Arrhythmia Mechanism and Dynamics in a Humanized Mouse Model of Inherited Cardiomyopathy Caused by Phospholamban R14del Mutation

Abstract

Background: Arginine (Arg) 14 deletion (R14del) in the calcium regulatory protein phospholamban (hPLN^R14del) has been identified as a disease-causing mutation in patients with an inherited cardiomyopathy. Mechanisms underlying the early arrhythmogenic phenotype that predisposes carriers of this mutation to sudden death with no apparent structural remodeling remain unclear.

Methods: To address this, we performed high spatiotemporal resolution optical mapping of intact hearts from adult knock-in mice harboring the human PLN^WT (wildtype [WT], n=12) or the heterozygous human PLN^R14del mutation (R14del, n=12) before and after ex vivo challenge with isoproterenol and rapid pacing.

Results: Adverse electrophysiological remodeling was evident in the absence of significant structural or hemodynamic changes. R14del hearts exhibited increased arrhythmia susceptibility compared with wildtype. Underlying this susceptibility was preferential right ventricular action potential prolongation that was unresponsive to β-adrenergic stimulation. A steep repolarization gradient at the left ventricular/right ventricular interface provided the substrate for interventricular activation delays and ultimately local conduction block during rapid pacing. This was followed by the initiation of macroreentrant circuits supporting the onset of ventricular tachycardia. Once sustained, these circuits evolved into high-frequency rotors, which in their majority were pinned to the right ventricle. These rotors exhibited unique spatiotemporal dynamics that promoted their increased stability in R14del compared with wildtype hearts.

Conclusions: Our findings highlight the crucial role of primary electric remodeling caused by the hPLN^R14del mutation. These inherently arrhythmogenic features form the substrate for adrenergic-mediated VT at early stages of PLN^R14del induced cardiomyopathy.

Keywords: arrhythmia; arrhythmogenic cardiomyopathy; dilated cardiomyopathy; dynamics; phospholamban; spiral wave reentry; sudden death; ventricular tachycardia.

Figure 1:. Arrhythmia induction in ex-vivo retrograde perfused hPLN^R14del (R14del) and hPLN^WT (WT) murine hearts.

a. Schematic describing the generation of the mouse models with knock-in hPLN^WT and hPLN^R14del. H2BGFP expressed under the control of endogenous mouse PLN promoter. b. Pacing protocol used on the ex-vivo perfused hearts. The median of the pacing frequency range in the second segment of the protocol (with ISO) was chosen as the cut-off frequency (i.e. at 22.5Hz). c. Representative 2-sec optical AP recordings showing the initiation of sustained VT in R14del but not WT hearts in response to an identical challenge. d. Up to a cut-off frequency of 22.5Hz, sustained VT was induced in 67% of R14del hearts vs. 25% of WT hearts, exclusively upon exposure to ISO.

Figure 2:. Preferential APD prolongation in the RV and increased APD dispersion in R14del hearts.

a. Representative AP traces measured from the RV and LV of ex-vivo R14del (black) and WT (blue) hearts, showing preferential APD prolongation in the RV vs LV of mutant hearts at 10Hz. b. Summary data showing the average right ventricular APD₇₅ and c. P_5–95, an index of the epicardial APD dispersion, in all WT and R14del hearts. *** p <0.001, ** p <0.01. d. R14del hearts exhibited a rightward shift in APD histograms relative to WT, with a bimodal distribution resulting in a marked increase in APD dispersion. δ₁ and δ₂ indicate the median of the distribution for WT and R14del hearts, respectively.

Figure 3:. Impaired APD adaptation to β-adrenergic stimulation in R14del hearts.

a. Representative AP traces recorded from the LV and RV before (blue for WT, black for R14del) and after (red) challenging hearts with ISO at 10Hz. b. A blunted response to ISO is observed across all R14del hearts in both LV and RV. c. Colormaps showing APD₇₅ values of individual pixels from the segmented left and right masks over the posterior epicardium of WT (top) and R14del (bottom) hearts before and after ISO showing a relatively static response to ß-adrenergic stimulation in R14del compared to WT as well as marked APD dispersion in the former vs latter. d. Representative histograms showing the epicardial APD₇₅ distribution in WT and R14del hearts at baseline (white) and after isoproterenol (blue). A leftward shift in the WT (median shift δ₁ → δ’₁) indicates an overall decrease in average APD₇₅ vs. a much-reduced response in R14del hearts (δ₂ → δ’₂). ** p <0.01, *** p <0.001, **** p <0.0001.

Figure 4:. Mechanism of arrhythmia initiation and maintenance in the R14del heart.

a. Representative activation maps measured during pacing at 10, 15, and 20Hz that illustrate the rate-dependent development of severe local conduction slowing/block in R14del but not WT hearts. b. Repolarization maps for the same WT and R14del hearts. R14del hearts exhibit a marked interventricular repolarization gradient compared to WT. Arrows indicate site of high repolarization gradient that coincides with the loci of severe conduction slowing/block in R14del.

Figure 5:. Sustained rotors preferentially pin to the RV in R14del hearts.

a. Snapshots showing the instantaneous phase for all pixels. These sequential maps show rotation around a PS (filled circle) with the arrow showing the direction of rotation (chirality). b. A lower f_Crit in R14del vs WT hearts (left) documents their significantly higher vulnerability to arrhythmia. Spirals entrained the ventricles at higher f_Dom in R14del vs WT hearts (right). c. Composite plot of the ratio of f_Crit/f_Dom and the VT duration of 9 WT and 10 R14del hearts. The ratio f_Crit/f_Dom is < 1 in R14del hearts and > 1 in WT hearts, resulting in a clear separation between the two groups. d. Phase density maps showing the probability of finding a PS at a certain location at a random time point during the VT episode in representative WT and R14del hearts. e. In R14del hearts, PS were located within the RV in more than 80% of the time vs. less than 25% of the time in WT hearts. ** p <0.01.

Figure 6:. CV as a function of distance from the tip during reentry in WT and R14del hearts.

a. Velocity field calculated from the phase gradient in a WT heart during VT. Inset shows a magnified view around the PS. b.& c. Velocity maps in the WT (top) and R14del hearts (bottom). Lowest velocities are recorded in the vicinity of the PS, progressively increasing in magnitude away from it. Corresponding velocity vs. distance (0 to 4mm) plots are also shown. d. Increased meandering range and faster V_Drift were calculated across R14del vs WT hearts. ** p <0.01. e. Weighted slopes (±95% confidence interval) of the velocity vs distance relationships for all WT and R14del hearts are 0.105±0.005 (R² = 0.99) and 0.137±0.006 (R = 0.99), respectively. ** p <0.01, *** p <0.001, ^# p <0.0001. f. Spiral waves in R14del hearts exhibited a considerably faster f_Pseudo compared to WT. The ratio of f_Crit/f_Pseudo is significantly lower in R14del hearts. ** p <0.01, **** p <0.0001. g. f_Dom vs. f_Pseudo plot. The red dotted line represents the best fit (slope = 0.81±0.06, R² = 0.94), as f_Dom correlates nicely with f_Pseudo in WT hearts. Such property is lost in R14del hearts (R² = 0.44).

Figure 7:. RV and LV expression of Kir2.1, Nav1.5, and Cx43 in WT (n=5) and R14del (n=4) hearts.

Representative western blots and quantification of RV vs LV protein expression of Kir2.1 (a), Nav1.5 (b), and Cx43 (c) in WT and R14del. GAPDH was used as an internal loading control.