Modified mRNA Treatment Restores Cardiac Function in Desmocollin-2–Deficient Mouse Models of Arrhythmogenic Right Ventricular Cardiomyopathy

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  Figure 1.. Identification of a novel DSC2 sequence variation in a family with arrhythmogenic right ventricular cardiomyopathy. A, A family with a recessive inheritance pattern of arrhythmogenic right ventricular cardiomyopathy (ARVC). Marriages between G3-3 and G3-4, and G4-1 and G4-2, were consanguineous. Squares indicate male; circles, female; black symbols, affected; white symbols, unaffected; gray symbols, undetermined status; +/+, normal; +/A138fs, heterozygous DSC2 sequence variation; and A138fs/A138fs, homozygous DSC2 sequence variation. B, Echocardiograms depict the 4-chamber view of the heart from individuals G5-4 (+/A138fs) and G5-2 (A138fs/A138fs). C, Representative cardiac magnetic resonance imaging in the 4-chamber view of the heart (left) and ventricular short-axis position (right). D, Twelve-lead ECG recording from patient G5-2. E, Schematic diagram illustrating the process of screening for pathogenic variants, encompassing 6 steps: (1) retaining only variants located in exonic or alternative splicing regions; (2) focusing on functional variant sites; (3) preserving variants that exhibit cosegregation within families; (4 and 5) concentrating on variants with a frequency <1%…

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  Figure 2.. Dsc2 loss-of-function mouse models develop arrhythmogenic right ventricular cardiomyopathy. A, Western blots showing DSC2 (desmocollin-2) and DSG2 (desmoglein-2) protein levels in the right ventricular (RV) tissue of 6-week-old wild-type (WT), Dsc2 knock-out (KO), and Dsc2 knock-in (KI) mice (n=4 mice per group). GAPDH served as a loading control. Quantification shown in B. C, DSC2 concentrations in 6-week-old WT, Dsc2 KO, and Dsc2 KI (variant) mouse sera detected by ELISA (n=5 mice per group). D, Representative image showing heart sizes of 12-week-old WT (left), Dsc2 KO (middle), and Dsc2 KI (variant; right) mice. E, Statistical analysis of heart weight/tibia length (HW/TL) in WT, Dsc2 KO, and Dsc2 KI (variant) mice at different time points (1, 2, 4, 6, and 12 weeks; n=8 mice per group). F through H, Echocardiographic analysis of RV function: RV fractional area change (RVFAC; F [left] and G) and tricuspid annular plane systolic excursion (TAPSE; F [right] and H) at different time points (6, 8, 10, and 12 weeks) in WT, Dsc2 KO, and Dsc2 KI (variant) mice (n=8 mice per group). I and J, Hemodynamic analysis of RV contractile or diastolic function by evaluation of dP/dtmax (I) and…

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  Figure 3.. Defective MLC2a expression in Dsc2 loss-of-function mice. A, Volcano plot showing the fold change and Padjusted of upregulated (red), downregulated (blue), and not significant (gray) genes in Dsc2 knock-out (KO) mice compared with wild-type (WT) mice (n=3 mice per group). Padjusted ≤0.05 and log2 fold change ≥2 were used for the cutoff. B, Quantitative polymerase chain reaction analysis of Myl7 mRNA expression in the right ventricles of WT and Dsc2 KO or Dsc2 knock-in (KI; variant) mice (n=6 mice per group) at 6 weeks of age. C, Representative Western blot showing the levels of MLC2a (myosin light chain-2a) protein in the right ventricles of 6-week-old WT and Dsc2 KO or Dsc2 KI (variant) mice (n=5 mice per group); quantification shown in D. E, Representative immunostaining of desmin (green), MLC2a (red), and DAPI (blue) in isolated right ventricular (RV) cardiomyocytes from 12-week-old Dsc2 KO or Dsc2 KI (variant) mice. Scale bar=10 μm. F, MLC2a concentrations in the serum of 6-week-old WT, Dsc2 KO, and Dsc2 KI (variant) mice (n=8 mice per group), as determined by ELISA. G, Representative images show the heart sizes of WT and Dsc2 KO mice after injection of adeno-associated virus…

