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Intermittent fasting (IF) extends life span via pleotropic mechanisms, but one important molecular mediator is adenosine monophosphate–activated protein kinase (AMPK). AMPK enhances lipid metabolism and modulates microtubule dynamics. Dysregulation of these molecular pathways causes right ventricular (RV) failure in patients with pulmonary arterial hypertension. In rodent pulmonary arterial hypertension, IF activates RV AMPK, which restores mitochondrial and peroxisomal morphology and restructures mitochondrial and peroxisomal lipid metabolism protein regulation. In addition, IF increases electron transport chain protein abundance and activity in the right ventricle. Echocardiographic and hemodynamic measures of RV function are positively associated with fatty acid oxidation and electron transport chain protein levels. IF also combats heightened microtubule density, which normalizes transverse tubule structure.

Introduction: Mechanical stress (e.g., pressure overload), cytokines, growth factors, and neurohumoral mediators stimulate cardiac fibroblast (CF) activation, which promotes extracellular matrix (ECM) protein synthesis leading to cardiac fibrosis and diastolic dysfunction. Therefore, CFs are an attractive target for reducing pathological cardiac remodeling, and understanding the underlying mechanisms of these processes is key to developing 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 provide a powerful tool for understanding CF biology in vivo in addition to the periostin promoter-driven myofibroblast-specific KO system. In addition to these transgenic mouse lines, one group recently reported the overexpression of Yes-associated protein (YAP) in CFs using an adeno-associated virus (AAV)-based system (Francisco J et al., Sci Rep. 2021). Aim: To test the feasibility of CF-selective gene expression and deletion in vivo using an AAV-based approach. Methods: The pAAV-hTCF21 promoter vector was generated by cloning the hTCF21 promoter fragment into a mammalian gene expression AAV vector (Vectorbuilder). AAV vectors were packaged into AAV serotype 9 (AAV9) and the viral particles were introduced into adult rats and mice via tail vein and retro-orbital injections, respectively. Gene expression or deletion in adult rats and mice were assessed 5-6-weeks after AAV9 injection. Results: First, tissue-specific gene delivery was evaluated in rats by injecting AAV9-hTCF21-GFP. GFP mRNA expression was highest in liver and heart tissues compared to other organs assessed by real-time qPCR. CF-specific GFP expression was confirmed by co-immunostaining ventricular tissue sections with antibodies against GFP and the CF marker vimentin. GFP was not detectable in cardiomyocytes, another major cell type in the heart. Next, we injected AAV9-hTCF21-iCre and -luciferase (as a control) into the mice in which critical exons of the protein kinase D1 (PKD1) gene are flanked with loxP sites (PKD1 fl/fl mice). PKD expression levels in CFs were quantified by co-immunostaining ventricular sections with PKD and vimentin antibodies. PKD was detectable in both CFs and cardiomyocytes in control hearts from AAV9-hTCF21-luciferase-injected PKD1 fl/fl mice. In contrast, significant decrease in PKD expression was observed only in CFs, but not in the cardiomyocyte area after AAV9-hTCF21-iCre injection. Conclusion: AAV9-mediated hTCF21-driven gene expression and deletion in CFs in vivo may be a powerful experimental platform in addition to the generation of CF-specific conditional KO mouse lines. R01HL136757 (to J.O.-U), AHA 18CDA34110091 (to B.S.J) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Introduction: Direct infection and viral protein expression of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the respiratory system, and other organs including the heart, contribute to multi-organ dysfunction during COVID-19. We previously showed that the protein encoded by the open reading frame 3a (ORF3a) gene of SARS-CoV-2, which is critical for viral replication, can be localized in the mitochondria and potentially increase oxidative stress and apoptotic signaling. Importantly, a clinical report suggests that one of the ORF3a variants, ORF3a-Q57H, is associated with increased transmission, but lower patient mortality. Computational structural modeling predicts that the Q57H variant destabilizes the ORF3a protein structure. Hypothesis: The SARS-CoV-2-ORF3a-Q57H variant produces less protein expression in host cells compared to the original Wuhan-type (WT) virus due to its unstable structure, thus exhibits less oxidative stress and apoptosis, which may contribute to decreased mortality. Aim: To investigate the impact of ORF3a-Q57H on apoptotic signaling cascades. Methods: Wuhan-type (WT) ORF3a, mutant ORF3a-Q57H, or their control vector were transiently introduced in HEK293T cells and H9c2 cardiac myoblasts, which were used for biochemical assays and live cell imaging by confocal microscopy. Results: Contrary to computational predictions, we found that ORF3a-Q57H exhibited relatively higher protein expression in our cell lines compared to WT ORF3a. Caspase-8, which is mainly regulated by extrinsic pathways, was significantly activated by WT, but not by Q57H ORF3a, assessed by live cell imaging using a fluorescent marker, caspase-8 inhibitor IETD-FMK conjugated to sulfo-rhodamine. Caspase-9, which is mainly regulated by intrinsic and mitochondrial pathways, was activated in both WT- and Q57H-transfected cells, measured by a fluorescent marker, caspase-9 inhibitor LEHD-FMK conjugated to sulfo-rhodamine. Q57H showed equal ability for mitochondrial trafficking as the WT, assessed by the subcellular localization of GFP-tagged WT- and Q57H constructs. Lastly, caspase-3 activity, downstream of both intrinsic and extrinsic apoptotic pathways, was significantly increased in WT compared to Q57H ORF3a, quantified by the number of positively-stained nuclei with a fluorogenic DNA dye coupled to the caspase-3/7 recognition sequence. Conclusion: The SARS-CoV-2-ORF3a-Q57H variant exhibits higher protein expression compared to WT, but induces less apoptosis in host cells due to the lack of extrinsic apoptotic pathway activation. The relatively mild phenotype of the Q57H variant may result from alterations to ORF3a functions by this mutation, rather than its protein expression levels in host cells. IEM Annual Conference Pilot Project Grant (to I.P.), OACA COVID19 Response Grants (to J.O.-U. and B.S.J.), and IEM COVID19 Response Grant (to J.O.