Tissue-based T cell activation and viral RNA persist for up to 2 years after SARS-CoV-2 infection.

The mechanisms of postacute medical conditions and unexplained symptoms after SARS-CoV-2 infection [Long Covid (LC)] are incompletely understood. There is growing evidence that viral persistence, immune dysregulation, and T cell dysfunction may play major roles. We performed whole-body positron emission tomography imaging in a well-characterized cohort of 24 participants at time points ranging from 27 to 910 days after acute SARS-CoV-2 infection using the radiopharmaceutical agent [18F]F-AraG, a selective tracer that allows for anatomical quantitation of activated T lymphocytes. Tracer uptake in the postacute COVID-19 group, which included those with and without continuing symptoms, was higher compared with prepandemic controls in many regions, including the brain stem, spinal cord, bone marrow, nasopharyngeal and hilar lymphoid tissue, cardiopulmonary tissues, and gut wall. T cell activation in the spinal cord and gut wall was associated with the presence of LC symptoms. In addition, tracer uptake in lung tissue was higher in those with persistent pulmonary symptoms specifically. Increased T cell activation in these tissues was also observed in many individuals without LC. Given the high [18F]F-AraG uptake detected in the gut, we obtained colorectal tissue for in situ hybridization of SARS-CoV-2 RNA and immunohistochemical studies in a subset of five participants with LC symptoms. We identified intracellular SARS-CoV-2 single-stranded spike protein-encoding RNA in rectosigmoid lamina propria tissue in all five participants and double-stranded spike protein-encoding RNA in three participants up to 676 days after initial COVID-19, suggesting that tissue viral persistence could be associated with long-term immunologic perturbations.

Podoplanin Positive Cell-derived Extracellular Vesicles Contribute to Cardiac Amyloidosis After Myocardial Infarction.

Background: Amyloidosis is a major long-term complication of chronic disease; however, whether it represents one of the complications of post-myocardial infarction (MI) is yet to be fully understood. Methods: Using wild-type and knocked-out MI mouse models and characterizing in vitro the exosomal communication between bone marrow-derived macrophages and activated mesenchymal stromal cells (MSC) isolated after MI, we investigated the mechanism behind Serum Amyloid A 3 (SAA3) protein overproduction in injured hearts. Results: Here, we show that amyloidosis occurs after MI and that amyloid fibers are composed of macrophage-derived SAA3 monomers. SAA3 overproduction in macrophages is triggered by exosomal communication from a subset of activated MSC, which, in response to MI, acquire the expression of a platelet aggregation-inducing type I transmembrane glycoprotein named Podoplanin (PDPN). Cardiac MSC PDPN+ communicate with and activate macrophages through their extracellular vesicles or exosomes. Specifically, MSC PDPN+ derived exosomes (MSC PDPN+ Exosomes) are enriched in SAA3 and exosomal SAA3 protein engages with Toll-like receptor 2 (TRL2) on macrophages, triggering an overproduction and impaired clearance of SAA3 proteins, resulting in aggregation of SAA3 monomers as rigid amyloid deposits in the extracellular space. The onset of amyloid fibers deposition alongside extra-cellular-matrix (ECM) proteins in the ischemic heart exacerbates the rigidity and stiffness of the scar, hindering the contractility of viable myocardium and overall impairing organ function. Using SAA3 and TLR2 deficient mouse models, we show that SAA3 delivered by MSC PDPN+ exosomes promotes post-MI amyloidosis. Inhibition of SAA3 aggregation via administration of a retro-inverso D-peptide, specifically designed to bind SAA3 monomers, prevents the deposition of SAA3 amyloid fibrils, positively modulates the scar formation, and improves heart function post-MI. Conclusion: Overall, our findings provide mechanistic insights into post-MI amyloidosis and suggest that SAA3 may be an attractive target for effective scar reversal after ischemic injury and a potential target in multiple diseases characterized by a similar pattern of inflammation and amyloid deposition. NOVELTY AND SIGNIFICANCE: What is known? Accumulation of rigid amyloid structures in the left ventricular wall impairs ventricle contractility.After myocardial infarction cardiac Mesenchymal Stromal Cells (MSC) acquire Podoplanin (PDPN) to better interact with immune cells.Amyloid structures can accumulate in the heart after chronic inflammatory conditions. What information does this article contribute? Whether accumulation of cumbersome amyloid structures in the ischemic scar impairs left ventricle contractility, and scar reversal after myocardial infarction (MI) has never been investigated.The pathophysiological relevance of PDPN acquirement by MSC and the functional role of their secreted exosomes in the context of post-MI cardiac remodeling has not been investigated.Amyloid structures are present in the scar after ischemia and are composed of macrophage-derived Serum Amyloid A (SAA) 3 monomers, although mechanisms of SAA3 overproduction is not established. SUMMARY OF NOVELTY AND SIGNIFICANCE: Here, we report that amyloidosis, a secondary phenomenon of an already preexisting and prolonged chronic inflammatory condition, occurs after MI and that amyloid structures are composed of macrophage-derived SAA3 monomers. Frequently studied cardiac amyloidosis are caused by aggregation of immunoglobulin light chains, transthyretin, fibrinogen, and apolipoprotein in a healthy heart as a consequence of systemic chronic inflammation leading to congestive heart failure with various types of arrhythmias and tissue stiffness. Although chronic MI is considered a systemic inflammatory condition, studies regarding the possible accumulation of amyloidogenic proteins after MI and the mechanisms involved in that process are yet to be reported. Here, we show that SAA3 overproduction in macrophages is triggered in a Toll-like Receptor 2 (TLR2)-p38MAP Kinase-dependent manner by exosomal communication from a subset of activated MSC, which, in response to MI, express a platelet aggregation-inducing type I transmembrane glycoprotein named Podoplanin. We provide the full mechanism of this phenomenon in murine models and confirm SAA3 amyloidosis in failing human heart samples. Moreover, we developed a retro-inverso D-peptide therapeutic approach, "DRI-R5S," specifically designed to bind SAA3 monomers and prevent post-MI aggregation and deposition of SAA3 amyloid fibrils without interfering with the innate immune response.

