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Cardiovascular Research 1998 37(1):187-201; doi:10.1016/S0008-6363(97)00205-8
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Altered cardiac hormone and contractile protein messenger RNA levels following left ventricular myocardial infarction in the rat: an in situ hybridization histochemical study

Richard L Young*, Andrew L Gundlach and William J Louis

The University of Melbourne, Clinical Pharmacology and Therapeutics Unit, Department of Medicine, Austin and Repatriation Medical Centre, Heidelberg, Victoria 3084, Australia

* Corresponding author. Tel: +61 3 9496 3420; Fax: +61 3 9459 3510; E-mail: richardy@ariel.ucs.unimelb.edu.au

Received 30 January 1997; accepted 6 August 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Cardiac remodeling secondary to myocardial infarction is associated with hypertrophy of surviving myocardium and altered cardiac gene expression. The present study examined the spatiotemporal expression of cardiac contractile protein and peptide hormone mRNA following left ventricular myocardial infarction (LVMI) in the rat heart. Methods: LVMI was produced in Wistar rats by ligation of the left anterior descending coronary artery and mRNA levels of cardiac {alpha}-actin (cACT), skeletal {alpha}-actin (sACT), ventricular myosin light chain-2 (MLC-2v), β-myosin heavy chain (β-MHC) and pre-proatrial natriuretic peptide (ppANP) were examined at 24 h, 1 and 4 weeks) post-LVMI by in situ hybridization histochemistry with 35S-labeled oligonucleotide probes. Results: Infarct size, determined at 1 week post-LVMI, was 44.5±2.7% of the combined left ventricular epi- and endocardial surface area. Myocyte fiber width, reflecting cellular hypertrophy, was increased in left ventricular, mid-septal and mid-right ventricular muscle fibers by 11–20% at 1 week post-LVMI (P<0.05) and by 24–29% at 4 weeks (P<0.05). At 24 h, 1 and 4 weeks post-LVMI, heart- and lung/body weight ratios were significantly elevated compared to sham-operated rats (1.3–1.8-fold, P<0.01 and 1.6–2.9-fold, P<0.005, respectively). PpANP mRNA levels in the left ventricle were increased 3.8- and 3.3-fold at 1 and 4 weeks (P<0.05), with highest levels in the epicardium, papillary muscle, infundibulum and apex of the chamber. Septal and right ventricular ppANP mRNA levels were highest at 24 h post-LVMI (2.1- and 2.6-fold increase, P<0.05) and remained elevated at 4 weeks, with maximum levels at the left endocardial surface of the septum and apex of the chambers. Atrial levels of cACT mRNA were increased 1.9-fold at 1 week post-LVMI (P<0.05) and remained elevated at 4 weeks. Skeletal ACT mRNA, not normally expressed in the adult rat heart, was induced as early as 24 h post-LVMI in both atria, the septum and right ventricle, with discrete hybridization signal detected at the apex of the chambers and in the right ventricular free-wall, and later (1 week) in the left ventricular epicardium. MLC-2v mRNA levels were unaltered post-LVMI, except for a transitory loss of expression at 24 h in the left atria, ventricle and apical septum. In contrast, ventricular β-MHC mRNA was markedly induced in regions containing increased ppANP mRNA, with a maximal 3.0- and 4.0-fold induction (P<0.05) seen at 1 and 4 weeks in the left ventricle and a 3.7-fold induction at 4 weeks in the septum and right ventricle (P<0.05). Conclusion: The regional increases in induced cardiac hormone and contractile protein mRNA in similar subchamber regions of the rat heart post-LVMI implies mutual activation by mechanical and/or neuroendocrine stimuli in the transcriptional response to myocardial overload.

KEYWORDS Myocardial infarction; Cardiac hypertrophy; Atrial natriuretic peptide; Actin; Myosin; In situ hybridization histochemistry; Wistar rat


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Alterations in mRNA levels of actin and myosin isoforms have been described in a variety of in vitro and in vivo models of cardiac myocyte hypertrophy. Phenotypic conversion from the constitutive cardiac {alpha}-actin (cACT) and ventricular {alpha}-myosin heavy chain forms of mRNA in the rat to the inducible skeletal {alpha}-actin (sACT) and β-myosin heavy chain (β-MHC) forms has been reported following stimulation of cultured neonatal rat cardiocytes [1], subsequent to aortic stenosis [2–5], and following aortocaval fistula [6], chronic coronary artery constriction [7]and left ventricular myocardial infarction (LVMI) in the rat [8]. Switching between myosin heavy chain protein forms has the potential to alter the contractile properties of the recombinant cardiac muscle fiber due to slower adenosine triphosphate (ATP) cycling of the induced β-MHC protein, producing a slower, more energy-efficient contraction [9]. Recent evidence that over-expression of sACT mRNA in BALB/c mice correlated with increased myocardial contractility [10], confirms the potential for transcriptional control of contraction in hypertrophic myocardium.

Increased mRNA expression and cellular accumulation of constitutive ventricular myosin light chain-2 (MLC-2v) and re-expression of ventricular pre-proatrial natriuretic peptide mRNA (ppANP) are also reported as features of the hypertrophic process [11–13]. Myosin LC-2v protein has been shown to regulate heavy chain myosin ATPase in vitro [14], and its phosphorylation is known to affect the Ca2+-sensitivity of atrial and ventricular muscle fibers [15]. Re-expression of ventricular ppANP mRNA may serve to attenuate increased pre- and after-load, secondary to cardiac dysfunction [16].

