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Cardiovascular Research 2005 66(1):74-83; doi:10.1016/j.cardiores.2004.12.009
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Copyright © 2004, European Society of Cardiology

Oxidized LDL induces ventricular myocyte damage and abnormal electrical activity–role of lipid hydroperoxides

Klaus Zorn-Paulya, Peter Schaffera,*, Brigitte Pelzmanna, Eva Bernharta, Guofeng Weib,c, Petra Langa, Gerhard Ledinskib, Joachim Greilbergerb, Bernd Koidla and Günther Jürgensb

aCenter of Physiological Medicine, Institute of Biophysics, Medical University Graz, Harrachgasse 21/4, A-8010 Graz, Austria
bCenter of Physiological Medicine, Institute of Physiological Chemistry, Medical University Graz, Graz, Austria
cDepartment of Cardiology, 2nd Hospital of Dalian Medical University, Dalian, PR China

* Corresponding author. Tel.: +43 316 385 72027; fax: +43 316 385 72034. Email address: peter.schaffer{at}meduni-graz.at

Received 14 July 2004; revised 25 November 2004; accepted 14 December 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: It was our aim to investigate effects of human LDL, copper-, or AAPH-oxidized over different periods of time to different degrees (ox-LDL), on viability and electrophysiological parameters of isolated ventricular myocytes of guinea pigs.

Methods: Guinea pig ventricular myocytes were incubated with ox-LDL or native LDL (at 0.5 mg/ml) for 12 h, and afterwards myocyte damage, action potentials, and transmembrane ion currents were studied (at 37 °C).

Results: Ox-LDL was found to induce severe myocyte damage, whereas native LDL had no effect. Myocyte damage was dependent on the content of total lipid hydroperoxides in both copper-oxidized and AAPH-oxidized LDL. Incubation with ox-LDL led to intense contractile and electrophysiological effects including prolongation of action potential duration, depolarization of resting membrane potential, spontaneous activity, generation of afterdepolarizations, and modification of transmembrane ion currents (e.g. inward rectifier, calcium, and background currents).

Conclusions: Ox-LDL induced cell damage and irregular electrical activity in ventricular myocytes. These effects were dependent on the lipid hydroperoxide content of ox-LDL and were similar to oxidative stress (OS) induced by various OS-generating systems. The observed effects may play a role for functional cardiac abnormalities in patients with increased ox-LDL levels.

KEYWORDS Lipoproteins; Oxygen radicals; Repolarization; Membrane currents; Ion channels


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Oxidized LDL (ox-LDL) is considered to exert various deleterious cellular effects that are thought to play a role in atherogenesis [1,2]. Some of these pathophysiological alterations seem to depend on lipid peroxidation products (LPO) of ox-LDL [3–6]. These include (i) alterations in intracellular calcium content, (ii) cell necrosis, and (iii) apoptosis; effects which were studied in vascular myocytes and endothelial cells [3–6]. However, the LPO-related deleterious effects of ox-LDL may also involve alterations in the cellular electrophysiological properties as reported for endothelial cells [7]. Recently, the plasma level of ox-LDL was shown to be a prognostic predictor of mortality in chronic congestive heart failure patients, whereby a significant negative correlation between ox-LDL level and left ventricular ejection fraction has been reported [8]. Hence, in addition to atherogenic effects, ox-LDL may also affect cardiac myocytes directly leading to an alteration of ventricular function. Thus, it was our aim to investigate the effects of native LDL and ox-LDL, oxidized to different degrees (i.e. different content of LPO), on viability, electrophysiology, and contractility of ventricular myocytes. Our data show that ox-LDL induces severe electrophysiological and contractile alterations as well as myocyte damage, which were depending on the LPO content of ox-LDL. Some of these effects may play a role for the clinically observed harmful effects of ox-LDL. Preliminary results of this study were published in abstract form [9].


