Engineered Heart Slice Model of Arrhythmogenic Cardiomyopathy Using Plakophilin-2 Mutant Myocytes

Abstract

Genetic heart diseases such as arrhythmogenic cardiomyopathy (AC), a common genetic cause of sudden cardiac death, can be modeled using patient-specific induced pluripotent stem cell-derived cardiac myocytes (CMs). However, it is important to culture these cells in a multicellular syncytium with exposure to surrounding matrix cues to create more accurate and robust models of the disease due to the importance of cell-cell and cell-matrix interactions. The engineered heart slice, constructed by seeding CMs on intact decellularized matrix slices, allows molecular and functional studies on an aligned multilayered syncytium of CMs. This study reveals the potential for an improved disease-in-a-dish model of AC.

Keywords: arrhythmogenic cardiomyopathy; cardiomyocyte; decellularized matrix; disease modeling; engineered tissues; induced pluripotent stem cells.

FIG. 1.

Characterization of AC-CMs. AC-CMs (A, top) and H9-CMs (A, bottom) were stained for cardiac troponin I (magenta), plakoglobin (green), and DAPI (blue) to visualize myocytes. AC-CMs (B, top) and H9-CMs (B, bottom) were stained for integrin β1 (green), Nile Red (red), and DAPI (blue) to visualize matrix interactions and the presence of lipid droplets. Expression of mRNA transcripts associated with ion channels, cell junctional proteins, calcium-handling machinery, proteins involved in lipid metabolism, and apoptosis in AC-CM monolayers compared to that in H9-CM monolayers (C). Western blots of plakoglobin, Cx43, and Na_V1.5 in H9-CMs and AC-CMs (D). Expression of each protein was normalized to GAPDH (loading) and then to α-actinin and plotted for H9-CM monolayers (blue bars) and AC-CM monolayers (red bars). Error bars denote standard deviations. * Represents p < 0.05. n = 3 for each group. AC, arrhythmogenic cardiomyopathy; CMs, cardiac myocytes; DAPI, 4′,6-diamidino-2 phenylindole, dihydrochloride; mRNA, messenger RNA. Color images are available online.

FIG. 2.

Characteristics of AC EHS. Decellularized slices were stained for collagen I (green) and laminin (red) to illustrate the composition and arrangement of the extracellular matrix (A). Collagen fibers were visualized using second harmonic generation imaging (yellow), and AC-CMs were stained for α-actinin (magenta) and DAPI (blue) to illustrate the interaction between the cells and matrix in EHS (B). A higher magnification view of α-actinin and DAPI shows the orderly arrangement of sarcomeres in EHS (C). AC monolayers and EHS were compared (D) for staining for nuclei (blue), cTnI (magenta), and plakoglobin (green). All scale bars within (D) share the same value. cTnI, cardiac troponin I; EHS, engineered heart slice. Color images are available online.

FIG. 3.

Gene expression analysis of AC EHS. Expression of mRNA transcripts associated with structural proteins, ion channels, cell junctional proteins, calcium-handling machinery, proteins involved in lipid metabolism, and apoptosis in AC-CM EHS (n = 3) compared to that in AC monolayers. Fold changes are visualized on a log scale. n = 3 batches for EHS, n = 4 batches for monolayers; each batch with three to eight replicates. Color images are available online.

FIG. 4.

Beat rate in AC EHS and AC monolayers. Example images (top) and traces (bottom) for AC EHS (A) and AC monolayers (B). Average beat rate for EHS (red trace) and monolayers (black trace) over a period of about 24 h measured 1 week after EHS and monolayer seeding (C). Comparison of average 24-h beat rates between EHS and monolayers measured 1 and 2 weeks after seeding (D). Error bars denote standard deviations. * Represents p < 0.05 (ANOVA with Tukey's post hoc test for multiple comparisons). n = 11 for EHS and n = 18 for monolayers. ANOVA, analysis of variance. Color images are available online.

FIG. 5.

Wavefront propagation in AC EHS and AC monolayers. Sample activation maps illustrate propagation of action potentials in an AC EHS (A, left) and AC monolayer (A, right) paced at 1300 ms cycle length. Black lines indicate isochrones at 10 ms intervals, and rectangular symbols indicate the pacing sites. Spatially averaged action potential traces are shown for EHS (B, red trace) and monolayer (B, black trace) paced at 1300 ms cycle length. Conduction velocities (C) and APD₈₀ (D) at different pacing rates are plotted for EHS (red traces) and monolayers (black traces). Error bars denote standard deviations. n = 2–8 for AC EHS and n = 4–8 for AC monolayers. * Denotes p < 0.05 when comparing EHS with monolayers. APD₈₀, action potential duration, measured at 80% repolarization. Color images are available online.

FIG. 6.

Reentrant activity in AC EHS. Pacing with a line electrode from the bottom of the EHS at a cycle length of 2000 ms produced linear propagation of an activation wavefront across the EHS (A.a). Pacing with a point electrode in the middle of the EHS, at a site indicated by the rectangular symbol, 480 ms after the last line electrode stimulus resulted in a clockwise spiral wave (A.b–l) that terminated after one rotation (A.m, n). Voltage maps in (A.b–n) are 50 ms apart, and white arrows show local direction of the activation wavefront (A). Area where electrode was placed is blanked out. The optical voltage recording throughout the time series is shown by the black trace in (B) for the location indicated by the magenta dot in (A). The pacing pulses are indicated by the blue trace, and the time points of each snapshot (A.a–n) are indicated by the gray tick marks (B). White areas are parts of the mapping field blocked from imaging by the S2 electrode. Color images are available online.