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Syndecan-4 deficiency accelerates the transition from compensated hypertrophy to heart failure following pressure overload
Cardiovascular Pathology 28 (2017) 74–79
Contents lists available at ScienceDirect
Cardiovascular Pathology
Original Article
Syndecan-4 deficiency accelerates the transition from compensated hypertrophy to heart failure following pressure overload Guannan Li, Jun Xie, Jianzhou Chen, Ran Li, Han Wu, Xinlin Zhang, Qinhua Chen, Rong Gu ⁎, Biao Xu ⁎ Department of Cardiology, Affiliated Drum Tower Hospital, Nanjing University Medical School, Nanjing, China
a r t i c l e
i n f o
Article history: Received 3 September 2016 Received in revised form 27 March 2017 Accepted 27 March 2017 Available online xxxx Keywords: Syndecan-4 Angiogenesis Cardiac hypertrophy Heart failure PKCα
a b s t r a c t Increasing evidence suggests that a mismatch between angiogenesis and myocardial growth contributes to the transition from adaptive cardiac hypertrophy to heart failure following pressure overload. Syndecan-4 is a transmembrane proteoglycan that binds to growth factors and extracellular matrix proteins and is critical in focal adhesion formation. However, its effects on coronary angiogenesis during pressure overload-induced heart failure have not been studied. Here, we hypothesize that syndecan-4 modulates cardiac remodeling in response to pressure overload through its ability to regulate adaptive angiogenesis. Syndecan-4 knockout (syndecan-4 KO) and wild-type (WT) mice were subjected to pressure overload induced by transverse aortic constriction (TAC). Syndecan-4 KO mice exhibited reduced capillary density, attenuated cardiomyocyte size, and worsened left ventricular cardiac function after TAC surgery compared with WT mice. Moreover, syndecan-4 KO mice showed a significant decrease in protein kinase C alpha expression. Our data suggest that syndecan-4 is essential for the compensated hypertrophy and the maintenance of cardiac function during the process of heart failure following pressure overload. © 2017 Published by Elsevier Inc.
1. Introduction Heart failure is a common consequence of most heart diseases and is one of the leading causes of morbidity and mortality worldwide [1–3]. Cardiac hypertrophy is an adaptive response to preserve cardiac function in several forms of increased afterload, such as aortic stenosis and elevated blood pressure [4]. However, sustained afterload eventually leads to a state of decompensated hypertrophy resulting in cardiac dilation and contractile dysfunction [5,6]. During the development of cardiac hypertrophy, a mismatch occurs between the number of capillaries and the size of cardiomyocytes, contributing to the progression from compensated hypertrophy to contractile dysfunction and heart failure [7,8]. Related studies have observed the reduction of cardiac vessel density during pathological hypertrophy in pressure overload-induced mice models of heart failure [9–11]. In addition, stimulation of angiogenesis could improve cardiac function and delay the progress of heart failure [12–14]. Several recent studies indicate a relationship between
This work was supported by grants from the National Natural Science Foundation of China (81200148, 81270281, and 8120092), Jiangsu Province Special Program of Medical Science (BL2012014), the Peak of Six Personnel in Jiangsu Province (2013-WSN-008), Funds for Distinguished Young Scientists in Nanjing (JQX13006), Key Program of Science Foundation in Nanjing (ZKX13023), and the funds from Nanjing Medical Science and Technique Development Foundation (QRX11158). ⁎ Corresponding authors at: Department of Cardiology, Affiliated Drum Tower Hospital, Nanjing University Medical School, Zhongshan Road, Nanjing 210008, China. Tel./fax: +86 25 68182812. E-mail addresses: [email protected] (R. Gu), [email protected] (B. Xu). http://dx.doi.org/10.1016/j.carpath.2017.03.008 1054-8807/© 2017 Published by Elsevier Inc.
cardiac angiogenesis, cardiac hypertrophy, and cardiac function [8]. However, the molecular mechanisms involved in the regulation of cardiac angiogenesis in pressure overload-induced heart failure are not understood. Syndecan-4 is a transmembrane proteoglycan that plays an important role in a variety of cellular functions, including cell proliferation, cell–matrix and cell–cell adhesion [15–17]. Syndecan-4 regulates a wide range of pathophysiological processes in cardiovascular diseases, including cardiac myofibroblast differentiation and endothelial alignment in atherosclerosis [17]. Previous work in our laboratory demonstrated that sustained syndecan-4 overexpression in the myocardium promotes neovascularization and suppresses inflammation and fibrosis in the rat model of myocardial infarction, resulting in improvements in cardiac function and remodeling [18]. However, it is unclear if syndecan-4 mediates angiogenesis in pressure overload-induced cardiac dysfunction. Here, we hypothesize that syndecan-4 deficiency affects the transition from cardiac adaptive hypertrophy to heart failure during pressure overload by regulating cardiac angiogenesis. This is important to understand the relationship between angiogenesis and cardiac hypertrophy, and to help guide therapies for heart failure. 2. Materials and methods All experimental procedures and research were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (National Institutes of
G. Li et al. / Cardiovascular Pathology 28 (2017) 74–79
Health publication No. 8023, revised 1978), and approved by the Institutional Ethics Committee of Nanjing Drum Tower Hospital.
2.1. Experimental animals Syndecan-4 KO mice, backcrossed more than 10 times into the C57BL/6 background, were obtained from the Center for Animal Resources and Development at Kumamoto University (Japan). Twelveweek-old male syndecan-4 KO mice and age-matched male wild-type (WT) mice on a C57BL/6 background were used in the present study. All WT mice were obtained from the Model Animal Research Center of Nanjing University.
2.2. Experimental protocols Pressure overload was induced by transverse aortic constriction (TAC), as described previously [19]. Briefly, mice were anesthetized with 10% chloral hydrate (0.3 ml/100 g) intraperitoneally. The chest was opened and the thoracic aorta was identified after blunt dissection through the intercostal muscles. A 6-0 silk suture was placed around the transverse aorta and tied around a 26-gauge blunt needle, which was then removed. After banding, the chest and overlying skin were closed.
2.3. Echocardiographic measurements Cardiac function was evaluated by serial transthoracic echocardiography at 0, 7, 14, and 28 days after TAC. The measurements were performed by an observer blinded to the treatment. Studies were recorded with a dynamic focused 30-MHz probe echocardiography machine (Visual Sonics Vevo2100, Toronto, Canada). Briefly, mice were anesthetized using isoflurane gas and transferred to an imaging stage equipped with a warming pad for controlled maintenance of mouse body temperature at 37 °C and a built-in electrocardiography system for monitoring continuous heart rate and respiratory rate. The Mmode measurements of left ventricular (LV) dimensions were averaged from more than 3 cycles. The end-diastolic LV inner diameter (LVIDd), end-systolic LV inner diameter (LVIDs), and end-diastolic interventricular septum thickness (IVSd) as well as LV mass were measured. Percentage of LV fractional shortening (FS) was calculated as follows: %FS= (LVIDd−LVIDs)/LVIDd*100. LV ejection fraction (LVEF) was calculated as follows: %EF=(LVIDd2−LVIDs2)/LVIDd2*100.
