How Physical Stress Affects Mitochondrial Recycling in Older Muscle Cells

Cell survival and adaptation in response to cellular stress need high-energy production, which is achieved via the function of organelles called mitochondria [1,2]. These cellular organelles have the capacity to change their function and morphology in response to different stimuli, originating both from within, as well as outside the cell [3]. Mitochondria are synthesized anew, arising from the growth and division of pre-existing mitochondria through the process known as mitochondrial biogenesis. This process requires an increase in cellular respiration, metabolism, and ATP production [4,5], while mitochondrial selective clearance is realized through autophagic degradation, known as mitophagy [6]. The interaction and regulation between the opposing processes of mitochondrial biogenesis and mitophagy are essential for the cellular adaptation to environmental and intracellular signals, e.g., mechanical loading and metabolic stress in skeletal muscle cells [1,7].

Mitochondrial biogenesis and mitophagy are balanced through the opposing processes of fusion (elongation) and fission (division) [8]. Specifically, fusion promotes mitochondrial DNA (mtDNA) complementation and thus the repair of defective mitochondria, while fission removes those parts of mitochondria that have been damaged, preventing major wasting of the mitochondrial network in its entirety [8,9]. The specific part of the mitochondrion that has been compromised is separated from the rest and is marked via ubiquitylation [10]. This works as a signal for mitophagy to commence through activation of mitochondrial autophagic receptors, which in turn forms autophagic membranes [11]. These membranes encase the damaged parts of the mitochondrion (or an entire mitochondrion), creating what is known as an autophagosome, which will fuse with a lysosome, finally leading to mitochondrial degradation [11,12].

In skeletal muscle, the transition from each of these steps to the next, as well as the molecular mechanisms that induce the individual processes, have not yet been decrypted [7]. Moreover, while several mechanisms regulate quality control in mitochondria, aging seems to adversely affect the interplay between mitochondrial biogenesis and mitophagy the most [13] (Figure 1). The accrual of protein damage during aging seems to functionally impair the mitochondria in skeletal muscle. When multiple damaged or dysfunctional mitochondria are accumulated in the aging muscle, mitophagy may become impaired [14,15]. Various studies on aged skeletal muscle have revealed higher levels of fission (Fis-1, Opa-1) compared to fusion proteins (Mfn1, Mfn2, and Drp-1) [16]. To compensate for this age-associated increase in the fission process, it has been suggested that mitochondria elongate due to intrinsic defects for promoting fusion [3]. Additionally, the mitochondrial dysfunction observed in aged skeletal muscle is not entirely caused by impaired mitophagy but also as a result of reduced mitochondrial biogenesis, as these two cellular processes are inextricably linked [1,17].

The impact that aging has on skeletal muscle mitophagy pathways has only scarcely been examined [18]. It could be assumed that the aging-induced mitochondrial dysfunction and fragmentation would result in increased mitophagy signaling to promote the removal of these dysfunctional organelles, and previous studies have reported increased expression of mitophagy proteins, such as Pink1 and Parkin, in aged muscle. The status of these proteins could indicate whether the mitophagy cascades are activated; nevertheless, their expression has not yet been examined in senescent muscle cells [19,20,21]. In addition, although the Pink1–Parkin pathway remains the most studied ubiquitylation mechanism in mitochondria, its necessity for skeletal muscle mitophagy has not been defined [6]. Specifically, Parkin is a basic component of signaling axis both in mitophagy and mitochondrial biogenesis, but limited evidence supports the existence of Pink1–Parkin-mediated mitophagy in skeletal muscle [10]. Nevertheless, a decreased Parkin expression and ubiquitylation in aged mouse muscles has been reported, suggesting that the Pink1–Parkin mitophagy response could occur in senescence [10].

Mitochondrial fission and mitophagy coordination are suggested to be regulated by AMPK [22]. Early studies have also shown that ULK-1 phosphorylation (Ser 555), mediated by AMPK, facilitates mitophagy [23]. When AMPK is inactive, basal mitophagy may occur, indicating that other, currently unknown, pathways could regulate mitophagy as a response to various stimuli [7]. In addition, AMPK activates both Parkin and Pink1 for mitophagy induction but not in C2C12 skeletal muscle myotubes. Despite the fact that the Pink1–Parkin pathway induces mitophagy, it is not necessary for the activation of AMPK-mediated mitophagy in skeletal muscle [10,21,24]. Lately, AMPK has emerged as an important mitophagy regulator in skeletal muscle, as its activation enhances mitochondrial fission before promoting the engulfment of damaged/dysfunctional mitochondria by the autophagosomes [22,25]. In mitochondrial biogenesis, the activation of AMPK appears to act as a promoter via the phosphorylation of PGC-1α [4].

