Junctophilin-2 physically interacts with ryanodine receptor type 2 for peripheral coupling of mouse cardiomyocytes

Background: Ryanodine receptor type 2 (RyR2) mediate Ca 2+ release from the endoplasmic and sarcoplasmic reticulum (ER and SR), which is involved in the peripheral coupling of mouse cardiomyocytes, and thereby plays an important role in cardiac contraction. Junctophilin-2 (JPH2, JP2) is anchored to the plasma membrane (PM) and membranes of the ER and SR, and modulates intracellular Ca 2+ handling through regulation of RyR2. However, the potential RyR2 binding region of JPH2 is poorly understood. Methods: The interaction of JPH2 with RyR2 was studied using LC-MS/MS , bioinformatic analysis,co-immunoprecipitation studies in cardiac SR vesicles. GST-pull down analysis was performed to investigate the physical interaction between RyR2 and JPH2 fragments. Immunofluorescent staining was carried out to determine the colocalization of RyR2 and JPH2 in isolated mouse cardiomyocytes. Ion Optix photometry system was used to measure the levels of intracellular Ca 2+ transients in cardiomyocytes isolated from JPH2 knock down mice. Results: We report that (i) JPH2 interacts with RyR2 and (ii) the C terminus of the JPH2 protein can pull down RyR2 receptors. Confocal immunofluorescence imaging indicated that the majority of JPH2 and RyR2 proteins were colocalized near Z-lines. A decrease in the levels of JPH2 expression reduced the amplitude of Ca 2+ transients in cardiomyocytes. Conclusions: This study suggests that the C terminus domain of JPH2 is required for interactions with RyR2 in the context of peripheral coupling of mouse cardiomyocytes, which provide a molecular mechanism for looking for Ca 2+ - related diseases prevention strategies.

JPHs are proteins of a family of junctional membrane complex (JMC) located and facilitating contact between the PM and ER membrane in all excitable cells [9]. Skeletal muscles express both JHP1 and JPH2, cardiac muscles only express JPH2, JPH3 and JPH4 are mainly expressed across parts of the central nervous system [9,10]. All JPHs contain eight membrane occupation and recognition nexus (MORN) domains which is able to interact with PM and are followed by an α helical region, a divergent region, and which includes C-terminal domain anchoring proteins located in the ER/SR [9,11]. The mice with knocked out JPH2 do not survive and die in embryo phase due to heart failure (HF) caused by decoupling of the excitation contraction, which indicates that JPH2 is essential for functional crosstalk between VGCC and RyR2 for proper functioning of the embryonic heart [9]. As JPH2 was down-regulated in cases of cardiomyopathy and HF, it was found that a subsequent induction of levels of overexpression of JPH2 corrected cardiac function for mice with early stage heart failure, inhibited SR Ca 2+ leaks induced by RyR2 [12,13]. Quantitative single-molecule localization microscopy also indicated that colocalization between RyR2 and JPH2 in JPH2 knocked down cardiomyocytes was reduced. In contrast, for cases of induced overexpression of JPH2 in cardiomyocytes, there was colocalization of RyR2 with JPH2 at levels that were significantly increased [14].However, the binding domain between JPH2 and RyR2 channel has not been fully elucidated.
In the present study, we sought to further examine the molecular regulation of RyR2 channel by JPH2 protein in the peripheral coupling of mouse cardiomyocytes, and to determine whether interaction sites were localized to the C terminus of JPH2 proteins.

Materials And Methods Animals
We used 3-month-old mature C57BL/6 mice of both sexes obtained from Beijing Weitong Lihua Experimental Animal Technology, Limited, China (No. SCXK Jing 2012-0001). Ethics for animal care followed guidelines from the Committee on the Ethics of Animal Experiments of the University of Zhengzhou, China (No. SYXK-2010-0001). Procedures for experimental use of animals followed guidelines from the National Institutes of Health and Institution. All mice were group housed on 12 hr light/dark cycles with food and water available ad libitum.

