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Am J Physiol Heart Circ Physiol 294: H1597-H1608, 2008. First published January 18, 2008; doi:10.1152/ajpheart.00484.2007
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Modulation of late sodium current by Ca2+, calmodulin, and CaMKII in normal and failing dog cardiomyocytes: similarities and differences

Victor A. Maltsev, Vitaliy Reznikov, Nidas A. Undrovinas, Hani N. Sabbah, and Albertas Undrovinas

Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan

Submitted 23 April 2007 ; accepted in final form 11 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Augmented and slowed late Na+ current (INaL) is implicated in action potential duration variability, early afterdepolarizations, and abnormal Ca2+ handling in human and canine failing myocardium. Our objective was to study INaL modulation by cytosolic Ca2+ concentration ([Ca2+]i) in normal and failing ventricular myocytes. Chronic heart failure was produced in 10 dogs by multiple sequential coronary artery microembolizations; 6 normal dogs served as a control. INaL fine structure was measured by whole cell patch clamp in ventricular myocytes and approximated by a sum of fast and slow exponentials produced by burst and late scattered modes of Na+ channel gating, respectively. INaL greatly enhanced as [Ca2+]i increased from "Ca2+ free" to 1 µM: its maximum density increased, decay of both exponentials slowed, and the steady-state inactivation (SSI) curve shifted toward more positive potentials. Testing the inhibition of CaMKII and CaM revealed similarities and differences of INaL modulation in failing vs. normal myocytes. Similarities include the following: 1) CaMKII slows INaL decay and decreases the amplitude of fast exponentials, and 2) Ca2+ shifts SSI rightward. Differences include the following: 1) slowing of INaL by CaMKII is greater, 2) CaM shifts SSI leftward, and 3) Ca2+ increases the amplitude of slow exponentials. We conclude that Ca2+/CaM/CaMKII signaling increases INaL and Na+ influx in both normal and failing myocytes by slowing inactivation kinetics and shifting SSI. This Na+ influx provides a novel Ca2+ positive feedback mechanism (via Na+/Ca2+ exchanger), enhancing contractions at higher beating rates but worsening cardiomyocyte contractile and electrical performance in conditions of poor Ca2+ handling in heart failure.

heart failure; arrhythmia

AUGMENTED AND SLOWED late Na+ current (INaL) in heart failure (HF) (21, 46) contributes to action potential (AP) duration variability and development of early afterdepolarizations (EADs) in human and dog ventricular cardiomyocytes (VCs) isolated from failing hearts (16, 21, 46). Interestingly, the amount of Na+ carried by large but short (a few ms) transient Na+ current (INaT) is almost equal to that carried by a much smaller but much longer INaL (hundreds of ms) (21, 23). Since INaT is reportedly decreased (19, 54) but INaL is increased in HF, the latter likely provides an important contribution to the increase in intracellular Na+ concentration ([Na+]i) and to a higher cell Ca2+ load in HF (26). Because of its pathophysiological significance, INaL recently emerged as a plausible target for cardioprotection therapies (see Refs. 8, 26 for reviews).

HF-related mechanisms leading to the INaL augmentation and slower decay are not clear. All previous comparisons of INaL in normal and failing hearts were performed at low intracellular Ca2+ in the presence of strong Ca2+ buffers; thus only Ca2+-independent changes have been compared so far. Taking into account that Ca2+ handling in HF is altered (see Ref. 6 for review), an intriguing question is then whether Ca2+ provides any modulatory feedback mechanism to INaL in HF.

The present study tested whether intracellular Ca2+ modulates INaL in VCs and whether this modulation is different in normal and failing hearts. For this purpose we used an established model of canine chronic HF produced by multiple sequential coronary artery microembolizations (34). This model produces an array of morphological and functional changes similar to that for humans. To get a mechanistic insight into INaL modulation by Ca2+, we tested how signaling via Ca2+/calmodulin (CaM)/Ca2+-dependent CaM kinase II (CaMKII) affects the whole cell INaL in patch-clamped VCs isolated from normal and failing dog hearts. We found that while intracellular Ca2+ signaling enhances INaL in both normal and failing VCs, its modulation range is larger in HF. This result indicates that previous evaluations of the functional importance of INaL in normal and especially in failing hearts might be underestimates because they were based on Ca2+-independent INaL function. Preliminary data from this study have been reported in abstract form (18).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
HF Model and Myocyte Isolation

The study conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and was approved by the Institutional Animal Care and Use Committee (IACUC) of the Henry Ford Health System. Chronic HF that is similar by a vast array of functional and pathophysiological parameters (33) to that in humans was produced in 10 dogs by multiple sequential coronary artery microsphere embolizations as previously described (34). Six normal dogs served as a control. At the time of harvesting the hearts (~3 mo after last embolization), left ventricular (LV) ejection fraction was approximately ~25%. Cardiomyocytes were enzymatically isolated from the apical LV midmyocardial slices as previously reported (16). The yield of viable rod-shaped, Ca2+-tolerant VCs varied from 40% to 70%.

