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Pressure Overload Induces Early Morphological Changes in the Heart

2012; Elsevier BV; Volume: 181; Issue: 4 Linguagem: Inglês

10.1016/j.ajpath.2012.06.015

1525-2191

Colby Souders, Thomas K. Borg, Indroneal Banerjee, Troy A. Baudino,

Cardiac electrophysiology and arrhythmias

Cardiac hypertrophy, whether pathological or physiological, induces a variety of additional morphological and physiological changes in the heart, including altered contractility and hemodynamics. Events exacerbating these changes are documented during later stages of hypertrophy (usually termed pathological hypertrophy). Few studies document the morphological and physiological changes during early physiological hypertrophy. We define acute cardiac remodeling events in response to transverse aortic constriction (TAC), including temporal changes in hypertrophy, collagen deposition, capillary density, and the cell populations responsible for these changes. Cardiac hypertrophy induced by TAC in mice was detected 2 days after surgery (as measured by heart weight, myocyte width, and wall thickness) and peaked by day 7. Picrosirius staining revealed increased collagen deposition 7 days after TAC; immunostaining and flow cytometry indicated a concurrent increase in fibroblasts. The findings correlated with angiogenesis in TAC hearts; a decrease in capillary density was observed at day 2, with recovery to sham-surgery levels by day 7. Increased pericyte levels, which were observed 2 days after TAC, may mediate this angiogenic transition. Gene expression suggests a coordinated response in growth, extracellular matrix, and angiogenic factors to mediate the observed morphological changes. Our data demonstrate that morphological changes in response to cardiovascular injury occur rapidly, and the present findings allow correlation of specific events that facilitate these changes. Cardiac hypertrophy, whether pathological or physiological, induces a variety of additional morphological and physiological changes in the heart, including altered contractility and hemodynamics. Events exacerbating these changes are documented during later stages of hypertrophy (usually termed pathological hypertrophy). Few studies document the morphological and physiological changes during early physiological hypertrophy. We define acute cardiac remodeling events in response to transverse aortic constriction (TAC), including temporal changes in hypertrophy, collagen deposition, capillary density, and the cell populations responsible for these changes. Cardiac hypertrophy induced by TAC in mice was detected 2 days after surgery (as measured by heart weight, myocyte width, and wall thickness) and peaked by day 7. Picrosirius staining revealed increased collagen deposition 7 days after TAC; immunostaining and flow cytometry indicated a concurrent increase in fibroblasts. The findings correlated with angiogenesis in TAC hearts; a decrease in capillary density was observed at day 2, with recovery to sham-surgery levels by day 7. Increased pericyte levels, which were observed 2 days after TAC, may mediate this angiogenic transition. Gene expression suggests a coordinated response in growth, extracellular matrix, and angiogenic factors to mediate the observed morphological changes. Our data demonstrate that morphological changes in response to cardiovascular injury occur rapidly, and the present findings allow correlation of specific events that facilitate these changes. The process of cardiac remodeling is responsible for changes in cardiac morphology and function. Left ventricular hypertrophy, which is observed in response to a variety of pathophysiological signals, is a typical response to pressure overload or any disease state that increases cardiac wall stress and marks an adaptive response to compensate to the unfavorable conditions. Both mechanical and chemical stressors induce cardiac remodeling, and over time the adaptive response concedes to cardiac dilatation and the ensuing remodeling process becomes maladaptive, leading to dysfunction,1Dorn 2nd, G.W. Robbins J. Sugden P.H. Phenotyping hypertrophy: eschew obfuscation.Circ Res. 2003; 92: 1171-1175Crossref PubMed Scopus (222) Google Scholar possibly as a result of enhanced catecholamine