Background-In ventricular myocytes, the majority of structures that couple excitation to the systolic rise of Ca 2ϩ are located at the transverse tubular (t-tubule) membrane. In the failing ventricle, disorganization of t-tubules disrupts excitation contraction coupling. The t-tubule membrane is virtually absent in the atria of small mammals resulting in spatiotemporally distinct profiles of intracellular Ca 2ϩ release on stimulation in atrial and ventricular cells. The aims of this study were to determine (i) whether atrial myocytes from a large mammal (sheep) possess t-tubules, (ii) whether these are functionally important, and (iii) whether they are disrupted in heart failure. Methods and Results-Sheep left atrial myocytes were stained with di-4-ANEPPS. Nearly all control cells had an extensive t-tubule network resulting in each voxel in the cell being nearer to a membrane (sarcolemma or t-tubule) than would otherwise be the case. T-tubules decrease the distance of 50% of voxels from a membrane from 3.35Ϯ0.15 to 0.88Ϯ0.04 m. During depolarization, intracellular Ca 2ϩ rises simultaneously at the cell periphery and center. In heart failure induced by rapid ventricular pacing, there was an almost complete loss of atrial t-tubules. The distance of 50% of voxels from a membrane increased to 2.04Ϯ0.08 m, and there was a loss of early Ca 2ϩ release from the cell center. Conclusion-Sheep atrial myocytes possess a substantial t-tubule network that synchronizes the systolic Ca 2ϩ transient. In heart failure, this network is markedly disrupted. This may play an important role in changes of atrial function in heart failure. (Circ Heart Fail. 2009;2:482-489.)

Richards MA, Clarke JD, Saravanan P, Voigt N, Dobrev D, Eisner DA, Trafford AW, Dibb KM. Transverse tubules are a common feature in large mammalian atrial myocytes including human. Am J Physiol Heart Circ Physiol 301: H1996 -H2005, 2011. First published August 12, 2011; doi:10.1152 doi:10. /ajpheart.00284.2011 tubules are surface membrane invaginations that are present in all mammalian cardiac ventricular cells. The apposition of L-type Ca 2ϩ channels on t tubules with the sarcoplasmic reticulum (SR) constitutes a "calcium release unit" and allows close coupling of excitation to the rise in systolic Ca 2ϩ . T tubules are virtually absent in the atria of small mammals, and therefore Ca 2ϩ release from the SR occurs initially at the periphery of the cell and then propagates into the interior. Recent work has, however, shown the occurrence of t tubules in atrial myocytes from sheep. As in the ventricle, Ca 2ϩ release in these cells occurs simultaneously in central and peripheral regions. T tubules in both the atria and the ventricle are lost in disease, contributing to cellular dysfunction. The aim of this study was to determine if the occurrence of t tubules in the atrium is restricted to sheep or is a more general property of larger mammals including humans. In atrial tissue sections from human, horse, cow, and sheep, membranes were labeled using wheat germ agglutinin. As previously shown in sheep, extensive t-tubule networks were present in horse, cow, and human atrial myocytes. Analysis shows half the volume of the cell lies within 0.64 Ϯ 0.03, 0.77 Ϯ 0.03, 0.84 Ϯ 0.03, and 1.56 Ϯ 0.19 m of t-tubule membrane in horse, cow, sheep, and human atrial myocytes, respectively. The presence of t tubules in the human atria may play an important role in determining the spatio-temporal properties of the systolic Ca 2ϩ transient and how this is perturbed in disease.atria; t-tubule heart T TUBULES ARE INVAGINATIONS of the surface membrane that penetrate deep within the cell. They occur at the z-line and are present in ventricular myocytes of all mammalian species studied to date. Many of the proteins involved in excitation contraction coupling are located on, or in close proximity to, the t-tubule membrane (16). In cardiac muscle, excitation contraction coupling is initiated by opening of L-type Ca 2ϩ channels and subsequent Ca 2ϩ entry (I Ca,L ) triggering release of Ca 2ϩ from the intracellular Ca 2ϩ store, the sarcoplasmic reticulum (SR). T tubules allow close coupling of I Ca,L to ryanodine receptors (RyRs) on the SR membrane resulting in rapid triggered Ca 2ϩ release in the cell interior upon electrical excitation. Thus, in ventricular cells, which have a regular t-tubule network penetrating the entire cell, the rise in intracellular Ca 2ϩ responsible for contraction is both rapid and synchronous throughout the entire cell. Chemically induced t-tubule removal with formamide results in this initial rise in intracellular Ca 2ϩ concentration being localized to the periphery and then propagating to the cell center (46).To ...

