Background:Neutrophils are the most rapid and abundant immune cells to infiltrate the myocardium following myocardial ischemia/reperfusion injury (MI/R). Neutrophil heterogeneity has not been well characterized in MI/R, and studies have shown conflicting results regarding the impact of neutrophil depletion on cardiac injury. We thus aim to study the impact of neutrophils with enriched type I interferon signature and the role of STING (stimulator of interferon genes) signaling in neutrophils on cardiac reperfusion injury. Methods:We utilized single-cell RNA sequencing to study neutrophil heterogeneity in response to MI/R. We generated a neutrophil-specific STING knockout mouse to assess the role of neutrophil STING in a model of MI/R. We examined cardiac function following injury via echocardiography and assessed the immune cell trajectory following injury utilizing flow cytometry. Results:We identified a population of neutrophils with enriched type I interferon signaling and response to type I interferon following MI/R. We found that genetic deletion of neutrophil-specific STING led to worsened cardiac function following MI/R. Further investigation of the immune response by flow cytometry revealed decreased neutrophil infiltration into the myocardium and a shift in macrophage polarization. Conclusions:Our findings suggest that neutrophil-specific STING is cardioprotective in MI/R, partly due to its effects on downstream immune cells. These results demonstrate that early alterations or therapeutic interventions can influence key events in the resolution of inflammation following MI/R.

Glycation is a posttranslational modification of proteins that contributes to the vast array of biological information that can be conveyed via a singular proteome. Understanding the role of advanced glycation end-products (AGEs) in human health and pathophysiology can be difficult, as the physiological effects of AGEs have been associated with multiple biological processes and disease state development, including acute myocardial ischemia-reperfusion injury, heart failure, and atherosclerosis, as well as tumor cell migration. The critical role of the glyoxalase system in the detoxification of methylglyoxal and other AGEs has been well established. Recently, evidence has emerged that DJ-1 displays antiglycative activity and may contribute to another mechanism of protection against protein glycation outside of the glyoxalase system. Identification of potential substrates of DJ-1 and determination of the pathways in which DJ-1 operates, is needed to fully understand the role of this protein in modulating biological homeostasis and the development of disease.

Although cardiac lymphatic vessels have received increasing attention in recent years, there is still a knowledge gap between cardiac lymphatics and heart homeostasis in a normal heart. In the present study, we established a mouse model of cardiac lymphatic insufficiency ablating cardiac lymphatic collector vessels to reveal the crucial role of cardiac lymphatic vessels in maintaining cardiac homeostasis and the impact on cardiac function both in physiological and pathologic settings. Furthermore, therapeutic lymphangiogenesis improved the adverse effect on cardiac morphologic changes and functions. These findings suggest that the cardiac lymphatic system would be a novel therapeutic target for heart disease.

At least seven cell death programs are activated during myocardial infarction (MI), but which are most important in causing heart damage is not understood. Two of these programs are mitochondrial-dependent necrosis and apoptosis. The canonical function of the pro-cell death BCL-2 family proteins BAX and BAK is to mediate permeabilization of the outer mitochondrial membrane during apoptosis allowing apoptogen release. BAX has also been shown to sensitize cells to mitochondrial-dependent necrosis, although the underlying mechanisms remain ill-defined. Genetic deletion of Bax or both Bax and Bak in mice reduces infarct size following reperfused myocardial infarction (MI/R), but the contribution of BAK itself to cardiomyocyte apoptosis and necrosis and infarction has not been investigated. In this study, we use Bak-deficient mice and isolated adult cardiomyocytes to delineate the role of BAK in the pathogenesis of infarct generation and post-infarct remodeling during MI/R and non-reperfused MI. Generalized homozygous deletion of Bak reduced infarct size ∼50% in MI/R in vivo, which was attributable primarily to decreases in necrosis. Protection from necrosis was also observed in BAK-deficient isolated cardiomyocytes suggesting that the cardioprotection from BAK loss in vivo is at least partially cardiomyocyte-autonomous. Interestingly, heterozygous Bak deletion, in which the heart still retains ∼28% of wild type BAK levels, reduced infarct size to a similar extent as complete BAK absence. In contrast to MI/R, homozygous Bak deletion did not attenuate acute infarct size or long-term scar size, post-infarct remodeling, cardiac dysfunction, or mortality in non-reperfused MI. We conclude that BAK contributes significantly to cardiomyocyte necrosis and infarct generation during MI/R, while its absence does not appear to impact the pathogenesis of non-reperfused MI. These observations suggest BAK may be a therapeutic target for MI/R and that even partial pharmacological antagonism may provide benefit.

