Protein synthesis inside the sarcomere

Introduction

Reduced sarcomeric content and sarcomeric protein levels are seen in the failing heart, suggesting a failure in the maintenance of the sarcomere. Yet, the mechanisms of sarcomere maintenance are poorly understood. We aimed to develop a new method to identify the translation sites and turnover of sarcomeric proteins in order to study sarcomere maintenance.

Material and method

We expressed tagged sarcomere proteins, and fluorescently labeling them in a pulse chase experiment with two colors, to differentiate the pre-existing proteins from the proteins synthesized during the chase. We expressed sarcomeric proteins fused to HaloTag in cultured neonatal and adult cardiomyocytes and in vivo using AAV vectors. We pulsed with a red fluorophore ligand that covalently binds the existing Halo tagged sarcomeric proteins, washed, and chased with green fluorophore at variable intervals to label the newly synthesized proteins and image them. In addition, we have implemented the SINAPS system for live imaging of single translation sites. This system is based on mRNA probe with 3`UTR of sarcomeric gene, and accumulation of GFP signal on active translated transcripts.

Results and discussion

Our technique can efficiently differentiate and image old and new sarcomeric proteins in cardiomyocytes, and can be used in vivo. We used it for imaging of the sarocmeric proteins tropomyosin, alpha actinin, mlc2-v, Myomesin and Mybpc3. We show that translation of Tropomyosin occur on both sides of the Z-disc, in agreement with our previous data that suggested that sarcomeric proteins are translated inside the sarcomere. The SINAPS APPROACH showed that the active translation of cardiac alpha actin is localized to the Z-disc in living adult cardiomyocytes.

Conclusion

We developed a novel technique to track and image the translation of sarocmeric proteins in cultured cardiomyocytes and in vivo. We show that sarcomeric proteins are translated in the sarcomere and are continuously turned-over.