During aging, PGC-1α has a major role in crosstalk modulation of signaling pathways that control mitochondrial quality, and it also regulates mitochondrial biogenesis [13]. For instance, the expression of Mfn2, a major player in the PINK–Mfn2–Parkin axis, is controlled by PGC-1α [26]. This is important because the mitophagic process is significantly impacted by alterations in mitochondrial dynamics. A disruption in mitochondrial homeostasis due to a PGC-1α-reduced expression could lead to increased ROS production, FOXO nuclear sequestration, and mitophagy during aging [13,27].

In addition, BNIP3L/NIX, an outer membrane protein, functions as a receptor for mitophagy and has been found to play a crucial role in receptor-mediated mitophagy, facilitating muscle health [28,29,30]. Indeed, deficiencies in this protein in muscle cells have been associated with increased inflammation and an impairment in mitochondrial function during aging [30,31,32]. Moreover, in the absence of Pink1/ Parkin, BNIP3L/NIX appears to be a key factor in mitochondrial clearance during muscle cell differentiation [33].

Mitophagy also plays a major role in repairing/forming new muscle cells through myogenesis and muscle regeneration [1]. Myogenesis is induced through different signaling pathways regulating a variety of factors, particularly the myogenic regulatory factors (MRF’s), Myf5 and myogenin, which together control the growth and development of skeletal muscle [18,34]. Interestingly, mitophagy and myogenesis are important muscle cellular processes, quite possibly associated, and they have both been shown to become impaired during the aging process [1].

Utilizing in vitro models to simulate senescence in skeletal muscle cells could greatly improve our knowledge regarding the cellular and molecular aspects of aging-induced alterations [35]. Moreover, mechanical loading is able to cause adaptations in skeletal muscle cells, highlighting muscle’s plasticity and its ability to alter its mass and contractile phenotype [14,36] (Figure 1). While mechanical stimuli have been shown to improve the impaired myogenesis in aged muscle [37], their effect on the myogenesis-associated mitophagy and the dysfunctional degradation of mitochondria associated with aging (mitophaging) is unclear [38]. Hence, in vitro mechanical loading models in aged muscle cells can be of particular importance for investigating the role of mechanical stimuli in mitophaging [36,39,40]. Therefore, the aim of this study was to examine the effects of mechanical stretching on mitophagy and myogenic potential in aged myoblasts, in vitro.

This study investigated the effect of mechanical loading on the process of mitophagy and myogenic regulation in senescent myoblasts, in vitro. Our group has previously characterized how aging affects myogenic lineage [42], as well as examined the mechanical loading protocol(s) that most effectively induces myogenic differentiation and survival in these cells [37]. However, less was known concerning the influence of mechanical loading on myogenesis- and aging-associated mitophagy (mitophaging) [38]. Herein, we found that implementing a specific mechanical stretching protocol, which has previously been characterized as high-strain loading [37], did induce significant alterations in multiple mitophagy-related factors, modulating mitophagy signaling pathways, mitochondrial biogenesis-associated regulators, and early differentiation markers in aged myoblasts, while it downregulated these cellular processes in the control cells.

In the signaling of mitophagy, AMPK possesses a distinct regulatory function to maintain control of mitochondrial quality in muscle cells [46]. On the other hand, dependent on AMPK activity and essential for targeting the damaged mitochondria to lysosomes, p-ULK1 is a critical factor in mitophagy process in skeletal muscle [47]. The present study revealed the downregulation of p-ULK1 and AMPK in the control (non-senescent) cells and the induction of previously undetected expression/activation of these proteins in the aged cells, as a result of the loading protocol used. These findings suggest that mechanical loading modulates key regulators involved in mitophagy initiation signaling, indicating the possible effects this loading exerts on mitophagy signaling. Interestingly, these effects appear to be beneficial for the mitophagy pathway in the aged cells and detrimental in the non-senescent cells.

Similarly, a significant reduction in the expression of the mitophagy receptor BNIP3L/NIX, which mediates the clearance of damaged mitochondria [28], and of Parkin, which likewise facilitates the degradation of damaged mitochondria [19], were observed in the control cells, along with an increased expression of Parkin and the detection of the previously undetected BNIP3L/NIX receptor in the aged cells. The increase in Parkin expression in the aged cells is noteworthy, since this factor also plays an important role in basal mitophagy [33,48]. Overall, the same pattern of response to mechanical loading, as in mitophagy signaling, was further observed with the aged cells exhibiting upregulation of these crucial for mitophagy activation factors and the control cells showing a decline in the expression levels of the same proteins, confirming that the specific loading protocol used differentially affected mitophagy-associated pathways in the aged cells compared to the control cells.

