Peripheral limitations for performance: Muscle capillarization

Sufficient delivery of oxygen and metabolic substrates, together with removal of waste products, are key elements of muscle performance. Capillaries are the primary site for this exchange in skeletal muscle and the degree of muscle capillarization affects diffusion conditions by influencing mean transit time, capillary surface area and diffusion distance. Muscle capillarization may thus represent a limiting factor for performance. Exercise training increases the number of capillaries per muscle fiber by about 10%–20% within a few weeks in untrained subjects, whereas capillary growth progresses more slowly in well‐trained endurance athletes. Studies show that capillaries are tortuous, situated along and across the length of the fibers with an arrangement related to muscle fascicles. Although direct data is lacking, it is possible that years of training not only enhances capillary density but also optimizes the positioning of capillaries, to further improve the diffusion conditions. Muscle capillarization has been shown to increase oxygen extraction during exercise in humans, but direct evidence for a causal link between increased muscle capillarization and performance is scarce. This review covers current knowledge on the implications of muscle capillarization for oxygen and glucose uptake as well as performance. A brief overview of the process of capillary growth and of physical factors, inherent to exercise, which promote angiogenesis, provides the foundation for a discussion on how different training modalities may influence muscle capillary growth. Finally, we identify three areas for future research on the role of capillarization for exercise performance.

gradient, the area for diffusion, mean transit time and the diffusion distance between the capillary and the mitochondria.Consequently, an increase in the number of capillaries in the muscle as well as an enhanced mitochondrial capacity are beneficial adaptation to endurance training.For glucose delivery and removal of metabolic waste products such as lactate, diffusion limitations are similar to those of gas exchange, although a major difference is that nutrients are not membrane permeable, unlike oxygen and carbon dioxide.Capillaries are composed of a single layer of endothelial cells, structurally strengthened by a basement membrane and by pericytes.The thin capillary wall allows for optimal diffusion conditions for oxygen and other compounds.The capillary endothelium includes small gaps between the endothelial cells, allowing for diffusion of larger, nonpermeable compounds.Both the architecture and size of the capillary network surrounding the skeletal muscle fibers are likely to have direct implications for exchange of oxygen and substrates and thus, ultimately performance.This review summarizes existing evidence on the importance of the skeletal muscle capillary network for performance.Based on current knowledge of the functional implications of capillaries, how they adapt to the level of muscle activity, combined with data from animal and human models of capillary exchange limitations, it is discussed to what extent the capillary network is a limiting factor for elite performance.Moreover, evidence from longitudinal exercise training studies, as well as from cross sectional studies, including studies on top athletes, is discussed to present the current position and provide future directions.

