Daily muscle stretching enhances blood flow, endothelial function, capillarity, vascular volume and connectivity in aged skeletal muscle

Abstract

Key points: In aged rats, daily muscle stretching increases blood flow to skeletal muscle during exercise. Daily muscle stretching enhanced endothelium-dependent vasodilatation of skeletal muscle resistance arterioles of aged rats. Angiogenic markers and capillarity increased in response to daily stretching in muscles of aged rats. Muscle stretching performed with a splint could provide a feasible means of improving muscle blood flow and function in elderly patients who cannot perform regular aerobic exercise.

Abstract: Mechanical stretch stimuli alter the morphology and function of cultured endothelial cells; however, little is known about the effects of daily muscle stretching on adaptations of endothelial function and muscle blood flow. The present study aimed to determine the effects of daily muscle stretching on endothelium-dependent vasodilatation and muscle blood flow in aged rats. The lower hindlimb muscles of aged Fischer rats were passively stretched by placing an ankle dorsiflexion splint for 30 min day-1 , 5 days week-1 , for 4 weeks. Blood flow to the stretched limb and the non-stretched contralateral limb was determined at rest and during treadmill exercise. Endothelium-dependent/independent vasodilatation was evaluated in soleus muscle arterioles. Levels of hypoxia-induced factor-1α, vascular endothelial growth factor A and neuronal nitric oxide synthase were determined in soleus muscle fibres. Levels of endothelial nitric oxide synthase and superoxide dismutase were determined in soleus muscle arterioles, and microvascular volume and capillarity were evaluated by microcomputed tomography and lectin staining, respectively. During exercise, blood flow to plantar flexor muscles was significantly higher in the stretched limb. Endothelium-dependent vasodilatation was enhanced in arterioles from the soleus muscle from the stretched limb. Microvascular volume, number of capillaries per muscle fibre, and levels of hypoxia-induced factor-1α, vascular endothelial growth factor and endothelial nitric oxide synthase were significantly higher in the stretched limb. These results indicate that daily passive stretching of muscle enhances endothelium-dependent vasodilatation and induces angiogenesis. These microvascular adaptations may contribute to increased muscle blood flow during exercise in muscles that have undergone daily passive stretch.

Keywords: endothelium; exercise hyperemia; hypoxia inducible factor-1 alpha; nitric oxide synthase.

Conflict of interest statement

The authors declare that they have no competing interests.

© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.

Figures

Figure 1. Muscle stretch using splint

An ankle dorsiflexion splint was applied to hold the left ankle joint at 30° of dorsiflexion. The ankle dorsiflexion splint elongated ankle plantar flexor muscles (soleus, plantaris, flexor hallucis longus and flexor digitorum longus) and shortened ankle dorsiflexor muscles (tibialis anterior and extensor digitorum longus). [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 2. Blood flow at rest and during exercise after 4 weeks of muscle stretching

A, blood flow at rest was not different between any muscles from the stretched (grey) and non‐stretched contralateral limbs (black). B, blood flow to SOL, PLA, FDL and FHL during treadmill exercise was higher in the stretched limb (grey) compared to the non‐stretched contralateral limb (black); however, blood flow to TA and EDL was not different between the stretched and non‐stretched contralateral limb. SOL, soleus muscle; PLA, plantaris muscle; FDL, flexor digitorum longus muscle; FHL, flexor hallucis longus muscle; TA red, red portion of tibialis anterior muscle; TA white, white portion of tibialis anterior muscle; EDL, extensor digitorum longus muscle; n, number of rats. *P < 0.05 vs. non‐stretched contralateral limb. Values are the mean ± SE.

Figure 3. Skeletal muscle blood flow during and immediately after acute muscle stretching

A, blood flow to SOL, PLA, FDL and FHL during acute muscle stretching was lower in the stretched limb (grey) compared to the non‐stretched contralateral limb (black); however, blood flow to TA and EDL was not different between the stretched and non‐stretched contralateral limbs. B, skeletal muscle blood flow was not different between any muscles from the stretched and non‐stretched contralateral limbs (B). SOL, soleus muscle; PLA, plantaris muscle; FDL, flexor digitorum longus muscle; FHL, flexor hallucis longus muscle; TA red, red portion of tibialis anterior muscle; TA white, white portion of tibialis anterior muscle; EDL, extensor digitorum longus muscle; n, number of rats; *P < 0.05 vs. non‐stretched contralateral limb. Values are the mean ± SE.

Figure 4. Endothelium‐dependent and independent vasodilatation of soleus muscle arterioles

A, ACh‐induced vasodilatation was significantly greater in soleus muscle arterioles from the stretched limb (grey) compared to dilatation of arterioles from the non‐stretched contralateral limb (black) or limbs of sham control rats. B, inhibition with l‐NAME eliminated differences in ACh‐induced dilatation of soleus muscle arterioles from the stretched limb and the non‐stretched contralateral limb. C, Dea‐NONOate‐induced vasodilatation was not different between soleus muscle arterioles from the stretched limb and the non‐stretched contralateral limb stretch or limbs of sham control rats. n, number of rats; *P < 0.05 vs. non‐stretched contralateral limb; +P < 0.05 vs. sham control.

Figure 5. eNOS and SOD proteins in soleus muscle arterioles after 4 weeks of muscle stretching

A, representative images of soleus muscle arterioles from the stretched and non‐stretch contralateral limbs. B, average pixel intensity of eNOS staining was higher in soleus muscle arterioles from the stretched limb. SOD staining in soleus muscle arterioles was not different between the stretched and the non‐stretched contralateral limb. n, number of rats; *P < 0.05 vs. non‐stretched contralateral limb. Scale bar = 25 μm. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 6. Capillarity and angiogenic proteins in soleus muscles after 4 weeks of muscle stretching

A, representative images of staining with lectin, HIF‐1α, VEGF‐A and nNOS in soleus muscles from stretched and non‐stretched contralateral limbs. B, the number of capillaries per muscle fibre was higher in the soleus muscle from the stretched limb. Levels of HIF‐1α and VEGF‐A were higher in the soleus muscle from the stretched limb compared to the non‐stretched contralateral limb; however, the level of nNOS was not different between the stretched limb and the non‐stretched contralateral limb. n, number of rats; *P < 0.05 vs. non‐stretched contralateral limb. Scale bar = 25 μm. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 7. Micro‐CT analysis of microvascular parameters after 4 weeks of muscle stretching

A, representative images of the soleus muscle microvasculature from the stretched and non‐stretched contralateral hindlimb. B, absolute vessel volume (mm3), normalized vascular volume (vascular volume/total muscle volume; mm3/mm3 and vessel connectivity in soleus muscles from stretched and non‐stretched contralateral limbs. n, number of rats; *P < 0.05 vs. non‐stretched contralateral limb. [Color figure can be viewed at http://wileyonlinelibrary.com]