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. LDL preparation and oxidation
LDL was isolated from the plasma of normolipidemic young male donors, as described previously [10]. All donors gave informed consent prior blood sampling. Oxidation of LDL (1.5 mg/ml total lipoprotein) was performed at 37 °C with 10 µM CuCl2 for 2 to 24 h to obtain different degrees of oxidation. The reaction was stopped by adding EDTA at a final concentration of 1 mg/ml. For studying cellular viability, LDL was also alternatively oxidized with the free radical-producer 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH; Polysciences Inc.). AAPH-mediated oxidation of LDL (5 mM AAPH) was performed at 37 °C. Prior to oxidation, native LDL was dialyzed against 10 mM PBS (pH 7.4) containing 0.1 M diethylenetriaminepentaacetic acid (DTPA). At intervals between 2 and 24 h, the reaction was terminated by adding butylated hydroxytoluene (BHT) to a final concentration of 10 µM. The samples were saturated with nitrogen and dialyzed against Tyrode solution at 4 °C for 24 h.

The REM (electrophoretic mobility relative to non-modified native LDL) was measured using the Lipidophor system (Immuno, Vienna, Austria) and the LPO content was determined for each LDL sample by an assay developed in this laboratory [11].

2.2. Myocyte isolation
Guinea pig ventricular myocytes were isolated by Langendorff perfusion. The isolated myocytes were stored in a cell culture medium (M-199, Sigma, St-Louis, MO, USA), supplemented with 5 µg/ml penicillin and 5 IU/ml streptomycin and were kept in an incubator at 37 °C.

2.3. Incubation procedure
The isolated myocytes were incubated with native LDL, copper-oxidized, or AAPH-oxidized LDL (at 0.5 mg LDL/ml cell culture medium) for 12 h before cell damage and electrophysiological parameter evaluation. Myocyte viability was estimated by determining the percentage of rod shaped (i.e. viable) myocytes for each experimental condition (control myocytes, myocytes incubated with ox-LDL (ox-LDL myocytes) and native LDL (n-LDL myocytes)). At least 800 myocytes were counted for each incubation fraction. The percentage of rod-shaped myocytes was normalized to control to compensate for variations in the percentage of rod shaped myocytes from different myocyte preparations. Under control conditions about 50% of the myocytes were found to be rod shaped. For all electrophysiological and contraction recordings copper-oxidized LDL was used (4 h of oxidation if not indicated otherwise).

2.4. Electrophysiological recordings
The isolated myocytes were placed in an experimental chamber mounted on the stage of an inverted microscope and were superfused with extracellular saline (composition in mM: NaCl 137, KCl 5.4, CaCl2 1.8, MgCl2 1.1, NaHCO3 0.4, HEPES/Na+ 10, d(+)-glucose 5.6, adjusted to a pH of 7.4 with NaOH) at a temperature of 36–37 °C and a flow rate of ~3 ml/min. Only rod-shaped myocytes were used for electrophysiological experiments. Action potentials (AP) and transmembrane currents were recorded as described previously [12] by the patch clamp technique in the whole cell mode by the use of a L/M EPC-7 amplifier (List, Darmstadt, Germany), and a Digidata 1200 interface (Axon Instruments, Foster City, CA, USA). When filled with standard internal solution (composition in mM: KCl 110, ATP/K+ 4.3, MgCl2 2, CaCl2 1, EGTA 11, HEPES/K+ 10 adjusted with KOH to a pH of 7.4 (estimated free [Ca2+] <10–8 M) the patch-electrodes had a tip-resistance of 2–3 M{Omega}. In some experiments Ba2+ (0.5 mM) was added to the external solution in order to inhibit the inward rectifier current IK1. For studying L-type calcium current (ICaL) KCl was replaced by equimolar CsCl in both, the external and the internal solution. Using these CsCl solutions and adding CdCl2 (200 µM) to the external solution, enabled the recording of time independent currents in the absence of K+- and Ca2+-currents. The sodium calcium exchanger current (INCX) was measured as the NiCl2 (5 mM)-sensitive current fraction [13,14] in the above-mentioned CsCl solutions, with 10 mM Na+ added to the pipette solution. To allow a comparison of current amplitudes between different cells, currents are displayed as current density (current divided by membrane capacitance). The contractile activity of single ventricular myocytes was studied by using an edge detection system (Axiomat, Zeiss). Contraction was elicited by field stimulation (electrode distance, 1 cm; pulse duration, 5 ms; super-threshold pulse amplitude at ~4 V/cm) at a frequency of 1 Hz (bath temperature of ~36.5 °C, solution as used for AP recordings).