Background: Cardiomyocyte (CM) proliferation decreases after birth but the underlying mechanism is poorly understood. Chromobox 7 (CBX7) regulates the cell cycle but its role in CM proliferation is unknown. Methods: We profiled CBX7 expression in the mouse hearts via qRT-PCR, western blotting, and immunohistochemistry. We knocked down CBX7 by using constitutive and inducible conditional knockout mice (Tnnt2-cre;Cbx7 fl/+ and Myh6-MCM;Cbx7 fl/fl , respectively). We then measured CM proliferation by immunostaining. To examine the role of CBX7 in cardiac regeneration, we employed neonatal cardiac apical resection and adult myocardial infarction (MI) models. We examined the mechanism of CBX7-mediated repression of CM proliferation via co-immunoprecipitation, mass spectrometry, and other molecular techniques. Results: The mRNA expression of Cbx7 was increased at the perinatal stage and sustained in the postnatal heart. The haplodeficiency of Cbx7 (Tnnt2-Cre;Cbx7 fl/+ ) promoted proliferation of neonatal CMs in vivo , leading to increased myocardial wall thickness. Genetic deletion of Cbx7 in CMs ( Cbx7 iCKO, Myh6-MCM;Cbx7 fl/fl ) at P0 resulted in increased CM proliferation (% of Ki67 + CMs: vehicle, 9.5 vs. tamoxifen, 18.3, P < 0.01). In response to cardiac apical resection surgery at P7, Cbx7 iCKO mice exhibited better cardiac function (% of LVEF: vehicle, 44.1 vs. tamoxifen, 54.7, P < 0.05) and less fibrosis at P28 compared to the control. Following the MI surgery, adult (5-months-old) Cbx7 iCKO mice exhibited better cardiac function (% of LVEF: vehicle, 41.4 vs. tamoxifen, 51.5, P < 0.05) and less fibrosis at one month post-MI compared to the control. Mechanistically, CBX7 interacted with TARDBP and positively regulated its downstream target, RBM38, an inducer of the cell cycle arrest, in a TARDBP-dependent manner. Rbm38 was perinatally upregulated in the mouse hearts and overexpression of Rbm38 reduced proliferation of neonatal CMs. Conclusions: CBX7 expression is perinatally increased and inhibits proliferation of CMs by controlling TARDBP/Rbm38 pathway. Genetic ablation of Cbx7 promoted regeneration of neonatal and adult hearts. This is the first study to uncover the role of CBX7 in regulation of CM proliferation and cardiac regeneration.

Introduction: Inflammation following myocardial ischemia/reperfusion (MI/R) plays a significant role in damaging cardiomyocytes and influencing infarct size, with neutrophils being the most rapid and numerous cells in the post-MI/R heart at 24 hours post reperfusion. Preliminary single-cell RNA sequencing data shows type I interferon (IFN) responding neutrophils are present in the hearts of mice 24 hours post-MI/R. The following study aims to address how the type I IFN responding neutrophil phenotype identified in single-cell RNA sequencing data affects cardiac inflammation in MI/R. We hypothesize that STING and type I IFN activation in neutrophils in the early stages of inflammation can exacerbate inflammation, causing further damage to the heart in MI/R. Methods: Male C57BL/6J mice (10-11 weeks old) underwent 60 minutes of ischemia followed by reperfusion, and were dosed with either vehicle, a STING inhibitor (H151), or an IFNAR1 antibody (ab) at reperfusion and at 24 hours following reperfusion. Cardiac function was assessed two weeks post-reperfusion via echocardiography and tissue samples were collected for histological analysis. Results: Mice treated with IFNAR1 ab showed significant improvement in ejection fraction (44.9% ± 6.8) compared to mice treated with vehicle (31.9% ± 6.2; One-way ANOVA, N=5-6, p<0.05). Additionally, mice treated with IFNAR1 ab showed significant improvements in left ventricular end systolic volume (LVESD) (3.16mm ± 0.52) compared to vehicle treated mice (3.89mm ± 0.40; One-way ANOVA, N=5-6, p<0.05). Mice treated with H151 showed significant improvement in ejection fraction (43.8% ± 7.1) compared to mice treated with vehicle (31.9% ± 6.2; One-way ANOVA, N=5-6, p<0.05). Treatment with H151 or IFNAR1 ab reduced scar size (7.22% ± 3.6; 7.88% ± 3.1%) compared to mice treated with vehicle (9.74% ± 3.7; N=5-6). Conclusion: These data show that targeting the early IFN response in MI/R can improve cardiac function, and provide a basis for studies identifying the effects of the neutrophil specific STING and type I IFN activation following MI/R. If correct, this pathway could be a powerful and clinically feasible point of intervention to improve cardiac recovery after MI/R.