CAPILLARY NETWORK
The capillary network in skeletal muscle has traditionally been described as a group of parallel-flowing capillaries that originate from a terminal arteriole and that drain into a venule, known as a capillary module or capillary unit.In 2021, this paradigm was updated by Asher Mendelson, Christopher Ellis and coworkers with the description of so-called capillary fascicles, with capillaries organized in relation to the skeletal muscle fascicle structure. 4The capillary fascicle structure consists of largely parallel capillary units which are connected and thereby extend along the length of the muscle fiber. 4nterestingly, the pressure difference over the capillary fascicles was reported to be as little as ~3 mmHg and there was a substantial functional as well as structural heterogeneity for red blood cell haemodynamics. 4,5The low pressure gradient over the length of the capillary also implies that microvascular pressures are important and very precisely regulated.On average, the typical length of capillaries is 500-1000 um 6 and the diameter of capillaries are on average ~ 5 um 7 but can be as small as 3 um. 8The small diameter of capillaries means that erythrocytes with a diameter of about 7-8 um in size must be highly flexible to pass through the capillary lumen, but the advantage of the narrow lumen is that diffusion distance to the capillary wall is minimal.
An increase in the capillary density can improve diffusion conditions for molecules from the blood to tissue by affecting diffusion distance, capillary surface area for diffusion and the mean transit time in the capillary bed, that is, the time it takes for a red blood cell carrying oxygen to pass from one end of a capillary to the other.Mean transit time is dictated by the velocity of blood and, for the same volume flow of blood, the velocity is reduced with a larger cross sectional area of the capillary bed through a higher number of parallel capillaries (Figure 1).
In a cross section of the skeletal muscle, the number of capillaries can be determined as number of capillaries per muscle fiber (capillary to fiber ratio) or as capillaries per section area expressed in mm 2 (capillary density).In healthy untrained subjects, there are typically 1.3-1.8capillaries per muscle fiber and in endurance athletes this number can reach ~3-3.5. 9The magnitude of capillary density is in the range of ~300-600 capillaries per mm 2 in sedentary to endurance athletes.These measures are, however, dependent on how the muscle sample is treated and stained; the mode of staining and counting of capillaries as well as tissue fixation and cutting of the samples, where the latter two influence the true cross sectional area and thus will affect the calculated capillarization. 10While capillary to fiber ratio is a useful indicator of growth of the capillary network over time, capillary density is considered to be a better indicator of the diffusion conditions for oxygen in a muscle sample.This follows as more capillaries per muscle cross sectional area lowers the average diffusion distance from capillary lumen to muscle mitochondria.Mathematical modeling furthermore suggests that the position of capillaries in a muscle influences oxygen diffusion. 11ross sectional quantification of capillarization is the most feasible method for the determination of capillarization in a muscle, but it is important to recognize that capillaries are not straight tubes but rather tortuous, which means that they also are oriented more perpendicular to the muscle fiber.At the physiological level, capillary tortuosity has important implications for the exchange surface area but at the methodological level, it raises the question of whether the common determination of two-dimensional capillarization provides the full picture of diffusion capacity in muscle.Both metrics of capillary density and capillary to fiber ratio, derived from cross sectional muscle sections, are somewhat limited measures of diffusion capacity in that these measures are insensitive to capillary tortuosity. 12It should also be noted that the current knowledge about the arrangement of capillaries is mostly derived from animal research.Both the concept of capillary fascicles and the tortuous arrangement of capillaries are yet to be thoroughly confirmed in human skeletal muscle tissue but there is no reason to expect the structural arrangement to be different in human skeletal muscle.Methods for the determination of three dimensional capillarization and capillary volume are emerging and may provide valuable for such confirmations.

| IMPORTANCE OF CAPILLARY DENSITY FOR OXYGEN UPTAKE AND PERFORMANCE
In theory, a greater capillary network in skeletal muscle would result in improved diffusion conditions for both oxygen and nutrients, but direct in vivo evidence for this relationship is weak.Nevertheless, there is indirect evidence for a coupling between oxidative metabolism and F I G U R E 1 Illustration of the impact of exercise training on skeletal muscle capillarization and performance.In previously untrained subjects, weeks of training leads to increased number of capillaries in the skeletal muscle which in turn increases red blood cell mean transit time and the area for diffusion while decreasing the diffusion distance from red blood cell to myocyte.As a result, oxygen extraction and glucose uptake are improved leading to the ability to maintain a higher percentage of maximal oxygen uptake for a longer period of time.In addition, handling of metabolic waste products like lactate are improved.In already well-trained individuals, weeks of training is not found to change capillarization but after years of training, capillary number and distribution is improved.Years of training may also lead to improved capillary organization within the muscle, effectively improving diffusion condition potentially without changing capillary number.Along with mitochondrial capacity, these are peripheral factors that may be limiting for performance and that should be considered along with maximal cardiac output and gross efficiency.capillarization.For example, studies in skeletal muscle and cardiac muscle of different animals show that capillary volume density correlates well with mitochondrial volume. 13Data on men and women of varying ages, reveal a relationship between maximal aerobic capacity and capillary density, although, the relationship is moderate. 14,15 moderate rather than strong relationship between these variables is, nevertheless, to be expected as cardiac output, rather than oxygen extraction, is the primary determinant of maximal oxygen uptake. 2,3,16A determining factor in endurance performance is also the ability to perform at a high percentage of VO 2 max. 17Although, direct evidence for the role of capillarization for this ability is lacking, Coyle et al. reported a relationship between capillary density and time to fatigue at 88% of VO 2 max in competitive cyclist with a fitness level close to 70 mL/min/ kg. 18Moreover, Mitchell et al. 2018 reported an association between capillary density and critical power also in competitive cyclist and triathletes. 19These observations suggest that capillarization may be of importance also for aerobic capacity.
Exercise training interventions can lead to parallel increases in capillarization and oxygen extraction thereby providing an indication of the role of capillarization in skeletal muscle. 20However, as endurance training can influence many peripheral properties such as mitochondrial capacity, the sole effect of increased capillarization per se cannot be deduced from such interventions.This was illustrated in a study by Skattebo et al. 2020 that showed that the enhanced oxygen extraction after one leg training correlated with markers of mitochondrial capacity (citrate synthase and COX-IV) but not with the capillary to fiber ratio. 21ne study has attempted to more directly determine the role of capillaries for oxygen extraction in skeletal muscle by elevating the level of capillarization in young healthy men by treatment with an αadrenergic receptor blocker, terazosin. 22The study showed that an increased capillarization, induced by 4 weeks of treatment, was associated with an increase in oxygen extraction, determined as the femoral arterio-venous difference of oxygen during exercise.The finding was paralleled by a lowering of blood flow in accordance with unaltered muscle oxygen demand during the exercise.However, the change in capillarization and oxygen extraction did not influence peak workload, maximal oxygen uptake during cycling or peak workload during knee extensor exercise. 22part from its role for oxidative capacity, muscle capillarization can be of importance for more high intensity exercise performance, as indicated by an increase in capillaries with electrostimulation of glycolytic muscle fibers in rodents 23 and by a relationship between capillary density and performance at 380 watts of cycling. 24The role of capillarization for performance at higher intensities may be related to removal of metabolites such as lactate and during high intensity exercise of intermittent nature like soccer, improved peripheral oxygen diffusion capacity may also be important for metabolic recovery between bouts.

