CLIPPER Regulates LPIN1-Mediated Mitochondrial Biogenesis and Heart Regeneration

The adult mammalian heart lacks the significant regenerative potential needed to cope with the massive loss of cardiomyocytes following myocardial infarction. Ultimately, irreversible cardiac damage leads to heart failure, which is associated with a very poor prognosis. Given this, reactivating dormant regenerative processes in the injured heart represents an attractive therapeutic approach. When regeneration does occur, newly formed cardiomyocytes are derived from preexisting ones. We aimed to identify novel regulators of cardiomyocyte proliferation. In this context, the genome is transcribed for a large part into RNAs with little or no protein-coding potential. Among noncoding RNAs, long noncoding (lnc)RNAs represent the most diverse class of molecules, and are implicated in numerous epigenetic mechanisms, making them ideal targets for controlling cell identity and behavior. In this project, we developed a high-throughput screening assay to identify lncRNAs that promote cardiomyocyte proliferation upon knockdown. Using a stringent selection pipeline, we identified Clipper, an enhancer-associated lncRNA regulating the expression of its cognate protein-coding gene Lpin1 in cis. Clipper was found to control mitochondrial biogenesis via LPIN1. Specifically, productive mitochondrial division, characterized by fission site positioning at the midzone of the mitochondrion, was stimulated by Clipper or Lpin1 silencing. The process was associated with a change in mitochondrial bioenergetics, particularly decreased oxidative metabolism, reduced production of reactive oxygen species, and dampened DNA damage, creating favorable conditions for cardiomyocyte proliferation. Importantly, Clipper knockdown in vivo following myocardial infarction stimulated cardiac regeneration in the damaged myocardium leading to the restoration of heart function. Importantly, CLIPPER is positionally and functionally conserved in humans. Our data identify CLIPPER as a promising therapeutic target for heart regeneration, acting through control of LPIN1-dependent mitochondrial biogenesis and cardiomyocyte proliferation. Overall design: Human ESCs were grown to 80% confluency before being seeded for differentiation. Beating hESC-CMs were transfected 9 days after the initiation of differentiation with 50 nM of either Control or CLIPPER-GapmeRs using Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher #13778150), and harvested 48 hours later for transcriptomic analysis. Total RNA was extracted using Trizol Reagent (15596026, ThermoFisher) and Direct-Zol RNA miniprep Kit (Zymo Research, R2050) according to manufacturer's protocol. RNA quality and yield was accessed using Agilent RNA 6000 pico kit (50167-1513, Agilent) for quality. Bulk RNA-seq library preparations were prepared using TruSeq Stranded Total RNA library Prep HMR kit (20020596, Illumina). cDNA libraries were quantified using the Agilent DNA1000 kit (5067-1505, Agilent), prior to sequencing on Illumina NextSeq Sequencing platform by Macrogen Asia Pacific Pte. Ltd. Raw Fastq files were trimmed using the trim-galore tool (v0.65) from Babraham Bioinformatics, to sequence length of 50. Paired-end reads were mapped and aligned to the human reference genome GrCh38/hg38 using STAR aligner (v2.7.0) as per default settings, and using gene annotations from GENCODE v38. Aligned gene counts were mapped using Htseq-count, and normalized to Counts per Million (CPM) by sequencing depth. The normalized gene expression count matrix was normalized by gene length.