Heterogeneity of Exercising Muscle Blood Flow and O2
Heterogeneity of Exercising Muscle Blood Flow and O2
Studying muscle blood flow distribution and heterogeneity in humans has been challenging. Attempts to do so have used PET or MRI during small muscle mass exercise such as one leg knee extension. When PET is being used, O-H2O radiowater tracer, the autoradiographic method, and a one-compartment model have been applied to calculate blood flow voxel-by-voxel and portrayed as parametric blood flow images (Fig. 5). The method has been validated against the radiolabeled microsphere technique in dogs in vivo, which demonstrated high accuracy in measuring regional muscle blood flow.
(Enlarge Image)
Figure 5.
A typical PET blood flow image from the middle thigh region at rest and during one leg exercise of low- to moderate-intensity exercise. Only the quadriceps femoris muscle as a whole muscle group is illustrated as a region of interest for the sake of clarity. At rest, blood flow is generally highest (4–12 mL·[100 mL of muscle] ·min) in vastus intermedius muscle, which is the muscle closest to the bone (lowest perfusion region in the middle of the images) and generally regarded to consist mostly of type I muscle fibers, whereas blood flow is generally much lower (1–6 mL·[100 mL of muscle] ·min) in the three other superficial muscles of the knee extensors. During exercise, this blood flow distribution pattern is largely maintained, and in some areas of vastus intermedius muscle, blood flow can reach values close to 200 mL·[100 mL of muscle] ·min (red areas) even during low- to moderate-intensity exercise, but due to much lower perfusion in superficial muscles, the mean blood flow in whole knee extensors is hardly ever more than 50 mL·[100 mL of muscle]·min in this low-intensity exercise setting. Moreover, blood flow in hamstring muscles does not change from rest to one-leg knee extensor exercise, at least up to low- to moderate-intensity exercise. Altogether, blood flow is thus markedly heterogeneous within and among human skeletal muscles, especially during knee extensor exercise.
PET facilitates blood flow measurement in any organ or tissue (e.g.,). Common utilization of the one-leg knee extensor exercise model has focused attention on the four heads of the quadriceps femoris muscle (Fig. 5), specifically, the vastus intermedius muscle (nearest to the bone) and the three superficial muscles, RF, vastus medialis (VM), and vastus lateralis (VL) muscles. In addition, other parts of the leg, such as the calf, and tissues such as adipose tissue, bone, and skin have been evaluated. As an index of blood flow heterogeneity (relative dispersion) within the muscles, the coefficient of variation of the voxel values of the defined regions are calculated as CV = SD/mean × 100%. Blood flow heterogeneity can be resolved down to voxel sizes of 2.6 × 2.6 × 2.4 mm, and thus, blood flow is measured in sample volumes of 16 mm. Heterogeneity among the quadriceps femoris muscle is calculated as previously mentioned, but instead of voxel values, the coefficient of variation of the separate blood flow values of its four heads is used. Thus, PET can precisely evaluate spatial blood flow heterogeneity, but as the frame sampling frequency is usually always >5 s and conditions and results represent steady-state situations, addressing the temporal heterogeneities of blood flow with high resolution in humans requires other methods such as Doppler ultrasound.
One of the most consistent findings regarding blood flow distribution in the thigh musculature, the most heavily assessed limb area in human studies, is that blood flow is often highest in the vastus intermedius muscle compared with the other three superficial muscles of the quadriceps femoris muscle (e.g.,), Fig. 5). This muscle is located deep within a muscle group and is in an anatomical position to resist gravity and is thus "designed" and responsible for body posture maintenance. It is generally regarded to consist mostly of Type I muscle fibers being highly oxidative and rich in capillaries, making it ideal for repetitive low-intensity muscle work. Therefore, it is natural that the highest blood flow is generally observed in this muscle, at least during low- to moderate-intensity exercise. However, the fact that O2 delivery (and also muscle V̇O2) is the highest in this muscle even at rest (i.e., not standing/moving) when the muscle is quiescent suggests, intriguingly, that the inherent metabolism of highly oxidative muscle (fibers) is greater than that of less oxidative muscles, comprised predominantly of Type II glycolytic fibers. This phenomenon raises questions regarding whether the differing degree of muscle fiber-type recruitment, or these inherent characteristics, represent the driving force controlling blood flow distribution and its changes among and within muscles in response to exercise.
