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Roberta A Gottlieb

Publications and source records attributed to Roberta A Gottlieb.

At least 19 recordsLinked to original sources
PubMed2007

Identification of targets of phosphorylation in heart mitochondria.

Whereas most strategies in proteomics deal with changes in protein levels, posttranslational modifications often represent pathological consequences at the molecular level without changes in abundance and, therefore represent a critical phenomenon to study. The present report elucidates an approach to studying one such posttranslational modification, phosphorylation, which is an important event in a variety of signaling cascades initiated in the heart in response to physiological and pathological stimuli. Comparison of phosphorylation profiles in different conditions, including drug treatments, receptor stimulation or blockade, and so forth, may lead to identification of relevant targets of signal transduction cascades. Furthermore, it may be possible to devise similar schemes to detect the subset of proteins modified by ubiquitinylation, lipid acylation, and so forth, and to compare differential patterns. Phosphorylation of mitochondrial targets is increasingly being recognized. Mitochondria are critical targets of a number of signaling pathways and therefore it is important to develop suitable methods to detect phosphorylation of mitochondrial targets. One approach is to perform in vitro phosphorylation in a reconstituted system of cytosol and mitochondria.

Animals↗
PubMed2006

Autophagy as a protective response to Bnip3-mediated apoptotic signaling in the heart.

Bnip3 is a member of the 'BH3-only' Bcl-2 subfamily which has been implicated in apoptotic,(1) necrotic(2) and autophagic cell death.(3,4) We recently reported that Bnip3 is a key mediator of mitochondrial dysfunction and cell death in the ex vivo heart following ischemia/reperfusion (I/R).(5) Moreover, we found that Bnip3 was involved in upregulation of autophagy in I/R and that Bnip3-mediated mitochondrial dysfunction correlated with upregulation of autophagy. Using a model of simulated I/R and overexpression of Bnip3 in HL-1 cardiac myocytes, we determined that Bnip3-mediated upregulation of autophagic activity constituted a protective response against Bnip3 death signaling. Here we present additional evidence that enhanced autophagic activity functions as a cytoprotective pathway to oppose ischemia/reperfusion-related apoptosis.

Animals↗
PubMed2006

Bcl-2 family members and apoptosis, taken to heart.

Loss of myocardial cells via apoptosis has been observed in many cardiovascular diseases and has been shown to contribute to the initiation and progression of heart failure. The Bcl-2 family members are important regulators of the mitochondrial pathway of apoptosis. These proteins decide whether the mitochondria should initiate the cell death program and release proapoptotic factors such as cytochrome c. The Bcl-2 proteins consist of anti- and proapoptotic members and play a key role in regulating apoptosis in the myocardium. The antiapoptotic proteins have been demonstrated to protect against various cardiac pathologies, whereas the antiapoptotic proteins have been reported to contribute to heart disease. This review summarizes the current understanding of the role of Bcl-2 proteins in the heart.

Animals↗
PubMed2006

Enhancing macroautophagy protects against ischemia/reperfusion injury in cardiac myocytes.

Cardiac myocytes undergo programmed cell death as a result of ischemia/reperfusion (I/R). One feature of I/R injury is the increased presence of autophagosomes. However, to date it is not known whether macroautophagy functions as a protective pathway, contributes to programmed cell death, or is an irrelevant event during cardiac I/R injury. We employed simulated I/R of cardiac HL-1 cells as an in vitro model of I/R injury to the heart. To assess macroautophagy, we quantified autophagosome generation and degradation (autophagic flux), as determined by steady-state levels of autophagosomes in relation to lysosomal inhibitor-mediated accumulation of autophagosomes. We found that I/R impaired both formation and downstream lysosomal degradation of autophagosomes. Overexpression of Beclin1 enhanced autophagic flux following I/R and significantly reduced activation of pro-apoptotic Bax, whereas RNA interference knockdown of Beclin1 increased Bax activation. Bcl-2 and Bcl-x(L) were protective against I/R injury, and expression of a Beclin1 Bcl-2/-x(L) binding domain mutant resulted in decreased autophagic flux and did not protect against I/R injury. Overexpression of Atg5, a component of the autophagosomal machinery downstream of Beclin1, did not affect cellular injury, whereas expression of a dominant negative mutant of Atg5 increased cellular injury. These results demonstrate that autophagic flux is impaired at the level of both induction and degradation and that enhancing autophagy constitutes a powerful and previously uncharacterized protective mechanism against I/R injury to the heart cell.

