<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>

<div><h4>Brain α2δ-1-Bound NMDA Receptors Drive Calcineurin Inhibitor-Induced Hypertension.</h4><i>Zhou JJ, Shao JY, Chen SR, Pan HL</i><br /><b>Background</b><br />Calcineurin is highly enriched in immune T cells and in the nervous system. Calcineurin inhibitors, including cyclosporine and tacrolimus (FK506), are the cornerstone of immunosuppressive regimens for preserving transplanted organs and tissues. However, these drugs often cause persistent hypertension owing to excess sympathetic outflow, which is maintained by N-methyl-D-aspartate receptor (NMDAR)-mediated excitatory input to the hypothalamic paraventricular nucleus (PVN). It is unclear how calcineurin inhibitors increase NMDAR activity in the PVN to augment sympathetic vasomotor activity. α2δ-1 (encoded by the <i>Cacna2d1</i> gene), known colloquially as a calcium channel subunit, is a newly discovered NMDAR-interacting protein. Here, we determined whether α2δ-1 plays a role in calcineurin inhibitor-induced synaptic NMDAR hyperactivity in the PVN and hypertension development.<br /><b>Methods and results</b><br />Immunoblotting and coimmunoprecipitation assays revealed that prolonged treatment with FK506 in rats significantly increased protein levels of α2δ-1, GluN1 (the obligatory NMDAR subunit), and the α2δ-1-GluN1 complex in PVN synaptosomes. These effects were blocked by inhibiting α2δ-1 with gabapentin or interrupting the α2δ-1-NMDAR interaction with an α2δ-1 C-terminus peptide. Whole-cell recordings in brain slices showed that treatment with FK506 potentiated the activity of presynaptic and postsynaptic NMDARs in spinally projecting PVN neurons; such effects were abolished by gabapentin, <i>Cacna2d1</i> knockout, or α2δ-1 C-terminus peptide. Furthermore, microinjection of α2δ-1 C-terminus peptide into the PVN diminished renal sympathetic nerve discharges and arterial blood pressure that had been increased by FK506 treatment. Remarkably, telemetry recording showed that concurrent administration of gabapentin prevented the development of FK506-induced hypertension in rats. Additionally, FK506 treatment induced sustained hypertension in wild-type mice but not in <i>Cacna2d1</i> knockout mice.<br /><b>Conclusions</b><br />These findings indicate that α2δ-1 is essential for calcineurin inhibitor-induced increases in synaptic NMDAR activity in PVN presympathetic neurons and sympathetic outflow. Thus, α2δ-1 and α2δ-1-bound NMDARs represent new targets for treating calcineurin inhibitor-induced hypertension.<br /><br /><br /><br /><small>Circ Res: 22 Aug 2023; epub ahead of print</small></div>

<div><h4>Endothelial FIS1 DeSUMOylation Protects Against Hypoxic Pulmonary Hypertension.</h4><i>Zhou X, Jiang Y, Wang Y, Fan L, ... Simons M, Yu L</i><br /><b>Background</b><br />Hypoxia is a major cause and promoter of pulmonary hypertension (PH), a representative vascular remodeling disease with poor prognosis and high mortality. However, the mechanism underlying how pulmonary arterial system responds to hypoxic stress during PH remains unclear. Endothelial mitochondria are considered signaling organelles on oxygen tension. Results from previous clinical research and our studies suggested a potential role of posttranslational SUMOylation in endothelial mitochondria in hypoxia-related vasculopathy.<br /><b>Methods</b><br />Chronic hypoxia mouse model and Sugen/hypoxia rat model were employed as PH animal models. Mitochondrial morphology and subcellular structure were determined by transmission electron and immunofluorescent microscopies. Mitochondrial metabolism was determined by mitochondrial oxygen consumption rate and extracellular acidification rate. SUMOylation and protein interaction were determined by immunoprecipitation.<br /><b>Results</b><br />The involvement of SENP1 (sentrin-specific protease 1)-mediated SUMOylation in mitochondrial remodeling in the pulmonary endothelium was identified in clinical specimens of hypoxia-related PH and was verified in human pulmonary artery endothelial cells under hypoxia. Further analyses in clinical specimens, hypoxic rat and mouse PH models, and human pulmonary artery endothelial cells and human embryonic stem cell-derived endothelial cells revealed that short-term hypoxia-induced SENP1 translocation to endothelial mitochondria to regulate deSUMOylation of mitochondrial fission protein FIS1 (mitochondrial fission 1), which facilitated FIS1 assembling with fusion protein MFN2 (mitofusin 2) and mitochondrial gatekeeper VDAC1 (voltage-dependent anion channel 1), and the membrane tethering activity of MFN2 by enhancing its oligomerization. Consequently, FIS1 deSUMOylation maintained the mitochondrial integrity and endoplasmic reticulum-mitochondria calcium communication across mitochondrial-associated membranes, subsequently preserving pulmonary endothelial function and vascular homeostasis. In contrast, prolonged hypoxia disabled the FIS1 deSUMOylation by diminishing the availability of SENP1 in mitochondria via inducing miR-138 and consequently resulted in mitochondrial dysfunction and metabolic reprogramming in pulmonary endothelium. Functionally, introduction of viral-packaged deSUMOylated FIS1 within pulmonary endothelium in mice improved pulmonary endothelial dysfunction and hypoxic PH development, while knock-in of SUMO (small ubiquitin-like modifier)-conjugated FIS1 in mice exaggerated the diseased cellular and tissue phenotypes.<br /><b>Conclusions</b><br />By maintaining endothelial mitochondrial homeostasis, deSUMOylation of FIS1 adaptively preserves pulmonary endothelial function against hypoxic stress and consequently protects against PH. The FIS1 deSUMOylation-SUMOylation transition in pulmonary endothelium is an intrinsic pathogenesis of hypoxic PH.<br /><br /><br /><br /><small>Circ Res: 17 Aug 2023; epub ahead of print</small></div>