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2.5. Western blot analysis The hearts of mice were harvested at day 0 (baseline), day 7, day 14 and day 28 after TAC (n=4/genotype/timepoint). Western blotting analysis was performed and the expression of proteins was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primary antibodies used were anti-CD31(1:1000; Abcam, Cambridge, UK),anti-protein kinase C alpha (PKCα; 1:500; BD), anti-atrial natriuretic factor (anti-ANF; 1:500; BD), anti-synd4(1:500; LSbio,Seattle, WA,USA), and anti-GAPDH (1:1000; Millipore, Billerica, MA, USA). 2.6. Statistical analysis Data are presented as mean±S.D. All data analyses were performed with the use of SPSS 16.0 software. Group differences were analyzed by two-tailed Student's t test or analysis of variance. A value of Pb.05 was considered statistically significant. 3. Results 3.1. Syndecan-4 deficiency exacerbates cardiac dysfunction after pressure overload To induce cardiac hypertrophy, male mice were subjected to TAC surgery. Multiple time points (0, 7, 14, and 28 days) were utilized to examine the gradual transition from a compensated to a decompensated stage of cardiac function (Fig. 1a). No significant difference was found between the two groups at baseline. Serial echocardiographic measurements revealed a progressive decline in the LVEF and FS after TAC surgery both in WT and KO mice (Fig. 1b and c). A significant increase in the LVIDd and LVIDs (Fig. 1d and e) was concurrently observed. However, the reduction of LVEF and FS, and the increase in LVIDd and LVIDs were significantly more pronounced in syndecan-4 KO mice compared with WT mice at 2 and 4 weeks after TAC (Fig. 1b–e). Interestingly, we also found that syndecan-4 KO mice showed a larger diameter in LVIDs than WT mice at day 7 (Fig. 1e), suggesting that the syndecan-4 KO mice had decreased contractile function in the early adaptable stage following TAC. The expression of ANF in the myocardium after TAC was examined by Western blot showed no difference at baseline between the WT and KO groups, but syndecan-4 KO mice showed increased ANF expression from day 7 to day 28 after TAC, compared with WT mice (Fig. 1f). These data indicate that syndecan-4 deficiency exacerbated cardiac dysfunction after pressure overload in mice. 3.2. Syndecan-4 deficiency prevents the development of cardiac hypertrophy
2.4. Histological examination For morphological and immunohistochemical experiments, mice were sacrificed and their hearts were collected at day 0 (baseline), day 7, day 14, and day 28 after TAC (n=4/genotype/timepoint). All hearts were weighed and fixed with formalin, embedded in paraffin, and cut into 4-μm slices for subsequent immunohistochemical histological analysis. The micrographs were then analyzed using the Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, USA). To determine the cross-sectional area (CSA) of a cardiomyocyte, cross sections of the LV area between the apices and bases of the hearts were stained with hematoxylin and eosin, and histological studies were performed. At least 100 cardiomyocytes per heart were measured for myocyte CSA calculation (magnification,×400). To determine the capillary density, tissue sections were stained with anti-CD31 antibody (1:200; BD, Franklin Lakes, NJ, USA) and the numbers of CD31-positive cells per cardiomyocyte were measured. These were obtained from 20 randomly chosen microscopic fields from three different sections in each tissue block. Hematoxylin was used for counterstaining.
To gain further insight into the potential effect of syndecan-4 deletion on the development of cardiac hypertrophy, we assessed the heart both in vivo and in vitro. Echocardiography showed that the mean IVSd and LV mass in syndecan-4 KO mice was significantly attenuated compared to the WT group from day 7 to day 28 (Fig. 2a and b). This observation was validated by determining the ratio of heart weight to body weight (HW/BW) (Fig. 2c). Hearts were also processed for histological analyses. The mean single cardiomyocyte CSA was significantly increased after TAC in both genotypes, although to a lesser extent in syndecan-4 KO mice (Fig. 2d). Representative photomicrographs of cardiomyocytes are shown in Fig. 2e. Taken together, these data suggest that deletion of syndecan-4 prevented the development of cardiac hypertrophy and resulted in the deterioration of cardiac function after pressure overload in mice. 3.3. Syndecan-4 deficiency inhibits cardiac angiogenesis in pressure overload-induced cardiac hypertrophy To investigate the extent of cardiac microvessel formation following pressure overload, capillary density was measured by staining for CD31
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Fig. 1. The knockout of syndecan-4 results in exacerbation of pressure overloaded-induced heart failure compared with wild-type (WT) mice. (a) Representative echocardiographic images of WT and syndecan-4 KO mice before and after TAC surgery. (b–e) Echocardiographic analysis of the end-diastolic left ventricular inner diameter (LVID;d), end-systolic left ventricular inner diameter (LVID;s), ejection fraction (EF), and fractional shortening (FS) for WT and KO mice. N=10 at day 0, N=24 at day 7, N=16 at day 14, N=8 at day 28. (f) Myocardial expression of ANF protein in WT and KO mice were determined by Western blot analysis. Representative ANF bands with corresponding NAPDH bands and quantitative analysis of ANF protein levels are shown. Data are shown as mean±S.D.; *Pb.05 vs. day 0(baseline) and #Pb.05 vs. corresponding WT mice.
in histological heart sections (Fig. 3a). Quantitative analysis of CD31positive endothelial cells in WT mice showed that the capillary density was increased after TAC, peaked at day 7, and gradually decreased from day 14 to day 28 (Fig. 3b). However, the analysis of CD31positive endothelial cells in the hearts of syndecan-4 KO mice revealed a progressive decrease in functional capillaries compared with WT mice at all time points after TAC (Fig. 3b). Furthermore, Western blot analyses suggested that angiogenesis was significantly attenuated in syndecan-4 KO mice compared with WT mice at days 7, 24, and 28
(Fig. 3c). Reduced myocardial capillary density in syndecan-4 KO mice subjected to pressure overload was crucial in preserving cardiac function as well as in developing cardiac hypertrophy. 3.4. Syndecan-4 KO mice after TAC showed decreased PKCα levels A recent study reported that syndecan-4 is involved in modulation of prostaglandin E2-dependent proangiogenic effects via activation of PKCα [20]. To examine potential mechanisms underlying the
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Fig. 2. Syndecan-4 KO mice exhibit attenuated cardiac hypertrophy after TAC. (a–b) Echocardiographic analysis of the end-diastolic inter-ventricular septum thickness (IVS;d) and LV mass was performed before and after TAC. (c) The ratio of heart weight to body weight (HW/BW). (d) Quantitative analysis of the cross-sectional area (CSA) of cardiomyocytes from each group. (e) Representative histological LV sections from WT and KO mice stained with HE. Data are shown as mean±S.D.; *Pb.05 vs. day 0 and #Pb.05 vs. corresponding WT mice; scale bars, 50 μm.
antiangiogenic effects of syndecan-4 during pressure overload-induced heart failure, we examined syndecan-4 and PKCα protein levels in the harvested hearts. TAC surgery significantly increased cardiac syndecan-4 expression in the first week in WT mice as seen by Western blot analyses, but decreased dramatically in the second week and the fourth week (Fig. 4a). Consistent with the levels of syndecan-4, the expression of PKCα was upregulated in the early phase and decreased in the late phase, in WT mice (Fig. 4b). However, the increase in PKCα in the syndecan-4 KO mice was diminished at all time points compared with WT mice following TAC (Fig. 4b).