Background:Shortly after birth, cardiomyocytes exit the cell cycle and cease proliferation. At present, the regulatory mechanisms for this loss of proliferative capacity are poorly understood. CBX7 (chromobox 7), a polycomb group (PcG) protein, regulates the cell cycle, but its role in cardiomyocyte proliferation is unknown. Methods:We profiled CBX7 expression in the mouse hearts through quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. We overexpressed CBX7 in neonatal mouse cardiomyocytes through adenoviral transduction. We knocked down CBX7 by using constitutive and inducible conditional knockout mice (Tnnt2-Cre;Cbx7(fl/+) and Myh6-MCM;Cbx7(fl/fl), respectively). We measured cardiomyocyte proliferation by immunostaining of proliferation markers such as Ki67, phospho-histone 3, and cyclin B1. To examine the role of CBX7 in cardiac regeneration, we used neonatal cardiac apical resection and adult myocardial infarction models. We examined the mechanism of CBX7-mediated repression of cardiomyocyte proliferation through coimmunoprecipitation, mass spectrometry, and other molecular techniques. Results:We explored Cbx7 expression in the heart and found that mRNA expression abruptly increased after birth and was sustained throughout adulthood. Overexpression of CBX7 through adenoviral transduction reduced proliferation of neonatal cardiomyocytes and promoted their multinucleation. On the other hand, genetic inactivation of Cbx7 increased proliferation of cardiomyocytes and impeded cardiac maturation during postnatal heart growth. Genetic ablation of Cbx7 promoted regeneration of neonatal and adult injured hearts. Mechanistically, CBX7 interacted with TARDBP (TAR DNA-binding protein 43) and positively regulated its downstream target, RBM38 (RNA Binding Motif Protein 38), in a TARDBP-dependent manner. Overexpression of RBM38 inhibited the proliferation of CBX7-depleted neonatal cardiomyocytes. Conclusions:Our results demonstrate that CBX7 directs the cell cycle exit of cardiomyocytes during the postnatal period by regulating its downstream targets TARDBP and RBM38. This is the first study to demonstrate the role of CBX7 in regulation of cardiomyocyte proliferation, and CBX7 could be an important target for cardiac regeneration.