<div><h4>CCND2 Modified mRNA Activates Cell Cycle of Cardiomyocytes in Hearts With Myocardial Infarction in Mice and Pigs.</h4><i>Sun J, Wang L, Matthews RC, Walcott GP, ... Zangi L, Zhang J</i><br /><b>Background</b><br />Experiments in mammalian models of cardiac injury suggest that the cardiomyocyte-specific overexpression of CCND2 (cyclin D2, in humans) improves recovery from myocardial infarction (MI). The primary objective of this investigation was to demonstrate that our specific modified mRNA translation system (SMRTs) can induce CCND2 expression in Cardiomyocytes and replicate the benefits observed in other studies of cardiomyocyte-specific CCND2 overexpression for myocardial repair.<br /><b>Methods</b><br />The CCND2-cardiomyocyte-specific modified mRNA translation system (cardiomyocyte SMRTs) consists of 2 modRNA constructs: 1 code for CCND2 and contains a binding site for L7Ae, and the other codes for L7Ae and contains recognition elements for the cardiomyocyte-specific microRNAs miR-1 and miR-208. Thus, L7Ae suppresses CCND2 translation in noncardiomyocytes but is itself suppressed by endogenous miR-1 and -208 in cardiomyocytes, thereby facilitating cardiomyocyte-specific CCND2 expression. Experiments were conducted in both mouse and pig models of MI, and control assessments were performed in animals treated with an SMRTs coding for the cardiomyocyte-specific expression of luciferase or GFP, in animals treated with L7Ae modRNA alone or with the delivery vehicle, and in Sham-operated animals.<br /><b>Results</b><br />CCND2 was abundantly expressed in cultured, postmitotic cardiomyocytes 2 days after transfection with the CCND2-cardiomyocyte SMRTs, and the increase was accompanied by the upregulation of markers for cell-cycle activation and proliferation (eg, Ki67 and Aurora B kinase). When the GFP-cardiomyocyte SMRTs were intramyocardially injected into infarcted mouse hearts, the GFP signal was observed in cardiomyocytes but no other cell type. In both MI models, cardiomyocyte proliferation (on day 7 and day 3 after treatment administration in mice and pigs, respectively) was significantly greater, left-ventricular ejection fractions (days 7 and 28 in mice, days 10 and 28 in pigs) were significantly higher, and infarcts (day 28 in both species) were significantly smaller in animals treated with the CCND2-cardiomyocyte SMRTs than in any other group that underwent MI induction.<br /><b>Conclusions</b><br />Intramyocardial injections of the CCND2-cardiomyocyte SMRTs promoted cardiomyocyte proliferation, reduced infarct size, and improved cardiac performance in small and large mammalian hearts with MI.<br /><br /><br /><br /><small>Circ Res: 11 Aug 2023; epub ahead of print</small></div>

<div><h4>PROX1 Inhibits PDGF-B Expression to Prevent Myxomatous Degeneration of Heart Valves.</h4><i>Ho YC, Geng X, O\'Donnell A, Ibarrola J, ... Yutzey KE, Srinivasan RS</i><br /><b>Background</b><br />Cardiac valve disease is observed in 2.5% of the general population and 10% of the elderly people. Effective pharmacological treatments are currently not available, and patients with severe cardiac valve disease require surgery. PROX1 (prospero-related homeobox transcription factor 1) and FOXC2 (Forkhead box C2 transcription factor) are transcription factors that are required for the development of lymphatic and venous valves. We found that PROX1 and FOXC2 are expressed in a subset of valvular endothelial cells (VECs) that are located on the downstream (fibrosa) side of cardiac valves. Whether PROX1 and FOXC2 regulate cardiac valve development and disease is not known.<br /><b>Methods</b><br />We used histology, electron microscopy, and echocardiography to investigate the structure and functioning of heart valves from <i>Prox1</i><sup><i>ΔVEC</i></sup> mice in which <i>Prox1</i> was conditionally deleted from VECs. Isolated valve endothelial cells and valve interstitial cells were used to identify the molecular mechanisms in vitro, which were tested in vivo by RNAScope, additional mouse models, and pharmacological approaches. The significance of our findings was tested by evaluation of human samples of mitral valve prolapse and aortic valve insufficiency.<br /><b>Results</b><br />Histological analysis revealed that the aortic and mitral valves of <i>Prox1</i><sup><i>ΔVEC</i></sup> mice become progressively thick and myxomatous. Echocardiography revealed that the aortic valves of <i>Prox1</i><sup><i>ΔVEC</i></sup> mice are stenotic<i>. FOXC2</i> was downregulated and PDGF-B (platelet-derived growth factor-B) was upregulated in the VECs of <i>Prox1</i><sup><i>ΔVEC</i></sup> mice. Conditional knockdown of FOXC2 and conditional overexpression of PDGF-B in VECs recapitulated the phenotype of <i>Prox1</i><sup><i>ΔVEC</i></sup> mice. PDGF-B was also increased in mice lacking FOXC2 and in human mitral valve prolapse and insufficient aortic valve samples. Pharmacological inhibition of PDGF-B signaling with imatinib partially ameliorated the valve defects of <i>Prox1</i><sup><i>ΔVEC</i></sup> mice.<br /><b>Conclusions</b><br />PROX1 antagonizes PDGF-B signaling partially via FOXC2 to maintain the extracellular matrix composition and prevent myxomatous degeneration of cardiac valves.<br /><br /><br /><br /><small>Circ Res: 09 Aug 2023; epub ahead of print</small></div>

