scholarly journals Enhanced activity of ventricular Na+-HCO3− cotransport in pressure overload hypertrophy

2007 ◽  
Vol 293 (2) ◽  
pp. H1254-H1264 ◽  
Author(s):  
Taku Yamamoto ◽  
Takeshi Shirayama ◽  
Tomohiko Sakatani ◽  
Tomosaburo Takahashi ◽  
Hideo Tanaka ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Single Cells ◽  
Ischemia Reperfusion ◽  
Pressure Overload ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Aortic Constriction ◽  
Ph Indicator ◽  
Adult Rat ◽  
Acid Extrusion

The Na+-HCO3− cotransporter (NBC) plays a key role in intracellular pH (pHi) regulation in normal ventricular muscle. However, the state of NBC in nonischemic hypertrophied hearts is unresolved. In this study, we examined functional and molecular properties of NBC in adult rat ventricular myocytes. The cells were enzymatically isolated from both normal and hypertrophied hearts. Ventricular hypertrophy was induced by pressure overload created by suprarenal abdominal aortic constriction of 50% for 7 wk. pHi was measured in single cells using the fluorescent pH indicator 2′,7′-bis(2-carboxyethyl)5-( 6 )carboxyfluorescein. Real-time PCR analysis was used to quantitatively assess expression of NBC-encoding mRNA, including SLC4A4 (encoding electrogenic NBC, NBCe1) and SLC4A7 (electroneutral NBC, NBCn1). Our results demonstrate that: 1) mRNA levels of both the electrogenic NBCe1 (SLC4A4) and electroneutral NBCn1 (SLC4A7) forms of NBC were increased by aortic constriction, 2) the onset of NBC upregulation occurred within 3 days after constriction, 3) normal and hypertrophied ventricles displayed regional differences in NBC expression, 4) acid extrusion via NBC ( JNBC) was increased significantly in hypertrophied myocytes, 5) although acid extrusion via Na+/H+ exchange was also increased in hypertrophied myocytes, the relative enhancement of JNBC was larger, 6) membrane depolarization markedly increased JNBC in hypertrophied myocytes, and 7) losartan, an ANG II AT1 receptor antagonist, significantly attenuated the upregulation of both NBCs induced by 3 wk of aortic constriction. Enhanced NBC activity during hypertrophic development provides a mechanism for intracellular Na+ overload, which may render the ventricles more vulnerable to Ca2+ overload during ischemia-reperfusion.

Na+/H+ exchanger inhibitor cariporide attenuates the mitochondrial Ca2+ overload and PTP opening

2007 ◽  
Vol 293 (6) ◽  
pp. H3517-H3523 ◽  
Author(s):  
Takako Toda ◽  
Toshie Kadono ◽  
Minako Hoshiai ◽  
Yu Eguchi ◽  
Shinpei Nakazawa ◽  
...  
Keyword(s):  
Mitochondrial Permeability Transition ◽  
Ventricular Myocytes ◽  
Ischemia Reperfusion ◽  
Permeability Transition ◽  
Laser Illumination ◽  
Adult Rat ◽  
Myocardial Ischemia Reperfusion ◽  
Ptp Opening ◽  
And Oxidative Stress ◽  
Tetramethylrhodamine Methyl Ester

