ABSTRACT
UNLABELLED: Entry of calcium into cardiomyocyte via L-type calcium channel (LTCC) is fundamental to cardiac contraction. CACNA1C, a type of LTCC and a hallmark of a matured ventricular myocyte, is developmentally regulated. Here, we identified 138 potential transcription factors by a comparative genomic study on 5-kb promoter regions of CACNA1C gene across eight vertebrate species, and showed that six factors were developmentally regulated with the expression of Cacna1c in mouse P19cl6 in vitro cardiomyocyte differentiation model. We further demonstrated that the nuclear factor of activated T cells 5 (Nfat5) bound to a consensus sequence TGGAAGCGTTC and activated the transcription of Cacna1c. The siRNA-mediated knockdown of Nfat5 suppressed the expression of Cacna1c and decreased L-type calcium current in mouse neonatal cardiomyocytes. Furthermore, morpholino-mediated knockdown of nfat5 in zebrafish prohibited the expression of cacna1c and resulted in a non-contractile ventricle, while over-expression of either cacna1c or nfat5 rescued this impaired phenotype. Thus, NFAT5-mediated expression of CACNA1C is evolutionarily conserved and critical for cardiac electrophysiological development and maturation of cardiomyocyte. KEY MESSAGE: Nfat5 binds to a consensus sequence TGGAAGCGTTC in the promoter of Cacna1c. Nfat5 activates the transcription of Cacna1c. Nfat5 knockdown suppresses Cacna1c expression, decreases L-type calcium current, and results in non-beating ventricle. NFAT5-mediated expression of CACNA1C is evolutionarily conserved. NFAT5-mediated CACNA1C expression is critical for cardiac electrophysiological development and maturation.
Subject(s)
Calcium Channels, L-Type/genetics , Cell Differentiation , Electrophysiological Phenomena , Gene Expression Regulation , Myocytes, Cardiac/cytology , Myocytes, Cardiac/physiology , Transcription Factors/metabolism , Animals , Base Sequence , Binding Sites , Biomarkers , Cell Differentiation/genetics , Cell Line , Cells, Cultured , Consensus Sequence , Electrophysiological Phenomena/genetics , Gene Knockdown Techniques , Humans , Mice , Promoter Regions, Genetic , Protein Binding , Rats , Transcription Factors/genetics , ZebrafishABSTRACT
Zebrafish is an important vertebrate model organism for the study of embryonic development and the underlying genetic mechanism. Numerous mutants and transgenics have been generated in recent years, long-term and safe storage of these fish lines is of crucial importance for every zebrafish community/lab. Sperm cryopreservation and revival has become a preferred method for this purpose, which provides extra and reliable security as a backup for the cost-effective maintenance of genetic stocks in addition to reducing space demanding for housing large amount of live fish. This is especially critical for invaluable fish lines against accidental loss. Generally, the sperm are obtained by either squeezing the male fish or dissecting out and homogenizing the testes, then they are mixed with the freezing medium before gradually frozen as aliquots in liquid nitrogen. They can be easily revived through in vitro fertilization whenever necessary. This technique was introduced into zebrafish research three decades ago and has gradually become mature and more reliable following the im-provement of many critical factors and steps, including cryoprotectants and conditions for freezing and revival. Base on pioneers' work, our lab has established and improved a simple method for sperm cryopreservation and revival which shows high recovery rate after relatively long storage time. Here we briefly summarize the history and development of the methods for sperm cryopreservation and revival in zebrafish and present a brief protocol for the practice of sperm cryopreservation and revival, which is routinely used in our lab.
