Peptidyl-Prolyl Isomerase ppiB Is Essential for Proteome Homeostasis and Virulence in Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to Southeast Asia and northern Australia. Mortality rates in these areas are high even with antimicrobial treatment, and there are few options for effective therapy. Therefore, there is a need to identify antibacterial targets for the development of novel treatments. Cyclophilins are a family of highly conserved enzymes important in multiple cellular processes. Cyclophilins catalyze the cis-trans isomerization of xaa-proline bonds, a rate-limiting step in protein folding which has been shown to be important for bacterial virulence. B. pseudomallei carries a putative cyclophilin B gene, ppiB, the role of which was investigated. A B. pseudomalleiΔppiB (BpsΔppiB) mutant strain demonstrates impaired biofilm formation and reduced motility. Macrophage invasion and survival assays showed that although the BpsΔppiB strain retained the ability to infect macrophages, it had reduced survival and lacked the ability to spread cell to cell, indicating ppiB is essential for B. pseudomallei virulence. This is reflected in the BALB/c mouse infection model, demonstrating the requirement of ppiB for in vivo disease dissemination and progression. Proteomic analysis demonstrates that the loss of PpiB leads to pleiotropic effects, supporting the role of PpiB in maintaining proteome homeostasis. The loss of PpiB leads to decreased abundance of multiple virulence determinants, including flagellar machinery and alterations in type VI secretion system proteins. In addition, the loss of ppiB leads to increased sensitivity toward multiple antibiotics, including meropenem and doxycycline, highlighting ppiB inhibition as a promising antivirulence target to both treat B. pseudomallei infections and increase antibiotic efficacy.

Cyclophilin D deficiency attenuates mitochondrial F1Fo ATP synthase dysfunction via OSCP in Alzheimer's disease.

Mitochondrial dysfunction is pivotal in inducing synaptic injury and neuronal stress in Alzheimer's disease (AD). Mitochondrial F1Fo ATP synthase deregulation is a hallmark mitochondrial defect leading to oxidative phosphorylation (OXPHOS) failure in this neurological disorder. Oligomycin sensitivity conferring protein (OSCP) is a crucial F1Fo ATP synthase subunit. Decreased OSCP levels and OSCP interaction with amyloid ß (Aß) constitute key aspects of F1Fo ATP synthase pathology in AD-related conditions. However, the detailed mechanisms promoting such AD-related OSCP changes have not been fully resolved. Here, we have found increased physical interaction of OSCP with Cyclophilin D (CypD) in AD cases as well as in an AD animal model (5xFAD mice). Genetic depletion of CypD mitigates OSCP loss via ubiquitin-dependent OSCP degradation in 5xFAD mice. Moreover, the ablation of CypD also attenuates OSCP/Aß interaction in AD mice. The relieved OSCP changes by CypD depletion in 5xFAD mice are along with preserved F1Fo ATP synthase function, restored mitochondrial bioenergetics as well as improved mouse cognition. The simplest interpretation of our results is that CypD is a critical mediator that promotes OSCP deficits in AD-related conditions. Therefore, to block the deleterious impact of CypD on OSCP has the potential to be a promising therapeutic strategy to correct mitochondrial dysfunction for AD therapy.

Mitochondrial Dysfunction, Through Impaired Autophagy, Leads to Endoplasmic Reticulum Stress, Deregulated Lipid Metabolism, and Pancreatitis in Animal Models.

