Altered melusin pathways involved in cardiac remodeling following acute myocardial infarction

Cardiovascular Pathology 21 (2012) 105 – 111

Original Article

Altered melusin pathways involved in cardiac remodeling following acute myocardial infarction Rong Gu, Di Zheng, Jian Bai, Jun Xie, Qing Dai, Biao Xu⁎ Department of Cardiology, Drum Tower Hospital, Nanjing University Medical School, Nanjing 210008, China Received 13 January 2010; received in revised form 1 February 2011; accepted 11 March 2011

Abstract Background: Melusin, a muscle-specific integrin-linked protein, has been reported to be a biomechanical sensor and to protect the heart from pressure overload. In the present study, we investigated the possible role that melusin plays during cardiac remodeling after myocardial infarction (MI). Methods: We constructed a heart failure model of rats induced by left anterior descending coronary artery ligation. At different time points (1, 2, 3, 4, 6, and 8 weeks) following the operation, cardiac function was monitored by echocardiography and hemodynamic assessment; cardiac morphology was measured using hematoxylin–eosin-stained sections. Melusin expression, as well as pAkt, Akt, and one of the Rho small GTPase family members, CDC42, was determined dynamically by Western blotting analysis during the postinfarction cardiac remodeling. Results: Progressive increase in left ventricular (LV) end-systolic dimension and LV end-diastolic dimension and decrease in percent LV fractional shortening (%FS) and LVdp/dtmax demonstrated gradually deteriorated cardiac function in rats following MI operation. Morphological analysis revealed cardiac remodeling in MI animals, including increased LV diameter and decreased border zone thickness. We also showed a dynamic change in melusin during heart failure progression; it had an initial decline which was evident at 3 weeks and increased subsequently, reaching peak levels at 6 weeks. This dynamic change in melusin was significantly correlated with %FS and LVdp/dtmax. p-Akt/Akt and CDC42 protein expression was correlated with melusin content. Conclusions: The altered melusin pathways and CDC42 parallel the cardiac function progression during cardiac remodeling post-MI. The dynamic change of them during this procedure may represent an important molecular mechanism underlying postinfarction cardiac remodeling and provide potential therapeutic targets. © 2012 Elsevier Inc. All rights reserved. Keywords: Melusin; CDC42; Cardiac remodeling; Myocardial infarction; Heart failure

1. Introduction Ischemic heart disease, in particular myocardial infarction (MI), is the leading cause of heart failure in the world now [1]. Myocardial infarction may initiate a process known as cardiac remodeling, which is a dynamic progressing process believed to be a compensatory mechanism to maintain

This work was supported in part by grants from the National Natural Science Foundation of China (Research Grant 81070195) and from the Jiangsu Key Laboratory for Molecular Medicine, Nanjing University (Research Grant 2008). ⁎ Corresponding author. Department of Cardiology, Affiliated Drum Tower Hospital, Nanjing University Medical School, Nanjing 210008, China. Tel.: +86 25 831 052 05; fax: +86 25-833-0 80-59. E-mail address: [email protected] (B. Xu). 1054-8807/11/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.carpath.2011.03.002

cardiac output initially but contribute to the development of heart failure eventually [2]. A cascade of biochemical signaling pathways has been reported to modulate this process [3,4]; however, the mechanism underlying this definite regulation of cardiac remodeling is believed to be complex and remains largely unknown. We know that prevention or attenuation of cardiac remodeling is the main target to prevent heart failure, so better addressing the signaling pathways regulating the progress is of definite importance to develop novel strategies. Melusin, a muscle-specific integrin β1-interacting protein, is expressed in skeletal and cardiac muscles [5,6]. In physiological conditions, melusin-null mice showed normal anatomy structure and cardiac function, while an abnormal cardiac remodeling was exhibited following pressure overload which led to dilated cardiomyopathy and contractile

