Defective Desmosomal Adhesion Causes Arrhythmogenic Cardiomyopathy by Involving an Integrin-αVß6/TGF-ß Signaling Cascade.

BACKGROUND: Arrhythmogenic cardiomyopathy (ACM) is characterized by progressive loss of cardiomyocytes with fibrofatty tissue replacement, systolic dysfunction, and life-threatening arrhythmias. A substantial proportion of ACM is caused by mutations in genes of the desmosomal cell-cell adhesion complex, but the underlying mechanisms are not well understood. In the current study, we investigated the relevance of defective desmosomal adhesion for ACM development and progression. METHODS: We mutated the binding site of DSG2 (desmoglein-2), a crucial desmosomal adhesion molecule in cardiomyocytes. This DSG2-W2A mutation abrogates the tryptophan swap, a central interaction mechanism of DSG2 on the basis of structural data. Impaired adhesive function of DSG2-W2A was confirmed by cell-cell dissociation assays and force spectroscopy measurements by atomic force microscopy. The DSG2-W2A knock-in mouse model was analyzed by echocardiography, ECG, and histologic and biomolecular techniques including RNA sequencing and transmission electron and superresolution microscopy. The results were compared with ACM patient samples, and their relevance was confirmed in vivo and in cardiac slice cultures by inhibitor studies applying the small molecule EMD527040 or an inhibitory integrin-αVß6 antibody. RESULTS: The DSG2-W2A mutation impaired binding on molecular level and compromised intercellular adhesive function. Mice bearing this mutation develop a severe cardiac phenotype recalling the characteristics of ACM, including cardiac fibrosis, impaired systolic function, and arrhythmia. A comparison of the transcriptome of mutant mice with ACM patient data suggested deregulated integrin-αVß6 and subsequent transforming growth factor-ß signaling as driver of cardiac fibrosis. Blocking integrin-αVß6 led to reduced expression of profibrotic markers and reduced fibrosis formation in mutant animals in vivo. CONCLUSIONS: We show that disruption of desmosomal adhesion is sufficient to induce a phenotype that fulfils the clinical criteria to establish the diagnosis of ACM, confirming the dysfunctional adhesion hypothesis. Deregulation of integrin-αVß6 and transforming growth factor-ß signaling was identified as a central step toward fibrosis. A pilot in vivo drug test revealed this pathway as a promising target to ameliorate fibrosis. This highlights the value of this model to discern mechanisms of cardiac fibrosis and to identify and test novel treatment options for ACM.

Desmosomal COP9 regulates proteome degradation in arrhythmogenic right ventricular dysplasia/cardiomyopathy.

Dysregulated protein degradative pathways are increasingly recognized as mediators of human disease. This mechanism may have particular relevance to desmosomal proteins that play critical structural roles in both tissue architecture and cell-cell communication, as destabilization/breakdown of the desmosomal proteome is a hallmark of genetic-based desmosomal-targeted diseases, such as the cardiac disease arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C). However, no information exists on whether there are resident proteins that regulate desmosomal proteome homeostasis. Here, we uncovered a cardiac constitutive photomorphogenesis 9 (COP9) desmosomal resident protein complex, composed of subunit 6 of the COP9 signalosome (CSN6), that enzymatically restricted neddylation and targeted desmosomal proteome degradation. CSN6 binding, localization, levels, and function were affected in hearts of classic mouse and human models of ARVD/C affected by desmosomal loss and mutations, respectively. Loss of desmosomal proteome degradation control due to junctional reduction/loss of CSN6 and human desmosomal mutations destabilizing junctional CSN6 were also sufficient to trigger ARVD/C in mice. We identified a desmosomal resident regulatory complex that restricted desmosomal proteome degradation and disease.

Immunosuppression of Macrophages Underlies the Cardioprotective Effects of CST (Catestatin).

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Desmoplakin maintains gap junctions by inhibiting Ras/MAPK and lysosomal degradation of connexin-43.

Desmoplakin (DP) is an obligate component of desmosomes, intercellular adhesive junctions that maintain the integrity of the epidermis and myocardium. Mutations in DP can cause cardiac and cutaneous disease, including arrhythmogenic cardiomyopathy (ACM), an inherited disorder that frequently results in deadly arrhythmias. Conduction defects in ACM are linked to the remodeling and functional interference with Cx43-based gap junctions that electrically and chemically couple cells. How DP loss impairs gap junctions is poorly understood. We show that DP prevents lysosomal-mediated degradation of Cx43. DP loss triggered robust activation of ERK1/2-MAPK and increased phosphorylation of S279/282 of Cx43, which signals clathrin-mediated internalization and subsequent lysosomal degradation of Cx43. RNA sequencing revealed Ras-GTPases as candidates for the aberrant activation of ERK1/2 upon loss of DP. Using a novel Ras inhibitor, Ras/Rap1-specific peptidase (RRSP), or K-Ras knockdown, we demonstrate restoration of Cx43 in DP-deficient cardiomyocytes. Collectively, our results reveal a novel mechanism for the regulation of the Cx43 life cycle by DP in cardiocutaneous models.

