Manuel Gutiérrez-Aguilar

Genetic manipulation of the cardiac mitochondrial phosphate carrier does not affect permeability transition

The Mitochondrial Permeability Transition (MPT) pore is a voltage-sensitive unselective channel known to instigate necrotic cell death during cardiac disease. Recent models suggest that the isomerase cyclophilin D (CypD) regulates the MPT pore by binding to either the F0F1-ATP synthase lateral stalk or the mitochondrial phosphate carrier (PiC). Here we confirm that CypD, through its N-terminus, can directly bind PiC. We then generated cardiac-specific mouse strains overexpressing or with decreased levels of mitochondrial PiC to assess the functionality of such interaction. While PiC overexpression had no observable pathologic phenotype, PiC knockdown resulted in cardiac hypertrophy along with decreased ATP levels. Mitochondria isolated from the hearts of these mouse lines and their respective non-transgenic controls had no divergent phenotype in terms of oxygen consumption and Ca(2+)-induced MPT, as assessed by swelling and Ca(2+)-retention measurements. These results provide genetic evidence indicating that the mitochondrial PiC is not a critical component of the MPT pore.

Physiological and pathological roles of mitochondrial SLC25 carriers

The mitochondrion relies on compartmentalization of certain enzymes, ions and metabolites for the sake of efficient metabolism. In order to fulfil its activities, a myriad of carriers are properly expressed, targeted and folded in the inner mitochondrial membrane. Among these carriers, the six-transmembrane-helix mitochondrial SLC25 (solute carrier family 25) proteins facilitate transport of solutes with disparate chemical identities across the inner mitochondrial membrane. Although their proper function replenishes building blocks needed for metabolic reactions, dysfunctional SLC25 proteins are involved in pathological states. It is the purpose of the present review to cover the current knowledge on the role of SLC25 transporters in health and disease.

Structural mechanisms of cyclophilin D-dependent control of the mitochondrial permeability transition pore.

BACKGROUND Opening of the mitochondrial permeability transition pore is the underlying cause of cellular dysfunction during diverse pathological situations. Although this bioenergetic entity has been studied extensively, its molecular componentry is constantly debated. Cyclophilin D is the only universally accepted modulator of this channel and its selective ligands have been proposed as therapeutic agents with the potential to regulate pore opening during disease. SCOPE OF REVIEW This review aims to recapitulate known molecular determinants necessary for Cyclophilin D activity regulation and binding to proposed pore constituents thereby regulating the mitochondrial permeability transition pore. MAJOR CONCLUSIONS While the main target of Cyclophilin D is still a matter of further research, permeability transition is finely regulated by post-translational modifications of this isomerase and its catalytic activity facilitates pore opening. GENERAL SIGNIFICANCE Complete elucidation of the molecular determinants required for Cyclophilin D-mediated control of the mitochondrial permeability transition pore will allow the rational design of therapies aiming to control disease phenotypes associated with the occurrence of this unselective channel. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.

Mitochondrial Unselective Channels throughout the eukaryotic domain.

Mitochondria from diverse species can undergo a massive permeability increase known as the permeability transition, a process first thought to be an artifact. It is currently accepted that in the inner mitochondrial membrane there is a Mitochondrial Unselective Channel (MUC), also known as the permeability transition pore. Regardless of the species, MUC opening leads to uncoupling of oxidative phosphorylation. In each species, MUC regulation appears to be different, probably as a result of the adaptation of each organism to its specific environment. To date, the components and the putative physiological role of MUCs are still a matter of debate. Current hypothesis suggests that proteins normally participating in diverse metabolic functions constitute MUCs. Among these proteins, the Adenine Nucleotide Translocase and the phosphate carrier have been proposed as putative MUC components in mammalian and yeast mitochondria. In this review, the characteristics of MUCs from different species and strains are discussed. The data from the literature reinforce the current notion that these channels are preserved through evolution albeit with different control factors. We emphasize the knowledge available of Mitochondrial Unselective Channels from different yeast species.

In Saccharomyces cerevisiae, the phosphate carrier is a component of the mitochondrial unselective channel.

