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Am J Physiol Heart Circ Physiol 283: H1157-H1168, 2002. First published May 2, 2002; doi:10.1152/ajpheart.00518.2001
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Vol. 283, Issue 3, H1157-H1168, September 2002

Myocardial infarction in rat eliminates regional heterogeneity of AP profiles, Ito K+ currents, and [Ca2+]i transients

Roger Kaprielian3, Rajan Sah1, Tin Nguyen1, Alan D. Wickenden1, and Peter H. Backx1,2

1 Heart and Stroke Richard Lewar Centre and Departments of Physiology and Medicine, University of Toronto; 2 Division of Cardiology, University Health Network, Toronto, Ontario, Canada M5G 2C4; and 3 Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129-0060


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Transient outward K+ current density (Ito) has been shown to vary between different regions of the normal myocardium and to be reduced in heart disease. In this study, we measured regional changes in action potential duration (APD), Ito, and intracellular Ca2+ concentration ([Ca2+]i) transients of ventricular myocytes derived from the right ventricular free wall (RVW) and interventricular septum (SEP) 8 wk after myocardial infarction (MI). At +40 mV, Ito density in sham-operated hearts was significantly higher (P < 0.01) in the RVW (15.0 ± 0.8 pA/pF, n = 47) compared with the SEP (7.0 ± 1.1 pA/pF, n = 18). After MI, Ito density was not reduced in SEP myocytes but was reduced (P < 0.01) in RVW myocytes (8.7 ± 1.0 pA/pF, n = 26) to levels indistinguishable from post-MI SEP myocytes. These changes in Ito density correlated with Kv4.2 (but not Kv4.3) protein expression. By contrast, Kv1.4 expression was significantly higher in the RVW compared with the SEP and increased significantly after MI in RVW. APD measured at 50% or 90% repolarization was prolonged, whereas peak [Ca2+]i transients amplitude was higher in the SEP compared with the RVW in sham myocytes. These regional differences in APD and [Ca2+]i transients were eliminated by MI. Our results demonstrate that the significant regional differences in Ito density, APD, and [Ca2+]i between RVW and SEP are linked to a variation in Kv4.2 expression, which largely disappears after MI.

right ventricle; septum; heart disease; contraction


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THERE ARE MARKED DIFFERENCES in the action potential duration (APD) in different regions of the mammalian ventricle (4, 15, 18, 39, 58). This electrical heterogeneity in normal myocardium correlates with regional differences in the Ca2+-independent transient outward K+ current density (Ito) (5, 15, 18, 34, 35, 43) as well as in gene expression of K+ channels (8, 16, 58). APD prolongation and reductions in Ito density occur in rat heart after left anterior descending coronary artery ligation (2, 43, 58), aortic banding (5, 22, 55), as well as after treatment with either catecholamine (11) or monocrotaline (32, 33). Depending on the model, the extent of Ito density changes in disease may not be uniform throughout the ventricle (2, 5, 11, 22, 55), thereby leading to possible losses of electrical heterogeneity and increased susceptibility to arrhythmias (3).

Aside from electrical heterogeneity, regional differences in other myocardial properties also exist. For example, systolic intracellular Ca2+ concentration ([Ca2+]i) is higher in the endocardium than in the epicardium (19, 54), consistent with the notion that the endocardium may play a more important role in contraction compared with the epicardium. The underlying basis for the regional differences in contraction is currently unknown but may be related to a heterogeneous transmural expression of Ca2+ handling proteins (26, 31). Alternatively, APD might also play a key role because the action potential profile is an important determinant of the inotropic state of the heart in both normal (7, 42, 54) and hypertrophied (10, 30) rat ventricular myocytes.

In this study, we examined the regional changes in APD, Ito density, and [Ca2+]i transient magnitude and MI and correlated these differences with the expression of K+ channel genes encoding for Ito (i.e., Kv1.4, Kv4.2, and Kv4.3). Our results show that gradients in APD, Ito density, Kv4.2 expression, and peak [Ca2+]i exist between the right ventricular free wall (RVW) and interventricular septum (SEP), and these differences are largely eliminated after myocardial infarction (MI).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Induction of MI and isolation of ventricular myocytes. Male Lewis Brown Norway rats (Harlan; Indianapolis, IN) weighing 220-250 g underwent left anterior coronary artery ligation, as described previously (40). Sham-operated rats were handled in the same manner except the coronary artery was not ligated. After the surgical procedure, the rats were housed in a climate-controlled environment at an ambient temperature of 21°C with 12:12-h light/dark cycle. Water and standard Purina rat chow were given ad libitum. Eight weeks after surgery, the animals were euthanized and the myocytes isolated as described in our earlier study (30). After enzymatic digestion, the RVW and SEP were carefully dissected from sham-operated and post-MI rat hearts. In a few experiments, left ventricular epicardial cells were also isolated from the left ventricular free wall of sham-operated hearts. All ventricular tissues were minced, triturated, and stored in a Kraftbrühe solution containing 50 mg/ml bovine serum albumin. Infarct size was assessed by dissection of the left ventricular free wall and measurement of the fraction of the left ventricular free wall, which was replaced with fibrous tissue (30).

