Spatially resolved RNA-sequencing of the injured murine heart

Myocardial infarction results in a massive loss of cardiomyocytes which will not be regenerated. Recent studies using models of regeneration show that cardiomyocytes at the border of the injury are most prone to proliferate. Which processes occur in this region and make these cells act as progenitors, remains to be elucidated. In addition, we lack molecular markers for the identification of these cells in the human heart. To create a transcriptional profile of the murine border zone, identifying local processes and enabling the discovery of marker genes which will allow the identification of human cardiomyocytes with potential proliferative capacity. Myocardial infarction (MI) was induced in mice and 3, 7 or 14 days later ventricular tissue was isolated ranging from the injured area into the remote myocardium. Tomo-sequencing was performed on these cardiac tissue samples, which comprises of tissue sectioning together with RNA-sequencing on individual sections. This resulted in spatially resolved transcriptome maps containing a dynamic, transcriptionally distinct border zone with reduced expression of genes involved in mitochondrial oxidative phosphorylation, fatty acid metabolism and sarcomere function and increased expression of myofibroblast and proliferation genes. Genes shared between the murine and zebrafish border zone, such as ANKRD1, UCHL1 and DES, were validated as molecular markers in injured human hearts. Unexpectedly, pronounced expression of these marker genes was found in surviving cardiomyocytes located at the sub-endocardium. The border zone is an evolutionary conserved, transcriptionally distinct and dynamic region. With the use of newly identified markers, we discovered the existence of sub-endocardial cardiomyocytes in ischemic human hearts that share transcriptional characteristics with potential progenitor cells. This might have far reaching implications for the development of strategies to stimulate the regenerative capacity of patients suffering from ischemic injury. Under a fluorescent stereoscope, injured mouse hearts were isolated and cut open longitudinally and tissue was selected based on katushka signal. The katushka signal in these hearts is expressed by a Nppb promotor, thereby marking stressed tissue which was used to orient the tissue. Tissue was isolated from the injured hearts (1x 3dpi, 2x 7dpi, 2x 14dpi) containing part of the injury, katushka signal and part of the remote myocardium respectively. Tomo-seq was conducted as previously described (Junker, Noël, et al., 2014) whereby the tissue was embedded in Jung tissue freezing medium (Leica), sectioned at 20um from the injured area to the remote myocardium, of which every fifth section was collected into a single tube. RNA was extracted from each tube using Trizol (Ambion) after adding a defined amount of spike-in RNA to correct for technical variations during the downstream processing. RNA-seq was performed as previously described (Junker, Noël, et al., 2014) including the reverse transcription using primers containing a tube-specific barcode. The barcoding of the cDNA allowed for the pooling of material, since the barcode could later be used to determine the positional origin of the labeled transcript. The pooling of cDNA was followed by linear amplification and sequencing.