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Am J Physiol Heart Circ Physiol 283: H1225-H1236, 2002. First published May 16, 2002; doi:10.1152/ajpheart.00162.2001
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Vol. 283, Issue 3, H1225-H1236, September 2002

Normal contractions triggered by ICa,L in ventricular myocytes from rats with postinfarction CHF

Ivar Sjaastad1,2, Janny Bøkenes1, Fredrik Swift1, J. Andrew Wasserstrom3, and Ole M. Sejersted1

1 Institute for Experimental Medical Research, University of Oslo; 2 Department of Cardiology, Heart and Lung Center, Ullevaal University Hospital, 0407 Oslo, Norway; and 3 Cardiology Division, Department of Medicine, and the Feinberg Cardiovascular Research Institute, Northwestern University Medical School, Chicago, Illinois 60611


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Attenuated L-type Ca2+ current (ICa,L), or current-contraction gain have been proposed to explain impaired cardiac contractility in congestive heart failure (CHF). Six weeks after coronary artery ligation, which induced CHF, left ventricular myocytes from isoflurane-anesthetized rats were current or voltage clamped from -70 mV. In both cases, contraction and contractility were attenuated in CHF cells compared with cells from sham-operated rats when cells were only minimally dialyzed using high-resistance microelectrodes. With patch pipettes, cell dialysis caused attenuation of contractions in sham cells, but not CHF cells. Stepping from -50 mV, the following variables were not different between sham and CHF, respectively: peak ICa,L (4.5 ± 0.3 vs. 3.8 ± 0.3 pApF-1 at 23°C and 9.4 ± 0.5 vs. 8.4 ± 0.5 pApF-1 at 37°C), the bell-shaped voltage-contraction relationship in Cs+ solutions (fractional shortening, 15.2 ± 1.0% vs. 14.3 ± 0.7%, respectively, at 23°C and 7.5 ± 0.4% vs. 6.7 ± 0.5% at 37°C) and the sigmoidal voltage-contraction relationship in K+ solutions. Caffeine-induced Ca2+ release and sarcoplasmic reticulum Ca2+-ATPase-to-phospholamban ratio were not different. Thus CHF contractions triggered by ICa,L were normal, and the contractile deficit was only seen in undialyzed cardiomyocytes stimulated from -70 mV.

electrophysiology; myocardial infarction; sarcoplasmic reticulum Ca2+ ATPase; phospholamban; caffeine; L-type Ca2+ current; congestive heart failure


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ONE OF THE MOST COMMON FORMS of congestive heart failure (CHF) clinically evolves secondary to myocardial infarction (MI), and in this study CHF refers to postinfarction CHF unless otherwise noted (8). Echocardiographic studies on patients suffering from CHF have demonstrated impaired myocardial contractile function (5). Experiments on isolated cardiomyocytes in human CHF have offered alternative explanations for the contractile defects (24), and the controversies and varying results might in part be due to heterogeneous patient populations. For this reason, a rat model of MI has been used for numerous studies on excitation-contraction (EC) coupling (1, 7, 11, 15, 19, 21, 23, 25-27, 29, 33, 34, 36-40), but surprisingly few studies (1, 7, 11, 19, 21, 26, 29, 33) have focused on myocardial contractile abnormalities. Moreover, the studies have also been performed with various techniques. Most of the investigators have used electrical field stimulation of isolated cells, which gives no control of membrane potential (1, 7, 15, 19, 21, 25, 26, 29, 38, 39). There are few data on EC coupling in voltage-clamped cardiomyocytes from MI animals (11, 15, 21, 34), and sarcoplasmic reticulum (SR) Ca2+ load in CHF has not been assessed in any study under voltage clamp. Moreover, few studies (1, 15, 29, 34) have been performed on post-MI myocytes from animals that had clear evidence of CHF and not merely MI without any contractile deficit of the surviving myocardium. Others have not used sham-operated animals as controls (11). Differences in severity of symptoms, undocumented pathophysiology, variable SR load, and highly variable experimental conditions makes it difficult to conclude from existing studies that EC coupling or gain of EC coupling is really altered in postinfarction CHF. We therefore carried out the present study on a standardized rat model of severe CHF by using single cell current- and voltage-clamp techniques under various conditions with control of SR Ca2+ load.

In this rat model of CHF, we have previously found lower myocardial contractility and fractional shortening (FS) in vivo (33). Field-stimulated isolated cardiomyocytes showed slow and attenuated contraction (15). This contraction deficit may be due a priori to a defective trigger of contraction or to reduced myofilament Ca2+ sensitivity. However, because myofilament Ca2+ sensitivity has previously been found to be normal in this CHF model (15), the contraction deficit is most likely due to a defective trigger mechanism of contraction.