Patch-Clamp Technique and Data Analysis

INaL was measured with a whole cell patch-clamp technique (16, 46). INaL was assessed by 2-s membrane depolarizations to various potentials from a holding potential of –130 mV applied with a stimulation frequency of 0.2 Hz. INaL was presented in terms of current density (pA/pF), i.e., INaL = (whole cell INaL)/Cm, where Cm is cell electric capacitance that was measured by a voltage ramp (19) in each cell. The composition of bath and pipette solutions is shown in Table 1. Experiments were performed at room temperature (22–24°C). All measurements were made 8–25 min after the membrane rupture to complete cell dialysis with intracellular recording solutions (24, 28, 31). As shown in previous studies peptides or small proteins such as protein kinase catalytic subunits (14) can quickly (within 5 min) reach their targets in adult ventricular myocytes during whole cell dialysis via patch pipette.


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  		Table 1. Composition of bath and pipette solutions

 
The fine structure of the INaL time course has been approximated by a double exponential fit to INaL decay starting at 40 ms after the onset of depolarization to –30 mV as previously suggested (23):

Formula 1(1)
where {tau}1 and {tau}2 are the time constants, I40 is INaL instant value 40 ms after membrane depolarization, and k1 and k2 are the contributions of each exponent (k1 + k2 = 1), respectively. Five to fifteen experimental traces were averaged to improve the quality of analysis.

The steady-state inactivation (SSI) was evaluated by a double-pulse protocol with 2-s-duration prepulses (Vp) ranging from –130 mV to –40 mV, followed by a testing pulse to –30 mV. INaL amplitudes were normalized to that measured at Vp = –130 mV, and the data points were fitted to a Boltzmann function A(Vp):

Formula 2(2)
where V1/2A and kA are the midpoint and the slope of the Boltzmann function. The steady-state activation (SSA) parameters were determined from the current-voltage relationships by fitting data points of the normalized current with the function (19)

Formula 3(3)
Where Gmax is a normalized maximum Na+ conductance, Vr is a reversal potential, Vt is testing voltage, and V1/2G and kG are the midpoint and the slope of the respective Boltzmann function underlying the steady-state Na+ channel activation. The INaL data points in the current-voltage relationships and SSI were measured as the averaged current density within 200–220 ms after depolarization onset (vertical bar in Figs. 1, A and B, 3, A and B, and 5, A and B).


Figure 1
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  		Fig. 1. Ca2+modulates late Na+ current (INaL) kinetics and amplitude in ventricular cardiomyocytes of normal and failing dog hearts. Representative raw traces were recorded at different intracellular Ca2+ concentrations ([Ca2+]i) in normal (A) and failing (B) cells. Exponential fits (Eq. 1, see MATERIALS AND METHODS) are shown by solid lines together with their parameters. Inset shows voltage-clamp protocol. Vertical bars indicate (here and in Figs. 3 and 5) the time window (200–220 ms of depolarization) used to evaluate INaL density. Statistical data for the decay time constants {tau}1 (C) and {tau}2 (D) at different [Ca2+]i in normal and failing hearts are also shown. Bars represent data means ± SE for n cells (hearts). Statistical significant difference (P < 0.05, ANOVA followed by Bonferroni's post hoc test): *,**normal and failing heart 0 Ca2+ vs. 1 µM Ca2+, respectively; ¶,§normal vs. failing heart at 0 Ca2+ and 1 µM Ca2+, respectively. Vh, holding potential; Vm, membrane potential.

 

Figure 3
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  		Fig. 3. Inhibition of CaMKII accelerates INaL decay in normal and failing hearts. Representative raw traces were recorded at high [Ca2+]i alone or in the presence of CaMKII inhibitor KN93 (10 µM) in normal (A) and failing (B) cells. Exponential fits (Eq. 1, see MATERIALS AND METHODS) are shown by solid lines together with their parameters. Inset shows voltage-clamp protocol. C and D: statistical data for the decay time constants {tau}1 (C) and {tau}2 (D) in normal and failing hearts. Bars represent data means ± SE for n cells (hearts). For decay time constants (C, D) statistical significant difference (P < 0.05, ANOVA followed by Bonferroni's post hoc test): *,**normal and failing heart 1 µM Ca2+ vs. 1 µM Ca2+ + KN93, respectively; ¶normal vs. failing heart at 1 µM Ca2+; §normal vs. failing heart at 1 µM Ca2+ + KN93, respectively.

 

Figure 5
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  		Fig. 5. Peptide P290-309, antagonist of Ca2+-dependent CaM binding, accelerates INaL decay and reduces its amplitude in normal and failing hearts. Representative raw traces were recorded at high [Ca2+]i alone or in the presence of P290-309 in normal (A) and failing (B) cells. Exponential fits (Eq. 1, see MATERIALS AND METHODS) are shown by solid lines together with their parameters. Inset shows voltage-clamp protocol. Statistical data for decay time constants {tau}1 (C) and {tau}2 (D) at different [Ca2+]i in normal and failing hearts are also shown. Bars represent data means ± SE for n cells (hearts). Statistically significant difference (P < 0.05, ANOVA followed by Bonferroni's post hoc test): *,**normal and failing heart 1 µM Ca2+ vs. 1 µM Ca2+ + P290-309, respectively; ¶normal vs. failing heart at 1 µM Ca2+ (C, D). No significant difference: §normal vs. failing heart at 1 µM Ca2+ + P290-309 (C, D).