Animals↗
PubMed2006

Alterations in oxidative phosphorylation complex proteins in the hearts of transgenic mice that overexpress the p38 MAP kinase activator, MAP kinase kinase 6.

Ischemia-reperfusion (I/R) has critical consequences in the heart. Recent studies on the functions of I/R-activated kinases, such as p38 mitogen-activated protein kinase (MAPK), showed that I/R injury is reduced in the hearts of transgenic mice that overexpress the p38 MAPK activator MAPK kinase 6 (MKK6). This protection may be fostered by changes in the levels of many proteins not currently known to be regulated by p38. To examine this possibility, we employed the multidimensional protein identification technology MudPIT to characterize changes in levels of proteins in MKK6 transgenic mouse hearts, focusing on proteins in mitochondria, which play key roles in mediating I/R injury in the heart. Of the 386 mitochondrial proteins identified, the levels of 58 were decreased, while only 2 were increased in the MKK6 transgenic mouse hearts. Among those that were decreased were 21 mitochondrial oxidative phosphorylation complex proteins, which was unexpected because p38 is not known to mediate such decreases. Immunoblotting verified that proteins in each of the five oxidative phosphorylation complexes were reduced in MKK6 mouse hearts. On assessing functional consequences of these reductions, we found that MKK6 mouse heart mitochondria exhibited 50% lower oxidative respiration and I/R-mediated reactive oxygen species (ROS) generation, both of which are predicted consequences of decreased oxidative phosphorylation complex proteins. Thus the cardioprotection observed in MKK6 transgenic mouse hearts may be partly due to decreased electron transport, which is potentially beneficial, because damaging ROS are known to be generated by mitochondrial complexes I and III during reoxygenation.

Animals↗
PubMed2006

Proapoptotic BCL-2 family members and mitochondrial dysfunction during ischemia/reperfusion injury, a study employing cardiac HL-1 cells and GFP biosensors.

The objective of this study was to evaluate mitochondrial alterations in a cell-based model of myocardial ischemia/reperfusion (I/R) injury. Using GFP-biosensors and fluorescence deconvolution microscopy, we investigated mitochondrial morphology in relation to Bax and Bid activation in the HL-1 cardiac cell line. Mitochondria underwent extensive fragmentation during ischemia. Bax translocation from cytosol to mitochondria was initiated during ischemia and proceeded during reperfusion. However, Bax translocation was not sufficient to induce cell death or mitochondrial dysfunction. Bid processing was caspase-8 dependent, and Bid translocation to mitochondria occurred after Bax translocation and clustering, and minutes before cell death. Clustering of Bax into distinct regions on mitochondria could be prevented by CsA, an inhibitor of the mitochondrial permeability transition pore, and also by SB203580, an inhibitor of p38 MAPK. Surprisingly, mitochondrial fragmentation which occurred during ischemia and before Bax translocation could be reversed by the addition of the p38 inhibitor SB203580 at reperfusion. Taken together, these results implicate p38 MAPK in the mitochondrial remodeling response to I/R that facilitates Bax recruitment to mitochondria.

Apoptosis↗
PubMed2005

Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death.

Mitochondria play a critical role in mediating both apoptotic and necrotic cell death. The mitochondrial permeability transition (mPT) leads to mitochondrial swelling, outer membrane rupture and the release of apoptotic mediators. The mPT pore is thought to consist of the adenine nucleotide translocator, a voltage-dependent anion channel, and cyclophilin D (the Ppif gene product), a prolyl isomerase located within the mitochondrial matrix. Here we generated mice lacking Ppif and mice overexpressing cyclophilin D in the heart. Ppif null mice are protected from ischaemia/reperfusion-induced cell death in vivo, whereas cyclophilin D-overexpressing mice show mitochondrial swelling and spontaneous cell death. Mitochondria isolated from the livers, hearts and brains of Ppif null mice are resistant to mitochondrial swelling and permeability transition in vitro. Moreover, primary hepatocytes and fibroblasts isolated from Ppif null mice are largely protected from Ca2+-overload and oxidative stress-induced cell death. However, Bcl-2 family member-induced cell death does not depend on cyclophilin D, and Ppif null fibroblasts are not protected from staurosporine or tumour-necrosis factor-alpha-induced death. Thus, cyclophilin D and the mitochondrial permeability transition are required for mediating Ca2+- and oxidative damage-induced cell death, but not Bcl-2 family member-regulated death.