| Capillary density and importance for glucose uptake
Evidence for the role of skeletal muscle capillarization for glucose uptake is emerging.Capillaries may impact muscle glucose uptake by affecting both glucose and insulin transport from the blood to the muscle.6][27] With regard to insulin, due to the relatively large size of the insulin molecule, transit from capillaries to the muscle likely occurs via vesicular trafficking through the endothelial cells rather than through the junctions between the endothelial cells. 28Specifically, how this trafficking occurs remains unclear and overall findings are both scarce and divergent, however it is likely that a greater capillary surface area would enhance the possibility for insulin trafficking. 28To date, no studies have specifically assessed the impact of muscle capillary density on glucose uptake in the context of endurance performance.It is well known that endurance trained individuals have faster and larger resynthesis of glycogen after exercise, 29 where one of the contributing factors to the glucose uptake could be the greater capillarization.Assessments of glucose uptake during exercise indicate that glucose uptake is higher in trained than untrained individuals at high workloads. 30However, a causal relationship has not been established in athletes.Further indirect evidence for the role of capillaries for glucose uptake stems from a relationship between insulin resistance and the level of capillarization, where cross sectional data indicate that insulin resistant individuals have lower levels of capillarization compared to healthy individuals. 31,32In animals, genetic modifications of mice to knock down the important capillary growth factor vascular endothelial growth factor (VEGF) in muscle tissue, have demonstrated that a consequent 50%-60% lower capillary density is associated with a significantly reduced peripheral glucose uptake. 33Moreover, treatment of rats with the αadrenergic receptor blocker prazosin for 3 weeks, leading to an approximate 20% increase in muscle capillarization was paralleled by an ~25% increase in whole body insulin sensitivity, determined as a hyperinsulemic euglycemic clamp and a 30% increase in glucose disposal. 34Of note, these studies reported that muscle insulin signaling was not influenced by the prazosin treatment, indicating a direct role of a change in capillarization.Although endurance training is well known to influence insulin sensitivity of skeletal muscle in parallel with increased capillarization, the role of capillarization per se cannot be discerned due to the many other training induced changes related to glucose uptake, such as an increase in glucose transporters. 35

