Cardiovascular Research

Cardiovascular Research 1998 37(2):467-477; doi:10.1016/S0008-6363(97)00278-2
© 1998 by European Society of Cardiology

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Copyright © 1998, European Society of Cardiology

Early changes in excitation-contraction coupling: transition from compensated hypertrophy to failure in Dahl salt-sensitive rat myocytes

Kohzo Nagata, Ronglih Liao, Franz R Eberli, Naoya Satoh, Brigitte Chevalier, Carl S Apstein and Thomas M Suter^*

Cardiac Muscle Research Laboratory, Whitaker Cardiovascular Institute, Boston University School of Medicine, 715 Albany Street, W-611, Boston, MA 02118, USA

^* Corresponding author. Tel. (+1-617) 638 4033; Fax (+1-617) 638 4031.

Received 31 July 1997; accepted 3 November 1997

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: The aims were to (1) define the early changes in excitation-contraction coupling during the transition from cardiac hypertrophy to heart failure, and (2) to clarify the causal relationship between mechanical dysfunction and abnormal Ca²⁺ handling in the Dahl salt-sensitive rat model. Methods: Myocardial contractile function was assessed in whole heart perfusion studies. In separate experiments, isolated left ventricular myocytes from Dahl salt-sensitive (DS) and Dahl salt-resistant (DR) rats were paced at a physiological rate of 5Hz and cell shortening (CS) and [Ca²⁺]_i measured simultaneously by video-edge detection and fura-2 fluorescence. Results: DS hearts developed hypertrophy after 4 weeks of a high-salt diet (4WHSD), as indicated by a 26% increase (p<0.01) in the heart to body weight ratio and a 21% increase (p<0.01) in cell width. Heart failure developed after 12 weeks of a high-salt diet (12WHSD), as indicated by an 11% increase (p<0.01) in the lung wet to dry weight ratio. Furthermore, in DS-12WHSD hearts, the diastolic pressure-volume relationship had shifted rightward. DR rats did not develop hypertension and served as age-matched controls. A 31% (p<0.05) increase in the %CS in DS-4WHSD myocytes compared to DR-4WHSD myocytes with a trend of a parallel increase in Ca²⁺ transient amplitude was found. There was no difference in the Ca²⁺ transient parameters between DR and DS at 12WHSD, but an 18% (p<0.01) decrease occurred in peak [Ca²⁺]_i in DS myocytes between 4WHSD and 12WHSD. In DS-12WHSD, the time to peak shortening and the time from peak shortening to 50% and 90% relaxation was significantly prolonged by 27%, 44%, and 38%, respectively, as compared to the age-matched DR myocytes. Conclusion: Our results indicated that: (1) normal Ca²⁺ homeostasis is preserved at the stage of compensated hypertrophy; (2) the early signs of isolated myocyte dysfunction were a prolongation of the shortening and relaxation time course without an abnormal time course of the Ca²⁺ transient. Thus, in the hypertensive Dahl salt rat model, abnormal Ca²⁺ handling appears neither to precede nor initiate the transition to failure.

KEYWORDS Dahl rat; Hypertrophy; Heart failure; Myocyte; Fura-2; Calcium handling; Excitation-contraction coupling

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Chronic pressure overload due to hypertension can lead to myocardial hypertrophy and heart failure, and is a major cause of death in many developed countries. However, the cellular mechanisms responsible for the transition from compensated left ventricular (LV) hypertrophy to congestive heart failure have not yet been completely elucidated. Cardiac hypertrophy is thought to be an adaptive mechanism of the heart to overcome hemodynamic overload and normalize wall stress [1]and is accompanied by biochemical and molecular changes in cardiac myocytes. The sarcoplasmic reticulum (SR) is an important determinant of myocyte contraction and relaxation because of its ability to regulate cytosolic Ca²⁺ [2]. To elucidate the alteration in excitation-contraction coupling, several studies on the intracellular calcium transient and contractility from failing human hearts [3, 4]and from animal models of heart failure [5, 6]have been performed. They consistently report a significant prolongation in the time course of the Ca²⁺ transient and a decrease in contractility. However, it is unclear whether contractile dysfunction and abnormal Ca²⁺ handling occur in conjunction, or if one event precedes the other as the heart progresses to failure. The correlation of physiological, biochemical, and molecular data in myocardial hypertrophy and failure is hampered by the lack of a good animal model that can consistently demonstrate compensated LV hypertrophy and overt, clinically relevant congestive heart failure [7–9].

