H2O2-induced Ca2+ overload in NRVM involves ERK1/2 MAP kinases: role for an NHE-1-dependent pathway

2002 ◽  
Vol 283 (2) ◽  
pp. H598-H605 ◽  
Author(s):  
Emily C. Rothstein ◽  
Kenneth L. Byron ◽  
Ryan E. Reed ◽  
Larry Fliegel ◽  
Pamela A. Lucchesi
Keyword(s):  
Map Kinases ◽  
Ventricular Myocytes ◽  
Ischemia Reperfusion ◽  
Neonatal Rat ◽  
Dependent Manner ◽  
Low Doses ◽  
Extracellular Signal ◽  
Intracellular Ca ◽  
Neonatal Rat Ventricular Myocytes ◽  
The Relationship

Generation of reactive oxygen species (ROS) and intracellular Ca2+ overload are key mechanisms involved in ischemia-reperfusion (I/R)-induced myocardial injury. The relationship between I/R injury and Ca2+overload has not been fully characterized. The increase in Na+/H+ exchanger (NHE-1) activity observed during I/R injury is an attractive candidate to link increased ROS production with Ca2+ overload. We have shown that low doses of H2O2 increase NHE-1 activity in an extracellular signal-regulated kinase (ERK)-dependent manner. In this study, we examined the effect of low doses of H2O2 on intracellular Ca2+ in fura 2-loaded, spontaneously contracting neonatal rat ventricular myocytes. H2O2 induced a time- and concentration-dependent increase in diastolic intracellular Ca2+ concentration that was blocked by inhibition of ERK1/2 activation with 5 μM U-0126 (88%) or inhibition of NHE-1 with 5 μM HOE-642 (50%). Increased NHE activity was associated with phosphorylation of the NHE-1 carboxyl tail that was blocked by U-0126. These results suggest that H2O2 induced Ca2+ overload is partially mediated by NHE-1 activation secondary to phosphorylation of NHE-1 by the ERK1/2 MAP kinase pathway.

Glucosamine inhibits angiotensin II-induced cytoplasmic Ca2+ elevation in neonatal cardiomyocytes via protein-associated O-linked N-acetylglucosamine

2006 ◽  
Vol 290 (1) ◽  
pp. C57-C65 ◽  
Author(s):  
Tamas Nagy ◽  
Voraratt Champattanachai ◽  
Richard B. Marchase ◽  
John C. Chatham
Keyword(s):  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Ang Ii ◽  
Neonatal Cardiomyocytes ◽  
Hexosamine Biosynthesis ◽  
Intracellular Ca ◽  
Glcnac Transferase ◽  
Neonatal Rat Ventricular Myocytes ◽  
The Relationship ◽  
Hexosamine Biosynthesis Pathway

We previously reported that glucosamine and hyperglycemia attenuate the response of cardiomyocytes to inositol 1,4,5-trisphosphate-generating agonists such as ANG II. This appears to be related to an increase in flux through the hexosamine biosynthesis pathway (HBP) and decreased Ca2+ entry into the cells; however, a direct link between HBP and intracellular Ca2+ homeostasis has not been established. Therefore, using neonatal rat ventricular myocytes, we investigated the relationship between glucosamine treatment; the concentration of UDP- N-acetylglucosamine (UDP-GlcNAc), an end product of the HBP; and the level of protein O-linked N-acetylglucosamine ( O-GlcNAc) on ANG II-mediated changes in intracellular free Ca2+ concentration ([Ca2+]i). We found that glucosamine blocked ANG II-induced [Ca2+]i increase and that this phenomenon was associated with a significant increase in UDP-GlcNAc and O-GlcNAc levels. O-(2-acetamido-2-deoxy-d-glucopyranosylidene)-amino- N-phenylcarbamate, an inhibitor of O-GlcNAcase that increased O-GlcNAc levels without changing UDP-GlcNAc concentrations, mimicked the effect of glucosamine on the ANG II-induced increase in [Ca2+]i. An inhibitor of O-GlcNAc-transferase, alloxan, prevented the glucosamine-induced increase in O-GlcNAc but not the increase in UDP-GlcNAc; however, alloxan abrogated the inhibition of the ANG II-induced increase in [Ca2+]i. These data support the notion that changes in O-GlcNAc levels mediated via increased HBP flux may be involved in the regulation of [Ca2+]i homeostasis in the heart.

