Cardiovascular Research

Cardiovascular Research 2000 48(1):77-88; doi:10.1016/S0008-6363(00)00160-7
© 2000 by European Society of Cardiology

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Copyright © 2000, European Society of Cardiology

Myocyte contractile function is intact in the post-infarct remodeled rat heart despite molecular alterations

Sudhir Gupta, Arun J.C Prahash and Inder S Anand^*

Division of Cardiology, Department of Medicine, VA Medical Center 111C and University of Minnesota, 1 Veterans Drive, Minneapolis, MN 55417, USA

^* Corresponding author. Tel.: +1-612-725-2000; fax: +1-612-727-5668 anand001{at}tc.umn.edu

Received 11 April 2000; accepted 8 June 2000

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Limitations 6 Conclusion References

 
Objective: To investigate the cellular mechanisms underlying global and regional LV dysfunction in the post-infarct (MI) remodeled rat hearts. Methods: LV remodeling and function were quantified by echocardiography, morphometry, in vivo hemodynamics, and isolated perfused heart studies in 6 weeks post-MI and sham-operated rats. LV myocytes from sham and MI hearts were used for morphometric and functional studies. Myocyte contractile function and intracellular calcium kinetics were measured at different stimulation frequencies (0.2–2 Hz), temperatures (30 and 37°C), and external viscous load (1, 15, 200 and 300 centipoise). Myocyte apoptosis was measured by DNA laddering; BCL-2, BAX, Na⁺–Ca²⁺ exchanger, and SERCA-2 proteins by western blot; and brain natriuretic peptide (BNP), SERCA-2 mRNA by RT-PCR. Results: MI hearts were remodeled (Echo LV diameter 7.3±0.38 vs. 5.9±0.16 mm, P<0.03), and showed global (Echo % fractional shortening 30±2.4 vs. 58±3, P<0.001), and regional contractile dysfunction of non-infarcted myocardium (Echo % systolic posterior wall thickening 36±2 vs. 57±1.7, P<0.001). In vivo hemodynamic and isolated heart function studies confirmed depressed LV systolic and diastolic function and increased volumes. Whereas, myocytes isolated from infarcted hearts were remodeled (40% longer and 10% wider), their contractile function and calcium kinetics under basal conditions and at high stimulation frequency, temperature and viscous load were similar to sham myocytes. The mRNA for BNP was increased whereas that for SERCA-2 decreased, but the SERCA-2 protein was normal. Despite myocyte hypertrophy, ventricular septal thickness was reduced in infarcted hearts (2.2±0.1 vs. 2.6±0.07 mm, P<0.01), and showed increased apoptosis. Conclusions: Myocytes from remote non-infarcted myocardium of the remodeled hearts have normal contractile function, despite structural remodeling and altered gene expression. Non-myocyte factors may be more important in genesis of contractile dysfunction in the remodeled heart, for up to 6 weeks after MI.

KEYWORDS Calcium (cellular); Contractile function; Gene expression; Heart failure; Hypertrophy; Infarction; Myocytes; Remodeling; Ventricular function

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Limitations 6 Conclusion References

 
Following a large myocardial infarction (MI), the heart undergoes a series of structural changes collectively known as ventricular remodeling. Salient features of early remodeling include expansion and thinning of the infarcted segment, which increases left ventricular (LV) volume [1,2]. While these changes may initially help to maintain cardiac performance, progressive remodeling due to myocyte hypertrophy and slippage lengthens the non-infarct segment. This leads to further increase in LV volume and decrease in LV function. Later, during the remodeling process, the remote non-infarcted myocardium also develops contractile dysfunction [2]. The cellular and molecular changes contributing to the global and regional contractile dysfunction of the remodeled myocardium are poorly understood. An important issue to be resolved is whether, and at what stage, intrinsic myocyte contractile dysfunction develops in remodeled myocytes. Besides structural remodeling, a number of significant biochemical and molecular alterations have also been observed at the level of the surviving myocyte. These include changes in bioenergetics [3], myosin isoforms [4], cytoskeletal proteins [5], and ion transport systems. If these changes have functional significance then a defect in myocyte contractile function would be expected. However, in an earlier study we were unable to demonstrate any significant abnormalities in the contractile function of unloaded myocytes isolated from the non-infarct segments of remodeled and dysfunctional hearts, for up to 6 weeks after MI [6].

