bluuebunny/crossref_metadata_2025 huggingface dataset:
Akt/PI3K signaling sustains the acute alcoholic effects in isolated rat cardicytes
Authors: Umoh Nsini Alease , Walker Robin K. , Jeffress Miara , Cousins Valerie , AlRubaiee Mustafa , Haddad Georges E.
Published: June 2021

Cardiovascular diseases are the leading causes of increased mortality and morbidity rates. High alcohol consumption has been shown to negatively impact the heart function into having reduced contractility with cardiomyopathy. However, low alcohol consumption is shown to have beneficial effects on the heart. Prior studies in our lab have linked the Akt/PI3K pathway to cardiac dysfunction during eccentric hypertrophy. In this study, we investigate the role of Akt activity (western blot) that contributes to changes in cardiac contraction (Ionoptix), cell survival (Cell Viability Assay), and caspase activity (caspase 3/7 assay), associated with low (5mM) and high (100mM) alcohol consumption. Our results demonstrate that Akt overexpression increased cardiocyte survival during low alcohol exposure. However, the effects of Akt are diminished with high alcohol exposure. Low alcohol also decreased caspase activity and increased cellular and sarcomere contraction. Knockout of the Akt gene negated the above effects of Akt, and therefore decreased cell survival, and increased caspase activity. Our data suggests that Akt signaling plays an important role in supporting the beneficial effects of low alcohol which is diminished with high alcohol exposure having detrimental effects on cardiocyte function. This work was supported in part by grants NIH/NIAAA/1R15AAA019816–01A1, and 2G12 RR003048 RCMI/NCRR/NIH.
Treatment of tumor‐induced cardiovascular dysfunction with losartan, an angiotensin II receptor antagonist
Authors: Devine Raymond D , Stevens Sarah , Youtz Dane , Bicer Sabahattin , Clark Yvonne , Wold Loren E
Published: June 2021

Tumor burden elicits a multitude of pathological responses from multiple organ systems. Part of this pathological response is cachexia; generalized as host tissue wasting. Angiotensin II(Ang II) has been implicated in the activation of the proteasome. Losartan is a well‐known selective and competitive Ang II receptor antagonist, which is hypothesized to lessen the pathological burden. Raised levels of mRNA of biomarkers for ubiquitin pathway such as MuRF and Bnip3 are present in tumor bearing mice, implicating the role of the ubiqutin pathway. Degradation of myosin heavy chain (MHC) also occurs in the ventricles of the heart. The heart has been observed using echocardiography, which shows a decrease in left ventricle function. Ex vivo cardiomyocyte isolations of tumor burdened mice show deficits in function. Losartan treatment ameliorates the pathology state; it decreases ubiquitin markers coupled with reductions of Bnip3, MuRF, and IL‐6 lessening the effect of cardiac degradation and preserving function. In cardiomyocyte isolations losartan normalized the contractions from tumor bearing mice, this mechanism will need to be explored further. Losartan successfully showed an improvement in cardiac function and normalization of single cardiomyocyte, more experiments are planned in the future to further elucidate the mechanisms of cachexia and the action of losartan on them.
Genetic truncation of PLA2g6 leads to the impairment of endogenous store‐operated Ca2+ entry (SOCE): new molecular mechanism of STIM1 interaction with PLA2g6 and its critical role for endogenous SOCE
Authors: Bolotina Victoria M. , Zhou Qingde , Rymarczyk Grzegorz , Schafer Claudia , Kirber Michael T.
Published: June 2021

Ca2+‐independent phospholipase A2β (PLA2g6) is important for endogenous store‐operated Ca2+ entry (SOCE), but the molecular mechanism of its involvement is unknown. Here we report that genetic truncation of N terminus of PLA2g6 does not change its catalytic activity, but leads to a total loss of PLA2g6 activation by depletion of intracellular Ca2+ stores and ablation of PLA2g6‐ dependent SOCE in mouse embryonic fibroblasts (MEFs). Rescue experiments showed that plasma membrane‐associated (L), but not cytosolic (S) variant of PLA2g6 is required for endogenous SOCE. Co‐IP and pull‐down experiments identified N terminus of PLA2g6(L) to be critical for its molecular interaction with STIM1. Proximity ligation in situ assay revealed store depletion‐induced co‐localization of endogenous STIM1 with PLA2g6(L) in control MEFs, but not in MEFs in which N terminus of PLA2g6 was deleted. Thus, molecular interaction of STIM1 with N terminus of specific plasma membrane‐associated PLA2g6(L) presents an essential signaling step in the mechanism of endogenous SOCE activation. The loss of interaction between STIM1 and PLA2g6(L) results in impairment of endogenous SOCE and sustained depletion of intracellular Ca2+ stores.
Molecular Basis for Anabolic Resistance in Disuse Atrophy of Skeletal Muscle
Authors: Kelleher Andrew Ryan , Kimball Scot R , Dennis Michael D , Schilder Rudolf J , Jefferson Leonard S
Published: June 2021

The present study employed a rat handlimb immobilization model of disuse atrophy to assess responses of mTORC1 signaling and protein synthesis, both in the basal state and after a nutrient stimulus. In the basal state, mTORC1 signaling and protein synthesis were repressed within 24h in the soleus of an immobilized compared to a non‐immobilized hindlimb. These responses were accompanied by a concomitant induction in expression of the mTORC1 repressors REDD1/2. The nutrient stimulus produced an elevation of similar magnitude in mTORC1 signaling in both the immobilized and non‐immobilized muscle. In contrast, phosphorylation of S6K1 on T229 and T389 in response to the nutrient stimulus was severely blunted. This result indicated an immobilization‐induced defect in PDK1 activity, which is required for phosphorylation of T229 as a prerequisite for phosphorylation of T389 by mTORC1. In conclusion, the results show an immobilization‐induced attenuation of mTORC1 signaling mediated by REDD1/2 that is nonetheless responsive to a nutrient stimulus. More importantly, they suggest that phosphorylation of S6K1 on T389, which is required for full activation of the kinase, fails to respond appropriately to the stimulus. This lack of response is apparently due to a defect in PDK1 activity and would explain the phenomenon of “anabolic resistance” referred to in other models of disuse atrophy.

