Patent Application: US-21817408-A

Abstract:
the invention relates to the field of muscle pathologies , more particularly to the field of diseases where skeletal muscle degeneration occurs . the invention describes transgenic mice that do not produce prolyl - hydroxylase - 1 , - 2 or - 3 . it is revealed that the phenotype of the prolyl - hydroxylase 1 knock - out mouse is characterized by a protection of skeletal muscle atrophy due to a variety of muscle damages , especially ischemic insults . the invention thus relates to the use of molecules that can bind to prolyl - hydroxylase - 1 for the prevention and / or treatment of skeletal muscle degeneration .

Description:
the following examples more fully describe the invention , but are not intended to limit the invention . all of the starting materials and reagents disclosed below are known to those skilled in the art and are available commercially or can be prepared using well - known techniques . 1 . generation of phd1 −/− , phd2 −/− and phd3 −/− mice to inactivate the phd1 and phd2 genes , we constructed a targeting vector in which the second exon of each gene , which encodes part of the catalytic domain conferring the prolyl hydroxylase activity , 15 was replaced by a neomycin phosphotransferase selection cassette ( fig1 , panel a ). to inactivate the phd3 gene , a targeting vector was designed to delete exon 1 , as well as the transcription initiation site and the proximal 5 ′ promoter region . each of these targeting vectors was electroporated in embryonic stem ( es ) cells and correct recombination of these targeting vectors was confirmed by southern blot analysis ( fig1 , panel b ). targeted es cell clones were used to generate chimeric mice , which sired viable and healthy offspring , lacking a single allele of phd1 ( phd1 +/− ), phd2 ( phd2 +/− ) or phd3 ( phd3 +/− ). these heterozygously deficient mice were used to establish a colony of phd1 −/− , phd2 −/− and phd3 −/− mice . by northern and western blot analysis of mouse embryonic fibroblasts ( mefs ), phd1 , phd2 and phd3 transcript and protein levels were detectable in wild - type ( wt ) cells , while no transcripts or protein of the inactivated phd gene could be detected in the respective homozygous phd - deficient cells ( fig1 , panel c ). in order to evaluate whether the phds might have distinct or complementary roles , we determined the expression of each of these phds in mefs of the various knock - out lines . as expected , the expression of phd1 , phd2 and phd3 was up - regulated by hypoxia in wt cells . loss of phd2 compensatorily up - regulated the expression of phd1 and phd3 in normoxic conditions ( fig1 , panel c ). by contrast , deficiency of phd1 or phd3 did not affect the expression of the residual phd genes ( fig1 , panel c ). of note , hif - 1α protein levels were increased in normoxic phd2 −/− cells , while hif - 2α protein levels were enhanced in hypoxic phd1 −/− and phd3 −/− cells ( fig1 , panel c ), suggesting a specific regulation of hif - 2α expression by phd1 and phd3 . we first determined whether loss of a phd affected the inheritance ( table 1 ). phd1 −/− mice were born at the expected mendelian frequencies and were healthy and fertile . phd3 −/− mice were slightly underrepresented at birth ( the precise cause of this remains to be analyzed ), but the surviving mice appeared healthy and were fertile . in contrast , loss of phd2 resulted in embryonic lethality at mid - gestation , indicating that the critical role of phd2 during embryonic development cannot be compensated by phd1 and phd3 . further phenotyping revealed that phd2 −/− embryos succumbed because of severe placentation defects . phd2 +/− mice were healthy , fertile and gained normal weight . in this study , we analyzed the distinct role of each phd in ischemic disease in adult phd1 −/− , phd2 +/− and phd3 −/− mice . 2 . ischemic skeletal muscle injury is attenuated in phd1 −/− mice to determine the role of the phds in the cellular response to hypoxia , we focused on the skeletal muscle , as ligation of the femoral artery provides a simple and reproducible model of hind limb ischemia , which has been extensively characterized previously . for a phd to have a functional role in skeletal muscle , it should be expressed in this tissue . immunoblotting revealed that phd1 and phd3 were expressed in skeletal muscle from wt mice . immunohistochemistry studies confirmed that nuclei of skeletal myocytes expressed phd1 and phd3 . in contrast , phd2 expression was undetectable in skeletal muscle ; this was not due to technical limitations as phd2 was readily detectable by western blot analysis in cardiac muscle . in baseline conditions , muscle development appeared normal in wt , phd1 −/− , and in phd2 +/− and phd3 −/− mice , no genotypic differences in myofiber size or muscle mass were detected . in order to determine the specific role of phd1 , phd2 and phd3 in ischemic muscle disease , we ligated the femoral artery . transverse sections through the gastrocnemius muscle revealed extensive ischemic tissue damage in wt mice at seven days after femoral artery ligation . crural muscles from phd2 +/− and phd3 −/− hind limbs also developed a comparable degree of ischemic muscle necrosis ( table 2 ). in sharp contrast , ischemic phd1 −/− muscles predominantly contained viable skeletal muscle fibers and were largely devoid of necrotic areas ( table 2 ). fibrotic scar tissue was also dramatically reduced in phd1 −/− hind limbs (% fibrotic area / total transverse section area : 3 . 7 ± 1 . 9 % in phd1 −/− versus 74 . 4 ± 12 . 9 % in wt ; n = 8 ; p & lt ; 0 . 