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1Weis Center for Research and 2Department of Medicine, Geisinger Medical Center, Danville, Pennsylvania 17822
Submitted 20 January 2004 ; accepted in final form 29 March 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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exercise training; gene transfer; antisense; primary cardiac myocyte culture; excitation-contraction coupling
In myocytes isolated from rat hearts 3–9 wk post-MI, contractile amplitudes were significantly higher at 0.6 mM extracellular Ca2+ concentration ([Ca2+]o) but lower at 5.0 mM [Ca2+]o compared with those isolated from rats that had received sham operations (24, 27). At intermediate [Ca2+]o (1.8 mM), there were no differences in contractile behavior between Sham and MI myocytes. We interpreted this abnormal pattern of contractile behavior observed in MI myocytes as due to altered Ca2+ influx and efflux pathways (27). Of the major Ca2+ influx and efflux pathways in our rat MI model, only NCX1 (7, 32), but not L-type Ca2+ channel (26), was depressed in MI myocytes. It should be recalled that the NCX1 can mediate both Ca2+ influx (3 Na+ out:1 Ca2+ in) and efflux (3 Na+ in:1 Ca2+ out) during an action potential, although the extent and duration of Ca2+ influx during systole remain uncertain and controversial (1, 21). Circumstantial evidence supports the hypothesis that altered NCX1 function may largely account for the contractile dysfunction observed in rat MI myocytes. For example, NCX1 downregulation in normal rat myocytes resulted in contractile dysfunction similar to that observed in MI myocytes (19). In addition, overexpression of NCX1 rescued contractile abnormalities in rat MI myocytes (30). It is therefore tempting to hypothesize that HIST, by enhancing Na+/Ca2+ exchange (28), resulted in improvement in contractile function in rat MI myocytes (24). The present study was undertaken to test this hypothesis.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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250 g) were randomly assigned into three groups: sham-sedentary (Sham), MI-sedentary (MISed), and MI-exercised (MIHIST). To induce MI, the left main coronary artery of each anesthetized (2% isoflurane-98% O2), intubated, and ventilated rat was ligated 3–5 mm distal to its origin from the ascending aorta (3, 24–28, 30, 32, 33). Sham operation was identical to MI, except that the coronary artery was not ligated. In our hands, sham-operated rats had close to 100% survival, whereas the mortality for animals that underwent the coronary ligation procedure was 30% within 24 h of the operation. Rats that survived the first 24 h of operation would go on to complete the experimental protocol. All surviving rats (Sham, n = 10; MISed, n = 10; and MIHIST, n = 6) received rat chow and water ad libitum and were maintained on a 12:12-h light-dark cycle. Survivors typically had 36 ± 3% of myocardium infarcted as determined histologically (3). In addition, despite no overt signs of heart failure in MI rats, we observed at 1 and 3 wk postinfarction 20% lower LV systolic pressure in MI hearts when perfused in vitro (3). Two weeks after operation, all rats were introduced to the treadmill (0° grade, 10 m/min, 10 min/day, 5 days/wk) to acclimatize for 1 wk. Sham and MISed rats continued at the same speed and degree of incline twice per week for another 7–9 wk until heart excision. For the entire training period, the training protocol consisted of 5 consecutive 1-min running bouts daily, 5 days/wk, and each running bout was interspersed with 60 s of rest. During the first week of training, treadmill speed was set at 40 m/min, and grade was set at 15°. During the second week of training, treadmill speed was progressively increased to 60 m/min. The treadmill grade and speed were then held constant for the remaining 6–8 wk of the training period (total: 7–9 wk). The protocols for induction of myocardial infarction, treadmill exercise training and myocyte isolation were approved by the Institutional Animal Care and Use Committee.
