Biomedical subjects

Roberta A Gottlieb

Publications and source records attributed to Roberta A Gottlieb.

26 records · Page 2Linked to original sources

PubMed2003

The mitochondrial voltage-dependent anion channel (VDAC) as a therapeutic target for initiating cell death.

Voltage-dependent anion channels (VDAC) are major constituents of the outer mitochondrial membrane. Over the past few years, several hypotheses and mechanisms have been forwarded for and against a role for VDAC in outer mitochondrial membrane permeability and the subsequent release of apoptosis promoting factors. The current review outlines current knowledge related to the role of VDAC in the regulation of cell death and speculates on possible therapeutic applications through VDAC modulation.

Animals↗

PubMed2002

Calcineurin transgenic mice have mitochondrial dysfunction and elevated superoxide production.

Introduction of the constitutively active calcineurin gene into neonatal rat cardiomyocytes by adenovirus resulted in decreased mitochondrial membrane potential (P < 0.05). Infection of H9c2 cells with calcineurin adenovirus resulted in increased superoxide production (P < 0.001). Transgenic mice with cardiac-specific expression of a constitutively active calcineurin cDNA (CalTG mice) exhibit a two- to threefold increase in heart size that progresses to heart failure. We prepared mitochondria enriched for the subsarcolemmal population from the hearts of CalTG mice and transgene negative littermates (control). Intact, well-coupled mitochondria prepared from one to two mouse hearts at a time yielded sufficient material for functional studies. Mitochondrial oxygen consumption was measured with a Clark-type oxygen electrode with substrates for mitochondrial complex II (succinate) and complex IV [tetramethylpentadecane (TMPD)/ascorbate]. CalTG mice exhibited a maximal rate of electron transfer in heart mitochondria that was reduced by approximately 50% (P < 0.002) without a loss of respiratory control. Mitochondrial respiration was unaffected in tropomodulin-overexpressing transgenic mice, another model of cardiomyopathy. Western blotting for mitochondrial electron transfer subunits from mitochondria of CalTG mice revealed a 20-30% reduction in subunit 3 of complex I (ND3) and subunits I and IV of cytochrome oxidase (CO-I, CO-IV) when normalized to total mitochondrial protein or to the adenine nucleotide transporter (ANT) and compared with littermate controls (P < 0.002). Impaired mitochondrial electron transport was associated with high levels of superoxide production in the CalTG mice. Taken together, these data indicate that calcineurin signaling affects mitochondrial energetics and superoxide production. The excessive production of superoxide may contribute to the development of cardiac failure.

Adenosine Diphosphate↗

PubMed2002

TAT protein transduction into isolated perfused hearts: TAT-apoptosis repressor with caspase recruitment domain is cardioprotective.

BACKGROUND: Linkage of the 11-amino-acid transduction domain of HIV TAT to a heterologous protein allows the protein to be transduced readily into cells. METHODS AND RESULTS: In this study, we inserted the apoptosis repressor with caspase recruitment domain (ARC) or beta-galactosidase (beta-gal) cDNA into the pTAT-hemagglutinin bacterial expression vector to produce genetic in-frame TAT-ARC or TAT-beta-gal fusion proteins for use in cell culture and in Langendorff perfusion of adult rat hearts. TAT-beta-gal and TAT-ARC were conjugated with Texas Red and could be detected in >95% of cells. TAT-ARC was able to protect H9c2 cells against cell death mediated by hydrogen peroxide, as measured by protection against the loss of mitochondrial membrane potential and preservation of nuclear morphology. Isolated adult hearts were perfused with recombinant TAT-beta-gal or TAT-ARC (20 nmol/L) for 15 minutes and then subjected to 30 minutes of global no-flow ischemia, followed by 2 hours of reperfusion. Protein transduction was assessed by Western blotting of cell lysates and cytosolic and mitochondrial fractions and by fluorescence microscopy of Texas Red-conjugated TAT proteins. TAT-beta-gal and TAT-ARC readily transduced into perfused hearts and were homogeneously distributed. Infarct size was determined by 2,3,5-triphenyltetrazolium chloride staining, and creatine kinase release was measured. Transduction of TAT-ARC was cardioprotective when administered before global ischemia and reperfusion. CONCLUSIONS: Our results demonstrate that TAT-linked fusion protein transduction into the myocardium is feasible and that transduction of TAT-ARC is protective in cell culture and in the perfused heart.

Animals↗

PubMed2002

Mitochondria: regulators of cell death and survival.

The past 5 years has seen an intense surge in research devoted toward understanding the critical role of mitochondria in the regulation of cell death. Apoptosis can be initiated by a wide array of stimuli, inducing multiple signaling pathways that, for the most part, converge at the mitochondrion. Although classically considered the powerhouses of the cell, it is now understood that mitochondria are also "gatekeepers" that ultimately determine the fate of the cell. The mitochondrial decision as to whether a cell lives or dies is complex, involving protein-protein interactions, ionic changes, reactive oxygen species, and other mechanisms that require further elucidation. Once the death process is initiated, mitochondria undergo conformational changes, resulting in the release of cytochrome c (cyt c), caspases, endonucleases, and other factors leading to the onset and execution of apoptosis. The present review attempts to outline the complex milieu of events regulating the mitochondrial commitment to and processes involved in the implementation of the executioner phase of apoptotic cell death.

Animals↗

PubMed2002

Calpain and mitochondria in ischemia/reperfusion injury.