BACKGROUND & AIMS: Little is known about the signaling pathways that initiate and promote acute pancreatitis (AP). The pathogenesis of AP has been associated with abnormal increases in cytosolic Ca2+, mitochondrial dysfunction, impaired autophagy, and endoplasmic reticulum (ER) stress. We analyzed the mechanisms of these dysfunctions and their relationships, and how these contribute to development of AP in mice and rats. METHODS: Pancreatitis was induced in C57BL/6J mice (control) and mice deficient in peptidylprolyl isomerase D (cyclophilin D, encoded by Ppid) by administration of L-arginine (also in rats), caerulein, bile acid, or an AP-inducing diet. Parameters of pancreatitis, mitochondrial function, autophagy, ER stress, and lipid metabolism were measured in pancreatic tissue, acinar cells, and isolated mitochondria. Some mice with AP were given trehalose to enhance autophagic efficiency. Human pancreatitis tissues were analyzed by immunofluorescence. RESULTS: Mitochondrial dysfunction in pancreas of mice with AP was induced by either mitochondrial Ca2+ overload or through a Ca2+ overload-independent pathway that involved reduced activity of ATP synthase (80% inhibition in pancreatic mitochondria isolated from rats or mice given L-arginine). Both pathways were mediated by cyclophilin D and led to mitochondrial depolarization and fragmentation. Mitochondrial dysfunction caused pancreatic ER stress, impaired autophagy, and deregulation of lipid metabolism. These pathologic responses were abrogated in cyclophilin D-knockout mice. Administration of trehalose largely prevented trypsinogen activation, necrosis, and other parameters of pancreatic injury in mice with L-arginine AP. Tissues from patients with pancreatitis had markers of mitochondrial damage and impaired autophagy, compared with normal pancreas. CONCLUSIONS: In different animal models, we find a central role for mitochondrial dysfunction, and for impaired autophagy as its principal downstream effector, in development of AP. In particular, the pathway involving enhanced interaction of cyclophilin D with ATP synthase mediates L-arginine-induced pancreatitis, a model of severe AP the pathogenesis of which has remained unknown. Strategies to restore mitochondrial and/or autophagic function might be developed for treatment of AP.

Cyclophilin D deficiency protects against the development of mitochondrial ROS and cellular inflammation in aorta.

INTRODUCTION: Inflammation and oxidative stress are closely correlated in the pathology of cardiovascular disease. Mitochondrial cyclophilin D (CypD), the important modulator for mPTP opening, is increasingly recognized as a key regulator of cellular ROS generation. Besides, its association with cell inflammation is also being discovered. However, the effects of CypD in modulating vascular inflammatory response is unknown. We sought to investigate whether CypD deficiency attenutes vascular inflammation under physical conditions. METHODS AND RESULTS: We adopted CypD KO mouse and their littermate controls to observe the effects of CypD deficiency on aortic mitochondrial functions and vascular inflammation. As we found in our study, we confirmed that under physical conditions, CypD deficiency enhanced mouse whole body metabolic status, increased aortic mitochondrial complex III activity and decreased mitochondrial ROS generation. Functionally, CypD deficiency also attenuated inflammatory molecules expression, including VCAM-1, IL-6 and TNF-α in mouse aorta. CONCLUSIONS: Our results review that mitochondrial CypD is involved in the regulation of inflammation in aorta and provide insights that blocking mitochondrial CypD enhances vascular resistance to inflammatory injuries.

Estrogen receptor beta modulates permeability transition in brain mitochondria.

Recent evidence highlights a role for sex and hormonal status in regulating cellular responses to ischemic brain injury and neurodegeneration. A key pathological event in ischemic brain injury is the opening of a mitochondrial permeability transition pore (MPT) induced by excitotoxic calcium levels, which can trigger irreversible damage to mitochondria accompanied by the release of pro-apoptotic factors. However, sex differences in brain MPT modulation have not yet been explored. Here, we show that mitochondria isolated from female mouse forebrain have a lower calcium threshold for MPT than male mitochondria, and that this sex difference depends on the MPT regulator cyclophilin D (CypD). We also demonstrate that an estrogen receptor beta (ERß) antagonist inhibits MPT and knockout of ERß decreases the sensitivity of mitochondria to the CypD inhibitor, cyclosporine A. These results suggest a functional relationship between ERß and CypD in modulating brain MPT. Moreover, co-immunoprecipitation studies identify several ERß binding partners in mitochondria. Among these, we investigate the mitochondrial ATPase as a putative site of MPT regulation by ERß. We find that previously described interaction between the oligomycin sensitivity-conferring subunit of ATPase (OSCP) and CypD is decreased by ERß knockout, suggesting that ERß modulates MPT by regulating CypD interaction with OSCP. Functionally, in primary neurons and hippocampal slice cultures, modulation of ERß has protective effects against glutamate toxicity and oxygen glucose deprivation, respectively. Taken together, these results reveal a novel pathway of brain MPT regulation by ERß that could contribute to sex differences in ischemic brain injury and neurodegeneration.