Therefore, the aim of the present study was to identify possible defects in EC coupling in CHF that corresponded to the reduction in contractility found in vivo (33). Using high-resistance pipettes, we show that the contractile defect was preserved in isolated cardiomyocytes under current-clamp conditions. However, we did not find a contractile defect under current-clamp conditions by using suction pipettes. Subsequently, we focused on L-type Ca2+ current (ICa,L)-triggered contraction with a voltage-clamp protocol. We used Cs+-containing solutions and protocols that would reveal a defect in contraction due to defective coupling between ICa,L and the ryanodine receptor (11, 12). Previous studies have often been performed at unphysiological temperatures. We therefore explored the possible influence of temperature on contraction defects. In addition to other factors, the availability of the different trigger mechanisms is dependent on the composition of the experimental solutions (20). For this reason, Cs+ was substituted with K+ in some of the present experiments. We did not find a contractile defect using voltage-clamp protocols and a postconditioning potential (VPC) of -50 mV. SR Ca2+-ATPase (SERCA2) to phospholamban ratio and SR Ca2+ load were used as parameters of SR function.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals were cared for according to the Norwegian Animal Welfare Act, which conforms to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996). Two animals were kept in each cage and housed in a temperature-regulated room on a 12:12-h day/night cycle. The animals were given access to food and water ad libitum.

Induction of MI and cell isolation. Male Wistar rats (Møllegaard Breeding and Research Center; Skensved, Denmark) weighing ~320 g were intubated and ventilated on a Zoovent ventilator (Triumph Technical Services; Milton Keynes, UK) with 68% N2O-29% O2-3% isoflurane (Abbott Laboratories). An extensive MI was induced by proximal ligation of the left coronary artery. The sham-operated animals (sham) were subjected to the same surgical procedure, but the coronary artery was not ligated. Six weeks later, the rats were anesthetized and ventilated with 2.2% isoflurane. Left ventricular pressures were measured with a 2-Fr micromanometer-tipped catheter (model SPR-407, Millar Instruments), and CHF rats were included in the study if left ventricular end-diastolic pressure (LVEDP) was >15 mmHg. The infarct comprised most of the left ventricular free wall, and echocardiography has previously demonstrated a substantial depression of myocardial function and CHF in these selected rats (33).

Left ventricular myocytes were isolated with the use of a Langendorff setup and perfused with a collagenase solution, as described by Holt and Christensen (14). Care was taken to exclude the scar tissue and adjacent fibrotic area. Isolated myocytes were placed on laminin-coated coverslips that were used as the floor of the experimental chamber on the stage of an inverted microscope (Diaphot-TMD, Nikon). The chamber (200 µl) was superfused with prewarmed solution at a rate of 4 ml/min. A custom-made rapid switcher allowed us to change the solution in the chamber within 500 ms. All of the experiments were performed at 23°C or 37°C, and only rod-shaped cells with preserved striation and no blebs were used.

Electrophysiological measurement and analysis. Current- and voltage-clamp experiments were performed using two methods. First, isolated cardiomyocytes were impaled with high-resistance microelectrodes (15-20 MOmega , filled with 2.7 M KCl) to minimize cell dialysis and buffering of intracellular Ca2+. Recordings were made with an Axoclamp-2A amplifier (Axon Instruments; Foster City, CA) in bridge mode for current-clamp experiments or in discontinuous single-electrode voltage-clamp mode (switching rate ~11 kHz). In current-clamp experiments, cells were stimulated at 1 Hz with current pulses 1.5× threshold. Membrane potential and contractions were recorded and analyzed at steady state. In the voltage-clamp experiments, test steps increasing by 10 mV were preceded by 10 conditioning pulses (50 ms) from -80 to 0 mV and delivered at 2 Hz, followed by the return to a VPC for 500 ms. Both current and membrane potential were recorded in these experiments. The switching circuit was monitored during the voltage clamp to ensure adequate settling time for accurate voltage measurement. In these experiments, we used a superfusing solution composed of (in mM) 4 KCl, 1 MgCl2, 145 NaCl2, 10 HEPES, 10 glucose, 1.8 CaCl2, 0.4 NaH2PO4, 1.0 4-aminopyridine, and 0.25 lidocaine, pH 7.4.

Second, the cells were patched using suction pipettes with a resistance of 2.5-4.5 MOmega . The pipettes were filled with either a K+-containing solution composed of (in mM) 120 K-aspartate, 25 KCl, 0.5 MgCl2, 6 NaCl, 4 K2-ATP, 0.06 EGTA, 10 HEPES, and 10 glucose, and pH adjusted to 7.2 with KOH, or a Cs+-containing solution composed of (in mM) 130 CsCl, 0.33 MgCl2, 4 Mg-ATP, 0.06 EGTA, 10 HEPES, and 20 tetraethylammonium, with pH adjusted to 7.20 with CsOH. In the K+ experiments, the superfusing solution contained (in mM) 5.4 KCl, 0.5 MgCl2, 140 NaCl, 5 HEPES, 11 glucose, 1.8 CaCl2, and 0.4 NaH2PO4, and pH adjusted to 7.4 with NaOH. In the Cs+ experiments, the cells were superfused with a solution containing (in mM) 20 CsCl, 1 MgCl2, 135 NaCl, 10 HEPES, 10 glucose, 1 4-aminopyridine, and 1 CaCl2, and pH adjusted to 7.40 with NaOH. We used an amplifier (Axopatch 200B, Axon Instruments) in current-clamp mode or in the continuous whole cell configuration, compensating for series resistance. Current-clamp experiments were carried out as described above. In voltage-clamp mode, the suction pipette experiments started with a train of four prepulses from -80 to 0 mV (50 ms) at 1 Hz and a VPC of -50 mV (1 s). Test potentials ranged from -50 to 80 mV (200 ms). Current and contraction were recorded in all experiments. Cs+ solutions were chosen to recreate the conditions of Gomez et al. (12) to allow direct comparisons between experimental models of hypertrophy and failure. K+ solutions were used to mimic more physiological experimental conditions that we have published previously (34). In all current-clamp experiments the cells were held at -70 mV (using 0 to 0.3 nA) to avoid influence of a variable resting membrane potential on cellular Ca2+ loading.

