bluuebunny/crossref_metadata_2025 huggingface dataset:
Overexpression and nuclear translocation of glyceraldehyde‐3‐phosphate dehydrogenase in acetaminophen acutely intoxicated rats
Authors: Delfante Amalia , Ruiz María Laura , DiCarlo María Biatriz , Perazzo Juan Carlos , Mottino Aldo Domingo , Ghanem Carolina Inés
Published: June 2021

The mechanism of acetaminophen (AAP) hepatotoxicity involves glutathione depletion, oxidative stress and mitochondrial dysfunction leading to necrosis. Recently, glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was highlighted as a mediator of cell death.

Aim

To evaluate GAPDH expression and subcellular distribution in liver of AAP acute intoxicated rats.

M&M

Male Wistar rats were injected i.p. with 1 g/kg b.w. of AAP (AAP group) or vehicle (C group) (n=4). After 24 h, liver GAPDH expression (mRNA and protein) was studied by RT‐PCR and Western‐Blot (WB). Nuclear, mitochondrial, and cytosolic GAPDH content was evaluated in hepatic subcellular fractions by WB. Results were expressed as mean±SD and statistically analized with Student‐t‐test (* p<0.05 vs C).

Results

GAPDH mRNA (AAP:408±221*; C:100±26) and protein expression (AAP:292±127*; C:100±7) were significantly increased in treated rats. Subcellular distribution of GAPDH changed for nucleus (AAP:232±95*; C:100±49) but not for mitochondrial and cytosolic fractions.

Conclusion

AAP acute intoxication increased GAPDH expression due to a transcriptional mechanism, which questions its use as a housekeeping gene for either RT‐PCR or WB studies. Under AAP overdose, the GAPDH increase its content in the nucleus sugesting a role in AAP toxicity.
Potential role of FasL in the development of AZT‐mediated Hepatotoxicity
Authors: Donde Hridgandh , Ghare Smita , Chen Wei-Yang , Joshi-Barve Swati , McClain Craig , Barve Shirish
Published: June 2021

Highly Active Antiretroviral Therapy (HAART) has led to a significant increase in the life expectancy of HIV patients. Hepatotoxicity due to HAART is also common. Importantly, alcohol addiction is a common feature among HIV patient and HAART/alcohol interactions are emerging as significant contributing factors to HAART‐hepatotoxicity. The mechanisms involved in HAART‐hepatotoxicity, particularly in the context of alcohol abuse remain undetermined. Hepatocytes are particularly sensitive to FasL‐mediated cell death Using relevant in vitro and in vivo model systems we investigated the role of Fas/FasL apoptotic signaling in the induction of HAART‐ mediated hepatotoxicity and its exacerbation by chronic alcohol consumption. Treatment of a hepatic cell line (HepG2) with AZT (3′‐azido‐3′‐deoxythymidine) showed decreased cell survival (apoptosis) along with a robust up‐regulation of FasL at both mRNA and protein level. AZT also led to the down regulation of cellular levels of FLIP (FLICE inhibitory protein) which prevents FasL‐mediated apoptotic signaling and death. In vivo AZT administration led to the development of significant hepatic steatosis which was further compounded by chronic ethanol administration. The present studies have begun to elucidate the potential mechanisms in HAART mediated hepatotoxicity.
Decreased Calcineurin Activity Suppresses Protein Synthesis and Activates AMP Dependent Kinase (AMPK)
Authors: Franch Harold A , Ding Changlin , Zoromsky Sarah , Gooch Jennifer
Published: June 2021

CAβ knock out mice (CAβ−/−) kidneys have normal development and function but do not hypertrophy to diabetes. We examined calcineurinˈs role in protein metabolism and in Akt, mitogen associated kinase (MAPK) and AMPK signaling in proximal tubule cells in epidermal growth factor (EGF) and/or cyclosporin A (CYA) treated NRK‐52E renal epithelial cells and SV40 immortalized cell lines created from CAβ−/− and wildtype litter mate proximal tubules. CYA (4μM) decreased protein content/well (48h) by 14±2% and protein synthesis (18h) by 51±2% of control in NRK‐52E cells. In contrast, protein degradation(0h–28h) was decreased by 10±1%. Inhibitors upstream of MAPK or Akt reduced protein synthesis by 48±3% or 56±2%. However, CYA increased both basal and EGF‐induced MAPK and AKT phosphorylation (1h) by ~3‐fold. AMPK, which acts to inhibit the mammalian target of rapamycin, phosphorylation was 6 fold higher with CYA compared to control. CAβ −/− tubular cells had lower protein per well (48h, 20±2%) and EGF‐induced protein synthesis than CAβ +/+ cells. In H2O2 treated CAβ −/− cells, AMPK phosphorylation was >10 fold higher in CAβ −/− compared with CAβ +/+ cells. Decreased calcineurin activity reduces protein accumulation in renal tubular cells via reduced protein synthesis possibly through activation of AMPK.
Electrophysiological properties of cardiac myocytes in regenerating zebrafish hearts
Authors: Yamada Toshiki , Denton Jerod , Yin Viravuth P. , Strange Kevin
Published: June 2021

Zebrafish hearts fully regenerate within 30–60 days post‐amputation (dpa) of up to 20% of the ventricle. Lineage studies have shown regeneration is mediated by proliferation of existing myocytes. Genes encoding ion channel proteins undergo transcriptional changes during regeneration suggesting that electrical properties of the heart may be remodeled as part of the regenerative process. In these studies, we isolated myocytes from hearts 7 dpa, a time of peak myocyte proliferation, and characterized their electrical properties by patch clamp. Myocytes from uninjured and 30 dpa hearts had similar Na+, K+ and Ca2+ currents and action potentials. However, 7 dpa myocytes exhibited decreased T‐ and L‐type Ca2+ current amplitudes and a tendency to generate short duration action potentials. The T‐ and L‐type Ca2+ channel subunits CACNA1G, CACNA1C and CACN1B have reduced expression at 7 dpa. Our studies are the first to characterize ion channels in regenerating hearts. Reduced Ca2+ currents and shortened action potentials will reduce cardiac contractile forces, which may be required for regeneration. Changes in myocyte electrical activity and Ca2+ signaling may also play a role in regulating the regenerative process. Collectively, our studies document that dynamic changes in ion channel activity accompanies the transformation of quiescent myocytes into highly proliferative cardiac cells during regeneration.
Changes in myofilament lattice spacing accounts for differences in systolic strains in Vinculin deficient mouse hearts
Authors: Tangney Jared Rylan , Janssen Matthew S , Liao Peter , Zemljic-Harpf Alice , Chuang Joyce , Hoshijima Masahiko , McCulloch Andrew D , Ross Robert S , Omens Jeffrey H
Published: June 2021