<div><h4>SparkMaster 2: A New Software for Automatic Analysis of Calcium Spark Data.</h4><i>Tomek J, Nieves-Cintron M, Navedo MF, Ko CY, Bers DM</i><br /><b>Background</b><br />Calcium (Ca) sparks are elementary units of subcellular Ca release in cardiomyocytes and other cells. Accordingly, Ca spark imaging is an essential tool for understanding the physiology and pathophysiology of Ca handling and is used to identify new drugs targeting Ca-related cellular dysfunction (eg, cardiac arrhythmias). The large volumes of imaging data produced during such experiments require accurate and high-throughput analysis.<br /><b>Methods</b><br />We developed a new software tool SparkMaster 2 (SM2) for the analysis of Ca sparks imaged by confocal line-scan microscopy, combining high accuracy, flexibility, and user-friendliness. SM2 is distributed as a stand-alone application requiring no installation. It can be controlled using a simple-to-use graphical user interface, or using Python scripting.<br /><b>Results</b><br />SM2 is shown to have the following strengths: (1) high accuracy at identifying Ca release events, clearly outperforming previous highly successful software SparkMaster; (2) multiple types of Ca release events can be identified using SM2: Ca sparks, waves, miniwaves, and long sparks; (3) SM2 can accurately split and analyze individual sparks within spark clusters, a capability not handled adequately by prior tools. We demonstrate the practical utility of SM2 in 2 case studies, investigating how Ca levels affect spontaneous Ca release, and how large-scale release events may promote release refractoriness. SM2 is also useful in atrial and smooth muscle myocytes, across different imaging conditions.<br /><b>Conclusions</b><br />SparkMaster 2 is a new, much-improved user-friendly software for accurate high-throughput analysis of line-scan Ca spark imaging data. It is free, easy to use, and provides valuable built-in features to facilitate visualization, analysis, and interpretation of Ca spark data. It should enhance the quality and throughput of Ca spark and wave analysis across cell types, particularly in the study of arrhythmogenic Ca release events in cardiomyocytes.<br /><br /><br /><br /><small>Circ Res: 09 Aug 2023; epub ahead of print</small></div>

<div><h4>T-Cell MyD88 Is a Novel Regulator of Cardiac Fibrosis Through Modulation of T-Cell Activation.</h4><i>Bayer AL, Smolgovsky S, Ngwenyama N, Hernández-Martínez A, ... Sharma S, Alcaide P</i><br /><b>Background</b><br />Cardiac inflammation in heart failure is characterized by the presence of damage-associated molecular patterns, myeloid cells, and T cells. Cardiac damage-associated molecular patterns provide continuous proinflammatory signals to myeloid cells through TLRs (toll-like receptors) that converge onto the adaptor protein MyD88 (myeloid differentiation response 88). These induce activation into efficient antigen-presenting cells that activate T cells through their TCR (T-cell receptor). T-cell activation results in cardiotropism, cardiac fibroblast transformation, and maladaptive cardiac remodeling. T cells rely on TCR signaling for effector function and survival, and while they express MyD88 and damage-associated molecular pattern receptors, their role in T-cell activation and cardiac inflammation is unknown.<br /><b>Methods</b><br />We performed transverse aortic constriction in mice lacking MyD88 in T cells and analyzed remodeling, systolic function, survival, and T-cell activation. We profiled WT versus <i>Myd88</i><sup><i>-/-</i></sup> mouse T cells at the transcript and protein level and performed several functional assays.<br /><b>Results</b><br />Analysis of single-cell RNA-sequencing data sets revealed that MyD88 is expressed in mouse and human cardiac T cells. MyD88 deletion in T cells resulted in increased levels of cardiac T-cell infiltration and fibrosis in response to transverse aortic constriction. We discovered that TCR-activated <i>Myd88</i><sup><i>-/-</i></sup> T cells had increased proinflammatory signaling at the transcript and protein level compared with WT, resulting in increased T-cell effector functions such as adhesion, migration across endothelial cells, and activation of cardiac fibroblast. Mechanistically, we found that MyD88 modulates T-cell activation and survival through TCR-dependent rather than TLR-dependent signaling.<br /><b>Conclusions</b><br />Our results outline a novel intrinsic role for MyD88 in limiting T-cell activation that is central to tune down cardiac inflammation during cardiac adaptation to stress.<br /><br /><br /><br /><small>Circ Res: 26 Jul 2023; epub ahead of print</small></div>

