Active Wnt signaling in response to cardiac injury

Although the contribution of Wnt signaling in infarct healing is suggested, its exact role after myocardial infarction (MI) still needs to be unraveled. We evaluated the cardiac presence of active Wnt signaling in vivo following MI, and investigated in which cell types active Wnt signaling was present by determining Axin2 promoter-driven LacZ expression. C57BL/6 Axin2-LacZ reporter mice were sacrificed at days 0, 1, 3, 7, 14, and 21 after LAD ligation. Hearts were snap-frozen for immunohistochemistry (IHC) or enzymatically digested to obtain a single cell suspension for flow cytometric analysis. For both FACS and IHC, samples were stained for β-galactosidase and antibodies against Sca-1, CD31, ckit, and CD45. Active Wnt signaling increased markedly in the myocardium, from 7 days post-MI onwards. Using Sca-1 and CD31, to identify progenitor and endothelial cells, a significant increase in LacZ+ cells was found at 7 and 14 days post-MI. LacZ+ cells also increased in the ckit+ and CD45+ cell population. IHC revealed LacZ+ cells co-expressing Sca, CD31, CD45, vWF, and αSMA in the border zone and the infarcted area. Wnt signaling increased significantly after MI in Sca+- and CD31+-expressing cells, suggesting involvement of Wnt signaling in resident Sca+ progenitor cells, as well as endothelial cells. Moreover, active Wnt signaling was present in ckit+ cells, leukocytes, and fibroblast. Given its broad role during the healing phase after cardiac injury, additional research seems warranted before a therapeutic approach on Wnt to enhance cardiac regeneration can be carried out safely

Innate immunity and remodelling

A wide variety of cardiac disease states can induce remodelling and lead to the functional consequence of heart failure. These complex disease states involve a plethora of parallel signal transduction events, which may be associated with tissue injury or tissue repair. Innate immunity is activated in hearts injured in different ways, evident as cytokine release from the heart, activation of toll-like receptors involved in recognizing danger, and activation of the transcription factor nuclear factor kappa B. Nuclear factor kappa B regulates gene programmes involved in inflammation as well as the resolution of inflammation. The impact of this is an enigma; while cytokines, toll-like receptors, and nuclear factor kappa B appear to elicit myocardial protection in studies of preconditioning, the literature strongly indicates a detrimental role for activation of innate immunity in studies of acute ischaemia–reperfusion injury. The impact of activation of cardiac innate immunity on the long-term outcome in in vivo models of hypertrophy and remodelling is less clear, with conflicting results as to whether it is beneficial or detrimental. More research using genetically engineered mice as tools, different models of evoking remodelling, and long-term follow-up is required for us to conclude whether activation of the innate immune system is good, bad, or unimportant in chronic injury models

Role of beta-catenin in adult cardiac remodeling

The adult heart has a uniform cellular response to adapt to injury after infarct or increased wall stress in chronic hypertension: hypertrophy of adult cardiomyocytes increases muscle fiber mass while at the same time apoptosis of cardiomyocytes may lead to further loss of contractile mass. The existence and quantitative amount of endogenous cardiac regeneration is currently under intense dispute, no clear picture has yet emerged. Recently, cardiac precursor cells and the signaling pathways controlling their differentiation in the adult organ have come into focus. In heart development, beta-catenin was identified to play a biphasic role in cardiomyocyte differentiation. While initially WNT/beta-catenin activation is required to commit mesenchymal cells to the cardiac lineage, downregulation of beta-catenin is needed for cardiomyocyte differentiation at later stages. Recent genetic data published by our lab suggest beta-catenin downregulation to be beneficial for adult cardiac remodeling. Here we discuss these data in the context of beta-catenin's role in adult cardiomyocyte hypertrophy, apoptosis and possibly regeneration

Cutting-free application of CRISPR-mediated endogenous gene activation in human induced pluripotent stem cell derived cardiomyocytes and engineered human myocardium

Abstract Background Imbalanced transcriptional networks characterize cardiomyocyte stress and result in cardiac remodelling. We hypothesize that re-establishing homeostatic gene networks in cardiomyocytes will prevent further tissue damage. To tackle this challenge, we applied CRISPR-based endogenous gene activation (CRISPRa) in vivo and in vitro. Methods We employ precision transcriptome editing tools based on CRISPR/Cas9 with enzymatically inactive Cas9 (dCas9) fused to transcriptional activators (VPR) to induce target gene expression by directing dCas9VPR to promoter regions by guide RNAs (gRNA). Results Homozygous CRISPRa hiPSC cell lines were generated by targeted integration of a CAG promoter driven dCas9VPR-T2A-tdTomato expression cassette into the AAVS1 locus by CRISPR/Cas9 editing and homology directed repair. Expression of dCas9VPR was evaluated by immunoblotting and co-expressed reporter fluorescence in spontaneously beating hiPSC-CM. We previously identified a crosstalk between WNT signalling and Krueppel-like factor 15 (KLF15) necessary for controlling cardiac homeostasis. We designed and tested 8 non-overlapping gRNAs in the –400 bp region upstream of the KLF15 transcriptional start site (TSS) and tested individual gRNA effectiveness for gene activation in HEK293T cells. Five gRNAs were identified inducing KLF15 transcript levels between 2- and 5-fold compared to non-targeted (NT) gRNA transfected cells (n=3 experiments). The single most effective gRNA was transduced by lentiviral particles into CRISPRa hiPSC-CM increasing KLF15 transcript levels to 1.5-fold compared to NT-gRNA control. Synergistic effects of 3 instead of single gRNA increased KLF15 transcript levels by 3-fold compared to controls (n≥3 experiments). We hypothesized that dCas9VPR expression could be harnessed as an additional option for gene dose titration and we generated hiPSC lines with enhanced dCas9VPR expression (v2.0). We observed up to 5-fold KLF15 gene activation when triple gRNA and v2.0 were combined (n≥4 experiments). Engineered human myocardium (EHM) was generated consisting of CRISPRa cardiomyocytes, fibroblasts and collagen and we observed similar contractility in 4-week cultured EHM suggesting innocuous dCas9VPR and gRNA expression. CRISPRa component expression was maintained over the entire culture period as evaluated by dCas9VPR immunoblotting and KLF15 transcriptional activation (1.4 fold, v1.0 CRISPRa hiPSC-CM, n≥8 tissues) indicating sustained gene activition. Conclusions Targeted gene activation with CRISPR/Cas9 is a precise and effective tool for transcriptional activation in hiPSC-CM. We observed titratability of gene activation by 1.) dCas9VPR expression levels and 2.) single versus multiple gRNA use. We furthermore elucidated general rules for effective gRNA targeting within the 5' TSS of genes of interest which confirmed a dependency of baseline gene activity as a limiting factor for endogenous gene activation. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German Research Foundation (DFG) - Collaborative Research Center 1002German Center for Cardiovascular Research (DZHK) </jats:sec