Division of Cardiovascular Medicine

MAJOR AREAS OF RESEARCH

Ischemic Preconditioning

Oxidative Stress

Gene Therapy

Regeneration of Cardiac Myocytes

Genetically-engineered Animal Models of Cardiovascular Diseases

The basic research program of the Division of Cardiology represents a multidisciplinary, comprehensive effort aimed at unraveling the mechanism of tissue injury during acute myocardial ischemia and reperfusion and developing innovative therapeutic strategies.  The two main focuses are the investigation of ischemic preconditioning and the development of intracoronary gene therapy.

1. Ischemic Preconditioning

Ischemic preconditioning is the phenomenon whereby a brief episode of myocardial ischemia renders the heart resistant to subsequent more severe ischemic insults.  Ischemic preconditioning is the most powerful form of protection against ischemic injury that has been discovered thus far, and has been described in all species examined, including man.  There are two phases of ischemic preconditioning: an early phase, which develops within minutes from the ischemic insult and lasts for 2-4 hours, and a late phase, which develops 12-24 hours after the ischemic insult and lasts for 3-5 days.  Because of its remarkable effectiveness, the exploitation of ischemic preconditioning in patients with coronary artery disease could represent a novel powerful strategy to protect the heart against the injury incurred during acute ischemia and reperfusion.  If we can understand the precise cellular mechanisms underlying ischemic preconditioning, it should be possible to develop drugs that can reproduce these effects and can be given to patients at risk for heart attacks.

The research program in Cardiology focuses on the mechanism of the late phase of ischemic preconditioning.  Following is a list of projects that are underway to address this issue.

Role of protein kinase C in ischemic preconditioning.  We have completed a number of projects focusing on the role of protein kinase C in mediating the protection afforded by ischemic preconditioning.  Using Western blotting, we have demonstrated that the rabbit heart expresses ten different isoforms of protein kinase C and that ischemic preconditioning selectively translocates two of these isoforms (epsilon and eta) from the cytosolic to the particulate fraction, indicating that they are activated.  We have also demonstrated that inhibitors of protein kinase C block the development of ischemic preconditioning and, at the same time, block the translocation of PKC epsilon, indicating that this isoform is specifically responsible for the protection afforded by ischemic preconditioning.  This is the first time that translocation of protein kinase C has been demonstrated in an in vivo model of ischemic preconditioning.  This is also the first time that the specific isoform of protein kinase C (the epsilon isoform) responsible for ischemic preconditioning has been identified.  Finally, this is the first time that the effect of a protein kinase C inhibitor on the development of preconditioning has been correlated with its effect on the activity of protein kinase C isoforms.  The identification of epsilon as the specific protein kinase C isoform involved in ischemic preconditioning now opens the way for the development of drugs that can selectively activate this isoform and therefore reproduce the protection afforded by ischemic preconditioning in patients.  We have also developed mutant forms of protein kinase C epsilon and expressed them in isolated myocytes to identify the downstream pathways that are initiated by protein kinase C.  Using this approach, we have demonstrated that protein kinase C epsilon activates the mitogen-activated protein kinases.  This is the first time that such a signaling mechanism has been demonstrated in the heart.  We have also developed transgenic mouse lines overexpressing either the wild-type or mutant types of protein kinase C epsilon and characterized the signaling events that are caused by these genetic manipulations.  This is the first time that a transgenic mouse line overexpressing epsilon has been developed and carefully characterized.  Such an approach will be very powerful to determine the role of protein kinase C epsilon in ischemic preconditioning as well as in many other pathophysiological processes involving the heart.

Role of nitric oxide in ischemic preconditioning.  We have demonstrated that the generation of NO during the preconditioning ischemia is the trigger that initiates the development of ischemic preconditioning.  We have also demonstrated that the generation of NO during the subsequent (second) ischemic episode 24 hours later mediates the cardioprotective effects of ischemic preconditioning.  This is the first time that NO has been discovered to play a role as a trigger of ischemic preconditioning.  This is also the first time that NO has been found to play a role as a mediator of ischemic preconditioning.  We have also demonstrated, for the first time, that administration of drugs that release NO (NO donors) in the absence of ischemia can produce a preconditioning effect similar to that produced by ischemia; that is, NO donors can mimic the protection of ischemic preconditioning.  On the basis of these discoveries, we have proposed, for the first time, the nitric oxide hypothesis of late preconditioning, which postulates that NO acts both as the trigger and the mediator of this powerful cardioprotective effect.  This hypothesis has major pathophysiological and clinical implications, because it predicts that NO donors could be used in patients to chronically precondition the heart against heart attacks.