A mathematical model of NCX [15] was used to estimate the effects of alterations in intracellular calcium concentration ([Ca2+]i) on the I/V relationship of INCX. Computation of INCX was done with Matlab 6.5 (Math Works Inc., USA) running on a personal computer. Since we took this model only for qualitative comparison we used the fixed model parameters as given in [15] but setting temperature and ion concentration as used in our experiments. Maximal flux (Vmax) and [Ca2+]i were estimated as described.

Data are presented as mean ± S.E.M., n=number of cells/number of hearts used. Pearson's correlation coefficient and t-test were calculated by using SPSS software (SPSS Inc. Chicago, IL, USA). A value of P<0.05 was considered significant.

The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996) and conforms with the principles outlined in the Declaration of Helsinki (Cardiovascular Research 1997; 35:2–3).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Ox-LDL-induced cell damage
The myocytes were examined under the microscope 15–20 min after addition of either native or ox-LDL in order to study acute effects. Spontaneous contractions were seen in approximately 50% of ox-LDL myocytes but not in control and n-LDL myocytes.

Cell damage was determined 12 h after start of LDL or ox-LDL incubation by determining the percentage of rod-shaped (i.e. viable) myocytes. Ox-LDL induced pronounced myocyte damage whereas native LDL had no influence. Ox-LDL myocyte damage varied considerably depending on the duration of the oxidation and was found to depend on the LPO content. Both, the myocyte damage, as well as the total LPO content were maximal with 4 h copper-ox-LDL, whereas in case of 24 h copper-ox-LDL the content of LPO was similar to native LDL due to the decomposition of LPO (Fig. 1A). In case of AAPH-ox-LDL, both cell damage and total LPO content were highest with 24 h AAPH-ox-LDL (Fig. 1B). The relation of LPO content of ox-LDL (both copper-oxidized and AAPH-oxidized) and myocyte damage is demonstrated in Fig. 1C. This figure suggests that copper oxidation renders LDL more cytotoxic at a given LPO content than AAPH-oxidation. REM as an indicator of negative net charge did not show a relation to myocyte damage (not shown).


Figure 1
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  		Fig. 1 (A) Ox-LDL-induced cell damage and LPO content in dependence of the duration of copper-induced LDL oxidation. (bullet) Percentage of rod shaped myocytes in ox-LDL-incubated myocyte fractions (each data point represents the mean of four different ox-LDL preparations; data were normalized to untreated myocytes). ({square}) Mean LPO-content. Four copper-ox-LDL preparations were used for these experiments (n=4 hearts). (B) AAPH-ox-LDL-induced cell damage and LPO in dependence of the duration of AAPH-induced LDL oxidation. ({blacktriangleup}) Percentage of rod shaped myocytes in ox-LDL-incubated myocyte fractions (mean of 3 ox-LDL preparations, normalized). ({triangleup}) Mean LPO-content of the three ox-LDL preparations used for these experiments (n=3 hearts). (C) Cell damage in dependence of the LPO content of copper-oxidized (bullet) and AAPH-induced ox-LDL ({blacktriangleup}), r: represents the correlation coefficient.

 
3.2. Ox-LDL-induced AP alterations
All further experiments were performed with copper oxidized LDL only. Since ox-LDL induced spontaneous contractions we further investigated whether incubation with ox-LDL leads to electrophysiological alterations.

Fig. 2 shows representative APs from a control myocyte (A) and myocytes incubated with 4 h ox-LDL recorded after 12 to 16 h of incubation (B, C). In ox-LDL myocytes, early afterdepolarizations (EADs, Fig. 2B), delayed afterdepolarizations (DADs), and abnormal spontaneous activity (suprathreshold DAD triggered; Fig. 2C) were frequently seen, whereas in control myocytes and n-LDL myocytes, none of these alterations could be observed. In general, ox-LDL led to AP prolongation compared to control myocytes or n-LDL myocytes.


Figure 2
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  		Fig. 2 Action potentials from a control myocyte (A) and typical examples from ox-LDL-incubated myocytes showing EADs (B) and spontaneous activity (C). ({blacktriangleup}) Electrical stimulation; (x) EADs; (*) DADs; (#) spontaneously induced action potentials.

