors focus on CMY5 when many other protein that affect Ca signalling are associated with the gene product. I am struck by the fact that the highest ranked protein was phospholambam… This raises serious concerns as to causation in these experiments as many higher ranked protein are known to affect many aspect of EC coupling.
We prioritized mass spect hits that had no detected signal in controls. This was not well reflected in the original figure, and we revised it accordingly (revised Fig. 1f). We further prioritized hits that were consistent between Junctin and Triadin and for which relatively less was known about function in EC coupling.

Revised text:
Biotinylated proteins were purified using immobilized streptavidin and analyzed by mass spectrometry (Fig. 1f). We overlapped the genes present in both BirA*-ASPH and BirA*-TRDN groups and excluded those with similar signals in the AAV9-GFP control group. Among the proteins highly enriched in the BioID groups were RYR2, JPH2, ASPH, and TRDN ( Fig. 1f; Suppl. Data 1). Recovery of these known dyadic proteins validated our experimental strategy.
We prioritized proteins found in both BioID groups that lacked signal in the control group. Among these proteins, one of the most highly enriched in the BioID groups was CMYA5, a protein expressed in striated muscle and neurons. 23,24 398: Who the authors say "GCaMP6f-junctin16and srCES29 were kind gifts from Dr. Heping Cheng, Peking University" when Cheng is a co-author?
We deleted this statement. 388: "Aortic banding was performed on male mice between 25 and 30 g, using a modification of our previously described protocol35" The citation does not describe their protocol but is a general review of techniques by other authors.
We corrected this reference .   Fig 1c. "Expression of BirA* dyadic biosensors in myocardium. Heart sections were stained for the myc epitope tag. The majority of cardiomyocytes were immunoreactive". Without quantification this is an overstatement of the result which show ~50% stained.
We quantified the staining and now include more representative images (Fig. 1c). Fig 1E: Where are the MW markers? It is clear that the biotinylation hits many proteins and there are clear differences between triadin and junctin. Why is this ignored? I would like to have seen an overlay of the ASPH expression or at least have the putative band corresponding to the mw of ASPH indicated.
In Fig. 1E, we now include molecular weight markers and label the bands that have the appropriate molecular weight (Fig. 1e). Triadin and Junctin are distinct proteins and would be expected to have different but overlapping interactomes. Experimental factors (incomplete sensitivity, false positives) can also lead to imperfect overlap between mass spect results for each bait. We prioritized interacting proteins that were consistent between Triadin-and Junctin baits.

Reviewer #2 (Remarks to the Author):
These authors use an AAV-based targeted proteome biosensor to identify by mass spectrometry new proteins associated to dyads in the P1 mouse heart. CMYA5 was a protein highly enriched in virus-transduced cardiomyocytes. CMYA5 co-localizes with RYR2 in cardiomyocytes. Using PLA, the authors determine that RYR2 and CMYA5 are in close proximity, showing a striated pattern throughout cardiomyocytes. These data suggest that CMYA5 is a dyadic protein associated with RYR2. Generation of null mice by Crispr-Cas9 gene edition reveals that mutants show reduced systolic function and ventricular dilatation, indicating that CMYA5 is essential for heart function.
Dyads are critical for E-C coupling and examination of T tubules system shows that they are disrupted. T tubule disruption is a cell autonomous phenotype occurring in the absence of cardiac dysfunction, as AAV-mediated mosaic ablation of CMYA5 in a low number of cardiomyocytes indicates. TEM reveals changes in T tubules shape and dyad architecture. The authors then study the interrelationships between T-tubules, jSR, and Z-lines in wild type and Cmya5KO adult atria and P7 and foetal ventricular cardiomyocytes. Their results show that dyads are built on scaffolding provided by sarcomeres so that CMYA5 localizes to Z-lines, and subsequently tethers jSR adjacent to these structures. T-tubules subsequently form and co-localize with jSR, yielding organized, properly positioned dyads. Then, they assay CMYA5MD9 (C-terminal region) and FSD2, using AAV vectors, for their ability to rescue Cmya5KO hearts, without success. The authors claim that the N-terminal region of CMYA5 (aa 1-2730) isessential for its localization at Z-lines.
Visualization of Ca2+ dynamics in contracting wild type and Cmya5KO cardiomyocytes indicated that CMYA5 is required to coordinate E-C coupling and regulate RYR2 activity. Finally, these authors use a pressure overload (TAC) model to establish that CMYA5 stabilizes dyad structure and function to biomechanical stress. Overall, the paper is very complete and has very carefully performed and elegant experiments.
We thank the reviewer for the positive and encouraging comments.
One suggestion to complement the negative results with CMYA5MD9 C-terminal region is to test if, as predicted, the N-terminal region of CMYA5 (aa 1-2730) is essential for its localization at Z-lines.
Since AAV has a limited capacity, we were not able to test the entire 2730 amino acids that are N-terminal to CMYA5-MD9. Most of the N-terminal 2500 amino acids are predicted to be unstructured and are poorly conserved between species. However, the very N-terminal portion of CMYA5 (~aa 70-320) is conserved. However, we tested the N-terminal regions [aa1-450] and [aa1-1200], both encompassing the conserved region, for localization at Z-lines by using AAV to express them fused to HA epitope tag. As shown in revised Suppl. Fig. 6c, we found that both proteins overlapped well with Z line marker ACTN2, suggesting that the Nterminus contributes to CMYA5 localization to Z lines.

Reviewer #3 (Remarks to the Author):
Review on Lu et al. "CMYA5 establishes cardiac dyad architecture and positioning" The authors applied an innovative proximity proteomics approach in intact, living hearts to identify proteins enriched near dyads. Proteins being biotinylated in close proximity to the known transmembrane jSR proteins junctin and triadin 1 were enriched by streptavidin and identified by mass spectrometry in comparison to a control setup. Results are visualized in Figure 1C and compiled in Supplemental data 1. Data were uploaded to PRIDE database and thus will be publicly available.
The analysis looks sound. However, the authors do not comment at all on the proteins identified beside CMYA5. Phospholamban (rank 1) or Striated muscle-specific serine/threonine-protein kinase (rank 2) seem to be much more interesting candidates, because CMYA5 was found only at rank 448. If the authors have used proximity proteomics only to confirm that the already -based on literature data-selected candidate CMYA5 is part of the dyad, the results part should be rewritten in this way. In the current version of the first section of the results, the selection of the candidate on which the experiments of the whole manuscript concentrates, seems to be artificial. At least the authors should explain, why no other candidates have been taken into consideration, but that other proteins might be important in maintenance of the dyad architecture and cell integrity.
Our proximity proteomic study was designed to identify proteins localized at or near dyads. We prioritized proteins with no signal in control samples, that were identified by both baits, and that were relatively understudied in excitation-contraction coupling. The original figure summarizing the proteomics data did not explain the important place that lack of signal in control played in prioritization of candidates. This is better illustrated in the revised figure, in which candidates are ranked by the ratio of bait signal to control signal. Using this metric, CMYA5 ranks 16 th .
No information is provided on protein sample preparation and the number of replicates being analysed. No information at all is provided on the LC-MS/MS method used and therefore sensitivity of the analysis cannot be evaluated.