Rhythmic handgrip exercise elevates arterial shear‐rate and increases indices of endothelial cell autophagy and nitric oxide synthase activation in humans

Abstract

Autophagy is a process whereby damaged cellular cargo is delivered to the lysosome for degradation and eventual recycling or removal. Knowledge concerning the importance of autophagy to endothelial cell (EC) metabolism is evolving. Previously we reported that 180‐min × 20 dyne · cm 2 shear‐stress initiates autophagy, activates EC nitric oxide (NO) synthase (eNOS) at serine 1177 (p‐eNOSS1177), and generates NO in immortalized bovine and human arterial endothelial cells (ECs) (Bharath et al., Arterioscler Thromb Vasc Biol, 2017). Importantly, when EC autophagy was repressed using genetic or pharmacological procedures, the ability of shear‐stress to generate NO was prevented. Here we report data from the first step to eventually determine whether these findings obtained in vitro can be translated to primary human ECs. After familiarization with laboratory procedures (visit 1), five male subjects (23±3 yr) completed a maximal voluntary contraction (MVC) test and a flow‐mediated vasodilation (FMD) assessment during a second visit (visit 2). Within 1‐week, a 20 g catheter was placed into the radial artery (RA) during visit 3. Next, ECs were collected via j‐wire biopsy from the RA catheter, transferred to dissociation buffer, recovered by washing and centrifugation, placed onto previously prepared glass slides, and frozen at −80°C. Thirty‐min after collecting pre‐exercise ECs, subjects performed rhythmic handgrip exercise for 60‐min at a contraction intensity that elevated (p<0.05) arterial shear‐rate 2.7 ± 0.3‐fold from baseline. Shear‐rate [8Vmean/brachial artery (BA) diameter] was obtained by direct and continuous assessment of BA diameter, BA blood flow velocity (Vmean), and BA blood flow [(Vmeanπ (vessel diameter/2)2 × 60)] using Doppler ultrasound. Heart rate (HR, beats/min; 3‐lead ECG), stroke volume (SV, ml/beat), cardiac output (CO, L/min), and mean arterial pressure (MAP, mmHg; automated plethysmography), respectively, were not different when pre‐exercise values (56±1, 101±6, 5.6±0.3, and 91±3) were compared to 60‐min values (60±2, 104±6, 6.2±0.4, and 94±2). At 60‐min of handgrip exercise, ECs were collected from the RA, prepared, and stored as described. Using quantitative immunofluorescence, primary ECs (75 ECs per endpoint) were identified by positive co‐staining for VE‐Cadherin and DAPI via confocal microscopy. Immortalized human arterial endothelial cells (P4–6) were processed and stained in parallel using identical procedures to serve as fluorescence intensity controls. Relative to pre‐exercise values, ECs obtained at 60‐min displayed increased expression of beclin‐1 (p<0.01), microtubule associated protein light chain 3 (LC3 II; p=0.05), autophagy‐related gene 3 (Atg3; p=0.06), lysosomal associated membrane protein 2a (LAMP2a; p<0.004), and p‐eNOSS1177 (p<0.02), and decreased expression (i.e., enhanced degradation) of the adapter protein p62/SQSTM1 (p<0.02). Taken together, these findings provide novel evidence in primary human ECs that elevated shear‐rate evoked by functional hyperemia initiates autophagy and activates p‐eNOSS1177. Ongoing studies in our laboratory are determining whether this response is dysregulated in older humans and in patients with type 2 diabetes, conditions associated with arterial dysfunction.Support or Funding InformationAmerican Heart Association (AHA)17POST33670663 to SKP; AHA 16GRNT31050004 and National Institutes of Health NIH:RO3AGO52848 to JDS; Veteran Affairs Rehabilitation Research and Development Grant IK2RX001215 CDA2 and American Heart Association Grant 14SDG1850039 to JDT.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.