Passive Force Analysis of Single Sarcomeres from Muscles Lacking Arginyl-tRNA-Protein Transferase (Ate1)

Abstract

Introduction: Arginylation is a post-translational process mediated by the enzyme arginyl-tRNA transferase (Ate1). Recent findings from our laboratory suggest an important role of arginylation on the architecture and function of skeletal and cardiac muscles. In this study, we investigated the passive force development of single sarcomeres isolated from soleus muscles of mice lacking Ate1.Methods: Soleus muscles from Ate1 KO and wild type (WT) mice were homogenized to myofibrils. Single sarcomeres in relaxing solution were tested using a pair of micro-needles. The sarcomeres were stretched in steps of 300 nm, starting in a sarcomere length of 2.2μm (±0.2μm). The force produced by the sarcomeres was plotted against the sarcomere length. Mass spectrometry was used to evaluate the presence of arginylation sites on titin. Gel electrophoresis was used to test for isoform modifications due to the lack of Ate1.Results: Single sarcomeres of Ate KO developed less passive force than sarcomere from WT. Mass Spectrometry reviewed 4 sites for arginylation within titin's A-band structure. Electrophoresis gels showed no difference in the titin N2A isoform.Conclusion: Post-translation arginylation of titin A-band does not change titin's isoform, but is required for normal passive force development in soleus muscles.