The current study was, therefore, designed to identify whether increasing load, stimulation frequency, and temperature could unmask any latent abnormalities in myocyte contractile function and intracellular calcium ([Ca²⁺]_i) kinetics. We also examined whether expression of [Ca²⁺]_i handling proteins had become abnormal by 6 weeks post-MI. Finally, we evaluated whether inadequate compensatory hypertrophy or increased apoptosis in the remaining non-infarcted myocardium could have contributed to global and regional wall dysfunction.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Limitations 6 Conclusion References

 
2.1 Experimental myocardial infarction
Myocardial infarction was produced in adult male Sprague–Dawley rats (150–200 g) by ligating the left coronary artery [6]. Mortality from the procedure was approximately 40%. In sham-operated rats (sham) a ligature was passed around the artery but not tied. Seventy animals (sham=36 and MI=34) surviving the procedure were studied 5–6 weeks after surgery in separate groups (Fig. 1). The study conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

View larger version (31K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Flow chart showing the experimental protocol, number of animals studied, and the intervention performed in the three groups of rats.

 
2.2 Echocardiography
Echocardiography (2-D) was done in all animals (sham 36, MI 34) at 1 and 5 weeks after coronary ligation or sham procedure, and was performed with a 7.5-MHz phased array transducer (SONOS 2500 Hewlett-Packard) in anesthetized animals (50 mg/kg ketamine, intramuscular) [2]. Short axis views of left ventricular (LV) cavity were obtained at the level of the papillary muscle. Global LV function was assessed by calculating LV% fractional shortening. Anterior and posterior LV wall thicknesses were measured in systole and diastole (AWTs or d and PWTs or d). The percentage posterior wall thickening in systole (% PWT=[PWTs–PWTd]/PWTdx100) and relative wall thickness (RWT=2xPWTd/LVIDd) were calculated. All measurements were made off-line by an observer blinded to the study group.

2.3 In vivo hemodynamics
Rats (group 1; sham 11, MI 8) were anesthetized with pentobarbital (50 mg/kg, i.p.), intubated and ventilated. The carotid artery was cannulated with a saline filled polyethylene catheter, and arterial, left ventricular systolic (LVSP), and end-diastolic (LVEDP) pressures recorded [6]. This group of rats was then used for whole heart morphometry.

2.4 Whole heart morphometry
After hemodynamic studies, rats were sacrificed and hearts removed, weighed and perfusion fixed in 10% phosphate-buffered formaldehyde for 24 h. After fixation, each heart was cut serially into 2-mm thick slices, from apex to the AV groove. Histological sections were obtained from the two mid-slices, and stained with Masson's trichrome and hematoxylin–eosin. Images were acquired into a digitizing software (Metamorph, Universal Imaging, Westchester, PA) and the epicardial and endocardial circumferences and areas, septal, and RV free wall thickness and infarct size were calculated from each slice and averaged. Infarct size was expressed as a percent of total LV epicardial circumference [7].

2.5 Isolated heart function
Isolated heart function was studied in a separate group of 15 MI and 14 sham-operated rats (group 2). After pentobarbital anesthesia (50 mg/kg, i.p.) animals were heparinized (1000 IU/kg, i.p.), hearts excised and perfused retrogradely via the aorta (Langendorff mode) with modified Krebs–Henseleit buffer and isovolumic left ventricular pressure was measured [6].

2.6 Pressure volume relationship
After studying isolated heart function, passive diastolic pressure–volume relationship (P–V) was recorded in cardioplegia arrested hearts [6]. Three sets of P–V curves were recorded from each heart and the LV volume corresponding to a filling pressure of 10 mmHg (LVV₁₀) was calculated from each curve and averaged. These hearts were then used for isolation of ventricular myocytes.

2.7 Myocyte isolation and morphometry
Ventricular myocytes were isolated using enzymatic digestion for functional and morphometric studies from group 2 rats using retrograde aortic perfusion [6]. In the MI rats, the infarct area and a 2 mm rim of the adjoining normal appearing myocardium was removed and discarded, and myocytes were only obtained from the non-infarct segment of LV. In sham hearts, myocytes were isolated from the whole LV. The % viable myocytes (Trypan Blue exclusion) were 75±4% for sham and 70±4% for MI hearts (P = ns). For morphometric analysis, LV myocytes were fixed in 1.5% glutaraldehyde. From each heart, length and width of 100 randomly chosen LV myocytes were measured (Total n = 1400 sham and 1500 MI myocytes) by phase contrast microscopy [6].