Cardiac SR membrane vesicles isolation
Cardiac SR membrane vesicles were isolated using differential centrifugation as previously described [16,17]. Mice were anaesthetized using chloralhydrate. Whole hearts were quickly removed by careful dissection and stored at -80 °C. Heart samples were minced and homogenized in a cold lysis buffer containing 0.25 M sucrose, 10 M Tris-HCl (pH 7.0), and 1 mM EDTA. Individual homogenates were centrifuged at 5000 g for 10 min. Supernatant was removed and samples were centrifuged at 40,000 g for 45 min. The resultant pellet representative of the SR vesicles was suspended in 0.6 M KCl solution and centrifuged at 40,000 g for 45 min. The pellet was suspended in a cold lysis buffer and then stored at -80 °C until future use.

Co-immunoprecipitation
Solubilized SR vesicles or transfected HEK293 cells lysis were incubated with anti-JPH2 or RyR2 antibodies at 4 °C overnight, followed by incubation with protein A/G sepharose (Santa Cruz) for 6 h at 4 °C. Next, the beads were washed for 10 min with washing buffer, collected after each wash and resuspended in SR vesicle sample buffer by boiling for 5 min. Extracted precipitated proteins were used for SDS-PAGE separation and Coomassie blue staining described in steps below.
Protein Digestion and LC-MS/MS analysis Immunoprecipitated complexes were separated using SDS-PAGE gradient (4%-8%-12%) followed by Coomassie-staining. The sectioned portion of each vertical lane from each gel was placed in Trypsin for digestion and peptide based extraction performed as previously described [18]. Every gel lane was chopped, destained using 25 mM NH 4 HCO 3 and 50% acetonitrile, and was dried in a speedVac.
Individual dessicated samples were added together to perform another gel-based digestion wherein we used a trypsin digestion buffer for 24 h at 37 °C. Peptides were extracted by using 60% acetonitrile and 5% trifluoroacetic acid solutions and identified by using LC-MS/MS and liquid chromatography quadruopole time-of-flight (LC-QqTOF) mass spectrometry (Waters). Tandem measurements of mass were obtained from the resultant raw files, and we used Mass lynx 4.0 software to identify and characterize peptide sequences. Identified proteins were submitted to the National Center of Biotechnology Information non-redundant Database (NCBInr).

Functional categorization and network analysis
Proteins that were identified using LC-MS/MS analyses were screened by making comparisons to available published literature. The resultant selected proteins were submitted to Gene Ontology (http://www.geneontology.org/) for functional categorization. We used the string mapping tool (http://www.string embl.de/) to analyze the levels of interaction between the different proteins.
GST pull-down assays Construction of the GST-JPH2 fragment plasmids and implement of GST-pull down assays were performed in our previous study [15]. Briefly, plasmids were transformed into E. coli BL-21 (DE3), and were induced into samples by using 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) at 20 °C with 40r overnight. The resultant immobilized GST, GST-JPH2-N1, GST-JPH2-N2, and GST-JPH2-C were incubated with the protein samples prepared from SR vesicles at 4 °C overnight. The pulldown complexes were eluted using elution buffer as part of the Pierce GST protein interaction pull-down kit. Eluted products were boiled for 5 min at 95 °C, and then analyzed by western blot analysis.

Single cardiomyocyte isolation
Single cardiomyocyte samples were prepared as described in a previous study [15]. After application of anesthesia using sodium pentobarbital, whole hearts from adult mice were rapidly dissected and washed with ice-cold Ca 2+ -free modified Tyrode's solution (140 mM NaCl, 5.4 mM KCl, 1 mM MgCl 2 , 10 mM HEPES, and 10 mM glucose at pH 7.4). Each heart sample was cannulated and mounted onto a JPH2 knockdown by RNA interference (TTCTCCGAACGTGTCACGT) used for our study were previously successfully constructed. We applied a total dose of 1 × 10 9 PFU adenovirous with control siRNA and JPH2 siRNA by means of delivery into mice as previously described [15].
To determine quantitative levels of JPH2 and RyR2 mRNA expression, total RNA was isolated from infected myocardium using Trizol (Thermo Fisher Scientific) and was analyzed using Real-time PCR as described [19]. PCR primer used to detect levels of expression of JPH2, RyR2, and GAPDH were listed in Table1. Each primer sets were designed and synthesized by Shanghai Genechem Biotechnology, Shanghai, China. The relative gene expression levels of JPH2 and RyR2 were normalized to the GAPDH gene. The mRNA expression levels were calculated by the 2 −ΔΔCt method.
To determine levels of expression of JPH2 and RyR2 proteins, infected samples of mouse myocardium were lysed in RIPA buffer. Total protein concentrations were determined using BCA kits (Thermo Fisher Scientific). Precipitated proteins were analyzed with western blotting methodologies.

