E-cigarette aerosol impairs male mouse skeletal muscle force development and prevents recovery from injury.

To date, there has been a lag between the rise in E-cigarette use and an understanding of the long-term health effects. Inhalation of E-cigarette aerosol delivers high doses of nicotine, raises systemic cytokine levels, and compromises cardiopulmonary function. The consequences for muscle function have not been thoroughly investigated. The present study tests the hypothesis that exposure to nicotine-containing aerosol impairs locomotor muscle function, limits exercise tolerance, and interferes with muscle repair in male mice. Nicotine-containing aerosol reduced the maximal force produced by the extensor digitorum longus (EDL) by 30%-40% and, the speed achieved in treadmill running by 8%. Nicotine aerosol exposure also decreased adrenal and increased plasma epinephrine and norepinephrine levels, and these changes in catecholamines manifested as increased muscle and liver glycogen stores. In nicotine aerosol exposed mice, muscle regenerating from overuse injury only recovered force to 80% of noninjured levels. However, the structure of neuromuscular junctions (NMJs) was not affected by e-cigarette aerosols. Interestingly, the vehicle used to dissolve nicotine in these vaping devices, polyethylene glycol (PG) and vegetable glycerin (VG), decreased running speed by 11% and prevented full recovery from a lengthening contraction protocol (LCP) injury. In both types of aerosol exposures, cardiac left ventricular systolic function was preserved, but left ventricular myocardial relaxation was altered. These data suggest that E-cigarette use may have a negative impact on muscle force and regeneration due to compromised glucose metabolism and contractile function in male mice.NEW & NOTEWORTHY In male mice, nicotine-containing E-cigarette aerosol compromises muscle contractile function, regeneration from injury, and whole body running speeds. The vehicle used to deliver nicotine, propylene glycol, and vegetable glycerin, also reduces running speed and impairs the restoration of muscle function in injured muscle. However, the predominant effects of nicotine in this inhaled aerosol are evident in altered catecholamine levels, increased glycogen content, decreased running capacity, and impaired recovery of force following an overuse injury.

IL-33/ST2 receptor-dependent signaling in the development of pulmonary hypertension in Sugen/hypoxia mice.

Pulmonary arterial hypertension (PAH) is associated with significant morbidity and mortality. PAH is characterized by pulmonary artery remodeling, elevated right ventricular pressure (RVP) and, ultimately, cardiac failure. Pulmonary endothelial cells can sense danger or damage caused by mechanical injury or pathogens through alarmin cytokines. These cytokines can signal proliferation to restore barrier integrity or aberrant hyperproliferation and remodeling. We hypothesized that IL-33 signals pulmonary artery endothelial cells to proliferate under hypertensive conditions during the remodeling response and rise in RVP. To test this hypothesis, pulmonary hypertension (PH) was induced in C57Bl/6J, IL-33 receptor gene deleted (ST2-/- ) and MYD88 gene deleted (MYD88-/- ) mice by exposure to 10% O2 and SU5416 injections (SUHX). RVP, arterial wall thickness, endothelial cell proliferation and IL-33 levels and signaling were evaluated. In response to SUHX. RVP increased in C57Bl/6J mice in response to SUHX (49% male and 70% female; p < 0.0001) and this SUHX response was attenuated in ST2-/- mice (29% male p = 0.003; 30% female p = 0.001) and absent in MYD88-/- mice. Wall thickness was increased in SUHX C57Bl/6J mice (p = 0.005), but not in ST2-/- or MYD88-/- mice. Proliferating cells were detected in C57Bl/6J mice by flow cytometry (CD31+ /BrDU+ ; p = 0.02) and immunofluorescence methods (Ki-67+). IL-33 was increased by SUHX (p = 0.03) but a genotype effect was not observed (p = 0.76). We observed that in hPAECs, IL-33 expression is regulated by both IL-33 and DLL4. These data suggest IL-33/ST2 signaling is essential for the endothelial cell proliferative response in PH.

