Objective: Humans are unique among hominids in that they are endurance athletes. Inactivation of the CMP-Neu5Ac hydroxylase (CMAH) gene, which encodes a hydroxylase converting sialic acid Neu5Ac to Neu5Gc, is thought to contribute to this exceptional capacity for endurance running. Recently, our group demonstrated improved fatigue resistance and preserved intracellular O2, estimated by NAD(P)H levels in contracting mouse Cmah−/− flexor digitorum brevis (FDB) single fibers under low oxygen tension. Hypothesis: Effcient regulation of mitochondrial oxygen consumption contributes to improved skeletal myofiber fatigue resistance in Cmah−/− mice. Methods: The mitochondrial bioenergetic potential of permeabilized FDB and soleus fiber bundles from wild type (WT) and Cmah−/− mice was evaluated using a physiological creatine kinase clamp method and high-resolution respirometer. Experiments were conducted with pyruvate and malate with or without branched chain amino acids (BCAAs) as the substrates. Respiratory chain conductance and maximal respiration were measured. Results: FDB Cmah−/− fibers from male but not female mice revealed an increase conductance above WT fibers independent of substrate (male: genotype, p=0.006, substrate, ns; female: genotype, ns, substrate, ns). Maximal respiration was also increased only in male FDBs fibers and was even greater when BCAAs were included in the respiration media. (male: genotype, p=0.003, substrate, p=0.0007, interaction, p=0.002.; female: genotype, ns, substrate, ns). Soleus Cmah−/− fibers from male and female mice revealed an increase conductance above WT fiber bundles that was also greater in the presence of BCAAs but only for male mice (male: genotype, p=0.003, substrate, p=0.05.; female: genotype, p=0.009, substrate, ns). Maximal respiration was increased only in male Cmah−/− soleus fibers independent of substrate. (male: genotype, p=0.002, substrate, ns.; female: genotype, ns, substrate, ns). Summary: Fibers from both the FDB and soleus of male Cmah−/− mice have a greater capacity for mitochondrial respiration under a metabolically active state that is similar to myofibers stimulated to repeatedly contraction in a hypoxic environment. Conclusion: These data suggest that effcient regulation of mitochondrial respiration utilizing BCAAs as a substrate, particularly in males, could contribute to an elite endurance phenotype. This project was funded through the UC San Diego Academic Senate Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background and Purpose: Traumatic brain injury (TBI) imposes life-long physical, psychological, and financial burdens on affected individuals. The current study investigated the effects of chronic nicotine exposure via E-cigarette (E-cig) on TBI-associated behavioral and biochemical changes. Experimental Approach: Adult C57/BL6J male mice were subjected to controlled cortical impact (CCI) followed by daily exposure to E-cigarette (E-Cig) vapor for six weeks. The effects of chronic nicotine exposure on sensorimotor functions, locomotion, and sociability were evaluated by nesting, open field, and social approach, respectively. Immunoblots were performed to assess changes of mature brain-derived neurotrophic factor (mBDNF) and associated downstream signaling proteins (p-Akt and p-Erk). Histological analyses of the cortex were performed to evaluate the effects of chronic nicotine exposure on Microglia-mediated neuroinflammation. Key Results: Post-injury chronic nicotine exposure significantly improved nesting performance in CCI mice. Histology analysis revealed that chronic nicotine exposure increased the survival of cortical neurons in the perilesion cortex. Immunoblots of cortical tissue revealed that chronic nicotine exposure significantly upregulated mBDNF expression, P-Erk, and p-Akt in the perilesion cortical tissue of CCI mice. Additional IF microscopy revealed elevated mBDNF and p-Akt expression was predominantly localized in cortical neurons of CCI mice. Furthermore, immunolabeling of Iba1 showed that chronic nicotine exposure attenuates microglia-mediated chronic neuroinflammation in the perilesional cortex of CCI mice. Conclusions and Implications: Post-injury chronic nicotine exposure via vaping facilitates sensorimotor function recovery by upregulating neuroprotective mBDNF/TrkB/Akt/Erk signaling. Results from this study support the neuroprotective properties of nicotine, further investigation is needed due to its highly addictive nature.

