Changes in proteolytic enzyme mRNAs relevant for meat quality during myogenesis of primary porcine satellite cells

Abstract

The objective was to study the regulation of proteolytic enzyme mRNA’s in porcine satellite cells during proliferation and differentiation. Beyond 80% confluence, cells were grown in absence or presence of 1 μM insulin. The temporal changes in transcription of micro molar-, milli molar- and muscle specific calpains (p94), calpastatin and caspase 3 in response to insulin was evaluated and myogenin transcription and creatine kinase activity was determined to indicate differentiation. The housekeeping genes (GAPDH and β-actin) were slightly affected by developmental stage and transiently by the insulin treatment but this did not affect the conclusions. The mRNA abundance of micro molar calpain, p94 and calpastatin increased from proliferation to differentiation. Milli molar calpain- and caspase 3-transcriptions were up-regulated in two steps, suggesting these two enzymes are involved in two distinct processes. Insulin stimulated differentiation as indicated by elevated creatine kinase activity but did not affect myogenin transcription. Insulin down-regulated milli molar calpain and calpastatin transcription and tended to down-regulate caspase 3 transcription but did not affect p94 or micro molar calpain. In conclusion, proteolytic enzymes relevant for post-mortem tenderisation are regulated at the gene level during myogenesis, indicating they are involved in muscle cell and muscle fibre development. Thus, a porcine satellite cell culture may be a model system to study regulation and relative contribution to proteolysis by the calpains.