Role of IL-33 receptor (ST2) deletion in diaphragm contractile and mitochondrial function in the Sugen5416/hypoxia model of pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease of the pulmonary vasculature that leads to right ventricular failure. Skeletal muscle maladaptations limit physical activity and may contribute to disease progression. The role of alarmin/inflammatory signaling in PAH respiratory muscle dysfunction is unknown. We hypothesized that diaphragm mitochondrial and contractile functions are impaired in SU5416/hypoxia-induced pulmonary hypertension due to increased systemic IL-33 signaling. We induced pulmonary hypertension in adult C57Bl/6 J (WT) and ST2 (IL1RL1) gene ablated mice by SU5416/hypoxia (SuHx). We measured diaphragm fiber mitochondrial respiration, inflammatory markers, and contractile function ex vivo. SuHx reduced coupled and uncoupled permeabilized myofiber respiration by ∼40 %. During coupled respiration with complex I substrates, ST2-/- attenuated SuHx inhibition of mitochondrial respiration (genotype × treatment interaction F[1,67] = 3.3, p = 0.07, η2 = 0.04). Flux control ratio and coupling efficiency were not affected by SuHx or genotype. A higher substrate control ratio for succinate was observed in SuHx fibers and attenuated in ST2-/- fibers (F[1,67] = 5.3, p < 0.05, η2 = 0.07). Diaphragm TNFα, but not IL-33 or NFkB, was increased in SuHx vs. DMSO in both genotypes (F[1,43] = 4.7, p < 0.05, η2 = 0.1). Diaphragm force-frequency relationships were right-shifted in SuHx vs. WT (F[3,440] = 8.4, p < 0.05, η2 = 0.0025). There was no effect of ST2-/- on the force-frequency relationship. Force decay during a fatigue protocol at 100 Hz, but not at 40 Hz, was attenuated by SuHx vs. DMSO in both genotypes (F[1,41] = 5.6, p < 0.05, η2 = 0.11). SuHx mice exhibit a modest compensation in diaphragm contractility and mitochondrial dysfunction during coupled respiration; the latter partially regulated through ST2 signaling.

Hepatocyte HIF-1 and Intermittent Hypoxia Independently Impact Liver Fibrosis in Murine Nonalcoholic Fatty Liver Disease.

Obstructive sleep apnea is associated with insulin resistance, lipid dysregulation, and hepatic steatosis and fibrosis in nonalcoholic fatty liver disease (NAFLD). We have previously shown that hepatocyte HIF-1 (hypoxia-inducible factor-1) mediates the development of liver fibrosis in a mouse model of NAFLD. We hypothesized that intermittent hypoxia (IH) modeling obstructive sleep apnea would worsen hepatic steatosis and fibrosis in murine NAFLD, via HIF-1. Mice with hepatocyte-specific deletion of Hif1a (Hif1a-/-hep) and wild-type (Hif1aF/F) controls were fed a high trans-fat diet to induce NAFLD with steatohepatitis. Half from each group were exposed to IH, and the other half were exposed to intermittent air. A glucose tolerance test was performed just prior to the end of the experiment. Mitochondrial efficiency was assessed in fresh liver tissue at the time of death. The hepatic malondialdehyde concentration and proinflammatory cytokine levels were assessed, and genes of collagen and fatty acid metabolism were examined. Hif1a-/-hep mice gained less weight than wild-type Hif1a mice (-2.3 g, P = 0.029). There was also a genotype-independent effect of IH on body weight, with less weight gain in mice exposed to IH (P = 0.003). Fasting glucose, homeostatic model assessment for insulin resistance, and glucose tolerance test results were all improved in Hif1a-/-hep mice. Liver collagen was increased in mice exposed to IH (P = 0.033) and was reduced in Hif1a-/-hep mice (P < 0.001), without any significant exposure/genotype interaction being demonstrated. Liver TNF-α and IL-1ß were significantly increased in mice exposed to IH and were decreased in Hif1a-/-hep mice. We conclude that HIF-1 signaling worsens the metabolic profile and hastens NAFLD progression and that IH may worsen liver fibrosis. These effects are plausibly mediated by hepatic inflammatory stress.

Synergistic effect of vascular endothelial growth factor gene inactivation in endothelial cells and skeletal myofibres on muscle enzyme activity, capillary supply and endurance exercise in mice.

