The Desmoglein 2 interactome in primary neonatal cardiomyocytes

The Desmoglein 2 interactome in primary neonatal cardiomyocytes | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results The Desmoglein 2 interactome in primary neonatal cardiomyocytes Yang Li , Alexandra P. Campbell , Sahana Balasubramanian , Xuemei Zeng , Emma Porter , Pamela S. Cantrell , Mai Sun , View ORCID Profile Alexandra S. Mattheyses , View ORCID Profile Adam V. Kwiatkowski doi: https://doi.org/10.1101/2025.06.09.658637 Yang Li 1 Department of Cell Biology, University of Pittsburgh School of Medicine , Pittsburgh, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alexandra P. Campbell 1 Department of Cell Biology, University of Pittsburgh School of Medicine , Pittsburgh, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sahana Balasubramanian 1 Department of Cell Biology, University of Pittsburgh School of Medicine , Pittsburgh, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Xuemei Zeng 2 Biomedical Mass Spectrometry Center, University of Pittsburgh Schools of the Health Sciences , Pittsburgh, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Emma Porter 1 Department of Cell Biology, University of Pittsburgh School of Medicine , Pittsburgh, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pamela S. Cantrell 2 Biomedical Mass Spectrometry Center, University of Pittsburgh Schools of the Health Sciences , Pittsburgh, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mai Sun 2 Biomedical Mass Spectrometry Center, University of Pittsburgh Schools of the Health Sciences , Pittsburgh, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alexandra S. Mattheyses 3 Department of Cell, Developmental, and Integrative Biology, The University of Alabama at Birmingham , Birmingham, AL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alexandra S. Mattheyses Adam V. Kwiatkowski 2 Biomedical Mass Spectrometry Center, University of Pittsburgh Schools of the Health Sciences , Pittsburgh, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Adam V. Kwiatkowski For correspondence: adamkwi{at}pitt.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Mechanical coupling and chemical communication between cardiomyocytes are facilitated through a specialized adhesive structure known as the intercalated disc (ICD). The ICD is essential for heart organization and contraction. Yet, the network of adhesion, adaptor, and signaling proteins that form the ICD remains poorly defined. Here, we combined proximity labeling and quantitative mass spectrometry to identify proteins associated with the desmosomal cadherin, Desmoglein 2 (DSG2), in cultured neonatal cardiomyocytes. We identified over 300 proteins in the DSG2 interactome; half of which are shared with the N-cadherin (CDH2) interactome in cardiomyocytes. Proteins unique to DSG2 include the gap junction protein connexin 43 and the plakin family of cytolinker proteins. Comparison of the cardiomyocyte DSG2 interactome with the interactomes of desmosomal proteins from epithelia revealed only a small number of shared proteins. In cardiomyocytes, plakoglobin (JUP) and plakophilin 2 (PKP2) were the most abundant shared proteins between the DSG2 and CDH2 interactomes. PKP2 is a dynamic protein whose membrane recruitment in cardiomyocytes is tension-dependent. Our analysis of the DSG2 interactome provides a critical new dimension to the proteomic atlas of the essential molecular complexes required for cardiomyocyte adhesion. Introduction The heart is formed from a massive, interconnected network of individual cardiac muscle cells, known as cardiomyocytes, that function collectively to pump blood throughout the body. Cardiomyocytes are connected end-to-end by a specialized adhesive structure called the intercalated disc (ICD), which enables both mechanical and electrochemical coupling between cardiomyocytes ( Nielsen et al., 2023 ; Pruna and Ehler, 2020 ). The ICD is comprised of three adhesion complexes: adherens junctions (AJs) and desmosomes, which link the actin and intermediate filament (IF) cytoskeletons of adjoining cells, respectively; and gap junctions, which are membrane pores that permit the exchange of ions and small metabolites between cells. The establishment, maintenance, and function of all three adhesion complexes are essential for proper heart function, as mutations in the proteins that form these complexes are linked to heart disease, notably arrhythmogenic cardiomyopathy (AC) ( Nielsen et al., 2023 ). Yet, we lack a complete understanding of the proteins that form these adhesion complexes and how they work together to establish and maintain intercellular adhesion in the heart under demanding mechanical conditions unique to this organ. The AJ and desmosome are well-studied adhesion complexes, best understood for their function in epithelia ( Campas et al., 2024 ; Rubsam et al., 2018 ; Yeruva and Waschke, 2023 ). The core of the cardiac desmosome is formed by a pair of transmembrane proteins, desmosomal cadherins, Desmoglein 2 (DSG2) and Desmocollin 2 (DSC2, Fig. 1A ). The extracellular (EC) domains of DSG2 and DSC2 engage in homo- and hetero-typic interactions with the EC domains of desmosomal cadherins on opposing cells. Heterotypic binding (i.e., DSG2 on one cell binds DSC2 on the adjacent cell) is postulated as the primary adhesion mechanism. Cytoplasmic linker proteins mediate the linkage of desmosomal cadherins to IFs ( Harmon and Green, 2013 ). Plakoglobin (JUP) and plakophilin 2 (PKP2) are Armadillo (ARM) repeat domain-containing proteins that bind directly to the tails of DSG2 and DSC2. JUP and PKP2, in turn, recruit desmoplakin, which links the desmosome complex to IFs ( Fig. 1A ). Download figure Open in new tab Figure 1. Desmosome organization and dynamics at neonatal cardiomyocyte cell-cell contacts. (A) Cartoon schematic of the cardiac desmosome showing Desmocollin 2 (DSC2), Desmoglein 2 (DSG2), Plakophilin 2 (PKP2), Plakoglobin (JUP), Desmoplakin (DSP), and Desmin (DES). (B) Thin section electron microscopy image of neonatal cardiomyocyte cell-cell junction. The cell-cell contact is highlighted in purple. Desmosomes (DE), adherens junctions (AJ), and gap junction (GJ) labeled. Scale bar is 1 micron. (C) Top, cartoon schematic of exon organization and splicing at the 3’ end of desmocollin 2 in mouse chromosome 18. Splicing of exons 15 and 17 produces variant Dsc2a with a longer C-terminal cytoplasmic tail. In contrast, splicing of exons 15, 16, and 17 introduces a new stop codon to create variant Dsc2b with a truncated C-terminal tail. PCR primer pair locations for set 1 and set 2 are shown as arrows. Bottom, PCR results from cDNA isolated from cultured neonatal cardiomyocytes and adult mouse heart. Variant Dsc2b is the primary transcript in cultured cardiomyocytes and the adult heart. (D) Images from FRAP experiments showing pre-bleach, post-bleach, and recovery after 10 minutes in cardiomyocytes expressing a GFP-tagged mDSG2, mDSC2b, hDSP, and hPKP2. Yellow arrowheads point at the FRAP region along a cell-cell contact. Scale bar is 5 microns. (E) Plots of FRAP recovery fraction over 10 minutes. Black squares define the mean, and black vertical lines mark the standard deviation. All recovery data was fit to a double exponential curve (orange line). Values in gray indicate the number of experiments and FRAP contacts quantified for each protein. (F) The mobile fraction (percentage) and recovery halftimes (seconds, sec) for the fast and slow pools. The 95% confidence interval (CI) is listed for the mobile fraction and slow pool recovery. The fast pool percentage of the total mobile fraction is also listed. AC is often described as a desmosomal disease, as mutations in all desmosomal proteins are linked to AC in humans ( Delmar and McKenna, 2010 ). Most AC-linked mutations are found in PKP2 and DSG2 ( Zhang et al., 2023 ), underscoring the importance of both proteins in cardiovascular health. Loss of DSG2 function in the developing mouse heart prevents desmosome formation along the ICD and leads to AC ( Kant et al., 2015 ). Likewise, deletion of extracellular (EC) regions 1 and 2 in DSG2 caused AC-like phenotypes in mice ( Kant et al., 2012 ; Krusche et al., 2011 ), as did overexpression of the Dsg2 mutant N271S (corresponding to the AC-linked Dsg2 N266S mutation in humans) ( Pilichou et al., 2009 ). Thus, DSG2 