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Am J Physiol Heart Circ Physiol 289: H160-H167, 2005. First published February 25, 2005; doi:10.1152/ajpheart.00895.2004
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Increased tolerance to hypoxic metabolic inhibition and reoxygenation of cardiomyocytes from apolipoprotein E-deficient mice

Martin Dworschak,1,2 Livius V. d'Uscio,1 Dirk Breukelmann,1,3 and James D. Hannon1

1Department of Anesthesia Research, Mayo Foundation, Rochester, Minnesota; 2Division of Cardiothoracic and Vascular Anesthesia and Intensive Care, University Hospital Vienna, Vienna, Austria; and 3Department of Anaesthesiology and Intensive Care, University of Münster, Münster, Germany

Submitted 25 August 2004 ; accepted in final form 23 February 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although hypercholesterolemia is a strong risk factor for cardiovascular disease, it has in some instances paradoxically been associated with reduced infarct size and preserved contractile function in isolated hearts after ischemia and reperfusion. To elucidate potential cellular protective mechanisms, myocytes of hypercholesterolemic apolipoprotein E-deficient (ApoE–/–) and wild-type mice were subjected to hypoxic metabolic inhibition (I) with subsequent reoxygenation (R). Intracellular Ca2+ concentration ([Ca2+]i) and pH (pHi) were monitored as well as cell length and arrhythmic events. Force measurements in papillary muscles were also recorded, and myocardial expression of Na+/H+ exchanger 1 (NHE1) and three Ca2+ handling proteins [sarco(endo)plasmic reticulum Ca2+-ATPase, Na+/Ca2+ exchanger, and plasma membrane Ca2+-ATPase] was quantified. After 30 min of I and 35 min of R, Ca2+ overload was more pronounced in wild-type cells (P < 0.05). In these myocytes, pHi also dropped faster and remained below those values determined in ApoE–/– cells (P < 0.05). Furthermore, more wild-type myocytes remained in a contracted state (P < 0.05). This group also showed a higher incidence of arrhythmic events during R (P < 0.05). No group difference was found in the expression of the Ca2+ handling proteins. However, NHE1 protein was downregulated in hearts of ApoE–/– mice (P < 0.05). Histological results depict hyperplasia in ApoE–/– hearts without atherosclerosis of the coronaries. Contractile dysfunction was not observed in papillary muscles from ApoE–/– hearts. Our results suggest that downregulated myocardial NHE1 expression in hypercholesterolemic ApoE–/– mice could have contributed to increased tolerance to I/R. It remains to be elucidated whether NHE1 downregulation is a unique feature of these genetically altered animals.

hypoxic metabolic inhibition and reoxygenation; intracellular Ca2+ and pH handling; Na+/H+ exchanger; ventricular myocytes

HYPERCHOLESTEROLEMIA is an important risk factor for cardiovascular disease and has to be treated aggressively. Hyperlipemia is usually linked to decreased tolerance to ischemia and loss of the beneficial effect of preconditioning (for a detailed review, see Ref. 9). Paradoxically, some "cardioprotective" effects have occasionally been reported in hearts of hypercholesterolemic animals that were subjected to ischemia and consecutive reperfusion. Long-term hypercholesterolemia with excessively high total serum cholesterol levels (3,200 mg/dl), for example, induced protective mechanisms from ischemia-reperfusion injury in low-density-lipoprotein (LDL) receptor-deficient mice (11). Additionally, after 30 min of global normothermic myocardial ischemia, contractile recovery was improved and creatine phosphokinase release was decreased in isolated hearts from rabbits that were fed a 2% cholesterol diet in relation to hearts from rabbits that received a standard chow (25).

Apolipoprotein E-deficient (ApoE–/–) mice frequently serve as a model to study the effects of hypercholesterolemia on the cardiovascular system. The development and distribution of atherosclerotic plaques in ApoE–/– mice are quite similar to those in humans (45). However, even before microscopic abnormalities are detectable, endothelial dysfunction is present in the aorta and carotid arteries (7, 37). As in humans, hypercholesterolemia-induced endothelial dysfunction in ApoE–/– mice is associated with increased production of superoxide anions and impaired nitric oxide-mediated endothelium-dependent relaxation (5, 6, 31, 44). In addition, it has recently been shown that superoxide formation is elevated in cardiac extracts in ApoE–/– mice (12). Cardioprotection from ischemic preconditioning was also more pronounced in hearts from double knockout ApoE/LDL–/– mice compared with wild-type mice (26).

