Stem Cell Antigen-1 in Skeletal Muscle Function

Stem cell antigen-1 (Sca-1) is a member of the Ly-6 multigene family encoding highly homologous, glycosyl-phosphatidylinositolanchored membrane proteins. Sca-1 is expressed on muscle-derived stem cells and myogenic precursors recruited to sites of muscle injury. We previously reported that inhibition of Sca-1 expression stimulated myoblast proliferation in vitro and regulated the tempo of muscle repair in vivo. Despite its function in myoblast expansion during muscle repair, a role for Sca-1 in normal, post-natal muscle has not been thoroughly investigated. We systematically compared Sca-1-/(KO) and Sca-1+/+ (WT) mice and hindlimb muscles to elucidate the tissue, contractile, and functional effects of Sca-1 in young and aging animals. Comparison of muscle volume, fibrosis, myofiber cross-sectional area, and Pax7+ myoblast number showed little differences between ages or genotypes. Exercise protocols, however, demonstrated decreased stamina in KO versus WT mice, with young KO mice achieving results similar to aging WT animals. In addition, KO mice did not improve with practice, while WT animals demonstrated conditioning over time. Surprisingly, myomechanical analysis of isolated muscles showed that KO young muscle generated more force and experienced less fatigue. However, KO muscle also demonstrated incomplete relaxation with fatigue. These findings suggest that Sca-1 is necessary for muscle conditioning with exercise, and that deficient conditioning in Sca-1 KO animals becomes more pronounced with age.


INTRODUCTION
In response to skeletal muscle damage, resident myogenic progenitors undergo activation to form a pool of proliferating myoblasts.These mononuclear myoblasts differentiate and fuse, forming multinucleated myocytes, which repair or replace the damaged tissue 1 , 2 , 3 .This programmed series of events is essential to maintaining tissue homeostasis during exercise and aging, and to ensuring recovery from muscle trauma 4 .While the balance between myoblast proliferation and differentiation is critical to muscle repair, its regulation is incompletely understood.
We previously identified Stem cell antigen-1 (Sca-1; also known as Ly-6A/E) during an expression screen to identify genes regulating myoblast cell cycle withdrawal during differentiation 5 .Sca-1 is a member of the Ly-6 multigene family encoding a number of highly homologous, glycosyl-phosphatidylinositol (GPI)-anchored surface membrane proteins, and is widely used as a marker of murine hematopoietic stem cells 6 , 7 , 8 .Beyond its role as a stem cell marker, it has been shown that overexpression of Sca-1 inhibits proliferation of CD4 + T-cells (9), as well as differentiation of hematopoietic stem cells 6 , 10 , 11 , 12 .Sca-1 -/-mice are viable, however, they exhibit immune and hematopoietic defects 6 , 10 , 11 , 12 .Specifically, these mice demonstrate a lymphocytosis and thrombocytopenia, and isolated Sca-1 -/-T-cells undergo prolonged hyperproliferation with stimulation in vitro 11 .Consistent with a role in progenitor cell maintenance, Sca-1-null animals have a reduced ability to re-populate bone marrow after serial transplantation 6 , 12 and develop age-related failure of osteogenesis 10 .
Sca-1 also is expressed on the surface of muscle-derived stem cells 13 , 14 and myogenic precursors recruited to sites of skeletal or cardiac muscle injury 13 , 15 , 16 , 17 , 18 .We previously reported that inhibition of Sca-1 expression by antisense or Sca-1 interference with blocking antibodies stimulated myoblast proliferation and delayed myoblast fusion in vitro 19 .Subsequently, others observed sustained proliferation in Sca-1 -/-myoblasts cultured ex vivo 20 .Using a myonecrotic injury model in Sca-1 -/- and Sca-1 +/+ mice, we then showed that Sca-1 regulates the tempo of muscle repair by controlling the balance between proliferation and differentiation of activated myoblasts 21 .

INTRODUCTION
In response to skeletal muscle damage, resident myogenic progenitors undergo activation to form a pool of proliferating myoblasts.These mononuclear myoblasts differentiate and fuse, forming multinucleated myocytes, which repair or replace the damaged tissue 1 , 2 , 3 .This programmed series of events is essential to maintaining tissue homeostasis during exercise and aging, and to ensuring recovery from muscle trauma 4 .While the balance between myoblast proliferation and differentiation is critical to muscle repair, its regulation is incompletely understood.
We previously identified Stem cell antigen-1 (Sca-1; also known as Ly-6A/E) during an expression screen to identify genes regulating myoblast cell cycle withdrawal during differentiation 5 .Sca-1 is a member of the Ly-6 multigene family encoding a number of highly homologous, glycosyl-phosphatidylinositol (GPI)-anchored surface membrane proteins, and is widely used as a marker of murine hematopoietic stem cells 6 , 7 , 8 .Beyond its role as a stem cell marker, it has been shown that overexpression of Sca-1 inhibits proliferation of CD4 + T-cells (9), as well as differentiation of hematopoietic stem cells 6 , 10 , 11 , 12 .Sca-1 -/-mice are viable, however, they exhibit immune and hematopoietic defects 6 , 10 , 11 , 12 .Specifically, these mice demonstrate a lymphocytosis and thrombocytopenia, and isolated Sca-1 -/-T-cells undergo prolonged hyperproliferation with stimulation in vitro 11 .Consistent with a role in progenitor cell maintenance, Sca-1-null animals have a reduced ability to re-populate bone marrow after serial transplantation 6 , 12 and develop age-related failure of osteogenesis 10 .
Sca-1 also is expressed on the surface of muscle-derived stem cells 13 , 14 and myogenic precursors recruited to sites of skeletal or cardiac muscle injury 13 , 15 , 16 , 17 , 18 .We previously reported that inhibition of Sca-1 expression by antisense or Sca-1 interference with blocking antibodies stimulated myoblast proliferation and delayed myoblast fusion in vitro 19 .Subsequently, others observed sustained proliferation in Sca-1 -/-myoblasts cultured ex vivo 20 .Using a myonecrotic injury model in Sca-1 -/- and Sca-1 +/+ mice, we then showed that Sca-1 regulates the tempo of muscle repair by controlling the balance between proliferation and differentiation of activated myoblasts 21 .

INTRODUCTION
In response to skeletal muscle damage, resident myogenic progenitors undergo activation to form a pool of proliferating myoblasts.These mononuclear myoblasts differentiate and fuse, forming multinucleated myocytes, which repair or replace the damaged tissue 1 , 2 , 3 .This programmed series of events is essential to maintaining tissue homeostasis during exercise and aging, and to ensuring recovery from muscle trauma 4 .While the balance between myoblast proliferation and differentiation is critical to muscle repair, its regulation is incompletely understood.
We previously identified Stem cell antigen-1 (Sca-1; also known as Ly-6A/E) during an expression screen to identify genes regulating myoblast cell cycle withdrawal during differentiation 5 .Sca-1 is a member of the Ly-6 multigene family encoding a number of highly homologous, glycosyl-phosphatidylinositol (GPI)-anchored surface membrane proteins, and is widely used as a marker of murine hematopoietic stem cells 6 , 7 , 8 .Beyond its role as a stem cell marker, it has been shown that overexpression of Sca-1 inhibits proliferation of CD4 + T-cells (9), as well as differentiation of hematopoietic stem cells 6 , 10 , 11 , 12 .Sca-1 -/-mice are viable, however, they exhibit immune and hematopoietic defects 6 , 10 , 11 , 12 .Specifically, these mice demonstrate a lymphocytosis and thrombocytopenia, and isolated Sca-1 -/-T-cells undergo prolonged hyperproliferation with stimulation in vitro 11 .Consistent with a role in progenitor cell maintenance, Sca-1-null animals have a reduced ability to re-populate bone marrow after serial transplantation 6 , 12 and develop age-related failure of osteogenesis 10 .
Sca-1 also is expressed on the surface of muscle-derived stem cells 13 , 14 and myogenic precursors recruited to sites of skeletal or cardiac muscle injury 13 , 15 , 16 , 17 , 18 .We previously reported that inhibition of Sca-1 expression by antisense or Sca-1 interference with blocking antibodies stimulated myoblast proliferation and delayed myoblast fusion in vitro 19 .Subsequently, others observed sustained proliferation in Sca-1 -/-myoblasts cultured ex vivo 20 .Using a myonecrotic injury model in Sca-1 -/- and Sca-1 +/+ mice, we then showed that Sca-1 regulates the tempo of muscle repair by controlling the balance between proliferation and differentiation of activated myoblasts 21 .
Despite its function in stem cell proliferation in general, and more specifically in myoblast expansion during secondary myogenesis, a role for Sca-1 in normal, post-natal muscle function has not been apparent.To explore this, we undertook the systematic comparison of Sca-1 -/-and Sca-1 +/+ mice and hindlimb muscles to elucidate the tissue, mechanical, and functional effects of Sca-1 in young adult and aging animals.

