bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of age‐induced hyperglycaemia on structural remodelling in the left ventricle of the type 2 Goto‐Kakizaki rat
Authors: Singh Jaipaul , Moore Caronda J , Bidasee Keshore R , D'Souza Alicia
Published: June 2021

Chronic hyperglycaemia (HG) induced by diabetes mellitus can elicit adverse structural remodelling in the heart. This study investigated the hypothesis whether pre‐diabetes and mild chronic HG in Goto‐Kakizaki (G‐K) type 2 diabetic rats can cause similar adverse remodelling of the heart. The study employed the left ventricles of 2 month (pre‐diabetes) and 18 month (mild chronic) old G‐K rats compared to age‐matched controls to investigate structural remodelling and associated changes transforming growth factor beta 1 (TGF‐beta1), brain natriuretic peptide (BNP), atrial natriuretic factor (ANP). pro‐hyperthropic markers Akt‐mTOR‐p70S6K1 and gene expressions for a number of extracellular matrix (EMC) factors which are associated with fibrosis. The results show that both groups of G‐K rats had moderate increases in blood fasting glucose compared to controls. They also had significantly (p<0.05) higher levels of blood glucose following GTT compared to controls confirming their diabetic status. G‐K rats had significantly (p<0.05) increased heart weights, heart weight to body weight ratio, left ventricle wall (LVW) to body weight ratio, LV free wall thickness and myocyte diameter compared to controls, an indication of marked hypertrophy. The changes were accompanied by significant (p<0.05) increases in TGF‐beta 1, BNP, ANP, Akt‐mTOR:p70S6K and gene expressions for ECM factors and their regulators in the left ventricles of G‐K rats compared to controls. The results reveal that both pre‐diabetes and mild chronic HG can evoke severe adverse remodelling of the LV.
CaM Kinase II and Ca2+ sensitization mediate enhanced KCl constriction in collateral‐dependent coronary arteries of both sedentary and exercise‐trained pigs
Authors: Robles Juan Carlos , Heaps Cristine
Published: June 2021

The purpose of this study was to determine mechanisms underlying enhanced KCl‐mediated constriction in collateral‐dependent coronary arteries of sedentary (SED) and exercise‐trained (ET) pigs subjected to chronic occlusion. Ameroid constrictors were surgically placed around the left circumflex artery of female Yucatan miniature swine. Eight weeks post‐operatively, pigs were randomized into SED (pen‐confined) or ET (treadmill run; 5 days/week; 14 weeks) groups. Arteries (150–300μm luminal diameter) were isolated from the collateral‐dependent and control (nonoccluded) myocardial regions. Contractile tension and simultaneous measures of tension and intracellular free Ca2+ levels ([Ca2+]i; fura‐2) were measured in response to increasing concentrations of KCl. Chronic occlusion enhanced contractile responses to KCl and increased tension at comparable levels of [Ca2+]i in collateral‐dependent compared to control arteries of both SED and ET pigs. Inhibition of CaMKII (1μM), but not of Rho‐kinase (Y27632; 10μM or hydroxyfasudil; 30μM) or PKC (calphostin C; 1μM), abolished the enhanced contractile responses to KCl. Taken together, these data reveal that chronic coronary occlusion leads to enhanced contractile responses to KCl in collateral‐dependent coronary arteries likely via an increase in CaMKII‐mediated Ca2+ sensitization that is not corrected with ET.
Arteriolar remodeling limits maximal perfusion after percutaneous revascularization of a chronic stenosis in pigs with hibernating myocardium
Authors: Weil Brian Raymond , Page Brian J , Banas Michael D , Suzuki Gen , Canty John M
Published: June 2021

Chronic myocardial ischemia results in remodeling of the coronary microcirculation in hibernating myocardium but the reversibility of these changes is unknown. Accordingly, we evaluated parameters of arteriolar structure and measured transmural myocardial perfusion during adenosine vasodilation before and up to one month after percutaneous coronary intervention (PCI) of a chronic LAD stenosis in swine (n=12). Regional function was depressed at rest (LAD wall thickening 38.6±4.0 vs. 92.2±7.5%, p<0.05). Immediately after PCI, subendocardial flow during adenosine vasodilation increased from 1.5±0.3 to 4.6±0.8 ml/min/g, but fell to 2.7±0.3 ml/min/g one month later without evidence of restenosis. Histological assessment of subendocardial resistance vessel remodeling is shown below:

Group
Subendocardial Arterioles

Lumen Area (μm2)
Density (arterioles/mm2)
WT/Lumen Diameter (%)

Hibernating (n=148)
721±67*
10.7±1.8*
37.3±1.5*

Revascularized (n=54)
659±88*
5.8±0.4
40.3±2.2*

Sham (n=54)
1184±173
6.2±0.3
28.7±1.3

Values are mean±SEM; n=total number of vessels analyzed; WT=wall thickness;

p<0.05 vs. Sham.

These data indicate that there is subendocardial microvascular remodeling in hibernating myocardium with an increase in arteriolar density compensating for a reduction in arteriolar lumen area. After revascularization, arteriolar density returns to normal but lumen area remains reduced and decreases maximal perfusion. These chronic microvascular structural changes could precipitate subendocardial ischemia in the absence of coronary restenosis. Supported by NIH HL61610.
CaR‐mediated vasodilatation: Role of endothelium TRPC channels
Authors: Albert Anthony Paul , Shi Jian , Mclough Jessica , Greenberg Harry , Preet Chadha , Greenwood Iain , Ho Vanessa
Published: June 2021

There is increasing evidence that Ca2+‐sensing G‐protein‐coupled receptors (CaR) have an important role in modulating vascular activity; both CaR‐mediated contraction and dilation responses have been proposed. However, there is little information on mechanisms which govern CaR‐mediated responses. The aim of the present work was to investigate endothelium‐dependent actions of CaR stimulation on canonical transient receptor channels (TRPC) channel activity using patch clamp electrophysiology and functional studies. In cell‐attached patches from freshly isolated endothelium cells of the rabbit mesenteric artery, bath application of CaCl2 (1–10 mM), and the CaR mimics calindol and AC265347 activated two distinct TRPC channels with TRPC1 and TRPC3/6/7 subtype properties. TRPC channel activities were inhibited by the negative modulator of CaR, calhex, and the phospholipase C inhibitor U73122. Wire myograph studies showed that CaR stimulation relaxed methoxyamine‐contracted rabbit mesenteric arteries through an endothelium‐dependent mechanism. Immunocytochemistry revealed CaR expression in both vascular smooth muscle and endothelium cells. The present work indicates that endothelium‐dependent vasodilatations mediated by CaR are likely to involve activation of TRPC channels.

