<div><h4>Cooperative Response to Endocardial Notch Reveals Interaction With Hippo Pathway.</h4><i>Luna-Zurita L, Flores-Garza BG, Grivas D, Siguero-Álvarez M, de la Pompa JL</i><br /><b>Background</b><br />The endocardium is a crucial signaling center for cardiac valve development and maturation. Genetic analysis has identified several human endocardial genes whose inactivation leads to bicuspid aortic valve formation and calcific aortic valve disease, but knowledge is very limited about the role played in valve development and disease by noncoding endocardial regulatory regions and upstream factors.<br /><b>Methods</b><br />We manipulated Notch signaling in mouse embryonic endocardial cells by short-term and long-term coculture with OP9 stromal cells expressing Notch ligands and inhibition of Notch activity. We examined the transcriptional profile and chromatin accessibility landscape for each condition, integrated transcriptomic, transcription factor occupancy, chromatin accessibility, and proteomic datasets. We generated in vitro and in vivo models with CRISPR-Cas9-edited deletions of various noncoding regulatory elements and validated their regulatory potential.<br /><b>Results</b><br />We identified primary and secondary transcriptional responses to Notch ligands in the mouse embryonic endocardium, and a NOTCH-dependent transcriptional signature in valve development and disease. By defining the changes in the chromatin accessibility landscape and integrating with the landscape in developing mouse endocardium and adult human valves, we identify potential noncoding regulatory elements, validated selected candidates, propose interacting cofactors, and define the timeframe of their regulatory activity. Additionally, we found cooperative transcriptional repression with Hippo pathway by inhibiting nuclear Yap (Yes-associated protein) activity in the endocardium during cardiac valve development.<br /><b>Conclusions</b><br />Sequential Notch-dependent transcriptional regulation in the embryonic endocardium involves multiple factors. Notch activates certain noncoding elements through these factors and simultaneously suppresses elements that could hinder cardiac valve development and homeostasis. Biorxviv: https://www.biorxiv.org/content/10.1101/2023.03.23.533882v1.full.<br /><br /><br /><br /><small>Circ Res: 13 Nov 2023; epub ahead of print</small></div>

<div><h4>Nitric Oxide Modulates Ca Leak and Arrhythmias via S-Nitrosylation of CaMKII.</h4><i>Power AS, Asamudo EU, Worthington LPI, Alim CC, ... Bers DM, Erickson JR</i><br /><b>Background</b><br />Nitric oxide (NO) has been identified as a signaling molecule generated during β-adrenergic receptor stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca<sup>2+</sup> release via <i>S</i>-nitrosylation of CaMKIIδ (Ca<sup>2+</sup>/calmodulin kinase II delta) is emerging. NO donors are routinely used clinically for their cardioprotective effects on the heart, but it is unknown how NO donors modulate the proarrhythmic CaMKII to alter cardiac arrhythmia incidence. We test the role of <i>S</i>-nitrosylation of CaMKIIδ at the Cysteine-273 inhibitory site and cysteine-290 activating site in cardiac Ca<sup>2+</sup> handling and arrhythmogenesis before and during β-adrenergic receptor stimulation.<br /><b>Methods</b><br />We measured Ca<sup>2+</sup>-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the <i>S</i>-nitrosylation site on CaMKIIδ at cysteine-273 or cysteine-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, <i>S</i>-nitrosoglutathione (GSNO; 150 μM), sodium nitroprusside (200 μM), and β-adrenergic agonist isoproterenol (100 nmol/L).<br /><b>Results</b><br />Both WT and CaMKIIδ-KO cardiomyocytes responded to isoproterenol with a full inotropic and lusitropic Ca<sup>2+</sup> transient response as well as increased Ca<sup>2+</sup> spark frequency. However, the increase in Ca<sup>2+</sup> spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from isoproterenol-induced Ca<sup>2+</sup> sparks and waves was mimicked by GSNO pretreatment in WT cardiomyocytes but lost in CaMKIIδ-C273S cardiomyocytes. When GSNO was applied after isoproterenol, this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pretreatment limited isoproterenol-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after isoproterenol sustained or exacerbated arrhythmic events.<br /><b>Conclusions</b><br />We conclude that prior <i>S</i>-nitrosylation of CaMKIIδ at cysteine-273 can limit subsequent β-adrenergic receptor-induced arrhythmias, but that <i>S</i>-nitrosylation at cysteine-290 might worsen or sustain β-adrenergic receptor-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.<br /><br /><br /><br /><small>Circ Res: 13 Nov 2023; epub ahead of print</small></div>

<div><h4>NAT10 Is Involved in Cardiac Remodeling Through ac4C-Mediated Transcriptomic Regulation.</h4><i>Shi J, Yang C, Zhao K, Zhang J, ... Chen L, Kong X</i><br /><b>Background</b><br />Heart failure, characterized by cardiac remodeling, is associated with abnormal epigenetic processes and aberrant gene expression. Here, we aimed to elucidate the effects and mechanisms of NAT10 (N-acetyltransferase 10)-mediated N4-acetylcytidine (ac4C) acetylation during cardiac remodeling.<br /><b>Methods</b><br />NAT10 and ac4C expression were detected in both human and mouse subjects with cardiac remodeling through multiple assays. Subsequently, acetylated RNA immunoprecipitation and sequencing, thiol (SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq), and ribosome sequencing (Ribo-seq) were employed to elucidate the role of ac4C-modified posttranscriptional regulation in cardiac remodeling. Additionally, functional experiments involving the overexpression or knockdown of NAT10 were conducted in mice models challenged with Ang II (angiotensin II) and transverse aortic constriction.<br /><b>Results</b><br />NAT10 expression and RNA ac4C levels were increased in in vitro and in vivo cardiac remodeling models, as well as in patients with cardiac hypertrophy. Silencing and inhibiting NAT10 attenuated Ang II-induced cardiomyocyte hypertrophy and cardiofibroblast activation. Next-generation sequencing revealed ac4C changes in both mice and humans with cardiac hypertrophy were associated with changes in global mRNA abundance, stability, and translation efficiency. Mechanistically, NAT10 could enhance the stability and translation efficiency of <i>CD47</i> and <i>ROCK2</i> transcripts by upregulating their mRNA ac4C modification, thereby resulting in an increase in their protein expression during cardiac remodeling. Furthermore, the administration of Remodelin, a NAT10 inhibitor, has been shown to prevent cardiac functional impairments in mice subjected to transverse aortic constriction by suppressing cardiac fibrosis, hypertrophy, and inflammatory responses, while also regulating the expression levels of CD47 and ROCK2.<br /><b>Conclusions</b><br />Therefore, our data suggest that modulating epitranscriptomic processes, such as ac4C acetylation through NAT10, may be a promising therapeutic target against cardiac remodeling.<br /><br /><br /><br /><small>Circ Res: 12 Nov 2023; epub ahead of print</small></div>

<div><h4>DWORF Extends Life Span in a PLN-R14del Cardiomyopathy Mouse Model by Reducing Abnormal Sarcoplasmic Reticulum Clusters.</h4><i>Stege NM, Eijgenraam TR, Oliveira Nunes Teixeira V, Feringa AM, ... de Boer RA, Silljé HHW</i><br /><b>Background</b><br />The p.Arg14del variant of the <i>PLN</i> (phospholamban) gene causes cardiomyopathy, leading to severe heart failure. Calcium handling defects and perinuclear PLN aggregation have both been suggested as pathological drivers of this disease. Dwarf open reading frame (DWORF) has been shown to counteract PLN regulatory calcium handling function in the sarco/endoplasmic reticulum (S/ER). Here, we investigated the potential disease-modulating action of DWORF in this cardiomyopathy and its effects on calcium handling and PLN aggregation.<br /><b>Methods</b><br />We studied a PLN-R14del mouse model, which develops cardiomyopathy with similar characteristics as human patients, and explored whether cardiac DWORF overexpression could delay cardiac deterioration. To this end, R14<sup>Δ/Δ</sup> (homozygous PLN-R14del) mice carrying the DWORF transgene (R14<sup>Δ/Δ</sup>DWORF<sup>Tg</sup> [R14<sup>Δ/Δ</sup> mice with a copy of the DWORF transgene]) were used.<br /><b>Results</b><br />DWORF expression was suppressed in hearts of R14<sup>Δ/Δ</sup> mice with severe heart failure. Restoration of DWORF expression in R14<sup>Δ/Δ</sup> mice delayed cardiac fibrosis and heart failure and increased life span >2-fold (from 8 to 18 weeks). DWORF accelerated sarcoplasmic reticulum calcium reuptake and relaxation in isolated cardiomyocytes with wild-type PLN, but in R14<sup>Δ/Δ</sup> cardiomyocytes, sarcoplasmic reticulum calcium reuptake and relaxation were already enhanced, and no differences were detected between R14<sup>Δ/Δ</sup> and R14<sup>Δ/Δ</sup>DWORF<sup>Tg</sup>. Rather, DWORF overexpression delayed the appearance and formation of large pathogenic perinuclear PLN clusters. Careful examination revealed colocalization of sarcoplasmic reticulum markers with these PLN clusters in both R14<sup>Δ/Δ</sup> mice and human p.Arg14del PLN heart tissue, and hence these previously termed aggregates are comprised of abnormal organized S/ER. This abnormal S/ER organization in PLN-R14del cardiomyopathy contributes to cardiomyocyte cell loss and replacement fibrosis, consequently resulting in cardiac dysfunction.<br /><b>Conclusions</b><br />Disorganized S/ER is a major characteristic of PLN-R14del cardiomyopathy in humans and mice and results in cardiomyocyte death. DWORF overexpression delayed PLN-R14del cardiomyopathy progression and extended life span in R14<sup>Δ/Δ</sup> mice, by reducing abnormal S/ER clusters.<br /><br /><br /><br /><small>Circ Res: 12 Nov 2023; epub ahead of print</small></div>

