Myofibre injury induces capillary disruption and regeneration of disorganized microvascular networks.

Injury to skeletal muscle disrupts myofibres and their microvascular supply. While the regeneration of myofibres is well described, little is known of how the microcirculation is affected by skeletal muscle injury or its recovery during regeneration. Nevertheless, the microvasculature must also recover to restore skeletal muscle function. We aimed to define the nature of microvascular damage and time course of repair during muscle injury and regeneration induced by the myotoxin BaCl2 . To test the hypothesis that microvascular disruption occurred secondary to myofibre injury, isolated microvessels were exposed to BaCl2 or the myotoxin was injected into the gluteus maximus (GM) muscle of mice. In isolated microvessels, BaCl2 depolarized smooth muscle cells (SMCs) and endothelial cells while increasing intracellular calcium in SMCs but did not elicit death of either cell type. At 1 day post-injury (dpi) of the GM, capillary fragmentation coincided with myofibre degeneration while arteriolar and venular networks remained intact; neutrophil depletion before injury did not prevent capillary damage. Perfused capillary networks reformed by 5 dpi in association with more terminal arterioles and were dilated through 10 dpi. With no change in microvascular area or branch point number in regenerating capillary networks, fewer capillaries aligned with myofibres and were no longer organized into microvascular units. By 21 dpi, capillary orientation and microvascular unit organization were no longer different from uninjured GM. We conclude that following their disruption secondary to myofibre damage, capillaries regenerate as disorganized networks that remodel into microvascular units as regenerated myofibres mature. KEY POINTS: Skeletal muscle regenerates after injury; however, the nature of microvascular damage and repair is poorly understood. Here, the myotoxin BaCl2 , a standard experimental method of acute skeletal muscle injury, was used to investigate the response of the microcirculation to local injury of intact muscle. Intramuscular injection of BaCl2 induced capillary fragmentation with myofibre degeneration; arteriolar and venular networks remained intact. Direct exposure to BaCl2 did not kill microvascular endothelial cells or smooth muscle cells. Dilated capillary networks reformed by 5 days post-injury (dpi) in association with more terminal arterioles. Capillary orientation remained disorganized through 10 dpi. Capillaries realigned with myofibres and reorganized into microvascular units by 21 dpi, which coincides with the recovery of vasomotor control and maturation of nascent myofibres. Skeletal muscle injury disrupts its capillary supply secondary to myofibre degeneration. Reorganization of regenerating microvascular networks accompanies the recovery of blood flow regulation.

Serine 319 phosphorylation is necessary and sufficient to induce a Cx37 conformation that leads to arrested cell cycling.

Connexin 37 (Cx37; protein product of GJA4) expression profoundly suppresses proliferation of rat insulinoma (Rin) cells in a manner dependent on gap junction channel (GJCh) functionality and the presence and phosphorylation status of its C-terminus (CT). In Rin cells, growth is arrested upon induced Cx37 expression and serine 319 (S319) is frequently phosphorylated. Here, we show that preventing phosphorylation at this site (alanine substitution; S319A) relieved Cx37 of its growth-suppressive effect whereas mimicking phosphorylation at this site (aspartate substitution; S319D) enhanced the growth-suppressive properties of Cx37. Like wild-type Cx37 (Cx37-WT), Cx37-S319D GJChs and hemichannels (HChs) preferred the closed state, rarely opening fully, and gated slowly. In contrast, Cx37-S319A channels preferred open states, opened fully and gated rapidly. These data indicate that phosphorylation-dependent conformational differences in Cx37 protein and channel function underlie Cx37-induced growth arrest versus growth-permissive phenotypes. That the closed state of Cx37-WT and Cx37-S319D GJChs and HChs favors growth arrest suggests that rather than specific permeants mediating cell cycle arrest, the closed conformation instead supports interaction of Cx37 with growth regulatory proteins that result in growth arrest.

Functionalizing biomaterials to promote neurovascular regeneration following skeletal muscle injury.

During embryogenesis, blood vessels and nerves develop with similar branching structure in response to shared signaling pathways guiding network growth. With both systems integral to physiological homeostasis, dual targeting of blood vessels and nerves to promote neurovascular regeneration following injury is an emerging therapeutic approach in biomedical engineering. A limitation to this strategy is that the nature of cross talk between emergent vessels and nerves during regeneration in an adult is poorly understood. Following peripheral nerve transection, intraneural vascular cells infiltrate the site of injury to provide a migratory pathway for mobilized Schwann cells of regenerating axons. As Schwann cells demyelinate, they secrete vascular endothelial growth factor, which promotes angiogenesis. Recent advances point to concomitant restoration of neurovascular architecture and function through simultaneous targeting of growth factors and guidance cues shared by both systems during regeneration. In the context of traumatic injury associated with volumetric muscle loss, we consider the nature of biomaterials used to engineer three-dimensional scaffolds, functionalization of scaffolds with molecular signals that guide and promote neurovascular growth, and seeding scaffolds with progenitor cells. Physiological success is defined by each tissue component of the bioconstruct (nerve, vessel, muscle) becoming integrated with that of the host. Advances in microfabrication, cell culture techniques, and progenitor cell biology hold great promise for engineering bioconstructs able to restore organ function after volumetric muscle loss.