Studies of ischemia/reperfusion (I/R) injury and preconditioning have shown that ion homeostasis, particularly calcium homeostasis, is critical to limiting tissue damage. However, the relationship between ion homeostasis and specific cell death pathways has not been investigated in the context of I/R. Previously we reported that calpain cleaved Bid in the absence of detectable caspase activation (1). In this study, we have shown that an inhibitor of the sodium/hydrogen exchanger prevented calpain activation after I/R. Calpain inhibitors prevented cleavage of Bid as well as the downstream indices of cell death, including DNA strand breaks, creatine kinase (CK) release, and infarction measured by triphenyl tetrazolium chloride (TTC) staining. In contrast, the broad spectrum caspase inhibitor IDN6734 was not protective in this model. To ascertain whether mitochondrial dysfunction downstream of these events was a required step, we utilized a peptide corresponding to residues 4-23 of Bcl-x(L) conjugated to the protein transduction domain of HIV TAT (TAT-BH4), which has been shown to protect mitochondria against Ca2+-induced deltaPsi(m) loss (2). TAT-BH4 attenuated CK release and loss of TTC staining, demonstrating the role of mitochondria and a pro-apoptotic Bcl-2 family member in the process leading to cell death. We propose the following pathway. (i) Reperfusion results in sodium influx followed by calcium accumulation. (ii) This leads to calpain activation, which in turn leads to Bid cleavage. (iii) Bid targets the mitochondria, causing dysfunction and release of pro-apoptotic factors, resulting in DNA fragmentation and death of the cell. Ischemia/reperfusion initiates a cell death pathway that is independent of caspases but requires calpain and mitochondrial dysfunction.

Animals↗

PubMed2002

Inhibition of mitochondrial calcium-independent phospholipase A2 (iPLA2) attenuates mitochondrial phospholipid loss and is cardioprotective.

Calcium-independent phospholipase A(2) (iPLA(2)) is the predominant phospholipase A(2) present in myocardium, and its pathophysiological role in acute myocardial infarction has been suggested by the rapid increase in membrane-associated iPLA(2) activity during myocardial ischaemia and reperfusion (I/R). We therefore examined iPLA(2) in mitochondrial fractions prepared from Langendorff-perfused adult rabbit hearts. Our studies indicate that iPLA(2)beta is present in rabbit heart mitochondrial inner membranes with no apparent translocation during ischaemia, I/R or preconditioning. Mitochondrion-associated iPLA(2) was catalytically competent and exhibited 2-, 3- and 2.5-fold increases in measured iPLA(2) activity following ischaemia, I/R and preconditioning, respectively, when compared with the activity of iPLA(2) measured in mitochondria from control hearts. Mitochondrial phospholipids are essential for maintaining the ordered structure and function of the organelle. I/R resulted in a rapid overall decrease in phosphatidylcholine and phosphatidylethanolamine glycerophospholipid species, as determined by electrospray ionization MS, that was partially alleviated by pretreatment of hearts with the iPLA(2)-specific inhibitor, bromoenol lactone (BEL). Pretreatment of I/R hearts with 10 microM BEL significantly reduced the infarct size almost to that of continuously perfused hearts and was cardioprotective only when administered prior to ischaemia. Cardioprotection by BEL was reversed by the simultaneous perfusion of 100 microM 5-hydroxydecanoate, implicating the mitochondrial K(ATP) channel in BEL-mediated protection from I/R. Preconditioning also significantly reduced the infarct size in response to I/R but protection was lost by concurrent perfusion of 10 microM arachidonic acid. Taken together, these data strongly implicate mitochondria-associated iPLA(2) in the signal transduction of myocardial I/R injury.

Animals↗

PubMed2002

Analyzing mitochondrial changes during apoptosis.

Mitochondria play a central role in programmed cell death through the release of cytochrome c and other proapoptotic factors. Fluorescence microscopy is used to visualize cytochrome c translocation and loss of mitochondrial membrane potential. Flow cytometry can also be used to measure mitochondrial membrane potential. Cytochrome c content in cytosol and mitochondria can be determined by immunoblotting after subcellular fractionation or selective permeabilization with digitonin. Isolated mitochondria can be used to study the mechanism of cytochrome c release. This article summarizes some of the more widely used methods to assess mitochondrial alterations in apoptosis.

Apoptosis↗

PubMedDate not supplied

A wave of reactive oxygen species (ROS)-induced ROS release in a sea of excitable mitochondria.

Once considered simply as the main source of ATP, mitochondria are now implicated in the control of many additional aspects of cell physiology, such as calcium signaling, and pathology, as in injury incurred on ischemia and subsequent reperfusion (I/R). Mitochondrial respiration is ordinarily accompanied by low-level ROS production, but they can respond to elevated ROS concentrations by increasing their own ROS production, a phenomenon termed ROS-induced ROS release (RIRR). Two modes of RIRR have been described. In the first mode of RIRR, enhanced ROS leads to mitochondrial depolarization via activation of the MPTP, yielding a short-lived burst of ROS originating from the mitochondrial electron transport chain (ETC). The second mode of RIRR is MPTP independent but is regulated by the mitochondrial benzodiazepine receptor (mBzR). Increased ROS in the mitochondrion triggers opening of the inner mitochondrial membrane anion channel (IMAC), resulting in a brief increase in ETC-derived ROS. Both modes of RIRR have been shown to transmit localized mitochondrial perturbations throughout the cardiac cell in the form of oscillations or waves but are kinetically distinct and may involve different ROS that serve as second messengers. In this review, we discuss the mechanisms of these different modes of RIRR.

Animals↗