Michael Oellermann

Mitochondrial dynamics underlying thermal plasticity of cuttlefish (Sepia officinalis) hearts

SUMMARY In the eurythermal cuttlefish Sepia officinalis, performance depends on hearts that ensure systemic oxygen supply over a broad range of temperatures. We therefore aimed to identify adjustments in energetic cardiac capacity and underlying mitochondrial function supporting thermal acclimation and adaptation that could be crucial for the cuttlefishs competitive success in variable environments. Two genetically distinct cuttlefish populations were acclimated to 11, 16 and 21°C. Subsequently, skinned and permeabilised heart fibres were used to assess mitochondrial functioning by means of high-resolution respirometry and a substrate–inhibitor protocol, followed by measurements of cardiac citrate synthase and cytosolic enzyme activities. Temperate English Channel cuttlefish had lower mitochondrial capacities but larger hearts than subtropical Adriatic cuttlefish. Warm acclimation to 21°C decreased mitochondrial complex I activity in Adriatic cuttlefish and increased complex IV activity in English Channel cuttlefish. However, compensation of mitochondrial capacities did not occur during cold acclimation to 11°C. In systemic hearts, the thermal sensitivity of mitochondrial substrate oxidation was high for proline and pyruvate but low for succinate. Oxygen efficiency of catabolism rose as temperature changed from 11 to 21°C via shifts to oxygen-conserving oxidation of proline and pyruvate and via reduced relative proton leak. The changes observed for substrate oxidation, mitochondrial complexes, relative proton leak and heart mass improve energetic efficiency and essentially seem to extend tolerance to high temperatures and reduce associated tissue hypoxia. We conclude that cuttlefish sustain cardiac performance and, thus, systemic oxygen delivery over short- and long-term changes of temperature and environmental conditions by multiple adjustments in cellular and mitochondrial energetics.

Transmural differences in respiratory capacity across the rat left ventricle in health, aging, and streptozotocin-induced diabetes mellitus: evidence that mitochondrial dysfunction begins in the subepicardium.

In diabetic cardiomyopathy, ventricular dysfunction occurs in the absence of hypertension or atherosclerosis and is accompanied by altered myocardial substrate utilization and depressed mitochondrial respiration. It is not known if mitochondrial function differs across the left ventricular (LV) wall in diabetes. In the healthy heart, the inner subendocardial region demonstrates higher rates of blood flow, oxygen consumption, and ATP turnover compared with the outer subepicardial region, but published transmural respirometric measurements have not demonstrated differences. We aim to measure mitochondrial function in Wistar rat LV to determine the effects of age, streptozotocin-diabetes, and LV layer. High-resolution respirometry measured indexes of respiration in saponin-skinned fibers dissected from the LV subendocardium and subepicardium of 3-mo-old rats after 1 mo of streptozotocin-induced diabetes and 4-mo-old rats following 2 mo of diabetes. Heart rate and heartbeat duration were measured under isoflurane-anesthesia using a fetal-Doppler, and transmission electron microscopy was employed to observe ultrastructural differences. Heart rate decreased with age and diabetes, whereas heartbeat duration increased with diabetes. While there were no transmural respirational differences in young healthy rat hearts, both myocardial layers showed a respiratory depression with age (30-40%). In 1-mo diabetic rat hearts only subepicardial respiration was depressed, whereas after 2 mo diabetes, respiration in subendocardial and subepicardial layers was depressed and showed elevated leak (state 2) respiration. These data provide evidence that mitochondrial dysfunction is first detectable in the subepicardium of diabetic rat LV, whereas there are measureable changes in LV mitochondria after only 4 mo of aging.

Simultaneous high-resolution pH and spectrophotometric recordings of oxygen binding in blood microvolumes

Oxygen equilibrium curves have been widely used to understand oxygen transport in numerous organisms. A major challenge has been to monitor oxygen binding characteristics and concomitant pH changes as they occur in vivo, in limited sample volumes. Here we report a technique allowing highly resolved and simultaneous monitoring of pH and blood pigment saturation in minute blood volumes. We equipped a gas diffusion chamber with a broad-range fibre-optic spectrophotometer and a micro-pH optode and recorded changes of pigment oxygenation along oxygen partial pressure (PO2) and pH gradients to test the setup. Oxygen binding parameters derived from measurements in only 15 μl of haemolymph from the cephalopod Octopus vulgaris showed low instrumental error (0.93%) and good agreement with published data. Broad-range spectra, each resolving 2048 data points, provided detailed insight into the complex absorbance characteristics of diverse blood types. After consideration of photobleaching and intrinsic fluorescence, pH optodes yielded accurate recordings and resolved a sigmoidal shift of 0.03 pH units in response to changing PO2 from 0 to 21 kPa. Highly resolved continuous recordings along pH gradients conformed to stepwise measurements at low rates of pH changes. In this study we showed that a diffusion chamber upgraded with a broad-range spectrophotometer and an optical pH sensor accurately characterizes oxygen binding with minimal sample consumption and manipulation. We conclude that the modified diffusion chamber is highly suitable for experimental biologists who demand high flexibility, detailed insight into oxygen binding as well as experimental and biological accuracy combined in a single setup.

