Potential biomarkers for ischemic heart damage identified in mitochondrial proteins by comparative proteomics

We used proteomics to detect regional differences in protein expression levels from mitochondrial fractions of control, ischemia-reperfusion (IR), and ischemic preconditioned (IPC) rabbit hearts. Using 2-DE, we identified 25 mitochondrial proteins that were differentially expressed in the IR heart compared with the control and IPC hearts. For three of the spots, the expression patterns were confirmed by Western blotting analysis. These proteins included 3-hydroxybutyrate dehydrogenase, prohibitin, 2-oxoglutarate dehydrogenase, adenosine triphosphate synthases, the reduced form of nicotinamide adenine dinucleotide (NADH) oxidoreductase, translation elongation factor, actin alpha, malate dehydrogenase, NADH dehydrogenase, pyruvate dehydrogenase and the voltage-dependent anion channel. Interestingly, most of these proteins are associated with the mitochondrial respiratory chain and energy metabolism. The successful use of multiple techniques, including 2-DE, MALDI-TOF-MS and Western blotting analysis demonstrates that proteomic analysis provides appropriate means for identifying cardiac markers for detection of ischemia-induced cardiac injury.     

Combined blockade of the Na+ channel and the Na+/H+ exchanger virtually prevents ischemic Na+ overload in rat hearts

Blocking either the Na⁺ channel or the Na⁺/H⁺ exchanger (NHE) has been shown to reduce Na⁺ and Ca²⁺ overload during myocardial ischemia and reperfusion, respectively, and to improve post-ischemic contractile recovery. The effect of combined blockade of both Na⁺ influx routes on ionic homeostasis is unknown and was tested in this study. [Na⁺]_i, pH_i and energy-related phosphates were measured using simultaneous ²³Na- and ³¹P-NMR spectroscopy in isolated rat hearts. Eniporide (3 μM) and/or lidocaine (200 μM) were administered during 5 min prior to 40 min of global ischemia and 40 min of drug free reperfusion to block the NHE and the Na⁺ channel, respectively. Lidocaine reduced the rise in [Na⁺]_i during the first 10 min of ischemia, followed by a rise with a rate similar to the one found in untreated hearts. Eniporide reduced the ischemic Na⁺ influx during the entire ischemic period. Administration of both drugs resulted in a summation of the effects found in the lidocaine and eniporide groups. Contractile recovery and infarct size were significantly improved in hearts treated with both drugs, although not significantly different from hearts treated with either one of them.     

Fatty acid composition of mitochondrial phospholipids of the ischemic heart

Gas liquid chromatography was used to study fatty acid (FA) composition of lysophosphatidylcholine (lyso-PC) and phosphatidylcholine in isolated rabbit heart mitochondria. In control, lyso-PC and PC were found to contain 95 and 66% of unsaturated FA, respectively. At 1 hour of ischemia (autolysis at 37 degrees C) the percentage of saturated FA in lyso-PC noticeably increased whereas FA composition remained unchanged. It is concluded that changes in FA composition of lyso-PC are caused by phospholipase A2 action.     

The role of mitochondrial membrane potential in ischemic heart failure

The molecular events occurring during myocardial infarction and cardioprotection are described with an emphasis on the changes of the mitochondrial membrane potential (ΔΨ_m). The low ΔΨ_m values of the normal beating heart (100–140 mV) are explained by the allosteric ATP-inhibition of cytochrome c oxidase (CcO) through feedback inhibition by ATP at high [ATP]/[ADP] ratios. During ischemia the mechanism is reversibly switched off by signaling through reactive oxygen species (ROS). At reperfusion high ΔΨ_m values cause a burst of ROS production leading to apoptosis and/or necrosis. Ischemic preconditioning is suggested to cause additional phosphorylation of CcO, protecting the enzyme from immediate dephosphorylation via ROS signaling.     

