Interact CardioVasc Thorac Surg 2008;7:27-31. doi:10.1510/icvts.2007.165563
© 2008 European Association of Cardio-Thoracic Surgery
Work in progress report - Assisted circulation |
Partial left ventricular unloading reverses contractile dysfunction and helps recover gene expressions in failing rat hearts
Jian Wang,
Akira Marui,
Tadashi Ikeda and
Masashi Komeda*
Department of Cardiovasular Surgery, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
Received 23 August 2007;
received in revised form 5 October 2007;
accepted 9 October 2007
 Presented at the 21st Annual Meeting of the European Association for Cardio-thoracic Surgery, Geneva, Switzerland, September 16–19, 2007.
*Corresponding author. Tel.: +81-75-751-3784; fax: +81-75-751-4960.
E-mail address: komelab{at}kuhp.kyoto-u.ac.jp (M. Komeda).
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Abstract
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We investigated the effects of partial left ventricular unloading on failing rat hearts by using heterotopic heart-lung transplantation model. Heart failure (HF) was induced in Lewis rats by ligating the left anterior descending artery. After four weeks, the infarcted hearts and lungs were harvested and transplanted into the recipient rats by anastomosing donor's ascending aorta to recipient's abdominal aorta. Therefore, coronary venous blood entered the left ventricle (LV) and LV was partially unloaded (HF-PU group). Normal and infarcted heart rats (HF group) without transplantation served as control animals. After two weeks' unloading, the infarcted LV in HF-PU group significantly decreased its weight and myocardial diameter compared with HF group and they were close to normal levels. Developed tension of posterior papillary muscle was significantly increased in HF-PU group compared with HF group. The mRNA expressions of brain natriuretic peptide (BNP), sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a), β1 and β2-adrenergic receptors (β1 and β2-AR) in LV tissue were almost normalized in HF-PU group. Partial left ventricular unloading regressed myocardial hypertrophy, reversed contractile dysfunction and normalized the mRNA (BNP, SERCA2a, β1 and β2-AR) expressions of failing rat hearts.
Key Words: Heart failure; Heart-assist device; Myocardial contraction; Remodeling; Genes
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1. Introduction
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Recently, myocardial recovery due to ventricular unloading and workload reduction with a left ventricular assist device (LVAD) has been noted [1]. LVAD provides profound volume and pressure unloading of the left ventricle (LV) and restores systemic blood pressure and flow to near normal levels, which leads to a normalization of the neurohormonal and local cytokine milieu contributing to myocardial recovery [2]. Although the structural and functional outcomes of LVAD-induced unloading are described, clinical success of achieving sustained recovery of the end-stage failing heart has been sporadic and the use of an LVAD as a bridge to recovery remains controversial. Therefore, a better understanding of the ‘reverse remodeling’ process may lead to a novel hemodynamic support strategy toward myocardial recovery and potentially successful weaning from LVADs without heart transplantation.
The rat heterotopic heart transplantation (i.e. complete unloading) model was reported in the 1960s [3], which was commonly used for the study of LV mechanical unloading. However, LV was completely unloaded in that model and therefore that model did not reflect the situation of patients with LVADs support. Recently, Mizuno et al. [4] reported a new heterotopic heart-lung transplantation (i.e. partial unloading) model for simulation of LVAD. In this model, LV was loaded with coronary venous blood, which creates partial unloading. The objective of this study was to examine the effects of partial unloading on failing rat hearts by using the heterotopic heart-lung transplantation model.
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2. Materials and methods
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2.1. Induction of heart failure (HF) and LV functional assessment
Lewis rats weighing 160–200 g (Japan SLC Inc., Hamamat-su, Japan) were used. All procedures were conducted according to Kyoto University's guidelines for animal care, and followed guidelines set forth in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Myocardial infarction (MI) was induced through ligation of the left anterior descending (LAD) artery as previously described [5]. Four weeks after the LAD ligation, and heart function and infarct size were evaluated by echocardiography with a 12 MHz phased array transducer (HP SONOS 4500, Agilent Technologies, Andover, MA). M-mode tracings were recorded through the anterior and posterior LV end diastolic dimension (LVDd) and LV end systolic dimension (LVSd). Fractional shortening (FS) was calculated as (LVDd–LVSd)/LVDdx100 and used as an index of LV systolic function. Infarct size (%) was calculated as (infarct length in LV diastolic phase)/(LV diastolic circumference)x100. Three measurements were taken and averaged to calculate these parameters during each examination.
2.2. Experimental protocol
Four weeks after LAD liagtion, 20 rats with ischemic cardiomyopathy (infarct size >30%) were randomly divided into two groups: in HF-partial unloading group (HF-PU group, n=10), the infarcted hearts and lungs were heterotopically transplanted into the abdomen of recipient rats; in HF group (n=10), the hearts were not transplanted. Normal Lewis rats were as control group (Nor group, n=8). After two weeks, the transplanted infarcted heart, non-transplanted hearts, and normal hearts were excised and analyzed.