The Na+/H+ exchanger (NHE) inhibitor cariporide has a cardioprotective effect in various animal models of myocardial ischemia-reperfusion. Recent studies have suggested that cariporide interacts with mitochondrial Ca2+ overload and the mitochondrial permeability transition (MPT); however, the precise mechanisms remain unclear. Therefore, we examined whether cariporide affects mitochondrial Ca2+ overload and MPT. Isolated adult rat ventricular myocytes were used to study the effects of cariporide on hypercontracture induced by ouabain or phenylarsine oxide (PAO). Mitochondrial Ca2+ concentration ([Ca2+]m) and the mitochondrial membrane potential (ΔΨm) were measured by loading myocytes with rhod-2 and JC-1, respectively. We also examined the effect of cariporide on the MPT using tetramethylrhodamine methyl ester (TMRM) and oxidative stress generated by laser illumination. Cariporide (1 μM) prevented ouabain-induced hypercontracture (from 40 ± 2 to 24 ± 2%, P < 0.05) and significantly attenuated ouabain-induced [Ca2+]m overload (from 149 ± 6 to 121 ± 5% of the baseline value, P < 0.05) but did not affect ΔΨm. These results indicate that cariporide attenuates the [Ca2+]m overload without the accompanying depolarization of ΔΨm. Moreover, cariporide increased the time taken to induce the MPT (from 79 ± 11 to 137 ± 20 s, P < 0.05) and also attenuated PAO-induced hypercontracture (from 59 ± 3 to 50 ± 4%, P < 0.05). Our data indicate that cariporide attenuates [Ca2+]m overload and MPT. Thus these effects might potentially contribute to the mechanisms of cardioprotection afforded by NHE inhibitors.

Differential response to myocardial reperfusion injury in eNOS-deficient mice

2002 ◽  
Vol 282 (6) ◽  
pp. H2422-H2426 ◽  
Author(s):  
Brent R. Sharp ◽  
Steven P. Jones ◽  
David M. Rimmer ◽  
David J. Lefer
Keyword(s):  
Ischemia Reperfusion ◽  
Myocardial Necrosis ◽  
In Vivo Model ◽  
Pcr Analysis ◽  
Inos Mrna ◽  
Mrna Levels ◽  
Wild Type ◽  
Significant Difference ◽  
Deficient Mice

Two strains of endothelial nitric oxide synthase (eNOS)-deficient (−/−) mice have been developed that respond differently to myocardial ischemia-reperfusion (MI/R). We evaluated both strains of eNOS−/− mice in an in vivo model of MI/R. Harvard (Har) eNOS−/− mice ( n = 12) experienced an 84% increase in myocardial necrosis compared with wild-type controls ( P < 0.05). University of North Carolina (UNC) eNOS−/−( n = 10) exhibited a 52% reduction in myocardial injury versus wild-type controls ( P < 0.05). PCR analysis of myocardial inducible NO synthase (iNOS) mRNA levels revealed a significant ( P < 0.05) increase in the UNC eNOS−/− mice compared with wild-type mice, and there was no significant difference between the Har eNOS−/− and wild-type mice. UNC eNOS−/− mice treated with an iNOS inhibitor (1400W) exacerbated the extent of myocardial necrosis. When treated with 1400W, Har eNOS−/− did not exhibit a significant increase in myocardial necrosis. These data demonstrate that two distinct strains of eNOS−/− mice display opposite responses to MI/R. Although the protection seen in the UNC eNOS−/− mouse may result from compensatory increases in iNOS, other genes may be involved.

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.

scholarly journals Redox-Resistant SERCA [Sarco(endo)plasmic Reticulum Calcium ATPase] Attenuates Oxidant-Stimulated Mitochondrial Calcium and Apoptosis in Cardiac Myocytes and Pressure Overload–Induced Myocardial Failure in Mice

Circulation ◽  
2020 ◽  
Vol 142 (25) ◽  
pp. 2459-2469
Author(s):  
Jena B. Goodman ◽  
Fuzhong Qin ◽  
Robert J. Morgan ◽  
Jordan M. Chambers ◽  
Dominique Croteau ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Calcium Concentration ◽  
Ventricular Myocytes ◽  
Calcium Content ◽  
Mitochondrial Calcium ◽  
Oxygen Species ◽  
Wild Type ◽  
Aortic Constriction ◽  
Rat Ventricular Myocytes ◽  
Adult Rat