2.8 Myocyte contractile and intracellular calcium response
LV myocytes loaded with the calcium sensitive dye FURA-2 acetoxymethyl ester (FURA-2AM) were randomly chosen, and simultaneous recording of contractile and [Ca²⁺]_i transients made using a video edge detector and a high speed camera coupled to a dual excitation fluorescence system [6]. The [Ca²⁺]_i transient during each contraction cycle was expressed as FURA-2 360/380 nm fluorescence ratio. In vivo calibration was done according to Cheung et al. [8]. Myocytes were perfused with HEPES buffer (2 ml/min) and stimulated by a bipolar field stimulator (5-ms pulse width, 20% above threshold voltage). The shortening–frequency relationship, and effects of external load were studied in two separate protocols.

2.8.1 Shortening–frequency response of isolated ventricular myocytes
Myocyte shortening–frequency relationship (0.2, 0.3, 0.5, 1, and 2 Hz) was studied in 70 MI and 76 sham myocytes (eight rats each). Each myocyte was tested randomly at both 30 and 37°C. Myocytes were exposed to each stimulation frequency until a new steady state was reached, and 6–8 consecutive beats were recorded. All digitized waveforms were stored and analyzed subsequently using a custom designed software. At frequencies higher than 2 Hz, myocytes from sham and MI hearts demonstrated a gradual reduction in their resting length and did not recover completely.

2.8.2 Effects of increasing external load on myocyte function
These studies were performed on 268 myocytes from MI (n = 7) and 144 myocytes from sham operated animals (n = 6). After baseline measurements (HEPES buffer with 4 mM [Ca²⁺]_o), myocytes were bathed in methyl cellulose solution of increasing viscosity as described by Kent et al. [9]. After initial experiments to standardize the viscosity gradient, three viscosities 15, 200 and 300 centipoise (cp) were identified for further studies. Effects of varying viscosity were studied in different batches of myocytes as technical constraints prevented studying the same cell in different viscous milieu.

2.9 Molecular biology studies
In group 3 rats (11 sham and 11 MI hearts), we evaluated changes in expression of a number of molecular markers of cardiac hypertrophy and failure (BNP, SERCA-2 Na⁺–Ca²⁺ exchanger and myocyte apoptotic gene products BCL-2, BAX) in LV myocardium of sham and non-infarcted LV of MI hearts. Apoptosis was further characterized by identifying DNA fragmentation by DNA laddering in the remote myocardium.

2.9.1 Expression of SERCA-2 and BNP
Total cellular RNA was isolated using TRIZOL reagent (Life Technologies). The integrity of RNA was confirmed by visualization of the 28S and 18S ribosomal electrophoretic bands. For reverse transcription, each sample containing 10 µg total RNA, 50 mmol/l Tris–HCl, pH8.3, 75 mmol/l KCl, 0.5 mmol/l MgCl₂, 10 mmol/l dithiothreitol, 0.5 nmol/l each dNTP, 20 U RNAse inhibitor, 100 pmol/l random hexamer, and 200 U reverse transcriptase in a final volume of 33 µl was incubated at 37°C for 60 min. For PCR each sample containing 50 pmol upstream and downstream primers (Table 1), 200 nmol/l dNTP, 50 mmol/l KCl, 10 mmol/l Tris–HCl, pH8.3, 10 mmol/l MgCl₂, 2.5 U Taq DNA polymerase in a final volume of 50 µl was amplified for 30 cycles. The amplification profile involved denaturation at 94°C for 45 s, primer annealing at 55°C for 45 s and primer extension at 74°C for 45 s. After the last cycle, samples were incubated at 74°C for 15 min to extend incomplete products. The PCR product was analyzed on 2% agarose gel and semi-quantified using Image Quant software.

