(melle-4)cyclosporin and Disease-Models--Animal

The opening of the mitochondrial permeability transition pore (PTP), which is regulated by the matrix protein cyclophilin D (CypD), plays a key role in the pathophysiology of post-cardiac arrest (CA) syndrome. We hypothesized that therapeutic hypothermia could prevent post-CA syndrome through a CypD-mediated PTP inhibition in both heart and brain. In addition, we investigated whether specific pharmacological PTP inhibition would confer additive protection to cooling. Adult male New Zealand White rabbits underwent 15 min of CA followed by 120 min of reperfusion. Five groups (n = 10-15/group) were studied: control group (CA only), hypothermia group (HT, hypothermia at 32-34 °C induced by external cooling at reperfusion), NIM group (injection at reperfusion of 2.5 mg/kg NIM811, a specific CypD inhibitor), HT + NIM, and sham group. The following measurements were taken: hemodynamics, echocardiography, and cellular damage markers (including S100β protein and troponin Ic). Oxidative phosphorylation and PTP opening were assessed on mitochondria isolated from both brain and heart. Acetylation of CypD was measured by immunoprecipitation in both the cerebral cortex and myocardium. Hypothermia and NIM811 significantly prevented cardiovascular dysfunction, pupillary areflexia, and early tissue damage. Hypothermia and NIM811 preserved oxidative phosphorylation, limited PTP opening in both brain and heart mitochondria and prevented increase in CypD acetylation in brain. There were no additive beneficial effects in the combination of NIM811 and therapeutic hypothermia. In conclusion, therapeutic hypothermia limited post-CA syndrome by preventing mitochondrial permeability transition mainly through a CypD-dependent mechanism.

Topics: Animals; Brain; Brain Diseases; Cardiopulmonary Resuscitation; Combined Modality Therapy; Cyclophilins; Cyclosporine; Disease Models, Animal; Enzyme Inhibitors; Heart Arrest; Hemodynamics; Hypothermia, Induced; Male; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocardium; Oxidative Phosphorylation; Peptidyl-Prolyl Isomerase F; Rabbits; Signal Transduction; Syndrome

Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) are inherited muscle diseases due to mutations in the genes encoding the extracellular matrix protein collagen (Col) VI. Opening of the cyclosporin A-sensitive mitochondrial permeability transition pore (PTP) is a causative event in disease pathogenesis, and a potential target for therapy. Here, we have tested the effect of N-methyl-4-isoleucine-cyclosporin (NIM811), a non-immunosuppressive cyclophilin inhibitor, in a zebrafish model of ColVI myopathy obtained by deletion of the N-terminal region of the ColVI α1 triple helical domain, a common mutation of UCMD. Treatment with antisense morpholino sequences targeting col6a1 exon 9 at the 1-4 cell stage (within 1 h post fertilization, hpf) caused severe ultrastructural and motor abnormalities as assessed by electron and fluorescence microscopy, birefringence, spontaneous coiling events and touch-evoked responses measured at 24-48 hpf. Structural and functional abnormalities were largely prevented when NIM811--which proved significantly more effective than cyclosporin A--was administered at 21 hpf, while FK506 was ineffective. Beneficial effects of NIM811 were also detected (i) in primary muscle-derived cell cultures from UCMD and BM patients, where the typical mitochondrial alterations and depolarizing response to rotenone and oligomycin were significantly reduced; and (ii) in the Col6a1(-/-) myopathic mouse model, where apoptosis was prevented and muscle strength was increased. Since the PTP of zebrafish shares its key regulatory features with the mammalian pore, our results suggest that early treatment with NIM811 should be tested as a potential therapy for UCMD and BM.