Induction of Na+ / K+ exchange in swollen heart mitochondria

Heart mitochondria swollen passively in nitrate salts contract in a respiration-dependent reaction which can be attributed to an endogenous cation/H⁺ exchange component (or components). The rate of contraction increases with increased extent of passive swelling in both Na⁺ and K⁺ salts. Since nearly constant internal cation concentrations are maintained during osmotic swelling, this result suggests that both Na⁺/H⁺ and K⁺/H⁺ exchange is enhanced by increased matrix volume. Endogenous Mg²⁺ is also lost with increased matrix volume, and this observation, in conjunction with other evidence available in the literature, suggests that monovalent cation/H⁺ exchanges may be regulated by divalent cations. Passive exchange of Na⁺/K⁺,⁴²K⁺/K⁺, and²⁴Na⁺/Na⁺ can be readily demonstrated in mitochondria swollen in nitrate. All these exchanges are low or not detectable in unswollen control mitochondria, and it appears that they are manifestations of the activated cation/H⁺ component (or components) functioning in the absence of pH.     

Characterization of the mitochondrial Na+-H+ exchange. The effect of amiloride analogues

The kinetic properties and inhibitor sensitivity of the Na+-H+ exchange activity present in the inner membrane of rat heart and liver mitochondria were studied. (1) Na+-induced H+ efflux from mitochondria followed Michaelis-Menten kinetics. In heart mitochondria, the Km for Na+ was 24 +/- 4 mM and the Vmax was 4.5 +/- 1.4 nmol H+/mg protein per s (n = 6). Basically similar values were obtained in liver mitochondria (Km = 31 +/- 2 mM, Vmax = 5.3 +/- 0.2 nmol H+/mg protein per s, n = 4). (2) Li+ proved to be a substrate (Km = 5.9 mM, Vmax = 2.3 nmol H+/mg protein per s) and a potent competitive inhibitor with respect to Na+ (Ki approximately 0.7 mM). (3) External H+ inhibited the mitochondrial Na+-H+ exchange competitively. (4) Two benzamil derivatives of amiloride, 5-(N-4-chlorobenzyl)-N-(2',4'-dimethyl)benzamil and 3',5'-bis(trifluoromethyl)benzamil were effective inhibitors of the mitochondrial Na+-H+ exchange (50% inhibition was attained by approx. 60 microM in the presence of 15 mM Na+). (5) Three 5-amino analogues of amiloride, which are very strong Na+-H+ exchange blockers on the plasma membrane, exerted only weak inhibitory activity on the mitochondrial Na+-H+ exchange. (6) The results indicate that the mitochondrial and the plasma membrane antiporters represent distinct molecular entities.     

Role of Na+-K+-Cl- cotransport and Na+/Ca2+ exchange in mitochondrial dysfunction in astrocytes following in vitro ischemia

Na⁺-K⁺-Cl^– cotransporter isoform 1 (NKCC1) and reverse mode operation of the Na⁺/Ca²⁺ exchanger (NCX) contribute to intracellular Na⁺ and Ca²⁺ overload in astrocytes following oxygen-glucose deprivation (OGD) and reoxygenation (REOX). Here, we further investigated whether NKCC1 and NCX play a role in mitochondrial Ca²⁺ (Ca_m²⁺) overload and dysfunction. OGD/REOX caused a doubling of mitochondrial-releasable Ca²⁺ (P < 0.05). When NKCC1 was inhibited with bumetanide, the mitochondrial-releasable Ca²⁺ was reduced by 42% (P < 0.05). Genetic ablation of NKCC1 also reduced Ca_m²⁺ accumulation. Moreover, OGD/REOX in NKCC1^+/+ astrocytes caused dissipation of the mitochondrial membrane potential (_m) to 42 ± 3% of controls. In contrast, when NKCC1 was inhibited with bumetanide, depolarization of _m was attenuated significantly (66 ± 10% of controls, P < 0.05). Cells were also subjected to severe in vitro hypoxia by superfusion with a hypoxic, acidic, ion-shifted Ringer buffer (HAIR). HAIR/REOX triggered a secondary, sustained rise in intracellular Ca²⁺ that was attenuated by reversal NCX inhibitor KB-R7943. The hypoxia-mediated increase in Ca_m²⁺ was accompanied by loss of _m and cytochrome c release in NKCC1^+/+ astrocytes. Bumetanide or genetic ablation of NKCC1 attenuated mitochondrial dysfunction and astrocyte death following ischemia. Our study suggests that NKCC1 acting in concert with NCX causes a perturbation of Ca_m²⁺ homeostasis and mitochondrial dysfunction and cell death following in vitro ischemia. intracellular calcium ion; mitochondrial membrane potential; sodium ion influx; bumetanide; cytochrome c; glial cell death     

