Cardiovascular Research

Cardiovascular Research 2002 53(2):372-381; doi:10.1016/S0008-6363(01)00487-4
© 2002 by European Society of Cardiology

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Copyright © 2001, European Society of Cardiology

Hypertrophy and functional alterations in hyperdynamic phospholamban-knockout mouse hearts under chronic aortic stenosis

Helen Kiriazis^a, Yoji Sato^b, Vivek J Kadambi^c, Albrecht G Schmidt^a,1, Michael J Gerst^a, Brian D Hoit^d and Evangelia G Kranias^a,*

^aDepartment of Pharmacology and Cell Biophysics, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267-0575, USA
^bDivision of Xenobiotics, Metabolism and Disposition, National Institute of Health Sciences, Setagaya, Tokyo 158-8501, Japan
^cDepartment of Cardiovascular Biology, Millennium Pharmaceuticals Inc., Cambridge, MA 02139, USA
^dDivision of Cardiology, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, OH 44106-5029, USA

litsa.kranias{at}uc.edu

^* Corresponding author. Tel.: +1-513-5582-377; fax: +1-513-5582-269

Received 23 April 2001; accepted 1 October 2001

	Abstract

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

 
Objective: To determine whether the hyperdynamic phospholamban-knockout hearts are capable of withstanding a chronic aortic stenosis. Methods: The transverse section of the aorta was banded in phospholamban-knockout and their isogenic wild-type mice, which were followed with echocardiography in parallel, along with sham-operated mice, before and at 2.5, 5 and 10 weeks after surgery. Results: Cardiac decompensation was evidenced by the presence of lung congestion in some banded knockouts and wild-types, giving rise to a subset of non-failing and failing hearts within each group. The incidence of heart failure was not genotype-dependent but rather associated with higher heart rates before surgery. The development of left ventricular hypertrophy was similar between knockouts and wild-types and longitudinal assessment of end-diastolic dimension indicated progressive increases after banding, with a greater dilation in failing mice. Fractional shortening was reduced in failing knockouts and wild-types to a similar degree, with an earlier onset in the knockouts. In addition, fractional shortening was decreased in non-failing knockouts but not wild-types. Ejection times shortened after aortic banding particularly for failing hearts. Assessment of the SR Ca²⁺-ATPase protein levels indicated similar downregulation for failing knockouts and wild-types, while the phospholamban levels were not significantly altered in wild-types. Conclusion: The hyperdynamic phospholamban-knockout hearts are able to compensate against a sustained aortic stenosis similar to wild-types.

KEYWORDS Ca-pump; Heart failure; Hypertrophy; SR (function); Ventricular function

	1. Introduction

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

 
The heart undergoes hypertrophy in response to a chronic hemodynamic stress, and when the primary stimulus is a pressure overload (e.g., aortic stenosis, hypertension), peak systolic wall stress is normalized by an increase in left ventricular wall thickness with little or no change in cavity radius [1]. Despite this compensatory concentric remodeling, the overloaded heart eventually fails to pump effectively [2], although the mechanisms underlying the transition to a decompensated state remain an enigma.