View this table: [in this window] [in a new window]   Table 1 Primer sequences used for PCR assisted mRNA amplification

 
2.9.2 Western blot analysis
Expressions of SERCA-2 and Na⁺–Ca²⁺ exchanger gene products were determined by Western blot analysis. Total proteins were isolated from LV myocardium and concentration determined by the Lowry method. First, 50 µg protein was separated on 10% SDS-polyacrylamide gel, electrophoresed, and transferred onto nitrocellulose membranes. The blots were blocked with PBS containing 3% dry milk and 0.05% Tween-20 and probed with either anti-rat SERCA-2 antibody (1/5000 dilution, ARB) or Na⁺–Ca²⁺ exchanger polyclonal anti-canine NCX antibody (1/500 dilution, a kind gift from Dr K.D. Philipson), for 3 h at 37°C. Subsequently, blots were incubated with HRP-conjugated rabbit anti-rat antibodies or sheep anti-mouse IgG (1/10 000) for 1 h at 37°C and developed using enhanced chemiluminescence method (Amersham). SERCA-2 protein was detected as a 110-kDa band and Na⁺–Ca²⁺ exchanger protein as a 120-kDa band. Blots were semi-quantified using Image Quant software.

2.10 Markers of apoptosis
2.10.1 DNA fragmentation and estimation of BCL-2 and BAX proteins
DNA fragmentation was assessed with agarose gel electrophoresis. Frozen myocardial samples were homogenized, fixed in 70% ethanol, and re-suspended in 40 µl of phosphate-citrate buffer, containing 192 parts 0.2 mol/l Na₂HPO₄ and 8 parts 0.1 mol/l citric acid (pH 7.8). After centrifugation at 1000xg for 5 min, the supernatant was concentrated by vacuum. A 3-µl aliquot of 0.25% Nonidet NP-40 (Sigma Chemicals Co.) in distilled water was then added, followed by 3 µl of a solution of RNase, 1 mg/ml in water. After 30 min incubation at 37°C, 3 µl of proteinase K (1 mg/ml) was added and extract incubated for an additional 1 h at 37°C. After incubation, 12 µl of loading buffer containing 0.25% bromophenol blue and 30% glycerol were added and samples were electrophoresed on 2% agarose gel with 5 µg/ml ethidium bromide. The DNA in the gels was visualized under UV light.

For BCL-2 and BAX, 50 µg protein from the myocardium was separated on 15% SDS-polyacrylamide gel and transfered onto nitrocellulose membranes as described above. The primary and secondary antibodies used were: Rabbit anti-rat BCL-2 or BAX antibody (1/5000) and HRP-conjugated anti-rabbit IgG (1/10 000), respectively. BCL-2 protein was as a 25-kDa band, and BAX as a 21-kDa band.

2.11 Statistical analysis
All data are expressed as mean±S.E.M. A Student's t-test was used for pair-wise comparison of dependent variables between two groups. Repeated variables were analyzed using a mixed design repeated measures analysis of variance (ANOVA), followed by a post-hoc test to identify between group differences if indicated. Myocyte parameters in viscosity studies were analyzed using a two-way ANOVA (group, viscosity). A significant interaction term was followed with a Bonferroni post-hoc procedure. A P value of 0.05 or less was considered statistically significant. Statistical analysis was performed using commercial software (SPSS for Windows version 7.5, Evanston, IL).

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Limitations 6 Conclusion References

 
3.1 Echocardiography
Echocardiographic examination was done on all animals. There was a marked increase in LV diastolic (LVIDd) and systolic (LVIDs) dimensions in MI as compared to sham hearts 1 week after coronary ligation (Table 2, P<0.001, MI vs. sham). Thereafter, only a small further increase in these dimensions was seen at 5 weeks. LV fractional shortening was significantly decreased in MI hearts at 1 week with no further change at 5 weeks. The anterior wall thickness (infarcted area) was significantly reduced in MI hearts at 1 week and remained unchanged at 5 weeks. The posterior wall thickness in diastole (PWTd) tended to increase in both groups with time: by 16% in sham and only 3% in MI hearts. Whereas, the PWTd was similar in both groups at 1 week, it was significantly thinner in MI as compared to sham hearts at 5 weeks. Relative wall thickness was significantly decreased in the MI hearts at 1 week with no further change at 5 weeks (P<0.05). The posterior wall thickening during systole (% PW thickening) was similar in both groups at 1 week but decreased significantly at 5 weeks in post-MI hearts.

