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Skeletal Muscle–Specific Genetic Determinants Contribute to the Differential Strain-Dependent Effects of Hindlimb Ischemia in Mice

Open AccessPublished:March 23, 2012DOI:https://doi.org/10.1016/j.ajpath.2012.01.032
      Genetics plays an important role in determining peripheral arterial disease (PAD) pathology, which causes a spectrum of clinical disorders that range from clinically silent reductions in blood flow to limb-threatening ischemia. The cell-type specificity of PAD pathology, however, has received little attention. To determine whether strain-dependent differences in skeletal muscle cells might account for the differential responses to ischemia observed in C57BL/6 and BALB/c mice, endothelial and skeletal muscle cells were subjected to hypoxia and nutrient deprivation (HND) in vitro, to mimic ischemia. Muscle cells were more susceptible to HND than were endothelial cells. In vivo, C57BL/6 and BALB/c mice displayed strain-specific differences in myofiber responses after hindlimb ischemia, with significantly greater myofiber atrophy, greater apoptosis, and attenuated myogenic regulatory gene expression and stress-responsive signaling in BALB/c mice. Strain-specific deficits were recapitulated in vitro in primary muscle cells from both strains after HND. Muscle cells from BALB/c mice congenic for the C57BL/6 Lsq-1 quantitative trait locus were protected from HND-induced atrophy, and gene expression of vascular growth factors and their receptors was significantly greater in C57BL/6 primary muscle cells. Our results indicate that the previously identified specific genetic locus regulating strain-dependent collateral vessel density has a nonvascular or muscle cell-autonomous role involving both the myogenic program and traditional vascular growth factor receptor expression.
      Peripheral arterial disease (PAD) results from atherosclerosis of peripheral arteries, most commonly in the lower extremities, and causes a spectrum of clinical disorders that range from clinically silent reductions in blood flow to limb-threatening ischemia for which amputation is often required.
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      TASC II Working Group
      Inter-Society consensus for the management of peripheral arterial disease (TASC II).
      In patients with intermittent claudication, arterial occlusive disease results in reduced blood flow manifested as pain with exertion, whereas in patients with critical limb ischemia (CLI) the blood flow is inadequate to meet the resting demands of the limb and results in pain at rest and/or tissue necrosis. Although less common than claudication, CLI results in significantly higher morbidity and mortality; patients with CLI have a risk of major amputation or death that approaches 40% in 1 year.
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      Comparison of interventional outcomes according to preoperative indication: a single center analysis of 2,240 limb revascularizations.
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      • Vig S.
      The fate of patients with critical leg ischemia.
      Although it was long held that CLI represents the natural progressive deterioration of PAD in patients with claudication, this appears not to be the case. In fact, only a small percentage of patients with claudication eventually develop symptoms of CLI, and a substantial number of patients with CLI deny prior symptoms of claudication.
      • Mätzke S.
      • Lepäntalo M.
      Claudication does not always precede critical leg ischemia.
      Furthermore, patients with the same degree of lower extremity athero-occlusive disease can present with either intermittent claudication or CLI. For these reasons, it appears that intermittent claudication and CLI represent distinct phenotypic manifestations of the same underlying atherosclerotic disease process, likely because of differences in genetic susceptibility. Thus, identifying the genetic modifiers that predispose individuals to develop CLI remains an important area of investigation in PAD.
      Mouse models of limb ischemia provide useful tools with which to investigate the mechanisms regulating the ischemic response.
      • Couffinhal T.
      • Silver M.
      • Kearney M.
      • Sullivan A.
      • Witzenbichler B.
      • Magner M.
      • Annex B.
      • Peters K.
      • Isner J.M.
      Impaired collateral vessel development associated with reduced expression of vascular endothelial growth factor in ApoE-/- mice.
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      • Silver M.
      • Zheng L.P.
      • Kearney M.
      • Witzenbichler B.
      • Isner J.M.
      Mouse model of angiogenesis.
      It is well established that different inbred strains of mice display markedly different responses to surgically induced hindlimb ischemia (HLI).
      • Chalothorn D.
      • Clayton J.A.
      • Zhang H.
      • Pomp D.
      • Faber J.E.
      Collateral density, remodeling, and VEGF-A expression differ widely between mouse strains.
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      • Keum S.
      • Hazarika S.
      • Li Y.
      • Lamonte G.M.
      • Wheeler F.
      • Marchuk D.A.
      • Annex B.H.
      A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.
      • Helisch A.
      • Wagner S.
      • Khan N.
      • Drinane M.
      • Wolfram S.
      • Heil M.
      • Ziegelhoeffer T.
      • Brandt U.
      • Pearlman J.D.
      • Swartz H.M.
      • Schaper W.
      Impact of mouse strain differences in innate hindlimb collateral vasculature.
      In particular, the C57BL/6 (BL6) and BALB/c strains have frequently been compared because of their markedly different responses to ischemia: BL6 mice display significantly better collateral artery formation and limb perfusion and less tissue damage than BALB/c mice after HLI.
      • Helisch A.
      • Wagner S.
      • Khan N.
      • Drinane M.
      • Wolfram S.
      • Heil M.
      • Ziegelhoeffer T.
      • Brandt U.
      • Pearlman J.D.
      • Swartz H.M.
      • Schaper W.
      Impact of mouse strain differences in innate hindlimb collateral vasculature.
      Nonetheless, little is known about the genetic mechanisms responsible for these differences in phenotype. Chalothorn et al
      • Chalothorn D.
      • Clayton J.A.
      • Zhang H.
      • Pomp D.
      • Faber J.E.
      Collateral density, remodeling, and VEGF-A expression differ widely between mouse strains.
      demonstrated significantly lower expression of vascular endothelial growth factor A (VEGF-A) in response to HLI in BALB/c mice compared with BL6, suggesting that insufficient angiogenesis or collateralization is responsible for the poor recovery of BALB/c mice. In that study, a bioinformatics approach was used to identify a putative expression quantitative trait locus (QTL) for VEGF-A expression on mouse chromosome 17, suggesting a polymorphism in BALB/c mice that may be responsible for reduced VEGF-A expression.
      To investigate the genetic mechanisms responsible for the ischemic response in more detail, our group recently performed genome-wide scanning with polymorphic markers in BL6×BALB/c offspring.
      • Dokun A.O.
      • Keum S.
      • Hazarika S.
      • Li Y.
      • Lamonte G.M.
      • Wheeler F.
      • Marchuk D.A.
      • Annex B.H.
      A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.
      A QTL linked to perfusion recovery and limb necrosis was identified on chromosome 7 (Lsq-1) with an extremely high degree of statistical significance [logarithm of the odds (LOD) score > 7.9]. Using haplotype mapping in BL6, BALB/c, and A/J mice, in which the ischemic response is similar to that of BALB/c, this QTL was narrowed to a region encompassing 37 genes. The same locus was subsequently linked to the regulation of pial collateral vessel number and limitation of cerebral infarct size after middle cerebral artery occlusion.
      • Wang S.
      • Zhang H.
      • Dai X.
      • Sealock R.
      • Faber J.E.
      Genetic architecture underlying variation in extent and remodeling of the collateral circulation.
      • Keum S.
      • Marchuk D.A.
      A locus mapping to mouse chromosome 7 determines infarct volume in a mouse model of ischemic stroke.
      Notably, however, none of the candidate genes in this QTL has a previously defined role in vascular growth, suggesting an as yet undiscovered vascular function for at least one of these genes, and/or that genetic influences on other cellular processes play an important role in the response to ischemia.
      Numerous studies of the effects of ischemia on muscle tissue have centered on vascular cell responses or collateral vessel density, perhaps in part because targeting the vasculature through therapeutic angiogenesis holds promise as a potential treatment for ischemic diseases such as PAD.
      • Simons M.
      Angiogenesis: where do we stand now.
      Although studies have examined the effects of ischemia on other aspects of the limb muscle response, including mitochondrial biogenesis and the transition of muscle fibers to more ischemia-tolerant phenotypes,
      • Arany Z.
      • Foo S.Y.
      • Ma Y.
      • Ruas J.L.
      • Bommi-Reddy A.
      • Girnun G.
      • Cooper M.
      • Laznik D.
      • Chinsomboon J.
      • Rangwala S.M.
      • Baek K.H.
      • Rosenzweig A.
      • Spiegelman B.M.
      HIF-independent regulation of VEGF and angiogenesis by the transcriptional coactivator PGC-1alpha.
      • van Weel V.
      • Deckers M.M.L.
      • Grimbergen J.M.
      • van Leuven K.J.M.
      • Lardenoye J.H.P.
      • Schlingemann R.O.
      • van Nieuw Amerongen G.P.
      • van Bockel J.H.
      • van Hinsbergh V.W.M.
      • Quax P.H.A.
      Vascular endothelial growth factor overexpression in ischemic skeletal muscle enhances myoglobin expression in vivo.
      • Williams R.S.
      • Annex B.H.
      Plasticity of myocytes and capillaries: a possible coordinating role for VEGF.
      little is known about genetic modifiers of the skeletal muscle cell response to ischemia. Furthermore, it is unclear whether vascular cells or muscle cells are more susceptible to ischemia-induced injury. In the present study, we used both in vitro and in vivo models to investigate the genetic influence on the skeletal muscle cell response to ischemia. Here we demonstrate mouse strain-dependent differences in the myogenic regulatory program in response to HLI in vivo and show that these differences are recapitulated in isolated primary skeletal muscle cells in vitro. Furthermore, we demonstrate that the muscle cell-specific expression of vascular growth factors and their cognate receptors in vivo and in vitro in response to ischemia is genetically determined. These results provide novel insights into the genetic determinants of severe limb ischemia, such as that caused by CLI, by demonstrating that the same genetic locus linked to strain-dependent collateral vessel density also has a nonvascular or muscle cell-autonomous role. These findings establish that muscle–specific responses play a greater role than previously thought in determining pathological outcomes in response to ischemia.