 
Fig. 3A shows that AP prolongation was dependent on the LPO content of LDL, being most pronounced for 2 and 4 h ox-LDL. A clear dependence of APD90 (AP duration at 90% of repolarization) on the LPO-content of ox-LDL (r=0.97; p<0.01) is shown in Fig. 3B. To allow a reliable comparison, all data in Fig. 3 were obtained from myocytes of one heart using ox-LDL from a single preparation (n=4/1 for each group). Similar results were obtained from two additional ox-LDL preparations in myocytes from two further hearts. APs with EADs or spontaneously induced APs were not used for APD-analysis.


Figure 3
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  		Fig. 3 Action potential duration (APD90) in dependence of duration of LDL oxidation (A) and LPO content (B); see text for details.

 
3.3. Ion currents in ox-LDL-treated myocytes
All further experiments were performed with ox-LDL copper oxidized for 4 h. The L-type calcium current (ICaL) which is thought to underlay EADs was studied using a holding potential of –80 mV a prepulse to –40 mV (50 ms; in order to activate and voltage inactivate sodium current) and subsequent steps to potentials between –40 and +90 mV (400 ms). Representative traces elicited by depolarization to 0 mV and +90 mV from a control and an ox-LDL-incubated myocyte are shown in Fig. 4A. Determining ICaL as the difference between the maximum inward current and the current at the end of the pulse we constructed the mean current–density/voltage relationship (Fig. 4B) which revealed a significant ox-LDL-induced increase in calcium current density. At 0 mV ICaL density was –8.44 ± 0.49 pA/pF in control myocytes (n=8/4) and –11.93 ± 0.90 pA/pF in ox-LDL myocytes (n=8/4; P<0.05). As shown in panel A, the steady-state current (at the end of the pulse) is enhanced in ox-LDL-incubated myocytes suggesting the activation of a background conductance. Voltage dependence of inactivation of ICaL was studied by using the following protocol. A test pulse to +10 mV (400 ms) was preceded by conditioning pulses to potentials between –40 mV and +60 mV (increment 5 mV, duration 400 ms). The amplitude of ICaL elicited by the test pulse was determined and plotted against the voltage of the conditioning pulse and fitted with the sum of two Boltzmann equations to account for inactivation and re-activation (Fig. 4C). Both, half maximal inactivation (V1/2) and slope factor (k) of inactivation were not affected by ox-LDL (V1/2: –18.4 ± 0.32 mV, k: 4.6 ± 0.26 mV under control conditions; V1/2: –17.4 ± 1.09 mV, k: 4.81 ± 0.18 mV with ox-LDL; NS). However, at potentials positive to +20 mV ox-LDL enhanced re-activation of ICaL (amplitude: 0.31 ± 0.02; V1/2: 44.6 ± 0.56 mV; k: 10.20 ± 0.12 in control; amplitude: 0.56 ± 0.03; V1/2: 45.9 ± 0.89 mV, k: 11.08 ± 0.11 mV for ox-LDL; P<0.01 for amplitude) which, in combination with the observed increase in current density may favor the incidence of EADs.


Figure 4
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  		Fig. 4 Ox-LDL-induced alterations of L-type calcium current. (A) Typical recordings from a control and an ox-LDL-incubated myocyte at 0 and +90 mV. (B) Current–density to voltage relationship for control ({circ}) and ox-LDL-incubated myocytes (bullet). (C) Steady-state inactivation of calcium current in control and ox-LDL myocytes. The solid line represents the result of fitting the sum of two Boltzmann equations to the data-points; *P<0.05, **P<0.01.

