Localized translation and sarcomere maintenance requires ribosomal protein SA in mice
Rami Haddad, … , Jolanda van der Velden, Izhak Kehat
Rami Haddad, … , Jolanda van der Velden, Izhak Kehat
Published May 14, 2024
Citation Information: J Clin Invest. 2024;134(13):e174527. https://doi.org/10.1172/JCI174527.
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Localized translation and sarcomere maintenance requires ribosomal protein SA in mice

Abstract

Cardiomyocyte sarcomeres contain localized ribosomes, but the factors responsible for their localization and the significance of localized translation are unknown. Using proximity labeling, we identified ribosomal protein SA (RPSA) as a Z-line protein. In cultured cardiomyocytes, the loss of RPSA led to impaired local protein translation and reduced sarcomere integrity. By employing CAS9-expressing mice, along with adeno-associated viruses expressing CRE recombinase and single-guide RNAs targeting Rpsa, we knocked out Rpsa in vivo and observed mislocalization of ribosomes and diminished local translation. These genetic mosaic mice with Rpsa knockout in a subset of cardiomyocytes developed dilated cardiomyopathy, featuring atrophy of RPSA-deficient cardiomyocytes, compensatory hypertrophy of unaffected cardiomyocytes, left ventricular dilation, and impaired contractile function. We demonstrated that RPSA C-terminal domain is sufficient for localization to the Z-lines and that if the microtubule network is disrupted RPSA loses its sarcomeric localization. These findings highlight RPSA as a ribosomal factor essential for ribosome localization to the Z-line, facilitating local translation and sarcomere maintenance.

Authors

Rami Haddad, Omer Sadeh, Tamar Ziv, Itai Erlich, Lilac Haimovich-Caspi, Ariel Shemesh, Jolanda van der Velden, Izhak Kehat

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Figure 6

Rpsa knockout disrupts local sarcomeric protein translation and sarcomere integrity in vivo.

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Rpsa knockout disrupts local sarcomeric protein translation and sarcome...
(A–D) OPP-injected mosaic Rpsa-knockout and control mice were stained for OPP (white) and HA-CRE (red) to label nascent proteins and targeted cardiomyocytes, respectively. Representative images show OPP localized sarcomeric translation pattern in control mice (A). In Rpsa-knockout mice, the localized sarcomeric translation is lost only in targeted cardiomyocytes (B). Yellow arrowheads mark targeted nuclei. DAPI (blue). Scale bar: 10 μm. (C) Line-scan quantification in targeted cardiomyocytes confirms the loss of localized sarcomeric translation in Rpsa-knockout cardiomyocytes. n = 2 mice per group; n = 57 and 88 cardiomyocytes. (D) The translational activity in Rpsa-knockout cardiomyocytes, assessed by OPP cytoplasmic integrated density, is reduced yet higher than the negative control samples (NC.1- no OPP or NC.2- no Click labeling). n = 2 mice per group; n = 63, 64, 70, and 75 cardiomyocytes. (E–J) Representative images and analysis showing disrupted ACTN2 and MYH staining in Rpsa-knockout cardiomyocytes, quantified by the increased percentage of the sarcomere-free area. n = 2 mice for control sgRNA, n = 3 mice for the Rpsa-knockout group, n = 56 and 64 (I), n = 39 and 42 (J) cardiomyocytes. (K and L) Western blot and densitometry analysis after Rpsa siRNA–mediated knockdown in NRVMs showing significantly reduced sarcomeric protein levels (MYBPC3, ACTN2, TNNI3) and α-tubulin (TUBA) but not in the nonsarcomeric proteins (GAPDH, VDAC1, SDHA). n = 4 biological samples from 3 independent experiments. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001, by 1-way ANOVA test (D) or Student’s t test (C, I, J, and L). Data are presented as individual values, with box plots displaying the median with 25th and 75th percentiles, and bar graph (mean ± SD).