Olga Hudlicka

The effects of different patterns of muscle activity on capillary density, mechanical properties and structure of slow and fast rabbit muscles.

SummaryWhen rabbit fast muscles were chronically stimulated at a frequency naturally occurring in nerves to slow muscles (10 Hz), there was a transformation towards a slow muscle type such as an increase of capillary density, increased activity of the oxidative enzyme, succinic dehydrogenase, and a decrease of muscle fibre diameters. After 28 days the intensity and distribution of SDH and the capillary density were similar to those of soleus.The increases in capillary density preceded the changes in activity of SDH; there was a significantly greater capillary/muscle fibre ratio and number of capillaries/mm2 in muscles stimulated for only 4 days at which time no change could be detected in SDH. These changes were induced by slow frequency stimulation only, and not by an overall increase of activity.Stimulation of fast muscles for 4 days at a higher frequency naturally occurring in the nerves to fast muscles (short bursts of tetani), with the same total number of stimuli as that used in slow frequency stimulation did not produce any changes in capillary density, activity of SDH or contraction times. No changes were observed in either fast or slow muscles stimulated with short bursts of tetani (and lower total number of impulses) up to 28 days.Activation of fast muscles at 5 Hz continuously or 10 Hz intermittently also caused an increase in capillary density.It is therefore concluded that only low frequency activation of fast muscles brings about a transformation of the muscle fibres towards a slow type and that the first noticeable change is an increase in the capillary density.

Modulation of physiological angiogenesis in skeletal muscle by mechanical forces: Involvement of VEGF and metalloproteinases

Growth factors are involved in physiological angiogenesis in female reproductive organs but their role in capillary growth in skeletal muscles during activity or exercise training is not proven. Evidence suggests that increases in muscle blood flow and accompanying capillary shear stress and/or wall tension, or mechanical stress due to sarcomere length changes during contraction/relaxation cycles are closely linked with angiogenesis. Time-dependent studies of rat muscles in models with increased shear stress (chronic vasodilator treatment with α1 antagonist prazosin), altered sarcomere length (stretch-induced overload with no increase in blood flow), or both (chronic electrical muscle stimulation) showed a similar increase in capillary supply in all models but by different modes of growth. With prazosin, it occurred by intra-luminal splitting of vessels, with stretch by abluminal sprouting, and in stimulated muscles by both methods. Whole muscle matrix metalloproteinase-2 (MMP-2) was elevated during sprouting growth induced by extravascular tensile forces but not during splitting growth induced by shear. Vascular endothelial growth factor (VEGF) protein was elevated at capillary sites in all three models but with different time courses. With shear as the stimulus, the increase occurred early although there was little capillary proliferation; it matched the rise in proliferation in stretched muscles but lagged behind proliferation in stimulated muscles. Mechanical forces therefore influence MMP and VEGF expression and capillary growth patterns in skeletal muscle differentially depending upon whether they act intra- or ab-luminally. In exercise-trained muscles, the type of capillary growth remains to be determined but the most likely stimuli for angiogenesis are increased blood flow and shear forces to vessel supplying the active fibres, probably linked with metabolic factors.

Unorthodox angiogenesis in skeletal muscle

OBJECTIVE The morphological pattern of angiogenesis occurring in mature, differentiated skeletal muscle in response to chronically increased muscle blood flow, muscle stretch or repetitious muscle contractions was examined to determine (a) whether capillary neoformation follows the generally accepted temporal paradigm, and (b) how the growth pattern is influenced by mechanical stimuli. METHODS Adult rats were treated for a maximum of 14 days either with the vasodilator prazosin, to elevate skeletal muscle blood flow, or underwent surgical removal of one ankle flexor, to induce compensatory overload in the remaining muscles, or had muscles chronically stimulated by implanted electrodes. Extensor digitorum longus and/or extensor hallucis proprius muscles were removed at intervals and processed for electron microscopy. A systematic examination of capillaries and their ultrastructure characterised the sequence of morphological changes indicative of angiogenesis, i.e., basement membrane disruption, endothelial cell (EC) sprouting and proliferation [immunogold labelling after bromodeoxyuridine (BrdU) incorporation]. RESULTS Capillary growth in response to increased blood flow occurred by luminal division without sprouting or basement membrane (BM) breakage. In stretched muscles, EC proliferation and abluminal sprouting gave rise to new capillaries, with BM loss only at sprout tips. These distinct mechanisms appear to be additive as in chronically stimulated muscles (increased blood flow with repetitive stretch and shortening during muscle contractions) both forms of capillary growth occurred. Endothelial cell numbers per capillary profile, mitotic EC nuclei, and BrdU labelling confirmed cell proliferation prior to overt angiogenesis. CONCLUSIONS Physiological angiogenesis within adult skeletal muscle progresses by mechanisms that do not readily conform to the consensus view of capillary growth, derived mainly from observations made during development, pathological vessel growth, or from in vitro systems. The temporal and spatial pattern of growth is determined by the polarity of the mechanical stimulus, i.e., by intra-luminal (increased shear stress) or abluminal (external stretch) stimuli.