is essential for desmosome formation in cardiomyocytes, and the loss or disruption of DSG2 function leads to the development of cardiovascular disease. In polarized epithelia, the desmosome organizes as a distinct, well-defined structure below (basal to) the AJ and connects the keratin networks of adjacent cells ( Broussard et al., 2020 ; Nekrasova and Green, 2013 ; Yeruva and Waschke, 2023 ). Cardiomyocytes, in contrast, lack apical-basal polarity, and AJs and desmosomes are interspersed along the ICD. AJ and desmosome components can also be intermingled at cardiomyocyte cell-cell contacts, especially during ICD development ( Borrmann et al., 2006 ; Franke et al., 2006 ; Franke et al., 2007 ). Furthermore, there is evidence that the desmosomal protein PKP2 binds a core component of the cardiac AJ, αT-catenin, to form a unique hybrid junction at the ICD termed the area compositae ( Goossens et al., 2007 ; Li et al., 2012 ; van Hengel et al., 2013 ). Previously, we used proximity proteomics to define the N-cadherin (CDH2) interactome in neonatal cardiomyocytes and identified adaptor and adhesion proteins unique to the cardiomyocyte AJ ( Li et al., 2019 ). Among the most abundant hits were two proteins associated with the desmosome, JUP and PKP2. The enrichment of desmosomal proteins at the AJ could reflect the proximity of the two adhesion complexes at developing junctions or the mixing of components in hybrid junctions. The blending of AJ and desmosomal proteins, along with the existence of a hybrid junction, underscores the uniqueness of cardiomyocyte cell-cell adhesion and the potential for mechanical and signaling crosstalk between desmosomes and AJs. Here, we define the DSG2 interactome in cardiomyocytes, gaining insight into the composition and organization of cardiomyocyte adhesion complexes. We demonstrate that cardiomyocyte desmosome proteins are largely stable, albeit with varying dynamics that may reflect distinct roles at junctions. We used proximity proteomics to identify proteins associated with DSG2. Approximately 50% of the DSG2 interactome overlapped with the CDH2 interactome ( Li et al., 2019 ), reflecting the proximity of the two complexes and shared cellular pathways. Nonetheless, we identified 153 proteins unique to DSG2, suggesting the two adhesion proteins assemble specific complexes at developing contacts. Finally, we provide evidence that PKP2 is a primary component of both the desmosome and the AJ in cardiomyocytes. Our results provide insight into the cardiomyocyte desmosome and the molecular complexes assembled at cardiomyocyte cell-cell contacts. Results The desmosome in neonatal cardiomyocytes Neonatal cardiomyocytes can reestablish cell-cell junctions when grown in culture ( Li et al., 2019 ; Merkel et al., 2019 ). Newly formed junctions contain the three ICD adhesion complexes – AJs, desmosomes, and gap junctions – intermingled along the contact membrane ( Fig. 1B ). The AJ is the primary complex in these nascent junctions (or pre-ICD), where it functions to couple the cortical cytoskeletons and link contractile myofibrils of adjacent cardiomyocytes. Desmosomes are organized adjacent to and between myofibril-coupled AJs along the pre-ICD and are often proximal to small gap junctions ( Fig.1B ). Though neonatal cardiomyocytes lack mature ICDs and the classic bipolar, rod-shaped morphology observed in the adult heart, the pre-ICD shares many of the organizational and architectural hallmarks of the ICD. Two isoforms of DSC2 are generated by alternative splicing ( Fig. 1C ) ( Parker et al., 1991 ). Isoform DSC2a is recognized as the canonical protein, a 901 amino acid (aa) protein formed by splicing exons 15 and 17. Isoform DSC2b is formed by splicing exons 15 and 16, which removes a large part of the C-terminal tail (aa837-901), including part of the catenin-binding domain (CBD), and replaces it with a short, unique 11 aa peptide (aa837-847) ( Parker et al., 1991 ; Troyanovsky et al., 1994 ). We isolated RNA from cultured neonatal cardiomyocytes and adult mouse hearts to determine which Dsc2 isoforms are expressed in cardiac tissue. We used RT-PCR to amplify two fragments of the Dsc2 mRNA containing exon 16 ( Fig. 1C ). If exon 16 was absent (as in Dsc2a), a fragment of ∼150 base pairs (bps, primer set 1) or ∼100 bps (primer set 2) would be generated. In contrast, if exon 16 were spliced downstream of exon 15 (as in Dsc2b), a fragment of ∼190 bps (primer set 1) or 140 bps (primer set 2) would be observed. In cardiomyocytes and heart samples, we observed both Dsc2a and Dsc2b isoforms, but Dsc2b was the predominant isoform in both samples ( Fig. 1C ). Given that the partial deletion of the CBD in DSCb variants removes much of the predicted JUP binding interface ( Pasani et al., 2024 ) and prevents JUP binding in cells ( Troyanovsky et al., 1994 ), we speculate that DSG2 forms the primary link between the desmosomal complex and IFs and regulates desmosome cytoplasmic signaling in the mouse heart. Consistent with this, loss of DSG2 function in the murine heart prevents desmosome formation, induces cardiomyocyte necrosis and fibrosis, and causes cardiomyopathy ( Kant et al., 2015 ). In contrast, desmosomes organize normally in DSC2-deficient hearts, and DSC2 mutant mice exhibit no major defects in heart organization and function ( Rimpler, 2014 ). Desmosome protein dynamics We next examined the dynamics of DSG2, DSC2, PKP2, and DSP in cardiomyocytes. The dynamics of JUP, a protein known to bind directly to classical cadherins as part of the AJ, were previously described by our group ( Li et al., 2019 ). Fluorescent protein (FP)-tagged proteins were transfected individually into cultured neonatal cardiomyocytes. As expected, all fusion constructs localized to cell-cell contacts ( Fig. 1D ). Protein dynamics were measured by fluorescence recovery after photobleaching (FRAP) in semi-confluent cells plated for 48-72 hours ( Fig. 1D ). Fluorescence recovery over ten minutes was quantified, plotted, and fit to a double exponential curve ( Fig. 1E ). The mobile fractions of DSG2 (29.4%) and DSP (28.8%) were nearly identical ( Fig. 1F ) and similar to the mobile fraction of cardiomyocyte JUP (26.5%) as well as the core AJ proteins CDH2, CTNNB1, CTNNA1, and CTNNA3 (32.3 – 36.1%; ( Li et al., 2019 )). The fact that more than 70% of DSG2, JUP, and DSP are immobile suggests a highly stable desmosome core, similar to the cardiomyocyte adherens junction (AJ). Note also that the mobile fractions of DSG2 and DSP (29.4% and 28.8%, respectively) were similar to DSG2 and DSP mobile fractions measured in Ca 2+ -dependent epithelial desmosomes (∼30%) ( Bartle et al., 2020 ). In contrast, the mobile fractions of DSC2b (46.9%) and PKP2 (50.7%) were markedly larger, indicating these two proteins were more dynamic than the other adhesion proteins. Notably, the PKP2 mobile fraction in MDCK epithelial cells was significantly higher than other desmosomal proteins (75% vs. 30-50% ( Fulle et al., 2021 )), possibly reflecting PKP2 functions independent of cytoskeletal coupling ( Bass-Zubek et al., 2009 ). The increased dynamics of DSC2b could reflect a weaker connection to the IF cytoskeleton. We then assessed the mobile fraction recovery rates for both the fast and slow pools for each of these key desmosomal components, DSG2, DSC2b, DSP, and PKP2 ( Fig. 1F ). The fast pool recovery halftimes of cytosolic DSP and PKP2 (6.4, 7.3 sec) could reflect an unbound population of protein near cell contacts, or it could be caused by reversible photobleaching or photoswitching ( Mueller et al., 2012 ; Sinnecker et al., 2005 ). The fast pool recovery rates of DSG2 and DSC2b (13.7, 5.7 sec) likely represent photoswitching, as the diffusion of new transmembrane protein in this time frame is unlikely. Notably, the fast pool is relatively small for all desmosome proteins (3–15%); thus, the slow pool represents the dynamics of most of the junction population. The half-times of fluorescence recovery for the slow pools were similar: DSG2 (229.1 sec), DSC2b (250.2 sec), DSP (287.3 sec), and PKP2 (214.7 sec) ( Fig. 1F ). The rates were also in the range of what we observed previously with JUP and the AJ core: JUP (237.6 sec), CDH2 (315.2 sec), CTNNB1 (255.2 sec), CTNNA1 (360.0 sec), and CTNNA3 (221.5 sec) ( Li et al., 2019 ). These slow rates reflect the strong