Regulation of Skeletal Muscle Sarcomere Length through Titin Changes in iMSBmal1 Mice

Abstract

Prior research shows that sarcomere length correlates with isometric force production, but the specific structural elements regulating resting sarcomere length are not well understood. Based on the assumption that sarcomeres operate similarly throughout a muscle fiber, heterogeneity in sarcomere lengths should decrease maximum active tension production. One structural protein that has been proposed to maintain sarcomere length is titin. Our lab developed an inducible skeletal muscle specific Bmal1 knockout mouse, which exhibits muscle weakness and a shift to longer titin isoform. My project takes a histological approach to study phenotypic changes in sarcomere length and A‐band centrality in the iMSBmal1−/− skeletal muscle. I hypothesized that the iMSBmal1−/− mice would exhibit an increased variance in sarcomere length, as well as a change in the A‐band centrality index (i.e. the distance of peak fluorescence of the myosin filament from the center of the sarcomere). After collecting tibilalis anterior (TA) muscle from these mice at 5 weeks after recombination, the muscles were frozen in OCT compound using liquid nitrogen cooled isopentane. These samples (n=3 animals/group) were then longitudinally cryosectioned at 6 microns. The samples were stained using primary antibodies that target α‐actinin and myosin heavy chain followed by fluorescent secondary antibodies. α‐actinin labels the Z‐lines, allowing us to measure sarcomere lengths in the longitudinal sections, while myosin heavy chain allows us to visualize the A‐band. I measured the peak fluorescence of both fluorescent secondary antibodies and measured where the myosin heavy chain peak was in relation to the Z‐line on either side to determine an A‐band centering index. The data was compiled by sample (~450 sarcomeres measured per sample) and then averaged both by sample and then by comparing iMSBmal1+/+ to iMSBmal1−/−. Results of iMSBmal1+/+ to iMSBmal1−/− showed a trend toward a longer sarcomere length from 2.64 μm to 2.82 μm (not significant, p=0.1), but the variance did not change. Since the variance of sarcomere lengths did not change, my hypothesis that altered titin expression correlates to more variable sarcomere length was not supported; however, longer titin could lead to the observed increased sarcomere length and my sample number is currently small (n=3/group). The mean and variance of the A‐band centrality index between these groups were not found to be statistically significant (p = 0.7769 and 0.3672, respectively). The lack of change in the A‐band centrality index does not support my hypothesis that a shift would occur. However, there were some technical issues with the antibody that I am repeating and further studying. Further investigations will include increasing my sample size with a focus on sarcomere length. I will also be using site specific titin antibodies to look at the inclusion of longer titin isoforms through the sarcomeres in muscle. Understanding the relationship among titin isoforms, sarcomere length and structure and muscle force will be important for understanding causes of muscle weakness.Support or Funding InformationThis work was made possible by the University of Florida and the funding of the American Physiological Society Undergraduate Summer Research Fellowship Program. Special thanks to Lance A. Riley and Karyn A. Esser for their support and guidance through this process. Thanks also to David Hammers and Cora Coker for their help with microscopy.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.