Association between Shear Stress, Angiogenesis, and VEGF in Skeletal Muscles In Vivo

Objective: To investigate the hypothesis that capillary proliferation in skeletal muscles, induced by a long‐term increase in blood flow which elevates capillary shear stress, is associated with capillary expression of vascular endothelial growth factor (VEGF).

The effect of long-term stimulation of fast muscles on their blood flow, metabolism and ability to withstand fatigue.

SummaryChronic stimulation of fast rabbit muscles (tibialis anterior, extensor digitorum longus and the peroneal muscle group) at a frequency naturally occurring in nerves to slow muscles increased their ability to withstand fatigue. Isometric tension decreased during a 10-min period of contractions at 4 Hz by 75% in control muscles, but only 55% in muscles chronically stimulated for 4 days, and 23% in muscles stimulated for 28 days.Chronic stimulation had little effect on resting blood flow, oxygen or glucose consumption. The output or consumption of lactate and free fatty acids (FFA) at rest were also unaffected. The glycogen content was regularly increased, and was apparent after only 2 days of stimulation. The activity of fatty acid activating enzyme was increased after 28 days.During a 10-min period of isometric contractions at 4 Hz, there was a markedly greater increase in blood flow and oxygen consumption in muscles stimulated for 14–28 days than in control muscles; lactic acid output was lower in muscles stimulated for 28 days, and the uptake of FFA was significantly higher. It is therefore suggested that muscles chronically stimulated for 14–28 days use fats as the main source of energy during isometric contractions. The predominantly oxidative metabolism is probably facilitated by the higher density of capillaries. The latter also enables more efficient delivery of oxygen, and therefore smaller fatiguability, already after 4 days of chronic stimulation.

Capillary growth in chronically stimulated adult skeletal muscle as studied by intravital microscopy and histological methods in rabbits and rats.

Abstract Rabbit tenuissimus and rat extensor hallucis proprius (EHP) muscles were stimulated, via implanted stainless steel electrodes, at 10 Hz, 8 hr a day for 7 to 14 days. After stimulation the dimensions of the microvascular bed were measured by intravital microscopy. The contralateral normal muscles or nonstimulated muscles with implanted electrodes served as controls. All the stimulated muscles showed an increased capillary diameter. This increase was most prominent in the venular end of the capillaries in the tenuissimus muscles stimulated for 7 days and amounted to 9.5 ± 0.40 μm (mean ± SE) as compared to 6.1 ± 0.15 μm in the control muscles. The surface area of individual capillaries increased by 10% in the tenuissimus muscle and by 3% in the EHP after 7 days of stimulation, and by 6% in the tenuissimus after 12 to 14 days. Formation of new capillaries was observed in stimulated but not in control or sham-operated muscles and appeared as “sprouts” emanating from the wall of preexisting capillaries. The proportion of sprouts to preexisting capillaries was 30 ± 4% after 7 days and 40 ± 4% after 14 days of stimulation, in both rabbit and rat muscles. Capillary density was evaluated using histochemical staining of the endothelial cells in cryostat cross sections. In 14 days, the average capillary muscle fiber ratios of stimulated EHP muscle increased from 0.99 ± 0.01 to 1.36 ± 0.02. Combination of in vivo studies and histochemical techniques enabled us to calculate the total capillary surface area in EHP muscle. This was found to be 2.08 ± 0.09 m 2 /100 g of muscle after 12 to 14 days of stimulation and 1.81 ± 0.048 m 2 /100 g in muscles stimulated for 7 days, representing increases of 46 and 29%, respectively, when compared to an area of 1.41 ± 0.03 m 2 /100 g for control muscles.