      Materials and Methods

      Animals

      Experiments were conducted on 6- to 8-week-old adult male C57BL/6 or BALB/c mice (Jackson Laboratory, Bar Harbor, ME) and were approved by the Duke University Institutional Animal Care and Use Committee. Surgical hindlimb ischemia was performed as described previously.
      • Couffinhal T.
      • Silver M.
      • Zheng L.P.
      • Kearney M.
      • Witzenbichler B.
      • Isner J.M.
      Mouse model of angiogenesis.
      • Dokun A.O.
      • Keum S.
      • Hazarika S.
      • Li Y.
      • Lamonte G.M.
      • Wheeler F.
      • Marchuk D.A.
      • Annex B.H.
      A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.
      Briefly, ischemia was induced by anesthetizing mice by injection of ketamine (90 mg/kg i.p.) and xylazine (10 mg/kg i.p.), and unilateral hindlimb ischemia was surgically induced by ligation and excision of the femoral artery from its origin just above the inguinal ligament to its bifurcation at the origin of the saphenous and popliteal arteries. The inferior epigastric, lateral circumflex, and superficial epigastric artery branches were also isolated and ligated. Mice were closely monitored during the postoperative period, and perfusion in the ischemic and contralateral nonischemic limbs was measured immediately after surgery to verify successful ischemia.

      Necrosis Score

      The extent of necrosis in ischemic hindlimbs, if any, was recorded postoperatively using a previously described semiquantitative scale
      • Dokun A.O.
      • Keum S.
      • Hazarika S.
      • Li Y.
      • Lamonte G.M.
      • Wheeler F.
      • Marchuk D.A.
      • Annex B.H.
      A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.
      : grade 0, no necrosis in ischemic limb; grade I, necrosis limited to toes; grade II, necrosis extending to dorsum pedis; grade III, necrosis extending to crus; and grade IV, necrosis extending to thigh or complete limb necrosis.

      Immunofluorescence Microscopy

      Immunofluorescence microscopy was used for visualization of muscle morphology, capillary density, apoptosis, and monocyte infiltration in ischemic and control skeletal muscle. Transverse sections (8 μm thick) were cut from tibialis anterior (TA) muscle frozen in liquid nitrogen-cooled isopentane in optimum cutting temperature (OCT) medium. Sections were allowed to come to room temperature and then were fixed/permeabilized with ice-cold acetone for 10 minutes at 4°C. Sections were allowed to air dry for 5 minutes at room temperature and then were rehydrated in 1× PBS before blocking in 5% normal goat serum (Sigma-Aldrich, St. Louis, MO) in 1× PBS at room temperature for 45 minutes. Slides were then incubated overnight at 4°C in a primary antibody solution containing either rat anti-CD31 (1:50; MCA-1364; AbD Serotec, Raleigh, NC) or mouse IgG2b anti-CD11 (1:100; MAS 034; Harlan Laboratories, Indianapolis, IN) and mouse IgG2a anti-dystrophin supernatant (1:5; MANDYS1 3B7; developed by G.E. Morris and obtained from the Developmental Studies Hybridoma Bank under the auspices of the NIH National Institute of Child Health and Human Development and maintained by the University of Iowa Department of Biology, Iowa City, IA). Slides were then washed three times in 1× PBS at room temperature and incubated for 1 hour at room temperature in the dark in a secondary antibody solution containing Alexa Fluor 488-, 568-, or 633-conjugated secondary antibodies in blocking solution (all at 1:250 dilution). Sections were then washed three times for 5 minutes each in the dark with 1× PBS at room temperature. Coverslips were mounted using Vectashield HardSet mounting medium with DAPI (H-1500; Vector Laboratories, Burlingame, CA). TUNEL immunofluorescence staining (Invitrogen; Life Technologies, Carlsbad, CA) was performed according to the manufacturer's recommendations. Images were captured using a Zeiss Axio Observer inverted laser scanning microscope LSM 510 using Zeiss LSM 510 software version 4.2 and were analyzed offline using ImageJ software version 1.43u (NIH, Bethesda, MD).

      Myofiber Cross-Sectional Area

      Frozen sections from TA muscle samples were stained with H&E. Digital images were obtained at ×20 magnification, and myofiber cross-sectional area (in μm2) was quantified by a single blinded investigator from approximately 300 myofibers per animal, using NIH ImageJ image analysis software version 1.43u.

      Cell Lines and Culture

      Immortalized murine C2C12 and rat L6 skeletal muscle cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). Cells were propagated in growth medium [Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% penicillin/streptomycin and 0.2% amphotericin B, and 10% fetal bovine serum]. Differentiation was stimulated by serum withdrawal in differentiation medium (DMEM supplemented with 2% horse serum, 1% penicillin/streptomycin, 0.2% amphotericin B, and 0.01% human insulin/transferrin/selenium). Immortalized EC-RF24 (ECRF) cells (described by Fontijn et al
      • Fontijn R.
      • Hop C.
      • Brinkman H.J.
      • Slater R.
      • Westerveld A.
      • van Mourik J.A.
      • Pannekoek H.
      Maintenance of vascular endothelial cell-specific properties after immortalization with an amphotrophic replication-deficient retrovirus containing human papilloma virus 16 E6/E7 DNA.
      ) were a gift from Ruud Fontijn and were propagated in DMEM supplemented with 1% penicillin/streptomycin, 0.2% amphotericin B, and 10% fetal bovine serum. Human umbilical vein endothelial cells (HUVECs) were isolated from donor placental umbilical veins and were used before passage 6. HUVECs were propagated on 0.1% gelatin-coated dishes in endothelial basal medium (Lonza, Walkersville, MD) supplemented with 20% fetal bovine serum, 1% penicillin/streptomycin and 0.2% amphotericin B, and an EGM-MV bullet kit (Lonza, Walkersville, MD) containing epidermal growth factor, bovine brain extract, hydrocortisone, and gentamicin. To evaluate the effects of ischemia/hypoxia in vitro, we used an established model of cellular hypoxia in which cells are subjected to 0% O2 and deprived of nutrients in Hank's balanced salt solution
      • Arany Z.
      • Foo S.Y.
      • Ma Y.
      • Ruas J.L.
      • Bommi-Reddy A.
      • Girnun G.
      • Cooper M.
      • Laznik D.
      • Chinsomboon J.
      • Rangwala S.M.
      • Baek K.H.
      • Rosenzweig A.
      • Spiegelman B.M.
      HIF-independent regulation of VEGF and angiogenesis by the transcriptional coactivator PGC-1alpha.
      to mimic the local environment resulting from severe ischemia in PAD, referred to hereafter as hypoxia plus nutrient deprivation (HND).

