bluuebunny/crossref_metadata_2025_split huggingface dataset:
KvBeta2 Alters Kv1 Surface Expression and Modulates Electrophysiologic Properties in Ventricular Cardiomyocytes
Authors: Kilfoil Peter J , Raucci Frank J , Tipparaju Srinivas , Bhatnagar Aruni 
Published: November 2023

IntroductionThe voltage‐gated potassium (Kv) channels regulate the resting membrane potential and the repolarization phase of the cardiac action potential. Members of the Kv1 and Kv4 family associate with ancillary subunits, such as Kvβ 1–3, that modify channel kinetics, gating and trafficking. Although Kvβ1 has been shown to regulate Ito and IKslow currents in the heart, the role of Kvβ2 in regulating myocardial potassium conductances is unknown. In heterologous expression systems, Kvβ2 increases surface expression of Kv1 channels, shifts the activation potential of Kv1 currents to more polarized voltages, and increases the inactivation of inactivating Kv1 currents, but its role in regulating native cardiac channels remains unknown. Hence, to identify the role of Kvβ2 in the heart, we examined the distribution and activity of Kv1 and Kv4 channels in cardiac myocytes from wild‐type (WT) and Kvβ2‐null mice (KO).MethodsWestern blotting and confocal microscopy were used to determine the abundance and localization of Kvβ2, Kv1.4 and Kv1.5 proteins in heart and isolated cardiomyocytes. Action potentials and whole‐cell Kv currents were recorded from freshly isolated mouse cardiac myocytes using standard patch clamp methodologies.ResultsImmunoprecipitation of Kvβ2 from the heart resulted in co‐immunoprecipitation of Kv1.5 and Kv1.4. In comparison with WT cardiac myocytes, the surface expression of Kv1.5 was reduced in KO myocytes. Ventricular myocytes from KO mice had significantly lower peak Kv current density (p<0.01) and component Kv current densities as determined by triexponential curve fitting suggesting reduced IKur and Ito (p<0.01, n=34 cells, N=10 mice), consistent with a decrease in Kv1.4 and Kv1.5 channel density and a shift in the voltage‐dependence of the activation of the current. The duration of action potentials (APD 20, APD90) recorded from myocytes isolated from the right ventricle was significantly prolonged in KO mice (p<0.01, n=32 cells, N=11 mice) when compared with WT cardiac myocytes. Simulation of action potentials in CellML using experimental voltage clamp data resulted in similarly prolonged APs, as did partial pharmacological blockade of Kv1 channels with 4‐aminopyridine.ConclusionMouse cardiac myocytes express Kvβ2, which interacts primarily with Kv1.4 and Kv1.5 channels. Association with Kvβ2 regulates Kv1 expression and function in the sarcolemma, and thereby, the repolarization phase of the action potential.
Immature Endothelial Cells Initiate Endothelin‐Mediated Constriction of Newborn Arteries
Authors: Chang Fumin , Flavahan Sheila , Flavahan Nicholas A 
Published: November 2023

The early postnatal period is associated with remarkable changes in the morphology and function of endothelial cells. Newborn arterial endothelial cells have a highly unusual phenotype, which includes minimal NO activity, heightened Rho/ROCK signaling, prominent actin stress fibers and poorly developed cellular junctions. During the immediate postnatal period, arterial endothelial cells then acquire characteristics normally associated with mature protective cells including exuberant NO‐mediated dilation, complex endothelial cellular junctions and reorganization of stress fibers to a cortical actin network. Indeed, the atypical phenotype of immature newborn endothelial cells resembles dysfunctional endothelial cells that contribute to the development of vascular disease. The present experiments were performed to determine whether newborn endothelial cells might express and release endothelin‐1 (ET‐1) and initiate endothelium‐dependent constriction, hallmark features of pathological endothelial dysfunction. Carotid arteries were isolated from newborn (postnatal day 1, P1), P7 and P21 mice and mounted in pressure myographs at a constant transmural pressure of 20 mmHg (mean blood pressure for a P1 mouse). Exogenous ET‐1 caused similar concentration‐dependent constrictions of P1, P7 and P21 arteries. Endothelial stimulation with A23187 or thrombin caused powerful constriction in P1 arteries, no significant change in arterial diameter in P7 arteries, and powerful dilatation in P21 arteries. In P1 arteries, constriction to thrombin or A23187 was inhibited by endothelial denudation, by ET‐1 receptor antagonists (BQ123 or BQ123 plus BQ788), or by inhibition of endothelin converting enzyme (ECE) (phosphoramidon or SM19712). ET‐1 receptor antagonism did not significantly affect responses to thrombin or A23187 in more mature P7 and P21 arteries. Endothelial expression of ET‐1 peptides, assessed by immunofluorescent analysis, was significantly increased in P1 compared to P21 arteries. Endothelial stimulation with thrombin rapidly increased the endothelium‐dependent release of ET‐1 from P1 but not P21 arteries. Therefore, newborn endothelial cells express high levels of ET‐1 peptides, rapidly release ET‐1 in response to endothelial stimulation and initiate ET‐1 mediated endothelium‐dependent constriction. This activity is lost as the endothelium matures in the immediate postnatal period. The unusual phenotype of newborn endothelium including heightened activity of ET‐1 is reminiscent of pathological endothelial dysfunction, which may reflect reemergence of the newborn phenotype.Support or Funding InformationNIH R01 HD078639
The Exon Junction Complex proteins are non‐AMP kinase dependent sensors of energetic stress and regulate contractility and sarcomere structure in cardiac myocytes
Authors: Boateng Samuel , Pierrat Olivier , Paudyal Anju , koroleva Olga 
Published: November 2023

