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Cardiovascular Research Advance Access first published online on October 22, 2008
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Cardiovascular Research, doi:10.1093/cvr/cvn285
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org.

Sarcolemmal Ca2+-ATPase ability to transport Ca2+ gradually diminishes after myocardial infarction in the rat

Urszula Mackiewicz1,*, Michal Maczewski1, Anna Konior1, James O. Tellez2, Dominika Nowis3, Halina Dobrzynski2, Mark R. Boyett2 and Bohdan Lewartowski1

1 Department of Clinical Physiology, Postgraduate Medical School, Marymoncka Str 99/101, 01-813 Warsaw, Poland
2 Cardiovascular Research Group, University of Manchester, Manchester, UK
3 Department of Immunology, Center of Biostructure Research, Medical University of Warsaw, Warsaw, Poland

* Corresponding author. Tel: +48 225693840; fax: +48 225693712. E-mail address: urszulam{at}cmkp.edu.pl

Received 16 April 2008; revised 10 October 2008; accepted 15 October 2008

Time for primary review: 28 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: Plasmalemmal Ca2+-ATPase (PMCA) is involved in Ca2+ handling and the regulation of intracellular signalling pathways in the heart. However, there is no information on its functioning in heart hypertrophy and failure. We aimed to investigate the Ca2+-transporting ability of PMCA, Na+/Ca2+ exchanger (NCX), and sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a), as well as the amplitude of Ca2+ transients and cell shortening in myocytes isolated from rat hearts at various time intervals after myocardial infarction (MI).

Methods and results: The rate of Ca2+ transport by PMCA, NCX, and SERCA2a was estimated from the rate constants of decay of electrically and caffeine-evoked Ca2+ transients in left ventricular myocytes isolated 1 week, 1 month, and 3 months after MI. One week, 1 month, and 3 months after MI, the transporting function of PMCA decreased by 27, 41, and 67%, respectively, compared with that in time-matched sham animals. This was accompanied by increased amplitude of Ca2+ transients, cell shortening, and SR Ca2+ content. Carboxyeosin, a blocker of PMCA, increased the amplitude of shortening in cells extracted from control hearts. This effect was absent 1 and 3 months after MI. PMCA1, 2, and 4 mRNAs were unchanged in the ventricular muscle 3 months after MI when compared with time-matched sham animals. The transporting function of NCX was increased by 65% only 3 months after MI, whereas that of SERCA2a was decreased by ~18% at all three time points after MI.

Conclusion: The ability of PMCA to transport Ca2+ progressively decreases over 3 months after MI. This decrease may contribute to the increase in amplitude of Ca2+ transients and myocyte shortening.

KEYWORDS Sarcolemmal Ca2+-ATPase; Calcium handling; Myocardial infarction; Heart remodelling


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
The changes of expression and/or activity of Ca2+ handling proteins have been extensively investigated in cardiac myocytes in many models of cardiac hypertrophy and failure.1–3 However, the plasmalemmal Ca2+-ATPase (PMCA) has not received the same attention. To our knowledge, only PMCA mRNA expression was investigated in spontaneously hypertensive rats (SHR)4 and human explanted hearts.5 The lack of evidence concerning transporting activity of PMCA in cardiac hypertrophy and failure contrasts with cumulating knowledge on its important role in regulation of cellular Ca2+ handling and intracellular signalling pathways.

PMCA is a high-affinity, low-capacity enzyme transporting Ca2+ out of the cells in exchange for protons.6 There are four isoforms of PMCA (PMCA1–4). PMCA1, PMCA4, and, to a lesser extent, PMCA2 were detected in the heart muscle.4

Since PMCA exhibits high affinity for Ca2+ and low transport velocity, it has been proposed to be engaged primarily in subtle regulation of diastolic [Ca2+]i, while its contribution to outward Ca2+ transport has been thought to be of minor importance.7 However, experiments with a specific PMCA blocker, carboxyeosin (CE),8 suggest that PMCA is important not only for the control of the diastolic Ca2+ concentration9,10 but may also contribute significantly to relaxation11–15 in ferret, rabbit, rat, guinea-pig, and sheep ventricular myocytes. PMCA may also affect sarcoplasmic reticulum (SR) Ca2+ content and amplitude of Ca2+ transients in rat, ferret, and guinea-pig ventricular myocytes.9,13,15

Recent studies indicate that PMCA is also a modulator of intracellular pathways involved in ventricular remodelling in cardiac hypertrophy and failure. Nitric oxide synthase I (NOSI, nNOS),16 calcineurin, and proapoptotic tumour suppressor Ras-associated factor 1 (RASSF1) have been shown to bind to cytoplasmic loop of PMCA.17,18 Binding of calcineurin to PMCA results in calcineurin inhibition.17 Binding of nNOS to PMCA decreases NO production,16 whereas binding of RASSF1 to PMCA impairs activation of extracellular signal-regulated kinase (ERK) pathway and its target, transcription factor AP-1.18,19 Thus, PMCA might be an important organizer of protein complexes and controller of their activity.

