Muscle progenitor cells are required for skeletal muscle regeneration and prevention of adipogenesis after limb ischemia.

Skeletal muscle injury in peripheral artery disease (PAD) has been attributed to vascular insufficiency, however evidence has demonstrated that muscle cell responses play a role in determining outcomes in limb ischemia. Here, we demonstrate that genetic ablation of Pax7+ muscle progenitor cells (MPCs) in a model of hindlimb ischemia (HLI) inhibited muscle regeneration following ischemic injury, despite a lack of morphological or physiological changes in resting muscle. Compared to control mice (Pax7WT), the ischemic limb of Pax7-deficient mice (Pax7Δ) was unable to generate significant force 7 or 28 days after HLI. A significant increase in adipose was observed in the ischemic limb 28 days after HLI in Pax7Δ mice, which replaced functional muscle. Adipogenesis in Pax7Δ mice corresponded with a significant increase in PDGFRα+ fibro/adipogenic progenitors (FAPs). Inhibition of FAPs with batimastat decreased muscle adipose but increased fibrosis. In vitro, Pax7Δ MPCs failed to form myotubes but displayed increased adipogenesis. Skeletal muscle from patients with critical limb threatening ischemia displayed increased adipose in more ischemic regions of muscle, which corresponded with fewer satellite cells. Collectively, these data demonstrate that Pax7+ MPCs are required for muscle regeneration after ischemia and suggest that muscle regeneration may be an important therapeutic target in PAD.

Coupled myovascular expansion directs cardiac growth and regeneration.

Heart regeneration requires multiple cell types to enable cardiomyocyte (CM) proliferation. How these cells interact to create growth niches is unclear. Here, we profile proliferation kinetics of cardiac endothelial cells (CECs) and CMs in the neonatal mouse heart and find that they are spatiotemporally coupled. We show that coupled myovascular expansion during cardiac growth or regeneration is dependent upon VEGF-VEGFR2 signaling, as genetic deletion of Vegfr2 from CECs or inhibition of VEGFA abrogates both CEC and CM proliferation. Repair of cryoinjury displays poor spatial coupling of CEC and CM proliferation. Boosting CEC density after cryoinjury with virus encoding Vegfa enhances regeneration. Using Mendelian randomization, we demonstrate that circulating VEGFA levels are positively linked with human myocardial mass, suggesting that Vegfa can stimulate human cardiac growth. Our work demonstrates the importance of coupled CEC and CM expansion and reveals a myovascular niche that may be therapeutically targeted for heart regeneration.

Myoepithelial Cells of Submucosal Glands Can Function as Reserve Stem Cells to Regenerate Airways after Injury.

Cells demonstrate plasticity following injury, but the extent of this phenomenon and the cellular mechanisms involved remain underexplored. Using single-cell RNA sequencing (scRNA-seq) and lineage tracing, we uncover that myoepithelial cells (MECs) of the submucosal glands (SMGs) proliferate and migrate to repopulate the airway surface epithelium (SE) in multiple injury models. Specifically, SMG-derived cells display multipotency and contribute to basal and luminal cell types of the SMGs and SE. Ex vivo expanded MECs have the potential to repopulate and differentiate into SE cells when grafted onto denuded airway scaffolds. Significantly, we find that SMG-like cells appear on the SE of both extra- and intra-lobular airways of large animal lungs following severe injury. We find that the transcription factor SOX9 is necessary for MEC plasticity in airway regeneration. Because SMGs are abundant and present deep within airways, they may serve as a reserve cell source for enhancing human airway regeneration.

BAG3 (Bcl-2-Associated Athanogene-3) Coding Variant in Mice Determines Susceptibility to Ischemic Limb Muscle Myopathy by Directing Autophagy.