Vinculin has been previously shown to play a significant role in mechanotransduction and may affect myocardial mechanical function in a directionally‐dependent manner. Cardiac MRI tagging in a vinculin knockout (VclKO) mouse model revealed significant decreases in systolic sheet‐normal shear strain (P<0.05) and systolic sheet strain (P<0.05) in KO mice compared with littermate wildtype (WT) controls. We hypothesized that an increase in transverse systolic stress development may explain these observations, and changes in myofilament lattice spacing in the VclKO could alter systolic stress in the myocytes. Actin‐myosin lattice spacing was then measured and normalized to sarcomere length in barium‐contracted WT and VclKO hearts with values of 19.1±1.51 nm and 21.8±1.39 nm, respectively. These results were used as inputs in a structural model of crossbridge geometry similar to that of Schoenberg (1980), which showed a significant increase in the radial component of sacromere force in the VclKO compared with WT. The calculated distribution of forces was then used in a three‐dimensional finite element model in which end systolic sheet strains were predicted. The results matched the trends seen experimentally between the WT and VclKO mice. These results suggest that the change in systolic strains seen experimentally in the VclKO mice can be explained by the increase in lattice spacing seen in the knockout mice.
Effects of Long Term Exercise on Cardiac Hemodynamic and Contractility Measures in Aging Rats
Authors: Akins Tiffany L , Bates Melissa , McKiernan Susan , Aiken Judd , Diffee Gary
Published: June 2021

Pressure‐volume (P‐V) analysis is a robust approach for the study of in‐vivo ventricular function. In this study, we characterized P‐V alterations that occur with advanced age, with and without long‐term exercise training. Adult (24 month) male Fischer 344 x Brown Norway (FBN) rats were randomly divided into 3 groups; high intensity exercise (HI), moderate intensity exercise (MI), and sedentary (S). MI and HI animals trained at 13 m/min for 30 min/day, with the HI group at 5% incline. Animals were trained daily for 12 months until 36 months of age. Hemodynamic measurements were conducted via a P‐V catheter in the left ventricle (LV) in 36‐month old animals. As a result of the exercise training, rats in the MI group had significant increases (compared to S) in indices of contractility such as End Systolic Pressure‐ Volume relationship (ESPVR), and dP/dtmax. HI animals showed no such improvement and, in fact, had decreases in resting cardiac parameters such as stroke volume, cardiac output, stroke work, and ejection fraction, when compared to MI and S animals. End‐ Diastolic Pressure‐Volume Relationship (EDPVR) was higher in S compared to MI and HI groups. We conclude that long‐term exercise training altered cardiac function in aged hearts but that the response is dependent on exercise intensity. Supported by: NIH AG030423
Functional Assessment of Stenosis Severity by Contrast‐Induced Submaximal Hyperemia
Authors: Nolte F , van de Hoef T P , de Klerk W , Spaan J AE , Piek J J , Siebes M
Published: June 2021

Introduction

The hemodynamic effect of a stenosis is uniquely characterized by the relationship between pressure drop across (ΔP) and velocity (v) distal to it (ΔP‐v relationship). Normally this relationship is assessed after a bolus injection of adenosine (ADO). However, ADO is not readily available everywhere. We therefore aimed to establish whether vasodilation by contrast medium (CM) results in the same ΔP‐v relationship or is affected by factors like viscosity.

Methods

In 21 patients scheduled for percutaneous coronary intervention, measurements were performed in a stenotic vessel before treatment. Aortic pressure (Pa), intracoronary pressure (Pd) and v were recorded during sequential periods covering control and response to ADO and CM injections. ΔP‐v relationships were fitted using the equation ΔP=0+Av+Bv2. The similarity between CM and ADO curves was established by comparing the predicted pressure drop at v=50 cm/s (ΔPv50).

Results

ΔP‐v relationships showed good agreement between CM and ADO. Hyperemic v was 35.5±17.6 cm/s during ADO and reduced to 81% after CM (p<0.01). ΔPv50 was 50.5±12.5 with ADO and 76.8±24.6 mmHg after CM (NS).

Conclusion

CM does not alter the shape of the ΔP‐v relationship. Consequently, although CM does not result in full vasodilation it can be used to assess functional stenosis severity based on the shape of this curve.

Supported by NHF grant 2006B186
miR‐mediated regulation of coronary collateral growth in the metabolic syndrome
Authors: HUTCHESON REBECCCA LYNN , SMITH ERIKA , MUSIYENKO ALLA , ROCIC PETRA
Published: June 2021

In response to transient, repetitive coronary artery occlusion, native collateral vessels enlarge. We stimulate coronary collateral growth (CCG) in the rat model by transient, repetitive LAD occlusion. CCG is impaired in the metabolic syndrome, both in the human and in the rat model (JCR). We evaluated CCG by measurements of coronary blood flow (microspheres). We hypothesized that lack of CCG in the JCR rat is in part due to aberrant phenotypic modulation of vascular smooth muscle cells (VSMCs). VSMC phenotype is regulated by transcription factors myocardin and Klf‐4, which are regulated by microRNA (miR)‐145. We hypothesized that decreased miR‐145 in the JCR rat leads a less contractile VSMC phenotype resulting in an inability to form functional collaterals. Our results show that expression of VSMC‐specific contractile proteins, myocardin, and miR‐145 are decreased in the JCR rat heart while Klf‐4 is increased in the later stages of CCG versus normal rats. These findings suggest that decreased miR‐145 expression in the metabolic syndrome correlates with a less contractile VSMC phenotype which may account for impaired CCG. Support: RO1HL093052 and AHA11PRE7690011.
Effect of Mental Stress on Coronary Blood Flow in Humans
Authors: Sauder Charity L , Thompson Alison E , Myers Terrell , Ray Chester A
Published: June 2021

