Physiology of the Myocyte Cell Membrane

The human body is composed predominantly of salts and water. This pool of salts and water is segregated into functional units (cells and their intracellular components) that locally alter the concentration of the salts they contain. Hydrophobic phospholipid membranes surrounding the cells (and intracellular compartments) regulate the exit/entrance of water-soluble salts and allow the cells to maintain this individualized environment (Fig. 23-1).¹ Units within these phospholipid membranes actively adjust the ion content within the cell (or intracellular compartment). For example, an energy-dependent sodium (Na⁺), potassium (K⁺) pump extrudes Na⁺ ions from the cell and takes K⁺ ions into the cell at an exchange of three Na⁺ ions extruded per two K⁺ ions taken in. This allows cells to raise intracellular K⁺ concentrations and lower intracellular Na⁺. Another pump extrudes calcium (Ca²⁺) in exchange for Na⁺. This decreases Ca²⁺ concentrations in the intracellular (compared with the extracellular) fluid, but allows Na⁺ to reenter the cell. This pump is driven by the Na⁺ gradient created by the energy-dependent Na⁺, K⁺ pump. Other channels within the membrane act as passages that intermittently open to allow transit of a specific ion through the cell membrane. Regulation of pump activation and channel opening is vital for the proper functioning of the cell. Similarly, intracellular compartments called the sarcoplasmic reticulum (SR) contain energy-dependent ion pumps in their surrounding membranes that allow them to collect the majority of the intracellular Ca²⁺. Moving ions against their concentration gradient to create a relative intracellular deficit or abundance requires energy. In the cell, this energy is supplied in the form of adenosine triphosphate (ATP).

Myocyte Depolarization

Relative to the extracellular fluid, the intracellular fluid in the resting state has a lower ratio of positively to negatively charged ions (i.e., the myocyte membrane is polarized). These electrical and ionic gradients across the myocyte membrane when at rest create the potential for rapid ionic shifts if restrictions to ion movement across the membrane are relaxed. Transmembrane ion movement is facilitated by a series of events precipitated by a small decrease in the magnitude of the electrical polarity across one point of the myocyte membrane. The resultant ion shifts increase calcium concentrations at critical intracellular sites, allowing myocyte contraction.²

When the transmembrane electrical potential, –90 mV, at rest, becomes less negative (i.e., depolarizes), sodium channels on the cell membrane are triggered to open (Fig. 23-2). Some of these sodium channels are also triggered at the time of membrane depolarization to close, although the onset of closure is (of course) delayed until after the channel has been opened. Other channels, like L-type calcium channels, also open when the cell membrane depolarizes. The initiation of ion passage through calcium channels is delayed until after the Na⁺ channels have begun to close. The opening of these Na⁺ and Ca²⁺ channels results in the entrance of cations into the cell, decreasing the magnitude of the membrane potential. As the membrane potential reaches a nadir, K⁺ channels are triggered to open. Their opening increases the rate of exit of K⁺ from the intracellular space into which it had been concentrated. The loss of cations from the intracellular space helps return the transmembrane potential back to the resting state. This K⁺ channel is inwardly rectified (i.e., turned off when cell is depolarized) and is responsible for maintaining the resting membrane potential. This basic sequence is somewhat modified by channels for Cl⁻ and transiently open channels for K⁺ (activated after depolarization to allow Cl⁻ entrance into cell and K⁺ efflux from cell, beginning to reverse to return the transmembrane potential toward –90 mV).

As a new transmembrane electrical gradient develops at one point on the sarcolemma (i.e., membrane of a muscle fiber), ion channels traversing the adjacent sarcolemma, closed to the passage of ions under resting transmembrane potentials, become exposed to the new transmembrane potential of the adjacent sarcolemma. This exposure increases the open probability of the nearby channels. The passage of ions through these newly open channels changes the membrane potential surrounding the channel, exposing an additional section of the cell membrane to a different transmembrane potential. In this way, the new transmembrane potential propagates along the surface of the myocytes.