Tipifarnib Reduces Extracellular Vesicles and Protects From Heart Failure.

BACKGROUND: Heart failure (HF) is one of the leading causes of mortality worldwide. Extracellular vesicles, including small extracellular vesicles or exosomes, and their molecular cargo are known to modulate cell-to-cell communication during multiple cardiac diseases. However, the role of systemic extracellular vesicle biogenesis inhibition in HF models is not well documented and remains unclear. METHODS: We investigated the role of circulating exosomes during cardiac dysfunction and remodeling in a mouse transverse aortic constriction (TAC) model of HF. Importantly, we investigate the efficacy of tipifarnib, a recently identified exosome biogenesis inhibitor that targets the critical proteins (Rab27a [Ras associated binding protein 27a], nSMase2 [neutral sphingomyelinase 2], and Alix [ALG-2-interacting protein X]) involved in exosome biogenesis for this mouse model of HF. In this study, 10-week-old male mice underwent TAC surgery were randomly assigned to groups with and without tipifarnib treatment (10 mg/kg 3 times/wk) and monitored for 8 weeks, and a comprehensive assessment was conducted through performed echocardiographic, histological, and biochemical studies. RESULTS: TAC significantly elevated circulating plasma exosomes and markedly increased cardiac left ventricular dysfunction, cardiac hypertrophy, and fibrosis. Furthermore, injection of plasma exosomes from TAC mice induced left ventricular dysfunction and cardiomyocyte hypertrophy in uninjured mice without TAC. On the contrary, treatment of tipifarnib in TAC mice reduced circulating exosomes to baseline and remarkably improved left ventricular functions, hypertrophy, and fibrosis. Tipifarnib treatment also drastically altered the miRNA profile of circulating post-TAC exosomes, including miR 331-5p, which was highly downregulated both in TAC circulating exosomes and in TAC cardiac tissue. Mechanistically, miR 331-5p is crucial for inhibiting the fibroblast-to-myofibroblast transition by targeting HOXC8, a critical regulator of fibrosis. Tipifarnib treatment in TAC mice upregulated the expression of miR 331-5p that acts as a potent repressor for one of the fibrotic mechanisms mediated by HOXC8. CONCLUSIONS: Our study underscores the pathological role of exosomes in HF and fibrosis in response to pressure overload. Tipifarnib-mediated inhibition of exosome biogenesis and cargo sorting may serve as a viable strategy to prevent progressive cardiac remodeling in HF.

Epigenetic mechanisms regulate sex differences in cardiac reparative functions of bone marrow progenitor cells.

Historically, a lower incidence of cardiovascular diseases (CVD) and related deaths in women as compared with men of the same age has been attributed to female sex hormones, particularly estrogen and its receptors. Autologous bone marrow stem cell (BMSC) clinical trials for cardiac cell therapy overwhelmingly included male patients. However, meta-analysis data from these trials suggest a better functional outcome in postmenopausal women as compared with aged-matched men. Mechanisms governing sex-specific cardiac reparative activity in BMSCs, with and without the influence of sex hormones, remain unexplored. To discover these mechanisms, Male (M), female (F), and ovariectomized female (OVX) mice-derived EPCs were subjected to a series of molecular and epigenetic analyses followed by in vivo functional assessments of cardiac repair. F-EPCs and OVX EPCs show a lower inflammatory profile and promote enhanced cardiac reparative activity after intra-cardiac injections in a male mouse model of myocardial infarction (MI). Epigenetic sequencing revealed a marked difference in the occupancy of the gene repressive H3K9me3 mark, particularly at transcription start sites of key angiogenic and proinflammatory genes in M-EPCs compared with F-EPCs and OVX-EPCs. Our study unveiled that functional sex differences in EPCs are, in part, mediated by differential epigenetic regulation of the proinflammatory and anti-angiogenic gene CCL3, orchestrated by the control of H3K9me3 by histone methyltransferase, G9a/Ehmt2. Our research highlights the importance of considering the sex of donor cells for progenitor-based tissue repair.

