University of Groningen
Analysis of Cre-mediated genetic deletion of Gdf11 in cardiomyocytes of young mice Garbern, Jessica; Kristl, Amy C; Bassaneze, Vinicius; Vujic, Ana; Schoemaker, Henk; Sereda, Rebecca; Peng, Liming; Ricci-Blair, Elisabeth M; Goldstein, Jill M; Walker, Ryan G
Published in:
American Journal of Physiology - Heart and Circulatory Physiology DOI:
10.1152/ajpheart.00615.2018
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.
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Final author's version (accepted by publisher, after peer review)
Publication date: 2019
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Garbern, J., Kristl, A. C., Bassaneze, V., Vujic, A., Schoemaker, H., Sereda, R., Peng, L., Ricci-Blair, E. M., Goldstein, J. M., Walker, R. G., Bhasin, S., Wagers, A. J., & Lee, R. T. (2019). Analysis of Cre-mediated genetic deletion of Gdf11 in cardiomyocytes of young mice. American Journal of Physiology - Heart and Circulatory Physiology. https://doi.org/10.1152/ajpheart.00615.2018
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1 2
Analysis of Cre-mediated genetic deletion of Gdf11 in cardiomyocytes of young mice
3 4 5
Jessica C. Garberna,b, Amy C. Kristla, Vinicius Bassanezea,c, Ana Vujica, Henk Schoemakerc, 6
Rebecca Seredaa, Liming Pengd, Elisabeth Ricci-Blaira, Jill M. Goldsteina, Ryan G. Walkera, 7
Shalender Bhasind, Amy J. Wagersa,e,f, Richard T. Leea,c
8 9 10
Author contributions: 11
J.C.G. Designed and conducted experiments, wrote manuscript; A.C.K. Designed and conducted 12
experiments; V.B. Designed and conducted experiments, edited manuscript; A.V. Designed and 13
conducted experiments, edited manuscript; H.S. Designed and conducted experiments; R.S. 14
Conducted experiments; L.P. Conducted experiments; E.R-B. Conducted experiments; J.M.G. 15
Designed experiments, edited manuscript; R.G.W. Designed experiments, edited manuscript; 16
S.B. conducted experiments; A.J.W. Designed experiments, edited manuscript; R.T.L. Designed 17
experiments, edited manuscript 18
19 20
Author affiliations: 21
aDepartment of Stem Cell and Regenerative Biology and the Harvard Stem Cell Institute,
22
Harvard University, 7 Divinity Ave, Cambridge, MA 02138 23
bDepartment of Cardiology, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA
24
02115 25
cDivision of Cardiovascular Medicine, Department of Medicine, Brigham and Women’s Hospital
26
and Harvard Medical School, 75 Francis St, Boston, MA 02115 27
dBrigham Research Assay Core, Brigham and Women’s Hospital, 221 Longwood Ave, Boston,
28
MA 02115 29
ePaul F. Glenn Center for the Biology of Aging, Harvard Medical School, 77 Louis Pasteur
30
Avenue, Boston, MA 02115 31
fSection on Islet Cell and Regenerative Biology, Joslin Diabetes Center, One Joslin Place,
32
Boston, MA 02215 33
34
Abbreviated title: Gdf11 deletion in cardiomyocytes 35 36 Corresponding author: 37 Dr. Richard T. Lee 38
Department of Stem Cell and Regenerative Biology 39 Harvard University 40 7 Divinity Ave 41 Cambridge, MA 02138 42 Phone: 617-496-5394 43 Fax: 617-496-8351 44 richard_lee@harvard.edu 45
Abstract
46
Administration of active growth differentiation factor 11 (GDF11) to aged mice can reduce 47
cardiac hypertrophy, and low serum levels of GDF11 measured together with the related protein, 48
myostatin (also known as GDF8), predict future morbidity and mortality in coronary heart 49
patients. Using mice with a loxP-flanked (“floxed”) allele of Gdf11 and Myh6-driven expression 50
of Cre recombinase to delete Gdf11 in cardiomyocytes, we tested the hypothesis that cardiac-51
specific Gdf11 deficiency might lead to cardiac hypertrophy in young adulthood. We observed 52
that targeted deletion of Gdf11 in cardiomyocytes does not cause cardiac hypertrophy but rather 53
leads to left ventricular dilation when compared to control mice carrying only the Myh6-cre or 54
Gdf11-floxed alleles, suggesting a possible etiology for dilated cardiomyopathy. However, the
55
mechanism underlying this finding remains unclear due to multiple confounding effects 56
associated with the selected model. First, whole heart Gdf11 expression did not decrease in 57
Myh6-cre;Gdf11-floxed mice, possibly due to upregulation of Gdf11 in non-cardiomyocytes in
58
the heart. Second, we observed Cre-associated toxicity, with lower body weights and increased 59
global fibrosis in Cre-only control male mice compared to flox-only controls, making it 60
challenging to infer which changes in Myh6-cre;Gdf11-floxed mice were due to Cre toxicity 61
versus deletion of Gdf11. Third, we observed differential expression of cre mRNA in Cre-only 62
controls compared to the cardiomyocyte-specific knockout mice, also making comparison 63
between these two groups difficult. Thus, targeted Gdf11 deletion in cardiomyocytes may lead to 64
left ventricular dilation without hypertrophy, but alternative animal models are necessary to 65
understand the mechanism for these findings. 66
67 68
New and Noteworthy
69
We observed that targeted deletion of Gdf11 in cardiomyocytes does not cause cardiac 70
hypertrophy but rather leads to left ventricular dilation when compared to control mice carrying 71
only the Myh6-cre or Gdf11-floxed alleles. However, the mechanism underlying this finding 72
remains unclear due to multiple confounding effects associated with the selected mouse model. 73
74
Keywords: Growth differentiation factor 11, myostatin, Myh6-cre, cardiomyocytes,
75
cardiomyopathy 76
Introduction
77
Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor 78
β (TGF-β) superfamily, best known for its morphogenic roles during development (14). The role 79
of GDF11 in cardiac aging has been controversial (9, 21, 24, 25). We initially demonstrated that 80
GDF11 is a circulating factor that when administered to aged mice can decrease age-related 81
cardiac hypertrophy as indicated by a decrease in heart weight to tibia length ratios (12). Others 82
have shown that GDF11 administration may be beneficial following myocardial infarction (6). 83
However, following our initial report, contrasting work indicated that GDF11 administration to 84
aged mice does not affect heart weight to body weight ratios (24), or, that GDF11 85
supplementation impairs cardiac function in concert with reducing cardiomyocyte size and 86
cardiac mass (21). Furthermore, additional controversy has been raised due to a lack of 87
specificity of antibody and aptamer reagents used to detect and quantify GDF11 separate from 88
the closely related protein, myostatin (also known as GDF8 and encoded by the gene, Mstn). 89
