Dataset DOI: 10.5061/dryad.z612jm6r8

Description of the data and file structure

The details about all of the experiments that led to data in this file are provided in the main manuscript

Files and variables

All sheets within the file Source_Data.xlsx are also provided as a .CSV file.

File: Source_Data.xlsx

Description: The file is presented in a Microsoft Excel format. Where applicable, the formulas for converting raw values into relative values have been included so that readers can determine how the values were derived for each experiment. In some cases, the file also contains highlighted cells so that readers can identify which samples came from which animals.

Figure 1: C57BL/6J mice were subjected to a mechanical overload (MOV) or sham (control) surgery and injected daily with either rapamycin (RAP) or the vehicle DMSO (VEH) as a control condition. After 16 days, the plantaris (PLT) muscle from one leg was weighed, cross-sectioned at the mid-belly, and analyzed via immunohistochemistry (IHC), while the PLT from the contralateral leg was weighed and then processed for single fiber isolation and analysis. Measurements of the (B) PLT muscle mass to body mass ratio expressed as a percent of the average control ratio, (C) whole muscle cross-sectional area (CSA) expressed as a percent of the average control CSA, (D) the mean CSA of the Type II fibers expressed as a percent of the average control Type II fiber CSA, and (E) number of fibers per whole muscle cross-section expressed as a percent of the control group average. Measurement of the (G) average fiber length expressed as a percent of the average control fiber length, and (H) number of in-series sarcomeres per fiber expressed as a percent of the average number of in-series sarcomeres per fiber in the control group.

Figure 1 Raw Values: The measured values that were used to calculate the ratios and percent of control values presented in Figure 1. The data are displayed in an Excel file and, where applicable, the formulas used to calculate the relative values are embedded in the spreadsheet to aid the reader in understanding how these values were derived.

Figure 2: C57BL/6J mice were subjected to a mechanical overload (MOV) or sham (control) surgery. The plantaris muscles were collected at 4, 8, or 16 days after surgery and processed for single muscle fiber isolation. Measurements of the average (A) muscle fiber length expressed as a percent of the average control fiber length, (B) sarcomere length expressed as a percent of the average control sarcomere length, and (C) number of in-series sarcomeres per fiber expressed as a percent of the average number of in-series sarcomeres per fiber in the control group. For the sham samples, no significant difference was observed in any of the variables across the different time points. Accordingly, all of the sham samples were consolidated into a single group (i.e., 0 days of MOV).

Figure 2 Raw Values: These are all of the measured values that were used to calculate the ratios and percent of control values presented in Figure 2. The data are displayed in an Excel file and, where applicable, the formulas used to calculate the relative values are embedded in the spreadsheet to aid the reader in understanding how these values were derived.

Figure 3: C57BL/6J mice were subjected to a mechanical overload (MOV) or sham (control) surgery. At 8 days after surgery, the plantaris muscle from one leg was collected, and longitudinal sections were analyzed via immunohistochemistry for α-actinin. This staining was used to trace A-bands across the width of the fibers. (C) Manual measurements of the number of sarcomere transverse splits normalized to the width of the muscle fiber. (E) Measurements of the continuous Z-line length from the same images that were analyzed in C.

Figure 4: MetRS^L274G+/+ mice were subjected to a mechanical overload (MOV) or sham (control) surgery. After 7 days, the mice were injected with ANL or the vehicle (PBS), and the plantaris muscles were collected 24 hr later. A “click” reaction with a fluorophore bearing a reactive DBCO or alkyne group was used to label the azide on the ANL and identify newly synthesized proteins (NSPs). (D) Quantification of the in-gel NSP to total protein ratio expressed relative to the mean of the control samples. (F) The NSP to total protein ratio for each muscle cross-section was expressed relative to the mean of the control samples.

Figure 5: MetRS^L274G+/+ mice were subjected to a mechanical overload (MOV) or sham (control) surgery. After 7 days, the mice were injected with ANL or the vehicle (PBS), and the plantaris muscles were collected 24 hr later. A “click” reaction with a fluorophore bearing a reactive alkyne group was used to label the azide on the ANL and identify newly synthesized proteins (NSPs). (B) The percentage of fibers within each sample that were positive for NSP hot spots. (C) Regions of interest (ROI) within the fibers from the MOV samples that were positive for NSP hot spots were classified as having either a “normal” or a “disarrayed” Z-line configuration. An equivalent number of “normal” and “disarrayed” ROIs within each fiber were then analyzed for the number of hot spots per ROI. The mean values for the “normal” and “disarrayed” ROIs within each fiber were calculated, and then the mean values for each type of ROI within each fiber were reported as paired values.

