The retinoic acid response is a minor component of the cardiac phenotype in H9c2 myoblast differentiation.

The H9c2 myoblast cell line, isolated from the left ventricular tissue of rat, is currently used in vitro as a mimetic for skeletal and cardiac muscle due to its biochemical, morphological, and electrical/hormonal signaling properties. During culture, H9c2 cells acquire a myotube phenotype, where a critical component is the inclusion of retinoic acid (RA). The results from some authors on H9c2 suggested that thousands of genes respond to RA stimuli, while others report hundreds of genes responding to RA over different cell types. In this article, using a more appropriate experimental design, we first confirm the H9c2 cardiac phenotype with and without RA and report transcriptomic and physiological changes regarding calcium handling, bioenergetics, and other biological concepts. Interestingly, of the 2360 genes showing a transcriptional change, 622 genes were statistically associated with the RA response. Of these genes, only 305 were RA-specific, and the rest also showed a culture-time component. Thus, the major expression changes (from 74 to 87%) were indeed due to culture conditions over time. Unexpectedly, only a few components of the retinol pathway in KEGG responded to RA. Our results show the role of RA in the H9c2 cultures impacting the interpretation using H9c2 as an in vitro model.

In yeast, cardiolipin unsaturation level plays a key role in mitochondrial function and inner membrane integrity.

Cardiolipin is the signature phospholipid of the mitochondrial inner membrane. It participates in shaping the inner membrane as well as in modulating the activity of many membrane-bound proteins. The acyl chain composition of cardiolipin is finely tuned post-biosynthesis depending on the surrounding phospholipids to produce mature or unsaturated cardiolipin. However, experimental evidence showing that immature and mature cardiolipin are functionally equivalents for mitochondria poses doubts on the relevance of cardiolipin remodeling. In this work, we studied the role of cardiolipin acyl chain composition in mitochondrial bioenergetics, including a detailed bioenergetic profile of yeast mitochondria. Cardiolipin acyl chains were modified by genetic and nutritional manipulation. We found that both the bioenergetic efficiency and osmotic stability of mitochondria are dependent on the unsaturation level of cardiolipin acyl chains. It is proposed that cardiolipin remodeling and, consequently, mature cardiolipins play an important role in mitochondrial inner membrane integrity and functionality.

Origin and diversification of the cardiolipin biosynthetic pathway in the Eukarya domain.

Cardiolipin (CL) and its precursor phosphatidylglycerol (PG) are important anionic phospholipids widely distributed throughout all domains of life. They have key roles in several cellular processes by shaping membranes and modulating the activity of the proteins inserted into those membranes. They are synthesized by two main pathways, the so-called eukaryotic pathway, exclusively found in mitochondria, and the prokaryotic pathway, present in most bacteria and archaea. In the prokaryotic pathway, the first and the third reactions are catalyzed by phosphatidylglycerol phosphate synthase (Pgps) belonging to the transferase family and cardiolipin synthase (Cls) belonging to the hydrolase family, while in the eukaryotic pathway, those same reactions are catalyzed by unrelated homonymous enzymes: Pgps of the hydrolase family and Cls of the transferase family. Because of the enzymatic arrangement found in both pathways, it seems that the eukaryotic pathway evolved by convergence to the prokaryotic pathway. However, since mitochondria evolved from a bacterial endosymbiont, it would suggest that the eukaryotic pathway arose from the prokaryotic pathway. In this review, it is proposed that the eukaryote pathway evolved directly from a prokaryotic pathway by the neofunctionalization of the bacterial enzymes. Moreover, after the eukaryotic radiation, this pathway was reshaped by horizontal gene transfers or subsequent endosymbiotic processes.

ATP regulates the activity of an alternative oxidase in Trypanosoma brucei.

The reduced mitochondrial respiratory chain from the bloodstream forms of Trypanosoma brucei is composed of only a membrane-bound glycerol-3-phosphate dehydrogenase and an alternative oxidase. Since these enzymes are not proton pumps, their functions are restricted to the maintenance of the redox balance in the glycosome by means of the dihydroxyacetone phosphate/glycerol-3-phosphate shuttle. Additionally, an F1 Fo -ATP synthase functions as an ATP-hydrolysing enzyme to establish the proton motive force necessary to maintain the basic functions of mitochondria. In this report, we studied the interplay between the alternative oxidase and ATP synthase, and we found that, in addition to its role as a proton pump, ATP synthase contributes to maintain safe levels of ATP to prevent the inhibition of the alternative oxidase by ATP.

Mitochondrial alternative oxidase is determinant for growth and sporulation in the early diverging fungus Blastocladiella emersonii.

