蛋白激酶D(PKD)酶在调节心肌收缩中起重要作用,肥大,和重塑。PKD磷酸化的蛋白质之一是肌动蛋白,参与肌丝功能。在这项研究中,我们旨在研究氧化应激条件下PKD在心肌细胞功能中的作用。要做到这一点,我们使用了心肌细胞特异性敲除Prkd1的小鼠,该小鼠编码PKD1(Prkd1loxP/loxP;αMHC-Cre;PKD1cKO),以及野生型同窝动物对照(Prkd1loxP/loxP;WT)。我们从PKD1cKO小鼠中分离出透化的心肌细胞,发现它们表现出增加的被动僵硬(Fpassive),这与增加的titin氧化有关,但肌动蛋白泛素化没有变化。此外,PKD1cKO小鼠显示肌丝钙(Ca2)敏感性(pCa50)增加,最大Ca2激活张力降低。这些变化伴随着小肌丝蛋白心肌肌球蛋白结合蛋白C(cMyBPC)的氧化增加和磷酸化减少,以及肌钙蛋白I(TnI)不同磷酸位点的磷酸化水平改变。当我们用还原型谷胱甘肽(GSH)处理分离的透化心肌细胞时,增加的Fpassive和pCa50以及减少的最大Ca2激活张力被逆转。这表明肌丝蛋白氧化有助于心肌细胞功能障碍。此外,PKD1cKO小鼠表现出氧化应激增加,促炎标志物白细胞介素(IL)-6,IL-18和肿瘤坏死因子α(TNF-α)表达增加.氧化应激和炎症都通过抑制PKD1cKO小鼠肌细胞中雷帕霉素的哺乳动物靶标(mTOR)而导致微管相关蛋白1轻链3(LC3)-II水平和热休克反应的增加。这些发现揭示了PKD1在调节舒张被动特性中的先前未知的作用,肌丝Ca2+敏感性,和在氧化应激条件下的最大Ca2激活张力。最后,我们强调了PKD1在自噬中维持氧化应激和炎症平衡的重要性,以及心肌细胞功能。
Protein kinase D (PKD) enzymes play important roles in regulating myocardial contraction, hypertrophy, and remodeling. One of the proteins phosphorylated by PKD is titin, which is involved in myofilament function. In this study, we aimed to investigate the role of PKD in cardiomyocyte function under conditions of oxidative stress. To do this, we used mice with a cardiomyocyte-specific knock-out of Prkd1, which encodes PKD1 (Prkd1loxP/loxP; αMHC-Cre; PKD1 cKO), as well as wild type littermate controls (Prkd1loxP/loxP; WT). We isolated permeabilized cardiomyocytes from PKD1 cKO mice and found that they exhibited increased passive stiffness (Fpassive), which was associated with increased oxidation of titin, but showed no change in titin ubiquitination. Additionally, the PKD1 cKO mice showed increased myofilament calcium (Ca2+) sensitivity (pCa50) and reduced maximum Ca2+-activated tension. These changes were accompanied by increased oxidation and reduced phosphorylation of the small myofilament protein cardiac myosin binding protein C (cMyBPC), as well as altered phosphorylation levels at different phosphosites in troponin I (TnI). The increased Fpassive and pCa50, and the reduced maximum Ca2+-activated tension were reversed when we treated the isolated permeabilized cardiomyocytes with reduced glutathione (GSH). This indicated that myofilament protein oxidation contributes to cardiomyocyte dysfunction. Furthermore, the PKD1 cKO mice exhibited increased oxidative stress and increased expression of pro-inflammatory markers interleukin (IL)-6, IL-18, and tumor necrosis factor alpha (TNF-α). Both oxidative stress and inflammation contributed to an increase in microtubule-associated protein 1 light chain 3 (LC3)-II levels and heat shock response by inhibiting the mammalian target of rapamycin (mTOR) in the PKD1 cKO mouse myocytes. These findings revealed a previously unknown role for PKD1 in regulating diastolic passive properties, myofilament Ca2+ sensitivity, and maximum Ca2+-activated tension under conditions of oxidative stress. Finally, we emphasized the importance of PKD1 in maintaining the balance of oxidative stress and inflammation in the context of autophagy, as well as cardiomyocyte function.