01 ). we initially thought that the healthy appearance of the phd1 −/− muscles after ischemia was attributable to enhanced tissue regeneration . however , staining for cd56 , a marker of activated myogenic precursor cells , revealed that most satellite cells were quiescent in post - ischemic limbs of phd1 −/− animals , whereas multiple activated cd56 - positive myogenic precursor cells were observed in the regenerating crural muscles of wt mice . moreover , myocellular brdu uptake was not enhanced in phd1 −/− muscles following ischemia , and muscle fibers did not have a central nucleus , a characteristic histo - pathological feature of muscle regeneration . we therefore suspected that the observed effect was caused by tissue protection , rather than by enhanced muscle regeneration . indeed , analysis of semi - thin sections at four hours after induction of ischemia revealed progressive myofiber edema and sarcomere loss in wt limbs , whereas striated muscle fibers appeared intact and healthy in phd1 −/− mice . consistently , extensive coagulation necrosis dominated the histological picture in wt but not in phd1 −/− mice at 48 hours after induction of ischemia . moreover , in wt mice , labeling of the intermediate filament desmin was absent or weak , indicative of filament protein breakdown due to impaired skeletal muscle viability . in contrast , desmin was detected throughout all fibers of the ischemic phd1 −/− gastrocnemius muscle at 48 hours after induction of ischemia . morphometric quantification after 48 hours of ischemia revealed more desmin - immunopositive areas and more intense staining in phd1 −/− than in wt gastrocnemius muscle (% relative desmin - positive area / optical field : 7 . 1 ± 1 . 2 % in phd1 −/− versus 1 . 7 ± 0 . 7 % in wt ; n = 4 ; p & lt ; 0 . 01 ). thus , loss of phd1 , but not of phd2 or phd3 , significantly protected the mice against ischemic muscle necrosis . 3 . vascular perfusion of the hind limb is not altered in phd1 −/− mice to elucidate the mechanisms of muscle protection in phd1 −/− mice , we first sought to determine whether deficiency of phd1 increased vascular perfusion of the hind limbs in baseline conditions or after ischemia ( a likely mechanism when considering that loss of phd1 increased the levels of the pro - angiogenic hif - 2α ). in baseline conditions , the mean capillary density in the gastrocnemius muscle was comparable in wt and phd1 −/− mice ( capillary / muscle fiber ratio : 1 . 93 ± 0 . 1 in phd1 −/− versus 1 . 97 ± 0 . 1 in wt mice ; n = 7 ). vascular microcomputed tomography ( ct ) imaging further revealed a normal anatomical pattern of the crural arterial branches in hind limbs of phd1 −/− mice , without evidence for supernumerary collateral vessels . accordingly , functional vascular perfusion of the gastrocnemius muscle , as assessed by administration of fluorescent microspheres , was similar in phd1 −/− and wt mice ( microspheres in the gastrocnemius muscle ml × g − 1 × min − 1 at baseline conditions : 0 . 35 ± 0 . 03 in phd1 −/− versus 0 . 39 ± 0 . 02 in wt mice ; p = 0 . 3 ; n = 6 ). revascularization of the ischemic hind limb involves growth of collateral arteries in the adductor muscle in the upper hind limb and capillary vessel growth in the gastrocnemius muscle in the lower hind limb . morphometric analysis of collateral arterial growth at seven days after hind limb ischemia revealed that the total number of collateral side branches was not increased in the adductor region of phd1 −/− limbs ( second - generation collateral side branches / mm 2 : 22 . 5 ± 5 in phd1 −/− versus 22 . 7 ± 3 . 1 in wt limbs ; n = 7 ). the collateral perfusion area , representing the sum of the luminal areas of all secondary and tertiary collaterals , was also not significantly altered in phd1 −/− limbs ( total collateral perfusion area in μm 2 / mm 2 : 2 , 560 ± 241 in phd1 −/− versus 2 , 290 ± 282 in wt limbs ; n = 7 ; p = n . s .). the mean capillary density in the gastrocnemius muscle was comparable in wt and phd1 −/− mice in viable muscle areas after seven days of ischemia ( capillaries / mm 2 : 702 ± 56 in phd1 −/− versus 721 ± 80 in wt mice ; n = 7 ). vascular microcomputed tomography ( ct ) imaging further revealed a comparable degree of residual perfusion of the popliteal artery via collateral vessels after femoral artery ligation in both genotypes . residual blood supply to the lower hind limb was additionally determined by administration of fluorescent microspheres at various time points following vascular ligation , and was comparable in phd1 −/− and wt mice ( microspheres in the gastrocnemius muscle ml × g − 1 × min − 1 after 2 hours ischemia : 0 . 15 ± 0 . 03 in phd1 −/− versus 0 . 13 ± 0 . 04 in wt mice ; p = 0 . 74 ; n = 4 ). thus , loss of phd1 did not result in an increased vascular supply . collectively , these data suggest that skeletal muscle protection in phd1 −/− mice was not attributable to an increased baseline perfusion or an enhanced compensatory angiogenic response after ischemia . instead , inactivation of the phd1 gene preserved skeletal muscle against ischemic insult via an angiogenesis - independent mechanism . 