 
Statistical Analysis

Multiple comparisons between treatment groups were made by one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. Data are reported as means ± SE. The significance of SSA or SSI changes was evaluated with an F test (StatMost, DataMost, Salt Lake City, UT) for tabulated values predicted by the model (Eqs. 2 and 3) at a confidence level of 0.95. Differences for both experimental data and model predictions were considered statistically significant at P < 0.05.

Chemicals

Collagenase type II (291 U/mg) was from Worthington (Freehold, NJ). CaM, CaM binding domain, peptide P290-309, and CaMKII inhibitor KN93 and its inactive analog KN92 were purchased from Calbiochem (Darmstadt, Germany). All other chemicals and enzymes were purchased from Sigma (St. Louis, MO).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Elevated Intracellular Ca2+ Slows Decay of INaL, Increases Density, and Shifts SSI in Both Normal and Failing Hearts

First we compared INaL decay time course, density, SSA, and SSI at low (depicted as 0 Ca2+) and high (1 µM Ca2+) intrapipette Ca2+ concentration ([Ca2+]i; solution compositions are depicted in Table 1). Two intracellular Ca2+ buffering agents were tested: EGTA and BAPTA (solutions P1 and P2 in Table 1). Although BAPTA provides more stringent Ca2+ buffering and more rapid Ca2+ binding kinetics (50), no difference was found for INaL decay kinetics, density, or SSI when using these buffers (not shown). Accordingly, "0 Ca2+" refers to EGTA buffer. Elevated [Ca2+]i substantially increased amplitude and slowed inactivation of INaL in both normal and failing hearts (Fig. 1). Both {tau}1 and {tau}2 significantly increased in the elevated Ca2+ (Fig. 1, C and D). For example, at –30 mV (maximum INaL) their average values in 0 Ca2+ and 1 µM Ca2+ in normal hearts were as follows: {tau}1 = 31.3 ± 0.7, {tau}2 = 417.7 ± 9.1 ms [n = 27 VCs (4 hearts)] vs. {tau}1 = 48.6 ± 2.1, {tau}2 = 487 ± 18 ms [n = 11 (2)] (P < 0.005, ANOVA), respectively. For HF, {tau}1 = 35.2 ± 1.9, {tau}2 = 505.7 ± 19.2 ms [n = 12 (4)] vs. {tau}1 = 57.4 ± 3.7, {tau}2 = 625.6 ± 42 ms [n = 28 (8)] (P < 0.005), respectively. Interestingly, both time constants were significantly different at the same [Ca2+]i in normal vs. failing VCs. However, the relative contribution of these exponents remained almost unchanged [normal heart: k2 = 0.43 ± 0.02 vs. 0.46 ± 0.05, 0 Ca2+ vs. 1 µM Ca2; HF: k2 = 0.41 ± 0.05 vs. 0.43 ± 0.04, respectively; not significant (NS)]. Elevated [Ca2+]i significantly increased INaL density (Table 2) in a wide range of voltages, including potentials of the AP plateau in both normal and failing hearts (Fig. 2, A and B). Another noticeable effect of the Ca2+ elevation was a shift (~5.8 and ~4.4 mV for normal and failing hearts, respectively) of the SSI midpotential toward positive potentials (Fig. 2, C and D, Table 3) without changes in the SSA midpotential (Fig. 2, A and B).


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  		Table 2. Effects of intracellular Ca2+ signaling on density of INaL in failing dog cardiomyocytes

 

Figure 2
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  		Fig. 2. Elevation of intracellular Ca2+ from 0 to 1 µM uncouples the steady-state activation (SSA) and inactivation (SSI) of INaL in both normal and failing hearts. A and B: INaL-voltage relationship at different [Ca2+]i in normal (A) and failing (B) hearts. Data represent means ± SE pooled from 4–11 cells. Solid lines show theoretical curves of SSA (Eq. 3, MATERIALS AND METHODS) fitted to data points. Maximum INaL conductance (Gmax) significantly increased from 2.34 to 3.68 pS/pF (normal hearts) and from 3.65 to 5.38 pS/pF (failing hearts) (P < 0.001, F test). Other fit parameters [midpotential (V1/2) and slope (k) are shown above traces] remained almost unchanged in augmented [Ca2+]i. Action potential (AP) plateau range from –15 to 20 mV (46) is depicted by a box (B). SSI was significantly shifted rightward in response to the elevated [Ca2+]i in both normal (C) and failing (D) hearts (P < 0.001, F test). Solid lines represent theoretical curve fit (Eq. 2, MATERIALS AND METHODS). Statistical comparison of all SSI parameters for experiments is given in the text. Data points were pooled from 5–20 cells [3 normal and 6 heart failure (HF) dogs]. Inset in D shows voltage-clamp protocol. Vp, prepulse voltage.