The particular mechanism through which hypercholesterolemia may potentially convey greater tolerance to ischemia-reperfusion injury is still unresolved. Generation of oxygen or peroxynitrite species could play an important part (11, 32). Reports are accumulating that acute oxidative stress increases the resistance to subsequent myocardial ischemia-reperfusion injury (2, 40).

Several studies have shown that altered Ca2+ and pH handling plays a crucial role in mediating protection against ischemia-reperfusion injury (1, 39, 48, 49). Multiple interactions between reactive oxygen species with cellular ion transport mechanisms have been proposed. This interaction could account for potential changes in the activity of certain ionic transporters that may further affect intracellular Ca2+ and intracellular pH (pHi) handling properties of myocytes of ApoE–/– mice under ischemia-reperfusion stress (1, 22). Furthermore, hypercholesterolemia could alter sarcoplasmic and intracellular membranes by the incorporation of lipids. These changes can additionally modulate the activity of various ion channels.

Studies investigating the cellular response of cardiomyocytes from hypercholesterolemic animals undergoing ischemic injury and recovery are still lacking. We used a slightly modified cellular model of simulated ischemia-reperfusion that has previously been published. It employs hypoxic metabolic inhibition followed by reoxygenation for this purpose (42). With the help of this model, we investigated whether ventricular myocytes from ApoE–/– mice are less vulnerable to hypoxic metabolic inhibition and subsequent reoxygenation [inhibition/reoxygenation (I/R)] than myocytes from wild-type mice and whether intracellular Ca2+ and pHi handling is different. Because anatomic and hemodynamic alterations that are reported in ApoE–/– mice could be potential confounding variables, we also include mechanical, histological, and cytological data (16).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animals. Male wild-type (C57BL/6J) mice and homozygous ApoE–/– (C57BL/6J-ApoETm1Unc) mice were obtained at the age of 4–5 wk from Jackson Laboratories (Bar Harbor, ME). Mice from both groups were fed a high-fat Western-type diet (TD88137, Harlan Teklad; Madison, WI) for 6 mo to accelerate the development of aortic atherosclerosis (45). This diet contains 0.15% cholesterol and 42% milk fat by weight. The hearts of these mice that were used for a different investigation were provided by Dr. Katusic's laboratory (6). Body weight of the mice was measured with triple beam balance (Ohaus; Florham Park, NJ). Experimental protocols were approved by the Institutional Animal Care and Use Committee of the Mayo Clinic, and the investigation conforms with the American Physiological Society's "Guiding Principles in the Care and Use of Animals."

Noninvasive blood pressure measurement. In a subgroup of mice, blood pressure was determined noninvasively by tail-cuff plethysmography before the study (Harvard Apparatus; Kent, UK). Measurements were performed after several trials to accustom the mice to this device (19).

Cholesterol determination. Blood samples were obtained through puncture of the right ventricle. The blood was immediately transferred to a tube containing heparin (1,000 units) and centrifuged at 4°C for 10 min. Plasma was separated immediately at 4°C and kept at –80°C until assayed. Total and LDL cholesterol was determined using a colorimetric-based assay on a Cobas Mira system.

Anatomic and histological parameters. Hearts from each group were cut interventricularly (short-axis view) to give two transverse sections that were hematoxylin-eosin and van Gieson stained. Cell diameter from 55 single ventricular myocytes per group was measured and documented. Furthermore, the degree of myocardial fibrosis of the left ventricular free wall was determined morphometrically after the van Gieson-stained slides were digitized using Adobe Photoshop software. Gross body and heart weights were measured for the calculation of heart-to-body weight ratios. Additionally, hearts were checked for transventricular scars and sclerotic alterations of the aorta as described previously (12). Furthermore, the coronary vessels were screened for obvious occluding thrombi or plaques, intimal thickening, and perivascular fibrosis.

Force measurements. Papillary muscles of the left ventricle were dissected, and isometric force development after electrical stimulation was determined as previously described by our laboratory (18). The stimulation rate was 0.25 Hz, and the voltage applied was 20% above threshold.

