Intracellular Ca2+ overloading is a well-known factor contributing to apoptosis[16]. Under pathological stimuli, the highly organized network of Ca2+ signaling is disturbed, leading to uncontrolled Ca2+ accumulation. There were studies reporting mechanical stress provoked intracellular Ca2+ overloading and resultant apoptosis in various cell types[17–20]. However, the mechanism through which mechanical stimuli perturb the balanced Ca2+ influx and outflux network in myoblasts is unknown. In this study, we confirmed the feedback loop between CRT and PMCA1 was evoked by intensive mechanical stretch in myoblasts, which potentiated Ca2+ overloading and prompted apoptosis. To our knowledge, this is the first report revealing that stretch-induced intracellular Ca2+ accumulation was ascribed to the cross-talk between two critical components of Ca2+ signaling system, CRT and PMCA1.
CRT, a major Ca2+ binding chaperone inside the endoplasmic reticulum (ER), plays a fundamental role in many processes that maintain cellular Ca2+ homeostasis, such as ER Ca2+ storage and release[21], activation of store-operated Ca2+ influx[22]. It is implicated in regulation of apoptosis, with its deficiency protecting cells from apoptosis while its overexpression sensitizing cells to proapoptotic stresses[23, 24]. This is consistent with our present study, which also demonstrated that CRT overexpression and knockdown potentiated and dampened stretch-induced myoblast apoptosis to some extent. Considering the Ca2+ buffering nature of CRT, studies by others and us all agree with the proapoptotic nature of abnormally high level of CRT[25]. It has been revealed that mechanical loading (compressive or tensile) could trigger CRT expression, while mechanical unloading ameliorated it, in various cell types[26–29]. However, the mechanism through which mechanical stimuli could modulate CRT expression in these cells have not been elucidated. p38 MAPK has been manifested to be the upstream pathway of CRT induction by ER stress and hypoxic condition[30–32]. Our present proposed that stretch incurred to enhanced CRT level via p38 MAPK activation. This conclusion can be substantiated by the following evidences: 1) elevated CRT level and activation of p38 MAPK correlated well with each other in myoblasts exposed to mechanical stretching; 2) blockage of p38 MAPK by SB20350 partially suppressed stretch-elevated CRT expression.
PMCA1 belongs to the family of plasma membrane Ca2+ transport ATPases (PMCAs), which are fundamental in maintaining the intracellular Ca2+ level in the physiological range. Up to date, there are no studies exploring the effect of mechanical stimuli on cellular PMCAs level and activity. We found that PMCA1 expression and activity were both diminished in mechanically stretched myoblasts. It has been testified that transcriptional factors, such as c-myb and EGR1, were involved in transcriptional control of PMCAs expression in different cells and conditions[33–36]. Whether these factors are involved in stretching stimuli-inhibited PMCA1 level in myoblasts awaits to be explored. In contrast to their expressions, the activity of PMCAs was orchestrated via different patterns. For example, PMCAs could be auto-inhibited by binding of the inhibitory unit to the N- and A-domains, which is competed by calmodulin (CaM), the main activator of PMCAs[37]. Our study manifested that CaM mediated stretch-inhibited PMCA1 activity, evidenced by the fact that decreased CaM level correlated with reduced PMCA1 activity in stretched myoblasts, and pretreatment with CaM inhibitor further prohibited PMCA1 activity. It should be noted that caspase-3 was also able to cleave the inhibitory binding domains in PMCA proteins, thus favoring PMCAs activation[38, 39]. However, the exact role of caspase-3 in modulating PMCAs activity is still a matter of debate, with the outcome largely depending on the given cell type, stimulus and conditions[40]. In addition, the actin cytoskeleton is another important modulator of PMCAs activity[41, 42], and PMCAs can regulate actin dynamics through feedback regulations[43]. Given that mechanical stretching stimuli could undoubtably affect the actin cytoskeleton of cells, and caspase-3 was shown to be activated in stretched myoblasts in our study, it will be interesting to explore the possible involvement of caspase-3 and actin in stretch-inhibited PMCA1 activity in the future.
