New FindingsWhat is the central question of this study?Are biomarkers of endothelial function, oxidative stress and inflammation altered by non-freezing cold injury (NFCI)?What is the main finding and its importance?Baseline plasma [interleukin-10] and [syndecan-1] were elevated in individuals with NFCI and cold-exposed control participants. Increased [endothelin-1] following thermal challenges might explain, in part, the increased pain/discomfort experienced with NFCI. Mild to moderate chronic NFCI does not appear to be associated with either oxidative stress or a pro-inflammatory state. Baseline [interleukin-10] and [syndecan-1] and post-heating [endothelin-1] are the most promising candidates for diagnosis of NFCI. Plasma biomarkers of inflammation, oxidative stress, endothelial function and damage were examined in 16 individuals with chronic NFCI (NFCI) and matched control participants with (COLD, n = 17) or without (CON, n = 14) previous cold exposure. Venous blood samples were collected at baseline to assess plasma biomarkers of endothelial function (nitrate, nitrite and endothelin-1), inflammation [interleukin-6 (IL-6), interleukin-10 (IL-10), tumour necrosis factor alpha and E-selectin], oxidative stress [protein carbonyl, 4-hydroxy-2-nonenal (4-HNE), superoxide dismutase and nitrotyrosine) and endothelial damage [von Willebrand factor, syndecan-1 and tissue type plasminogen activator (TTPA)]. Immediately after whole-body heating and separately, foot cooling, blood samples were taken for measurement of plasma [nitrate], [nitrite], [endothelin-1], [IL-6], [4-HNE] and [TTPA]. At baseline, [IL-10] and [syndecan-1] were increased in NFCI (P P = 0.015, respectively) and COLD (P = 0.033 and P = 0.030, respectively) compared with CON participants. The [4-HNE] was elevated in CON compared with both NFCI (P = 0.002) and COLD (P < 0.001). [Endothelin-1] was elevated in NFCI compared with COLD (P < 0.001) post-heating. The [4-HNE] was lower in NFCI compared with CON post-heating (P = 0.032) and lower than both COLD (P = 0.02) and CON (P = 0.015) post-cooling. No between-group differences were seen for the other biomarkers. Mild to moderate chronic NFCI does not appear to be associated with a pro-inflammatory state or oxidative stress. Baseline [IL-10] and [syndecan-1] and post-heating [endothelin-1] are the most promising candidates for diagnosing NFCI, but it is likely that a combination of tests will be required.

New FindingsWhat is the central question of this study?Does non-freezing cold injury (NFCI) alter normal peripheral vascular function?What is the main finding and its importance?Individuals with NFCI were more cold sensitive (rewarmed more slowly and felt more discomfort) than controls. Vascular tests indicated that extremity endothelial function was preserved with NFCI and that sympathetic vasoconstrictor response might be reduced. The pathophysiology underpinning the cold sensitivity associated with NFCI thus remains to be identified. The impact of non-freezing cold injury (NFCI) on peripheral vascular function was investigated. Individuals with NFCI (NFCI group) and closely matched controls with either similar (COLD group) or limited (CON group) previous cold exposure were compared (n = 16). Peripheral cutaneous vascular responses to deep inspiration (DI), occlusion (PORH), local cutaneous heating (LH) and iontophoresis of acetylcholine and sodium nitroprusside were investigated. The responses to a cold sensitivity test (CST) involving immersion of a foot in 15 degrees C water for 2 min followed by spontaneous rewarming, and a foot cooling protocol (footplate cooled from 34 degrees C to 15 degrees C), were also examined. The vasoconstrictor response to DI was lower in NFCI compared to CON (toe: 73 (28)% vs. 91 (17)%; P = 0.003). The responses to PORH, LH and iontophoresis were not reduced compared to either COLD or CON. During the CST, toe skin temperature rewarmed more slowly in NFCI than COLD or CON (10 min: 27.4 (2.3)degrees C vs. 30.7 (3.7)degrees C and 31.7 (3.9)degrees C, P < 0.05, respectively); however, no differences were observed during the footplate cooling. NFCI were more cold-intolerant (P < 0.0001) and reported colder and more uncomfortable feet during the CST and footplate cooling than COLD and CON (P < 0.05). NFCI showed a decreased sensitivity to sympathetic vasoconstrictor activation than CON and greater cold sensitivity (CST) compared to COLD and CON. None of the other vascular function tests indicated endothelial dysfunction. However, NFCI perceived their extremities to be colder and more uncomfortable/painful than the controls.