Western blot analysis. Briefly, ventricles of murine hearts from mice after a 6-mo high-fat diet were excised and homogenized on ice in lysis buffer [50 mM Tris·HCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% SDS, 0.1% deoxycholate, 1% IGEPAL, and a 1,000-fold dilution of protease inhibitor (Sigma), pH 7.4]. One hundred micrograms of protein per lane were separated by SDS-PAGE and then transferred to a nitrocellulose membrane (Amersham). Mouse monoclonal anti-Na+/H+ exchanger 1 (anti-NHE1; 1:500, Chemicon; Temecula, CA), mouse monoclonal anti-sarco(endo)plasmic reticulum Ca2+-ATPase 2 (anti-SERCA2; 1:2,500, ABR; Golden, CO), rabbit polyclonal anti-Na+/Ca2+ exchanger (1:1,000, Research Diagnostics; Flanders, NJ), anti-plasma membrane Ca2+-ATPase 2a (anti-PMCA 2a; 1:1,000, customized polyclonal rabbit antibody), and mouse monoclonal anti-inducible nitric oxide synthase (anti-iNOS; 1:100; Transduction Laboratories) were used as primary antibodies. Bands were visualized by chemiluminescence (Amersham). Blots were rehybridized with mouse monoclonal anti-actin (1:50,000, Sigma). Optical density was determined by NIH Image 1.26 [National Institutes of Health (NIH); Bethesda, MD] and normalized to the corresponding myocardial actin content (12).

Cardiomyocyte preparation. Mice were anesthetized with pentobarbital (60 mg/kg ip, Nembutal, Abbott Laboratories; North Chicago, IL), and the hearts were rapidly excised. Cardiomyocytes were dissociated as previously described (15). In brief, hearts were perfused via aortic cannulation with a cold, calcium-free oxygenated buffer solution containing (in mM) 75 NaCl, 2.4 KCl, 1 MgCl2, 10 HEPES, 58 sucrose, 10 dextrose, 5 NaHCO3, and 2.5 glutamic acid (pH 7.2 with KOH) until clear of blood. Ventricles were dissected, minced, and washed in the same solution. After 15 min, the tissue was cut into smaller pieces and transferred to a beaker, where 0.7 mg/ml collagenase II (Sigma) and 1 mg/ml BSA (Sigma) had been added to the buffer. The cellular suspension was then placed into a vial and centrifuged for 2.5 min at 35°C and 900 rpm (60 g). The supernatants were withdrawn, and the cell pellet was resuspended in a modified Tyrode solution that was composed of (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 5 HEPES (pH 7.4 with KOH).

Loading with fluorescent indicators. After a resting period, aliquots of the cell suspension were simultaneously loaded at 35°C with two ratiometric dyes (both from Molecular Probes; Eugene, OR): 5 µM fura-2 AM to measure the intracellular Ca2+ concentration ([Ca2+]i) and 1.25 µM 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) to monitor pHi. After an incubation period of 30 min, cardiomyocytes were washed twice with Tyrode solution to remove dye that had not entered the cells. A minimum of 30 min was allowed until the start of the experiments to guarantee sufficient deesterification of intracellular AMs. Cells were kept in Tyrode solution at room temperature until final use.

Fluorescence and cell length measurements. Aliquots of 200 µl of the cell suspension were introduced into a 300-µl flow-through perfusion chamber on the stage of an inverted microscope (Nikon Eclipse TE 300). A laminin-coated coverslip served as the bottom of the chamber. Cardiomyocytes were superfused at 3 ml/min with Tyrode solution, and electrical field stimulation was applied. Cells were stimulated (Grass S88 Stimulator, Grass Instruments; W. Warwick, RI) throughout the whole experiment at 0.25 Hz with a pulse duration of 5 ms and a voltage of 20% above threshold.

Cardiomyocytes were positioned in the light path of the inverted epifluorescence microscope and alternatingly excited at 340 and 380 nm for fura-2 and 450 and 490 nm for BCECF, respectively. Emitted light was collected at 520 nm from a small area around a single fluorescent cell through a x40 oil lens (Nikon S Fluor x40/1.3) by the photomultiplier of the laser system (C&L Instruments; Hummelstown, PA). The BCECF ratio was calibrated using 10 µg/min of the K+/H+ ionophore nigericin, which causes rapid equilibration of extracellular pH and pHi. The pH values of the incubating Tyrode solution served as the calibration standards for the pH signal. To prevent formation of a pH gradient, the Tyrode solution was prepared with 130 mM K+ (38).

[Ca2+]i is expressed as the fura-2 ratio of the light emitted at the two excitation wavelengths (340/380 nm). We prefer to report fura-2 ratios because of problems that arise from in vivo calibration of the fura-2 signal and the changes in Kd of fura-2 during alterations of pHi (36). The fura-2 ratios change in the same direction as [Ca2+]i. The rate constant of the fura-2 transient decay ({lambda}) was determined using the following monoexponential function:

where a and b represent the amplitude of the Ca2+ transient and the resting [Ca2+]i, respectively.

