The Ski/Zeb2/Meox2 pathway provides a novel mechanism for regulation of the cardiac 1myofibroblast phenotype 2

3

Ryan H. Cunnington1,3†, Josette M. Northcott 2,3†, Saeid Ghavami 1,3, Krista L. Bathe 1,3, 4Fahmida Jahan2,3, Morvarid Kavosh1,3, Jared Davies1,3, Jeffrey T. Wigle 2,3, and Ian M.C. 5

Dixon1,3* 6

1Department of Physiology, University of Manitoba; 2Department of Biochemistry and Medical 7Genetics and 3 Institute of Cardiovascular Sciences University of Manitoba, Winnipeg, Canada 8

Running title: Ski, Zeb2, Meox2, and cardiac fibroblast phenotype 9 10

Word count: ~7428 11 12

† Co-first authorship 13*Address for correspondence: 14 15Ian M.C. Dixon, PhD, FIACS 16Professor of Physiology 17Director, Molecular Cardiology Lab 18Institute of Cardiovascular Sciences 19St. Boniface Research Centre 20351 Tache Avenue 21Winnipeg, Manitoba, Canada 22R2H 2A6 23 24idixon@sbrc.ca 25www.sbrc.ca 26 27 28 29 30

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© 2013. Published by The Company of Biologists Ltd.Jo

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JCS Advance Online Article. Posted on 23 October 2013

Summary 32

Background: Cardiac fibrosis is linked to fibroblast to myofibroblast phenoconversion and 33

proliferation; mechanisms underlying this phenoconversion are poorly understood. c-Ski (Ski) is 34

a negative regulator of TGF-β/Smad signaling in myofibroblasts, and may redirect the 35

myofibroblast phenotype back to fibroblasts. Meox2 may alter TGF-β-mediated cellular 36

processes and is repressed by Zeb2. Hypothesis: Ski diminishes the myofibroblast phenotype by 37

de-repressing Meox2 expression and function via repression of Zeb2 expression. Results: Meox1 38

and Meox2 mRNA expression, Meox2 protein expression are reduced during phenoconversion 39

of fibroblasts to myofibroblasts. Meox2 over-expression shifts the myofibroblasts to fibroblasts, 40

whereas the Meox2 DNA-binding mutant has no effect on myofibroblast phenotype. Ski over-41

expression partially restores Meox2 mRNA expression levels to those in cardiac fibroblasts. 42

Expression of Zeb2 increased during phenoconversion and Ski over-expression reduces Zeb2 43

expression in first-passage myofibroblasts. Meox2 expression is decreased in scar following 44

myocardial infarction, whereas Zeb2 protein expression increases in the infarct scar. Thus Ski 45

modulates the cardiac myofibroblast phenotype and function via suppression of Zeb2 by up-46

regulating Meox2. This cascade may regulate cardiac myofibroblast phenotype and presents 47

therapeutic options for treatment of cardiac fibrosis. 48

49

Keywords: myofibroblasts; heart; phenoptype switch; fibrosis; TGF-β50

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Introduction 51

Fibroblast to myofibroblast phenoconversion in the heart is a critical event in the onset of many 52

cardiovascular diseases. Cardiac fibroblasts are an understudied cell despite the fact that they 53

constitute the majority of cells in the heart (Weber and Brilla, 1991). These relatively quiescent 54

cells reside in the cardiac interstitium and synthesize cardiac fibrillar collagen types (e.g., types I 55

and III), as well as the majority of other extracellular matrix proteins. 56

Cardiac stress and injury both induce differentiation of fibroblasts into hypersecretory 57

and contractile myofibroblasts which can persist in the myocardium for months (and even years) 58

after the initial pathological insult (Willems et al., 1994). The myofibroblast is associated with 59

fibrillar collagen deposition leading to reduced cardiac efficiency and, ultimately, 60

decompensated heart failure. Initially, increased collagen deposition is beneficial, but when left 61

to persist for years, it may transform healthy myocardium with normal extracellular matrix 62

complement to overtly fibrosed tissue with abnormal expansion of the interstitium (Freed et al., 63

2005). 64

Fibroblast-to-myofibroblast phenoconversion is marked by increased expression of α-65

smooth muscle actin (α-SMA) (Darby et al., 1990), extra domain A (ED-A) fibronectin (Serini et 66

al., 1998), and non-muscle myosin heavy chain b (SMemb) (Frangogiannis et al., 2000). 67

Although not well understood, this process is typically thought of as being a “one-way” pathway. 68

However, Hinz and colleagues have provided data that both forward and reverse 69

phenoconversion are possible in these cells depending upon the compressibility of the underlying 70

substrate (Hinz et al., 2001). Still, the molecular mechanisms for fibroblast/myofibroblast inter-71

conversion have yet to be fully elucidated. 72

The canonical TGF-β signaling cascade has been implicated as an important inducer of 73

phenoconversion. High levels of this cytokine are associated with cardiac fibrosis (Brooks and 74

Conrad). The diverse functions of TGF-β necessitate the tight control of membrane-to-nucleus 75

signal transduction; in particular, levels of the endogenous inhibitors Smad7 and Ski. The proto-76

oncoprotein Ski has a complex set of functions and is linked to skeletal muscle hypertrophy 77

