Background
Cardiac fibroblasts play a vital role in maintaining cardiac integrity, adaptation to mechanical stress, cardiac signaling, and in cardiac excitability. Following coronary occlusion, the expression of ANP/BNP, cardiac hypertrophic responses, and the development of cardiac arrhythmia partly depend on fibroblasts. Potassium channels may contribute to the membrane potential of fibroblasts, and thus regulate fibroblast function.We recently found that neonatal rat atrial fibroblasts express high levels of mRNA for certain subunits of the KATP channel, in particular KIR6.1 and SUR2B. Others have found fibroblast expression of ANP/BNP.
Working Hypothesis
KATP channels and ANP/BNP secretory pathways exist in cardiac fibroblasts and significantly influence impulse conduction and cardiac hypertrophy.
Aims
1. Compare mRNA/protein levels and confocal images of KATP channel subunits and ANP/BNP vesicles in purified fibroblasts, myofibroblasts, and myocytes; these cells are derived separately from the four chambers of the rat heart, and from right atrial appendage biopsies of the infant heart.
2. Characterize the KATP channel in stretched rat and human cardiac (myo)fibroblasts by patch clamp recordings.
3. Determine ANP/BNP secretory vesicle formation in stretch-stimulated (myo)fibroblasts.
4. Measure the stretch-stimulated gene expression (cDNA arrays) in (myo)fibroblasts, myocytes and co-cultures during pharmacological activation or blockade of KATP channels.
5. Test the role of (myo)fibroblast KATP channels in myocyte hypertrophy, and electrical impulse conduction.
Methods
Right atrial appendage human tissues are obtained with informed consent.The four heart chambers from neonate rats are microdissected under the microscope. Myocytes and fibroblasts are purified, cultured on flexible membranes (for stretch) or glass, and characterized. Imaging is performed by confocal microscopy. Protein and mRNA extracts are quantified in Western blots and real time PCR, respectively. KATP function is ascertained electrophysiologically. Collaborator Pierre Philip-Couderc is experienced in cDNA array technology. Collaborator Cécile Brun is competent in creating the various constructs coding for the EGFP fusion proteins required for these projects. Collaborator Angela Roatti performs cell culture, Western blots, immunocytochemistry, cDNA electroporation, and hormone assays. Studies on hypertrophy and impulse conduction are carried out in collaboration with other laboratories. All methods are available for this project.
Expected Value of the Proposed Project
KATP channels and ANP/BNP vesicle formation in fibroblasts, including myofibroblasts, could profoundly affect cardiac function, and impact on atrial fibrillation, cardiac hypertrophy, and the recovery from a heart attack. This study addresses the first elements in a chain of mechanisms that link fibroblast stress to the cardiac adaptation to metabolic and mechanical challenges.
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