Skeletal muscle dysfunction in survivors of pneumonia disproportionately affects older individual... more Skeletal muscle dysfunction in survivors of pneumonia disproportionately affects older individuals in whom it causes substantial morbidity. We found that skeletal muscle recovery was impaired in old compared with young mice after influenza A virus‐induced pneumonia. In young mice, recovery of muscle loss was associated with expansion of tissue‐resident skeletal muscle macrophages and downregulation of MHC II expression, followed by a proliferation of muscle satellite cells. These findings were absent in old mice and in mice deficient in Cx3cr1. Transcriptomic profiling of tissue‐resident skeletal muscle macrophages from old compared with young mice showed downregulation of pathways associated with phagocytosis and proteostasis, and persistent upregulation of inflammatory pathways. Consistently, skeletal muscle macrophages from old mice failed to downregulate MHCII expression during recovery from influenza A virus‐induced pneumonia and showed impaired phagocytic function in vitro. Like old animals, mice deficient in the phagocytic receptor Mertk showed no macrophage expansion, MHCII downregulation, or satellite cell proliferation and failed to recover skeletal muscle function after influenza A pneumonia. Our data suggest that a loss of phagocytic function in a CX3CR1+ tissue‐resident skeletal muscle macrophage population in old mice precludes satellite cell proliferation and recovery of skeletal muscle function after influenza A pneumonia.
Molecular oxygen and carbon dioxide are the primary substrate and product of oxidative metabolism... more Molecular oxygen and carbon dioxide are the primary substrate and product of oxidative metabolism, respectively. Hypoxia (low oxygen) and hypercapnia (high carbon dioxide) are co-incidental features of the tissue microenvironment in a range of pathophysiologic states including acute and chronic respiratory diseases. The hypoxia inducible factor (HIF) is the master regulator of the transcriptional response to hypoxia, however little is known about the impact of hypercapnia on gene transcription. Because of the relationship between hypoxia and hypercapnia, we investigated the effect of hypercapnia on the HIF pathway. Hypercapnia suppressed HIF-α protein stability and HIF target gene expression both in mice and in cultured cells in a manner that was at least in part independent of the canonical O2-dependent HIF degradation pathway. The suppressive effects of hypercapnia on HIF-α protein stability could be mimicked by reducing intracellular pH at constant pCO2. Bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase that blocks lysosomal degradation, prevented the hypercapnic suppression of HIF-α protein. Based on these results, we hypothesize that hypercapnia counter-regulates activation of the HIF pathway by reducing intracellular pH and promoting lysosomal degradation of HIF-α subunits. Therefore, hypercapnia may play a key role in the pathophysiology of diseases where HIF is implicated.
The c-Jun amino-terminal kinase (JNK) plays a role in inflammation, proliferation, apoptosis and ... more The c-Jun amino-terminal kinase (JNK) plays a role in inflammation, proliferation, apoptosis and cell adhesion and cell migration by phosphorylating paxillin and β-catenin. We have reported that JNK phosphorylation down-stream of AMP-activated protein kinase (AMPK) activation is required for high CO2 (hypercapnia)-induced Na,K-ATPase endocytosis in alveolar epithelial cells. Here, we provide evidence that during hypercapnia, JNK promotes the phosphorylation of LMO7b, a scaffolding protein, in vitro and in intact cells. LMO7b phosphorylation was blocked exposing the cells to the JNK inhibitor SP600125 and by infecting cells with dominant negative JNK or AMPK adenovirus. Knockdown of the endogenous LMO7b or over-expression of mutated LMO7b with alanine substitutions of five potential JNK phosphorylation sites (LMO7b-5SA) or only the Ser-1295 rescued both LMO7b phosphorylation and the hypercapnia-induced Na,K-ATPase endocytosis. Moreover, high CO2 promoted the co-localization and interaction of LMO7b and the Na,K-ATPase α1-subunit at the plasma membrane which were prevented by SP600125 or by transfecting cells with LMO7b-5SA. Collectively, our data suggest that hypercapnia leads to JNK-induced LMO7b phosphorylation at Ser-1295, which facilitates the interaction of LMO7b with the Na,K-ATPase at the plasma membrane promoting the endocytosis of Na,K-ATPase in alveolar epithelial cells.