The trigger to opening the sarcolemmal ion channels is often the potential or ionic concentration gradient of the surrounding milieu. Thus, transmembrane channels are frequently described as being either voltage-gated (opening probability is increased if the transmembrane potential of the surrounding membrane is within certain parameters) or ion-gated (opening probability is increased if there is a sudden change in the concentration of an ion in the surrounding fluid). Some channels, described as rectified channels, alter the resistance imposed by the channel-to-ion flow through the channel as the surrounding electrical or ion potential is changing. Speaking of K⁺ channels, an ion whose exit from the depolarized cell tends to restore the resting membrane potential (by increasing the relative concentration of negatively charged ions intracellularly), outward rectified channels increase the resistance to K⁺ passage as the membrane potential returns to resting state. This type of channel tends to promote restoration of the repolarized state. By contrast, inward rectifying K⁺ channels are relatively more resistant to ion passage as the membrane is depolarized. This type of channel tends to promote the maintenance of the depolarized state (Fig. 23-2).

T Tubules and Sarcoplasmic Reticulum

T tubules are invaginations in the cell membrane that penetrate into the cell to lie in close proximity to the myocyte contractile proteins. These T tubules act as extensions of the surface membrane and allow changes in the cell surface to effect changes deep within the myocyte.

Compared with the membrane on the surface of the cell, the membrane of T tubules has a relatively high concentration of L-type calcium channels. These T tubules extend to close approximation with the myofibrils—the contractile apparatus of the myocyte. Also located intracellularly near the junction of the myofibrils and the T tubules are compartments known as SR. The membrane of the SR contains ion pumps that both concentrate calcium within the SR and channels that, when open, allow the sequestered calcium to exit the SR. The stimulus for opening of the calcium channels on the SR is a rise in calcium in the cytoplasm surrounding the SR. Thus, as membrane depolarization propagates from the cell surface down the invaginations known as the T tubules, it stimulates L-type calcium channels in the T tubules to open. This allows calcium to enter the cell at a site near the SR, raising cytosolic calcium concentrations near the SR. These high calcium concentrations near the SR stimulate calcium release from the SR, a phenomenon known as calcium-induced calcium release. The rise in calcium concentrations near the myofibrils reorients the troponin in the thin filaments, moving the tropomyosin (attached to the troponin) and making it sterically possible for the actin of the thin filament to bind to myosin. Contraction follows. Contraction is terminated (and relaxation initiated) by the closing of calcium channels in the SR and the reuptake of calcium by the SR as a result of pumps activated on the SR. This reduces calcium concentrations surrounding the thin/thick filaments, so tropomyosin filaments return to a position preventing the interaction of actin and myosin filaments.

Regional Differences in Cardiac Myocyte Depolarization

The ion channel(s) predominating in cardiac myocytes differ by the myocyte’s location in the heart (atria/ventricles vs sinus node/AV node) (Fig 23-3).³ This difference in predominating ion channel(s) helps explain the special features of different sites of the heart. Fast-conducting sodium channels predominate in the atria and ventricles, allowing for rapid depolarization and expediting conduction. These channels are less prominent in the SA node and AV node, reducing rate of conduction through these sites.

Additional features of the SA and AV node are responsible for the automaticity (i.e., spontaneous depolarization) that is characteristic of these cells. The resting membrane potential of contracting atrial and ventricular myocytes is maintained by an inward rectifying (i.e., shuts off when cell is depolarized) potassium channel that maintains resting membrane potential of around –90 mV. In the nodal tissues, additional channels (voltage-dependent slow Na channel that allows Na to slowly enter cell as depolarization reaches –70 to –90 mV and, possibly an outward rectifying K channel that reduces K flow as repolarization progresses) result in an unstable “resting” membrane potential. Thus, the resting membrane potential of nodal cells will spontaneously diminish from about –70 to –50 mV, at which time voltage-dependent Ca²⁺ channels will open and allow complete depolarization.

Finally, nodal tissue (predominantly AV node) contains a K⁺ channel that is central to pharmacologic interventions commonly used to treat dysrhythmias. This channel, activated by adenosine and acetylcholine (I_Kach,Ado), allows K⁺ to exit the cell upon stimulation by either adenosine or acetylcholine. The exit of K⁺ makes the membrane potential more negative, hyperpolarizing the cell and making depolarization more difficult. Thus, acetylcholine reduces sinus node spontaneous depolarization and adenosine blocks transmission through the AV node.

