Endurance training-induced cardiac remodeling in a guinea pig athlete's heart model.

Besides the health benefits of regular exercise, high-level training-above an optimal level-may have adverse effects. In this study, we investigated the effects of long-term vigorous training and its potentially detrimental structural-functional changes in a small animal athlete's heart model. Thirty-eight 4-month-old male guinea pigs were randomized into sedentary and exercised groups. The latter underwent a 15-week-long endurance-training program. To investigate the effects of the intense long-term exercise, in vivo (echocardiography, electrocardiography), ex vivo, and in vitro (histopathology, patch-clamp) measurements were performed. Following the training protocol, the exercised animals exhibited structural left ventricular enlargement and a significantly higher degree of myocardial fibrosis. Furthermore, resting bradycardia accompanied by elevated heart rate variability occurred, representing increased parasympathetic activity in the exercised hearts. The observed prolonged QTc intervals and increased repolarization variability parameters may raise the risk of electrical instability in exercised animals. Complex arrhythmias did not occur in either group, and there were no differences between the groups in ex vivo or cellular electrophysiological experiments. Accordingly, the high parasympathetic activity may promote impaired repolarization in conscious exercised animals. The detected structural-functional changes share similarities with the human athlete's heart; therefore, this model might be useful for investigations on cardiac remodeling.

Correction: Experimentally-Based Computational Investigation into Beat-To-Beat Variability in Ventricular Repolarization and Its Response to Ionic Current Inhibition.

[This corrects the article DOI: 10.1371/journal.pone.0151461.].

Experimentally-Based Computational Investigation into Beat-To-Beat Variability in Ventricular Repolarization and Its Response to Ionic Current Inhibition.

Beat-to-beat variability in repolarization (BVR) has been proposed as an arrhythmic risk marker for disease and pharmacological action. The mechanisms are unclear but BVR is thought to be a cell level manifestation of ion channel stochasticity, modulated by cell-to-cell differences in ionic conductances. In this study, we describe the construction of an experimentally-calibrated set of stochastic cardiac cell models that captures both BVR and cell-to-cell differences in BVR displayed in isolated canine action potential measurements using pharmacological agents. Simulated and experimental ranges of BVR are compared in control and under pharmacological inhibition, and the key ionic currents determining BVR under physiological and pharmacological conditions are identified. Results show that the 4-aminopyridine-sensitive transient outward potassium current, Ito1, is a fundamental driver of BVR in control and upon complete inhibition of the slow delayed rectifier potassium current, IKs. In contrast, IKs and the L-type calcium current, ICaL, become the major contributors to BVR upon inhibition of the fast delayed rectifier potassium current, IKr. This highlights both IKs and Ito1 as key contributors to repolarization reserve. Partial correlation analysis identifies the distribution of Ito1 channel numbers as an important independent determinant of the magnitude of BVR and drug-induced change in BVR in control and under pharmacological inhibition of ionic currents. Distributions in the number of IKs and ICaL channels only become independent determinants of the magnitude of BVR upon complete inhibition of IKr. These findings provide quantitative insights into the ionic causes of BVR as a marker for repolarization reserve, both under control condition and pharmacological inhibition.

ORM-10103, a novel specific inhibitor of the Na+/Ca2+ exchanger, decreases early and delayed afterdepolarizations in the canine heart.

BACKGROUND AND PURPOSE: At present there are no small molecule inhibitors that show strong selectivity for the Na(+) /Ca(2+) exchanger (NCX). Hence, we studied the electrophysiological effects of acute administration of ORM-10103, a new NCX inhibitor, on the NCX and L-type Ca(2+) currents and on the formation of early and delayed afterdepolarizations. EXPERIMENTAL APPROACH: Ion currents were recorded by using a voltage clamp technique in canine single ventricular cells, and action potentials were obtained from canine and guinea pig ventricular preparations with the use of microelectrodes. KEY RESULTS: ORM-10103 significantly reduced both the inward and outward NCX currents. Even at a high concentration (10 µM), ORM-10103 did not significantly change the L-type Ca(2+) current or the maximum rate of depolarization (dV/dtmax ), indicative of the fast inward Na(+) current. At 10 µM ORM-10103 did not affect the amplitude or the dV/dtmax of the slow response action potentials recorded from guinea pig papillary muscles, which suggests it had no effect on the L-type Ca(2+) current. ORM-10103 did not influence the Na(+) /K(+) pump or the main K(+) currents of canine ventricular myocytes, except the rapid delayed rectifier K(+) current, which was slightly diminished by the drug at 3 µM. The amplitudes of pharmacologically- induced early and delayed afterdepolarizations were significantly decreased by ORM-10103 (3 and 10 µM) in a concentration-dependent manner. CONCLUSIONS AND IMPLICATIONS: ORM-10103 is a selective inhibitor of the NCX current and can abolish triggered arrhythmias. Hence, it has the potential to be used to prevent arrhythmogenic events.

