Regulation of cardiac excitation-contraction coupling by action potential repolarization: role of the transient outward potassium current (Ito)

doi:10.1113/jphysiol.2002.026468

Regulation of cardiac excitation-contraction coupling by action potential repolarization: role of the transient outward potassium current (I_to)

Rajan Sah, Rafael J. Ramirez, Gavin Y. Oudit, Dominica Gidrewicz, Maria G. Trivieri, Carsten Zobel * and Peter H. Backx

Departments of Physiology and Medicine, University of Toronto, Heart & Stroke/Richard Lewar Centre, University of Toronto, Room 68, Fitzgerald Building, 150 College Street, Toronto, Ontario, M5S 3E2, Canada and * Laboratory of Muscle Research and Molecular Cardiology, University of Cologne, Cologne, Germany

ABSTRACT

Top
Abstract
Introduction
Methods
Results
Discussion
References

The cardiac action potential (AP) is critical for initiating and coordinating myocyte contraction. In particular, the early repolarization period of the AP (phase 1) strongly influences the time course and magnitude of the whole-cell intracellular Ca²⁺ transient by modulating trans-sarcolemmal Ca²⁺ influx through L-type Ca²⁺ channels (I_Ca,L) and Na-Ca exchangers (I_Ca,NCX). The transient outward potassium current (I_to) has kinetic properties that make it especially effective in modulating the trajectory of phase 1 repolarization and thereby cardiac excitation-contraction coupling (ECC). The magnitude of I_to varies greatly during cardiac development, between different regions of the heart, and is invariably reduced as a result of heart disease, leading to corresponding variations in ECC. In this article, we review evidence supporting a modulatory role of I_to in ECC through its influence on I_Ca,L, and possibly I_Ca,NCX. We also discuss differential effects of I_to on ECC between different species, between different regions of the heart and in heart disease.

(Received 12 June 2002; accepted after revision 12 November 2002; first published online 29 November 2002)
Corresponding author P. H. Backx: Departments of Physiology and Medicine, University of Toronto,Heart and Stroke/Richard Lewar Centre of Excellence, Room 68, Fitzgerald Building, 150 College Street, Toronto, Ontario, M5S 3E2, Canada. Email: p.backx{at}utoronto.ca

INTRODUCTION

Top
Abstract
Introduction
Methods
Results
Discussion
References

Myocardial contraction is activated by a transient rise in cytosolic free calcium concentration ([Ca²⁺]_i) to about 1 µM from a resting diastolic [Ca²⁺]_i of about 0.1 µM. Despite a rather modest rise in the average free [Ca²⁺]_i, activation of the contractile proteins typically requires the binding of between 40 and 60 µM total Ca²⁺ (reviewed in Bers, 2002a,b). The bulk of this Ca²⁺ required for activation of contraction originates from the sarcoplasmic reticulum (SR), with Ca²⁺ influx through L-type Ca²⁺ channels and Na⁺-Ca²⁺ exchangers (NCX) making more minor contributions, although the relative contribution varies between species (reviewed in Bers, 2002a,b). Relaxation of myocyte contraction involves the extrusion of Ca²⁺ from the cytosol. At steady state, the majority of relaxation involves Ca²⁺ reuptake into the SR by the Ca²⁺-ATPase (SERCA2a), with the remaining Ca²⁺ (equivalent to the amount entering via L-type Ca²⁺ channels) being extruded from the myocyte via the NCX (5-10 % in rats and mice versus 25-30 % in larger species) (reviewed in Bers, 2002a,b).

Since the SR is the primary source of Ca²⁺ necessary for contraction, factors modulating SR Ca²⁺ release strongly influence the amplitude and time course of the whole-cell Ca²⁺ transient. The release of Ca²⁺ from the SR following membrane depolarization and subsequent Ca²⁺-induced myofilament activation is referred to as excitation- contraction coupling (ECC). A central feature of ECC is the gating of SR Ca²⁺ release channels (called ryanodine or RyR2 receptors), where gating refers to the opening (or 'activation') and the closing of individual RyR2 channels located within the terminal cisternae of the SR. Gating of RyR2 channels is controlled primarily by elevations of [Ca²⁺]_i in the subsarcolemmal space between the T-tubular membrane of the sarcolemma and the terminal cisternae of the SR, which occurs following the opening of sarcolemmal L-type Ca²⁺ channels in response to membrane depolarization (Lopez-Lopez et al. 1994, 1995; Cannell et al. 1995) (Fig. 1). T-type Ca²⁺ channels and/or reverse mode NCX can also contribute to elevations of subsarcolemmal Ca²⁺ levels following membrane depolarization, although the relative contribution is generally small but can vary with experimental conditions, pathological state and species (Leblanc & Hume, 1990; Nuss & Houser, 1993; Levi et al. 1994; Wasserstrom & Vites, 1996; Martinez et al. 1999), as well as between different regions of the heart (Zhou & January, 1998; Huser et al. 2000; Lipsius et al. 2001). The amplification process whereby elevated subsarcolemmal [Ca²⁺] 'triggers' further Ca²⁺ release from the SR is called Ca²⁺-induced Ca²⁺ release (CICR). Recent studies have suggested that Ca²⁺ release from the SR may also be activated under certain experimental conditions by direct voltage-dependent coupling between sarcolemmal L-type Ca²⁺ channels and SR Ca²⁺ release channels (Ferrier & Howlett, 2001), although the validity of this release mechanism remains controversial (Piacentino et al. 2000) and might be explained by involvement of other transporters like the NCX (Viatchenko-Karpinski & Gyorke, 2001).

View larger version
[in this window]
[in a new window]

Figure 1. Sites of regulation of SR Ca²⁺ release in cardiac myocytes
SR Ca²⁺ load (1) is maintained by Ca²⁺ uptake via the SR Ca²⁺ ATPase (SERCA2a), which is modulated through its interaction with phospholamban (PLN) and sarcolipin (SLN). Passive leak through ryanodine receptors (RyR) also plays a role in regulating steady-state SR Ca²⁺ content. The SR Ca²⁺ load determines the amount of Ca²⁺ released and the sensitivity of the RyR release mechanism to trigger Ca²⁺. This trigger Ca²⁺ enters primarily via L-type Ca²⁺ channels (2), while Ca²⁺ influx via reverse-mode Na⁺-Ca²⁺ exchange (3) may secondarily contribute to Ca²⁺-induced Ca²⁺-release. Since both L-type Ca²⁺ channel gating and Na⁺-Ca²⁺ exchange activity are voltage dependent, we propose that altering early repolarization of the action potential via changes in transient outward potassium current (I_to) magnitude may play a role in modulating SR Ca²⁺ release.

The activities of L-type and T-type Ca²⁺ channels, as well as NCXs, are tightly controlled by membrane voltage. Consistent with this, factors that modulate AP profile can significantly affect contractile strength (Bouchard et al. 1995; Fiset et al. 1997; Kaprielian et al. 1999; Sah et al. 2001). Alterations in the early repolarization period are particularly effective in modulating SR Ca²⁺ release by influencing the duty cycle of I_Ca,L, the primary determinant of CICR (Bouchard et al. 1995; Kaprielian et al. 1999; Sah et al. 2001, 2002b). In this review we describe how altered repolarization of the AP by differences in the transient outward potassium current (I_to) affects SR Ca²⁺ release and ECC. We also review the significance of these observations with respect to regional control of myocardial contractility and heart disease.

Regulation of SR Ca²⁺ release by CICR

The amplification of the trans-sarcolemmal Ca²⁺ signal via CICR was initially proposed and characterized by Fabiato (Fabiato & Fabiato, 1979; Fabiato, 1985). This mechanism was later predicted to be controlled within discrete regions of the diadic cleft, by a mechanism termed 'local control' (Stern, 1992) which was subsequently verified experimentally (Cheng et al. 1993; Lopez-Lopez et al. 1994, 1995; Cannell et al. 1995; Shacklock et al. 1995). According to local control theory, the overall amplitude and time course of the whole-cell intracellular Ca²⁺ transient results from the recruitment and temporal summation of fundamental Ca²⁺ release events in these discrete regions, termed Ca²⁺ sparks (O'Neill et al. 1990; Cleemann et al. 1998; Wier & Balke, 1999). These discrete regions are comprised of anatomical units containing a cluster of 10-400 RyR2 channels, with each RyR2 channel making physical contact with up to four other release channels (Bridge et al. 1999; Franzini-Armstrong et al. 1999). SR clusters are found in close proximity and are colocalized with one or several sarcolemmal L-type Ca²⁺ channels within the diads or junctional complexes of ventricular cardiomyocytes (Cheng et al. 1993; Lopez-Lopez et al. 1994, 1995; Cannell et al. 1995; Shacklock et al. 1995). The anatomical relationship between RyR2 clusters and Na⁺-Ca²⁺ exchangers, T-type Ca²⁺ channels and Na⁺ channels is less certain, although a recent report has demonstrated that Na⁺-Ca²⁺ exchangers do not colocalize with RyR2 in rat ventricular myocytes, even though both are located within the triads (Scriven et al. 2000). Regardless of the Ca²⁺ source, however, the activation of RyR2 clusters requires relatively high elevations of Ca²⁺ levels within the subsarcolemmal space of the diads, which is necessary to prevent cross-activating geographically distinct RyR2 clusters (Stern, 1992).