Because GDF11 and GDF8 share approximately 90% identity in their active domains, it has been 90
challenging to quantify levels of each protein independently (7). 91
Human clinical data indicate that low serum levels of the GDF11 + myostatin pool at 92
study entry predict increased mortality in adult patients with heart disease over the subsequent 93
eight years (16). These human data point to low levels of the circulating pool of GDF11 + 94
myostatin as a potential pathogenic factor in human heart disease. However, conflicting human 95
data have also been published suggesting that higher levels of GDF11 in adults with severe aortic 96
stenosis are associated with an increased risk of adverse events following valve replacement 97
surgery (22). Thus, further studies are needed to understand how perturbations to the GDF11 and 98
myostatin system might contribute to cardiovascular risk. 99
The effect of GDF11 deficiency on the heart also remains unclear, but prior studies have 100
evaluated the effect of myostatin deficiency on the heart. Mstn-/- mice are viable and have 101
increased skeletal muscle weight as well as increased heart weight and heart weight to tail length, 102
but they show no increase in heart weight to body weight ratio (10). Mstn-/- mice also have 103
mildly increased left ventricular volumes and mildly reduced systolic function at baseline but 104
have an enhanced response to isoproterenol stress with a higher percent change in fractional 105
shortening compared to wild type mice (10). Inducible cardiomyocyte-specific deletion of Mstn 106
leads to transient chamber dilation and impairment of systolic function; however, upregulation of 107
Mstn in non-cardiomyocytes occurs leading to an unexpected overall increase in Mstn expression
108
in whole heart extracts of knockout mice (3). Conversely, overexpression of Mstn leads to 109
decreased heart weights in males but not females (20). Despite similarities in sequence between 110
mature myostatin and GDF11, there are structural differences between the two proteins that 111
could lead to distinct effects with deletion of Gdf11 in cardiomyocytes that have not previously 112
been described with cardiomyocyte-specific Mstn deletion (25). 113
We sought to test the effects of reduced levels of GDF11 in the heart during young 114
adulthood in mice. Because systemic germline deletion in the mouse (Gdf11-/-) results in a
115
number of developmental defects and is lethal by 1 day of age, likely due to renal agenesis (14), 116
we generated a mouse model to specifically delete Gdf11 only within cardiomyocytes. We chose 117
to delete Gdf11 from cardiomyocytes for our initial study as opposed to other cardiac cell types 118
given the demonstrated phenotype seen with cardiomyocyte deletion of Mstn and challenges of 119
deleting Gdf11 only in the heart if targeting other cell types. By crossing a cardiomyocyte-120
specific Myh6-cre allele with a Gdf11-floxed allele (13), we were able to induce genomic 121
excision of the regions encoding mature GDF11 protein exclusively in cardiomyocytes. Our 122
experiment was designed to test the hypothesis that postnatal Gdf11 deficiency in 123
cardiomyocytes would promote cardiac hypertrophy. We found instead that targeted 124
cardiomyocyte deletion of Gdf11 during young adulthood, using the Myh6-cre system, does not 125
result in cardiac hypertrophy and rather leads to progressive left ventricular dilation that is 126
apparent in both females and males by 6 months of age. However, because of multiple adverse 127
effects from Cre recombinase itself on the heart and potential differential expression of the cre 128
gene across genotypes, we are unable to define the molecular mechanism of the dilated 129
cardiomyopathy phenotype that develops when the Gdf11 gene is removed from cardiomyocytes 130
using this mouse model. 131
132
Materials and Methods
133 134
Animals – constitutively active Cre
135
Mice expressing constitutively active Cre recombinase driven by the Myh6 promoter 136
(B6.FVB-Tg(Myh6-cre)2182Mds/J) were obtained from The Jackson Laboratory. Mice 137
containing a floxed (flanking loxP) Gdf11 allele with loxP sites flanking exons 2 and 3 of Gdf11 138
were generously provided by Dr. Se-Jin Lee (Johns Hopkins University) (13). Mice were bred to 139
obtain three genotypes used in this study: Myh6cre/wt; Gdf11fl/fl (experimental genotype), 140
Myh6cre/wt; Gdf11wt/wt (Cre-only control genotype, referred to as Myh6cre/wt), and Myh6wt/wt;
141
Gdf11fl/fl (flox-only control genotype and littermate control, referred to as Gdf11fl/fl). All mice
142
were on a mixed C57Bl/6J and 6N background. 143
Young adult male and female mice of all three genotypes were weighed weekly starting 144
at 2 months of age. Echocardiograms were performed in all mice at 1-2 and 6 months of age in a 145
blinded manner. Additional echocardiogram time points at 3 or 4 months of age were also 146
performed in a subset of mice. Mice were observed for up to 6 months, and then animals were 147
euthanized and serum and tissues harvested for further analysis. We also determined tibia length 148
as a normalizing parameter because GDF11 administration has been shown to decrease body 149
weight (17). Tissues for downstream DNA, RNA and protein analysis were flash frozen in liquid 150
nitrogen and stored at -70˚C until further processing was performed. All experiments were 151
conducted according to the Guide for the Use and Care of Laboratory Animals and approved by 152
the Institutional Animal Care and Use Committee of Harvard University Faculty of Arts and 153
Sciences. 154
155
Animals – tamoxifen-inducible Cre
156 157
Mice expressing tamoxifen-inducible Cre recombinase (Mer-Cre-Mer, abbreviated 158
MCM) driven by the Myh6 promoter (B6.FVB(129)-A1cfTg(Myh6-cre/Esr1*)1Jmk/J) were obtained
159
from The Jackson Laboratory (backcrossed to a C57Bl/6J agouti background per vendor). Mice 160
were bred with Gdf11fl/fl mice to obtain two genotypes used in this experiment: Myh6MCM/wt; 161
Gdf11fl/fl (experimental genotype), and Myh6wt/wt; Gdf11fl/fl (flox control genotype). Male and
162
female mice were stratified based on gender then randomized at 5 months of age to receive 163
injections of either 4-hydroxytamoxifen (4OH-tamoxifen, 75 mg/kg) or vehicle (10% ethanol, 164
90% sunflower oil) via intraperitoneal (i.p.) injection for 5 days. Echocardiography was 165
performed at baseline (at 5 months of age) and 4 weeks later (at 6 months of age) prior to harvest 166
