Immuno-Spin Trapping-Based Detection of Oxidative Modifications in Cardiomyocytes and Coronary Endothelium in the Progression of Heart Failure in Tgαq*44 Mice.

Recent studies suggest both beneficial and detrimental role of increased reactive oxygen species and oxidative stress in heart failure (HF). However, it is not clear at which stage oxidative stress and oxidative modifications occur in the endothelium in relation to cardiomyocytes in non-ischemic HF. Furthermore, most methods used to date to study oxidative stress are either non-specific or require tissue homogenization. In this study, we used immuno-spin trapping (IST) technique with fluorescent microscopy-based detection of DMPO nitrone adducts to localize and quantify oxidative modifications of the hearts from Tgαq*44 mice; a murine model of HF driven by cardiomyocyte-specific overexpression of Gαq* protein. Tgαq*44 mice and age-matched FVB controls at early, transition, and late stages of HF progression were injected with DMPO in vivo and analyzed ex vivo for DMPO nitrone adducts signals. Progressive oxidative modifications in cardiomyocytes, as evidenced by the elevation of DMPO nitrone adducts, were detected in hearts from 10- to 16-month-old, but not in 8-month-old Tgαq*44 mice, as compared with age-matched FVB mice. The DMPO nitrone adducts were detected in left and right ventricle, septum, and papillary muscle. Surprisingly, significant elevation of DMPO nitrone adducts was also present in the coronary endothelium both in large arteries and in microcirculation simultaneously, as in cardiomyocytes, starting from 10-month-old Tgαq*44 mice. On the other hand, superoxide production in heart homogenates was elevated already in 6-month-old Tgαq*44 mice and progressively increased to high levels in 14-month-old Tgαq*44 mice, while the enzymatic activity of catalase, glutathione reductase, and glutathione peroxidase was all elevated as early as in 4-month-old Tgαq*44 mice and stayed at a similar level in 14-month-old Tgαq*44. In summary, this study demonstrates that IST represents a unique method that allows to quantify oxidative modifications in cardiomyocytes and coronary endothelium in the heart. In Tgαq*44 mice with slowly developing HF, driven by cardiomyocyte-specific overexpression of Gαq* protein, an increase in superoxide production, despite compensatory activation of antioxidative mechanisms, results in the development of oxidative modifications not only in cardiomyocytes but also in coronary endothelium, at the transition phase of HF, before the end-stage disease.

Vascular Nitric Oxide-Superoxide Balance and Thrombus Formation after Acute Exercise.

INTRODUCTION: An acute bout of strenuous exercise in humans results in transient impairment of nitric oxide (NO)-dependent function, but it remains unknown whether this phenomenon is associated with increased risk of thrombotic events after exercise. This study aimed to evaluate effects of a single bout of exhaustive running in mice on the balance of vascular NO/reactive oxygen species production, and on thrombogenicity. METHODS: At different time points (0, 2, and 4 h) after exercise and in sedentary C57BL/6 mice, the production of NO and superoxide (O2) in aorta was measured by electron paramagnetic resonance spin trapping and by dihydroethidium/high-performance liquid chromatography-based method, respectively, whereas collagen-induced thrombus formation was analyzed in a microchip-based flow-chamber system (total thrombus-formation analysis system). We also measured pre- and postexercise plasma concentration of nitrite/nitrate and 6-keto-PGF1α. RESULTS: An acute bout of exhaustive running in mice resulted in decreased production of NO and increased production of O2 in aorta, with maximum changes 2 h after completion of exercise when compared with sedentary mice. However, platelet thrombus formation was not changed by exercise as evidenced by unaltered time to start of thrombus formation, capillary occlusion time, and total thrombogenicity (area under the flow pressure curve) as measured in a flow-chamber system. Strenuous exercise increased the plasma concentration of nitrite but did not affect nitrate and 6-keto-PGF1α concentrations. CONCLUSION: An acute bout of strenuous exercise in mice reduced NO and in parallel increased O2 production in aorta. This response was most pronounced 2 h after exercise. Surprisingly, the reduced NO and increased O2 production in mice after exercise did not result in increased platelet-dependent thrombogenicity. These results show that transient reduction in NO bioavailability does not modify thromboresistance in healthy mice after exercise.