bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lack of an effect of collecting duct‐derived adenylyl cyclase 3 or 4 knockout on renal sodium and water excretion and arterial pressure (860.2)
Authors: Kittikulsuth Wararat , Stuart Deborah , Mironova Elena , Stockand James , Kohan Donald 
Published: June 2021

Adenylyl cyclase (AC)‐stimulated cAMP is a key mediator of collecting duct (CD) Na and water transport. AC isoforms 3, 4 and 6 are expressed in the CD; while AC6 is involved in modulating CD Na and water transport, the roles of AC3 and AC4 in this regard are unknown. Therefore, we generated mice with loxP‐flanked critical exons in AC3 and AC4 genes and bred with mice expressing the AQP‐2/Cre transgene to yield CD principal cell (PC)‐specific knockout (KO) of AC3 or AC4. Isolated inner medullary CD (IMCD) showed 100% genomic target gene recombination in both KO lines, while renal papillary AC3 or AC4 mRNA were significantly reduced in CD AC3 or AC4 KO mice, respectively. CD AC3 or AC4 KO had no effect on AVP‐stimulated cAMP generation in isolated IM, although AngII‐stimulated cAMP was reduced in IM from CD AC3 KO mice. CD AC3 or AC4 KO did not alter water intake, urine volume, urine osmolality or plasma AVP during baseline or water restriction as compared to control mice. Na intake, urinary Na excretion, plasma renin concentration and blood pressure on a normal or high Na diet were not affected by CD AC3 or AC4 KO. Further, CD AC3 KO did not alter baseline or AVP‐stimulated ENaC activity as assessed by patch clamp of split‐open isolated cortical CD. In summary, these data suggest that AC3 and AC4 do not play a key role in physiologic regulation of CD Na and water handling.Grant Funding Source: a Merit Review from the Department of Veterans Affairs and by NIH R01 DK097007
Dehydration induced reduction in body fat content (860.21)
Authors: McKinley Michael , Mathai Michael 
Published: June 2021

The aim of this study was to determine the feasibility of body fat being metabolized by dehydrated rats thereby providing an endogenous source of body water to partially ameliorate dehydration. We measured total body fat mass (TBF) and total body water (TBW) using magnetic resonance relaxation analysis with an EchoMRI 100/900 Body Composition Analyser (Echo Medical Instruments, Singapore) before and after depriving rats (n = 6) of water for 24 h. When rats fed a normal pelleted lab diet (Na content of 0.5%) were water deprived, body mass (BM) was reduced from 400 ± 29 to 368 ± 22 g (mean ± SEM) and TBW from 264 ± 15 to 242 ± 15 g, while TBF fell from 29.6 ± 4.3 to 24.7 ± 4.1 g (p < 0.01). Food intake fell from 32.7 ± 1.2 to 20.9 ± 0.7 g/24h. Following rehydration, BM (405 ± 23 g) and TBW (268 ± 14.3 g) had returned to pre‐dehydrational levels by 24 h, whereas, TBF (28.1 ± 4.5 g) was still reduced in 5 of 6 rats at this time, but had recovered (29.1 ± 5.0 g) after 48 h of rehydration. When water replete rats were restricted to the same low intake of food for 24 h, the reduction of TBF (1.1 ± 0.5 g) during 24 h was significantly less than in dehydrated rats (4.9 ± 0.9 g). We also deprived rats (n = 4) of water for 24 h that were fed a diet of low NaCl content (0.02%). No reduction in TBF occurred (37.5 ± 8.2 pre‐dehydration versus 37.6 ± 8.6 g post‐dehydration), whereas TBW fell from 304 ± 8 to 285 ± 8 g. These data show that TBF is metabolized in dehydrated rats, but the effect is dependent on sodium status. They show also that it is feasible that body fat may be metabolized to water in dehydrated rats.Grant Funding Source: NHMRC of Australia
HDAC1 is a novel regulator of nitric oxide synthase 3 in endothelial cells (860.3)
Authors: Hyndman Kelly , Ho Dao , Sega Martiana , Pollock Jennifer 
Published: June 2021

