Lea K. Seidlmayer

Inorganic polyphosphate is a potent activator of the mitochondrial permeability transition pore in cardiac myocytes.

Mitochondrial dysfunction caused by excessive Ca2+ accumulation is a major contributor to cardiac cell and tissue damage during myocardial infarction and ischemia–reperfusion injury (IRI). At the molecular level, mitochondrial dysfunction is induced by Ca2+-dependent opening of the mitochondrial permeability transition pore (mPTP) in the inner mitochondrial membrane, which leads to the dissipation of mitochondrial membrane potential (ΔΨm), disruption of adenosine triphosphate production, and ultimately cell death. Although the role of Ca2+ for induction of mPTP opening is established, the exact molecular mechanism of this process is not understood. The aim of the present study was to test the hypothesis that the adverse effect of mitochondrial Ca2+ accumulation is mediated by its interaction with inorganic polyphosphate (polyP), a polymer of orthophosphates linked by phosphoanhydride bonds. We found that cardiac mitochondria contained significant amounts (280 ± 60 pmol/mg of protein) of short-chain polyP with an average length of 25 orthophosphates. To test the role of polyP for mPTP activity, we investigated kinetics of Ca2+ uptake and release, ΔΨm and Ca2+-induced mPTP opening in polyP-depleted mitochondria. polyP depletion was achieved by mitochondria-targeted expression of a polyP-hydrolyzing enzyme. Depletion of polyP in mitochondria of rabbit ventricular myocytes led to significant inhibition of mPTP opening without affecting mitochondrial Ca2+ concentration by itself. This effect was observed when mitochondrial Ca2+ uptake was stimulated by increasing cytosolic [Ca2+] in permeabilized myocytes mimicking mitochondrial Ca2+ overload observed during IRI. Our findings suggest that inorganic polyP is a previously unrecognized major activator of mPTP. We propose that the adverse effect of polyphosphate might be caused by its ability to form stable complexes with Ca2+ and directly contribute to inner mitochondrial membrane permeabilization.

Mitochondria-mediated cardioprotection by trimetazidine in rabbit heart failure

Trimetazidine (TMZ) is used successfully for treatment of ischemic cardiomyopathy, however its therapeutic potential in heart failure (HF) remains to be established. While the cardioprotective action of TMZ has been linked to inhibition of free fatty acid oxidation (FAO) via 3-ketoacyl CoA thiolase (3-KAT), additional mechanisms have been suggested. The aim of this study was to evaluate systematically the effects of TMZ on calcium signaling and mitochondrial function in a rabbit model of non-ischemic HF and to determine the cellular mechanisms of the cardioprotective action of TMZ. TMZ protected HF ventricular myocytes from cytosolic Ca(2+) overload and subsequent hypercontracture, induced by electrical and ß-adrenergic (isoproterenol) stimulation. This effect was mediated by the ability of TMZ to protect HF myocytes against mitochondrial permeability transition pore (mPTP) opening via attenuation of reactive oxygen species (ROS) generation by the mitochondrial electron transport chain (ETC) and uncoupled mitochondrial nitric oxide synthase (mtNOS). The majority of ROS generated by the ETC in HF arose from enhanced complex II-mediated electron leak. TMZ inhibited the elevated electron leak at the level of mitochondrial ETC complex II and improved impaired activity of mitochondrial complex I, thereby restoring redox balance and mitochondrial membrane potential in HF. While TMZ decreased FAO by ~15%, the 3-KAT inhibitor 4-bromotiglic acid did not provide protection against palmitic acid-induced mPTP opening, indicating that TMZ effects were 3-KAT independent. Thus, the beneficial effect of TMZ in rabbit HF was not linked to FAO inhibition, but rather associated with reduced complex II- and uncoupled mtNOS-mediated oxidative stress and decreased propensity for mPTP opening.

Distinct mPTP activation mechanisms in ischaemia-reperfusion: contributions of Ca2+, ROS, pH, and inorganic polyphosphate.

