Na ⁺ -Ca ²⁺ Exchange Activity Is Localized in the T-Tubules of Rat Ventricular Myocytes

Abstract

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.

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

“…It is well established that a major mechanism for recovery of Ca 2+ transients in ventricular myocytes is sarcolemmal Na + /Ca 2+ exchange, which has been suggested to be concentrated within Ttubule membranes [5,34]. The reduced recovery of Ca 2+ transients in detubulated ventricular myocytes observed in this study (Fig.…”

Quantification of t-tubule area and protein distribution in rat cardiac ventricular myocytes

“…Membrane capacitance is a function of membrane area, and decreases by ~32% after detubulation Brette et al, 2004a;Brette et al, 2004b;Brette & Orchard, 2006a;Despa et al, 2003;Kawai et al, 1999;Komukai et al, 2002;Yang et al, 2002). This is similar to the fraction of cell membrane estimated to be in the ttubules using electron microscopy (Page, 1978).…”

Reduced synchrony of Ca2+ release with loss of T-tubules—a comparison to Ca2+ release in human failing cardiomyocytes

“…SR Ca 2+ content, measured as the total charge extruded by the Na/Ca exchanger during caffeine application, was comparable for control (92F23 pC, n cells =9), 24 h (105F16 pC, n cells =14), and 72 h of culture (83F14 pC, n cells =11). The amplitude of the caffeine-induced [Ca 2+ ] i transient was also not significantly different ( F/F 0 of control 3.26F0.21, n cells =7; at 24 h 2.82F0.19, n cells =13; at 72 h 2.59F0.43, n cells =11), though relaxation was markedly slower after 24 and 72 h, consistent with loss of NCX in the T-tubules [15,26,27].…”

“…Acute detubulation of adult rat ventricular myocytes produces a similar pattern [15]. In guinea-pig ventricular myocytes which had lost Ttubules following culture, a wavelike propagation of the [Ca 2+ ] i transient was observed [8].…”