The inbred strain of Dahl salt-sensitive rats was derived from noninbred stock of Brookhaven Dahl salt-sensitive rats [10, 11]. When fed a high-salt diet, the Dahl salt-sensitive rat develops hypertension with low renin and aldosterone levels, compensated LV hypertrophy, followed by congestive heart failure [12]. Dahl salt-resistant rats, the contrasting strain, derived from the same colony with a 95% genetic homology with Dahl salt-sensitive rats, developed neither hypertension, nor hypertrophy, and can serve as age-matched controls. Based on their consistent demonstration of the transition from hypertrophy to congestive heart failure due to hypertension, the Dahl salt-sensitive rat has been proposed as an ideal animal model to study hypertension induced hypertrophy and heart failure [12].

Our study is the first to use myocytes, isolated from Dahl rats, paced at a physiological rate of 5Hz. We, (1) measured the serial relationship between cell shortening and the Ca²⁺ transient in the LV myocytes; and (2) compared the results between the hypertrophy and heart failure stages and the age-matched controls. The data indicate that alterations in excitation-contraction coupling occur in LV myocytes isolated from DS rats during the transition from compensated hypertrophy to failure. However, our results suggest that, in this model, abnormalities in mechanical function are not secondary to abnormal intracellular Ca²⁺ handling.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Animal model
Male inbred DS and DR rats, obtained from Harlan Sprague Dawley, were housed, one rat per cage, in the Animal Facility of Boston University Medical Center as per the Guide for the Care and Use of Laboratory Animals. We followed the protocol described by Inoko et al. [12]with the following modifications: The rats arrived at 8–9 weeks of age, and were fed a low-salt diet (0.12% NaCl) for one week for acclimatization. At the end of the acclimatization period, the rats were fed a high-salt diet (7.8% NaCl) and water ad libitum for 4 or 12 weeks. Body weight was measured weekly. Systemic blood pressures were measured by the tail-cuff method [13]in unanesthetized rats at 30°C at the time point of study.

Two cohorts of animals were used in this study. One cohort of animals underwent a whole heart perfusion protocol to assess LV remodeling and contractile performance by establishing diastolic and systolic pressure-volume relationships. The other cohort was used for an isolated myocyte study to assess mechanical function and Ca²⁺ homeostasis. The isolated myocytes were studied at two stages: (1) 4WHSD: after hypertension and concentric LV hypertrophy developed, but before heart failure occurred (after 4 weeks of a high-salt diet); and (2) 12WHSD: between the LV hypertrophy stage and end-stage heart failure (after 12 weeks of a high-salt diet).

2.2 Whole heart perfusion study
To characterize hemodynamic changes and LV remodeling, whole heart perfusion studies were performed in an isolated isovolumically beating (balloon-in-LV) heart preparation perfused with red blood cells as previously described [14, 15]. Briefly, rats were injected intraperitoneally with sodium pentobarbital (1 ml, 15 mg/ml). The thorax was rapidly opened and the heart was excised. A short perfusion cannula was inserted into the aortic root and the hearts retrogradely perfused within 10 s. The left ventricle was vented of the Thebesian drainage with an apical cannula. All coronary venous effluent was collected by a cannula in the ligated pulmonary artery and measured by timed collection. Two pacing electrodes were placed on the epicardial surface and the hearts were paced at 5Hz (Grass Instruments, Model 59, Quincy, MA). A fluid-filled latex balloon connected to a Statham P23Db pressure transducer (Statham Instrument, Hato Rey, Puerto Rico) by a short, stiff polyethylene tubing was then placed into the left ventricle through an incision in the left atrium. Coronary perfusion pressure was measured via a Statham P23Db pressure transducer connected to the aortic cannula. Coronary pressure, left ventricular pressure and its derivative were recorded on a multichannel recorder (Gould Cleveland, Ohio).

Coronary perfusion pressure was 80 mmHg for the DR hearts and 100 mmHg for the DS hearts. A higher perfusion pressure was used in the DS hearts to compensate for the known increased coronary resistance, and to provide comparable levels of myocardial perfusion to both groups. At 4WHSD, the levels of myocardial perfusion were 2.70±0.15 vs. 2.45±0.15 ml/min/gm LV for the DR vs. DS hearts respectively (p=NS). At 12WHSD, they were 2.79±0.11 vs. 2.27±0.06 ml/min/gm (p<0.01) indicating slightly higher perfusion levels in the DR group. Despite this slight disparity of perfusion, the DS group maintained a higher level of systolic function than the DR group at 12WHSD (see Result section).

The perfusion system consisted of a ‘venous’ reservoir, a variable flow pump, an oxygenator, a water jacketed ‘arterial’ reservoir, and a filter of 20 µm pore size. The red blood cell perfusate consisted of bovine red blood cells at a final hematocrite of 40% in Krebs–Henseleit buffer (in mM: NaCl 118, KCl 4.7, CaCl₂ 2.0, KH₂PO₄ 1.2, MgSO₄ 1.2, NaHCO₃ 26.6, glucose 5.5, lactate 1.0, palmitic acid 0.4 [as a source for free fatty acid metabolism], and 40 g/l bovine serum albumin). Gentamicin (0.02 g/l) was present to retard bacterial growth. The perfusate was equilibrated with 20% O₂-3% CO₂-77% N₂ to achieve a pO₂ of 120–140 mmHg and maintained at 37°C and pH 7.4.

