Determining Maximal Muscle Strength in Mice: Validity and Reliability of an Adapted Swimming Incremental Overload Test.

Gouvêa, AL, Martinez, CG, and Kurtenbach, E. Determining maximal muscle strength in mice: validity and reliability of an adapted swimming incremental overload test. J Strength Cond Res 34(8): 2360-2368, 2020-At present, there are no reliable methods to determine maximal muscle strength in small rodents. Here, we established an adapted swimming incremental overload test (SIOT) as an instrument for this purpose. First, to validate the test, BALB/c mice received 20 mg·kg·d of dexamethasone (DEXA group) or water (control group). After 14 days, with a cumulative dose of 120 mg·kg of dexamethasone, the SIOT could detect a decrease of approximately 7% in muscle strength. In addition, this decrease was consistent with a significant reduction in body (above 13.5%) and muscle (approximately 15%) weight in DEXA atrophic animals. To establish the SIOT reliability, another group of animals was evaluated for 5 consecutive days. In this second protocol, the SIOT was executed with an initial load corresponding to 12% of the mouse body weight (BW) fixed to the tail. Increments between 1 and 5% of the BW were added during each attempt to obtain the highest load that was tolerated for a time interval of 5-7 seconds. On the last day, the SIOT reliability test was performed by 2 different raters to obtain the inter-rater reproducibility. The adapted SIOT was shown to be reliable when measured by the same rater (intraclass correlation coefficient [ICC] = 0.939) and by 2 different raters (ICC = 0.830). The Bland-Altman graphical representation did not demonstrate heteroscedastic errors. Therefore, the SIOT proved to be a sensitive and reliable method to measure muscle strength, and it can be applied to small animals in different models of muscle atrophy.

Complex SUMO-1 regulation of cardiac transcription factor Nkx2-5.

Reversible post-translational protein modifications such as SUMOylation add complexity to cardiac transcriptional regulation. The homeodomain transcription factor Nkx2-5/Csx is essential for heart specification and morphogenesis. It has been previously suggested that SUMOylation of lysine 51 (K51) of Nkx2-5 is essential for its DNA binding and transcriptional activation. Here, we confirm that SUMOylation strongly enhances Nkx2-5 transcriptional activity and that residue K51 of Nkx2-5 is a SUMOylation target. However, in a range of cultured cell lines we find that a point mutation of K51 to arginine (K51R) does not affect Nkx2-5 activity or DNA binding, suggesting the existence of additional Nkx2-5 SUMOylated residues. Using biochemical assays, we demonstrate that Nkx2-5 is SUMOylated on at least one additional site, and this is the predominant site in cardiac cells. The second site is either non-canonical or a "shifting" site, as mutation of predicted consensus sites and indeed every individual lysine in the context of the K51R mutation failed to impair Nkx2-5 transcriptional synergism with SUMO, or its nuclear localization and DNA binding. We also observe SUMOylation of Nkx2-5 cofactors, which may be critical to Nkx2-5 regulation. Our data reveal highly complex regulatory mechanisms driven by SUMOylation to modulate Nkx2-5 activity.