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dysfunction [6]. On the other hand, overexpression of melusin in hearts of transgenic mice induced left ventricular (LV) hypertrophy in basal conditions, while there was resistance towards heart failure in response to long-standing pressure overload [7]. The mRNA and protein expression of melusin was diminished in human aortic stenosis hearts, while pAKT/AKT was paralleled with its content [8]. Under mechanical stress, melusin, as well as many other proteins which were all associated with integrin, triggers intracellular signaling cascade executing the hypertrophy program leading to cardiomyocyte hypertrophy [9]. Taken together, these researches demonstrated that melusin is a necessary component in ventricular hypertrophy and should exhibit an important role during cardiac remodeling. CDC42 is a member of the small-molecular-weight GTPbinding proteins of Rho family which plays an important role in regulating cell shape, adhesion, motility, and actin organization in various types of cells [10–12], especially in sarcomere organization of cardiac myocytes [13]. Previous studies have shown that CDC42 can also be activated by integrin as mediating cell spreading [14]. In line with these, we speculate that CDC42 also participates in cardiac remodeling and may have some correlation with melusin during this procedure. Until now, the dynamic alteration of melusin pathway as well as CDC42 expression during cardiac remodeling process following MI remains largely unknown. The purpose of the present study was therefore to investigate the temporal progression of melusin pathways as well as CDC42 in the rat model of MI, which we believe to be important to better understand the regulation mechanism of cardiac remodeling and show some significance to the guidance of heart failure therapy.

ligation of LAD. We confirmed the success of cardiac ischemia by 2,3,4-triphenyltetrazolium chloride dye (TTC; Sigma-Aldrich) staining and hematoxylin–eosin (H–E) staining 3 days after the operation. 2.2. Echocardiography and hemodynamic assessment We monitored the cardiac function using transthoracic echocardiography and hemodynamic assessment at varying intervals (before and 1, 2, 3, 4, 6, and 8 weeks postoperation). After anesthesia as described above and placing on a warm blanket, echocardiography was performed using a cardiovascular ultrasound system (SONOS model 5500, HewlettPackard Co.) with an 8-MHz linear-array transducer. We obtained the long axis and short axis two-dimensional views as well as the M-mode tracings. The measurements of left ventricular end-systolic dimension (LVESD) and end-diastolic dimension (LVEDD) as well as the interventricular septal thickness in diastole (IVSd) and LV posterior wall thickness (LVPWT) were all obtained in the M-mode tracings at the papillary muscle level by an observer blinded to the surgery, which were repeated for at least three consecutive pulsation cycles. The averaged data were used for analysis. Percent LV fractional shortening (%FS) was obtained as follows: %FS ¼ ðLVEDD −LVESDÞ = LVEDD × 100 ð%Þ:

2. Methods

Following echocardiography, a polyethylene catheter (PE 50, Becton-Dickinson) was introduced via the right carotid artery into the left ventricle which connected to a pressure transducer (MPU-0.5, Nihon Kohden, Japan) to obtain the hemodynamic indexes as LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), maximum positive and negative dP/dt (+dP/dtmax, −dP/dtmax), and heart rate (HR). All measurements were performed by an observer blinded to treatment.

2.1. Animal model of acute MI

2.3. Tissue sample preparation

Animal researches that were performed conformed to 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). We constructed an acute MI model of rats (n=8 operation group and n=5 sham operation for different time points) as previously described [15]. Briefly, after intraperitoneal administration of ketamine and diazepam mixture (the concentration of which was 50 and 5 mg/kg, respectively), 200–250-g male SpragueDawley rats aged 8 weeks were incised in the fourth intercostal space under the protection of ventilator (Jiangxi Teli, China). After ligation of the left anterior descending coronary artery (LAD) 1–2 mm below the left atrial appendage, we inflated the lung fully with positive endexpiratory pressure. The chest cavity, muscles, and skin were sutured. The sham-operation group was also set up which followed the same procedure as described above except for