Oral choline supplementation for postoperative pain.

BACKGROUND: Activation of nicotinic receptors with nicotine has been shown to reduce post-surgical pain in clinical and preclinical studies. Choline is a selective agonist at α7-type nicotinic receptors that does not have addictive or sympathetic activating properties. It is anti-nociceptive in animal studies. We conducted a double-blind randomized trial of oral choline supplementation with lecithin to aid in the treatment of pain after gynaecological surgery. METHODS: Sixty women having open gynaecological surgery were randomly assigned to receive 20 g of lecithin before surgery or placebo. Plasma choline concentration and tumour necrosis factor (TNF) were measured. Pain report was the primary outcome measure. RESULTS: We achieved a small but statistically significant increase in choline after surgery with oral supplementation. Plasma TNF was not decreased and pain report was not different between groups at rest or with movement. There were no adverse effects of treatment. CONCLUSIONS: Oral supplementation with lecithin during the perioperative period resulted in very slow absorption and thus only a small increase in plasma choline was achieved. This concentration was inadequate to reduce TNF as has been shown in other studies. The absence of an anti-inflammatory effect was likely related to our failure to demonstrate efficacy in pain reduction.

Biochemomechanics of intraluminal thrombus in abdominal aortic aneurysms.

Most computational models of abdominal aortic aneurysms address either the hemodynamics within the lesion or the mechanics of the wall. More recently, however, some models have appropriately begun to account for the evolving mechanics of the wall in response to the changing hemodynamic loads. Collectively, this large body of work has provided tremendous insight into this life-threatening condition and has provided important guidance for current research. Nevertheless, there has yet to be a comprehensive model that addresses the mechanobiology, biochemistry, and biomechanics of thrombus-laden abdominal aortic aneurysms. That is, there is a pressing need to include effects of the hemodynamics on both the development of the nearly ubiquitous intraluminal thrombus and the evolving mechanics of the wall, which depends in part on biochemical effects of the adjacent thrombus. Indeed, there is increasing evidence that intraluminal thrombus in abdominal aortic aneurysms is biologically active and should not be treated as homogeneous inert material. In this review paper, we bring together diverse findings from the literature to encourage next generation models that account for the biochemomechanics of growth and remodeling in patient-specific, thrombus-laden abdominal aortic aneurysms.

[Invasive pulmonary aspergillosis and esophageal candidiasis in a patient with decompensated liver cirrhosis due to chronic hepatitis C and alcohol]. / Invazívna pl'úcna aspergilóza a kandidová ezofagitída u pacienta s dekompenzáciou cirhózy pecene na podklade chronickej hepatitídy C a alkoholu.

We present a rare case of combined fungal infection in a critically ill 47 year-old patient with chronic hepatitis C at the stage of liver cirrhosis. The patient was admitted for signs of decompensated cirrhosis caused by hepatitis C and increased alcohol consumption. After 2 week hospital stay, his condition was complicated by a pulmonary infiltrate and rapid deterioration followed. Despite intensive care, the patient died. Autopsy findings showed invasive pulmonary aspergillosis. The aim of this case report is to point to a broad differential diagnosis of jaundice and pulmonary infiltrates, thus stressing the value of interdisciplinary cooperation and the need to consider the possibility of invasive fungal infections when caring for liver cirrhosis patients. In addition, several risk factors contributing to the development of fungal diseases in these patients are discussed in the article.

Atrial remodeling and novel pharmacological strategies for antiarrhythmic therapy in atrial fibrillation.