to evaluate left ventricular size and function. Tissues were harvested and processed for histology 167
or qPCR. 168
Echocardiography
170
Mice were sedated with 0.1-0.5% inhaled isoflurane for echocardiography, with the dose 171
titrated to maintain heart rates of >500 beats per minute for acquired images. Mice were placed 172
on a heating pad, and echocardiograms were obtained with the Vevo770 (Visualsonics, Toronto, 173
Ontario, Canada). M-mode was used to measure left ventricular (LV) interventricular septal 174
(IVS) wall thickness, LV posterior wall thickness (LVPW), and LV internal diameter (LVID) 175
during both systole and diastole. Fractional shortening (%), LV mass and LV volumes were 176
calculated with the Visualsonics software package. 177
178
Cardiomyocyte and non-cardiomyocyte isolation from adult hearts
179
Cardiomyocytes and non-cardiomyocytes were isolated from adult male and female mice 180
using a Langendorff-free method as previously described (1). We modified the original protocol 181
to include blebbistatin (5 µM, Sigma) instead of 2-3-butanedione monoxime in the culture 182
media. In addition, we used a peristaltic pump rather than hand injection to better control the 183
flow rate of the perfused solutions. Briefly, mice were anesthetized with isoflurane then the chest 184
cavity was opened. The descending aorta and inferior vena cava were cut followed by perfusion 185
of 7 mL of EDTA buffer into the apex of the right ventricle. The aorta was then clamped and cut 186
distal to the clamp, and the heart was removed. EDTA buffer (10 mL) was then perfused into the 187
apex of the left ventricle followed by injection of 3 ml of perfusion buffer then 30-40 mL of 188
collagenase buffer delivered into the left ventricular apex. The clamp was removed and the heart 189
was manually dissociated. Stop buffer (perfusion buffer + 5% fetal bovine serum) was added, 190
cells were passed through a 300 µm strainer then cardiomyocytes were allowed to gravity settle 191
for 20 minutes. The supernatant containing non-cardiomyocytes and debris was plated into an 192
uncoated tissue culture plate in DMEM:F12/10% FBS for 3 hours. The cardiomyocyte fraction in 193
the pellet then underwent sequential gravity settling with low speed centrifugation (12 g x 3 min) 194
with calcium re-introduction followed by plating into laminin-coated plates for 3 hours. After 3 195
hours, both cardiomyocytes and non-cardiomyocytes were washed and harvested in Trizol. 196
Samples were frozen at -70 deg C until RNA extraction by QIAcube. 197
198
PCR
199
DNA was extracted from harvested tissue using the REDExtract-N-Amp Tissue PCR Kit 200
(Sigma). Table 1 shows the primers that were used to amplify Myh6, Myh6-cre and Gdf11 PCR 201
products and their expected product sizes. PCR products were run on a 2% agarose gel in TAE 202
buffer at 100V for 45-60 minutes and gels were imaged in a Gel Doc EZ system (Bio-Rad). 203
204
Quantitative PCR
205
RiboZol reagent (VWR) and the E.Z.N.A. Total RNA I kit (Omega) were used to isolate 206
RNA from homogenized whole organ tissue, followed by the High Capacity cDNA Reverse 207
Transcription kit (Thermo Fisher Scientific) to reverse transcribe mRNA to cDNA according to 208
the manufacturers’ instructions. Quantitative PCR (qPCR) was performed using TaqMan probes 209
for Nppb (Mm01255770_g1), Gdf11 (Mm01159973_m1, spanning exons 1-2), Gdf15 210
(Mm00442228_m1), Inhba (activin A, Mm00434339_m1), Mstn (Mm01254559_m1), and 211
Tgfbr1 (Mm00436964_m1), with TATA-binding protein (Tbp) used as the housekeeping gene
212
(Mm01277042_m1) using a Bio-Rad CFX384 Real-Time System. Due to low yields of isolated 213
cardiomyocytes and non-cardiomyocytes from adult hearts, RNA was extracted using the Qiagen 214
QIACube from Trizol (Thermo) followed by reverse transcription to cDNA using the 215
SuperScript VILO cDNA synthesis kit (Thermo) per the manufacturer’s instructions. The 216
isolated cDNA (10-20 ng) was then pre-amplified using the Taqman PreAmp Master Mix 217
(Thermo) for 14 cycles. The pre-amplified cDNA was then diluted 1:20 for quantification of 218
Gdf11 expression in isolated cardiomyocytes and non-cardiomyocytes by qPCR on the
219
QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems). 220
221
Flow cytometry
222
Flow cytometry was performed to quantify the percentage of cardiomyocytes in the cell 223
population obtained immediately after the final step fo the extraction procedure. In brief, the 224
cardiomyocyte fraction was fixed with 1 mL of 70% ethanol then stored at -20 deg C. The non-225
cardiomyocyte fraction was plated into a six-well plate and allowed to expand for seven days in 226
DMEM/F12 + 10% fetal bovine serum + 1% penicillin/streptomycin. Cells were dissociated with 227
trypsin then fixed in 70% ethanol. The fixative was removed via low speed centrifugation (20 g x 228
3 min) and the cells were washed then permeabilized in 0.1% Triton in PBS. Cells were labeled 229
overnight at 4 deg C using an antibody to cardiac troponin T (Abcam ab8295, diluted 1:250 in 230
phosphate buffered saline (PBS) supplemented with 10% goat serum) to detect cardiomyocytes, 231
and an antibody to vimentin (Abcam ab92547, diluted 1:200 in PBS supplemented woith 10% 232
goat serum), which is highly expressed in fibroblasts. After washing steps and incubation with 233
the corresponding AlexaFluor 568 conjugated secondary antibody for 2h at room temperature 234
(Thermo, Mouse IgG1 and Rabbit IgG, respectively), cells were analyzed by flow cytometry 235
(MoFlo Astrios, Beckman Coulter, using the nozzle of 200µm). Data were analyzed with FlowJo 236
software (version 10.0.8). 237
239
Histology
240
Harvested tissues were fixed in 4% paraformaldehyde for 24 hours then exchanged for 241
70% ethanol for 3 days prior to embedding in paraffin. Sections were stained with Masson’s 242
trichrome staining as previously described (8). Global fibrosis was quantified using a Python 243
script written to quantify the percentage of blue pixels out of the total pixels from a cross-244
sectional image of the heart (https://github.com/jgarbern/global-fibrosis). Average 245
cardiomyocyte cross-sectional area for each mouse was quantified in a blinded manner with a 246
total of 40 cardiomyocytes measured from multiple sections from each heart. 247
248
Quantitative mass spectrometry
249
Blood was obtained by retro-orbital collection at the time of harvest and transferred to 250
serum separator tubes (SST). Tubes were spun at 2000 g for 5 min and serum was transferred to 251