MATERIALS AND METHODS
Animals.All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of California, San Francisco, in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International guidelines.Mice heterozygous at the Sca-1 locus were graciously provided by Patrick Flood (University of North Carolina) 11 , and backcrossed to BALB/c strain for ten generations.Sca-1 +/-littermates were bred to homozygosity.Genotypes were confirmed for all experimental animals by Southern blot analysis, as previously described 21 .All experiments were performed on 12-16 week-old or 47-50 week-old female mice.Wild type (Sca-1 +/+ ) female BALB/c littermates from heterozygous matings were used as controls in all experiments.
Muscle volume analysis.Animals were euthanized with intraperitoneal injection of pentobarbital followed by cervical dislocation.Following euthanization, skinned hindlimbs were fixed in 10% paraformaldehyde, then dehydrated in ethanol and embedded in paraffin.Hindlimbs were sectioned at 5 ?m perpendicular to the myofiber through the length of the limb.A series of every tenth section was selected and processed for staining.Sections were deparaffinized and stained with hematoxylin and eosin to identify tissue architecture.Using accepted anatomic boundaries, the relevant muscle was traced at low power (4X) using the live image generated with a Zeiss Axiovision fluorescent microscope (Carl Zeiss; Thornwood, NY) equipped with a Hamamatsu Orca2 digital camera (Hamamatsu; Bridgewater, NJ).Volume estimates were calculated using the Cavalieri principle 22 and contours traced at low power.Both hindlimbs from at least 3 animals were analyzed for each genotype/age.Myofiber CSA and perimeter analysis.Tissue sections were prepared as described above.A series of every tenth section was selected and processed for staining.Sections were deparaffinized in xylene and dehydrated with ethanol before antigen retrieval with proteinase K for 10 minutes at room temperature (RT).After washing out proteinase K, sections were incubated with 3% H 2 O 2 in methanol for 10 min at RT, Rodent Block M (BioCare; Concord, CA) for 30 min at RT, DAKO antibody diluent (DAKO; Carpinteria, CA) for 30 min at RT, rabbit anti-laminin (Sigma-Aldrich; St. Louis, MO) at 1:100 dilution in DAKO antibody diluent (DAKO; Carpinteria, CA) overnight at 4°C, then washed with phosphate-buffered saline.To develop peroxidase, sections were incubated with Rabbit HRP Polymer (BioCare; Concord, CA), then DAB substrate (BioCare; Concord, CA) according to the manufacturer's instructions.Sections were counterstained with hematoxylin before dehydration with ethanol (80, 90, 100%).To quantitate myofiber cross-sectional area (CSA) and perimeter within a section, ?300 myofibers within a centrally located field (20X; ~300,000 µm 2 ) within the tibialis anterior, extensor digitorum longus, or soleus muscle were analyzed using Stereo Investigator software (Microbrightfield; Williston, VT).Both hindlimbs from at least 3 animals were analyzed for each genotype/age.Pax7 analysis.Paraffin-embedded tissue sections were prepared as described above.Sections were incubated with Rodent Block M (BioCare; Concord, CA) for 30 min at RT, blocking buffer (2% goat serum, 1% bovine serum albumin, 0.1% fish gelatin, 0.1% Triton X-100, 0.1% glycine) for 30 min at RT, 10 µg/ml Alex488-conjugated anti-Pax7 (R&D Systems; Minneapolis, MN) in blocking buffer without glycine for 60 min at RT, then washed with phosphate-buffered saline, counterstained with DAPI, and dehydrated with ethanol (70, 80, 90, 100%).Immunostained sections were imaged by randomly placing the 20X objective within the tibialis anterior muscle within the section.Following imaging, the field was manually moved a fixed distance of approximately 600 µm in the horizontal and then vertical axis, resulting in 4-6 counting images per section.DAPI + Pax7 + nuclei localized beneath the basal lamina of myofibers were confirmed at 60X magnification, then counted and expressed as a percentage of total DAPI + nuclei.Both hindlimbs from at least 3 animals were analyzed for each genotype/age.
Arteriole number and CSA analysis.Tissue sections were prepared as described above.Sections were incubated with Rodent Block M (BioCare; Concord, CA) for 30 min at RT, DAKO antibody diluent (DAKO; Carpinteria, CA) for 30 min at RT, rat anti-CD31 (BioCare; Concord, CA) at 1:25 dilution in DAKO antibody diluent (DAKO) overnight at 4°C, then washed with phosphatebuffered saline.To develop peroxidase, sections were incubated with Rat HRP Polymer (BioCare; Concord, CA) followed by DAB substrate (BioCare; Concord, CA) according to the manufacturer's instructions.After rinsing with phosphate-buffered saline, sections were then re-blocked with Rodent Block M (BioCare; Concord, CA) for 30 min at RT and DAKO antibody diluent (DAKO; Carpinteria, CA) for 30 min at RT, and incubated with mouse anti-SMA (BioCare; Concord, CA) at 1:50 dilution for 60 min at RT.Sections were then incubated with Mouse AP Polymer (BioCare; Concord, CA) and developed with Vulcan Red (BioCare; Concord, CA) according to the manufactorer's instructions.Sections were counterstained with hematoxylin before dehydration with ethanol (70, 80, 90, 100%).Immunostained sections were imaged using the 4X objective.Arteriole number was calculated as the total number of SMA + CD31 + vessels per section.Each vessel was confirmed at 40X.To quantitate arteriolar CSA, all identified arterioles were analyzed using Stereo Investigator software (Microbrightfield; Williston, VT).Both hindlimbs from at least 3 animals were analyzed for each genotype/age.
Fibrosis.Tissue sections were prepared as described above.A series of every tenth section was selected and processed for staining.Sections were deparaffinized in xylene and dehydrated with ethanol before staining with aniline blue, hematoxylin, and Despite its function in stem cell proliferation in general, and more specifically in myoblast expansion during secondary myogenesis, a role for Sca-1 in normal, post-natal muscle function has not been apparent.To explore this, we undertook the systematic comparison of Sca-1 -/-and Sca-1 +/+ mice and hindlimb muscles to elucidate the tissue, mechanical, and functional effects of Sca-1 in young adult and aging animals.