Recent studies have identified that acetylation of lysines on NOS3 regulates NO production; however, there is little information regarding deacetylation of NOS3. Histone deacetylases (HDACs) remove acetyl groups from histone and non‐histone proteins. Recently it was reported that HDAC3 antagonizes aspirin‐induced NOS3 acetylation and reduces NO production in HUVECs. The role of other HDAC isoforms in mediating NOS3 deacetylation and/or NO production has not been determined. We tested the hypothesis that HDAC1 interacts with NOS3 leading to a decrease in lysine acetylation and NO production. Bovine Aortic Endothelial Cells (BAECs) were transfected with empty vector (N = 6) or human HDAC1 (N = 6) and lysine acetylation and NO production determined. In BAECs, we found that HDAC1 and NOS3 interact, and cells over‐expressing (OE) HDAC1 expressed reduced NOS3 acetylation (vector control = 1.0 ± 0.04, OE HDAC1 = 0.6 ± 0.04 A.U.; P < 0.001). We observed no differences in NOS3 phosphorylation at T495, S633, or S1177. Basal nitrite production was also reduced in the OE HDAC1 cells (356 ± 15 pmol/mg prt/h vs 223 ± 35 pmol/mg prt/h; P < 0.05). Stimulation of BAECs by 100 nM endothelin‐1 resulted in blunted nitrite production in OE HDAC1 cells (607± 115 pmol/mg prt/h vs 321 ± 61 pmol/mg prt/h; P < 0.05). In conclusion, HDAC1 is a novel inhibitor of NOS3 acetylation and NO production.Grant Funding Source: Supported by NIH to JSP
Role of the H,K‐ATPase type 2 in the regulation of blood pressure during K+ depletion (860.8)
Authors: Lamouroux Christine , Modyanov Nikolai , Crambert Gilles 
Published: June 2021

Alteration of blood pressure (BP) level is one of the side effects associated to K+‐depletion. However, the mechanisms linking K+ balance to BP remain unsettled and debated. Adaptation to K+‐depletion requires ability to retain K+. In kidney and colon, this is handled by the stimulation of H,K‐ATPase type 2 (HKA2) and its absence leads to severe K+‐depletion under low‐K+ diet condition. Our aim is to investigate whether HKA2 may contribute to determination of BP level under low‐K+ diet. Wild‐type mice (WT) displayed a decrease of BP after 10 days of low‐K+ diet (from 113 to 103 mm Hg). In HKA2‐null mice, the decrease of BP (109 to 100 mm Hg) occurred much earlier (day 2‐3). At early stage of K+‐depletion, plasma volume remained constant in WT but decreased by 20% in HKA2‐null mice, indicating that decrease of BP is linked to a problem of volemia. However, this decrease of BP and volemia are not related to a leak of Na+ and fluid by the kidney. On the contrary, this hypovolemic state stimulates urinary Na+ retention through thiazide‐ and amiloride‐sensitive Na+ transport. Measurements of tissue Na+ contents suggest that the origin of the hypovolemia could be a transfer of fluid from the extracellular space toward the intracellular compartment. Presence of HKA2 is therefore required to limit the decrease of plasma K+ value and to protect against development of hypotension.Grant Funding Source: Supported by the Fondation du Rein
Early post‐natal ventricle resection is accompained by neoformed cardiomyocytes and maintenance of cardiac function under stress in adult rats (862.1)
Authors: Zogbi Camila , de Carvalho Ana Elisa , Nakamuta Juliana , Caceres Viviane , Rochitte Carlos , Krieger Jose 
Published: June 2021

