Visualizing sarcomere and cellular dynamics in skeletal muscle to improve cell therapies
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eLife Assessment
This study offers valuable information on how titin derived from different nuclei within the syncytium is organized and integrated during skeletal muscle development and remodeling. The authors developed a novel mCherry titin knock-in mice with the fluorophore mCherry inserted into titin's Z-disk region to track the titin during cell fusion. The approach using mcherry adds to understanding of the role and localization of titin in controlling stiffness of striated muscles and fine tuning contraction. The results demonstrate that the integration of titin into the sarcomere is tightly regulated, with its unexpected mobility aiding in the uniform distribution of titin post-cell fusion. Although the experimental approach is convincing, the work is very qualitative in its approaches, and the data needs rigorous statistical analysis. There is a need for some clarification concerning numbers of animals and control groups. Future studies will need more rigorous data analysis and interpretation.
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Abstract
The giant striated muscle protein titin integrates into the developing sarcomere to form a stable myofilament system that is extended as myocytes fuse. The logistics underlying myofilament assembly and disassembly have started to emerge with the possibility to follow labeled sarcomere components. Here, we generated the mCherry knock-in at titin’s Z-disk to study skeletal muscle development and remodeling. We find titin’s integration into the sarcomere tightly regulated and its unexpected mobility facilitating a homogeneous distribution of titin after cell fusion – an integral part of syncytium formation and maturation of skeletal muscle. In adult mCherry-titin mice, treatment of muscle injury by implantation of titin-eGFP myoblasts reveals how myocytes integrate, fuse, and contribute to the continuous myofilament system across cell boundaries. Unlike in immature primary cells, titin proteins are retained at the proximal nucleus and do not diffuse across the whole syncytium with implications for future cell-based therapies of skeletal muscle disease.
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Author response:
Reviewer #1 (Public Review):
In this work, the authors aimed to understand how titins derived from different nuclei within the syncytium are organized and integrated after cell fusion during skeletal muscle development and remodeling. The authors developed mCherry titin knock-in mice with the fluorophore mCherry inserted into titin's Z-disk region to track the titin during cell fusion. The results suggested that titin exhibited homogenous distribution after cell fusion. The authors also probed on how titin behaves during muscle injury by implantation of titin-eGFP myoblasts into adult mCherry-titin mice. Interestingly, titin is retained at the proximal nucleus and does not diffuse across the whole syncytium in this system. The findings of the study are novel and interesting. The experimental approaches are …
Author response:
Reviewer #1 (Public Review):
In this work, the authors aimed to understand how titins derived from different nuclei within the syncytium are organized and integrated after cell fusion during skeletal muscle development and remodeling. The authors developed mCherry titin knock-in mice with the fluorophore mCherry inserted into titin's Z-disk region to track the titin during cell fusion. The results suggested that titin exhibited homogenous distribution after cell fusion. The authors also probed on how titin behaves during muscle injury by implantation of titin-eGFP myoblasts into adult mCherry-titin mice. Interestingly, titin is retained at the proximal nucleus and does not diffuse across the whole syncytium in this system. The findings of the study are novel and interesting. The experimental approaches are appropriate. The results are described well. However, the manuscript needs revisions to enhance its clarity.
(1) In this work, the authors have not described the statistical analysis appropriately. In most of the figures, significance levels are not described. The information on the biological and technical replicates is missing in almost all the figures. This information is critical for understanding the strength of the experimentation.
Thank you for this feedback, added the missing information to the figure legends.
(2) The in vivo experiments are underpowered. The authors have used only 3 animals in the cardiotoxin injury experiment and eliminated another 3 animals from the analysis. How did they determine insufficient myoblast integration?
The experimental design was targeted at using transplantation of myoblasts into skeletal muscle to obtain information on the ability of transplanted cells to fuse with cells in the injured area – and if those myoblasts could provide titin protein beyond the confinement of the transplanted cells (as would be expected after cell fusion). The goal was not to optimize cell transplantation with improved force generation of lesioned muscle. For this, we agree, the experiments would be underpowered.