<div><h4>Interaction of Filamin C With Actin Is Essential for Cardiac Development and Function.</h4><i>Zhou X, Fang X, Ithychanda SS, Wu T, ... Qin J, Chen J</i><br /><b>Background</b><br />FLNC (filamin C), a member of the FLN family predominantly expressed in striated muscles, plays a crucial role in bridging the cytoskeleton and ECM (extracellular matrix) in cardiomyocytes, thereby maintaining heart integrity and function. Although genetic variants within the N-terminal ABD (actin-binding domain) of FLNC have been identified in patients with cardiomyopathy, the precise contribution of the actin-binding capability to FLNC\'s function in mammalian hearts remains poorly understood.<br /><b>Methods</b><br />We conducted in silico analysis of the 3-dimensional structure of mouse FLNC to identify key amino acid residues within the ABD that are essential for FLNC\'s actin-binding capacity. Subsequently, we performed coimmunoprecipitation and immunofluorescent assays to validate the in silico findings and assess the impact of these mutations on the interactions with other binding partners and the subcellular localization of FLNC. Additionally, we generated and analyzed knock-in mouse models in which the FLNC-actin interaction was completely disrupted by these mutations.<br /><b>Results</b><br />Our findings revealed that F93A/L98E mutations completely disrupted FLNC-actin interaction while preserving FLNC\'s ability to interact with other binding partners ITGB1 (β1 integrin) and γ-SAG (γ-sarcoglycan), as well as maintaining FLNC subcellular localization. Loss of FLNC-actin interaction in embryonic cardiomyocytes resulted in embryonic lethality and cardiac developmental defects, including ventricular wall malformation and reduced cardiomyocyte proliferation. Moreover, disruption of FLNC-actin interaction in adult cardiomyocytes led to severe dilated cardiomyopathy, enhanced lethality and dysregulation of key cytoskeleton components.<br /><b>Conclusions</b><br />Our data strongly support the crucial role of FLNC as a bridge between actin filaments and ECM through its interactions with actin, ITGB1, γ-SAG, and other associated proteins in cardiomyocytes. Disruption of FLN-actin interaction may result in detachment of actin filaments from the extracellular matrix, ultimately impairing normal cardiac development and function. These findings also provide insights into mechanisms underlying cardiomyopathy associated with genetic variants in FLNC ABD and other regions.<br /><br /><br /><br /><small>Circ Res: 26 Jul 2023; epub ahead of print</small></div>

<div><h4>Age at Menopause, Leukocyte Telomere Length, and Coronary Artery Disease in Postmenopausal Women.</h4><i>Schuermans A, Nakao T, Uddin MM, Hornsby W, ... Natarajan P, Honigberg MC</i><br /><b>Background</b><br />Premature menopause is a risk factor for accelerated cardiovascular aging, but underlying mechanisms remain incompletely understood. This study investigated the role of leukocyte telomere length (LTL), a marker of cellular aging and genomic instability, in the association of premature menopause with cardiovascular disease.<br /><b>Methods</b><br />Participants from the UK Biobank and Women\'s Health Initiative with complete reproductive history and LTL measurements were included. Primary analyses tested the association between age at menopause and LTL using multivariable-adjusted linear regression. Secondary analyses stratified women by history of gynecologic surgery. Mendelian randomization was used to infer causal relationships between LTL and age at natural menopause. Multivariable-adjusted Cox regression and mediation analyses tested the joint associations of premature menopause and LTL with incident coronary artery disease.<br /><b>Results</b><br />This study included 130 254 postmenopausal women (UK Biobank: n=122 224; Women\'s Health Initiative: n=8030), of whom 4809 (3.7%) had experienced menopause before age 40. Earlier menopause was associated with shorter LTL (meta-analyzed ß=-0.02 SD/5 years of earlier menopause [95% CI, -0.02 to -0.01]; <i>P</i>=7.2×10<sup>-12</sup>). This association was stronger and significant in both cohorts for women with natural/spontaneous menopause (meta-analyzed ß=-0.04 SD/5 years of earlier menopause [95% CI, -0.04 to -0.03]; <i>P</i><2.2×10<sup>-16</sup>) and was independent of hormone therapy use. Mendelian randomization supported a causal association of shorter genetically predicted LTL with earlier age at natural menopause. LTL and age at menopause were independently associated with incident coronary artery disease, and mediation analyses indicated small but significant mediation effects of LTL in the association of menopausal age with coronary artery disease.<br /><b>Conclusions</b><br />Earlier age at menopause is associated with shorter LTL, especially among women with natural menopause. Accelerated telomere shortening may contribute to the heightened cardiovascular risk associated with premature menopause.<br /><br /><br /><br /><small>Circ Res: 25 Jul 2023; epub ahead of print</small></div>