 

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  		Table 3. Effects of intracellular Ca2+ signaling pathways on steady-state inactivation parameters in normal and failing dog cardiomyocytes

 
Infusion of CaM Does Not Affect INaL

To test whether intrinsic intracellular concentration of CaM ([CaM]i) can limit effects of elevated [Ca2+]i on INaL, we infused additional CaM molecules in the presence of [Ca2+]i of 1 µM. The INaL decay, density, and SSI (not shown) parameters were not significantly affected by the intracellular infusion of CaM into the cells via the patch pipette. These data indicated that [CaM]i either is not involved in Ca2+ modulation of INaL or remains high enough during the whole cell recordings to mediate Na+ channel modulation by Ca2+. Thus the absence of any effect of CaM infusion served as a control for our further experiments with peptide P290-309, which were designed to clarify the role of CaM in INaL modulation by testing effects of CaM inhibition (see below).

Effects of CaMKII Inhibitor KN93

In the presence of high [Ca2+]i, CaMKII inhibition by KN93 caused two major effects (see examples of recordings in Fig. 3, A and B): 1) INaL decay acceleration (both fast and slow components of INaL) in both normal and failing hearts blunting the difference in terms of INaL inactivation kinetics between these two specimens (Fig. 3, C and D) and 2) significant INaL density reduction observed at various levels of testing voltages within the range of action potential plateau (as the whole cell conductance decreased) in failing but not in normal hearts (Fig. 4, A and B, Table 2). The midpotential V1/2 of the SSA in both normal and failing hearts changed insignificantly. There was no significant effect on SSI by KN93 in either normal or failing hearts compared with the augmented [Ca2+]i (Fig. 4, C and D, Table 3).


Figure 4
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  		Fig. 4. Effects of CaMKII inhibition on SSA and SSI of INaL in both normal and failing hearts. A and B: INaL-voltage relationship at high [Ca2+]i and in the presence of CaMKII inhibitor KN93 (10 µM) in normal (A) and failing (B) cells. Data represent means ± SE pooled from 4 or 5 cells (2 normal and 2 failing hearts). Solid lines show theoretical curves of SSA (Eq. 3, MATERIALS AND METHODS) fitted to data points. In response to CaMKII inhibition Gmax did not change significantly, 3.7 vs. 4.0 pS/pF in normal hearts (A) but significantly reduced from 5.4 to 4.62 pS/pF in failing hearts (B) (P < 0.05, F test). Other fit parameters (V1/2 and k) are shown above the traces. Asterisks in B show statistically significant (P < 0.05) differences in data points. SSI was not affected by CaMKII inhibition in either normal (C) or failing hearts (D). Detailed statistical comparison of all SSI parameters for large number of experiments is given in Table 3. Data points were pooled from 5–10 cells (2 normal and 3 HF dogs). Inset in D shows voltage-clamp protocol.

 
To provide a control for the substantial KN93 effect on INaL kinetics, we also tested the effect of KN92 (an inactive analog of KN93): in the presence of 1 µM [Ca2+]i and 10 µM KN92 the INaL kinetics and SSA remained unchanged [n = 16 cells (4 hearts) HF, not shown]. However, we did not compare effects of KN93 and KN92 on SSI for two reasons, 1) the fact that CaMKII blockade by KN93 had no effect on SSI and 2) the well-known nonspecific effects of KN92 on SSI (9).

Effects of Interference to Ca2+-Dependent CaM Binding

Infusion of P290-309, a potent CaM antagonist, in the presence of high [Ca2+]i caused two effects described above for KN93, i.e., INaL decay acceleration and density decrease (Fig. 5, Table 2), and this was expected because inhibition of CaM is a less specific intervention that includes CaMKII inhibition (CaMKII function is by definition CaM dependent). However, a specific effect of CaM inhibition that was not observed under KN93 was a significant shift of SSI curve toward positive potentials in failing but not in normal hearts (Fig. 6, C and D, Table 3).


Figure 6
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  		Fig. 6. Effects of blockade of Ca2+-dependent CaM binding by the peptide P290-309 on SSA and SSI of INaL in both normal and failing hearts. A and B: INaL-voltage relationship at high [Ca2+]i alone or in the presence of P290-309 in normal (A) and failing (B) hearts. Data represent means ± SE pooled from 4–10 cells (2 normal and 2 failing hearts). Solid lines show theoretical curves of SSA (Eq. 3, MATERIALS AND METHODS) fitted to data points. Gmax significantly decreased from 3.68 to 2.17 pS/pF (normal hearts) and from 5.38 to 4.12 pS/pF (HF hearts) (P < 0.001, F test) in the presence of P290-309. Other fit parameters (V1/2 and k are shown at the traces) remained almost unchanged in augmented [Ca2+]i. AP plateau range from –15 to 20 mV (46) is depicted by a box (B). The theoretical curve of SSI was significantly shifted rightward in the presence of P290-309 in HF (D; P < 0.001, F test) but not normal (C) hearts. Statistical comparison of all SSI parameters for experiments is given in the text. Data points were pooled from 5–20 cells (3 normal and 6 HF dogs). Inset in D shows voltage-clamp protocol.

