Ivan A. Sammut

Targeting an antioxidant to mitochondria decreases cardiac ischemia-reperfusion injury

Mitochondrial oxidative damage contributes to a wide range of pathologies, including cardiovascular disorders and neurodegenerative diseases. Therefore, protecting mitochondria from oxidative damage should be an effective therapeutic strategy. However, conventional antioxidants have limited efficacy due to the difficulty of delivering them to mitochondria in situ. To overcome this problem, we developed mitochondria‐targeted antioxidants, typified by MitoQ, which comprises a lipophilic triphenylphosphonium (TPP) cation covalently attached to a ubiquinol antioxidant. Driven by the large mitochondrial membrane potential, the TPP cation concentrates MitoQ several hundred‐fold within mitochondria, selectively preventing mitochondrial oxidative damage. To test whether MitoQ was active in vivo, we chose a clinically relevant form of mitochondrial oxidative damage: cardiac ischemia‐reperfusion injury. Feeding MitoQ to rats significantly decreased heart dysfunction, cell death, and mitochondrial damage after ischemia‐reperfusion. This protection was due to the antioxidant activity of MitoQ within mitochondria, as an untargeted antioxidant was ineffective and accumulation of the TPP cation alone gave no protection. Therefore, targeting antioxidants to mitochondria in vivo is a promising new therapeutic strategy in the wide range of human diseases such as Parkinsons disease, diabetes, and Friedreichs ataxia where mitochondrial oxidative damage underlies the pathology. Adlam, V. J., Harrison, J. C., Porteous, C. M., James, A. M., Smith, R. A. J., Murphy, M. P., Sammut, I. A. Targeting an antioxidant to mitochondria decreases cardiac ischemia‐reperfusion injury. FASEB J. 19, 1088–1095 (2005)

Carbon monoxide is a major contributor to the regulation of vascular tone in aortas expressing high levels of haeme oxygenase-1

1 The contribution of haeme oxygenase‐derived carbon monoxide (CO) to the regulation of vascular tone in thoracic aorta was investigated following induction of the inducible isoform of haeme oxygenase (HO‐1). 2 Isometric smooth muscle contractions were recorded in isolated rat aortic ring preparations. Rings were incubated in the presence of the nitric oxide (NO) donor S‐nitroso‐N‐acetyl penicillamine (SNAP, 500 μm) for 1 h, then repetitively washed and maintained for a further 4 h prior to producing a concentration‐response curve to phenylephrine (PE, 1–3000 nm). 3 Treatment with SNAP resulted in increased mRNA and protein expression of aortic HO‐1 and was associated with a significant suppression of the contractile response to PE (P<0.05 vs control). Immunohistochemical staining procedures revealed marked HO‐1 expression in the endothelial layer and, to a lesser extent, in smooth muscle cells. 4 Induction of HO‐1 in SNAP‐treated rings was associated with a higher 14CO release compared to control, as measured by scintillation counting after incubation of aortas with [2‐14C]‐L‐glycine, the precursor of haeme. Guanosine 3′,5′‐monophosphate (cyclic GMP) content was also greatly enhanced in aortas expressing high levels of HO‐1. 5 Incubation of aortic rings with the NO synthase inhibitor, NG‐monomethyl‐L‐arginine (100 μm), significantly (P<0.05) increased the contractile response to PE in controls but failed to restore PE‐mediated contractility in SNAP‐treated rings. In contrast, the selective inhibitor of haeme oxygenase, tin protoporphyrin IX (SnPP‐IX, 10 μm), restored the pressor response to PE in SNAP‐treated rings whilst markedly reducing CO and cyclic GMP production. 6 We conclude that up‐regulation of the HO‐1/CO pathway significantly contributes to the suppression of aortic contractility to PE. This effect appears to be mediated by the elevation of cyclic GMP levels and can be reversed by inhibition of the haeme oxygenase pathway.