One of the major characteristics of the failing heart is an abnormal Ca²⁺ homeostasis, manifested by a prolonged time course of intracellular Ca²⁺ transients and changes in systolic/diastolic Ca²⁺ levels [3] which, at least in part, may be linked to depressed sarcoplasmic reticulum (SR) Ca²⁺ uptake [4]. Furthermore, it has been suggested that reduced levels of SR Ca²⁺-ATPase mRNA may be a marker of the transition from compensated hypertrophy to heart failure [5]. Thus, various therapeutic approaches targeting the SR Ca²⁺-ATPase or its endogenous inhibitor, phospholamban, have been recently applied as means to rescue the failing heart. Specifically, adenoviral transfection techniques have been used to increase the SR Ca²⁺-ATPase or decrease the phospholamban protein levels in failing human myocytes and failing pressure-overloaded rat hearts, resulting in improved function [6–9]. Further support has been gained from genetic approaches that have targeted phospholamban in embryonic stem cells, generating mice with reduced or ablated phospholamban in the heart [10]. The phospholamban-knockout mice displayed a hyperdynamic cardiac function and attenuated inotropic/lusitropic responses to β-adrenergic agonists [10–12]. Thus, it was suggested that phospholamban inhibition might constitute an important therapeutic target in the treatment of depressed cardiac function. Along these lines, ablation of phospholamban from the embryonic stage in some cardiomyopathic mouse models restored cardiac function [13] and prevented heart failure [14], while another heart failure model with enhanced Ca²⁺ cycling was not rescued [15]. However, to evaluate the significance of long-term phospholamban inhibition in heart failure, a fundamental question must be addressed: Can phospholamban deficient hearts cope with a chronic stress? It may be argued that enhanced SR Ca²⁺ cycling comes at an energetic cost. Indeed, the hyperdynamic phospholamban-knockout hearts have metabolic adaptations to meet the increased ATP demand [16,17], but their energy reserve via the creatine phosphokinase reaction is diminished [16] and, therefore, phospholamban deficient hearts may be less likely to compensate when challenged with a chronic stress.

Thus, the present study was designed to test whether the hyperdynamic phospholamban-knockout hearts with the enhanced basal energetic profile could endure a transverse aortic coarctation. Although a short-term duration ( 3 weeks) of transverse aortic banding does not appear to induce a decompensated state in wild-type mice [18–20], signs of heart failure are evident after a longer duration (9 weeks) of the pressure overload [21]. Using non-invasive techniques, we were able to progressively follow the time–course of cardiac hypertrophy and left ventricular performance in sham-operated and aortic-banded phospholamban-knockout and wild-type male mice for up to a period of 10 weeks, and identify animals in heart failure. Our findings indicate that phospholamban ablation does not compromise the heart's capacity to tolerate a chronic aortic stenosis.

	2. Methods

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

 
This study was approved by the ethics committee of the University of Cincinnati. The investigation conforms 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), and the Responsible Care and Use of Animals in Research and Education manual published by the University of Cincinnati (Cincinnati, OH, USA). Inbred 129SvJ mice were used. Some wild-type and phospholamban-knockout 129SvJ mice were bred at Taconic (Germantown, NY, USA) under support by Merck Laboratories.

2.1 Production of hypertrophy
Adult (12–16 weeks) male phospholamban-knockout and age-matched isogenic wild-type mice underwent sham or transverse aortic banding surgery [18,19]. Briefly, mice were anesthetized (100 mg/kg ketamine, 5 mg/kg xylazine and 2.5 mg/kg morphine, i.p.), intubated and ventilated [0.5 ml tidal volume (room air), 100 cycles per min, Harvard apparatus]. The chest cavity was entered at the second intercostal space at the left upper sternal border through a small incision. The transverse section of the aorta was freed and a suture [7-0 TI-CRON (Davis+Geck)] was placed around the aorta between the right innominate and left common carotid arteries, and a tight ligature was tied against a 27-gauge needle; the needle was subsequently promptly removed. The lungs were re-expanded and the thoracotomy was repaired. Sham operations were concurrently performed using an identical procedure except that the suture around the transverse aorta was removed. The 5-week operative mortalities of banded wild type (bWT) and banded phospholamban KO (bKO) mice were 22 and 33%, respectively. Most died within the first 2 weeks; these animals were excluded from the study.

2.2 Pressure gradient across the band
At 2.5 weeks after banding the aorta, a subset of mice were anesthetized, as described above, intubated and the right and left carotid arteries were cannulated with flame-stretched polyethylene tubing. The catheters were connected to calibrated fluid-filled pressure transducers, allowing the simultaneous monitoring of pressure in both carotids. The pressure gradient across the band was measured at a steady hemodynamic state.