clean low binding microcentrifuge tubes and stored at -70 deg C until further processing. 252
Samples (100 µL) were submitted to the Brigham Research Assay Core (BRAC) at Brigham and 253
Women’s Hospital for quantitative mass spectrometry. Mouse serum was denatured, reduced and 254
alkylated, followed by pH based fractionation using cation ion exchange solid phase extraction 255
(SPE); appropriate elution fraction was digested with trypsin. After desalting and concentrating 256
of tryptic digest, the peptide mixture was separated and eluted by liquid chromatography 257
followed by mass spectrometric analysis operated in positive electrospray ionization mode. The 258
most intensive and unique proteotypic peptides from GDF11 and myostatin as surrogated 259
peptides along with heavy-labeled unique peptides as internal standards were used for 260
quantitative determination of GDF11 and myostatin. 261
262
Statistical analysis
263
Data are expressed as mean ± standard error of the mean (SEM) unless noted otherwise. 264
Data were evaluated with the D’Agostino and Pearson omnibus normality test; normally 265
distributed data were evaluated with Student’s t-test or two-way ANOVA with Tukey post-hoc 266
analysis, while data that were not normally distributed were analyzed with Mann-Whitney or 267
Kruskal-Wallis with Dunn’s multiple comparisons test as indicated. Kaplan-Meier survival 268
curves were evaluated the Mantel-Cox log-tank test. Body weight trends with time were 269
analyzed with a two-way repeated measures ANOVA with Tukey multiple comparisons test. A 270
p-value < 0.05 was considered statistically significant. 271
272
Results
273 274
Cardiomyocyte-specific deletion of Gdf11 leads to left ventricular dilation that progresses with
275
age
276
Due to known sexual dimorphism in body weight and heart size in mice, we analyzed 277
body weight, heart weight, and heart weight/body weight ratio data from males and females 278
separately. Myh6cre/wt; Gdf11fl/fl mice had progressive left ventricular dilation with a significant 279
increase in left ventricular end diastolic volume (Figures 1A (1-2 months), 1B (3-4 months), and 280
1C (6 months)), a significant decrease in septal thickness (Figure 1J) and a non-significant 281
decrease in left ventricular posterior wall thickness (Figure 1L) by echocardiography that was 282
apparent in both genders by 6 months of age. Male Myh6cre/wt; Gdf11fl/fl mice also had 283
significantly increased left ventricular internal diameters at 6 months of age compared to sex-284
matched Myh6cre/wt mice (Figure 1K). Cardiomyocyte-specific deletion of Gdf11 also led to a 285
decrease in left ventricular function with decreased fractional shortening in Myh6cre/wt; Gdf11fl/fl 286
females compared to sex-matched Gdf11fl/fl controls, and in Myh6cre/wt; Gdf11fl/fl males when 287
compared to sex-matched Myh6cre/wt controls (Figure 1G). The chamber dilation and functional 288
decline were not associated with differences in either estimated left ventricular mass by 289
echocardiography, or heart weight compared to either the Cre-only control genotype (Myh6cre/wt) 290
or the flox-only control genotype (Gdf11fl/fl) at 6 months of age (Figure 1H, 1I, 1F, respectively). 291
Heart weight to body weight ratios were not different across groups at any age (Figures 1D (1-2 292
months), 1E (3-4 months), and 1F (6 months)). There were no significant differences in tibia 293
lengths or heart weight to tibia length ratios at 6 months of age (Figures 1N and 1O, 294
respectively). 295
We also utilized a tamoxifen-inducible cardiomyocyte deletion model with 296
administration of 4OH-tamoxifen or vehicle to 6 month old Gdf11fl/fl or Myh6MCM/wt; Gdf11fl/fl 297
mice for 5 days at 75 mg/kg delivered intraperitoneally, but saw no significant differences in left 298
ventricular end diastolic volume, estimated left ventricular mass, body weight, heart weight, or 299
heart weight to body weight ratio with this treatment regimen (Figures 2E, 2F, 2G, 2I, 2J 300
respectively) despite evidence of recombination by PCR (Figure 2C). We did not see significant 301
changes in Gdf11 mRNA expression following this treatment regimen by qPCR (Figure 2D). 302
However, we observed significant differences in survival across all groups in males (Figure 2B) 303
but not females (Figure 2A). The etiology for the survival difference in 4OH-tamoxifen-treated 304
males is unclear, although we presume it reflects tamoxifen toxicity given the acute nature of the 305
deaths. Survival of only the “healthiest” mice may also confound our long-term results. 4OH-306
tamoxifen significantly increased tibia length, therefore we did not evaluate heart weight to tibia 307
length ratios in this model (Figure 2H); the effect of tamoxifen on long bone length has been 308
reported previously (26). We also found significantly increased activin A (Inbha) mRNA 309
expression within the spleens of 4OH-TAM treated Myh6MCM/wt; Gdf11fl/fl male mice, and a 310
similar non-significant trend in other TAM-treated groups (Figure 2K), consistent with prior 311
reports that tamoxifen may activate TGF-β signaling (4, 5, 15). We did not study this model in 312
further depth due to the absence of a cardiac phenotype at the age selected (6 months of age). 313
We note that the different findings observed between tamoxifen-inducible Myh6-MCM 314
versus constitutively active Myh6-cre models may be due to the different ages at which Gdf11 315
knockdown occurs in the two models. In addition, application of stress such as with pressure 316
overload could be considered to enhance any potential phenotype caused with tamoxifen-induced 317
Myh6-MCM expression. In the context of increased mortality in mice injected with 4OH-TAM 318
even without surgery, and concerns for confounding effects from 4OH-TAM administration on 319
TGF-β signaling, we chose not to apply pressure overload stress (e.g. transverse aortic 320
constriction model) or repeat this experiment at a younger age in the tamoxifen-inducible 321
cardiomyocyte deletion model and instead focused on the constitutively active Myh6-cre model 322
for the remainder of this study. 323
324
Cardiomyocyte-specific deletion of Gdf11 is not sufficient to decrease Gdf11 mRNA
325
expression in the whole heart
326
Myh6 Cre-induced genomic recombination of Gdf11 was evident in the heart in neonatal
327
mice (Figure 3A). Gdf11 recombination was observed only in the heart by PCR and, as expected, 328
was not seen in the lung, liver, kidney, spleen, or skeletal muscle at 6 months of age (Figure 3B). 329