Mental stress elicits diverse vascular responses in humans. The coronary circulation is responsible for providing the heart with adequate blood supply during stress. Psychological stress has been linked to the development of coronary artery disease. The effect of mental stress on coronary vascular conductance (CVC) was examined in both men (n=9) and women (n=8). Coronary blood flow velocity (Doppler ultrasound), MAP, and HR were monitored continuously before, during, and after 3 min of mental arithmetic. Mean arterial blood pressure (MAP; 85±2 and 84±4 mmHg) and heart rate (HR; 68±4 and 73±5 beats/min) at rest were not significantly different between men and women, respectively. Mental arithmetic increased HR and MAP (Δ17±3 beats/min and Δ17±2 mmHg, respectively) in all subjects. No sex differences in HR and MAP responses to mental arithmetic were observed (P>0.05). When all subjects were analyzed together, coronary blood velocity increased (17.9±1.4 to 22.5±1.5 cm/s; P=0.001) but CVC did not change with mental stress (0.27±0.02 to 0.29±0.02 cm/s/mmHg; P=0.38). However, when analyzed separately, men demonstrated a significant increase in CVC (Δ21±1%; P<0.002) and women elicited no change in CVC (Δ3±1%; P=0.63). These data indicate that women, unlike men, do not augment CVC during mental stress. Therefore, these results suggest that women may be more susceptible to adverse cardiac events during mental stress.
The causal role of PKC in Reduced Endothelial Ca2+ Signaling and Impaired EDHF‐Mediated Uterine Vasodilation in Diabetic Pregnancy
Authors: Gokina Natalia I , Linder Erika
Published: June 2021

Increased PKC activity is an essential mechanism of diabetic vascular complications. To characterize the role of PKC in uterine vascular dysfunction during diabetic pregnancy we: (1) defined the effects of PKC activation (PDBu) or inhibition (bisindolylmaleimide, BIS) on EDHF‐mediated responses to ACh; (2) elucidated PKC dependence of ACh‐induced endothelial cell (EC) [Ca2+]i.

Diabetes was induced by streptozotocin (STZ) injection. ACh was tested in uterine arteries from control and diabetic pregnant rats in the presence of L‐NNA and indomethacin. Fura‐2 based SMC or EC [Ca2+]i responses were studied as well. Diabetes reduced EDHF‐mediated vasodilation from 90 ± 4 % to 26 ± 9 % at 10 μM ACh. SMC and EC [Ca2+]i responses were markedly reduced as well. PDBu inhibited ACh‐evoked vasodilation (14 ± 5 %) and associated SMC [Ca2+]i responses of control vessels. BIS partially restored EDHF‐mediated vasodilation (to 72 ± 10 %) and SMC [Ca2+]i in diabetic vessels. PDBu reduced EC [Ca2+]i responses of control vessels to ACh by 50 ± 7 %. BIS significantly restored EC [Ca2+]i elevations to ACh in diabetic arteries.

PKC‐dependent reduction in EC Ca2+ signaling underlies impaired EDHF‐mediated uterine vasodilation during diabetic pregnancy. Inhibition of PKC may serve as a therapeutic strategy to improve uterine vasodilation in pregnancies complicated with diabetes. Supported by NIH HL088245
Nuclear Factor kappa B (NFkB) Inhibition Improves Vascular Function in Type 2 Diabetic Mice
Authors: Choi Soo-Kyoung , Kassan Modar , Umezawa Kazuo , Trebak Mohamed , Matrougui Khalid
Published: June 2021

Type 2 diabetes (T2D) is associated with microvascular dysfunction. We hypothesized that increased NFκB signaling contributes to vascular dysfunction in T2D. Type 2 diabetic (db−/db−) and control (db−/db+) mice were treated with two NFκB inhibitors (DHMEQ, 6mg/kg, twice a week and IKK‐NBD peptide, 500ug/kg/day) for four weeks. Isolated coronary arterioles (CA) and mesenteric resistance arteries (MRA) were mounted in an arteriograph and a myograph. Pressure‐induced myogenic tone (MT) was significantly potentiated, while endothelium‐dependent relaxation (EDR) was significantly attenuated in CA and MRA from diabetic compared to control mice. These results were associated with increased NFκB activity. Interestingly, diabetic mice treated with NFκB inhibitors significantly reduced MT potentiation and improved EDR. Western blot analysis revealed a decrease in eNOS expression and phosphorylation, increase in p‐65NFκB phosphorylation and COX2 expression in diabetic mice, which were normalized with NFκB inhibition. Thus, we demonstrated that increased NFκB activity disrupts the basic mechanism of microvascular function in T2D. Therefore, NFκB could be a potential target to overcome diabetic microvascular complications.
Carbonylation Induces Ryanodine Receptor Dysregulation During Diabetes
Authors: Bidasee Keshore R , Shao Chun-Hong , Tian Chengju , Moore Caronda J , Singh Jaipaul , Rozanski George
Published: June 2021

We and others earlier found that cardiac ryanodine receptor Ca2+ release channels (RyR2) becomes dysregulated during diabetes and suggested this defect to be a contributing cause for increased incidence of heart failure (HF) and fatal ventricular arrhythmias in individuals with diabetes mellitus. To date, mechanisms responsible for RyR2 dysregulation during diabetes remain poorly understood. This study assesses whether carbonylation (modification by reactive carbonyl species, RCS) is an underlying cause. After 8 weeks of diabetes, spontaneous Ca2+ released was enhanced and evoked Ca2+ release became dyssynchronous (non‐uniform) in ventricular myocytes from diabetic rats. RyR2 protein level remained unchanged but its capacity to bind the Ca2+‐dependent ligand [3H]ryanodine was significantly reduced. Western blots and mass spectroscopic analyses revealed carbonyl adducts on select basic residues of RyR2. Altering charge and increasing bulk on affected residues to mimic carbonylation resulted in channels with low to exaggerated cytoplasmic Ca2+ responses, akin to that seen during diabetes. Expressing carbonylation‐mimicking mutants in RyR2‐null HEK‐ 293T cells triggered abnormal intracellular Ca2+ cycling. Exposing healthy rat ventricular myocytes to the reactive carbonyl species methylglyoxal (MGO) induced aberrant SR Ca2+ release, similar to that seen in diabetic myocytes. MGO also altered the binding of [3H]ryanodine and the open probability of RyR2. Treating diabetic rats with scavengers of reactive carbonyl species reduced carbonylation of RyR2 and aberrant SR Ca2+ release. From these new data we conclude that carbonylation is a specific mechanism that contributes to RyR2 dysregulation during diabetes. Funded in part by NIH
Hypertension‐induced endothelial cell angiopoietin‐2 release is inhibited by angiopoietin‐1
Authors: Korff Thomas , Ernst Evelyn , Nobiling Rainer , Feldner Anja , Reiss Yvonne , Plate Karl H , Fiedler Ulrike , Augustin Hellmut G , Hecker Markus
Published: June 2021

