Barium chloride injures myofibers through calcium-induced proteolysis with fragmentation of motor nerves and microvessels.

BACKGROUND: Local injection of BaCl2 is an established model of acute injury to study the regeneration of skeletal muscle. However, the mechanism by which BaCl2 causes muscle injury is unresolved. Because Ba2+ inhibits K+ channels, we hypothesized that BaCl2 induces myofiber depolarization leading to Ca2+ overload, proteolysis, and membrane disruption. While BaCl2 spares resident satellite cells, its effect on other tissue components integral to contractile function has not been defined. We therefore asked whether motor nerves and microvessels, which control and supply myofibers, are injured by BaCl2 treatment. METHODS: The intact extensor digitorum longus (EDL) muscle was isolated from male mice (aged 3-4 months) and irrigated with physiological salt solution (PSS) at 37 °C. Myofiber membrane potential (Vm) was recorded using sharp microelectrodes while intracellular calcium concentration ([Ca2+]i) was evaluated with Fura 2 dye. Isometric force production of EDL was measured in situ, proteolytic activity was quantified by calpain degradation of αII-spectrin, and membrane disruption was marked by nuclear staining with propidium iodide (PI). To test for effects on motor nerves and microvessels, tibialis anterior or gluteus maximus muscles were injected with 1.2% BaCl2 (50-75 µL) in vivo followed by immunostaining to evaluate the integrity of respective tissue elements post injury. Data were analyzed using Students t test and analysis of variance with P ≤ 0.05 considered statistically significant. RESULTS: Addition of 1.2% BaCl2 to PSS depolarized myofibers from - 79 ± 3 mV to - 17 ± 7 mV with a corresponding rise in [Ca2+]i; isometric force transiently increased from 7.4 ± 0.1 g to 11.1 ± 0.4 g. Following 1 h of BaCl2 exposure, 92 ± 3% of myonuclei stained with PI (vs. 8 ± 3% in controls) with enhanced cleavage of αII-spectrin. Eliminating Ca2+ from PSS prevented the rise in [Ca2+]i and ameliorated myonuclear staining with PI during BaCl2 exposure. Motor axons and capillary networks appeared fragmented within 24 h following injection of 1.2% BaCl2 and morphological integrity deteriorated through 72 h. CONCLUSIONS: BaCl2 injures myofibers through depolarization of the sarcolemma, causing Ca2+ overload with transient contraction, leading to proteolysis and membrane rupture. Motor innervation and capillarity appear disrupted concomitant with myofiber damage, further compromising muscle integrity.

Recovery of blood flow regulation in microvascular resistance networks during regeneration of mouse gluteus maximus muscle.

KEY POINTS: Skeletal muscle regenerates after injury, however the recovery of its microvascular supply is poorly understood. We injured the gluteus maximus muscle in mice aiming to investigate the recovery of blood flow regulation in microvascular resistance networks. We hypothesized that blood flow regulation recovers in concert with myofibre regeneration. Microvascular perfusion ceased within 1 day post injury and was restored at 5 days coincident with the appearance of new myofibres; however, the resistance network was dilated and unresponsive to vasoactive agents. Spontaneous vasomotor tone, endothelium-dependent dilatation and adrenergic vasoconstriction increased at 10 days in concert with myofibre regeneration. Vasomotor control recovered at 21 days, when regenerated myofibres matured and active force production stabilized. Functional vasodilatation in response to muscle contraction recovered at 35 days. Physiological integrity of microvascular smooth muscle and endothelium recovers in parallel with myofibre regeneration. Additional time is required to restore the efficacy of signalling between myofibres and microvascular networks controlling their oxygen supply. ABSTRACT: Myofibre regeneration after skeletal muscle injury is well-studied, although little is known about how microvascular perfusion is restored. The present study aimed to evaluate the recovery of blood flow regulation during skeletal muscle regeneration. In anaesthetized male C57BL/6J mice (aged 4 months), the gluteus maximus muscle (GM) was injured by local injection of barium chloride solution (1.2%, 75 µL). Functional integrity of the resistance network was evaluated at 5, 10, 21 and 35 days post-injury vs. Control by measuring internal diameter of feed arteries, first-, second- and third-order arterioles supplying the GM using intravital microscopy. The resting diameters of all branch orders were significantly greater (P < 0.05) than Control at 5 and 10 days and recovered to Control by 21 days, as did spontaneous vasomotor tone. Vasodilatation to ACh and vasoconstriction to phenylephrine (10-9 to 10-5  m) were absent at 5 days, increased at 10 days and recovered to Control by 21 days; reactivity improved in a distal-to-proximal gradient. Across branch orders, functional vasodilatation to single tetanic contraction (100 Hz, 500 ms) and to rhythmic twitch contractions (4 Hz, 30 s) was impaired at 5 days, improved through 21 days and was not different from Control at 35 days. Peak force development (g) was 60% of Control at 10 days and recovered by 21 days. Diminished vasomotor tone during the initial stages of regeneration promotes tissue perfusion as myofibre recovery begins. Recovery of tone and vasomotor responses to agonists occur in concert with myofibre regeneration. Delayed recovery of functional vasodilatation indicates that additional time is required to restore signalling between contracting myofibres and their vascular supply.

