During the experiments, muscle fibers and muscles were randomly assigned to the control and test (probenecid, carbenoxolone, or other treatment) groups. When comparing results between control muscle fibers and muscle fibers treated under a given pharmacological condition, groups of fibers from the same animal were considered as technical replicates and statistical comparison was achieved using a nested analysis, as described by Eisner (2021) (link). When determining the extent of change in a given parameter produced by the acute application of a pharmacological compound (with the initial measurement from the same muscle or muscle fiber being the control value), each experiment was considered independent, and statistics were based on the number of fibers.
The number of necessary experiments was estimated on the basis of our previous experience with each protocol. A sample size estimation (with α = 0.05 and β = 0.80) was made from our pilot data showing a 55% reduction of maximum SR Ca2+ release in the presence of 1 mM probenecid, giving a total of six per group.
Experiments on single muscle fibers and isolated muscles require a great extent of specific expertise, preparation, dexterity, and watchfulness, with the operator on duty being in charge from beginning to end. Under these conditions, further complexification by blinding was not implemented to prevent any related risk of misidentification error. Blinding would also have required two dedicated persons on duty on each experimental day, which was not routinely possible.
Data and statistical analysis complied with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2018 (link)). Electrophysiological and fluorescence data were processed with Clampfit 10.0 (Molecular Devices) and ImageJ software (National Institutes of Health), respectively, and Origin 7.0 (OriginLab Corporation). Statistical analysis was performed with GraphPad Prism 9.1 (GraphPad Software). Data values are presented as means ± SD for either n muscle fibers or n muscles. Individual datapoints for the parameters measured from each and every muscle/fiber tested are also presented in the figures. Statistical comparison was made only on measurements conducted under a given experimental condition on a minimum group of either five distinct muscles or five distinct muscle fibers. No search was made for outliers within datasets. The level of probability (p) deemed to constitute the threshold for statistical significance was defined as P < 0.05. Exact P values are indicated on the graphs in the figures.
For single muscle fiber experiments designed to test the effect of chronic exposure to a pharmacological compound (Figs. 1, 2, and 4), the normality of the distribution of each Boltzmann fit parameter was assessed using Shapiro-Wilk test, and the statistical difference between the control group and either the probenecid group or the carbenoxolone group was determined using the hierarchical (nested) analysis or linear mixed modeling described by Eisner (2021) (link), taking into account the number of fibers from each mouse.
For single muscle fiber experiments designed to test the effect of acute exposure to probenecid, statistical differences between the control group and the probenecid group in terms of the extent of change in (1) baseline rhod-2 fluorescence, (2) resting membrane conductance, and (3) peak SR Ca2+ release flux during the test period and after wash-out, was assessed using unpaired Mann–Whitney Wilcoxon test (Figs. 6 and 9).
For muscle force measurements in control conditions and in the presence of probenecid, a Mann–Whitney Wilcoxon test was used to compare the amplitude of tetanic force at a given time point of the experiment. Friedman’s nonparametric test for repeated measurements followed by Dunn’s post-hoc test was used to compare the time-dependent changes in the tetanic force amplitude in the two groups.