-U.) from University of Minnesota This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Introduction: A mitochondrial fusion protein, Mitofusin 2 (Mfn2) serves as one of the major components for tethering the mitochondria and endoplasmic reticulum (ER), which regulates the exchange of lipids, Ca2+, and reactive oxygen species (ROS) across the two organelles. Several post-translational modifications (PTMs) of Mfn2 have been identified via mass spectroscopy, such as tyrosine phosphorylation (P-Tyr). However, little information is available whether any PTMs of Mfn2 can regulate mitochondrial and ER tethering function under physiological and pathological conditions. Hypothesis: P-Tyr of Mfn2 regulates Mfn2 function such as ER-mitochondrial tethering. Methods: To achieve stable activation of c-Src, we stably overexpressed c-Src or an shRNA targeting the shRNA targeted to C-terminal Src kinase (CSK), a specific negative regulator of c-Src, in HEK293T cells. Stable cell lines were used for biochemical, cell biological, and physiological assays. Results: Two computational program for phosphorylation site prediction, NetPhos and Group-Based Prediction System, predicted at least five c-Src-specific P-Tyr sites within the Mfn2 structure. Using immunoprecipitation and a phosphate-affinity electrophoresis technique (Phos-tag SDS-PAGE), we found that overexpression of c-Src, but not other Src family members, leads to P-Tyr of Mfn2. Moreover, dephospho-mimetic mutations (replacing Tyr with Phe) in Mfn2 significantly decreased the c-Src-dependent P-Tyr in Mfn2. Stable c-Src activation decreased the distance between the ER and outer mitochondrial membrane (OMM) as assessed by transmission electron microscopy and Förster resonance energy transfer between an OMM-targeted cyan fluorescent protein and an ER membrane-targeted yellow fluorescent protein the ER membrane measured by confocal microscopy. Mitochondrial Ca2+ (mtCa2+) uptake in response to cytosolic Ca2+ elevation increased with faster kinetics under c-Src activation, followed by increased mitochondrial ROS, and decreased oxygen consumption rate. Knocking down of endogenous Mfn2 by stable expression of shRNA targeting 3’ untranslated region of Mfn2 increased the distance between ER and OMM, and decreased mtCa2+ uptake. The reintroduction of wild-type Mfn2 in Mfn2-knockdown cells reversed these changes to the levels observed in cells expressing control shRNA. However, re-expression of a dephspho-mimetic mutant of Mfn2 in Mfn2-knockdown cells failed to rescue the changes in mtCa2+ uptake profile. Conclusion: P-Tyr of Mfn2 by c-Src modulates the distance between the OMM and ER, resulting in mtCa2+ elevation and oxidative stress. NIH/NHLBI R01HL136757 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Introduction: Discovery of the molecular identity of the mitochondrial Ca2+ (mtCa2+) uniporter complex (mtCUC) provides rapid progress in characterizing mtCa2+ handling and its roles in regulating cellular functions. MCU, a pore-forming subunit of mtCUC, which resides at the inner mitochondrial membrane, forms the major mtCa2+ influx pathway. Human MCU gene is predicted to produce mRNA that codes a shorter transcript variant (termed MCU-S) in addition to canonical full-length MCU transcript (renamed as MCU-L). However, its functional relevance in human cells is not fully investigated. Hypothesis: MCU-S variant can form Ca2+-permeable channels in addition to canonical MCU-L. Methods: Human platelets from adult healthy donors, human megakaryoblastic leukemia cell line MEG-01, and HEK293T cells were used for biomechanical, cell biological, and physiological assays. Ca2+ permeability via MCU-S was assessed by patch clamp and Ca2+ imaging under confocal microscope. Results: The existence of the mRNA and protein of MCU-S was confirmed in various human cells and tissues. Specifically, highest MCU-S mRNA levels was observed in human platelets compared to other cells and tissues including HEK293T, MEG-01 cell lines and the heart tissues. Expression of MCU and several other mtCUC components in the plasma membrane (PM) was confirmed by cell-surface protein biotinylation. Immunostaining of the human platelets exhibited extra-mitochondrial expression of MCU protein in addition to mitochondria. Overexpressed MCU-S is localized at the PM rather than in the mitochondria, while overexpressed MCU-L is exclusively expressed in the mitochondria, as assessed by live cell imaging and cell-surface protein biotinylation. Conventional whole-cell patch clamping showed that MCU-S expression HEK293T cells (but not control cells) exhibit an inward Ca2+ current sensitive to Ru360, the conventional MCU blocker. MEG-01 overexpressing MCU-S (but not the cells expressing GFP as a control or dominant-negative pore-forming mutant of MCU-S, termed MCU-S-DN) showed Ru360-sensitive PM Ca2+ permeabilization after switching the extracellular Ca2+ concentration from 0 mM to physiological range in the presence of BTP2, a selective inhibitor for the store-operated Ca2+ entry (SOCE). There is no significant difference in the magnitude of SOCE between cells expressing MCU-S, MCU-S-DN, and GFP. Lastly, we confirmed that human platelets also possess the Ru360-sensitive, but BTP-insensitive Ca2+ permeabilization at the PM, which can significantly alter the cytosolic Ca2+ concentration. Conclusion: MCU-S variant is capable of forming Ca2+-permeable channels at the PM. Elucidation of the role of MCU transcript variants may provide new insights into the molecular basis of mitochondrial and cellular Ca2+ handling in human platelet activation under physiological and pathological conditions. NIH/NHLBI R01HL136757 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Intermittent fasting (IF) extends lifespan via pleotropic mechanisms, but one important molecular mediator of the beneficial effects of IF is AMP-kinase (AMPK). AMPK enhances lipid metabolism and modulates microtubule dynamics. Dysregulation of these two molecular pathways causes right ventricular (RV) failure in pulmonary arterial hypertension (PAH). In two models of rodent PAH, we show IF activates RV AMPK, which restores mitochondrial morphology and peroxisomal density and restructures mitochondrial/peroxisomal lipid metabolism protein regulation. IF also increases electron transport chain (ETC) protein abundance and activity in the RV. Echocardiographic and hemodynamic measures of RV function are positively associated with fatty acid oxidation and ETC protein levels in correlational heatmapping analyses. IF also combats heightened microtubule density, which normalizes t-tubule structure. In summation, we demonstrate IF-mediated AMPK signaling counteracts two key molecular drivers of RV failure. Thus, IF may be a novel treatment approach for RV dysfunction, a currently untreatable and lethal consequence of PAH.Highlights-Intermittent fasting activates AMPK to restructure right ventricular mitochondrial and peroxisomal fatty acid fatty acid metabolism in two rodent models of PAH.-Intermittent fasting prevents downregulation of multiple electron transport chain proteins in both monocrotaline and Sugen-hypoxia RVs.-Pathological microtubule-mediated junctophilin-2 dysregulation and subsequent t-tubule remodeling is mitigated by intermittent fasting.-Intermittent fasting suppresses the induction of both the canonical and peroxisomal ferroptosis pathways in RV failure.