A new haemocyanin in cuttlefish (Sepia officinalis) eggs: sequence analysis and relevance during ontogeny

BackgroundHaemocyanin is the respiratory protein of most of the Mollusca. In cephalopods and gastropods at least two distinct isoforms are differentially expressed. However, their physiological purpose is unknown. For the common cuttlefish Sepia officinalis, three isoforms are known so far, whereas for only two of them the complete mRNA sequences are available. In this study, we sequenced the complete mRNA of the third haemocyanin isoform and measured the relative expression of all three isoforms during embryogenesis to reveal a potential ontogenetic relevance.ResultsThe cDNA of isoform 3 clearly correlates to the known Sepia officinalis haemocyanin subunits consisting of eight functional units and an internal duplicated functional unit d. Our molecular phylogenetic analyses reveal the third isoform representing a potentially ancestral haemocyanin isoform, and the analyses of the expression of haemocyanin type 3 reveal that haemocyanin type 3 only can be observed within eggs and during early development. Isoforms 1 and 2 are absent at these stages. After hatching, isoform 3 is downregulated, and isoform 1 and 2 are upregulated.ConclusionsOur study clearly shows an embryonic relevance of the third isoform, which will be further discussed in the light of the changes in the physiological function of haemocyanin during ontogeny. Taken together with the fact that it could also be the isoform closest related to the common ancestor of cuttlefish haemocyanin, the phylogeny of cuttlefish haemocyanin may be recapitulated during its ontogeny.

Complex patterns of actin molecular evolution in the red alga Stylonema alsidii (Stylonematophyceae, Rhodophyta)

The Stylonematophyceae is an early diverging red algal class that contains organisms in which sexual reproduction has not been reported. Previous studies have hypothesized a single copy of the actin gene in this class. Amplification of the actin gene in members of the Stylonematophyceae, including several isolates of Stylonema alsidii, reveals a more complex evolutionary history. The data support independent gene duplications in Goniotrichopsis reniformis and Stylonema alsidii. Three of the seven isolates of S. alsidii had three copies of actin. Analyses indicate that all copies are functional and not pseudogenes. Due to our lack of knowledge of the ploidy level in Stylonema and the asexual nature of these organisms, an ancient change in ploidy level, producing diploid or even triploid organisms, cannot be ruled out to explain two copies of actin within these organisms. Within Stylonema alsidii two of the actin copies (Groups 1 and 2) show similar levels of variation; p‐distances and the number of synonymous and non‐synonymous sites are roughly the same. Yet the changes are distributed differently. One group showed little shared variation among the samples, mutations therefore represent autoapomorphies, while variation in the other group is usually shared among samples (parsimony‐informative). Variation in the other group is usually shared among samples (parsimony‐informative). A third group of actin genes also observed in S. alsidii are highly divergent from the other two copies, yet they maintain all of the signatures of functional proteins. Our data reveal a complex evolutionary history of the actin gene in these species and highlight the lack of knowledge into the basic biology (ploidy level, life cycle characteristics) of this class.

Blue blood on ice: modulated blood oxygen transport facilitates cold compensation and eurythermy in an Antarctic octopod

