High-Throughput Screening Platform in Postnatal Heart Cells and Chemical Probe Toolbox to Assess Cardiomyocyte Proliferation

Restoring lost heart muscle is an
attractive goal for cardiovascular
regenerative medicine. One appealing strategy is the therapeutic stimulation
of cardiomyocyte proliferation, which inter alia remains
challenging due to available assay technologies capturing the complex
biology. Here, a high-throughput-formatted phenotypic assay platform
was established using rodent whole heart-derived cells to preserve
the cellular environment of cardiomyocytes. Several readouts allowed
the quantification of cycling cardiomyocytes, including a transgenic
H2B-mCherry system for unequivocal, automated detection of cardiomyocyte
nuclei. A chemical genetics approach revealed pronounced species differences
and furnished pan-kinase inhibitors 5 and 36 as potent and robust inducers of endoreplication and acytokinetic
mitosis. Combined profiling of the commonly used p38 MAPK inhibitors
SB203580 (1), SB239063 (2) and a novel set
of skepinone-L (6) derivatives pointed to off-target
effects beyond p38 that might be critical for effective cardiomyocyte
cytokinesis. Kinome-focused screening eventually furnished TG003 (38) as a novel candidate for stimulating cardiomyocyte proliferation.

修复受损心肌是心血管再生医学领域极具吸引力的研究目标。其中一种颇具潜力的策略是通过治疗手段刺激心肌细胞（cardiomyocyte）增殖，但受限于当前检测技术难以精准捕捉其复杂的生物学过程，该策略至今仍面临诸多挑战。本研究采用啮齿类动物全心脏来源的细胞构建了适配高通量的表型检测平台，以最大程度保留心肌细胞的原生细胞微环境。该平台搭载多种检测读端以量化增殖中的心肌细胞，其中包括可精准、自动化检测心肌细胞核的转基因H2B-mCherry系统。通过化学遗传学研究策略，本研究发现了显著的物种差异，并筛选出泛激酶抑制剂5与36，二者可强效且稳定地诱导心肌细胞发生核内复制与无胞质分裂的有丝分裂。对常用p38丝裂原活化蛋白激酶（p38 MAPK）抑制剂SB203580（1）、SB239063（2）以及全新的skepinone-L（6）衍生物进行联合靶点谱分析，结果表明这些抑制剂存在p38以外的脱靶效应，而该效应可能对心肌细胞的有效胞质分裂至关重要。基于激酶组的筛选最终得到TG003（38），其可作为刺激心肌细胞增殖的全新候选化合物。