As we demonstrated in the present study, stretching stimuli activated two pathways in myoblasts, p38MAPK-CRT and CaM-PMCA1. The former led to raised CRT level, while the latter impeded PMCA1 activity. Intriguingly, there seemed to be connections between these two pathways. Firstly, CRT was demonstrated to be an upstream factor of CaM, with CRT knock-down causing expression of CaM to increase[44–47]. Consistently, our data extended this assumption by verifying that declined CaM level in stretched myoblasts partially resulted from CRT elevation. In this way, CRT was confirmed to negatively modulate PMCA1 activity in myoblasts under stretching stimuli. Secondly, the impaired PMCA1 activity and level inevitably caused intracellular Ca2+ level to accrue, which is a potential factor contributing to p38 MAPK activation[48–50]. Thus, PMCA1 could possibly manipulate p38 MAPK via accumulating intracellular Ca2+. This assumption was evidenced by the fact that PMCA1 overexpression or knock-down dephosphorylated or phosphorylated p38 MAPK in stretched myoblasts. Accordingly, these data implied that PMCA1 could also negatively modulate CRT expression through p38MAPK in myoblasts under stretching stimuli. Taken together, the present study proposed the bidirectional regulation between CRT and PMCA1 in myoblasts exposed to stretching stimuli, forming a vicious cycle that propelled increased CRT expression, decreased CaM level, activated p38MAPK and reduced PMCA1 activity (Fig. 8).
The Ca2+ signaling system is a highly complicated network consisting of many Ca2+ channels, buffering proteins and pumps, residing in different subcellular components such as plasma membrane, nuclei, endoplasmic reticulum and mitochondria. Normally, the intracellular Ca2+ homeostasis was coordinated by cross-talks among Ca2+ signaling components. For example, Abell et al demonstrated the cross-talk among stromal interaction molecule (STIM)-Orai Ca2+ influx pathway, Sarco/ER Ca2+-ATPase (SERCA) pumps and PMCA. Any alteration to one of them led to compensative adjustment of the other two. These intracellular negative feedback processes stabilized and maintained basal cytosolic and ER Ca2+ levels in response to extracellular stimuli, which protected cells from disturbed Ca2+ homeostasis[51]. In contrast to this, our present study indicated a completely opposite mode of Ca2+ signaling responses to mechanical stretching stimuli. We manifested a positive feedback loop between two Ca2+ signaling proteins, CRT and PMCA1, with the elevated expression of former and diminished activity of latter potentiating each other. This vicious cycle contributed to intracellular Ca2+ overloading and myoblast apoptosis under mechanical stretching.
There are two major questions remaining to be answered in our study. First, what are the primary triggering factors that initiate this vicious cycle in stretched myoblasts? We supposed that any component of this cycle could be the primary triggering point (Fig. 8). Thus, future studies should conduct mechanical stretch more briefly on cells (within seconds to minutes) and focus on the earliest changes to CRT, PMCA1, CaM and p38MAPK, in order to uncover the primary driving force of this feedback loop. It will also be interesting to investigate whether blocking this assumed primary factor could potentially prevent the vicious loop from forming.
Second, is there any other Ca2+ channels that mediate stretch-induced Ca2+ overloading in myoblasts, in addition to PMCA1? So far, members of the transient receptor potential (TRP) family (TRPV2, TRPV4, TRPC6, and TRPM7) and Piezo1/2 have been suggested as classical mechanosensitive ion channels[52–54]. However, the expressions and mechanosensitive activity of these channels are cell- and organ-specific[55]. Moreover, even in the same cell type, there existed functional shift among these channels in response to distinct stretching magnitude[56]. Therefore, our future study will elucidate whether some of these mechanosensitive channels are also involved in potentiating Ca2+ influx in stretched myoblasts. Notably, even though Ca2+ overloading was illustrated to be the consequence of CRT-PMCA1 feedback loop in our study, it could also be an alternative factor propelling this vicious loop. This is because one component of this loop, p38MAPK, could be activated by intracellular Ca2+ accumulation (Fig. 8). Accordingly, elaboration of the second question could also partially answer the first question.
In summary, the positive feedback loop between CRT and PMCA1 was activated by intensive mechanical stretch in myoblasts. Circulation of this vicious close cycle facilitated intracellular Ca2+ accumulation and resultant myoblast apoptosis under stretching stimuli.