Cyclophilin B Deficiency Causes Abnormal Dentin Collagen Matrix.

Cyclophilin B (CypB) is an endoplasmic reticulum-resident protein that regulates collagen folding, and also contributes to prolyl 3-hydroxylation (P3H) and lysine (Lys) hydroxylation of collagen. In this study, we characterized dentin type I collagen in CypB null (KO) mice, a model of recessive osteogenesis imperfecta type IX, and compared to those of wild-type (WT) and heterozygous (Het) mice. Mass spectrometric analysis demonstrated that the extent of P3H in KO collagen was significantly diminished compared to WT/Het. Lys hydroxylation in KO was significantly diminished at the helical cross-linking sites, α1/α2(I) Lys-87 and α1(I) Lys-930, leading to a significant increase in the under-hydroxylated cross-links and a decrease in fully hydroxylated cross-links. The extent of glycosylation of hydroxylysine residues was, except α1(I) Lys-87, generally higher in KO than WT/Het. Some of these molecular phenotypes were distinct from other KO tissues reported previously, indicating the dentin-specific control mechanism through CypB. Histological analysis revealed that the width of predentin was greater and irregular, and collagen fibrils were sparse and significantly smaller in KO than WT/Het. These results indicate a critical role of CypB in dentin matrix formation, suggesting a possible association between recessive osteogenesis imperfecta and dentin defects that have not been clinically detected.

Ablation of Cyclophilin D Results in an Activation of FAK, Akt, and ERK Pathways in the Mouse Heart.

Cyclophilin D (CypD) is a mitochondrial chaperone that regulates the mitochondrial permeability transition pore. Metabolically, deletion of Ppif (the gene encoding CypD) in mice is associated with elevated levels of mitochondrial matrix Ca2+ that leads to increased glucose as relative to fatty acid oxidation. Here, we characterized the adaptive mechanisms involved in the regulation of glucose metabolism including the regulation of Akt and ERK kinases that we evaluated by Western blot analysis of Ppif-/- in comparison to wild type (WT) mouse hearts. CypD loss led to adaptive mechanisms in the heart resulting in an upregulation of focal adhesion kinase (phosphorylated at Tyr925) and increased phosphorylation of Akt at S473. The increased activity of this pathway (pAktS473 increased to 170% and 145% in Ppif-/- versus WT males and females, respectively) could be responsible for the observed metabolic switch towards glycolysis. Furthermore, the phosphorylation of ERK1/2 proteins was elevated following CypD ablation. In addition, we observed differences in protein expression and activity in male versus female hearts that were independent of CypD expression. This included an upregulation of pAktS473 (to 273% and 269% in Ppif-/- and WT females as compared to their corresponding males, respectively). Furthermore, decreased levels of endothelial nitric oxide synthase (eNOS) inhibitor asymmetric dimethylarginine were accompanied by an upregulation of eNOS in female mice. The higher extent of kinases phosphorylation may be responsible for the reported lowered tolerance of CypD animals to stress. Moreover, the higher nitric oxide production could be responsible for the cardioprotective properties observed only in female hearts. J. Cell. Biochem. 118: 2933-2940, 2017. © 2017 Wiley Periodicals, Inc.

The mitochondrial permeability transition pore regulates endothelial bioenergetics and angiogenesis.

