The Impact of Acute Exercise on Hemostasis and Angiogenesis Mediators in Patients With Continuous-Flow Left Ventricular Assist Devices: A Prospective Observational Pilot Study.

Impaired primary hemostasis and dysregulated angiogenesis, known as a two-hit hypothesis, are associated with gastrointestinal (GI) bleeding in patients with continuous-flow left ventricular assist devices (CF-LVADs). Exercise is known to influence hemostasis and angiogenesis in healthy individuals; however, little is known about the effect in patients with CF-LVADs. The objective of this prospective observational study was to determine whether acute exercise modulates two-hit hypothesis mediators associated with GI bleeding in patients with a CF-LVAD. Twenty-two patients with CF-LVADs performed acute exercise either on a cycle ergometer for approximately 10 minutes or on a treadmill for 30 minutes. Blood samples were taken pre- and post-exercise to analyze hemostatic and angiogenic biomarkers. Acute exercise resulted in an increased platelet count (p < 0.00001) and platelet function (induced by adenosine diphosphate, p = 0.0087; TRAP-6, p = 0.0005; ristocetin, p = 0.0009). Additionally, high-molecular-weight vWF multimers (p < 0.00001), vWF collagen-binding activity (p = 0.0012), factor VIII (p = 0.034), angiopoietin-1 (p = 0.0026), and vascular endothelial growth factor (p = 0.0041) all increased after acute exercise. This pilot work demonstrates that acute exercise modulated two-hit hypothesis mediators associated with GI bleeding in patients with CF-LVADs.

Modulation of red blood cell nitric oxide synthase phosphorylation in the quiescent and exercising human forearm.

In vitro investigations demonstrate that human erythrocytes synthesize nitric oxide via a functional isoform of endothelial nitric oxide synthase (NOS) (RBC-NOS). We tested the hypothesis that phosphorylation of RBC-NOS at serine residue 1177 (RBC-NOS1177) would be amplified in blood draining-active skeletal muscle. Furthermore, given hypoxemia modulates local blood flow and thus shear stress, and nitric oxide availability, we performed duplicate experiments under normoxia and hypoxia. Nine healthy volunteers performed rhythmic handgrip exercise at 60% of individualized maximal workload for 3.5 min while breathing room air (normoxia) and after being titrated to an arterial oxygen saturation ≈80% (hypoxemia). We measured brachial artery blood flow by high-resolution duplex ultrasound, while continuously monitoring vascular conductance and mean arterial pressure using finger photoplethysmography. Blood was sampled during the final 30 s of each stage from an indwelling cannula. Blood viscosity was measured to facilitate calculation of accurate shear stresses. Erythrocytes were assessed for levels of phosphorylated RBC-NOS1177 and cellular deformability from blood collected at rest and during exercise. Forearm exercise increased blood flow, vascular conductance, and vascular shear stress, which coincided with a 2.7 ± 0.6-fold increase in RBC-NOS1177 phosphorylation (P < 0.0001) and increased cellular deformability (P < 0.0001) under normoxia. When compared with normoxia, hypoxemia elevated vascular conductance and shear stress (P < 0.05) at rest, while cellular deformability (P < 0.01) and RBC-NOS1177 phosphorylation (P < 0.01) increased. Hypoxemic exercise elicited further increases in vascular conductance, shear stress, and cell deformability (P < 0.0001), although a subject-specific response in RBC-NOS1177 phosphorylation was observed. Our data yield novel insights into the manner that hemodynamic force and oxygen tension modulate RBC-NOS in vivo.

Accelerated Hemocompatibility Testing of Rotary Blood Pumps.