Blastocladiella emersonii is an early diverging fungus of the phylum Blastocladiomycota. During the life cycle of the fungus, mitochondrial morphology changes significantly, from a fragmented form in sessile vegetative cells to a fused network in motile zoospores. In this study, we visualize these morphological changes using a mitochondrial fluorescent probe and show that the respiratory capacity in zoospores is much higher than in vegetative cells, suggesting that mitochondrial morphology could be related to the differences in oxygen consumption. While studying the respiratory chain of the fungus, we observed an antimycin A and cyanide-insensitive, salicylhydroxamic (SHAM)-sensitive respiratory activity, indicative of a mitochondrial alternative oxidase (AOX) activity. The presence of AOX was confirmed by the finding of a B. emersonii cDNA encoding a putative AOX, and by detection of AOX protein in immunoblots. Inhibition of AOX activity by SHAM was found to significantly alter the capacity of the fungus to grow and sporulate, indicating that AOX participates in life cycle control in B. emersonii.

Fasting promotes functional changes in liver mitochondria.

Overnight fasting of rodents is commonly adopted in protocols to obtain isolated liver mitochondria, but the effects of fasting itself on mitochondrial function are poorly characterized. In this study we show that overnight fasting (15 h) promotes a shift in the liver mitochondrial bioenergetic profile, with a reduction in ADP-stimulated and maximal respiration, lower membrane potentials and lower resistance to Ca2+-induced mitochondrial permeability transition. Short term fasting (4 h) promoted similar changes, suggesting that this is a physiological shift in mitochondrial function associated with fasting, but not torpor. Our results suggest that the widely adopted liver mitochondrial isolation technique using fasted animals should be reconsidered, and also uncover physiological changes in bioenergetic function associated to nutritional status.

Topological characterization of the mitochondrial phospholipid scramblase 3.

Scramblases redistribute phospholipids in biological membranes. Phospholipid scramblase 3 (PLSCR3), which is located in mitochondria, has been reported to be involved in cardiolipin distribution from the inner to the outer membrane, thus regulating cellular processes such as apoptosis or mitophagy. However, the localization and topology of this protein has not been convincingly addressed to support a role in intermembrane phospholipid transfer. Here, we studied PLSCR3 topology within mitochondria. We show that PLSCR3 inserts in the inner membrane (IM) via its C-terminal transmembrane helix, whereas its N-terminal portion is oriented toward the intermembrane space where it is activated by calcium. Our results suggest that PLSCR3, via its C-terminal transmembrane domain, participates in the bidirectional movement of phospholipids within the IM.

Calorie restriction promotes cardiolipin biosynthesis and distribution between mitochondrial membranes.

Calorie restriction (CR) has been amply demonstrated to modify mitochondrial function. However, little is known regarding the effects of this dietary regimen on mitochondrial membranes. We isolated phospholipids from rat liver mitochondria from animals on CR or ad libitum diets and found that mitochondria from ad libitum animals present an increased content of lipoperoxides and the content of cardiolipin. Cardiolipin is the main anionic phospholipid present in mitochondrial membranes, and plays a key role in mitochondrial function, signaling and stress response. Expression levels of the enzymes involved in cardiolipin biosynthesis and remodeling were quantified and found to be upregulated in CR animals. Interestingly, when mitochondrial membranes were fractionated, the outer membrane presented a higher content of cardiolipin, indicating a redistribution of this phospholipid mediated by a phospholipid scramblase in CR. This change is associated with Drp1-mediated mitochondrial fragmentation and autophagy. Overall, we find that CR promotes extensive mitochondrial membrane remodeling, decreasing oxidatively damaged lipids, and increasing cardiolipin levels and redistributing cardiolipin. These changes in membrane properties are consistent with and may be causative of changes in mitochondrial morphology, function and turnover previously found to occur in CR.

Caloric restriction increases brain mitochondrial calcium retention capacity and protects against excitotoxicity.

Caloric restriction (CR) protects against many cerebral pathological conditions that are associated with excitotoxic damage and calcium overload, although the mechanisms are still poorly understood. Here we show that CR strongly protects against excitotoxic insults in vitro and in vivo in a manner associated with significant changes in mitochondrial function. CR increases electron transport chain activity, enhances antioxidant defenses, and favors mitochondrial calcium retention capacity in the brain. These changes are accompanied by a decrease in cyclophilin D activity and acetylation and an increase in Sirt3 expression. This suggests that Sirt3-mediated deacetylation and inhibition of cyclophilin D in CR promote the inhibition of mitochondrial permeability transition, resulting in enhanced mitochondrial calcium retention. Altogether, our results indicate that enhanced mitochondrial calcium retention capacity underlies the beneficial effects of CR against excitotoxic conditions. This protection may explain the many beneficial effects of CR in the aging brain.

Phosphatidylglycerol-derived phospholipids have a universal, domain-crossing role in stress responses.

Phosphatidylglycerol and phospholipids derived from it are widely distributed throughout the three domains of life. Cardiolipin is the best characterized of these phospholipids, and plays a key role in the response to environmental variations. Phosphatidylglycerol-derived phospholipids confer cell membranes with a wide range of responses, including changes in surface charge, fluidity, flexibility, morphology, biosynthesis and remodeling, that adapt the cell to these situations. Furthermore, the synthesis and remodeling of these phospholipids is finely regulated, highlighting the importance of these lipids in cell homeostasis and responses during stressful situations. In this article, we review the most important roles of these anionic phospholipids across domains, focusing on the biophysical basis by which these phospholipids are used in stress responses.