After hemodynamic evaluation, hearts were arrested in diastolic phase by intravenous administration of 2 mol/L KCl. The middle part of the myocardium was sliced into several sections of 5 mm each for histologic analysis and fixed in 4% neural formalin for 24 h at room temperature, followed by embedding in paraffin wax and then slicing into 5-µm slices for subsequent H–E staining. Then we divided the remaining left ventricle of rats into three zones: infarct, peri-infarct (border, 2 mm of myocardium around the infarct area), and remote zones; the remote zone of the left ventricle was snap-frozen in liquid nitrogen and stored at −80°C for subsequent Western blotting analysis. 2.4. Histological analysis Paraffin-embedded slices 5 µm thick were stained with H–E for morphologic examination. The LV diameter was

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measured at the level of the papillary muscle as described [16]. Infarct scar size was expressed as the sum of the epicardial and endocardial scar circumferences divided by the sum of the LV epicardial and endocardial circumferences [17]. 2.5. Protein expression during the cardiac remodeling Following hemodynamic assessment, the heart was frozen in liquid nitrogen until Western blotting analysis. Frozen hearts were homogenized using a Dounce tissue grinder in lysis buffer containing the following composition: 10 mmol/L Tris/HCl (pH 7.2), 150 mmol/L NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate, 5 mmol/L EDTA, 50 mmol/L NaF, 1 mmol/L sodium orthovanadate, 1% (vol/vol) Triton X-100, protease inhibitor cocktail (Roche), and phosphatase inhibitor (Roche). Protein concentrations were quantified by BCA protein assay kit (Pierce). Fifty micrograms protein per sample was separated using SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). After incubation with mouse anti-melusin antibody (1:2000, BD), mouse anti-PKB/Akt antibody (1:250, BD), mouse anti-p-Akt (pS472/pS473) antibody (1:500, BD), rabbit anti-CDC42 antibody (1:1000, Millipore), or mouse anti-GAPDH (1:3000, Kangchen Bio-tech), horseradish-peroxidase-conjugated goat anti-mouse IgG (1:5000, Santa Cruz Biotechnology) or goat anti-rabbit IgG (1:5000, Jackson ImmunoResearch) was used as secondary antibody. Bands were visualized by an enhanced chemiluminescence detection system (Pierce Biotechnology Inc, Rockford, IL, USA). The densitometric analysis of each bands exhibited on BioMax films (Kodak) was conducted using QuantityOne (Bio-Rad) software. Results were presented as the ratio of density values of objective bands to GAPDH band or ratio of pAkt to Akt.

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Table 1 physical characteristics of rats at respective time points post-MI Time Basal Sham MI Week 1 Sham MI Week 2 Sham MI Week 3 Sham MI Week 4 Sham MI Week 6 Sham MI Week 8 Sham MI

Body weight (g)

Heart weight (g)

Heart weight/body weight (‰)

232±5 238±4

0.52±0.03 0.55±0.04

2.25±0.15 2.31±0.19

0±0 41±3

282±8 246±5 ⁎

0.68±0.08 0.72±0.13 ⁎

2.39±0.29 2.83±0.35 ⁎

0±0 40±2

295±9 255±7 ⁎

0.94±0.18 0.83±0.09 ⁎

3.17±0.42 3.23±0.19 ⁎

0±0 46±3

324±10 273±8 ⁎

0.91±0.15 0.86±0.11 ⁎

2.80±0.27 3.13±0.33 ⁎

0±0 42±2

344±11 303±9 ⁎

1.02±0.20 1.12±0.23 ⁎

2.93±0.20 3.71±0.41 ⁎

0±0 44±3

362±10 315±9 ⁎

1.08±0.21 1.19±0.23 ⁎

2.93±0.22 3.77±0.52 ⁎

0±0 43±2

371±12 326±11 ⁎

1.10±0.25 1.12±0.18 ⁎

2.93±0.15 3.46±0.26 ⁎

MI (%) 0±0 0±0

⁎ Pb.05 vs. sham.

obviously higher than control. Gross anatomic inspection of myocardium after TTC staining demonstrated the ischemia of cardiac muscle exhibiting pale color as compared with red-stained viable tissue. Only infarct scar size larger than 40% in different groups was included in the consequent analysis (also exclusion of those that died during the operation or postoperation procedure, n=6 operation group and n=5 sham-operation for different time points analyzed subsequently). The H–E-stained sections of myocardium revealed evidence for increased LV diameter and decreased peri-infarct (border) zone thickness in operation animals as compared with controls. Progressive increase in LV diameter was also noticed at 1, 4, and 8 weeks post-MI (Fig. 1).