4. Discussion In this study, we examined the importance of syndecan-4 in cardiac angiogenesis and hypertrophy during heart failure. Our main finding is that syndecan-4 deficiency prevents adaptive cardiac hypertrophy and accelerates the progression from compensated hypertrophy to heart failure, partly via impaired angiogenesis.
Angiogenesis is the formation of new blood vessels, and is thought to be important in many physiologic and pathophysiological processes [21]. The relationship between angiogenesis and myocardium hypertrophy has attracted considerable attention in the past few years [11,22–25]. In our study, we confirmed that angiogenesis is involved in preserving cardiac function as well as in developing cardiac hypertrophy in the TAC model. To investigate the chronic process of cardiac function, we observed longer time points (0, 7, 14, and 28 days) after surgery compared to previous studies. At 1 week after TAC, we found that the increase in new blood vessels was accompanied with cardiomyocyte growth. Furthermore, cardiac function is preserved, similar to what is observed in the clinical stage of heart failure with preserved EF. We speculate that angiogenesis plays a major role during this early adaptive phase of the TAC model. The rapid increase in the vasculature provides adequate oxygen and nutrition promoting the transition from adaptive hypertrophy to compensatory hypertrophy. With time, the accumulation and overexpression of other cytokines, including p38-MAPK and ERKs [26,27], turn into the major factors that decrease the expression of angiogenic factors and reduce the capillary density. These are
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Fig. 3. Syndecan-4 deficiency causes decreased cardiac angiogenesis following pressure overload. (a) Representative immunohistochemical images of CD31-stained heart sections from WT and KO mice before and after TAC. (b) Quantitative analysis of capillary density by measuring the number of CD31-positive cells per cardiomyocyte from each group. (c) Representative Western blot of CD31 protein of myocardium in different groups and quantitative analysis of CD31 protein levels. Data are shown as mean±S.D.; *Pb.05; scale bars, 50 μm.
insufficient to maintain the function of the hypertrophied myocardium and can eventually develop to eccentric hypertrophy and heart failure. Syndecan-4 is a transmembrane heparin sulfate proteoglycan and is a central mediator of cell adhesion, migration, proliferation, endocytosis, and mechanotransduction [17]. Previous studies have shown that neovascularization mediated by syndecan-4 plays an important role in animal models of skin wound healing and limb ischemia [28]. In the present study, we observed that in pressure overload-induced heart failure, syndecan-4 KO mice exhibited reduced capillary density compared with WT mice after TAC. Furthermore, associated with the drop in myocardial capillary number, syndecan-4 KO mice also displayed impaired cardiac function from the second week detected by echocardiography, suggesting that a serious imbalance between oxygen supply and myocardial demand led to worsening cardiac function. In addition, the hypertrophy of the heart is partly inhibited without the support of the microvasculature and is subsequently accompanied by impaired cardiac function. These results demonstrate that lack of syndecan-4 promotes the progression from compensated hypertrophy to heart failure by inhibiting cardiac angiogenesis in response to TAC. The levels of cardiac syndecan-4 were measured in WT mice following the 0, 1, 2, and 4 weeks of TAC, and we observed that syndecan-4 expression was increased at 1 week after TAC, then decreased dramatically in the following 2 to 4 weeks. Consistent with previous work, the expression of syndecan-4 in cardiac myocytes is increased in the initial stage of heart failure [29], followed by the shedding of its ectodomain, which was mediated by proinflammatory signals, including tumor necrosis factor-α, interleukin-1β, and nuclear factor-kappa B. It has been reported that syndecan-4 activates PKCα in the presence of phosphatidylinositol 4, 5-bisphosphate (PIP2) [30,31], and PKCα has
been shown to participate in angiogenesis [32,33]. Hence, we propose that the syndecan-4 may regulate angiogenesis through the downstream-PKCα following pressure overload. We, therefore, checked the PKCα in the myocardium of the two groups before and after TAC, and we found that the activation of PKCα is consistent with the expression of syndecan-4 in WT mice. We also detected that PKC activation was diminished in the syndecan-4 KO mice compared with WT at all time points. Thus, syndecan-4 appears to control cardiac angiogenesis via a PKCα-dependent mechanism following pressure overloaded heart failure. Our present study indicates that syndecan-4 is essential for the compensated hypertrophy and the maintenance of cardiac function during the process of heart failure following pressure overload. These results suggest a specific target to prevent or reverse heart failure through stimulating angiogenesis. However, there are many unresolved problems about cardiac hypertrophy and angiogenesis at the cellular and molecular levels. We acknowledge that syndecan-4 is only a tip of the iceberg, and further investigations will extend these observations and provide a solid proof of concept between angiogenesis and heart failure treatment. Conflict of interest None. Author contributions Guannan Li, Jun Xie, Rong Gu, and Biao Xu conceived and organized the project and wrote the manuscript. Guannan Li, Jianzhou Chen, Ran
G. Li et al. / Cardiovascular Pathology 28 (2017) 74–79
Fig. 4. Syndecan-4 and PKCα expressions in the heart were determined by Western blot analysis. (a) Expressions of syndecan-4 in the WT mice were assessed before and after TAC, and the quantitative results are shown. (b) PKCα expression was assessed at four time points both in WT and KO groups. Representative images and analysis of protein levels are shown. Data are shown as mean±S.D.; *Pb.05.
Li, Han Wu, Xinlin Zhang, Qinhua Chen, and Rong Gu contributed to experiments and data analysis. All authors discussed the results and commented on the manuscript.
References [1] McMurray JJ, Adamopoulos S, Anker SD, Auricchio A, Bohm M, Dickstein K, et al. ESC guidelines for the diagnosis and treatment of acute and chronic heart failure 2012: the Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2012 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association (HFA) of the ESC. Eur Heart J 2012;33:1787–847. [2] Yancy CW, Jessup M, Bozkurt B, Butler J, Casey DE, Drazner MH, et al. 2013 ACCF/ AHA guideline for the management of heart failure: a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines. Circulation 2013;128:e240–327. [3] Lloyd-Jones D, Adams R, Carnethon M, De Simone G, Ferguson TB, Flegal K, et al. Heart disease and stroke statistics— 2009 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 2009;119:480–6.