NEW FINDINGS: What is the central question of this study? Does vascular endothelial growth factor (VEGF) expressed by both endothelial cells and skeletal myofibres maintain the number of skeletal muscle capillaries and regulate endurance exercise? What is the main finding and its importance? VEGF expressed by both endothelial cells and skeletal myofibres is not essential for maintaining capillary number but does contribute to exercise performance. ABSTRACT: Many chronic diseases lead to exercise intolerance, with loss of skeletal muscle capillaries. While many muscle cell types (myofibres, satellite cells, endothelial cells, macrophages and fibroblasts) express vascular endothelial growth factor (VEGF), most muscle VEGF is stored in myofibre vesicles which can release VEGF to signal VEGF receptor-expressing cells. VEGF gene ablation in myofibres or endothelial cells alone does not cause capillary regression. We hypothesized that simultaneously deleting the endothelial cell (EC) and skeletal myofibre (Skm) VEGF gene would cause capillary regression and impair exercise performance. This was tested in adult mice by simultaneous conditional deletion of the VEGF gene (Skm/EC-VEGF-/- mice) through the use of VEGFLoxP, HSA-Cre-ERT2 and PDGFb-iCre-ERT2 transgenes. These double-deletion mice were compared to three control groups - WT, EC VEGF gene deletion alone and myofibre VEGF gene deletion alone. Three weeks after initiating gene deletion, Skm/EC-VEGF-/- mice, but not SkmVEGF-/- or EC-VEGF-/- mice, reached exhaustion 40 min sooner than WT mice in treadmill tests (P = 0.002). WT, SkmVEGF-/- and EC-VEGF-/- , but not Skm/EC-VEGF-/- , mice gained weight over the 3 weeks. Capillary density, fibre area and capillary: fibre ratio in soleus, plantaris, gastrocnemius and cardiac papillary muscle were similar across the groups. Phosphofructokinase and pyruvate dehydrogenase activities increased only in Skm/EC-VEGF-/- mice. These data suggest that deletion of the VEGF gene simultaneously in endothelial cells and myofibres, while reducing treadmill endurance and despite compensatory augmentation of glycolysis, is not required for muscle capillary maintenance. Reduced endurance remains unexplained, but may possibly be related to a role for VEGF in controlling perfusion of contracting muscle.

Skeletal myofiber VEGF deficiency leads to mitochondrial, structural, and contractile alterations in mouse diaphragm.

Diaphragm dysfunction accompanies cardiopulmonary disease and impaired oxygen delivery. Vascular endothelial growth factor (VEGF) regulates oxygen delivery through angiogenesis, capillary maintenance, and contraction-induced perfusion. We hypothesized that myofiber-specific VEGF deficiency contributes to diaphragm weakness and fatigability. Diaphragm protein expression, capillarity and fiber morphology, mitochondrial respiration and hydrogen peroxide (H2O2) generation, and contractile function were compared between adult mice with conditional gene ablation of skeletal myofiber VEGF (SkmVEGF-/-; n = 12) and littermate controls (n = 13). Diaphragm VEGF protein was ~50% lower in SkmVEGF-/- than littermate controls (1.45 ± 0.65 vs. 3.04 ± 1.41 pg/total protein; P = 0.001). This was accompanied by an ~15% impairment in maximal isometric specific force (F[1,23] = 15.01, P = 0.001) and a trend for improved fatigue resistance (P = 0.053). Mean fiber cross-sectional area and type I fiber cross-sectional area were lower in SkmVEGF-/- by ~40% and ~25% (P < 0.05). Capillary-to-fiber ratio was also lower in SkmVEGF-/- by ~40% (P < 0.05), and thus capillary density was not different. Sarcomeric actin expression was ~30% lower in SkmVEGF-/- (P < 0.05), whereas myosin heavy chain and MAFbx were similar (measured via immunoblot). Mitochondrial respiration, citrate synthase activity, PGC-1α, and hypoxia-inducible factor 1α were not different in SkmVEGF-/- (P > 0.05). However, mitochondrial-derived reactive oxygen species (ROS) flux was lower in SkmVEGF-/- (P = 0.0003). In conclusion, myofiber-specific VEGF gene deletion resulted in a lower capillary-to-fiber ratio, type I fiber atrophy, actin loss, and contractile dysfunction in the diaphragm. In contrast, mitochondrial respiratory function was preserved alongside lower ROS generation, which may play a compensatory role to preserve fatigue resistance in the diaphragm.NEW & NOTEWORTHY Diaphragm weakness is a hallmark of diseases in which oxygen delivery is compromised. Vascular endothelial growth factor (VEGF) modulates muscle perfusion; however, it remains unclear whether VEGF deficiency contributes to the onset of diaphragm dysfunction. Conditional skeletal myofiber VEGF gene ablation impaired diaphragm contractile function and resulted in type I fiber atrophy, a lower number of capillaries per fiber, and contractile protein content. Mitochondrial function was similar and reactive oxygen species flux was lower. Diaphragm VEGF deficiency may contribute to the onset of respiratory muscle weakness.