Objective: Some marine mammals of the order pinniped and cetacean can dive for long durations to forage for food and travel over distances. During these extended dives both pinnipeds and cetaceans experience extreme tissue hypoxia. Numerous adaptations have evolved to allow these species to effciently deliver and utilize oxygen (O2). One genetic difference, which our laboratory has proposed to play an important role in O2 transport effciency in modern hominids, is the inactivation of the CMP-Neu5AC hydroxylase (CMAH) gene. Pinnipeds inactivated Cmah ~ 40 million years ago but, cetaceans still express this N-glycan modifying enzyme. CMAH converts the sialic acid, N-acetylneuraminic acid (Neu5Ac), to its hydroxylated form, N-glycolylneuraminic acid (Neu5Gc). These sialic acids are located at the end of branched sugar chains on the surface of cell membranes. As a result, a change in composition could affect cell membrane charge and hydrophobicity, as well as potentially alter O2 diffusion laterally or across cell membranes. Hypothesis: In this study we hypothesized that O2 transfer from hemoglobin (Hb) in red blood cells (RBCs) to myoglobin (Mb) would differ between cetaceans (CMAH+) and pinnipeds (CMAH-). Methods: This hypothesis was tested using optical absorption spectroscopy to measure O2 exchange between oxygenated Hb in RBC and deoxygenated Mb collected in several species of mustelids and pinniped (CA sea otter, CA sea lion, and walrus) and cetaceans (Bottlenose Dolphin, Beluga, and Orca). Measurements of cell diameters (diffusion length) were also measured. Results: The mean oxygen transfer (O2D/A410) was 47% lower in pinnipeds than cetaceans (p<0.01). The amount of O2 transferred in cetaceans increased with longer reported maximal dive durations. There was a trend for overall RBC diameters to be larger in cetaceans than pinnipeds (p=0.054). Summary: The RBC O2 transfer data in these hypoxia tolerant marine mammals suggest a transfer mechanism that is more effcient in cetaceans in a normoxic environment. Conclusions: More effcient oxygen transfer in cetaceans, who retained the Cmah gene, compared to pinnipeds and mustelids, who did not retain the Cmah gene, suggests that the gene knockout may not be the only factor that facilitates delivery of O2 in these marine mammals. A trend for larger RBC diameters in cetaceans than pinnipeds also suggests that diffusion length is not a main contributing factor. Future experiments to measure oxygen transfer from RBC O2-Hb to Mb under different O2 environments are needed to more closely model what may occur during long duration dives. NSF1929325. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract The use of tobacco cigarettes produces locomotor muscle weakness and fatigue intolerance. Also, smokers and chronic obstructive pulmonary disease patients have a greater incidence of muscle injury and a deficient myogenic response. However, the effects of smoke exposure on the recovery from eccentric exercise‐induced muscle injuries are unknown. Mice were exposed daily to cigarette smoke (CS) or room air (Air) for 4 months; the anterior crural muscles from one limb were injured by a lengthening contractions protocol (LCP) and recovered for 7 days. Lung compliance was greater, and body weights were lower, in CS‐exposed than in the Air group. In LCP‐subjected limbs, CS exposure lowered tibialis anterior myofiber cross‐sectional area, decreased the size of centrally nucleated myofibers, and decreased extensor digitorum longus (EDL) mass, but did not affect EDL force from both limbs. CS exposure upregulated the mRNA levels of several myogenic (Pax7, Myf5, nNOS) genes in the EDL. The combination of CS exposure and LCP decreased Myf5 and nNOS mRNA levels and exacerbated pro‐inflammatory mRNA levels. These data suggest that smoke exposure leads to an excessive pro‐inflammatory response in regenerating muscle that is associated with a lower muscle mass recovery from a type of injury that often occurs during strenuous exercise.