BACKGROUND: Critical limb ischemia is a manifestation of peripheral artery disease that carries significant mortality and morbidity risk in humans, although its genetic determinants remain largely unknown. We previously discovered 2 overlapping quantitative trait loci in mice, Lsq-1 and Civq-1, that affected limb muscle survival and stroke volume after femoral artery or middle cerebral artery ligation, respectively. Here, we report that a Bag3 variant (Ile81Met) segregates with tissue protection from hind-limb ischemia. METHODS: We treated mice with either adeno-associated viruses encoding a control (green fluorescent protein) or 2 BAG3 (Bcl-2-associated athanogene-3) variants, namely Met81 or Ile81, and subjected the mice to hind-limb ischemia. RESULTS: We found that the BAG3 Ile81Met variant in the C57BL/6 (BL6) mouse background segregates with protection from tissue necrosis in a shorter congenic fragment of Lsq-1 (C.B6-Lsq1-3). BALB/c mice treated with adeno-associated virus encoding the BL6 BAG3 variant (Ile81; n=25) displayed reduced limb-tissue necrosis and increased limb tissue perfusion compared with Met81- (n=25) or green fluorescent protein- (n=29) expressing animals. BAG3Ile81, but not BAG3Met81, improved ischemic muscle myopathy and muscle precursor cell differentiation and improved muscle regeneration in a separate, toxin-induced model of injury. Systemic injection of adeno-associated virus-BAG3Ile81 (n=9), but not BAG3Met81 (n=10) or green fluorescent protein (n=5), improved ischemic limb blood flow and limb muscle histology and restored muscle function (force production). Compared with BAG3Met81, BAG3Ile81 displayed improved binding to the small heat shock protein (HspB8) in ischemic skeletal muscle cells and enhanced ischemic muscle autophagic flux. CONCLUSIONS: Taken together, our data demonstrate that genetic variation in BAG3 plays an important role in the prevention of ischemic tissue necrosis. These results highlight a pathway that preserves tissue survival and muscle function in the setting of ischemia.

Methods for Acute and Subacute Murine Hindlimb Ischemia.

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably reproduce the human disease are necessary to develop new therapies for this disease. The mouse hindlimb ischemia model has been widely used for this purpose, but the standard practice of inducing acute limb ischemia by ligation of the femoral artery can result in substantial tissue necrosis, compromising investigators' ability to study the vascular and skeletal muscle tissue responses to ischemia. An alternative approach to femoral artery ligation is the induction of gradual femoral artery occlusion through the use of ameroid constrictors. When placed around the femoral artery in the same or different locations as the sites of femoral artery ligation, these devices occlude the artery over 1 - 3 days, resulting in more gradual, subacute ischemia. This results in less substantial skeletal muscle tissue necrosis, which may more closely mimic the responses seen in human PAD. Because genetic background influences outcomes in both the acute and subacute ischemia models, consideration of the mouse strain being studied is important in choosing the best model. This paper describes the proper procedure and anatomical placement of ligatures or ameroid constrictors on the mouse femoral artery to induce subacute or acute hindlimb ischemia in the mouse.

Subacute limb ischemia induces skeletal muscle injury in genetically susceptible mice independent of vascular density.

OBJECTIVE: The primary preclinical model of peripheral artery disease, which involves acute limb ischemia (ALI), can result in appreciable muscle injury that is attributed to the acuity of the ischemic injury. A less acute model of murine limb ischemia using ameroid constrictors (ACs) has been developed in an attempt to mimic the chronic nature of human disease. However, there is currently little understanding of how genetics influence muscle injury following subacute arterial occlusion in the mouse. METHODS: We investigated the influence of mouse genetics on skeletal muscle tissue survival, blood flow, and vascular density by subjecting two different mouse strains, C57BL/6 (BL6) and BALB/c, to ALI or subacute limb ischemia using single (1AC) or double (2AC) AC placement on the femoral artery. RESULTS: Similar to ALI, the 2AC model resulted in significant tissue necrosis and limb perfusion deficits in genetically susceptible BALB/c but not BL6 mice. In the 1AC model, no outward evidence of tissue necrosis was observed, and there were no differences in limb blood flow between BL6 and BALB/c. However, BALB/c mice displayed significantly greater muscle injury, as evidenced by increased inflammation and myofiber atrophy, despite having no differences in CD31(+) and SMA(+) vascular density and area. BALB/c mice also displayed significantly greater centralized myonuclei, indicating increased muscle regeneration. CONCLUSIONS: The susceptibility of skeletal muscle to ischemia-induced injury is at least partly independent of muscle blood flow and vascular density, consistent with a muscle cell autonomous response that is genetically determined. Further development of preclinical models of peripheral artery disease that more accurately reflect the nature of the human disease may allow more accurate identification of genetic targets for therapeutic intervention.