IntroductionThe Antarctic Ocean hosts a rich and diverse fauna despite inhospitable temperatures close to freezing, which require specialist adaptations to sustain animal activity and various underlying body functions. While oxygen transport has been suggested to be key in setting thermal tolerance in warmer climates, this constraint is relaxed in Antarctic fishes and crustaceans, due to high levels of dissolved oxygen. Less is known about how other Antarctic ectotherms cope with temperatures near zero, particularly the more active invertebrates like the abundant octopods. A continued reliance on the highly specialised blood oxygen transport system of cephalopods may concur with functional constraints at cold temperatures. We therefore analysed the octopod’s central oxygen transport component, the blue blood pigment haemocyanin, to unravel strategies that sustain oxygen supply at cold temperatures.ResultsTo identify adaptive compensation of blood oxygen transport in octopods from different climatic regions, we compared haemocyanin oxygen binding properties, oxygen carrying capacities as well as haemolymph protein and ion composition between the Antarctic octopod Pareledone charcoti, the South-east Australian Octopus pallidus and the Mediterranean Eledone moschata. In the Antarctic Pareledone charcoti at 0°C, oxygen unloading by haemocyanin was poor but supported by high levels of dissolved oxygen. However, lower oxygen affinity and higher oxygen carrying capacity compared to warm water octopods, still enabled significant contribution of haemocyanin to oxygen transport at 0°C. At warmer temperatures, haemocyanin of Pareledone charcoti releases most of the bound oxygen, supporting oxygen supply at 10°C. In warm water octopods, increasing oxygen affinities reduce the ability to release oxygen from haemocyanin at colder temperatures. Though, unlike Eledone moschata, Octopus pallidus attenuated this increase below 15°C.ConclusionsAdjustments of haemocyanin physiological function and haemocyanin concentrations but also high dissolved oxygen concentrations support oxygen supply in the Antarctic octopus Pareledone charcoti at near freezing temperatures. Increased oxygen supply by haemocyanin at warmer temperatures supports extended warm tolerance and thus eurythermy of Pareledone charcoti. Limited haemocyanin function towards colder temperatures in Antarctic and warm water octopods highlights the general role of haemocyanin oxygen transport in constraining cold tolerance in octopods.

Positive selection in octopus haemocyanin indicates functional links to temperature adaptation

BackgroundOctopods have successfully colonised the world’s oceans from the tropics to the poles. Yet, successful persistence in these habitats has required adaptations of their advanced physiological apparatus to compensate impaired oxygen supply. Their oxygen transporter haemocyanin plays a major role in cold tolerance and accordingly has undergone functional modifications to sustain oxygen release at sub-zero temperatures. However, it remains unknown how molecular properties evolved to explain the observed functional adaptations. We thus aimed to assess whether natural selection affected molecular and structural properties of haemocyanin that explains temperature adaptation in octopods.ResultsAnalysis of 239 partial sequences of the haemocyanin functional units (FU) f and g of 28 octopod species of polar, temperate, subtropical and tropical origin revealed natural selection was acting primarily on charge properties of surface residues. Polar octopods contained haemocyanins with higher net surface charge due to decreased glutamic acid content and higher numbers of basic amino acids. Within the analysed partial sequences, positive selection was present at site 2545, positioned between the active copper binding centre and the FU g surface. At this site, methionine was the dominant amino acid in polar octopods and leucine was dominant in tropical octopods. Sites directly involved in oxygen binding or quaternary interactions were highly conserved within the analysed sequence.ConclusionsThis study has provided the first insight into molecular and structural mechanisms that have enabled octopods to sustain oxygen supply from polar to tropical conditions. Our findings imply modulation of oxygen binding via charge-charge interaction at the protein surface, which stabilize quaternary interactions among functional units to reduce detrimental effects of high pH on venous oxygen release. Of the observed partial haemocyanin sequence, residue 2545 formed a close link between the FU g surface and the active centre, suggesting a role as allosteric binding site. The prevalence of methionine at this site in polar octopods, implies regulation of oxygen affinity via increased sensitivity to allosteric metal binding. High sequence conservation of sites directly involved in oxygen binding indicates that functional modifications of octopod haemocyanin rather occur via more subtle mechanisms, as observed in this study.

Transmural differences in respiratory capacity across the rat left ventricle in aging and diabetes: mitochondrial dysfunction begins in the subepicardium

In diabetic cardiomyopathy, ventricular dysfunction occurs in the absence of hypertension or atherosclerosis and is accompanied by altered myocardial substrate utilization and depressed mitochondrial respiration. It is not known if mitochondrial function differs across the left ventricular (LV) wall in diabetes. In the healthy heart, the inner subendocardial region demonstrates higher rates of blood flow, oxygen consumption, and ATP turnover compared with the outer subepicardial region, but published transmural respirometric measurements have not demonstrated differences. We aim to measure mitochondrial function in Wistar rat LV to determine the effects of age, streptozotocin-diabetes, and LV layer. High-resolution respirometry measured indexes of respiration in saponin-skinned fibers dissected from the LV subendocardium and subepicardium of 3-mo-old rats after 1 mo of streptozotocin-induced diabetes and 4-mo-old rats following 2 mo of diabetes. Heart rate and heartbeat duration were measured under isoflurane-anesthesia using a fetal-Doppler, and transmission electron microscopy was employed to observe ultrastructural differences. Heart rate decreased with age and diabetes, whereas heartbeat duration increased with diabetes. While there were no transmural respirational differences in young healthy rat hearts, both myocardial layers showed a respiratory depression with age (30-40%). In 1-mo diabetic rat hearts only subepicardial respiration was depressed, whereas after 2 mo diabetes, respiration in subendocardial and subepicardial layers was depressed and showed elevated leak (state 2) respiration. These data provide evidence that mitochondrial dysfunction is first detectable in the subepicardium of diabetic rat LV, whereas there are measureable changes in LV mitochondria after only 4 mo of aging.