The aim of our study was to investigate PMCA Ca2+-transporting ability and gene expression in myocytes isolated from rat hearts at various time intervals after myocardial infarction (MI). This was confronted with the transporting function and expression of SR Ca2+-ATPase (SERCA2a) and Na+/Ca2+ exchanger (NCX).


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
2.1 Study design
The investigation conforms with 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 was approved by the local Ethics Committee.

One hundred and seven male 12-week-old Wistar-Kyoto rats were included in the study. Five rats served as time 0 controls (intact), 73 rats were subjected to MI, and 29 rats were sham operated (sham). Twenty-four hours after the surgery, rats were randomly assigned to three groups, examined 1 week (9 MI and 5 sham), 1 month (11 MI and 5 sham), and 3 months (34 MI and 19 sham) after surgery. Nineteen MI rats and 0 sham rats died between surgery and randomization. At the conclusion of the experiment, rats were euthanized, ex vivo cardiac mechanics was assessed, and myocytes from the left ventricle were isolated. Three months after the surgery, left ventricular (LV) muscle from 16 MI rats and 14 sham rats was dissected for the qPCR, immunofluorescence, and western blot studies.

2.2 Experimental myocardial infarction
Rats were anaesthetized with ketamine HCl and xylazine (100 mg/5 mg/kg body weight, ip), left thoracotomy was performed, the heart was externalized, and a suture (5-0 silk) was placed around proximal left coronary artery. In sham-operated animals, it was left loose, and in MI animals, it was tied. The heart was immediately internalized, the chest was closed, and pneumothorax was reduced with negative pressure.

To estimate infarct size, LV tissue was spread on the sheet of blotting paper, and surface areas of healthy tissue and the scar were measured. Infarct size was expressed as the ratio of the scar area to the total area of the left ventricle. For the final experiments, only rats with large MI (>40%) were used.

2.3 In vivo and in vitro haemodynamic measurements
In a total of nine animals 3 months after the surgery, under light anaesthesia (ketamine HCl and xylazine, 75 mg and 3,5 mg/kg body weight, ip), a micromanometer-tipped catheter (Millar Instruments) was advanced through the right carotid artery into the LV for recording of LV pressures.

One week, 1 month, or 3 months after the surgery, the animals were injected with heparin (1000 IU/kg), subjected to pentobarbital anaesthesia (50 mg/kg), and the heart was removed and retrogradely perfused with Krebs–Henseleit buffer. Saline-filled latex balloon, connected to a pressure transducer (Hugo Sachs), was introduced into the left ventricle and gradually inflated. The LV diastolic and systolic pressures were measured for each volume of the balloon and LV diastolic and systolic pressure–volume curves were plotted. The left ventricular end-diastolic volume (LVEDV) was read from the LV diastolic pressure–volume curve at 5 mmHg (average LV end-diastolic pressure measured in vivo in healthy rats) and at 15 mmHg (average pressure measured in rats after large MI) (Table 1). The left ventricular maximal developed pressure (LVDevPmax) was read from the LV developed pressure–volume curve. Heart and lungs were weighed and their ratio to body mass was calculated.


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  		Table 1 Weights, haemodynamics measurements, and myocyte dimensions

 
2.4 Myocytes isolation
Immediately after completion of recording of the heart mechanical performance, Krebs–Henseleit buffer was changed for the Tyrode solution (TS) in order to commence the procedure of the enzymatic myocytes isolation as described in detail previously.20 After perfusion of the heart with collagenase (Boehringer)- and protease (Sigma)-containing solution, right ventricle was separated and discarded. Infarct size was evaluated and myocytes of the healthy LV tissue underwent further isolation procedure.20

2.5 Ca2+ transients and cell shortening
The myocytes were incubated for 15 min with 10 µM Indo-1 acetoxymethyl ester as described by Spurgeon et al.21 The ratio of 405 to 495 nm Indo-1 fluorescence for diastolic and systolic [Ca2+]i was obtained from the output of Dual Channel Ratio Fluorometer (Biomedical Instrumentation Group, University of Pennsylvania). The difference between the systolic and diastolic Indo-1 ratios was used as a measure of the amplitude of Ca2+ transients. Cell shortening was recorded with video edge detector (Cardiovascular Laboratories, School of Medicine, UCLA).