Adenoviridae↗
PubMed2005

TAT-mediated protein transduction: delivering biologically active proteins to the heart.

TAT protein transduction is a novel method of delivering biologically active proteins into cells and tissues through the fusion of a protein transduction domain to the protein of interest. The present chapter outlines the methodology pertaining to the preparation of TAT-fusion proteins and how to efficiently transduce these proteins into cultured cells or isolated rat hearts perfused in Langendorff mode.

Animals↗
PubMed2004

Ischemia/reperfusion injury at the intersection with cell death.

Since its description in myocardial ischemia/reperfusion a decade ago, the process of programmed cell death has been the subject of intense study by investigators in cardiology. Comparison with other systems has identified parallels as well as features that are unique to the heart. An understanding of the molecular events that transpire during ischemia should inform our analysis of the relevant pathways of cell death. The intersection of ischemic preconditioning and cell death can provide illumination to both signal transduction pathways.

Animals↗
PubMed2004

Apoptosis repressor with caspase recruitment domain protects against cell death by interfering with Bax activation.

Myocardial ischemia/reperfusion (I/R) is associated with an extensive loss of myocardial cells. The apoptosis repressor with caspase recruitment domain (ARC) is a protein that is highly expressed in heart and skeletal muscle and has been demonstrated to protect the heart against I/R injury (Gustafsson, A. B., Sayen, M. R., Williams, S. D., Crow, M. T., and Gottlieb, R. A. (2002) Circulation 106, 735-739). In this study, we have shown that transduction of TAT-ARCL31F, a mutant of ARC in the caspase recruitment domain, did not reduce creatine kinase release and infarct size after I/R. TAT-ARCL31F also failed to protect against hydrogen peroxide-mediated cell death in H9c2 cells, suggesting that the caspase recruitment domain is important in mediating ARC's protective effects. In addition, we report that ARC co-immunoprecipitated with the pro-apoptotic protein Bax, which causes cytochrome c release when activated. TAT-ARC, but not TAT-ARCL31F, prevented Bax activation and cytochrome c release in hydrogen peroxide-treated H9c2 cells. TAT-ARC was also effective in blocking cytochrome c release after ischemia and reperfusion, whereas TAT-ARCL31F had no effect on cytochrome c release. In addition, recombinant ARC protein abrogated Bax-induced cytochrome c release from isolated mitochondria. This suggests that ARC can protect against cell death by interfering with activation of the mitochondrial death pathway through the interaction with Bax, preventing mitochondrial dysfunction and release of pro-apoptotic factors.

Animals↗
PubMed2004

Reduction of ischemia and reperfusion-induced myocardial damage by cytochrome P450 inhibitors.

Ischemia and reperfusion both contribute to tissue damage after myocardial infarction. Although many drugs have been shown to reduce infarct size when administered before ischemia, few have been shown to be effective when administered at reperfusion. Moreover, although it is generally accepted that a burst of reactive oxygen species (ROS) occurs at the onset of reperfusion and contributes to tissue damage, the source of ROS and the mechanism of injury is unclear. We now report the finding that chloramphenicol administered at reperfusion reduced infarct size by 60% in a Langendorff isolated perfused rat heart model, and that ROS production was also substantially reduced. Chloramphenicol is an inhibitor of mitochondrial protein synthesis and is also an inhibitor of a subset of cytochrome P450 monooxygenases (CYPs). We could not detect any effect on mitochondrial encoded proteins or mitochondrial respiration in chloramphenicol-perfused hearts, and hypothesized that the effect was caused by inhibition of CYPs. We tested additional CYP inhibitors and found that cimetidine and sulfaphenazole, two CYP inhibitors that have no effect on mitochondrial protein synthesis, were also able to reduce creatine kinase release and infarct size in the Langendorff model. We also showed that chloramphenicol reduced infarct size in an open chest rabbit model of regional ischemia. Taken together, these findings implicate CYPs in myocardial ischemia/reperfusion injury.