2.3. Contraction of isolated papillary muscle
Papillary muscle function was examined at experiment end points. The rats were anesthetized and heparinized. After opening the abdomen, the heart was rapidly removed. The heart was placed into normal Tyrode's solution. Posterior papillary muscle was carefully ligated with a silk thread, dissected from the LV wall and mounted in a tissue bath containing Krebs-Henseleit solution. The bath was maintained at a constant temperature of 37 °C and bubbled with 95% O2 and 5% CO2. The papillary muscle was stimulated at 1 Hz with impulses of 5-ms duration and five-millisecond pluses at a voltage approximately 10% above threshold level were used. The papillary muscle was stretched to the length at which maximal tension occurred. After stabilization, isometric tension was recorded digitally at the maximum tension position. Developed tension (DT) was normalized to cross-sectional area.
After removal of the papillary muscle from the heart, the LV myocardium was transversely sliced into 2 mm-diameter sections at the base of the papillary muscle and fixed in 10% buffered formalin. The remaining LV myocardium was frozen at –80 °C until analyzed.
2.4. Pathological studies
Transverse sections of LV myocardium were stained with Hematoxylin-Eosin. The mean cardiomyocyte diameters were calculated by measurement of 50 cells in the remote myocardium under microscopy (magnificationx400) as before [5].
Other transverse sections were stained with Picrosirius red [5]. In each section, 20 separate parts of the remote area were scanned under microscopy (magnificationx200), and the images were analyzed using an IPLabTM for Windows image system (Solution Systems, Inc., Japan). The percentage of myocardial fibrosis was obtained by calculating the mean ratio of the fibrotic area in 20 separate parts of the LV remote myocardium.
2.5. Analysis of mRNA expression
Total mRNA was prepared from the frozen LV pieces with TRIzol (Life Technologies Inc.) reagent, reverse-transcribed, and amplified with the ABI PRISMTM 7700 sequence detector (Applied Biosystems). Polymerase chain reaction (PCR) conditions were 40 cycles of denaturing at 94 °C for 20 s and primer annealing/extension at 62 °C for 60 s. The PCR sequences of brain natriuretic peptide (BNP), sarco(endo)plasmic reticulum Ca2+-ATPase2a (SERCA2a), β1 and β2-adrenergic receptors (AR) were previously reported in our studies [5, 6]. The TaqMan rodent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) control reagent was used to detect rat GAPDH as the internal standard. Expression levels of the target gene were normalized to the GAPDH level in each sample.
2.6. Statistical analysis
All data are described as mean±S.E.M. Differences among groups were evaluated by one-way analysis of variance (ANOVA) followed by Scheffe's F test. All statistical analyses were performed using the Statview® for Windows version 5.0 (SAS Institute Inc., Cary, NC). A value of P<0.05 was considered significant.
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3. Results
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3.1. Baseline echocardiographic data
As shown in Table 1, LVDd and FS in HF-PU and HF groups were significantly impaired compared with Nor group. Accordingly, LAD ligation induced severe HF. There was no significant difference at baseline and at pretreatment in LVDd, FS and MI size between HF-PU and HF groups.
3.2. LV weight, cardiomyocyte diameter and myocardial fibrosis
At experiment endpoint, LV in HF-PU group suffered a significant loss of weight compared with HF group (0.56±0.02 vs. 0.85±0.01 g, P<0.05), but no difference with Nor group (0.57±0.01 g, NS). Histologic analysis is shown in Fig. 1. There was a substantial decrease in myocyte diameter of HF-PU group compared with HF group (20.63±0.54 vs. 26.77±0.48 µm, P<0.05), but no difference with Nor group (19.64±0.39 µm, NS). The percentage of myocardial fibrosis significantly increased in HF-PU group compared with HF and Nor groups (5.37±0.22% vs. 3.75±0.14 and 2.24±0.12%, P<0.05).
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Fig. 1. (a) Hematoxylin-Eosin-stained sections of the non-infarcted LV myocardium demonstrating myocyte diameter (original magnificationx400). Normalization of myocyte diameter is evident after partial unloading. (b) Picrosirius red-stained sections of the non-infarcted LV myocardium demonstrating myocardial fibrosis (original magnificationx200). The percentage of myocardial fibrosis increased after partial unloading. All values are expressed as the mean±S.E.M. *P<0.05 vs. Nor group;  P<0.05 vs. HF group.
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3.3. Contraction of isolated papillary muscle
As shown in Fig. 2, there was no difference in DT between HF-PU and Nor group (0.16±0.09 vs. 0.21±0.02 g/mm2, NS), but significant improvement compared with HF group (0.02±0.01 g/mm2, P<0.01).
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Fig. 2. Developed tension of posterior papillary muscle. All values are expressed as the mean±S.E.M. *P<0.01 vs. Nor group; P<0.01 vs. HF group.