Supported by NIH grant DK‐15658.
Contributions of endothelial nitric oxide synthase, norepinephrine, and neuropeptide Y to local warming‐induced cutaneous vasodilation in humans
Authors: Carter Stephen Jordan , Hodges Gary James
Published: June 2021

Despite being somewhat counter‐intuitive, our previous studies have assigned a role for sympathetic neurotransmitters during vasodilation in the cutaneous vessels in response to increased local skin temperature. We examined whether norepinephrine (NE), neuropeptide Y (NPY), and endothelial nitric oxide synthase (eNOS) were involved in maintaining the cutaneous vasodilator response to increased local skin temperature. Participant forearms were fitted with microdialysis fibers, local skin heaters, and laser‐Doppler probes. Skin sites were heated to 42 °C and allowed to stabilize before drug infusion was initiated. Site 1 was perfused with yohimbine, propranolol, and BIBP3226 antagonising NE and NPY, while site 2 was perfused with L‐NAA inhibiting eNOS. The combination of sympathetic antagonists did not affect skin blood flow (P >; 0.05), yet L‐NAA perfusion resulted in a pronounced reduction (P < 0.05). These data indicate that sympathetic neurotransmitters do not play a role in the persistent vasodilator response to local skin warming.
Adrenergic agonists do not modify in vivo cholinergic eccrine sweating dose‐response relations
Authors: Wilson Thad E. , Jurovcik Andrew J. , Mann Sarah , Metzler‐Wilson Kristen , Simpson Chris
Published: June 2021

Muscarinic and α‐ and β‐adrenergic receptors have been identified on secretory clear cells of human eccrine sweat glands. Potential interactions of these receptor populations in an intact in vivo model are unclear. To probe for subadditive, additive, or synergistic multiple agonist relations, 15 healthy subjects had 6 intradermal microdialysis probes placed in dorsal forearm skin perfused with: 1 & 2) 6 log M doses of acetylcholine (ACh); 3 & 4) ACh doses plus 200 or 80 μM isoproterenol; 5) ACh doses plus 3.16 μM norepinephrine and 1 mM propranolol; 6) ACh doses plus 100 μM phenylephrine. Sweat rate was measured via capacitance hygrometry and dose causing ½ maximal response (ED50) was identified by logistic modeling. ACh alone produced dose‐response relations with ED50 of −1.2±0.2. The addition of a β‐adrenergic agonist did not significantly alter the ED50 (200 μM = −1.3±0.1 and 80 μM = −1.1±0.3). Neither the addition of an α1‐adrenergic agonist nor α1‐ and α2‐adrenergic agonists co‐perfused with a β‐adrenergic antagonist significantly affected ED50 (−1.2±0.1 and −1.4±0.1, respectively). Combined, these data indicate that functional doses of adrenergic agonists do not modify the in vivo cholinergic eccrine sweating dose‐response relations. These results do not provide evidence for multiple agonist combinations causing augmented sweating responses in vivo.

Funding: Osteopathic Heritage Foundation
Thermoregulatory adjustments induced by exercise in rats with low brain angiotensinogen
Authors: Leite Laura Hora Rios , Santiago Henrique Pereira , Rodrigues Quezia Teixeira , Campagnole‐Santos Maria José , Coimbra Cândido Celso
Published: June 2021

The effect of exercise on heat balance and performance were assessed in TGR(ASrAogen)‐680 rats (TG) with low glial angiotensinogen production. Sprague‐Dawley (SD) and TG rats (n=7/group) performed an incremental running test to determine their maximal performance. All animals run at 60% of maximal workload until fatigue while body (Tb) and tail (Ttail) temperatures were recorded. Workload (W), the rates of body heating (BHR) and heat storage (HSR) and Tb threshold for tail vasodilation (TTbV) were calculated. The speed that corresponded to 60% of maximal workload was lower in TG rats in comparison with SD animals (19.6 ± 0.5 m.min‐1, SD vs. 15.0 ± 0.3 m.min‐1, TG; p < 0.01). After 13 min of exercise, TG animals showed a greater rise in Tb (13 min: 38.2 ± 0.1 °C, SD vs. 38.5 ± 0.1 °C, TG; p < 0.01) due to the delay in tail skin vasodilation (7.7 ± 0.9 min, SD vs. 11.3 ± 0.7 min, TG; p < 0.01) as well as the higher TTbV (38.0 ± 0.1 °C, SD vs. 38.4 ± 0.1 °C, TG; p < 0.01). The BHR and the HSR of TG animals were, respectively, 39% and 41% higher (p<0.03), both of which closely related with their decreased time to fatigue (p<0.05). The data bring evidence that central angiotensin‐mediated pathways are involved in thermoregulatory heat loss during exercise since low brain angiotensinogen levels attenuate heat dissipation, leading to a faster exercise‐induced increase in Tb associated with decreased performance. Supported by CNPq, FAPEMIG and CAPES
Muscle Ring Finger‐1 (MuRF1) inhibits PPARα through mono‐ubiquitination of specific lysines adjacent to a novel nuclear export sequence (NES)
Authors: Willis Monte S , Liao Jie Ying
Published: June 2021