Physiological functions of mitochondrial Na+-Ca2+ exchanger,NCLX, in lymphocytes

Roles of mitochondrial Na⁺-Ca²⁺ exchanger, NCLX, were studied in B lymphocytes such as heterozygous NCLX knockout DT40 cells, NCLX knockdown A20 cells, and native mouse spleen B lymphocytes treated with a NCLX blocker, CGP-37157. Cytosolic Ca²⁺ response to B cell receptor stimulation was impaired in these B lymphocytes, demonstrating importance of mitochondria-ER Ca²⁺ recycling via NCLX and sarco/endoplasmic reticulum Ca²⁺-ATPase SERCA, and interaction with store-operated Ca²⁺ entry. NCLX was also associated with motility and chemotaxis of B lymphocyte. Contrary to B lymphocytes, contribution of NCLX in mouse spleen T lymphocytes was minor.     

Kappa-opioid receptor stimulation increases the expression of Na+-H+ exchange gene in the heart

Kappa-opioid receptor (OR) stimulation increases intracellular pH (pHi) via activating the Na+-H+ exchange (NHE). In the present study, we determined the expression of the gene of NHE1, the predominant NHE isoform in the heart, and intracellular pH (pHi) upon kappa-OR stimulation in the rat heart. We found that 1 microM U50,488H (trans-3,4-dichloro-N-methyl-N-(2-(1 pyrrolidinyl)cyclohexyl)benzeneacetamide), a selective kappa-OR agonist, increased the expression of the NHE1 gene. We also found that U50,488H dose-dependently increased pHi in the heart. The effects were abolished by 1 microM nor-binaltorphimine (nor-BNI), a selective kappa-OR antagonist, indicating that the events were kappa-OR mediated. The effects on both NHE1 gene expression and pHi were also abolished by 5 microM chelerythrine and 5 microM BSM (bisyndolylmaleimide), protein kinase C (PKC) inhibitors, indicating that PKC mediated the actions. In addition, the effect of U50,488H on pHi was blocked by 10 microM EIPA (ethylisopropyl amiloride), a NHE1 inhibitor, indicating that NHE1 also mediated the action of U50,488H. The present study provides evidence for the first time that kappa-OR stimulation increased the NHE1 gene expression in the heart via a PKC dependent pathway. Kappa-OR stimulation also increases pHi via PKC and NHE in the heart.     

Stimulation of Na+-Ca2+ exchange in heart sarcolemma by insulin

Insulin was found to stimulate Na+-dependent Ca2+ uptake in dog heart sarcolemma in a concentration dependent manner (0.001 to 1 milliunits/ml). Maximal stimulation (160 to 170%) was seen at 0.1 to 1 milliunits/ml of insulin. Unlike Na+-dependent Ca2+ uptake, ATP-dependent Ca2+ uptake was unaltered by 1 microunit/ml of insulin. However, high concentrations of insulin (0.01 to 1 milliunits/ml) significantly increased the ATP-dependent Ca2+ uptake activity of heart sarcolemma; maximal increase (60%) was observed at 1 milliunit/ml of insulin. The Na+ K+-ATPase activity did not change upon incubating sarcolemma with insulin. The membrane preparation exhibited specific insulin binding characteristics. The Scatchard plot analysis of the data indicated two binding sites for insulin; the association constants for the high and low affinity sites were 2 X 10(9) M-1 and 4.4 X 10(8) M-1, respectively. These results support the view regarding the presence of insulin receptors in the heart cell membrane and indicate a dramatic effect of insulin on the sarcolemmal Ca2+ transport systems.     

Detection of early ischemic damage by analysis of mitochondrial function in skinned fibers