Electrical stimulation of a normal ventricular myocyte causes contraction that is preceded by a distinct Ca2+ transient. Spontaneous, i.e., stimulation-independent, fura-2 transients and stimulations not followed by a normal transient were defined as cellular arrhythmic events. Microscopic images of the cardiomyocytes were recorded by a video camera and stored for later off-line determination of diastolic cell length and cell surface area using NIH Image 1.26. In the case of hypercontracted cells, the dimension of the previous longitudinal axis was used for cell length (38).

Experimental procedures. Only fura-2- and BCECF-loaded, rod-shaped cells without blebs and clear striation that contracted upon electrical stimulation from 10 mice of each group were used for the I/R experiments. They were all carried out at room temperature to minimize the effect of cellular processes that transport negatively charged Ca2+ indicators from the cell. In addition, increased metabolism and faster intracellular Ca2+ accumulation by higher temperatures and stimulation rates would result in earlier cell death and preclude observation of group differences that develop at a slower pace.

The detailed I/R protocol has already been described elsewhere (8). In brief, hypoxic conditions (PO2 < 15 mmHg) were achieved by vigorously bubbling the solution with nitrogen and introducing nitrogen under the hood of the perfusion chamber as has been demonstrated by Henderson and Brutsaert (17). PO2 of the perfusate in the chamber was measured in a gas analyzer (ABL). Metabolic inhibition was initiated by lowering the pH of the glucose-free Tyrode solution to 6.3 and adding 10 mM deoxyglucose to reversibly inhibit glycolysis. After 30 min of hypoxic metabolic inhibition, cardiomyocytes were again reoxygenated at normal ambient PO2 and pH (7.4) for another 35 min with superfusion of a Tyrode solution that again contained glucose (42).

Statistical analysis. ANOVA for repeated measures, two-way ANOVA, {chi}2-tests, and unpaired Student's t-tests were used for statistical analysis. Appropriate post hoc tests (Bonferoni or Tukey test) were performed when statistically significant differences were obtained by ANOVA. Point-to-point comparisons were made with t-tests, and Sigma Stat 2.0 was the software we employed. P < 0.05 was considered statistically significant. Data are presented as means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Noninvasive blood pressure measurements. Noninvasive systolic, mean, and diastolic blood pressures were not different between ApoE–/– and wild-type mice (Table 1).


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  		Table 1. Anatomic and histological characterization

 
Serum cholesterol levels. Total and LDL serum cholesterol was significantly higher in ApoE–/– mice [24.6 ± 3.6 vs. 6.3 ± 0.5 mmol/l in wild-type mice (P < 0.05) and 15.7 ± 2.8 vs. 0.8 ± 0.1 mmol/l in wild-type mice (P < 0.05), respectively].

Anatomy and histology data. The body weight of wild-type mice was significantly greater than that of ApoE–/– mice (P < 0.001; Table 1), whereas heart weight was significantly lower in this group (P < 0.05; Table 1). Consequently, the heart-to-body weight ratio of ApoE–/– mice was two times higher than controls (P < 0.001; Table 1). The diameter of the ApoE–/– hearts at the midventricular transverse section was also significantly greater than that of controls (P < 0.05; Table 1), and the interventricular septum was thicker in ApoE–/– mice (P < 0.0001; Table 1). In contrast, cell length, cell surface area, and cell diameter (and thus also cell volume) were not different between groups (Table 1). In the 10 hearts that were examined per group, no increased subendocardial fibrosis or transmural scarring was evident in the van Gieson-stained hearts in both groups (Fig. 1).



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  		Fig. 1. Hematoxylin-eosin-stained cross sections of wild-type (WT; A) and apolipoprotein E-deficient (ApoE–/–; B) hearts (original magnification x25) with representative van Gieson-stained epicardial coronary vessels with perivascular tissue from the corresponding WT (C) and ApoE–/– (D) hearts (original magnification x100). No significant signs of chronic ischemia, perivascular fibrosis, or luminal narrowing of coronaries could be observed in hearts from ApoE–/– mice.

 
Furthermore, there were no vascular alterations (intimal thickening, perivascular fibrosis, and atherosclerotic plaques) or thrombotic material visible in epicardial coronaries and smaller coronary vessels from wild-type and ApoE–/– mice (Fig. 1). However, multiple atherosclerotic plaques were detectable in the aortic arch as well as throughout the thoracic and abdominal aorta of ApoE–/– mice (data not shown). Such extended lesions in the aorta have previously been described by other investigators in the same strain being fed a Western-type diet for 24 wk (23).