 
Interpretation of Experimental Data in Terms of Ca2+ Modulation of INaL Gating Modes and INaL-Mediated Na+ Influx

While the experimental data (Figs. 3–6) of various interventions to Ca2+ regulatory pathways provide mechanistic insights into INaL regulation by those pathways (biochemical insights), additional analysis is required to evaluate numerically modulation by those pathways of various INaL gating modes (23, 45) (biophysical insights) and of INaL-mediated Na+ influx into the cells (physiological insights). In our experiments INaL density was measured 200 ms after membrane depolarization, and INaL kinetics were evaluated starting from 40 ms. Thus the observed changes in the density can be due to either INaL slowing or a decrease in the number of Na+ channels operating in the late gating modes. To address this question, the fine structure of the entire INaL time course was further analyzed based on INaL approximation by a double exponential function in Eq. 2, which allows an easy interpretation of the whole cell data in terms of gating modes of late Na+ channel openings. Contrary to a prediction of a noninactivating INaL by a nonunique model of Na+ channel gating (11), patch-clamp studies in human ventricular myocytes showed that both the burst mode (BM) and the late scattered mode (LSM) openings do inactivate (29, 45)! A recent unique numerical model (23) of Na+ channel gating based on the single-channel data of late openings describes the entire INaL time course as a sum of two exponentials: BM generates the fast, exponentially decaying INaL component (IBM), whereas LSM openings generate a relatively slow decaying INaL component (ILSM):

Formula 4(4)

Formula 5(5)
where I40, k1, k2, {tau}1, and {tau}2 are from Eq. 1. Using the above approximations, we evaluated the following important parameters: amplitudes IBM(0) and ILSM(0) and integrals QBM and QLSM of the fast and slow INaL components, respectively:

Formula 6(6)

Formula 7(7)
The amplitudes thus reflect the total number of channels in respective gating modes, and the integrals reflect the respective Na+ influxes transferred by each mode. Since we found a complex modulation of SSI by Ca2+ and CaM, we also evaluated the total integral for INaL elicited from a physiological resting potential (Vrest) of –80 mV:

Formula 8(8)
where A(–80) is Na+ channel steady-state availability at –80 mV calculated with Eq. 2. The results of the analysis are summarized in Table 4 and can be interpreted for each modulation pathway as follows.


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  		Table 4. Results of numerical evaluation and comparison of different Ca2+-dependent mechanisms of INaL modulation in normal and failing cardiomyocytes

 
CaMKII slows decay kinetics of both BM and LSM openings, with the effect being greater in failing VCs, resulting in a substantially larger regulation range of QLSM by CaMKII in the failing cells, 56.8% vs. 35% [CaMKII modulates ILSM(0), i.e., the number of LSM channels, nonsignificantly]. At the same time, CaMKII substantially decreases IBM(0), i.e., the number of BM channels, with QBM remaining almost unchanged in both normal and failing cells.

CaM produces no additional modulatory effects on the fine structure of INaL than those described above for CaMKII modulation. Hence, the only major and specific effect of CaM on INaL is the negative SSI shift in the failing cells.

Ca2+ itself, independently of CaM, increases ILSM(0) (reflecting the number of LSM channels) by ~30.7% in failing cells but produces no effect on this parameter in normal cells. Ca2+ also (independently of CaM) shifts SSI to negative potentials. The combined effect of this shift and ILSM(0) increase on Qtot was substantially larger in failing cells vs. normal cells (150% vs. 61.6%). Interestingly, that CaMKII contribution to Qtot modulation is minor (only ~21%) in the failing cells.

Net Ca2+ effect on INaL and its integral is strong in both normal and failing VCs (Fig. 7). The absolute modulation range of INaL is larger in failing cells, with the difference being greater at the beginning of depolarization (shaded area in Fig. 7C). The relative instant increase of INaL produced by Ca2+ was larger in failing cells; in both cell types it exhibits an N-like shape with a local maximum at ~120 ms (Fig. 7D). CaMKII-mediated slowing of LSM decay and rightward shift of SSI by Ca2+ (CaM independent) turn out to be the major contributors to the INaL integral change (Fig. 7, E–G, Table 4). In HF VCs, while Ca2+ increases the number of LSM channels and shifts SSI to more positive voltages these effects are offset by CaM-induced negative SSI shift, so that the net Ca2+-induced increase of Qtot is only moderately higher in HF (135% vs. 101%, respectively, Table 4; see also shaded area in Fig. 7G).