Thus far, however, no studies have used in situ hybridization histochemistry to examine the spatiotemporal distribution of these cardiac mRNAs following LVMI. An advantage of this technique compared to Northern blot analysis, is the ability to determine, in serial sections, the regional localization of mRNA in atrial, left and right ventricular and interventricular septal chambers. We have therefore employed in situ hybridization histochemistry using specific 35S-labeled oligonucleotide probes to examine the distribution of several cardiac mRNA species 24 h, 1 and 4 weeks after LVMI-induced cardiac overload. The oligonucleotide probes chosen represent both constitutive (‘adult’; cACT, MLC-2) and inducible (‘fetal’; sACT, β-MHC) molecular markers of the cardiac contractile phenotype, as well as ppANP, a known marker of ventricular hypertrophy [16].

A preliminary account of this work was reported to the 15th Scientific Meeting of the International Society of Hypertension, Melbourne, Australia [17].


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Animals
Wistar rats were obtained from the Biological Research Laboratories, Austin and Repatriation Medical Centre (Heidelberg, VIC, Australia). All studies were performed in agreement with, and according to, the Prevention of Cruelty to Animals Act (1986) and the NH & MRC/CSIRO/AAC Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (1990) and with the approval of the Austin and Repatriation Medical Centre Animal Welfare Committee.

2.2 Left ventricular myocardial infarction
Female Wistar rats with a mean weight of 189±3 g were subjected to left anterior descending coronary artery (LAD) ligation to produce LVMI, as described by Pfeffer et al. [18], with slight modifications. Rats were rapidly anesthetized with ether, orally intubated and artificially ventilated with 2% halothane in oxygen (Fluothane, ICI Pharmaceuticals, Melbourne, VIC, Australia). A left thoracotomy was performed within the fourth intercostal space, the pericardium gently ruptured and the heart exteriorized. A 1-mm long polypropylene suture was placed between the pulmonary outflow tract and the left atrium, permanently ligating the LAD 2–3 mm from its origin. The heart was then returned to the thoracic cavity, the overlying pectoral muscles sutured with a purse-string, plain gut suture and the lungs hyper-inflated with positive end-expiratory pressure. The skin was then closed with a continuous silk suture and the animals allowed to recover. Sham-operated animals were treated similarly, except that the ligature around the coronary artery was not tied.

Upon recovery, rats were assigned to one of three groups — 24 h, 1 week and 4 weeks post-LVMI — and had access to standard rat chow and tap water ad libitum. Body weight was measured pre-LVMI, 24 h post-LVMI and thereafter at weekly intervals.

Rats were killed by cervical dislocation and decapitation and the heart was rapidly removed, placed in ice-cold isotonic saline, gently palpated to remove excess blood and dissected free of associated adipose and connective tissue. The left ventricle was flushed via the aorta and the heart blotted dry and weighed. Similarly, the lungs, liver and right kidney were removed, dissected free of associated adipose and connective tissue and weighed. The heart was then filled via the aorta with Tissue-tek OTC mounting media (Miles Laboratories, Elkhart, IN, USA) and quickly frozen over liquid N2 (<1 min). Frozen hearts were stored (<2 weeks) at –70°C until further processed.

In the study groups, LVMI was confirmed macroscopically and cardiac hypertrophy by changes in heart and lung weights expressed either as g wet weight, or as a proportion of body weight (Table 1). In previous studies conducted in our laboratory [19], rats possessing infarcts occupying ≥40% of the left ventricular endocardial circumference could be identified from their heart- and lung/body weight ratio changes. For this reason, only rats with evidence of LVMI and increased heart- and lung/body weight ratio ≥29% and ≥59%, respectively, were selected for evaluation of cardiac mRNA levels.


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  		Table 1 Effect of sham- and LVMI-surgery on body and wet organ weight

 
2.3 Tissue preparation
Serial coronal sections (14 µm) of heart tissue were cut on a cryostat at –18°C with the cutting plane oriented parallel to the left–right atria axis. Heart sections containing complete atrial and ventricular chamber profiles were thaw-mounted onto poly-L-lysine-coated slides, air-dried and stored at –20°C (<2 days). Slides were then washed in 1xSSC (1xSSC: 0.15 M NaCl, 15 mM Na citrate, pH 7.0) to remove extraneous blood and mounting compound and dehydrated in serial ethanol washes (70%, 95%, 100%) prior to storage in 100% ethanol at 4°C (<2 weeks).

2.4 Infarct size determination
In an additional experiment (n = 6 rats), representative transverse infarct size was determined at 1 week post-LVMI. Four to five fresh-frozen, transverse sections (5 µm) were cut parallel to the atrioventricular groove, with sections taken at 1–1.2-mm intervals from the apex. Sections were stained with either hematoxylin and eosin, or Masson's trichrome and subjected to computer-assisted planimetry (MCID, Imaging Research, St. Catharines, Ontario, Canada), according to the method of Pfeffer and co-workers [18]. Briefly, the epi- and endocardial circumferences of the left ventricle and scar were measured for each digitized section image, summed separately for each animal, and expressed as a ratio of scar length-to-left ventricular circumference for each surface. These two ratios were averaged and expressed as a percentage, representative of the combined left ventricular epi- and endocardial surface area occupied by the infarct scar (infarct size).