4 . ischemic phd1 −/− limbs maintain sufficient levels of energy - rich phosphates the lack of any vascular or regenerative defects in phd1 −/− limbs prompted us to consider that loss of phd1 might perhaps change the metabolism and thereby protect against muscle ischemia . high energy phosphates , in particular atp , are critical to secure cell functioning and viability . in ischemic conditions , skeletal muscle atp levels are initially maintained through a rapid hydrolyzation of phosphocreatine ( pcr ) by creatine kinase , an enzyme transferring high - energy phosphates from pcr to adp to generate atp . to estimate the availability and turnover of energy - rich phosphates in the hind limb musculature of phd1 −/− and wt mice , we recorded in vivo 31 phosphorus magnetic resonance spectra . in wt mice , pcr levels progressively declined within 30 - 120 minutes after onset of ischemia , while inorganic phosphate ( pi ) levels steadily increased , reflecting continuous atp utilization and insufficient atp regeneration . in phd1 −/− limbs , by contrast , muscular pcr contents also decreased but only to 50 % of its baseline levels and only during the initial 30 minutes after onset of ischemia , but subsequently stabilized and even seemed to recover during prolonged ischemia . accordingly , pcr - and atp - levels were substantially higher in phd1 −/− than in wt limbs at 24 hours after the ischemic insult . thus , loss of phd1 prevents complete energy exhaustion in ischemic limbs and thereby protects skeletal muscle against ischemic damage . 5 . normal anaerobic glycolysis in ischemic phd1 −/− skeletal muscle we therefore studied the mechanisms whereby phd1 −/− myofibers were capable of restoring their energy sources in conditions of low oxygen . we first hypothesized that anaerobic glycolysis was enhanced in phd1 −/− skeletal muscle and , therefore , studied , by micro - pet imaging , the uptake of blood - borne glucose into the gastrocnemius muscle . muscular glucose uptake was markedly increased at one to two hours after vascular ligation in both wt and phd1 −/− mice , reflecting a reactive response to compensate for insufficient energy supply after femoral artery ligation . however , this compensatory response was comparable in both genotypes ( glucose uptake as % injected radioactivity / g : 0 . 39 ± 0 . 07 in phd1 −/− versus 0 . 39 ± 0 . 04 in wt in baseline conditions ; 0 . 9 ± 0 . 3 in phd1 −/− versus 1 . 1 ± 0 . 3 in wt at one to two hours post - ligation ; n = 4 ; p = ns ). compared to baseline conditions , glucose uptake was increased by 2 . 4 ± 0 . 4 fold in phd1 −/− mice versus 2 . 78 ± 0 . 84 fold in wt mice ( n = 5 ; p = ns ). accordingly , baseline mrna levels of the skeletal muscle glucose transporters glut1 and glut4 also did not differ significantly in phd1 −/− and wt mice ( mrna copies per 1 , 000 mrna copies of β - actin : for glut1 , 14 ± 3 in phd1 −/− versus 24 ± 5 in wt ; for glut4 , 13 ± 1 . 5 in phd1 −/− versus 12 . 8 ± 2 . 5 in wt ; n = 6 ; p = ns ). by immunostaining , translocation of insulin - dependent glut4 - positive vesicles to the sarcolemma was comparably increased in myofibers of both genotypes at six hours after induction of ischemia . moreover , blood glucose levels were similar in phd1 −/− mice shortly after ischemia ( mg / dl : 413 . 8 ± 75 . 3 in phd1 −/− versus 373 . 8 ± 80 . 1 in wt ; n = 5 ; p = ns ). thus , genotypic changes in glucose uptake could not explain the preservation of high energy phosphate stores in phd1 −/− mice . in order to address whether glycolysis itself was enhanced in ischemic phd1 −/− muscle , in a first experiment , we assessed levels of the final anaerobic glycolysis metabolite lactate by proton spectroscopy . lactate levels were considerably induced after one to two hours of ischemia in both phd1 −/− and wt mice ( lactate content in baseline gastrocnemius : 11 . 7 ± 1 . 1 in phd1 −/− versus 12 . 7 ± 0 . 7 in wt ; n = 5 ; p = ns ; gastrocnemius lactate content one to two hours post - ligation : 79 . 4 ± 2 . 8 in phd1 −/− versus 80 . 2 ± 14 . 06 in wt ; n = 3 ; p = ns ). altogether , these data reveal a rapid compensatory induction of anaerobic glycolysis to enhance muscular atp generation in acute ischemia , but this response was not significantly more effective in phd1 −/− mice . as mitochondria are critical for proper electron transfer and production of atp , and ischemia may cause mitochondrial damage , we also analyzed whether loss of phd1 might affect ( i . e ., preserve better ) mitochondrial function using various complementary methods . we first analyzed whether the elevated lactate levels returned to baseline levels at later stages after ischemia , as persistent lactate accumulation might indirectly reflect mitochondrial dysfunction . notably , when analyzed at six hours post - ischemia , elevated lactate levels persisted in ischemic wt muscle , but returned to baseline levels in phd1 −/− mice ( lactate content in ischemic gastrocnemius at six hours post - ligation : 12 . 4 ± 0 . 4 in phd1 −/− versus 95 . 7 ± 43 . 1 in wt ; n = 3 ). we also determined succinate levels in the ischemic gastrocnemius muscle by proton spectroscopy , as succinate accumulates when the mitochondrial respiratory chain complex ii fails to use the fadh2 , generated from the conversion of succinate to fumarate in the krebs cycle . in wt mice , succinate levels accumulated to high levels at six hours after ischemia ; by contrast , succinate levels in phd1 −/− muscle did not increase above baseline levels ( succinate content : 0 . 42 ± 0 . 096 in phd1 −/− versus 0 . 22 ± 0 . 048 in wt in baseline conditions ; 0 . 36 ± 0 . 03 in phd1 −/− versus 1 . 92 ± 0 . 58 in wt in ischemia ; n = 3 ; p = ns ). biochemical measurements further revealed that the mitochondrial complex i activity in the gastrocnemius muscle was reduced in wt mice but preserved in phd1 −/− mice at six hours after femoral artery ligation . ischemia is well known to induce morphological changes reminiscent of mitochondrial myopathies , including the presence of ragged red fibers , identifiable by enhanced histochemical succinate dehydrogenase ( sdh ) staining . 