Excitation Contraction Coupling

atria and ventricles, which is known as the Wenckebach effect [139]. A fast re-excitation of the atria, e.g. due to atrial fibrillation, is not conducted to the ventricle with the same frequency but only every 1 – 4 excitations.

Calcium is considered to be the most important ion involved in the cardiac ECC [146].

The inward Ca²⁺ current contributes to the AP plateau, increases intracellular calcium, and induces the activation of the contraction (fig. 4.9 inset). Measurements with the patch clamp technique revealed that Ca²⁺ current reaches its peak value in approximately 2 – 3 ms after begin of the depolarization. The combination of Ca²⁺influx from extracellular space and Ca²⁺

release from SR raises the free intracellular Ca²⁺ concentration ([Ca²⁺]i) and allows Ca²⁺

to bind to the thin filament protein TnC. The TnC-Ca²⁺ complex enables the contraction process of the myofilaments. However, contraction depends not solely on [Ca²⁺]i, but also on other indispensable factors.

4.6.1 Involved Ionic Channels

Several calcium specific proteins are involved in cardiac ECC and the calcium removal pro-cedure (fig. 4.9). They are located in the sarcolemma and intracellular organelles, e.g. the SR and mitochondria. This section describes channels that contribute to the increasing of [Ca²⁺]i, i.e. the L-type Ca²⁺ channel (DHPR) and the SR Ca²⁺ release channel (RyR). Four pathways involved in Ca²⁺ transport out of the myoplasm are known, i.e. the SR Ca²⁺ -ATPase, the sarcolemmal Ca²⁺-ATPase, and the mitochondrial Ca²⁺ uniport. The Na/Ca exchanger was already explained in section 4.3.3.

Dihydropyridine Receptors (DHPR) are voltage-dependent L-type Ca²⁺channels. They are sensitive to dihydropyridines and are activated by depolarization of the cell membrane.

DHPRs are found mostly adjacent to the junctional segment of the SR (section 2.1.2.3).

Fig. 4.9.Transmembrane channels for calcium fluxes can be divided into two groups, i.e. channels which increase (red-colored) and decrease (green-colored)[Ca²⁺]i. Increasing[Ca²⁺]iproteins are DHPR (denoted asICa) and RyR. Decreasing[Ca²⁺]i proteins are SR Ca²⁺-ATPase, sarcolemmal Na⁺/Ca²⁺ exchanger (NCX), sarcolemmal Ca²⁺-ATPase (ATP), and mitochondrial uniport (The mitochodrion is the ellipsoid shaped part). Calcium binds to proteins of the myofilament during high [Ca²⁺]i. (Inset) Time course of transmembrane voltage, intracellular concentration of calcium, and contraction. Fig. from [147].

4.6. Excitation Contraction Coupling 43 Studies showed that the Ca²⁺-induced Ca²⁺-release mechanism is initiated and mediated by the L-type Ca²⁺ channel current [147]. The selectivity of L-type Ca²⁺channel in ion transport is mediated by preferential binding of two principal competitors, i.e. Ca²⁺ over Na⁺ [148].

Ryanodine Receptors (RyR) are channels that releases Ca²⁺ from the SR. RyRs are located at the junctional SR (JSR). The Ca²⁺ buffer calsequestrin participates in the active Ca²⁺ release process by modulating and inhibiting the function of RyRs [48, 49, 149].

SR Ca²⁺-ATPase: The free myoplasmic Ca²⁺ concentration is kept below 0.1 µM during the resting phase, primarily by Ca²⁺ pumps [150, 151]. Ca²⁺ pumps are mainly located along the membrane of the network SR (NSR) moving Ca²⁺ continually from the myoplasm into the SR. Because of this activity, the SR acts as an intracellular reservoir for calcium. The transport mechanism starts with the reaction of two calcium ions and one ATP molecule, binding with high affinity to the myoplasmic sites of the pump. The phosphate terminal of ATP is transferred to a receptor and ATP gets hydrolyzed. Calcium ions are adsorbed and obstructed in the pump. The hydrolysis reduces the affinity of calcium ions, which can be released to the lumen of the SR. The cardiac SR Ca²⁺-ATPase is endogenously inhibited by the integral membrane protein phospholamban [152].