2.2.1 Perfusion protocol
After 30 min of baseline perfusion, hemodynamic measurements were made over a range of balloon filling volumes to determine the systolic and diastolic pressure-volume relationships. Using an air-tight Hamilton syringe containing saline, left ventricular balloon volume was increased by 0.02 ml increments. To compare the diastolic pressure-volume relationship between groups, left ventricular volumes/kg body weight were determined from each pressure-volume curve at 5 mmHg intervals up to an end-diastolic pressure of 30 mmHg as previously described [14]. Similarly, a systolic pressure-volume curve was established. Since in both hypertensive animals and older animals the systolic and diastolic pressure-volume curves were shifted to the right, the following calculations were made to compare contractile force, i.e. left ventricular developed pressure, between the different groups. Diastolic wall stress was estimated by multiplying left ventricular volumes and corresponding end-diastolic pressures [LV volume (ml)xLVEDP (mmHg)] [16]. Left ventricular developed pressure was then plotted against the corresponding diastolic pressure-volume product. This allowed comparison of left ventricular developed pressure at approximately identical preloads.

2.3 Isolated myocyte experiments
2.3.1 Cell isolation
DS and DR rats were anesthetized (pentobarbital sodium, 50mg/kg i.p.) and heparinized (200 IU i.v.). Hearts were quickly excised and immediately immersed in an ice-cold, modified cardioplegic KB solution (in mM: 85 KOH, 30 KCl, 30 KH₂PO₄, 3 MgSO₄, 0.5 EGTA, 10 N-2-hydroxyethylpiperadine-Nñ-2-ethanesulfonic acid [HEPES], 50 L-glutamic acid, 20 taurine, and 10 glucose, pH 7.4). The hearts were cannulated via the aorta and perfused in the Langendorff mode with a constant perfusion pressure of 90 cm H₂O and 120 cm H₂O for the hearts excised from DR and DS rats respectively. The hearts were first perfused for 5 min with non-recirculating 1.8 mM Ca²⁺ Tyrode solution (in mM: 137 NaCl, 5.4 KCl, 1.8 CaCl₂, 0.5 MgCl₂, 10 HEPES, 10 glucose, pH 7.4) and Ca²⁺-free Tyrode solution for another 5 min. They were then perfused with a circulating digestion solution containing 0.08% collagenase A (Boehringer Mannheim, Indianapolis, MN) and 0.02% protease type XIV (Sigma Chemicals, St. Louis, MO). After the hearts were palpably flaccid, the digestion solution was then washed out with KB solution for 5 min. The left ventricle (including the septum) was cut into small pieces and gently agitated, allowing the cells to be dispersed in the KB solution. After 60 min, the cells were resuspended in 1.2 mM Ca²⁺ Tyrode for subsequent experimental use. All perfusion solutions were equilibrated with 100% oxygen and the temperature of the solutions was maintained at 37°C throughout the isolation process. The yield of rod-shaped, Ca²⁺-tolerant myocytes was approximately 50–75% of isolated cells in all groups, except DS rats after 12WHSD, which produced only 20–30%.

2.3.2 Fura-2/AM loading and distribution
Freshly dissociated LV myocytes were incubated in the solution containing 1 µM of membrane-permeant fura-2/AM (Molecular Probes, Eugene, OR) for 10 min at room temperature. After washing out the fura-2/AM in the loading solution, an additional 40 min was allowed for the deesterification of the fura-2 ester in the cells. 500 µM probenecid was included throughout this procedure to prevent the leakage of fura-2 from the cells. The extent of loading of fura-2 into non-cytosolic compartments was tested by the MnCl₂ quenching method [17]. The results indicated that more than 90% of the recorded fura-2 fluorescence emanated from the cytosolic space. Fura-2 is a Ca²⁺ chelator and is known to buffer cytosolic Ca²⁺. This Ca²⁺-buffering effect may affect cytosolic Ca²⁺ concentrations and myocyte function. Therefore, we selected a fura-2 loading concentration that did not affect overall cell shortening by comparing the % cell shortening with and without fura-2 loading. In addition, the fura-2 loaded myocytes were required to have consistent fura-2 dependent fluorescence (the ratio of 360 nm excited fura-2 fluorescence to 360 nm excited cell auto fluorescence was always around 4–5).

2.3.3 Cell inclusion criteria
The cells were electrically stimulated at a physiological rate of 5 Hz and superfused with 1.2 mM Ca²⁺ Tyrode solution at 37°C. Myocytes included in this study were selected using the following criteria: (1) rod-shaped with a clear striation pattern (no granulation and no cauliflower-shaped cell ends); (2) quiescent when unstimulated; and (3) stable mechanical behavior at 5 Hz and 37°C for 10–15 min. No cells were used past 6 h post-isolation.