BACKGROUND:Hydrogen sulfide (H2S) exerts mitochondria-specific actions that include the preservation of oxidative phosphorylation, biogenesis, and ATP synthesis, while inhibiting cell death. 3-MST (3-mercaptopyruvate sulfurtransferase) is a mitochondrial H2S-producing enzyme whose functions in the cardiovascular disease are not fully understood. In the current study, we investigated the effects of global 3-MST deficiency in the setting of pressure overload-induced heart failure. METHODS:Human myocardial samples obtained from patients with heart failure undergoing cardiac surgeries were probed for 3-MST protein expression. 3-MST knockout mice and C57BL/6J wild-type mice were subjected to transverse aortic constriction to induce pressure overload heart failure with reduced ejection fraction. Cardiac structure and function, vascular reactivity, exercise performance, mitochondrial respiration, and ATP synthesis efficiency were assessed. In addition, untargeted metabolomics were utilized to identify key pathways altered by 3-MST deficiency. RESULTS:Myocardial 3-MST was significantly reduced in patients with heart failure compared with nonfailing controls. 3-MST KO mice exhibited increased accumulation of branched-chain amino acids in the myocardium, which was associated with reduced mitochondrial respiration and ATP synthesis, exacerbated cardiac and vascular dysfunction, and worsened exercise performance following transverse aortic constriction. Restoring myocardial branched-chain amino acid catabolism with 3,6-dichlorobenzo1[b]thiophene-2-carboxylic acid (BT2) and administration of a potent H2S donor JK-1 ameliorates the detrimental effects of 3-MST deficiency in heart failure with reduced ejection fraction. CONCLUSIONS:Our data suggest that 3-MST derived mitochondrial H2S may play a regulatory role in branched-chain amino acid catabolism and mediate critical cardiovascular protection in heart failure.

Background Accumulating evidence suggests that hydrogen sulfide ( H 2 S ), an endogenously produced gaseous molecule, plays a critical role in the regulation of cardiovascular homeostasis. However, little is known about its role in lymphangiogenesis. Thus, the current study aimed to investigate the involvement of H 2 S in lymphatic vessel growth and lymphedema resolution using a murine model and assess the underlying mechanisms. Methods and Results A murine model of tail lymphedema was created both in wild‐type mice and cystathionine γ‐lyase–knockout mice, to evaluate lymphedema up to 28 days after lymphatic ablation. Cystathionine γ‐lyase–knockout mice had greater tail diameters than wild‐type mice, and this phenomenon was associated with the inhibition of reparative lymphangiogenesis at the site of lymphatic ablation. In contrast, the administration of an H 2 S donor, diallyl trisulfide, ameliorated lymphedema by inducing the formation of a considerable number of lymphatic vessels at the injured sites in the tails. In vitro experiments using human lymphatic endothelial cells revealed that diallyl trisulfide promoted their proliferation and differentiation into tube‐like structures by enhancing Akt (protein kinase B) phosphorylation in a concentration‐dependent manner. The blockade of Akt activation negated the diallyl trisulfide–induced prolymphangiogenic responses in lymphatic endothelial cells. Furthermore, the effects of diallyl trisulfide treatment on lymphangiogenesis in the tail lymphedema model were also negated by the inhibition of phosphoinositide 3'‐kinase (P13K)/Akt signaling. Conclusions H 2 S promotes reparative lymphatic vessel growth and ameliorates secondary lymphedema, at least in part, through the activation of the Akt pathway in lymphatic endothelial cells. As such, H 2 S donors could be used as therapeutics against refractory secondary lymphedema.

Phospholipids are ligands for nuclear hormone receptors (NRs) and regulate transcriptional programs relevant to normal physiology and disease. Here, we demonstrate that mimicking phospholipid-NR interactions greatly improves agonists of liver receptor homolog-1 (LRH-1), a promising therapeutic target for diabetes and colitis. Conventional LRH-1 modulators partially occupy the binding pocket, leaving vacant a region important for phospholipid binding and allostery. Therefore, we constructed a set of hybrid molecules with elements of natural phospholipids appended to a synthetic LRH-1 agonist. The phospholipid-mimicking group improves binding affinity, increases LRH-1 transcriptional activity, promotes coregulator recruitment, and interacts with the targeted LRH-1 residues in crystal structures. The best new agonist markedly improves colonic histopathology and disease-related weight loss in a humanized LRH-1 murine T-cell transfer model of colitis. This is the first evidence of in vivo efficacy for an LRH-1 modulator in colitis, a leap forward in agonist development.

