Activation Of Permeability Transition Pore Research Articles

Proline‐rich Tyrosine Kinase 2 Phosphorylates Mitochondrial Calcium Uniporter and Regulates Mitochondrial Calcium Uptake

Mitochondrial Ca2+ (mtCa2+) uptake via the mtCa2+ uniporter (MCU) complex is a critical factor in determining cell survival or death. We previously reported that the activation of a Ca2+‐ and reactive oxygen species (ROS)‐sensitive protein tyrosine kinase (PTK), proline‐rich tyrosine kinase 2 (Pyk2), under Gq protein–coupled receptor (GqPCR) stimulation increases tyrosine phosphorylation (P‐Y) of MCU and mtCa2+ uptake, followed by the activation of mitochondrial permeability transition pore, and apoptotic cell death in cardiomyocytes. However, further investigation is required to determine 1) the identity of Pyk2‐specific phosphorylation sites within the MCU structure and 2) the functional relevance of P‐Y to MCU channel function as well as mitochondrial functions both in situ and in vivo. In this project, we determined specific Pyk2 phosphorylation site(s) in MCU and tested whether P‐Y at these site(s) modulates MCU channel function using cell lines stably expressing wild‐type (WT)‐ or dephosphomimetic mutants of MCU (MCU‐YFs). Furthermore, we investigated the role of MCU P‐Y in vivo using transgenic (TG) mice with cardiac‐specific overexpression of constitutively active Gαq protein (Gαq TG mice). Through phosphorylation prediction programs, we identified three tyrosine residues as candidate PTK phosphorylation sites, which are conserved across all eukaryotic species. In vitro kinase assays showed that purified active Pyk2 phosphorylated purified full‐length MCU. Next, P‐Y levels at candidate sites were biochemically detected after GqPCR stimulation using cell lines stably expressing Flag‐tagged WT‐ or MCU‐YFs. This in situ assay revealed that only two tyrosine sites increased P‐Y levels in response to GqPCR stimulation. We further assessed mtCa2+ uptake profiles in response to cytosolic Ca2+ elevation in cells stably expressing WT‐ and MCU‐YFs via live cell imaging using mitochondria‐targeted Ca2+‐sensitive biosensors. Using GFP‐tagged MCU‐YFs, we confirmed that all mutant MCUs, like WT‐MCU, were exclusively expressed in mitochondria. Overexpression of one MCU‐YF failed to increase mtCa2+ uptake in response to cytosolic Ca2+ elevation, although the overexpression of WT‐ and the other two MCU‐YFs significantly accelerated mtCa2+ uptake compared to non‐transfected cells. Finally, we demonstrated that P‐Y of MCU occurs in Gαq TG mouse hearts, concomitant with higher Pyk2 activation and apoptotic signaling. In summary, MCU contains Pyk2‐specific phosphorylation site(s) and Pyk2‐dependent P‐Y of MCU increase mtCa2+ uptake via the MCU complex. Moreover, GqPCR‐Pyk2 signaling may induce P‐Y of MCU and cardiomyocyte death in vivo. These findings suggest that inhibition of GqPCR‐Pyk2‐MCU signaling may be a novel therapeutic target to prevent mitochondrial Ca2+ overload, oxidative stress, and cardiomyocyte death during pathophysiological conditions such as heart failure, in which chronic GqPCR stimulation occurs.Support or Funding InformationB.S.J was supported by NIH/NIGMS U54GM115677 and P30GM1114750. U.M. was supported by NIH/NHLBI R01HL114784. S.S.S was supported by NIH/NHLBI R01HL093671 and R01HL122124. J.O.‐U. was supported by NIH/NHLBI R01HL136757, NIH/NIGMS P30GM1114750, American Heart Association (AHA) 4BGIA18830032, AHA 16SDG27260248, Rhode Island Foundation #20164376 Medical Research Grant, and American Physiological Society (APS) 2017 Shih‐Chun Wang Young Investigator Award.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cardioprotection in mice with a knock-in mutation in Cyclophilin D (CypD-C202S): a site of S-nitrosylation