A pivotal autocrine mechanism following endothelial cell activation is the release of angiopoietin‐2 (Ang‐2) which subsequently antagonizes the binding of Ang‐1 to the Tie‐2 receptor. In this context, we noted that hypertension in mice reduces the abundance of Ang‐2 stored in arterial endothelial cells and hypothesized that this Ang‐2 release depends on an increase in circumferential wall tension or stretch – a hallmark of hypertension. In fact, stretching isolated perfused mouse arteries or human cultured endothelial cells triggered rapid Ang‐2 release in a calcium‐dependent manner. Interestingly, Ang‐1 abolished stretch‐induced Ang‐2 release both in the isolated perfused mouse arteries and in the cultured endothelial cells. Subsequent analyses of the latter indicated that pre‐treatment with Ang‐1 robustly inhibits their stretch‐induced calcium response as well as the stretch‐mediated expression of pro‐inflammatory adhesion molecules. Collectively, our results suggest that stretch induces the release of Ang‐2 from human cultured endothelial cells and that Ang‐1 counteracts these Ang‐2 functions by attenuating the stretch‐dependent intracellular calcium release which may at least in part explain its anti‐inflammatory effect in stretch‐stimulated endothelial cells.
Mechanosensitive TRPV4 channels mediate cardiac fibroblast differentitation to myofibroblasts
Authors: Thodeti Charles K , Adapala Ravi k , Luther Daniel J , Thoppil Roslin , Chilian William , Meszaros J. Gary
Published: June 2021

Cardiac fibroblasts play critical role in cardiac remodeling and during development. Cardiac remodeling following myocardial infarction involves the migration, proliferation and differentiation of cardiac fibroblasts to hypersecretory myofibroblasts. Myofibroblasts facilitate wound repair in the myocardium by secreting and organizing extra cellular matrix (ECM) and fibrosis. However, the molecular mechanisms involved in myofibroblast differentiation are not well known. Because the infarcted/ischemic myocardium is known to undergo mechanical stretch and the muscle phenotypic modulator TGF‐β is also increased in ischemia, we hypothesized the mechanotransduction and TGF‐β signaling pathway play active role in the differentiation of cardiac fibroblasts to myofibroblasts. Here, we show that the mechanosensitve ion channel TRPV4 is required for TGF‐β‐induced differentiation of cardiac fibroblasts to myofibroblasts. We found that cardiac fibroblasts functionally express TRPV4 channels and the TRPV4‐specific antagonist, AB159908 significantly inhibited TGFβ‐induced differentiation as measured by incorporation of α‐SMA into actin stress fibers (p < 0.05). Importantly, we demonstrate that TGF‐β‐induced myofibroblast differentiation is regulated by the variations in ECM elasticity in a TRPV4 dependent manner. Further, we found that TGF‐β treatment increased the expression of TRPV4. Finally, calcium imaging experiments with Fluo‐4 revealed that TGF‐β treated fibroblasts exhibit enhanced TRPV4‐dependent calcium influx compared to untreated controls ( p< 0.05). Taken together these results suggest, for the first time, that a mechanosensitive ion channel, TRPV4 regulates cardiac fibroblast differentiation to myofibroblasts which could be used as a novel therapeutic target for the treatment of ischemia and myocardial infarction.
The inflammatory‐fibrotic phenotype of right ventricular cardiac fibroblasts in hypoxia‐induced pulmonary hypertension
Authors: Brown R Dale , Li Min , Stewart Charles T. , McKeon B. Alexandre , Walker Lori A. , Buttrick Peter M. , Stenmark Kurt R.
Published: June 2021

We hypothesized that cardiac fibroblasts isolated from RV could recapitulate in part the inflammatory‐fibrotic signature of the remodeling ventricle in a calf model of pulmonary hypertension. One‐day old neonatal calves were exposed to chronic hypobaric hypoxia (14d, simulated elevation 4200 m) leading to pulmonary hypertension compared to normoxic controls. Cardiac fibroblasts (pro‐collagen I+, SM myosin‐ ) were cultured from tissue explants of RV free wall. Production of inflammatory‐fibrotic mRNAs was quantitated by RealTime PCR. Stimulation of cultured cardiac fibroblasts with bacterial lipopolysaccharide LPS (1 μg/ml, 48 hr) increases production of pro‐inflammatory mRNAs (IL‐6, IL‐1 receptor antagonist, MCP‐1). Cardiac fibroblasts derived from hypertensive animals exhibit modest (~2‐fold) enhancement of constitutive and LPS‐stimulated cytokine production, suggesting a preserved pro‐inflammatory phenotype. Cardiac fibroblasts respond to autocrine‐paracrine factors released from hypertensive pulmonary‐adventitial fibroblasts by producing important inflammatory‐fibrotic mRNAs (IL‐18, IL‐6, MCP‐1, Osteopontin, Tenascin C) that were previously identified in the hypertensive RV. We conclude that activated cardiac fibroblasts contribute significantly to the inflammatory‐fibrotic signature of the hypertensive RV in PH. Supported by The Children's Hospital‐Denver and NIH.
Cyclic AMP induces necroptotic cell death in cardiac fibroblasts
Authors: Yun Hongruo , Wilderman Andrea Scarpa , Lu David , Aroonsakool Nakon , Insel Paul A.
Published: June 2021

