This study examined and compared the effects of 3 different in vitro cell stretching protocols on gene expression and signalling responses associated with the myogenic lineage of differentiated H9C2 cardiomyoblasts, in order to reveal potential loading-specific, detrimental or beneficial effects on cardiac myotubes, depending on the loading characteristics of the different protocols. The expression of myogenic, anabolic, atrophy, pro-apoptotic and inflammatory factors, as well as the activation of major intracellular signaling cascades were measured 12 hours after the completion of each stretching protocol, to check durable, persistently triggered rather than short signaling and transcriptional responses. Our main findings revealed that a low strain (2.7% elongation), low frequency (0.25 Hz) of an intermediate duration (12 hrs) mechanical stretching protocol was overall the most effective in inducing a hypertrophic response in cardiac myotubes, by increasing the expression of the anabolic factor IGF-1 and the phosphorylation of Akt and Erk 1/2 signaling proteins, while downregulating atrophy, pro-apoptotic and inflammation-related factors. Furthermore, the present study revealed that the late myogenic factor MRF4 exhibited differential responses to mechanical loading compared to the other two MRFs examined, MyoD and Myogenin.
The ability of cardiomyocytes to sense external mechanical stimuli (mechanosensing) and convert them into electrochemical and biochemical signals is critical for the maintenance of their homeostasis as well as for cardiac muscle tissue adaptation to mechanical loading [1, 2, 4, 5]. In this context, in vitro models of cell stretching are virtually the main if not the only experimental approach to meticulously study the intracellular molecular events in cardiomyocytes as a result of mechanical stimuli [36, 37].
Mechanical loading of skeletal and cardiac muscle cells both in vivo and in vitro can lead to the upregulation of many growth factors, including IGF-1, and the activation of signaling pathways associated with protein synthesis and cell growth, eventually leading to muscle hypertrophy [10, 24–26, 38, 39]. Indeed, IGF-1 upregulation and signaling have been implicated in the mechanical loading-induced adaptive cardiac hypertrophy [10, 25, 26], while potentially differential actions of IGF-1 isoforms in myocardial repair/remodeling process have been proposed [27–29, 40]. To the authors’ best knowledge, this is the first study investigating the distinct expression profiles of IGF-1 isoforms following mechanical loading of cardiac myotubes, in vitro. Interestingly, our findings showed that both isoforms were upregulated by low strain/frequency and long durations stretching protocols, with the more pronounced responses being exhibited after the intermediate duration (12 hrs) protocol. Inversely, the high strain/frequency of short duration stretching of cardiomyotubes resulted in a tendency of decreased IGF-1 isoforms expression (Fig. 2). These findings suggest that both IGF-1 isoforms need prolonged, low strain/frequency loading to be activated in differentiated cardiac cells, in vitro.
Furthermore, two primary mechanosensitive intracellular pathways have been associated with the IGF-1 actions in skeletal and cardiac muscle physiology [26, 34]; the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, the activation of which is involved in cellular processes such as protein synthesis, hypertrophy and protection from apoptosis, and the Ras/Raf/MEK/Erk 1/2 signaling pathway, which has been shown to increase muscle cell proliferation. The outcomes of these two pathways are based on complex interactions that require comprehensive identification, since in some cell types, PI3K and Erks appear to act in concert, e.g., both PI3K and Erks are possibly required for the myogenic differentiation of myoblasts [26, 41]. In particular, the PI3K/Akt has been implicated in myocardial cells survival and their protection against reperfusion-induced injury , while both the IGF-1 receptor (IGF-1R)/PI3K/Akt  and the Ras/Raf/Erk 1/2 signaling pathways have been shown to be essential for myocardial hypertrophy .
Our findings showed a loading-specific activation of these two pathways in cardiomyotubes in vitro; specifically and similarly to the uperegulation of IGF-1, only the intermediate duration (12 hrs) low strain/frequency protocol induced the activation of both signaling proteins, Akt and Erk1/2 (Fig. 5). These findings suggest that the loading-induced activation of these signaling mediators appears not to be mutually exclusive and may be depended on the loading characteristics of mechanical stretching and possibly on the stretching-induced IGF-1 upregulation. Further studies are needed to determine whether these pathways are activated through the same or different mechanosensors of cardiac muscle cells. Overall, we found that the low strain/frequency of intermediate duration stretching protocol was the more effective in inducing an anabolic/anti-apoptotic response in the differentiated cardiomyocytes.