Atrial fibrillation (AF) is the most common arrhythmia in clinical practice. It can occur at any age, however, it becomes extremely common in the elderly, with a prevalence approaching more than 20% in patients older than 85 years. AF is associated with a wide range of cardiac and extra-cardiac complications and thereby contributes significantly to morbidity and mortality. Present therapeutic approaches to AF have major limitations, which have inspired substantial efforts to improve our understanding of the mechanisms underlying AF, with the premise that improved knowledge will lead to innovative and improved therapeutic approaches. Our understanding of AF pathophysiology has advanced significantly over the past 10 to 15 years through an increased awareness of the role of "atrial remodeling". Any persistent change in atrial structure or function constitutes atrial remodeling. Both rapid ectopic firing and reentry can maintain AF. Atrial remodeling has the potential to increase the likelihood of ectopic or reentrant activity through a multitude of potential mechanisms. The present paper reviews the main novel results on atrial tachycardia-induced electrical, structural and contractile remodeling focusing on the underlying pathophysiological and molecular basis of their occurrence. Special attention is paid to novel strategies and targets with therapeutic significance for atrial fibrillation.

Mechanism of reverse rate-dependent action of cardioactive agents.

Class 3 antiarrhythmic agents exhibit reverse rate-dependent lengthening of the action potential duration (APD), i.e. changes in APD are greater at longer than at shorter cycle lengths. In spite of the several theories developed to explain this reverse rate-dependency, its mechanism has been clarified only recently. The aim of the present study is to elucidate the mechanisms responsible for reverse rate-dependency in mammalian ventricular myocardium. Action potentials were recorded using conventional sharp microelectrodes from human, canine, rabbit, guinea pig, and rat ventricular myocardium in a rate-dependent manner. Rate-dependent drug-effects of various origin were studied using agents known to lengthen or shorten action potentials allowing thus to determine the drug-induced changes in APD as a function of the cycle length. Both drug-induced lengthening and shortening of action potentials displayed reverse rate-dependency in human, canine, and guinea pig preparations, but not in rabbit and rat myocardium. Similar results were obtained when repolarization was modified by injection of inward or outward current pulses in isolated canine cardiomyocytes. In contrast to reverse rate-dependence, drug-induced changes in APD well correlated with baseline APD values (i.e. that measured before the superfusion of drug or injection of current) in all of the preparations studied. Since the net membrane current (I(net)), determined from the action potential waveform at the middle of the plateau, was inversely proportional to APD, and consequently to cycle length, it is concluded that that reverse rate-dependency may simply reflect the inverse relationship linking I(net) to APD. In summary, reverse rate-dependency is an intrinsic property of drug action in the hearts of species showing positive APD - cycle length relationship, including humans. This implies that development of a pure K(+) channel blocking agent without reverse rate-dependent effects is not likely to be successful.

Analysis of the contribution of I(to) to repolarization in canine ventricular myocardium.

BACKGROUND AND PURPOSE: The contribution of the transient outward potassium current (I(to)) to ventricular repolarization is controversial as it depends on the experimental conditions, the region of myocardium and the species studied. The aim of the present study was therefore to characterize I(to) and estimate its contribution to repolarization reserve in canine ventricular myocardium. EXPERIMENTAL APPROACH: Ion currents were recorded using conventional whole-cell voltage clamp and action potential voltage clamp techniques in canine isolated ventricular cells. Action potentials were recorded from canine ventricular preparations using microelectrodes. The contribution of I(to) to repolarization was studied using 100 µM chromanol 293B in the presence of 0.5 µM HMR 1556, which fully blocks I(Ks). KEY RESULTS: The high concentration of chromanol 293B used effectively suppressed I(to) without affecting other repolarizing K(+) currents (I(K1), I(Kr), I(p)). Action potential clamp experiments revealed a slowly inactivating and a 'late' chromanol-sensitive current component occurring during the action potential plateau. Action potentials were significantly lengthened by chromanol 293B in the presence of HMR 1556. This lengthening effect induced by I(to) inhibition was found to be reverse rate-dependent. It was significantly augmented after additional attenuation of repolarization reserve by 0.1 µM dofetilide and this caused the occurrence of early afterdepolarizations. The results were confirmed by computer simulation. CONCLUSIONS AND IMPLICATIONS: The results indicate that I(to) is involved in regulating repolarization in canine ventricular myocardium and that it contributes significantly to the repolarization reserve. Therefore, blockade of I(to) may enhance pro-arrhythmic risk.