<div><h4>Cyclin D-CDK4 Disulfide Bond Attenuates Pulmonary Vascular Cell Proliferation.</h4><i>Knight H, Abis G, Kaur M, Green HLH, ... Eaton P, Rudyk O</i><br /><b>Background</b><br />Pulmonary hypertension (PH) is a chronic vascular disease characterized, among other abnormalities, by hyperproliferative smooth muscle cells and a perturbed cellular redox and metabolic balance. Oxidants induce cell cycle arrest to halt proliferation; however, little is known about the redox-regulated effector proteins that mediate these processes. Here, we report a novel kinase-inhibitory disulfide bond in cyclin D-CDK4 (cyclin-dependent kinase 4) and investigate its role in cell proliferation and PH.<br /><b>Methods</b><br />Oxidative modifications of cyclin D-CDK4 were detected in human pulmonary arterial smooth muscle cells and human pulmonary arterial endothelial cells. Site-directed mutagenesis, tandem mass-spectrometry, cell-based experiments, in vitro kinase activity assays, in silico structural modeling, and a novel redox-dead constitutive knock-in mouse were utilized to investigate the nature and definitively establish the importance of CDK4 cysteine modification in pulmonary vascular cell proliferation. Furthermore, the cyclin D-CDK4 oxidation was assessed in vivo in the pulmonary arteries and isolated human pulmonary arterial smooth muscle cells of patients with pulmonary arterial hypertension and in 3 preclinical models of PH.<br /><b>Results</b><br />Cyclin D-CDK4 forms a reversible oxidant-induced heterodimeric disulfide dimer between C7/8 and C135, respectively, in cells in vitro and in pulmonary arteries in vivo to inhibit cyclin D-CDK4 kinase activity, decrease Rb (retinoblastoma) protein phosphorylation, and induce cell cycle arrest. Mutation of CDK4 C135 causes a kinase-impaired phenotype, which decreases cell proliferation rate and alleviates disease phenotype in an experimental mouse PH model, suggesting this cysteine is indispensable for cyclin D-CDK4 kinase activity. Pulmonary arteries and human pulmonary arterial smooth muscle cells from patients with pulmonary arterial hypertension display a decreased level of CDK4 disulfide, consistent with CDK4 being hyperactive in human pulmonary arterial hypertension. Furthermore, auranofin treatment, which induces the cyclin D-CDK4 disulfide, attenuates disease severity in experimental PH models by mitigating pulmonary vascular remodeling.<br /><b>Conclusions</b><br />A novel disulfide bond in cyclin D-CDK4 acts as a rapid switch to inhibit kinase activity and halt cell proliferation. This oxidative modification forms at a critical cysteine residue, which is unique to CDK4, offering the potential for the design of a selective covalent inhibitor predicted to be beneficial in PH.<br /><br /><br /><br /><small>Circ Res: 12 Nov 2023; epub ahead of print</small></div>

<div><h4>Myeloid Cell Derived IL1β Contributes to Pulmonary Hypertension in HFpEF.</h4><i>Agrawal V, Kropski JA, Gokey JJ, Kobeck E, ... West JD, Hemnes AR</i><br /><b>Background</b><br />Pulmonary hypertension (PH) in heart failure with preserved ejection fraction (HFpEF) is a common and highly morbid syndrome, but mechanisms driving PH-HFpEF are poorly understood. We sought to determine whether a well-accepted murine model of HFpEF also displays features of PH, and we sought to identify pathways that might drive early remodeling of the pulmonary vasculature in HFpEF.<br /><b>Methods</b><br />Eight-week-old male and female C57BL/6J mice received either N<sup>γ</sup>-nitro-L-arginine methyl ester and high-fat diet or control water and diet for 2, 5, and 12 weeks. The db/db mice were studied as a second model of HFpEF. Early pathways regulating PH were identified by bulk and single-cell RNA sequencing. Findings were confirmed by immunostain in lungs of mice or lung slides from clinically performed autopsies of patients with PH-HFpEF. ELISA was used to verify IL-1β (interleukin-1 beta) in mouse lung, mouse plasma, and also human plasma from patients with PH-HFpEF obtained at the time of right heart catheterization. Clodronate liposomes and an anti-IL-1β antibody were utilized to deplete macrophages and IL-1β, respectively, to assess their impact on pulmonary vascular remodeling in HFpEF in mouse models.<br /><b>Results</b><br />N<sup>γ</sup>-nitro-L-arginine methyl ester/high-fat diet-treated mice developed PH, small vessel muscularization, and right heart dysfunction. Inflammation-related gene ontologies were overrepresented in bulk RNA sequencing analysis of whole lungs, with an increase in CD68<sup>+</sup> cells in both murine and human PH-HFpEF lungs. Cytokine profiling showed an increase in IL-1β in mouse and human plasma. Finally, clodronate liposome treatment in mice prevented PH in N<sup>γ</sup>-nitro-L-arginine methyl ester/high-fat diet-treated mice, and IL-1β depletion also attenuated PH in N<sup>γ</sup>-nitro-L-arginine methyl ester/high-fat diet-treated mice.<br /><b>Conclusions</b><br />We report a novel model for the study of PH and right heart remodeling in HFpEF, and we identify myeloid cell-derived IL-1β as an important contributor to PH in HFpEF.<br /><br /><br /><br /><small>Circ Res: 06 Nov 2023; epub ahead of print</small></div>

<div><h4>Shear Forces Induced Platelet Clearance Is a New Mechanism of Thrombocytopenia.</h4><i>Rauch A, Dupont A, Rosa M, Desvages M, ... Casari C, Susen S</i><br /><b>Background</b><br />Thrombocytopenia has been consistently described in patients with extracorporeal membrane oxygenation (ECMO) and associated with poor outcome. However, the prevalence and underlying mechanisms remain largely unknown, and a device-related role of ECMO in thrombocytopenia has been hypothesized. This study aims to investigate the mechanisms underlying thrombocytopenia in ECMO patients.<br /><b>Methods</b><br />In a prospective cohort of 107 ECMO patients, we investigated platelet count, functions, and glycoprotein shedding. In an ex vivo mock circulatory ECMO loop, we assessed platelet responses and VWF (von Willebrand factor)-GP Ibα (glycoprotein Ibα) interactions at low- and high-flow rates, in the presence or absence of red blood cells. The clearance of human platelets subjected or not to ex vivo perfusion was studied using an in vivo transfusion model in NOD/SCID (nonobese diabetic/severe combined Immunodeficient) mice.<br /><b>Results</b><br />In ECMO patients, we observed a time-dependent decrease in platelet count starting 1 hour after device onset, with a mean drop of 7%, 35%, and 41% at 1, 24, and 48 hours post-ECMO initiation (<i>P</i>=0.00013, <i>P</i><0.0001, and <i>P</i><0.0001, respectively), regardless of the type of ECMO. This drop in platelet count was associated with a decrease in platelet GP Ibα expression (before: 47.8±9.1 versus 24 hours post-ECMO: 42.3±8.9 mean fluorescence intensity; <i>P</i>=0.002) and an increase in soluble GP Ibα plasma levels (before: 5.6±3.3 versus 24 hours post-ECMO: 10.8±4.1 µg/mL; <i>P</i><0.0001). GP Ibα shedding was also observed ex vivo and was unaffected by (1) red blood cells, (2) the coagulation potential, (3) an antibody blocking VWF-GP Ibα interaction, (4) an antibody limiting VWF degradation, and (5) supraphysiological VWF plasma concentrations. In contrast, GP Ibα shedding was dependent on rheological conditions, with a 2.8-fold increase at high- versus low-flow rates. Platelets perfused at high-flow rates before being transfused to immunodeficient mice were eliminated faster in vivo with an accelerated clearance of GP Ibα-negative versus GP Ibα-positive platelets.<br /><b>Conclusions</b><br />ECMO-associated shear forces induce GP Ibα shedding and thrombocytopenia due to faster clearance of GP Ibα-negative platelets. Inhibiting GP Ibα shedding could represent an approach to reduce thrombocytopenia during ECMO.<br /><br /><br /><br /><small>Circ Res: 27 Oct 2023; 133:826-841</small></div>