The work was supported by the British Heart foundation.
Dysfunctional mitochondrial topoisomerases mediates diabetic induced myocardial mitochondrial DNA damage.
Authors: Edwards John G. , Hicks Steven , Piteo Brian , Laurent Dimitri , Mathew Jamie , Labinskyy Nazar
Published: June 2021

We have reported previously that chronically elevated glucose induced mtDNA damage that was linked to dysfunctional mitochondrial topoisomerase activity. The present study extends those observations to the type 2 diabetic Goto‐Kakizaki (GK) rat. Diabetes significantly decreased cytochrome oxidase activity in GK LV compared to Wistar LV. Mitochondrial dysfunction was associated with significantly increased mtDNA damage without a change in mitochondrial copy number. TTGE analysis of the Cytochrome Oxidase Subunit 3 identified several different nucleotide substitutions in the GK LV that altered the primary protein sequence; none were observed in Wistar LV. In isolated mitochondria, diabetes significantly increased mitochondrial dependent DNA cleavage. Immunoprecipitation with a mitochondrial topoisomerase I antibody partially blocked mitochondrial dependent DNA cleavage indicating a functional role for this enzyme. Incubation of mitochondrial extracts with 50 μM H2O2 increased mitochondrial topoisomerase dependent DNA cleavage, implicating ROS as a modifier of topoisomerase function. Separate from a direct impact of oxidative stress on mtDNA, ROS‐induced alteration of mitochondrial topoisomerase function propagated mtDNA damage. These findings indicate a significant role for mitochondrial topoisomerase function in the development and complications of diabetic cardiomyopathy.
Atorvastatin Improves Cardiovascular Status, but Does Not Prevent the Development of Dilated Cardiomyopathy in Streptozotocin‐induced Diabetic Rats
Authors: Quidgley Jose , Cruz Nildris , Lopez Jose Carlos , Ramirez Manuel A. , Crespo Maria J.
Published: June 2021

Therapy with statins is associated with a significant reduction of cardiovascular (CV) events in normo‐cholesterolemic diabetic patients. The mechanisms, however, remain unclear. We assessed the effects of 4‐week administration of atorvastatin (ATV, 10 mg/kg/day) on CV function in streptozotocin‐induced diabetic rats. Age‐matched, non‐diabetic rats were used as controls (CT). Systolic blood pressure (SBP) and echocardiographic parameters were evaluated. Previous reports from our laboratory showed that SBP is significantly higher in diabetic rats than in non‐diabetic rats. In the current study, ATV decreased SBP in diabetic rats by 14% (P<0.05), whereas the drug did not modify this parameter in CT rats. ATV also increased stroke volume (from 0.20±0.02 to 0.51±0.06 ml, n=5, P<0.05), ejection fraction (from 44.9±3 to 59.9%, n=5, P<0.05), and cardiac output (from 63.6 ±7 to 145.5 ±17 ml/min, n=5, P<0.05) in diabetic rats. In addition, ATV reduced left ventricular end systolic volume by 27% (P<0.05), but failed to reduce left ventricular end diastolic volume. These results indicate that although ATV does not reverse ventricular dilatation, it has a positive hemodynamic effect on the CV system of diabetic rats. Thus, type 1 diabetic patients may benefit from ATV treatment beyond the reduction of cholesterol levels. Supported by MBRS‐RISE Grant R25‐GM061838.
Chromium downregulates a potent proatherogenic protein, thrombospondin‐1, in vascular smooth muscle cells via reduced O‐glycosylation and oxidative stress
Authors: Raman Priya , Ganguly Rituparna , Haney Rebecca M , Chavez Ronaldo J
Published: June 2021

Trivalent chromium (Cr), alternative therapy for diabetes, is proposed to have therapeutic potential in diabetic vascular complications. However, precise mechanisms of Cr action are not known. The present study is the first evidence that Cr downregulates a potent antiangiogenic and proatherogenic matricellular protein, thrombospondin‐1 (TSP‐1), known to facilitate initiation of atherosclerosis. Specifically, Cr decreased TSP‐1 expression induced by high glucose (30mM) or glycosylation activators in HASMC. We reported earlier that glycosylation mediates TSP‐1 upregulation by glucose. We now show that Cr‐mediated inhibition of TSP‐1 is accompanied by decreased nuclear and cytosolic O‐glycosylation, shown by immunoblotting and immunocytochemistry, coupled with a reduction in O‐GlcNAc transferase (OGT) expression, the enzyme catalyzing protein O‐glycosylation. Cr also decreased TSP‐1 expression in H2O2‐treated HASMC. Finally, using XF24 Extracellular Flux Analyzer, we demonstrated that Cr prevents high glucose‐induced decrease in maximal oxygen consumption rate, an index for mitochondrial metabolic activity, suggesting a role to combat oxidative stress. Collectively, our data suggest that decreased O‐glycosylation and attenuation of oxidative stress by Cr downregulates TSP‐1 expression, providing a novel anti‐atherogenic effect of Cr in diabetic atherosclerotic complications.
Endoplasmic reticulum stress‐associated oxidative stress and autophagy in the RVLM in neurogenic hypertension
Authors: Chan Julie YH , Chao Yun-Mei
Published: June 2021

