Targeted Gene Deletion or Antagonism of the Prostaglandin E2 EP3 Receptor Protects Against Cardiac Injury Postmyocardial Infarction.

BACKGROUND: Prostaglandin E2 acts through 4 G-protein-coupled receptors (EP1-EP4). We previously reported that activation of the EP3 receptor reduces cardiac contractility, and its expression increases after a myocardial infarction (MI), mediating the reduction in cardiac function. In contrast, cardiac overexpression of the EP4 receptor in MI substantially improves cardiac function. Moreover, we recently reported that mice overexpressing EP3 have heart failure under basal conditions and worsened cardiac function after MI. Thus, the deleterious effects of the prostaglandin E2 EP receptors in the heart are mediated via its EP3 receptor. We, therefore, hypothesized that cardiomyocyte-specific knockout (CM-EP3 KO) or antagonism of the EP3 receptor protects the heart after MI. METHODS: To test our hypothesis, we made the novel CM-EP3 KO mouse and subjected CM-EP3 KO or controls to sham or MI surgery for 2 weeks. In separate experiments, C57BL/6 mice were subjected to 2 weeks of MI and treated with either the EP3 antagonist L798 106 or vehicle starting 3 days post-MI. RESULTS: CM-EP3 KO significantly prevented a decline in cardiac function after MI compared with WT animals and prevented an increase in hypertrophy and fibrosis. Excitingly, mice treated with L798 106 3 days after MI had significantly better cardiac function compared with vehicle-treated mice. CONCLUSIONS: Altogether, these data suggest that EP3 may play a direct role in regulating cardiac function, and pharmaceutical targeting of the EP3 receptor may be a therapeutic option in the treatment of heart failure.

Prostaglandin E2 affects mitochondrial function in adult mouse cardiomyocytes and hearts.

Prostaglandin E2 (PGE2) signals differently through 4 receptor subtypes (EP1-EP4) to elicit diverse physiologic/pathologic effects. We previously reported that PGE2 via its EP3 receptor reduces cardiac contractility and male mice with cardiomyocyte-specific deletion of the EP4 receptor (EP4 KO) develop dilated cardiomyopathy. The aim of this study was to identify pathways responsible for this phenotype. We performed ingenuity pathway analysis (IPA) and found that genes differentiating WT mice and EP4 KO mice were significantly overrepresented in mitochondrial (adj. p value = 6.28 × 10-26) and oxidative phosphorylation (adj. p value = 1.58 × 10-27) pathways. Electron microscopy from the EP4 KO hearts show substantial mitochondrial disarray and disordered cristae. Not surprisingly, isolated adult mouse cardiomyocytes (AVM) from these mice have reduced ATP levels compared to their WT littermates and reduced expression of key genes involved in the electron transport chain (ETC) in older mice. Moreover, treatment of AVM from C57Bl/6 mice with PGE2 or the EP3 agonist sulprostone resulted in changes of various genes involved in the ETC, measured by the Mitochondrial Energy Metabolism RT2-profiler assay. Lastly, the EP4 KO mice have reduced expression of superoxide dismuatse-2 (SOD2), whereas treatment of AVM with PGE2 or sulprostone increase superoxide production, suggesting increased oxidative stress levels in these EP4 KO mice. Altogether the current study supports the premise that PGE2 acting via its EP4 receptor is protective, while signaling through its other receptors, likely EP3, is deleterious.

Prostaglandin E2 and myocarditis; friend or foe?

This review article summarizes the role of prostaglandin E2 (PGE2) and its receptors (EP1-EP4) as it relates to the inflammatory cardiomyopathy, myocarditis. During the COVID-19 pandemic, the onset of myocarditis in a subset of patients prompted a debate on the use of nonsteroidal anti-inflammatory drugs (NSAIDs), like ibuprofen, which act to inhibit the actions of prostaglandins. This review aims to further understanding of the role of PGE2 in the pathogenesis or protection of the myocardium in myocarditis. Inflammatory cardiomyopathies encompass a broad spectrum of disorders, all characterized by cardiac inflammation. Therefore, for the purpose of this review, the authors have placed particular emphasis on etiologies of myocarditis where effects of PGE2 have been documented.

Deleterious effects of cardiomyocyte-specific prostaglandin E2 EP3 receptor overexpression on cardiac function after myocardial infarction.