Cigarette Smoke Triggers IL-33-associated Inflammation in a Model of Late-Stage Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a worldwide threat. Cigarette smoke (CS) exposure causes cardiopulmonary disease and COPD and increases the risk for pulmonary tumors. In addition to poor lung function, patients with COPD are susceptible to bouts of dangerous inflammation triggered by pollutants or infection. These severe inflammatory episodes can lead to additional exacerbations, hospitalization, further deterioration of lung function, and reduced survival. Suitable models of the inflammatory conditions associated with CS, which potentiate the downward spiral in patients with COPD, are lacking, and the underlying mechanisms that trigger exacerbations are not well understood. Although initial CS exposure activates a protective role for vascular endothelial growth factor (VEGF) functions in barrier integrity, chronic exposure depletes the pulmonary VEGF guard function in severe COPD. Thus, we hypothesized that mice with compromised VEGF production and challenged with CS would trigger human-like severe inflammatory progression of COPD. In this model, we discovered that CS exposure promotes an amplified IL-33 cytokine response and severe disease progression. Our VEGF-knockout model combined with CS recapitulates severe COPD with an influx of IL-33-expressing macrophages and neutrophils. Normally, IL-33 is quickly inactivated by a post-translational disulfide bond formation. Our results reveal that BAL fluid from the CS-exposed, VEGF-deficient cohort promotes a significantly prolonged lifetime of active proinflammatory IL-33. Taken together, our data demonstrate that with the loss of a VEGF-mediated protective barrier, the CS response switches from a localized danger to an uncontrolled long-term and long-range, amplified, IL-33-mediated inflammatory response that ultimately destroys lung function.

Human-like Cmah inactivation in mice increases running endurance and decreases muscle fatigability: implications for human evolution.

Compared to other primates, humans are exceptional long-distance runners, a feature that emerged in genus Homo approximately 2 Ma and is classically attributed to anatomical and physiological adaptations such as an enlarged gluteus maximus and improved heat dissipation. However, no underlying genetic changes have currently been defined. Two to three million years ago, an exon deletion in the CMP-Neu5Ac hydroxylase (CMAH) gene also became fixed in our ancestral lineage. Cmah loss in mice exacerbates disease severity in multiple mouse models for muscular dystrophy, a finding only partially attributed to differences in immune reactivity. We evaluated the exercise capacity of Cmah-/- mice and observed an increased performance during forced treadmill testing and after 15 days of voluntary wheel running. Cmah-/- hindlimb muscle exhibited more capillaries and a greater fatigue resistance in situ Maximal coupled respiration was also higher in Cmah null mice ex vivo and relevant differences in metabolic pathways were also noted. Taken together, these data suggest that CMAH loss contributes to an improved skeletal muscle capacity for oxygen use. If translatable to humans, CMAH loss could have provided a selective advantage for ancestral Homo during the transition from forest dwelling to increased resource exploration and hunter/gatherer behaviour in the open savannah.

Cigarette smoke directly impairs skeletal muscle function through capillary regression and altered myofibre calcium kinetics in mice.

KEY POINTS: Cigarette smoke components directly alter muscle fatigue resistance and intracellular muscle fibre Ca2+ handling independent of a change in lung structure. Changes in muscle vascular structure are associated with a depletion of satellite cells. Sarcoplasmic reticulum Ca2+ uptake is substantially impaired in myofibres during fatiguing contractions in mice treated with cigarette smoke extract. ABSTRACT: Cigarette smokers exhibit exercise intolerance before a decline in respiratory function. In the present study, the direct effects of cigarette smoke on limb muscle function were tested by comparing cigarette smoke delivered to mice by weekly injections of cigarette smoke extract (CSE), or nose-only exposure (CS) 5 days each week, for 8 weeks. Cigarette smoke delivered by either route did not alter pulmonary airspace size. Muscle fatigue measured in situ was 50% lower in the CSE and CS groups than in control. This was accompanied by 34% and 22% decreases in soleus capillary-to-fibre ratio of the CSE and CS groups, respectively, and a trend for fewer skeletal muscle actin-positive arterioles (P = 0.07). In addition, fewer quiescent satellite cells (Nes+Pax7+) were associated with soleus fibres in mice with skeletal myofibre VEGF gene deletion (decreased 47%) and CS exposed (decreased 73%) than with control fibres. Contractile properties of isolated extensor digitorum longus and soleus muscles were impaired. In flexor digitorum brevis myofibres isolated from CSE mice, fatigue resistance was diminished by 43% compared to control and CS myofibres, and this was accompanied by a pronounced slowing in relaxation, an increase in intracellular Ca2+ accumulation, and a slowing in sarcoplasmic reticulum Ca2+ uptake. These data suggest that cigarette smoke components may impair hindlimb muscle vascular structure, fatigue resistance and myofibre calcium handling, and these changes ultimately affect contractile efficiency of locomotor muscles independent of a change in lung function.

Chronic inhalation of e-cigarette vapor containing nicotine disrupts airway barrier function and induces systemic inflammation and multiorgan fibrosis in mice.

