Translational control of Ybx1 expression regulates cardiac function in response to pressure overload in vivo.

RNA-protein interactions are central to cardiac function, but how activity of individual RNA-binding protein is regulated through signaling cascades in cardiomyocytes during heart failure development is largely unknown. The mechanistic target of rapamycin kinase is a central signaling hub that controls mRNA translation in cardiomyocytes; however, a direct link between mTOR signaling and RNA-binding proteins in the heart has not been established. Integrative transcriptome and translatome analysis revealed mTOR dependent translational upregulation of the RNA binding protein Ybx1 during early pathological remodeling independent of mRNA levels. Ybx1 is necessary for pathological cardiomyocyte growth by regulating protein synthesis. To identify the molecular mechanisms how Ybx1 regulates cellular growth and protein synthesis, we identified mRNAs bound to Ybx1. We discovered that eucaryotic elongation factor 2 (Eef2) mRNA is bound to Ybx1, and its translation is upregulated during cardiac hypertrophy dependent on Ybx1 expression. Eef2 itself is sufficient to drive pathological growth by increasing global protein translation. Finally, Ybx1 depletion in vivo preserved heart function during pathological cardiac hypertrophy. Thus, activation of mTORC1 links pathological signaling cascades to altered gene expression regulation by activation of Ybx1 which in turn promotes translation through increased expression of Eef2.

Muscle-specific Cand2 is translationally upregulated by mTORC1 and promotes adverse cardiac remodeling.

The mechanistic target of rapamycin (mTOR) promotes pathological remodeling in the heart by activating ribosomal biogenesis and mRNA translation. Inhibition of mTOR in cardiomyocytes is protective; however, a detailed role of mTOR in translational regulation of specific mRNA networks in the diseased heart is unknown. We performed cardiomyocyte genome-wide sequencing to define mTOR-dependent gene expression control at the level of mRNA translation. We identify the muscle-specific protein Cullin-associated NEDD8-dissociated protein 2 (Cand2) as a translationally upregulated gene, dependent on the activity of mTOR. Deletion of Cand2 protects the myocardium against pathological remodeling. Mechanistically, we show that Cand2 links mTOR signaling to pathological cell growth by increasing Grk5 protein expression. Our data suggest that cell-type-specific targeting of mTOR might have therapeutic value against pathological cardiac remodeling.

Saraf-dependent activation of mTORC1 regulates cardiac growth.

Pathological cardiac hypertrophy is an independent risk for heart failure (HF) and sudden death. Deciphering signaling pathways regulating intracellular Ca2+ homeostasis that control adaptive and pathological cardiac growth may enable identification of novel therapeutic targets. The objective of the present study is to determine the role of the store-operated calcium entry-associated regulatory factor (Saraf), encoded by the Tmem66 gene, on cardiac growth control in vitro and in vivo. Saraf is a single-pass membrane protein located at the sarco/endoplasmic reticulum and regulates intracellular calcium homeostasis. We found that Saraf expression was upregulated in the hypertrophied myocardium and was sufficient for cell growth in response to neurohumoral stimulation. Increased Saraf expression caused cell growth, which was associated with dysregulation of calcium-dependent signaling and sarcoplasmic reticulum calcium content. In vivo, Saraf augmented cardiac myocyte growth in response to angiotensin II and resulted in increased cardiac remodeling together with worsened cardiac function. Mechanistically, Saraf activated mTORC1 (mechanistic target of rapamycin complex 1) and increased protein synthesis, while mTORC1 inhibition blunted Saraf-dependent cell growth. In contrast, the hearts of Saraf knockout mice and Saraf-deficient myocytes did not show any morphological or functional alterations after neurohumoral stimulation, but Saraf depletion resulted in worsened cardiac function after acute pressure overload. SARAF knockout blunted transverse aortic constriction cardiac myocyte hypertrophy and impaired cardiac function, demonstrating a role for SARAF in compensatory myocyte growth. Collectively, these results reveal a novel link between sarcoplasmic reticulum calcium homeostasis and mTORC1 activation that is regulated by Saraf.

Identification of dynamic RNA-binding proteins uncovers a Cpeb4-controlled regulatory cascade during pathological cell growth of cardiomyocytes.

RNA-binding proteins (RBPs) control critical aspects of cardiomyocyte function, but the repertoire of active RBPs in cardiomyocytes during the growth response is largely unknown. We define RBPs in healthy and diseased cardiomyocytes at a system-wide level by RNA interactome capture. This identifies 67 cardiomyocyte-specific RBPs, including several contractile proteins. Furthermore, we identify the cytoplasmic polyadenylation element-binding protein 4 (Cpeb4) as a dynamic RBP, regulating cardiac growth both in vitro and in vivo. We identify mRNAs bound to and regulated by Cpeb4 in cardiomyocytes. Cpeb4 regulates cardiac remodeling by differential expression of transcription factors. Among Cpeb4 target mRNAs, two zinc finger transcription factors (Zeb1 and Zbtb20) are discovered. We show that Cpeb4 regulates the expression of these mRNAs and that Cpeb4 depletion increases their expression. Thus, Cpeb4 emerges as a critical regulator of cardiomyocyte function by differential binding to specific mRNAs in response to pathological growth stimulation.

m6A-mRNA methylation regulates cardiac gene expression and cellular growth.

Conceptually similar to modifications of DNA, mRNAs undergo chemical modifications, which can affect their activity, localization, and stability. The most prevalent internal modification in mRNA is the methylation of adenosine at the N6-position (m6A). This returns mRNA to a role as a central hub of information within the cell, serving as an information carrier, modifier, and attenuator for many biological processes. Still, the precise role of internal mRNA modifications such as m6A in human and murine-dilated cardiac tissue remains unknown. Transcriptome-wide mapping of m6A in mRNA allowed us to catalog m6A targets in human and murine hearts. Increased m6A methylation was found in human cardiomyopathy. Knockdown and overexpression of the m6A writer enzyme Mettl3 affected cell size and cellular remodeling both in vitro and in vivo. Our data suggest that mRNA methylation is highly dynamic in cardiomyocytes undergoing stress and that changes in the mRNA methylome regulate translational efficiency by affecting transcript stability. Once elucidated, manipulations of methylation of specific m6A sites could be a powerful approach to prevent worsening of cardiac function.