(Sutrave et al., 1990a; Sutrave et al., 1990b), as well as developmental defects in the neural tube 78

and cranial mesenchyme (Berk et al., 1997). Ski binds to R-Smads (Sun et al., 1999; Suzuki et 79

al., 2004; Ueki and Hayman, 2003; Xu et al., 2000) and may inhibit TGF-β mediated effects 80

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through both nuclear and cytosolic mechanisms (Akiyoshi et al., 1999; Ferrand et al., 2010; 81

Nagata et al., 2006; Suzuki et al., 2004). We have previously demonstrated the anti-fibrotic and 82

anti-contractile effects of Ski over-expression in TGF-β1 stimulated primary cardiac 83

myofibroblasts, and have shown these inhibitory effects to be orchestrated at the nuclear level 84

(Cunnington et al., 2011). 85

Mesenchyme homeobox 2 (Meox2, also known as growth arrest specific homeobox 86

protein, Gax) and Meox1 comprise the Meox family of homeodomain proteins. Meox1 and 87

Meox2 proteins share 95% sequence identity within the homeodomain region, but are otherwise 88

highly divergent (Douville et al., 2011). Both Meox1 and Meox2 are required for proper bone 89

and muscle formation in developing mouse embryos (Mankoo et al., 1999; Mankoo et al., 2003). 90

Meox2 expression is decreased by mitogen stimulation of vascular smooth muscle cells (Gorski 91

et al., 1993) and in mechanically damaged arteries (Weir et al., 1995), indicating that Meox2 92

expression is sensitive to a range of stimuli. Meox2 may enhance TGF-β mediated inhibition of 93

cell proliferation (Valcourt et al., 2007) or, conversely, may block TGF-β induced epithelial-to-94

mesenchymal transition (EMT) (Valcourt et al., 2007). Meox2 expression is repressed by the 95

Zeb2 protein (also called Smad interacting protein 1 (Sip1 or Zfhx1b)), a zinc-finger E-box 96

binding protein (Chen et al., 2010). Zeb2 expression itself has been shown to be positively 97

regulated by TGF-β signaling during EMT (Comijn et al., 2001). 98

In this study we provide data to support a novel mechanism through which Ski regulates 99

the cardiac fibroblast phenotype. We show for the first time that both Meox1 and Meox2 are 100

down-regulated during fibroblast to myofibroblast phenoconversion, and that over-expression of 101

Ski can rescue Meox2 expression. Moreover, our data establish a link between increased Meox2 102

expression and diminution of the myofibroblast phenotype. Finally, we identify a putative 103

signaling cascade that may modulate fibroblast to myofibroblast phenoconversion through the 104

differential expression of Ski, Zeb2 and Meox2 proteins. 105

106

Results 107

Meox1 and Meox2 are down-regulated during phenoconversion – Marker proteins frequently 108

used to monitor fibroblast-to-myofibroblast phenoconversion include an increase in expression 109

of α-SMA (Darby et al., 1990), ED-A fibronectin (Serini et al., 1998) and SMemb 110

(Frangogiannis et al., 2000). Myofibroblasts in the infarct scar may arise from a range of 111

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different processes, e.g., fibroblast migration (Gabbiani, 1996), differentiation of bone marrow 112

progenitor cells (Mollmann et al., 2006; van Amerongen et al., 2008) and EMT (van Tuyn et al., 113

2007; Zhou et al., 2010). As Meox2 is associated with inhibition of TGF-β1 induced EMT 114

(Valcourt et al., 2007), we examined the potential link between Meox2 and induction of the 115

myofibroblast phenotype in rat primary cardiac fibroblasts, and in first and second passage 116

myofibroblasts. To our knowledge, Meox1, closely related to Meox2, has not been implicated in 117

EMT or TGF-β1 signaling. Therefore we also examined its putative role in fibroblast to 118

myofibroblast phenoconversion. We observed a 96 and 99% down-regulation of Meox2 mRNA 119

expression during the phenoconversion (Figure 1A) of primary fibroblasts (P0) to first and 120

second passage myofibroblasts (P1 and P2), respectively. Meox1 mRNA decreased with 121

increasing passage in an identical manner to that of Meox2 (Figure 1A). Western analysis 122

demonstrated that Meox2 protein expression is also significantly decreased during fibroblast to 123

myofibroblast phenoconversion (Figure 1B). 124

Meox2 expression is decreased in post-myocardial infarct (post-MI) scar – To assess the 125

relevance of Meox2 expression to myofibroblast phenotype in vivo, we performed 126

immunohistochemistry on 4 week post-MI hearts (Figure 1C). Vimentin staining (red color in 127

composite) intensity was increased in the infarct scar region showing the predominance of 128

mesenchymal cells (e.g., myofibroblasts) in the infarct scar. We observed a substantial reduction 129

in Meox2 staining (green color in composite) in the scar portion of the heart compared to the 130

remnant heart. Nuclei were stained with DAPI (blue color). 131

Meox2, but not Meox1, over-expression diminishes the myofibroblast phenotype – As Meox1 and 132

Meox2 mRNA expression is down-regulated during fibroblast-to-myofibroblast differentiation, 133

we hypothesized that re-introduction of these proteins in first passage myofibroblasts would 134

restore the fibroblastic phenotype. Using an adenoviral system for over-expression of Meox1 and 135

Meox2, we observed that Meox2 was able to reduce the expression of the myofibroblast markers 136

