INTRODUCTION

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IT HAS BEEN WIDELY ACCEPTED that the cardiac sarcolemmal Na⁺/Ca²⁺ exchanger gene represents an important exchange mechanism for Ca²⁺ and Na⁺ transport across the sarcolemma. The Na⁺/Ca²⁺ exchanger gene can operate in both a forward mode (Ca²⁺ out, Na⁺ in) and a reverse mode (Na⁺ out, Ca²⁺ in), and the Na⁺/Ca²⁺ exchanger does so with a stoichiometry of 3:1; i.e., it exchanges 3 Na⁺ ions for every 1 Ca²⁺ ion (22). In rodent myocardium, the sarcoplasmic reticulum (SR) is thought to handle ~90% of cellular Ca²⁺ during a contraction-relaxation cycle, whereas up to 10% of Ca²⁺ movement can be attributed to the Na⁺/Ca²⁺ exchanger gene (5). However, in failing hearts, the Na⁺/Ca²⁺ exchanger expression and function are changed. Myocardium from dogs with pacing-induced heart failure exhibits significant downregulation of the SR Ca²⁺-ATPase (SERCA), whereas the Na⁺/Ca²⁺ exchanger protein is increased twofold compared with control animals (20). In human heart failure, the expression of Ca²⁺ regulatory proteins has been shown to be abnormal with decreased levels of SERCA and increased levels of the Na⁺/Ca²⁺ exchanger protein (12, 23). Given that SR function is decreased and Na⁺/Ca²⁺ exchanger function is increased during heart failure, Ca²⁺ influx via the reverse mode and Ca²⁺ efflux via the forward mode of the Na⁺/Ca²⁺ exchanger may become a more significant process in the contraction and relaxation of failing myocytes. Increased Ca²⁺ influx via reverse-mode exchange activity during the action potential could be a source of intracellular Ca²⁺ to help supplant the SR as a mechanism for the activation of the myofibrils. Litwin and Bridge (14) suggested that an increase of Na⁺/Ca²⁺ exchanger proteins in infarcted rabbit hearts might promote Ca²⁺ entry and enhance SR Ca²⁺ loading and release in damaged heart. However, the functional significance of enhanced gene expression of the Na⁺/Ca²⁺ exchanger in postinfarcted myocardium remains speculative. The availability of specific enhanced gene expression of the cardiac Na⁺/Ca²⁺ exchanger in transgenic (TG) mice (1) provides an opportunity to investigate the effects of overexpression of the Na⁺/Ca²⁺ exchanger gene in postinfarcted cardiac dysfunction. The purpose of this study was to investigate myocardial function and SR Ca²⁺ content in TG mice with overexpression of the Na⁺/Ca²⁺ exchanger gene compared with wild-type (WT) mice 5 wk after myocardial infarction.

	MATERIALS AND METHODS

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Animals. The study was composed of TG mice with overexpression of the Na⁺/Ca²⁺ exchanger (n = 20) and WT mice (n = 24). The investigation conformed to the Guide for the Care and Use of Laboratory Animals, DHEW Publication No. (NIH) 85-23, Revised 1986, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20205. Heterozygous TG mice used in this study were developed and characterized by Philipson's group (1). The Na⁺/Ca²⁺ exchanger activity in TG hearts was shown to be 150-300% of that in WT mice (1).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Hemodynamics (A) and isometric contractility to cumulative isoproterenol stimulation (B) from transgenic (TG) and wild-type (WT) mouse myocardium at baseline without myocardial infarction (MI; n = 5 for each). No differences were found in both hemodynamics and isometric contraction between TG and WT mouse hearts. LVSP, left ventricular (LV) systolic pressure; LVEDP, LV end-diastolic pressure; +dP/dtmax, maximum rising rate of LV pressure.

