Plakophilin-2 truncating variants impair cardiac contractility by disrupting sarcomere stability and organization

Abstract

Progressive loss of cardiac systolic function in arrhythmogenic cardiomyopathy (ACM) has recently gained attention as an important clinical consideration in managing the disease. However, the mechanisms leading to reduction in cardiac contractility are poorly defined. Here, we use CRISPR gene editing to generate human induced pluripotent stem cells (iPSCs) that harbor plakophilin-2 truncating variants (PKP2tv), the most prevalent ACM-linked mutations. The PKP2tv iPSC–derived cardiomyocytes are shown to have aberrant action potentials and reduced systolic function in cardiac microtissues, recapitulating both the electrical and mechanical pathologies reported in ACM. By combining cell micropatterning with traction force microscopy and live imaging, we found that PKP2tvs impair cardiac tissue contractility by destabilizing cell-cell junctions and in turn disrupting sarcomere stability and organization. These findings highlight the interplay between cell-cell adhesions and sarcomeres required for stabilizing cardiomyocyte structure and function and suggest fundamental pathogenic mechanisms that may be shared among different types of cardiomyopathies.

Fig. 1.. CRISPR-engineered human iPSC-CMs with PKP2tvs demonstrate decreased expression of PKP2.

(A) MiSeq readout aligned to the reference PKP2 genome shows 5-bp deletion at the end of exon2 in the PKP2tv^+/− and PKP2tv^−/− cell lines. (B) Schematic representation of PKP2 protein showing the location of patient mutations p.R79X and p.Q133X, and the CRISPR-engineered mutation p.D109Afs in two WT iPSC lines (WT1 versus PKP2tv^+/− and WT2 versus PKP2tv^−/−). AA, amino acid. (C) Representative images of iPSC-CM monolayers fixed and stained for nuclei (blue), α–actinin-2 (gray; top), and PKP2 (gray; bottom). PKP2 fluorescence is not detectable in PKP2tv^−/−. Scale bars, 20 μm. (D) Relative RNA expression of PKP2 gene (2^−ΔΔCT) in iPSC-CMs measured by reverse transcription quantitative polymerase chain reaction (PCR): 63.2 ± 13.9% in PKP2tv^+/− (n = 4) compared to WT1 (n = 3) and 32.5 ± 6.2% in PKP2tv^−/− (n = 3) compared to WT2 (n = 3), data presented as means ± SEM. (E) Western blots of PKP2 protein in iPSC-CMs. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (F) Mean fluorescence intensity of junctional PKP2 shows a significant reduction in PKP2tv^+/− (n = 10) versus WT1 (n = 11) and in PKP2tv^−/− (n = 5) versus WT2 (n = 5). a.u., arbitrary units. Statistics: Individual data points across three independent experiments are shown with means ± SEM on plots; Student’s t test, α = 0.05. Immunostains are representative of at least three independent experiments.

Fig. 2.. PKP2tvs result in decreased junctional localization of desmoplakin and Cx43.

(A) Representative images of iPSC-CM monolayers fixed and stained for nuclei (blue), N-cadherin (gray; top), and desmoplakin (DSP; gray; bottom). Scale bars, 20 μm. (B) Representative images of iPSC-CM monolayers fixed and stained for nuclei (blue), N-cadherin (gray; top), and Cx43 (gray; bottom). Scale bars, 20 μm. (C) Mean fluorescence intensity of junctional N-cadherin is comparable in PKP2tv^+/− (n = 13) versus WT1 (n = 13) and in PKP2tv^−/− (n = 5) versus WT2 (n = 5). n.s., not significant. (D) Mean fluorescence intensity of junctional desmoplakin at cell-cell contacts (defined by junctional N-cadherin signal) is significantly lower in PKP2tv^+/− (n = 11) versus WT1 (n = 10) and in PKP2tv^−/− (n = 5) versus WT2 (n = 5). (E) Mean fluorescence intensity of junctional Cx43 at cell-cell contacts (defined by junctional N-cadherin signal) is significantly lower in PKP2tv^+/− (n = 13) versus WT1 (n = 13) and in PKP2tv^−/− (n = 8) versus WT2 (n = 7). Statistics: Individual data points across three independent experiments are shown with means ± SEM on plots; Student’s t test, α = 0.05. Immunostains are representative of at least three independent experiments.

Fig. 3.. PKP2tvs lead to prolongation of action potential.

(A and C) Action potential traces of iPSC-CMs expressing Archon1 measured by normalized Archon1 fluorescence intensity, while iPSC-CM monolayers are electrically paced at 1 Hz. Data are shown as means (solid lines) ± SD (bars), and curves are aligned at peaks for (A) PKP2tv^+/− (n = 30) versus WT1 (n = 30) and (C) PKP2tv^−/− (n = 27) versus WT2 (n = 25). (B and D) Quantification of APD at 30, 50, and 80% below the peak of action potential curve (APD30, APD50, and APD80) shows significant increase in APD of (B) PKP2tv^+/− (n = 30) versus WT1 (n = 30) and (D) PKP2tv^−/− (n = 27) versus WT2 (n = 25). Statistics: Individual data points across three independent experiments are shown with means ± SEM on plots, unless otherwise specified; Student’s t test, α = 0.05.

