Inhibition of matrix metalloproteinases prevents peroxynitrite-induced contractile dysfunction in the isolated cardiac myocyte.
OBJECTIVE
The potent oxidant peroxynitrite (ONOO(-)) induces mechanical dysfunction in the intact heart in part through activation of matrix metalloproteinase-2 (MMP-2). This effect may be independent of the proteolytic actions of MMPs on extracellular matrix proteins. The purpose of this study was to examine the effects of ONOO(-) on contractile function at the level of the single cardiac myocyte and whether this includes the action of MMPs.


METHODS
Freshly isolated ventricular myocytes from adult rats were superfused with Krebs-Henseleit buffer at 21 degrees C and paced at 0.5 Hz. Contractility was measured using a video edge-detector. ONOO(-) or decomposed ONOO(-) (vehicle control) were co-infused over 40 min to evaluate the contraction cease time (CCT). The effects of ONOO(-) on intracellular [Ca(2+)] were determined in myocytes loaded with calcium green-1 AM. MMP-2 activity was measured by gelatin zymography.


RESULTS
ONOO(-) (30-600 microM) caused a concentration-dependent reduction in CCT. Myocytes subjected to 300 microM ONOO(-) had a shorter CCT than decomposed ONOO(-) (14.9+1.5 vs 32.2+3.5 min, n=7-8; P<0.05) and showed increased MMP-2 activity. The MMP inhibitors doxycycline (100 microM) or PD 166793 (2 microM) reduced the decline in CCT induced by 300 microM ONOO(-). ONOO(-) caused shorter calcium transient cease time and significant alterations in intracellular [Ca(2+)] homoeostasis which were partially prevented by doxycycline.


CONCLUSIONS
This is the first demonstration that inhibition of MMPs protects the cardiac myocyte from ONOO(-)-induced contractile failure via an action unrelated to proteolysis of extracellular matrix proteins.
León
H
H
Cardiovascular Research Group, University of Alberta, Edmonton, Alberta, Canada.
Baczkó
I
I
Sawicki
G
G
Light
P E
PE
Schulz
R
R
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002304:Cardiac Pacing, Artificial; D004318:Doxycycline / Q000494:pharmacology*; D004789:Enzyme Activation; D006706:Homeostasis; D006877:Hydroxamic Acids / Q000494:pharmacology*; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D009200:Myocardial Contraction / Q000187:drug effects*; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology*; D030421:Peroxynitrous Acid / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
C370242:(R)-2-(4'-bromo-biphenyl-4-sulfonyl-amino)-3-methyl-butyric acid; D006877:Hydroxamic Acids; D061965:Matrix Metalloproteinase Inhibitors; D009842:Oligopeptides; D011480:Protease Inhibitors; D030421:Peroxynitrous Acid; D020778:Matrix Metalloproteinase 2; C522363:Mmp2 protein, rat; D004318:Doxycycline; D002118:Calcium