The peroxisomal enzyme, FAR1, is induced during ER stress in an ATF6-dependent manner in cardiac myocytes

2021 ◽  
Author(s):  
Kayleigh G. Marsh ◽  
Adrian Arrieta ◽  
Donna J. Thuerauf ◽  
Erik A. Blackwood ◽  
Lauren MacDonnell ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Er Stress ◽  
Cardiac Myocytes ◽  
Ventricular Myocytes ◽  
Ischemia Reperfusion ◽  
Cell Types ◽  
Fatty Acyl ◽  
Neonatal Rat ◽  
Dependent Manner ◽  
Peroxisomal Enzyme

While peroxisomes have been extensively studied in other cell types, their presence and function have gone virtually unexamined in cardiac myocytes. Here, in neonatal rat ventricular myocytes (NRVM) we showed that several known peroxisomal proteins co-localize to punctate structures with a morphology typical of peroxisomes. Surprisingly, we found that the peroxisomal protein, fatty acyl-CoA reductase 1 (FAR1), was upregulated by chemical and pathophysiological ER stress induced by tunicamycin (TM) and simulated ischemia/reperfusion (sI/R), respectively. Moreover, FAR1 induction in NRVM was mediated by the ER stress-sensor, activating transcription factor 6 (ATF6). Functionally, FAR1 knockdown reduced myocyte death during oxidative stress induced by either sI/R or hydrogen peroxide (H2O2). Thus, Far1 is an ER stress-inducible gene, which encodes a protein that localizes to peroxisomes of cardiac myocytes, where it reduces myocyte viability during oxidative stress. Since FAR1 is critical for plasmalogen synthesis, these results imply that plasmalogens may exert maladaptive effects on the viability of myocytes exposed to oxidative stress.

Nav1.5-dependent persistent Na+ influx activates CaMKII in rat ventricular myocytes and N1325S mice

2011 ◽  
Vol 301 (3) ◽  
pp. C577-C586 ◽  
Author(s):  
Lina Yao ◽  
Peidong Fan ◽  
Zhan Jiang ◽  
Serge Viatchenko-Karpinski ◽  
Yuzhi Wu ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Polymorphic Ventricular Tachycardia ◽  
Inhibitory Peptide ◽  
Rat Ventricular Myocytes ◽  
Protein Protein Interaction ◽  
Intracellular Ca ◽  
Dependent Protein ◽  
Neonatal Rat Ventricular Myocytes ◽  
Camkii Activation

Late Na+ current ( INaL) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) are both increased in the diseased heart. Recently, CaMKII was found to phosphorylate the Na+ channel 1.5 (Nav1.5), resulting in enhanced INaL. Conversely, an increase of INaL would be expected to cause elevation of intracellular Ca2+ and activation of CaMKII. However, a relationship between enhancement of INaL and activation of CaMKII has yet to be demonstrated. We investigated whether Na+ influx via Nav1.5 leads to CaMKII activation and explored the functional significance of this pathway. In neonatal rat ventricular myocytes (NRVM), treatment with the INaL activators anemone toxin II (ATX-II) or veratridine increased CaMKII autophosphorylation and increased phosphorylation of CaMKII substrates phospholamban and ryanodine receptor 2. Knockdown of Nav1.5 (but not Nav1.1 or Nav1.2) prevented ATX-II-induced CaMKII phosphorylation, providing evidence for a specific role of Nav1.5 in CaMKII activation. In support of this view, CaMKII activity was also increased in hearts of transgenic mice overexpressing a gain-of-function Nav1.5 mutant (N1325S). The effects of both ATX-II and the N1325S mutation were reversed by either INaL inhibition (with ranolazine or tetrodotoxin) or CaMKII inhibition (with KN93 or autocamtide 2-related inhibitory peptide). Furthermore, ATX-II treatment also induced CaMKII-Nav1.5 coimmunoprecipitation. The same association between CaMKII and Nav1.5 was also found in N1325S mice, suggesting a direct protein-protein interaction. Pharmacological inhibitions of either CaMKII or INaL also prevented ATX-II-induced cell death in NRVM and reduced the incidence of polymorphic ventricular tachycardia induced by ATX-II in rat perfused hearts. Taken together, these results suggest that a Nav1.5-dependent increase in Na+ influx leads to activation of CaMKII, which in turn phosphorylates Nav1.5, further promoting Na+ influx. Pharmacological inhibition of either CaMKII or Nav1.5 can ameliorate cardiac dysfunction caused by excessive Na+ influx.

scholarly journals Glucosamine protects neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein O-GlcNAc and increased mitochondrial Bcl-2

2008 ◽  
Vol 294 (6) ◽  
pp. C1509-C1520 ◽  
Author(s):  
Voraratt Champattanachai ◽  
Richard B. Marchase ◽  
John C. Chatham
Keyword(s):  
Protective Effect ◽  
Ventricular Myocytes ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Neonatal Rat ◽  
Cellular Injury ◽  
Protective Effects ◽  
Neonatal Cardiomyocytes ◽  
Glcnac Transferase ◽  
Neonatal Rat Ventricular Myocytes

