Abstract
Skeletal muscle function is highly dependent on the ability to regenerate, however, during ageing or disease, the proliferative capacity is reduced, leading to loss of muscle function. We have previously demonstrated the presence of vitamin K2 in bovine skeletal muscles, but whether vitamin K has a role in muscle regulation and function is unknown. In this study, we used primary bovine skeletal muscle cells, cultured in monolayers in vitro, to assess a potential effect of vitamin K2 (MK-4) during myogenesis of muscle cells. Cell viability experiments demonstrate that the amount of ATP produced by the cells was unchanged when MK-4 was added, indicating viable cells. Cytotoxicity analysis show that MK-4 reduced the lactate dehydrogenase (LDH) released into the media, suggesting that MK-4 was beneficial to the muscle cells. Cell migration, proliferation and differentiation was characterised after MK-4 incubation using wound scratch analysis, immunocytochemistry and real-time PCR analysis. Adding MK-4 to the cells led to an increased muscle proliferation, increased gene expression of the myogenic transcription factor myod as well as increased cell migration. In addition, we observed a reduction in the fusion index and relative gene expression of muscle differentiation markers, with fewer complex myotubes formed in MK-4 stimulated cells compared to control cells, indicating that the MK-4 plays a significant role during the early phases of muscle proliferation. Likewise, we see the same pattern for the relative gene expression of collagen 1A, showing increased gene expression in proliferating cells, and reduced expression in differentiating cells. Our results also suggest that MK-4 incubation affect low density lipoprotein receptor-related protein 1 (LRP1) and the low-density lipoprotein receptor (LDLR) with a peak in gene expression after 45 min of MK-4 incubation. Altogether, our experiments show that MK-4 has a positive effect on muscle cell migration and proliferation, which are two important steps during early myogenesis.
Highlights
Vitamin K is a fat-soluble vitamin present in many foods including vegetables, fish, meat, cheese and eggs
Our experiments demonstrate that the amount of ATP produced by the cells was unchanged when MK-4 was added to the cells, indicating that the muscle cells were viable and metabolically active (Fig 1A)
It has previously been shown that MK-4 plays a function in mitochondria by speeding up the transfer of electrons, which results in more efficient ATP production [25]
Summary
Vitamin K is a fat-soluble vitamin present in many foods including vegetables, fish, meat, cheese and eggs. Comparison of the relative gene expression (fold change) between control (vehicle EtOH only) and treated cells (10 μM MK-4) was derived by using the comparative Ct method. It has been shown that after 72 hours of MK-4 treatment, there was a significant increase in muscle cell proliferation, compared to control cells (Fig 2A), with 10 μM MK4 showing the best effect (Figs 1B and 2A).
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