背景

心肌病是一类以心肌结构和功能异常为特征的疾病，通常与基因突变密切相关，主要包括扩张型心肌病和肥厚型心肌病。随着基因检测技术的普及，基因突变已成为心肌病诊断的重要依据。然而，基因型与表型之间的关系复杂，许多突变的致病性尚未明确，这限制了精准治疗的发展。此外，现有的研究模型难以全面模拟心肌病的病理特征，导致缺乏有效的体外心肌病分型方法。人多能干细胞分化的心肌细胞因其与人类心肌细胞的高度相似性，为研究心肌病的分子机制提供了理想的实验平台。

目的

本研究旨在利用人多能干细胞构建基因敲除和突变人源心肌模型，通过系统评估心肌细胞的结构、收缩功能和钙瞬变活动，建立一种基于张力曲线计算的体外心肌病分型方法，为心肌病的精准诊断和治疗提供新的工具和理论依据。

方法

使用CRISPR/Cas9技术在 H9 人胚胎干细胞中敲除 MYH7、TPM1、TNNC1、TNNT2、NEXN、CSRP3 等6个心肌病相关基因，随后将干细胞定向分化为心肌样细胞。通过免疫荧光检测、收缩力检测、钙瞬变检测等多种方法，系统评估基因敲除心肌的表型。在敲除心肌细胞中，通过慢病毒转染过表达相应野生型蛋白，筛选出 TNNC1、NEXN、CSRP3 三个可通过过表达恢复表型的基因用于后续研究。进一步在敲除心肌细胞中过表达突变蛋白，构建不同致病类型的突变心肌模型，建立基于张力曲线的体外心肌病分型方法。随后，在 JPH2 心肌敲除模型中，过表达多种突变蛋白构建突变心肌模型，通过收缩力、钙瞬变和定量PCR检测验证该方法的普适性和准确性。

结果

本研究成功构建了 MYH7、TPM1、TNNC1、TNNT2、NEXN 和 CSRP3 基因敲除的心肌细胞模型，基因敲除未影响干细胞的多能性和心肌分化效率，分化率达75%以上。敲除心肌在结构和功能上出现显著异常。在 TNNC1、NEXN、CSRP3 敲除心肌中，过表达相应野生型蛋白可恢复其收缩表型。选择TNNC1、NEXN、CSRP3的不同致病性突变各3个构建变体心肌模型，在构建的不同致病类型和良性突变模型中，扩张型心肌病和肥厚型心肌病相关突变表现出收缩功能的显著差异，而良性突变无明显变化。通过计算张力指数，发现不同类型突变的张力指数具有显著差异，即肥厚型心肌病变异张力指数呈正值，而扩张型心肌病张力指数呈负值，这一差异可应用于体外心肌病的分型。随后使用JPH2突变心肌模型进行普适性验证，JPH2 突变心肌模型上的钙瞬变及分子表达结果进一步验证了基于张力指数的分型方法的有效性和普适性。

结论

本研究建立并验证了一种基于人多能干细胞分化心肌细胞的体外心肌病分型方法，通过张力指数可有效区分扩张型心肌病和肥厚型心肌病，为心肌病的精准诊断和治疗提供了新的工具。这一方法不仅有助于深入理解基因突变与心肌病表型之间的关系，还为未来开发个性化治疗方案奠定了理论基础。

Background

Cardiomyopathy is a group of disorders characterized by structural and functional abnormalities of the myocardium, often closely associated with genetic mutations, and primarily includes dilated cardiomyopathy and hypertrophic cardiomyopathy. The widespread adoption of gene sequencing technologies has positioned genetic mutations as a cornerstone of cardiomyopathy diagnosis. However, the intricate relationship between genotype and phenotype, coupled with the uncertain pathogenicity of numerous mutations, continues to impede advancements in precision therapy. Furthermore, current research models often fall short of fully replicating the pathological characteristics of cardiomyopathy, leading to a deficiency in effective in vitro classification methods for cardiomyopathy. Human pluripotent stem cell-derived cardiomyocytes, owing to their high degree of similarity to native human cardiomyocytes, offer an optimal experimental platform for elucidating the molecular mechanisms of cardiomyopathy.

Purpose

This study seeks to leverage human pluripotent stem cells to develop gene knockout and mutant human cardiomyocyte models. Through a systematic evaluation of their structure, contractile function, and calcium transient dynamics, we aim to devise an in vitro cardiomyopathy classification method based on tension curve analysis, thereby providing innovative tools and a theoretical framework for the precise diagnosis and treatment of cardiomyopathy.

Method

Employing CRISPR/Cas9 gene editing technology, we knocked out six cardiomyopathy-associated genes—MYH7, TPM1, TNNC1, TNNT2, NEXN and CSRP3—in H9 human embryonic stem cells, subsequently inducing their differentiation into cardiomyocyte-like cells. The phenotypes of these knockout cardiomyocytes were comprehensively assessed via immunofluorescence, contractile force measurements, and calcium transient analyses. In the knockout cardiomyocytes, lentiviral transfection facilitated overexpression of the corresponding wild-type proteins, identifying TNNC1, NEXN, and CSRP3 as genes whose phenotypes could be rescued for further investigation. Mutant proteins were then overexpressed in these knockout cardiomyocytes to establish models representing various pathogenic mutation types, enabling the development of an in vitro cardiomyopathy classification method based on tension curves. Additionally, in a JPH2 knockout cardiomyocyte model, we overexpressed multiple mutant proteins to construct mutation-specific models, validating the universality and precision of this classification method through contractile force, calcium transient, and quantitative PCR assessments.

Result

We successfully established cardiomyocyte models with knockouts of MYH7, TPM1, TNNC1, TNNT2, NEXN, and CSRP3 genes, with gene knockout not affecting the pluripotency and cardiomyocyte differentiation efficiency of stem cells, achieving a differentiation rate of over 75%. The knockout cardiomyocytes displayed pronounced structural and functional anomalies. In TNNC1, NEXN, and CSRP3 knockout cardiomyocytes, overexpression of their respective wild-type proteins effectively rescued the pathological phenotypes. Three pathogenic variants and benign variants of TNNC1, NEXN, and CSRP3 were selected to construct variant cardiomyocyte models. In these models, mutations associated with dilated cardiomyopathy- and hypertrophic cardiomyopathy-associated mutations exhibited marked differences in contractile function, while benign mutations showed no notable deviations. Through the quantitative analysis of the tension index, we found that different types of mutations exhibited significant differences in tension indices. Specifically, dilated cardiomyopathy-associated variants displayed negative values, whereas hypertrophic cardiomyopathy-associated variants showed positive tension index values. This distinct pattern can be applied for the in vitro classification of cardiomyopathies. Thereafter, the JPH2 variant cardiomyocyte model was employed to validate the universality of the approach. The results of calcium transients and molecular expression in the JPH2 model provided further evidence supporting the efficacy and broad applicability of the tension index-based classification method.

Conclusion

This study developed and validated an in vitro cardiomyopathy classification method utilizing human pluripotent stem cell-derived cardiomyocytes. The tension index proved effective in differentiating dilated cardiomyopathy from hypertrophic cardiomyopathy, offering a novel tool for the precise diagnosis and management of cardiomyopathy. This approach not only deepens our understanding of the interplay between genetic mutations and cardiomyopathy phenotypes but also establishes a theoretical basis for the future development of personalized therapeutic strategies.