2.6 Ca2+ transport by plasmalemmal Ca2+-ATPase, Na+/Ca2+ exchanger, and sarcoplasmic reticulum Ca2+-ATPase and sarcoplasmic reticulum Ca2+ content
The elimination of Ca2+ from the cytosol resulting in decaying phase of electrically stimulated Ca2+ transients is attributed to parallel function of SERCA2a, NCX, and PMCA working at given rates (rSERCA, rNCX, and rPMCA, respectively). The rate of Ca2+ transport by each transporter was estimated from the rate constants (r) of single exponential curves fitted to decaying part of the electrically or caffeine-evoked Ca2+ transients, according to Choi and Eisner.9

In order to estimate the rate of Ca2+ transport by PMCA, contribution of SERCA2a and NCX was eliminated as shown in Figure 1A (top panel). The myocytes superfused at 37°C with normal TS were paced at 1 Hz and Ca2+ transients were recorded. Stimulation was stopped and TS was changed to Na+, Ca2+ -free TS (0Na0Ca). After 60 s of superfusion, 10 mM caffeine (dissolved in 0Na0Ca) was applied. Caffeine releases Ca2+ from the SR and prevents its reaccumulation in SR (Ca2+ transport by SERCA2a = 0) and 0Na0Ca superfusion blocks Ca2+ transport by NCX (Ca2+ transport by NCX = 0). Therefore, under these conditions, the rate constant of decline of caffeine-evoked Ca2+ transient (rPMCA) reflects the rate of Ca2+ transport by PMCA and possibly by mitochondrial uptake. In order to dissect the rate of Ca2+ transport realized exclusively by PMCA, myocytes were incubated for 30 min with 10 µM Ru360 (Calbiochem), which specifically blocks Ca2+ uptake into mitochondria.22 Ten micromolars of Ru360 was also included in 0Na0Ca solution. Ru360 did not affect the rate of decline of caffeine-evoked Ca2+ transients (0.27 ± 0.01 in control myocytes vs. 0.30 ± 0.03 s–1 in myocytes incubated with Ru360, n = 9) (Figure 1B). The same result was obtained with 1 µM mitochondrial uncoupler FCCP (carbonyl cyanide p-(trifluoromethoxy) phenylhydrazoner) (Sigma) included in 0Na0Ca solution (data not shown, n = 9). Thus, mitochondrial Ca2+ uptake did not affect the trace of the decay of caffeine-evoked Ca2+ transients under our experimental conditions and rPMCA reflects solely the rate of Ca2+ transport by PMCA. Indeed, preincubation of myocytes for 15 min with 5 µM CE (Sigma-Fluka), a blocker of PMCA, completely inhibited decay of caffeine-evoked Ca2+ transient in myoctes superfused with 0Na0Ca solution (Figure 1C). Under some conditions, CE might inhibit all P-type ATPases, including PMCA, SERCA2a, and Na/K-ATPase and H/K-ATPase.8,23 However, SERCA2a, Na/K-ATPase, and H/K-ATPase are inhibited by CE competing with ATP, whereas blockade of PMCA by CE does not depend on ATP. Due to these differences in mechanism of inhibition, SERCA2a, Na/K-ATPase, and H/K-ATPase, but not PMCA, are protected against CE by sarcoplasmic ATP concentrations8,23 ranging from 5 to 10 mM.24


Figure 1
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  		Figure 1 Experimental protocol used to assess Ca2+ transport by PMCA, NCX, and SERCA. (A) The monoexponential curves were fitted to decaying part of electrically or caffeine-evoked Ca2+ transients to calculate rPMCA, rTOTAL, and rSL rate constants. The rPMCA was taken as the rate of Ca2+ transport by PMCA. The rate of Ca2+ transport by SERCA and NCX was estimated according to formulas: rSERCA=rTOTAL–rSL and rNCX=rSL–rPMCA, respectively (see Section 2). (B and C) Representative traces of normalized Ca2+ transients in normal myocytes (Control) or preincubated for 30 min with 10 µM Ru360 or for 15 min with 5 µM carboxyeosine, isolated from the heart of intact rat.

 
The rate of Ca2+ transport by NCX or SERCA2a was estimated according to protocol shown in Figure 1A (bottom panel). The myocytes superfused with TS were paced at 1 Hz and Ca2+ transients were recorded. Thereafter, the stimulation was stopped and 10 mM caffeine dissolved in TS was immediately applied. The rate constant of decline of the electrically activated Ca2+ transients reflects the rate of combined transport of Ca2+ by SERCA2a, NCX, and PMCA (rTOTAL). The rate constant of decline of the Ca2+ transient initiated by caffeine (which blocks SR uptake) reflects the rate of combined transport of Ca2+ by NCX and PMCA through sarcolemma (rSL). In order to estimate the rate of Ca2+ transport by SERCA2a and NCX, rSL was subtracted from rTOTAL (rSERCA=rTOTAL–rSL) and rPMCA was subtracted from rSL (rNCX=rSL–rPMCA), respectively. SR content was estimated from the amplitude of caffeine-evoked Ca2+ transients in myocytes superfused with 0Na0Ca solution (Figure 1A, top panel).