2.6. Statistical analysis 3.2. Cardiac function All results were expressed as mean±S.E.M. Comparisons of data among different time points were performed by analysis of variance with Dunnett's posttest. Associations between two variables were examined by linear regression analysis. All data were analyzed using SPSS 13.0 software (SPSS, Inc, Chicago, IL, USA). Statistical significance was defined as Pb.05 (two-tailed).

3. Results 3.1. Cardiac morphology As shown in Table 1, the body weight of rats at respective time points postoperation is lower than that of sham-operation group, while heart weight/body weight is

As shown in Fig. 2, systolic function, evaluated by %FS, showed an obvious deterioration compared with control. The difference between the two groups was already notable at 1 week, and it showed a mild fluctuation thereafter. In parallel with this, LVESD and LVEDD were both higher in different time point as compared with control. There is no significant difference between the two groups in IVSd or LVPWT. Hemodynamic analysis also confirmed the deterioration in the operation group. An increase in the LVEDP and a decrease in LVSP were seen in operation animals (data not shown). A lower maximum +dP/dt and a greater maximum −dP/dt than the control animals were also notified between the two groups, while no difference was seen in HR.

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Fig. 1. Cardiac morphology post-MI. The H–E-stained sections of myocardium post-MI revealed increased LV diameter and decreased peri-infarct (border) zone thickness in operation animals as compared with controls. Progressive increase in LV diameter were also noticed at 1, 4, and 8 weeks post-MI.

Fig. 2. Cardiac function postoperation monitored by echocardiography and hemodynamic analysis. Serial changes are shown in cardiac function following operation, indicating a progressive increase in LVESD and LVEDD and a progressive decrease in FS% and LVdp/dtmax in rat with MI. (A) FS%. (B) LVdp/dtmax. (C) LVESD. (D) LVEDD. n=6 operation group and n=5 sham-operation for per different time points. ∗Pb.05 vs. sham-operation group.

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Fig. 3. Protein expression of melusin pathway and CDC42 at respective time points post-MI. (A) Western blotting showing melusin expression. (B) Western blotting showing pAKT/AKT expression. (C) Western blotting showing CDC42 expression. Dynamic changes of melusin and CDC42 were observed during this process. The protein expression decreased in the first 3 weeks after the infarction and then increased until 6 weeks to decrease again afterwards, and the expression was even lower than baseline at 8 weeks. Its downstream phosphorylation of Akt had a similar expression pattern with melusin. GAPDH expression was also shown as a housekeeping protein. n: sham-operation control. n=6 operation group and n=5 sham-operation for different time points. ∗Pb.05 vs. shamoperation group. ∗∗Pb.01 vs. sham-operation group.

3.3. Protein expression of melusin pathway and CDC42 at different time points following the operation and their correlation with cardiac function To investigate melusin pathway and CDC42 expression at different time points (1, 2, 3, 4, 6, and 8 weeks) following MI operation, we quantified the protein expression using

Western blotting. As shown in Fig. 3, melusin and CDC42 expression exhibited dynamic changes during this process: decreased in the first 3 weeks after the infarction and then increased while peaking at 6 weeks. Afterwards, the expression began to decrease, and it was even lower than baseline at 8 weeks. We also noticed that there is a modest fluctuation at 3 weeks. The dynamic change of melusin was

Fig. 4. Melusin expression was significantly correlated with LV function during LV remodeling procedure post-MI. pAkt/Akt and CDC42 were also correlated with melusin content. (A and B) Dynamic expression of melusin was positively correlated with FS% (r=0.332, Pb.05) and LVdp/dtmax change (r=0.684, Pb.01). (C, D) Protein expressions of pAkt/Akt and CDC42 were both positively correlated with melusin content (r=0.907, Pb.01; r=0.837, Pb.01). n=36.