2.3 Serial echocardiographic studies
In vivo left ventricular function and chamber dimensions were blindly assessed with M-mode and Doppler echocardiography under anesthesia (0.01 ml/g, i.p. of 2.5% Avertin), using an Interspec Apogee CX-200 ultrasonograph (Interspec-ATL) [22]. This non-invasive technique allowed the same animals to be assessed at baseline (prior to surgery, 0 weeks) and at 2.5, 5 and 10 weeks after surgery. At any particular time point, echocardiography was performed on sham and banded wild-type and phospholamban-knockout mice in parallel. For the bWT group, echocardiographic data were serially collected from 16 mice up to 5 weeks post-banding, at which point a portion of the group was terminated for heart sampling (see below); nine animals were carried through to 10 weeks (excluding one animal which died prior to this time point). In the bKO group, there were 13 animals up to the 5-week time point and eight animals at 10 weeks. In addition, echocardiographic data were obtained from sham-operated wild-type mice (n=5–10) and phospholamban-knockout mice (n=5–9).

Freeze frames of M-mode images were captured on-line and printed on a color video printer (UP-5200, Sony Corporation). From the hard-copies, M-mode measurements of left ventricular end-diastolic dimension (EDD), left ventricular end-systolic dimension, and interventricular septal wall thickness (IVS) and posterior wall thickness at end-diastole, were made by using the leading-edge convention of the American Society of Echocardiography and by using the steepest continuous endocardial echoes. Aortic outflows were recorded on S-VHS videotapes and ejection times (time from aortic valve opening to closure) were obtained upon playback of videotapes using a commercially available image analysis system (Tomtec). Left ventricular fractional shortening (FS), velocity of circumferential fiber shortening corrected for heart rate (Vcf_c), ratio of wall thickness to cavity radius (h/r) and echocardiographic left ventricular mass were calculated, as previously described [22,23].

2.4 Morphological studies
Mice were terminated following their final cardiac echocardiographic assessment. Hearts were quickly excised and rinsed in cold phosphate-buffered saline (pH 7.4). The atria, right ventricular free wall, left ventricle (cut open) and lungs were blotted and weighed. In all animals that had undergone aortic banding surgery, the band was visually inspected and was found to be intact.

2.5 Quantitative immunoblotting
Left ventricles were homogenized and quantitative immunoblotting for phospholamban, SR Ca²⁺-ATPase, and calsequestrin was performed after sodium dodecyl sulfate–polyacrylamide gel electrophoresis [24].

2.6 Statistics
The data are expressed as means±S.E. Statistical analyses were performed using SigmaStat 2.03S and SYSTAT 10 (SPSS). Unless otherwise specified, one-way analyses of variance (ANOVA), and two-way repeated-measures ANOVA designs were employed. Differences were considered significant at p<0.05. When the ANOVA indicated a statistically significant difference, the Student–Newman–Keuls test was subsequently employed as a post hoc test.

	3. Results

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

 
3.1 Baseline echocardiographic measurements
In this study, phospholamban-knockout and wild-type mice in the 129SvJ background were utilized. Only male mice were used, eliminating any gender-specific effects of pressure overload [25]. The baseline cardiac phenotype was assessed prior to sham or aortic banding surgery. The mean heart rate was not significantly different between the two animal groups (phospholamban-knockout: 412±10 beats/min, n=22; wild-type: 407±11 beats/min, n=26). Left ventricular dimensions were not changed upon ablation of phospholamban (see below), but ejection time was significantly shorter (55±1 ms, n=22 vs. 68±2 ms, n=26) and Vcf_c was augmented (7.76±0.23 circ/s, n=22 vs. 6.39±0.26 circ/s, n=26) in phospholamban-knockout vs. wild-type mice (unpaired t-tests). FS was not different between the genotypes (phospholamban-knockout: 35±1%, n=22; wild-type: 35±1%, n=26). These data are in agreement with previous observations, using the original phospholamban-knockout model in the 129SvJ+CF-1 background [22].