Ex vivo hemocompatibility testing is a vital element of preclinical assessment for blood-contacting medical devices. Current approaches are resource intensive; thus, we investigated the feasibility of accelerating hemocompatibility testing by standardizing the number of pump exposures in loops of various sizes. Three identical blood loops were constructed, each with a custom-molded reservoir able to facilitate large-volume expansion. Using the HVAD rotary blood pump operating at 5 L·min -1 and 100 mmHg, three test volumes (80, 160, and 320 ml) were circulated for 4000 pump exposures. Blood sampling was performed at individualized intervals every one-sixth of total duration for the assessment of hemolysis and von Willebrand Factor (vWF) degradation. While steady increases in hemolysis (~24 mg·dl -1 ) were identified in all tests at completion, loop volume was not a primary discriminator. The normalized index of hemolysis did not vary significantly between loops (4.2-4.9 mg·100 L -1 ). vWF degradation progressively occurred with duration of testing to a similar extent under all conditions. These data support an accelerated approach to preclinical assessment of ex vivo blood damage. Adopting this approach enables: enhanced efficiency for rapid prototyping; reduced ex vivo blood aging, and; greater utility of blood, which is presently limited if 450 ml loops are desired.

Single-night continuous positive airway pressure treatment improves blood fluid properties in individuals recently diagnosed with obstructive sleep apnoea.

Obstructive sleep apnoea (OSA) is a prevalent disorder that causes repetitive, temporary collapses of the upper airways during sleep, resulting in intermittent hypoxaemia and sleep fragmentation. Given those with OSA also exhibit decreased blood fluidity, this clinical population is at heightened risk for cardiovascular disease (CVD) development. Continuous positive airway pressure (CPAP) remains a primary therapy in OSA, which improves sleep quality and limits sleep fragmentation. While CPAP effectively ameliorates nocturnal hypoxic events and associated arousals, it remains unclear whether CVD risk factors are positively impacted. The aim of the present study was thus to assess the effects of an acute CPAP therapy on sleep quality and the physical properties of blood that determine blood fluidity. Sixteen participants with suspected OSA were recruited into the current study. Participants attended the sleep laboratory for two visits: an initial diagnostic visit that included confirmation of OSA severity and comprehensive assessments of blood parameters, followed by a subsequent visit where participants were administered an individualised, acute CPAP therapy session and had their blood assessments repeated. Holistic appraisal of blood rheological properties included assessment of blood and plasma viscosity, red blood cell (RBC) aggregation, deformability, and osmotic gradient ektacytometry. Acute CPAP treatment significantly improved sleep quality parameters, which were associated with decreased nocturnal arousals and improved blood oxygen saturation. Whole blood viscosity was significantly decreased following acute CPAP treatment, which might be explained by the improved RBC aggregation during this visit. Although an acute increase in plasma viscosity was observed, it appears that the alterations in RBC properties that mediate cell-cell aggregation, and thus blood viscosity, overcame the increased plasma viscosity. While deformability of RBC was unaltered, CPAP therapy had mild effects on the osmotic tolerance of RBC. Collectively, novel observations demonstrate that a single CPAP treatment session acutely improved sleep quality, which was accompanied by improved rheological properties.

Red Blood Cell Sublethal Damage: Hemocompatibility Is not the Absence of Hemolysis.

Blood is a complex fluid owing to its two-phase suspension of formed cellular elements within a protein-rich plasma. Vital to its role in distributing nutrients throughout the circulatory system, the mechanical properties of blood - and particularly red blood cells (RBC)-primarily determine bulk flow characteristics and microcirculatory flux. Various factors impair the physical properties of RBC, including cellular senescence, many diseases, and exposure to mechanical forces. Indeed, the latter is increasingly relevant following the advent of modern life support, such as mechanical circulatory support (MCS), which induce unique interactions between blood and artificial environments that leave blood cells with the signature of aging, albeit accelerated, and crucially underlie various serious complications, including death. Accumulating evidence indicates that these complications appear to be associated with mechanical shear forces present within MCS that are not extreme enough to overtly rupture cells, yet may still induce "sublethal" injury and "fatigue" to vital blood constituents. Impaired RBC physical properties following elevated shear exposure-a hallmark of sublethal injury to blood-are notable and may explain, at least in part, systemic complications and premature mortality associated with MCS. Design of optimal next-generation MCS devices thus requires consideration of biocompatibility and blood-device interactions to minimize potential blood complications and promote clinical success. Presented herein is a contemporary understanding of "blood damage," with emphasis on shear exposures that alter microrheological function but do not overtly destroy cells (ie, sublethal damage). Identification of key cellular factors perturbed by supraphysiological shear exposure are examined, offering potential pathways to enhance design of MCS and blood-contacting medical devices.