Dysfunctional APPL1-Mediated Epigenetic Regulation in Diabetic Vascular Injury.

BACKGROUND: APN (adiponectin) and APPL1 (adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1) are potent vasculoprotective molecules, and their deficiency (eg, hypoadiponectinemia) contributes to diabetic vascular complications. However, the molecular mechanisms that govern their vasculoprotective genes as well as their alteration by diabetes remain unknown. METHODS: Diabetic medium-cultured rat aortic endothelial cells, mouse aortic endothelial cells from high-fat-diet animals, and diabetic human aortic endothelial cells were used for molecular/cellular investigations. The in vivo concept-prove demonstration was conducted using diabetic vascular injury and diabetic hindlimb ischemia models. RESULTS: In vivo animal experiments showed that APN replenishment caused APPL1 nuclear translocation, resulting in an interaction with HDAC (histone deacetylase) 2, which inhibited HDAC2 activity and increased H3Kac27 levels. Based on transcriptionome pathway-specific real-time polymerase chain reaction profiling and bioinformatics analysis, Angpt1 (angiopoietin 1), Ocln (occludin), and Cav1 (caveolin 1) were found to be the top 3 vasculoprotective genes suppressed by diabetes and rescued by APN in an APPL1-dependent manner. APN reverses diabetes-induced inhibition of Cav1 interaction with APPL1. APN-induced Cav1 expression was not affected by Angpt1 or Ocln deficiency, whereas APN-induced APPL1 nuclear translocation or upregulation of Angpt1/Ocln expression was abolished in the absence of Cav1 both in vivo and in vitro, suggesting Cav1 is upstream molecule of Angpt1/Ocln in response to APN administration. Chromatin immunoprecipitation-qPCR (quantitative polymerase chain reaction) demonstrated that APN caused significant enrichment of H3K27ac in Angpt1 and Ocln promoter region, an effect blocked by APPL1/Cav1 knockdown or HDAC2 overexpression. The protective effects of APN on the vascular system were attenuated by overexpression of HDAC2 and abolished by knocking out APPL1 or Cav1. The double knockdown of ANGPT1/OCLN blunted APN vascular protection both in vitro and in vivo. Furthermore, in diabetic human endothelial cells, HDAC2 activity is increased, H3 acetylation is decreased, and ANGPT1/OCLN expression is reduced, suggesting that the findings have important translational implications. CONCLUSIONS: Hypoadiponectinemia and dysregulation of APPL1-mediated epigenetic regulation are novel mechanisms leading to diabetes-induced suppression of vasculoprotective gene expression. Diabetes-induced pathological vascular remodeling may be prevented by interventions promoting APPL1 nuclear translocation and inhibiting HDAC2.

GSK-3α aggravates inflammation, metabolic derangement, and cardiac injury post-ischemia/reperfusion.

Reperfusion after acute myocardial infarction further exaggerates cardiac injury and adverse remodeling. Irrespective of cardiac cell types, loss of specifically the α isoform of the protein kinase GSK-3 is protective in chronic cardiac diseases. However, the role of GSK-3α in clinically relevant ischemia/reperfusion (I/R)-induced cardiac injury is unknown. Here, we challenged cardiomyocyte-specific conditional GSK-3α knockout (cKO) and littermate control mice with I/R injury and investigated the underlying molecular mechanism using an in vitro GSK-3α gain-of-function model in AC16 cardiomyocytes post-hypoxia/reoxygenation (H/R). Analysis revealed a significantly lower percentage of infarct area in the cKO vs. control hearts post-I/R. Consistent with in vivo findings, GSK-3α overexpression promoted AC16 cardiomyocyte death post-H/R which was accompanied by an induction of reactive oxygen species (ROS) generation. Consistently, GSK-3α gain-of-function caused mitochondrial dysfunction by significantly suppressing mitochondrial membrane potential. Transcriptomic analysis of GSK-3α overexpressing cardiomyocytes challenged with hypoxia or H/R revealed that NOD-like receptor (NLR), TNF, NF-κB, IL-17, and mitogen-activated protein kinase (MAPK) signaling pathways were among the most upregulated pathways. Glutathione and fatty acid metabolism were among the top downregulated pathways post-H/R. Together, these observations suggest that loss of cardiomyocyte-GSK-3α attenuates cardiac injury post-I/R potentially through limiting the myocardial inflammation, mitochondrial dysfunction, and metabolic derangement. Therefore, selective inhibition of GSK-3α may provide beneficial effects in I/R-induced cardiac injury and remodeling. KEY MESSAGES: GSK-3α promotes cardiac injury post-ischemia/reperfusion (I/R). GSK-3α regulates inflammatory and metabolic pathways post-hypoxia/reoxygenation (H/R). GSK-3α overexpression upregulates NOD-like receptor (NLR), TNF, NF-kB, IL-17, and MAPK signaling pathways in cardiomyocytes post-H/R. GSK-3α downregulates glutathione and fatty acid metabolic pathways in cardiomyocytes post-H/R.