associations between core components of the desmosome ( Al-Jassar et al., 2013 ; Choi et al., 2009 ; Kami et al., 2009 ). Interestingly, in both cardiomyocytes and MDCK cells, DSG2, DSC2a, and JUP all recovered at similar rates (60-76 sec) ( Fulle et al., 2021 ). However, the MDCK recovery rates for the trio were roughly four times faster than those observed in cardiomyocytes. The difference in dynamics between cardiomyocytes and epithelial cells may reflect inherent differences in the molecular composition of the desmosome, the mechanical environment (e.g., the load on the complex), or other post-translational modifications. Notably, the FRAP results indicate that the neonatal cardiomyocyte desmosome in general, and DSG2 in particular, is stable and amenable to proximity labeling. DSG2-BioID2 labels proteins at cardiomyocyte cell-cell contacts We sought to define the proteins associated with the desmosome using proximity proteomics, similar to how we defined the cardiomyocyte N-cadherin interactome ( Li et al., 2019 ). We decided to use DSG2 as the probe because 1) exogenous DSG2 tagged at the C-terminus with EGFP was stably expressed and localized to cell-cell contacts ( Fig. 1D ), and 2) DSC2b lacks a complete CBD and is more dynamic than DSG2 ( Fig. 1C-F ), possibly compromising biotin labeling. We fused the biotin ligase BioID2 ( Kim et al., 2016 ) to the C-terminal tail of DSG2 ( Fig. 2A ) and cloned the DSG2-BioID2 fusion into an adenoviral expression system to make DSG2-BioID2 adenovirus. This allowed us to infect primary cardiomyocytes and express low levels of DSG2-BioID2 for imaging and protein analysis ( Fig. 2B,C ). When expressed in cardiomyocytes and supplied with biotin in the media, DSG2-BioID2 biotinylated proteins at cell-cell contacts (Streptavidin [SA] stain in Fig. 2B , asterisks mark expressing cells). To further assess protein labeling, we precipitated biotinylated proteins from lysates of cardiomyocytes infected with either Dsg2-BioID2 or Cdh2-BioID2 and cultured with biotin and analyzed biotinylation by western blotting. DSG2-BioID2 was stably expressed ( Fig. 2C , HA blot) and biotinylated a large pool of proteins ( Fig. 2C , SA blot), similar to CDH2-BioID2. Download figure Open in new tab Figure 2. DSG2–BioID2 localizes to cell-cell contacts and labels junctional proteins. (A) Illustration of DS2G-BioID. The BioID2 biotin ligase with a C-terminal HA tag was fused to the end of the DSG2 C-terminal tail. (B) Neonatal cardiomyocytes infected with DSG2-BioID adenovirus and stained for CDH2 (green in merge) and labeled with streptavidin conjugated to Cy3 (magenta in merge) to identify biotinylated proteins. White asterisks mark infected cells. Scale bar is 10 microns. (C) Streptavidin western blot of pulldowns from control (uninfected), CDH2-BioID2 infected, or DSG2-BioID2 infected cardiomyocytes. Streptavidin-precipitated material was separated by SDS-PAGE and blotted for hemagglutinin (HA, left blot) or streptavidin (SA, right blot). Quantitative proximity proteomics reveals the cardiomyocyte DSG2 interactome Next, we used quantitative mass spectrometry (MS) to define the DSG2 interactome. For each replicate, 4 million cells were infected with Dsg2-BioID2 adenovirus 24 h post-plating. The next day (48 h post-plating), 50 µM biotin (final concentration) was added to the media, and the cells were harvested the following day (72 h post-plating). Uninfected control samples were treated identically to Dsg2-BioID samples (i.e., 50 µM biotin was added 48 h post-plating, and cells were harvested 24 h after biotin addition). In total, six Dsg2-BioID2 replicates (each containing 4 million cells, for a total of 24 million cells) and six control replicates (each containing 4 million cells, for a total of 24 million cells) were collected and analyzed. MS sample analysis revealed 5809 peptides from 924 proteins ( Fig. 3A,B ). When proteins with single unique peptides were excluded, the list was reduced to 687 proteins ( Fig. 3B ). To define Dsg2-BioiD2 enriched proteins, we established thresholds of fold change ≥ 10 and p < 0.001 ( Fig. 3A , dashed lines). These thresholds culled the list to a final 331 proteins from 325 genes ( Fig. 3B ). Download figure Open in new tab Figure 3. Quantitative mass spectrometry identifies the DSG2 interactome. (A) Plot of p-value (−log10) versus fold-change (log2) of identified proteins. Dashed lines mark p=0.001 (y-axis) and fold-change of 10 (x-axis). (B) Summary of identified peptide and protein number at each stage of condition stringency. (C) Rank plot of abundance (iBAQ mass, log2). Proteins of interest are marked with colored circles and labeled. Circle color corresponds to the primary adhesion complex: green (desmosome), orange (adherens junction), and purple (gap junction). (D) Protein distribution by assigned category based on number (top pie chart) or abundance (iBAQ) (bottom pie chart). (E) IPA enrichment analysis of DSG2 hits in canonical signaling pathways. The relative abundance of these 331 proteins is plotted in Fig. 3C , and the 35 most abundant proteins are listed in Table 1 . Among the most abundant proteins were the core components of the desmosome ( Fig. 3C , highlighted in green), DSG2, JUP, PKP2, and DSC2. The AJ proteins ( Fig. 3C , highlighted in yellow) CTTND1 (p120-catenin), CTNNB1, CTNNA3, and CDH2 were also enriched, as was the α-catenin ligand AFDN (afadin). Finally, the gap junction protein GJA1 (connexin-43, Fig. 3C , highlighted in purple) was also identified. The abundance of AJ proteins could reflect the proximity of desmosomes to AJs in the developing junctions. For example, DSG2 and PKP2 were abundant hits in the CDH2 interactome ( Li et al., 2019 ). Alternatively, AJ proteins proximal to DSG2 could indicate the intermingling of specific junctional components in hybrid junctions, as previously proposed ( Borrmann et al., 2006 ; Franke et al., 2006 ). Notably, CTNNA3, considered a key component of the hybrid junction ( Vite and Radice, 2014 ), was observed in the DSG2 interactome, whereas CTNNA1 was not. View this table: View inline View popup Download powerpoint Table 1. The top 35* most abundant proteins in the DSG2-BioID2 interactome. We next classified all 325 genes in the DSG2-BioID2 interactome as belonging to one of 20 functional categories based on Uniprot, GeneCards, and Entrez designations ( Fig. 3D ). The categories with the most hits by number were Adaptor (15%), Trafficking/Golgi/ER (13%), Metabolic Enzyme (11%), and Actin-Binding Adaptor (11%). The top categories based on protein abundance (iBAQ) were Adaptor (35%), RNA/Ribosome/Translation (14%), and Metabolic Enzyme (13%) ( Fig. 3D ). We then performed enrichment analysis using Ingenuity Pathway Analysis (IPA) to determine the canonical signaling pathways represented in the DSG2 interactome. The eight most enriched pathways are depicted in Fig. 3E . Four themes emerged from IPA: 1) Rho signaling ( Rho GTPase cycle ), 2) trafficking ( neutrophil degranulation, clathrin-mediated endocytosis ), 3) cell-cell organization and signaling ( cell junction organization, SC-GC junction signaling, integrin signaling, GC-SC junction ), and 4) cardiomyopathy ( dilated cardiomyopathy ). Given the desmosome’s established role in adhesion and links to heart disease, the cell-cell junction and cardiomyopathy signaling pathways were expected. The Rho GTPase and trafficking signaling pathways likely reflect the prevalence of adaptor, actin-binding adaptor, and trafficking proteins in the DSG2 interactome. Notably, the IPA results highlight the core adhesive and signaling functions ascribed to the desmosome. Comparing DSG2 and CDH2 interactomes We compared the DSG2 interactome with the previously reported cardiomyocyte CDH2 interactome ( Li et al., 2019 ). There were 172 hits shared between the two interactomes ( Fig. 4A ) and 153 hits unique to the DSG2 interactome. We then compared the abundance of the shared pool between the two interactomes by plotting the iBAQ abundance (Log2) of each hit in the DSG2 interactome (x-axis) versus the CDH2 interactome (y-axis) ( Fig. 4B ). Core AJ and desmosome proteins are marked in red. Linear regression analysis revealed a positive correlation in abundance. Note that JUP and PKP2 are heavily enriched in both interactomes. JUP was not surprising: it binds directly to both classical and desmosomal cadherins and is thus common to both adhesion complexes. PKP2, however, is a core component of the desmosome and is not typically associated with the AJ; yet, like JUP, we found it to be highly enriched in both datasets. CTNND1 (p120-catenin), another ARM-repeat protein like PKP2, was also enriched in both interactomes. CTNNA3 was shared between the interactomes, whereas the more abundant CTNNA1 (αE-catenin) was not (it was present in the DSG2 interactome but was not enriched above background). This suggests DSG2 preferentially associates with, or organizes near, specific AJs containing CTNNA3. Download figure Open in new tab Figure 4. DSG2 and CDH2 interactome comparison. (A) Venn diagram of cardiomyocyte DSG2 interactome (325 hits, green) versus cardiomyocyte CDH2 interactome (353 hits, orange). 