Changes in gene expression during the formation of bioengineered heart muscle.
A three-dimensional bioengineered heart muscle (BEHM) construct model had been previously developed, exhibiting contractile forces up to 800 microN. The interest of this study was to determine gene expression levels of biologic markers involved in calcium-handling between BEHM, cell monolayer, and neonatal heart. Cardiac cells were isolated from one litter of F344 rats and organized into groups (n = 5): 4-, 7-, 10-day BEHM and cell monolayer; BEHM was evaluated for cell viability and contractility. Groups were then analyzed for mRNA expression of calcium-handling proteins: myosin heavy chain (MHC) alpha and beta, Sarcoplasmic reticulum Ca++ ATPase (SERCA) 2, phospholamban (PBL), and ryanodine receptor. BEHM exhibited electrically stimulated active force (208 +/- 12 microN day 4, 361 +/- 22 microN day 7, and 344 +/- 29 microN day 10) and no decrease in cell number. Real-time polymerase chain reaction (PCR) showed an increase in gene expression of all calcium-handling proteins in BEHM at 7 and 10 days compared with monolayers, for example, comparing BEHM to monolayer (7 and 10 days, respectively), MHC-alpha: 2600-fold increase and a 100-fold increase; MHC-beta: 70-fold increase at 10 days; ryanodine receptor: 74-fold increase at 10 days; SERCA: 19-fold increase and sixfold increase; PBL: 158-fold increase and 24-fold increase. It was concluded that a three-dimensional environment is a better culturing condition of cardiac cells than a monolayer. Also, BEHM constructs demonstrated a high similarity to a native myocardium, and is, thus, a good starting foundation for engineered heart muscle.
Khait
Luda
L
Section of Cardiac Surgery, University of Michigan, Ann Arbor, MI, USA.
Birla
Ravi K
RK
D000818:Animals; D000831:Animals, Newborn; D015415:Biomarkers; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D015870:Gene Expression; D009200:Myocardial Contraction; D009206:Myocardium / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D013778:Tetrazolium Salts / Q000378:metabolism; D023822:Tissue Engineering; D000099211:Phospholamban
D015415:Biomarkers; D002118:Calcium; D002135:Calcium-Binding Proteins; D018995:Myosin Heavy Chains; D019837:Ryanodine Receptor Calcium Release Channel; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013778:Tetrazolium Salts; D000099211:Phospholamban; C110656:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; C506948:Atp2a2 protein, rat; C000594638:MYH7 protein, rat