Exaggerated endoplasmic reticulum (ER) stress has been demonstrated to participate in pathogenesis of various cardiovascular diseases. We tested the hypothesis that ER stress in the rostral ventrolateral medulla (RVLM), location of the sympathetic premotor neurons, contributes to neurogenic hypertension via induction of oxidative stress and autophagy. In RVLM of normotensive WKY rats, expressions of GRP78 and the phosphorylated eukaryotic initiation factor 2α (eIF2α), markers of ER stress, as well as tissue level of superoxide anion and the conversion of LC3‐I to LC3‐II, molecular marker of autophagy, were significantly increased following intracisternal infusion of an ER stress inducer, tunicamycin. In the RVLM of the SHR, where oxidative stress plays an active role in neural mechanism of hypertension, microinjection bilaterally into the nucleus of an ER stress inhibitor, salubrinal, ameliorated the enhanced tissue level of superoxide anion and suppressed the conversion of LC3‐I to LC3‐II. The same treatment also promoted antihypertension in SHR. Moreover, microinjection of an autophagosome inhibitor, 3‐methyladenie (3‐MA), into the bilateral RVLM promoted antihypertension. Collectively these results suggest that ER stress in RVLM may contribute to neurogenic hypertension via induction of oxidative stress and autophagy.
ATP‐mediated vasodilation occurs via vascular hyperpolarization in humans
Authors: Crecelius Anne R. , Kirby Brett S. , Luckasen Gary J. , Larson Dennis G. , Dinenno Frank A.
Published: June 2021

ATP possesses unique vasomotor properties and has been hypothesized to contribute to the control of vascular tone under a variety of physiological conditions; however, in humans, the underlying vasomotor signaling mechanisms remain unknown. We sought to determine whether ATP‐mediated vasodilation occurs via vascular hyperpolarization in young healthy humans. In Protocol 1 (n=8), progressive doses of ATP and potassium chloride (KCl) were infused via brachial artery catheter. Forearm vascular conductance (FVC) was calculated from forearm blood flow (venous occlusion plethysmography) and intra‐arterial blood pressure to quantify local vasodilation. Vasodilatory responses (%ΔFVC) to ATP infusions were unchanged following combined inhibition of nitric oxide (NO) and prostaglandins (PGs; P>0.05) whereas the response to KCl was significantly greater (P<0.05). In Protocol 2 (n=21), infusion of both ouabain (to inhibit Na+/K+ ATPase) and barium chloride (BaCl2; to inhibit KIR channels) abolished KCl‐mediated vasodilation (%ΔFVC= 134±13 vs 4±5%; P<0.05) indicating effective inhibition of vasodilation mediated via hyperpolarization. On average, forearm vasodilation to 3 doses of ATP was inhibited ~60% following combined ouabain+BaCl2 infusion. Our novel results indicate that the majority of vasodilation in response to ATP occurs via vascular hyperpolarization in humans. Supported by HL102720
Myoendothelial Contacts Within the Skeletal Muscle Arterial Network: A Potential Mediator for Divergent Control of Vascular Tone
Authors: Kirby Brett S , Bruhl Allison , Earley Scott
Published: June 2021

Holes within the internal elastic lamina (IEL) of blood vessels, referred to as myoendothelial contacts (MECs), are sites of fenestration allowing for passage of diffusible vasoactive substances and interface of endothelial cell membrane projections with underlying vascular smooth muscle. Endothelial projections are sites of dynamic Ca2+ events leading to endothelial cell hyperpolarization‐mediated relaxation and the activity of these events increase as vessel diameter decreases. Given that arterioles are the primary site of vascular resistance within skeletal muscle, we tested the hypothesis that fenestration within the IEL would be greater in distal vs. proximal arteries of the skeletal muscle arterial network. Popliteal arteries, subsequent gastrocnemius feed arteries, and intramuscular 1A arterioles from the Sprague Dawley rat hindlimb were isolated, cut and fixed longitudinally, and imaged using confocal microscopy. Quantitative analysis revealed a larger MEC total area of 1A vs. feed and popliteal arteries (20.7% vs. 5.8% and 2.8%; N = 10 images/artery), due to a noticeably greater average size of MEC (39.6μm2 vs. 11.1μm2 and 7.6μm2). These initial observations highlight the superior MEC area of intramuscular arteries and are suggestive that the anatomical architecture for distinct vasodilatory signaling is present.

Supported by RO1HL091905
Scleraxis works synergistically with Smads to regulate collagen gene expression
Authors: Bagchi Rushita , Czubryt Michael
Published: June 2021

Elevated type I collagen expression in cardiac fibrosis impairs heart function. We have shown that the transcription factor scleraxis (Scx) is expressed by cardiac fibroblasts and myofibroblasts, and is sufficient to regulate human collagen Iα2 (COLIα2) expression. Here we studied the interaction of Scx with the fibrotic Smad signaling pathway. We identified Scx binding sites (E‐boxes) within the COLIα2 proximal gene promoter, examined promoter occupancy by Scx using ChIP assay, and measured promoter activation by Scx and/or Smads via luciferase gene reporter assays. Scx augmented TGF‐β1 transactivation of the promoter. Scx and Smad3 synergistically up‐regulated COLIα2 promoter transactivation. Mutation of the promoter Smad binding element significantly reduced transactivation by Scx. A similar effect of E‐box mutations on Smad3‐mediated activation was also observed, suggesting that cross‐talk in these pathways is required for maximal gene activity. Scx expression was up‐regulated by Smad3 and down‐regulated by Smad7, further supporting cross‐pathway signaling. A dominant negative Scx mutant completely abrogated COLIα2 gene expression by TGF‐β1. Our results indicate that Scx and Smads synergistically co‐regulate collagen gene expression, and that interference with this interaction may represent a novel avenue for anti‐fibrotic therapy development.