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3.4. Expression of mRNAs
LV mRNA expressions of BNP, SERCA2a, β1 and β2-AR are shown in Fig. 3. BNP increased significantly in HF group compared with Nor group, but this expression of BNP was normalized after unloading in HF-PU group. In HF group, mRNA expressions of SERCA2a, β1 and β2-AR decreased significantly compared with Nor group. After two weeks unloading, expressions of SERCA2a, β1 and β2-AR were normalized in HF-PU group.
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Fig. 3. Expressions of BNP, SERCA2a, β1 and β2-AR mRNA in non-infarcted myocardium. BNP, brain natriuretic peptide; SERCA2a, saco(endo)plasmic reticulum Ca2+-ATPase2a; AR, adrenergic receptor. All values are expressed as the mean±S.E.M. *P<0.05 vs. Nor group; P<0.05 vs. HF group.
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4. Discussion
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In the present study, after two weeks' unloading, infarcted LV decreased in weight and myocyte diameter toward normal size. In some patients, LVAD support leads to improvement in global pump function to allow removal of the device. Although long-term outcome was poor in some patients [7], LVAD may play a role of a ‘bridge to recovery’, because of studies demonstrating that circulatory support with an LVAD results in regression of cellular hypertrophy and a tendency toward normalization of myocyte size and shape despite equally advanced cardiomyopathy [1, 8].
LVAD support is also associated with changes in the collagen fibers. Several studies show that myocardial collagen content increases during mechanical unloading above the already abnormal levels observed in the chronic failing state [9, 10]. However, a few studies indicated the opposite results [1, 11]. In our present study, the percentage of myocardial fibrosis significantly increased in HF-PU group compared with HF and Nor groups. One of the possible reasons for increased fibrosis in the HF-PU group may be a decrease in myocyte size by unloading, which would help increase the relative percentage of interstitial collagen content. In addition, the half-life of fibrous tissue turnover is between 80 and 120 days [8], and thus unloading for two weeks may not be sufficient time to allow for significant alteration in fibrosis deposition or resorption.
We examined the BNP mRNA expression in LV tissue. As a result, increased BNP mRNA expression was found in HF group. In HF-PU group, the BNP level was lower than that in HF group and normalized. BNP is a cardiac neurohormone secreted from the ventricles in response to cardiomyocyte stress and stretch. Bruggink et al. [12] have shown that BNP mRNA and protein expression significantly decreased in heart tissue of patients with LVAD support. LVAD reduced the LV pressure and the stretch of the cardiomyocyte and therefore may have a profound effect on the BNP production by the cardiomyocyte.
To monitor LV function recovery after unloading, we also examined the DT of posterior papillary muscle. In the present study, we found that DT was significantly increased in HF-PU group compared with HF group, which suggests that the contractility of myocardium in HF was improved after partial unloading. The improvement seen in DT in unloaded infarcted rat hearts may be related to several possible mechanisms, including: 1) increased SERCA2a activity and Ca2+ uptake, or 2) an increased β-AR signaling system in myocardium. Myocardial contractile dysfunction in HF of either etiology has been linked to alteration of Ca2+ cycling and downregulated gene expression of SERCA2a appears to correlate with contractile dysfunction. In this study, SERCA2a mRNA expression significantly decreased in HF group compared with Nor group. After two weeks' unloading, SERCA2a mRNA expression was nearly normalized in HF-PU group. Takaseya et al. [13] demonstrated that mechanical unloading by heterotopic transplantation of the hearts with doxorubicin-induced cardiomyopathy increased SERCA2a, which resulted in improved contractility and intracellular free Ca2+ level dynamic in rats.
We found that partial unloading can restore myocardial β1 and β2-AR mRNA expression to near normal in HF-PU group. Ogletree-Hughes et al. [14] demonstrated that by mechanically supporting the failing human hearts with an LVAD can reverse the downregulation of β-ARs and restore the ability of cardiac muscle to respond to inotropic stimulation by the sympathetic nervous system. In our previous study [5], only β2-AR mRNA expression was normalized in failing rat hearts under complete unloading. Nakahara et al. [15] also demonstrated that LV mechanical unloading (complete unloading model) restored β2-AR, but not β1-AR mRNA expression in the failing rat hearts. However, the mechanism(s) of this difference are not clear. Appropriate loading may contribute to the improvement of both β1 and β2-AR in partial unloading of failing rat hearts. Further investigations are needed to focus on the difference of β-AR mRNA expression in these two models. Moreover, we only examined for two weeks' partial unloading, additional experiments will be required to investigate longer duration.
In summary, partial unloading regressed myocardial hypertrophy, reversed contractile dysfunction of the papillary muscle and normalized the BNP, SERCA2a, β1 and β2-AR mRNA expressions in failing rat hearts. Appropriate loading may contribute to recovery of LV function toward ‘bridge to recovery’ by a LVAD.
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Acknowledgements
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The authors thank Dr. Tsukashita, Dr. Yoshikawa, Dr. Muranaka and Dr. Nishina for their technical assist and Ms. Kataoka and Ms. Kominami for their histologic studies and biochemical analysis. We aslo thank Ms. Ishii and Ms. Yamamot for their excellent secretarial work.
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Abstract
1. Introduction