22 consistent with our other results that mitochondrial function was better preserved in ischemic phd1 −/− muscle , we found reduced numbers of sdh - positive fibers in phd1 −/− muscle at six hours after onset of ischemia (% sdh - positive fibers / ischemic muscle area : 125 ± 10 . 3 in phd1 −/− versus 89 . 2 ± 6 . 88 in wt mice ; n = 6 ; p = 0 . 01 ). transmission electron microscopy studies of ischemic gastrocnemius muscle further revealed vacuolization , matrix clarification and disruption of mitochondrial crystae in ischemic wt fibers , while such signs of mitochondrial degeneration were not detectable in phd1 −/− fibers . taken together , in the absence of phd1 , mitochondrial integrity and performance were better preserved , thereby enabling the ischemic muscle to maintain a critical supply of atp . 7 . ischemia - induced oxidative stress is attenuated in ischemic phd1 −/− muscle we next sought to determine the mechanisms underlying the mitochondrial protection in ischemic phd1 −/− muscle . mitochondrial dysfunction in ischemic conditions may result from excessive oxidative stress . we therefore analyzed whether phd1 −/− muscle fibers more efficiently counteracted the oxidative stress . when assessing protein carbonylation as a surrogate marker of the oxidative stress - induced damage by using the oxyblot , we found that ischemia induced considerably more protein oxidation in cytoplasmic or mitochondrial preparations in ischemic wt than phd1 −/− gastrocnemius . densitometric quantification of the total amount of carbonylated protein revealed that protein oxidation was increased in ischemic wt muscle , but only insignificantly in ischemic phd1 −/− muscle fibers . use of the ferricytochrome c reduction assay on submitochondrial particle preparations further revealed much higher production levels of reactive superoxide species ( ros ) in ischemic wt than phd1 −/− gastrocnemius muscle . apart from a reduced generation of ros , the decreased cellular oxidative stress in phd1 −/− muscle could also result from an enhanced antioxidant capacity and superoxide clearance . indeed , we found that protein levels of the antioxidant enzymes sod1 and sod2 ( the latter being the most relevant antioxidant mechanism in mitochondria ) were significantly higher in the mitochondria of phd1 −/− than wt muscle after ischemia . upon densitometric quantification , sod2 levels were increased in ischemic phd1 −/− muscle but only insignificantly in ischemic wt muscle fibers . thus , in the absence of phd1 , generation of ros and protein peroxidation were reduced , while ros clearance was enhanced , resulting in a reduced oxidative stress for the mitochondria with better preservation of mitochondrial function and supply of high energy phosphates after ischemia . 8 . phd1 −/− myoblast cultures are protected against cellular stress conditions the possibility was also considered that the absence of phd1 might alter the intrinsic property of skeletal myocytes to sustain better hypoxic stress . we therefore cultured phd1 −/− and wt myoblasts and determined cell survival after subjecting them to hypoxia , serum deprivation , or a combination of both stress conditions , to mimic the ischemic situation in vivo . undifferentiated wt myoblasts detached from the culture dish and died in hypoxic conditions and , even more so , after serum deprivation , or when both stress conditions were combined . in contrast , a larger number of phd1 −/− myoblasts survived the hypoxic or serum - deprivation stress . in order to determine whether this protective effect was cell - autonomous , we analyzed whether conditioned medium of cultured phd1 −/− myoblasts , exposed to oxygen and serum deprivation , could increase the survival of stressed wt cells . indeed , conditioned medium from phd1 −/− myoblasts enhanced the survival of wt myoblasts after hypoxia and serum deprivation , thus suggesting that a secreted humoral factor ( s ) provided the protection . interestingly , conditioned medium from phd1 −/− myoblasts was also capable of reducing the oxidative stress in wt myoblasts . 9 . skeletal muscle protection in phd1 −/− mice is mediated by hif - 2α loss of hif - 2α leads to skeletal myopathy due to mitochondrial dysfunction , indicating that hif - 2α has an important role in mounting an anti - oxidant response . 23 it was therefore evaluated whether hif - 2α might be a downstream target of phd1 , mediating the skeletal muscle resistance to ischemia . protein expression analysis revealed no genotypic differences in the ischemic induction of hif1 - α in the skeletal muscle of phd1 −/− and wt mice . by contrast , hif - 2α levels were more elevated in ischemic phd1 −/− than in wt muscle . additional evidence supporting a predominant role for phd1 in the regulation of hif - 2α was provided by the accumulation of hif - 2α , but not hif - 1α , in phd1 −/− mefs ( fig1 , panel c ). to assess whether ischemic muscle protection in phd1 −/− mice was indeed dependent on hif - 2α , we compared the response to muscle ischemia in viable hif - 2α +/− mice ( hif - 2α −/− mice are lethal ) and phd1 −/− hif - 2α +/− mice ( lacking both phd1 alleles and a single hif - 2αallele ), obtained by intercrossing hif - 2α +/− and phd1 −/− mice . phd1 −/− hif - 2α +/− mice were healthy , fertile , gained weight normally and had no muscle defects . similar to wt mice , hif - 2α +/− mice exhibited severe signs of ischemic muscle damage ( table 2 ). notably , however , phd1 −/− hif - 2α +/− mice , lacking both phd1 alleles and a single hif - 2α allele , also suffered the same degree of ischemic muscle necrosis as wt mice ( table 2 ). these genetic findings thus suggest that hif - 2α is a ( but , therefore , not the only ) critical downstream mediator of the myoprotective activity of phd1 . prompted by our