2.7 Quantitative polymerase chain reaction
Total RNA isolation was performed with Qiagen RNeasy Mini spin columns, from tissue dissected from the LV of MI and sham-operated rats. Two micrograms of total RNA was reverse transcribed with superscript III reverse transcriptase (Invitrogen) using random hexamer priming. qPCR was performed using an Applied Biosystems 7900HT instrument, with Power SYBR green master mix (Applied Biosystems). Primer assays were purchased from Qiagen (PMCA1: QT00182210; PMCA2: QT01082081; PMCA4: QT01583274; NCX1: QT01592451; SERCA2: QT01081500). Atrial natriuretic peptide (ANP) primer sequences used were: forward, 5'-AGTGAGCCGAGACAGCAAACAT-3' and reverse, 5'-GCAGGTTCTTGAAATCCATCAGA-3'. Relative expression levels were calculated using the {Delta}{Delta}Ct method;25 28S was used as a reference gene; the primer sequences used were: forward, 5'-GTTGTTGCCATGGTAATCCTGCTCAGTACG-3' and reverse, 5'-TCTGACTTAGAGGCGTTCAGTCATAATCCC-3'.

2.8 Western blot analysis
Protein levels of SERCA2a and phospholamban (PLB) were examined by standard western blot analysis. The LV tissue from MI (n = 3) and sham (n = 2) hearts was frozen in liquid nitrogen and kept at –80°C until used. Crude membrane preparations from each LV were prepared according to Rannou et al.26

The proteins were separated on 7.5% (for SERCA2a) or 12% (for PLB) SDS–polyacrylamide gels and transferred to PVDF membrane. The membranes were blocked in 5% fat-free milk in TBST for 1 h at room temperature and incubated overnight at 4°C with rabbit polyclonal anti-SERCA (1:5000, Badrilla), mouse monoclonal anti-PLB (1:5000, Badrilla), or rabbit polyclonal anti-calsequestrin antibodies (1:5000, Affinity BioReagents) followed by incubation for 2 h at room temperature with alkaline phosphatase (AP)-conjugated secondary antibodies (Pierce). The AP reaction was detected by One Step NBT/BCIP reagent (Pierce). The membranes were scanned at 300 dpi and subjected to densitometric analysis using the Image Quant 5.2 software (Amersham Bioscience, Piscataway, NJ, USA).

2.9 Immunofluorescence
Immunofluorescence experiments were carried out on 12 µm tissue sections cut through the interventricular septum from four MI and four sham-operated rats as described previously.27 Images were captured using a confocal scanning microscope (Zeiss LSM5, Germany). To visualize the primary antibodies, rabbit anti-Serca2a antibody (1:100, Badrilla) and mouse anti-PLB antibody (1:100, Badrilla) attached to their corresponding proteins, appropriate secondary antibodies conjugated to fluorescence marker (Cy3) were used at concentration of 1:400. Signal intensity for both Serca2a and PLB was measured using Volocity software (Improvision, UK).

2.10 Solutions
For myocytes isolation and superfusion, TS containing (in mM) 144 NaCl, 5 KCl, 1 MgCl2, 0.43 NaH2PO4, 10 HEPES, and 11 glucose was used (1 mM CaCl2 was added only to the superfusion TS). pH of TS was adjusted with NaOH to 7.3 for myocytes isolation and to 7.4 for myocytes superfusion. In order to block the NCX, NaCl in TS was replaced with LiCl and NaH2PO4 and CaCl2 were omitted. One millimolar of EGTA was added to 0Na0Ca solution in order to accelerate Ca2+ removal. The pH of this solution was adjusted with KOH to 7.4.

2.11 Statistical analysis
All results are given as means ± SEM. Normal distribution of data was verified using Shapiro–Wilk test. Student’s t-test for unpaired samples was used to compare two experimental groups. To compare more than two experimental groups, one-way ANOVA (Statistica software) was used. P < 0.05 was accepted as a level of significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
3.1 Post-infarction ventricular remodelling
The mean infarct size did not differ between the experimental groups (Table 1). There were no differences in body weight between MI and sham-operated animals at any time point after the surgery. The increase in heart and lung weight corrected for body weight was evident 1 month after MI and it did not progress thereafter. One and 3 months after MI, the LV myocytes were longer by 16 and 25%, respectively, when compared with sham-operated animals, while myocytes width did not change over 3 months of follow-up (Table 1).

