Titin's cardiac-specific N2B Element is Critical to Mechanotransduction during Volume Overload of the heart

The heart has the ability to detect and respond to changes in mechanical load through a process called mechanotransduction. In this study, we focused on investigating the role of the cardiac-specific N2B element within the spring region titin, which has been proposed to function as a mechanosensor. To assess its significance, we conducted experiments using N2B knockout (KO) mice and wildtype (WT) mice, subjecting them to three different conditions: 1) cardiac pressure overload induced by transverse aortic constriction (TAC), 2) volume overload caused by aortocaval fistula (ACF), and 3) exercise-induced hypertrophy through swimming. Under conditions of pressure overload (TAC), both genotypes exhibited similar hypertrophic responses. However, in contrast, WT mice displayed robust left ventricular hypertrophy after one week of volume overload (ACF), while the KO mice failed to undergo hypertrophy and experienced a high mortality rate. Similarly, swim exercise-induced hypertrophy was significantly reduced in the KO mice. RNA-Seq analysis revealed an abnormal beta-adrenergic response to volume overload in the KO mice, as well as a diminished response to isoproterenol-induced hypertrophy. Because it is known that the N2B element interacts with the four-and-a-half LIM domains 1 and 2 (FHL1 and FHL2) proteins, both of which have been associated with mechanotransduction, we evaluated these proteins as well. Interestingly, while FHL1 protein expression levels were comparable between KO and WT mice, FHL2 protein levels were reduced by over 90% in the KO mice compared to WT. This suggests that in response to volume overload FHL2 may act as a potential signaling mediator between the N2B element and downstream signaling pathways. Overall, our study highlights the importance of the N2B element in mechanosensing during volume overload, both in physiological and pathological settings.

心脏能够通过一种被称为机械转导（mechanotransduction）的过程感知并响应机械负荷的变化。本研究聚焦于探究肌联蛋白弹性区内的心脏特异性N2B元件的作用，该元件被认为可作为机械传感器。为评估其重要性，我们使用心脏特异性N2B元件基因敲除（KO）小鼠与野生型（WT）小鼠开展实验，将小鼠分为三种不同处理条件：1）通过主动脉弓缩窄（TAC）构建心脏压力超负荷模型；2）通过主动脉腔静脉瘘（ACF）构建容量超负荷模型；3）通过游泳运动诱导心肌肥厚。在压力超负荷（TAC）条件下，两种基因型小鼠均表现出相似的肥厚反应。然而，与之形成对比的是，野生型小鼠在容量超负荷（ACF）造模一周后出现显著的左心室肥厚，而基因敲除小鼠未发生肥厚反应且死亡率较高。类似地，基因敲除小鼠的运动诱导心肌肥厚程度显著降低。RNA测序（RNA-Seq）分析显示，基因敲除小鼠对容量超负荷存在异常的β肾上腺素能反应，同时对异丙肾上腺素诱导的肥厚反应减弱。已知N2B元件可与四个半LIM结构域蛋白1和2（FHL1和FHL2）结合，这两种蛋白均与机械转导相关，因此我们也对这两种蛋白进行了检测。有趣的是，尽管基因敲除与野生型小鼠的FHL1蛋白表达水平相当，但基因敲除小鼠的FHL2蛋白表达水平较野生型小鼠降低了90%以上。这表明，在应对容量超负荷时，FHL2可能作为N2B元件与下游信号通路之间的潜在信号介质发挥作用。总体而言，本研究揭示了N2B元件在生理与病理状态下容量超负荷过程中的机械传感重要性。