Voltage-clamp protocols were written in pCLAMP6 software (Axon Instruments). The data were digitized with a Digidata 1200B and sampled and stored on a computer. Current and contraction were analyzed with pCLAMP6 software. ICa,L was measured as the difference between peak inward and steady-state currents at the end of the 200-ms test step in the high-resistance pipette experiments and in the experiments using Cs+-containing solutions. Peak ICa,L was normalized to cell capacitance, and data are presented as current density. Cell capacitance was estimated by integrating the capacitive currents elicited by short hyperpolarizing steps. In some experiments, verapamil (20 µM) was added to block ICa,L. The interfering Na+ current was blocked with 150 µM lidocaine in voltage-clamp experiments. Unloaded cell shortening was measured using a charge-coupled device camera and a video edge detector (Crescent Electronics; Sandy, UT). Peak contraction was normalized to cell length and presented as FS. Time to peak contraction (TTP) and time to 50% relaxation (R50) were calculated. Maximal cell shortening velocity (SV) was normalized to cell length (cell length/s).

SR Ca2+ load was assessed in the Cs+ experiments by applying 10 mM caffeine to the myocytes for 10 s after the standard prepulse protocol and a VPC of -50 mV and measuring the contraction and integral of the inward Na+-Ca2+ exchange current (INa,Ca) that ensued.

Western blot analysis. Western blot analysis was performed on homogenate (phospholamban) and membrane fraction (SERCA2) of left ventricular tissue of CHF and sham hearts. After a standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 20 µg of protein per lane, the proteins were transferred to polyvinylidene difluoride membranes and blocked with dry milk in Tris-buffered saline with 0.1% Tween 20. Primary antibodies for phospholamban (mouse monoclonal antibody A1, Phosphoprotein Research Fluorescence; Leeds, UK) and SERCA2 (mouse monoclonal antibody, Affinity BioReagents; Golden, CO) were used. An anti-mouse IgG conjugated to horseradish peroxidase was used as secondary antibody. The immunoreactive bands were detected by the enhanced chemiluminescence method, whereas luminescence was detected by a LAS-1000 video system (Fujifilm; Stockholm, Sweden) and quantified with the Image Gauge software (Fujifilm).

Statistics. Data are expressed as group means ± SE unless noted otherwise. Comparisons between groups were done with analysis of variance (Statistica, Jandel), and P < 0.05 was considered significant. All protocols were performed on a minimum of three animals, and n represents the number of myocytes used.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal and cell characteristics. The infarcted rats showed signs of heart failure with tachypnoe, pleural effusion, and pulmonary congestion, all due to large anteriolateral infarctions. Heart and lung weights were increased and the hemodynamic data demonstrated diastolic and systolic cardiac failure (Table 1), with increased LVEDP and low left ventricular systolic pressure. We have previously demonstrated depressed in vivo cardiac function and failure in the CHF group (33). Cell length was 136 ± 3 µm in CHF and 118 ± 2 µm in sham (P < 0.001), and cell capacitance was 217 ± 14 pF in CHF (n = 20) and 176 ± 8 pF in sham (n = 25, P < 0.01).

                              
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  		Table 1.   Animal characteristics

Contractions triggered by action potentials at 37°C. The contractile defect present in vivo was examined in isolated cardiomyocytes under "physiological" conditions, i.e., the use of high-resistance electrodes and current clamp. Representative tracings from CHF and sham are shown in Fig. 1A, illustrating attenuated contraction magnitude and a low shortening velocity in CHF compared with sham. Specific characteristics of cell shortening were investigated further. TTP was 14% longer in CHF (P < 0.01) than in sham (Table 2, protocol 1). TTP increases with increasing FS, even when contractility is unchanged, and should therefore be normalized to FS. Relative TTP (RTTP) is the time the cell uses to shorten by 1%. RTTP was 71% longer in CHF than in sham (P < 0.01). SV was reduced by 41% in CHF compared with sham (P < 0.01). Also, R50 and R50 normalized to FS (RR50) were significantly slower in CHF compared with sham (Table 2, protocol 1). Thus, compared with sham, the undialyzed current-clamped CHF cells showed a contractile deficit akin to that seen in vivo. Action potentials were recorded, and 50% repolarization duration (APD50) was nearly doubled in CHF compared with sham (Fig. 1 and Table 2, protocol 1).