MuRF1 inhibits fatty acid oxidation while increasing glucose metabolism by its specific inhibition of PPARα activity. Surprisingly, MuRF1 does not degrade PPARα through poly‐ubiquitination. Alternatively, we identified that MuRF1 mono‐ubiquitinates PPARα. To identify the underlying mechanism of MuRF1's inhibition of PPARα, we increased MuRF1 expression and determined PPARα's nuclear localization by confocal microscopy. Increasing MuRF1 cleared nuclear PPARα in 80% of the cells (vs. <0.1% of controls), leading to the hypothesis that MuRF1 enhances PPARα nuclear export. Using the nuclear export inhibitor leptomycin B, we prevented MuRF1‐dependent PPARα nuclear export, suggesting a role of the nuclear export machinery. We next identified two potential non‐canonical nuclear export signals in PPARα and mutated their leucines to confirm their role in nuclear export. Only the PPARα NES1 mutant was enriched in its nuclear localization. We next mutated the three adjacent lysines to PPARα's NES1 to determine their role in MuRF1's ubiquitination and activity. Mutating any one of the three lysines around NES1 prevented MuRF1‐dependent nuclear export of PPARα. These data suggest that the increased MuRF1 that occurs in cardiac stress may drive the mono‐ubiquitination of PPARα, resulting in enhanced nuclear export and inhibition of PPARα activity.
The regulation of mitochondrial movement within muscle cells
Authors: Iqbal Sobia , Hood David A.
Published: June 2021

Mitochondria are dynamic organelles and their movement and morphology are controlled by their interaction with the cytoskeleton. Our purpose was to investigate regulatory events governing mitochondrial movement within muscle cells. Thus, we transfected C2C12 myoblasts with DsRED2‐Mito DNA to visualize mitochondria using realtime imaging. Destabilization of microtubules (MT) using nocodazole reduced organelle velocity and total path length traveled by 72–75%. The amount of time that mitochondria remained stationary increased ~2‐fold when MTs were destabilized. Downregulation of the MT motor protein, Kif5B by siRNA reduced organelle velocity by 33%. Mitochondrial movement was not affected by actin disruption. Thus, the distribution of mitochondria within muscle cells is dependent on a MT‐based transportation system. Using ionomycin to induce an increase in cytoplasmic calcium, we found that organelle velocity was suppressed by 48%, a situation which was reversible upon the chelation of calcium with EGTA. Mitochondrial velocity was also reduced by 22% when cytoplasmic calcium was elevated using thapsigargin, and it returned to basal levels upon the addition of BAPTA‐AM. By understanding mitochondrial movements we can elucidate the underlying basis for organelle interactions, leading to the formation of the mitochondrial reticulum and the distribution of energy in muscle cells. Supported by NSERC.
Cold acclimation increases the contribution of brown adipose tissue‐derived thermogenesis in adult humans
Authors: Blondin Denis P. , Labbé Sébastien M. , Turcotte Éric E. , Richard Denis , Carpentier André C. , Haman François
Published: June 2021

Recent studies examining brown adipose tissue (BAT) metabolism in adult humans have provided convincing evidence that it can significantly contribute to cold‐induced thermogenesis under acute mild‐cold exposure. Although, many mammalian models have demonstrated the adaptability of this tissue through chronic cold exposure, little is known about its plasticity in humans. Using electromyography combined with positron emission tomography with 11C‐acetate and 18F‐fluorodeoxyglucose, shivering intensity and BAT oxidative metabolism and glucose uptake prior to and following four weeks of cold acclimation were examined. Non‐acclimated men were exposed to 10°C, two hours daily for four weeks (5 days/week), using a liquid‐conditioned suit (LCS). Preliminary data suggests a 5‐fold increase in BAT oxidative metabolism (from 0.005±0.004to 0.025±0.007 sec−1, n=3) resulting in a ~40% decrease in shivering intensity (from 3.2±1.4 to 2.0±1.3 %MVC, n=3), through four weeks of cold‐acclimation, despite similar 1.8‐fold increases in cold‐induced energy expenditure (10.6±1.4 and 10.3±1.3 kJ·min−1, pre‐and postacclimation, respectively, n=3). Fractional and net glucose uptake in BAT increased 2.2‐fold (from 0.017±0.007 to 0.037±0.015 min−1 and from 81±35 to 183±82 nmol·g−1·min−1) following cold‐acclimation. Our preliminary results demonstrate an increased capacity of BAT‐derived thermogenesis during acute cold exposure after a 4‐week cold acclimation period in healthy men. This research was supported by the Natural Sciences and Engineering Research Council of Canada and the Canadian Diabetes Association (grant OG 3–10‐2970‐AC).
Comparison of hemodynamic responses to lower body negative pressure and hemorrhage in baboons
Authors: Hinojosa‐Laborde Carmen , Shade Robert , Muniz Gary , Bauer Cassondra , Convertino Victor
Published: June 2021

Lower body negative pressure (LBNP), an experimental model of hemorrhage (Hem), causes shifts in blood volume to the legs and elicits central hypovolemia. This study defined the levels of LBNP which equate to actual Hem. Male baboons (n=8) were sedated and instrumented for measurement of arterial pressure (AP), pulse pressure (PP), and central venous pressure (CVP). Stroke volume (SV) was calculated from the AP waveform. Blood was removed in four steps (7 min/step): 6.25%, 12.5%, 18.75%, and 25% of total blood volume. Shed blood was returned and the animals were allowed to recover. Four weeks after the Hem protocol, the same animals were subjected to four levels of LBNP which elicited equivalent changes in PP and/or CVP observed during Hem. Hemodynamic responses to Hem and matched LBNP levels are shown below (mean±SD).

Hem (% blood vol)
Delta CVP (mm Hg)
Delta PP (mm Hg)
Delta SV (% baseline)
Matched LBNP (mm Hg)

6.25
−2.4 ± 0.7
−3 ± 3
−6 ± 11
−22 ± 7

12.5
−3.6 ± 1.2
−10 ± 4
−26 ± 14
−40 ± 9

18.75
−4.1 ± 1.5
−16 ± 5
−41 ± 11
−53 ± 13

25
−4.7 ± 1.2
−20 ± 7
−51 ± 10
−72 ± 8

These results indicate that stepwise Hem elicited hemodynamic changes which could be mimicked with distinct levels of LBNP. We conclude that LBNP levels can be used as surrogates for actual blood loss. This study supports the validity of LBNP as a human model of central hypovolemia associated with Hem. Funding Source: U.S. Army MRMC Combat Casualty Care Research Program.
Changes in Body Composition and Aerobic Capacity after High Altitude Exposure in Elite Climbers
Authors: Carlson Alex Robert , Kasak Alexander , Issa Amine , Taylor Bryan , Summerfield Douglas , Miller Andrew , Fuglestad Andrew , Johnson Bruce
Published: June 2021

BACKGROUND

High altitude (HA) exposure influences appetite, protein synthesis and breakdown, and aerobic capacity. Thus, HA exposure typically causes weight (wt) loss, while the impact on aerobic capacity upon return to sea level is more variable.