We observed progressive increase in the LVEDV beginning as early as 1 month after MI, whereas LVDevPmax was depressed already 1 week after infarction and its decrease did not progress further thereafter (Table 1).

Thus, in our model, hearts that underwent large MI exhibited hypertrophy, progressive ventricular dilation and profound systolic LV dysfunction reflected also by increased corrected lungs weight. Large increase in myocardial mRNA ANP expression (Figure 6B) and significantly increased LV end-diastolic pressure (19.2 ± 3.3 vs. 4.8 ± 1.2 mmHg in MI vs. sham operated, respectively) 3 months after the MI, support diagnosis of advanced heart failure in our model.

3.2 The rate of Ca2+ transport by plasmalemmal Ca2+-ATPase, Na+/Ca2+ exchanger, and sarcoplasmic reticulum Ca2+-ATPase after myocardial infarction
Analysis of variance did not show statistically significant differences between rPMCA, rNCX, and rSERCA measured in myocytes isolated from intact animals and from sham-operated animals in all time groups (Figures 2B and 3). As shown in Figure 2A, the rate of Ca2+ transport by PMCA is clearly slower in the myocyte isolated from MI rat 3 months after surgery when compared with that in the myocyte isolated from respective sham animal. Mean rPMCA measured in myocytes from MI hearts 1 week, 1 month, and 3 months after the surgery decreased when compared with that in myocytes from the sham animals by 27, 41, and 67%, respectively (Figure 2B).


Figure 2
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  		Figure 2 The rate of Ca2+ transport by PMCA after myocardial infarction (MI). (A) Representative traces of normalized Ca2+ transients in myocytes isolated from heart of sham-operated or infracted (MI) animals 3 months after the surgery. Ca2+ transients were evoked by 10 mM caffeine according to the protocol illustrated in the top panel of Figure 1A. (B) Average values of the rate constants (rPMCA) of decay of Ca2+ transients evoked by 10 mM caffeine (Figure 1A, top panel) in left ventricular myocytes obtained from hearts 1 week, 1 month, and 3 months after MI or sham operation and from hearts of intact animals (mean±SEM, n = 20–29). *Significantly different (P < 0.05) from sham-operated animals from the same time group.

 


Figure 3
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  		Figure 3 The rate of Ca2+ transport by NCX and SERCA2a after myocardial infarction (MI). The rate constants rNCX (A) and rSERCA (B) calculated from formulas: rNCX=rSL–rPMCA and rSERCA=rTOTAL–rSL, respectively. rTOTAL, rSL, and rPMCA were measured (according protocol presented in Figure 1A) in left ventricular myocytes isolated from rat hearts 1 week, 1 month, and 3 months after MI or sham operation and from hearts of intact animals (mean±SEM, n = 27–50). *Significantly different (P < 0.05) from sham-operated animals from the same time group.

 
The rate of Ca2+ transport by NCX (rNCX) was greatly (by ~65%) increased 3 months after MI when compared with that in myocytes from hearts of sham-operated animals. One week or 1 month after the surgery, rNCX did not differ between MI and sham-operated rats (P > 0.05) (Figure 3A).

The rate of Ca2+ transport by SERCA2a (rSERCA) was decreased already at 1 week after MI (by ~18%) and the decrease was similar 1 and 3 months after MI (Figure 3B).

In the above experiments, the rate of Ca2+ transport by PMCA, NCX, and SERCA2a was evaluated in myocytes stimulated at 1 Hz, which is well below the physiological frequency (4–6 Hz). This was necessary because in most myocytes stimulated at the rate of 4 Hz the upstroke of Ca2+ transient occurred when the decay of preceding Ca2+ transient was not completed. In these myocytes, fitting of monoexponential curve to the decaying part of Ca2+ transient was not precise enough, which precluded reliable calculation of rTOTAL and evaluation of SERCA2a function (see Section 2 and Figure 1A).