2.3.4 Measurement of cell shortening
Myocytes were viewed using a Nikon Diaphot microscope (Nikon, Melville, NY). The cell image, collected by the Nikon 40x oil immersion objective lens (Nikon, Melville, NY) was diverted to the microscope's side port and transmitted to a video camera (MyoCam^TM IonOptix, Milton, MA). Cell length and contractile amplitude of myocytes were recorded in real time on an IBM-compatible PC with a video edge detector and specialized data acquisition software (SoftEdge^TM Acquisition System and IonWizard^TM, IonOptix, Milton, MA). The camera was specially adapted to acquire images at 240 Hz and a cell length measurement time resolution of 4.2 ms. The signal to noise ratios were significantly improved by averaging 10 sequential runs. Calibration of the system was accomplished with a micrometer and data acquisition software.

2.3.5 Measurement of [Ca²⁺]_i
Cytosolic calcium was measured by the fluorescent calcium indicator fura-2 (Molecular Probes, Eugene, OR), using a dual fluorescence, calcium ion sensing system (IonOptix, Milton, MA). The fura-2-loaded myocytes were excited at 360±6.5 nm and 380±6.5 nm. Emission fluorescence was measured at 510±15 nm. The fluorescence ratio, F₃₆₀/F₃₈₀, was independent of the intracellular fura-2 concentration, cell geometry and excitation light intensity, and reflected the intracellular calcium concentration [18].

Myocytes were alternately excited with an ultraviolet (UV) xenon lamp at wavelengths of 360 and 380 nm through filters installed on a rotary wheel controlled by the data acquisition system. The excitation light was transmitted to the cell through a dichroic mirror (cutoff 430 nm, Omega Optical, VT) and an epifluorescence 40x oil immersion objective lens. The emission fluorescence was collected by the objective transmitted through the previously described dichroic mirror, reflected to the side port of the microscope, where another dichroic mirror (cutoff 550 nm) reflected the light (<550 nm) through a barrier filter, 510±15 nm, to a photomultiplier tube (Hamamatsu, NJ). An adjustable aperture stop was used to restrict the collected light to the observed cell. These photons, counted by the photomultiplier tube, were collected simultaneously with the cell shortening signal by the data acquisition system provided by IonOptix. In order to take into account any inference effects of background fluorescence, the background fluorescence value was subtracted from the entire recording. The background fluorescence was measured at 4 points adjacent to the myocyte being measured. Myocytes were always more than two myocyte lengths apart. The average of the fluorescence signal from these 4 adjacent points thus assessed a cell-free area of approximately two myocyte lengths distance between myocytes.

2.3.6 Fluorescence ratio F₃₆₀/F₃₈₀ and time resolution of the system
The fluorescence excited by 380 nm was decreased upon binding of Ca²⁺, whereas the fluorescence excited by 360 nm was independent of [Ca²⁺]_i changes. During a single data collection run, the myocytes were excited by 360 nm for 120 ms at the beginning and the end of the run, which lasted 500 ms. Between these two points, the myocytes were only excited by 380 nm wavelength. The fluorescence signal was acquired by the system at a sampling rate of 500 Hz. At each of these sampling points, a calculated 360 nm excited fluorescence was determined, using an interpolation of the 360 nm excited fluorescence collected at the beginning and the end of the data collection run. The data from 10 sequential runs were averaged to increase signal to noise ratio. This technique had a time resolution of 2 ms.

2.3.7 In situ calibration of Fura-2 fluorescence
A subset of cells from both groups at each stage was calibrated in situ by sequential exposure to calibration buffers according to the modified method of Borzak [19]. Briefly, cells were superfused with nominally Ca²⁺-free Tyrode buffer containing 40 mM 2,3-Butanedionemonoxime (BDM, Sigma) followed by the same buffer with the addition of 1 mM EGTA and 10 µM ionomycin, and finally an identical buffer with 4 mM Ca²⁺ substituted for EGTA. We used BDM to uncouple myocyte excitation and contraction, thereby preventing the fluorescence altered by hypercontracture [19, 20]. Background fluorescence was determined by measuring fluorescence from 4 points of adjacent cell-free area and subtracting this average value from the entire recording. Intracellular Ca²⁺ concentrations ([Ca²⁺]_i) were determined by the equation of Grynkiewicz et al. [18]:

where K_d is the dissociation constant, 224 nM [18], β is the ratio of fluorescence intensity at 380 nm when the fura-2 is in the Ca²⁺-free (F_{380, free}) form to the fluorescence intensity when the fura-2 is in the Ca²⁺-bound (F_{380, bound}) condition; and R is the ratio of fluorescence intensity measured at an excitation wavelength of 360 to 380 nm; R_min is the limiting value of the ratio R when fura-2 is in the Ca²⁺-free form; and R_max is the limiting value of the ratio R when fura-2 is in the Ca²⁺-bound condition.