chemical signaling by monoamine oxidases.2Kaludercic N. Takimoto E. Nagayama T. Feng N. Lai E.W. Bedja D. Chen K. Gabrielson K.L. Blakely R.D. Shih J.C. Pacak K. Kass D.A. Di Lisa F. Paolocci N. Monoamine oxidase A-mediated enhanced catabolism of norepinephrine contributes to adverse remodeling and pump failure in hearts with pressure overload.Circ Res. 2010; 106: 193-202Crossref PubMed Scopus (168) Google Scholar Factors associated with cardiac remodeling include myocyte hypertrophy, increased extracellular matrix (ECM) deposition, and abnormalities of the coronary vasculature.3Drazner M.H. The progression of hypertensive heart disease.Circulation. 2011; 123: 327-334Crossref PubMed Scopus (474) Google Scholar, 4Goldsmith E.C. Borg T.K. The dynamic interaction of the extracellular matrix in cardiac remodeling.J Card Fail. 2002; 8: S314-S318Abstract Full Text PDF PubMed Scopus (37) Google Scholar The latter two conditions often combine to create perivascular fibrosis, and previous studies have demonstrated that reducing myocardial fibrosis improves coronary hemodynamics.5Susic D. Varagic J. Frohlich E.D. Pharmacologic agents on cardiovascular mass, coronary dynamics and collagen in aged spontaneously hypertensive rats.J Hypertens. 1999; 17: 1209-1215Crossref PubMed Scopus (62) Google Scholar In addition, proliferation of nonmyocyte constituents (ie, fibroblasts, endothelial cells, immune cells, and smooth muscle cells) encourages disorganized tissue heterogeneity,4Goldsmith E.C. Borg T.K. The dynamic interaction of the extracellular matrix in cardiac remodeling.J Card Fail. 2002; 8: S314-S318Abstract Full Text PDF PubMed Scopus (37) Google Scholar which is initially adaptive but subsequent overcompensation induces pathological cardiac remodeling.6Weber K.T. Fibrosis and hypertensive heart disease.Curr Opin Cardiol. 2000; 15: 264-272Crossref PubMed Scopus (251) Google Scholar Two principal elements of pathological hypertrophic remodeling that lead to malfunction are accumulation of collagen and vascular remodeling.7Schwartzkopff B. Motz W. Frenzel H. Vogt M. Knauer S. Strauer B.E. Structural and functional alterations of the intramyocardial coronary arterioles in patients with arterial hypertension.Circulation. 1993; 88: 993-1003Crossref PubMed Scopus (362) Google Scholar An increase in collagen deposition stiffens the heart, resulting in systolic and diastolic dysfunction,8Burak M. Frangogiannis N.G. The role of TGF-b signaling in myocardial infarction and cardiac remodeling.Cardiovasc Res. 2007; 74: 184-195Crossref PubMed Scopus (730) Google Scholar whereas insufficient angiogenesis deprives the hypertrophic myocardium of oxygenation because of low capillary density.9Frey N. Olson E.N. Cardiac hypertrophy: the good, the bad, and the ugly.Annu Rev Physiol. 2003; 65: 45-79Crossref PubMed Scopus (1182) Google Scholar, 10de Boer R.A. Pinto Y.M. van Veldhuisen D.J. The imbalance between oxygen demand and supply as a potential mechanism in the pathophysiology of heart failure: the role of microvascular growth and abnormalities.Microcirculation. 2003; 10: 113-126Crossref PubMed Google Scholar, 11Sano M. Minamino T. Toko H. Miyauchi H. Orimo M. Qin Y. Akazawa H. Tateno K. Kayama Y. Harada M. Shimizu I. Asahara T. Hamada H. Tomita S. Molkentin J.D. Zou Y. Komuro I. p53-induced inhibition of Hif-1 causes cardiac dysfunction during pressure overload.Nature. 2007; 446: 444-448Crossref PubMed Scopus (712) Google Scholar Numerous studies have attempted to alter these remodeling processes, with varying success. Some authors have identified a relationship between the degree of hypertrophy and prognosis: higher survival rates were observed in patients treated before left ventricular end systolic diameter reaches 40 mm,12Tribouilloy C. Grigioni G. Avierinos J.F. Barbieri A. Rusinaru D. Szymanski C. Ferlito M. Tafanelli L. Bursi F. Trojette F. Branzi A. Habib G. Modena M.G. Enriquez-Sarano M. MIDA InvestigatorsSurvival implications of left ventricular end-systolic diameter in mitral regurgitation due to flail leaflets: a long-term follow-up multicenter study.J Am Coll Cardiol. 