Differential α-adrenergic modulation of rapid onset vasodilatation along resistance networks of skeletal muscle in old versus young mice.

KEY POINTS: Rapid onset vasodilatation (ROV) initiates functional hyperaemia upon skeletal muscle contraction and is attenuated during ageing via α-adrenoreceptor (αAR) stimulation, but it is unknown where this effect predominates in resistance networks. In gluteus maximus muscles of young (4 months) and old (24 months) male C57BL/6 mice, tetanic contraction while observing feed arteries and arterioles initiated ROV, which increased with contraction duration, peaked later in upstream versus downstream vessel branches and was attenuated throughout networks with advanced age. With no effect on muscle force production, inhibiting αARs improved ROV in old mice while activating αARs attenuated ROV in young mice. Modulating ROV through αARs was greater in upstream feed arteries and arterioles compared to downstream arterioles, with α2 ARs more effective than α1 ARs. ROV is coordinated along resistance networks and modulated differentially between young and old mice via αARs; with advanced age, attenuated dilatation of upstream branches will restrict muscle blood flow. ABSTRACT: Rapid onset vasodilatation (ROV) in skeletal muscle is attenuated during advanced age via α-adrenoreceptor (αAR) activation, but it is unknown where such effects predominate in the resistance vasculature. Studying the gluteus maximus muscle (GM) of anaesthetized young (4 months) and old (24 months) male C57BL/6 mice, we tested the hypothesis that attenuation of ROV during advanced age is most effective in proximal branches of microvascular resistance networks. Diameters of a feed artery (FA) and first- (1A), second- (2A) and third- (3A) order arterioles were studied in response to single tetanic contractions (100 Hz, 100-1000 ms). ROV began within 1 s and peaked sooner in 2A and 3A (∼3 s) than in 1A or FA (∼4 s). Relative amplitudes of dilatation increased with contraction duration and with vessel branch order (FA<1A<2A<3A). In old mice, attenuation of ROV was greater in FA and 1A compared to 2A and 3A. With no effect on muscle force production, inhibiting αARs (phentolamine; 10-6  m) improved ROV in FA and 1A of old mice while subthreshold stimulation of αARs in young mice (noradrenaline; 10-9  m) depressed ROV most effectively in FA and 1A. In young mice, stimulating α1 ARs (phenylephrine; 10-7  m) and α2 ARs (UK 14304; 10-7  m) attenuated ROV primarily in FA. In old mice, inhibiting α2 ARs (rauwolscine; 10-7  m) restored ROV more effectively in FA and 1A than did inhibiting α1 ARs (prazosin; 10-8  m). We conclude that, with temporal and spatial coordination along resistance networks, attenuation of ROV with advanced age is most effective in proximal branches via constitutive activation of α2 ARs.

Attenuated rapid onset vasodilation with greater force production in skeletal muscle of caveolin-2-/- mice.

Caveolin-2 (Cav2) is a major protein component of caveolae in membranes of vascular smooth muscle and endothelium, yet its absence alters the ultrastructure of skeletal muscle fibers. To gain insight into Cav2 function in skeletal muscle, we tested the hypothesis that genetic deletion of Cav2 would alter microvascular reactivity and depress contractile function of skeletal muscle in vivo. In the left gluteus maximus muscle (GM) of anesthetized Cav2(-/-) and wild-type (WT) male mice (age, 6 mo), microvascular responses to physiological agonists and to GM contractions were studied at 34°C. For feed arteries (FA), first- (1A), second- (2A) and third-order (3A) arterioles, respective mean diameters at rest (45, 35, 25, 12 µm) and during maximal dilation (65, 55, 45, 30 µm) were similar between groups. Cumulative dilations to ACh (10(-9) to 10(-5) M) and constrictions to norepinephrine (10(-9) to 10(-5) M) were also similar between groups, as were steady-state dilations during rhythmic twitch contractions (2 and 4 Hz; 30 s). For single tetanic contractions (100 Hz; 100, 250, and 500 ms), rapid onset vasodilation (ROV) increased with contraction duration throughout networks in GM of both groups but was reduced by nearly half in Cav2(-/-) mice compared with WT mice (P < 0.05). Nevertheless, maximal force during tetanic contraction was ∼40% greater in GM of Cav2(-/-) vs. WT mice (152 ± 14 vs. 110 ± 3 mN per square millimeter, respectively; P < 0.05). Thus, while structural and functional properties of resistance networks are well maintained in the GM of Cav2(-/-) mice, diminished ROV with greater force production reveals novel physiological roles for Cav2 in skeletal muscle.