Accumulation of oxidized extracellular matrix (ECM) between endothelium and muscle is an important risk factor in the endothelium-myocytes (E-M) uncoupling in congestive heart failure (CHF). Although ventricular remodeling is accompanied by increased matrix metalloproteinase-9 (MMP-9) activity, it is unclear whether MMP-9 plays a role in endothelial apoptosis in chronic volume overload CHF. We tested the hypothesis that in chronic volume overload, myocardial dysfunction involves endocardial endothelial (EE) apoptosis in response to MMP-9 activation, ECM accumulation and E-M uncoupling. Methods: Arteriovenous fistula (AVF) was created in control (FVB/NJ, AVF group) and MMP-9 knockout (FVB.Cg-Mmp9^tm1Tvu/J, MMP9KO+AVF group) mice. Sham surgery was used as control. Mice were grouped as follows: WT, n=3 (sham control), MMP9KO, n=3 (sham), AVF, n=3, and MMP9KO+AVF (n=3). Heart function was analyzed by M-mode and Doppler echocardiography, and with a pressure tipped Millar catheter placed in the left ventricle (LV) of anesthetized mice 8 wks after AVF. Apoptosis was detected by measuring caspase-3, TUNEL and CD-31 by immuno-labeling. Protease activated receptors-1 (PAR-1), connexin-43 and a disintegrin and metalloproteinase-12 (ADAM-12) expression were measured by Western blot analyses. MMP-2 and MMP-9 expression were measured by Q-RT-PCR. Results: Compared to control, AVF caused an increase in LVEDP and decrease in -dP/dt. In contrast, in MMP-9KO+AVF group, these variables were changed toward control levels. Increased EE apoptosis (caspase-3 activation and TUNEL/CD-31 co-labeling) in AVF mice was prevented in the MMP-9KO+AVF group. PAR-1, connexin-43 and ADAM-12 were induced in AVF. MMP-9 gene ablation ameliorated the induction. Conclusions: The results suggest that impaired cardiac function in volume overload is associated with EE apoptosis, cardiac remodeling and E-M uncoupling in response to MMP-9 activation.