Background: SERCA [sarco(endo)plasmic reticulum calcium ATPase] is regulated by oxidative posttranslational modifications at cysteine 674 (C674). Because sarcoplasmic reticulum (SR) calcium has been shown to play a critical role in mediating mitochondrial dysfunction in response to reactive oxygen species, we hypothesized that SERCA oxidation at C674 would modulate the effects of reactive oxygen species on mitochondrial calcium and mitochondria-dependent apoptosis in cardiac myocytes. Methods: Adult rat ventricular myocytes expressing wild-type SERCA2b or a redox-insensitive mutant in which C674 is replaced by serine (C674S) were exposed to H 2 O 2 (100 µmol/Lμ). Free mitochondrial calcium concentration was measured in adult rat ventricular myocytes with a genetically targeted fluorescent probe, and SR calcium content was assessed by measuring caffeine-stimulated release. Mice with heterozygous knock-in of the SERCA C674S mutation were subjected to chronic ascending aortic constriction. Results: In adult rat ventricular myocytes expressing wild-type SERCA, H 2 O 2 caused a 25% increase in mitochondrial calcium concentration that was associated with a 50% decrease in SR calcium content, both of which were prevented by the ryanodine receptor inhibitor tetracaine. In cells expressing the C674S mutant, basal SR calcium content was decreased by 31% and the H 2 O 2 -stimulated rise in mitochondrial calcium concentration was attenuated by 40%. In wild-type cells, H 2 O 2 caused cytochrome c release and apoptosis, both of which were prevented in C674S-expressing cells. In myocytes from SERCA knock-in mice, basal SERCA activity and SR calcium content were decreased. To test the effect of C674 oxidation on apoptosis in vivo, SERCA knock-in mice were subjected to chronic ascending aortic constriction. In wild-type mice, ascending aortic constriction caused myocyte apoptosis, LV dilation, and systolic failure, all of which were inhibited in SERCA knock-in mice. Conclusions: Redox activation of SERCA C674 regulates basal SR calcium content, thereby mediating the pathologic reactive oxygen species–stimulated rise in mitochondrial calcium required for myocyte apoptosis and myocardial failure.

scholarly journals Endothelin downregulates SERCA2 gene and protein expression in adult rat ventricular myocytes: regulation by pertussis toxin-sensitive Gi protein and cAMP

2009 ◽  
Vol 296 (3) ◽  
pp. H728-H734 ◽  
Author(s):  
Randa Hilal-Dandan ◽  
Huaping He ◽  
Jody L. Martin ◽  
Laurence L. Brunton ◽  
Wolfgang H. Dillmann
Keyword(s):  
Gene Expression ◽  
Pertussis Toxin ◽  
Ventricular Myocytes ◽  
Intracellular Camp ◽  
Mrna Levels ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
Protein Levels ◽  
Gene And Protein Expression ◽  
Gi Protein

Downregulation of the sarcoplasmic reticulum calcium ATPase (SERCA2) is associated with diastolic dysfunction in the failing heart. Elevated plasma endothelin-1 (ET) levels are correlated with congestive heart failure suggesting that ET may play a pathophysiological role. We have investigated the ability of ET to regulate SERCA2 gene expression in isolated adult rat ventricular myocytes. We find that ET enhances net protein synthesis by ∼40% but significantly downregulates SERCA2 mRNA expression, time dependently, by ∼30–50%, and the expression of SERCA2 protein by ∼ 50%. In myoyctes, ET binds to ETA receptor that couples to Gq and Gi proteins. Inhibition of Gq-PLC-induced phosphoinositide (PI) hydrolysis with U73122 (1 μM) or inhibition of Gi protein with pertussis toxin (PTX) abolishes the ability of ET to downregulate SERCA2 mRNA gene expression. Further investigation suggests that ET coupling to PTX-sensitive Gi with consequent lowering of cAMP is required for downregulation of SERCA2 mRNA levels. Increasing intracellular cAMP quantity using cAMP-specific PDE inhibitor Ro20-1724 or cAMP analog dibutyryl-cAMP reverses ET-induced downregulation of SERCA2 mRNA levels. The data indicate that, in adult myocytes, ET downregulates SERCA2 mRNA and protein levels, and the effect requires cross-talk between Gq and PTX-sensitive Gi pathways.

scholarly journals Quantitative Real-Time RT—PCR Analysis of Inflammatory Gene Expression Associated with Ischemia—Reperfusion Brain Injury