View this table: [in this window] [in a new window]   Table 2 Serial echocardiography in sham-operated and MI ratsa

 
3.2 Body weight, heart weight, and in vivo hemodynamics
Whereas the body weights were similar in group 1 MI and sham-operated rats, the heart weights, heart weight/body weight ratios, and heart weight/tibial length ratios were significantly greater in MI rats (Table 3, P<0.01). Similar findings were seen in group 2 rats except for a higher body weight in the sham-operated rats. Heart rate and LVEDP were higher (P<0.05), and mean arterial pressure lower (P<0.05) in MI as compared to sham operated rats.

View this table: [in this window] [in a new window]   Table 3 Body weight, heart weight, in vivo hemodynamics and isolated heart function in sham-operated and MI ratsa

 
3.3 Isolated perfused heart function and diastolic P–V relationship
Isovolumic left ventricular developed pressure (LVDP=LV systolic pressure–LV end-diastolic pressure) was measured under similar conditions of left ventricular end-diastolic pressure (7.5 mmHg) and coronary flow (10 ml/g/min). MI rats, had significantly lower LVDP, and peak positive and negative LV dP/dt (Table 3, P<0.01). Left ventricular volume at 10 mmHg LVEDP (LVV₁₀) was significantly higher in the MI as compared to sham (P = 0.03).

3.4 Whole heart morphometry
Heart morphometry was done in group 1 rats. All MI rats (n = 8) had large transmural infarcts and no infarct was seen in sham rats (n = 11). The average infarct size was 28.2±1.8% of LV epicardial circumference (Table 4). Although the epicardial circumference of MI and sham hearts were not different, the endocardial circumference of MI hearts was 36% greater (P<0.0001), and the mean LV diameter 27% larger (P<0.001) than sham-operated hearts. A marked thinning of infarct segment (1.26±0.16 vs. 2.6±0.38 mm, P<0.0001), a 18% decrease in septum thickness (2.2±0.1 vs. 2.6±0.07 mm, P<0.01), and lengthening of the non-infarcted segments contributed to the increase in LV diameter. The RV free wall thickness was significantly increased in MI hearts (P<0.005).

View this table: [in this window] [in a new window]   Table 4 Infarct size, and myocardial morphometry on two middle slices of the heart

 
3.5 Myocyte morphology
LV myocytes from MI rats were 40% longer (168±0.95 vs. 120±1.43 µm, P<0.001) and 10% wider (22±0.15 vs. 20±0.18 µm, P<0.001) than myocytes from sham hearts. The average length of LV myocytes chosen for the contractile function were similarly remodeled (MI myocytes 156±1.7 µm vs. Sham 115±1.2 myocytes; P<0.01).

3.6 Isolated myocyte function studies
3.6.1 Shortening–frequency relationship
3.6.1.1 Contractile parameters
A representative trace of a shortening–frequency response in a sham myocyte is shown in Fig. 2A. The effects of temperature (30 and 37°C) and stimulation frequency (0.2, 0.3, 0.5, 1, and 2 Hz) were studied in 70 MI and 76 sham myocytes at 2 mM [Ca²⁺]_o. The % shortening, and mean velocity of shortening showed a frequency-dependent decrease in myocytes from MI and sham hearts at both 30 and 37°C (Table 5, P<0.05, ANOVA). The decrease in % shortening, and mean velocity of shortening, with increasing stimulation frequency were not different between MI and sham cells at both temperatures.

View larger version (35K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 (A) A representative trace showing the effect of increasing stimulation frequency (0.2–2 Hz, [Ca2+]o, 2 mM) on [Ca2+]i kinetics (top) and cell shortening (below) in a myocyte from a sham-operated rat. A frequency dependent decrease in [Ca2+]i and myocyte shortening is seen. At high frequency diastolic [Ca2+]i increased and length decreased. (B) A representative trace showing decrease in myocyte shortening in response to loading with solutions of increasing viscosity.