We have previously reported that glucosamine protected neonatal rat ventricular myocytes against ischemia-reperfusion (I/R) injury, and this was associated with an increase in protein O-linked- N-acetylglucosamine ( O-GlcNAc) levels. However, the protective effect of glucosamine could be mediated via pathways other that O-GlcNAc formation; thus the initial goal of the present study was to determine whether increasing O-GlcNAc transferase (OGT) expression, which catalyzes the formation of O-GlcNAc, had a protective effect similar to that of glucosamine. To better understand the potential mechanism underlying O-GlcNAc-mediated cytoprotection, we examined whether increased O-GlcNAc levels altered the expression and translocation of members of the Bcl-2 protein family. Both glucosamine (5 mM) and OGT overexpression increased basal and I/R-induced O-GlcNAc levels, significantly decreased cellular injury, and attenuated loss of cytochrome c. Both interventions also attenuated the loss of mitochondrial membrane potential induced by H2O2 and were also associated with an increase in mitochondrial Bcl-2 levels but had no effect on Bad or Bax levels. Compared with glucosamine and OGT overexpression, NButGT (100 μM), an inhibitor of O-GlcNAcase, was less protective against I/R and H2O2 and did not affect Bcl-2 expression, despite a 5- to 10-fold greater increase in overall O-GlcNAc levels. Decreased OGT expression resulted in lower basal O-GlcNAc levels, prevented the I/R-induced increase in O-GlcNAc and mitochondrial Bcl-2, and increased cellular injury. These results demonstrate that the protective effects of glucosamine are mediated via increased formation of O-GlcNAc and suggest that this is due, in part, to enhanced mitochondrial Bcl-2 translocation.

scholarly journals Signaling responses after exposure to 5α-dihydrotestosterone or 17β-estradiol in norepinephrine-induced hypertrophy of neonatal rat ventricular myocytes

2010 ◽  
Vol 108 (3) ◽  
pp. 686-696 ◽  
Author(s):  
Yevgeniya E. Koshman ◽  
Mariann R. Piano ◽  
Brenda Russell ◽  
Dorie W. Schwertz
Keyword(s):  
Cell Size ◽  
Sex Hormones ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Heart Cells ◽  
Adrenergic Stimulation ◽  
Rat Ventricular Myocytes ◽  
Extracellular Signal ◽  
17Β Estradiol ◽  
Neonatal Rat Ventricular Myocytes

Androgens appear to enhance, whereas estrogens mitigate, cardiac hypertrophy. However, signaling pathways in cells for short (3 min) and longer term (48 h) treatment with 17β-estradiol (E2) or 5α-dihydrotestosterone (DHT) are understudied. We compared the effect of adrenergic stimulation by norepinephrine (NE; 1 μM) alone or in combination with DHT (10 nM) or E2 (10 nM) treatment in neonatal rat ventricular myocytes (NRVMs) by cell area, protein synthesis, sarcomeric structure, gene expression, phosphorylation of extracellular signal-regulated (ERK), and focal adhesion kinases (FAK), and phospho-FAK nuclear localization. NE alone elicited the expected hypertrophy and strong sarcomeric organization, and DHT alone gave a similar but more modest response, whereas E2 did not alter cell size. Effects of NE dominated when used with either E2 or DHT with all combinations. Both sex hormones alone rapidly activated FAK but not ERK. Long-term or brief exposure to E2 attenuated NE-induced FAK phosphorylation, whereas DHT had no effect. Neither hormone altered NE-elicited ERK activation. Longer term exposure to E2 alone reduced FAK phosphorylation and reduced nuclear phospho-FAK, whereas its elevation was seen in the presence of NE with both sex hormones. The mitigating effects of E2 on the NE-elicited increase in cell size and the hypertrophic effect of DHT in NRVMs are in accordance with results observed in whole animal models. This is the first report of rapid, nongenomic sex hormone signaling via FAK activation and altered FAK trafficking to the nucleus in heart cells.