Nevertheless in order to test the function of Ca2+-transporting proteins under more physiological conditions, myocytes isolated from three rats 1 month after MI and from three respective sham rats were paced at 4 Hz instead 1 Hz before caffeine application (Figure 1A). We did not find any frequency-dependent changes in rPMCA in myocytes from sham animals (0.29 ± 0.02 at 1 Hz vs. 0.27 ± 0.02 s–1 at 4 Hz) or in myocytes from MI hearts (0.21 ± 0.02 at 1 Hz vs. 0.20 ± 0.01 s–1 at 4 Hz). The MI-induced decrease in rPMCA also did not depend on frequency of stimulation (27% at 1 Hz vs. 25% at 4 Hz). Furthermore, there were no frequency-dependent differences in rNCX (sham: 0.74 ± 0.07 at 1 Hz and 0.63 ± 0.07 s–1 at 4 Hz; MI: 0.85 ± 0.08 at 1 Hz and 0.76 ± 0.07 s–1 at 4 Hz) and rSERCA (sham: 9.82 ± 0.21 at 1 Hz and 10.66 ± 0.5 s–1 at 4 Hz; MI: 7.67 ± 0.23 at 1 Hz and 7.84 ± 0.37 s–1 at 4 Hz). rSERCA was evaluated only in myocytes in which monoexponential could be precisely fitted to Ca2+ transient decay at 4 Hz.

Additionally, we measured rPMCA at 2 mM (instead of 1 mM used in remaining experiments) of extracellular Ca2+ concentration in myocytes stimulated at 1 and 4 Hz. We did not find frequency-dependent differences in rPMCA (sham: 0.41 ± 0.03 at 1 Hz and 0.38 ± 0.06 s–1 at 4 Hz; MI: 0.23 ± 0.04 at 1 Hz and 0.20 ± 0.03 s–1 at 4 Hz) and even higher (but also frequency independent –44% at 1 Hz vs. 47% at 4 Hz) MI-induced decrease in rPMCA when compared with that measured at 1 mM of extracellular Ca2+ concentration.

3.3 Amplitude of Ca2+ transient, rate of Ca2+ transient decay, and sarcoplasmic reticulum Ca2+ content after myocardial infarction
Analysis of variance did not show statistically significant differences in amplitudes of Ca2+ transients and SR Ca2+ content between myocytes isolated from intact animals and from sham-operated animals in all time groups (Figure 4A–C).


Figure 4
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  		Figure 4 Parameters of Ca2+ transients and sarcoplasmic reticulum Ca2+ content after myocardial infarction (MI). The amplitude of Ca2+ transient (A) and the rate of Ca2+ transient decay (rTOTAL; Figure 1A) (B) were measured in electrically paced left ventricular (LV) myocytes obtained from hearts 1 week, 1 month, and 3 months after MI or sham operation and from hearts of intact animals (mean±SEM, n = 73–164). (C) Amplitude of caffeine-evoked Ca2+ transient in myocytes superfused with 0Na0Ca (an index of SR Ca2+ content) (Figure 1A, top panel) was measured in LV myocytes obtained from hearts 1 week, 1 month, and 3 months after MI or sham operation and from hearts of intact animals (mean±SEM, n = 20–36). * Significantly different (P < 0.05) from sham-operated animals from the same time group; f.u., fluorescence unit.

 
The amplitude of electrically evoked Ca2+ transients (Figure 4A), as well as SR Ca2+ content measured as amplitude of caffeine-evoked Ca2+ transient in myocytes superfused with 0Na0Ca solution (Figure 4C), progressively increased in myocytes from MI hearts when compared with that in myocytes from hearts of sham-operated animals, while the rate of Ca2+ transient decay (rTOTAL) was decreased similarly in myocytes from MI hearts at all time group (Figure 4B).

3.4 The effect of carboxyeosin on cell shortening after myocardial infarction
Increase in amplitude of Ca2+ transients and SR Ca2+ content in MI rats was accompanied by increased amplitude of cell shortening. It was significantly larger in myocytes isolated 1 and 3 months after MI when compared with that in myocytes from the hearts of intact animals (Figure 5, open bars).


Figure 5
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  		Figure 5 The effect of carboxyeosine (CE) on cell shortening after myocardial infarction (MI). Cell shortening (expressed as % of resting length) of myocytes treated or not treated by 5 µM CE was measured in left ventricular myocytes obtained from hearts of intact animals or from hearts 1 week, 1 month, and 3 months after MI (mean±SEM, n = 19–36). *Significantly different (P < 0.05) from cell shortening of myocytes not treated by CE; #Significantly different (P < 0.05) from cell shortening of myocytes not treated by CE from intact animals.

