Depletion of mammalian target of rapamycin (mTOR) via siRNA mediated knockdown leads to stabilization of β-catenin and elicits distinct features of cardiomyocyte hypertrophy

AbstractCardiac myocyte growth is under differential control of mammalian target of rapamycin (mTOR) and glycogen-synthase-kinase-3β (GSK3β). Whereas active GSK3β negatively regulates growth and down-regulates cellular protein synthesis, activation of the mTOR pathway promotes protein expression and cell growth. Here we report that depletion of mTOR via siRNA mediated knockdown causes marked down-regulation of GSK3β protein in cardiac myocytes. As a result, GSK3β target protein β-catenin becomes stabilized and translocates into the nucleus. Moreover, mTOR knockdown leads to increase in cardiac myocyte surface area and produces an up-regulation of the fetal gene program. Our findings suggest a new type of convergence of mTOR and GSK3β activities, indicating that GSK3β-dependent stabilization of β-catenin in cardiac myocytes is influenced by mTOR

DYRK2 Negatively Regulates Cardiomyocyte Growth by Mediating Repressor Function of GSK-3β on eIF2Bε

<div><p>Background</p><p>A prerequisite of hypertrophic response of the myocardium is an increase in protein synthesis. A central regulator of translation initiation is Eukaryotic initiation factor 2B (eIF2B). Here we assessed the hypothesis that regulation of protein synthesis via eIF2Bε is essential to cardiac hypertrophic response in vivo.</p><p>Methods</p><p>Two transgenic mouse lines were generated with cardiac restricted overexpression of eIF2Bε or its mutant eIF2Bε-eIFS⁵³⁵A, which cannot be inactivated by phosphorylation through GSK-3β.</p><p>Results</p><p>(1) Under baseline conditions eIF2Bε transgenic mice showed no difference in cardiac phenotype compared to wild type, whereas in the mutant eIF2Bε-S⁵³⁵A an increase in LV/tibia length (7.5±0.4 mg/mm vs. 6.2±0.2 mg/mm, p<0.001) and cardiomyocyte cross sectional area (13004±570 vs. 10843±347 RU, p<0.01) was observed. (2) Cardiac overexpression of eIF2Bε did not change the response of the heart to pathologic stress induced by chronic isoproterenol treatment. (3) Cardiac overexpression of the eIF2Bε transgene was followed by overexpression of DYRK2 which is known to prime the inhibitory action of GSK-3β on eIF2Bε, while DYRK1A and GSK-3β itself were not increased. (4) In C57BL/6 mice after 48 h of isoproterenol-stimulation or aortic banding, eIF2Bε was increased and DYRK2 was concomitantly decreased. (5) In line with these in vivo findings, siRNA knockdown of DYRK2 in cultured cardiomyocytes resulted in decreased levels of p(S535)- eIF2Bε, (6) whereas adenoviral induced overexpression of DYRK2 was accompanied by clearly increased phosphorylation of eIF2Bε, indicating a coordinated response pattern (7) Adenoviral induced overexpression of DYRK2 leads to significantly reduced cardiomyocyte size and diminishes hypertrophic response to adrenergic stimulation.</p><p>Conclusions</p><p>The interaction of GSK-3β and its priming kinase DYRK2 regulate the activity of eIF2Bε in cardiac myocytes. DYRK2 is a novel negative regulator of cardiomyocyte growth. DYRK2 could serve as a therapeutic option to regulate myocardial growth.</p></div

Levels of phospho(S535)-eIF2Bε and DYRK2 expression show strict correlation, indicating a coordinated response pattern following pathologic injury.

<p><b>A:</b> In acute myocardial response 48 h after hypertrophic stimulation by isoproterenol or aortic banding of C57BL/6 wild type mice eIF2Bε levels were elevated whereas phospho(S535)-eIF2Bε and DYRK2 levels were significantly decreased. <b>B:</b> siRNA mediated knockdown of DYRK2 lead to reduced phospho(S535)-eIF2Bε levels in vitro. Exemplary immunoblots of three individual experiments.</p

Expression of DYRK2 is elevated in left ventricular tissue in eIF2Bε-TG. GSK-3β, DYRK1A and eIF2α were not different between groups.

<p>In each case, sections are exemplary details of the same blot <b>(A)</b>. <b>B:</b> Immunoblots of co-immunoprecipitation demonstrating physical interaction of eIF2Bε and DYRK2 in left ventricular tissue of eIF2Bε-TG and eIF2Bε-S⁵³⁵A-TG.</p

Adenoviral induced overexpression of DYRK2 in cultivated cardiomyocytes reduces baseline cell surface area and diminishes hypertrophic response to phenylephrine stimulation.

<p><b>A:</b> Overexpression of DYRK2 is accompanied by increased levels of phospho(S535)-eIF2Bε, and GSK-3β. <b>B:</b> Representative cultivated cardiomyocytes with reduced cell size and density of cytoskeleton following DYRK2 overexpression under baseline conditions. <b>C:</b> Cardiomyocyte size was significantly reduced in DYRK2 overexpressing cells compared to negative control under baseline conditions. Further, DYRK2 overexpression could almost inhibit the hypertrophic response to phenylephrine stimulation (n = 117 ad-Glox, n = 125 ad-DYRK2, n = 51 ad-Glox+PE, n = 68 ad-DYRK2+PE, *** <0.0001).</p

Morphological and echocardiographic parameters at the age of 20 months.

<p>All values of eIF2Bε-TG showed no significant difference to wild type. ^ap  =  eIF2Bε-S⁵³⁵A-TG vs. wild type. AWTD: Diastolic anterior wall thickness; PWTD: Diastolic posterior wall thickness.</p

Biometric and functional measurements at the age of 3–4 months.

<p>AWTD: Diastolic anterior wall thickness; PWTD: Diastolic posterior wall thickness; LVESP: left ventricular end systolic pressure; LVEDP: left ventricular end diastolic pressure. All values of eIF2Bε-TG showed no significant difference to wild type. ^ap  =  eIF2Bε-S⁵³⁵A-TG vs. wild type.</p

Morphological and echocardiographic parameters after administration of isoproterenol or isovolumic vehicle for 14 days.

<p>AWTD: Diastolic anterior wall thickness; PWTD: Diastolic posterior wall thickness.</p>a<p>p  =  isoproterenol: wild type vs eIF2Bε-TG; ^bp  =  isoproterenol: wild type vs eIF2Bε-S⁵³⁵A-TG; ^cp  =  isoproterenol: eIF2Bε-TG vs eIF2Bε-S⁵³⁵A-TG. ^dp  =  wild type: isoproterenol vs. vehicle; ^ep  =  eIF2Bε-TG: isoproterenol vs. vehicle; ^fp  =  eIF2Bε-S⁵³⁵A-TG: isoproterenol vs. vehicle.</p