Fig. 4.. PKP2tvs cause reduction in systolic contractility in CMTs.

(A) Top view of a CMT suspended between two elastic polydimethylsiloxane (PDMS) pillars with spherical caps at day 10 after seeding. Scale bar, 200 μm. (B) Representative force-over-time curve measured from a CMT made with WT2 cells, while the tissue is electrically paced at 1 Hz. (C) Quantification of peak systolic forces of CMTs shows a significant reduction in force generation in PKP2tv^+/− (n = 22) versus WT1 (n = 25) and in PKP2tv^−/− (n = 27) versus WT2 (n = 20). (D) Quantification of peak systolic stresses of CMTs shows comparable stress levels in PKP2tv^+/− (n = 22) versus WT1 (n = 25) and a significant reduction of systolic stress in PKP2tv^−/− (n = 27) versus WT2 (n = 20). Statistics: Individual data points across three independent experiments are shown with means ± SEM on plots; Student’s t test, α = 0.05.

Fig. 5.. Single CMs harboring pkp2tvs exhibit increased contractility.

(A) Representative image of PAA gel surface micropatterned with rectangular islands of fluorescently labeled fibronectin (FN–555 nm; shown in orange). Each fibronectin island has a 1:7 aspect ratio with an area of 2000 μm² and allows a single CM to attach and spread on it when seeding density is properly controlled. Scale bar, 100 μm. (B) Representative phase image of a single iPSC-CM spread on a rectangular fibronectin island on PAA gel surface. Scale bar, 20 μm. (C) Representative stress vector maps at the peak systole of single CMs from traction force microscopy (TFM) measurements comparing PKP2tv^−/− with WT2. Boundaries of cell area/fibronectin island are marked by white dotted lines. Scale bars, 20 μm. (D) Quantification of root mean square (RMS) stress at peak systole of single CM contractions shows a significantly higher stress level in PKP2tv^+/− (n = 20) versus WT1 (n = 20) and in PKP2tv^−/− (n = 48) versus WT2 (n = 47). (E) Quantification of work at peak systole of single CM contractions shows a significantly higher work production in PKP2tv^+/− (n = 20) versus WT1 (n = 20) and in PKP2tv^−/− (n = 48) versus WT2 (n = 47). Statistics: Individual data points across three independent experiments are shown with means ± SEM on plots; Student’s t test, α = 0.05.

Fig. 6.. PKP2tvs impair multicellular cardiac contractility.

(A) Representative image of PAA gel surface micropatterned with square islands of fluorescently labeled fibronectin (FN–555 nm; shown in orange). Each fibronectin island is 100 μm by 100 μm and allows around six to eight CMs to attach and form a multicellular cardiac patch on it when seeding density is properly controlled. Scale bar, 100 μm. (B) Representative phase image of a cardiac patch formed by multiple iPSC-CMs spread on a square fibronectin island on PAA gel surface. Scale bar, 50 μm. (C) Representative stress vector maps at the peak systole of cardiac patches from TFM measurements comparing PKP2tv^−/− with WT2. Boundaries of cardiac patch area/fibronectin island are marked by white dotted lines. Scale bars, 50 μm. (D) Quantification of RMS stress at peak systole of cardiac patches shows a significantly lower stress level in PKP2tv^+/− (n = 17) versus WT1 (n = 21) and in PKP2tv^−/− (n = 59) versus WT2 (n = 65). (E) Quantification of work at peak systole of cardiac patches shows a significantly lower work production in PKP2tv^+/− (n = 17) versus WT1 (n = 21) and in PKP2tv^−/− (n = 51) versus WT2 (n = 64). Statistics: Individual data points across three independent experiments are shown with means ± SEM on plots; Student’s t test, α = 0.05.

Fig. 7.. PKP2tvs alter molecular stability and turnover of junctional N-cadherin.

(A) Representative images of junctional N-cadherin before, right after, and 30 min after photobleaching in monolayer cultures of iPSC-CMs. Scale bars, 5 μm. (B) Percentage junctional N-cadherin FRAP over 30 min in monolayer cultures of iPSC-CMs comparing PKP2tv^+/− (n = 12) versus WT1 (n = 12) and PKP2tv^−/− (n = 25) versus WT2 (n = 19). Data are shown as means (dots) ± SD (bars) with the solid lines showing data fitted in a two-phase association model. (C) Mobile fraction of junctional N-cadherin measured as the plateau of FRAP recovery curves shows a significant reduction in monolayer cultures of PKP2tv^+/− (n = 12) versus WT1 (n = 12) and in PKP2tv^−/− (n = 25) versus WT2 (n = 19). Data across three independent experiments are shown as means ± SEM; Student’s t test, α = 0.05.