Figure 7
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  		Fig. 7. Theoretical evaluation of difference in the cumulative effects of the elevated Ca2+ on INaL and its integral (~Na+ influx) in cardiomyocytes of normal dogs (Norm) and dogs with chronic heart failure (HF). Parameters for idealized, 2-exponential INaL decay time course were assigned from the average experimental data measured in 2 extreme conditions: [Ca2+]i = 0 and [Ca2+]i = 1 µM (see RESULTS for details). The average maximum INaL density at –30 mV was corrected for SSI voltage-dependence assuming a resting/holding potential of –80 mV. A and B: idealized INaL in normal and failing ventricular myocytes. C and D: absolute and relative ranges of Ca2+ regulation of INaL are larger in failing cells (absolute difference is shown by shaded area in C). E and F: substantial modulation of INaL-related Na+ influx by Ca2+ assessed as an absolute value of INaL integral in normal and failing myocytes. G: modulation of INaL-related Na+ influx by Ca2+ is greater in failing myocytes (shaded area HF-Norm).

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study has demonstrated that INaL in ventricular myocytes of normal and failing dog hearts is strongly enhanced by intracellular Ca2+ and this modulation is linked to complex Ca2+/CaM/CaMKII signaling (summarized in Table 4 and schematically illustrated in Fig. 8). We identified Ca2+-dependent modulation mechanisms, which are common and different in normal and failing cells. The common mechanisms include 1) CaMKII slows the INaL decay kinetics and decreases the amplitude of the fast/burst INaL component and 2) direct Ca2+ binding shifts the SSI curve to more positive potentials. INaL modulation in failing VCs differs from that in normal cells: 1) slowing of both INaL components by CaMKII is greater; 2) Ca2+ shifts SSI to more positive voltages; 3) CaM shifts the SSI curve to negative potentials; and 4) Ca2+ increases the amplitude of the INaL slow/LSM component.


Figure 8
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  		Fig. 8. Simplified hypothetical diagram of the intracellular Ca2+ signaling pathways modulating INaL in normal and failing canine ventricular myocytes based on the results of the present study. Pathways 1, 2, and 3 (marked by black numbers) represent Ca2+ binding to the E–F domain, CaM-binding site (IQ motif) on COOH terminus of Na channel, as well as CaM/CaMKII complex, respectively. Arrows with +/– signs indicate modulation in terms of increase/decrease of INaL integral. Biophysical mechanisms of the modulations are shown as follows: SSI, shift of the voltage dependence of steady-state inactivation; #LSM and #BM, no. of channels in late scattered or burst mode, respectively; gating kinetics, slowing inactivation gating for LSM and BM. Note that pathways shown by dashed lines are effective only in failing myocytes. Also shown are the inhibitory sites of the CaM antagonist peptide P290-309 and KN93, the inhibitor of CaMKII employed in the present study to test the signaling. A color version of this figure may be found linked with the online version of this article.

 
Molecular Mechanisms of INaL Modulation by Ca2+-CaM-CaMKII Pathways

Previous studies of Na+ channels have established several pathways for Na+ channel modulation by intracellular Ca2+: 1) direct binding of Ca2+ ions to Ca2+ binding EF-hand motif found in Nav1.5 COOH terminus (50), 2) direct CaM binding to IQ motif of NaV1.5 COOH terminus (39), and 3) activation of CaMKII resulting in phosphorylation of the channel protein (9, 49). Ca2+ modulation of Na+ channels is a rather complex phenomenon and continues to be a matter of debate (see Ref. 1 for review), especially when heterologous expression systems and native myocytes are compared. Moreover, the conclusions of the aforementioned studies are mainly based on recordings of the transient Na+ current that may or may not be relevant to INaL modulation.

All three of the above molecular mechanisms can be potentially involved in the effects of complex Ca2+/CaM/CaMKII modulation of INaL characteristics found in the present study (Table 4, Fig. 8). More specifically, we found that late Na+ channel gating is regulated by CaMKII (Fig. 3) in both normal (in line with Ref. 49) and failing hearts and that Ca2+ shifts SSI toward positive potentials (Fig. 1, C and D) (in line with Ref. 50). The latter effect is likely independent of CaM and CaMKII in normal myocytes because inhibition of either CaM or CaMKII has no effect on SSI (Fig. 6C).

However, specifically in failing cells CaM (independently of CaMKII) offsets, at least in part, the SSI shift associated with the major CaM-independent effect. The negative shift of SSI by CaM-dependent regulation is in line with Tan et al. (39). However, Wagner et al. (49) reported an opposite effect (compared with Ref. 50 and our study) of [Ca2+]i elevation on SSI in rabbit and rat cardiomyocytes, including those in which CaMKII{delta}C was overexpressed. The difference in results could be due to 1) different cell types, e.g., tsA201 (50) cells vs. cardiomyocytes; 2) perturbation of Ca2+ signaling by an overexpression of the signaling proteins; 3) different currents used to assess SSI, i.e., INaT or INaL (our study); or 4) different recording conditions, e.g., 10 mM (49) vs. 140 mM of bath [Na+]. In fact, occupancy of the Na+ channel pore by the permeant ion substantially affects slow inactivation of the channel (3).

Why Is Ca2+ Modulation of INaL Different in Heart Failure?

