Abstract
Background: Myocardial infarction leads to a massive loss of cardiomyocytes (CM) and cardiac remodeling, which results in reduced cardiac function and, ultimately, heart failure. Adult mammalian CMs cannot spontenously proliferate, and thus repair the cardiac injury, however in our previous study we showed that simultaneous knock down of Rb1 and Meis2 induced CM cell cycle activation that resulted in improved cardiac function. Moreover, most significant cardioprotective effects were due to CM mediated paracrine mechanisms including angiogenesis and CM cell survival. Thus, this study aims to identify these indirect cardioprotective pathways dependent on CM cell cycle. Methods and Results: We utilized genetically modified FUCCI mice, which allows identifying the cycling vs. non-cycling CM for transcriptome analysis. Adult mouse CM was isolated through the Langendorff heart perfusion method, using our modified isolation protocol, and cultured in the modified DMEM media. Adult CMs were transfected with equimolar (50uM each) combination of siRb1 and siMeis2 (siRNA-cocktail), cel-miR-67 served as control. CMs were harvested, and RNA isolation was performed on day seven after transfection. Rb1 and Meis2 knock-down were validated through CM proliferation analysis and RT-PCR based expression profiling for selected markers of proliferation, angiogenesis, cell survival, and structural genes. Our analysis showed a significant down-regulation of Rb1 and Meis2 after siRNA treatment.Further, we observed an up-regulation of pro-angiogenic and pro-survival genes without any significant alteration in the structural genes. After validating our samples, we performed a whole-genome transcriptome analysis using a high-throughput Illumina NextSeq platform. Our RNAseq analysis revealed the activation of pathways, which may induce the CM rejuvenation. The identified potential targets will be further validated through in vivo experiments. Conclusions: Our results suggest the activation of CM rejuvenation after knocking-down Rb1 and Meis2 to improve the cardioprotection after injury.
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