New FindingsWhat is the central question of this study?Is peripheral sensory function impaired in the chronic phase of non-freezing cold injury (NFCI)?What is the main finding and its importance?Warm and mechanical detection thresholds are elevated and intraepidermal nerve fibre density is reduced in individuals with NFCI in their feet when compared to matched controls. This indicates impaired sensory function in individuals with NFCI. Interindividual variation was observed in all groups, and therefore a diagnostic cut-off for NFCI has yet to be established. Longitudinal studies are required to follow NFCI progression from formation to resolution The aim of this study was to compare peripheral sensory neural function of individuals with non-freezing cold injury (NFCI) with matched controls (without NFCI) with either similar (COLD) or minimal previous cold exposure (CON). Thirteen individuals with chronic NFCI in their feet were matched with the control groups for sex, age, race, fitness, body mass index and foot volume. All undertook quantitative sensory testing (QST) on the foot. Intraepidermal nerve fibre density (IENFD) was assessed 10 cm above the lateral malleolus in nine NFCI and 12 COLD participants. Warm detection threshold was higher at the great toe in NFCI than COLD (NFCI 45.93 (4.71)degrees C vs. COLD 43.44 (2.72)degrees C, P = 0.046), but was non-significantly different from CON (CON 43.92 (5.01)degrees C, P = 0.295). Mechanical detection threshold on the dorsum of the foot was higher in NFCI (23.61 (33.59) mN) than in CON (3.83 (3.69) mN, P = 0.003), but was non-significantly different from COLD (10.49 (5.76) mN, P > 0.999). Remaining QST measures did not differ significantly between groups. IENFD was lower in NFCI than COLD (NFCI 8.47 (2.36) fibre/mm(2) vs. COLD 11.93 (4.04) fibre/mm(2), P = 0.020). Elevated warm and mechanical detection thresholds may indicate hyposensitivity to sensory stimuli in the injured foot for individuals with NFCI and may be due to reduced innervation given the reduction in IENFD. Longitudinal studies are required to identify the progression of sensory neuropathy from the formation of injury to its resolution, with appropriate control groups employed.

Chronic heart failure (CHF) results in central and peripheral derangements that ultimately reduce skeletal muscle O2 delivery and impair exercise tolerance. Dietary nitrate (NO3 (-)) supplementation improves skeletal muscle vascular function and tolerance to exercise. We tested the hypothesis that NO3 (-) supplementation would elevate exercising skeletal muscle blood flow (BF) and vascular conductance (VC) in CHF rats. Myocardial infarction (MI) was induced (coronary artery ligation) in young adult male rats. After 21 days of recovery, rats randomly received 5 days of NO3 (-)-rich beetroot juice (CHF + BR, n = 10) or a placebo (CHF, n = 10). Mean arterial pressure (carotid artery catheter) and skeletal muscle BF (radiolabeled microspheres) were measured during treadmill exercise (20 m/min, 5% grade). CHF-induced dysfunction, as determined by myocardial infarction size (29 ± 3% and 33 ± 4% in CHF and CHF + BR, respectively) and left ventricular end-diastolic pressure (18 ± 2 and 18 ± 2 mmHg in CHF and CHF + BR, respectively), and exercising mean arterial pressure (131 ± 3 and 128 ± 4 mmHg in CHF and CHF + BR, respectively) were not different (P > 0.05) between groups. Total exercising hindlimb skeletal muscle BF (95 ± 5 and 116 ± 9 ml·min(-1)·100 g(-1) in CHF and CHF + BR, respectively) and VC (0.75 ± 0.05 and 0.90 ± 0.05 ml·min(-1)·100 g(-1)·mmHg(-1) in CHF and CHF + BR, respectively) were 22% and 20% greater in BR-supplemented rats, respectively (P < 0.05). During exercise, BF in 9 and VC in 10 hindlimb muscles and muscle portions were significantly greater in the CHF + BR group. These results provide strong evidence that dietary NO3 (-) supplementation improves skeletal muscle vascular function during exercise in rats with CHF and, thus, support the use of BR as a novel therapeutic modality for the treatment of CHF.

