Deciphering the Endothelial Cell-Cardiomyocyte Crosstalk in LMNA Cardiomyopathy

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PROJECT SUMMARY Summary of Parent R01: Dilated cardiomyopathy (DCM) is a leading cause of heart failure and the leading reason for heart transplantation. Major gaps exist in our understanding of the pathophysiology of DCM and mutations in the gene that encodes the nuclear envelope proteins lamin A and C (LMNA) are considered to be the most common cause of DCM. However, the molecular mechanisms that underlie “cardiolaminopathy” remain elusive, and it is unknown why mutations in this ubiquitously expressed gene have such a disproportionate effect on the heart. Using induced pluripotent stem cell (iPSCs)-derived endothelial cells (iPSC-ECs), we recently studied a family affected by DCM due to a frameshift variant in LMNA, which showed endothelial dysfunction (Sayed et al. Science Translational Medicine, 2020). This EC dysfunction could be reversed by upregulating Krüppel-like Factor 2 (KLF2) by treatment of iPSC-ECs with a subset of statins, including lovastatin. Importantly, this improvement in EC dysfunction had a positive effect on co-cultured iPSC-cardiomyocytes (iPSC-CMs) from cardiolaminopathy patients, indicating an intricate crosstalk between the ECs and CMs in LMNA cardiomyopathy. Despite impressive progress, little attention has been given to the potential importance of cell-to-cell signaling between ECs and CMs, despite the fact that ECs serve a paracrine function to enhance signaling in CMs, especially in context to pharmacological stimulation. This knowledge gap impedes our comprehensive understanding of organ dysfunction at a multi-cellular level. The overarching goal of our proposal is to use a multidisciplinary approach that integrates human iPSCs, bioengineering tools, genome editing, and NGS to gain novel insights into the pathogenesis of DCM. Using human iPSCs, we propose to decipher the impaired cross-talk between ECs and CMs in LMNA cardiomyopathy and elucidate the beneficial class effects of statins in improving the EC-CM signaling as a key factor in regulating cardiac function. We will pursue three specific aims. In Aim 1: we will establish an experimental platform to study the genotype-phenotype association of LMNA mutations on ECs and CMs. For this, we will recapitulate the EC-CM crosstalk in LMNA iPSC-derived cells with 3D engineered heart tissues (EHTs). In Aim 2: we will decipher the mechanism of EC-CM crosstalk in LMNA iPSC-derived EHTs using single-cell approaches (scRNA-seq and scATAC-seq). In Aim 3: we will validate the key regulatory players of EC-CM crosstalk in LMNA cardiomyopathy by using CRISPR technology and zebrafish animal model. We have provided compelling preliminary data to support the soundness of our hypothesis-driven research proposal, and we are well positioned to achieve the project goals within five years. If successful, our studies will provide a new paradigm for understanding the pathogenesis and treatment of familial DCM. Proposed Supplement: The African American community, which represents 12.1% of the US population, is the second largest racial/ethnic minority group in the United States. When compared to other race/ethnic groups, African Americans have the highest incidence and prevalence of heart failure (HF) as well as the worst clinical outcomes. Moreover, when compared to Caucasians, they have a ~3-fold increased risk for developing dilated cardiomyopathy (DCM), and ~2-fold increased risk of death after diagnosis that is not explained by socioeconomic status and hypertension. In the proposed diversity supplement, we will extend the scope of our parent R01 to exclusively include additional patients from the African American cohort to understand this disparity at the molecular level. Specifically, we will investigate 10 additional individuals that belong to the African American community. The goal of this supplement grant would be to investigate the impact of variants, specifically in the LMNA gene, on the cardiac tissue and determine the cell-type specific signature responsible for this impaired function. For this, we will generate 3D engineered heart tissue (EHTs) from iPSC-ECs and iPSC-CMs from African American patients to characterize the effect of LMNA mutation on CM and EC function. These generated EHTs will be investigated at the single cell level to decipher the transcriptomic landscape and the impact of EC dysfunction on CMs. Furthermore, high-throughput affinity- based proteomics will be conducted to identify any secreted factors potentially involved in EC-CM crosstalk in LMNA DCM. The experiments will be carried out by Ms. Naima Turbes.