genetic findings that loss of phd1 prevents ischemic skeletal muscle damage , we sought to determine whether knock - down of phd1 expression might offer therapeutic opportunities to counteract ischemic fiber injury by using rnai , previously recognized as an efficient tool to silence gene expression in vivo and in vitro . in order to interfere with endogenous phd1 expression in skeletal muscle , we transferred phd1 sirna in gastrocnemius fibers by using adenovirus - driving transcription of small hairpin phd1 irna ( shphd1irna ). the short hairpin sequence against phd1 ( 5 ′- cac cgc tgc atc acc tgt atc tat ttc tct tga aaa tag ata cag gtg atg cag c - 3 ′ ( seq id no : 5 )) was cloned into the pad / block - it gateway ™ ( invitrogen ) vector system according to the manufacturer &# 39 ; s instructions . as a control , we used an empty e1 - deleted recombinant adenovirus ( adrr5 ). for viral injection , the leg was depilated at the injection site . after incision of the skin , the gastrocnemius muscle was injected with adenovirus containing shphd1 or adrr5 at a volume of 60 μl per muscle , using a hamilton microsyringe . ligation of the femoral vessels was performed two days after viral injection , and necrotic tissue areas quantified morphometrically two days after induction of ischemia . rt - pcr analysis revealed that gastrocnemius phd1 mrna levels were reduced by 60 - 80 % after intramuscular injection of adeno - shphd1irna , compared to mice injected with adenovirus control . in order to increase our chance to abrogate phd1 expression , we treated phd1 +/− mice . interestingly , wt and phd1 +/− mice treated three days in advance with sh - phd1 sirna showed a significant protection against fiber damage monitored two days after femoral ligation ( see fig2 ). these data show that the inhibition of phd1 activity is a therapeutic strategy to treat lower limb ischemic diseases as well as to treat pathologies in which muscle degeneration occurs . in a next step , short hairpin constructs against phd1 were delivered by electroporation . we used dna constructs designed to produce short hairpin interference rna to knock - down phd1 expression : shphd1 kd ( 5 ′- caccgctgcatcacctgtatctatttctcttgaaaatagatacaggtgatgcagc - 3 ′ ( seq id no : 6 )) and a control shphd1 ctr ( 5 ′- caccgcttaacccgtattgcctatttctcttgaaaataggcaatacgggttaagc - 3 ′ ( seq id no : 7 )), which differed by a mismatch of ten nucleotides in the phd1 - specific sequence . the efficiency and specificity of shphd1 kd and shphd1 ctr were first tested in cultured murine c2c12 myoblast cells . compared to non - transfected cells , transfection of c2c12 cells with shphd1 ctr did not affect phd1 transcript levels ( mrna copies phd1 / 10 3 mrna copies β - actin : 12 . 0 ± 1 . 0 after vehicle versus 11 . 3 ± 0 . 3 after shphd1 ctr ; n = 4 ; p = n . s .). in contrast , shphd1 kd efficiently lowered phd1 mrna levels by 72 % ( mrna copies phd1 / 10 3 mrna copies β - actin : 3 . 4 ± 0 . 5 after shphd1 kd ; n = 4 ; p & lt ; 0 . 05 versus shphd1 ctr ). shphd1 kd did not knock down phd2 or phd3 . we then injected and electroporated these constructs into the muscle fibers in vivo , as this method is very efficient and causes negligible muscle damage and inflammation ( j . m . mcmahon et al ., 2001 , gene ther . 8 , 1264 - 70 ). the constructs were electroporated five days prior to the ligation of the femoral artery for two reasons : ( i ) to achieve sufficient elimination of the pre - existing phd1 transcripts prior to the induction of ischemia ; and ( ii ) to avoid death of ischemic myofibers before their phd1 transcript levels would be sufficiently reduced to provide protection . both the right and left hind limb muscles were electroporated , but only the right femoral artery was ligated , permitting us to use the right legs for histological scoring of muscle necrosis and the left muscles for rna analysis . to obtain sufficiently low endogenous phd1 levels after phd1 knock - down , we used phd1 +/− mice , which only expressed 50 % of the phd1 transcript levels present in wt mice . electroporation of shphd1 kd in phd1 +/− muscle reduced phd1 transcripts to 23 ± 4 % ( n = 13 ; p & lt ; 0 . 001 ) of the levels normally detected in wt muscle , while a similar dose of shphd1 ctr insignificantly reduced phd1 levels to 44 ± 5 % of wt levels ( n = 13 ; p = 0 . 20 ). histological analysis at two days after femoral artery ligation revealed that electroporation of shphd1 ctr insignificantly reduced phd1 +/− muscle necrosis ( necrotic plantaris muscle area : 5 . 5 ± 1 . 2 × 10 − 1 mm 2 in non - electroporated versus 3 . 7 ± 1 . 1 × 10 − 1 mm 2 after shphd1 ctr ; n = 13 ; p = n . s . versus non - electroporated ). by contrast , electroporation of shphd1 kd reduced phd1 +/− myofiber necrosis by 83 % and 74 % as compared to non - electroporated or shphd1 ctr - injected muscle , respectively ( necrotic plantaris muscle area : 0 . 9 ± 0 . 3 × 10 − 1 mm 2 after shphd1 kd ; n = 13 ; p & lt ; 0 . 005 versus non - electroporated and p & lt ; 0 . 