He H, Meyer M, Martin J L, McDonough P M, Ho P, Lou X, Lew W Y, Hilal-Dandan R, Dillmann W H
Department of Medicine, University of California San Diego, La Jolla 92093-0618, USA.
Circulation. 1999 Aug 31;100(9):974-80. doi: 10.1161/01.cir.100.9.974.
The delayed cardiac relaxation in failing hearts has been attributed to a reduced activity of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2). Phospholamban (PLB) inhibits SERCA2 activity and is therefore a potential target to improve the cardiac performance in heart failure.
Mutants of PLB (Adv/mPLB) or antisense RNA of PLB (Adv/asPLB) was expressed in cardiac myocytes by recombinant adenovirus, and their effects on SERCA2 activity and myocyte contractility were studied. One mPLB, K3E/R14E, pentamerized with endogenous PLB in neonatal myocytes and resulted in a 45% increase in the affinity of SERCA2 for Ca(2+) and 27% faster diastolic Ca(2+) decline as determined by SR (45)Ca uptake assays and by indo 1-facilitated Ca(2+) transient measurement, respectively. Edge-detection analysis of adult myocyte contractility showed a 74% increase in fractional shortening, accompanied by 115% increase in velocity of relengthening and 25% decrease in time to half-maximal relengthening. In parallel, infection of neonatal cardiac myocytes by Adv/asPLB decreased the endogenous PLB level by 54%, which was associated with a 35% increase in Ca(2+) affinity of SERCA2 and 21% faster diastolic Ca(2+) decline. However, in adult cardiac myocytes, Adv/asPLB failed to significantly alter the endogenous PLB level, the SERCA2 activity, or most of the contractile parameters.
K3E/R14E is a dominant negative mutant of PLB that disrupts the structural integrity and function of the endogenous PLB and consequently enhances SERCA2 activity and myocyte contractility. In neonatal myocytes, the decrease in steady-state abundance of PLB by asPLB also leads to increased SERCA2 activity.
衰竭心脏中延迟的心脏舒张归因于肌浆网Ca(2+)-ATP酶（SERCA2）活性降低。受磷蛋白（PLB）抑制SERCA2活性，因此是改善心力衰竭心脏功能的潜在靶点。
通过重组腺病毒在心肌细胞中表达PLB突变体（Adv/mPLB）或PLB反义RNA（Adv/asPLB），并研究它们对SERCA2活性和心肌细胞收缩性的影响。一种mPLB，K3E/R14E，在新生心肌细胞中与内源性PLB形成五聚体，通过肌浆网（45）Ca摄取试验和indo 1介导的Ca(2+)瞬时测量分别确定，导致SERCA2对Ca(2+)的亲和力增加45%，舒张期Ca(2+)下降速度加快27%。对成年心肌细胞收缩性的边缘检测分析显示缩短分数增加74%，同时再延长速度增加115%，达到最大再延长时间的一半减少25%。同时，Adv/asPLB感染新生心肌细胞使内源性PLB水平降低54%，这与SERCA2的Ca(2+)亲和力增加35%和舒张期Ca(2+)下降速度加快21%相关。然而，在成年心肌细胞中，Adv/asPLB未能显著改变内源性PLB水平、SERCA2活性或大多数收缩参数。
K3E/R14E是PLB的显性负突变体，破坏内源性PLB的结构完整性和功能，从而增强SERCA2活性和心肌细胞收缩性。在新生心肌细胞中，asPLB使PLB的稳态丰度降低也导致SERCA2活性增加。