Our previous study in mouse embryonic fibroblasts showed that cysteine 202 of cyclophilin D (CyPD) is necessary for redox stress-induced activation of the mitochondrial permeability transition pore (mPTP). To further investigate the essential function of this cysteine residue in situ, we used CRISPR to develop a knock-in mouse model (C57BL/6N strain), where CyPD cysteine 202 was mutated to a serine (C202S-KI). The amount of total CyPD expressed in the CyPD C202S-KI did not differ compared to the wild-type (WT). However, the CyPD C202S-KI mouse hearts elicit a significant cardioprotective effect against ischemia-reperfusion (I/R) injury in the Langendorff perfused heart model. After 20 min of global ischemia followed by 90 min of reperfusion, the post-ischemic recovery of rate pressure product (RPP= heart rate x LVDP) was 45.0±4.2% in CyPD WT and 59.6±4.0% in CyPD C202S-KI mice (p=0.0455). Myocardial infarct size was decreased in CyPD C202S-KI mouse hearts versus CyPD WT mice (24.5±4.7% vs 49.8±2.7%, p=0.0095). Isolated heart mitochondria from CyPD C202-KI mice had a higher calcium retention capacity compared to CyPD WT mice (213.3±16.67 vs 140.0±20.82 umol Ca+2/g protein, p=0.0371). However, in contrast to CyPD knockout mice which exhibit more pronounced cardiac hypertrophy in response to pressure overload stimulation than control mice, CyPD C202S-KI mice developed a comparable level of hypertrophy to their WT littermate in an angiotensin II-induced hypertrophy model delivered by implanted osmotic minipumps. In conclusion, these results show that mutated CyPD C202S affords cardioprotection against I/R injury, suggesting that the redox-modification of cysteine 202 might play an important role in the regulation of CyPD and its downstream targets such as mPTP.

Two mutations in mitochondrial ATP6 gene of ATP synthase, related to human cancer, affect ROS, calcium homeostasis and mitochondrial permeability transition in yeast

The relevance of mitochondrial DNA (mtDNA) mutations in cancer process is still unknown. Since the mutagenesis of mitochondrial genome in mammals is not possible yet, we have exploited budding yeast S. cerevisiae as a model to study the effects of tumor-associated mutations in the mitochondrial MTATP6 gene, encoding subunit 6 of ATP synthase, on the energy metabolism. We previously reported that four mutations in this gene have a limited impact on the production of cellular energy. Here we show that two mutations, Atp6-P163S and Atp6-K90E (human MTATP6-P136S and MTATP6-K64E, found in prostate and thyroid cancer samples, respectively), increase sensitivity of yeast cells both to compounds inducing oxidative stress and to high concentrations of calcium ions in the medium, when Om45p, the component of porin complex in outer mitochondrial membrane (OM), was fused to GFP. In OM45-GFP background, these mutations affect the activation of yeast permeability transition pore (yPTP, also called YMUC, yeast mitochondrial unspecific channel) upon calcium induction. Moreover, we show that calcium addition to isolated mitochondria heavily induced the formation of ATP synthase dimers and oligomers, recently proposed to form the core of PTP, which was slower in the mutants. We show the genetic evidence for involvement of mitochondrial ATP synthase in calcium homeostasis and permeability transition in yeast. This paper is a first to show, although in yeast model organism, that mitochondrial ATP synthase mutations, which accumulate during carcinogenesis process, may be significant for cancer cell escape from apoptosis.

The path from mitochondrial ROS to aging runs through the mitochondrial permeability transition pore.

SummaryExcessive production of mitochondrial reactive oxygen species (mROS) is strongly associated with mitochondrial and cellular oxidative damage, aging, and degenerative diseases. However, mROS also induces pathways of protection of mitochondria that slow aging, inhibit cell death, and increase lifespan. Recent studies show that the activation of the mitochondrial permeability transition pore (mPTP), which is triggered by mROS and mitochondrial calcium overloading, is enhanced in aged animals and humans and in aging‐related degenerative diseases. mPTP opening initiates further production and release of mROS that damage both mitochondrial and nuclear DNA, proteins, and phospholipids, and also releases matrix NAD that is hydrolyzed in the intermembrane space, thus contributing to the depletion of cellular NAD that accelerates aging. Oxidative damage to calcium transporters leads to calcium overload and more frequent opening of mPTP. Because aging enhances the opening of the mPTP and mPTP opening accelerates aging, we suggest that mPTP opening drives the progression of aging. Activation of the mPTP is regulated, directly and indirectly, not only by the mitochondrial protection pathways that are induced by mROS, but also by pro‐apoptotic signals that are induced by DNA damage. We suggest that the integration of these contrasting signals by the mPTP largely determines the rate of cell aging and the initiation of cell death, and thus animal lifespan. The suggestion that the control of mPTP activation is critical for the progression of aging can explain the conflicting and confusing evidence regarding the beneficial and deleterious effects of mROS on health and lifespan.