Nitrite (NO2-) has been implicated as a major storage pool of nitric oxide (NO) in vivo and thus potentially plays a critical role in skeletal muscle vascular and metabolic function. This nitric oxide synthase (NOS) independent pathway of NO production may have therapeutic applications for those with cardiovascular diseases hallmarked by compromised NOS function. PURPOSE: We tested the hypothesis that NO2- infusion would reduce mean arterial pressure (MAP) and increase skeletal muscle blood flow (BF) and vascular conductance (VC) during exercise, in the face of NOS blockade via NG-nitro-L arginine methyl ester (L-NAME). This simulates the most extreme NOS downregulation that could be caused by disease. METHODS: Male Sprague-Dawley rats (3-6 months) exercised without (control, n=8) and after (n=8) infusion with L-NAME (10 mg/kg: L-NAME) and sodium NO2- (7 mg/kg: L-NAME + NO2-). MAP and hindlimb skeletal muscle BF and VC (radiolabeled microsphere infusions) were measured during submaximal treadmill running (20 m/min, 5% grade). RESULTS: Following an L-NAME induced increase in exercising MAP, NO2- infusion restored MAP to levels observed in healthy control animals (control: 137 ± 3 L-NAME: 157 ± 7, L-NAME + NO2-: 136 ± 5 mmHg). Relative to control, L-NAME significantly reduced BF and VC during exercise (P<0.05). NO2- infusion restored VC to levles observed in control animals (control: 0.77 ± 0.18, L-NAME: 0.57 ± 0.03, L-NAME + NO2-: 0.69 ± 0.04 ml/min/100g/mmHg). Furthermore, following NO2- infusion BF was not significantly different when compared to healthy control animals (control: 105 ± 10, L-NAME: 88 ± 3, L-NAME + NO2-: 94 ± 6 ml/min/100g, P=0.38 L-NAME vs. L-NAME + NO2-). CONCLUSION: These results suggest that, in the face of NOS blockade, NO2- infusion can restore MAP and skeletal muscle vascular control to levels reported in healthy young adult rats (with intact NOS function). Individuals with diseases that impair NOS activity, and thus tolerance to exercise, may benefit from a NO2- based therapy in which NO bioavailability is elevated in a NOS independent manner.

Chronic heart failure (CHF) results in central and peripheral derangements that ultimately reduce skeletal muscle O2 delivery and impair exercise tolerance. Dietary nitrate (NO3‐) supplementation improves skeletal muscle vascular function and improves tolerance to exercise. We tested the hypothesis that NO3‐ supplementation would elevate exercising skeletal muscle blood flow (BF) and vascular conductance (VC) in CHF rats. Myocardial infarction (MI) was induced (coronary artery ligation) in young‐adult male rats. After 21‐days of recovery, rats randomly received NO3‐ rich beetroot juice (CHF+BR, n=10) or a placebo (CHF, n=10). Mean arterial pressure (MAP, carotid artery catheter) and skeletal muscle BF (radiolabeled microspheres) was measured during treadmill exercise (20 m/min, 5% grade). CHF parameters (MI size, CHF: 29 ± 3, CHF+BR: 33 ± 4%, LVEDP, CHF: 18 ± 2, CHF+BR: 18 ± 2 mmHg) and exercising MAP (CHF: 131 ± 3, CHF +BR: 128 ± 4 mmHg) were not different (P<0.05) between groups. Total hindlimb skeletal muscle BF (CHF: 95 ± 5, CHF+BR: 116 ± 9 ml/min/100g) and VC (CHF: 0.75 ± 0.05, CHF+BR: 0.90 ± 0.05) were greater (P<0.05) in BR supplemented rats. The increases in BF and VC (P<0.05) were found preferentially in muscles and muscle parts containing 蠅70% type IIb + d/x muscle fibers which resulted in a ~22% increase in total skeletal muscle BF and a 20% increase in total VC during exercise. These results provide strong evidence that dietary NO3‐ supplementation improves skeletal muscle vascular function during exercise in rats with CHF and suggest that BR may provide a novel therapeutic modality for the treatment of CHF. NIH HL‐108328