Excitation: Contraction Coupling

The cascade of events linking myocyte membrane depolarization to myocte contraction is termed excitation:contraction coupling. It can be summarized as follows (Figs. 23-2 and 23-3):

Phase 1 depolarization, mediated by opening of voltage-gated fast Na channels, depolarizes the membrane to a point at which L-type Ca²⁺ channels can open (Fig. 23-3). The influx of calcium through these (dihydropyridine-sensitive) voltage-gated channels, especially through those located along the T tubules, increases intracellular calcium concentration in the vicinity of the SR. (The calcium channels on the SR are termed ryanodine-sensitive calcium channels because the compound ryanodine induces their opening.) This triggers the opening of calcium channels in the SR membrane, releasing calcium stored in the SR. The result of calcium entrance into the cell via L-type calcium channel opening, combined with release of calcium from SR stores, is a dramatic increase in calcium concentration surrounding the contractile myofilaments. High concentrations of calcium allows binding of calcium to troponin C of the thin filament, modulating the troponin such that tropomyosin (attached to troponin) gets moved from its position of preventing interaction between actin (of the thin filament) and myosin. The strength of bond between actin and myosin is decreased by acidosis, high concentrations of Mg²⁺, or high concentrations of phosphate. This bond is enhanced by caffeine and β-adrenergic stimulation. β-Adrenergic stimulation increases actin–myosin bonding through a cascade that results in the activation of myosin-binding protein C.

Regulation of Myocyte Function by the Myocyte Cell-Surface Receptor

Beta Receptors

The beta receptor is a protein that is incorporated within the cell membrane. It spans the entire depth of the membrane. It has both components that extend into the cytoplasm and components exposed on the surface of the cell.⁴ When an extracellular agonist stimulates the portion of the receptor exposed on the cell surface, a conformational change in the beta receptor allows the portion of the receptor protruding into the cytoplasm to bind to a G protein (i.e., it is a G protein-coupled receptor). G proteins have multiple components, and stimulation of the G protein causes dissociation of the subunits so that each subunit is free to regulate its particular effector. One such effector is adenylatecyclase, which is activated to increase intracellular concentrations of cyclic adenosine monophosphate (cAMP) and thereby activate protein kinase A (PKA). Activated PKA is then capable of phosphorylating multiple intermediaries of excitation:contraction. For example, it increases intracellular calcium concentrations by facilitating calcium entrance into the cell (via L-type calcium channels) as well as calcium release from compartments within the cell (i.e., the SR).

β-Agonist stimulation also expedites relaxation. Relaxation results when cytosolic calcium is in low concentration or is prevented from binding to troponin C. If troponin C is not bound to calcium, tropomyosin returns to a location that inhibits interaction between actin and myosin. Cytosolic calcium content drops when calcium leaves the myocyte through the sodium–calcium exchanger on the cell surface. The predominant method of lowering cytosolic calcium levels, though, is reuptake by the SR. The pump mediating calcium reuptake by the SR is dependent on ATP and regulated by the phosphorylation of phospholamban. Beta stimulation facilitates relaxation by two mechanisms. First, it results in phosphorylation of troponin I by PKA, expediting dissociation of calcium from troponin C. Beta stimulation also results in phosphorylation of phospholamban, which speeds up calcium reuptake into the SR.

Muscarinic Receptors

Muscarinic receptors sensitive to acetylcholine are also present on the myocardium. In particular, the heart contains the M2 subtype of muscarinic receptor.⁵ Like beta receptors, muscarinic receptors are G protein-coupled, which are incorporated within the cell membrane, span the entire depth of the membrane, and contain components exposed to both the extracellular fluid and the intracellular fluid. The G protein to which the M2 receptor is coupled has inhibitory effects on adenylatecyclase and is sensitive to pertussis toxin. In fact, the negative inotropic effects of M2 receptor stimulation appear to be indirect; that is, they can be demonstrated only in the setting of baseline stimulation adenylatecyclase. Therefore, it appears that their negative inotropy results entirely from this inhibitory effect on adenylatecyclase.