Blood flow heterogeneity in the whole knee-extensor muscle group decreases with increasing workload: an effect that can be explained primarily by the decreased variability in mean blood flow values of the individual quadriceps femoris muscle with the increasing exercise intensity resulting disproportionately from a larger blood flow increase in VL muscle than that in the other muscles (toward the values observed in the three other muscles). This could reflect that VL muscle is recruited proportionally more in this exercise model when intensity is increased. Interestingly, VL muscle is also the only individual quadriceps femoris muscle in which heterogeneity decreases from the lowest to the highest exercise intensity. It has previously been shown that when exercise intensity is increased, blood flow is directed to newly recruited muscle fibers rather than to fibers already engaged in exercise, and this is the most likely reason for the decreased blood flow heterogeneity observed solely in VL muscle as well as for the decreased variability among the quadriceps femoris muscles. Moreover, it is pertinent that the hyperemia and blood flow distribution induced by drug infusion may not be similar to that induced by exercise, although mean blood flow can be similar among muscles. The current evidence suggests that the often-observed exercise-induced changes in blood flow heterogeneity probably reflect true changes in blood flow linked to muscle fiber recruitment and increased blood flow within defined vascular units.
To assess the coupling of muscle blood flow and V̇O2, these variables must be assessed independently. One powerful aspect of PET is that it permits direct measurement of blood flow, O2 extraction, and V̇O2in separatum in skeletal muscle using inhaled [O]-O2 tracer. The most suitable approach to measure muscle O2 extraction and V̇O2 at rest is the steady-state method because, when inhaled as a bolus, tissue time activity curves remain stable (initial rise followed by steady state) even after 5–6 min of inhalation because of the relatively low V̇O2. From a general physiological perspective, this finding supports that when O2 is extracted from the blood, it first binds to myoglobin rather than being consumed directly by the muscle. The steady-state method has revealed that when measured directly from thigh musculature, resting muscle O2 extraction approaches 60%. Thus, it is two to three times greater than is usually obtained from the determinations of femoral artery and mixed venous samples (20%–25%), meaning that O2 extraction is much lower in skin, adipose tissue, and bone than that in muscle itself. Moreover, although muscle V̇O2 is increased for a period after exhaustive exercise (the so-called excess postexercise O2 consumption), fractional O2 extraction is reduced, reflecting that blood flow decreases more slowly than V̇O2. This raises the likelihood that sustained the elevation of blood flow (and O2 delivery) postexercise, while raising microvascular and intramyocyte PO2, also subserves key functions of heat and metabolite removal.
Direct PET determinations confirm that fractional O2 extraction is enhanced in endurance-trained athletes compared with untrained subjects during exercise. Lower blood flow heterogeneity, potentially longer red blood cell (RBC) capillary transit times, overall improved vascular control, and increased muscle O2 diffusing capacity are thought to contribute to this finding. However, when blood flow and V̇O2 are examined at the individual muscle level, their relationship evinces substantial variability. Yet matching appears to be better in endurance athletes than that in untrained subjects. Indeed, in untrained subjects, muscle blood flow heterogeneity correlates positively with fractional O2 extraction (arterio-venous sampling over the limb) only at rest, but not during exercise. Moreover, blood flow heterogeneity does not correlate with the excess muscle V̇O2 (analogous to the V̇O2 slow component) during high-intensity exercise. In addition, when Richardson et al. investigated the coupling of muscle blood flow (with a resolution of 0.25–0.35 cm, image acquisition every 5–6 s) and V̇O2 (1–1.5 cm, 10 min) by MRI, they found that matching was far from ideal during submaximal exercise. These findings may be partially explained by the fact that, anatomically, microvascular units do not closely approximate muscle motor units. Consequently, to ensure blood flow to contracting muscle fibers during exercise, some vascular units (i.e., those abutting noncontracting nonrecruited muscle fibers) may be relatively overperfused. It also appears that this "luxurious" blood flow in relation to O2 demand may be especially prominent in untrained healthy subjects performing small muscle mass exercise. One potential benefit of this situation is that the high O2 delivery–V̇O2 ratio will elevate microvascular PO2 and, consequently, intramyocyte PO2, thereby enhancing metabolic control. Obviously, having the greatest microvascular PO2 possible in those capillaries in close proximity to the working muscle fibers would be expected to better facilitate this process.
