Bradley J. Behnke

Mitochondrial dysfunction and sarcopenia of aging: from signaling pathways to clinical trials.

Sarcopenia, the age-related loss of muscle mass and function, imposes a dramatic burden on individuals and society. The development of preventive and therapeutic strategies against sarcopenia is therefore perceived as an urgent need by health professionals and has instigated intensive research on the pathophysiology of this syndrome. The pathogenesis of sarcopenia is multifaceted and encompasses lifestyle habits, systemic factors (e.g., chronic inflammation and hormonal alterations), local environment perturbations (e.g., vascular dysfunction), and intramuscular specific processes. In this scenario, derangements in skeletal myocyte mitochondrial function are recognized as major factors contributing to the age-dependent muscle degeneration. In this review, we summarize prominent findings and controversial issues on the contribution of specific mitochondrial processes - including oxidative stress, quality control mechanisms and apoptotic signaling - on the development of sarcopenia. Extramuscular alterations accompanying the aging process with a potential impact on myocyte mitochondrial function are also discussed. We conclude with presenting methodological and safety considerations for the design of clinical trials targeting mitochondrial dysfunction to treat sarcopenia. Special emphasis is placed on the importance of monitoring the effects of an intervention on muscle mitochondrial function and identifying the optimal target population for the trial. This article is part of a Directed Issue entitled: Molecular basis of muscle wasting.

Ageing diminishes endothelium-dependent vasodilatation and tetrahydrobiopterin content in rat skeletal muscle arterioles.

Ageing reduces endothelium‐dependent vasodilatation through an endothelial nitric oxide synthase (NOS) signalling pathway. The purpose of this study was to determine whether arginase activity diminishes endothelium‐dependent vasodilatation in skeletal muscle arterioles from old rats, and whether NOS substrate (l‐arginine) and cofactor (tetrahydrobiopterin; BH4) concentrations are reduced. First‐order arterioles were isolated from the soleus muscle of young (6 months old) and old (24 months old) male Fischer 344 rats. In vitro changes in luminal diameter in response to stepwise increases in flow were determined in the presence of the NOS inhibitor NG‐nitro‐l‐arginine methyl ester (l‐NAME, 10−5 mol l−1), the arginase inhibitor Nω‐hydroxy‐nor‐l‐arginine (NOHA, 5 × 10−4 mol l−1), exogenous l‐arginine (3 × 10−3 mol l−1) or the precursor for BH4 synthesis sepiapterin (1 μmol l−1). Arteriolar l‐arginine and BH4 content were determined via HPLC. Ageing decreased flow‐mediated vasodilatation by 52%, and this difference was abolished with NOS inhibition. Neither inhibition of arginase activity nor addition of exogenous l‐arginine had any effect on flow‐mediated vasodilatation; arteriolar l‐arginine content was also not different between age groups. BH4 content was lower in arterioles from old rats (94 ± 8 fmol (mg tissue)−1) relative to controls (234 ± 21 fmol (mg tissue)−1), and sepiapterin elevated flow‐mediated vasodilatation in arterioles from old rats. These results demonstrate that the impairment of endothelium‐dependent vasodilatation induced by old age is due to an altered nitric oxide signalling mechanism in skeletal muscle arterioles, but is not the result of increased arginase activity and limited l‐arginine substrate. Rather, the age‐related deficit in flow‐mediated vasodilatation appears to be the result, in part, of limited BH4 bioavailability.

Aging blunts the dynamics of vasodilation in isolated skeletal muscle resistance vessels

Aging is associated with an altered ability to match oxygen delivery (QO2) to consumption ((.)VO2) in skeletal muscle and differences in the temporal profile of vasodilation may provide a mechanistic basis for the QO2-to-(.)VO2 mismatching during the rest-to-exercise transition. Therefore, we tested the hypothesis that the speed of vasodilation will be blunted in skeletal muscle first-order arterioles from old vs. young rats. Arterioles from the soleus and the red portion of the gastrocnemius (Gast(Red)) muscles were isolated from young (Y, 6 mo; n = 9) and old (O, 24 mo; n = 9) Fischer 344 rats and studied in vitro. Vessels were exposed to acetylcholine (ACh; 10(-6) M), sodium nitroprusside (SNP; 10(-4) M), and increased intraluminal flow, and the subsequent vasodilation was recorded at 30 frames/s. The data were fit to a monoexponential model and the dynamics of vasodilation [i.e., time delay, time constant (tau), and rate of change (delta/tau)] were calculated. With old age, the rate of vasodilation was significantly blunted in resistance vessels from the soleus to ACh (Y, 27.9 +/- 3.6; O, 8.8 +/- 2.6 microm/s) and flow (Y, 12.8 +/- 2.1; O, 3.1 +/- 0.9 microm/s). In the Gast(Red) the old age-associated impairment of endothelium-dependent vasodilator dynamics was even greater than that of the soleus. With SNP neither the magnitude nor time constant of vasodilation was affected by age in either muscle. The results indicate that aging impairs the dynamics of vasodilation in resistance vessels from the soleus and Gast(Red) muscles mediated, in part, through the endothelium. Thus the old age-associated slower rate and magnitude of vasodilation could inhibit the delivery of O2 during the critical transition from rest to exercise in moderate to highly oxidative skeletal muscle.

