Cardioprotection of Immature Heart by Simultaneous Activation of PKA and EPAC : A Role for the Mitochondrial Permeability Transition Pore

Metabolic and ionic changes during ischaemia predispose the heart to the damaging effects of reperfusion. Such changes and the resulting injury differ between immature and adult heart. Therefore, cardioprotective strategies for adults need to be tested in immature heart. We have recently shown that simultaneous activation of PKA and EPAC confers marked cardioprotection in adult hearts. The aim of this study is to investigate the efficacy of this intervention in immature hearts and determine whether the mitochondrial permeability transition pore (MPTP) is involved. Isolated perfused Langendorff hearts from both adult and immature rats were exposed to global ischaemia and reperfusion injury (I/R) following control perfusion or perfusion after an equilibra-tion period with activators of PKA and/or EPAC. Functional outcome and reperfusion injury were measured and in parallel, mitochondria were isolated following 5 min reperfusion to determine whether cardioprotective interventions involved changes in MPTP opening behaviour. Perfusion for 5 minutes preceding ischaemia of injury- matched adult and immature hearts with 5 µM 8-Br (8-Br-cAMP-AM), an activator of both PKA and EPAC, led to significant reduction in post-reperfusion CK release and infarct size. Perfusion with this agent also led to a reduction in MPTP opening propensity in both adult and immature hearts. These data show that immature hearts are innately more resistant to I/R injury than adults, and that this is due to a reduced ten-dency to MPTP opening following reperfusion. Further, simultaneous stimulation of PKA & EPAC causes cardioprotection which is additive to the innate resistance.

SIRT3 Protects Against Cognitive Dysfunction Induced by Sepsis-associated Encephalopathy Via JNK/p66Shc-Regulated Mitochondrial Apoptosis in Mice

Background: Sepsis-associated encephalopathy (SAE) is one of the severe central nervous system complications. Oxidative stress and synaptic dysfunction were involved in cognitive impairment induced by SAE. The mitochondrial nicotinamide adenine dinucleotide (NAD+) dependent deacetylase, sirtuin3 (SIRT3), plays a critical role in regulating mitochondrial function. The aim of this study was to evaluate the effect of SIRT3 in cognitive dysfunction induced by SAE.Methods: Mice were treated with lipopolysaccharide (LPS, 10 mg/kg, i.p.). Contextual and cue memory were evaluated by fear conditioning test in wild-type (WT) and SIRT3-deficient (SIRT3-/-) mice. Synapse-associated proteins and mitochondrial apoptosis-associated protein were examined by western blotting. In vitro studies, acetylation levels of cyclophilin D (CypD) were detected with different SIRT3 deacetylase activity in HT22 cells after LPS-induced microglia supernatant (Mi-sup) exposure. Oxidative stress was detected by reactive oxygen species (ROS) staining, and mitochondrial membrane potential (MMP) was detected by JC-1 staining, and mitochondrial membrane permeability transition pore (MPTP) opening was detected by Calcein and Co2+ staining. Furthermore, the phosphorylation levels of mitochondrial p66Shc and JNK were evaluated by western blotting.Results: SIRT3 expression was diminished in hippocampus of mice after LPS treatment. SIRT3-deficiency contributed to more severe contextual memory loss and synaptic dysfunction, decreased ratio of Bcl-2/Bax and increased Cyt C release to cytoplasm in hippocampus compared with wild-type controls. In HT22 cells, lysine acetylation levels of CypD were significantly increased after Mi-sup exposure and further enhanced with 3-TYP (SIRT3 deacetylation inhibitor) pretreatment, in association with the accumulation of ROS, declined MMP and increased MPTP opening, as well as the increased mitochondrial Cyt C release and phosphorylation levels of mitochondrial JNK and p66Shc-Ser36. SIRT3 overexpression restored CypD lysine acetylation levels and MPTP opening in HT22 cells after Mi-sup exposure and reduced mitochondrial JNK and p66Shc activation. Conclusions: Taken together, our results showed that SIRT3-mediated CypD deacetylation was involved in LPS-induced hippocampal synaptic dysfunction, via ROS accumulation, declined MMP, increased MPTP opening, mitochondrial Cyt C release and mitochondrial apoptosis of hippocampal neuron via JNK/p66Shc pathway. Our results revealed that SIRT3 may be a promising therapeutic and diagnostic target for cognitive dysfunction induced by SAE.