RATIONALE: The mitochondrial permeability transition pore is a well-known initiator of cell death that is increasingly recognized as a physiological modulator of cellular metabolism. OBJECTIVE: We sought to identify how the genetic deletion of a key regulatory subunit of the mitochondrial permeability transition pore, cyclophilin D (CypD), influenced endothelial metabolism and intracellular signaling. METHODS AND RESULTS: In cultured primary human endothelial cells, genetic targeting of CypD using siRNA or shRNA resulted in a constitutive increase in mitochondrial matrix Ca(2+) and reduced nicotinamide adenine dinucleotide (NADH). Elevated matrix NADH, in turn, diminished the cytosolic NAD(+)/NADH ratio and triggered a subsequent downregulation of the NAD(+)-dependent deacetylase sirtuin 1 (SIRT1). Downstream of SIRT1, CypD-deficient endothelial cells exhibited reduced phosphatase and tensin homolog expression and a constitutive rise in the phosphorylation of angiogenic Akt. Similar changes in SIRT1, phosphatase and tensin homolog, and Akt were also noted in the aorta and lungs of CypD knockout mice. Functionally, CypD-deficient endothelial cells and aortic tissue from CypD knockout mice exhibited a dramatic increase in angiogenesis at baseline and when exposed to vascular endothelial growth factor. The NAD(+) precursor nicotinamide mononucleotide restored the cellular NAD(+)/NADH ratio and normalized the CypD-deficient phenotype. CypD knockout mice also presented accelerated wound healing and increased neovascularization on tissue injury as monitored by optical microangiography. CONCLUSIONS: Our study reveals the importance of the mitochondrial permeability transition pore in the regulation of endothelial mitochondrial metabolism and vascular function. The mitochondrial regulation of SIRT1 has broad implications in the epigenetic regulation of endothelial phenotype.

Cyclophilin D Modulates the Cardiac Mitochondrial Target of Isoflurane, Sevoflurane, and Desflurane.

BACKGROUND: Volatile anesthetics are known to limit myocardial ischemia-reperfusion injuries. Mitochondria were shown to be major contributors to cardioprotection. Cyclophilin D (CypD) is one of the main regulators of mitochondria-induced cell death. We compared the effect of isoflurane, sevoflurane, and desflurane in the presence or absence of CypD, to clarify its role in the mechanism of cardioprotection induced by these anesthetics. METHODS: Oxidative phosphorylation, mitochondrial membrane potential, and H2O2 production were measured in isolated mitochondria from wild-type (WT) or CypD knockout mice in basal conditions and after hypoxia-reoxygenation in the presence or absence of volatile anesthetics. RESULTS: All volatile anesthetics inhibited mitochondrial state 3 of complex I, decreased membrane potential, and increased adenosine diphosphate consumption duration in both WT and CypD knockout mice. However, they differently modified H2O2 production after stimulation by succinate: CypD ablation reduced H2O2 production, isoflurane decreased H2O2 level in WT but not in CypD knockout mice, sevoflurane affected both lines whereas desflurane increased H2O2 production in CypD knockout and had no effect on WT mice. CONCLUSIONS: This study showed different effects of isoflurane, sevoflurane, and desflurane on mitochondrial functions and highlighted the implication of CypD in the regulation of adenosine diphosphate consumption and complex I-induced radical oxygen species production.

Phenotypic Characterization of a Leishmania donovani Cyclophilin 40 Null Mutant.

Protozoan parasites of the genus Leishmania adapt to their arthropod and vertebrate hosts through the development of defined life cycle stages. Stage differentiation is triggered by environmental stress factors and has been linked to parasite chaperone activities. Using a null mutant approach we previously revealed important, nonredundant functions of the cochaperone cyclophilin 40 in L. donovani-infected macrophages. Here, we characterized in more detail the virulence defect of cyp40-/- null mutants. In vitro viability assays, infection tests using macrophages, and mixed infection experiments ruled out a defect of cyp40-/- parasites in resistance to oxidative and hydrolytic stresses encountered inside the host cell phagolysosome. Investigation of the CyP40-dependent proteome by quantitative 2D-DiGE analysis revealed up regulation of various stress proteins in the null mutant, presumably a response to compensate for the lack of CyP40. Applying transmission electron microscopy we showed accumulation of vesicular structures in the flagellar pocket of cyp40-/- parasites that we related to a significant increase in exosome production, a phenomenon previously linked to the parasite stress response. Together these data suggest that cyp40-/- parasites experience important intrinsic homeostatic stress that likely abrogates parasite viability during intracellular infection.

