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European Journal of Heart Failure 2004 6(2):161-172; doi:10.1016/j.ejheart.2003.11.004
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© 2003 European Society of Cardiology

Left–right asymmetric ventricular expression of CARP in the piglet heart: regional response to experimental heart failure{star}

Mario Torradoa, Eduardo Lópezb, Alberto Centenob, Alfonso Castro-Beirasb and Alexander T. Mikhailova,*

a Developmental Biology Unit, Institute of Health Sciences University of La Coruña, Campus de Oza, Building ‘El Fortín’, Las Xubias s/n, La Coruña 15006, Spain
b University Hospital ‘Juan Canalejo’ Las Xubias de arriba 58, La Coruña 15006, Spain

* Corresponding author. Tel.: +34-981-167-000; fax: +34-981-138-714. E-mail address: margot{at}udc.es


   Abstract
Top
 Notes
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background and aim: Cardiac ankyrin repeat protein (CARP), whose expression is down-regulated in response to doxorubicin (Dox) in vitro, has been proposed to be a marker of experimentally-induced cardiac hypertrophy in rodent models. In piglets, the rapid hypertrophy rate of the left ventricle (LV) as compared to that of the right ventricle (RV) represents a natural model of asymmetric ventricular enlargement. We tested whether CARP expression correlates with postnatal ventricular hypertrophy and to what extent CARP can be sensitive to Dox treatment in vivo.

Methods: CARP mRNA and protein levels were quantified (by Northern blot hybridization, semi-quantitative RT-PCR and Western blot) in the piglet heart, both during early postnatal development and upon Dox-induced cardiomyopathy (Dox-CM).

Results: The study revealed: (1) significantly augmented CARP mRNA and protein levels in the LV compared to the RV resulting in left vs. right asymmetry in ventricular CARP expression throughout early postnatal development; (2) dose- and chamber-dependent CARP mRNA and protein enrichment in ventricular myocardium in response to Dox; and (3) abolishment of asymmetric patterns of ventricular CARP expression at heart failure resulting from Dox-CM.

Conclusions: (1) CARP is differentially regulated in the LV and RV during both postnatal development and disease; and (2) monitoring of ventricular CARP expression patterns can be used for further analysis of transition from compensated to overt heart failure.

Key Words: Cardiac left–right asymmetry • Gene expression • CARP • Heart failure • Cardiomyopathy • Doxorubicin

Received May 15, 2003; Revised July 15, 2003; Accepted November 12, 2003


   1. Introduction
Top
 Notes
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The heart reacts to increased demands for mechanical stretch, volume overload or injury via the activation of an adaptive hypertrophic response that is associated with the up-regulation of a defined subset of cardiac-specific genes [1–7]. Although compensatory in nature, this response can lead to cardiomyopathy and heart failure [8–10]. The neonatal heart is a conventional model for the study of distinct patterns in ventricular hypertrophy. In neonatal piglets, the right (RV) and left (LV) ventricles of the same heart display such a different degree of hypertrophy that the RV can be used as a slower heart-matched control for the more rapidly thickening LV [11,12].

We are interested in developing a new model of cardiomyopathy with low-output cardiac failure in neonatal piglets using an antineoplastic agent, Doxorubicin (Dox). In most experimental studies, Dox clearly causes both diastolic and systolic congestive heart failure [13,14]. Dox cardiotoxicity is associated with changes in cardiac gene expression in vivo and in vitro [15–18]. CARP, cardiac ankyrin repeat protein, has been identified as a cardiac target for Dox [19]. In primary cultures of neonatal rat ventricular myocytes, CARP expression was severely down-regulated in response to Dox exposure [19,20]. In contrast, in a rabbit model of Dox-induced cardiomyopathy, CARP mRNA levels were variable, and only in four out of 12 animals the transcript was up-regulated as compared to controls [21].

Increased CARP expression characterizes many forms of cardiac hypertrophy and disease. Ventricular CARP up-regulation has been reported in distinct models of cardiac hypertrophy in rats [4] and mice [6,22], in the mouse models of dilated cardiomyopathy and hypertrophy [8,23], in the pig model of heart ischemia/reperfusion [24], in pacing-induced canine heart failure (in the absence of hypertrophy) and in failing left ventricular myocardium from patients with dilated and ischemic cardiomyopathy [25].