Single cell physiological studies. Current densities and action potentials were recorded using the whole cell patch-clamp technique with an amplifier (Axopatch 200A, Axon Instruments). Microelectrodes were prepared with the use of a 1.5-mm thin-walled borosilicate glass (World Precision Instruments, Sarasota, FL). After the pipettes were polished, the typical resistances were 3-4 MOmega when filled with the pipette solutions. Series resistance compensation was typically ~75-85%. After membrane rupture, cell capacitance was estimated by integrating the area of the capacitance transients after a 5-mV step from a holding potential of -70 mV. The measured currents were divided by the cell capacitance to normalize currents for cell size.

To measure L-type Ca2+ currents (ICa,L), voltage-clamp recordings were made in myocytes superfused with a modified Tyrode solution composed of (in mmol/l) 140 NaCl, 1 MgCl2, 10 HEPES, 4 CsCl, 1 CaCl2, and 10 D-glucose, pH adjusted to 7.4 with NaOH. The pipette solution for ICa,L recordings contained (in mmol/l) 150 CsCl, 10 HEPES, 1 MgCl2, 5 EGTA, and 5 MgATP, pH adjusted to 7.2 with CsOH. ICa,L was estimated as the Cd2+-sensitive current (0.3 mmol/l CdCl2). For K+ currents and action potential recordings, myocytes were superfused with the same modified Tyrode solution, except that CsCl was replaced with KCl. CdCl2 (0.3 mmol/l) was routinely added to block ICa,L when recording K+ currents. Pipette solutions for K+ current and action potential recordings contained (in mmol/l) 130 K-aspartate, 20 KCl, 10 HEPES, 1 MgCl2, 5 NaCl, 5 EGTA, and 5 MgATP, pH adjusted to 7.2 with Trizma base. Action potentials were corrected, but voltage-clamp recordings were not corrected for the measured liquid junction potential (-8.6 mV) between the pipette and the bath solution.

[Ca2+]i was recorded with the same pipette and extracellular solutions used in the K+ current measurements except that intracellular EGTA was replaced with 75 µM fura 2 pentopotassium salt and CdCl2 was absent. Fluorescence measurements were performed using light from a 75-W xenon lamp (Oriel; Stratford, CT) passed through band-pass filters (Omega Optical) centered at 340 or 380 nm via an epifluorescence port and a ×40 Fluor objective microscope (Nikon; Tokyo, Japan). The emitted fluorescence was collected by the objective and passed through a 510-nm filter to a photomultiplier detection unit (Hamamatsu; Bridgewater, NJ). The photomultiplier output was filtered at 100 Hz and stored in the computer for later analysis. In our experimental setup, background fluorescence was measured at both wavelengths after a gigaohm seal formation and before rupturing the cell membrane. The ratio of the background subtracted fluorescent signal (340/380) was used to estimate [Ca2+]i using the equation (24)
[Ca<SUP>2+</SUP>]<SUB>i</SUB> = <IT>K</IT>′<SUB>d</SUB>·(R − R<SUB>min</SUB>)/(R<SUB>max</SUB> − R)
where K'd is the apparent dissociation constant and R is the ratio of the background-subtracted fluorescence at 340-nm excitation to that at 380-nm excitation. The effective K'd was 1.99 µM, Rmax was 6.43, and Rmin was 0.20 in our studies. [Ca2+]i was estimated in current-clamp and voltage-clamp conditions, as previously described (30). In voltage-clamp studies, myocytes were depolarized to +10 mV (100 ms in duration) from a holding potential of -80 mV. All experiments were performed at room temperature (19-21°C) within 18 h of cell isolation. [Ca2+]i measurements were always made under steady-state conditions by stimulating the myocyte at 0.25 Hz and recording fluorescence at both wavelengths between the 17th and 20th beat.

Ribonuclease protection assay. Immediately after removing the hearts, the right ventricle and septum were dissected (5 rats per group), rinsed briefly in 0.9% NaCl (wt/vol) and snap-frozen in liquid nitrogen. Ventricular tissue was powdered and RNA extracted by the one-step acid guanidium phenol method. The concentration of RNA was measured spectrophotometrically and confirmed by agarose gel electrophoresis. RNase protection assays were performed as previously described (30, 59).

Western blot analysis. Rat hearts were quickly removed and retrogradely perfused with Tyrode solution for 20 s. Immediately after this procedure, the RVW and SEP were dissected (4 rats per group) and stored at -80°C before isolation of membrane proteins, as previously described (58). Total heart protein (50-100 µg) and 10-20 µg of total brain protein were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. After transfer, the membrane was rinsed with Tris-buffered saline (TBS; 150 mM NaCl and 20 mM Tris, pH 7.5). Blots were blocked with 10% (Carnation) instant milk powder in TBS for 1 h and probed with anti-Kv4.2, Kv4.3, and Kv1.4 antibodies diluted in 3% milk-TBS overnight at 4°C. After the membrane was washed with TBS to remove excess primary antibody, blots were incubated with secondary antibody (donkey anti-rabbit-IgG conjugated to horseradish peroxidase, Amersham) in blocking buffer for 1 h at room temperature. The membrane was washed again with TBS containing 0.05% Tween 20 and 1% Triton X-100, and developed by enhanced chemiluminescence (ECL reagent, Amersham). We checked the gel loading by staining total proteins with Ponceau S, whereas molecular weights were determined using prestained markers (Kaleidoscope, Bio-Rad). Western blots were repeated 2-3 times per sample. Protein abundance was quantified by integrated densitometry of the bands (GS670 Imaging Densitometer, Bio-Rad). The integrated density of the protein samples was normalized by the corresponding value in the RVW of sham hearts for comparison.