Moreover, PGC-1α, a key regulator of mitochondrial biogenesis [13], was shown in this study to also respond to mechanical loading, with increased expression in the aged cells and its downregulation in the control cells, providing further evidence of a loading-specific differential response of the aged cells compared to the controls. These findings suggest that high-strain loading can induce mitochondrial biogenesis in senescent muscle cells while diminishing it in control cells. Overall, it can be postulated that the cell stretching protocol utilized was associated with the activation of signaling molecules implicated in mitophagy initiation, induction, and mitochondrial biogenesis, thus facilitating a possibly beneficial mitophagy response and subsequently mitochondrial regeneration by augmenting mitochondrial biogenesis in the aged cells but generating a detrimental effect in mitochondrial renewal by negatively impacting mitophagy and mitochondrial biogenesis pathways in the control cells.

In addition, the present study examined the effects of mechanical loading on myogenic differentiation by checking the mRNA expression of two key muscle cell differentiation factors responsible for controlling muscle growth and development, the MRFs Myf5 and myogenin [18,34]. It was found that, while the early differentiation factor, Myf5, showed an augmented expression in response to mechanical stretching in the senescent cells compared to their non-stretched counterparts, the opposite response was observed in the control cells, revealing that the stretching protocol employed may also positively influence muscle cell differentiation in senescence whilst downregulating this process in control cells. The late differentiation factor, myogenin, presented a reduced expression in both the control and the aged myoblasts after mechanical loading, implying that the high-strain protocol implemented could disturb the completion of myogenic differentiation not only in the context of senescence, but also in non-senescent cells. It should be emphasized that these observations were obtained in proliferating myoblasts under growth medium conditions and therefore reflect modulation of myogenic regulatory pathway rather than differentiation outcomes.

Overall, the aforementioned findings indicate that mechanical loading can indeed affect mitophagy in both control and aged myoblasts by influencing the expression of key factors that regulate the mitophagy activation and signaling. Furthermore, mechanical loading appeared to also have an effect on mitochondrial renewal, as it impacts the expression of a major regulator of mitochondrial biogenesis. It has been previously reported that aging diminishes muscle cell sensitivity to mechanical stimuli, reducing the efficiency of mitophagy [49,50]. On the other hand, non-senescent muscle cells are more sensitive to mechanical stimuli, and high amounts of loading can negatively affect mitophagy [47]. Intriguingly, in this study, the manifestations of mechanical loading in the regulation of all the factors examined indicate that a high-strain loading protocol is potent enough to enhance mitophagy in aged cells, but it downregulates it in control cells. It therefore could be assumed that the protocol used unfavorably altered mitophagy-related pathway responses in the control cells while enhancing them in aged cells, suggesting a differential response of myoblasts to mechanical loading dependent on whether cellular senescence has occurred. Similarly, the high-strain mechanical loading exerted different effects on myogenic potential of senescent vs. control cells, inducing beneficial effects on early-stage differentiation in aged myoblasts and detrimental effects in control myoblasts, while disrupting late-stage myogenic differentiation regardless of cell senescence.

A limitation of the present study is that mitophagy and mitochondrial biogenesis were assessed at the level of signaling and protein expression rather than through direct measurement of mitophagic flux or mitochondrial turnover. Therefore, the present findings reflect a modulation of mitophagy-related regulatory pathways rather than direct quantification of functional mitophagy activity. Future studies employing flux-based assays and morphological approaches will be required to further validate the functional consequences of the observed molecular changes. Another limitation is the use of deformable collagen-coated, flexible-bottomed culture plates, required for cyclic mechanical loading. Due to the nature of the deformable membranes of those plates, high-resolution fluorescence imaging or ultrastructural transmission electron microscopy analysis could not be performed within the same experimental setup.

Mitochondria produce the necessary energy for cell survival and adaptation and have been shown to respond to intracellular or extracellular stimuli. The cell’s fate is determined by the interaction between mitochondrial biogenesis and selective clearance known as mitophagy. Aging is known to adversely affect both mitophagy (mitophaging) and biogenesis in muscle cells; however, mechanical stimuli have been shown to cause adaptations in skeletal muscle and improve myogenesis, though their effects on myogenesis-associated mitophagy remain inconclusive. This study utilized an in vitro mechanical loading protocol to assess the mitophagy responses in control and senescent myoblasts and revealed a differential modulation of mitophagy signaling and initiation, as well as mitochondrial biogenesis proteins dependent on cellular senescence. The findings of the study indicate that mechanical loading influences key molecular regulators associated with impaired mitophagy and early differentiation in aged myoblasts, as suggested by mitophagy initiation and the promotion of mitochondrial biogenesis in these cells, in contrast to the downregulation of mitophagy and myogenesis in the control myoblasts, indicative of loading-specific differential responses of these cells compared to the aged cells. These findings might serve as a valuable source for further research into skeletal muscle mitophaging and the development of different experimental models for studying the effects of mechanical loading in senescent skeletal muscle mitochondrial function.