The nitric oxide synthase (NOS)-independent pathway of nitric oxide (NO) production in which nitrite (NO2−) is reduced to NO may have therapeutic applications for those with cardiovascular diseases in which the NOS pathway is downregulated. We tested the hypothesis that NO2− infusion would reduce mean arterial pressure (MAP) and increase skeletal muscle blood flow (BF) and vascular conductance (VC) during exercise in the face of NOS blockade via L-NAME. Following infusion of L-NAME (10 mg kg−1, L-NAME), male Sprague-Dawley rats (3-6 months, n = 8) exercised without NG-nitro-L arginine methyl ester (L-NAME) and after infusion of sodium NO2− (7 mg kg−1; L-NAME + NO2−). MAP and hindlimb skeletal muscle BF (radiolabeled microsphere infusions) were measured during submaximal treadmill running (20 m min−1, 5% grade). Across group comparisons were made with a published control data set (n = 11). Relative to L-NAME, NO2− infusion significantly reduced MAP ( P < 0.03). The lower MAP in L-NAME+NO2− was not different from healthy control animals (control: 137 ± 3 L-NAME: 157 ± 7, L-NAME + NO2−: 136 ± 5 mm Hg). Also, NO2− infusion significantly increased VC when compared to L-NAME ( P < 0.03), ultimately negating any significant differences from control animals (control: 0.78 ± 0.05, L-NAME: 0.57 ± 0.03, L-NAME + NO2−; 0.69 ± 0.04 mL min−1 100 g−1 mm Hg−1) with no apparent fiber-type preferential effect. Overall, hindlimb BF was decreased significantly by L-NAME; however, in L-NAME + NO2−, BF improved to a level not significantly different from healthy controls (control: 108 ± 8, L-NAME: 88 ± 3, L-NAME + NO2−: 94 ± 6 mL min−1 100 g−1, P = 0.38 L-NAME vs L-NAME + NO2−). Individuals with diseases that impair NOS activity, and thus vascular function, may benefit from a NO2−-based therapy in which NO bioavailability is elevated in an NOS-independent manner.

Chronic heart failure (CHF) causes deficiencies in skeletal muscle blood flow resulting in compromised skeletal muscle capillary red blood cell (RBC) distribution and hemodynamics. These impairments are thought to be due, in part, to reductions in nitric oxide (NO) bioavailability within skeletal muscle. In healthy animals, nitrate (NO 3 ‐ ) supplementation via beetroot juice (BR) elevates skeletal muscle blood flow and raises the pressure head for capillary‐myocyte O 2 flux during exercise presumably following a stepwise reduction to NO in vivo . We tested the hypothesis that BR supplementation would increase the % of capillaries supporting RBC flow at rest and during contractions in CHF rats. CHF was induced in young adult male Sprague‐Dawley rats via myocardial infarction (MI). Following a 5‐week recovery period rats were given BR ([NO 3 ‐ ] 1 mmol/kg/day, CHF+BR) or water (CHF) for 5 days. MI size was not different between groups (CHF: 28 ± 5, CHF+BR: 28 ± 6 %). Intravital microscopy was used to study the in vivo spinotrapezius muscle microcirculation at rest and during 180 s 1Hz twitch contractions (6‐8 V). The percentage of capillaries supporting continuous RBC flow was elevated in CHF+BR rats at rest (CHF: 65 ± 6, CHF+BR: 76 ± 2 %, P 蠄0.05) and during contractions (CHF: 78 ± 6, CHF+BR 82: ± 2 %, P <0.05). The improvements seen herein are likely due to enhanced arteriolar vasodilation mediated, in part, by elevated NO bioavailability. These results have important implications and suggest that NO 3 ‐ supplementation via BR may constitute a viable therapeutic modality that improves muscle vascular and potentially metabolic function in CHF. Grant Funding Source : Supported by NIH‐HL108328