Statistical analysis and curve fitting. All data are expressed as means ± SE. Steady-state activation (g) and inactivation (hproportional to ) data were fit with the following Boltzmann functions
g=g<SUB>max</SUB><IT>/</IT>{1<IT>′+</IT>exp[<IT>−</IT>(<IT>V−V</IT><SUB>½</SUB>)<IT>/k</IT>]}

h<SUB>∝</SUB>=1/{1+exp[(<IT>V−V</IT><SUB>½</SUB>)<IT>/k</IT>]}
where V is the step or conditioning potential, V1/2 is the midpoint of the function, and k is the slope factor. Biexponential functions were used to fit recovery from inactivation data using the equation below
I/I<SUB>to</SUB> = 100 − [<IT>A</IT><SUB>fast</SUB> exp(−<IT>x/&tgr;</IT><SUB>fast</SUB>)]

<IT>+</IT>[(100<IT>−A</IT><SUB>fast</SUB>) × exp(−<IT>x/&tgr;</IT><SUB>slow</SUB>)]
where I/Ito is the fraction of current recovered, Afast and tau fast are the amplitude and time constant for the fast component of recovery, 100 - Afast (i.e., Aslow) and tau slow represent the amplitude and time constant for the slow component, and x is the time spent at the recovery potential. Correlation between APD and [Ca2+]i was performed by linear regression. Statistical comparisons were made with one-way analysis of variance (ANOVA) using the SPSS program (version 7.0 for Windows, SPSS). When ANOVA showed statistical significance with the use of the F test, intergroup comparisons were made with the Student-Newman-Keuls procedure. A value of P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The effects of MI in rat hearts were assessed 8 wk after left anterior descending coronary artery ligation. Left ventricular free wall infarct sizes of hearts used in our studies were 47.5 ± 2.6% (range 34.4-64.0%). Hearts with infarct sizes <30% were not included in our analysis because small infarcts are not associated with significant hemodynamic changes (30, 47). As summarized in Table 1, MI was associated with global and cellular hypertrophy. Specifically, tissue weight-to-body weight ratios and myocyte capacitance were increased in both the RVW and SEP after MI (Table 1).

                              
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  		Table 1.   Changes associated with MI in RVW and SEP myocytes

Regional changes in membrane potential after MI. Figure 1 shows representative action potentials measured in RVW and SEP myocytes derived from sham-operated (Fig. 1A) and post-MI (Fig. 1B) hearts. For sham-operated hearts, 50% and 90% APD (APD50 and APD90, respectively) in RVW myocytes (APD50 = 4.8 ± 0.6 ms, n = 31, and APD90 = 28.9 ± 2.7 ms, n = 31) were shorter (P < 0.01) in duration than in SEP myocytes (APD50 = 9.7 ± 1.1 ms, n = 26, and APD90 = 49.4 ± 4.5 ms, n = 26). In SEP myocytes, APD50 was unchanged (12.2 ± 1.3 ms, n = 25, P = 0.1), whereas APD90 was slightly increased (71.2 ± 7.0 ms, n = 25, P < 0.05) after MI. In RVW myocytes both APD50 (13.2 ± 1.6 ms, n = 26, P < 0.05) and APD90 (70.9 ± 7.0, n = 26, P < 0.05) were increased by MI. More important, MI entirely eliminated differences in APD between RVW and SEP myocytes.


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  		Fig. 1.   Representative action potential traces in right ventricular free wall (RVW) and intraventricular septum (SEP) myocytes derived from sham (A) and postmyocardial infarcted (post-MI) (B) rats. Action potentials were elicited by a brief (5 ms) suprathreshold (2× threshold) current injection applied at 0.2 Hz. Recordings were made with 5 mM EGTA in the pipette. Frequency distribution at 50% action potential duration (APD50) is shown for myocytes derived from sham (C) and post-MI (D) hearts. The solid bars represent the results for RVW myocytes, whereas the open bars are for the SEP myocytes. The short bars in A and B indicate 0 mV.

