Muscle regeneration is improved by hot water immersion but unchanged by cold following a simulated musculoskeletal injury in humans

Abstract

Cryotherapy is a popular strategy for the treatment of skeletal muscle injuries. However, its effect on post-injury human muscle regeneration remains unclear. In contrast, promising results recently emerged using heat therapy to facilitate recovery from muscle injury. This study aimed to examine the effect of three different thermal treatments on muscle recovery and regeneration following a simulated injury in humans. Thirty-four participants underwent a muscle damage protocol induced by electrically stimulated eccentric contractions triggering regenerative processes following myofibre necrosis. Thereafter, participants were exposed to daily lower body water immersion for 10 days in cold (CWI, 15 min at 12°C), thermoneutral (TWI, 30 min at 32°C) or hot water immersion (HWI, 60 min at 42°C). Muscle biopsies were sampled before and at +5 (D5) and +11 (D11) days post-damage. None of the water immersions differed in recovery of force-generating capacity (P = 0.108). HWI induced a lower perceived muscle pain than TWI (P = 0.035) and lower levels of circulating creatine kinase (P ≤ 0.012) and myoglobin (P < 0.001) than TWI and CWI. Contrary to our hypothesis, CWI did not improve perceived muscle pain or reduce circulating markers of muscle damage (P ≥ 0.207). Expression of heat shock proteins 27 and 70 was significantly increased in HWI (P < 0.038) at D11 and appeared blunted using CWI. Furthermore, nuclear factor-κB expression significantly increased in all conditions except HWI, while interleukin-10 was upregulated only in HWI at D11 (P = 0.014). In conclusion, our results support the use of HWI but not cold, to improve muscle regeneration following an injury. KEY POINTS: Cryotherapy and heat therapy are popular strategies in the treatment of skeletal muscle injury; however, existing literature is equivocal, and their effects on human muscle regeneration remain unknown. We investigated the effect of three thermal treatments (cold water immersion (CWI): 15 min at 12°C; thermoneutral water immersion (TWI): 30 min at 32°C; or hot water immersion (HWI): 60 min at 42°C) performed daily for 10 days following electrically stimulated eccentric muscle damage inducing regenerative mechanisms. CWI did not improve chronic perceived muscle pain nor reduce circulating markers of muscle damage. HWI limited chronic perceived pain and circulating markers of muscle damage, potentially influenced inflammatory mechanisms, and increased the expression of heat shock proteins. HWI appears more beneficial than CWI in improving muscle regeneration after a muscle injury.

Keywords: CWI; HWI; damage; electrically stimulated eccentric contraction; muscle injury.

Figure 1. Schematic overview of the study

Participants were allocated to their thermal intervention group on day (D) 0 after completing the muscle damage protocol of the right leg. The thermal intervention consisted of 15 min at 15°C cold water immersion (CWI) or 30 min at 32°C thermoneutral water immersion (TWI) or 60 min at 42°C hot water immersion (HWI). PRE biopsy was sampled from left leg and D5/D11 biopsies were sampled from right leg.

Figure 2. Microscopy images of histological staining illustrating the effect of the muscle damage protocol at D5 and D11 in comparison to PRE

H&E, laminin (green), nuclei (blue) and dystrophin (orange) staining are displayed. The asterisk identifies the same fibre across the different stains for each sampling time. Examples of damaged fibres are identified by arrows at D5 and D11; these fibres present a swollen aspect with a loss of normal polygonal outline, mononuclear cell infiltration, internal nuclei or a loss of dystrophin immunoreactivity of their membrane. A large number of similar fibres presenting necrosis are visible in the D11 illustration. Scale bars, 50 µm.

Figure 3. Vastus lateralis muscle temperature at 1, 2 and 3 cm depth from skin surface after CWI, TWI and HWI interventions

Data are presented as boxplot with median and interquartile range along with individual data. *Significantly different between group (P < 0.05); ^#significantly different from all depths in the group (P < 0.05); ^†significantly different from 1 cm in the same group (P < 0.05).

Figure 4. Maximal voluntary contraction force

Data are presented as means and standard deviation of the percentage decrease of MVC from PRE. ^#Significant time effect (P < 0.05).

Figure 5. Muscle pain measured during self‐palpation (A) and after three squats (B) using an arbitrary unit scale (0–20) where 0 represented no pain and 20 extremely painful

Data are presented as means and standard deviation of the perceived pain score. PE: post‐exercise, PI: post water immersion. ^#Significant time effect showing differences from PRE (P < 0.05); *significant group effect between HWI and TWI (P < 0.05).

Figure 6. The blood markers creatine kinase (CK) (A) and myoglobin (B)

Data are presented as means and standard deviation along with individual data. CK at D4 is displayed in the graph but data were excluded from statistical analysis. ^#Significantly different from PRE (P < 0.05); ^###significantly different from PRE (P < 0.001); ^†significantly different from D4 (P < 0.05); ^†††significantly different from D4 (P < 0.001); *significantly different between groups (P < 0.05); ***significantly different between groups (P < 0.001).

Figure 7. Representative blots and quantification of HSP 27 (A, B) and HSP 70 (C, D) protein expression at PRE, D5 and D11 in CWI, TWI and HWI groups

Data are presented as medians along with individual data. ^#Significantly different from PRE (P < 0.05); *significantly different between groups (P < 0.05).

Figure 8. Representative blots and quantification of phosphorylated NF‐κB p65 (A, B) and IL‐10 (C, D) protein expression at PRE, D5 and D11 in CWI, TWI and HWI groups

Data are presented as medians along with individual data. ^#Significantly different from PRE (P < 0.05); ^##significantly different from PRE (P < 0.01); ^†significantly different from D5 (P < 0.05); *significantly different between groups (P < 0.05).

Figure 9. Representative blots and quantification of phosphorylated mTOR (A, B), phosphorylated‐S6 (C, D) and phosphorylated‐p70 (E, F) protein expression at PRE, D5 and D11 in CWI, TWI and HWI groups

Data are presented as medians along with individual data. ^#Significantly different from PRE (P < 0.05); ^##significantly different from PRE (P < 0.01); ^†significantly different from D5 (P < 0.05); *significantly different between groups (P < 0.05).

Figure 10. Vascular endothelial growth factor (VEGF) representative blots (A) and quantification (B) of protein expression at PRE, D5 and D11 in CWI, TWI and HWI groups

Data are presented as median along with individual data. ^#Significantly different from PRE (P < 0.05); ^##significantly different from PRE (P < 0.01); ^†significantly different from D5 (P < 0.05); *significantly different between groups (P < 0.05).

Figure 11. Transforming growth factor‐β1 (TGF‐β1) representative blots (A) and quantification (B) of protein expression at PRE, D5 and D11 in CWI, TWI and HWI groups

Data are presented as median along with individual data.