Role of antioxidant enzymes in ischemic preconditioning.  One of the major hypotheses regarding ischemic preconditioning is that it is mediated by an increase in antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, etc.  To test this hypothesis, we have measured the activity of antioxidant enzymes after preconditioning and found that there was no increase.  Therefore, we have been able to refute one of the major leading hypotheses regarding preconditioning.

Role of adenosine A3 receptors in ischemic preconditioning.  Adenosine A3 receptors are a novel subtype of adenosine receptors that has been discovered in the last few years and holds great promise for therapeutic application.  Activation of these receptors does not have the undesirable hemodynamic effects associated with activation of adenosine A1 receptors, such as decrease in heart rate, blood pressure, etc.  We have already demonstrated, for the first time, that activation of A3 receptors protects the heart against both myocardial infarction and myocardial stunning without hemodynamic effects.  This suggests that A3 receptor agonists could be used clinically to protect the heart in patients with coronary artery disease.  We have developed a transgenic mouse line that overexpresses the A3 receptor to study the effect of this receptor on myocardial ischemia/reperfusion injury.  We are now examining the mechanism whereby A3 receptor activation effects protection of the ischemic myocardium.  We are studying these mechanisms in isolated cardiomyocytes as well as transgenic mice.  These data could have great importance for therapeutic strategies aimed at protecting ischemic myocardium in humans.

Role of other kinases in ischemic preconditioning.  Besides protein kinase C, a number of other kinases have been suggested to contribute to ischemic preconditioning, including mitogen activated protein (MAP) kinases, tyrosine kinases, etc.  We have demonstrated, for the first time, that two subfamilies of the mitogen activated protein kinase family (the ERK1 and ERK2 subfamily and the JNK/SAPK subfamily) are activated by ischemic preconditioning in vivo.  We have also demonstrated, for the first time, that the activation of these subfamilies of mitogen activated protein kinases is mediated by protein kinase C.  Specifically, we have shown in isolated myocytes that overexpression of the ? isoform of protein kinase C causes activation of ERK1 and ERK2.  This is a novel signaling mechanism that could have major implications not only for ischemic preconditioning, but also for many other pathophysiological processes.  We are now administering inhibitors of mitogen activated protein kinases to see whether they block the protection afforded by late preconditioning.  We have also demonstrated that the mechanism for the activation of mitogen activated protein kinases is the activation of these kinases in the cytosol and their subsequent translocation to the nucleus.  The elucidation of the signaling mechanisms underlying ischemic preconditioning is essential to understand the mechanism of this phenomenon and to develop therapeutic strategies.

Role of nuclear factor kappa-B in ischemic preconditioning.  The antioxidant-sensitive nuclear transcription factor NF-kappa-B plays a pivotal role in many cellular responses to stress.  We have demonstrated, for the first time, that ischemic preconditioning activates this factor and that the activation of this factor is essential for the development of the protective response, by causing upregulation of NOS.  We have also characterized the signaling factors that lead to NF-kappa-B activation.  We are currently developing transgenic mice overexpressing mutant forms of this factor to conclusively establish its role in ischemic preconditioning.

Role of Cyclooxygenase 2 (COX-2) in ischemic preconditioning. One molecular consequence of ischemic preconditioning is the up regulation of one of the key enzymes in prostaglandin synthesis, COX-2. The importance of COX-2 in late PC has been defined in mice and rabbit models of preconditioning by pharmacologically inhibiting COX-2. In these studies preconditioning was unable to decrease the extent of reperfusion injury after COX-2 inhibition. COX-2 is clearly a mediator of protection as a result of ischemic preconditioning. This protective mechanism is dependent on the induction of iNOS and the formation of NO and is downstream of the JAK – STAT signaling pathway. Although the role of COX-2 in ischemic late preconditioning is clear, the exact mechanism by which this enzyme is able to impart its protective effect is unclear and the focus of ongoing research. COX-2 is at the top the arachidonic acid metabolism cascade that leads to the formation of prostaglandin and thromboxane metabolites. These metabolites then have multiple actions including the activation of a family of prostanoid receptors comprised of at least 8 subtypes. Ongoing studies are using genetically modified mice to delineate how each prostanoid receptor subtype is involved in COX-2 mediated preconditioning, The importance of completely understanding the role of COX-2 in preconditioning and its underlying mechanisms has recently been underscored by the withdrawal of COX-2 inhibitors that had served as popular and effective relief for those with arthritis due to increased cardiovascular risk with long term use.