<div><h4>EGFR/IGF1R Signaling Modulates Relaxation in Hypertrophic Cardiomyopathy.</h4><i>Algül S, Schuldt M, Manders E, Jansen V, ... van der Velden J, Kuster DWD</i><br /><b>Background</b><br />Diastolic dysfunction is central to diseases such as heart failure with preserved ejection fraction and hypertrophic cardiomyopathy (HCM). However, therapies that improve cardiac relaxation are scarce, partly due to a limited understanding of modulators of cardiomyocyte relaxation. We hypothesized that cardiac relaxation is regulated by multiple unidentified proteins and that dysregulation of kinases contributes to impaired relaxation in patients with HCM.<br /><b>Methods</b><br />We optimized and increased the throughput of unloaded shortening measurements and screened a kinase inhibitor library in isolated adult cardiomyocytes from wild-type mice. One hundred fifty-seven kinase inhibitors were screened. To assess which kinases are dysregulated in patients with HCM and could contribute to impaired relaxation, we performed a tyrosine and global phosphoproteomics screen and integrative inferred kinase activity analysis using HCM patient myocardium. Identified hits from these 2 data sets were validated in cardiomyocytes from a homozygous <i>MYBPC3</i><sub>c.2373insG</sub> HCM mouse model.<br /><b>Results</b><br />Screening of 157 kinase inhibitors in wild-type (n=33) cardiomyocytes (n=24 563) resulted in the identification of 17 positive inotropes and 21 positive lusitropes, almost all of them novel. The positive lusitropes formed 3 clusters: cell cycle, EGFR (epidermal growth factor receptor)/IGF1R (insulin-like growth factor 1 receptor), and a small Akt (α-serine/threonine protein kinase) signaling cluster. By performing phosphoproteomic profiling of HCM patient myocardium (n=24 HCM and n=8 donors), we demonstrated increased activation of 6 of 8 proteins from the EGFR/IGFR1 cluster in HCM. We validated compounds from this cluster in mouse HCM (n=12) cardiomyocytes (n=2023). Three compounds from this cluster were able to improve relaxation in HCM cardiomyocytes.<br /><b>Conclusions</b><br />We showed the feasibility of screening for functional modulators of cardiomyocyte relaxation and contraction, parameters that we observed to be modulated by kinases involved in EGFR/IGF1R, Akt, cell cycle signaling, and FoxO (forkhead box class O) signaling, respectively. Integrating the screening data with phosphoproteomics analysis in HCM patient tissue indicated that inhibition of EGFR/IGF1R signaling is a promising target for treating impaired relaxation in HCM.<br /><br /><br /><br /><small>Circ Res: 21 Jul 2023; epub ahead of print</small></div>

<div><h4>Danicamtiv Increases Myosin Recruitment and Alters Cross-Bridge Cycling in Cardiac Muscle.</h4><i>Kooiker KB, Mohran S, Turner KL, Ma W, ... Davis J, Moussavi-Harami F</i><br /><b>Background</b><br />Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known that danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking.<br /><b>Methods</b><br />Permeabilized porcine cardiac tissue and myofibrils were used for X-ray diffraction and mechanical measurements. A mouse model of genetic dilated cardiomyopathy was used to evaluate the ability of danicamtiv to correct the contractile deficient.<br /><b>Results</b><br />Danicamtiv increased force and calcium sensitivity via increasing the number of myosins in the on state and slowing cross-bridge turnover. Our detailed analysis showed that inhibition of ADP release results in decreased cross-bridge turnover with cross bridges staying attached longer and prolonging myofibril relaxation. Danicamtiv corrected decreased calcium sensitivity in demembranated tissue, abnormal twitch magnitude and kinetics in intact cardiac tissue, and reduced ejection fraction in the whole organ.<br /><b>Conclusions</b><br />As demonstrated by the detailed studies of Danicamtiv, increasing myosin recruitment and altering cross-bridge cycling are 2 mechanisms to increase force and calcium sensitivity in cardiac muscle. Myosin activators such as Danicamtiv can treat the causative hypocontractile phenotype in genetic dilated cardiomyopathy.<br /><br /><br /><br /><small>Circ Res: 20 Jul 2023; epub ahead of print</small></div>

