Max Armour

BME 728 Organs on Chips

Spring 2017
March 2nd, 2017

Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent

stem cell and heart-on-chip technologies

Background: Barth syndrome (BTHS) is a genetic disease almost exclusively found in males. It
is responsible for cardiomyopathy, skeletal muscle weakness, growth delays and neutropenia.
The cause of the disease is rooted in a mutation of the TAZ gene, which encodes for the
phospholipid-lysophospholipid transacylase tazaffin. This enzyme is responsible for the
acylation of cardiolipin, the major phospholipid of mitochondrial inner membrane (~20%). It
regulates the aggregate structure of membrane, serves as a proton trap during cellular respiration,
translocate cholesterol and can trigger apoptosis. The exact transition from abnormal cardiolipin
biogenesis to cardiomyopathy is unclear; therefore, a platform consisting of patient-derived
iPSCs, genome editing and heart on a chip technology was used to replicate the pathophysiology
of BTHS in tissue constructs.
Experiment: Two iPSCs lines from two individuals with BTHS were generated using modRNA
and retroviral approaches (BTH-C, BTH-H), with three normal iPSC lines used as controls
(WT1, WT2, WT3). A skin biopsy was taken to retrieve fibroblasts, which were converted into
iPSCs using Yamanaka factors. The iPSCs were then differentiated into cardiomyocytes which
formed sheets and beat spontaneously once plated.
In BTHS, mature cardiolipin does not form causing monolysocardiolipin, the immature form, to
accumulate. Using mass spectroscopy, the monolysocardiolipin to cardiolipin ratio was
measured. The ratio exceeded 0.3, the clinically used threshold for BTHS.
Mitochondrial morphology and function was assessed using FACS. The BTHS iPSCs showed
mitochondrial numbers similar to the WT groups; however, they were smaller due to increased
fragmentation as compared to the more structurally organized WT mitochondria. ATP levels
were measured using galactose-based medium which forces the mitochondria to produce ATP via
glycolysis. ATP levels were much lower in the BTHS iPSCs and AMP-dependent kinase was
much higher due to increased cellular energy deprivation. Function was also assessed using an
extracellular flux analyzer, an instrument that enables the real-time measurement of the two
central pathways used by cells to generate ATP: glycolysis and oxidative phosphorylation.
BTHS iPSCs exhibited an elevated oxygen consumption rate (OCR) caused by an increase in
both F1F0 ATP synthase oxygen consumption (inefficient synthase activity) and hydrogen ion
leakage (decreased respiratory capacity caused by a hindered electron transport chain). Using
glucose based cultures, it was seen that glucose resorts basal ATP levels through increased
glycolysis but does not correct underlying defects in the electron transport chain.
TAZ mutation causation was assessed using three different approaches, taking into account the
genetic differences between control iPSCs and those derived from patients with BTHS. The first
strategy used adenoviral delivery of TAZ specific using short hairpin RNA to deplete TAZ in rat
ventricular cardiomyocytes. The same results were recorded as those in previous experiments.
The second strategy used modRNA due to the refractory nature of cardiomyocytes to standard
transfection methods.
The last strategy used Cas9-mediated genome editing which proved to be the most accurate
method in TAZ mutation. Together, the results indicated that TAZ mutation alone is sufficient to
cause said defects in an otherwise unaffected genetic background.
Sarcomere architecture was the next parameter studied. Mitochondria regulate cardiomyocyte
maturation, which is dependent on the proper assembly of organized arrays of sarcomeres.
BTHS iPSC-CMs assembled irregular sarcomeres compared to controls. Engineered
rectangular shaped iPSC-CM were made by seeding cells on a micropatterend fibronectin coated
rectangle designed to mimic the dimensions of mature cardiomyocytes (length to width ratio of
7:1). Alpha actin was used as the immunostaining marker for a testing platform called sarcomere
spacing, which used a 2D Fourier spectra of the stains to determine spacing. Sarcomeres
orientation in the control group was uniform, while it was intermittent in the BTHS group. It
was determined that sarcomerogenesis is sensitive to mitochondrial function independently of
ATP levels.
Heart-on-chip assays were used to replicate the contractile pathophysiology of BTHS in an in
vitro model of engineered myocardium. Cells were seeded onto thin elastomers micropatterend
with fibronectin and supported by glass coverslips. After 5 days the cells organized into laminar
anisotropic myocardium. The diastolic and peak systolic stresses were calculated using movies
of contracting MTFs (muscular thin filmed) tissues. The radii of curvature were measured and
plugged into a modified version of Stoneys equation. The rhythm of contraction was measured
using electric stimulation, with contraction frequency between 1-5 Hz. Treatment with TAZ
modRNA for 5 days restored the tissues to WT characteristics.
Potential therapies include bromoenol lactone (inhibitor of phospholipase A which catabolizes
mature cardiolipin), linoleic acid (essential unsaturated fatty acid precursor of mature
cardiolipin) and arginine plus cysteine. The latter had no effect, while the previous two showed
partial corrections of the monolysocardiolipin to cardiolipin ratio. Overall LA was the best
treatment option.
Critique: The use of iPSCs from patients with BTHS compared to other types of human stem
cells or standard cardiomyocytes conveyed the forthcoming power of personalized medicine for
drug testing. One drawback to this approach is the genetic variation between iPSC cell lines,
introducing variables that are both difficult to identify and control. The number of available in
vitro tissue models are also extremely low, with most systems relying on single cell to represent
entire tissues. The MTF tissue construct created could better mimic cardiac physiology in vivo
by creating a 3D structure or incorporating other cell types such as pacemaker cells.
Future Directions: Because sarcomere assembly is only one of several factors that leads to
reduced twitch stress, additional studies can be performed to identify other possible variables.
The system created by the authors can also be used for testing in cases of ischemia and aging.
ROS scavenging can also be used as a potential treatment method.