To the best of our knowledge, to date no studies have been performed to compare CARP mRNA levels in both LV and RV during mammalian postnatal development and transition from compensated to overt heart failure. It is still unclear whether CARP gene up-regulation is specific for cardiac hypertrophy in both ventricles and whether CARP expression is induced by Dox. To test the hypothesis that proposed chamber-dependent patterns [26] of CARP cardiac expression can be used as a sensitive marker for progression not only of normal ventricular hypertrophy but also of ventricular contractile dysfunction, and of a predisposition to heart failure, we analyzed LV vs. RV CARP gene expression during postnatal development and at different stages and degrees of Dox-induced heart dysfunction in neonatal piglets. Quantification of mRNA levels for brain natriuretic peptide (BNP) was also performed in order to monitor the severity of cardiac damage [27–29] and Dox cardiotoxicity [18].

The present study reports three novel findings, namely: (1) significantly augmented CARP mRNA and protein levels in the LV compared to the RV resulting in left vs. right asymmetry in ventricular CARP expression throughout early postnatal development; (2) dose- and chamber-dependent CARP mRNA and protein enrichment in ventricular myocardium in response to Dox; and (3) abolishment of asymmetric patterns of ventricular CARP expression at heart failure.


   2. Methods
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Animals and Dox treatment
Animals were treated and cared for in accordance with the European commission directive 86/609/EEC on the protection of animals used for experimental and other scientific purposes, and all animal protocols were approved by the ethical research committee of Galicia (Spain). Litters of ‘Large White’ domestic pigs were obtained from a local commercial breeder (La Coruña, Galicia) and maintained in an automatic nursery system (Nütinger System). Thirty 8–10-day-old piglets were randomized in five groups and assigned to receive intravenous (i.v.) Doxorubicin (Tedec-Meiji Farma) or a normal isotonic saline (NIS) vehicle. All i.v. Dox infusions (15 ml each) were administered through a marginal ear vein over a period of 30 min. To avoid possible irritant effects, 15 ml of NIS were additionally infused through the same vein after each Dox administration. Control animals were injected with 30 ml of NIS alone. On the 20th day after the last Dox or NIS infusion (i.e. on postnatal day 30), the animals were anesthetized, the thoracic cavity was opened through a median sternotomy, and the entire heart was rapidly removed, weighed and photographed while still beating.

2.2. Tissue preparation and morphometry
The isolated hearts were partially sectioned at the midpoint of the LV length and photographs of the open ventricular chambers were taken. The LV and RV free walls (FWs) and the left and right auricles were dissected, flash frozen in liquid nitrogen and stored at –85 °C until study. The thickness of the LV and RV FW was measured using digitized photoimages of the ventricular-chamber cross-sections, and the mean and standard error of the mean (±S.E.M.) were calculated.

2.3. RNA isolation and Northern blot hybridization
Deep-frozen tissue samples (150–200 mg) were directly disrupted in RLT buffer (Qiagen) using a high-speed rotor-stator homogenizer (Ultra-Turrax T8, Germany), digested with Proteinase K (Qiagen), loaded into a RNeasy Midi column (Qiagen), subjected to on-column digestion of DNA with RNase-free DNase (Qiagen) and proceeded in accordance with the manufacturer's recommendations. For Northern blot analyses, 4 µg of total RNA was electrophoresed, transferred onto Nytran (Amersham) membranes, UV-cross-linked, prehybridized for 1 h at 68 °C in ExpressHyb solution (Clontech), and then hybridized overnight with a random-primed, [32P]dCTP-labeled porcine CARP probe (i.e. the full-length coding sequence obtained by digestion of the pCAL-nFLAG-FS-CARP plasmid with restriction enzymes used for cloning). Membranes were consecutively washed in 2xSSC-0.1% SDS, 1xSSC-0.1% SDS and 0.1xSSC-0.1% SDS solutions at 68 °C prior to autography with intensifying screen at –80 °C.

