Respiration of permeabilized cardiomyocytes from mice: no sex differences, but substrate-dependent changes in the apparent ADP-affinity

Abstract

Sex differences in cardiac physiology are getting increased attention. This study assessed whether isolated, permeabilized cardiomyocytes from male and female C57BL/6 mice differ in terms of their respiration with multiple substrates and overall intracellular diffusion restriction estimated by the apparent ADP-affinity of respiration. Using respirometry, we recorded 1) the activities of respiratory complexes I, II and IV, 2) the respiration rate with substrates fuelling either complex I, II, or I + II, and 3) the apparent ADP-affinity with substrates fuelling complex I and I + II. The respiration rates were normalized to protein content and citrate synthase (CS) activity. We found no sex differences in CS activity (a marker of mitochondrial content) normalized to protein content or in any of the respiration measurements. This suggests that cardiomyocytes from male and female mice do not differ in terms of mitochondrial respiratory capacity and apparent ADP-affinity. Pyruvate modestly lowered the respiration rate, when added to succinate, glutamate and malate. This may be explained by intramitochondrial compartmentalization caused by the formation of supercomplexes and their association with specific dehydrogenases. To our knowledge, we show for the first time that the apparent ADP-affinity was substrate-dependent. This suggests that substrates may change or regulate intracellular barriers in cardiomyocytes.

Figure 1

Assessment of the activities of the respiratory complexes I, II and IV in permeabilized cardiomyocytes from male and female mice. A representative trace of the oxygen consumption recorded during SUIT protocol 1 is shown in a, where the oxygen concentration in the chamber is shown in blue, and the oxygen consumption rate, i.e. the slope of the blue line, is shown in red. Panels (b,c) show the averaged data (9 males and 10 females) normalized to the protein content and CS activity, respectively. The individual datapoints are shown with pale dots, and the data are summarized with the box-and-whisker plots, where the black, solid line shows the mean, the box borders show the interquartile range from 25% to 75%, and the whiskers correspond to the Tukey box plot notation.

Figure 2

Assessment of respiration rate with multiple substrates activating both complexes I and II in permeabilized cardiomyocytes from male and female mice. Representative traces of oxygen consumption during SUIT protocol 2 and 3 are shown in (a,b), respectively. The blue line shows the oxygen concentration in the chamber, and the red line is the oxygen consumption rate, i.e. the slope of the blue line. Panels c–f show the averaged data from 9 males and 10 females. The individual datapoints are shown with pale dots, and the data are summarized with the box-and-whisker plots, where the black, solid line shows the mean, the box borders show the interquartile range from 25% to 75%, and the whiskers correspond to the Tukey box plot notation. In (c,d), the data from SUIT 2 and 3, respectively, are normalized to the protein content. In (e,f), the data from SUIT 2 and 3, respectively, are normalized to the CS activity.

Figure 3

Assessment of the ADP-dependency of respiration in permeabilized cardiomyocytes from male and female mice. Representative traces of oxygen consumption during ADP_GM- and ADP_SGMP-titration are shown in (a,b), respectively. The blue line shows the oxygen concentration in the chamber, and the red line is the oxygen consumption rate, i.e. the slope of the blue line. Panels c and d show the averaged data (10 males and 10 females) normalized to the protein content and CS activity, respectively. The data points show mean ± SEM, and the lines show the hyperbolic curve obtained when the average V_max and K_M are inserted into the Michaelis-Menten equation.