Supported by CIHR and MHRC.
Reversal of cardiac fibroblast‐to‐myofibroblast transformation by cyclic AMP
Authors: Aroonsakool Nakon , Yokoyama Utako , Lu David , Murray Fiona , Insel Paul A.
Published: June 2021

Fibrosis, which is characterized by scarring due to excessive production, deposition and contraction of the extracellular matrix, is a key event that occurs after injury to the heart: the transformation of cardiac fibroblasts (CFb) to myofibroblasts (myoFb) contributes to remodeling. We have previously shown that cyclic AMP (cAMP) can attenuate fibroblast‐to‐myofibroblast transformation when increased during the administration of pro‐fibrotic stimuli such as TGF‐β. We sought to investigate if cAMP not only inhibits CFb‐to‐myoFb transformation, but reverses established myoFb phenotype. Incubation of primary cultures of rat CFbs with TGF‐β (10ng/ml, 48h) significantly increased mRNA and protein expression of α‐smooth muscle actin (α‐SMA), plasminogen activator inhibitor‐1 (PAI‐1) and collagen type I, all of which characterize myoFb. Treatment of these myoFb with Forskolin (Fsk, 10 μM, the direct adenylyl cyclase activator) for an additional 48h completely reversed the TGF‐β‐induced increased expression of all the markers of CFb transformation. Moreover, gel contraction (by 3D cultures of CFb in collagen gels), which was induced by TGF‐β (10 ng/ml) after 6 days, returned to that of untreated CFbs after treatment with 10 μM Fsk for a further 2 days. These findings suggest that increasing cAMP can reverse the pro‐fibrotic state of CFbs and thus may provide a means to reverse cardiac fibrosis. Funded by NIH.
Effective Teaching and Mentoring While Publishing and (Hopefully) Having a Life
Authors: Wykoff Dennis D
Published: June 2021

Being successful at an undergraduate institution that values both teaching and research requires a balancing of priorities. Working with undergraduates can be challenging but incredibly rewarding. I will highlight important tips for dynamic teaching including: incorporating student‐centered learning, challenging boredom in the classroom, integrating relevant examples, and getting useful feedback from students. Forcing student‐student and student‐teacher interactions is vital for learning, even in a class of more than 100 students. I will also give tips for running a laboratory with undergraduates so that data can be publishable. Finally, I will discuss the “controversial” topic of perfectionism being your own worst enemy at a primarily undergraduate institution.
Enhanced Proliferation and Cardiogenic Differentiation of Cardiac Progenitor Cells Treated with a Naturally Derived Cardiac Extracellular Matrix
Authors: French Kristin M , DeQuach Jessica A , Christman Karen L , Davis Michael E
Published: June 2021

Adverse remodeling of the myocardium after myocardial infarction speeds progression to heart failure. Some of the major shortcomings in cell therapy are the growth and differentiation of implanted cells. In patients, cells are injected to an area comprised mostly of noncontractile collagen, far different from their native microenvironment. This study aims to determine whether a naturally derived porcine cardiac extracellular matrix (cECM) enhances proliferation and differentiation in cardiac progenitor cells (CPCs).

CPCs were isolated via magnetic sorting of c‐kit+ cells in cardiac tissue homogenates and were grown on plates coated with either cECM or collagen I (COL). Greater serum‐induced proliferation was seen on cECM as compared to COL (3.17 vs. 1.99‐fold) as measured by Coulter counting at 2 days. Further, a trend toward increased expression of cardiac markers (Nkx2.5 and GATA4) was seen at this early time point. At 7 days, protein levels of Nkx2.5 and GATA4 were increased. Finally, fibroblastic markers were decreased at 7 days, while the environment of the cells had become oxidative, compatible with reports of redox regulation during differentiation.

Our data demonstrate that a naturally derived matrix may provide enhanced proliferation and cardiogenic differentiation of CPCs compared to collagen and thus may be a suitable vehicle to deliver cells in vivo. This study was supported by internal funding.
Age‐dependent alterations to the cardiac extracellular matrix in heart failure: differences between ventricular and atrial remodeling
Authors: Horn Margaux Anne , Graham Helen , Bode Elizabeth , Dibb Katharine , Trafford Andrew
Published: June 2021

Structural remodeling of the ventricles and the atria are features of both myocardial disease and aging and the extracellular matrix (ECM) plays a pertinent role in this process. This study sought to determine if aging exacerbates structural remodeling of the myocardium in heart failure (HF).

HF was induced in young (18 months) and old (>8 years) sheep by ventricular tachypacing. Echocardiography was performed consciously and collagen content assessed histologically. Matrix metalloproteinases (MMPs) were quantified by gelatin zymography whereas tissue inhibitor of metalloproteinases (TIMPs) were assessed by immunoblotting.

HF led to left ventricular (LV) dilatation and reduced contractility in both young and old animals, although changes were more pronounced in old. In contrast, HF increased left atrial (LA) diameter to a similar extent in both groups. Age‐associated changes to LV ECM remodeling occurred in HF with collagen accumulation in young and depletion in old. In the LA however, fibrosis was present with HF in both young and old animals. LV MMP‐2 activity was elevated in young HF but to no greater extent in old HF, however TIMP levels were reduced only in aged HF. In the LA, MMP‐2:TIMP ratio was elevated in both young and old HF.

This study demonstrates that age‐dependent alterations to LV ECM remodeling occur in HF, but HF‐induced remodeling of the LA is largely unaffected by age.