Non-technical summary Heart failure is where the heart is unable to pump sufficient blood in order to meet the requirements of the body. Symptoms of heart failure often first present during exercise. During exercise the blood levels of a hormone, noradrenaline, increase and activate receptors on the muscle cells of the heart known as β-receptors causing the heart to contract more forcefully. We show that in heart failure the response to β-receptor stimulation is reduced and this appears to be due to a failure of the β-receptor to signal correctly to downstream targets inside the cell. However, by-passing the β-receptor and directly activating one of the downstream targets, an enzyme known as adenylyl cyclase, inside the cell restores the function of the muscle cells in failing hearts. These observations provide a number of potential targets for therapies to improve the function of the heart in patients with heart failure.Abstract Reduced inotropic responsiveness is characteristic of heart failure (HF). This study determined the cellular Ca 2+ homeostatic and molecular mechanisms causing the blunted β-adrenergic (β-AR) response in HF. We induced HF by tachypacing in sheep; intracellular Ca 2+ concentration was measured in voltage-clamped ventricular myocytes. In HF, Ca 2+ transient amplitude and peak L-type Ca 2+ current (I Ca-L ) were reduced (to 70 ± 11% and 50 ± 3.7% of control, respectively, P < 0.05) whereas sarcoplasmic reticulum (SR) Ca 2+ content was unchanged. β-AR stimulation with isoprenaline (ISO) increased Ca 2+ transient amplitude, I Ca-L and SR Ca 2+ content in both cell types; however, the response of HF cells was markedly diminished (P < 0.05). Western blotting revealed an increase in protein phosphatase levels (PP1, 158 ± 17% and PP2A, 188 ± 34% of control, P < 0.05) and reduced phosphorylation of phospholamban in HF (Ser16, 30 ± 10% and Thr17, 41 ± 15% of control, P < 0.05). The β-AR receptor kinase GRK-2 was also increased in HF (173 ± 38% of control, P < 0.05). In HF, activation of adenylyl cyclase with forskolin rescued the Ca 2+ transient, SR Ca 2+ content and SR Ca 2+ uptake rate to the same levels as control cells in ISO. In conclusion, the reduced responsiveness of the myocardium to β-AR agonists in HF probably arises as a consequence of impaired phosphorylation of key intracellular proteins responsible for regulating the SR Ca 2+ content and therefore failure of the systolic Ca 2+ transient to increase appropriately during β-AR stimulation.

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Heart failure (HF) is commonly associated with reduced cardiac output and an increased risk of atrial arrhythmias particularly during β-adrenergic stimulation. The aim of the present study was to determine how HF alters systolic Ca2 + and the response to β-adrenergic (β-AR) stimulation in atrial myocytes. HF was induced in sheep by ventricular tachypacing and changes in intracellular Ca2 + concentration studied in single left atrial myocytes under voltage and current clamp conditions. The following were all reduced in HF atrial myocytes; Ca2 + transient amplitude (by 46% in current clamped and 28% in voltage clamped cells), SR dependent rate of Ca2 + removal (kSR, by 32%), L-type Ca2 + current density (by 36%) and action potential duration (APD90 by 22%). However, in HF SR Ca2 + content was increased (by 19%) when measured under voltage-clamp stimulation. Inhibiting the L-type Ca2 + current (ICa-L) in control cells reproduced both the decrease in Ca2 + transient amplitude and increase of SR Ca2 + content observed in voltage-clamped HF cells. During β-AR stimulation Ca2 + transient amplitude was the same in control and HF cells. However, ICa-L remained less in HF than control cells whilst SR Ca2 + content was highest in HF cells during β-AR stimulation. The decrease in ICa-L that occurs in HF atrial myocytes appears to underpin the decreased Ca2 + transient amplitude and increased SR Ca2 + content observed in voltage-clamped cells.

The atria contribute 25% to ventricular stroke volume and are the site of the commonest cardiac arrhythmia, atrial fibrillation (AF). The initiation of contraction in the atria is similar to that in the ventricle involving a systolic rise of intracellular Ca(2+) concentration ([Ca(2+)](i)). There are, however, substantial inter-species differences in the way systolic Ca(2+) is regulated in atrial cells. These differences are a consequence of a well-developed and functionally relevant transverse (t)-tubule network in the atria of large mammals, including humans, and its virtual absence in smaller laboratory species such as the rat. Where T-tubules are absent, the systolic Ca(2+) transient results from a 'fire-diffuse-fire' sequential recruitment of Ca(2+) release sites from the cell edge to the centre and hence marked spatiotemporal heterogeneity of systolic Ca(2+). Conversely, the well-developed T-tubule network in large mammals ensures a near synchronous rise of [Ca(2+)](i). In addition to synchronizing the systolic rise of [Ca(2+)](i), the presence of T-tubules in the atria of large mammals, by virtue of localization of the L-type Ca(2+) channels and Na(+)-Ca(2+) exchanger antiporters on the T-tubules, may serve to, respectively, accelerate changes in the amplitude of the systolic Ca(2+) transient during inotropic manoeuvres and lower diastolic [Ca(2+)](i). On the other hand, the presence of T-tubules and hence wider cellular distribution of the Na(+)-Ca(2+) exchanger may predispose the atria of large mammals to Ca(2+)-dependent delayed afterdepolarizations (DADs); this may be a determining factor in why the atria of large mammals spontaneously develop and maintain AF.

Using the baculovirus system, the skeletal muscle chloride channel, CIC-1 (rat), and a point mutant replacing arginine 304 with glutamic acid were expressed at high levels in cultured Sf-9 insect cells. Whole-cell patch-clamping revealed large inwardly rectifying currents with maxima up to 15 nA inward and 2.5 nA outward. Saturation was evident at voltage steps positive to +40 mV whilst steps negative to -60 mV produced inactivating currents made up of a steady state component and two exponentially decaying components with tau 1 = 6.14+/- 0.92 ms, tau 2 = 36.5+/- 3.29 ms (S.D) n = 7 for steps to -120 mV. Currents recorded in the outside-out patch configuration were often unexpectedly large and up to 5% of whole-cell currents obtained in the same cell, suggesting an uneven channel distribution in the plasmalemma of Sf-9 cells. The pharmacology of a number of chloride channel blockers, including anthracene-9-carboxylate (A9C), niflumate, and perrhenate, was investigated and showed for the first time that perrhenate is an effective blocker of C1C-1 and that it has a complex mechanism of action. Further, the potency of A9C was found to be dependent on external chloride concentration. As in studies on muscle cells themselves, blockade was rapidly effective and easily reversible, except when applying the indanyloxyacetate derivative, IAA94/95, which took up to 10 min to act, and, consistent with an intracellular site of action, was difficult to reverse by washing. Mutation of the highly conserved arginine at position 304 to a glutamic acid did not significantly alter the behaviour of the channel.

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