2002 ◽  
Vol 22 (9) ◽  
pp. 1068-1079 ◽  
Author(s):  
Rossana Berti ◽  
Anthony J. Williams ◽  
John R. Moffett ◽  
Sarah L. Hale ◽  
Luisa C. Velarde ◽  
...  
Keyword(s):  
Gene Expression ◽  
Brain Injury ◽  
Real Time ◽  
Adhesion Molecules ◽  
Ischemia Reperfusion ◽  
Fold Increase ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Time Points ◽  
Time Point

Ischemia-reperfusion brain injury initiates an inflammatory response involving the expression of adhesion molecules and cytokines, some of which are regulated by the nuclear transcription factor NF-κB. In this study the authors examined mRNA expression levels for several important genes associated with inflammation at five time points (3, 6, 12, 24, and 72 hours) after transient middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats. A sensitive and quantitative technique (TaqMan real-time QRT-PCR) was used to simultaneously measure mRNA levels for key cell adhesion molecules and inflammatory cytokines. Gene expression increased significantly in the injured hemisphere for interleukin (IL)-1β (12-fold increase at 24 hours), IL-6 (25-fold increase at 6 hours) and ICAM-1 (4-fold increase at 24 hours), and the in-terhemispheric differences for these genes were significant for every time point examined ( P < 0.05 for all values). Tumor necrosis factor-α mRNA was upregulated in the injured versus uninjured hemisphere from 3 to 24 hours (5-fold increase at 6 hours), while E-selectin showed a significant increase in mRNA levels from 6 to 24 hours after MCAO (10-fold increase at 6 hours) ( P < 0.05 for all values). VCAM-1 mRNA levels did not respond differentially to injury at any time point between the two brain hemispheres. At all time points examined, activated NF-κB immunoreactivity was observed in cells throughout the infarct-damaged tissue. These results are consistent with the proinflammatory properties of the induced molecules, which are involved in the initiation of the inflammatory cascade, and may thus contribute to secondary cellular responses that lead to further brain damage.

scholarly journals Fenofibrate inhibits aldosterone-induced apoptosis in adult rat ventricular myocytes via stress-activated kinase-dependent mechanisms

2009 ◽  
Vol 296 (6) ◽  
pp. H1983-H1993 ◽  
Author(s):  
Deepa S. De Silva ◽  
Richard M. Wilson ◽  
Christoph Hutchinson ◽  
Peter C. Ip ◽  
Anthony G. Garcia ◽  
...  
Keyword(s):  
Cardiac Remodeling ◽  
Ventricular Myocytes ◽  
Pressure Overload ◽  
In Vivo Studies ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
Induced Apoptosis

Aldosterone induces extracellular signal-regulated kinase (ERK)-dependent cardiac remodeling. Fenofibrate improves cardiac remodeling in adult rat ventricular myocytes (ARVM) partly via inhibition of aldosterone-induced ERK1/2 phosphorylation and inhibition of matrix metalloproteinases. We sought to determine whether aldosterone caused apoptosis in cultured ARVM and whether fenofibrate ameliorated the apoptosis. Aldosterone (1 μM) induced apoptosis by increasing terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive nuclei in ARVM. Spironolactone (100 nM), an aldosterone receptor antagonist, but not RU-486, a glucocorticoid receptor, inhibited aldosterone-mediated apoptosis, indicating that the mineralocorticoid receptor (MR) plays a role. SP-600125 (3 μM)—a selective inhibitor of c-Jun NH2-terminal kinase (JNK)—inhibited aldosterone-induced apoptosis in ARVM. Although aldosterone increased the expression of both stress-activated protein kinases, pretreatment with fenofibrate (10 μM) decreased aldosterone-mediated apoptosis by inhibiting only JNK phosphorylation and the aldosterone-induced increases in Bax, p53, and cleaved caspase-3 and decreases in Bcl-2 protein expression in ARVM. In vivo studies demonstrated that chronic fenofibrate (100 mg·kg body wt−1·day−1) inhibited myocardial Bax and increased Bcl-2 expression in aldosterone-induced cardiac hypertrophy. Similarly, eplerenone, a selective MR inhibitor, used in chronic pressure-overload ascending aortic constriction inhibited myocardial Bax expression but had no effect on Bcl-2 expression. Therefore, involvement of JNK MAPK-dependent mitochondrial death pathway mediates ARVM aldosterone-induced apoptosis and is inhibited by fenofibrate, a peroxisome proliferator-activated receptor (PPAR)α ligand. Fenofibrate mediates beneficial effects in cardiac remodeling by inhibiting programmed cell death and the stress-activated kinases.

