Search

Cardiac t-tubules (TTs) form a network of complex surface membrane invaginations that is essential for proper excitation-contraction coupling. Although electron and optical microscopy studies provided a wealth of important information about the structure of TTs, assessing their functional properties remains a challenge. In this study, we investigated the diffusional accessibility of TTs in intact isolated adult mouse ventricular myocytes using, to our knowledge, a novel fluorescence-based assay. In this approach, a small part of TTs is first locally filled with fluorescent dextran and then its diffusion out of TTs is monitored after rapid removal of extracellular dextran. In normal cells, diffusion of 3 kDa dextran is characterized by an average time constant of 3.9 ± 1.2 s with the data ranging from 1.8 to 10.5 s. The data are consistent with essentially free diffusion of dextran in TTs although measurable contribution of binding is also evident. TT fluorescence is abolished in cells treated with high concentration of formamide or after hyposmotic stress. Importantly, the assay we use allows for quantitative, repetitive measurements of subtle dynamic changes in TT structure of the same cell that are not possible to observe with other approaches. In particular, dextran diffusion rate decreases two-to-threefold during cell swelling, suggesting significant structural remodeling of TTs. Computer modeling shows that diffusional accessibility and electrical properties of TTs are primarily determined by the constrictions and dilations of individual TTs and that, from a functional perspective, TTs cannot be considered as a network of cylinders of the same average diameter. Constriction/dilation model of cardiac TTs is in a quantitative agreement with previous high-resolution microscopy studies of TT structure and alternative measurements of diffusional and electrical time constants of TTs. The data also show that the apparent electrical length constant of cardiac TTs is likely several-fold smaller than that estimated in earlier studies.

T-tubules are membrane invaginations of cardiac myocytes critical for effective excitation-contraction coupling and are significantly altered in various diseases. However, the mechanisms underlying t-tubule remodeling remain largely unknown, particularly due to a number of limitations of currently available technologies. Therefore, we have developed an assay that employs the diffusion of fluorescent 3 KD dextran out of the t-tubule network of live intact isolated ventricular cardiomyocytes. To significantly improve the quality of measurements, dextran is applied locally using a patch-clamp pipette (∼2-3 μm diameter) under constant flow of washing solution through 0.5 mm ID capillary glass, which limits dextran filling of t-tubules to a small area of the cardiomyocyte. With 40X objective and small pinholes in the light paths, excitation is limited to ∼9-10 μm diameter area and fluorescence emission is measured from ∼5-6 μm diameter region using a photomultiplier. Upon cessation of dye application, fluorescence decline displays 2 major components: a fast component (τ ∼5-50 ms) corresponding to dye washout from the solution surrounding the cell and a slow component corresponding to dye diffusion out of the t-tubule system. In healthy mouse ventricular myocytes the slow component had a time constant of 1304 ± 98 ms (n=24). In contrast, “detubulation” by acute hyposmotic stress significantly decreased the time constant to 848 ± 99 ms (n=27, p

It has been recently shown that various stress-inducing manipulations in isolated ventricular cardiac myocytes may lead to significant remodeling of t-tubules. Osmotic stress is one of the most common complications in various experimental and clinical settings, and therefore, this study was designed to test a hypothesis that osmotic challenge may affect the integrity of t-tubules. T-tubular remodeling in mouse ventricular myocytes in response to various osmotic challenges was studied using two approaches: (1) electrophysiologically, by measuring membrane capacitance and IK1 tail currents originating from K+ accumulation in ttubules, and (2) using confocal microscopy of fluorescent dextrans trapped in vesiculated t-tubules. In particular, application and removal of 0.6 T (60% of NaCl) hypo-osmotic solution to myocytes led to ∼30% reduction in membrane capacitance, ∼3-fold reduction in the amplitude of IK1 tail current and ∼2-fold reduction in so-called IK1 ‘inactivation' (due to depletion of t-tubular K+) at negative membrane potentials – all being consistent with strong detubulation. Importantly, confocal imaging experiments showed that extracellularly applied dextrans become trapped inside the myocytes only upon removal of hypo-osmotic solutions (i.e. during shrinking phase) but not during initial swelling period. In light of these data some relevant previous studies, including those on EC coupling phenomena during hypo-osmotic stress, may need to be reinterpreted and the experimental design of future experiments should take into account the novel findings.

