Sarcolipin overexpression improves muscle energetics and reduces fatigue

Abstract

Sarcolipin (SLN) is a regulator of sarcoendoplasmic reticulum calcium ATPase in skeletal muscle. Recent studies using SLN-null mice have identified SLN as a key player in muscle thermogenesis and metabolism. In this study, we exploited a SLN overexpression (Sln(OE)) mouse model to determine whether increased SLN level affected muscle contractile properties, exercise capacity/fatigue, and metabolic rate in whole animals and isolated muscle. We found that Sln(OE) mice are more resistant to fatigue and can run significantly longer distances than wild-type (WT). Studies with isolated extensor digitorum longus (EDL) muscles showed that Sln(OE) EDL produced higher twitch force than WT. The force-frequency curves were not different between WT and Sln(OE) EDLs, but at lower frequencies the pyruvate-induced potentiation of force was significantly higher in Sln(OE) EDL. SLN overexpression did not alter the twitch and force-frequency curve in isolated soleus muscle. However, during a 10-min fatigue protocol, both EDL and soleus from Sln(OE) mice fatigued significantly less than WT muscles. Interestingly, Sln(OE) muscles showed higher carnitine palmitoyl transferase-1 protein expression, which could enhance fatty acid metabolism. In addition, lactate dehydrogenase expression was higher in Sln(OE) EDL, suggesting increased glycolytic capacity. We also found an increase in store-operated calcium entry (SOCE) in isolated flexor digitorum brevis fibers of Sln(OE) compared with WT mice. These data allow us to conclude that increased SLN expression improves skeletal muscle performance during prolonged muscle activity by increasing SOCE and muscle energetics.

Keywords: Ca2+ ATPase; muscle fatigue; muscle metabolism.

Fig. 1.

The effect of sarcolipin (SLN) overexpression on sarcoplasmic reticulum (SR) and myofilament proteins and muscle morphology. A: fast- and slow-twitch skeletal muscles from SLN overexpression (Sln^OE) mouse have higher expression of SLN than wild-type (WT) muscles (WT, 20 μg protein; Sln^OE, 2 μg protein), but expression in atria and ventricle is not different (atria, 5 μg protein; ventricle, 20 μg protein). EDL, extensor digitorum longus. B: expression levels of sarcoendoplasmic reticulum calcium ATPase (SERCA) and calsequestrin (CSQ) isoforms are not affected in Sln^OE muscles (10 μg protein for all muscles). GAPDH is the loading control. C: mRNA expression pattern of myosin heavy chain isoforms is similar in EDL muscle from WT and Sln^OE mouse. Expression was normalized to that of hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA level. D: morphology of diaphragm, soleus, and quadriceps muscle is unchanged in WT and Sln^OE mouse, as seen by representative hematoxylin and eosin-stained histological sections (n = 4 for WT and Sln^OE for each experiment).

Fig. 2.

Overexpression of SLN does not affect the basal metabolic characteristics of Sln^OE mice. A: rate of O₂ consumption of Sln^OE mice is not different from their WT littermates. B: respiratory exchange ratio (RER), which is the ratio of volume of carbon dioxide (V̇co₂) produced to volume of oxygen consumed (V̇o₂), is also unchanged between the 2 genotypes. C: basal activity is similar in WT and Sln^OE mice (n = 9 for WT and Sln^OE).

Fig. 3.

Overexpression of SLN improves muscle endurance exercise capacity and increases mitochondrial fatty acid transporter carnitine palmitoyl transferase 1 (CPT1) expression. A: Sln^OE mice run significantly longer distances than the WT mice during a prolonged treadmill run. B: maximal oxygen consumption capacity (V̇o₂ max) is similar for the 2 groups of mice. C: Western blot analysis of CPT1 protein showing that, compared with WT, its expression is higher in both EDL (fast glycolytic) and soleus (slow oxidative) muscles of Sln^OE mice (EDL, 30 μg; soleus, 30 μg protein loaded). D: densitometric analyses of Western blot showing that CPT1 expression (normalized to GAPDH) is significantly higher in EDL and soleus muscles of Sln^OE mice. AU, arbitrary units. E: Western blotting of specific subunit proteins of the mitochondrial electron transport chain complexes (CI, CII, CIII, CIV, and CV) shows that their expression is not altered in Sln^OE mouse soleus (5 μg protein). F: expression of citrate synthase, a regulatory protein of Krebs cycle, is similar in WT and Sln^OE mouse soleus (5 μg protein). GAPDH is the loading control. G: Western blot analysis showing protein expression of lactate dehydrogenase (LDH) in Sln^OE and WT EDL (5 μg protein loaded). H: LDH expression is significantly higher in Sln^OE EDL than in WT EDL. GAPDH is the loading control (n = 4 for all Western blot data) (*P < 0.05, **P < 0.01, and ****P < 0.0001).

Fig. 4.

Overexpression of SLN increases twitch force and tetanus force (at lower frequencies) in isolated EDL. A: twitch profile of WT and Sln^OE isolated EDL in buffer containing 10 mM pyruvate or 10 mM glucose as substrate. B: isolated EDL from Sln^OE mouse produces significantly higher twitch force in 10 mM pyruvate than WT EDL. C: SLN overexpression does not affect the force-frequency curve at higher frequencies (>70 Hz), but it shifts the curve further to the left at lower frequencies (especially 30 and 50 Hz) in 10 mM pyruvate compared with 10 mM glucose. D: 50-Hz-specific force traces showing that the highest force is produced by Sln^OE EDL in 10 mM pyruvate. E: Sln^OE EDL has a significantly higher average specific force in pyruvate than in glucose at 50 Hz. The thick lines in A and D are the mean, and the thinner dashed lines are the range of SE (*P < 0.05, ***P < 0.001).

Fig. 5.

The contractile properties of soleus are not altered in Sln^OE mice. A: twitch force produced by WT and Sln^OE mouse isolated soleus is similar in 10 mM glucose, and potentiation of force by pyruvate is also not different. B: overexpression of SLN does not affect the force-frequency curve of soleus, in both 10 mM glucose and 10 mM pyruvate, compared with WT soleus.

Fig. 6.

SLN overexpression significantly reduces fatigue in isolated EDL and soleus muscles. A: during a 10-min fatigue, WT EDL has significantly higher reduction in % force than Sln^OE muscle. B: isolated soleus muscle from Sln^OE mice produces significantly higher % force than WT soleus during a 10-min fatigue protocol. C: sum of average specific forces over the 10-min fatigue is significantly greater for Sln^OE EDL than WT EDL. D: sum of average specific forces over the 10-min fatigue is significantly greater for Sln^OE soleus than WT soleus (*P < 0.05, **P < 0.01).

Fig. 7.

Overexpression of SLN increases store-operated calcium entry (SOCE) in isolated flexor digitorum brevis (FDB) fibers. A: representative trace of SOCE from WT (gray trace) and Sln^OE (black trace) FDB fiber showing increased Ca²⁺ entry in fibers overexpressing SLN after depletion of SR stores. Black arrow shows addition of caffeine/ryanodine/thapsigargin buffer to deplete stores (WT has similar response as Sln^OE). Gray arrow shows addition of Ca²⁺ buffer after SR store has been completely depleted. Inset: decline in SOCE after calcium release-activated Ca²⁺ channels have been blocked by addition of 2-aminoethoxydiphenyl borate (2-APB). B: FDB fibers from Sln^OE show significantly greater SOCE response compared with WT FDB fibers (n = 8) (*P < 0.05).