Contact sites between the mitochondria and endoplasmic reticulum (ER) are irregulates the exchange of lipids, Ca2+, and reactive oxygen species (ROS) across the two organelles. Mitofusin 2 (Mfn2) has been shown as one of the major components tethering these two organelles. Several post-translational modifications (PTMs) of Mfn2 have been identified to modulate canonical (i.e., mitochondrial fusion) and non- canonical functions, such as mitophagy and activation of ER stress signaling, however there is little information whether any PTMs can regulate mitochondrial and ER tethering. Basal tyrosine phosphorylation of Mfn2 was detected by mass spectroscopy, but it is unknown whether Mfn2 is a substrate of mitochondria-localized tyrosine kinases. Here we show that the mitochondria-localized Src family tyrosine kinases including c-Src can phosphorylate Mfn2, which decreases distance between the mitochondria and ER, and increases Ca2+ transfer from the ER to mitochondria, followed by changes in ROS generation and mitochondrial bioenergetics. Our findings suggest that tyrosine phosphorylation of Mfn2 may uniquely work to fine-tune ER- mitochondrial Ca2+ transport under physiological conditions, without activating mitophagy or ER stress signaling.

Introduction: We have described previously an arrhythmic mechanism in nonischemic heart failure (HF) involving an increased mitochondrial Ca 2+ uptake that results in QT prolongation and lethal arrhythmias during cardiomyopathy. This arrhythmic mechanism is associated with mitochondrial Ca 2+ uniporter (MCU) tyrosine phosphorylation (p-Tyr). Hypothesis: Here, we determined which kinase was responsible for MCU phosphorylation in ischemic cardiomyopathy. Methods: Ischemic HF was induced in wild type FVB/NJ mice for three weeks by ligated left anterior descending coronary artery. Western blot, immunoprecipitation, fluorescence resonance energy transfer (FRET) and patch-clamp techniques were employed in isolated mouse cardiomyocytes. Results: When compared to sham hearts, the protein level of p-Src increased significantly in ischemic HF mouse hearts. In a heterologous expression system, c-Src could bind MCU and tyrosine phosphorylate MCU. This phosphorylation was blocked by a c-Src inhibitor, PP1. Overexpression of constitutive active c-Src (Src-Y527F) significantly increased MCU inward current at -160 mV. Downregulation of C-terminal Src kinase (CSK), a regulatory kinase inversely related to c-Src activity, increased p-Src, enhanced mitochondrial and SR contact, and facilitated mitochondrial Ca 2+ uptake in ischemic HF mouse cardiomyocytes. Protein expression of CaMKII, PyK2, and p-PyK2 did not change during ischemic HF. MI activated c-Src by S-nitrosylation of caveolin-1 with subsequent release of the kinase. c-Src activation was associated with APD prolongation and arrhythmia following myocardial infarction in mice. Human heart tissue showed that ischemic HF patients had significantly increased p-Src associated with increased QTc prolongation and arrhythmia. Conclusions: During ischemic HF, c-Src activation directly increased MCU current that contributed to QT prolongation and arrhythmic risk in ischemic cardiomyopathy.