Is Physiological Angiogenesis in Skeletal Muscle Regulated by Changes in Microcirculation

Physiological angiogenesis occurs in female reproductive organs, in growing antlers as a result of long‐term exposure to cold and possibly hypoxia, and due to increased activity (training) in skeletal and cardiac muscle. The common denominator is increased blood flow, which may result in increased velocity of flow and/or diameters in arterioles and capillaries, increased capillary pressure and increased capillary hematocrit. Increased velocity would lead to increased shear stress, while increased pressure and/or diameters would increase wall tension. Either of these factors may cause a disturbance of the endothelium on the luminal side of vessels. In addition, increased contractile activity during training could cause changes on the abluminal side (for example, modification of the capillary basement membrane or the extracellular matrix induced by stretch/relaxation). In order to elucidate the role of these individual factors in angiogenesis, microcirculation was studied in skeletal muscles which were exposed to: (a) increased activity by chronic electrical stimulation; (b) long‐term increase in blood flow by various vasodilators; (c) long‐term administration of CoCl2 to increase hematocrit; and (d) long‐term stretch, achieved by removal of agonist muscles. Capillary growth, demonstrated as an increased capillary/fiber ratio, as determined by histochemical staining and by electron microscopy, occurred in (a), (b), and (d), but not (c). Capillary proliferation, estimated by labeling index for bromodeoxyuridine of capillary‐linked nuclei, occurred in (a), but not in (b).

Internal division of capillaries in rat skeletal muscle in response to chronic vasodilator treatment with alpha1-antagonist prazosin.

Abstract Chronic vasodilatation represents a stimulus for capillary growth associated with increased luminal shear stress. We have examined the ultrastructure of more than 2000 capillaries to establish whether the sequence of angiogenesis in response to this stimulus is similar to that described during development and under pathological circumstances. Administration of the α1-blocker prazosin to rats for 2 weeks led to a greater capillary length density in extensor hallucis proprius muscles without any change in capillary tortuosity: Jv(c,f)=262±54 compared with 350±17 mm–2, control compared with prazosin (P<0.002). There were obvious signs of endothelial cell (EC) activation after prazosin treatment, including an increased proportion of capillaries with rough endoplasmic reticulum, large cytoplasmic vacuoles, thickened endothelium and an irregular luminal surface. Capillaries from control muscles had a maximum of three ECs in cross section, whereas four ECs were noted in 0.8+0.5% of capillaries after 1 week (n.s.) and 2.5±0.9% after 2 weeks (P<0.01) of treatment. This could be due to elongation and/or migration of ECs, as cell proliferation has not been described at these time points. There was also an increase in the proportion of capillaries having a narrow, slit-like lumen (1.7±0.8% of controls; 7.1±1.9% at 1 week; 8.8±2.5% at 2 weeks; P<0.02), some of which were smaller in size (less than 2 μm diameter) than in controls (3–5 μm) and/or “seamless”, i.e. lacking EC junctions. These may represent newly formed vessels. Focal discontinuity of the basement membrane and abluminal EC processes were rarely seen, and capillary growth by abluminal sprouting appeared to be very infrequent (less than 0.001% of profiles). Of more importance was growth starting from the luminal side. Significantly more thin cytoplasmic processes were observed protruding into the lumen of capillaries after 1 week (47.5±6.2%, P<0.001) and 2 weeks of prazosin (34.2±5.5%, P<0.05) than in control vessels (16.7±3.9%). Some of these traversed the entire lumen and connected with endothelium of the opposite side, probably involving membrane fusion, resulting in the appearance of a double lumen. Individual capillaries with a complete double lumen were observed after 2 weeks’ prazosin but comparatively rarely, in only four out of six muscles. These findings indicate a pattern of luminal growth which is completely different from intussusceptive growth previously described during development, and from the abluminal capillary sprouting seen under pathological circumstances.