2. Oxidative Stress

The major focus of our research is to elucidate the mechanisms by which oxidative stress affects cardiovascular function. We are investigating the biochemical pathways that metabolize and detoxify reactive aldehydes generated by lipid peroxidation. We are studying the kinetic and structural properties of the enzymes that are involved in aldehyde metabolism. Our aim is to identify and characterize aldehyde metabolites in cardiovascular tissue and to delineate the signaling pathways that regulate the expression and activity of aldehyde-metabolizing enzymes. Using rodent models, we are assessing the contribution of lipid peroxidation to myocardial ischemia and reperfusion and atherosclerosis. Our hypothesis is that aldehydes derived from lipid peroxidation are important signaling molecules that regulate cell growth and apoptosis. Therefore, we are currently investigating the cellular and molecular mechanisms by which lipid-derived aldehydes affect intracellular signaling and cause cardiovascular dysfunction.

3. Gene Therapy

 Gene therapy is emerging as a revolution in medicine that offers almost unlimited potential for innovative therapeutic approaches for a variety of disorders.  We are developing techniques to deliver genes via the intracoronary route.  Using a cineangiography apparatus generously donated by the Jewish Hospital, we have developed miniaturized catheters to inject adenoviral vectors containing selected genes into the left coronary artery of rabbits.  We have already found that bolus injection of adenoviral vectors results in transfection of both endothelial cells and myocytes within the heart.  We are now attempting to increase the efficiency of gene transfer, so that the majority of myocytes can be transduced after the intracoronary injection.  In addition, we are developing a new generation of adenoviral vectors that will be less immunogenic, and therefore will not cause inflammatory reactions.  We are the only laboratory in the world that can administer selective intracoronary infusions of genes or drugs in a rabbit using cardiac catheterization.  The long-term goal of research is to develop a technique that will enable us to deliver cardioprotective genes to the heart via the intracoronary route.  Presumably, these will be the same genes that we will have identified as the mediators of protection during the late phase of preconditioning.  This will enable us to exploit the powerful cardioprotective mechanisms of ischemic preconditioning to protect the heart by directly delivering the genes to the myocardium.  We have already demonstrated the principle that gene therapy can protect against both myocardial stunning and myocardial infarction.  By administering a recombinant form of extracellular superoxide dismutase by the intravenous route, we have demonstrated that this gene is expressed in the liver, where it leads to the overexpression of extracellular superoxide dismutase which can then be redistributed to the heart by giving heparin.  This results in marked protection against myocardial stunning and myocardial infarction.

4. Regeneration of Cardiac Myocytes

A major focus of research for the IMC is the study of cardiac and bone marrow stem cells to regenerate dead myocardium following myocardial infarction.  IMC investigators have demonstrated that the bone marrow contains cells that are committed to cardiac differentiation and are responsible for cardiac repair following acute ischemia/reperfusion injury.  The IMC investigators have also used cardiac stem cells (i.e., stem cells that are resident in the heart) to repair the myocardium following an infarction.  State-of-the-art facilities are available for confocal microscopy, echocardiographic studies of mice and rats, and cell sorting, in collaboration with Dr. Mariusz Ratajczak at the Brown Cancer Center.  Current areas of work include the utility of intracoronary infusion of cardiac stem cells for regeneration of myocardium after infarction and the function of circulating bone marrow-derived cells that are committed to cardiac lineage. 

5. Genetically-Engineered Animal Models of Cardiovascular Disease

The IMC has utilized a plethora of transgenic and knockout mouse models to study cardiovascular disease.  The IMC investigators have generated many novel mouse lines.  Under the direction of Dr. Yiru Guo, the Murine Physiology Laboratory has become a leading lab worldwide in murine physiology, having studied over 11,000 mice since 1997.  Under the direction of Dr. Gregg Rokosh, the Transgenic/Knockout Core has developed several novel transgenic and knockout animals, including cardiac-restricted transgenic and knockout mice.  These models are being utilized to probe the molecular basis of preconditioning and cardioprotection.