Tethered Jagged-1 Synergizes with Culture Substrate Stiffness to Modulate Notch-Induced Myogenic Progenitor Differentiation

Abstract

Introduction

Notch signaling is amongst the key intrinsic mechanisms regulating satellite cell fate, promoting the transition of activated satellite cells to highly proliferative myogenic progenitor cells and preventing their premature differentiation. Although much is known about the biochemical milieu that drives myogenic progression, less is known about the spatial cues providing spatiotemporal control of skeletal muscle repair in the context of Notch signaling.

Methods

Using a murine injury model, we quantified in vivo biophysical changes that occur within the skeletal muscle during regeneration. Employing tunable poly(ethylene glycol)—based hydrogel substrates, we modeled the measured changes in bulk stiffness in the context of Notch ligand signaling, which are present in the regenerative milieu at the time of injury.

Results

Following injury, there is a transient increase in the bulk stiffness of the tibialis anterior muscle that may be explained in part by changes in extracellular matrix deposition. When presented to primary myoblasts, Jagged-1, Jagged-2, and Dll1 in a tethered format elicited greater degrees of Notch activity compared to their soluble form. Only tethered Jagged-1 effects were tuned by substrate stiffness, with the greatest Notch activation observed on stiff hydrogels matching the stiffness of regenerating muscle. When exposed to tethered Jagged-1 on stiff hydrogels, fewer primary myoblasts expressed myogenin, and pharmacological inhibitor studies suggest this effect is Notch and RhoA dependent.

Conclusion

Our study proposes that tethered Jagged-1 presented in the context of transient tissue stiffening serves to tune Notch activity in myogenic progenitors during skeletal muscle repair and delay differentiation.

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Abbreviations

bFGF:

Basic fibroblast growth factor

Dll1:

Delta-like 1

DM:

Differentiation media

ECM:

Extracellular matrix

GM:

Growth media

IgG1:

Immunoglobulin G subtype 1

Jag1:

Jagged-1

Jag2:

Jagged-2

MyoG:

Myogenin

NICD:

Notch intracellular domain

PEG:

Polyethylene glycol

PBS:

Phosphate buffered saline

ROCK:

Rho-associated, coiled-coil containing protein kinase

TBST:

Tris-buffered saline plus Tween-20

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Acknowledgments

We are grateful to Dr. Yasuhiro Matsumura (National Cancer Center Hospital, Kashiwa City, Japan) for providing us with his custom antibody to analyze fibrin clot formation in our study.

Author Contributions

HS, MAB, CAS, and PMG conceived the study and designed experiments. HS, SD, RYC, AJM, and EWL performed experiments, analyzed data, performed statistical analyses, and prepared figures. HS, MAB, SD, RYC, AJM, EWL, CAS, and PMG wrote, assembled, and revised the manuscript. All authors reviewed and approved the submission.

Funding

This study was funded by the Natural Sciences and Engineering Research Council (USRA fellowship to H.S., CREATE ToEP fellowship to S.D., RGPIN 327627-06 to C.A.S., RGPIN 435724-13 and Canada Research Chair 950-231201 to P.M.G.); Toronto Musculoskeletal Centre (Graduate Scholarships to M.A.B. and R.Y.C.); Barbara and Frank Milligan Foundation (R.Y.C.); Ontario Provincial Government (OGS-visa to M.A.B., 31390 and ER15-11-073 to P.M.G.); Canada Foundation for Innovation (31390 to P.M.G.); Krembil Foundation (Scholarship to M.A.B.); Toronto Western Arthritis Program (to P.M.G.); and Canadian Institutes of Health Research (MOP-302041 to C.A.S. and ONM-137370 to P.M.G.)

Conflict of interest

All authors declared that they have no conflict of interest.

Ethical Approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.

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Correspondence to Penney M. Gilbert.

Additional information

Penney Gilbert is an Assistant Professor at the University of Toronto in the Institute of Biomaterials and Biomedical Engineering. She holds cross-appointments in the Department of Biochemistry and the Donnelly Centre for Cellular and Biomolecular Research. Gilbert received her PhD from the University of Pennsylvania under the mentorship of Valerie Weaver and then pursued Postdoctoral studies with Helen Blau at Stanford University. Her research team is focused on engineering human skeletal muscle culture models and uncovering niche cues that drive muscle stem cell fate changes in vivo. She is recipient of an Ontario Early Researcher Award and a BMES-CMBE Conference ‘Rising Star’ Award. She holds a Tier II Canada Research Chair in Endogenous Repair.

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This article is part of the 2017 CMBE Young Innovators special issue.

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Safaee, H., Bakooshli, M.A., Davoudi, S. et al. Tethered Jagged-1 Synergizes with Culture Substrate Stiffness to Modulate Notch-Induced Myogenic Progenitor Differentiation. Cel. Mol. Bioeng. 10, 501–513 (2017). https://doi.org/10.1007/s12195-017-0506-7

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Keywords

  • Skeletal muscle
  • Regeneration
  • Biophysical cues
  • Biochemical cues
  • Spatiotemporal
  • Niche
  • Compression testing
  • Extracellular matrix
  • Hydrogel
  • Ligand presentation