Increased levels of the second messenger cyclic AMP (cAMP) can promote cell loss of a variety of cell types. The precise mechanism(s) for this loss is/are not clearly defined, although cAMP can promote apoptosis of certain types of cells (Insel et al, Acta Physiol in press). In the present study, we used adult rat cardiac fibroblasts (rCFs) to test the hypothesis that cAMP induces necroptosis. Our results from morphological and biochemical studies provide evidence that cAMP induces necroptosis in rCFs and is mediated by protein kinase A and not Epac, another cAMP effector. The findings include destruction of the plasma membrane of rCFs, which differs from apoptotic shrinking of the membrane, and a prominent increase in expression of HMGB1, a marker of necrosis, following treatment with cAMP‐elevating agents. In addition, such agents induce death in a caspase‐PARP independent manner. The pan‐caspase inhibitor, z‐VAD‐fmk increases cell death induced by CPT‐cAMP but the necroptosis inhibitors necrostatin‐1 and cyclosporine A reduce this effect. Taken together, these findings lead us to conclude that cAMP induces necroptosis in rCFs and that this occurs via a PKA‐mediated mechanism. cAMP/PKA‐promoted necroptosis may contribute to decrease in number of CFs in vivo and may be a mechanism of cAMP‐promoted cell death in other types of cells. (Supported by research and training grants from NIH).
Resveratrol prevents accumulation and secretion of the pro‐hypertrophic high molecular weight FGF‐2 by rat and human cardiac myofibroblasts
Authors: Santiago Jon-Jon , McNaughton Leslie J , Fandrich Robert F , Arora Rakesh C , Kardami Elissavet
Published: June 2021

Background

Resveratrol (Resv) is known to prevent cardiac hypertrophy by a mechanism attributed to direct effects on cardiomyocytes. Cardiac fibroblasts are known to mediate the paracrine induction of hypertrophy, by secreting bioactive agents including the pro‐hypertrophic high molecular weight (Hi) FGF‐2. Here, we examined the effects of Resv on the angiotensin II (Ang II)‐induced HiFGF‐2 upregulation and release by cardiac myofibroblasts.

Results

Cardiac non‐myocytes, isolated from the hearts of neonatal rats or from human cardiac atrial tissue, expressed proteins characteristic of myofibroblastic phenotype at passage P2, as indicated by expression of extra domain A‐fibronectin, non‐muscle myosin heavy chain, and α‐smooth muscle actin. Stimulation with Ang II promoted the upregulation of both total and exported HiFGF‐2. Secreted HiFGF‐2 was capable of inducing cardiomyocyte hypertrophy in culture. Resv prevented both total and exported HiFGF‐2 upregulation, in both rat and human heart cells. Studies have shown that the effects of Resv can be mediated by inhibition of ERK and MMP‐2 activities; in agreement, we found that inhibition of these pathways diminished the Ang II‐induced HiFGF2 upregulation and release.

Conclusions

Our data suggest that the anti‐hypertrophic effect of Resv may be mediated in part by its effects on cardiac fibroblasts, preventing HiFGF‐2 accumulation and secretion.

Grant Funding Source: CIHR
Mechanical Stress activates NFATc4 in Cardiac Fibroblasts via Syndecan‐4
Authors: Herum Kate Louise Møller , Lunde Ida Gjervold , Behmen Dina , Florholmen Geir , Carlson Cathrine Rein , Christensen Geir
Published: June 2021

Cardiac fibroblasts differentiate into myofibroblasts in response to left ventricular pressure overload. Myofibroblasts have contractile properties and produce excessive amounts of extracellular matrix, thereby increasing myocardial stiffness. The objective of this study was to investigate the role of the focal adhesion transmembrane protein syndecan‐4 as a stress‐sensor activating nuclear factor of activated T‐cells (NFAT) and thereby inducing myofibroblast differentiation. Aortic banding increased mRNA levels of the myofibroblast marker gene smooth muscle α‐actin, as well as collagen I and III in wild‐type left ventricles but not in the syndecan‐4 knockout (KO) hearts. Stretch caused translocation of GFP‐NFATc4 fusion proteins to the nucleus indicating activation of this isoform. Furthermore, NFATc4 was dephosphorylated (activated) following stretch in a calcineurin‐and syndecan‐4‐dependent manner. Finally, overexpressing NFATc4 caused an increase in the myofibroblast‐associated myocardin‐related transcription factor A. In conclusion, we show that syndecan‐4 activates NFATc4 signaling following stretch and this may cause myofibroblast differentiation.

Source of research support: Norwegian Health Association
Knockout of type VI collagen improves cardiac function and remodeling following myocardial infarction
Authors: Luther Daniel J. , Thodeti Charles K. , Weihrauch Dorothee , Patel Hemal H. , Niesman Ingrid R. , Bonaldo Paolo , Chilian William M. , Meszaros J. Gary
Published: June 2021

Cardiac remodeling is a dynamic process that is accelerated following myocardial infarction (MI) injury. We have previously reported that in addition to type I and III collagen, type VI collagen deposition also increases in response to MI. The goal of this project was to determine whether type VI collagen influences post‐MI remodeling using mice lacking this collagen. We induced MI by occluding the LAD in wild type (WT) and collagen VI null mice (Col6KO) and studied remodeling 1–8 weeks following MI. Ejection fraction was significantly preserved (43.9 ± 3.3% vs. 29.1 ± 4.3% for WT) and LV chamber dilation was attenuated in the Col6KO MI group (25.8 ± 7.9% increase vs. 62.6 ± 16.5% for WT). Apoptosis was significantly decreased in Col6KO mice at day 14 compared to WT. Isolectin staining indicated baseline blood vessel density in Col6KO mice was greater than WT mice. Interstitial collagen levels were reduced 39.6 ± 9.8% and electron microscopy shows more dramatic mitochondrial and nuclear ultrastructural morphology changes in Col6KO MI mice. We conclude that the absence of type VI collagen preserves cardiac function and limits aberrant remodeling of the myocardium following MI.
Cardiac‐myocyte Ablation of Talin1 Results in a Blunted Hypertrophic Response to Chronic Pressure Overload
Authors: Manso Ana Maria , Li Ruixia , Cruz Nathalia M , Gu Yusu , Chang Sharon M , Rasmussen Brett J , Peterson Kirk L , Abel Dale , Monkley Susan J , Critchley David R , Ross Robert S
Published: June 2021