Study of the relationship between AGEs and oxidative stress damage to trophoblast cell mitochondria.

To study the influence of AGEs on placental trophoblast mitochondria oxidative stress, and to explore the possible pathogenesis which may participate in pre-eclampsia. Human trophoblast cells from early pregnancy were cultured by an enzyme-digestion method. When trophoblast cells reached approximately 70-80% after passages, they were incubated with pre-eclampsia serum for 24 hours. A fluorescent dye assay was applied to measure the mitochondrial membrane potential; ELISA was used to measure the activity of the mitochondrial permeability transition pore. mtDNA was detected by Real-time fluorescence quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR). We continued to culture one group of cells with pre-eclampsia maternal serum, and other cells were pulsed with 600 mg/L AGEs. Cells were incubated for 16 hours before assaying the levels of mitochondrial oxidative stress damage. The levels of mitochondria oxidative stress damage in the AGEs group were higher than in the pre-eclampsia group 1 and pre-eclampsia group 2. There was no statistically significant difference in mitochondrial oxidative stress damage between the pre-eclampsia group 1 and group 2. The AGEs are involved in the pathogenesis of pre-eclampsia, possibly through the enhancement of mito-chondrial oxidative stress damage.

Osmotic regulation of the mitochondrial permeability transition pore investigated by light scattering, fluorescence and electron microscopy techniques

Mitochondrial permeability transition (PT) is a phenomenon of an increase of the inner membrane permeability in response to an excessive matrix calcium accumulation. PTP is caused by the opening of the large weakly selective channel. Molecular composition and regulation of permeability transition pore (PTP) are not well understood. Here we used isolated mitochondria to investigate dependence of PTP activation on the osmotic pressure. We found that in low osmotic strength solution calcium-induced PTP is significantly inhibited. We propose that this effect is linked to the changes in the curvature of the mitochondrial inner membrane. This interpretation is consistent with the idea about the importance of ATP synthase dimerization in modulation of the PTP activity.

The mitochondrial Na+/Ca2+ exchanger is essential for Ca2+ homeostasis and viability.

Mitochondrial calcium (mCa2+) has a central role in both metabolic regulation and cell death signalling, however its role in homeostatic function and disease is controversial. Slc8b1 encodes the mitochondrial Na+/Ca2+ exchanger (NCLX), which is proposed to be the primary mechanism for mCa2+ extrusion in excitable cells. Here we show that tamoxifen-induced deletion of Slc8b1 in adult mouse hearts causes sudden death, with less than 13% of affected mice surviving after 14 days. Lethality correlated with severe myocardial dysfunction and fulminant heart failure. Mechanistically, cardiac pathology was attributed to mCa2+ overload driving increased generation of superoxide and necrotic cell death, which was rescued by genetic inhibition of mitochondrial permeability transition pore activation. Corroborating these findings, overexpression of NCLX in the mouse heart by conditional transgenesis had the beneficial effect of augmenting mCa2+ clearance, preventing permeability transition and protecting against ischaemia-induced cardiomyocyte necrosis and heart failure. These results demonstrate the essential nature of mCa2+ efflux in cellular function and suggest that augmenting mCa2+ efflux may be a viable therapeutic strategy in disease.

Absence of physiological Ca2+ transients is an initial trigger for mitochondrial dysfunction in skeletal muscle following denervation

