Macroautophagy, herein referred to as autophagy, is an essential and highly regulated mechanism for the bulk degradation of cytosolic proteins and organelles in mammalian cells. While the basal levels of autophagy might be tissue- or cell type-dependent, the induction of autophagy by stress is highly conserved (Mizushima and Komatsu, 2011). Hypoxia, oxidative stress, nutrient depletion, and exercise are just some of the many stressors that have been shown to increase autophagic activity (Vainshtein and Hood, 2016). This increase in autophagy serves as an adaptive response mechanism, essential for the cell’s survival and return to homeostasis.

Autophagy has been shown to be critical for skeletal muscle homeostasis (Masiero et al., 2009; Grumati et al., 2010). A deficiency in autophagic flux results in an accumulation of aggregated proteins and damaged organelles, leading to skeletal muscle atrophy, and weakness (Masiero et al., 2009; Kim et al., 2013). Several myopathies, including muscular dystrophy and glycogen storage disease have an abnormal reduction in autophagy that contributes to disease progression (Malicdan et al., 2008). Similarly, skeletal muscle autophagy is dysregulated in metabolic diseases, including insulin resistant and diabetes (Hotamisligil, 2010; He et al., 2012; Møller et al., 2017). Importantly, in some models, structure and function of skeletal muscle can be restored by pharmacological reactivation of autophagy (De Palma et al., 2012, 2014; Pauly et al., 2012). Thus, strategies to increase skeletal muscle autophagic flux may have important implications in the context of muscle myopathies, obesity, insulin resistance, and aging, among others.

The contraction of skeletal muscle generates a large amount of physical and metabolic stress, which instigates high levels of protein turnover (Röckl et al., 2007; Poortmans et al., 2012). Muscular contraction during exercise induces a state of metabolic stress in skeletal muscle, decreasing ATP content, altering molecular messengers, and inducing protein and organelle damage (Röckl et al., 2007), and upregulation of autophagy (Sandri, 2010; He et al., 2012). This increase in autophagy is essential for exercise-induced benefits in glucose homeostasis (He et al., 2012), and likely other homeostatic processes. During exercise, skeletal muscle temperature increases by several degrees (Brooks et al., 1971; Saltin et al., 1972), which appears to account for at least some of the biochemical responses to exercise. Exposure to short bouts of heat, mimicking temperatures achieved during exercise, recapitulates many of the beneficial effects exercise, including protection against muscle atrophy (Naito et al., 2000; Selsby and Dodd, 2005), and improvements in glucose homeostasis (Hooper, 1999; Gupte et al., 2009, 2011) and vascular function (Brunt et al., 2016a, b). and may be an important component of the beneficial effects of exercise. While there is literature supporting the adverse effects of prolonged heating on skeletal muscle autophagy (Brownstein et al., 2017), less is known about the effects of acute heating on autophagy signaling in skeletal muscle.

The purpose of the present study was to investigate the effects of an acute, short bout of HT on autophagic signaling in skeletal muscle cells. To do this, C2C12 myotubes were exposed to 1 h of HT at 40°C, with and without recovery. We hypothesized that a single short bout of HT would increase autophagic signaling and the heat shock response in skeletal muscle cells.

Heat treatment can provide many benefits to skeletal muscle, such as reduced oxidative stress, improved insulin signaling, and increased mitochondrial biogenesis (Gupte et al., 2009; Liu and Brooks, 2012). Acute heating of C2C12 skeletal muscle cells at 40°C for 1 h has previously been shown to increase the phosphorylation of AMPK at Thr172, leading to transcription of PGC-1α, and activation of mitochondrial biogenesis (Liu and Brooks, 2012). In agreement, our data suggest that acute HT of 1 h upregulated autophagy initiation signaling, reflected by changes in the phosphorylation of AMPK at Thr172. Additionally, as expected, HT activated the heat shock response. These results support our hypothesis that a single bout of mild heat stress for 1 h can stimulate autophagy initiation signaling in addition to the heat shock response, in skeletal muscle cells.