2.3.8 Data analysis
The data was collected and analyzed using a data acquisition system developed by IonOptix, Milton, MA. The system allowed for the simultaneous acquisition of fluorescence signal and cell shortening. The absolute twitch amplitude was the difference between the systolic cell length and the diastolic cell length. The cell shortening (%CS) was expressed as the ratio of absolute twitch amplitude to diastolic cell length. The duration of twitches was measured as the time from onset of contraction to peak shortening and the time from peak shortening to 50% and 90% relaxation. Similarly, the amplitude and the time course of Ca²⁺ transients were analyzed accordingly. The instrument, was calibrated weekly by using a fura-2-Ca²⁺ calibration kit (Molecular Probes, Eugene, OR) to ensure the stability of the fluorometer.

2.4 Statistical analysis
Statistical differences between the mean values for two groups were evaluated by the unpaired Student's t test. Where multiple means were compared, an ANOVA analysis was performed. Data are expressed as mean±SEM. A p value less than 0.05 was considered statistically significant.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Animal and cardiac characteristics
Fig. 1 shows body weight, systolic blood pressure, heart to body weight ratio, and lung wet to dry weight ratio in DS and DR rats used for the isolated myocytes study. As expected, the high-salt diet resulted in hypertension (systolic blood pressure>200 mmHg) that was maintained throughout the entire experimental period. In DS rats, the heart weight to body weight ratio, an index of cardiac hypertrophy, was increased by 26% (p<0.01) after four weeks of the high-salt diet (4WHSD) and 39% (p<0.01) after 12 weeks of the high-salt diet (12WHSD), as compared to that of the age-matched DR rats. Many of the DS rats became less active and developed respiratory distress after 8–9 weeks of high-salt feeding, whereas DR rats showed normal activity and no respiratory distress. The lung wet to dry weight ratio, an index of pulmonary congestion, was not different between DS and DR rats after 4WHSD. The ratio was increased by 11% (p<0.01) when compared to age-matched DR rats after 12WHSD, indicating pulmonary congestion in DS after 12WHSD.

View larger version (68K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Animal and cardiac characteristics of Dahl salt-sensitive (DS) and salt-resistant (DR) rats. After 4 weeks of the high-salt diet, DS rats developed systemic hypertension and myocardial hypertrophy, but no pulmonary congestion. After 12 weeks of the high-salt diet, DS rats maintained a high blood pressure but developed pulmonary congestion. Solid bar, DS; Slashed bar, DR. (Panel A) BW=body weight, (Panel B) SBP=systolic blood pressure, (Panel C) HW/BW=heart weight to body weight ratio, (Panel D) lung WW/DW=lung wet weight to dry weight ratio. *p<0.05, **p<0.01 vs. age-matched DR, p<0.05, p<0.01 vs. baseline, ¶p<0.01 vs. 4WHS.

 
Animal characteristics of the groups of rats used in the whole heart perfusion studies were comparable to animals used for isolated myocyte studies. Pre-hypertensive animals had similar LV/BW ratios (DR-baseline vs. DS-baseline=2.20±0.05 vs. 2.41±0.10 mg/g; p=NS). After 4WHSD, LV/BW was significantly higher in DS rats (DR-4WHSD vs. DS-4WHSD=2.29±0.06 vs. 3.07±0.09; p<0.05) and had further increased after 12WHSD (DR-12WHSD vs. DS-12WHSD=2.56±0.07 vs. 3.79±0.23; p<0.01), indicating increased LV hypertrophy. After 4WHSD, there was no pulmonary congestion as indicated by a similar lung wet to dry weight ratio (DR-4WHSD vs. DS-4WHSD=4.52±0.13 vs. 4.54±0.03; NS). However, lung wet to dry weight ratio was increased after 12WHSD (DR-12WHSD vs. DS-12WHSD=4.50±0.19 vs. 7.62±1.13; p<0.05).

3.2 Myocyte morphology
Fig. 2 shows the measured cell width and cell length in fresh isolated LV myocytes from DS and the age-matched DR rats at each stage. In DS rats, the cell width was significantly increased both after 4WHSD (21%) and after 12WHSD (25%), when compared to age-matched DR rats. The cell length, however, was only significantly increased (25%) after 12WHSD. In contrast, DR rats exhibited a slight increase (p<0.01) in cell width overtime but no changes in cell length.

