Mapping Ryanodine Receptor Arrangement and Function
I. Experimental Goal
During the heartbeat, calcium is released via a large channel known as the Ryanodine Receptor (RyR) in cardiac myocytes, and contraction is triggered. However, the precise manner by which RyRs collaborate is unclear, since it has not been previously possible to image RyRs and Ca simultaneously. This important goal will be attained in the present project using a gene-modified mouse with a super-resolution appropriate fluorophore encoded on the RyR. We will first examine healthy cardiomyocytes, to determine how plasticity of RyR localization affects Ca homeostasis. In follow-up work, we will examine how changing RyR localization during heart failure leads to impaired calcium homeostasis, decreased contractility, and arrhythmia which are hallmarks of this condition. Thus the work will provide unprecedented understanding of RyR configuration and function in both normal and failing cardiomyocytes.
II. Expected detrimental effects on animal welfare
The gene-modified mice will be employed in several types of experiments. Some of these are acute experiments, where animals are sacrificed and cells isolated from the heart and imaged. In other experiments, animals will be subjected to surgery under anesthesia (infarction or aortic banding) to induce the development of heart failure. Moderate pain can be experienced either immediately following surgery or during heart failure progression. In both cases, pain will be treated with analgesics.
III. Expected scientific news value
The relationship between RyR organization and function has remained elusive, placing high news value on the current work. Equally important are the planned experiments aimed at understanding how this structure/function relationship changes during heart failure. We anticipate that our work will lead to an improved understanding of the mechanisms of this disease and the discovery of novel drug targets for heart failure patients.
IV. Number of animals and species
1236 gene-modified mice with “knock-in” labeling of the RyR will be employed.
V. Fulfillment of the 3Rs
Replacement: Experiments on animals can unfortunately not yet replace the experiments described in this study. However, we hope that our results will provide sufficient insight for others in the field to avoid similar experiments in the future. As part of our study, we are providing attained data to mathematical modelers in an ongoing effort to create models of calcium homeostasis which will help reduce the use of experimental animals.
Reduction: The number of required animals has been minimized. Indeed, cells isolated from hearts will be simultaneously used by several researchers which will make data collection more efficient, allowing examination of more cells from each heart. In the case of early successful results, the number of employed mice can be immediately reduced.
Refinement: Our previous experience in this field has enabled refinement of the experimental protocols to minimize pain and suffering caused to animals. The time points examined after myocardial infarction or aortic banding are chosen based on previous findings which have indicated that these represent distinct, progressive stages of heart failure development. Trained surgical staff reproducibly induce heart failure with minimum animal suffering.
During the heartbeat, calcium is released via a large channel known as the Ryanodine Receptor (RyR) in cardiac myocytes, and contraction is triggered. However, the precise manner by which RyRs collaborate is unclear, since it has not been previously possible to image RyRs and Ca simultaneously. This important goal will be attained in the present project using a gene-modified mouse with a super-resolution appropriate fluorophore encoded on the RyR. We will first examine healthy cardiomyocytes, to determine how plasticity of RyR localization affects Ca homeostasis. In follow-up work, we will examine how changing RyR localization during heart failure leads to impaired calcium homeostasis, decreased contractility, and arrhythmia which are hallmarks of this condition. Thus the work will provide unprecedented understanding of RyR configuration and function in both normal and failing cardiomyocytes.
II. Expected detrimental effects on animal welfare
The gene-modified mice will be employed in several types of experiments. Some of these are acute experiments, where animals are sacrificed and cells isolated from the heart and imaged. In other experiments, animals will be subjected to surgery under anesthesia (infarction or aortic banding) to induce the development of heart failure. Moderate pain can be experienced either immediately following surgery or during heart failure progression. In both cases, pain will be treated with analgesics.
III. Expected scientific news value
The relationship between RyR organization and function has remained elusive, placing high news value on the current work. Equally important are the planned experiments aimed at understanding how this structure/function relationship changes during heart failure. We anticipate that our work will lead to an improved understanding of the mechanisms of this disease and the discovery of novel drug targets for heart failure patients.
IV. Number of animals and species
1236 gene-modified mice with “knock-in” labeling of the RyR will be employed.
V. Fulfillment of the 3Rs
Replacement: Experiments on animals can unfortunately not yet replace the experiments described in this study. However, we hope that our results will provide sufficient insight for others in the field to avoid similar experiments in the future. As part of our study, we are providing attained data to mathematical modelers in an ongoing effort to create models of calcium homeostasis which will help reduce the use of experimental animals.
Reduction: The number of required animals has been minimized. Indeed, cells isolated from hearts will be simultaneously used by several researchers which will make data collection more efficient, allowing examination of more cells from each heart. In the case of early successful results, the number of employed mice can be immediately reduced.
Refinement: Our previous experience in this field has enabled refinement of the experimental protocols to minimize pain and suffering caused to animals. The time points examined after myocardial infarction or aortic banding are chosen based on previous findings which have indicated that these represent distinct, progressive stages of heart failure development. Trained surgical staff reproducibly induce heart failure with minimum animal suffering.