Inhibition of MMP-2 expression affects metabolic enzyme expression levels: proteomic analysis of rat cardiomyocytes.
In this study we examined the effect of inhibition of MMP-2 expression, using siRNA, on the cardiomyocyte proteome. Isolated cardiomyocytes were transfected with MMP-2 siRNA and incubated for 24h. Control cardiomyocytes from the same heart were transfected with scrambled siRNA following the same protocol. Comparison of control cardiomyocyte proteomes with proteomes from MMP-2 suppressed cardiomyocytes revealed 13 protein spots of interest (9 protein spots increased; 4 decreased). Seven protein spots were identified as mitochondrial enzymes involved in energy production and represent: ATP synthase beta subunit, dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, cytochrome c oxidase subunit 5A, electron transfer flavoprotein subunit beta, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 5 and a fragment of mitochondrial precursor of long-chain specific acyl-CoA dehydrogenase. Furthermore, precursor of heat shock protein 60 and Cu-Zn superoxide dismutase were identified. Two protein spots corresponding to MLC1 were also detected. In addition, ATP synthase activity was measured and was increased by approximately 30%. Together, these results indicate that MMP-2 inhibition represents a novel cardioprotective therapy by promoting alterations in the levels of mitochondrial enzymes for improved energy metabolism and by preventing degradation of contractile proteins needed for normal excitation-contraction coupling.


CONCLUSIONS
During ischemia and reperfusion of cardiomyocytes, abnormality in excitation-contraction coupling and decreased energy metabolism often lead to myocardial infarction, but the cellular mechanisms are not fully elucidated. We show for the first time that intracellular inhibition of MMP-2 in cardiomyocytes increases contractility of aerobically perfused myocytes, which was accompanied by increased expression of contractile proteins (e.g., MLC-1). We also showed that MMP-2 inhibition produced a cardiomyocyte proteome that is consistent with improved mitochondrial energy metabolism (e.g., increased expression and activity of mitochondrial beta ATP synthase). Thus, MMP-2 appears to be involved in homeostatic regulation of protein turnover. Our results are significant since they point to targeting MMP-2 activity as a novel therapeutic option to limit myocardial damage by decreasing proteolytic degradation of mitochondrial metabolic enzymes and myocardial contractile proteins during ischemia. In addition, the development of novel pharmacological agents that selectively targets cardiac MMP-2 represents a novel approach to treat and prevent other heart diseases.
Department of Pharmacology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
Han-Bin
HB
Lin
Department of Pharmacology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
Keshav
K
Sharma
Department and Clinic of Cardiology, Medical University of Wroclaw, Wroclaw, Poland.
Dariusz
D
Bialy
Department of Medical Emergency, Medical University of Wroclaw, Wroclaw, Poland.
Magdalena
M
Wawrzynska
National Research Council of Canada, Saskatoon, Saskatchewan, Canada.
Randy
R
Purves
Department of Physiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
Francisco S
FS
Cayabyab
Department of Pharmacology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Department of Clinical Chemistry, Medical University of Wroclaw, Wroclaw, Poland.
Mieczyslaw
M
Wozniak
Department of Pharmacology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Department of Clinical Chemistry, Medical University of Wroclaw, Wroclaw, Poland. Electronic address: greg.sawicki@usask.ca.
Grzegorz
G
Sawicki
D000595:Amino Acid Sequence; D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D008297:Male; D013058:Mass Spectrometry; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D061965:Matrix Metalloproteinase Inhibitors / Q000737:chemistry*; D008928:Mitochondria / Q000378:metabolism; D025261:Mitochondrial Proton-Translocating ATPases / Q000378:metabolism; D008969:Molecular Sequence Data; D009200:Myocardial Contraction; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D040901:Proteomics; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid
D061965:Matrix Metalloproteinase Inhibitors; D034741:RNA, Small Interfering; D020778:Matrix Metalloproteinase 2; C522363:Mmp2 protein, rat; D025261:Mitochondrial Proton-Translocating ATPases
ATP synthase; Cardiomyocytes; Contractility; Matrix metalloproteinase-2; Metabolic enzymes; siRNA