In contrast to healthy individuals, patients with congestive heart failure have a low (and limiting) cardiac output, which is distributed according to pathophysiological constraints, such as preventing a catastrophic fall in blood pressure rather than regulating muscle blood flow and its distribution as in health. Patients with congestive heart failure may have high fractional O2 extractions, not because there is a superior matching of muscle O2 delivery–V̇O2 or a high O2 diffusing capacity but rather because muscle O2 delivery is very low relative to muscle V̇O2 requirements. In contrast to endurance training where blood flow distribution to highly oxidative fibers (muscles) may be improved (see previous section), aging redistributes available blood flow toward more fast glycolytic muscles at the expense of their more oxidative counterparts, at least in rats. It is worth noting that, as introduced in the Dynamic Heterogeneity of Exercising Muscle V̇O2 section, during maximal exercise, individuals with a higher V̇O2max demonstrate superior fractional O2 extractions according to the approximation:
where Δ CaO2–CvO2 is arterial–mixed venous O2 extraction (mL per 100 mL) and V̇O2 is V̇O2max in liters per minute. Thus, Δ CaO2–CvO2 increases as a hyperbolic function of V̇O2max (i.e., 13.3, 15, 16, and 16.7 mL per 100 mL for V̇O2max values of 2, 3, 4, and 5 L·min, respectively). The degree to which this is facilitated by reduced heterogeneity of O2 delivery–V̇O2 in the exercising muscles rather than improved diffusing capacity, for example, is unknown.
There are many, especially regulatory, aspects of blood flow heterogeneity, in addition to those identified earlier, that require further investigation. For instance, the differing degrees of α-adrenergic regulation and NO synthase contribution across different muscle fiber types demonstrated in experimental animals is not well explored in humans. Regarding α-adrenergic control, one indication for the possible differences in regulation among different human muscles stems from the finding that blood flow is consistently higher, and because perfusion pressure is similar in all muscles, resistance lower in the knee extensor muscles that are known to be more oxidative. This may be due to different α-adrenergic tone in different muscles as demonstrated in rats. However, a recent study with direct intra-arterial drug infusions of α-adrenergic agonist and antagonist drugs did not confirm this hypothesis in humans. Moreover, although it is evident from human and animal studies that NO plays a major role in regulating muscle blood flow at rest, human knee extensor studies suggest that NO is not as important during exercise in humans versus animals. In fact, the finding that NO synthesis inhibition reduces blood flow at rest, but not during exercise paradoxically, suggests that NO inhibition increases the absolute hyperemic response of exercise, making the comparison to animals about its relevance in fiber-type specific vasodilation challenging. However, this conclusion is tempered by the fact that a far more complete NO synthase blockade is possible in animals than humans. Finally, as previously mentioned, muscles in humans are not as defined with respect to solely oxidative or glycolytic fibers as clearly as seen in animals ( Table 2 ), which suggests that human muscle-specific differences are not as pronounced and easily detectable as shown in animals. In addition to these regulatory aspects, it would also be crucial in the future to separate inherent vascular/metabolic regulatory differences from those resulting from contrasting patterns of muscle activation/recruitment.
In summary, human studies indicate a marked heterogeneity among and within muscles with respect to blood flow, O2 delivery, and V̇O2. However, before parallels can be drawn to the animal data, heterogeneity due to varying recruitment patterns must be assessed carefully. Once this is done, it is likely that the less extreme stratification or "clumping" of fiber types in humans will mean that these differences are less pronounced than found in animals. Notwithstanding this, the regulatory aspects of muscle-specific blood flow and their applicability to animal findings remain largely uncharacterized in humans, presenting the opportunity for mechanistically valuable PET and MRI investigations in health and disease.
