Targeted overexpression of tissue inhibitor of matrix metalloproteinase-4 modifies post-myocardial infarction remodeling in mice

Abstract

Rationale: Myocardial infarction (MI) causes an imbalance between matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases (TIMPs) and is associated with adverse left ventricular (LV) remodeling. A uniform reduction in TIMP-4 post-MI has been observed.

Objective: To examine post-MI remodeling with cardiac-restricted overexpression of TIMP-4, either through a transgenic or viral delivery approach.

Methods and results: MI was induced in mice and then randomized to targeted injection of an adenoviral construct (10 μL; 8×10(9) plaque forming units/mL) encoding green fluorescent protein (GFP) and the full-length human TIMP-4 (Ad-GFP-TIMP4) or GFP. A transgenic construct with cardiac-restricted overexpression TIMP-4 (hTIMP-4exp) was used in a parallel set of studies. LV end-diastolic volume, an index of LV remodeling, increased by >60% from baseline at 5 days post-MI and by >100% at 21 days post-MI in the Ad-GFP only group. However, LV dilation was reduced by ≈50% in both the Ad-GFP-TIMP4 and hTIMP-4exp groups at these post-MI time points. LV ejection fraction was improved with either Ad-GFP-TIMP-4 or hTIMP-4exp. Fibrillar collagen expression and content were increased within the MI region with both TIMP-4 interventions, suggestive of matrix stabilization.

Conclusions: This study is the first to demonstrate that selective myocardial targeting for TIMP-4 induction through either a viral or transgenic approach favorably altered the course of adverse LV remodeling post-MI. Thus, localized induction of endogenous matrix metalloproteinase inhibitors, such as TIMP-4, holds promise as a means to interrupt the progression of post-MI remodeling.

Keywords: extracellular matrix; myocardial infarction; tissue inhibitor of metalloproteinases; ventricular function.

Figure 1

(A) Representative echocardiograms of the LV long axis taken at end diastole at the baseline study (pre-MI time point) and in terminal studies performed at 5 or 21 days post-MI in mice injected with either Ad-GFP only (Top Panel) or the Ad-GFP-TIMP-4 construct (Middle Panel). In a second set of studies, mice with transgenic overexpression of human TIMP-4 (hTIMP-4exp) were treated in identical fashion (Bottom Panel). Significant LV dilation was observed in all groups at 5 and 21 days post-MI, but the relative degree of LV dilation appeared attenuated in both the Ad-GFP-TIMP4 and hTIMP-4exp groups. Summary data are presented in Table 1. (B) Key LV response variables as a function from baseline. LV ejection fraction significantly fell in all groups at 5 and 21 days post-MI, but this relative reduction was attenuated in the Ad-GFP-TIMP4 or hTIMP-4exp groups. LV end-diastolic volume and LV mass/volume ratio, both indices of post-MI remodeling, were significantly reduced in the Ad-GFP-TIMP-4 or hTIMP-4exp groups. (C) LV myocardial levels of GFP were expressed at equivalent levels in both adenoviral injection groups at both 5 and 21 days post-MI. In the hTIMP-4exp line, consistent human TIMP-4 levels were obtained in the non-MI referent control mice and these levels were higher at 5 and 21 days post MI. (D) LV myocardial mRNA levels for human TIMP-4 were detectable in the Ad-GFP-TIMP4 and hTIMP-4exp groups at 5 and 21 days post-MI. While endogenous mouse TIMP-4 was reduced at 5 days post-MI, expression levels were higher at 21 days post-MI with the notable exception in the hTIMP-4 group. [*p<0.05 compared to baseline (or referent non-MI values), # compared to Ad-GFP only group, Pp<0.05 vs Ad-GFP-TIMP-4 group].

Figure 2

(A) Distribution plots for myocyte cross sectional area (CSA) for referent control and within the LV of the remote, viable myocardium at 5 and 21 days post-MI in the Ad-GFP, Ad-GFP-TIMP-4, and the hTIMP-4exp groups. A significant increase in myocyte CSA occurred post-MI in all groups, which appeared higher in the Ad-GFP-TIMP-4 group at 21 days post-MI. (B) LV relative collagen content was computed in both the MI and remote regions in all groups at 5 and 21 days post-MI using morphometric measurements on PSR stained sections. While collagen content was increased within the MI region as well as in the remote region in all groups, consistent with a post-MI fibrotic response, these values were higher in both TIMP-4 induction groups. [*p<0.05 compared to referent non-MI values, +p<0.05 vs respective 5 day values, # compared to Ad-GFP only group, $p<0.05 vs Ad-GFP-TIMP-4 group; sample sizes are n>10/group/time point].

