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Muscular dystrophies (MDs) are caused by dysregulation of over 40 proteins but commonly share features of muscle weakness, myofiber death and regeneration, loss of ambulation and premature death. A MD involves a broken link anywhere in the connection from extracellular matrix through the sarcolemma to the sarcomere. Thus, any protein which is a part of this link causes MD if misfolded, dysregulated or absent. In MD, the most common causes of death are cardiac or respiratory failure, when the muscles involved in these processes fail. Although MDs affect 1:3500-5000 births worldwide there are currently no cures available. Extraocular muscles (EOMs) are strikingly not affected by MDs, however, the mechanisms behind this native resistance remain elusive. We have recently shown that the EOMs cytoskeleton differs significantly from that of other muscles and hypothesized that investigation of their cytoskeleton in MD models would provide important clues. Furthermore, we hypothesized that application of the EOMs strategies to trunk muscle tissue would decrease the detrimental impact of MD overall.

The zebrafish model system has recently increased vastly in popularity, and has quickly become a MD model. Due to its compatibility with the CRISPR/Cas9 method, genetic knockout studies can be utilized to generate novel mutant lines tailored to fit various aspects in studies of the zebrafish skeletal muscle. In this thesis I present nine new zebrafish lines which I used to study muscle biology processes, including muscle regeneration and the EOM cytoskeleton. 

Our results clearly demonstrate the need for understanding compensatory mechanisms in biology. Interestingly, pax3 and pax7 were shown to functionally compensate for each other both in appendicular muscle formation and in muscle regeneration, respectively, two processes where these individual genes have great impact in other organisms. This finding would also prove to be important in aiding our understanding of the EOM biology in adaptive strategies towards MDs. Furthermore, our results show that zebrafish EOMs are a good model to study cytoskeletal composition, as they share important features with human EOMs. Utilizing the CRISPR/Cas9 genome editing technique, I developed several knockout models of cytoskeletal proteins (desmin, obscurin, plectin) and studied their importance for the function of the EOMs.  In studies of zebrafish EOMs lacking obscurin, we found that EOMs functionally adapt their myosin composition over time via upregulation of myh7, a cardiac specific myosin. Furthermore, an RNA-sequencing screen on a CRISPR/Cas9 induced desminopathy model (desma; desmb double mutant) identified several protective genes of interest. We show that a four and a half LIM-domain protein (Fhl2) is upregulated in EOMs in several muscular dystrophy models and that fhl2b protects EOMs from excessive myonuclei turnover and hypertrophy. Furthermore, its ectopic expression in trunk muscle can also protect an additional muscle dystrophy model (dmd) from acute early death, improve myofiber function and stabilize neuromuscular junctions. Importantly, this protein was also detected in both human and mouse EOMs, indicating a potentially conserved role in the EOMs across species.

In summary, we identified several novel strategies of adaptation to disease progression in the EOMs. Together, these findings have contributed significantly to a better understanding of the EOMs and suggest new treatment strategies for MD that may have important future clinical applications.