Strenuous exercise often induces skeletal muscle damage, which results in impaired performance. Sphingolipid metabolism contributes to various cellular processes, including apoptosis, stress response, and inflammation. However, the relationship between exercise-induced muscle damage and ceramide (a key component of sphingolipid metabolism), is rarely studied. The present study aimed to explore the regulatory role of sphingolipid metabolism in exercise-induced muscle damage.
Mice were subjected to strenuous exercise by treadmill running with gradual increase in intensity. The blood and gastrocnemius muscles (white and red portion) were collected immediately after and 24 h post exercise. For 3 days, imipramine was intraperitoneally injected 1 h prior to treadmill running.
Interleukin 6 (IL-6) and serum creatine kinase (CK) levels were enhanced immediately after and 24 h post exercise (relative to those of resting), respectively. Acidic sphingomyelinase (A-SMase) protein expression in gastrocnemius muscles was significantly augmented by exercise, unlike, serine palmitoyltransferase-1 (SPT-1) and neutral sphingomyelinase (N-SMase) expressions. Furthermore, imipramine (a selective A-SMase inhibitor) treatment reduced the exercise-induced CK and IL-6 elevations, along with a decrease in cleaved caspase-3 (Cas-3) of gastrocnemius muscles.
We found the crucial role of A-SMase in exercise-induced muscle damage.
Sphingolipids, including ceramide (Cer), are important bioactive molecules that are involved in diverse biological processes, including apoptosis, proliferation, differentiation, growth arrest, and inflammation
Exercise-induced muscle damage (EIMD) holds importance in the field of exercise and sports and is caused by unaccustomed or strenuous exercise involving a large amount of eccentric muscle contractions
However, studies focusing on the direct relationship between exercise-induced muscle damage and sphingolipid metabolism remain limited. In the present study, we explored whether the rate-limiting enzymes of sphingolipid metabolism, such as SPT-1 and SMases contribute to EIMD in gastrocnemius muscle after acute strenuous exercise.
Seven week-old C57BL/6 male mice were acquired from Daehan Biolink, Co., Ltd. (Eumsung, Chungbuk, Korea) and housed in clear plastic cages under specific pathogen-free conditions and a 12:12 h light-dark cycle (light period: 08:00-20:00). The mice had free access to standard irradiated chow diet (Purina Mills, Seoul, Korea). All of the experimental procedures involving animals were approved by the Animal Care and Use Committee of Ewha Women’s University.
The mice were acclimatized to treadmill-running (Myung Jin Instruments Co., Seoul, Korea) by pre-exercising (Pre-Ex) at 10 m/min for 20 min/day (5% grade) for 3 days (each group, N = 9). The animals were placed on a restricted daily diet, 4 h prior to exercise. The protocol of exercise-induced muscle damage was modified according to a previous study
Tissues dissected from the mice were homogenized. The western blot analysis was performed based on the protocol described in our previous study
The IL-6 and CK levels in serum were determined using a mouse IL-6 ELISA Kit (Ray Biotech, GA, USA) and EnzyChromTM Creatine Kinase Assay Kit (BioAssay Systems, CA, USA), respectively, according to manufacturer's instructions. The following protocol was followed for measuring the serum CK levels: R1 reagent buffer (100 μl) containing substrate was added to 6 μl of serum sample, and then incubated for 10 min at 37ºC. Following incubation, absorbance was determined at 340 nm wavelength. Serum IL-6 levels were measured as follows: Sample (100 μl) was added into 96-well plate coated with anti-IL-6 antibody and was incubated for 2.5 h at R/T. Next, the sample was incubated with biotin antibody at R/T for 1 h, followed by incubation with streptavidin incubation for 45 min. Absorbance was measured at 450 nm after 30 min of incubation with TMB One-Step Substrate.
Significant differences between the groups were determined using independent paired
Serum CK and IL levels were measured to confirm the relevance of the protocol to EIMD model. Serum CK level at 24 h after treadmill running was noted as significantly increased, but not immediately after the exercise (F2, 24 = 42.15,
To identify the relationship between Cer production by de novo synthesis and EIMD, the expression of SPT-1, which is a key rate-limiting enzyme of de novo synthesis, was measured in gastrocnemius muscles. SPT-1 protein expression did not vary from time to time in gastrocnemius muscles regardless of the time points (for GR: F2, 9 = 0.39,
To identify the relationship between Cer production by sphingomyelin hydrolysis and EIMD, the expression of the two SMases (key rate-limiting enzyme of de novo synthesis) was noted in gastrocnemius muscles. A-SMase protein expression was significantly enhanced between 0–24 h after exercise in both gastrocnemius muscles (for GR: F2, 9 =21.28,
The quantitative analysis of serum (A) CK levels and (B) IL-8 levels in response to strenuous treadmill running, (C) representative images of western blot for SPT-1, N-SMase, and A-SMase. The quantitative analysis of (D) SPT-1, (E) N-SMase, and (F) A-SMase. The quantitative analysis of serum (G) CK levels and (H) IL-6 levels based on imipramine in response to strenuous treadmill running. Representative images of western blot for (I) caspase-3 and the quantitative analysis of (J) caspase-3. Data are presented as mean ± SE. * and ** denote
The enhanced A-SMase expression in gastrocnemius muscle during EIMD condition led us to explore whether suppressing A-SMase activity reduced EIMD. We found that upon imipramine treatment, serum CK levels significantly reduced 24 h after exercise (for Resting: t14 = -0.32,
The current study demonstrated that EIMD might be partly responsible for cellular Cer production by enhancing A-SMase expression against strenuous exercise. Moreover, suppressing A-SMase activity can alleviate skeletal muscle damage in response to intensive exercise.
Strenuous or unaccustomed exercise causes skeletal muscle damage characterized by muscle soreness and decreased muscle function
Cer, a key molecule of sphingolipid metabolism, contributes to diverse biological processes, including apoptosis, proliferation, differentiation, growth arrest, and inflammation
In addition to the
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