Introduction

In clinical and animal studies, Hypertrophic Cardiomyopathy (HCM) shares many similarities with non-inherited cardiac hypertrophy induced by pressure overload (hypertension). This suggests a potential role for mechanical stress in priming tissues with mutation-induced changes in the sarcomere to develop phenotypes associated with HCM, including hypercontractility and aberrant calcium handling. Here, we tested the hypothesis that heterozygous loss of function of Myosin Binding Protein C (MYBCP3^+/−, mutations in which account for almost 50% of inherited HCM) combines with environmental stiffness to drive HCM phenotypes.

Methods

We differentiated isogenic control (WTC) and MYBPC3^+/− iPSC into cardiomyocytes using small molecule manipulation of Wnt signaling, and then purified them using lactate media. The purified cardiomyocytes were seeded into “dog bone” shaped stencil molds to form micro-heart muscle arrays (μHM). To mimic changes in myocardial stiffness stemming from pressure overload, we varied the rigidity of the substrates μHM contract against. Stiffness levels ranged from those corresponding to fetal (5 kPa), healthy (15 kPa), pre-fibrotic (30 kPa) to fibrotic (65 kPa) myocardium. Substrates were embedded with a thin layer of fluorescent beads to track contractile force, and parent iPSC were engineered to express the genetic calcium indicator, GCaMP6f. High speed video microscopy and image analysis were used to quantify calcium handling and contractility of μHM.

Results

Substrate rigidity triggered physiological adaptation for both genotypes. However, MYBPC3^+/− μHM showed a lower tolerance to substrate stiffness with the peak traction on 15 kPa, while WTC μHM had peak traction on 30 kPa. MYBPC3^+/− μHM exhibited hypercontractility, which was exaggerated by substrate rigidity. MYBPC3^+/− μHM hypercontractility was associated with longer rise times for calcium uptake and force development, along with higher overall Ca²⁺ intake.

Conclusion

We found MYBPC3^+/− mutations cause iPSC-μHM to exhibit hypercontractility, and also a lower tolerance for mechanical stiffness. Understanding how genetics work in combination with mechanical stiffness to trigger and/or exacerbate pathophysiology may lead to more effective therapies for HCM.

中文翻译：

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基因型和基质硬度在 iPSC-微心肌阵列中驱动肥厚型心肌病表型中的相互作用

介绍

在临床和动物研究中，肥厚性心肌病 (HCM) 与压力超负荷（高血压）引起的非遗传性心脏肥大有许多相似之处。这表明机械应力在启动组织中肌节突变诱导的变化中的潜在作用，以发展与 HCM 相关的表型，包括过度收缩和异常的钙处理。在这里，我们测试了这样的假设：肌球蛋白结合蛋白 C（MYBCP3 ^+/-，突变占遗传性 HCM 的近 50%）杂合性功能丧失与环境僵化相结合，驱动 HCM 表型。

方法

我们使用 Wnt 信号传导的小分子操作将等基因对照 (WTC) 和 MYBPC3 ^+/- iPSC 分化为心肌细胞，然后使用乳酸培养基纯化它们。将纯化的心肌细胞接种到“狗骨”形模板模具中，形成微心肌阵列（μHM）。为了模拟压力超负荷引起的心肌硬度变化，我们改变了 μHM 收缩基质的硬度。硬度水平范围从对应于胎儿（5 kPa）、健康（15 kPa）、纤维化前（30 kPa）到纤维化（65 kPa）心肌的硬度水平。基质中嵌入了一层薄薄的荧光珠来追踪收缩力，亲本 iPSC 被设计为表达遗传钙指示剂 GCaMP6f。使用高速视频显微镜和图像分析来量化 μHM 的钙处理和收缩性。

结果

基质刚性引发了两种基因型的生理适应。然而，MYBPC3 ^+/- μHM 对基底刚度的耐受性较低，峰值牵引力为 15 kPa，而 WTC μHM 的峰值牵引力为 30 kPa。MYBPC3 ^+/- μHM 表现出超收缩性，这种收缩性因基质刚性而加剧。MYBPC3 ^+/- μHM 过度收缩性与钙摄取和力量发展的较长上升时间以及较高的总体 Ca ²⁺摄入量相关。

结论

我们发现 MYBPC3 ^+/-突变导致 iPSC-μHM 表现出过度收缩性，并且对机械刚度的耐受性较低。了解遗传学如何与机械刚度相结合来触发和/或加剧病理生理学可能会带来更有效的 HCM 治疗方法。