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  		Fig. 1.   Contractions triggered in current clamp at 37°C. A: representative action potential (top) and contraction (bottom) from current-clamped congestive heart failure (CHF) and sham cardiomyocytes using high-resistance electrodes (HRE; ~20 MOmega ). B: representative action potential (top) and contraction (bottom) from current-clamped CHF and sham cardiomyocytes using low-resistance electrode (LRE) suction pipettes (~2-3 MOmega ). All cells were held at -70 mV.


                              
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  		Table 2.   Contraction characteristics

When the same experiments were repeated using low-resistance suction pipettes, we could not detect any significant differences between CHF and sham cells with regard to any contractile properties (Fig. 1B, Table 2, protocol 5). It is likely that switching from high-resistance to low-resistance electrodes significantly altered the properties of the sham cells but not the CHF cells. In the sham cells, FS was reduced by 46% (P < 0.05), RTTP was longer by 48% (P < 0.05), SV faster by 62% (P < 0.05), and RR50 longer by 25% (P < 0.05) using low-resistance compared with high-resistance electrodes (Table 2, compare protocols 1 and 5).

Also, action potential configuration was different with low-resistance pipettes. APD50, and especially the duration at 90% repolarization (APD90), were significantly shorter in both sham and CHF cells (Fig. 1 and Table 2, protocol 1 vs. protocol 5). However, with both types of electrodes APD50 was nearly doubled in CHF compared with sham.

These data indicate that with regard to size and velocity of cell shortening, sham cells are more sensitive to the switch of the experimental conditions than CHF cells, possibly because of dialysis of small cytosol constituents. However, using low-resistance patch pipettes, Gomez et al. (11, 12) have claimed that the gain of ICa,L-induced contractions is reduced in CHF, and we therefore investigated ICa,L-induced contractions in more detail using standard voltage-clamp protocols.

EC coupling at room temperature. We used the same experimental conditions as Gomez et al. (12) to study EC coupling in CHF and sham. Figure 2A shows representative tracings of current and contraction with a large contraction at 0 mV and a minimal contraction at 60 mV in both CHF and sham. The current-voltage relationship of ICa,L was similar in CHF and sham at 23°C (Fig. 2B) and had the typical bell shape (12). The current density was not significantly different between the groups. Thus ICa,L potentially constituted the same trigger of SR Ca2+ release in the two groups. To test the efficacy of the trigger, contraction was also measured. The contraction-voltage relationship was also bell shaped in both CHF and sham, and FS was not significantly different between the groups at any voltage.


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  		Fig. 2.   Contractions and currents at 23°C. A: representative contraction and current tracings from CHF and sham using a postconditioning potential of -50 mV and 200 ms test potentials to 0 and 60 mV in Cs+-containing solutions during control conditions (Ctr) and after addition of µM verapamil (Vp) to the same cardiomyocytes. Voltage protocol shown on top lanes. Contraction was normalized to cell length. B: summary data for fractional shortening and L-type Ca2+ current (ICa,L) density in sham (, n = 17) and CHF (, n = 13). There was no significant difference between the groups. Addition of 20 µM verapamil reduced contraction and current to <5% of control value in sham (open circle , n = 5) and CHF (, n = 4).

Analysis of contraction characteristics showed 11% reduction of TTP in CHF (P < 0.05) at a test potential of 0 mV. However, RTTP was not significantly different in CHF and sham. SV, R50, and RR50 were not significantly different between CHF and sham (Table 2, protocol 3).

Because we used 0 mM Na+ in the pipette, reverse-mode INa,Ca could not account for the contractions in these experiments. To confirm this, we added 20 µM verapamil, which reduced ICa,L to ~5% of peak value in both CHF and sham (Fig. 2, A and B). This current elicited a contraction of ~5% of the baseline value in both groups. Thus ICa,L was the trigger of contraction in the absence of verapamil.

The data mentioned above indicate that ICa,L density is similar in CHF and sham at 23°C, and that there are no major differences in the contractions elicited by ICa,L in the two groups. Thus we could not reproduce the contraction defect demonstrated by echocardiography (33) in field-stimulated cells (15) and in undialyzed current-clamped cells. However, the gain of the EC coupling is profoundly affected by temperature (3), possibly because the various processes involved in EC coupling have different temperature sensitivities (20, 35). Hence, a difference between CHF and sham could have been concealed by low temperature.

Ca2+-induced Ca2+ release contractions at 37°C. The voltage-clamp experiments were therefore repeated at 37°C, at which temperature ICa,L was ~50% higher than at 23°C in both CHF and sham, whereas FS was ~50% lower in both groups. Both ICa,L and the contractions were large at 0 mV and minimal at 60 mV in both CHF and sham (Fig. 3), and showed bell-shaped voltage relationships that were not significantly different between CHF and sham (Fig. 4A). Verapamil (20 µM) reduced both ICa,L and FS to ~5% of baseline value (Figs. 3 and 4A). TTP, RTTP, SV, R50, and RR50 were not significantly different between CHF and sham (Table 2, protocol 4). Accordingly, our data did not show a defect in ICa,L and Ca2+-induced Ca2+ release (CICR)-induced contractions at 37°C. The lack of consistency between the contraction data in field-stimulated and undialyzed cells on one hand, and in patch-clamped cells on the other hand, could be due to low SR Ca2+ load in the sham cells. For this reason, we measured SR protein abundance and SR Ca2+ content.


