Dietary nitrate supplementation: impact on skeletal muscle vascular control in exercising rats with chronic heart failure

Chronic heart failure (CHF) impairs skeletal muscle vascular control and tolerance to exercise. Dietary nitrate supplementation elevates skeletal muscle blood flow and improves exercise performance in some populations. Here we demonstrate that nitrate supplementation via beetroot juice elevates skeletal muscle blood flow during exercise in rats with moderate CHF.

Abstract

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.

NEW & NOTEWORTHY

Chronic heart failure (CHF) impairs skeletal muscle vascular control and tolerance to exercise. Dietary nitrate supplementation elevates skeletal muscle blood flow and improves exercise performance in some populations. Here we demonstrate that nitrate supplementation via beetroot juice elevates skeletal muscle blood flow during exercise in rats with moderate CHF.

chronic heart failure (CHF) results in severe exercise intolerance, consequent to the impaired pumping capacity of the heart and reduced skeletal muscle vascular and metabolic function. At the onset of exercise, an increase in O₂ delivery (Q̇o₂) is mandated to meet the rapidly rising O₂ demand (V̇o₂) of the skeletal muscle. This is accomplished by a complex array of central cardiac and local humoral components that ultimately become dysfunctional in CHF (reviewed in Ref. 54). Of the local humoral components, nitric oxide (NO), a primary controller of skeletal muscle blood flow (BF), is greatly diminished in CHF due, in part, to impaired NO synthase (NOS) function (12, 31). Thus the ability to elevate Q̇o₂ relative to V̇o₂ is severely compromised, increasing the reliance on glycolytic metabolism, which ultimately contributes to the premature fatigue evident in CHF.

Although once believed to be inert end products of NO metabolism, it is now well understood that nitrate (NO₃⁻) and nitrite (NO₂⁻) can be recycled to produce NO via a step-wise, NOS-independent mechanism, whereby NO₃⁻ is reduced to NO₂⁻ and, finally, to NO. Crucially, the reduction of NO₂⁻ to NO is facilitated in the low-Po₂/pH environments common in systemic diseases, making the NO₃⁻-NO₂⁻-NO pathway a prime candidate for a therapy aimed at pathologically induced local and systemic hypoxia (reviewed in Refs. 45 and 46). Our laboratory recently showed that, in healthy rats, direct infusion of NO₂⁻ reduces mean arterial pressure (MAP) and improves skeletal muscle vascular control during exercise in the face of NOS blockade via N^G-nitro-l-arginine methyl ester (l-NAME) (17). l-NAME presents the most extreme model of NOS inhibition and reduced NOS-related NO bioavailability. Additionally, other investigations utilizing murine models have revealed that NO₂⁻ reverses aging-induced vascular endothelial dysfunction (56) and renal injury in l-NAME-induced hypertension (34).

In humans, ingestion of NO₃⁻-rich foodstuffs, such as beetroot juice (BR), also increases circulating NO₂⁻ concentration ([NO₂⁻]), reduces the O₂ cost of submaximal exercise (41, 42), and can result in improved tolerance to exercise in healthy (reviewed by Refs. 32 and 48) and patient (36, 61) populations. Of particular relevance, Coggan et al. (11) recently demonstrated that dietary NO₃⁻ supplementation via BR improves muscle contractile function in patients with heart failure. Further mechanistic insight gained from animal models reveals that BR increases exercising skeletal muscle BF and raises the Q̇o₂-to-V̇o₂ ratio [thereby increasing microvascular Po₂ (Po_2mv), the pressure head required for blood-myocyte O₂ flux] within fast-twitch, but not slow-twitch, skeletal muscles (19-21). This phenomenon further substantiates the therapeutic use of dietary NO₃⁻, especially when considering that phosphocreatine and glycogen depletion and, thus, fatigue are greater in fast- than slow-twitch muscles (25).

Given that CHF reduces BF primarily in slow-twitch (type I) muscles (53), which is expected to increase reliance on fast-twitch (type II) muscles during submaximal exercise and the lower fatigue resistance evident in type II than type I muscles (25), dietary NO₃⁻ may ameliorate the vascular dysfunction caused by CHF. Consequently, the purpose of the present investigation was to test the hypothesis that 5 days of dietary NO₃⁻ supplementation via BR would elevate skeletal muscle BF and vascular conductance (VC) during submaximal treadmill exercise in rats with moderate CHF. We anticipated that BR would elevate skeletal muscle BF and VC and that the elevations would occur preferentially in muscle with a high proportion of type IIb + IId/IIx fibers.

METHODS

Ethical approval.

Young (∼3-mo-old) adult male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were maintained at accredited animal facilities at Kansas State University on a 12:12-h light-dark cycle with food and water provided ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee of Kansas State University and conducted according to the National Research Council Guide for the Care and Use of Laboratory Animals.

Myocardial infarction protocol and treadmill exercise acclimatization.