Topics: Animals; Apoptosis; Cells, Cultured; Collagen Type VI; Cyclosporine; Disease Models, Animal; Humans; Mice; Mitochondria; Muscle Strength; Muscular Dystrophies; Zebrafish; Zebrafish Proteins

Operation on the infrarenal aorta and large arteries of the lower extremities may cause rhabdomyolysis of the skeletal muscle, which in turn may induce remote kidney injury. NIM-811 (N-metyl-4-isoleucine-cyclosporine) is a mitochondria specific drug, which can prevent ischemic-reperfusion (IR) injury, by inhibiting mitochondrial permeability transition pores (mPTP).. Our aim was to reduce damages in the skeletal muscle and the kidney after IR of the lower limb with NIM-811.. Wistar rats underwent 180 minutes of bilateral lower limb ischemia and 240 minutes of reperfusion. Four animal groups were formed called Sham (receiving vehicle and sham surgery), NIM-Sham (receiving NIM-811 and sham surgery), IR (receiving vehicle and surgery), and NIM-IR (receiving NIM-811 and surgery). Serum, urine and histological samples were taken at the end of reperfusion. NADH-tetrazolium staining, muscle Wet/Dry (W/D) ratio calculations, laser Doppler-flowmetry (LDF) and mean arterial pressure (MAP) monitoring were performed. Renal peroxynitrite concentration, serum TNF-α and IL-6 levels were measured.. Less significant histopathological changes were observable in the NIM-IR group as compared with the IR group. Serum K+ and necroenzyme levels were significantly lower in the NIM-IR group than in the IR group (LDH: p<0.001; CK: p<0.001; K+: p = 0.017). Muscle mitochondrial viability proved to be significantly higher (p = 0.001) and renal function parameters were significantly better (creatinine: p = 0.016; FENa: p<0.001) in the NIM-IR group in comparison to the IR group. Serum TNF-α and IL-6 levels were significantly lower (TNF-α: p = 0.003, IL-6: p = 0.040) as well as W/D ratio and peroxynitrite concentration were significantly lower (p = 0.014; p<0.001) in the NIM-IR group than in the IR group.. NIM-811 could have the potential of reducing rhabdomyolysis and impairment of the kidney after lower limb IR injury.

Topics: Acute Kidney Injury; Animals; Biomarkers; Cell Survival; Cyclosporine; Disease Models, Animal; Hemodynamics; Interleukin-6; Kidney Function Tests; Lower Extremity; Male; Microcirculation; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Muscle Fibers, Skeletal; Rats; Reperfusion Injury; Rhabdomyolysis; Tumor Necrosis Factor-alpha

Ischemic postconditioning (IPoC) modulates the reperfusion maneuver to mitigate ischemia-reperfusion (I/R) injury. This study aims to investigate the effects and protective mechanism of IPoC on intestinal I/R injury.. Intestinal I/R was induced by occluding the superior mesenteric artery for 30 min followed by reperfusion for 60 min on male Wistar rats. IPoC was elicited by three cycles of 30-sec reperfusion and reocclusion of superior mesenteric artery at the initiation of reperfusion. Carboxyatractyloside (CATR), a mitochondrial permeability transition pore (mPTP) opener, and N-methyl-4-isoleucine cyclosporine (NIM811), an mPTP inhibitor, were administered separately in selected groups. The serum and intestinal sections were collected for analysis.. IPoC and the administration of NIM811 significantly diminished the expression of intestinal-type fatty acid-binding protein and lactate dehydrogenase (3427±236.8 U/L for I/R, 1190.5±36.7 U/L for IPoC, 1399.3±295.6 U/L for I/R+NIM811, and 2002±370.9 IU/L for IPoC+CATR) in portal blood, the release of cytosolic cytochrome c, and the cleaved caspase 9 expression in intestinal mucosa after intestinal I/R injury (P<0.05). Histopathologically, IPoC and NIM811 mitigated mucosal damage after I/R as well (Chiu's score, 3.8±0.4 for I/R, 0.2±0.2 for IPoC, 0.4±0.2 for I/R+NIM811, and 4.2±0.2 for IPoC+CATR; apoptotic index, 59.5%±4.6% for I/R, 15.7%±15.7% for I/R+IPoC, 3.5%±3.5% for I/R+NIM811, and 67.1%±9.3% in IPoC+CATR). CATR negated the protection conferred by IPoC.. IPoC and NIM811 attenuate intestinal I/R injury. The addition of CATR negated the effects of IPoC, indicating that the protective mechanism of IPoC was associated with the modulation of mPTP opening.

