Laser microdissection and capture of pure cardiomyocytes and fibroblasts from infarcted heart regions: perceived hyperoxia induces p21 in peri-infarct myocytes.
Myocardial infarction caused by ischemia-reperfusion in the coronary vasculature is a focal event characterized by an infarct-core, bordering peri-infarct zone and remote noninfarct zone. Recently, we have reported the first technique, based on laser microdissection pressure catapulting (LMPC), enabling the dissection of infarction-induced biological responses in multicellular regions of the heart. Molecular mechanisms in play at the peri-infarct zone are central to myocardial healing. At the infarct site, myocytes are more sensitive to insult than robust fibroblasts. Understanding of cell-specific responses in the said zones is therefore critical. In this work, we describe the first technique to collect the myocardial tissue with a single-cell resolution. The infarcted myocardium was identified by using a truncated hematoxylin-eosin stain. Cell elements from the infarct, peri-infarct, and noninfarct zones were collected in a chaotropic RNA lysis solution with micron-level surgical precision. Isolated RNA was analyzed for quality by employing microfluidics technology and reverse transcribed to generate cDNA. Purity of the collected specimen was established by real-time PCR analyses of cell-specific genes. Previously, we have reported that the oxygen-sensitive induction of p21/Cip1/Waf1/Sdi1 in cardiac fibroblasts in the peri-infarct zone plays a vital role in myocardial remodeling. Using the novel LMPC technique developed herein, we confirmed that finding and report for the first time that the induction of p21 in the peri-infarct zone is not limited to fibroblasts but is also evident in myocytes. This work presents the first account of an analytical technique that applies the LMPC technology to study myocardial remodeling with a cell-type specific resolution.
Kuhn
Donald E
DE
Laboratory of Molecular Medicine, Department of Surgery, Davis Heart & Lung Research Institute, The Ohio State University Medical Center, Columbus, OH 43210, USA.
Roy
Sashwati
S
Radtke
Jared
J
Khanna
Savita
S
Sen
Chandan K
CK
D000199:Actins / Q000235:genetics; D000818:Animals; D004195:Disease Models, Animal; D005347:Fibroblasts / Q000648:ultrastructure*; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D018496:Hyperoxia / Q000503:physiopathology*; D007834:Lasers; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D042282:Microdissection / Q000379:methods*; D032342:Muscle Cells / Q000648:ultrastructure*; D009203:Myocardial Infarction / Q000473:pathology*; D009206:Myocardium / Q000473:pathology* / Q000648:ultrastructure; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000199:Actins; D012313:RNA; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)