Correction: Adult Cardiac Expression of the Activating Transcription Factor 3, ATF3, Promotes Ventricular Hypertrophy.

[This corrects the article DOI: 10.1371/journal.pone.0068396.]

Adult Cardiac Expression of the Activating Transcription Factor 3, ATF3, Promotes Ventricular Hypertrophy

<div><p>Cardiac hypertrophy is an adaptive response to various mechanophysical and pathophysiological stresses. However, when chronic stress is sustained, the beneficial response turns into a maladaptive process that eventually leads to heart failure. Although major advances in the treatment of patients have reduced mortality, there is a dire need for novel treatments for cardiac hypertrophy. Accordingly, considerable efforts are being directed towards developing mice models and understanding the processes that lead to cardiac hypertrophy. A case in point is ATF3, an immediate early transcription factor whose expression is induced in various cardiac stress models but has been reported to have conflicting functional significance in hypertrophy. To address this issue, we generated a transgenic mouse line with tetracycline-regulated ATF3 cardiac expression. These mice allowed us to study the consequence of ATF3 expression in the embryo or during the adult period, thus distinguishing the effect of ATF3 on development versus pathogenesis of cardiac dysfunction. Importantly, ATF3 expression in adult mice resulted in rapid ventricles hypertrophy, heart dysfunction, and fibrosis. When combined with a phenylephrine-infusion pressure overload model, the ATF3 expressing mice displayed a severe outcome and heart dysfunction. In a complementary approach, ATF3 KO mice displayed a lower level of heart hypertrophy in the same pressure overload model. In summary, ectopic expression of ATF3 is sufficient to promote cardiac hypertrophy and exacerbates the deleterious effect of chronic pressure overload; conversely, ATF3 deletion protects the heart. Therefore, ATF3 may serve as an important drug target to reduce the detrimental consequences of heart hypertrophy.</p> </div

Adult-ATF3 expressing mice display higher fibrosis and lower heart function following a 2-week pressure overload model.

<p>RT-qPCR analysis for cDNA derived from either wild-type or ATF3 transgenic mice. mRNA was extracted from ventricles from wild-type (black) or ATF3 transgenic (gray) and RT-qPCR was performed with the indicated specific primers: <b>A</b>. Col1α <b>B</b>. TGFβ <b>C</b>. connective tissue growth factor (cTGF). The results represent the mean and SEM relative to GAPDH expression of the indicated number of animals (n). <b>D</b>. Masson trichrome staining of paraffin embedded sections of wild-type and adult-ATF3 expressing mice, either untreated (control) or after 2 weeks of PE infusion. <b>E</b>. Quantification of fibrosis of the indicated number of mice (n). At least five sections for the indicated number of mice (n) were analyzed <b>F</b>. Adult-ATF3 expressing mice treated as indicated were examined by micro-ultrasound and measurements were recorded to determine fractional shortening (FS) percentage in order to assess heart function. Maximal left ventricles end-diastolic (LVDd) and end-systolic (LVDs) dimensions parameters were measured in short-axis M-mode images. Fractional shortening (FS) was calculated as: FS (%) = [(LVDd-LVDs)/LVDd] X 100. The results represent the mean and SEM of the indicated number of animals (n). Asterisks (*/**) indicate a P value <0.05 or <0.01 respectively of a one-tailed t-test compared to wild-type mice.</p

Adult-ATF3 expressing mice display increased Vw/Bw growth ratio in basal and following PE infusion.

<p><b>A</b>. RT-qPCR analysis for cDNA derived from either wild-type or ATF3 transgenic mice. mRNA was extracted from ventricles derived from wild-type (black) or adult-ATF3 expressing (gray) and RT-qPCR was performed with the hATF3 specific primers. <b>B</b>. Mice ventricles weight (Vw) relative to mouse body weight (Bw) is calculated (mg/gr). The results represent the mean and SEM of the indicated number of animals (n). The mean and SEM of the absolute values is provided (right panel). <b>C</b>–<b>E</b>. RT-qPCR with cDNA from A was performed with the indicated specific primers: <b>C</b>. ANP. <b>D</b>. BNP. <b>E</b>. Skeletal actin (Acta1). The results represent the mean expression relative to GAPDH of the indicated number of animals (n). Asterisks (*/**) indicate a P value <0.05 or <0.01 respectively of a one-tailed t-test compared to wild-type mice.</p

Adult-ATF3 expression promotes hypertrophy.