In the atria and nodal myocytes, M2 receptors appear to have a direct effect (i.e., not dependent on baseline stimulation) on the inward rectifying potassium channel that maintains phase 4 (resting) membrane potential. By opening this channel through a direct action of the G protein upon the channel, M2 receptor stimulation hyperpolarizes the resting membrane to slow the automaticity rate of the cells.

Adenosine Receptors

Adenosine receptors are also G-coupled protein receptors. There are three basic subtypes of adenosine receptors (A₁, A₂, A₃). A₁ and A₃ subtypes inhibit adenylatecyclase via inhibitory G proteins whereas A₂ receptors increase adenylatecyclase via stimulatory G proteins.⁶ These receptors also regulate other pathways via G proteins. Thus, A₁ and A₃ subtypes mediate the catabolism of phospholipids whereas A₂ receptors regulate phosphoinositide metabolism.

Two clinical effects of exogenous adenosine can be traced back to the distribution and effects of various adenosine receptors. A₁ receptors are plentiful in the nodal tissues. A₁ receptor stimulation opens the inward rectifying potassium channel that maintains phase 4 (resting) membrane potential. Like the effects of M2 receptor stimulation of this channel, opening inward rectifying potassium channels via A₁ receptor stimulation hyperpolarizes the resting membrane to slow the automaticity rate of the cells. In the coronary vasculature, A₂ receptors predominate. Their stimulation results in vasodilation, likely through G protein–mediated activation of intracellular adenylatecyclase.

α-Adrenergic Receptors

There is growing evidence that α-adrenergic stimulation of myocytes results in a positive inotropic effect.⁷ The mechanism and clinical importance of this is unclear. It may also be coupled to myocardial hypertrophy.

Cardiac myocytes possess the capacity to contract because they contain a series of protein filaments (myofibrils) oriented along the longitudinal axis of the cell (see later). For myocytes to shorten, these longitudinally oriented myofibrils must interact with each other and slide along each other in the longitudinal axis. A rise in intracellular (cytosolic) calcium is critical for this myofibril interaction. Calcium enters the cytosolic space from two sources. First, extracellular calcium crosses the plasma membrane to enter the cell. This rise in intracellular calcium then induces the release of calcium, the second source—intracellular calcium storage pools (calcium-induced calcium release). At baseline, extracellular calcium concentrations are higher than intracellular calcium concentrations because calcium is actively extruded across the cell’s phospholipid plasma membrane. The entrance of calcium back into the cell (across the plasma membrane) is a passive process, driven by the concentration gradient and allowed by the brief opening of channels through the calcium-impermeant phospholipid plasma membrane. The impetus for the stimulation is a change in the transmembrane electrical gradient (see Myocyte Cell Membrane Physiology section).

The Sarcomere as a Contractile Element

Contractile Apparatus

The sarcomere is the contractile unit of the myocyte. A sarcomere contains two types of coaxially aligned filaments that differ in their component protein (actin/tropomyosin/troponin vs myosin) as well as in thickness (actin/tropomyosin/troponin filaments are thinner, myosin filaments are thicker). Thin and thick filaments are arranged as follows. Each thin filament is axially aligned with, but not attached to, another thin filament. Each pair of thin filaments is straddled by a single thick filament lying parallel to it. Within the myocyte, the contractile unit (a pair of thin filaments and their associated thick filament) is bound on the ends by a Z band (Fig. 23-4). The Z band (for Zuckung, the German word for “contraction”) is a dark band visible by electron microscopy and corresponding to the site to which the ends of a series of parallel thick and thin myosin filaments are anchored. This defines the ends of a sarcomere. Under appropriate stimulation, the members of an axially aligned thin-filament pair will move closer to each other through an interaction with the thick filament that straddles them. Because each member of a thin filament pair is anchored to Z bands, this coaxial movement draws the Z bands within the cell closer to one another, shortening the cell.^1,8

Each end of the thick myosin filament is attached to the Z band by a protein called titin or connectin. This protein contains a nonmalleable portion that anchors the filament to the Z band as well as a distensible portion that enfolds on itself if the sarcomere is not stretched. As the sarcomere stretches, the Z bands are pulled farther apart from one another and the enfolded portion of the titin protein is extended. When stretching is relieved, the elastic recoil of the titin molecule helps to actively restore the sarcomere to its resting length.