1 channels (VDCC), enhances Ca 2 1 influx through VDCC, and increases cytosolic Ca 2 1 concentration ([Ca 2 1 ] cyt ) in PASMCs. A rise in [Ca 2 1 ] cyt causes PASMC contraction and thus pulmonary vasoconstriction and stimulates PASMC migration and proliferation that contributes to the development and progression of concentric pulmonary vascular remodeling and muscularization of pulmonary arteriole and capillary. Increased ( " ) I K as a result, for example, of activation of K ATP channels by cromakalim and diazoxide, causes membrane hyperpolarization or repolarization that subsequently closes VDCC. The resultant inhibition of Ca 2 1 influx through VDCC and decreases in [Ca 2 1 ] cyt lead to pulmonary vasodilation and regression of remodeled pulmonary arteries and arterioles. Furthermore, activation of K 1 efflux through K ATP channels (and other types of K 1 channels) would relieve K 1 -mediated inhibition of caspase and nuclease activity and enhance PASMC apoptosis. Activation of K 1 efflux through K ATP channels would also facilitate apoptotic volume decrease, an early hallmark of apoptosis, and induce PASMC apoptosis. The inhibitory effects of K ATP channel activation (via Kir6 and/or SUR1) on pulmonary vasoconstriction and vascular remodeling and the apoptotic effect on highly proliferated cells in the remodeled distal arteries all contribute to the potential therapeutic effects of the K ATP channel activators.

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.

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 x treatment interaction F[1,67] = 3.3, p = 0.07, eta 2 = 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, eta 2 = 0.07). Diaphragm TNF alpha, but not IL-33 or NFkB, was increased in SuHx vs. DMSO in both genotypes (F[1,43] = 4.7, p < 0.05, eta 2 = 0.1). Diaphragm force-frequency relationships were right-shifted in SuHx vs. WT (F[3,440] = 8.4, p < 0.05, eta 2 = 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, eta 2 = 0.11). SuHx mice exhibit a modest compensation in diaphragm contractility and mitochondrial dysfunction during coupled respiration; the latter partially regulated through ST2 signaling.

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.

Excessive pulmonary artery (PA) smooth muscle cell (PASMC) proliferation and migration are implicated in the development of pathogenic pulmonary vascular remodeling characterized by concentric arterial wall thickening and arteriole muscularization in patients with pulmonary arterial hypertension (PAH). Pulmonary artery smooth muscle cell contractile-to-proliferative phenotypical transition is a process that promotes pulmonary vascular remodeling. A rise in cytosolic Ca2+ concentration [(Ca2+) cyt ] in PASMCs is a trigger for pulmonary vasoconstriction and a stimulus for pulmonary vascular remodeling. Here, we report that the calcium homeostasis modulator (CALHM), a Ca2+ (and ATP) channel that is allosterically regulated by voltage and extracellular Ca2+, is upregulated during the PASMC contractile-to-proliferative phenotypical transition. Protein expression of CALHM1/2 in primary cultured PASMCs in media containing serum and growth factors (proliferative PASMC) was significantly greater than in freshly isolated PA (contractile PASMC) from the same rat. Upregulated CALHM1/2 in proliferative PASMCs were associated with an increased ratio of pAKT/AKT and pmTOR/mTOR and an increased expression of the cell proliferation marker PCNA, whereas serum starvation and rapamycin significantly downregulated CALHM1/2. Furthermore, CALHM1/2 were upregulated in freshly isolated PA from rats with monocrotaline (MCT)-induced PH and in primary cultured PASMC from patients with PAH in comparison to normal controls. Intraperitoneal injection of CGP 37157 (0.6 mg/kg, q8H), a non-selective blocker of CALHM channels, partially reversed established experimental PH. These data suggest that CALHM upregulation is involved in PASMC contractile-to-proliferative phenotypical transition. Ca2+ influx through upregulated CALHM1/2 may play an important role in the transition of sustained vasoconstriction to excessive vascular remodeling in PAH or precapillary PH. Calcium homeostasis modulator could potentially be a target to develop novel therapies for PAH.

