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. 2024 Apr 30;12(5):985.
doi: 10.3390/biomedicines12050985.

Endurance Training Provokes Arrhythmogenic Right Ventricular Cardiomyopathy Phenotype in Heterozygous Desmoglein-2 Mutants: Alleviation by Preload Reduction

Larissa Fabritz  1   2   3   4 , Lisa Fortmueller  1   2   4 , Katja Gehmlich  3   5 , Sebastian Kant  6 , Marcel Kemper  3   4 , Dana Kucerova  4 , Fahima Syeda  3 , Cornelius Faber  7 , Rudolf E Leube  6 , Paulus Kirchhof  1   2   3 , Claudia A Krusche  6
Affiliations

Endurance Training Provokes Arrhythmogenic Right Ventricular Cardiomyopathy Phenotype in Heterozygous Desmoglein-2 Mutants: Alleviation by Preload Reduction

Larissa Fabritz et al. Biomedicines. .

Abstract

Desmoglein-2 mutations are detected in 5-10% of patients with arrhythmogenic right ventricular cardiomyopathy (ARVC). Endurance training accelerates the development of the ARVC phenotype, leading to earlier arrhythmic events. Homozygous Dsg2 mutant mice develop a severe ARVC-like phenotype. The phenotype of heterozygous mutant (Dsg2mt/wt) or haploinsufficient (Dsg20/wt) mice is still not well understood. To assess the effects of age and endurance swim training, we studied cardiac morphology and function in sedentary one-year-old Dsg2mt/wt and Dsg20/wt mice and in young Dsg2mt/wt mice exposed to endurance swim training. Cardiac structure was only occasionally affected in aged Dsg20/wt and Dsg2mt/wt mice manifesting as small fibrotic foci and displacement of Connexin 43. Endurance swim training increased the right ventricular (RV) diameter and decreased RV function in Dsg2mt/wt mice but not in wild types. Dsg2mt/wt hearts showed increased ventricular activation times and pacing-induced ventricular arrhythmia without obvious fibrosis or inflammation. Preload-reducing therapy during training prevented RV enlargement and alleviated the electrophysiological phenotype. Taken together, endurance swim training induced features of ARVC in young adult Dsg2mt/wt mice. Prolonged ventricular activation times in the hearts of trained Dsg2mt/wt mice are therefore a potential mechanism for increased arrhythmia risk. Preload-reducing therapy prevented training-induced ARVC phenotype pointing to beneficial treatment options in human patients.

Keywords: arrhythmogenic cardiomyopathy; arrhythmogenic right ventricular cardiomyopathy (ARVC); desmoglein 2; desmosome; endurance exercise; intercalated disk; mouse model; preload-reducing therapy.

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Conflict of interest statement

