Donald M. Bers

Chapter Outline

Excitation-Contraction Coupling and Relationship to Action Potentials

Cardiac excitation-contraction coupling (ECC) refers to the process by which the electrical activation of cardiac myocytes leads to the activation of contraction.1–3 In its broadest use, ECC refers to everything from the initial membrane depolarization through the action potential (AP) and activation of the Ca transient, including how the myofilaments respond to the Ca transient to produce contraction. This chapter will focus on the initiation of normal and abnormal Ca transients in myocytes, Ca transport and buffering mechanisms, and the interaction of Ca signaling with the AP, arrhythmias, and gene regulation. In recent years, it has become increasingly clear that Ca signaling and cardiac electrophysiology are inextricably interrelated, making it essential to understand myocyte Ca regulation to understand arrhythmogenesis.

Figure 16-1 shows the structure and key mediators of ECC in myocytes. Ca influx via Ca current (I_Ca) and Ca release from the ryanodine receptor (RyR) in the sarcoplasmic reticulum (SR) are central to myofilament activation. Ventricular myocytes have a network of transverse or T-tubules that dive into the cell center, perpendicular to the long axis of the myocyte. This T-tubular system also exhibits longitudinal extension in some myocytes. The role of the T-tubular system is to synchronize the sarcolemmal electrical signal (AP) at junctions throughout the cell, where the plasma membrane and SR are close together and mediate Ca-induced Ca release. In heart failure (HF), there is evidence that the T-tubule network is less extensive and less well organized,^4,5 which can reduce efficacy of the overall ECC process.

Atrial myocytes have fewer T-tubules,⁵ and specialized conduction fibers (sinoatrial and atrioventricular nodes and Purkinje fibers) have almost no T-tubules. In cells or regions thereof that lack T-tubules, I_Ca initiates SR Ca release only at the surface membrane junctions. Then depending on the conditions discussed later, activation can more slowly propagate as a wave of Ca-induced Ca release to the center of the myocyte (via a chain of RyR clusters) or fail to propagate such that the surface release produces only a small and slow [Ca] elevation near the center of the cell.

It is this rise in intracellular [Ca] ([Ca]_i) that activates the myofilaments to contract. Ca binds to troponin C on the thin filaments, which induces a conformational change that allows the heads of myosin molecules that stick out along the thick filament to bind to actin molecules, which form the body of the thin filament (see Figure 16-1). The myosin head uses energy stored in adenosine triphosphate (ATP) to tilt the head (pulling on the actin filament), thus creating force or sarcomere shortening, which is responsible for isovolumic contraction and ejection of blood from the heart. The synchronization of local Ca transients throughout the heart is therefore essential for synchronous ventricular contraction. The strength of contraction is directly related to the [Ca] surrounding the myofilaments. Thus a myocyte (or region thereof) that has a small Ca transient will be weaker than an adjacent area and can damp the strength of the stronger region (i.e., the strong cell can expend its strength, stretching a weak neighbor, and thus not contribute to cardiac output). Readily appreciable consequences of this mechanical dyssynchrony include ischemia ventricular fibrillation and spatially discordant cardiac alternans.

Another point worth mentioning is that Ca signaling is highly local, and in that sense it contrasts with electrical signals. As a result, the distribution of Ca entry and SR Ca release must be uniform, even at each region within a single myocyte, to ensure proper function. In contrast, because of the long electrical space constant in the heart, there could be potassium channels in only every other myocyte, and electrical signaling could be normal.

For cardiac relaxation and ventricular refilling to occur, [Ca]_i must decline, allowing Ca to dissociate from troponin C and terminate myofilament cross-bridge cycling. This [Ca]_i decline is driven by Ca transport, mainly via the SR Ca–adenosine triphosphatase (ATPase) and sarcolemmal Na/Ca exchanger (NCX; discussed later). Because Ca movement across the sarcolemma via Ca channels and the 3Na:1Ca electrogenic NCX carries a charge, it affects the AP and excitability. There are other Ca-sensitive channels whose electrophysiologic impact is regulated by the local [Ca]_i near those channels. Thus, it is important to integrate our understanding of Ca handling into our electrophysiologic framework.