      Primary Myoblast Isolation and Culture

      Primary murine muscle precursor cells (mouse myoblasts) derived from diaphragm and hindlimb muscles were prepared as described previously.
      • Lees S.J.
      • Rathbone C.R.
      • Booth F.W.
      Age-associated decrease in muscle precursor cell differentiation.
      • Mitchell P.O.
      • Pavlath G.K.
      Skeletal muscle atrophy leads to loss and dysfunction of muscle precursor cells.
      Briefly, hindlimb and diaphragm muscles were extracted from 4- to 6-week-old female C57BL/6 or BALB/c mice, digested with pronase (0.2%) for 60 minutes at 37°C, and then triturated to release precursor cells. Individual cells were washed in DMEM and PBS and then preplated for 3 hours on tissue culture polystyrene dishes; the cellular supernatant was transferred into collagen-coated (1 mg/mL in DMEM) plates and maintained in growth medium (Ham's F10 medium supplemented with 1% penicillin/streptomycin and 0.2% amphotericin B, 20% fetal bovine serum, and 2.5 ng/mL hFGF) for 4 days, which allowed myoblasts to attach to the substratum and proliferate. Myoblasts were then propagated in growth medium and used in experiments within four passages of isolation. Differentiation was stimulated by serum withdrawal in differentiation medium (DMEM supplemented with 2% horse serum, 1% penicillin/streptomycin, 0.2% amphotericin B, and 0.01% human insulin/transferrin/selenium) and verified by light microscopy and quantitative RT-PCR for the myogenic regulatory factor myogenin. Analysis of the effects of recombinant VEGF on myoblast differentiation was performed by plating equal numbers of myoblasts on plates coated with entactin, collagen, and laminin and allowing cells to reach 90% confluence. Cells were then treated with PBS or recombinant human vascular endothelial growth factor (rH-VEGF; 50 ng/mL) in growth medium for 24 hours and analyzed by quantitative RT-PCR.

      DNA Fragmentation

      DNA fragmentation was analyzed on 1% agarose gels using 2 μg genomic DNA isolated from cells using an apoptotic-DNA ladder kit (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's instructions.

      Myotube Diameter

      Muscle myotube diameter was quantified as described previously.
      • Menconi M.
      • Gonnella P.
      • Petkova V.
      • Lecker S.
      • Hasselgren P.O.
      Dexamethasone and corticosterone induce similar, but not identical, muscle wasting responses in cultured L6 and C2C12 myotubes.
      Briefly, images of myotubes after treatment were acquired by phase contrast microscopy at ×100 magnification on an Olympus IX70 inverted microscope connected to a PAXCam ARC digital camera system (MIS, Franklin Park, IL). Diameters were measured in ∼100 myotubes from at least 10 random fields (a number chosen by determining no additional change in standard deviation) using NIH ImageJ image analysis software version 1.43u. Each myotube analyzed was measured at three points along the length of the myotube in a blinded fashion, and results are expressed as a percentage of the control treatment diameter.

      Mitochondrial DNA Content

      Cellular mitochondrial DNA content was analyzed in total cellular DNA by quantitative RT-PCR using genomic DNA isolated from cells using an apoptotic-DNA ladder kit (Roche Diagnostics) according to the manufacturer's instructions. A primer set against NADH dehydrogenase subunit 2 (Nd2) was used to quantify the mitochondrial genome and was corrected for the nuclear-specific gene Nme1 by quantitative real-time PCR using an ABI 7300 Real-Time PCR System (Applied Biosystems; Life Technologies, Foster City, CA) as described previously.
      • Zhang H.
      • Bosch-Marce M.
      • Shimoda L.A.
      • Tan Y.S.
      • Baek J.H.
      • Wesley J.B.
      • Gonzalez F.J.
      • Semenza G.L.
      Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia.

      Quantitative RT-PCR

      Total RNA was extracted from mouse gastrocnemius muscles, primary murine skeletal myoblasts, C2C12 murine myoblasts, L6 rat myoblasts, ECRF cells, and HUVECs using TRIzol (Invitrogen) phenol/chloroform extraction. RNA (5 μg) was reverse-transcribed using SuperScript III Reverse Transcriptase and random primers (Invitrogen). Reactions were incubated at 50°C for 50 minutes and at 85°C for 5 minutes. Real-time PCR was performed using an ABI 7300 system (Applied Biosystems). Relative quantification of GADD45, p21, MyoD, myogenin, paired box gene (Pax7), Pgc1-α, Ang-1, Ang-2, Tie1, Tie2, VEGF, VEGFR-1/Flt, VEGFR-2/Flk, and NRP-1 mRNA levels were determined using the comparative threshold cycle (ΔΔCT) method using FAM TaqMan gene expression assays (Applied Biosystems) specific for each of these genes run in complex (multiplex) with a VIC-labeled GAPDH control primer.

      Muscle Cell Hypoxia Response

      Strain-specific primary myotube hypoxia responses were determined in vitro using an adenoviral hypoxia response element-luciferase reporter construct.
      • Cao Y.
      • Li C.Y.
      • Moeller B.J.
      • Yu D.
      • Zhao Y.
      • Dreher M.R.
      • Shan S.
      • Dewhirst M.W.
      Observation of incipient tumor angiogenesis that is independent of hypoxia and hypoxia inducible factor-1 activation.
      • Lima B.
      • Lam G.K.
      • Xie L.
      • Diesen D.L.
      • Villamizar N.
      • Nienaber J.
      • Messina E.
      • Bowles D.
      • Kontos C.D.
      • Hare J.M.
      • Stamler J.S.
      • Rockman H.A.
      Endogenous S-nitrosothiols protect against myocardial injury.
      Cells were adenovirally transduced with a hypoxia response element-luciferase reporter construct and subjected to HND; luciferin substrate (Promega, Madison, WI) was added and luciferase activity was measured by bioluminescence imaging (IVIS; Xenogen, Hopkinton, MA).

      Immunoblotting

      Cell cultures or gastrocnemius muscles were washed twice with ice-cold PBS and lysed on ice in a lysis buffer consisting of 50 mmol/L HEPES, 150 mmol/L NaCl, 100 mmol/L NaF, 5 mmol/L EDTA, 0.5% Triton X-100, and protease inhibitors (5 mg/mL aprotinin, 2 mg/mL leupeptin, and 100 mmol/L phenylmethylsulfonyl fluoride). Lysates were centrifuged at 1000 × g for 5 minutes, and the supernatant (30 to 80 μg total protein) was boiled for 5 minutes in Laemmli sample buffer, and proteins were separated by SDS-PAGE and analyzed by Western blotting. Antibodies were against phospho-ERK1/2 (Thr202/Tyr204), total ERK1/2, phospho-Akt (Thr308/Ser473), total Akt, phospho-mTOR (Ser2481), phospho-p70S6K1 (Thr389), total p70S6K1, phospho-FoxO3a (Ser253), Bax, Bcl-2, and total and cleaved caspase-3 (all purchased from Cell Signaling Technology, Danvers, MA). Loading and transfer of equal amounts of protein was confirmed by Ponceau staining and stripping the membranes and reprobing with antibodies against α-tubulin (for in vitro cell lysate analysis) (Sigma-Aldrich) or GAPDH (for in vivo gastrocnemius tissue analysis) (Novus Biologicals, Littleton, CO).

      Statistical Analysis

      Statistical analysis of within-group differences was performed using one-way analysis of variance; between-group comparisons were performed using Student's t-test. A P value of ≤0.05 was considered statistically significant.

      Results

      Skeletal Muscle Cells, but Not Endothelial Cells, Display Nuclear and Mitochondrial Damage after Hypoxia and Nutrient Deprivation