Right ventricular (RV) fibrosis is a key feature of maladaptive RV hypertrophy and dysfunction and is associated with poor outcomes in pulmonary hypertension (PH). However, mechanisms and therapeutic strategies to mitigate RV fibrosis remain unrealized. Previously, we identified that cardiac fibroblast α7 nicotinic acetylcholine receptor (α7 nAChR) drives smoking-induced RV fibrosis. Here, we sought to define the role of α7 nAChR in RV dysfunction and fibrosis in the settings of RV pressure overload as seen in PH. We show that RV tissue from PH patients has increased collagen content and ACh expression. Using an experimental rat model of PH, we demonstrate that RV fibrosis and dysfunction are associated with increases in ACh and α7 nAChR expression in the RV but not in the left ventricle (LV). In vitro studies show that α7 nAChR activation leads to an increase in adult ventricular fibroblast proliferation and collagen content mediated by a Ca2+/epidermal growth factor receptor (EGFR) signaling mechanism. Pharmacological antagonism of nAChR decreases RV collagen content and improves RV function in the PH model. Furthermore, mice lacking α7 nAChR exhibit improved RV diastolic function and have lower RV collagen content in response to persistently increased RV afterload, compared with WT controls. These finding indicate that enhanced α7 nAChR signaling is an important mechanism underlying RV fibrosis and dysfunction, and targeted inhibition of α7 nAChR is a potentially novel therapeutic strategy in the setting of increased RV afterload.

Introduction COVID‐19 caused by SARS‐CoV‐2 is an ongoing global health emergency. Pre‐existing cardiovascular diseases (CVD) including hypertension have a higher risk for severe illness, cardiac damage, and death. Therefore, strategies for cardiovascular protection in COVID‐19 are urgently needed, in addition to the development of vaccines against COVID‐19. Previously, it has been reported that several SARS‐CoV‐1 genes including encoded Envelope (E) protein, open reading frame (ORF) 3a, are capable of forming viral channels in mammalian cells and damaging the cells via activation of apoptotic or endoplasmic reticulum (ER) stress signaling. In addition, SARS‐CoV‐1 patients are found to possess antibodies against ORF3a, indicating that this protein was expressed at the surface of the host cells. Moreover, a recent clinical report confirmed that SARS‐CoV‐2 can directly infect cardiomyocytes. However, the specific molecular mechanisms underlying the linkage between SARS‐CoV‐2 infection and cardiac risk are unclear. Hypothesis SARS‐CoV‐2 genes including ORF3a can be expressed in cardiomyocytes after virus infection, and subsequently dysregulate cardiac functions. Methods HEK293T cells and H9c2 cardiac myoblasts were transfected with ORF3a and utilized for biochemical, cell biological, and electrophysiological assays. Numerical model of rabbit ventricular action potential was used to evaluate the role of ORF3a on cardiac electrophysiology and arrhythmia. Results Although ORF3a sequences from SARS‐CoV‐1 and ‐CoV‐2 have 72% similarity, ORF3a from SARS‐CoV‐2 maintains high homology with SARS‐CoV‐1 in the protein sequences of the transmembrane domains and the pore, which are critical for forming ion channels. We found that SARS‐CoV‐2‐ORF3a is expressed at plasma membrane and outer mitochondrial membrane as assessed by protein fractionation, mitochondrial digestion, and live‐cell imaging. Whole cell patch clamp revealed that ORF3a can form K+‐permeable channels at the plasma membrane with similar channel properties as the endogenous transient outward K+ current (Ito). Moreover, numerical simulations suggest that increased Ito amplitude (mimicking increase of ORF3a expression) may dysregulate Ca2+ transient and increase the risk for early afterdepolarization in response to increased heart rate. Lastly, we found that ORF3a promotes apoptotic signaling activation, but not ER stress signaling, assessed by caspase activity and the expression of major ER chaperones, respectively. Conclusion ORF3a from SARS‐CoV‐2 forms K+‐permeable channels at the plasma membrane and dysregulates cardiac Ca2+ handling and electrophysiology. ORF3a can also be expressed at the mitochondria and activate oxidative stress signaling, which exacerbates the risk of arrhythmia. Targeting SARS‐CoV‐2 viroporins including ORF3a may reduce the risk of sudden cardiac death and cardiac damage in COVID‐19 patients with pre‐existing CVDs.