The RyR2 channels within a SR Ca²⁺ release cluster are not only physically coupled (Franzini-Armstrong et al. 1999) but their activation and inactivation properties (in response to trigger Ca²⁺) appear to be functionally coupled as well (Stern et al. 1999; Guatimosim et al. 2002; Sobie et al. 2002). This functional coupling between RyR2 channels in a cluster is hypothesized to involve direct coupled gating, possibly through other regulatory proteins that localize with RyR2 channels in the junctional complex (Marx et al. 2000; Bers, 2002a; Sobie et al. 2002). Specifically, loss or dissociation of the FK506-binding protein, FKBP12.6, results in decoupling of neighbouring RyR2 channels leading to enhanced CICR and elevated [Ca²⁺]_i (Xin et al. 2002). PKA-dependent hyperphosphorylation of RyR2 has also been proposed to chronically increase the open probability of the SR release complex and thereby deplete SR Ca²⁺ load in heart disease by promoting dissociation of FKBP12.6 from RyR2 channel complexes (Marx et al. 2000, 2002). On the other hand, functional coupling is also expected to occur as a consequence of Ca²⁺-dependent cross-talk between RyR2 channels in a cluster (Stern, 1992; Stern et al. 1999; Guatimosim et al. 2002; Sobie et al. 2002). Interestingly, this type of coupling involves increased RyR2 channel opening in response to elevated Ca²⁺ levels on both the luminal and the cytosolic faces of RyR2 channels (Stern, 1992; Gyorke, 1998; Lukyanenko et al. 1999; Sobie et al. 2002).

As might be expected from the colocalization of L-type Ca²⁺ channels with RyR2 clusters (Scriven et al. 2000), SR Ca²⁺ flux and the amplitude of the intracellular Ca²⁺ transient are controlled by the frequency, amplitude and time course of L-type Ca²⁺ channel openings (Isenberg & Han, 1994; Bassani et al. 1995; Lopez-Lopez et al. 1995). Indeed, in cardiomyocytes from transgenic mice overexpressing the $alpha$ 1 subunit of the L-type Ca²⁺ channel in the heart, SR Ca²⁺ release is enhanced due to increases in trigger I_Ca,L without any change in SR Ca²⁺ load (Song et al. 2002). These findings are consistent with previous studies establishing that the timing of Ca²⁺ sparks correlates well with the time course of L-type Ca²⁺ current (Lopez-Lopez et al. 1994, 1995; Cannell et al. 1995; Cleemann et al. 1998; Collier et al. 1999) and that the Ca²⁺ spark probability (frequency) correlates strongly with the first latency of L-type Ca²⁺ channel opening (Isenberg & Han, 1994; Lopez-Lopez et al. 1995; Santana et al. 1996; Cannell & Soeller, 1999; Wier & Balke, 1999). Consequently, the kinetic properties of trigger I_Ca,L will influence the amplitude as well as the time course of SR Ca²⁺ release. Furthermore, recent studies have demonstrated that the connection between I_Ca,L and synchronization of SR Ca²⁺ release events is a critical determinant of peak systolic Ca²⁺ in normal rat myocytes (Song et al. 1998), following $beta$ -adrenergic stimulation (Song et al. 2001) and in myocytes from infarcted rabbit myocardium (Litwin et al. 2000). Because of the strong coupling between I_Ca,L and SR Ca²⁺ release, ECC has often been characterized by estimating the ECC gain which represents the ratio between the amount of Ca²⁺ released from the SR (or the probability of SR Ca²⁺ cluster activation) and the quantity of trigger Ca²⁺ entering via I_Ca,L (Stern, 1992; Wier et al. 1994; Shannon et al. 2000; Cheng & Wang, 2002). The utility of this estimated parameter obviously depends critically on whether other sources of Ca²⁺ participate in evoking SR Ca²⁺ release (see below).

T-type Ca²⁺ currents (I_Ca,T) can also evoke SR Ca²⁺ release, although Ca²⁺ entry through these channels appears to be far less effective as a trigger for SR Ca²⁺ release (Sipido et al.1998; Zhou & January, 1998). In addition, I_Ca,T is either absent or small compared to I_Ca,L in most myocardial regions such as the atria and ventricles of many mammalian species (Richard et al. 1998). These observations suggest that T-type Ca²⁺ channels play a relatively minor role in ECC. However, I_Ca,T might work synergistically with I_Ca,L in ECC, especially at more negative potentials, while reliance on I_Ca,T for the maintenance of ECC may be heightened in diseased myocardium, where T-type Ca²⁺ channel expression is often increased (Richard et al. 1998). Moreover in the pacemaker of the heart, I_Ca,T is more prominent where it appears to modulate spontaneous firing rates by controlling SR Ca²⁺ release (Huser et al. 2000; Lipsius et al. 2001). Thus, T-type Ca²⁺ channels might play a significant role in regulating ECC, depending on the region of the heart, species and disease state.

Ca²⁺ influx via reverse-mode Na⁺-Ca²⁺ exchanger activity has also been suggested to directly trigger SR Ca²⁺ release (Levi et al. 1993; Wasserstrom & Vites, 1996; Weber et al. 2002). A subsarcolemmal space with restricted diffusion has been proposed to assist the NCX in triggering SR Ca²⁺ release and making release dependent on both Na⁺ channel and Na⁺-K⁺-ATPase activation (Lipp & Niggli, 1994; Su et al. 2001; Bers, 2002a; Han et al. 2002) (Fig. 1). However, the efficacy of this trigger over the physiological voltage range and in response to APs remains to be determined. Recent studies suggest that insufficient quantities of Ca²⁺ enter through reverse-mode Na⁺-Ca²⁺ exchanger activity in order to initiate SR Ca²⁺ release (Sipido et al. 1997; Weber et al. 2002). Nevertheless, reverse mode Na⁺-Ca²⁺ exchange activity may act synergistically with I_Ca,L to trigger SR Ca²⁺ release (Litwin et al. 1998; Cordeiro et al. 2001; Viatchenko-Karpinski & Gyorke, 2001), particularly at depolarized membrane potentials where L-type Ca²⁺ channel currents are small. This concept is supported by studies demonstrating that NCX activity depends strongly on Ca²⁺ provided by I_Ca,L (Haworth & Goknur, 1991; Haworth et al. 1991), presumably via allosteric regulation of NCX by Ca²⁺ (Hilgemann, 1990). This synergistic interaction between L-type Ca²⁺ channels and NCX appears to underlie the observation that, in the presence of $beta$ -adrenergic activation, SR Ca²⁺ release does not decline with I_Ca,L at positive potentials in the presence of physiological levels of Na⁺ (Viatchenko-Karpinski & Gyorke, 2001).

Elevations in SR Ca²⁺ load have also been shown to increase the absolute, as well as the relative, amount of Ca²⁺ released from the SR for a given amount of trigger I_Ca,L (Han et al. 1994; Bassani et al. 1995; Janczewski et al. 1995; Santana et al. 1997; Shannon et al. 2000; Trafford et al. 2002). The underlying basis for the enhanced ECC gain as the SR Ca²⁺ load increases probably results from a combination of elevated trans-SR Ca²⁺ flux and increased open probabilities of RyR2s in response to stimulation by triggered Ca²⁺ due to elevated SR luminal [Ca²⁺] (Sitsapesan & Williams, 1994; Lukyanenko et al. 1996, 1999; Trafford et al. 2002; Guatimosim et al. 2002; Sobie et al. 2002). The enhanced probability of RyR2 opening in response to elevated SR Ca²⁺ also increases the frequency and amplitude of unstimulated spontaneous Ca²⁺ release (i.e. resting Ca²⁺ sparks) which is linked to the genesis of arrhythmias (Santana et al. 1997; Satoh et al. 1997; Guatimosim et al. 2002).