Carbon dioxide (CO2) is a product of oxidative metabolism in humans. The arterial CO2 partial pre... more Carbon dioxide (CO2) is a product of oxidative metabolism in humans. The arterial CO2 partial pressure (PO2) can be represented by equation (1): $ PO_2 = K \cdot (VCO_2 /V + PiCO_2 $ PO_2 = K \cdot (VCO_2 /V + PiCO_2 (1) where K is a proportion constant, VCO 2 the CO2 production, V A the alveolar ventilation, and PCO 2 is the inhaled CO2. Aveolar ventilation (VA) is a component of the total ventilation (VT) minus the dead space ventilation (VD), which is VA= VT − VD or: $ V_A = V_T (1 - V_D /V_T ) $ V_A = V_T (1 - V_D /V_T ) (2) therefore, by combining equations 1 and 2, the following equation results: $ PCO_2 = K \cdot (VCO_2 /V_T (1 - V_D /V_T ) + PiCO_2 $ PCO_2 = K \cdot (VCO_2 /V_T (1 - V_D /V_T ) + PiCO_2 (3) Consistent with this equation, we can identify four causes for hypercapnia: A) increased CO2 production (VCO2); B) hypoventilation (increased value of 1/VT); C) increased dead space ventilation (VD); and D) increased inhaled CO2 (PiCO2) [1].
Development of the enteric nervous system (ENS) requires interactions between migrating neural cr... more Development of the enteric nervous system (ENS) requires interactions between migrating neural crest cells and the nascent gastrointestinal tract that are dependent upon genes expressed by both cell compartments. Hlx, a homeobox transcription factor gene that is expressed in mouse intestinal and hepatic mesenchyme, is required for normal embryonic growth of intestine and liver, and the Hlx-/- genotype is embryonic lethal. We hypothesized that Hlx is required for ENS development. Enteric neurons were identified in Hlx+/+ and Hlx-/- mouse embryos by immunostaining of embryo sections for the neural markers PGP9.5 and Phox2b, or by staining for beta-galactosidase in whole-mount embryos containing the dopamine beta-hydroxylase-nLacZ transgene. In Hlx+/+ embryos, neural crest cells/enteric neurons have moved from the stomach into the intestine by E10.5. By contrast, neural crest cells/enteric neurons remain largely restricted to the lateral stomach mesenchyme of Hlx-/- embryos, with only ...
Patients with chronic obstructive pulmonary disease (COPD), acute lung injury and critical care i... more Patients with chronic obstructive pulmonary disease (COPD), acute lung injury and critical care illness may develop hypercapnia. Many of these patients often have muscle dysfunction which increases morbidity and impairs their quality of life. Here, we investigated whether hypercapnia leads to skeletal muscle atrophy. Mice exposed to high CO2 had decreased skeletal muscle wet weight, fiber diameter, and strength. Cultured myotubes exposed to high CO2 had reduced fiber diameter, protein/DNA ratio and anabolic capacity. High CO2 induced the expression of MuRF1 in vivo and in vitro, while MuRF1-/- mice exposed to high CO2 did not develop muscle atrophy. AMPK, a metabolic sensor, was activated in myotubes exposed to high CO2 and loss-of-function studies showed that AMPK-α2 isoform is necessary for MuRF1 up-regulation and myofiber size reduction. High CO2 induced AMPK-α2 activation, triggering the phosphorylation and nuclear translocation of FoxO3a, and leading to an increase in MuRF1 expression and myotube atrophy. Accordingly, we provide evidence that high CO2 activates skeletal muscle atrophy via AMPKα2-FoxO3a-MuRF1, which is of biological and potentially clinical significance in patients with lung diseases and hypercapnia.