Hypoadiponectinemia-induced upregulation of microRNA449b downregulating Nrf-1 aggravates cardiac ischemia-reperfusion injury in diabetic mice.

Diabetes enhances myocardial ischemic/reperfusion (MI/R) injury via an incompletely understood mechanism. Adiponectin (APN) is a cardioprotective adipokine suppressed by diabetes. However, how hypoadiponectinemia exacerbates cardiac injury remains incompletely understood. Dysregulation of miRNAs plays a significant role in disease development. However, whether hypoadiponectinemia alters cardiac miRNA profile, contributing to diabetic heart injury, remains unclear. Methods and Results: Wild-type (WT) and APN knockout (APN-KO) mice were subjected to MI/R. A cardiac microRNA profile was determined. Among 23 miRNAs increased in APN-KO mice following MI/R, miR-449b was most significantly upregulated (3.98-fold over WT mice). Administrating miR-449b mimic increased apoptosis, enlarged infarct size, and impaired cardiac function in WT mice. In contrast, anti-miR-449b decreased apoptosis, reduced infarct size, and improved cardiac function in APN-KO mice. Bioinformatic analysis predicted 73 miR-449b targeting genes, and GO analysis revealed oxidative stress as the top pathway regulated by these genes. Venn analysis followed by luciferase assay identified Nrf-1 and Ucp3 as the two most important miR-449b targets. In vivo administration of anti-miR-449b in APN-KO mice attenuated MI/R-stimulated superoxide overproduction. In vitro experiments demonstrated that high glucose/high lipid and simulated ischemia/reperfusion upregulated miR-449b and inhibited Nrf-1 and Ucp3 expression. These pathological effects were attenuated by anti-miR-449b or Nrf-1 overexpression. In a final attempt to validate our finding in a clinically relevant model, high-fat diet (HFD)-induced diabetic mice were subjected to MI/R and treated with anti-miR-449b or APN. Diabetes significantly increased miR-449b expression and downregulated Nrf-1 and Ucp3 expression. Administration of anti-miR-449b or APN preserved cardiac Nrf-1 expression, reduced cardiac oxidative stress, decreased apoptosis and infarct size, and improved cardiac function. Conclusion: We demonstrated for the first time that hypoadiponectinemia upregulates miR-449b and suppresses Nrf-1/Ucp3 expression, promoting oxidative stress and exacerbating MI/R injury in this population. Dysregulated APN/miR-449b/oxidative stress pathway is a potential therapeutic target against diabetic MI/R injury.

Novel roles for G protein-coupled receptor kinases in cardiac injury and repair.

G protein-coupled receptors (GPCRs) are key modulators of cell signaling. Multiple GPCRs are present in the heart where they regulate cardiac homeostasis including processes such as myocyte contraction, heart rate and coronary blood flow. GPCRs are pharmacological targets for several cardiovascular disorders including heart failure (HF) such as beta-adrenergic receptor (ßAR) blockers and angiotensin II receptor (AT1R) antagonists. The activity of GPCRs are finely regulated by GPCR kinases (GRKs), which phosphorylate agonist-occupied receptors and start the process of desensitization. Among the seven members of the GRK family, GRK2 and GRK5 are predominantly expressed in the heart, where they exhibit both canonical and non-canonical functions. Both kinases are known to be increased in cardiac pathologies and contribute to pathogenesis through their roles in different cellular compartments. Lowering or inhibiting their actions mediate cardioprotective effects against pathological cardiac growth and failing heart. Therefore, given their importance in cardiac dysfunction, these kinases are drawing attention as promising targets for the treatment of HF, which needs improved therapies. Over the past three decades, broad knowledge on GRK inhibition in HF has been gained by studies using genetically engineered animal models or through gene therapy with peptide inhibitors or using small molecule inhibitors. In this mini review, we summarize the work focusing on GRK2 and GRK5 but also discuss a couple of the non-abundant cardiac subtypes and their multi-functional roles in the normal and diseased heart and the potential and therapeutic targets.

Nitrative Modification of Caveolin-3: A Novel Mechanism of Cardiac Insulin Resistance and a Potential Therapeutic Target Against Ischemic Heart Failure in Prediabetic Animals.