Abstract 318: Targeting of Tp53inp1 by Mir-221 to Reduce P62-mediated Autophagy is Cardioprotective in Ischemia / Reperfusion Injury

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Peipei Wang ◽  
Qiying Chen ◽  
Arthur M Richards
Keyword(s):  
Protective Effect ◽  
Cell Count ◽  
Cell Injury ◽  
Ventricular Myocytes ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Neonatal Rat ◽  
Autophagosome Formation ◽  
Direct Targeting ◽  
Neonatal Rat Ventricular Myocytes

Purpose: Tumor protein 53-induced nuclear protein 1 (Tp53inp1) acts as a tumor suppressor by inducing cell death. Tp53inp1 mRNA is a predicted target of miR-221. Whether targeting Tp53inp1 plays a role in miR-221-mediated cardioprotection has not been investigated. We hypothesized that miRNA-221 directly targets Tp53inp1 to reduce ischemia/reperfusion (I/R)-induced autophagy. Method: Myoblast H9c2 cells underwent 16 hours 0.2% O 2 hypoxia followed by 2 hours re-oxygenation (H-R, simulating I/R). H9c2 were transfected with miRNA-221 mimic (25 nmol) and scrambled mimic control (miR-221 and MC). Cell count/viability, WST assay, cell injury-induced LDH release, and GFP-LC3 labeled autophagosome formation were measured. Cells were collected for RT-qPCR and western blot (WB) analyses. pCMV-Myc-Tp53inp1 and pcDNA3.1-Flag-p62 plasmids were cloned and transfected into H9c2 for recovery and immuno-precipitation (IP) studies. The effects of miRNA-221 inhibitor in H9c2 were also assessed. Results: miR-221 significantly reduced H-R injury as indicated by higher cell count/viability and WST activity, and reduced LDH (miR-221 vs. MC p<0.05). qPCR confirmed that (1) miRNA-221 expression was reduced in H-R; (2) RISC-loaded (IP pull-down Ago-2) miRNA-221 increased by ~80 fold and reduced by 95% following mimic and inhibitor transfection respectively; (3) Increased Tp53inp1 following H-R was reversed by miR-221. miR-221 inhibited H-R induced autophagosome formation (GFP-LC3). WB indicated (1) increase of LC3-I/II ratio and p62, indicators of reduced autophagy, and (2) decrease of Tp53inp1 by miR-221. IP pull-down Myc-Tp53inp1 indicated the formation of p62-Tp53inp1 complex. The protective effect of miR-221 was abolished by Tp53inp1 overexpression (pCMV-Myc-Tp53inp1 and miRNA-221 mimic co-transfection). The protective effect was corroborated in neonatal rat ventricular myocytes (NRVM). MiRNA-221 inhibitor induced reverse effects. Conclusion: The cardioprotection of miR-221 entails direct targeting of Tp53inp1 which reducing p62-Tp53inp1 complex formation and inhibiting H-R-induced autophagy.

Phosphoinositide 3-kinase regulates excitation-contraction coupling in neonatal cardiomyocytes

2004 ◽  
Vol 286 (2) ◽  
pp. H796-H805 ◽  
Author(s):  
Susan A. McDowell ◽  
Eileen McCall ◽  
William F. Matter ◽  
Thomas B. Estridge ◽  
Chris J. Vlahos
Keyword(s):  
Steady State ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Dependent Manner ◽  
Neonatal Cardiomyocytes ◽  
Reverse Mode ◽  
Ec Coupling ◽  
Intracellular Ca ◽  
Phosphoinositide 3 Kinase ◽  
State Contraction

The phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 decreased steady-state contraction in neonatal rat ventricular myocytes (NRVM). To determine whether the effect on steady-state contraction could be due to decreased intracellular Ca2+ content, Ca2+ content was assessed with fluorescent plate reader analysis by using the caffeine-releasable Ca2+ stores as an index of sarcoplasmic reticulum (SR) Ca2+ content. Caffeine-releasable Ca2+ content was diminished in a dose-dependent manner with LY-294002, suggesting that the decrease in steady-state contraction was due to diminished intracellular Ca2+ content. Activation of the L-type Ca2+ channel by BAY K 8644 was attenuated by LY-294002, suggesting the effect of LY-294002 is to reduce Ca2+ influx at this channel. To investigate whether additional proteins involved in excitation-contraction (EC) coupling are likewise regulated by PI3K activity, the effects of compounds acting at sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a), the ryanodine receptor, and the Na/Ca exchanger (NCX) were compared with LY-294002. Inhibition of SERCA2a by thapsigargin increased basal Ca2+ levels in contrast to LY-294002, indicating that SERCA2a activity is sustained in the presence of LY-294002. Ryanodine decreased SR Ca2+ content. The additive effect with coadministration of LY-294002 could be attributed to a decrease in Ca2+ influx at the L-type Ca2+ channel. The NCX inhibitor Ni2+ was used to investigate whether the decrease in intracellular Ca2+ content with LY-294002 could be due to inhibition of the NCX reverse-mode activity. The minimal effect of LY-294002 with Ni2+ suggests that the primary effect of LY-294002 on EC coupling occurs through inhibition of PI3K-mediated L-type Ca2+ channel activity.

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