2009; 54: 1961-1968Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar thus illustrating the importance of early intervention. Others have succeeded in correlating markers of fibrosis with left ventricular hypertrophy and clinical heart failure,13Ahmed S.H. Clark L.L. Pennington W.R. Webb C.S. Bonnema D.D. Leonardi A.H. McClure C.D. Spinale F.G. Zile M.R. Matrix metalloproteinases/tissue inhibitors of metalloproteinases: relationship between changes in proteolytic determinants of matrix composition and structural, functional, and clinical manifestations of hypertensive heart disease.Circulation. 2006; 113: 2089-2096Crossref PubMed Scopus (344) Google Scholar, 14Martos R. Baugh J. Ledwidge M. O'Loughlin C. Murphy N.F. Conlon C. Patle A. Donnelly S.C. McDonald K. Diagnosis of heart failure with preserved ejection fraction: improved accuracy with the use of markers of collagen turnover.Eur J Heart Fail. 2009; 11: 191-197Crossref PubMed Scopus (98) Google Scholar indicating that the combination of hypertrophy and fibrosis results in cardiac dysfunction. In addition, it has been shown that treatment to increase capillary density after a pathological insult is accompanied by improved cardiac function, even if delayed treatment is unable to decrease myocardial infarct size,15van der Meer P. Lipsic E. Henning R.H. Boddeus K. van der Velden J. Voors A.A. van Veldhuisen D.J. van Gilst W.H. Schoebaker R.G. Erythropoietin induces neovascularisation and improves cardiac function in rats with heart failure after myocardial infarction.J Am Coll Cardiol. 2005; 46: 125-133Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar suggesting that capillary density may be more of a determining factor than is tissue remodeling. One overlooked aspect is the time course within which these changes take place after pathological insult and their progression in relation to each other. Mainly, studies have investigated only later time points (day 7 and later), when advanced stages of remodeling, adaption, and pathology have already manifested in the heart.16Konstam M.A. Kramer D.G. Patel A.R. Maron M.S. Udelson J.E. Left ventricular remodeling in heart failure: current concepts in clinical significance and assessment.JACC Cardiovasc Imaging. 2011; 4: 98-108Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar Limited studies examining early responses to pathology have uncovered important, cell-specific acute responses,17Stewart J.A. Massey E.P. Fix C. Zhu J. Goldsmith E.C. Carver W. Temporal alterations in cardiac fibroblast function following induction of pressure overload.Cell Tissue Res. 2010; 340: 117-126Crossref PubMed Scopus (37) Google Scholar but additional research is needed for a better understanding of the immediate response by the heart to injury. In the present study, we analyzed the acute morphological response within the first week after pathological cardiac insult, to determine the early progression of cardiac remodeling and correlate these remodeling events to better understand the complex coordination and how it relates to cardiac function. Here, we illustrate immediate changes in cardiac hypertrophy, angiogenesis, fibrosis, and alterations in the cell populations contributing to these events. We also propose possible gene-specific changes that may guide these morphological changes. Transverse aortic constriction (TAC) was performed in 12-week-old male C57/BL6 mice, as described previously.18Tsujita Y. Muraski J. Shiraishi I. Kato T. Kajstura J. Anversa P. Sussman M.A. Nuclear targeting of Akt antagonizes aspects of cardiomyocyte hypertrophy.Proc Natl Acad Sci USA. 2006; 103: 11946-11951Crossref PubMed Scopus (82) Google Scholar Mice were anesthetized under 3% isoflurane via intubation, the chest was opened, the aortic arch was visualized, and 6–0 silk suture was passed under the arch between the innominate and left common carotid arteries. The suture was secured around both the aorta and a 28-gauge needle, the needle was removed, the chest was closed, and the mouse was extubated. Sham-surgery mice underwent an identical procedure except for the aortic ligation. Mice were provided buprenorphine via intraperitoneal injection before recovery. Echocardiographic analysis using a Vevo2100 digital imaging system (VisualSonics, Toronto, ON, Canada) was performed under 1% isoflurane at indicated time points (0, 2, 7, 14, and 28 days), with mid ventricle M and B mode measurements acquired in the parasternal short-axis view at the