BACKGROUND: Myocardial insulin resistance is a hallmark of diabetic cardiac injury. However, the underlying molecular mechanisms remain unclear. Recent studies demonstrate that the diabetic heart is resistant to other cardioprotective interventions, including adiponectin and preconditioning. The "universal" resistance to multiple therapeutic interventions suggests impairment of the requisite molecule(s) involved in broad prosurvival signaling cascades. Cav (Caveolin) is a scaffolding protein coordinating transmembrane signaling transduction. However, the role of Cav3 in diabetic impairment of cardiac protective signaling and diabetic ischemic heart failure is unknown. METHODS: Wild-type and gene-manipulated mice were fed a normal diet or high-fat diet for 2 to 12 weeks and subjected to myocardial ischemia and reperfusion. Insulin cardioprotection was determined. RESULTS: Compared with the normal diet group, the cardioprotective effect of insulin was significantly blunted as early as 4 weeks of high-fat diet feeding (prediabetes), a time point where expression levels of insulin-signaling molecules remained unchanged. However, Cav3/insulin receptor-ß complex formation was significantly reduced. Among multiple posttranslational modifications altering protein/protein interaction, Cav3 (not insulin receptor-ß) tyrosine nitration is prominent in the prediabetic heart. Treatment of cardiomyocytes with 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride reduced the signalsome complex and blocked insulin transmembrane signaling. Mass spectrometry identified Tyr73 as the Cav3 nitration site. Phenylalanine substitution of Tyr73 (Cav3Y73F) abolished 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride-induced Cav3 nitration, restored Cav3/insulin receptor-ß complex, and rescued insulin transmembrane signaling. It is most important that adeno-associated virus 9-mediated cardiomyocyte-specific Cav3Y73F reexpression blocked high-fat diet-induced Cav3 nitration, preserved Cav3 signalsome integrity, restored transmembrane signaling, and rescued insulin-protective action against ischemic heart failure. Last, diabetic nitrative modification of Cav3 at Tyr73 also reduced Cav3/AdipoR1 complex formation and blocked adiponectin cardioprotective signaling. CONCLUSIONS: Nitration of Cav3 at Tyr73 and resultant signal complex dissociation results in cardiac insulin/adiponectin resistance in the prediabetic heart, contributing to ischemic heart failure progression. Early interventions preserving Cav3-centered signalsome integrity is an effective novel strategy against diabetic exacerbation of ischemic heart failure.

ß3AR-Dependent Brain-Derived Neurotrophic Factor (BDNF) Generation Limits Chronic Postischemic Heart Failure.

BACKGROUND: Loss of brain-derived neurotrophic factor (BDNF)/TrkB (tropomyosin kinase receptor B) signaling accounts for brain and cardiac disorders. In neurons, ß-adrenergic receptor stimulation enhances local BDNF expression. It is unclear if this occurs in a pathophysiological relevant manner in the heart, especially in the ß-adrenergic receptor-desensitized postischemic myocardium. Nor is it fully understood whether and how TrkB agonists counter chronic postischemic left ventricle (LV) decompensation, a significant unmet clinical milestone. METHODS: We conducted in vitro studies using neonatal rat and adult murine cardiomyocytes, SH-SY5Y neuronal cells, and umbilical vein endothelial cells. We assessed myocardial ischemia (MI) impact in wild type, ß3AR knockout, or myocyte-selective BDNF knockout (myoBDNF KO) mice in vivo (via coronary ligation [MI]) or in isolated hearts with global ischemia-reperfusion (I/R). RESULTS: In wild type hearts, BDNF levels rose early after MI (<24 hours), plummeting at 4 weeks when LV dysfunction, adrenergic denervation, and impaired angiogenesis ensued. The TrkB agonist, LM22A-4, countered all these adverse effects. Compared with wild type, isolated myoBDNF KO hearts displayed worse infarct size/LV dysfunction after I/R injury and modest benefits from LM22A-4. In vitro, LM22A-4 promoted neurite outgrowth and neovascularization, boosting myocyte function, effects reproduced by 7,8-dihydroxyflavone, a chemically unrelated TrkB agonist. Superfusing myocytes with the ß3AR-agonist, BRL-37344, increased myocyte BDNF content, while ß3AR signaling underscored BDNF generation/protection in post-MI hearts. Accordingly, the ß1AR blocker, metoprolol, via upregulated ß3ARs, improved chronic post-MI LV dysfunction, enriching the myocardium with BDNF. Last, BRL-37344-imparted benefits were nearly abolished in isolated I/R injured myoBDNF KO hearts. CONCLUSIONS: BDNF loss underscores chronic postischemic heart failure. TrkB agonists can improve ischemic LV dysfunction via replenished myocardial BDNF content. Direct cardiac ß3AR stimulation, or ß-blockers (via upregulated ß3AR), is another BDNF-based means to fend off chronic postischemic heart failure.

Pepducin ICL1-9-Mediated ß2-Adrenergic Receptor-Dependent Cardiomyocyte Contractility Occurs in a Gi Protein/ROCK/PKD-Sensitive Manner.

