Optimizing Mechanical Stretching Protocols for Hypertrophic and Anti-apoptotic Responses in H9c2 Cardiomyocytes

Abstract

Background: Cardiomyocytes are sensitive to mechanical loading, possessing the ability to respond to mechanical stimuli by reprogramming their gene expression. In this study, signaling as well as expression responses of myogenic, anabolic, inflammatory, atrophy and pro-apoptotic genes to different mechanical stretching protocols were examined in differentiated cardiomyocytes.Methods: H9C2 cardiomyoblasts were cultured on elastic membranes up to their 5th day of differentiation (myotubes) and then subjected to three different stretching protocols by altering their strain, frequency and duration characteristics, using an in vitro cell tension system. cells were harvested and lysed 24 hours after the completion of each stretching protocol and Real Time-PCR was used to monitor changes in mRNA expression of myogenic regulatory factors (MyoD, Myogenin, MRF4), the IGF-1 isoforms (IGF-1Ea, IGF-1Eb), as well as atrophy (Atrogin-1), pro-apoptotic (FoxO, Fuca), and inflammatory (IL-6) factors in response to the different mechanical loading conditions. The activation of Akt and Erk 1/2 signaling proteins following the various stretching protocols was also evaluated by Western blot analysis.Results: We documented that the low strain (2.7% elongation), low frequency (0.25 Hz) and intermediate duration (12 hrs) stretching protocol was overall the most effective in inducing beneficial responses in differentiated cardiomyoblasts as it increased the expression of IGF-1 isoforms and phosphorylation of Akt and Erk1/2 (p<0.05), while it provoked the downregulation of all the other factors examined (p<0.05-0.001). Conclusion: These findings demonstrated that a low strain, low frequency of intermediate duration stretching protocol is the most effective in inducing a hypertrophic and anti-apoptotic response in H9C2 cardiomyotubes, in vitro.

The Effects of Muscle Cell Aging on Myogenesis

“…Myogenic differentiation is driven by multiple signal transduction and mechanotransduction pathways which coordinate the balance between muscle growth and atrophy, or protein synthesis and protein degradation [ 27 , 59 , 60 , 61 ]. Our findings documented a delayed or reduced ability of aged myoblasts to differentiate, as demonstrated by the expression time course of the myogenic transcription factors (MRFs) and the morphological characteristics of these cells during the differentiation process.…”