2.4. Semi-quantitative RT-PCR
A two-step semi-quantitative RT-PCR was used [30]. The gene under study and the internal standard gene were amplified in separate or in the same tubes (multiplex PCR). The pig ribosomal protein L19 (RPL19) cDNA (accession number BI181894 [GenBank] ) was used to design oligonucleotides 36 (5'-AAC TCC CGT CAG CAG ATC CG-3') and 65 (5'-CTT GGT CTC TTC CTC CTT GGA-3') for a 480-bp PCR product. Pig glyceraldehyde-3-phospho-dehydrogenase (GAPDH) was amplified with primers 15 (5'-TCC TGC ACC ACC AAC TGC TTA-3') and 16 (5'-GGA CAC GGA AGG CCA TGC CA-3') generating a 256-bp PCR product. A 399-bp fragment of the pig CARP was amplified with oligonucleotides 27p (5'-TTA ATA GAA GCT GGA GCC CAG-3') and 28p (5'-GTC AAA TAT TGC TTT GGT TCC AT-3'). A 223-bp fragment of the pig BNP was amplified with oligonucleotide 78 (5'-CTA GGA TGC CGT TCC CAT CCA-3') and 83 (5'-GGA CTT GGA AGA TGC TAC TGC-3'). Two non-overlapping sequences (accession numbers: BF708430 [GenBank] and BF710105 [GenBank] ) of the pig eHAND were used to design oligonucleotides 23 (5'-GAA AGG CTC AGG ACC CAA GAA-3') and 24 (5'-CTG GGC CCA GGG CAG GAG-3') for a 296-bp PCR fragment. A 272-bp fragment of the dHAND gene was amplified with oligonucleotides 25 (5'-CGC AAG GAG CGG CGC AGG-3') and 26 (5'-TTG CTG CTC ACT GTG CTT TTC-3'). The complete coding sequence of the porcine myosin light chain-2v (MLC2-v; accession number AW785064 [GenBank] ) was used to design oligonucleotides 69 (5'-TCA TGG ACC AGA ACA GGG ATG-3') and 70 (5'-GTG ATG ATG TGC ACC AGG TTC-3') for a 376-bp PCR product. PCR products were analyzed in the linear range of amplification by agarose gel electrophoresis and intensity of bands was estimated by videodensitometry (GelDoc 2000 and VersaDoc 1000, Bio-Rad) and computer software (Quantity One, Bio-Rad).

2.5. Isolation of porcine CARP full-length cDNA
Primers 27 (5'-TTA ATG GAA GCT GGA GCC CAG-3') and 28 (5'-GTC GAA TAT TGC TTT GGT TCC AT-3') designed on a relatively conserved cDNA region of known CARP sequences were used in RT-PCR reactions with the RNA isolated from the LV FW of 30-day-old piglets. A single 399-bp RT-PCR product of the expected size was obtained, purified and sequenced in both directions. This partial porcine CARP cDNA sequence was used to design primers for 5'- and 3'-RACE reactions to complete a full-size porcine CARP cDNA, using the SMART RACE technology under conditions recommended by the manufacturer (Clontech).

2.6. Cloning and expression of tagged-CARP fusion proteins
The full-size (FS, amino acid residues 1–319), N-terminal (NT, amino acid residues 1–151), and the C-terminal part (CT, amino acid residues 152–319) of porcine CARP protein were RT-PCR cloned into the 5'BamHI and 3'NcoI sites of the pCAL-n-FLAG expression vector (Stratagene) and expressed in BL21 E. coli cells (Stratagene). CARP proteins fused to the 5-kDa tag, which included the calmodulin-binding peptide (CBP) and FLAG peptide, were purified from bacterial extracts, using calmodulin-affinity resin (Stratagene). Affinity purified CBP-FLAG-CARP fusion proteins (without or with 10 mM DTT) were separated by SDS-PAGE followed by Coomassie staining.

2.7. SDS-PAGE and Western blot analysis of cardiac samples
Cardiac muscle lysates were fractionated by SDS-PAGE, stained with Coomassie or transferred to PVDF membranes (Millipore). Isolated protein bands were subjected to in-gel digestion with trypsin for peptide fingerprint analysis by MALDI-TOF. Western blots were probed with rabbit anti-CARP polyclonal antibodies (1:1000) and detected using horse-peroxidase-conjugated anti-rabbit IgG (1:10 000; Sigma) and the SuperSignal West Pico chemiluminescent substrate (Pierce Biotechnology) according to the manufacturer's instructions. Polyclonal antibodies against porcine NT-CARP were raised in rabbits by Davids Biotechnologie (Regenburg, Germany).