3.2 Pressure gradient across the band
Wall stress is a function of the cardiac chamber geometry and pressure gradient between the chamber and its afterload system and, like contractility, it is an important factor that determines myocardial work and energy consumption. Therefore, we examined whether the pressure gradient across the induced stenosis, assessed by the differences in systolic pressures in the right and left carotid arteries at 2.5 weeks, was similar between bKO and bWT mice. The pressure gradient across the band in bWT mice was 53±3 mmHg (n=4), which is similar to that reported previously [19]. However, the same stenosis applied to phospholamban-knockout mice resulted in a significantly higher pressure gradient of 97±9 mmHg (n=4).

3.3 Evidence of failing and non-failing banded animals
Since the duration of the transverse aortic constriction was relatively long-term, we were expecting to find some animals in failure, as previously reported [21]. Indeed, inspection of lungs from banded animals revealed that some were enlarged, edematous and pale, while other lungs looked normal at the gross morphologic level (Fig. 1A). Furthermore, a plot of lung/body mass ratio against the left ventricular/body mass ratio identified a subset of transverse aortic banded animals that had an increased lung mass, and these animals also had a relatively greater degree of left ventricular hypertrophy (Fig. 1B). Specifically, aortic banded animals with a lung/body mass ratio greater than three standard deviations of the mean lung/body mass ratio of sham-operated wild-type or phospholamban-knockout mice {8.51=5.51(mean)+[1.00(S.D.)x3] mg/g, n=22}, were considered to have congested lungs and, hence, to be in heart failure. Based on this criterion, banded animals were split into failing and non-failing groups, and there appeared to be a trend for a greater number of bKOs to develop heart failure. This may be expected from the finding of a relatively larger gradient across the band for bKOs vs. bWTs, however, Fisher's exact test indicated that the incidence of heart failure was not significantly different between bKOs and bWTs (p=0.274).

View larger version (48K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Identification of failing and non-failing hearts. (A) Hearts and lungs from phospholamban-knockout (KO) sham-operated (left) and banded (right) mice at 10 weeks after surgery. Bar=1 cm. (B) Lung/body mass against left ventricular (LV)/body mass for sham-operated and aortic banded wild-type (WT) and KO mice. A subset of banded mice with lung/body mass greater than three standard deviations of the mean lung/body mass of sham-operated mice (dashed line), were considered to be in heart failure. Based on this criterion, the banded mice were divided into two groups: failing and non-failing. Note that one banded wild-type animal died prior to the 10-week echocardiogram and, while morphological data were not obtained on this animal, it was included in the failing group since the 5-week echocardiogram revealed poor left ventricular function.

 
Heart rate is another important factor for myocardial energy consumption and an independent risk factor of cardiovascular mortality [26], therefore it was of special interest to examine whether individual differences in basal heart rate could be associated with lung congestion. Although, mean in vivo heart rate is not different between phospholamban-knockout and wild-type mice, as shown in this and previous studies [22,27], it does vary from animal to animal within each group. Quinlan's C4.5 algorithms [28] were applied to generate a decision tree for the outcome after banding (ie, non-failing, failing) using genotype and baseline heart rate as the non-categorical attributes. The analysis indicated that the informational gain ratio by a partition of the data at a specific heart rate between 375 and 415 beats/min was higher (>0.1) than that based on genotype (0.038) for the prediction of the outcome following aortic stenosis (Fig. 2). Furthermore, discriminant analysis for the outcome (non-failing vs. failing) indicated that F-values of genotype (bKO, bWT) and basal heart rate (continuous variable) were 1.52 and 4.08, respectively. These results suggest that basal heart rate is more critical than genotype as a predictor of the outcome. Differences in heart rate were not only evident at baseline. Heart rate was higher in failing vs. non-failing mice throughout the protocol (p<0.001, two-way repeated-measures ANOVA across 0–10 weeks).

