Essential role of obscurin kinase-1 in cardiomyocyte coupling via N-cadherin phosphorylation

Obscurins are giant cytoskeletal proteins with structural and regulatory roles. Obscurin-B (~870 kDa), the largest known isoform, contains 2 enzymatically active Ser/Thr kinase (kin) domains, kin1 and kin2, which belong to the myosin light chain kinase family. Kin1 binds to and phosphorylates N-cadherin, a major component of the intercalated disc, the unique sarcolemmal microdomain that mediates the mechanochemical coupling of adjacent cardiomyocytes. Obscurin-B containing kin1 and N-cadherin colocalize at cell junctions in embryonic rat ventricular myocytes (ERVMs), and their codistribution is regulated by Ca2+. Phosphoproteomics analysis revealed that obscurin-kin1 phosphorylates N-cadherin at Ser-788 located within the juxtamembrane region of its cytoplasmic domain, with an apparent Kcat of approximately 5.05 min–1. Overexpression of obscurin-kin1 or phosphomimic-Ser-788-Glu N-cadherin in ERVMs markedly increases cell adhesion and chemical coupling. Importantly, phosphomimic Ser-788-Glu N-cadherin exhibits significantly reduced binding to p120-catenin, while overexpression of phosphoablated Ser-788-Ala N-cadherin increases RhoA activity. Consistent with an essential role of the obscurin-kin1/N-cadherin axis in cardiomyocyte coupling, it is deregulated in end-stage human heart failure. Given the nearly ubiquitous expression of obscurin and N-cadherin, our findings may have broad applicability in deciphering the obscurin-kin1/N-cadherin axis that likely mediates cell coupling in diverse tissues and organs.

constructs containing the cytoplasmic domain of N-cadherin were generated with the QuickChange II site-directed mutagenesis kit (#200521, Agilent), following the manufacturer's instructions.Protein expression was induced by 0.5 mM IPTG for 3-4 hours at 37°C, and recombinant proteins were affinity-purified using glutathione-sepharose beads (GE Healthcare Life Sciences, Marlborough, MA).A shorter portion of the cytoplasmic domain of N-cadherin (aa 786-880, accession # P15116, Uniprot: https://www.uniprot.org/uniprot/P15116;Ncad786-880) enriched in potential Ser/Thr phospho-sites was subcloned into the pET21a vector (Addgene) at NdeI/XhoI sites.Protein expression was induced by 1 mM IPTG for 3-4 hours at 37°C, and the recombinant protein was affinity-purified using the Ni-NTA His Bind Superflow resin (EMD Millipore, Billerica, MA).Moreover, a fragment of mouse p120-catenin (aa 311-747, accession # P30999, Uniprot: https://www.uniprot.org/uniprot/P30999)containing armadillo domains 1-8 was cloned into the pMAL-c2E vector at EcoRI/HindIII sites.Protein expression was induced by 1 mM IPTG overnight at 18°C, and the recombinant protein was affinity-purified using the pMAL protein fusion and purification system kit (#E8200S, New England Biolabs, Ipswich, MA).The authenticity of all constructs was verified by sequencing before proceeding to further experimentation.The primer sets used for the generation of the indicated constructs are provided in Supplemental Table 1.

Preparation and culturing of embryonic rat ventricular myocytes (ERVM)
ERVM were obtained from hearts of embryonic day 21 (E21) Sprague-Dawley rats, as previously reported (3); of note, ERVM form cell junctions, which are considered "primordial ICDs" (4), allowing the study of de novo formation of cell junctions rather than disassembly and reassembly of ICDs that takes place in adult cardiomyocyte cultures.In brief, euthanasia of pregnant dams via carbon dioxide (CO2) asphyxiation was confirmed by cervical dislocation, in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.E21 embryos were decapitated on ice, the hearts were excised, and the atria were removed.Ventricles were rinsed in PBS, followed by 5-6 rounds of successive digestions of 10-15 min each with a mixture of collagenase II (Worthington Biomedical Corporation, Lakewood, NJ) and pancreatin (Sigma-Aldrich, St. Louis, MO) dissolved in 116 mM NaCl, 20 mM HEPES, 1 mM NaH2PO4, 5.5 mM Glucose, 5.4 mM KCl, 0.8 mM MgSO4, and phenol red, pH 7.4.The supernatants (from the series of successive digestions) were removed from the tissue "chunk" and each placed in a 15 ml conical tube containing 10% heat-inactivated horse serum (Thermo Fisher Scientific).After centrifugation, cardiomyocyte fractions were resuspended in 100% horse serum, combined, and filtered through a sterile cell strainer with mesh size of 70 μm (Thermo Fisher Scientific).The filtrate was subsequently centrifuged at 2,000 rpm for 5 min, and the cell pellet was resuspended in the proper volume of plating medium containing DMEM-F12 (Thermo Fisher Scientific), 10% heat-inactivated FBS (Thermo Fisher Scientific), 200 U/ml penicillin and 200 U/ml streptomycin (Thermo Fisher Scientific) to obtain a final cell density in the range of 0.5-1 x 10 6 cells/ml.Twenty-four hours after plating, the plating medium was replaced with growth medium containing DMEM-F12, 5% FBS, 100 U/ml penicillin and 100 U/ml streptomycin.