The overall experimental protocol is outlined in Fig. 1. Myocardial infarction (MI) was induced in rats (n = 20) by surgical ligation of the left main coronary artery (53). Briefly, rats were initially anesthetized with a 5% isoflurane-O₂ mixture (Butler Animal Health Supply, Elk Grove Village, IL; Linweld, Dallas, TX) and maintained on an ∼2.5% isoflurane-O₂ mixture and then intubated and mechanically ventilated with a rodent respirator (model 680, Harvard Instruments, Holliston, MA) for the duration of the surgical procedure. A left thoracotomy was performed to expose the heart through the fifth intercostal space, and the left main coronary artery was ligated 1–2 mm distal to the edge of the left atrium with a 6-0 braided polyester suture. The thorax was then closed with 2-0 gut and the skin with 3-0 silk. Bupivacaine (1.5 mg/kg sc), ampicillin (50 mg/kg im), and buprenorphine (∼0.03 mg/kg im) were administered to alleviate pain and reduce the risk of infection. After the rats were removed from mechanical ventilation and anesthesia, they were monitored closely for ≥6 h postsurgery. After ≥21 days of recovery for complete remodeling of necrotic myocardial tissue and development of compensated CHF (23, 53), all rats were familiarized with running on a custom-built, motor-driven treadmill for ∼5 min daily for 5 consecutive days. All rats ran at a speed of 20 m/min up a 5% incline (∼65% maximal V̇o₂) (49).

Fig. 1.

Top: experimental protocol for rats with chronic heart failure (CHF) and rats with CHF treated with beetroot juice (CHF + BR). Rats were supplemented with NO₃⁻-free (placebo) or NO₃⁻-rich BR juice (1 mmol·kg⁻¹·day⁻¹) for 5 days. During the supplementation period, rats were familiarized with treadmill running for ∼5 min/day. BF, blood flow. Bottom: detailed timeline for measurement of skeletal muscle BF and vascular conductance (VC) during exercise in CHF and CHF + BR rats. A ∼0.3-ml blood sample was taken from the carotid artery catheter prior to termination of exercise, and resting BF was measured following ∼1 h of recovery from exercise prior to euthanasia (not shown).

BR supplementation.

Rats were assigned randomly to receive 5 days of NO₃⁻-rich (NO₃⁻ dose = 1 mmol·kg⁻¹·day⁻¹; Beet it Sport, James White Drinks, Ipswich UK; CHF + BR, n = 10) or NO₃⁻-depleted (Placebo Beet it Sport, James White Drinks; CHF, n = 10) BR. Daily doses of the placebo and NO₃⁻-rich BR were diluted in 50 ml of tap water. This dose has been used by our laboratory and has been demonstrated to elicit significant physiological effects (18–21). In addition, this dose is also based on that used in humans by Jones et al. (3, 4, 57, 58, 60), accounting for the higher resting metabolic rate of the rat (∼7 times greater than humans) (49), and results in substantial elevations of blood plasma NO₃⁻ concentration ([NO₃⁻]) and [NO₂⁻] of ∼120 μM and ∼600 nM, respectively (20). We also ensured that the tap water did not provide any substantial [NO₃⁻] (3.7 μM) or [NO₂⁻] (∼100 nM).

Surgical instrumentation.

On the day of the experiment, rats were anesthetized with a 5% isoflurane-O₂ mixture and maintained subsequently on ∼3% isoflurane-O₂. The right carotid artery was isolated and cannulated for the advancement of a 2-Fr catheter-tipped pressure transducer (Millar Instruments, Houston, TX) into the left ventricle (LV) for measurement of systolic and diastolic pressures, LV end-diastolic pressure (LVEDP), and the rate of LV pressure rise over time (LV dP/dt). The 2-Fr pressure transducer was removed, and the artery was recannulated with a catheter (PE-10 connected to PE-50, Intra-Medic polyethylene tubing, Clay Adams, Becton, Dickinson, Sparks, MD) for the measurement of MAP and heart rate (HR) and the infusion of radiolabeled microspheres (model 200, DigiMed BPA, Louisville, KY; see below). A second catheter was placed in the caudal (tail) artery as described previously (53) for arterial blood sampling. Both catheters were tunneled subcutaneously through the dorsal aspect of the cervical region and exteriorized via a puncture wound in the skin. The incisions were then closed, anesthesia was terminated, and the rats were given ≥60 min to recover (24). After recovery, the rats were placed on the motor-driven treadmill, and the carotid artery catheter was connected to a pressure transducer (model P23ID, Gould Statham, Valley View, OH) maintained at the same height as the animal. Rats were allowed to stabilize for ∼15 min before the final experimental protocol was initiated while HR and MAP were monitored continuously using the carotid artery catheter.

Measurement of hindlimb skeletal muscle BF during exercise.