Fig. 8.. PKP2tvs destabilize N-cadherin junctions at cell-cell contact.

(A) Representative time-lapse images of N-cadherin junctions at 0, 2, 4, 6, and 8 hours into the live imaging of monolayer cultures of iPSC-CMs comparing PKP2tv^+/− versus WT1. Scale bars, 10 μm. (B) Temporal color-coded hyperstacks of representative N-cadherin time-lapse movies showing stable N-cadherin junctions in monolayer cultures of WT1 and unstable cell-cell contact in PKP2tv^+/−. Scale bars, 10 μm. (C) Net displacement of junctional N-cadherin in time-lapse movies measured by optical flow tracking shows a significant increase in monolayer cultures of PKP2tv^+/− (n = 14) versus WT1 (n = 18) and in PKP2tv^−/− (n = 14) versus WT2 (n = 12). (D) Mean displacement per frame of junctional N-cadherin in time-lapse movies measured by optical flow tracking shows a significant increase in monolayer cultures of PKP2tv^+/− (n = 14) versus WT1 (n = 18) and in PKP2tv^−/− (n = 14) versus WT2 (n = 12). (E) Ramble of junctional N-cadherin in time-lapse movies measured by optical flow tracking shows an increase in monolayer cultures of PKP2tv^+/− (n = 14) versus WT1 (n = 18) (not significant) and in PKP2tv^−/− (n = 14) versus WT2 (n = 12) (significant). Statistics: Individual data points across three independent experiments are shown with means ± SEM on plots; Student’s t test, α = 0.05.

Fig. 9.. PKP2tvs destabilize sarcomeres.

(A) Representative time-lapse images of sarcomeric α–actinin-2 at 0, 2, 4, 6, and 8 hours into the live imaging of monolayer cultures of iPSC-CMs comparing PKP2tv^+/− versus WT1. Scale bars, 10 μm. (B) Temporal color-coded hyperstacks of representative sarcomeric α–actinin-2 time-lapse movies showing stable sarcomere structures in in monolayer cultures of WT1 and unstable sarcomeres in PKP2tv^+/−. Scale bars, 10 μm. (C) Net displacement of sarcomeric α–actinin-2 in time-lapse movies measured by optical flow tracking shows a significant increase in monolayer cultures of PKP2tv^+/− (n = 13) versus WT1 (n = 18) and in PKP2tv^−/− (n = 13) versus WT2 (n = 12). (D) Mean displacement per frame of junctional N-cadherin in time-lapse movies measured by optical flow tracking shows comparable mean displacement per frame in monolayer cultures of PKP2tv^+/− (n = 13) versus WT1 (n = 18) and in PKP2tv^−/− (n = 13) versus WT2 (n = 12). (E) Ramble of junctional N-cadherin in time-lapse movies measured by optical flow tracking shows a significant decrease in monolayer cultures of PKP2tv^+/− (n = 13) versus WT1 (n = 18) and in PKP2tv^−/− (n = 13) versus WT2 (n = 12). Statistics: Individual data points across three independent experiments are shown with means ± SEM on plots; Student’s t test, α = 0.05.

Fig. 10.. PKP2tvs impair sarcomere content and organization in multicellular cardiac patches.

(A) Representative images of cardiac patches immunostained for sarcomeric α–actinin-2 (gray) and nuclei (blue). Scale bars, 50 μm. (B) Quantification of the number of α–actinin-2–positive sarcomeric z-discs in each cardiac patch shows significantly less sarcomere content in PKP2tv^+/− (n = 25) versus WT1 (n = 25) and in PKP2tv^−/− (n = 11) versus WT2 (n = 8). (C) Order parameter (O.P.) for sarcomere alignment plotted over distance from any sarcomere z-disc comparing cardiac patches formed with PKP2tv^+/− (n = 25) versus WT1 (n = 25) and PKP2tv^−/− (n = 1) versus WT2 (n = 8). Data are shown as means (dots) ± SD (bars) with the solid lines showing data fitted in an exponential decay model. (D) Quantification of O.P. at 50 μm away from any sarcomere z-disc shows a significant decrease in sarcomere alignment in cardiac patches formed with PKP2tv^+/− (n = 25) versus WT1 (n = 25) and in PKP2tv^−/− (n = 11) versus WT2 (n = 8). (E) Quantification of the plateau of O.P. decay model shows comparable levels of sarcomere alignment in long range in cardiac patches formed with PKP2tv^+/− (n = 25) versus WT1 (n = 25) and significantly lower levels of sarcomere alignment in PKP2tv^−/− (n = 11) versus WT2 (n = 8). Statistics: Individual data points across three independent experiments are shown with means ± SEM on plots, unless otherwise specified; Student’s t test, α = 0.05. Immunostains are representative of at least three independent experiments.