Role of slow delayed rectifier K+-current in QT prolongation in the alloxan-induced diabetic rabbit heart.

AIM: In diabetes mellitus, several cardiac electrophysiological parameters are known to be affected. In rodent experimental diabetes models, changes in these parameters were reported, but only limited relevant information is available in other species, having cardiac electrophysiological properties more resembling the human, including the rabbit. The present study was designed to analyse the effects of experimental type 1 diabetes on ventricular repolarization and the underlying transmembrane potassium currents in rabbit hearts. METHODS: Diabetes was induced by a single injection of alloxan (145 mg kg(-1) i.v.). After the development of diabetes (3 weeks), electrophysiological studies were performed using whole cell voltage clamp and ECG measurements. RESULTS: The QT(c) interval in diabetic rabbits was moderately but statistically significantly longer than measured in the control animals (155 +/- 1.8 ms vs. 145 +/- 2.8 ms, respectively, n = 9-10, P < 0.05). This QT(c)-lengthening effect of diabetes was accompanied by a significant reduction in the density of the slow delayed rectifier K(+) current, I(Ks) (from 1.48 +/- 0.35 to 0.86 +/- 0.17 pA pF(-1) at +50 mV, n = 19-21, P < 0.05) without changes in current kinetics. No differences were observed either in the density or in the kinetics of the inward rectifier K(+) current (I(K1)), the rapid delayed rectifier K(+) current (I(Kr)), the transient outward current (I(to)) and the L-type calcium current (I(CaL)) between the control and alloxan-treated rabbits. CONCLUSION: It is concluded that type 1 diabetes mellitus, although only moderately, lengthens ventricular repolarization. Diabetes attenuates the repolarization reserve by decreasing the density of I(Ks) current, and thereby may enhance the risk of sudden cardiac death.

Pathophysiologic role of oxidative stress-induced poly(ADP-ribose) polymerase-1 activation: focus on cell death and transcriptional regulation.

PARP-1 is a nuclear enzyme activated by DNA breaks. Activated PARP-1 cleaves NAD into nicotinamide and ADP-ribose and polymerizes the latter covalently coupled to nuclear acceptor proteins. Poly(ADP-ribosyl)ation has been implicated in the regulation of a diverse array of cellular processes ranging from DNA repair, chromatin organization, transcription, replication to protein degradation. On the 'dark side' of poly(ADP-ribosyl)ation, PARP-1 activation has been shown to contribute to tissue injury in shock, diabetes, myocardial or cerebral ischemia reperfusion and various forms of inflammation, as proven by pharmacological studies as well as experiments utilizing PARP-1 knockout animals. To our current knowledge, two mechanisms are responsible for the beneficial effects of PARP inhibitors in inflammatory, neurodegenerative and ischemia-reperfusion-based diseases: (i) inhibition of cell death caused by over-activation of PARP-1; (ii) inhibition of inflammatory signal transduction and production of inflammatory mediators. Here we review the possible regulatory mechanisms (e.g. calcium signaling, metabolism, density-dependent signaling, kinase cascades) of the PARP-1-mediated cell death pathway and discuss recent developments shedding new light on the complex role of PARP-1 in the regulation of the expression of inflammatory mediators.

Effect of the antifibrillatory compound tedisamil (KC-8857) on transmembrane currents in mammalian ventricular myocytes.

The cellular mechanism of action of tedisamil (KC-8857) (TED), a novel antiarrhythmic/antifibrillatory compound, was studied on transmembrane currents in guinea pig, rabbit and dog ventricular myocytes by applying the patch-clamp and the conventional microelectrode technique. In guinea pig myocytes the rapid component of the delayed rectifier potassium current (IKr) was largely diminished by 1 microM TED (from 0.88+/-0.17 to 0.23+/-0.07 pA/pF, n=5, p<0.05), while its slow component (IKs) was reduced only by 5 microM TED (from 8.1+/-0.3 to 4.23+/-0.07 pA/pF, n=5, p<0.05). TED did not significantly change the IKr and IKs kinetics. In rabbit myocytes 1 microM TED decreased the amplitude of the transient outward current (I(to)) from 20.3+/-4.9 to 13.9+/-2.8 pA/pF (n=5, p<0.05), accelerated its fast inactivation time constant from 8.3+/-0.6 to 3.5+/-0.5 ms (n=5, p<0.05) and reduced the ATP-activated potassium current (IKATP) from 38.2+/-11.8 to 18.4+/-4.7 pA/pF (activator: 50 microM cromakalim; n=5, p<0.05). In dog myocytes 2 microM TED blocked the fast sodium current (INa) with rapid onset and moderately slow offset kinetics, while the inward rectifier potassium (IK1), the inward calcium (ICa) and even the I(to) currents were not affected by TED in concentration as high as 10 microM. The differences in I(to) responsiveness between dog and rabbit are probably due to the different alpha-subunits of I(to) in these species. It is concluded that inhibition of several transmembrane currents, including IKr, IKs, I(to), IKATP and even INa, can contribute to the high antiarrhythmic/antifibrillatory potency of TED, underlying predominant Class III combined with I A/B type antiarrhythmic characteristics.