The heart is known to undergo significant alterations in gene expression during stress and sometimes this adaptation can lead to heart failure. The mechanism by which cells select specific mRNAs for translation is not fully understood, but the Exon Junction Complex (EJC) plays a major role in this process in eukaryotes. The EJC selects the mRNA isoforms to be exported to the cytoplasm and determines whether they are then translated into protein. In this study we hypothesised that the EJC is involved in the regulation of gene expression during the stress response in cardiac myocytes. Using cultured rat neonatal myocytes and subcellular fractionation we examined the cellular distribution of two major components of the EJC in response to metabolic stress. In cardiac myocytes we found that there was a significant relocalisation of eIF4A3 and mago from the nucleus to cytoplasm following 18 hours of hypoxia. To examine the effect of metabolic stress on the EJC in more detail we treated cardiac myocytes with 50mM NaN3 for 4 hours to mimic the metabolic stress induced by hypoxia. Following NaN3 treatment there was a significant relocalisation of eIF4A3 and mago to the cytoplasm and this was associated with the formation of cytoplasmic stress granules. However, immunocytochemical staining with the stress granule marker PABP1 showed that cytoplasmic mago did not colocalise with stress granules. To examine whether the effects of metabolic stress on the EJC proteins was dependent on the metabolic sensor AMP kinase (AMPK), we treated myocytes with 1μM dorsomorphin in combination with NaN3. Dorsomorphin augmented the translocation of mago and eIF4A3 from the nucleus to the cytoplasm. This indicates that the EJC proteins respond to metabolic stress independently of AMPK activity. Moreover, the inhibition of AMPK was associated with increased metabolic cellular stress resulting in the augmented subcellular translocation of EJC proteins. Constructs of eIF4A3 tagged with green fluorescence protein on the N terminus resulted in a predominantly cytosolic protein distribution that did not relocalise in response to NaN3 treatment. This suggests that the N‐terminus of eIF4A3 is necessary for subcellular localisation. Finally, to determine the function of eIF4A3 in cardiac myocytes we knocked down the protein with siRNA. Protein knockdown of eIF4A3 resulted in cessation of cell contractility 96 hours post treatment. There was also a significant reduction in the number of intact sarcomeres suggesting that the contractile filaments were degrading in response to reduced eIF4A3. Taken together, these data suggest that eIF4A3 and other EJC members play an important role in the myocyte stress response, cell contractility and morphology.Support or Funding InformationThis work was supported by the British Heart Foundation project grant PG/10/64/28520 and PG/10/97/28666
Reperfusion Therapy with Rapamycin Prevents Myocardial Injury and restores Plasma Levels of Stress‐Sensitive microRNA‐191 in Conscious Diabetic Rabbits
Authors: Samidurai Arun , Ockaili Ramzi , Kukreja Rakesh C , Das Anindita 
Published: November 2023