 

View this table: [in this window] [in a new window]   Table 5 Myocyte contractile function in sham-operated and MI ratsa

 
3.6.1.2 [Ca²⁺]_i parameters
Increasing stimulation frequency increased resting [Ca²⁺]_i (basal ratio) in both groups of myocytes (P<0.05 ANOVA). The increase in diastolic [Ca²⁺]_i was significantly greater in MI as compared to sham cells at 30°C (Table 6, P<0.05, MI vs. sham). A frequency-dependent decrease in the amplitude of [Ca²⁺]_i ratio was seen in both group of myocytes at 30 and 37°C. Mean velocity of rise in [Ca²⁺]_i decreased with increasing frequency in MI at 30 and 37°C but not in sham cells, and was significantly different between sham and MI cells at both 30 and 37°C.

View this table: [in this window] [in a new window]   Table 6 Myocyte intracellular Ca2+ transient analysis in sham-operated and MI ratsa

 
3.6.2 Effect of temperature
3.6.2.1 Contractile parameters
Temperature had no significant effect on % myocyte shortening in sham or MI cells (P interaction term 0.06 and 0.14, respectively, Table 5). However, the mean velocity of shortening, was significantly higher in both groups at the higher temperature (P interaction term <0.05).

3.6.2.2 [Ca²⁺]_i parameters
Temperature had no significant effect on the basal ratio, and ratio amplitude, in MI and sham cells. However, higher temperature caused a significant increase in the mean velocity of rise in [Ca²⁺]_i in MI but not sham cells (Table 6).

3.6.3 Effect of viscous load on myocyte function
A representative trace of cell shortening in response to increasing viscous load is shown in Fig. 2B. Myocyte shortening, and mean velocity of shortening, showed a viscosity-dependent decrease in both sham and MI cells (Table 7). No differences in % myocyte shortening, was seen between sham and MI cells at 1 cp. However, the mean velocity of was higher in MI cells at 1 cp (P<0.05, unpaired t test). The main findings of these experiments were unexpected, and showed that sham, and not MI myocytes, showed a significantly greater viscosity-dependent decrease in all these contractile parameters (interaction between group and viscosity terms, two-way ANOVA, P<0.05). Another interesting finding was that although the extent of myocyte shortening decreased gradually with increasing viscosity in both groups, the decline in the mean velocity of shortening was most marked at low viscosity (15 cp). At higher viscosities there was little further decline in these parameters, especially in the MI myocytes.

View this table: [in this window] [in a new window]   Table 7 Effect of external viscous load on myocyte contractile functiona

 
3.7 Apoptotic markers
3.7.1 DNA laddering, BCL-2 and BAX proteins
A diffuse smear pattern of DNA was observed in samples obtained from the infarcted myocardium, whereas a clear laddering pattern was seen in the non-infarcted regions of MI hearts (Fig. 3A). No laddering was seen in the LV myocardium of sham rats. There was a 60% decrease in BCL-2 protein in myocardium from the non-infarct segments of MI as compared with sham mycardium (MI 325±33 vs. sham 830±22 arbitrary units, P<0.01). In contrast, the expression of BAX protein increased by 46% in the same MI region (MI 536±28 vs. sham 367±22 arbitrary units, P<0.01) (Fig. 3B).

View larger version (49K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 (A) Electrophoretic pattern of DNA fragments extracted from the infarcted and remote LV myocardium (6 weeks post MI), and corresponding region of sham myocardium. A diffuse smear like pattern is seen in the infarct scar, whereas, distinct DNA bands (arrows) are seen in the tissue from remote myocardium of the same heart. No DNA smear pattern is seen in the sham myocardium. (B) Western blot analysis of BCL-2 and BAX products in myocytes from remote non-infarcted myocardium of 6 weeks post MI rat hearts and corresponding regions of sham hearts. Bar graph show protein density in arbitrary units. There was a decrease in expression of BCL-2 and increase in BAX protein in MI myocardium as compared to sham.

 
3.7.2 SERCA-2 and BNP mRNA
SERCA-2 mRNA levels decreased by 31% in the remote non-infarcted LV of MI hearts (MI 180±20 vs. sham 260±18 arbitrary units, P<0.01), whereas the mRNA for BNP increased by 31% (MI 230±18 vs. sham 176±12 arbitrary units, P<0.005, Fig. 4), as compared with corresponding areas in sham animals.

View larger version (44K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 RT-PCR analysis of SERCA-2 and BNP mRNA expression in the LV myocardium of sham and remote non-infarcted myocardium of 6 weeks post MI rat hearts. -Actin served as positive transcription control. Bar graph show band density in arbitrary units. There was a decrease in expression of SERCA-2 and increase in expression of BNP mRNA in MI myocardium as compared to sham.

