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Cardiac fibroblasts (CFs) are an attractive target for reducing pathological cardiac remodeling, and understanding the underlying mechanisms of these processes is the key to develop successful therapies for treating the pressure‐overloaded heart. CF‐specific knockout (KO) mouse lines with a Cre recombinase under the control of human TCF21 (hTCF21) promoter and/or an adeno‐associated virus serotype 9 (AAV9)‐hTCF21 system provide a powerful tool for understanding CF biology in vivo. Although a variety of rat disease models are vital for the research of cardiac fibrosis similar to mouse models, there are few rat models that employ cardiac cell‐specific conditional gene modification, which has hindered the development and translational relevance of cardiac disease models. In addition, to date, there are no reports of gene manipulation specifically in rat CFs in vivo. Here, we report a simplified CF‐specific rat transgenic model using an AAV9‐hTCF21 system that achieved a CF‐specific expression of transgene in adult rat hearts. Moreover, we successfully applied this approach to specifically manipulate mitochondrial morphology in quiescent CFs. In summary, this model will allow us to develop fast and simple rat CF‐specific transgenic models for studying cardiovascular diseases in vivo. [ABSTRACT FROM AUTHOR]

The cover image is based on the Short Report Adeno‐associated virus‐based approach for genetic modification of cardiac fibroblasts in adult rat hearts by Bridget Nieto et al., https://doi.org/10.14814/phy2.15989 [ABSTRACT FROM AUTHOR]

R4128 --> 732.9 --> Introduction: The Ca2+‐activated Cl‐ channel Anoctamin‐1 (Ano1) regulates multiple cell functions including cell proliferation, survival, and migration. We previously reported that overexpression of Ano1 is associated with hyperproliferation of pulmonary artery endothelial cells isolated from patients with idiopathic pulmonary arterial hypertension. We also showed that Ano1 is expressed not only in the plasma membrane (PM), but also in the mitochondria. However, the physiological and pathological roles of mitochondrial Ano1 have not been fully investigated. Aim: To investigate the role of mitochondria‐localized Ano1 on the regulation of mitochondrial dynamics and reactive oxygen species (ROS) generation. Methods: Stable overexpression and knockdown of Ano1 were employed in HEK293T cells and mouse embryonic fibroblasts (MEFs), respectively. Cells were used for biochemical and cell biological assays. Results: First, mitochondrial localization of Ano1 in addition to the PM was confirmed by live cell imaging of HEK293T cells expressing GFP‐tagged Ano1 and immunostaining of endogenous Ano1 in MEFs. Next, a co‐immunoprecipitation assay showed that overexpressed Ano1 in HEK293T cells was associated with optic atrophy 1 (OPA1) which is an inner mitochondrial membrane (IMM) protein and a critical regulator of mitochondrial fusion, cristae formation, and bioenergetics. We also confirmed the interaction between endogenous Ano1 and OPA1 using wild‐type and OPA1‐knockout MEFs. Overexpression of wild‐type Ano1 in HEK293T cells facilitated proliferation, but a pore‐dead mutant (Ano1‐R621E) did not. Importantly, Ano1 overexpressing HEK293T cells have higher cell death rates in response to oxidative stress compared to control cells. To further understand the role of endogenous Ano1 expression on mitochondrial functions, we next employed MEFs stably overexpressing shRNA targeting Ano1. Quantifying mitochondrial morphology from live MEFs expressing mitochondria‐matrix targeted GFP revealed that Ano1‐knockdown (Ano1‐KD) MEFs contained more elongated mitochondria compared to cells stably expressing control shRNA. Importantly, we also found higher expression of mitofusin 2 and long‐form OPA1 in Ano1‐KD MEFs compared to control cells. The expression levels of other fission/fusion proteins were not altered by Ano1‐KD. Lastly, significantly lower levels of mitochondrial superoxide in Ano1‐KD cells and a tendency towards lower cellular oxidative levels in Ano1‐KD cells compared to controls were observed by live cell imaging with mitochondrial superoxide‐sensitive dye MitoSOX Red and protein carbonyl quantification in the whole cell lysates, respectively. Summary: Ano1, expressed in both the IMM and the PM, is involved in the maintenance of mitochondrial morphology and ROS, possibly by modulating fusion protein expression levels. Future studies will include the development of genetically engineered mitochondria‐targeted Ano1 to more precisely dissect the roles of IMM‐localized Ano1 in mitochondrial functions. [ABSTRACT FROM AUTHOR]

