For mTom/mGFP analysis, sucrose-protected samples were visualized at 10x. Collectively, our findings demonstrate that effective SC fusion is required for optimal muscle hypertrophy following a load-induced stimulus. All Teq products and services can categorized using the filters below. Is there evidence of lacz+ (or lacz-) muscle progenitor cell apoptosis during this interval, or are the cells simply shutting down expression of myomaker? We conclude that myonuclear accretion is required for different facets of exercise-induced adaptive responses, impacting both muscle repair and hypertrophic growth. Data presented is representative of the average number of LacZ+ non-myofibers, and the percentage of LacZ-expressing myofibers, quantified from several non-overlapping fields of view. After 14 days of overload in control mice, BrdU+ nuclei within a dystrophin-stained myofiber were readily detected (Figure 4B). We also reveal that in response to increased muscle workload, myomaker is primarily upregulated in activated SCs, which is required for fusion with a myofiber. Your revised article has been favorably evaluated by Sean Morrison (Senior Editor), and Amy Wagers (Reviewing Editor). Thus, in this system LacZ serves as a readout for myomaker transcription but not myomaker localization. There are however a number of issues related to the strength with which the authors' data support the conclusions drawn and the presentation of these data that need to be addressed before publication. However, the detailed molecular and cellular mechanisms underlying its endosomal recycling are largely unknown. What is not fully understood is whether this fusion process also helps undamaged adult muscles to increase in size (for example, in response to exercise). Per the reviewers’ request, we have also included a line in our manuscript on the need to further uncover the mechanisms by which satellite cell fusion drives muscle hypertrophy (Discussion, third paragraph). Plantaris muscles were homogenized with a bead homogenizer (TissueLyser II; Qiagen) in a lysis buffer [10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, 0.5% Triton X-100, 2.1 mg/ml NaF] containing protease and phosphatase inhibitor cocktails (5 μl/ml; Sigma-Aldrich), and following centrifugation, the amount of protein in supernatants was determined through Bradford protein assay. Quantification of LacZ+ non-myofibers shows a significant increase at day 7 in myomaker scKO mice compared to control mice because myomaker scKO are fusion defective and thus remain outside the myofiber (n = 3–5 mice). Cryosections were fixed in 1% PFA/PBS; permeabilized with 0.2% Triton X-100/PBS or denatured with 2 M HCL in 0.5%Triton X-100/PBS and subsequently neutralized with TBS (pH 8.4) for BrdU labeling. In 8 month-old mice, HIIT led to progressive myonuclear accretion throughout the protocol, and functional muscle hypertrophy. To understand the myogenic state of the different types of LacZ+ cells we stained serial sections with either x-gal or embryonic myosin (myh3), a marker of muscle differentiation. (D) Fiber number was quantitated at the mid-belly of the plantaris, and reveals a significant inhibition of new fiber formation in plantaris muscle when myomaker was ablated in satellite cells (n = 7–11 mice). Overall, the findings presented by Goh and Millay reveal that the fusion of muscle stem cells is an important event for adult muscle growth. Analysis was performed using FACSDiva software. Thus, fusion may provide new nuclei to facilitate enhanced transcription of contractile proteins necessary for hypertrophy, as supported by our findings that myofibers are not able to activate protein synthesis in the absence of fusion. Particularly, Figure 3D did not show appreciable difference between the fiber CSA of myomakerscKO MOV and Sham controls but a clear shift towards larger myofiber sizes of myomakerscKO MOV is observed in 3E. Fuse is a standalone 3D character creator that enables you to make unique characters to use in your game. In contrast, the most direct assessment of hypertrophy is CSA, which we have emphasized in the revised manuscript. 