Force measurements. Although peak isometric force was slightly lower in papillary muscles from hearts from ApoE–/– mice, the difference did not reach statistical significance (P = 0.772). The time to peak force was nominally and the time to 50% peak force after electrical stimulation was significantly longer (P = 0.042) in papillary muscles of ApoE–/– mice (Table 2).


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  		Table 2. Isometric force measurements in mouse papillary muscle

 
Western blot analysis. There was no apparent difference in the expression of the three major intracellular Ca2+ handling proteins, SERCA2, Na+/Ca2+ exchanger, and PMCA 2a (Fig. 2) even when protein content was normalized to the corresponding myocardial actin content (data not shown). Additionally, expression of iNOS was similar in ventricular tissue of ApoE–/– mice compared with wild-type mice. A significant difference, however, was found in the expression of NHE1, which was downregulated in hearts of ApoE–/– mice (P < 0.05; Fig. 2).



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  		Fig. 2. Western blot analysis of Na+/H+ exchanger 1 (NHE1), sarco(endo)plasmic reticulum Ca2+-ATPase 2 (SERCA2), Na+/Ca2+ exchanger (Na/Ca Ex), plasma membrane Ca2+-ATPase 2a (PMCA 2a), inducible nitric oxide synthase (iNOS), and actin determined in myocardial ventricular tissue of WT (lanes 1–3, n = 3) and ApoE–/– mice (lanes 4–6, n = 3). The corresponding molecular masses (in kDa) are shown at the righthand side of each lane. Significant downregulation of NHE1 was detected in hearts of ApoE–/– mice (P < 0.05, unpaired t-test).

 
Resting and peak cytosolic [Ca2+]i and amplitude and rate constant of the Ca2+ transient. In both groups, we observed a slow but steady rise of resting, end-diastolic fura-2 ratio during I/R. The increase of resting [Ca2+]i was significantly more pronounced in the wild-type group, particularly during reoxygenation (P < 0.05; Fig. 3A).



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  		Fig. 3. A: changes of resting cytosolic intracellular Ca2+ concentration ([Ca2+]i) over time depicted as the fura-2 ratio (340/380 nm). The abscissa indicates the corresponding time (in min). A more marked rise of diastolic cytoplasmic [Ca2+]i can be seen during hypoxic metabolic inhibition and subsequent reoxygenation in the WT group (n = 26) compared with the ApoE–/– group (n = 29, *P < 0.05 by ANOVA). B: peak fura-2 ratio (340/380 nm) during inhibition and reoxygenation. Peak [Ca2+]i values increase to a greater extent in the WT group during reoxygenation (*P < 0.05 by ANOVA).

 
Peak [Ca2+]i followed a similar pattern as diastolic [Ca2+]i, also showing higher Ca2+ concentrations in the wild-type group during reoxygenation (P < 0.05; Fig. 3B).

The amplitude of the Ca2+ transient, i.e., the difference between peak and resting end-diastolic Ca2+ level, was not significantly different between the two groups at baseline. It decreased in both groups during hypoxic metabolic inhibition, which was more pronounced in the ApoE–/– group (P < 0.05; Fig. 4). A brief overshoot was noted immediately after institution of reoxygenation in both groups until 10 min of reoxygenation. However, it leveled out toward the end of the reoxygenation phase without apparent group differences. The rate constant of the Ca2+ transient decay, i.e., cytosolic Ca2+ removal during diastole, was higher in ventricular myocytes from ApoE–/– mice at baseline (P < 0.05). During ischemia, it initially decreased in both groups but then increased again to approximately baseline values during reoxygenation, however, without significant group differences. Figure 4 further shows an example of excessive Ca2+ overload in a hypercontracting myocyte of the wild-type group, where rigor contractions occurred more often.



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  		Fig. 4. Ca2+ transients from a myocyte of the WT (top) and ApoE–/– groups (bottom) at baseline (left), at 30 min of hypoxic metabolic inhibition (middle), and reoxygenation (right), respectively. At baseline, the amplitude of the Ca2+ transient was not different between groups; however, the rate of the Ca2+ transient decay was higher in ApoE–/– myocytes. ApoE–/– cells also showed a lower incidence of arrhythmic events during hypoxic metabolic inhibition and reoxygenation. Furthermore, typical arrhythmic events that occurred in both groups can be seen exemplary in the depicted myocyte from the WT group (top middle and right). Also note the different scale in the top middle and right as cytosolic [Ca2+]i rose rapidly in this particular myocyte before it hypercontracted.