Na+ channel environment and composition. The function of Na+ channels (especially their late openings) is complex and not fully determined by their protein structure but also greatly depends on interactions with multiple molecules of the channel environment, the function of which could be, in turn, also Ca2+ dependent and greatly altered in HF. In addition to the pore-forming {alpha}-subunit and its auxiliary β-subunits, the multiprotein Na+ channel complex includes components of cytoskeleton, regulatory kinases and phosphatases, trafficking proteins, and extracellular matrix proteins embedded into lipid bilayer plasma membrane (see Refs. 1 and 25 for reviews). Subsarcolemmal actin-fodrin cytoskeleton is involved in modulation of the Nav1.5 inactivation gating (44, 47). Breakdown of actin-fodrin cytoskeleton integrity leads to activation-inactivation uncoupling (22) similar to that reported here (Fig. 2, A and B vs. C and D). The fodrin breakdown that occurs in some heart disease states featuring poor Ca2+ handling can be mediated by Ca2+/CaM via the enzymes calpain and caspase (32, 51). Accordingly, these indirect mechanisms can contribute to the CaM effect on SSI in failing myocytes reported here.

Possible specific mechanisms for differences. While the greater modulation range for CaMKII in failing cells found in the present study is likely due to a higher expression of CaMKII in HF (see Ref. 53 for review), the mechanism of the effect of CaM on SSI in failing hearts is not clear. As to Nav1.5, it is still ambiguous whether its SSI is (39) or is not (9, 50, 52) regulated by CaM. Our data in normal cardiomyocytes rather support the latter hypothesis. Thus possibilities for the specific INaL regulation by CaM in HF include an indirect effect of CaM (via molecules of the Na+ channel complex, see above) or expression of other Na+ channel isoforms modulated by CaM. Different transcripts of highly TTX-sensitive neuronal isoforms (Nav1.1, -1.3, -1.6) have indeed been identified in mouse and dog heart (Nav1.1, -1.2, -1.3) (12, 15). These neuronal Na+ channel isoforms are responsible for 10–20% of the Na+ current peak in myocardial and Purkinje cells, respectively (12). CaM also shifts SSI curve for the skeletal muscle isoform Nav1.4 (9, 52). Furthermore, four splice variants of the SCN5A gene that encodes Nav1.5 have been reported in humans, and they differ in their biophysical properties (38). The contributions of these variants and/or isoforms to cardiac INaL and their modulation by different pathological conditions are not yet understood and need further studies.

Significance of INaL Modulation by Ca2+ for Normal Heart Function

Ca2+ modulation of INaL likely contributes to cell Na+/Ca2+ balance, which is critical for normal and enhanced cardiomyocyte performance. More specifically, Ca2+-dependent increase of INaL may provide a novel mechanism contributing to a well-known Bowditch-Treppe effect postulating that an increase in the heart rate leads to an increase in the force of contraction (inotropic effect of rate). This fundamental law defines the net heart performance when demand for blood pumping increases. Higher cell Ca2+ levels, which are required for stronger contractions, can be preserved with increased Na+ influx via INaL during AP plateau by the Ca2+-dependent positive feedback mechanism discovered in the present study. The contribution of this mechanism is thus linked to a higher cellular Na+ concentration, which, in turn, decreases Ca2+ extrusion by Na+/Ca2+ exchanger (NCX).

Significance of INaL and Its Modulation by Ca2+ in Heart Failure

Chronic HF is characterized by both cardiac arrhythmias and reduced contractility, and INaL likely contributes to these problems, because partial blockade of INaL normalizes AP repolarization, inhibits EADs, and greatly improves contractility of failing VCs (16, 21, 46). INaL decay is significantly slowed and INaL density is increased in VCs of chronic human and canine HF (21, 46). The importance of INaL relative to other currents can be greater in HF, because K+ current and INaT are downregulated in failing myocardium (7, 19, 48, 54). For example, after 500 ms of membrane depolarization INaL becomes comparable with late Ca2+ current (ICaL) (INaL = 0.68 x ICaL) in canine failing VCs (46). Thus INaL greatly contributes to the ion current balance on the AP plateau (36, 45) as well as to cell Na+ influx (21), and thereby indirectly to cell Ca2+ regulation via NCX (Refs. 17, 41; see Ref. 5 for review).

The increased net INaL provides additional Na+ influx in failing cardiomyocytes by ~54–58% (21, 23), and the total INaL-mediated Na+ influx is almost equal to that mediated by INaT (23). The rise of INaL-related Na+ influx can explain, at least in part, the increased cell Na+ load documented in HF (4, 10, 30). The INaL contribution might be even larger, because INaL was always measured at low [Ca2+]i in the previous studies, but the present study discovered strong positive INaL modulation by [Ca2+]i (Fig. 1, Table 2). The effect of Ca2+ modulation was observed within the voltage range of AP plateau (Fig. 2B). Accordingly, the Na+ overload, in turn, is expected to shift the operation of the NCX [upregulated in HF (37)] during the AP plateau from the forward to the reverse mode of operation, leading to a higher cell Ca2+ load.

