We examined the phosphorylation state of desmin in healthy people and tested whether this can be specifically influenced. More concretely, we have investigated whether acute resistance exercise and continued resistance training will impact desmin phosphorylation and as a functional consequence also affect desmin cleavage. We showed that: 1.) Desmin phosphorylation at several sites is a constitutive state in healthy muscle. 2.) This phosphorylation state can transiently be specifically influenced by acute resistance exercise, resulting in a dephosphorylation of desmin. 3.) The acute exercise, and most likely the dephosphorylation of desmin caused by it, renders the protein less prone to cleavage. 4.) Chronic contractile stress induced by resistance training increases total desmin content, augments the acute exercise-induced dephosphorylation and alters the baseline phosphorylation state of the protein differentially, i.e.: tunes up pDesS31, while downregulating pDesS17 and pDesT60.
Desmin is constitutively phosphorylated in healthy human skeletal muscle.
Before, the phosphorylation of desmin was studied primarily in vitro [26, 34, 44] and in a pathophysiological context such as ischemic heart failure [1, 11, 44, 56] or due to denervation and catabolic states [16, 67]. In these contexts, increased phosphorylation of desmin is associated with destabilization or depolymerization of the desmin IF-system, as well as the formation of amyloid-like oligomers [1, 6, 12, 56]. Thus, especially pDesS31, but also the other investigated sites like S60, T17, as well as T76/77, were regarded as hallmarks of pathophysiology or diseased muscle, respectively [1, 56, 68], with a focus on cardiac [1, 10] or animal skeletal muscle [67].
Here we show that phosphorylation of desmin occurs regularly in healthy human skeletal muscle, a highly dynamic tissue undergoing constant remodeling, even in the absence of stress. According to Agnetti et al. [2], the desmin-IF system is subject to this ongoing remodeling process, too. This involves constant depolymerization and reconstruction by newly synthesized monomers, which may contribute to IF integrity and cellular homeostasis. Furthermore, mitochondrial trafficking, an ongoing process in muscle, depends on desmin-IF disassembly, driven by pDesS31 [27]. Hence, the constitutive PTM of desmin might facilitate this dynamic exchange.
However, it cannot be excluded that the degree of phosphorylation in diseased or aged skeletal muscle is notably higher than what we have seen here in healthy muscle. Indeed, some studies performed on failing hearts of dogs and humans [1] and in mouse skeletal muscle due to denervation and fasting [67], suggest this. Yet, especially for human skeletal muscle, there is a lack of data, and this needs to be elucidated in upcoming research. Nevertheless, our observations provide a new perspective on the PTM of IFs, considering its role under physiological conditions.
Acute resistance exercise is a tool that can transiently modulate desmin phosphorylation.
Despite the growing evidence for the relevance of desmin phosphorylation in muscle cell or fiber homeostasis, to date, there is no known strategy to influence desmin phosphorylation in vivo, according to Agnetti et al. [2]. However, information on how to influence desmin phosphorylation or dephosphorylation may be a promising strategy to counteract muscle wasting. Here, we demonstrate that acute resistance exercise results in a transient decrease in phosphorylation or dephosphorylation of desmin. To our knowledge, we are the first to show a possibility of specifically intervening in the regulation of specific phospho-sites of desmin-IFs.
Phosphorylation of desmin preferentially occurs in its head domain, and an overall increase in phosphorylated desmin is associated with loosening of the IFs structure, making desmin accessible for ubiquitination, calpain-dependent cleavage, and degradation [2, 5, 6]. In the present study, the two serine sites 31 and 60 were sensitive to acute loading. Previously, the sites were associated with myoblast fusion and cell division [39, 68], IF-disassembly[27] and amyloid-like oligomer formation [1, 56], further highlighting their influence on IF stability by modulating desmin reorganization [44, 47].
While systematic RT unequivocally has positive effects on muscle function, mass, and overall health [20, 31, 32], due to the high mechanical strain, acute resistance exercise can also induce myofibrillar damage, especially in an unadapted state and when eccentric contractions are conducted [24, 42, 43]. Such myofibrillar damage is characterized by Z-line streaming, membrane damage, and disrupted cytoskeletal organization [22, 23, 61]. As desmin-IFs play a crucial role in orchestrating the cytoskeletal integrity as a main strain-transmitting component, it is not surprising that the protein is affected by acute, unaccustomed resistance exercise. In this context, a loss of desmin immunostaining has been noted following eccentric loading of the tibialis anterior muscle in rats[7, 38] and rabbits [43], indicating degradation of the protein. Thus, our finding of a reduction in desmin phosphorylation one hour post-acute RE was somewhat surprising, as one would expect an increase in phosphorylated desmin as a prerequisite for protein degradation, based on the aforementioned studies. The opposing findings may imply the following: It is possible that a reduction in phosphorylation as a result of acute loading represents a mechanism that transiently protects the IF system from destabilization during phases of increased stress. This could be suggested by the fact that in the aforementioned studies, an initial loss of immunostaining for desmin was observed very soon after the end of the exercise [43], but intensified over time and peaked after 12 hours [7]. Nevertheless, in order to gain more clarity on this, desmin’s PTM should be examined more closely in a time window of about 24 hours in future studies.