Mitigation effect of cell exclusion on blood damage in spiral groove bearings.

Cell exclusion in spiral groove bearing (SGB) excludes red blood cells from high shear regions in the bearing gaps and potentially reduce haemolysis in rotary blood pumps. However, this mechanobiological phenomenon has been observed in ultra-low blood haematocrit only, whether it can mitigate blood damage in a clinically-relevant blood haematocrit remains unknown. This study examined whether cell exclusion in a SGB alters haemolysis and/or high-molecular-weight von Willebrand factor (HMW vWF) multimer degradation. Citrated human blood was adjusted to 35 % haematocrit and exposed to a SGB (n = 6) and grooveless disc (n = 3, as a non-cell exclusion control) incorporated into a custom-built Couette test rig operating at 2000RPM for an hour; shearing gaps were 20, 30, and 40 µm. Haemolysis was assessed via spectrophotometry and HMW vWF multimer degradation was detected with gel electrophoresis and immunoblotting. Haemolysis caused by the SGB at gaps of 20, 30 and 40 µm were 10.6 ± 3.3, 9.6 ± 2.7 and 10.5 ± 3.9 mg/dL.hr compared to 23.3 ± 2.6, 12.8 ± 3.2, 9.8 ± 1.8 mg/dL.hr by grooveless disc. At the same shearing gap of 20 µm, there was a significant reduced in haemolysis (P = 0.0001) and better preserved in HMW vWF multimers (p < 0.05) when compared SGB to grooveless disc. The reduction in blood damage in the SGB compared to grooveless disc is indicative of cell exclusion occurred at the gap of 20 µm. This is the first experimental study to demonstrate that cell exclusion in a SGB mitigates the shear-induced blood damage in a clinically-relevant blood haematocrit of 35 %, which can be potentially utilised in future blood pump design.

Physical Properties of Blood and their Relationship to Clinical Conditions.

It has been long known that blood health heavily influences optimal physiological function. Abnormalities affecting the physical properties of blood have been implicated in the pathogenesis of various disorders, although the exact mechanistic links between hemorheology and clinical disease manifestations remain poorly understood. Often overlooked in current medical practice, perhaps due to the promises offered in the molecular and genetic era, the physical properties of blood which remain a valuable and definitive indicator of circulatory health and disease. Bridging this gap, the current manuscript provides an introduction to hemorheology. It reviews the properties that dictate bulk and microcirculatory flow by systematically dissecting the biomechanics that determine the non-Newtonian behavior of blood. Specifically, the impact of hematocrit, the mechanical properties and tendency of red blood cells to aggregate, and various plasma factors on blood viscosity will be examined. Subsequently, the manner in which the physical properties of blood influence hemodynamics in health and disease is discussed. Special attention is given to disorders such as sickle cell disease, emphasizing the clinical impact of severely abnormal blood rheology. This review expands into concepts that are highly topical; the relation between mechanical stress and intracellular homeostasis is examined through a contemporary cell-signaling lens. Indeed, accumulating evidence demonstrates that nitric oxide is not only transported by erythrocytes, but is locally produced by mechanically-sensitive enzymes, which appears to have intracellular and potentially extracellular effects. Finally, given the importance of shear forces in the developing field of mechanical circulatory support, we review the role of blood rheology in temporary and durable mechanical circulatory support devices, an increasingly utilized method of life support. This review thus provides a comprehensive overview for interested trainees, scientists, and clinicians.