Recent discoveries of the molecular identity of mitochondrial Ca(2+) influx/efflux mechanisms have placed mitochondrial Ca(2+) transport at center stage in views of cellular regulation in various cell-types/tissues. Indeed, mitochondria in cardiac muscles also possess the molecular components for efficient uptake and extraction of Ca(2+). Over the last several years, multiple groups have taken advantage of newly available molecular information about these proteins and applied genetic tools to delineate the precise mechanisms for mitochondrial Ca(2+) handling in cardiomyocytes and its contribution to excitation-contraction/metabolism coupling in the heart. Though mitochondrial Ca(2+) has been proposed as one of the most crucial secondary messengers in controlling a cardiomyocyte’s life and death, the detailed mechanisms of how mitochondrial Ca(2+) regulates physiological mitochondrial and cellular functions in cardiac muscles, and how disorders of this mechanism lead to cardiac diseases remain unclear. In this review, we summarize the current controversies and discrepancies regarding cardiac mitochondrial Ca(2+) signaling that remain in the field to provide a platform for future discussions and experiments to help close this gap.

Introduction: We have described previously an arrhythmic mechanism in nonischemic heart failure (NI-HF) involving an increased mitochondrial Ca 2+ uptake that results in QT prolongation and lethal arrhythmias. This arrhythmic mechanism is associated with mitochondrial Ca 2+ uniporter (MCU) tyrosine phosphorylation (p-Tyr). Hypothesis: Here, we determined which kinase was responsible for MCU phosphorylation in cardiomyopathy. Methods: NI-HF was induced by hypertension in Wt C57BL6 mice by unilateral nephrectomy, deoxycorticosterone acetate (DOCA) treatment and substituting drinking water with 1% saline for six weeks. Western blot, immunoprecipitation, fluorescence resonance energy transfer (FRET) and patch-clamp techniques were employed in isolated mouse cardiomyocytes. Results: When compared to control hearts, the protein level of p-Src S17 increased significantly accompanied by significant enhancement of tyrosine but not serine/threonine phosphorylation of MCU in NI-HF mouse hearts. In a heterologous expression system, c-Src could bind MCU and tyrosine phosphorylate MCU. This phosphorylation was blocked by a c-Src inhibitor, PP1. Overexpression of constitutive active c-Src (Src-Y527F) significantly increased MCU inward current at -160 mV. It suggested that c-Src activation was directly related to mitochondrial-mediated arrhythmic risk. Downregulation of C-terminal Src kinase (CSK), a regulatory kinase inversely related to c-Src activity, increased p-Src, enhanced mitochondrial and SR contact, and facilitated mitochondrial Ca 2+ uptake in NI-HF mouse cardiomyocytes. Proline-rich tyrosine kinase 2 (Pyk2) did not contribute more phosphorylated MCU during cardiomyopathy as the protein expression of PyK2, p-PyK2 Y402 and p-PyK2 Y579/580 did not change during NI-HF. Correlation using human heart tissue showed that NI-HF patients had significantly increased p-Src S17 associated with an increased incidence of QTc elongation and arrhythmia. Conclusions: During cardiomyopathy, c-Src activation directly tyrosine phosphorylated MCU, increased MCU current, and increased mitochondrial/SR contact, suggesting this kinase is responsible for the increase in mitochondria-mediated arrhythmic risk seen in NI-HF.

Introduction: COVID-19 patients with pre-existing cardiovascular diseases (CVDs) have a higher risk for severe illness, cardiac damage, and death. Therefore, strategies for cardiovascular protection in COVID-19 are urgently needed until a reliable and effective treatment for COVID-19 (e.g., vaccine) is established. Because angiotensin-converting enzyme 2 (ACE2) is overexpressed in the cardiovascular system during CVD, COVID-19 patients with pre-existing CVDs may have higher risk for direct SARS-CoV-2 infection into the heart. However, the specific molecular mechanisms linking SARS-CoV-2 and cardiac risk are still unclear. Previous studies for SARS suggest that several SARS-CoV-1 genes [Envelope (E) protein, open reading frame (ORF) 3a, and ORF8a] have the potential to form viral channels (i.e., “viroporins”) in host cells and dysregulate host-cell functions. Hypothesis: SARS-CoV-2 genes encode viroporins, which can be expressed in the heart after infection and dysregulate cardiac electrophysiology and contractility. Methods: Individual SARS-CoV-2 genes were expressed in HEK293T cells via plasmids vector and the transfected cells were used for biochemical and electrophysiological assays. Results: By database analysis, we found that ORF3a and E from SARS-CoV-2 share over 85% similarity to those in SARS-CoV and maintain the critical sequences for ion channel functions. Both SARS-CoV-2-ORF3a and E protein form K + channels with similar channel properties of the endogenous transient outward K + current ( I to ), which can be activated during the initial repolarization phase of an action potential (AP) in cardiomyocytes. A computational model of human ventricular myocytes (O'Hara-Virag-Varro-Rudy Model) with increased I to amplitude suggests that the expression of these viroporins may increase Ca 2+ transient (CaT) amplitude at the baseline, but decrease CaT in response to increased heart rate. Conclusion: SARS-CoV-2 genes encode K + channels, which may dysregulate AP and Ca 2+ handing in cardiomyocytes, thus promoting decreased cardiac contractility and increased susceptibility to arrhythmias. Targeting these viroporins may reduce the risk of sudden cardiac death and cardiac damage in COVID-19 patients with pre-existing CVDs.