05 versus shphd1 ctr by t - test and mann whitney test ). thus , similar to knock - out of phd1 , knock - down of phd1 provided protection of myofibers against ischemic necrosis . two human shphd1kd sequences that gave nice knock - down results of phd1 in human cell lines are : human shphd1 kd - 1 ( 5 ′- caccgctgcatcacctgtatctatttctcttgaaaatagatacaggtgatgcagc - 3 ′ ( seq id no : 8 )), human shphd1kd - 2 ( 5 ′- caccgccaacatcgagccactcttttctcttgaaaaagagtggctcgatgttggc - 3 ′ ( seq id no : 9 )). note that the human shphd1 kd - 1 has the same sequence as the mouse shphd1kd sequence . we used this sequence for sirna oligos design . these sirna oligos were used for in vitro studies in human cell lines and this sirna phd1 oligo gave nice results . based on the proprietary rules from the proligo company , we have designed an antisense lna to knock down phd1 in skeletal muscles . this approach can confirm the results obtained in the ko mice , and will allow us to develop a therapeutic approach in order to apply these findings in skeletal muscle degenerative disorders . one example of an lna that is directed to the murine phd1 sequence , coding sequence span from position 284 to position 1543 : the chosen strategy to deliver the lna in skeletal muscles is via in vivo electroporation . skeletal muscle - targeting via in vivo electroporation induces minimal damage of the skeletal muscle fiber and a mild degree of influx of inflammatory cells . we deliver the lna inside the skeletal muscle fiber just before the limb ischemia protocol during the same anesthetic procedure . prior to intramuscular injection , mice are anesthetized . the gastrocnemius anterior muscles of anesthetized animals are injected with hyaluronidase two hours prior to injection and electrotransfer of the lna as is described by mcmahon et al ., 2001 , gene ther . august ; 8 ( 16 ): 1264 - 70 . we perform the experiment on male sv129 mice , ranging in age from 11 to 18 weeks old . animals are housed in a disease - free facility with food and water ad libitum . control experiments with fluorescent scrambled lna were previously done in order to find the best conditions for the lna delivery . forty - eight hours after electroporation , longitudinal sections of gastrocnemius anterior are examined under fluorescent microscope ; we chose the condition that shows both homogeneous distribution and high intrafiber fluorescence . the experimental conditions are : 8 μg phd1 - lna or phd1 src lna was injected in normal saline in a final volume of 25 μl in the gastrocnemius anterior ; an electrical field was applied to the muscle immediately . the injection of lna and the electrotransfer ( using a btx ecm 830 electroporator ) are carried out under isofluorane inhalation anesthesia . immediately afterwards , limb ischemia is induced as described in materials and methods . the limb ischemia is checked by doppler analysis six hours after the surgical protocol . seven days after the electroporation , transverse sections are derived from the gastrocnemius after ischemia ; they are subjected to histological and morphometric analysis . the phd1 targeting vector contains , from 5 ′ to 3 ′, the following fragments cloned in pkoscramblerntkv - 1908 : a 5 - kb kpni / hindiii fragment located upstream of exon2 as 5 ′ homology ; a neo resistance cassette in opposite orientation ; a 1 . 6 - kb bamhi / ecorv fragment located downstream of exon2 as the 3 ′ homology , and a thymidine kinase selection cassette . the phd2 targeting vector contains , from 5 ′ to 3 ′, the following fragments cloned in ppntlox2 : a 5 . 2 - kb bamhi / hindiii fragment located upstream of exon2 as 5 ′ homology ; a neo resistance cassette ; a 2 . 5 - kb xbai / bamhi fragment located downstream of exon2 as the 3 ′ homology , and a thymidine kinase selection cassette . the phd3 targeting vector contains , from 5 ′ to 3 ′, the following fragments cloned in ppntlox2 : a 5 - kb kpni / hindiii fragment located upstream of exon2 as 5 ′ homology ; a neo resistance cassette in opposite orientation ; a 1 . 6 - kb bamhi / ecorv fragment located downstream of exon2 as the 3 ′ homology ; and a thymidine kinase selection cassette . es cells ( 129 svev background ) were electroporated with linearized targeting vectors for phd1 , phd2 or phd3 as described . 24 resistant clones were screened for correct homologous recombination by appropriate southern blot and pcr ( see supplemental information ). several phd1 +/− , phd2 +/− and phd3 +/− positive clones were obtained . correctly recombined es cell clones were aggregated with morula embryos as described 24 to generate chimeric mice that were intercrossed with wild - type swiss females to obtain get phd1 +/− , phd2 +/− and phd3 +/− germline offspring ( 50 % swiss / 50 % 129 background ). subsequently , heterozygous breeding pairs were established to generate homozygous phd1 −/− , phd2 −/− and phd3 −/− progeny . gene expression was quantified by real time rt - pcr , relative to the expression level of alfa - actin , using the following forward ( f ) and reverse primers ( r ) and probes ( p ), labeled with fluorescent dye ( fam or joe ) and quencher ( tamra ). for western blot analysis , gastrocnemius samples were homogenized on ice using lysis buffer ( 8 m urea , 1 / 10 v / v glycerol , 1 / 20 v / v 20 % sds , 1 / 200 v / v 1 m dtt , 1 / 100 v / v 0 . 5 m tris ( ph 6 . 8 )) containing a cocktail of protease inhibitors ( complete mini , roche ). then samples were centrifuged 13 , 000 rpm at 4 ° c . and supernatants were collected . protein concentration was determined and 100 μg were fractionated by sds - page electrophoresis and transferred onto nitrocellulose membranes ( hybond - ecl , amersham biosciences ). membranes were incubated with specific antibodies against phd1 , phd2 , phd3 , hif - 1α ( novus biologicals , littleton , usa ), hif - 2α ( novus biologicals ), glut - 1 ( alpha diagnostic international , san antonio , usa ) and glut - 4 ( alpha diagnostic international ). bound primary antibody was visualized with species - specific horseradish peroxidase - conjugated secondary antibody ( dakocytomation ) and chemiluminescence system ( pierce ). limb ischemia was induced by high unilateral right or bilateral ligation of the femoral artery and vein , and of the cutaneous vessels branching from the caudal femoral artery , sparing the femoral nerve . crural muscles were dissected and processed for histological analysis two days or seven days after ligation . for assessment of re - vascularization , bismuth gelatino - angiography was carried out , and arterioles branching directly from pre - existing collaterals , connecting the femoral and saphenous artery in the adductor region of the thigh , were quantified by morphometry as described on histological cross sections after seven days . 