Animals↗
PubMed2003

Cytochrome P450: major player in reperfusion injury.

While attention has historically focused on mitochondria as the primary source of ROS in myocardial ischemia/reperfusion injury, recent evidence has implicated cytochrome P450 monooxygenases (CYPs) as a significant factor. CYPs represent a large family of enzymes that catalyze the oxidation of endogenous and exogenous compounds. They catalyze arachidonic acid oxidation to a variety of biologically active eicosanoids that regulate ion channels and protein kinases, with effects on vasomotor tone and cardiac inotropy. They also represent a significant source of reactive oxygen species that may target cellular homeostatic mechanisms and mitochondria. In this review, we will consider the contribution of cytochrome P450 enzymes to reperfusion injury and will speculate on whether the mechanism of injury is due to CYP-mediated ROS production or arachidonic acid metabolites.

Animals↗
PubMed2003

Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis.

During apoptosis, the permeabilization of the mitochondrial outer membrane allows the release of cytochrome c, which induces caspase activation to orchestrate the death of the cell. Mitochondria rapidly lose their transmembrane potential (Delta Psi m) and generate reactive oxygen species (ROS), both of which are likely to contribute to the dismantling of the cell. Here we show that both the rapid loss of Delta Psi m and the generation of ROS are due to the effects of activated caspases on mitochondrial electron transport complexes I and II. Caspase-3 disrupts oxygen consumption induced by complex I and II substrates but not that induced by electron transfer to complex IV. Similarly, Delta Psi m generated in the presence of complex I or II substrates is disrupted by caspase-3, and ROS are produced. Complex III activity measured by cytochrome c reduction remains intact after caspase-3 treatment. In apoptotic cells, electron transport and oxygen consumption that depends on complex I or II was disrupted in a caspase-dependent manner. Our results indicate that after cytochrome c release the activation of caspases feeds back on the permeabilized mitochondria to damage mitochondrial function (loss of Delta Psi m) and generate ROS through effects of caspases on complex I and II in the electron transport chain.

Amino Acid Chloromethyl Ketones↗
PubMed2003

Mitochondrial signaling in apoptosis: mitochondrial daggers to the breaking heart.

Mitochondria respond to signals from the cytoplasmic environment, including energy needs, substrate and oxygen availability, ion fluxes, as well as kinase and proteolytic signals. They integrate these complex, often conflicting signals with a coordinated response, whether for growth, hibernation, or apoptosis. In this review we consider the panoply of signals that impact the mitochondria, and focus particularly on the apoptotic pathways in which the mitochondria stand at the crossroads.

Animals↗
PubMed2003

Cytochrome c dissociation and release from mitochondria by truncated Bid and ceramide.

We have examined the effects of truncated Bid (tBid) and ceramide on mitochondrial membrane integrity and cytochrome c release, using mitochondria with intact outer membranes. While tBid permeabilizes the outer membrane and efficiently stimulates cytochrome c release, digitonin is unable to cause cytochrome c release in the absence of salt. Ceramides did not permeabilize the mitochondrial outer membrane, and stimulated cytochrome c release only in the presence of digitonin. Taken together, these observations support a model for cytochrome c release in which the first step is dissociation from the inner membrane followed by transit across the outer membrane.

Journal Article↗
PubMed2003

Mechanisms of apoptosis in the heart.

Apoptosis is a complex and highly regulated form of cell death, and believed to contribute to the continuous decline of ventricular function in heart failure. Apoptotic cell death is observed in a variety of cardiovascular diseases, including myocardial infarction, ischemia-reperfusion injury, end-stage heart failure, arrhythmias, and adriamycin cardiomyopathy. There are several pathways leading to programmed cell death. Apoptosis can be initiated by extracellular or intracellular stimuli, leading to the activation of caspases and subsequent cell death. A better understanding of the process of apoptosis in the heart is clearly important as it may lead to the identification of novel therapies for cardiovascular disease. This review is focused on the basic cellular mechanisms of apoptosis, as well as our current understanding of this process in the heart.

Animals↗