Krüger Martina, Linke Wolfgang A
Physiology and Biophysics Unit, University of Muenster, Schlossplatz 5, D-48149, Muenster, Germany.
J Muscle Res Cell Motil. 2006;27(5-7):435-44. doi: 10.1007/s10974-006-9090-5. Epub 2006 Aug 4.
Protein kinase-A (PKA) is activated during beta-adrenergic stimulation of the heart and is known to phosphorylate several sarcomeric proteins including the giant polypeptide titin. A PKA phosphorylation site on titin is located within the N2B-unique sequence, which is present in the elastic segment of the two major isoforms of cardiac titin, N2B and N2BA, but not in the skeletal-muscle isoforms of the N2A-type. In bovine and rat cardiomyocytes, PKA-mediated phosphorylation decreases passive tension (PT), an effect ascribed to titin phosphorylation. Whether titin is phosphorylated by PKA upon beta-adrenergic stimulation in human heart has not been shown to date. Here we report that PKA induces phosphorylation of N2B and N2BA titin isoforms, as well as a characteristic proteolytic fragment of titin, T2, in human donor hearts. The PKA-induced phosphorylation signals were stronger when myofilaments were first de-phosphorylated by protein phosphatase-1, suggesting inherent phosphorylation of titin in human heart. Titin phosphorylation was associated with a reduction in PT of skinned human cardiac strips; the relative decrease was higher at shorter than at longer physiological sarcomere lengths. The PKA-dependent PT drop was substantially larger when fibers were pre-treated with protein phosphatase-1, indicating that inherent phosphorylation of titin is important for the basal myocardial PT level. Mechanical measurements on isolated myofibrils from rat heart confirmed the PKA effect on passive stiffness and also showed a more pronounced effect in the presence of reducing agent, DTT. In contrast, PKA did not alter the PT of single skinned rat diaphragm muscle fibers; however, the kinase was still able to phosphorylate the skeletal N2A-titin isoform, which lacks the N2B-unique sequence. Thus, an additional phosphorylation site in titin may exist outside the cardiac N2B-unique sequence. We conclude that PKA mediates phosphorylation of titin in normal human myocardium. Titin phosphorylation lowers titin-based passive stiffness in heart but not in skeletal muscle.
蛋白激酶A（PKA）在心脏的β-肾上腺素能刺激过程中被激活，已知它能使包括巨型多肽肌联蛋白在内的多种肌节蛋白磷酸化。肌联蛋白上的一个PKA磷酸化位点位于N2B独特序列内，该序列存在于心脏肌联蛋白两种主要同工型N2B和N2BA的弹性段中，但不存在于N2A型的骨骼肌同工型中。在牛和大鼠心肌细胞中，PKA介导的磷酸化降低了被动张力（PT），这一效应归因于肌联蛋白的磷酸化。迄今为止，尚未证明在人类心脏中β-肾上腺素能刺激时肌联蛋白是否会被PKA磷酸化。在此我们报告，PKA在人类供体心脏中诱导N2B和N2BA肌联蛋白同工型以及肌联蛋白的一个特征性蛋白水解片段T2发生磷酸化。当肌丝首先被蛋白磷酸酶-1去磷酸化时，PKA诱导的磷酸化信号更强，这表明人类心脏中肌联蛋白存在固有磷酸化。肌联蛋白磷酸化与脱细胞人心脏条带的PT降低有关；在较短的生理肌节长度下，相对降低幅度高于较长肌节长度时。当纤维用蛋白磷酸酶-1预处理时，PKA依赖性的PT下降幅度更大，这表明肌联蛋白的固有磷酸化对基础心肌PT水平很重要。对大鼠心脏分离的肌原纤维进行的力学测量证实了PKA对被动僵硬度的影响，并且在存在还原剂二硫苏糖醇（DTT）的情况下显示出更明显的效应。相比之下，PKA并未改变单个脱细胞大鼠膈肌肌纤维的PT；然而，该激酶仍然能够使缺乏N2B独特序列的骨骼肌N2A-肌联蛋白同工型磷酸化。因此，肌联蛋白中可能存在一个位于心脏N2B独特序列之外的额外磷酸化位点。我们得出结论，PKA在正常人类心肌中介导肌联蛋白的磷酸化。肌联蛋白磷酸化降低了心脏中基于肌联蛋白的被动僵硬度，但在骨骼肌中则不然。