The authors declare no direct conflicts of interest to the work of this submission but have disclosed all interests as below. L.F. (Larissa Fabritz) received institutional research grants for basic, translational, and clinical research projects from the European Union, the British Heart Foundation, the Medical Research Council (UK), National Institute of Health Research, DZHK, DFG, and several biomedical companies. L.F. (Larissa Fabritz) is listed as the inventor of two patents held by the University of Birmingham (Atrial Fibrillation Therapy WO 2015140571 and Markers for Atrial Fibrillation WO 2016012783). L.F. (Larissa Fabritz) is part of the advisory board of an ARVC patient initiative (no honoraria). The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Summarizes the rationale for the models and time points used in this study. (A) The time points of ARVC manifestation in human patients (age ± SD) [14]. (B) The corresponding mouse ages (in weeks) according to the study by Wang and coworkers [60] and the time points of structural ARVC onset with cardiomyocyte death, inflammation, and fibrotic remodeling that we observed in homozygous Dsg2 mutant [48] and cardiomyocyte-specific Dsg2 knockout mice [42]. These mouse models mimicked ARVC patients with disease onset in early childhood but not in patients with disease onset during their second to fifth decade of life. (C,D) We therefore investigated haploinsufficient Dsg20/wt mice [59] and heterozygous mutant Dsg2mt/wt mice [48] at the indicated ages (bars in orange) to assess whether they were useful models for the concealed phase and/or an ARVC onset in adulthood. We first analyzed the histomorphology and cardiac function of sedentary one-year-old mice, i.e., an age corresponding to 40- to 50-year-old humans. We then assessed sedentary Dsg2mt/wt mice at 16–29 weeks, which corresponded to 25- to 30-year-old humans. Since no obvious ARVC phenotype was detected in this age range, a seven-week-long endurance swim training, which is an aerobic exercise known to induce physiological cardiac growth (blue bar), was initiated at the age of 8–12 weeks to provoke an ARVC phenotype.
Figure 2
Figure 2
A structural ARVC phenotype is detectable only in 8% of one-year-old Dsg20/wt haploinsufficient mice. (A,D,G,J) Hearts of one-year-old Dsg2WT mice, (B,E,H,K) heart sections of haploinsufficient Dsg20/wt mice without a structural phenotype, and (C,F,I,L) heart sections of one-year-old Dsg20/wt mice displaying an ARVC-like structural phenotype (Dsg20/wt fib mice; 8% of mice with Dsg2 haploinsufficiency). (A–C) Heidenhain’s AZAN staining detects fibrosis and scars (blue: pf = physiological periarterial fibrosis; if = interstitial fibrosis; rf = replacement fibrosis; n = necrotic cardiomyocytes). (D–F) Tenascin C immunostaining (arrows) highlights early cardiac remodeling. (G–I) CD44 immunostaining detects active inflammation (arrowheads indicate CD44 immune cells; asterisks (*) mark areas with necrotic and calcified cardiomyocytes, which appear empty due to material loss during heat-mediated antigen retrieval). (J–M) CX43 mislocalization to the lateral plasma membrane and CX43-positive cytoplasmic dots are detected predominantly in Dsg20/wt mice with a structural phenotype (white arrowheads; ** p < 0.01). (N,O) Immunoblot analysis reveals reduced cardiac DSG2 protein levels in haploinsufficient Dsg20/wt mice compared with mice harboring two functional Dsg2 alleles (*** p < 0.001). Extended data are shown in Supplemental Figure S2, and original immunoblots are depicted in Supplemental Figure S3.
Figure 3
Figure 3
Sedentary heterozygous one-year-old Dsg2mt/wt mice have a higher incidence of CX43 mislocalization than their wild-type counterparts but do not show cardiac fibrosis or inflammation. (A,B) Representative Heidenhain’s Azan trichrome-stained sections of one-year-old Dsg2mt/wt (A) and Dsg2WT (B) hearts. The results of connective tissue semiquantification are presented in Supplemental Figure S2. (C,D) Only single foci of TnC-positive interstitial cells (arrow) were detected within entire heart cross sections of Dsg2WT and Dsg2mt/wt hearts. (E,F) CD44-positive immune cells (arrowheads) were rarely found in the hearts of one-year-old Dsg2WT and Dsg2mt/wt mice. (G–I) CX43 immunolocalization. Dsg2mt/wt hearts harbor more cardiomyocytes with mislocalized CX43 (arrowheads; (H) with image detail magnified 2.6 fold) than wild-type hearts (* p < 0.05). (J,K) Transmission electron microscopy reveals that desmosomes (white triangles) are discernable in Dsg2mt/wt hearts. White arrows, fascia adherens; *, gap junctions. Extended data are shown in Supplemental Figure S5.
Figure 4
Figure 4