Frequency histograms of APD50 for myocytes are summarized in Fig. 1C and reveal notable differences in the distribution of APD50 magnitudes between RVW (solid bars) and SEP (open bars) myocytes derived from sham-operated hearts. Indeed, for the sham group ~65% of the RVW myocytes had APD50 magnitudes <5 ms compared with <25% for SEP myocytes. A comparison of the distribution of APD50 magnitudes reveals that substantial changes occur in RVW, but not SEP, myocytes after MI. Consequently, the APD50 frequency histograms of RVW and SEP myocytes become very similar after MI with only 12% of RVW myocytes compared with 23% of SEP myocytes exhibiting APD50 values <5 ms. These results suggest that action potential profiles in RVW myocytes take on characteristics of SEP myocytes in response to MI. Similar patterns were observed for the APD90 magnitudes (data not shown). These findings are consistent with the APD changes observed previously after MI (55) and aortic stenosis (22) and demonstrate that MI leads to a loss of electrical heterogeneity of repolarization.

Despite regional differences of APD, no differences in resting membrane potential were observed between RVW myocytes and SEP myocytes. However, post-MI RVW myocytes were somewhat more depolarized (corrected for junction potentials) compared with sham RVW myocytes (sham, -84.1 ± 1.3 mV, n = 31; post-MI, -76.9 ± 1.1 mV, n = 26, P < 0.01) (see Table 1).

Ito and the sustained current. To investigate the basis for the loss of electrical heterogeneity between the RVW and SEP after MI, we initially focused on voltage-dependent K+ currents because our previous studies revealed that K+ channel expression is altered in this heart disease (30). Figure 2A shows that Ito density, measured in response to depolarizing steps to +40 mV, was larger in RVW myocytes compared with SEP myocytes [15.0 ± 0.8 pA/pF (n = 47) vs. 7.0 ± 0.9 pA/pF (n = 18)]. After MI, Ito density was reduced far more in RVW myocytes to 8.7 ± 1.0 pA/pF (n = 26, P < 0.01) than in SEP myocytes (5.1 ± 0.6 pA/pF, n = 20) compared to sham.


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  		Fig. 2.   Representative normalized traces of the transient outward current (Ito) and sustained current (Isus) in RVW and SEP myocytes derived from sham (A) and post-MI (B) rats elicited by 500-ms voltage steps over the range of -30 to +70 mV in +10-mV increments from a holding potential of -80 mV (see inset). Arrows indicate 0 pA/pF. Ito in RVW myocytes (C) and SEP myocytes (D) and Isus in RVW myocytes (E) and SEP myocytes (F) were normalized to membrane capacitance and plotted against the test potential for sham and post-MI groups. G and H: frequency distribution of Ito density evaluated at +40 mV for RVW myocytes and SEP myocytes, respectively. Dagger P < 0.05, RVW sham myocytes vs. RVW post-MI myocytes; *P < 0.05, SEP sham myocytes vs. SEP post-MI myocytes. Myocytes were depolarized every 5 s.

As summarized in Table 2, the variation in Ito density at +40 mV between the groups was not related to measurable differences in activation and inactivation gating parameters (i.e., V1/2 and k), suggesting that differences in the maximal conductance of Ito (Gmax) exist. Indeed, Gmax was reduced (P < 0.05) from 138.3 ± 7.6 pS/pF (n = 47) to 78.1 ± 8.1 pS/pF (n = 26) in RVW myocytes, whereas Gmax was not changed (P = 0.12) in the SEP (sham, 67.8 ± 3.6 pS/pF, n = 18, and post-MI, 48.6 ± 5.9 pS/pF, n = 20) (Table 2).

                              
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  		Table 2.   Regional differences in characteristics and biophysical properties of transient outward current in RVW and SEP myocytes after MI

These regional changes in Ito density after MI mirrored the changes in APD. Inspection of the Ito density frequency histograms in Fig. 2G reveals that only ~17% of the RVW myocytes (solid bars) compared with 83% of SEP myocytes (open bars) exhibited current densities <9 pA/pF in sham-operated hearts. After MI, 67% of the RVW myocytes and 100% for SEP myocytes (Fig. 2H) displayed Ito densities <9 pA/pF, suggesting again a convergence of cell populations after MI.

Figure 3 shows typical recovery from inactivation traces from sham-operated (Fig. 3A) and post-MI (Fig. 3B) hearts using a double-pulse protocol. Table 2 summarizes the mean data for recovery kinetics after fitting the data using a biexponential function. No differences were uncovered in the magnitudes of tau fast or tau slow between the different groups. In sham-operated hearts, the fast recovering component (i.e., tau fast) accounted for virtually all of the recovering current in RVW myocytes versus in SEP myocytes where it accounts for only 91.4 ± 0.9% (n = 14) of the recovering current. In post-MI hearts, tau fast accounted for only 92.7 ± 0.8% (n = 14) of the current in RVW myocytes, which was not statistically different (P = 0.2) from SEP myocytes (92.4 ± 0.9%, n = 17).


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  		Fig. 3.   Representative recovery properties of Ito in RVW and SEP myocytes derived from sham (A) and post-MI (B) rats using a two-pulse protocol with two identical depolarizing pulses to +60 mV separated by variable (recovery) intervals spanning from 10 ms to 10,000 ms. This protocol was repeated every 15 s. Plot of peak amplitude of Ito as a function of the recovery interval in myocytes derived from sham (C) and post-MI (D) rats, respectively. The data in these figures were fit to a biexponential function (see METHODS) in RVW myocytes () and SEP myocytes (open circle ).