The cellular electrophysiologic effect of a new amiodarone like antiarrhythmic drug GYKI 16638 in undiseased human ventricular muscle: comparison with sotalol and mexiletine.

The cellular electrophysiologic effect of GYKI 16638, a new antiarrhythmic compound was studied and compared with that of sotalol and mexiletine in undiseased human right ventricular muscle preparation by applying the conventional microelectrode technique. GYKI 16638 (5 microM), at stimulation cycle length of 1000 ms, lengthened action potential duration (APD(90)) from 338.9 +/- 28.6 ms to 385.4 +/- 24 ms (n = 9, p > 0.05). This APD lengthening effect, unlike that of sotalol (30 microM), was rate-independent. GYKI 16638, contrary to sotalol and like mexiletine (10 microM), exerted a use-dependent depression of the maximal rate of depolarization (V(max)) which amounted to 36.4 +/- 11.7% at cycle length of 400 ms (n = 5, p < 0.05) and was characterised with an offset kinetical time constant of 298.6 +/- 70.2 ms. It was concluded that GYKI 16638 in human ventricular muscle shows combined Class IB and Class III antiarrhythmic properties, resembling the electrophysiological manifestation seen after chronic amiodarone treatment.

Activation of poly(ADP-ribose) polymerase contributes to development of doxorubicin-induced heart failure.

Activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) by oxidant-mediated DNA damage is an important pathway of cell dysfunction and tissue injury in conditions associated with oxidative stress. Increased oxidative stress is a major factor implicated in the cardiotoxicity of doxorubicin (DOX), a widely used antitumor anthracycline antibiotic. Thus, we hypothesized that the activation of PARP may contribute to the DOX-induced cardiotoxicity. Using a dual approach of PARP-1 suppression, by genetic deletion or pharmacological inhibition with the phenanthridinone PARP inhibitor PJ34, we now demonstrate the role of PARP in the development of cardiac dysfunction induced by DOX. PARP-1+/+ and PARP-1-/- mice received a single injection of DOX (25 mg/kg i.p). Five days after DOX administration, left ventricular performance was significantly depressed in PARP-1+/+ mice, but only to a smaller extent in PARP-1-/- ones. Similar experiments were conducted in BALB/c mice treated with PJ34 or vehicle. Treatment with a PJ34 significantly improved cardiac dysfunction and increased the survival of the animals. In addition PJ34 significantly reduced the DOX-induced increase in the serum lactate dehydrogenase and creatine kinase activities but not metalloproteinase activation in the heart. Thus, PARP activation contributes to the cardiotoxicity of DOX. PARP inhibitors may exert protective effects against the development of severe cardiac complications associated with the DOX treatment.

Partial protection by poly(ADP-ribose) polymerase inhibitors from nitroxyl-induced cytotoxity in thymocytes.