Neuroprotective effects of (–)-epigallocatechin gallate following hypoxia-ischemia-induced brain damage: novel mechanisms of action

(−)‐Epigallocatechin gallate (EGCG) is a potent antioxidant that is neuroprotective against ischemia‐induced brain damage. However, the neuroprotective effects and possible mechanisms of action of EGCG after hypoxia‐ischemia (HI) have not been investigated. Therefore, we used a modified “Levine” model of HI to determine the effects of EGCG. Wistar rats were treated with either 0.9% saline or 50 mg/kg EGCG daily for 1 day and 1 h before HI induction and for a further 2 days post‐HI. At 26‐days‐old, both groups underwent permanent left common carotid artery occlusion and exposure to 8% oxygen/92% nitrogen atmosphere for 1 h. Histological assessment showed that EGCG significantly reduced infarct volume (38.0±16.4 mm3) in comparison to HI + saline (99.6±15.6 mm3). In addition, EGCG significantly reduced total (622.6±85.8 pmol l‐[3H]citrulline/30 min/mg protein) and inducible nitric oxide synthase (iNOS) activity (143.2±77.3 pmol l‐[3H]citrulline/30 min/mg protein) in comparison to HI+saline controls (996.6±113.6 and 329.7±59.6 pmol l‐[3H]citrulline/30 min/mg protein for total NOS and iNOS activity, respectively). Western blot analysis demonstrated that iNOS protein expression was also reduced. In contrast, EGCG significantly increased endothelial and neuronal NOS protein expression compared with HI controls. EGCG also significantly preserved mitochondrial energetics (complex I‐V) and citrate synthase activity. This study demonstrates that the neuroprotective effects of EGCG are, in part, due to modulation of NOS isoforms and preservation of mitochondrial complex activity and integrity. We therefore conclude that the in vivo neuroprotective effects of EGCG are not exclusively due to its antioxidant effects but involve more complex signal transduction mechanisms.

Heat Stress Contributes to the Enhancement of Cardiac Mitochondrial Complex Activity

Hyperthermic stress is known to protect against myocardial dysfunction after ischemia-reperfusion injury. It is unclear however, what energetic mechanisms are affected by the molecular adaptation to heat stress. We hypothesized that mild hyperthermic stress can increase mitochondrial respiratory enzyme activity, affording protection to mitochondrial energetics during prolonged cardiac preservation for transplantation. Rat hearts were excised after heat-stress or sham treatment and subjected to cold cardioplegic arrest and ischemia followed by reperfusion in an ex vivo perfusion system. Cardiac function, mitochondrial respiratory, and complex activities were assessed before and after ischemia. Heat shock protein (Hsp 32, 60, and 72) expression was increased in heat-stressed hearts. This was associated with increased mitochondrial complex activities in heat-stress versus sham-treated groups for complex I-V. During reperfusion, higher complex activities and respiratory control ratios were observed in heat-stressed versus sham-treated groups. Recovery of ventricular function was improved in heat-stressed hearts. Furthermore, mitochondria in reperfused heat-stressed myocardium exhibited intact membranes with packed, parallel, lamellar cristae, whereas in sham-treated myocardium, mitochondria were severely disrupted. This study provides the first evidence of heat-stress-mediated enhancement of mitochondrial energetic capacity. This is associated with increased tolerance to ischemia-reperfusion injury. Protection by heat stress against myocardial dysfunction may be partially due to enhancement of mitochondrial energetics.

Neuroprotective effects of spermine following hypoxic-ischemic-induced brain damage: A mechanistic study

