Temperature-dependent development of porcine skeletal muscle cells in an <em>in-vitro</em> model

Abstract

Satellite cells are quiescent myogenic stem cells and it is possible to derive an in-vitro model of these cells. This in-vitro model can be used to represent myogenesis using proliferating myoblasts and differentiating myotubes. Due to climate change and the associated occurrence of extreme temperature events, various cultivation temperatures are currently being investigation regarding the stimulation of myogenesis. Thermal stress can be triggered by both cold and heat and can affect farm animals’ muscle growth, health, and welfare. The aim of the present PhD thesis is to establish a satellite-cell-derived porcine primary muscle cell culture and investigate the effects of various cultivation temperatures on muscle cell growth in this in -vitro model. Three studies were published for this purpose. The first study describes the comparative investigation of three muscle cell pools, along with their unpooled muscle cells. In piglets, the perinatal/early postnatal period is associated with high mortality, partially due to their immature thermoregulation. To account for the thermoregulatory properties of donor piglets, two cell pools were established using the M. rhomboideus of thermolabile 5-day-old (Pool 5) and thermostable 20-day-old piglets (Pool 20). No differences were found between the cell pools and their corresponding unpooled muscle cells regarding proliferative growth behavior and differentiation ability and demonstrate that the use of cell pools is an appropriate method. Two subsequent studies investigated the effect of permanent cultivation below (35°C) and above (39°C, 41°C) the standard temperature of 37°C on the two muscle cell pools. All temperatures were found to be appropriate for the proliferation of porcine muscle cells. Muscle cells cultured at 35°C are smaller and express more of satellite cell marker PAX7’s mRNA. These results suggest that they are inhibited in their myogenesis as compared to myoblasts cultivated at higher temperatures. In addition, genes related to biological processes and signaling pathways of the immune system, were found which contribute to increased metabolic flux. When cultured at the physiological body temperature of piglets (39°C), as compared with 37°C, increased DNA content and differences in the expression of genes associated with DNA replication and cell growth were revealed. Despite similar growth characteristics, these differences indicate an adaptive mechanism and temperature threshold. A cultivation at 41°C demonstrated a heat-stress response, as well as the downregulation of many biological processes and signaling pathways related to proliferative ability. These cells were larger and expressed less of “early” muscle-specific transcription factors, suggesting a more differentiated cell type as compared to 37°C. The cell pools also reflect the different thermoregulatory properties of their donor piglets. Cells from Pool 5 are smaller and express more PAX7 than those from Pool 20. The most transcription-related differences between the two muscle cell pools were found at the standard temperature of 37°C, suggesting that both pools can access their full developmental potential at this temperature.