Female sex and Western-style diet protect mouse resistance arteries during acute oxidative stress.

A Western-style diet (WD; high in fat and carbohydrates) increases vascular oxidative stress. We hypothesized that vascular cells adapt to a WD by developing resilience to oxidative stress. Male and female C57BL/6J mice (4 wk of age) were fed a control diet (CD) or a WD for 16-20 wk. Superior epigastric arteries (SEAs; diameter, ~125 µm) were isolated and pressurized for study. Basal reactive oxygen species production was greatest in SEAs from males fed the WD. During exposure to H2O2 (200 µM, 50 min), propidium iodide staining identified nuclei of disrupted endothelial cells (ECs) and smooth muscle cells (SMCs). For mice fed the CD, death of SMCs (21%) and ECs (6%) was greater (P < 0.05) in SEAs from males than females (9% and 2%, respectively). WD consumption attenuated cell death most effectively in SEAs from males. With no difference at rest, H2O2 increased intracellular Ca2+ concentration ([Ca2+]i) to the greatest extent in SEAs from males, as shown by fura 2 fluorescence. Selective disruption of the endothelium (luminal air bubble) increased [Ca2+]i and SMC death during H2O2 exposure irrespective of sex; the WD reduced both responses most effectively in males. Nonselective transient receptor potential (TRP) channel inhibition (ruthenium red, 5 µM) attenuated the rise of [Ca2+]i, as did selective inhibition of TRP vanilloid type 4 (TRPV4) channels (HC-067047, 1 µM), which also attenuated cell death. In contrast, inhibition of voltage-gated Ca2+ channels (diltiazem, 50 µM) was without effect. Thus, for resistance arteries during acute oxidative stress: 1) ECs are more resilient than (and can protect) SMCs, 2) vessels from females are inherently more resilient than those from males, and 3) a WD increases vascular resilience by diminishing TRPV4 channel-dependent Ca2+ entry.

Advanced age protects resistance arteries of mouse skeletal muscle from oxidative stress through attenuating apoptosis induced by hydrogen peroxide.

KEY POINTS: Vascular oxidative stress increases with advancing age. We hypothesized that resistance vessels develop resilience to oxidative stress to protect functional integrity and tested this hypothesis by exposing isolated pressurized superior epigastric arteries (SEAs) of old and young mice to H2 O2 . H2 O2 -induced death was greater in smooth muscle cells (SMCs) than endothelial cells (ECs) and lower in SEAs from old vs. young mice; the rise in vessel wall [Ca2+ ]i induced by H2 O2 was attenuated with ageing, as was the decline in noradrenergic vasoconstriction; genetic deletion of IL-10 mimicked the effects of advanced age on cell survival. Inhibiting NO synthase or scavenging peroxynitrite reduced SMC death; endothelial denudation or inhibiting gap junctions increased SMC death; delocalization of cytochrome C activated caspases 9 and 3 to induce apoptosis. Vascular cells develop resilience to H2 O2 during ageing by preventing Ca2+ overload and endothelial integrity promotes SMC survival. ABSTRACT: Advanced age is associated with elevated oxidative stress and can protect the endothelium from cell death induced by H2 O2 . Whether such protection occurs for intact vessels or differs between smooth muscle cell (SMC) and endothelial cell (EC) layers is unknown. We tested the hypothesis that ageing protects SMCs and ECs during acute exposure to H2 O2 (200 µm, 50 min). Mouse superior epigastric arteries (SEAs; diameter, ∼150 µm) were isolated and pressurized to 100 cmH2 O at 37˚C. For SEAs from young (4 months) mice, H2 O2 killed 57% of SMCs and 11% of ECs in males vs. 8% and 2%, respectively, in females. Therefore, SEAs from males were studied to resolve the effect of ageing and experimental interventions. For old (24 months) mice, SMC death was reduced to 10% with diminished accumulation of [Ca2+ ]i in the vessel wall during H2 O2 exposure. In young mice, genetic deletion of IL-10 mimicked the protective effect of ageing on cell death and [Ca2+ ]i accumulation. Whereas endothelial denudation or gap junction inhibition (carbenoxolone; 100 µm) increased SMC death, inhibiting NO synthase (l-NAME, 100 µm) or scavenging peroxynitrite (FeTPPS, 5 µm) reduced SMC death along with [Ca2+ ]i . Despite NO toxicity via peroxynitrite formation, endothelial integrity protects SMCs. Caspase inhibition (Z-VAD-FMK, 50 µm) attenuated cell death with immunostaining for annexin V, cytochrome C, and caspases 3 and 9 pointing to induction of intrinsic apoptosis during H2 O2 exposure. We conclude that advanced age reduces Ca2+ influx that triggers apoptosis, thereby promoting resilience of the vascular wall during oxidative stress.