   3. Results
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Cloning and characterization of the porcine CARP gene
The nucleotide sequence of the full-length porcine CARP cDNA obtained by 3'- and 5'-RACE was deposited into GenBank (accession number AY248702 [GenBank] ). The size of the pig CARP mRNA is about of 2.0 kb as determined by Northern blot hybridization (see Fig. 1a). The transcript has an open reading frame encoding a putative protein of 319 amino acids with a calculated molecular weight of 36.1 kDa. Both the coding DNA and deduced amino acid sequences of porcine CARP show a high homology to human (90.6 and 95%), rabbit (89.1 and 91.9%), rat (86.3 and 90.9%), and mouse (85.9 and 89.3%) CARP. Despite having two nuclear localization signals, the pig CARP is predicted to be a cytoplasmic protein as revealed by PSORT [31] and SubLoc [32] programs. Computer analysis by COILS 2.2 [33] and PairCoil [34] programs of the porcine CARP amino acid sequence revealed a high-predicted coiled-coil region located at the NT part of the protein, which spans from the amino acid residues 67–97. Secondary structure prediction analyses (by GOR IV [35] and DSC [36] programs) revealed that the most probable porcine CARP conformation state is alpha-helices. Proteins form dimers through coiled-coil association of the alpha-helices [37]. To determine whether the N-terminus of porcine CARP is able to form dimers, the FS-, NT- and CT-CARP-containing constructs were expressed in E. coli and purified as single polypeptides (see Fig. 1f). Co-electrophoresis of the DTT-free and DTT-treated samples demonstrated that the NT-CARP (but not the CT) could form dimers. A partial conversion of the NT peptide to a stable dimer occurs in the absence of reducing agent.


Figure 1
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  		Fig. 1 Estimation of mRNA and protein levels for CARP and other genes in ventricles (LV, RV) and atria (LA, RA) of 12-h-old piglets (C5, C6, C7). (a) Northern blot hybridization: total RNA (4 µg per lane) from each heart chamber was hybridized with pig CARP-specific probe. Loaded RNA was visualized by staining with ethidium bromide (b). (c) Semi-quantitative RT-PCR after normalization of the cDNA template to GAPDH and RPL19. (d) Quantification of CARP mRNA signals obtained by Northern blot hybridization normalized to 28S and 18S rRNA (white boxes) and those obtained by semi-quantitative RT-PCR normalized to RPL19 (black boxes). Values relative to the CARP transcript level in the RV (taken as 1). (e) equal amounts of total protein extracted from four cardiac chambers were separated by SDS-PAGE. Proteins identified by MALDI-TOF: β-MHC – beta-myosin heavy chain; ACT-{alpha}2 – actinin-alpha 2 form; DES – desmin; {alpha}-AC – cardiac alpha-actin; TPM-1 – cardiac tropomyosin-1 alpha chain; MLC-1v – myosin light chain form 1 ventricular; *- MLC-2v. (f) Western blot from replica-gel probed with anti-NT-CARP antibodies. FS-cloned full-size porcine CARP. 30–94 – MW values, kDa. (g) Quantification of CARP protein signal relative to that in the RV.

 
3.2. CARP is asymmetrically expressed in the heart of newborn piglets
The levels of the CARP mRNA in four chambers of the 12-h-old piglet heart (Fig. 1a, piglet C5) were analyzed and quantified by Northern blot hybridization. The following CARP mRNA level values relative to those in the RV (taken as 1) were estimated by film scanning and densitometry: LV – 9, RV – 1, LA – 25, and RA – 15 (Fig. 1d, white boxes). To verify this distribution of the CARP transcript in the heart, semi-quantitative RT-PCR with RNAs isolated from four cardiac chambers of the first (i.e. C5) and two other 12-h-old piglets (i.e. C6 and C7) were performed (Fig. 1c), yielding nearly identical results for the three hearts studied. Note that the relative CARP mRNA values estimated for each cardiac chamber by semi-quantitative RT-PCR were very similar to those obtained by Northern blot analyses (Fig. 1d, black boxes). In contrast to CARP, expression of several other genes involved in ventricular chamber specification (MLC-2v, dHAND, eHAND) was found to be similar in both the RV and LV (see Fig. 1c).