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  Figure 4.. Modified mRNA treatment for Dsc2 deficiency–mediated arrhythmogenic right ventricular cardiomyopathy in mice. A, Images show ultrasound-guided intracardiac injections of modified mRNA. Three injection sites are circled with black lines. B, Schematics depict the experimental strategy for Dsc2 mRNA/lipid nanoparticle (LNP)–Dsc2 mRNA treatment (10 µg) in 2-month-old wild-type (WT) or Dsc2 knock-out (KO) mice after baseline right ventricular (RV) cardiac function was measured by cardiac ultrasonography. RV cardiac function monitoring was performed by echocardiography once a month for 6 months. C and D, Echocardiographic images of RV fractional area change (RVFAC) and tricuspid annular plane systolic excursion (TAPSE) of WT and Dsc2 KO mice at 0, 3, and 6 months after Dsc2 mRNA/LNP-Dsc2 mRNA treatment. E and F, Quantification of RVFAC and TAPSE at 0, 1, 2, 3, 4, 5, and 6 months after Dsc2 mRNA/LNP-Dsc2 mRNA treatment (n=8 mice per group). G, Quantification of heart weight/tibia length (HW/TL) in WT and Dsc2 KO mice (n=24 mice per group) after Dsc2 mRNA/LNP-Dsc2 mRNA at different time points after treatment. H, Development of fibrosis and ventricular wall thinning in right ventricles…

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  Figure 5.. Comparison of Dsc2 mRNA treatment with valsartan or fosinopril. A, A schematic diagram depicting the experimental strategy for 1-dose injection of Dsc2 mRNA or lipid nanoparticle (LNP)–Dsc2 mRNA, or daily intake of fosinopril or valsartan, in 8-week-old Dsc2 knock-out (KO) mice. B, Overview images showing heart size after 8 weeks with different treatments (fosinopril, valsartan, Dsc2 mRNA, or LNP-Dsc2 mRNA). C, Quantification of heart weight/tibia length (HW/TL) in Dsc2 KO mice (n=6 mice per group) after treatments. D and E, Hemodynamics analysis of right ventricular (RV) systolic and diastolic function by measuring dP/dtmax and −dP/dtmax in Dsc2 KO mice (n=4 mice per group) 8 weeks after treatments. F and G, RV fractional area change (RVFAC) and tricuspid annular plane systolic excursion (TAPSE) measurements at 0, 1, and 2 months after treatment (n=8 mice per group). H, Schematic diagram depicts the experimental procedure for coronary artery injection of mRNA. I, Dsc2 KO mice were used as a blank control to prepare a standard curve, dividing the hearts of Dsc2 KO+LNP-Dsc2 mRNA mice into right ventricle, interventricular septum (IVS), and left ventricle according to different…

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  Figure 6.. Loss of DSC2 expression in dilated cardiomyopathy with right ventricular dysfunction. A through F, Western blots show the levels of DSP (desmoplakin), DSG2 (desmoglein-2), DSC2 (desmocollin-2), MLC2a (myosin light chain-2a), and MLC2v (myosin light chain-2v) proteins in the myocardium of controls (n=6) and patients with dilated cardiomyopathy (DCM; n=6). G and H, ELISA was used to measure the levels of DSC2 and MLC2a proteins in the sera of healthy controls (n=32), patients with DCM without abnormal systolic function (n=62), patients with DCM with left ventricular (LV) systolic dysfunction (LV ejection fraction [LVEF] <50%; n=37), and patients with DCM with LV (LVEF <50%) and right ventricular (RV; tricuspid annular plane systolic excursion [TAPSE] ≤15 mm) systolic dysfunction (n=32). I, Correlation analysis of serum levels of DSC2 and MLC2a. The Pearson correlation coefficient (r) was calculated using the R function cor.test. J, Representative parasternal long-axis views, M-mode images, and TAPSE were obtained from 8-week-old wild-type (WT) mice after 8 weeks of transverse aortic constriction (TAC). K through O, Echocardiographic analysis was performed to assess LVEF, LV…