<div><h4>Chylomicrons Regulate Lacteal Permeability and Intestinal Lipid Absorption.</h4><i>Zarkada G, Chen X, Zhou X, Lange M, ... Eichmann A, Zhang F</i><br /><b>Background</b><br />Lymphatic vessels are responsible for tissue drainage, and their malfunction is associated with chronic diseases. Lymph uptake occurs via specialized open cell-cell junctions between capillary lymphatic endothelial cells (LECs), whereas closed junctions in collecting LECs prevent lymph leakage. LEC junctions are known to dynamically remodel in development and disease, but how lymphatic permeability is regulated remains poorly understood.<br /><b>Methods</b><br />We used various genetically engineered mouse models in combination with cellular, biochemical, and molecular biology approaches to elucidate the signaling pathways regulating junction morphology and function in lymphatic capillaries.<br /><b>Results</b><br />By studying the permeability of intestinal lacteal capillaries to lipoprotein particles known as chylomicrons, we show that ROCK (Rho-associated kinase)-dependent cytoskeletal contractility is a fundamental mechanism of LEC permeability regulation. We show that chylomicron-derived lipids trigger neonatal lacteal junction opening via ROCK-dependent contraction of junction-anchored stress fibers. LEC-specific ROCK deletion abolished junction opening and plasma lipid uptake. Chylomicrons additionally inhibited VEGF (vascular endothelial growth factor)-A signaling. We show that VEGF-A antagonizes LEC junction opening via VEGFR (VEGF receptor) 2 and VEGFR3-dependent PI3K/AKT activation of the small GTPase RAC1, thereby restricting RhoA/ROCK-mediated cytoskeleton contraction.<br /><b>Conclusions</b><br />Our results reveal that antagonistic inputs into ROCK-dependent cytoskeleton contractions regulate the interconversion of lymphatic junctions in the intestine and in other tissues, providing a tunable mechanism to control the lymphatic barrier.<br /><br /><br /><br /><small>Circ Res: 18 Jul 2023; epub ahead of print</small></div>

<div><h4>Brown Adipose Tissue and BMP3b Decrease Injury in Cardiac Ischemia-Reperfusion.</h4><i>Martí-Pàmies Í, Thoonen R, Morley M, Graves L, ... Bloch DB, Scherrer-Crosbie M</i><br /><b>Background</b><br />Despite advances in treatment, myocardial infarction (MI) is a leading cause of heart failure and death worldwide, with both ischemia and reperfusion (I/R) causing cardiac injury. A previous study using a mouse model of nonreperfused MI showed activation of brown adipose tissue (BAT). Recent studies showed that molecules secreted by BAT target the heart. We investigated whether BAT attenuates cardiac injury in I/R and sought to identify potential cardioprotective proteins secreted by BAT.<br /><b>Methods</b><br />Myocardial I/R surgery with or without BAT transplantation was performed in wild-type (WT) mice and in mice with impaired BAT function (uncoupling protein 1 [<i>Ucp1</i>]-deficient mice). To identify potential cardioprotective factors produced by BAT, RNA-seq was performed in BAT from WT and <i>Ucp1</i><sup><i>-</i></sup><sup><i>/-</i></sup> mice. Subsequently, myocardial I/R surgery with or without BAT transplantation was performed in <i>Bmp3b</i> (bone morphogenetic protein 3b)-deficient mice, and WT mice subjected to myocardial I/R were treated using BMP3b.<br /><b>Results</b><br />Dysfunction of BAT in mice was associated with larger MI size after I/R; conversely, augmenting BAT by transplantation decreased MI size. We identified Bmp3b as a protein secreted by BAT after I/R. Compared with WT mice, <i>Bmp3b</i>-deficient mice developed larger MIs. Increasing functional BAT by transplanting BAT from WT mice to <i>Bmp3b</i>-deficient mice reduced I/R injury whereas transplanting BAT from <i>Bmp3b</i>-deficient mice did not. Treatment of WT mice with BMP3b before reperfusion decreased MI size. The cardioprotective effect of BMP3b was mediated through SMAD1/5/8. In humans, the plasma level of BMP3b increased after MI and was positively correlated with the extent of cardiac injury.<br /><b>Conclusions</b><br />The results of this study suggest a cardioprotective role of BAT and BMP3b, a protein secreted by BAT, in a model of I/R injury. Interventions increasing BMP3b levels or targeting Smad 1/5 may represent novel therapeutic approaches to decrease myocardial damage in I/R injury.<br /><br /><br /><br /><small>Circ Res: 18 Jul 2023; epub ahead of print</small></div>

<div><h4>Loss of the Atrial Fibrillation-Related Gene, , Results in Atrial Dilation and Arrhythmias.</h4><i>Jameson HS, Hanley A, Hill MC, Xiao L, ... Milan DJ, Ellinor PT</i><br /><b>Background</b><br /><i>ZFHX3</i>, a gene that encodes a large transcription factor, is at the second-most significantly associated locus with atrial fibrillation (AF), but its function in the heart is unknown. This study aims to identify causative genetic variation related to AF at the <i>ZFHX3</i> locus and examine the impact of <i>Zfhx3</i> loss on cardiac function in mice.<br /><b>Methods</b><br />CRISPR-Cas9 genome editing, chromatin immunoprecipitation, and luciferase assays in pluripotent stem cell-derived cardiomyocytes were used to identify causative genetic variation related to AF at the <i>ZFHX3</i> locus. Cardiac function was assessed by echocardiography, magnetic resonance imaging, electrophysiology studies, calcium imaging, and RNA sequencing in mice with heterozygous and homozygous cardiomyocyte-restricted <i>Zfhx3</i> deletion (<i>Zfhx3</i> Het and knockout, respectively). Human cardiac single-nucleus ATAC-sequencing data was analyzed to determine which genes in atrial cardiomyocytes are directly regulated by <i>ZFHX3</i>.<br /><b>Results</b><br />We found SNP rs12931021 modulates an enhancer regulating <i>ZFHX3</i> expression, and the AF risk allele is associated with decreased <i>ZFHX3</i> transcription. We observed a gene-dose response in AF susceptibility with Zfhx3 knockout mice having higher incidence, frequency, and burden of AF than <i>Zfhx3</i> Het and wild-type mice, with alterations in conduction velocity, atrial action potential duration, calcium handling and the development of atrial enlargement and thrombus, and dilated cardiomyopathy. <i>Zfhx3</i> loss results in atrial-specific differential effects on genes and signaling pathways involved in cardiac pathophysiology and AF.<br /><b>Conclusions</b><br />Our findings implicate <i>ZFHX3</i> as the causative gene at the 16q22 locus for AF, and cardiac abnormalities caused by loss of cardiac <i>Zfhx3</i> are due to atrial-specific dysregulation of pathways involved in AF susceptibility. Together, these data reveal a novel and important role for <i>Zfhx3</i> in the control of cardiac genes and signaling pathways essential for normal atrial function.<br /><br /><br /><br /><small>Circ Res: 14 Jul 2023; epub ahead of print</small></div>