OBJECTIVE

To examine the influence of hypobaric hypoxia on body composition and exercise capacity in elite HA climbers (n=4, 2 female) acclimating at ≥5,150 m over 2 mo. relative to trekkers (n=8, 0 female) acclimating at 5,150 m over 2 wks (Mt. Everest, Nepal). Each elite climber successfully summited Mt. Everest.

METHODS

Body composition (DEXA, skin folds, wt) and exercise capacity (treadmill) were assessed before and within 2 wks post HA.

RESULTS

HA exposure increased Hct and Hb, with peak values of 52±5% and 18±2 g/dl, and 50±3% and 17±1 g/dl for the climbers and trekkers, respectively. HA decreased lean and fat tissue in both the climbers (lean −4.4±2.3%, fat −8.2±4.0%) and trekkers (lean −1.8±1.6%, fat −5.8±6.1%) (all P≤0.06). No change was observed in skin folds for either group. Wt decreased in the climbers (−5±2%, P<0.03) and trekkers (−3±1%, P<0.01). There was no change in VO2max pre‐to post‐HA in either group (climbers 3.26 ± 0.37 vs. 3.27 ± 0.61 L/min; trekkers 3.60 ± 0.54 vs. 3.66 ± 0.43 L/min).

CONCLUSION

HA results in both lean and fat tissue loss in well acclimatized climbers and trekkers with little subsequent impact on aerobic capacity. Mayo Clinic & TNF.
Dysfunctional autophagy and mitochondrial bioenergetics in a model of type I diabetes
Authors: Mitchell Tanecia , Chacko Balu , Johnson Michelle , Koizumi Akio , Ramanadham Sasanka , Darley‐Usmar Victor
Published: June 2021

Maintenance of mitochondrial quality and function is essential for pancreatic β‐cell insulin release. During type I diabetes (TID) progression, increased mitochondrial reactive oxygen species generation contributes to mitochondrial and cellular damage. However, the exact mechanisms leading to β‐cell injury are not clearly defined. The purpose of this study was to investigate mitochondrial quality and the role of autophagy in TID. We hypothesized that impaired cellular bioenergetics, mitochondrial protein assembly, and autophagy contribute to β‐cell injury. To test this, we used pancreatic β‐cell lines generated from wild type Ins2+/+ (WT) and Akita+/Ins2 mice (a spontaneous ER stress‐induced model of TID). Mitochondrial oxygen consumption rates (OCR) were measured using Seahorse Biosciences XF24 technology, while microscopy, immunoblotting, and mass spectrometry were used to evaluate mitochondrial protein and autophagy. Our data reveal that Akita+/Ins2β‐cells have a significant decrease in OCR, significant up‐regulation of ATP synthase α‐subunit and MnSOD; and down‐regulation of complex III core 1 and citrate synthase compared to WT β‐cells. In addition, clearance of damaged mitochondria was impaired in Akita β‐cells. These findings suggest that dysfunctional mitochondrial bioenergetics and autophagy may play a role in TID β‐cell injury. This work was supported by NIH T32 DK007545.
Impact of mitochondria phospholipid hydroperoxide glutathione peroxidase (mPHGPx) overexpression on the type 1 diabetic heart
Authors: Lewis Sara E , Dabkowski Erinne R , Baseler Walter A , Shepherd Danielle L , Croston Tara L , Nichols Cody E , Hollander John M
Published: June 2021

Mitochondrial dysfunction is a major contributor to diabetic cardiomyopathy during type 1 diabetes mellitus (DM). In type 1 DM, interfibrillar mitochondria (IFM) display changes in reactive oxygen species production and oxidative damage. The goal of this study was to determine whether overexpression of mitochondria phospholipid hydroperoxide glutathione peroxidase (mPHGPx), an antioxidant enzyme, could attenuate cardiac and mitochondrial function associated with type 1 DM. MPHGPx transgenic mice and littermate controls were made diabetic and five weeks post diabetic onset, cardiac contractile function was measured. Ejection fraction, fractional shortening and cardiac output were significantly decreased in diabetic versus control hearts (P<0.05 for all three), with mPHGPx overexpression restoring function to control levels (p<0.05). Mitochondrial respiration rates (state 3 and 4) were significantly decreased in diabetic IFM versus control (p<0.05) and were restored with mPHGPx overexpression. Reactive oxygen species and oxidative stress levels were higher in diabetic IFM compared to control (p<0.05), which were attenuated with mPHGPx overexpression (p<0.05). In summary, overexpression of mPHGPx restored cardiac and mitochondrial function normally lost during a type 1 DM insult by preserving IFM function. (Support: NIH DP2DK083095, NIH T32HL090610, AHA 10PRE3420006, NIH S10 RR‐026378)
Reduced Mitochondrial Na+/H+ Exchanger 1 Protein Expression Contribute to the Functional Remodeling of the Hypertrophic Rat Heart Mitochondria
Authors: ALVAREZ BERNARDO V , Vargas Lorena A
Published: June 2021