Cardiac t-tubules are critical for efficient excitation-contraction coupling and undergo significant remodeling during various experimental and clinical stress conditions including heart failure. Recently, we have shown that recovery of ventricular myocytes after a brief hyposmotic shock is associated with significant sealing of t-tubules. Dimethyl Sulfoxide (DMSO) is a commonly used solvent and has been reported to have cardioprotective properties although the mechanisms underlying these protective effects remain largely unknown. Therefore, we tested whether the cardioprotective effects of DMSO are mediated by its action on t-tubular remodeling. We found that application of DMSO at the time of resolution of hyposmotic stress, when t-tubule sealing occurs, reduced the amount of fluorescent dextran trapped within sealed t-tubules. The effect of DMSO displayed sharp biphasic concentration dependence with 1% being the most effective dose (∼4-fold reduction of dextran trapping; p

Cardiac ventricular myocytes possess an extensive t-tubule system which plays a number of important roles including facilitation of membrane potential propagation across the cell body. It has been shown that ion fluxes at a highly restricted t-tubular space may lead to significant accumulation/depletion of specific ions which in turn may affect t-tubular, as well as whole-cell, membrane potential. The extent of ion accumulation/depletion depends on the current densities and the volume/structure of t-tubules. In this study we used the whole-cell patch-clamp technique to monitor t-tubular accumulation of potassium (caused by outward potassium currents in response to 400 ms voltage step to +50 mV) by measuring inward rectifier IK1 tail currents (Itail) at −75 mV. At room temperatures of ∼21-23 °C the amplitude of Itail current measured 9-10 minutes after the establishment of whole-cell configuration was essentially unchanged (93.6% of initial value), but declined to 53.5% upon application (within 2-5 minutes after the beginning of cell dialysis ) of 1 mM cyanide, a blocker of oxidative phosphorylation (n=9 and 6, respectively; p

Inward rectifier potassium channels IK1 underlie major resting ionic conductance in the heart. A wealth of research on IK1 has been conducted on isolated cells using voltage-clamp techniques but studying IK1 in intact hearts is limited to ion flux assays. Additionally, in the past, flux studies were primarily performed in beating hearts thus eliminating the possibility of measuring any resting conductance. We have implemented a novel approach based on recently discovered thallium-dependent fluorescence of a low-affinity calcium-sensititive dye (BTC). Langendorff-perfused isolated mouse hearts were (i) loaded with membrane permeant BTC and (ii) stopped using verapamil and lidocaine, in order to achieve stable hyperpolarized membrane potential across the heart (thus silencing voltage-dependent potassium conductances). (iii) 1 mM thallium was then applied and (iv) fluorescence of BTC captured using a camera. In all hearts the time course of the relative increase in fluorescence, ΔF, was characterized by (1) well-defined fast (tau∼50÷150 sec) and (2) highly variable slow components. The total initial rate of ΔF was significantly increased in transgenic hearts with upregulated IK1 (up to 2-fold vs control). The initial rates of both components of ΔF were strongly IK1 dependent. Both, transgenic down regulation of IK1 and blockade of IK1 with Ba2+ ions led to a ∼10-20% decrease in the rate of ΔF (not statistically significant) suggesting other major pathways for thallium influx (e.g. Na+/K+ ATPase). Confocal microscopy of isolated ventricular myocytes revealed significant accumulation of BTC in the mitochondria consistent with the hypothesis that the slow component of ΔF reflects mitochondrial uptake of thallium. Results of this study show the possibility of studying sarcolemmal and mitochondrial K+ conductances including IK1 and other pathways (KATP, Na+/K+ ATPase, Na+/K+/CL- symporter) in intact isolated hearts with high temporal resolution.