Introduction: During the first surge of the COVID-19 pandemic, healthcare utilization changed. We sought to examine the impact of the first COVID-19 surge on the outcomes of patients whose elective surgeries for diverticulitis were postponed and those who underwent urgent surgery during the surge. Materials and methods: This was a retrospective study from a single tertiary center in the Northeast of the US. Patients whose elective surgeries were delayed, or who underwent urgent surgery for diverticulitis during the first COVID-19 surge (3/16/2020 to 8/1/2020) were included. A cohort from 2019 was used for comparison. Variables were compared between groups including: procedure, death, length of stay, disposition, stoma rate, technique for surgery, and leak rate. Results: Forty-five patients were included in the COVID-19 group and 44 patients in the 2019 group. Twentyseven patients had elective surgeries delayed during the COVID-19 surge. Ten (37%) required more urgent surgery, 80% with complicated disease. Six (22%) were admitted to the hospital and 13 (48%) required additional antibiotics. Eight (30%) patients postponed their surgeries indefinitely and 7 (26%) had surgery once permitted. There were no observed differences between the two groups in the rate of complicated disease, leaks, technique for surgery or stoma rate. Conclusions: During the first COVID-19 surge, over 1/3 of patients whose elective diverticulitis surgeries were postponed required urgent surgery, a majority of whom had complicated disease. There were no apparent differences in outcomes when compared to a pre-pandemic cohort, highlighting the importance of a triage system with the ability to escalate surgery in a timely manner.

BackgroundObstructive sleep apnea (OSA) is associated with metabolic dysfunction, including insulin resistance, lipid dysregulation, and hepatic steatosis and fibrosis in nonalcoholic fatty liver disease (NAFLD). Mechanisms underlying the association between OSA and NAFLD are unclear, but we have previously shown that hepatocyte hypoxia inducible factor‐1 (HIF‐1) mediates the development of liver fibrosis in a mouse model of NAFLD. Moreover, chronic intermittent hypoxia (IH) as a model of OSA has been shown to increase hepatic steatosis and fibrosis in rodent models. Here, we aimed to query the interactive effect of IH and hepatocyte HIF‐1. We hypothesized that IH would increase liver steatosis and fibrosis in murine NAFLD, in a HIF‐1‐dependent manner.MethodsMice were generated to have a hepatocyte‐specific deletion of Hif1a (Hif1a−/−hep) by a Cre‐recombinase system. Cre‐negative Hif1aflox mice were used as wild‐type control. Eight‐week‐old male mice from each genotype were fed a high trans‐fat diet to induce characteristics similar to human NAFLD. At week 20, the mice were exposed either to intermittent hypoxia (IH, FiO2 ranging from 21% to 6%, once per minute, for 12 hours during the light phase) or intermittent air (IA). Intraperitoneal glucose tolerance test (IPGTT) was done at week 25, one week before sacrifice. At sacrifice, liver fibrosis, the primary outcome of interest, was determined by hydroxyproline assay. Mitochondrial function was assessed in fresh liver tissue, and the respiratory control ratio (RCR) was calculated. Malondialdehyde and triglycerides were quantified in liver samples using commercial assays. Aminotransferase levels were quantified using veterinary laboratory services. Data were analyzed by two‐way ANOVA.ResultsLiver fibrosis was increased in IH versus IA (9% increase in hydroxyproline in IH, p=0.022), and was decreased in Hif1a−/−hep mice relative to Hif1aflox (20% decrease in hydroxyproline in Hif1a−/−hep, p<0.001). There was no significant interaction between genotype and hypoxic exposure. Hif1a−/−hep mice had an improved metabolic profile, with lower fasting glucose and area under the IPGTT curve. However, Hif1a−/−hep mice had increased oxidative stress (malondialdehyde increased 55% in Hif1a−/−hep, p=0.004), and worsened mitochondrial efficiency (32% reduction in RCR in Hif1a−/−hep, p=0.002). Hepatic triglycerides and aminotransferase levels were similar between groups.ConclusionsHepatocyte HIF‐1 appears to be a more important mediator of metabolic outcomes, liver fibrosis, liver oxidative stress, and hepatic mitochondrial function than hypoxia in this model of NAFLD and superimposed IH. The impact of HIF‐1 may be hypoxia‐independent, or the effect of hypoxia may be mediated via non‐HIF‐1 pathways.Support or Funding InformationThis research was funded by grants from the American Academy of Sleep Medicine Foundation (177‐PA‐17), the American Thoracic Society Foundation (ATS‐2017‐19), the University of California San Diego (RS295R), and the National Institutes of Health (1K08HL143140).