Skeletal muscle requires efficient autophagic flux to allow for proper nutrient recycling and the removal of protein aggregates or dysfunctional organelles (Neel et al., 2013). It is well established that mTOR and AMPK are opposing regulators of ULK1, the main protein attributed to autophagy initiation is ULK1 (Kim et al., 2011; Russell et al., 2013). In the current study, we did not observe a HT effect on phosphorylation levels of mTOR at Ser2448, which is indicative of its activation. It has been suggested that phosphorylation of ULK1 at Ser555 levels may be a more efficient indicator of ULK1 activity, than levels of phosphorylation at Ser757 (Laker et al., 2017). With prolonged heat stress (HS) of 12 h, the phosphorylation of AMPK at Thr172 and ULK1 at Ser555 were significantly elevated in porcine skeletal muscle, but no changes were seen in the phosphorylation of ULK1 at Ser757 (Ganesan et al., 2017a). Here we show a similar response with a much shorter bout of heat, and a tendency (p = 0.10) for an increase in p-ULK1 at Ser555, with a large effect size (Cohen’s f = 0.41). Similarly, the ratio of ULK1 Ser555 to Ser757, an indicator of its activation state, tended to increase (p = 0.08), and had a large effect size (Cohen’s f = 0.59). These initiation events resemble those seen with exercise (Laker et al., 2017). Cells exposed to Bafilomycin A1 maintained the effects of HT on phosphorylated AMPK (p < 0.05; data not shown), further supporting changes in an initial step toward autophagy initiation by HT.

Here, we show that 1 h of HT significantly increased levels of Beclin-1 and LC3 II after 2 h of recovery. As expected, changes in LC3 II occurred sequentially after the elevation in autophagic initiation markers. Despite heat-induced changes in LC3 II, no effects on p62 were observed, suggesting an increase in overall autophagic flux or enhanced degradation. The ratio of p62/Beclin-1 has been suggested as another indicator of autophagic turnover (Klionsky et al., 2016). Our data support a time-dependent heat effect of reduced p62/Beclin-1 (p = 0.03), suggestive of turnover. This is in contrast to prolonged HS, in which both LC3 II and p62 were elevated simultaneously, signifying that degradation was inhibited, causing a buildup of autophagosomes in skeletal muscle (Ganesan et al., 2017a).

Using BafA1 to chemically inhibit autophagasome degradation, we observed that 1 h of HT decreased the BafA1-induced increase in LC3 II immediately after HT, until at least 2 h of recovery. It is possible that this short duration of heat is increasing initiation signals, without inducing high levels of cellular stress that would further drive autophagic flux, as supported by reduction in p38 and JNK phosphorylation, and no change in NF-κB signaling. Our finding is consistent with the effects of both muscle contraction and exercise to reduce autophagasome accumulation in the presence of chemical inhibition of autophagy, despite increased basal levels of autophagy (Jiang et al., 2014; Parousis et al., 2018). This effect is speculated to reflect a normalization of autophagic degradation, although the mechanism underlying this effect remains unclear.

Many of the proteins involved in autophagasome nucleation and elongation, such as Atg3, Atg16L1, and PI3K Class III, were unaffected by HT. Despite important roles in this process, these proteins are unlikely to be consumed during autophagy or be a rate-limiting factor in autophagic activity. However, 1 h of HT significantly increased Beclin-1 by 2 h of recovery, lasting until at least 24 h after HT. When Beclin-1 is bound to Bcl-2, the pro-autophagy activity of Beclin-1 is inhibited (He et al., 2012). The rise in Beclin-1 could be due to an increased need for unbound Beclin-1, to further propagate autophagy signaling. However, in porcine skeletal muscle exposed to 12 h of HS, PI3K Class III and Atg3 were significantly elevated compared to TN (Ganesan et al., 2017a). This may suggest that the proteins involved in autophagy nucleation and elongation may only be altered by a greater degree of stress, imposed by a longer duration of heat.