In parallel with highlighting the anti-apoptotic/anabolic profile of the cardiac myotubes in response to different loading conditions, this study also examined the expression responses of pro-apoptotic and muscle atrophy genes to the various mechanical stimuli. While many studies have suggested potentially beneficial effects of mechanical stretching on cardiomyocytes structure and function [1, 44–46], nevertheless, excessive mechanical stimuli have been reported to induce cardiac cell apoptosis and maladaptive hypertrophy, which promote upregulation of atrophy and inflammation factors [7, 36, 47].
Various pro-apoptotic factors may potentially be involved in the myogenic program of myoblasts; FoxO is a fate decider within the myogenic lineage as opposed to an inducer of the myogenic differentiation , while Fuca inhibits cell growth and induces cell death . Moreover, muscle-specific atrophy genes, such as Atrogin-1, are considered to play an important role in driving an atrophic phenotype through the ubiquitin-proteasome pathway , although the defined mechanisms of their action remain to be fully elucidated.
Interestingly and in contrast with the anabolic signaling and IGF-1 responses, our study showed that the stretching protocol characterized by low strain/frequency for an intermediate duration resulted in decreased expression of the atrophy (Atrogin-1), pro-apoptotic (FoxO, Fuca) and inflammation-related (IL-6) genes examined. Moreover, it is worth mentioning that increasing the duration of the low strain/frequency stretching led to significant increase in the expression of Fuca, Atrogin-1 and IL-6 compared with the intermediate duration protocol (Figs. 3 and 4).
These findings suggest a multiple beneficial effect of the low strain/frequency of intermediate duration mechanical stretching, which simultaneously upregulates anabolic/survival program and downregulates muscle atrophy and pro-apoptotic factors in advanced differentiation cardiomyocytes . Moreover, our findings indicate that there might be a threshold (or range) of duration of low strain/frequency mechanical loading for the induction of beneficial or detrimental effects on cardiomyotubes [45, 47], (Fig. 2–5).
The differentiation of myoblasts into myotubes has become a model for understanding the molecular mechanisms that regulate the antagonistic phenomena of cell proliferation and differentiation. Myogenic differentiation of myoblasts is regulated by MRFs  and it has been established that MyoD is already present in the proliferating myoblasts and is involved in the myogenic determination, while Myogenin and MRF4 are expressed in a subsequent phase and are involved in the terminal differentiation of myoblasts into non-proliferating myotubes. Nevertheless, MyoD can further trigger muscle differentiation by activating the expression of myogenin and other muscle-specific genes that are key factors of the myogenic lineage progression [22, 23, 51–53]. Moreover, studies have revealed that mechanical stimuli affect the expression of these myogenic determination factors [54, 55]. Nevertheless, the specific responses of MRFs to mechanical loading in cardiomyocytes remain largely unknown.
In our study, MRFs exhibited differential responses to the various stretching protocols applied on the differentiated cardiomyotubes. Specifically, the 12-hr, low strain/frequency mechanical loading resulted in significant decrease in the expression of the late differentiation factor MRF4, while the same low strain/frequency protocol applied for a longer duration (24 hrs) led to the upregulation of MyoD and Myogenin. Interestingly, the MRFs responses to low strain/frequency loading in differentiated cardiomyocytes and, thus, the regulation of their myogenic lineage appears also to be time-dependent (Fig. 1A-C).
The effects of the higher strain/frequency for a short duration (15 min) loading protocol on signaling and gene expression responses of cardiomyoblasts found, overall, to be mild and limited, resulting only in the downregulation of the pro-apoptotic factor FoxO. Nevertheless, this appeared to be a common effect of all the stretching protocols used in the present study, regardless of their specific loading characteristics (Fig. 3A).