BackgroundDiabetes is a major risk factor for myocardial infarction (MI). Persistent activation of the mammalian target of rapamycin (mTOR) leads to diabetic complications and plays a critical role in myocardial reperfusion injury following ischemia. mTOR inhibition with rapamycin at reperfusion preserves cardiac function with reduction of myocardial infarction in type 2 diabetic mice. To demonstrate the clinical potential of these novel observations, we examined the effect of rapamycin in a conscious diabetic rabbit model of MI. In addition we examined whether rapamycin influences the plasma levels of stress‐sensitive microRNA‐191.Methods and ResultsTo induce diabetes, alloxan monohydrate (125 mg/kg, i.v.) was administered in New Zealand white rabbits (n=10; average 3kg, 4 month old). Blood glucose levels increased to 345±66 mg/dL (n=10) following 2 weeks of alloxan administration. Subsequently, balloon occluders were implanted and 7 days later, these rabbits were subjected to a 30‐min ischemia and 3 days of reperfusion. Rapamycin (0.25 mg/kg, i.v.) or DMSO (vehicle) was infused 5 min before reperfusion. Plasma was collected at baseline, 1, 2, 4 and 24 hours during the procedure. The results show that rapamycin significantly reduced myocardial infarction in diabetic rabbit following I/R (Figure 1A) as assessed by triphenyltetrazolium chloride staining. Rapamycin also reduced cardiac troponin I level in blood at 1 hr and 2 hr of reperfusion as compared to vehicle alone (Figure 1B). Moreover the plasma levels of microRNA‐191 were decreased at 1 and 2 hr after I/R which were significantly elevated with rapamycin treatment.ConclusionThe current translational study suggests that rapamycin could be a promising drug in attenuation of reperfusion injury in diabetic patients following acute MI. Also, micro‐RNA‐191 may have a role in attenuation of I/R injury following rapamycin treatment.Support or Funding InformationNIH R37 HL051045, RO1 HL079424, RO1 HL093685, RO1 HL118808 (RCK)A.D. Williams’ Fund of VCU UL1RR031990 (AD)VCU National Center for Advancing Translational Sciences (UL1TR000058) and CCTR Endowment Fund (AD)
HIF‐1α Stabilization Inhibits Basal and Maximal Mitochondrial Respiration in Fetal Cardiac and Skeletal Myocytes
Authors: Saharan Sunil , Espinoza Herbert M , Brown Laura D , Jonker Sonnet S 
Published: November 2023

IntroductionHypoxia is the most common stress experienced by fetuses, and chronic hypoxia affects cardiac function as well as growth of fetal striated muscle. Cardiac muscle is active in utero, while skeletal muscle is relatively inactive, thus the metabolic effects of hypoxia may be differently mediated in these cells. Hypoxia signaling is mediated through both HIF‐1α and depletion of high‐energy phosphates, but the relative regulatory roles of these two signals on metabolism in fetal striated muscle is unknown.HypothesisChronic HIF‐1α stabilization and reduced mitochondrial proton gradient suppress oxidative respiration and promote glycolysis in fetal sheep cardiac and skeletal myocytes.MethodsNear‐term fetal sheep cardiomyocytes and biceps femoris myoblasts were cultured (n=4) and myoblasts were differentiated to myotubes. Cells were treated for 48h with cobalt (II) chloride (to stabilize HIF‐1α) or carbonyl cyanide 4‐trifluoromethoxy phenylhydrazone (FCCP; to diminish the mitochondrial proton gradient). Doses were selected to achieve 80% cell survival; compounds and detached cells were washed out prior to metabolic assessment. The Seahorse XFe96 metabolic analyzer measured oxygen consumption rate (respiration) and H+ production rate (glycolysis), which were normalized to protein content. Data were analyzed within cell type by one‐way analysis of variance followed by Tukey's multiple comparisons test, and between cell type by Student's t‐test. Noted differences were significant at p<0.05.ResultsBasal ATP‐producing respiration (excluding proton leak) was 2.5 fold higher in cardiomyocytes than skeletal myotubes, and maximal respiration was 5.4 fold higher, while glycolysis was not different between cardiac and skeletal myocytes. In skeletal myotubes, basal ATP‐producing respiration was reduced 24% by FCCP and 77% by cobalt. In cardiomyocytes, ATP‐producing respiration was reduced 39% by cobalt. No significant changes occurred in glycolytic capacity following chronic cobalt or FCCP treatment.ConclusionMetabolic changes following treatment were similar between cardiac and skeletal muscle, although the metabolic capacity of fetal cardiomyocytes was many‐fold higher than skeletal myotubes. HIF‐1α signaling, as occurs in chronic hypoxia, significantly reduces the mitochondrial oxidative respiratory capacity of fetal striated muscle. This change may affect cardiac and skeletal muscle function and growth during and following birth.Support or Funding InformationThis research was supported by NICHD under awards numbered R01HD071068 and P01HD034430.
Hypoxic inhibition of mitochondrial oxygen consumption depends on HIF‐1α
Authors: Mairbaurl Heimo , Baloglu Emel , Zhang Ying 
Published: November 2023