Nitrate (NO3(-)) supplementation via beetroot juice (BR) preferentially improves vascular conductance and O2 delivery to contracting skeletal muscles comprised predominantly of type IIb + d/x (i.e. highly glycolytic) fibers following its reduction to nitrite and nitric oxide (NO). To address the mechanistic basis for NO3(-) to improve metabolic control we tested the hypothesis that BR supplementation would elevate microvascular PO2 (PO2mv) in fast twitch but not slow twitch muscle. Twelve young adult male Sprague-Dawley rats were administered BR ([NO3(-)] 1 mmol/kg/day, n = 6) or water (control, n = 6) for 5 days. PO2mv (phosphorescence quenching) was measured at rest and during 180 s of electrically-induced 1-Hz twitch contractions (6-8 V) of the soleus (9% type IIb +d/x) and mixed portion of the gastrocnemius (MG, 91% type IIb + d/x) muscles. In the MG, but not the soleus, BR elevated contracting steady state PO2mv by ~43% (control: 14 ± 1, BR: 19 ± 2 mmHg (P < 0.05)). This higher PO2mv represents a greater blood-myocyte O2 driving force during muscle contractions thus providing a potential mechanism by which NO3(-) supplementation via BR improves metabolic control in fast twitch muscle. Recruitment of higher order type II muscle fibers is thought to play a role in the development of the VO2 slow component which is inextricably linked to the fatigue process. These data therefore provide a putative mechanism for the BR-induced improvements in high-intensity exercise performance seen in humans.

Effective blood‐muscle O2 flux demands a sufficient microvascular O2 driving pressure (PO2mv) which is set by the ratio of O2 delivery‐O2 utilization. Smooth muscle cell hyperpolarization contributes to exercise induced increases in skeletal muscle O2 delivery mediated, in part, by ATP‐sensitive K+ (KATP) channels. We hypothesized that KATP channel blockade via glibenclamide (GLI) would speed the fall of PO2mv following the onset of skeletal muscle contractions. Spinotrapezius PO2mv (phosphorescence quenching) was measured in 12 adult male Sprague Dawley rats at rest and during 180 s of 1 Hz twitch contractions (~7 V) under control (CON) and GLI (5 mg/kg) conditions. GLI increased mean arterial pressure (ΔCON: 2 ± 1, ΔGLI: 17 ± 4 mmHg, p < 0.05) and decreased heart rate (ΔCON: 3 ± 2, ΔGLI: ‐9 ± 3 bpm, p < 0.05) but did not change baseline PO2mv (CON: 34.0 ± 2.2, GLI: 33.7 ± 1.6 mmHg, p > 0.05). Following the onset of contractions the time constant, mean response time and contracting steady‐state PO2mv were not different between conditions (p > 0.05 for all). However, a clearly defined undershoot (p < 0.05) of the contracting steady‐state PO2mv was evident with GLI (8.0 ± 2.6 %) but not during CON (1.6 ± 1.1 %). Our data indicate that blockade of KATP channels does not impact PO2mv kinetics parameters during small muscle mass electrical stimulation, but can cause transient mismatch of O2 delivery‐O2 utilization prior to stabilizing at the contracting steady‐state PO2mv. This suggests that KATP channels contribute substantially to skeletal muscle microvascular function during the crucial rest to contraction transition and therefore have the potential to mediate skeletal muscle performance decrements evident in disease states.Grant Funding Source: Supported by AHA Midwest Affiliate 0750090Z, NIH HL‐108328