2.5 Myocyte morphometry
In three further sets of animals, (n = 6 per time point) infarcted as outlined above, myocyte fiber width was determined in 5 µm sections taken from 24 h, 1 and 4 weeks post-LVMI rats. Following dissection, hearts were weighed and immediately placed in 10% buffered formalin (18–24 h, RT) cryoprotected in 30% sucrose (12–18 h, 4°C) and quickly frozen over liquid N2. In mid-ventricular transverse sections, cut and stained with Masson's trichrome as outlined above (see Section 2.4), computer-assisted planimetry (VideoPro 32, Leading Edge, Adelaide, SA, Australia) was performed at x200 magnification. In a 0.65-mm2 field, the width of 30–50 myocyte fibers displaying discrete, non-apoptotic nuclei were measured. In order to minimize variation, measurements were made across the central nuclear region of the myocytes. In this manner, the width of transversely oriented, papillary muscle fibers (representative of non-infarcted left ventricular myocardium) and longitudinally oriented, mid-septal and mid-right ventricular fibers were averaged for each section, animal and time point.

2.6 Oligonucleotide probes
To hybridize the various cardiac mRNA species, DNA oligonucleotide probes (38–45-mers; 50–60% GC content) were prepared to nucleotides 163–207 of the ppANP cDNA [20](5'-CGCTTCATCGGTCGTCTCGCTCAGGGCCTGCGGAGGCATGACCTC-3') by Dr. Paul Pearce (Prince Henry's Institute for Medical Research, Melbourne, VIC, Australia); and to nucleotides 52–90 of the cACT cDNA [21](5'-CAAACTGTACAATGACTGATGGGAGATGGGAGAGGGCC-3'); nucleotides 43–88 of the sACT cDNA [22](5'-CGGAGGATGGGGCCACCCTGCAACCATAGCACGATGGTCGATTGT-3'); nucleotides 57–101 of the MLC-2v cDNA [23](5'-CATGGAGAACACGTTGGAGCTTCCGCCCTCTAACCTCTTCTTGGC-3'); and nucleotides 22–67 of the β-MHC cDNA [24](5'-GATTTTCGCAGGAAGGGGGCTCCGGCCCCAAATGCAGCCATCTCT-3') by Biotech International, Perth, WA, Australia. The specificity of all probes for the intended mRNA target was confirmed by Basic Local Alignment Search Tool (BLAST) analysis of GenBank, EMBL, DDBJ and PDB sequence databases [25]. All probes were synthesized on an Applied Biosystems model 308B DNA Synthesizer and were purified prior to labeling using OPC cartridges (Applied Biosystems, Foster City, CA, USA) or NAP-25 columns (Pharmacia Biotechnology LKB, Uppsala, Sweden). Throughout this study, cardiac tissue was batch harvested, sectioned and concurrently hybridized as previously described [26]to minimize inter-assay variation.

2.7 In situ hybridization histochemistry
Fresh, unfixed tissue was used for detection of cardiac gene mRNAs according to published methods [27]. Oligonucleotide probes were 3'-end labeled with [{alpha}-35S]dATP (1200 Ci/mmol, Dupont-NEN, Boston, MA, USA) using terminal deoxynucleotidyl transferase (AMRAD Pharmacia Biotech, Sydney, NSW, Australia) to a specific activity of 1.0–2.5x109 dpm/µg [28]. Probes (2 pg/µl, 60–70 µl per slide) were hybridized to heart sections overnight at 42°C in a buffer containing 50% formamide/10% dextran sulfate/4xSSC and 200 mM dithiothreitol. ‘Non-specific hybridization’ was measured in the presence of a 100-fold excess of unlabeled probe. Slides were washed in 1xSSC (containing 20 mM β-mercaptoethanol) for 60 min at 55°C, rinsed in room temperature 1xSSC, 0.1xSSC, serially dehydrated in ethanol and air dried. Sections were then exposed to X-ray film (Kodak XAR-5, Ajax Chemicals, Melbourne, VIC, Australia) for 1–20 days depending on hybridization signal strength, prior to development.

2.8 Quantification of cardiac mRNAs
Cardiac mRNA levels were quantified using computer-assisted, densitometric image analysis (MCID). Relative optical densities (ROD) of autoradiographic images were converted to dpm/cm2 by interpolation from standard curves generated on individual films, from labeled brain paste standards co-exposed with the tissue sections [26]. As hybridization signal strength differed for each probe, films were exposed for different lengths of time with a single set of standards designed to encompass and exceed the dynamic range of the X-ray film over a wide range of dpm/cm2 values (eg. 3–30 000 dpm/cm2 for sACT). These standards were used to define the linear portion of the standard curve for each individual film after adjusting for 35S-decay. Duplicate film exposures of sections and standards were only required for ppANP mRNA, where 1-day (atria) and 3-day (ventricles) exposures were performed due to significant differences in hybridization signal strength (high in atria, lower in ventricles). Total hybridization signal of [{alpha}-35S]dATP-labeled probes was determined by manually outlining discrete cardiac chambers and averaging total hybridization signal measured in three consecutive serial sections from each animal. Total hybridization values less than the mean+2 s.d. of the ‘non-specific hybridization’ level measured on two consecutive serial sections were defined as undetectable (UD).

