ERK1/2 inhibition promotes robust myotube growth via CaMKII activation resulting in myoblast-to-myotube fusion

Tamar Eigler; Giulia Zarfati; Emmanuel Amzallag; Sansrity Sinha; Nadav Segev; Yishaia Zabary; Assaf Zaritsky; Avraham Shakked; Kfir-Baruch Umansky; Eyal D Schejter; Douglas P Millay; Eldad Tzahor; Ori Avinoam

doi:10.1016/j.devcel.2021.11.022

ERK1/2 inhibition promotes robust myotube growth via CaMKII activation resulting in myoblast-to-myotube fusion

Tamar Eigler, Giulia Zarfati, Emmanuel Amzallag, Sansrity Sinha, Nadav Segev, Yishaia Zabary, Assaf Zaritsky, Avraham Shakked, Kfir-Baruch Umansky, Eyal D Schejter, Douglas P Millay, Eldad Tzahor^*, Ori Avinoam^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

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Abstract

Myoblast fusion is essential for muscle development and regeneration. Yet, it remains poorly understood how mononucleated myoblasts fuse with preexisting fibers. We demonstrate that ERK1/2 inhibition (ERKi) induces robust differentiation and fusion of primary mouse myoblasts through a linear pathway involving RXR, ryanodine receptors, and calcium-dependent activation of CaMKII in nascent myotubes. CaMKII activation results in myotube growth via fusion with mononucleated myoblasts at a fusogenic synapse. Mechanistically, CaMKII interacts with and regulates MYMK and Rac1, and CaMKIIδ/γ knockout mice exhibit smaller regenerated myofibers following injury. In addition, the expression of a dominant negative CaMKII inhibits the formation of large multinucleated myotubes. Finally, we demonstrate the evolutionary conservation of the pathway in chicken myoblasts. We conclude that ERK1/2 represses a signaling cascade leading to CaMKII-mediated fusion of myoblasts to myotubes, providing an attractive target for the cultivated meat industry and regenerative medicine.

Original language	English
Pages (from-to)	3349-3363.e6
Number of pages	22
Journal	Developmental Cell
Volume	56
Issue number	24
DOIs	https://doi.org/10.1016/j.devcel.2021.11.022
Publication status	Published - 20 Dec 2021

Bibliographical note

CaMK2δ fl/fl /γ fl/fl mice were kindly provided by Eric Olson and Johannes Backs. Histology sections were prepared by Calanit Raanan. This study was supported by grants to E.T. from the AFM ( #21655 ), the Weizmann Institute Hellen and Martin Kimmel Stem Cell grant, the European Research Council ( ERC StG #281289 , ERC AdG #788194 ), the Israel Science Foundation (ISF), and Minerva Foundation with funding from the Federal German Ministry for Education and Research (to E.T. and O.A.). This project also received funding from the European Research Council (ERC StG # 851080 to O.A.). O.A. also acknowledges funding from the David Barton Center for Research on the Chemistry of Life and the Ruth and Herman Albert Scholarship Program for New Scientists as well as the Estate of Fannie Sherr. O.A. is an incumbent of the Miriam Berman Presidential Development Chair.