The high permeability of K+ channels to monovalent thallium (Tl+) ions and the low solubility of thallium bromide salt were used to develop a simple yet very sensitive approach to the study of membrane localization of potassium channels. K+ channels (Kir1.1, Kir2.1, Kir2.3, Kv2.1), were expressed in Xenopus oocytes and loaded with Br− ions by microinjection. Oocytes were then exposed to extracellular thallium. Under conditions favoring influx of Tl+ ions (negative membrane potential under voltage clamp, or high concentration of extracellular Tl+), crystals of TlBr, visible under low-power microscopy, formed under the membrane in places of high density of K+ channels. Crystals were not formed in uninjected oocytes, but were formed in oocytes expressing as little as 5μS K+ conductance. The number of observed crystals was much lower than the estimated number of functional channels. Based on the pattern of crystal formation, K+ channels appear to be expressed mostly around the point of cRNA injection when injected either into the animal or vegetal hemisphere. In addition to this pseudopolarized distribution of K+ channels due to localized microinjection of cRNA, a naturally polarized (animal/vegetal side) distribution of K+ channels was also frequently observed when K+ channel cRNA was injected at the equator. A second novel “agarose-hemiclamp” technique was developed to permit direct measurements of K+ currents from different hemispheres of oocytes under two-microelectrode voltage clamp. This technique, together with direct patch-clamping of patches of membrane in regions of high crystal density, confirmed that the localization of TlBr crystals corresponded to the localization of functional K+ channels and suggested a clustered organization of functional channels. With appropriate permeant ion/counterion pairs, this approach may be applicable to the visualization of the membrane distribution of any functional ion channel.

Potassium conduction through unblocked inwardly rectifying (IRK1, Kir2.1) potassium channels was measured in inside-out-patches from Xenopus oocytes, after removal of polyamine-induced strong inward rectification. Unblocked IRK1 channel current-voltage (I-V) relations show very mild inward rectification in symmetrical solutions, are linearized in nonsymmetrical solutions that bring the K+ reversal potential to extreme negative values, and follow Goldman-Hodgkin-Katz constant field equation at extreme positive E alpha. When intracellular K+ concentration (KIN) was varied, at constant extracellular K+ concentration (KOUT) the conductance at the reversal potential (GREV) followed closely the predictions of the Goldman-Hodgkin-Katz constant field equation at low concentrations and saturated sharply at concentrations of > 150 mM. Similarly, when KOUT was varied, at constant KIN, GREV saturated at concentrations of > 150 mM. A square-root dependence of conductance on KOUT is a well-known property of inward rectifier potassium channels and is a property of the open channel. A nonsymmetrical two-site three-barrier model can qualitatively explain both the I-V relations and the [K+] dependence of conductance of open IRK1 (Kir2.1) channels.

Cardiac t-tubules are central players in excitation-contraction coupling. It has been shown that in ventricular myocytes t-tubules can be sealed during various experimental conditions including metabolic inhibition and osmotic stress. Sealing of t-tubules is associated with trapping of extracellular solution inside the myocytes but the fate of various trapped ions and the consequences of potential trans-tubular ion fluxes remain unknown. In this study we aimed to determine the movements of Ca2+ during t-tubular sealing induced by resolution of hyposmotic stress. T-tubular and cytosolic concentrations of Ca2+ in mouse ventricular myocytes were assessed using Rhod-5N and Fluo-3 Ca2+ indicators, respectively. We show that under normal conditions the concentration of Ca2+ in sealed t-tubules is below 100 μM. The latter strongly suggests the flux of trapped t-tubular Ca2+ into the myocytes during sealing process. Blockade of voltage-dependent Ca2+ channels with 10 μM nicardipine led to several fold increase in concentration of Ca2+ in sealed t-tubules. Alternatively, release of Ca2+ from sarcoplasmic reticulum (SR) in response to 10 mM caffeine also led to restoration of t-tubular Ca2+ towards extracellular levels within few seconds. T-tubular sealing under normal ionic conditions led to occasional and sporadic intracellular Ca2+ transients, consistent with influx of trapped Ca2+. Accordingly, sealing of t-tubules in the presence of 10 mM caffeine (to prevent Ca2+ uptake by SR) was characterized by significant long lasting increase in intracellular Ca2+. The effect was completely abolished in the absence of extracellular Ca2+ and significantly reduced in pre-detubulated myocytes. This study shows that sealed t-tubules are capable of highly regulated transport of Ca2+ and present a major route for Ca2+ influx into myocytes during sealing process.