10) Figure 5 seems to be very superficial, although the control of hypertrophy by myomaker is the main point of this manuscript. Quantification of the number of LacZ+ non-myofibers indicates myomaker is robustly activated in MPs at day 3 and day 7 of MOV but the number is reduced at day 10 and day 14 (n = 3–5 mice). Given that this is one level of a particular muscle, if a BrdU myonucleus was above or below this plane then that fiber would not be counted as a positive fusion event. Expression of myomaker peaked 7 days after MOV, and was down-regulated at later time points. For Pax7 immunostaining, the above protocol was used in addition to antigen retrieval (1x Antigen Retrieval Citra Solution (Biogene # HK086-9K) and boiled for 30 min), followed by incubation with Pax 7antibodies overnight. b) Myofibers that are both LacZ+ and myh3+, which represent de novo fibers because they express embryonic myosin. Mice were subjected to MOV and myomaker expression was assessed through qPCR analysis 7 days post-surgery showing that myomaker was efficiently deleted in myomaker scKO muscle (n = 3–4 independent mice). IGF-1 directly activates the Akt pathway (Rommel et al., 2001) while myostatin is a negative regulator of muscle mass through SMAD-dependent inhibition of Akt (Sartori et al., 2009; Trendelenburg et al., 2009). We used antigen-specific affinity purified products for western analysis. Provide a password for the new account in both fields. Our finding of increased fibrosis in muscles of myomaker scKO mice after overload further suggests a protective role for SC fusion on myofiber stability in response to mechanical strain, however the cellular circuitry driving fibrosis development remains unknown. The stem cells subsequently differentiate and fuse with one another or to existing muscle fibers to restore the muscle. Fibers isolated from vehicle-treated mice were used to establish an impartial baseline for auto-fluorescence, and the threshold for GFP intensity was set at a constant value. While BrdU analysis revealed approximately 30% of fibers to undergo a fusion event, findings from the fluorescent reporter model demonstrate that the majority of myofibers fuse (mGFP+), indicating the BrdU strategy is an underestimation of the fusion process during muscle hypertrophy. STEM represents science, technology, engineering and maths. Nonetheless, the behavior and fate of fusion-incompetent MPs requires future investigation. Prior to muscle overload surgery, 3-month old mice were administered five daily doses of TAM at 0.075 mg/g/day through intraperitoneal injections. The item you just added is unavailable. However, results from this study could have been confounded by incomplete SC ablation and/or by indirect effects of dying SCs. However, the functionality of the antibodies to detect myomaker in vivo on sections is not clear as we observe staining in myomakerscKO samples that we have definitively shown are negative for myomaker mRNA. We also co-immunostained these sections with embryonic myosin (myh3) to evaluate the relative contribution of protein synthesis from de novo and existing myofibers. DPM, Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing, "This ORCID iD identifies the author of this article:". We agree with the reviewers that there are caveats associated with our FACS analysis, which is why we originally used a secondary method (Pax7 immunostaining) to confirm these data. Adult muscles also contain stem cells, which are normally dormant, but activate when the muscle is damaged. Does this mean that myomaker is required for the increase in fiber number? The e-mail address is not made public and will only be used if you wish to receive a new password or wish to receive certain news or notifications by e-mail. 1) Ideally, the impact of fusion blockade by ablation of myomaker in muscle progenitor cells would be further supported by evaluation of muscle contractile function. We interpret the small LacZ+ myh3- cells to be MPs given that myomaker is muscle-specific. (Available for grades K-5), Game:ITExpose your students to a wide range of concepts and technical skills that will greatly help them succeed in the future. The reviewers make an excellent point that our initial description of LacZ+ cells did not accurately distinguish mononuclear cells and fully differentiated myocytes. We think the fate of fusion-defective MPs is an extremely important question that will require more extensive analysis prior to a definitive conclusion. READ:ITA fully digital and interactive RTI (response to intervention) based reading program. We agree that findings of associated reductions in Akt/mTOR signaling in fusion-incompetent muscles that demonstrate impaired hypertrophy following mechanical overload would be strengthened by more experiments. The initial SC DTA experiments indicated that SCs are not necessary for hypertrophy, where CSA was increased 10% in control mice with normal SC activity. Thus, myomaker exhibits a contrasting expression pattern in MPs compared to the myofiber compartment in response to a load-induced stimulus.