Animals.
All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of California, San Francisco, in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International guidelines.Mice heterozygous at the Sca-1 locus were graciously provided by Patrick Flood (University of North Carolina) 11 , and backcrossed to BALB/c strain for ten generations.Sca-1 +/-littermates were bred to homozygosity.Genotypes were confirmed for all experimental animals by Southern blot analysis, as previously described 21 .All experiments were performed on 12-16 week-old or 47-50 week-old female mice.Wild type (Sca-1 +/+ ) female BALB/c littermates from heterozygous matings were used as controls in all experiments.
Muscle volume analysis.Animals were euthanized with intraperitoneal injection of pentobarbital followed by cervical dislocation.Following euthanization, skinned hindlimbs were fixed in 10% paraformaldehyde, then dehydrated in ethanol and embedded in paraffin.Hindlimbs were sectioned at 5 ?m perpendicular to the myofiber through the length of the limb.A series of every tenth section was selected and processed for staining.Sections were deparaffinized and stained with hematoxylin and eosin to identify tissue architecture.Using accepted anatomic boundaries, the relevant muscle was traced at low power (4X) using the live image generated with a Zeiss Axiovision fluorescent microscope (Carl Zeiss; Thornwood, NY) equipped with a Hamamatsu Orca2 digital camera (Hamamatsu; Bridgewater, NJ).Volume estimates were calculated using the Cavalieri principle 22 and contours traced at low power.Both hindlimbs from at least 3 animals were analyzed for each genotype/age.Myofiber CSA and perimeter analysis.Tissue sections were prepared as described above.A series of every tenth section was selected and processed for staining.Sections were deparaffinized in xylene and dehydrated with ethanol before antigen retrieval with proteinase K for 10 minutes at room temperature (RT).After washing out proteinase K, sections were incubated with 3% H 2 O 2 in methanol for 10 min at RT, Rodent Block M (BioCare; Concord, CA) for 30 min at RT, DAKO antibody diluent (DAKO; Carpinteria, CA) for 30 min at RT, rabbit anti-laminin (Sigma-Aldrich; St. Louis, MO) at 1:100 dilution in DAKO antibody diluent (DAKO; Carpinteria, CA) overnight at 4°C, then washed with phosphate-buffered saline.To develop peroxidase, sections were incubated with Rabbit HRP Polymer (BioCare; Concord, CA), then DAB substrate (BioCare; Concord, CA) according to the manufacturer's instructions.Sections were counterstained with hematoxylin before dehydration with ethanol (80, 90, 100%).To quantitate myofiber cross-sectional area (CSA) and perimeter within a section, ?300 myofibers within a centrally located field (20X; ~300,000 µm 2 ) within the tibialis anterior, extensor digitorum longus, or soleus muscle were analyzed using Stereo Investigator software (Microbrightfield; Williston, VT).Both hindlimbs from at least 3 animals were analyzed for each genotype/age.Pax7 analysis.Paraffin-embedded tissue sections were prepared as described above.Sections were incubated with Rodent Block M (BioCare; Concord, CA) for 30 min at RT, blocking buffer (2% goat serum, 1% bovine serum albumin, 0.1% fish gelatin, 0.1% Triton X-100, 0.1% glycine) for 30 min at RT, 10 µg/ml Alex488-conjugated anti-Pax7 (R&D Systems; Minneapolis, MN) in blocking buffer without glycine for 60 min at RT, then washed with phosphate-buffered saline, counterstained with DAPI, and dehydrated with ethanol (70, 80, 90, 100%).Immunostained sections were imaged by randomly placing the 20X objective within the tibialis anterior muscle within the section.Following imaging, the field was manually moved a fixed distance of approximately 600 µm in the horizontal and then vertical axis, resulting in 4-6 counting images per section.DAPI + Pax7 + nuclei localized beneath the basal lamina of myofibers were confirmed at 60X magnification, then counted and expressed as a percentage of total DAPI + nuclei.Both hindlimbs from at least 3 animals were analyzed for each genotype/age.
Arteriole number and CSA analysis.Tissue sections were prepared as described above.Sections were incubated with Rodent Block M (BioCare; Concord, CA) for 30 min at RT, DAKO antibody diluent (DAKO; Carpinteria, CA) for 30 min at RT, rat anti-CD31 (BioCare; Concord, CA) at 1:25 dilution in DAKO antibody diluent (DAKO) overnight at 4°C, then washed with phosphatebuffered saline.To develop peroxidase, sections were incubated with Rat HRP Polymer (BioCare; Concord, CA) followed by DAB substrate (BioCare; Concord, CA) according to the manufacturer's instructions.After rinsing with phosphate-buffered saline, sections were then re-blocked with Rodent Block M (BioCare; Concord, CA) for 30 min at RT and DAKO antibody diluent (DAKO; Carpinteria, CA) for 30 min at RT, and incubated with mouse anti-SMA (BioCare; Concord, CA) at 1:50 dilution for 60 min at RT.Sections were then incubated with Mouse AP Polymer (BioCare; Concord, CA) and developed with Vulcan Red (BioCare; Concord, CA) according to the manufactorer's instructions.Sections were counterstained with hematoxylin before dehydration with ethanol (70, 80, 90, 100%).Immunostained sections were imaged using the 4X objective.Arteriole number was calculated as the total number of SMA + CD31 + vessels per section.Each vessel was confirmed at 40X.To quantitate arteriolar CSA, all identified arterioles were analyzed using Stereo Investigator software (Microbrightfield; Williston, VT).Both hindlimbs from at least 3 animals were analyzed for each genotype/age.
Fibrosis.Tissue sections were prepared as described above.A series of every tenth section was selected and processed for staining.Sections were deparaffinized in xylene and dehydrated with ethanol before staining with aniline blue, hematoxylin, and Despite its function in stem cell proliferation in general, and more specifically in myoblast expansion during secondary myogenesis, a role for Sca-1 in normal, post-natal muscle function has not been apparent.To explore this, we undertook the systematic comparison of Sca-1 -/-and Sca-1 +/+ mice and hindlimb muscles to elucidate the tissue, mechanical, and functional effects of Sca-1 in young adult and aging animals.

MATERIALS AND METHODS
Animals.All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of California, San Francisco, in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International guidelines.Mice heterozygous at the Sca-1 locus were graciously provided by Patrick Flood (University of North Carolina) 11 , and backcrossed to BALB/c strain for ten generations.Sca-1 +/-littermates were bred to homozygosity.Genotypes were confirmed for all experimental animals by Southern blot analysis, as previously described 21 .All experiments were performed on 12-16 week-old or 47-50 week-old female mice.Wild type (Sca-1 +/+ ) female BALB/c littermates from heterozygous matings were used as controls in all experiments.
Muscle volume analysis.Animals were euthanized with intraperitoneal injection of pentobarbital followed by cervical dislocation.Following euthanization, skinned hindlimbs were fixed in 10% paraformaldehyde, then dehydrated in ethanol and embedded in paraffin.Hindlimbs were sectioned at 5 ?m perpendicular to the myofiber through the length of the limb.A series of every tenth section was selected and processed for staining.Sections were deparaffinized and stained with hematoxylin and eosin to identify tissue architecture.Using accepted anatomic boundaries, the relevant muscle was traced at low power (4X) using the live image generated with a Zeiss Axiovision fluorescent microscope (Carl Zeiss; Thornwood, NY) equipped with a Hamamatsu Orca2 digital camera (Hamamatsu; Bridgewater, NJ).Volume estimates were calculated using the Cavalieri principle 22 and contours traced at low power.Both hindlimbs from at least 3 animals were analyzed for each genotype/age.Myofiber CSA and perimeter analysis.Tissue sections were prepared as described above.A series of every tenth section was selected and processed for staining.Sections were deparaffinized in xylene and dehydrated with ethanol before antigen retrieval with proteinase K for 10 minutes at room temperature (RT).After washing out proteinase K, sections were incubated with 3% H 2 O 2 in methanol for 10 min at RT, Rodent Block M (BioCare; Concord, CA) for 30 min at RT, DAKO antibody diluent (DAKO; Carpinteria, CA) for 30 min at RT, rabbit anti-laminin (Sigma-Aldrich; St. Louis, MO) at 1:100 dilution in DAKO antibody diluent (DAKO; Carpinteria, CA) overnight at 4°C, then washed with phosphate-buffered saline.To develop peroxidase, sections were incubated with Rabbit HRP Polymer (BioCare; Concord, CA), then DAB substrate (BioCare; Concord, CA) according to the manufacturer's instructions.Sections were counterstained with hematoxylin before dehydration with ethanol (80, 90, 100%).To quantitate myofiber cross-sectional area (CSA) and perimeter within a section, ?300 myofibers within a centrally located field (20X; ~300,000 µm 2 ) within the tibialis anterior, extensor digitorum longus, or soleus muscle were analyzed using Stereo Investigator software (Microbrightfield; Williston, VT).Both hindlimbs from at least 3 animals were analyzed for each genotype/age.Pax7 analysis.Paraffin-embedded tissue sections were prepared as described above.Sections were incubated with Rodent Block M (BioCare; Concord, CA) for 30 min at RT, blocking buffer (2% goat serum, 1% bovine serum albumin, 0.1% fish gelatin, 0.1% Triton X-100, 0.1% glycine) for 30 min at RT, 10 µg/ml Alex488-conjugated anti-Pax7 (R&D Systems; Minneapolis, MN) in blocking buffer without glycine for 60 min at RT, then washed with phosphate-buffered saline, counterstained with DAPI, and dehydrated with ethanol (70, 80, 90, 100%).Immunostained sections were imaged by randomly placing the 20X objective within the tibialis anterior muscle within the section.Following imaging, the field was manually moved a fixed distance of approximately 600 µm in the horizontal and then vertical axis, resulting in 4-6 counting images per section.DAPI + Pax7 + nuclei localized beneath the basal lamina of myofibers were confirmed at 60X magnification, then counted and expressed as a percentage of total DAPI + nuclei.Both hindlimbs from at least 3 animals were analyzed for each genotype/age.
Arteriole number and CSA analysis.Tissue sections were prepared as described above.Sections were incubated with Rodent Block M (BioCare; Concord, CA) for 30 min at RT, DAKO antibody diluent (DAKO; Carpinteria, CA) for 30 min at RT, rat anti-CD31 (BioCare; Concord, CA) at 1:25 dilution in DAKO antibody diluent (DAKO) overnight at 4°C, then washed with phosphatebuffered saline.To develop peroxidase, sections were incubated with Rat HRP Polymer (BioCare; Concord, CA) followed by DAB substrate (BioCare; Concord, CA) according to the manufacturer's instructions.After rinsing with phosphate-buffered saline, sections were then re-blocked with Rodent Block M (BioCare; Concord, CA) for 30 min at RT and DAKO antibody diluent (DAKO; Carpinteria, CA) for 30 min at RT, and incubated with mouse anti-SMA (BioCare; Concord, CA) at 1:50 dilution for 60 min at RT.Sections were then incubated with Mouse AP Polymer (BioCare; Concord, CA) and developed with Vulcan Red (BioCare; Concord, CA) according to the manufactorer's instructions.Sections were counterstained with hematoxylin before dehydration with ethanol (70, 80, 90, 100%).Immunostained sections were imaged using the 4X objective.Arteriole number was calculated as the total number of SMA + CD31 + vessels per section.Each vessel was confirmed at 40X.To quantitate arteriolar CSA, all identified arterioles were analyzed using Stereo Investigator software (Microbrightfield; Williston, VT).Both hindlimbs from at least 3 animals were analyzed for each genotype/age.
Fibrosis.Tissue sections were prepared as described above.A series of every tenth section was selected and processed for staining.Sections were deparaffinized in xylene and dehydrated with ethanol before staining with aniline blue, hematoxylin, and scarlet acid fuchsin with Masson's Trichrome 2000 stain kit (American MasterTech, Lodi, CA) according to the manufacturer's instructions.Stained sections dehydrated with alcohol, cleared with xylene, and imaged using the 4X objective.To quantitate CSA of fibrosis, all stained areas were analyzed using Stereo Investigator software (Microbrightfield; Williston, VT).Both hindlimbs from at least 3 animals were analyzed for each genotype/age.Differences between genotype/age groups were tested for significance by one-way analysis of variance.Ki67 immunostaining.Tissue sections were prepared as described above.A series of every tenth section was selected and processed for staining.Sections were deparaffinized in xylene and dehydrated with ethanol before antigen retrieval with citrate buffer for 20 minutes at 37°C.After washing with phosphate-buffered saline, sections were incubated with 3% H 2 O 2 in methanol for 20 min at RT, Rodent Block M (BioCare; Concord, CA) for 30 min at RT, DAKO antibody diluent (DAKO; Carpinteria, CA) for 30 min at RT, rat anti-Ki67 (DAKO; Carpinteria, CA) at 1:10 dilution in DAKO antibody diluent (DAKO; Carpinteria, CA) overnight at 4°C, then washed with phosphate-buffered saline.To develop peroxidase, sections were incubated with Rat HRP Polymer (BioCare; Concord, CA), then DAB substrate (BioCare; Concord, CA) according to the manufacturer's instructions.Sections were counterstained with hematoxylin then washed with phosphate-buffered saline, counterstained with DAPI, and dehydrated with ethanol.Immunostained sections were imaged by randomly placing the 20X objective within the section.Following imaging, the field was manually moved a fixed distance of approximately 600 µm in the horizontal and then vertical axis, resulting in 4-6 counting images per section.DAPI + Ki67 + nuclei localized beneath the basal lamina of myofibers were confirmed at 60X magnification, then counted and expressed as a percentage of total DAPI + nuclei.Both hindlimbs from at least 3 animals were analyzed for each genotype/age.Differences between genotype/age groups were tested for significance by one-way analysis of variance.
Exercise.To evaluate voluntary exercise, mice were allowed to run at will during normal light-dark cycles as established at the UCSF Laboratory Animal Resource Center.Distance and duration over consecutive 24h periods were recorded with individual FX10 cycle computers (E3 Cycling; Chapel Hill, NC).To evaluate forced exercise, mice were forced to run on each day over a 37d period on a Lafayette Mouse Forced Exercise Run/Walk Wheel System (Lafayette Instrument; Lafayette, IN) at 8 meters/min for five 10 min intervals with 30s rests between intervals.Over the next five 10 min intervals immediately following during that day's exercise session, speeds were progressively increased until mice reached their maximum rate, as determined by the maximum speed (meters/min) at which mice continued to run on the wheel without falling off or hanging onto the wheel.

