Genetic landscape of cardiomyopathies – Landscardio

Introduction of rare variants of interest in two cell lines THP-1 and AC16.
CRISPR-Cas9 genome editing is changing the landscape of genomics due to its ease of use and ability to create double-stranded breaks (DSB) at almost any locus of interest. Genome stability in eukaryotic cells requires a mechanism for the efficient repair of DNA lesions such as the homology-directed repair (HDR) pathway, which faithfully copies the genetic information from a related sequence and seals the DSB in an error-free manner.

Generate Inducible pluripotent stem cells (iPSC)-derived cardiomyocytes from patients and controls. Peripheral blood mononuclear cells (PBMC) from one CCC (carrying gene variant) and one ASY (wild-type) member of each family will be collected. Inducible pluripotent stem cells (iPS) will be generated from peripheral blood erythroblasts.

Assess whether cells carrying pathogenic variants display increased mitochondrial dysfunction as compared to «wild-type«cells, in the presence or absence of IFN? and TNFa. Using the IPSC-derived CMs, EBV immortalized PBMCs, AC16 cardiomyocytes and THP-1 with or without the pathogenic variants, we will assess energy metabolism and mitochondrial dysfunction in the presence or absence of IFN? and TNFa.

Assess whether the rare deleterious variants in IPSC-derived cardiomyocytes, EBV immortalized PBMCs, AC16 cardiomyocytes and THP-1 cells induce major transcriptomic pattern changes and increases NF-kB transcriptional activity and in the presence or absence of IFN? and TNFa.
Wild-type or mutant cells will be treated by inflammatory cytokines (IFN? +TNFa). We expect that loss-of-function variants in mitochondrial genes may increase mitochondrial susceptibility to IFN? and TNFa-induced damage, leading to mitochondrial dysfunction and release of NF-kB activating molecules such as ROS and mitochondrial DNA.

Assess and compare the genetic landscape of unrelated individuals with various heart disease phenotypes.

not available yet

The characterization of rare deleterious variants located in mitochondrial genes in cell lines and biological samples in heart disease is innovative. The identification of the genetic landscape of CCC and other cardiomyopathies will help to decipher the pathogenic process.

Ouarhache M, Marquet S, Frade AF, Ferreira AM, Ianni B, Almeida RR, Nunes JPS, Ferreira LRP, Rigaud VO, Cândido D, Mady C, Zaniratto RCF, Buck P, Torres M, Gallardo F, Andrieux P, Bydlowsky S, Levy D, Abel L, Cardoso CS, Santos- Junior OR, Oliveira LC, Oliveira CDL, Nunes MDC, Cobat A, Kalil J, Ribeiro AL, Sabino EC, Cunha-Neto E, Chevillard C. Rare Pathogenic Variants in Mitochondrial and Inflammation-Associated Genes May Lead to Inflammatory Cardiomyopathy in Chagas Disease. J Clin Immunol. 2021 Mar 3. doi: 10.1007/s10875-021-01000-y. Epub ahead of print. PMID: 33660144.

Cardiomyopathies are an important worldwide public health problem. Available therapies are insufficient and do not fully address the cardiac molecular abnormalities. Mitochondrial dysfunction is the source of heart energy deprivation in cardiomyopathies and heart failure. Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is a neglected disease affecting around 8 million people, with no effective antiparasitic drugs or vaccines. About 30% of Chagas disease patients develop chronic Chagas disease cardiomyopathy (CCC), an inflammatory dilated cardiomyopathy that occurs decades after the initial infection, while most infected patients (60%) remain asymptomatic/indeterminate (ASY). Death results from heart failure or arrhythmia in a subset of CCC patients. Survival in CCC is worse than in other cardiomyopathies like inflammatory dilated (DCM) and ischemic cardiomyopathy. Distinct from other cardiomyopathies, CCC displays a Th1-T cell rich myocarditis with abundant IFN? and TNFa, and selectively lower levels of mitochondrial energy metabolism enzymes in the heart. A genetic component to disease susceptibility was suggested by familial aggregation of CCC cases, as well as case-control studies that identified gene polymorphisms associated to CCC development. Assessment of rare variants with whole exome sequencing (WES) in nuclear families with multiple CCC/ASY cases has disclosed rare heterozygous pathogenic variants in mitochondrial and inflammatory genes associated to CCC cases. In each family, the identified variants were shared only by family members with CCC, but absent from siblings with the ASY form of disease. Indeed, from the 25 CCC-associated heterozygous pathogenic mutations, 22 were in genes playing a role in mitochondria and inflammation. This suggests that similar mechanisms may occur in other families. IFN? and TNFa /NF-kB signaling negatively affect mitochondrial function. We thus hypothesize that the mechanism underlying this finding is that Chagas disease patients carrying these gene variants displayed increased myocardial mitochondrial dysfunction, inflammatory responses and therefore develop CCC, while relatives not carrying the variants will not develop these processes and remain asymptomatic. We also hypothesize the genetic landscape of unrelated patients across the spectrum of Chagas disease (ASY, moderate and severe CCC) will be different from that of other cardiomyopathies such as idiopathic dilated (DCM) and noncompaction cardiomyopathy (LVNC). To test these hypotheses, our objectives are: 1) to functionally characterize the rare variants in the mitochondrial and inflammatory genes identified in CCC cases in two multicase families using patients’ iPS-derived cardiomyocytes and immortalized lymphoblastoid cell lines and cell lines with point mutations. 2) Assess whether these variants induce mitochondrial dysfunction in the presence or absence of IFN? and TNFa. 3) Assess whether these variants induce major transcriptomic pattern changes in the presence or absence of IFN? and TNFa. 4) Assess whether cells carrying these variants display differential cytokine production and differential gene expression profile after stimulation with IFN?/TNFa. 5) to identify the genetic landscape of unrelated Chagas patients with varying clinical severity by whole exome sequencing of DNAs from unrelated individuals (ASY, moderate and severe CCC phenotypes (arrhythmia/heart failure) as well as that of non-CCC cardiomyopathies (DCM and LVNC). This project is set up by three groups that are used to work together. We have a unique biological sample collection that is already available. The characterization of rare deleterious variants located in mitochondrial genes in cell lines and biological samples in heart disease is innovative. The identification of the genetic landscape of CCC and other cardiomyopathies will help to decipher the pathogenic process.