After the stabilization period, the caudal artery catheter was connected to a 1-ml syringe chambered in an infusion/withdrawal pump (model 907, Harvard Instruments). Exercise was then initiated up a 5% incline with speed progressing to 20 m/min within the first 30 s. The rat continued at this speed for another 2.5 min until a total time of 3 min was reached. During this time, radiolabeled microspheres (⁵⁷Co or ⁸⁵Sr in random order; Perkin Elmer, Waltham, MA) were thoroughly mixed by a vortex agitator (Fisher Scientific, Hampton, NH). At the 3-min exercise mark, the carotid artery catheter was disconnected from the pressure transducer, and 0.5–0.6 × 10⁶ radiolabeled microspheres (15 μm diameter, ∼0.10 ml) were infused into the aortic arch. Simultaneously, the pump connected to the caudal artery catheter was activated and blood withdrawal was initiated at a rate of 0.25 ml/min. Blood withdrawal was terminated 30 s following the microsphere infusion, and ∼0.3 ml of blood was sampled from the carotid artery catheter for the determination of blood lactate concentration ([lactate]), pH, Pco₂, Po₂, %O₂ saturation, and hematocrit (Nova Stat Profile M, Nova Biomedical, Waltham, MA). Exercise was then terminated.

After ≥30 min of recovery, a second microsphere infusion was performed (radiolabeled differently from the first microsphere infusion) while the rat sat quietly on the treadmill for the determination of resting BF, HR, and MAP. This experimental strategy, whereby exercise measurements were made before rest, mitigates the potential influence of the preexercise anticipatory response on resting skeletal muscle BF and MAP measurements (2).

Determination of BF and VC.

After the resting microsphere protocol had been completed, rats were euthanized via pentobarbital sodium overdose (≥50 mg/kg administered into the carotid artery catheter). The thorax of each rat was opened, and accurate placement of the carotid artery catheter was confirmed before the heart, lungs, select internal organs, and 28 individual muscles and muscle parts of the hindlimb were excised. Upon removal, tissues were blotted, weighed, and placed promptly into counting vials. The radioactivity of each tissue was determined with a gamma scintillation counter (model 5230, Auto Gamma Spectrometer, Packard, Downers Grove, IL). Tissue BF was then calculated using the reference sample method (53) and expressed as ml·min⁻¹·100 g tissue⁻¹. Adequate mixing of the microspheres was verified for each microsphere infusion as demonstrated by a <15% difference in BF to the right and left kidneys or to the right and left hindlimb musculature. VC was then calculated by normalizing BF to MAP measured at the time of microsphere infusion and expressed as ml·min⁻¹·100 g tissue⁻¹·mmHg⁻¹. The LV, right ventricle (RV), and lungs were normalized to body weight, and MI size of the LV was measured via planimetry as described previously by our laboratory (22). The necrotic zone caused by ischemia was determined as the actual infarct size. Specifically, the LV was excised and opened from base to apex and flattened onto a wax sheet using pins. Under transillumination, the remodeled region appears white and distinct from the healthy cardiac muscle. Color photographs were taken, and the area infarcted as a percentage of the total (i.e., healthy tissue + infarcted) area was measured via planimetry by cutting out the respective sections and weighing the paper.

Blood sampling and measurement of plasma [NO₃⁻] and [NO₂⁻].

A blood sample was collected from CHF and CHF + BR rats for the measurement of plasma [NO₃⁻] and [NO₂⁻]. These samples were collected following instrumentation and prior to regional BF measurements. Approximately 0.8 ml of blood was drawn from the caudal artery catheter and quickly (i.e., <2 min) centrifuged at 5,000 g at 4°C for 6 min. Plasma was subsequently extracted and frozen at −80°C for later analysis of [NO₃⁻] and [NO₂⁻].

All measurements of plasma NO₃⁻ and NO₂⁻ were performed within 30 min of thawing via chemiluminescence with an Ionic/Sievers NO analyzer (NOA 280i, Sievers Instruments, Boulder, CO). To obtain plasma NO₂⁻ levels and avoid potential reduction of NO₃⁻, potassium iodide in acetic acid was used as a reductant. This reductant possesses the ability to reduce NO₂⁻ to NO but is incapable of reducing higher oxides of nitrogen (i.e., NO₃⁻), thus increasing the specificity for NO₂⁻. Plasma NO₃⁻ concentrations were then obtained using the same apparatus with the stronger reductant vanadium chloride in hydrochloric acid at 95°C. This stronger reductant reduces the sum of all nitrogen oxides with an oxidation state of +2 or higher [predominantly NO₃⁻ (μM)] but also includes NO₂⁻ and nitrosothiols (nM).

Statistical analyses.

Indexes of CHF were compared using unpaired Student's t-tests. All other results were compared within (rest vs. exercise) and among (CHF vs. CHF + BR) groups using mixed two-way ANOVAs and Student-Newman-Keuls post hoc tests where appropriate. Pearson's product-moment correlations and linear regressions were used to determine relationships among variables. Muscle fiber type composition was based on the percentage of type I, IIa, IIb, and IId/IIx fibers in the individual muscles and muscle parts of the rat hindlimb according to Delp and Duan (15). Values are means ± SE; significance was set at P < 0.05.

RESULTS

Indexes of CHF.