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  		Fig. 3.   Contractions and currents at 37°C. Representative contraction and current tracings from sham and CHF using a VPC of -50 mV and 200-ms test potentials to 0 (A and B) or 60 mV (C and D) in Cs+-containing solutions during control conditions in A and C, and after addition of 20 µM verapamil in B and D. Voltage protocol shown on top of A and C. Contraction was normalized to cell length.



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  		Fig. 4.   Contraction, current, and sarcoplasmic reticulum (SR) Ca2+ load at 37°C. A: summary data for fractional shortening and inward current density in sham () and CHF () using a VPC of -50 mV and 200-ms test potentials with 10-mV increment in Cs-containing solutions. Representative contraction and current tracings are shown in Fig. 3. Addition of 20 µM verapamil reduced contraction and current to <5% of control value in sham (open circle ) and CHF (). B: SR Ca2+ load assessed by caffeine (10 mM for 10 s) induced inward Na+/Ca2+ exchange current (INa,Ca) and contractions. Representative tracings are shown. C: caffeine-induced inward INa,Ca and contractions, mean data (sham, n = 6; CHF, n = 8, both not significant).

SR function. The abundance of SERCA2 was lower in CHF (n = 6) compared with sham (n = 6) (68 ± 9% and 100 ± 9%; P < 0.05), whereas the phospholamban protein abundance (monomer 95 ± 12% and 100 ± 12%, pentamer 93 ± 5% and 100 ± 6%) and the SERCA2-to-phospholamban ratio (0.165 ± 0.026 and 0.204 ± 0.019, calculated from immunolabeling density where the pentamer and monomer bands, were summed) were not significantly different (Fig. 5). However, this does not rule out that there might be a functional difference between these proteins that would influence SR Ca2+ content. For this reason, we assessed SR Ca2+ load by applying 10 mM caffeine to CHF (n = 6) and sham (n = 8) myocytes for 10 s, and measured the contractures and inward INa,Ca (7) (Fig. 4B). There was no significant difference in either caffeine-induced contractures (23.4 ± 2.3% and 21.7 ± 1.7% for CHF and sham, respectively) or current integral (1.4 ± 0.2 and 1.5 ± 0.3 pCpF-1 for CHF and sham, respectively) between the two groups (Fig. 4C).


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  		Fig. 5.   SR Ca2+ ATPase (SERCA2) and phospholamban protein abundance. Representative Western blot lanes with the use of SERCA2 (A) and phospholamban antibody (B) are shown. C and D: quantitative results from sham (n = 6) and CHF (n = 6) hearts normalized to the mean values for sham samples. S, sham; C, CHF. * P < 0.05.

Contractions in Na+- and K+-containing solutions at 37°C. To reestablish the difference between CHF and sham cells with voltage-clamp protocols, we first omitted Cs+ from the solutions. Cs+ is known to influence EC coupling (20), and we therefore used a Na+- and K+-containing solution. In Fig. 6A, representative tracings from CHF and sham are shown at test potentials of 0 and 80 mV from a VPC of -50 mV. Contractions were large at both 0 and 80 mV as opposed to the contractions at positive potentials in Cs+-containing solutions. The contraction-voltage relationship in K+-containing solutions was sigmoidal (Fig. 6B), whereas it was bell shaped in Cs+-containing solutions. However, there were still no significant differences in FS, TTP, RTTP, SV, and R50 between CHF and sham at 0 mV (Table 2, protocol 6).


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  		Fig. 6.   Fractional shortening in K+-containing solutions at 37°C. A: representative contraction and current tracings from sham and CHF in K+-containing solutions using a VPC of -50 mV and 200-ms test potentials to 0 and 80 mV. Top, voltage protocols. Contraction was normalized to cell length. B: mean contraction data in sham (, n = 8) and CHF (, n = 9).

Contractions triggered under voltage clamp from -70 mV at 37°C. Finally, if cell dialysis were the cause of the reduced contractility of the sham cells, voltage clamp using high-resistance electrodes would be expected to reveal the difference in contractility between the two cell types. Representative tracings and summary data from CHF and sham are shown in Fig. 7 from a VPC of -70 mV. As expected, we now detected attenuated contraction magnitude and a slow shortening velocity in CHF compared with sham. With a step to 10 mV, TTP was 19% longer in CHF (P < 0.01) than in sham. RTTP was 53% longer in CHF than in sham (P < 0.01). SV was reduced by 47% in CHF compared with sham (P < 0.01), whereas R50 and RR50 were not significantly different between CHF and sham (Table 2, protocol 2). Thus the attenuated contraction demonstrated in vivo and in undialyzed current-clamped CHF cardiomyocytes was also present in undialyzed voltage-clamped cardiomyocytes.