      To evaluate the individual responses of different cell types (endothelial and muscle cells) primarily affected in the ischemic limb, we used an established in vitro model of cellular hypoxia in which cells are subjected to hypoxia and nutrient deprivation (HND) by incubation in 0% O2 and Hank's balanced salt solution,
      • Arany Z.
      • Foo S.Y.
      • Ma Y.
      • Ruas J.L.
      • Bommi-Reddy A.
      • Girnun G.
      • Cooper M.
      • Laznik D.
      • Chinsomboon J.
      • Rangwala S.M.
      • Baek K.H.
      • Rosenzweig A.
      • Spiegelman B.M.
      HIF-independent regulation of VEGF and angiogenesis by the transcriptional coactivator PGC-1alpha.
      in an attempt to reproduce the local O2 and nutrient-depleted environment resulting from severe ischemia in PAD. Initially, we examined effects of HND on skeletal myotube and endothelial cell survival. Three hours of HND induced DNA fragmentation (Figure 1A) in two different immortalized muscle lines, as well as in primary skeletal myotubes. DNA fragmentation was not increased in either of two different types of endothelial cells (Figure 1B). Mitochondrial DNA content decreased with HND in all muscle cell types tested (Figure 1C). Notably, this reduced mitochondrial:nuclear DNA ratio was a primary mitochondrial effect, in that there was a loss of mitochondrial Nd2 without change in total genomic DNA (nuclear-specific Nme1). Somewhat surprisingly, mitochondrial DNA content was either unchanged (immortalized ECRF cells) or increased significantly (primary HUVECs) in endothelial cells (Figure 1D). Although this finding is consistent with the lack of apoptosis, it is opposite that of the skeletal muscle response and was due to an increase in copy number of the mitochondrial gene Nd2. In addition, Western blotting revealed an increase in the Bax/Bcl-2 protein expression ratio and caspase-3 cleavage in muscle cells (Figure 1E), but no change in either measure in endothelial cells (Figure 1F), further supporting a lack of endothelial cell injury at this time point. These findings demonstrate that endothelial cells and myotubes display markedly different responses to the same duration of HND insult, including different effects on myonuclear apoptosis and mitochondrial content.
      Figure thumbnail gr1
      Figure 1Muscle and endothelial cells respond differently to hypoxia and nutrient deprivation in vitro. Changes in DNA fragmentation (A and B), mitochondrial DNA content (C and D), and expression of Bax, Bcl-2, and cleaved caspase-3 (E and F) were assessed in immortalized (mouse C2C12 or rat L6) or primary BL6 myotubes (A, C, and E) and in immortalized human ECRF and primary HUVECs (B, D, and F) after 3 hours of hypoxia and nutrient deprivation (3 hours HND). *P < 0.05 versus cell-type control (Con).
      To further characterize the cachectic signaling responses of muscle to ischemia such as that occurring in PAD, mature myotubes were subjected to 3 hours HND and then were analyzed morphologically. All three muscle cell lines underwent significant atrophy (Figure 2, A and B). Consistent with this apparent loss of cellular protein, the phosphorylation of signaling proteins important for cell growth and protein synthesis, Akt and p70 S6 kinase, was markedly reduced (Figure 2C). Similarly, FoxO3a, a critical transcriptional regulator of proteolysis and atrophy in skeletal muscle,
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      FoxO3 controls autophagy in skeletal muscle in vivo.
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      Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy.
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      • Lecker S.H.
      • Goldberg A.L.
      FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells.
      was also dephosphorylated, consistent with its activation (Figure 2C). Accordingly, two ubiquitin ligases involved in atrophic skeletal muscle signaling, muscle atrophy F-box protein (MAFbx, or atrogin-1) and muscle-specific RING finger protein 1 (MuRF-1),
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      • Jagoe R.T.
      • Navon A.
      • Goldberg A.L.
      Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy.
      were transcriptionally up-regulated in response to 3 hours HND in L6 myotubes (Figure 2D). Taken together, these data demonstrate that muscle cells rapidly undergo atrophy in response to the HND insult.
      Figure thumbnail gr2
      Figure 2The myotube response to hypoxia and nutrient deprivation in vitro includes atrophic signaling pathways. Immortalized mouse C2C12 and rat L6 and primary BL6 myotubes were induced to differentiate, subjected to 3 hours HND, and analyzed for traditional protein synthetic and degradative responses. A: Representative images of differentiated C2C12 myotubes before (Con) and after 3 hours HND. B: Myotube diameter was quantified in each cell type before (Con) and after 3 hours HND. C: Cell lysates from control (Con) and 3-hour HND-treated myotubes were assessed by Western blot for proteins involved in synthetic (pAkt, p-p70S6K) and degradative (pFoxO3a) signaling; α-tubulin was used as a loading control. D: Changes in mRNA expression of the muscle-specific ubiquitin ligases MAFbx and MuRF-1 were quantified in C2C12 myotubes after control or 3-hour HND treatment. *P < 0.05 versus cell-type control.

      Strain-Specific Alterations in the Muscle Recovery Program Dictate the Pathological Response to Ischemia in Vivo

      Previously published reports have definitively established the critical influence of genetics on the differential responses to HLI in C57BL/6 (BL6) and BALB/c mice in vivo.
      • Chalothorn D.
      • Clayton J.A.
      • Zhang H.
      • Pomp D.
      • Faber J.E.
      Collateral density, remodeling, and VEGF-A expression differ widely between mouse strains.
      • Dokun A.O.
      • Keum S.
      • Hazarika S.
      • Li Y.
      • Lamonte G.M.
      • Wheeler F.
      • Marchuk D.A.
      • Annex B.H.
      A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.
      • Helisch A.
      • Wagner S.
      • Khan N.
      • Drinane M.
      • Wolfram S.
      • Heil M.
      • Ziegelhoeffer T.
      • Brandt U.
      • Pearlman J.D.
      • Swartz H.M.
      • Schaper W.
      Impact of mouse strain differences in innate hindlimb collateral vasculature.
      Specifically, perfusion recovers almost completely in BL6 mice, whereas BALB/c mice suffer severe limb necrosis. To expand on these findings and to extend our in vitro results in myocytes and endothelial cells, we examined whether strain-specific differences in the limb muscle response to ischemia suggest a myocyte-specific deficit. BALB/c mice responded to ischemia with significantly greater semiquantitative necrosis scores (Figure 3A), although capillary density (Figure 3B) was not significantly different in the two strains at baseline or after 7 days of HLI. Differential patterns of TUNEL staining were observed, with rampant nuclear apoptosis 1 day after HLI in both strains that decreased by day 7 in BL6 but not in BALB/c muscle (Figure 3C). Notably, entire BALB/c myofibers frequently stained TUNEL+, an effect not observed at any time point in the BL6 muscle. Immunofluorescence staining for monocytes revealed inflammatory cell infiltration in both strains, peaking at day 3 and subsequently declining by day 7 after HLI only in the BL6 muscle (Figure 3D). Immunofluorescence and H&E staining also revealed striking strain-dependent morphological differences during the initial week of recovery.
      Figure thumbnail gr3
      Figure 3BL6 and BALB/c mice display differential strain-dependent responses to ischemia in vivo. BL6 and BALB/c mice were subjected to hindlimb ischemia (HLI) or sham surgery (n = 3 per group) and allowed to recover for 1, 3, or 7 days. A: Limbs were analyzed for necrosis score at the three time points. NR, none recorded. B: Capillary density was assessed by CD31 staining of TA muscle and quantified as capillaries per fiber. C: Representative images of TA muscle from mice at 1-, 3-, and 7-day recovery times stained for dystrophin (myofibers, red), TUNEL (apoptosis, green), and DAPI (nuclei, blue). D: Representative images from TA muscle costained for dystrophin (red) and CD11b (monocytes, green). In C and D, note the greater and persistent apoptosis and monocyte infiltration. *P < 0.05 versus strain-specific control (Sham). Original magnification: ×200 (C and D).
      By day 7 after HLI, BL6 muscle showed clear signs of regeneration, with restoration of immunoreactive dystrophin staining and abundant centralized myonuclei (Figure 4A). In contrast, BALB/c muscle displayed continued loss of dystrophin immunoreactivity and lack of morphological indices of muscle regeneration. Although significant muscle atrophy, measured by changes in gastrocnemius muscle wet weight, was observed in both strains on day 7 after HLI, BALB/c muscle displayed a significant increase in weight at day 3, consistent with increased edema, followed by atrophy and necrosis (Figure 4B). Although gastrocnemius muscle fiber diameters were not significantly different between BALB/c and BL6 mice at baseline or at 7 days after HLI (Figure 4C), the number of myofibers with centralized nuclei, a histological marker of ongoing muscle regeneration, was markedly attenuated at day 7 of HLI in BALB/c mouse muscle (Figure 4D).
      Figure thumbnail gr4
      Figure 4BL6 and BALB/c myofibers differentially recover from ischemia in vivo. BL6 and BALB/c mice were subjected to hindlimb ischemia (HLI) or sham surgery (n = 3 per group) and allowed to recover for 7 days. A: Representative images from TA muscle stained for dystrophin (myofibers, red) and DAPI (nuclei, blue) or H&E for morphology. Note the attenuated myofiber recovery in BALB/c versus BL6. B: Gastrocnemius muscle tissue was removed at 1, 3, or 7 days after HLI and analyzed for wet weight. C: Mean myofiber cross-sectional area (CSA) was assessed in nonischemic (Sham) gastrocnemius muscle and 7 days after HLI. D: Percentage of TA muscle fibers with centralized nuclei was assessed 7 days after HLI. *P < 0.05 versus strain-specific control (Sham). Original magnification: ×200 (A, left column); ×300 (A, right column).
      Western blotting of gastrocnemius muscle lysates during the initial week of HLI revealed decreases (days 1 and 3) and subsequent recovery or increases (day 7) in the phosphorylation of protein synthetic signaling components (Akt, mTOR, p70 S6 kinase) in BL6 mice (Figure 5A). Similarly, dephosphorylation of FoxO3a, dephosphorylation of p42/44 MAPK, and a relative increase in the Bax/Bcl-2 protein ratio all occurred between days 1 and 3 but were rescued by day 7 of HLI in BL6 mice. BALB/c mice, however, demonstrated similar changes in each of these markers early, but failed to show recovery by day 7 (Figure 5A).
      Figure thumbnail gr5
      Figure 5The strain-dependent limb tissue response to ischemia in vivo alters the myogenic regulatory program. BL6 and BALB/c mice were subjected to HLI or sham surgery and allowed to recover for 1, 3, or 7 days. A: Gastrocnemius muscle homogenates were assessed by Western blot for markers of protein synthesis and degradation, proliferation (p42/44 ERK), and apoptosis (Bax/Bcl-2). B: Gastrocnemius muscle RNA was harvested and the fold change in mRNA expression of the muscle cell cycle regulatory genes (GADD45, p21, MyoD, myogenin) was determined by quantitative RT-PCR. C: Samples shown in panel B were analyzed by quantitative RT-PCR for fold changes in mRNA for myostatin, the muscle-specific ubiquitin ligases (MAFbx and MuRF-1), and the metabolic regulator PGC1-α. *P < 0.05 versus strain-specific control (Sham); P < 0.05 versus all other strain-specific time points; P < 0.05 versus BL6 control (Sham).
      Last of all, we analyzed the genetic program of muscle precursor cell differentiation and maturation in gastrocnemius muscle from both strains of mice after HLI (Figure 5B). Increases in mRNA expression of the cell cycle withdrawal factors GADD45 and p21 as well as the myogenic regulatory factors MyoD and myogenin were significantly attenuated in BALB/c mice in the initial week of HLI. Similarly, expression of myostatin, the ubiquitin ligases MAFbx and MuRF-1, and Pgc1-α was significantly suppressed in BALB/c mice during the initial week of HLI (Figure 5C). These gene expression data suggest that the normal atrophic and myogenic transcriptional programs activated by ischemia in BL6 mice are disrupted in BALB/c mice, potentially contributing to overall limb necrosis. Collectively, these results demonstrate an organized recovery of skeletal muscle after HLI in BL6 mice that is lacking in BALB/c mice, because of genetic variability.