Effect of I_to on early AP repolarization, I_Ca,L and SR Ca²⁺ release

Ventricular AP profiles vary widely among different species as well as within different regions of the ventricle of the same species (Linz & Meyer, 2000; Nerbonne et al. 2001; Oudit et al. 2001) and depend strongly on the recording conditions. Typical ventricular APs of rodents (rats and mice) have spike-like morphologies with a brief late plateau at potentials negative to -20 mV and are significantly shorter (Kaprielian et al. 1999; Wickenden et al. 1999) than those of other species such as humans (Nabauer et al. 1996; Li et al. 1998), rabbits (Linz & Meyer, 2000) or dogs (Kaab et al. 1996; O'Rourke et al. 1999) (Fig. 2). APs in these larger species show distinct phase 1 and phase 2 periods of repolarization that are generally separated by a notch whose prominence varies in a region-dependent manner. Guinea-pig ventricular myocytes exhibit even longer APs with a prominent plateau (phase 2), a limited phase 1 repolarization and the absence of a notch (Linz & Meyer, 2000). These interspecies differences in AP profile could reflect variations in the types, kinetics and amplitudes of both inward (depolarizing) and outward (repolarizing) currents. While only a few studies have been published directly comparing depolarizing currents, it is evident that there are few species-dependent differences in I_Ca,L and I_Na. By contrast, differences in outward K⁺ currents due to variations in K⁺ channel and/or accessory subunit(s) expressions appear to be the primary determinants of species- and region-dependent variations in AP morphology (Nerbonne et al. 2001; Oudit et al. 2001; Rosati et al. 2001). Of particular interest are the differences in the (fast) transient outward potassium current (I_to,f), encoded by Kv4.2/4.3 genes, which contribute significantly to both interspecies and region-dependent variation in AP profile (Nerbonne et al. 2001; Oudit et al. 2001). The presence of relatively large I_to current densities in rodent myocytes (compared to larger mammals) overpowers the depolarizing effects of I_Ca,L, thereby minimizing the plateau of the AP in these species (Greenstein et al. 2000), while in canine, human and rabbit myocytes, where I_to is less dominant, the balance and interplay of I_to, I_Ca,L and other potassium channels establishes a clear plateau (phase 2) which is often preceded by a prominent notch (Greenstein et al. 2000). Importantly, even in these large species, reductions in I_to lead to progressive slowing of phase 1 repolarization, elimination of the notch and elevation of the AP plateau (Greenstein et al. 2000; Oudit et al. 2001) (Fig. 2). In the absence of I_to, APs in these larger species (Priebe & Beuckelmann, 1998; Greenstein et al. 2000) and rodents (Wickenden et al. 1997; Gaughan et al. 1998) resemble those of the guinea-pig, which lacks I_to entirely.

View larger version
[in this window]
[in a new window]

Figure 2. Effect of transient outward potassium current elimination on murine and canine AP profile
A, AP measured in current-clamp mode from a control mouse myocyte (left) and Kv4.2N transgenic mouse myocyte (right). Kv4.2N-expressing myocytes have reduced I_to densities and prolonged APs compared to controls. B, AP generated from a canine computer model (Greenstein et al. 2000) with control I_to levels (conductance = 0.12 nS pF^-1; left) and elimination of I_to (right). Removal of I_to markedly slows early repolarization, abolishes the AP notch, and shortens the overall AP duration (APD₉₀,_G_{= 0.12} = 300 ms, APD₉₀,_G_{= 0} = 250 ms) in canine APs. Note the difference in time scale between the canine and murine APs. Canine APs were generated using the interactive version of the Winslow-Rice-Jafri canine myocyte model (Greenstein et al. 2000).

Variations in AP repolarization associated with I_to differences lead to profound changes in the magnitude and time course of I_Ca,L, resulting in corresponding changes in SR Ca²⁺ loading, SR Ca²⁺ release and contractility. For example, there are increases in net Ca²⁺ entry via I_Ca,L along with increased Ca²⁺ transient amplitudes and myocyte contractility in rat myocytes following treatment with 4-AP (Bouchard et al. 1995), following myocardial infarction (Kaprielian et al. 1999; Sah et al. 2001) or in endocardial myocytes compared to epicardial myocytes (Kaprielian et al. 2002), as well as in transgenic mouse myocytes overexpressing dominant-negative I_to (Sah et al. 2002a). Similarly, prolongation of square-wave voltage steps (2 to 20 ms; Isenberg & Han, 1994) or short repolarizing ramps from 5 to 50 ms (Fig. 3) also leads to increased Ca²⁺ entry and elevated Ca²⁺ transients in rat myocytes. The connection between increased Ca²⁺ entry and elevated Ca²⁺ transients can originate from larger triggers for SR Ca²⁺ release or enhanced SR Ca²⁺ loading or both. Indeed, SR load is increased in guinea-pig (Terracciano & MacLeod, 1997; Terracciano et al. 1997) and rat myocytes (Sah et al. 2001) following AP prolongation. In myocytes stimulated with prolonged APs, as observed in heart disease, the increased Ca²⁺ transient amplitudes could be traced, almost exclusively, to enhanced SR Ca²⁺ release (Brooksby et al. 1993; Kaprielian et al. 1999, 2002; Sah et al. 2001). These findings are consistent with studies using step depolarizations showing that changes in trigger Ca²⁺ via I_Ca,L can cause pronounced effects on triggered SR Ca²⁺ release while having minimal effects on SR Ca²⁺ loading (Trafford et al. 2000, 2002). On the other hand under some circumstances, changes in I_Ca,L do affect SR Ca²⁺ content (Han et al. 1994; Bouchard et al. 1995; Kaprielian et al. 1999), leading to corresponding alterations in RyR2 open probabilities and SR Ca²⁺ release (Stern, 1992; Gyorke, 1998; Lukyanenko et al. 1999; Sobie et al. 2002). Regardless, the degree of enhancement of SR Ca²⁺ release with reduced I_to correlates strongly with increases in net Ca²⁺ entry via I_Ca,L with modest effects on ECC gain (Sah et al. 2001; R. Sah & P. H. Backx, unpublished observations). Thus, while numerous results have shown that changes in SR Ca²⁺ load can affect the amount and efficiency of Ca²⁺ released from the SR, the increased SR Ca²⁺ release and Ca²⁺ transient amplitude associated with slowing of early AP repolarization via reductions in I_to in rodents appear to be primarily due to enhancements in I_Ca,L, leading to proportional increases in SR Ca²⁺ release (Kaprielian et al. 1999; Sah et al. 2001).

View larger version
[in this window]
[in a new window]

Figure 3. Effect of repolarization rate on I_Ca,L and [Ca²⁺]
A, stimulus ramps of 5, 50, 200 and 500 ms duration were applied to rat ventricular myocytes. B, representative I_Ca,L traces triggered by voltage ramps shown in A above. C, peak intracellular Ca²⁺ triggered by the voltage ramps above show a biphasic relationship with respect to repolarization rate, increasing from the 5 ms to 50 ms ramp and decreasing thereafter. The rates of rise of the Ca²⁺ transients are also slowed as repolarization is prolonged. Note the difference in time scale between panel C and panels A and B.

Slowing early repolarization of a human ventricular AP by reducing I_to conductance can also lead to increased trans-sarcolemmal Ca²⁺ entry (Sah et al. 2002b). However in contrast to rodent APs, slowing of membrane repolarization, as occurs with I_to inhibition in larger mammals, leads to blunted Ca²⁺ transients with slower rise times (Fig. 3C, right), reduced numbers of Ca²⁺ spikes (Fig. 5C), impaired myocyte contractility and decreased efficiency of excitation-contraction coupling (Sah et al. 2002b). It should be emphasized that these observations might depend critically on the region of the heart from which the myocytes are derived, since AP profiles vary quite broadly due to variation in many currents including I_to (Schram et al. 2002). Nevertheless, alterations in the magnitude and profile of SR Ca²⁺ spikes following I_to-associated AP changes in larger species still correlate closely with the amplitude and kinetics of I_Ca,L, as observed with AP clamps in rat myocytes (Sah et al. 2002b).

Biphasic dependence of SR Ca²⁺ release by membrane repolarization rate: a hypothesis

While the dichotomy in the observed effects of I_to alterations on SR Ca²⁺ release between rodents versus larger species could originate from several sources, the relationship between I_Ca,L properties and SR Ca²⁺ release is likely to be of central importance (Cannell & Soeller, 1999). Systematic decreases in amplitude and increases in duration of I_Ca,L are observed in myocytes from rat to rabbit to guinea-pig (Linz & Meyer, 2000) in response to progressive decreases in I_to and early repolarization. Furthermore, similar changes in I_Ca,L occur within each species when I_to and early repolarization rates are decreased, as observed between myocytes derived from different regions of the heart or following heart disease. The same pattern of changes in I_Ca,L profiles can be observed by varying membrane repolarization rate using ramp protocols, suggesting that these changes are primarily dependent on repolarization rate (Fig. 3). Based on these considerations, it is apparent that in order to understand the regulation of myocyte contraction by I_to, the quantitative dependence of SR Ca²⁺ release on AP profile and I_Ca,L needs to be considered. Previous studies have established that, at relatively negative membrane potentials, the probability of a single L-type Ca²⁺ channel activating an individual SR Ca²⁺ release unit (i.e. P_O,RU) is determined by the product of C(i,t) and P_O,Ca, where P_O,Ca is the open channel probability of the L-type Ca²⁺ channel and C(i,t) is a coupling factor (ranging from 0 to 1), quantifying SR release unit activation in response to L-type Ca²⁺ channel opening. C(i,t) is proportional to the square of the unitary current of L-type Ca²⁺ channels, i² (Santana et al. 1996) and depends linearly on the mean open time of L-type Ca²⁺ channels, $tau$ . At more positive potentials, there is an increased probability of simultaneous stochastic openings of adjacent L-type Ca²⁺ channels feeding Ca²⁺ into the same cluster of RyR2 channels. Consequently, at more positive potentials, P_O,RU becomes proportional to P_O,Ca² which explains the relative lack of decline in ECC gain at positive potentials (Santana et al. 1996; Song et al. 2001; Guatimosim et al. 2002).

