Peipei Ping

Intracoronary gene transfer of fibroblast growth factor–5 increases blood flow and contractile function in an ischemic region of the heart

Increased coronary blood vessel development could potentially benefit patients with ischemic heart disease. In a model of stress–induced myocardial ischemia, intracoronary injection of a recombinant adenovirus expressing human fibroblast growth factor–5 (FCF–5) resulted in messenger RNA and protein expression of the transferred gene. Two weeks after gene transfer, regional abnormalities in stress–induced function and blood flow were improved, effects that persisted for 12 weeks. Improved blood flow and function were associated with evidence of angiogenesis. This report documents, for the first time, successful amelioration of abnormalities in myocardial blood flow and function following in vivo gene transfer.

Role of the Mitochondrial Permeability Transition in Myocardial Disease

Mitochondria play a key role in determining cell fate during exposure to stress. Their role during ischemia/reperfusion is particularly critical because of the conditions that promote both apoptosis by the mitochondrial pathway and necrosis by irreversible damage to mitochondria in association with mitochondrial permeability transition (MPT). MPT is caused by the opening of permeability transition pores in the inner mitochondrial membrane, leading to matrix swelling, outer membrane rupture, release of apoptotic signaling molecules such as cytochrome c from the intermembrane space, and irreversible injury to the mitochondria. During ischemia (the MPT priming phase), factors such as intracellular Ca2+ accumulation, long-chain fatty acid accumulation, and reactive oxygen species progressively increase mitochondrial susceptibility to MPT, increasing the likelihood that MPT will occur on reperfusion (the MPT trigger phase). Because functional cardiac recovery ultimately depends on mitochondrial recovery, cardioprotection by ischemic and pharmacological preconditioning must ultimately involve the prevention of MPT. Investigations into this area are beginning to unravel some of the mechanistic links between cardioprotective signaling and mitochondria.

Protein Kinase Cε Interacts With and Inhibits the Permeability Transition Pore in Cardiac Mitochondria

Abstract— Although functional coupling between protein kinase C&egr; (PKC&egr;) and mitochondria has been implicated in the genesis of cardioprotection, the signal transduction mechanisms that enable this link and the identities of the mitochondrial proteins modulated by PKC&egr; remain unknown. Based on recent evidence that the mitochondrial permeability transition pore may be involved in ischemia/reperfusion injury, we hypothesized that protein-protein interactions between PKC&egr; and mitochondrial pore components may serve as a signaling mechanism to modulate pore function and thus engender cardioprotection. Coimmunoprecipitation and GST-based affinity pull-down from mouse cardiac mitochondria revealed interaction of PKC&egr; with components of the pore, namely voltage-dependent anion channel (VDAC), adenine nucleotide translocase (ANT), and hexokinase II (HKII). VDAC1, ANT1, and HKII were present in the PKC&egr; complex at ≈2%, ≈0.2%, and ≈1% of their total expression, respectively. Moreover, in vitro studies demonstrated that PKC&egr; can directly bind and phosphorylate VDAC1. Incubation of isolated cardiac mitochondria with recombinant PKC&egr; resulted in a significant inhibition of Ca2+-induced mitochondrial swelling, an index of pore opening. Furthermore, cardiac-specific expression of active PKC&egr; in mice, which is cardioprotective, greatly increased interaction of PKC&egr; with the pore components and inhibited Ca2+-induced pore opening. In contrast, cardiac expression of kinase-inactive PKC&egr; did not affect pore opening. Finally, administration of the pore opener atractyloside significantly attenuated the infarct-sparing effect of PKC&egr; transgenesis. Collectively, these data demonstrate that PKC&egr; forms physical interactions with components of the cardiac mitochondrial pore. This in turn inhibits the pathological function of the pore and contributes to PKC&egr;-induced cardioprotection.