PURPOSE: ß-Adrenergic receptors (ßAR) are essential targets for the treatment of heart failure (HF); however, chronic use of ßAR agonists as positive inotropes to increase contractility in a Gs protein-dependent manner is associated with increased mortality. Alternatively, we previously reported that allosteric modulation of ß2AR with the pepducin intracellular loop (ICL)1-9 increased cardiomyocyte contractility in a ß-arrestin (ßarr)-dependent manner, and subsequently showed that ICL1-9 activates the Ras homolog family member A (RhoA). Here, we aimed to elucidate both the proximal and downstream signaling mediators involved in the promotion of cardiomyocyte contractility in response to ICL1-9. METHODS: We measured adult mouse cardiomyocyte contractility in response to ICL1-9 or isoproterenol (ISO, as a positive control) alone or in the presence of inhibitors of various potential components of ßarr- or RhoA-dependent signaling. We also assessed the contractile effects of ICL1-9 on cardiomyocytes lacking G protein-coupled receptor (GPCR) kinase 2 (GRK2) or 5 (GRK5). RESULTS: Consistent with RhoA activation by ICL1-9, both Rho-associated protein kinase (ROCK) and protein kinase D (PKD) inhibition were able to attenuate ICL1-9-mediated contractility, as was inhibition of myosin light chain kinase (MLCK). While neither GRK2 nor GRK5 deletion impacted ICL1-9-mediated contractility, pertussis toxin attenuated the response, suggesting that ICL1-9 promotes downstream RhoA-dependent signaling in a Gi protein-dependent manner. CONCLUSION: Altogether, our study highlights a novel signaling modality that may offer a new approach to the promotion, or preservation, of cardiac contractility during HF via the allosteric regulation of ß2AR to promote Gi protein/ßarr-dependent activation of RhoA/ROCK/PKD signaling.

Binding potential changes of SERT in patients with depression are associated with remission: A prospective [¹²³I]ß-CIT-SPECT study.

The status of remission in patients with major depressive disorder treated with selective serotonin reuptake inhibitors (SSRIs) is mostly evaluated with clinical rating scales. Morphological correlates of the remission status remain a rare event. Addressing this challenge, we investigated functional correlates of remission by assessment of serotonin and dopamine transporter availability (SERT and DAT) using single-photon emission computed tomography (SPECT). Our purpose was to identify changes in the SERT/DAT binding potential in accordance with the clinical improvement. Nineteen drug-naïve patients with a Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) diagnosis of major depression were included. [¹²³I]2ß-carbomethoxy-3ß-(4-iodophenyl)tropane(ß-CIT) SPECT was obtained from each participant before (baseline) and after 6 weeks (follow-up) of standardized treatment with escitalopram. The [¹²³I]ß-CIT-SPECT recordings were acquired 4 hr (SERT-weighted) and 20-24 hr p.i (DAT-weighted), and binding potentials (˜BPND: baseline, follow-up, and rate of change) were calculated for thalamus, midbrain, pons (SERT), and striatum (DAT). From all study participants, neuropsychiatric symptoms were assessed using Hamilton depression (HAM-D) and Beck Depression Inventory scores. At follow-up, patients were divided into responders and nonresponders (as well as remitters and nonremitters). Compared to nonremitted, remitted patients showed over the course of 6 weeks a significantly higher loss of SERT binding potential in the thalamus (p = .036) and in the midbrain (p = .019). Additionally, the correlation of HAM-D with SERT binding potential in the thalamus showed a trend toward significance (p = .057) with higher HAM-D scores (at baseline) leading to lower SERT binding potential. No significant associations were identified for the analysis of baseline prediction of therapy response with SERT and DAT. Our results suggest that patients who remit from their depressive symptoms under escitalopram are characterized by stronger decreases of SERT, indicating that escitalopram blocking of SERT leads to clinical improvement. Therefore, this study shows that measuring SERT availability with SPECT could be an efficient and applicable technique to illustrate a possible underlying pathophysiology of symptom remission in response to treatment. In addition, the present results could help to stimulate new treatment approaches based on SERT and DAT binding. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Beta-3 adrenergic receptor overexpression reverses aortic stenosis-induced heart failure and restores balanced mitochondrial dynamics.

Aortic stenosis (AS) is associated with left ventricular (LV) hypertrophy and heart failure (HF). There is a lack of therapies able to prevent/revert AS-induced HF. Beta3 adrenergic receptor (ß3AR) signaling is beneficial in several forms of HF. Here, we studied the potential beneficial effect of ß3AR overexpression on AS-induced HF. Selective ß3AR stimulation had a positive inotropic effect. Transgenic mice constitutively overexpressing human ß3AR in the heart (c-hß3tg) were protected from the development of HF in response to induced AS, and against cardiomyocyte mitochondrial dysfunction (fragmented mitochondria with remodeled cristae and metabolic reprogramming featuring altered substrate use). Similar beneficial effects were observed in wild-type mice inoculated with adeno-associated virus (AAV9) inducing cardiac-specific overexpression of human ß3AR before AS induction. Moreover, AAV9-hß3AR injection into wild-type mice at late disease stages, when cardiac hypertrophy and metabolic reprogramming are already advanced, reversed the HF phenotype and restored balanced mitochondrial dynamics, demonstrating the potential of gene-therapy-mediated ß3AR overexpression in AS. Mice with cardiac specific ablation of Yme1l (cYKO), characterized by fragmented mitochondria, showed an increased mortality upon AS challenge. AAV9-hß3AR injection in these mice before AS induction reverted the fragmented mitochondria phenotype and rescued them from death. In conclusion, our results step out that ß3AR overexpression might have translational potential as a therapeutic strategy in AS-induced HF.