BackgroundMotor neurons control muscle contraction by initiating action potentials in muscle. Denervation of muscle from motor neurons leads to muscle atrophy, which is linked to mitochondrial dysfunction. It is known that denervation promotes mitochondrial reactive oxygen species (ROS) production in muscle, whereas the initial cause of mitochondrial ROS production in denervated muscle remains elusive. Since denervation isolates muscle from motor neurons and deprives it from any electric stimulation, no action potentials are initiated, and therefore, no physiological Ca2+ transients are generated inside denervated muscle fibers. We tested whether loss of physiological Ca2+ transients is an initial cause leading to mitochondrial dysfunction in denervated skeletal muscle.MethodsA transgenic mouse model expressing a mitochondrial targeted biosensor (mt-cpYFP) allowed a real-time measurement of the ROS-related mitochondrial metabolic function following denervation, termed “mitoflash.” Using live cell imaging, electrophysiological, pharmacological, and biochemical studies, we examined a potential molecular mechanism that initiates ROS-related mitochondrial dysfunction following denervation.ResultsWe found that muscle fibers showed a fourfold increase in mitoflash activity 24 h after denervation. The denervation-induced mitoflash activity was likely associated with an increased activity of mitochondrial permeability transition pore (mPTP), as the mitoflash activity was attenuated by application of cyclosporine A. Electrical stimulation rapidly reduced mitoflash activity in both sham and denervated muscle fibers. We further demonstrated that the Ca2+ level inside mitochondria follows the time course of the cytosolic Ca2+ transient and that inhibition of mitochondrial Ca2+ uptake by Ru360 blocks the effect of electric stimulation on mitoflash activity.ConclusionsThe loss of cytosolic Ca2+ transients due to denervation results in the downstream absence of mitochondrial Ca2+ uptake. Our studies suggest that this could be an initial trigger for enhanced mPTP-related mitochondrial ROS generation in skeletal muscle.

Advanced glycation end products regulate anabolic and catabolic activities via NLRP3‐inflammasome activation in human nucleus pulposus cells

Intervertebral disc degeneration is widely recognized as a cause of lower back pain, neurological dysfunction and other musculoskeletal disorders. The major inflammatory cytokine IL‐1β is associated with intervertebral disc degeneration; however, the molecular mechanisms that drive IL‐1β production in the intervertebral disc, especially in nucleus pulposus (NP) cells, are unknown. In some tissues, advanced glycation end products (AGEs), which accumulate in NP tissues and promote its degeneration, increase oxidative stress and IL‐1β secretion, resulting in disorders, such as obesity, diabetes mellitus and ageing. It remains unclear whether AGEs exhibit similar effects in NP cells. In this study, we observed significant activation of the NLRP3 inflammasome in NP tissues obtained from patients with degenerative disc disease compared to that with idiopathic scoliosis according to results detected by Western blot and immunofluorescence. Using NP cells established from healthy tissues, our in vitro study revealed that AGEs induced an inflammatory response in NP cells and a degenerative phenotype in a NLRP3‐inflammasome‐dependent manner related to the receptor for AGEs (RAGE)/NF‐κB pathway and mitochondrial damage induced by mitochondrial reactive oxygen species (mtROS) generation, mitochondrial permeability transition pore (mPTP) activation and calcium mobilization. Among these signals, both RAGE and mitochondrial damage primed NLRP3 and pro‐IL‐1β activation as upstream signals of NF‐κB activity, whereas mitochondrial damage was critical for the assembly of inflammasome components. These results revealed that accumulation of AGEs in NP tissue may initiate inflammation‐related degeneration of the intervertebral disc via activation of the NLRP3 inflammasome.

Genetic Rescue of Mitochondrial Calcium Efflux in Alzheimer's Disease Preserves Mitochondrial Function and Protects against Neuronal Cell Death

Background. Alzheimer's disease (AD) is characterized by neurodegeneration, specifically the progressive loss of neuronal populations in the frontal cortex and hippocampus. Numerous studies have shown that neuronal cell death and metabolic dysregulation are fundamental cellular mechanisms driving the progression of AD and other dementia-related diseases. Previous studies have suggested numerous mechanisms whereby intracellular Ca2+ load is increased in AD and thereby likely significantly impacts mCa2+ signaling. Methods. To discern if mCa2+ signaling is causative in the progression of AD, human AD brain samples, an AD mutant mouse model (3xTg) and an AD-mutant cell line (N2a/APPswe) were examined for mitochondrial alterations in: Ca2+ handling, ROS generation, membrane potential (ΔΨ), permeability transition pore activation, OxPhos, APP metabolism and cell death. Results. 3xTg-AD mice and human AD brain samples displayed significant alterations in the expression of key mCa2+ exchange genes, most notably a reduction in the expression of the mitochondrial Na+/Ca2+ exchanger (mNCX, SLC8B1), the major efflux pathway in excitable cells. We discovered that mCa2+ efflux and mCa2+ retention capacity was severely impaired in N2a/APPswe cells. Rescue of mCa2+ extrusion, via adenoviral expression of mNCX, enhanced the clearance of pathogenic mCa2+, recovered (ΔΨ), enhanced OxPhos, reduced extracellular Aβ1-40 levels and protected from ionomycin-, glutamate- and ROS-induced cell death. Conclusions. Our data suggest that impaired mCa2+ exchange is a central contributor to neuronal cell death in AD and that mNCX represents a new therapeutic target to inhibit or reverse AD progression.