Extracts of each cardiac chamber of 12-h-old piglets were subjected to SDS-PAGE (Fig. 1e) followed by Western blot with antibodies against porcine NT-CARP. On Western blots, these antibodies recognized a single 40-kD band (Fig. 1f), which corresponds to the apparent molecular mass of mouse [38] and rat [39] CARP. In the LV myocardium, CARP was approximately 15 times more abundant than in the RV where a weak signal was observed only after a prolonged film exposure. In the RA and LA, CARP content was, respectively, two and four times higher that that in the LV (Fig. 1g). Remarkably, the overall distribution patterns of the protein and CARP mRNA across four cardiac chambers were similar indicating the transcript is actually being translated into the protein product. In spite of fairly significant differences in the CARP transcript and protein level in the LV and RV myocardium of newborn piglets, their overall protein composition, as judged by SDS-PAGE, was identical, with equal levels of the β-myosin heavy chain, alpha-actin, alpha-actinin 2, MLC-1v, MLC-2v, cardiac tropomyosin, and desmin in both ventricles (see Fig. 1e).

3.3. Asymmetric patterns of ventricular CARP-side expression are maintained throughout early postnatal development
Given previous studies demonstrating a disproportional myocardial growth of the LV in neonatal piglets [12,40–42], we determined LV and RV FW thickness during the first 60 days of postnatal pig development. At birth, the FW thickness was slightly higher in the LV (5.5±0.2 mm) as compared to the RV (4.5±0.2 mm), but at the of 1 month the LV FW was found to be four-fold thicker than the RV FW (Fig. 2a, b, and c). The dynamics of the LV to RV FW-thickness ratio during the first 60 days of life is shown in Fig. 2d. The levels of CARP mRNA in both ventricular chambers of neonatal piglets were analyzed by Northern blot hybridization (Fig. 2e,f). In the LV, the CARP transcript subsequently increased as postnatal development proceeded, reaching the highest level in 60-day-old piglets. In the RV, the CARP transcript was very low at the 12-h-stage, increasing significantly on day 8, maintaining nearly the same level up to day 30, and then increasing again at the 60-day-stage. As a result, the asymmetric patterns of LV/RV CARP expression were observed at each stage studied (see Fig. 3f).


Figure 2
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  		Fig. 2 Developmental dynamics of ventricular free wall thickness (VFWT) and CARP mRNA levels in the LV and RV of neonatal piglets. Cross-sections through ventricular chambers of 12-h-old (a) and 30-day-old (b) piglets (Bar – 1 cm). (c, d) Developmental dynamics of L/R VFWT values and L-VFWT/R-VFWT ratio, respectively. (e) Northern blot hybridization: total RNA (4 µg per lane) normalized to 28S rRNA was hybridized with CARP-specific probe. (f) Quantification of CARP mRNA signal relative to that in the RV (taken as 1).

 


Figure 3
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  		Fig. 3 CARP mRNA levels in the LV and RV myocardium of the control (C31, C32, C33) and experimental piglets (D23–D28) exposed to 4 mg/kg of Dox. (a) Multiplex RT-PCR for CARP and RPL19. (b) The intensity value of the CARP bands shown in A normalized to that of the RPL19 are plotted as a CARP mRNA levels (grey – LV; black – RV) relative to those in the RV of control 30-day-old piglets. (c) Representative Northern blot of CARP expression signals in the RV normalized to 18S and 28S rRNA (D).

 
3.4. Asymmetric patterns of ventricular CARP-side expression are specifically altered upon Dox- exposure in vivo
At the highest dose used (6 mg/kg; DOX-I group), Dox caused a significant reduction in body weight from the first week after the last injection as compared to controls. The DOX-I group was also characterized by a high mortality rate. At post mortem, augmented thickness of the LV FW was observed in these piglets. Administration of decreasing Dox amounts to neonatal piglets resulted in a significantly higher survival rate. No case of premature death was observed in the DOX-II (4 mg/kg), DOX-III (2 mg/kg), and DOX-IV (1 mg/kg) experimental groups. However, heart failure/arrest was developed during anesthesia procedure in 50% and 30% of the animals in the DOX-II (4 mg/kg) and DOX-III (2 mg/kg) group, respectively. In contrast, animals of the DOX-IV (1 mg/kg) group did not display either signs of cardiac impairment or clinical discomfort throughout the experiment. The results indicated that the critical Dox dose for 8–10-day-old piglets is 4 mg/kg, because a higher Dox dose caused the rapid development of heart failure leading to premature death.