Hyperproliferative endothelial cells (ECs) play an important role in the pathogenesis of pulmonary arterial hypertension (PAH). Anoctamin (Ano)-1, a calcium-activated chloride channel, can regulate cell proliferation and cell cycle in multiple cell types. However, the expression and function of Ano1 in the pulmonary endothelium is unknown. We examined whether Ano1 was expressed in pulmonary ECs and if altering Ano1 activity would affect EC survival. Expression and localization of Ano1 in rat lung microvascular ECs (RLMVECs) was assessed using immunoblot, immunofluorescence, and subcellular fractionation. Cell counts, flow cytometry, and caspase-3 activity were used to assess changes in cell number and apoptosis in response to the small molecule Ano1 activator, Eact. Changes in mitochondrial membrane potential and mitochondrial reactive oxygen species (mtROS) were assessed using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine, iodide (mitochondrial membrane potential dye) and mitochondrial ROS dye, respectively. Ano1 is expressed in RLMVECs and is enriched in the mitochondria. Activation of Ano1 with Eact reduced RLMVEC counts through increased apoptosis. Ano1 knockdown blocked the effects of Eact. Ano1 activation increased mtROS, reduced mitochondrial membrane potential, increased p38 phosphorylation, and induced release of apoptosis-inducing factor. mtROS inhibition attenuated Eact-mediated p38 phosphorylation. Pulmonary artery ECs isolated from patients with idiopathic PAH (IPAH) had higher expression of Ano1 and increased cell counts compared with control subjects. Eact treatment reduced cell counts in IPAH cells, which was associated with increased apoptosis. In summary, Ano1 is expressed in lung EC mitochondria. Activation of Ano1 promotes apoptosis of pulmonary ECs and human IPAH-pulmonary artery ECs, likely via increased mtROS and p38 phosphorylation, leading to apoptosis.

Key points Abnormal mitochondrial morphology and function in cardiomyocytes are frequently observed under persistent Gq protein-coupled receptor (Gq PCR) stimulation. Cardiac signalling mechanisms for regulating mitochondrial morphology and function under pathophysiological conditions in the heart are still poorly understood. We demonstrate that a downstream kinase of Gq PCR, protein kinase D (PKD) induces mitochondrial fragmentation via phosphorylation of dynamin-like protein 1 (DLP1), a mitochondrial fission protein. The fragmented mitochondria enhance reactive oxygen species generation and permeability transition pore opening in mitochondria, which initiate apoptotic signalling activation. This study identifies a novel PKD-specific substrate in cardiac mitochondria and uncovers the role of PKD on cardiac mitochondria, with special emphasis on the molecular mechanism(s) underlying mitochondrial injury with abnormal mitochondrial morphology under persistent Gq PCR stimulation. These findings provide new insights into the molecular basis of cardiac mitochondrial physiology and pathophysiology, linking Gq PCR signalling with the regulation of mitochondrial morphology and function. Abstract Regulation of mitochondrial morphology is crucial for the maintenance of physiological functions in many cell types including cardiomyocytes. Small and fragmented mitochondria are frequently observed in pathological conditions, but it is still unclear which cardiac signalling pathway is responsible for regulating the abnormal mitochondrial morphology in cardiomyocytes. Here we demonstrate that a downstream kinase of Gq protein-coupled receptor (Gq PCR) signalling, protein kinase D (PKD), mediates pathophysiological modifications in mitochondrial morphology and function, which consequently contribute to the activation of apoptotic signalling. We show that Gq PCR stimulation induced by α1 -adrenergic stimulation mediates mitochondrial fragmentation in a fission- and PKD-dependent manner in H9c2 cardiac myoblasts and rat neonatal cardiomyocytes. Upon Gq PCR stimulation, PKD translocates from the cytoplasm to the outer mitochondrial membrane (OMM) and phosphorylates a mitochondrial fission protein, dynamin-like protein 1 (DLP1), at S637. PKD-dependent phosphorylation of DLP1 initiates DLP1 association with the OMM, which then enhances mitochondrial fragmentation, mitochondrial superoxide generation, mitochondrial permeability transition pore opening and apoptotic signalling. Finally, we demonstrate that DLP1 phosphorylation at S637 by PKD occurs in vivo using ventricular tissues from transgenic mice with cardiac-specific overexpression of constitutively active Gαq protein. In conclusion, Gq PCR-PKD signalling induces mitochondrial fragmentation and dysfunction via PKD-dependent DLP1 phosphorylation in cardiomyocytes. This study is the first to identify a novel PKD-specific substrate, DLP1 in mitochondria, as well as the functional role of PKD in cardiac mitochondria. Elucidation of these molecular mechanisms by which PKD-dependent enhanced fission mediates cardiac mitochondrial injury will provide novel insight into the relationship among mitochondrial form, function and Gq PCR signalling.