The average body mass of the 20 CHF rats [n = 10 (CHF) and n = 10 (CHF + BR)] that completed the investigation was 483 ± 11 g, with no significant between-group differences (P > 0.05). There were also no between-group differences in MI size (29 ± 3% and 33 ± 4% of LV area for CHF and CHF + BR, respectively, P > 0.05), LVEDP (18 ± 2 and 18 ± 2 mmHg for CHF and CHF + BR, respectively, P > 0.05), LV dP/dt (6,311 ± 259 and 6,438 ± 436 mmHg/s for CHF and CHF + BR, respectively, P > 0.05), lung weight-to-body mass ratio (5.43 ± 1.55 and 5.35 ± 0.80 mg/g for CHF and CHF + BR, respectively, P > 0.05), or RV-to-body mass ratio (0.66 ± 0.03 and 0.75 ± 0.06 mg/g for CHF and CHF + BR, respectively, P > 0.05). All these values are consistent with moderate CHF (16).

Effect of BR on plasma [NO₂⁻].

Plasma [NO₂⁻] was significantly greater in CHF + BR than CHF rats (345 ± 59 vs. 569 ± 81 nM, P < 0.05).

Hemodynamic responses.

There were no significant between-group differences in MAP at rest (125 ± 5 and 118 ± 3 mmHg for CHF and CHF + BR, respectively, P > 0.05) or during exercise (131 ± 3 and 128 ± 4 mmHg for CHF and CHF + BR, respectively, P > 0.05). In addition, there were no significant between-group differences in HR at rest (409 ± 12 and 413 ± 9 beats/min for CHF and CHF + BR, respectively, P > 0.05) or during exercise (518 ± 10 and 507 ± 5 beats/min for CHF and CHF + BR, respectively, P > 0.05). No between-group differences in arterial Pco₂, Po₂, %O₂ saturation, or hematocrit were present (P > 0.05 for all), and there were no between-group differences in exercising blood [lactate] (2.1 ± 0.2 and 2.1 ± 0.2 mM for CHF and CHF + BR, respectively, P > 0.05).

Impact of BR on skeletal muscle BF and VC at rest and during exercise.

There were no effects of BR on total resting hindlimb skeletal muscle BF or VC (P > 0.05; Fig. 2), nor were there any significant differences in resting BF or VC in any of the 28 hindlimb muscles or muscle portions (Table 1). However, during exercise, total hindlimb skeletal muscle BF and VC were significantly greater in CHF + BR than CHF rats (Fig. 2). Specifically, BR resulted in significantly greater BF in 9 and VC in 10 of the 28 individual hindlimb muscles or muscle parts (Table 2). Significant differences in BF were found in muscles comprised of 9–100% type IIb + IId/IIx fibers, whereas changes in VC were found in muscles comprised of 35–100% type IIb + IId/IIx fibers. There were no significant correlations between the BR-induced changes in BF and VC and muscle fiber type composition (P > 0.05 for both BF and VC); however, spatial distribution of BF increase among muscles was directed preferentially toward ankle and knee extensors (Fig. 3).

Fig. 2.

Effects of 5 days of dietary NO₃⁻ supplementation via BR on total hindlimb skeletal muscle blood flow (BF) and vascular conductance (VC) at rest and during treadmill exercise in rats with CHF. *P < 0.05.

Table 1.

Effects of BR supplementation on resting hindlimb skeletal muscle BF and VC in CHF and CHF + BR rats