Schisandrin B Antagonizes Cardiotoxicity Induced by Pirarubicin by Inhibiting Mitochondrial Permeability Transition Pore (mPTP) Opening and Decreasing Cardiomyocyte Apoptosis

Objective: Pirarubicin (THP), one of the anthracycline anticancer drugs, is widely used in the treatment of various cancers, but its cardiotoxicity cannot be ignored. Schisandrin B (SchB) has the ability to upregulate cellular antioxidant defense mechanism and promote mitochondrial function and antioxidant status. However, it has not been reported whether it can resist THP-induced cardiotoxicity. The aim of this study was to investigate the effect of SchB on THP cardiotoxicity and its mechanism.Methods: The rat model of cardiotoxicity induced by THP was established, and SchB treatment was performed at the same time. The changes of ECG, cardiac coefficient, and echocardiogram were observed. The changes of myocardial tissue morphology were observed by H&E staining. Apoptosis was detected by TUNEL. The levels of LDH, BNP, CK-MB, cTnT, SOD, and MDA in serum were measured to observe the heart damage and oxidative stress state of rats. The expression of cleaved-caspase 9, pro/cleaved-caspase 3, Bcl-2/Bax, and cytosol and mitochondrial Cyt C and Bax was evaluated by western blot. H9c2 cardiomyocytes were cocultured with THP, SchB, and mPTP inhibitor CsA to detect the production of ROS and verify the above signaling pathways. The opening of mPTP and mitochondrial swelling were detected by mPTP kit and purified mitochondrial swelling kit.Results: After 8 weeks, a series of cardiotoxicity manifestations were observed in THP rats. These adverse effects can be effectively alleviated by SchB treatment. Further studies showed that SchB had strong antioxidant and antiapoptotic abilities in THP cardiotoxicity.Conclusion: SchB has an obvious protective effect on THP-induced cardiotoxicity. The mechanism may be closely related to the protection of mitochondrial function, inhibition of mPTP opening, and alleviation of oxidative stress and apoptosis of cardiomyocytes.

Pioglitazone Is a Mild Carrier-Dependent Uncoupler of Oxidative Phosphorylation and a Modulator of Mitochondrial Permeability Transition

Pioglitazone (PIO) is an insulin-sensitizing antidiabetic drug, which normalizes glucose and lipid metabolism but may provoke heart and liver failure and chronic kidney diseases. Both therapeutic and adverse effects of PIO can be accomplished through mitochondrial targets. Here, we explored the capability of PIO to modulate the mitochondrial membrane potential (ΔΨm) and the permeability transition pore (mPTP) opening in different models in vitro. ΔΨm was measured using tetraphenylphosphonium and the fluorescent dye rhodamine 123. The coupling of oxidative phosphorylation was estimated polarographically. The transport of ions and solutes across membranes was registered by potentiometric and spectral techniques. We found that PIO decreased ΔΨm in isolated mitochondria and intact thymocytes and the efficiency of ADP phosphorylation, particularly after the addition of Ca2+. The presence of the cytosolic fraction mitigated mitochondrial depolarization but made it sustained. Carboxyatractyloside diminished the PIO-dependent depolarization. PIO activated proton transport in deenergized mitochondria but not in artificial phospholipid vesicles. PIO had no effect on K+ and Ca2+ inward transport but drastically decreased the mitochondrial Ca2+-retention capacity and protective effects of adenine nucleotides against mPTP opening. Thus, PIO is a mild, partly ATP/ADP-translocase-dependent, uncoupler and a modulator of ATP production and mPTP sensitivity to Ca2+ and adenine nucleotides. These properties contribute to both therapeutic and adverse effects of PIO.

A Cardiac Mitochondrial FGFR1 Mediates the Antithetical Effects of FGF2 Isoforms on Permeability Transition

Mitochondria, abundant organelles in high energy demand cells such as cardiomyocytes, can determine cell death or survival by regulating the opening of mitochondrial permeability transition pore, mPTP. We addressed the hypothesis that the growth factor FGF2, known to reside in intracellular locations, can directly influence mitochondrial susceptibility to mPTP opening. Rat cardiac subsarcolemmal (SSM) or interfibrillar (IFM) mitochondrial suspensions exposed directly to rat 18 kDa low molecular weight (Lo-) FGF2 isoform displayed increased resistance to calcium overload-induced mPTP, measured spectrophotometrically as “swelling”, or as cytochrome c release from mitochondria. Inhibition of mitochondrial protein kinase C epsilon abrogated direct Lo-FGF2 mito-protection. Exposure to the rat 23 kDa high molecular weight (Hi) FGF2 isoform promoted cytochrome c release from SSM and IFM under nonstressed conditions. The effect of Hi-FGF2 was prevented by mPTP inhibitors, pre-exposure to Lo-FGF2, and okadaic acid, a serine/threonine phosphatase inhibitor. Western blotting and immunoelectron microscopy pointed to the presence of immunoreactive FGFR1 in cardiac mitochondria in situ. The direct mito-protective effect of Lo-FGF2, as well as the deleterious effect of Hi-FGF2, were prevented by FGFR1 inhibitors and FGFR1 neutralizing antibodies. We propose that intracellular FGF2 isoforms can modulate mPTP opening by interacting with mito-FGFR1 and relaying isoform-specific intramitochondrial signal transduction.