Traumatic brain injury-induced axonal phenotypes react differently to treatment.

Injured axons with distinct morphologies have been found following mild traumatic brain injury (mTBI), although it is currently unclear whether they reflect varied responses to the injury or represent different stages of progressing pathology. This complicates evaluation of therapeutic interventions targeting axonal injury. To address this issue, we assessed axonal injury over time within a well-defined axonal population, while also evaluating mitochondrial permeability transition as a therapeutic target. We utilized mice expressing yellow fluorescent protein (YFP) in cortical neurons which were crossed with mice which lacked Cyclophilin D (CypD), a positive regulator of mitochondrial permeability transition pore opening. Their offspring were subjected to mTBI and the ensuing axonal injury was assessed using YFP expression and amyloid precursor protein (APP) immunohistochemistry, visualized by confocal and electron microscopy. YFP(+) axons initially developed a single, APP(+), focal swelling (proximal bulb) which progressed to axotomy. Disconnected axonal segments developed either a single bulb (distal bulb) or multiple bulbs (varicosities), which were APP(-) and whose ultrastructure was consistent with ongoing Wallerian degeneration. CypD knock-out failed to reduce proximal bulb formation but decreased the number of distal bulbs and varicosities, as well as a population of small, APP(+), callosal bulbs not associated with YFP(+) axons. The observation that YFP(+) axons contain several pathological morphologies points to the complexity of traumatic axonal injury. The fact that CypD knock-out reduced some, but not all, subtypes highlights the need to appropriately characterize injured axons when evaluating potential neuroprotective strategies.

Cyclophilin D modulates mitochondrial acetylome.

RATIONALE: Mice lacking cyclophilin D (CypD(-/-)), a mitochondrial chaperone protein, have altered cardiac metabolism. As acetylation has been shown to regulate metabolism, we tested whether changes in protein acetylation might play a role in these metabolic changes in CypD(-/-) hearts. OBJECTIVE: Our aim was to test the hypothesis that loss of CypD alters the cardiac mitochondrial acetylome. METHODS AND RESULTS: To identify changes in lysine-acetylated proteins and to map acetylation sites after ablation of CypD, we subjected tryptic digests of isolated cardiac mitochondria from wild-type and CypD(-/-) mice to immunoprecipitation using agarose beads coupled to antiacetyl lysine antibodies followed by mass spectrometry. We used label-free analysis for the relative quantification of the 875 common peptides that were acetylated in wild-type and CypD(-/-) samples and found 11 peptides (10 proteins) decreased and 96 peptides (48 proteins) increased in CypD(-/-) samples. We found increased acetylation of proteins in fatty acid oxidation and branched-chain amino acid metabolism. To evaluate whether this increase in acetylation might play a role in the inhibition of fatty acid oxidation that was previously reported in CypD(-/-) hearts, we measured the activity of l-3-hydroxyacyl-CoA dehydrogenase, which was acetylated in the CypD(-/-) hearts. Consistent with the hypothesis, l-3-hydroxyacyl-CoA dehydrogenase activity was inhibited by ≈50% compared with the wild-type mitochondria. CONCLUSIONS: These results implicate a role for CypD in modulating protein acetylation. Taken together, these results suggest that ablation of CypD leads to changes in the mitochondrial acetylome, which may contribute to altered mitochondrial metabolism in CypD(-/-) mice.

Enhancing mitochondrial calcium buffering capacity reduces aggregation of misfolded SOD1 and motor neuron cell death without extending survival in mouse models of inherited amyotrophic lateral sclerosis.