View larger version (38K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Potential association of heart rate with incidence of heart failure post aortic stenosis. Lung/body mass ratio after chronic aortic stenosis against heart rate (HR) before surgery for banded wild-type (bWT) and phospholamban-knockout (bKO) mice. The horizontal dashed line indicates three standard deviations of the mean lung/body mass ratio of sham-operated mice; animals above this line have lung congestion. The shaded grey box marks the heart rate range of 375–415 beats/min, identified using Quinlan algorithms [28]. Based on the analysis, partitioning the data using a heart rate falling within this range was suggested to be more important than genotype for the prediction of outcome (non-failing, failing) post banding.

 
The atrial, right ventricular, left ventricular and lung/body mass ratios of sham-operated and banded mice at the time of termination are shown in Fig. 3, along with body mass. The findings are qualitatively similar between wild-type and phospholamban-knockout mice. Relative to the non-failing groups, the degree of left ventricular hypertrophy was greater in the failing animals. In addition to an increase in lung mass, atrial hypertrophy and right ventricular hypertrophy were evident in these groups and further supported the notion that their hearts were indeed decompensated. Body mass was not significantly affected by the treatment (sham vs. band).

View larger version (42K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Gravimetric data. Body mass (right) and atrial, right ventricular (RV), left ventricular (LV) and lung mass per body mass ratios (left, center) for wild-type and phospholamban-knockout animals. Mice were grouped as sham-operated, non-failing, and failing. Means±S.E; n=4–12; *p<0.05 vs. sham-operated; #p<0.05 vs. non-failing.

 
3.4 Time course of hypertrophy
Left ventricular dimensions were similar between wild-type and phospholamban-knockout mice at baseline (Figs. 4 and 5), as reported previously [22]. Examination of echocardiographic left ventricular/body mass ratios against time post banding indicated that the hypertrophic process was comparable between bKOs and bWTs (Fig. 4). Left ventricular hypertrophy was evident in the first 2.5 weeks in aortic-banded mice. Moreover, the failing hearts developed a significantly greater degree of left ventricular hypertrophy compared to their non-failing counterparts, for both bKOs and bWTs, which was in agreement with the findings at autopsy (Fig. 3).

View larger version (28K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Development of hypertrophy post banding. Echo-derived left ventricular (LV) mass normalized for body mass against time post banding for wild-type (upper) and phospholamban-knockout (lower) sham, non-failing and failing mice. Means±S.E; wild-type: n=5–10 (sham), 6–11 (non-failing) and 3–5 (failing); knockout: n=5–9 (sham), 3–6 (non-failing) and 5–7 (failing); *p<0.05 vs. sham-operated and #p<0.05 vs. non-failing, at specific time points indicated; vertical lines at the end of the plots indicate which groups (as a whole) were significantly different (p<0.05).

 

View larger version (42K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Left ventricular cavity dimensions post banding. End-diastolic dimension (EDD), interventricular septal wall thickness (IVS) and wall thickness to cavity radius ratio (h/r) for wild-type (left) and phospholamban-knockout (right) sham, non-failing and failing hearts. Means±S.E; wild-type: n=5–10 (sham), 6–11 (non-failing) and 3–5 (failing); knockout: n=5–9 (sham), 3–6 (non-failing) and 5–7 (failing); #p<0.05 vs. failing, at specific time points indicated; vertical lines at the end of the plots indicate which groups (as a whole) were significantly different (p<0.05). Time-dependent changes discussed in text.

 
More information was obtained from the left ventricular cavity size and wall thickness at end-diastole, shown in Fig. 5. The left ventricular chamber progressively dilated after 2.5 weeks banding, with the degree of dilation significantly greater for failing vs. non-failing bKOs and bWTs. IVS increased in the first 2.5 weeks and thereafter remained relatively plateau. There were no significant differences in IVS between non-failing and failing groups (Fig. 5). A significant time-dependent increase in the h/r ratio was evident for both bWTs and bKOs mice, indicating the presence of concentric hypertrophy in the early stages. However, group differences in the h/r ratio were only significant between non-failing bWTs vs. sham-operated mice (Fig. 5). Despite an initial increase in the h/r ratio of failing bWTs similar to their non-failing counterparts, its value subsequently declined towards the sham group (non-failing bWT vs. sham, p=0.056). This decline was due to further increases in EDD while IVS remained unchanged (also evident in failing bKOs), suggesting eccentric hypertrophy at the later time-points.

