Cardioprotective effects of lysyl oxidase inhibition against volume overload-induced extracellular matrix remodeling

Abstract

A hallmark of heart failure (HF) is adverse extracellular matrix (ECM) remodeling, which is regulated by the collagen cross-linking enzyme, lysyl oxidase (LOX). In this study, we evaluate the efficacy of LOX inhibition to prevent adverse left ventricular (LV) remodeling and dysfunction using an experimental model of HF. Sprague-Dawley rats were subjected to surgically induced volume overload (VO) by creation of aortocaval fistula (ACF). A LOX inhibitor, beta-aminopropionitrile (BAPN; 100 mg/kg/day), was administered to rats with ACF or sham surgery at eight weeks postsurgery. Echocardiography was used to assess progressive alterations in cardiac ventricular structure and function. Left ventricular (LV) catheterization was used to assess alterations in contractility, stiffness, LV pressure and volume, and other indices of cardiac function. The LV ECM alterations were assessed by: (a) histological staining of collagen, (b) protein expression of collagen types I and III, (c) hydroxyproline assay, and (d) cross-linking assay. LOX inhibition attenuated VO-induced increases in cardiac stress, and attenuated increases in interstitial myocardial collagen, total collagen, and protein levels of collagens I and III. Both echocardiography and catheterization measurements indicated improved cardiac function post-VO in BAPN treated rats vs. untreated. Inhibition of LOX attenuated VO-induced decreases in LV stiffness and cardiac function. Overall, our data indicate that LOX inhibition was cardioprotective in the volume overloaded heart.

Keywords: Fibrosis; cardiac function; cardioprotection; collagen; heart failure.

Figure 1

Experimental timeline. Echocardiography was used to assess cardiac function at the time points indicated. A baseline echo was performed a week before surgery (−1 week). At 14 weeks, left ventricular pressure–volume catheterization was performed and tissue was collected. (A color version of this figure is available in the online journal)

Figure 2

Lysyl oxidase (LOX) inhibition attenuated volume overload (VO)-induced increases in cardiac wall stress. TOP: A ratio between left ventricular (LV) mass and end diastolic volume (EDV) was calculated to assess cardiac stress. LV mass was determined from wet weight and LV EDV was determined by pressure–volume catheterization. The ratio decreased in VO, indicative of increased cardiac stress. LOX inhibition with beta-aminoproprionitrile (BAPN;B) attenuated these decreases. Similar results and statistical significance were obtained using LV mass and volume estimated by echocardiography (data not shown). BOTTOM: A calculation of diastolic stress using Equation 1 confirms that LOX inhibition attenuated VO-induced increases in cardiac stress. Statistical significance denoted as P < 0.05 vs. Sham (*) and VO (†); n = 4–8 per group

Figure 3

Echocardiography assessment determined that lysyl oxidase (LOX) inhibition attenuated cardiac dysfunction following volume overload (VO). VO induced significant increases in left ventricle chamber size (LVIDd), but had no significant effect on wall thickness (LVPWd). VO caused a significant decrease in fractional shortening (FS) when compared to SHAM. LOX inhibition (B) did not significantly alter LVPWd or LVIDd in either SHAM or VO rats. However, LOX inhibition did significantly attenuate the decrease in %FS caused by VO. Statistical significance denoted as P < 0.05 vs. Sham (*) and VO (†). (A color version of this figure is available in the online journal)

Figure 4

Representative pressure–volume loops for each group collected by left ventricular (LV) pressure–volume catheterization. Sham-operated (SHAM) controls were not significantly altered by the lysyl oxidase inhibitor (B). Volume overload (VO) caused significant LV dilatation (rightward shift), which was not prevented by lysyl oxidase inhibition (B)

Figure 5

Left ventricular (LV) pressure–volume catheterization indicates that lysyl oxidase (LOX) inhibition using beta-aminoproprionitrile (BAPN; B) improved contractility and cardiac function in the volume overloaded heart. Volume overload (VO) induced significant decreases in (A) end systolic pressure–volume relationship (ESPVR), (B) end diastolic pressure–volume relationship (EDPVR), (C) preload recruited stroke work (PRSW), and (D) the rate of LV pressure change per unit time (dP/dt max) vs. end diastolic volume (EDV). LOX inhibition attenuated the VO-induced decreases in each of these parameters. Statistical significance denoted as P < 0.05 vs. Sham (*) and VO (†)

Figure 6

Lysyl oxidase (LOX) inhibition attenuated volume overload (VO)-induced increases in interstitial collagen. Representative picrosirius red (PSR)-stained left ventricular sections at 14 weeks postsurgery and calculated collagen volume fraction (CVF) are shown for each group. CVF was increased in the VO group compared to SHAM. LOX inhibition (B) attenuated the VO-induced increases in collagen. Statistical significance denoted as P < 0.05 vs. Sham (*) and VO(†)

Figure 7

Lysyl oxidase (LOX) inhibition (B) attenuated volume overload (VO)-induced increases in left ventricular (LV) total collagen and collagen cross-linking. Hydroxyproline (Hpro) concentration in mid-LV free wall was significantly increased in the VO group, but not different from SHAM in VO + B group. Pyridinoline (PYD) levels are a measure of LOX-dependent collagen cross-links. Fourteen weeks of VO caused a significant increase in LV PYD levels. Treatment with the LOX inhibitor attenuated the increase in PYD (P < 0.05 vs. Sham (*) and VO(†))

Figure 8

Lysyl oxidase (LOX) inhibition (B) attenuated volume overload (VO)-induced increases in collagen I and III, but did not alter elastin expression. Protein expression of collagen I and collagen III in left ventricular (LV) extracts was determined by western blot analysis (n = 4 to 6 per group). VO significantly increased LV expression of both collagen types I and III, when compared to SHAM. Rats with volume overload that were treated with LOX inhibitor had collagen I and III levels similar to SHAM. LV elastin expression was similar among all groups (P < 0.05 vs. Sham (*) and VO(†))

Figure 9

The beneficial effects of lysyl oxidase (LOX) inhibition in the rat model of volume overload. Volume overload (VO)-induced cardiac stress increases both LOX and collagen expression. The increased deposition of collagen results in extracellular matrix (ECM) remodeling and progressive cardiac dysfunction. Inhibiting LOX with beta-aminoproprionitrile (BAPN), attenuates cross-linking and ECM remodeling, leading to reduced ventricular wall stress, which halts further progression of cardiac dysfunction. (A color version of this figure is available in the online journal)