CLIC4 localizes to mitochondrial-associated membranes and mediates cardioprotection.

Mitochondrial-associated membranes (MAMs) are known to modulate organellar and cellular functions and can subsequently affect pathophysiology including myocardial ischemia-reperfusion (IR) injury. Thus, identifying molecular targets in MAMs that regulate the outcome of IR injury will hold a key to efficient therapeutics. Here, we found chloride intracellular channel protein (CLIC4) presence in MAMs of cardiomyocytes and demonstrate its role in modulating ER and mitochondrial calcium homeostasis under physiological and pathological conditions. In a murine model, loss of CLIC4 increased myocardial infarction and substantially reduced cardiac function after IR injury. CLIC4 null cardiomyocytes showed increased apoptosis and mitochondrial dysfunction upon hypoxia-reoxygenation injury in comparison to wild-type cardiomyocytes. Overall, our results indicate that MAM-CLIC4 is a key mediator of cellular response to IR injury and therefore may have a potential implication on other pathophysiological processes.

Ectonucleoside triphosphate diphosphohydrolase-1 (CD39) impacts TGF-ß1 responses: insights into cardiac fibrosis and function following myocardial infarction.

Extracellular purine nucleotides and nucleosides released from activated or injured cells influence multiple aspects of cardiac physiology and pathophysiology. Ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1; CD39) hydrolyzes released nucleotides and thereby regulates the magnitude and duration of purinergic signaling. However, the impact of CD39 activity on post-myocardial infarction (MI) remodeling is incompletely understood. We measured the levels and activity of ectonucleotidases in human left ventricular samples from control and ischemic cardiomyopathy (ICM) hearts and examined the impact of ablation of Cd39 expression on post-myocardial infarction remodeling in mice. We found that human CD39 levels and activity are significantly decreased in ICM hearts (n = 5) compared with control hearts (n = 5). In mice null for Cd39, cardiac function and remodeling are significantly compromised in Cd39-/- mice following myocardial infarction. Fibrotic markers including plasminogen activator inhibitor-1 (PAI-1) expression, fibrin deposition, α-smooth muscle actin (αSMA), and collagen expression are increased in Cd39-/- hearts. Importantly, we found that transforming growth factor ß1 (TGF-ß1) stimulates ATP release and induces Cd39 expression and activity on cardiac fibroblasts, constituting an autocrine regulatory pathway not previously appreciated. Absence of CD39 activity on cardiac fibroblasts exacerbates TGF-ß1 profibrotic responses. Treatment with exogenous ectonucleotidase rescues this profibrotic response in Cd39-/- fibroblasts. Together, these data demonstrate that CD39 has important interactions with TGF-ß1-stimulated autocrine purinergic signaling in cardiac fibroblasts and dictates outcomes of cardiac remodeling following myocardial infarction. Our results reveal that ENTPD1 (CD39) regulates TGF-ß1-mediated fibroblast activation and limits adverse cardiac remodeling following myocardial infarction.NEW & NOTEWORTHY We show that CD39 is a critical modulator of TGF-ß1-mediated fibroblast activation and cardiac remodeling following myocardial infarction via modulation of nucleotide signaling. TGF-ß1-induced CD39 expression generates a negative feedback loop that attenuates cardiac fibroblast activation. In the absence of CD39 activity, collagen deposition is increased, elastin expression is decreased, and diastolic dysfunction is worsened. Treatment with ecto-apyrase attenuates the TGF-ß1-induced profibrotic cardiac fibroblast phenotype, revealing a novel approach to combat post-myocardial infarction cardiac fibrosis.

GRK2 regulates ADP signaling in platelets via P2Y1 and P2Y12.

The critical role of G protein-coupled receptor kinase 2 (GRK2) in regulating cardiac function has been well documented for >3 decades. Targeting GRK2 has therefore been extensively studied as a novel approach to treating cardiovascular disease. However, little is known about its role in hemostasis and thrombosis. We provide here the first evidence that GRK2 limits platelet activation and regulates the hemostatic response to injury. Deletion of GRK2 in mouse platelets causes increased platelet accumulation after laser-induced injury in the cremaster muscle arterioles, shortens tail bleeding time, and enhances thrombosis in adenosine 5'-diphosphate (ADP)-induced pulmonary thromboembolism and in FeCl3-induced carotid injury. GRK2-/- platelets have increased integrin activation, P-selectin exposure, and platelet aggregation in response to ADP stimulation. Furthermore, GRK2-/- platelets retain the ability to aggregate in response to ADP restimulation, indicating that GRK2 contributes to ADP receptor desensitization. Underlying these changes in GRK2-/- platelets is an increase in Ca2+ mobilization, RAS-related protein 1 activation, and Akt phosphorylation stimulated by ADP, as well as an attenuated rise of cyclic adenosine monophosphate levels in response to ADP in the presence of prostaglandin I2. P2Y12 antagonist treatment eliminates the phenotypic difference in platelet accumulation between wild-type and GRK2-/- mice at the site of injury. Pharmacologic inhibition of GRK2 activity in human platelets increases platelet activation in response to ADP. Finally, we show that GRK2 binds to endogenous Gßγ subunits during platelet activation. Collectively, these results show that GRK2 regulates ADP signaling via P2Y1 and P2Y12, interacts with Gßγ, and functions as a signaling hub in platelets for modulating the hemostatic response to injury.