172 hits are shared between the two interactomes (tan). Note that the original CDH2 interactome listed 354 genes, not 353. PALM2 and PALM2-AKAP were initially designated as separate genes, but they are classified as one gene (PAKAP) in mouse. (B) Scatter plot of shared hits (black dots) based on abundance (iBAQ mass, log2) in DSG2 (x-axis) and CDH2 (y-axis) interactomes. Core desmosome and adherens junction proteins are marked in red. Linear regression analysis calculated the slope (orange line), 95% confidence intervals (dashed orange lines flanking the slope), and R 2 value. Slope deviation from zero was analyzed for significance (p-value). (C) IPA enrichment analysis of DSG2 only hits (left), DSG2-CDH2 shared hits (middle), and CDH2 only hits (right) in canonical signaling pathways. To gain further insight into the potential similarities and differences between the DSG2 and CDH2 interactomes, we performed enrichment analysis using IPA. We examined the DSG2 unique (i.e., proteins not shared with CHD2), CDH2 unique (i.e., proteins not shared with DSG2), and DSG2/CDH2 shared sets for enrichment in canonical signaling pathways. The DSG2/CDH2 shared set was enriched for Rho signaling, endocytosis, and cell-cell organization/signaling ( Fig. 4C ). The top two canonical signaling pathways in the DSG2/CDH2 shared set — Rho GTPase cycle and clathrin-mediated endocytosis — were also the top two pathways in the CDH2 only set. Actin cytoskeleton and muscle contraction pathways were enriched in the CDH2 set, but not in the shared or DSG2-only sets, reflecting the role of the AJ in connecting the actin networks of adjacent cells. Finally, trafficking, mitosis, and Rho signaling pathways were enriched in the DSG2-only hits ( Fig. 4C ). These results suggest that DSG2 recruits a distinct collection of adhesion and signaling proteins from CDH2. Shared desmosome components Next, we compared the cardiomyocyte DSG2 interactome to two recent proteomic analyses of the epithelial desmosome: the DSP interactome in keratinocytes ( Badu-Nkansah and Lechler, 2020 ), and the compiled JUP, PKP2, and DSC2a Ca2+-dependent interactomes in MDCK cells ( Fulle et al., 2024 ). The overlap between the three interactomes is shown as a Venn diagram in Fig. 5A . Surprisingly, only 16 proteins were shared between the multiple interactomes (listed in Table 2 ). Only two desmosome core proteins, DSG2 and JUP, were among the shared hits. This is partly due to differences in Plakophilin isoform expression between cell types. All interactomes contained at least one Plakophilin protein: the DSG2 and DM interactomes shared PKP2, whereas the DSP and DM interactomes shared PKP1 and PKP3. DSP was observed in the DSG2 interactome but not in the DM combined interactome, which included proteins associated with DSC2a. In this study and the work from Fulle and colleagues ( Fulle et al., 2024 ), the biotin ligase BioID2 was fused to the end of the C-terminal cytoplasmic tail in both desmosomal cadherins. However, the DSG2 cytoplasmic tail is significantly longer than the DSC2a tail and is thus more likely to promote labeling of distal desmosome proteins, such as DSP. Other proteins shared between all interactomes include the adaptor proteins CTNND1 (p120-catenin), AFDN (afadin), and MSN (moesin), as well as a collection of chaperone and trafficking proteins essential for desmosome protein folding, delivery, and recycling. Download figure Open in new tab Figure 5. Desmosome interactome comparison. (A) Venn diagram of cardiomyocyte DSG2 interactome (325 hits), DSP interactome in keratinocytes (682 hits), and the combined JUP, PKP2, and DSC2a Ca2+-dependent interactome in MDCK cells (labeled DM for desmosome; 194 hits in total). Sixteen proteins are shared between the interactomes. (B) IPA enrichment analysis of DSG2-DSP shared hits (top left), DSG2-DM shared hits (top right), DSG2-DSP-DM shared hits (bottom right), and DSG2 unique hits (bottom right) in canonical signaling pathways. View this table: View inline View popup Download powerpoint Table 2. DSG2-DSP-DM Interactomes Shared Hits We then performed enrichment analysis using IPA to gain insight into the proteins shared between the interactomes. We examined the DSG2-DSP (129 shared hits), DSG2-DM (31 shared hits), and DSG2-DSP-DM (16 shared hits) sets for enrichment in canonical signaling pathways ( Fig. 5B ). The top pathways in the DSG2-DSP set were neutrophil degranulation, apoptotic execution phase, and Rho GTPase cycle. The neutrophil degranulation and apoptotic execution phase pathways likely reflect shared trafficking and degradation proteins. The Rho GTPase cycle pathway was enriched in all shared sets ( Fig. 5B ), demonstrating the importance of CTNND1 and PKP proteins in regulating Rho signaling ( Anastasiadis and Reynolds, 2001 ; Hatzfeld et al., 2014 ). The top pathways in the DSG2-DM set were Rho GTPase cycle, SC-GC (Sertoli cell – germ cell) junction signaling, and L1CAM interactions. SG-GC junction signaling (as well as GC-SC junction signaling) pathways were enriched in all sets, reflecting the importance of the desmosome in Sertoli cell-germ cell adhesion. Finally, the top pathways enriched in the shared DSG2-DSP-DM set were L1CAM interactions, GC-SC junction signaling, and Rho GTPase cycle. The L1CAM interactions pathway, also enriched in the DSG2-DM set, likely represents the prevalence of trafficking and adaptor proteins required for cell-cell adhesion. Overall, the analysis of enriched canonical signaling pathways shared between epithelial and cardiomyocyte desmosome interactomes revealed a common reliance on adaptor, cytoskeletal remodeling, and trafficking proteins that mediate intercellular adhesion across cell types. We then analyzed hits unique to the cardiomyocyte DSG2 interactome ( Fig. 5B ). The top enriched pathway was dilated cardiomyopathy (CM) signaling, followed by ROBO signaling and neddylation. The prevalence of unique hits associated with dilated CM signaling was not surprising given the difference in cell type (cardiac versus epithelial) and the well-recognized role of the desmosome in CM. ROBO signaling is best known for its importance in axon guidance, but it also plays a critical role in heart development ( Zhao and Mommersteeg, 2018 ). Likewise, neddylation regulates protein degradation and plays a role in heart development and function ( Li et al., 2020 ). Rho GTPase cycle signaling was also enriched and was the only pathway observed in all four sets. The fact that Rho signaling was enriched in proteins unique to DSG2 could reflect a cardiomyocyte-specific repertoire of Rho regulatory proteins, possibly necessitated by the unique cytoskeletal organization and physical demands of cardiomyocyte cell-cell adhesion. DSG2 interactome protein network We connected and organized the DSG2 interactome to gain a deeper understanding of the molecular complexes assembled at cardiomyocyte desmosomes and how these complexes may differ from those at cardiomyocyte AJs. ( Fig. 6A ). We classified all interactors as either unique to DSG2 (green circles) or shared between DSG2 and CDH2 (yellow circles). The top 30 hits in the DSG2 interactome are circled in red. We then constructed a hierarchical classification with DSG2 at the top (see Methods for details). We separated the interacting network into four tiers: 11 primary, 138 secondary, 127 tertiary, and 10 quaternary ( Fig. 6A ). All protein-protein interactions were based on published experimental data. Thirty-nine of the DSG2-BioID hits could not be