level of the papillary muscles. In addition, echocardiographic analysis and blood pressure measurements were taken before surgery in all animals, to ensure an identical baseline. Adequate levels of anesthesia were monitored by toe pinch, and euthanasia was administered via cervical dislocation under 5% isoflurane. Experiments were approved by the Institutional Animal Care and Use Committee (Scott & White Hospital and Texas A&M Health Science Center) and conform to the Guide for the Care and Use of Laboratory Animals (2011, 8th edition). Hearts (n = 4) were removed at indicated time points (2, 7, and 28 days) and were flushed in ice-cold PBS. The mid left ventricular free wall and septum were isolated in 4% buffered glutaraldehyde and sectioned into 1-mm blocks. After the initial fixation, samples were postfixed in OsO4 with 2% tannic acid and then were dehydrated, embedded, and sectioned as described previously.19Nakagawa M. Price R.L. Chintanawonges C. Simpson D.G. Horacek M.J. Borg T.K. Terracio L. Analysis of heart development in cultured rat embryos.J Mol Cell Cardiol. 1997; 29: 369-379Abstract Full Text PDF PubMed Scopus (21) Google Scholar Samples were stained with uranyl acetate and lead hydroxide, and at least four separate grids were imaged for each block with a JEOL 200CX transmission electron microscope at 180 kV (JEOL, Tokyo, Japan). Low-magnification imaging was followed by high-magnification imaging, and representative images were acquired. Picrosirius-Fast Green staining was modified from a previous protocol.20Houghton P.E. Keefer K.A. Diegelmann R.F. Krummel T.M. A simple method to assess the relative amount of collagen deposition in wounded fetal mouse limbs.Wound Repair Regen. 1996; 4: 489-495Crossref PubMed Scopus (12) Google Scholar Briefly, heart sections (10 μm) were fixed in 4% paraformaldehyde, stained in a solution of 0.1% Fast Green (Acros; Thermo Fisher Scientific, Pittsburgh, PA) and Sirius Red 0.1% in saturated picric acid (Electron Microscopy Sciences, Hatfield, PA) and imaged at ×20 magnification using a Nikon Eclipse TS100 inverted microscope with a Digital Sight DS-2Mv camera and NIS-Elements software version 3.20. Images (≥6 per animal) of mid left ventricle and septum were analyzed using ImageJ software version 1.46 (NIH, Bethesda, MD). The ratio of collagen to total tissue area was calculated. Frozen heart sections (10 μm thick) were fixed and immunostained with rat anti-CD31 antibody (BD Pharmingen, San Diego, CA), anti-3G5 antibody isolated from a mouse B-lymphocyte hybridoma cell line (CRL-1814; ATCC, Manassas, VA), and a fibroblast-specific rabbit polyclonal antibody (1611) that was described in a previous report from our laboratory.21Baudino T.A. McFadden A. Fix C. Hastings J. Price R. Borg T.K. Cell patterning: interaction of cardiac myocytes and fibroblasts in three-dimensional culture.Microsc Microanal. 2008; 14: 117-125Crossref PubMed Scopus (53) Google Scholar, 22Bowers S.L.K. McFadden W.A. Borg T.K. Baudino T.A. Desmoplakin is important for proper cardiac cell-cell interactions.Microsc Microanal. 2012; 18: 107-114Crossref PubMed Scopus (9) Google Scholar Sections (≥6 per animal) from the mid left ventricle and septum were counterstained with DAPI and phalloidin and then were sequentially imaged under a TCS SP5 white light laser confocal microscope (Leica Microsystems, Wetzlar, Germany) at ×40 and ×20 magnification. ImageJ software was used for quantitative analysis, and total antibody staining was normalized to DAPI or phalloidin. Whole hearts from TAC and sham surgery (n = 4 each) were prepared at indicated time points (0, 3, 7, and 14 days), as described previously.23Banerjee I. Fuseler J.W. Price R.L. Borg T.K. Baudino T.A. Determination of cell types and numbers during cardiac development in the neonatal and adult rat and mouse.Am J Physiol Heart Circ Physiol. 