2.9 Statistical analysis
All data are given as mean±s.e.m. Differences between groups were evaluated by 2-way analysis of variance (ANOVA; CLR-ANOVA, Clear Lake Research, Clear Lake, TX, USA) with surgery (sham, LVMI) and time (24 h, 1 week, 4 weeks) as the main effects. Body and organ weights were further analyzed by simple effects testing to determine interaction terms with an interaction considered significant at the P<0.05 level. Differences in myocyte width were evaluated by 1-way ANOVA, with surgery and time main effects, followed by Tukey–Kramer multiple comparisons tests. Total hybridization signal differences were analyzed by two-way ANOVA with the error mean square from the ANOVA used to calculate 95% confidence intervals of the ratio of LVMI/naive levels. An interval range that did not include a 100% value was considered significant at the P<0.05 level.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Assessment of LVMI
3.1.1 Infarct size and myocyte morphometry
Infarct size in a representative group of rats with increased heart- and lung/body weight ratio ≥29% and ≥59%, respectively at 1 week post-LVMI, was 44.5±2.7% of the combined left ventricular epi- and endocardial surface area. In mid-ventricular transverse sections, myocyte fiber width was increased in transverse-oriented, left ventricular-papillary myocyte fibers by 11% and 24% at 1 and 4 weeks post-LVMI, respectively (Fig. 1A). Longitudinally-oriented, mid-septal myocyte fibers were similarly increased by 20% and 25% (P<0.05, Fig. 1B) at 1 and 4 weeks, and in mid-right ventricular fibers by 22% (P<0.05) and 29% (P<0.01, Fig. 1C).


Figure 1
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  		Fig. 1 Effects of LVMI on cardiomyocyte fiber width (µm) in sham- and LVMI-operated hearts at 24 h, 1 and 4 weeks post-LVMI (n = 6). Fiber width was determined by computer-assisted, light microscopic (x200) planimetry of transverse (A, papillary muscle) and longitudinally (B, mid-septal, C, mid-right ventricular) oriented fibers in 5 µm mid-ventricular transverse sections (means±s.e.m.). *P<0.05, **P<0.01 relative to sham myocyte width (ANOVA, Tukey–Kramer multiple comparisons test).

 
3.1.2 Heart, lung weight changes
In situ hybridization studies were performed in groups of 5 rats at 24 h, 1 week and 4 weeks post-LVMI and in groups of 3–6 sham-operated rats at each time point. Macroscopic evidence of left ventricular free-wall infarction (wall thinning, fibrous plaque) was present in all ligated hearts, with no evidence of infarction in sham-operated hearts. Heart/body wet weight ratios were significantly elevated at 24 h (1.3-fold), largely as a result of the characteristic acute inflammation (interstitial edema, infiltration) and ischemic cardiomyocyte swelling seen at this early time [29]. At 1 and 4 weeks post-LVMI, heart/body wet weight increased 1.3–1.8-fold relative to sham controls, reflecting the development of concentric and eccentric hypertrophy of overloaded myocardium. Similarly, lung/body wet weight ratios were increased from 24 h post-LVMI (1.6-fold) relative to sham controls and increased further over 1 and 4 weeks (1.9–2.9-fold), due to the characteristic pulmonary edema secondary to left ventricular dysfunction [29](Table 1). Body weight was unaltered as a result of LVMI (Table 1) and liver and kidney weights were similarly unaffected (data not shown).

3.2 Atrial natriuretic peptide mRNA
3.2.1 Sham levels
Atrial ppANP mRNA levels were, on average, 14.6-fold higher than ventricular levels, which were low, but detectable (Table 2). Localized ppANP mRNA expression appeared transiently induced by sham-surgery in the left ventricular free-wall at 24 h, but returned to basal levels by 1 week (Fig. 2A,C).


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  		Table 2 Effect of sham- and LVMI-surgery on cardiac pre-pro-atrial natriuretic peptide mRNA levels

 

Figure 2
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  		Fig. 2 Distribution of ppANP mRNA in sham- and LVMI-operated hearts at 24 h, 1 and 4 weeks post-LVMI. Photographs are printed directly from representative autoradiographs of whole-heart serial sections hybridized with specific 35S-labeled oligonucleotide against ppANP mRNA with white areas indicating hybridization. ‘Non-specific hybridization’ was determined in the presence of a 100-fold excess (100xs) of unlabeled probe. (A) 24 h sham, total hybridization; (B) 1 week LVMI; (C) 1 week sham; (D) 1 week LVMI; (E) 4 weeks LVMI, ‘non-specific hybridization’; (F) 4 weeks LVMI. LA, left atria; RA, right atria; Ao, aorta; LV, left ventricle; IS, interventricular septum; RV, right ventricle; pm, papillary muscle. Approximate ligature position is indicated by a long arrow. Scale bar=10 mm.

 
3.2.2 LVMI levels
Twenty-four hours post-LVMI, left, but not right, atrial ppANP mRNA levels, measured as dpm/cm2, were significantly reduced (55% reduction; P<0.05; Table 2, Fig. 2B). In the left ventricular free-wall, ppANP mRNA expression was restricted to the endocardial surface surrounding the infarcted area, with no mRNA detectable in the remaining free-wall. In contrast, infundibular, interventricular septal and right ventricular ppANP mRNA levels were markedly induced at 24 h (2.7-, 2.1-fold; P<0.05), with the highest levels expressed in the septal endocardium, at the apex and in regions of the right ventricle (Fig. 2B). At 1 week post-LVMI, atrial ppANP mRNA levels were indistinguishable from sham levels, while left ventricular levels were further increased (3.8-fold; P<0.05; Table 2), particularly in the epicardium bordering the infarct area and in the infundibulum and papillary muscle (Fig. 2D). High ppANP mRNA levels were evident at the apex of the heart and in the septum (2.5-fold increase; P<0.05) where there was a gradient of expression that was maximal at the left ventricular endocardial surface. Expression of ppANP mRNA was still evident in discrete regions of the right ventricle at 1 week. By 4 weeks, considerable remodeling of the ventricles had occurred with the infarcted area of the left ventricular free-wall now thinned, dilated and fibrotic, with few remaining myocytes. At this time, atrial ppANP mRNA levels were at sham levels, while the infundibulum, papillary muscle and endocardium bordering the infarcted area accounted for a 3.3-fold (P<0.05) increase in left ventricular ppANP mRNA levels. PpANP mRNA levels in the septal wall and right ventricular chamber also remained elevated at 4 weeks (2.4-, 1.7-fold; P<0.05; Fig. 2F), with less evidence of a gradient across the septum.