One‐day‐old mice hold a brief and robust capacity of heart regeneration after apex resection. We tested whether the rat displays comparable response and its impact in adult cardiac overall performance. Apical resection in rats at postnatal day 1 (P1) and 7 (P7) rendered comparable loss of cardiac tissue (16 vs.18%) estimated by magnetic resonance imaging. P1 was associated with robust cardiac regeneration at 21 and 60 days while in P7 rats there was essentially scar tissue replacement. Cardiac tissue perfusion in the neoformed region decreased in both P1 and P7 groups at 21 and 60 days using SPECT scanning. General cardiac function at 60 days was comparable among the groups, but under hemodynamic stress the degree of change on stroke work decreased significantly in P7 (sham: 18.4±4.0; P1: 47.0±17,8; P7: ‐0.5±7.5; P<0.05). Furthermore, P7 displayed increased chamber stiffness and interstitial collagen (P1: 6.9±0.6; P7: 11.4±0.6; P<0.05). Taken together, we provide evidence that adult P1 rats undergo robust neoformation of cardiomyocytes enabling preservation of overall cardiac function under hemodynamic stress despite decreased tissue perfusion. In contrast, P7 rats display mainly scar tissue replacement and compromised overall cardiac function.Grant Funding Source: FAPESP/Capes
Dysfunctional calcium homeostasis in aged mice primary tenocytes ő a potential functional link to tendon disorders (863.10)
Authors: Vallejo Julian , Spence Madoka , Brotto Leticia , Brotto Marco , Abreu Eduardo 
Published: June 2021

Musculoskeletal diseases (MSKs) afflict more than 1.7 billion people worldwide. In the US, tendon disorders account for a surprising 30‐50% of all sports‐related injuries and close to half of all occupational illnesses. These diseases are further complicated by aging. Tendon disorders are difficult to treat in part because of our poor understanding of tendon physiology and pathophysiology at the cellular and molecular levels. Historically, more attention has been given to other cells of the musculoskeletal unit (bone‐tendon‐muscle) than to tendon cells (i.e., tenocytes), maybe because we lack good functional tests for tenocytes. Calcium plays an important role in tendon physiology and its response to mechanical load, and as a result calcium studies can be a useful tool to evaluate tenocytes. We compared the resting levels of calcium as well as the responses to Ionomycin in young and aged mouse primary tenocytes and found magnitude and the time to the maximal response were reduced in aged tenocytes. We are now conducting systematic studies to dissect the potential mechanisms for these differences. We postulate that dysfunctional calcium homeostasis might be a critical functional link between tenocytes and the organismal manifestation of tendon and MSKs.Grant Funding Source: Supported by Dale & Dorothy Thompson Endowment Fund (MB)
Diaphragm muscle sarcopenia and neuromuscular junction morphology (863.6)
Authors: Greising Sarah , Stowe Jessica , Mantilla Carlos , Sieck Gary 
Published: June 2021

We recently determined the presence of sarcopenia in the diaphragm muscle (DIAm), specifically an age‐related reduction in force and muscle fiber cross‐sectional area, the latter being more pronounced at type IIx and/or IIb DIAm fibers. In limb muscles, age‐related changes at neuromuscular junctions (NMJs) include retraction of presynaptic terminals, disaggregation and fragmentation of the motor end‐plate. However, it is presently not clear whether aging impacts the morphology of DIAm NMJs. Adult male mice (n=14) at 6 and 24 months of age, representing survival rates of 100 and 75%, respectively, were used to examine DIAm NMJ structure, pre‐ and post‐synaptic morphology and overlap as measured in 3D by confocal microscopy (643 NMJs). No age related differences were evident in 2D planar area (mean: 328 µm2) or complexity (i.e., fragmentation) of motor end‐plates. Pre‐ and post‐synaptic volumes were also similar across age groups. Evidence of partial or full denervation was present in ~6% of NMJs in both age groups. Thus, these results indicate that by 24 months of age, DIAm sarcopenia is not associated with evidence of ongoing denervation. Examination of earlier age groups will be necessary to rule out the contribution of NMJ denervation to DIAm sarcopenia given the loss of type IIx and/or IIb fibers by 24 months of age.Grant Funding Source: Supported by R01‐AG044615, T32‐HL105355, and the Mayo Clinic.
Striatin: a novel regulator of cardiomyocyte calcium homeostasis and contraction (864.3)
Authors: Nader Moni , Khalil Bariaa , Kvietys Peter , Bakheet Dana , Dzimiri Nduna 
Published: June 2021

