Dysfunctional crosstalk of cardiomyocytes and cardiac fibroblasts in a pluripotent stem cell model of dilated cardiomyopathy
Authors: Maurer W , Koehne S , Maus A , Tiburcy M , Schlick S , Zibat A , Zimmermann W.H , Sossalla S , Wollnik B , Streckfuss-Boemeke K 
Published: November 2020

Abstract

Background/Purpose
Dilated cardiomyopathy (DCM) is characterized by left ventricular dilation and contractile dysfunction. Fibrosis is one major phenotypic result in DCM, pointing to the contribution of both, cardiomyocytes (CM) and cardiac fibroblasts (cFB) to DCM. The molecular basis of most DCM cases remains unknown. Nevertheless, it is known that up to 35% of all cases have a family history, linked to mutations in more than 30 gene loci. The aim of this study is to analyse the crosstalk of iPSC-CM and cFB and the underlying genetic and molecular causes in a patient-specific induced pluripotent stem cell (iPSC) model of DCM.

Methods and results
For this purpose a 4-member family was recruited containing 2 patients (father and daughter) with severe DCM and heart transplantation. iPSCs of all family members were generated and differentiated into iPSC-CMs. All iPSC-CMs express general cardiac markers, e.g. βMHC, α-actinin. Interestingly, αMHC expression was decreased in diseased iPSC-CMs in comparison to control cells. Additionally, the sarcomeric regularity was decreased in diseased iPSC-CMs. As we found significantly increased fibrosis (22%) in explanted myocardium of the diseased father compared to healthy myocardium (8%), both cFB and CM seem to play an important role. From the same myocardium primary cFBs were isolated and shown to express typical cFB markers clearly distinguishing these cells from non-fibroblasts as well as from fibroblasts with different origin. To analyse the contribution of cFBs and CMs to DCM on a functional level, 3D engineered heart muscles (EHMs) were generated in different diseased/healthy cell combinations. EHMs composed of both or either one affected DCM-iPSC-CMs and/or DCM-cFBs in comparison to healthy control EHMs did not produce any measurable force, indicating that the DCM-EHM phenotype is clearly diseased. Evaluation of tissues' viscoelasticity showed that DCM-cFB, DCM-iPSC-CMs and DCM-EHMs were stiffer than healthy control EHMs. Thus these data suggest that apart from the obvious dysfunction of DCM CMs, DCM cFBs clearly contribute to the contractile pathophysiology in DCM EHMs. Furthermore, whole exome sequencing of iPSCs was conducted to identify disease-causing variants. This analyses point towards a new genetic variant in the FLNc gene coding for a protein important in development, stabilization and maintenance of myofibrils. Rescue of this variant by CRISPR Cas9 genome editing will shed more light onto the role of this variant during DCM development in the future.

Conclusion
Using a ps-iPSC-CM model of a 4-member family with two severe DCM patients, we could demonstrate a clear contribution of both cell types, iPSC-CMs and cFB, to the contractile pathophysiology of DCM. We identified a potentially disease-causing new variant in the FLNc gene, which may contribute to the impaired functionality within the diseased iPSC-CMs and EHM. This makes FLNc a new therapeutic target for DCM.

Funding Acknowledgement
Type of funding source: Public grant(s) – National budget only. Main funding source(s): IRTG1816