connected to any protein in the network (listed in Table S3 ). We then calculated the percent distribution of DSG2 and DSG2/CDH2 hits within each tier ( Fig. 6B ). The percentage of DSG2/CDH2 shared hits was highest in the primary (73%), tertiary (61%), and quaternary tiers (80%), but noticeably reduced in the secondary tier (38%). The high percentage of shared hits in the primary tier likely reflects shared core components (e.g., JUP, CTNNA3, PKP2). In contrast, the large percentage of shared hits in the tertiary and quaternary tiers could indicate shared ancillary proteins required for cadherin trafficking, stability, and degradation, among other functions. DSG2-only hits dominated the secondary tier, and we speculate that these include specific, specialized interactors recruited to the desmosome core complex to promote adhesion and initiate signaling. Download figure Open in new tab Figure 6. Cardiomyocyte DSG2 interactome network. (A) DSG2 interactome interaction network organized into four tiers based on IPA. Published experimental data support all protein-protein interactions. Hierarchical classification was done manually around DSG2. Primary interactors bind DSG2 directly. Secondary interactors bind to primary interactors, but not to DSG2. Tertiary interactors bind secondary interactors, and quaternary interactors bind tertiary interactors. Green circles signify hits unique to the DSG2 interactome; yellow circles mark hits shared with the CDH2 interactome. The 30 most abundant DSG2 interactome proteins are outlined in orange. 39 proteins not connected to the network are listed in Table S1 . (B) Percent distribution of DSG2 unique (green bar) and DSG2/CDH2 shared (yellow bar) hits within each tier. View this table: View inline View popup Download powerpoint Supplemental Table 1 PKP2 recruitment to cell-cell contacts is tension-regulated The interactome network analysis revealed that 73% of the proteins thought to interact directly with DSG2 (primary tier) are also associated with CDH2, though most do not bind directly to CDH2. One notable exception is JUP, which binds with high affinity to the tails of both classical cadherins and desmosomal cadherins. Not surprisingly, JUP was one of the most abundant hits in both the DSG2 and CDH2 interactomes ( Fig. 3C , 4B , Table 1 (L i et al., 2019) ) . Nearly as abundant in both interactomes was PKP2 ( Fig. 3C , 4B , Table 1 , ( Li et al., 2019 )). PKP2 is primarily regarded as a desmosome protein, although it has been shown to associate with the AJ in cardiac and non-muscle cells ( Borrmann et al., 2006 ; Chen et al., 2002 ; Franke et al., 2006 ). We decided to examine the localization of endogenous PKP2 in neonatal cardiomyocytes and compare it with that of other cell-cell junction proteins. Confluent cultures of cardiomyocytes were fixed and stained for PKP2 and CTBNNB1 (β-catenin), CTNNA1, CTNNA3, JUP (plakoglobin), AFDN, and GJA1 ( Fig. 7A-F ) . PKP2 was enriched at cell-cell contacts, and the localization looked nearly identical to an AJ protein. Pearson’s colocalization analysis confirmed this: we measured high Pearson’s correlation coefficients between PKP2 and CTNNB1, CTNNA1, and JUP ( Fig. 7G ). We also measured high, though significantly lower, Pearson’s coefficients between PKP2 and CTNNA3 and AFDN ( Fig. 7G ). A significantly lower correlation was noted between PKP2 and GJA1, as well as between PKP2 and F-actin. Based on these staining results and our mass spec data, we suggest that PKP2 should be considered a component of both the desmosome and the AJ in cardiomyocytes, similar to JUP. Download figure Open in new tab Figure 7. PKP2 is recruited to cardiomyocyte AJs under tension. (A-F) Cardiomyocytes were cultured for 72-96 hours, fixed, and stained for PKP2, F-actin, and CTNNB1 (A), CTNNA1 (B), CTNNA3 (C), JUP (D), AFDN (E), or GJA1 (F). All images are maximum projections of 1 micron z-stacks. Merge images show overlap between PKP2 (green) and adhesion protein (magenta) channels. (G) Pearson’s coefficient was calculated between PKP2 and individual adhesion protein channels at cell-cell contacts. Scatter plot of all data points shown. The thick horizontal blue line marks the median, and the blue error bars define the interquartile range. Compact letter display defines statistically indistinguishable coefficients (one-way ANOVA with Tukey’s multiple comparisons). (H-J) Cardiomyocytes were cultured for 72 hours, incubated for 1 hour in DMSO (control, H) or 100 μM blebbistatin (I and J), fixed, and stained for JUP, PKP2, and F-actin. (K). PKP2 average fluorescence signal intensity at cell-cell contacts divided by the average intensity in the cytoplasm (PKP contact/cytoplasm ratio) in cells treated with DMSO or blebbistatin. A scatter plot of all data points is shown, and the median and interquartile range are marked in (G). Blebbistatin treatment caused a significant decrease in PKP2 enrichment at cell-cell contacts (p = 0.008, Welch’s t-test). Scale bar in A-F, H-J is 20 microns. It has been proposed that PKP is specifically recruited to the middle (M) domain of CTNNA3 to form a hybrid junction in the heart ( Goossens et al., 2007 ; Li et al., 2012 ). The CTNNA3 M-domain, like that of CTNNA1, is mechanosensitive in that applied load opens the domain to reveal a binding site for VCL (vinculin) ( Pang et al., 2019 ). While there are differences in M-region regulation between CTNNA1 and CTNNA3 ( Choi et al., 2012 ; Heier et al., 2021 ), both are force-sensitive, and mechanical load is required to promote VCL binding ( Le et al., 2019 ; Maki et al., 2018 ; Pang et al., 2019 ; Seddiki et al., 2018 ; Yao et al., 2014 ; Yonemura et al., 2010 ). We questioned if PKP2 recruitment to cardiomyocyte cell-cell junctions was tension-dependent. Cultured cardiomyocytes were treated with dimethyl sulfoxide (DMSO) alone or DMSO plus 100 μM blebbistatin to suppress myosin activity for one hour. Cells were then fixed and stained for PKP2, JUP, and F-actin ( Fig. 7, H-J ). PKP2 at cell-cell junctions was significantly reduced in blebbistatin-treated cells after one hour ( Fig. 7 , compare PKP2 channel I, J to H, quantification in K ), consistent with a requirement for tension to recruit PKP2, possibly to α-catenin. While PKP2 junction levels decreased after blebbistatin treatment, a large pool of PKP2 remained localized to cell-cell junctions. We speculate that this tension-insensitive pool of PKP2 could be bound directly to desmosomal proteins (DSG2, DSC2, JUP, and DSP). Together, these results show that, in neonatal cardiomyocytes, PKP2 is a ubiquitous junction protein recruited, in part, through tension. Discussion We describe the first detailed analysis of the DSG2 interactome in cardiomyocytes. Our results provide insight into DSG2 molecular associations in developing cardiomyocyte cell-cell adhesions. We uncovered an extensive set of shared and DSG2-specific interactions that highlight the unique mechanical environment of cardiomyocyte intercellular adhesions and the interplay between the AJ and desmosome. The DSG2 interactome This work builds off our past study of the CDH2 interactome in cardiomyocytes ( Li et al., 2019 ) and complements more recent studies of the desmosome proteome in epithelia ( Badu-Nkansah and Lechler, 2020 ; Fulle et al., 2024 ; Hegazy et al., 2022 ) as well as past studies of the epithelial AJ CDH1 (E-cadherin) interactome ( Guo et al., 2014 ; Shafraz et al., 2020 ; Van Itallie et al., 2014 ). Proteomic studies to define the desmosome interactome in epithelia used similar biotinylation-based approaches to define the interactomes of DSP ( Badu-Nkansah and Lechler, 2020 ) and DSG1 ( Hegazy et al., 2022 ) in keratinocytes, and DSC2a, JUP, and PKP2a in MDCK cells ( Fulle et al., 2024 ). Here we add the DSG2 interactome to these datasets, expanding our appreciation of desmosomes by defining DSG2 interactions in the context of an adhesion system under load with unique organizational and mechanical demands. The limited number of shared hits between the multiple desmosomal protein interactomes was somewhat surprising. We speculate that the dearth of shared proteins reflects cell-specific or tension-dependent requirements for desmosome adhesion, trafficking, turnover, and/or cytoskeletal integration. Nonetheless, a complex network of adhesion protein interactions has emerged, varying