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  		Fig. 7.   Contractions triggered from -70 mV at 37°C. A: representative contraction and current tracings from CHF and sham using a VPC of -70 mV and 200-ms test pulses to 10 mV. Performed with high-resistance electrodes. Top, voltage protocol. The dot in front of current tracings shows 0 nA. Contraction was normalized to cell length. B: summary data for fractional shortening, CHF, n = 9, and sham, n = 10. * P < 0.01.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We report that in a rat model of CHF we were not able to detect any defect of contractions triggered by ICa,L in isolated cardiomyocytes using low-resistance patch pipettes and standard voltage-clamp techniques and solutions. However, with high-resistance electrodes, both FS and velocity of contraction were reduced in CHF cells compared with sham cells, both during current-clamp and voltage-clamp from a VPC of -70 mV conditions. The latter finding is in accordance with echocardiographic data of CHF rats compared with sham (33), and field stimulation of CHF cardiomyocytes that exhibit a defect both in Ca2+ transients and contractions (15). Interestingly, ICa,L was not significantly different between CHF and sham using voltage clamp, a VPC of -50 mV, and dialyzed cells, and because contractions were not altered, the gain of the CICR seemed to be normal. Moreover, the SERCA2 to phospholamban ratio was not significantly different, and SR Ca2+ content was similar in CHF and sham. Both ICa,L and contractile function of the cardiomyocytes were predictably dependent on the temperature and ionic composition of the solution used. The contraction-voltage relationship changed from sigmoidal to bell shaped when Cs+ was substituted with K+ and Na+ to the pipette solution; however, CHF cells were not significantly different from sham. The experiments suggest that a defect in CICR probably does not account for the contractile defect of in CHF cardiomyocytes.

Experimental model of CHF secondary to MI. The model of postinfarction CHF in rats is well characterized in vivo in our laboratory (33). To secure a safe CHF diagnosis in the experimental animals, we used the same clinical and hemodynamic inclusion criteria in the present study as in our echocardiographic study. The myocytes under study from CHF animals also exhibited a defect in EC coupling (15). However, most other cellular studies (19, 21, 25, 26, 37-40) on MI models have been performed on myocytes from animals without a definitive diagnosis of CHF. This fact has to be taken into consideration when the data from the present study are compared with data from previous studies.

Contractions triggered by action potentials using high- and low-resistance pipettes cardiomyocytes. The cell isolation procedure did not remove the contractile defect present in CHF in vivo because it could be detected in isolated cardiomyocytes when contractions were activated both under current and voltage-clamp conditions using high-resistance electrodes. However, we were not able to detect any contractile deficit in CHF cardiomyocytes using low-resistance patch electrodes. Although our data should be cautiously interpreted, they indicate that the contractile properties of the sham cells, and not of the CHF cells, were seriously altered when comparing data obtained with the two types of electrodes. This finding calls for further experiments but could point to important methodological aspects of using patch pipettes that allow for exchange of small molecules between the cytosol and the pipette solution. One could speculate that cell dialysis of sham cells will alter the phosphorylation level of important proteins at one or several levels of the EC coupling, whereas the phosphorylation level in CHF cells was already low so that dialysis could not reduce it further (30).

Also action potential configuration was different using the two types of pipettes, most notably because APD90 was much shorter using low-resistance electrodes compared with high-resistance electrodes. However, in contrast to the cell-shortening parameters, the effect was present in both sham and CHF cells, which points to another explanation than for the contractility parameters. Because it will have no consequences for the main conclusions of this study, we have not attempted to sort out the reasons for the different shapes of action potentials; however, intracellular and subsarcolemmal concentrations of Na+, Ca2+, and K+ may be different in the two experimental situations. For instance, late repolarization is highly influenced by inward INa,Ca, the size of which is governed by these ions (5). Interestingly, there was consequently no overall association between action potential duration and magnitude of cell shortening. Bouchard et al. (5) reported that during otherwise similar conditions magnitude of cell shortening increased with increasing action potential duration due to higher SR Ca2+ load. However, in the present study, various experimental conditions like high- and low-resistance pipettes and cells from sham and CHF animals seem to be more important determinants of cell shortening than action potential duration. The independent effect of action potential duration in our model of CHF should be examined more closely.