Nitroxyl (NO(-)/HNO), has been proposed to be one of the NO(*)-derived cytotoxic species. Although the biological effect of nitroxyl is largely unknown, it has been reported to cause DNA breakage and cytotoxicity. We have therefore investigated whether NO(-)/HNO-induced DNA single-strand breakage activates the nuclear nick sensor enzyme poly(ADP-ribose) polymerase (PARP) and whether PARP activation affects the mode of NO(-)/HNO- induced cell death. NO(-)/HNO generated from Angeli's salt (AS, sodium trioxodinitrate) (0-300 microM) induced DNA single-strand breakage, PARP activation, and a concentration-dependent cytotoxicity in murine thymocytes. AS-induced cell death was also accompanied by decreased mitochondrial membrane potential and increased secondary superoxide production. The cytotoxicity of AS, as measured by propidium iodide uptake, was abolished by electron acceptors potassium ferricyanide, TEMPOL, the intracellular calcium chelator BAPTA-AM, and by PARP inhibitors 3-aminobenzamide (3-AB) and PJ-34. The cytoprotective effect of 3-AB was paralleled by increased output of AS-induced apoptotic parameters such as phosphatidylserine exposure, caspase activation, and DNA fragmentation. No significant increase in tyrosine nitration could be observed in AS-treated thymocytes as opposed to peroxynitrite-treated cells, indicating that tyrosine nitration is not likely to contribute to NO(-)/HNO-induced cytotoxicity. Our results demonstrate that NO(-)/HNO-induced PARP activation shifts the default apoptotic cell death toward necrosis in thymocytes. However, as total PARP inhibition resulted only in 30% cytoprotection, PARP-independent mechanisms dominate NO(-)/HNO-induced cytotoxicity in thymocytes.

Dexmedetomidine decreases extracellular dopamine concentrations in the rat nucleus accumbens.

The effects of dexmedetomidine, a highly selective alpha(2)-adrenoceptor agonist, on extracellular dopamine (DA) concentrations in the nucleus accumbens of awake rats were collected via in vivo cerebral microdialysis and measured using HPLC with electrochemical detection. The administration of dexmedetomidine (DEX) at a low dose (2 microg/kg bolus i.v. over 2 min followed by a continuous infusion of 0.1 microg/kg per min) and a high dose (20 microg/kg bolus i.v. over 2 min followed by a continuous infusion of 1 microg/kg per min), significantly decreased extracellular dopamine concentrations in the nucleus accumbens. The observed decrease was dose-dependent, occurring sooner and to a greater magnitude in the rats receiving a high dose of DEX. This inhibitory modulation of accumbal dopamine was receptor-specific, as the decrease in extracellular DA produced by DEX was no longer evident following pre-treatment and co-infusion with the highly selective alpha(2)-adrenoceptor antagonist, atipamezole (ATZ). Thus, these data suggest that adrenoceptor agonists and antagonists may modulate dopaminergic neurotransmission via mechanisms that are specific to the alpha(2)-adrenoceptor.

Suppression of poly (ADP-ribose) polymerase activation by 3-aminobenzamide in a rat model of myocardial infarction: long-term morphological and functional consequences.

1. Recent studies demonstrated that inhibition or genetic inactivation of the enzyme poly (ADP-ribose) polymerase (PARP) is beneficial in myocardial reperfusion injury. PARP activation in the reperfused myocardium has been assumed, but not directly demonstrated. Furthermore, the issue whether pharmacological PARP inhibition affords long-term functional benefit in the reperfused myocardium has not been explored. These questions were addressed in the present study. 2. In a rat model of myocardial ischemia (1 h) and reperfusion (up to 24 h), there was a marked and significant activation of PARP in the ischemic borderzone, as determined by poly(ADP-ribose) (PAR) immunohistochemistry. PAR localized to the nuclei of myocytes and infiltrating mononuclear cells. In the core of the infarction, necrotic tissues and diffuse PAR staining were observed. PARP activation remained markedly detectable 24 h after reperfusion. The PARP inhibitor 3-aminobenzamide (20 mg kg(-1) intraperitoneally 10 min before reperfusion, and every 2 h thereafter for 6 h) markedly reduced the activation of the enzyme in myocytes. 3. 3-aminobenzamide significantly protected against myocardial morphological and functional alterations at 24 h post-reperfusion. Notably, infarct size was reduced, circulating creatine kinase activity was attenuated, and myocardial contractility (dP dt(-1)) was restored by 3-aminobenzamide. 4. Our results demonstrate a significant and prolonged activation of PARP in the reperfused myocardium, localizing to the necrotic area and the ischaemic borderzone. Furthermore, the studies demonstrate that PARP inhibition affords long-term beneficial morphological and functional effects in the reperfused myocardium. These data strengthen the notion that pharmacological PARP inhibition is a viable novel experimental approach for protection against myocardial reperfusion injury.