GRK2 in cardiovascular disease and its potential as a therapeutic target.

Cardiovascular diseases (CVDs) represent the leading cause of death globally. Despite major advances in the field of pharmacological CVD treatments, particularly in the field of heart failure (HF) research, case numbers and overall mortality remain high and have trended upwards over the last few years. Thus, identifying novel molecular targets for developing HF therapeutics remains a key research focus. G protein-coupled receptors (GPCRs) are critical myocardial signal transducers which regulate cardiac contractility, growth, adaptation and metabolism. Additionally, GPCR dysregulation underlies multiple models of cardiac pathology, and most pharmacological therapeutics currently used in HF target these receptors. Currently-approved treatments have improved patient outcomes, but therapies to stop or reverse HF are lacking. A recent focus on GPCR intracellular-regulating proteins such as GPCR kinases (GRKs) has uncovered GRK2 as a promising target for combating HF. Current literature strongly establishes increased levels and activity of GRK2 in multiple models of CVD. Additionally, the GRK2 interactome includes numerous proteins which interact with differential domains of GRK2 to modulate both beneficial and deleterious signaling pathways in the heart, indicating that these domains can be targeted with a high level of specificity unique to various cardiac pathologies. These data support the premise that GRK2 should be at the forefront of a novel investigative drug search. This perspective reviews cardiac GPCRs, describes the structure and functions of GRK2 in cardiac function and maladaptive pathology, and summarizes the ongoing and future research for targeting this critical kinase across cellular, animal and human models of cardiac dysfunction and HF.

Diabetes impairs cardioprotective function of endothelial progenitor cell-derived extracellular vesicles via H3K9Ac inhibition.

Background and Purpose: Myocardial infarction (MI) in diabetic patients results in higher mortality and morbidity. We and others have previously shown that bone marrow-endothelial progenitor cells (EPCs) promote cardiac neovascularization and attenuate ischemic injury. Lately, small extracellular vesicles (EVs) have emerged as major paracrine effectors mediating the benefits of stem cell therapy. Modest clinical outcomes of autologous cell-based therapies suggest diabetes-induced EPC dysfunction and may also reflect their EV derivatives. Moreover, studies suggest that post-translational histone modifications promote diabetes-induced vascular dysfunctions. Therefore, we tested the hypothesis that diabetic EPC-EVs may lose their post-injury cardiac reparative function by modulating histone modification in endothelial cells (ECs). Methods: We collected EVs from the culture medium of EPCs isolated from non-diabetic (db/+) and diabetic (db/db) mice and examined their effects on recipient ECs and cardiomyocytes in vitro, and their reparative function in permanent ligation of left anterior descending (LAD) coronary artery and ischemia/reperfusion (I/R) myocardial ischemic injuries in vivo. Results: Compared to db/+ EPC-EVs, db/db EPC-EVs promoted EC and cardiomyocyte apoptosis and repressed tube-forming capacity of ECs. In vivo, db/db EPC-EVs depressed cardiac function, reduced capillary density, and increased fibrosis compared to db/+ EPC-EV treatments after MI. Moreover, in the I/R MI model, db/+ EPC-EV-mediated acute cardio-protection was lost with db/db EPC-EVs, and db/db EPC-EVs increased immune cell infiltration, infarct area, and plasma cardiac troponin-I. Mechanistically, histone 3 lysine 9 acetylation (H3K9Ac) was significantly decreased in cardiac ECs treated with db/db EPC-EVs compared to db/+ EPC-EVs. The H3K9Ac chromatin immunoprecipitation sequencing (ChIP-Seq) results further revealed that db/db EPC-EVs reduced H3K9Ac level on angiogenic, cell survival, and proliferative genes in cardiac ECs. We found that the histone deacetylase (HDAC) inhibitor, valproic acid (VPA), partly restored diabetic EPC-EV-impaired H3K9Ac levels, tube formation and viability of ECs, and enhanced cell survival and proliferative genes, Pdgfd and Sox12, expression. Moreover, we observed that VPA treatment improved db/db EPC-mediated post-MI cardiac repair and functions. Conclusions: Our findings unravel that diabetes impairs EPC-EV reparative function in the ischemic heart, at least partially, through HDACs-mediated H3K9Ac downregulation leading to transcriptional suppression of angiogenic, proliferative and cell survival genes in recipient cardiac ECs. Thus, HDAC inhibitors may potentially be used to restore the function of diabetic EPC and other stem cells for autologous cell therapy applications.