between adhesion types, adhesion states, and tissue sources. Defining how these interactions regulate the mechanical and signaling properties of the desmosome and AJ, as well as potential hybrid and transitory adhesion complexes, will be the focus of future work. Our quantitative analysis revealed that the most abundant proteins associated with DSG2 ( Fig. 3D , Table 1 ) are foundational desmosome proteins: JUP, PKP2, DSP, and DSC2. We conclude that the early cardiac desmosome is fundamentally similar to the epithelial desmosome. The CDH2 interactome in neonatal cardiomyocytes contains many specific adaptor and linker proteins that likely function to strengthen cytoskeletal connections to cardiac AJs under increased load. We presume the cardiomyocyte desmosome experiences high tension. Consistent with this, a DSG2 FRET-based tension sensor showed that DSG2 was under more load in contracting cardiomyocytes compared to MDCK cells ( Baddam et al., 2018 ). However, we found that DSG2 interactome largely lacks unique secondary scaffolding, adaptor, and/or cytoskeletal proteins that could assist in mechanical adhesion. An exception was two plakin proteins in addition to DSP, plectin (PLEC) and periplakin (PPL). Plakin proteins are large, versatile linker proteins that connect IFs to junctional complexes ( Leung et al., 2002 ), and mutations in PLEC, like DSP, are associated with cardiomyopathy and cardiac disease ( Bolling et al., 2010 ; Villa et al., 2015 ). PLEC can bind both actin and IFs ( Outla et al., 2025 ), and thus link multiple cytoskeletal attachments to the desmosome, similar to the hybrid junction (discussed more below). Actin and IF integration at cardiomyocyte cell-cell junctions is poorly understood yet could play a critical role in ICD organization and function. While desmosomes form between cultured neonatal cardiomyocytes ( Fig. 1B ), the cell-cell junctions are not mature in this model. We often refer to these junctions as pre-ICDs to distinguish them from mature ICDs, which are only observed in vivo . Thus, these newly formed desmosomes may lack elements of specialization required in mature cardiac tissue. Desmosome assembly is also more complicated than AJ assembly in epithelia (( Beggs et al., 2022 ; Bharathan et al., 2024 ; Kowalczyk and Green, 2013 ; Nekrasova and Green, 2013 )), though desmosome maturation is largely undefined in CMs. Finally, between labeling time length (24 h) and variability in adhesion maturation within the culture, biotin labeling “captures” the DSG2 interactome at multiple stages of desmosome formation. Future experiments with faster ligases (TurboID, ( Branon et al., 2018 )), different culture conditions, and in vivo cardiac labeling could add deeper insight into desmosome organization and function. DSG2 is the primary mechanical link to the cytoskeleton DSG and DSC function together to mediate intercellular adhesion in mature desmosomes. While homotypic trans interactions (i.e., DSG-DSG) can occur and may have a physiological role, heterotypic trans interactions (i.e., DSG-DSC) are much stronger in vitro and likely represent the dominant interface in vivo ( Harrison et al., 2016 ). DSG cadherins possess a long cytoplasmic tail with established binding sites for JUP and PKP, as well as additional ligands ( Bharathan et al., 2024 ). DSC cadherins, on the other hand, exist as two spliceforms: an ‘a’ isoform with a cytoplasmic tail that binds JUP and PKP proteins, and a ‘b’ isoform with a truncated tail that lacks the complete CBD and does not bind JUP. In the mouse cardiomyocytes, we found that both Dsc2a and Dsc2b are expressed, but Dsc2b is the predominant isoform ( Fig. 1C ). A similar expression pattern was observed in the adult human heart ( De Bortoli et al., 2010 ). We conclude that DSC2b is the predominant DSC2 isoform in the heart. Heterotypic trans interactions between DSG2 and DSC2b would potentially create a mechanical imbalance within the intercellular complex based on the asymmetric recruitment of JUP. Such an imbalance could be advantageous in contractile tissues, allowing the desmosome to absorb mechanical strain. Alternatively, an imbalance could be remedied by cis interactions (extracellular or intracellular) between neighboring desmosome complexes or the ability of DSC2b to connect to IFs through PKP2 recruitment. While there are notable differences in ligand binding between PKP homologs ( Bonne et al., 2003 ), the structural remodeling suggests PKP1 binds a region of DSC1 tail that is also present in DSC1b ( Pasani et al., 2024 ). DSC2a could also provide sufficient mechanical linkage, despite being the minor isoform. However, DSC2b can compensate for Dsc2 mutations that prevent DSC2a tail expression in humans, suggesting that DSC2b is sufficient for desmosome function in the heart ( De Bortoli et al., 2010 ). Note that DSG2 expression in the rat heart is roughly 5X that of DSC2, suggesting that DSG2 may possess functions independently of DSC2. Consistent with this, there are AC-linked mutations in surface residues of the DSG2 EC3 domain, well outside the trans -binding interface ( Harrison et al., 2016 ). Furthermore, DSG2 has been shown to engage in Ca 2+ -dependent trans interactions with CDH1 (E-cadherin) and CDH2 ( Fuchs et al., 2022 ), as well as cis interactions with CDH1 ( Shafraz et al., 2018 ). Much remains to be learned about the DSG2-DSC2 adhesion complex and the roles of both DSG2 and DSC2 in the heart. PKP2 is recruited to AJs and desmosomes in the heart JUP and PKP2 were among the most abundant hits in both the CDH2 and DSG2 interactomes. JUP is a homolog of β-catenin and binds directly to the cytoplasmic tails of classical cadherins and desmosomal cadherins. PKP2, however, binds directly to JUP, DSP, DSG, and DSC, and is primarily considered a core component of the desmosome. There is, however, evidence for PKP2 associations outside the desmosome, and it has been suggested that PKP proteins coordinate AJ and desmosome assembly ( Bass-Zubek et al., 2009 ). Overexpressed PKP2 in keratinocytes localized to cell-cell contacts in a continuous, rather than the punctate, pattern, suggesting membrane recruitment independent of the desmosome. When overexpressed in COS cells, β-catenin co-precipitated with PKP2 ( Chen et al., 2002 ). Likewise, endogenous PKP2 co-precipitated with N-cadherin from bovine heart lysates ( Borrmann et al., 2006 ). Further, immunoelectron microscopy studies of mammalian heart tissue revealed PKP2 localization along the entire ICD ( Franke et al., 2006 ). The intermingling of desmosomal and AJ components along the ICD observed by Franke and colleagues led to the concept of the area compositae or hybrid junction. We observed a continuous pattern of PKP2 localization at cardiomyocyte cell-cell contacts. PKP2 membrane recruitment was indistinguishable from JUP, CTTNA1, CTNNA3, and CTNNB1, reflected in the high Pearson’s colocalization coefficients ( Fig. 6G ). This is consistent with past immunoelectron imaging of adult mammalian hearts and immunofluorescent studies of cultured rat neonatal cardiomyocytes ( Franke et al., 2006 ; Franke et al., 2007 ). Combined with our current and past mass spec results, we propose that PKP2 is a component of the cardiomyocyte desmosome and AJ. As noted above, neonatal cardiomyocytes do not form mature ICDs, and we don’t consider these mixed junctions as true area compositae. However, Franke and colleagues showed that PKP2 is an integral component of both CDH2 and DSG2 complexes and suggest that component mixing is an intrinsic property of both developing and mature cardiomyocyte cell-cell contacts ( Borrmann et al., 2006 ; Franke et al., 2006 ; Franke et al., 2007 ). This wider, desmosome-independent recruitment of PKP2 to cardiomyocyte adhesions is essential when considering PKP2 function in cardiac health and disease. Finally, we showed that PKP2 recruitment to junctions is tension-dependent. Force is required to unfurl the mechanosensitive α-catenin M-region and reveal binding sites for ligands such as VCL, and we speculate that force is also required to promote PKP2 binding to α-catenin. Note, however, that a pool of PKP2 remained membrane-bound in the absence of tension. This tension-independent PKP2 pool could reflect head domain binding to other ligands (e.g., JUP) and/or binding of the ARM repeats to the DSG2 