Introduction: Cardiac fibrosis persists in patients with heart failure (HF) even during treatment with conventional HF therapies designed to control volume/pressure overload and restore cardiomyocyte function. This suggests a critical need to develop novel and effective anti-fibrotic therapies specifically targeting cardiac fibroblasts (CFs). CFs contribute to pathophysiological remodeling such as cardiac fibrosis after cardiac injury and/or during repair in response to various profibrotic stimuli in vivo such as angiotensin II (Ang II). Oxidative stress promotes pathological CF proliferation, but the detailed molecular mechanisms linking mitochondria and CF proliferation remain unclear. Hypothesis: Ang II stimulation promotes mitochondrial ROS (mROS) generation via mitochondrial Ca 2+ (mCa 2+ ) uptake, which subsequently activates mROS-dependent proliferative signaling in CFs. Methods: In CFs isolated from rat hearts, mCa 2+ uptake, mROS levels and mitochondrial morphology were measured using confocal microscopy with a mitochondria-targeted Ca 2+ biosensor mtRCamp1h, a mitochondrial superoxide-sensitive dye MitoSOX, and mitochondria-targeted GFP, respectively.. Results: Ang II (≥1 μM) stimulation induces Ca 2+ release from the endoplasmic reticulum (ER) followed by increased mCa 2+ , mROS, and mitochondrial fragmentation. In addition, Ang II activates ERK1/2- and p38-mediated proliferative pathways, which was inhibited by pretreatment with losartan, an Ang II receptor antagonist. Overexpression of a dominant-negative mCa 2+ uniporter (MCU) with pore domain mutations abolished Ang II-mediated mCa 2+ uptake and mROS generation, and prevented the activation of proliferative pathways. Pretreatment with a mitochondria-targeted antioxidant, mitoTEMPO, also significantly inhibited Ang II-mediated activation of the proliferative pathways without affecting ER-Ca 2+ release or mCa 2+ -uptake. Conclusion: The mROS generation via mCa 2+ accumulation and mitochondrial fragmentation resulting from Ang II stimulation activates mROS-dependent proliferative signaling pathways in CFs. The MCU-dependent mROS generation may serve as an important molecular switch for CF proliferation during cardiac stress.

Mitochondrial Ca2+homeostasis, the Ca2+influx-efflux balance, is responsible for the control of numerous cellular functions, including energy metabolism, generation of reactive oxygen species, spatiotemporal dynamics of Ca2+signaling, and cell growth and death. Recent discovery of the molecular identity of the mitochondrial Ca2+uniporter (MCU) provides new possibilities for application of genetic approaches to study the mitochondrial Ca2+influx mechanism in various cell types and tissues. In addition, the subsequent discovery of various auxiliary subunits associated with MCU suggests that mitochondrial Ca2+uptake is not solely regulated by a single protein (MCU), but likely by a macromolecular protein complex, referred to as the MCU-protein complex (mtCUC). Moreover, recent reports have shown the potential role of MCU posttranslational modifications in the regulation of mitochondrial Ca2+uptake through mtCUC. These observations indicate that mtCUCs form a local signaling complex at the inner mitochondrial membrane that could significantly regulate mitochondrial Ca2+handling, as well as numerous mitochondrial and cellular functions. In this review we discuss the current literature on mitochondrial Ca2+uptake mechanisms, with a particular focus on the structure and function of mtCUC, as well as its regulation by signal transduction pathways, highlighting current controversies and discrepancies.

Protein kinase C (PKC) plays key roles in the regulation of signal transduction and cellular function in various cell types. At least ten PKC isoforms have been identified and intracellular localization and trafficking of these individual isoforms are important for regulation of enzyme activity and substrate specificity. PKC can be activated at downstream of Gq-protein coupled receptor (GqPCR) signaling and translocated to the various cellular compartments including plasma membrane (PM). Recent reports suggested that a different type of GqPCRs would activate different PKC isoforms (classic, novel and atypical PKCs) with different trafficking patterns. However, the knowledge of isoform-specific activation of PKC by each GqPCR is limited. α1-Adrenoceptor (α1-AR) is the one of the GqPCR highly expressed in the cardiovascular system. In this study, we examined the isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-AR stimulation (α1-ARS). Rat PKCα, βI, βII, δ, ε and ζ fused with GFP at C-term were co-transfected with human α1A-AR into HEK293T cells. The isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-ARS using phenylephrine was measured by confocal microscopy. Before stimulation, GFP-PKCs were localized at cytosolic region. α1-ARS strongly and rapidly translocated a classical PKC (cPKC), PKCα, (< 30s) to PM, with PKCα returning diffusively into the cytosol within 5 min. α1-ARS rapidly translocated other cPKCs, PKCβI and PKCβII, to the PM (

Mitochondrial Ca2+ uptake is crucial for an array of cellular functions while an imbalance can elicit cell death. In this chapter, we briefly reviewed the various modes of mitochondrial Ca2+ uptake and our current understanding of mitochondrial Ca2+ homeostasis in regards to cell physiology and pathophysiology. Further, this chapter focuses on the molecular identities, intracellular regulators as well as the pharmacology of mitochondrial Ca2+ uniporter complex.