25 side branches were categorized as second or third generation according to their luminal area (& gt ; 300 μm , & lt ; 300 μm , respectively ). for micro - ct angiography , hind limbs were perfused with a solution containing 30 % barium and 5 % gelatin , dissected in toto , and the crural vasculature was imaged using a high - resolution micro - ct imaging system ( skyscan - 1172 , skyscan , aartselaar , belgium ), with a voltage of 50 kvp and a current of 200 μa . three - dimensional morphometric analysis was performed to assess vessel volume , number , and thickness in a volume of interest ( voi ) defined as the calf area . blood flow in non - operated and ligated limbs was measured applying 15 μm fluorescent microspheres ( 1 × 10 6 beads per ml , molecular probes , eugene , oreg .) after maximal vasodilation with sodium nitroprusside ( 50 ng / ml , sigma ) as described . 26 , 27 for immunostaining , the crural flexor muscle group was dissected , fixed in 4 % pfa , dehydrated , embedded in paraffin , and sectioned at a 10 μm thickness . briefly , after deparaffinization and rehydration , sections were digested with 0 . 2 % trypsin ( sigma ), blocked , and incubated overnight with a rat anti - mouse cd31 antibody ( bd pharmingen ; dilution 1 / 500 ), a rabbit anti - desmin antibody ( abcam ; dilution 1 / 100 ), an anti - phd1 antibody or with an anti - glut4 antibody ( alpha diagnostic international ). sections were subsequently incubated with appropriate secondary antibodies , developed with 3 , 3 ′- diaminobenzidine ( dab , sigma ) as a chromogen substrate and counterstained with harris hematoxilin . for detection of hypoxic cells , mice were treated with pimonidazole ( chemicon ) one hour prior to dissection of muscle specimens , and staining with the hydroxyprobe - 1 antibody ( chemicon ) was carried out according to the manufacturer &# 39 ; s instructions . microscopic analysis was performed with a zeiss axioplan 2 imaging microscope , equipped with an axiocam hrc camera and ks300 morphometry software ( zeiss ). capillary density , fiber size , and the cross - sectional area of viable and necrotic zones were quantified on eight entire sections ( each 320 μm apart ) of the crural muscle package . for assessment of metabolic fiber properties , consecutive cryostat sections ( 12 μm ) of snap frozen crural muscles were subjected to histochemical sdh ( succinate dehydrogenase , reveals oxidative vs . non - oxidative fibers ), glycogen phosphorylase ( reveals glycogenolytic potential ), and myofibrillar atpase stainings ( ph 4 . 1 ; reveals slow versus fast fibers ) according to standard protocols . pas staining was carried out to assess glycogen content . in vivo 31 phosphorus mrs experiments on lower limbs were performed at 188 mhz in a bruker biospec ( karlsruhe , germany ), equipped with a horizontal 4 . 7 tesla superconducting magnet with 30 cm bore , using a 10 mm transmit - receive surface coil . prior to the measurement , the mice were anesthetized with intraperitoneally ( i . p .) injected sodium pentobarbital ( 1 − 1g body weight nembutal , sanofi , belgium ). mice were placed on a perspex plate such that lower limbs was positioned directly inside the circular surface coil . serial 31 phosphorus nmr spectra were acquired every 13 minutes during 20 minutes to two hours ( 90 degree pulse of 11 seconds , total repetition time ( tr )= 0 . 75 seconds , number of averages ( na )= 1024 , spectral width = 27 khz ; acquisition size = 2048 points ; no proton decoupling ). the signals for pcr , pi , atp resonances were processed by zero - filling to 4096 points and exponential multiplication of 6 hz . during the in vivo 31 phosphorus mrs measurements , the body temperature of the mice was kept at 36 ° c . with the use of warm air ventilation in the magnet bore . in vitro analysis of gastrocnemius extracts from operated limbs in wt and phd i −/− mice were performed in a high resolution amx 360 ( 8 . 4 tesla ) spectrometer ( bruker , karlsruhe , germany ). specific settings for the tissue extracts ( 278 k ) were , tr = 2 . 5 seconds , na = 3072 or 12288 , 1 h waltz - 16 - decoupling during the one - second acquisition ; and for the lysates ( 295 k ), tr = 2 . 5 seconds , na = 256 , no proton decoupling . the tissue extracts were measured at 278 k to exploit the substantially shorter relaxation time at low temperature ( typically a factor three ). 28 absolute lactate concentrations were determined using a phantom sample of 5 - fu in water ( 2 . 8 mm , 500 l ) with a coaxial fba reference sample insert . purification of the primary muscle - derived cells was performed using a previously described protocol . 29 the hind limbs were removed from wt or phd1 −/− mice , and the bone was dissected . the remaining muscle mass was minced into a coarse slurry using razor blades . cells were enzymatically dissociated by adding 0 . 2 % collagenase - type b for 45 minutes at 37 ° c ., dispase ( grade ii , 2 . 