The hypertrophic response tended to be delayed in non-failing bKOs. However, direct comparisons of left-ventricular dimensions between (i) non-failing bKOs and non-failing bWTs, and (ii) failing bKOs and failing bWTs, did not reveal any significant differences indicating that the hypertrophic response was independent of phospholamban.

3.5 Effects of aortic banding on cardiac function in vivo
To assess the effects of aortic banding on left ventricular performance, FS, ejection time, and Vcf_c, were plotted against time after surgery (Fig. 6). FS was significantly different between the non-failing and failing bWTs with the failing bWT hearts having a much-reduced FS at 5 and 10 weeks post banding. There was no such distinction between failing and non-failing bKO hearts. For both, FS was attenuated relative to the sham group and the onset of the decrease in FS was earlier in the bKOs, compared with the failing bWTs (2.5 vs. 5 weeks) (Fig. 6). Nonetheless, at the 5- and 10-week time points, FS was not significantly different between failing bKOs and bWTs. Prolonged aortic stenosis led to a decrease in the time for ventricular ejection. Failing bWTs had ejection times lower than non-failing and sham groups, and phospholamban-knockout hearts, which started with a lower ejection time relative to wild-types, had a significant decrease at 10 weeks post banding (i.e., there was a time-dependent effect). The patterns of change in Vcf_c were similar to FS, although not significantly different, due to changes in ejection time post banding. Note that the levels of Vcf_c at the 5- and 10-week time points were lower in the failing bWTs, compared to the failing bKOs, which is presumably due to the difference in ejection time between the two groups (Fig. 6).

View larger version (41K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Left ventricular function post banding. Fractional shortening (FS), ejection time and velocity of circumferential fiber shortening corrected for heart rate (Vcfc) for wild-type (left) and phospholamban-knockout (right) sham, non-failing and failing hearts. Means±S.E; wild-type: n=5–10 (sham), 6–11 (non-failing) and 3–5 (failing); knockout: n=5–9 (sham), 3–6 (non-failing) and 5–7 (failing); #p<0.05 vs. failing, at specific time points indicated; vertical lines at the end of the plots indicate which groups (as a whole) were significantly different (p<0.05). Time-dependent changes discussed in text.

 
3.6 Sarcoplasmic reticulum associated proteins
Various studies have shown changes in SR proteins and particular the Ca²⁺-ATPase in hypertrophy and heart failure [8,29–32], which may underlie functional alterations. Thus, to examine whether the observed functional changes between failing and non-failing hearts could be linked to changes at the SR level, the SR transport and storage proteins were assessed. For simplicity, protein levels in the non-failing and failing groups were normalized to the mean of their respective sham-operated groups. Calsequestrin was not significantly altered upon banding [wild-type (n=3–6): 1.00±0.05, 0.92±0.08 and 0.88±0.06; phospholamban-knockout (n=3–5): 1.00±0.03, 0.98±0.04 and 0.89±0.06, for sham, non-failing and failing, respectively]. However, there was a marked decrease (50%) in the Ca²⁺-ATPase levels for both non-failing and failing bKO and the failing bWT hearts (Fig. 7). This protein was not as severely downregulated in the non-failing bWT hearts (24% decrease). As this group seemed to have preserved function after aortic banding, whereas the other banded animals had relatively depressed function (Fig. 6), it was of interest to examine whether the SR Ca²⁺-ATPase levels correlated with fractional shortening. Indeed, SR Ca²⁺-ATPase protein levels had a significant correlation [r²=0.310 (n=36, p<0.001)] with fractional shortening of banded and sham-operated animals. Assessment of phospholamban levels in wild-type hearts indicated no significant changes upon banding, although they tended to be higher in the non-failing group (p=0.058) (Fig. 7). In addition, the phospholamban to Ca²⁺-ATPase ratio was somewhat increased in both failing and non-failing bWT hearts (p=0.055) (Fig. 7).

