Quantification of Calcium Entry at the T-Tubules and Surface Membrane in Rat Ventricular Myocytes

Abstract

The action potential of cardiac ventricular myocytes is characterized by its long duration, mainly due to Ca flux through L-type Ca channels. Ca entry also serves to trigger the release of Ca from the sarcoplasmic reticulum. The aim of this study was to investigate the role of cell membrane invaginations called transverse (T)-tubules in determining Ca influx and action potential duration in cardiac ventricular myocytes. We used the whole cell patch clamp technique to record electrophysiological activity in intact rat ventricular myocytes (i.e., from the T-tubules and surface sarcolemma) and in detubulated myocytes (i.e., from the surface sarcolemma only). Action potentials were significantly shorter in detubulated cells than in control cells. In contrast, resting membrane potential and action potential amplitude were similar in control and detubulated myocytes. Experiments under voltage clamp using action potential waveforms were used to quantify Ca entry via the Ca current. Ca entry after detubulation was reduced by approximately 60%, a value similar to the decrease in action potential duration. We calculated that Ca influx at the T-tubules is 1.3 times that at the cell surface (4.9 vs. 3.8 micromol/L cytosol, respectively) during a square voltage clamp pulse. In contrast, during a cardiac action potential, Ca entry at the T-tubules is 2.2 times that at the cell surface (3.0 vs. 1.4 micromol/L cytosol, respectively). However, more Ca entry occurs per microm(2) of junctional membrane at the cell surface than in the T-tubules (in nM/microm(2): 1.43 vs. 1.06 during a cardiac action potential). This difference is unlikely to be due to a difference in the number of Ca channels/junction at each site because we estimate that the same number of Ca channels is present at cell surface and T-tubule junctions ( approximately 35). This study provides the first evidence that the T-tubules are a key site for the regulation of action potential duration in ventricular cardiac myocytes. Our data also provide the first direct measurements of T-tubular Ca influx, which are consistent with the idea that cardiac excitation-contraction coupling largely occurs at the T-tubule dyadic clefts.

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

“…Consistent with this, it has been shown that the peripheral VOCC/RyR couplings in ventricular myocytes are functionally distinct from their T-tubule-located counterparts. They may be less well arranged for EC-coupling, but provide more Ca 2+ influx during depolarisation for SR reloading [35,36]. It therefore appears that following detubulation, ventricular myocytes rely on relatively weaker VOCC/RyR couplings in their periphery, as compared to the deliberate physiological peripheral VOCC/RyR couplings of atrial myocytes.…”

Is overall blockade superior to selective blockade of adrenergic receptor subtypes in suppressing left ventricular remodeling in spontaneously hypertensive rats?

“…Cell pellets were collected by centrifugation and resuspended in 0.5 ml PBS. 15 Then, 0.5 ml of propidium iodide solution (100 mg ml À1 in PBS) was added and the mixture was allowed to stand on ice for 30 min. The myocardial cells (1Â10 6 ml À1 ) were analyzed with a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA) and distribution of the cell cycle phases was determined by means of ModFit LT software (Verity Software House, Topsham, ME, USA).…”

Voltage Dependence of Cardiac Excitation–Contraction Coupling

“…It is thus not surprising that reducing NP o rapidly reduces the fraction of active junctions from the relatively large value at V m ϭ0 mV (Figure 6a). We did not study the Ϸ30% of LCCs on the surface sarcolemma, 32 but these likely also colocalize with RyRs at surface junctions. 5 Although Brette et al 32,33 measured slower I Ca inactivation for surface versus transverse tubular I Ca , they reported similar ECC gain for both.…”