Myocyte isolation and culture. Cardiac myocytes were isolated from the septum and LV free wall of rat hearts by successive perfusion with collagenase and hyaluronidase (4). Freshly isolated myocytes were seeded on laminin-coated coverslips (5), and a portion was used within 2 h of isolation for contractility measurements. The remaining myocytes were cultured in modified, serum-free medium 199 ([Ca2+]o = 1.8 mM) as described previously (18, 19, 31). After 2 h, media were changed to remove nonadherent myocytes. Myocytes were incubated for an additional 3–4 h before initiation of pacing (1-Hz, 5-ms pulses of alternating polarity, field strength of 4 V/cm) as described previously (31). Culture media were changed daily. Our laboratory (18) and others (2) have previously demonstrated that continuous pacing of adult rat cardiac myocytes in culture maintained normal contractile function for at least 72 h.
Adenoviral infection of cardiac myocytes. Recombinant, replication-defective adenovirus expressing either green fluorescent protein (GFP) alone (Adv-GFP), or GFP and rat NCX1 (each under a separate cytomegalovirus promoter) (Adv-GFP-NCX1), were constructed as described previously (31). The coding sequence of rat heart NCX1 (3,067 bp) was released from pcDNA3.1(+) by sequential digestion with HindIII and XhoI, and inserted in the antisense (AS) direction into the shuttle vector pAdTrack-CMV, using the same HindIII and XhoI restriction sites on the shuttle vector. The resulting shuttle vector plasmid was used to construct recombinant, replication-defective adenovirus expressing GFP and ASNCX1 (Adv-GFP-ASNCX1) (19). Two hours after isolation, myocytes were infected with either Adv-GFP, Adv-GFP-NCX1, or Adv-GFP-ASNCX1 at a multiplicity of infection of 2–5 for 3–4 h. Media were then changed, and myocytes were studied after 72 h in continuous pacing culture. Over 95% of myocytes fluoresced green (excitation 478 nm, emission 535 nm) within 12 h, indicating successful adenoviral infection and GFP expression. For brevity, Sham myocytes infected with Adv-GFP are referred to as Sham-GFP myocytes; MISed myocytes infected with Adv-GFP and Adv-GFP-NCX1 are referred to as MISed-GFP and MISed-NCX1 myocytes, respectively; and MIHIST myocytes infected with Adv-GFP and Adv-GFP-ASNCX1 are referred to as MIHIST-GFP and MIHIST-ASNCX1 myocytes, respectively.
Myocyte shortening experiments. Cell contraction was measured in myocytes incubated in HEPES-buffered (20 mM, pH 7.4) medium 199 (37°C), containing either 0.6, 1.8, or 5.0 mM [Ca2+]o, by using a charge-coupled device video camera and edge-detection software (Ionoptix, Milton, MA) as described previously (18, 19, 24, 27, 30, 31). The low (0.6 mM) and high (5.0 mM) [Ca2+]o were chosen to favor Ca2+ efflux and influx pathways, respectively (19), and to accentuate the differences in contractility between Sham and MISed myocytes (3, 24, 27, 30).
NCX1, SERCA2, and calsequestrin immunoblotting. Lysates from cultured myocytes in SDS sample buffer containing 10 mM N-ethylmaleimide were subjected to 7.5% polyacrylamide gel electrophoresis (19, 28, 30, 31). Fractionated proteins were transferred onto Immunoblot polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). NCX1 was detected with a mouse monoclonal antibody (1:1,500 dilution; R3F1, Swant; Bellinzona, Switzerland), SERCA2 was detected with another monoclonal antibody (1:2,500; MA3-919, Affinity Bioreagents; Golden, CO), and sheep anti-mouse antibody (1:2,000; Amersham, Buckinghamshire, UK) was used as the secondary antibody in both cases. To detect calsequestrin, a rabbit anti-calsequestrin antibody (1:3,500, Swant) and donkey anti-rabbit IgG (1:5,000, Amersham) were used. Immunoreactive proteins were detected with enhanced chemiluminescence-Western blotting system. Protein band signal intensities were quantitated by scanning autoradiograms of the blots with a PhosphorImager (Molecular Dynamics; Sunnyvale, CA).