View larger version (39K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Morphological characteristics of myocytes from Dahl salt-sensitive (DS) and salt-resistant (DR) rats. After 4 weeks of the high-salt diet, DS rats demonstrated increased LV myocyte width (Panel A) but no increase in LV myocyte length (Panel B). After 12 weeks of the high-salt diet, DS rats demonstrated increased LV myocyte length, as well as further increased width. Solid bar, DS; Slashed bar, DR. **p<0.01 vs. age-matched DR, p<0.01 vs. baseline, p<0.05, ¶p<0.01 vs. 4WHS.

 
3.3 Hemodynamics of whole heart perfusion study
The diastolic pressure-volume curves were indistinguishable between DS and DR hearts at the baseline stage (Fig. 3A). After 4WHSD, the LV diastolic pressure-volume curve of DS hearts was shifted to the left of that of age-matched DR hearts (Fig. 3B), consistent with concentric LV hypertrophy. After 12WHSD, for any given LV diastolic pressure, LV volume of DS hearts was increased almost threefold compared to DS-4WHSD. This resulted in a rightward shift of the diastolic pressure-volume curve of DS-12WHSD hearts compared to that of the age-matched DR hearts (Fig. 3C).

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Diastolic pressure-volume relationship of Dahl salt-resistant () and salt-sensitive () rats. At the prehypertensive stage (Panel A), LV volume between the two groups (n=6/group) was identical. After 4 weeks of a high salt diet (Panel B) salt-sensitive rats (n=8) developed concentric hypertrophy resulting in a significantly smaller LV volume compared to salt-resistant rats. After 12 weeks of a high salt diet (Panel C) the pressure-volume relationship had shifted to the right in salt-resistant rats (n=8), and to a greater extent in the salt-sensitive rats (n=12), indicating beginning of LV dilation at this stage of hypertrophy. *p<0.05 (DR-4WHSD vs. DS-4WHSD); **p<0.01 (DR-12WHSD vs. DS-12WHSD).

 
At the baseline stage, LV contractile function, as assessed by the developed pressure-volume relationship, was similar between DS and DR rats (Fig. 4A). After 4WHSD, the contractile performance of DS-4WHSD rats was improved compared to DR-4WHSD, i.e., for any given preload DS-4WHSD hearts developed a greater pressure (Fig. 4B). After 12WHSD, when pulmonary congestion was present, LV systolic function in the DS-12WHSD rats was still greater than that of DR-12WHSD hearts (Fig. 4C), albeit decreased compared to DS-4WHSD.

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Systolic pressure-volume relationship of Dahl salt-resistant () and salt-sensitive () rats. Diastolic wall stress was estimated by multiplying LV volume and diastolic pressure to compare systolic function of hearts of different sizes. Compared to prehypertensive hearts (Panel A), concentric hypertrophied hearts of Dahl salt-sensitive rats (Panel B) showed an increased contractile function compared to nonhypertensive salt-resistant rats. After 12 weeks of a high salt diet (Panel C), when hypertrophied hearts had started to dilate, contractile function was reduced compared to the concentric hypertrophied hearts, but was still better than in non-hypertrophied control hearts. **p<0.01 (DR vs. DS)

 
3.4 Mechanical performance of myocytes
A representative tracing of cell shortening is shown in Fig. 5 (Trace A). The results of mechanical function of myocytes are shown in Table 1. After 4WHSD, the percent cell shortening (%CS) was higher (p<0.05) in DS myocytes than in the age-matched DR myocytes. There were no significant differences in the time from onset of contraction to peak shortening and the time from peak to 50% and 90% relaxation after 4WHSD between DS and the age-matched DR myocytes. The %CS after 12WHSD in DS myocytes was not different as compared to the age-matched DR myocytes, but was significantly depressed as compared to DS-4WHSD myocytes. After 12WHSD, the time to peak shortening and the time from peak to 50% and 90% relaxation was significantly prolonged by 27%, 44%, and 38%, respectively, as compared to the age-matched DR myocytes, indicating a slowing of contractile kinetics. 12WHSD myocytes in DR rats showed a 20% increase in the time to peak shortening and a 35% increase in the time from peak to 50% relaxation, as compared to 4WHSD myocytes in DR rats, indicating age-dependent changes of contractile function.

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Representative tracings of simultaneously recorded twitch amplitude (Trace A) and intracellular Ca2+ concentrations (Trace B). This tracing shows typical data from DS-4WHSD.