Dispase assay
Confluent ERVM monolayers transfected with the indicated plasmids were gently washed with PBS, followed by treatment with 10 μM blebbistatin for 30 min to inhibit contraction, and then incubated with 2.5 U/ml dispase at 37°C/5%CO2 for 4-6 hr.The detached cell monolayer was subsequently subjected to mechanical stress achieved using an orbital shaker (70 rpm) for 30 min.
Fragmentation of monolayers was imaged with a HAMAMATSU Digital Camera (ORCA-Flash4.0LT;MODEL: C11440-42U; S/No.000452; HAMAMATSU Photonics) connected to a Leica MZ10F Stereo Microscope (MODEL MSV269; S/NO 5828800), and evaluation of cell fragment numbers was performed with ImageJ software.

Dye transfer assay
Seventy-two hr post-transfection, GFP-positive ERVM were randomly assigned to either donor or recipient groups.Donor cells (1 x 10 6 cells/ml) were labeled with 10 μM of the GJ transferrable calcein deep red acetate dye (AAT Bioquest, Sunnyvale, CA), while recipient cells (1 x 10 6 cells/ml) were labeled with the GJ non-transferable CytoTell Red 590 dye (1:500x dilution; AAT Bioquest) in a 37°C/5% CO2 incubator for 1 hr.Following labeling, cells were gently washed three times with PBS, and subsequently mixed and incubated at 37°C/5% CO2 for 2 hr.Evidence of calcein deep red acetate transfer from donor to recipient cells was evaluated by flow cytometry (BD LSR II; BD Biosciences).The rate of dye transfer was calculated with the following formula: number of cells positive for both calcein deep red and CytoTell Red 590/number of cells positive for CytoTell Red 590 x 100% (5).

Expression and purification of His-tagged obscurin kinase-1 from insect cells
The catalytic domain of the mouse obscurin-kin1 (aa 7401-7669, accession # A2AAJ9, Uniprot: https://www.uniprot.org/uniprot/A2AAJ9)conjugated to the 6xHis tag (6xHis-ObscKin1-CA) (1) was subcloned into the pUC57 baculovirus expression vector (GenScript, Piscataway, NJ).Following sequence verification, the recombinant plasmid was transfected into DH10Bac competent cells for production of recombinant bacmid (rbacmid), which was transfected into Sf9 insect cells using Cellfectin II (Thermo Fisher Scientific).Sf9 cells were subsequently incubated in Sf-900 II serum free media for 5-7 days at 27°C.At the end of the incubation period, Sf9 cells were centrifuged, and the supernatant was collected and designated as baculovirus P1 virus stock (GenScript).The P1 viral stock was used to infect a new batch of Sf9 cells to scale-up production and obtain viral stock P2.6xHis-ObscKin1-CA recombinant protein was purified from viral stock P2 72 hours post-infection by pelleting, harvesting, and lysing the Sf9 cells in a buffer containing 50 mM Na3PO4, 300 mM NaCl, 20 mM imidazole, 8 M Urea, and 0.5 mM DTT supplemented with a cOmplete TM protease inhibitor cocktail (Roche, Mannheim, Germany), pH 7.4.The clarified Sf9 cell lysate was subsequently affinity-purified under denaturing conditions through a His60 Ni Superflow Resin (Clontech Laboratories, Mountain View, CA), according to the manufacturer's instructions.In brief, the Sf9 cell lysate was added to His60 Ni superflow resin column and gently rocked at 4°C for 1 hour to allow binding of the 6xHis-ObscKin1-CA protein.
Following extensive washes with a buffer containing 50 mM Na3PO4, 300 mM NaCl, 60 mM imidazole, 8 M Urea, the recombinant protein was eluted by adding successively elution buffer A (5 column volumes): 50 mM Na3PO4, 300 mM NaCl, 150 mM imidazole, 8 M Urea, and buffer B (5 column volumes): 50 mM Na3PO4, 300 mM NaCl, 300 mM imidazole, 8 M Urea.A total of 10 eluant fractions (5 fractions from buffer A and another 5 from buffer B) were collected, which were subjected to dialysis in PBS, followed by buffer exchange and concentration via Amicon centricon (EMD Millipore, Burlington, MA) in 50 mM Tris-HCl, 150 mM NaCl, and 1 mM TCEP-HCl in the presence of cOmplete TM protease inhibitors.Non-infected Sf9 cells went through the same purification procedure as 6xHis-ObscKin1-CA expressing cells, and the final product served as negative control in subsequent experiments.