3.3 Cardiac {alpha}-actin mRNA
3.3.1 Sham levels
Cardiac ACT mRNA was homogeneously expressed throughout the chambers of the adult heart, with up to 2-fold higher levels in atria, compared to ventricles (Table 3, Fig. 3A).


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  		Table 3 Effect of sham- and LVMI-surgery on cardiac and skeletal {alpha}-actin mRNA levels

 

Figure 3
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  		Fig. 3 Distribution of cACT and sACT mRNA in sham- and LVMI-operated hearts at 24 h, 1 week and 4 weeks post-LVMI. ‘Non-specific hybridization’ was determined in the presence of a 100-fold excess (100xs) of unlabeled cACT and sACT probe. (A) 4 weeks sham (cACT), total hybridization; (B) 1 week sham (sACT); (C) 1 week LVMI (cACT), ‘non-specific hybridization’; (D) 1 week LVMI (sACT), ‘non-specific hybridization’; (E) 24 h LVMI (cACT), total hybridization; (F) 1 week LVMI (cACT); (G) 4 weeks LVMI (cACT); (H) 24 h LVMI (sACT); (I) 1 week LVMI (sACT); (J) 4 weeks LVMI (sACT). Approximate ligature position is indicated by a long arrow. Scale bar=10 mm.

 
3.3.2 LVMI levels
At 24 h, left and right atrial cACT mRNA levels were not significantly different from sham, while left ventricular levels were significantly reduced (57% decrease; P<0.05, Table 3), with cACT mRNA detected in the infundibulum, papillary muscle and apex of the heart, but not in the infarcted area (Fig. 3E). At this time, right ventricular cACT mRNA levels were unchanged, although elevated levels were present in areas of the right ventricular free-wall (Fig. 3E). By 1 week post-LVMI, cACT mRNA levels had increased 1.9-fold (P<0.05) in both atria compared to sham hearts (Table 3). Left ventricular cACT mRNA levels, however, were not altered, although increased expression in regions of the free-wall and apex were now apparent (Fig. 3F). Septal and right ventricular cACT mRNA levels remained at sham levels at this time. Four weeks post-LVMI, atrial cACT mRNA levels remained elevated, while ventricular levels were not statistically different from sham, except for reduced expression in the thin, dilated free-wall of the left ventricle (Fig. 3G).

3.4 Skeletal {alpha}-actin mRNA
3.4.1 Sham levels
Skeletal ACT mRNA could not be detected in the heart at any time point following sham-operation (Table 3, Fig. 3B), reflecting the quiescence of this fetal {alpha}-actin transcript.

3.4.2 LVMI levels
At 24 h, sACT mRNA was detected in both atria and ventricles, in particular in the right ventricular free-wall and epicardium and at the apex of the heart, where it was expressed in a punctate pattern (Table 3, Fig. 3H). At 1 and 4 weeks, sACT mRNA levels remained elevated (Table 3), particularly at 1 week in the left atria, in the infundibular epicardium and septal endocardium of the left ventricular chamber and in the right ventricular free-wall and apex (Fig. 3I). At 4 weeks, sACT mRNA was evident in both atria and in a punctate distribution throughout the right ventricular free-wall and apical regions (Fig. 3J). Interestingly, unlike other transcripts, sACT mRNA expression was evident in the infarcted area of the left ventricular free-wall at 1 and 4 weeks.

3.5 Myosin light chain-2 mRNA
3.5.1 Sham levels
Myosin light chain-2v mRNA was expressed at high levels throughout ventricular chambers of the heart, with a graded increase peaking at the apex. Atrial levels were low, approximately twice the limit of detection (Table 4, Fig. 4A,C).


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  		Table 4 Effect of sham- and LVMI-surgery on cardiac myosin light chain-2mRNA levels

 

Figure 4
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  		Fig. 4 Distribution of MLC-2v mRNA in sham- and LVMI-operated hearts at 24 h, 1 week and 4 weeks post-LVMI (A–F). ‘Non-specific hybridization’ was determined in the presence of a 100-fold excess (100xs) of unlabeled MLC-2v probe. (A) 24 h sham, total hybridization; (B) 1 week LVMI; (C) 1 week sham; (D) 1 week LVMI; (E) 4 weeks LVMI, ‘non-specific hybridization’; (F) 4 weeks LVMI. Approximate ligature position is indicated by a long arrow. Scale bar=10 mm.

 
3.5.2 LVMI levels
Myosin LC-2v mRNA levels in atria changed little during the 4-week study period (Table 4). At 24 h, MLC-2v mRNA expression was not detectable in the ischemic region of the left ventricular free-wall, and was largely restricted to the infundibulum, where it was present in highest density at the endocardial surface (Fig. 4B). As a consequence, left ventricular MLC-2v mRNA was reduced by 56% (P<0.05), as were septal levels (28%; P<0.05), due to the extension of the ischemic region into the septal myocardium. In contrast, right ventricular MLC-2v mRNA levels were unaltered at this time. Except for the left ventricular free-wall infarct area at 1 and 4 weeks, MLC-2v mRNA levels were homogeneous and relatively high throughout ventricular chambers, although at levels not significantly different from those of sham (Table 4). There was however, as with other mRNAs, increased MLC-2v mRNA levels evident at 1 week in the infundibulum, epicardium, septal endocardium and papillary muscle of the left ventricular chamber and in the apex (Fig. 4D). At 4 weeks, MLC-2v mRNA levels were maximal in the left ventricular free-wall endocardium and at the apex of the heart (Fig. 4F).

