Localized translation and sarcomere maintenance requires ribosomal protein SA in mice
Rami Haddad, Omer Sadeh, Tamar Ziv, Itai Erlich, Lilac Haimovich-Caspi, Ariel Shemesh, Jolanda van der Velden, Izhak Kehat
Rami Haddad, Omer Sadeh, Tamar Ziv, Itai Erlich, Lilac Haimovich-Caspi, Ariel Shemesh, Jolanda van der Velden, Izhak Kehat
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Research Article Cardiology

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 5

Cardiomyocyte cell autonomous loss of Rpsa results in cardiac dysfunction and cardiomyocyte atrophy.

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Cardiomyocyte cell autonomous loss of Rpsa results in cardiac dysfunctio...
(A–F) Echocardiographic assessment of mice with Lox-stop-Lox-CAS9 transduced with AAVs encoding for sgRNAs targeting Rpsa or control sgRNAs and troponin promoter–driven HA-CRE expression to knockout Rpsa specifically in cardiomyocytes. Analysis shows Rpsa knockout results in cardiac dilation with increased LVIDd, decreased IVSd and LVPWd, and decreased contractile function with increased LVIDs and decreased FS percentage. HR was unchanged. n = 8, 12, 13, 14, 15, 19, and 22 mice per experimental group; mean of 3 measurements per mouse. (G–P) Gene expression analysis by RT-qPCR shows an increase in markers of cardiac stress and hypertrophy (Nppa, Nppb, Myh7, Myh7/Myh6 ratio) as well as in markers of fibrosis Fn1 and Col1a1 in Rpsa-knockout hearts. Tnni1 and Tnni3 gene expression had no statistically significant alteration. Expression data are normalized to Gapdh. n = 2, 3, and 4 mice per age and experimental group; 2–3 technical replicates per mouse. (Q–T) Representative images and quantitative analysis in Rpsa-knockout hearts showing decreased cardiomyocyte width, length, and area in targeted HA-CRE–positive cardiomyocytes, indicating atrophy, and an increase in nontargeted HA-CRE–negative cardiomyocytes, indicating hypertrophy. Sections were stained with WGA (light green), HA-tag antibody (red) to identify nuclear HA-CRE, and DAPI (blue) for nuclei. n = 51, 53, 54, 57, 58, and 60 cells from 2–3 mice per group. Scale bar: 20 μm. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, by Student’s t test (A–P) or 1-way ANOVA test (R–T). Data are presented as individual values, with box plots displaying the median with 25th and 75th percentiles.