Integrated Bioinformatics Analysis Confirms the Diagnostic Value of Nourin-Dependent miR-137 and miR-106b in Unstable Angina Patients.

The challenge of rapidly diagnosing myocardial ischemia in unstable angina (UA) patients presenting to the Emergency Department (ED) is due to a lack of sensitive blood biomarkers. This has prompted an investigation into microRNAs (miRNAs) related to cardiac-derived Nourin for potential diagnostic application. The Nourin protein is rapidly expressed in patients with acute coronary syndrome (ACS) (UA and acute myocardial infarction (AMI)). MicroRNAs regulate gene expression through mRNA binding and, thus, may represent potential biomarkers. We initially identified miR-137 and miR-106b and conducted a clinical validation, which demonstrated that they were highly upregulated in ACS patients, but not in healthy subjects and non-ACS controls. Using integrated comprehensive bioinformatics analysis, the present study confirms that the Nourin protein targets miR-137 and miR-106b, which are linked to myocardial ischemia and inflammation associated with ACS. Molecular docking demonstrated robust interactions between the Nourin protein and miR137/hsa-miR-106b, involving hydrogen bonds and hydrophobic interactions, with -10 kcal/mol binding energy. I-TASSER generated Nourin analogs, with the top 10 chosen for structural insights. Antigenic regions and MHCII epitopes within the Nourin SPGADGNGGEAMPGG sequence showed strong binding to HLA-DR/DQ alleles. The Cytoscape network revealed interactions of -miR137/hsa-miR--106b and Phosphatase and tensin homolog (PTEN) in myocardial ischemia. RNA Composer predicted the secondary structure of miR-106b. Schrödinger software identified key Nourin-RNA interactions critical for complex stability. The study identifies miR-137 and miR-106b as potential ACS diagnostic and therapeutic targets. This research underscores the potential of miRNAs targeting Nourin for precision ACS intervention. The analysis leverages RNA Composer, Schrödinger, and I-TASSER tools to explore interactions and structural insights. Robust Nourin-miRNA interactions are established, bolstering the case for miRNA-based interventions in ischemic injury. In conclusion, the study contributes to UA and AMI diagnosis strategies through bioinformatics-guided exploration of Nourin-targeting miRNAs. Supported by comprehensive molecular analysis, the hypoxia-induced miR-137 for cell apoptosis (a marker of cell damage) and the inflammation-induced miR-106b (a marker of inflammation) confirmed their potential clinical use as diagnostic biomarkers. This research reinforces the growing role of miR-137/hsa-miR-106b in the early diagnosis of myocardial ischemia in unstable angina patients.

Cyclocreatine Phosphate: A Novel Bioenergetic/Anti-Inflammatory Drug That Resuscitates Poorly Functioning Hearts and Protects against Development of Heart Failure.

Irreversible myocardial injury causes the exhaustion of cellular adenosine triphosphate (ATP) contributing to heart failure (HF). Cyclocreatine phosphate (CCrP) was shown to preserve myocardial ATP during ischemia and maintain cardiac function in various animal models of ischemia/reperfusion. We tested whether CCrP administered prophylactically/therapeutically prevents HF secondary to ischemic injury in an isoproterenol (ISO) rat model. Thirty-nine rats were allocated into five groups: control/saline, control/CCrP, ISO/saline (85 and 170 mg/kg/day s.c. for 2 consecutive days), and ISO/CCrP (0.8 g/kg/day i.p.) either administrated 24 h or 1 h before ISO administration (prophylactic regimen) or 1 h after the last ISO injection (therapeutic regimen) and then daily for 2 weeks. CCrP protected against ISO-induced CK-MB elevation and ECG/ST changes when administered prophylactically or therapeutically. CCrP administered prophylactically decreased heart weight, hs-TnI, TNF-α, TGF-ß, and caspase-3, as well as increased EF%, eNOS, and connexin-43, and maintained physical activity. Histology indicated a marked decrease in cardiac remodeling (fibrin and collagen deposition) in the ISO/CCrP rats. Similarly, therapeutically administered CCrP showed normal EF% and physical activity, as well as normal serum levels of hs-TnI and BNP. In conclusion, the bioenergetic/anti-inflammatory CCrP is a promising safe drug against myocardial ischemic sequelae, including HF, promoting its clinical application to salvage poorly functioning hearts.

Impedimetric Sensors for Cyclocreatine Phosphate Determination in Plasma Based on Electropolymerized Poly(o-phenylenediamine) Molecularly Imprinted Polymers.

