From the Institute of Molecular Cardiology (R.R.E.A., A.B.M.S., Q.O., X.-L.T., M.S., Y.G., Y.N., K.M.K., S.K.H., R.B., T.M.A.M.), University of Louisville, KY.
Faculty of Medicine (A.B.M.S.), Zagazig University, Egypt.
Circulation. 2022 Apr 26;145(17):1339-1355. doi: 10.1161/CIRCULATIONAHA.121.057641. Epub 2022 Jan 21.
The regenerative capacity of the heart after myocardial infarction is limited. Our previous study showed that ectopic introduction of 4 cell cycle factors (4F; CDK1 [cyclin-dependent kinase 1], CDK4 [cyclin-dependent kinase 4], CCNB [cyclin B1], and CCND [cyclin D1]) promotes cardiomyocyte proliferation in 15% to 20% of infected cardiomyocytes in vitro and in vivo and improves cardiac function after myocardial infarction in mice.
Using temporal single-cell RNA sequencing, we aimed to identify the necessary reprogramming stages during the forced cardiomyocyte proliferation with 4F on a single cell basis. Using rat and pig models of ischemic heart failure, we aimed to start the first preclinical testing to introduce 4F gene therapy as a candidate for the treatment of ischemia-induced heart failure.
Temporal bulk and single-cell RNA sequencing and further biochemical validations of mature human induced pluripotent stem cell-derived cardiomyocytes treated with either LacZ or 4F adenoviruses revealed full cell cycle reprogramming in 15% of the cardiomyocyte population at 48 hours after infection with 4F, which was associated mainly with sarcomere disassembly and metabolic reprogramming (n=3/time point/group). Transient overexpression of 4F, specifically in cardiomyocytes, was achieved using a polycistronic nonintegrating lentivirus (NIL) encoding 4F; each is driven by a TNNT2 (cardiac troponin T isoform 2) promoter (TNNT2-4Fpolycistronic-NIL). TNNT2-4Fpolycistronic-NIL or control virus was injected intramyocardially 1 week after myocardial infarction in rats (n=10/group) or pigs (n=6-7/group). Four weeks after injection, TNNT2-4Fpolycistronic-NIL-treated animals showed significant improvement in left ventricular ejection fraction and scar size compared with the control virus-treated animals. At 4 months after treatment, rats that received TNNT2-4Fpolycistronic-NIL still showed a sustained improvement in cardiac function and no obvious development of cardiac arrhythmias or systemic tumorigenesis (n=10/group).
This study provides mechanistic insights into the process of forced cardiomyocyte proliferation and advances the clinical feasibility of this approach by minimizing the oncogenic potential of the cell cycle factors owing to the use of a novel transient and cardiomyocyte-specific viral construct.
心肌梗死后心脏的再生能力有限。我们之前的研究表明，异位引入 4 种细胞周期因子（4F；CDK1[周期蛋白依赖性激酶 1]、CDK4[周期蛋白依赖性激酶 4]、CCNB[周期蛋白 B1]和 CCND[周期蛋白 D1]）可促进体外和体内感染的心肌细胞中 15%至 20%的心肌细胞增殖，并改善小鼠心肌梗死后的心脏功能。
我们旨在通过单细胞 RNA 测序，从单细胞基础上确定在强制心肌细胞增殖过程中所需的重编程阶段。我们使用大鼠和猪缺血性心力衰竭模型，旨在开始首次临床前测试，将 4F 基因治疗作为治疗缺血性心力衰竭的候选药物。
对用 LacZ 或 4F 腺病毒处理的成熟人诱导多能干细胞衍生的心肌细胞进行的时间批量和单细胞 RNA 测序以及进一步的生化验证显示，在感染 4F 后 48 小时，心肌细胞群体中有 15%的细胞完全进入细胞周期重编程，这主要与肌节解体和代谢重编程有关（n=3/时间点/组）。使用编码 4F 的多顺反子非整合慢病毒（NIL）瞬时过表达 4F，该病毒特异性在心肌细胞中表达；每个都由 TNNT2（心肌肌钙蛋白 T 同工型 2）启动子（TNNT2-4F 多顺反子-NIL）驱动。在大鼠（n=10/组）或猪（n=6-7/组）心肌梗死后 1 周，心肌内注射 TNNT2-4F 多顺反子-NIL 或对照病毒。注射后 4 周，与对照病毒处理的动物相比，TNNT2-4F 多顺反子-NIL 处理的动物的左心室射血分数和疤痕大小明显改善。治疗后 4 个月，接受 TNNT2-4F 多顺反子-NIL 治疗的大鼠仍持续改善心功能，且无明显的心脏心律失常或全身肿瘤发生发展（n=10/组）。
这项研究提供了强制心肌细胞增殖过程的机制见解，并通过使用新型瞬时和心肌细胞特异性病毒构建体，最小化了细胞周期因子的致癌潜能，从而推进了该方法的临床可行性。