Electrocardiogram measurement. Electrocardiograms (ECGs) were recorded in conscious WT and TG mice with Na⁺/Ca²⁺ exchanger gene overexpression at 2 days, 3 wk, and 5 wk after MI as in our previous report (7). Briefly, mice were gently positioned on an ECG recording platform embedded with an array of gel-coated ECG pads spaced to provide contact between the pads and the animals' paws (AnonyMOUSE ECG System, Mouse Specifics; Boston, MA). The signals were digitized with 12-bit precision at a sampling rate of 2,000 samples/s. Only data from continuous recordings of 20-30 ECG signals were used in the analyses. Data were transmitted to the mousespecifics.com Internet site using standard file-transfer protocols for ECG signal analyses by e-MOUSE. Fourier analyses and linear time-invariant digital filtering of frequencies <2 and >100 Hz were used to minimize signal environmental disturbances. The software plots its interpretation of the P, Q, R, S, and T waves for each beat so that spurious data resulting from unfiltered noise or motion artifacts may be rejected.

MI and infarct size determination. Five weeks after MI and before death, in vivo left ventricular (LV) pressure measurements were performed using a previously described method (8). With the mice under pentobarbital sodium anesthesia (60 mg/kg ip), the LV apex was punctured with an 18-gauge needle that was connected to a Statham pressure transducer (Gould Instruments; Eastlake, OH) via short, stiff, polyethylene tubing. LV systolic pressure (LVSP) and end-diastolic pressure (LVEDP) and the maximum rising rate of LV pressure (+dP/dt_max) were recorded.

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Rapid cooling contractures and protein levels of SERCA2 and Na+/Ca²⁺ exchanger. Rapid cooling contractures (RCCs) were used to investigate the Ca²⁺ content of the SR (3, 4, 6, 15) in muscle preparations isolated from another cohort of animals (TG, n= 4; WT, n = 6 mice). Briefly, the posterior LV papillary muscle was dissected and fixed to a muscle holder with a spring clip and then connected to a force transducer. Bath temperature was measured on a chart-strip recorder. RCCs were evoked by a rapid switch to low-temperature superfusate via valves at the chamber inlet. The cold solution was maintained at 2°C by a cooling bath (RMT, MGW Lauda; Brinkmann Instrument), which cools the solution and surrounds the tubing that is connected to the bath chamber. With this setup, it is possible to cool the surface of a muscle to 5°C in 300 ms and the core of a muscle with a diameter of 400 µm in <2 s (3, 6). Muscles were not stimulated during the cooling period. To investigate the influence of rest, rest intervals between 2 and 240 s (2, 10, 30, 60, 120, and 240 s) were conducted from a basal stimulation rate of 0.5 Hz. The first twitch after resumption of stimulation (postrest twitch) was compared with the last steady-state twitch. Additionally, RCCs induced after the same rest intervals (i.e., 2, 10, 30, 60, 120, and 240 s) were compared with the RCC induced 2 s after interruption of 0.5-Hz stimulation.

Statistical analysis. All values are presented as means ± SE. Data were evaluated by one-way ANOVA with repeated measurements. Differences between individual groups were compared using unpaired Student's t-test. Survival during the 5-wk trial was analyzed by standard Kaplan-Meier analysis, and a statistical comparison between survival curves was made using the log-rank test. The criterion for statistical significance was accepted at the level of P < 0.05.

	RESULTS

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Two days after MI, the ST segment recorded in conscious mouse hearts was significantly depressed in both WT and TG mouse hearts (Fig. 2). No significant ventricular arrhythmia was found in the mice during the measurement. The depressed ST segment was persistent at 3 and 5 wk after MI (data not shown). There is a trend for mortality to be reduced in TG (3/15) compared with WT (4/13) mice, which the Kaplan-Meier test suggested to have weak significance (² = 3.6; P = 0.08). Additional animals will be required to see if overexpression of Na⁺/Ca²⁺ exchanger could reduce the post-MI mortality with long-term follow up.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Electrocardiogram from a conscious postinfarcted WT mouse (A) and a conscious postinfarcted TG mouse (B) with overexpression of the Na+/Ca2+ exchanger gene. ST segments were significantly depressed in both WT and TG mice.

