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

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, inammatory, 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 5 th 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 inammatory (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 benecial 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 ndings 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.

cardiomyocyte growth, in both physiological and pathological conditions [8,10] can also affect their differentiation [13,14], which is driven by multiple signal transduction pathways that coordinate the balance between protein synthesis and protein degradation, or muscle growth and atrophy [18].
Speci cally, myogenic differentiation is regulated by four transcription factors, the myogenic regulatory factors (MRFs) MyoD, Myf5, Myogenin and MRF4 [19,20]; these transcriptional activators share the ability to convert various differentiated cell types to myogenic [21], while recent studies have documented that they play a similar role in cardiomyocyte myogenic differentiation [22,23]. In addition, insulin-like growth factor-1 (IGF-1) signaling has been implicated in skeletal and cardiac cell growth through the activation of extracellular signal-regulated kinases (Erk) 1/2 [24] and in the loading-induced adaptive cardiac hypertrophy through the activation of Akt (protein kinase B) [11,25,26]. Moreover, potentially differential actions of IGF-1 isoforms in cardiac myoblasts growth as well as in the myocardial repair/remodeling process have been proposed [27][28][29].
Thus, it remains a challenge to better understand the contribution of the particular inputs of such factors on cardiac cell adaptation to mechanical loading, depending on the speci c loading characteristics.
Given the complexity of the in vivo models of cardiac adaptations [33] in response to mechanical loading, ex vivo and in vitro models of muscle mechanical loading [34] applied on myocardial cells are crucial for understanding the cellular and molecular mechanisms that mediate loading-induced adaptations.
The aim of the present study was to investigate and compare the effects of various in vitro mechanical stretching protocols on signaling and gene expression responses of differentiated H9C2 cardiomyoblasts, associated with anabolic/atrophy, pro-apoptotic and in ammation-related factors that are involved in their myogenic lineage. We set the hypothesis that the signaling and expression responses elicited would differ depending on the loading characteristics of the protocols used, thus indicating loading-speci c, detrimental or bene cial effects on cardiomyotubes in vitro.

H9C2 Cell Culture
The H9C2 cell line of embryonic rat heart-derived ventricular cells was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured as previously described [25]. Brie y, cells grown in Dulbecco's modi ed Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin at 37 o C in a humidi ed atmosphere of 5% CO 2 in air, while medium was changed every other day. The H9C2 cardiomyoblasts were seeded onto 6-well exible-bottomed culture plates coated with Collagen I (Flex I Culture Plates Collagen I; Flexcell International, Hillborough, NC, USA) and maintained in growth media until 70-80% con uent, then switched to differentiation media (2% horse serum, 1% of penicillin/streptomycin in DMEM). Cardiomyoblasts were allowed to differentiate into multinucleated myotubes for a 5-day period during which media was changed every other day before stretching, as described below.
Cardiomyocyte mechanical loading Differentiated H9C2 (myotubes) were stretched using the Flexcell FX-4000 strain unit (Flexcell International) that produces isotropic two-dimensional (biaxial) strain of cells cultured on the exible surface (silicone membrane) of the culture plates, again at 37 °C in a humidi ed atmosphere of 5% CO 2 .
Brie y, cardiomyotubes were subjected to three different stretching protocols: a) 12.7% elongation (strain), at a frequency of 0.5 Hz for 15 min (high strain/short duration protocol); b) 2.7% elongation, 0.25 Hz for 12 h (low strain/intermediate duration protocol), or c) 2.7% elongation, 0.25 Hz for 24 h (low strain/long duration protocol). It is worth mentioning that in order to use experimental approaches the more physiological possible, in each protocol the pattern (waveform) of the tension applied on the cardiomyocytes in each stretching cycle was mimicking the pressure uctuations of a heart beat in vivo.
Cell lysis and RNA extraction Cell extracts were obtained by cell lysis using NucleoZOL (Mecherey-Nagel, German) 12 hrs after the completion of the stretching protocol, while control (non-stretched) myotubes were also harvested 12 hrs after the end of each stretching protocol used for the stretched cardiomyotubes. Total RNA was isolated from the lysates according to the manufacturer's recommendations. The extracted RNA was dissolved in RNAases free water (Invitrogen) and the concentration and purity were determined spectrophotometrically (ThermoNanodrop 2000) by absorption at 260 and 280 nm. Integrity of total RNA was con rmed by visual inspection of the electrophoretic pattern of 18S and 28S ribosomal RNA in ethidium bromidestained 1% agarose gels under ultraviolet (UV) light. The total RNA samples were stored at − 80 °C until further analyses for the determination of the mRNA levels of the genes of interest by reverse transcription and semi-quantitative real-time PCR procedures.