Sources and Sinks of Ca in Myocytes: Sarcolemma, Sarcoplasmic Reticulum, Mitochondria

In reference to the cytosolic compartment in which the myofilaments reside and with which the sarcolemma, SR, mitochondria, and nucleus interface, the sources of Ca that cause cytosolic [Ca] to rise and sink, which remove Ca from the cytosol, can be considered. Ca channels (L-type and T-type) and the NCX are the main pathways of Ca entry. However, with the huge electrochemical driving force for Ca influx caused by a 20,000-fold concentration gradient (2 mM outside: diastolic [Ca]_i = 100 nM) and −80 mV membrane potential (E_m), even a slight Ca permeability other than these known transporters would constitute an additional non-negligible leak pathway. During diastole, the Ca channels are tightly closed and NCX (which is reversible) functions predominantly in the Ca extrusion mode (inward current).

During the AP, voltage-dependent Ca channels open and allow Ca entry down its electrochemical gradient, causing an inward depolarizing current (see Chapter 2 for Ca-channel gating details). In ventricular myocytes, the I_Ca is virtually all mediated by L-type Ca channels (LTCC), in particular by the Cav1.2 isoform. Atrial, and especially pacemaker, cells also express the Cav1.3 LTCC isoform, which activates at a more negative E_m, and thus can better contribute to phase 4 depolarization in pacemakers and recruit additional Cav1.2 channels.

T-type Ca channels are also seen in atrial, pacemaker, and conduction fibers, but not in ventricular myocytes (except in neonatal myocytes and in some pathophysiologic settings). These channels activate at even more negative E_m but also inactivate much more rapidly than LTCCs. They can contribute to triggered or pacemaker activity, but even when they are present they provide less peak I_Ca and integrated Ca influx than LTCC and cannot effectively substitute for LTCC in triggering SR Ca release. The latter is probably because they do not target to the sarcolemmal-SR junctions, where Ca-induced Ca release occurs during ECC.

I_Ca is activated by depolarization and exhibits E_m– and Ca-dependent inactivation (VDI and CDI), and CDI is by far the dominant physiologic mode of inactivation.⁶ When CDI is abrogated experimentally, AP duration (APD) becomes extremely long and Ca tends to overload in cells. There appears to be calmodulin (CaM) constitutively bound to the Cav1.2 in myocytes and it senses local [Ca]_i elevation and induces inactivation.⁷ Although Ca entry via the channel itself can contribute to CDI, most LTCCs in cardiac myocytes are localized at junctions with the SR and RyR, and the Ca-induced Ca release is even more powerful in causing CDI. The integrated amount of Ca influx via I_Ca in ventricular myocytes during a normal AP is 5 to 10 µmol/L cytosol but is about twice as large if SR Ca release is blocked. In context, in this Ca flux the amount of SR Ca release in mammalian ventricular myocytes is normally three to ten times larger than this, depending on species and conditions. The regulation of SR Ca release will be discussed in more detail.

NCX can also contribute to the rise in [Ca]_i, but Ca entry via NCX is normally small (<1 µmol/L cytosol) and constrained largely to the very early part of the AP. NCX can bring Ca in or out (outward or inward current) during the AP, depending on the trans-sarcolemmal [Na] and [Ca] gradients and E_m (Figure 16-2). During diastole, the E_m is negative to the electrophysiologic reverse potential for NCX (E_NCX = 3E_Na − 2E_Ca), so Ca efflux is thermodynamically favored (see Figure 16-2, A). However, the low diastolic [Ca]_i kinetically limits the amount of transport (low substrate concentration). The peak of the AP exceeds E_NCX and briefly favors Ca influx (an outward current). However, as soon as I_Ca and SR Ca release begin, the local [Ca]_i in the cleft and submembrane space ([Ca]_sm) is much higher than [Ca]_i and drives Ca extrusion via NCX (see Figure 16-2, B and C). The declining E_m during repolarization also more strongly favors Ca efflux, and that persists throughout [Ca]_i decline and diastole (see Figure 16-2, A). Thus, NCX is mainly considered a Ca efflux mechanism. An exception to this is certain pathophysiologic conditions like HF, in which [Na]_i is elevated, NCX expression may be elevated, Ca transients are small, and APD is prolonged (see Figure 16-2, C). All these factors shift NCX more in favor of Ca influx, and thus in HF, Ca entry via NCX can persist for most of the AP plateau and significantly contribute to the Ca transient. In a sense, this limits the extent of cardiac dysfunction in HF by bringing Ca in and by indirectly helping load the SR with Ca.

