Margaret D. Brown

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.

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.

Inspiratory muscle training using an incremental endurance test alleviates dyspnea and improves functional status in patients with chronic heart failure.

Background The benefits of inspiratory muscle training (IMT) in patients with chronic heart failure (CHF) have been inadequately studied. Design and methods Using a prospective, age and sex-matched controlled study, we investigated 35 patients with moderate to severe CHF (NYHA class II–III and left ventricular ejection fraction 24.4 ± 1.3% [mean ± SEM]). An incremental respiratory endurance test using a fixed respiratory workload was provided by software with an electronic mouth pressure manometer interfaced with a computer. The training group (n = 20) exercised at 60% of individual sustained maximal inspiratory pressure (SMIP) and the control group (n = 15) at 15% of SMIP. All patients exercised three times weekly for 10 weeks. Pulmonary function, exercise capacity, dyspnea and quality of life were assessed, pre- and post-training. Results The training group significantly increased both maximum inspiratory pressure (Pimax), (111 ± 6.8 versus 83 ± 5.7cmH2O, P<0.001), and SMIP (527822 ± 51358 versus 367360 ± 41111 cmH2O/sec × 10-1, P < 0.001). Peak VO2 increased after training (17.8 ± 1.2 versus 15.4 ± 0.9 ml/kg/min, P< 0.005), as did the six-minute walking distance (433 ± 16 versus 367 ± 22 meters, P < 0.001). Perceived dyspnea assessed using the Borg scale was reduced for both the treadmill (12.7 ± 0.57 versus 14.2 ± 0.48, P < 0.005) and the walking (9 ± 0.48 versus 10.5 ± 0.67, P< 0.005) exercise tests and the quality of life score was also improved (21.1 ± 3.5 versus 25.2 ± 4, P < 0.01). Resting heart rate was significantly reduced with training (77 ± 3.3 versus 80 ± 3beats/min, P < 0.05). The control group significantly increased Pimax (86.6 ± 6.3 versus 78.4 ± 6.9cmH2O, P < 0.05), but decreased SMIP (274972 ± 32399 versus 204661 ± 37184cmH2O/sec × 101, P < 0.005). No other significant effect on exercise capacity, heart rate, dyspnea, or quality of life was observed in this group. Conclusion Inspiratory muscle training using an incremental endurance test, successfully increases both inspiratory strength and endurance, alleviates dyspnea and improves functional status in CHF.

Postnatal growth of the heart and its blood vessels.

Although rapid growth of the heart during early postnatal development ceases with maturation of the organism, the potential for cardiomyocyte growth is not lost and may be observed even in senescent hearts. Rapid developmental heart growth is accompanied by a proportional growth of capillaries but not always of larger vessels, and thus coronary vascular resistance gradually increases. Growth of adult hearts can be enhanced by thyroid hormones, catecholamines and the renin-angiotensin system hormones, but these do not always stimulate growth of coronary vessels. Likewise, chronic exposure to hypoxia leads to growth, mainly of the right ventricle and its vessels but without vascular growth elsewhere in the heart. On the other hand, ischaemia is a potent stimulus for the release of various growth factors involved in the development of collateral circulation. Heart hypertrophy develops in response to training, pressure or volume overload. Training usually leads to growth of larger coronary vessels but little growth of capillaries, except in young animals. However, growth of the capillary bed, but not the resistance vasculature capacity, can be induced by either increased coronary blood flow, bradycardia (electrically or pharmacologically induced) or increased inotropism, all of which are involved in the training stimulus. Thus, what actually promotes growth of larger vessels as opposed to capillaries in training is unclear. Pressure overload hypertrophy is mediated by both the renin-angiotensin system and the response of cardiomyocytes to stretch; both lead to activation of early oncogenes (c-fos, c-jun, c-myc) and angiotensin II activates several protein kinases involved in cell growth. In this condition, growth of larger vessels is inadequate, although some capillary growth may occur. Volume overload leads to cardiomyocyte hypertrophy and hyperplasia and some increase in vascular supply. Deficits in capillary supply in pressure or volume overload hypertrophy can be reversed by chronic administration of ACE inhibitors, dipyridamole, the bradycardic drug alinidine or pacing-induced bradycardia respectively, but in neither case is training effective. Mechanical and humoral factors are involved in growth of cardiomyocytes and vessels. For cardiomyocytes, stretch is most important, activating oncogenes, protein kinases and possibly the inositol phosphate pathway, but not ion channels, with regulation by the balance of angiotensin II, TGF-beta 1 and IGF-1, but not FGFs. For vessels, growth is stimulated by stretch and shear stress, possibly with involvement of VEGF. Increased shear stress disrupts the glycocalyx on the luminal side of vessels and releases plasminogen activator and metalloproteinases which disrupt the basement membrane and enable endothelial cell migration and proliferation. It also causes rearrangement of the endothelial cytoskeleton and transmission of mechanical signals to the abluminal side disturbing extracellular matrix and causing distortion of capillary basement membrane. Stretch acting from the abluminal side has a similar effect resulting also in basement membrane disruption and endothelial cell proliferation.

Arterial blood pressure and forearm vascular conductance responses to sustained and rhythmic isometric exercise and arterial occlusion in trained rock climbers and untrained sedentary subjects

Abstract Cardiovascular responses to sustained and rhythmic (5 s on, 2 s off ) forearm isometric exercise to fatigue at 40% maximal voluntary contraction (MVC) and to a period of arterial occlusion were investigated in elite rock climbers (CLIMB) as a trained population compared to non-climbing sedentary subjects (SED). Blood pressure (BP), monitored continuously by Finapres, and forearm blood flow, by venous occlusion plethysmography, were measured and used to calculate vascular conductance. During sustained exercise, times to fatigue were not different between CLIMB and SED. However, peak increases in systolic (S) BP were significantly lower in CLIMB [25 (13) mmHg; (3.3 (1.7) kPa] than in SED [48 (17) mmHg; (6.4 (2.3) kPa] (P<0.05), with a similar trend for increases in diastolic (D) BP. Immediately after sustained exercise, forearm conductance was higher in CLIMB than SED (P<0.05) for up to 2 min. During rhythmic exercise, times to fatigue were two fold longer in CLIMB than SED [853 (76) vs 420 (69) s, P<0.05]. Increases in SBP were not different between groups except during the last quarter of exercise when they fell in CLIMB. Conductance both during and after rhythmic exercise was higher in CLIMB than in SED. Following a 10-min arterial occlusion, peak vascular conductance was significantly greater in CLIMB than SED [0.597 (0.084) vs 0.431 (0.035) ml · min−1 · 100 ml−1 · mmHg−1; P<0.05]. The attenuated BP response to sustained isometric exercise could be due in part to enhanced forearm vasodilatory capacity, which also supports greater endurance during rhythmic exercise by permitting greater functional hyperaemia in between contraction phases. Such adaptations would all facilitate the ability of rock climbers to perform their task of making repetitive sustained contractions.