To investigate the molecular basis for the changes in Ito density, we examined the expression of Kv4.2, Kv4.3, and Kv1.4alpha subunits, which represent candidate voltage-dependent K+ channels encoding for Ito-like currents previously shown to be expressed in rat heart (16). Figure 4 shows typical Western blots for Kv4.2, Kv4.3, and Kv1.4. In sham-operated hearts, Kv4.2 expression levels were significantly lower in SEP (0.77 ± 0.05, n = 4, P < 0.01) compared with RVW. MI induced a more than twofold reduction (0.46 ± 0.11, n = 4, P < 0.01) in Kv4.2 protein within the RVW with more modest reductions seen in SEP (0.56 ± 0.05, n = 4, P < 0.01), thereby eliminating the differences in Kv4.2 protein between the RVW and SEP. Unlike Kv4.2, Kv4.3 protein expression did not differ between the RVW and SEP myocytes and was not changed after MI. The regional differences in Kv4.2 and Kv4.3 channel expression are identical to that recently reported in normal rat heart (45, 58).


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  		Fig. 4.   Representative comparison of candidate K+ channel alpha -subunits encoding the transient outward current in the RVW and SEP after MI. Each blot shows side-by-side Western blots for Kv4.2 (A), Kv4.3 (B), and Kv1.4 (C) in the RVW and SEP derived from sham and post-MI rats. The bars show mean changes in Kvalpha subunits in tissues derived from RVW sham (solid bars), SEP sham (open bars), RVW post-MI (gray bars), and SEP post-MI (hatched bars). Values have been normalized to sham RVW samples and are expressed as percentage units. dagger P < 0.05 for RVW sham myocytes vs. RVW post-MI myocytes; Dagger P < 0.05 SEP sham myocytes vs. SEP post-MI myocytes; §P < 0.05 RVW sham myocytes vs. SEP sham myocytes.

The relative amount of Kv1.4 protein expression in SEP myocytes (i.e., 0.60 ± 0.04, n = 4) was significantly (P < 0.05) different than in RVW myocytes. After MI, Kv1.4 protein increased significantly in both RVW (2.09 ± 0.4, n = 4, P < 0.05) and SEP (1.29 ± 0.39, n = 4, P < 0.05). The pattern of Kv1.4 channel expression changes correlated with the presence of a slow component in the recovery from inactivation, suggesting that Kv1.4 encodes for the slowly recovering component of Ito in rat myocytes (58).

Unlike Ito, no regional differences in Isus density, recorded at the end of a 500-ms depolarizing pulse, existed between RVW and SEP myocytes. After MI, Isus density at +40 mV was reduced by similar extents in SEP (sham, 6.6 ± 0.4 pA/pF, n = 18, and post-MI, 4.7 ± 0.4 pA/pF, n = 20, P < 0.05) and RVW (sham, 7.5 ± 0.6 pA/pF, n = 47, and post-MI, 5.7 ± 0.5 pA/pF, n = 23, P > 0.05) myocytes. These results are consistent with a previous study showing that Isus is significantly reduced in the left ventricular endocardium after short-term infarction (61). The molecular correlates of Isus remains uncertain, but three candidate K+ channel genes (Kv1.2, Kv1.5, and Kv2.1) are expressed in the rat heart (16, 51, 53). In the right ventricle, RNase protection assays (data not shown) revealed that the percentage reductions in mRNA levels after MI did not reach significance for Kv1.2, (23.7 ± 15.3%, n = 5, P = 0.19), Kv1.5 (22.4 ± 11.4%, n = 5, P = 0.11) or Kv2.1 (27.0 ± 9.8%, n = 5, P = 0.06) genes. In SEP, mRNA levels for Kv1.2 and Kv2.1 were decreased by 39.7 ± 4.5% (n = 5, P < 0.01) and 22.2 ± 7.5% (n = 5, P < 0.05), respectively, whereas Kv1.5 mRNA levels were unchanged (n = 5, 18.7 ± 11.8, P = 0.17) after MI. Collectively, these RNase protection assay results suggest that there is a general reduction in the expression of candidate genes encoding for Isus in both ventricles. The relevance of these observations will require further investigation.

The inward rectifier and ICa,L. The differences in APD between the groups might also be associated with variations in other currents. Unlike the differences observed in Ito density, inward rectifier current (IK1) density was not different between RVW and SEP myocytes. Similar to Ito, no significant change in IK1 density (evaluated at -130 mV) was observed in SEP myocytes, whereas IK1 density was decreased (P < 0.05) in RVW myocytes (sham, -16.0 ± 0.6 pA/pF, n = 28, and post-MI, -12.2 ± 1.0 pA/pF, n = 20). These differences in IK1 density assessed at -130 mV may explain the depolarized resting membrane potentials of post-MI RVW myocytes compared with RVW sham myocytes provided, of course, these differences reflect corresponding changes at more positive potentials above the equilibrium potential for K+. However, we cannot rule out changes in other currents, such as chloride, the electrogenic Na+-K+ pump currents, or others that can also affect resting membrane potential under our recording conditions.