Mitochondrial dysfunction underlies the causes of numerous cardiac diseases including decompensated cardiac hypertrophy and heart failure. Irreversible opening of the mitochondrial permeability transition pore (MPTP) with the concomitant mitochondrial swelling (MS) characterizes the mitochondrial disturbance. Recently, we revealed the expression of the mitochondrial NHE1 Na+/H+ exchanger (mtNHE1) and demonstrated that inhibition of mtNHE1 protects heart mitochondria from Ca2+‐induced MS and MPTP opening. Mitochondria were isolated from normal (NH) and hypertrophic rat hearts (HH), heart weight/body weight ratio 2.8±0.2, and 3.8±0.2, respectively (n=12). MS was measured in mitochondria as a decrease in the light scattering (LS) signal induced by addition of 200 μM CaCl2, and calculated as the difference between LS values before and after addition of Ca2+. The HH mitochondria showed a significant reduction in MS compared to the NH mitochondria, 53±7%, and 100±9%, respectively (n=5, P<0.05). mtNHE1 expression in HH and NH mitochondrial lysates was analyzed by SDS‐PAGE. Immunoblots revealed a reduced mtNHE1 expression in the HH compared to NH (~40% reduction, n=5). The decreased mtNHE1 protein expression which accompanied the increase in threshold to open the MPTP appears as the manifestation of an endogenous protective mechanism, participating in the functional remodeling of the HH mitochondria.
Substrate ‐dependent Action of Isoflurane on Electron Transport Chain Complexes
Authors: Agarwal Bhawana , Dash Ranjan K , Bosnjak Zeljko J , Stowe David F , Camara Amadou K.S.
Published: June 2021

The volatile anesthetic isoflurane (ISO) alters mitochondrial electron transport activity, which, in part, may underlie ISO‐induced cell protection. To unravel the mechanism of ISO effects on mitochondrial bioenergetics (ΔΨm, NADH and respiration), rat isolated cardiac mitochondria were energized with complex I (K+ ‐ pyruvate/malate; KPM) or complex II (K+ ‐succinate; KSuc) substrates ± sub‐maximal concentrations of ISO and the complex I and complex III blockers rotenone (ROT) and antimycin A (AA). Results showed that: (a) ISO decreased state 3 NADH oxidation and respiration, and increased the duration of state 3 ΔΨm with KPM; (b) ISO decreased state 3 respiration and the fall in ΔΨm, but did not decrease NADH oxidation with KSuc; (c) ISO increased state 3 ΔΨm duration but enhanced respiration with KSuc and ROT. These data indicate that: (1) ISO decreases the magnitude, while enhancing the duration, of state 3 NADH oxidation, ΔΨm and respiration, in part by inhibiting complex I activity with KPM; (2) ISO decreases KSuc supported production of NADH by attenuating complex I activity; (3) ISO increases state 3 ΔΨm duration but enhances respiration by attenuating complex III activity), with KSuc and ROT. Thus, isoflurane direct substrate‐dependent attenuation of complex I and complex III activities suggests a mitochondrial bioenergetic mechanism of cardioprotection during anesthetic exposure. (NIH Grants)
Cytoplasmic Hsp70 and Hsp90 regulate functionally distinct ER quality control checkpoints during thiazide‐sensitive NaCl cotransporter biogenesis
Authors: Donnelly Bridget F. , Needham Patrick G. , Snyder Avin C. , Roy Ankita , Khadem Shaheen , Brodsky Jeffrey L. , Subramanya Arohan R.
Published: June 2021

Mutations of the thiazide‐sensitive NaCl cotransporter (NCC) cause Gitelman syndrome, a recessive renal salt‐wasting disorder whose carrier state protects against essential hypertension. Given its complex topology, NCC is inefficiently processed and prone to ER‐associated degradation (ERAD). In Gitelman syndrome, misfolded mutants are targeted for ERAD more strongly, highlighting the importance of NCC quality control in human disease and hypertension resistance. Previously, we reported that the cytoplasmic chaperone Hsp70 stimulates NCC ERAD. Here, we identify Hsp90 as a chaperone that promotes NCC folding. In MDCK cells, disruption of the Hsp90 chaperone cycle accelerated NCC ERAD, and augmenting NCC transfer from Hsp70 to Hsp90 by coexpressing the organizer cochaperone HOP stabilized NCC turnover. In a yeast expression system, Hsp70/Ssa1 inactivation effectively stabilized NCC, while ablation of Hsp90 or HOP homologs only mildly increased NCC turnover, suggesting that Hsp70 selects NCC for ERAD prior to Hsp90 engagement. Consistent with these findings, adjusting the folding environment in MDCK and HEK cells via reduced temperature (25°C) enhanced NCC ER export, increased Hsp90‐NCC interaction, and diminished Hsp70 association. These observations indicate that Hsp70 and Hsp90 comprise distinct checkpoints that sequentially monitor the early stages of NCC biogenesis. Support: VA, AHA, NIH
Renal mechanisms driving K excretion after a K containing meal
Authors: Rengarajan Srinivas , Youn Jang H , Lee Donna H. , McDonough Alicia A
Published: June 2021

We aimed to determine the molecular mechanisms leading to renal K excretion after an acute meal induced K load by studying ion transporter regulation.

Method

Male Sprague Dawley rats (n=8) were fasted overnight with free access to water and then fed a meal with no K+ (0K) or 2% K+ (2K) for 3 hours in metabolic cages. Urine was collected and after anesthesia (ketamine‐xylazine), blood was sampled, kidneys removed and cortex and medulla homogenates were prepared. Transporter abundance and phosphorylation were assessed by quantitative immunoblots with specific antibodies.

Results

A single K rich meal increased plasma [K+] within the normal range in the 2K versus 0K group: 5.18±0.16mEq versus 4.01±0.12mEq (p<0.01), and sharply increased urine K+ in the 2K versus 0K group: 713±48 versus 55±15μmol (p<0.01). Urine volume tended to increase in the 2K versus 0K group: 3.63±0.16 versus 1.97±0.54ml/3 hr, but urine Na was unchanged. Cortical NCC, NCC‐P and SPAK‐P abundance were decreased by 24±4% (p<0.01), 63±4% (p<0.01) and 38±5% (p<0.01) respectively in 2K versus 0K rats. NHE3, ENaC and ROMK were unchanged by 2K diet feeding.