Statistics. All results are expressed as means ± SE. In experiments in which maximal contraction amplitudes were measured as function of group (Sham vs. MISed), [Ca2+]o, and days in culture, three-way ANOVA was performed to determine significance of differences. A linear model-fitted standard least squares analysis (JMP version 4, SAS Institutes, Cary, NC) was used. For analysis of contraction amplitude as a function of group and [Ca2+]0, two-way ANOVA was used. Significance of differences among the means of proteins (NCX1, SERCA2, calsequestrin) in the five groups was determined by one-way ANOVA. A priori comparisons of means of any two groups were then performed by using F-tests as tests of significance. In all analyses, P < 0.05 was taken to be statistically significant.
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RESULTS |
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Effects of HIST on MI myocyte contractile function. Our laboratory has previously shown that 6–8 wk of HIST instituted shortly after induction of MI in the rat was efficacious in restoring near normal contractility (24). Our present study demonstrated that the beneficial effects of HIST on MI myocyte contractility were still evident after adenoviral infection followed by 3 days of continuous pacing culture. Compared with MISed-GFP myocytes, maximal contractile amplitudes were significantly lower at 0.6 mM [Ca2+]o but higher at 5.0 mM [Ca2+]o in MIHIST-GFP myocytes (Tables 4 and 5, comparison B). In addition, comparing contractile amplitudes between Sham-GFP and MIHIST-GFP myocytes revealed no statistically significant differences (Tables 4 and 5, comparison B), indicating that HIST was effective in restoring contractile amplitudes in MI myocytes to normal.
In contrast to our laboratory's previous observations in freshly isolated Sham, MISed, and MIHIST myocytes that contractile dynamics (cell shortening and relengthening velocities) were improved by HIST (24), the results of our present study on adenoviral-infected and cultured myocytes showed no differences in contractile dynamics between MISed-GFP and MIHIST-GFP myocytes (Table 4; P < 0.4).
Effects of NCX1 downregulation on MIHIST myocyte contractile function.
After exposure to Adv-GFP-ASNCX1 for 3 days, NCX1 protein levels in MIHIST-ASNCX1 myocytes were downregulated by 26% compared with MIHIST-GFP myocytes (Fig. 1 and Table 3), although the difference did not reach statistical significance (P < 0.14). In previous studies (19), normal rat myocytes infected with Adv-GFP-ASNCX1 showed a significant (P < 0.01) 30% decrease in NCX1 after 72 h. Functionally, compared with MIHIST-GFP myocytes, NCX1 downregulation in MIHIST-ASNCX1 myocytes resulted in maximal contraction amplitudes that were significantly increased at 0.6 mM [Ca2+]o but decreased at 5.0 mM [Ca2+]o (Tables 4 and 5, comparison D). Indeed, comparison of maximal contraction amplitudes between MIHIST-ASNCX1 and MISed-GFP myocytes showed no statistically significant differences (Tables 4 and 5, comparison E), indicating the salutary effects of HIST on MI myocyte contraction amplitude (Tables 4 and 5, comparison B) were lost when NCX1 was downregulated. This conclusion can also be arrived at independently by the significant differences in contraction amplitudes between Sham-GFP and MIHIST-ASNCX1 myocytes (Tables 4 and 5, comparison D), whereas no such differences were observed between Sham-GFP and MIHIST-GFP myocytes (Tables 4 and 5, comparison D).
Downregulating NCX1 in MIHIST myocytes was associated with maximal shortening velocities that were higher at 0.6 mM [Ca2+]o but slightly slower at 5.0 mM [Ca2+]o (Table 4, P < 0.002). Comparisons of maximal relengthening velocities between MIHIST-GFP and MIHIST-ASNCX1 myocytes generally followed similar trends, although the differences did not reach statistical significance (Table 4, P < 0.13).
Effects of MI and HIST on NCX1, SERCA2, and calsequestrin abundance.
Our laboratory has previously demonstrated that, in freshly isolated myocytes, despite decreases in NCX1 current 3 wk after MI (32) and its partial restoration by HIST (28), there were no detectable differences in NCX1 protein when the rabbit polyclonal antibody to NCX1 was used (
11–13, Swant) (28). In the present study, however, which used 3-day cultured myocytes and the murine monoclonal antibody to NCX1 (R3F1, Swant), NCX1 protein levels were significantly decreased in MISed-GFP myocytes compared with Sham-GFP myocytes (Fig. 1 and Table 3). HIST was effective in partially restoring the depressed NCX1 levels (Fig. 1 and Table 3).
