Engineered Tissue for Biomechanical Phenotyping of Cardiomyopathy Patients

描述(由申请者提供)：心肌病是一种危及生命的心脏肌肉疾病，常见于家族。这种疾病有多种形式，所有这些都会使患者面临病理性心脏重塑、心源性猝死和心力衰竭的风险。虽然目前还没有治愈的方法，但如果在疾病的早期阶段被发现，医生可以减少疾病的影响。早期检测计划目前依赖于基因测试，以确定一个人是否携带已知的1400种心肌病突变之一。如果新的心肌病患者碰巧携带已知的突变，他们的家庭成员可以进行有意义的筛查。携带突变的人将得到预防性护理，而非携带者可以安全地免除临床随访，并缓解不必要的焦虑。不幸的是，只有40%-60%的心肌病患者的检测结果是阳性的，这是经过验证的突变。其余患者约占所有患者家庭的一半，除非采取进一步措施，否则他们无法从早期诊断的基因测试中受益。这类家庭的选择是有限的。共分离分析可以在足够大的家系中进行，并可能识别出新的基因突变，但这一结果并不能得到保证。如果这个家庭很小，或者不适合进行共分离研究，所有的一级亲属必须定期接受新出现的疾病迹象的检测--即使他们有50%的可能性没有携带突变。因此，在基因检测结果不确定的情况下，迫切需要一种更强大的、单次重复的方法来早期检测心肌病遗传。诱导多能干细胞(IPSC)是通过表达多能干细胞因子从人自身的体细胞中衍生出来的，可能提供一种解决方案。我们设想了一种方法，在这种方法中，IPSC来源的心肌细胞(IPSC-CMS)从患者身上获得，并形成工程心脏组织(EHT)，用于体外研究。我们假设，在EHTS中测量的异常行为将反映存在的任何突变的直接影响。因此，这些表型可以作为遗传的替代标记。通过测试每个家庭成员的EHTS，可以建立遗传模式，而不考虑受试者的年龄，并且不需要像在共分离分析中那样需要一个大家庭。考虑到这一特定的目标，我们开发了一种新的策略来产生理想的生物力学表型。我们通过将心肌细胞种植到经过激光切割的猪去细胞心肌薄片中来制作这些带状标本。对新生大鼠心室肌细胞的初步测试已经产生了自发跳动的EHTS，这种EHTS产生可测量的收缩力量，并可以在培养中存活数月。该项目的目标是评估这项令人兴奋的新技术的真正潜力，通过从对照和疾病受试者中使用IPSC-CMS产生EHTS。然后，我们可以测试我们的系统重复检测即使是临床上轻微突变的表型后果的能力。我们的目标的成功完成将是朝着替代基因检测的重要一步，以早期识别有发展成心肌病风险的个人。

DESCRIPTION (provided by applicant): Cardiomyopathies are life threatening heart muscle disorders that often run in families. The disease takes many forms, all of which place patients at risk for pathological heart remodeling, sudden cardiac death, and heart failure. While no cure exists, physicians can reduce the impact of the disease if it is caught in its early stages. Early detection schemes currently rely on genetic testing, to see if an individual carries one of the ~1400 known cardiomyopathy mutations. If a new cardiomyopathy patient happens to carry a known mutation, their family members can be meaningfully screened. Those carrying the mutation will receive preventative care, while non-carriers can be safely excused from clinical follow-ups and relieved of undue anxiety. Unfortunately, only 40-60% of cardiomyopathy patients test positive for a verified mutation. The rest, comprising roughly half of all patient families, cannot benefit from genetic testing for early diagnosis unless further steps are taken. The options for such families are limited. Co-segregation analysis can be performed in families of sufficient size and may identify a novel gene mutation, but this result is not guaranteed. If th family is small or otherwise unsuited for co-segregation study, all first-degree relatives must be periodically tested for signs of emerging disease - even though there is a 50% chance that they do not carry the mutation. Hence, in cases where genetic testing results are indeterminate, a more robust, single-repetition method for early detection of cardiomyopathy inheritance is badly needed. Induced pluripotent stem cells (iPSC), derived from a person's own somatic cells by expression of pluripotent stem cell factors, may provide a solution. We envision an approach in which iPSC-derived cardiomyocytes (iPSC-CMs) are generated from a patient and formed into engineered heart tissue (EHT) for in vitro study. We hypothesize that abnormal behavior measured in EHTs will reflect direct effects of any mutations present. As such, these phenotypes could act as surrogate markers of inheritance. By testing EHTs of each family member, the pattern of inheritance could be established regardless of the subjects' ages, and without requiring a large family, as in co-segregation analysis. With this specific goal in mind, w have developed a novel strategy to generate EHTs that are ideal for biomechanical phenotyping. We produce these ribbon-like specimens by seeding cardiac cells into thin, laser-cut sections of decellularized porcine myocardium. Initial tests with neonatal rat ventricular myocytes have yielded spontaneously beating EHTs that produce measurable contraction forces and can survive months in culture. The goal of this project is to assess the true potential of this exciting new technology by generating EHTs with iPSC-CMs from control and diseased subjects. We can then test the ability of our system to reproducibly detect the phenotypic consequences of even clinically mild mutations. The successful completion of our aims will be an important step toward an alternative to genetic testing for the early identification of individuals who are at risk of developing cardiomyopathy.