Mitochondrial PKCε and MAPK Form Signaling Modules in the Murine Heart. Enhanced Mitochondrial PKCε-MAPK Interactions and Differential MAPK Activation in PKCε-Induced Cardioprotection

Although activation of protein kinase C (PKC) epsilon and mitogen-activated protein kinases (MAPKs) are known to play crucial roles in the manifestation of cardioprotection, the spatial organization of PKCepsilon signaling modules in naïve and protected myocardium remains unknown. Based on evidence that mitochondria are key mediators of the cardioprotective signal, we hypothesized that PKCepsilon and MAPKs interact, and that they form functional signaling modules in mitochondria during cardioprotection. Both immunoblotting and immunofluorescent staining demonstrated that PKCepsilon, ERKs, JNKs, and p38 MAPK co-localized with cardiac mitochondria. Moreover, transgenic activation of PKCepsilon greatly increased mitochondrial PKCepsilon expression and activity, which was concomitant with increased mitochondrial interaction of PKCepsilon with ERKs, JNKs, and p38 as determined by co-immunoprecipitation. These complex formations appeared to be independent of PKCepsilon activity, as the interactions were also observed in mice expressing inactive PKCepsilon. However, although both active and inactive PKCepsilon bound to all three MAPKs, increased phosphorylation of mitochondrial ERKs was only observed in mice expressing active PKCepsilon but not in mice expressing inactive PKCepsilon. Examination of potential downstream targets of mitochondrial PKCepsilon-ERK signaling modules revealed that phosphorylation of the pro-apoptotic protein Bad was elevated in mitochondria. Together, these data show that PKCepsilon forms subcellular-targeted signaling modules with ERKs, leading to the activation of mitochondrial ERKs. Furthermore, formation of mitochondrial PKCepsilon-ERK modules appears to play a role in PKCepsilon-mediated cardioprotection, in part by the phosphorylation and inactivation of Bad.

The crystal structure of mouse VDAC1 at 2.3 Å resolution reveals mechanistic insights into metabolite gating

The voltage-dependent anion channel (VDAC) constitutes the major pathway for the entry and exit of metabolites across the outer membrane of the mitochondria and can serve as a scaffold for molecules that modulate the organelle. We report the crystal structure of a β-barrel eukaryotic membrane protein, the murine VDAC1 (mVDAC1) at 2.3 Å resolution, revealing a high-resolution image of its architecture formed by 19 β-strands. Unlike the recent NMR structure of human VDAC1, the position of the voltage-sensing N-terminal segment is clearly resolved. The α-helix of the N-terminal segment is oriented against the interior wall, causing a partial narrowing at the center of the pore. This segment is ideally positioned to regulate the conductance of ions and metabolites passing through the VDAC pore.

The nitric oxide hypothesis of late preconditioning

Abstract Ischemic preconditioning (PC) occurs in two phases: an early phase, which lasts 2–3 h, and a late phase, which begins 12–24 h later and lasts 3–4 days. The mechanism for the late phase of PC has been the subject of intensive investigation. We have recently proposed the “NO hypothesis of late PC”, which postulates that NO plays a prominent role both in initiating and in mediating this cardioprotective response. The purpose of this essay is to review the evidence supporting the NO hypothesis of late PC and to discuss its implications. We propose that, on day 1, a brief ischemic stress causes increased production of NO (probably via eNOS) and ·O2–, which then react to form ONOO–, ONOO–, in turn, activates the ɛ isoform of protein kinase C (PKC); either directly or via its reactive byproducts such as ·OH. Both NO and secondary species derived from ·O2– could also stimulate PKC ɛ independently. PKC ɛ activation triggers a complex signaling cascade that involves tyrosine kinases (among which Src and Lck appear to be involved) and probably other kinases, the transcription factor NF-κB, and most likely other as yet unknown components, resulting in increases transcription of the iNOS gene and increased iNOS activity on day 2, which is responsible for the protection during the second ischemic challenge. Tyrosine kinases also appear to be involved on day 2, possibly by modulating iNOS activity. According to this paradigm, NO plays two completely different roles in late PC: on day 1, it initiates the development of this response, whereas on day 2, it protects against myocardial ischemia. We propose that two different NOS isoforms are sequentially involved in late PC, with eNOS generating the NO that initiates the development of the PC response on day 1 and iNOS then generating the NO that protects against recurrent ischemia on day 2. The NO hypothesis of late PC puts forth a comprehensive paradigm that can explain both the initiation and the mediation of this complex phenomenon. Besides its pathophysiological implications, this hypothesis has potential clinical reverberations, since NO donors (i.e., nitrates) are widely used clinically and could be used to protect the ischemic myocardium in patients.