α-SMA and ED-A fibronectin (Figure 2A and 2B). In contrast, Meox1 had no effect on 137

expression of these markers (Figure 2). Notably, over-expression of Meox1 or Meox2 did not 138

affect SMemb expression (Figure 2). Finally, over-expression of a DNA binding deficient 139

version of Meox2 (Meox2DBDmt) (Douville et al., 2011) was unable to alter the myofibroblast 140

phenotype (Figure 2C and 2D). 141

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Meox2 over-expression induces myofibroblast proliferation - We have previously shown that 142

Meox2 expression induces cell cycle arrest and senescence in vascular endothelial cells 143

(Douville et al., 2011). However, in cardiac myofibroblasts, Meox2 over-expression induces 144

significant (p <0.01, p < 0.001) proliferation (Figure 3) indicating that Meox2-mediated effects 145

on cellular proliferation are cell type dependent. 146

Ski diminishes, but does not fully reverse, the myofibroblast phenotype - Our lab has previously 147

shown the ability of Ski over-expression to diminish expression of the myofibroblast phenotypic 148

marker α-SMA in first passage myofibroblasts (Cunnington et al., 2011). To extend this finding, 149

we tested the expression of additional markers of phenoconversion by Western analysis. 150

Following Ad-Ski transduction for 24 hours, we observed that Ski over-expression was able to 151

significantly reduce ED-A fibronectin expression, yet had no effect on the expression of SMemb 152

(Figure 4), as we had observed following over-expression of Meox2 (Figure 1C and 1D). Thus, 153

while the fibroblastic phenotype was not fully restored, the myofibroblastic phenotype was 154

significantly diminished. 155

Ski increases Meox2, but not Meox1, mRNA expression – Given the identical effects of Ski and 156

Meox2 over-expression on diminution of the myofibroblast phenotype, we hypothesized that a 157

link may exist between these two proteins. We therefore transduced first passage cardiac 158

myofibroblasts with Ad-Ski and then measured the mRNA expression levels of Meox1 and 159

Meox2 with and without TGF-β1 stimulation (10 ng/ml for 24 h). In the absence of TGF-β1 160

stimulation, Ad-Ski over-expression resulted in a ~5.5 fold increase in Meox2 mRNA expression 161

levels over controls, but produced no discernible effect on Meox1 levels (Figure 5). Following 162

TGF-β1 stimulation, Meox1 mRNA levels were, again, unaltered, whereas those of Meox2 163

increased ~7.5 fold above control levels. However, this increase was not found to be 164

significantly different when compared with the Meox2 levels attained by Ad-Ski over-expression 165

in the absence of TGF-β1 stimulation (Figure 5). 166

Zeb2 expression increases during fibroblast-to-myofibroblast differentiation – As down-167

regulation of Meox2 mRNA and protein is associated with phenoconversion, we examined the 168

potential role of Zeb2 (a repressor of Meox2 transcription), in fibroblast-to-myofibroblast 169

differentiation. Proteins were isolated from primary cardiac fibroblasts and first and second 170

passage myofibroblasts and tested for Zeb2 protein expression. We observed a significant up-171

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regulation of Zeb2 protein in P1 and P2 myofibroblasts as compared to primary fibroblasts 172

(Figure 6A, C). 173

Over-expression of Ski reduces Zeb2 expression – Since the Zeb2 protein has been shown to 174

repress Meox2 expression in human umbilical vein endothelial cells (Chen et al., 2010), we 175

examined Ski over-expressing cells for Zeb2 protein expression. Following 48 hours of 176

incubation with Ad-Ski, we observed a significant reduction in Zeb2 protein expression, as 177

compared with non-transduced and Ad-LacZ transduced P1 myofibroblasts (Figure 6 B, D). 178

These data provide a link between Ski, Zeb2 and Meox2 expression levels in modulating cardiac 179

fibroblast/myofibroblast phenotype. 180

Meox2 decreases while Zeb2 increases in cytoplasm following myocardial infarction (MI)-We 181

have previously shown that Ski accumulates in the cytoplasm after myocardial infarction 182

(Cunnington et al., 2011). We examined Meox2 and Zeb2 expression in an in vivo coronary 183

ligation model of MI. We observed a significant decrease in Meox2 expression in the cytosolic 184

fraction at 48 h and 4 weeks post-MI scar portion of the heart as compared to sham and viable 185

tissue. While expression of Meox2 at 2 weeks post-MI also decreased in the scar, the change was 186

not significant (Figure 7). Zeb2 expression was significantly increased in the cytosolic fractions 187

48 h and 2 weeks post-MI, but was decreased at 4 weeks post-MI in the scar portion of the heart 188

compared with sham heart and viable tissue (Figure 7). 189

Discussion 190

Cardiac fibroblast-to-myofibroblast phenoconversion constitutes an important area of 191

investigation due to the long-term deleterious consequences of chronic fibrosis in the heart. 192

However, the mechanism(s) by which this phenomenon occurs is/are poorly understood. 193

Myofibroblasts arise in the heart in response to the stress/injury associated with MI, 194

hypertension, diabetic cardiomyopathy, as well as other cardiomyopathies (Gonzalez-Vilchez et 195

al., 2005; Gonzalez et al., 2002; Ihm et al., 2007; Pelouch et al., 1993). These cells contribute to 196

inappropriate myocardial remodeling that is characterized by the deposition of excessive fibrillar 197

collagens which impair overall cardiac function and hasten the progression into heart failure. 198