In normal rat or mouse myocytes, particularly of epicardial origin, large L-type Ca²⁺ currents with a very brief duration are evoked during an AP (Bridge et al. 1999; Kaprielian et al. 1999; Volk et al. 1999; Linz & Meyer, 2000). This rapid membrane repolarization effectively limits the probability of L-type Ca²⁺ channel opening (i.e. P_O,Ca) by restricting the time allowed for channel activation (typically about 5 ms) (Bers, 2002b) while also reducing the mean open time by accelerating channel closure via deactivation (Bouchard et al. 1995; Kaprielian et al. 1999), as seen with tail currents in response to short voltage steps. On the other hand, rapid membrane repolarization in rodents ensures that the unitary currents (i) (and thus C(i,t)) are relatively large (for those L-type Ca²⁺ channels that remain open during the repolarization) resulting in Ca²⁺ release occurring primarily during the repolarization phase in rodents (Bridge et al. 1999; Kaprielian et al. 1999). Since I_to densities are lowered and early repolarization is slowed in rodent myocytes, as seen in diseased hearts or in myocytes from endocardial regions, L-type Ca²⁺ channel inactivation, rather than deactivation, dominates the rate of I_Ca,L decay (Kaprielian et al. 1999; Volk et al. 1999; Linz & Meyer, 2000), leading to marked increases in both P_O,Ca and $tau$ . Consequently, the total amount of Ca²⁺ entering via I_Ca,L per pulse is increased as I_to is decreased. Provided the rate of repolarization remains sufficiently rapid when I_to is reduced, repetitive openings will remain infrequent. Thus, because RyR2 clusters are primarily activated by the first latency distribution of L-type Ca²⁺ channel opening (Isenberg & Han, 1994; Lopez-Lopez et al. 1995; Cannell & Soeller, 1999; Wier & Balke, 1999), any increases in I_Ca,L as a result of reduced I_to should couple efficiently and directly to enhanced SR Ca²⁺ release, provided the unitary currents remain sufficiently large (Fig. 4, left dashed arrows). On the other hand, augmenting P_O,Ca and $tau$ following slowing of repolarization will also increase the simultaneous stochastic opening of adjacent L-type Ca²⁺ channels and cooperatively activate the adjacent underlying cluster of RyR2s (Santana et al. 1996; Guatimosim et al. 2002). This latter mechanism will tend to offset the reduction in ECC associated with decreased unitary currents of L-type Ca²⁺ channels that are expected to occur with AP prolongation. The slowed time course of I_Ca,L following prolongation of early repolarization in rodents also causes subtle increases in the dispersion of SR Ca²⁺ release, which has insignificant effects on the time course of the whole-cell Ca²⁺ transients (P. H. Backx and D. Gidrewicz, unpublished results).

View larger version
[in this window]
[in a new window]

Figure 4. Putative mechanism responsible for biphasic dependence of SR Ca²⁺ release on repolarization rates
The mechanisms describing the ascending portion of the relationship (grey dashed lines; left side of diagram) may dominate in species with short, triangular APs, while species with longer, notched AP morphologies may operate primarily on the descending portion (black continuous lines; right side of diagram) and may be governed by different parameters of I_Ca,L.

In the larger species, I_to densities are generally much lower than in rodents (Linz & Meyer, 2000). As expected, reductions of I_to in these species also slow early AP repolarization thus diminishing I_Ca,L peak amplitude and prolonging the time to peak of I_Ca,L (Fig. 3B and Fig. 5B) (Linz & Meyer, 2000; Sah et al. 2002b). In this case, while $tau$ and P_O,Ca are large during the plateau of the AP, C(i,t) is small, which decreases P_O,RU and SR Ca²⁺ release. Furthermore, the slowed I_Ca,L time course desynchronizes the SR Ca²⁺ release events and reduces their degree of summation, thereby further decreasing the peak intracellular Ca²⁺ transient (Sah et al. 2002b) (Fig. 4, right black continuous arrows). Indeed, when phase 1 AP repolarization is slowed in a human AP model, the reduced amplitude and prolonged kinetics of trigger I_Ca,L result in asynchronous SR Ca²⁺ release events with diminished intensity and frequency (Fig. 5C; Sah et al. 2002b). In addition, excessive slowing of repolarization allows L-type Ca²⁺ channels to undergo repetitive opening, leading to increased Ca²⁺ entry via I_Ca,L without coupling to RyR2 cluster activation, thus causing reductions in ECC gain (Isenberg & Han, 1994; Lopez-Lopez et al. 1995; Cannell & Soeller, 1999; Wier & Balke, 1999). These data suggest that, in normal human and canine myocytes where AP durations are typically of the order of 500 ms (Coltart & Meldrum, 1970; Beuckelmann et al. 1993; Kaab et al. 1996), I_to and the associated AP notch may be required to transiently increase I_Ca,L in order to synchronize and optimize SR Ca²⁺ release. These effects of slowed early repolarization in human and canine myocytes would be offset, of course, by the increased tendency of evoking simultaneous opening of L-type Ca²⁺ channels that are anatomically associated with the same RyR2 cluster (Santana et al. 1996; Guatimosim et al. 2002). However, this does not appear to be sufficient to maintain SR Ca²⁺ release when the early repolarization notch disappears (Sah et al. 2002b).

View larger version
[in this window]
[in a new window]

Figure 5. Effect of fast and slow phase 1 AP repolarization on I_Ca,L and Ca²⁺ spikes
A, fast (left) and slow (right) phase 1 AP waveforms were applied to rat ventricular myocytes. B, representative I_Ca,L traces in myocytes stimulated with the AP shown above. C, 600 ms surface plots generated from confocal line scans show loss of recruitment and temporal synchronization of Ca²⁺ release events in myocytes stimulated with slow phase 1 APs.

The results from AP-clamp studies suggest that SR Ca²⁺ release is most robust at repolarization rates where an optimal combination of P_O,Ca, i, and $tau$ can be achieved (Fig. 4, bottom) with the precise relationship depending critically on the anatomy and stochiometry between the RyR2 cluster and L-type Ca²⁺ channels. While the specific position of the optimal rate is expected to vary among different species due to differences in the type, quantity and properties of various ion channels and transporters, it appears that reductions in I_to and AP prolongation in rats and mice operate primarily on the ascending limb of the relationship between $Delta$ [Ca²⁺]_i and repolarization time, while human and canine myocytes may operate predominantly on the descending limb. Of course, due to known regional heterogeneities in channel expression and repolarization rates within the heart, the alterations in the contractile response of myocytes to changes in I_to may vary widely within the ventricle. For example, excessive reductions in I_to in rodent myocytes could broaden and reduce SR Ca²⁺ release in a manner resembling the response in larger species even in the absence of a notch in the AP. Nevertheless, the effects of early repolarization on Ca²⁺ release may have important implications for the consequences of variations in I_to expression in the context of regional heterogeneity of repolarization as well as in heart disease.

Implications of changes in cardiac I_to on Ca²⁺ handling and regional variability of contraction

Although I_to downregulation and reduced expression of Kv4.2/4.3 genes are regarded as hallmark features of diseased myocardium in humans (Beuckelmann et al. 1993; Kaab et al. 1998) and numerous other animal models (Cerbai et al. 1994; Coulombe et al. 1994; Le Grand et al. 1994; Kaab et al. 1996; Chouabe et al. 1997; McIntosh et al. 1998; Kaprielian et al. 1999; Tsuji et al. 2000), the contribution of these changes to alterations in Ca²⁺ handling are often not considered. Typically, altered Ca²⁺ homeostasis in disease has been linked to changes in the expression pattern of several Ca²⁺ handling proteins such as SERCA2a, phospholamban, the Na⁺-Ca²⁺ exchanger and a myriad of other genetic and molecular changes. These changes often lead to reductions in SR Ca²⁺ load, reduced RyR2 sensitivity, reduced trigger I_Ca,L, and altered spatial remodelling between L-type Ca²⁺ channel and RyR2 s (Table 1). The end result is often blunted Ca²⁺ transients with slowed rising and declining phases in either field-stimulated or voltage-clamped myocytes, particularly in advanced heart disease. In myocytes from diseased rodent hearts, the positive inotropic effect of I_to reduction and AP prolongation increases both SR Ca²⁺ load and triggered SR Ca²⁺ release (Kaprielian et al. 1999), thereby compensating for impaired Ca²⁺ cycling and contractility. However, in failing canine and human myocytes, I_to downregulation may actually contribute to further impairment of SR Ca²⁺ release. Indeed, slowing the rate of repolarization using ramps (Fig. 6A) reproduces many features of whole-cell Ca²⁺ transients observed in field stimulated post-MI (myocardial infarction) rabbit myocytes (Litwin et al. 2000) (Fig. 6B), including the slow rise, diminished Ca²⁺ transient amplitude and late occurring Ca²⁺ sparks. These findings suggest that I_to reductions, and the associated slowing of phase 1 repolarization, as a result of diminished Kv4.2/4.3 expression, might exacerbate the coexisting defects in Ca²⁺ handling seen in diseased myocardium. If correct, therapeutic strategies designed to restore normal I_to densities and/or accelerate phase 1 repolarization in failing myocytes may potentially improve systolic function.

