Functional analysis of Na+/K+-ATPase isoform distribution in rat ventricular myocytes

2007 ◽  
Vol 293 (1) ◽  
pp. C321-C327 ◽  
Author(s):  
Sanda Despa ◽  
Donald M. Bers
Keyword(s):  
Cardiac Myocyte ◽  
Ventricular Myocytes ◽  
Α Subunit ◽  
Rat Ventricular Myocytes ◽  
High Affinity ◽  
Functional Density ◽  
T Tubules ◽  
Subunit Isoforms ◽  
Ouabain Sensitivity ◽  
Cell Capacitance

The Na+/K+-ATPase (NKA) is the main route for Na+ extrusion from cardiac myocytes. Different NKA α-subunit isoforms are present in the heart. NKA-α1 is predominant, although there is a variable amount of NKA-α2 in adult ventricular myocytes of most species. It has been proposed that NKA-α2 is localized mainly in T-tubules (TT), where it could regulate local Na+/Ca2+ exchange and thus cardiac myocyte Ca2+. However, there is controversy as to where NKA-α1 vs. NKA-α2 are localized in ventricular myocytes. Here, we assess the TT vs. external sarcolemma (ESL) distribution functionally using formamide-induced detubulation of rat ventricular myocytes, NKA current (IPump) measurements and the different ouabain sensitivity of NKA-α1 (low) and NKA-α2 (high) in rat heart. Ouabain-dependent IPump inhibition in control myocytes indicates a high-affinity NKA isoform (NKA-α2, K1/2 = 0.38 ± 0.16 μM) that accounts for 29.5 ± 1.3% of IPump and a low-affinity isoform (NKA-α1, K1/2 = 141 ± 17 μM) that accounts for 70.5% of IPump. Detubulation decreased cell capacitance from 164 ± 6 to 120 ± 8 pF and reduced IPump density from 1.24 ± 0.05 to 1.02 ± 0.05 pA/pF, indicating that the functional density of NKA is significantly higher in TT vs. ESL. In detubulated myocytes, NKA-α2 accounted for only 18.2 ± 1.1% of IPump. Thus, ∼63% of IPump generated by NKA-α2 is from the TT (although TT are only 27% of the total sarcolemma), and the NKA-α2/NKA-α1 ratio in TT is significantly higher than in the ESL. The functional density of NKA-α2 is ∼4.5 times higher in the T-tubules vs. ESL, whereas NKA-α1 is almost uniformly distributed between the TT and ESL.

Validation of formamide as a detubulation agent in isolated rat cardiac cells

2002 ◽  
Vol 283 (4) ◽  
pp. H1720-H1728 ◽  
Author(s):  
Fabien Brette ◽  
Kimiaki Komukai ◽  
Clive H. Orchard
Keyword(s):  
Cell Function ◽  
Ventricular Myocytes ◽  
Cardiac Cells ◽  
Direct Effects ◽  
Microtubule Network ◽  
Rat Ventricular Myocytes ◽  
Ventricular Cells ◽  
T Tubules ◽  
Cell Capacitance ◽  
Action Potential Configuration

Kawai M, Hussain M, and Orchard CH. Am J Heart Circ Physiol 277: H603–H609, 1999 developed a technique to detubulate rat ventricular myocytes using formamide and showed that detubulation results in a decrease in cell capacitance, Ca2+ current density, and Ca2+ transient amplitude. We have investigated the mechanism of this detubulation and possible direct effects of formamide. Staining ventricular cells with di-8-ANEPPS showed that the t tubule membranes remain inside the cell after detubulation; trapping of FITC-labeled dextran within the t tubules showed that detubulation occurs during formamide washout and that the t tubules appear to reseal within the cell. Detubulation had no effect on the microtubule network but resulted in loss of synchronous Ca2+ release on electrical stimulation. In contrast, formamide treatment of atrial cells did not significantly change cell capacitance, Ca2+ current amplitude, action potential configuration, the Ca2+ transient or the response of the Ca2+ transient to isoprenaline. We conclude that formamide washout induces detubulation of single rat ventricular myocytes, leaving the t tubules within the cell, but without direct effects on cell proteins that might alter cell function.

p38 MAP kinase inhibitor reverses stress-induced cardiac myocyte dysfunction

2005 ◽  
Vol 98 (1) ◽  
pp. 77-82 ◽  
Author(s):  
Hong Kan ◽  
Dale Birkle ◽  
Abnash C. Jain ◽  
Conard Failinger ◽  
Sherry Xie ◽  
...  
Keyword(s):  
Map Kinase ◽  
Cardiac Myocyte ◽  
Kinase Inhibitor ◽  
Ventricular Myocytes ◽  
Myocardial Dysfunction ◽  
P38 Map Kinase ◽  
Border Detection ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
Fractional Shortening