Cyclocreatine and its water-soluble derivative, cyclocreatine phosphate (CCrP), are potent cardioprotective drugs. Based on recent animal studies, CCrP, FDA-awarded Orphan Drug Designation, has a promising role in increasing the success rate of patients undergoing heart transplantation surgery by preserving donor hearts during transportation and improving the recovery of transplanted hearts in recipient patients. In addition, CCrP is under investigation as a promising treatment for creatine transporter deficiency, an X-linked inborn error resulting in a poor quality of life for both the patients and the caregiver. A newly designed molecularly imprinted polymer (MIP) material was fabricated by the anodic electropolymerization of o-phenylenediamine on screen-printed carbon electrodes and was successfully applied as an impedimetric sensor for CCrP determination to dramatically reduce the analysis time during both the clinical trial phases and drug development process. To enhance the overall performance of the proposed sensor, studies were performed to optimize the electropolymerization conditions, incubation time, and pH of the background electrolyte. Scanning electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry were used to characterize the behavior of the developed ultrathin MIP membrane. The CCrP-imprinted polymer has a high recognition affinity for the template molecule because of the formation of 3D complementary cavities within the polymer. The developed MIP impedimetric sensor had good linearity, repeatability, reproducibility, and stability within the linear concentration range of 1 × 10-9 to 1 × 10-7 mol/L, with a low limit of detection down to 2.47 × 10-10 mol/L. To verify the applicability of the proposed sensor, it was used to quantify CCrP in spiked plasma samples.

Nourin-Associated miRNAs: Novel Inflammatory Monitoring Markers for Cyclocreatine Phosphate Therapy in Heart Failure.

BACKGROUND: Cyclocreatine phosphate (CCrP) is a potent bioenergetic cardioprotective compound known to preserve high levels of cellular adenosine triphosphate during ischemia. Using the standard Isoproterenol (ISO) rat model of heart failure (HF), we recently demonstrated that the administration of CCrP prevented the development of HF by markedly reducing cardiac remodeling (fibrosis and collagen deposition) and maintaining normal ejection fraction and heart weight, as well as physical activity. The novel inflammatory mediator, Nourin is a 3-KDa formyl peptide rapidly released by ischemic myocardium and is associated with post-ischemic cardiac inflammation. We reported that the Nourin-associated miR-137 (marker of cell damage) and miR-106b-5p (marker of inflammation) are significantly upregulated in unstable angina patients and patients with acute myocardial infarction, but not in healthy subjects. OBJECTIVES: To test the hypothesis that Nourin-associated miR-137 and miR-106b-5p are upregulated in ISO-induced "HF rats" and that the administration of CCrP prevents myocardial injury (MI) and reduces Nourin gene expression in "non-HF rats". METHODS: 25 male Wistar rats (180-220 g) were used: ISO/saline (n = 6), ISO/CCrP (0.8 g/kg/day) (n = 5), control/saline (n = 5), and control/CCrP (0.8 g/kg/day) (n = 4). In a limited study, CCrP at a lower dose of 0.4 g/kg/day (n = 3) and a higher dose of 1.2 g/kg/day (n = 2) were also tested. The Rats were injected SC with ISO for two consecutive days at doses of 85 and 170 mg/kg/day, respectively, then allowed to survive for an additional two weeks. CCrP and saline were injected IP (1 mL) 24 h and 1 h before first ISO administration, then daily for two weeks. Serum CK-MB (U/L) was measured 24 h after the second ISO injection to confirm myocardial injury. After 14 days, gene expression levels of miR-137 and miR-106b-5p were measured in serum samples using quantitative real-time PCR (qPCR). RESULTS: While high levels of CK-MB were detected after 24 h in the ISO/saline rats indicative of MI, the ISO/CCrP rats showed normal CK-MB levels, supporting prevention of MI by CCrP. After 14 days, gene expression profiles showed significant upregulation of miR-137 and miR-106b-5p by 8.6-fold and 8.7-fold increase, respectively, in the ISO/saline rats, "HF rats," compared to the control/saline group. On the contrary, CCrP treatment at 0.8 g/kg/day markedly reduced gene expression of miR-137 by 75% and of miR-106b-5p by 44% in the ISO/CCrP rats, "non-HF rats," compared to the ISO/Saline rats, "HF rats." Additionally, healthy rats treated with CCrP for 14 days showed no toxicity in heart, liver, and renal function. CONCLUSIONS: Results suggest a role of Nourin-associated miR-137 and miR-106b-5p in the pathogenesis of HF and that CCrP treatment prevented ischemic injury in "non-HF rats" and significantly reduced Nourin gene expression levels in a dose-response manner. The Nourin gene-based mRNAs may, therefore, potentially be used as monitoring markers of drug therapy response in HF, and CCrP-as a novel preventive therapy of HF due to ischemia.

Nourin-Dependent miR-137 and miR-106b: Novel Biomarkers for Early Diagnosis of Myocardial Ischemia in Coronary Artery Disease Patients.