ICa,L and ICa,L-triggered contractions. We found similar ICa,L density in CHF compared with sham. However, there was an effect of temperature, and in both groups ICa,L was ~100% larger at 37°C than at 23°C. Also, in previous studies, ICa,L has been found to be unchanged in MI and CHF (11, 34, 37, 40), although in one study, ICa,L was found to be reduced in CHF (31). In the present study, the contractions triggered by ICa,L were not significantly different in CHF and sham with regard to FS. However, the contraction amplitudes were substantially smaller in both groups at 37°C than at 23°C. Thus ICa,L and FS exhibited inverse responses with regard to temperature changes, which is illustrated in Fig. 8. Independent of temperature, CHF and sham had similar current-contraction "gain" functions. However, the triggering efficacy of ICa,L was substantially reduced at 37°C compared with 23°C for both groups. Figure 8 also shows that a given ICa,L density at constant temperature triggered contractions of similar magnitude, whether the test potential was below or >0 mV (on the "ascending" or "descending" limb of the bell-shaped voltage-contraction curve). This observation also provides indirect evidence for ICa,L being the only trigger of contraction in these experiments. Furthermore, it fits with the absence of reverse-mode INa,Ca at high test potentials because the cells were dialyzed with 0 Na+. The assumption that ICa,L was the only trigger of the contractions was also supported by applying 20 µM verapamil, which blocked both ICa,L and contractions >95%. In contrast to our results, Gomez et al. (11, 12) reported that in cardiomyocytes from rats with heart failure, ICa,L exhibit reduced gain with regard to its capacity to trigger intracellular Ca2+ release and contraction, despite unchanged SR Ca2+ load. However, in both of these studies, they assessed SR Ca2+ load under experimental conditions that were quite different from those used in their studies of EC coupling, and thus the reported reduction in gain of CICR might be due to undetected differences in SR Ca2+ load. Furthermore, the experiments were carried out 6 mo after induction of MI so the disease had progressed further, but LVEDP was not as high as in the present study. A recent review by Houser et al. (16) also points out that there is poor evidence in the literature of reduced gain function in hypertrophy and failure.


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  		Fig. 8.   ICa,L contraction relationship. ICa,L is plotted against contraction for the data acquired at 23°C in sham () and CHF (), and at 37°C (sham, open circle  and CHF, ). Data are from Fig. 2B and 4A.

Physiological temperature and test solutions. Our study as well as those by Ferrier et al. (10) and Wassersrtom and Vites (35) point to important consequences of temperature and ionic composition of solutions on the EC coupling. In some of the experiments we included Na+ in the pipette solution and substituted K+ for Cs+. Because the Na+/Ca2+ exchanger (NCX) has a high Q10 and thus is sensitive to temperature changes, we also used a physiological temperature in these experiments (3, 35). Under these experimental conditions, the contraction-voltage relationship was sigmoidal (Fig. 5). This is in accordance with previous studies, in which solutions allowing reverse-mode INa,Ca, were used (22, 35). K+- and Na+-containing solutions are more physiological than solutions containing Cs+ and 4-aminopyridine (20). Our results therefore support that the physiological contraction-voltage relationship is sigmoidal rather than bell shaped and indicate a contribution of the NCX to EC coupling as previously pointed out. We (34) have shown that verapamil is a much less efficient blocker of contraction at high voltages in CHF than in sham, probably due to increased NCX activity in CHF cells. Thus we cannot rule out that there might be a reduced efficiency of ICa,L at high voltages when using only physiological ions and temperature because it would be concealed by the increased NCX.

Is contractility or relaxation altered? FS, TTP, and R50 are used as parameters of mechanical cardiomyocyte function. FS and TTP are interrelated; at the same rate of cell shortening, TTP is longer in cells with high FS than in cells with low FS. TTP should, for this reason, be corrected for variations in FS. RTTP gives the time it takes for the cell to shorten by 1%. This correction has not been performed in previous studies. We found that both TTP and RTTP were prolonged in CHF during current clamp and voltage clamp with high-resistance electrodes.

In patch-clamp experiments we found, as most others, that TTP was significantly longer in CHF at low temperatures (15, 21, 25). However, RTTP was not significantly different between CHF and sham. At 37°C, both TTP and RTTP were similar in CHF and sham. This applied both to voltage-clamp experiments using suction pipettes and Cs+- or K+-containing solutions and to current clamp experiments using suction pipettes.

SV has been measured by several investigators (1, 7, 19, 25, 26, 29), but only a few have normalized it to cell length (7, 26). In our hands, normalized SV was substantially lower in CHF than in sham in undialyzed cardiomyocytes. Interestingly, normalized SV did not differ significantly between CHF and sham, neither in Cs+- nor K+-containing solutions, when patch pipettes were used from a VPC of -50 mV. Normalized SVs have been reported in MI animals without CHF (7, 26). The unchanged SV found by them might be due to normal myocardial function because the animals at study did not show evidence of CHF. On the basis of the standard deviation and the number of cells in the K+-experiments with patch electrodes, we expected to be able to detect a difference (with a power of 0.80) of >31% in FS, >29% in SV and >35% in RTTP. We have previously found that in vivo left ventricular posterior shortening velocity, a parameter of contractility, was 49% lower in CHF than in sham, and that posterior wall thickening, which reflects FS, was 52% lower in CHF (33). In the high-resistance pipette experiments, SV was 47% slower in CHF than in sham, and RTTP was 53% longer in CHF. Accordingly, if a similar difference in contractility were present in the experiments with low-resistance electrodes, we should have been able to detect it.

