Induced pluripotent stem cell-derived 3D models to unravel the phenotypic diversity of arrhythmogenic cardiomyopathy

Sudden cardiac death (SCD) induced by ventricular arrhythmias is one of the
leading causes of death worldwide. Myocardial loss of mechanical continuity,
the underlying pathogenesis of arrhythmogenic cardiomyopathy (ACM) is one of
the major players involved in SCD. ACM is an inherited disease most often
caused by mutations in components of the desmosome, the organelle involved in
cell-cell adhesion. The cardiac desmosome consists of five different components
and a deteriorated function of one these component can disintergrate the entire
desmosome. The latter results in the loss of electromechanical coupling between
cardiomyocytes. Patients with ACM therefore display a loss of mechanical
continuity.
The early phase of ACM is in general mostly asymptomatic with
occasional arrhythmias triggered by mechanical strain. Patients are usually
diagnosed during the late phase of the disease, where inflammation, fibrosis
and adipocytes have infiltrated the myocard. These systemic changes are
irreversible and complicate the investigation of underlying mechanisms that
characterize the early phase of ACM. Unraveling these mechanisms is important
because mutation-carriers in the ACM associated genes display extreme diverse
phenotypes. This diversity is even depicted in patients displaying identical
mutations and no explanation has thus far been found that might explain the
latter. By reprogramming patient-derived fibroblast to induced pluripotent stem
cells, differentiated cardiomyocytes can be generated. With this model we
believe we can investigate the patient-specific development of ACM on a
cellular level.

Our main objective is to investigate whether the diversity between identical
mutations is merely caused by environmental factors, or whether genetic factors
also underlie the manifestation of the disease. With this study, we aim to use
ACM patient-derived iPSCs to generate 3D cardiac tissues as a specific model to
test our main objective as well as the effects of environmental factors such as
exercise induced elements (stretch, altered substrate metabolism) on the
mechanical coupling of cardiac tissues.

Primary (or first) Objective: Generate iPSC-derived cardiac structures from
patients with ACM in order to establish the typical ACM associated phenotype.

Secondary Objective(s):
- Introduce the identical mutation in a well established and characterized H9
hESC line and repair the mutation in the patient-derived iPSC line using the
CRISPR/Cas9 genetic editing tool. Genetically unaltered and altered H9
ESCs-derived cardiac structures will be phenotypically compared to the
genetically unaltered and altered iPSC derived cardiac structures.
- Additionally, use the cell lines generated above to generate 3D cardiac
tissues as a specific model to test the effects of environmental factors such
as exercise induced elements (stretch, altered substrate metabolism).

Non-therapeutic study, exploring ACM patients, one visit research.

For the whole study, fibroblasts from 16 ACM patients, 4 patients per ACM
associated mutation, will be used. After patient selection, a skin punch biopsy
(6mm) will be performed according to standard procedures (10) under sterile
conditions and after local anesthesia at the cardiology outpatient clinic,
UMCG. During the same visit, a blood sample (~30 ml) will be collected by
standard venipuncture. The skin biopsies will be collected in sterile
physiological salt solution and transported to the lab of the dept. of
Experimental Cardiology, UMCG. After tissue dissociation, fibroblasts will be
cultured and multiplied under fibroblast specific/selective culture conditions.
Part of these fibroblasts will be stored frozen; another part will be used for
reprogramming into iPSCs and subsequently differentiated into cardiomyocytes
according to procedures developed in the dept. of Experimental Cardiology,
UMCG. Subsequently, the iPSC-derived cardiac structures will be subjected to
extensive analyses. These characteristics will be compared with cardiac tissues
generated from the same iPSC line that has been genetically repaired, as well
as genetically altered and unaltered H9 lines. IPSC lines will be frozen and
stored for a period of 15 years and can be included in later research projects.
Moreover, there will be exchange of iPSC lines with other research institutes,
including a department that is specialized in bio-engineering.

Harvesting one skin punch biopsy (diameter 6mm) from the inner side of the
upper arm under local anesthesia. Blood samples will be collected from
patients. If a heart transplant has been performed and the heart is available
for research purposes, we apply to perform immunohistochemistry staining of the
right (and left) ventricle.

The burden or risks associated with this study is minimal and may only include
complications of the skin punch biopsy and venipuncture. Infection from a skin
biopsy is a rare event occurring less than 1% of the time. A venipuncture very
rarely results in a hematoma or infection. Complications will be handled at the
time that they arise. The study will not provide a direct benefit to the
patients, but may prove beneficial to the research community with potential new
insights in HF pathogenesis and new treatment approaches.