Liang Jingsheng, Kazmierczak Katarzyna, Veerasammy Melanie, Yadav Sunil, Takeuchi Lauro, Kanashiro-Takeuchi Rosemeire, Szczesna-Cordary Danuta
Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, USA.
Interdisciplinary Stem Cell Institute University of Miami Miller School of Medicine, Miami, Florida, USA.
Cytoskeleton (Hoboken). 2024 Dec;81(12):806-814. doi: 10.1002/cm.21854. Epub 2024 Mar 17.
We investigated the impact of the phosphomimetic (Ser15 → Asp15) myosin regulatory light chain (S15D-RLC) on the Super-Relaxed (SRX) state of myosin using previously characterized transgenic (Tg) S15D-D166V rescue mice, comparing them to the Hypertrophic Cardiomyopathy (HCM) Tg-D166V model and wild-type (WT) RLC mice. In the Tg-D166V model, we observed a disruption of the SRX state, resulting in a transition from SRX to DRX (Disordered Relaxed) state, which explains the hypercontractility of D166V-mutated myosin motors. The presence of the S15D moiety in Tg-S15D-D166V mice restored the SRX/DRX balance to levels comparable to Tg-WT, thus mitigating the hypercontractile behavior associated with the HCM-D166V mutation. Additionally, we investigated the impact of delivering the S15D-RLC molecule to the hearts of Tg-D166V mice via adeno-associated virus (AAV9) and compared their condition to AAV9-empty vector-injected or non-injected Tg-D166V animals. Tg-D166V mice injected with AAV9 S15D-RLC exhibited a significantly higher proportion of myosin heads in the SRX state compared to those injected with AAV9 empty vector or left non-injected. No significant effect was observed in Tg-WT hearts treated similarly. These findings suggest that AAV9-delivered phosphomimetic S15D-RLC modality mitigates the abnormal Tg-D166V phenotype without impacting the normal function of Tg-WT hearts. Global longitudinal strain analysis supported these observations, indicating that the S15D moiety can alleviate the HCM-D166V phenotype by restoring SRX stability and the SRX ↔ DRX equilibrium.
我们使用先前表征的转基因（Tg）S15D-D166V拯救小鼠，研究了拟磷酸化（Ser15→Asp15）肌球蛋白调节轻链（S15D-RLC）对肌球蛋白超松弛（SRX）状态的影响，并将它们与肥厚型心肌病（HCM）Tg-D166V模型和野生型（WT）RLC小鼠进行比较。在Tg-D166V模型中，我们观察到SRX状态的破坏，导致从SRX状态转变为DRX（无序松弛）状态，这解释了D166V突变的肌球蛋白马达的过度收缩性。Tg-S15D-D166V小鼠中S15D部分的存在将SRX/DRX平衡恢复到与Tg-WT相当的水平，从而减轻了与HCM-D166V突变相关的过度收缩行为。此外，我们研究了通过腺相关病毒（AAV9）将S15D-RLC分子递送至Tg-D166V小鼠心脏的影响，并将它们的状况与注射AAV9空载体或未注射的Tg-D166V动物进行比较。与注射AAV9空载体或未注射的小鼠相比，注射AAV9 S15D-RLC的Tg-D166V小鼠在SRX状态下的肌球蛋白头部比例显著更高。对Tg-WT心脏进行类似处理未观察到显著影响。这些发现表明，AAV9递送的拟磷酸化S15D-RLC方式减轻了异常的Tg-D166V表型，而不影响Tg-WT心脏的正常功能。整体纵向应变分析支持了这些观察结果，表明S15D部分可以通过恢复SRX稳定性和SRX↔DRX平衡来减轻HCM-D166V表型。
