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

Abstract

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.

Comparison of the T-tubule system in adult rat ventricular and atrial myocytes, and its role in excitation–contraction coupling and inotropic stimulation

“…However, much less is known about the expression and function of sarcolemmal tubules in atrial cells. Although it is commonly cited that mammalian atrial myocytes do not possess T-tubules [5,9,26], it has been suggested that these cells possess a rudimentary transverse-axial tubular system [21][22][23]25,33]. Results obtained from immunostaining (Fig.…”

“…Orchard and colleagues have established a method for detubulating myocytes using brief application of 1.5 M formamide solution to cause a rapid osmotic shock [26,27]. Formamide treatment did not alter the distributions of type 2 RyRs or L-type VOCCs in atrial myocytes ( Fig.…”

“…2A illustrate the well-known T-tubule system within adult ventricular myocytes [26]. The presence of the tubules allows the action potential to activate L-type VOCCs, and their juxtaposed RyRs, throughout a ventricular cell leading to a homogenous global Ca 2+ increase.…”

“…In this study, we characterised the T-tubules within adult rat ventricular and atrial myocytes, and examined the effect of their presence on the characteristics of Ca 2+ signalling and responses to cardioactive hormones. For comparison, we examined Ca 2+ signalling in control myocytes with an intact tubular system and cells that had been detubulated using an established technique involving osmotic swelling [26,27].…”

Endothelin-1 Stimulates the Na + /Ca 2+ Exchanger Reverse Mode Through Intracellular Na + (Na + i )–Dependent and Na + i -Independent Pathways

“…Although we demonstrated that both interventions increase contractility by NCX in reverse, and that for a given increase in Na ϩ i , ET-1 induced a greater increase in contractility than inhibition of the Na ϩ /K ϩ ATPase, the following possibilities should be analyzed: (1) Despite an equal Na ϩ i in the bulk of the cytosol, the increase of Na ϩ i induced by ET-1 would be higher in a fuzzy space close to NCX. However, a colocalization of NCX and NHE, 20,21 and also of NCX and the Na ϩ /K ϩ ATPase, 22 have been reported; (2) ET-1 could induce a greater prolongation of the action potential duration (APD) than low K ϩ o , then the influx of Ca 2ϩ through the NCX reverse mode would be greater than that induced by Na ϩ /K ϩ ATPase inhibition. Although the increase in APD by ET-1 has been reported, 23,24 low K ϩ o can also induce a prolongation of APD, 25 and the differences between both interventions are difficult to evaluate; (3) The way selected by us to increase Na ϩ i (low K ϩ o ), by hyperpolarizing the cell, may promote Ca 2ϩ efflux through the forward mode of NCX, thus reducing PIE.…”