Myomechanical analysis.
Ex vivo muscle analysis was performed as previously described 23 .Following euthanization, the EDL muscle was extracted and placed in a bath containing Krebs Hensleit buffer within 15 min.The muscle was attached by opposing tendons to a DMT Model 820MS force transducer (Danish Myo Technology; Ann Arbor, MI) filled with Krebs Hensleit buffer prewarmed to 25 o C and bubbled with O 2 /CO 2 (95%/5%) for ?15 min prior to use.During the mounting process, the muscle was only handled through the suture, without direct contact to the muscle to prevent damage to the muscle fiber.The muscle was stimulated by a Grass Model S48 square pulse electrical stimulator (Grass Technologies; West Warwick, RI) and the data analyzed and projected using a custom acquisition platform (ADInstruments PowerLab Data Acquisition System and LabChart software; ADInstruments; Colorado Springs, CO).
Twitch tension (P t ) was recorded by first stretching the EDL muscle until there was no laxity in the muscle fiber.A square stimulation of 0.5 ms duration was used to induce twitch.The voltage was increase incrementally until maximal twitch tension was achieve and then the voltage was set at 20% above the maximum to induce a supramaximal stimulus (mean supramaximal stimulus was 40 volts).Optimum length of the muscle was determined by carefully stretching the muscle and recording the twitch response after square stimulation until maximal twitch was recorded.The muscle was left to equilibrate at the optimal length for 3 min before another supramaximal stimulus was applied and the output recorded as the twitch force.Tetanic tension (P o ) was recorded by applying a train of supramaximal stimuli for 300 ms at 150 Hz.
Force-frequency fatigue was measured by exposing muscle to a supramaximal stimulus train of 3-5 pulses (300 msec duration separated by 3 sec) at successive frequencies (30, 60, 100 and 140 Hz), with 5 min intervals between stimulations.For a given frequency stimulus and muscle, the maximum pulse was chosen and then normalized relative to the peak response over all pulse frequencies.An average force-frequency diagram was then constructed from all normalized muscle responses.Forcefrequency stimulation produces a maximum response at a given frequency that may fall off precipitously at other frequencies.The fatigue characteristic of a given group would be interpreted as a significant reduction in the peak relative to the other groups at the various frequencies.
Low-frequency time-fatigue was measured by supramaximal tetanic muscle stimulation at low frequency (60 Hz) for a duration of 300 msec, repeated every 3 seconds for a period of 10 min.The low-frequency time-fatigue curve produces a peak response at a given time that is followed by a multi-exponential decay in contraction force.Fatigue in a given group would be interpreted as a significant reduction in percent maximum contraction force over time, assuming that the different groups experienced the same average peak force at the onset.We sampled the percent of maximal force of contraction at given periods (0.5 , 1, 1.5, 2-10 min in 1 min increments).
Muscle mass, cross-sectional area, and length were measured after the fatigue analysis to avoid handling prior to analysis.
Statistical analysis.For volume, CSA, perimeter, and cell number analyses, differences between genotype/age groups were tested for significance by one-way analysis of variance (ANOVA), followed by unpaired t-test with Bonferroni correction to scarlet acid fuchsin with Masson's Trichrome 2000 stain kit (American MasterTech, Lodi, CA) according to the manufacturer's instructions.Stained sections dehydrated with alcohol, cleared with xylene, and imaged using the 4X objective.To quantitate CSA of fibrosis, all stained areas were analyzed using Stereo Investigator software (Microbrightfield; Williston, VT).Both hindlimbs from at least 3 animals were analyzed for each genotype/age.Differences between genotype/age groups were tested for significance by one-way analysis of variance.Ki67 immunostaining.Tissue sections were prepared as described above.A series of every tenth section was selected and processed for staining.Sections were deparaffinized in xylene and dehydrated with ethanol before antigen retrieval with citrate buffer for 20 minutes at 37°C.After washing with phosphate-buffered saline, sections were incubated with 3% H 2 O 2 in methanol for 20 min at RT, Rodent Block M (BioCare; Concord, CA) for 30 min at RT, DAKO antibody diluent (DAKO; Carpinteria, CA) for 30 min at RT, rat anti-Ki67 (DAKO; Carpinteria, CA) at 1:10 dilution in DAKO antibody diluent (DAKO; Carpinteria, CA) overnight at 4°C, then washed with phosphate-buffered saline.To develop peroxidase, sections were incubated with Rat HRP Polymer (BioCare; Concord, CA), then DAB substrate (BioCare; Concord, CA) according to the manufacturer's instructions.Sections were counterstained with hematoxylin then washed with phosphate-buffered saline, counterstained with DAPI, and dehydrated with ethanol.Immunostained sections were imaged by randomly placing the 20X objective within the section.Following imaging, the field was manually moved a fixed distance of approximately 600 µm in the horizontal and then vertical axis, resulting in 4-6 counting images per section.DAPI + Ki67 + nuclei localized beneath the basal lamina of myofibers were confirmed at 60X magnification, then counted and expressed as a percentage of total DAPI + nuclei.Both hindlimbs from at least 3 animals were analyzed for each genotype/age.Differences between genotype/age groups were tested for significance by one-way analysis of variance.
Exercise.To evaluate voluntary exercise, mice were allowed to run at will during normal light-dark cycles as established at the UCSF Laboratory Animal Resource Center.Distance and duration over consecutive 24h periods were recorded with individual FX10 cycle computers (E3 Cycling; Chapel Hill, NC).To evaluate forced exercise, mice were forced to run on each day over a 37d period on a Lafayette Mouse Forced Exercise Run/Walk Wheel System (Lafayette Instrument; Lafayette, IN) at 8 meters/min for five 10 min intervals with 30s rests between intervals.Over the next five 10 min intervals immediately following during that day's exercise session, speeds were progressively increased until mice reached their maximum rate, as determined by the maximum speed (meters/min) at which mice continued to run on the wheel without falling off or hanging onto the wheel.

