Citations

Acclimation to high altitude was characterized in nine non‐athletic college aged students aged 19 to 24 during a trek in the Himalayas. A three minute step test (40.6 cm step and a 24 step/minute rate) was performed at 1654 (am, day 1), 3696 (pm, day 1), 3865 (day 8), 3688 (day 10), 4115 (day 13) and again at 3688 meters (day 14). Resting heart rate and oxygen saturation (SaO 2 ) was measured prior to the step test and 20 seconds after exercise cessation (recovery). The heart rate difference (percent change) between pre‐ and post‐exercise cessation was used to measure hypoxic stress at altitude and significant acclimation could not be documented until day 14 (3688 m). The percent difference between pre‐ and post‐step test SaO 2 at 1654 m was significantly different from tests performed at 3696, 3865, 3688, and 4115 m. However, at day 14 (3688 m) the percent change in SaO 2 was no longer significantly different from the 1654 m value at day 1. Pre‐trip response to acute hypoxia (12.5 % O 2 ) was examined by performing the same step test at 402 m, with inconclusive results. This study suggests that 14 days are required for non‐athletic students to achieve acclimation to a step test.

Rewarming shock following accidental hypothermia is a potentially fatal scenario involving cardiac dysfunction. Previously our lab demonstrated that rewarming shock results in a decrease in Ca 2+ sensitivity of contraction in cardiac muscle due to an increase in cTnI phosphorylation. In this study, we hypothesized that rewarming shock initiates a cascade of intracellular events including reactive oxygen species (ROS) formation, endoplasmic reticulum (ER) stress, and mitochondrial disruption. Isolated cardiomyocytes from Sprague‐Dawley rats were exposed to a hypothermia/rewarming protocol (cooled for 1 h, held at 17 o C for 30 min, rewarmed for 1 h) that results in contractile dysfunction indicated by an increase in cytosolic Ca 2+ and reduced cell shortening measured using an IonOptix system. Rewarming shock increased ROS levels measured by an HPLC‐based dihydroethidium assay and by MitoSox fluorescence. Hypothermia/rewarming was also associated with ER stress indicated by increased expression of ER stress markers (Grp78, PERK, and CHOP) measured by Western blot. Finally, rewarming shock was associated with mitochondrial disruption evidenced by mitochondrial membrane depolarization (decreased in TMRM fluorescence) and by an increase in cytosolic cytochrome c (measured by Western blot). Our results support our overall hypothesis and suggest that these ER and mitochondrial intracellular events may underlie myocardial contractile dysfunction following hypothermia/rewarming. Supported by NIH Grant 5T32HL105355‐02 (GCS)