Reverse Transcription and Real-time PCR
Total RNA from each sample was used for the production of single-stranded cDNA by reverse transcription using reverse transcriptase ProtoScript II (NEB) and the resultant cDNAs were utilized in realtime PCR. More speci cally, for the reverse transcription 1 µg of total RNA from each sample was mixed with random primers mix (300 ng/reaction), oligod(T) 23 VN (300 ng/reaction) and nuclease-free water in a total volume of 8 µl, heated at 65 o C for 5 min and then placed on ice. Next, the samples were mixed with 10 µl ProtoScript II Reaction Mix and 2 µl Protoscript II Enzyme mix and incubated consecutively at 25 o C for 5 min and at 45 o C for 1 hour according to manufacturer's recommendations. At the nal step of the reverse transcription, the samples were heated at 80 o C for 5 min, to inactivate the reaction, and stored at -20 o C.
The primer set sequences used for the speci c detection of IGF-1 isoforms (IGF-1Ea, IGF-1Eb), myogenic regulatory factors (MyoD, Myogenin, MRF4), pro-apoptotic (Foxο, FUCA), atrophy (Atrogin-1) and in ammation-related (IL-6) factors are shown in Table 1. To prevent detection of genomic DNA, the primer sets were designed to lie within different exons while, particularly, each set of primers for the detection of the IGF-1 isoforms was speci c to detect only one IGF-1 transcript variant. Each PCR reaction contained 50 ng of cDNA, 12.5 µl SYBR green master mix, 0.4 µM of each primer, and nuclease free water to a total volume of 20 µl. The real-time PCR parameters were the following: initial denaturation at 95 °C for 5 min followed by 40 cycles of 30 sec at 95 °C, 30 seconds at 62 °C for annealing, and 30 seconds at 72 °C for extension. Transcript levels of the genes of interest were assessed by automatically calculating the threshold cycle (Ct) as the number of cycles at which the measured uorescence exceeds the threshold for detection. To normalize the amount of total RNA present in each PCR reaction and the mRNA expression (relative quanti cation-dCt) of the genes of interest, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as housekeeping gene (internal standard). Each sample was analyzed in duplicate, and the resulting data were averaged. A melting curve (Tm) was also generated by the Bio-Rad iQ5 Real-Time PCR Detection System software after the nal cycle for each experimental sample, by continuous monitoring the Bio-Rad SYBR uorescence throughout the temperature ramp from 70 °C to 95 °C. The speci city of the primers for the corresponding transcript was also con rmed by the melting curve analysis of samples, where there was only one melting curve for each sample, while electrophoretic analysis of the real-time PCR products further veri ed the speci city of the transcript of each gene of interest. Control for speci city included cDNA-free and template-free reactions. Table 1 The sequence of the speci c sets of primers used for RT-PCR analyses.