 
3.7.3 SERCA-2 and Na⁺–Ca²⁺ exchanger proteins
Whereas SERCA-2 mRNA fell significantly in the remote non-infarcted LV of MI hearts, the level of SERCA-2 protein from these areas remained unchanged (Fig. 5, Sham 112±9 vs. MI 115±11 arbitrary units). However, the Na⁺–Ca²⁺ exchanger protein increased by 41% in the remote non-infarcted LV of MI hearts (Fig. 5, Sham 341±20 vs. MI 482±31 arbitrary units, P<0.001).

View larger version (35K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Western blot analysis of SERCA-2 and Na+–Ca2+ exchanger proteins in myocytes from remote non-infarcted myocardium of 6 weeks post MI hearts and corresponding regions of sham hearts. SERCA-2 protein levels were similar in sham and MI myocardium, whereas there was an increase in the expression of Na+–Ca2+ exchanger in the remote non-infarcted myocardium of MI as compared to sham hearts.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Limitations 6 Conclusion References

 
The main objective of this study was to identify whether intrinsic abnormalities of myocyte contraction and related genotype could explain the global and regional LV dysfunction seen in the remodeled rat heart 6 weeks after MI. Infarcted hearts in our study were characterized by marked LV dilatation, and systolic dysfunction, which are the hallmark of post-MI ventricular remodeling. A less known but significant feature in this model is the presence of regional contractile dysfunction in the remote non-infarcted myocardium [2]. Whereas this was not evident 1 week post-MI, contractile dysfunction had developed in the non-infarcted regions 5 weeks following MI. Contractile function of papillary muscle from the non-infarcted myocardium has been shown to be abnormal [10]. However, the presence of contractile abnormalities in myocytes isolated from those areas remains controversial [6,8,11–15]. In this study, myocytes from the remote myocardium were hypertrophied, and showed significant alterations in gene expression. However, their contractile function, even under conditions of increase in stimulation frequency, temperature and external load were normal.

4.1 Shortening–frequency relationship in remodeled ventricular myocytes
The force–frequency response in isolated muscle preparations is qualitatively different, and often blunted in heart failure [16,17]. In contrast to the positive force–frequency relationship observed in most mammalian myocardium, this relationship is mostly negative in normal rat hearts [18]. Rate-dependent variations in the ion transport components of E–C coupling have been proposed as possible explanations for this variation in the rat [19]. In our study myocyte shortening and [Ca²⁺]_i showed a rate-dependent reduction, which was nearly parallel between remodeled and sham myocytes. Isolated myocytes, unlike trabeculae and papillary muscle preparations, provide a medium to explore intrinsic contractile abnormalities without the confounding influence of non-myocyte factors. However, few studies have reported the shortening–frequency relationship in isolated myocytes from models of post-MI heart failure [13,20,21]. In the only other study in the rat post-MI model [13], the shortening–frequency relationship in MI myocytes was normal at physiological [Ca²⁺]_o but abnormal at 5 mM [Ca²⁺]_o. Abnormalities at such supraphysiologic concentration confound their significance.

We measured SERCA-2 and Na⁺–Ca²⁺ exchanger because changes in these two important modulators of [Ca²⁺]_i could alter myocyte contractility. In our study, while the Na⁺–Ca²⁺ exchanger protein was increased, there was no difference in the level of SERCA-2 protein, although SERCA-2 mRNA fell. Increase in Na⁺–Ca²⁺ exchanger expression has been reported in the rat post-MI [22] and other models of heart failure [20]. It has been proposed that increase in Na⁺–Ca²⁺ exchanger could compensate for impaired SERCA-2 calcium reuptake by aiding calcium extrusion [23]. As to SERCA-2, the levels of mRNA and protein have been reported to be either increased, decreased or unchanged [14,24–26]. An explanation for these different results is not available but may be related to the time point at which measurements were made. Linck et al. [27] found a decrease in SERCA-2 mRNA and normal protein levels as seen in our study. It should also be noted, however, that SR calcium uptake and SERCA-2 activity could be reduced despite the presence of normal protein levels and vice versa [28]. In the present study despite an increase in Na⁺–Ca²⁺ exchanger and unchanged SERCA-2 protein, isolated myocytes from the remodeled myocardium did not demonstrate any contractile abnormalities, even under increasing stimulation frequency. These findings, therefore, underscore the fact that molecular alterations in myocytes are not necessarily associated with functional abnormalities, as is often implied. It is possible, however, that changes in gene expression occur early and that myocyte contractile dysfunction may evolve later in the natural history of remodeling. Further studies may help to resolve this issue.