View larger version (39K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Quantitative immunoblotting for SR Ca2+-ATPase (SERCA) and phospholamban (PLB). (A) Representative Western blots. A control homogenate from pooled wild-type hearts was used to generate a standard line for the quantitation of SERCA and PLB protein levels in wild-type (WT) and phospholamban-knockout (KO) sham (S), non-failing (NF) and failing (F) left ventricles. For PLB, solubilized samples were boiled for 5 min prior to loading to dissociate the protein into monomers. (B) Bar graphs showing SERCA protein levels for WTs and KOs, and PLB protein levels and PLB/SERCA ratios for WTs. Values were normalized to the average level of the specific protein in the respective sham-operated group. Means±S.E; n=3–10; *p<0.05 vs. sham-operated, #p<0.05 vs. non-failing (one-way ANOVA).

 

	4. Discussion

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

 
The present longitudinal study provides evidence that phospholamban-knockout hearts are able to tolerate an aortic stenosis similar to the wild-types despite their hyperdynamic nature and, hence, increased energy demand. Furthermore, the aortic coarctation was carried out for a long-term, allowing the evaluation of non-failing and failing phospholamban-knockout hearts which exhibited, in the most part, similar functional alterations to the respective wild-type groups. The identification of failing hearts was based on lung congestion, which increased the afterload on the right ventricle and produced right ventricular enlargement, as previously observed [20,21,33]. In addition, atrial mass was increased in the failing groups, similar to previous reports [21,33]. Atrial hypertrophy was also observed in non-failing hearts, which may be a result of assisting the filling of the hypertrophic left ventricle during diastole. The degree of left ventricular hypertrophy was greater in the failing bKO and bWT hearts, relative to their respective non-failing groups, as revealed by gravimetric analyses. This finding agreed with the echocardiographic data, which allowed not only the left ventricular mass, but also cavity size and wall thickness to be progressively monitored for the duration of the aortic stenosis. The development of hypertrophy was similar between bKOs and bWTs, and both failing and non-failing hearts showing an initial concentric hypertrophic response, which for the failing hearts, progressed towards eccentric hypertrophy.

Although the transverse aorta was constricted to a similar degree in bKOs and bWTs, a greater gradient across the band was evident in bKOs, which may in part be due to alterations in the vasculature that is also devoid of phospholamban [34]. Moreover, the increased peak aortic velocity in phospholamban-knockouts [22] per se could contribute to a greater gradient (based on Poiseuille's law, where the pressure gradient across a resistance is directly proportional to flow). Despite the increased gradient, the incidence of heart failure was not significantly different between bKO and bWT groups. Although cardiac parameters were evaluated under anesthesia in this study, the high incidence of heart failure in mice with a relatively higher heart rate suggests that basal heart rate itself or some heart rate-related factors, rather than phospholamban deficiency (and concomitant hyperdynamic profiles), are more critical for the development of heart failure during aortic stenosis. The underlying factors contributing to variances in the basal heart rate in vivo are unknown, but possibly may be due to individual differences in basal autonomic nervous tone, plasma catecholamine levels, thyroid status or expression of pacemaker-related genes (e.g., isoforms of potassium and pacemaker channels). In contrast to our observations in phospholamban-knockout mice, elevated cardiac β-adrenergic activity in β₂-adrenoceptor overexpressing mice has been reported to result in more deleterious consequences after aortic stenosis, compared to the wild-types [21,33]. Excessive expression of β₂-adrenoceptors enhances myocardial contractility but also leads to a considerable increase in basal heart rate. Therefore, it is interesting to speculate that the increased heart rate in β₂-adrenoceptors overexpressing mice, rather than their enhanced myocardial contractility, accounts for their high susceptibility to pressure overload.