The AMP‐activated protein kinase (AMPK) couples cellular energy demand and ATP production by increasing PGC‐1α‐dependent expression of mitochondrial proteins. Hypoxia decreases mitochondrial activity. Here we tested the hypothesis that stimulation of AMPK, which indicates increased metabolic demand, blunts the impaired mitochondrial function in hypoxia to prevent functional impairment of cardiomyocytes. H‐10 cardiomyocytes were treated with AICAR to stimulate AMPK in normoxia and hypoxia (1.5 % O2).AICAR, hypoxia, and their combination increased phosphorylated AMPK indicating its stimulation. AICAR induced a 3x increase in mitochondrial oxygen consumption (JO2), whereas hypoxia decreased JO2 (−20%) and prevented the AICAR‐induced increase. Hypoxia did not prevent the AICAR‐induced increase in pyruvate dehydrogenase (PDH) activity despite increased PDK mRNA expression. The HIF‐dependent genes GAPD (1.5x) and BNIP3 (4x) were increased in hypoxia, also in the presence of AICAR. Silencing HIF‐1α prevented the hypoxia‐induced inhibition of mitochondrial respiration in control and AICAR‐treated cells and significantly reduces upregulation of HIF‐dependent genes.These results indicate that hypoxia prevents the AMP‐ and AMPK‐dependent stimulation of mitochondrial metabolism by HIF‐1α dependent mechanisms thus disproving our hypothesis. The inhibition by hypoxia seems to be caused by decreasing the expression of enzymes of the electron transfer chain by blocking signaling downstream of AMPK rather than by decreasing PDH‐mediated substrate flux into mitochondria. Together this indicates that hypoxia decreases cardiomyocyte oxidative metabolism despite increased metabolic demand.Support or Funding InformationDFG Ma‐1503/29‐1
Pregnancy activates HIF‐1a and PPARγ in left ventricle of rats
Authors: Godoy‐Lugo Jose Arquimides , Hernandez‐Palomares Magally Luisa Elena , Rodriguez‐Martinez Daniel , Rosas‐Rodriguez Jesus Alfredo , Ortiz Rudy M , Soñanez‐Organis Jose Guadalupe 
Published: November 2023

Pregnancy‐induced physiologic cardiac hypertrophy with important changes in the energetic metabolism to support its contractile demands and maintain viability. HIF‐1α and PPARγ activates glycolytic and glycerol‐lipid biosynthesis genes in response to pathologic hypertrophy, respectively. Therefore, these changes increase the rate of glycolysis and glucose‐to‐lipid ratio via glycerol‐3‐phosphate (G3P) pathway. However, the contribution of HIF‐1α and PPARγ to the pregnancy‐induced hypertrophy and its reversible process (postpartum) to heart metabolism are not defined. We evaluated HIF‐1α and PPARγ transcriptional activity, and quantified their target genes, key enzymes, and metabolic intermediates of glycolysis and lipid metabolism in the left ventricle of rats before, during, and after pregnancy. HIF‐1α and PPARγ activity increased 1.2‐ and 1.6‐fold, respectively, during pregnancy, and decreased to basal levels during postpartum. Expressions of mRNA for glycolytic (HK2, PFKM and GAPDH) and glycerol‐lipid biosynthesis (GPAT and GPD1) genes increased 1.6‐ to 14‐fold during pregnancy and returned to basal levels postpartum. While the increase in GPD1 expression translated to an increase in its activity, such was not the case for GAPDH suggestive of differential, post‐translational regulation of these proteins. Glycolytic (glucose, lactate and DHAP) and lipid biosynthesis (G3P and FFA) intermediates increased with pregnancy and were maintained postpartum. Results demonstrate that pregnancy induces the expression of glycolytic and glycerol‐lipid biosynthesis genes that translates to a shift in cardiac metabolism, which is sustained, at least acutely, postpartum and is associated with the activation of HIF‐1α and PPARγ.Support or Funding InformationUniversity of California Institute for Mexico and the United States and Mexico's National Council for Science and Technology (UC MEXUS‐CONACYT) and The Integral Program for Institutional Strengthening (PIFI).
Long‐term Sugar‐sweetened Beverage Intake elicits Early Metabolic Perturbations in Rat Hearts: a Bitter‐sweet Taste?
Authors: Driescher Natasha , Benade Janina , Joseph Danzil , Marnewick Jeanine , Bester Dirk , Essop M. Faadiel 
Published: November 2023