Cyclophilin D Acetylation Regulates Cardiac Myocyte Differentiation

INTRODUCTION: Protein acetylation is an important regulator of cellular function. Acetylation of cyclophilin D (CyPD) at lysine 166 increases its ability to open the mitochondria permeability transition pore (PTP), which is an important regulator of mitochondrial metabolism and myocyte differentiation. HYPOTHESIS: Acetylation of CyPD controls PTP activity, mitochondrial structure and function, and myocyte differentiation in the developing heart. METHODS: We use immunoblotting and immunoprecipitation to determine the global protein acetylation state and that of CyPD during embryonic cardiac development in wild type (WT) and CyPD-null mice. We also re-expressed WT and mutant CyPD in CyPD-null embryonic and neonatal cardiac myocyte cultures to determine effects on mitochondrial structure and function and myocyte differentiation. RESULTS: Immunoblotting revealed that global protein acetylation was high in the embryo but fell during later development. In contrast, CyP-D was highly acetylated in the early embryonic heart, but this decreased dramatically during subsequent development. When re-expressed in cultures of CyPD-null myocytes, WT CyPD recapitulated the WT cell phenotype of fragmented mitochondrial structure, low mitochondrial membrane potential (Δψm), and decreased differentiation. Re-expression of a CyPD acetylation mimic mutant (K166Q) had similar or greater effects on these parameters than WT CyPD, while an inactivating (R96G) and de-acetylation mimic (K166R) CyPD mutant did not. CONCLUSION: These data suggest that myocyte differentiation during cardiac development is associated with a decrease in overall protein acetylation, perhaps because of the changes that occur in metabolism as the heart develops. However, specific acetylation of CyPD appears to be particularly important for maturation of mitochondrial function and regulation of cardiac myocyte differentiation.

Penehyclidine hydrochloride prevents anoxia/reoxygenation injury and induces H9c2 cardiomyocyte apoptosis via a mitochondrial pathway

Penehyclidine hydrochloride (PHC) is an anticholinergic drug that has been widely used in the clinic for years, evaluating for anesthetic premedication, anti-muscarinic, or improving microcirculation. However, very little is known about its protective effects against anoxia/reoxygenation (A/R) injury in myocardiocytes. The aim of our study was to investigate the protective effects of PHC pretreatment on A/R injury, as well as the underlying mechanisms involved. To investigate apoptosis, we used the cell viability assay, Annexin-V/PI assay, and lactate dehydrogenase (LDH) and creatine kinase (CK) activity measurements. Intracellular Ca2+ concentrations, reactive oxygen species (ROS), malonaldehyde (MDA), superoxide dismutase (SOD), and mitochondrial membrane potential were also measured. To analyze the molecular mechanisms, mitochondrial permeability transition pore (MPTP) activity and mRNA expressions of Bcl-2, Bax, cytochrome C (Cyt-C), caspase-3, and voltage-dependent anion channel (VDAC) were assessed. Pretreatment with PHC significantly increased cell viability and decreased the percentage of apoptotic cells, as well as LDH and CK activity, which was accompanied by a reduction in intracellular Ca2+ concentration, ROS, and MDA, as well as an increase in SOD levels. PHC pretreatment also restored mitochondrial membrane potential. Moreover, pre-incubation with PHC significantly attenuated MPTP activity and mRNA expressions of Bax, Cyt-C, caspase-3, and VDAC. Our results showed that PHC pretreatment protected H9c2 cells against A/R injury by modulating the mitochondrial apoptosis pathway.

The use of the Petri net method in the simulation modeling of mitochondrial swelling.

Using photon correlation spectroscopy, which allows investigating changes in the hydrodynamic dia­meter of the particles in suspension, it was shown that ultrahigh concentrations of Ca2+ (over 10 mM) induce swelling of isolated mitochondria. An increase in hydrodynamic diameter was caused by an increase of non-specific mitochondrial membrane permeability to Ca ions, matrix Ca2+ overload, activation of ATP- and Ca2+-sensitive K+-channels, as well as activation of cyclosporin-sensitive permeability transition pore. To formalize the experimental data and to assess conformity of experimental results with theoretical predictions we developed a simulation model using the hybrid functional Petri net method.

