Exercise triggers CAPN1-mediated AIF truncation, inducing myocyte cell death in arrhythmogenic cardiomyopathy

Abstract

Myocyte death occurs in many inherited and acquired cardiomyopathies, including arrhythmogenic cardiomyopathy (ACM), a genetic heart disease plagued by the prevalence of sudden cardiac death. Individuals with ACM and harboring pathogenic desmosomal variants, such as desmoglein-2 (DSG2), often show myocyte necrosis with progression to exercise-associated heart failure. Here, we showed that homozygous Dsg2 mutant mice (Dsg2mut/mut), a model of ACM, die prematurely during swimming and display myocardial dysfunction and necrosis. We detected calcium (Ca2+) overload in Dsg2mut/mut hearts, which induced calpain-1 (CAPN1) activation, association of CAPN1 with mitochondria, and CAPN1-induced cleavage of mitochondrial-bound apoptosis-inducing factor (AIF). Cleaved AIF translocated to the myocyte nucleus triggering large-scale DNA fragmentation and cell death, an effect potentiated by mitochondrial-driven AIF oxidation. Posttranslational oxidation of AIF cysteine residues was due, in part, to a depleted mitochondrial thioredoxin-2 redox system. Hearts from exercised Dsg2mut/mut mice were depleted of calpastatin (CAST), an endogenous CAPN1 inhibitor, and overexpressing CAST in myocytes protected against Ca2+ overload–induced necrosis. When cardiomyocytes differentiated from Dsg2mut/mut embryonic stem cells (ES-CMs) were challenged with β-adrenergic stimulation, CAPN1 inhibition attenuated CAPN1-induced AIF truncation. In addition, pretreatment of Dsg2mut/mut ES-CMs with an AIF-mimetic peptide, mirroring the cyclophilin-A (PPIA) binding site of AIF, blocked PPIA-mediated AIF-nuclear translocation, and reduced both apoptosis and necrosis. Thus, preventing CAPN1-induced AIF-truncation or barring binding of AIF to the nuclear chaperone, PPIA, may avert myocyte death and, ultimately, disease progression to heart failure in ACM and likely other forms of cardiomyopathies.

Altered Electrical, Biomolecular, and Immunologic Phenotypes in a Novel Patient-Derived Stem Cell Model of Desmoglein-2 Mutant ARVC

“…We identified eight cytokines in DSG2 Mut hiPSC-CMs that are involved in the innate immune response and complement component system that were upregulated in DSG2 Mut hiPSC-CM lysates (Figure 4A-C,H). Included among these was the receptor for advanced glycation end products (RAGE), which binds to nuclear highmobility group box-1 (HMGB1) to promote cell death [44]; this is consistent with our recent work demonstrating that HMGB1-nuclear loss leads to myocyte cell necroptosis in Dsg2-mutant mice [14]. We identified numerous upregulated pleiotropic cytokines and chemokines involved in mobilizing and recruiting immune cells (Figure 4D,E), and members of the potent, proinflammatory interleukin (IL)-family (Figure 4F,G).…”

“…Considering the numerous chemotactic cytokines and inflammation-associated gene transcripts upregulated in purified DSG2 Mut hiPSC-CM cultures when compared to wild-type cells, this intrinsic pathway may be the driving force for immune cell recruitment to ARVC hearts. Specifically, our RNA-Seq data supports a causative role for canonical NFκB signaling through an IRAK-TAB/TAK-RELA pathway and non-canonical NFκB signaling through a TRAF-IKKa (CHUK) -RELB pathway [ 14 ]. More broadly, the cytokine profiles in these DSG2 Mut cells indicates a global upregulation of inflammation machinery and immune cell recruitment.…”

“…It is not yet known whether these inflammatory periods are the result of extrinsic factors (e.g., immune cell infiltration) or intrinsic CM dysregulation (e.g., NFĸB-mediated cytokine secretion from CMs), but they likely involve both. Specifically, prior reports show infiltration of immune cells in hearts of animal models of ARVC [ 14 , 15 ] and patients with ARVC [ 58 ], indicating that extrinsic inflammatory pathways are activated. Yet, our cytokine data in DSG2 Mut hiPSC-CMs largely corroborates prior reports in PKP2-mutant hiPSC-CMs [ 15 ] and clearly demonstrate an intrinsic, NFĸB-mediated inflammatory pathway that is dysregulated in hiPSC-CMs, independent of nonmyocyte infiltration.…”

“…And while patients with advanced ARVC develop severe myocardial scarring and cardiomyocyte loss, which can contribute to arrhythmic risk, substantial evidence demonstrates that cardiac electrical abnormalities and elevated risk of dangerous arrhythmias—develop well before any overt structural deterioration can be detected [ 11 , 12 , 13 ]. Several animal models of ARVC have been established, including in mice [ 14 , 15 , 16 , 17 ] and dogs [ 18 , 19 ], which have proven useful in studying specific pathophysiologic processes, but these models are limited due to substantial differences in cardiac electrophysiology between animals and humans [ 20 ]. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) present a powerful model for studying cardiac disease, and ARVC patient-specific hiPSC-CMs have been shown to recapitulate key characteristics observed in human disease [ 21 ].…”

Desmosomes: emerging pathways and non-canonical functions in cardiac arrhythmias and disease

“…2) (Kim et al 2019). Interestingly, recent studies have drawn a connection between the desmosomal protein, DSG2, and mitochondrial-mediated calcium overload in ARVC in exercise settings via the calpain-1 pathway and shown that its inhibition can circumvent cardiomyocyte death by interfering with cleavage of apoptosis inducing factor (Chelko et al 2021). These latter studies reveal the possibility that mitochondrial calcium overload induced cell death may generate arrhythmogenic substrates and further contribute to arrhythmias.…”