Five weeks after MI, there was no significant difference in body weight and heart weight between WT and TG mice [body wt: WT, 37 ± 3.3; TG, 33 ± 3.5 g; P = not significant (NS); heart wt: WT, 0.24 ± 0.02; TG, 0.24 ± 0.01 g; P = NS; heart wt-to-body wt ratio: WT, 6.7 ± 2.2; TG, 7.5 ± 1.3 mg/g; P = NS]. Additionally, infarct size was similar in both groups (WT, 36.2 ± 1.8; TG, 34.5 ± 1.6%; P = NS). Compared with the WT mouse hearts, hearts with overexpression of the Na⁺/Ca²⁺ exchanger gene demonstrated significantly greater LVSP and +dP/dt_max values as well as significantly lower LVEDP values at 5 wk after MI (Fig. 3).

View larger version (49K): [in this window] [in a new window]   Fig. 3.   Continuous chart-strip recordings of hemodynamic measurements (A) in postinfarcted mice from a WT mouse and a TG mouse with overexpression of the Na+/Ca2+ exchanger gene. Summarized data are presented (B). Overexpression of the Na+/Ca2+ exchanger gene significantly attenuated ventricular dysfunction compared with WT mice. Measurements were performed in 6 mouse hearts for each group; *P < 0.05, TG vs. WT.

The baseline values of isometric contraction in papillary muscles isolated from TG and WT mouse hearts were not significantly different (see Fig. 1). However, -adrenergic stimulation with cumulatively increasing concentrations of isoproterenol induced a pronounced increase in developed tension in muscle preparations isolated from TG hearts with overexpression of the Na⁺/Ca²⁺ exchanger gene (Fig. 4). In contrast, papillary muscle isolated from WT mouse hearts had only a mildly positive inotropic response to isoproterenol stimulation.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Inotropic response of papillary muscles to isoproterenol stimulation. Original representative recordings (A) show inotropic responsiveness during isoproterenol stimulation in papillary muscles isolated from a WT mouse and a TG mouse 5 wk after MI. Summarized data are shown (B). Measurements were conducted in 6 papillary muscles for each group; *P < 0.05, TG vs. WT.

Normalized change to the values obtained from steady-state conditions (i.e., 2-s rest interval) of postrest twitch force and postrest RCCs were shown in Fig. 5. Isometric twitch force increased in both TG and WT muscle preparations after 10 s of rest and was more pronounced after 30 s of rest. The twitch force in papillary muscles from TG mice became greater after 60 s of rest and reached maximal amplitudes after 120 s of rest. In contrast, the twitch force in muscles isolated from WT mice was reduced after 60 s of rest and showed a further decrease after 120 s of rest. Similarly, RCC amplitudes in muscle strips from TG mice were enhanced with an increase of the rest intervals. However, twitch force of the papillary muscles isolated from WT mice were continuously reduced with an increase of the rest period. The greater amplitudes of RCCs in papillary muscles from TG mice were significantly different from those from the muscle strips from WT mice.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Rest-dependent changes in isometric twitch force (A) and rapid cooling contractures (RCCs; B) in papillary muscles isolated from TG (n = 4) and WT (n = 6) mouse hearts 5 wk after MI. All values are normalized to the values measured at steady-state conditions (i.e., 2-s rest intervals). *P < 0.05, TG vs. WT.

The cardiac protein levels of SERCA2 and Na⁺/Ca²⁺ exchanger after MI were measured with Western blotting in additional ventricles from TG (n = 4) and WT (n = 6) mice. As shown in Fig. 6, there was no significant change in SERCA2 levels between TG and WT left ventricles 5 wk after MI. As expected, a strong immunoreactivity was observed in TG cardiac tissues when we used anti-Na⁺/Ca²⁺ exchange antibodies, which confirmed overexpression of the Na⁺/Ca²⁺ exchanger.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Protein levels of sarcoplasmic reticulum Ca2+-ATPase-2 (SERCA2; A) and Na+/Ca2+ exchanger (B) from TG (n = 4) and WT (n = 6) left ventricles 5 wk after MI were assessed using the Western blot technique (top). Densitometric analyses of SERCA2/GAPDH and Na+/Ca2+ exchanger/GAPDH are shown separately (bottom). *P < 0.05, TG vs. WT.

