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Mechanisms of Innate Immune Injury in Arrhythmogenic Cardiomyopathy

ABSTRACT: Introduction: We have reported that iPSC-cardiac myocytes from patients with arrhythmogenic cardiomyopathy (ACM) mount an intense innate immune response under basal conditions in vitro. In addition, blocking innate immune signaling via NFκB prevents disease in a mouse model of ACM (Dsg2mut/mut mice). Here, we defined the relative pathogenic roles of immune signaling in cardiac myocytes vs. infiltrating inflammatory cells in Dsg2mut/mut mice. Methods: To define the role of immune signaling in cardiac myocytes, we bred Dsg2mut/mut mice with mice with cardiac myocyte-specific expression of a dominant-negative form of IκBα (DN-IκBα), which prevents nuclear translocation and, thereby, activation of NFκB signaling only in cardiac myocytes. To define the role of inflammatory cells in ACM, we focused on cells expressing C-C motif chemokine receptor-2 (CCR2+ cells), known to mediate adverse cardiac remodeling and fibrosis. We bred Dsg2mut/mut mice with germline deletion of Ccr2 mice (Ccr2-/- mice). We compared phenotypes in Dsg2mut/mut mice with double-mutant Dsg2mut/mut X DN-IκB and Dsg2mut/mut X Ccr2-/- lines. Results: Dsg2mut/mut mice develop marked myocardial fibrosis, reduced LV ejection fraction (EF) and many PVCs. These disease features were all normalized in Dsg2mut/mut X DN-IκB mice. Hearts of Dsg2mut/mut and Dsg2mut/mut X DN-IκB mice contained equal numbers of CD68+ macrophages, but Dsg2mut/mut hearts had many more CCR2+ cells. Cardiac myocyte expression of Tlr4 which activates NFκB, was increased in Dsg2mut/mut mice but not in Dsg2mut/mut X Ccr2-/- mice. Dsg2mut/mut X Ccr2-/- mice showed greatly reduced myocardial fibrosis and PVCs but LV EF was markedly depressed. This suggested that contractile dysfunction in ACM is due not only to loss of muscle but to immune signaling in viable cardiac myocytes as well. To test this hypothesis, we treated Dsg2mut/mut and Dsg2mut/mut X Ccr2-/- mice with the NFκB inhibitor Bay 11-7082. Before treatment, LV EFs were significantly reduced in both groups. Untreated Dsg2mut/mut mice had ongoing deterioration of LV function, whereas treated Dsg2mut/mut mice showed no further disease progression and actually had some improvement in contractile function. However, LV function in treated Dsg2mut/mut X Ccr2-/- mice was virtually normalized. Conclusions: NFκB signaling in cardiac myocytes drives myocardial injury, LV dysfunction and arrhythmias in Dsg2mut/mut mice. It also mobilizes CCR2+ cells to the heart, where they promote myocardial injury and arrhythmias. CCR2+ cells alter gene expression in cardiac myocytes. LV dysfunction in Dsg2mut/mut mice is caused not only by muscle loss but also by negative inotropic effects of inflammation mediated by NFκB in viable cardiac myocytes. These results have obvious implications for ACM patients and suggest that anti-inflammatory therapy may be beneficial even in patients with established disease.

ORGANISM(S): Mus musculus

PROVIDER: GSE228048 | GEO | 2024/02/28

REPOSITORIES: GEO

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Project description:Background: Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disorder, characterized by cardiomyocyte hypertrophy, cardiomyocyte disarray and fibrosis, which has a prevalence of ~1:200-500 and predisposes individuals to sudden death and heart failure. The mechanisms through which diverse HCM-causing mutations cause cardiac dysfunction remain mostly unknown and their identification may reveal new therapeutic avenues. MicroRNAs have emerged as critical regulators of gene expression and disease phenotype in various pathologies. We explored whether miRNAs could play a role in HCM pathogenesis and offer potential therapeutic targets. Methods and Results: Using high-throughput miRNA expression profiling and qPCR analysis in two distinct mouse models of HCM, we found that miR-199a-3p expression levels are upregulated in mutant mice compared to age- and treatment-matched wild-type mice. We also found that miR-199a-3p expression is enriched in cardiac non-myocytes compared to cardiomyocytes. When we expressed miR-199a-3p mimic in cultured primary cardiac non-myocytes and analyzed the conditioned media by proteomics, we found that several ECM proteins (e.g., TSP2, FBLN3, COL11A1, LYOX) were differentially expressed. We confirmed our proteomics findings by qPCR analysis of selected mRNAs and demonstrated that miR-199a-3p mimic expression in cardiac non-myocytes drives upregulation of ECM genes including Tsp2, Fbln3, Pcoc1, Col1a1 and Col3a1. To examine the role of miR-199a-3p in vivo, we inhibited its function using lock-nucleic acid (LNA)-based inhibitors (antimiR-199a-3p) in an HCM mouse model. Our results revealed that progression of cardiac fibrosis is attenuated when miR-199a-3p function is inhibited in mild-to-moderate HCM. Finally, guided by computational target prediction algorithms, we identified mRNAs Cd151 and Itga3 as direct targets of miR-199a-3p and have shown that miR-199a-3p mimic expression negatively regulates AKT activation in cardiac non-myocytes. Conclusions: Altogether, our results suggest that miR-199a-3p may contribute to cardiac fibrosis in HCM through its actions in cardiac non-myocytes. Thus, inhibition of miR-199a-3p in mild-to-moderate HCM may offer therapeutic benefit in combination with complementary approaches that target the primary defect in cardiac myocytes.

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Mechanisms of Innate Immune Injury in Arrhythmogenic Cardiomyopathy

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