      Strain-Specific Alterations in Muscle Precursor Cell Activation Regulate the Ischemic Response

      Our earlier in vitro data demonstrated that HND has a greater effect on muscle cells than on endothelial cells, suggesting that the inability of BALB/c mice to mount an effective survival and/or recovery response to HLI may be due to a deficit in muscle precursor cell activation. To investigate the strain-specific effects of ischemia/hypoxia on skeletal muscle precursor cells, we isolated primary satellite cells from both BL6 and BALB/c mice and subjected them to HND in vitro, subsequently allowing cells from both strains to recover under identical oxygenation and nutritional conditions. In this manner, we effectively eliminated any contribution of the vasculature to the muscle precursor cell regeneration or differentiation program. The overall capacity to mount a hypoxia response, as evidenced using a hypoxia response element-luciferase reporter, was similar for primary muscle cells from both strains (data not shown). Morphologically, primary myoblasts and myotubes from BL6 and BALB/c mice looked similar and differentiated similarly (Figure 6A). Myotubes from both strains displayed a similar degree of atrophy after 3 hours HND, but BALB/c precursor cells failed to recover over 5 days (Figure 6, A and B). This morphological defect was accompanied by attenuated expression of the anti-apoptotic protein Bcl-2 (Figure 6C), as well as blunted phosphorylation of Akt and p70 S6 kinase and sustained dephosphorylation of FoxO3a (Figure 6D). The genetic program of precursor cell activation and differentiation during recovery from HND revealed similarities in the expression patterns of the cyclin D1, GADD45, and p21 cell cycle regulatory genes, suggesting no differences in satellite cell proliferative capacity. However, significant deficits were observed in BALB/c muscle cells in the expression of the activity marker PAX7 and the muscle-specific myogenic regulatory factors MyoD and myogenin (Figure 6F). Expression of the hypoxia-responsive atrophic gene myostatin increased early in BALB/c myotubes, and the patterns of MAFbx and MuRF-1 induction remained similar to those of BL6 during recovery (Figure 6F). Together, these data demonstrate that BALB/c muscle precursor cells exhibit significant defects in the activation/regeneration program necessary for recovery from hypoxia and nutrient deprivation.
      Figure thumbnail gr6
      Figure 6Primary skeletal muscle cells display strain-dependent differences in the myogenic regulatory program in vitro. Primary myoblasts were isolated from BL6 and BALB/c mice, induced to differentiate, then subjected to 3 hours HND and allowed to recover for various lengths of time. A: Representative images of cultured skeletal myotubes subjected to 3 hours HND and 72 hours recovery (Rec) in normal O2 and nutrients. B: Primary myotube diameters were quantified. C and D: Primary myotube lysates were Western blotted with antibodies to assess changes in apoptosis (C) and regulators of protein synthesis (D) during HND and recovery. E: Diameters of primary myotubes from congenic BALB/cBL6 QTL7 mice were quantified. F: RNA was harvested from primary myotubes and used for quantitative RT-PCR to quantify fold changes in mRNA abundance of nine cell cycle or myogenic regulatory genes. *P < 0.05 versus strain-specific control (Con). P < 0.05 versus all other strain-specific time points; P < 0.05 versus BL6 control (Sham).
      To investigate whether the observed differences in the satellite cell response to ischemia are due to the previously identified chromosome 7 QTL, we used primary myoblasts isolated from congenic BALB/c mice in which the 37-gene Lsq-1 QTL was selectively replaced with the same locus from BL6 mice (BALB/cBL6 QTL7).
      • Dokun A.O.
      • Keum S.
      • Hazarika S.
      • Li Y.
      • Lamonte G.M.
      • Wheeler F.
      • Marchuk D.A.
      • Annex B.H.
      A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.
      Primary myoblasts and myotubes from these mice appeared morphologically similar to those of the other two strains and differentiated normally (data not shown). HND-induced atrophy of BALB/cBL6 QTL7 myotubes was similar to that of both parental strains after 3 hours; however, unlike BALB/c primary cells, these cells recovered fully over 5 days, like the BL6 myocytes (Figure 6E). These findings confirm that genes within the Lsq-1 QTL are responsible for the muscle cell-specific response to ischemia.

      Muscle Precursor Cells Display Strain-Specific Differences in Expression of Vascular Growth Factors and their Receptors