This raises the question: How much Ca enters the cytosol during the AP and where does it go? The sum of Ca coming from I_Ca, NCX, and SR Ca release is between 50 and 100 µmol/L cytosol. Notably, this is the total amount of Ca entering the myoplasm that contains many Ca buffers (e.g., troponin C, the SR Ca-ATPase, CaM, membrane lipids, ATP). Quantitatively, the most important Ca buffers are troponin C and the SR Ca-ATPase, which are present at approximately 70 and 50 µmol/L cytosol, respectively, and have affinity in the 0.5-µM range. Overall, the cytosolic buffering is approximately 100 : 1, such that 50 to 100 µmol/L cytosol of added Ca only raises the free [Ca]_i from 100 nM to approximately 500 to 1000 nM. However, that is sufficient to partially saturate troponin C and activate contraction. Notably, Ca buffering does not directly remove Ca from the cytosol, and energy-dependent Ca transport is required to return [Ca]_i and cytosolic buffers back to their diastolic levels.

There are four transporters that work in parallel to bring [Ca]_i down and drive relaxation: (1) the SR Ca-ATPase, (2) NCX, (3) the plasma membrane Ca-ATPase, and (4) mitochondrial Ca uptake.1,2 We have analyzed quantitatively how these processes compete in myocytes from different mammalian species. In rabbit (and similarly in human, canine, feline, ferret, and guinea pig), the percent contribution to [Ca]_i decline is roughly 70%, 28%, 1%, and 1%, respectively. Thus, the SR Ca-ATPase is the dominant transporter by a factor of 3 over the NCX. In rat and mouse ventricular myocytes, these numbers are 92%, 7%, 0.5%, and 0.5%, so the system is much more SR-dominated (14-fold more than NCX). In HF, there is generally a downregulation of SR Ca-ATPase and upregulation of NCX, such that in rabbit and human HF, the SR and sarcolemmal fluxes become more equally balanced. There are fewer data available in atrial myocytes, but for human atrial myocytes, we estimate 66% and 25% of Ca removal occur via SR Ca-ATPase and NCX, respectively. This predominance of SR Ca-ATPase over NCX in sinus rhythm is converted to an equal contribution of these Ca-removal processes (46% vs. 44%) in chronic atrial fibrillation.^8,9

In mitochondria, there is uptake of Ca during the normal heartbeat via the mitochondrial Ca uniporter,¹⁰ but it can be inferred that this is only about 0.5 µmol/L cytosol (~1 µmol/L mitochondria). Significant Ca buffering is expected in this compartment, which is packed with proteins (as in the cytosol), so the rise in free [Ca] in the mitochondria at each beat is rather small.¹¹ The extrusion of Ca from mitochondria is mediated mainly by a mitochondrial Na/Ca exchanger (NCLX),¹² which is different from the sarcolemmal NCX. This Ca extrusion is very slow, such that at higher heart rates, the diastolic mitochondrial [Ca] gradually accumulates to a higher steady-state level.

One important effect of increased mitochondrial [Ca] is that it binds to and stimulates several dehydrogenase enzymes in the mitochondria to increase the production of NADH and consequently increases ATP production.³ This is thought to be an important feedback pathway, where the mitochondria increase their ATP production under conditions in which the myocyte is using more ATP (i.e., when Ca transients are more frequent and/or of higher amplitude). When heart rate is reduced, the diastolic Ca efflux has a better opportunity to keep up with the less frequent influx pulses, and mitochondrial [Ca] gradually falls.

Under severe cellular stress, mitochondria can store large amounts of Ca, in part by progressively increasing their buffering power.¹³ This is thought to provide temporary protection in the myocyte from the dangers of Ca overload. However, too much Ca in mitochondria can lead to opening of the permeability transition pore and loss of proteins, which can lead to mitochondrial demise.