R4149 --> 567.1 --> Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) primarily targets the respiratory system. However, direct SARS‐CoV‐2 infection and viral protein expression have also been reported in other organs, which potentially contributes to multi‐organ dysfunction and increased mortality in COVID‐19. Since the original Wuhan‐type genome was sequenced, several genetic variants of SARS‐CoV‐2 have emerged with differing pathophysiological properties such as the levels of transmissibility, disease severity, and mortality. We previously reported that the protein encoded by open reading frame 3a (ORF3a), a critical protein for SARS‐CoV‐2 replication and release, is found in the mitochondria of host cells and could increase oxidative stress and apoptotic signaling. ORF3a‐Q57H is a highly recurrent variation and the most commonly found variant of ORF3a. Interestingly, the Q57H variant is associated with increased transmissibility, but lower mortality. Aim: To investigate the impact of the ORF3a‐Q57H variant on host cell damage. Methods: Plasmids carrying ORF3a‐Q57H were generated by PCR‐based site mutagenesis using Wuhan‐type ORF3a (ORF3a‐WT) as a template. Whole cell lysates were prepared from HEK293T cells and H9c2 cardiac myoblasts expressing ORF3a‐WT or the mutant ORF3a‐Q57H and used for biochemical assays. Live cell imaging for assessing subcellular localization of ORF3a/ORF3a‐Q57H, mitochondrial reactive oxygen species (mROS), and caspase 3 activity in H9c2 cells was performed by confocal microscopy. Results: In whole cell lysates, we found that ORF3a‐Q57H exhibits significantly higher protein expression compared to ORF3a‐WT. However, there is no significant difference in the ability of mitochondrial trafficking between ORF3a‐WT and ORF3a‐Q57H assessed by live cell imaging using GFP‐tagged ORF3a/ORF3a‐Q57H with mitochondria‐targeted DsRed. Next, we investigated the effects of ORF3a expression on apoptotic and mitophagy signaling by quantifying the caspase 3 activity and LC3A/B ratio. We found that ORF3a‐Q57H has significantly lower apoptosis and mitophagy signaling compared to WT despite its higher protein expression levels. ER‐stress signaling was not activated in either ORF3a‐WT or ORF3a‐Q57H, as assessed by markers including Glucose‐regulated protein 78/94 and C/EBP‐homologous protein. Lastly, live cell imaging using the mitochondrial superoxide‐sensitive dye Mitosox Red revealed that ORF3a‐WT significantly increases mROS levels, but ORF3a‐Q57H expression does not. Conclusion: SARS‐CoV‐2‐ORF3a‐Q57H causes lower oxidative stress and cell damage compared to the Wuhan‐type variant, which could help explain the decreased mortality associated with the ORF3a‐Q57H variant. These results provide novel insights on how genetic variations of SARS‐CoV‐2 influence the pathophysiology and clinical severity of COVID‐19. [ABSTRACT FROM AUTHOR]

R4274 --> Anoctamin‐1 (Ano1) forms a Ca2+‐activated chloride channel found at the plasma membrane (PM) of several different cell types, including endothelial cells (ECs). We previously showed Ano1 overexpression in pulmonary artery ECs isolated from patients with idiopathic pulmonary arterial hypertension (PAH), which may be associated with hyperproliferative phenotype of pulmonary artery ECs in PAH. However, the detailed molecular mechanisms explaining how Ano1 modulates proliferative signaling are still not clear. In addition, recent proteomics studies showed that Ano1 may interact with inner mitochondrial membrane (IMM) proteins in addition to plasma membrane (PM)‐localized proteins. However, the expression levels of Ano1 and its functions in other cellular compartments including mitochondria are not fully understood. Here, we report that Ano1 is expressed in the mitochondria in pulmonary artery ECs and may be involved in EC proliferation by interacting with an IMM protein, optic atrophy 1 (OPA1) which is a critical regulator of mitochondrial bioenergetics and cristae formation. First, mitochondrial localization of Ano1 in addition to PM was found by protein fractionation and immunostaining of Ano1 in rat lung microvascular ECs (RLMVECs). We also confirmed mitochondrial Ano1 expression by live cell imaging of HEK293T cells expressing GFP‐tagged Ano1. Next, a co‐immunoprecipitation assay showed that overexpressed Ano1 interacts with OPA1 in HEK293T cells, indicating that Ano1 is expressed at IMM. We also confirmed that endogenous Ano1 interacts with OPA1 in mouse embryonic fibroblasts. Overexpression of Ano1 in HEK293T cells showed higher proliferation rate compared to controls (cells stably expressing GFP) at rest, but showed higher cell death rates in response to oxidative stress compared to control compared to control cells. Lastly, we found that treatment of a small molecule Ano1 activator Eact promotes apoptosis in pulmonary artery ECs from patients with idiopathic PAH, which likely occurs via depolarization of mitochondrial membrane potential by opening of mitochondrial Ano1. In summary, we found that 1) Ano1 is expressed in both the IMM and the PM, 2) Ano1 and OPA1 physically interact at IMM, 3) overexpression of Ano1 does increase proliferation, but Ano1 activation promotes cellular apoptosis. Interactions between Ano1 and OPA1 may provide a molecular mechanism underlying the hyperproliferation of pulmonary artery ECs during PAH. In addition, Ano1 activation may serve as a new therapeutic tool in PAH associated with hyperproliferative ECs. [ABSTRACT FROM AUTHOR]