DENND3-p.R534S disrupts dyadic microdomain architecture to drive potentially pro-arrhythmic calcium and electrophysiologic instability

Figures

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  Figure 1. DENND3-p.R534S drives multicellular electrical instability and electromechanical uncoupling in iPSC-CMs. (A) Representative MEA recordings demonstrating stable, rhythmic electrical activity in IC monolayers and irregular, heterogeneous waveforms with intermittent abnormal deflections in DENND3-p.R534S iPSC-CMs. Corresponding Poincaré plots show tight clustering of beat intervals in ICs vs. marked dispersion in DENND3-p.R534S CMs, indicating impaired beat-to-beat temporal stability. (B) Quantification of beat period coefficient of variation (CoV), percentage of electrodes exhibiting arrhythmic activity, conduction velocity, and excitation-contraction delay. DENND3-p.R534S monolayers exhibit increased rhythm variability, elevated arrhythmogenic burden, slowed conduction, and prolonged electromechanical delay. Data were obtained from three independent cardiomyocyte differentiation batches (N = 3). Each data point represents one independent iPSC-CM monolayer. Data are presented as mean ± SEM, *P < 0.05, **P < 0.01, ****P < 0.0001.

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  Figure 2. DENND3-p.R534S induces heterogeneous and dyssynchronous calcium cycling independent of Ca2+ handling protein expression. (A) Representative Fluo-4 AM calcium line-scan recordings from paced iPSC-CMs showing spatially uniform and synchronized calcium transients in IC-CMs vs. irregular, dyssynchronous calcium release in DENND3-p.R534S CMs. (B) Proportion of cells exhibiting abnormal calcium transients and representative patterns of abnormal calcium behaviour observed in DENND3-p.R534S iPSC-CMs, including irregular and dyssynchronous calcium release. DENND3-p.R534S I–III represent representative examples of distinct abnormal calcium transient patterns. (C) Quantification of calcium transient duration at 90% decay (CTD90), peak-to-90% decay time, calcium transient amplitude (ΔF/F0), and maximal upstroke velocity (Vmax), demonstrating impaired calcium handling in DENND3-p.R534S CMs. (D) Representative immunoblot analysis and densitometric quantification of RyR2, SERCA2a, PLN, and NCX1 protein expression. Calcium imaging data were obtained from N = 3 independent cardiomyocyte differentiation batches. Each data point represents one individual cardiomyocyte. Immunoblot analyses were…

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  Figure 3. DENND3-p.R534S disrupts BIN1-dependent membrane architecture and pre-T-tubule organization. (A) Representative Airyscan super-resolution images (4× expansion microscopy) showing organized, striated BIN1-positive membrane structures and coordinated Cav1.2 localization in IC-CMs, compared with dispersed BIN1 organization and heterogeneous Cav1.2 distribution in DENND3-p.R534S CMs. (B) Quantification of membrane organization using directionality analysis, demonstrating increased dispersion of BIN1-positive structures in DENND3-p.R534S CMs. (C) Representative immunoblot and densitometric quantification of total BIN1 expression. Imaging data were obtained from N = 3 independent cardiomyocyte differentiation batches. Each data point in (B) represents one individual cardiomyocyte. Immunoblot analyses were performed using protein collected from N = 7 independent cardiomyocyte differentiation batches, and each data point in (C) represents one independent differentiation batch. Data are presented as mean ± SEM. Scale bars as indicated. Scale bars = 10 μm, ****P < 0.0001.

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  Figure 4. BIN1 disruption is associated with nanoscale uncoupling of CRUs. (A) Representative Airyscan super-resolution images (4× expansion microscopy) of Cav1.2 and RyR2 localization. IC-CMs exhibit close spatial apposition of Cav1.2 and RyR2 along BIN1-positive pre-T-tubule structures, whereas DENND3-p.R534S CMs display disrupted architecture and loss of coordinated spatial alignment. Insets show higher magnification. (B) Three-dimensional rendering of the regions shown in (A) and quantitative nearest-neighbour analysis demonstrating increased Cav1.2-RyR2 separation distance in DENND3-p.R534S CMs. Imaging data were obtained from N = 3 independent cardiomyocyte differentiation batches. Each data point represents one individual cardiomyocyte. Data are presented as mean ± SEM. Scale bars as indicated. Scale bars = 10 μm, ****P < 0.0001.

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  Figure 5. Genetic or pharmacologic inhibition of Rab11b rescues membrane architecture, dyadic coupling, calcium handling, and electrical stability. (A) Representative super-resolution images (4× expansion microscopy) and quantification of BIN1 organization showing restoration of BIN1 membrane organization following Rab11b inhibition using either dominant-negative Rab11b or the small-molecule CDKI-73 in DENND3-p.R534S iPSC-CMs. (B) Quantification of Cav1.2-RyR2 nearest-neighbour distance demonstrating normalization of nanoscale dyadic coupling following Rab11b inhibition. (C) Quantification of calcium transient duration at 90% decay (CTD90), peak-to-90% decay time, calcium transient amplitude (ΔF/F0), and maximal upstroke velocity (Vmax) showing restoration of calcium-handling kinetics following Rab11b inhibition. (D) Quantification of beat period coefficient of variation (CoV) and excitation-contraction delay demonstrating restoration of multicellular electrical stability following Rab11b inhibition. Imaging data were obtained from N = 3 independent cardiomyocyte differentiation batches. Each data point in (A) and (B) represents one individual cardiomyocyte. Calcium imaging data in (C)…