Kovács Árpád, Herwig Melissa, Budde Heidi, Delalat Simin, Kolijn Detmar, Bódi Beáta, Hassoun Roua, Tangos Melina, Zhazykbayeva Saltanat, Balogh Ágnes, Czuriga Dániel, Van Linthout Sophie, Tschöpe Carsten, Dhalla Naranjan S, Mügge Andreas, Tóth Attila, Papp Zoltán, Barta Judit, Hamdani Nazha
Division of Clinical Physiology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary.
Institut für Forschung und Lehre (IFL) Molecular and Experimental Cardiology, St. Josef-Hospital, Ruhr University Bochum, 44801 Bochum, Germany.
Antioxidants (Basel). 2021 Jun 16;10(6):964. doi: 10.3390/antiox10060964.
Standard heart failure (HF) therapies have failed to improve cardiac function or survival in HF patients with right ventricular (RV) dysfunction suggesting a divergence in the molecular mechanisms of RV vs. left ventricular (LV) failure. Here we aimed to investigate interventricular differences in sarcomeric regulation and function in experimental myocardial infarction (MI)-induced HF with reduced LV ejection fraction (HFrEF). MI was induced by LAD ligation in Sprague-Dawley male rats. Sham-operated animals served as controls. Eight weeks after intervention, post-ischemic HFrEF and Sham animals were euthanized. Heart tissue samples were deep-frozen stored ( = 3-5 heart/group) for ELISA, kinase activity assays, passive stiffness and Ca-sensitivity measurements on isolated cardiomyocytes, phospho-specific Western blot, and PAGE of contractile proteins, as well as for collagen gene expressions. Markers of oxidative stress and inflammation showed interventricular differences in post-ischemic rats: TGF-β1, lipid peroxidation, and 3-nitrotyrosine levels were higher in the LV than RV, while hydrogen peroxide, VCAM-1, TNFα, and TGF-β1 were increased in both ventricles. In addition, nitric oxide (NO) level was significantly decreased, while FN-1 level was significantly increased only in the LV, but both were unchanged in RV. CaMKII activity showed an 81.6% increase in the LV, in contrast to a 38.6% decrease in the RV of HFrEF rats. Cardiomyocyte passive stiffness was higher in the HFrEF compared to the Sham group as evident from significantly steeper F vs. sarcomere length relationships. In vitro treatment with CaMKIIδ, however, restored cardiomyocyte passive stiffness only in the HFrEF RV, but had no effect in the HFrEF LV. PKG activity was lower in both ventricles in the HFrEF compared to the Sham group. In vitro PKG administration decreased HFrEF cardiomyocyte passive stiffness; however, the effect was more pronounced in the HFrEF LV than HFrEF RV. In line with this, we observed distinct changes of titin site-specific phosphorylation in the RV vs. LV of post-ischemic rats, which may explain divergent cardiomyocyte stiffness modulation observed. Finally, Ca-sensitivity of RV cardiomyocytes was unchanged, while LV cardiomyocytes showed increased Ca-sensitivity in the HFrEF group. This could be explained by decreased Ser-282 phosphorylation of cMyBP-C by 44.5% in the RV, but without any alteration in the LV, while Ser-23/24 phosphorylation of cTnI was decreased in both ventricles in the HFrEF vs. the Sham group. Our data pointed to distinct signaling pathways-mediated phosphorylations of sarcomeric proteins for the RV and LV of the post-ischemic failing rat heart. These results implicate divergent responses for oxidative stress and open a new avenue in targeting the RV independently of the LV.
标准的心力衰竭（HF）治疗方法未能改善右心室（RV）功能不全的HF患者的心脏功能或生存率，这表明RV与左心室（LV）衰竭的分子机制存在差异。在此，我们旨在研究实验性心肌梗死（MI）诱导的左心室射血分数降低的心力衰竭（HFrEF）中肌节调节和功能的心室间差异。通过结扎Sprague-Dawley雄性大鼠的左前降支（LAD）诱导MI。假手术动物作为对照。干预8周后，对缺血后HFrEF和假手术动物实施安乐死。将心脏组织样本深度冷冻保存（每组3 - 5个心脏），用于酶联免疫吸附测定（ELISA）、激酶活性测定、分离心肌细胞的被动僵硬度和钙敏感性测量、磷酸化特异性蛋白质印迹法以及收缩蛋白的聚丙烯酰胺凝胶电泳（PAGE），以及胶原基因表达分析。氧化应激和炎症标志物在缺血后大鼠中表现出心室间差异：转化生长因子-β1（TGF-β1）、脂质过氧化和3 - 硝基酪氨酸水平在LV中高于RV，而过氧化氢、血管细胞黏附分子-1（VCAM-1）、肿瘤坏死因子α（TNFα）和TGF-β1在两个心室中均升高。此外，一氧化氮（NO）水平显著降低，而纤连蛋白-1（FN-1）水平仅在LV中显著升高，但在RV中两者均无变化。与假手术组相比，HFrEF大鼠LV中的钙/钙调蛋白依赖性蛋白激酶II（CaMKII）活性增加了81.6%，而RV中则降低了38.6%。从F与肌节长度关系明显更陡峭可以看出，HFrEF组中心肌细胞的被动僵硬度高于假手术组。然而，用CaMKIIδ进行体外处理仅恢复了HFrEF RV中心肌细胞的被动僵硬度，而对HFrEF LV没有影响。与假手术组相比，HFrEF组两个心室中的蛋白激酶G（PKG）活性均较低。体外给予PKG可降低HFrEF心肌细胞的被动僵硬度；然而，该作用在HFrEF LV中比HFrEF RV中更明显。与此一致，我们观察到缺血后大鼠RV与LV中肌联蛋白位点特异性磷酸化的明显变化，这可能解释了观察到的不同的心肌细胞僵硬度调节。最后，RV心肌细胞的钙敏感性未改变，而HFrEF组中LV心肌细胞的钙敏感性增加。这可以通过RV中肌球蛋白结合蛋白C（cMyBP-C）的Ser-282磷酸化降低44.5%来解释，但在LV中没有任何改变，而与假手术组相比，HFrEF组两个心室中肌钙蛋白I（cTnI）的Ser-23/24磷酸化均降低。我们的数据表明，缺血后衰竭大鼠心脏的RV和LV中，肌节蛋白的磷酸化由不同的信号通路介导。这些结果提示氧化应激的不同反应，并为独立于LV靶向RV开辟了一条新途径。
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