Electronic (e)-cigarettes theoretically may be safer than conventional tobacco. However, our prior studies demonstrated direct adverse effects of e-cigarette vapor (EV) on airway cells, including decreased viability and function. We hypothesize that repetitive, chronic inhalation of EV will diminish airway barrier function, leading to inflammatory protein release into circulation, creating a systemic inflammatory state, ultimately leading to distant organ injury and dysfunction. C57BL/6 and CD-1 mice underwent nose only EV exposure daily for 3-6 mo, followed by cardiorenal physiological testing. Primary human bronchial epithelial cells were grown at an air-liquid interface and exposed to EV for 15 min daily for 3-5 days before functional testing. Daily inhalation of EV increased circulating proinflammatory and profibrotic proteins in both C57BL/6 and CD-1 mice: the greatest increases observed were in angiopoietin-1 (31-fold) and EGF (25-fold). Proinflammatory responses were recapitulated by daily EV exposures in vitro of human airway epithelium, with EV epithelium secreting higher IL-8 in response to infection (227 vs. 37 pg/ml, respectively; P < 0.05). Chronic EV inhalation in vivo reduced renal filtration by 20% ( P = 0.017). Fibrosis, assessed by Masson's trichrome and Picrosirius red staining, was increased in EV kidneys (1.86-fold, C57BL/6; 3.2-fold, CD-1; P < 0.05), heart (2.75-fold, C57BL/6 mice; P < 0.05), and liver (1.77-fold in CD-1; P < 0.0001). Gene expression changes demonstrated profibrotic pathway activation. EV inhalation altered cardiovascular function, with decreased heart rate ( P < 0.01), and elevated blood pressure ( P = 0.016). These data demonstrate that chronic inhalation of EV may lead to increased inflammation, organ damage, and cardiorenal and hepatic disease.

Skeletal myofiber vascular endothelial growth factor is required for the exercise training-induced increase in dentate gyrus neuronal precursor cells.

KEY POINTS: Peripheral vascular endothelial growth factor (VEGF) is necessary for exercise to stimulate hippocampal neurogenesis. Here we report that skeletal myofiber VEGF directly or indirectly regulates exercise-signalled proliferation of neuronal precursor cells. Our results found skeletal myofiber VEGF to be necessary for maintaining blood flow through hippocampal regions independent of exercise training state. This study demonstrates that skeletal myofiber VEGF is required for the hippocampal VEGF response to acute exercise. These results help to establish the mechanisms by which exercise, through skeletal myofiber VEGF, affects the hippocampus. ABSTRACT: Exercise signals neurogenesis in the dentate gyrus of the hippocampus. This phenomenon requires vascular endothelial growth factor (VEGF) originating from outside the blood-brain barrier, but no cellular source has been identified. Thus, we hypothesized that VEGF produced by skeletal myofibers plays a role in regulating hippocampal neuronal precursor cell proliferation following exercise training. This was tested in adult conditional skeletal myofiber-specific VEGF gene-ablated mice (VEGFHSA-/- ) by providing VEGFHSA-/- and non-ablated (VEGFf/f ) littermates with running wheels for 14 days. Following this training period, hippocampal cerebral blood flow (CBF) was measured by functional magnetic resonance imaging (fMRI), and neuronal precursor cells (BrdU+/Nestin+) were detected by immunofluorescence. The VEGFf/f trained group showed improvements in both speed and endurance capacity in acute treadmill running tests (P < 0.05). The VEGFHSA-/- group did not. The number of proliferating neuronal precursor cells was increased with training in VEGFf/f (P < 0.05) but not in VEGFHSA-/- mice. Endothelial cell (CD31+) number did not change in this region with exercise training or skeletal myofiber VEGF gene deletion. However, resting blood flow through the hippocampal region was lower in VEGFHSA-/- mice, both untrained and trained, than untrained VEGFf/f mice (P < 0.05). An acute hypoxic challenge decreased CBF (P < 0.05) in untrained VEGFf/f , untrained VEGFHSA-/- and trained VEGFHSA-/- mice, but not trained VEGFf/f mice. VEGFf/f , but not VEGFHSA-/- , mice were able to acutely run on a treadmill at an intensity sufficient to increase hippocampus VEGF levels. These data suggest that VEGF expressed by skeletal myofibers may directly or indirectly regulate both hippocampal blood flow and neurogenesis.

Functional magnetic resonance imaging for in vivo quantification of pulmonary hypertension in the Sugen 5416/hypoxia mouse.

