Novel junctophilin-2 mutation A405S is associated with basal septal hypertrophy and diastolic dysfunction

Abstract

Background: Hypertrophic cardiomyopathy (HCM), defined as asymmetric left ventricular hypertrophy, is a leading cause of cardiac death in the young. Perturbations in calcium (Ca²⁺) handling proteins have been implicated in the pathogenesis of HCM. JPH2-encoded junctophilin 2 is a major component of the junctional membrane complex, the subcellular microdomain involved in excitation-contraction coupling. We hypothesized that a novel JPH2 mutation identified in patients with HCM is causally linked to HCM, and alters intracellular Ca²⁺ signaling in a pro-hypertrophic manner.

Objectives: To determine using a transgenic mouse model whether a JPH2 mutation found in a HCM patient is responsible for disease development.

Methods: Genetic interrogation of a large cohort of HCM cases was conducted for all coding exons of JPH2. Pseudo-knock-in (PKI) mice containing a novel JPH2 variant were subjected to echocardiography, cardiac MRI, hemodynamic analysis, and histology.

Results: A novel JPH2 mutation, A405S, was identified in a genotype-negative proband with significant basal septal hypertrophy. Although initially underappreciated by traditional echocardiographic imaging, PKI mice with this JPH2 mutation (residue A399S in mice) were found to exhibit similar basal hypertrophy using a newly developed echo imaging plane, and this was confirmed using cardiac MRI. Histological analysis demonstrated cardiomyocyte hypertrophy and disarray consistent with HCM.

Conclusions: Variant A405S is a novel HCM-associated mutation in JPH2 found in a proband negative for mutations in the canonical HCM-associated genes. Studies in the analogous mouse model demonstrated for the first time a causal link between a JPH2 defect and HCM. Moreover, novel imaging approaches identified subvalvular septal hypertrophy, specific findings also reported in the human JPH2 mutation carrier.

Keywords: Calcium; hypertrophic cardiomyopathy; junctophilin-2; magnetic resonance imaging.

Graphical abstract

Figure 1

JPH2 Mutation A405S Occurred De Novo and Affects a Conserved Residue (A) Pedigree of HCM proband with JPH2 mutation A405S. (B) Cartoon showing that the JPH2 mutation A405S is localized within the alpha helix domain. (C) Alignment showing conservation of JPH2 sequences across species, the mutant residue is marked by the red box. HCM = hypertrophic cardiomyopathy; JPH2 = junctophilin-2; LVOTO = left ventricular outflow tract obstruction; MORN = membrane occupation and recognition nexus; PM = plasma membrane; RyR2 = ryanodine receptor type-2; SR = sarcoplasmic reticulum; TMD = transmembrane domain; VGCC = voltage-gated calcium channel.

Figure 2

Patients With JPH2 Mutation A405S Develop HCM With LVOTO (A and B) Echocardiography of a patient with HCM JPH2 mutation A405S showing asymmetrical septal hypertrophy denoted by a red arrow, and (C) LVOTO evidenced by (D) color Doppler flow mapping. (E to H) Bright-blood CMR depicting left ventricular basal septal hypertrophy (red arrow) in diastole (E) and (F) systole, with corresponding transverse CMR in diastole (G) and systole (H). Scale bars = 2.0 cm. CMR = cardiac magnetic resonance imaging; other abbreviations as in Figure 1.

Figure 3

Standard Echocardiographic Analysis of A399S Mice (A) Representative echocardiograms of traditional mouse SAX mid-level B-modes; asterisks mark papillary muscles with corresponding standard M-mode tracings. Quantifications revealed no differences in (B) left ventricular outer diastolic diameter (LVOD;d) or (C) ejection fraction (EF) between A399S and wild-type (WT) mice. Circles in box plots represent outliers. IVS = intraventricular septum; LV = left ventricle; N = number of animals; SAX = short axis.

Figure 4

Novel Echocardiographic Imaging Plane Reveals Asymmetrical Interventricular Septal Hypertrophy in A399S Mice (A) Representative modified long-axis (mLAX) echocardiograms were used to visualize the LV and IVS; red stars mark papillary regions. White boxes indicate region of zoom shown in panels below. Dotted lines delineate the traditional mouse echocardiography plane, for comparison. (B) Quantification showing significantly reduced LV chamber area in diastole (endoArea;d) in A399S mice compared with WT and (C) increased maximal interventricular septum diameter in diastole (IVSD;d_max) in A399S mice compared with WT. Circle in box plot represents outlier. *p < 0.05, ***p < 0.001. Ao = aorta; other abbreviations as in Figure 3.

Figure 5

Confirmation of IVS Hypertrophy in A399S-PKI Mice Using CMR (A) Representative long-axis view (LAX) magnetic resonance images demonstrating IVS hypertrophy. Quantifications of (B) left ventricular end-diastolic mass (LV Mass) and (C) maximal IVSD;d_max. Circles in box plot represent outliers. *p < 0.05, ***p < 0.001. FW = free wall; PKI = pseudo-knockin; RV = right ventricle; other abbreviations as in Figures 2, 3, and 4.

Figure 6

Cardiomyocyte Hypertrophy and Disarray in IVS of A399S-PKI Mice (A) Long-axis hematoxylin and eosin–stained paraffin sections. (B) Box plots showing median IVS diameter and (C) LV free wall diameter (FWD). (D) Representative images of wheat germ agglutinin–stained cardiomyocyte cross sections at 40× magnification from the IVS and LV FW with (E and F) quantification in bar graphs. (G) Representative 40× magnifications of Masson’s Trichrome–stained paraffin sections of cardiomyocytes showing disarray and interstitial fibrosis with (H) quantification in bar graph. For B and C, n = number animals. For E, F, and H, n = number images, and number in parentheses = animals. ***p < 0.001. IVSD = interventricular septum diameter; other abbreviations as in Figures 3 and 5.

Figure 7

Unaltered SR Calcium Handling, But Reduced TT Power, in A399S-PKI Mice (A) Representative fluorescent tracings during 1-Hz pacing showing Ca²⁺ transient amplitudes and summary results (B). The SERCA2a activity, defined as the inverse of the decay time constant of the caffeine-induced SR Ca²⁺ dump, was increased in A399S-PKI mice (C). Representative confocal line-scan images of individual Ca²⁺ sparks (D) and summary results (E). Di-8-ANEPPS staining of ventricular myocytes isolated from WT and A399S mice showing a reduction in T-tubule (TT) power (F) and summary results (G). N = cells, and number in parentheses = animals. **p < 0.01. Abbreviations as in Figures 1, 3, and 5.