AIMS: Prostaglandin E2 (PGE2) is a lipid hormone that signals through 4 different G-protein coupled receptor subtypes which act to regulate key physiological processes. Our laboratory has previously reported that PGE2 through its EP3 receptor reduces cardiac contractility at the level of isolated cardiomyocytes and in the isolated working heart preparation. We therefore hypothesized that cardiomyocyte specific overexpression of the PGE2 EP3 receptor further decreases cardiac function in a mouse model of heart failure produced by myocardial infarction. MAIN METHODS: Our study tested this hypothesis using EP3 transgenic mice (EP3 TG), which overexpress the porcine analogue of human EP3 in the cardiomyocytes, and their wildtype (WT) littermates. Mice were analyzed 2 wks after myocardial infarction (MI) or sham operation by echocardiography, RT-PCR, immunohistochemistry, and histology. KEY FINDINGS: We found that the EP3 TG sham controls had a reduced ejection fraction, reduced fractional shortening, and an increased left ventricular dimension at systole and diastole compared to the WT sham controls. Moreover, there was a further reduction in the EP3 TG mice after myocardial infarction. Additionally, single-cell analysis of cardiomyocytes isolated from EP3 TG mice showed reduced contractility under basal conditions. Overexpression of EP3 significantly increased cardiac hypertrophy, interstitial collagen fraction, macrophage, and T-cell infiltration in the sham operated group. Interestingly, after MI, there were no changes in hypertrophy but there were changes in collagen fraction, and inflammatory cell infiltration. SIGNIFICANCE: Overexpression of EP3 reduces cardiac function under basal conditions and this is exacerbated after myocardial infarction.

Rapid Treatment with Intramuscular Magnesium Sulfate During Cardiopulmonary Resuscitation Does Not Provide Neuroprotection Following Cardiac Arrest.

Brain injury is the most common cause of death for patients resuscitated from cardiac arrest. Magnesium is an attractive neuroprotective compound which protects neurons from ischemic injury by reducing neuronal calcium overload via NMDA receptor modulation and preventing calcium-induced mitochondrial permeability transition. Intramuscular (IM) delivery of MgSO4 during CPR has the potential to target these mechanisms within an early therapeutic window. We hypothesize that IM MgSO4 administrated during CPR could achieve therapeutic serum magnesium levels within 15 min after ROSC and improve neurologic outcomes in a rat model of asphyxial cardiac arrest. Male Long Evans rats were subjected to 8-min asphyxial cardiac arrest and block randomized to receive placebo, 107 mg/kg, 215 mg/kg, or 430 mg/kg MgSO4 IM at the onset of CPR. Serum magnesium concentrations increased rapidly with IM delivery during CPR, achieving twofold to fourfold increase by 15 min after ROSC in all magnesium dose groups. Rats subjected to cardiac arrest or sham surgery were block randomized to treatment groups for assessment of neurological outcomes. We found that IM MgSO4 during CPR had no effect on ROSC rate (p > 0.05). IM MgSO4 treatment had no statistically significant effect on 10-day survival with good neurologic function or hippocampal CA1 pyramidal neuron survival compared to placebo treatment. In conclusion, a single dose IM MgSO4 during CPR achieves up to fourfold baseline serum magnesium levels within 15 min after ROSC; however, this treatment strategy did not improve survival, recovery of neurologic function, or neuron survival. Future studies with repeated dosing or in combination with hypothermic targeted temperature management may be indicated.

Prostaglandin E2 EP receptors in cardiovascular disease: An update.

This review article provides an update for the role of prostaglandin E2 receptors (EP1, EP2, EP3 and EP4) in cardiovascular disease. Where possible we have reported citations from the last decade although this was not possible for all of the topics covered due to the paucity of publications. The authors have attempted to cover the subjects of ischemia-reperfusion injury, arrhythmias, hypertension, novel protein binding partners of the EP receptors and their pathophysiological significance, and cardiac regeneration. These latter two topics bring studies of the EP receptors into new and exciting areas of research that are just beginning to be explored. Where there is peer-reviewed literature, the authors have placed particular emphasis on clinical studies although these are limited in number.

Mitochondrial fission and mitophagy are independent mechanisms regulating ischemia/reperfusion injury in primary neurons.

