Mutations in JPH2-encoded junctophilin-2 associated with hypertrophic cardiomyopathy in humans

Abstract

Junctophilin-2 (JPH2) is a cardiac specific member of the junctophilins, a newly characterized family of junctional membrane complex proteins important in physically approximating the plasmalemmal L-type calcium channel and the sarcoplasmic reticulum ryanodine receptor for calcium-induced calcium release. JPH2 knockout mice showed disrupted calcium transients, altered junctional membrane complex formation, cardiomyopathy, and embryonic lethality. Furthermore, JPH2 gene expression is down-regulated in murine cardiomyopathy models. To this end, we explored JPH2 as a novel candidate gene for the pathogenesis of hypertrophic cardiomyopathy (HCM) in humans. Using polymerase chain reaction, denaturing high performance liquid chromatography, and direct DNA sequencing, comprehensive open reading frame/splice site mutational analysis of JPH2 was performed on DNA obtained from 388 unrelated patients with HCM. HCM-associated JPH2 mutations were engineered and functionally characterized using immunocytochemistry, cell morphometry measurements, and live cell confocal calcium imaging. Three novel HCM-susceptibility mutations: S101R, Y141H and S165F, which localize to key functional domains, were discovered in 3/388 unrelated patients with HCM and were absent in 1000 ethnic-matched reference alleles. Functionally, each human mutation caused (i) protein reorganization of junctophilin-2, (ii) perturbations in intracellular calcium signaling, and (iii) marked cardiomyocyte hyperplasia. The molecular and functional evidence implicates defective junctophilin-2 and disrupted calcium signaling as a novel pathogenic mechanism for HCM and establishes HCM as the first human disease associated with genetic defects in JPH2. Whether susceptibility for other cardiomyopathies, such as dilated cardiomyopathy, can be conferred by mutations in JPH2 warrants investigation.

Figure 1. Junctophilin-2 linear topology and location of identified mutations

Approximate location of HCM-associated mutations as well as exons boundaries and putative functional domains of junctophilin-2.

Figure 2. JPH2 primary sequence mutations are conserved across species or may effect protein phosphorylation

Sequence conservation analysis for the observed JPH2 mutations demonstrates that Y141H and S165F are conserved between multiple divergent species, while S101R localizes to the conserved MORN motif of JPH2.

Figure 3. JPH2 mutants are mis-localized and induce cardiomyocyte hypertrophy

(A) Western blot analysis for JPH2 expression in H9c2 cells. H9c2 cells do not express endogenous JPH2 (vector lane); however, expression of all mutants was found when cells were harvested 24 hours or 72 hours post transfection. Markers indicate molecular weight in kiloDaltons (kDa). (B) Confocal images of H9c2 cells expressing full-length wild type and mutant JPH2 stained with anti-JPH2 antibody. Intracellular vacuolization and disrupted localization of the JPH2 gene product are present in cells expressing mutant JPH2. Scale bar indicates 10 μm. (C) Confocal images of H9c2 cells expressing full-length wild type and mutant JPH2 stained with anti-calreticulin antibody to indicate the presence of an intact SR network (green). Red indicates expression of red fluorescent protein from a separate promoter on the plasmid that servers to mark transfected cells as well as to stain the cytsolic compartment. Blue indicates nuclei as stained by DAPI. Scale bar indicates 10 μm. (D, E) Live-cell confocal images of HL-1 cells transfected with JPH2-GFP fusion proteins at 24 (C) and 48 (D) hours. These fusion proteins do not contain the C-terminal transmembrane domain (TM) which normally allows the JPH2 gene product to association with the SR. Deletion of the TM allows for JPH2-GFP fusion proteins (JPH2-ΔTM-GFP) to associate with the plasma membrane via the MORN motifs. Expression of JPH2-ΔTM-GFP and mutant constructs did not have any obvious detrimental effects on cell morphology. Scale bar indicates 20 μm. (F) Hypertrophy of H9c2 cells transfected with mutant JPH2 was observed when cell size was measured at 5 days post-transfection. Results are representative from one trial of three conducted. In this trial control H9c2 cells, (n=148, mean=63.3 μm², SE=5.3 μm²), JPH2-WT (n=182, mean=66.4 μm², SE=4.0 μm²), JPH2-N101R (n=118, mean=78.1 μm², SE=7.2 μm²), JPH2-Y141H, (n=108, mean=96.6 μm², SE=8.5 μm²), and JPH2-S165F (n=132, mean=97.7 μm², SE=16.0 μm²) transfected cells were examined. Arrows indicate vacuolization of intracellular structure. Data listed as mean ± SE. * p<0.05

Figure 4. JPH2 mutants disrupt calcium signaling in HL-1 cells

(A) HL-1 cells were loaded with Fluo-4-AM (top panel) at 5 days post-transfection. Transfected cells were selected by the presence of a red fluorescent protein marker (bottom panel overlay image) from a separate promoter of the expression vector backbone. Scale bar indicates 10 μm. Boxed areas are representative regions of interest (ROI) used for analysis. (B) ROI were selected in transfected cells and signal was collected by xy scan for 5–10 minutes. Representative traces for each mutant construct are provided. Scale bars indicate 1.0 for the ratio of fluorescence change (ΔF/F₀) for the y-axis and 200 seconds for the x-axis. (C) The ratio of fluorescence change (ΔF/F₀), as an indication of global calcium flux, is significantly attenuated in HL-1 cells expressing JPH2 mutants. The ΔF/F₀ was measured in HL-1 (n=29, mean=0.933 ΔF/F₀, SE=0.142), JPH2-WT (n=11, mean=0.884 ΔF/F₀, SE=0.0831), JPH2-N101R (n=5, mean=0.474 ΔF/F₀, SE=0.124), JPH2-Y141H, (n=7, mean=0.200 ΔF/F₀, SE=0.0309), and JPH2-S165F (n=7, mean=0.269 ΔF/F₀, SE=0.0971) transfected cells were examined. Data listed as mean ± SE. * p<0.05.