Decreased NO signaling leads to enhanced vasoconstrictor responsiveness in skeletal muscle arterioles of the ZDF rat prior to overt diabetes and hypertension

Approximately 40% of patients with type 2 diabetes present with concurrent hypertension at the time of diabetes diagnosis. Increases in peripheral vascular resistance and correspondingly enhanced vasoconstrictor capacity could have profound implications for the development of hypertension and the progression of insulin resistance to overt diabetes. The purpose of this study was to determine whether skeletal muscle arteriolar vasoconstrictor dysfunction precedes or occurs concurrently with the onset of diabetes and hypertension. Male Zucker diabetic fatty (ZDF) rats were studied at 7, 13, and 20 wk of age to represent prediabetic and short-term and long-term diabetic states, respectively. Conscious mean arterial pressure (MAP), fasted plasma insulin and glucose, vasoconstrictor responses, and passive mechanical properties of isolated skeletal muscle arterioles were measured in prediabetic, diabetic, and age-matched control rats. Elevated MAP was manifest in short-term diabetes (control 117 +/- 1, diabetic 135 +/- 3 mmHg) and persisted with long-term diabetes (control 113 +/- 2, diabetic 135 +/- 3 mmHg). This higher MAP was preceded by augmented arteriolar vasoconstrictor responses to norepinephrine and endothelin-1 and followed by diminished beta-adrenergic vasodilation and enhanced myogenic constriction in long-term diabetes. Furthermore, we demonstrate that diminished nitric oxide (NO) signaling underlies the increases in vasoconstrictor responsiveness in arterioles from prediabetic and diabetic rats. Arteriolar stiffness was not different between control and prediabetic or diabetic rats at any time point studied. Collectively, these results indicate that increases in vasoconstrictor responsiveness resulting from diminished NO signaling in skeletal muscle arterioles precede the development of diabetes and hypertension in ZDF rats.

Spaceflight-induced alterations in cerebral artery vasoconstrictor, mechanical, and structural properties: implications for elevated cerebral perfusion and intracranial pressure

Evidence indicates that cerebral blood flow is both increased and diminished in astronauts on return to Earth. Data from ground‐based animal models simulating the effects of microgravity have shown that decrements in cerebral perfusion are associated with enhanced vasoconstriction and structural remodeling of cerebral arteries. Based on these results, the purpose of this study was to test the hypothesis that 13 d of spaceflight [Space Transportation System (STS)‐135 shuttle mission] enhances myogenic vasoconstriction, increases medial wall thickness, and elicits no change in the mechanical properties of mouse cerebral arteries. Basilar and posterior communicating arteries (PCAs) were isolated from 9‐wk‐old female C57BL/6 mice for in vitro vascular and mechanical testing. Contrary to that hypothesized, myogenic vasoconstrictor responses were lower and vascular distensibility greater in arteries from spaceflight group (SF) mice (n=7) relative to ground‐based control group (GC) mice (n=12). Basilar artery maximal diameter was greater in SF mice (SF: 236±9 μm and GC: 215±5 μm) with no difference in medial wall thickness (SF: 12.4±1.6 μm; GC: 12.2±1.2 μm). Stiffness of the PCA, as characterized via nanoindentation, was lower in SF mice (SF: 3.4±0.3 N/m; GC: 5.4±0.8 N/m). Collectively, spaceflight‐induced reductions in myogenic vasoconstriction and stiffness and increases in maximal diameter of cerebral arteries signify that elevations in brain blood flow may occur during spaceflight. Such changes in cerebral vascular control of perfusion could contribute to increases in intracranial pressure and an associated impairment of visual acuity in astronauts during spaceflight.—Taylor, C. R., Hanna, M., Behnke, B. J., Stabley, J. N., McCullough, D. J., Davis III, R. T., Ghosh, P., Papadopoulos, A., Muller‐Delp, J. M., Delp, M. D. Spaceflight‐induced alterations in cerebral artery vasoconstrictor, mechanical, and structural properties: implications for elevated cerebral perfusion and intracranial pressure. FASEB J. 27, 2282–2292 (2013). www.fasebj.org