<div><h4>CTLA-4 Pathway Is Instrumental in Giant Cell Arteritis.</h4><i>Régnier P, Le Joncour A, Maciejewski-Duval A, Darrasse-Jèze G, ... Salem JE, Saadoun D</i><br /><b>Background</b><br />Giant cell arteritis (GCA) causes severe inflammation of the aorta and its branches and is characterized by intense effector T-cell infiltration. The roles that immune checkpoints play in the pathogenesis of GCA are still unclear. Our aim was to study the immune checkpoint interplay in GCA.<br /><b>Methods</b><br />First, we used VigiBase, the World Health Organization international pharmacovigilance database, to evaluate the relationship between GCA occurrence and immune checkpoint inhibitors treatments. We then further dissected the role of immune checkpoint inhibitors in the pathogenesis of GCA, using immunohistochemistry, immunofluorescence, transcriptomics, and flow cytometry on peripheral blood mononuclear cells and aortic tissues of GCA patients and appropriated controls.<br /><b>Results</b><br />Using VigiBase, we identified GCA as a significant immune-related adverse event associated with anti-CTLA-4 (cytotoxic T-lymphocyte-associated protein-4) but not anti-PD-1/PD-L1 treatment. We further dissected a critical role for the CTLA-4 pathway in GCA by identification of the dysregulation of CTLA-4-derived gene pathways and proteins in CD4<sup>+</sup> T cells (and specifically regulatory T cells) present in blood and aorta of GCA patients versus controls. While regulatory T cells were less abundant and activated/suppressive in blood and aorta of GCA versus controls, they still specifically upregulated CTLA-4. Activated and proliferating CTLA-4<sup>+</sup> Ki-67<sup>+</sup> regulatory T cells from GCA were more sensitive to anti-CTLA-4 (ipilimumab)-mediated in vitro depletion versus controls.<br /><b>Conclusions</b><br />We highlighted the instrumental role of CTLA-4 immune checkpoint in GCA, which provides a strong rationale for targeting this pathway.<br /><br /><br /><br /><small>Circ Res: 12 Jul 2023; epub ahead of print</small></div>

<div><h4>P53 Regulates the Extent of Fibroblast Proliferation and Fibrosis in Left Ventricle Pressure Overload.</h4><i>Liu X, Burke RM, Lighthouse JK, Baker CD, ... Ashton JM, Small EM</i><br /><b>Background</b><br />Cardiomyopathy is characterized by the pathological accumulation of resident cardiac fibroblasts that deposit ECM (extracellular matrix) and generate a fibrotic scar. However, the mechanisms that control the timing and extent of cardiac fibroblast proliferation and ECM production are not known, hampering the development of antifibrotic strategies to prevent heart failure.<br /><b>Methods</b><br />We used the Tcf21 (transcription factor 21)<sup>MerCreMer</sup> mouse line for fibroblast-specific lineage tracing and tumor protein p53 (<i>p53</i>) gene deletion (called p53-cardiac fibroblasts knockout). We characterized cardiac physiology and used single-cell RNA-sequencing and in vitro studies to investigate the p53-dependent mechanisms regulating cardiac fibroblast cell cycle and fibrosis in left ventricular pressure overload induced by transaortic constriction.<br /><b>Results</b><br />Cardiac fibroblast proliferation occurs primarily between days 7 and 14 following transaortic constriction in mice, correlating with alterations in p53-dependent gene expression. P53 deletion in fibroblasts led to a striking accumulation of Tcf21-lineage cardiac fibroblasts within the normal proliferative window and precipitated a robust fibrotic response to left ventricular pressure overload. However, excessive interstitial and perivascular fibrosis does not develop in p53-cardiac fibroblasts knockout mice until after cardiac fibroblasts exit the cell cycle. Single-cell RNA sequencing revealed p53 null fibroblasts unexpectedly express lower levels of genes encoding important ECM proteins while they exhibit an inappropriately proliferative phenotype. in vitro studies establish a role for p53 in suppressing the proliferative fibroblast phenotype, which facilitates the expression and secretion of ECM proteins. Importantly, <i>Cdkn2a</i> expression and the p16<sup>Ink4a</sup>-retinoblastoma cell cycle control pathway is induced in <i>p53</i> null cardiac fibroblasts, which may eventually contribute to cell cycle exit and fulminant scar formation.<br /><b>Conclusions</b><br />This study reveals a mechanism regulating cardiac fibroblast accumulation and ECM secretion, orchestrated in part by p53-dependent cell cycle control that governs the timing and extent of fibrosis in left ventricular pressure overload.<br /><br /><br /><br /><small>Circ Res: 06 Jul 2023; epub ahead of print</small></div>

