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.

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 (VEGF^HSA-/- ) by providing VEGF^HSA-/- and non-ablated (VEGF^f/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 VEGF^f/f trained group showed improvements in both speed and endurance capacity in acute treadmill running tests (P < 0.05). The VEGF^HSA-/- group did not. The number of proliferating neuronal precursor cells was increased with training in VEGF^f/f (P < 0.05) but not in VEGF^HSA-/- 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 VEGF^HSA-/- mice, both untrained and trained, than untrained VEGF^f/f mice (P < 0.05). An acute hypoxic challenge decreased CBF (P < 0.05) in untrained VEGF^f/f , untrained VEGF^HSA-/- and trained VEGF^HSA-/- mice, but not trained VEGF^f/f mice. VEGF^f/f , but not VEGF^HSA-/- , 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.

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.

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.

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.

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 β-hydroxyacyl CoA dehydrogenase (β-HAD)] training response; β-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.

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.

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.

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.

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.