Optical System for Assessment of Atrial and Ventricular Mechanics in Langendorff-perfused Mouse Hearts

Summary

This protocol describes the procedure for mouse heart extraction and cannulation, as well as the preparation and configuration of the Langendorff retrograde perfusion system. The experimental setup is coupled with a high-resolution optical system designed to evaluate the mechanical responses of the atria and ventricles while preserving their natural dynamics.

Abstract

The mouse heart serves as a widely used model in cardiovascular research, facilitated by advances in engineering technologies. However, assessment of atrial and ventricular contractility remains challenging due to the small size of the mouse heart. This study introduces a novel methodology for evaluating contractility and heart rate in an intact, isolated heart. Using a Langendorff-perfused mouse heart in combination with a high-resolution optical system enables evaluation of atrial and ventricular contractility. The experimental setup addresses challenges associated with small heart size and rapid contraction rates while acquiring high-resolution signals. Synchronization between stimulus frequency and heart contraction frequency in the range of 420 to 660 beats per minute (bpm) was consistent with results from the auxotonic method. Additionally, measurements of ventricular shortening and atrial dynamic signals allow for the determination of atrioventricular contraction. This method is suitable for assessing contractility and heart rate in an intact, isolated heart without pharmacological or systemic influences. These results demonstrate that the optical system enables measurement of ventricular and atrial shortening responses.

Introduction

There is much to discover in cardiac physiology; key processes, such as cardiac pace-making and excitation-contraction coupling, are central tenets that are continually researched¹. Although the effects of biological noise on cardiac contractility² and pace-making activity remain poorly understood¹, new optical imaging methods have allowed the presentation of new paradigms^3,4.

Two experimental methodologies using the Langendorff system have made invaluable contributions to measuring cardiac function. First, the ....

Protocol

All animal procedures were conducted in accordance with Federal Regulations for Animal Experimentation and Care and were approved by the Animal Care Committee of CINVESTAV (CINVESTAV Zacatenco, D.F., Mexico). In this study, C57BL/6J male mice are used, weighing 28–34 g, and aged 8–14 weeks. All materials and equipment used in the protocol are listed in the Table of Materials. Supplementary Files 1–11 provide supporting diagrams, software guides, analysis programs, pacing data, and application files for the protocol.

1. Stock preparation

1. Prepare two stock solutions in advance an....

Results

The cardiac mechanical function experiments are performed using the Langendorff apparatus, as shown in Figure 1A (Top). The monitoring and optical systems enable video recording when illumination control is triggered, as shown in Figure 1A (Bottom). Figure 1C illustrates the external view of the thermostatically controlled cabinet, designed to maintain the heart isolated from ambient thermal fluctua.......

Discussion

The mouse heart is very sensitive to temperature variations^2,9,10,11,22. In this experiment, the perfused heart was vertically positioned. We included a slight modification to minimize loss of perfusate temperature and motion artifacts in the Langendorff perfusion method. A recirculation pump was added to the serpentine to minimize loss of perfusate temperatur.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mL syringe	BD Plastipak	302485	Surgical procedure
20 G needle	NIPRO	AH-2032	Aortic cannula
25 mm adjustable lens with 8x-100x magnification	Generic	N/A	Optical system camera lens; cited generically as adjustable camera lens
27 G needle	BD PrecisionGlide	302358	Surgical procedure
3D-designed support pieces	CINVESTAV	N/A	Custom 3D-designed support pieces
4-0 silk suture	AMERICAN SUTURE	TGSN0901	Aortic cannula
40-A power supply	STEREN	PRL-25	LED power supply
Air stone	N/A	N/A	Tyrode's solution oxygenation
CaCl2 (2H2O)	Sigma-Aldrich	C3306	Tyrode's solution
D-Glucose	J.T. Baker	1916-01	Tyrode's solution
Drainage pump	CINVESTAV	N/A	Custom drainage pump; cited generically as drainage pump
F = 25.4 mm, plano-convex lens	THORLABS	LA1951-A	Optical system illumination lens; cited generically as plano-convex illumination lens
Glacial acetic acid	Sigma-Aldrich	695092	Langendorff system cleaning
Heparin	PiSA	060M91	Anticoagulant
HEPES	Sigma-Aldrich	H3375	Tyrode's solution
High-power LEDs	Luminius	CBT-90-G-L11-C12	LED light source
Illumination system	CINVESTAV	N/A	Custom illumination system; cited generically as illumination system
KCl	CTR SCIENTIFIC	CTR02196	Tyrode's solution
LabVIEW software version 2018	NATIONAL INSTRUMENTS	N/A	Software used to run the monitoring and pacing application; cited generically as monitoring and pacing application
MATLAB software	The MathWorks	N/A	Computational software for video normalization, segmentation, and analysis programs; cited generically as computational software
Medical-grade oxygen	GRUPO INFRA	N/A	Tyrode's solution oxygenation
MgCl2 (6H2O)	CTR SCIENTIFIC	CTR02104	Tyrode's solution
Mini-Pump variable flow	CONTROL COMPANY	3385	Perfusion pump
Mouse Surgical Kit	Kent Scientific	INSMOUSEKIT	Surgical procedure
NaCl	Sigma-Aldrich	S5886	Tyrode's solution
NaH2PO4	DEQ	DEQF290400250	Tyrode's solution
NaOH	CTR SCIENTIFIC	CTR03108	pH adjustment
NI USB-6009	NATIONAL INSTRUMENTS	779026-01	Acquisition card
Platinum electrodes, diam. 0.25 mm	Sigma-Aldrich	349402	Electrical stimulation electrodes
Power supply (12 V, 30 A)	Karnick	PTA-12V30A	Recirculator power supply
Pressure monitor	CINVESTAV	N/A	Custom pressure monitor; cited generically as pressure monitor
Pressure transducer	LA BOUVET	B17295	Langendorff system pressure monitor
Pure ethyl alcohol	Sigma-Aldrich	493511	Ethanol for system cleaning
Recirculating thermal bath (LXC)	PolyScience	L113A0412	Reservoir recirculator
Saline solution	PiSA	111848	0.9% sodium chloride (NaCl) solution
Serpentine thermal bath	CINVESTAV	N/A	Custom serpentine recirculator/thermal bath
Stereomicroscope	N/A	N/A	Surgical procedure
Stereomicroscope illumination	N/A	N/A	Surgical procedure
uEye Cockpit software	IDS Imaging Development System GmBH	N/A	Camera acquisition software; cited generically as camera acquisition software
UI-3360CP-NIR-GL Rev.2	IDS	AB00624	High-speed CMOS camera; cited generically as CMOS camera
Xylazine	PiSA	4001211	Anesthetic
Zoletil 100	Virbac	83048907	Tiletamine-zolazepam anesthetic; cited generically as anesthetic mixture