3.4.1. Control group
CARP transcript levels in the LV and RV of the 30-day-old piglet control group were determined by semi-quantitative RT-PCR and Northern blot hybridization. The LV/RV CARP mRNA ratio was on average 3.2±0.5:1±0.2 (see Fig. 2e,f). Western blot analysis of the same cardiac muscle samples (see Fig. 5a) revealed that CARP was enriched approximately five-fold in the LV compared to the RV. BNP transcript levels were very low in both the LV and RV of the control group (see Fig. 4a).


Figure 4
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  		Fig. 4 CARP and BNP mRNA levels in the LV and RV myocardium of the control (C14, C31, C32) and experimental piglets (D34–D38) exposed to 2 mg/kg of Dox. (a) RT-PCR for CARP, BNP and RPL19. (b) The intensity value of the CARP bands shown in A normalized to that of the RPL19 are plotted as CARP mRNA levels (grey – LV; black – RV) relative to those in the RV of control 30-day-old piglets. (c) Representative Northern blot of CARP expression signals in the RV normalized to 18S and 28S rRNA (d).

 
3.4.2. DOX-II group (4 mg/kg)
The semi-quantitative RT-PCR analysis showed that all animals of the DOX-II group displayed the augmented CARP mRNA levels in the RV ranging from a 2.3- (D28) to 6.3-fold (D26) transcript enrichment over controls (Fig. 3a,b). Similar results were obtained by Northern blot hybridization (Fig. 3c,d). In contrast, in the LV the CARP mRNA was augmented in only three (i.e. D23, D26 and D27) of the six animals studied (see Fig. 3a,b). The average fold increase of the CARP mRNA content in the LV and RV was 1.3 and 4.2, respectively. Thus, cardiac toxicity induced by Dox at a dose of 4 mg/kg resulted in preferential CARP up-regulation in the RV myocardium. In turn, owing to the different up-regulation of the gene, the total CARP mRNA levels became practically equal (i.e. 1:1) in the LV and the RV in Dox-treated animals, in clear contrast to the asymmetric pattern of transcript presence in the LV vs. RV of control 30-day-old piglets (i.e. 3.2:1). Particularly important was the finding that enriched CARP protein levels are nearly similar in both ventricles of Dox-treated piglets as judged by Western blot analysis (see Fig. 5a).


Figure 5
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  		Fig. 5 Western blot analysis of myocardium lysates obtained from the LV and RV of control and Dox-treated (4 and 2 mg/kg) piglets. Equal amounts of total protein were separated by SDS-PAGE (b) followed by Western blot (a) with anti-NT-CARP polyclonal antibodies. 200-14.4 – MW values, kDa.

 
3.4.3. DOX-III group (2 mg/kg)
All Dox-treated animals displayed increased CARP mRNA levels in both the RV and LV as demonstrated by RT-PCR (Fig. 4a,b) and Northern blot hybridization (Fig. 4c,d). The average fold-increase of the CARP mRNA content in the LV was 1.8 and 2.6 in the RV as compared to controls. As a result of this, the LV/RV CARP mRNA ratio in this group (i.e. 2.2:1) changed in comparison to that of DOX-II (i.e. 1:1), but it was still different from control values (i.e. 3.2:1). In these animals, similar proportions in CARP protein enrichment in the LV vs. RV were observed on the corresponding Western blots (see Fig. 5a). Of note, Dox provoked significant BNP up-regulation in both ventricular chambers being more pronounced in the LV myocardium. In both ventricles, the levels of BNP mRNA positively correlated with those of the CARP transcript, suggesting that CARP and BNP responded similarly across ventricular regions to Dox treatment (Fig. 4A).

3.4.4. DOX-IV group (1 mg/kg)
Dox, at a single dose of 1 mg/kg, led to a 1.6-fold increase in CARP mRNA of the LV and a 1.3-fold of the RV. As a result, the LV/RV CARP mRNA ratio in this experimental group (i.e. 3.8:1.0) was slightly amplified compared to controls (i.e. 3.2:1.0). In the LV, BNP mRNA levels were significantly elevated in all Dox-treated animals of this group, while in the RV a lower increase in the BNP transcript was observed in only two out of five piglets. Considering pronounced BNP up-regulation as a marker of the severity of heart damage [43,44], it seems likely that a relatively more serious myocardial impairment developed in the LV of these animals.

