Project 9

Figure 1 legend: A. Expression of Igf1 and WT1 by myocardial and pericardial cell clusters. B: Immunohistochemical detection of td-tomato expression in peri/epicardium after transient Tx-induced activation of CreERT2 integrated into the WT1⁺ locus leading to persistent td-tomato expression in WT1+ cells and descendants. basal: 5d Cre activation followed by recovery for 5 days leads to abundant tomato expression in pericardium but not epicardium. I/R: On day 6 post MI (induced 5 days after last Tx injection) pericardial thickness and tomato expression increased. Note that epicardium was thickened and contains abundant tomato expressing cells

Background and preliminary work: Insulin-like growth factor I (Igf1) plays a pivotal role in cardiac tissue repair and remodeling. The Gödecke lab investigates the role of Igf1 in the heart ¹ and demonstrated that a 3 days Igf1 application starting at reperfusion after AMI reduces scar size, and improves angiogenesis and pump function far beyond the time course of Igf1 application ². This protective effect involves an Igf1-dependent phenotypic modulation of myeloid cells, particularly NF and MF, resulting in an attenuated pro-inflammatory phenotype of both cell types in vivo ³. Furthermore, others discovered that the endogenous expression of Igf1 in the heart is crucial for driving the hypertrophic response of cardiac myocytes in pressure overload, emphasizing the role of Igf1 in modifying the CM phenotype ⁴. 

Based on these data, Gödecke and coworkers aim for elucidating the role of Igf1, released locally in the myocardium and pericardium, in cardiac remodeling post MI. scSeq of the cardiac non-myocyte fraction and of the pericardium after MI revealed that Igf1 is highly expressed by resident MF and FB clusters in the heart. Further, MF, adipocytes, and, particularly WT1⁺ mesothelial cells in the pericardium express Igf1 (Fig.4A). This leads to the hypothesis, that Igf1 derived from pericardial and cardiac cell populations modulates cellular phenotypes and thereby improves the outcome post MI. Notably, WT1⁺ cells have the potential to undergo epithelial-to-mesenchymal transition ⁵, and can give rise to FB, SMC, and in some cases to CM after migration into the myocardium. By lineage tracing using tamoxifen-(Tx)-inducible expression of the fluorescent td-tomato protein in WT1⁺ pericardial cells (WT1⁺PC) and their descendants, we found that the pericardium, but not the epicardium, contains high numbers of WT1⁺ expressing cells under basal conditions. As shown in Fig.4B, WT1⁺PC proliferate after MI, leading to thickening of the pericardial sac, and tomato⁺ cells were also found in the activated epicardium and the scar area. Thus, in contrast to the current assumption that epicardium is the main source of WT1⁺ cells post MI, we hypothesize that WT1⁺PC or their differentiated descendants migrate across the pericardial cavity into the epi- and myocardium. The cell fate of migrated WT1⁺PC cells and their role in healing post-AMI is unclear.

The lack of sufficient myocardial regeneration is the core problem of myocardial infarction, causing the loss of contractile myocardium. However, the group of Matthew Wolf, by using a sophisticated double recombinase system (sequential Dre and Cre-mediated recombination) for advanced lineage tracing, has provided crucial insights. They have demonstrated that cardiac myocytes reenter the cell cycle post-MI, leading to an increased ploidy due to endoreplication. In some cases, these myocytes even underwent cytokinesis, giving rise to newly formed CM ^{6, 7}. This finding has profound implications for the field of myocardial regeneration post-MI, as it highlights the reactivation of the cell cycle in cardiomyocytes as a key mechanism in post-infarction remodeling ⁸. Moreover, RNA sequencing of cycling cardiomyocytes ⁹ revealed extensive transcriptional changes, including a signature similar to that of epicardial or pericardial WT1⁺ progenitor cells and a striking 28-fold upregulation of Igf1 expression (Fig.5A).

Hypothesis: We hypothesize that the endogenous expression of Igf1 by various cell populations in the heart is a major determinant, which alters cell fate post-AMI via autocrine and/or paracrine mechanisms. Thereby, endogenous Igf1 contributes to cardiac adaptation by modulating the phenotype of pericardial cells, macrophages, neutrophils, and cardiomyocytes, particularly cycling cardiomyocytes.