 

View this table: [in this window] [in a new window]   Table 1 Characteristics of myocyte mechanical function

 
3.5 Ca²⁺ transient of myocytes
Fig. 5 (Trace B) shows a representative tracing of the Ca²⁺ transient. Table 2 shows the Ca²⁺ transient data of myocytes from DS and age-matched DR rats. After 4WHSD, there was a trend toward an increased Ca²⁺ transient amplitude of 32% for DS myocytes compared to DR myocytes. The diastolic [Ca²⁺]_i in DS myocytes after 12WHSD was similar to that of the age-matched DR myocytes. In DS myocytes after 12WHSD, the peak [Ca²⁺]_i was decreased by 18%, and the Ca²⁺ transient amplitude was decreased by 28%, as compared to those after 4WHSD (p<0.01), but these parameters were not different from those of the age-matched DR myocytes. The time to peak [Ca²⁺]_i and the time to 50% and 90% decline from peak [Ca²⁺]_i were not changed in DS myocytes after 12WHSD compared to those of the age-matched DR myocytes. Similarly, DR myocytes had no change in the measured parameters during this period.

View this table: [in this window] [in a new window]   Table 2 Characteristics of intracellular Ca2+ transient

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
4.1 Animal model
The Dahl rat model was chosen because of its clear and reproducible transition from hypertension to compensated LV hypertrophy to overt heart failure following a high-salt diet in DS rats [12]. DR rats, which are 95% genetically identical to DS rats, do not develop hypertension, hypertrophy, or heart failure, and served as age-matched controls. In this model, the level of hypertension and the progression from hypertrophy to failure were dictated both by the time of initiating the high-salt diet and by the amount of salt in the diet [12, 21]. When the high-salt diet was started in 5 week old rats, the animals showed a sharp increase in blood pressure and a rapid progression from myocardial hypertrophy to heart failure. In contrast, the onset of myocardial hypertrophy and heart failure occurred later in life and less sudden when the high-salt diet was started at an animal age of 8–9 weeks. Since we were interested in the pathophysiological changes in the early stages of heart failure and wished to achieve a slower progression from hypertrophy to heart failure, we started the high-salt diet in 9–10 weeks old rats. This might explain some of the pathophysiological differences observed by other investigators using the same model [12, 22].

4.2 Development of hypertension and compensated cardiac hypertrophy
In the present study, DS rats developed significant hypertension after 2 weeks of a high-salt diet resulting in concentric myocardial hypertrophy after 4WHSD, as reflected by the increased heart to body weight ratio and leftward shift of the diastolic pressure-volume curve. The presence of myocardial hypertrophy was further supported by a significant increase in cell width and no changes in resting cell length, when compared to age-matched DR rats after 4WHSD. Inconsistent changes in myocyte size have been reported in several models of pressure-overload hypertrophy [23, 24]. However, most of these studies support the idea that increased myocyte cross-sectional area will be more pronounced than increased cell length in concentric hypertrophy. No changes were found in the lung wet to dry weight ratio at this stage. As demonstrated in Fig. 4B and Table 1, contractile function was better in DS than DR rats at this stage. Therefore, we believe that these data provide sufficient evidence that the DS myocytes, at 4WHSD, were isolated from compensated hypertrophic and non-failing myocardium.

4.3 Mechanical function and intracellular Ca²⁺ handling at the stage of compensated hypertrophy
Controversial findings have been reported relating to mechanical function and Ca²⁺ homeostasis in various animal models of pressure-overload hypertrophy. Some [6, 9, 25–27]observed mechanical dysfunction and abnormal Ca²⁺ dynamics in pressure-overload hypertrophy, suggesting impaired SR function. Others have reported that there was an enhancement in Ca²⁺ cycling by the SR Ca²⁺ pump in mild cardiac hypertrophy due to pressure-overload [28]. Moreover, a recent study, using the Dahl rat model [22], has shown that the plasma membrane calcium current density and the SR calcium release channels were normal. However, a defect appears as a change in the spatial relationship between the SR calcium-release and the sarcolemmal calcium channels.

We found that DS-4WHSD hearts generated a greater LV developed pressure for any given preload compared to DR-4WHSD hearts (Fig. 4B). This finding is consistent with our observed 31% increase in the %CS in DS myocytes compared to age-matched DR myocytes (Table 1). These results indicate that the molecular remodeling occurring during compensated hypertrophy, at least in the Dahl rat model, is a beneficial alteration in response to hypertension. A 31% increase (p<0.05) in the %CS of the DS cells, relative to DR, occurred with a parallel trend of increase of the Ca²⁺ transient amplitude (Table 2). This observation indicated that DS-4WHSD myocytes maintained normal Ca²⁺ homeostasis. The difference in our finding from others with more advanced heart failure [3]is most likely due to the fact that the molecular remodeling process is dependent on the degree, and/or duration and/or specific model of hemodynamic load.