or DSC2b tail. Summary Building on our previous work on the cardiomyocyte CDH2 interactome, we have identified over 500 proteins that comprise the core of the cardiac adherens junction (AJ) and desmosome. This interconnected array of proteins regulates all aspects of cadherin biology, including trafficking, organization, adhesion, post-translational modification, and downstream signaling. Defining how these cadherin-associated proteins function, individually and collectively, to regulate desmosome, AJ, ICD biology in the heart remains the next challenge. Materials and Methods Plasmids To create the mouse Dsc2b and Dsg2 (mDsc2b and mDsg2) constructs, RNA was first isolated and purified from adult mouse heart using the RNeasy Fibrous Tissue Mini kit (Qiagen) and reverse transcribed to create cDNA using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche). Gene specific primers were designed against the 5’ and 3’ ends of each gene to generate full-length clones by PCR. PCR products were cloned directly into pCR-BluntII-TOPO (Thermo-Fisher) and sequence verified. mDsg2 and mDsc2b CDS were cloned into pEGFP-N1 to create EGFP fusions. Human Desmoplakin-GFP was a gift from Kathleen Green (Addgene plasmid # 32227 ; http://n2t.net/addgene:32227 ; RRID:Addgene_32227). Human PKP2a was cloned from Plakophilin 2a-Flag (a gift from Kathleen Green, Addgene plasmid # 32230 ; http://n2t.net/addgene:32230 ; RRID:Addgene_32230) into pEGFP-C1. PKP2a fragments were cloned by PCR into pEGFP-C1. All constructs were sequence verified. Antibodies Primary antibodies used for immunostaining were: anti-αE-catenin (1:100, Enzo Life Science, ALX-804-101-C100), anti-αT-catenin (1:100, rat, a kind gift from Jolanda Van Hengel, Ghent University), anti-β-Catenin (1:100, Cell Signaling, D10A8), anti-I-Afadin (1:500, Sigma, A0349), anti-γ-Catenin (1:100, Cell Signaling Technology, 2309), anti-Connexin-43 (1:100, Proteintech, 15386-1-AP), and anti-Plakophilin 2 (1:10, Progen, 651101). Streptavidin-Cy3 (1:300, Jackson Immunoresearch, 016-160-084) was used to label biotinylated proteins. Secondary antibodies used were goat anti-mouse or anti-rabbit IgG labeled with Alexa Fluor 647 (1:250, Thermo Fisher Scientific). Dsc2 isoform analysis RNA was isolated from adult mouse heart or cultured neonatal cardiomyocytes and reverse transcribed to create cDNA using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche) for heart RNA or the High Capacity RNA to cDNA Kit (Thermo Fisher Scientic) for cardiomyocyte RNA. Purified cDNA was then used as template for PCR to amplify the Dsc2 region surrounding exon 16. Cardiomyocyte isolation and culture All animal work was approved by the University of Pittsburgh Division of Laboratory Animal Resources. Primary cardiomyocytes were isolated from Swiss Webster mice as described ( Ehler et al., 2013 ). For protein isolation, cardiomyocytes were plated onto 35 mm dishes (1 x 10 6 cells/dish) coated with Collagen Type I (Millipore). For immunostaining, cardiomyocytes were plated onto 35 mm MatTek dishes with 10 mm insets coated with Collage Type I. Cardiomyocytes were plated in plating media: 65% high glucose DMEM (Thermo Fisher Scientific), 19% M-199 (Thermo Fisher Scientific), 10% horse serum (Thermo Fisher Scientific), 5% FBS (Atlanta Biologicals) and 1% penicillin-streptomycin (Thermo Fisher Scientific). Media was replaced 16 hours after plating with maintenance media: 78% high glucose DMEM, 17% M-199, 4% horse serum, 1% penicillin-streptomyocin, 1 μM AraC (Sigma) and 1 μM Isoproternol (Sigma). Cells were cultured in maintenance media for 2-4 days until lysis or fixation. Immunostaining and confocal microscopy Cells were fixed in 4% EM grade paraformaldehyde in PHEM buffer (60 mM PIPES pH 7.0, 25 mM HEPES pH 7.0, 10 mM EGTA, pH 8.0, 2 mM MgCl2 and 0.12 M Sucrose) or PHM (no EGTA) buffer for 10 minutes, washed twice with PBS and then stored at 4°C until staining. Cells were permeabilized with 0.2% Triton X-100 in PBS for 5 minutes and washed twice with PBS. Cells were then blocked for 1 hour at room temperature in PBS + 10% BSA (Sigma), washed 2X in PBS, incubated with primary antibodies in PBS + 1% BSA for 1 hour at room temperature, washed 2X in PBS, incubated with secondary antibodies in PBS + 1% for 1 hour at room temperature, washed 2X in PBS and then mounted in ProLong Glass (Thermo Fisher Scientific). All samples were cured at least 24 hours before imaging. For blebbistatin experiments, cardiomyocytes (72 hours post-plating) were treated with 100 μM blebbistatin in DMSO or DMSO alone for 1 hour. Cells were incubated at 37°C during treatment. After incubation, cells were fixed and labeled as described Cells were imaged on a Nikon Eclipse Ti inverted microscope outfitted with a Prairie swept field confocal scanner, Agilent monolithic laser launch and Andor iXon3 camera using NIS-Elements (Nikon) imaging software. Maximum projections of 3-5 µm image stacks were created and deconvolved (3D Deconvolution) in NIS-Elements (Nikon) for presentation. Image analysis To measure colocalization between PKP2 and adhesion proteins in cardiomyocytes, a one-micron maximum projection containing the cell-cell contacts was created for analysis in in ImageJ (NIH). IsoJ Dark thresholding was then used to create a mask of the PKP2 staining channel to define the region of analysis (cell-cell contacts). The Pearson’s coefficient was calculated between PKP2 and an individual adhesion protein channel within the masked region and plotted in Prism (Graphpad). One-way ANOVA with Tukey’s multiple comparisons was used to calculate significance. To measure average PKP2 fluorescence signal intensity at cell–cell contacts in the blebbistatin experiments, a single plane was selected from the z-stack where the cell– cell contacts were most in focus for analysis in ImageJ (NIH). IsoJ Dark thresholding was used to create a mask of the plakoglobin channel to define the region of analysis (cell–cell contacts). The average PKP2 signal intensity was measured in the masked region and divided by the average intensity in the cytoplasm to calculate a PKP2 contact/cytoplasm ratio. Ratios were plotted using Prism software (GraphPad). Statistical significance was determined using Welch’s t-test FRAP experiments FRAP experiments were conducted on a Nikon swept field confocal microscope (describe above) outfitted with a Tokai Hit cell incubator and Bruker miniscanner. Actively contracting cells were maintained at 37°C in a humidified, 5% CO2 environment. User-defined regions along cell-cell contacts (DSP, DSC2, PKP2, DSG) were bleached with a 405 laser and recovery images collected every 10 seconds for 10 minutes. FRAP data was quantified in ImageJ (NIH). Individual recovery data was analyzed in Prism (GraphPad) via nonlinear regression (curve fit) two phase association with two constraints: plateau must be between zero and one, and the y-intercept must be equal to zero. Following analysis, individual recovery plots were excluded if the r 2 value was below 0.85. The calculated plateau was then compared to the average of the last four data points in the set. If the difference between the calculated plateau and the average was greater than 0.25, the recovery plot was excluded. The remaining individual recovery plots were placed into Excel (Microsoft) to calculate the average and standard deviation. The FRAP average and standard deviation were plotted in Prism (Graphpad). FRAP recovery plots represent data from at least three separate transfections of unique cell preps. The plotted data were fit to a two-phase association curve to determine the mobile fraction and half-time of recovery using Prism (GraphPad). Electron microscopy Cardiomyocytes were cultured on collagen-coated MatTek dishes and fixed as described above. After fixation and washing, cells were incubated with 1% OsO4 for one hour. After several PBS washes, dishes were dehydrated through a graded series of 30% to 100% ethanol and then infiltrated for 1 hour in Polybed 812 epoxy resin (Polysciences). After several changes of 100% resin over 24 hours, cells were embedded in inverted Beem capsules, cured at 37°C overnight and then hardened for 2 days at 65°C. Blocks were removed from the glass dish via a freeze/thaw method by alternating liquid Nitrogen and 100°C water. Ultrathin (60nm) sections were collected on to 200-mesh copper