4.2 External load and isolated myocyte function
External load is commonly applied to identify contractile abnormalities of isolated muscle preparations. In many models of heart failure, the slope of the negative load–velocity relationship has been shown to be shifted down and to the left, suggesting an underlying intrinsic myocardial contractile defect. Altering the viscous milieu, a novel but standardized method of applying an external load while studying isolated myocyte function, has been found to be analogous to the force–frequency relationship [9]. In the current study we found a load-dependent reduction in myocyte shortening that was less prominent in MI as compared to sham myocytes. These data present further evidence against the presence of intrinsic contractile abnormalities in the remodeled myocytes.

4.3 Global contractile dysfunction without intrinsic myocyte dysfunction
Mechanisms underlying global LV contractile dysfunction could be attributed to alterations of myocyte and non-myocyte factors. A key myocyte factor that could explain global contractile dysfunction is the reduction in number of viable myocytes due to the infarction process itself, and due to accelerated apoptosis. Our study supports the finding of an acceleration of pro-apoptotic gene program found in other models of heart failure as evidenced by an increase in BAX with an accompanying reduction in BCL-2, and DNA fragmentation [29–31]. Besides myocyte loss, presence of contractile abnormalities in the remaining viable myocytes could further lead to the development of global LV dysfunction. While abnormalities of isolated myocyte function have been demonstrated in various models of hypertrophy and heart failure [32–35], its role in the post-MI LV dysfunction remains unclear [8,11,12,36,37]. Results the current study supports our previous finding that factors other then myocyte contractile dysfunction could play a more significant role in contributing to global and regional contractile dysfunction than previously considered [6,15].

Important non-myocyte factors include geometric remodeling and alterations of extracellular matrix. While a detailed evaluation of these factors was beyond the scope of this study, we did find evidence of abnormal wall thinning in the non-infarcted myocardium (15% reduction in septal thickness) which could impair global and regional LV contractility by worsening wall stress [38]. It is interesting that wall thinning occurred despite considerable myocyte hypertrophy (40% longer; 10% wider). Although the exact mechanisms are unclear, our data support the presence of myocyte loss due to apoptosis. This could contribute to wall thinning by reducing the number of myocytes across the thickness of the non-infarcted myocardium. However, the time course of apoptosis in this model remains to be determined. In addition, the mechanical effects of LV remodeling lead to a change in shape of the heart from an ellipsoidal configuration to a more spherical appearance. This change in geometry leads to the development of functional mitral regurgitation and places the heart at a considerable mechanical disadvantage, independent of any changes at the cellular level.

Finally, alterations in the extracellular matrix include an increase in interstitial fibrosis [39,40], and a loss of fibrillar scaffolding to which myocytes are anchored [41]. Either of these conditions could cause myocardial dysfunction by over- or under-tethering myocytes, despite normal intrinsic contractile function.

	5 Limitations

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Limitations 6 Conclusion References

 
A significant limitation, common to all isolated myocyte studies is that cells studied may not be representative of the myocytes in the myocardium. The isolation procedure could inadvertently affect the yield of viable but dysfunctional myocytes.

	6 Conclusion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Limitations 6 Conclusion References

 
In the rat infarct model, global ventricular dysfunction is accompanied by contractile dysfunction in the remote noninfarcted myocardium. Myocytes isolated from this area are structurally remodeled and show altered gene expression. Despite this, however, the remodeled myocytes do not demonstrate contractile dysfunction either at basal state or when subjected to increased stimulation frequency, higher temperature and external load for up to 6 weeks post-MI. Accelerated apoptosis is evident in the remote myocardium that may contribute to wall thinning. Non-myocyte factors such as increased wall stress and alterations of the extracellular matrix could be more important in the early stages of development of ventricular dysfunction in post-MI remodeling.

Time for primary review 24 days.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Limitations 6 Conclusion References

 

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