Capillary growth in overloaded, hypertrophic adult rat skeletal muscle: An ultrastructural study

We examined the early stages of angiogenesis in overloaded m. extensor digitorum longus following extirpation of the agonist m. tibialis anterior. Capillary‐to‐fibre ratio increased after 1 week (1.54 ± 0.02) vs. control (1.38 ± 0.06; P < 0.01) and resulted in a greater tortuosity of the capillary bed at 2 weeks, indicating the presence of lateral sprouts or anastomoses. Capillary endothelial cells (ECs) showed ultrastructural signs of activation, were thickened, and had irregular luminal and abluminal surfaces. The proportion of ECs with abluminal processes increased after overload (13.5 ± 0.6% vs. 2.0 ± 1.5%, 1 week vs. contralateral, P < 0.01; 12.5 ± 2.6% vs. 3.5 ± 0.6%, 2 weeks vs. contralateral, P < 0.01), whereas there was no significant change in proportion of luminal processes. Abluminal processes occurred in approximately 13% of capillaries in overloaded muscles (P < 0.01 v. control and contralateral), and most were associated with focal breakage of the basement membrane (BM). Small sprouts (<3 μm in diameter) comprised of one or two ECs sometimes lacked a lumen, and others had a slitlike or vacuolelike lumen between adjacent ECs or vacuolelike lumen formed by fusion of vesicles within a single EC. Endothelial mitosis was occasionally seen in nonsprouting capillaries with intact BM, increasing the average number of ECs per capillary from approximately 1.7 in control muscles to 2.1 after 1 week of overload (P < 0.05) when bromodeoxyuridine incorporation was also higher (P < 0.001). We conclude that muscle overload induces capillary growth by sprouting of existing capillaries, probably due to mechanical stretch acting from the abluminal side of the vessels. Anat. Rec. 252:49–63, 1998.

Relationship between mitochondria and oxygen consumption in isolated cat muscles.

1. Oxygen consumption, mitochondrial content and composition, intracellular lipid stores and fibre size were studied in isolated cat muscles: predominantly glycolytic gracilis, purely oxidative soleus and gracilis transformed into an oxidative muscle by chronic low‐frequency (10 Hz) electrical stimulation. 2. Oxygen consumption in control gracilis at rest (0.303 +/‐ 0.050 ml O2 min‐1 100 g‐1; mean +/‐ S.E. of mean) was three to five times lower than in either stimulated gracilis (1.16 +/‐ 0.40) or soleus (1.57 +/‐ 0.56); it was about two times lower during maximal contractions in control gracilis (5.15 +/‐ 0.24) than in either stimulated gracilis (11.6 +/‐ 2.0) or soleus (9.34 +/‐ 0.78). 3. The volume density of mitochondria in control gracilis (2.75 +/‐ 0.12%) was half that of soleus (6.23 +/‐ 0.76) and only one‐third that of stimulated gracilis (8.35 +/‐ 0.71). Subsarcolemmal mitochondria represented a significantly smaller fraction of the total mitochondrial volume in control gracilis than in either soleus or stimulated gracilis. 4. The surface area of inner and outer mitochondrial membranes per unit volume of mitochondria ranged from 23.4 to 26.1 and from 14.0 to 16.5 m2 cm‐3, respectively. Mean values of these variables were not significantly different among experimental groups. 5. The volume density of the intracellular lipid stores in control gracilis (0.232 +/‐ 0.041%) was one‐fourth of that in stimulated gracilis (0.860 +/‐ 0.12) and one‐fifth of that in soleus (1.17 +/‐ 0.27). 6. The fibre cross‐sectional area was 1670 +/‐ 260 micron 2 in control gracilis, 2250 +/‐ 280 in stimulated gracilis and 2390 +/‐ 110 in soleus. The difference was statistically significant only between control gracilis and soleus. 7. There was a significant correlation between the volume density of mitochondria and maximal oxygen consumption for all three muscles combined. 8. It was found that mitochondrial structure was similar in muscles with different oxidative capacities and that equal amounts of mitochondria consumed equal amounts of oxygen under limiting conditions of maximal in vivo respiration.