The mitochondrial permeability transition (PT) involves the opening of a mitochondrial unselective channel (MUC) resulting in membrane depolarization and increased permeability to ions. PT has been observed in many, but not all eukaryotic species. In some species, PT has been linked to cell death, although other functions, such as matrix ion detoxification or regulation of the rate of oxygen consumption have been considered. The identification of the proteins constituting MUC would help understand the biochemistry and physiology of this channel. It has been suggested that the mitochondrial phosphate carrier is a structural component of MUC and we decided to test this in yeast mitochondria. Mersalyl inhibits the phosphate carrier and it has been reported that it also triggers PT. Mersalyl induced opening of the decavanadate-sensitive Yeast Mitochondrial Unselective Channel (YMUC). In isolated yeast mitochondria from a phosphate carrier-null strain the sensitivity to both phosphate and mersalyl was lost, although the permeability transition was still evoked by ATP in a decavanadate-sensitive fashion. Polyethylene glycol (PEG)-induced mitochondrial contraction results indicated that in mitochondria lacking the phosphate carrier the YMUC is smaller: complete contraction for mitochondria from the wild type and the mutant strains was achieved with 1.45 and 1.1 kDa PEGs, respectively. Also, as expected for a smaller channel titration with 1.1 kDa PEG evidenced a higher sensitivity in mitochondria from the mutant strain. The above data suggest that the phosphate carrier is the phosphate sensor in YMUC and contributes to the structure of this channel.

Physiological uncoupling of mitochondrial oxidative phosphorylation. Studies in different yeast species

Under non-phosphorylating conditions a high proton transmembrane gradient inhibits the rate of oxygen consumption mediated by the mitochondrial respiratory chain (state IV). Slow electron transit leads to production of reactive oxygen species (ROS) capable of participating in deleterious side reactions. In order to avoid overproducing ROS, mitochondria maintain a high rate of O2 consumption by activating different exquisitely controlled uncoupling pathways. Different yeast species possess one or more uncoupling systems that work through one of two possible mechanisms: i) Proton sinks and ii) Non-pumping redox enzymes. Proton sinks are exemplified by mitochondrial unspecific channels (MUC) and by uncoupling proteins (UCP). Saccharomyces. cerevisiae and Debaryomyces hansenii express highly regulated MUCs. Also, a UCP was described in Yarrowia lipolytica which promotes uncoupled O2 consumption. Non-pumping alternative oxido-reductases may substitute for a pump, as in S. cerevisiae or may coexist with a complete set of pumps as in the branched respiratory chains from Y. lipolytica or D. hansenii. In addition, pumps may suffer intrinsic uncoupling (slipping). Promising models for study are unicellular parasites which can turn off their aerobic metabolism completely. The variety of energy dissipating systems in eukaryote species is probably designed to control ROS production in the different environments where each species lives.

The Saccharomyces cerevisiae mitochondrial unselective channel behaves as a physiological uncoupling system regulated by Ca2+, Mg2+, phosphate and ATP.

It is proposed that the Saccharomyces cerevisiae the Mitochondrial Unselective Channel (ScMUC) is tightly regulated constituting a physiological uncoupling system that prevents overproduction of reactive oxygen species (ROS). Mg2+, Ca2+ or phosphate (Pi) close ScMUC, while ATP or a high rate of oxygen consumption open it. We assessed ScMUC activity by measuring in isolated mitochondria the respiratory control, transmembrane potential (ΔΨ), swelling and production of ROS. At increasing [Pi], less [Ca2+] and/or [Mg2+] were needed to close ScMUC or increase ATP synthesis. The Ca2+-mediated closure of ScMUC was prevented by high [ATP] while the Mg2+ or Pi effect was not. When Ca2+ and Mg2+ were alternatively added or chelated, ScMUC opened and closed reversibly. Different effects of Ca2+ vs Mg2+ effects were probably due to mitochondrial Mg2+ uptake. Our results suggest that ScMUC activity is dynamically controlled by both the ATP/Pi ratio and divalent cation fluctuations. It is proposed that the reversible opening/closing of ScMUC leads to physiological uncoupling and a consequent decrease in ROS production.