	BF, ml·min−1·100 g−1		VC, ml·min−1·100 g−1·mmHg−1
	CHF	CHF + BR	CHF	CHF + BR
Ankle extensors
Soleus (9%)	102 ± 19	62 ± 17	0.85 ± 0.17	0.54 ± 0.16
Plantaris (80%)	9 ± 1	13 ± 1	0.07 ± 0.01	0.11 ± 0.04
Gastrocnemius
Red (14%)	44 ± 10	32 ± 9	0.36 ± 0.08	0.27 ± 0.09
White (100%)	7 ± 1	7 ± 2	0.05 ± 0.01	0.06 ± 0.01
Mixed (91%)	13 ± 2	12 ± 3	0.10 ± 0.02	0.10 ± 0.03
Tibialis posterior (73%)	9 ± 1	20 ± 1	0.08 ± 0.01	0.17 ± 0.07
Flexor digitorum longus (68%)	13 ± 5	26 ± 10	0.11 ± 0.04	0.22 ± 0.08
Flexor halicus longus (71%)	9 ± 2	9 ± 2	0.07 ± 0.01	0.08 ± 0.02
Ankle flexors
Tibialis anterior
Red (63%)	15 ± 6	8 ± 1	0.12 ± 0.05	0.06 ± 0.01
White (80%)	13 ± 2	11 ± 3	0.10 ± 0.02	0.09 ± 0.02
Extensor digitorum longus (76%)	7 ± 3	12 ± 5	0.06 ± 0.02	0.10 ± 0.04
Peroneals (67%)	12 ± 3	9 ± 2	0.10 ± 0.02	0.07 ± 0.02
Knee extensors
Vastus intermedius (4%)	67 ± 16	48 ± 12	0.57 ± 0.15	0.41 ± 0.10
Vastus medialis (82%)	15 ± 3	9 ± 3	0.13 ± 0.03	0.08 ± 0.02
Vastus lateralis
Red (35%)	38 ± 10	31 ± 8	0.32 ± 0.09	0.26 ± 0.07
White (100%)	7 ± 1	8 ± 3	0.06 ± 0.01	0.06 ± 0.02
Mixed (89%)	10 ± 2	10 ± 3	0.08 ± 0.02	0.08 ± 0.02
Rectus femoris
Red (66%)	16 ± 5	11 ± 2	0.14 ± 0.05	0.09 ± 0.02
White (100%)	8 ± 1	7 ± 2	0.06 ± 0.01	0.07 ± 0.02
Knee flexors
Biceps femoris
Anterior (100%)	6 ± 1	7 ± 2	0.05 ± 0.01	0.06 ± 0.02
Posterior (92%)	7 ± 1	10 ± 2	0.06 ± 0.01	0.08 ± 0.02
Semitendinosus (83%)	10 ± 2	11 ± 2	0.08 ± 0.01	0.09 ± 0.02
Semimembranosus
Red (72%)	9 ± 1	13 ± 3	0.07 ± 0.01	0.11 ± 0.03
White (100%)	7 ± 1	8 ± 2	0.06 ± 0.08	0.07 ± 0.02
Thigh adductors
Adductor longus (5%)	126 ± 14	109 ± 19	1.03 ± 0.13	0.93 ± 0.17
Adductor magnus and brevis (89%)	10 ± 1	9 ± 2	0.08 ± 0.01	0.07 ± 0.01
Gracilis (77%)	12 ± 2	26 ± 13	0.10 ± 0.02	0.23 ± 0.11
Pectineus (69%)	23 ± 4	21 ± 4	0.19 ± 0.04	0.18 ± 0.04

Values are means ± SE; n = 10 in each group. CHF, congestive heart failure; BF, blood flow; VC, vascular conductance. Values in parentheses indicate %type IIb + IId/IIx according to Delp and Duan (15). BR, beetroot juice.

Table 2.

Effects of BR supplementation on exercising hindlimb skeletal muscle BF and VC in CHF and CHF + BR rats

	BF, ml·min−1·100 g−1		VC, ml·min−1·100 g−1·mmHg−1
	CHF	CHF + BR	CHF	CHF + BR
Ankle extensors
Soleus (9%)	262 ± 36	337 ± 28*	2.05 ± 0.32	2.62 ± 0.16
Plantaris (80%)	155 ± 11	204 ± 20*	1.18 ± 0.08	1.58 ± 0.14*
Gastrocnemius
Red (14%)	442 ± 49	407 ± 60	3.45 ± 0.49	3.14 ± 0.40
White (100%)	28 ± 3	40 ± 6*	0.21 ± 0.02	0.31 ± 0.05*
Mixed (91%)	131 ± 9	156 ± 10*	1.00 ± 0.07	1.21 ± 0.05*
Tibialis posterior (73%)	97 ± 13	114 ± 17	0.75 ± 0.10	0.90 ± 0.13
Flexor digitorum longus (68%)	52 ± 9	61 ± 13	0.39 ± 0.06	1.24 ± 0.77
Flexor halicus longus (71%)	47 ± 4	80 ± 16*	0.36 ± 0.03	0.62 ± 0.12*
Ankle flexors
Tibialis anterior
Red (63%)	299 ± 22	319 ± 33	2.29 ± 0.18	2.50 ± 0.26
White (80%)	93 ± 11	102 ± 14	0.71 ± 0.07	0.79 ± 0.10
Extensor digitorum longus (76%)	61 ± 26	98 ± 44	0.45 ± 0.18	0.78 ± 0.35
Peroneals (67%)	120 ± 8	138 ± 24	0.92 ± 0.07	1.07 ± 0.18
Knee extensors
Vastus intermedius (4%)	360 ± 52	415 ± 40	2.79 ± 0.42	3.22 ± 0.24
Vastus medialis (82%)	153 ± 13	170 ± 15	1.18 ± 0.12	1.30 ± 0.10
Vastus lateralis
Red (35%)	340 ± 20	411 ± 45	2.62 ± 0.19	3.18 ± 0.27*
White (100%)	18 ± 3	24 ± 5	0.14 ± 0.02	0.19 ± 0.04
Mixed (89%)	136 ± 9	160 ± 11*	1.05 ± 0.10	1.24 ± 0.07*
Rectus femoris
Red (66%)	232 ± 18	279 ± 33	1.80 ± 0.17	2.16 ± 0.23
White (100%)	91 ± 5	108 ± 9*	0.70 ± 0.04	0.85 ± 0.06*
Knee flexors
Biceps femoris
Anterior (100%)	31 ± 5	50 ± 9*	0.23 ± 0.04	0.39 ± 0.06*
Posterior (92%)	77 ± 5	95 ± 8*	0.59 ± 0.04	0.74 ± 0.06*
Semitendinosus (83%)	43 ± 4	43 ± 7	0.33 ± 0.03	0.34 ± 0.05
Semimembranosus
Red (72%)	107 ± 10	124 ± 9	0.82 ± 0.08	0.98 ± 0.07
White (100%)	24 ± 3	33 ± 5	0.18 ± 0.02	0.26 ± 0.04*
Thigh adductors
Adductor longus (5%)	264 ± 23	298 ± 48	2.03 ± 0.19	2.29 ± 0.33
Adductor magnus & brevis (89%)	61 ± 5	73 ± 11	0.46 ± 0.03	0.57 ± 0.09
Gracilis (77%)	33 ± 5	36 ± 8	0.25 ± 0.03	0.28 ± 0.06
Pectineus (69%)	42 ± 10	53 ± 10	0.31 ± 0.08	0.42 ± 0.08

