Abstract：
Altered mechanical load in response to injury is a main driver of myocardial interstitial fibrosis. No current in vitro model can precisely modulate mechanical load in a multicellular environment while maintaining physiological behaviour. Living myocardial slices (LMS) are a 300 μm-thick cardiac preparation with preserved physiological structure and function. Here we apply varying degrees of mechanical preload to rat and human LMS to evaluate early cellular, molecular, and functionality changes related to myocardial fibrosis.
Left ventricular LMS were obtained from Sprague Dawley rat hearts and human cardiac samples from healthy and failing (dilated cardiomyopathy) hearts. LMS were mounted on custom stretchers and two degrees of diastolic load were applied: physiological sarcomere length (SL) (SL = 2.2 μm) and overload (SL = 2.4 μm). LMS were maintained for 48 h under electrical stimulation in circulating, oxygenated media at 37°C. In overloaded conditions, LMS displayed an increase in nucleus translocation of Yes-associated protein (YAP) and an up-regulation of mechanotransduction markers without loss in cell viability. Expression of fibrotic and inflammatory markers, as well as Collagen I deposition were also observed. Functionally, overloaded LMS displayed lower contractility (7.48 ± 3.07 mN mm-2 at 2.2 SL vs. 3.53 ± 1.80 mN mm-2 at 2.4 SL). The addition of the profibrotic protein interleukin-11 (IL-11) showed similar results to the application of overload with enhanced fibrosis (8% more of collagen surface coverage) and reduced LMS contractility at physiological load. Conversely, treatment with the Transforming growth factor β receptor (TGF-βR) blocker SB-431542, showed down-regulation of genes associated with mechanical stress, prevention of fibrotic response and improvement in cardiac function despite overload (from 2.40 ± 0.8 mN mm-2 to 4.60 ± 1.08 mN mm-2 ).
The LMS have a consistent fibrotic remodelling response to pathological load, which can be modulated by a TGF-βR blocker. The LMS platform allows the study of mechanosensitive molecular mechanisms of myocardial fibrosis and can lead to the development of novel therapeutic strategies.
Left ventricular LMS were obtained from Sprague Dawley rat hearts and human cardiac samples from healthy and failing (dilated cardiomyopathy) hearts. LMS were mounted on custom stretchers and two degrees of diastolic load were applied: physiological sarcomere length (SL) (SL = 2.2 μm) and overload (SL = 2.4 μm). LMS were maintained for 48 h under electrical stimulation in circulating, oxygenated media at 37°C. In overloaded conditions, LMS displayed an increase in nucleus translocation of Yes-associated protein (YAP) and an up-regulation of mechanotransduction markers without loss in cell viability. Expression of fibrotic and inflammatory markers, as well as Collagen I deposition were also observed. Functionally, overloaded LMS displayed lower contractility (7.48 ± 3.07 mN mm-2 at 2.2 SL vs. 3.53 ± 1.80 mN mm-2 at 2.4 SL). The addition of the profibrotic protein interleukin-11 (IL-11) showed similar results to the application of overload with enhanced fibrosis (8% more of collagen surface coverage) and reduced LMS contractility at physiological load. Conversely, treatment with the Transforming growth factor β receptor (TGF-βR) blocker SB-431542, showed down-regulation of genes associated with mechanical stress, prevention of fibrotic response and improvement in cardiac function despite overload (from 2.40 ± 0.8 mN mm-2 to 4.60 ± 1.08 mN mm-2 ).
The LMS have a consistent fibrotic remodelling response to pathological load, which can be modulated by a TGF-βR blocker. The LMS platform allows the study of mechanosensitive molecular mechanisms of myocardial fibrosis and can lead to the development of novel therapeutic strategies.
摘要：
响应于损伤的改变的机械负荷是心肌间质纤维化的主要驱动因素。目前没有体外模型可以在多细胞环境中精确调节机械负荷，同时保持生理行为。活心肌切片（LMS）是300μm厚的心脏制剂，具有保留的生理结构和功能。在这里，我们对大鼠和人类LMS应用不同程度的机械预负荷来评估早期细胞，分子，以及与心肌纤维化相关的功能变化。
从SpragueDawley大鼠心脏和来自健康和衰竭(扩张型心肌病)心脏的人心脏样品获得左心室LMS。将LMS安装在定制担架上，并施加两个舒张负荷：生理肌节长度（SL）（SL=2.2μm）和过载（SL=2.4μm）。LMS在循环电刺激下维持48小时，37°C的充氧介质在过载条件下，LMS显示Yes相关蛋白（YAP）的核易位增加，机械转导标志物上调，而细胞活力没有损失。纤维化和炎症标志物的表达，以及胶原蛋白I沉积也被观察到。功能上,过载LMS显示较低的收缩性(7.48±3.07mNmm-2在2.2SL与2.4SL时3.53±1.80mNmm-2)。促纤维化蛋白白介素-11(IL-11)的添加显示出与在生理负荷下具有增强的纤维化(胶原表面覆盖率增加8%)和降低的LMS收缩性的超负荷应用类似的结果。相反，用转化生长因子β受体（TGF-βR）阻断剂SB-431542治疗，显示与机械应激相关的基因下调，尽管超负荷（从2.40±0.8mNmm-2到4.60±1.08mNmm-2），仍可预防纤维化反应并改善心功能。
LMS对病理负荷有一致的纤维化重塑反应，可以通过TGF-βR阻断剂调节。LMS平台允许研究心肌纤维化的机械敏感性分子机制，并可导致开发新的治疗策略。
从SpragueDawley大鼠心脏和来自健康和衰竭(扩张型心肌病)心脏的人心脏样品获得左心室LMS。
LMS对病理负荷有一致的纤维化重塑反应，