Mitochondria have been proposed as targets for toxicity in amyotrophic lateral sclerosis (ALS), a progressive, fatal adult-onset neurodegenerative disorder characterized by the selective loss of motor neurons. A decrease in the capacity of spinal cord mitochondria to buffer calcium (Ca(2+)) has been observed in mice expressing ALS-linked mutants of SOD1 that develop motor neuron disease with many of the key pathological hallmarks seen in ALS patients. In mice expressing three different ALS-causing SOD1 mutants, we now test the contribution of the loss of mitochondrial Ca(2+)-buffering capacity to disease mechanism(s) by eliminating ubiquitous expression of cyclophilin D, a critical regulator of Ca(2+)-mediated opening of the mitochondrial permeability transition pore that determines mitochondrial Ca(2+) content. A chronic increase in mitochondrial buffering of Ca(2+) in the absence of cyclophilin D was maintained throughout disease course and was associated with improved mitochondrial ATP synthesis, reduced mitochondrial swelling, and retention of normal morphology. This was accompanied by an attenuation of glial activation, reduction in levels of misfolded SOD1 aggregates in the spinal cord, and a significant suppression of motor neuron death throughout disease. Despite this, muscle denervation, motor axon degeneration, and disease progression and survival were unaffected, thereby eliminating mutant SOD1-mediated loss of mitochondrial Ca(2+) buffering capacity, altered mitochondrial morphology, motor neuron death, and misfolded SOD1 aggregates, as primary contributors to disease mechanism for fatal paralysis in these models of familial ALS.

Depressing mitochondria-reticulum interactions protects cardiomyocytes from lethal hypoxia-reoxygenation injury.

BACKGROUND: Under physiological conditions, Ca(2+) transfer from the endoplasmic reticulum (ER) to mitochondria might occur at least in part at contact points between the 2 organelles and involves the VDAC1/Grp75/IP3R1 complex. Accumulation of Ca(2+) into the mitochondrial matrix may activate the mitochondrial chaperone cyclophilin D (CypD) and trigger permeability transition pore opening, whose role in ischemia/reperfusion injury is well recognized. We questioned here whether the transfer of Ca(2+) from ER to mitochondria might play a role in cardiomyocyte death after hypoxia-reoxygenation. METHODS AND RESULTS: We report that CypD interacts with the VDAC1/Grp75/IP3R1 complex in cardiomyocytes. Genetic or pharmacological inhibition of CypD in both H9c2 cardiomyoblasts and adult cardiomyocytes decreased the Ca(2+) transfer from ER to mitochondria through IP3R under normoxic conditions. During hypoxia-reoxygenation, the interaction between CypD and the IP3R1 Ca(2+) channeling complex increased concomitantly with mitochondrial Ca(2+) content. Inhibition of either CypD, IP3R1, or Grp75 decreased protein interaction within the complex, attenuated mitochondrial Ca(2+) overload, and protected cells from hypoxia-reoxygenation. Genetic or pharmacological inhibition of CypD provided a similar effect in adult mice cardiomyocytes. Disruption of ER-mitochondria interaction via the downregulation of Mfn2 similarly reduced the interaction between CypD and the IP3R1 complex and protected against hypoxia-reoxygenation injury. CONCLUSIONS: Our data (1) point to a new role of CypD at the ER-mitochondria interface and (2) suggest that decreasing ER-mitochondria interaction at reperfusion can protect cardiomyocytes against lethal reperfusion injury through the reduction of mitochondrial Ca(2+) overload via the CypD/VDAC1/Grp75/IP3R1 complex.

Involvement of the mitochondrial permeability transition pore in chronic ethanol-mediated liver injury in mice.