Stress is gaining increasing acceptance as an independent risk factor contributing to adverse cardiovascular outcomes. Potential mechanisms responsible for the deleterious effects of stress on the development and progression of cardiovascular disease remain to be elucidated. An established animal model of stress in humans is the prenatally stressed (PS) rat. We stressed rats in their third trimester of pregnancy by daily injections of saline and moving from cage to cage. Male offspring of these stressed dams (PS) and age-matched male control offspring (control) were further subjected to restraint stress (R) at 6 and 7 wk of age. Echocardiography revealed a significant decrease in fractional shortening in PS + R vs. controls + R (45.8 ± 3.9 vs. 61.9 ± 2.4%, PS + R vs. controls + R; P < 0.01; n = 12). Isolated adult rat ventricular myocytes from PS + R also revealed diminished fractional shortening (6.7 ± 0.8 vs. 12.7 ± 1.1%, PS + R vs. controls + R; P < 0.01; n = 24) and blunted inotropic responses to isoproterenol ( P < 0.01; n = 24) determined by automated border detection. The p38 mitogen-activated protein (MAP) kinase inhibitor SB-203580 blocked p38 MAP kinase phosphorylation, reversed the depression in fractional shortening, and partially ameliorated the blunted adrenergic signaling seen in adult rat ventricular myocytes from PS + R. Phosphorylation of p38 MAP kinase in cardiac myocytes by stress may be sufficient to lead to myocardial dysfunction in animal models and possibly humans.

Differential MAP kinase activation and Na+/H+exchanger phosphorylation by H2O2 in rat cardiac myocytes

2001 ◽  
Vol 281 (5) ◽  
pp. C1542-C1550 ◽  
Author(s):  
Shan Wei ◽  
Emily C. Rothstein ◽  
Larry Fliegel ◽  
Louis J. Dell'Italia ◽  
Pamela A. Lucchesi
Keyword(s):  
Reactive Oxygen Species ◽  
Map Kinase ◽  
Map Kinases ◽  
Cardiac Myocyte ◽  
Ventricular Myocytes ◽  
Ischemia Reperfusion ◽  
Oxygen Species ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
Jnk Activation

Bursts in reactive oxygen species production are important mediators of contractile dysfunction during ischemia-reperfusion injury. Cellular mechanisms that mediate reactive oxygen species-induced changes in cardiac myocyte function have not been fully characterized. In the present study, H2O2 (50 μM) decreased contractility of adult rat ventricular myocytes. H2O2 caused a concentration- and time-dependent activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38, and c-Jun NH2-terminal kinase (JNK) mitogen-activated protein (MAP) kinases in adult rat ventricular myocytes. H2O2(50 μM) caused transient activation of ERK1/2 and p38 MAP kinase that was detected as early as 5 min, was maximal at 20 min (9.6 ± 1.2- and 9.0 ± 1.6-fold, respectively, vs. control), and returned to baseline at 60 min. JNK activation occurred more slowly (1.6 ± 0.2-fold vs. control at 60 min) but was sustained at 3.5 h. The protein kinase C inhibitor chelerythrine completely blocked JNK activation and reduced ERK1/2 and p38 activation. The tyrosine kinase inhibitors genistein and PP-2 blocked JNK, but not ERK1/2 and p38, activation. H2O2-induced Na+/H+ exchanger phosphorylation was blocked by the MAP kinase kinase inhibitor U-0126 (5 μM). These results demonstrate that H2O2-induced activation of MAP kinases may contribute to cardiac myocyte dysfunction during ischemia-reperfusion.

Nitric oxide triggers programmed cell death (apoptosis) of adult rat ventricular myocytes in culture

1999 ◽  
Vol 277 (3) ◽  
pp. H1189-H1199 ◽  
Author(s):  
David J. Pinsky ◽  
Walif Aji ◽  
Matthias Szabolcs ◽  
Eleni S. Athan ◽  
Youping Liu ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cell Death ◽  
Programmed Cell Death ◽  
Cardiac Myocytes ◽  
Cardiac Myocyte ◽  
Ventricular Myocytes ◽  
No Production ◽  
P53 Expression ◽  
Rat Ventricular Myocytes ◽  
Adult Rat

Excessive nitric oxide (NO) production within the heart is implicated in the pathogenesis of myocyte death, but the mechanism whereby NO kills cardiac myocytes is not known. To determine whether NO may trigger programmed cell death (apoptosis) of adult rat ventricular myocytes in culture, the NO donor S-nitroso- N-acetylpenicillamine (SNAP) was shown to kill purified cardiac myocytes in a dose-dependent fashion. In situ analysis of ventricular myocytes plated on chamber slides using nick-end labeling of DNA demonstrated that SNAP induces cardiac myocyte apoptosis, which was confirmed by the identification of oligonucleosomal DNA fragmentation on agarose gel electrophoresis. Similarly, treatment of cardiac myocytes with cytokines that induce inducible NO synthase was shown to cause an NO-dependent induction of apoptosis. Addition of reduced hemoglobin to scavenge NO liberated by SNAP extinguished both the increase in percentage of apoptotic cells and the appearance of DNA ladders. Treatment with SNAP (but not with N-acetylpenicillamine or SNAP + hemoglobin) not only induced apoptosis but resulted in a marked increase in p53 expression in cardiac myocytes detected by Western blotting and immunohistochemistry. These data indicate that NO has the capacity to kill cardiac myocytes by triggering apoptosis and suggest the involvement of p53 in this process.

