Calcium biology of the transverse tubules in heart

Abstract

Ca(2+) sparks in heart muscle are activated on depolarization by the influx of Ca(2+) through dihydropyridine receptors in the sarcolemmal (SL) and transverse tubule (TT) membranes. The cardiac action potential is thus able to synchronize the [Ca(2+)](i) transient as Ca(2+) release is activated throughout the cell. Increases in the amount of Ca(2+) within the sarcoplasmic reticulum (SR) underlie augmented Ca(2+) release globally and an increase in the sensitivity of the ryanodine receptors (RyRs) to be triggered by the local [Ca(2+)](i). In a similar manner, phosphorylation of the RyRs by protein kinase A (PKA) increases the sensitivity of the RyRs to be activated by local [Ca(2+)](i). Heart failure and other cardiac diseases are associated with changes in SR Ca(2+) content, phosphorylation state of the RyRs, [Ca(2+)](i) signaling defects and arrhythmias. Additional changes in transverse tubules and nearby junctional SR may contribute to alterations in local Ca(2+) signaling. Here we briefly discuss how TT organization can influence Ca(2+) signaling and how changes in SR Ca(2+) release triggering can influence excitation-contraction (EC) coupling. High speed imaging methods are used in combination with single cell patch clamp experiments to investigate how abnormal Ca(2+) signaling may be regulated in health and disease. Three issues are examined in this presentation: (1) normal Ca(2+)-induced Ca(2+) release and Ca(2+) sparks, (2) abnormal SR Ca(2+) release in disease, and (3) the triggering and propagation of waves of elevated [Ca(2+)](i).

FIGURE 1

Transverse tubules (TTs) in rat heart cells. (A) A wide field transmitted light image of a rat ventricular myocyte. (B) A confocal fluorescence image of the TTs in a living rat ventricular myocyte stained with the membrane marker di-8 ANEPPS imaged with 488 nm excitation. The myocyte was exposed to 10 μM dye for 10 min. The striation or TT separation along the long axis of the cell is 1.8 μm.

FIGURE 2

Transverse tubules (TTs) and other related structures in the sarcomere. (A) The TT, schematic diagram of the TT, ryanodine receptor (RyR) cluster, and junctional sarcoplasmic reticulum (jSR). (B) The intermyofibrillar mitochondrion. Relationship between TT structures and the intermyo-fibrillar mitochondrion. (C) The reticular longitudinal sarcoplasmic reticulum (SR). The longitudinal SR wraps around the mitochondrion and is linked to the jSR through diffusion-restricted connections [in color in Annals Online].

FIGURE 3

The transverse tubule (TT) array in a mammalian ventricular myocyte. (A) Diagram of a fully populated TT system (array) at the level of the nucleus (arrow). The TTs flare open at the sarcolemmal surface and do not penetrate the nucleus. The TTs are approximately 1.2 microns apart. (B) An array of TTs are found at every Z-line and are sparsely connected to the neighboring Z-line array by longitudinal tubules (lines at 45 degrees, shown in red in Annals Online). There are three Z-line arrays shown in this diagram. (C) Diagram of the placement of the array of TTs in an intact cell. (D) Diagram of a TT elementary unit. An elementary unit of TTs is composed of a square (1.2 microns on an edge) in successive Z-line arrays. The small sphere (shown in green online) represents the most distant location in the elementary unit from a TT, and it is approximately 1.17 microns from the nearest TT, assuming the distance between Z-lines is 2 microns. Yellow lines (seen online) connect opposite corners and go through the center of the small sphere [in color in Annals Online].

FIGURE 4

Organization of key channel and transporter proteins and sarcoplasmic reticulum (SR) structures near the transverse tubule (TT). The IP3 receptors (IP3Rs) are separated from the ryanodine receptors (RyRs) in this diagram because of recent immunofluorescence data. However, both IP3Rs and RyRs are thought to be SR structures. The key proteins are labeled in the figure. The extensive array of cytoskeletal proteins, signaling receptors, adaptor proteins, and contractile filaments are not shown. SL, sacrolemmal; NCX, Na⁺/Ca²⁺ exchanger; DHPR, dihydropyridine receptors; JSR, junctional sarcoplasmic reticulum [in color in Annals Online].

FIGURE 5

Calcium profile across the diameter of the cell during excitation–contraction (EC) coupling. (A) A [Ca²⁺]_i transient shown using a linescan image of a heart cell taken across the diameter of the cell. Note that there is virtually synchronous activation of Ca²⁺ release at all points. (B) Same as A but after the application of ryanodine (RY) and thapsigargin (TG) to completely block sarcoplasmic reticulum (SR) Ca²⁺ release and re-uptake respectively. (C) The [Ca²⁺]_i transients from A and B. (D) Time-course of the [Ca²⁺]_i transient due to I_Ca alone and I_Ca plus triggered release are nearly identical during the early phase. (E) Effect of diffusion distance and diffusion half-time. (A–C modified from Cheng et al. Used with permission.) TT, transverse tubule; STIM, stimulus [in color in Annals Online.]

FIGURE 6

Images of the initiation of a propagated wave of elevated [Ca²⁺]_i in a rat ventricular myocyte. Frames are numbered sequentially 1–24. Ca²⁺ sparks can be seen in virtually every frame. In frame 2, two of several Ca²⁺ sparks are identified with arrows. In Frames 4 and 5, the arrows (shown in yellow in Annals Online) mark a Ca²⁺ spark that initiates the wave of propagating elevated [Ca²⁺]_i. It is clear in the frames (starting in frame 4) that there is a local instability over a region of about 8 microns, where sparks activate neighboring regions and then form an increasing cloud of activating and spreading elevated [Ca²⁺]_i that by frame 24 has started to exit the region. This wave propagates throughout the cell in frames that follow these first 24. The cell was loaded with the Ca²⁺ sensitive indicator fluo-4 and was exposed to a normal extracellular solution with [Ca²⁺]_o = 3 mM. Sequential high-resolution images acquired at 30 ms intervals using the very fast confocal microscope, the Zeiss LSM 5 Live [in color in Annals Online].