 
Changes in pHi. There was no significant difference in baseline pHi before the initiation of I/R (P = 0.21). During hypoxic metabolic inhibition, pHi dropped accordingly. It decreased significantly from baseline in both groups within the first few minutes of hypoxic metabolic inhibition. However, this decline occurred faster in wild-type myocytes (P < 0.05). During reoxygenation, it showed a better recovery in ApoE–/– myocytes. In the wild-type group, it did not reach the baseline value again but remained at a lower level (P < 0.05; Fig. 5A).



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  		Fig. 5. A: temporal changes in pHi during hypoxic metabolic inhibition and reoxygenation. The abscissa indicates the corresponding time (in min). During hypoxic metabolic inhibition, pHi declined to a much greater extend in the WT group (n = 26) and did not recover to the same degree as pHi in the ApoE–/– group (n = 29) during reoxygenation (*P < 0.05 by ANOVA). B: time course of end-diastolic cell length during simulated inhibition and reoxygenation. Mean baseline cell length during complete relengthening was 97 µm in both groups. It decreased to a significantly greater extent by hypoxic metabolic inhibition and reoxygenation in the WT group (n = 26) compared with ApoE–/– myocytes (n = 29, *P < 0.05 by ANOVA).

 
Changes of relative cell length in the course of I/R. Enzymatic dissociation produced the same yield of contracting, morphologically normal ventricular myocytes in both groups. The initial cell length was ~97 µm and not different between groups. Loading the ventricular myocytes with fura-2 and BCECF neither affected systolic cell shortening nor diastolic relaxation. Diastolic relengthening was decreased and remained depressed in both groups after the initiation of hypoxic metabolic inhibition and subsequent recovery. However, diastolic cellular relaxation was more severely impaired in the wild-type group (P < 0.05; Fig. 5B).

We also noted a higher incidence of hypercontracture, i.e., diastolic cell length < 55% of initial cell length, in the wild-type group (31% vs. 21%, P < 0.05; Fig. 4). Rigor contraction, which preceded imminent autolysis of the myocyte, occurred exclusively during reoxygenation.

Arrhythmia. The incidence of arrhythmia detected in myocytes from the ApoE–/– and wild-type groups is given in Fig. 6.



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  		Fig. 6. Percentage of arrhythmic events in each group throughout the course of simulated inhibition and reoxygenation. *P < 0.05 by unpaired t-test, ApoE–/– vs. WT.

 
As can be seen, arrhythmia was most pronounced during hypoxic metabolic inhibition, with only insignificant group differences at each time point including baseline. During reoxygenation, however, a greater percentage of wild-type myocytes remained arrhythmic (P < 0.05 by two-way ANOVA), i.e., they either showed stimulation independent Ca2+ transients (Fig. 4, top middle) or no transient despite electrical stimulation (Fig. 4, top right).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This is the first study to examine the effects of I/R on isolated cardiomyocytes of chronically hypercholesterolemic ApoE–/– mice. In this investigation, we demonstrated that single ventricular myocytes from these severely hyperlipemic ApoE–/– mice are more resistant to immediate injury from I/R. Cytosolic Ca2+ accumulation, development of intracellular acidosis, reoxygenation arrhythmia, and hypercontraction in the course of I/R are prevented or delayed in cardiomyocytes of these animals.