The skinned fibers technique was applied for studies of the effects of global acute ischemia (1 h at 37°C) and long time (15 h) hypothermic (4°C) preservation of isolated rat hearts under different conditions (immersion or low-flow perfusion) on mitochondrial function in the cells in vivo. Skinned fibers were obtained by using saponin for permeabilization of the sarcolemma in separated fiber bundles cut from left ventricle. The experimental protocol of the respiration rate determination included a cytochrome c test to check the intactness of the outer mitochondrial membrane. The apparent K_m for ADP and the effect of creatine on the mitochondrial activity were also evaluated in these permeabilized fibers, taken from different groups of hearts. The preservation of low-flow perfused hearts resulted only in a slight decrease of creatine (20 mM) stimulated respiration at 0.1 mM ADP. The fibers from ischemic hearts or from hearts preserved by immersion showed a decrease of the apparent K_m for ADP, and a complete loss of the stimulatory effect of creatine. In these fibers, we could observe that the outer mitochondrial membrane was damaged. In conclusion, the results of this study show that assessment of mitochondrial parameters sensitive to organelles swelling – intactness of outer membrane and functionally coupled creatine kinase reaction – are the most sensitive indicators of early hypoxic or ischemic damage to mitochondria. Their determination in biopsy samples could be used for evaluation of the efficiency of the cardiac protection in heart surgery. (Mol Cell Biochem 174: 79–85, 1997)     

Effects of chronic caloric restriction on mitochondrial respiration in the ischemic reperfused rat heart

Dietary restriction increases life span and delays the development of age-related diseases in rodents. We have recently demonstrated that chronic dietary restriction is beneficial on recovery of heart function following ischemia. We studied whether the metabolic basis of this benefit is associated with alterations in mitochondrial respiration. Male Wistar rats were assigned to an ad libitum-fed (AL) group and a food restricted (FR) group, in which food intake was reduced to 55% of the amount consumed by the AL group. Following an 8-month period of restricted caloric intake, isolated working hearts perfused with glucose and high levels of fatty acids were subjected to global ischemia followed by reperfusion. At the end of reperfusion, total heart mitochondria was respiration was assessed in the presence of pyruvate, tricarboxylic acid intermediates, and palmitoylcarnitine. Recovery of heart function following ischemia was greater in FR hearts compared to AL hearts. Paralleling these changes in heart function was in increase in state 3 respiration with pyruvate. The respiratory control ratios in the presence of pyruvate and tricarboxylic acid intermediates were higher in FR hearts compared to AL hearts, indicating well-coupled mitochondria. Overall energy production, expressed as the ADP:O ratio and the oxidative phosphorylation rate, was also improved in FR hearts. Our results indicate that the beneficial effect of FR on recovery of heart function following ischemia is associated with changes in mitochondrial respiration.     

Na+ effects on mitochondrial respiration and oxidative phosphorylation in diabetic hearts

Intracellular Na+ is approximately two times higher in diabetic cardiomyocytes than in control. We hypothesized that the increase in Na+i activates the mitochondrial membrane Na+/Ca2+ exchanger, which leads to loss of intramitochondrial Ca2+, with a subsequent alteration (generally depression) in bioenergetic function. To further evaluate this hypothesis, mitochondria were isolated from hearts of control and streptozotocin-induced (4 weeks) diabetic rats. Respiratory function and ATP synthesis were studied using routine polarography and 31P-NMR methods, respectively. While addition of Na+ (1-10 mM) decreased State 3 respiration and rate of oxidative phosphorylation in both diabetic and control mitochondria, the decreases were significantly greater for diabetic than for control. The Na+ effect was reversed by providing different levels of extramitochondrial Ca2+ (larger Ca2+ levels were needed to reverse the Na+ depressant effect in diabetes mellitus than in control) and by inhibiting the Na+/Ca2+ exchanger function with diltiazem (a specific blocker of Na+/Ca2+ exchange that prevents Ca2+ from leaving the mitochondrial matrix). On the other hand, the Na+ depressant effect was enhanced by Ruthenium Red (RR, a blocker of mitochondrial Ca2+ uptake, which decreases intramitochondrial Ca2+). The RR effect on Na+ depression of mitochondrial bioenergetic function was larger in diabetic than control. These findings suggest that intramitochondrial Ca2+ levels could be lower in diabetic than control and that the Na+ depressant effect has some relation to lowered intramitochondrial Ca2+. Conjoint experiments with 31P-NMR in isolated superfused mitochondria embedded in agarose beads showed that Na+ (3-30 mM) led to significantly decreased ATP levels in diabetic rats, but produced smaller changes in control. These data support our hypothesis that in diabetic cardiomyocytes, increased Na+ leads to abnormalities of oxidative processes and subsequent decrease in ATP levels, and that these changes are related to Na+ induced depletion of intramitochondrial Ca2+.