View larger version
[in this window]
[in a new window]

Figure 6. Slowed repolarization produces a similar pattern of Ca²⁺ release to that observed in disease
A, 1.6 s line scans taken during 50 ms (left) and 500 ms (right) voltage ramps following a train of eight 100 ms steps to +10 mV in rat ventricular myocytes. With the 500 ms ramp, Ca²⁺ release was sufficiently asynchronous to resolve individual Ca²⁺ sparks and the rise and decay of the Ca²⁺ transient (continuous line below) was significantly slowed relative to that of the 50 ms ramp. This pattern of Ca²⁺ release is strikingly similar to recent findings in myocytes from infarcted rabbit hearts (MI) (Litwin, 2000), shown in panel B below. B, confocal line scans and derived Ca²⁺ transients (below) from control (left) and MI (right) rabbit myocytes. Reproduced with permission from Circulation Research.

Variations in I_to also have important implications for the observed regional differences in myocardial contractility (Volk et al. 1999; Kaprielian et al. 2002). For example, a ventricular gradient of increasing I_to and early repolarization rates exists from the endocardium to the epicardium (Schram et al. 2002). This I_to gradient follows the order of transmural depolarization, suggesting a potential role in ensuring global synchronization of ventricular contraction and optimization of mechanical pump function. Our hypothesis predicts that in heart disease, impaired myocardial contractility in response to I_to reductions (due to slowed rise of [Ca²⁺]_i) complements other molecular changes, associated with impaired cardiac systolic function, such as increased $beta$ -myosin and reduced SERCA2a expression. Similarly, the relatively rapid repolarization rate of rodent myocytes, due to high levels of I_to, ensures rapid Ca²⁺ release and contraction processes to match the rapid heart rates in these animals, while slower repolarization in larger species coincides with much slower heart rates and increased contraction duration.

Another feature of Ca²⁺ transients measured in normal myocytes stimulated with slowly repolarizing stimulus waveforms is a subtle decrease in the rate of decay of the intracellular Ca²⁺ transient (Sah et al. 2002b). This can be attributed to very late SR Ca²⁺ release events that interfere with relaxation of the Ca²⁺ transient, or reduced forward-mode Ca²⁺ extrusion by Na⁺-Ca²⁺ exchanger resulting from the sustained membrane depolarization that occurs with longer ramps. The contribution of forward-mode Na⁺-Ca²⁺ exchange to Ca²⁺ efflux during relaxation of the Ca²⁺ transient may be especially important in the setting of heart failure where SERCA2a expression/function is reduced (Ito et al. 1974; Gwathmey et al. 1987; Beuckelmann et al. 1992; Kuo et al. 1992; Arai et al. 1993; Flesch et al. 1996b; Zarain-Herzberg et al. 1996; O'Rourke et al. 1999) and NCX expression and activity is elevated (Studer et al. 1994; Flesch et al. 1996a; Reinecke et al. 1996; Litwin & Bridge, 1997; O'Rourke et al. 1999; Pogwizd et al. 1999; Ahmmed et al. 2000; Wasserstrom et al. 2000). Clearly, elevated membrane potentials during late repolarization will reduce Ca²⁺ efflux via the Na⁺-Ca²⁺ exchanger. Furthermore, there is evidence to suggest that Ca²⁺ influx via reverse-mode Na⁺-Ca²⁺ exchange may contribute to slowed decay of the Ca²⁺ transient in failing human myocytes (Dipla et al. 1999) and in failing rabbit myocytes with elevated intracellular [Na⁺]_i (Despa et al. 2002).

Implications of altered AP repolarization on Na⁺-Ca²⁺ exchange-mediated SR Ca²⁺ release

In addition to modulating I_Ca,L-triggered Ca²⁺ release, AP repolarization may also influence Na⁺-Ca²⁺ exchange-mediated SR Ca²⁺ release (Fig. 1). During a normal AP in rodents, relatively little time is spent at positive membrane potentials due to rapid phase 1 repolarization, and thus Ca²⁺ influx via I_Ca,NCX may be transient, and possibly insufficient to independently trigger Ca²⁺ release. However, slowing of AP repolarization will prolong the time spent at depolarized membrane potentials, enhance reverse-mode Na⁺-Ca²⁺ exchange, and increase the potential contribution of NCX in triggering SR Ca²⁺ release. In addition to enhancing Ca²⁺ influx via I_Ca,NCX, AP prolongation also shifts the voltage at which I_Ca,L peaks to more positive, depolarized values (ie. from -35 to +30 mV) (Kaprielian et al. 1999), which might enhance potential synergistic interactions between I_Ca,L and I_Ca,NCX in triggering SR Ca²⁺ release by simultaneously activating these independent Ca²⁺ influx pathways. The suggestion that NCX can work synergistically with L-type Ca²⁺ channels in stimulating SR Ca²⁺ release is consistent with several previous studies. (Haworth & Goknur, 1991; Haworth et al. 1991; Cordeiro et al. 2001; Viatchenko-Karpinski & Gyorke, 2001).

In the context of heart disease, Na⁺-Ca²⁺ exchanger expression and function has been shown to be increased in numerous species (Studer et al. 1994; Flesch et al. 1996a; Reinecke et al. 1996; Litwin & Bridge, 1997; O'Rourke et al. 1999; Pogwizd et al. 1999; Ahmmed et al. 2000; Wasserstrom et al. 2000). In some studies, enhanced reverse-mode Na⁺-Ca²⁺ exchange has been demonstrated to provide positive inotropic support to failing myocardium (Flesch et al. 1996b; Gaughan et al. 1999; Sipido et al. 2000; Pogwizd et al. 2001; Despa et al. 2002). Since I_to downregulation and AP prolongation is also associated with heart failure, it is plausible that the combination of slowed AP repolarization and enhanced Na⁺-Ca²⁺ exchanger expression may act complementarily to increase I_Ca,NCX and ultimately systolic [Ca²⁺]_i directly through enhanced triggered SR Ca²⁺ release and/or indirectly via elevated SR Ca²⁺ loading.

In summary, alterations to I_to and AP profile should not be considered independently from changes in ECC in either physiological or pathophysiological states. In large species, such as humans and dogs, rapid early repolarization may be necessary physiologically for adequate recruitment and synchronization of Ca²⁺ release events that contribute to the whole-cell Ca²⁺ transient. Loss of early AP repolarization, as occurs in heart failure, reduces trigger I_Ca,L and disperses Ca²⁺ release, potentially exacerbating impaired Ca²⁺ cycling in disease. Restoration of I_to and rapid phase 1 repolarization in these failing myocytes might prove to be a beneficial therapeutic strategy for optimizing SR Ca²⁺ release. In species with much shorter APs, like rats and mice, we hypothesize that an inverse relationship exists between SR Ca²⁺ release and repolarization rate. In this case, enhanced $Delta$ [Ca²⁺]_i resulting from AP prolongation is predicted to result from increased recruitment of SR Ca²⁺ release units via increased probability of activation and mean open time of individual Ca²⁺ channels. Finally, we speculate that altering early AP repolarization by changes in I_to may also modulate the effective contribution of the reverse-mode Na⁺-Ca²⁺ exchanger to ECC, particularly in diseased states when Na⁺-Ca²⁺ exchanger expression is elevated.

REFERENCES

Top
Abstract
Introduction
Methods
Results
Discussion
References

Ahmmed GU, Dong PH, Song G, Ball NA, Xu Y, Walsh RA & Chiamvimonvat N (2000). Changes in Ca(2+) cycling proteins underlie cardiac action potential prolongation in a pressure-overloaded guinea pig model with cardiac hypertrophy and failure. Circ Res 86, 558-570 [Abstract/Full Text]

Arai M, Alpert NR, MacLennan DH, Barton P & Periasamy M (1993). Alterations in sarcoplasmic reticulum gene expression in human heart failure. A possible mechanism for alterations in systolic and diastolic properties of the failing myocardium. Circ Res 72, 463-469 [Abstract]

Bassani JW, Yuan W & Bers DM (1995). Fractional SR Ca release is regulated by trigger Ca and SR Ca content in cardiac myocytes. Am J Physiol 268, C1313-1319 [Medline]

Bers DM, (2002a). Cardiac excitation-contraction coupling. Nature 415, 198-205 [Medline]

Bers DM, (2002b). Excitation-Contraction Coupling and Cardiac Contractile Force. Kluwer Academic Press, Dordrecht, Netherlands

Beuckelmann DJ, Nabauer M & Erdmann E (1992). Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation 85, 1046-1055 [Abstract]

Beuckelmann DJ, Nabauer M & Erdmann E (1993). Alterations of K⁺ currents in isolated human ventricular myocytes from patients with terminal heart failure. Circ Res 73, 379-385 [Abstract]

Beuckelmann DJ, Nabauer M, Kruger C & Erdmann E (1995). Altered diastolic [Ca²⁺]_i handling in human ventricular myocytes from patients with terminal heart failure. Am Heart J 129, 684-689 [Medline]

Bouchard RA, Clark RB & Giles WR (1995). Effects of action potential duration on excitation-contraction coupling in rat ventricular myocytes. Action potential voltage-clamp measurements. Circ Res 76, 790-801 [Abstract/Full Text]

Bridge JH, Ershler PR & Cannell MB (1999). Properties of Ca²⁺ sparks evoked by action potentials in mouse ventricular myocytes. J Physiol 518, 469-478 [Abstract/Full Text]

Brooksby P, Levi AJ & Jones JU (1993). Investigation of the mechanisms underlying the increased contraction of hypertrophied ventricular myocytes isolated from the spontaneously hypertensive rat. Cardiovasc Res 27, 1268-1277 [Medline]

Cannell MB, Cheng H & Lederer WJ (1995). The control of calcium release in heart muscle Science 268, 1045-1049.