SIRT3 Protects Against Cognitive Dysfunction Induced by Sepsis-Associated Encephalopathy via JNK/p66Shc-Regulated Mitochondrial Apoptosis in Mice

Background Sepsis-associated encephalopathy (SAE) is one of the severe central nervous system complications. Oxidative stress and synaptic dysfunction were involved in cognitive impairment induced by SAE. The mitochondrial nicotinamide adenine dinucleotide (NAD+) dependent deacetylase, sirtuin3 (SIRT3), plays a critical role in regulating mitochondrial function. The aim of this study was to evaluate the effect of SIRT3 in cognitive dysfunction induced by SAE. Methods Mice were treated with lipopolysaccharide (LPS, 10 mg/kg, i.p.). Contextual and cue memory were evaluated by fear conditioning test in wild-type (WT) and SIRT3-deficient (SIRT3-/-) mice. Synapse-associated proteins and mitochondrial apoptosis-associated protein were examined by western blotting. In vitro studies, acetylation levels of cyclophilin D (CypD) were detected with different SIRT3 deacetylase activity in HT22 cells after LPS-induced microglia supernatant (Mi-sup) exposure. Oxidative stress was detected by reactive oxygen species (ROS) staining, and mitochondrial membrane potential (MMP) was detected by JC-1 staining, and mitochondrial membrane permeability transition pore (MPTP) opening was detected by Calcein and Co2+ staining. Furthermore, the phosphorylation levels of mitochondrial p66Shc and JNK were evaluated by western blotting. Results SIRT3 expression was diminished in hippocampus of mice after LPS treatment. SIRT3-deficiency contributed to more severe contextual memory loss and synaptic dysfunction, decreased ratio of Bcl-2/Bax and increased Cyt C release to cytoplasm in hippocampus compared with wild-type controls. In HT22 cells, lysine acetylation levels of CypD were significantly increased after Mi-sup exposure and further enhanced with 3-TYP (SIRT3 deacetylation inhibitor) pretreatment, in association with the accumulation of ROS, declined MMP and increased MPTP opening, as well as the increased mitochondrial Cyt C release and phosphorylation levels of mitochondrial JNK and p66Shc-Ser36. SIRT3 overexpression restored CypD lysine acetylation levels and MPTP opening in HT22 cells after Mi-sup exposure and reduced mitochondrial JNK and p66Shc activation. Conclusion Taken together, our results showed that SIRT3-mediated CypD deacetylation was involved in LPS-induced hippocampal synaptic dysfunction, via ROS accumulation, declined MMP, increased MPTP opening, mitochondrial Cyt C release and mitochondrial apoptosis of hippocampal neuron via JNK/p66Shc pathway. Our results revealed that SIRT3 may be a promising therapeutic and diagnostic target for cognitive dysfunction induced by SAE.

Mitochondrial Dysfunction and Alterations in Mitochondrial Permeability Transition Pore (mPTP) Contribute to Apoptosis Resistance in Idiopathic Pulmonary Fibrosis Fibroblasts

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by increased activation of fibroblasts/myofibroblasts. Previous reports have shown that IPF fibroblasts are resistant to apoptosis, but the mechanisms remain unclear. Since inhibition of the mitochondrial permeability transition pore (mPTP) has been implicated in the resistance to apoptosis, in this study, we analyzed the role of mitochondrial function and the mPTP on the apoptosis resistance of IPF fibroblasts under basal conditions and after mitomycin C-induced apoptosis. We measured the release of cytochrome c, mPTP opening, mitochondrial calcium release, oxygen consumption, mitochondrial membrane potential, ADP/ATP ratio, ATP concentration, and mitochondrial morphology. We found that IPF fibroblasts were resistant to mitomycin C-induced apoptosis and that calcium, a well-established activator of mPTP, is decreased as well as the release of pro-apoptotic proteins such as cytochrome c. Likewise, IPF fibroblasts showed decreased mitochondrial function, while mPTP was less sensitive to ionomycin-induced opening. Although IPF fibroblasts did not present changes in the mitochondrial membrane potential, we found a fragmented mitochondrial network with scarce, thinned, and disordered mitochondria with reduced ATP levels. Our findings demonstrate that IPF fibroblasts are resistant to mitomycin C-induced apoptosis and that altered mPTP opening contributes to this resistance. In addition, IPF fibroblasts show mitochondrial dysfunction evidenced by a decrease in respiratory parameters.