Myomechanical analysis.
Ex vivo muscle analysis was performed as previously described 23 .Following euthanization, the EDL muscle was extracted and placed in a bath containing Krebs Hensleit buffer within 15 min.The muscle was attached by opposing tendons to a DMT Model 820MS force transducer (Danish Myo Technology; Ann Arbor, MI) filled with Krebs Hensleit buffer prewarmed to 25 o C and bubbled with O 2 /CO 2 (95%/5%) for ?15 min prior to use.During the mounting process, the muscle was only handled through the suture, without direct contact to the muscle to prevent damage to the muscle fiber.The muscle was stimulated by a Grass Model S48 square pulse electrical stimulator (Grass Technologies; West Warwick, RI) and the data analyzed and projected using a custom acquisition platform (ADInstruments PowerLab Data Acquisition System and LabChart software; ADInstruments; Colorado Springs, CO).
Twitch tension (P t ) was recorded by first stretching the EDL muscle until there was no laxity in the muscle fiber.A square stimulation of 0.5 ms duration was used to induce twitch.The voltage was increase incrementally until maximal twitch tension was achieve and then the voltage was set at 20% above the maximum to induce a supramaximal stimulus (mean supramaximal stimulus was 40 volts).Optimum length of the muscle was determined by carefully stretching the muscle and recording the twitch response after square stimulation until maximal twitch was recorded.The muscle was left to equilibrate at the optimal length for 3 min before another supramaximal stimulus was applied and the output recorded as the twitch force.Tetanic tension (P o ) was recorded by applying a train of supramaximal stimuli for 300 ms at 150 Hz.
Force-frequency fatigue was measured by exposing muscle to a supramaximal stimulus train of 3-5 pulses (300 msec duration separated by 3 sec) at successive frequencies (30, 60, 100 and 140 Hz), with 5 min intervals between stimulations.For a given frequency stimulus and muscle, the maximum pulse was chosen and then normalized relative to the peak response over all pulse frequencies.An average force-frequency diagram was then constructed from all normalized muscle responses.Forcefrequency stimulation produces a maximum response at a given frequency that may fall off precipitously at other frequencies.The fatigue characteristic of a given group would be interpreted as a significant reduction in the peak relative to the other groups at the various frequencies.
Low-frequency time-fatigue was measured by supramaximal tetanic muscle stimulation at low frequency (60 Hz) for a duration of 300 msec, repeated every 3 seconds for a period of 10 min.The low-frequency time-fatigue curve produces a peak response at a given time that is followed by a multi-exponential decay in contraction force.Fatigue in a given group would be interpreted as a significant reduction in percent maximum contraction force over time, assuming that the different groups experienced the same average peak force at the onset.We sampled the percent of maximal force of contraction at given periods (0.5 , 1, 1.5, 2-10 min in 1 min increments).
Muscle mass, cross-sectional area, and length were measured after the fatigue analysis to avoid handling prior to analysis.
Statistical analysis.For volume, CSA, perimeter, and cell number analyses, differences between genotype/age groups were tested for significance by one-way analysis of variance (ANOVA), followed by unpaired t-test with Bonferroni correction to scarlet acid fuchsin with Masson's Trichrome 2000 stain kit (American MasterTech, Lodi, CA) according to the manufacturer's instructions.Stained sections dehydrated with alcohol, cleared with xylene, and imaged using the 4X objective.To quantitate CSA of fibrosis, all stained areas were analyzed using Stereo Investigator software (Microbrightfield; Williston, VT).Both hindlimbs from at least 3 animals were analyzed for each genotype/age.Differences between genotype/age groups were tested for significance by one-way analysis of variance.Ki67 immunostaining.Tissue sections were prepared as described above.A series of every tenth section was selected and processed for staining.Sections were deparaffinized in xylene and dehydrated with ethanol before antigen retrieval with citrate buffer for 20 minutes at 37°C.After washing with phosphate-buffered saline, sections were incubated with 3% H 2 O 2 in methanol for 20 min at RT, Rodent Block M (BioCare; Concord, CA) for 30 min at RT, DAKO antibody diluent (DAKO; Carpinteria, CA) for 30 min at RT, rat anti-Ki67 (DAKO; Carpinteria, CA) at 1:10 dilution in DAKO antibody diluent (DAKO; Carpinteria, CA) overnight at 4°C, then washed with phosphate-buffered saline.To develop peroxidase, sections were incubated with Rat HRP Polymer (BioCare; Concord, CA), then DAB substrate (BioCare; Concord, CA) according to the manufacturer's instructions.Sections were counterstained with hematoxylin then washed with phosphate-buffered saline, counterstained with DAPI, and dehydrated with ethanol.Immunostained sections were imaged by randomly placing the 20X objective within the section.Following imaging, the field was manually moved a fixed distance of approximately 600 µm in the horizontal and then vertical axis, resulting in 4-6 counting images per section.DAPI + Ki67 + nuclei localized beneath the basal lamina of myofibers were confirmed at 60X magnification, then counted and expressed as a percentage of total DAPI + nuclei.Both hindlimbs from at least 3 animals were analyzed for each genotype/age.Differences between genotype/age groups were tested for significance by one-way analysis of variance.
Exercise.To evaluate voluntary exercise, mice were allowed to run at will during normal light-dark cycles as established at the UCSF Laboratory Animal Resource Center.Distance and duration over consecutive 24h periods were recorded with individual FX10 cycle computers (E3 Cycling; Chapel Hill, NC).To evaluate forced exercise, mice were forced to run on each day over a 37d period on a Lafayette Mouse Forced Exercise Run/Walk Wheel System (Lafayette Instrument; Lafayette, IN) at 8 meters/min for five 10 min intervals with 30s rests between intervals.Over the next five 10 min intervals immediately following during that day's exercise session, speeds were progressively increased until mice reached their maximum rate, as determined by the maximum speed (meters/min) at which mice continued to run on the wheel without falling off or hanging onto the wheel.

