Manasa Kalluri 1 Louise Reilly PhD 1 and Lee Eckhardt MD 1 1 Cellular and Molecular Arrhythmia Research Program University of Wisconsin School of Medicine and Public Health Madison WI ID: 812341
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Analysis of the electrophysiological arrhythmia mechanisms of ARVC via multiparametric fluorescent optical mapping
Manasa Kalluri,
1 Louise Reilly, PhD1 and Lee Eckhardt, MD1
1: Cellular and Molecular Arrhythmia Research Program, University of Wisconsin School of Medicine and Public Health, Madison, WI
IntroductionChannelopathies are well recognized in neuro-muscular and cardiovascular disorders. Excitable tissue with voltage-gated ion channels are integral to these systems. Common channelopathy phenotypes include epilepsy, migraines, paralysis, and cardiac arrhythmias (Abriel et al, 2012). Cardiac contraction is controlled by the regulated spread of cell to cell electrical impulses. In arrhythmias, electrical impulses are abnormal and lead to irregular heart rhythms. A trigger for cardiac contraction is Ca+2 influx, as Ca+2 is needed for the action potential of cardiomyocytes to enable them to contract. Excitation-contraction coupling in cardiomyocyte tissue is regulated by the mechanism of Ca+2 -induced Ca+2 release (CICR) (Priori et. al, 2011). Mutations of genes encoding for cardiac ion channels disrupt ionic currents responsible for cardiac action potentials, causing severe arrhythmias.Arrhythmogenic right ventricular cardiomyopathy (ARVC), can result from mutations in several genes. Many are desmosome genes, providing instructions for making components of desmosomes. Desmosomes attach cardiomyocytes to one another, providing strength to the myocardium and playing a role in signaling (includes Ca+2 signaling) between neighboring cells. (Austin et. al, 2019)Prevalence of ARVC: Between 1:1,000 and 1:5,000 This project uses patient-specific induced pluripotent stem cell derived cardiomyocytes (iPS-CMs) from an ARVC patient in Dr. Lee Eckhardt’s UW Inherited Arrhythmia Clinic, to analyze the patient’s ARVC mechanism on a cellular scale using single cell optical mapping.
Clinical Background: Patient Model
ObjectiveCharacterize Ca+2 dynamics between iPS-CM patient model and control iPS-CMs to localize and analyse arrhythmia mechanisms of a patient-specific unknown variant of ARVC
ResultsCa2+ handling and β-adrenergic response of WT and ARVC iPS-CMs
Figure 6: Above depicted are bar graphs with error bars, depicting mean CaD (top) and RS (bottom), for WT control and ARVC iPS-CM groups, under both treatment conditions, paced at 0.5Hz.
Future Directions1. Analysing Ca+2 activity of arrhythmogenic, VF+ iPS-CMs
Figure 7: Examples of arrhythmogenic Ca+2 transient traces that visually exemplify strong evidence of VF+ cells recorded during experimentation.. A: Ca+2 transient of a VF+ cell depicted by top red trace. B: Ca+2 transient of a VF+ cell depicted by top black cell.
DiscussionThe individualized specificity provided by the iPS-CM patient model is promising in its adaptability to mimic the patient phenotype. This is crucial in potentially identifying cellular mechanisms of ARVC and its variants on an individualized basis.Protocol may prove to not only be reproducible, but adaptable. If future results suggest localized origins of electrophysiological abnormalities related to patient-specific ARVC symptoms, it could lead to a platform to test of a variety of drugs or treatment methods for ARVC and other arrhythmias. In the future, in conjunction with this cellular level of optical mapping, whole-heart optical mapping can be applied, in which Ca+2 dynamics can be analyzed on an anatomical basis, providing more evidence for localization of Ca+2 handling.
AcknowledgementsI would like to acknowledge Dr. Lee Eckhardt, Dr. Louise Reilly, and the Eckhardt Lab of the University of Wisconsin-Madison’s Cellular and Molecular Arrhythmia Research Program (CMARP) for their guidance and support in conducting this study. Additionally, I would like to thank the Glukhov Lab, also part of CMARP at the University of Wisconsin-Madison, for their guidance in single-cell optical mapping technique/protocol and data analysis.