Integrin signaling has been reported to be essential for cardiac myocyte hypertrophy. Talin (Tln) is an integrin‐actin linker that has been shown to be essential for integrin activation. Vertebrates contain two talin genes, tln1 and tln2, which encode very similar proteins (74% amino acid sequence identity). Tln2 is the main Tln isoform in postnatal cardiac muscle but under pressure overload conditions, Tln1 expression is increased as it localizes in the costameres, with integrins. To test Tln1 contribution to the cardiac hypertrophic response in vivo, we generated Tln1 cardiac myocyte specific knockout mice (Tln1cKO). Tln1cKO mice were viable and fertile and show normal cardiac structure and function under baseline conditions. When Tln1cKO mice were subjected to 8 weeks pressure overload (POL) they showed a blunted response, with less hypertrophy, less fibrosis and better preservation of heart function compared to controls. Acute responses following mechanical stress (10 min POL) indicated that normal activation of ERK1/2, p38, Akt and GSK3b was strongly blunted in Tln1cKO mice. These data demonstrate the critical role of Tln1 in the mechanotransductive response of the cardiac myocyte. (Supported by NIH grants HL088390 and HL46345)
The matricellular proteins thrombospondin‐2, osteonectin and osteoglycin modulate cardiac inflammation, injury and function during viral myocarditis.
Authors: Heymans Stephane , Rienks Marieke , Vanhoutte Davy , Swinnen Melissa , Westermann Dirk , VandenDriessche Thierry , Lijnen Roger , Schroen Blanche , Papageorgiou Anna , Carmeliet Peter
Published: June 2021

Non‐structural matri‐cellular proteins (MCPs) modulate the process of heart failure (HF) by acting on different cardiac cellular compartments. Here, we investigated whether MCPs, including TSP2, SPARC or osteoglycin (OGN), may affect cardiac inflammation. Therefore, TSP‐2, SPARC and OGN‐null animals and their WT littermates were submitted to human Coxsackievirus B3 (CVB3)‐induced myocarditis. Whereas the absence of TSP‐2 or SPARC increased cardiac inflammation, necrosis and overall mortality as compared to WT at 14 days, resulting in systolic dysfunction at 35 days, lack of OGN was protective. Increased cardiac inflammation and injury was caused by decreased activation of the anti‐inflammatory T‐regulatory cells (TSP‐2‐null) and increased trans‐endothelial leukocyte migration (SPARC‐null). Decreased cytokine activation caused less cardiac inflammation in OGN‐null. Finally, over expression of TSP‐2 or SPARC in WT hearts with AAV9‐TSP2 or AdVSPARC significantly reduced cardiac inflammation and injury. In conclusion, whereas increased SPARC or TSP‐2 protect against cardiac necrosis and failure during viral myocarditis, OGN is detrimental.
Pharmacological activators of Kv7 potassium channels attenuate methacholine‐induced constriction of rat airways
Authors: Brueggemann Lioubov I , Tate Shawn , Byron Kenneth L
Published: June 2021

We recently discovered that guinea pig airway smooth muscle cells have Kv7 potassium currents that are suppressed by the muscarinic receptor agonist methacholine. In the present study we evaluated the constriction of airways in rat precision‐cut lung slices. Methacholine induced constriction of small bronchi with an EC50 of 217 ± 44 nM. Verapamil (an L‐type Ca2+ channel blocker, 10 μM) completely reversed the airway constriction induced by 230 nM methacholine. Methacholine‐induced bronchoconstriction was also attenuated by 30% in the presence of retigabine (10 μM), a selective activator of Kv7.2–7.5 potassium channels. Zinc pyrithione and 2,5‐dimethylcelecoxib, other activators of Kv7.2–7.5 potassium channels, were also effective in reducing 230 nM methacholine‐induced bronchoconstriction. These findings indicate that activation of L‐type VSCCs is the primary mechanism responsible for bronchoconstriction at sub‐maximal concentrations of a muscarinic receptor agonist and support a role for Kv7 potassium channels in the regulation of airway diameter. Our results also further establish Kv7 potassium channels as potential pharmacological targets for bronchodilatory therapy.
Alveolar type II cell‐specific deletion of p120‐catenin in mice demonstrates the crucial role of Type II cell‐localized p120‐ catenin in regulating alveolar epithelial barrier function
Authors: Liu Yuru , Reynolds Albert B , Malik Asrar
Published: June 2021

Maintenance of alveolar epithelial barrier integrity is essential for normal alveolar gas exchange function and lung fluid balance. The normal alveolar epithelium is composed of two types of cells: flat type I cells comprising ~95% of the gas‐exchange surface, and cuboidal type II cells, which secrete surfactant. Type II cells also function as facultative progenitor cells in that they can proliferate and trans‐differentiate into type I cells during homeostasis as well as post alveolar epithelial injury. However, molecular mechanisms of maintenance and repair of alveolar barrier are not clear. p120‐ catenin is located at epithelial adherens junction (AJ) where is required for stabilizing E‐Cadherin. Here we addressed whether p120‐catenin contributes to alveolar barrier function in lung. We made an alveolar type II cell specific p120‐Catenin knock‐out mouse line. These mice displayed a persistently leaky alveolar barrier and low grade pulmonary edema and chronic lung inflammation characterized by infiltration of inflammatory cells. These findings indicate that while type II cells make up only 5% of alveolar epithelial surface area, type II cell p120‐catenin plays a critical role in maintenance of alveolar epithelial barrier and homeostasis.
First detection of Ca2+ responses in alveolar macrophages in situ
Authors: Westphalen Kristin , Islam Mohammad Naimul , Gusarova Galina , Bhattacharya Jahar
Published: June 2021

