3.1. Effect of Culture Temperature on Viability and Growth of Hanwoo Cells and C2C12 Cells
Hanwoo satellite cells and C2C12 cells in flasks were cultured at 37 ℃ or 39 ℃ for 4 days and 5 days (Fig. 1a-a’’’), respectively. Live cell count and viability were measured. Doubling time was calculated to compare differences by culture temperature. For both Hanwoo cells and C2C12 cells, more live cells were obtained at 37 ℃ than at 39 ℃ based on live cell count. Cell viability was also found to be higher at 37 ℃ than at 39 ℃ (Fig. 1c-e). Heat stress is known to negatively affect skeletal muscle growth by converting energy and nutrients to body temperature maintenance. Acute heat stress has been found to affect cell function by increasing proteolysis and reducing protein synthesis (Febbraio, 2001). Basavaraj et al. (2019) have shown that when C2C12 cells are cultured at 37 ℃ or 39 ℃, total protein content of C2C12 cells cultured at 39 ℃ is lower than that of cells cultured at 37 ℃. In addition, the present study found that values of maximum resolution (MR) and spare resolution capacity (SRC) as indicators of mitochondrial function were significantly reduced in cells cultured at 39 ℃ than in cells cultured at 37 ℃, indicating that 39 ℃ caused damage to C2C12 cells.
Hanwoo cells and C2C12 cells confluent in flask were differentiated with differentiation medium. It was confirmed that Hanwoo cells differentiated at 37 ℃ had root canal formation after culturing for 2 days and 3 days. They were highly differentiated after culturing for 4 days and 5 days. The root canal tore off after culturing for 6 days. On the other hand, Hanwoo cells cultured at 39 ℃ were differentiated after 1 day of culture. They were highly differentiated after culturing for 2 days and 3 days. The root canal quickly tore off after culturing for 4 days. In the case of C2C12 cells, it was confirmed that root canal was formed after culturing for 3 days. The root canal was highly differentiated after culturing for 5 days. It tore off after culturing for 7 days. C2C12 cells cultured at 39 ℃ showed differentiation after 2 days. The root canal was highly differentiated after culturing for 4 days. It tore off after culturing for 7 days (Fig. 1b-b’’’).
3.2. Immunofluorescence Staining
Paired box 7 (Pax7), a transcription factor that controls the proliferation and differentiation of satellite cells during muscle formation, is an essential factor for muscle formation and differentiation of satellite cells into myoblasts (Maltzahn et al., 2013). Hoechst dye is the most popular dye that can distinguish cell nuclei due to their high affinity and specificity for DNA. To indirectly compare proliferative power of cultured Hanwoo cells and C2C12 cells, immunofluorescence staining was performed to observe cells and cell nuclei using Pax7 antibodies and Hoechst. When immunofluorescence staining was performed for Hanwoo cells cultured for 3 days using Pax7 antibodies and Hoechst, the blue color indicated nucleus and the green color indicated Pax7 (Fig. 2a-d’’). When the number of nuclei was calculated based on stained cells and nuclei, the distribution of Hanwoo cells cultured at 37 ℃ for 3 days appeared to be significantly higher (p < 0.05) that that cultured at 39 ℃. Distribution of C2C12 cells cultured at 37 ℃ for 4 days was also higher than that cultured at 39 ℃ (p < 0.05) (Fig. 2e).
Immunofluorescence staining was performed using myosin antibodies and Hoechst to indirectly compare differentiation power of differentiated Hanwoo cells and C2C12 cells. Myosin is a representative structural protein of muscle cells and functional protein that enables muscle contraction and relaxation as the main component of the myosin filament of root fiber. It is judged as an appropriate marker for the degree of cell differentiation (SIN et al., 1986). When dyed area was observed under a microscope, the blue color indicated the nucleus and the green color indicated myosin (Fig. 3a-d’’). When immunofluorescence staining was performed to calculate root width, number of nuclei, and fusion index, it was found that Hanwoo cells differentiated at 39 ℃ and 37 ℃, showed no significant difference in root thickness and fusion index of cells (Fig. 3f, g). However, cells differentiated at 37 ℃ had significantly higher (p < 0.05) number of nuclei (Fig. 3e). In addition, C2C12 cells differentiated for 4 days at 39 ℃ had a slightly higher number of nuclei that those differentiated for 4 days at 37 ℃ (p < 0.05) (Fig. 3e), although there was no significant difference in the thickness of differentiated root canal and fusion index between cells cultured at 39 ℃ and those cultured at 37 ℃ (Fig. 3f, g).
3.3. Western Blot
Western blot was performed to determine myosin and cytochrome C levels in Hanwoo cells differentiated for 3 days at 37 ℃ or 39 ℃ (Fig. 4a-d’). Cytochrome C, a water-soluble peripheral membrane protein, is known to be an essential component of mitochondrial respiration (Boyer et al., 1977). Protein levels was measured to determine the degree of differentiation into root canals. When western blot results of Hanwoo cells differentiated at 37 ℃ and 39 ℃ were compared, levels of myosin and cytochrome C protein in Hanwoo cells differentiated at 39 ℃ were measured to be relatively higher than those at 37 ℃, although such differences were not statistically significant (Fig. 4e, f). Levels of myosin in C2C12 cells differentiated at 39 ℃ for 3 days were also measured to be relatively higher than those at 37 ℃, although there was no significant difference in the relative fluorescence intensity of the band (Fig. 4g). A similar pattern was found for myotubes based on immunofluorescence staining. To confirm the difference in protein expression levels for cells cultured at 37 ℃ and 39 ℃, it is necessary to proceed with additional experiments using myosin and cytochrome C as well as other protein antibodies. According to Kanatous & Mammen (2010), high levels of actin and myosin are induced by electrical stimulation of C2C12 cells during differentiation. Even in this case, it is necessary to find the factor that can enhance the expression of these proteins. According to Liu & Brooks (2011), expression levels of several genes including transcription factors involved in mitochondrial biogenesis are increased during mild heat shock when the temperature not excessively high, suggesting that there is an additional reason for the absence of cytochrome C protein. Kang (2005) has shown that mild heat stress plays a beneficial role in organisms through facilitating growth factor-mediated cell survival and proliferation. Mild heat stress may act as one physicochemical signal to regulate activities of membrane proteins by affecting membrane fluidity. In additional experiments, C2C12 cells were cultured until passage no. 9 by subculture after proliferating for 4 days. After differentiation through DM for 5 days, western blotting result revealed that myosin protein expression in cells cultured at 39°C was significantly increased (p < 0.05) than in cells cultured at 37 °C. It seems that a certain amount of subculture and time are needed for a differentiation to occur in this experiment through a temperature change.