Post-fatigue ability to activate muscle is compromised across a wide range of torques during acute hypoxic exposure.

The purpose of this study was to assess how severe acute hypoxia alters the neural mechanisms of muscle activation across a wide range of torque output in a fatigued muscle. Torque and electromyography responses to transcranial and motor nerve stimulation were collected from 10 participants (27 years ± 5 years, 1 female) following repeated performance of a sustained maximal voluntary contraction that reduced torque to 60% of the pre-fatigue peak torque. Contractions were performed after 2 h of hypoxic exposure and during a sham intervention. For hypoxia, peripheral blood oxygen saturation was titrated to 80% over a 15-min period and remained at 80% for 2 h. Maximal voluntary torque, electromyography root mean square, voluntary activation and corticospinal excitability (motor evoked potential area) and inhibition (silent period duration) were then assessed at 100%, 90%, 80%, 70%, 50% and 25% of the target force corresponding to the fatigued maximal voluntary contraction. No hypoxia-related effects were identified for voluntary activation elicited during motor nerve stimulation. However, during measurements elicited at the level of the motor cortex, voluntary activation was reduced at each torque output considered (P = .002, ηp 2  = .829). Hypoxia did not impact the correlative linear relationship between cortical voluntary activation and contraction intensity or the correlative curvilinear relationship between motor nerve voluntary activation and contraction intensity. No other hypoxia-related effects were identified for other neuromuscular variables. Acute severe hypoxia significantly impairs the ability of the motor cortex to voluntarily activate fatigued muscle across a wide range of torque output.

Piezo1 regulates shear-dependent nitric oxide production in human erythrocytes.

Mature circulating red blood cells (RBCs) are classically viewed as passive participants in circulatory function, given erythroblasts eject their organelles during maturation. Endogenous production of nitric oxide (NO) and its effects are of particular significance; however, the integration between RBC sensation of the local environment and subsequent activation of mechano-sensitive signaling networks that generate NO remain poorly understood. The present study investigated endogenous NO production via the RBC-specific nitric oxide synthase isoform (RBC-NOS), connecting membrane strain with intracellular enzymatic processes. Isolated RBCs were obtained from apparently healthy humans. Intracellular NO was compared at rest and following shear (cellular deformation) using semiquantitative fluorescent imaging. Concurrently, RBC-NOS phosphorylation at its serine1177 (Ser1177) residue was measured. The contribution of cellular deformation to shear-induced NO production in RBCs was determined by rigidifying RBCs with the thiol-oxidizing agent diamide; rigid RBCs exhibited significantly impaired (up to 80%) capacity to generate NO via RBC-NOS during shear. Standardizing membrane strain of rigid RBCs by applying increased shear did not normalize NO production, or RBC-NOS activation. Calcium imaging with fluo-4 revealed that diamide-treated RBCs exhibited a 42% impairment in Piezo1-mediated calcium movement when compared with untreated RBCs. Pharmacological inhibition of Piezo1 with GsMTx4 during shear inhibited RBC-NOS activation in untreated RBCs, whereas Piezo1 activation with Yoda1 in the absence of shear stimulated RBC-NOS activation. Collectively, a novel, mechanically activated signaling pathway in mature RBCs is described. Opening of Piezo1 and subsequent influx of calcium appear to be required for endogenous production of NO in response to mechanical shear, which is accompanied by phosphorylation of RBC-NOS at Ser1177.NEW & NOTEWORTHY The mechano-sensitive ion channel Piezo1 is expressed in enucleated red blood cells and provides a mechanism of shear-induced red cell nitric oxide production via nitric oxide synthase phosphorylation. Thiol oxidation of red cells decreases Piezo1-dependent calcium movement and thus impairs nitric oxide generation in response to mechanical force. The emerging descriptions of exclusively posttranslational signaling networks in circulating red cells as acute regulators of cell function support that these cells play an important role in cardiovascular physiology that extends beyond passive oxygen transport.