Furthermore, it raises the question of the molecular mechanism for the reduction in phosphorylated desmin signal following acute resistance exercise. Is it a result of decreased phosphorylation or increased dephosphorylation? Recently, Aweida et al. [6] demonstrated that desmin is a substrate of glycogen synthase kinase 3-β (GSK3-β). GSK3-β-dependent phosphorylation of desmin was shown to prime the protein for depolymerization and subsequent myofibril disassembly or atrophy. Conversely, inhibition of GSK3-β prevented desmin phosphorylation, depolymerization, and atrophy [5, 6]. Although we did not assess GSK3-β activity or phosphorylation in the present study, our findings of reduced phosphorylation following acute resistance exercise can be explained by previous studies covering GSK3-β regulation as a result of exercise. These studies show a decrease in GSK3-β activity following submaximal and maximal endurance-type exercise, downhill running, and passive stretch in rodent [3, 4, 45, 59] as well as human skeletal muscle [58]. GSK3-β is constitutively active and can be deactivated upon phosphorylation at serine 9 by protein kinase B (PKB/Akt) [18, 64]. PKB/Akt activity, which relies on phosphorylation at T308 and S473, is also sensitive to exercise-induced stress [25, 28, 58, 59]. It is conceivable that desmin undergoes perpetual phosphorylation by constitutively active GSK3-β. Subsequently, exercise-induced activation of the PKB/Akt axis may phosphorylate and deactivate GSK3-β. Consequently, this cascade is suggested to influence or decrease desmin phosphorylation, potentially through a more dominant influence of phosphatase activity.
Taken together, the discussed signaling connections establish a plausible link between physical activity, the activation/deactivation of key kinases, and the observed reduction in desmin phosphorylation following acute resistance exercise. However, empirical evidence is needed to substantiate these theoretical connections in future studies.
The acute exercise-induced dephosphorylation of desmin renders the protein less prone to cleavage.
Increased desmin phosphorylation is associated with a higher susceptibility of the IFS to calpain 1-dependent cleavage, and a well-established mechanism proposes the following sequence of events: Initial phosphorylation of desmin by GSK3-β is necessary for facilitating Trim32-dependent ubiquitination [15]. Subsequently, ATAD1 binds phosphorylated and ubiquitinated desmin IFs, along with its interaction partners PLAA and UBXN4, leading to the dissociation of IFs. This process extracts desmin from the tight IF-network, exposing calpain 1-specific cleavage sites on desmin. Consequently, calpain 1 cleaves desmin, rendering it accessible to the ubiquitin-proteasome system for degradation [5].
When desmin phosphorylation is increased, the protein becomes more prone to cleavage and degradation. Conversely, decreased desmin phosphorylation should render it less susceptible to degradation. Our findings support this notion: Muscle previously subjected to acute resistance exercise, displaying reduced desmin phosphorylation, exhibited lower susceptibility to cleavage compared to muscle at rest, which exhibited higher levels of phosphorylated desmin.
Desmin fragmentation or cleavage has been extensively studied, with Nelson and Traub [50] providing a detailed description of the characteristic fragment band pattern of purified desmin cleaved by calpain 1. Notably, the N-terminal part is particularly vulnerable and is cleaved rapidly by Ca2+-dependent proteases, resulting in a small and a large fragment with rod- and tail-domain comprising approximately 49 kDa [50]. Interestingly, the small 9 kDa N-terminal fragment is crucial for the assembly of 10 nm desmin filaments and for their binding to nucleic acids [50, 65]. This indicates that the removal of this part by the protease is not just a matter of desmin turnover but rather serves the regulation of specific cell functions [50]. The fragmentation, particularly the approximately 49 kDa fragment, has been observed in numerous studies [6, 10, 13, 56, 67].
