Amy J. Davidoff, Ph.D

Research Interests

My laboratory is currently engaged in defining the intracellular signaling pathways associated with the development of abnormal excitation-contraction (E-C) coupling in heart failure. We are particularly interested in the effects of diabetes and the factors that contribute to the onset of diabetic cardiomyopathy. This diabetic cardiomyopathy is a dysfunction directly associated with abnormal ventricular muscle cells rather than simply a response to coronary artery disease. We use both in vivo and in vitro models of type 1 and type 2 diabetes to better understand the adverse effects on the heart, and we employ biophysical techniques to evaluate E-C coupling in single ventricular myocytes, and protein biochemistry to evaluate changes in intracellular signaling.

Biophysical Assessments of E-C coupling
The major focus of my research is determining the role played by diabetes in altering the function and expression of Ca²⁺ regulating mechanisms (e.g., L-type calcium channel, sarco/endoplasmic reticulum ATPase (SERCA), and the Na-Ca exchanger (NCX)). We have begun to delineate the effects of diabetes on Ca²⁺ homeostasis in cardiac myocytes. Using freshly isolated ventricular myocytes from diabetic rats, as well as myocytes cultured in a "diabetic medium", we are able to examine changes in the expression, function and regulation of these excitation-contraction coupling mechanisms. We utilize whole-cell current and voltage clamp techniques (i.e., patch clamp methods) to characterize changes in action potential configuration and associated ionic currents, and a computerized video based-detection system coupled with spectrofluorometry (fura-2 and fluo-4) to assess changes in contractility and Ca²⁺ transients in single cells. We investigate Ca²⁺ regulation in intact cardiomyocytes by employing real-time spectrofluorometry using Ca²⁺ sensitive, membrane permeable dyes, inconjunction with rapid (msec) switching perfusion techniques.

Signal Transduction Assessments
A major focus of our research is to determine whether there is a link between cellular insulin resistance (i.e., impaired insulin signaling) and abnormal E-C coupling. We have recently demonstrated that either high extracellular glucose and elevated fatty acids (common to both type 1 and type 2 diabetes) have profound impacts on cardiomyocyte E-C coupling and insulin signaling. We are currently investigating the underlying cellular mechanisms associated with glucose- and fatty acid-induced changes in the signaling mechanisms contributing to these changes in cardiac myocytes. For example, glucose toxicity appears to depress myocyte PKA levels and elevate PKC activity, both of which impact E-C coupling mechanisms (e.g., depress SERCA function and transient outward potassium current). Elevated PKC also blunts insulin-stimulated glucose uptake (assessed by ³H-deoxyglucose uptake measurements). Fatty acid metabolism may alter these processes in a similar manner. We also employ immunoblot techniques to evaluate changes in the extent of phosphorylation and glycosylation of key cellular targets (e.g., insulin receptor substrates and ion channels).

Clinically Relevant Implications
We are also exploring the mechanisms which underlie the cardioprotective effects of specific antidiabetic agents. For example, both troglitazone (PPARagonist) and metformin (AMP Kinase activator) prevent most of the high glucose-induced dysfunctions in our cell culture system. We have recently shown that various clinically relevant treatment strategies (e.g., antidiabetic agents, exercise, dietary fish oil) are efficacious in preventing or reversing diabetic cardiomyopathy in insulin resistant rats. We have also begun to evaluate the efficacy of dietary supplements and natural products on preventing/reversing cardiovascular complications associated with diabetes.

 

9/19/05