Roberta Gottlieb

Affiliation: Scripps

Associate Member, Division of Hematology, Molecular and Experimental Medicine

Biography

Dr Gottlieb recieved her MD from Johns Hopkins University. Following post-doctoral work in the laboratory of Michael Karin, she joined the Scripps Research Institute, where she has been an associate professor in the department of molecular and experimental medicine since 1996. She received the American Society of Hematology Junior Faculty Scholar Award in 1997, was appointed a Pew Scholar's Program in the Biomedical Sciences in 1997, and became a fellow of the American Heart Association in 2001.

Research Summary

Myocardial infarctions result in the death of half a million Americans annually. Our lab is interested in understanding the molecular events that commit the cell to a death program following ischemia/reperfusion in the heart. While ischemia itself is deleterious due to energy depletion, further damage ensues upon reperfusion, when there is a burst of reactive oxygen species produced and when apoptosis, or programmed cell death, is activated in vulnerable cells. Because apoptosis is a tightly regulated program, there exists the potential to interfere with the process and salvage cardiac cells. We are defining the biochemical events of cell death in the heart in order to identify potential therapeutic targets to mitigate reperfusion injury.

Using isolated perfused rat hearts subjected to global ischemia and reperfusion, we have found that, calpain is activated during reperfusion, leading to cleavage of Bid, a pro-apoptotic member of the Bcl-2 family. Bid targets the mitochondria, resulting in energetic failure and release of pro-apoptotic factors that mediate destruction of survival factors and DNA. This process appears to be a form of caspase-independent apoptosis. We have used TAT protein transduction in the isolated perfused heart to define the role of mitochondria in the cell death pathway. When Bid binds to mitochondria, structural alterations of inner and outer mitochondrial membranes result in the rearrangement of the cristae, dissociation of cytochrome c from the inner membrane, and permeabilization of the outer mitochondrial membrane allowing the release of multiple intermembrane space constituents including cytochrome c, SMAC, and Endonuclease G. We believe that alterations to the unique mitochondrial phospholipid, cardiolipin, underlies some of the structural alterations of mitochondrial membranes, and work is ongoing to understand how Bid might affect cardiolipin structure and membrane architecture.

Apoptosis Repressor with Caspase Recruitment Domain (ARC) is expressed at high levels in cardiac and skeletal muscle, and has been shown to be strongly protective against cell death mediated by oxidative stress, including high levels of administered hydrogen peroxide. We are using TAT-mediated protein transduction of ARC to understand its mechanism of protection against ischemia/reperfusion injury in the heart. A major goal is to identify the relevant proteins that interact with ARC in the regulation of cell death. ARC is located in the cytosol and endoplasmic reticulum, and its protective function may be related to control of the endoplasmic reticulum stress response that is activated during ischemia/reperfusion. Overexpression of ARC leads to its association with mitochondria and increased cardioprotection, raising the possibility that ARC may also interact with mitochondria to mediate cardioprotection. Recent work has shown that ARC interacts with Bax to prevent apoptosis through the mitochondrial pathway.

Recently we showed that chloramphenicol and other inhibitors of cytochrome P450 monooxygenases are able to reduce ischemia/reperfusion injury in the heart. These drugs are protective even when administered after ischemia, suggesting that they may have therapeutic potential in the treatment of myocardial infarction. Cytochrome P450 monooxygenases in the heart participate in arachidonic acid metabolism, generating a variety of metabolites that regulate contractility and vasomotor tone. In addition, some P450 enzymes are potent sources of superoxide, which may contribute to reperfusion injury. We are investigating the basis for this protective effect of P450 inhibition and are focusing on vasoactive arachidonic acid metabolites generated by P450 enzymes, as well as the excessive production of superoxide. Efforts to identify additional cardioprotective compounds and to extend the studies to in vivo models are ongoing.

Fluorescence live-cell imaging of adult cardiomyocyte mitochondria. A freshly isolated adult rat cardiomyocyte was dual-labeled with the mitochondrial dye tetramethylrhodamine methyl ester (TMRM) and nuclear marker Hoechst 33342, and then imaged through a 60x lens of an inverted Nikon TE300 fluorescence microscope. Using no neighbor 2D deconvolution algorithms (MetaMorph 6.2, Universal Imaging) the captured fluorescent images were digitally processed for enhanced resolution. The cationic, lipophilic TMRM (red) accumulates electrophoretically in the mitochondrial matrix according to the Nernst equation and thus allows visualization of the polarized mitochondrial population. In the adult cardiomyocyte, the mitochondria account for about 35% of total cell volume and are arranged longitudinally along the myofibrils at the sarcomeric A-band, providing the majority of ATP needed for contraction and ion homeostasis. Hoechst 33342 (blue) stains the nuclei, revealing regions of dense chromatin. Adult cardiomyocytes are often bi-nucleate and their nuclei are surrounded by densely grouped mitochondria.

References

References From PubMed (NCBI)

Training for Careers in Biomedical Research