Renewal of the myocardium by preexisting cardiomyocytes is a powerful strategy for restoring the architecture and function of hearts injured by myocardial infarction. To advance this strategy, we show that combining two clinically approved drugs, but neither alone, muscularizes the heart through cardiomyocyte proliferation. Specifically, in adult murine cardiomyocytes, metoprolol, a cardioselective β 1 -adrenergic receptor blocker, when given with triiodothyronine (T3, a thyroid hormone) accentuates the ability of T3 to stimulate ERK1/2 phosphorylation and proliferative signaling by inhibiting expression of the nuclear phospho-ERK1/2-specific phosphatase, dual-specificity phosphatase-5. While short-duration metoprolol plus T3 therapy generates new heart muscle in healthy mice, in mice with myocardial infarction-induced left ventricular dysfunction and pathological remodeling, it remuscularizes the heart, restores contractile function and reverses chamber dilatation; outcomes that are enduring. If the beneficial effects of metoprolol plus T3 are replicated in humans, this therapeutic strategy has the potential to definitively address ischemic heart failure.

Background: Inflammation succeeding myocardial ischemia/reperfusion (MI/R) plays a significant role in damaging cardiomyocytes and influencing infarct size, with neutrophils being the most rapid and most numerous cells in the post-MI/R heart. While neutrophils play a role in wound healing, they can cause further damage through the generation of reactive oxygen species, proteolytic enzymes, and neutrophil extracellular traps (NETs). We thus hypothesize that double stranded DNA stemming from these NETs activates cGAS-STING signaling in a murine model of MI/R, leading to the production of type I interferons by neutrophils, which worsens reperfusion injury. Methods: Single-cell RNA sequencing was performed on neutrophils isolated from the hearts of mice 24 hours post-MI/R. Additionally, mice underwent MI/R and heart tissues were harvested 6- and 24- hours after reperfusion for ELISA and qPCR analysis. Results: Utilizing single-cell RNA sequencing to better characterize the role of neutrophils and NETs in MI/R, we identified a significant population of neutrophils with a strong type I interferon (IFN) signature recruited to the area of infarct 24 hours post MI/R in a murine model. To further investigate the IFN response seen in this population of neutrophils, we preformed protein and gene expression analysis on heart tissue samples isolated from mice at timepoints in which neutrophils would be present in the area of infarct. Analysis by qPCR shows an increased fold change in gene expression of IFNα (7.22 ± 6.05, n = 3; 3.83 ± 2.22, n = 4) and IFNβ (6.74 ± 5.39, n = 3; 3.64 ± 2.25, n = 4) at 6- and 24- hours post MI/R respectively, compared to sham mice. Furthermore, analysis by ELISA shows a significant increase in IFNβ protein levels in the heart after 6- hours (0.41pg/mg ± 0.04, n =8) compared to sham mice (0.16 pg/mg ± 0.06, n = 7; p ≤ 0.01, Student’s t-test). Conclusions: These data show a rapid increase in type I IFN in the hearts of mice post-MI/R, which implicates neutrophils may be participating in the IFN signaling pathway. Identifying neutrophils as an early source of IFN can provide a potentially new therapeutic target in treating MI/R.

Background: Secondary lymphedema is one of the serious clinical problems that can often occur after surgical resection of malignant tumors. However, no effective treatment options exist at present. Diallyl trisulfide (DATS) is an organic polysulfide found in garlic oil that liberates H2S under physiological conditions. We previously demonstrated that DATS augmented reparative angiogenesis and improved blood perfusion recovery in the HLI model. Here, we investigated the effects of DATS on lymphatic vessel growth and lymphedema in a mouse model and assessed its potential mechanism. Methods and Results: A mouse model of lymphedema was created by ablation of a tail surface lymphatic network, and the tail diameter was measured to estimate lymphedema at day 28 after induction of lymphedema. Lymphatic vessels were detected by immunohistochemical staining as LYVE-1 and podoplanin double-positive cells. The DATS group was intraperitoneally injected with DATS for up to 10 days following lymphedema induction. DATS treated mice (n=10) showed a smaller tail thickness compared with control wild-type (WT) mice (n=11) (p<0.001) which was accompanied by a greater number of lymphatic vessels in the injured tails (p<0.05). Interestingly, the distal lymphatic lumen area was dilated in the control group, whereas the DATS treatment group improve dilated lymphatic area at the distal site of the injured point (p<0.001). In vitro experiments revealed that treatment with DATS promoted the differentiation of human lymphatic endothelial cells (LECs) into tube-like structures (p<0.001) in a dose-dependent manner, when cultured on Matrigel matrix. DATS also stimulated the phosphorylation of Akt in LECs. Furthermore, blockade of Akt activation canceled the DATS-stimulated increase in LEC differentiation. Conclusions: Our data clearly demonstrate that DATS promotes reparative lymphatic vessel growth and ameliorates secondary lymphedema, at least in part, through activation of the Akt pathway within lymphatic endothelial cells.