Myofibroblasts persist in the injured heart for years (Willems et al., 1994) and the mechanism for 199

the rescue of the quiescent fibroblastic phenotype in vivo has yet to be determined. 200

The importance of mechanical tension in regulating fibroblast/myofibroblast 201

differentiation has been shown in vitro and in vivo (Hinz et al., 2001) (Wang et al., 2003); (Arora 202

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et al., 1999). Others have also identified stretch as being a modulator of myofibroblast phenotype 203

(Thayer et al., 2011). Additional factors have been shown to affect myofibroblast phenotype, for 204

example Wnt3a signaling (Carthy et al., 2011). Knowledge of the precise mechanisms mediating 205

this process is vital to creating an effective treatment for fibrotic diseases of the heart. 206

In this study, we have identified a novel signaling pathway that regulates the 207

differentiation of primary cardiac fibroblasts to myofibroblasts (Figure 8), and highlights the 208

involvement of three proteins that are associated with phenoconversion, namely, Ski, Meox2 and 209

Zeb2. Herein we provide evidence that Ski de-represses Meox2 via repression of Zeb2 to reduce 210

the myofibroblast phenotype (i.e., α-SMA and ED-A fibronectin expression) and, taken with our 211

previous study (Cunnington et al., 2011) thereby reduces collagen synthesis/secretion and 212

contracture. 213

Figure 3 provides evidence that Meox2 overexpression induces cardiac myofibroblast 214

proliferation. We postulate that the finding of decreasing myofibroblast marker expression does 215

not reflect a hypoproliferation of cells. Rather, this indicates the decreased averaged expression 216

of cardiac myofibroblast markers from each cell en masse. Seminal work by Gabbiani and 217

colleagues has led to the well accepted tenet that transition from so-called fibroblastic phenotype 218

to a differentiated supermature myofibroblast is not defined as an on/off event, but is a 219

continuum (Desmouliere et al., 2005). Thus one may observe altered expression of salient 220

markers and proliferative capacity in cells that retain the relative contractility and hypersecretory 221

characteristics of myofibroblasts. While they may not be supermature myofibroblasts, they 222

retain high expression of the specific markers. While Gabbiani et al carried out their work 223

largely in skin myofibroblasts, we have extended this work to cardiac myofibroblasts with the 224

work of (Santiago et al., 2010) which showed a similarly detailed graded expression of 225

myofibroblastic markers both in vivo and in vitro. Further, in our studies on Ski (Cunnington et 226

al., 2011) we found that Ski acts a molecular rheostat to “dial back” the expression of salient 227

markers for myofibroblasts in these cells. Thus, we interpret Figure 3 results to represent 228

increased proliferation of a population with an intermediate phenotype, still categorized as 229

myofibroblasts. 230

In addition to the changes in protein levels typically observed with the more commonly 231

used indicators of fibroblast-to-myofibroblast phenoconversion (i.e., α-SMA, ED-A fibronectin, 232

and SMemb) (Darby et al., 1990; Frangogiannis et al., 2000; Serini et al., 1998), we show that 233

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Meox1 and Meox2 mRNA levels are down-regulated during phenoconversion. Our observation 234

that a loss of Meox2 protein is associated with phenoconversion suggests they may be important 235

in maintaining the quiescent phenotype. Moreover, increased Zeb2 protein expression, associated 236

with increasing passage number, points to a direct molecular mechanism for the down-regulation 237

of Meox2 and subsequent change in cellular phenotype. Thus, Meox2 and Zeb2 form part of a 238

signal cascade that is regulated by Ski, and drives the differentiation of fibroblasts into 239

hypersynthetic myofibroblasts (Figure 8). Although fibroblast-to-myofibroblast phenoconversion 240

is a complex process that likely requires a myriad of signals to proceed, our current results shed 241

light on the nature of these pathways. 242

If treatment of primary cells with Ski or Meox2 adenoviruses were enriching a fibroblast 243

population (as opposed to reducing myofibroblast phenotype) then the expected result would be 244

a decrease in all myofibroblast markers. Our results do show a decrease in alpha-SMA and ED-A 245

fibronectin, but do not show a significant change in SMemb (a third myofibroblast marker). We 246

interpret this result to indicate a diminution of the myofibroblast cell phenotype rather than an 247

enrichment of a fibroblast sub-population. 248

Ad-Ski or Ad-Meox2 transduction was able to partially reverse the myofibroblastic 249

phenotype as indicated by a reduction in α-SMA and ED-A fibronectin expression levels. 250

However, exogenous application of these two proteins could not fully restore the fibroblastic 251

phenotype as characterized by steady levels of SMemb. Thus, we interpret this finding as 252

indicating that the cells convert to a proto-myofibroblastic phenotype. Although Meox1 and 253

Meox2 share a high degree of amino acid sequence identity within their homeodomains, they are 254

otherwise quite divergent from each other (Douville et al., 2011). Therefore, the observation that 255

Meox1and Meox2 did not exert the same effects on P1 myofibroblasts was not necessarily 256

surprising. Meox1 mRNA expression was dramatically reduced in first passage myofibroblasts 257

as compared to primary fibroblasts. However, over-expression of this protein did not modulate 258

the expression of any of the myofibroblastic phenotype markers analyzed. However, Meox1 may 259

affect the expression of other proteins related to phenoconversion that were not examined in this 260

study. Alternatively, Meox1 may not directly affect fibroblast/myofibroblast phenotype at all, but 261

is simply down-regulated as a “side effect” of other mechanisms operating during the process of 262

phenoconversion. The DNA binding deficient version of Meox2, Meox2DBDmt, was also 263

unable to elicit changes in myofibroblast phenotype, indicating that an interaction of Meox2 with 264

