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DIAPH1-MFN2 interaction regulates mitochondria-sarcoplasmic reticulum contact and modulates ischemic/hypoxic stress

ABSTRACT: Inter-organelle contact and communication between mitochondria (Mito) and endoplasmic reticulum (ER), also known as sarcoplasmic reticulum (SR) in cardiomyocytes (CM), play vital roles in the maintenance of Mito and SR properties and cellular homeostasis. Here, we tested the hypothesis that the formin, Diaphanous-1 (DIAPH1), which regulates actin dynamics and signal transduction, contributes to these processes. Mice bearing CM-specific deletion of Diaph1 displayed reduced injury and superior functional recovery after the induction of cardiac ischemia and reperfusion (I/R). Studies in DIAPH1-silenced human induced pluripotent stem cell-derived CMs (HiPSC-CMs) and murine hearts devoid of CM-Diaph1 revealed that DIAPH1 interacts directly with Mitofusin-2 (MFN2) to regulate Mito-SR contact, Mito turnover, mitophagy, and oxidative stress. Ischemic murine hearts and human heart biopsies revealed more robust DIAPH1-MFN2 interaction vs. that observed in non-ischemic hearts. Solution structure studies affirm this interaction and reveal strong pH-dependent interaction between the Diaphanous Inhibitory Domain (DID) and the cytosolic GTPase domain of MFN2. In HiPSC-CMs, introduction of synthetic linkers, which reduce the Mito-SR distance, mitigated the cardioprotective benefits of Diaph1 deletion. Finally, DIAPH1 binding to the cytoplasmic domain of receptor for advanced glycation endproduct (RAGE) supports signal transduction and contributes to I/R injury in the heart; in HiPSC-CMs, small molecule antagonism of RAGE-DIAPH1 reduced DIAPH1-MFN2 interaction and increased Mito-SR distance. This work establishes fundamental roles for DIAPH1-MFN2 interaction in the regulation of Mito-SR contact networks that control responses to ischemic stress. Targeting pathways that regulate DIAPH1-MFN2 may facilitate recovery from cardiac ischemic injury.

ORGANISM(S): Homo sapiens

PROVIDER: GSE214149 | GEO | 2022/09/28

REPOSITORIES: GEO

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Project description:ABSTRACT Background: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. Methods and Results: Human iPSC-CMs were infected with a luciferase-expressing mutant of the coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were used to characterize virally infected hiPSC-CMs. Viral proliferation on hiPSC-CMs was subsequently quantified using bioluminescence imaging. For drug screening, select antiviral compounds including interferon beta 1 (IFNβ1), ribavirin, pyrrolidine dithiocarbamate (PDTC), and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of some of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with the reported drug effects in previous studies. Finally, mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways within these hiPSC-CMs after IFNβ1 treatment. Conclusions: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to confirm antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that could be used to screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion. For this experiment, human induced pluripotent stem cell derived cardiomyocytes were infected with coxsackievirus at multiplicity of infection (MOI) of 5 for 8 hours. Cells were treated with and without interferon beta 1 in order to determine if treatment activates antiviral response genes and/or viral clearance pathways. 4 total samples (2 for each condition) were analyzed

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DIAPH1-MFN2 interaction regulates mitochondria-sarcoplasmic reticulum contact and modulates ischemic/hypoxic stress

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