Aims: Mitochondrial Ca2+ homeostasis is crucial for balancing cell survival and death. The recent discovery of the molecular identity of the mitochondrial Ca2+ uniporter pore (MCU) opens new possibilities for applying genetic approaches to study mitochondrial Ca2+ regulation in various cell types, including cardiac myocytes. Basal tyrosine phosphorylation of MCU was reported from mass spectroscopy of human and mouse tissues, but the signaling pathways that regulate mitochondrial Ca2+ entry through posttranslational modifications of MCU are completely unknown. Therefore, we investigated α1-adrenergic-mediated signal transduction of MCU posttranslational modification and function in cardiac cells. Results: α1-adrenoceptor (α1-AR) signaling translocated activated proline-rich tyrosine kinase 2 (Pyk2) from the cytosol to mitochondrial matrix and accelerates mitochondrial Ca2+ uptake via Pyk2-dependent MCU phosphorylation and tetrametric MCU channel pore formation. Moreover, we found that α1-AR stimulation increases reactive oxygen species production at mitochondria, mitochondrial permeability transition pore activity, and initiates apoptotic signaling via Pyk2-dependent MCU activation and mitochondrial Ca2+ overload. Innovation: Our data indicate that inhibition of α1-AR-Pyk2-MCU signaling represents a potential novel therapeutic target to limit or prevent mitochondrial Ca2+ overload, oxidative stress, mitochondrial injury, and myocardial death during pathophysiological conditions, where chronic adrenergic stimulation is present. Conclusion: The α1-AR-Pyk2-dependent tyrosine phosphorylation of the MCU regulates mitochondrial Ca2+ entry and apoptosis in cardiac cells. Antioxid. Redox Signal. 21, 863–879.

Abnormal mitochondrial morphology, especially fragmented mitochondria, and mitochondrial dysfunction are hallmarks of a variety of human diseases including heart failure (HF). Although emerging evidence suggests a link between mitochondrial fragmentation and cardiac dysfunction, it is still not well described which cardiac signaling pathway regulates mitochondrial morphology and function under pathophysiological conditions such as HF. Mitochondria change their shape and location via the activity of mitochondrial fission and fusion proteins. This mechanism is suggested as an important modulator for mitochondrial and cellular functions including bioenergetics, reactive oxygen species (ROS) generation, spatiotemporal dynamics of Ca2+ signaling, cell growth, and death in the mammalian cell- and tissue-specific manners. Recent reports show that a mitochondrial fission protein, dynamin-like/related protein 1 (DLP1/Drp1), is post-translationally modified via cell signaling pathways, which control its subcellular localization, stability, and activity in cardiomyocytes/heart. In this review, we summarize the possible molecular mechanisms for causing post-translational modifications (PTMs) of DLP1/Drp1 in cardiomyocytes, and further discuss how these PTMs of DLP1/Drp1 mediate abnormal mitochondrial morphology and mitochondrial dysfunction under adrenergic signaling activation that contributes to the development and progression of HF.

Mitochondria are the essential eukaryotic organelles that produce most cellular energy. The energy production and supply by mitochondria appear closely associated with the continuous shape change of mitochondria mediated by fission and fusion, as evidenced not only by the hereditary diseases caused by mutations in fission/fusion genes but also by aberrant mitochondrial morphologies associated with numerous pathologic insults. However, how morphological change of mitochondria is linked to their energy-producing activity is poorly understood. In this study, we found that perturbation of mitochondrial fission induces a unique mitochondrial uncoupling phenomenon through a large-scale fluctuation of a mitochondrial inner membrane potential. Furthermore, by genetically controlling mitochondrial fission and thereby inducing mild proton leak in mice, we were able to relieve these mice from oxidative stress in a hyperglycemic model. These findings provide mechanistic insight into how mitochondrial fission participates in regulating mitochondrial activity. In addition, these results suggest a potential application of mitochondrial fission to control mitochondrial reactive oxygen species production and oxidative stress in many human diseases.

Increased production of reactive oxygen species (ROS) from mitochondria is the main cause of hyperglycemic complications. We previously showed that hyperglycemic conditions induce mitochondrial fragmentation that is causal for ROS overproduction. This study was to identify signaling components that induce mitochondrial fragmentation in high-glucose stimulation. We found that exposing cells to the high-glucose concentration evokes increases in cytosolic Ca2+. Chelating Ca2+ in the high-glucose medium prevented not only the Ca2+ transient but also mitochondrial fragmentation and the ROS increase, indicating that the Ca2+ influx across the plasma membrane is an upstream event governing mitochondrial fission and the ROS generation in high-glucose stimulation. We found that the high-glucose-induced Ca2+ increase activates the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 (ERK1/2). The Ca2+ chelation prevented the ERK1/2 activation, and inhibition of the ERK1/2 phosphorylation decreased mitochondrial fragmentation as well as ROS levels in high-glucose stimulation. In addition, the level of the mitochondrial fission protein dynamin-like protein 1 in mitochondria increased in high-glucose incubation in a Ca2+-dependent manner. In vitro kinase assays showed that ERK1/2 is capable of phosphorylating dynamin-like protein 1. These results demonstrate that high-glucose stimulation induces the activation of mitochondrial fission via signals mediated by intracellular Ca2+ and ERK1/2. Antioxid. Redox Signal. 14, 425–437.