ARTICLE IN PRESS doi:10.1510/icvts.2007.165563
Interactive CardioVascular and Thoracic Surgery 7 (2008) 27–31 www.icvts.org
Work in progress report - Assisted circulation
Partial left ventricular unloading reverses contractile dysfunction and helps recover gene expressions in failing rat hearts夞 Jian Wang, Akira Marui, Tadashi Ikeda, 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
Abstract 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 Ca2q -ATPase (SERCA2a), b1 and b2 -adrenergic receptors (b1 and b2-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, b1 and b2-AR) expressions of failing rat hearts. 䊚 2008 Published by European Association for Cardio-Thoracic Surgery. All rights reserved. Keywords: Heart failure; Heart-assist device; Myocardial contraction; Remodeling; Genes
1. Introduction Recently, myocardial recovery due to ventricular unloading and workload reduction with a left ventricular assist device (LVAD) has been noted w1x. 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 w2x. 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 w3x, 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. w4x 夞 Presented at the 21st Annual Meeting of the European Association for Cardio-thoracic Surgery, Geneva, Switzerland, September 16–19, 2007. *Corresponding author. Tel.: q81-75-751-3784; fax: q81-75-751-4960. E-mail address:
[email protected] (M. Komeda). 䊚 2008 Published by European Association for Cardio-Thoracic Surgery
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. 2. Materials and methods 2.1. Induction of heart failure (HF) and LV functional assessment Lewis rats weighing 160–200 g (Japan SLC Inc., Hamamatsu, 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 w5x. 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 (LVDdyLVSd)yLVDd=100 and used as an index of LV systolic
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function. Infarct size (%) was calculated as (infarct length in LV diastolic phase)y(LV diastolic circumference)=100. 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, ns10), the infarcted hearts and lungs were heterotopically transplanted into the abdomen of recipient rats; in HF group (ns10), the hearts were not transplanted. Normal Lewis rats were as control group (Nor group, ns8). After two weeks, the transplanted infarcted heart, nontransplanted 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 8C and bubbled with 95% O2 and 5% CO2. The papillary muscle was stimulated at 1 Hz with impulses of 5-ms duration and fivemillisecond 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 y80 8C 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 (magnification=400) as before w5x. Other transverse sections were stained with Picrosirius red w5x. In each section, 20 separate parts of the remote area were scanned under microscopy (magnification=200), and the images were analyzed using an IPLab姠 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.
scribed, and amplified with the ABI PRISM姠 7700 sequence detector (Applied Biosystems). Polymerase chain reaction (PCR) conditions were 40 cycles of denaturing at 94 8C for 20 s and primer annealingyextension at 62 8C for 60 s. The PCR sequences of brain natriuretic peptide (BNP), sarco(endo)plasmic reticulum Ca2q-ATPase2a (SERCA2a), b1 and b2-adrenergic receptors (AR) were previously reported in our studies w5, 6x. 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䊛 for Windows version 5.0 (SAS Institute Inc., Cary, NC). A value of P-0.05 was considered significant. 3. Results 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 mm, P-0.05), but no difference with Nor group (19.64"0.39 mm, 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). 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 gymm2, NS), but significant improvement compared with HF group (0.02"0.01 gymm2, P-0.01). Table 1 Baseline echocardiographic data LVDd (mm)
FS (%)
Infarct size (%)
6.6"0.3 9.4"0.1* 9.6"0.2*
50.8"2.2 18.1"0.4* 18.2"0.3*
0 31.2"0.5* 31.5"0.4*
2.5. Analysis of mRNA expression
Nor group HF group HF-PU group
Total mRNA was prepared from the frozen LV pieces with TRIzol (Life Technologies Inc.) reagent, reverse-tran-
LVDd, left ventricular end diastolic dimension; FS, fractional shortening. *P-0.01 vs. Nor group.
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Fig. 1. (a) Hematoxylin-Eosin-stained sections of the non-infarcted LV myocardium demonstrating myocyte diameter (original magnification=400). Normalization of myocyte diameter is evident after partial unloading. (b) Picrosirius red-stained sections of the non-infarcted LV myocardium demonstrating myocardial fibrosis (original magnification=200). 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. Fig. 3. Expressions of BNP, SERCA2a, b1 and b2-AR mRNA in non-infarcted myocardium. BNP, brain natriuretic peptide; SERCA2a, saco(endo)plasmic reticulum Ca2q-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.
4. Discussion
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.
3.4. Expression of mRNAs LV mRNA expressions of BNP, SERCA2a, b1 and b2-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, b1 and b2-AR decreased significantly compared with Nor group. After two weeks unloading, expressions of SERCA2a, b1 and b2-AR were normalized in HF-PU group.
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 w7x, 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 w1, 8x. 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 w9, 10x. However, a few studies indicated the opposite results w1, 11x. 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 w8x, and thus unloading for
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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. w12x 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 Ca2q uptake, or 2) an increased b-AR signaling system in myocardium. Myocardial contractile dysfunction in HF of either etiology has been linked to alteration of Ca2q 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. w13x demonstrated that mechanical unloading by heterotopic transplantation of the hearts with doxorubicin-induced cardiomyopathy increased SERCA2a, which resulted in improved contractility and intracellular free Ca2q level dynamic in rats. We found that partial unloading can restore myocardial b1 and b2-AR mRNA expression to near normal in HF-PU group. Ogletree-Hughes et al. w14x demonstrated that by mechanically supporting the failing human hearts with an LVAD can reverse the downregulation of b-ARs and restore the ability of cardiac muscle to respond to inotropic stimulation by the sympathetic nervous system. In our previous study w5x, only b2-AR mRNA expression was normalized in failing rat hearts under complete unloading. Nakahara et al. w15x also demonstrated that LV mechanical unloading (complete unloading model) restored b2-AR, but not b1-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 b1 and b2-AR in partial unloading of failing rat hearts. Further investigations are needed to focus on the difference of bAR 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, b1 and b2-AR mRNA expressions in failing rat hearts. Appropriate loading may contribute to recovery of LV function toward ‘bridge to recovery’ by a LVAD.
