Objective: Brugada Syndrome is an autosomal dominant congenital heart disease that
is responsible for 20% of sudden deaths of patients with structurally normal hearts.
The majority of mutations involve the cardiac sodium channel gene SCN5A and give
rise to classical symptoms, which include an abnormal electrocardiogram with
ST segment elevation and a predisposition to ventricular fibrillation. To date, the
implantation of a cardioverter defibrillator is the only proven effective treatment of
the disease. The ability to reprogram dermal fibroblasts to induced pluripotent stem
(iPS) cells and to differentiate these into cardiomyocytes with the same genetic
background provides a novel approach to studying inherited cardiac channelopathies
with advantages over existing model systems. Whilst this technique has enormous
potential to model inherited channelopathies, such as Brugada Syndrome, the derived
cells have not been fully characterised and compared to foetal and adult
cardiomyocytes.
Methods: Dermal fibroblasts from a patient with Brugada syndrome (SCN5A;
c.1100G>A - pARG367HIS) and an age- and sex-matched control were
reprogrammed using episomal vectors. All newly derived iPS cell lines were fully
characterised using immunocytochemistry, flow cytometry, real-time quantitative
reverse transcription PCR and single nucleotide polymorphism analysis and were
compared to established human embryonic stem (hES) cell and in-house derived
healthy control iPS cell lines. The same control cell lines were used to compare the
efficiencies of several cardiac differentiation media. Spontaneously contracting areas,
derived from control as well as patient iPS cell lines, were disaggregated and single
cardiomyocytes were compared to foetal and adult cardiomyocytes isolated from
primary human tissue using immunocytochemistry, transmission electron
microscopy, membrane visualisation, calcium imaging and electrophysiology.
Results: Comparison of cardiac differentiation protocols using healthy control hES
and iPS cell lines found that despite significant inter-line variability with regard to
efficiency of cardiac formation guided differentiation protocols could be used to
reliably and efficiently generate beating bodies. Spontaneous contraction was
observed in stem cell-derived cardiomyocytes and human foetal cardiomyocytes.
Pluripotent stem cell-derived cardiomyocytes stained for markers of the cardiac
contractile apparatus such as α-actinin, cardiac troponin I and cardiac troponin T.
They also expressed functional voltage-activated sodium channels and exhibited
action potential triggered calcium-induced calcium release. Stem cell-derived
cardiomyocytes showed organisation of myofibrils, ultrastructure and calcium
handling more similar to foetal than adult cardiomyocytes. Brugada Syndrome
patient-specific cardiomyocytes were structurally indistinguishable from healthy
control iPS cell line-derived cardiomyocytes. Electrophysiological analysis of
sodium current density confirmed a ~50% reduction in patient-derived compared to
healthy control-derived cardiomyocytes.
Conclusion: Although iPS cells give rise to a mixture of immature and more mature
cardiomyocytes, they all express typical cardiac proteins and have functional cardiac
sodium channels. Results illustrate the ability of patient-specific iPS cell technology
to model the abnormal functional phenotype of an inherited channelopathy that is
independent of structural abnormalities and that the relative immaturity of iPS
cell-derived cardiomyocytes does not prevent their use as an accurate model system
for channelopathies affecting the cardiac sodium channel Nav1.5. This iPS cell based
model system for classical Brugada Syndrome allows for the first time to study the
mutation in its native environment and holds promise for further studies to
investigate disease mechanisms of known and unknown mutations and to develop
new therapies