NEW FINDINGS: What is the central question of this study? Non-invasive, quantitative methods to assess right cardiac function in mice with pulmonary hypertension have not been demonstrated. What is the main finding and its importance? This study shows the potential of magnetic resonance imaging to estimate right ventricular ejection fraction and measure spatial, dynamic changes in cardiac structure in the Sugen 5416/hypoxia mouse model of pulmonary hypertension. Pulmonary arterial hypertension (PAH) is characterized by elevated pulmonary artery pressures and right heart failure. Mouse models of PAH are instrumental in understanding the disease pathophysiology. However, few methods are available to evaluate right cardiac function in small animals. In this study, magnetic resonance imaging was used to measure in vivo cardiac dimensions in the Sugen 5416/hypoxia mouse model. Pulmonary hypertension (PH) was induced in C57BL/6 mice by 3 weeks of exposure to 10% oxygen and vascular endothelial growth factor receptor inhibition (20 mg kg-1 SU5416). Control mice were housed in room air and received vehicle (DMSO). Right ventricular pressures were recorded with a pressure-conductance transducer. Short-axis contiguous 1-mm-thick slices were acquired through the heart and great vessels using a fast low-angle shot (FLASH)-cine sequence. Thirteen images were collected throughout each cardiac cycle. Right ventricular systolic pressure was elevated in PH mice (23.6 ± 6 versus 41.0 ± 11 mmHg, control versus PH, respectively; P < 0.001, n = 5-11). Right ventricular wall thickness was greater in PH than in control mice at end diastole (0.30 ± 0.05 versus 0.48 ± 0.06 mm, control versus PH, respectively; P < 0.01, n = 6), but measurements were not different at end systole (control versus PH, 0.59 ± 0.11 versus 0.70 ± 0.11 mm, respectively). Right ventricular ejection fraction was decreased in PH mice (72 ± 3 versus 58 ± 5%, control versus PH, respectively; P < 0.04, n = 6). These data demonstrate that magnetic resonance imaging is a precise method to monitor right ventricular remodelling and cardiac output longitudinally in mouse models of PH.

Skeletal myofiber VEGF regulates contraction-induced perfusion and exercise capacity but not muscle capillarity in adult mice.

A single bout of exhaustive exercise signals expression of vascular endothelial growth factor (VEGF) in the exercising muscle. Previous studies have reported that mice with life-long deletion of skeletal myofiber VEGF have fewer capillaries and a severe reduction in endurance exercise. However, in adult mice, VEGF gene deletion conditionally targeted to skeletal myofibers limits exercise capacity without evidence of capillary regression. To explain this, we hypothesized that adult skeletal myofiber VEGF acutely regulates skeletal muscle perfusion during muscle contraction. A tamoxifen-inducible skeletal myofiber-specific VEGF gene deletion mouse (skmVEGF-/-) was used to reduce skeletal muscle VEGF protein by 90% in adult mice. Three weeks after inducing deletion of the skeletal myofiber VEGF gene, skmVEGF-/- mice exhibited diminished maximum running speed (-10%, P < 0.05) and endurance capacity (-47%; P < 0.05), which did not persist after 8 wk. In skmVEGF-/- mice, gastrocnemius complex time to fatigue measured in situ was 71% lower than control mice. Contraction-induced perfusion measured by optical imaging during a period of electrically stimulated muscle contraction was 85% lower in skmVEGF-/- than control mice. No evidence of capillary rarefication was detected in the soleus, gastrocnemius, and extensor digitorum longus (EDL) up to 8 wk after tamoxifen-induced VEGF ablation, and contractility and fatigue resistance of the soleus measured ex vivo were also unchanged. The force-frequency of the EDL showed a small right shift, but fatigue resistance did not differ between EDL from control and skmVEGF-/- mice. These data suggest myofiber VEGF is required for regulating perfusion during periods of contraction and may in this manner affect endurance capacity.

Selective Life-Long Skeletal Myofiber-Targeted VEGF Gene Ablation Impairs Exercise Capacity in Adult Mice.

Exercise is dependent on adequate oxygen supply for mitochondrial respiration in both cardiac and locomotor muscle. To determine whether skeletal myofiber VEGF is critical for regulating exercise capacity, independent of VEGF function in the heart, ablation of the VEGF gene was targeted to skeletal myofibers (skmVEGF-/-) during embryogenesis (∼ E9.5), leaving intact VEGF expression by all other cells in muscle. In adult mice, VEGF levels were decreased in the soleus (by 65%), plantaris (94%), gastrocnemius (74%), EDL (99%) and diaphragm (64%) (P < 0.0001, each muscle). VEGF levels were unchanged in the heart. Treadmill speed (WT 86 ± 4 cm/sec, skmVEGF-/- 70 ± 5 cm/sec, P = 0.006) and endurance (WT 78 ± 24 min, skmVEGF-/- 18 ± 4 min, P = 0.0004) were severely limited in skmVEGF-/- mice in contrast to minor effect of conditional skmVEGF gene deletion in the adult. Body weight was also reduced (WT 22.8 ± 1.6 g, skmVEGF-/-, 21.1 ± 1.5, P = 0.02), but the muscle mass/body weight ratio was unchanged. The capillary/fiber ratio was lower in skmVEGF-/- plantaris (WT 1.51 ± 0.12, skmVEGF-/- 1.16 ± 0.20, P = 0.01), gastrocnemius (WT 1.61 ± 0.08, skmVEGF-/- 1.39 ± 0.08, P = 0.01), EDL (WT 1.36 ± 0.07, skmVEGF-/- 1.14 ± 0.13, P = 0.03) and diaphragm (WT 1.39 ± 0.18, skmVEGF-/- 0.79 ± 0.16, P = 0.0001) but, not in soleus. Cardiac function (heart rate, maximal pressure, maximal dP/dt, minimal dP/dt,) in response to dobutamine was not impaired in anesthetized skmVEGF-/- mice. Isolated soleus and EDL fatigue times were 16% and 20% (P < 0.02) longer, respectively, in skmVEGF-/- mice than the WT group. These data suggest that skeletal myofiber VEGF expressed during development is necessary to establish capillary networks that allow maximal exercise capacity.