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. 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-ER(T2)and PDGFb-iCre-ER(T2)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.

The use of tobacco cigarettes is known to impair muscle contractile function and decrease muscle mass. Smokers also show a slower recovery following overuse‐induced muscle injury, but the effects of cigarette smoke exposure on muscle repair are not known. We tested whether a short‐term cigarette smoke exposure would affect the regeneration of locomotor muscles following injury by lengthening contractions. Wild‐type C57Bl/6J mice were nose‐only exposed to the smoke of 10 tobacco research cigarettes (1R6F) a day (CS, n=12 mice), or air‐exposed (Air, n=10 mice), for 5 days per week, lasting 8 weeks. CS mice gained less weight than their Air counterparts over the 8‐week period (2.0 ± 0.5 g vs 3.0 ± 0.4 g, respectively). On the seventh week of the exposure, the mice underwent an in vivo lengthening contraction procedure (LCP) to injure the tibialis anterior (TA) and the extensor digitorum longus (EDL) muscles of the right hindlimb (LCP leg), while the left hindlimb was used as a contralateral control (Control leg). During the LCP, the peroneal nerve of the right hindlimb from each mouse was electrically stimulated to evoke contractions of the anterior crural muscles and the foot was actively moved to produce plantar flexion (150 contractions with 20 s intervals). One week after the LCP, mice were euthanized and the EDL and TA muscles were harvested from both legs to test ex‐vivo contractility and to perform histochemistry of cross‐sections of the muscles. Force development in EDL muscles from the LCP legs were significantly smaller than the control legs for both groups (7 ± 4 % and 14 ± 6 % decrease, for CS Air and CS groups; P<0.01 vs control leg). Air and CS groups did not show differences in force in EDL muscles from the control legs (473 ± 25 vs 469 ± 13 kPa, respectively), but in muscles from the LCP legs, force in the CS group (398 ± 24 kPa) was significantly smaller than the Air group (444 ± 24 kPa; P<0.05). These data suggest that tobacco smoking impairs muscle regeneration when muscles are injured by lengthening contractions.Support or Funding InformationTobacco‐Related Disease Research Program

Humans are unusual among primates in being capable of sustained long-distance running, a key phenotype that emerged in genus Homo about 2 million years ago (Mya). While biomechanical factors have been defined, underlying molecular changes in myofibers that allow repetitive contractions remains obscure. We recently demonstrated that ablating the CMP N-glycolylneuraminic acid hydroxylase (CMAH) gene in mice, (replicating what occurred 2-3 Mya ago during the emergence of genus Homo), significantly increases myofiber oxygen flux.In this experiment single myofibers were used to monitor in real-time mitochondrial activation during a fatigue protocol. This intact single myofiber system provides a measure of the O2 uptake and utilization required as the force and energy requirements change during a bout of repeated contractions to fatigue. Experiments were performed in precisely control extracellular O2 environments over a range of physiological levels that are observed in vivo during the rest (∼15.7 ± 6.1 mmHg in the rat) to contraction (10.6 ± 5.2 mmHg) transition. The contraction-induced changes in intracellular NAD(P)H autofluorescence is a dynamic response between NAD(P)H production and oxidation, mostly occurring in mitochondria. The observed decay of fluorescence is suggestive of mitochondrial O2 consumption (indicative of the dissipation in mitochondrial membrane potential). In wild-type myofibers mitochondrial activation is delayed from ∼ 18 seconds to ∼ 30 seconds as the extracellular PO2 is decreased from 40.5 mmHg to 10 mmHg. In near-anoxia, a slow accumulation of fluorescent signal was observed in Cmah-/- fibers at 2.7 mmHg suggests ongoing NAD(P)H oxidation in mitochondria. In contrast there was almost no fall in NAD(P)H levels in the wild type fibers, and this response suggests minimal O2 utilization. Thus, mitochondrial activation appears to be still ongoing under very limited mitochondrial O2 availability in Cmah-/- fibers.