Figure 3

Relative myocardial mRNA levels for TIMP-1, TIMP-2, and TIMP-3, as well as for the fibrillar collagens, collagen type I and III, were computed in referent non-MI mice immediately following myocardial injection of Ad-GFP, Ad-GFP-TIMP4, or in hTIMP-4exp mice and at 5 and 21 days post-MI. TIMP-1 levels increased at 5 days post-MI and then fell at 21 days post-MI with no significant differences between groups. Relative TIMP-2 mRNA levels increased in the Ad-GFP-TIMP4 and hTIMP-4exp groups at 5 and 21 days post-MI. At 21 days post-MI, TIMP-3 mRNA levels were higher in the Ad-GFP-TIMP4 and hTIMP-4exp groups. At 5 days post-MI, increased mRNA levels for collagen I and III were equivalently increased in all groups. However, a persistent induction of collagen I and III occurred at 21 days post-MI in the hTIMP-4exp group. [*p<0.05 compared to referent non-MI values, +p<0.05 vs respective 5 day values, # compared to Ad-GFP only group, Pp<0.05 vs Ad-GFP-TIMP-4 group; sample sizes are n>10/group/time point].

Figure 4

(Left Panels) LV myocardial mRNA levels for MT1-MMP increased in all groups at 5 days post-MI and remained elevated at 21 days post-MI in the hTIMP-4exp group. Relative DDR2 mRNA levels appeared lower in the Ad-GFP-TIMP-4 group with no MI and at 5 days post-MI, whereas DDR2 mRNA levels were increased in the hTIMP-4exp group at 21 days post-MI. TGFbeta-R1 mRNA levels increased in all post-MI time points in all groups, but was highest in the hTIMP-4exp groups. (Middle Panels) LV myocardial IL-6 content increased in all groups at 5 days post-MI and reduced to referent control values by 21 days post-MI. However, IL-6 levels were lower in the hTIMP-4exp group at 5 days post-MI. LV myocardial IL-10 levels were detectable in referent control and Ad-GFP post-MI time points but were significantly reduced in the Ad-GFP-TIMP-4 group and non-detectable (ND) in the hTIMP-4exp group. MCP-1 was increased at 5 days post-MI in all groups but reduced in the hTIMP-4exp group. By 21 days post-MI, MCP-1 levels had fallen below referent control values. (Right Panels) LV myocardial expression levels for Bcl2 were higher at the non-MI and 21 day post-MI time point in the hTIMP-4exp group. A significant induction of Apaf1 and Ripk1 occurred at 5 days post-MI in the Ad-GFP group but not in either the Ad-GFP-TIMP-4 or hTIMP-4exp groups. [*p<0.05 compared to baseline (or referent non-MI values), +p<0.05 vs respective 5 day values, # compared to Ad-GFP only group, Pp<0.05 vs Ad-GFP-TIMP-4 group; sample sizes are n>6/group/time point].

Figure 5

(Top) Representative myocardial zymograms performed in referent non-MI mice immediately following myocardial injection of Ad-GFP, Ad-GFP-TIMP4, or in hTIMP-4exp mice and at 5 and 21 days post-MI. The latent form of MMP-9 (92 kDa) and the latent and active forms of MMP-2 (72/68 kDa, respectively) were identified and quantified by densitometry. (Bottom) Summary results from zymography were computed as a percent of referent non-MI values, whereby referent values were set to 100% (dashed line). In the Ad-GFP group, MMP-9 values increased at 5 days post-MI and returned to within normal ranges by 21 days post-MI. In the Ad-GFP-TIMP4 and hTIMP-4exp group, relative MMP-9 levels did not increase at 5 days post-MI and were actually reduced at specific post-MI time points. For MMP-2, a robust increase in active MMP-2 occurred in the Ad-GFP group at 5 and 21 days post-MI, which was significantly blunted in both the Ad-GFP-TIMP4 and hTIMP-4exp groups. [*p<0.05 compared to referent non-MI values, +p<0.05 vs respective 5 day values, # compared to Ad-GFP only group; sample sizes are n>10/group/time point]

Figure 6

(Left Panels) LV murine myocardial fibroblasts were transduced with either Ad-GFP or Ad-GFP-TIMP4, which yielded a uniform transduction efficiency of 70% with both vectors. LV fibroblasts were imaged for GFP fluorescence using confocal microscopy (Ziess LSM 510 Meta Confocal, Plan-Neofluar 43X, GFP excitation/emission 488/505–530 nm). (Right Panels) LV fibroblast cell density, indicative of cell proliferation, was higher at 24 hours post-transduction with TIMP-4. In addition, transduction of TIMP-4 in these LV fibroblast cultures resulted in significantly higher mRNA levels for Bcl2, collagen I, DDR2, and TGFbetaR1. [*p<0.05 compared to Ad-GFP only values, #p<0.05 fold change compared to Ad-GFP; n=3 with all studies performed in triplicate]