Cardiac myocyte adenosine A2a receptor activation fails to alter cAMP or contractility: role of receptor localization

2002 ◽  
Vol 282 (3) ◽  
pp. H1035-H1040 ◽  
Author(s):  
Eric L. Kilpatrick ◽  
Prakash Narayan ◽  
Robert M. Mentzer ◽  
Robert D. Lasley
Keyword(s):  
Cardiac Myocyte ◽  
Vascular Tissue ◽  
Ventricular Myocytes ◽  
Receptor Activation ◽  
Peptide Sequence ◽  
Rat Ventricular Myocytes ◽  
Receptor Localization ◽  
Immunoreactive Band ◽  
Adenosine A2a ◽  
Camp Levels

Adenosine A2a receptors are found in coronary vascular tissue although, their presence in myocardium is subject to investigation. Although there have been numerous studies on adenosine A2a receptor agonist effects on contractility and cAMP levels in ventricular myocytes, these have yielded conflicting results. Negative pharmacological studies have even led to the conclusion that A2a receptors are not present in cardiac myocytes. The purpose of this study was to determine whether A2a receptors are expressed in rat ventricular myocytes and what physiological effects are mediated via activation of these receptors. Western blot analysis with a polyclonal antibody raised against a peptide sequence specific to the carboxy terminus of the A2a receptor revealed the presence of a band at ∼45 kDa. However, the immunoreactivity was located in the nonmembrane fraction of the cell lysate. The membrane fraction only exhibited an immunoreactive band ≥50 kDa. Treatment of isolated myocytes with the adenosine A2a agonist 2-[4-[(2-carboxyethyl)-phenyl]ethylamino]-5′- N-ethylcarboxamidoadenosine (CGS-21680) exerted no effects on cAMP levels or myocyte twitch amplitude. These results indicate that although rat ventricular myocytes appear to express adenosine A2a receptors, stimulation with an A2a agonist exerts no functional effects, possibly because of the subcellular localization of the A2a receptor.

Contractile regulation by overexpressed ETArequires intact T tubules in adult rat ventricular myocytes

2008 ◽  
Vol 294 (5) ◽  
pp. H2391-H2399 ◽  
Author(s):  
Ka Young Chung ◽  
Misuk Kang ◽  
Jeffery W. Walker
Keyword(s):  
Actin Polymerization ◽  
Ventricular Myocytes ◽  
Morphological Changes ◽  
Receptor Subtype ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
Protein Levels ◽  
Intercalated Discs ◽  
T Tubules ◽  
G Protein Coupled

Endothelin (ET)-1 regulates the contractility and growth of the heart by binding G protein-coupled receptors of the ET type A receptor (ETA)/ET type B (ETB) receptor family. ETA, the predominant ET-1 receptor subtype in myocardium, is thought to localize preferentially within cardiac T tubules, but the consequences of mislocalization are not fully understood. Here we examined the effects of the overexpression of ETAin conjunction with T-tubule loss in cultured adult rat ventricular myocytes. In adult myocytes cultured for 3 to 4 days, the normally robust positive inotropic effect (PIE) of ET-1 was lost in parallel with T-tubule degeneration and a decline in ETAprotein levels. In these T tubule-compromised myocytes, an overexpression of ETAusing an adenoviral vector did not rescue the responsiveness to ET-1, despite the robust expression in the surface sarcolemma. The inclusion of the actin polymerization inhibitor cytochalasin D (CD) during culture prevented gross morphological changes including a loss of T tubules and a rounding of intercalated discs, but CD alone did not rescue the responsiveness to ET-1 or prevent ETAdownregulation. The rescue of a normal PIE in 3- to 4-day cultured myocytes required both an increased expression of ETAand intact T tubules (preserved with CD). Therefore, the activation of ETAlocalized in T tubules was associated with a strong PIE, whereas the activation of ETAin surface sarcolemma was not. The results provide insight into the pathological cardiac conditions in which ETAis upregulated and T-tubule morphology is altered.

scholarly journals Na + -Ca 2+ Exchange Activity Is Localized in the T-Tubules of Rat Ventricular Myocytes

2002 ◽  
Vol 91 (4) ◽  
pp. 315-322 ◽  
Author(s):  
Z. Yang ◽  
C. Pascarel ◽  
D.S. Steele ◽  
K. Komukai ◽  
F. Brette ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Rat Ventricular Myocytes ◽  
T Tubules ◽  
Exchange Activity

Relative role of T-tubules disruption and decreased SERCA2 on contractile dynamics of isolated rat ventricular myocytes

Life Sciences ◽  
2021 ◽  
Vol 264 ◽  
pp. 118700
Author(s):  
Antonio Celestino-Montes ◽  
Perla Pérez-Treviño ◽  
Maya D. Sandoval-Herrera ◽  
Norma L. Gómez-Víquez ◽  
Julio Altamirano
Keyword(s):  
Ventricular Myocytes ◽  
Relative Role ◽  
Rat Ventricular Myocytes ◽  
T Tubules ◽  
Isolated Rat
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