 
Voltage ramps (–100 to +60 mV, duration 20 s) were used to investigate the effect of ox-LDL on the steady state current. The averaged quasi steady state current–voltage relationship elicited by this ramp protocol in control myocytes (n=8/4) and ox-LDL–treated myocytes (n=11/4) are shown in Fig 5. Several alterations due to ox-LDL incubation are clearly visible. Outward current at potentials positive to +10 mV was increased and outward current at negative potentials was decreased or even inward directed. These alterations indicate that ox-LDL does not induce non-specific membrane damage (leaks) but affects several specific ion conductances. Since some of these alterations might be due to reduction of the inward rectifier potassium current (IK1) we studied IK1 as the Ba2+-sensitive current (obtained by digital subtraction). Fig. 6 shows mean IK1 traces elicited by hyperpolarization to –120 mV (n=8/4 for both groups). At this potential, peak IK1 was –36.8 ± 3.03 pA/pF in control and –25.5 ± 0.99 pA/pF in 4 h ox-LDL-incubated myocytes (P<0.05). Outward directed IK1 was also reduced (Ba2+ sensitive current at –60 mV was 1.88 ± 0.17 pA/pF in control myocytes and 1.08 ± 0.16 pA/pF in ox-LDL-incubated myocytes; P<0.05). To elucidate the increase in the steady state current at positive potentials we replaced external and internal KCl by equimolar CsCl, added 200 µM CdCl2 to the external solution and applied the voltage ramp protocol described above. Fig. 7 displays the ox-LDL-induced background current under these conditions (non-selective background current, INS). This trace was obtained by digital subtraction of INS in control myocytes (mean of 7 cells) from INS recorded in ox-LDL-incubated myocytes (n=7/4). This ox-LDL-induced current reverses around –10 mV and displays outward rectification. It has to be kept in mind that INS was recorded in the absence of K+-ions and might be of larger amplitude under physiological conditions.


Figure 5
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  		Fig. 5 Steady-state current–density to voltage relation in control ({circ}) and ox-LDL myocytes (bullet); *P<0.05, **P<0.01.

 

Figure 6
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  		Fig. 6 Ox-LDL decreases inward rectifier current IK1. Ba2+-sensitive inward current (IK1) at –120 mV (holding potential of –40 mV). Traces show means of 8/4 for both groups.

 

Figure 7
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  		Fig. 7 Ox-LDL induces a non-selective background current (INS) in the absence of external and internal K+. This trace was obtained by digital subtraction of INS obtained in control myocytes (means of 7/4) from INS recorded in ox-LDL myocytes (means of 7/4).

 
To study possible effects of ox-LDL on ventricular contractility we recorded contractions in field-stimulated single ventricular myocytes by using an edge detection system. These recordings shown in Fig. 8A, B reveal a significant slowing in the onset (determined as time to peak, TTP) and relaxation (50% and 90% relaxation, i.e. R50, R90) of contraction (n=6/2 for both groups).


Figure 8
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  		Fig. 8 Ox-LDL slows contractile activity of isolated myocytes. (A) Representative normalized contractions of a control and an ox-LDL-incubated myocyte. (B) Mean time to peak (TTP), 50% relaxation (R50), and 90% relaxation (R90) of contraction. *P<0.05, **P<0.01.

 
Since ox-LDL was reported to alter calcium handling in vascular and cardiac myocytes [17–20] we recorded the sodium–calcium exchanger (NCX) as a probe for [Ca2+]i. NCX is thought to transfer 3 Na+ for 1 Ca2+ resulting in a net transfer of one positive charge [for review see: [22]]. Thus, when Ca2+ is extruded from the cell an inward current can be recorded. Since NCX is activated by a rise in intracellular Ca2+ [15–21] INCX may serve as an indicator for alterations in [Ca2+]i. The protocol for recording INCX is illustrated in Fig. 9A (insert) and comprises a voltage ramp from +60 to –100 mV with a duration of 2 s from a holding potential of –40 mV. Application of NiCl2 (5 mM) blocked a large fraction of the ramp response (Fig. 9A) which was regarded as INCX [13,14] as shown in Fig. 9B (control cell). INCX reversed around –30 mV and was inward directed (indicating Ca2+ extrusion) at more negative potentials. The means of INCX current density recorded by this protocol are displayed in Fig. 9C (n=8/3 for each group) and showed no difference between control and ox-LDL myocytes suggesting that neither NCX nor resting calcium (due to buffering by the pipette solution) was altered.


Figure 9
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  		Fig. 9 Recording of INXC. (A) Typical current trace from a control myocyte in response to a descending voltage ramp (see inset) in the absence (control) and presence of NiCl2 (5 mM). (B) Digital subtraction revealed the Ni2+-sensitive current (INCX, data from panel A). (C) Mean current density of I(NCX) in control and ox-LDL myocytes.