ICa,L density recorded at 0 mV also did not differ between RVW and SEP myocytes and did not change after MI in the RVW (sham, -6.0 ± 0.4 pA/pF, n = 15, and post-MI, -5.5 ± 0.5 pA/pF, n = 14, P = 0.59) or SEP (sham, -5.8 ± 0.4 pA/pF, n = 16, and post-MI, -5.2 ± 0.3 pA/pF, n = 16, P = 0.59). There was no difference in the steady-state or kinetic gating properties of ICa,L between any of the groups (data not shown).

Action potentials and [Ca2+]i transients. Previous studies have shown that changes in Ito density and the corresponding changes in APD correlate with alterations in Ca2+ cycling. Figure 5 shows simultaneous records of action potentials and [Ca2+]i (under current-clamp conditions) in RVW and SEP myocytes derived from sham (Fig. 5A) and post-MI (Fig. 5B) rats under Ca2+ buffer conditions (i.e., 75 µM fura 2). As was the case with high Ca2+-buffering conditions (i.e., 5 mM EGTA), there were significant differences in APD50 and APD90 between RVW and SEP myocytes. After MI, APD50 (sham, 6.5 ± 1.4 ms, n = 17, and post-MI, 37.2 ± 5.8 ms, n = 16, P < 0.01) and ADP90 (sham, 72.3 ± 13.8 ms, and post-MI, 673.6 ± 89.9 ms, P < 0.05) were prolonged in RVW myocytes. As in studies with high EGTA, APD90 (sham, 154.2 ± 23.4 ms and post-MI, 592.3 ± 74.3 ms, P < 0.05) was prolonged in SEP myocytes after MI without changes in APD50 (sham, 24.2 ± 3.4 ms, n = 15 and post-MI, 33.3 ± 3.7 ms, n = 20, P = 0.09). It is important to note that the APD50 and especially the APD90 were generally prolonged in all groups when recorded in the presence of Ca2+ transients when low intracellular Ca2+ buffering was used compared to high Ca2+-buffering conditions.


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  		Fig. 5.   Representative action potentials and intracellular Ca2+ concentration ([Ca2+]i) derived from sham and post-MI rats. Action potentials (top traces) and their associated Ca2+ (bottom traces) transients in RVW and SEP myocytes are derived from sham (A) and post-MI (B) rats. The short bars in A indicate 0 mV, and the arrows in B indicate 0 nM [Ca2+]i. C: mean values for systolic [Ca2+]i in myocytes derived from RVW sham, SEP sham, RVW post-MI, and SEP post-MI. dagger P < 0.05 RVW sham myocytes vs. RVW post-MI myocytes; Dagger P < 0.05 SEP sham myocytes vs. SEP post-MI myocytes; §P < 0.05 RVW sham myocytes vs. SEP sham myocytes.

Associated with the observed differences in APD, the mean peak systolic [Ca2+]i was higher (P < 0.01) in the SEP (687.4 ± 84.9 nmol/l, n = 15) compared with the RVW (381.8 ± 44.7 nmol/l, n = 17). After MI, peak systolic [Ca2+]i levels were elevated (P < 0.01) more than twofold in RVW myocytes (1,056.4 ± 92.9 nmol/l, n = 16) compared with sham, with relatively smaller changes occurring in SEP (1,177.5 ± 127.5 nmol/l, n = 15) in response to MI. Remarkably, the peak systolic [Ca2+]i was not significantly different between RVW and SEP myocytes after MI, which correlated with the abolishment of regional differences in APD and Ito. Despite these regional differences in [Ca2+]i transient amplitudes and the changes that occur in response to MI, no differences in the time course of relaxation could be detected between the different groups (data not shown). By contrast to the [Ca2+]i transient amplitudes, diastolic [Ca2+]i did not differ between the groups studied (sham RVW, 81.3 ± 5.4 nmol/l, n = 17; sham SEP, 100.4 ± 8.8 nmol/l, n = 15; post-MI RVW, 93.5 ± 4.5 nmol/l, n = 16; post-MI SEP, 93.5 ± 10.5 nmol/l, n = 15), establishing that APD prolongation affects primarily peak [Ca2+]i transient amplitude (30).