RT-qPCR was performed to confirm quantitative gene amounts of myogenin, myosin heavy chain (MyHC), myogenic factor 4 (MRF4), and myoglobin in Hanwoo cells differentiated for 3 days at 37 ℃ and 39 ℃. Myogenin is one of MRFs. It regulates proliferation and differentiation of satellite cells, precursor cells of myofibers (Kim, 2009). That is, it is expressed in the nucleus of activated satellite cells for the regeneration of myofibers. It affects the process of myofiber growth by regulating the formation of myoblasts and myotubes (Grounds, 1992). MRF4 is a transcription factor that regulates myogenic and amnion cells involved in skeletal muscle formation. It is known to be abundant in adult muscle cells (Yaniv et al., 2010). MyHC is also an important factor in determining muscle contractility. It is a late-stage marker of muscle cell differentiation. Its expression level increases as muscle cell differentiation proceeds (Park et al., 1999). Myoglobin is a cytoplasmic protein expressed only in cardiac muscle cells and oxidized skeletal muscle fibers (Park et al., 1999). As an oxygen storage protein in muscle, it serves to buffer the concentration of intracellular oxygen and promote intracellular oxygen diffusion when muscle activity increases (Ordway & Garry, 2004). In addition, myoglobin is a meat pigment that determines the red color of meat. It has a heme ring containing iron atoms. Thus, when we eat meat, we feel a quick taste of blood (Yancey et al., 2006). Looking at the flow of differentiation, when Pax7 and Pax3 are expressed in satellite cells, they express Myf5 and MyoD and differentiate into dividing myoblasts. These myoblasts are differentiated into myocytes while expressing myogenin and MRF4. Then myocytes stop dividing and form multinucleated myotubes (Ministry of Health and Welfare, 2017). When expression levels of the above gene were measured to determine the degree of formation of differentiated myotubes, gene levels of myogenin in cells differentiated at 39 ℃ were higher than those in cells differentiated at 37 °C, although these differences were not statistically significant. Gene levels of MyHC (p < 0.05), MRF4, and myoglobin (p < 0.01) were significantly higher in cells cultured at 39 ℃ than in cells cultured at 37 ℃ (Fig. 5a). In the case of C2C12, gene levels of MyHC in cells differentiated at 39 ℃ were higher than in cells differentiated at 37 °C. However, such differences were not statistically significant. Relative gene levels of myogenin and myoglobin were significantly higher in cells cultured at 39 ℃ than in cells cultured at 37 ℃ (p < 0.05) (Fig. 5b). MyHC, MRF4, and myoglobin gene levels in Korean beef muscle satellite cells and myogenin and myoglobin gene levels in C2C12 cells were significantly higher in cells cultured at 39 ℃ than in cells cultured at 37 ℃. However, there was no specific difference, meaning that genes involved in differentiation were present but not expressed. According to Zak et al. (2016) and Shi et al. (2016), exposure to a high temperature or a low temperature did not have a significant effect on human skeletal muscle gene expression or affect gene expression of MyHC type in pig back muscles. Thus, temperature can have various effects depending on the temperature difference and the organism affected by the temperature. Previous experimental results have shown that differentiation of C2C12 cells cultured at a high temperature for a long time into root canals can be inhibited by heat shock (Bolus et al., 2018). In the case of a mild heat stress at a low temperature, cell proliferation, differentiation, and immunity can respond positively (Park et al., 2005). In the case of myocytes of quail, differentiation at 39 ℃ for a long period of time can increase the length and diameter of myotubes than incubation at 37 ℃. Protein contents of slow myosin heavy chain isoform and cytochrome C oxidase subunit IV are higher than others (Chli et al., 2016). In addition, there are experimental results showing that the amount of PGC-1α protein varies according to the duration of heat stress and the temperature (Yamaguchi et al., 2009). As an additional experiment, it was judged that it was necessary to extend the culture period of Hanwoo muscle satellite cells and C2C12 cells through subculture, considering the fact that root canals were thick and plenty in numbers. In addition, the amount of protein was increased when C2C12 cells were subcultured to passage number 9. In addition, heat shock protein (HSP), a defense mechanism to protect itself from external stress (Ellis, 1987), is an important cellular temperature resistance protein to prevent denaturation of polypeptides under heat stress. When cells face stress, intracellular material transport and protein misfolding will occur. HSP can prevent these phenomena, increasing cell viability and helping cells overcome heat stress (Mishra & Palai, 2014). The fact that expression of a specific protein was not observed, but was expressed after some subculture, was presumed to be related to HSP. A more accurate analysis will be possible if HSP expression level according to temperature and incubation time is checked.