The results of the comparative analysis of CARP expression (at mRNA and protein levels) in both ventricular chambers of neonatal piglets in response to Dox demonstrated that: (1) CARP was augmented in the ventricular myocardium of all animals; (2) Dox-induced CARP enrichment was chamber- and dose-dependent; (3) higher Dox doses led to preferential CARP up-regulation in the RV, while lower Dox doses resulted in primary transcript and protein enrichment in the LV; and (4) at a Dox dose of 4 mg/kg, the asymmetric LV/RV pattern of CARP distribution characteristic of controls was not observed and was progressively reconstructed upon decreasing Dox-dose.


   4. Discussion
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
4.1. CARP expression during early postnatal development
Cardiac CARP expression is characterized by distinct left- and right-sided patterns resulting in an overall left-right asymmetry of CARP distribution in the pig neonatal heart. The marked difference in the CARP transcript and protein level between LV and RV found in 12-h-old piglets does not correlate either with FW-thickness which is practically equal in both ventricles (this work), or with ventricular cell volume which is slightly higher for RV myocytes as compared to LV myocytes [12]. In prenatal circulation, the pressure between two ventricular chambers is equal, however, there is a relatively higher volume preload in the RV vs. the LV. This hemodynamic situation is reversed shortly after birth when the LV is exposed to a higher systemic workload in comparison to the RV. As a result, in neonatal piglets the LV undergoes rapid hypertrophic growth, while the RV displays a progressive wall thinning associated with increasing RV-chamber volume (see Fig. 2b,c). In the LV of neonates, the developmental dynamics of the CARP mRNA level strongly correlates with the rate of its hypertrophic growth [11,12]. In the RV, by contrast, the CARP transcript level did not significantly change on day 8 as compared to that on day 30 post partum, e.g. during a period when the RV porcine cardiomyocytes display morphological signs of moderate hypertrophy [12]. Therefore, it is difficult to associate the CARP ventricular enrichment only with cardiac myocyte hypertrophy.

Our findings raise two important questions concerning the mechanisms underlying the developmental CARP up-regulation in the ventricular myocardium. Firstly, what is the mechanism by which CARP functions as a protein specifically involved in myocardium maturation during postnatal development? To some extent, it may be possible to define CARP as a molecular adaptor required for correct supramolecular assembly of cardiac sarcomeres [39]. Identification of the coiled-coil domain in the N-terminus of CARP and demonstration of its dimer formation in vitro strongly suggests that CARP can be implicated in protein–protein interactions in different cellular compartments, both cytoplasmic and nuclear. Thus, in neonatal piglets CARP expression may be directly associated with structural and functional maturation of the sarcomere network of ventricular cardiomyocytes. Also secondly, which are the target genes that are regulated by overexpression of CARP in cardiac myocytes? Studies on cultured in vitro mouse and rat neonatal cardiomyocytes demonstrated that experimentally forced CARP expression inhibits synthesis of some contractile proteins [19,20] and could partly be responsible for the depressed contractile responsiveness of failing hearts to catecholamines [45]. Our comparative electrophoretic analysis of LV and RV myocardium biopsies from newborn piglets characterized by the 10–15-fold difference in the CARP transcript and protein presentation did not reveal noticeable differences in the contractile protein content between two ventricular chambers (see Fig. 1e). Thus, the identification of the CARP target genes still represent an important next step in understanding the physiological functions of the CARP gene in LV and RV chambers during early postnatal mammalian development.