Muscle Blood Flow Heterogeneity as Assessed by PET
Studying muscle blood flow distribution and heterogeneity in humans has been challenging. Attempts to do so have used PET or MRI during small muscle mass exercise such as one leg knee extension. When PET is being used, O-H2O radiowater tracer, the autoradiographic method, and a one-compartment model have been applied to calculate blood flow voxel-by-voxel and portrayed as parametric blood flow images (Fig. 5). The method has been validated against the radiolabeled microsphere technique in dogs in vivo, which demonstrated high accuracy in measuring regional muscle blood flow.
(Enlarge Image)
Figure 5.
A typical PET blood flow image from the middle thigh region at rest and during one leg exercise of low- to moderate-intensity exercise. Only the quadriceps femoris muscle as a whole muscle group is illustrated as a region of interest for the sake of clarity. At rest, blood flow is generally highest (4–12 mL·[100 mL of muscle] ·min) in vastus intermedius muscle, which is the muscle closest to the bone (lowest perfusion region in the middle of the images) and generally regarded to consist mostly of type I muscle fibers, whereas blood flow is generally much lower (1–6 mL·[100 mL of muscle] ·min) in the three other superficial muscles of the knee extensors. During exercise, this blood flow distribution pattern is largely maintained, and in some areas of vastus intermedius muscle, blood flow can reach values close to 200 mL·[100 mL of muscle] ·min (red areas) even during low- to moderate-intensity exercise, but due to much lower perfusion in superficial muscles, the mean blood flow in whole knee extensors is hardly ever more than 50 mL·[100 mL of muscle]·min in this low-intensity exercise setting. Moreover, blood flow in hamstring muscles does not change from rest to one-leg knee extensor exercise, at least up to low- to moderate-intensity exercise. Altogether, blood flow is thus markedly heterogeneous within and among human skeletal muscles, especially during knee extensor exercise.
PET facilitates blood flow measurement in any organ or tissue (e.g.,). Common utilization of the one-leg knee extensor exercise model has focused attention on the four heads of the quadriceps femoris muscle (Fig. 5), specifically, the vastus intermedius muscle (nearest to the bone) and the three superficial muscles, RF, vastus medialis (VM), and vastus lateralis (VL) muscles. In addition, other parts of the leg, such as the calf, and tissues such as adipose tissue, bone, and skin have been evaluated. As an index of blood flow heterogeneity (relative dispersion) within the muscles, the coefficient of variation of the voxel values of the defined regions are calculated as CV = SD/mean × 100%. Blood flow heterogeneity can be resolved down to voxel sizes of 2.6 × 2.6 × 2.4 mm, and thus, blood flow is measured in sample volumes of 16 mm. Heterogeneity among the quadriceps femoris muscle is calculated as previously mentioned, but instead of voxel values, the coefficient of variation of the separate blood flow values of its four heads is used. Thus, PET can precisely evaluate spatial blood flow heterogeneity, but as the frame sampling frequency is usually always >5 s and conditions and results represent steady-state situations, addressing the temporal heterogeneities of blood flow with high resolution in humans requires other methods such as Doppler ultrasound.
Human Muscle Blood Flow Heterogeneity at Rest and During Exercise
One of the most consistent findings regarding blood flow distribution in the thigh musculature, the most heavily assessed limb area in human studies, is that blood flow is often highest in the vastus intermedius muscle compared with the other three superficial muscles of the quadriceps femoris muscle (e.g.,), Fig. 5). This muscle is located deep within a muscle group and is in an anatomical position to resist gravity and is thus "designed" and responsible for body posture maintenance. It is generally regarded to consist mostly of Type I muscle fibers being highly oxidative and rich in capillaries, making it ideal for repetitive low-intensity muscle work. Therefore, it is natural that the highest blood flow is generally observed in this muscle, at least during low- to moderate-intensity exercise. However, the fact that O2 delivery (and also muscle V̇O2) is the highest in this muscle even at rest (i.e., not standing/moving) when the muscle is quiescent suggests, intriguingly, that the inherent metabolism of highly oxidative muscle (fibers) is greater than that of less oxidative muscles, comprised predominantly of Type II glycolytic fibers. This phenomenon raises questions regarding whether the differing degree of muscle fiber-type recruitment, or these inherent characteristics, represent the driving force controlling blood flow distribution and its changes among and within muscles in response to exercise.