Skeletal muscle dysfunction in survivors of pneumonia disproportionately affects older individual... more Skeletal muscle dysfunction in survivors of pneumonia disproportionately affects older individuals in whom it causes substantial morbidity. We found that skeletal muscle recovery was impaired in old compared with young mice after influenza A virus‐induced pneumonia. In young mice, recovery of muscle loss was associated with expansion of tissue‐resident skeletal muscle macrophages and downregulation of MHC II expression, followed by a proliferation of muscle satellite cells. These findings were absent in old mice and in mice deficient in Cx3cr1. Transcriptomic profiling of tissue‐resident skeletal muscle macrophages from old compared with young mice showed downregulation of pathways associated with phagocytosis and proteostasis, and persistent upregulation of inflammatory pathways. Consistently, skeletal muscle macrophages from old mice failed to downregulate MHCII expression during recovery from influenza A virus‐induced pneumonia and showed impaired phagocytic function in vitro. Like old animals, mice deficient in the phagocytic receptor Mertk showed no macrophage expansion, MHCII downregulation, or satellite cell proliferation and failed to recover skeletal muscle function after influenza A pneumonia. Our data suggest that a loss of phagocytic function in a CX3CR1+ tissue‐resident skeletal muscle macrophage population in old mice precludes satellite cell proliferation and recovery of skeletal muscle function after influenza A pneumonia.
Molecular oxygen and carbon dioxide are the primary substrate and product of oxidative metabolism... more Molecular oxygen and carbon dioxide are the primary substrate and product of oxidative metabolism, respectively. Hypoxia (low oxygen) and hypercapnia (high carbon dioxide) are co-incidental features of the tissue microenvironment in a range of pathophysiologic states including acute and chronic respiratory diseases. The hypoxia inducible factor (HIF) is the master regulator of the transcriptional response to hypoxia, however little is known about the impact of hypercapnia on gene transcription. Because of the relationship between hypoxia and hypercapnia, we investigated the effect of hypercapnia on the HIF pathway. Hypercapnia suppressed HIF-α protein stability and HIF target gene expression both in mice and in cultured cells in a manner that was at least in part independent of the canonical O2-dependent HIF degradation pathway. The suppressive effects of hypercapnia on HIF-α protein stability could be mimicked by reducing intracellular pH at constant pCO2. Bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase that blocks lysosomal degradation, prevented the hypercapnic suppression of HIF-α protein. Based on these results, we hypothesize that hypercapnia counter-regulates activation of the HIF pathway by reducing intracellular pH and promoting lysosomal degradation of HIF-α subunits. Therefore, hypercapnia may play a key role in the pathophysiology of diseases where HIF is implicated.
The c-Jun amino-terminal kinase (JNK) plays a role in inflammation, proliferation, apoptosis and ... more The c-Jun amino-terminal kinase (JNK) plays a role in inflammation, proliferation, apoptosis and cell adhesion and cell migration by phosphorylating paxillin and β-catenin. We have reported that JNK phosphorylation down-stream of AMP-activated protein kinase (AMPK) activation is required for high CO2 (hypercapnia)-induced Na,K-ATPase endocytosis in alveolar epithelial cells. Here, we provide evidence that during hypercapnia, JNK promotes the phosphorylation of LMO7b, a scaffolding protein, in vitro and in intact cells. LMO7b phosphorylation was blocked exposing the cells to the JNK inhibitor SP600125 and by infecting cells with dominant negative JNK or AMPK adenovirus. Knockdown of the endogenous LMO7b or over-expression of mutated LMO7b with alanine substitutions of five potential JNK phosphorylation sites (LMO7b-5SA) or only the Ser-1295 rescued both LMO7b phosphorylation and the hypercapnia-induced Na,K-ATPase endocytosis. Moreover, high CO2 promoted the co-localization and interaction of LMO7b and the Na,K-ATPase α1-subunit at the plasma membrane which were prevented by SP600125 or by transfecting cells with LMO7b-5SA. Collectively, our data suggest that hypercapnia leads to JNK-induced LMO7b phosphorylation at Ser-1295, which facilitates the interaction of LMO7b with the Na,K-ATPase at the plasma membrane promoting the endocytosis of Na,K-ATPase in alveolar epithelial cells.