Topics: Animals; Apoptosis; Atractyloside; Caspase 3; Cyclosporine; Cytochromes c; Disease Models, Animal; Enzyme Activation; Fatty Acid-Binding Proteins; Intestinal Mucosa; Intestine, Small; Ischemic Postconditioning; L-Lactate Dehydrogenase; Ligation; Male; Malondialdehyde; Mesenteric Artery, Superior; Mesenteric Vascular Occlusion; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Oxidative Stress; Rats; Rats, Wistar; Reperfusion Injury; Time Factors

Preventing cyclophilin D (cypD) translocation to the inner mitochondrial membrane can limit lethal reperfusion injury through the inhibition of the opening of the mitochondrial permeability transition pore. Inhibition or loss of function of cypD may also result into an endoplasmic reticulum (ER) stress that has been shown to alter cell survival. We therefore questioned whether ER stress might play a role in the protection induced by CypD deficiency or inhibition. CypD-KO and NIM811 (a CypD inhibitor)-treated mice were subjected to a prolonged ischemia-reperfusion (I/R). Area at risk and infarct size was measured using blue dye and triphenyltetrazolium chloride staining. ER stress markers were measured in the hearts during the reperfusion phase. As expected, cypD-KO mice exhibited a decreased infarct size when compared to wild-type mice (8 ± 1 vs. 20 ± 4% of left ventricular weight; p < 0.01). CypD-deficient mice displayed an increased expression of ER stress proteins such as eukaryotic initiation factor 2α (eIF2α) or glucose regulated protein 78 (Grp78 or Bip). The ER stress inhibitor TUDCA prevented the infarct size reduction afforded by the loss of cypD function (mean infarct size averaged 21 ± 4% of LV weight, p < 0.01 vs. cypD-KO). Similar results were obtained when NIM811, an analog of cyclosporine A, was used to pharmacologically (instead of genetically) inhibit cypD function. This study suggests that the ER stress induced by the inhibition of cypD function plays a key role in protecting the heart against lethal ischemia-reperfusion injury.

Topics: Animals; Cyclophilins; Cyclosporine; Cyclosporins; Disease Models, Animal; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Heart; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondrial Membranes; Myocardial Infarction; Myocardial Reperfusion Injury; Peptidyl-Prolyl Isomerase F; Taurochenodeoxycholic Acid

We tested our hypothesis that postischemic conditioning (PostC) is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP). In bilateral 8x13 cm pig latissimus dorsi muscle flaps subjected to 4 h ischemia, muscle infarction increased from 22+/-4 to 41+/-1% between 2 and 24 h reperfusion and remained unchanged at 48 (38+/-6%) and 72 (40+/-1%) h reperfusion (P<0.05; n=4 pigs). PostC induced by four cycles of 30-s reperfusion/reocclusion at the onset of reperfusion after 4 h ischemia reduced muscle infarction from 44+/-2 to 22+/-2% at 48 h reperfusion. This infarct protective effect of PostC was mimicked by intravenous injection of the mPTP opening inhibitor cyclosporin A or NIM-811 (10 mg/kg) at 5 min before the end of 4 h ischemia and was abolished by intravenous injection of the mPTP opener atractyloside (10 mg/kg) at 5 min before PostC (P<0.05; n=4-5 pigs). PostC or intravenous cyclosporin A injection at 5 min before reperfusion caused a decrease in muscle myeloperoxidase activity and mitochondrial free Ca2+ concentration and an increase in muscle ATP content after 4 h ischemia and 2 h reperfusion compared with the time-matched controls. These effects of PostC were abolished by intravenous injection of atractyloside at 5 min before PostC (P<0.05; n=6 pigs). These observations support our hypothesis that PostC is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mPTP.