To further explore whether factors other than membrane potential contribute to differences in systolic [Ca2+]i between the groups, [Ca2+]i transients were measured in response to step depolarizations to +10 mV for 100 ms from a holding potential of -80 mV. Peak systolic [Ca2+]i was not different in RVW and SEP myocytes derived from sham-operated (RVW, 648.9 ± 143.3 nmol/l, n = 10; SEP, 841 ± 127.7 nmol/l, n = 10) or post-MI (RVW, 736.8 ± 100.8 nmol/l, n = 10, SEP, 727.8 ± 124.3 nmol/l, n = 9) hearts. Similarly, diastolic [Ca2+]i did not differ between the various groups (RVW-sham, 91.7 ± 3.0 nmol/l, n = 10; SEP-sham, 101.9 ± 9.3 nmol/l, n = 10; RVW-MI: 84.7 ± 3.2 nmol/l, n = 10; SEP-MI, 82.0 ± 5.7 nmol/l, n = 9). In addition, when step depolarizations are used, no differences could be detected in the time course of the [Ca2+]i transient relaxation (data not shown). These results suggest that while many differences in Ca2+-handling proteins might exist between the groups, changes in action potential profile correlate with the observed alterations in [Ca2+]i transients.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our findings show that regional differences in APD exist in the normal rat myocardium with much shorter durations being observed in RVW versus SEP. These regional differences between RVW and SEP are very similar to those observed across the left ventricular free wall of the rat (15, 22, 48, 55). Regional gradients in APD have also been detected in other animal species (4, 18) and humans (37) and appear to be critical for orchestrating and coordinating ventricular repolarization, thereby minimizing life-threatening arrhythmias (3). The regional differences in APD across the left ventricular wall have been attributed to variations in Ito density (3, 5, 15, 22, 58). Our results show a strong correlation between APD and Ito density in both the RVW and SEP of rat myocardium. Clearly, variations in other currents could also conceivably contribute to regional differences in APD. However, in this study, no detectable differences were observed between RVW and SEP myocytes in Isus, IK1, or ICa,L, suggesting these currents are not responsible for APD heterogeneity as previously reported (2, 14). Nevertheless, possible differences in other currents to the regional variations in APD cannot be ruled out.

Our findings demonstrate high mRNA and protein expression of Kv4.2 in the RVW compared with the SEP, mirroring closely the Ito densities. Similar correlations between Kv4.x genes and Ito have been documented in humans (17, 28, 52) and other animal species (16, 17, 21, 25, 58, 59). By contrast, no regional differences in Kv4.3 expression were observed. These results suggest that varied expression of Kv4.2 channels might underlie the electrical heterogeneity observed in rat myocardium, as suggested previously (58). mRNA and protein expression of Kv1.4 was higher in the RVW versus SEP. The significance of the current produced by these channels remains unclear, but recent studies (25, 39, 58-60) have suggested that in rodents this channel encodes for the slow component of Ito (see below). If correct, the relative contribution of Kv1.4 channels to the total amplitude of Ito recorded at low rates of stimulation is relatively small in both the RVW and SEP even after MI.

Along with electrical heterogeneity, regional differences in mechanical function have also been previously reported (12, 29, 44). The basis for this has never been fully addressed. It is certainly conceivable that these regional differences in contractility are related (at least in part) to corresponding regional variations in APD. Indeed, APD prolongation in rat myocytes is associated with increased [Ca2+]i transient amplitudes (7, 10, 30, 54), particularly when APD prolongation is caused by reductions in Ito (46, 54). The link between changes in Ito density and [Ca2+]i in rat myocytes can be traced to the profound impact that the relatively fast activating and inactivating Ito has on the early repolarization period when ICa,L are also prominent (30, 46, 54). In our studies, peak [Ca2+]i was much lower in RVW versus SEP myocytes, which correlated strongly with higher Ito densities and shorter APDs. These findings suggest that variations in Ito underlie regional differences in myocardial contractility. Because the density of Ito in RVW rat myocytes is not significantly different from those recorded in epicardial myocytes from the left ventricle (16.2 ± 1.9 pA/pF at +40 mV, P = 0.5), our results can help explain the greater contractile force generated and larger contribution to pressure development by endocardial versus epicardial regions of the left ventricle (19, 54). Differences in peak magnitudes of the [Ca2+]i transients between the groups might also conceivably be related to variations in ICa,L density independent of changes in APD. However, ICa,L and [Ca2+]i transients did not differ between SEP and RVW in voltage-clamp experiments. Nevertheless, it is possible that regional differences in other proteins modulating Ca2+ handling might also vary between the different regions we studied.

Electrical and molecular changes after MI. Prolongation of APD, reductions in Ito density, decreased expression of Kv4.2 and increases in [Ca2+]i transient amplitudes have been reported previously in the rat myocardium for a number of models of cardiac hypertrophy (6, 9, 10, 30, 50). Although alterations in any number of currents could explain the changes in APD after infarction, only reductions in Ito without changes in ICa,L, Isus, or IK1 were observed in the two regions studied. We found that reductions in Ito density, the maximal conductance of Ito (Gmax) and Kv4.2 expression, as well as the degree of APD prolongation, were far larger in RVW myocytes after MI compared to SEP myocytes. These differential regional effects of MI eliminated the normal pattern of Kv4.2 expression and APD50 while reducing substantially the Ito density and Gmax between RVW and SEP. Similar effects on the normal regional electrical heterogeneity in rat hearts have been reported after MI (55) and other disease models (11, 22, 49). On the other hand, more uniform changes in the electrical properties between different regions in heart have also been reported previously (5, 13). These published discrepancies in the regional response of the myocardium may be related to differences in the type, severity or duration of the disease model under investigation.

