Abstract-The transverse tubules (t-tubules) of mammalian cardiac ventricular myocytes are invaginations of the surface membrane. Recent studies have suggested that the structure and function of the t-tubules are more complex than previously believed; in particular, many of the proteins involved in cellular Ca 2ϩ cycling appear to be concentrated at the t-tubule. Thus, the t-tubules are an important determinant of cardiac cell function, especially as the main site of excitation-contraction coupling, ensuring spatially and temporally synchronous Ca 2ϩ release throughout the cell. Changes in t-tubule structure and protein expression occur during development and in heart failure, so that changes in the t-tubules may contribute to the functional changes observed in these conditions. The purpose of this review is to provide an overview of recent studies of t-tubule structure and function in cardiac myocytes. Key Words: cardiac muscle Ⅲ t-tubules Ⅲ excitation-contraction coupling Ⅲ heart failure T he transverse tubules (t-tubules) of mammalian cardiac ventricular myocytes are invaginations of the surface membrane that occur at the Z line and have both transverse and longitudinal elements. Many of the proteins involved in excitation-contraction coupling appear to be concentrated at the t-tubules. Therefore, it has been suggested that the t-tubules play a central role in cell activation. In the present review, we will consider the immunohistochemical and functional evidence for protein localization at the t-tubules, potential problems in the interpretation of such data, and the functional consequences of such localization. We will also consider the possible role of the t-tubules in the functional changes that occur during cardiac development, hypertrophy, and failure. Occurrence and Morphology of the T-TubulesT-tubules are present in the cardiac tissue of all species of mammals so far investigated (eg, mice, 1 rats, 2 guinea pigs, 3 rabbits, 4 dogs, 5 pigs, 1 and humans 6 ) but appear to be absent in avian, 7 reptile 8 and amphibian 8 cardiac tissue. Within mammalian cardiac tissue, t-tubules occur predominantly in ventricular myocytes, being either absent or far less developed in atrial, pacemaking, and conducting tissue, 9 although a recent report has suggested that Ϸ50% of atrial myocytes possess a sparse irregular tubular system. 10 The following discussion will concentrate on mammalian ventricular myocytes.The t-tubules are invaginations of the sarcolemma and glycocalyx, which appears to remain associated with the sarcolemma within the t-tubules. 11 Early studies of cardiac muscle showed that they occur at the Z line, at the end of each sarcomere 12 ; therefore, they occur at intervals of Ϸ2 m along the longitudinal axis of the ventricular myocyte. Subsequent studies have shown that the t-tubular system also has longitudinal extensions. 13 Although the t-tubules leave the surface membrane at the Z line, forming an approximately rectangular array, only Ϸ60% of the tubular volume occurs near the Z line; the other 40% ...

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, Ca(2+) current density, and Ca(2+) 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 Ca(2+) release on electrical stimulation. In contrast, formamide treatment of atrial cells did not significantly change cell capacitance, Ca(2+) current amplitude, action potential configuration, the Ca(2+) transient or the response of the Ca(2+) 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.

Crude oil is known to disrupt cardiac function in fish embryos. Large oil spills, such as the Deepwater Horizon (DWH) disaster that occurred in 2010 in the Gulf of Mexico, could severely affect fish at impacted spawning sites. The physiological mechanisms underlying such potential cardiotoxic effects remain unclear. Here, we show that crude oil samples collected from the DWH spill prolonged the action potential of isolated cardiomyocytes from juvenile bluefin and yellowfin tunas, through the blocking of the delayed rectifier potassium current (I(Kr)). Crude oil exposure also decreased calcium current (I(Ca)) and calcium cycling, which disrupted excitation-contraction coupling in cardiomyocytes. Our findings demonstrate a cardiotoxic mechanism by which crude oil affects the regulation of cellular excitability, with implications for life-threatening arrhythmias in vertebrates.

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The Deepwater Horizon disaster drew global attention to the toxicity of crude oil and the potential for adverse health effects amongst marine life and spill responders in the northern Gulf of Mexico. The blowout released complex mixtures of polycyclic aromatic hydrocarbons (PAHs) into critical pelagic spawning habitats for tunas, billfishes, and other ecologically important top predators. Crude oil disrupts cardiac function and has been associated with heart malformations in developing fish. However, the precise identity of cardiotoxic PAHs, and the mechanisms underlying contractile dysfunction are not known. Here we show that phenanthrene, a PAH with a benzene 3-ring structure, is the key moiety disrupting the physiology of heart muscle cells. Phenanthrene is a ubiquitous pollutant in water and air, and the cellular targets for this compound are highly conserved across vertebrates. Our findings therefore suggest that phenanthrene may be a major worldwide cause of vertebrate cardiac dysfunction.