      Crosstalk between muscle precursor cells and the vasculature plays an important role in the recovery of muscle tissue from ischemia. Satellite cells, which reside in close proximity to myofiber capillaries, receive support from endothelial cells in the form of growth factors (bFGF, IGF-1, HGF, VEGF, and PDGF-BB). In return, satellite cells express VEGF to stimulate neovascularization.
      • Christov C.
      • Chretien F.
      • Abou-Khalil R.
      • Bassez G.
      • Vallet G.
      • Authier F.J.
      • Bassaglia Y.
      • Shinin V.
      • Tajbakhsh S.
      • Chazaud B.
      • Gherardi R.K.
      Muscle satellite cells and endothelial cells: close neighbors and privileged partners.
      • Hawke T.J.
      • Garry D.J.
      Myogenic satellite cells: physiology to molecular biology [Erratum appeared in J Appl Physiol 2001, 91:2414].
      Although evidence shows that receptors expressed predominantly on endothelial cells (Tie and VEGF receptors) are also expressed on muscle cells,
      • Germani A.
      • Di Carlo A.
      • Mangoni A.
      • Straino S.
      • Giacinti C.
      • Turrini P.
      • Biglioli P.
      • Capogrossi M.C.
      Vascular endothelial growth factor modulates skeletal myoblast function.
      • Abou-Khalil R.
      • Le Grand F.
      • Pallafacchina G.
      • Valable S.
      • Authier F.J.
      • Rudnicki M.A.
      • Gherardi R.K.
      • Germain S.
      • Chretien F.
      • Sotiropoulos A.
      • Lafuste P.
      • Montarras D.
      • Chazaud B.
      Autocrine and paracrine angiopoietin 1/Tie-2 signaling promotes muscle satellite cell self-renewal.
      it remains unclear whether these receptors and their ligands (angiopoietins and VEGF) play a role in determining the survival and recovery of myofibers after ischemia. We first investigated the strain-specific differences in ischemic muscle expression of vascular growth factors and their receptors in vivo. Analysis of gastrocnemius tissue from BL6 and BALB/c mice revealed similar patterns of expression of angiopoietins 1 and 2 (Ang-1 and Ang-2) and their receptor tyrosine kinases Tie1 and Tie2 (Figure 7, A, C, E, and G). Under ischemic conditions, limb muscle expression of the Ang-1 ligand and both Tie1 and Tie2 receptors decreased, whereas Ang-2 mRNA was significantly increased on day 1 of HLI. Because this analysis reflects expression from both muscle and nonmuscle cells, we next investigated expression of these factors in primary myoblasts from both BL6 and BALB/c mice subjected to HND. Notably, in vitro analysis revealed similar patterns of expression of Ang-1 (Figure 7B) and Tie2 (Figure 7H) for both strains, but significantly different expression patterns of Ang-2 (Figure 7D) and Tie1 mRNA (Figure 7F) during recovery from HND. These findings revealed a specific Ang-2/Tie1 ligand/receptor alteration in BALB/c precursor cells that was masked by in vivo analysis of whole limb muscle.
      Figure thumbnail gr7
      Figure 7Skeletal muscle cells exhibit strain-dependent differences in angiopoietin ligand and Tie receptor gene expression. RNA was harvested from gastrocnemius muscle in vivo (A, C, E, and G) and primary myotubes in vitro (B, D, F, and H) from BL6 and BALB/c mice, and the fold change in mRNA expression was determined by quantitative RT-PCR for Ang-1, Ang-2, Tie1, and Tie2. In all cases, BL6 Sham and BL6 control (Con) were normalized to 1.0. *P < 0.05 versus cell- or strain-specific control. P < 0.05 versus all other strain-specific time points; P < 0.05 versus BL6 control (Sham).
      VEGF plays a critical role in skeletal muscle angiogenesis and vascular survival and has been shown to regulate myoblast proliferation.
      • Germani A.
      • Di Carlo A.
      • Mangoni A.
      • Straino S.
      • Giacinti C.
      • Turrini P.
      • Biglioli P.
      • Capogrossi M.C.
      Vascular endothelial growth factor modulates skeletal myoblast function.
      • Tang K.
      • Breen E.C.
      • Gerber H.P.
      • Ferrara N.M.
      • Wagner P.D.
      Capillary regression in vascular endothelial growth factor-deficient skeletal muscle.
      • Lee S.
      • Chen T.T.
      • Barber C.L.
      • Jordan M.C.
      • Murdock J.
      • Desai S.
      • Ferrara N.
      • Nagy A.
      • Roos K.P.
      • Iruela-Arispe M.L.
      Autocrine VEGF signaling is required for vascular homeostasis.
      Therefore, we next investigated the strain-specific differences in VEGF expression in response to ischemia in vivo and HND in vitro. We subjected both HUVECs (Figure 8A) and differentiated C2C12 myotubes (Figure 8B) to sustained HND for 3, 8, or 24 hours and analyzed VEGF mRNA expression using quantitative RT-PCR. VEGF mRNA increased with all durations of HND in both HUVECs and C2C12 cells, although the degree of up-regulation was markedly greater (>40-fold) after 24 hours HND in C2C12 myotubes. Analysis of gastrocnemius muscle revealed significantly greater baseline VEGF mRNA expression in BALB/c mice, but no induction during the week of recovery from HLI, in contrast to a significant increase on day 1 in BL6 muscle (Figure 8C). In vitro analysis of muscle precursor cell-specific expression revealed no difference between strains in VEGF expression during recovery from HND (Figure 8D), suggesting that the differences observed in vivo might be attributable to other cell types, such as endothelial cells and inflammatory cells.
      Figure thumbnail gr8
      Figure 8Cell type- and strain-dependent differences in VEGF expression. A and B: HUVECs (A) and C2C12 myotubes (B) were subjected to HND for 3, 8 or 24 hours and the fold change in VEGF mRNA was determined by quantitative RT-PCR. C and D: RNA was harvested from gastrocnemius muscle (C) or primary myotubes (D) from BL6 and BALB/c mice. Fold change in VEGF mRNA was determined by quantitative RT-PCR. *P < 0.05 versus cell- or strain-specific control (Con). P < 0.05 versus all other strain-specific time points; P < 0.05 versus BL6 control (Sham).
      To use VEGF ligand, cells must express sufficient levels of VEGF receptors. In vivo analysis of gastrocnemius muscle demonstrated higher baseline expression of VEGFR-2 and NRP-1 in BALB/c muscle, but similar patterns of expression of VEGFR-1 (Figure 9A), VEGFR-2 (Figure 9C), and NRP-1 (Figure 9E) in BL6 and BALB/c mice after HLI. In analysis of primary muscle precursor cells in vitro, myoblasts from BL6 mice demonstrated significantly increased expression of VEGFR-1 (Figure 9B), VEGFR-2 (Figure 9D), and NRP-1 (Figure 9F) during recovery from HND. In stark contrast, BALB/c myoblast expression of all three VEGF receptors was not only significantly lower at baseline but also never increased during recovery, suggesting a strain-specific deficit in the ability of satellite cells to respond to VEGF ligand-induced signaling during recovery from ischemia.
      Figure thumbnail gr9
      Figure 9Skeletal muscle cells exhibit strain-dependent differences in VEGF receptor gene expression. RNA was harvested from gastrocnemius muscle (A, C, and E) or primary myotubes (B, D, and F) from BL6 and BALB/c mice. Fold change in VEGF receptor mRNA was determined by quantitative RT-PCR. *P < 0.05 versus cell- or strain-specific control (Con). P < 0.05 versus all other strain-specific time points; P < 0.05 versus BL6 control (Sham).
      To investigate the potential biological relevance of this deficit in VEGF receptor expression, we examined the effects of VEGF on skeletal myoblast differentiation, as would occur during satellite cell activation in response to muscle injury. Confluent myoblasts were treated with PBS or recombinant VEGF for 24 hours in normal growth medium. VEGF decreased expression of the cell cycle regulator cyclin D1 (Figure 10A) and significantly increased expression of the cell cycle withdrawal factor p21 (Figure 10B) and the myogenic regulatory factors MyoD (Figure 10C) and myogenin (Figure 10D). These findings demonstrate that VEGF induces a gene expression pattern consistent with myoblast differentiation under these conditions, and they also support the possibility that deficits in myoblast VEGF receptor expression could contribute to the impaired response to ischemia in BALB/c mice.
      Figure thumbnail gr10
      Figure 10Exogenous VEGF enhances myoblast differentiation. Primary mouse myoblasts were grown to confluence and treated with PBS or recombinant human vascular endothelial growth factor (rH-VEGF; 50 ng/mL) in growth medium for 24 hours, and RNA was harvested and analyzed by quantitative RT-PCR for expression of cyclin D1 (A), p21 (B), MyoD (C), and myogenin (D). *P < 0.05 versus PBS-treated myoblasts.