Bovine erythrocytes are poor surrogates for human when exposed to sublethal shear stress.

Animal blood products are routinely used as surrogates for human tissue in haemocompatibility testing of rotary blood pumps. Bovine blood is particularly attractive due to the animal's large blood volume; however, bovine red blood cells (RBC) differ substantially from those of human, both in biophysical properties and molecular composition. We aimed to determine whether differences also exist in the sensitivity of bovine RBC to a standardised shear stress protocol. Fresh blood from healthy human and bovine donors was exposed to discrete combinations of shear stress using a Couette shearing system, prior to assessment of cellular deformability and mechanical sensitivity. Each sample was exposed to 25 sublethal shear stress combinations (ranging 60-100 Pa × 5-300 s). While bovine RBC exhibited decreased maximal elongation in the absence of conditioning shear, overall deformability at lower shears was ~1.8-fold greater than human. When exposed to any conditioning shear stresses >80 Pa (or 60-70 Pa beyond 5 s), human RBC were significantly rigidified, with greater magnitudes and prolonged exposure compounding this effect. Significantly larger shears were required to rigidify bovine RBC; the most extreme shear condition (100 Pa × 300 s) resulted in approximately three-times more rigidification of human RBC than bovine (137% and 47% respectively). Bovine RBC have superior resilience to mechanical stress when compared with human. Using bovine blood in ex vivo evaluation of rotary blood pumps may thus misrepresent and overestimate device-blood success, and may also have flow-on effects for eventual users. Fresh human blood during early-phase ex vivo testing is thus recommended, given shear-inducing blood pumps are designed for humans - not cattle.

Protocol for inspecting blood cell dynamics with a custom ektacytometer-rheometer apparatus.

Investigating flowing red blood cell (RBC) morphology and orientation is important for elucidating physiology and disease; existing commercially available products are limited to observing cell populations or single cells. In this protocol, we create a custom apparatus that combines coaxial brightfield microscopy with laser diffractometry to inspect near-real-time deformability, morphology, and orientation of flowing RBCs. There are difficulties associated with building optical systems for biological inspection; however, this protocol provides a suitable framework for developing an "ektacytoscope" for studying blood cells. For complete details on the use and execution of this protocol, please refer to McNamee et al. (2020).

Enhanced Blood Plasma Extraction Utilising Viscoelastic Effects in a Serpentine Microchannel.

Plasma extraction from blood is essential for diagnosis of many diseases. The critical process of plasma extraction requires removal of blood cells from whole blood. Fluid viscoelasticity promotes cell migration towards the central axis of flow due to differences in normal stress and physical properties of cells. We investigated the effects of altering fluid viscoelasticity on blood plasma extraction in a serpentine microchannel. Poly (ethylene oxide) (PEO) was dissolved into blood to increase its viscoelasticity. The influences of PEO concentration, blood dilution, and flow rate on the performance of cell focusing were examined. We found that focusing performance can be significantly enhanced by adding PEO into blood. The optimal PEO concentration ranged from 100 to 200 ppm with respect to effective blood cell focusing. An optimal flow rate from 1 to 15 µL/min was determined, at least for our experimental setup. Given less than 1% haemolysis was detected at the outlets in all experimental combinations, the proposed microfluidic methodology appears suitable for applications sensitive to haemocompatibility.

Impact of small fractions of abnormal erythrocytes on blood rheology.