3.6 β-Myosin heavy chain mRNA
3.6.1 Sham levels
β-Myosin HC mRNA in sham-operated hearts was detectable at a diffuse, homogeneous level in ventricular chambers, but not in atria (Table 5, Fig. 5A,C).


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  		Table 5 Effect of sham- and LVMI-surgery on cardiac β-myosin heavy chain mRNA levels

 

Figure 5
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  		Fig. 5 Distribution of β-MHC mRNA in sham- and LVMI-operated hearts at 24 h, 1 week and 4 weeks post-LVMI. ‘Non-specific hybridization’ was determined in the presence of a 100-fold excess (100xs) of unlabeled probe. (A) 24 h sham, total hybridization; (B) 1 week LVMI; (C) 1 week sham; (D) 1 week LVMI; (E) 4 weeks LVMI, ‘non-specific hybridization’; (F) 4 weeks LVMI. Approximate ligature position is indicated by a long arrow. Scale bar=10 mm.

 
3.6.2 LVMI levels
Atrial levels of β-MHC mRNA remained undetectable until 4 weeks, when β-MHC was present at levels near the detection limit adopted for this study (see Section 2.8; Table 5). At 24 h, left and right ventricular β-MHC mRNA levels were unaltered compared to sham levels, although 2 out of 5 hearts displayed homogeneously increased mRNA throughout the right ventricular free-wall and apex (Fig. 5B). The endocardial surface of the interventricular septum also displayed increased β-MHC mRNA levels at this time. At 1 and 4 weeks, β-MHC mRNA levels in the left ventricle were significantly induced above those of sham (3.0–4.0-fold; P<0.05; Table 5), particularly in the epicardium bordering the infarct area, infundibulum and papillary muscle at 1 week, and in the endocardium and infundibulum at 4 weeks (Fig. 5D, F). Septal β-MHC mRNA levels were similarly increased at 1- (1.8-fold; P<0.05) and 4 weeks, particularly in the apex and at 1 week at the septal surface of the left ventricular endocardium. Right ventricular β-MHC mRNA levels at 1 week were not significantly different from sham, whilst levels were induced at 4 weeks (3.7-fold; P<0.05), with 3 out of 5 infarcted hearts displaying increased expression in the outflow tract (Fig. 5F).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Results of the present study extend our knowledge of the altered expression of myocardial protein mRNA following experimental infarction and during the development of cardiac hypertrophy and chronic heart failure. Using in situ hybridization histochemistry we have detailed the spatiotemporal distribution of constitutive and inducible mRNA transcripts for the major components of the contractile apparatus (actin, myosin) and for natriuretic peptide in atrial and ventricular regions of the post-LVMI rat heart. In particular, we demonstrated a ventricular re-expression of mRNA encoding the natriuretic peptide precursor ppANP, an induction of mRNA encoding the fetal contractile proteins sACT throughout the heart and β-MHC in the ventricles, and the up-regulation of cACT mRNA in the atria following LVMI.

4.1 Infarct size, myocyte morphometry
Infarct size (44.5±2.7% of combined left ventricular epi- and endocardial surface area) was examined in a representative group of rats 1 week post-LVMI, and found to be typical of ‘large’ infarcts, as classified by Pfeffer and coworkers [18]. Fiber width of left ventricular-papillary, mid-septal and mid-right ventricular myocytes was significantly increased as a result of LVMI from 1 week, reflecting the development of myocyte hypertrophy. These results are in accord with fiber width changes described in enzymatically dissociated myocytes following experimental infarction of up to 1 month duration [30]. In the current study, morphological changes were evident from 1 week in left (papillary, mid-septal) and right ventricular myocytes, and increased at 4 weeks in both ventricles, coinciding with the temporal mRNA changes described herein for β-MHC, but proceeding those described for ppANP and sACT.

4.2 Cardiac mRNA levels
The interpretation of cardiac mRNA levels post-LVMI is complicated by the fact that several processes are progressing simultaneously, namely a loss of mRNA expression in areas of the left ventricular free-wall, an associated inflammatory response, and the progressive development of acute, followed by chronic, cardiac decompensation and hypertrophy. Results obtained in the current study suggest that in non-compromised ventricular areas mRNA expression is induced as early as 24 h. By contrast, a transitory loss of all mRNAs studied was detected in the left ventricular free-wall and septal apex at 24 h (for example, Fig. 4B), presumably because of an acute down-regulation of myocardial metabolism [31]. By 1 week post-LVMI, mRNA expression was restored in peri-infarct areas, possibly due to angiogenesis and collateralized perfusion [32]. Subsequently this area forms the infarct scar, through apoptosis, necrosis [33]and lateral slippage of cardiomyocytes [34], leading to progressive left ventricular dilatation, this process of infarct expansion and chamber dilatation making interpretation of mRNA changes (measured as hybridization signal density per unit area), somewhat difficult. In contrast, the absence of scar formation in septal and right ventricular chambers during the development of progressive cardiac hypertrophy allows a clearer discrimination of mRNA changes.