Although cardiac arrhythmias are still a major health threat for human, there is paucity of information on the molecular mechanisms involved in this pathology. Striatin (STRN) is a multivalent protein with dynamic domains including caveolin (Cav), Calmodulin (CaM), and Protein Phosphatase 2A (PP2A) binding sites, all of which are important regulatory units of the L‐type Calcium Channels (LCC) in cardiomyocytes. Previous studies indicate that mutations/deletions of the striatin gene correlate with the development of cardiac arrhythmias in animal models. We aimed to investigate whether STRN regulates calcium homeostasis and cardiomyocyte contraction rate. Data indicate that STRN is highly expressed in atrial myocardium when compared to the ventricles. Overexpression of STRN elevated the contraction rate (~ 3 fold) and resulted in sustained levels of KCl‐induced increases in intracellular calcium in neonatal rat cardiomyocytes. Conversely, silencing (>80%) the STRN gene using shRNA was associated with decreased contraction rate (~50%) of cardiomyocytes. Interestingly, pharmacological challenge with isoproterenol (β‐adrenergic agonist) failed to improve the contraction rate of cardiomyocytes with low levels of striatin (shRNA‐STRN). Biochemical analysis (CaM pull down) revealed that the interaction between CaM/PP2A/Cav‐3/LCC was increased in shRNA‐STRN cardiomyocytes and reduced when overexpressing STRN. Collectively our data indicate that striatin is a novel regulator of cardiomyocyte calcium homeostasis and contraction rate by modulating the CaM/PP2A/Cav‐3/LCC complex and influencing the activity of the LCC in the myocardium.Grant Funding Source: King Faisal Specialist Hospital and Research Center
Activation of Toll‐like receptor 4 mediates bladder hypercontractility in diabetes (865.10)
Authors: Szasz Theodora , Nunes Kenia P. , Webb R. Clinton 
Published: June 2021

Diabetic bladder dysfunction (DBD) is a common lower urinary tract complication of diabetes. Manifestations of DBD progress from bladder hypercontractility and urge incontinence in the initial phase to bladder hypocontractility and overflow incontinence in the late phase. Activation of the innate immune system via Toll‐like receptors (TLRs) like TLR4 leads to inflammation and oxidative stress and was implicated in diabetes pathophysiology. We hypothesized that bladder hypercontractility in the initial phase of DBD is mediated by TLR4 activation. Urinary bladder strips without urothelium from male C57bl/6 mice treated with vehicle or streptozotocin (65 mg/kg twice) were used to evaluate contractile function and protein expression of TLR4 and downstream mediator MyD88. Bladder expression of TLR4 and MyD88 was increased in STZ compared to control mice (TLR4=178±26, MyD88=138±12 % control). Carbachol‐mediated contraction was increased in bladder strips from STZ mice. The TLR4 inhibitor CLI095 (10 μM) decreased the maximal CCh‐induced contraction in STZ, but not in control mice (CON= 67.6±2.3, STZ=93.7±11.6, CON+CLI=76.3±6.7, STZ+CLI=66.2±7.2 % KCl‐induced contraction). These data suggest that activation of TLR4 during diabetes mediates bladder hypercontractility and contributes to DBD.Grant Funding Source: NIH
Novel findings in membrane depolarization‐induced force development in vascular smooth muscle (865.11)
Authors: Ets Hillevi , Moreland Robert 
Published: June 2021

