Cardiomyocyte adhesion and hyper-adhesion differentially require ERK1/2 and plakoglobin Maria Shoykhet, … , Sunil Yeruva, Jens Waschke JCI Insight. 2020. https://doi.org/10.1172/jci.insight.140066. Research In-Press Preview Cardiology Graphical abstract Find the latest version: https://jci.me/140066/pdf 1 Research Article 2 Cardiomyocyte adhesion and hyper-adhesion differentially require ERK1/2 and 3 plakoglobin. 4 Maria Shoykhet1, Sebastian Trenz1, Ellen Kempf1, Tatjana Williams2, Brenda Gerull2, 5 Camilla Schinner1+, Sunil Yeruva§1 and Jens Waschke§1 6 1Faculty of Medicine, Ludwig-Maximilians-University (LMU) Munich, Munich, Germany 7 2Comprehensive Heart Failure Center and Department of Internal Medicine I, University 8 Hospital Würzburg, Würzburg, Germany 9 + Current address: Department of Biomedicine, University of Basel, Basel, Switzerland 10 §Corresponding authors: 11 Sunil Yeruva and Jens Waschke, Institute of Anatomy and Cell Biology, Department I, 12 Ludwig-Maximilians-University, Pettenkoferstraße 11, 80336 Munich, Germany; Phone: 13 +49-89-2180-72610; Fax: +49-89-2180-72602; [email protected], 14 [email protected] 15 16 Conflict of Interest 17 The authors declare that no conflict of interest exists. 18 1 19 Abstract: 20 Arrhythmogenic cardiomyopathy (AC) is a heart disease often caused by mutations in genes 21 coding for desmosomal proteins including desmoglein-2 (DSG2), plakoglobin (PG), and 22 desmoplakin (DP). Therapy is symptomatic to limit arrhythmia since the mechanisms by 23 which desmosomal components control cardiomyocyte function are largely unknown. A new 24 paradigm would be to stabilize desmosomal cardiomyocyte adhesion and hyper-adhesion, 25 which renders desmosomal adhesion independent from Ca2+. Here, we further characterized 26 the mechanisms behind enhanced cardiomyocyte adhesion and hyper-adhesion. Dissociation 27 assays performed in HL-1 cells and murine ventricular cardiac slice cultures allowed us to 28 define a set of signaling pathways regulating cardiomyocyte adhesion under basal and hyper- 29 adhesive conditions. Adrenergic signaling, activation of PKC and inhibition of p38MAPK 30 enhanced cardiomyocyte adhesion, referred to as positive adhesiotropy, and induced hyper- 31 adhesion. Activation of ERK1/2 paralleled positive adhesiotropy, whereas adrenergic 32 signaling induced Pg phosphorylation at S665 under both basal and hyper-adhesive 33 conditions. Adrenergic signaling and p38MAPK inhibition recruited DSG2 to cell junctions. 34 In PG-deficient mice with an AC phenotype, only PKC activation and p38MAPK inhibition 35 enhanced cardiomyocyte adhesion. Our results demonstrate that cardiomyocyte adhesion can 36 be stabilized by different signaling mechanisms, which are in part off-set in PG-deficient AC. 37 2 38 1. Introduction 39 In the heart, cardiomyocytes are coupled via intercalated disks (ICD) consisting of 40 desmosomes, adherens junctions (AJ) and gap junctions (GJ), which together allow 41 mechanical stability and electrical conduction of the heart muscle (1). Cardiomyocytes 42 express desmosomal cadherins desmoglein-2 (DSG2) and desmocollin-2 (DSC2) and the AJ 43 protein N-Cadherin (N-CAD) (2). The cytosolic part of these adhesion molecules connects to 44 the cytoskeletal network via the plaque proteins plakoglobin (PG), plakophilin (PKP), and 45 desmoplakin (DP) in desmosomes or to β-catenin and other plaque proteins in AJs, 46 respectively. Adjacent cardiomyocytes attach via DSG2, DSC2, and N-CAD, which bind to 47 their respective partners extracellularly in a Ca2+-dependent manner (3). Cardiomyocyte 48 function markedly relies on the proper function of desmosomal and AJ proteins since 49 mutations in these genes lead to an arrhythmic heart disorder called arrhythmogenic 50 cardiomyopathy (AC) (4, 5). The progressive loss of cardiomyocytes with cardiac fibrosis, 51 ventricular dilatation, and arrhythmia accompanied by a high risk for sudden cardiac death, 52 especially in young athletes, are the characteristics of AC. The mechanisms by which 53 desmosomal components serve heart function and how cardiomyocyte adhesion is regulated 54 in pathological conditions are poorly understood. Thus, therapy of AC is symptomatic and 55 focused on limiting arrhythmia in patients. In this context, desmosomal components are 56 thought to regulate conduction of excitation via GJ and the cardiac sodium channel complex 57 allowing ephaptic conduction (6, 7). Therefore, characterization of mechanisms regulating 58 desmosomal adhesion can help to establish new experimental approaches to treat desmosomal 59 diseases, as has been shown for pemphigus (1), a disease caused by autoantibodies primarily 60 against DSG1 and DSG3, leading to severe blistering of the skin and mucosa (8, 9). For 61 pemphigus, the mechanisms controlling keratinocyte adhesion have been studied extensively 62 and were found to focus on p38MAPK, PKC, ERK1/2, and cAMP-signaling (10-12), whereas 3 63 others, such as SRC appear not to be central for the disease (13). The context of pemphigus is 64 relevant for AC for two aspects: Firstly, AC is known to occur in syndromes, also affecting 65 the skin and its appendices when specific mutations in genes coding for PG or DP are 66 causative (14-16). Secondly, most recently, it has been proposed that different mutations in 67 desmosomal genes causing AC can induce the formation of autoantibodies against 68 cardiomyocyte antigens, including DSG2, which are pathogenic and thus may aggravate the 69 disease (17, 18). This would imply that the mechanisms causing AC and pemphigus may be at 70 least in part comparable. Therefore, in order to establish new therapeutic approaches for AC, 71 it is pivotal to characterize the molecular mechanisms regulating cardiomyocyte adhesion in 72 detail. Such therapeutic approaches might focus on enhancing cardiomyocyte adhesion and 73 thus prevent fibro-fatty replacement with the subsequent occurrence of arrhythmias. 74 Although desmosomal cadherins depend on the presence of extracellular Ca2+, it was shown 75 that in keratinocytes, desmosomes acquire a Ca2+-independent state, referred to as hyper- 76 adhesion, and which was proposed to be the primary adhesive state of desmosomes in intact 77 epidermis (19-21). This phenomenon was not explored further in other tissues. Indeed, in a 78 previous study, we found that in cardiomyocytes, adrenergic signaling leads to the 79 enhancement of basal cellular adhesion, which we referred to as positive adhesiotropy, and 80 also causes hyper-adhesion (22). Since cardiac muscle contractions depend on Ca2+, which is 81 tightly regulated intra- and extracellularly, it is possible that a hyper-adhesive state similar to 82 keratinocytes exists in the physiologic state of desmosomes in cardiomyocytes. Thus, the 83 signaling mechanisms regulating hyper-adhesion in cardiomyocytes need to be investigated. 84 In multiple heart diseases including myocardial dysfunction, diabetic cardiomyopathy, 85 hypertrophy, fibrosis, and heart failure, MAPK signaling pathways, such as ERK1/2, JNK, 86 and p38MAPK, are demonstrated to be involved in disease pathogenesis (23-25). Besides, 87 PKC has been shown to be important in the regulation of GJs in cardiomyocytes (26, 27). 88 Moreover, in previous studies, we found that activation of PKC (28) and adrenergic signaling 4 89 enhance cardiomyocyte adhesion, the latter of which is mediated through PKA pathway 90 induced PG phosphorylation at S665 (22). 91 In this study, we investigated the signaling pathways that enhance cardiomyocyte adhesion 92 and may induce hyper-adhesion of desmosomal contacts. Our results show that enhancement 93 of cAMP levels by adrenergic signaling, activation of PKC as well as inhibition of p38MAPK 94 enhance basal cardiomyocyte adhesion and cause hyper-adhesion. ERK1/2 was found to be 95 critical to enhance basal cardiomyocyte adhesion, i.e., for positive adhesiotropy, which was 96 dependent on PG, DP, and DSG2 in cultured cardiomyocytes. In ventricular cardiac slices 97 obtained from PG-deficient mice with an AC phenotype, adrenergic signaling failed to 98 enhance cardiomyocyte adhesion, whereas PKC activation and p38MAPK inhibition were 99 still effective. On the other hand, ERK1/2 activation did not play a role in hyper-adhesion 100 caused by the above signaling pathways, indicating that the mechanisms to enhance 101 cardiomyocyte adhesion at least in part are different between basal and hyper-adhesive 102 conditions. 5 103 2. Results 104 2.1.Adrenergic signaling, PKC and p38MAPK regulate cardiomyocyte adhesion in 105 HL-1 cells and murine ventricular cardiac slices 106 In keratinocytes, a Ca2+-independent desmosomal adhesion, which is called hyper-adhesion, 107 has been identified (19-21). In a previous study, we observed a similar phenomenon in 108 cultured HL-1 cardiomyocytes (22). In this study, we first determined the Ca2+-dependency of 109 cardiomyocyte adhesion using dissociation assays. In dissociation assays cells are treated with 110 dispase and collagenase to detach cell monolayers from the well bottoms and a standardized 111 mechanical stress is applied to the monolayers. The resulting fragmentation serves as an 112 indirect measurement of cell-cell cohesion; a higher amount of fragmentation indicates a 113 weaker cell-cell cohesion and vice versa. However, it has to be noted that the assay measures 114 overall cell cohesion and cannot discriminate between adhesion provided by desmosomal 115 cadherins and classical cadherins