Blood flow heterogeneity in the whole knee-extensor muscle group decreases with increasing workload: an effect that can be explained primarily by the decreased variability in mean blood flow values of the individual quadriceps femoris muscle with the increasing exercise intensity resulting disproportionately from a larger blood flow increase in VL muscle than that in the other muscles (toward the values observed in the three other muscles). This could reflect that VL muscle is recruited proportionally more in this exercise model when intensity is increased. Interestingly, VL muscle is also the only individual quadriceps femoris muscle in which heterogeneity decreases from the lowest to the highest exercise intensity. It has previously been shown that when exercise intensity is increased, blood flow is directed to newly recruited muscle fibers rather than to fibers already engaged in exercise, and this is the most likely reason for the decreased blood flow heterogeneity observed solely in VL muscle as well as for the decreased variability among the quadriceps femoris muscles. Moreover, it is pertinent that the hyperemia and blood flow distribution induced by drug infusion may not be similar to that induced by exercise, although mean blood flow can be similar among muscles. The current evidence suggests that the often-observed exercise-induced changes in blood flow heterogeneity probably reflect true changes in blood flow linked to muscle fiber recruitment and increased blood flow within defined vascular units.
Matching of Muscle Blood Flow to V̇O2 at Rest and During Exercise in Humans
To assess the coupling of muscle blood flow and V̇O2, these variables must be assessed independently. One powerful aspect of PET is that it permits direct measurement of blood flow, O2 extraction, and V̇O2in separatum in skeletal muscle using inhaled [O]-O2 tracer. The most suitable approach to measure muscle O2 extraction and V̇O2 at rest is the steady-state method because, when inhaled as a bolus, tissue time activity curves remain stable (initial rise followed by steady state) even after 5–6 min of inhalation because of the relatively low V̇O2. From a general physiological perspective, this finding supports that when O2 is extracted from the blood, it first binds to myoglobin rather than being consumed directly by the muscle. The steady-state method has revealed that when measured directly from thigh musculature, resting muscle O2 extraction approaches 60%. Thus, it is two to three times greater than is usually obtained from the determinations of femoral artery and mixed venous samples (20%–25%), meaning that O2 extraction is much lower in skin, adipose tissue, and bone than that in muscle itself. Moreover, although muscle V̇O2 is increased for a period after exhaustive exercise (the so-called excess postexercise O2 consumption), fractional O2 extraction is reduced, reflecting that blood flow decreases more slowly than V̇O2. This raises the likelihood that sustained the elevation of blood flow (and O2 delivery) postexercise, while raising microvascular and intramyocyte PO2, also subserves key functions of heat and metabolite removal.
Direct PET determinations confirm that fractional O2 extraction is enhanced in endurance-trained athletes compared with untrained subjects during exercise. Lower blood flow heterogeneity, potentially longer red blood cell (RBC) capillary transit times, overall improved vascular control, and increased muscle O2 diffusing capacity are thought to contribute to this finding. However, when blood flow and V̇O2 are examined at the individual muscle level, their relationship evinces substantial variability. Yet matching appears to be better in endurance athletes than that in untrained subjects. Indeed, in untrained subjects, muscle blood flow heterogeneity correlates positively with fractional O2 extraction (arterio-venous sampling over the limb) only at rest, but not during exercise. Moreover, blood flow heterogeneity does not correlate with the excess muscle V̇O2 (analogous to the V̇O2 slow component) during high-intensity exercise. In addition, when Richardson et al. investigated the coupling of muscle blood flow (with a resolution of 0.25–0.35 cm, image acquisition every 5–6 s) and V̇O2 (1–1.5 cm, 10 min) by MRI, they found that matching was far from ideal during submaximal exercise. These findings may be partially explained by the fact that, anatomically, microvascular units do not closely approximate muscle motor units. Consequently, to ensure blood flow to contracting muscle fibers during exercise, some vascular units (i.e., those abutting noncontracting nonrecruited muscle fibers) may be relatively overperfused. It also appears that this "luxurious" blood flow in relation to O2 demand may be especially prominent in untrained healthy subjects performing small muscle mass exercise. One potential benefit of this situation is that the high O2 delivery–V̇O2 ratio will elevate microvascular PO2 and, consequently, intramyocyte PO2, thereby enhancing metabolic control. Obviously, having the greatest microvascular PO2 possible in those capillaries in close proximity to the working muscle fibers would be expected to better facilitate this process.