CaMK2?fl/fl/?fl/fl mice were kindly provided by Eric Olson and Johannes Backs. Histology sections were prepared by Calanit Raanan. This study was supported by grants to E.T. from the AFM (#21655), the Weizmann Institute Hellen and Martin Kimmel Stem Cell grant, the European Research Council (ERC StG #281289, ERC AdG #788194), the Israel Science Foundation (ISF), and Minerva Foundation with funding from the Federal German Ministry for Education and Research (to E.T. and O.A.). This project also received funding from the European Research Council (ERC StG # 851080 to O.A.). O.A. also acknowledges funding from the David Barton Center for Research on the Chemistry of Life and the Ruth and Herman Albert Scholarship Program for New Scientists as well as the Estate of Fannie Sherr. O.A. is an incumbent of the Miriam Berman Presidential Development Chair. T.E. E.T. and O.A. conceived and designed the experiments. T.E. with help from G.Z. E.A. and S.S. carried out most of the experiments and analyzed the data. Specifically, G.Z. performed and analyzed the live-cell imaging data with assistance from N.S. and S.S. T.E. S.S. and E.A. performed qRT-PCR experiments. T.E. G.Z. and S.S. performed and analyzed the experiments comparing ERKi in PM and DM. T.E. and G.Z. performed immunohistochemistry. Y.Z. and A.Z. performed the simulations. T.E. and K.U. performed CTX injuries, and T.E. carried out all follow-up studies. E.S. contributed to experimental design and critical review of the manuscript. D.M. contributed to the design of the in vivo model and associated experiments. E.T. and O.A. supervised the project. T.E, E.T. and O.A. wrote the manuscript with editing contributions from all the authors. T.E, E.T. and O.A. hold a patent related to the scientific findings presented in this manuscript and are the founders of ProFuse Technology. T.E. is the CTO and E.T. and O.A. are the scientific advisors of ProFuse Technology.
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Eigler, T., Zarfati, G., Amzallag, E., Sinha, S., Segev, N., Zabary, Y., Zaritsky, A., Shakked, A., Umansky, K.-B., Schejter, E. D., Millay, D. P., Tzahor, E., & Avinoam, O. (2021). ERK1/2 inhibition promotes robust myotube growth via CaMKII activation resulting in myoblast-to-myotube fusion. Developmental Cell, 56(24), 3349-3363.e6. https://doi.org/10.1016/j.devcel.2021.11.022

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title = "ERK1/2 inhibition promotes robust myotube growth via CaMKII activation resulting in myoblast-to-myotube fusion",

abstract = "Myoblast fusion is essential for muscle development and regeneration. Yet, it remains poorly understood how mononucleated myoblasts fuse with preexisting fibers. We demonstrate that ERK1/2 inhibition (ERKi) induces robust differentiation and fusion of primary mouse myoblasts through a linear pathway involving RXR, ryanodine receptors, and calcium-dependent activation of CaMKII in nascent myotubes. CaMKII activation results in myotube growth via fusion with mononucleated myoblasts at a fusogenic synapse. Mechanistically, CaMKII interacts with and regulates MYMK and Rac1, and CaMKIIδ/γ knockout mice exhibit smaller regenerated myofibers following injury. In addition, the expression of a dominant negative CaMKII inhibits the formation of large multinucleated myotubes. Finally, we demonstrate the evolutionary conservation of the pathway in chicken myoblasts. We conclude that ERK1/2 represses a signaling cascade leading to CaMKII-mediated fusion of myoblasts to myotubes, providing an attractive target for the cultivated meat industry and regenerative medicine.",

author = "Tamar Eigler and Giulia Zarfati and Emmanuel Amzallag and Sansrity Sinha and Nadav Segev and Yishaia Zabary and Assaf Zaritsky and Avraham Shakked and Kfir-Baruch Umansky and Schejter, {Eyal D} and Millay, {Douglas P} and Eldad Tzahor and Ori Avinoam",