However, despite evidence of DNA recombination, we did not observe differences in Gdf11 330
mRNA expression in the whole heart at 6 months of age (Figure 3D), and in fact saw a 331
significant increase in Gdf11 mRNA expression in Myh6cre/wt; Gdf11fl/fl male mice at 3 months of 332
age (Figure 3C). 333
Since approximately half of the whole heart is composed of non-cardiomyocytes 334
(including endothelial, fibroblast and smooth muscle cells) (2), we isolated cardiomyocytes and 335
non-cardiomyocytes from adult (>2 months) mice to evaluate Gdf11 mRNA expression in the 336
two cell populations. In a representative batch, the cardiomyocyte population consisted of 84% 337
cTnT+ cardiomyocytes, while the non-cardiomyocyte population consisted of 95% vimentin+ 338
fibroblasts, as quantified by flow cytometry (data not shown). Gdf11 expression shows 339
substantial variability in Myh6cre/wt mice in this analysis; however, we detected a decreased signal 340
in cardiomyocytes from Myh6cre/wt; Gdf11fl/fl mice and a trend toward differences in Gdf11 341
expression in cardiomyocytes across genotypes (p-value 0.05 by Kruskal-Wallis test) (Figure 342
3E). 343
The high variability in Gdf11 expression in Myh6cre/wt mice suggested that this genotype 344
may be abnormal, which became evident in subsequent analysis. The Gdf11 signal in Myh6cre/wt; 345
Gdf11fl/fl mice was greater than zero likely due to contamination from non-cardiomyocytes in the
346
cardiomyocyte population, though it is possible this reflects incomplete recombination of the 347
floxed alleles. We did not observe any differences in Gdf11 expression in non-cardiomyocytes 348
across genotypes (Figure 3F). Direct comparison across cell types is challenging due to different 349
amounts of RNA per cell in different cell types. If starting with an equal amount of RNA, there 350
was a trend toward increased Gdf11 expression in non-cardiomyocytes compared to 351
cardiomyocytes in all genotypes (data not shown). However, we obtained much lower total RNA 352
after isolation from non-cardiomyocytes (predominantly cardiac fibroblasts) compared to an 353
equal number of cardiomyocytes, and therefore comparing Gdf11 expression from the same 354
amount of total RNA may be suboptimal. 355
Due to challenges in quantifying protein levels of GDF11 with antibody-based 356
approaches (7), we measured GDF11 and myostatin levels in serum by quantitative mass 357
spectrometry and found that Gdf11fl/fl females had significantly lower circulating levels of 358
GDF11 than Myh6cre/wt females; however, there were no significant differences in GDF11 serum 359
concentrations between Myh6cre/wt; Gdf11fl/fl mice and their sex-matched Cre- or flox-only 360
control groups (Figure 3G). Although the major source(s) of circulating GDF11 remains unclear, 361
prior work from our group demonstrated that the spleen has the highest mRNA levels among 362
organs studied (12); thus it is not unexpected that serum levels of GDF11 were not altered in 363
Myh6cre/wt; Gdf11fl/fl mice, as only cardiomyocyte Gdf11 was targeted for deletion in these
364
animals. Circulating myostatin levels did not differ across groups (Figure 3H). 365
366
Mechanism of left ventricular dilation confounded by adverse effects from Myh6-driven Cre
367
recombinase activity
368
Myh6cre/wt male mice were significantly smaller than both Gdf11fl/fl and Myh6cre/wt;
369
Gdf11fl/fl mice by 6 months of age (Figure 1M), with a plateau in the body weight curve in
370
Myh6cre/wt mice appearing at around 4 months of age in both genders (Figures 4C-D). In addition,
371
there was a non-significant trend toward decreased survival in Myh6cre/wt mice with death in 372
several mice at around 4 months of age (Figures 4A-B). 373
There was a significant increase in cardiac mRNA expression of Mstn in Myh6cre/wt;
374
Gdf11fl/fl mice compared to Myh6cre/wt mice at 2 months (Figure 5A), but not 6 months of age
375
(Figure 5E). In contrast, at 6 months but not 2 months of age, Nppb, which encodes B-type 376
natriuretic peptide (BNP), a clinically used marker of heart failure, had significantly higher 377
expression in hearts of Myh6cre/wt mice compared to Gdf11fl/fl mice, with the experimental 378
genotype (Myh6cre/wt; Gdf11fl/fl) having an intermediate expression profile (2 months, Figure 5B; 379
6 months, Figure 5F). Expression of Tgfbr1, which encodes one of the receptors for GDF11, was 380
also significantly lower in hearts of Gdf11fl/fl mice compared to both Myh6cre/wt mice and 381
Myh6cre/wt; Gdf11fl/fl mice at 6 months (Figure 5G) but not 2 months of age (Figure 5C). Finally,
382
expression of Gdf15, which was reported to be upregulated following administration of 383
supraphysiologic doses of GDF11 (11), was significantly increased in hearts of Myh6cre/wt mice 384
compared to both Gdf11fl/fl mice and Myh6cre/wt; Gdf11fl/fl mice at 6 months (Figure 5H) but not 2 385
months of age (Figure 5D). These results suggest that cardiomyocyte deletion of Gdf11 has an 386
early effect on Mstn expression, which precedes left ventricular dilation, and is followed later by 387
differential regulation on other proteins involved in TGF-β signaling. However, the differences 388
between the two control genotypes particularly at older ages suggest that the presence of Cre 389
recombinase has adverse effects on the heart and make it difficult to identify molecular 390
mechanisms to explain our echocardiographic findings in the experimental genotype. 391
392
Myh6-driven Cre recombinase expression leads to myocardial fibrosis and increased
393
cardiomyocyte size in males
394
Cardiotoxicity has been previously described by Pugach et al. with prolonged Myh6-395
driven Cre expression in this strain (18), and our results are consistent with these prior findings. 396
We observed a significant increase in global myocardial fibrosis in males compared to females in 397
the Cre-only control genotype (Myh6cre/wt) at 6 months of age, with males more sensitive than 398
females to adverse effects from Cre, consistent with Pugach et al. (18) (Figures 6A and B). There 399
was a similar non-significant trend observed in Myh6cre/wt; Gdf11fl/fl mice. In addition, there was 400
a non-significant trend of increased global myocardial fibrosis in male mice expressing Cre 401
recombinase compared to the flox-only control (Gdf11fl/fl). Furthermore, the cardiomyocyte 402
cross-sectional area was significantly increased in Myh6cre/wt male mice compared to either 403
Gdf11fl/fl or Myh6cre/wt; Gdf11fl/fl mice (Figure 6C).