Increased consumption of sugar‐sweetened beverages (SSB) is linked to the development of obesity, diabetes and heart diseases. However, the underlying mechanisms driving this process remain unclear. For this study we hypothesized that regular SSB intake elicits metabolic dysregulation and activates non‐oxidative glucose pathways (NOGPs), i.e. polyol pathway, hexosamine biosynthetic pathway (HBP), advanced glycation end‐products, and PKC; contributing to the onset of cardio‐metabolic complications. To further investigate this, we began by establishing and characterizing a rodent model of long‐term SSB consumption. Here male Wistar rats (~200 gr) were daily gavaged with 3–5.1 mL of a local SSB for a 3‐month period vs. two matched controls (water gavaging; caloric equivalent). Dietary behaviour and phenotypic changes were monitored on a weekly basis while we also measured metabolite levels, performed oral glucose tolerance tests (OGTT), determined the degree of oxidative stress and assessed NOGP activation in heart and liver tissues. These data demonstrate that SSB consumption did not cause significant changes in weight gain and food consumption, while fasting blood glucose levels remained unchanged. However, heart: body weight was higher vs. controls (p<0.05), while the OGTT analysis established that the area under the curve was significantly lower for the SSB group vs. controls. Myocardial cholesterol and triglyceride levels were not significantly altered. No differences were found between groups for oxidative stress markers (GSH/GSSG; malondialdehyde) evaluated in heart and liver tissues. Of note, SSB intake triggered the activation of the HBP and PKC pathway in heart tissues vs. controls (p<0.05). Our study successfully established a novel in vivo rat model of SSB consumption. These findings reveal that long‐term SSB intake results in dysregulation of potentially detrimental metabolic circuits (HBP, PKC pathway) in the heart that may place the organism at risk despite an apparently “healthy” phenotype.Support or Funding InformationResearch funded by the National Research Foundation and Stellenbosch University (South Africa).
Long‐term Effect of a Post‐natal Hyperoxia on Cardiac Autophagy
Authors: Hopp Alexandria L , Tetri Laura Hope , Kumari Santosh , Haraldsdottir Kristin , Pegelow David F , Braun Rudolf K , Gross Kara N , Eldridge Marlowe W 
Published: November 2023

IntroductionBronchopulmonary dysplasia (BPD) is a chronic lung disease affecting pre‐term infants characterized by decreased alveolarization and impaired vascular development, and may contribute to the development of pulmonary hypertension (PH) and subsequent right ventricular dysfunction. Currently, little is known about the long‐term cardiac outcomes of individuals with BPD. The aim of this project is to evaluate markers of autophagy and apoptosis in the right ventricle of aged rats exposed to brief postnatal hyperoxia. We hypothesize that the right ventricle from rats exposed to postnatal hyperoxia will demonstrate decreased or impaired autophagy with age.MethodsSprague‐Dawley rats were exposed to either 85% oxygen (HYP) (n=12, 6 males) or 21% oxygen (CTL) (n=8, 3 males) for 14 days within 12 hours following birth. After 14 days, these animals were allowed to age out for one year in room air. At one year, the rats were sacrificed and tissues collected. Markers of autophagy and apoptosis (BCL2, Bax, Beclin‐1, LC3B, LAMP2, and P62) in the right ventricle were evaluated using Western Blot analysis. Statistical analysis was done via t‐test.ResultsAged HYP rats demonstrate a persistent right ventricular hypertrophy, with the right ventricle to left ventricle plus septum ratio significantly elevated compared to CTL rats (0.21 ± 0.02 CTL versus 0.25 ± 0.03 HYP, p=0.002). Beclin‐1, a protein necessary for nucleation of autophagosomes, is significantly upregulated in HYP as compared to CTL (46580±1405 CTL HYP versus 34633±3008, p=.004). LC3B, a protein needed for elongation of autophagosomes, is significantly downregulated in HYP rats than in CTL rats (.036 ± .001 HYP versus .052 ±.006 CTL, p=.01). Lamp2, a membrane bound lysosomal protein needed for phagosomal and autophagsomal maturation, is significantly downregulated in HYP compared to CTL (.01 ± .0008 HYP versus .017 ± .001 CTL, p=.001). p62 and Bax were not significantly different in the right ventricles of HYP as compared to CTL.ConclusionThe purpose of this study was to determine if there are effects of post‐natal hyperoxic exposure in the right ventricle at one year of age. We found that the elevated Fulton Index persists which has previously been associated with pulmonary hypertension and right ventricular dysfunction. The significant upregulation of beclin‐1 in the aged HYP right ventricles suggests that there is an increase in autophagic activation. However, the downregulation of LC3B and LAMP2 in HYP may represent impaired phagosomal maturation and an accumulation of autophagosomes consistent with impaired completion of autophagy. The data suggests that while there is significant right ventricular hypertrophy and an upregulation of autophagic activation, this autophagy may not be completed in rats exposed to hyperoxia.Support or Funding InformationFunding source: NHLBI, R01 HL115061‐03, Suppl (Eldridge) & NHLBI, R01 HL115061 (Eldridge)