The polyamines (spermine, putrescine, and spermidine) can have neurotoxic or neuroprotective properties in models of neurodegeneration. However, assessment in a model of hypoxia– ischemia (HI) has not been defined. Furthermore, the putative mechanisms of neuroprotection have not been elucidated. Therefore, the present study examined the effects of the polyamines in a rat pup model of HI and determined effects on key enzymes involved in inflammation, namely, nitric oxide synthase (NOS) and arginase. In addition, effects on mitochondrial function were investigated. The polyamines or saline were administered i.p. at 10mg/kg/day for 6 days post‐HI. Histological assessment 7 days post‐HI revealed that only spermine significantly (P<0.01) reduced infarct size from 46.14 ± 10.4mm3 (HI + saline) to 4.9 ± 2.7 mm3. NOS activity was significantly increased following spermine treatment in the left (ligated) hemisphere compared with nonintervention controls (P<0.01) and HI + saline (P<0.05). In contrast, spermine decreased arginase activity compared with HI + saline but was still significantly elevated in comparison to nonintervention controls (P<0.01). Assessment of mitochondrial function in the HI + saline group, revealed significant and extensive damage to complex‐I (P<0.01) and IV (P<0.001) and loss of citrate synthase activity (P<0.05). No effect on complex II‐III was observed. Spermine treatment significantly prevented all these effects. This study has therefore confirmed the neuroprotective effects of spermine in vivo. However, for the first time, we have shown that this effect may, in part, be due to increased NOS activity and preservation of mitochondrial function.

Cardiac mitochondrial complex activity is enhanced by heat shock proteins

1. Prolonged ischaemia and reperfusion in heart transplantation results in mitochondrial dysfunction and loss of cardio‐energetics. Improved myocardial tolerance to ischaemia–reperfusion can be increased by de novo synthesis of heat shock protein (Hsp) groups, transiently expressed following mild hyperthermic or oxidative stress. Consideration of the roles of various Hsp in ischaemic–reperfused myocardium can provide new insights into potential therapeutic adjuncts to cardiac surgery.

Postnatal Development of the Complexes of the Electron Transport Chain in Synaptic Mitochondria from Rat Brain

The postnatal development of the complexes of the electron transport chain in mitochondria isolated from rat brain synaptosomes was investigated. Synaptosomal brain mitochondria were isolated from rats aged 10-60 days, and the activities of mitochondrial complex I, complex II-III, complex IV and complex V were measured. There was a significant increase in the activity of II-III from day 10 to day 15 and complex IV from day 10 to day 21, thereafter the activities of complexes I-III and IV did not change significantly. The activity of complex I did not change significantly during the period 10-60 days post partum. In synaptic mitochondria, complex V activity was higher than in non-synaptic mitochondria, whereas the activity of complex I was lower than in non-synaptic mitochondria. These data show that the complexes of the respiratory chain within synaptic mitochondria have activities different from those of non-synaptic mitochondria and may have major implications for the relative susceptibility of mitochondria in different brain cell types to neurotoxins such as MPP+, hypoxic/ischaemic damage and oxidative stress.

Time-dependent impairment of mitochondrial function after storage and transplantation of rabbit kidneys.

BACKGROUND The mitochondrial respiratory chain is implicated as a major target of kidney damage after ischemia-reperfusion. This study measures changes in integrated mitochondrial function and in the activity of enzymes of the respiratory chain after cold storage and transplantation-reperfusion in vivo. METHODS Mitochondrial oxygen consumption and activities of respiratory chain enzymes and citrate synthase were measured in cortical mitochondria isolated from rabbit kidneys after 1-48 hr of cold ischemia with or without transplantation-reperfusion. RESULTS State 4 mitochondrial oxygen consumption was significantly increased after 48 hr of ischemia or 24-48 hr of ischemia with transplantation. Prolonged (24 or 48 hr) ischemic storage with and without transplantation caused a significant decrease in state 3 oxygen consumption, as did transplantation after 1, 24, and 48 hr of cold storage. Complex I and complex II-III activity decreased after 24 or 48 hr of ischemia, with transplantation having little additional effect. Complex IV activity was significantly decreased after 48 hr of ischemia, this decrease being exacerbated by transplantation-reperfusion. Complex V activity decreased significantly after 1 hr of ischemia and continued to decrease after 24-48 hr of ischemia. Transplantation after 1-24 hr (but not 48 hr) of ischemia resulted in partial recovery of complex V activity. Citrate synthase activity was decreased significantly only after 48 hr of ischemia and reperfusion, consistent with the loss of mitochondrial membrane integrity seen in electron micrographs of the transplanted 48-hr group. CONCLUSIONS These data suggest that individual rabbit kidney mitochondrial complexes have different susceptibilities to cold ischemic and reperfusion damage.