Carbon dioxide (CO2) is a product of oxidative metabolism in humans. The arterial CO2 partial pre... more Carbon dioxide (CO2) is a product of oxidative metabolism in humans. The arterial CO2 partial pressure (PO2) can be represented by equation (1): $ PO_2 = K \cdot (VCO_2 /V + PiCO_2 $ PO_2 = K \cdot (VCO_2 /V + PiCO_2 (1) where K is a proportion constant, VCO 2 the CO2 production, V A the alveolar ventilation, and PCO 2 is the inhaled CO2. Aveolar ventilation (VA) is a component of the total ventilation (VT) minus the dead space ventilation (VD), which is VA= VT − VD or: $ V_A = V_T (1 - V_D /V_T ) $ V_A = V_T (1 - V_D /V_T ) (2) therefore, by combining equations 1 and 2, the following equation results: $ PCO_2 = K \cdot (VCO_2 /V_T (1 - V_D /V_T ) + PiCO_2 $ PCO_2 = K \cdot (VCO_2 /V_T (1 - V_D /V_T ) + PiCO_2 (3) Consistent with this equation, we can identify four causes for hypercapnia: A) increased CO2 production (VCO2); B) hypoventilation (increased value of 1/VT); C) increased dead space ventilation (VD); and D) increased inhaled CO2 (PiCO2) [1].
Development of the enteric nervous system (ENS) requires interactions between migrating neural cr... more Development of the enteric nervous system (ENS) requires interactions between migrating neural crest cells and the nascent gastrointestinal tract that are dependent upon genes expressed by both cell compartments. Hlx, a homeobox transcription factor gene that is expressed in mouse intestinal and hepatic mesenchyme, is required for normal embryonic growth of intestine and liver, and the Hlx-/- genotype is embryonic lethal. We hypothesized that Hlx is required for ENS development. Enteric neurons were identified in Hlx+/+ and Hlx-/- mouse embryos by immunostaining of embryo sections for the neural markers PGP9.5 and Phox2b, or by staining for beta-galactosidase in whole-mount embryos containing the dopamine beta-hydroxylase-nLacZ transgene. In Hlx+/+ embryos, neural crest cells/enteric neurons have moved from the stomach into the intestine by E10.5. By contrast, neural crest cells/enteric neurons remain largely restricted to the lateral stomach mesenchyme of Hlx-/- embryos, with only ...
Patients with chronic obstructive pulmonary disease (COPD), acute lung injury and critical care i... more Patients with chronic obstructive pulmonary disease (COPD), acute lung injury and critical care illness may develop hypercapnia. Many of these patients often have muscle dysfunction which increases morbidity and impairs their quality of life. Here, we investigated whether hypercapnia leads to skeletal muscle atrophy. Mice exposed to high CO2 had decreased skeletal muscle wet weight, fiber diameter, and strength. Cultured myotubes exposed to high CO2 had reduced fiber diameter, protein/DNA ratio and anabolic capacity. High CO2 induced the expression of MuRF1 in vivo and in vitro, while MuRF1-/- mice exposed to high CO2 did not develop muscle atrophy. AMPK, a metabolic sensor, was activated in myotubes exposed to high CO2 and loss-of-function studies showed that AMPK-α2 isoform is necessary for MuRF1 up-regulation and myofiber size reduction. High CO2 induced AMPK-α2 activation, triggering the phosphorylation and nuclear translocation of FoxO3a, and leading to an increase in MuRF1 expression and myotube atrophy. Accordingly, we provide evidence that high CO2 activates skeletal muscle atrophy via AMPKα2-FoxO3a-MuRF1, which is of biological and potentially clinical significance in patients with lung diseases and hypercapnia.
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Papers by Lynn Welch