Here, we use a different approach, and successfully demonstrate the integration of titin protein from transplanted cells into sarcomeres of host muscle fibers. Here, only an animal number of 5 per group was approved by the local authorities, in agreement with the scope of our proposed hypothesis on cell fusion contributing titin beyond the transplanted cell and in agreement with the 3R guidelines and the necessity to addressed our research question in as few animals as possible. We proposed the need for at least 3 animals per implantation group and included 2 additional animals for compensation in case there was insufficient myoblast integration (no detection of GFP+ cells). The resulting n=3 and n=4 animals provided enough fusion events to show that even after 3 weeks, titin protein is confined to the address our hypothesis: in case after cell fusion titin is homogenously distributed, we would have expected red and greed striation throughout the fiber. This was not the case. In 8 out of 8 fused cells we had a segregation of green and red titin molecules as depicted in figure 6 and S5.
(3) Similarly, the in vitro imaging experiments, especially the in vitro titin mobility assays used only 3 cells (Fig 2b) or 6-9 cells (Fig 2c-2e). The number of cells imaged is insufficient to derive a valid conclusion. What is the variability in the results between cells? Whether all the cells behave similarly in titin mobility assays?
For Figure 2 we had described our replicates insufficiently. Quantification in 2b-e consists of total 9 cells out of 3 independent experiments (3 per experiment). For 2d one outlier (Grubbs test) was excluded for the GFP signal. For 2e we only included cells that could be fitted with a two-phase association curve. That resulted in 6 cells for the GFP signal and 7 cells for the mCherry signal.
(4) Figure 1c-e, Figure 2a, Figure 3, Figure 4, Figure 5, Figure 6- please describe the replicates and also if possible, quantify the data and present them as separate figures.
Figure 1d (former 1c) is the validation that titin is properly integrated into the sarcomere and that the cherry signal localizes to the Z-disk, overlapping with actinin. This is qualitative, not quantitative information and replicated and confirmed in figure 2. 1e (former 1d) is a representative image for the quantification in 1f (former 1e) with 3 biological replicates (=cells) and 3 technical replicates (=Z-disks) each, for every time point significantly different with p<0.001, tested by 2-way ANOVA
2a: representative image (+regarding profile) for quantification in 2b (9 biological replicates(=cells) measured at 3 different experiment days) (see answer to 1-3)
Representative images: Cells were seeded on several cover slips and fusion was started. This was done on 4 occasions (=technical replicates) with different stainings (see supplement) and 30+ images were taken in total with at least 5 images per staining. The taken images of different fusion stadiums were later classified based on the distribution of the differentially labeled titin.
a-c: representative image that shows two independent fusion events; fusion experiments were performed at 4 days with a total of 13 fusion events captured (6 only immature cells, 7 with one mature cell). For quantification in d+e, very small (< 1000 μm2) and very large (> 10,000 μm2) syncytia were excluded to minimize the effect of large size differences of the syncytia, so that 5 immature and 4 mature fusion events remained for comparative analysis.
smFISH Experiment was repeated on 2 days and 6 images of fusion events were made. Since they were in different stages of fusion and 4 elements contributed to the images (mCherry-RNA, GFP-RNA, mCh-Titin protein, GFP-Titin protein), it was difficult to compare. However, we added the quantification to Fig. S4 (b and c) and added a regarding paragraph to the results. There seems to be a smaller overlap region for the RNA than for the protein signal.
Representative images with n=6 (but 3 excluded due to insufficient myoblast integration) biological replicates (mice) for the CTX+cells group (main experiment group) and n=4 for the only cells control and n=1 for the only CTX control, based on 3R regulation of animal experiments. From each mouse (n=11) the contralateral TA muscle was harvested as well to serve as an uninjured and without cell transplantation control.
(5) Figure 2- the authors excluded samples with an obvious decrease in cell quality during imaging from the analysis. How do the authors assess the cell quality? Simply by visual examination? Or were the samples that did not show fluorescence recovery eliminated? I am wondering what percentage of cells showed poor cell quality. How do they avoid the bias? I recommend that the authors include these cells also for the analysis of data presented in Figures 2b, 2c, and 2f.