BACKGROUND: Although cardiovascular imaging techniques are widely used to diagnose myocardial ischemia in patients with suspected stable coronary artery disease (CAD), they have limitations related to lack of specificity, sensitivity and "late" diagnosis. Additionally, the absence of a simple laboratory test that can detect myocardial ischemia in CAD patients, has led to many patients being first diagnosed at the time of the development of myocardial infarction. Nourin is an early blood-based biomarker rapidly released within five minutes by "reversible" ischemic myocardium before progressing to necrosis. Recently, we demonstrated that the Nourin-dependent miR-137 (marker of cell damage) and miR-106b-5p (marker of inflammation) can diagnose myocardial ischemia in patients with unstable angina (UA) and also stratify severity of ischemia, with higher expression in acute ST-segment elevation myocardial infarction (STEMI) patients compared to UA patients. Minimal baseline-gene expression levels of Nourin miRNAs were detected in healthy subjects. OBJECTIVES: To determine: (1) whether Nourin miRNAs are elevated in chest pain patients with myocardial ischemia suspected of CAD, who also underwent dobutamine stress echocardiography (DSE) or ECG/Treadmill stress test, and (2) whether the elevated levels of serum Nourin miRNAs correlate with results of ECHO/ECG stress test in diagnosing CAD patients. METHODS: Serum gene expression levels of miR-137, miR-106b-5p and their corresponding molecular pathway network were measured blindly in 70 enrolled subjects using quantitative real time PCR (qPCR). Blood samples were collected from: (1) patients with chest pain suspected of myocardial ischemia (n = 38) both immediately "pre-stress test" and "post-stress test" 30 min. after test termination; (2) patients with acute STEMI (n = 16) functioned as our positive control; and (3) healthy volunteers (n = 16) who, also, exercised on ECG/Treadmill stress test for Nourin baseline-gene expression levels. RESULTS: (1) strong correlation was observed between Nourin miRNAs serum expression levels and results obtained from ECHO/ECG stress test in diagnosing myocardial ischemia in CAD patients; (2) positive "post-stress test" patients with CAD diagnosis showed upregulation of miR-137 by 572-fold and miR-106b-5p by 122-fold, when compared to negative "post-stress test" patients (p < 0.001); (3) similarly, positive "pre-stress test" CAD patients showed upregulation of miR-137 by 1198-fold and miR-106b-5p by 114-fold, when compared to negative "pre-stress test" patients (p < 0.001); and (4) healthy subjects had minimal baseline-gene expressions of Nourin miRNAs. CONCLUSIONS: Nourin-dependent miR-137 and miR-106b-5p are promising novel blood-based biomarkers for early diagnosis of myocardial ischemia in chest pain patients suspected of CAD in outpatient clinics. Early identification of CAD patients, while patients are in the stable state before progressing to infarction, is key to providing crucial diagnostic steps and therapy to limit adverse cardiac events, improve patients' health outcome and save lives.

Nourin-Dependent miR-137 and miR-106b: Novel Early Inflammatory Diagnostic Biomarkers for Unstable Angina Patients.

BACKGROUND: Currently, no blood biomarkers exist that can diagnose unstable angina (UA) patients. Nourin is an early inflammatory mediator rapidly released within 5 min by reversible ischemic myocardium, and if ischemia persists, it is also released by necrosis. Nourin is elevated in acute coronary syndrome (ACS) patients but not in symptomatic noncardiac and healthy subjects. Recently, circulating microRNAs (miRNAs) have been established as markers of disease, including cardiac injury and inflammation. OBJECTIVES: To profile and validate the potential diagnostic value of Nourin-dependent miR-137 (marker of cell damage) and miR-106b-5p (marker of inflammation) as early biomarkers in suspected UA patients and to investigate the association of their target and regulating genes. METHODS: Using Nourin amino acid sequence, an integrated bioinformatics analysis was conducted. Analysis indicated that Nourin is a direct target for miR-137 and miR-106b-5p in myocardial ischemic injury. Two linked molecular networks of lncRNA/miRNAs/mRNAs were also retrieved, including CTB89H12.4/miR-137/FTHL-17 and CTB89H12.4/miR-106b-5p/ANAPC11. Gene expression profiling was assessed in serum samples collected at presentation to an emergency department (ED) from: (1) UA patients (n = 30) (confirmed by invasive coronary angiography with stenosis greater than 50% and troponin level below the clinical decision limit); (2) patients with acute ST elevation myocardial infarction (STEMI) (n = 16) (confirmed by persistent ST-segment changes and elevated troponin level); and 3) healthy subjects (n = 16). RESULTS: Gene expression profiles showed that miR-137 and miR-106b-5p were significantly upregulated by 1382-fold and 192-fold in UA compared to healthy, and by 2.5-fold and 4.6-fold in STEMI compared to UA, respectively. Healthy subjects showed minimal expression profile. Receiver operator characteristics (ROC) analysis revealed that the two miRNAs were sensitive and specific biomarkers for assessment of UA and STEMI patients. Additionally, Spearman's correlation analysis revealed a significant association of miRNAs with the associated mRNA targets and the regulating lncRNA. CONCLUSIONS: Nourin-dependent gene expression of miR-137 and miR-106b-5p are novel blood-based biomarkers that can diagnose UA and STEMI patients at presentation and stratify severity of myocardial ischemia, with higher expression in STEMI compared to UA. Early diagnosis of suspected UA patients using the novel Nourin biomarkers is key for initiating guideline-based therapy that improves patients' health outcomes.