Another important question is whether the functional alterations in non-failing and failing bKOs were similar to those in bWTs. Our observations indicated an early onset of the fractional shortening attenuation in the failing bKO mice, compared with the failing bWTs. In addition, although function of non-failing bWTs was preserved, non-failing bKOs had a depressed fractional shortening relative to shams. The heightened energy consumption in the phospholamban-knockout mice and/or a putative stressed oxygen supply, as a result of little or delayed angiogenesis during the hypertrophic process [35], could be, in part, responsible for this early drop in function for the bKO mice. Nonetheless, fractional shortening was comparable between failing bKOs and bWTs at 5 and 10 weeks after banding. The decrease in ejection time, evident for both failing bKO and failing bWT groups, could be partly due to the depressed fractional shortening leading to a poor cardiac output per beat. Decreases in ejection time have also been noted in human heart failure [36].

Due to distinct functional differences between the groups, it was of interest to assess the levels of SR Ca²⁺ handling proteins, and discern whether any of these could be underlying the functional changes. Changes in the SR Ca²⁺-ATPase and phospholamban, are more controversial, with most studies showing either no change or a decrease in human and animal end-stage heart failure [8,29–32]. Decreases in Ca²⁺-ATPase levels in human failing hearts have been directly linked to deteriorated contractile function [37]. In addition, an increase in the phospholamban to SR Ca²⁺-ATPase ratio was associated with depressed contractile reserve in hypertrophied failing mouse myocytes [38]. The present study showed no significant changes in bWT phospholamban levels, while a marked decrease in the SR Ca²⁺-ATPase was evident. Specifically, both failing and non-failing bKO hearts, as well as failing bWT hearts, had similar reductions in Ca²⁺-ATPase levels, whereas non-failing bWT hearts had a relatively smaller attenuation in Ca²⁺ pump protein. Furthermore, SR Ca²⁺-ATPase protein levels correlated with fractional shortening and this, at least in part, seemed to underlie the functional differences noted between the animal groups. Thus, the early drop of FS in bKO mice, observed in the failing group, may reflect the higher sensitivity of their SR Ca²⁺-ATPase expression to chronic aortic stenosis, compared to the bWTs. However, it must also be considered that the absence of phospholamban in the bKO hearts implies that the remaining Ca²⁺ pumps are fully uninhibited and, hence, cardiac contractility may be expected to be better than in the failing bWT group, which has an increased phospholamban to Ca²⁺-ATPase ratio, or more pumps in the inhibited state.

In summary, our results suggest: (i) the hyperdynamic phospholamban-knockout hearts are able to compensate against a sustained aortic stenosis similar to their wild-type counterparts, and (ii) basal heart rate or some heart rate-related factors may be more critical than inotropic state for the success of the adaptation. In the present study, phospholamban was ablated from embryonic stages and, therefore, it is not known whether inhibiting phospholamban during the compensated hypertrophy stage or, in particular, after the development of heart failure, would be beneficial for cardiac function. The future use of genetic models with tissue-specific and regulated phospholamban levels will address the potential clinical application of such a therapeutic strategy.

Time for primary review 28 days.

	Acknowledgements

 
This work was supported in part by US National Institute of Health Grants HL26057, HL52318, HL64018 and P40RR12358 (to E.G.K.), the Japan Health Sciences Foundation Grant No. 23004 and a Japan Ministry of Health, Labour and Welfare Grant (to Y.S.). We thank Dr. L.R. Jones (Indiana University, Indianapolis, IN, USA) for providing the dog polyclonal anti-calsequestrin antibody, Dr. D.P. Suresh (University of Cincinnati, Cincinnati, OH, USA) for his help with echocardiography, and Drs. R.G. Johnson, Jr. (Chiron Corporation, Emeryville, USA), G.W. Dorn, II (University of Cincinnati) and C.J. Barclay (Monash University, Melbourne, Australia) for valuable input and suggestions. Special thanks go to Ms. N. Ball for the instruction of the microsurgery techniques.

	Notes

 
¹ Current address: Zentrum Innere Medizin, Abteilung Kardiologie und Pneumologie, Georg-August-Universität Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany.

	References

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