4.3 Atrial natriuretic peptide mRNA
Changes in ppANP mRNA are of particular interest. Post-LVMI, atrial ppANP mRNA levels (measured as dpm/cm2) were essentially unaltered. However, as a consequence of the progressive development of atrial hypertrophy, ppANP mRNA hybridization signal (dpm) increased over the study period. A significant decrease in left atrial ppANP mRNA below sham levels was observed at 24 h post-LVMI, although this normalized by 1 week. While the exact reasons for this result are unclear, it is possible that acute ventricular dysfunction and the resultant acute left atrial distension immediately upon ligation of the LAD may cause the transitory loss of atrial myocyte transcription in a manner similar to that observed at 24 h in the ischemic areas of the left ventricular and septal myocardium [31]. This may also explain similar reductions evident in left atrial MLC-2 mRNA levels, and to a lesser extent cACT mRNA levels at this time.

In the ventricles, except for the ischemic areas of the free-wall and apex of the left ventricle at 24 h, ppANP mRNA was rapidly induced, a striking finding considering the normal quiescence of the ppANP gene in the adult ventricle. In the left ventricular chamber, ppANP mRNA levels reached their highest values by 1 week post-LVMI, presumably reflecting the activation of new protein synthesis mechanisms within the myocardium [35], and remained elevated at 4 weeks. Septal and right ventricular ppANP mRNA levels were also rapidly induced and remained elevated throughout the study period. As in the atria, significant ventricular hypertrophy also occurred over the 4-week study period (1.8-fold heart/body weight increase), largely due to septal and right ventricular hypertrophy. This was associated with a progressive 2.9-fold increase in lung/body weight ratio indicative of the development of chronic heart failure. Our data is therefore consistent with previous reports of elevated ventricular ppANP mRNA during the chronic phase of experimental infarction [35]. By contrast to the rapid induction reported here, an earlier report using this experimental model described a delayed appearance of immunofluorescent ANP peptide in the right ventricle 4–6 days, post-LVMI [36]. This suggests that either there is a marked post-transcriptional delay in the conversion of ppANP mRNA to ANP peptide, or alternatively, a difference in the sensitivity of the two techniques.

The spatial distribution of ppANP mRNA induction in the present study is also consistent with reports describing a gradient of ANP immunoreactivity extending from the endo- to epicardial surface of acute and chronic volume-overload ventricles, which in turn correlates with the distribution of transmural wall stress [37]. Thus, ppANP mRNA hybridization was consistently present at peak intensity at the apex of the heart, an area known to be subject to maximal increases in mid-wall circumferential stress, post-LVMI [38]. However, an intense hybridization signal was also present, post-LVMI, in the left ventricular epicardium bordering the scar and in the infundibulum, areas that are less subject to altered mid-wall stresses. This suggests that wall stress per se may not be entirely responsible for ppANP mRNA expression post-LVMI and this, coupled with the observation that primary cultures of atrial and ventricular myocytes exposed to neuroendocrine stimuli have previously shown rapid induction of ppANP mRNA [35], suggests that local auto- and/or paracrine and circulating endocrine stimuli may play a role in inducing this quiescent gene [39]. Indeed, ANP peptide itself has demonstrable anti-trophic effects on vascular endothelial [40]and smooth muscle cells [35], and the ability to inhibit DNA synthesis in cardiac fibroblasts [41], suggesting the potential for local anti-hypertrophic actions, post-LVMI.

4.4 Cardiac and skeletal {alpha}-actin mRNA
A striking feature of iso-actin mRNA expression post-LVMI was the up-regulation of adult cACT mRNA in both atria (2-fold) as well as in localized ventricular regions, and the induction of fetal sACT mRNA throughout the heart (Table 3). Regulation of atrial cACT mRNA in experimental cardiac overload has not been previously described, although in vivo studies in rat models of abdominal [2]and early thoracic aortic stenosis [4]and LVMI [8], and in vitro studies following {alpha}1-adrenergic receptor stimulation of cultured neonatal rat ventricular myocytes [1], suggest that the cACT gene is not a target for mechanisms that trigger ventricular hypertrophy. These studies however, employed Northern blotting, primer extension or endonuclease VII assays to measure cACT mRNA levels in ventricular muscle, whereas the use of in situ hybridization in the current study allowed identification of localized up-regulation of mRNA levels in the infundibulum, in areas bordering the infarct scar and at the apex of the heart post-LVMI. While this localized cACT mRNA increase was insufficient to increase left ventricular levels, due to the loss of expression in the infarct scar, or significantly elevate septal or right ventricular cACT mRNA, levels were increased in similar areas to those containing increased ppANP and sACT mRNA. This suggests a lesser, but common response to damage and/or functional, metabolic or hormonal change.

Skeletal ACT mRNA, which was undetectable in sham hearts throughout the study period, in line with previous ontogenic studies [42], was clearly evident post-LVMI early in the right atrium, ventricle and septum and later in the left atrium and ventricle. The spatiotemporal change in induced ventricular sACT mRNA was similar to that for ppANP mRNA, with peak hybridization signal consistently detected at the apex of the heart and in the left ventricular epicardium at 1 week, consistent with both mechanical [37]and local and/or circulating neuroendocrine stimuli initiating these effects [1, 43, 44]. To our knowledge, this study is the first to describe early right ventricular and septal induction and differential atrial induction of sACT mRNA, post-LVMI, although the latter phenomenon has been reported for the left atria in the early stages of experimental thoracic aortic stenosis [3]. The functional significance of sACT mRNA expression is uncertain, however it has been suggested that over-expression can be correlated to increased myocardial contractility in BALB/c mice [10]and may confer improved contractile efficiency in the setting of cardiac hypertrophy and failure.

The relevance of these changes to humans, where sACT represents 60% of the adult ventricular isoactin mRNA [45], in contrast to that of the rat (<20%), remains uncertain. Iso-actin switching in human cardiac hypertrophy has thus far only been studied in cardiac transplantation patients where no change in ventricular {alpha}-iso-actin composition was noted [46].