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[4] Cooper Gt. Cardiocyte adaptation to chronically altered load. Annu Rev Physiol 1987; 49:501–18. [5] Berenji K, Drazner MH, Rothermel BA, Hill JA. Does load-induced ventricular hypertrophy progress to systolic heart failure? Am J Physiol Heart Circ Physiol 2005;289: H8-16. [6] Frey N, Olson EN. Cardiac hypertrophy: the good, the bad, and the ugly. Annu Rev Physiol 2003;65:45–79. [7] Shiojima I, Sato K, Izumiya Y, Schiekofer S, Ito M, Liao R, et al. Disruption of coordinated cardiac hypertrophy and angiogenesis contributes to the transition to heart failure. J Clin Invest 2005;115:2108–18. [8] Oka T, Akazawa H, Naito AT, Komuro I. Angiogenesis and cardiac hypertrophy: maintenance of cardiac function and causative roles in heart failure. Circ Res 2014; 114:565–71. [9] Sano M, Minamino T, Toko H, Miyauchi H, Orimo M, Qin Y, et al. p53-induced inhibition of Hif-1 causes cardiac dysfunction during pressure overload. Nature 2007; 446:444–8. [10] Dorn GW. Myocardial angiogenesis: its absence makes the growing heart founder. Cell Metab 2007;5:326–7. [11] Anversa P, Capasso JM. Loss of intermediate-sized coronary arteries and capillary proliferation after left ventricular failure in rats. Am J Physiol 1991;260:H1552–60. [12] Zou Y, Li J, Ma H, Jiang H, Yuan J, Gong H, et al. Heat shock transcription factor 1 protects heart after pressure overload through promoting myocardial angiogenesis in male mice. J Mol Cell Cardiol 2011;51:821–9. [13] Gogiraju R, Xu X, Bochenek ML, Steinbrecher JH, Lehnart SE, Wenzel P, et al. Endothelial p53 deletion improves angiogenesis and prevents cardiac fibrosis and heart failure induced by pressure overload in mice. J Am Heart Assoc 2015;4. [14] Guo J, Mihic A, Wu J, Zhang Y, Singh K, Dhingra S, et al. Canopy 2 attenuates the transition from compensatory hypertrophy to dilated heart failure in hypertrophic cardiomyopathy. Eur Heart J 2015;36:2530–40. [15] Carey DJ. Syndecans: multifunctional cell-surface co-receptors. Biochem J 1997; 327(Pt 1):1–16. [16] Couchman JR, Gopal S, Lim HC, Norgaard S, Multhaupt HA. Syndecans: from peripheral coreceptors to mainstream regulators of cell behaviour. Int J Exp Pathol 2015;96:1–10. [17] Elfenbein A, Simons M. Syndecan-4 signaling at a glance. J Cell Sci 2013;126: 3799–804. [18] Xie J, Wang J, Li R, Dai Q, Yong Y, Zong B, et al. Syndecan-4 over-expression preserves cardiac function in a rat model of myocardial infarction. J Mol Cell Cardiol 2012;53: 250–8. [19] Takimoto E, Yao A, Toko H, Takano H, Shimoyama M, Sonoda M, et al. Sodium calcium exchanger plays a key role in alteration of cardiac function in response to pressure overload. FASEB J 2002;16:373–8. [20] Corti F, Finetti F, Ziche M, Simons M. The syndecan-4/protein kinase Calpha pathway mediates prostaglandin E2-induced extracellular regulated kinase (ERK) activation in endothelial cells and angiogenesis in vivo. J Biol Chem 2013;288:12712–21. [21] Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000;407: 249–57. [22] Giordano FJ, Gerber HP, Williams SP, VanBruggen N, Bunting S, Ruiz-Lozano P, et al. A cardiac myocyte vascular endothelial growth factor paracrine pathway is required to maintain cardiac function. Proc Natl Acad Sci U S A 2001;98:5780–5. [23] Shyu KG, Liou JY, Wang BW, Fang WJ, Chang H. Carvedilol prevents cardiac hypertrophy and overexpression of hypoxia-inducible factor-1alpha and vascular endothelial growth factor in pressure-overloaded rat heart. J Biomed Sci 2005;12:409–20. [24] Yoon YS, Uchida S, Masuo O, Cejna M, Park JS, Gwon HC, et al. Progressive attenuation of myocardial vascular endothelial growth factor expression is a seminal event in diabetic cardiomyopathy: restoration of microvascular homeostasis and recovery of cardiac function in diabetic cardiomyopathy after replenishment of local vascular endothelial growth factor. Circulation 2005;111:2073–85. [25] Tomanek RJ. Response of the coronary vasculature to myocardial hypertrophy. J Am Coll Cardiol 1990;15:528–33. [26] Heineke J, Molkentin JD. Regulation of cardiac hypertrophy by intracellular signalling pathways. Nat Rev Mol Cell Biol 2006;7:589–600. [27] Bernardo BC, Weeks KL, Pretorius L, McMullen JR. Molecular distinction between physiological and pathological cardiac hypertrophy: experimental findings and therapeutic strategies. Pharmacol Ther 2010;128:191–227. [28] Echtermeyer F, Streit M, Wilcox-Adelman S, Saoncella S, Denhez F, Detmar M, et al. Delayed wound repair and impaired angiogenesis in mice lacking syndecan-4. J Clin Invest 2001;107:R9-14. [29] Strand ME, Herum KM, Rana ZA, Skrbic B, Askevold ET, Dahl CP, et al. Innate immune signaling induces expression and shedding of the heparan sulfate proteoglycan syndecan-4 in cardiac fibroblasts and myocytes, affecting inflammation in the pressure-overloaded heart. FEBS J 2013;280:2228–47. [30] Lim ST, Longley RL, Couchman JR, Woods A. Direct binding of syndecan-4 cytoplasmic domain to the catalytic domain of protein kinase C alpha (PKC alpha) increases focal adhesion localization of PKC alpha. J Biol Chem 2003;278:13795–802. [31] Dovas A, Yoneda A, Couchman JR. PKC alpha-dependent activation of RhoA by syndecan-4 during focal adhesion formation. J Cell Sci 2006;119:2837–46. [32] Xu H, Czerwinski P, Hortmann M, Sohn HY, Forstermann U, Li H. Protein kinase C alpha promotes angiogenic activity of human endothelial cells via induction of vascular endothelial growth factor. Cardiovasc Res 2008;78:349–55. [33] Wang A, Nomura M, Patan S, Ware JA. Inhibition of protein kinase Calpha prevents endothelial cell migration and vascular tube formation in vitro and myocardial neovascularization in vivo. Circ Res 2002;90:609–16.