Chronic ethanol consumption increases sensitivity of the mitochondrial permeability transition (MPT) pore induction in liver. Ca(2+) promotes MPT pore opening, and genetic ablation of cyclophilin D (CypD) increases the Ca(2+) threshold for the MPT. We used wild-type (WT) and CypD-null (CypD(-/-)) mice fed a control or an ethanol-containing diet to investigate the role of the MPT in ethanol-mediated liver injury. Ca(2+)-mediated induction of the MPT and mitochondrial respiration were measured in isolated liver mitochondria. Steatosis was present in WT and CypD(-/-) mice fed ethanol and accompanied by increased terminal deoxynucleotidyl transferase dUTP-mediated nick-end label-positive nuclei. Autophagy was increased in ethanol-fed WT mice compared with ethanol-fed CypD(-/-) mice, as reflected by an increase in the ratio of microtubule protein 1 light chain 3B II to microtubule protein 1 light chain 3B I. Higher levels of p62 were measured in CypD(-/-) than WT mice. Ethanol decreased mitochondrial respiratory control ratios and select complex activities in WT and CypD(-/-) mice. Ethanol also increased CypD protein in liver of WT mice. Mitochondria from control- and ethanol-fed WT mice were more sensitive to Ca(2+)-mediated MPT pore induction than mitochondria from their CypD(-/-) counterparts. Mitochondria from ethanol-fed CypD(-/-) mice were also more sensitive to Ca(2+)-induced swelling than mitochondria from control-fed CypD(-/-) mice but were less sensitive than mitochondria from ethanol-fed WT mice. In summary, CypD deficiency was associated with impaired autophagy and did not prevent ethanol-mediated steatosis. Furthermore, increased MPT sensitivity was observed in mitochondria from ethanol-fed WT and CypD(-/-) mice. We conclude that chronic ethanol consumption likely lowers the threshold for CypD-regulated and -independent characteristics of the ethanol-mediated MPT pore in liver mitochondria.

ß3 adrenergic receptor selective stimulation during ischemia/reperfusion improves cardiac function in translational models through inhibition of mPTP opening in cardiomyocytes.

Selective stimulation of ß3 adrenergic-receptor (ß3AR) has been shown to reduce infarct size in a mouse model of myocardial ischemia/reperfusion. However, its functional long-term effect and the cardioprotective mechanisms at the level of cardiomyocytes have not been elucidated, and the impact of ß3AR stimulation has not been evaluated in a more translational large animal model. This study aimed at evaluating pre-perfusion administration of BRL37344 both in small and large animal models of myocardial ischemia/reperfusion. Pre-reperfusion administration of the ß3AR agonist BRL37344 (5 µg/kg) reduced infarct size at 2-and 24-h reperfusion in wild-type mice. Long-term (12-weeks) left ventricular (LV) function assessed by echocardiography and cardiac magnetic resonance (CMR) was significantly improved in ß3AR agonist-treated mice. Incubation with ß3AR agonist (BRL37344, 7 µmol/L) significantly reduced cell death in isolated adult mouse cardiomyocytes during hypoxia/reoxygenation and decreased susceptibility to deleterious opening of the mitochondrial permeability transition pore (mPTP), via a mechanism dependent on the Akt-NO signaling pathway. Pre-reperfusion BRL37344 administration had no effect on infarct size in cyclophilin-D KO mice, further implicating mPTP in the mechanism of protection. Large-white pigs underwent percutaneous coronary ischemia/reperfusion and 3-T CMR at 7 and 45 days post-infarction. Pre-perfusion administration of BRL37344 (5 µg/kg) decreased infarct size and improved long-term LV contractile function. A single-dose administration of ß3AR agonist before reperfusion decreased infarct size and resulted in a consistent and long-term improvement in cardiac function, both in small and large animal models of myocardial ischemia/reperfusion. This protection appears to be executed through inhibition of mPTP opening in cardiomyocytes.

CypD(-/-) hearts have altered levels of proteins involved in Krebs cycle, branch chain amino acid degradation and pyruvate metabolism.