Ca 2+ transient measurements
Infected adult cardiomyocytes were incubated with 2 µmol/L fura-2 for 30 min at 37 ℃, and then were washed twice with Tyrodes solution containing 1.8 mmol/L CaCl 2 . We stimulated cells at 1.0 Hz using field stimulation to evoke Ca 2+ transient. Levels of intracellular Ca 2+ were determined by calculation of the 340 to 380 nm (340/380) ratio of Fura-2 fluorescence excited at 510 nm using an IonOptix photometry system (PMT-300, USA). The SR based calcium content in cells was determined by using 10 mM caffeine. Data were collected and analyzed by using SignalAverager Software IonWizard 6.6 (IonOptix) and with all parameters set to manufacture defaults.

Statistical analysis
Data were expressed as the mean value ± standard error of the mean (SEM). Differences between the treatment groups were evaluated using one-way ANOVA followed by paired or unpaired Student's t tests as appropriate. Differences were considered significant when P values were 0.05.

Results
Search of the JPH2 interactome in the Cardiac SR membrane vesicles using LC-MS/MS analysis To define the functional interactome of JPH2 protein in the heart, we isolated cardiac SR vesicles and performed LC-MS/MS analysis, followed by bioinformatic analysis. The SR vesicles are known to possess junctional sarcoplasmic reticulum-plasmalemma complexes [20,21], and thus we assessed the presence of JPH2 and RyR2 in the SR vesicles via western blotting. Specific anti-JPH2 and anti-RyR2 antibodies were individually used for the recognition of JPH2 (MW ~ 97 kDa) and RyR2 (~ 565 kDa; Fig. 1A). Results revealed that JPH2 and RyR2 are present in SR vesicles. Furthermore, the JPH2 immunoprecipitate was pulled-down using anti-JPH2 antibodies with protein A/G plus agarose from SR vesicles and was separated by SDS-PAGE, digested in in-gel trypsin, and underwent LC-MS/MS analysis. JPH2 interacting proteins were approximately 35013 in 13 different target bands chosen by observations from the Coomassie bright blue stain results. Based on previous literature, we listed the important proteins that were found to interact with JPH2 in Table2. The selected proteins were classified on the Gene ontology website, according to the molecular function, it can be divided into binding protein, catalytic protein, receptor active protein, structural molecule active protein and transport active protein (Fig. 1B). According to the different proteins involved in physiological process, they can be divided into 12 classes, of which most of the proteins involved in cellular physiological movement, followed by some of the local function involved in cell metabolic process, cell developmental process and the others arranged in Fig. 1C .We further construct our version of the JPH2 interaction network among the selected 90 proteins using the STRING tool (http://string.embl.de/). By analyzing experimentally confirmed values and the predicted values in a statistically based three-dimensional assessment, we found that some proteins interacted with each other based on their genetic relationships (Fig. 1D). Meanwhile, we carried out additional biologically based validations and network analyses to better understand the dynamics behind the model of their interaction. Although JPH2 and RyR2 proteins had not been found in the most complete threedimensional model in our computer modeling assessments, the possibility of interactions between them can be hypothesized since their direct homologous proteins can be co-expressed in the same species (Fig. 1E).

JPH2 associates with RyR2 receptors in the Cardiac membrane SR vesicles
We examined the interaction between JPH2 and RyR2 in SR vesicles using co-immunoprecipitation assays. Solubilized proteins from SR vesicles were immunoprecipitated using the anti-JPH2 antibody and subsequently immunoblotted using the anti-RyR2 antibody, RyR2 selectively bound to endogenous JPH2 in SR vesicles (Fig. 2B, left). Similarly, in the reverse co-IP experiments using the anti-RyR2 antibody ( Fig. 2A, right), the JPH2 protein specifically bound to RyR2, whereas no specific immunoreaction was observed using an unrelated antibody ( Fig. 2A, left and right panels). These results imply that a specific interaction between JPH2 and RyR2 in native cardiac tissue.
We further observed that JPH2 selectively bound to RyR2 using GST pull down assay. The experiments were performed on three GST-fused constructs, including GST-JPH2-N1, GST-JPH2-N2, and GST-JPH2-C [15]. Purified GST or GST-JPH2 fragments fusion proteins were immobilized using glutathione Sepharose 4B affinity beads. Tissue lysates from SR vesicles were then incubated with glutathione Sepharose 4B bound to GST or GST-JPH2 fragments. As shown in Fig. 2B, JPH2-C (aa340-696 containing the C-terminal region of JPH2) selectively bound to RyR2. However, GST alone and GST-JPH2-N1 and GST-JPH2-N2 did not interact with RyR2. Our data indicated that the JPH2-C domain contains the majority of the binding sites for the RyR2 receptor in vitro, and ultimately suggest that this is a preliminary structural requirement for JPH2 to link to RyR2.