A unique phenomenon of vascular smooth muscle (VSM) is its ability to maintain force for a prolonged period of time. The exact mechanism responsible is not known but studies suggest a critical role for L‐type Ca2+ channels (LTCC). The purpose of this study was to investigate the consequences of prolonged LTCC opening with an agonist, Bay K8644, to elucidate the intracellular mechanisms responsible for maintaining VSM tone. Smooth muscle tissue isolated from swine carotid arteries was mounted for isometric force recording. Tissues were stimulated with Bay K8644 alone or following pretreatment with one of the following inhibitors: nifedipine (LTCC), ML‐7 (MLC kinase), bisindolylmaleimide (PKC), U0126 (MAP kinase), or H‐1152 (Rho kinase). The following results were obtained: • Bay K8644 increased MLC phosphorylation, PKC activity, MAP kinase activity, and force development but partially inhibited ROCK activity; CPI‐17 phosphorylation was not changed.• Bay K8644‐dependent activation of PKC inhibited MAP kinase activity; the Bay K8644 partial inhibition of ROCK activity was reversed by inhibition of LTCC with nifedipine .• Inhibition of MAP kinase activity significantly increased MLC kinase activity and inhibition of MLC kinase activity greatly increased MAP kinase activity.These results indicate that the maintenance of force in VSM involves an intricate signaling network with multiple modulatory steps.
The large conductance Ca2+‐activated K+ channel: a key intermediate in cholinergic regulation of human urinary bladder smooth muscle excitability (865.5)
Authors: Parajuli Shankar , Hristov Kiril , Cheng Qiuping , Malysz John , Rovner Eric , Petkov Georgi 
Published: June 2021

We investigated whether muscarinic receptor (MR) activation increases human urinary bladder smooth muscle (UBSM) excitability by directly or indirectly inhibiting the large conductance Ca2+‐activated K+ (BK) channels. We used the MR agonist, carbachol, to determine the changes in BK channel activity in freshly‐isolated human UBSM cells using the perforated whole cell and single BK channel patch‐clamp recordings. Carbachol inhibited the amplitude and frequency of BK channel‐mediated spontaneous transient outward currents and spontaneous transient hyperpolarizations, both triggered by ryanodine receptor‐dependent Ca2+ release. Carbachol also caused UBSM cell depolarization, which was not observed in the presence of iberiotoxin, a BK channel inhibitor. The potential direct effects of carbachol on BK channels were examined under conditions of removing all major cellular Ca2+ sources for BK channel activation with pharmacological inhibitors (thapsigargin, ryanodine, and nifedipine). In the presence of these inhibitors, carbachol did not affect the single BK channel open probability and mean BK channel conductance (cell‐attached configuration) or the whole cell BK currents. The data support the concept that MR activation triggers indirect BK channel inhibition mediated by intracellular Ca2+, thus increasing human UBSM cell excitability.Grant Funding Source: Supported by NIH R01 DK084284 to Georgi V. Petkov.
Novel insight into human urinary bladder function: the role of transient receptor potential melastatin 4 channels (865.6)
Authors: Hristov Kiril , Parajuli Shankar , Smith Amy , Malysz John , Malysz John , Petkov Georgi 
Published: June 2021

Transient receptor potential melastatin 4 (TRPM4) channels have been recently identified in urinary bladder smooth muscle (UBSM) in rodents. Here, we applied a combined experimental approach to study the role of TRPM4 channels in human UBSM function. We used RT‐PCR, Western blot, immunohistochemistry, immunocytochemistry, patch‐clamp electrophysiology, and isometric tension recordings of human UBSM isolated strips from donor patients. mRNA message for TRPM4 channels in whole tissue and single UBSM cells was detected. Western blot and immunohistochemistry revealed TRPM4 protein expression in whole UBSM tissue. Immunocytochemistry further confirmed TRPM4 protein expression in DSM single cells. Voltage‐clamp experiments showed that the TRPM4 channel inhibitor 9‐phenanthrol decreased the transient inward cation currents (TICCs) recorded at ‐70 mV. Inhibition of IP3‐receptors with xestospongin‐C also abolished the TICCs activity in human UBSM. 9‐phenanthrol hyperpolarized the UBSM membrane potential and attenuated the spontaneous phasic, carbachol‐induced and nerve‐evoked contractions in UBSM isolated strips. The results reveal that TRPM4 channels are present in human UBSM and are critical regulators of human UBSM excitability and contractility. UBSM TRPM4 channels should be considered further as new targets overactive bladder treatment.Grant Funding Source: Supported by R01 DK084284 to G. V. Petkov
Angiotensin II activates the proteasome and stimulates vascular smooth muscle cell proliferation (866.10)
Authors: Martin Doug , Wang Xuejun , Li Yifan , Ford Hannah , Kjellsen Barton , Li Shuai 
Published: June 2021