Cyclophilin D (CypD) is a mitochondrial chaperone that has been shown to regulate the mitochondrial permeability transition pore (MPTP). MPTP opening is a major determinant of mitochondrial dysfunction and cardiomyocyte death during ischemia/reperfusion (I/R) injury. Mice lacking CypD have been widely used to study regulation of the MPTP, and it has been shown recently that genetic depletion of CypD correlates with elevated levels of mitochondrial Ca(2+). The present study aimed to characterize the metabolic changes in CypD(-/-) hearts. Initially, we used a proteomics approach to examine protein changes in CypD(-/-) mice. Using pathway analysis, we found that CypD(-/-) hearts have alterations in branched chain amino acid metabolism, pyruvate metabolism and the Krebs cycle. We tested whether these metabolic changes were due to inhibition of electron transfer from these metabolic pathways into the electron transport chain. As we found decreased levels of succinate dehydrogenase and electron transfer flavoprotein in the proteomics analysis, we examined whether activities of these enzymes might be altered. However, we found no alterations in their activities. The proteomics study also showed a 23% decrease in carnitine-palmitoyltransferase 1 (CPT1), which prompted us to perform a metabolomics analysis. Consistent with the decrease in CPT1, we found a significant decrease in C4/Ci4, C5-OH/C3-DC, C12:1, C14:1, C16:1, and C20:3 acyl carnitines in hearts from CypD(-/-) mice. In summary, CypD(-/-) hearts exhibit changes in many metabolic pathways and caution should be used when interpreting results from these mice as due solely to inhibition of the MPTP.

Lack of cyclophilin B in osteogenesis imperfecta with normal collagen folding.

Osteogenesis imperfecta is a heritable disorder that causes bone fragility. Mutations in type I collagen result in autosomal dominant osteogenesis imperfecta, whereas mutations in either of two components of the collagen prolyl 3-hydroxylation complex (cartilage-associated protein [CRTAP] and prolyl 3-hydroxylase 1 [P3H1]) cause autosomal recessive osteogenesis imperfecta with rhizomelia (shortening of proximal segments of upper and lower limbs) and delayed collagen folding. We identified two siblings who had recessive osteogenesis imperfecta without rhizomelia. They had a homozygous start-codon mutation in the peptidyl-prolyl isomerase B gene (PPIB), which results in a lack of cyclophilin B (CyPB), the third component of the complex. The proband's collagen had normal collagen folding and normal prolyl 3-hydroxylation, suggesting that CyPB is not the exclusive peptidyl-prolyl cis-trans isomerase that catalyzes the rate-limiting step in collagen folding, as is currently thought.

Endoplasmic reticulum stress contributes to heart protection induced by cyclophilin D inhibition.

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.

Absence of FKBP10 in recessive type XI osteogenesis imperfecta leads to diminished collagen cross-linking and reduced collagen deposition in extracellular matrix.

Recessive osteogenesis imperfecta (OI) is caused by defects in genes whose products interact with type I collagen for modification and/or folding. We identified a Palestinian pedigree with moderate and lethal forms of recessive OI caused by mutations in FKBP10 or PPIB, which encode endoplasmic reticulum resident chaperone/isomerases FKBP65 and CyPB, respectively. In one pedigree branch, both parents carry a deletion in PPIB (c.563_566delACAG), causing lethal type IX OI in their two children. In another branch, a child with moderate type XI OI has a homozygous FKBP10 mutation (c.1271_1272delCCinsA). Proband FKBP10 transcripts are 4% of control and FKBP65 protein is absent from proband cells. Proband collagen electrophoresis reveals slight band broadening, compatible with ≈10% over-modification. Normal chain incorporation, helix folding, and collagen T(m) support a minimal general collagen chaperone role for FKBP65. However, there is a dramatic decrease in collagen deposited in culture despite normal collagen secretion. Mass spectrometry reveals absence of hydroxylation of the collagen telopeptide lysine involved in cross-linking, suggesting that FKBP65 is required for lysyl hydroxylase activity or access to type I collagen telopeptide lysines, perhaps through its function as a peptidylprolyl isomerase. Proband collagen to organics ratio in matrix is approximately 30% of normal in Raman spectra. Immunofluorescence shows sparse, disorganized collagen fibrils in proband matrix.