Values are means ± SE; n = 10 in each group. Values in parentheses indicate %type IIb + IId/IIx according to Delp and Duan (15).

^*P < 0.05 vs. control (CHF).

Fig. 3.

Effect of chronic BR supplementation on distribution of skeletal muscle blood flow (BF) among hindlimb muscles in exercising CHF rats. Left: lateral view of rat hindlimb. Middle: cross section of thigh and leg displaying distribution of BF increase. Coloring of muscles corresponds to their action: blue and red represent extensors and flexors, respectively. Right: percent increase in BF to individual muscles after BR compared with placebo.

Effects of BR on renal and splanchnic BF and VC at rest and during exercise.

Renal and splanchnic BF and VC values are presented in Table 3. At rest, BR-supplemented rats had significantly lower BF and VC in the large intestines (P < 0.05); during exercise, only BF was lower in the large intestines of CHF + BR rats (P < 0.05).

Table 3.

Effects of BR supplementation on resting and exercising BF and VC in kidneys and organs of the splanchnic region in CHF and CHF + BR rats.

	Rest				Exercise
	BF		VC		BF		VC
	CHF	CHF + BR	CHF	CHF + BR	CHF	CHF + BR	CHF	CHF + BR
Kidney	541 ± 25	436 ± 57	4.41 ± 0.28	3.64 ± 0.43	419 ± 34†	501 ± 38	3.19 ± 0.24†	3.91 ± 0.22
Stomach	103 ± 21	86 ± 18	0.87 ± 0.19	0.72 ± 0.14	74 ± 13	64 ± 5	0.57 ± 0.11	0.51 ± 0.04
Adrenals	595 ± 103	578 ± 82	4.71 ± 0.75	4.83 ± 0.65	311 ± 53†	430 ± 41	2.35 ± 0.37†	3.38 ± 0.33
Spleen	323 ± 21	305 ± 38	2.57 ± 0.13	2.55 ± 0.29	91 ± 19†	101 ± 17†	0.68 ± 0.14†	0.79 ± 0.13†
Pancreas	140 ± 17	121 ± 18	1.15 ± 0.16	1.02 ± 0.15	137 ± 24	155 ± 15	1.05 ± 0.18	1.22 ± 0.11
Intestine
Small	373 ± 48	324 ± 59	3.04 ± 0.41	2.72 ± 0.47	274 ± 33	299 ± 29	2.09 ± 0.23	2.35 ± 0.22
Large	289 ± 46	156 ± 30*	2.38 ± 0.43	1.31 ± 0.24*	245 ± 43	134 ± 17*	1.86 ± 0.31	1.05 ± 0.13
Liver (arterial, not portal)	33 ± 4	32 ± 6	0.26 ± 0.03	0.26 ± 0.04	22 ± 3	31 ± 5	0.17 ± 0.03	0.24 ± 0.03

Values (means ± SE) are expressed in ml·min⁻¹·100 g⁻¹ (BF) or ml·min⁻¹·100 g⁻¹·mmHg⁻¹ (VC).

^*P < 0.05 vs. CHF.

^†P < 0.05 vs. rest.

DISCUSSION

The principal original finding of the present investigation was that, in rats with CHF, 5 days of dietary NO₃⁻ supplementation via BR resulted in ∼22% greater total hindlimb skeletal muscle BF and VC during exercise than the placebo group. More specifically, BR resulted in elevated BF and VC to >54% of the total hindlimb skeletal muscle mass, with no distinct fiber type effects. Instead, the elevated BF and VC occurred preferentially in the locomotory muscles responsible for propulsion during running. This should reduce the CHF-induced metabolic perturbation within these tissues owing to an elevated O₂ availability (improved perfusive and diffusive O₂ transport), which, by definition, would be expected to raise intracellular Po₂ and reduce the dependence on glycolytic metabolism (30). Considering that the principal predictor of hospital readmission and mortality in CHF is exercise intolerance (35), these data provide compelling evidence to support the use of dietary NO₃⁻ as an effective therapeutic aimed at the skeletal muscle peripheral dysfunction caused by this disease.

