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

Abstract

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.

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

“…The results of the limited safety study, where healthy rats were treated with CCrP at a dose of 0.8 g/kg/day for 14 consecutive days (CCrP control), showed no toxicity in rat hearts as evidenced by the absence of cardiac inflammation, apoptosis, biomarkers, and remodeling, and that these rats continued to exhibit normal cardiac function and physical activity similar to the saline control rats. The lack of cardiac toxicity further confirms our recently reported studies that the administration of CCrP did not alter liver and renal function, suggesting that CCrP is a safe drug [ 12 ].…”

“…Previously, we have reported that CCrP treatment inhibited a cardiac molecular mechanism involved in the pathogenesis of HF via the autophagy-related Nourin-dependent miR-137 (a marker of myocardial ischemic damage which promotes cardiac remodeling in HF) and Nourin-dependent miR-106b (a marker of inflammation which promotes cardiac inflammation) [ 12 ]. Specifically, the Nourin-associated miR-137 and miR-106b were significantly upregulated in the ISO/saline rats, but not in the ISO/CCrP rats that did not develop HF [ 12 ], further supporting the anti-inflammatory property of CCrP.…”

“…Rats were injected with isoproterenol hydrochloride (Sigma Aldrich; Merck KGaA, Darmstadt, Germany, Cat. # 15627) subcutaneously (s.c.) for two consecutive days at specific doses of 85 and 170 mg/kg/day (the LD50 for s.c. isoproterenol in rats = 600 mg/kg), respectively, and then left for an additional two weeks [ 10 , 11 , 12 , 13 , 14 ]. The ISO/saline rats were treated with 1 mL saline via intraperitoneal (IP) injection both 24 h and 1 h before the first ISO administration, and then daily for two weeks.…”

“…Prior to use, solutions of CCrP were freshly prepared in saline. According to our previous studies, CCrP at a dose of 0.8 gm/kg/day is the most effective dose to prevent myocardial ischemic injury in intact canine and rat models of acute myocardial ischemia, global cardiac arrest, cardiopulmonary bypass, and heart transplantation [ 6 , 10 , 11 , 12 ].…”

“…The purpose of this study was to evaluate the cardioprotective benefits of CCrP in the standard ISO rat model of ischemia-induced HF [ 10 , 11 , 12 , 13 , 14 ]. We tested the hypothesis that CCrP treatment will prevent ischemic injury and the development of HF when administered prophylactically and will salvage poorly functioning hearts when administered therapeutically.…”