Cell Stress and Chaperones

, Volume 23, Issue 3, pp 399–410 | Cite as

Recurrent heat shock impairs the proliferation and differentiation of C2C12 myoblasts

  • Daniel J. Bolus
  • Gobinath Shanmugam
  • Madhusudhanan Narasimhan
  • Namakkal S. Rajasekaran
Original Paper


Heat-related illness and injury are becoming a growing safety concern for the farmers, construction workers, miners, firefighters, manufacturing workers, and other outdoor workforces who are exposed to heat stress in their routine lives. A primary response by a cell to an acute heat shock (HS) exposure is the induction of heat-shock proteins (HSPs), which chaperone and facilitate cellular protein folding and remodeling processes. While acute HS is well studied, the effect of repeated bouts of hyperthermia and the sustained production of HSPs in the myoblast-myotube model system of C2C12 cells are poorly characterized. In C2C12 myoblasts, we found that robust HS (43 °C, dose/time) significantly decreased the proliferation by 50% as early as on day 1 and maintained at the same level on days 2 and 3 of HS. This was accompanied by an accumulation of cells at G2 phase with reduced cell number in G1 phase indicating cell cycle arrest. FACS analysis indicates that there was no apparent change in apoptosis (markers) and cell death upon repeated HS. Immunoblot analysis and qPCR demonstrated a significant increase in the baseline expression of HSP25, 70, and 90 (among others) in cells after a single HS (43 °C) for 60 min as a typical HS response. Importantly, the repeated HS for 60 min each on days 2 and 3 maintained the elevated levels of HSPs compared to the control cells. Further, the continuous HS exposure resulted in significant inhibition of the differentiation of C2C12 myocytes to myotubes and only 1/10th of the cells underwent differentiation in HS relative to control. This was associated with significantly higher levels of HSPs and reduced expression of myogenin and Myh2 (P < 0.05), the genes involved in the differentiation process. Finally, the cell migration (scratch) assay indicated that the wound closure was significantly delayed in HS cells relative to the control cells. Overall, these results suggest that a repeated HS may perturb the active process of proliferation, motility, and differentiation processes in an in vitro murine myoblast-myotube model.


Heat shock proteins (HSP) Repeated heat shock Myoblasts C2C12 Proliferation Differentiation Myopathy 



The authors thank Drs. Sandeep Balu Shelar and Radhakrishnan Rajesh Kumar for their assistance with cell cultures.

Funding information

This study was supported by multiple awards/research funds from the NHLBI (R01# HL118067), NIA (R03#AG042860), the AHA (BGIA#0865015F), University of Utah center for Aging (Pilot grant#2009), the Division of Cardiovascular Medicine/Department of Medicine, University of Utah, and the start-up funds (for NSR) by the Department of Pathology and School of Medicine, the University of Alabama at Birmingham, AL. This research was partially supported by the SIBS-UPS Undergraduate Research Program funded by the UAB Department of Pathology for Bolus.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Cell Stress Society International 2017

Authors and Affiliations

  1. 1.Cardiac Aging & Redox Signaling Laboratory, Division of Molecular & Cellular Pathology, Department of PathologyThe University of Alabama at BirminghamBirminghamUSA
  2. 2.Department of Pharmacology and NeuroscienceTexas Tech University Health Sciences CenterLubbockUSA
  3. 3.Division of Cardiovascular Medicine, Department of MedicineThe University of Utah School of MedicineSalt Lake CityUSA
  4. 4.Center for Free Radical BiologyThe University of Alabama at BirminghamBirminghamUSA

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