Contents lists available at ScienceDirect
Cardiovascular Pathology
Original Article
Syndecan-4 deficiency accelerates the transition from compensated hypertrophy to heart failure following pressure overload Guannan Li, Jun Xie, Jianzhou Chen, Ran Li, Han Wu, Xinlin Zhang, Qinhua Chen, Rong Gu ⁎, Biao Xu ⁎ Department of Cardiology, Affiliated Drum Tower Hospital, Nanjing University Medical School, Nanjing, China
a r t i c l e
i n f o
Article history: Received 3 September 2016 Received in revised form 27 March 2017 Accepted 27 March 2017 Available online xxxx Keywords: Syndecan-4 Angiogenesis Cardiac hypertrophy Heart failure PKCα
a b s t r a c t Increasing evidence suggests that a mismatch between angiogenesis and myocardial growth contributes to the transition from adaptive cardiac hypertrophy to heart failure following pressure overload. Syndecan-4 is a transmembrane proteoglycan that binds to growth factors and extracellular matrix proteins and is critical in focal adhesion formation. However, its effects on coronary angiogenesis during pressure overload-induced heart failure have not been studied. Here, we hypothesize that syndecan-4 modulates cardiac remodeling in response to pressure overload through its ability to regulate adaptive angiogenesis. Syndecan-4 knockout (syndecan-4 KO) and wild-type (WT) mice were subjected to pressure overload induced by transverse aortic constriction (TAC). Syndecan-4 KO mice exhibited reduced capillary density, attenuated cardiomyocyte size, and worsened left ventricular cardiac function after TAC surgery compared with WT mice. Moreover, syndecan-4 KO mice showed a significant decrease in protein kinase C alpha expression. Our data suggest that syndecan-4 is essential for the compensated hypertrophy and the maintenance of cardiac function during the process of heart failure following pressure overload. © 2017 Published by Elsevier Inc.
1. Introduction Heart failure is a common consequence of most heart diseases and is one of the leading causes of morbidity and mortality worldwide [1–3]. Cardiac hypertrophy is an adaptive response to preserve cardiac function in several forms of increased afterload, such as aortic stenosis and elevated blood pressure [4]. However, sustained afterload eventually leads to a state of decompensated hypertrophy resulting in cardiac dilation and contractile dysfunction [5,6]. During the development of cardiac hypertrophy, a mismatch occurs between the number of capillaries and the size of cardiomyocytes, contributing to the progression from compensated hypertrophy to contractile dysfunction and heart failure [7,8]. Related studies have observed the reduction of cardiac vessel density during pathological hypertrophy in pressure overload-induced mice models of heart failure [9–11]. In addition, stimulation of angiogenesis could improve cardiac function and delay the progress of heart failure [12–14]. Several recent studies indicate a relationship between
This work was supported by grants from the National Natural Science Foundation of China (81200148, 81270281, and 8120092), Jiangsu Province Special Program of Medical Science (BL2012014), the Peak of Six Personnel in Jiangsu Province (2013-WSN-008), Funds for Distinguished Young Scientists in Nanjing (JQX13006), Key Program of Science Foundation in Nanjing (ZKX13023), and the funds from Nanjing Medical Science and Technique Development Foundation (QRX11158). ⁎ Corresponding authors at: Department of Cardiology, Affiliated Drum Tower Hospital, Nanjing University Medical School, Zhongshan Road, Nanjing 210008, China. Tel./fax: +86 25 68182812. E-mail addresses: [email protected] (R. Gu), [email protected] (B. Xu). http://dx.doi.org/10.1016/j.carpath.2017.03.008 1054-8807/© 2017 Published by Elsevier Inc.
cardiac angiogenesis, cardiac hypertrophy, and cardiac function [8]. However, the molecular mechanisms involved in the regulation of cardiac angiogenesis in pressure overload-induced heart failure are not understood. Syndecan-4 is a transmembrane proteoglycan that plays an important role in a variety of cellular functions, including cell proliferation, cell–matrix and cell–cell adhesion [15–17]. Syndecan-4 regulates a wide range of pathophysiological processes in cardiovascular diseases, including cardiac myofibroblast differentiation and endothelial alignment in atherosclerosis [17]. Previous work in our laboratory demonstrated that sustained syndecan-4 overexpression in the myocardium promotes neovascularization and suppresses inflammation and fibrosis in the rat model of myocardial infarction, resulting in improvements in cardiac function and remodeling [18]. However, it is unclear if syndecan-4 mediates angiogenesis in pressure overload-induced cardiac dysfunction. Here, we hypothesize that syndecan-4 deficiency affects the transition from cardiac adaptive hypertrophy to heart failure during pressure overload by regulating cardiac angiogenesis. This is important to understand the relationship between angiogenesis and cardiac hypertrophy, and to help guide therapies for heart failure. 2. Materials and methods All experimental procedures and research were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (National Institutes of
G. Li et al. / Cardiovascular Pathology 28 (2017) 74–79
Health publication No. 8023, revised 1978), and approved by the Institutional Ethics Committee of Nanjing Drum Tower Hospital.
2.1. Experimental animals Syndecan-4 KO mice, backcrossed more than 10 times into the C57BL/6 background, were obtained from the Center for Animal Resources and Development at Kumamoto University (Japan). Twelveweek-old male syndecan-4 KO mice and age-matched male wild-type (WT) mice on a C57BL/6 background were used in the present study. All WT mice were obtained from the Model Animal Research Center of Nanjing University.
2.2. Experimental protocols Pressure overload was induced by transverse aortic constriction (TAC), as described previously [19]. Briefly, mice were anesthetized with 10% chloral hydrate (0.3 ml/100 g) intraperitoneally. The chest was opened and the thoracic aorta was identified after blunt dissection through the intercostal muscles. A 6-0 silk suture was placed around the transverse aorta and tied around a 26-gauge blunt needle, which was then removed. After banding, the chest and overlying skin were closed.
2.3. Echocardiographic measurements Cardiac function was evaluated by serial transthoracic echocardiography at 0, 7, 14, and 28 days after TAC. The measurements were performed by an observer blinded to the treatment. Studies were recorded with a dynamic focused 30-MHz probe echocardiography machine (Visual Sonics Vevo2100, Toronto, Canada). Briefly, mice were anesthetized using isoflurane gas and transferred to an imaging stage equipped with a warming pad for controlled maintenance of mouse body temperature at 37 °C and a built-in electrocardiography system for monitoring continuous heart rate and respiratory rate. The Mmode measurements of left ventricular (LV) dimensions were averaged from more than 3 cycles. The end-diastolic LV inner diameter (LVIDd), end-systolic LV inner diameter (LVIDs), and end-diastolic interventricular septum thickness (IVSd) as well as LV mass were measured. Percentage of LV fractional shortening (FS) was calculated as follows: %FS= (LVIDd−LVIDs)/LVIDd*100. LV ejection fraction (LVEF) was calculated as follows: %EF=(LVIDd2−LVIDs2)/LVIDd2*100.