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DNA is necessary to alter the gene expression patterns that are essential for a diminished 265

myofibroblastic phenotype. The mechanism of these changes is currently unknown. 266

We found that Ski, Meox2 and Zeb2 are relatively unregulated in viable eg, noninfarcted 267

tissues at various times following myocardial infarction, and that regulation is significant in the 268

infarct scar in the viable region with these three genes at many of the times assessed in the data 269

from these two different papers. As we know that the infarct scar is populated mainly by 270

myofibroblasts in the infarct scar (Santiago et al., 2010), and the viable cardiac tissue is 271

composed of at least 4 different cell types including cardiac myocytes, we expect the diversity of 272

cell types may be at the basis of this disparity between regions. This cumulative finding may 273

also point to the existence of another regulatory network specific to the viable region. 274

In subcellular fractionation experiments designed to detect cytosolic Meox2, we observed 275

a significant reduction of expression in early (48 hr) and late (4 weeks) time points following 276

myocardial infarction (Figure 7). In contrast, Zeb2 was significantly elevated in early (48 hr and 277

2 weeks post-myocardial infarction) and reduced in late stage (4 weeks post-myocardial 278

infarction) relative to sham-operated and viable sample averages . These contrasting expression 279

patterns are consistent with our model of signaling response following myocardial injury (Figure 280

8). Further, our immunofluorescence data of infarcted rat heart revealed a striking relative 281

reduction of Meox2 (Figure 1C), which may underscore the pathophysiological relevance of this 282

pathway in this model of human heart disease, as the infarct scar is populated by myofibroblasts 283

(Santiago et al., 2010). 284

In the infarct scar, we found evidence for elevated cytosolic Zeb2. This finding may 285

point to a new mechanism for Zeb2-mediated regulation of Meox2. For example, Zeb2 could be 286

decreasing Meox2 indirectly via sequestration to the cytoplasm of transcriptional activators of 287

Meox2 expression, much as C184M sequesters receptor-activated Smad2 (Kokura et al., 2003). 288

The elucidation of this possible regulatory mode will be an object of our future studies. 289

We have previously shown that Ski expression increases as fibroblasts phenoconvert into 290

myofibroblasts, both in culture and during cardiac remodeling following myocardial infarction 291

(Cunnington et al., 2011). This result may seem counter intuitive in relation to the observed 292

effect of exogenous Ski application decreasing Zeb2 expression and diminishing the 293

myofibroblast phenotype. However, in the infarct scar, Ski localization shifts from nuclear to 294

cytoplasmic during the progression of cardiac remodeling and the associated fibroblast-to-295

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myofibroblast phenoconversion (Cunnington et al., 2011). Ad-Ski localizes to the nucleus and 296

causes diminution of myofibroblast phenotype. Thus, Ski localization is important in 297

determining its function. Within the nucleus, we propose that Ski, via inhibiting Smad signaling, 298

inhibits Zeb2 production thus maintaining an elevated level of Meox2 that helps to keep the 299

fibroblast in a quiescent state. When Ski translocates to the cytosol, however, the mechanisms 300

required to maintain the quiescent phenotype are disabled and the cell differentiates into a 301

myofibroblast. Thus, nuclear Ski may act as a cellular “brake” in preventing differentiation of 302

fibroblasts to myofibroblasts via its ability to inhibit Zeb2 expression, thereby relieving Zeb2's 303

inhibitory influence on Meox2 expression levels. In the damaged heart, however, that “brake” 304

may be released by the shuttling of Ski from the nucleus to the cytosol. 305

Understanding the specific mechanisms that orchestrate fibroblast-to-myofibroblast 306

phenoconversion is critical in developing effective strategies for the treatment of fibrotic heart 307

disease. The current results outline a novel signaling pathway that regulates phenoconversion 308

between cardiac fibroblasts and myofibroblasts. 309

310

Materials and Methods 311

Cell isolation/culture: Cardiac fibroblasts were isolated as described previously (Hao et al., 312

2000). Briefly, hearts from adult male Sprague-Dawley rats (150-200g) were subjected to 313

Langendorff perfusion with DMEM-F12 (Gibco, Carlsbad, CA, USA) followed by SMEM 314

(Gibco, Carlsbad, CA, USA). Perfused hearts were digested with 0.1% w/v collagenase type 2 315

(Worthington) in SMEM for 20 minutes, removed from the Langendorff apparatus, and 316

minced/triturated in dilute collagenase solution (0.05% w/v) for 15 minutes before addition of 317

growth media (DMEM-F12 supplemented with 10% fetal bovine serum (FBS), 100 U/ml 318

penicillin (Gibco BRL), 100 µg/ml streptomycin (Gibco, Carlsbad, CA, USA), and 1 µM 319

ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA)). Upon settling of large tissue pieces in a 50 320

ml conical tube, the supernatant was recovered and centrifuged at 750 x g for 5 minutes. Cell 321

pellets were resuspended in growth media and plated in 75 cm2 culture flasks. Cells were 322

allowed to adhere for 2-3 hours in a 5% CO2 37oC incubator, then washed twice with phosphate-323

buffered saline (PBS) followed by the addition of fresh growth media. The media was changed 324

the following day and cells allowed to grow for 3-4 days before passaging into P1 325

myofibroblasts. P1 myofibroblasts were infected with adenovirus at the time of plating. 326

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Transforming growth factor beta 1 (TGF-β1) cytokine (Cell Signaling, Danvers, MA, USA) (10 327

ng/ml) treatment was applied to cells following a 24 hr period of serum starvation. 328

Adenoviral constructs: Generation of the HA-Ski adenoviral vector was previously described 329

(Cunnington et al., 2011). Construction of the Meox1, Meox2 and Meox2 DNA binding deficient 330

adenoviruses (Meox2DBDmt) was previously described (Douville et al., 2011). 331

Protein isolation: Following treatment, cells were washed twice with PBS. RIPA lysis buffer 332

containing protease inhibitor cocktail (Sigma, St. Louis, MO, USA) and phosphatase inhibitors 333

(10 mmol/L NaF, 1 mmol/L sodium orthovanadate, and 20 mmol/L β-glycorophosphate) was 334

used to lyse cells and, following mechanical scraping, cell lysates were incubated on ice for 1 335

hour. Lysates were sonicated for 3 x 10 seconds and then centrifuged at 8765 x g at 4oC for 15 336

minutes. Supernatants were transferred to new tubes and protein assays performed using the 337

bicinchoninic acid method (Smith et al., 1985). 338

Western Blot analysis: SDS-PAGE of 20-40 ug of protein was performed on 6-8% gels using 339

SeeBlue Plus2 Pre-Stained Standard (BioRad, Hercules, CA, USA) molecular weight marker. 340

Proteins were transferred to 0.45 µM polyvinylidene difluoride (PVDF) membrane and blocked 341

in PBS with 0.2% Tween-20 (PBS-T) containing 10% skim milk for 1.5 hours at room 342

temperature with constant shaking. Primary antibodies were diluted in PBS-T with 3% skim milk 343

as follows: Zeb2 (1:1,000) (Sigma, St. Louis, MO, USA), β-tubulin (1:5,000) (Abcam, 344

Cambridge, UK), α-smooth muscle actin (1:5,000) (Sigma, St. Louis, MO, USA), eukaryotic 345

elongation factor 2 (eEF2) (1:3,000) (Cell Signaling, Danvers, MA, USA), ED-A fibronectin 346

(1:1,000) (Chemicon, Temecula, CA, USA), SMemb (1:1,000) (Abcam, Cambridge, UK), FLAG 347

[M2] (1:10,000) (Sigma, St. Louis, MO, USA), Meox2 [JJ-7] (1:1000) (Santa Cruz 348

Biotechnology. Santa Cruz, CA, USA), α-tubulin (1:2000) (Abcam, Cambridge, UK). 349

Membranes were incubated for 1.5 hours at room temperature or overnight at 4oC with shaking. 350

Secondary antibodies were horseradish peroxidase (HRP)-labeled anti-rabbit and HRP-labeled 351

anti-mouse antibodies, which were diluted 1:10,000 with PBS-T with 3% skim milk and 352

incubated for 1.5 hours at room temperature with shaking. Protein bands were visualized using 353

ECL or ECL Plus (Amersham, Arlington Heights, IL, USA) according to the manufacturer's 354

instructions, and developed on X-ray film. Equal protein loading was confirmed using β-tubulin , 355

α-tubulin or eEF2 staining following stripping and re-probing of the Western blots. 356

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RNA isolation: Cardiac fibroblasts/myofibroblasts in 100 mm dishes were harvested for RNA 357

using GenElute Mammalian Total RNA miniprep kits (Sigma, St. Louis, MO, USA) or RNeasy 358

Plus kits (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA was 359

diluted in 10 mM Tris (pH 7.5) and the absorbance at 260 nm (A260) was used to determine RNA 360

concentration. RNA purity was assessed using the ratio of absorbance at 260 nm to absorbance at 361

280 nm (A260/A280). 362

qPCR: Isolated RNA was treated with RQ1 RNase-Free DNase according to the manufacturer’s 363

instructions. One-step real-time PCR was performed using an iQ5 thermocycler (BioRad, 364

Hercules, CA, USA) and BR 1-Step SYBR Green qRT-PCR kits (Quanta, Gaithersburg, MD, 365

USA). Relative gene quantification (2-ΔΔCT method) was performed whereby the mRNA 366

expression levels of Meox1 and Meox2 were compared to the mRNA expression of GAPDH. 367

Primer sets for Meox1(amplicon = 123 bp): ratMEOX1forward - 5'-tcgcccaccataactacctg-3', 368

ratMEOX1reverse - 5'-ccttcacacgcttccacttc-3'; Meox2 (amplicon = 114 bp): ratMEOX2forward - 369

5'-ttagcgggctctgctcaaac-3', ratMEOX2reverse - 5'-ttcacgaaggtcccaaagtc-3'; GAPDH (amplicon = 370

87 bp): GAPDH forward - 5’-tgcaccaccaactgcttagc-3’, GAPDH reverse - 5’-371

ggcatggactgtggtcatgag-3’. Parameters for qPCR amplification steps available upon request. 372

EdU cell proliferation assay: P3 myofibroblasts (40% confluent) were infected with Meox2 and 373