Rationale: Doxorubicin is a widely used anticancer drug. However, its major side effect, cardiotoxicity, results from cardiomyocyte loss that causes left ventricle (LV) wall thinning, chronic LV dysfunction and heart failure. Cardiomyocyte number expansion by thyroid hormone (T3) during preadolescence is suppressed by the developmental induction of an ERK1/2-specific dual specificity phosphatase 5 (DUSP5). Here, we sought to determine if a brief course of combined DUSP5 suppression plus T3 therapy replaces cardiomyocytes lost due to preexisting doxorubicin injury and reverses heart failure. Methods: We used in vivo-jetPEI to deliver DUSP5 or scrambled siRNA to ~5-week-old C57BL6 mice followed by 5 daily injections of T3 (2 ng/µg body weight). Genetic lineage tracing using Myh6-MerCreMer::Rosa26fs-Confetti mice and direct cardiomyocyte number counting, along with cell cycle inhibition (danusertib), was used to test if this treatment leads to de novo cardiomyocyte generation and improves LV contractile function. Three doses of doxorubicin (20 µg/g) given at 2-weekly intervals, starting at 5-weeks of age in C57BL6 mice, caused severe heart failure, as evident by a decrease in LV ejection fraction. Mice with an ~40 percentage point decrease in LVEF post-doxorubicin injury were randomized to receive either DUSP5 siRNA plus T3, or scrambled siRNA plus vehicle for T3. Age-matched mice without doxorubicin injury served as controls. Results: In uninjured adult mice, transient therapy with DUSP5 siRNA and T3 increases cardiomyocyte numbers, which is required for the associated increase in LV contractile function, since both are blocked by danusertib. In mice with chronic doxorubicin injury, DUSP5 siRNA plus T3 therapy rebuilds LV muscle by increasing cardiomyocyte numbers, which reverses LV dysfunction and prevents progressive chamber dilatation. Conclusion: RNA therapies are showing great potential. Importantly, a GMP compliant in vivo-jetPEI system for delivery of siRNA is already in use in humans, as is T3. Given these considerations, our findings provide a potentially highly translatable strategy for addressing doxorubicin cardiomyopathy, a currently untreatable condition.

Objective: Lymphatic vessels are distributed throughout the body and tightly collaborate with blood vessels to maintain tissue homeostasis. However, the functional roles of lymphangiogenesis in the process of reparative angiogenesis in ischemic tissues are largely unknown. Accordingly, we investigated potential roles of lymphangiogenesis using a mouse model of ischemia-induced angiogenesis. Approach and Results: Male C57BL/6J mice were subjected to unilateral hindlimb ischemia, in which not only angiogenesis but also lymphangiogenesis was induced. Next, the excessive and prolonged tissue edema model significantly deteriorated reparative angiogenesis and blood perfusion recovery in ischemic limbs. Finally, implantation of adipose-derived regenerative cells augmented ischemia-induced lymphangiogenesis, which was accompanied by reduced tissue edema and inflammation, resulting in improving reparative angiogenesis and blood perfusion recovery. In addition, inhibition of lymphangiogenesis by MAZ51, a specific VEGFR3 (vascular endothelial cell growth factor receptor 3) inhibitor, resulted in enhanced inflammatory cell infiltration, gene expression of TNF (tumor necrosis factor)-α, IL (interleukin)-1β, IL-6, TGF (transforming growth factor)-β, angiostatin, vasohibin, and endostatin, and tissue edema, resulting in reduced angiogenesis. Conclusions: The lymphatic system may have a clearance role of tissue edema and inflammation, which contribute to functional reparative angiogenesis in response to tissue ischemia. Modulation of lymphangiogenesis would become a novel therapeutic strategy for severe ischemic disease in addition to ordinary vascular intervention and therapeutic angiogenesis.