Funded by BHF and EU “Normacor”.
In Vivo Application of Dynamic Hyaluronic Acid Material for Myocardial Infarction Therapy
Authors: Young Jennifer L , Schaefer Jacquelyn , Tuler Jeremy , Schup-Magoffin Pamela , Braden Rebecca , Christman Karen L , Engler Adam J
Published: June 2021

Tissue‐specific elasticity arises from developmental changes, e.g. ~10‐fold myocardial stiffening from E3 to E10 in the chick embryo. We have shown that pre‐cardiac mesodermal cells plated on top of a thiolated hyaluronic acid (HA) hydrogel engineered to mimic this time‐dependent stiffening improves cardiomyocyte maturation compared to cells on static matrices. Here we determined cell‐matrix interactions using in vivo injections and in vitro encapsulation assays. Prior to assembly, HA was injected subcutaneously or intramyocardially into rats. Histological analysis showed minimal infiltration of host cells and capsule formation around the matrix. Hematological analysis showed no significant systemic immune response was elicited in pre‐ vs. post‐injection animals for all time points. Atomic force microscopy of subcutaneously injected gels showed increasing hydrogel stiffness over time similar to that previously found in vitro. Improved pre‐cardiac and embryonic stem cell distribution and viability was observed when cells were encapsulated with HA and immobilized thiolated protein. These data indicate the presentation of developmentally appropriate matrix stiffness may improve cell differentiation for use in future studies for treating heart failure post‐myocardial infarction.

Funding: AHA (0865150F to A.J.E.), NIH (1DP02OD006460 to A.J.E.), ARCS (to J.L.Y.), AHA (10PRE4160143 to J.L.Y.)
Hsp90 inhibition attenuates the LPS‐mediated phosphorylation of CREB and STAT3 in human lung microvascular endothelial cells
Authors: Joshi Atul D , Thangjam Gagan , Snead Connie , Feldman Sara , Catravas John D
Published: June 2021

We have previously shown that Hsp90 inhibitors protect and restore bacterial lipopolysaccharide (LPS)‐mediated endothelial barrier dysfunction. However, the underlying mechanisms for this action of Hsp90 inhibitors remain unclear. CREB and STAT3 are important pro‐inflammatory transcription factors. Our aim here is to delineate the mechanisms of LPS‐mediated phosphorylation and activation of CREB and STAT3 and its regulation by the Hsp90 inhibitor 17AAG in human lung microvascular endothelial cells (HLMVEC). Pre‐treatment of HLMVEC with 17AAG attenuated phosphorylation of both CREB and STAT3. Furthermore, 17AAG attenuated expression of CREB target genes, GADD45B and NR4A2, and STAT3 target genes, cMyc and IL6, suggesting that 17AAG decreases LPS‐mediated transcriptional activation of both CREB and STAT3. Pre‐treatment with either the p38 MAPK inhibitor (SB203580) or the PKA/MSK1 inhibitor (H89) completely abolished the LPS‐mediated CREB and STAT3 phosphorylation. Furthermore, treatment with Src family kinase (SFK) inhibitors PP1 and PP2 exhibited partial attenuation of CREB and STAT3 phosphorylation. These data suggest that the Hsp90 inhibitor, 17AAG, attenuates LPS‐mediated phosphorylation and activation of both CREB and STAT3 via the p38 MAPK and SFK pathways. (Supported by HL093460 and HL101902)
Localized activation of Rac1 promotes IQGAP1‐dependent VE‐cadherin trans interaction: Role in junction stabilization
Authors: Daneshjou Nazila , Vogel Stephen M , Komarova Yulia A , Malik Asrar B
Published: June 2021

The monomeric RhoA‐GTPases play a critical role in regulating junctional permeability in the endothelium; however, the casual relationship between Rac1 activity at adherens junctions (AJs) and stability of Vascular Endothelial (VE)‐cadherin adhesion, the main adhesive complex of AJs, is not well understood. Utilizing a photoactivatable Rac1 probe (PA‐Rac1) we demonstrated that activation of PA‐Rac1, but not of a light insensitive probe, at the level of AJs induced VE‐cadherin clustering with a rate constant of 0.36 ± 0.22 min−1 in human dermal microvascular endothelial cells. Pretreatment of cells with inhibitory peptide which binds to a VEcadherin extracellular 1 module and prevents trans interaction abolished PA‐Rac1 effect, confirming stabilization of trans interaction downstream of Rac1 signaling. Interestingly, PA‐Rac1 activation also induced junctional accumulation of IQGAP1, a scaffold protein that sequesters GTP‐Rac1 and GTP‐Cdc42 at AJs. Furthermore, down regulation of IQGAP1 using siRNA inhibited clustering of VE‐cadherin upon PA‐Rac1 photo‐activation suggesting that Rac1‐induced VE‐cadherin trans interaction is IQGAP1‐dependent. These findings demonstrate the essential role of Rac1‐IQGAP1 signaling in stabilization of VE‐cadherin adhesion and AJ integrity. Supported by R01 45638 and 103922, and T32 HL 007829.
Signaling from the natriuretic peptide system to ENaC: implications in primary cilium of renal epithelial cells
Authors: Alli Abdel A , Guo Lai-Jing , Eaton Douglas C , Bao Hui-Fang
Published: June 2021

Natriuretic peptides (NPs) bind to and activate two different classes of natriuretic peptide receptors (NPRs). Previous investigations have targeted the effect of NPs on the regulation of the epithelial sodium channel (ENaC), which is located at the apical membrane of polarized epithelial cells. However, the expression of NPRs in polarized epithelial cells has not been thoroughly investigated and the exact mechanism for the NP response on ENaC has not been reported. We show by confocal microscopy the guanylyl cyclase class of NPRs (NPR‐A and –B) and the non‐guanylyl cyclase class of NPRs (NPR‐C) are both expressed at the apical membrane of 2F3 cells. We found NPR‐A is localized to the basal body, while NPR‐C is expressed in the cilia of 2F3 cells. We show by single channel patch clamp studies in 2F3 cells that administration of ANP or 8‐pCPT‐cGMP causes a decrease in the open probability of ENaC. Since CNP, the preferred NPR‐B ligand, and cANF, the selective NPR‐C agonist did not affect the open probability of ENaC, we infer that the mechanism primarily involves NPR‐A. We investigated if signaling pathways downstream or independent of NPR‐A were involved in the NP‐mediated regulation of ENaC activity, but found that inhibitors of sGC, PKG, iNOS, or an NO scavenger did not affect ENaC activity in cell‐attached patches. Taken together, our results help to elucidate a new mechanism for the regulation of ENaC.
Insulin regulates hSGLT2 expression via PKA and PKC activation
Authors: Ghezzi Chiara , Wright Ernest M
Published: June 2021