LacZ adenovirus (50 and 100 MOI). Click-iT™ EdU flow cytometry assay kit (Invitrogen,) was 374

used to further investigate Meox2 effects on primary rat myofibroblast proliferation. Briefly, 5-375

ethynyl-2´-deoxyuridine (EdU) reagent was added at a 10 µM concentration 16 after infection 376

and cells were harvested 8 h later. Cells were collected, fixed and then incubated with saponin-377

based permeabilization buffer. Click-iT reaction cocktail containing copper sulphate and 378

fluorescent dye azide (Alexa Fluor® 488) was freshly prepared and added to the samples. EdU 379

incorporation into DNA was assessed using flow cytometry. Simultaneously, cells were counted 380

using a hemocytometer to confirm the EdU FACS assay (Hesam Movassagh et al., 2013; In 381

press). 382

Experimental rat model of MI and cellular fractionation: Experimental protocols for animal 383

studies were approved by the Animal Care Committee of the University of Manitoba, Canada, 384

and conform to the guidelines established by the Canadian Institutes of Health Research and the 385

Canadian Council on Animal Care (2001). MI was induced in male Sprague-Dawley rats (150–386

175 g) by surgical occlusion of the left coronary artery, as described previously (Dixon et al., 387

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1990). The mortality of the animals operated on in this fashion was 30% within 48 hr. 388

Experimental animals were sacrificed after 48 hr (i.e., acutely-infarcted), and 2 or 4 weeks (i.e., 389

chronically-infarcted) post-MI, and cardiac tissues isolated from two, left ventricular (LV) 390

regions: remnant/viable (i.e., non-infarcted) LV free wall remote from the infarct scar and 391

septum), and infarct scar. Infarcted (i.e., pale, necrotic tissue in acutely-infarcted vs. overtly-392

healed scar tissue in chronically-infarcted animals) and non-infarcted regions were determined 393

visually, and compared with the same regions obtained from sham-operated rats. Cardiac tissues 394

from sham-operated LVs, viable LVs, and scar regions were homogenized in 100 mM Tris (pH 395

7.4) containing 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 4 µM leupeptin, 1 µM 396

pepstatin, 0.3 µM aprotinin. This homogenate was sonicated for 5 x 5 seconds. To isolate the 397

cytosolic fractions, the samples were centrifuged at 3000 x g for 10 min at 4 °C. The resulting 398

supernatant was further subjected to centrifugation at 48000 x g for 20 min at 4°C (Cunnington 399

et al., 2011). The resulting supernatant was used for the cytosolic Meox2 and Zeb2 protein 400

assays by Western blot. 401

402

Statistics. Data were analyzed using SigmaStat software and are expressed as mean ± SEM. 403

Groups were compared using one-way ANOVA with Student-Newman-Keuls post hoc test for 404

multiple group comparisons. * indicates P < 0.05 unless otherwise indicated. 405

406

Acknowledgements This work was supported by the Canadian Institutes for Health Research 407

(IMCD/JTW) and the Heart and Stroke Foundation of Manitoba (IMCD/JTW). JTW is a MHRC 408

Manitoba Research Chair. RHC is the recipient of the MHRC/CIHR RPP doctoral student 409

scholarship. SG is a Parker B. Francis scholar in Respiratory Diseases. 410

411

Disclosures None. 412

413

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558

Figure Legends 559

Figure 1. The role of Meox1 and Meox2 in cardiac fibroblast-to-myofibroblast 560phenoconversion. (Panel A) Meox1 and Meox2 RNA levels are reduced during fibroblast-to-561myofibroblast differentiation. RNA was harvested from P0, P1 and P2 cells and used for qPCR. 562GAPDH primers served as a control for loading. The data shown were obtained from n = 3 563independent experiments. * = P < 0.005. (Panel B) Primary fibroblasts (P0) and first (P1) and 564second (P2) passage myofibroblasts were isolated, total protein prepared, and Meox2 protein 565expression examined via Western blot using α-tubulin as a loading control. Representative 566Western blots for Meox2 and α-tubulin protein expression are shown above the histograms for 567Meox2 protein expression. The data shown are from n = 3 independent experiments; * P < 0.01 568vs. P0. (Panel C) Cryosections of 4 week post-MI rat heart tissue were stained for Meox2 569(green) and Vimentin (red) with DAPI nuclear stain (blue). Images are representative of n = 3 570hearts. 571

Figure 2. Rat cardiac myofibroblast phenotype is altered by Meox2 but not by Meox1 or 572Meox2 DNA binding mutant over-expression. (Panel A) Meox2 over-expression causes a 573reduction of the myofibroblast phenotype. P1 cardiac myofibroblasts were transduced with Ad-574LacZ (100 MOI) or Ad-FLAG-Meox2 (200 MOI). Non-transduced cells were cultured as an 575additional control. Cells were allowed to grow in the presence of virus for 48 hours before 576harvesting for Western blot analysis. Myofibroblast phenotype was assessed using α-SMA, ED-577A fibronectin and SMemb antibodies with β-tubulin expression used as a loading control. Images 578are representative of n = 4 independent experiments. (Panel B) Histographic representation of 579Western blots in (A). α-SMA, * = P < 0.01 vs. control; ED-A fibronectin, † = P < 0.05 vs. 580control. No significant differences among groups were detected in the SMemb assay data. Data 581were obtained from n = 4 independent experiments. (Panel C) First passage (i.e., P1) 582myofibroblasts were transduced with Ad-LacZ (100 MOI), Ad-FLAG-Meox1 (200 MOI), Ad-583FLAG-Meox2 DBDmt (200 MOI) or left non-transduced for 48 hours. Representative Western 584blots are shown α-SMA, ED-A fibronectin, Smemb and FLAG expression using eEF2 585expression as a loading control. (Panel D) Histographic representation of data in (A). The data 586shown are from n = 7 - 10 independent experiments. 587