Matrix metalloproteinases mediate β-adrenergic receptor-stimulated apoptosis in adult rat ventricular myocytes

2005 ◽  
Vol 289 (1) ◽  
pp. C168-C176 ◽  
Author(s):  
Bindu Menon ◽  
Mahipal Singh ◽  
Krishna Singh
Keyword(s):  
Matrix Metalloproteinases ◽  
Adrenergic Receptor ◽  
Ventricular Myocytes ◽  
Mrna Levels ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
Protein Levels ◽  
Activity Inhibition ◽  
Apoptotic Effects ◽  
Expression And Activity

Changes in the synthesis and activity of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are associated with myocardial remodeling. Here we measured the expression and activity of MMPs and TIMPs, and tested the hypothesis that increased MMP activity plays a proapoptotic role in β-adrenergic receptor (β-AR)-stimulated apoptosis of adult rat ventricular myocytes (ARVMs). β-AR stimulation (isoproterenol, 24 h) increased mRNA levels of MMP-2 and TIMP-1 while it decreased TIMP-2 mRNA levels as analyzed by real-time PCR. Western blot analysis, immunocytochemical analysis, in-gel zymography, and MMP-2 activity assay confirmed β-AR-stimulated increases in MMP-2 protein levels and activity. Inhibition of MMPs using GM-6001 (a broad-spectrum inhibitor of MMPs), SB3CT (inhibitor of MMP-2), and purified TIMP-2 inhibited β-AR-stimulated apoptosis as determined by TdT-mediated dUTP nick end labeling staining. Treatment with active MMP-2 alone increased the number of apoptotic cells. This increase in MMP-2-mediated apoptosis was inhibited by GM-6001 and SB3CT pretreatment. Coimmunoprecipitation studies indicated increased physical association of MMP-2 with β1-integrins after β-AR stimulation. Inhibition of MMP-2 using SB3CT or stimulation of β1-integrin signaling using laminin inhibited the increased association of MMP-2 with β1-integrins. β-AR stimulation increased poly-ADP-ribose-polymerase cleavage, which was inhibited by inhibition of MMP-2. These data suggest the following: 1) β-AR stimulation increases MMP-2 expression and activity and inhibits TIMP-2 expression; 2) inhibition of MMPs, most likely MMP-2, inhibits β-AR-stimulated apoptosis; and 3) the apoptotic effects of MMP-2 may be mediated, at least in part, via its interaction with β1 integrins and poly-ADP-ribose-polymerase cleavage.

Enhanced calcium mobilization in rat ventricular myocytes during the onset of pressure overload-induced hypertrophy

2006 ◽  
Vol 291 (4) ◽  
pp. H1803-H1813 ◽  
Author(s):  
Beatriz M. R. Carvalho ◽  
Rosana A. Bassani ◽  
Kleber G. Franchini ◽  
José W. M. Bassani
Keyword(s):  
Sham Group ◽  
Ventricular Myocytes ◽  
Pressure Overload ◽  
Group Development ◽  
Left Ventricular ◽  
Mrna Levels ◽  
Inotropic Effects ◽  
Adrenoceptor Agonist ◽  
Intracellular Ca ◽  
Rat Myocytes