Most, if not all, of these observations can be explained by downregulation of NHE1 in myocardial tissue of ApoE–/– mice. There is abundant evidence that inhibition of NHE1 protects against ischemia-reperfusion injury (20). Additionally, is has been shown that mice with a null mutation of the NHE1 are resistant to ischemia-reperfusion injury (43). Now, the question arises if NHE1 downregulation is specific to the heart of the genetically altered ApoE–/– mouse or a general phenomenon in the setting of hypercholesterolemia with concomitant chronic oxidative stress and endothelial dysfunction. NHE not only regulates pHi, but it also modifies cell growth and proliferation (20). Therefore, downregulation of the antiporter could possibly block myocardial hypertrophic stimuli in ApoE–/– mice. On the other hand, chronic intermittent hypoxia and ischemic preconditioning also decreases expression of NHE1, whereas myocardial ischemia and acute exposure to H2O2 and ·OH cause upregulation (4, 10, 27). Furthermore, hypercholesterolemia by altering membrane cholesterol and increasing the LDL fraction can additionally inhibit NHE1 activity (30, 34). Despite decreased NHE1 activity, Zicha and co-workers (47) found no difference in basal pHi and the buffering capacity in platelets from hereditary hypertriglyceremic rats compared with controls. This would explain why there was no difference in baseline pHi between ApoE–/– and wild-type myocytes in our investigation. However, with downregulated and potentially inhibited NHE1, one would expect a more severe intracellular acidosis in myocytes from ApoE–/– mice during simulated ischemia as NHE1 removes intracellular H+ in exchange for extracellular Na+. Contrary to this expectation, acidosis was attenuated in this study, which may primarily be explained by the fact that the cells were superfused with an acidic Tyrode solution during hypoxic metabolic inhibition that provokes reverse-mode activity of the NHE1 just as during metabolic acidosis (21). Additional pH-regulating mechanisms transmitted via the Na+/HCO3– symporter and the Cl–/HCO3– antiporter, although not studied here, could also have contributed to the pH changes we determined in the course of simulated ischemia. Mitigated acidosis in ApoE–/– myocytes at the beginning of reoxygenation, however, leads to a decreased intracellular Na+ load. Lower intracellular Na+ concentration reduces Ca2+ overload because the Na+/Ca2+ exchanger works in the reverse mode during reoxygenation, which consequently protects against diminished diastolic relengthening, hypercontracture, and arrhythmia.

Interestingly, these ionic alterations in cardiomyocytes from ApoE–/– mice are strikingly similar to those described by Steenbergen and colleagues (39) after preconditioning. Preconditioning in their study attenuated the increase in [Ca2+]i and the drop in pHi during ischemia. Although intriguing, there are several indexes that speak against preconditioning in ApoE–/– mice. First, compared with the atherosclerotic aortas of these mice (6), we did not find increased expression of iNOS, a marker of late preconditioning, in ApoE–/– hearts. Second, overproduction of oxygen free radicals and endothelial dysfunction in postinfarct remodeled hearts did not induce a better myocardial tolerance against infarction (29). Furthermore, hearts would probably show some form of tachyphylaxis when being chronically preconditioned (41).

No measurable upregulation of protein expression of PMCA 2a, Na+/Ca2+ exchanger, and SERCA2 could be detected in ApoE–/– mice. As myocardial dysfunction can "normalize" previously altered protein content, we also determined isometric force development upon electrical stimulation in papillary muscles of both groups. However, no decreased force development in ApoE–/– mice could be demonstrated. Because force = mass x acceleration, the time to reach peak and particularly 50% peak force was consequently longer in the larger papillary muscles of ApoE–/– mice in our study. These observations are in line with those reported by Kuhlencordt et al. (23), who did not report differences in fractional shortening between hearts from ApoE–/– and wild-type mice. Thus "pseudonormalization" most likely has not occurred.

Similar to rats, SERCA2 seems to be the most important protein for Ca2+ sequestration in mice (46). Before the initiation of cellular relaxation, Ca2+ is on the one hand extruded from the myocyte via PMCA 2a and, more importantly, the Na+/Ca2+ exchanger and, on the other hand, and quantitatively to a much greater extent, sequestered by SERCA2. As the protein content of these major Ca2+ handling proteins was equal in both groups, less Ca2+ accumulation in ApoE–/– mice can thus only be explained by an increased activity of extruding or sequestering proteins or a decreased influx. As already mentioned, chronic oxidative stress has been observed in hypercholesterolemic ApoE–/– mice and results in endothelial dysfunction (5). Moreover, increased superoxide anion formation has recently been detected in cardiac extracts from ApoE–/– mice (12). Oxidative stress can modify the activity of various ion transporters (1, 14, 22). Determining activity levels, however, was not in the scope of this investigation. Therefore, we cannot answer the question regarding the actual activity of myocardial proteins.