Tandem Mass Spectrometry
The in vitro kinase reaction mixtures were subjected to in-solution digestion as described in (6).Following pH adjustment to 8 with triethylammonium bicarbonate (TEAB) buffer, protein mixtures were reduced with 3 mg/ml DTT for 1 hr at 56°C, alkylated with 15 mg/ml iodoacetomide for 45 min at room temperature in the dark, and digested with 12.5 ng/μl trypsin/Lys-C mixture (Promega, Madison, WI) at 37°C overnight.Post-digestion, the peptides were acidified and desalted on Oasis μ-HLB plates (Waters Corporation, Milford, MA), and eluted with 60% acetonitrile (ACN) and 1% trifluoroacetic acid (TFA).The eluted peptides from each reaction mix were split into two halves and dried.One half was directly used for liquid chromatography tandemmass spectrometry (LC-MS/MS) and the other half was enriched for phosphopeptides using titanium dioxide (TiO2).Not enriched peptides were analyzed on an QExactive HF (Thermo Fisher Scientific) interfaced with a nano-Acquity LC system from Waters by reversed-phase chromatography using a 2%-90% acetonitrile in 0.1% formic acid gradient over 76 min at 300 nl/min on a 75 µm x 150 mm ProntoSIL-120-5-C18 H column 5 µm, 120Å (BISCHOFF; http://www.bischoff-chrom.com/hplc-prontosil-c18-h-c18-phasen.html).Eluting peptides were sprayed into the mass spectrometer through a 1 µm emitter tip (New Objective) at 2.2 kV.Survey scans (MS) of precursor ions were acquired from 350-1800 m/z at 120,000 resolution for 200 m/z, 3xe6 automatic gain control (AGC), 100 ms maximum injection time (IT).Precursor ions were individually isolated within m/z 1.6Da with 0.5Da offset by data dependent monitoring and 15s dynamic exclusion, and fragmented (MS/MS) using an HCD activation collision energy 28.MS/MS spectra were acquired using a 1e5 automatic gain control (AGC), 200 ms maximum injection time (IT) at 30,000 resolution at 200Da and a 271.1012 lock mass.Phosphopeptides, enriched by binding to TiO2 beads, were similarity analyzed except for the following changes.
Phosphoenriched peptides were analyzed on an QExactive Plus (Thermo Fisher Scientific) interfaced with an Easy-nLC1100 UPLC.Precursor ions were acquired at 70,000 resolution for 200 m/z and MS/MS spectra were acquired at 35,000 resolution at 200Da using a 271.1012 lock mass.Isotopically resolved masses in precursor (MS) and fragmentation (MS/MS) spectra were extracted using 3 nodes (without deconvolution, and with deconvolution using Xtract or MS2 Processor) in Proteome Discoverer (PD) software (v1.4,Thermo-Fisher Scientific).All extracted data were searched using Mascot (2.5.1; www.matrixscience.com)against the RefSeq2015 Mus musculus or RefSeq2015_mammals, each with an auto-concatenated decoy reversed database.The search criteria included: sample's species; trypsin as the enzyme, allowing one missed cleavage; cysteine carbamidomethylation as fixed modification; and methionine oxidation, and asparagine and glutamine deamidation as variable modifications.Proteome Discoverer uses only the peptide identifications with the highest Mascot score for the same peptide matched spectrum from the 3 different extract nodes and filtered the peptide identifications to 1% False Discovery Rate confidence based on the concatenated decoy database search.

RNA isolation and Reverse Transcription Polymerase Chain Reaction
50 mg of adult mouse (6-month old) heart tissue and human donor LV were treated with 1 mL of TRIZOL® Reagent (Thermo Fisher Scientific, USA) to obtain total RNA, which was reverse transcribed with the QuantiTect Reverse Transcription kit (Qiagen, Valencia, CA).250 ng of cDNA were used in a PCR reaction with the GoTaq Green Master Mix (Promega, Madison, WI, USA) for amplification of the unique COOH-terminus of obscurin-B (amplicon size: 976 nts) or control GAPDH (amplicon size: 332 nts).PCR products were resolved in 1% agarose gels.The relevant primer sets are included in Supplemental Table 1.

pS788-N-cad ab S788-N-cad ab Hsp90 ab
Note: Blots in Fig. 3B and E are presented in grayscale mode; red boxes indicate the lanes/bands used in Fig. 3B and E.
Note: Areas marked with white dashed boxes are shown in Fig. 3C.

Figure 3D
Note: Areas marked with white dashed boxes are shown in Fig. 3D.Coomassie Blue

Figure 4 1 :B 1 :
Figure 4 A Gels and blots in Fig.5A-B are presented in grayscale mode; red boxes indicate the lanes/bands used in Fig.5 A-B.