Reprogramming of gene expression in cultured cardiomyocytes and in explanted hearts by the myosin ATPase inhibitor butanedione monoxime.
BACKGROUND
Butanedione monoxime (BDM) is a reversible myosin ATPase inhibitor. Its use in transplantation medicine may be of benefit in the preservation of hearts. As little is known about its ability to prevent stress and metabolic deregulation, we wanted to investigate the genomic response in cultured cardiomyocytes and explanted, preserved hearts at the transcriptional level.


METHODS
We thus investigated the gene expression of the transcription factors GATA-4, Nkx2.5, MEF-2c, and Oct-1 and of the downstream target genes atrial and brain natriuretic peptide, alpha- and beta-myosin heavy chain, alpha-cardiac actin, and alpha-skeletal actin. Additionally, lactate dehydrogenase and creatine kinase enzyme activities were measured as markers for membrane integrity and metabolic deregulation of cardiomyocytes.


RESULTS
In untreated cardiomyocyte cultures, expression of GATA-4 and Nkx2.5 was increased 7- and 4-fold, 72 hr after isolation, but the gene expression of MEF-2c and Oct-1 was reduced to 10% and 70%, at day 3 in culture. We show atrial natriuretic peptide and brain natriuretic peptide gene expression to be maximal 24 and 72 hr after isolation, the level being 3- and 2-fold, when compared with freshly isolated cells. The gene expression of alpha- and beta-myosin heavy chain was reduced to approximately 30% at day 3 in culture and similar observations were made for alpha-cardiac and alpha-skeletal actin, which declined to approximately 20% and 10% of control values, 72 hr after isolation. BDM prevented at the transcriptional level enhanced expression of markers for stress and metabolic deregulation, and the activities of lactate dehydrogenase and creatine kinase were highly significantly reduced. Similar results were obtained when explanted hearts were stored in BDM-containing organ preservation solution.


CONCLUSIONS
Preservation of metabolic function in donor organs is of critical importance in transplantation medicine, and we show gene markers for stress and metabolic deregulation in cultures of cardiomyocytes and explanted hearts to be significantly reduced by BDM. Reprogramming of gene expression of nuclear transcription factors and downstream target genes may prolong the acceptable storage time between explantation and transplantation.
Centre of Drug Research and Medical Biotechnology, Fraunhofer Institute of Toxicology and Aerosol Research, Hannover, Germany.
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Borlak
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002478:Cells, Cultured; D003931:Diacetyl / Q000031:analogs & derivatives* / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects; D016027:Heart Transplantation / Q000502:physiology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D009206:Myocardium / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D009218:Myosins / Q000037:antagonists & inhibitors*; D019188:Organ Preservation Solutions / Q000494:pharmacology; D015800:Protozoan Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley
D019188:Organ Preservation Solutions; D015800:Protozoan Proteins; C004717:diacetylmonoxime; D007770:L-Lactate Dehydrogenase; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C033142:myosin-heavy-chain kinase; D009218:Myosins; D003931:Diacetyl