Influence of ageing and physical activity on vascular morphology in rat skeletal muscle

Key structural and functional properties of the skeletal muscle vasculature that underlie diminished vascular conductance with ageing remain obscure. The purpose of this investigation was to test the hypotheses that (1) reduced levels of spontaneous physical activity in old rats are associated with skeletal muscle vascular remodelling (e.g. arterial rarefaction), and (2) consequent to a vascular remodelling, calculated shear stress is maintained in feed arteries of aged muscle at levels commensurate with that in young. Activity during daily light and dark cycles (12–12 h) was measured at 30‐s intervals for 2 weeks in young (6 months; n= 9) and old (24 months; n= 9) Fisher 344 rats via telemetry. Subsequently, the gastrocnemius complex and soleus muscles were excised and all feed arteries were counted, isolated, cannulated and maximally dilated for measurement of luminal diameter. Resting blood flow was also measured to estimate vessel wall shear‐stress in the feed arteries perforating the soleus and gastrocnemius muscles. Overall, young rats were ∼1.6 times more active during dark periods and ∼4 times more active during light periods than old rats. In addition, young rats had approximately one additional feed artery perforating both the soleus (young, 3.3 ± 0.2; old, 2.6 ± 0.2 vessels; P < 0.05) and gastrocnemius (young, 8.8 ± 0.1; old, 7.5 ± 0.2 vessels, P < 0.05) muscles compared with old rats. However, average vessel wall shear stress at rest was similar between young and old rats (soleus: Y, 65 ± 5; O, 64 ± 5 dynes cm−2; gastrocnemius: Y, 329 ± 22; O, 327 ± 27 dynes cm−2) resulting from a larger vessel diameter in arteries from old rats. In conclusion, lower activity levels of old rats likely contribute to resistance artery rarefaction and, consequently, this provides a plausible mechanism for the altered blood flow patterns observed during exercise in aged skeletal muscle.

Modulation of Blood Flow, Hypoxia, and Vascular Function in Orthotopic Prostate Tumors During Exercise

BACKGROUND Previous studies have hypothesized that tumor blood flow may be elevated or reduced during exercise, which could impact the tumor microenvironment. However, to date technical limitations have precluded the measurement of tumor blood flow during exercise. Using an orthotopic preclinical model of prostate cancer, we tested the hypotheses that during exercise tumors would experience 1) diminished vascular resistance, 2) augmented blood flow, 3) increased numbers of perfused vessels, and 4) decreased tissue hypoxia and, furthermore, that the increased perfusion would be associated with diminished vasoconstriction in prostate tumor arterioles. METHODS Dunning R-3327 MatLyLu tumor cells were injected into the ventral prostate of male Copenhagen rats aged 4 to 6 months randomly assigned to tumor-bearing (n = 42) or vehicle control (n = 14) groups. Prostate tumor blood flow, vascular resistance, patent vessel number, and hypoxia were measured in vivo in conscious rats at rest and during treadmill exercise, and vasoconstrictor responsiveness of resistance arterioles was investigated in vitro. RESULTS During exercise there was a statistically significant increase in tumor blood flow (approximately 200%) and number of patent vessels (rest mean ± standard deviation [SD] = 12.7±1.3; exercise mean ± SD = 14.3±0.6 vessels/field; Student t test two-sided P = .02) and decreased hypoxia compared with measurements made at rest. In tumor arterioles, the maximal constriction elicited by norepinephrine was blunted by approximately 95% vs control prostate vessels. CONCLUSIONS During exercise there is enhanced tumor perfusion and diminished tumor hypoxia due, in part, to a diminished vasoconstriction. The clinical relevance of these findings are that exercise may enhance the delivery of tumor-targeting drugs as well as attenuate the hypoxic microenvironment within a tumor and lead to a less aggressive phenotype.

Exercise training and muscle microvascular oxygenation: functional role of nitric oxide