Skeletal myofiber VEGF is necessary for myogenic and contractile adaptations to functional overload of the plantaris in adult mice.

The ability to enhance muscle size and function is important for overall health. In this study, skeletal myofiber vascular endothelial growth factor (VEGF) was hypothesized to regulate hypertrophy, capillarity, and contractile function in response to functional overload (FO). Adult myofiber-specific VEGF gene-ablated mice (skmVEGF(-/-)) and wild-type (WT) littermates underwent plantaris FO or sham surgery (SHAM). Mass, morphology, in vivo function, IGF-1, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and Akt were measured at 7, 14, and 30 days. FO resulted in hypertrophy in both genotypes, but fiber sizes were 13% and 23% smaller after 14 and 30 days, respectively, and mass 15% less after 30 days in skmVEGF(-/-) than WT. FO increased isometric force after 30 days in WT and decreased in skmVEGF(-/-) after 7 and 14 days. FO also resulted in a reduction in specific force and this differed between genotypes at 14 days. Fatigue resistance improved only in 14-day WT mice. Capillary density was decreased by FO in both genotypes. However, capillary-to-fiber ratios were 19% and 15% lower in skmVEGF(-/-) than WT at the 14- and 30-day time points, respectively. IGF-1 was increased by FO at all time points and was 45% and 40% greater in skmVEGF(-/-) than WT after 7 and 14 days, respectively. bFGF, HGF, total Akt, and phospho-Akt, independent of VEGF expression, and VEGF levels in WT were increased after 7 days of FO. These findings suggest VEGF-dependent capillary maintenance supports muscle growth and function in overloaded muscle and is not rescued by compensatory IGF-1 expression.

Increase in relative deposition of fine particles in the rat lung periphery in the absence of gravity.

While it is well recognized that pulmonary deposition of inhaled particles is lowered in microgravity (µG) compared with gravity on the ground (1G), the absence of sedimentation causes fine particles to penetrate deeper in the lung in µG. Using quantitative magnetic resonance imaging (MRI), we determined the effect of gravity on peripheral deposition (DEPperipheral) of fine particles. Aerosolized 0.95-µm-diameter ferric oxide particles were delivered to spontaneously breathing rats placed in plethysmographic chambers both in µG aboard the NASA Microgravity Research Aircraft and at 1G. Following exposure, lungs were perfusion fixed, fluid filled, and imaged in a 3T MR scanner. The MR signal decay rate, R2*, was measured in each voxel of the left lung from which particle deposition (DEP) was determined based on a calibration curve. Regional deposition was assessed by comparing DEP between the outer (DEPperipheral) and inner (DEPcentral) areas on each slice, and expressed as the central-to-peripheral ratio. Total lung deposition tended to be lower in µG compared with 1G (1.01 ± 0.52 vs. 1.43 ± 0.52 µg/ml, P = 0.1). In µG, DEPperipheral was larger than DEPcentral (P < 0.03), while, in 1G, DEPperipheral was not significantly different from DEPcentral. Finally, central-to-peripheral ratio was significantly less in µG than in 1G (P ≤ 0.05). These data show a larger fraction of fine particles depositing peripherally in µG than in 1G, likely beyond the large- and medium-sized airways. Although not measured, the difference in the spatial distribution of deposited particles between µG and 1G could also affect particle retention rates, with an increase in retention for particles deposited more peripherally.

MRI-based measurements of aerosol deposition in the lung of healthy and elastase-treated rats.