Incremental endurance swim training increases the inducibility of ventricular arrhythmias and prolongs the maximal activation time in Dsg2mt/wt hearts. (A) Representative examples of original monophasic action potential (MAP) recordings from Dsg2WT (left) and Dsg2mt/wt (right) Langendorff-perfused hearts after endurance swim training. Ventricular tachycardia (VT) is induced by a single right ventricular extra stimulus S2 during right ventricular pacing in a Dsg2mt/wt heart (RV MAP (black): MAP measured in the RV wall; LV MAP (blue): MAP measured in the left ventricular wall; S MAP (red): MAP measured in the septum). (B) Number of Langendorff-perfused hearts with arrhythmias (dark blue) and without arrhythmias (light blue) in trained Dsg2WT and Dsg2mt/wt mice induced by a single right ventricular extra stimulus during right ventricular pacing show increased arrhythmia events in Dsg2mt/wt mice (Dsg2WT: n = 0 of n = 7; Dsg2mt/wt: n = 5 of n = 8; * p < 0.05). (C) Maximal ventricular activation time (max AT) measured in Langendorff-perfused Dsg2WT (blank) and Dsg2mt/wt (gray) hearts with a pacing cycle length of 100 ms. The activation time was prolonged in Dsg2mt/wt hearts compared with Dsg2WT hearts (n = 8 for Dsg2WT, n = 7 for Dsg2mt/wt; * p < 0.05).
Figure 5
Figure 5
Right ventricular enlargement in Dsg2mt/wt mice after endurance swim training is alleviated by preload-reducing therapy. (A–D) Representative echocardiographic images of the short-axis view during diastole after endurance swim training of Dsg2WT (A) and Dsg2mt/wt (B) mice treated with placebo and Dsg2WT (C) and Dsg2mt/wt (D) mice with preload-reducing treatment therapy. The blue dotted line surrounds the area measured of the right ventricle in the short-axis view. (A’–D’) MRI showing prospectively triggered cardiac Cine magnetic resonance images acquired at 9.4 T (groups and protocols as in (A–D)). (E) Bar graphs showing increased left ventricular mass/body weight (LVmass/BW) index after endurance swim training without (placebo) and with preload-reducing therapy (* p < 0.05). Augmentation of the index provides evidence that the training was effective. (F) Bar graphs representing the measured diastolic right ventricular (RV) area in the short axis view during echocardiography before and after training, without (placebo) and with preload reduction therapy in Dsg2WT and Dsg2mt/wt mice (* p < 0.05). After training, Dsg2mt/wt mice showed an increased RV short axis area. (G) Bar graphs showing calculated diastolic RV volume before and after training, without (placebo) and with preload reduction therapy in Dsg2WT and Dsg2mt/wt mice (* p < 0.05). After training, RV volume was enlarged in trained and placebo-treated Dsg2mt/wt mice compared with before training and with trained and untreated Dsg2WT mice. (H) Contractility of the RV shown by the fractional area change (FAC) measured in the short axis view (sav) before and after training, with placebo and with therapy in Dsg2WT and Dsg2mt/wt mice (* p < 0.05). After training, FAC RV sav was reduced in trained Dsg2mt/wt mice.
Figure 6
Figure 6
Hearts of trained Dsg2mt/wt mice show no obvious signs of fibrosis and/or inflammation in immunohistochemistry. (A–D) Representative images of cardiac sections stained with Heidenhain’s AZAN staining. (A’–D’) The typical localization of the sporadically detected CD44-positive inflammatory cells (black triangles). (A”–D”) Occasionally found foci of TGFBI protein expression (white arrows). The foci are typically located near or within the sites of heart valve attachment, whereas the endomysium and perimysium of the myocardium are devoid of TGFBI staining indicating the absence of fibrotic myocardial remodeling in these regions. Extended data are shown in Supplemental Figure S7.
Figure 7
Figure 7
DSG2 protein content is reduced in Dsg2mt/wt hearts after training, whereas the amount of the desmosomal proteins plakoglobin, plakophilin-2, and desmoplakin and the fascia adherens protein beta-catenin is not altered. (A) Immunoblots showing DSG2 and, as a control, cardiac actin levels (* on Ponceau S stain) in the left ventricle (LV) and right ventricle (RV) of Dsg2WT and Dsg2mt/wt hearts after endurance swim training. Bar graphs show the relative amounts of DSG2 normalized to cardiac actin. In the LV as well as in the RV, Dsg2mt/wt hearts showed a lower DSG2 level. Bar graphs present the mean ± SEM. n = 5 per genotype and heart chamber; ** p < 0.007. (B) Immunoblots showing the amount of the desmosomal proteins plakoglobin, desmoplakin, and beta-catenin in LVs and RVs of Dsg2WT and Dsg2mt/wt hearts after training. (C) Immunoblot showing plakophilin-2 protein levels in left and RVs of Dsg2WT and Dsg2mt/wt hearts after training. The amount of these proteins is unaffected in Dsg2mt/wt hearts compared with wild-type hearts (n = 5 per genotype and heart chamber). The original immunoblots are shown in Supplemental Figure S8.
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