SERCA2 protein levels were significantly lower in MISed-GFP myocytes compared with Sham-GFP myocytes (Fig. 1 and Table 3), in agreement with results of previous studies on freshly isolated myocytes (25). HIST was beneficial in partially restoring SERCA2 protein levels in MI myocytes toward control Sham-GFP levels (Table 3). It is important to note that manipulating NCX1 levels did not affect SERCA2 protein abundance in either MISed or MIHIST myocytes (Fig. 1 and Table 3). Calsequestrin levels, used as an internal standard for protein loading (31), were similar among the five groups of myocytes (Fig. 1 and Table 3).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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35%) LV infarction. In addition, high-intensity sprint training was efficacious in restoring contractile abnormalities of post-MI myocytes toward normal (24) by affecting a variety of subcellular pathways involved in beat-to-beat [Ca2+]i regulation (24, 25, 28, 33). To elucidate which one of the many beneficial effects of HIST was predominant in bringing about improved contractility in post-MI myocytes, we attempted to manipulate only one pathway (e.g., increase Na+/Ca2+ exchange) at a time in MISed myocytes and to evaluate whether such manipulation would result in improvement in myocyte contractility similar to that observed in MIHIST myocytes. Conversely, if the same pathway were manipulated in the opposite direction (e.g., decrease Na+/Ca2+ exchange) in MIHIST myocytes, then the beneficial effects of HIST on MI myocyte contractility would be expected to be lost. The first major finding is that MISed myocytes isolated from rat hearts 8–9 wk post-MI maintained their contractile abnormalities even after 3 days of continual pacing culture. This observation establishes the feasibility of an in vitro, cultured MI myocyte model system suitable for short-term manipulation of gene expression. In addition, adenovirus infection did not affect contractility in both Sham and MISed myocytes, indicating that it is possible to utilize the highly efficient adenovirus-mediated gene transfer technique to manipulate genes and study the resultant effects on myocyte contractile performance. Perhaps most relevant to the present study is the finding that the beneficial effects of HIST were sustained in MI myocytes after adenoviral infection and 72 h of culture. This important observation indicates, for the first time, that the cellular effects of exercise training could persist for at least 3 days in continual pacing culture.
The characteristic contractile abnormality in our rat MISed myocytes in which NCX1 function was depressed (7, 28, 32) is that, compared with control Sham myocytes, maximal contractile amplitude was higher at 0.6 mM [Ca2+]o, not different at 1.8 mM [Ca2+]o, but lower at 5 mM [Ca2+]o (24, 27). This abnormal contractile pattern was similar to that observed in normal rat myocytes in which NCX1 was downregulated (19). It should be noted that, in addition to action potential amplitude and duration, important Ca2+ transport pathways such as L-type Ca2+ currents and SR Ca2+ uptake were not affected in NCX1-downregulated myocytes (19), suggesting that the effects on contractility were due to NCX1 downregulation alone. The similarities in the pattern of contractile abnormalities in MISed and normal myocytes in which NCX1 was downregulated (when compared with their respective controls) suggest that decreased NCX1 function may partly account for the observed contractile dysfunction in MISed myocytes.
The second major finding of our present study is that overexpression of NCX1 rescued contractile abnormalities in MISed myocytes, indicating the important role of depressed NCX1 function (7, 28, 32) in mediating contractile abnormalities in our rat MI model. Our third major finding is that downregulation of NCX1 in MIHIST myocytes resulted in loss of contractile improvement by HIST in postinfarction rat myocytes. This observation suggests that restoration of normal contractility in post-MI myocytes by HIST (24) is at least partially mediated by enhanced NCX1 activity in MIHIST myocytes (28). In this light, it is interesting to note that NCX1 activity (especially in the 3 Na+ out:1 Ca2+ in mode) contributed importantly to [Ca2+]i transient, contraction, and relaxation in myocytes isolated from human end-stage cardiomyopathic hearts (6, 9).
