Smyd1 Facilitates Heart Development by Antagonizing Oxidative and ER Stress Responses

<div><p>Smyd1/Bop is an evolutionary conserved histone methyltransferase previously shown by conventional knockout to be critical for embryonic heart development. To further explore the mechanism(s) in a cell autonomous context, we conditionally ablated <i>Smyd1</i> in the first and second heart fields of mice using a knock-in (KI) <i>Nkx2</i>.<i>5-cre</i> driver. Robust deletion of <i>floxed-Smyd1</i> in cardiomyocytes and the outflow tract (OFT) resulted in embryonic lethality at E9.5, truncation of the OFT and right ventricle, and additional defects consistent with impaired expansion and proliferation of the second heart field (SHF). Using a transgenic (Tg) <i>Nkx2</i>.<i>5-cre</i> driver previously shown to not delete in the SHF and OFT, early embryonic lethality was bypassed and both ventricular chambers were formed; however, reduced cardiomyocyte proliferation and other heart defects resulted in later embryonic death at E11.5-12.5. Proliferative impairment prior to both early and mid-gestational lethality was accompanied by dysregulation of transcripts critical for endoplasmic reticulum (ER) stress. Mid-gestational death was also associated with impairment of oxidative stress defense—a phenotype highly similar to the previously characterized knockout of the Smyd1-interacting transcription factor, skNAC. We describe a potential feedback mechanism in which the stress response factor Tribbles3/TRB3, when directly methylated by Smyd1, acts as a co-repressor of Smyd1-mediated transcription. Our findings suggest that Smyd1 is required for maintaining cardiomyocyte proliferation at minimally two different embryonic heart developmental stages, and its loss leads to linked stress responses that signal ensuing lethality.</p></div

Deletion of <i>Smyd1</i> by <i>Tg-Nkx2</i>.<i>5-cre</i> leads to a delayed embryonic lethal cardiac phenotype.

<p><b>A.</b><i>Smyd1</i>^{<i>flox/flox</i>}; <i>Tg-Nkx2</i>.<i>5-cre</i> (Tg-CKO) embryos die at midgestation. Table numbers are total recovered embryos of each genotype. The number of dead or abnormal embryos is given in parentheses. <b>B, C.</b><i>Smyd1</i> mRNA expression at E10.5 assayed by RT-PCR (B) and real-time PCR (C). <b>D.</b> H&E-stained transverse sections of E11.5 control (Cx) and Tg-CKO embryos showing pericardial edema, thinned pericardium and decreased trabeculation. <b>E.</b> Decreased proliferation was observed in the hearts of E10.5 <i>Smyd1</i> Tg-CKO embryos. Representative images of Cx and Tg-CKO hearts stained with H&E (upper panels) and the mitosis marker phospho-histone H3 serine 10 (p-H3) (lower panels). <b>F.</b> Quantification of p-H3 positive cells within the heart from three sections for three independent embryos (n = 3). <b>G.</b> Comparison of <i>skNAC</i> knockout and <i>Smyd1 Tg-CKO</i> heart gene expression by real-time PCR at E11.5. Data, focused primarily on oxidative response deregulation, are presented as mean for each genotype (<i>skNAC</i>^-/-, n = 5; <i>Smyd1</i> Tg-CKO, n = 4). Error bars indicate SEM. <b>H.</b> Genes encoding mediators of ER stress are deregulated by loss of Smyd1. Real-time PCR data represents average of 3 biological replicates each with 3 technical replicates; error bars indicate SEM. Data were analyzed by Student’s t-test (*P < 0.05, **P <0.01, ***P < 0.001, ****P < 0.0001).</p

Loss of <i>Smyd1</i> using <i>Ki-Nkx2</i>.<i>5</i>^<i>cre/+</i> disrupts looping morphogenesis and chamber formation through perturbation of the SHF and activation of ER stress.

<p><b>A.</b> Gross morphological comparison of control (Cx: <i>Nkx2</i>.<i>5</i>^<i>+/+</i>; <i>Smyd1</i>^{<i>Flox/Flox</i>}) and <i>Smyd1</i> Ki-CKO (CKO: <i>Nkx2</i>.<i>5</i>^<i>cre/+</i>; <i>Smyd1</i>^{<i>Flox/Flox</i>}) hearts at E9.5. Scale bar = 200 μm. <b>B, C</b>. The lengths of the outflow tract (OFT) (B) and right ventricle (RV) (C) were significantly reduced in Ki-CKO embryos at E9.5 (n = 6/group). <b>D.</b> Representative results of microarray gene expression comparison of transcripts critical to SHF and chamber formation at E9.5. Data are presented as expression values of 2 independent biological replicas of each genotype averaged from 2 technical replicas. <b>E.</b> Confirmation of microarray for deregulated transcripts critical to SHF and chamber formation by real-time PCR using RNA from E9.5 heart/pharyngeal mesoderm (n = 9/group). <b>F</b>. Whole mount <i>in situ</i> hybridization comparison of selected SHF and chamber formation transcripts deregulated and/or mislocalized in CKO hearts at E9.5 (n = 3/group). Arrows denote areas of differential expression. <b>G.</b> Comparison of cell proliferation in the outflow tract of Control (Cx) and Ki-CKO by BrdU immunohistochemistry. PE, Pharyngeal Endoderm. OFT, outflow tract. V, ventricle. Scale bar = 100 μm. <b>H.</b> Quantification of anti-BrdU staining in the outflow tract (n = 6/group). <b>I.</b> Loss of Smyd1 leads to deregulation of genes critical to anti-proliferative responses to ER stress. Data are presented as a heat map with expression values of 2 independent biological replicas of each genotype averaged from 2 technical replicas plotted as log² expression values. For B, C, E and H, data were analyzed by Student’s t-test (*P < 0.05).</p

Expression of <i>Smyd1</i> during early heart development.

<p><b>A</b>. Whole mount <i>in situ</i> hybridization for <i>Smyd1</i> in E8.5 (top panel) and E9.5 embryos (bottom panel) are shown from the left and right side. Red arrows denote the boundaries of <i>Smyd1</i> expression at the atrial and venous poles. Scale bar = 500 μm. <b>B.</b><i>In situ</i> hybridization for <i>Smyd1</i> in transverse sections of E10.0 hearts. The box in the left panel is enlarged in the right panel. <i>Smyd1</i> mRNA was enriched in the myocardium and not detectable in either the epicardium or endocardium. Black arrow = epicardium; Black arrowhead = endocardium. <b>C.</b> Immunolocalization of Smyd1 protein in the heart at E13.5. Smyd1 was specifically detected in cardiomyocytes. No Smyd1 protein was detectable in the endocardium (red arrowhead), epicardium (yellow arrowhead) or coronary vasculature (white arrowhead). Scale bar = 50 μm.</p