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2.5. Western blot analysis The hearts of mice were harvested at day 0 (baseline), day 7, day 14 and day 28 after TAC (n=4/genotype/timepoint). Western blotting analysis was performed and the expression of proteins was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primary antibodies used were anti-CD31(1:1000; Abcam, Cambridge, UK),anti-protein kinase C alpha (PKCα; 1:500; BD), anti-atrial natriuretic factor (anti-ANF; 1:500; BD), anti-synd4(1:500; LSbio,Seattle, WA,USA), and anti-GAPDH (1:1000; Millipore, Billerica, MA, USA). 2.6. Statistical analysis Data are presented as mean±S.D. All data analyses were performed with the use of SPSS 16.0 software. Group differences were analyzed by two-tailed Student's t test or analysis of variance. A value of Pb.05 was considered statistically significant. 3. Results 3.1. Syndecan-4 deficiency exacerbates cardiac dysfunction after pressure overload To induce cardiac hypertrophy, male mice were subjected to TAC surgery. Multiple time points (0, 7, 14, and 28 days) were utilized to examine the gradual transition from a compensated to a decompensated stage of cardiac function (Fig. 1a). No significant difference was found between the two groups at baseline. Serial echocardiographic measurements revealed a progressive decline in the LVEF and FS after TAC surgery both in WT and KO mice (Fig. 1b and c). A significant increase in the LVIDd and LVIDs (Fig. 1d and e) was concurrently observed. However, the reduction of LVEF and FS, and the increase in LVIDd and LVIDs were significantly more pronounced in syndecan-4 KO mice compared with WT mice at 2 and 4 weeks after TAC (Fig. 1b–e). Interestingly, we also found that syndecan-4 KO mice showed a larger diameter in LVIDs than WT mice at day 7 (Fig. 1e), suggesting that the syndecan-4 KO mice had decreased contractile function in the early adaptable stage following TAC. The expression of ANF in the myocardium after TAC was examined by Western blot showed no difference at baseline between the WT and KO groups, but syndecan-4 KO mice showed increased ANF expression from day 7 to day 28 after TAC, compared with WT mice (Fig. 1f). These data indicate that syndecan-4 deficiency exacerbated cardiac dysfunction after pressure overload in mice. 3.2. Syndecan-4 deficiency prevents the development of cardiac hypertrophy
2.4. Histological examination For morphological and immunohistochemical experiments, mice were sacrificed and their hearts were collected at day 0 (baseline), day 7, day 14, and day 28 after TAC (n=4/genotype/timepoint). All hearts were weighed and fixed with formalin, embedded in paraffin, and cut into 4-μm slices for subsequent immunohistochemical histological analysis. The micrographs were then analyzed using the Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, USA). To determine the cross-sectional area (CSA) of a cardiomyocyte, cross sections of the LV area between the apices and bases of the hearts were stained with hematoxylin and eosin, and histological studies were performed. At least 100 cardiomyocytes per heart were measured for myocyte CSA calculation (magnification,×400). To determine the capillary density, tissue sections were stained with anti-CD31 antibody (1:200; BD, Franklin Lakes, NJ, USA) and the numbers of CD31-positive cells per cardiomyocyte were measured. These were obtained from 20 randomly chosen microscopic fields from three different sections in each tissue block. Hematoxylin was used for counterstaining.
To gain further insight into the potential effect of syndecan-4 deletion on the development of cardiac hypertrophy, we assessed the heart both in vivo and in vitro. Echocardiography showed that the mean IVSd and LV mass in syndecan-4 KO mice was significantly attenuated compared to the WT group from day 7 to day 28 (Fig. 2a and b). This observation was validated by determining the ratio of heart weight to body weight (HW/BW) (Fig. 2c). Hearts were also processed for histological analyses. The mean single cardiomyocyte CSA was significantly increased after TAC in both genotypes, although to a lesser extent in syndecan-4 KO mice (Fig. 2d). Representative photomicrographs of cardiomyocytes are shown in Fig. 2e. Taken together, these data suggest that deletion of syndecan-4 prevented the development of cardiac hypertrophy and resulted in the deterioration of cardiac function after pressure overload in mice. 3.3. Syndecan-4 deficiency inhibits cardiac angiogenesis in pressure overload-induced cardiac hypertrophy To investigate the extent of cardiac microvessel formation following pressure overload, capillary density was measured by staining for CD31
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Fig. 1. The knockout of syndecan-4 results in exacerbation of pressure overloaded-induced heart failure compared with wild-type (WT) mice. (a) Representative echocardiographic images of WT and syndecan-4 KO mice before and after TAC surgery. (b–e) Echocardiographic analysis of the end-diastolic left ventricular inner diameter (LVID;d), end-systolic left ventricular inner diameter (LVID;s), ejection fraction (EF), and fractional shortening (FS) for WT and KO mice. N=10 at day 0, N=24 at day 7, N=16 at day 14, N=8 at day 28. (f) Myocardial expression of ANF protein in WT and KO mice were determined by Western blot analysis. Representative ANF bands with corresponding NAPDH bands and quantitative analysis of ANF protein levels are shown. Data are shown as mean±S.D.; *Pb.05 vs. day 0(baseline) and #Pb.05 vs. corresponding WT mice.
in histological heart sections (Fig. 3a). Quantitative analysis of CD31positive endothelial cells in WT mice showed that the capillary density was increased after TAC, peaked at day 7, and gradually decreased from day 14 to day 28 (Fig. 3b). However, the analysis of CD31positive endothelial cells in the hearts of syndecan-4 KO mice revealed a progressive decrease in functional capillaries compared with WT mice at all time points after TAC (Fig. 3b). Furthermore, Western blot analyses suggested that angiogenesis was significantly attenuated in syndecan-4 KO mice compared with WT mice at days 7, 24, and 28
(Fig. 3c). Reduced myocardial capillary density in syndecan-4 KO mice subjected to pressure overload was crucial in preserving cardiac function as well as in developing cardiac hypertrophy. 3.4. Syndecan-4 KO mice after TAC showed decreased PKCα levels A recent study reported that syndecan-4 is involved in modulation of prostaglandin E2-dependent proangiogenic effects via activation of PKCα [20]. To examine potential mechanisms underlying the
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Fig. 2. Syndecan-4 KO mice exhibit attenuated cardiac hypertrophy after TAC. (a–b) Echocardiographic analysis of the end-diastolic inter-ventricular septum thickness (IVS;d) and LV mass was performed before and after TAC. (c) The ratio of heart weight to body weight (HW/BW). (d) Quantitative analysis of the cross-sectional area (CSA) of cardiomyocytes from each group. (e) Representative histological LV sections from WT and KO mice stained with HE. Data are shown as mean±S.D.; *Pb.05 vs. day 0 and #Pb.05 vs. corresponding WT mice; scale bars, 50 μm.
antiangiogenic effects of syndecan-4 during pressure overload-induced heart failure, we examined syndecan-4 and PKCα protein levels in the harvested hearts. TAC surgery significantly increased cardiac syndecan-4 expression in the first week in WT mice as seen by Western blot analyses, but decreased dramatically in the second week and the fourth week (Fig. 4a). Consistent with the levels of syndecan-4, the expression of PKCα was upregulated in the early phase and decreased in the late phase, in WT mice (Fig. 4b). However, the increase in PKCα in the syndecan-4 KO mice was diminished at all time points compared with WT mice following TAC (Fig. 4b).