grids, stained with 2% uranyl acetate in 50% methanol for 10 minutes and 1% lead citrate for 7 minutes. Samples were photographed with a JEOL JEM 1400 PLUS transmission electron microscope at 80kV with a Hamamatsu ORCA-HR side mount camera. Adenovirus production The mDsg2 was cloned into MCS-BioID2-HA (a gift from Kyle Roux, Addgene plasmid # 74224 ; http://n2t.net/addgene:74224 ; RRID:Addgene_74224) to fuse BioID2 to the C-terminal tail of DSG2. The Dsg2–BioID2 fusion was then subcloned into pAdTrack-CMV (a gift from Bert Vogelstein, Addgene plasmid # 16405 ; http://n2t.net/addgene:16405 ; RRID:Addgene_16405) ( He et al., 1998 ). Recombinant adenovirus was produced by transforming the pAdTrack-CMV-Dsg2-BioID2 plasmid into pAdEasier-1 E. coli cells (deposited by Bert Vogelstein, Addgene 16399) ( He et al., 1998 ). Virus packaging and amplification were performed as described ( Luo et al., 2007 ). Virus particles were purified using Vivapure AdenoPACK 20 Adenovirus (Ad5) purification and concentration kit (Sartorius). Adeno-X qPCR Titration Kit (Clontech) was used to calculate virus titer using quantitative PCR on an Applied Biosystems 7900HT. Adenovirus infection and biotin labeling Each experimental replicate included four 35 mm dishes with 1×10 6 cells each (4×10 6 total). Cardiomyocytes were infected one day after plating with Dsg2–BioID2 adenovirus at a multiplicity of infection (MOI) of 2-5. We reproducibly infected >90% of cardiomyocytes at a low MOIs. Twenty-four hours later (48 hours post-plating), the media was replaced with fresh maintenance media plus 50 μM biotin in both Dsg2– BioID2-infected and control uninfected samples. The next day (72 hours post-plating), cells were harvested for protein isolation and mass spectrometry. Cell lysate preparation and affinity purification were performed according to published protocols ( Kim et al., 2016 ; Le Sage et al., 2016 ). Western blotting Protein samples were separated on an 8% SDS-PAGE gel and transferred onto a PVDF membrane (Bio-Rad). The membrane was blocked in TBST+5% BSA, washed in TBST, incubated with IRDye 680RD Streptavidin (1:1000, LI-COR) in TBST, washed twice in TBST and washed once in PBS. The membrane was scanned using a LI-COR Odyssey Infrared Imager. Mass spectrometry and statistical analysis Protein samples were processed using the single-pot, solid-phase-enhanced sample-preparation (SP3) on-bead digestion protocol with trypsin, similar to ( Flanagan et al., 2024 ). In brief, samples were adjusted to a final volume of 200 μL in 100 mM Tris, pH 7.4, and then subjected to reduction with 10 mM dithiothreitol (DTT) and alkylation with 20 mM iodoacetamide (IAA). Subsequently, 1000 μL of 100% acetonitrile (ACN) and 30 μL of 20 mg/mL pre-washed SP3 paramagnetic bead mix (an equal mix of hydrophobic beads (GE Healthcare Bio-Sciences, Cat #65152105050250) and hydrophilic beads (GE Healthcare Bio-Sciences Cat #45152105050250) were added, followed by a 20-minute incubation with vigorous shaking (1400 rpm). The beads were then washed three times with 80% ethanol, all liquid was removed, and the beads were resuspended in 100 μL of 50 mM ammonium bicarbonate containing 1 μg of trypsin and incubated overnight at 37°C with vigorous shaking (1400 rpm). The supernatant, containing the resulting tryptic peptides, was transferred to a new tube. To ensure complete recovery of peptides, 100 μL of 0.1% TFA was added to wash the SP3 beads, and the wash was combined with the previous supernatant. Tryptic peptides were desalted on a vacuum manifold using 50-mg bed reverse-phase C18 solid phase columns (Supelco), speed-vac dried, and resuspended in 12 μL 0.1% formic acid (FA). A pooled instrument control (PIC) sample was prepared by combining 2 μL from each of the 12 samples and used to monitor instrument reproducibility. Tryptic peptides were analyzed by nLC-MS/MS using a nanoACQUITY (Waters Corporation) online coupled with an Orbitrap Velos Pro hybrid ion trap mass spectrometer (Thermo Fisher Scientific). For each nLC-MS/MS analysis, 1 µL of peptides was injected onto a C18 column PicoChip 25 cm column packed with Reprosil C18 3 µm 120 Å chromatography with a 75 µm ID and 15 µm tip (New Objective). Peptides were eluted off to the mass spectrometer with a 66 minute linear gradient of 2-35% ACN/0.1% formic acid at a flow rate of 300 nL/min. The full scan MS spectra were collected over mass range m/z 375-1800 in positive ion mode with an FTMS resolution setting of 60,000 at m/z 400 and AGC target 1,000,000 ms. The top 13 most intense ions were sequentially isolated for collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) in the ion trap with ITMS AGC target 5,000 ms. Dynamic exclusion (90s) was enabled to minimize the redundant selection of peptides previously selected for MS/MS fragmentation. The nLC-MS/MS data were analyzed with MaxQuant software ( Cox and Mann, 2008 ; Tyanova et al., 2016 ), version 1.6.6.0. Briefly, the proteomic features were quantified by high resolution full MS intensities after retention alignment and the corresponding MS/MS spectra were searched with Andromeda search engine against the Uniprot mouse database (release November 2017, 82,555 entries) ( UniProt, 2011 ). The mass tolerance was set at 20 ppm for the precursor ions and 0.8 Da for the ITMS fragment ions. Andromeda search included specific trypsin enzyme with maximum two missed cleavages, minimum of seven amino acids in length. Fixed modification carbamidomethyl (C), and variable modifications of oxidation (M), acetyl (Protein N-term), and deamidation (NQ) were considered. Protein identification threshold was set to 1% false discovery rate (FDR) as described previously ( Cox and Mann, 2008 ). Proteins that exhibit statistically significant abundance between DSG2-BioID2 to control were selected as follows. Proteins with a single peptide identification were excluded from the data analysis and Student’s t-test on log2 transformed protein intensity was used for the statistical inference to select DSG2-BioID2 interacting proteins. A protein was considered a significant candidate if the t-test p-value was 10 when compared to the control. As a surrogate for protein abundance, MaxQuant iBAQ values were used for label-free absolute quantification of identified proteins ( Schwanhausser et al., 2011 ). The average iBAQ value for each protein was determined from the six replicates in both DSG2 and control samples. The final iBAQ value was determined by subtracting the control average from the DSG2 average. Bioinformatics analysis CDH2 BioID proximity proteomics results were previously published from our group ( Li et al., 2019 ). DSP and the combined JUP, PKP2, and DSC2a Ca2+-dependent interactomes were from two previous studies ( Badu-Nkansah and Lechler, 2020 ; Fulle et al., 2024 ). A similar proximity proteomic screen to identify DSG1 interactors in differentiated keratinocytes ( Hegazy et al., 2022 )was excluded because it focused on trafficking proteins. Venn diagrams comparing protein lists were generated using BioVenn ( Hulsen et al., 2008 ). Canonical signaling p( Hulsen et al., 2008 )( Hulsen et al., 2008 )( Hulsen et al., 2008 )athway enrichment analysis was performed using Ingenuity Pathway Analysis (IPA) tools (Qiagen). Protein Network Analysis The protein interaction map was generated using Ingenuity Pathway Analysis (IPA, Qiagen). Only protein-protein interactions supported by published, experimental data in the manually curated Ingenuity Knowledge Base were considered for building the network. Hierarchical classification was done by grouping the proteins manually using DSG2 at the core. Proteins that bind DSG2 directly were designated as primary interactors. Proteins that bind to primary interactors but not DSG2 were classified as secondary interactors. Proteins that bind secondary interactors were designated as tertiary interactors. Finally, proteins that bind tertiary interactors or to outermost tier proteins were defined as quaternary interactors. 39 proteins could not be linked to the protein network. Funding This work was supported by National Institutes of Health R01 HL127711 to AVK. An American Heart Association Predoctoral Fellowship supported SB. Acknowledgments We thank Nathan Yates for his assistance with the mass spectrometry experiments. 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