In the present study, we first analyzed the expression levels of myogenic regulatory genes (MyoD, Myf5, MyoG and MRF4), myostatin-regulated genes (Mstna, Mstnb), and myoblast development-related genes (Pax7, Prmt5, Desmin and MYHC) in three adult zebrafish tissues (myocardium, dorsal muscle, and tail handle), and found that they participated in the development of zebrafish muscle; Secondly, by analyzing the expression profile of zebrafish during embryonic development, it was found that these 10 genes also participated in the regulation of the whole zebrafish embryonic development process, and it was determined that 30 days old was the key period for zebrafish muscle growth; Finally, it was found that water stress could increase the expression level of myogenic regulatory factors MyoD, MyoG, Pax7, Desmin in the dorsal muscles of zebrafish, and significantly reduce the mRNA level of myoblast development-related genes (Mstna and Mstnb). Furthmore, water flow stress increased energy metabolism of zebrafish muscle, such as ATP content, combined SDH-COX enzyme activities. Taken together, these results demonstrate that these 10 muscle growth regulator gene play an important role in the growth and development of fish, particularly in muscle development. Furthermore, our results demonstrate the significant impact of water stress on the expression levels of these genes.
In adult zebrafish, myogenic regulatory genes (MyoD), myostatin-regulated genes (Mstnb), myoblast development-related genes (Desmin and MYHC) were displayed high expression in zebrafish myocardium, dorsal muscle, and tail handle muscle. But Myf5 and Prmt5 were exhibited the highest expression levels in zebrafish myocardium. However, MYHC is displayed higher expression levels in dorsal muscle and tail handle muscle of zebrafish. Additionally, these 10 genes were highly expressed in the muscle effect stage during zebrafish embryonic development, and MYHC was exhibited the highest expression levels in this stage. Myogenic regulatory genes (MyoD) is involved in regulating the formation and differentiation of muscle cells(Lahmann et al, 2019). It is highly expressed in three types of muscle cells, indicating that MyoD is closely related to the structure and function of these three types of muscle cells. The myocardium is the main tissue that makes up the heart, responsible for its contraction and pumping of blood, ensuring the normal operation of the circulatory system. The high expression of MyoD gene may be involved in the formation of cardiomyocytes and the contraction of the heart. Dorsal muscle is one of the largest and most important muscle tissues in zebrafish, which is responsible for supporting and promoting body movement(Cutler et al, 2018). The high expression of MyoD gene may be related to the formation of dorsal muscle fibers and the regulation of contractility. The tail handle muscle, as the main muscle tissue of the tail, is responsible for regulating the movement and posture of the tail. The high expression of the MyoD gene may be involved in the regulation of the normal structure and motor function of the tail handle muscle. Desmin is an intermediate filament protein closely related to muscle tissue development and structural stability(Szymkowicz et al, 2018). The high expression of Desmin in zebrafish myocardium, dorsal muscle, and tail handle muscle may be involved in the tissue structure. This formation of a cellular scaffold provides support for the subsequent development and structural organization of muscle tissue. MYHC is the encoding gene for myosin heavy chain, a major component of muscle fibers(Gros et al, 2005; Relaix et al, 2005). The high expression of MYHC in three kinds of muscles and muscle effector stage of zebrafish may be associated with the development and functional performance of muscle fibers. MYHC is involved in muscle cell contraction and force generation and plays a crucial role in the formation and functional performance of muscle fibers(Gros et al, 2005). The high expression during muscle effector stage likely drives the formation of muscle fibers and further development of the tissue. Prmt5 is a methyltransferase involved in protein arginine methylation (Zhang et al, 2015). The high expression of Prmt5 in zebrafish myocardium and during the muscle effect stage of zebrafish may be associated with muscle cells differentiation and directed development. Prmt5 may participate in regulating the methylation modification of specific proteins, influencing the occurrence and regulation of cell differentiation (Chen et al, 2017; Dacwag et al, 2007). Therefore, the high expression of those genes may provide a foundation for the initiation and regulation of muscle cells differentiation processes.
Then, we investigated the relationship between muscle growth and expression of muscle development-related genes in fish. We observed two distinct populations with significantly different sizes during embryonic development in zebrafish embryos with the same genetic background and under the influence of biological and abiotic factors. We identified that the critical growth period of zebrafish to be around 30 day old, and these 30-day-old zebrafish were subjected for water flow exercise training. After 1 week of treatment, the expression of myogenic regulatory factors (MyoD and MyoG) was up-regulated in the dorsal muscle, while myostatin-regulated genes (Mstna and Mstnb) were significantly down-regulated in the dorsal muscle and up-regulated in the tail handle muscle. Additionally, the expression of myoblast development-related genes (Pax7 and Desmin) was significantly up-regulated in both the dorsal and tail handle muscles. Water flow is an important living environment factor for fish, as it affects fish movement and metabolism, ultimately influencing their growth and development(Mrnak et al, 2020; Ogata et al, 2000; Peteiro et al, 2019; Vanwalleghem et al, 2020). The contraction of skeletal muscles plays a crucial role in fish movement(Cutler et al, 2018), and muscle cells, particularly myofibers, are the primary cell type involved in this process (Li et al, 2014; Rescan, 2019). Early activation of the muscle cell proliferation through the chimeric growth stage is essential for muscle development and individual weight gain in fry and juvenile fish(McGuigan et al, 2004). Myogenic regulatory factors, such as MyoD, exhibited up-regulation in the dorsal and tail handle muscles after 1 and 2 weeks of stress, but down-regulation after 2 and 4 weeks. This suggests that water flow stress may activate muscle cell proliferation within the first two weeks. Myostatin-regulated genes, such as Mstna, showed down-regulation in the dorsal muscle and up-regulation in the tail handle muscle after 1 week of stress, followed by down-regulation in both muscles after 2 and 4 weeks. This indicates that appropriate water stress may also activate muscle stem cells. Pax7, a marker of muscle stem cells, exhibited up-regulation in both the dorsal and tail handle muscles after 1 week of stress, but down-regulation in the tail handle muscles after 2 and 4 weeks. These findings suggest that water flow stress can activate muscle stem cells and promote muscle development. Furthermore, swimming exercise improves skeletal muscle oxidative capacity(Brett et al, 2020), which is crucial for muscle function and dependent on ATP production, and also on the calcium cycle within the muscle cells(Little. et al, 2021). The study on highland sprinters also showed that only a certain intensity of exercise load caused significant changes in serum CK activity in athletes(Koch et al, 2014; Noyes et al, 2017). The observed changes in combined SDH-COX enzyme activities, which are indicators of oxidative capacity, suggest that water flow stress affects mitochondrial content in zebrafish muscle tissue(Anttila et al, 2008; Pinho et al, 2012; Rietbroek et al, 2007; Yamano et al, 2002). Increased ATP content, SDH activity, CK activity, and SDH-COX activity after water flow stress indicate enhanced energy metabolism in zebrafish.