Mitochondrial dynamics and mitophagy are constitutive and complex systems that ensure a healthy mitochondrial network through the segregation and subsequent degradation of damaged mitochondria. Disruption of these systems can lead to mitochondrial dysfunction and has been established as a central mechanism of ischemia/reperfusion (I/R) injury. Emerging evidence suggests that mitochondrial dynamics and mitophagy are integrated systems; however, the role of this relationship in the context of I/R injury remains unclear. To investigate this concept, we utilized primary cortical neurons isolated from the novel dual-reporter mitochondrial quality control knockin mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J) with conditional knockout (KO) of Drp1 to investigate changes in mitochondrial dynamics and mitophagic flux during in vitro I/R injury. Mitochondrial dynamics was quantitatively measured in an unbiased manner using a machine learning mitochondrial morphology classification system, which consisted of four different classifications: network, unbranched, swollen, and punctate. Evaluation of mitochondrial morphology and mitophagic flux in primary neurons exposed to oxygen-glucose deprivation (OGD) and reoxygenation (OGD/R) revealed extensive mitochondrial fragmentation and swelling, together with a significant upregulation in mitophagic flux. Furthermore, the primary morphology of mitochondria undergoing mitophagy was classified as punctate. Colocalization using immunofluorescence as well as western blot analysis revealed that the PINK1/Parkin pathway of mitophagy was activated following OGD/R. Conditional KO of Drp1 prevented mitochondrial fragmentation and swelling following OGD/R but did not alter mitophagic flux. These data provide novel evidence that Drp1 plays a causal role in the progression of I/R injury, but mitophagy does not require Drp1-mediated mitochondrial fission.

Plasma Proteomic Profile Predicts Survival in Heart Failure With Reduced Ejection Fraction.

BACKGROUND: It remains unclear whether the plasma proteome adds value to established predictors in heart failure (HF) with reduced ejection fraction (HFrEF). We sought to derive and validate a plasma proteomic risk score (PRS) for survival in patients with HFrEF (HFrEF-PRS). METHODS: Patients meeting Framingham criteria for HF with EF<50% were enrolled (N=1017) and plasma underwent SOMAscan profiling (4453 targets). Patients were randomly divided 2:1 into derivation and validation cohorts. The HFrEF-PRS was derived using Cox regression of all-cause mortality adjusted for clinical score and NT-proBNP (N-terminal pro-B-type natriuretic peptide), then was tested in the validation cohort. Risk stratification improvement was evaluated by C statistic, integrated discrimination index, continuous net reclassification index, and median improvement in risk score for 1-year and 3-year mortality. RESULTS: Participants' mean age was 68 years, 48% identified as Black, 35% were female, and 296 deaths occurred. In derivation (n=681), 128 proteins associated with mortality, 8 comprising the optimized HFrEF-PRS. In validation (n=336) the HFrEF-PRS associated with mortality (hazard ratio, 2.27 [95% CI, 1.84-2.82], P=6.3×10-14), Kaplan-Meier curves differed significantly between HFrEF-PRS quartiles (P=2.2×10-6), and it remained significant after adjustment for clinical score and NT-proBNP (hazard ratio, 1.37 [95% CI, 1.05-1.79], P=0.021). The HFrEF-PRS improved metrics of risk stratification (C statistic change, 0.009, P=0.612; integrated discrimination index, 0.041, P=0.010; net reclassification index=0.391, P=0.078; median improvement in risk score=0.039, P=0.016) and associated with cardiovascular death and HF phenotypes (eg, 6-minute walk distance, EF change). Most HFrEF-PRS proteins had little known connection to HFrEF. CONCLUSIONS: A plasma multiprotein score improved risk stratification in patients with HFrEF and identified novel candidates.

Machine learning-based classification of mitochondrial morphology in primary neurons and brain.

The mitochondrial network continually undergoes events of fission and fusion. Under physiologic conditions, the network is in equilibrium and is characterized by the presence of both elongated and punctate mitochondria. However, this balanced, homeostatic mitochondrial profile can change morphologic distribution in response to various stressors. Therefore, it is imperative to develop a method that robustly measures mitochondrial morphology with high accuracy. Here, we developed a semi-automated image analysis pipeline for the quantitation of mitochondrial morphology for both in vitro and in vivo applications. The image analysis pipeline was generated and validated utilizing images of primary cortical neurons from transgenic mice, allowing genetic ablation of key components of mitochondrial dynamics. This analysis pipeline was further extended to evaluate mitochondrial morphology in vivo through immunolabeling of brain sections as well as serial block-face scanning electron microscopy. These data demonstrate a highly specific and sensitive method that accurately classifies distinct physiological and pathological mitochondrial morphologies. Furthermore, this workflow employs the use of readily available, free open-source software designed for high throughput image processing, segmentation, and analysis that is customizable to various biological models.

The deleterious role of the prostaglandin E2 EP3 receptor in angiotensin II hypertension.