 
The above results made us hypothesize that progressive increase in amplitude of Ca2+ transients, the SR Ca2+ content, and amplitude of cell shortening in myocytes of the MI hearts are related to the progressive decrease in ability of their PMCA to transport Ca2+. In order to verify this hypothesis, we compared the effect of CE on the amplitude of cell shortening of myocytes isolated from MI hearts 1 week, 1 month, and 3 months after the surgery with that in myocytes of the hearts of intact animals. In myocytes of intact rats in which Ca2+ transport by PMCA was normal, CE increased mean amplitude of cell shortening by ~54%. In myocytes from hearts 1 week after MI in which the decrease in PMCA ability to transport Ca2+ was not very profound, CE increased the mean amplitude of cell shortening similarly as in myocytes from intact rats. One month after MI, CE tended to increase amplitude of cell shortening by ~22% (P = 0.07). In myocytes 3 months after MI, in which PMCA ability to transport Ca2+ was drastically diminished (~30% of normal value), CE did not significantly affect amplitude of cell shortening (Figure 5). However, in hearts 3 months after MI, the amplitude of cell shortening of myocytes not treated by CE was significantly increased when compared with intact group. It could be argued that in myocytes 3 months after MI, exhaustion of contractile reserve rather than low activity of their PMCA was responsible for their poor response to CE. However, these myocytes readily responded to inotropic factors other than CE. Five minutes superfusion of 10–8 M isoproterenol increased the contractile amplitude of electrically stimulated myocytes by 49.6 ± 10.0% (n = 10). Similarly, paired stimulation induced by second stimulus placed immediately after the refractory period increased the amplitude of shortening by 28.6 ± 5.1% (n = 10).

3.5 mRNA and protein expression
Our qPCR experiments showed that ANP mRNA expression is significantly increased in ventricular muscle from MI rats when compared with sham animals (Figure 6B). Furthermore, there was no difference in the expression of PMCA1, PMCA2, PMCA4, and NCX mRNAs but significant decrease in SERCA2a mRNA in LV muscle from rats 3 months after MI when compared with respective sham rats (Figure 6A). However, there was no difference in SERCA2a and PLB at the level of protein expression (Figure 7).


Figure 6
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  		Figure 6 qPCR analysis of expression of Ca2+ handling proteins and atrial natriuretic peptide (ANP) mRNAs in left ventricular muscle 3 months after myocardial infarction. (A) Expression of PMCA1, PMCA2, PMCA4, NCX1, and SERCA2a mRNAs. (B) Expression of ANP mRNA. Means±SEM (n = 8). *Significantly different (P < 0.05) from sham-operated rats.

 


Figure 7
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  		Figure 7 Sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) and phospholamban (PLB) protein expression. (A) Representative blots from left ventricular (LV) tissue from myocardial infarction (MI) (n = 3) and sham-operated rats (n = 2) 3 months after the surgery (upper panel). The protein levels of SERCA2a and PLB normalized to calsequestrin (lower panel). Means±SEM. (B) Immunoconfocal images showing expression of SERCA2a and PLB protein in LV tissue from MI (n = 4) and sham-operated rats (n = 4) (upper panels) and quantitative results (lower panel). Means ± SEM.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
The major finding of this study is that PMCA of LV myocytes isolated from post-MI rat hearts demonstrates significantly reduced ability to transport Ca2+. Decrease in Ca2+ transport rate by PMCA was observed as early as 1 week after MI and progressed over 3 months of follow-up.

Myocyte relaxation results from Ca2+ reuptake by the SR and outward Ca2+ transport by NCX and PMCA. Relative contribution of PMCA to relaxation in rat, guinea pig, ferret, and sheep measured by other authors using the method applied in our work did not exceed 5%.9,13–15 However, its contribution to the outward Ca2+ transport measured at room temperature by Choi and Eisner9 in rat cardiac myocytes was as large as 20%. In our experiments performed at 37°C, it was even larger, reaching 35%.15 The significant relative contribution of PMCA to outward Ca2+ transport results from low contribution of NCX in rat. The above results show that complete inhibition of PMCA would result in intracellular retention of ~35% Ca2+ normally removed from the myocyte at each contraction–relaxation cycle. Therefore, despite relative small contribution of PMCA to relaxation, its inhibition may importantly disturb the balance between Ca2+ influx and efflux. Indeed, we and others have demonstrated that complete inhibition of PMCA by its selective blocker CE increases diastolic [Ca2+]i, amplitude of Ca2+ transients, and cell shortening as well as increases the SR Ca2+ content.9,10,13,15

Therefore, it is conceivable that increased amplitude of Ca2+ transients and cell shortening was related in our experiments to the decrease in ability of PMCA to transport Ca2+. This assumption is supported by results presented in Figure 5. The positive effect of CE on cell shortening should progressively decrease with decreasing activity of PMCA. Indeed, CE significantly increased amplitude of cell shortening only 1 week after MI. One and 3 months after MI, the increase was absent despite the presence of contractile reserve in these myocytes demonstrated by ISO and paired stimulation.

Probably decreased function of PMCA is not the only mechanism, which may contribute to the increase in amplitude of Ca2+ transient and cell shortening in the post-MI setting. Prolonged action potential and resting depolarization, observed in the post-MI rats, promoting increased Ca2+ influx through reversed NCX and increasing Ca2+ influx through L-type28 calcium channels could also contribute to increased amplitude of cell shortening.