Exercise training induces multiple adaptations within skeletal muscle that may improve local O(2) delivery-utilization matching (i.e., Po(2)mv). We tested the hypothesis that increased nitric oxide (NO) function is intrinsic to improved muscle Po(2)mv kinetics from rest to contractions after exercise training. Healthy young Sprague-Dawley rats were assigned to sedentary (n = 18) or progressive treadmill exercise training (n = 10; 5 days/wk, 6-8 wk, final workload of 60 min/day at 35 m/min, -14% grade) groups. Po(2)mv was measured via phosphorescence quenching in the spinotrapezius muscle at rest and during 1-Hz twitch contractions under control (Krebs-Henseleit solution), sodium nitroprusside (SNP, NO donor; 300 μM), and N(G)-nitro-L-arginine methyl ester (l-NAME, nonspecific NO synthase blockade; 1.5 mM) superfusion conditions. Exercise-trained rats had greater peak oxygen uptake (Vo(2 peak)) than their sedentary counterparts (81 ± 1 vs. 72 ± 2 ml · kg(-1) · min(-1), respectively; P < 0.05). Exercise-trained rats had significantly slower Po(2)mv fall throughout contractions (τ(1); time constant for the first component) during control (sedentary: 8.1 ± 0.6; trained: 15.2 ± 2.8 s). Compared with control, SNP slowed τ(1) to a greater extent in sedentary rats (sedentary: 38.7 ± 5.6; trained: 26.8 ± 4.1 s; P > 0.05) whereas l-NAME abolished the differences in τ(1) between sedentary and trained rats (sedentary: 12.0 ± 1.7; trained: 11.2 ± 1.4 s; P < 0.05). Our results indicate that endurance exercise training leads to greater muscle microvascular oxygenation across the metabolic transient following the onset of contractions (i.e., slower Po(2)mv kinetics) partly via increased NO-mediated function, which likely constitutes an important mechanism for training-induced metabolic adaptations.

Effects of aging and exercise training on skeletal muscle blood flow and resistance artery morphology

With old age, blood flow to the high-oxidative red skeletal muscle is reduced and blood flow to the low-oxidative white muscle is elevated during exercise. Changes in the number of feed arteries perforating the muscle are thought to contribute to this altered hyperemic response during exercise. We tested the hypothesis that exercise training would ameliorate age-related differences in blood flow during exercise and feed artery structure in skeletal muscle. Young (6-7 mo old, n = 36) and old (24 mo old, n = 25) male Fischer 344 rats were divided into young sedentary (Sed), old Sed, young exercise-trained (ET), and old ET groups, where training consisted of 10-12 wk of treadmill exercise. In Sed and ET rats, blood flow to the red and white portions of the gastrocnemius muscle (Gast(Red) and Gast(White)) and the number and luminal cross-sectional area (CSA) of all feed arteries perforating the muscle were measured at rest and during exercise. In the old ET group, blood flow was greater to Gast(Red) (264 ± 13 and 195 ± 9 ml · min(-1) · 100 g(-1) in old ET and old Sed, respectively) and lower to Gast(White) (78 ± 5 and 120 ± 6 ml · min(-1) · 100 g(-1) in old ET and old Sed, respectively) than in the old Sed group. There was no difference in the number of feed arteries between the old ET and old Sed group, although the CSA of feed arteries from old ET rats was larger. In young ET rats, there was an increase in the number of feed arteries perforating the muscle. Exercise training mitigated old age-associated differences in blood flow during exercise within gastrocnemius muscle. However, training-induced adaptations in resistance artery morphology differed between young (increase in feed artery number) and old (increase in artery CSA) animals. The altered blood flow pattern induced by exercise training with old age would improve the local matching of O(2) delivery to consumption within the skeletal muscle.

Effects of spaceflight and ground recovery on mesenteric artery and vein constrictor properties in mice

Following exposure to microgravity, there is a reduced ability of astronauts to augment peripheral vascular resistance, often resulting in orthostatic hypotension. The purpose of this study was to test the hypothesis that mesenteric arteries and veins will exhibit diminished vasoconstrictor responses after spaceflight. Mesenteric arteries and veins from female mice flown on the Space Transportation System (STS)‐131 (n=11), STS‐133 (n=6), and STS‐135 (n=3) shuttle missions and respective ground‐based control mice (n=30) were isolated for in vitro experimentation. Vasoconstrictor responses were evoked in arteries via norepinephrine (NE), potassium chloride (KCl), and caffeine, and in veins through NE across a range of intraluminal pressures (2–12 cmH2O). Vasoconstriction to NE was also determined in mesenteric arteries at 1, 5, and 7 d postlanding. In arteries, maximal constriction to NE, KCl, and caffeine were reduced immediately following spaceflight and 1 d postflight. Spaceflight also reduced arterial ryanodine receptor‐3 mRNA levels. In mesenteric veins, there was diminished constriction to NE after flight. The results indicate that the impaired vasoconstriction following spaceflight occurs through the ryanodine receptor‐mediated intracellular Ca2+ release mechanism. Such vascular changes in astronauts could compromise the maintenance of arterial pressure during orthostatic stress.—Behnke, B. J., Stabley, J. N., McCullough, D. J., Davis, R. T., III, Dominguez, J. M., II, Muller‐Delp, J. M., Delp, M. D. Effects of spaceflight and ground recovery on mesenteric artery and vein constrictor properties in mice. FASEB J. 27, 399–409 (2013). www.fasebj.org