 
We then applied the following stimulatory protocol to induce cellular Ca2+ loading. Ten conditioning voltage clamp steps from a holding potential of –40 to 0 mV were applied (step duration 200 ms, interpulse interval 200 ms) to induce repetitive activation of ICaL and were followed by the ramp described above. In control cells, this pacing resulted in a small increase of INCX (Fig. 10A). Pacing of ox-LDL myocytes induced a much larger increase in INCX as shown in Fig. 10B and C where the mean INCX current density after pacing is shown (n=7/2 for control, n=6/2 for ox-LDL). In order to illustrate how a rise in cytosolic Ca2+ may affect INCX we performed a numerical simulation of INCX [15]. To adjust the INCX model to our experimental data, estimators of the two model parameters [Ca2+]i and Vmax were computed by using the least square method. The experimental data (open symbols) and the modeling result (line) are shown in Fig. 10D. The best fit was found at an [Ca2+]i of 200 nM and a Vmax of 30.16 A/F. Vmax was kept constant for all further simulations and [Ca2+]i was estimated by fitting the model to experimental data under pacing conditions (control and ox-LDL). The best fit was found at 219 nM [Ca2+]i for control myocytes (Fig. 10E) and 267 nM for ox-LDL myocytes (Fig. 10F). These data are of qualitative nature supporting our hypothesis of an increase of [Ca2+]i.


Figure 10
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  		Fig. 10 Pacing induced alterations in INCX, experiments and numerical simulation. (A) INCX of a representative control myocyte under control conditions (unpaced) and after pacing. (B) INCX of a representative ox-LDL-incubated myocyte under control conditions (unpaced) and after pacing. (C) Mean INCX current density after pacing in control ({circ}) and ox-LDL-incubated myocytes (bullet). (D) Mean INCX current density in control myocytes (symbols) and the result of the numerical simulation (line). (E) Mean INCX current density in control myocytes after pacing (symbols) and the result of the numerical simulation (line). (F) Mean INCX current density in ox-LDL-incubated myocytes after pacing (symbols) and the result of the numerical simulation (line).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
We demonstrate that ox-LDL induces cellular alterations in cardiomyocytes (cell damage, electrophysiological and contractile alterations) which depend on the content of total LPO but are independent of methods used for oxidation of LDL. A deleterious role of the amount of LPO in the ox-LDL fractions tested has also been reported for ox-LDL-induced apoptosis of smooth muscle cells [6]. LDL copper-oxidized for 4 h and AAPH-oxidized for 24 h were the most cell damaging fractions tested, but showed a completely different negative net charge (as determined by REM). Thus, this parameter, which is assumed to represent the modification of the protein moiety of LDL, does not play a role in the transfer of LPO from the oxidized lipoprotein to the cell membranes.

Our data show that visible cell damage is preceded by contractile and electrophysiological alterations whereby the observed slowing of contractile activity supports the clinical observation of a significant negative correlation between the plasma level of ox-LDL and left ventricular ejection fraction [8]. The observed electrophysiological alterations include EADs, DADs, and spontaneous activity which are known as potential triggers for arrhythmias. Furthermore, ox-LDL induced the activation of a non-selective (cation) background current as well as a reduction of IK1. All these effects have been described for free radical generating systems [33–35] and since ox-LDL is known to induce various radicals [4,32] it can be speculated that these mechanisms play a role for the observed effects. An alteration of electrophysiological parameters has already been described for 4-hydroxynonenal (HNE) [36], a breakdown product of LPO [37], suggesting a common mechanism of ion channel alteration which may take place via redox modification of SH groups [34,38].

An ox-LDL-induced rise in intracellular calcium content ([Ca2+]i) is a consistent finding in endothelial [16,17], smooth muscle [18], and cardiac muscle cells [19] whereby an inositol 1,4,5-triphosphate (IP3)-dependent release of Ca2+ from the sarcoplasmic reticulum underlies this effect in smooth muscle cells [25]. Interestingly, IP3-dependent Ca2+ release is also thought to contribute to electrophysiological alterations and cell death in cardiomyocytes (for review see: [26]) suggesting IP3 as a possible mediator of the observed ox-LDL-induced cardiomyocyte damage.

Although we did not monitor the intracellular calcium content, we present experimental evidence for ox-LDL induced alterations in calcium handling. We report an ox-LDL-induced increase in ICaL [20] as well as an enhanced re-availability of calcium channels assessed by steady-state inactivation characteristics. Both effects may favor an increase in [Ca2+]i and may also account for the observed EADs [27].