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correlated with cardiac function (such as FS% and LVdp/ dtmax) (r=0.332, Pb.05; r=0.684, Pb.01; Fig. 4A, B) but unexpectedly not with LV diameter (LVEDD and LVESD) evolution (data not shown). The pAkt/Akt, which represents the activity of melusin as well as the member of Rho small GTPase family CDC42, was also significantly correlated with melusin expression level (r=0.907, Pb.01; r=0.837, Pb.01; Fig. 4C, D).

4. Discussion Ventricular remodeling is a dynamic process which includes ventricular wall thinning [18], cardiomyocyte lengthening [19], myocyte hypertrophy or necrosis [20], and collagen accumulation [21]. The initial remodeling phase is thought to be beneficial, leading to reparation of the injured zone and maintenance of cardiac function. As remodeling progresses, the ejection fraction begins to decline and heart function deteriorates. Therapies should not only aim at symptom but also target underlying mechanisms so as to delay or reverse the disease progression. However, the exact story of all pathways involved in remodeling process is still uncertain. Melusin pathway plays an important role in integrinmediated mechanochemical signal transduction in the heart [7], the altered expression of which is believed to be paralleled with cardiac function [8]. CDC42 is a member of the Rho small GTPase family and mediates cell spreading and hypertrophy which was also activated by integrin. Both as the downstream effectors of integrin and transduce extracellular mechanical signals, we hypothesized that melusin and CDC42 may participate in cardiac remodeling and that there may be some correlation between them. However, previous studies just gave us the general opinion of their physiologic role, while neither the evolving pattern nor their relationship with progressive alteration of heart morphology or cardiac function has been systematically studied. Therefore, in the present study, we overcome the shortcomings by monitoring cardiac function and studying the expression of melusin, CDC42, as well as pAkt/Akt during cardiac remodeling using a dynamic perspective. It has been proposed that the stretch and tension exerted on the ventricular wall have caused the expression alteration of melusin pathway and CDC42. As described before, the process of ventricular remodeling post-MI began only a few hours after infarction and continued to progress [22]. The infarction expansion, wall thinning, as well as ventricular dilatation elevated the mural stress which activated intracellular signaling including melusin and CDC42 through mechanoreceptor integrin. The activated intracellular signaling induced hypertrophy of myocytes to compensate for the altered wall stress, and the cardiac function was maintained transiently. But as the ventricle continued to distend and change globally, the intracellular signaling cannot balance the mural stress and maintain the cardiac function. So, in our

experiment, we can see the dynamic alteration of melusin and CDC42 expression during this procedure: increased at first and peaking at 6 weeks to induce cardiomyocyte hypertrophy and reduce infarction expansion to maintain pulsation. Their expression decreased thereafter upon entering the decompensated period, while it is obviously lower as compared with control at 8 weeks. Since heart failure is frequently accompanied by increased fibrosis [22], we speculated this as one of the possible causes of reduced melusin level in our samples as increased scar tissue reduces the muscle component and consequently melusin level. In accordance with this, the cardiac function index FS% and LVdp/dtmax were relatively maintained from 1 week until 6 weeks postsurgery, while they were obviously exacerbated at 8 weeks. As we have expected, there is significant correlation between CDC42 and melusin expression during this process. It was also confirmed by our results that melusin content was significantly correlated with cardiac function indexes FS% and LVdp/dtmax. We also investigated Akt phosphorylation in this study. As we know, Akt is a serine/threonine protein kinase which has many cellular functions such as antiapoptosis, stimulating migration, promoting protein synthesis, and regulating angiogenesis [23–25]. Apoptosis is the key determinant to unfavorable ventricular remodeling [26]. Also, activated Akt is critical to increase cell size, leading to cardiac hypertrophy. Intriguingly, Akt exerts a dual function, that is, promoting physiological hypertrophy whilst suppressing pathological hypertrophy of heart [27]. In the present study, pAkt/Akt parallels with melusin content during cardiac remodeling, which was consistent with previous results [8]. Unexpectedly, the expression of melusin pathway and CDC42 expression has a modest decrease at 3 weeks postMI. We also noticed that, in our experiment, FS% and LVdp/ dtmax were relatively lower at 3 weeks compared with those of other time points from 1 week onward. This may be due to the relative great amount of cardiomyocyte death in the acute phase of MI while the compensative mechanism has not played its role fully yet, or may represent a particular phase during cardiac remodeling with an unknown mechanism. We should pay more attention to this and widen our samples in our future studies. In conclusion, we have shown the dynamic changes of melusin pathway and CDC42 involved in cardiac remodeling post-MI over an 8-week period. It is suggested that the melusin pathway was involved in cardiac remodeling postMI and that its overexpression strategy at a relatively early phase (take 6 weeks for example) may be of benefit to prevent cardiac remodeling evolution.