Recent evidence suggest that in the rat proximal tubule SGLT2 (Sodium dependent Glucose Transporter 2) is phosphorylated. To test if human SGLT2 is regulated by posphorylation we analyze the effect of PKA and PKC activation on HEK‐293T cells expressing hSGLT2 using patch clamp and radio tracer uptake. PKA activation was induced by incubation of cells in 8‐bromoadenosine 3′‐5′‐cyclic monophosphate (Br‐cAMP) and sn‐1,2‐dioctanoylglycerol (DOG) was use as activator of PKC. After 1 h incubation in Br‐cAMP we observed a 2.5 fold increase in [14 C]α‐MDG uptake confirmed by a similar increase in glucose induced current. Incubation in DOG resulted in a 50 % increase in [14C]α‐MDG uptake and glucose induced current. In both cases increase is caused by an increase in Imax and not in the change in glucose affinity. These effects were completely suppressed by mutation of the SGLT2 phosphorylation site (S625A). In order to determine the initial event triggering the phosphorylation, we measured the effect of insulin on [14C]α‐MDG uptake. Incubating cells expressing hSGLT2 wt or S625A mutant with 400 pM insulin induced a 3 fold increase in glucose transport in wild type hSGLT2 but not in the mutant. In conclusion our results indicate that insulin via PKA and PKC activation regulates hSGLT2 expression in a fast time scale by an increasing trafficking of hSGLT2 to the plasma membrane.

NIH grant DK077133 and SNSF grant PBZHP3‐135919.
Broad range neutral amino acid transporter (B0AT1) requires association with TMEM27 for surface expression in renal cells
Authors: Torrente Marta , Arps Lisa , Guetg Adriano , Camargo Simone M.R. , Verrey François
Published: June 2021

TMEM27 (collectrin), a homologue of angiotensin converting enzyme 2 (ACE2), was previously shown in renal proximal tubule to regulate amino acid uptake via interaction with the broad range neutral amino acid transporter B0AT1, whereas ACE2 was shown to associate with B0AT1 in small intestine. To investigate the role of collectrin interaction with B0AT1, we generated Madin‐Darby canine kidney (MDCK) cell lines constitutively expressing B0AT1 and observed that this expression was not tolerated. Therefore, using a KRAB repressor system and lentiviral transduction, we produced cells that inducibly express either B0AT1, collectrin or both. As in vivo, collectrin was shown to be required for efficient B0AT1 cell surface expression and function. Furthermore, we demonstrated that collectrin co‐expression increases B0AT1 protein stability. Interestingly, expression levels of both B0AT1 and collectrin were increased when amino acid concentrations in the culture medium were reduced to physiological levels. In conclusion, we generated cell lines expressing inducible B0AT1 and collectrin recapitulating the dependence of B0AT1 expression on collectrin co‐expression and thus representing a very good model for testing the role and the mechanism of B0AT1‐collectrin interaction and the impact of the amino acid concentration for the control of its expression.
Trafficking of renal betaine/GABA transporter (BGT1) is disrupted by mutation at T40
Authors: Day Christopher R , Gordon Sashana S , Williams Robert , Day Richard N , Kempson Stephen A
Published: June 2021

Cells in renal medulla accumulate several osmolytes to survive hypertonicity and this is reproduced in renal MDCK cells. In response to hypertonic stress (Hyp, 500 mOsm) for 24 h there is upregulation of BGT1 mRNA and protein synthesis followed by trafficking to the plasma membrane (PM) and a marked increase in betaine transport. PM trafficking motifs reside near the C‐terminus of BGT1 and truncation at K560 resulted in a protein that remained intracellular during Hyp stress, as expected. The K560Δ mutant expressed in MDCK cells was colocalized with an ER marker but not markers for endosomes or Golgi. Surprisingly, substitution of A at T40 of native BGT1, to block phosphorylation, also prevented trafficking to the PM during Hyp stress. The T40A mutant was not retained in the ER and colocalized with markers for Golgi and endosomes suggesting, indirectly, that it was not misfolded. Mutation of T40 to E or D, to mimic phosphorylation, restored normal trafficking to the PM. HEK cells transfected with K560Δ or T40A mutants had 10% of GABA transport activity of native BGT1, but normal transport activity was restored in cells expressing T40E. Data suggest that normal BGT1 trafficking (or PM retention) requires both C‐terminal motifs and phosphorylation at T40.
ROS production as a common mechanism of ENaC regulation by EGF, insulin and IGF‐1
Authors: Ilatovskaya Daria V. , Pavlov Tengis S. , Levchenko Vladislav , Staruschenko Alexander
Published: June 2021

Epithelial Sodium Channel (ENaC) is a key transporter participating in the fine‐tuning of Na+ reabsorption in the nephron. ENaC activity is acutely upregulated by EGF, insulin and IGF‐1. It was also proposed that ROS, including H2O2, have a stimulatory effect on ENaC. Here we studied whether effects of EGF, insulin and IGF‐1 correlate with ROS production in the mpkCCDc14 cells. Western Blotting confirmed the expression of the NADPH oxidase complex subunits in these cells. Short circuit current measurements showed an acute increase of ENaC‐mediated current through the mpkCCDc14 monolayer in response to 50 ng/ml EGF, 100 nM insulin or 100 ng/ml IGF‐1. Treatment of mpkCCDc14 cells with EGF, insulin or IGF‐1 also caused an increase in H2O2 production as measured by DCF fluorescence. Pretreatment with NADPH oxidase activity inhibitor apocynin blunted both H2O2 production and increase in ENaC‐mediated current in response to these drugs. To further test whether NADPH oxidase subunits are involved in the effect of EGF, we created a stable M‐1 cell line with a knockdown of Rac1, which is one of the key subunits of the NADPH oxidase complex, and measured amiloride‐senstitive currents in response to EGF. In contrast to control cells, EGF had no effect in Rac1 knockdowned cells. We hypothesize that EGF, insulin and IGF‐1 have a common stimulatory effect on ENaC mediated by ROS production.
Identification of ENaC intersubunit interfacing residues provides insight into conformational changes associated with channel gating
Authors: Collier Daniel M , Snyder Peter M
Published: June 2021