<p>A. Mice were mated in the presence of doxycycline (adult ATF3 expressing). Weaned newborn mice were either maintained with doxycycline containing water (0 weeks without doxycycline) or provided with regular water. Mice were sacrificed at the indicated number of weeks following doxycycline removal. Mice ventricles were weighed (Vw) to mouse body weight (Bw). The results represent the ratio of Vw/Bw (mg/gr) of ATF3 transgenic mice (gray) and wild-type (black) mice at the indicated time (weeks) following doxycycline removal. The results represent the mean and SEM of the indicated number of animals (n). <b>B</b>. Atria and ventricles weight relative to body weight at 6 weeks of age (mg/gr). <b>C</b>–<b>F</b>. Adult-ATF3 expressing mice show higher expression of hypertrophic markers. RT-qPCR analysis for cDNA derived from RNA extracted from ventricles of ATF3-transgenic and wild-type mice with the corresponding specific primers to the following genes: <b>C</b>. Atrial natriuretic peptide (ANP) D. Brain natriuretic peptide (BNP) <b>E</b>. β Myosin heavy chain (βMHC) F. Skeletal actin (Acta1). The results represent the mean expression relative to GAPDH of the indicated number of animals (n). <b>G</b>. Ventricles sections were stained with TRITC-labeled wheat germ aglutinin and the cell size was analyzed using the Image Pro Plus software. <b>H</b>. Quantification of cell size in G. The results represent the mean and SEM from five different sections derived from wild type (n=2) and adult ATF3 expressing (n=3) animals at the indicated time following doxycycline removal. Asterisks (*/**) represent P value <0.05 or <0.01 respectively of a one-tailed t-test compared with wild-type mice.</p

HA-ATF3 transient expression is tightly controlled by doxycycline.

<p><b>A</b>. pBI-G expression vector plasmid or HA-ATF3 expression plasmid (pBIG-HA-ATF3) were co-transfected with the tTA (tet-off) expression plasmid into HEK-293T cells in the presence (+) or absence (-) of doxycycline (Dox, 10µg/ml). Nuclear cell lysate was separated on 12.5% SDS-PAGE followed by Western blot analysis with anti-HA, anti-ATF3, and anti-α tubulin (loading control). <b>B</b>. Schematic representation of the mating cages. Gender matched heterozygotes αMHC-tTA driver mice were mated with ATF3-tg responder mice line. Mating in the presence of doxycycline represents ATF3-Off expression during embryonic development therefore, mice are designated adult-ATF3 expressing. Mating in the absence of doxycycline represents ATF3-On expression through embryonic development therefore, mice are designated embryonic-ATF3 expressing. <b>C</b>. RT-qPCR analysis for cDNA derived from atria and ventricles of either wild-type or ATF3 transgenic mice treated with doxycycline as indicated. The expression level of ATF3 was examined by either mouse- (black) or human-specific primers (gray). The results represent the mean expression relative to GAPDH of the indicated number of animals (n). Asterisks (*/**) represent P value <0.05 or <0.01 of a one-tailed t-test compared to wild-type mice. <b>D</b>. Representative Western blot analysis (Top panel) of cell lysates derived from ventricles of wild type mice (Wt.), adult-ATF3 expressing and wild type mice following 2 h PE injection. The membrane was probed with anti-ATF3 and GAPDH for loading control. The asterisks (*) represent non-specific cross reactive proteins (non-specific). Densitometry analysis (bottom panel) of ATF3 expression was normalized with the GAPDH level. The results represent the mean and SEM from six independent animals. <b>E</b>. Immunohistochemistry of left ventricle sections stained with αATF3 (1:200). Representative sections derived from mice positive for HA-ATF3 responder and αMHC driver (ATF3 Tg, right panel) and Wild-type mouse (left panel). The magnification shown is X20. The black arrow indicates an ATF3-stained nucleus.</p

The hearts derived from adult-ATF3 expressing mice display a higher level of inflammatory response.

<p>mRNA derived from ventricles of wild-type and ATF3 transgenic, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068396#pone-0068396-g002" target="_blank">Figure 2C</a>, was analyzed for the indicated inflammatory markers <b>A</b>. IL-6 <b>B</b>. F4/80 C. CD68. The results represent the mean expression relative to GAPDH of the indicated number of animals (n). Asterisks (*) indicate a P value <0.05 of a one-tailed t-test compared with wild-type mice.</p

The hearts derived from adult-ATF3 expressing mice display a higher level of fibrosis and lower heart function.

