Konhilas John P, Irving Thomas C, Wolska Beata M, Jweied Eias E, Martin Anne F, Solaro R John, de Tombe Pieter P
Program in Cardiovascular Sciences, Department of Physiology and Biophysics, Section of Cardiology, University of Illinois at Chicago, College of Medicine, Chicago, IL 60612, USA.
J Physiol. 2003 Mar 15;547(Pt 3):951-61. doi: 10.1113/jphysiol.2002.038117. Epub 2003 Jan 24.
Cyclic AMP-dependent protein kinase (PKA) targets contractile proteins, troponin-I (TnI) and myosin binding protein C (MyBP-C) in the heart and induces a decrease in myofilament Ca2+ sensitivity. Yet, the effect of sarcomere length (SL) change on Ca2+ sensitivity (length-dependent activation: LDA) following PKA-dependent phosphorylation is not clear. To clarify the role of PKA-dependent phosphorylation of TnI and MyBP-C on LDA in the heart, we examined LDA in skinned myocytes from a non-transgenic (NTG) and a transgenic murine model in which the native cardiac isoform (cTnI) was completely replaced by the slow skeletal isoform of TnI (ssTnI-TG) lacking the phosphorylation sites for PKA, while retaining PKA sites on MyBP-C. In NTG myocytes, PKA treatment decreased Ca2+ sensitivity at each SL, but enhanced the impact of SL change on Ca2+ sensitivity. Despite a greater sensitivity to Ca2+ and a reduction in LDA, neither Ca2+ responsiveness nor LDA was affected by PKA treatment in ssTnI-TG myocytes. To determine whether the above observations could be explained by the lateral separation between thick and thin filaments, as suggested by others, we measured interfilament spacing by X-ray diffraction as a function of SL in skinned cardiac trabeculae in the passive state from both NTG and ssTnI-TG models before and following treatment with PKA. Phosphorylation by PKA increased lattice spacing at every SL in NTG trabeculae. However, the relationship between SL and myofilament lattice spacing in ssTnI-TG was markedly shifted downward to an overall decreased myofilament lattice spacing following PKA treatment. We conclude: (1) PKA-dependent phosphorylation enhances length-dependent activation in NTG hearts; (2) replacement of native TnI with ssTnI increases Ca2+ sensitivity of tension but reduces length-dependent activation; (3) MyBP-C phosphorylation by PKA does not alter calcium responsiveness and induces a decrease in myofilament lattice spacing at all sarcomere lengths and (4) length-dependent activation in the heart cannot be entirely explained by alterations in myofilament lattice spacing.
环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）作用于心脏中的收缩蛋白肌钙蛋白I（TnI）和肌球蛋白结合蛋白C（MyBP-C），并导致肌丝对钙离子的敏感性降低。然而，在PKA依赖性磷酸化后，肌节长度（SL）变化对钙离子敏感性（长度依赖性激活：LDA）的影响尚不清楚。为了阐明TnI和MyBP-C的PKA依赖性磷酸化在心脏LDA中的作用，我们在非转基因（NTG）和转基因小鼠模型的脱细胞心肌细胞中检测了LDA。在转基因小鼠模型中，天然心脏同工型（cTnI）被缺乏PKA磷酸化位点的慢肌骨骼肌TnI同工型（ssTnI-TG）完全取代，而MyBP-C上保留了PKA位点。在NTG心肌细胞中，PKA处理降低了每个SL下的钙离子敏感性，但增强了SL变化对钙离子敏感性的影响。尽管ssTnI-TG心肌细胞对钙离子的敏感性更高且LDA降低，但PKA处理对其钙离子反应性和LDA均无影响。为了确定上述观察结果是否如其他人所建议的那样，可以用粗细肌丝之间的横向间距来解释，我们通过X射线衍射测量了NTG和ssTnI-TG模型在PKA处理前后被动状态下脱细胞心脏小梁中肌丝间距随SL的变化。PKA磷酸化增加了NTG小梁中每个SL下的晶格间距。然而，在PKA处理后，ssTnI-TG中SL与肌丝晶格间距之间的关系明显向下移动，导致肌丝晶格间距总体减小。我们得出以下结论：（1）PKA依赖性磷酸化增强了NTG心脏中的长度依赖性激活；（2）用ssTnI替代天然TnI可增加张力的钙离子敏感性，但降低长度依赖性激活；（3）PKA对MyBP-C的磷酸化不会改变钙反应性，并导致所有肌节长度下的肌丝晶格间距减小；（4）心脏中的长度依赖性激活不能完全用肌丝晶格间距的改变来解释。