Impact of BR on skeletal muscle BF during exercise.

The most striking finding of the present investigation was the greater skeletal muscle BF and VC in the CHF + BR than the CHF group. These results complement a recent study from our laboratory whereby BR resulted in a significant increase in skeletal muscle BF and VC during exercise in healthy rats (20). However, to our knowledge, this is the first investigation to show the impacts of dietary NO₃⁻ supplementation on vascular control in rats with CHF. A recent clinical investigation by Zamani et al. (61) demonstrated that a single dose of dietary NO₃⁻ raised maximum V̇o₂ by ∼10% and improved total work capacity by ∼13% in patients with preserved ejection fraction heart failure (HFpEF). The authors speculated that the beneficial effects of BR were likely due to targeted improvements in peripheral abnormalities, as suggested by the ∼25% reduction in systemic vascular resistance during exercise. These findings are corroborated by the present investigation, whereby BR resulted in a significant increase in BF and VC to approximately half (50% and 54% for BF and VC, respectively) of the total hindlimb skeletal muscle mass during exercise. Importantly, it is well appreciated that reduced ejection fraction heart failure (HFrEF, the model of CHF used here) results in temporal and spatial derangements in Po_2mv (the exclusive driving force for O₂ flux into the myocyte) during transitions in metabolic demand (6, 7, 12, 13, 16, 22, 29, 30), and while deficits in the skeletal muscle Q̇o₂-V̇o₂ relationship caused by HFpEF are not fully understood, decrements in convective and diffusive O₂ transport are thought to occur in HFpEF patients (37) and animal models (59). The augmented skeletal muscle vascular function in the present study and the functional improvements reported by Zamani et al. (61) suggest that dietary NO₃⁻ may partially restore physical functionality via improved skeletal muscle vascular and metabolic control in HFrEF and HFpEF.

Contrary to our original hypothesis, dietary NO₃⁻ supplementation augmented BF and VC across the full spectrum of muscle fiber types with no significant preferential effects. This could be due, at least in part, to the CHF-induced decrements in BF and VC in slow-twitch, but not fast-twitch, muscles. More specifically, CHF alters the heterogeneity of skeletal muscle Q̇o₂, such that BF to highly oxidative muscles is reduced whereas it may be better maintained in their fast-twitch counterparts (31, 47, 53), likely resulting in the faster fall in Po_2mv during the rest-contraction transition in slow-twitch, but not fast-twitch, muscles (6). Considering that slow-twitch muscles may require a much greater BF due to their substantially higher V̇o₂ and lower fractional O₂ extraction than fast-twitch muscles [nearly double for both BF and V̇o₂ (8)] and the evidence to suggest that NO₂⁻ bioconversion to NO is facilitated in low-Po₂/pH environments (14), it is possible that the CHF etiology may have produced an environment well suited for NO₂⁻ reduction in muscles that would otherwise, in health, fall above the critical Po₂ required to elicit vascular effects [a Po₂ approximating 25 mmHg (14)]. This idea is supported by a recent report from our laboratory, whereby direct NO₂⁻ infusion resulted in nonspecific elevations in skeletal muscle BF and VC in healthy rats following NOS inhibition via l-NAME (17). To this end, despite no statistically significant correlations, the “targeted effects” of dietary NO₃⁻ supplementation proposed by our group (33) as well as others (28, 32, 36, 60) may have afforded vascular effects in some, but not all, slow-twitch muscles due to the CHF-induced derangements in the Q̇o₂-to-V̇o₂ ratio and lowered Po_2mv.

Blood lactate.

We previously demonstrated that NO₃⁻ supplementation via BR reduces blood [lactate] during submaximal exercise (20); therefore, the expectation was that this effect would be associated with the enhanced BFs seen here. However, blood lactate was not different between CHF and CHF + BR. It is also notable that, in healthy animals under NOS blockade (l-NAME), blood [lactate] is increased by NO₂⁻ infusions during exercise. This occurs despite elevated BF to select muscles (17). The mechanism whereby elevated plasma [NO₂⁻] alters the balance between rates of appearance and disappearance of lactate in the arterial blood remains to be addressed.

Clinical relevance.

Exercise training elicits a full spectrum of beneficial adaptations to the O₂ transport system and, thus, represents an extremely effective therapy for treatment of the skeletal muscle dysfunction elicited by CHF (reviewed in Ref. 30). Of particular relevance to the present investigation is the fact that endurance exercise training produces a redistribution of BF within the hindlimb of the rat, such that BF appears to be directed away from fast-twitch glycolytic muscle toward its highly oxidative counterparts (43, 52). This redistribution occurs in the absence of changes in bulk Q̇o₂, yet it substantially augments skeletal muscle Q̇o₂-V̇o₂ matching during transitions in metabolic demand (27, 38, 39), providing the mechanistic basis for improved exercise tolerance and better quality of life (9) for CHF patients. Considering that 5 days of dietary NO₃⁻ supplementation via BR, a nonpharmacological intervention, increased bulk skeletal muscle Q̇o₂ (elevated BF and VC; Fig. 2), combinations of exercise-based rehabilitation programs with dietary NO₃⁻ supplementation and other NO-based therapies [i.e., sildenafil (44)] may represent an especially effective therapeutic strategy for patients with CHF. Indeed, even small increases in physical capacity may lead to an upregulation of endothelial NOS activity and vascular function (1). These data call for further clinical investigations to elucidate the potential for chronic dietary NO₃⁻ to improve exercise tolerance and the efficacy of (and adherence to) cardiac rehabilitation programs.