A transient elevation of cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) triggers a force response in excitation‐contraction coupling. The Ca 2+ sensitivity of muscle reflects the relationship between [Ca 2+ ] cyt and force, and in skeletal and cardiac muscle is often defined as the [Ca 2+ ] cyt at which 50% force is generated (ED50). In smooth muscle, Ca 2+ sensitivity has been assessed as the ratio of peak force to peak cytosolic [Ca 2+ ] cyt . However, this conventional measurement overlooks the dynamic nature of Ca 2+ regulation of force generation. In the present study, in order to provide a dynamic index of Ca 2+ sensitivity in airway smooth muscle (ASM), we simultaneously measured [Ca 2+ ] cyt and force and determined their temporal dependence using a phase loop plot. We examined the phase loop plot assessment of Ca 2+ sensitivity in ASM under the condition in which the conditional assessment indicated an increase ( Calyculin A treatment) in Ca 2+ sensitivity . Intact ASM strips were isolated from porcine tracheas obtained from local abattoir. ASM strips were loaded with Fura‐2 AM (Ca 2+ fluorescent indicator) and stimulated using 1 mM ACh. Isometric force and [Ca 2+ ] cyt responses were simultaneously measured using a Guth Muscle Research System. ASM strips were exposed to Calyculin A (phosphatase inhibitor; 100 nM for 10 min), which was previously reported to alter static Ca 2+ sensitivity. Phase loop plots were calculated from simultaneous [Ca 2+ ] cyt and force responses. The extent of rMLC phosphorylation was determined at different time periods after ACh stimulation using Phos‐tag SDS‐PAGE (SuperSep Phos‐tag; Wako Pure Chemical Industries, Ltd.) and Western blotting. Calyculin A increased ACh‐induced isometric force in ASM strips, but it had no effect on the amplitude of evoked [Ca 2+ ] cyt transients. These results suggested that Calyculin A increased static Ca 2+ sensitivity. However, the phase loop plots showed no significant shift in dynamic Ca 2+ sensitivity with Calyculin A. These results indicate that the phase loop plot provides a valuable tool to assess dynamic Ca 2+ sensitivity of isometric force generation in ASM, which better reflects the complex process of excitation‐contraction coupling in smooth muscle. These results further indicate that dynamic Ca 2+ sensitivity of force generation in ASM is independent of rMLC phosphorylation. Support or Funding Information Supported by NIH grant HL126451 (GCS) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Ca 2+ sensitivity plays a critical role in regulating cardiac contraction, and pathophysiological changes in Ca 2+ sensitivity of cardiac contraction may significantly impact cardiac function. For example, oxidative stress causes myocardial dysfunction, which is due in part to a decrease in Ca 2+ sensitivity. Oxidative stress (e.g., elevated H 2 O 2 ) underlies, at least in part, various conditions of cardiac dysfunction including ischemia‐reperfusion injury, heart failure, hypertension, and atherosclerosis. In cardiac muscle, Ca 2+ sensitivity can be dynamically assessed using a phase loop plot of concurrently measured cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) and sarcomere length shortening (Spurgeon et al J Physiol 447:83–102, 1992; Han et al Am J Physiol 298:890–897, 2010; Schaible et al Am J Physiol 310:1533–40, 2016). We hypothesized that oxidative stress reversibly reduces Ca 2+ sensitivity of cardiomyocytes within minutes. Cardiomyocytes were isolated from adult rat hearts (Sprague Dawley) using a modified Langendorff system. Cardiomyocytes were loaded with Fura‐2 AM (Ca 2+ fluorescent indicator; 1 mM) and electrically stimulated every 2 s. [Ca 2+ ] cyt and sarcomere shortening responses to stimulation were simultaneously measured using an IonOptix System. After stabilization for 30 min at 35°C, cardiomyocytes were exposed to PSS for 10 min, then to H 2 O 2 (200 μM) for 10 min and finally to either PSS washout or DTT (1 mM)‐induced reduction for 10 min. H 2 O 2 ‐induced oxidative stress increased basal [Ca 2+ ] cyt and the amplitude of the evoked [Ca 2+ ] cyt response. H 2 O 2 ‐induced oxidative stress also reduced the extent of sarcomere shortening and slowed the kinetics of shortening/relaxation. As a result, H 2 O 2 ‐induced oxidative stress reduced Ca 2+ sensitivity reflected by a rightward shift in the phase loop plots. Interestingly, with both PSS washout and DTT‐induced reduction Ca 2+ sensitivity was further reduced. These results support the hypothesis that H 2 O 2 ‐induced oxidative stress reduces Ca 2+ sensitivity of cardiomyocytes, but this effect was not immediately reversed, but indeed worsened by PSS washout or by DTT‐induced reduction. Together, these results provide insight into the dynamic effects of oxidative stress on Ca 2+ sensitivity of cardiomyocytes that may be reflected under pathological conditions. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Transforming growth factor beta (TGF‐β) plays a significant role in diverse biologic processes, including the regulation of cell growth, survival, differentiation, development, inflammation, immunity, hematopoiesis, and tissue remodeling. TGF‐β is essential for wound healing, stimulates matrix molecule deposition, and has been implicated in the pathogenesis of a variety of fibrotic disorders, including idiopathic pulmonary fibrosis, scleroderma, radiation‐induced pulmonary fibrosis, and asthma. Furthermore, activation of profibrotic TGF‐β signaling has a central role in glucose‐induced cell hypertrophy, induces glucose transporter 1, and regulates mitochondrial energetics. However, the mechanisms by which TGF‐β induces specific metabolic alterations in the fibrogenic process have not been fully defined. In this study, we demonstrate that TGF‐β stimulates type I TGF β receptor (TβRI) translocation to the mitochondria and induction of hexokinase 2 (HK2) via both Smad‐dependent and –independent pathways. Also, we showed that TbRI association with HK2 and the mitochondrial voltage‐dependent ion channel (VDAC). These findings suggest that TβRI plays a novel role in controlling mitochondrial function and regulation of glucose metabolism. Support or Funding Information This work was supported by Public Health Service grants GM‐055816 and GM‐054200 from the National Institutes of General Medical Sciences. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Recovery from accidental hypothermia followed by rewarming (H/R) is often unsuccessful due to heart failure. Although the causes for H/R‐induced heart failure remain elusive, studies at the cellular level suggest alterations in both cytoplasmic and mitochondrial Ca 2+ regulation. We hypothesize that H/R is associated with mitochondrial Ca 2+ overload, which leads to: 1) excessive reactive oxygen species (ROS) generation, 2) mitochondrial membrane depolarization, and 3) mitochondrial cytochrome c release. To test this hypothesis, isolated cardiomyocytes from adult male Sprague‐Dawley rats were subjected to an in vitro H/R protocol (1 h cooling to 15 o C held for 30 min followed by 1 h rewarming to 37 o C). Cytoplasmic (CM‐H 2 DCFDA fluorescence) and mitochondrial (MitoSOX fluorescence) ROS generation increased during cooling and remained elevated throughout the H/R protocol. Mitochondrial membrane potential (TMRM fluorescence) depolarized during rewarming suggesting mitochondrial permeability transition pore opening. After rewarming, cytochrome c content (Western blot) in cytoplasmic to mitochondrial fractions increased indicating release. These results suggest that Ca 2+ overload associated with H/R induces ROS generation leading to mitochondrial disruption. Grant Funding Source : Supported by NIH Grant 5T32HL105355‐02 (GCS)

Niclosamide is a salicylanilide derivative used as antihelminthic agent. Recently, Nic has been screened out as an autophagy modulator by us and other groups and was identified to be a structurally distinct mTORC1 signaling inhibitor. In this work, we measured the long‐lived protein degradation activity and found Niclosamide could inhibit the autophagic process. To elucidate the inhibition of Niclosamide on autophagy, we further tested the lysosomal activity using DQ‐BSA assay and measured protease activity such as Cathepsin B and Cathepsin D with fluorescent substrates. The lower degradation of DQ‐BSA and decreased protease activity after Niclosamide treatments indicated the impaired lysosome function. In addition, both LC‐II and P62 proteins accumulated significantly when treated by Niclosamide suggesting the late stage of autophagy was blocked. The increased pH of lysosome and release of Cathepsins from lysosome to cytosol demonstrated that Niclosamide might affect lysosome resulted from lysosomal membrane permeabilization. Collectively, we firstly disclosed Niclosamide not only inhibited mTORC1 activity but also, as other well‐known lysosome inhibitors, strongly blocked autophagy via lysosomal dysfunction. Grant Funding Source : Supported by Guandong Provincial Natural Science Foundation, China (S2013010015876)