One important feature of the electrical changes that occur after MI in our studies is the convergence of frequency distribution of APD and Ito densities between RVW and SEP myocytes. This suggests that myocytes become more uniform in their electrical properties after MI. This coincided with a complete loss of heterogeneity of the [Ca2+]i transients and the level of Kv4.2 expression between these regions. It will be interesting to see whether other cellular and biochemical properties of the myocardium also become more uniform after MI.

Along with the regional differences in Ito density, the recovery kinetics of Ito were also measurably slowed after MI (30) with a greater change in RVW myocytes versus SEP myocytes (Fig. 3). The significance of this observation is unclear. At the present time, the relative contribution of Kv1.4 versus Kv4.2 and Kv4.3 protein to adult Ito in cardiac myocytes remains unclear (36). However, as already mentioned, a series of recent studies has concluded that the fast inactivating/recovering component of Ito is produced by Kv4.2 and Kv4.3 genes while the slow inactivating/recovering portion of Ito is produced by Kv1.4 genes (20, 25, 27, 56, 58, 59). These conclusions suggest that the combination of marked downregulation of Kv4.2 coupled with a significant increase in Kv1.4 expression probably explain the emergence of a slowly recovering component of Ito after MI.

The loss of regional heterogeneity after MI has several important implications on the electrical and contractile properties of the whole heart. Previous studies have demonstrated that, as heart disease progresses toward heart failure, the amplitude of the [Ca2+]i decreases as a result of any number of potential molecular mechanisms, such as decreases in sarcoplasmic reticulum Ca2+-ATPase activity or expression (1, 6), uncoupling between ICa,L and the ryanodine receptors (23) or increases in Na+/Ca2+ exchange function (41). However, before the development of heart failure in rodents, [Ca2+]i transient amplitudes can actually be increased in hypertrophic heart disease as a result of APD prolongation (10, 30), thereby increasing contractility of the mechanically challenged heart. Our results demonstrate that after MI the [Ca2+]i transient amplitudes are increased far more in RVW myocytes than SEP myocytes mirroring precisely the alterations in APD, Ito density, and Kv4.2 expression. These observations suggest that the changes in electrical properties convert RVW myocytes to become more like the strongly contracting SEP myocytes (19, 54). This change in contractility of the RVW myocytes is expected to functionally compensate for the elevated work loads placed on the right heart after MI (30).

The normal electrical heterogeneity within the heart is expected to reduce the propensity of heart to develop (global) reentry type arrhythmias by synchronizing repolarization. Therefore, one possible consequence of the loss of the electrical heterogeneity between different regions of the heart, as observed in our study, might be a disturbance of the normal repolarization process, thereby favoring the onset of global reentry circuits in the myocardium. Whereas our experiments were limited to cellular studies, an earlier study (43) using the rat infarct model showed evidence for increased propensity of arrhythmias in this model. Action potential prolongation might also conceivably promote enhanced susceptibility to certain Ca2+-dependent triggered arrhythmias. Future studies will be necessary to assess whether and how this loss of electrical heterogeneity and action potential prolongation contributes to arrhythmias in this and other models of heart disease.

The extent to which our results are applicable to other regions of the heart or to other species remains uncertain. However, in the rat heart, the Ito density is identical between RVW myocytes and left ventricular epicardial myocytes (R. Kaprielian and P. H. Backx, unpublished observations), as reported previously (14), whereas the Ito density in SEP myocytes is similar to that observed in left ventricular endocardial myocytes (11, 49, 55, 61). Moreover, a recent study (55) in the rat aortic stenosis model showed a similar pattern of electrical changes between the epicardium and endocardium of the left ventricle between the epicardial RVW and the endocardial septum.

In conclusion, our studies demonstrate that differences in the expression of Kv4.2 channel protein between RVW myocytes and SEP myocytes in normal and diseased hearts account for regional changes in Ito density, APD, and [Ca2+]i transients. Our studies further suggest that the differences in Kv4.2 channel expression play a role in normal regional differences in myocardial contractility and changes in Kv4.2 expression might lead to changes in the regional differences in contractility and increase the susceptibility of the heart to arrhythmias.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the Tiffen Trust Fund and the Centre for Cardiovascular Research at the University of Toronto for providing funds for equipment purchases. The anti-Kv1.4, Kv4.2, Kv4.3 antibodies were kindly provided by Dr. Owen T. Jones in the Department of Pharmacology, University of Toronto.


    FOOTNOTES

This study was supported by a grant from the Canadian Institutes for Health Research (to P. H. Backx). R. Kaprielian holds a Medical Research Council Doctoral Research Award and P. H. Backx is a Career Investigator of the Heart and Stroke Foundation of Ontario.

Address for reprint requests and other correspondence: P. H. Backx, Heart and Stroke Richard Lewar Centre, Univ. of Toronto, Rm. 68, Fitzgerald Bldg., 150 College St., Toronto, Ontario, Canada M5G 2C4 (E-mail: p.backx{at}utoronto.ca).

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.

May 2, 2002;10.1152/ajpheart.00518.2001

Received 13 June 2001; accepted in final form 15 April 2002.


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