Randall Centre for Cell and Molecular Biophysics, and British Heart Foundation Centre of Research Excellence, King's College London, London, United Kingdom.
Randall Centre for Cell and Molecular Biophysics, and British Heart Foundation Centre of Research Excellence, King's College London, London, United Kingdom.
J Biol Chem. 2023 Jan;299(1):102767. doi: 10.1016/j.jbc.2022.102767. Epub 2022 Dec 5.
PKA-mediated phosphorylation of sarcomeric proteins enhances heart muscle performance in response to β-adrenergic stimulation and is associated with accelerated relaxation and increased cardiac output for a given preload. At the cellular level, the latter translates to a greater dependence of Ca sensitivity and maximum force on sarcomere length (SL), that is, enhanced length-dependent activation. However, the mechanisms by which PKA phosphorylation of the most notable sarcomeric PKA targets, troponin I (cTnI) and myosin-binding protein C (cMyBP-C), lead to these effects remain elusive. Here, we specifically altered the phosphorylation level of cTnI in heart muscle cells and characterized the structural and functional effects at different levels of background phosphorylation of cMyBP-C and with two different SLs. We found Ser22/23 bisphosphorylation of cTnI was indispensable for the enhancement of length-dependent activation by PKA, as was cMyBP-C phosphorylation. This high level of coordination between cTnI and cMyBP-C may suggest coupling between their regulatory mechanisms. Further evidence for this was provided by our finding that cardiac troponin (cTn) can directly interact with cMyBP-C in vitro, in a phosphorylation- and Ca-dependent manner. In addition, bisphosphorylation at Ser22/Ser23 increased Ca sensitivity at long SL in the presence of endogenously phosphorylated cMyBP-C. When cMyBP-C was dephosphorylated, bisphosphorylation of cTnI increased Ca sensitivity and decreased cooperativity at both SLs, which may translate to deleterious effects in physiological settings. Our results could have clinical relevance for disease pathways, where PKA phosphorylation of cTnI may be functionally uncoupled from cMyBP-C phosphorylation due to mutations or haploinsufficiency.
PKA 介导的肌球蛋白结合蛋白 C（cMyBP-C）磷酸化增强心脏肌肉对β-肾上腺素能刺激的反应性，与给定前负荷下的舒张加速和心输出量增加有关。在细胞水平上，后者表现为 Ca 敏感性和最大力对肌节长度（SL）的依赖性增加，即增强的长度依赖性激活。然而，PKA 对最显著的肌球蛋白结合蛋白 C（cMyBP-C）磷酸化靶标肌钙蛋白 I（cTnI）的磷酸化如何导致这些效应的机制仍不清楚。在这里，我们专门改变了心肌细胞中 cTnI 的磷酸化水平，并在不同背景 cMyBP-C 磷酸化水平和两种不同 SL 下，对其结构和功能效应进行了特征描述。我们发现 cTnI 的 Ser22/23 双磷酸化对于 PKA 增强长度依赖性激活是必不可少的，cMyBP-C 的磷酸化也是如此。cTnI 和 cMyBP-C 之间的这种高度协调可能表明它们的调节机制之间存在耦合。我们的研究结果进一步提供了证据，即我们发现心肌肌钙蛋白（cTn）可以在磷酸化和 Ca 依赖性的方式下直接与 cMyBP-C 在体外相互作用。此外，在存在内源性磷酸化 cMyBP-C 的情况下，Ser22/Ser23 的双磷酸化增加了长 SL 时的 Ca 敏感性。当 cMyBP-C 去磷酸化时，cTnI 的双磷酸化增加了两种 SL 下的 Ca 敏感性和降低了协同性，这可能在生理环境下产生有害影响。我们的研究结果可能与疾病途径有关，由于突变或单倍不足，PKA 对 cTnI 的磷酸化可能与 cMyBP-C 的磷酸化在功能上失去偶联。