2007; 293: H1883-H1891Crossref PubMed Scopus (455) Google Scholar Briefly, hearts were digested to single-cell suspensions with collagenase and then were labeled with the following antibodies conjugated to Qdot nanocrystals (Life Technologies-Invitrogen, Carlsbad, CA), according to the manufacturer's protocol: DDR2 (Santa Cruz Biotechnology, Santa Cruz, CA) for fibroblasts, α-myosin heavy chain (Abcam, Cambridge, MA) for myocytes, CD31 (Zymed Laboratories, South San Francisco, CA) for endothelial cells, and α-smooth muscle actin (R&D Systems, Minneapolis, MN) for vascular smooth muscle cells. An equal number of cells (20,000 events) for each sample were processed using an Epics XL fluorescence-activated cell sorting system (Beckman Coulter, Brea, CA) and were analyzed using the associated Expo 32 software. Total RNA was extracted at indicated time points (0, 2, and 7 days) from septa and left mid-ventricular regions of TAC and sham-surgery hearts (n = 4 each) using an RNeasy Plus mini kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. Isolated RNA was quantified and converted to cDNA with an SABiosciences RT2 first-strand kit (Qiagen), which was then quantified via quantitative real-time PCR using a StepOnePlus thermocycler (Life Technologies-Applied Biosystems, Foster City, CA). Primer sequences are available upon request. Echocardiographic, physiological, and immunofluorescent data were analyzed for significance using Student's t-test or an analysis of variance test with a Mann-Whitney test. All analyses were performed using Microsoft Excel version 12.3.3 and GraphPad Prism software version 4 (GraphPad Software, La Jolla, CA). Significance was set at P < 0.05. Hearts were examined for gross morphological changes at early (day 2) and late time points (days 7, 14, and 28) after induction of pressure overload by TAC surgery. All animals had similar baseline measurements of cardiac function and blood pressure. Survival rates after TAC were approximately 70% at 28 days after surgery, in accord with findings of similar studies from our laboratory and from others.18Tsujita Y. Muraski J. Shiraishi I. Kato T. Kajstura J. Anversa P. Sussman M.A. Nuclear targeting of Akt antagonizes aspects of cardiomyocyte hypertrophy.Proc Natl Acad Sci USA. 2006; 103: 11946-11951Crossref PubMed Scopus (82) Google Scholar Echocardiographic analysis revealed a significant increase in relative wall thickness just 48 hours after TAC surgery, which persisted through day 28 (Figure 1A). The ratio of heart weight to body weight also revealed significant cardiac growth in TAC hearts by day 2, and this increased further until by day 7 growth peaked and was maintained through day 28 (Figure 1B). To determine the extent of myocyte hypertrophy during this early growth period, myocyte width was measured in F-actin-stained cross-sections (Figure 1C). Cardiac growth 2 days after TAC cannot be attributed to myocyte growth, because no significant change in myocyte width was measured; however, a significant increase was observed at 7 days after TAC, indicating that myocyte hypertrophy is a contributing factor to the increase in heart weight and wall thickness observed by day 7. Taken together, these data indicate that cardiac growth in response to acute pressure overload is fully realized by day 7 after surgery. Examination of cardiac function revealed a lower ejection fraction observed by day 2 and a significant decrease in day 28 TAC hearts (Figure 1D). A decrease in cardiac function (ejection fraction) 28 days after TAC indicates that the hypertrophy observed here progresses into pathological decompensated cardiac hypertrophy. In addition, a lower (albeit not significantly lower) ejection fraction observed 2 days after TAC illustrates the importance of acute cardiac remodeling in response to pathological insult to quickly recover normal cardiac function and maintain cardiovascular output. Given the drastic functional changes that occur within the first week after induction of cardiac stress, we also investigated the morphological changes that occur during this early time period. In the normal murine myocardium, collagen accounts for very little of the overall tissue, which provides the malleability that the heart requires.24Weber K.T. Sun Y. Tyagi S.C. Cleutjens J.P.M. Collagen network of the myocardium: function, structural remodeling and regulatory mechanisms.J Mol Cell Cardiol. 1994; 26: 279-292Abstract Full Text PDF PubMed Scopus (431) Google Scholar, 25Banerjee I. Fuseler J.W. Intwala A.R. Baudino T.A. IL-6 causes ventricular dysfunction, fibrosis, reduced capillary density, and dramatically alters the cell populations of the developing and adult heart.Am J Physiol Heart Circ Physiol. 