Angiotensin II (ANG II) plays a key role in regulating blood pressure and inflammation. Prostaglandin E2 (PGE2) signals through four different G protein-coupled receptors, eliciting a variety of effects. We reported that activation of the EP3 receptor reduces cardiac contractility. More recently, we have shown that overexpression of the EP4 receptor is protective in a mouse myocardial infarction model. We hypothesize in this study that the relative abundance of EP3 and EP4 receptors is a major determinant of end-organ damage in the diseased heart. Thus EP3 is detrimental to cardiac function and promotes inflammation, whereas antagonism of the EP3 receptor is protective in an ANG II hypertension (HTN) model. To test our hypothesis, male 10- to 12-wk-old C57BL/6 mice were anesthetized with isoflurane and osmotic minipumps containing ANG II were implanted subcutaneously for 2 wk. We found that antagonism of the EP3 receptor using L798,106 significantly attenuated the increase in blood pressure with ANG II infusion. Moreover, antagonism of the EP3 receptor prevented a decline in cardiac function after ANG II treatment. We also found that 10- to 12-wk-old EP3-transgenic mice, which overexpress EP3 in the cardiomyocytes, have worsened cardiac function. In conclusion, activation or overexpression of EP3 exacerbates end-organ damage in ANG II HTN. In contrast, antagonism of the EP3 receptor is beneficial and reduces cardiac dysfunction, inflammation, and HTN.NEW & NOTEWORTHY This study is the first to show that systemic treatment with an EP3 receptor antagonist (L798,106) attenuates the angiotensin II-induced increase in blood pressure in mice. The results from this project could complement existing hypertension therapies by combining blockade of the EP3 receptor with antihypertensive drugs.

Prostaglandin E2 and an EP4 receptor agonist inhibit LPS-Induced monocyte chemotactic protein 5 production and secretion in mouse cardiac fibroblasts via Akt and NF-κB signaling.

BACKGROUND: Prostaglandin E2 (PGE2) signals through 4 separate G-protein coupled receptor sub-types to elicit a variety of physiologic and pathophysiological effects. We have previously reported that mice lacking the EP4 receptor in the cardiomyocytes develop heart failure with a phenotype of dilated cardiomyopathy. Also, these mice have increased levels of chemokines, like MCP-5, in their left ventricles. We have recently reported that overexpression of the EP4 receptor could improve cardiac function in the myocardial infarction model. Furthermore, we showed that overexpression of EP4 had an anti-inflammatory effect in the whole left ventricle. It has also been shown that PGE2 can antagonize lipopolysaccharide-induced secretion of chemokines/cytokines in various cell types. We therefore hypothesized that PGE2 inhibits lipopolysaccharide (LPS)-induced MCP-5 secretion in adult mouse cardiac fibroblasts via its EP4 receptor. METHODS AND RESULTS: Our hypothesis was tested using isolated mouse adult ventricular fibroblasts (AVF) treated with LPS. Pre-treatment of the cells with PGE2 and the EP4 agonist CAY10598 resulted in reductions of the pro-inflammatory response induced by LPS. Specifically, we observed reductions in MCP-5 secretion. Western blot analysis showed reductions in phosphorylated Akt and IκBα indicating reduced NF-κB activation. The anti-inflammatory effects of PGE2 and EP4 agonist signaling appeared to be independent of cAMP, p-44/42, or p38 pathways. CONCLUSION: Exogenous treatment of PGE2 and the EP4 receptor agonist blocked the pro-inflammatory actions of LPS. Mechanistically, this was mediated via reduced Akt phosphorylation and inhibition of NF-κB.

Overexpression of prostaglandin E2 EP4 receptor improves cardiac function after myocardial infarction.

BACKGROUND: Prostaglandin E2 (PGE2) signals through 4 separate G-protein coupled receptor sub-types to elicit a variety of physiologic and pathophysiological effects. We recently reported that PGE2 via its EP3 receptor could reduce cardiac contractility of isolated myocytes and the working heart preparation. We thus hypothesized that there is an imbalance in the EP3/EP4 ratio towards EP3 in the failing heart and that overexpression of EP4 in a mouse model of heart failure would improve cardiac function. METHODS AND RESULTS: Our hypothesis was tested in a mouse model of myocardial infarction (MI) with the use of AAV9-EP4 driven by the myosin heavy chain promoter to overexpress EP4 in the cardiac myocytes. Echocardiography was performed to assess cardiac function. We found that overexpression of EP4 improved shortening fraction (p = 0.0025), ejection fraction (p = 0.0003), and reduced left ventricular dimension at systole (p = 0.0013). Overexpression of EP4 also significantly reduced indices of cardiac hypertrophy and interstitial collagen fraction. Animals treated with AAV9-EP4 also had a significant decrease in TNFα mRNA expression and in the number of macrophages and T cells migrated post MI coupled with a reduction in the expression of iNOS. CONCLUSION: Overexpression of EP4 improves cardiac function post MI. This may be mediated through reductions in adverse cardiac remodeling or via inhibition of cytokine/chemokine production.