DOI: 10.5061/dryad.z612jm6r8
title: Mechanical loading induces the longitudinal growth of muscle fibers via a rapamycin-insensitive mechanism

description: Mechanical loading drives skeletal muscle growth, yet the mechanisms that
 regulate this process remain undefined. The data in this study show that
 an increase in mechanical loading induces muscle fiber growth through two
 distinct mechanisms. Radial growth, reflected by an increase in fiber
 cross-sectional area, is mediated through a rapamycin-sensitive signaling
 pathway, whereas longitudinal growth, marked by the in-series addition of
 sarcomeres, is mediated through a rapamycin-insensitive signaling pathway.
 To gain further insight into the events that drive longitudinal growth, we
 combined BONCAT-based labeling of newly synthesized proteins with
 high-resolution imaging and determined that the in-series addition of
 sarcomeres is mediated by a process that involves transverse splitting at
 the Z-lines of pre-existing sarcomeres. Collectively, our findings not
 only challenge the long-standing view that mechanically induced growth is
 uniformly governed by mTORC1, but they also lay the framework for a new
 understanding of the molecular and structural events that drive this
 process.  The dataset in this submission includes the values that
 were used to generate the main and supplemental figures in the manuscript
 titled “Mechanical Loading Induces the Longitudinal Growth of Muscle
 Fibers via a Rapamycin-Insensitive Mechanism”. All values are presented in
 a Microsoft Excel as well as a CSV format. Where applicable, the formulas
 for converting raw values into relative values have been included in the
 Microsoft Excel document so that readers can determine how the values were
 derived for each experiment.
description: # Mechanical loading induces the longitudinal growth of muscle fibers via
 a rapamycin-insensitive mechanism Dataset DOI:
 [10.5061/dryad.z612jm6r8](https://doi.org/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 µm^2^
 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