5. Summary In this MS, we reported a dynamic change that took place in melusin during heart failure progression, and this change was significantly correlated with FS% and LVdp/dtmax. p-

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Akt/Akt and CDC42 protein expression was correlated with melusin content. We therefore conclude that the dynamic change of melusin pathways and CDC42 may represent an important molecular mechanism underlying postinfarction cardiac remodeling. Acknowledgment We thank Dr. Di Xu for expert technical assistance in performing the experiments outlined here. References [1] Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and the acute coronary syndromes, II. N Engl J Med 1992;326:310–8. [2] Sutton MG, Sharpe N. Left ventricular remodeling after myocardial infarction: pathophysiology and therapy. Circulation 2000;101:2981–8. [3] Olivetti G, Capasso JM, Meggs LG, Sonnenblick EH, Anversa P. Cellular basis of chronic ventricular remodeling after myocardial infarction in rats. Circ Res 1991;68:856–69. [4] Yamazaki T, Komuro I, Yazaki Y. Molecular mechanism of cardiac cellular hypertrophy by mechanical stress. J Mol Cell Cardiol 1995;27: 133–40. [5] Brancaccio M, Fratta L, Notte A, Hirsch E, Poulet R, Guazzone S, De Acetis M, Vecchione C, Marino G, Altruda F, Silengo L, Tarone G, Lembo G. Melusin, a muscle-specific integrin β1-interacting protein, is required to prevent cardiac failure in response to chronic pressure overload. Nat Med 2003;9(1):68–75. [6] Brancaccio M, Guazzone S, Menini N, Sibona E, Hirsch E, De Andrea M, Rocchi M, Altruda F, Tarone G, Silengo L. Melusin is a new muscle-specific interactor for β1 integrin cytoplasmic domain. J Biol Chem 1999;274(41):29282–8. [7] De Acetis M, Notte A, Accornero F, Selvetella G, Brancaccio M, Vecchione C, Sbroggio M, Collino F, Pacchioni B, Lanfranchi G, Aretini A, Ferretti R, Maffei A, Altruda F, Silengo L, Tarone G, Lembo G. Cardiac overexpression of melusin protects from dilated cardiomyopathy due to long-standing pressure overload. Circ Res 2005;96: 1087–94. [8] Brokat S, Thomas J, Herda LR, Knosalla C, Pregla R, Brancaccio M, Accornero F, Tarone G, Hetzer R, Regitz-Zagrosek V. Altered melusin expression in the hearts of aortic stenosis patients. Eur J Heart Failure 2007;9:568–73. [9] Brancaccio M, Hirsch E, Notte A, Selvetella G, Lembo G, Tarone G. Integrin signalling: the tug-of-war in heart hypertrophy. Cardiovasc Res 2006;70:422–33. [10] Subauste MC, Von Herrath M, Benard V, Chamberlain CE, Chuang TH, Chu K, Bokoch GM, Hahn KM. Rho family proteins modulate rapid apoptosis induced by cytotoxic T lymphocytes and Fas. J Biol Chem 2000;275:9725–33. [11] Bazenet CE, Mota MA, Rubin LL. The small GTP-binding protein Cdc42 is required for nerve growth factor withdrawal-induced neuronal death. Proc Nat Acad Sci 1998;95:3984–9.

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