In contrast to healthy individuals, patients with congestive heart failure have a low (and limiting) cardiac output, which is distributed according to pathophysiological constraints, such as preventing a catastrophic fall in blood pressure rather than regulating muscle blood flow and its distribution as in health. Patients with congestive heart failure may have high fractional O2 extractions, not because there is a superior matching of muscle O2 delivery–V̇O2 or a high O2 diffusing capacity but rather because muscle O2 delivery is very low relative to muscle V̇O2 requirements. In contrast to endurance training where blood flow distribution to highly oxidative fibers (muscles) may be improved (see previous section), aging redistributes available blood flow toward more fast glycolytic muscles at the expense of their more oxidative counterparts, at least in rats. It is worth noting that, as introduced in the Dynamic Heterogeneity of Exercising Muscle V̇O2 section, during maximal exercise, individuals with a higher V̇O2max demonstrate superior fractional O2 extractions according to the approximation:
where Δ CaO2–CvO2 is arterial–mixed venous O2 extraction (mL per 100 mL) and V̇O2 is V̇O2max in liters per minute. Thus, Δ CaO2–CvO2 increases as a hyperbolic function of V̇O2max (i.e., 13.3, 15, 16, and 16.7 mL per 100 mL for V̇O2max values of 2, 3, 4, and 5 L·min, respectively). The degree to which this is facilitated by reduced heterogeneity of O2 delivery–V̇O2 in the exercising muscles rather than improved diffusing capacity, for example, is unknown.
Regulatory Aspects of Blood Flow and Its Heterogeneity in Humans
There are many, especially regulatory, aspects of blood flow heterogeneity, in addition to those identified earlier, that require further investigation. For instance, the differing degrees of α-adrenergic regulation and NO synthase contribution across different muscle fiber types demonstrated in experimental animals is not well explored in humans. Regarding α-adrenergic control, one indication for the possible differences in regulation among different human muscles stems from the finding that blood flow is consistently higher, and because perfusion pressure is similar in all muscles, resistance lower in the knee extensor muscles that are known to be more oxidative. This may be due to different α-adrenergic tone in different muscles as demonstrated in rats. However, a recent study with direct intra-arterial drug infusions of α-adrenergic agonist and antagonist drugs did not confirm this hypothesis in humans. Moreover, although it is evident from human and animal studies that NO plays a major role in regulating muscle blood flow at rest, human knee extensor studies suggest that NO is not as important during exercise in humans versus animals. In fact, the finding that NO synthesis inhibition reduces blood flow at rest, but not during exercise paradoxically, suggests that NO inhibition increases the absolute hyperemic response of exercise, making the comparison to animals about its relevance in fiber-type specific vasodilation challenging. However, this conclusion is tempered by the fact that a far more complete NO synthase blockade is possible in animals than humans. Finally, as previously mentioned, muscles in humans are not as defined with respect to solely oxidative or glycolytic fibers as clearly as seen in animals ( Table 2 ), which suggests that human muscle-specific differences are not as pronounced and easily detectable as shown in animals. In addition to these regulatory aspects, it would also be crucial in the future to separate inherent vascular/metabolic regulatory differences from those resulting from contrasting patterns of muscle activation/recruitment.
Summary
In summary, human studies indicate a marked heterogeneity among and within muscles with respect to blood flow, O2 delivery, and V̇O2. However, before parallels can be drawn to the animal data, heterogeneity due to varying recruitment patterns must be assessed carefully. Once this is done, it is likely that the less extreme stratification or "clumping" of fiber types in humans will mean that these differences are less pronounced than found in animals. Notwithstanding this, the regulatory aspects of muscle-specific blood flow and their applicability to animal findings remain largely uncharacterized in humans, presenting the opportunity for mechanistically valuable PET and MRI investigations in health and disease.
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