Pulmonary arterial hypertension (PAH) is a devastating disease that is often misdiagnosed and has a high mortality rate. It is characterized by remodeling of the small resistance arteries in the lungs that leads to elevated right ventricular pressure (RVP) and cardiac failure. Interleukin‐33 (IL‐33) is a cytokine that functions as an alarmin to sense danger or damage to endothelial cells, signal proliferation and restore barrier integrity. However, uncontrolled, active forms of IL‐33 could initiate endothelial hyperproliferation, a putative first step in the remodeling process. We hypothesize that IL‐33 initiates the hyperproliferative response of pulmonary artery endothelial cells and contributes to the wall remodeling and increased RVP observed in PAH. To test this, we induced pulmonary hypertension of C57Bl/6J (wild‐type), IL‐33 receptor knock‐out (ST2KO) and the adaptor protein, MyD88 knock‐out (MyD88KO) mice by exposure to 10% O2 and weekly SU5416 injections (SuHx). Hypertension was assessed by measuring RVP and arterial wall thickness (ratio of wall cross‐sectional area/total vessel cross‐sectional area). Proliferating pulmonary endothelial (CD31+/BrdU+) cells were detected by flow cytometry. Processed IL‐33 forms in the lung were analyzed by western blot. RVPs increased in wild‐type mice in response to SuHx (C57Bl/6J male, RA/DMSO 27.0 ± 3.0 mmHg, SuHx 40.5 ± 6.3 mmHg, p < 0.01; female, RA/DMSO 25.8 ± 3.3 mmHg, SuHx 43.8 ± 7.4 mmHg, p < 0.01) and, this response was attenuated in ST2KO mice (ST2KO male, RA/DMSO 25.0 ± 4.3 mmHg, SuHx 32.8 ± 6.9 mmHg, p<0.01; female, RA/DMSO 25.3 ± 3.5 mmHg, SuHx 32.8 ± 4.9 mmHg, p<0.01) and completely blocked in MyD88KO mice (MyD88KO male, RA/DMSO 19 ± 2.9 mmHg, SuHx 19.7 ± 5.9 mmHg, NS; female, RA/DMSO 20.2 ± 2.5 mmHg, SuHx 24.5 ± 7.1 mmHg, NS). Similarly, wall thickness increased in wild‐type mice, but not ST2KO and MyD88KO mice, under SuHx conditions (C57Bl/6J Male, RA/DMSO 0.35 ± 0.07, SuHx 0.53 ± 0.12, p < 0.01; Female RA/DMSO 0.34 ± 0.05, SuHx 0.41 ± 0.1, p=0.4; ST2KO Male, RA/DMSO 0.37 ± 0.07, SuHx 0.38 ± 0.13, NS; Female RA/DMSO 0.31 ± 0.02, SuHx 0.34 ± 0.03, NS; MyD88KO Male, RA/DMSO 0.37 ± 0.05, SuHx 0.41 ± 0.10, NS; Female RA/DMSO 0.34 ± 0.07, SuHx 0.38 ± 0.03, NS). The percentage of proliferating endothelial cells was increased in wild‐type mice under SuHx conditions (C57Bl/6J Male, RA/DMSO 1.9 ± 0.4, SuHx 4.8 ± 2.6, p< 0.01; Female RA/DMSO 0.3 ± 0.05, SuHx 0.4 ± 0.1, p= 0.07). This endothelial proliferative response was absent in ST2KO and Myd88KO mice (ST2KO Male, RA/DMSO 0.35 ± 0.07, SuHx 0.53 ± 0.12, NS; Female RA/DMSO 0.34 ± 0.05, SuHx 0.41 ± 0.1, NS). Proliferating (Ki‐67+) cells were detected in the intima layer of small arteries. The C‐terminal and caspase cleaved form of IL‐33 is increased in wild‐type and ST2KO mice but not in MyD88KO mice under SuHx conditions. IL‐33 signaling through the ST2 receptor is important for endothelial cell proliferation in the SuHx model. This response is associated with increased levels of both the C‐terminal IL‐1 like cytokine domain and caspase cleaved fragment of IL‐33 in lung tissue.Support or Funding InformationDepartment of Veteran Affairs