We postulate that the kaliuresis can be explained by a decrease in distal convoluted tubule (DCT) Na which would drive more Na to the cortical collecting duct (CCD) where Na reabsorption drives K secretion, analogous to the effect of an NCC inhibitor. Research support: NIH DK083785
Genetics of physical performance in U.S. Marines
Authors: Jensen Andrew , Congalton Robert , Niederberger Brenda , Jaworski Rebecca , Kelly Karen
Published: June 2021

This study evaluated genotypes of U.S. Marine Corps infantrymen on characteristics representative of physical performance. Whole blood was drawn from Marines (N=170; 19.9 ± 0.4 yrs, 81.2 ± 0.7 kg, 179.5 ± 0.8 cm) and DNA was extracted using the Gentra Puregene Blood Kit; genotyping was performed with TaqMan allelic discrimination technology. Results of the genotyping revealed that 3 of the 19 SNPs assessed were significantly associated with physical performance attributes. The C allele variant of the rs4149056 SNP was found to be associated with a higher body weight (CC/CT: n= 45, 84.2 ± 1.5; TT: n= 125, 80.1 ± 0.8; P= 0.02 kg). Muscle‐specific creatine kinase Ncol SNP, rs8111989, was associated with max number of pull‐ups performed; the TT genotype bested the C allele variants (CC/CT: n= 74, 13.0 ± 0.5; TT: n= 96, 14.4 ± 0.5; P= 0.05 max # pull‐ups). The interleukin‐6–174 SNP, rs1800795, was associated with 3‐mile run time; with the CC genotypes having lower run times (CC: n= 77, 20.78 ± 0.18; CG/GG: n= 93, 21.57 ± 0.17; P= 0.002 min). These data advocate a relationship between certain SNPs and physical performance in U.S. Marines; however, these data do not account for training and individual experiences that may influence performance. At this time, these results suggest that the genetic profiles of U.S. Marines are not necessarily indicative of physical performance, or more importantly combat performance.
An interactive quantitative temporal physiological model of glucose passage and absorption through the gastrointestinal tract and subsequent modulation of insulin and glucagon secretion in humans
Authors: Garan Steven A , Sun Shuzhang , Gurm Hashroop K , Chen Alexander , Govinthasamy Anensshiya , Efe Gizem , Chen Tiffany H. , Sachaphimukh Shivam , Plamthottam Sheba , Goitom Benyam , Banerjee Nabhojit , White Randall , Ahmed Behnaz , Sarcon Aida , Freitag Warren , van Loon Luc J C , Brooks George A
Published: June 2021

An interactive quantitative temporal model of glucose intake, absorption and utilization in humans has been assembled using the Center for Research and Education in Aging Language (CREAL). CREAL is a language that can be used to describe biological systems from a macro to a molecular scale. This model displays the concentration of glucose throughout the gastrointestinal (GI) tract in relation to time. The rate of absorption of glucose through the intestinal walls and subsequent modulation of insulin and glucagon secretion rates can be graphed in relation to time as well as the utilization and depletion of glucose by the various organ systems. The model can also be utilized as part of a more comprehensive system that can simulate the intake, absorption and utilization of other compounds, such as lipids, proteins, minerals and carbohydrates. The quantitative results from these large scale simulations can be used to validate results from higher resolution simulations such as those that take place at the cellular scale.
Modulation of BKCa channel activities by calcium sensing receptor in rat bronchopulmonary sensory neurons
Authors: Vysotskaya Zhanna , Gilbert Carolyn , Gu Qihai
Published: June 2021

Large‐conductance Ca2+‐activated potassium (BKCa) channels play an important role in shaping the neuronal excitability, but little is known about their expression and properties in bronchopulmonary sensory neurons. We have demonstrated recently the functional expression of extracellular calcium‐sensing receptors (CaSR) in rat vagal bronchopulmonary afferents. The aim of this study was to characterize the BKCa currents in isolated rat bronchopulmonary sensory neurons and to investigate the possible modulating effect of CaSR on these currents. Whole‐cell perforated patch‐clamp recordings were carried out and BKCa currents were isolated from total K+ currents with paxilline and iberiotoxin, two selective BKCa channel blockers. Our results showed that there was a large variability in BKCa current densities among individual neurons, and paxilline seems more potent in blocking these currents comparing to iberiotoxin. Pretreatment with CaSR antagonist NPS 2143 dramatically inhibited BKCa currents, whereas activation of CaSR with its agonist NPS R‐568 potentiated BKCa currents in these neurons. In summary, our data suggested that CaSR plays an integrative role in bronchopulmonary afferent signaling, at least partially through the regulation of BKCa channel activities. Funded by AHA grant and MUSM startup funds.
Microdialysis of a NK1R antagonist into the ventral medulla does not affect breathing frequency
Authors: Muere Clarissa , Miller Justin , Neumueller Suzanne , Olesiak Samantha , Mouradian Gary , Martino Paul , Pan Lawrence , Hodges Matthew , Forster Hubert
Published: June 2021

Previous studies in vivo have found an excitatory effect on breathing frequency (f) of microdialysis (MD) of Atropine into regions of the ventral medulla within the vicinity of the pre‐Bötzinger Complex (pre‐BötC) (FASEB J, 25: 1074.1, 2011). The increase in f was associated with increases in serotonin, substance P (SP), and glycine. These findings suggest that disrupting transmission in one neuromodulator system leads to compensatory changes in other neuromodulators. To determine the effect on breathing of blocking SP neurotransmission, the neurokinin‐1 receptor antagonist, Spantide (SPA), was unilaterally microdialyzed in one of two doses (133.5 uM or 267 uM) into sites in the ventral medulla in or near the preBötC of intact goats. Minute ventilation, f, and tidal volume were continuously monitored. Preliminary data show that SPA had minimal effects on these parameters at the lower dose, and suggest that this level of receptor blockade is not enough to stimulate compensatory mechanisms that appreciably affect breathing. However, one of two goats that received both doses of SPA had a dose‐dependent increase in breathing frequency with MD of SPA. This finding suggests that blockade of peptidergic neurotransmission at a currently unknown threshold level may also trigger compensatory changes in neurochemicals that stimulate breathing. Support: NIH HL25739 & Veterans Administration
The horizontal brainstem slice: a novel preparation to study cellular interactions of respiratory rhythmogenesis
Authors: Anderson Tatiana , Garcia Alfredo J. , Ramirez Jan‐Marino
Published: June 2021

