Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise

Abstract

High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca(2+) release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca(2+) leak at rest, and depressed force production due to impaired SR Ca(2+) release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca(2+)-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group.

Keywords: Ca2+; high-intensity exercise; reactive oxygen species; ryanodine receptor 1; skeletal muscle.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

HIIT exercise induces extensive RyR1 fragmentation in recreationally active subjects. (A) Representative Western blot reveals decreased expression of full-sized RyR1 (red arrow) 24 h after exercise, which was accompanied by the appearance of fragments of ∼375, 80, and 60 kDa (indicated by black arrows). (B) Mean relative distribution of native RyR1 and its fragments from four subjects; the total intensity of all four analyzed bands was set to 100% at each time point in each subject. (C) Representative Western blots and mean data of DHPR, SERCA2, CSQ1, DMD, and actin expression ∼10 min (n = 11) and 24 h (n = 5) after exercise; relative expression before exercise was set to 100% in each subject. Data are expressed as mean ± SEM.

Fig. 2.

HIIT exercise does not induce RyR1 fragmentation in elite endurance athletes. (A) Representative Western blots show no signs of RyR1 fragmentation after the cycling bouts in the elite athletes. Arrows indicate full-sized RyR1 (red arrow) and the location of ∼375-, 80-, and 60-kDa fragments (black arrows) observed 24 h after exercise in recreationally active subjects (Fig. 1A). (B) Mean data (± SEM) obtained from 14 elite athletes before (Pre) and ∼10 min (Post) and 24 h after exercise; total RyR1 expression was set to 100% at each time point in each subject. (C, Upper) Representative Western blots of SOD2, catalase, and DHPR from biopsies taken before the HIIT exercise in recreationally active subjects (Rec) and elite athletes (EA). DHPR did not differ between the two groups and was used as loading control. (C, Lower) Bar graphs show mean SOD2 and catalase expressions (± SEM; n = 7) relative to the mean in the Rec group, which was set to 100%. **P < 0.01 in unpaired t test. (D) Mean data (± SEM; n = 6–8) of the transcript levels of PGC-1α1 and -1α4 expressed relative to hypoxanthine guanine phosphoribosyl transferase (HPRT), which did not differ between the groups and was used as a housekeeping gene. #P < 0.05; ##P < 0.01; ###P < 0.001 vs. before exercise (one-way repeated measures ANOVA/Holm–Sidak post hoc test). PGC-1α4 was significantly higher before exercise in Rec than in EA (P < 0.05; unpaired t test).

Fig. 3.

Simulated HIIT exercise causes a ROS-dependent RyR1 fragmentation in mouse muscle. (A) Representative Western blots of RyR1, MDA adducts on RyR1, DHPR, and actin obtained from single-digit FDB muscles 5 min after being fatigued by six bouts of 30-s simulated HIIT exercise or kept at rest (Ctrl). The contractions had no effect on RyR1, DHPR, and actin expression, but it approximately doubled the amount of MDA adducts on RyR1. (B) Western blot of RyR1 on single-digit FDB muscles snap-frozen either at rest (Ctrl) or 3 h after exercise, which was performed in the absence or presence of NAC (20 mM). Shown is relative expression of the full-size RyR1 with the average Ctrl set to 100% (n = 5–12 muscles). (C) Calpain activity in single-digit mouse FDB muscles before (Ctrl) and 30 min and 3 h after simulated HIIT exercise (n = 4–6). Positive and negative controls were obtained by adding fully active calpain and calpain inhibitor, respectively. Data are expressed as relative fluorescence units (RFU) divided by muscle wet weight (w.w.); the mean value in Ctrl was set to 1.0. All data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 with unpaired t test (A) or one-way ANOVA (B and C).

Fig. 4.

Simulated HIIT exercise induces prolonged decrease in tetanic [Ca2+]i and increase in resting [Ca2+]i. (A, Upper) Representative records of [Ca2+]i during 100-Hz tetanic stimulation trains evoked at the start of the first and sixth simulated cycling bout in a single FDB fiber. (A, Lower) Mean [Ca2+]i in the initial tetanus of the six cycling bouts. (B) Force measured 5–120 min after the simulated HIIT exercise; data are expressed relative to the force before exercise, which in each fiber was set to 100% at both 40- and 120-Hz stimulation. (C) Tetanic [Ca2+]i before (PRE) and 5–120 min after exercise. (D) The relation between tetanic force and [Ca2+]i before (white circle; obtained by stimulating fibers at 15–150 Hz at 1-min intervals) and 120 min after exercise (black and red circles; data taken from B and C). (E) Resting [Ca2+]i before and 5–120 min after exercise. (F) Representative [Ca2+]i records and mean data obtained from 100-Hz tetanic stimulations in the presence of 5 mM caffeine produced before and 5 min after exercise. All data are expressed as mean ± SEM (n = 6–14 fibers). ***P < 0.001 vs. the first simulated cycling bout (A) or before exercise (B, C, and F) (one-way repeated measures ANOVA). *P < 0.05 in paired t test (F).