      Discussion

      It has become increasingly apparent that the overall degree of tissue damage in response to ischemia is mechanistically linked to individual genetic differences, and there is a critical need to identify the factors that regulate this response.
      • Dokun A.O.
      • Keum S.
      • Hazarika S.
      • Li Y.
      • Lamonte G.M.
      • Wheeler F.
      • Marchuk D.A.
      • Annex B.H.
      A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.
      • Keum S.
      • Marchuk D.A.
      A locus mapping to mouse chromosome 7 determines infarct volume in a mouse model of ischemic stroke.
      With the present study, we have demonstrated that the responses of skeletal muscle cells play a major role in tissue survival after ischemia. In particular, genetic differences in affected skeletal muscle cells may be a major factor determining the severity of disease in PAD. Our findings demonstrate that the transcriptional program of myogenic precursor cell activation is retarded in a strain-dependent manner after tissue ischemia in vivo and HND in vitro. These findings shed important light on the individual cellular responses to ischemia and suggest that myocytes themselves may in fact represent an effective target for therapeutic intervention in patients with CLI. Furthermore, we expanded on these observations by demonstrating that skeletal muscle cells are capable not only of inducing an angiogenic transcriptional program through the expression of vascular growth factors (VEGF and the angiopoietins) but also of responding to these factors in an autocrine manner via the expression of Tie and VEGF receptors. Although we observed few strain-dependent differences in angiogenic growth factor expression, differences in Tie1 and VEGF receptor expression were significant, suggesting that the autocrine responses of muscle cells to angiogenic signals may play an important role in the tissue response to ischemia. Taken together, our results suggest that the genetic susceptibility of skeletal muscle to ischemia may be mediated largely by the responses of skeletal myofibers and satellite cells.
      In PAD, perfusion is insufficient to meet the metabolic demands of the tissue and so PAD frequently results in skeletal myopathy and mitochondrial dysfunction.
      • Brass E.P.
      • Wang H.
      • Hiatt W.R.
      Multiple skeletal muscle mitochondrial DNA deletions in patients with unilateral peripheral arterial disease.
      • Brass E.P.
      • Hiatt W.R.
      Acquired skeletal muscle metabolic myopathy in atherosclerotic peripheral arterial disease.
      The degree of muscle survival or necrosis determines the ultimate clinical manifestation of PAD, although previous studies have demonstrated that pre-existing collateral vessels are critical determinants of the response to ischemia and that these collateral vessels are also genetically determined.
      • Helisch A.
      • Wagner S.
      • Khan N.
      • Drinane M.
      • Wolfram S.
      • Heil M.
      • Ziegelhoeffer T.
      • Brandt U.
      • Pearlman J.D.
      • Swartz H.M.
      • Schaper W.
      Impact of mouse strain differences in innate hindlimb collateral vasculature.
      • Wang S.
      • Zhang H.
      • Dai X.
      • Sealock R.
      • Faber J.E.
      Genetic architecture underlying variation in extent and remodeling of the collateral circulation.
      • Keum S.
      • Marchuk D.A.
      A locus mapping to mouse chromosome 7 determines infarct volume in a mouse model of ischemic stroke.
      • Scholz D.
      • Ziegelhoeffer T.
      • Helisch A.
      • Wagner S.
      • Friedrich C.
      • Podzuweit T.
      • Schaper W.
      Contribution of arteriogenesis and angiogenesis to postocclusive hindlimb perfusion in mice.
      Our data do not rule out the possibility of pre-existing or nascent collateral vessels as key players in the ischemic response. However, using a well-controlled in vitro model of hypoxia and nutrient deprivation in which collateral blood vessels are absent, we found that myotubes are more susceptible than endothelial cells to the ischemic/hypoxic insult. The rapid response of skeletal muscle cells in this model represented a shift toward cellular catabolism and apoptosis, which could be reversed with restoration of normal culture conditions. This response, coupled with the relative tolerance of endothelial cells, suggests that in vivo skeletal muscle is programmed to preserve vascular integrity and to sacrifice myofiber homeostasis during ischemia. Accordingly, apoptosis was more pronounced in muscle than in endothelial cells in the present study. Given the unique ability of muscle to regenerate, our findings lend credence to the idea of evolutionary selection for muscle plasticity as a protective mechanism in ischemia until blood flow can be restored by collateral vessel formation or angiogenesis. Although it is unclear whether the BALB/c genotype confers any particular selection advantage in muscle (eg, differences in baseline metabolic function or efficiency), it is possible that similar polymorphisms contribute to human CLI.
      Previous work by our group
      • Dokun A.O.
      • Keum S.
      • Hazarika S.
      • Li Y.
      • Lamonte G.M.
      • Wheeler F.
      • Marchuk D.A.
      • Annex B.H.
      A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.
      • Keum S.
      • Marchuk D.A.
      A locus mapping to mouse chromosome 7 determines infarct volume in a mouse model of ischemic stroke.
      and by others
      • Helisch A.
      • Wagner S.
      • Khan N.
      • Drinane M.
      • Wolfram S.
      • Heil M.
      • Ziegelhoeffer T.
      • Brandt U.
      • Pearlman J.D.
      • Swartz H.M.
      • Schaper W.
      Impact of mouse strain differences in innate hindlimb collateral vasculature.
      • Wang S.
      • Zhang H.
      • Dai X.
      • Sealock R.
      • Faber J.E.
      Genetic architecture underlying variation in extent and remodeling of the collateral circulation.
      • Chalothorn D.
      • Faber J.E.
      Strain-dependent variation in collateral circulatory function in mouse hindlimb.
      has demonstrated differential vascular responses in C57BL/6 and BALB/c mouse strains in response to limb or cerebral ischemia. In our earlier study linking tissue necrosis and perfusion recovery after HLI to Lsq-1 on chromosome 7,
      • Dokun A.O.
      • Keum S.
      • Hazarika S.
      • Li Y.
      • Lamonte G.M.
      • Wheeler F.
      • Marchuk D.A.
      • Annex B.H.
      A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.
      it was somewhat surprising to find that none of the 37 genes implicated by haplotype analysis within this QTL had a previously described function in angiogenesis (eg, encoding angiogenic growth factors or receptors). Nonetheless, a number of these gene products have well-defined roles in the immune response (integrin αM/CD11b), tissue remodeling (MMP21, ADAM12), or metabolism (cytochrome c oxidase), which could be involved in arteriogenesis, angiogenesis, or cellular survival. Baseline collateral vessel number in hindlimb and brain has been shown to be strain-dependent,
      • Wang S.
      • Zhang H.
      • Dai X.
      • Sealock R.
      • Faber J.E.
      Genetic architecture underlying variation in extent and remodeling of the collateral circulation.
      • Chalothorn D.
      • Faber J.E.
      Strain-dependent variation in collateral circulatory function in mouse hindlimb.
      • Chalothorn D.
      • Faber J.E.
      Formation and maturation of the native cerebral collateral circulation.
      • Zhang H.
      • Prabhakar P.
      • Sealock R.
      • Faber J.E.
      Wide genetic variation in the native pial collateral circulation is a major determinant of variation in severity of stroke.
      and the genetic locus (Canq-1) linked to collateral phenotype maps to the same position as Lsq-1,
      • Wang S.
      • Zhang H.
      • Dai X.
      • Sealock R.
      • Faber J.E.
      Genetic architecture underlying variation in extent and remodeling of the collateral circulation.
      which has been linked to tissue survival after hindlimb and cerebral ischemia.
      • Dokun A.O.
      • Keum S.
      • Hazarika S.
      • Li Y.
      • Lamonte G.M.
      • Wheeler F.
      • Marchuk D.A.
      • Annex B.H.
      A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.
      • Keum S.
      • Marchuk D.A.
      A locus mapping to mouse chromosome 7 determines infarct volume in a mouse model of ischemic stroke.
      It is important to note that genetically determined differences in collateral vessel density likely contribute to tissue protection from ischemia. Because we focused on the initial 7-day response, it is possible that we caught a glimpse into the biology of the ischemic response before the onset of any changes in vascularity (either capillary regression or angiogenesis). Blood flow to the ischemic limb recovers almost completely over 21 days only in the BL6 strain, although both strains recover a large percentage of their perfusion deficit in the initial week of HLI.
      • Dokun A.O.
      • Keum S.
      • Hazarika S.
      • Li Y.
      • Lamonte G.M.
      • Wheeler F.
      • Marchuk D.A.
      • Annex B.H.
      A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.
      In the context of the present study, this may suggest that pre-existing collateral vessels and capillaries may determine flow to the tissue in the initial week, but that longer periods of ischemic injury might result in capillary regression and collateral vessel failure in BALB/c mice, in part due to extensive tissue necrosis, whereas BL6 mice might mount an angiogenic response. Nonetheless, our present observations in isolated muscle cells in vitro, including cells from BALB/cBL6 QTL7 mice, indicate that this locus plays an important role in the plasticity and survival of muscle precursor cells and myotubes, independent of the vascular contribution to the ischemic response. Although it is possible that collateral vessel density and the muscle response to ischemia are genetically coregulated, our data suggest that effects on nonvascular cells may be equally important or more important than effects on angiogenesis in determining the ultimate outcome of ischemia.
      Strain-specific differences in the regeneration capacity of muscle have generally been overlooked. After intramuscular toxin injection, BALB/c mice displayed pronounced collagen deposition and blunted myofiber regeneration, findings that were not observed in BL6 mice.
      • Lagrota-Candido J.
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      • Lannes-Vieira J.
      • Quirico-Santos T.
      Characteristic pattern of skeletal muscle remodelling in different mouse strains.
      Separate studies also demonstrated a BALB/c strain-specific deficit in muscle regeneration after crush injury or isografting.