AIMS The mitochondrial permeability transition pore (mPTP) plays a central role for tissue damage and cell death during ischaemia-reperfusion (I/R). We investigated the contribution of mitochondrial inorganic polyphosphate (polyP), a potent activator of Ca(2+)-induced mPTP opening, towards mPTP activation and cardiac cell death in I/R. METHODS AND RESULTS A significant increase in mitochondrial free calcium concentration ([Ca(2+)]m), reactive oxygen species (ROS) generation, mitochondrial membrane potential depolarization (ΔΨm), and mPTP activity, but no cell death, was observed after 20 min of ischaemia. The [Ca(2+)]m increase during ischaemia was partially prevented by the mitochondrial Ca(2+) uniporter (MCU) inhibitor Ru360 and completely abolished by the combination of Ru360 and the ryanodine receptor type 1 blocker dantrolene, suggesting two complimentary Ca(2+) uptake mechanisms. In the absence of Ru360 and dantrolene, mPTP closing by polyP depletion or CSA decreased mitochondrial Ca(2+) uptake, suggesting that during ischaemia Ca(2+) can enter mitochondria through mPTP. During reperfusion, a burst of endogenous polyP production coincided with a decrease in [Ca(2+)]m, a decline in superoxide generation, and an acceleration of hydrogen peroxide (H2O2) production. An increase in H2O2 correlated with restoration of mitochondrial pHm and an increase in cell death. mPTP opening and cell death on reperfusion were prevented by antioxidants Trolox and MnTBAP [Mn (III) tetrakis (4-benzoic acid) porphyrin chloride]. Enzymatic polyP depletion did not affect mPTP opening during reperfusion, but increased ROS generation and cell death, suggesting that polyP plays a protective role in cellular stress response. CONCLUSIONS Transient Ca(2+)/polyP-mediated mPTP opening during ischaemia may serve to protect cells against cytosolic Ca(2+) overload, whereas ROS/pH-mediated sustained mPTP opening on reperfusion induces cell death.

Inorganic polyphosphate--an unusual suspect of the mitochondrial permeability transition mystery.

Inorganic polyphosphate (polyP) is a naturally occurring polyanion made of ten to several hundred orthophosphates (Pi) linked together by phosphoanhydride bonds. PolyP is ubiquitously present in all organisms from bacteria to humans. Specific physiological roles of polyP vary dramatically depending on its size, concentration, tissue and subcellular localization. Recently we reported that mitochondria of ventricular myocytes contain significant amounts (280 ± 60 pmol/mg of protein) of polyP with an average length of 25 orthophosphates, and that polyP is involved in Ca2+-dependent activation of the mitochondrial permeability transition pore (mPTP). Here we extend our study to demonstrate the involvement of mitochondrial polyP in cardiac cell death. Furthermore, we show that polyP levels depend on the activity of the respiratory chain and are lower in myocytes from failing hearts. We conclude that polyP is a dynamically regulated macromolecule that plays an important role in mPTP-dependent cell death pathway.

Impact of Thoracic Surgery on Cardiac Morphology and Function in Small Animal Models of Heart Disease: A Cardiac MRI Study in Rats

Background Surgical procedures in small animal models of heart disease might evoke alterations in cardiac morphology and function. The aim of this study was to reveal and quantify such potential artificial early or long term effects in vivo, which might account for a significant bias in basic cardiovascular research, and, therefore, could potentially question the meaning of respective studies. Methods Female Wistar rats (n = 6 per group) were matched for weight and assorted for sham left coronary artery ligation or control. Cardiac morphology and function was then investigated in vivo by cine magnetic resonance imaging at 7 Tesla 1 and 8 weeks after the surgical procedure. The time course of metabolic and inflammatory blood parameters was determined in addition. Results Compared to healthy controls, rats after sham surgery showed a lower body weight both 1 week (267.5±10.6 vs. 317.0±11.3 g, n<0.05) and 8 weeks (317.0±21.1 vs. 358.7±22.4 g, n<0.05) after the intervention. Left and right ventricular morphology and function were not different in absolute measures in both groups 1 week after surgery. However, there was a confined difference in several cardiac parameters normalized to the body weight (bw), such as myocardial mass (2.19±0.30/0.83±0.13 vs. 1.85±0.22/0.70±0.07 mg left/right per g bw, p<0.05), or enddiastolic ventricular volume (1.31±0.36/1.21±0.31 vs. 1.14±0.20/1.07±0.17 µl left/right per g bw, p<0.05). Vice versa, after 8 weeks, cardiac masses, volumes, and output showed a trend for lower values in sham operated rats compared to controls in absolute measures (782.2±57.2/260.2±33.2 vs. 805.9±84.8/310.4±48.5 mg, p<0.05 for left/right ventricular mass), but not normalized to body weight. Matching these findings, blood testing revealed only minor inflammatory but prolonged metabolic changes after surgery not related to cardiac disease. Conclusion Cardio-thoracic surgical procedures in experimental myocardial infarction cause distinct alterations upon the global integrity of the organism, which in the long term also induce circumscribed repercussions on cardiac morphology and function. This impact has to be considered when analyzing data from respective animal studies and transferring these findings to conditions in patients.