The rhythmically active transverse slice containing the preBötzinger complex (preBötC; Smith et al. 1991) has led to a wealth of detailed cellular and systems level insights into respiratory rhythm generation. However, the preBötC is only one of several interacting respiratory networks that extend along the entire ventral medulla. Prior to this report, the only in vitro slice approach capable of preserving a larger portion of the ventral respiratory column (VRC) is the so called semi‐sagittal slice (Paton et al. 1994). This preparation, however, isolates only one side of a normally tightly coupled, bilaterally organized network. Here we report a novel horizontal slice preparation (P6–10 CD1 neonatal mice) that: (1) retains the entire extent of the medullary VRC; (2) preserves inspiratory rhythmogenesis from the preBötC throughout the rostral‐caudal axis; and (3) maintains bilateral connectivity within the VRC. Ongoing experiments characterize the discharge patterns of respiratory neurons along the in vitro VRC while examining the effects of gas exchange and neuromodulation on rhythmogenesis. Differences between the characteristics of the horizontal and the more conventional transverse preBötC slice provide important functional insights into the interactions between the preBötC and other neuronal networks distributed throughout the VRC. Supported by funding from the NIH.
BICARBONATE PERMEABILITY OF PORCINE BRONCHIAL EPITHELIUM
Authors: Ballard Stephen T. , Walker Jessica , Townsley Mary I.
Published: June 2021

CFTR mediates secretion of bicarbonate by airway epithelia. Since CFTR function is lost in cystic fibrosis (CF), there is speculation that bicarbonate concentration in mucous liquid is reduced, leading to increased mucus viscosity and reduced mucociliary clearance. Elevating bicarbonate in airway liquid is being considered for CF treatments. Thus, we sought to determine the permeability of airway epithelium to this anion. Pig bronchi were cannulated, filled with either isosmotic NaHCO3 solution (156 meq HCO3−/l, n=6) or isosmotic NaCl solution (0 meq HCO3−/l, n=5), and immersed in normal bicarbonate‐buffered Krebs solution (24 meq HCO3−/l) at 37ݸC. The bath solution contained acetazolamide to inhibit bicarbonate synthesis. Permeabilities were calculated from the mean change in luminal bicarbonate concentration over the first 7.5 min exposure. Bicarbonate permeabilities were 1.7 × 10−5 cm/sec and 8.6 × 10−5 cm/sec for NaHCO3‐filled and NaCl‐filled bronchi, respectively. We conclude that the permeability of bronchial airway epithelium to bicarbonate is relatively high, comparable to the permeabilities for other small solutes of similar molecular weights.
Voltage‐activated K+ channel activity and its effects on bronchoconstriction in rat airways
Authors: Randhawa Devjit Singh , Haick Jennifer M , Brueggemann Lioubov I , Byron Kenneth L
Published: June 2021

Asthma and other airway diseases are often classified by symptoms that include airway hyperresponsiveness (AHR), the excessive contraction of airway smooth muscle cells (ASMCs) in response to a variety of stimuli. The hypercontraction of ASMCs may result from membrane depolarization followed by activation of voltage‐sensitive Ca2+ channels (VSCCs). K+ channels have a high open probability in the absence of ASMC hypercontraction and therefore are thought to oppose membrane depolarization. Using image analysis techniques to measure airway constriction in rat precision cut lung slices (PCLS), we found that blocking voltage‐activated K+ channels using 2mM 4‐aminopyridine (4‐AP) resulted in only 6.3 % constriction of rat airways whereas 230nM methacholine (MeCh) gave rise to 41% constriction. Both responses were blocked by 10μM verapamil. We hypothesized that in the presence of MeCh, K+ channel blockers would cause amplified constriction of the airways when compared to MeCh alone. However, on average, we found that blocking voltage‐gated K+ channels using 2mM 4‐AP in the presence of 230nM MeCh caused significantly less constriction than with MeCh alone in rat PCLS. These findings suggest that Kv channel activity is essential for optimal MeCh‐induced bronchoconstriction. Further work is required to elucidate the roles of Kv channels in bronchoconstriction. (Research supported by Loyola University Chicago.)
Rotten eggs’ H2S acting on human airway smooth muscle mechanics
Authors: Fitzgerald Robert , Lee Danielle Y , Panettieri Reynold A , Wang Rui , An Steven S
Published: June 2021

The well‐known and reported mechanisms of action of small molecular gases on the vasculature point to a possible therapeutic agent for patients suffering from hypertension and/or stroke. Hydrogen sulphide (H2S) is a new gas in this class. H2S relaxes vascular smooth muscle via the KATP channel in several species. Trachealis of dog and rat express ATP‐sensitive potassium channels. Here, we explored the impact of two agents capable of generating H2S (Na2S and GYY4137) on human airway smooth muscle (ASM). ASM cells were prepared from human bronchi obtained from lungs unsuitable for transplantation, and dynamic changes in stiffness were measured as an indicator of contraction and relaxation of isolated human ASM cells, using Magnetic Twisting Cytometry (MTC). Acute exposure to both Na2S and GYY4137 decreased cell stiffness in a dose‐dependent fashion, with maximal relaxation at 10mM and 5mM, respectively. In addition, cells exposed to varying doses of GYY4137 for 24 hours exhibited dose‐dependent decreases in cell stiffness that were substantially greater than acute responses (21% relaxation at 300s vs. 50% relaxation at 24h). These findings demonstrate that smooth muscle in the airway react quite similarly to that in the vasculature to H2S and suggest, thereby, this class of small gas molecules can be exploited for treating constricted human airways. We are currently validating the effects of H2S on human lung slices, as well as exploring for the presence of H2S synthesizing enzymes (cystathionine β‐synthase and cystathionine γ‐lyase) in human ASM and their mechanistic role using animal models.