Aerosolized drugs are increasingly being used to treat chronic lung diseases or to deliver therapeutics systemically through the lung. The influence of disease, such as emphysema, on particle deposition is not fully understood. With the use of magnetic resonance imaging (MRI), the deposition pattern of iron oxide particles with a mass median aerodynamic diameter of 1.2 µm was assessed in the lungs of healthy and elastase-treated rats. Tracheostomized rats were ventilated with particles, at a tidal volume of 2.2 ml, and a breathing frequency of 80 breaths/min. Maximum airway pressure was significantly lower in the elastase-treated (Paw = 7.71 ± 1.68 cmH2O) than in the healthy rats (Paw = 10.43 ± 1.02 cmH2O; P < 0.01). This is consistent with an increase in compliance characteristic of an emphysema-like lung structure. Following exposure, lungs were perfusion fixed and imaged in a 3T MR scanner. Particle concentration in the different lobes was determined based on a relationship with the MR signal decay rate, R2 (*). Whole lung particle deposition was significantly higher in the elastase-treated rats (CE,part = 3.03 ± 0.61 µm/ml) compared with the healthy rats (CH,part = 1.84 ± 0.35 µm/ml; P < 0.01). However, when particle deposition in each lobe was normalized by total deposition in the lung, there was no difference between the experimental groups. However, the relative dispersion [RD = standard deviation/mean] of R2 (*) was significantly higher in the elastase-treated rats (RDE = 0.32 ± 0.02) compared with the healthy rats (RDH = 0.25 ± 0.02; P < 0.01). These data show that particle deposition is higher and more heterogeneously distributed in emphysematous lungs compared with healthy lungs.

Skeletal myofiber VEGF is essential for the exercise training response in adult mice.

Vascular endothelial growth factor (VEGF) is exercise responsive, pro-angiogenic, and expressed in several muscle cell types. We hypothesized that in adult mice, VEGF generated within skeletal myofibers (and not other cells within muscle) is necessary for the angiogenic response to exercise training. This was tested in adult conditional, skeletal myofiber-specific VEGF gene-deleted mice (skmVEGF-/-), with VEGF levels reduced by >80%. After 8 wk of daily treadmill training, speed and endurance were unaltered in skmVEGF-/- mice, but increased by 18% and 99% (P < 0.01), respectively, in controls trained at identical absolute speed, incline, and duration. In vitro, isolated soleus and extensor digitorum longus contractile function was not impaired in skmVEGF-/- mice. However, training-induced angiogenesis was inhibited in plantaris (wild type, 38%, skmVEGF-/- 18%, P < 0.01), and gastrocnemius (wild type, 43%, P < 0.01; skmVEGF-/-, 7%, not significant). Capillarity was maintained (different from VEGF gene deletion targeted to multiple cell types) in untrained skmVEGF-/- mice. Arteriogenesis (smooth muscle actin+, artery number, and diameter) and remodeling [vimentin+, 5'-bromodeoxycytidine (BrdU)+, and F4/80+ cells] occurred in skmVEGF-/- mice, even in the absence of training. skmVEGF-/- mice also displayed a limited oxidative enzyme [citrate synthase and ß-hydroxyacyl CoA dehydrogenase (ß-HAD)] training response; ß-HAD activity levels were elevated in the untrained state. These data suggest that myofiber expressed VEGF is necessary for training responses in capillarity and oxidative capacity and for improved running speed and endurance.

Impaired exercise capacity and skeletal muscle function in a mouse model of pulmonary inflammation.

Pulmonary TNFα has been linked to reduced exercise capacity in a subset of patients with moderate to severe chronic obstructive pulmonary disease (COPD). We hypothesized that prolonged, high expression of pulmonary TNFα impairs cardiac and skeletal muscle function, and both contribute to exercise limitation. Using a surfactant protein C promoter-TNFα construct, TNFα was overexpressed throughout life in mouse lungs (SP-C/TNFα+). TNFα levels in wild-type (WT) female serum and lung were two- and threefold higher than in WT male mice. In SP-C/TNFα+ mice, TNFα increased similarly in both sexes. Treadmill exercise was impaired only in male SP-C/TNFα+ mice. While increases in lung volume and airspace size induced by TNFα were comparable in both sexes, pulmonary hypertension along with lower body and muscle mass were evident only in male mice. Left ventricular (LV) function (cardiac output, stroke volume, LV maximal pressure, and LV maximal pressure dP/dt) was not altered by TNFα overexpression. Fatigue measured in isolated soleus and EDL was more rapid only in soleus of male SP-C/TNFα+ mice and accompanied by a loss of oxidative IIa fibers, citrate synthase activity, and PGC-1α mRNA and increase in atrogin-1 and MuRF1 expression also only in male mice. In situ gastrocnemius fatigue resistance, reflecting both oxygen availability and contractility, was decreased similarly in female and male SP-C/TNFα+ mice. These data indicate that male, but not female, mice overexpressing pulmonary TNFα are susceptible to exercise limitation, possibly due to muscle wasting and loss of the oxidative muscle phenotype, with protection in females possibly due to estrogen.