This Na+-driven higher Ca2+ load limits depression of systolic function of failing VCs (5) and thus can be considered as an intrinsic, adaptive, digitalis-like effect with all the corresponding risks and benefits. Interestingly, a large burst mode component of the Na+ current has been identified in post-myocardial infarction-remodeled myocytes (13), i.e., in the transitional period from an infarction to HF. The increased INaL may indeed serve as an initial adaptation mechanism to match an increased contractility demand for the surviving VCs.

On the other hand, Ca2+ overload has substantial adverse effects in failing hearts including 1) arrhythmias via increased probability of spontaneous Ca2+ release and delayed afterdepolarizations and 2) failing contractility due to diastolic dysfunction, especially at high heart rates. In addition to the problems of Ca2+ overload, the increased INaL prolongs AP plateau and leads to beat-to-beat variability of AP duration and EADs (16, 21, 41, 46), which may also cause arrhythmias (40). Interestingly, decay kinetics of ICaL and INaL are regulated in the opposite ways by Ca2+, i.e., accelerating (2) and slowing (this study), respectively. Because failing VCs are loaded with Ca2+, the mechanism of the Ca2+-induced INaL increase may thus provide a positive feedback mechanism to increase further Na+ influx and facilitate cell Ca2+ overload, thus exaggerating further the aforementioned HF-related problems.

Ca2+ Modulates Gating Modes of Late Na+ Channel Openings

We interpret our results in terms of channel gating based on previous single-channel studies and numerical modeling of INaL (see Interpretation of Experimental Data in Terms of Ca2+ Modulation of INaL Gating Modes and INaL-Mediated Na+ Influx). CaMKII is expected to slow inactivation kinetics of both BM and LSM (to a greater extent in HF) and to decrease the number of BM channels; Ca2+ (independently of CaM) increases the number of LSM channels specifically in failing myocytes. The effect of Ca2+ on bursts is in line with the fact that cytoskeleton integrity and lysophosphatidylcholine affect the burst mode of Na+ channel openings (43, 47). These modulators are Ca2+ dependent: the cytoskeleton is affected by calpain and caspase (see Molecular Mechanisms of INaL Modulation by Ca2+-CaM-CaMKII Pathways), and lysophosphatidylcholine is a substrate for protein kinase C (27). We found that instant Ca2+ modulation in failing cells is much greater at the beginning of depolarization (shaded area in Fig. 7C), indicating that Ca2+ regulates not only Na+ total influx via INaL but also complex INaL dynamics, contributed by different gating modes of late channel openings. More specifically, while the integral of the burst mode (i.e., QBM) remains almost the same at various [Ca2+]i (Table 4), the dynamic contribution of bursts into INaL is greatly modulated by [Ca2+]i in HF. Since bursts are active early after depolarization onset (45), this modulation, in turn, could be important for early repolarization phase (the "notch" phase), which is altered in HF [in our canine model and in humans (16, 46)] and determines the synchronicity of the Ca2+ release (see Ref. 35 for review), and thereby the quality of excitation-contraction coupling.

Targeting INaL is a Novel Approach in Prevention of Ventricular Arrhythmia and Diastolic Dysfunction in HF

In light of the discovery that amiodarone effectively and selectively reduces INaL compared with INaT in human failing myocardium, we previously suggested (20) a new therapeutic strategy for cardioprotection by a selective inhibition of INaL. Our most recent finding (41) in failing VCs was that ranolazine blocks INaL even more selectively than amiodarone, and this new strategy of INaL blockade is now seriously considered by clinical cardiologists in future therapies to prevent cardiac arrhythmias and Ca2+ overload both in HF and cardiac ischemia (8). The results of the present study indicate that INaL can also be targeted indirectly via Ca2+ signaling. More specifically, because CaMKII was found to be upregulated in human HF (see Ref. 53 for review), INaL decay can potentially be accelerated by CaMKII inhibition as well as by intracellular Ca2+ buffering, or by prevention of CaM binding.

Study Limitations

Although the 1 µM [Ca2+]i used in the study is high (but still within a physiological range for VCs), its effect on INaL can be exaggerated because of a prolonged influence of Ca2+ at the steady state that is different from dynamic Ca2+ variations in intact beating cells. Studies of the dynamic modulation of INaL by Ca2+ (including numerical modeling of AP in HF) as well as of detailed molecular mechanisms of the modulation merit further consideration. Although we suggest INaL and its Ca2+ regulation as potential targets for cardioprotection, this should be considered with caution because INaL inhibition can decrease systolic function and in some cases can be proarrhythmic, when the INaL-mediated AP prolongation is associated with an antireentry effect.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grants HL-53819 and HL-074238 (A. Undrovinas) and by American Heart Association Grant-in-Aid 0350472Z (A. Undrovinas).


   ACKNOWLEDGMENTS
 
Present address of V. A. Maltsev: Gerontology Research Center, National Institute on Aging, NIH, 5600 Nathan Shock Drive, Baltimore, MD 21224.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Undrovinas, Henry Ford Hosp., Cardiovascular Research, Education & Research Bldg. Rm. 4015, 2799 West Grand Blvd., Detroit, MI 48202-2689 (e-mail: aundrov1{at}hfhs.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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