NIH Funding: HL107361, HL 050712–13
Extracellular matrix properties regulates the phenotypic and genetic features of human epidermal keratinocytes
Authors: KIM MINA , Shin Dong Wook , Nho Minsoo , Shin Jennifer Hyunjong
Published: June 2021

Keratinocytes, like most other cells, are sensitive to their physicochemical conditions of their microenvironment, being able to sense and respond to different rigidity as well as ECM (extracellular matrix) protein type. In this study, we investigated that how ECM protein type and rigidity regulate keratinocyte. To investigate the effects of ECM protein, normal human epidermal keratinocytes (NHEKs) were cultured on three different ECM (LN, FN, COL) coated glass for 13days. Keratinocytes undergo noticeable changes in the morphology and exhibit mesenchymal features typically observed in fibroblast on FN or COL coated glass. Real time PCR results showed that NHEKs on FN or COL coated glass showed mesenchymal features indicated by relative up‐and down‐regulation of vimentin and E‐cadherin, respectively. To investigate the applicability on therapeutic, NHEKs were cultured on three different ECM coated PDMS, biocompatible material. Keratinocyte on PDMS showed more mesenchymal features compared to ones cultured on glass. Phenotypic and genetic changes were observed in FN or COL coated PDMS cultured cells compared to LN coating. Considering the transition of keratinocyte from epithelial to mesenchymal feature occur during re‐epithelialization of wound healing, our results suggest that silicon including FN or COL plays a central role in wound repair and skin therapy.
Mass spectrometry‐based pseudo‐western blots
Authors: Liu Gary , Claxton J'Neka S. , Sandoval Pablo C. , Chou Chung‐Lin , Knepper Mark A.
Published: June 2021

Modern systems biology utilizes protein mass spectrometry (MS) to quantify thousands of proteins in a given tissue. The alternative to MS, western blotting, is low‐throughput, but gives quantitative information as a function of protein molecular weight, providing critical information about protein modifications. Here, we describe an approach that combines the advantages of both: “pseudo‐western blots” (pWBs). Protein extracts are separated by standard SDS‐PAGE, and each lane is sliced into 40 successive blocks followed by in‐gel trypsin digestion. Individual samples are analyzed by label‐free quantitative LC‐MS/MS (Velos‐Orbitrap). The resulting data were then translated into a western blot‐like format (abundance vs. slice number) using a heat map generator. To establish proof‐of‐principle, we analyzed kidney inner medullas from rats subjected to high‐ or low‐water intake for 3 days. The data were assembled into hundreds of pWBs. pWBs of the vasopressin‐regulated water channel aquaporin‐2 (Aqp2) and a urea channel (Slc14a2) matched corresponding antibody‐based western blots and reproduced changes. Our results show the feasibility of the pWB approach for systems biology applications. (G.L. supported by NIBIB Biomedical Engineering Student Internship Program, 2012)
SDF‐1 alpha Nanoglycan Complexes Exhibit Exended Retention Time and Beneficial Effect in Pulmonary Hypertension
Authors: Bader Andrew R. , Yin Tao , Kao Derrick , Hou Tim K. , Qian Ray , Kohane Daniel S. , Handy Diane E. , Loscalzo Joseph , Zhang Ying‐Yi
Published: June 2021

SDF‐1α has been known to be an important stem cell homing factor, and exhibits pro‐angiogenic activity in vivo. Exogenously delivered SDF‐1α, however, has limited retention time in tissue, which is thought to be related to protease degradation or rapid diffusion into the circulation. In the present study, we formulated SDF‐1α nanoglycan complexes by mixing SDF‐1α with dextran sulfate and chitosan. The resulting complex particles showed diameters of 600–800 nm and a zeta potential of ~−40mV. In vitro release of SDF‐1α from the nanoglycan particles was extremely slow, 1–2% after 7 days at 37°C. In cell culture studies, the nanoglycan‐incorporated SDF‐1α (SDF‐1α NP) showed the same chemotactic activity as that of free SDF‐1α. SDF‐1α NP also activated CXCR4‐mediated phosphorylation of Akt and ERK similarly as that of free of SDF‐1α. Delivery of SDF‐1α to rat lungs by intratracheal aerosolization revealed that the retention time of SDF‐1α NP in the lung was significantly longer than that of free SDF‐1α (half‐life 2.8 hr vs 36 hr). Aerosolization of SDF‐ 1α NP, but not free SDF‐1α, was found to attenuate monocrotaline‐induced pulmonary hypertension in rats. This study demonstrated that functional SDF‐1α NPs have extended retention time in the lung and is beneficial in the treatment of pulmonary hypertension in rats. Supported by NIH grant HL107192.
Klotho Regulation of TRPC Channels Involving Human Breast Cancer Cell Migration
Authors: Kong In Deok , Kim Ji‐Hee , Hwang Kyuhee , Jeong Yangsik , Cha Seung‐Kuy
Published: June 2021

Klotho is a mammalian aging‐suppression gene and function as a suppressor of cancer. Here, we investigated the molecular mechanism of Klotho regulation of TRPC channels involving human breast cancer cell migration. Serum‐induced activation of TRPC and cell migration was specifically blocked by TRPC channel inhibitor, SKF96365, but not by voltage‐gated Ca2+ channel inhibitor, nimodipine. Treatment of purified Klotho protein inhibited TRPC6 channel activity. Interestingly, serum treatment increased the surface expression of TRPC6 channel. Klotho suppressed cell surface abundance of TRPC6 channels, whereas did not affect gating and activation kinetics of the channel. In addition, treatment of PI3K inhibitor (Wortmannin) or Erk1/2 inhibitor (U0126) blocked the serum‐induced augmentation of TRPC6. Suppression of vesicular trafficking by treatment of brefeldin A or tetanus toxin significantly decreased TRPC6 activity and Klotho regulation.

Taken together, our data suggest that Klotho decreases surface expression of TRPC6 probably by reducing serum growth factor‐induced secretory pathway of the channel. Klotho may function as suppressor for human breast cancer cell migration caused by upregulation of TRPC6.

[This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2010–0024789)]