Muscle cell derived angiopoietin-1 contributes to both myogenesis and angiogenesis in the ischemic environment.

Recent strategies to treat peripheral arterial disease (PAD) have focused on stem cell based therapies, which are believed to result in local secretion of vascular growth factors. Little is known, however, about the role of ischemic endogenous cells in this context. We hypothesized that ischemic muscle cells (MC) are capable of secreting growth factors that act as potent effectors of the local cellular regenerative environment. Both muscle and endothelial cells (ECs) were subjected to experimental ischemia, and conditioned medium (CM) from each was collected and analyzed to assess myogenic and/or angiogenic potential. In muscle progenitors, mRNA expression of VEGF and its cognate receptors (Nrp1, Flt, Flk) was present and decreased during myotube formation in vitro, and EC CM or VEGF increased myoblast proliferation. Angiopoietin-1 (Ang-1), Tie1, and Tie2 mRNA increased during MC differentiation in vitro. Exogenous Ang-1 enhanced myogenic (MyoD and Myogenin) mRNA in differentiating myoblasts and increased myosin heavy chain protein. Myotube formation was enhanced by MC CM and inhibited by EC CM. Ang-1 protein was present in CM from MCs isolated from both the genetically ischemia-susceptible BALB/c and ischemia-resistant C57BL/6 mouse strains, and chimeric Tie2 receptor trapping in situ ablated Ang-1's myogenic effects in vitro. Ang-1 or MC CM enhanced myotube formation in a mixed isolate of muscle progenitors as well as a myoblast co-culture with pluripotent mesenchymal cells (10T1/2) and this effect was abrogated by viral expression of the extracellular domain of Tie2 (AdsTie2). Furthermore, mesh/tube formation by HUVECs was enhanced by Ang-1 or MC CM and abrogated by Tie2 chimeric receptor trapping. Our results demonstrate the ability of muscle and endothelial cell-derived vascular growth factors, particularly Ang-1, to serve as multi-functional stimuli regulating crosstalk between blood vessels and muscle cells during regeneration from ischemic myopathy.

Skeletal muscle-specific genetic determinants contribute to the differential strain-dependent effects of hindlimb ischemia in mice.

Genetics plays an important role in determining peripheral arterial disease (PAD) pathology, which causes a spectrum of clinical disorders that range from clinically silent reductions in blood flow to limb-threatening ischemia. The cell-type specificity of PAD pathology, however, has received little attention. To determine whether strain-dependent differences in skeletal muscle cells might account for the differential responses to ischemia observed in C57BL/6 and BALB/c mice, endothelial and skeletal muscle cells were subjected to hypoxia and nutrient deprivation (HND) in vitro, to mimic ischemia. Muscle cells were more susceptible to HND than were endothelial cells. In vivo, C57BL/6 and BALB/c mice displayed strain-specific differences in myofiber responses after hindlimb ischemia, with significantly greater myofiber atrophy, greater apoptosis, and attenuated myogenic regulatory gene expression and stress-responsive signaling in BALB/c mice. Strain-specific deficits were recapitulated in vitro in primary muscle cells from both strains after HND. Muscle cells from BALB/c mice congenic for the C57BL/6 Lsq-1 quantitative trait locus were protected from HND-induced atrophy, and gene expression of vascular growth factors and their receptors was significantly greater in C57BL/6 primary muscle cells. Our results indicate that the previously identified specific genetic locus regulating strain-dependent collateral vessel density has a nonvascular or muscle cell-autonomous role involving both the myogenic program and traditional vascular growth factor receptor expression.