By demonstrating that acute resistance training reduces desmin phosphorylation, leading to decreased susceptibility of the protein to cleavage of the N-terminal fragment, we establish a link between contractile activity, its influence on desmin modification, and the corresponding functional consequence. However, it is essential to consider several factors: First, we have not confirmed whether cleavage occurs in vivo or is solely a consequence of cell lysis. Second, it remains unclear whether in our study, in fact the head domain is specifically targeted for cleavage. While our assumptions align with previous evidence, we lack experimental confirmation. Nevertheless, the lack of recognition of the 49 kDa band by the phospho-antibodies raised against the N-terminal domain lends support to our hypothesis.
Resistance training not only enhances acute exercise-induced dephosphorylation of desmin, but also modifies the baseline phosphorylation state of the protein and increases its content.
As previously discussed, acute unaccustomed exercise can have detrimental effects on skeletal muscle, while systematic training unequivocally has positive adaptive effects. To validate the effectiveness of our training regimen, we demonstrated an increase in muscle force and fatigue resistance (Fig. 5A and B), without affecting muscle fiber type composition or size (Fig. 5C and D). Concurrently, we observed a significant upregulation in total desmin content (Fig. 7B). This increase following resistance training has been previously reported by our group [35] and others [21, 53, 69, 70]. Notably, an elevated desmin content may contribute to enhanced resilience of skeletal muscle fibers against mechanical stress [35]. Moreover, the increase in desmin content, despite the absence of fiber hypertrophy, has been reported previously [35]. This dynamic nature of desmin suggests that it surpasses most other proteins involved in fiber hypertrophy. While loss of desmin is considered a prerequisite for muscle atrophy, an increase in desmin content may not necessarily precede muscle hypertrophy. This was demonstrated by Joanne et al. [37], who showed that mice subjected to one month of resistance training, regardless of desmin knockout or wild-type status, exhibited similar increases in muscle weight. Thus, muscle hypertrophy can occur independently of desmin content increase, and vice versa, suggesting that these adaptive mechanisms are not interdependent.
In this study, we demonstrated that desmin is phosphorylated at all four investigated sites in resting skeletal muscle (Fig. 2). Interestingly, RT influenced this baseline phosphorylation, with S31 being upregulated while S60 and T17 were downregulated in the trained state (pre 14) compared to the untrained state (pre 1; Fig. 7). However, the functional significance of these differentially altered phosphorylation patterns remains unclear, and there are currently no studies from which precise conclusions can be drawn.
Yet, considering only pDesS31 (Fig. 6B) and following our previous argumentation, the increased baseline phosphorylation in the trained state might suggest an even higher susceptibility of IFs to destabilization and cleavage compared to the untrained state. This seemingly contradictory observation is hypothesized to indicate a mechanism that enhances the dynamics of the desmin-IFs, thereby promoting adaptation processes in this system. Additionally, we observed a reduction in phosphorylation of S31 following acute exercise in the trained state that was significantly more pronounced as compared to the untrained state. As discussed earlier, this acute exercise-induced dephosphorylation is considered to transiently protect the IF system from destabilization during periods of increased stress. Thus, the augmented acute loading-induced dephosphorylation in the trained state could represent a heightened adaptive response, providing greater protection for IF integrity.
However, this interpretation overlooks the behavior of baseline pDesS60 and pDesT17 in the trained state which, unlike S31, displayed reduced phosphorylation. The functional implications of this divergence are challenging to interpret, as previous studies have typically assumed a uniformly directed modification [39], they did not differentiate into specific sites [5, 6, 10, 11, 16, 67] or focused on one site only [27, 44, 56]. At present, we can only speculate that the differentially altered baseline phosphorylation of desmin may be associated with changes in protein turnover, potentially facilitating protein accrual. However, further research is needed to elucidate the underlying mechanisms and functional consequences of these phosphorylation changes for skeletal muscle.
In summary, our research highlights the importance of regulating pDes under physiological conditions in human skeletal muscle, focusing on non-pathological aspects of desmin-PTM. We demonstrated that acute RE transiently decreases desmin phosphorylation, rendering it less susceptible to cleavage. Furthermore, RT alters acute exercise-induced phospho-regulation as well as the protein’s baseline phosphorylation state. However, the functional significance of these alterations requires further elucidation.
In conclusion, our study underscores the effectiveness of acute resistance exercise and prolonged training in modulating desmin phosphorylation and the integrity of the protein within skeletal muscle. This modulation appears to play a crucial role in preserving proteostasis under both, acute and chronic stress conditions. However, further exploration into the underlying mechanisms and fundamental regulation of desmin PTMs under physiological conditions is warranted. Such insights are essential for the development of targeted interventions aimed at combating muscle atrophy processes and enhancing overall skeletal muscle health.