Figure 6: MetRS^L274G+/+ mice were subjected to a mechanical overload (MOV) or sham (control) surgery and injected daily with either rapamycin (RAP) or the vehicle (VEH) as a control condition. After 7 days, the mice were given a final dose of RAP or VEH and injected with ANL, and then the plantaris (PLT) muscles were collected 24 hr later. A “click” reaction with a fluorophore bearing a reactive DBCO or alkyne group was used to label the azide on the ANL and identify newly synthesized proteins (NSPs). (B) The number of fibers per whole muscle cross-section expressed as a percent of the control group average, (C) the PLT muscle mass to body mass ratio expressed as a percent of the average control group ratio, and (D) whole muscle cross-sectional area (CSA) expressed as a percent of the average control CSA. (G) Quantitative analysis of the NSP to total protein ratio expressed as a percent of the control group average.

Figure 6 Raw Values: These are the measured values that were used to calculate the ratios and percent of control values presented in Figure 6. The data are displayed in an Excel file and, where applicable, the formulas used to calculate the relative values are embedded in the spreadsheet to aid the reader in understanding how these values were derived.

Figure 7: MetRS^L274G+/+ mice were subjected to a mechanical overload (MOV) or sham (control) surgery and injected daily with either rapamycin (RAP) or the vehicle (VEH) as a control condition. After 7 days, the mice were given a final dose of RAP or VEH and injected with ANL, and then the plantaris (PLT) muscles were collected 24 hr later. A “click” reaction with a fluorophore bearing a reactive alkyne group was used to label the azide on the ANL and identify newly synthesized proteins (NSPs). (B) The percentage of fibers within each sample that were positive for NSP hot spots. (C) 100 µm² regions of interest (ROIs) within the fibers from the MOV samples that were positive for NSP hot spots were classified as having a “normal” or a “disarrayed” Z-line configuration. A “normal” and a “disarrayed” ROI within each fiber was then analyzed for the number of hot spots per ROI. The mean value for the “normal” and a “disarrayed” ROIs in each muscle was calculated and recorded as paired values. (D) The number of sarcomere transverse splits normalized to the width of the muscle fiber.

Figure 8: Three different models of in-series sarcomerogenesis and the expected morphology of the NSP hot spots that they would produce were identified. Briefly, the Rodier et al. model of sarcomere transverse Z-line splitting is expected to produce NSP hot spots that span the length of exactly two in-series sarcomeres. The Yu et al. model of sarcomere transverse Z-line splitting is expected to produce NSP hot spots that span the length of exactly one sarcomere. The Jahromi and Charlton model of sarcomere transverse H-zone splitting is expected to produce NSP hot spots that are confined to the adjacent inner halves in a pair of in-series sarcomeres. (D) MetRS^L274G+/+ mice were subjected to mechanical overload (MOV). After 7 days, the mice were injected with ANL, and the plantaris muscles were collected 24 hr later. Longitudinal sections were subjected to immunohistochemistry for α-actinin and a click reaction with alkyne-AZDye 555 to label the newly synthesized proteins (NSPs). The images were assessed for the presence of fibers that had qualifying NSP hot spots (see main text for details), and for each analyzed fiber, the proportion of the NSP hot spots whose morphology aligned with each of the models of in-series sarcomerogenesis was determined.

Supplemental Figure 1: C57BL/6J mice were subjected to a mechanical overload (MOV) or sham (control) surgery. After 8 days, the plantaris muscles were collected, and then mid-belly cross-sections were subjected to immunohistochemistry for SERCA1 to identify the periphery of the sarcomeres. The minimal and maximal Feret diameters of the sarcomeres within randomly selected fibers were determined using a custom CellProfiler program. Frequency distribution of the minimal and maximal Feret diameters of the sarcomeres in the control (C) and MOV (D) muscles. (E) The mean values for each of the muscles in C -D.

Supplemental Figure 4: MetRS^L274G+/+ mice were subjected to a mechanical overload, and after 7 days, the mice were injected with ANL. The plantaris muscles were collected 24 hr later, and longitudinal sections were subjected to immunohistochemistry for α-actinin and a click reaction with alkyne-AZDye 555 to label the newly synthesized proteins (NSPs). The images were assessed for the presence of fibers that had qualifying NSP hot spots, and for each analyzed fiber, the proportion of the NSP hot spots whose morphology aligned with the models of in-series sarcomerogenesis or one of the two atypical split types was determined.

Code/software

Microsoft excel

Access information

Other publicly accessible locations of the data:

• none

Data was derived from the following sources:

• mouse skeletal muscle