note = "CaMK2δ fl/fl /γ fl/fl mice were kindly provided by Eric Olson and Johannes Backs. Histology sections were prepared by Calanit Raanan. This study was supported by grants to E.T. from the AFM ( #21655 ), the Weizmann Institute Hellen and Martin Kimmel Stem Cell grant, the European Research Council ( ERC StG #281289 , ERC AdG #788194 ), the Israel Science Foundation (ISF), and Minerva Foundation with funding from the Federal German Ministry for Education and Research (to E.T. and O.A.). This project also received funding from the European Research Council (ERC StG # 851080 to O.A.). O.A. also acknowledges funding from the David Barton Center for Research on the Chemistry of Life and the Ruth and Herman Albert Scholarship Program for New Scientists as well as the Estate of Fannie Sherr. O.A. is an incumbent of the Miriam Berman Presidential Development Chair. CaMK2?fl/fl/?fl/fl mice were kindly provided by Eric Olson and Johannes Backs. Histology sections were prepared by Calanit Raanan. This study was supported by grants to E.T. from the AFM (#21655), the Weizmann Institute Hellen and Martin Kimmel Stem Cell grant, the European Research Council (ERC StG #281289, ERC AdG #788194), the Israel Science Foundation (ISF), and Minerva Foundation with funding from the Federal German Ministry for Education and Research (to E.T. and O.A.). This project also received funding from the European Research Council (ERC StG # 851080 to O.A.). O.A. also acknowledges funding from the David Barton Center for Research on the Chemistry of Life and the Ruth and Herman Albert Scholarship Program for New Scientists as well as the Estate of Fannie Sherr. O.A. is an incumbent of the Miriam Berman Presidential Development Chair. T.E. E.T. and O.A. conceived and designed the experiments. T.E. with help from G.Z. E.A. and S.S. carried out most of the experiments and analyzed the data. Specifically, G.Z. performed and analyzed the live-cell imaging data with assistance from N.S. and S.S. T.E. S.S. and E.A. performed qRT-PCR experiments. T.E. G.Z. and S.S. performed and analyzed the experiments comparing ERKi in PM and DM. T.E. and G.Z. performed immunohistochemistry. Y.Z. and A.Z. performed the simulations. T.E. and K.U. performed CTX injuries, and T.E. carried out all follow-up studies. E.S. contributed to experimental design and critical review of the manuscript. D.M. contributed to the design of the in vivo model and associated experiments. E.T. and O.A. supervised the project. T.E, E.T. and O.A. wrote the manuscript with editing contributions from all the authors. T.E, E.T. and O.A. hold a patent related to the scientific findings presented in this manuscript and are the founders of ProFuse Technology. T.E. is the CTO and E.T. and O.A. are the scientific advisors of ProFuse Technology. Publisher Copyright: {\textcopyright} 2021 The Author(s)",

year = "2021",

month = dec,

day = "20",

doi = "10.1016/j.devcel.2021.11.022",

language = "English",

volume = "56",

pages = "3349--3363.e6",

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Eigler, T, Zarfati, G, Amzallag, E, Sinha, S, Segev, N, Zabary, Y, Zaritsky, A, Shakked, A, Umansky, K-B, Schejter, ED, Millay, DP, Tzahor, E & Avinoam, O 2021, 'ERK1/2 inhibition promotes robust myotube growth via CaMKII activation resulting in myoblast-to-myotube fusion', Developmental Cell, vol. 56, no. 24, pp. 3349-3363.e6. https://doi.org/10.1016/j.devcel.2021.11.022

TY - JOUR

T1 - ERK1/2 inhibition promotes robust myotube growth via CaMKII activation resulting in myoblast-to-myotube fusion