      • Grounds M.D.
      Phagocytosis of necrotic muscle in muscle isografts is influenced by the strain, age, and sex of host mice.
      • Grounds M.D.
      • McGeachie J.K.
      A comparison of muscle precursor replication in crush-injured skeletal muscle of Swiss and BALBc mice.
      • McGeachie J.K.
      • Grounds M.D.
      Retarded myogenic cell replication in regenerating skeletal muscles of old mice: an autoradiographic study in young and old BALBc and SJL/J mice.
      • Mitchell C.A.
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      The genotype of bone marrow-derived inflammatory cells does not account for differences in skeletal muscle regeneration between SJL/J and BALB/c mice.
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      • Grounds M.D.
      The host environment determines strain-specific differences in the timing of skeletal muscle regeneration: cross-transplantation studies between SJL/J and BALB/c mice.
      A common hypothesis for this strain-dependent defect is that type 2 cytokine production (high IL-4) in BALB/c mice results in differential macrophage activity and delayed tissue phagocytosis and satellite cell activation.
      • Lagrota-Candido J.
      • Canella I.
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      • Santos-Silva L.P.
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      • Lannes-Vieira J.
      • Quirico-Santos T.
      Characteristic pattern of skeletal muscle remodelling in different mouse strains.
      The local environment likely also plays a critical role, with significant deficits observed in muscle precursor cell DNA synthesis, leukocyte infiltration, and myotube formation when BALB/c mice are used as transplantation recipients.
      • Roberts P.
      • McGeachie J.K.
      • Grounds M.D.
      The host environment determines strain-specific differences in the timing of skeletal muscle regeneration: cross-transplantation studies between SJL/J and BALB/c mice.
      BALB/c-specific delays in satellite cell activation after crush injury have been reported,
      • Grounds M.D.
      • McGeachie J.K.
      A comparison of muscle precursor replication in crush-injured skeletal muscle of Swiss and BALBc mice.
      • McGeachie J.K.
      • Grounds M.D.
      Retarded myogenic cell replication in regenerating skeletal muscles of old mice: an autoradiographic study in young and old BALBc and SJL/J mice.
      although it was subsequently determined that differences in bone marrow-derived inflammatory cells could not account for these findings.
      • Mitchell C.A.
      • Grounds M.D.
      • Papadimitriou J.M.
      The genotype of bone marrow-derived inflammatory cells does not account for differences in skeletal muscle regeneration between SJL/J and BALB/c mice.
      Our in vivo data demonstrate that macrophage infiltration proceeds along a similar timeline regardless of mouse strain, although inflammation persists longer in BALB/c muscle. Our in vitro data in primary myoblasts further support the idea that monocytes are not the primary cause of the muscle deficit and demonstrate that the genotype of the muscle cells themselves is a critical determinant of the myoblast differentiation program and resistance to hypoxia.
      Although the transcriptional response to hypoxia is classically linked to stabilization of hypoxia-inducible factor 1-α (HIF-1-α),
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      recent evidence has shown that VEGF expression and angiogenesis in limb ischemia are HIF-independent and peroxisome proliferator-activated receptor gamma coactivator (PGC) 1-α–dependent.
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      HIF-independent regulation of VEGF and angiogenesis by the transcriptional coactivator PGC-1alpha.
      Although this finding may be related to differential responses of endothelial and skeletal muscle cells, our results suggest a common program of angiopoietin and Tie receptor gene expression in limb muscle during the initial week of ischemia, independent of mouse strain. Whereas our results are consistent with previous studies demonstrating expression of the Tie receptors in myoblasts in vitro,
      • Abou-Khalil R.
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      Autocrine and paracrine angiopoietin 1/Tie-2 signaling promotes muscle satellite cell self-renewal.
      we observed a distinct expression pattern in BALB/c mice in which Ang-2 was significantly enhanced but Tie1 expression was suppressed during recovery from HND. Although Tie1 signaling has not been well studied,
      • Kontos C.D.
      • Cha E.H.
      • York J.D.
      • Peters K.G.
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      several reports have linked Ang-2 and Tie1 to common functions, including inflammation
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      Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation.
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      and lymphangiogenesis,
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      and recent structural evidence demonstrates differences in the interaction of Ang-2 with Tie1 versus Tie2.
      • Seegar T.C.
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      • Kolev M.V.
      • Henderson S.C.
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      Tie1-Tie2 interactions mediate functional differences between angiopoietin ligands.
      Although the exact role of this strain-dependent differential Ang-2/Tie1 expression in muscle cells is not yet known, it may be responsible for differences in angiogenesis, inflammation, and/or myoblast survival or differentiation and therefore may be part of the genetically determined response to limb ischemia.
      We observed a significant attenuation in limb muscle expression of VEGF at 1 day after HLI in BALB/c mice, although this deficit did not persist at later stages of recovery. Moreover, this in vivo difference did not translate into a strain-dependent difference in muscle cell VEGF mRNA expression in vitro, suggesting that the muscle precursor cell contribution to VEGF expression may not be a significant factor in the limb muscle response. However, there were striking differences in myotube expression of VEGF receptors in vitro after HND, which were significantly up-regulated in BL6 but not BALB/c myocytes. This effect was not observed in vivo after HLI, but could have been masked by the vascular response to ischemia. This muscle-specific change in VEGF receptor expression suggests an ability of muscle cells to respond to VEGF during recovery from ischemia and suggests that a growth factor traditionally linked to endothelial cell activation may serve a dual purpose by also promoting satellite cell and/or myofiber survival and recovery. Although relatively little is known about the biological role of VEGF receptors in muscle cells, this possibility is consistent with several previous reports demonstrating responses of myoblasts to VEGF and expression of VEGF receptors in muscle cells during ischemia.
      • van Weel V.
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      • Lardenoye J.H.P.
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      • van Bockel J.H.
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      • Quax P.H.A.
      Vascular endothelial growth factor overexpression in ischemic skeletal muscle enhances myoglobin expression in vivo.
      • Germani A.
      • Di Carlo A.
      • Mangoni A.
      • Straino S.
      • Giacinti C.
      • Turrini P.
      • Biglioli P.
      • Capogrossi M.C.
      Vascular endothelial growth factor modulates skeletal myoblast function.
      • Wagatsuma A.
      • Tamaki H.
      • Ogita F.
      Sequential expression of vascular endothelial growth factor, Flt-1, and KDR/Flk-1 in regenerating mouse skeletal muscle.
      In addition, muscle-derived stem cells expressing VEGFR-2 have been shown to contribute to skeletal muscle regeneration,
      • Lee J.Y.
      • Qu-Petersen Z.
      • Cao B.
      • Kimura S.
      • Jankowski R.
      • Cummins J.
      • Usas A.
      • Gates C.
      • Robbins P.
      • Wernig A.
      • Huard J.
      Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing.
      and expression of VEGF and VEGFR-2 have been localized to atrophic and regenerating muscle cells in ischemic muscle.
      • Rissanen T.T.
      • Vajanto I.
      • Hiltunen M.O.
      • Rutanen J.
      • Kettunen M.I.
      • Niemi M.
      • Leppänen P.
      • Turunen M.P.
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      • Arve K.
      • Alhava E.
      • Kauppinen R.A.
      • Ylä-Herttuala S.
      Expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 (KDR/Flk-1) in ischemic skeletal muscle and its regeneration.
      In support of previous data demonstrating a biological role for VEGF in myoblast differentiation,
      • Bryan B.A.
      • Walshe T.E.
      • Mitchell D.C.
      • Havumaki J.S.
      • Saint-Geniez M.
      • Maharaj A.S.
      • Maldonado A.E.
      • D'Amore P.A.
      Coordinated vascular endothelial growth factor expression and signaling during skeletal myogenic differentiation.
      our in vitro data on the promotion of the genetic myoblast differentiation program by exogenous VEGF strongly suggest that genetic differences in potential responsiveness to growth factors traditionally characterized as exclusively angiogenic, such as VEGF and the angiopoietins, may contribute to the ability of skeletal muscle to withstand and recover from ischemia.
      In conclusion, our data demonstrate an important and previously unrecognized genetic component to the skeletal muscle–specific response to ischemia in mice. In humans, similar genetic regulatory mechanisms may contribute to a patient's predisposition to develop CLI. Whereas our data suggest that endothelial cells are more ischemia-tolerant than muscle cells, our results did not address a genetic contribution to the effects of hypoxia or ischemia on endothelial cell survival or angiogenesis. Nonetheless, our observation of strain-dependent differences in muscle cell–specific expression of vascular growth factors and their receptors demonstrates that genetic differences in these cells can extend to the vascular response. Moreover, these findings suggest that, in addition to promoting vascular remodeling through the paracrine secretion of vascular growth factors like VEGF, muscle cells can potentially respond to these same factors in an autocrine fashion under conditions of stress or injury. Taken together, these results suggest that identifying genetic determinants of the skeletal muscle cell response to ischemia may lead to new targets for the treatment of patients with CLI.

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