Red blood cell (RBC) populations are inherently heterogeneous, given mature RBC lack the transcriptional machinery to re-synthesize proteins affected during in vivo aging. Clearance of older, less functional cells thus aids in maintaining consistent hemorheological properties. Scenarios occur, however, where portions of mechanically impaired RBC are re-introduced into blood (e.g., damaged from circulatory support, blood transfusion) and may alter whole blood fluid behavior. Given such perturbations are associated with poor clinical outcomes, determining the tolerable level of abnormal RBC in blood is valuable. Thus, the current study aimed to define the critical threshold of blood fluid properties to re-infused physically-impaired RBC. Cell mechanics of RBC were impaired through membrane cross-linking (glutaraldehyde) or intracellular oxidation (phenazine methosulfate). Mechanically impaired RBC were progressively re-introduced into the native cell population. Negative alterations of cellular deformability and high shear blood viscosity were observed following additions of only 1-5% rigidified RBC. Low-shear blood viscosity was conversely decreased following addition of glutaraldehyde-treated cells; high-resolution microscopy of these mixed cell populations revealed decreased capacity to form reversible aggregates and decreased aggregate size. Mixed RBC populations, when exposed to supraphysiological shear, presented with compounded mechanical impairment. Collectively, key determinants of blood flow behavior are sensitive to mechanical perturbations in RBC, even when only 1-5% of the cell population is affected. Given this fraction is well-below the volume of rigidified RBC introduced during circulatory support or transfusion practice, it is plausible that some adverse events following surgery and/or transfusion may be related to impaired blood fluidity.

Discrete responses of erythrocytes, platelets, and von Willebrand factor to shear.

Despite decades of technological advancements in blood-contacting medical devices, complications related to shear flow-induced blood trauma are still frequently observed in clinic. Blood trauma includes haemolysis, platelet activation, and degradation of High Molecular Weight von Willebrand Factor (HMW vWF) multimers, all of which are dependent on the exposure time and magnitude of shear stress. Specifically, accumulating evidence supports that when blood is exposed to shear stresses above a certain threshold, blood trauma ensues; however, it remains unclear how various constituents of blood are affected by discrete shears experimentally. The aim of this study was to expose blood to discrete shear stresses and evaluate blood trauma indices that reflect red cell, platelet, and vWF structure. Citrated human whole blood (n = 6) was collected and its haematocrit was adjusted to 30 ± 2% by adding either phosphate buffered saline (PBS) or polyvinylpyrrolidone (PVP). Viscosity of whole blood was adjusted to 3.0, 12.5, 22.5 and 37.5 mPa·s to yield stresses of 3, 6, 9, 12, 50, 90 and 150 Pa in a custom-developed shearing system. Blood samples were exposed to shear for 0, 300, 600 and 900 s. Haemolysis was measured using spectrophotometry, platelet activation using flow cytometry, and HMW vWF multimer degradation was quantified with gel electrophoresis and immunoblotting. For tolerance to 300, 600 and 900 s of exposure time, the critical threshold of haemolysis was reached after blood was exposed to 90 Pa for 600 s (P < 0.05), platelet activation and HMW vWF multimer degradation were 50 Pa for 600 s and 12 Pa for 300 s respectively (P < 0.05). Our experimental results provide simultaneous comparison of blood trauma indices and thus also the relation between shear duration and magnitude required to induce damage to red cells, platelets, and vWF. Our results also demonstrate that near-physiological shear stress (<12 Pa) is needed in order to completely avoid any form of blood trauma. Therefore, there is an urgent need to design low shear-flow medical devices in order to avoid blood trauma in this blood-contacting medical device field.

Erythrocyte morphological symmetry analysis to detect sublethal trauma in shear flow.