Moreover, an increased activity of INCX was seen after pacing induced Ca2+ loading (as also seen in numerical simulations of INCX). This data suggest a slowing of Ca2+-removal from the cytoplasm, whereby an impairment of the sarcoplasmic reticulum calcium ATPase (SERCA) [29], as reported for oxidative stress [31], is the most likely explanation. Such a SERCA impairment would subsequently lead to a decrease of calcium release which may account for the slowing in contraction onset, whereas a possible dysfunction of Ca2+ release [28] may also play a role.

In addition, the complex Na+-homeostasis of cardiac myocytes (for review see: [21]) may have also been altered by ox-LDL. The elucidation of such effects will require further extensive experiments and was thus not considered for the present investigation. In addition, our data do not allow to exclude alterations in calcium sensitivity of myofibrils by ox-LDL which may also play a role for the observed changes in contractility. Oxidized LDL induced a broad spectrum of effects ranging from slight AP-prolongation, severe AP-prolongation, occurrence of EADs, DADs, and abnormal spontaneous activity to cell death, even in the same incubation fraction. It will have to be determined in future experiments whether these different states of damage are linked via a progressive rise in internal calcium content.

The ox-LDL concentration chosen was considerably smaller than that reported in patients [30] but was able to induce the whole range of effects (lack of observable alterations up to severe damage), depending on the LPO concentration. However, myocytes in vivo are likely exposed to ox-LDL for longer durations and therefore even lower concentrations might be able to achieve similar effects.

Recently, a lectine-like ox-LDL receptor (LOX-1) has been shown to be up-regulated in cardiac myocytes during ischemia/reperfusion and activation of LOX-1 played a role for the extent of myocardial injury [39]. Activation of LOX-1 by ox-LDL has also been shown to induce oxidative stress in isolated cardiac myocytes [40] suggesting LOX-1 as a possible mediator of the effects observed in our study.

It is important to note that ox-LDL is chemically not well defined and thus the cell-damaging potential may vary strongly between different ox-LDL preparations [41] and depends on the method of oxidation. Although ox-LDL has been shown to be present in human ventricles [23] it is unknown which concentrations occur in the vicinity of cardiac myocytes. As discussed by Liu et al. [19] LDL cholesterol in the interstitial space, which is in contact with cardiomyocytes, may be in the range of 500 µg/ml (as used in our study) and ox-LDL plasma levels of up to 3.44 mg/dl have been reported in patients with acute myocardial infarction [42]. Since it is generally believed that LDL oxidation takes place outside of the vessel lumen [41], local tissue concentrations (vessel wall or even heart) may be significantly higher than plasma levels. In line with this a significantly higher concentration of ox-LDL was found in coronary sinus plasma compared to the aortic root or peripheral veins [43] of patients with dilated cardiomyopathy. This finding suggests that LDL oxidation may take place in the myocardium, which would further imply a higher concentration in the myocardium than in plasma.

At present we are not able to assign the effects observed in our experiments to individual oxidation products of LDL which were described to act cytotoxic [3,6,24]. Ox-LDL is well known to induce necrosis and apoptosis [5] but since cardiomyocyte damage occurred within 12 h after start of incubation, apoptosis is not likely to play a major role in our experiments.

4.1. Possible limitations
Since a Na+-free pipette solution was used for AP-recording, only sodium entering via sodium current or INCX was present in the cytosol. Thus, all ionic exchange procedures, which depend on intracellular Na+, will not have contributed substantially to the recorded electrical activity. For recording INCX, 10 mM Na+ were added to the pipette solution to ensure reverse mode activity of INCX.

It is important to note that oxidative stress does not result in a stationary alteration of electrophysiological parameters, but is a dynamic and time-dependent process where even opposed effects may occur within a short period of time [44].

In conclusion, our study is the first description of ox-LDL induced electrophysiological alterations and cell damage in cardiac myocytes. Both of these effects were critically depending on the LPO content of ox-LDL. Our data suggest that specific ion channels are first targets of ox-LDL induced myocyte alterations and these changes in excitability may promote further cell damage. The effects described may partly play a role for functional cardiac alterations in patients with elevated ox-LDL levels.


   Acknowledgements
 
The financial support by the Austrian Research Fund (P15403 [GenBank] -Med) is gratefully acknowledged.


   Notes
 
Time for primary review 26 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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