HSPs have many cellular functions, such as folding nascent or unfolded proteins, preventing protein aggregation, and degrading proteins (Bozaykut et al., 2014). The acute, transient hyperphosphorylation of HSF1, immediately after heating, signifies the activation of its transcription factor activity (Xia and Voellmy, 1997). We have previously seen that 15 min of recovery was enough for HSF1 hyperphosphorylation to return to thermoneutral (TN) levels (data not shown). An in vivo study reported no changes in skeletal muscle Hsp70 expression when pigs were exposed to 2, 4, or 6 h of HS (Ganesan et al., 2017b). Whereas we observed a significant duration-dependent elevation of Hsp70 during recovery. This is likely due, in part, to the lag in time between HSF1 activation of HSP transcription and the translation of Hsp70.

In contrast to the literature (Landry et al., 1992) our data demonstrate that the phosphorylation status of Hsp27, at Ser86, was acutely reduced by heat, which is known to alter its cellular function. When phosphorylated, Hsp27 is associated with cytoskeleton stabilization. Unphosphorylated Hsp27 is thought to form large oligomers, and play a role in anti-oxidant and chaperone activity, in addition to inhibition of apoptosis (Katsogiannou et al., 2014). This raises the possibility for the potential of heat to alter chaperone-mediated autophagy through this mechanism, which warrants further investigation.

Hsp27 can be phosphorylated by several kinases, including the MAP-kinases and PKCs (Marais et al., 2005), and appear to be most influenced by p38 MAPK (Larsen et al., 1997; Concannon et al., 2003). Unlike during exercise, in which mechanical stimulation increases p-p38 and subsequently increases p-Hsp27 (Hoffman et al., 2017), heat transiently suppressed the phosphorylation of p38, which may explain the reduced p-Hsp27 observed as this same time point. In addition to the altered HSP signaling, changes in MAPK signaling have been implicated in autophagy regulation (Sui et al., 2014). Interestingly, p38 MAPK has been suggested as both a negative and positive regulator of autophagy (Webber, 2010). Specifically, under conditions of starvation, an increase in p38 inhibits autophagy, whereas p38 depletion leads to an increase in LC3 (Webber, 2010). We observed a similar inverse pattern, in which the increase in LC3 II corresponded with reduced p-p38. In contrast, JNK activations is thought to promote autophagy, and thus does not appear to be a major factor responsible for heat-induced autophagy.

A standardized exposure to the autophagy inhibitor BafA1 (3 hr) was used to gain insight into autophagy flux, but resulted in varying degrees of autophagy inhibition occurring prior to (0 h and 2 h) or following heat exposure (24 h). An alternative approach of inhibiting autophagy for a consistent duration prior to the exposure to heat would result in varying degrees of autophagy inhibition, assay saturation, and durations exceeding recommendations (Klionsky et al., 2016). The data presented here are limited to heat-induced changes in the expression of autophagy-related proteins via Western blot, and are suggestive that the well-established nutrient sensor, AMPK, likely contributes to an upregulation in autophagy signaling in response to heat. The observed reduction in p-p38 may also contribute to autophagy. However, factors such as ER stress, oxidative stress, and lysosomal acidification and function may also be altered in response to heat. The role of these cell signaling changes on heat-induced autophagy cannot be ruled out. Future, mechanistic studies, incorporating immunocytochemistry of LC3 puncta and localization will be important to confirm and extend our findings.

In conclusion, our study showed that, in C2C12 skeletal muscle cells, a single 1 h bout of 40°C HT increased autophagy initiation signals, and indicators of autophagosomal accumulation, along with activation of the heat shock response (Figure 6). Our data stem from cell culture experiments, are limited to healthy cells, and lack the systemic response to heat that would likely impact in vivo cellular metabolism. However, these results suggest that autophagy is altered by HT in skeletal muscle cells, and suggest that HT could be a therapeutic approach to target autophagy in skeletal muscle. Strategies such as hot water emersion, sauna, or localized heat application may provide therapeutic benefit, particularly in conditions with dysfunctional autophagy. Further studies are needed to directly investigate the potential difference in autophagic responses to varying heat loads and whether the activation of autophagy by heat can prevent or improve skeletal muscle pathologies.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

CS and RV were responsible for the conception and design of the study, completed the data collection, and were responsible for data analysis and drafting the manuscript. Both authors approved the final version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.