[Frontiers In Bioscience, Landmark, 23, 298-309, January 1, 2018]

L-NAME improves doxycycline and ML-7 cardioprotection from oxidative stress

Iwona Bil-Lula1, Anna Krzywonos –Zawadzka1, Jolanta Sawicka2, Dariusz Bialy3, Magdalena Wawrzynska4, Mieczyslaw Wozniak1,2, Grzegorz Sawicki1,2

1Department of Clinical Chemistry, Wroclaw Medical University, Wroclaw, Poland, 2Department of Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Canada, 3Department and Clinic of Cardiology, Wroclaw Medical University, Wroclaw, Poland, 4Department of Emergency Medicine, Wroclaw Medical University, Wroclaw, Poland


1. Abstract
2. Introduction
3. Materials and methods
3.1. Isolated heart perfusion according to Langendorff
3.2. Protocol of global ischemia/reperfusion
3.3. Concentration of proteins
3.4. Determination of LDH activity
3.5. Immunoblott analysis of MLC1
3.6. Troponin I content in rat hearts and coronary effluents
3.7. Statistical analysis
4. Results
4.1. Determination of the protective threshold for cardiac mechanical function
4.2. Protective effect of co-administration of subthreshold concentration of Doxy, ML-7 and L- NAME against cardiac I/R injury
5. Discussion
6. Acknowledgements
7. References


Matrix metalloproteinase-2 (MMP-2) mediated degradation of myosin light chain 1 (MLC1) and troponin I (TnI) contributes to myocardial ischemia/reperfusion (I/R) injury. Modifications of MLC1 triggered by oxidative stress are mediated by myosin light chain kinase (MLCK), nitric oxide synthase (NOS), and MMP-2. Previous studies have shown that inhibiting both MLCK and MMP-2 protects against I/R injury. Here, we hypothesized that the addition of NOS inhibitor (L-NAME) at subprotective concentration to the mixture of subprotective concentrations of ML-7 and doxycycline (Doxy), will increase a synergistic cardioprotection of Doxy and ML-7 during I/R. Isolated rat hearts were subjected to global ischemia without or with administration of the mixture of inhibitors. Markers of I/R injury were measured in hearts and coronary effluents. Addition of L-NAME to the mixture of Doxy and ML-7 led to full recovery of heart contractility in comparison to combination of Doxy and ML-7. Improved heart contractility was associated with reduced degradation of TnI and MLC1. The combined administration of NOS, MMP-2 and MLCK inhibitors provides a novel strategy to protect heart from I/R injury.


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Abbreviations:CF: coronary flow; Doxy: doxycycline; HR: heart rate; H/R: hypoxia/reoxygenation; eNOS: endothelial nitric oxide synthase; iNOS: inducible NOS; I/R: ischemia/reperfusion; L-NAME: non-selective inhibitor of iNOS/eNOS; LVDP: left ventricular developed pressure; MLC- myosin light chain; MLC1: myosin light chain type 1; MLC2: myosin light chain type 2; MLCK: myosin light chain kinase; ML-7: inhibitor of MLCK; MMPs: matrix metalloproteinases; MMP-2: matrix metalloproteinase 2; MYL3: ventricular isoform of MLC1; NO: nitric oxide; NOS: nitric oxide synthase; ONOO-: peroxynitrate; PVDF: polyvinylidene fluoride; ROS: reactive oxygen species; RPP: the difference of systolic and diastolic ventricular pressures; TnI: troponin I;

Key Words: Ischemia, Reperfusion, Isolated rat heart, Doxycycline, ML-7, L-NAME

Send correspondence to: Grzegorz Sawicki, Department of Pharmacology, College of Medicine, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5 Canada., Telephone: 306-9666997, Fax: 306-9664298, E-mail: greg.sawicki@usask.ca