Work program: To study the role of Igf1 release by WT1⁺PC and cFB the Igf1 gene will be inactivated by Tx application either in pericardial WT1⁺PC (Wt1-CreERT2-deleter) ¹⁰ or in cFB using the Tcf21-mCm (merCremer) deleter ¹¹. Active Cre induces simultaneously the expression of td-tomato in cells expressing WT1 or Tcf21 at the time of Tx-injection allowing lineage tracing of WT1⁺PC  and cFB ± Igf1 expression post AMI. Serial echocardiography reveals the functional consequences of loss of Igf1 release by WT1⁺PC and cFB and their descendants, histological analyses (scar size, hypertrophy, capillary density, etc.) the cardiac remodeling. Tomato-expression serves as histological marker to identify the migration and differentiation of WT1⁺ pericardial cells after MI using co-staining of slices with EC, FB, CM, and MF markers and in FACS analyses aiming for measuring the cellular composition of the hearts post AMI. 

In an untargeted approach, use of tomato expression as molecular marker for lineage tracing in scSeq of total cardiac cell preparations will result in comprehensive information on the cell fate of WT⁺PC and CF derived cells. Taking into account the plasticity of WT1⁺PC and cFB, we expect to discover transition/differentiation of cells derived from both precursor cell types. In addition, scSeq allows analysis of Igf1-mediated phenotypic modulation all other cardiac cell populations. These analyses will elucidate (i) the transition/differentiation of WT1⁺PC and cFB in pericardium and myocardium, (ii) how Igf1 released by these cells affects the cellular composition of pericardium (mesothelial cells, macrophages, B-cells) and myocardium (immune cells, CM, EC,…), (iii) how the Igf1 pools promote plastic remodeling of other pericardial and cardiac cell populations (NF, MF, FB, EC,…), and (iv) how Igf1 alters the myocardial microenvironment post AMI as a major determinant for cardiac healing and functional adaptation.

The bioinformatic analysis (QC, transcript mapping, cell clustering, and differentially expressed genes) will be conducted using the Seurat or Scanpy packages. RNA velocity and trajectory inference (monocle3, slingshot) will be used in collaboration with P5 (Marschall) and P10 (Dörr) to generate models for differentiation pathways of WT1⁺PC and cFB post MI as well as that of other cell clusters in the heart affected by Igf1 released by WT1⁺PC or cFB. scSeq data for cFB will be re-used by P5 (Altschmied/Leitinger/ organ specific fibroblast differentiation) and P7 (Bottermann/Fisher/Harris/ fibroblast plasticity). 

In the complementing part of our project, Wolf and coworkers will utilize their Dre/cre-recombinase system to inactivate the Igf1 gene specifically in cycling CM expressing high levels of Igf1 and to induce GFP expression in the same set of cells. Histological analysis of hearts ± Igf1 knockout in cycling CM will provide data on the influence of Igf1 on the number of replicating cells (GFP⁺) and, through the quantification of paired GFP⁺ cells, on the numbers of CM that, most likely, completed cell division. Parameters such as scar size and fibrosis will also be obtained. A detailed functional analysis by echocardiography will reveal the functional effects of Igf1 deletion in cycling CM. A hallmark of cycling CM is their molecular signature of gene expression, similar to epicardial progenitor cells (Wolf et al., unpublished). RNA seq of enriched (GFP⁺) cycling CM will provide insight into the extent to which the molecular phenotype is determined by Igf1 expression. The Wolf lab has also developed new Dre/Cre lineage tracing mice that will label WT1⁺ cells in tdTomato and CMs derived from WT1⁺ precursor cells in eGFP (Fig.5B,C). This approach will enable us to quantify the spatial distribution and genetically ablate Igf1 in the population of CMs derived from WT1⁺ precursors. Given the presence of a “progenitor cell” signature in cycling cardiac myocytes, the data sets obtained by sequencing from the WT1⁺ derived pericardial cells together with those obtained from cycling CM will be analyzed to infer if WT1⁺ derived cells contribute to the pool of cycling CM. The potential implications of these findings for our understanding of cardiac regeneration and disease progression are significant.

Added value of the collaboration: Sharing of specific mouse models and complementary methods will cause scientific synergism, particularly during the internships in Charlottesville and Düsseldorf. The German graduate student will work on immune cell composition in hearts of mice with cycling CM-specific KO of Igf1 to study the role of this Igf1 pool particularly on myeloid cell modulation. In a complementary approach to Igf1 gene deletion, the doctoral researcher from Charlottesville will investigate the effect of an Igf1 receptor deletion in WT1⁺ cells post MI in mice available at Düsseldorf. In particular, the number and distribution of tomato⁺ cardiac myocytes derived from WT1⁺ cells will be in the focus of the analysis. Our collaborative project will shed new light on the impact of Igf1 and on origin and function of cycling CM in post MI cardiac repair and regeneration.