<div><h4>Microtubule-Mediated Regulation of βAR Translation and Function in Failing Hearts.</h4><i>Kwan Z, Nadappuram BP, Leung MM, Kondrashov A, ... Edel JB, Gorelik J</i><br /><b>Background</b><br />Beta-1 adrenergic receptor (β<sub>1</sub>AR)- and Beta-2 adrenergic receptor (β<sub>2</sub>AR)-mediated cyclic adenosine monophosphate signaling has distinct effects on cardiac function and heart failure progression. However, the mechanism regulating spatial localization and functional compartmentation of cardiac β-ARs remains elusive. Emerging evidence suggests that microtubule-dependent trafficking of mRNP (messenger ribonucleoprotein) and localized protein translation modulates protein compartmentation in cardiomyocytes. We hypothesized that β-AR compartmentation in cardiomyocytes is accomplished by selective trafficking of its mRNAs and localized translation.<br /><b>Methods</b><br />The localization pattern of β-AR mRNA was investigated using single molecule fluorescence in situ hybridization and subcellular nanobiopsy in rat cardiomyocytes. The role of microtubule on β-AR mRNA localization was studied using vinblastine, and its effect on receptor localization and function was evaluated with immunofluorescent and high-throughput Förster resonance energy transfer microscopy. An mRNA protein co-detection assay identified plausible β-AR translation sites in cardiomyocytes. The mechanism by which β-AR mRNA is redistributed post-heart failure was elucidated by single molecule fluorescence in situ hybridization, nanobiopsy, and high-throughput Förster resonance energy transfer microscopy on 16 weeks post-myocardial infarction and detubulated cardiomyocytes.<br /><b>Results</b><br />β<sub>1</sub>AR and β<sub>2</sub>AR mRNAs show differential localization in cardiomyocytes, with β<sub>1</sub>AR found in the perinuclear region and β<sub>2</sub>AR showing diffuse distribution throughout the cell. Disruption of microtubules induces a shift of β<sub>2</sub>AR transcripts toward the perinuclear region. The close proximity between β<sub>2</sub>AR transcripts and translated proteins suggests that the translation process occurs in specialized, precisely defined cellular compartments. Redistribution of β<sub>2</sub>AR transcripts is microtubule-dependent, as microtubule depolymerization markedly reduces the number of functional receptors on the membrane. In failing hearts, both β<sub>1</sub>AR and β<sub>2</sub>AR mRNAs are redistributed toward the cell periphery, similar to what is seen in cardiomyocytes undergoing drug-induced detubulation. This suggests that t-tubule remodeling contributes to β-AR mRNA redistribution and impaired β<sub>2</sub>AR function in failing hearts.<br /><b>Conclusions</b><br />Asymmetrical microtubule-dependent trafficking dictates differential β<sub>1</sub>AR and β<sub>2</sub>AR localization in healthy cardiomyocyte microtubules, underlying the distinctive compartmentation of the 2 β-ARs on the plasma membrane. The localization pattern is altered post-myocardial infarction, resulting from t-tubule remodeling, leading to distorted β<sub>2</sub>AR-mediated cyclic adenosine monophosphate signaling.<br /><br /><br /><br /><small>Circ Res: 23 Oct 2023; epub ahead of print</small></div>

<div><h4>Role of cAMP in Cardiomyocyte Viability: Beneficial or Detrimental?</h4><i>Zhang Y, Chen S, Luo L, Greenly S, ... Jiayuan Xu J, Yan C</i><br /><b>Background</b><br />3\', 5\'-cyclic AMP (cAMP) regulates numerous cardiac functions. Various hormones and neurotransmitters elevate intracellular cAMP (i[cAMP]) in cardiomyocytes through activating stimulatory GsPCRs (G protein [Gs]-coupled receptors) and membrane-bound ACs (adenylyl cyclases). Increasing evidence has indicated that stimulating different GsPCRs and ACs exhibits distinct, even opposite effects, on cardiomyocyte viability. However, the underlying mechanisms are not fully understood.<br /><b>Methods</b><br />We used molecular and pharmacological approaches to investigate how different GsPCR/cAMP signaling differentially regulate cardiomyocyte viability with in vitro, ex vivo, and in vivo models.<br /><b>Results</b><br />For prodeath GsPCRs, we explored beta1-adrenergic-receptor and histamine-H2-receptor. We found that their prodeath effects were similarly dependent on AC5 activation, ATP release to the extracellular space via PANX1 (pannexin-1) channel, and extracellular ATP (e[ATP])-mediated signaling involving in P2X7R (P2X purinoceptor 7) and CaMKII (Ca<sup>2+</sup>/calmodulin-dependent protein kinase II). PANX1 phosphorylation at Serine 206 by cAMP-dependent-PKA (protein-kinase-A) promoted PANX1 activation, which was critical in beta1-adrenergic-receptor- or histamine-H2-receptor-induced cardiomyocyte death in vitro and in vivo. Beta1-adrenergic-receptor or histamine-H2-receptor was localized proximately to PANX1, which permits ATP release. For prosurvival GsPCRs, we explored adenosine-A2-receptor (A2R), calcitonin-gene-related-peptide-receptor, and RXFP1 (relaxin-family peptide-receptor 1). Their prosurvival effects were dependent on AC6 activation, cAMP efflux via MRP4 (multidrug resistance protein 4), extracellular cAMP metabolism to adenosine (e[cAMP]-to-e[ADO]), and e[ADO]-mediated signaling. A2R, calcitonin-gene-related-peptide-receptor, or RXFP1 was localized proximately to MRP4, which enables cAMP efflux. Interestingly, exogenously increasing e[cAMP] levels by membrane-impermeable cAMP protected against cardiomyocyte death in vitro and in ex vivo and in vivo mouse hearts with ischemia-reperfusion injuries.<br /><b>Conclusions</b><br />Our findings indicate that the functional diversity of different GsPCRs in cardiomyocyte viability could be achieved by their ability to form unique signaling complexes (signalosomes) that determine the fate of cAMP: either stimulate ATP release by activating PKA or directly efflux to be e[cAMP].<br /><br /><br /><br /><small>Circ Res: 18 Oct 2023; epub ahead of print</small></div>

<div><h4>Nrg1 Regulates Cardiomyocyte Migration and Cell Cycle in Ventricular Development.</h4><i>Grego-Bessa J, Gómez-Apiñaniz P, Prados B, Gómez MJ, MacGrogan D, de la Pompa JL</i><br /><b>Background</b><br />Cardiac ventricles provide the contractile force of the beating heart throughout life. How the primitive endocardium-layered myocardial projections called trabeculae form and mature into the adult ventricles is of great interest for biology and regenerative medicine. Trabeculation is dependent on the signaling protein Nrg1 (neuregulin-1). However, the mechanism of action of Nrg1 and its role in ventricular wall maturation are poorly understood.<br /><b>Methods</b><br />We investigated the functions and downstream mechanisms of Nrg1 signaling during ventricular chamber development using confocal imaging, transcriptomics, and biochemical approaches in mice with cardiac-specific inactivation or overexpression of Nrg1.<br /><b>Results</b><br />Analysis of cardiac-specific <i>Nrg1</i> mutant mice showed that the transcriptional program underlying cardiomyocyte-oriented cell division and trabeculae formation depends on endocardial Nrg1 to myocardial ErbB2 (erb-b2 receptor tyrosine kinase 2) signaling and phospho-Erk (extracellular signal-regulated kinase; pErk) activation. Early endothelial loss of Nrg1 and reduced pErk activation diminished cardiomyocyte Pard3 and Crumbs2 protein and altered cytoskeletal gene expression and organization. These alterations are associated with abnormal gene expression related to mitotic spindle organization and a shift in cardiomyocyte division orientation. Nrg1 is crucial for trabecular growth and ventricular wall thickening by regulating an epithelial-to-mesenchymal transition-like process in cardiomyocytes involving migration, adhesion, cytoskeletal actin turnover, and timely progression through the cell cycle G2/M phase. Ectopic cardiac Nrg1 overexpression and high pErk signaling caused S-phase arrest, sustained high epithelial-to-mesenchymal transition-like gene expression, and prolonged trabeculation, blocking compact myocardium maturation. Myocardial trabecular patterning alterations resulting from above- or below-normal Nrg1-dependent pErk activation were concomitant with sarcomere actin cytoskeleton disorganization. The Nrg1 loss- and gain-of-function transcriptomes were enriched for Yap1 (yes-associated protein-1) gene signatures, identifying Yap1 as a potential downstream effector. Furthermore, biochemical and imaging data reveal that Nrg1 influences pErk activation and Yap1 nuclear-cytoplasmic distribution during trabeculation.<br /><b>Conclusions</b><br />These data establish the Nrg1-ErbB2/ErbB4 (erb-b2 receptor tyrosine kinase 4)-pErk axis as a crucial regulator of cardiomyocyte cell cycle progression and migration during ventricular development.<br /><br /><br /><br /><small>Circ Res: 17 Oct 2023; epub ahead of print</small></div>

