Identifying Features of Cardiac Disease Phenotypes Based on Mechanical Function in a Catecholaminergic Polymorphic Ventricular Tachycardia Model

Abstract

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is characterized by an arrhythmogenic mechanism involving disruption of calcium handling. This genetic disease can lead to sudden death in children and young adults during physical or emotional stress. Prior CPVT studies have focused on calcium handling, but mechanical functionality has rarely been investigated in vitro. In this research we combine stem cell-derived cardiomyocytes from a CPVT patient (RyR2-H2464D mutation) and a healthy familial control with an engineered culture platform to evaluate mechanical function of cardiomyocytes. Substrates with Young's modulus ranging from 10 to 50 kPa were used in conjunction with microcontact printing of ECM proteins into defined patterns for subsequent attachment. Digital Image Correlation (DIC) was used to evaluate collections of contracting cells. The amplitude of contractile strain was utilized as a quantitative indicator of functionality and disease severity. We found statistically significant differences: the maximum contractile strain was consistently higher in patient samples compared to control samples on all substrate stiffnesses. Additionally, the patient cell line had a statistically significantly slower intrinsic contraction rate than the control, which agrees with prior literature. Differences in mechanical strain have not been previously reported, and hypercontractility is not a known characteristic of CPVT. However, functional changes can occur as the disease progresses, thus this observation may not represent behavior observed in adolescent and adult patients. These results add to the limited studies of mechanical function of CPVT CMs reported in literature and identify functional differences that should be further explored.

Keywords: 2D cell culture; DIC; contraction rate; disease model; iPSC-cardiomyocytes; mechanical strain; microcontact printing.

FIGURE 1

(A) Chevron pattern of 30 µm lanes connected at 15°: (i) CAD drawing of the pattern; (ii) Matrigel stained with laminin; (iii) Pattern seeded with control CPVT stem-cell derived cardiomyocytes. Scale bars are 100 µm. (B) Timeline of cell culture, differentiation, purification, and experiments. Day 0 denotes the start of CM differentiation and E0 denotes the start of experiments after cells are seeded onto a micropatterned substrate.

FIGURE 2

Quantification of contractile strain for control (blue) and patient (red) cell lines. Stars indicate relaxed state and peak contraction of a sample on a 10 kPa substrate. The full-field second principal strain for these two states is shown in the strain heatmaps (A). Representative samples at each substrate stiffness for control (B) and patient (D) cell line. The maximum contractile strain for the three substrate stiffness conditions for the control (C) and patient (E) cell lines.

FIGURE 3

Comparison of peak contractile strain for control and patient across all three substrate stiffnesses are shown. **p < 0.001, ***p < 0.0001, one-way ANOVA with post hoc Tukey tests.

FIGURE 4

Quantification of contraction rate for patient and control cell lines (A) Contraction rate was calculated by the distance between mid-points of the intersection of 60% peak strain and average contractile strain plots and converted to beats per minute. (B) Overlaid histograms of the contraction rate for all samples of blue (control) and patient (red) cell lines. (C) Rate of spontaneous contraction across all samples is significantly slower in the patient line than the control p < 0.0001.