Modulation of Titin-Based Stiffness in Hypertrophic Cardiomyopathy via Protein Kinase D
Modulation of Titin-Based Stiffness in Hypertrophic Cardiomyopathy via Protein Kinase D
复制标题
DOI：
10.3389/fphys.2020.00240
复制
发表时间：
2020-04-15
影响因子：
4
通讯作者：
Hamdani, Nazha
中科院分区：
文献类型：
--
作者：
Herwig, Melissa;Kolijn, Detmar;Hamdani, Nazha
巨蛋白titin在肌节中执行结构保存功能，对心肌细胞的被动僵硬（f -被动）很重要。蛋白激酶D （PKD）酶在调节心肌收缩、肥大和重构中起着至关重要的作用。PKD磷酸化肌丝蛋白，但尚不清楚巨蛋白titin是否也是PKD的底物。在这里，我们的目的是确定PKD是否磷酸化titin，从而调节正常和衰竭心肌的心肌细胞f -被动。在心肌细胞特异性PKD敲除小鼠（cKO）和人类心脏中，使用磷酸化丝氨酸/苏氨酸和磷酸化特异性titin抗体的免疫印迹法评估了titin的磷酸化。通过稳定同位素标记的细胞培养（SILAC）小鼠心脏蛋白裂解物与野生型对照（WT）或cKO小鼠心脏分离物混合，通过质谱法定量体内pkd依赖性位点特异性titin磷酸化。记录单渗透心肌细胞在PKD和HSP27给药前后的f -被动水平。与WT心脏相比，cKO的全titin磷酸化水平降低。定量质谱法在titin的z盘区、a带区和m带区检测到多个保守的pkd依赖性磷酸位点，而在弹性titin i带区检测到的许多pkd依赖性磷酸位点在cKO中显著减少。对titin位点特异性磷酸化的分析显示，与匹配的WT心脏相比，cKO的磷酸化没有改变或上调。与WT心肌细胞相比，cKO心肌细胞的f -被动水平升高，PKD降低了WT和cKO心肌细胞的f -被动水平。与对照心脏相比，肥厚性心肌病（HCM）患者的心肌细胞表现出更高的f -被动，而PKD治疗后的f -被动水平显著降低。此外，我们发现与对照心脏相比，HCM中camkii依赖的titin位点的磷酸化水平更高。HCM心脏中PKD底物HSP27的表达和磷酸化水平升高，这与PKD表达和磷酸化水平升高有关。HSP27在HCM中远离肌肉z盘和i带的重新定位表明，HSP27未能发挥其对titin可扩展性的保护作用。然而，这种保护作用可以通过给药HSP27恢复，HCM心肌细胞中的F-passive显著降低。这些发现确立了PKDin调节健康和患病心脏舒张被动特性的一个以前未知的作用。
The giant protein titin performs structure-preserving functions in the sarcomere and is important for the passive stiffness (F-passive) of cardiomyocytes. Protein kinase D (PKD) enzymes play crucial roles in regulating myocardial contraction, hypertrophy, and remodeling. PKD phosphorylates myofilament proteins, but it is not known whether the giant protein titin is also a PKD substrate. Here, we aimed to determine whether PKD phosphorylates titin and thereby modulates cardiomyocyte F-passive in normal and failing myocardium. The phosphorylation of titin was assessed in cardiomyocyte-specific PKD knock-out mice (cKO) and human hearts using immunoblotting with a phosphoserine/threonine and a phosphosite-specific titin antibody. PKD-dependent site-specific titin phosphorylation in vivo was quantified by mass spectrometry using stable isotope labeling by amino acids in cell culture (SILAC) of SILAC-labeled mouse heart protein lysates that were mixed with lysates isolated from hearts of either wild-type control (WT) or cKO mice. F-passive of single permeabilized cardiomyocytes was recorded before and after PKD and HSP27 administration. All-titin phosphorylation was reduced in cKO compared to WT hearts. Multiple conserved PKD-dependent phosphosites were identified within the Z-disk, A-band and M-band regions of titin by quantitative mass spectrometry, and many PKD-dependent phosphosites detected in the elastic titin I-band region were significantly decreased in cKO. Analysis of titin site-specific phosphorylation showed unaltered or upregulated phosphorylation in cKO compared to matched WT hearts. F-passive was elevated in cKO compared to WT cardiomyocytes and PKD administration lowered F-passive of WT and cKO cardiomyocytes. Cardiomyocytes from hypertrophic cardiomyopathy (HCM) patients showed higher F-passive compared to control hearts and significantly lower F-passive after PKD treatment. In addition, we found higher phosphorylation at CaMKII-dependent titin sites in HCM compared to control hearts. Expression and phosphorylation of HSP27, a substrate of PKD, were elevated in HCM hearts, which was associated with increased PKD expression and phosphorylation. The relocalization of HSP27 in HCM away from the sarcomeric Z-disk and I-band suggested that HSP27 failed to exert its protective action on titin extensibility. This protection could, however, be restored by administration of HSP27, which significantly reduced F-passive in HCM cardiomyocytes. These findings establish a previously unknown role for PKDin regulating diastolic passive properties of healthy and diseased hearts.