The chimeric origin of the cardiolipin biosynthetic pathway in the Eukarya domain.

Cardiolipin (CL) and phosphatidylglycerol (PG) are the main anionic phospholipids present in the Eukarya and Bacteria domains. They participate in energy transduction by activating and stabilizing the components of the oxidative phosphorylation machinery. Experimental evidence shows that they are synthesized by two different mechanisms which indicate that both pathways evolved convergently. Former studies on the lipid composition of archaeal membranes showed the absence of CL in these organisms, consequently, restricting it to the Eukarya and Bacteria domains. Interestingly, recent studies have demonstrated that both CL and PG are present as constitutive components of membranes of the haloarchaea group. However, this scenario complicates the analysis of the evolutionary origin of this biosynthetic pathway. Here I suggest that a phospholipid biosynthetic pathway in Eukarya probably arose from a chimeric event between bacterial and archaeal enzymes during the endosymbiosis event. Phylogenetic analyses support the different evolutionary origin of the enzymes comprising this pathway in bacteria and Eukarya. Based on protein domain analyses, orthologous proteins in the Archaea domain were identified. An integrative analysis of the proteins found demonstrates that CL biosynthesis in major clades of the Eukarya domain originated by chimerism between the bacteria and archaea pathways. Moreover, primary and secondary endosymbiontic events in plants and chromoalveolata respectively, reshaped this pathway again. The implications and advantages that these new enzymatic orders conferred to the Eukarya domain are discussed.

Cardiolipin is a key determinant for mtDNA stability and segregation during mitochondrial stress.

Mitochondria play a key role in adaptation during stressing situations. Cardiolipin, the main anionic phospholipid in mitochondrial membranes, is expected to be a determinant in this adaptive mechanism since it modulates the activity of most membrane proteins. Here, we used Saccharomyces cerevisiae subjected to conditions that affect mitochondrial metabolism as a model to determine the possible role of cardiolipin in stress adaptation. Interestingly, we found that thermal stress promotes a 30% increase in the cardiolipin content and modifies the physical state of mitochondrial membranes. These changes have effects on mtDNA stability, adapting cells to thermal stress. Conversely, this effect is cardiolipin-dependent since a cardiolipin synthase-null mutant strain is unable to adapt to thermal stress as observed by a 60% increase of cells lacking mtDNA (ρ0). Interestingly, we found that the loss of cardiolipin specifically affects the segregation of mtDNA to daughter cells, leading to a respiratory deficient phenotype after replication. We also provide evidence that mtDNA physically interacts with cardiolipin both in S. cerevisiae and in mammalian mitochondria. Overall, our results demonstrate that the mitochondrial lipid cardiolipin is a key determinant in the maintenance of mtDNA stability and segregation.

Deletion of the transcriptional regulator opi1p decreases cardiolipin content and disrupts mitochondrial metabolism in Saccharomyces cerevisiae.

Cardiolipin, the main anionic phospholipid in the inner mitochondrial membrane, provides shape, charge and osmotic support to this membrane due to its biophysical properties. In addition, it helps form respiratory supercomplexes and provides functionality to mitochondrial proteins. Defects in the biosynthesis or remodeling of cardiolipin have been related to severe diseases, such as Barth syndrome. Opi1p, a transcriptional repressor for most enzymes in phospholipid biosynthesis found in Saccharomyces cerevisiae, has been demonstrated not to affect the biosynthesis of this mitochondrial phospholipid. However, we found that opi1 deletion compromises mitochondrial metabolism producing severe respiratory defects. The mechanism producing this phenotype was explored and found to be a mitochondrial cardiolipin depletion of almost 50%, resulting in low cytochrome content and high mitochondrial DNA instability. The origin of this low cardiolipin content strongly correlated with the overproduction of inositol, an intrinsic phenotype of this mutation. Overall, our results show that adequate regulation of phospholipid synthesis is essential for the maintenance of mitochondrial function.

Uncoupling proteins (UCP) in unicellular eukaryotes: true UCPs or UCP1-like acting proteins?

Uncoupling proteins belong to the superfamily of mitochondrial anion carriers. They are apparently present throughout the Eukarya domain in which only some members have an established physiological function, i.e. UCP1 from brown adipose tissue is involved in non-shivering thermogenesis. However, the proteins responsible for the phenotype observed in unicellular organisms have not been characterized. In this report we analyzed functional evidence concerning unicellular UCPs and found that true UCPs are restricted to some taxonomical groups while proteins conferring a UCP1-like phenotype to fungi and most protists are the result of a promiscuous activity exerted by other mitochondrial anion carriers. We describe a possible evolutionary route followed by these proteins by which they acquire this promiscuous mechanism.