Apart from the diminished Ca2+ influx modulated by NHE1 during reoxygenation, oxygen radicals are reported to depress L-type Ca2+ currents and alter the function of many regulatory proteins that may consequently result in less Ca2+ efflux from the sarcoplasmic reticulum and less Ca2+ cycling, which could prevent a substantial rise of [Ca2+]i (22). Lower amplitudes of Ca2+ transients during hypoxic metabolic inhibition in ApoE–/– myocytes, i.e., less Ca2+-induced Ca2+ release from the sarcoplasmic reticulum, could also be recorded in this investigation. Additionally, cytosolic Ca2+ removal was augmented in ApoE–/– cells as depicted by a higher rate constant of the Ca2+ transient decay at baseline. The superoxide anion, for example, has been shown to activate SERCA (14). Enhanced Na+/Ca2+ exchange in ventricular myocytes has also been induced by free radicals (13). Interestingly in this regard, cholesterol incorporation in sarcolemmal vesicles stimulated Na+/Ca2+ exchange and thus facilitated Ca2+ extrusion (24). In the present study, Ca2+ removal declined during early ischemia while [Ca2+]i increased in both groups. This general finding can be explained by a critical fall of the energy supply of the cell after this brief period of ischemia as SERCA function is ATP dependent (3).

Besides the decreased expression of NHE1 and the prooxidative environment that has been reported in ApoE–/– mice, various other factors could potentially induce similar protective mechanisms. Myocardial hypertrophy, for example, is associated with impaired coronary vascular reserve (33). Although the hearts of ApoE–/– mice weighed more than controls and the heart-to-body weight ratio was significantly greater in these animals, the cell volume of cardiomyocytes was not significantly different from controls. This would be uncommon in myocardial hypertrophy. Therefore, hyperplasia seems to be the major reason for this disproportionate heart-to-body weight ratio (33). Stimulation of cell proliferation by oxygen radicals has previously been reported (35). Hyperplasia, however, is not accompanied by a decreased ratio of capillary surface area to myocyte cell volume (33). Furthermore, we could neither observe luminal narrowing of epicardial and smaller coronary vessels nor any alterations in the vascular wall of these vessels despite the marked arteriosclerosis that was found throughout the aortic wall. These findings are in accordance with results obtained by other investigators in the same species (23). In addition, there were no signs of previous infarctions evident as increased fibrosis of the ventricular endocardial layer or transmural scars visible on the epicardium. Therefore, we assume that major flow-limited myocardial ischemia did not predominate in ApoE–/– mice. Because blood pressure was identical in both groups, increased afterload in these animals can be ruled out as well. Severe hypercholesterolemia does, however, affect blood viscosity, which yet should not determine afterload to a great extent. Both of these factors could induce preconditioning. As already mentioned, preconditioning most likely was not responsible for the increased resistance to I/R stress of cardiomyocytes from ApoE–/– mice. However, another interesting observation in this context was made by Mathew and Lerman (28), who found endothelial dysfunction in a model of hypercholesterolemic pigs to be associated with an enhanced response of coronary ATP-sensitive K+ channels that do not only modulate the coronary vasomotor tone but facilitate ischemic preconditioning as well. This could explain why the effect of ischemic preconditioning was most pronounced in hearts from ApoE/LDL–/– mice compared with wild-type mice in the study by Li and colleagues (26).

In conclusion, ventricular myocytes from severely hypercholesterolemic ApoE–/– mice are more tolerant to I/R. Decreased expression of NHE1 in myocardial tissue of these mice could account for this finding. In addition, endothelial dysfunction induced by a prooxidative environment could also precipitate cellular alterations and chronic oxidative stress may further directly modify the function of Ca2+ and pHi handling proteins. The cause of NHE1 downregulation in ApoE–/– mice remains to be determined. If it should be unique to these genetically altered animals, ApoE–/– mice may not be a useful model to study myocardial alterations in hypercholesterolemia.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institute of General Medical Sciences Grant GM-57891 (to J. D. Hannon), by institutional funding from the Division of Cardiothoracic and Vascular Anesthesia and Intensive Care, University of Vienna, Austria (to M. Dworschak), and the Mayo Foundation. J. V. d'Uscio is a recipient of a stipend from the Swiss National Sciences Foundation and a postdoctoral fellowship from the American Heart Association, Northland Affiliate.


   ACKNOWLEDGMENTS
 
We thank Dr. David X. Sun for performing the Western blot for PMCA 2a and Dr. Y. S. Prakash for allowing us the use of the Na+/Ca2+ exchanger antibody and for stimulating discussions. We are indebted to Dr. Katusic for providing us with the murine hearts.

Results from this study were presented in part at the annual meeting of the European Society of Cardiology 2003, Vienna, Austria, and published as an abstract (Eur Heart J 24: 1318, 2003).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Dworschak, Div. of Cardiothoracic and Vascular Anesthesia and Intensive Care, Univ. Hospital Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria (E-mail: martin.dworschak{at}meduniwien.ac.at)

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


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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