Cyclocreatine protects against ischemic injury and enhances cardiac recovery during early reperfusion.

Introduction: A critical mechanism of how hypoxia/ischemia causes irreversible myocardial injury is through the exhaustion of adenosine triphosphate (ATP). Cyclocreatine (CCr) and its water-soluble salt Cyclocreatine-Phosphate (CCrP) are potent bioenergetic agents that preserve high levels of ATP during ischemia. Areas covered: CCr and CCrP treatment prior to the onset of ischemia, preserved high levels of ATP in ischemic myocardium, reduced myocardial cell injury, exerted anti-inflammatory and anti-apoptotic activities, and restored contractile function during reperfusion in animal models of acute myocardial infarction (AMI), global cardiac arrest, cardiopulmonary bypass, and heart transplantation. Medline and Embase (1970 - Feb 2019), the WIPO databank (up to Feb 2019); no language restriction. Expert opinion: This review provides the basis for a number of clinical applications of CCrP and CCr to minimize ischemic injury and necrosis. One strategy is to administer CCrP to AMI patients in the pre-hospital phase, as well as during, or after Percutaneous Coronary Intervention (PCI) procedure to potentially achieve protection of the myocardium, reduce infarcted-size, and, thus, limit the progression to heart failure. Another clinical applications are in predictable myocardial ischemia where pretreatment with CCrP would likely improve outcome and quality of life of patients who will undergo cardiopulmonary bypass for coronary revascularization and end-stage heart failure patients scheduled for heart transplantation.

CYCLOSPORIN H: A NOVEL ANTI-INFLAMMATORY THERAPY FOR INFLUENZA FLU PATIENTS.

Relatively little is known about the inflammatory mediators and mechanisms that drive the progression of influenza flu infection to cytokine storm, lung dysfunction, organ failure, and ultimately death. Vaccines and antiviral medications cannot control the excessive host inflammatory response associated with severe influenza flu infection. Studies by Elgebaly et al demonstrated the rapid release of a potent inflammatory mediator, recently named Nourin, by local mammalian tissues in response to injury and infection. Nourin is a formyl peptide that acts through the formyl peptide receptor (FPR) on phagocytic leukocytes. As an initial signal in the innate immunity, Nourin stimulates leukocyte chemotaxis, induces acute and chronic inflammation, and stimulates the release of a number of the cytokine storm mediators from monocytes, neutrophils and endothelial cells. Furthermore, Nourin detected in plasma samples from patients with severe influenza infection was much higher compared to moderate influenza. The Nourin antagonist, Cyclosporin H, is a potent anti-inflammatory compound, which acts as a specific competitive antagonist of formyl peptides on the formyl peptide receptor (FPR) on phagocytic: leukocytes. Cyclosporin H completely blocked neutrophil chemotaxis induced by: (a) the standard formyl peptide, f-MLF, (b) the Staphylococcus aureus bacteria-derived formyl peptide Phenol-soluble modulins, such as PSM3a, plus(c) the host-derived Nourin released by: (1) cultured epithelial cells infected with the PR8 HINI influenza virus for 6.to 24 hours, (2),Nourin detected in the serum of mouse model of HINI Swine flu influenza infection for 6 hours , along with (3) Nourin detected in plasma samples collected from severe and moderate influenza pa- tients. Furthermore, in-vivo treatment by Cyclosporin H in the mouse model of HINI Swine flu influenza infection for 5 days markedly reduced lung inflammation and endothelial cell damage. Thus, two clinical applications for Nourin and its antagonist Cyclosporin H are proposed: Diagnostic Application: The blood Nourin test can be used as a key inflammatory biomarker for "early" detection and monitoring of influenza flu patients proceeding to hyperactive inflammation and, thus, permitting early crucial anti-inflammatory therapy. Therapeutic Application: Cyclosporin H will specifically block Nourin as an important initial stimulant of cytokine mediators, and thus can control the development and progression of cytokine storm plus organ inflammation, which usually initiates 3 to 8 days post influenza. Since Cyclosporin H does not target the virus,, it will not develop drug resistance,and will reduce the host uncontrpolled inflammatory response, induced by both new strains of flu viruses and existing viruses with mutations.