Formamide-induced detubulation of rat ventricular myocytes was used to investigate the functional distribution of the Na/Ca exchanger (NCX) and Na/K-ATPase between the t-tubules and external sarcolemma. Detubulation resulted in a 32% decrease in cell capacitance, whereas cell volume was unchanged. Thus, the surface-to-volume ratio was used to assess the success of detubulation. NCX current (I(NCX)) and Na/K pump current (I(pump)) were recorded using whole-cell patch clamp, as Cd-sensitive and K-activated currents, respectively. Both inward and outward I(NCX) density was significantly reduced by approximately 40% in detubulated cells. I(NCX) density at 0 mV decreased from 0.19 +/- 0.03 to 0.10 +/- 0.03 pA/pF upon detubulation. I(pump) density was also lower in detubulated myocytes over the range of voltages (-50 to +100 mV) and internal [Na] ([Na](i)) investigated (7-22 mM). At [Na](i) = 10 mM and -20 mV, I(pump) density was reduced by 39% in detubulated myocytes (0.28 +/- 0.02 vs. 0.17 +/- 0.03 pA/pF), but the apparent K(m) for [Na](i) was unchanged (16.9 +/- 0.4 vs. 17.0 +/- 0.3 mM). These results indicate that although thet-tubules represent only approximately 32% of the total sarcolemma, they contribute approximately 60% to the total I(NCX) and I(pump). Thus, the functional density of NCX and Na/K pump in the t-tubules is 3-3.5-fold higher than in the external sarcolemma.

Abstract-We have characterized modulation of I Ca by Ca 2ϩ at the t-tubules (ie, in control cells) and surface sarcolemma (ie, in detubulated cells) of cardiac ventricular myocytes, using the whole-cell patch clamp technique to record I Ca . I Ca inactivation was significantly slower in detubulated cells than in control cells (27.1Ϯ7.8 ms, nϭ22, versus 16.4Ϯ7.9 ms, nϭ22; PϽ0.05). In atrial myocytes, which lack t-tubules, I Ca inactivation was not changed by the treatment used to produce detubulation. In the presence of ryanodine or BAPTA, or when Ba 2ϩ was used as the charge carrier, the rate of inactivation was not significantly different in control and detubulated cells. Frequency-dependent facilitation occurred in control cells but not in detubulated cells, and was abolished by ryanodine. These results suggest that Ca 2ϩ released from the SR has a greater effect on I Ca in the t-tubules than at the surface sarcolemma. This does not appear to be due to differences in local Ca 2ϩ release from the SR, because the gain of Ca 2ϩ release was not significantly different in control and detubulated cells. These data suggest that the t-tubules are a key site for the regulation of transsarcolemmal releases. 2 The transverse (t-) tubules of ventricular myocytes play an important role in this process. These tubules are invaginations of the sarcolemma that occur at the Z-line, perpendicular to the longitudinal axis of the cell (see review). 3 Functional and immunohistochemical data suggest that I Ca occurs predominantly in the t-tubules, adjacent to RyRs, which are also located predominantly at the t-tubules. 4 -6 Thus, it appears that the t-tubules are the major site of Ca 2ϩ entry and hence Ca 2ϩ release in cardiac ventricular myocytes. Conversely, Ca 2ϩ released by the SR can modulate I Ca ; this plays an important role in cellular Ca 2ϩ homeostasis, controlling Ca 2ϩ entry via negative feedback. 7,8 However, it is unknown whether the efficacy of coupling between SR Ca 2ϩ release and I Ca is the same in the t-tubule and surface membranes, so that the relative importance of these sites in cellular Ca 2ϩ homeostasis is unknown. We have, therefore, investigated the regulation of I Ca by Ca 2ϩ released from the SR in normal ventricular myocytes, in which I Ca triggers Ca 2ϩ release predominantly at the t-tubules, and in myocytes in which the t-tubules have been physically and functionally uncoupled from the surface membrane (detubulated), 5 in which Ca 2ϩ release occurs predominantly at the surface membrane. 9

Detubulation of rat ventricular myocytes has been used to investigate the role of the t-tubules in Ca2+ cycling during excitation-contraction coupling in rat ventricular myocytes. Ca2+ was monitored using fluo-3 and confocal microscopy. In control myocytes, electrical stimulation caused a spatially uniform increase in intracellular [Ca2+] across the cell width. After detubulation, [Ca2+] rose initially at the cell periphery and then propagated into the center of the cell. Application of caffeine to control myocytes resulted in a rapid and uniform increase of intracellular [Ca2+]; the distribution and amplitude of this increase was the same in detubulated myocytes, although its decline was slower. On application of caffeine to control cells, there was a large, rapid, and transient rise in extracellular [Ca2+] as Ca2+ was extruded from the cell; this rise was significantly smaller in detubulated cells, and the remaining increase was blocked by the sarcolemmal Ca2+ ATPase inhibitor carboxyeosin. The treatment used to produce detubulation had no significant effect on Ca2+ efflux in atrial cells, which lack t-tubules. Detubulation of ventricular myocytes also resulted in loss of Na+-Ca2+ exchange current, although the density of the fast Na+ current was unaltered. It is concluded that Na+-Ca2+ exchange function, and hence Ca2+ efflux by this mechanism, is concentrated in the t-tubules, and that the concentration of Ca2+ flux pathways in the t-tubules is important in producing a uniform increase in intracellular Ca2+ on stimulation.