Functional comparison of phosphomimetic S15D and T160D mutants of myosin regulatory light chain exchanged in cardiac muscle preparations of HCM and WT mice.
Functional comparison of phosphomimetic S15D and T160D mutants of myosin regulatory light chain exchanged in cardiac muscle preparations of HCM and WT mice.
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DOI：
10.3389/fcvm.2022.988066
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发表时间：
2022
影响因子：
3.6
通讯作者：
Szczesna-Cordary, Danuta
中科院分区：
文献类型：
--
作者：
Kazmierczak, Katarzyna;Liang, Jingsheng;Gomez-Guevara, Michelle;Szczesna-Cordary, Danuta
关键词：
在这项研究中，我们研究了肌球蛋白调节轻链(RLC，MYL2基因)的两个拟磷突变体S15D和T160D RLCs的拯救潜力。S15D-RLC模拟人心脏RLC已建立的丝氨酸-15位点的磷酸化。T160D-RLC模拟苏氨酸-160的磷酸化，计算分析确定苏氨酸-160是肌球蛋白RLC的高分磷酸化位点。从先前建立的肥厚型心肌病(HCM)模型、TG-R58Q和TG-野生型(WT)小鼠中分离出心肌肌球蛋白和左心室乳头肌(LVPM)纤维。肌肉标本首先去除内源性RLC，然后用重组人心脏S15D和T160D仿磷RLCs重建。以重组人心脏WT-RLC和R58Q-RLC重组制剂为对照。然后测试小鼠肌球蛋白的肌动蛋白激活的肌球蛋白ATPase活性和LVPM纤维的稳态力量发展和钙敏感性的力量。结果表明，与T160D-RLC或WT-RLC重组制剂相比，S15D-RLC显著提高肌球蛋白ATPase活性。S15D和T160D两种拟磷脂RLC均能挽救重组R58Q-RLC重组的TG-R58Q肌球蛋白的Vmax，但S15D-RLC的作用比T160D-RLC更明显。从Tg-R58Q小鼠体内观察到的R58Q-RLC重组LVPM的低张力可被两种拟磷脂RLC同等地拯救。在HCM TG-R58Q心肌中，S15D-RLC引起从超松弛(SRX)状态到无序松弛(DRX)状态的转变，并增加了容易与肌动蛋白相互作用并产生作用力的头部数量。同时，T160D-RLC将SRX状态稳定在与R58Q-RLC重组光纤相似的水平。我们在这里报道了人心脏RLC的S15磷酸化位点与C末端T160-RLC相比的功能优势，S15D-RLC在减轻由TG-R58Q心肌的低收缩行为所强调的非规范的HCM行为方面显示出治疗潜力。
In this study, we investigated the rescue potential of two phosphomimetic mutants of the myosin regulatory light chain (RLC, MYL2 gene), S15D, and T160D RLCs. S15D-RLC mimics phosphorylation of the established serine-15 site of the human cardiac RLC. T160D-RLC mimics the phosphorylation of threonine-160, identified by computational analysis as a high-score phosphorylation site of myosin RLC. Cardiac myosin and left ventricular papillary muscle (LVPM) fibers were isolated from a previously generated model of hypertrophic cardiomyopathy (HCM), Tg-R58Q, and Tg-wild-type (WT) mice. Muscle specimens were first depleted of endogenous RLC and then reconstituted with recombinant human cardiac S15D and T160D phosphomimetic RLCs. Preparations reconstituted with recombinant human cardiac WT-RLC and R58Q-RLC served as controls. Mouse myosins were then tested for the actin-activated myosin ATPase activity and LVPM fibers for the steady-state force development and Ca2+-sensitivity of force. The data showed that S15D-RLC significantly increased myosin ATPase activity compared with T160D-RLC or WT-RLC reconstituted preparations. The two S15D and T160D phosphomimetic RLCs were able to rescue Vmax of Tg-R58Q myosin reconstituted with recombinant R58Q-RLC, but the effect of S15D-RLC was more pronounced than T160D-RLC. Low tension observed for R58Q-RLC reconstituted LVPM from Tg-R58Q mice was equally rescued by both phosphomimetic RLCs. In the HCM Tg-R58Q myocardium, the S15D-RLC caused a shift from the super-relaxed (SRX) state to the disordered relaxed (DRX) state, and the number of heads readily available to interact with actin and produce force was increased. At the same time, T160D-RLC stabilized the SRX state at a level similar to R58Q-RLC reconstituted fibers. We report here on the functional superiority of the established S15 phospho-site of the human cardiac RLC vs. C-terminus T160-RLC, with S15D-RLC showing therapeutic potential in mitigating a non-canonical HCM behavior underlined by hypocontractile behavior of Tg-R58Q myocardium.
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影响因子：
2.4
作者：
Schmid M;Toepfer CN
通讯作者：
Toepfer CN
影响因子：
3.4
作者：
Garfinkel AC;Seidman JG;Seidman CE
通讯作者：
Seidman CE
DOI：
10.1073/pnas.1600633113
发表时间：
2016-07-05
影响因子：
11.1
作者：
Chang, Audrey N.;Mahajan, Pravin;Stull, James T.
通讯作者：
Stull, James T.
影响因子：
3.9
作者：
Muthu, Priya;Liang, Jingsheng;Szczesna-Cordary, Danuta
通讯作者：
Szczesna-Cordary, Danuta
DOI：
10.1073/pnas.1602776113
发表时间：
2016-05-24
影响因子：
11.1
作者：
Kampourakis, Thomas;Sun, Yin-Biao;Irving, Malcolm
通讯作者：
Irving, Malcolm