Our observations indicate that the CARP transcript content in the porcine ventricular myocardium gradually increases from newborn to adult. A similar tendency in the CARP mRNA enrichment in the heart has been demonstrated in rats [4] and mice [6,8]. In addition, in mice [38] and rats [7,23] comparable levels of cardiac CARP expression have been found in both neonates and adults. These results are in a sharp contrast to what has been described previously in mice according to which CARP is gradually down-regulated in the heart from neonate to adult stages [19] and a CARP promoter is inactive in the normal adult heart [46]. The present study demonstrates that in the neonatal porcine heart the CARP transcript is abundant as a housekeeping RPL19. In the adult pig heart, the CARP mRNA and protein was detected at the same or at an even higher level (Torrado and Mikhailov, unpublished). In the adult human heart, numerous CARP-expressing myocytes were found throughout the ventricle and atrium myocardium [47]. All of these situations can be viewed as various aspects of the same possible role of CARP as one of the molecular players involved in cardiomyocyte myofibril assembly [39,47,48] both during development and in adult state.

4.2. A model of Dox-induced heart failure in neonatal piglets
Dox-induced cardiotoxicity remains a major limitation for its use as an antitumor agent. In humans, this toxicity appears to be dose- and age-dependent [14]. In neonatal piglets, the severity of Dox-induced cardiac impairments was clearly dose-dependent as it has been demonstrated at functional (heart failure and survival rates), morphometric (heart weight and ventricular FW measurements) and molecular (CARP and BNP expression) levels. Our findings demonstrate, for the first time, that cardiomyopathy produced by Dox could be associated with specific alterations in the expression patterns of the CARP gene whose protein product seems to be important for the maintenance of adequate structural integrity of cardiac cell sarcomeres [39]. At the dose 4–6 mg/kg, Dox led to development of severe heart growth retardation, myocardial dysfunction and heart failure accompanied by a marked CARP up-regulation in both ventricles. It should be noted that the normal asymmetric LV/RV pattern of CARP mRNA and protein distribution was completely abolished in response to these Dox doses. Dox-dose lessening resulted in obvious improvement of general and cardiac parameters with consequential restoring of CARP LV/RV asymmetric patterns against the persistent gene up-regulation in both ventricles.

4.3. Doxorubicin cardiotoxicity
Our results indicate that a distinction should be made between the response to Dox in vivo and that elicited by exposure to Dox in vitro. Dox administration to neonatal piglets led to significant CARP and BNP up-regulation in the ventricular myocardium, while incubation of neonatal rat ventricular myocytes with Dox in vitro rapidly decreased CARP [19,20] and BNP [18,20,21] mRNA levels. A possible interpretation of this discrepancy is that we determined CARP and BNP levels in the ventricle myocardium 20 days after Dox injections to piglets, whereas in culture experiments mentioned above the corresponding expression measurements were performed 1–24 h after adding Dox.

Children younger than 2 years seem to be more susceptible than adults to cardiotoxic side-effects of Dox [13,49]. Our study demonstrated that a single injection of Dox (2 mg/kg) in 10-day-old piglets led to onset of a heart failure in approximately 30% and to significant CARP and BNP up-regulation in all animals studied (see Fig. 4). However, administration of the same dose of Dox to 20-day-old animals did not result in either morphological signs of cardiac damage or CARP up-regulation in ventricular myocardium (Torrado and Mikhailov, unpublished). Of note, the Dox dose used in these experiments (i.e. 2 mg/kg) is approximately equivalent to 40–50 mg/m2 in humans and it is at least three times lower than Dox doses (150–200 mg/m2) commonly used in pediatric practice for children of 1–4 years of age. In this context, 3-week-old piglets received Dox in a cumulative dose of approximately 300–400 mg/m2 display altered diastolic and systolic functions [50].

Collectively, the apparently direct association between LV/RV CARP up-regulation and severity of Dox-cardiotoxicity makes our assays on neonatal piglets an attractive model for studying the process of heart failure development.


   Acknowledgements
 
This work was supported by a long-time grant (SAF2001-0910) from the Spanish Ministry of Science and Technology, by funding from the Institute of Health Sciences (University of La Coruña, Spain), and by infrastructure funding (2002) from the Autonomic Government of Galicia. We are grateful to Dr Beatriz Nespereira for assistance in Western blot experiments and to Dr Esperanza Cerdán for welcoming our Northern blot hybridization experiments in her laboratory.


   Notes
Top
 Notes
Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
{star} For detailed access on porcine CARP domain structure, CARP dimer formation, design of the Dox treatment protocol, and cardiac parameters of control and Dox-treated piglets see the online-only Figure Supplement Data (Fig. 6, 7 and 8) at http://www.elsevier.com/locate/ejheart. Back


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

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