Rosmarinic Acid suppressed high glucose-induced apoptosis in H9c2 cells by ameliorating the mitochondrial function and activating STAT3

Mitochondrial injury characterized by intracellular reactive oxygen species (ROS) accumulation plays a critical role in hyperglycemia-induced myocardium dysfunction. Previous studies have demonstrated that Rosmarinic Acid (RA) treatment and activating Signal transducer and activator of transcription 3 (STAT3) signaling pathway have protective effects on mitochondrial dysfunction in cardiomyocyte, but there is little data regarding cardiomyocyte under condition of high-glucose. The present study was undertaken to determine the relationship between RA and STAT3 activation, as well as their effects on high glucose-induced mitochondrial injury and apoptosis in H9c2 cardiomyocyte. Our results revealed that RA pretreatment suppressed high glucose-induced apoptosis in H9c2 cells. Moreover, the effect of RA on apoptosis was related with improved mitochondrial function, which was demonstrated by that RA attenuated high glucose-induced ROS generation, inhibited mitochondrial permeability transition pore (MPTP) activation, suppressed cytochrome c release and caspase-3 activation. In addition, the phosphorylation of STAT3 in H9c2 cells was inhibited under condition of high-glucose, but RA improved STAT3 phosphorylation. Importantly, inhibition of STAT3 expression by using STAT3-siRNA partly suppressed the effect of RA on high glucose-induced apoptosis. Taken together, pretreatment with RA suppressed high glucose-induced apoptosis in cardiomyocyte by ameliorating mitochondrial function and activating STAT3.

Comprehensive analysis of mitochondrial permeability transition pore activity in living cells using fluorescence-imaging-based techniques.

Mitochondrial permeability transition (mPT) refers to a sudden increase in the permeability of the inner mitochondrial membrane. Long-term studies of mPT revealed that this phenomenon has a critical role in multiple pathophysiological processes. mPT is mediated by the opening of a complex termed the mPT pore (mPTP), which is responsible for the osmotic influx of water into the mitochondrial matrix, resulting in swelling of mitochondria and dissipation of the mitochondrial membrane potential. Here we provide three independent optimized protocols for monitoring mPT in living cells: (i) measurement using a calcein-cobalt technique, (ii) measurement of the mPTP-dependent alteration of the mitochondrial membrane potential, and (iii) measurement of mitochondrial swelling. These procedures can easily be modified and adapted to different cell types. Cell culture and preparation of the samples are estimated to take ∼1 d for methods (i) and (ii), and ∼3 d for method (iii). The entire experiment, including analyses, takes ∼2 h.

In vitro and in vivo activation of mitochondrial membrane permeability transition pore using triiodothyronine.

Using a novel method for evaluating mitochondrial swelling (Drahota et al. 2012a) we studied the effect of calcium (Ca(2+)), phosphate (P(i)), and triiodothyronine (T(3)) on the opening of mitochondrial membrane permeability transition pore and how they interact in the activation of swelling process. We found that 0.1 mM P(i), 50 microM Ca(2+) and 25 microM T(3) when added separately increase the swelling rate to about 10 % of maximal values when all three factors are applied simultaneously. Our findings document that under experimental conditions in which Ca(2+) and P(i) are used as activating factors, the addition of T(3) doubled the rate of swelling. T(3) has also an activating effect on mitochondrial membrane potential. The T(3) activating effect was also found after in vivo application of T(3). Our data thus demonstrate that T(3) has an important role in opening the mitochondrial membrane permeability pore and activates the function of the two key physiological swelling inducers, calcium and phosphate ions.

Role of inorganic polyphosphate in mammalian cells: from signal transduction and mitochondrial metabolism to cell death.

Inorganic polyphosphate (polyP) is a polymer compromised of linearly arranged orthophosphate units that are linked through high-energy phosphoanhydride bonds. The chain length of this polymer varies from five to several thousand orthophosphates. PolyP is distributed in the most of the living organisms and plays multiple functions in mammalian cells, it is important for blood coagulation, cancer, calcium precipitation, immune response and many others. Essential role of polyP is shown for mitochondria, from implication into energy metabolism and mitochondrial calcium handling to activation of permeability transition pore (PTP) and cell death. PolyP is a gliotransmitter which transmits the signal in astrocytes via activation of P2Y1 receptors and stimulation of phospholipase C. PolyP-induced calcium signal in astrocytes can be stimulated by different lengths of this polymer but only long chain polyP induces mitochondrial depolarization by inhibition of respiration and opening of the PTP. It leads to induction of astrocytic cell death which can be prevented by inhibition of PTP with cyclosporine A. Thus, medium- and short-length polyP plays role in signal transduction and mitochondrial metabolism of astrocytes and long chain of this polymer can be toxic for the cells.

