Characterization Of The Different Expression Patterns And Transcription Regulations Of Calsarcins In Muscle

Calcineurin is a calmodulin dependent, calcium-activated protein phosphatase thatplays an important role in transducing calcium-dependent signals in a variety of celltypes. Calsarcins comprise a novel family of muscle-specific calcineurin-interactingproteins and play an important role in modulating both the function and substratespecificity of calcineurin in muscle cells. The expression of calsarcin-1 is restricted toslow-twitch skeletal muscle fibres, whereas that of both calsarcin-2 and calsarcin-3 isenriched in fast-twitch fibres. The skeletal muscle fiber type can be controlled by acalcineurin-dependent, transcriptional pathway, and the knowledge of which factorsinteract with calcineurin and their modulation (and thus, the influence of myofiber clusterpattern formation) will contribute to the understanding and improvement of pork quality.In this study, we cloned and characterized the porcine calsarcins and investigated theexpression and cellular localization of calsarcin-1 and calsarcin-2, in order to providesome information for the further study of calsarcins in relate to the porcine muscledevelopment and their applications in the animal breeding to improve pork quality andquantity. In addition, calsarcins are selectively expressed in slow or fast fibers, thetranscriptional control of this fiber type specified expression has also been partlyelucidated in this study. These results provide new insights into the molecularmechanisms governing transcription in specific muscle fiber cells. The main results areas follows:1) The full-length of coding sequence of porcine calsarcin-1, 2, 3 were cloned andtheeir mRNA and deduced protein sequences'characteristics were well defined.2) The IMpRH panel was employed in the mapping of these genes. The porcinecalsarcin-1, 2, 3 were assigned to porcine chromosome 8, 14 and 2, respectively.3) The real-time PCR confirmed the porcine calsarcins were muscle specific genes. Thetemporal expression data indicate that porcine calsarcin-1 and calsarcin-2 mRNAexpression patterns are similar during postnatal development but not duringembryonic development.4) The porcine calarcin-1 and calsarcin-2 GFP fusion plasmids were transfected withPK15 cells. The cellular localization of porcine calsarcin-2 protein is predominantlynuclear and is predicted to contain a nuclear localization signal, while calsarcin-1 hasa more complicated localization pattern.5) One calsarcin-1 SNP (A/G247) and one calsarcin-2 SNP (A/G451) were assessed by Restriction Fragment Length Polymorphism (RFLP) in almost 200 unrelated pigs,Genotyping results showed great variation in allele frequency between Chineseindigenous and introduced commercial breeds.6) The real-time RT-PCR analyses suggest that the expression of calarcin-1 andcalsarcin-2 is increased during the myogenic differentiation of mouse C2C12 cells.7) The promoter regions of these mouse calsarcin genes by long-PCR. In order toidentify their cis-regulatory elements, a series of deletion constructs from the5'-flanking regions were analyzed. For calsarcin-1, a construct containing aputative NF-κB site demonstrated the highest promoter activity levels. We thusspeculated that this potential binding site for NF-κB and other putative transcriptionfactor sites in this region would be important for the expression of the calsarcin-1gene. However, a putative NF-κB site is also located in the core region of calsarcin-2promoter, but our deletion reporter analysis did not reveal any significantinvolvement of this element in its transcriptional control.8) EMSA experiments authentized the NFkB site in calsarcin-1 promoter but not in thecalsarcin-2.9) Treatment with an NF-κB inhibitor (PDTC) resulted in a dramatic reduction inreporter gene activity of the deletion fragment of calsarcin-1 promoter, whichharbours the NF-κB binding element. In contrast, none of the reporter constructs ofcalsarcin-2 showed any PDTC responsiveness.10) The overexpression of NFATc4 induces both the calsarcin-1 and calsarcin-2promoters, whereas MEF2C only activates calsarcin-1 indicated that the regulationof the calsarcins is mediated also by the NFAT and MEF2 transcription factors.