<div><h4>Single-Nuclei RNA-Sequencing of the Gastrocnemius Muscle in Peripheral Artery Disease.</h4><i>Pass C, Palzkill V, Tan J, Kim K, ... Berceli SA, Ryan TE</i><br /><b>Background</b><br />Lower extremity peripheral artery disease (PAD) is a growing epidemic with limited effective treatment options. Here, we provide a single-nuclei atlas of PAD limb muscle to facilitate a better understanding of the composition of cells and transcriptional differences that comprise the diseased limb muscle.<br /><b>Methods</b><br />We obtained gastrocnemius muscle specimens from 20 patients with PAD and 12 non-PAD controls. Nuclei were isolated and single-nuclei RNA-sequencing was performed. The composition of nuclei was characterized by iterative clustering via principal component analysis, differential expression analysis, and the use of known marker genes. Bioinformatics analysis was performed to determine differences in gene expression between PAD and non-PAD nuclei, as well as subsequent analysis of intercellular signaling networks. Additional histological analyses of muscle specimens accompany the single-nuclei RNA-sequencing atlas.<br /><b>Results</b><br />Single-nuclei RNA-sequencing analysis indicated a fiber type shift with patients with PAD having fewer type I (slow/oxidative) and more type II (fast/glycolytic) myonuclei compared with non-PAD, which was confirmed using immunostaining of muscle specimens. Myonuclei from PAD displayed global upregulation of genes involved in stress response, autophagy, hypoxia, and atrophy. Subclustering of myonuclei also identified populations that were unique to PAD muscle characterized by metabolic dysregulation. PAD muscles also displayed unique transcriptional profiles and increased diversity of transcriptomes in muscle stem cells, regenerating myonuclei, and fibro-adipogenic progenitor cells. Analysis of intercellular communication networks revealed fibro-adipogenic progenitors as a major signaling hub in PAD muscle, as well as deficiencies in angiogenic and bone morphogenetic protein signaling which may contribute to poor limb function in PAD.<br /><b>Conclusions</b><br />This reference single-nuclei RNA-sequencing atlas provides a comprehensive analysis of the cell composition, transcriptional signature, and intercellular communication pathways that are altered in the PAD condition.<br /><br /><br /><br /><small>Circ Res: 12 Oct 2023; epub ahead of print</small></div>

<div><h4>MRP1-Dependent Extracellular Release of Glutathione Induces Cardiomyocyte Ferroptosis After Ischemia-Reperfusion.</h4><i>Ichihara G, Katsumata Y, Sugiura Y, Matsuoka Y, ... Ieda M, Sano M</i><br /><b>Background</b><br />The membrane components of cardiomyocytes are rich in polyunsaturated fatty acids, which are easily oxidized. Thus, an efficient glutathione-based lipid redox system is essential for maintaining cellular functions. However, the relationship between disruption of the redox system during ischemia-reperfusion (IR), oxidized lipid production, and consequent cell death (ferroptosis) remains unclear. We investigated the mechanisms underlying the disruption of the glutathione-mediated reduction system related to ferroptosis during IR and developed intervention strategies to suppress ferroptosis.<br /><b>Methods</b><br />In vivo fluctuations of both intra- and extracellular metabolite levels during IR were explored via microdialysis and tissue metabolome analysis. Oxidized phosphatidylcholines were assessed using liquid chromatography high-resolution mass spectrometry. The areas at risk following IR were assessed using triphenyl-tetrazolium chloride/Evans blue stain.<br /><b>Results</b><br />Metabolomic analysis combined with microdialysis revealed a significant release of glutathione from the ischemic region into extracellular spaces during ischemia and after reperfusion. The release of glutathione into extracellular spaces and a concomitant decrease in intracellular glutathione concentrations were also observed during anoxia-reperfusion in an in vitro cardiomyocyte model. This extracellular glutathione release was prevented by chemical inhibition or genetic suppression of glutathione transporters, mainly MRP1 (multidrug resistance protein 1). Treatment with MRP1 inhibitor reduced the intracellular reactive oxygen species levels and lipid peroxidation, thereby inhibiting cell death. Subsequent in vivo evaluation of endogenously oxidized phospholipids following IR demonstrated the involvement of ferroptosis, as levels of multiple oxidized phosphatidylcholines were significantly elevated in the ischemic region 12 hours after reperfusion. Inhibition of the MRP1 transporter also alleviated intracellular glutathione depletion in vivo and significantly reduced the generation of oxidized phosphatidylcholines. Administration of MRP1 inhibitors significantly attenuated infarct size after IR injury.<br /><b>Conclusions</b><br />Glutathione was released continuously during IR, primarily in an MRP1-dependent manner, and induced ferroptosis. Suppression of glutathione release attenuated ferroptosis and reduced myocardial infarct size following IR.<br /><br /><br /><br /><small>Circ Res: 11 Oct 2023; epub ahead of print</small></div>

<div><h4>Body Mass Index, Adverse Pregnancy Outcomes, and Cardiovascular Disease Risk.</h4><i>Khan SS, Petito LC, Huang X, Harrington K, ... Grobman WA, NICHD nuMoM2b and NHLBI nuMoM2b Heart Health Study Networks</i><br /><b>Background</b><br />Obesity is a well-established risk factor for both adverse pregnancy outcomes (APOs) and cardiovascular disease (CVD). However, it is not known whether APOs are mediators or markers of the obesity-CVD relationship. This study examined the association between body mass index, APOs, and postpartum CVD risk factors.<br /><b>Methods</b><br />The sample included adults from the nuMoM2b (Nulliparous Pregnancy Outcomes Study: Monitoring Mothers-To-Be) Heart Health Study who were enrolled in their first trimester (6 weeks-13 weeks 6 days gestation) from 8 US sites. Participants had a follow-up visit at 3.7 years postpartum. APOs, which included hypertensive disorders of pregnancy, preterm birth, small-for-gestational-age birth, and gestational diabetes, were centrally adjudicated. Mediation analyses estimated the association between early pregnancy body mass index and postpartum CVD risk factors (hypertension, hyperlipidemia, and diabetes) and the proportion mediated by each APO adjusted for demographics and baseline health behaviors, psychosocial stressors, and CVD risk factor levels.<br /><b>Results</b><br />Among 4216 participants enrolled, mean±SD maternal age was 27±6 years. Early pregnancy prevalence of overweight was 25%, and obesity was 22%. Hypertensive disorders of pregnancy occurred in 15%, preterm birth in 8%, small-for-gestational-age birth in 11%, and gestational diabetes in 4%. Early pregnancy obesity, compared with normal body mass index, was associated with significantly higher incidence of postpartum hypertension (adjusted odds ratio, 1.14 [95% CI, 1.10-1.18]), hyperlipidemia (1.11 [95% CI, 1.08-1.14]), and diabetes (1.03 [95% CI, 1.01-1.04]) even after adjustment for baseline CVD risk factor levels. APOs were associated with higher incidence of postpartum hypertension (1.97 [95% CI, 1.61-2.40]) and hyperlipidemia (1.31 [95% CI, 1.03-1.67]). Hypertensive disorders of pregnancy mediated a small proportion of the association between obesity and incident hypertension (13% [11%-15%]) and did not mediate associations with incident hyperlipidemia or diabetes. There was no significant mediation by preterm birth or small-for-gestational-age birth.<br /><b>Conclusions</b><br />There was heterogeneity across APO subtypes in their association with postpartum CVD risk factors and mediation of the association between early pregnancy obesity and postpartum CVD risk factors. However, only a small or nonsignificant proportion of the association between obesity and CVD risk factors was mediated by any of the APOs, suggesting APOs are a marker of prepregnancy CVD risk and not a predominant cause of postpartum CVD risk.<br /><br /><br /><br /><small>Circ Res: 10 Oct 2023; epub ahead of print</small></div>

<div><h4>Disrupted Binding of Cystathionine γ-Lyase to p53 Promotes Endothelial Senescence.</h4><i>Hu J, Leisegang MS, Looso M, Drekolia MK, ... Fleming I, Bibli SI</i><br /><b>Background</b><br />Advanced age is unequivocally linked to the development of cardiovascular disease; however, the mechanisms resulting in reduced endothelial cell regeneration remain poorly understood. Here, we investigated novel mechanisms involved in endothelial cell senescence that impact endothelial cell transcription and vascular repair after injury.<br /><b>Methods</b><br />Native endothelial cells were isolated from young (20±3.4 years) and aged (80±2.3 years) individuals and subjected to molecular analyses to assess global transcriptional and metabolic changes. in vitro studies were conducted using primary human and murine endothelial cells. A murine aortic reendothelialization model was used to examine endothelial cell regenerative capacity in vivo.<br /><b>Results</b><br />RNA sequencing of native endothelial cells revealed that aging resulted in p53-mediated reprogramming to express senescence-associated genes and suppress glycolysis. Reduced glucose uptake and ATP contributed to attenuated assembly of the telomerase complex, which was required for endothelial cell proliferation. Enhanced p53 activity in aging was linked to its acetylation on K120 due to enhanced activity of the acetyltransferase MOZ (monocytic leukemic zinc finger). Mechanistically, p53 acetylation and translocation were, at least partially, attributed to the loss of the vasoprotective enzyme, CSE (cystathionine γ-lyase). CSE physically anchored p53 in the cytosol to prevent its nuclear translocation and in its absence inhibited Akt-mediated MOZ phosphorylation, which in turn increased MOZ activity and subsequently p53 acetylation. In mice, the endothelial cell-specific deletion of CSE activated p53, induced premature endothelial senescence, and arrested vascular repair after injury. In contrast, the adeno-associated virus 9-mediated reexpression of an active CSE mutant retained p53 in the cytosol, maintained endothelial glucose metabolism and proliferation, and prevented endothelial cell senescence. Adenoviral overexpression of CSE in native endothelial cells from aged individuals maintained low p53 activity and reactivated telomerase to revert endothelial cell senescence.<br /><b>Conclusions</b><br />Aging-associated impairment of vascular repair is partly determined by the vasoprotective enzyme CSE.<br /><br /><br /><br /><small>Circ Res: 06 Oct 2023; epub ahead of print</small></div>

<div><h4>Myocardial Recovery in Recent Onset Dilated Cardiomyopathy: Role of and Cardiac Fibrosis.</h4><i>Liu D, Wang M, Murthy V, McNamara DM, ... Pereira NL, IMAC-2 Investigators</i><br /><b>Background</b><br />Dilated cardiomyopathy (DCM) is a major cause of heart failure and carries a high mortality rate. Myocardial recovery in DCM-related heart failure patients is highly variable, with some patients having little or no response to standard drug therapy. A genome-wide association study may agnostically identify biomarkers and provide novel insight into the biology of myocardial recovery in DCM.<br /><b>Methods</b><br />A genome-wide association study for change in left ventricular ejection fraction was performed in 686 White subjects with recent-onset DCM who received standard pharmacotherapy. Genome-wide association study signals were subsequently functionally validated and studied in relevant cellular models to understand molecular mechanisms that may have contributed to the change in left ventricular ejection fraction.<br /><b>Results</b><br />The genome-wide association study identified a highly suggestive locus that mapped to the 5\'-flanking region of the <i>CDCP1</i> (CUB domain containing protein 1) gene (rs6773435; <i>P</i>=7.12×10<sup>-</sup><sup>7</sup>). The variant allele was associated with improved cardiac function and decreased <i>CDCP1</i> transcription. CDCP1 expression was significantly upregulated in human cardiac fibroblasts (HCFs) in response to the PDGF (platelet-derived growth factor) signaling, and knockdown of CDCP1 significantly repressed HCF proliferation and decreased AKT phosphorylation. Transcriptomic profiling after CDCP1 knockdown in HCFs supported the conclusion that CDCP1 regulates HCF proliferation and mitosis. In addition, CDCP1 knockdown in HCFs resulted in significantly decreased expression of soluble ST2, a prognostic biomarker for heart failure and inductor of cardiac fibrosis.<br /><b>Conclusions</b><br /><i>CDCP1</i> may play an important role in myocardial recovery in recent-onset DCM and mediates its effect primarily by attenuating cardiac fibrosis.<br /><br /><br /><br /><small>Circ Res: 06 Oct 2023; epub ahead of print</small></div>

<div><h4>TMEM215 Prevents Endothelial Cell Apoptosis in Vessel Regression by Blunting BIK-Regulated ER-to-Mitochondrial Ca Influx.</h4><i>Zhang P, Yan X, Zhang X, Liu Y, ... Liang L, Han H</i><br /><b>Background</b><br />In developmental and pathological tissues, nascent vessel networks generated by angiogenesis require further pruning/regression to delete nonfunctional endothelial cells (ECs) by apoptosis and migration. Mechanisms underlying EC apoptosis during vessel pruning remain elusive. TMEM215 (transmembrane protein 215) is an endoplasmic reticulum-located, 2-pass transmembrane protein. We have previously demonstrated that TMEM215 knockdown in ECs leads to cell death, but its physiological function and mechanism are unclear.<br /><b>Methods</b><br />We characterized the role and mechanism of TMEM215 in EC apoptosis using HUVECs by identifying its interacting proteins with immunoprecipitation-mass spectrometry. The physiological function of TMEM215 in ECs was assessed by establishing a conditional knockout mouse strain. The role of TMEM215 in pathological angiogenesis was evaluated by tumor and choroidal neovascularization models. We also tried to evaluate its translational value by delivering a <i>Tmem215</i> siRNA using nanoparticles in vivo.<br /><b>Results</b><br /><i>TMEM215</i> knockdown in ECs induced apoptotic cell death. We identified the chaperone BiP as a binding partner of TMEM215, and TMEM215 forms a complex with and facilitates the interaction of BiP with the BH (BCL-2 homology) 3-only proapoptotic protein BIK (BCL-2 interacting killer). <i>TMEM215</i> knockdown triggered apoptosis in a BIK-dependent way and was abrogated by BCL-2. Notably, <i>TMEM215</i> knockdown increased the number and diminished the distance of mitochondria-associated endoplasmic reticulum membranes and increased mitochondrial calcium influx. Inhibiting mitochondrial calcium influx by blocking the IP<sub>3</sub>R (inositol 1,4,5-trisphosphate receptor) or MCU (mitochondrial calcium uniporter) abrogated <i>TMEM215</i> knockdown-induced apoptosis. <i>TMEM215</i> expression in ECs was induced by physiological laminar shear stress via <i>EZH2</i> downregulation. In EC-specific <i>Tmem215</i> knockout mice, induced <i>Tmem215</i> depletion impaired the regression of retinal vasculature characterized by reduced vessel density, increased empty basement membrane sleeves, and increased EC apoptosis. Moreover, EC-specific <i>Tmem215</i> ablation inhibited tumor growth with disrupted vasculature. However, <i>Tmem215</i> ablation in adult mice attenuated lung metastasis, consistent with reduced <i>Vcam1</i> expression. Administration of nanoparticles carrying <i>Tmem215</i> siRNA also inhibited tumor growth and choroidal neovascularization injury.<br /><b>Conclusions</b><br /><i>TMEM215</i>, which is induced by blood flow-derived shear stress via downregulating <i>EZH2</i>, protects ECs from BIK-triggered mitochondrial apoptosis mediated by calcium influx through mitochondria-associated ER membranes during vessel pruning, thus providing a novel target for antiangiogenic therapy.<br /><br /><br /><br /><small>Circ Res: 26 Sep 2023; epub ahead of print</small></div>

<div><h4>PPP1R12C Promotes Atrial Hypocontractility in Atrial Fibrillation.</h4><i>Perike S, Gonzalez-Gonzalez FJ, Abu-Taha I, Damen FW, ... Wehrens XHT, McCauley MD</i><br /><b>Background</b><br />Atrial fibrillation (AF)-the most common sustained cardiac arrhythmia-increases thromboembolic stroke risk 5-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C (protein phosphatase 1 regulatory subunit 12C)-the PP1 (protein phosphatase 1) regulatory subunit targeting MLC2a (atrial myosin light chain 2)-causes hypophosphorylation of MLC2a and results in atrial hypocontractility.<br /><b>Methods</b><br />Right atrial appendage tissues were isolated from human patients with AF versus sinus rhythm controls. Western blots, coimmunoprecipitation, and phosphorylation studies were performed to examine how the PP1c (PP1 catalytic subunit)-PPP1R12C interaction causes MLC2a dephosphorylation. In vitro studies of pharmacological MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with electrophysiology studies.<br /><b>Results</b><br />In human patients with AF, PPP1R12C expression was increased 2-fold versus sinus rhythm controls (<i>P</i>=2.0×10<sup>-</sup><sup>2</sup>; n=12 and 12 in each group) with >40% reduction in MLC2a phosphorylation (<i>P</i>=1.4×10<sup>-</sup><sup>6</sup>; n=12 and 12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF (<i>P</i>=2.9×10<sup>-2</sup> and 6.7×10<sup>-3</sup>, respectively; n=8 and 8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a and dephosphorylation of MLC2a. Mice treated with lentiviral PPP1R12C vector demonstrated a 150% increase in left atrial size versus controls (<i>P</i>=5.0×10<sup>-</sup><sup>6</sup>; n=12, 8, and 12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in mice treated with lentiviral PPP1R12C vector was significantly higher than in controls (<i>P</i>=1.8×10<sup>-2</sup> and 4.1×10<sup>-2</sup>, respectively; n=6, 6, and 5).<br /><b>Conclusions</b><br />Patients with AF exhibit increased levels of PPP1R12C protein compared with controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key determinant of atrial contractility in AF.<br /><br /><br /><br /><small>Circ Res: 22 Sep 2023; epub ahead of print</small></div>

<div><h4>Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension.</h4><i>Ranasinghe ADCU, Holohan M, Borger KM, Donahue DL, ... Castellino FJ, Schwarz MA</i><br /><b>Background</b><br />Epigenetic regulation of vascular remodeling in pulmonary hypertension (PH) is poorly understood. Transcription regulating, histone acetylation code alters chromatin accessibility to promote transcriptional activation. Our goal was to identify upstream mechanisms that disrupt epigenetic equilibrium in PH.<br /><b>Methods</b><br />Human pulmonary artery smooth muscle cells (PASMCs), human idiopathic pulmonary arterial hypertension (iPAH):human PASMCs, iPAH lung tissue, failed donor lung tissue, human pulmonary microvascular endothelial cells, iPAH:PASMC and non-iPAH:PASMC RNA-seq databases, NanoString nCounter, and cleavage under targets and release using nuclease were utilized to investigate histone acetylation, hyperacetylation targets, protein and gene expression, sphingolipid activation, cell proliferation, and gene target identification. SPHK2 (sphingosine kinase 2) knockout was compared with control C57BL/6NJ mice after 3 weeks of hypoxia and assessed for indices of PH.<br /><b>Results</b><br />We identified that Human PASMCs are vulnerable to the transcription-promoting epigenetic mediator histone acetylation resulting in alterations in transcription machinery and confirmed its pathological existence in PH:PASMC cells. We report that SPHK2 is elevated as much as 20-fold in iPAH lung tissue and is elevated in iPAH:PASMC cells. During PH pathogenesis, nuclear SPHK2 activates nuclear bioactive lipid S1P (sphingosine 1-phosphate) catalyzing enzyme and mediates transcription regulating histone H3K9 acetylation (acetyl histone H3 lysine 9 [Ac-H3K9]) through EMAP (endothelial monocyte activating polypeptide) II. In iPAH lungs, we identified a 4-fold elevation of the reversible epigenetic transcription modulator Ac-H3K9:H3 ratio. Loss of SPHK2 inhibited hypoxic-induced PH and Ac-H3K9 in mice. We discovered that pulmonary vascular endothelial cells are a priming factor of the EMAP II/SPHK2/S1P axis that alters the acetylome with a specificity for PASMC, through hyperacetylation of histone H3K9. Using cleavage under targets and release using nuclease, we further show that EMAP II-mediated SPHK2 has the potential to modify the local transcription machinery of pluripotency factor KLF4 (Krüppel-like factor 4) by hyperacetylating KLF4 Cis-regulatory elements while deletion and targeted inhibition of SPHK2 rescues transcription altering Ac-H3K9.<br /><b>Conclusions</b><br />SPHK2 expression and its activation of the reversible histone H3K9 acetylation in human pulmonary artery smooth muscle cell represent new therapeutic targets that could mitigate PH vascular remodeling.<br /><br /><br /><br /><small>Circ Res: 12 Sep 2023; epub ahead of print</small></div>

<div><h4>Cardiac Aging Is Promoted by Pseudohypoxia Increasing p300-Induced Glycolysis.</h4><i>Serio S, Pagiatakis C, Musolino E, Felicetta A, ... Condorelli G, Papait R</i><br /><b>Rationale</b><br />Heart failure is typical in the elderly. Metabolic remodeling of cardiomyocytes underlies inexorable deterioration of cardiac function with aging: glycolysis increases at the expense of oxidative phosphorylation, causing an energy deficit contributing to impaired contractility. Better understanding of the mechanisms of this metabolic switching could be critical for reversing the condition.<br /><b>Objective</b><br />To investigate the role of 3 histone modifications (H3K27ac, H3K27me3, and H3K4me1) in the metabolic remodeling occurring in the aging heart.<br /><b>Results</b><br />We report a set of species-conserved enhancers associated with transcriptional changes underlying age-related metabolic remodeling in cardiomyocytes. Activation of the enhancer region of <i>Hk2</i>-a key glycolysis pathway gene-was fostered in old age-onset mouse heart by pseudohypoxia, wherein hypoxia-related genes are expressed under normal O<sub>2</sub> levels, via increased activity of the transcriptional coactivator p300 (E1A-associated binding protein p300)/CBP (CREB-binding protein). Pharmacological inhibition of p300/CBP before the onset of cardiac aging led to a more aerobic, less glycolytic, metabolic state, improved heart contractility, and overall blunting of cardiac decline.<br /><b>Conclusions</b><br />Taken together, our results suggest how epigenetic dysregulation of glycolysis pathway enhancers could potentially be targeted to treat heart failure in the elderly.<br /><br /><br /><br /><small>Circ Res: 08 Sep 2023; epub ahead of print</small></div>

<div><h4>Extracellular Perinexal Separation Is a Principal Determinant of Cardiac Conduction.</h4><i>Adams WP, Raisch TB, Zhao Y, Davalos R, ... Gourdie RG, Poelzing S</i><br /><b>Background</b><br />Cardiac conduction is understood to occur through gap junctions. Recent evidence supports ephaptic coupling as another mechanism of electrical communication in the heart. Conduction via gap junctions predicts a direct relationship between conduction velocity (CV) and bulk extracellular resistance. By contrast, ephaptic theory is premised on the existence of a biphasic relationship between CV and the volume of specialized extracellular clefts within intercalated discs such as the perinexus. Our objective was to determine the relationship between ventricular CV and structural changes to micro- and nanoscale extracellular spaces.<br /><b>Methods</b><br />Conduction and Cx43 (connexin43) protein expression were quantified from optically mapped guinea pig whole-heart preparations perfused with the osmotic agents albumin, mannitol, dextran 70 kDa, or dextran 2 MDa. Peak sodium current was quantified in isolated guinea pig ventricular myocytes. Extracellular resistance was quantified by impedance spectroscopy. Intercellular communication was assessed in a heterologous expression system with fluorescence recovery after photobleaching. Perinexal width was quantified from transmission electron micrographs.<br /><b>Results</b><br />CV primarily in the transverse direction of propagation was significantly reduced by mannitol and increased by albumin and both dextrans. The combination of albumin and dextran 70 kDa decreased CV relative to albumin alone. Extracellular resistance was reduced by mannitol, unchanged by albumin, and increased by both dextrans. Cx43 expression and conductance and peak sodium currents were not significantly altered by the osmotic agents. In response to osmotic agents, perinexal width, in order of narrowest to widest, was albumin with dextran, 70 kDa; albumin or dextran, 2 MDa; dextran, 70 kDa or no osmotic agent, and mannitol. When compared in the same order, CV was biphasically related to perinexal width.<br /><b>Conclusions</b><br />Cardiac conduction does not correlate with extracellular resistance but is biphasically related to perinexal separation, providing evidence that the relationship between CV and extracellular volume is determined by ephaptic mechanisms under conditions of normal gap junctional coupling.<br /><br /><br /><br /><small>Circ Res: 08 Sep 2023; epub ahead of print</small></div>