Protein extraction and Immunoblotting analysis
Total proteins were extracted from H9C2 myotubes as previously described [27]. Brie y, cardiomyotubes were washed with ice-cold PBS before lysing in 150 µL of RIPA buffer (Cell signaling) supplemented with protease and phosphatase inhibitors cocktails (Cell signaling). Lysates were incubated on ice under shaking for 20 minutes in order to ensure complete lysis of the cells, centrifuged at 15,000 rpm for 20 min at 4 °C and the supernatants retained. Protein content was determined using a BCA protein assay kit (Thermo Scienti c). Samples were stored in aliquots at − 80˚C until Western blot analysis as previously described [27,35]. Brie y, equal amounts of protein extracts ( Cruz Biotechnology) in TBS-T containing 2.5% BSA, for 1 h at room temperature. The expected bands were visualized by exposure of the membranes to x-ray lm after incubation with an enhanced chemiluminescent substrate for 3 min (ECL Supersignal west picoThermo scienti c). Glyceraldehyde 3phosphate dehydrogenase (GAPDH) (1:2,000 dilution; Santa Cruz Biotechnology) was used as an internal standard to correct for potential variation in the protein loading and to normalize the protein measurements on the same immunoblot. Band intensity was then semi-quanti ed using the Image J software.

Statistical analysis
One-way analysis of variance (ANOVA) with Dunn's Multiple Comparison post-hoc test was used for statistical analyses of gene expression and Student's t test for signaling data analyses, utilizing GraphPad Prism 5. All experiments were performed in triplicate and data are presented as mean ± standard error of the mean (SE). The level of statistical signi cance was set at p < 0.05.

Myogenic Regulatory Factors
In order to investigate the potential effects of mechanical loading on the myogenic lineage of differentiated cardiomyoblasts, we examined the expression levels of both early (MyoD) and late (Myogenin, MRF4) differentiation factors in cardiomyocytes. We found that only the low frequency (0.25 Hz), low elongation (2.7%) of longer durations stretching protocols induced signi cant changes in the expression levels of those MRFs compared either to control (no stretch) or the higher elongation/frequency and short duration condition (Fig. 1A-C). Moreover, the 24 hrs stretching resulted in a signi cant upregulation of MyoD and Myogenin compared to the intermediate duration (12 hrs) stretching protocol (Fig. 1A, B). Interestingly, the 12 hrs protocol induced a signi cant decrease in the expression of MRF4 compared to both the control and the higher elongation/frequency and short duration condition (Fig. 1C).

IGF-1 isoforms
IGF-1 is a key factor in the regulation of cardiomyocytes development and growth; thus, we examined the effects of different stretching protocols on the particular expression of IGF-1 isoforms in differentiated cardiomyocytes. Interestingly, a similar pro le was revealed for both IGF-1 isoforms regarding their responses to the different mechanical loading protocols. We found that the low frequency (0.25 Hz), low elongation (2.7%) of longer durations stretching protocols induced signi cant increases in the expression levels of both isoforms compared to the higher elongation/frequency and short duration condition ( Fig. 2A, B). However, only the 12 hrs loading protocol induced a signi cant increase in the expression of IGF-1Eb isoform compared to control (Fig. 1B).

Pro-apoptotic factors
In parallel with the effects of mechanical loading on the anabolic/survival factor IGF-1, we also examined the effects of different stretching protocols on the expression of apoptosis-related factors in cardiomyotubes. It was found that all three mechanical loading protocols resulted in a similar, signi cant downregulation of FoxO compared to the control condition (Fig. 3A). Moreover, the intermediate duration (12 hrs) protocol induced a signi cant decrease in the expression levels of Fuca compared to the long duration condition (Fig. 3B).

Muscle atrophy and in ammation-related factors
We further examined the loading-induced regulation of Atrogin-1 and IL-6 in cardiomyotubes. Similarly to the responses of the pro-apoptotic factor Fuca to the various mechanical loading protocols, the 12 hrs stretching of cardiomyoblasts resulted in a signi cant downregulation of the cardiac and skeletal muscle atrophy factor Atrogin-1 and the in ammation factor IL-6 compared to the 24 hrs duration stretching as well as to control condition (Fig. 4A, B). Particularly for IL-6, the long duration (24 hrs) and low elongation (2.7%)/frequency (0.25 Hz) protocol induced signi cantly higher responses compared to the short duration (15 min) and higher elongation (12.7%)/frequency (0.5 Hz) stretching protocol (Fig. 4A).

