TXLNB is a Novel Regulator of Cardiac Proteostasis
Authors: McLendon Jared M. , Zhang Xiaoming , Stein Colleen , Abouassaly Gabrielle , Witmer Nathan , Boudreau Ryan L. 
Published: June 2021

Cardiac hypertrophy is necessary to maintain effective cardiac function during increased ventricular workload. Hypertrophy is hallmarked by robust changes in gene expression, increased protein synthesis, and cell size. Proteostasis – the integrated regulation of protein synthesis, degradation, and quality control mechanisms – is dysregulated during cardiac hypertrophy and failure. The mechanisms of cardiac proteostasis are not fully understood and continued studies are needed to identify and characterize novel regulators of these pathways.

To address this, we performed a bioinformatic analysis of publicly available gene expression data to identify understudied cardiac‐enriched genes, with dynamic expression during cardiac stress, and without well‐defined functions. We tested 45 candidate genes to determine if they regulate proteostasis and cardiomyocyte hypertrophy in neonatal rat cardiomyocytes (NRCMs) stimulated with phenylephrine (PE). The assay we developed uses dual luciferase activities to quantify increases in protein expression and measure cardiomyocyte size in cells co‐transfected with a gene of interest. Several lead candidate genes emerged from this NRCM‐PE assay, including TXLNB, which encodes a coiled‐coil domain protein that increases luciferase protein expression in NRCMs and amplifies the PE induced response. Surprisingly, overexpression of TXLNB blocks the PE induced increase in cell size, suggesting that TXLNB may have a primary role regulating protein homeostasis during cardiac hypertrophy.

We next investigated the role of TXLNB at various mechanistic levels of protein homeostasis. Adenoviral‐TXLNB overexpression in PE‐NRCMs increased de novo protein synthesis (puromycin incorporation into polypeptides, Fig. 1A), decreased protein ubiquitination (pan‐ubiquitin blot, Fig. 1B), increased proteasome degradation (26Sβ5 activity assay, Fig. 1C) and altered autophagic flux (i.e. decreased p62 and increased LC3‐II, Fig. 1D), indicating that TXLNB plays a multifaceted role in protein homeostasis.

To determine the role of TXLNB in vivo, we used CRISPR‐Cas9 to delete TXLNB‐exon 3 in mice, and show that TXLNB knockout (KO) mice have increased cardiac protein ubiquitination (pan‐ubiquitin blot, Fig. 1E), and decreased proteasome degradation (26Sβ5 activity assay, Fig. 1F), consistent with defects in cardiac proteostasis. Surprisingly, TXLNB KO mice have normal cardiac structure and function, as measured by echocardiography. To evaluate if TXLNB is necessary for cardiac stress responses, we subjected mice to mild transverse aortic constriction (a hypertrophy model that does not progress to heart failure); this caused ~20% increase in HW:BW in male and female mice of both genotypes. Interestingly, TXLNB KO females had decreased ejection fractions (p=0.012).

Altogether, these data demonstrate that TXLNB is a novel regulator of cardiac proteostasis, operating via diverse coordination of protein synthesis, ubiquitination, proteasomal degradation, and autophagy. Notably, our bioinformatic analyses indicate that TXLNB mRNA is consistently up‐regulated in failing human hearts across multiple independent patient cohorts, further supporting the need for follow‐up studies to evaluate the effects of TXLNB KO on heart failure in mice and to define precise mechanisms of how TXLNB controls the removal and replacement of cardiac proteins during hypertrophy.

Support or Funding Information

This work was supported by 14SDG18590008 (RLB) and T32HL007121 (JMM).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.