Induced Pluripotent Stem Cells for Cardiovascular Disease Modeling and Precision Medicine: A Scientific Statement From the American Heart Association.

Induced pluripotent stem cells (iPSCs) offer an unprece-dented opportunity to study human physiology and disease at the cellular level. They also have the potential to be leveraged in the practice of precision medicine, for example, personalized drug testing. This statement comprehensively describes the provenance of iPSC lines, their use for cardiovascular disease modeling, their use for precision medicine, and strategies through which to promote their wider use for biomedical applications. Human iPSCs exhibit properties that render them uniquely qualified as model systems for studying human diseases: they are of human origin, which means they carry human genomes; they are pluripotent, which means that in principle, they can be differentiated into any of the human body's somatic cell types; and they are stem cells, which means they can be expanded from a single cell into millions or even billions of cell progeny. iPSCs offer the opportunity to study cells that are genetically matched to individual patients, and genome-editing tools allow introduction or correction of genetic variants. Initial progress has been made in using iPSCs to better understand cardiomyopathies, rhythm disorders, valvular and vascular disorders, and metabolic risk factors for ischemic heart disease. This promising work is still in its infancy. Similarly, iPSCs are only just starting to be used to identify the optimal medications to be used in patients from whom the cells were derived. This statement is intended to (1) summarize the state of the science with respect to the use of iPSCs for modeling of cardiovascular traits and disorders and for therapeutic screening; (2) identify opportunities and challenges in the use of iPSCs for disease modeling and precision medicine; and (3) outline strategies that will facilitate the use of iPSCs for biomedical applications. This statement is not intended to address the use of stem cells as regenerative therapy, such as transplantation into the body to treat ischemic heart disease or heart failure.

Connexin defects underlie arrhythmogenic right ventricular cardiomyopathy in a novel mouse model.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) termed a 'disease of the desmosome' is an inherited cardiomyopathy that recently underwent reclassification owing to the identification of left-dominant and biventricular disease forms. Homozygous loss-of-function mutations in the desmosomal component, desmoplakin, are found in patients exhibiting a biventricular form of ARVC; however, no models recapitulate the postnatal hallmarks of the disease as seen in these patients. To gain insights into the homozygous loss-of-function effects of desmoplakin in the heart, we generated cardiomyocyte-specific desmoplakin-deficient mice (DSP-cKO) using ventricular myosin light chain-2-Cre mice. Homozygous DSP-cKO mice are viable but display early ultrastructural defects in desmosomal integrity leading to a cardiomyopathy reminiscent of a biventricular form of ARVC, which includes cell death and fibro-fatty replacement within the ventricle leading to biventricular dysfunction, failure and premature death. DSP-cKO mice also exhibited ventricular arrhythmias that are exacerbated with exercise and catecholamine stimulation. Furthermore, DSP-cKO hearts exhibited right ventricular conduction defects associated with loss of connexin 40 expression and electrical wavefront propagation defects associated with loss of connexin 43 expression. Dose-dependent assessment of the effects of loss of desmoplakin in neonatal ventricular cardiomyocytes revealed primary loss of connexin 43 levels, phosphorylation and function independent of the molecular dissociation of the mechanical junction complex and fibro-fatty manifestation associated with ARVC, suggesting a role for desmoplakin as a primary stabilizer of connexin integrity. In summary, we provide evidence for a novel mouse model, which is reminiscent of the postnatal onset of ARVC while highlighting mechanisms underlying a biventricular form of human ARVC.

Embryonic survival and severity of cardiac and craniofacial defects are affected by genetic background in fibroblast growth factor-16 null mice.