Alveolar macrophages (AMs) play a major role in ventilator‐induced lung injury (VILI). However, it is not clear whether AMs respond directly to lung expansion. Here we tested this hypothesis in intact alveoli of the mouse lung. To determine the time course of the AM response to lung expansion, we used the cre/lox recombinase system to specifically express Enhanced Yellow Fluorescent Protein (EYFP) in CD11c‐positive AMs (AMYFP). We isolated lungs from AMYFP–expressing mice and blood‐perfused them at constant pulmonary artery, left atrial and airway pressures of 10, 3 and 5 cmH2O, respectively. We viewed the lungs by means of confocal microscopy. To determine cytosolic Ca2+ in AMYFP, we microinfused alveoli with the Ca2+ fluorophore, fluo‐4. At baseline, AMYFP displayed slow Ca2+ oscillations at an amplitude of 11±2% of mean (n=5). Following a 15s inflation to 15 cmH2O, the oscillation amplitude increased by 60±10% (P<.05) within 5 min, and remained elevated for 20 min. The oscillation frequency was unchanged. We conclude, AMs respond rapidly to lung expansion by augmenting Ca2+ oscillations. In VILI, crosstalk signaling between the stretched alveolar epithelium and local AMs may initiate proinflammatory mechanisms leading to increased permeability of the alveolo‐capillary barrier (HL78645, HL64896).
Cadherin ectodomains and cadherin‐actin linkages regulate the endothelial barrier
Authors: QUADRI SADIQA K , Sun Li , Bhattacharya Jahar
Published: June 2021

Cadherins, the components of endothelial barrier‐regulating adherens junctions (AJs), are stabilized by ectodomain trans‐interactions and by linkages to cortical actin. To determine the role of cadherin stabilization on the endothelial barrier, we viewed by confocal microscopy, monolayers of rat lung microvascular endothelial cells (RLMEC) expressing wild type (WT) E‐cadherin‐GFP or a mutant E‐cadherin‐GFP (W2A) that blocks trans‐interactions, or monolayers co‐expressing WT E‐cadherin‐GFP and an α‐actinin‐1 mutant (Y12F) that delinks E‐cadherin from actin. We quantified cadherin mobility by fluorescence recovery after photobleaching (FRAP) of GFP, and permeability at junctional microdomains by z‐axis microcopy of fluorescent dextran of size 70 kD (FD70). Both the W2A and Y12F mutations increased cadherin mobility and junctional permeability to FD70, indicating that delinking cadherin at the ectodomain or at the actin tether had similar effects on barrier function. By contrast, hyperosmolar sucrose decreased both cadherin mobility and FD70 permeability. We conclude that in the lung endothelial barrier (1) cadherin mobility and macromolecular permeability relate inversely; (2) barrier permeability is determined by cadherin tethers. We propose that the selectivity filter of the endothelial barrier is determined by dynamic regulation of cadherin tethers (HL36024).
Mobile phone radiation effects into ERK1, ERK2 and PKCα signaling pathway of Wistar rats pituitary
Authors: de Caires Luiz Carlos , da Silveira Goulart Guimarães Ernesto , Musso Camila Manso , Vasconcellos Rebecca , Assis Diego , de Mendoça José Paulo Rodrigues Furtado , Garcia Raúl Marcel González
Published: June 2021

Over the last few years, the mobile communication system has increased significantly. Some researches about the radiation emitted by mobile phones have been trying to verify possible modifications on gland functions. ERK1, ERK2 and PKCα enzymes have pivotal role on controlling synthesis and secretion of pituitary and hypothalamic hormones. Thus, the aim of this work was to evaluate the effect of the radiation emitted by mobile phones into the expression and activity of ERK1 and ERK2 and expression of PKCα in Wistar rats pituitary cells. Wistar male rats, 60 days old, were exposed to the radiation emitted by mobile phone (1800 MHz) during 1 or 3 hours uninterrupted. The pituitary gland of the animals was removed in order to analyze the expression of those enzymes and activity of ERK1 and ERK2, by Western Blot. After 1 hour of exposure, it was not observed any significant modification in the expression of ERK1, ERK2 and PKCα. The activity of ERK1 and ERK2 was shown to be reduced in the exposed animals. After 3 hours of exposure, the expression of ERK1 and ERK2 was not altered, although the activity of these enzymes and expression of PKCα was reduced in the exposed animals. According to these data, the exposure to this kind of radiation may alter the signaling pathways in which ERK's and PKCα are involved, and, consequently, the normal physiology of pituitary cells.

Financial Support: CNPq, Capes, Fapemig and UFJF.
Recruitment of OCRL and Inpp5B to phagosomes by Rab5 and APPL1 depletes phosphoinositides and attenuates Akt signaling
Authors: Bohdanowicz Michal , Balkin Daniel M , De Camilli Pietro , Grinstein Sergio
Published: June 2021

Sealing of phagosomes is accompanied by the disappearance of phosphatidylinositol (4,5)‐bisphosphate (PtdIns(4,5)P2) from their cytoplasmic leaflet. Elimination of PtdIns(4,5)P2, which is required for actin remodeling during phagosome formation, has been attributed to hydrolysis by phospholipase C and phosphorylation by phosphatidylinositol 3‐kinase. We found that two inositol 5‐phosphatases, OCRL and Inpp5B, become associated with nascent phagosomes. Both phosphatases, which are Rab5 effectors, associate with the adaptor protein APPL1, which is itself recruited to the phagosomes by active Rab5. Knock‐down of APPL1 or inhibition of Rab5 impair association of OCRL and Inpp5B with phagosomes and prolong the presence of PtdIns(4,5)P2 and actin on their membranes. Even though APPL1 can serve as an anchor for Akt, its depletion accentuated the activation of the kinase, likely by increasing the amount of PtdIns(4,5)P2 available to generate phosphatidylinositol (3,4,5)‐trisphosphate (PtdIns(3,4,5)P3). Thus, inositol 5‐phosphatases are important contributors to the phosphoinositide remodeling and signaling that are pivotal for phagocytosis.
Cytosolic phospholipase A2 mediates mechanical stretch‐induced apoptosis via caspase‐3 in human renal proximal tubular epithelial (HK‐2) cells
Authors: Alexander Larry D
Published: June 2021