404 405
Cre mRNA expression is higher in Myh6cre/wt mice compared to Myh6cre/wt; Gdf11fl/fl mice
406
We evaluated whether Cre is differentially expressed across genotypes as a possible 407
explanation for why Myh6cre/wt mice have a more pronounced phenotype in terms of body 408
weight, myocardial fibrosis and expression of selected genes associated with heart failure such as 409
Nppb. We found that cre mRNA expression is significantly greater in Myh6cre/wt mice compared
410
to Myh6cre/wt; Gdf11fl/fl mice (Figure 6D). In contrast to RNA levels, there were no differences 411
noted in Cre protein expression in Myh6cre/wt mice compared to Myh6cre/wt; Gdf11fl/fl mice (Figure 412
6E). No cre protein expression was detected in Gdf11fl/fl mice. 413
414
Discussion
415 416
We observed a significant increase in left ventricular end diastolic volume in mice with 417
Cre-mediated genetic deletion of Gdf11 in cardiomyocytes. This finding was consistent in both 418
males and females, with LV end diastolic volume significantly increased in Myh6cre/wt; Gdf11fl/fl 419
mice compared to both Myh6cre/wt and Gdf11fl/fl control mice. Left ventricular dilation was
420
associated with a decrease in left ventricular systolic function, suggesting a possible role for 421
GDF11 signaling in dilated cardiomyopathy. We observed that Mstn and Gdf11 both transiently 422
increase in RNA extracted from whole hearts of young adult Myh6cre/wt; Gdf11fl/fl mice prior to 423
the onset of ventricular dilation, which suggests that there is a developmental component to the 424
phenotype observed. However, our experimental design met numerous unanticipated challenges 425
that prohibit clear interpretation of the underlying molecular mechanisms to explain these 426
findings. First, we did not observe a significant decrease in Gdf11 expression in whole heart 427
extracts from Myh6cre/wt; Gdf11fl/fl mice, suggesting that counter-regulation in non-myocytes may 428
buffer cardiomyocyte-specific loss of Gdf11 in the heart. Second, mice expressing Cre 429
recombinase had a different phenotype from mice not expressing Cre, with decreased body 430
weights and increased global myocardial fibrosis in males, an effect only made clear because we 431
utilized two control genotypes. Third, cre mRNA expression was different between the Cre 432
control genotype and the Cre-containing experimental genotype. Although Cre protein levels 433
were not different by western analysis, in the context of Cre-associated toxicity, further study of 434
protein levels at different ages should be performed in future work to elucidate which 435
perturbations to the cardiac system should be attributed to differences in Cre levels versus Gdf11 436
deletion. 437
Although we observed a non-significant trend toward decreased Gdf11 expression in 438
isolated adult cardiomyocytes of Myh6cre/wt; Gdf11fl/fl mice (complete absence of Gdf11 could not 439
be shown likely due to contamination from non-cardiomyocytes), we did not observe a 440
significant decrease in Gdf11 expression in the whole heart despite constitutively active Myh6-441
driven Cre expression and PCR evidence of Gdf11 recombination. In fact, we observed a 442
transient increase in Gdf11 expression in whole heart extracts from male Myh6cre/wt; Gdf11fl/fl
443
mice compared to male Gdf11fl/fl control mice at 3 months of age and a similar non-significant 444
trend compared to male Myh6cre/wt control mice. A similar effect was previously reported when 445
Mstn was targeted for deletion in cardiomyocytes using mice with tamoxifen-inducible
Myh6-446
driven Cre expression, where Mstn mRNA expression increased in whole heart extracts due to 447
upregulation in non-myocytes despite cardiomyocyte deficiency of myostatin (3). Due to 448
different amounts of total RNA in cardiomyocytes versus non-cardiomyocytes, and low 449
expression levels of Gdf11 in cardiomyocytes requiring pre-amplification of cDNA to detect a 450
reliable signal by qPCR, we were unable to directly compare expression of Gdf11 in 451
cardiomyocytes to non-cardiomyocytes. Nonetheless, in non-cardiomyocytes, we did not observe 452
a difference in Gdf11 expression levels across genotypes. Taken together, this suggests that 453
Gdf11 may act locally in cardiomyocytes, given the presence of a dilated phenotype even in the
454
absence of expression differences at the organ level. In addition, we observed a significant 455
increase in Mstn mRNA expression in whole heart extracts of 2 month old mice. This suggests 456
that Cre-mediated cardiomyocyte deletion of Gdf11 leads to downstream signaling effects during 457
development which precede the observed phenotype of left ventricular dilation starting at 3-4 458
months of age. We also did not examine other organs given the lack of serum differences in 459
GDF11 at 6 months. Alternative models used in future work should focus on early downstream 460
signaling changes in the heart and other organs (such as the spleen which has higher baseline 461
mRNA expression) as well as measuring circulating GDF11 levels at younger ages to better 462
understand how these changes affect cardiac phenotype at later time points. 463
Myh6cre/wt mice have previously been shown to develop progressive myocardial fibrosis
464
and inflammation due to DNA damage at endogenous “loxP-like” or “pseudo-loxP” sites in the 465
myocardium (18). In that study, the authors identified 227 loxP-like sites within genes that could 466
be potentially recognized by Cre recombinase, when tolerating ≤4 mismatches in the canonical 467
loxP sequence. Of these 227 degenerate loxP sites, 55 are expressed in the heart, leading to
numerous off-target effects from Cre recombinase including myocardial fibrosis and 469
upregulation of apoptotic markers such as p53 and Bax (18). This previous study also found that 470
males appear to be more sensitive to the off-target effects of Cre expression in cardiomyocytes, 471
with an increased heart weight to body weight ratio in Myh6cre/wt compared to wild type control 472
(C57Bl/6J) male mice at 6 months of age (18). Although we did not have wild type mice as a 473
control in our study, we also observed that Myh6cre/wt males had significantly increased global 474
fibrosis compared to females and Myh6cre/wt males had significantly increased cardiomyocyte 475
cross-sectional area compared to Gdf11fl/fl control mice and Myh6cre/wt; Gdf11fl/fl knockout mice.