In our experiments, the decrease in transporting activity of PMCA in LV myocytes is not due to a decrease in PMCA1, PMCA2, and PMCA4 mRNA expression. Similarly, Zwadlo and Borlak4 observed no change in PMCA1 nor PMCA4 mRNA in the SHR rats. On the other hand, mRNA for PMCA1 and PMCA4 diminished in end-stage human heart failure.5 It is possible, therefore, that there are species differences in the changes of these transcripts in the failing heart. It is not known if there are any changes in the expression of the corresponding PMCA isoforms at the protein level. We could not investigate these proteins because of the lack of suitable commercially available antibodies.

It should be also considered how the changes in the transporting activity of NCX and SERCA2a found in our experiments after MI would affect the amplitude of Ca2+ transient and cell shortening. In myocytes of rodent hearts, high intracellular Na+ concentration promote NCX working in reversed mode for a large fraction of contraction–relaxation cycle, contributing significantly to Ca2+ influx.29 This might increase amplitude of Ca2+ transients and cell shortening. However, in our experiments, the increase in the rate of Ca2+ transport by NCX was observed only 3 months after MI, whereas increase in amplitude of Ca2+ transients was observed already 1 week after MI (paralleling decreased function of PMCA).

We found moderate decrease in transporting function of SERCA2a, despite unchanged expression of SERCA2a and PLB at protein level. Therefore, neither the changes in SERCA2a nor NCX function account for increase in amplitude of Ca2+ transient and SR Ca2+ content. One possible explanation of changes in intracellular Ca2+ signalling is that sarcoplasmic Ca2+ concentration was increased in our post-MI myocytes at least partly due to decrease in PMCA function. Since it has been shown that the amount of Ca2+ transported to the SR is highly dependent on sarcoplasmic Ca2+ concentration,30 this may account for increase in amplitude of Ca2+ transient and SR Ca2+ content, despite the moderate decrease in SERCA2a function.

We found in our rats with large MI a marked progressive LV dilation and relatively mild cardiac hypertrophy (as reflected by increase in corrected heart weight). This was accompanied by progressive increase in the wall stress in the post-MI hearts that should have resulted in progressive worsening of LV systolic function. However, in our experiments, LVDevPmax was depressed already 1 week after MI when ventricular dilatation was negligible and depression did not progress with increasing LV dilatation (Table 1). One possible explanation for this discrepancy is that LV remodelling was accompanied by increased contractility of individual myocytes. Indeed, cell shortening increased progressively since 1 week after MI presumably being due to decreasing PMCA activity. Increase in contractile force could act to compensate for detrimental LV dilation and support haemodynamic function of post-MI hearts.

The implications of changes in activity of PMCA should not be restricted to regulation of Ca2+ handling in myocytes.31 As pointed out in Section 1, PMCA is involved in the regulation of the signalling pathways important in heart hypertrophy and failure.

In particular, it has been shown that PMCA is localized (together with nNOS) to the syntrophin–dystrophin complex of cytoskeleton. On the one hand, this provides the physical and functional link with the nNOS which is inhibited by PMCA, whereas on the other hand, it exposes PMCA to the mural forces changed in the infarcted heart. Indeed, it has been proposed that activity of PMCA may be regulated by dystrophin via interaction with {alpha}–1 syntrophin as is the case with the L-type Ca2+ channel.32

Similarly, interaction of PMCA with the catalytic unit of calcineurin results in its Ca2+-dependent inhibition.17 Thus, it may be expected that reduced ability of PMCA to transport Ca2+ results in increased Ca2+ concentration in subsarcolemmal domains, enhancing activity of calcineurin/nuclear factor of activated T-cells (NFAT) pathway. On the other hand, increased calcineurin activity reported in experimental models as well as in hypertrophied and failing human hearts33 might inhibit PMCA ability to transport Ca2+.

In summary, we show for the first time that transporting function of PMCA is decreased in the myocytes from the post-MI rat heart and suggest that it might contribute to increased amplitude of myocyte shortening, supporting contractile function of post-MI rat hearts. Further investigations are necessary to understand the role of PMCA in modulation of calcineurin/NFAT, ERK, and nNOS signalling pathways after MI.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
This work has been supported by the grant (501-1-1-27-13/03) of Medical Center of Postgraduate Education and by the grant (N401 107 32/2257) of Ministry of Science and Higher Education.


   Acknowledgement
 
We thank Ms Alicja Protasowicka for expert and devoted technical contribution.

Conflict of interest: none declared.


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

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