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. 2023 May 8;15(9):2224.
doi: 10.3390/nu15092224.

Effects of Creatine Supplementation on the Myostatin Pathway and Myosin Heavy Chain Isoforms in Different Skeletal Muscles of Resistance-Trained Rats

Marianna Rabelo de Carvalho  1 , Ellen Fernandes Duarte  1 , Maria Lua Marques Mendonça  1 , Camila Souza de Morais  1 , Gabriel Elias Ota  1 , Jair José Gaspar-Junior  1 , Wander Fernando de Oliveira Filiú  2 , Felipe Cesar Damatto  3 , Marina Politi Okoshi  3 , Katashi Okoshi  3 , Rodrigo Juliano Oliveira  1 , Paula Felippe Martinez  1   4 , Silvio Assis de Oliveira-Junior  1   4
Affiliations

Effects of Creatine Supplementation on the Myostatin Pathway and Myosin Heavy Chain Isoforms in Different Skeletal Muscles of Resistance-Trained Rats

Marianna Rabelo de Carvalho et al. Nutrients. .

Abstract

Creatine has been used to maximize resistance training effects on skeletal muscles, including muscle hypertrophy and fiber type changes. This study aimed to evaluate the impact of creatine supplementation on the myostatin pathway and myosin heavy chain (MyHC) isoforms in the slow- and fast-twitch muscles of resistance-trained rats. Twenty-eight male Wistar rats were divided into four groups: a sedentary control (Cc), sedentary creatine supplementation (Cr), resistance training (Tc), and resistance training combined with creatine supplementation (Tcr). Cc and Tc received standard commercial chow; Cr and Tcr received a 2% creatine-supplemented diet. Tc and Tcr performed a resistance training protocol on a ladder for 12 weeks. Morphology, MyHC isoforms, myostatin, follistatin, and ActRIIB protein expressions were analyzed in soleus and white gastrocnemius portion samples. The results were analyzed using two-way ANOVA and Tukey's test. Tc and Tcr exhibited higher performance than their control counterparts. Resistance training increased the ratio between muscle and body weight, the cross-sectional area, as well as the interstitial collagen fraction. Resistance training alone increased MyHC IIx and follistatin while reducing myostatin (p < 0.001) and ActRIIB (p = 0.040) expressions in the gastrocnemius. Resistance training induced skeletal muscle hypertrophy and interstitial remodeling, which are more evident in the gastrocnemius muscle. The effects were not impacted by creatine supplementation.

Keywords: creatine; muscle anabolism; muscle fibers; myostatin; strength training.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Maximum carrying capacity in climbing test before (A) and after (B) the training protocol. Cc (n = 7): sedentary control; Cr (n = 7): sedentary creatine supplementation; Tc (n = 7): resistance training; Tcr (n = 7): resistance training combined with creatine supplementation. Two-way ANOVA and Tukey test. ‡ p < 0.05, resistance training effect; * p < 0.05 vs. Cc; † p < 0.05 vs. Cr.
Figure 2
Figure 2
Cross-sectional area and representative transverse histological sections of the gastrocnemius (A) and soleus (B) muscles (400-fold magnification) stained with hematoxylin-eosin. Cc (n = 7): sedentary control; Cr (n = 7): sedentary creatine supplementation; Tc (n = 7): resistance training; Tcr (n = 7): resistance training combined with creatine supplementation. Two-way ANOVA and Tukey test. ‡ p < 0.05, resistance training effect; * p < 0.05 vs. Cc.
Figure 3
Figure 3
Collagen interstitial fraction and representative transverse histological sections from gastrocnemius (A) and soleus (B) muscles (400-fold magnification) stained with Picrosirius red. Cc (n = 7): sedentary control; Cr (n = 7): sedentary creatine supplementation; Tc (n = 7): resistance training; Tcr (n = 7): resistance training combined with creatine supplementation. Two-way ANOVA and Tukey test. ‡ p < 0.05, resistance training effect; * p < 0.05 vs. Cc; † p < 0.05 vs. Cr.
Figure 4
Figure 4
Representative bands of myosin heavy chain isoforms (MyHC IIx and MyHC IIb) (A); relative proportions (%) of MyHC IIx (B), and MyHC IIb (C) isoforms, and MyHC IIx/MyHC IIb ratio (D) in the gastrocnemius muscle. Isolated factors: Sedentary; Trained; Control; Creatine. Groups: Cc (n = 6): sedentary control; Cr (n = 6): sedentary creatine supplementation; Tc (n = 6): resistance training; Tcr (n = 6): resistance training combined with creatine supplementation. Two-way ANOVA and Tukey test. ‡ p < 0.05, resistance training effect; * p < 0.05 vs. Cc.
Figure 5
Figure 5
Representative bands of myosin heavy chain isoforms (MyHC IIa and MyHC I) (A); relative proportions (%) of MyHC IIa (B), and MyHC I (C) isoforms, and MyHC IIa/MyHC I ratio (D) in the soleus muscle. Groups: Cc (n = 6): sedentary control; Cr (n = 6): sedentary creatine supplementation; Tc (n = 6): resistance training; Tcr (n = 6): resistance training combined with creatine supplementation. Two-way ANOVA (p > 0.05).
Figure 6
Figure 6
Protein levels and representative Western blots of follistatin (A) and myostatin (B) expression in the gastrocnemius muscle. Protein levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Isolated factors: Sedentary; Trained; Control; Creatine. Groups: Cc (n = 7): sedentary control; Cr (n = 7): sedentary creatine supplementation; Tc (n = 7): resistance training; Tcr (n = 7): resistance training combined with creatine supplementation. Two-way ANOVA and Tukey test. ‡ p < 0.05, resistance training effect; * p < 0.05 vs. Cc; † p < 0.05 vs. Cr; # p < 0.05 vs. Tc.
Figure 7
Figure 7
Protein levels and representative Western blots of follistatin (A) and myostatin (B) expression in the soleus muscle. Protein levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Isolated factors: Sedentary; Trained; Control; Creatine. Groups: Cc (n = 7): sedentary control; Cr (n = 7): sedentary creatine supplementation; Tc (n = 7): resistance training; Tcr (n = 7): resistance training combined with creatine supplementation. Two-way ANOVA and Tukey test. ‡ p < 0.05, resistance training effect; † p < 0.05 vs. Cr.
Figure 8
Figure 8
Protein levels and representative Western blots of ActRIIB in (A) gastrocnemius and (B) the soleus muscle. Protein levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Isolated factors: Sedentary; Trained; Control; Creatine. Groups: Cc (n = 7): sedentary control; Cr (n = 7): sedentary creatine supplementation; Tc (n = 7): resistance training; Tcr (n = 7): resistance training combined with creatine supplementation. Two-way ANOVA and Tukey test. ‡ p < 0.05, resistance training effect; * p < 0.05 vs. Cc.
Figure 9
Figure 9
Differential effects of ladder resistance training and creatine monohydrate supplementation on gastrocnemius (white portion) and soleus muscles. Resistance training-induced skeletal muscle remodeling is greater in the fast-twitch than the slow-twitch muscle type.
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