Blue Blood on Ice : Cephalopod haemocyanin function and evolution in a latitudinal cline

The Southern Ocean hosts a rich and diverse fauna that required specialist adaptations to colonize and persist at temperatures close to freezing. While much has been revealed about key adaptations in Antarctic fishes little is known about evolutionary strategies of other Antarctic ectotherms, particularly the abundant benthic incirrate octopods. Their oxygen demand is largely fuelled by the blue oxygen transporter haemocyanin that however, due to its increasing functional failure towards colder temperatures, poses a prime target for cold-adaptive adjustment. While haemocyanin structure has been well understood it remains unclear which molecular features and evolutionary trajectories explain functional properties of octopod haemocyanin. This thesis thus aimed to unravel cold-adaptive features of octopod haemocyanin and the underlying molecular features that evolved to sustain oxygen supply at sub-zero temperatures. Haemocyanin function is best assessed by oxygen binding experiments, which however was challenged due to the minute haemolymph volumes non-model organisms like Antarctic octopods, yield. I thus upgraded an oxygen diffusion chamber with a broad range fibre optic spectrophotometer and a micro-pH optode and tested the setup for Octopus vulgaris, the Antarctic eelpout and a Baikal amphipod. This technical advancement enabled simultaneous recordings of pH and oxygen dependent pigment absorbance in only 15 µl of sample. Results were highly reproducible and accurate and provided detailed insight to the complex and dynamic spectral features of three diverse blood-types. To identify cold-adaptive functional traits of blood oxygen transport this study compared haemocyanin oxygen binding properties, oxygen carrying capacities and haemolymph protein and ion composition between the Antarctic octopod Pareledone charcoti, the temperate Octopus pallidus and the subtropical Eledone moschata. Compared to octopods from warmer climates, Pareledone charcoti showed incomplete but significantly reduced oxygen affinity, which together with increased haemocyanin concentrations and high physically dissolved oxygen levels supported oxygen supply at 0°C. Therefore, unlike many Antarctic fishes Pareledone charcoti continued to rely on an oxygen transporting pigment. High temperature sensitivity of oxygen binding enabled Pareledone charcoti to utilise most of the oxygen bound by haemocyanin at 10°C. The concomitant relief for the circulatory system at warmer temperatures promotes warm tolerance and thus eurythermy in Pareledone charcoti. Underlying molecular mechanisms were studied by comparing 239 partial haemocyanin sequences of the functional unit f and g of 28 octopods species of polar, temperate, subtropical and tropical origin. Despite high conservation of these haemocyanin regions several sites were positively selected for their charge properties at the molecule’s surface. Net surface charges were generally elevated in polar octopods suggesting that charge-charge interactions raise intrinsic pK values to stabilise quaternary structure against higher ambient pH present in cold waters. The presence of at least two haemocyanin isoforms and high allelic variation in polar octopods indicate sustained genetic diversity of haemocyanin and thus the genetic potential to regulate blood oxygen transport in the cold. Further, amino acid variability located within a potential metal binding site suggests regulation of blood oxygen transport in octopods via altered intrinsic sensitivity to allosteric ligands In conclusion, this study revealed significant adaptations of octopod haemocyanin at the functional and molecular level that support oxygen supply at near freezing temperatures. However, ‘imperfect’ functional adaptation and ensuing reliance on high haemocyanin levels in Pareledone charcoti seems to add to the various design constraints of octopods compared to fishes. Hence, at second glance functional oxygen reserves indicate a higher capacity to sustain oxygen transport at warmer temperatures and together with a potential ability to regulate and reverse cold-adaptive molecular traits may be key to determine future winners and losers in an ecosystem facing radical environmental change.

Erratum to: Positive selection in octopus haemocyanin indicates functional links to temperature adaptation

1 Erratum The original version of this article [1] unfortunately contained a mistake. The presentation of Table 1 along with the table legend and footnote was incorrect in the HTML and PDF versions of this article. The corrected version is given below. permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver