<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>