AU - Eigler, Tamar

AU - Zarfati, Giulia

AU - Amzallag, Emmanuel

AU - Sinha, Sansrity

AU - Segev, Nadav

AU - Zabary, Yishaia

AU - Zaritsky, Assaf

AU - Shakked, Avraham

AU - Umansky, Kfir-Baruch

AU - Schejter, Eyal D

AU - Millay, Douglas P

AU - Tzahor, Eldad

AU - Avinoam, Ori

N1 - CaMK2δ fl/fl /γ fl/fl mice were kindly provided by Eric Olson and Johannes Backs. Histology sections were prepared by Calanit Raanan. This study was supported by grants to E.T. from the AFM ( #21655 ), the Weizmann Institute Hellen and Martin Kimmel Stem Cell grant, the European Research Council ( ERC StG #281289 , ERC AdG #788194 ), the Israel Science Foundation (ISF), and Minerva Foundation with funding from the Federal German Ministry for Education and Research (to E.T. and O.A.). This project also received funding from the European Research Council (ERC StG # 851080 to O.A.). O.A. also acknowledges funding from the David Barton Center for Research on the Chemistry of Life and the Ruth and Herman Albert Scholarship Program for New Scientists as well as the Estate of Fannie Sherr. O.A. is an incumbent of the Miriam Berman Presidential Development Chair. CaMK2?fl/fl/?fl/fl mice were kindly provided by Eric Olson and Johannes Backs. Histology sections were prepared by Calanit Raanan. This study was supported by grants to E.T. from the AFM (#21655), the Weizmann Institute Hellen and Martin Kimmel Stem Cell grant, the European Research Council (ERC StG #281289, ERC AdG #788194), the Israel Science Foundation (ISF), and Minerva Foundation with funding from the Federal German Ministry for Education and Research (to E.T. and O.A.). This project also received funding from the European Research Council (ERC StG # 851080 to O.A.). O.A. also acknowledges funding from the David Barton Center for Research on the Chemistry of Life and the Ruth and Herman Albert Scholarship Program for New Scientists as well as the Estate of Fannie Sherr. O.A. is an incumbent of the Miriam Berman Presidential Development Chair. T.E. E.T. and O.A. conceived and designed the experiments. T.E. with help from G.Z. E.A. and S.S. carried out most of the experiments and analyzed the data. Specifically, G.Z. performed and analyzed the live-cell imaging data with assistance from N.S. and S.S. T.E. S.S. and E.A. performed qRT-PCR experiments. T.E. G.Z. and S.S. performed and analyzed the experiments comparing ERKi in PM and DM. T.E. and G.Z. performed immunohistochemistry. Y.Z. and A.Z. performed the simulations. T.E. and K.U. performed CTX injuries, and T.E. carried out all follow-up studies. E.S. contributed to experimental design and critical review of the manuscript. D.M. contributed to the design of the in vivo model and associated experiments. E.T. and O.A. supervised the project. T.E, E.T. and O.A. wrote the manuscript with editing contributions from all the authors. T.E, E.T. and O.A. hold a patent related to the scientific findings presented in this manuscript and are the founders of ProFuse Technology. T.E. is the CTO and E.T. and O.A. are the scientific advisors of ProFuse Technology. Publisher Copyright: © 2021 The Author(s)

PY - 2021/12/20

Y1 - 2021/12/20

N2 - Myoblast fusion is essential for muscle development and regeneration. Yet, it remains poorly understood how mononucleated myoblasts fuse with preexisting fibers. We demonstrate that ERK1/2 inhibition (ERKi) induces robust differentiation and fusion of primary mouse myoblasts through a linear pathway involving RXR, ryanodine receptors, and calcium-dependent activation of CaMKII in nascent myotubes. CaMKII activation results in myotube growth via fusion with mononucleated myoblasts at a fusogenic synapse. Mechanistically, CaMKII interacts with and regulates MYMK and Rac1, and CaMKIIδ/γ knockout mice exhibit smaller regenerated myofibers following injury. In addition, the expression of a dominant negative CaMKII inhibits the formation of large multinucleated myotubes. Finally, we demonstrate the evolutionary conservation of the pathway in chicken myoblasts. We conclude that ERK1/2 represses a signaling cascade leading to CaMKII-mediated fusion of myoblasts to myotubes, providing an attractive target for the cultivated meat industry and regenerative medicine.

AB - Myoblast fusion is essential for muscle development and regeneration. Yet, it remains poorly understood how mononucleated myoblasts fuse with preexisting fibers. We demonstrate that ERK1/2 inhibition (ERKi) induces robust differentiation and fusion of primary mouse myoblasts through a linear pathway involving RXR, ryanodine receptors, and calcium-dependent activation of CaMKII in nascent myotubes. CaMKII activation results in myotube growth via fusion with mononucleated myoblasts at a fusogenic synapse. Mechanistically, CaMKII interacts with and regulates MYMK and Rac1, and CaMKIIδ/γ knockout mice exhibit smaller regenerated myofibers following injury. In addition, the expression of a dominant negative CaMKII inhibits the formation of large multinucleated myotubes. Finally, we demonstrate the evolutionary conservation of the pathway in chicken myoblasts. We conclude that ERK1/2 represses a signaling cascade leading to CaMKII-mediated fusion of myoblasts to myotubes, providing an attractive target for the cultivated meat industry and regenerative medicine.

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U2 - 10.1016/j.devcel.2021.11.022

DO - 10.1016/j.devcel.2021.11.022

M3 - Article

C2 - 34932950

SN - 1534-5807

VL - 56

SP - 3349-3363.e6

JO - Developmental Cell

JF - Developmental Cell

IS - 24

ER -

ERK1/2 inhibition promotes robust myotube growth via CaMKII activation resulting in myoblast-to-myotube fusion

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