Cardiac-restricted overexpression or deletion of tissue inhibitor of matrix metalloproteinase-4: differential effects on left ventricular structure and function following pressure overload-induced hypertrophy.

Historically, the tissue inhibitors of matrix metalloproteinases (TIMPs) were considered monochromatic in function. However, differential TIMP profiles more recently observed with left ventricular (LV) dysfunction and matrix remodeling suggest more diverse biological roles for individual TIMPs. This study tested the hypothesis that cardiac-specific overexpression (TIMP-4OE) or deletion (knockout; TIMP-4KO) would differentially affect LV function and structure following pressure overload (LVPO). LVPO (transverse aortic constriction) was induced in mice (3.5 ± 0.1 mo of age, equal sex distribution) with TIMP-4OE (n = 38), TIMP-4KO (n = 24), as well as age/strain-matched wild type (WT, n = 25), whereby indexes of LV remodeling and function such as LV mass and ejection fraction (LVEF) were determined at 28 days following LVPO. Following LVPO, both early (7 days) and late (28 days) survival was ~25% lower in the TIMP-4KO group (P < 0.05). While LVPO increased LV mass in all groups, the relative hypertrophic response was attenuated with TIMP-4OE. With LVPO, LVEF was similar between WT and TIMP-4KO (48 ± 2% and 45 ± 3%, respectively) but was higher with TIMP-4OE (57 ± 2%, P < 0.05). With LVPO, LV myocardial collagen expression (type I, III) increased by threefold in all groups (P < 0.05), but surprisingly this response was most robust in the TIMP-4KO group. These unique findings suggest that increased myocardial TIMP-4 in the context of a LVPO stimulus may actually provide protective effects with respect to survival, LV function, and extracellular matrix (ECM) remodeling. These findings challenge the canonical belief that increased levels of specific myocardial TIMPs, such as TIMP-4 in and of themselves, contribute to adverse ECM accumulation following a pathological stimulus, such as LVPO.

Enhancing the peroxidatic activity of KatG by deletion mutagenesis.

Catalase-peroxidase (KatG) enzymes use a peroxidase active site to facilitate robust catalase activity, an ability all other members of its superfamily lack. KatG's have a Met-Tyr-Trp covalent adduct that is essential for catalatic but not peroxidatic turnover. The tyrosine (Y226 in E. coli KatG) is supplied by a large loop (LL1) that is absent from all other plant peroxidases. Elimination of Y226 from the KatG structure, either by site directed mutagenesis (i.e., Y226F KatG) or by deletion of larger portions of LL1 invariably eliminates catalase activity, but deletion variants were substantially more active as peroxidases, up to an order of magnitude. Moreover, the deletion variants were more resistant to H(2)O(2)-dependent inactivation than Y226F KatG. Stopped-flow evaluation of reactions of H(2)O(2) with Y226F KatG and the most peroxidase active deletion variant (KatG[Δ209-228]) produced highly similar rate constants for formation of compounds I and II, and about a four-fold faster formation of compound III for the deletion variant as opposed to Y226F. Conversely, single turnover experiments showed a 60-fold slower return of Y226F KatG to its ferric state in the presence of the exogenous electron donor 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) than was determined for KatG(Δ209-228). Our data suggest that the peroxidatic output of KatG cannot be optimized simply by elimination of catalase activity alone, but also requires modifications that increase electron transfer between exogenous electron donors and the heme prosthetic group.