Early cardiovascular changes evoked by pressure overload (PO) may reveal adaptive strategies that allow immediate survival to the increased hemodynamic load. In this study, systolic and diastolic Ca2+ cycling was analyzed in left ventricular rat myocytes before ( day 2, PO-2d group) and after ( day 7, PO-7d group) development of hypertrophy subsequent to aortic constriction, as well as in myocytes from time-matched sham-operated rats (sham group). Ca2+ transient amplitude was significantly augmented in the PO-2d group. In the PO-7d group, intracellular Ca2+ concentration ([Ca2+]i) was reduced during diastole, and mechanical twitch relaxation (but not [Ca2+]i decline) was slowed. In PO groups, fractional sarcoplasmic reticulum (SR) Ca2+ release at a twitch, SR Ca2+ content, SR Ca2+ loss during diastole, and SR-dependent integrated Ca2+ flux during twitch relaxation were significantly greater than in sham-operated groups, whereas the relaxation-associated Ca2+ flux carried by the Na+/Ca2+ exchanger was not significantly changed. In the PO-7d group, mRNA levels of cardiac isoforms of SR Ca2+-ATPase (SERCA2a), phospholamban, calsequestrin, ryanodine receptor, and NCX were not significantly altered, but the SERCA2a-to-phospholamban ratio was increased 2.5-fold. Moreover, greater sensitivity to the inotropic effects of the β-adrenoceptor agonist isoproterenol was observed in the PO-7d group. The results indicate enhanced Ca2+ cycling between SR and cytosol early after PO imposition, even before hypertrophy development. Increase in SR Ca2+ uptake may contribute to enhancement of excitation-contraction coupling (augmented SR Ca2+ content and release) and protection against arrhythmogenesis due to buildup of [Ca2+]i during diastole.

scholarly journals Developmental analysis reveals mismatches in the expression of K+ channel alpha subunits and voltage-gated K+ channel currents in rat ventricular myocytes.

1996 ◽  
Vol 108 (5) ◽  
pp. 405-419 ◽  
Author(s):  
H Xu ◽  
J E Dixon ◽  
D M Barry ◽  
J S Trimmer ◽  
J P Merlie ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Developmental Expression ◽  
K Channel ◽  
Mrna Levels ◽  
K Channels ◽  
Voltage Gated ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
Alpha Subunits ◽  
Channel Currents

In the experiments here, the developmental expression of the functional Ca(2+)-independent, depolarization-activated K+ channel currents, Ito and IK, and of the voltage-gated K+ channel (Kv) alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2 in rat ventricular myocytes were examined quantitatively. Using the whole-cell patch clamp recording method, the properties and the densities of Ito and IK in ventricular myocytes isolated from postnatal day 5 (P5), 10 (P10), 15 (P15), 20 (P20), 25 (P25), 30 (P30), and adult (8-12 wk) rats were characterized and compared. These experiments revealed that mean Ito densities increase fourfold between birth and P30, whereas IK densities vary only slightly. Neither the time- nor the voltage-dependent properties of the currents vary measurably, suggesting that the subunits underlying functional Ito and IK channels are the same throughout postnatal development. In parallel experiments, the developmental expression of each of the voltage-gated K+ channel alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2, was examined quantitatively at the mRNA and protein levels using subunit-specific probes. RNase protection assays revealed that Kv1.4 message levels are high at birth, increase between P0 and P10, and subsequently decrease to very low levels in adult rat ventricles. The decrease in message is accompanied by a marked reduction in Kv1.4 protein, consistent with our previous suggestion that Kv1.4 does not contribute to the formation of functional K+ channels in adult rat ventricular myocytes. In contrast to Kv1.4, the mRNA levels of Kv1.2, Kv1.5, Kv2.1, and Kv4.2 increase (three- to five-fold) between birth and adult. Western analyses, however, revealed that the expression patterns of these subunits proteins vary in distinct ways: Kv1.2 and Kv4.2, for example, increase between P5 and adult, whereas Kv1.5 remains constant and Kv2.1 decreases. Throughout development, therefore, there is a mismatch between the numbers of Kv alpha subunits expressed and the functional voltage-gated K+ channel currents distinguished electrophysiologically in rat ventricular myocytes. Alternative experimental approaches will be required to define directly the Kv alpha subunits that underlie functional voltage-gated K+ channels in these (and other) cells. In addition, the finding that Kv alpha subunit protein expression levels do not necessarily mirror mRNA levels suggests that caution should be exercised in attempting functional interpretations of observed changes in mRNA levels alone.

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