The relaxation rate was not significantly different in CHF and sham in the present study, except in the experiments using high-resistance microelectrodes and current clamp. There is no consensus regarding the relaxation rate in MI, probably due to lack of uniform experimental conditions (1, 15, 19, 21, 25, 26, 29, 34). In the relaxation phase, the NCX is important, as is the SERCA2 function (3). In the Cs+ experiments we used 0 mM Na+ in the pipette, promoting forward mode INa,Ca and thus cell relaxation. CHF cells have more NCX protein than sham in this experimental model (34). Accordingly, we expected that the CHF cells would relax more rapidly than the sham cells compared with the physiological situation where the intracellular Na+ concentration is about 10 mM. However, Cs+ is known to interfere with EC coupling, and for this reason it is difficult to conclude anything about the contribution of INa,Ca to relaxation in the Cs+ experiments with 0 Na+ (20, 35). In the K+ experiments, Na+ was present in the pipette and no Cs+ was used. Under such conditions we detected no difference in R50 and RR50, which is in accordance with echocardiographic data on relaxation velocity (I. Sjaastad and R. Bjørnerheim, unpublished data).

SR function. Previous studies (13) have offered inconclusive evidence as to whether SERCA2 and phospholamban protein abundance is altered in CHF. In the present study, we found no significant changes in the SERCA2 to phospholamban ratio, whereas the SERCA2 immunolabeling density was lower in CHF than in sham. The SERCA2 Ca2+-pumping capacity is influenced by both protein abundances and the phosphorylation state of phospholamban. In CHF there is evidence of reduced Ser16 phosphorylation, which might reduce SR Ca2+ load (18, 30). Few of the previous studies on EC coupling have assessed SR Ca2+ load in CHF cells under voltage-clamp conditions. We measured SR releasable Ca2+ by applying caffeine and found no significant difference in SR Ca2+ load in CHF and sham using the same preconditioning pulse protocols. This observation does not exclude an altered phosphorylation level of phospholamban. The caffeine application procedure has some limitations, but is still commonly used to assess SR Ca2+ load (6). Taken together, our results show that with standard patch-clamp techniques we cannot detect significant differences in CICR-induced EC coupling in CHF cells when SR load is constant.

Trigger defect in CHF that is not unraveled in present study? Three triggers of contraction have been proposed: ICa,L, reverse-mode NCX, and the voltage-sensitive release mechanism (VSRM). The VSRM is analogous to the SR Ca2+-release mechanism in skeletal muscle, and provided this mechanism exists in the heart, it could be a trigger of SR Ca2+ release also in cardiomyocytes (9). However, contribution of this mechanism under physiological conditions is not generally accepted (28). Of the three proposed triggers of contraction, we have ruled out that ICa,L as measured by conventional techniques and reverse-mode NCX can explain the contractile deficit observed in CHF. However, we have revealed a significant effect of experimental conditions, probably cell dialysis, that could alter EC coupling in the sham cells. Hence, we cannot rule out that CICR by ICa,L is different with a higher gain in sham cells under more physiological conditions. Another possibility could be that the VSRM might be defective in CHF. The VSRM was recently shown to be defective in cardiomyopathic hamsters (17). The VSRM is suggested to interact with ICa,L to trigger more rapid and larger SR Ca2+ release (9). However, the VSRM is not detectable in dialyzed cells. The VSRM has been reported to be present in undialyzed cells from a VPC of -70 mV. We have preliminary data that suggest that even under physiological conditions CICR is unaltered in CHF cells, whereas contractions triggered from negative potentials seem to be selectively attenuated (32).

In conclusion, the present study is the first to perform voltage-clamp experiments in postinfarction CHF at 37°C with adequate assessment of SR Ca2+ load. In undialyzed CHF cardiomyocytes, a contraction deficit was present in both voltage clamp from a VPC of -70 mV and in current clamp. In dialyzed CHF cardiomyocytes, current clamped or voltage clamped using a VPC of -50 mV, we did not detect a difference in ICa,L or in EC coupling. There were no indications of altered current-contraction gain in CHF. The contraction-voltage relationship, although similar in CHF and sham, changed from bell shaped in Cs+-containing solutions to sigmoid in K+- and Na+-containing solutions. The SERCA2-to-phospholamban ratio and SR Ca2+ load were unchanged in CHF. The experiments suggest that a defect in ICa,L-triggered contractions does not account for the contractile defect observed in vivo and in undialyzed cardiomyocytes isolated from animals with a safe diagnosis of CHF.


    ACKNOWLEDGEMENTS

The authors are grateful to Bjørn Amundsen, Kathrine Andersen, Morten Eriksen, Ingvild Bartnes Hoen, Severin Leraand, Birthe Mikkelsen, Hilde Olsen, Line Solberg, Gerd Torgersen, and Annlaug Ødegaard for technical assistance.


    FOOTNOTES

This study was supported by The Norwegian Council for Cardiovascular Diseases, Anders Jahre's Fund for the Promotion of Science, Rakel and Otto Kr. Bruun's Fund, and by National Heart, Lung, and Blood Institute Grant HL-30724 (to J. A. Wasserstrom).

Address for reprint requests and other correspondence: I. Sjaastad, Institute for Experimental Medical Research, Univ. of Oslo, Ullevaal University Hospital, Kirkevn 166, 0407 Oslo, Norway (E-mail: ivar.sjaastad{at}ioks.uio.no).

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 16, 2002;10.1152/ajpheart.00162.2001

Received 8 March 2001; accepted in final form 8 May 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 283(3):H1225-H1236
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