4.4 Development of congestive heart failure
An increased lung wet to dry weight ratio in DS rats was observed after 12WHSD, suggesting the development of congestive heart failure. In addition, there was a significant increase in resting cell length and width, compared to age-matched DR myocytes. This myocyte lengthening indicates that myocytes are stretched and disproportionate to their normal growth. It has been suggested that myocyte lengthening is closely associated with the development of heart failure [29]. The results from our whole heart perfusion studies showed that, for any given LV diastolic pressure, LV volume was increased almost threefold in the DS-12WHSD hearts compared to DS-4WHSD. This suggests that LV dilation developed in the DS rats after 12WHSD. However, despite the occurrence of the ventricular dilation and congestive failure, the LV systolic function of isolated DS-12HSD hearts and isolated myocytes was preserved compared to DR rats, albeit decreased compared to DS-4WHSD. Therefore, the heart failure, i.e. pulmonary congestion, in our model appears to be largely due to diastolic dysfunction. This is also supported by the observation that these animals when exposed to ischemia and reperfusion develop a marked ischemic diastolic dysfunction when compared to DR-12WHSD rats, but recovery of systolic function is preserved [30]. Thus, the initial stage of congestive heart failure in this rat model appears to be predominantly caused by diastolic dysfunction, although the occurrence of LV dilation implies some degree of systolic dysfunction relative to afterload in vivo.

4.5 Mechanical performance and intracellular Ca²⁺ handling at heart failure stage
Previous studies have shown that a prolonged time course of the Ca²⁺ transient is directly associated with a prolongation of contraction in myocardium of patients with end-stage heart failure [3, 4], as well as in animal models of heart failure [5, 6, 8]. Molecular biological studies have demonstrated that the SR Ca²⁺-ATPase mRNA and protein levels [31–33]and ryanodine receptor mRNA levels [32]are decreased in end-stage heart failure. Moreover, the SR Ca²⁺ transport function is depressed [34]and voltage dependent Ca²⁺ channels are downregulated [35]in human failing hearts. Nevertheless, the causal relationship between these parameters of Ca²⁺ homeostasis and progression to heart failure is unclear. Our data indicate that a prolongation in contraction and relaxation was not associated with a prolonged time course in the Ca²⁺ transient, whereas others have implicated alterations in intracellular Ca²⁺ handling as a possible cause of myocardial dysfunction [5]. Abnormal calcium handling can be masked by slow pacing. Since we paced our myocytes at a physiologic rate of 5Hz, it is unlikely that such a phenomenon confounded our results. Our observations are undoubtedly related to the specific time point of the transition stage from compensatory hypertrophy to failure and/or because a different pressure-overload animal model was used. Our results do not exclude the participation of abnormal Ca²⁺ handling in the progression to end-stage heart failure. The slower speed of contraction that occurred in DR-12WHSD myocytes may be similar to the mechanism of preserving the amplitude of cell shortening in elderly rats [36].

4.6 Mechanisms of altered myocyte contractile function
Our results suggest that a prolongation of contraction and relaxation may be an early indicator of abnormalities in myocyte mechanical function during the transition from compensated hypertrophy to heart failure. In rats, a shift in myosin heavy chain (MHC) isoforms from the V₁ to the V₃ isoform was observed during the development of cardiac hypertrophy or failure [37, 38]. Altered catalytic hydrolysis of ATP by changes in the activity of myosin ATPase will principally affect maximal rates of energy liberation during the cross-bridge cycle [39]. Thus, this isoform shift may affect the time course of the contraction and represent a compensatory mechanism to maintain the contraction amplitude [6, 40]. Changes of other subcellular elements, e.g. an increase in tubulin [41], or other as yet determined myofilament protein changes may also contribute to the shortening and relaxation changes we observed. As the disease progresses, a decrease in contractility [12]will, eventually, be observed when the prolongation of contraction time course can no longer preserve the adequate contractile function.

4.7 Conclusions
The present study demonstrates that alterations in excitation-contraction coupling occur in LV myocytes isolated from DS rats during the transition from compensated hypertrophy to heart failure. During the compensated hypertrophy stage, increased contractile function was associated with a parallel trend of an increase of peak systolic [Ca²⁺]_i. With the transition to failure, the initial increase in peak [Ca²⁺]_i abated, but all parameters of the Ca²⁺ transient were comparable to those from non-failing myocytes. Thus, in this model abnormal Ca²⁺ handling neither precedes nor initiates the transition from compensated hypertrophy to failure.

Time for primary review 20 days.

	Acknowledgements

 
This study was supported by the National Heart, Lung, and Blood Institute Grant HL 55993 (CSA) and the American Heart Association Grant-in-Aid 95-014870 (TMS), 96-015220 (FRE). KN is supported by a fellowship from the Fukuda and the Yokoyama foundations. RL is the recipient of a NHLBI Minority Faculty Development Award (HL03377). FRE is the recipient of a NHLBI Mentored Clinical Scientist Development Award (HL03574). We are grateful to Drs. William H. Barry and Bruno Podesser for critical readings and helpful suggestions on the manuscript. We would like to thank Mr. Soeun Ngoy and Ms. Susan Hope for technical assistance and Ms. Ellen P. Wiklanski for the expert administrative assistance.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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