Activation of the signaling proteins Akt and Erk 1/2
Along with the various gene expression responses to mechanical stretching of cardiomyotubes, we also investigated the effects of the different loading protocols on the phosphorylation of important intracellular signaling mediators. Interestingly, only the intermediate duration (12 hrs) and low elongation (2.7%)/frequency (0.25 Hz) stretching protocol induced the activation of both signaling proteins, Akt and Erk1/2, while a tendency towards the downregulation of phosphorylation of these proteins was observed after either the short duration (15 min) and higher elongation (12.7%)/frequency (0.5 Hz), or the long duration (24 hrs) and low elongation (2.7%)/frequency (0.25 Hz) stretching protocol (Fig. 5A, B).

Discussion
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-speci c, detrimental or bene cial effects on cardiac myotubes, depending on the loading characteristics of the different protocols. The expression of myogenic, anabolic, atrophy, pro-apoptotic and in ammatory 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 ndings 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 in ammation-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][28][29]40]. To the authors' best knowledge, this is the rst study investigating the distinct expression pro les of IGF-1 isoforms following mechanical loading of cardiac myotubes, in vitro. Interestingly, our ndings 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 ndings 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 identi cation, 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 [42], while both the IGF-1 receptor (IGF-1R)/PI3K/Akt [43] and the Ras/Raf/Erk 1/2 signaling pathways have been shown to be essential for myocardial hypertrophy [24].
Our ndings showed a loading-speci c activation of these two pathways in cardiomyotubes in vitro; speci cally 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 ndings 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 pro le 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 bene cial effects of mechanical stretching on cardiomyocytes structure and function [1,[44][45][46], nevertheless, excessive mechanical stimuli have been reported to induce cardiac cell apoptosis and maladaptive hypertrophy, which promote upregulation of atrophy and in ammation 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 [48], while Fuca inhibits cell growth and induces cell death [32]. Moreover, muscle-speci c atrophy genes, such as Atrogin-1, are considered to play an important role in driving an atrophic phenotype through the ubiquitin-proteasome pathway [49], although the de ned 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 in ammation-related (IL-6) genes examined. Moreover, it is worth mentioning that increasing the duration of the low strain/frequency stretching led to signi cant increase in the expression of Fuca, Atrogin-1 and IL-6 compared with the intermediate duration protocol (Figs. 3 and 4).
These ndings suggest a multiple bene cial 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 [17]. Moreover, our ndings indicate that there might be a threshold (or range) of duration of low strain/frequency mechanical loading for the induction of bene cial 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 [50] 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-speci c genes that are key factors of the myogenic lineage progression [22,23,[51][52][53]. Moreover, studies have revealed that mechanical stimuli affect the expression of these myogenic determination factors [54,55]. Nevertheless, the speci c 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. Speci cally, the 12-hr, low strain/frequency mechanical loading resulted in signi cant 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 speci c loading characteristics (Fig. 3A).

Conclusions
Cardiomyocytes, as skeletal muscle cells, are mechanosensitive and respond to mechanical signals in order to maintain their homeostasis and adapt to external loading. The development of in vitro models of cell mechanical loading can greatly contribute to shed more light on the cellular and molecular responses of cardiac muscle cells to loading. The present study demonstrated that varying the characteristics of mechanical loading (i.e., strain, frequency, duration) applied on advanced differentiation cardiomyocytes in vitro resulted in different effects on their myogenic lineage, indicating speci c features of loading for regulating the anabolic/survival program in these cells. These ndings may be a valuable resource for developing more focused in vitro experimental designs to characterize the particular inputs of speci c factors and mechanotransduction pathways on cardiac cell adaptation to mechanical loading.

Con icts of Interest
The Authors declare no con icts of interest.
Authors' contributions