High resolution respirometry of isolated mitochondria from adult Octopus maya (Class: Cephalopoda) systemic heart

Abstract

Mitochondrial respirometry is key to understand how environmental factors model energetic cellular process. In the case of ectotherms, thermal tolerance has been hypothesized to be intimately linked with mitochondria capability to produce enough adenosine triphosphate (ATP) to respond to the energetic demands of animals in high temperatures. In a recent study made in Octopus maya was proposed the hypothesis postulating that high temperatures could restrain female reproduction due to the limited capacity of the animals' heart to sustain oxygen flow to the body, affecting in this manner energy production in the rest of the organs, including the ovarium Meza-Buendia AK et al. (2021). Unfortunately, until now, no reports have shown temperature effects and other environmental variables on cephalopod mitochondria activity because of the lack of a method to evaluate mitochondrial respiratory parameters in those species' groups. In this sense and for the first time, this study developed a method to obtain mitochondrial respirometry data of adult Octopus maya's heart. This protocol illustrates a step-by-step procedure to get high yield and functional mitochondria of cephalopod heart and procedure for determining the corresponding respiratory parameters. The procedure described in this paper takes approximately 3 to 4 hours from isolation of intact mitochondria to measurement of mitochondrial oxygen consumption.

Fig 1. Respiratory control parameter (RC) of isolated mitochondria from the systemic heart of Octopus maya.

The RC of systemic heart mitochondria was obtained from the ratio of respiratory states 3 (S3) and 4o (S4o) once corrected for the respiratory state ROX (S3 = S3’-ROX and S4o = S4’o-ROX). The RC is calculated individually (black dots) for each adult O. maya specimens (N = 4, mean weight 1 348 ± 263.49 g) using a mitochondrial concentration of 377± 0.02 μg ml ^-1. GROUP; represents the mean RC value of the four specimens (red dot), ±SD (gray box).

Fig 2. Respiratory parameter of state 3 (S3) of isolated mitochondria from the systemic heart of Octopus maya.

The S3 of the systemic heart mitochondria was obtained from the correction of the respiratory state 3’ (S3’) and ROX (S3 = S3’-ROX). To obtain S3’, adenosine diphosphate (ADP) was added at a final concentration of 1.25 mM. The S3 was obtained individually (black dots) for each adult O. maya specimens (N = 4, mean weight 1 348 ± 263.49 g) using a mitochondrial concentration of 377 ± 0.02 μg ml ^-1. GROUP; represents the mean S3 value of the four specimens (red dot), ±SD (gray box).

Fig 3. Phosphorylation state parameter (P) of isolated mitochondria from the systemic heart of Octopus maya.

P of systemic heart mitochondria was obtained by subtracting the respiratory parameter S3 from the respiratory parameter S4o (P = S3-S4o; S3 and S4 are ROX-corrected values: S3 = S3’-ROX y S4o = S4’o-ROX, respectively). The P was obtained individually (black dots) for each adult O. maya specimens (mean weight 1 348 ± 263.49 g) using a mitochondrial concentration of 377 ± 0.02 μg ml -1. GROUP; represents the mean P value of the four specimens (red dot), +SD (gray box).

Fig 4. Respiratory parameter of state 4 (S4o) of isolated mitochondria from the systemic heart of Octopus maya.

S4o in O. maya systemic heart mitochondria were obtained from the correction of respiratory state 4’ oligomycin-induced (S4’o) y ROX: S4o = S4’o-ROX. The S4o was obtained individually (black dots) for each adult O. maya specimens (mean weight 1 348 ± 263.49 g) using a mitochondrial concentration of 377 ± 0.02 μg ml ^-1. GROUP; represents the mean S4o value of the four specimens (red dot), +SD (gray box).