476
It remains unclear whether the potency of Cre toxicity is identical in the presence or absence of 477
loxP, or whether in the presence of loxP, there might be fewer off-target effects due to the
478
stoichiometry of Cre:loxP versus Cre:loxP-like or pseudo-loxP sites. These results underscore the 479
importance of inclusion of Cre control mice as well as evaluation of both genders when using 480
Cre-lox technology. 481
We utilized both Myh6cre/wt and Gdf11fl/fl controls to attempt to account for adverse
482
effects from Cre recombinase. Our results demonstrate that without inclusion of both controls, 483
our data would have been easily misinterpreted with potentially incorrect conclusions drawn – a 484
lesson that was learned from experience after we failed to include the Myh6MCM/wt control line in 485
the tamoxifen-inducible study described in Results above. For example, without Gdf11fl/fl 486
controls, we may have incorrectly concluded that deletion of Gdf11 leads to a decrease in Nppb 487
expression. Conversely, without Myh6cre/wt controls, we may have incorrectly concluded that 488
deletion of Gdf11 leads to an increase in Nppb expression. However, with inclusion of both 489
control genotypes, we see that there is actually a confounding effect with significant differences 490
in expression of Nppb between Gdf11fl/fl and Myh6cre/wt control mice. It is challenging to
reconcile these differences among the different genotypes. It appears that the presence of Cre 492
induces stress on the mouse (with increased mortality, decreased body weight, increased Nppb 493
expression, and increased cardiomyocyte size). Comparing the Myh6cre/wt control mice to 494
Myh6cre/wt; Gdf11fl/fl mice, one might conclude that cardiomyocyte deletion of Gdf11 is actually
495
cardioprotective (with increased survival, increased body weight, decreased Nppb expression and 496
decreased cardiomyocyte size). However, other explanations are possible as well, such as 497
differential off target effects in the presence or absence of true loxP sites, or differential 498
expression of Cre in the two genotypes. Given that comparison of Gdf11fl/fl control mice to 499
Myh6cre/wt; Gdf11fl/fl mice leads to different conclusions than when comparing Myh6cre/wt control
500
mice to Myh6cre/wt; Gdf11fl/fl mice, we are unable to definitively determine the mechanisms by 501
which Gdf11 deletion in cardiomyocytes leads to left ventricular dilation. Inclusion of multiple 502
control groups admittedly adds significant costs and time required to maintain a larger animal 503
colony, but careful selection of control groups is necessary to obtain meaningful results. 504
We observed higher cre mRNA but not protein expression in Myh6cre/wt compared to 505
Myh6cre/wt; Gdf11fl/fl mice in this study. Variable cre RNA expression in different generations has
506
been reported in other Cre lines (19, 23). For example, in an albumin-Cre model, despite 507
transmission of albumin-cre in genomic DNA of successive generations, some mice did not 508
express cre in the liver (23). The authors speculated that that this could be due to multiple 509
homologous recombination events leading to segregation of inactive copies of the transgene, 510
binding of transcriptional inhibitors, or post-transcriptional silencing of cre (23). In addition, 511
cytosine methylation of loxP sites following Cre recombination of a parent can lead to inhibition 512
of Cre-mediated recombination in subsequent generations (19). Finally, although all mice were 513
on a mixed C57Bl/6J and 6N background, there may be generational differences due to different 514
degrees of backcrossing to the underlying genetic background. It is possible that there is a more 515
complex interaction with Cre and the underlying genetic background which will be difficult to 516
uncover. To try to address this confounding effect, we used littermate controls to compare 517
Gdf11fl/fl with Myh6cre/wt; Gdf11fl/fl mice. However, our breeding strategy paired Myh6cre/wt with
518
wild type (C57Bl/6J) mice and Gdf11fl/fl with Myh6cre/wt; Gdf11fl/fl thus Myh6cre/wt were not
519
littermates with Myh6cre/wt; Gdf11fl/fl mice and may have had varying degrees of methylation or 520
varying genetic backgrounds in the breeders. Future work to understand how Cre mRNA and 521
protein levels change with age in both genotypes is necessary to interpret whether variable Cre 522
levels might be confounding the phenotype seen with cardiomyocyte Gdf11 deletion. 523
In conclusion, deletion of the Gdf11 gene from cardiomyocytes may lead to left 524
ventricular dilation and decreased systolic function, consistent with a dilated cardiomyopathy 525
phenotype. However, due to numerous confounding factors associated with the selected, and 526
commonly used, Cre-lox system as well as challenges in working with Gdf11 itself and its 527
complicated regulatory system, we are unable to attribute a mechanism to this phenotype. These 528
data highlight the importance of using appropriate control groups when using the Cre 529
recombinase system, as opposite conclusions could have been drawn had only one of the two 530
control genotypes been used for comparison. Further work to develop alternative animal models 531
that avoid Cre toxicity as well as investigate alternative proteins involved in regulation of 532
GDF11 expression are warranted. 533
534
Acknowledgements
535
The authors acknowledge Catherine MacGillivray and Diane Faria from the Department of Stem 536
Cell and Regenerative Biology Histology Core for histology processing and staining. 537
538
Sources of Funding
539
J.C.G. was supported by the John S. LaDue Memorial Fellowship in Cardiology from Harvard 540
Medical School and an NIH T32 fellowship (5T32HL007572). V.B. was supported by the 541
Lemann Foundation Cardiovascular Research Postdoctoral Fellowship through Harvard 542
University/Brigham and Women’s Hospital. J.M.G was supported by an NIH F32 postdoctoral 543
fellowship (F32AG050395). R.G.W. was supported by a T32 fellowship from the NIH 544
(T32HL007208-39). This work was supported by grants from the NIH (AG047131, HL119230, 545
AG048917, and AG057428 to RTL and AG048917 and AG057428 to AJW) and from the Glenn 546
Foundation (to AJW). 547
548
Disclosures
549
Richard Lee, Ryan Walker, and Amy Wagers are co-founders of, members of the scientific 550
advisory board for, and hold private equity in Elevian, Inc, a company that aims to develop 551
medicines to restore regenerative capacity. Elevian also provides sponsored research support to 552
the Lee Lab and Wagers Lab. 553
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Figure Captions
643 644
Figure 1. Targeted cardiac Gdf11 deletion leads to left ventricular dilation. (A-C) Left
645
ventricular end diastolic volume by echocardiography at 1-2 months (A), 3-4 months (B), and 6 646
months of age (C). (D-F) Heart weight/body weight ratio at 1-2 months (D), 3-4 months (E), and 647
6 months of age (F). (G) Fractional shortening by echocardiography at 6 months of age. (H) 648
Estimated left ventricular mass by echocardiography at 6 months of age. (I) Heart weight at 6 649
