Differential Modulation of L-type Ca
            2+
            Current by SR Ca
            2+
            Release at the T-Tubules and Surface Membrane of Rat Ventricular Myocytes

Formamide-induced osmotic shock has been used to detubulate isolated adult rat ventricular myocytes (i.e., disrupt the surface membrane-T tubule junction). Cell volume, calculated from cell length and width, rapidly decreased and increased upon application and removal of formamide, respectively. After treatment with formamide, membrane capacitance decreased by 26.4% (from 199.4 ± 18.7 pF in control cells to 146.7 ± 6.4 pF in formamide-treated cells; n = 13, P < 0.05). However, the amplitude of the L-type Ca2+ current ( I Ca) decreased by a greater extent (from 0.75 ± 0.14 to 0.18 ± 0.03 nA; n = 5, P < 0.05) so that the density of I Ca decreased by 74.5%. Simultaneous measurements of I Ca and Ca2+ transients (monitored using fura 2) showed that both decreased rapidly upon removal of formamide. However, the Ca2+ content of the sarcoplasmic reticulum showed little change. Cross-striations, visualized with the fluorescent dye di-8-aminonaphthylethenylpyridinium, were sparse or absent in cells that had been treated with formamide, suggesting that formamide can successfully detubulate cardiac cells and that I Ca is concentrated in the T tubules, which therefore play an important role in excitation-contraction coupling.

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Quantification of Calcium Entry at the T-Tubules and Surface Membrane in Rat Ventricular Myocytes

Biophysical Journal ◽

10.1529/biophysj.105.069013 ◽

2006 ◽

Vol 90 (1) ◽

pp. 381-389 ◽

Cited By ~ 62

Author(s):

F. Brette ◽

L. Sallé ◽

C.H. Orchard

Keyword(s):

Ventricular Myocytes ◽

Surface Membrane ◽

Calcium Entry ◽

Rat Ventricular Myocytes ◽

T Tubules

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

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00347.2002 ◽

2002 ◽

Vol 283 (4) ◽

pp. H1720-H1728 ◽

Cited By ~ 77

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.

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

AJP Cell Physiology ◽

10.1152/ajpcell.00597.2006 ◽

2007 ◽

Vol 293 (1) ◽

pp. C321-C327 ◽

Cited By ~ 49

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.

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Ca efflux via the sarcolemmal Ca ATPase occurs only in the t-tubules of rat ventricular myocytes

Journal of Molecular and Cellular Cardiology ◽

10.1016/j.yjmcc.2010.10.012 ◽

2011 ◽

Vol 50 (1) ◽

pp. 187-193 ◽

Cited By ~ 28

Author(s):

Anabelle Chase ◽

Clive H. Orchard

Keyword(s):

Ventricular Myocytes ◽

Rat Ventricular Myocytes ◽

T Tubules

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

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00011.2008 ◽

2008 ◽

Vol 294 (5) ◽

pp. H2391-H2399 ◽

Cited By ~ 9

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.

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Sarcolemmal distribution of ICa and INCX and Ca2+ autoregulation in mouse ventricular myocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00117.2017 ◽

2017 ◽

Vol 313 (1) ◽

pp. H190-H199 ◽

Cited By ~ 6

Author(s):

Hanne C. Gadeberg ◽

Cherrie H. T. Kong ◽

Simon M. Bryant ◽

Andrew F. James ◽

Clive H. Orchard

Keyword(s):

Steady State ◽

Sarcoplasmic Reticulum ◽

Cardiac Myocytes ◽

Ventricular Myocytes ◽

Surface Membrane ◽

Ryanodine Receptors ◽

Cell Voltage ◽

Intact Cells ◽

T Tubules

The balance of Ca2+ influx and efflux regulates the Ca2+ load of cardiac myocytes, a process known as autoregulation. Previous work has shown that Ca2+ influx, via L-type Ca2+ current ( ICa), and efflux, via the Na+/Ca2+ exchanger (NCX), occur predominantly at t-tubules; however, the role of t-tubules in autoregulation is unknown. Therefore, we investigated the sarcolemmal distribution of ICa and NCX current ( INCX), and autoregulation, in mouse ventricular myocytes using whole cell voltage-clamp and simultaneous Ca2+ measurements in intact and detubulated (DT) cells. In contrast to the rat, INCX was located predominantly at the surface membrane, and the hysteresis between INCX and Ca2+ observed in intact myocytes was preserved after detubulation. Immunostaining showed both NCX and ryanodine receptors (RyRs) at the t-tubules and surface membrane, consistent with colocalization of NCX and RyRs at both sites. Unlike INCX, ICa was found predominantly in the t-tubules. Recovery of the Ca2+ transient amplitude to steady state (autoregulation) after application of 200 µM or 10 mM caffeine was slower in DT cells than in intact cells. However, during application of 200 µM caffeine to increase sarcoplasmic reticulum (SR) Ca2+ release, DT and intact cells recovered at the same rate. It appears likely that this asymmetric response to changes in SR Ca2+ release is a consequence of the distribution of ICa, which is reduced in DT cells and is required to refill the SR after depletion, and NCX, which is little affected by detubulation, remaining available to remove Ca2+ when SR Ca2+ release is increased. NEW & NOTEWORTHY This study shows that in contrast to the rat, mouse ventricular Na+/Ca2+ exchange current density is lower in the t-tubules than in the surface sarcolemma and Ca2+ current is predominantly located in the t-tubules. As a consequence, the t-tubules play a role in recovery (autoregulation) from reduced, but not increased, sarcoplasmic reticulum Ca2+ release.

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