Cannell MB & Soeller C (1999). Mechanisms underlying calcium sparks in cardiac muscle. J Gen Physiol 113, 373-376.

Cerbai E, Barbieri M, Li Q & Mugelli A (1994). Ionic basis of action potential prolongation of hypertrophied cardiac myocytes isolated from hypertensive rats of different ages. Cardiovasc Res 28, 1180-1187 [Medline]

Cheng H, Lederer WJ & Cannell MB (1993). Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science 262, 740-744

Cheng H , & Wang SQ (2002). Calcium signaling between sarcolemmal calcium channels and ryanodine receptors in heart cells. Front Biosci 7, d1867-1878 [Medline]

Chouabe C, Espinosa L, Megas P, Chakir A, Rougier O, Freminet A & Bonvallet R (1997). Reduction of I(Ca,L) and I(to1) density in hypertrophied right ventricular cells by simulated high altitude in adult rats. J Mol Cell Cardiol 29, 193-206 [Medline]

Cleemann L, Wang W & Morad M (1998). Two-dimensional confocal images of organization, density, and gating of focal Ca²⁺ release sites in rat cardiac myocytes. Proc Natl Acad Sci U S A 95, 10984-10989 [Abstract/Full Text]

Collier ML, Thomas AP & Berlin JR (1999). Relationship between L-type Ca²⁺ current and unitary sarcoplasmic reticulum Ca²⁺ release events in rat ventricular myocytes. J Physiol. 516, 117-128 [Abstract/Full Text]

Coltart DJ , & Meldrum SJ (1970). Hypertrophic cardiomyopathy. An electrophysiological study. Br Med J 4, 217-218 [Medline]

Cordeiro J, Cannell M & Bridge JHB (2001). Non-linear signal transduction and excitation-contraction coupling in ventricular myocytes. Circulation 104 (suppl.), 244 [Full Text]

Coulombe A, Momtaz A, Richer P, Swynghedauw B & Coraboeuf E (1994). Reduction of calcium-independent transient outward potassium current density in DOCA salt hypertrophied rat ventricular myocytes. Pflugers Arch 427, 47-55 [Medline]

Despa S, Islam MA, Weber CR, Pogwizd SM & Bers DM (2002). Intracellular Na(+) concentration is elevated in heart failure but Na/K pump function is unchanged. Circulation 105, 2543-2548 [Abstract/Full Text]

Dipla K, Mattiello JA, Margulies KB, Jeevanandam V & Houser SR (1999). The sarcoplasmic reticulum and the Na⁺/Ca²⁺ exchanger both contribute to the Ca²⁺ transient of failing human ventricular myocytes. Circ Res 84, 435-444 [Abstract/Full Text]

Fabiato A, (1985). Rapid ionic modifications during the aequorin-detected calcium transient in a skinned canine cardiac Purkinje cell. J Gen Physiol 85, 189-246 [Abstract]

Fabiato A , & Fabiato F (1979). Use of chlorotetracycline fluorescence to demonstrate Ca²⁺-induced release of Ca²⁺ from the sarcoplasmic reticulum of skinned cardiac cells. Nature 281, 146-148 [Medline]

Ferrier GR , & Howlett SE (2001). Cardiac excitation-contraction coupling: role of membrane potential in regulation of contraction. Am J Physiol 280, H1928-1944

Fiset C, Clark RB, Larsen TS & Giles WR (1997). A rapidly activating sustained K⁺ current modulates repolarization and excitation- contraction coupling in adult mouse ventricle. J Physiol 504, 557-563 [Abstract]

Flesch M, Schwinger RH, Schiffer F, Frank K, Sudkamp M, Kuhn-Regnier F, Arnold G & Bohm M (1996a). Evidence for functional relevance of an enhanced expression of the Na(+)-Ca²⁺ exchanger in failing human myocardium. Circulation 94, 992-1002 [Abstract/Full Text]

Flesch M, Schwinger RH, Schnabel P, Schiffer F, Van Gelder I, Bavendiek U, Sudkamp M, Kuhn-Regnier F & Bohm M (1996b). Sarcoplasmic reticulum Ca²⁺ATPase and phospholamban mRNA and protein levels in end-stage heart failure due to ischemic or dilated cardiomyopathy. J Mol Med 74, 321-332 [Medline]

Franzini-Armstrong C, Protasi F & Ramesh V (1999). Shape, size, and distribution of Ca(2+) release units and couplons in skeletal and cardiac muscles. Biophys J 77, 1528-1539 [Abstract/Full Text]

Gaughan JP, Furukawa S, Jeevanandam V, Hefner CA, Kubo H, Margulies KB, McGowan BS, Mattiello JA, Dipla K, Piacentino V 3rd, Li S & Houser SR (1999). Sodium/calcium exchange contributes to contraction and relaxation in failed human ventricular myocytes. Am J Physiol 277, H714-724 [Medline]

Gaughan JP, Hefner CA, Houser SR (1998). Electrophysiological properties of neonatal rat ventricular myocytes with alpha1-adrenergic-induced hypertrophy. Am J Physiol 275, H577-590 [Medline]

Gomez AM, Guatimosim S, Dilly KW, Vassort G & Lederer WJ (2001). Heart failure after myocardial infarction: altered excitation-contraction coupling. Circulation 104, 688-693 [Abstract/Full Text]

Gomez AM, Valdivia HH, Cheng H, Lederer MR, Santana LF, Cannell MB, McCune SA, Altschuld RA & Lederer WJ (1997). Defective excitation-contraction coupling in experimental cardiac hypertrophy and heart failure [see comments]. Science 276, 800-806

Greenstein JL, Wu R, Po S, Tomaselli GF & Winslow RL (2000). Role of the calcium-independent transient outward current I(to1) in shaping action potential morphology and duration. Circ Res 87, 1026-1033 [Abstract/Full Text]

Guatimosim S, Dilly K, Ferno Santana L, Saleet Jafri M, Sobie E, Lederer W (2002). Local Ca(2+) signaling and EC coupling in heart: Ca(2+). Sparks and the regulation of the [Ca(2+)](i) transient. J Mol Cell Cardiol 34, 941 [Medline]

Gwathmey JK, Copelas L, MacKinnon R, Schoen FJ, Feldman MD, Grossman W & Morgan JP (1987). Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure. Circ Res 61, 70-76 [Abstract]

Gyorke S, (1998). Regulation of the cardiac ryanodine receptor channel by luminal Ca²⁺ involves luminal Ca²⁺ sensing sites. Biophys J 75, 2801-2810 [Abstract/Full Text]

Han S, Schiefer A & Isenberg G (1994). Ca²⁺ load of guinea-pig ventricular myocytes determines efficacy of brief Ca²⁺ currents as trigger for Ca²⁺ release. J Physiol 480, 411-421 [Abstract]

Han C, Tavi P & Weckstrom M (2002). Role of the Na(+)-Ca(2+) exchanger as an an alternative trigger of CICR in mammalian cardiac myocytes. Biophys J 82, 1483-1496 [Abstract/Full Text]

Haworth RA , & Goknur AB (1991). Control of the Na-Ca exchanger in isolated heart cells. II Beat-dependent activation in normal cells by intracellular calcium. Circ Res 69, 1514-1524 [Abstract]

Haworth RA, Goknur AB, Hunter DR (1991). Control of the Na-Ca exchanger in isolated heart cells. I Induction of Na-Na exchange in sodium-loaded cells by intracellular calcium. Circ Res 69, 1506-1513 [Abstract]

Hilgemann DW, (1990). Regulation and deregulation of cardiac Na(+)-Ca²⁺ exchange in giant excised sarcolemmal membrane patches. Nature 344, 242-245 [Medline]

Hobai, IA & O'Rourke B (2001). Decreased sarcoplasmic reticulum calcium content is responsible for defective excitation-contraction coupling in canine heart failure. Circulation 103, 1577-1584 [Abstract/Full Text]

Huser J, Blatter LA & Lipsius SL (2000). Intracellular Ca²⁺ release contributes to automaticity in cat atrial pacemaker cells. J Physiol 524, 415-422 [Abstract/Full Text]

Isenberg G , & Han S (1994). Gradation of Ca(2+)-induced Ca²⁺ release by voltage-clamp pulse duration in potentiated guinea-pig ventricular myocytes. J Physiol 480, 423-438 [Abstract]

