Cardiac Surgery Research Laboratory
The Cardiac Surgery Research Program comprises approximately 4,000 square feet in the North Campus Research Complex. These laboratories support the majority of basic research in the Department of Cardiac Surgery.
The Cardioprotection Research Lab currently studies the inflammatory and oxidative stress pathways involved in cardiac fibrosis and heart failure progression. Mitral regurgitation, mitral insufficiency or mitral incompetence are disorders of the heart in which the mitral valve does not close properly when the heart pumps out blood. It is the abnormal leaking of blood from the left ventricle through the mitral valve and into the left atrium when the left ventricle contracts resulting in regurgitation of blood back into the left atrium. Mitral regurgitation (MR) is the most common form of valvular heart disease, and unmanaged MR often results in heart failure in the elderly. Heart failure is currently the #1 health care expense in in the US.
If MR develops slowly over years, the individual may exist in a compensated phase of the disease. The left ventricle develops eccentric hypertrophy to manage the larger than normal stroke volume. The eccentric hypertrophy and the increased diastolic volume combine to increase stroke volume so that the forward stroke volume (cardiac output) approaches normal levels. Volume overload enlarges the left atrium which reduces left atrium filling pressure; this improves drainage from the pulmonary veins and decreases pulmonary congestion. Individuals in this chronic compensated phase may be asymptomatic for signs of heart failure, but they may display abnormal diastolic function that can be detected clinically.
Individuals with MR and chronic volume overload (as from unmanaged hypertension) are at risk for decompensation to left ventricular dysfunction and symptomatic heart failure. In this phase, the left ventricle cannot contract adequately to compensate for MR-induced volume overload and left ventricle stroke volume will decrease. This effect decreases cardiac output and increases end-systolic volume. Increased end-systolic volume increases left ventricle filling pressures and pulmonary venous congestion. The individual then typically becomes symptomatic for "congestive" heart failure like exercise intolerance and shortness of breath. The left ventricle further dilates causing dilatation of the mitral valve annulus and worsening of MR.
It is currently unclear what causes cardiac decompensation during heart failure progression; these pathways and means to influence them are of interest to our lab. Our research uses rodent models of hypertension and chronic volume overload that result in heart failure. As with human MR-associated heart failure, our rat models first develop a compensated cardiac pathology (eccentric hypertrophy, increased diastolic volume, increased stroke volume, enlarged left atria, and reduced atrial filling pressure, and normal ejection fraction). Eventually, the chronic volume overload results in decompensated heart failure. The rats also then become symptomatic for congestive heart failure including severe pulmonary congestion, labored breathing, and cachexia.
Research in our lab is currently focused on both drugs and natural products (diet, dietary supplements) that may affect the severity and trajectory of heart failure. Basic research has focused on the alterations of the inflammatory and oxidative stress pathways involved in cardiac fibrosis and heart failure progression.
The Cardiac Myocyte Structure and Function Laboratory is investigating the role of troponin I (TnI) in the adult cardiac myocyte contractile response to protein kinase C. Protein kinase C (PKC) is activated in myocardium by multiple acute physiological stimuli and under several important chronic pathophysiological conditions, including ischemia. Acute activation of PKC by physiological agonists generally increases contractile function in cardiac myocytes. The cardiac isoform of TnI (cTnI) is one of the target proteins phosphorylated by PKC, and this regulatory thin filament phosphoprotein is postulated to have an important role in the positive inotropic response to PKC agonists. Recently, our laboratory determined that cTnI phosphorylation by PKC causes accelerated relaxation in cardiac myocytes. This enhancement in the rate of relaxation is removed in myocytes expressing the slow skeletal isoform of TnI, which is not phosphorylated by protein kinase C. Ongoing studies are in the process of defining the cTnI site(s) phoshorylated by protein kinase C in the intact myofilament. Information gained from these studies can now be used to determine whether TnI phosphorylation and/or the TnI-mediated enhancement of relaxation produced in response to PKC is altered under physiological and pathophysiological conditions.