<p>mRNA described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068396#pone-0068396-g002" target="_blank">Figure 2C</a> was analyzed for the indicated fibrosis markers A. Collagen 1α (Col1α) <b>B</b>. Transforming growth factor β (TGFβ). The results represent the mean expression relative to GAPDH of the indicated number of animals (n). <b>C</b>. Representative Masson Trichrome staining of paraffin embedded sections of wild-type and adult-ATF3 expressing mice at the indicated time following doxycycline removal <b>D</b>. Quantification of fibrosis of the indicated number of mice (n). At least five sections per mice were analyzed. <b>E</b>. Adult-ATF3 expressing mice treated as indicated were examined by micro-ultrasound and measurements were recorded to determine the fractional shortening (FS) percentage. Maximal left ventricles end-diastolic (LVDd) and end-systolic (LVDs) dimensions parameters were measured in short-axis M-mode images. Fractional shortening (FS) was calculated as: FS (%) = [(LVDd-LVDs)/LVDd] X 100. Echocradiography measurements were performed at the indicated number of weeks following doxycycline removal. The results represent the mean and SEM of the indicated number of animals (n). Asterisks (*/**) represent P value <0.05 or <0.01 respectively of a one-tailed t-test compared with wild-type mice.</p

JDP2 and ATF3 deficiencies dampen maladaptive cardiac remodeling and preserve cardiac function.

c-Jun dimerization protein (JDP2) and Activating Transcription Factor 3 (ATF3) are closely related basic leucine zipper proteins. Transgenic mice with cardiac expression of either JDP2 or ATF3 showed maladaptive remodeling and cardiac dysfunction. Surprisingly, JDP2 knockout (KO) did not protect the heart following transverse aortic constriction (TAC). Instead, the JDP2 KO mice performed worse than their wild type (WT) counterparts. To test whether the maladaptive cardiac remodeling observed in the JDP2 KO mice is due to ATF3, ATF3 was removed in the context of JDP2 deficiency, referred as double KO mice (dKO). Mice were challenged by TAC, and followed by detailed physiological, pathological and molecular analyses. dKO mice displayed no apparent differences from WT mice under unstressed condition, except a moderate better performance in dKO male mice. Importantly, following TAC the dKO hearts showed low fibrosis levels, reduced inflammatory and hypertrophic gene expression and a significantly preserved cardiac function as compared with their WT counterparts in both genders. Consistent with these data, removing ATF3 resumed p38 activation in the JDP2 KO mice which correlates with the beneficial cardiac function. Collectively, mice with JDP2 and ATF3 double deficiency had reduced maladaptive cardiac remodeling and lower hypertrophy following TAC. As such, the worsening of the cardiac outcome found in the JDP2 KO mice is due to the elevated ATF3 expression. Simultaneous suppression of both ATF3 and JDP2 activity is highly beneficial for cardiac function in health and disease

Tumor Treating Fields (TTFields) Hinder Cancer Cell Motility through Regulation of Microtubule and Actin Dynamics

Tumor Treating Fields (TTFields) are noninvasive, alternating electric fields within the intermediate frequency range (100&ndash;300 kHz) that are utilized as an antimitotic cancer treatment. TTFields are loco-regionally delivered to the tumor region through 2 pairs of transducer arrays placed on the skin. This novel treatment modality has been FDA-approved for use in patients with glioblastoma and malignant pleural mesothelioma based on clinical trial data demonstrating efficacy and safety; and is currently under investigation in other types of solid tumors. TTFields were shown to induce an anti-mitotic effect by exerting bi-directional forces on highly polar intracellular elements, such as tubulin and septin molecules, eliciting abnormal microtubule polymerization during spindle formation as well as aberrant cleavage furrow formation. Previous studies have demonstrated that TTFields inhibit metastatic properties in cancer cells. However, the consequences of TTFields application on cytoskeleton dynamics remain undetermined. In this study, methods utilized in combination to study the effects of TTFields on cancer cell motility through regulation of microtubule and actin dynamics included confocal microscopy, computational tools, and biochemical analyses. Mechanisms by which TTFields treatment disrupted cellular polarity were (1) interference with microtubule assembly and directionality; (2) altered regulation of Guanine nucleotide exchange factor-H1 (GEF-H1), Ras homolog family member A (RhoA), and Rho-associated coiled-coil kinase (ROCK) activity; and (3) induced formation of radial protrusions of peripheral actin filaments and focal adhesions. Overall, these data identified discrete effects of TTFields that disrupt processes crucial for cancer cell motility