2009; 296: H1694-H1704Crossref PubMed Scopus (99) Google Scholar Approximately 3% of total cardiac tissue was composed of collagen in sham-surgery mice, as indicated by Picrosirius-Fast Green staining (Figure 2, A and C) and quantification (Figure 2E). Similar levels were measured in day 2 TAC hearts, indicating that collagen deposition is not an immediate physiological reaction to pressure overload (Figure 2, B and E). Conversely, a significant increase in collagen deposition was observed in day 7 TAC hearts (Figure 2, D and E), and interstitial fibrosis was evident along with dispersed focal fibrosis throughout the left ventricle and interventricular septum. Transmission electron microscopy was performed on day 2 and day 7 sham-surgery and TAC hearts and demonstrated a qualitative increase in the amount of collagen and fibroblasts present in TAC hearts, compared with sham-surgery controls (Figure 3). We also qualitatively observed dramatic increases in the mitochondria in myocytes in TAC hearts at both time points. Furthermore, in the TAC samples, the fibroblasts appeared to be more active, with abundant rough endoplasmic reticulum and enlarged Golgi apparatus, indicating high levels of secretion. These data corroborated increased collagen deposition and increased presence of secreted growth factors and cytokines in the TAC hearts. Given previous studies uncovering a strong functional link between ECM and vascular remodeling, we next examined the course of angiogenesis after TAC surgery. For long-term survival of the hypertrophic heart, increased vasculature must accompany the myocardial expansion, to maintain tissue health.26Shiojima I. Sato K. Izumiya Y. Schiekofer S. Ito M. Liao R. Colucci W.S. Walsh K. Disruption of coordinated cardiac hypertrophy and angiogenesis contributes to the transition to heart failure.J Clin Invest. 2005; 115: 2108-2118Crossref PubMed Scopus (743) Google Scholar However, the early vascular changes in response to pathological insult are poorly understood. Immunostaining and subsequent quantification (Figure 4) of the endothelial cell marker CD31 revealed a significant decrease in capillary density just 48 hours after TAC, which returned to sham-surgery levels by day 7 (Figure 4). These data indicate that the first stage of angiogenesis, destabilization of the existing vasculature,27Senger D.R. Davis G.E. Angiogenesis.Cold Spring Harb Perspect Biol. 2011; 3: a005090Crossref Scopus (216) Google Scholar occurs within 48 hours after induction of hypertrophy and that the ensuing capillary growth takes place in the days that follow, a time period that also correlates with the observed increase in ECM deposition (Figure 2, Figure 3). Pericytes are involved in vascular remodeling after injury in the heart,28Ren G. Michael L.H. Entman M.L. Frangogiannis N.G. Morphological characteristics of the microvasculature in healing myocardial infarcts.J Histochem Cytochem. 2002; 50: 71-79Crossref PubMed Scopus (139) Google Scholar and their presence represents the end of a window of vascular plasticity.29Benjamin L.E. Hemo I. Keshet E. A plasticity window for blood vessel remodeling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF.Development. 1998; 125: 1591-1598Crossref PubMed Google Scholar We therefore examined the heart for pericytes during the early remodeling process in response to TAC. A correlation with vascular remodeling was observed, with a significant increase in pericyte association with the vasculature 2 days after TAC, which returned to sham-surgery levels by 7 days (Figure 4). Although previous research using a myocardial infarction model found that pericytes were involved in long-term remodeling,28Ren G. Michael L.H. Entman M.L. Frangogiannis N.G. Morphological characteristics of the microvasculature in healing myocardial infarcts.J Histochem Cytochem. 