months of age. (J) Interventricular septal thickness during diastole at 6 months of age. (K) Left 650
ventricular internal diameter during diastole at 6 months of age. (L) Left ventricular posterior 651
wall thickness during diastole at 6 months of age. (M) Body weight at 6 months of age (just prior 652
to harvest). (N) Tibia length at 6 months of age. (O) Heart weight to tibia length ratio at 6 653
months of age. Sample sizes at 1-2 months: Myh6cre/wt (n=5 females, n=9 males), Gdf11fl/fl (n=11 654
females, n=21 males), and Myh6cre/wt; Gdf11fl/fl (n=12 females, n=15 males) mice; n=4/group for 655
heart weight to body weight ratio parameter. Sample sizes at 3-4 months: Myh6cre/wt (n=9 656
females, n=12 males), Gdf11fl/fl (n=9 females, n=13 males), and Myh6cre/wt; Gdf11fl/fl (n=13 657
females, n=14 males) mice for echocardiography; n=4/group for heart weight to body weight 658
ratio parameter. Sample sizes at 6 months: Myh6cre/wt (n=6 females, n=16 males), Gdf11fl/fl (n=3 659
females, n=5 males), and Myh6cre/wt; Gdf11fl/fl (n=6 females, n=9 males) mice, *p<0.05, 660
**p<0.01 by Kruskal-Wallis test with Tukey post-hoc analysis. 661
662
Figure 2. Survival curves, echocardiographic, heart weight, and body weight parameters in mice
663
with tamoxifen-inducible Myh6-driven Cre expression (Myh6-MCM). (A and B) Kaplan-Meier 664
survival analysis in Gdf11fl/fl (gray), and Myh6MCM/wt; Gdf11fl/fl (black) female (A) and male (B) 665
mice. Male mice have significant differences between groups with p<0.01 by the Mantel-Cox 666
test. n=12-16/group at start of study. (C) Recombination of Gdf11 gene seen in the heart but not 667
liver, kidney, spleen or skeletal muscle following administration of 4-hydroxytamoxifen (75 668
mg/kg i.p. x 5 days) by polymerase chain reaction (PCR). (D) Gdf11 expression in RNA 669
extracted from whole heart 1 month after 4-hydroxytamoxifen administration in female and male 670
mice by quantitative PCR (qPCR), n=3/group. Data are normalized to TATA-binding protein 671
(Tbp) then to female Gdf11fl/fl vehicle control. For echo and harvest data in (E) to (J), n=7-672
16/group, data collected 1 month after initiation of 4OH-tamoxifen injection (6 months of age). 673
(E) Left ventricular end diastolic volume (µl) by echocardiography. (F) Estimated left ventricular 674
mass (mg) by echocardiography. (G) Body weight (g) at harvest. (H) Tibia length, **p<0.01, 675
***p<0.001 by two-way ANOVA. (I) Heart weight and (J) Heart weight/body weight ratio. (K) 676
Activin A mRNA expression in spleen is significantly increased in male Myh6MCM/wt; Gdf11fl/fl 677
mice 1 month after initiation of 4OH-tamoxifen injection (6 months of age), n=4-6/group, 678
*p<0.05 by Kruskal-Wallis with Dunn’s multiple comparisons test. Data are normalized to Tbp 679
then to female Gdf11fl/fl vehicle control. 680
681
Figure 3. Targeted cardiomyocyte deletion of Gdf11 does not decrease total Gdf11 mRNA
682
expression in mouse hearts. (A) Representative agarose gel image depicting Myh6 (wild type 683
band at 894 bp, Myh6-cre band at 300 bp) and Gdf11 (wild type band at 359 bp, flox band at 393 684
bp, and ∆2-3 (post-recombination) band at 300 bp) alleles seen in the heart on day of life 0-1 by 685
polymerase chain reaction (PCR) in Myh6cre/wt, Gdf11fl/fl, and Myh6cre/wt; Gdf11fl/fl pups. (B)
686
Recombination of Gdf11 at 6 months of age in females and males in the heart but not in lung, 687
liver, kidney, spleen or skeletal muscle by PCR. (C and D) Gdf11 expression in RNA extracted 688
from whole heart in (C) 3 month old (n=4/group) or (D) 6 month old (n=3-4/group) female and 689
male mice by quantitative PCR (qPCR). Data are normalized to TATA-binding protein (Tbp) 690
then to female Myh6cre/wt control. (E and F) Gdf11 expression in RNA extracted from isolated 691
adult (>2 months old) cardiomyocytes (E) or non-cardiomyocytes (F), with each data point 692
(shown with open symbols) representing isolated cells from a single mouse of the same 693
genotype, n=3 per group (2 males, 1 female). Data normalized to Tbp then Myh6cre/wt control. 694
Cardiomyocytes (E), p-value = not significant (n.s., 0.05); non-cardiomyocytes (F), p-value = 695
n.s. (0.3) by Kruskal-Wallis test. (G and H) Serum levels of GDF11 (G) and myostatin (H) as 696
determined by quantitative mass spectrometry in 6-month-old mice. n=3-4/group, *p<0.05 by 697
Kruskal-Wallis test followed by Tukey’s multiple comparisons test. 698
699
Figure 4. Cre recombinase has adverse effects on survival and body weight. (A and B)
Kaplan-700
Meier survival analysis in (A) female and (B) male Myh6cre/wt (dark gray, dashed line, n=12 701
females, n=24 males), Gdf11fl/fl (light gray, dotted line, n=5 females, n=11 males), and 702
Myh6cre/wt; Gdf11fl/fl (black solid line, n=12 females, n=15 males) mice. (C and D) Body weight
703
versus age in weeks in (C) female and (D) male Myh6cre/wt (gray circles with dashed line, n=5
704
females, n=13 males), Gdf11fl/fl (gray squares with dotted line, n=5 females, n=11 males), and 705
Myh6cre/wt; Gdf11fl/fl (black tringles with solid line, n=12 females, n=15 males) mice. *p<0.05,
706
**p<0.01 by two-way repeated measures ANOVA with Tukey’s multiple comparisons test. 707
708
Figure 5. Gene expression analysis of in 2 month old (A-D) and 6 month old (E-H) from whole
709
hearts of (A and E) Mstn, (B and F) Nppb, (C and G) Tgfbr1, and (D and H) Gdf15 by qPCR. 710
Data are normalized to Tbp expression then to Myh6cre/wt control. No significant differences were 711
observed between genders therefore data represent combined male and female data. n=7-8/group, 712
*p<0.05, **p<0.01, ***p<0.001 by Kruskal-Wallis analysis with Dunn’s multiple comparisons 713
test. 714
715
Figure 6. Cre mRNA expression is higher in Myh6cre/wt compared to Myh6cre/wt; Gdf11fl/fl mice 716
and is associated with myocardial fibrosis and increased cardiomyocyte size. (A) Representative 717
histology sections of male and female Myh6cre/wt, Gdf11fl/fl, and Myh6cre/wt; Gdf11fl/fl mice stained 718
with Masson’s trichrome stain. (B) Global fibrosis (% blue pixels) shows increased fibrosis in 719
male (n=5-9/group) Myh6cre/wt mice compared to females (n=3-5/group). (C) Cardiomyocyte 720
cross-sectional area is increased in male Myh6cre/wt mice compared to male Gdf11fl/fl and 721
Myh6cre/wt; Gdf11fl/fl mice. Cross-sectional area from 40 cardiomyocytes per mouse were
722
averaged for each mouse then average cardiomyocyte cross-sectional area by mouse data were 723
analyzed with n=3=6 female mice, n=5-10 male mice, *p<0.05, **p<0.01 by two-way ANOVA 724
with Tukey post-hoc analysis. (D) Cre expression by qPCR from male and female 3-6 month old 725
mice. No significant differences were detected between genders therefore data depict combined 726
male and female data. Data are normalized to Tbp then to Myh6cre/wt control. n=16/group,
727
*p<0.05 by Mann-Whitney test. (E) Cre protein detected by Western analysis in Myh6cre/wt, 728
Gdf11fl/fl, and Myh6cre/wt; Gdf11fl/fl 4 month old male mice. Band densitometry analysis
729
comparing Myh6cre/wt mice to Myh6cre/wt; Gdf11fl/fl (not shown) is not significant with p-value 0.2 730
by Mann-Whitney test. 731