Acknowledgments 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. References w1x Xydas S, Rosen RS, Ng C, Mercando M, Cohen J, DiTullio M, Magnano A, Marboe CC, Mancini DM, Naka Y, Oz MC, Maybaum S. Mechanical unloading leads to echocardiographic, electrocardiographic, neurohormonal, and histologic recovery. J Heart Lung Transplant 2006;25:7–15. w2x Barbone A, Holmes JW, Heerdt PM, The’ AH, Naka Y, Joshi N, Daines M, Marks AR, Oz MC, Burkhoff D. Comparison of right and left ventricular responses to left ventricular assist device support in patients with severe heart failure: a primary role of mechanical unloading underlying reverse remodeling. Circulation 2001;104:670–675. w3x Ono K, Lindsey ES. Improvement technique of heart transplantation in rats. J Thorac Cardiovasc Surg 1969;57:225–229. w4x Mizuno T, Weisel RD, Li RK. Reloading the heart: a new animal model of left ventricular assist device removal. J Thorac Cardiovasc Surg 2005;130:99–106. w5x Tsuneyoshi H, Oriyahan W, Kanemitsu H, Shiina R, Nishina T, Ikeda T, Nishimura K, Komeda M. Heterotopic transplantation of the failing rat heart as a model of left ventricular mechanical unloading toward recovery. ASAIO J 2005;51:116–120. w6x Tsuneyoshi H, Oriyanhan W, Kanemitsu H, Shiina R, Nishina T, Matsuoka S, Ikeda T, Komeda M. Does the b2 -agonist clenbuterol help to maintain myocardial potential to recover during mechanical unloading? Circulation 2005;112wsuppl Ix:I-51–I-56. w7x Hetzer R, Mueller J, Weng J, Wallukat G, Spiegelsberger S, Loebe M. Cardiac recovery in dilated cardiomyopathy by unloading with a left ventricular assist device. Ann Thorac Surg 1999;68:742–749. w8x Madigan JD, Barbone A, Choudhri AF, Morales DL, Cai B, Oz MC, Burkhoff D. Time course of reverse remodeling of the left ventricle during support with a left ventricular assist device. J Thorac Cardiovasc Surg 2001;121:902–908. w9x McCarthy PM, Nakatani S, Vargo R, Kottke-Marchant K, Harasaki H, James KB, Savage RM, Thomas JD. Structural and left ventricular histologic changes after implantable LVAD insertion. Ann Thorac Surg 1995;59:609–613. w10x Li YY, Feng Y, McTiernan CF, Pei M, Moravec CS, Wang P, Rosenblum W, Kormos RL, Feldman AM. Downregulation of matrix metalloproteinases and reduction in collagen damage in the failing human heart after support with left ventricular assist devices. Circulation 2001;104:1147– 1152. w11x Bruckner BA, Stetson SJ, Farmer JA, Radovancevic B, Frazier OH, Noon GP, Entman ML, Torre-Amione G, Youker KA. The implication for cardiac recovery of left ventricular assist device support on myocardial collagen content. Am J Surg 2000;180:498–501. w12x Bruggink AH, de Jonge N, van Oosterhout MF, Van Wichen DF, de Koning E, Lahpor JR, Kemperman H, Gmelig-Meyling FH, de Weger RA. Brain natriuretic peptide is produced both by cardiomyocytes and cells infiltrating the heart in patients with severe heart failure supported by a left ventricular assist device. J Heart Lung Transplant 2006;25:174– 180. w13x Tkasaya T, Ishimatsu M, Tayama E, Nishi A, Akasu T, Aoyagi S. Mechanical unloading improves intracellular Ca2q regulation in rats with doxorubicin-induced cardiomyopathy. J Am Coll Cardiol 2004;44:2239–2246. w14x Ogletree-Hughes ML, Stull LB, Sweet WE, Smedira NG, McCarthy PM, Moravec CS. Mechanical unloading restores b-adrenergic responsiveness and reverses receptor downregulation in the failing human heart. Circulation 2001;104:881–886. w15x Nakahara K, Horimoto H, Nakai Y, Mieno S, Normura Y, Sasaki S. Left ventricular mechanical unloading restores beta-2-adrenergic receptor mRNA expression and decreases susceptibility to ischemia and reperfusion in the failing heart. Eur Surg Res 2003;35:108–114.
Conference discussion Dr. J. Horisberger (Lausanne, Switzerland): I think it’s always interesting to see if there is a way of weaning assisted patients off of a ventricular device. Did you do any comparisons to a completely unloaded heart?
ARTICLE IN PRESS J. Wang et al. / Interactive CardioVascular and Thoracic Surgery 7 (2008) 27–31 Dr. Wang: Sorry? Dr. Horisberger: You have a control with partial unloading. Would you think it would be a good idea to do partial unloading with a fully unloaded heart to see the difference?
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Dr. Wang: Yes. We held a study of full unloading on the failing rat hearts, but in that study we only found that the b2-adrenergic receptor mRNA normalized but not the b1, so I think this is the difference, and further studies are needed to focus on the differences between the two models.