The movement of thin filaments is an energy-requiring process regulated by local calcium concentrations within the cell. Two thin filaments move toward each other in the axial plane by each “pulling” itself along a shared myosin filament. Under “resting” conditions, physical contact between actin and myosin filaments is prohibited by the tropomyosin component of the thin filament. Tropomyosin is a protein strand that can interdigitate between actin and myosin, preventing their physical contact. Tropomyosin can be moved out of place by the actions of the third protein of the thin filament, the troponin protein. The presence of calcium sterically alters the troponin, causing it to reorient itself within the thin filament and thereby move the tropomyosin strand to which it is attached. The movement of the tropomyosin strand removes the physical barrier preventing actin–myosin interaction and allows the thin filaments to move closer to each other along a shared myosin chain.

Myocardial Contractile Physiology

Contractility is the ability of a myocyte to shorten, measured at a given preload and a given afterload. Changing the preload will alter contractility due to the length–tension relationship. Changing the afterload will alter contractility because it mandates generation of a different wall stress to achieve shortening.

Length–Tension (Frank–Starling) Relationships

The force of contraction of a cardiac myocyte increases when the sarcomere is stretched (within the lengths of 1.7–2.4 μm).⁹ Mechanistically, this increased contractility can be divided into two parts: (1) an immediate increase in force that is unaccompanied by a change in intracellular calcium concentration and (2) an additional increase in force of delayed (minutes) onset that is associated with an increase in intracellular calcium.¹⁰ The increase in intracellular calcium causing the second phase is probably mediated by changes in the cell membrane. The first phase, which apparently involves a change in myofilament sensitivity to calcium, is the Frank–Starling phenomenon (named for Otto Frank and Ernest Starling, who initiated the concept based on experimental findings they made at the beginning of the 20th century). It was originally hypothesized that sarcomere stretching improved myosin–actin cross-bridge formation. However, the increase in contractile force is observed even if the sarcomere is stretched to distances that would begin to reduce the potential for actin and myosin crossbridge formation. An alternative explanation is that stretching the myocyte narrows the sarcomere, reducing the spacing between myosin and actin filaments. This facilitates myosin–actin interaction. Studies in which the interfilament distance is reduced by cellular dehydration result in an increased calcium sensitivity of contractile strength, similar to that seen with myocyte stretch. A yet unexplained mechanism of stretch-induced change in myofilament sensitivity to calcium remains a third possible explanation.

Force–Velocity Relationships

To measure contractile force free of compounding influences from preload, afterload, or heart rate, the concept of measuring the velocity of shortening in a myocyte with no afterload has been developed.¹¹ In such a scenario, the maximal velocity of shortening (V_max) would be a measure of the inotropy of a myocyte. Practically speaking, it is difficult to completely remove external resistance from a contracting myocyte. The value of V_max must therefore be extrapolated from the maximal velocities observed when a myocyte is made to contract against a series of different afterloads. At one extreme of this series would be the minimal afterload at which the myocyte is no longer able to shorten at all (V_max = 0). This relationship is hyperbolic rather than linear (Fig. 23-5). Although the reason for this hyperbolic relationship remains unclear, it is felt to be due in part to shortening inactivation and in part to elastic forces that passively resist stretch (at long sarcomere length) or shortening (at short sarcomere length). Such passive elastic forces could be contributed by titin.

“Anrep Effect”

This is a two-phase response of myocytes to an abrupt stretch.¹² The first phase is the Frank–Starling effect—an increase in contractility, which occurs without a change in cytosolic calcium. There is a delayed second phase, which is associated with an increase in cytosolic calcium. The change in intracellular calcium seems to be independent of the SR, although the exact mechanism behind the rise remains obscure.

“Treppe Effect”

The Treppe effect (Treppe, German for “step”) is the observed increase in strength of contraction when heart rate is increased. It is associated with an increase in intracellular calcium concentrations,¹⁰ which itself may be due to an inability of the SR and myocyte calcium-extruding pumps to completely return cytosolic calcium to baseline levels between sequential cell depolarizations. The Treppe effect may also be influenced by endocardial–myocyte cross-talk through a process involving endothelin-I.

Myocyte Cytoskeleton