EUK-8, a Superoxide Dismutase and Catalase Mimetic, Reduces Cardiac Oxidative Stress and Ameliorates Pressure Overload-Induced Heart Failure in the Harlequin Mouse Mutant
Vanessa P.M. van Empel, MD*,
Anne T. Bertrand, PhD*,
Ralph J. van Oort, MSc*,
Roel van der Nagel, BS*,
Markus Engelen, MD ,
Harold V. van Rijen, PhD,
Pieter A. Doevendans, MD, PhD ,
Harry J. Crijns, MD, PhD ,
Susan L. Ackerman, PhD||,
Wim Sluiter, PhD¶ and
Leon J. De Windt, PhD*,*
* Hubrecht Laboratory and Interuniversity Cardiology Institute Netherlands, Royal Netherlands Academy of Sciences, Utrecht, the Netherlands
Department of Medical Physiology, University Medical Center Utrecht, Utrecht, the Netherlands
Heart Lung Center Utrecht, University Medical Center Utrecht, Utrecht, the Netherlands
Department of Cardiology, University Hospital Maastricht, Maastricht, the Netherlands
|| Howard Hughes Medical Institute, Jackson Laboratory, Bar Harbor, Maine
¶ Departments of Biochemistry and Genetics, Erasmus Medical Center, Rotterdam, the Netherlands
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Figure 1 (A) Dihydroethidium (DHE) was used to visualize and quantify the production of superoxide in situ. Wild-type (WT) and harlequin (Hq) hearts demonstrated a significantly higher level of superoxide production after aortic banding; this increase was attenuated by administration of antioxidant EUK-8. (B) Left ventricular myocardial sections were subjected to immunostaining with 8-OHdG, a marker of oxidative DNA damage, which was increased in nontreated mice after aortic banding; however, EUK-8–treated mice had similar levels of 8-OHdG staining as sham-operated mice.
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Figure 2 Kaplan-Meier survival curve demonstrates increased post-transverse aortic constriction (TAC) mortality in harlequin (Hq) mutant mice compared with wild-type (WT), which was attenuated by EUK-8 treatment.
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Figure 3 (A) Representive M-mode echocardiography images of wild-type (WT) and harlequin (Hq) mice, either treated with vehicle or with EUK-8, following sham operation or 7 or 14 days after transverse aortic constriction (TAC). (B) Fractional shortening (FS), a measure of systolic contractility, was similar in sham-operated mutant and WT mice but was progressively and significantly decreased in Hq mutant mice compared with WT mice after 4 weeks of TAC. (C) The heart weight (HW)/tibial length (TL) ratios increased in untreated WT and Hq mice after 4 weeks of TAC compared with corresponding sham-operated groups, although the percentage increase was more pronounced in Hq mutant mice. The WT and Hq mice treated with EUK-8 did not demonstrate a significant increase in HW/TL ratio after TAC compared with corresponding sham groups.
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Figure 4 (A) Wild-type (WT) and harlequin (Hq) hearts, subjected to 28 days of mechanical load, displayed increased myocyte apoptosis as scored by cleaved caspase-3–positive myocytes. The percentage increase in Hq mutants was more pronounced and was attenuated by treatment with EUK-8. (B) The WT and Hq hearts demonstrated increased cleaved PARP-positive myocytes after transverse aortic constriction (TAC), with the percentage increase in Hq mutants being more pronounced than in WT. Treatment with EUK-8 significantly reduced the amount of PARP-positive myocytes in Hq-TAC hearts.
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Figure 5 (A) Representive hematoxylin and eosin (H&E) sections at 400x magnification of wild-type (WT) and harlequin (Hq) hearts, treated with vehicle or EUK-8, either after sham treatment or 28 days of transverse aortic constriction (TAC). The Hq-TAC mice demonstrated a marked increase in myocyte fiber diameter compared with other experimental groups; this increase was attenuated by treatment with EUK-8. (B) Quantification of cross-sectional area of myofibers from indicated groups shows an accentuated myocyte hypertrophy response in Hq-TAC mice. (C, D) Sirius red staining demonstrates significant fibrosis in Hq mice after TAC, which is diminished by EUK-8 administration.
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