Cells were not excluded for their recovery status, but only if they showed signs of cell death (collapse of sarcomere structures, membrane bubbling, etc). All cells that stayed alive during the imaging showed a fluorescence recovery. Cells that had only a slower or uncomplete recovery were not excluded from the complete analysis. One cell was excluded from the comparison of exchange half-life (Fig. 2d), since it was a significant outlier. For Figure 2e (Fast phase) only cells could be included, where we were able to fit a two-phase association curve.
(6) It is unclear how the authors identified the different stages of cell fusion in the microscopy images i.e. early fusion, distribution, and complete distribution.
Early fusion was characterized when two cells made connection with their membranes, but differentially labeled titin has not yet mixed. Distribution was characterized when titin mixing has started but is not yet complete.
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Reviewer #3 (Public Review):
Hüttemeister et. al. describe a study where researchers utilized a genetic modification technique to knockin a red fluorescence protein variant mCherry into titin, a giant muscle protein, at the Z-disk in order to investigate skeletal muscle development and remodeling. The study revealed that titin's integration into the sarcomere is tightly regulated during muscle development, and its mobility allows for a homogeneous distribution of titin after cell fusion, which is crucial for syncytium formation and skeletal muscle maturation. Furthermore, in adult mice with mCherry-tagged titin, the researchers observed the process of muscle injury treatment by implanting myoblasts containing titin tagged with another fluorescent protein, eGFP. This experiment provided insights into how myocytes integrate, fuse, and …
Reviewer #3 (Public Review):
Hüttemeister et. al. describe a study where researchers utilized a genetic modification technique to knockin a red fluorescence protein variant mCherry into titin, a giant muscle protein, at the Z-disk in order to investigate skeletal muscle development and remodeling. The study revealed that titin's integration into the sarcomere is tightly regulated during muscle development, and its mobility allows for a homogeneous distribution of titin after cell fusion, which is crucial for syncytium formation and skeletal muscle maturation. Furthermore, in adult mice with mCherry-tagged titin, the researchers observed the process of muscle injury treatment by implanting myoblasts containing titin tagged with another fluorescent protein, eGFP. This experiment provided insights into how myocytes integrate, fuse, and contribute to the continuous myofilament system across cell boundaries during muscle regeneration. Interestingly, the behavior of titin proteins differed between immature primary cells and adult muscle tissue. The manuscripts point our interesting observation that develop treatment protocols that target the early postnatal patient or consider in utero cell therapy approaches based on controlling the ratio of therapeutic to diseased cells. though the approach is very interesting, the paper is very qualitative in its approaches. Community will benefit from better quantification of data as most of them are microscopic data that requires quantification.
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eLife Assessment
This study offers valuable information on how titin derived from different nuclei within the syncytium is organized and integrated during skeletal muscle development and remodeling. The authors developed a novel mCherry titin knock-in mice with the fluorophore mCherry inserted into titin's Z-disk region to track the titin during cell fusion. The approach using mcherry adds to understanding of the role and localization of titin in controlling stiffness of striated muscles and fine tuning contraction. The results demonstrate that the integration of titin into the sarcomere is tightly regulated, with its unexpected mobility aiding in the uniform distribution of titin post-cell fusion. Although the experimental approach is convincing, the work is very qualitative in its approaches, and the data needs rigorous statistical …
eLife Assessment
This study offers valuable information on how titin derived from different nuclei within the syncytium is organized and integrated during skeletal muscle development and remodeling. The authors developed a novel mCherry titin knock-in mice with the fluorophore mCherry inserted into titin's Z-disk region to track the titin during cell fusion. The approach using mcherry adds to understanding of the role and localization of titin in controlling stiffness of striated muscles and fine tuning contraction. The results demonstrate that the integration of titin into the sarcomere is tightly regulated, with its unexpected mobility aiding in the uniform distribution of titin post-cell fusion. Although the experimental approach is convincing, the work is very qualitative in its approaches, and the data needs rigorous statistical analysis. There is a need for some clarification concerning numbers of animals and control groups. Future studies will need more rigorous data analysis and interpretation.