Regulation of Cx37 channel and growth-suppressive properties by phosphorylation.

Growth suppression mediated by connexin 37 (Cx37; also known as GJA4) requires interaction between its C-terminus and functional pore-forming domain. Using rat insulinoma cells, we show that Cx37 induces cell death and cell cycle arrest, and slowed cell cycling. Whether differential phosphorylation might regulate intramolecular interactions, and consequently the growth-suppressive phenotype, is unknown. Protein kinase C inhibition increased the open state probability of low-conductance gap junction channels (GJChs) and reduced GJCh closed state probability. Substituting alanine at serine residues 275, 302 and 328 eliminated Cx37-induced cell death, supported proliferation and reduced the GJCh closed state probability. With additional alanine for serine substitutions at residues 285, 319, 321 and 325, Cx37-induced cell death was eliminated and the growth arrest period prolonged, and GJCh closed state probability was restored. With aspartate substitution at these seven sites, apoptosis was induced and the open state probability of large conductance GJChs (and hemichannels) was increased. These data suggest that differential phosphorylation of the C-terminus regulates channel conformation and, thereby, cell cycle progression and cell survival.

Angiogenesis precedes myogenesis during regeneration following biopsy injury of skeletal muscle.

BACKGROUND: Acute injury to skeletal muscle damages myofibers and fragment capillaries, impairing contractile function and local perfusion. Myofibers and microvessels regenerate from satellite cells and from surviving microvessel fragments, respectively, to restore intact muscle. Established models of injury have used myotoxins and physical trauma to demonstrate the concurrence of myogenesis and angiogenesis during regeneration. In these models, efferocytosis removes cellular debris while basal laminae persist to provide guidance during myofiber and microvessel regeneration. It is unknown whether the spatiotemporal coupling between myofiber and microvascular regeneration persists when muscle tissue is completely removed and local guidance cues are lost. METHODS: To test whether complete removal of skeletal muscle tissue affects the spatiotemporal relationship between myogenesis and angiogenesis during regeneration, subthreshold volumetric muscle loss was created with a biopsy punch (diameter, 2 mm) through the center of the gluteus maximus (GM) in adult mice. Regeneration into the void was evaluated through 21 days post-injury (dpi). Microvascular perfusion was evaluated in vivo by injecting fluorescent dextran into the circulation during intravital imaging. Confocal imaging and histological analyses of whole-mount GM preparations and tissue cross-sections assessed the growth of microvessels and myofibers into the wound. RESULTS: A provisional matrix filled with PDGFRα+ and CD45+ cells spanned the wound within 1 dpi. Regenerating microvessels advanced from the edges of the wound into the matrix by 7 dpi. Nascent microvascular networks formed by 10 dpi with blood-perfused networks spanning the wound by 14 dpi. In striking contrast, the wound remained devoid of myofibers at 7 and 10 dpi. Myogenesis into the wound was apparent by 14 dpi and traversed the wound by 21 dpi. Regenerated myofibers and microvessels were disorganized compared to the uninjured muscle. CONCLUSIONS: Following punch biopsy of adult skeletal muscle, regenerating microvessels span the wound and become perfused with blood prior to myofiber regeneration. The loss of residual guidance cues with complete tissue removal disrupts the spatiotemporal correspondence between microvascular and myofiber regeneration. We conclude that angiogenesis precedes myogenesis during regeneration following subthreshold volumetric muscle loss.

Phosphorylation-Dependent Intra-Domain Interaction of the Cx37 Carboxyl-Terminus Controls Cell Survival.

Differential phosphorylation of the carboxyl-terminus of connexin 37 (Cx37-CT) regulates phenotypic switching between cell growth phenotypes (cell death, cell cycle arrest, proliferation). The specific phosphorylation events in the Cx37-CT that are necessary for these growth regulatory effects are currently unknown. Through the combined use of deletion and site specific (de)phospho-mimetic Cx37-CT mutants, our data suggest a phosphorylation-dependent interaction between the mid-tail (aa 273⁻317) and end-tail (aa 318⁻333) portions of the Cx37-CT that regulates cell survival. As detected by mass spectrometry, Cx37 was phosphorylated at serines 275, 321, and 328; phosphomimetic mutations of these sites resulted in cell death when expressed in rat insulinoma cells. Alanine substitution at S328, but not at S275 or S321, also triggered cell death. Cx37-S275D uniquely induced the death of only low density, non-contact forming cells, but neither hemichannel open probability nor channel conductance distinguished death-inducing mutants. As channel function is necessary for cell death, together the data suggest that the phosphorylation state of the Cx37-CT controls an intra-domain interaction within the CT that modifies channel function and induces cell death.

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.