Inorganic polyphosphate in cardiac myocytes: from bioenergetics to the permeability transition pore and cell survival.

Inorganic polyphosphate (polyP) is a linear polymer of Pi residues linked together by high-energy phosphoanhydride bonds as in ATP. PolyP is present in all living organisms ranging from bacteria to human and possibly even predating life of this planet. The length of polyP chain can vary from just a few phosphates to several thousand phosphate units long, depending on the organism and the tissue in which it is synthesized. PolyP was extensively studied in prokaryotes and unicellular eukaryotes by Kulaev's group in the Russian Academy of Sciences and by the Nobel Prize Laureate Arthur Kornberg at Stanford University. Recently, we reported that mitochondria of cardiac ventricular myocytes contain significant amounts (280±60pmol/mg of protein) of polyP with an average length of 25 Pi and that polyP is involved in Ca(2+)-dependent activation of the mitochondrial permeability transition pore (mPTP). Enzymatic polyP depletion prevented Ca(2+)-induced mPTP opening during ischaemia; however, it did not affect reactive oxygen species (ROS)-mediated mPTP opening during reperfusion and even enhanced cell death in cardiac myocytes. We found that ROS generation was actually enhanced in polyP-depleted cells demonstrating that polyP protects cardiac myocytes against enhanced ROS formation. Furthermore, polyP concentration was dynamically changed during activation of the mitochondrial respiratory chain and stress conditions such as ischaemia/reperfusion (I/R) and heart failure (HF) indicating that polyP is required for the normal heart metabolism. This review discusses the current literature on the roles of polyP in cardiovascular health and disease.

Absence of Physiological Calcium Transients Triggers Mitochondrial ROS Production in Skeletal Muscle Following Denervation

Motoneurons control muscle contraction by initiating action potentials in muscle fibers. Denervation of muscle fiber from the motoneuron will lead to muscle paralysis and atrophy, which is often linked to mitochondrial dysfunction. It is known that denervation induces increased ROS production in muscle, whereas the initial cause of mitochondrial ROS production in denervated muscle remains elusive. In this study we examined whether physiological Ca2+ signaling plays a role in initiating mitochondrial ROS production. We generated a transgenic mouse model expressing a mitochondrial targeted biosensor (mt-cpYFP) that reports dynamic ROS production inside mitochondria termed “mitoflash”. We found that following 24-hours of denervation, muscle fibers showed ∼4-fold increase in mitoflash events. The denervation-induced mitoflash activity was associated with an increased activity of mitochondrial permeability transition pore (PTP), as the mitoflash signal was attenuated by application of cyclosporin A. Since severing the sciatic nerve isolates the muscle from the motoneuron and deprives it from any electric stimulation, no action potentials are initiated in the denervated muscle, and therefore, physiological Ca2+ transients are absent inside muscle fibers. Restoring physiological Ca2+ transients in denervated muscle fibers by electric stimulation eliminated the effect of denervation on mitoflash signal within seconds. In addition, the electric stimulation seemed to have a more potent effect on the activity of PTP than cyclosporine A. Furthermore, we demonstrated that the Ca2+ transient inside mitochondria follows the time course of the cytosolic Ca2+ transient, suggesting that mitochondrial PTP may respond to changes in both cytosolic and mitochondrial Ca2+ levels, and that loss of Ca2+ transients is an initial trigger for mitochondrial dysfunction in skeletal muscle following denervation. Supported by MDA and NIAMS.

Mitochondrial Function during and Regulation of Cardiac Development

The roles played by mitochondria during cardiac development are poorly understood. However, recent data has suggested that mitochondria are vitally important for normal formation and function of the heart in the embryo, the fetus, and the neonate. As the heart develops, mitochondrial ATP production becomes increasingly important, while mitochondrial regulation of the cellular redox state is important for cardiac myocyte differentiation. The mechanisms that control mitochondrial function during this period are becoming clearer and may include changes in protein acetylation, permeability transition pore activity, assembly of the electron transport chain, and mitochondrial dynamics and biogenesis.