	DISCUSSION

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The present study demonstrates that enhanced expression of the Na⁺/Ca²⁺ exchanger gene attenuates cardiac dysfunction after MI. Our findings in TG mice engineered to have increased abundance of the Na⁺/Ca²⁺ exchanger gene point to the functional relevance of upregulation of the Na⁺/Ca²⁺ exchanger gene in a pathological setting in which the SR Ca²⁺-regulating function is impaired. The inotropic response to -adrenergic stimulation was better preserved in papillary muscles isolated from postinfarcted TG than WT mouse hearts 5 wk after MI. The greater rest-dependent twitch force and the SR Ca²⁺ content, which are reflected by RCCs, might explain the beneficial effects on cardiac function with overexpression of the Na⁺/Ca²⁺ exchanger gene after MI.

It has been widely accepted that intracellular Ca²⁺ plays a central role as a second messenger in cardiac excitation-contraction coupling (18, 19). There is accumulating evidence to suggest that reduced expression and/or function of SERCA2 are major changes that contribute to altered Ca²⁺ homeostasis in failing hearts from postinfarction rats (2) and failing human myocardium (23). Enhanced Ca²⁺ efflux by the Na⁺/Ca²⁺ exchanger has been suggested to partially compensate impaired diastolic Ca²⁺ removal in failing human myocardium (13). Terracciano et al. (25) reported that the Na⁺/Ca²⁺ exchanger reverses and brings Ca²⁺ into mouse ventricular myocytes during the latter part of the decline in the intracellular Ca²⁺ transient. Furthermore, TG mice with overexpression of the Na⁺/Ca²⁺ exchanger revealed significant enhancement of the SR Ca²⁺ content in mouse ventricular myocytes (25). However, functional performance of injured ventricular function with overexpression of the Na⁺/Ca²⁺ exchanger is still unclear.

Our previous study showed no differences in peak systolic or diastolic intracellular Ca²⁺ levels at baseline between WT and TG mouse hearts with overexpression of the Na⁺/Ca²⁺ exchanger (11). The present study confirmed that there is no difference in myocardial function at baseline between TG and WT mouse hearts. Normal handling of intracellular Ca²⁺ was preserved in TG hearts during the early stage of ischemia but was significantly disturbed in WT hearts (11). Additionally, TG hearts maintained 40% of pressure-generating capacity during early ischemia, whereas WT hearts maintained only 25%. Our present results confirmed the hypothesis that overexpression of the Na⁺/Ca²⁺ exchanger attenuates cardiac dysfunction after MI although the infarct size was similar between TG and WT mouse hearts 5 wk after MI. It has been recognized that there are abnormalities in the release and reuptake of intracellular Ca²⁺ by the SR in surviving myocardium after MI (12, 18, 19). Failure to adequately augment intracellular Ca²⁺ availability may contribute to the impaired inotropic response seen during -adrenoceptor stimulation in infarcted hearts. Preservation of systolic and diastolic intracellular Ca²⁺ in TG hearts was a consequence of a greater number of Na⁺/Ca²⁺ exchangers operating in both the reverse and forward modes. The present study found greater SR Ca²⁺ content from TG mouse myocardium with RCC measurement, but cardiac protein levels of SERCA2 were not different between TG and WT mouse hearts. This suggested that an increase of Na⁺/Ca²⁺ exchanger gene expression could promote Ca²⁺ entry through the reverse mode and enhance SR Ca²⁺ loading, thereafter partially preserving cardiac function in postinfarcted hearts. A prominent role for reverse-mode Na⁺/Ca²⁺ exchangers in supporting contraction of myocytes from failing human cells has been indicated by Dipla et al. (9): they demonstrated that a tonic component of action potential-evoked Ca²⁺ transients and contraction of myocytes were insensitive to SR inhibition but sensitive to a Na⁺/Ca²⁺ exchanger inhibitor compound. The underlying mechanism might be partially related to overexpression of the Na⁺/Ca²⁺ exchanger compensating for impaired SR function in the condition of cardiac dysfunction after MI and its consequent contribution to the modification of intracellular Ca²⁺ handling and improvement of myocardial performance. Further experiments are required to investigate intracellular Ca²⁺ handling in post-MI myocardium with overexpression of the Na⁺/Ca²⁺ exchanger.