An Experimental Model of Myocardial Infarction for Studying Cardiac Repair and Remodeling in Knockout Mice

Summary

Here, we describe an experimental model of myocardial infarction, an echocardiography procedure to study cardiac remodeling and function, and procedures for quantifying fibrosis and hypertrophy in picrosirius red-stained and rhodamine-stained sections, as well as the infarct size and expansion index in slices stained with Masson’s trichrome.

Abstract

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. This paper describes a protocol for studying the role of galectin 3 (Gal-3) in the temporal evolution of cardiac healing and remodeling in an experimental animal model of MI.

The procedures described include an experimental model of MI with a permanent coronary ligature in male C57BL/6J (control) and Gal-3 knockout (KO) mice, an echocardiography procedure to study cardiac remodeling and systolic function in vivo, a histological evaluation of interstitial myocardial fibrosis with picrosirius red-stained and rhodamine-conjugated lectin-stained sections for studying myocyte hypertrophy by the cross-sectional area (MCSA), and the quantification of infarct size and cardiac remodeling (scar thinning, septum thickness, and expansion index) by planimetry in slices stained with Masson’s trichrome and triphenyl tetrazolium chloride. Gal-3 KO mice with MI showed disrupted cardiac remodeling and an increase in the scar thinning ratio and the expansion index. At the onset of MI, myocardial function and cardiac remodeling were also severely affected. At 4 weeks post MI, the natural evolution of fibrosis in infarcted Gal-3 KO mice was also affected.

In summary, the experimental model of MI is a suitable model for studying the temporal evolution of cardiac repair and remodeling in mice with the genetic deletion of Gal-3 and other animal models. The lack of Gal-3 affects the dynamics of cardiac repair and disrupts the evolution of cardiac remodeling and function after MI.

Introduction

Myocardial infarction (MI) is the most prevalent form of cardiovascular disease. After MI, the myocardium undergoes serial morphological and functional changes, including the healing of the MI infarct zone, ventricular remodeling (VR), and myocardial dysfunction¹. The healing of MI is a dynamic and well-orchestrated process associated with profound inflammatory infiltration that ends in the formation of a fibrotic scar^2,3. The experimental model of MI in mice is currently used for studying cardiac remodeling under pathological conditions^4,5, and awareness of the precise surgical protocol is essential to develop a reproducible and effective procedure for inducing a permanent coronary ligature. This method is needed to study the healing of MI and its relevance in the temporal evolution of left ventricular remodeling (LVR) and the cardiac dysfunction associated with MI.

Galectins are a group of lectins that recognize specific carbohydrates in intracellular ligands, membrane receptors, and extracellular glycoproteins. Galectin 3 (Gal-3) is a member of this family that acts through the recognition and cross-linking of N- and O-glycans in glycoconjugates on the cell surface, and it is widely expressed in the immune system⁶. Previous studies have investigated the role of Gal-3 as a regulator of inflammation and fibrosis in cardiovascular diseases^{7,8,9,10,11,12}. As targeting the regulatory factors of inflammation during healing is highly relevant because inflammation can notably affect the evolution of remodeling, we aimed to describe a protocol for studying the temporal evolution of post-MI ventricular remodeling and the steps and methods for determining how the genetic mutation of Gal-3 modifies the temporal evolution of healing in MI and affects cardiac remodeling and function in mice.

Protocol

NOTE: All the experiments described in this protocol were approved by the Animal Care and Research Committee of the University of Buenos Aires (CICUAL), in line with the National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals13. For the experiments, use male, age-matched C57BL/6J and Gal-3 KO mice (8-10 weeks old) weighing 30-35 g, which allow for better manipulation for surgery. Allow the animals access to water and food ad libi…

Representative Results

Post-MI survival and necropsy Over 4 weeks of follow-up, 17% (4/23) of the C57 mice versus 40% (8/20) of the Gal-3 KO mice were found dead. The necropsy was performed; the dead Gal-3 KO mice showed larger hearts than the C57 mice (Figure 1), and 38% of the C57 mice compared with 32% of the Gal-3 KO mice had macroscopic chest clots that were directly associated with cardiac rupture. The latter demonstrated that the cause of death was not relat…

Discussion

The experimental model of MI by permanent coronary artery ligature is used for studying a wide variety of pathophysiological mechanisms of cardiac repair and remodeling^5,14,17. This article summarizes different methods currently used in this laboratory for studying the temporal evolution of cardiac repair and its effects on post-MI ventricular remodeling^14,17. Two models…

Materials

8-0 silk suture	Ethicon
C57BL/6J mice	Department of Bioresources of the Faculty of Veterinary of the University of Buenos Aires, Argentina
Forceps
Hardvard 386 respirator	Hardvard company
Heating pad			maintain animal's temperature during surgery
Image Pro-Plus 6.0	Media Cybernetics		Image Analysis Software
Ketamine	Holiday
Masson Trichrome	BIOPUR
Picrosirius red	BIOPUR
Retractors
Rodent Ventilator Model 683	Harvard Apparatus		Mechanical ventilator
Scissors
Stereoscopic magnifying glass	Arcano
Vivid 7 machine (General Electric Medical Systems, Horten, Norway)	General Electric		Any tracking software can be utilized with this protocol
WGA no. RL-1022, Vector Laboratories, Burlingame	Vector Laboratories
Xylazine	Pro-Ser