Topics: Adenosine Triphosphate; Animals; Atractyloside; Calcium; Cyclosporine; Disease Models, Animal; Infarction; Injections, Intravenous; Mitochondria, Muscle; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Muscle, Skeletal; Peroxidase; Reperfusion Injury; Swine; Time Factors

Ca(2+) is the main trigger for mitochondrial permeability transition pore opening, which plays a key role in cardiomyocyte death after ischemia-reperfusion. We investigated whether a reduced accumulation of mitochondrial Ca(2+) might explain the attenuation of lethal reperfusion injury by postconditioning. Anesthetized New Zealand White rabbits underwent 30 min of ischemia, followed by either 240 (infarct size protocol) or 60 (mitochondria protocol) min of reperfusion. They received either no intervention (control), preconditioning by 5-min ischemia and 5-min reperfusion, postconditioning by four cycles of 1-min reperfusion and 1-min ischemia at the onset of reflow, or pharmacological inhibition of the transition pore opening by N-methyl-4-isoleucine-cyclosporin (NIM811; 5 mg/kg iv) given at reperfusion. Area at risk and infarct size were assessed by blue dye injection and triphenyltetrazolium chloride staining. Mitochondria were isolated from the risk region for measurement of 1) Ca(2+) retention capacity (CRC), and 2) mitochondrial content of total (atomic absorption spectrometry) and ionized (potentiometric technique) calcium concentration. CRC averaged 0.73 +/- 0.16 in control vs. 4.23 +/- 0.17 mug Ca(2+)/mg proteins in shams (P < 0.05). Postconditioning, preconditioning, or NIM811 significantly increased CRC (P < 0.05 vs. control). In the control group, total and free mitochondrial calcium significantly increased to 2.39 +/- 0.43 and 0.61 +/- 0.10, respectively, vs. 1.42 +/- 0.09 and 0.16 +/- 0.01 mug Ca(2+)/mg in sham (P < 0.05). Surprisingly, whereas total and ionized mitochondrial Ca(2+) decreased in preconditioning, it significantly increased in postconditioning and NIM811 groups. These data suggest that retention of calcium within mitochondria may explain the decreased reperfusion injury in postconditioned (but not preconditioned) hearts.

Topics: Animals; Calcium; Cardiovascular Agents; Cell Respiration; Cyclophilins; Cyclosporine; Disease Models, Animal; Ischemic Preconditioning, Myocardial; Male; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Peptidyl-Prolyl Isomerase F; Rabbits; Time Factors

Cerebral ischemia followed by reperfusion activates numerous pathways that lead to cell death. One such pathway involves the release of large quantities of the excitatory amino acid glutamate into the synapse and activation of N-methyl-D-aspartate receptors. This causes an increase in mitochondrial calcium levels ([Ca(2+)](m)) and a production of reactive oxygen species (ROS), both of which may induce the mitochondrial permeability transition (MPT). As a consequence, there is eventual mitochondrial failure culminating in either apoptotic or necrotic cell death. Thus, agents that inhibit MPT might prove useful as therapeutic interventions in cerebral ischemia. In this study, we have investigated the neuroprotective efficacy of the novel compound NIM811. Similar in structure of its parent compound cyclosporin A, NIM811 is a potent inhibitor of the MPT. Unlike cyclosporin A, however, it is essentially void of immunosuppressive actions, allowing the role of MPT to be clarified in ischemia/reperfusion injury. The results of these studies demonstrate that NIM811 provides almost 40% protection in a model of transient focal cerebral ischemia. This was associated with a nearly 10% reduction in mitochondrial reactive species formation and 34% and 38% reduction of cytochrome c release in core and penumbra, respectively. Treatment with NIM811 also increased calcium retention capacity by approximately 20%. Interestingly, NIM811 failed to improve ischemia-induced impairment of bioenergetics. The neuroprotective effects of NIM811 were not due to drug-induced alterations in cerebral perfusion after ischemia. Activation of MPT appears to be an important process in ischemia/reperfusion injury and may be a therapeutic target.

Topics: Animals; Brain Infarction; Brain Ischemia; Calcium Signaling; Cell Death; Cyclosporine; Cytochromes c; Disease Models, Animal; Energy Metabolism; Male; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Nerve Degeneration; Neurons; Neuroprotective Agents; Oxidative Stress; Rats; Rats, Inbred SHR; Reactive Oxygen Species; Treatment Outcome