Eya4 Induces Hypertrophy via Regulation of p27kip1

Background—E193, a heterozygous truncating mutation in the human transcription cofactor Eyes absent 4 (Eya4), causes hearing impairment followed by dilative cardiomyopathy. Methods and Results—In this study, we first show Eya4 and E193 alter the expression of p27kip1 in vitro, suggesting Eya4 is a negative regulator of p27. Next, we generated transgenic mice with cardiac-specific overexpression of Eya4 or E193. Luciferase and chromatin immunoprecipitation assays confirmed Eya4 and E193 bind and regulate p27 expression in a contradictory manner. Activity and phosphorylation status of the downstream molecules casein kinase-2&agr; and histone deacetylase 2 were significantly elevated in Eya4- but significantly reduced in E193-overexpressing animals compared with wild-type littermates. Magnetic resonance imaging and hemodynamic analysis indicate Eya4-overexpression results in an age-dependent development of hypertrophy already under baseline conditions with no obvious functional effects, whereas E193 animals develop onset of dilative cardiomyopathy as seen in human E193 patients. Both cardiac phenotypes were aggravated on pressure overload. Finally, we identified a new heterozygous truncating Eya4 mutation, E215, which leads to similar clinical features of disease and a stable myocardial expression of the mutant protein as seen with E193. Conclusions—Our results implicate Eya4/Six1 regulates normal cardiac function via p27/casein kinase-2&agr;/histone deacetylase 2 and indicate that mutations within this transcriptional complex and signaling cascade lead to the development of cardiomyopathy.

Inorganic Polyphosphates in the Mitochondria of Mammalian Cells

This review discusses the current literature on existence and critical roles of the inorganic polyphosphate (polyP) in the mitochondria of mammalian cells. Inorganic polyP is a linear polymer of orthophosphate (Pi) residues linked together by high-energy phosphoanhydride bonds as in ATP. While the length of polyP chain can vary from a few phosphates to several thousands phosphate units long, only short-chain polyPs are detected in mammalian mitochondria. Mitochondrial Ca2+ is an essential signaling molecule required for the activation of Ca2+-dependent dehydrogenases and energy production; however, in excess, it could also trigger cell death. PolyP affects mitochondrial Ca2+-transporting systems and mitochondrial metabolism in several ways: (i) it is a potent activator of Ca2+-dependent mitochondrial permeability transition pore (mPTP) and possibly even compose Ca2+-transporting core of the mPTP via formation of the poly-beta-hydroxybutyrate (PHB)-Ca2+-polyP complex in the inner mitochondrial membrane; (ii) reduction of polyP levels increases mitochondrial Ca2+-uptake capacity and decreases the probability of the mPTP opening, and (iii) it is a chelator of Ca2+, among other divalent ions, and therefore it can modify mitochondrial matrix Ca2+-buffering capacity. Furthermore, changes in polyP levels can modulate mitochondrial bioenergetics, generation of the mitochondrial membrane potential, and ATP production by the F0F1-ATPase, which can also affect mitochondrial Ca2+-uptake capacity. PolyP concentration is dynamically changed during activation of the mitochondrial respiratory chain and stress conditions such as ischemia-reperfusion and heart failure indicating that polyP is an important component of the normal cell metabolism.

29 Inorganic polyphosphate is a potent activator of the mitochondrial permeability transition pore in cardiac myocytes

Mitochondrial dysfunction caused by mitochondrial permeability transition pore opening (mPTP) and excessive calcium accumulation is a major contributor to cell and tissue damage during myocardial infarction and ischaemia-reperfusion injury. Although the role of calcium in induction of the mPTP opening is established, exact molecular mechanism of this process is not understood. We hypothesise that toxic effect of calcium accumulation is mediated by its interaction with inorganic polyphosphate, a biological polymer made of 10 to 100 orthophosphates. To test this hypothesis we investigated kinetics of the mPTP opening in permeabilised and intact cardiac cells depleted of polyphosphate under conditions of calcium overload. Polyphosphate depletion was achieved by targeted expression of specific polyphosphate hydrolysing enzyme using viral infection system. mPTP kinetics were estimated from the rates of TMRM, calcein red and X-rhod-1 release from mitochondria using confocal fluorescent microscopy approach. We found that although depletion of mitochondria of cardiac cells of polyphosphate did not affect their ability to accumulate calcium, it significantly inhibited mPTP opening. Inhibitory effect was observed in polyphosphate-depleted in experiments when calcium was added directly to mitochondria of permeabilised cells (80% decrease in rate of calcein red release, n=27; p<0.01 and 70% decrease in rate of X-Rhod-1 release n=17, p<0.01) as well as in experiments with intact cells when calcium overload was induced by creating conditions mimicking ischaemia-reperfusion injury (55% decrease in rate of calcein red release, n=19; p<0.05). Our findings suggest that inorganic polyphosphate is previously unrecognised major activator of mPTP during conditions of ischaemia-reperfusion injury.