Ito Y, Suko J & Chidsey C A (1974). Intracellular calcium and myocardial contractility. V Calcium uptake of sarcoplasmic reticulum fractions in hypertrophied and failing rabbit hearts. J Mol Cell Cardiol 6, 237-247 [Medline]

Janczewski AM, Spurgeon HA, Stern MD & Lakatta EG (1995). Effects of sarcoplasmic reticulum Ca²⁺ load on the gain function of Ca²⁺ release by Ca²⁺ current in cardiac cells. Am J Physiol 268, H916-920 [Medline]

Kaab S, Dixon J, Duc J, Ashen D, Nabauer M, Beuckelmann DJ, Steinbeck G, McKinnon D & Tomaselli GF (1998). Molecular basis of transient outward potassium current downregulation in human heart failure: a decrease in Kv43 mRNA correlates with a reduction in current density. Circulation 98, 1383-1393 [Abstract/Full Text]

Kaab S, Nuss HB, Chiamvimonvat N, O'Rourke B, Pak PH, Kass DA, Marban E & Tomaselli GF (1996). Ionic mechanism of action potential prolongation in ventricular myocytes from dogs with pacing-induced heart failure. Circ Res 78, 262-273 [Abstract/Full Text]

Kaprielian R, Sah R, Nguyen TT, Wickenden AD & Backx PH (2002). Myocardial infarction in rats eliminates heterogeneity of action potential profiles, transient outward K⁺ channel expression and [Ca²⁺]_i transients in rat myocardium. Am J Physiol 283, H1157-1168

Kaprielian R, Wickenden AD, Kassiri Z, Parker TG, Liu PP & Backx PH (1999). Relationship between K⁺ channel down-regulation and [Ca²⁺]_i in rat ventricular myocytes following myocardial infarction. J Physiol 517, 229-245 [Abstract/Full Text]

Kuo TH, Tsang W, Wang KK & Carlock L (1992). Simultaneous reduction of the sarcolemmal and SR calcium ATPase activities and gene expression in cardiomyopathic hamster. Biochim Biophys Acta 14, 343-349

Leblanc N , & Hume JR (1990). Sodium current-induced release of calcium from cardiac sarcoplasmic reticulum. Science 248, 372-376

Le Grand BL, Hatem S, Deroubaix E, Couetil JP & Coraboeuf E (1994). Depressed transient outward and calcium currents in dilated human atria. Cardiovasc Res 28, 548-556 [Medline]

Levi AJ, Brooksby P & Hancox JC (1993). A role for depolarisation-induced calcium entry on the Na-Ca exchange in triggering intracellular calcium release and contraction in rat ventricular myocytes. Cardiovasc Res 27, 1677-1690 [Medline]

Levi AJ, Spitzer KW, Kohmoto O & Bridge JH (1994). Depolarization-induced Ca entry via Na-Ca exchange triggers SR release in guinea pig cardiac myocytes. Am J Physiol 266, H1422-1433 [Medline]

Li GR, Feng J, Yue L & Carrier M (1998). Transmural heterogeneity of action potentials and Ito1 in myocytes isolated from the human right ventricle. Am J Physiol 275, H369-377 [Medline]

Lindner M, Erdmann E & Beuckelmann DJ (1998). Calcium content of the sarcoplasmic reticulum in isolated ventricular myocytes from patients with terminal heart failure. J Mol Cell Cardiol 30, 743-749 [Medline]

Linz KW , & Meyer R (2000). Profile and kinetics of L-type calcium current during the cardiac ventricular action potential compared in guinea-pigs, rats and rabbits. Pflugers Arch 439, 588-599 [Medline]

Lipp P , & Niggli E (1994). Sodium current-induced calcium signals in isolated guinea-pig ventricular myocytes. J Physiol 474, 439-446 [Abstract]

Lipsius SL, Huser J, Blatter LA (2001). Intracellular Ca²⁺ release sparks atrial pacemaker activity. News Physiol Sci 16, 101-106 [Medline]

Litwin SE , & Bridge J H (1997). Enhanced Na(+)-Ca²⁺ exchange in the infarcted heart. Implications for excitation-contraction coupling. Circ Res 81, 1083-1093 [Abstract/Full Text]

Litwin SE, Li J & Bridge JH (1998). Na-Ca exchange and the trigger for sarcoplasmic reticulum Ca release: studies in adult rabbit ventricular myocytes. Biophys J 75, 359-371 [Abstract/Full Text]

Litwin SE, Zhang D & Bridge JH (2000). Dyssynchronous Ca(2+) sparks in myocytes from infarcted hearts. Circ Res 87, 1040-1047 [Abstract/Full Text]

Lopez-Lopez JR, Shacklock PS, Balke CW & Wier WG (1994). Local, stochastic release of Ca²⁺ in voltage-clamped rat heart cells: visualization with confocal microscopy. J Physiol 480, 21-29 [Abstract]

Lopez-Lopez JR, Shacklock PS, Balke CW & Wier WG (1995). Local calcium transients triggered by single L-type calcium channel currents in cardiac cells. Science 268, 1042-1045

Lukyanenko V, Gyorke I & Gyorke S (1996). Regulation of calcium release by calcium inside the sarcoplasmic reticulum in ventricular myocytes. Pflugers Arch 432, 1047-1054 [Medline]

Lukyanenko V, Subramanian S, Gyorke I, Wiesner TF & Gyorke S (1999). The role of luminal Ca²⁺ in the generation of Ca²⁺ waves in rat ventricular myocytes. J Physiol 518, 173-186 [Abstract/Full Text]

McIntosh MA, Cobbe SM, Kane KA & Rankin AC (1998). Action potential prolongation and potassium currents in left-ventricular myocytes isolated from hypertrophied rabbit hearts. J Mol Cell Cardiol 30, 43-53 [Medline]

Marks AR, Reiken S & Marx SO (2002). Progression of heart failure: is protein kinase a hyperphosphorylation of the ryanodine receptor a contributing factor? Circulation 105, 272-275 [Full Text]

Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N & Marks AR (2000). PKA phosphorylation dissociates FKBP126 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell 101, 365-376 [Medline]

Nabauer M, Beuckelmann DJ, Uberfuhr P & Steinbeck G (1996). Regional differences in current density and rate-dependent properties of the transient outward current in subepicardial and subendocardial myocytes of human left ventricle. Circulation 93, 168-177 [Abstract/Full Text]

Nerbonne JM, Nichols CG, Schwarz TL & Escande D (2001). Genetic manipulation of cardiac K(+) channel function in mice: what have we learned, and where do we go from here? Circ Res 89, 944-956 [Abstract/Full Text]

O'Neill SC, Mill JG, Eisner DA (1990). Local activation of contraction in isolated rat ventricular myocytes. Am J Physiol 258, C1165-1168 [Medline]

O'Rourke B, Kass DA, Tomaselli GF, Kaab S, Tunin R & Marban E (1999). Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart failure, I: experimental studies. Circ Res 84, 562-570 [Abstract/Full Text]

Oudit GY, Kassiri Z, Sah R, Ramirez RJ, Zobel C & Backx PH (2001). The molecular physiology of the cardiac transient outward potassium current (i(to)) in normal and diseased myocardium. J Mol Cell Cardiol 33, 851-872 [Medline]

Piacentino V 3rd, Dipla K, Gaughan JP & Houser SR (2000). Voltage-dependent Ca²⁺ release from the SR of feline ventricular myocytes is explained by Ca²⁺-induced Ca²⁺ release. J Physiol 523, 533-548 [Abstract/Full Text]

Pieske B, Maier LS, Bers DM & Hasenfuss G (1999). Ca²⁺ handling and sarcoplasmic reticulum Ca²⁺ content in isolated failing and nonfailing human myocardium. Circ Res 85, 38-46 [Abstract/Full Text]

Pogwizd SM, Qi M, Yuan W, Samarel AM & Bers DM (1999). Upregulation of Na(+)/Ca(2+) exchanger expression and function in an arrhythmogenic rabbit model of heart failure. Circ Res 85, 1009-1019 [Abstract/Full Text]

Pogwizd SM, Schlotthauer K, Li L, Yuan W & Bers DM (2001). Arrhythmogenesis and contractile dysfunction in heart failure: Roles of sodium-calcium exchange, inward rectifier potassium current, and residual beta-adrenergic responsiveness. Circ Res 88, 1159-1167 [Abstract/Full Text]

Priebe L , & Beuckelmann DJ (1998). Simulation study of cellular electric properties in heart failure. Circ Res 82, 1206-1223 [Abstract/Full Text]

Reinecke H, Studer R, Vetter R, Holtz J & Drexler H (1996). Cardiac Na⁺/Ca²⁺ exchange activity in patients with end-stage heart failure. Cardiovasc Res 31, 48-54 [Medline]

Richard S, Leclercq F, Lemaire S, Piot C, Nargeot J (1998). Ca²⁺ currents in compensated hypertrophy and heart failure. Cardiovasc Res 37, 300-311 [Medline]

Rosati B, Pan Z, Lypen S, Wang HS, Cohen I, Dixon JE & McKinnon D (2001). Regulation of KChIP2 potassium channel beta subunit gene expression underlies the gradient of transient outward current in canine and human ventricle. J Physiol 533, 119-125 [Abstract/Full Text]