4. Discussion In this study, we examined the importance of syndecan-4 in cardiac angiogenesis and hypertrophy during heart failure. Our main finding is that syndecan-4 deficiency prevents adaptive cardiac hypertrophy and accelerates the progression from compensated hypertrophy to heart failure, partly via impaired angiogenesis.
Angiogenesis is the formation of new blood vessels, and is thought to be important in many physiologic and pathophysiological processes [21]. The relationship between angiogenesis and myocardium hypertrophy has attracted considerable attention in the past few years [11,22–25]. In our study, we confirmed that angiogenesis is involved in preserving cardiac function as well as in developing cardiac hypertrophy in the TAC model. To investigate the chronic process of cardiac function, we observed longer time points (0, 7, 14, and 28 days) after surgery compared to previous studies. At 1 week after TAC, we found that the increase in new blood vessels was accompanied with cardiomyocyte growth. Furthermore, cardiac function is preserved, similar to what is observed in the clinical stage of heart failure with preserved EF. We speculate that angiogenesis plays a major role during this early adaptive phase of the TAC model. The rapid increase in the vasculature provides adequate oxygen and nutrition promoting the transition from adaptive hypertrophy to compensatory hypertrophy. With time, the accumulation and overexpression of other cytokines, including p38-MAPK and ERKs [26,27], turn into the major factors that decrease the expression of angiogenic factors and reduce the capillary density. These are
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Fig. 3. Syndecan-4 deficiency causes decreased cardiac angiogenesis following pressure overload. (a) Representative immunohistochemical images of CD31-stained heart sections from WT and KO mice before and after TAC. (b) Quantitative analysis of capillary density by measuring the number of CD31-positive cells per cardiomyocyte from each group. (c) Representative Western blot of CD31 protein of myocardium in different groups and quantitative analysis of CD31 protein levels. Data are shown as mean±S.D.; *Pb.05; scale bars, 50 μm.
insufficient to maintain the function of the hypertrophied myocardium and can eventually develop to eccentric hypertrophy and heart failure. Syndecan-4 is a transmembrane heparin sulfate proteoglycan and is a central mediator of cell adhesion, migration, proliferation, endocytosis, and mechanotransduction [17]. Previous studies have shown that neovascularization mediated by syndecan-4 plays an important role in animal models of skin wound healing and limb ischemia [28]. In the present study, we observed that in pressure overload-induced heart failure, syndecan-4 KO mice exhibited reduced capillary density compared with WT mice after TAC. Furthermore, associated with the drop in myocardial capillary number, syndecan-4 KO mice also displayed impaired cardiac function from the second week detected by echocardiography, suggesting that a serious imbalance between oxygen supply and myocardial demand led to worsening cardiac function. In addition, the hypertrophy of the heart is partly inhibited without the support of the microvasculature and is subsequently accompanied by impaired cardiac function. These results demonstrate that lack of syndecan-4 promotes the progression from compensated hypertrophy to heart failure by inhibiting cardiac angiogenesis in response to TAC. The levels of cardiac syndecan-4 were measured in WT mice following the 0, 1, 2, and 4 weeks of TAC, and we observed that syndecan-4 expression was increased at 1 week after TAC, then decreased dramatically in the following 2 to 4 weeks. Consistent with previous work, the expression of syndecan-4 in cardiac myocytes is increased in the initial stage of heart failure [29], followed by the shedding of its ectodomain, which was mediated by proinflammatory signals, including tumor necrosis factor-α, interleukin-1β, and nuclear factor-kappa B. It has been reported that syndecan-4 activates PKCα in the presence of phosphatidylinositol 4, 5-bisphosphate (PIP2) [30,31], and PKCα has
been shown to participate in angiogenesis [32,33]. Hence, we propose that the syndecan-4 may regulate angiogenesis through the downstream-PKCα following pressure overload. We, therefore, checked the PKCα in the myocardium of the two groups before and after TAC, and we found that the activation of PKCα is consistent with the expression of syndecan-4 in WT mice. We also detected that PKC activation was diminished in the syndecan-4 KO mice compared with WT at all time points. Thus, syndecan-4 appears to control cardiac angiogenesis via a PKCα-dependent mechanism following pressure overloaded heart failure. Our present study indicates that syndecan-4 is essential for the compensated hypertrophy and the maintenance of cardiac function during the process of heart failure following pressure overload. These results suggest a specific target to prevent or reverse heart failure through stimulating angiogenesis. However, there are many unresolved problems about cardiac hypertrophy and angiogenesis at the cellular and molecular levels. We acknowledge that syndecan-4 is only a tip of the iceberg, and further investigations will extend these observations and provide a solid proof of concept between angiogenesis and heart failure treatment. Conflict of interest None. Author contributions Guannan Li, Jun Xie, Rong Gu, and Biao Xu conceived and organized the project and wrote the manuscript. Guannan Li, Jianzhou Chen, Ran
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Fig. 4. Syndecan-4 and PKCα expressions in the heart were determined by Western blot analysis. (a) Expressions of syndecan-4 in the WT mice were assessed before and after TAC, and the quantitative results are shown. (b) PKCα expression was assessed at four time points both in WT and KO groups. Representative images and analysis of protein levels are shown. Data are shown as mean±S.D.; *Pb.05.
Li, Han Wu, Xinlin Zhang, Qinhua Chen, and Rong Gu contributed to experiments and data analysis. All authors discussed the results and commented on the manuscript.
References [1] McMurray JJ, Adamopoulos S, Anker SD, Auricchio A, Bohm M, Dickstein K, et al. ESC guidelines for the diagnosis and treatment of acute and chronic heart failure 2012: the Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2012 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association (HFA) of the ESC. Eur Heart J 2012;33:1787–847. [2] Yancy CW, Jessup M, Bozkurt B, Butler J, Casey DE, Drazner MH, et al. 2013 ACCF/ AHA guideline for the management of heart failure: a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines. Circulation 2013;128:e240–327. [3] Lloyd-Jones D, Adams R, Carnethon M, De Simone G, Ferguson TB, Flegal K, et al. Heart disease and stroke statistics— 2009 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 2009;119:480–6.