Growing evidence implicates an important role of the proteasome in cardiovascular disease. However, the role of the proteasome in hypertension is understudied. We showed previously that proteasome inhibition attenuated angiotensin hypertension‐induced aortic hypertrophy in vivo. This study tested the hypothesis that proteasome inhibition attenuates angiotensin‐induced vascular smooth muscle proliferation.Experiments were performed on a rat embryonic aortic cell line, A7r5 (ATCC) and human umbilical artery vascular smooth muscle primary cells (Cell Applications) (HUAVSM). The cells were treated for 48 hours with vehicle (0.01% DMSO), angiotensin II (AngII) (100 nM), a proteasome inhibitor, bortezomib (Bort) (5 nM) or both (AngII+Bort). Cell number and viability were assessed by flow cytometry. Cells were also stained for Ki67, a marker of proliferating cells, and DAPI to stain DNA. Adenoviral infection was used to co‐express a green fluorescent protein fused with a ubiquitination signal sequence (GFPu) and a red fluorescent protein (RFP) in the cell. The GFPu to RFP ratio was taken as an inverse index of ubiquitin‐proteasome system (UPS) activity.Compared to vehicle, Ang II treatment increased UPS activity. Cell number was increased by AngII in both A7r5 and HUAVSM cells. AngII treatment increased Ki67 staining compared to vehicle in both A7r5 and HUAVSM cells. Treatment with Bort alone did not affect cell viability or cell number in either A7r5 or HUAVSM cells. On the other hand, Bort treatment abolished the AngII‐induced increase in UPS activity, cell number and Ki67 staining.Collectively, these data suggest that AngII‐induced vascular smooth muscle cell proliferation is mediated, at least in part, via activation of the UPS. This effect may contribute to AngII hypertension induced vascular remodeling.Grant Funding Source: Supported by HL072166, HL085629 (X.J.W.), HL109964 (Y.L.), NIGMS: P20GM103443 SD BRIN (D.M., H.F.)
Vascular oxidative stress promotes aortic stiffening (867.2)
Authors: Wu Jing , Itani Hanaa , Chen Wei , Roberts L. Jackson , Harrison David 
Published: June 2021

Oxidative stress correlates with arterial stiffness in clinical studies. Reactive oxygen species (ROS) contribute to blood pressure elevation, vascular smooth muscle hypertrophy and vascular dysfunction in hypertension. We hypothesized that vascular ROS induce aortic stiffening, which in turn promotes systolic hypertension with aging. We created mice overexpressing p22phox in smooth muscle (tgsm/p22phox mice), which have a constitutive increase in vascular ROS production. Six month old tgsm/p22phox mice were normotensive at baseline (118±6 systolic, mmHg), but had increased aortic stiffness as indicated by the leftward shift on stress‐strain curves. Aortic collagen level, as detected by hydroxyproline content, was increased in tgsm/p22phox mice compared to age‐matched C57Bl/6 mice (76.3±11.9 vs. 43.2±7.2 ng/μg total protein). At 9 months of age, aortic stiffening did not further increase but these mice developed mild hypertension (137±11 systolic), demonstrating that excessive ROS first promotes aortic stiffening and subsequently hypertension. Tempol, a superoxide dismutase mimetic, normalized blood pressure (117±10 systolic), reduced vascular collagen deposition (37.5±9.5 ng/μg) and prevented aortic stiffening in these mice, suggesting a role of vascular ROS in aging‐induced aortic stiffening. ROS oxidize arachidonic acid to produce isoketals, known to cross‐link lysines and promote protein cross‐linking, which could lead to vascular stiffening. Immunohistochemistry indicated a marked increase of isoketals in the aorta and peri‐aortic fat of tgsm/p22phox mice, which was prevented by 2‐hydroxybenzylamine (2‐HOBA), a scavenger of isoketals. 2‐HOBA also reduced blood pressure, prevented collagen deposition and aortic stiffening in tgsm/p22phox mice treated from 3 months to 9 months of age. We conclude that ROS promote aortic stiffening and that this might in part be due to isoketal formation and protein crosslinking.Grant Funding Source: Supported by NIH R01 grant HL105294