Colocalization of JPH2 and RyR2 in adult mouse cardiomyocytes
To determine whether JPH2 and RyR2 proteins were colocalized in cardiomyocytes, we carried out immunofluorescent staining of isolated mouse cardiomyocytes. Figures 3A and 3B indicate that JPH2 and RyR2 maintained the same patterns of striation in singly isolated cardiac cells, even in areas where Z-lines were present in ventricular myocytes (Fig. 3B) or areas with Z-tubules in atrial myocytes (Fig. 3A). Results from controls in which staining with the secondary antibodies were used indicated that co-localization of JPH2 and RyR2 in cardiomyocytes was a valid result (Fig. 3C). Scale bars, 10 µm.

JPH2 mutation disrupts the interaction between JPH2 and RyR2
To examine whether the mutation in JP2-C disrupted the binding between the two proteins, we applied Co-IP in HEK293 cells transfected RyR2 + JPH2-C or RyR2 + JPH2-Cmut, the expression of RyR2 channel and JPH2 protein in immunoprecipitation complex was detected by western blot. Figures 4 show that compared with HEK293 cells expressing RyR2 + JPH2-C plasmid, the expression of the pulled down RyR2 channels by JPH2 is significantly decreased in HEK293 cells transfected RyR2 + JPH2-C mut plasmids. The results suggest that JPH2 mutation may affect its binding to RyR2 channels.

Knockdown of JPH2 depresses intracellular Ca 2+ transient
To test whether JPH2 modulated RyR2 receptor function, we used the approach of RNA based interference and recorded levels of intracellular Ca2 + transients. After transfection of adenovirus vector with specific siJPH2 through tail vein injection, the levels of JPH2 mRNA (Fig. 5A) and proteins (Fig. 5C ,5D)in adult mouse myocardium infected with Ad-siJP2 were significantly suppressed compared with the control groups (Ad-NC). However, knockdown of JPH2 did not change the mRNA heart) (Fig. 6B). We further investigated whether knockdown of JPH2 interfered with the function of the RyR2 receptor. The similar results demonstrated the amplitude of Ca2 + transients was significantly decreased in the Ad-siJPH2 treatment group compared with the control group where we used 10 mmol /L caffeine in cell suspension(0.51 ± 0.02 vs 0.87 ± 0.03 ratio unites ,n = 32 from 9 heart, P < 0.05) (Fig. 6D).