An unexpected observation was the lower BF at rest and during exercise in the large intestine. This effect is contrary to previous findings that the low intestinal perfusion, and thus Po₂, of the intestine in CHF is thought to promote the reduction of NO₂⁻ to NO, and, thus, increase, rather than decrease, BF to the gastrointestinal tract (40, 46). It has been speculated that the reason for this effect is that BR resulted in a redistribution of BF to a vascular bed that was not measured in the present investigation.

Experimental considerations and future directions.

The techniques used to measure intra- and intermuscular BF during dynamic exercise represent a major strength of the present investigation. While current technologies preclude these measurements in humans because of technical and ethical limitations, these findings complement those of Zamani et al. (61) by providing crucial mechanistic insight into the physiological benefits of dietary NO₃⁻ supplementation in patients with CHF. The lack of sham-operated controls could be considered a potential limitation of the present investigation. However, in many comparisons between controls and sham-operated animals, Musch et al. (5, 16, 50, 51, 53) found no differences. In addition, the measurements of skeletal muscle BF and VC in rats receiving the placebo BR are consistent with previous reports from our laboratory in rats with moderate CHF (53, 55), further supporting the results observed here. However, while the elevations in skeletal muscle BF and VC observed in the present study suggest an improvement in microvascular oxygenation (Po_2mv), this was not directly measured in the present investigation, nor was exercise tolerance or key parameters of exercise performance (i.e., critical velocity and pulmonary V̇o₂ kinetics). To this point, the potential outcomes of dietary NO₃⁻ supplementation on these key parameters of physical function remain speculative and, thus, should be the focus of future investigations utilizing murine (for measurements of Po_2mv) and patient populations. Notwithstanding these limitations, Fick's law of diffusion dictates that an elevation of the diffusion pressure gradient, controlled by the Q̇o₂-V̇o₂ relationship (of which Q̇o₂ was increased ∼22% in the present study), will increase blood myocyte O₂ flux and, thus, enhance oxidative metabolism while reducing fatigue-associated metabolite accumulation (26, 58). Thus this investigation represents an important step in uncovering the mechanistic basis for the benefits of dietary NO₃⁻ supplementation reported in CHF (61) and other patient populations [i.e., pulmonary and peripheral artery diseases (10, 36)]. Finally, because of technical limitations, plasma [NO₃⁻] could not be obtained here. However, as NO₂⁻ is the key active intermediate between NO₃⁻ and NO and the magnitude of the biological consequences most tightly relate to plasma [NO₂⁻], we believe that measurements of this anion provide the crucial mechanistic linkage between BR consumption and the augmented vascular control observed in the present study.

Conclusions.

This is the first investigation to demonstrate the impact of dietary NO₃⁻ supplementation on intra- and intermuscular BF and VC at rest and during exercise in rats with CHF. Five days of NO₃⁻-rich BR supplementation resulted in robust increases in total hindlimb skeletal muscle BF and VC, with significant increases in BF and VC in ≥50% of the total hindlimb muscle mass. The finding that no distinct fiber type preferential effects were observed, despite previous investigations demonstrating BR-induced improvements in fast-twitch muscle function (33), suggests that BR improves vascular function in the full spectrum of muscle fiber types in CHF. Collectively, these results demonstrate the efficacy of a relatively short-term dietary intervention on vascular function in CHF and provide evidence to support implementation of BR as an effective addition to exercise-based cardiac rehabilitation programs.

GRANTS

These experiments were funded by a Kansas State University SMILE award to T. I. Musch and American Heart Association Midwest Affiliate Grant 10GRNT4350011 and National Heart, Lung, and Blood Institute Grant HL-108328 to D. C. Poole.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.K.F., J.C.C., A.M.J., J.D.A., T.I.M., and D.C.P. developed the concept and designed the research; S.K.F., C.T.H., T.D.C., J.L.W., A.F., and T.I.M. performed the experiments; S.K.F. analyzed the data; S.K.F., J.C.C., A.M.J., and D.C.P. interpreted the results of the experiments; S.K.F. and J.C.C. prepared the figures; S.K.F. and D.C.P. drafted the manuscript; S.K.F., C.T.H., T.D.C., J.C.C., A.M.J., J.D.A., T.I.M., and D.C.P. edited and revised the manuscript; S.K.F., C.T.H., T.D.C., J.L.W., J.C.C., A.F., A.M.J., J.D.A., T.I.M., and D.C.P. approved the final version of the manuscript.