Division of Cardiology, Department of Medicine and Clinical Science, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan.
Cardiovasc Res. 2011 Jan 1;89(1):79-88. doi: 10.1093/cvr/cvq252. Epub 2010 Jul 31.
protein phosphatase 1 (PP1) is the major isotype of serine/threonine phosphatase in cardiomyocytes, and its activity has been thought to be important for heart failure progression. The PP1 catalytic subunits consist of three distinct genes, PP1α, PP1β/δ, and PP1γ. To date, the function of each PP1 isoform is not well characterized in cardiomyocytes. We sought to determine the functional contribution of each PP1 isoform to sarcoplasmic reticulum (SR)-mediated Ca(2+) cycling in isolated adult rat cardiomyocytes.
adenoviral vectors encoding short hairpin RNA for each PP1 isoform were transfected into isolated rat cardiomyocytes, and this was followed by analysis of cell shortening, Ca(2+) transients, and the phosphorylation levels of Ca(2+) regulatory proteins. Physical interactions between each PP1 isoform and SR Ca(2+) regulatory proteins were characterized in isolated cardiomyocytes expressing green fluorescent protein (GFP)-tagged PP1 catalytic subunits, and also in canine junctional and longitudinal SR preparations. Successful PP1 isoform knockdown was achieved for each isoform without affecting the expression of the other isoforms. PP1β knockdown most significantly enhanced the Ca(2+) transient and cell shortening by augmenting phospholamban (PLN) phosphorylation at baseline and with low-dose isoproterenol stimulation (10 nM). Interestingly, PP1β was preferentially associated with sarco-endoplasmic ATPase and PLN in GFP-PP1-transfected cardiomyocytes, as well as in canine longitudinal SR preparations.
these findings indicate that PP1β is the most significant PP1 isoform involved in regulating SR Ca(2+) cycling in rat cardiomyocytes.
蛋白磷酸酶 1（PP1）是心肌细胞中丝氨酸/苏氨酸磷酸酶的主要同工型，其活性被认为对心力衰竭的进展很重要。PP1 的催化亚基由三个不同的基因组成，PP1α、PP1β/δ 和 PP1γ。迄今为止，每种 PP1 同工型在心肌细胞中的功能尚未得到很好的描述。我们试图确定每种 PP1 同工型对分离的成年大鼠心肌细胞肌浆网（SR）介导的 Ca2+循环的功能贡献。
腺病毒载体编码针对每种 PP1 同工型的短发夹 RNA 转染到分离的大鼠心肌细胞中，然后分析细胞缩短、Ca2+瞬变和 Ca2+调节蛋白的磷酸化水平。在表达 GFP 标记的 PP1 催化亚基的分离心肌细胞中以及在犬连接和纵向 SR 制剂中，表征了每种 PP1 同工型与 SR Ca2+调节蛋白的物理相互作用。成功实现了每种同工型的 PP1 同工型敲低，而不影响其他同工型的表达。PP1β 敲低在基础状态和低剂量异丙肾上腺素刺激（10 nM）下通过增强肌球蛋白轻链磷酸酶（PLN）磷酸化最显著地增强了 Ca2+瞬变和细胞缩短。有趣的是，PP1β 在 GFP-PP1 转染的心肌细胞以及犬纵向 SR 制剂中与肌浆网-内质网 ATP 酶和 PLN 优先相关。
这些发现表明，PP1β 是调节大鼠心肌细胞 SR Ca2+循环的最重要的 PP1 同工型。