Discussion
JPH2 is the cardiac isoform of the junctophilin family and acts as a molecular bridge for signal transduction by anchoring PM and ER/SR membrane systems [9]. In cardiomyocytes, a system of tubules is distributed around the myofibrils and is called diad or peripheral coupling. An association with T-tubules forms by invaginations of the sarcolemmal membrane which is closely juxtaposed with a single terminal cistern of the SR [22]. In the present study, cardiac SR membrane vesicles possessing peripheral coupling from mice were isolated by differential separation, and specific anti-JPH2 and anti-RyR2 antibodies were recognized JPH2 and RyR2 in the SR vesicles.
JPH2 has been previously suggested to interact with RyR2 and Cav1.2 such as to regulate their gating channel functions [23,24]. Previous observations suggested that both JPH2 and RyR2 labeling puncta strongly overlapped, and that the amount of JPH2 associated with RyR2 clusters was greatly reduced after JPH2 was manually knocked down [14,25]. In the present study, the interaction between JPH2 and RyR2 channels can be possibly predicted by LC-MS/MS and biochemical analysis in vitro and in living cells. We used co-immunoprecipitation assays and results suggested that RyR2 was interacting with JPH2 proteins in SR vesicles, and suggested that the majority of both JPH2 and RyR2 proteins were colocalized near Z-lines in adult mouse cardiomyocytes by confocal immunofluorescent imaging analysis. Thus, we provided further evidence that JPH2, a member of the JMC family of proteins found in cardiomyocytes, interacts with RyR2 receptors.
Previous research had predicted that JPHs have a short, C-terminal domain important for anchoring proteins into ER/SR as well as having repeated N-terminal MORN domains which are able to interact with the PM, thereby causing a junctional association of these two membrane systems [9]. Results from a related study indicated that the N-terminal domain of JPH2 is responsible for a physical and functional interaction with small-conductance Ca 2+ -activated K + channels subtype 2 (SK2) in PM [15].
We found that the region of JPH2 (amino acid residues 340-696) interacted with RyR2 in mouse cardiomyocytes. Recent reports have suggested that the N terminus region of JPH2 (amino acid residues 1-565) interacted with RyR2 and Cav1.2, but did not interact with the C terminus region of JPH2 (amino acid residues 566-end), and indicated it was not sufficient to restore Ca 2+ transients in knocked down JPH2 cardiomyocytes [26]. According to the different cleavage products found in the study compared to our results, we inferred that the overlapping region 340-565 may contain the determinants for the binding between JPH2 and RyR2. Meanwhile, a recent repot identified a novel HCM-associated mutation A405S in JPH2, and alters intracellular Ca2 + signaling in a pro-hypertrophic manner [27]. We then confirmed the mutation A405S in JPH2 affect its biding with RyR2 channel, indicating that the C terminus of JPH2 proteins was essential for the interaction. In another study, Beavers et al. found that E169K mutation in JPH2 specifically disrupts its binding domain with RyR2 due to perturbed Ca 2+ handling, indicating residue E169 within a crucial domain of JPH2 that modulates RyR2 channel [28] .E169 residue in JPH2 is located in a flexible `joining domain' between two MORN domains that attach JPH2 to the sarcolemma [9,10].
Recent studies have also demonstrated that cardiac-specific knockdown of JPH2 triggers an SR Ca 2+ leak by directly increasing the probability of RyR2 receptors being open in cardiomyocytes [14,23,28]. Moreover, downregulation of JPH2 has been observed in patients with hypertrophic cardiomyopathy, for some rodent based model analyses of hypertrophic cardiomyopathy, overexpression of JPH2 which could correct cardiac function with early stage heart failure, for approaches to inhibition of SR Ca 2+ leak induced by RyR2 [12,13]. Thus, these results suggest JPH2 could modulate intracellular Ca 2+ handling through regulation of RyR2. In the present study, we further confirmed that a siRNA based knockdown of JPH2 resulted in reduced amplitudes of Ca 2+ transient in cardiomyocytes, but the respective levels of expression of RyR2 mRNA and proteins showed no significant changes in our reports [15]. This result is consistent with previous findings from similarly oriented analyses by authors of this study and other researchers [14,15,23,28], and was a result likely due to decreasing levels of SR Ca 2+ stores which resulted in an SR Ca 2+ leak by directly increasing the probability that RyR2 receptors would be open.
To summarize, our data confirm that JPH2 plays a critical role in regulating intracellular calcium level by binding to and modulating RyR2 through its C-terminal domain. Nevertheless, further research is warranted to unequivocally evaluate the specific mechanisms by which JP2 proteins influence RyR2 channel function in vivo to determine the direct link between aberrant ion channel function and cardiac arrhythmias.

Conclusion
In conclusion, we provide more insights into the possible functional regulation of RyR2 channels by the JP2 protein and prove the interaction sites were localized to the C terminus of JPH2 proteins, which consequently plays important roles in maintaining intracellular calcium homeostasis and excitation-

Availability of data and materials
All data generated or analyzed during this study are included in this manuscript.   JPH2 mutation decrease its binding with RyR2 channel. A. Protein precipitated IP with JPH2 antibody from HEK293 cells transfected with RyR2+JPH2-C or RyR2+JPH2-Cmut plasmids was detected by western blot using JPH2 antibody and RyR2 antibody respectively. B.

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