Sah R, Oudit GY, Nguyen TT, Lim HW, Wickenden AD, Wilson GJ, Molkentin JD & Backx PH (2002a). Inhibition of calcineurin and sarcolemmal Ca²⁺ influx protects cardiac morphology and ventricular function in K(v)42N transgenic mice. Circulation 105, 1850-1856 [Abstract/Full Text]

Sah R, Ramirez RJ & Backx PH (2002b). Modulation of Ca(2+) release in cardiac myocytes by changes in repolarization rate: role of phase-1 action potential repolarization in excitation- contraction coupling. Circ Res 90, 165-173 [Abstract/Full Text]

Sah R, Ramirez RJ, Kaprielian R & Backx PH (2001). Alterations in action potential profile enhance excitation-contraction coupling in rat cardiac myocytes. J Physiol 533, 201-214 [Abstract/Full Text]

Santana LF, Cheng H, Gomez AM, Cannell MB & Lederer WJ (1996). Relation between the sarcolemmal Ca²⁺ current and Ca²⁺ sparks and local control theories for cardiac excitation-contraction coupling. Circ Res 78, 166-171 [Abstract/Full Text]

Santana LF, Kranias EG & Lederer WJ (1997). Calcium sparks and excitation-contraction coupling in phospholamban-deficient mouse ventricular myocytes. J Physiol 503, 21-29 [Abstract]

Satoh H, Blatter LA & Bers DM (1997). Effects of [Ca²⁺]_i, SR Ca²⁺ load, and rest on Ca²⁺ spark frequency in ventricular myocytes. Am J Physiol 272, H657-668 [Medline]

Schram G, Pourrier M, Melnyk P & Nattel S (2002). Differential distribution of cardiac ion channel expression as a basis for regional specialization in electrical function. Circ Res 90, 939-950 [Abstract/Full Text]

Scriven DR, Dan P & Moore ED (2000). Distribution of proteins implicated in excitation-contraction coupling in rat ventricular myocytes. Biophys J 79, 2682-2691 [Abstract/Full Text]

Shacklock PS, Wier WG & Balke CW (1995). Local Ca²⁺ transients (Ca²⁺ sparks) originate at transverse tubules in rat heart cells. J Physiol 487, 601-608 [Abstract]

Shannon TR, Ginsburg KS & Bers DM (2000). Potentiation of fractional sarcoplasmic reticulum calcium release by total and free intra-sarcoplasmic reticulum calcium concentration. Biophys J 78, 334-343 [Abstract/Full Text]

Sipido KR, Carmeliet E & Van de Werf F (1998). T-type Ca²⁺ current as a trigger for Ca²⁺ release from the sarcoplasmic reticulum in guinea-pig ventricular myocytes. J Physiol 508, 439-451 [Abstract/Full Text]

Sipido KR, Maes M & Van de Werf F (1997). Low efficiency of Ca²⁺ entry through the Na(+)-Ca²⁺ exchanger as trigger for Ca²⁺ release from the sarcoplasmic reticulum A comparison between L-type Ca²⁺ current and reverse-mode Na(+)-Ca²⁺ exchange. Circ Res 81, 1034-1044 [Abstract/Full Text]

Sipido KR, Volders PG, de Groot SH, Verdonck F, Van de Werf F, Wellens HJ & Vos MA (2000). Enhanced Ca(2+) release and Na/Ca exchange activity in hypertrophied canine ventricular myocytes: potential link between contractile adaptation and arrhythmogenesis. Circulation 102, 2137-2144 [Abstract/Full Text]

Sitsapesan R , & Williams AJ (1994). Regulation of the gating of the sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel by luminal Ca²⁺. J Membr Biol 137, 215-226 [Medline]

Sobie EA, Dilly KW, Dos Santos Cruz J, Lederer WJ & Jafri MS (2002). Termination of cardiac Ca(2+) sparks: an investigative mathematical model of calcium-induced calcium release. Biophys J 83, 59-78 [Abstract/Full Text]

Song LS, Guia A, Muth JN, Rubio M, Wang SQ, Xiao RP, Josephson IR, Lakatta EG, Schwartz A & Cheng H (2002). Ca(2+) signaling in cardiac myocytes overexpressing the alpha(1) subunit of L-type Ca(2+) channel. Circ Res 90, 174-181 [Abstract/Full Text]

Song LS, Sham JS, Stern MD, Lakatta EG & Cheng H (1998). Direct measurement of SR release flux by tracking 'Ca²⁺ spikes' in rat cardiac myocytes. J Physiol 512, 677-691 [Abstract/Full Text]

Song LS, Wang SQ, Xiao RP, Spurgeon H, Lakatta EG & Cheng H (2001). Beta-adrenergic stimulation synchronizes intracellular ca(2+) release during excitation-contraction coupling in cardiac myocytes. Circ Res 88, 794-801 [Abstract/Full Text]

Stern MD, (1992). Theory of excitation-contraction coupling in cardiac muscle. Biophys J 63, 497-517 [Abstract]

Stern MD, Song LS, Cheng H, Sham JS, Yang HT, Boheler KR & Rios E (1999). Local control models of cardiac excitation-contraction coupling. A possible role for allosteric interactions between ryanodine receptors. J Gen Physiol 113, 469-489 [Abstract/Full Text]

Studer R, Reinecke H, Bilger J, Eschenhagen T, Bohm M, Hasenfuss G, Just H, Holtz J & Drexler H (1994). Gene expression of the cardiac Na(+)-Ca²⁺ exchanger in end-stage human heart failure. Circ Res 75, 443-453 [Abstract]

Su Z, Sugishita K, Ritter M, Li F, Spitzer KW & Barry WH (2001). The sodium pump modulates the influence of I(Na) on [Ca²⁺]_i transients in mouse ventricular myocytes. Biophys J 80, 1230-1237 [Abstract/Full Text]

Terracciano C M , & MacLeod K T (1997). Measurements of Ca²⁺ entry and sarcoplasmic reticulum Ca²⁺ content during the cardiac cycle in guinea pig and rat ventricular myocytes. Biophys J 72, 1319-1326 [Abstract]

Terracciano CM, Tweedie D & MacLeod K T (1997). The effects of changes to action potential duration on the calcium content of the sarcoplasmic reticulum in isolated guinea-pig ventricular myocytes. Pflugers Arch 433, 542-544 [Medline]

Trafford AW, Diaz ME, O'Neill SC & Eisner DA (2002). Integrative analysis of calcium signalling in cardiac muscle. Front Biosci 7, d843-852 [Medline]

Trafford AW, Diaz ME, Sibbring GC & Eisner DA (2000). Modulation of CICR has no maintained effect on systolic Ca²⁺: simultaneous measurements of sarcoplasmic reticulum and sarcolemmal Ca²⁺ fluxes in rat ventricular myocytes. J Physiol 522, 259-270 [Abstract/Full Text]

Tsuji Y, Opthof T, Kamiya K, Yasui K, Liu W, Lu Z & Kodama II (2000). Pacing-induced heart failure causes a reduction of delayed rectifier potassium currents along with decreases in calcium and transient outward currents in rabbit ventricle. Cardiovasc Res 48, 300-309 [Medline]

Viatchenko-Karpinski S , & Gyorke S (2001). Modulation of the Ca(2+)-induced Ca(2+) release cascade by beta-adrenergic stimulation in rat ventricular myocytes. J Physiol 533, 837-848 [Abstract/Full Text]

Volk T, Nguyen TH, Schuktz JH & Ehmke H (1999). Relationship between transient outward K⁺ current and Ca ²⁺ influx in rat myocardium of endo- and epicardial origin. J Physiol 519, 841-850 [Abstract/Full Text]

Wasserstrom JA, Holt E, Sjaastad I, Lunde PK, Odegaard A & Sejersted OM (2000). Altered E-C coupling in rat ventricular myocytes from failing hearts 6 wk after MI. Am J Physiol Heart Circ Physiol 279, H798-807 [Medline]

Wasserstrom JA , & Vites AM (1996). The role of Na⁺-Ca²⁺ exchange in activation of excitation-contraction coupling in rat ventricular myocytes. J Physiol 493, 529-542 [Abstract]

Weber CR, Piacentino V 3rd, Ginsburg KS, Houser SR & Bers DM (2002). Na(+)-Ca(2+) exchange current and submembrane [Ca(2+)] during the cardiac action potential Circ Res 90,182-189.

Wier WG & Balke CW, (1999). Ca(2+) release mechanisms, Ca(2+) sparks, and local control of excitation�contraction coupling in normal heart muscle. Circ Res 85, 770-776.

Wier WG, Egan TM, Lopez-Lopez JR & Balke CW (1994). Local control of excitation-contraction coupling in rat heart cells. J Physiol 474, 463-471 [Abstract]

Wickenden AD, Kaprielian R, Parker TG, Jones OT & Backx PH (1997). Effects of development and thyroid hormone on K⁺ currents and K⁺ channel gene expression in rat ventricle. J Physiol 504, 271-286