Although several studies have been performed investigating the effects of cyclic strain on apoptosis in renal proximal tubular cells, its mechanism to induce apoptosis is not fully understood. Phospholipase A2 (PLA2) is a signaling molecule that regulates arachidonic acid production and participates in inflammatory responses. The aim of this study was to investigate whether cPLA2 could modulate cyclic stretch‐mediated cell death in human renal proximal tubular cells (HK‐2 cells) and to examine the mechanism(s) of any effects observed. Stretch‐induced apoptosis was detected by measuring histone associated DNA fragments using ELISA and caspase assays. The application of cyclic stretch significantly increased apoptosis and caspase‐3 activity in HK‐2 cells. Inhibition of cPLA2 by AACOCF3 and MAFP or by treatment of cPLA2 short interfering RNAs (siRNAs) attenuated both apoptosis and caspase‐3 activation by cyclic stretch, supporting the importance of cPLA2 as a mediator of stretch‐induced apoptosis in renal proximal tubular cells. In addition, inhibition of caspase‐3 prevented stretch‐induced apoptosis. Furthermore, arachidonic acid itself induced time‐ and dose‐dependent increases in capase‐3 activity and apoptosis in HK‐2 cells. These findings suggest that cPLA2‐mediated activation of caspase‐3 plays a key role in cyclic stretch‐induced renal cell apoptosis.
Blue native PAGE resolution of renal sodium transporters
Authors: Lee Donna H , McDonough Alicia A.
Published: June 2021

Blue native polyacrylamide gel electrophoresis (BN‐PAGE) is used to separate protein complexes from biological membranes under native conditions.

Aim

Determine size of native complexes of renal Na+ transporters and their putative associated regulators by BN‐PAGE (Invitrogen). 15–20 μg of rat cortical membranes (200K x g pellet) were resolved on 3–12% Bis‐Tris gels and blotted onto PVDF.

Results and Conclusions

NHE3 (mw ~75kD) is detected at 460 kD with a minor band at 550 kD. NHE3‐pS552 is also detected at 460 kD. The NHE3 regulator DPPIV (100kDa), is detected at: 430 kD (40% of the signal) near NHE3, at 525 kD (10%) and 330 kD (50%). Membrane bound Myosin VI (150kD), an actin‐based motor associated with NHE3‐DPPIV, resolves at 340 – 400 kDa, below NHE3. NKCC (150kD) is detected at 625 kD. Anti‐NKCC‐pT96,pT101 detects two bands: with NKCC at 625kD (55% of signal) and at 400 kD raising the possibility that the anti‐NKCC epitope is masked in the 400 kD complex. NCC (150 kD) is detected at both 610 kD (66% of signal) and 380 kD (33%). NCCpS71 or pT53 were detected only at 610 kD suggesting phosphorylation provokes 610kD complex (or –p epitopes are masked at 380kD). γ‐Adducin (100 kD), an auxiliary protein that interacts with NCC, is detected at 600kDa (40% of signal) near NCC and NCC‐p, and at 700 kD (60%). Na,K‐ATPase α1 (100kD) is detected at 285 kD. Na,K‐ATPase β, ENaCs and SPAK were not detected using this amount of protein. DK0837854
A novel physiological role of miR‐192 in renal handling of fluid balance
Authors: Mladinov Domagoj , Liu Yong , Liang Mingyu
Published: June 2021

miRNAs are gene regulators that reduce target protein abundance. Previously we found miR‐192 to be highly expressed in kidney cortex, and computational analysis predicted it targeted the β‐1 subunit (Atp1b1) of Na/K‐ATPase (NKA). We hypothesize that miR‐192 modulates Atp1b1 expression and affects renal handling of fluid balance. Over‐expression of miR‐192 in renal cells resulted in decreased Atp1b1 protein and NKA enzymatic activity (to ~50% and ~75% of control, respectively; p<0.05, n=6). Oppositely, suppression of miR‐192 with Anti‐miR‐192 oligonucleotides increased Atp1b1 protein and NKA activity (to ~250% and ~210% of control, respectively; p<0.05, n=6). In mice, injection of Anti‐miR‐192 substantially suppressed miR‐192 in kidney cortex (to ~30% of control) and up‐regulated the Atp1b1 protein (to ~160% of control; p<0.05, n=7). When mice were fed with high‐salt diet, Anti‐miR‐192 reduced urine output by ~30% in the first 24h of high salt intake, compared to the anti‐scramble control (p<0.05, n=8). Water intake was not significantly altered by the anti‐miR‐192 treatment at this time point. The study suggests a novel physiological mechanism in which miR‐192 suppresses NKA and contributes to renal handling of fluid balance on a high‐salt diet. Research support: NIH and AHA Predoctoral Fellowship.
The Transcription Factor ATF4 Is an Essential Mediator of Skeletal Muscle Atrophy
Authors: Ebert Scott Matthew , Kunkel Steven D , Dyle Michael C , Bullard Steven A , Adams Christopher M
Published: June 2021

Skeletal muscle atrophy is a common and debilitating condition that requires widespread changes in skeletal muscle gene expression. However, the transcriptional regulatory factors that mediate muscle atrophy are incompletely understood. Here, we examined the potential role of ATF4, a stress‐responsive bZIP transcription factor that is induced in atrophying muscle. We found that ATF4 overexpression in mouse skeletal muscle was sufficient to induce muscle fiber atrophy. To determine if ATF4 is required for muscle atrophy, we generated and studied mice lacking ATF4 specifically in striated muscle (ATF4 mKO mice). ATF4 mKO mice were phenotypically normal under baseline conditions, however they lost ≈ 50% less muscle in response to two distinct muscle atrophy stimuli (fasting and hindlimb immobilization), indicating resistance to muscle atrophy. To determine if ATF4 promotes muscle atrophy by altering protein metabolism, we performed metabolic labeling studies in C2C12 skeletal myotubes, and found that ATF4 reduced protein synthesis and increased proteolysis, leading to myotube atrophy. Collectively, these data indicate that ATF4 is an essential mediator of muscle atrophy, and suggest that ATF4 reduces protein synthesis, induces proteolysis and promotes muscle atrophy by inducing one or more downstream target genes. This work was supported by the NIH and Department of Veterans Affairs.