The extracellular domain of human ENaC functions as a sensor that fine tunes channel activity in response to changes in pH. In previous work, we determined that mutations at γE455 and βE446 decreased the response to acidic pH while αK477A increased the response to acidic pH. Here, we investigated potential intersubunit interactions. Based on the ASIC1a structure, we predict that αK477 and γE455 interact with βV85 and αL120, respectively. To test this, we substituted these residues with Cys and measured mutant channel activity in Xenopus oocytes. We found that covalent modification of Cys mutations at βV85 and αL120 with MTSET+ and MTSESaltered ENaC activity in a manner that was charge dependent. Modification with like charges (i.e. αK477 and MTSET+) increased current while opposite charges decreased current (i.e γE455 and MTSET+) – suggesting an electrostatic interaction between the two residues. To further test for an interaction, we cross‐linked the subunits. In cells expressing αK477CβV85CγENaC, treatment with long cysteine reactive cross‐linking reagents increased current, but short reagents did not. Intriguingly, treatment with either long or short reagents disrupted channel gating, reducing Na+ self‐inhibition. The data provide a potential link between pH sensing, extracellular domain conformational changes, and channel gating. PMS is supported by NIH HL072256, DMC is supported by AHA 10PRE2610282
Osmotic pressure of the cutaneous surface fluid of Rana esculenta
Authors: Larsen Erik Hviid , Ramløv Hans
Published: June 2021

The osmotic pressure of the cutaneous surface fluid (CSF) in vivo was measured for investigating whether evaporative water loss (EWL) derives from water diffusing through the skin or fluid secreted by exocrine subepidermal mucous glands. EWL was stimulated by subjecting R. esculenta to 30–34 °C. A solute‐free paper disc was placed on the skin for sampling of the clear CSF uniformly covering the body surface. The osmolality measured in a Wescor Vapro Vapor Osmometer was, 173 ± 9 mosmol/Kg, mean ± se, n = 21 samples. The osmolality of CSF of isoproterenol injected frogs at 20 °C was, 149 ± 5 mosmol/Kg, n = 16. Osmolality of lymph was, 239 ± 4 mosmol/Kg, n = 8. Thus the flow of water across the epidermis would be in the direction from CSF to the interstitial fluid driven by the above osmotic gradients and/or coupled to the inward active Na+ flux via the slightly hyperosmotic paracellular compartment [EH Larsen et al. (2009) Acta Physiologica 195: 171–186]. It is concluded that the source of EWL of the frog on land is the fluid secreted by the mucous glands and not water diffusing through the skin. The study supports the hypothesis [EH Larsen (2011) Acta Physiologica 202: 435–464] that volume and composition of CSF are regulated by subepidermal exocrine gland secretion balanced by EWL into the atmosphere and solute coupled fluid uptake by the epidermal epithelium, respectively. Supported by the Carlsberg Foundation.
Evaluation of salt appetite in brain specific 11β hydroxysteroid dehydrogenase type 2 knockout (11βHSD2−/−) mice
Authors: Evans Louise Christine , Holmes Megan , Mullins John , Bailey Matthew
Published: June 2021

Male brain specific 11βHSD2−/− mice and floxed controls were individually housed in metabolic cages and basal water turnover assessed over 4 days. In separate studies mice were given access to 2 bottles containing water and 1.5% NaCl and salt preference measured over 7 days. The role mineralocorticoid receptor (MR) in salt appetite was evaluated using spironolactone s.c.

Basal water and sodium turnover and plasma sodium concentrations were not altered by the deletion of 11βHSD2 in the brain. When given access to NaCl brain specific 11βHSD2−/− mice displayed a significant salt preference, 70% of total fluid intake was accounted for by NaCl. Salt appetite was significantly reduced by MR blockade. Sodium was not retained in the null mice; increased salt intake resulted in significant natriuresis and polyuria.

Deletion of 11βHSD2 in the brain, which is specifically expressed in the nucleus of the solitary tract (NTS) in mice, resulted in an increased salt appetite. Activation of MR is implicated since spironolactone significantly reduced NaCl intake. These data are the first demonstration of salt appetite occurring in the absence of sodium depletion and with normal renal function. They suggest that activation of MR on 11βHSD2 positive neurons in the NTS increases salt appetite. Funded by the British Heart Foundation.
Response of a marine population of three‐spine sticklebacks (Gasterosteus aculeatus) to salinity stress
Authors: Li Johnathon , Kueltz Dietmar
Published: June 2021

Three‐spine sticklebacks are euryhaline fish that can live in marine and limnic environments. Populations of this species have evolved the ability to invade freshwater and become landlocked many times independently in Europe, Asia, and North America. They represent an excellent model for studying adaptive constraints leading to parallel evolution. We are interested in how salinity tolerance as a physiological trait evolved during such parallel evolution in plasma‐hyper vs. plasma‐hyposmotic environments. Thus, we are characterizing the osmotolerance of different stickleback populations starting with an ancestral marine population established at Bodega Harbor, CA. Osmotolerance in these fish was assessed by exposing them to gradual salinity change (3 ppt/ day) and monitoring physiological parameters including ventilation rate and behavioral parameters (feeding, swimming behavior). We also collected osmoregulatory tissues from animals gradually acclimated to different salinities ranging from FW to 70 ppt and performed molecular phenotyping of these tissues using proteomics. The resulting data provide a comprehensive physiological baseline for comparing salinity stress responses of this marine population to that of land‐locked limnic populations.
Blood oxygen depletion in California sea lions
Authors: McDonald Birgitte I , Ponganis Paul J
Published: June 2021