Disruption of the X-chromosome fibroblast growth factor 16 (Fgf-16) gene, a member of the FGF-9 subfamily with FGF-20, was linked with an effect on cardiac development in two independent studies. However, poor trabeculation with lethality by embryonic day (E) 11.5 was associated with only one, involving maintenance in Black Swiss (Bsw) versus C57BL/6 mice. The aim of this study was to examine the potential influence of genetic background through breeding the null mutation onto an alternate (C57BL/6) background. After three generations, 25% of Fgf-16(-/Y) mice survived to adulthood, which could be reversed by reducing the contribution of the C57BL/6 genetic background by back crossing to another strain. There was no significant difference between FGF-9 and FGF-20 RNA levels in Fgf-16 null versus wild-type mice regardless of strain. However, FGF-8 RNA levels were reduced significantly in Bsw but not C57BL/6 mice. FGF-8 is linked to anterior heart development and like the FGF-9 subfamily is reportedly expressed at E10.5. Like FGF-16, neuregulin as well as signaling via ErbB2 and ErbB4 receptors have been linked to trabeculae formation and cardiac development around E10.5. Basal neuregulin, ErbB2, and ErbB4 as well as FGF-8, FGF-9, and FGF-16 RNA levels varied in Bsw versus C57BL/6 mice. These data are consistent with the ability of genetic background to modify the phenotype and affect embryonic survival in Fgf-16 null mice.

Deletion of Shp2 tyrosine phosphatase in muscle leads to dilated cardiomyopathy, insulin resistance, and premature death.

The intracellular signaling mechanisms underlying the pathogenesis of cardiac diseases are not fully understood. We report here that selective deletion of Shp2, an SH2-containing cytoplasmic tyrosine phosphatase, in striated muscle results in severe dilated cardiomyopathy in mice, leading to heart failure and premature mortality. Development of cardiomyopathy in this mouse model is coupled with insulin resistance, glucose intolerance, and impaired glucose uptake in striated muscle cells. Shp2 deficiency leads to upregulation of leukemia inhibitory factor-stimulated phosphatidylinositol 3-kinase/Akt, Erk5, and Stat3 pathways in cardiomyocytes. Insulin resistance and impaired glucose uptake in Shp2-deficient mice are at least in part due to impaired protein kinase C-zeta/lambda and AMP-kinase activities in striated muscle. Thus, we have generated a mouse line modeling human patients suffering from cardiomyopathy and insulin resistance. This study reinforces a concept that a compound disease with multiple cardiovascular and metabolic disturbances can be caused by a defect in a single molecule such as Shp2, which modulates multiple signaling pathways initiated by cytokines and hormones.

Fibroblast growth factor 2 isoforms and cardiac hypertrophy.

Fibroblast growth factor 2 (FGF-2), a multifunctional polypeptide that affects cell growth and differentiation and becomes upregulated by stress, is expressed as AUG-initiated 18 kDa FGF-2 or CUG-initiated 21-34 kDa (hi-FGF-2) isoforms. Animal models have provided strong evidence that FGF-2 is essential for the manifestation of overload- and angiotensin-induced cardiac hypertrophy. Nevertheless, studies to-date have not discriminated between the activities of 18 kDa FGF-2 and hi-FGF-2. Our recent work has pointed to a potent pro-hypertrophic effect of added hi-FGF-2, and a pro-apoptotic effect of sustained intracrine hi-FGF-2 signaling. In the future, it will be important to differentiate between the activities of the different FGF-2 isoforms in the context of adaptive and maladaptive myocardial hypertrophy and heart failure. Based on all available evidence, we propose that while the 18-kDa FGF-2 is a component of an adaptive trophic response, a switch to hi-FGF-2 accumulation would exacerbate hypertrophy and contribute to cell death, thus driving the myocardium towards a maladaptive phenotype.

Biological activities of fibroblast growth factor-2 in the adult myocardium.

Fibroblast growth factor-2 (FGF-2) is a potent regulator of many cellular functions and phenomena, including cell proliferation, differentiation, survival, adhesion, migration, motility and apoptosis, and processes such as limb formation, wound healing, tumorigenesis, angiogenesis, vasculogenesis and blood vessel remodeling. In the adult myocardium, FGF-2 is expressed by various cell types, including cardiomyocytes, fibroblasts and smooth muscle cells. The biological effects of FGF-2 in the myocardium are mediated by the high-affinity tyrosine kinase receptor FGFR-1, the major FGF receptor in the heart. Here, we give an overview of current insights into the multiple roles of FGF-2 in the myocardium, as they pertain to two basic phenomena: ischemia-reperfusion injury and cardiac hypertrophy. The first category includes roles for FGF-2 in cardioprotection, the inflammatory response, angiogenesis and vascular remodeling, while the second includes myocyte hypertrophy, fibrosis, and gap junction functioning (conduction). Given the strong evidence for FGF-2 as both a cardioprotective and angiogenic agent, the therapeutic potential of FGF-2 in the ischemic myocardium is discussed.