4 u / ml ) for 30 minutes . the muscle cell extract was preplated on collagen - coated flasks . we isolated different populations of muscle - derived cells based on the number of preplates performed on collagen - coated flasks . preplate ( pp ) 1 represented a population of muscle - derived cells that adhered in the first hour after isolation , pp2 in the next two hours , pp3 in the next 18 hours , and the subsequent preplates were obtained at 24 - hour intervals ( pps 4 - 6 ). the myogenic population in each flask was evaluated by desmin staining and on differentiation ability when cultured in a fusion medium . the proliferation medium was f12 - ham - supplemented with 20 % fbs ( growth medium , gm ) and 1 % penicillin / streptomycin ; the fusion medium was f12 - ham supplemented with 2 % hs and 1 % antibiotic solution ( penicillin / streptomycin ) ( differentiation medium , dm ). all the culture medium supplies were purchased through gibco ® cell culture products ( invitrogen , merelbeke , belgium ). for cellular treatment , cells seeded at 10 , 000 cells / cm 2 were grown in 48 - well plates and exposed either to normoxia or hypoxia in gm or in serum - deprived medium during 24 , 48 or 72 hours . hypoxia was induced by placing the culture plates into a 37 ° c . incubator in a humidified atmosphere perfused with 95 % n 2 / 5 % co 2 . serum deprivation was induced by replacing the gm by serum - free f12 - ham . the oxygen level was 2 %. co - cultures of wt myogenic cells and phd1 −/− myogenic cells were done after fluorescent labeling of each cell type with celltracker ™ red cmtpx for wt cells and celltracker ™ green cmfda ( 5 - chloromethylfluorescein diacetate ) for phd1 −/− cells ( molecular probes ™, invitrogen , merelbeke , belgium ). in direct co - cultures , survival and growth of each cell type ( 5000 cells / cm 2 each ) were monitored by numeration of fluorescent cells under microscope ( only attached cells are considered as alive ). in indirect co - cultures , phd1 −/− cells were seeded in inserts ( 0 . 4 - μm diameter pores ; falcon ™, bd biosciences ) placed over the wt cell - containing wells . using online shrna oligo sequence selector tool ( invitrogen rnai designer ), shrna templates were designed to match non - conserved nucleotide sequences within the mouse phd1 targeting sequence ( genbank accession no . nm — 053208 ). the sequences of the oligonucleotides are designed to use them in the pentr ™/ u6 vector ( invitrogen ), a vector that allows efficient transient expression of short hairpin rna ( shrna ) or stable expression of shrna following recombination with a suitable destination vector in mammalian cells . the shrna oligos were chemically synthesized by invitrogen . the shrnas design was based on a 21 nucleotide sense sequence derived from the target gene , followed by a short spacer of nine nucleotides ( i . e ., loop ) and a 21 nucleotide sequence that is the reverse complement of the initial target sequence . the three sets of shrnas phd1 targeting sequences correspond to the coding region : 1224 - 1244 ( gct gca tca cct gta tct att ( seq id no : 3 )), 1319 - 1339 ( gcc aac atc gag cca ctc ttt ( seq id no : 3 )) and 1490 - 1510 ( ggt gtt caa gta cca gta tca ( seq id no : 3 )) relative to the start codon . a mutated version was designed ( mutated sequence ) to be used as negative control . the three shphd1 were cloned into the pentr ™/ u6 vector according to the manufacturer &# 39 ; s instructions . the orientations of the short hairpins were confirmed by sequencing . efficiency of shphd1_pentr ™/ 6 vectors was tested by transfection into mouse embryonic fibroblasts ( mefs ), embryonic stem ( es ) cells and mouse myoblast cell line ( c2c12 ). cells were transfected with 5 μg shphd1_pentr ™/ u6 plasmid or gfp - plasmid ( as control ) by using fugene 6 ( roche ), according to the manufacturer &# 39 ; s instructions . after 48 hours , cells were lysed and decline in phd1 rna expression was referred to percentage of gpf positive cells . shphd1 cassettes were transferred from pentr ™/ u6 plasmid to plenti6 / block - it - dest expression plasmid for lentiviral production that was done as described . 30 briefly , 3 μg of lentiviral vector ( plenti6 / block - it - dest shphd1 expression plasmid ) and 9 μg virapower ™ packaging mix were mixed in opti - mem i medium according to the manufacturer &# 39 ; s instructions ( invitrogen ). packaging mix and lentiviral vector were co - transfected in 293ft cells by using the lipofectamine ™ 2000 transfection reagent ( invitrogen ). virus - containing supernatans was collected 48 to 72 hours post - transfection , filtered through a 0 . 22 μm filter , and used for infection . infection protocol . alternatively , skeletal muscle fibers were transfected with shphd1_pentr ™/ u6 plasmid by in vivo electroporation . the shphd1 pentr ™/ u6 plasmids were delivered to the skeletal muscle of phd1 +/− mice via in vivo electroporation , a procedure that induces only minimal fiber damage and inflammation . ep was carried out five days prior to induction of limb ischemia as follows . the crural muscles of anesthetized animals were injected with hyaluronidase enzyme ( 60 μl ; 0 . 4 u / μl ) one hour prior to plasmid injection . thereafter , 40 μg of shphd1 kd or shphd1 ctr pentr ™/ u6 plasmid ( final volume : 30 μl in normal saline ) was injected in the crural muscles . injected muscles were immediately electroporated with a strain of eight pulses ( 120v ; duration 20 ms ; intervals 1 / second ), using an electroporation system ( btx ecm 830 electroporator ; genetronix , inc .) with tweezertrode circular electrodes ( diameter 7 mm ). induction of hind limb ischemia , analysis of muscle necrosis and rna analysis were performed as described above . the data are represented as mean ± s . e . m . of the indicated number of measurements . standard t - 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