79
[4] Cooper Gt. Cardiocyte adaptation to chronically altered load. Annu Rev Physiol 1987; 49:501–18. [5] Berenji K, Drazner MH, Rothermel BA, Hill JA. Does load-induced ventricular hypertrophy progress to systolic heart failure? Am J Physiol Heart Circ Physiol 2005;289: H8-16. [6] Frey N, Olson EN. Cardiac hypertrophy: the good, the bad, and the ugly. Annu Rev Physiol 2003;65:45–79. [7] Shiojima I, Sato K, Izumiya Y, Schiekofer S, Ito M, Liao R, et al. Disruption of coordinated cardiac hypertrophy and angiogenesis contributes to the transition to heart failure. J Clin Invest 2005;115:2108–18. [8] Oka T, Akazawa H, Naito AT, Komuro I. Angiogenesis and cardiac hypertrophy: maintenance of cardiac function and causative roles in heart failure. Circ Res 2014; 114:565–71. [9] Sano M, Minamino T, Toko H, Miyauchi H, Orimo M, Qin Y, et al. p53-induced inhibition of Hif-1 causes cardiac dysfunction during pressure overload. Nature 2007; 446:444–8. [10] Dorn GW. Myocardial angiogenesis: its absence makes the growing heart founder. Cell Metab 2007;5:326–7. [11] Anversa P, Capasso JM. Loss of intermediate-sized coronary arteries and capillary proliferation after left ventricular failure in rats. Am J Physiol 1991;260:H1552–60. [12] Zou Y, Li J, Ma H, Jiang H, Yuan J, Gong H, et al. Heat shock transcription factor 1 protects heart after pressure overload through promoting myocardial angiogenesis in male mice. J Mol Cell Cardiol 2011;51:821–9. [13] Gogiraju R, Xu X, Bochenek ML, Steinbrecher JH, Lehnart SE, Wenzel P, et al. Endothelial p53 deletion improves angiogenesis and prevents cardiac fibrosis and heart failure induced by pressure overload in mice. J Am Heart Assoc 2015;4. [14] Guo J, Mihic A, Wu J, Zhang Y, Singh K, Dhingra S, et al. Canopy 2 attenuates the transition from compensatory hypertrophy to dilated heart failure in hypertrophic cardiomyopathy. Eur Heart J 2015;36:2530–40. [15] Carey DJ. Syndecans: multifunctional cell-surface co-receptors. Biochem J 1997; 327(Pt 1):1–16. [16] Couchman JR, Gopal S, Lim HC, Norgaard S, Multhaupt HA. Syndecans: from peripheral coreceptors to mainstream regulators of cell behaviour. Int J Exp Pathol 2015;96:1–10. [17] Elfenbein A, Simons M. Syndecan-4 signaling at a glance. J Cell Sci 2013;126: 3799–804. [18] Xie J, Wang J, Li R, Dai Q, Yong Y, Zong B, et al. Syndecan-4 over-expression preserves cardiac function in a rat model of myocardial infarction. J Mol Cell Cardiol 2012;53: 250–8. [19] Takimoto E, Yao A, Toko H, Takano H, Shimoyama M, Sonoda M, et al. Sodium calcium exchanger plays a key role in alteration of cardiac function in response to pressure overload. FASEB J 2002;16:373–8. [20] Corti F, Finetti F, Ziche M, Simons M. The syndecan-4/protein kinase Calpha pathway mediates prostaglandin E2-induced extracellular regulated kinase (ERK) activation in endothelial cells and angiogenesis in vivo. J Biol Chem 2013;288:12712–21. [21] Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000;407: 249–57. [22] Giordano FJ, Gerber HP, Williams SP, VanBruggen N, Bunting S, Ruiz-Lozano P, et al. A cardiac myocyte vascular endothelial growth factor paracrine pathway is required to maintain cardiac function. Proc Natl Acad Sci U S A 2001;98:5780–5. [23] Shyu KG, Liou JY, Wang BW, Fang WJ, Chang H. Carvedilol prevents cardiac hypertrophy and overexpression of hypoxia-inducible factor-1alpha and vascular endothelial growth factor in pressure-overloaded rat heart. J Biomed Sci 2005;12:409–20. [24] Yoon YS, Uchida S, Masuo O, Cejna M, Park JS, Gwon HC, et al. Progressive attenuation of myocardial vascular endothelial growth factor expression is a seminal event in diabetic cardiomyopathy: restoration of microvascular homeostasis and recovery of cardiac function in diabetic cardiomyopathy after replenishment of local vascular endothelial growth factor. Circulation 2005;111:2073–85. [25] Tomanek RJ. Response of the coronary vasculature to myocardial hypertrophy. J Am Coll Cardiol 1990;15:528–33. [26] Heineke J, Molkentin JD. Regulation of cardiac hypertrophy by intracellular signalling pathways. Nat Rev Mol Cell Biol 2006;7:589–600. [27] Bernardo BC, Weeks KL, Pretorius L, McMullen JR. Molecular distinction between physiological and pathological cardiac hypertrophy: experimental findings and therapeutic strategies. Pharmacol Ther 2010;128:191–227. [28] Echtermeyer F, Streit M, Wilcox-Adelman S, Saoncella S, Denhez F, Detmar M, et al. Delayed wound repair and impaired angiogenesis in mice lacking syndecan-4. J Clin Invest 2001;107:R9-14. [29] Strand ME, Herum KM, Rana ZA, Skrbic B, Askevold ET, Dahl CP, et al. Innate immune signaling induces expression and shedding of the heparan sulfate proteoglycan syndecan-4 in cardiac fibroblasts and myocytes, affecting inflammation in the pressure-overloaded heart. FEBS J 2013;280:2228–47. [30] Lim ST, Longley RL, Couchman JR, Woods A. Direct binding of syndecan-4 cytoplasmic domain to the catalytic domain of protein kinase C alpha (PKC alpha) increases focal adhesion localization of PKC alpha. J Biol Chem 2003;278:13795–802. [31] Dovas A, Yoneda A, Couchman JR. PKC alpha-dependent activation of RhoA by syndecan-4 during focal adhesion formation. J Cell Sci 2006;119:2837–46. [32] Xu H, Czerwinski P, Hortmann M, Sohn HY, Forstermann U, Li H. Protein kinase C alpha promotes angiogenic activity of human endothelial cells via induction of vascular endothelial growth factor. Cardiovasc Res 2008;78:349–55. [33] Wang A, Nomura M, Patan S, Ware JA. Inhibition of protein kinase Calpha prevents endothelial cell migration and vascular tube formation in vitro and myocardial neovascularization in vivo. Circ Res 2002;90:609–16.