Functional Recovery in Traumatic Spinal Cord Injury after Transplantation of Multineurotrophin-Expressing Glial-Restricted Precursor Cells

Demyelination contributes to the physiological and behavioral deficits after contusive spinal cord injury (SCI). Therefore, remyelination may be an important strategy to facilitate repair after SCI. We show here that rat embryonic day 14 spinal cord-derived glial-restricted precursor cells (GRPs), which differentiate into both oligodendrocytes and astrocytes, formed normal-appearing central myelin around axons of cultured DRG neurons and had enhanced proliferation and survival in the presence of neurotrophin 3 (NT3) and brain-derived neurotrophin factor (BDNF). We infected GRPs with retroviruses expressing the multineurotrophin D15A (with both BDNF and NT3 activities) and then transplanted them into the contused adult thoracic spinal cord at 9 d after injury. Expression of D15A in the injured spinal cord is five times higher in animals receiving D15A-GRP grafts than ones receiving enhanced green fluorescent protein (EGFP)-GRP or DMEM grafts. Six weeks after transplantation, the grafted GRPs differentiated into mature oligodendrocytes expressing both myelin basic protein (MBP) and adenomatus polyposis coli (APC). Ultrastructural analysis showed that the grafted GRPs formed morphologically normal-appearing myelin sheaths around the axons in the ventrolateral funiculus (VLF) of spinal cord. Expression of D15A significantly increased the percentage of APC+ oligodendrocytes of grafted GRPs (15-30%). Most importantly, 8 of 12 rats receiving grafts of D15A-GRPs recovered transcranial magnetic motor-evoked potential responses, indicating that conduction through the demyelinated VLF axons was restored. Such electrophysiological recovery was not observed in rats receiving grafts of EGFP-GRPs, D15A-NIH3T3 cells, or an injection of an adenovirus expressing D15A. Recovery of hindlimb locomotor function was also significantly enhanced only in the D15A-GRP-grafted animals at 4 and 5 weeks after transplantation. Therefore, combined treatment with neurotrophins and GRP grafts can facilitate functional recovery after traumatic SCI and may prove to be a useful therapeutic strategy to repair the injured spinal cord.

Isoform-Selective Activation of Protein Kinase C by Nitric Oxide in the Heart of Conscious Rabbits A Signaling Mechanism for Both Nitric Oxide–Induced and Ischemia-Induced Preconditioning

