Regeneration of muscle in the damaged myocardium is a major objective of cardiovascular research, for which purpose many investigators utilize mice containing transgenes encoding Cre-recombinase to recombine loxP-flanked target genes. An unfortunate side-effect of the Cre-loxP model is the propensity of Cre-recombinase to inflict off-target DNA damage, which has been documented in various eukaryotic cell-types including cardiomyocytes (CMs). In the heart, reported effects of Cre-recombinase include contractile dysfunction, fibrosis, cellular infiltration, and induction of the DNA damage response (DDR). During experiments on adult mice containing a widely used Myh6-merCremer transgene, the protein product of which is activated by tamoxifen, we observed large, transient off-target effects of merCremer, some of which have not been previously reported. On Day 3 after the first of three daily tamoxifen injections, immunofluorescent microscopy of heart sections revealed that the presence of merCremer protein in myonuclei was nearly uniform, thereafter diminishing to near extinction by Day 6; during this time, cardiac function was depressed as determined by echocardiography. On Day 5, peaks of apoptosis and expression of DDR regulatory genes were observed, highlighted by >25-fold increased expression of Brca1; concomitantly, the expression of genes encoding Cyclin A2, Cyclin B1 and Cdk1, which regulate the G₂/S cell-cycle transition, were dramatically increased (>50-100-fold). Importantly, immunofluorescent staining revealed that this was accompanied by peaks of Ki67, 5'-bromodeoxyuridine, and phosphohistone H3 labeling in non-CMs, as well as CMs. We further document that tamoxifen-induced activation of merCremer exacerbates cardiac dysfunction following MI. These findings, when considered in the context of previous reports, indicate that the presence of merCremer in the nucleus induces DNA damage and unscheduled cell-cycle activation. Although these effects are transient, the inclusion of appropriate controls, coupled with an awareness of defects caused by Cre-recombinase, are required to avoid misinterpreting results when using Cre-loxP models for cardiac regeneration studies.

中文翻译：

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在缺乏 loxP 位点的情况下，Myh6 驱动的 Cre 重组酶可激活心肌中的 DNA 损伤反应和细胞周期。

受损心肌中肌肉的再生是心血管研究的一个主要目标，为此，许多研究人员利用含有编码 Cre 重组酶的转基因的小鼠来重组 loxP 侧翼的靶基因。Cre-loxP 模型的一个不幸的副作用是 Cre 重组酶容易造成脱靶 DNA 损伤，这种情况已在包括心肌细胞 (CM) 在内的各种真核细胞类型中得到记录。在心脏中，据报道 Cre 重组酶的作用包括收缩功能障碍、纤维化、细胞浸润和诱导 DNA 损伤反应 (DDR)。在对含有广泛使用的Myh6-merCremer转基因（其蛋白质产物由他莫昔芬激活）的成年小鼠进行的实验中，我们观察到 merCremer 存在较大的、短暂的脱靶效应，其中一些效应以前从未报道过。每日三次他莫昔芬注射中的第一次注射后的第 3 天，心脏切片的免疫荧光显微镜显示，肌核中 merCremer 蛋白的存在几乎是均匀的，此后到第 6 天减少到接近消失；在此期间，超声心动图确定心脏功能下降。第 5 天，观察到细胞凋亡和 DDR 调节基因表达的峰值，其中Brca1表达增加了 25 倍以上；与此同时，调节G ₂ /S细胞周期转变的编码Cyclin A2、Cyclin B1和Cdk1的基因的表达显着增加(>50-100倍)。重要的是，免疫荧光染色显示，在非 CM 和 CM 中，这都伴随着 Ki67、5'-溴脱氧尿苷和磷酸组蛋白 H3 标记的峰。我们进一步证明他莫昔芬诱导的 merCremer 激活会加剧 MI 后的心脏功能障碍。结合之前的报告考虑，这些发现表明细胞核中 merCremer 的存在会诱导 DNA 损伤和非计划的细胞周期激活。尽管这些影响是短暂的，但在使用 Cre-loxP 模型进行心脏再生研究时，需要纳入适当的对照，并认识到 Cre-重组酶引起的缺陷，以避免误解结果。