Controlled Heat Stress Promotes Myofibrillogenesis during Myogenesis

RESEARCH ARTICLE Controlled Heat Stress Promotes Myofibrillogenesis during Myogenesis Qiongyu Guo1, Devin Miller1, Hongying An2, Howard Wang1, Joseph Lopez1, Denver Lough1, Ling He2, Anand Kumar1* 1 Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 2 Divisions of Metabolism and Endocrinology, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America * a11111 OPEN ACCESS Citation: Guo Q, Miller D, An H, Wang H, Lopez J, Lough D, et al. (2016) Controlled Heat Stress Promotes Myofibrillogenesis during Myogenesis. PLoS ONE 11(11): e0166294. doi:10.1371/journal. pone.0166294 Editor: Atsushi Asakura, University of Minnesota Medical Center, UNITED STATES Received: February 22, 2016 Accepted: October 26, 2016 Published: November 8, 2016 Copyright: © 2016 Guo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This study was funded by Johns Hopkins School of Medicine Faculty Start Up Research Grant (AK). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract Hyperthermia therapy has recently emerged as a clinical modality used to finely tune heat stress inside the human body for various biomedical applications. Nevertheless, little is known regarding the optimal timing or temperature of heat stress that is needed to achieve favorable results following hyperthermia therapy for muscle regeneration purposes. The regeneration of skeletal muscle after injury is a highly complex and coordinated process that involves a multitude of cellular mechanisms. The main objective of this study was to characterize the effects of hyperthermal therapy on the overall behavior of myoblasts during myogenic differentiation. Various cellular processes, including myogenesis, myofibrillogenesis, hypertrophy/atrophy, and mitochondrial biogenesis, were studied using systematic cellular, morphological, and pathway-focused high-throughput gene expression profiling analyses. We found that C2C12 myoblasts exhibited distinctive time and temperature-dependence in biosynthesis and regulatory events during myogenic differentiation. Specifically, we for the first time observed that moderate hyperthermia at 39˚C favored the growth of sarcomere in myofibrils at the late stage of myogenesis, showing universal upregulation of characteristic myofibril proteins. Characteristic myofibrillogenesis genes, including heavy polypeptide 1 myosin, heavy polypeptide 2 myosin, alpha 1 actin, nebulin and titin, were all significantly upregulated (p<0.01) after C2C12 cells differentiated at 39˚C over 5 days compared with the control cells cultured at 37˚C. Furthermore, moderate hyperthermia enhanced myogenic differentiation, with nucleus densities per myotube showing 2.2-fold, 1.9-fold and 1.6-fold increases when C2C12 cells underwent myogenic differentiation at 39˚C over 24 hours, 48 hours and 72 hours, respectively, as compared to the myotubes that were not exposed to heat stress. Yet, atrophy genes were sensitive even to moderate hyperthermia, indicating that strictly controlled heat stress is required to minimize the development of atrophy in myotubes. In addition, mitochondrial biogenesis was enhanced following thermal induction of myoblasts, suggesting a subsequent shift toward anabolic demand requirements for energy production. This study offers a new perspective to understand and utilize the time and temperature-sensitive effects of hyperthermal therapy on muscle regeneration. Competing Interests: The authors have declared that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0166294 November 8, 2016 1 / 15 Controlled Heat Stress Promotes Myofibrillogenesis during Myogenesis Introduction Skeletal muscle accounts for 40% of total body mass and demonstrates an innate self-repair capability in response to minor tissue damage or injury [1, 2]. However, regenerating muscle tissues elements capable of spanning segmental muscle gaps or defects following severe injury remains a clinical challenge [3]. Recently, hyperthermal therapy has attracted increasing attention in the fields of tissue engineering and cancer chemo-therapeutics due to its potential to modify the extracellular microenvironment, and thus regulate localized tissue responses including immunological reaction, tissue perfusion, and tissue oxygenation [4, 5]. Although controlled thermal delivery of heat has shown some beneficial effects on myogenesis during skeletal muscle repair in both in vitro [6–8] and in vivo studies [9–11], the detailed and coordinated effects of thermal treatment on muscle regeneration remain under characterized, limiting the development of a tailored hyperthermia treatment protocol for muscle regeneration. Skeletal muscle provides structural support and controls motor movements through highly organized long, tubular muscular cells or myofibers. Myofibers contain contractile fibril structures known as myofibrils that are composed of repeating units of sarcomeres. Sarcomeres primarily consist of thick filaments of myosin, thin filaments of actin, and elastic filaments of titin [12, 13]. Myofibrillogenesis, the development of the myofibril during myogenesis, plays a critical role in controlling the contractile strength of skeletal muscles [14, 15]. Recently, Yamaguchi et al. [6] and Oishi et al. [9] reported a fast-to-slow fiber-type shift in myotubes or myofibers during myogenesis in their in vitro and in vivo studies, respectively. Yet, their work solely focused on analyzing the expressions of myosin heavy chains. The effect of heat stress on myofibrillogenesis, including the expressions of various structural and regulatory proteins assembled in sarcomeres other than myosin such as actin, titin, and titin complexes, remains under characterized to date. Further investigation into thermal therapy applications on these fundamental functional proteins and resulting myogenic ultrastructure is of great importance to understanding temperature-induced alterations in muscle regeneration. Myogenesis involves the orchestration of multiple biological processes including myofibrillogenesis, the hypertrophy/atrophy of cellular entities as well as mitochondrial biogenesis, all of which are critical to the development of proper muscular function. Myocytic hypertrophy is associated with a mass increase of myofibers through stimulating protein synthesis, whereas atrophy is related to protein breakdown through activating protein degradation pathways [16]. Mitochondrial biogenesis, while not only coupled with m (...truncated)