Hoge-resolutie optische kaart brengen van de Muis sinusknoop
Summary
Hier presenteren we een protocol voor optische kaart brengen van de elektrische activiteit van de muis rechteratrium en vooral de sinusknoop, bij een hoge ruimtelijke en temporele resolutie.
Abstract
Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The development of novel pharmacological therapies to cure these patients relies on the thorough understanding of both normal physiology and pathophysiology of the SAN. Among the studies of cardiac pacemaking, the mouse SAN is widely used due to its feasibility for modifications in the expression of different genes that encode SAN ion channels or calcium handling proteins. Emerging evidence from electrophysiological and histological studies has also proved the representativeness and similarity of the mouse SAN structure and functions to larger mammals, including the presence of specialized conduction pathways from the SAN to the atrium and a complex pacemakers’ hierarchy within the SAN. Recently, the technique of optical mapping has greatly facilitated the exploration and investigation of the origin of excitation and conduction within and from the mouse SAN, which in turn has extended the understanding of the SAN and benefited clinical treatments of SAN dysfunction associated diseases. In this manuscript, we have described in detail how to perform the optical mapping of the mouse SAN from the intact, Langendorff-perfused heart and from the isolated atrial preparation. This protocol is a useful tool to enhance the understanding of mouse SAN physiology and pathophysiology.
Introduction
Novel scientific breakthroughs that lead to leaps in the understanding of human physiology are often preceded by technological advances. Fluorescent optical mapping, for example, enables investigation of multiple physiological parameters in both cells and tissues.1,2 It significantly improved our understanding of how the anatomical structure is associated with electrophysiological functions and dysfunctions. In the heart, the natural pacemaker and conduction systems such as the sinoatrial node (SAN) and the atrioventricular junction consist of nodal myocytes that are insulated by the surrounding atrial myocytes.3-5 Such organization creates complex three-dimensional structures with specialized electrical properties. Both structural and functional remodeling of these pacemaker structures has been recognized to form significant electrophysiological heterogeneities.6,7 Understanding the mechanism underlying how such heterogeneities result in SAN dysfunctions and atrial arrhythmogenesis will dramatically benefit the clinical treatment of these diseases. It requires a technique to visualize the propagation of electrical signals at the tissue level, such as optical mapping.
Recently, accumulating evidence has proven the advance of optical mapping in studies of atrial electrophysiology and pathology.2 However, novel and rigorous studies are dependent on the accurate interpretation of experimental data, whose validity and stability rely on careful experiment protocols. Genetically modified mice are extensively used for research as animal models of human diseases including sick sinus syndrome, pacemaker abnormalities and atrial arrhythmias.6,8-11 Thus, a combination of fluorescent optical mapping with transgenic mouse models provides a powerful tool to study cardiac electrical abnormalities associated with various pathologies. In this paper, we present a protocol for high-resolution optical mapping of the mouse SAN and atrium. Specifically, we discuss and compare different dye loading approaches, time effects on dye bleaching and heart rate stability during the experiment.
Protocol
Representative Results
Discussion
Hier presenteerden wij twee soorten muizen SAN preparaten: 1) intact SAN in het Langendorff-geperfuseerde hele hart, en 2) SAN in het geïsoleerde, open atriale preparaat. Deze twee types van de voorbereiding dienen verschillende experimentele doeleinden. In de Langendorff-geperfuseerde hele hartsvoorbereiding, wordt de intacte atriale structuur behouden waardoor het mogelijk complexe atriale aritmieën zoals atriumfibrillatie en interacties tussen de SAN en atrium bestuderen bij inspringende tachycardie. 6 d…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We are supported by the University of Wisconsin-Madison Medical School start-up (A.V.G.).
Materials
| water jacket | Radnoti | 1660 Series Tissue Bath for Large Organ or Single Cell Isolation Procedures | |
| water bath / circulator | Fisher Scientific | 1016S | |
| pressure amplifier | AD Instruments | MLT0670 | |
| EMD Millipore Nylon Net Filters | Fisher Scientific | NY1102500 | |
| Pressure transducer | AD Instruments | MLT0670 | |
| Stainless Steel Minutien Pins – 0.1mm Diameter | Fine Science Tools | 26002-10 | |
| Perfusion pump | World Precision Instruments | PERIPRO-4LS | |
| Superfusion pump | World Precision Instruments | PERIPRO-4HS | |
| Vannas Tubingen scissors | World Precision Instruments | 503379 | |
| Dumont forceps | World Precision Instruments | 501201, 500085 | |
| Mayo scissors | World Precision Instruments | 501750 | |
| Kelly hemostatic forceps | World Precision Instruments | 501241 | |
| Iris forceps | World Precision Instruments | 15917 | |
| Iris scissors | World Precision Instruments | 501263 | |
| ECG 12 mm needle (29-gauge) electrodes (monopolar) | AD Instruments | MLA1203 | |
| in-line Luer injection port | Ibidi | 10820 | |
| Ultima-L CMOS camera | SciMedia | MiCAM-05 | |
| halogen lamp | Moritex USA Inc | MHAB-150W | |
| NaCl | Fisher Scientific | S271-1 | |
| CaCl2 (2H2O) | Fisher Scientific | C79-500 | |
| KCl | Fisher Scientific | S217-500 | |
| MgCl2 (6H2O) | Fisher Scientific | M33-500 | |
| NaH2PO4 (H2O) | Fisher Scientific | S369-500 | |
| NaHCO3 | Fisher Scientific | S233-3 | |
| D-Glucose | Fisher Scientific | D16-1 | |
| Blebbistatin | Tocris Bioscience | 1760 | |
| RH237 | ThermoFisher Scientific | S1109 | |
| Dimethyl sulphoxide (DMSO) | Sigma-Aldrich | D2650 | |
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