MUSCLE CAPILLARIES GROW WITH TRAINING?
Since the early observations by Per Andersen at the August Krogh Institute in Copenhagen (1975) 36 of adaptations to the capillary bed after a period of exercise training, and concurrent work by Per Brodal and colleagues in Oslo, describing higher capillarization in endurance trained subjects compared to untrained, 37 it has been well-established that exercise training induces growth of new capillaries in human skeletal muscle. 99][40] The importance of mechanical factors for skeletal muscle capillary growth has since been confirmed in humans. 22,41In addition, given the relatively limited increase in capillarization induced by mechanical stimulation alone in humans, 22,41 it is likely that a component of muscle contraction and/or metabolism add an additional stimulus to the mechanical factors, to increase capillary growth.
Growth of new capillaries from existing capillaries, termed angiogenesis, can occur either by sprouting, where a capillary grows out of an existing capillary or longitudinal splitting where the capillary splits into two, originally parallel, capillaries. 38,39Both forms of capillary growth are tightly regulated processes involving several pro-and antiangiogenic factors where mechanical and metabolic signals affect their expression and release.Studies in animals show that shear stress primarily induces longitudinal splitting whereas passive muscle stretch induces sprouting. 39A key factor for both splitting and sprouting angiogenesis in skeletal muscle is VEGF. 40,42VEGF is present in many cell types in muscle tissue, including endothelial cells and pericytes, but skeletal muscle fibers appear to be the largest and most important source. 434][45] Once released to the extracellular space, VEGF acts on the VEGF receptor 2 on the capillary endothelial cells and promotes angiogenesis. 46,47lthough important, VEGF is one of a large number of angiogenic factors that have been described to participate in skeletal muscle angiogenesis.For a detailed description the reader is referred to comprehensive overviews which cover several of these pro-and antiangiogenic factors. 10,46,48

| ACUTE, CHRONIC, AND LONG-TERM TRAINING-INDUCED CAPILLARY GROWTH
0][51] The transient upregulation of both positive and negative angiogenic regulators is an indication of well-regulated growth which serves to secure that growth occurs in a tissue only if and where required.In rodents, it is possible to detect signs of division of endothelial cells and capillary growth within the first few days of electrical stimulation or exercise training. 524][55] A recent meta-analysis of 47 controlled exercise intervention trials, showed that a mean difference of 0.33 (95% CI 0.30-0.37)capillaries per muscle fiber can be expected after a training intervention of more than 2 weeks in previously untrained subjects. 9][58][59][60][61][62][63] However, it should be noted that there is a limited number of studies on adaptations to training in already trained individuals and longitudinal data on how years of training progressively affects skeletal muscle capillarization is, to our knowledge, completely lacking.From cross sectional studies it is however evident that continued regular training over many years leads to an expansion of the capillary network compared to what can be seen in short term training interventions.On average, the trained individuals included in the meta-analysis have a capillary to fiber ratio of 2.74 (95% CI, 2.61-2.86)compared to 1.70 (95% CI, 1.68-1.72) in untrained subjects. 95][66] In amateur cyclist, international level cyclist and team pursuit riders, capillary to fiber ratio have been reported to be 2.4 ± 0.3, 2.9 ± 0.3 and 3.2 ± 0.5, respectively. 66Elite road cyclist, studied at the age of 25, were found to have 35% higher capillarization compared to their 4-year younger elite competitors 67 suggesting that the capillary network continues to adapt to training, even in very well-trained subjects.To what extent training also induces significant changes to capillary positioning in relation to the muscle fibers and to their tortuous arrangement is difficult to study in humans but there are indications 68 and, from a physiological perspective, these are expected adaptations to exercise training.Technical developments, including 3-dimensional assessments, for example, in fluorescently stained samples or potentially new development of in vivo methods as well as more extensive use of modeling, is likely to further our understanding in this area. 69n summary, muscle capillary net expansion occurs rapidly with regular endurance training in untrained individuals and continues to increase at a slower pace if training is continued for many years, although levels are unlikely to exceed 3.5 capillaries per muscle fiber in most subjects.

