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    Cardiovascular Engineering

    One Month Exercise Training Enhances Endothelial and Red Blood Cell Nitric Oxide Production

    (G-274) One Month Exercise Training Enhances Endothelial and Red Blood Cell Nitric Oxide Production

    Saturday, October 14, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row G - Poster # 274

      Presenting Author(s)

    • PB

      Paige Boyland

      Undergraduate Reseacher
      Fischell Department of Bioengineering, University of Maryland
      Gaithersburg, Maryland, United States

      • Co-Author(s)

    • GS

      Gurneet S. Sangha, PhD (he/him/his)

      Postdoctoral Fellow
      University of Maryland, Fischell Department of Bioengineering, Maryland, United States

      • Primary Investigator(s)

    • AC

      Alisa M. Clyne

      Professor
      University of Maryland, Fischell Department of Bioengineering, United States

    Introduction::

    Nitric oxide (NO) is released by both endothelial and red blood cells (RBCs) to promote healthy vascular function [1]. NO is a potent vasodilator, regulating blood vessel tone and promoting healthy blood flow [2]. Decreased vascular NO bioavailability promotes endothelial dysfunction and thus initiates complex mechanisms that lead to cardiovascular disease. We know that exercise increases endothelial NO bioavailability [2], but it is unclear how exercise affects RBC NO production. Understanding how chronic exercise regulates endothelial and RBC NO could lead to novel therapies for cardiovascular disease. Here, I hypothesized that treadmill training in mice would increase NO production in both RBC and endothelial cells.


     



    Materials and Methods::
    C57BI/6J mice (n=4) were treadmill trained for one month. Mice exercised five days per week for one hour at a speed of 20 cm/s (Harvard Apparatus). Untrained mice were used as controls (n=4). We performed ultrasound imaging of mouse carotid arteries at baseline and after one-month training. Pulse wave Doppler was used to measure mean and peak blood flow velocity, motion modes (m-mode) to measure arterial compliance, and 3D images were segmented to quantify arterial volume and diameter. At the conclusion of the study, mice were euthanized and the carotid arteries and RBCs were isolated. Carotid arteries were cannulated, pressurized to 50 mmHg, and then constricted with 10-5 M phenylephrine. Endothelial-dependent vasodilation was measured by treating carotid arteries with increasing doses of acetylcholine (10-9 M - 10-5 M). We then used the mouse RBCs to compare eNOS phosphorylation (p-eNOS) in exercise-trained and untrained mice by Western blot. RBCs were lysed in RIPA containing protease phosphatase inhibitors and EDTA. We loaded 200ug of RBC lysate into BOLT 4-12% Bis-Tris protein gels. RBC lysate from each mouse was run in duplicate. Proteins were then transferred to polyvinylidene difluoride membranes and probed using primary antibodies for p-eNOS and eNOS (1:500). GAPDH (1:1000) was used as a housekeeper.


    Results, Conclusions, and Discussions::

    Our pressure myograph data showed that carotid arteries from exercise-trained mice had greater endothelial-dependent vasodilation than untrained animals (Fig 1a). Maximum vasodilation was significantly greater in carotid arteries from exercise-trained mice (105.2 ± 11.1%) than untrained mice (86.2 ± 17.4%) treated with 10-5 M acetylcholine (Fig 1b; p< 0.05). However, we did not observe significant changes in carotid artery morphology, blood flow velocity, or compliance between exercise-trained and untrained mice by ultrasound imaging. The Western blots showed that changes in RBC  p-eNOS were variable. In one Western blot, we found that RBCs from exercise-trained mice had significantly greater p-eNOS than untrained mice (Fig 1c; p< 0.05). In our second Western blot, conducted using different animals from our study, we did not observe differences between exercise-trained and untrained RBC p-eNOS (Fig 1d). Our data suggest that RBC p-eNOS increases in some animals but not all animals after one month of exercise training. More exercise training may be needed to cause robust adaptations in RBC p-eNOS. For example, it takes three months to observe robust changes in RBC HbA1C, a standard biomarker to diagnose diabetes [3]. Future work will investigate whether RBCs show p-eNOS adaptations after several months of exercise training.

    This study showed that one month of exercise training increased mouse carotid artery endothelial-dependent vasodilation compared to untrained mice, but these adaptations did not translate to improved carotid artery morphology, blood flow velocity, or compliance. RBCs had variable responses in p-eNOS, suggesting that exercise may enhance RBC NO production in some animals. In the future, we will focus on extended exercise training with larger sample size. 



    Acknowledgements (Optional): : Funded was provided to G.S.S through the AHA Postdoctoral Fellowship (916512) and A.M.C. by NIH (R01HL140239-0).

    References (Optional): : [1] Leo, F., Circulation, 114(2021):870-889, [2] Green, D.J., 8(2018):a029819, [3] Sherwani, S.I., 11(2016):95–104