It may be instructive to compare the effects of HIST on cell size, [Ca2+]i homeostasis, and contractile function in normal and postinfarction myocytes. In normal adult rat myocytes, 5–7 wk of HIST induced a modest (5%) but significant increase in cell length with no change in cell width (29). In contrast, HIST reversed the pathological hypertrophy in post-MI rat myocytes (24, 28, 33). Maximal contraction and [Ca2+]i transient amplitudes were decreased in normal myocytes (29) but enhanced in post-MI myocytes by HIST (24, 25). Elevated diastolic [Ca2+]i levels in post-MI myocytes were lowered toward normal by HIST (25). Because NCX1 was necessary to reach end-diastolic [Ca2+]i levels (22) and because diastolic [Ca2+]i levels were lower in myocytes overexpressing NCX1 (31) and higher in myocytes in which NCX1 was downregulated (19), lower diastolic [Ca2+]i levels in MIHIST myocytes (as compared with MISed myocytes) suggest that HIST may improve NCX1 function in post-MI myocytes. Indeed, both the depressed NCX1 current (28) and protein levels (Table 3) in post-MI rat myocytes were restored toward normal by HIST. By contrast, NCX1 protein levels in normal myocytes were decreased by HIST (29). In addition, decreased NCX1 activity in normal myocytes by HIST was suggested by the observation that diastolic [Ca2+]i values were consistently elevated in HIST myocytes (29). These paradoxical effects of HIST on normal and post-MI myocytes suggest that the signaling mechanisms by which HIST modulates cardiac size and function under physiological and pathological conditions are different.
The activity and/or protein levels of NCX1 in different models of heart failure and hypertrophy have been reported to be increased, unchanged, or decreased (for review, see Ref. 17). Focusing on animal models of ischemic heart disease, early reports indicated that Na+-dependent Ca2+ uptake in sarcolemmal vesicles (7) and whole cell Na+/Ca2+ exchange current (INaCa) (32) were depressed in rat myocytes studied 3–9 wk postinfarction. In addition, NCX1 protein levels were either unchanged (28) or decreased in MISed myocytes (Fig. 1). By contrast, other reports suggested doubling of INaCa in rat myocytes isolated from rat hearts 6 wk (20) to 6 mo (10) postinfarction. In rabbit myocytes examined 8–9 wk post-MI, INaCa was increased by 32% in one study (13) but decreased by 25% in another study (16). In canine subepicardial cells isolated from 5-day infarcted hearts, INaCa was not different compared with noninfarcted hearts (15). It is likely that the discrepancies in the results on NCX1 activity in post-MI myocytes are due to absence (3, 14, 27) or presence (13, 20) of overt heart failure and to different stages of ischemic cardiomyopathy at which myocyte function was examined. In failing human myocardium from dilated cardiomyopathy and ischemic cardiomyopathy, NCX1 protein levels were reported to be increased (11) or unchanged (11, 12). Whether the activity of NCX1 was altered in human failing myocardium, compared with that in normal human myocardium, is unclear at present. In contrast to our rat MI model of compensated failure, a common theme in the studies demonstrating an increase in INaCa and/or NCX1 protein is the presence of severe, overt heart failure. Viewed in this context, increases in NCX1 in these heart failure models may be regarded as an important compensatory mechanism for decreased contractile function (6, 9).