<div><h4>Epoxyeicosatrienoic Acids Prevent Cardiac Dysfunction in Viral Myocarditis via IFN-I Signaling.</h4><i>Zhou Z, Zhang M, Zhao C, Gao X, ... Hu J, Wang DW</i><br /><AbstractText>Myocarditis is a challenging inflammatory disease of the heart, and better understanding of its pathogenesis is needed to develop specific drug therapies. Epoxyeicosatrienoic acids (EETs), active molecules synthesized by CYP (cytochrome P450) enzymes from arachidonic acids and hydrolyzed to less active dihydroxyeicosatrienoic acids by sEH (soluble epoxide hydrolase), have been attributed anti-inflammatory activity. Here, we investigated whether EETs have immunomodulatory activity and exert protective effects on coxsackie B3 virus-induced myocarditis. Viral infection altered eicosanoid epoxide and diol levels in both patients with myocarditis and in the murine heart and correlated with the increased expression and activity of sEH after coxsackie B3 virus infection. Administration of a sEH inhibitor prevented coxsackie B3 virus-induced cardiac dysfunction and inflammatory infiltration. Importantly, EET/sEH inhibitor treatment attenuated viral infection or improved viral resistance by activating type I IFN (interferon) signaling. At the molecular level, EETs enhanced the interaction between GSK3β (glycogen synthase kinase-3 beta) and TBK1 (TANK-binding kinase 1) to promote IFN-β production. Our findings revealed that EETs and sEH inhibitors prevent the progress of coxsackie B3 virus-induced myocarditis, particularly by promoting viral resistance by increasing IFN production.</AbstractText><br /><br /><br /><br /><small>Circ Res: 08 Sep 2023; epub ahead of print</small></div>

<div><h4>ADAMTS-7 Modulates Atherosclerotic Plaque Formation by Degradation of TIMP-1.</h4><i>Amin Sharifi M, Wierer M, An Dang T, Milic J, ... Schunkert H, Kessler T</i><br /><b>Background</b><br />The <i>ADAMTS7</i> locus was genome-wide and significantly associated with coronary artery disease. Lack of the ECM (extracellular matrix) protease ADAMTS-7 (A disintegrin and metalloproteinase-7) was shown to reduce atherosclerotic plaque formation. Here, we sought to identify molecular mechanisms and downstream targets of ADAMTS-7 mediating the risk of atherosclerosis.<br /><b>Methods</b><br />Targets of ADAMTS-7 were identified by high-resolution mass spectrometry of atherosclerotic plaques from Apoe<sup>-/-</sup> and Apoe<sup>-/-</sup>Adamts7<sup>-/-</sup> mice. ECM proteins were identified using solubility profiling. Putative targets were validated using immunofluorescence, in vitro degradation assays, coimmunoprecipitation, and Förster resonance energy transfer-based protein-protein interaction assays. <i>ADAMTS7</i> expression was measured in fibrous caps of human carotid artery plaques.<br /><b>Results</b><br />In humans, <i>ADAMTS7</i> expression was higher in caps of unstable as compared to stable carotid plaques. Compared to Apoe<sup>-/-</sup> mice, atherosclerotic aortas of Apoe<sup>-/-</sup> mice lacking Adamts-7 (Apoe<sup>-/-</sup>Adamts7<sup>-/-</sup>) contained higher protein levels of Timp-1 (tissue inhibitor of metalloprotease-1). In coimmunoprecipitation experiments, the catalytic domain of ADAMTS-7 bound to TIMP-1, which was degraded in the presence of ADAMTS-7 in vitro. ADAMTS-7 reduced the inhibitory capacity of TIMP-1 at its canonical target MMP-9 (matrix metalloprotease-9). As a downstream mechanism, we investigated collagen content in plaques of Apoe<sup>-/-</sup> and Apoe<sup>-/-</sup>Adamts7<sup>-/-</sup> mice after the Western diet. Picrosirius red staining of the aortic root revealed less collagen as a readout of higher MMP-9 activity in Apoe<sup>-/-</sup> as compared to Apoe<sup>-/-</sup> Adamts7<sup>-/-</sup> mice. To facilitate high-throughput screening for ADAMTS-7 inhibitors with the aim of decreasing TIMP-1 degradation, we designed a Förster resonance energy transfer-based assay targeting the ADAMTS-7 catalytic site.<br /><b>Conclusions</b><br />ADAMTS-7, which is induced in unstable atherosclerotic plaques, decreases TIMP-1 stability reducing its inhibitory effect on MMP-9, which is known to promote collagen degradation and is likewise associated with coronary artery disease. Disrupting the interaction of ADAMTS-7 and TIMP-1 might be a strategy to increase collagen content and plaque stability for the reduction of atherosclerosis-related events.<br /><br /><br /><br /><small>Circ Res: 07 Sep 2023; epub ahead of print</small></div>

<div><h4>SIRT3 Regulates Clearance of Apoptotic Cardiomyocytes by Deacetylating Frataxin.</h4><i>Gao J, Huang C, Kong L, Zhou W, ... Wei T, Shen W</i><br /><b>Background</b><br />Efferocytosis is an activity of macrophages that is pivotal for the resolution of inflammation in hypertension. The precise mechanism by which macrophages coordinate efferocytosis and internalize apoptotic cardiomyocytes remains unknown. The aim of this study was to determine whether SIRT3 (sirtuin-3) is required for both apoptotic cardiomyocyte engulfment and anti-inflammatory responses during efferocytosis.<br /><b>Methods</b><br />We generated myeloid SIRT3 knockout mice and knock-in mice carrying an acetylation-defective lysine to arginine K189R mutation (FXN<sup>K189R</sup>). The mice were given Ang II (angiotensin II) infusion for 7 days. We analyzed cardiac macrophages\' mitochondrial iron levels, efferocytosis activity, and phenotype both in vivo and in vitro.<br /><b>Results</b><br />We showed that SIRT3 deficiency exacerbated Ang II-induced downregulation of the efferocytosis receptor MerTK (c-Mer tyrosine kinase) and proinflammatory cytokine production, accompanied by disrupted mitochondrial iron homeostasis in cardiac macrophages. Quantitative acetylome analysis revealed that SIRT3 deacetylated FXN (frataxin) at lysine 189. Ang II attenuated SIRT3 activity and enhanced the acetylation level of FXN K189. Acetylated FXN further reduced the synthesis of ISCs (iron-sulfur clusters), resulting in mitochondrial iron accumulation. Phagocytic internalization of apoptotic cardiomyocytes increased myoglobin content, and derived iron ions promoted mitochondrial iron overload and lipid peroxidation. An iron chelator deferoxamine improved the levels of MerTK and efferocytosis, thereby attenuating proinflammatory macrophage activation. FXN<sup>K189R</sup> mice showed improved macrophage efferocytosis, reduced cardiac inflammation, and suppressed cardiac fibrosis.<br /><b>Conclusions</b><br />The SIRT3-FXN axis has the potential to resolve cardiac inflammation by increasing macrophage efferocytosis and anti-inflammatory activities.<br /><br /><br /><br /><small>Circ Res: 30 Aug 2023; epub ahead of print</small></div>