The viscoelastic properties of red blood cells (RBC) facilitate flexible shape change in response to extrinsic forces. Their viscoelasticity is intrinsically linked to physical properties of the cytosol, cytoskeleton, and membrane-all of which are highly sensitive to supraphysiological shear exposure. Given the need to minimise blood trauma within artificial organs, we observed RBC in supraphysiological shear through direct visualisation to gain understanding of processes leading to blood damage. Using a custom-built counter-rotating shear generator fit to a microscope, healthy red blood cells (RBC) were directly visualised during exposure to different levels of shear (10-60 Pa). To investigate RBC morphology in shear flow, we developed an image analysis method to quantify (a)symmetry of deforming ellipsoidal cells-following RBC identification and centroid detection, cell radius was determined for each angle around the circumference of the cell, and the resultant bimodal distribution (and thus RBC) was symmetrically compared. While traditional indices of RBC deformability (elongation index) remained unaltered in all shear conditions, following ~100 s of exposure to 60 Pa, the frequency of asymmetrical ellipses and RBC fragments/extracellular vesicles significantly increased. These findings indicate RBC structure is sensitive to shear history, where asymmetrical morphology may indicate sublethal blood damage in real-time shear flow.

Severe acute hypoxia impairs recovery of voluntary muscle activation after sustained submaximal elbow flexion.

The purpose of this study was to determine how severe acute hypoxia alters neural mechanisms during, and following, a sustained fatiguing contraction. Fifteen participants (25 ± 3.2 years, six female) were exposed to a sham condition and a hypoxia condition where they performed a 10 min elbow flexor contraction at 20% of maximal torque. For hypoxia, peripheral blood oxygen saturation ( SpO2 ) was titrated to 80% over a 15 min period and maintained for 2 h. Maximal voluntary contraction torque, EMG root mean square, voluntary activation, rating of perceived muscle fatigue, and corticospinal excitability (motor-evoked potential) and inhibition (silent period duration) were then assessed before, during and for 6 min after the fatiguing contraction. No hypoxia-related effects were identified for neuromuscular variables during the fatigue task. However, for recovery, voluntary activation assessed by motor point stimulation of biceps brachii was lower for hypoxia than sham at 4 min (sham: 89% ± 7%; hypoxia: 80% ± 12%; P = 0.023) and 6 min (sham: 90% ± 7%; hypoxia: 78% ± 11%; P = 0.040). Similarly, voluntary activation (P = 0.01) and motor-evoked potential area (P = 0.002) in response to transcranial magnetic stimulation of the motor cortex were 10% and 11% lower during recovery for hypoxia compared to sham, respectively. Although an SpO2 of 80% did not affect neural activity during the fatiguing task, motor cortical output and corticospinal excitability were reduced during recovery in the hypoxic environment. This was probably due to hypoxia-related mechanisms involving supraspinal motor circuits. KEY POINTS: Acute hypoxia has been shown to impair voluntary activation of muscle and alter the excitability of the corticospinal motor pathway during exercise. However, little is known about how hypoxia alters the recovery of the motor system after performing fatiguing exercise. Here we assessed hypoxia-related responses of motor pathways both during active contractions and during recovery from active contractions, with transcranial magnetic stimulation and motor point stimulation of the biceps brachii. Fatiguing exercise caused reductions in voluntary activation, which was exacerbated during recovery from a 10 min sustained elbow flexion in a hypoxic environment. These results suggest that reductions in blood oxygen concentration impair the ability of motor pathways in the CNS to recover from fatiguing exercise, which is probably due to hypoxia-induced mechanisms that reduce output from the motor cortex.

Rate-Pressure Product Responses to Static Contractions Performed at Various Altitudes.