The rate and magnitude of O2 store depletion is critical to the dive performance and foraging ecology of breath hold divers. We investigated blood O2 depletion with a backpack recorder for the partial pressure of O2 (PO2) in nine California sea lions during maternal foraging trips. During routine shallow dives, arterial and venous minimum PO2 were routinely greater than 30 mmHg (~50% hemoglobin saturation). However in deep dives greater than 4 minutes in duration, minimum venous PO2 reached values below 10 mmHg (~ 5% saturation), and in dives over 7 min, were as low at 5 mmHg. During deep dive bouts, both arterial and venous PO2 increased during ascent and minimum arterial PO2 was never below 30 mmHg (~ 40–50% saturation), even in dive durations greater than 5 min. In contrast to shallow dives, venous PO2 values less than 10 mmHg for over two minutes during long dives are consistent with near complete blood O2 depletion. During deep dives, the increase in arterial and venous PO2 during ascent suggests that re‐expansion of collapsed lungs and resumption of gas exchange during the “ascent tachycardia” allows for lung‐to‐blood O2 transfer. We suggest that the O2 reservoir remaining in the collapsed lungs at depth serves to supplement blood O2 levels during ascent, and prevent shallow water black out at the end of deep dives.
The effects of temperature on cardiac E‐C coupling and intracellular Ca2+ buffering in trout cardiomyocytes
Authors: Warren Daniel E. , Shiels Holly A.
Published: June 2021

To investigate the effects of acute temperature change on cardiac excitation‐contraction in fish heart, atrial and ventricular myocytes were isolated from rainbow trout acclimated to 12°C and studied using voltage clamp recording while simultaneously recording [Ca2+]i at 7°C, 14°C, and 21°C. Cells were stimulated in the presence or absence of sarcoplasmic reticulum (SR) inhibitors and SR Ca2+ load and intracellular Ca2+ buffering were determined with caffeine. Atrial and ventricular cells showed increased ICa density, faster ICa inactivation, and smaller gain at 14°C and 21°C compared to 7°C. SR inhibition was most effective at 7°C, where it decreased [Ca2+]i rise slope and amplitude by 58% and 44%, respectively, and prolonged recovery of [Ca2+]i by 1.6‐fold and decreased gain by 40%. An opposite trend emerged in ventricular cells in which SR inhibition had its largest effect at 21°C, where it decreased [Ca2+]i rise slope and amplitude by 60% and 75%, respectively, resulting in a 56% decrease in gain. Bmax for Ca2+ buffering did not differ between atrium and ventricle but was higher at 7°C (1392 ± 274 μM) than at 21°C (451 ± 274 μM) while Kd was unaffected by tissue or temperature (0.27 and 0.30 μM for atrium and ventricle, respectively). Thus, atrial myocytes show a particular sensitivity to cold and increased reliance on the SR to maintain [Ca2+]i in the face of reduce ICa, while ventricular myocytes do not. Maintained [Ca2+]i at cold temperatures in both cell types is maintained by a larger SR Ca2+ load.
The Effects of Dietary Fatty Acid Composition on Energy and Dry Matter Digestibility in Siberian hamsters (P. Sungorus)
Authors: Vatnick Itzick , Blatteau Elizabeth , Batter Victoria
Published: June 2021

Dietary lipids affect many physiological processes such as aging, neurological susceptibility to hypoxia, cancer, and cardiovascular and inflammatory disease. In hibernating mammals the fatty acid composition of the diet may effect several parameters of torpor including, minimum body temperature during torpor, duration of torpor bouts and metabolic rate during torpor. Many experiments alter diet fatty acid composition assuring that the diets remain isocaloric. However, the effects of fatty acid composition of the diet on digestibility of dry matter and energy are not known. Therefore, we designed an experiment that allowed us to discern whether isocaloric diets of different fatty acid saturation are assimilated equally and whether there are sex differences in digestive performance of a well‐studied hibernator, Siberian hamsters (P. Sungorus). Preliminary data suggest that the digestibility of the diets supplemented with saturated fats is lower than the diet supplemented with unsaturated fats, in male hamsters but not in females. These are surprising results; therefore we are in the midst of a second round of experiments to ascertain the accuracy of these results.
Phytoecdysteroid treatment reduces Notch signaling in aged skeletal muscle
Authors: Arthur S. T. , Zwetsloot K. A. , Lawrence M. M. , Nieman D. C. , Lila M. A. , Grace M. , Nowicki C. R. , Cooley I. D. , Shanely R. A.
Published: June 2021

Dysfunctional Notch and Wnt signaling contributes to poor muscle regeneration and increased fibrosis in aged skeletal muscle. 20‐ hydroxyecdysone (20E), a phytoecdysteroid produced by the plant Ajuga turkestanica, increases muscle strength in young rats. The objective of this study was to determine if an extract from A. turkestanica (ATE), enriched in phytoecdysteroids, affects Notch and Wnt signaling in skeletal muscle of aged mice. Aged male C57BL/6 mice (20 mo) received ATE, 20E, or vehicle (CT) for 28 days. Immunohistochemistry on tricep brachii was performed using antibodies directed to Notch and Wnt signaling markers. Delta ligand expression in the triceps of ATE‐treated mice decreased relative to CT (p = 0.024) and 20E‐treated mice (p = 0.039). There was a non significant decrease of active beta‐catenin expression (33%) in triceps of 20E‐treated mice, compared to ATE or CT. H&E staining revealed fewer centrally‐located nuclei with 20E treatment compared to CT or ATE, suggesting fewer cycles of muscle regeneration with 20E. In conclusion, phytoecdysteroids, individually or in an enriched extract, did not rejuvenate Notch signaling or influence Wnt signaling in aged sedentary mouse skeletal muscle. However, 20E appears to attenuate the age‐related muscle regeneration process as evidenced by fewer centrallyn‐ucleated fibers. Supported in part by a USDA Kannapolis Scholars Fellowship.