Cardiac Response to β-Adrenergic Stimulation Determined by Pressure-Volume Loop Analysis

Summary

Here we describe a cardiac pressure-volume loop analysis under increasing doses of intravenously infused isoproterenol to determine the intrinsic cardiac function and the β-adrenergic reserve in mice. We use a modified open-chest approach for the pressure-volume loop measurements, in which we include ventilation with positive end-expiratory pressure.

Abstract

Determination of the cardiac function is a robust endpoint analysis in animal models of cardiovascular diseases in order to characterize effects of specific treatments on the heart. Due to the feasibility of genetic manipulations the mouse has become the most common mammalian animal model to study cardiac function and to search for new potential therapeutic targets. Here we describe a protocol to determine cardiac function in vivo using pressure-volume loop measurements and analysis during basal conditions and under β-adrenergic stimulation by intravenous infusion of increasing concentrations of isoproterenol. We provide a refined protocol including ventilation support taking into account the positive end-expiratory pressure to ameliorate negative effects during open-chest measurements, and potent analgesia (Buprenorphine) to avoid uncontrollable myocardial stress evoked by pain during the procedure. All together the detailed description of the procedure and discussion about possible pitfalls enables highly standardized and reproducible pressure-volume loop analysis, reducing the exclusion of animals from the experimental cohort by preventing possible methodological bias.

Introduction

Cardiovascular diseases typically affect cardiac function. This issue points out the importance in assessing in vivo detailed cardiac function in animal disease models. Animal experimentation is surrounded by a frame of the three Rs (3Rs) guiding principles (Reduce/Refine/Replace). In case of understanding complex pathologies involving systemic responses (i.e., cardiovascular diseases) at the current developmental level, the main option is to refine the available methods. Refining will also lead to a reduction of the required animal numbers due to less variability, which improves the power of the analysis and conclusions. In addition, combination of cardiac contractil....

Protocol

All animal experiments were approved and performed according to the regulations of the Regional Council of Karlsruhe and the University of Heidelberg (AZ 35-9185.82/A-2/15, AZ 35-9185.82/A-18/15, AZ 35-9185.81/G131/15, AZ 35-9185.81/G121/17) conform to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Data shown in this protocol are derived from wild type C57Bl6/N male mice (17 ± 1.4 weeks of age). Mice were maintained under specified .......

Discussion

Here, we provide a protocol to analyze the in vivo cardiac function in mice under increasing β-adrenergic stimulation. The procedure can be used to address both, baseline parameters of cardiac function and the adrenergic reserve (e.g., inotropy and chronotropy) in genetically modified mice or upon interventions. The most prominent advantage of pressure-volume loop (PVL) measurements as compared to other means of determining cardiac function is the analysis of intrinsic, load-independent cardiac function. All other m.......

Materials

Name	Company	Catalog Number	Comments
1.4F SPR-839 catheter	Millar Instruments, USA	840-8111
1 ml syringes	Beckton Dickinson, USA	REF303172
Bio Amplifier	ADInstruments, USA	FE231
Bridge-Amplifier	ADInstruments, USA	FE221
Bovine Serum Albumin	Roth, Germany	8076.2
Buprenorphine hydrochloride	Bayer, Germany	4007221026402
Calibration cuvette	Millar, USA	910-1049
Differential pressure transducer MPX	Hugo Sachs Elektronik- Harvard Apparatus, Germany	Type 39912
Dumont Forceps #5/45	Fine Science tools Inc.	11251-35
Dumont Forceps #7B	Fine Science tools Inc.	11270-20
Graefe Forceps	Fine Science tools Inc.	11051-10
GraphPad Prism	GraphPad Software	Ver. 8.3.0
EcoLab-PE-Micotube	Smiths, USA	004/310/168-1
Etomidate Lipuro	Braun, Germany	2064006
Excel	Microsoft
Heparin	Ratiopharm, Germany	R26881
Hot plate and control unit	Labotec, Germany	Hot Plate 062
Isofluran	Baxter, Germany	HDG9623
Isofluran Vaporizer	Abbot	Vapor 19.3
Isoprenalinhydrochloride	Sigma-Aldrich, USA	I5627
Fine Bore Polythene tubing 0.61 mm OD, 0.28 mm ID	Smiths Medical International Ltd, UK	Ref. 800/100/100
MiniVent ventilator for mice	Hugo Sachs Elektronik- Harvard Apparatus, Germany	Type 845
MPVS Ultra PVL System	Millar Instruments, USA
NaCl	AppliChem, Germany	A3597
NaCl 0.9% isotonic	Braun, Germany	2350748
Pancuronium-bromide	Sigma-Aldrich, USA	BCBQ8230V
Perfusor 11 Plus	Harvard Apparatus	Nr. 70-2209
Powerlab 4/35 control unit	ADInstruments, USA	PL3504
Rechargeable cautery-Set	Faromed, Germany	09-605
Scissors	Fine Science tools Inc.	140094-11
Software LabChart 7 Pro	ADInstruments, USA	LabChart 7.3 Pro
Standard mouse food	LASvendi GmbH, Germany	Rod18
Stereo microscope	Zeiss, Germany	Stemi 508
Surgical suture 8/0	Suprama, Germany	Ch.B.03120X
Venipuncture-cannula Venflon Pro Safty 20-gauge	Beckton Dickinson, USA	393224
Vessel Cannulation Forceps	Fine Science tools Inc.	00574-11
Water bath	Thermo Fisher Scientific, USA
Syringe filter (Filtropur S 0.45)	Sarstedt, Germany	Ref. 83.1826