Figure 3. Meox2 overexpression induces rat ventricular myofibroblast proliferation. Rat 588ventricular myofibroblasts (passage 3) were infected with Meox2 and LacZ adenoviral vectors 589(50 MOI) and cellular proliferation was investigated by FACS analysis using EdU (Figure 3 590panels A and B) and hemocytometry (Figure 3C and 3D) at 24 h post infection. (p < 0.01, p < 5910.001). In Figure 3 Panel A and Panel B the gate is indicated by the trapezoidal box, and the Meox2 592

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treated cells show higher incidence of EdU-positive staining. Cell number estimation in Panel C 593reveals a significant increase in Meox2 treatment vs. control which corresponds to the results of the 594EdU detection study. The histographic data are based on three independent experiments in two 595different populations of rat ventricular P3 myofibroblasts. 596

Figure 4. Ski over-expression affects myofibroblast phenotype. (A) ED-A fibronectin is 597decreased in Ski over-expressing cells. P1 cardiac myofibroblasts were infected with either Ad-598LacZ (100 MOI) or Ad-HA-Ski (50, 100 MOI) for 24 hours. Non-transduced cells served as a 599control. Total ED-A fibronectin expression was examined by Western blot using β-tubulin as a 600loading control. (Panel B) Histographic representation of data obtained in (Panel A). The image 601shown is representative of n = 4 independent experiments. *P < 0.01 vs. control; † P < 0.01 vs. 60250 MOI. (Panel C) SMemb is not significantly altered with Ad-HA-Ski transduction. P1 cardiac 603myofibroblasts were infected with either Ad-LacZ (100 MOI) or Ad-HA-Ski (100 MOI) for 48 604hours. Non-infected cells served as a control. Total SMemb expression was examined by 605Western blot analysis using β-tubulin expression as a loading control. (Panel D) Histographic 606representation of data obtained in Panel C. The image shown is representative of n = 3 607independent experiments. 608

Figure 5. Over-expression of Ski upregulates Meox2 mRNA expression. P1 cardiac 609myofibroblasts were either transduced with Ad-LacZ (100 MOI), Ad-HA-Ski (100 MOI) or not 610transduced (control). Cells were serum-starved for 24 hours and then were either left untreated or 611were stimulated with TGF-β1 (10 ng/ml for 24 h). Total RNA was isolated and one-step qPCR 612was performed with Meox1 and Meox2 primers. GAPDH primers were used to control for 613loading. * = P < 0.005 vs. control. The data shown are from n = 4 independent experiments. 614

Figure 6. Increased Zeb2 expression in rat ventricular myofibroblasts can be attenuated by 615Ski overexpression. Primary fibroblasts (P0) and first (P1) and second (P2) passage 616myofibroblasts were isolated, total protein prepared, and Zeb2 protein expression examined via 617Western blot using β-tubulin and α-tubulin as a loading control. (Panel A) Representative 618Western blots for Zeb2 and α-tubulin protein expression. (Panel C) Histographic representation 619of data in Panel A. The data shown are from n = 3 independent experiments; * P < 0.01 vs. P0 ; † 620P < 0.01 vs. P1. (Panel B) First passage (i.e., P1) myofibroblasts were transduced with Ad-LacZ 621(100 MOI), Ad-HA-Ski (100 MOI) or left non-transduced for 48 hours. Total protein was 622isolated followed by Western blot analysis. Representative Western blots for Zeb2 using β-623tubulin as a loading control are shown. (Panel D) Histographic representation of data in Panel B. 624The data shown are from n = 3 independent experiments. * P<0.01 vs. control. 625

Figure 7. Expression of Meox2 and Zeb2 in post-myocardial infarction (MI) cardiac tissues. 626Representative Western blots show cytosolic Meox2 (Panel A and histographic representation 627in Panel B) and Zeb2 (Panel C with histographic representation in Panel D) protein obtained 628from sham-operated control hearts and remnant tissue, as well as tissue from the infarct zone in 62948 hr, 2 week, and 4 week post-myocardial infarction left ventricular samples. 630

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Figure 8. Model of the signaling pathway for Ski modulation of myofibroblast phenotype. 631We have previously demonstrated that Ski over-expression leads to decreased collagen secretion 632in cardiac myofibroblasts. Based on the data generated in the current study, we propose that Ski 633represses expression of the Zeb2 protein through unknown mechanisms (bottom of Figure). 634Inhibition of Zeb2 protein then de-represses Meox2, thereby partially restoring the fibroblastic 635phenotype and decreasing myofibroblastic function (e.g.. collagen type I synthesis/secretion). 636After injury (top segment of Figure) we suggest that cytosolic movement of Ski is causal to 637elevated Zeb2 expression and repression of Meox2, thus facilitating fibroblast to myofibroblast 638phenoconversion. 639

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