There are limitations to the present study. The first is that we did not measure [Ca2+]i dynamics in myocytes from the five groups of myocytes. Previous studies comparing Sham, MISed, and MIHIST myocytes indicated that differences in contraction amplitudes paralleled those in [Ca2+]i transients (24, 25). In addition, changes in contractile behavior in both NCX1 overexpressed (31) and downregulated (19) myocytes, compared with control myocytes, closely correlated with changes in [Ca2+]i dynamics. Furthermore, in our previous studies, manipulating NCX1 levels by gene transfer did not affect action potential morphology, L-type Ca2+ current, SR Ca2+ uptake activity, and SERCA2 levels (19, 31). Therefore, it is reasonable to assume in the present study that changes in contractile behavior observed with NCX1 overexpression in MISed myocytes and NCX1 downregulation in MIHIST myocytes were due to changes in Ca2+ fluxes mediated by NCX1. It is important to note that we confirmed in the present study that changing NCX1 levels did not alter SERCA2 protein abundance. Another limitation is that we did not directly measure NCX1 function, i.e., NCX1 currents or relaxation from caffeine-induced contracture, but only NCX1 protein levels by Western blot. Although our laboratory has previously demonstrated reasonable correlation between NCX1 protein levels and NCX1 activity as estimated by half-times of relaxation from caffeine-induced contractures (19, 31), there are serious reservations about drawing firm conclusions on potential NCX1 activity on the basis of NCX1 protein levels. This is because the narrow range of linearity of densitometry measurements with protein concentrations precludes precise quantitation of protein levels. In addition, although distribution of endogenous NCX1 in normal adult cardiac myocytes has been shown to be limited to sarcolemma, transverse tubules, and intercalated discs (8), it is not known whether the overexpressed NCX1 is only present in sarcolemma (and participates in ion transport) or partly sequestered in some intracellular compartments (and unable to mediate Na+/Ca2+ exchange). It is obvious that performing Western blots on myocyte lysates cannot differentiate between the NCX1 that has been correctly inserted in the sarcolemma and the NCX1 that is intracellular. Therefore, NCX1 protein levels in the present study should properly be viewed as an indication that we have successfully overexpressed or downregulated NCX1 levels in appropriate myocyte groups. A third limitation is that in this study we focused on the role of NCX1 on contractile abnormalities in post-MI rat myocytes and ignored the other important steps involved in excitation-contraction coupling that are altered in post-MI myocytes (3, 7, 10, 13, 15, 20, 25, 28, 32, 33). The importance of alterations in these other pathways (e.g., SR Ca2+ uptake, myosin heavy chain isoenzyme distribution, increased phospholemman expression, etc.) in causing contractile dysfunction in post-MI myocytes needs to be addressed in future studies.
In summary, we have established in vitro myocyte model systems in which the phenotypes associated with MI and exercise training were preserved in culture. Adenoviral infection did not affect the phenotypic differences between Sham and MISed myocytes, thus allowing gene manipulation to be performed on our myocyte culture model system. Overexpression of NCX1 in MISed myocytes restored contractile function to that observed in Sham myocytes, whereas NCX1 downregulation in MIHIST myocytes abolished the beneficial effects of HIST on post-MI myocyte contractility. We suggest that decreased NCX1 activity played a significant role in abnormal contractility in postinfarction myocytes and that high-intensity exercise training mediated its salutary effects on contractile function in post-MI myocytes at least partially by restoring Na+/Ca2+ exchange activity toward normal.
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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-adrenergic receptor antagonists have improved ventricular myocyte calcium regulatory protein abundance. Circulation 104: 1012–1018, 2001.This article has been cited by other articles:
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  M. Maczewski and U. Mackiewicz Effect of metoprolol and ivabradine on left ventricular remodelling and Ca2+ handling in the post-infarction rat heart Cardiovasc Res, July 1, 2008; 79(1): 42 - 51. [Abstract] [Full Text] [PDF]
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 J. R. Libonati, Z. V. Kendrick, and S. R. Houser Sprint training improves postischemic, left ventricular diastolic performance J Appl Physiol, December 1, 2005; 99(6): 2121 - 2127. [Abstract] [Full Text] [PDF]
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B. A. Ahlers, J. Song, J. Wang, X.-Q. Zhang, L. L. Carl, G. M. Tadros, L. I. Rothblum, and J. Y. Cheung Effects of sarcoplasmic reticulum Ca2+-ATPase overexpression in postinfarction rat myocytes J Appl Physiol, June 1, 2005; 98(6): 2169 - 2176. [Abstract] [Full Text] [PDF]
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