<div><h4>Proteomic Atlas of Atherosclerosis: The Contribution of Proteoglycans to Sex Differences, Plaque Phenotypes, and Outcomes.</h4><i>Theofilatos K, Stojkovic S, Hasman M, van der Laan SW, ... Wojta J, Mayr M</i><br /><b>Background</b><br />Using proteomics, we aimed to reveal molecular types of human atherosclerotic lesions and study their associations with histology, imaging, and cardiovascular outcomes.<br /><b>Methods</b><br />Two hundred nineteen carotid endarterectomy samples were procured from 120 patients. A sequential protein extraction protocol was employed in conjunction with multiplexed, discovery proteomics. To focus on extracellular proteins, parallel reaction monitoring was employed for targeted proteomics. Proteomic signatures were integrated with bulk, single-cell, and spatial RNA-sequencing data, and validated in 200 patients from the Athero-Express Biobank study.<br /><b>Results</b><br />This extensive proteomics analysis identified plaque inflammation and calcification signatures, which were inversely correlated and validated using targeted proteomics. The inflammation signature was characterized by the presence of neutrophil-derived proteins, such as S100A8/9 and myeloperoxidase, whereas the calcification signature included fetuin-A, osteopontin, and gamma-carboxylated proteins. The proteomics data also revealed sex differences in atherosclerosis, with large-aggregating proteoglycans versican and aggrecan being more abundant in females and exhibiting an inverse correlation with estradiol levels. The integration of RNA-sequencing data attributed the inflammation signature predominantly to neutrophils and macrophages, and the calcification and sex signatures to smooth muscle cells, except for certain plasma proteins that were not expressed but retained in plaques, such as fetuin-A. Dimensionality reduction and machine learning techniques were applied to identify 4 distinct plaque phenotypes based on proteomics data. A protein signature of 4 key proteins (calponin, protein C, serpin H1, and versican) predicted future cardiovascular mortality with an area under the curve of 75% and 67.5% in the discovery and validation cohort, respectively, surpassing the prognostic performance of imaging and histology.<br /><b>Conclusions</b><br />Plaque proteomics redefined clinically relevant patient groups with distinct outcomes, identifying subgroups of male and female patients with elevated risk of future cardiovascular events.<br /><br /><br /><br /><small>Circ Res: 30 Aug 2023; epub ahead of print</small></div>

<div><h4>Neutrophils for Revascularization Require Activation of CCR6 and CCL20 by TNFα.</h4><i>Lörchner H, Cañes Esteve L, Góes ME, Harzenetter R, ... Pöling J, Braun T</i><br /><b>Background</b><br />Activation of immune-inflammatory pathways involving TNFα (tumor necrosis factor alpha) signaling is critical for revascularization and peripheral muscle tissue repair after ischemic injury. However, mechanisms of TNFα-driven inflammatory cascades directing recruitment of proangiogenic immune cells to sites of ischemia are unknown.<br /><b>Methods</b><br />Muscle tissue revascularization after permanent femoral artery ligation was monitored in mutant mice by laser Doppler imaging and light sheet fluorescence microscopy. TNFα-mediated signaling and the role of the CCL20 (C-C motif chemokine ligand 20)-CCR6 (C-C chemokine receptor 6) axis for formation of new vessels was studied in vitro and in vivo using bone marrow transplantation, flow cytometry, as well as biochemical and molecular biological techniques.<br /><b>Results</b><br />TNFα-mediated activation of tumor necrosis factor receptor TNFR (tumor necrosis factor receptor) 1 but not TNFR2 was found to be required for postischemic muscle tissue revascularization. Bone marrow-derived CCR6<sup>+</sup> neutrophil granulocytes were identified as a previously undescribed TNFα-induced population of proangiogenic neutrophils, characterized by increased expression of VEGFA (vascular endothelial growth factor A). Mechanistically, postischemic activation of TNFR1 induced expression of the CCL20 in vascular cells and promoted translocation of the CCL20 receptor CCR6 to the cell surface of neutrophils, essentially conditioning VEGFA-expressing proangiogenic neutrophils for CCL20-dependent recruitment to sites of ischemia. Moreover, impaired revascularization of ischemic peripheral muscle tissue in diabetic mice was associated with reduced numbers of proangiogenic neutrophils and diminished CCL20 expression. Administration of recombinant CCL20 enhanced recruitment of proangiogenic neutrophils and improved revascularization of diabetic ischemic skeletal muscles, which was sustained by sequential treatment with fluvastatin.<br /><b>Conclusions</b><br />We demonstrate that site-specific activation of the CCL20-CCR6 axis via TNFα recruits proangiogenic VEGFA-expressing neutrophils to sites of ischemic injury for initiation of muscle tissue revascularization. The findings provide an attractive option for tissue revascularization, particularly under diabetic conditions.<br /><br /><br /><br /><small>Circ Res: 29 Aug 2023; epub ahead of print</small></div>

<div><h4>Cardiac Vagal Nerve Activity Increases During Exercise to Enhance Coronary Blood Flow.</h4><i>Shanks J, Pachen M, Chang JW, George B, Ramchandra R</i><br /><b>Background</b><br />The phrase complete vagal withdrawal is often used when discussing autonomic control of the heart during exercise. However, more recent studies have challenged this assumption. We hypothesized that cardiac vagal activity increases during exercise and maintains cardiac function via transmitters other than acetylcholine.<br /><b>Methods</b><br />Chronic direct recordings of cardiac vagal nerve activity, cardiac output, coronary artery blood flow, and heart rate were recorded in conscious adult sheep during whole-body treadmill exercise. Cardiac innervation of the left cardiac vagal branch was confirmed with lipophilic tracer dyes (DiO). Sheep were exercised with pharmacological blockers of acetylcholine (atropine, 250 mg), VIP (vasoactive intestinal peptide; [4Cl-D-Phe6,Leu17]VIP 25 µg), or saline control, randomized on different days. In a subset of sheep, the left cardiac vagal branch was denervated.<br /><b>Results</b><br />Neural innervation from the cardiac vagal branch is seen at major cardiac ganglionic plexi, and within the fat pads associated with the coronary arteries. Directly recorded cardiac vagal nerve activity increased during exercise. Left cardiac vagal branch denervation attenuated the maximum changes in coronary artery blood flow (maximum exercise, control: 63.5±5.9 mL/min, n=8; cardiac vagal denervated: 32.7±5.6 mL/min, n=6, <i>P=</i>2.5×10<sup>-</sup><sup>7</sup>), cardiac output, and heart rate during exercise. Atropine did not affect any cardiac parameters during exercise, but VIP antagonism significantly reduced coronary artery blood flow during exercise to a similar level to vagal denervation.<br /><b>Conclusions</b><br />Our study demonstrates that cardiac vagal nerve activity actually increases and is crucial for maintaining cardiac function during exercise. Furthermore, our findings show the dynamic modulation of coronary artery blood flow during exercise is mediated by VIP.<br /><br /><br /><br /><small>Circ Res: 29 Aug 2023; epub ahead of print</small></div>

<div><h4>S14-Phosphorylated RPN6 Mediates Proteasome Activation by PKA and Alleviates Proteinopathy.</h4><i>Yang L, Parajuli N, Wu P, Liu J, Wang X</i><br /><b>Background</b><br />A better understanding of the regulation of proteasome activities can facilitate the search for new therapeutic strategies. A cell culture study shows that cAMP-dependent PKA (protein kinase A) activates the 26S proteasome by pS14-Rpn6 (serine14-phosphorylated Rpn6), but this discovery and its physiological significance remain to be established in vivo.<br /><b>Methods</b><br />Male and female mice with Ser14 of Rpn6 mutated to Ala (S14A [Rpn6/Psmd11<sup>S14A</sup>]) or Asp (S14D) to respectively block or mimic pS14-Rpn6 were created and used along with cells derived from them. cAMP/PKA were manipulated pharmacologically. Ubiquitin-proteasome system functioning was evaluated with the GFPdgn reporter mouse and proteasomal activity assays. Impact of S14A and S14D on proteotoxicity was tested in mice and cardiomyocytes overexpressing the misfolded protein R120G-CryAB (R120G).<br /><b>Results</b><br />PKA activation increased pS14-Rpn6 and 26S proteasome activities in wild-type but not S14A embryonic fibroblasts (mouse embryonic fibroblasts), adult cardiomyocytes, and mouse hearts. Basal 26S proteasome activities were significantly greater in S14D myocardium and adult mouse cardiomyocytes than in wild-type counterparts. S14D::GFPdgn mice displayed significantly lower myocardial GFPdgn protein but not mRNA levels than GFPdgn mice. In R120G mice, a classic model of cardiac proteotoxicity, basal myocardial pS14-Rpn6 was significantly lower compared with nontransgenic littermates, which was not always associated with reduction of other phosphorylated PKA substrates. Cultured S14D neonatal cardiomyocytes displayed significantly faster proteasomal degradation of R120G than wild-type neonatal cardiomyocytes. Compared with R120G mice, S14D/S14D::R120G mice showed significantly greater myocardial proteasome activities, lower levels of total and K48-linked ubiquitin conjugates, and of aberrant CryAB protein aggregates, less fetal gene reactivation, and cardiac hypertrophy, and delays in cardiac malfunction.<br /><b>Conclusions</b><br />This study establishes in animals that pS14-Rpn6 mediates the activation of 26S proteasomes by PKA and that the reduced pS14-Rpn6 is a key pathogenic factor in cardiac proteinopathy, thereby identifying a new therapeutic target to reduce cardiac proteotoxicity.<br /><br /><br /><br /><small>Circ Res: 29 Aug 2023; epub ahead of print</small></div>