Short-term heat acclimation is effective and may be enhanced rather than impaired by dehydration

Most heat acclimation data are from regimes longer than 1 week, and acclimation advice is to prevent dehydration. Objectives: We hypothesized that (i) short‐term (5‐day) heat acclimation would substantially improve physiological strain and exercise tolerance under heat stress, and (ii) dehydration would provide a thermally independent stimulus for adaptation. Methods: Nine aerobically fit males heat acclimated using controlled‐hyperthermia (rectal temperature 38.5°C) for 90 min on 5 days; once euhydrated (EUH) and once dehydrated (DEH) during acclimation bouts. Exercising heat stress tests (HSTs) were completed before and after acclimations (90‐min cycling in Ta 35°C, 60% RH). Results: During acclimation bouts, [aldosterone]plasma rose more across DEH than EUH (95%CI for difference between regimes: 40–411 pg ml−1; P = 0.03; n = 5) and was positively related to plasma volume expansion (r = 0.65; P = 0.05), which tended to be larger in DEH (CI: −1 to 10%; P = 0.06; n = 9). In HSTs, resting forearm perfusion increased more in DEH (by 5.9 ml 100 tissue ml−1 min−1: −11.5 to −1.0; P = 0.04) and end‐exercise cardiac frequency fell to a greater extent (by 11 b min−1: −1 to 22; P = 0.05). Hydration‐related effects on other endocrine, cardiovascular, and psychophysical responses to HSTs were unclear. Rectal temperature was unchanged at rest but was 0.3°C lower at end exercise (P < 0.01; interaction: P = 0.52). Conclusions: Short‐term (5‐day) heat acclimation induced effective adaptations, some of which were more pronounced after fluid‐regulatory strain from permissive dehydration, and not attributable to dehydration effects on body temperature. Am. J. Hum. Biol. 26:311–320, 2014.

Cell Damage Following Carbon Monoxide Releasing Molecule Exposure: Implications for Therapeutic Applications

The cytoprotective properties of carbon monoxide (CO) gas and CO‐releasing molecules (CORMs) are well established. Despite promising pre‐clinical results, little attention has been paid to the toxicological profile of CORMs. The effects of CORM‐2 and its CO‐depleted molecule (iCORM‐2) (20–400 μM) were compared in primary rat cardiomyocytes and two cell lines [human embryonic kidney (HeK) and Madine‐Darby canine kidney Cells (MDCK)]. Cells were assessed for cell viability, apoptosis, necrosis, cytology, mitochondrial energetics, oxidative stress and cell cycle arrest markers. In separate experiments, the anti‐apoptotic effects of CORM‐2 and i‐CORM‐2 treatment were compared against CO gas treatment in HeK and MDCK lines. H2O2‐induced cellular damage, measured by lactate dehydrogenase (LDH) release from primary cardiomyocytes, was reduced by 20 μM CORM‐2; LDH activity, however, was directly inhibited by 400 μM CORM‐2. Both CORM‐2/iCORM‐2 and CO gas decreased cisplatin‐induced caspase‐3 activity in MDCK and HeK cells suggesting an anti‐apoptotic effect. Conversely, both CORM‐2 and iCORM‐2 induced significant cellular toxicity in the form of decreased cell viability, abnormal cell cytology, increased apoptosis and necrosis, cell cycle arrest and reduced mitochondrial enzyme activity. Comparison of these markers after CO gas administration to MDCK cells found significantly less cellular toxicity than in 100 μM CORM‐2/iCORM‐2‐treated cells. CO gas did not have an adverse effect on mitochondrial energetics and integrity. Release of CO by low concentrations of intact CORM‐2 molecules provides cytoprotective effects. These results show, however, that the ruthenium‐based CORM by‐product, iCORM‐2, is cytotoxic and suggest that the accumulation of iCORM‐2 would seriously limit any clinical application of the ruthenium‐based CORMs.