4.5 Myosin light chain-2 mRNA
Following LVMI, MLC-2v mRNA levels were transiently decreased in left atria, ventricle and septum and thereafter normalized to sham levels. MLC-2v mRNA levels were unaltered in all other chambers. The apparent lack of ventricular MLC-2v mRNA modulation post-LVMI contrasts with the increases in ppANP and sACT mRNAs, and again may reflect different regulatory mechanisms to those required for the induction of fetal gene phenotypes. Indeed, the MLC-2v promoter is known to be regulated by a complex E-box independent mechanism, unlike that for the {alpha}-iso-actin and β-MHC genes [47]. However, like cACT mRNA, MLC-2v mRNA levels were elevated in similar discrete regions post-LVMI, without increasing overall chamber values, suggesting a lesser response to common stimuli. This localized up-regulation is consistent with results in a murine model harboring a MLC-2-luciferase transgene subject to 4 days of aortic banding, which displayed a 3–5-fold increase in left ventricular MLC-2v mRNA [48], and with results following abdominal aortic stenosis in the rat, where [3H]leucine incorporation into the phosphorylated form of MLC-2v protein significantly increased in the hypertrophic heart [49]. Furthermore, in human cardiac hypertrophy and in renal-hypertensive baboons, MLC-2v protein levels have been shown to increase in hypertrophied atria and correlate with the degree of overload [50, 51]. Again, as with ppANP and sACT mRNA, results from in vitro studies indicate that both mechanical [52]and neuroendocrine [49]stimuli up-regulate MLC-2v mRNA expression, highlighting the potential for common stimuli to initiate gene expression post-LVMI.

4.6 β-Myosin heavy chain mRNA
β-Myosin HC mRNA was undetectable in atria until 4 weeks post-LVMI, in line with reports describing a delayed appearance in the left atria 30 days after thoracic aortic stenosis [3]. In the ventricles, however, β-MHC mRNA was induced within 1 week, initially in the septum and left ventricle and subsequently in the right ventricle.

The spatial distribution of β-MHC mRNA was similar to that described for sACT mRNA, although the punctate hybridization signal of sACT mRNA contrasted with the relatively homogeneous signal of β-MHC mRNA. Moreover, an endo- to epicardial gradient of β-MHC mRNA in the ventricular wall was apparent, consistent with previous reports in the rat following early thoracic- and chronic abdominal aortic stenosis [3, 53]and closely correlated with reported alterations in wall stresses post-LVMI [37, 38]. However, we were unable to consistently observe the high β-MHC mRNA levels surrounding intramyocardial vessels described by Schiaffino and coworkers [3], presumably because, unlike their stenosis model, passive mechanical stretch due to elevated coronary perfusion pressure is not a major factor following LVMI. In addition to wall stress, neuroendocrine stimuli are also known to be candidates for inducing expression of the β-MHC gene in vitro [54–56].

Functionally, expression of β-MHC mRNA and recombination of its encoded protein into the sarcomere may depress contractility, as observed both in vitro and in vivo, due to a slower rate of ATP cycling by β-MHC [9]resulting in a slower, more economic contraction. Human ventricles are normally composed of 90% β-MHC isomyosin and switching has been reported in patients with cardiac hypertrophy due to chronic mitral valve stenosis, where ventricular {alpha}-MHC protein (normally expressed at 5–15% of total heavy myosin levels) disappeared in response to long-standing pressure-overload [57]. Similarly, in human atria which predominantly express {alpha}-MHC protein, exposure to chronic pressure-overload induced an almost complete reversion to β-MHC, which correlated with the degree of atrial pressure-overload [58]. It is of interest that although major differences exist between rats and humans in the relative proportions of sACT and β-MHC mRNA in ventricular muscle, parallel increases in the levels of these transcripts occur in both species during cardiac overload.

The current study did not address the regulation of the translation of proteins encoded for by the induced mRNAs, since previous evidence indicates that molecular regulation of these genes is largely pre-translational [47], with parallel accumulation of corresponding proteins widely reported following pressure- and volume-overload. Increased levels of cardiac protein arising from the translation of induced mRNA post-LVMI may confer adaptability to the contractile demands of the hemodynamically overloaded myocardium through incorporation into recombinant sarcomeres (sACT, β-MHC), and may have the potential for local anti-hypertrophic actions (ppANP) [36, 40, 41]. Interestingly, although variations existed between individual mRNA levels, the spatial distribution was similar for all induced genes post-LVMI, particularly in the ventricle (apex, left ventricular endocardial surface, infundibulum), suggesting that similar cis- and/or trans-activating factors may direct their myocardial expression. Indeed, altered diastolic wall stress due to redistribution of cardiac loading post-LVMI appears a primary candidate for initiating fetal gene induction [37], although increases in local para- and/or autocrine stimuli such as catecholamines, angiotensin-II and endothelin may also account for altered mRNA levels following experimental infarction ([16, 47]; for review see [59]).

Time for primary review 18 days.


   Acknowledgements
 
This research was supported by a grant from SmithKline Beecham Pharmaceuticals (Australia) and an equipment grant from the Austin Hospital Medical Research Foundation. R.L.Y. is the recipient of a National Health and Medical Research Council of Australia Dora Lush Biomedical Postgraduate Scholarship. The authors wish to thank Dr. ST Chou, Mr. Simon Eades, Dr. Paul Martinello and Dr. Duncan McGregor (Department of Anatomical Pathology, Austin and Repatriation Medical Centre) for assistance and advice with tissue staining and morphometry measurement.


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

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