Although isoform-selective translocation of protein kinase C (PKC) epsilon appears to play an important role in the late phase of ischemic preconditioning (PC), the mechanism(s) responsible for such translocation remains unclear. Furthermore, the signaling pathway that leads to the development of late PC after exogenous administration of NO in the absence of ischemia (NO donor-induced late PC) is unknown. In the present study we tested the hypothesis that NO activates PKC and that this is the mechanism for the development of both ischemia-induced and NO donor-induced late PC. A total of 95 chronically instrumented, conscious rabbits were used. In rabbits subjected to ischemic PC (six 4-minute occlusion/4-minute reperfusion cycles), administration of the NO synthase inhibitor Nomega-nitro-L-arginine (group III), at doses previously shown to block the development of late PC, completely blocked the ischemic PC-induced translocation of PKCepsilon but not of PKCeta, indicating that increased formation of NO is an essential mechanism whereby brief ischemia activates the epsilon isoform of PKC. Conversely, a translocation of PKCepsilon and -eta quantitatively similar to that induced by ischemic PC could be reproduced pharmacologically with the administration of 2 structurally unrelated NO donors, diethylenetriamine/NO (DETA/NO) and S-nitroso-N-acetylpenicillamine (SNAP), at doses previously shown to elicit a late PC effect. The particulate fraction of PKCepsilon increased from 35+/-2% of total in the control group (group I) to 60+/-1% after ischemic PC (group II) (P<0.05), to 54+/-2% after SNAP (group IV) (P<0.05) and to 52+/-2% after DETA/NO (group V) (P<0.05). The particulate fraction of PKCeta rose from 66+/-5% in the control group to 86+/-3% after ischemic PC (P<0.05), to 88+/-2% after SNAP (P<0.05) and to 85+/-1% after DETA/NO (P<0.05). Neither ischemic PC nor NO donors had any appreciable effect on the subcellular distribution of PKCalpha, -beta1, -beta2, -gamma, -delta, - micro, or -iota/lambda; on total PKC activity; or on the subcellular distribution of total PKC activity. Thus, the effects of SNAP and DETA/NO on PKC closely resembled those of ischemic PC. The DETA/NO-induced translocation of PKCepsilon (but not that of PKCeta) was completely prevented by the administration of the PKC inhibitor chelerythrine at a dose of 5 mg/kg (group VI) (particulate fraction of PKCepsilon, 38+/-4% of total, P<0.05 versus group V; particulate fraction of PKCeta, 79+/-2% of total). The same dose of chelerythrine completely prevented the DETA/NO-induced late PC effect against both myocardial stunning (groups VII through X) and myocardial infarction (groups XI through XV), indicating that NO donors induce late PC by activating PKC and that among the 10 isozymes of PKC expressed in the rabbit heart, the epsilon isotype is specifically involved in the development of this form of pharmacological PC. In all groups examined (groups I through VI), the changes in the subcellular distribution of PKCepsilon protein were associated with parallel changes in PKCepsilon isoform-selective activity, whereas total PKC activity was not significantly altered. Taken together, the results provide direct evidence that isoform-selective activation of PKCepsilon is a critical step in the signaling pathway whereby NO initiates the development of a late PC effect both after an ischemic stimulus (endogenous NO) and after treatment with NO-releasing agents (exogenous NO). To our knowledge, this is also the first report that NO can activate PKC in the heart. The finding that NO can promote isoform-specific activation of PKC identifies a new biological function of this radical and a new mechanism in the signaling cascade of ischemic PC and may also have important implications for other pathophysiological conditions in which NO is involved and for nitrate therapy.

Transgenic Overexpression of Constitutively Active Protein Kinase C ε Causes Concentric Cardiac Hypertrophy

Abstract—To test the hypothesis that activation of the protein kinase C (PKC) e isoform leads to cardiac hypertrophy without failure, we studied transgenic mice with cardiac-specific overexpression...

Functional Proteomic Analysis of Protein Kinase C ε Signaling Complexes in the Normal Heart and During Cardioprotection

Abstract— Using two-dimensional electrophoresis, mass spectrometry, immunoblotting, and affinity pull-down assays, we found that myocardial protein kinase C &egr; (PKC&egr;) is physically associated with at least 36 known proteins that are organized into structural proteins, signaling molecules, and stress-responsive proteins. Furthermore, we found that the cardioprotection induced by activation of PKC&egr; is coupled with dynamic modulation and recruitment of PKC&egr;-associated proteins. The results suggest heretofore-unrecognized functions of PKC&egr; and provide an integrated framework for the understanding of PKC&egr;-dependent signaling architecture and cardioprotection. (Circ Res. 2001;88:59-62.)