Simmonds, Michael J., Surendran Sabapathy, and Jean-Marc Hero. Rate-pressure product responses to static contractions performed at various altitudes. High Alt Med Biol. 22: 166-173, 2021. Background: Adventure tourism has led to an unprecedented number of individuals being exposed to altitude, including those with subclinical cardiometabolic disorders. The disproportionate hemodynamic challenge associated with small-muscle static activities is potentially dangerous at altitude as these may compound the risk for cardiac events. We thus examined the cardiovascular response to, and during recovery from, static exercise performed at altitude. Methods: Eighteen individuals completed this study at three altitudes (sea level; ∼1,500 m; ∼3,000 m) in central Nepal. At each altitude, individuals performed two handgrip contractions for 2 minutes at the same intensity (30% maximal voluntary contraction [MVC]), with two distinct recovery periods: during control recovery was completed quietly at rest, while during ischemic challenge recovery was conducted with a cuff occluding the upper limb. Results: Oxygen saturation decreased during ascent to 1,500 m (-2%) and 3,000 m (-8%), compared with sea level. Handgrip MVC was not affected by altitude, although heart rate at rest (∼70 beat/min), during static exercise (range ∼90-95 beat/min), and during recovery in both conditions (each ∼70 beat/min) was significantly increased by ∼15% at 3,000 m, but not 1,500 m. The magnitude of the muscle metaboreflex during recovery from static exercise was unaffected by altitude; however, the rate-pressure product was significantly elevated by ∼10% during and following static exercise at 3,000 m. Conclusions: A significant increase in the rate-pressure product during static exercise was observed at altitude, which persisted during recovery. Individuals at risk for cardiac events should use awareness of static contractions while at altitude, especially considering that stress induced by static exercise is additive to that of dynamic activities such as hiking.

Sub-Fractions of Red Blood Cells Respond Differently to Shear Exposure Following Superoxide Treatment.

Red blood cell (RBC) deformability is an essential component of microcirculatory function that appears to be enhanced by physiological shear stress, while being negatively affected by supraphysiological shears and/or free radical exposure. Given that blood contains RBCs with non-uniform physical properties, whether all cells equivalently tolerate mechanical and oxidative stresses remains poorly understood. We thus partitioned blood into old and young RBCs which were exposed to phenazine methosulfate (PMS) that generates intracellular superoxide and/or specific mechanical stress. Measured RBC deformability was lower in old compared to young RBCs. PMS increased total free radicals in both sub-populations, and RBC deformability decreased accordingly. Shear exposure did not affect reactive species in the sub-populations but reduced RBC nitric oxide synthase (NOS) activation and intriguingly increased RBC deformability in old RBCs. The co-application of PMS and shear exposure also improved cellular deformability in older cells previously exposed to reactive oxygen species (ROS), but not in younger cells. Outputs of NO generation appeared dependent on cell age; in general, stressors applied to younger RBCs tended to induce S-nitrosylation of RBC cytoskeletal proteins, while older RBCs tended to reflect markers of nitrosative stress. We thus present novel findings pertaining to the interplay of mechanical stress and redox metabolism in circulating RBC sub-populations.

Shear Stress and RBC-NOS Serine1177 Phosphorylation in Humans: A Dose Response.

Red blood cells (RBC) express a nitric oxide synthase isoform (RBC-NOS) that appears dependent on shear stress for Serine1177 phosphorylation. Whether this protein is equally activated by varied shears in the physiological range is less described. Here, we explored RBC-NOS Serine1177 phosphorylation in response to shear stress levels reflective of in vivo conditions. Whole blood samples were exposed to specific magnitudes of shear stress (0.5, 1.5, 4.5, 13.5 Pa) for discrete exposure times (1, 10, 30 min). Thereafter, RBC-NOS Serine1177 phosphorylation was measured utilising immunofluorescence labelling. Shear stress exposure at 0.5, 1.5, and 13.5 Pa significantly increased RBC-NOS Serine1177 phosphorylation following 1 min (p < 0.0001); exposure to 4.5 Pa had no effect after 1 min. RBC-NOS Serine1177 phosphorylation was significantly increased following 10 min at each magnitude of shear stress (0.5, 1.5, 13.5 Pa, p < 0.0001; 4.5 Pa, p = 0.0042). Shear stress exposure for 30 min significantly increased RBC-NOS Serine1177 phosphorylation at 0.5 Pa and 13.5 Pa (p < 0.0001). We found that RBC-NOS phosphorylation via shear stress is non-linear and differs for a given magnitude and duration of exposure. This study provides a new understanding of the discrete relation between RBC-NOS and shear stress.