-
Reviewer #1 (Public Review):
In this work, the authors aimed to understand how titins derived from different nuclei within the syncytium are organized and integrated after cell fusion during skeletal muscle development and remodeling. The authors developed mCherry titin knock-in mice with the fluorophore mCherry inserted into titin's Z-disk region to track the titin during cell fusion. The results suggested that titin exhibited homogenous distribution after cell fusion. The authors also probed on how titin behaves during muscle injury by implantation of titin-eGFP myoblasts into adult mCherry-titin mice. Interestingly, titin is retained at the proximal nucleus and does not diffuse across the whole syncytium in this system. The findings of the study are novel and interesting. The experimental approaches are appropriate. The results are …
Reviewer #1 (Public Review):
In this work, the authors aimed to understand how titins derived from different nuclei within the syncytium are organized and integrated after cell fusion during skeletal muscle development and remodeling. The authors developed mCherry titin knock-in mice with the fluorophore mCherry inserted into titin's Z-disk region to track the titin during cell fusion. The results suggested that titin exhibited homogenous distribution after cell fusion. The authors also probed on how titin behaves during muscle injury by implantation of titin-eGFP myoblasts into adult mCherry-titin mice. Interestingly, titin is retained at the proximal nucleus and does not diffuse across the whole syncytium in this system. The findings of the study are novel and interesting. The experimental approaches are appropriate. The results are described well. However, the manuscript needs revisions to enhance its clarity.
(1) In this work, the authors have not described the statistical analysis appropriately. In most of the figures, significance levels are not described. The information on the biological and technical replicates is missing in almost all the figures. This information is critical for understanding the strength of the experimentation.
(2) The in vivo experiments are underpowered. The authors have used only 3 animals in the cardiotoxin injury experiment and eliminated another 3 animals from the analysis. How did they determine insufficient myoblast integration?
(3) Similarly, the in vitro imaging experiments, especially the in vitro titin mobility assays used only 3 cells (Fig 2b) or 6-9 cells (Fig 2c-2e). The number of cells imaged is insufficient to derive a valid conclusion. What is the variability in the results between cells? Whether all the cells behave similarly in titin mobility assays?
(4) Figure 1c-e, Figure 2a, Figure 3, Figure 4, Figure 5, Figure 6- please describe the replicates and also if possible, quantify the data and present them as separate figures.
(5) Figure 2- the authors excluded samples with an obvious decrease in cell quality during imaging from the analysis. How do the authors assess the cell quality? Simply by visual examination? Or were the samples that did not show fluorescence recovery eliminated? I am wondering what percentage of cells showed poor cell quality. How do they avoid the bias? I recommend that the authors include these cells also for the analysis of data presented in Figures 2b, 2c, and 2f.
(6) It is unclear how the authors identified the different stages of cell fusion in the microscopy images i.e. early fusion, distribution, and complete distribution. -
Reviewer #2 (Public Review):
The titin protein, a large component of striated muscle, plays a crucial role in the formation of the sarcomere during muscle development. As myocytes merge, titin integrates into the sarcomere structure, creating a stable myofilament system. The authors of the present study have shed light on the intricate process of myofilament assembly and disassembly, which is made possible by tracking labeled sarcomere components. In this study, they introduced the mCherry marker into titin's Z-disk to investigate its role in skeletal muscle development and remodeling. Their findings demonstrate that the integration of titin into the sarcomere is tightly regulated, with its unexpected mobility aiding in the uniform distribution of titin post-cell fusion. This distribution is crucial for the formation and maturation of …
Reviewer #2 (Public Review):
The titin protein, a large component of striated muscle, plays a crucial role in the formation of the sarcomere during muscle development. As myocytes merge, titin integrates into the sarcomere structure, creating a stable myofilament system. The authors of the present study have shed light on the intricate process of myofilament assembly and disassembly, which is made possible by tracking labeled sarcomere components. In this study, they introduced the mCherry marker into titin's Z-disk to investigate its role in skeletal muscle development and remodeling. Their findings demonstrate that the integration of titin into the sarcomere is tightly regulated, with its unexpected mobility aiding in the uniform distribution of titin post-cell fusion. This distribution is crucial for the formation and maturation of skeletal muscle syncytium. In adult mice with mCherry-labeled titin, treating muscle injuries by introducing titin-eGFP myoblasts illustrates how myocytes integrate, fuse, and contribute to a seamless myofilament system across cell boundaries. The manuscript is well written, and the study is very novel.
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