| WHAT IS THE BEST TYPE OF TRAINING TO INDUCE CAPILLARY GROWTH?
On the assumption that capillarization is a limiting factor for performance, it is obviously important to know what training modality that is most effective in improving muscle capillarization.Currently, no studies have attempted to determine the most optimal type of training for capillary growth but meta-analysis of training studies reporting changes in muscle capillarity after well-controlled training interventions, provides some insight.Such analysis suggested that training intensity is the most important factor for inducing capillary growth and less so is training duration or number of training sessions 9 This notion fits well with the knowledge that mechanical factors, in particular shear stress, and metabolism, which are increased with increasing exercise intensity, are key effectors for capillary growth.However, intensity is a relative term and there appears to be a Janus-faced role of high intensity exercise in relation to capillary growth. 702][73] This discrepancy clearly points at a need for controlled training studies investigating the impact of various training forms on capillary growth in already well-trained subjects.When comparing the angiogenic response to moderate intensity, high volume cycling, to low volume high intensity interval cycling and sprint cycling, the angiogenic stimuli, measured as changes in proangiogenic mRNA response, was greatest in the high shear stress and high metabolic perturbations conditions. 74owever, in an attempt to examine whether a shift from 4 weeks of moderate training to an additional 4 weeks with more intense training, would potentiate muscle capillary growth, no such effect was observed, 72 and in already well trained soccer players and endurance runners, the shift to lower volume and higher intensity resulted in reduced angiogenesis. 59,73Thus, with the current body of evidence it is not possible to provide solid recommendations for which type of training that is most efficient in inducing capillary growth.

| FINAL CONSIDERATIONS - TIME TO UPDATE OUR UNDERSTANDING OF OXYGEN DIFFUSION CONDITIONS DURING DYNAMIC EXERCISE
Given that a causal link between muscle capillarization and performance is still lacking there is a need for a critical look on the way we understand limitations to oxygen delivery during exercise.Based on electron microscopy studies, Sullivan and Pittman (1987) suggested that oxygen supply to an individual muscle fiber should take into account the percent of the muscle fiber perimeter in contact with a capillary. 70Interestingly, however Mathieu-Costello later reported that, although capillary perimeter in transverse sections increases when the muscle contracts, muscle fiber perimeter is similarly increased, leaving the ratio between the two, close to unaltered. 72Capillaries are mainly arranged along the length but also perpendicular to the muscle fibers and by being tortuously arranged they can accommodate the variation in fiber length during contraction cycles. 7This also means that capillary tortuosity and thus exchange area, as well as diffusion distance due to the change in fiber cross sectional area, continuously change as the muscle fibers contract and relax. 12Moreover, intramuscular pressure increases dramatically (>100 mmHg) during muscle contraction, 75 which partly, or completely, blocks capillary perfusion until the relaxation phase.During dynamic exercise like running and cycling, skeletal muscle diffusion conditions are therefore extremely dynamic and we have to recognize that even the best model of 3D imaging is not likely to be able to evaluate to true diffusion conditions during exercise or how these are affected by training status.
| CONCLUSION AND PERSPECTIVES Direct evidence for an isolated role of capillarization for performance is lacking.However, muscle capillary density influences diffusion conditions for oxygen as well as glucose, thus an increase in the capillary network is likely to be a beneficial adaptation to endurance exercise, in parallel with an increase in cardiac output and enhanced mitochondrial capacity.
Based on the present overview of the current knowledge on muscle capillarization and performance we have identified three issues that is to be addressed in future research projects.i) To ascertain to what extent skeletal muscle capillarization is a limiting factor for endurance performance, there is a need for cleverly designed interventions that can manipulate skeletal muscle capillarization without also inducing other training adaptations to the heart, the blood, and the muscle.This can be done with pharmacological interventions that increase shear stress but how this compares to an integrative angiogenic response to hemodynamic and metabolic disturbances is unknown.ii) Methods to study the tortuous arrangement of the capillaries along the muscle fibers as well as the position of capillaries in the muscle are emerging and while this is difficult to study in humans, capillary arrangement might be an important factor for diffusion.iii) The long term temporal response of training induced capillary growth is still largely unknown and there is a need for intervention studies with serial muscle biopsies over months and years of training.Moreover, which training modality that is the most efficient for inducing capillary adaptations remains unclear and there is a need for studies comparing the effects of various training interventions.
While the focus of this paper has been on the importance of capillarization for endurance performance.It would be very interesting to study how improved diffusion conditions in the skeletal muscle affects sports of a more intermittent nature, like soccer, where the ability to recover in between bouts is of greatest importance to performance.