2002; 50: 71-79Crossref PubMed Scopus (139) Google Scholar the correlation observed here suggests that pericytes play a role during the acute, early destabilization process and/or immediate reformation of the coronary vasculature in response to injury. Indeed, recent studies suggest that pericytes are attempting to stabilize vessels during the destabilization process,30Davis G.E. Saunders W.B. Molecular balance of capillary tube formation versus regression in wound repair: role of matrix metalloproteinases and their inhibitors.J Investig Dermatol Symp Proc. 2006; 11: 44-56Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar and it may be this balance that facilitates early capillary growth. In addition, great vessels lacked pericyte association only in day 7 TAC hearts (Figure 4), further indicating that large-scale vascular plasticity is present at this time point in TAC animals, but not in the earlier day 2 TAC or sham-surgery animals. The cardiac fibroblast is another cell that has been identified as an important angiogenic potentiator.31Liu H. Chen B. Lilly B. Fibroblasts potentiate blood vessel formation partially through secreted factor TIMP-1.Angiogenesis. 2008; 11: 223-234Crossref PubMed Scopus (83) Google Scholar, 32Souders C.A. Bowers S.L.K. Baudino T.A. Cardiac fibroblast: the renaissance cell.Circ Res. 2009; 105: 1164-1176Crossref PubMed Scopus (711) Google Scholar Immunostaining with the fibroblast-specific antibody 161121Baudino T.A. McFadden A. Fix C. Hastings J. Price R. Borg T.K. Cell patterning: interaction of cardiac myocytes and fibroblasts in three-dimensional culture.Microsc Microanal. 2008; 14: 117-125Crossref PubMed Scopus (53) Google Scholar, 22Bowers S.L.K. McFadden W.A. Borg T.K. Baudino T.A. Desmoplakin is important for proper cardiac cell-cell interactions.Microsc Microanal. 2012; 18: 107-114Crossref PubMed Scopus (9) Google Scholar revealed a close association of fibroblasts with the microvasculature and myocardium under all experimental conditions (Figure 4). However, quantification revealed a significant up-regulation of the fibroblast population 7 days after TAC, which was not observed at the day 2 time point (Figure 4). Fibroblast-specific staining with 1611 was confirmed by staining with anti-vimentin and anti-DDR2 antibodies (data not shown). As expected, this rise in the fibroblast population is concomitant with fibrosis (Figure 2), but it may also be critical to the angiogenic process in terms of stimulating revascularization of the tissue after the observed early capillary regression. Given the extensive changes in cardiac cell populations, we analyzed cell proliferation to further infer growth rate and cell origin. In response to injury, new nonmyocyte cells arise within the heart via recruitment of circulating progenitors (fibrocytes), proliferation of resident cells, or differentiation of other cell types.33Norris R.A. Borg T.K. Butcher J.T. Baudino T.A. Banerjee I. Markwald R.R. Neonatal and adult cardiovascular pathophysiological remodeling and repair: developmental role of periostin.Ann N Y Acad Sci. 2008; 1123: 30-40Crossref PubMed Scopus (110) Google Scholar, 34Nikam V.S. Schermuly R.T. Dumitrascu R. Weissmann N. Kwapiszewska G. Morrell N. Klepetko W. Fink L. Seeger W. Voswinckel R. Treprostinil inhibits the recruitment of bone marrow-derived circulating fibrocytes in chronic hypoxic pulmonary hypertension.Eur Respir J. 2010; 36: 1302-1314Crossref PubMed Scopus (35) Google Scholar To determine the amount of resident cardiac cell proliferation and growth in response to TAC, we analyzed phosphorylated histone H3 (p-histone H3) expression at 2, 7, and 28 days after surgery (Figure 5, A–F). Although significant proliferation and/or growth was observed in the total cardiac cell population at day 2 in TAC hearts, compared with sham-surgery counterparts, a further increase was observed in day 7 TAC hearts (Figure 5G). In addition, the proportion of p-histone H3-positive cells shifted from a 1:1 ratio of myocytes to nonmyocytes to 1:3 by day 7 after TAC, indicating that the increased proliferative rates are due mainly to nonmyocyte populations. Interestingly, both total proliferating cells and the myocyte/nonmyocyte ratio returned to sham-surgery levels by day 28 (Figure 5G). To quantitatively analyze the changes in specific cell populations, we used flow cytometry to measure cell populations in TAC and sham-surgery animals at 3, 7, and 14 days after surgery. In accord with our immunostaining findings, the fibroblast population does not increase until 7 days after TAC, at which point it remains elevated through day 14 (Figure 6). Although it appears that the myocyte population increases, absolute quantities do not actually change, and it is only th