A new twist on an old idea part 2: cyclosporine preserves normal mitochondrial but not cardiomyocyte function in mini‐swine with compensated heart failure

We recently developed a clinically relevant mini‐swine model of heart failure with preserved ejection fraction (HFpEF), in which diastolic dysfunction was associated with increased mitochondrial permeability transition (MPT). Early diastolic function is ATP and Ca2+‐dependent, thus, we hypothesized chronic low doses of cyclosporine (CsA) would preserve mitochondrial function via inhibition of MPT and subsequently maintain normal cardiomyocyte Ca2+ handling and contractile characteristics. Left ventricular cardiomyocytes were isolated from aortic‐banded Yucatan mini‐swine divided into three groups; control nonbanded (CON), HFpEF nontreated (HF), and HFpEF treated with CsA (HF‐CsA). CsA mitigated the deterioration of mitochondrial function observed in HF animals, including functional uncoupling of Complex I‐dependent mitochondrial respiration and increased susceptibility to MPT. Attenuation of mitochondrial dysfunction in the HF‐CsA group was not associated with commensurate improvement in cardiomyocyte Ca2+ handling or contractility. Ca2+ transient amplitude was reduced and transient time to peak and recovery (tau) prolonged in HF and HF‐CsA groups compared to CON. Alterations in Ca2+ transient parameters observed in the HF and HF‐CsA groups were associated with decreased cardiomyocyte shortening and shortening rate. Cellular function was consistent with impaired in vivo systolic and diastolic whole heart function. A significant systemic hypertensive response to CsA was observed in HF‐CsA animals, and may have played a role in the accelerated the development of heart failure at both the whole heart and cellular levels. Given the significant detriment to cardiac function observed in response to CsA, our findings suggest chronic CsA treatment is not a viable therapeutic option for HFpEF.

Saxagliptin and Tadalafil Differentially Alter Cyclic Guanosine Monophosphate (cGMP) Signaling and Left Ventricular Function in Aortic‐Banded Mini‐Swine

Background Cyclic guanosine monophosphate‐protein kinase G‐phosphodiesterase 5 signaling may be disturbed in heart failure (HF) with preserved ejection fraction, contributing to cardiac remodeling and dysfunction. The purpose of this study was to manipulate cyclic guanosine monophosphate signaling using the dipeptidyl‐peptidase 4 inhibitor saxagliptin and phosphodiesterase 5 inhibitor tadalafil. We hypothesized that preservation of cyclic guanosine monophosphate cGMP signaling would attenuate pathological cardiac remodeling and improve left ventricular (LV) function. Methods and Results We assessed LV hypertrophy and function at the organ and cellular level in aortic‐banded pigs. Concentric hypertrophy was equal in all groups, but LV collagen deposition was increased in only HF animals. Prevention of fibrotic remodeling by saxagliptin and tadalafil was correlated with neuropeptide Y plasma levels. Saxagliptin better preserved integrated LV systolic and diastolic function by maintaining normal LV chamber volumes and contractility (end‐systolic pressure‐volume relationship, preload recruitable SW) while preventing changes to early/late diastolic longitudinal strain rate. Function was similar to the HF group in tadalafil‐treated animals including increased LV contractility, reduced chamber volume, and decreased longitudinal, circumferential, and radial mechanics. Saxagliptin and tadalafil prevented a negative cardiomyocyte shortening‐frequency relationship observed in HF animals. Saxagliptin increased phosphodiesterase 5 activity while tadalafil increased cyclic guanosine monophosphate levels; however, neither drug increased downstream PKG activity. Early mitochondrial dysfunction, evident as decreased calcium‐retention capacity and Complex II‐dependent respiratory control, was present in both HF and tadalafil‐treated animals. Conclusions Both saxagliptin and tadalafil prevented increased LV collagen deposition in a manner related to the attenuation of increased plasma neuropeptide Y levels. Saxagliptin appears superior for treating heart failure with preserved ejection fraction, considering its comprehensive effects on integrated LV systolic and diastolic function.

The mitochondrial unselective channel in Saccharomyces cerevisiae.

Opening of the mitochondrial permeability transition (MPT) pore mediates the increase in the unselective permeability to ions and small molecules across the inner mitochondrial membrane. MPT results from the opening of channels of unknown identity in mitochondria from plants, animals and yeast. However, the effectors and conditions required for MPT to occur in different species are remarkably disparate. Here we critically review previous and recent findings concerning the mitochondrial unselective channel of the yeast Saccharomyces cerevisiae to determine if it can be considered a counterpart of the mammalian MPT pore.