Myomechanical analysis.
Ex vivo muscle analysis was performed as previously described 23 .Following euthanization, the EDL muscle was extracted and placed in a bath containing Krebs Hensleit buffer within 15 min.The muscle was attached by opposing tendons to a DMT Model 820MS force transducer (Danish Myo Technology; Ann Arbor, MI) filled with Krebs Hensleit buffer prewarmed to 25 o C and bubbled with O 2 /CO 2 (95%/5%) for ?15 min prior to use.During the mounting process, the muscle was only handled through the suture, without direct contact to the muscle to prevent damage to the muscle fiber.The muscle was stimulated by a Grass Model S48 square pulse electrical stimulator (Grass Technologies; West Warwick, RI) and the data analyzed and projected using a custom acquisition platform (ADInstruments PowerLab Data Acquisition System and LabChart software; ADInstruments; Colorado Springs, CO).
Twitch tension (P t ) was recorded by first stretching the EDL muscle until there was no laxity in the muscle fiber.A square stimulation of 0.5 ms duration was used to induce twitch.The voltage was increase incrementally until maximal twitch tension was achieve and then the voltage was set at 20% above the maximum to induce a supramaximal stimulus (mean supramaximal stimulus was 40 volts).Optimum length of the muscle was determined by carefully stretching the muscle and recording the twitch response after square stimulation until maximal twitch was recorded.The muscle was left to equilibrate at the optimal length for 3 min before another supramaximal stimulus was applied and the output recorded as the twitch force.Tetanic tension (P o ) was recorded by applying a train of supramaximal stimuli for 300 ms at 150 Hz.
Force-frequency fatigue was measured by exposing muscle to a supramaximal stimulus train of 3-5 pulses (300 msec duration separated by 3 sec) at successive frequencies (30, 60, 100 and 140 Hz), with 5 min intervals between stimulations.For a given frequency stimulus and muscle, the maximum pulse was chosen and then normalized relative to the peak response over all pulse frequencies.An average force-frequency diagram was then constructed from all normalized muscle responses.Forcefrequency stimulation produces a maximum response at a given frequency that may fall off precipitously at other frequencies.The fatigue characteristic of a given group would be interpreted as a significant reduction in the peak relative to the other groups at the various frequencies.
Low-frequency time-fatigue was measured by supramaximal tetanic muscle stimulation at low frequency (60 Hz) for a duration of 300 msec, repeated every 3 seconds for a period of 10 min.The low-frequency time-fatigue curve produces a peak response at a given time that is followed by a multi-exponential decay in contraction force.Fatigue in a given group would be interpreted as a significant reduction in percent maximum contraction force over time, assuming that the different groups experienced the same average peak force at the onset.We sampled the percent of maximal force of contraction at given periods (0.5 , 1, 1.5, 2-10 min in 1 min increments).
Muscle mass, cross-sectional area, and length were measured after the fatigue analysis to avoid handling prior to analysis.
Statistical analysis.For volume, CSA, perimeter, and cell number analyses, differences between genotype/age groups were tested for significance by one-way analysis of variance (ANOVA), followed by unpaired t-test with Bonferroni correction to isolate specific differences.A value of p <0.05 was considered significant.For exercise studies, analysis of co-variance was used to test for differences between animals within groups.Mean slopes of each group were then compared using Tukey's test.
For myo-mechanical analysis, variables for twitch, tetanus and fatigue were first subjected to a nested ANOVA to evaluate whether left and right leg measurements associated with each genotype (WT vs. KO) and age (Young vs. Aging) were similar.If so, then the left and right leg variables of an animal were averaged and the genotype and age groups were evaluated by twoway ANOVA.For the tetanus analysis, a repeated measure ANOVA was performed on the basis of the stimulus frequency of contraction (30, 60, 100, 160 Hz), which included interactions between the genotype, age and frequency.The fatigue analysis included time of maximal contraction (0.5, 1, 1.5, 2-10 min in 1 min increments).If significant differences were present between the means of groups, a multiple comparison test was performed utilizing multilple t-tests with the number of comparisons adjusted by Bonferroni correction.A value of p <0.05 was considered significant.

Morphometric analysis of hindlimb muscle from wild type and Sca-1 KO mice.
To examine the effects of Sca-1 on body mass and muscle homeostasis over the lifespan, we evaluated 12-16 week-old adult Sca-1 -/-(KO) and Sca-1 +/+ (WT) mice compared with 47-50 week-old aging KO and WT mice.There was no significant difference in body weight (BW) or hindlimb weight (HW) between KO and WT mice at either age (KO young vs. WT young: BW 29.7±0.9 vs. 27.6±1.7 gms, p>0.05,HW 0.41±0.06 vs. 0.49±0.06gms, p>0.05;KO aging vs. WT aging: BW 28.1±2.1 vs. 28.7±3.4gms, p>0.05,HW 0.46±0.06 vs. 0.40±0.09gms, p>0.05), or between young and aging mice (KO young vs. KO aging: BW 29.7±0.9 vs. 28.1±2.1 gms, p>0.05,HW 0.41±0.06 vs. 0.46±0.06gms, p>0.05;WT young vs. WT aging: BW 27.6±1.7 vs. 28.7±3.4gms, p>0.05,HW 0.49±0.06 vs. 0.40±0.09gms, p>0.05) (Fig. 1).We also compared volumes of tibialis anterior (TA) and extensor digitorum longus (EDL) muscles between groups with unbiased stereology using the Cavalieri method.This demonstrated no significant difference between young and aging mice (TA-KO young vs. KO aging: 16.8±2.Animals were weighed immediately following euthanasia.Then hindlimbs were severed at the knee and ankle joints and weighed.No significant differences were observed between genotypes or ages.isolate specific differences.A value of p <0.05 was considered significant.For exercise studies, analysis of co-variance was used to test for differences between animals within groups.Mean slopes of each group were then compared using Tukey's test. For myo-mechanical analysis, variables for twitch, tetanus and fatigue were first subjected to a nested ANOVA to evaluate whether left and right leg measurements associated with each genotype (WT vs. KO) and age (Young vs. Aging) were similar.If so, then the left and right leg variables of an animal were averaged and the genotype and age groups were evaluated by twoway ANOVA.For the tetanus analysis, a repeated measure ANOVA was performed on the basis of the stimulus frequency of contraction (30, 60, 100, 160 Hz), which included interactions between the genotype, age and frequency.The fatigue analysis included time of maximal contraction (0.5, 1, 1.5, 2-10 min in 1 min increments).If significant differences were present between the means of groups, a multiple comparison test was performed utilizing multilple t-tests with the number of comparisons adjusted by Bonferroni correction.A value of p <0.05 was considered significant.

Morphometric analysis of hindlimb muscle from wild type and Sca-1 KO mice.
To examine the effects of Sca-1 on body mass and muscle homeostasis over the lifespan, we evaluated 12-16 week-old adult Sca-1 -/-(KO) and Sca-1 +/+ (WT) mice compared with 47-50 week-old aging KO and WT mice.There was no significant difference in body weight (BW) or hindlimb weight (HW) between KO and WT mice at either age (KO young vs. WT young: BW 29.7±0.9 vs. 27.6±1.7 gms, p>0.05,HW 0.41±0.06 vs. 0.49±0.06gms, p>0.05;KO aging vs. WT aging: BW 28.1±2.1 vs. 28.7±3.4gms, p>0.05,HW 0.46±0.06 vs. 0.40±0.09gms, p>0.05), or between young and aging mice (KO young vs. KO aging: BW 29.7±0.9 vs. 28.1±2.1 gms, p>0.05,HW 0.41±0.06 vs. 0.46±0.06gms, p>0.05;WT young vs. WT aging: BW 27.6±1.7 vs. 28.7±3.4gms, p>0.05,HW 0.49±0.06 vs. 0.40±0.09gms, p>0.05) (Fig. 1).We also compared volumes of tibialis anterior (TA) and extensor digitorum longus (EDL) muscles between groups with unbiased stereology using the Cavalieri method.This demonstrated no significant difference between young and aging mice (TA-KO young vs. KO aging: 16.8±2.Animals were weighed immediately following euthanasia.Then hindlimbs were severed at the knee and ankle joints and weighed.No significant differences were observed between genotypes or ages.isolate specific differences.A value of p <0.05 was considered significant.For exercise studies, analysis of co-variance was used to test for differences between animals within groups.Mean slopes of each group were then compared using Tukey's test. For myo-mechanical analysis, variables for twitch, tetanus and fatigue were first subjected to a nested ANOVA to evaluate whether left and right leg measurements associated with each genotype (WT vs. KO) and age (Young vs. Aging) were similar.If so, then the left and right leg variables of an animal were averaged and the genotype and age groups were evaluated by twoway ANOVA.For the tetanus analysis, a repeated measure ANOVA was performed on the basis of the stimulus frequency of contraction (30, 60, 100, 160 Hz), which included interactions between the genotype, age and frequency.The fatigue analysis included time of maximal contraction (0.5, 1, 1.5, 2-10 min in 1 min increments).If significant differences were present between the means of groups, a multiple comparison test was performed utilizing multilple t-tests with the number of comparisons adjusted by Bonferroni correction.A value of p <0.05 was considered significant.
Sections were stained with anti-SMA (brown) and -CD31 (pink) antibodies.Typical micrographs are shown (40X) (left).A significant increase in the number of vessels, accompanied by a decrease in total vascular cross-sectional area (CSA) was observed with age, however, this was independent of genotype (right).

Fibrosis and myoblast proliferation in hindlimb muscle of wild type and Sca-1 KO mice.
Previously, we had shown that KO myoblasts activated during the response to injury have a prolonged proliferative phase with resulting hyperplasia during healing 21 .To determine whether muscle homeostasis, or secondary myogenesis with normal use, resulted in a difference in fibrosis with age, we compared the extent of fibrotic tissue in cross-sections from young and aging, KO and WT hind limb muscle following a 37 day course of voluntary and forced exercise (described below; Figs. 8, 9).We found no difference between genotypes or at different ages (Fig. 6).To determine whether upregulated proliferation during secondary myogenesis in KO animals leads to exhaustion of proliferating myoblasts with age, we compared the number of Ki67 + proliferating cells in the TA muscles of these mice, and found no significant differences (Fig. 7).Hindlimbs were skinned, fixed and embedded in paraffin, and sectioned prior to staining with Masson's trichrome.Typical hindlimb sections are shown; muscle and intercellular fiber (red), collagen (blue), nuclei (black).Unbiased stereology was used to measure scar (blue) volumes in all genotypes and ages by the Cavalieri method (data not shown).No significant differences were observed between groups.Exercise capacity in wild type and Sca-1 KO mice.To assess the functional significance of the difference in myofiber size between young and aging KO versus WT mice, we evaluated exercise capacity of these animals during voluntary ( Fig. 8) and forced (Fig. 9) exercise.Mice were allowed to run at will during normal light-dark cycles over consecutive 24h periods.For voluntary distance, analysis of co-variance demonstrated no statistically significant differences between animals within any group, but the mean slopes of all groups (Day versus Distance: WT young 64.4,KO young 43.6, WT aging 41.9, KO aging 29.5) were statistically significantly different from each other (p<0.0001),except for WT aging and KO young, which were statistically the same (p>0.05)(Fig. 8).For voluntary duration, analysis of co-variance demonstrated no statistically significant differences Sections were stained with anti-SMA (brown) and -CD31 (pink) antibodies.Typical micrographs are shown (40X) (left).A significant increase in the number of vessels, accompanied by a decrease in total vascular cross-sectional area (CSA) was observed with age, however, this was independent of genotype (right).

Fibrosis and myoblast proliferation in hindlimb muscle of wild type and Sca-1 KO mice.
Previously, we had shown that KO myoblasts activated during the response to injury have a prolonged proliferative phase with resulting hyperplasia during healing 21 .To determine whether muscle homeostasis, or secondary myogenesis with normal use, resulted in a difference in fibrosis with age, we compared the extent of fibrotic tissue in cross-sections from young and aging, KO and WT hind limb muscle following a 37 day course of voluntary and forced exercise (described below; Figs. 8, 9).We found no difference between genotypes or at different ages (Fig. 6).To determine whether upregulated proliferation during secondary myogenesis in KO animals leads to exhaustion of proliferating myoblasts with age, we compared the number of Ki67 + proliferating cells in the TA muscles of these mice, and found no significant differences (Fig. 7).Exercise capacity in wild type and Sca-1 KO mice.To assess the functional significance of the difference in myofiber size between young and aging KO versus WT mice, we evaluated exercise capacity of these animals during voluntary ( Fig. 8) and forced (Fig. 9) exercise.Mice were allowed to run at will during normal light-dark cycles over consecutive 24h periods.For voluntary distance, analysis of co-variance demonstrated no statistically significant differences between animals within any group, but the mean slopes of all groups (Day versus Distance: WT young 64.4,KO young 43.6, WT aging 41.9, KO aging 29.5) were statistically significantly different from each other (p<0.0001),except for WT aging and KO young, which were statistically the same (p>0.05)(Fig. 8).For voluntary duration, analysis of co-variance demonstrated no statistically significant differences Sections were stained with anti-SMA (brown) and -CD31 (pink) antibodies.Typical micrographs are shown (40X) (left).A significant increase in the number of vessels, accompanied by a decrease in total vascular cross-sectional area (CSA) was observed with age, however, this was independent of genotype (right).

Fibrosis and myoblast proliferation in hindlimb muscle of wild type and Sca-1 KO mice.
Previously, we had shown that KO myoblasts activated during the response to injury have a prolonged proliferative phase with resulting hyperplasia during healing 21 .To determine whether muscle homeostasis, or secondary myogenesis with normal use, resulted in a difference in fibrosis with age, we compared the extent of fibrotic tissue in cross-sections from young and aging, KO and WT hind limb muscle following a 37 day course of voluntary and forced exercise (described below; Figs. 8, 9).We found no difference between genotypes or at different ages (Fig. 6).To determine whether upregulated proliferation during secondary myogenesis in KO animals leads to exhaustion of proliferating myoblasts with age, we compared the number of Ki67 + proliferating cells in the TA muscles of these mice, and found no significant differences (Fig. 7).Exercise capacity in wild type and Sca-1 KO mice.To assess the functional significance of the difference in myofiber size between young and aging KO versus WT mice, we evaluated exercise capacity of these animals during voluntary ( Fig. 8) and forced (Fig. 9) exercise.Mice were allowed to run at will during normal light-dark cycles over consecutive 24h periods.For voluntary distance, analysis of co-variance demonstrated no statistically significant differences between animals within any group, but the mean slopes of all groups (Day versus Distance: WT young 64.4,KO young 43.6, WT aging 41.9, KO aging 29.5) were statistically significantly different from each other (p<0.0001),except for WT aging and KO young, which were statistically the same (p>0.05)(Fig. 8).For voluntary duration, analysis of co-variance demonstrated no statistically significant differences between animals within any group, but the mean slopes of all groups (Day versus Duration: WT young 127.0,KO young 105.5, WT aging 81.5, KO aging 60.0) were statistically significantly different from each other (p<0.01)(Fig. 8).Voluntary exercise showed that young KO mice achieved significantly less distance and duration than their WT counterparts, and resembled aging WT animals.Similarly, aging KO mice achieved significantly less distance and duration than aging WT mice.Mice were allowed to run at will during normal light-dark cycles.Each line represents data from one animal.Distance and duration over consecutive 24 hr periods were recorded.Distance (meters/day; top) and duration (min/day; bottom) were plotted for individual animals over a 37 d period.For voluntary distance (top), analysis of co-variance demonstrated no statistically significant differences between animals within any group, but the mean slopes of all groups (Day versus Distance) were statistically significantly different from each other as determined using Tukey's test, except for WT aging and KO young, which were the same.For voluntary duration (bottom), analysis of co-variance demonstrated no statistically significant differences between animals within any group, but the mean slopes of all groups (Day versus Duration) were statistically significantly different from each other as determined by Tukey's test.
On each day over a 37d period, mice also were forced to run using a protocol where speeds were progressively increased until mice reached their maximum rate.Analysis of co-variance demonstrated no statistically significant differences between animals within any group, but the mean slopes of all groups (log(Day) versus Maximum Rate 2 : WT young 45.6, KO young 41.1, WT aging 32.4,KO aging 21.6) were statistically significantly different from each other (p<0.01)(Fig. 9).Forced exercise similarly demonstrated that young KO mice achieved a lower maximum exercise rate than young WT mice, as did aging KO animals between animals within any group, but the mean slopes of all groups (Day versus Duration: WT young 127.0,KO young 105.5, WT aging 81.5, KO aging 60.0) were statistically significantly different from each other (p<0.01)(Fig. 8).Voluntary exercise showed that young KO mice achieved significantly less distance and duration than their WT counterparts, and resembled aging WT animals.Similarly, aging KO mice achieved significantly less distance and duration than aging WT mice.Mice were allowed to run at will during normal light-dark cycles.Each line represents data from one animal.Distance and duration over consecutive 24 hr periods were recorded.Distance (meters/day; top) and duration (min/day; bottom) were plotted for individual animals over a 37 d period.For voluntary distance (top), analysis of co-variance demonstrated no statistically significant differences between animals within any group, but the mean slopes of all groups (Day versus Distance) were statistically significantly different from each other as determined using Tukey's test, except for WT aging and KO young, which were the same.For voluntary duration (bottom), analysis of co-variance demonstrated no statistically significant differences between animals within any group, but the mean slopes of all groups (Day versus Duration) were statistically significantly different from each other as determined by Tukey's test.
On each day over a 37d period, mice also were forced to run using a protocol where speeds were progressively increased until mice reached their maximum rate.Analysis of co-variance demonstrated no statistically significant differences between animals within any group, but the mean slopes of all groups (log(Day) versus Maximum Rate 2 : WT young 45.6, KO young 41.1, WT aging 32.4,KO aging 21.6) were statistically significantly different from each other (p<0.01)(Fig. 9).Forced exercise similarly demonstrated that young KO mice achieved a lower maximum exercise rate than young WT mice, as did aging KO animals between animals within any group, but the mean slopes of all groups (Day versus Duration: WT young 127.0,KO young 105.5, WT aging 81.5, KO aging 60.0) were statistically significantly different from each other (p<0.01)(Fig. 8).Voluntary exercise showed that young KO mice achieved significantly less distance and duration than their WT counterparts, and resembled aging WT animals.Similarly, aging KO mice achieved significantly less distance and duration than aging WT mice.Mice were allowed to run at will during normal light-dark cycles.Each line represents data from one animal.Distance and duration over consecutive 24 hr periods were recorded.Distance (meters/day; top) and duration (min/day; bottom) were plotted for individual animals over a 37 d period.For voluntary distance (top), analysis of co-variance demonstrated no statistically significant differences between animals within any group, but the mean slopes of all groups (Day versus Distance) were statistically significantly different from each other as determined using Tukey's test, except for WT aging and KO young, which were the same.For voluntary duration (bottom), analysis of co-variance demonstrated no statistically significant differences between animals within any group, but the mean slopes of all groups (Day versus Duration) were statistically significantly different from each other as determined by Tukey's test.
On each day over a 37d period, mice also were forced to run using a protocol where speeds were progressively increased until mice reached their maximum rate.Analysis of co-variance demonstrated no statistically significant differences between animals within any group, but the mean slopes of all groups (log(Day) versus Maximum Rate 2 : WT young 45.6, KO young 41.1, WT aging 32.4,KO aging 21.6) were statistically significantly different from each other (p<0.01)(Fig. 9).Forced exercise similarly demonstrated that young KO mice achieved a lower maximum exercise rate than young WT mice, as did aging KO animals compared to aging WT controls.On each day over a 37 d period, mice were forced to run at 8 meters/min for five 10 min intervals with 30 sec rests between intervals.Over the next five 10 min intervals immediately following that day's exercise session, speeds were progressively increased until mice reached their maximum rate, as determined by the maximum speed (meters/min) at which mice continued to run on the wheel without falling off or hanging onto the wheel.Each line represents data from one animal.Analysis of co-variance demonstrated no statistically significant differences between animals within any group, but the mean slopes of all groups (log(Day) versus Maximum Rate2) were statistically significantly different from each other as determined using Tukey's test.