Additional studies are focused on understanding the role individual protein kinase C isoforms play in the contractile response. In ongoing studies, we are examining the effects of protein kinase C isoform over-expression using viral-mediated gene transfer into adult rat myocytes. The effects of dominant negative mutants for each isoform on contractile performance are also being examined in these cells. In our initial studies, increased expression of both wildtype and dominant negative PKCalpha is observed within 48 hrs post-gene transfer, and cellular distribution appears similar to control myocytes. In myocytes expressing elevated levels of PKCalpha, basal contractile function is d significantly decreased, while function is enhanced in myocytes expressing the dominant negative analog. In ongoing studies it appears that PKCalpha acts to decrease phospholamban phosphorylation via its effects on protein phosphatase inhibitor 1. Future studies will focus on whether a similar mechanism operates in higher mammalian myocytes, including rabbit and human myocytes from non-failing hearts.
Another project underway in our laboratory is focused on investigating the role of PKC signaling in human myocytes from failing hearts before and after implantation of ventricular assist devices (VAD). Preliminary studies have provided evidence that PKCalpha expression diminishes in many hearts after VAD implantation. Experiments are currently underway to evaluate basal and PKC-mediated contractile responses in myocytes isolated from failing hearts. Future studies will focus on the ability of agonists to induce phosphorylation of target proteins, such as troponin I.
Many of the above basic research projects are being performed in collaboration with other departments including: Internal Medicine, Physiology, and Pharmacology.
- Recovery and non-recovery surgery of animals
- Microvascular surgery
- Isolated, perfused adult and neonatal rabbit hearts (both "working" and isovolumic preparations)
- Preparation of histologic specimens and slides
- High performance liquid chromatography (HPLC)
- Myocardial function measured in the animal models with sonomicrometry
- Studies of blood flow using the tracer labeled microsphere technique
- Pulsed doppler flowmetry
- Clearance of iodinated albumin to measure lung injury
- Myeloperoxide assay
- Intramyocardial pH measurements
- Triphenyltetrazolium technique for measurement of infarct size
- DNA sequencing
- DNA, RNA and protein electrophoresis
- RT-PCR analysis
- Cell culture
- Flow cytometry
- EIA and RIA assay
- Western Blot analysis
- Isolation of adult cardiac myocytes
- Sarcomere shortening assay
- Phosphorylation and back-phosphorylation studies
- To add to DNA, RNA, and protein electrophoresis - Western blot analysis, IEF
- Operating rooms with separate surgeon and animal preparation areas (in accordance with AAALAC guidelines), inhalation or intravenous anesthetic equipment and surgical instruments for procedures in large animals
- Cardiopulmonary bypass pumps; and other equipment to perform surgical procedures in large animals
- Microsurgery laboratory with operating microscope, support equipment and microsurgical instruments
- Equipment for preparing histologic specimens and slides
- Glassware, tubing arrangements, pumps and recording equipment for performing experiments on isolated hearts from small animals
- High performance liquid chromatography (HPLC) unit - Waters Millenium System
- Sonomicrometer, pulsed Doppler flowmeters, transducers and recording equipment for performance of acute and chronic experiments of myocardial function in large animals
- Gamma counter and PC dedicated to blood flow studies using tracer-labeled microspheres
- PC-based on-line analog to digital conversion of data
- Radiometer Blood gas
- Brandel Cell Harvester
- Labconco Lyophilizer
- Cell culture hoods and incubators
- Polyacrylimide and agarose electrophoresis units
- Flourescence and light microscopes
- Microplate Reader
- Steven F. Bolling, M.D.
- Jonathan W. Haft, M.D.
- Francis D. Pagani, M.D., Ph.D.
- Margaret V. Westfall, Ph.D.
- Bo Yang, M.D., Ph.D
North Campus Research Center (NCRC)
2800 Plymouth Road
Ann Arbor, MI 48109