Methods
Generation and culture of
Induced-pluripotent stem cell derived cardiomyocytes (
iPS
-CMs):
iPSC seeded and cultured via “
GiWi
” protocol for cardiomyocyte differentiation
Glucose-depleted culture medium allowed for yield of up to 99%
Control:
control (WT) iPS-CMs differentiated from the 19_9_ stem cell line Mutant: mutant (ARVC) iPS-CMs differentiated from cells of patient (blood sample). Figure 3: Timeline of cell differentiation process, with day 10 depicted as the approximate day differentiation into cardiomyocytes is achieved. iPS-CMs were used after day 30. Under this timeline is flow cytometry data, providing evidence of a high yield of mature cardiomyocytes. 2) Characterizing Ca2+ handling of WT and ARVC iPS-CMs via Single-Cell Optical Mapping Experimental Protocol): 1) Stain iPS-CMs with fluo-4 ABCAM (Ca+2 sensitive) 2) Follow pacing protocols (0.5Hz)Baseline pacingAdminister isoproterenol Post-isoproterenol pacingExtracting and analyzing mean CaD from recorded traces of single cell Ca+2 activity for WT and ARVC iPS-CMsRS (ms): Time to peak of Ca+2 transient, amount of time it takes for Ca+2 to be released from SRCaD (ms): Duration of Ca+2 transient, amount of time Ca+2 stays in the extracellular space before reuptake by sarcolemma
References:
Maizels
, L., et. al,
Circulation Research
, 2017,
doi
: 10.1161/CIRCEP.116.004725 Asimaki, A., et. al, Canadian Journal of Cardiology, 2015, doi: 10.1016/j.cjca.2015.04.012Austin, K.M., et. al, Nat Rev Cardiol, 2019, doi: 10.1038/s41569-019-0200-7Glukhov, A.V., et. al, 2015, doi: 10.1161/CIRCULATIONAHA.115.018131 Tohyama S., et. al, Cell Stem Cell, 2012, doi: 10.1016/j.stem.2012.09.013.Zipes, Douglas P., et. al, 2018, Cardiac Electrophysiology from Cell to Bedside.
Figure 2: Above depicts background for the patient model the ARVC-mutant iPSC in this experiment are from. Left: A pedigree of the patient’s family health history. The patient is depicted with gray shading. He is referred to as the “proband,” as he is the first individual in his family line to present with the listed arrhythmogenic symptoms. Right: An electrocardiogram (ECG) reading from the proband, depicting irregular heart rhythms upon aerobic exercise.
Figure 1: Schematic diagram depicting cellular components implicated in ARVC. Categories of protein in which ARVC-causing mutations occur are labelled accordingly. From: Austin et. al, 2019
2. Statistically significant differences for mean CaD and RS at baseline conditions, with no statistically significant differences evident upon administration of isoproterenol. (Sidak’s multiple comparisons test for equal means)At baseline conditions, on average, ARVC iPS-CMs exhibited a longer RS and a shorter CaD than WT. Upon administration of isoproterenol, no significant differences in both CaD and RS were exhibited for both WT and ARVC
Results
Figure 5:
The above images depict representative Ca+2 transient traces from WT cells and patient cells, at both baseline and under treatment with isoproterenol.
A:
Representative traces under both treatments from WT iPS-CMs. B: Representative traces under both treatment conditions from ARVC iPS-CMs.
Figure 4: From Glukhov et al, 2015 study. Left: fluorescent images of cells during a single-cell optical mapping experiment. Depicts Ca+2-activated fluorescence of T-tubule networks in ventricular cardiomyocytes. Right: Ca+2 transient activity collected on single ventricular cardiomyocytes.
A
B
Baseline
+ Iso
Proband is Caucasian male athlete, presented to UW-Madison hospital in 2008 after experiencing near syncope while playing soccer
Presented in initial ECG with bidirectional VT (
BiVT
)
Phenotypically demonstrated exercise-related
BiVT
with reproducible polymorphic ventricular tachycardia (PMVT) with exercise led to genetic testing for catecholaminergic polymorphic ventricular tachycardia (CPVT)
No pathogenic variants in related cardiomyocyte membrane proteins (
RyR2, CASQ2, KCNJ2
) were detected
Comprehensive arrhythmia genetic testing for variants of genes related to LQTS, CPVT, ARVC, SQTS,
BrS
Patient found to have a
PKP2
variant of unknown significance
Patient does not meet criteria for ARVC based on ECG and MRI, but his VT morphology and gene variant are suggestive of ARVC