Sarcolemmal Ca²⁺-ATPases function as a maintainer of low [Ca²⁺]i during the resting phase [153]. Transport of one Ca²⁺ is driven by the hydrolysis of one ATP. Sarcolemmal Ca²⁺-ATPase possesses a high affinity to Ca²⁺, but its transport rate is very slow. A sole removal of intracellular calcium through this pump would take about 20 – 40 s for the myocyte to relax [13]. In comparison to the SR Ca²⁺-ATPase, the number of sarcolemmal Ca²⁺ -ATPases in the cell membrane represents only a minor portion of the total membrane proteins.

The sarcolemmal Ca²⁺-ATPase has a higher affinity for Ca²⁺ than the sarcolemmal Na/Ca exchanger, but the sarcolemmal exchanger has a higher maximal throughput of Ca²⁺. Mitochondrial Ca²⁺ uniport: The mitochondria feature Ca²⁺ removal channels, the so-called mitochondrial Ca²⁺ uniports. They remove myoplasmic Ca²⁺ into the mitochondria, but the removal rate is very slow. The required energy is mostly replenished by mitochondrial oxidative phosphorylation. Slow cumulative changes in intra-mitochondrial calcium ions can increase the production of ATP to match increased energetic demands [154]. Since both, ATP and changes of [Ca²⁺]iare important for the cardiac contraction and relaxation, mitochondrial calcium dynamic plays an important role during ECC.

4.6.2 Intracellular Calcium Transients

The transsarcolemmal Ca²⁺ influx through the DHPR channels does not activate the my-ofilaments directly, but through the CICR from the SR. This process describes how a small elevation of the dyadic calcium concentration leads to a larger Ca²⁺ release from the SR [13].

CICR does not occur in an all-or-none fashion and can be graded with the magnitude of Ca²⁺ influx current through the sarcolemma i.e. modulated by DHPR [155]. The amplitude of CICR also depends on the rate at which the calcium concentration near to the SR changes.

A high concentration of Ca²⁺ in the dyadic space inactivates CICR.

4.6.2.1 Ca²⁺ Sparks

Ca²⁺ sparks are spontaneous local Ca²⁺ transients from RyRs (fig. 4.10), which are lo-cated at the junctional couplon, thus mostly at the dyadic spaces along the T-tubules (sec-tion 2.1.2.3) [147]. Ca²⁺ sparks are occuring nearly synchronous in a cluster of about 6 – 20

(a) (b)

Fig. 4.10.Ca²⁺sparks occur at certain locations and rise to a peak in about 10ms. Ca²⁺sparks decline in about 20ms. They have a spatial diameter of 2.5µm. (a) Recorded in intact mouse ventricular myocytes.

(b) Permeabilized cells. Fig. from [147].

RyRs. They can be recorded with a rising time to their peak of about 10 ms and a declining time of about 20 ms. In a resting myocyte, Ca²⁺ sparks are rare stochastic events, which are distributed equally to all RyRs. If the Ca²⁺ concentration in the dyadic space or the SR is high enough, stochastic releases of Ca²⁺ at a couplon can activate a neighboring couplon.

During the activation of the cell, sparks of all participating RyRs are synchronously evoked in time by depolarization [156]. The sychronization leads to an overlap in time and space of the Ca²⁺ transients and [Ca²⁺]i raises [157]. Since the number of involved RyRs is high, the measured Ca²⁺ transients appear spatially uniform and of high amplitude.

4.6.2.2 Activation and Deactivation of Calcium Release

Usually, a cluster of L-type Ca²⁺ channels is involved synchronizing RyR clusters. But even one single opened local L-type Ca²⁺ channel in one couplon can trigger synchronous sparks of the involved RyRs. This single opened DHPR is sufficient to fully activate the release process at that couplon. Other neighboring RyRs can be activated either by high local Ca²⁺ (> 10 µM) or by coupling mechanisms between RyRs.

Figure 4.11 describes the gating mechanism of RyRs in presence of Ca²⁺. A RyR is equipped with two Ca²⁺ binding sites including a coupling channel key, which functions as a release valve. One of the Ca²⁺ binding sites has a low affinity, the other a higher affinity. When [Ca²⁺]i in the dyadic space increases, Ca²⁺ binds to the lower affinity site. This site has a

Fig. 4.11.Schematic diagram of the activation and inactivation mechanism of RyR. The binding of Ca²⁺

to the low affinity activation site (1) leads to a rapid activation of RyR. Binding of Ca²⁺ to the high affinity site (2) inactivates RyR. Fig. from [13].

4.7. Tissue Specific Electrophysiology 45