Myomechanical analysis of hindlimb muscle from wild type and Sca-1 KO mice.
To quantify changes in muscle function observed during exercise, we performed myomechanical analysis of isolated EDL muscle from KO and WT young and aging mice.There were no significant differences in muscle mass or muscle cross-sectional area between ages and genotypes, consistent with data from isolated hind limbs (Fig. 1) and unbiased stereology assessment of muscle volumes by Cavalieri method (Fig. 2).Aging animals (KO aging 3.4±0.4x 10 4 , WT aging 3.9±0.6x 10 4 ) had significantly lower muscle:body mass ratios compared with young animals (KO young 3.9±0.6x 10 4 , WT young 5.5±0.5 x 10 4 ; p<0.05), however, this difference was similar for both KO and WT mice.
Force-frequency analysis of isolated EDL muscle from young and aging KO and WT mice showed no significant difference between genotypes or ages (Fig. 10 left).A low-frequency fatigue protocol, however, showed a significant difference between KO young muscle and all other groups (p<0.05), with KO young muscle generating a greater percent of maximal force, and less fatigue, at all time points over 10 min (Fig. 10 right).We also examined the percent maximum force generated with relaxation during low-frequency fatigue, to determine whether there was any difference in the return to baseline between contractions between the groups.Although the force generated during relaxation trended upward over time (i.e., incomplete relaxation with fatigue), this showed no significant differences between groups (Fig. 10 right).
compared to aging WT controls.On each day over a 37 d period, mice were forced to run at 8 meters/min for five 10 min intervals with 30 sec rests between intervals.Over the next five 10 min intervals immediately following that day's exercise session, speeds were progressively increased until mice reached their maximum rate, as determined by the maximum speed (meters/min) at which mice continued to run on the wheel without falling off or hanging onto the wheel.Each line represents data from one animal.Analysis of co-variance demonstrated no statistically significant differences between animals within any group, but the mean slopes of all groups (log(Day) versus Maximum Rate2) were statistically significantly different from each other as determined using Tukey's test.

Myomechanical analysis of hindlimb muscle from wild type and Sca-1 KO mice.
To quantify changes in muscle function observed during exercise, we performed myomechanical analysis of isolated EDL muscle from KO and WT young and aging mice.There were no significant differences in muscle mass or muscle cross-sectional area between ages and genotypes, consistent with data from isolated hind limbs (Fig. 1) and unbiased stereology assessment of muscle volumes by Cavalieri method (Fig. 2).Aging animals (KO aging 3.4±0.4x 10 4 , WT aging 3.9±0.6x 10 4 ) had significantly lower muscle:body mass ratios compared with young animals (KO young 3.9±0.6x 10 4 , WT young 5.5±0.5 x 10 4 ; p<0.05), however, this difference was similar for both KO and WT mice.
Force-frequency analysis of isolated EDL muscle from young and aging KO and WT mice showed no significant difference between genotypes or ages (Fig. 10 left).A low-frequency fatigue protocol, however, showed a significant difference between KO young muscle and all other groups (p<0.05), with KO young muscle generating a greater percent of maximal force, and less fatigue, at all time points over 10 min (Fig. 10 right).We also examined the percent maximum force generated with relaxation during low-frequency fatigue, to determine whether there was any difference in the return to baseline between contractions between the groups.Although the force generated during relaxation trended upward over time (i.e., incomplete relaxation with fatigue), this showed no significant differences between groups (Fig. 10 right).
compared to aging WT controls.On each day over a 37 d period, mice were forced to run at 8 meters/min for five 10 min intervals with 30 sec rests between intervals.Over the next five 10 min intervals immediately following that day's exercise session, speeds were progressively increased until mice reached their maximum rate, as determined by the maximum speed (meters/min) at which mice continued to run on the wheel without falling off or hanging onto the wheel.Each line represents data from one animal.Analysis of co-variance demonstrated no statistically significant differences between animals within any group, but the mean slopes of all groups (log(Day) versus Maximum Rate2) were statistically significantly different from each other as determined using Tukey's test.

Myomechanical analysis of hindlimb muscle from wild type and Sca-1 KO mice.
To quantify changes in muscle function observed during exercise, we performed myomechanical analysis of isolated EDL muscle from KO and WT young and aging mice.There were no significant differences in muscle mass or muscle cross-sectional area between ages and genotypes, consistent with data from isolated hind limbs (Fig. 1) and unbiased stereology assessment of muscle volumes by Cavalieri method (Fig. 2).Aging animals (KO aging 3.4±0.4x 10 4 , WT aging 3.9±0.6x 10 4 ) had significantly lower muscle:body mass ratios compared with young animals (KO young 3.9±0.6x 10 4 , WT young 5.5±0.5 x 10 4 ; p<0.05), however, this difference was similar for both KO and WT mice.
Force-frequency analysis of isolated EDL muscle from young and aging KO and WT mice showed no significant difference between genotypes or ages (Fig. 10 left).A low-frequency fatigue protocol, however, showed a significant difference between KO young muscle and all other groups (p<0.05), with KO young muscle generating a greater percent of maximal force, and less fatigue, at all time points over 10 min (Fig. 10 right).We also examined the percent maximum force generated with relaxation during low-frequency fatigue, to determine whether there was any difference in the return to baseline between contractions between the groups.Although the force generated during relaxation trended upward over time (i.e., incomplete relaxation with fatigue), this showed no significant differences between groups (Fig. 10 right).Extensor digitorum longus muscle was dissected, mounted in a force transducer, and stimulated as decribed in Methods.Data shown are mean percent maximum force (N=12 young WT; N=2 young KO; N=3 aging WT; N=9 aging KO).No differences were observed in force-frequency relationships between genotypes or ages (left).Young KO animals demonstrated significantly greater maximum contraction (C) force (less fatigue) over time compared with other groups, although relaxation (R) force was the same between groups (right).Absolute peak force at the beginning of the study was statistically similar for all groups (KO Young 198±40 mN, KO Aging 234±66 mN, WT Young 208±52 mN, WT Aging 186±30 mN).*, p

Fig. 6 :
Fig. 6: Hindlimb fibrosis in wild type and Sca-1 KO mice.Hindlimbs were skinned, fixed and embedded in paraffin, and sectioned prior to staining with Masson's trichrome.Typical hindlimb sections are shown; muscle and intercellular fiber (red), collagen (blue), nuclei (black).Unbiased stereology was used to measure scar (blue) volumes in all genotypes and ages by the Cavalieri method (data not shown).No significant differences were observed between groups.

Fig. 6 :
Fig. 6: Hindlimb fibrosis in wild type and Sca-1 KO mice.Hindlimbs were skinned, fixed and embedded in paraffin, and sectioned prior to staining with Masson's trichrome.Typical hindlimb sections are shown; muscle and intercellular fiber (red), collagen (blue), nuclei (black).Unbiased stereology was used to measure scar (blue) volumes in all genotypes and ages by the Cavalieri method (data not shown).No significant differences were observed between groups.