<div><h4>Stretch Harmonizes Sarcomere Strain Across the Cardiomyocyte.</h4><i>Li J, Sundnes J, Hou Y, Laasmaa M, ... de Tombe PP, Louch WE</i><br /><b>Background</b><br />Increasing cardiomyocyte contraction during myocardial stretch serves as the basis for the Frank-Starling mechanism in the heart. However, it remains unclear how this phenomenon occurs regionally within cardiomyocytes, at the level of individual sarcomeres. We investigated sarcomere contractile synchrony and how intersarcomere dynamics contribute to increasing contractility during cell lengthening.<br /><b>Methods</b><br />Sarcomere strain and Ca<sup>2+</sup> were simultaneously recorded in isolated left ventricular cardiomyocytes during 1 Hz field stimulation at 37 °C, at resting length and following stepwise stretch.<br /><b>Results</b><br />We observed that in unstretched rat cardiomyocytes, differential sarcomere deformation occurred during each beat. Specifically, while most sarcomeres shortened during the stimulus, ≈10% to 20% of sarcomeres were stretched or remained stationary. This nonuniform strain was not traced to regional Ca<sup>2+</sup> disparities but rather shorter resting lengths and lower force production in systolically stretched sarcomeres. Lengthening of the cell recruited additional shortening sarcomeres, which increased contractile efficiency as less negative, wasted work was performed by stretched sarcomeres. Given the known role of titin in setting sarcomere dimensions, we next hypothesized that modulating titin expression would alter intersarcomere dynamics. Indeed, in cardiomyocytes from mice with titin haploinsufficiency, we observed greater variability in resting sarcomere length, lower recruitment of shortening sarcomeres, and impaired work performance during cell lengthening.<br /><b>Conclusions</b><br />Graded sarcomere recruitment directs cardiomyocyte work performance, and harmonization of sarcomere strain increases contractility during cell stretch. By setting sarcomere dimensions, titin controls sarcomere recruitment, and its lowered expression in haploinsufficiency mutations impairs cardiomyocyte contractility.<br /><br /><br /><br /><small>Circ Res: 04 Jul 2023; epub ahead of print</small></div>

<div><h4>Mitochondrial GSNOR Alleviates Cardiac Dysfunction via ANT1 Denitrosylation.</h4><i>Tang X, Zhao S, Liu J, Liu X, ... Han Y, Ji Y</i><br /><b>Background</b><br />The cardiac-protective role of GSNOR (S-nitrosoglutathione reductase) in the cytoplasm, as a denitrosylase enzyme of S-nitrosylation, has been reported in cardiac remodeling, but whether GSNOR is localized in other organelles and exerts novel effects remains unknown. We aimed to elucidate the effects of mitochondrial GSNOR, a novel subcellular localization of GSNOR, on cardiac remodeling and heart failure (HF).<br /><b>Methods</b><br />GSNOR subcellular localization was observed by cellular fractionation assay, immunofluorescent staining, and colloidal gold particle staining. Overexpression of GSNOR in mitochondria was achieved by mitochondria-targeting sequence-directed adeno-associated virus 9. Cardiac-specific knockout of GSNOR mice was used to examine the role of GSNOR in HF. S-nitrosylation sites of ANT1 (adenine nucleotide translocase 1) were identified using biotin-switch and liquid chromatography-tandem mass spectrometry.<br /><b>Results</b><br />GSNOR expression was suppressed in cardiac tissues of patients with HF. Consistently, cardiac-specific knockout mice showed aggravated pathological remodeling induced by transverse aortic constriction. We found that GSNOR is also localized in mitochondria. In the angiotensin II-induced hypertrophic cardiomyocytes, mitochondrial GSNOR levels significantly decreased along with mitochondrial functional impairment. Restoration of mitochondrial GSNOR levels in cardiac-specific knockout mice significantly improved mitochondrial function and cardiac performance in transverse aortic constriction-induced HF mice. Mechanistically, we identified ANT1 as a direct target of GSNOR. A decrease in mitochondrial GSNOR under HF leads to an elevation of S-nitrosylation-ANT1 at cysteine 160 (C160). In accordance with these findings, overexpression of either mitochondrial GSNOR or ANT1 C160A, non-nitrosylated mutant, significantly improved mitochondrial function, maintained the mitochondrial membrane potential, and upregulated mitophagy.<br /><b>Conclusions</b><br />We identified a novel species of GSNOR localized in mitochondria and found mitochondrial GSNOR plays an essential role in maintaining mitochondrial homeostasis through ANT1 denitrosylation, which provides a potential novel therapeutic target for HF.<br /><br /><br /><br /><small>Circ Res: 28 Jun 2023; epub ahead of print</small></div>