Cyclosporine A-enhanced cardioplegia preserves mitochondrial basal respiration after ischemic arrest

Background: Mitochondrial permeability transition pore (mPTP) opening plays a crucial role in cell death during ischemia-reperfusion injury (IRI). Cyclosporine A (CsA) inhibits mPTP opening. This study aimed to investigate the effects of CsA treatment during cardioplegia on the mitochondrial function and cardiac IRI. Methods: Landrace pigs (52.9 ± 3.7 kg) were subjected to midline sternotomy, cardiopulmonary bypass at 34°C and 90 minutes of cardiac arrest. They received either a single shot of standard 4°C cold histidine-tryptophan-α-ketoglutarate (HTK)-Bretschneider solution ( n = 11) or HTK-Bretschneider plus 1.2 mg/L CsA (histidine-tryptophan-α-ketoglutarate plus cyclosporine A (HTK/CsA); n = 11). During reperfusion global left-ventricular function was assessed and myocardial biopsies were harvested at baseline, during ischemia and 45 minutes following reperfusion. High-resolution respirometry and hydrogen peroxide production were measured. Immunohistochemical stainings for apoptosis-inducing factor and hypoxia-inducible factor-1α as well as a flow cytometry-based JC-1 mitochondrial membrane potential assay were performed. Results: Hemodynamic parameters were comparable between both groups. The cytochrome C release (HTK: 930.3 ± 804.4 pg/mg, HTK/CsA: 699.7 ± 394.0 pg/mg, p = 0.457) as well as PGC1α content (HTK: 66.7%, HTK/CsA: 33.3%, p = 0.284) was lower in the HTK/CsA group. Respiratory measurements revealed that the oxygen flux under basal respiration was higher in the HTK/CsA group (8.2 ± 1.3 pmol·O2·s−1·mg−1·ww) than in the HTK group (3.8 ± 1.4 pmol·O2·s−1·mg−1·ww, p = 0.045). There were no significant differences regarding histological surrogates of apoptosis and necrosis. Conclusions: Supplementing cardioplegic solutions with CsA enhances the basal mitochondrial respiration thereby exerting a cardioprotective effect and diminishing IRI-induced damage. CsA seems to preserve mitochondrial function via non-ROS related pathways.

Resveratrol Prevents Right Ventricle Dysfunction, Calcium Mishandling, and Energetic Failure via SIRT3 Stimulation in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is characterized by pulmonary vessel remodeling; however, its severity and impact on survival depend on right ventricular (RV) failure. Resveratrol (RES), a polyphenol found in red wine, exhibits cardioprotective effects on RV dysfunction in PAH. However, most literature has focused on RES protective effect on lung vasculature; recent finding indicates that RES has a cardioprotective effect independent of pulmonary arterial pressure on RV dysfunction, although the underlying mechanism in RV has not been determined. Therefore, this study is aimed at evaluating sirtuin-3 (SIRT3) modulation by RES in RV using a monocrotaline- (MC-) induced PAH rat model. Myocyte function was evaluated by confocal microscopy as cell contractility, calcium signaling, and mitochondrial membrane potential ( Δ Ψ m ); cell energetics was assessed by high-resolution respirometry, and western blot and immunoprecipitation evaluated posttranslational modifications. PAH significantly affects mitochondrial function in RV; PAH is prone to mitochondrial permeability transition pore (mPTP) opening, thus decreasing the mitochondrial membrane potential. The compromised cellular energetics affects cardiomyocyte function by decreasing sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) activity and delaying myofilament unbinding, disrupting cell relaxation. RES partially protects mitochondrial integrity by deacetylating cyclophilin-D, a critical component of the mPTP, increasing SIRT3 expression and activity and preventing mPTP opening. The preserved energetic capability rescues cell relaxation by maintaining SERCA activity. Avoiding Ca2+ transient and cell contractility mismatch by preserving mitochondrial function describes, for the first time, impairment in excitation-contraction-energetics coupling in RV failure. These results highlight the importance of mitochondrial energetics and mPTP in PAH.