Mapping L-type calcium channel arrangement and function
1. Purpose
During the heartbeat, L-type calcium channels (LTCCs) in the surface membrane open and trigger additional calcium (Ca) release via Ryanodine Receptors (RyR) in myocytes, initiating contraction. The interaction between LTCCs and RyRs ensures well-regulated Ca release. However, the precise manner by which LTCCs collaborate is unclear, since it has not been previously possible to image LTCCs and Ca simultaneously at nanoscale resolution. This important goal will be attained in the present project using a gene-modified mouse with a super-resolution appropriate fluorophore encoded on the LTCC. We will examine healthy cardiomyocytes to determine how plasticity of LTCC localization affects Ca homeostasis. In follow-up work, we will examine how changing LTCC localization during heart failure (HF) leads to impaired Ca homeostasis, decreased contractility, and arrhythmia, which are hallmarks of this condition. Thus the work will provide unprecedented understanding of LTCC configuration and function in both normal and failing cardiomyocytes.
2. Distress
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 heart and imaged. In other experiments, animals will be subjected to surgery under anesthesia (infarction or aortic banding) to induce HF development. Pain can be experienced immediately following surgery, as indicated by raised fur, and decreased mobility. Animals exhibiting progression to HF may also exhibit distress, including signs of irregular, laboured breathing. In both the acute and chronic situation, signs of pain will be treated with analgesics.
3. Expected benefit
The relationship between LTCC 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 HF. We anticipate that our work will lead to an improved understanding of this disease's mechanisms and the discovery of novel drug targets for HF patients.
4. Number of animals and species
637 gene-modified mice with “knock-in” labeling of LTCCs will be employed.
5. Fulfillment of the 3Rs
Replacement: Experiments on animals can unfortunately not yet replace the experiments described in this study. However, 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 data to mathematical modelers to create calcium homeostasis models, which will help reduce the use of experimental animals.
Reduction: The number of required animals has been minimized. 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 timepoints examined after myocardial infarction or aortic banding are chosen based on previous findings which have indicated that these represent distinct, progressive stages of HF development. Trained surgical staff reproducibly induce HF with minimum animal suffering.
During the heartbeat, L-type calcium channels (LTCCs) in the surface membrane open and trigger additional calcium (Ca) release via Ryanodine Receptors (RyR) in myocytes, initiating contraction. The interaction between LTCCs and RyRs ensures well-regulated Ca release. However, the precise manner by which LTCCs collaborate is unclear, since it has not been previously possible to image LTCCs and Ca simultaneously at nanoscale resolution. This important goal will be attained in the present project using a gene-modified mouse with a super-resolution appropriate fluorophore encoded on the LTCC. We will examine healthy cardiomyocytes to determine how plasticity of LTCC localization affects Ca homeostasis. In follow-up work, we will examine how changing LTCC localization during heart failure (HF) leads to impaired Ca homeostasis, decreased contractility, and arrhythmia, which are hallmarks of this condition. Thus the work will provide unprecedented understanding of LTCC configuration and function in both normal and failing cardiomyocytes.
2. Distress
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 heart and imaged. In other experiments, animals will be subjected to surgery under anesthesia (infarction or aortic banding) to induce HF development. Pain can be experienced immediately following surgery, as indicated by raised fur, and decreased mobility. Animals exhibiting progression to HF may also exhibit distress, including signs of irregular, laboured breathing. In both the acute and chronic situation, signs of pain will be treated with analgesics.
3. Expected benefit
The relationship between LTCC 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 HF. We anticipate that our work will lead to an improved understanding of this disease's mechanisms and the discovery of novel drug targets for HF patients.
4. Number of animals and species
637 gene-modified mice with “knock-in” labeling of LTCCs will be employed.
5. Fulfillment of the 3Rs
Replacement: Experiments on animals can unfortunately not yet replace the experiments described in this study. However, 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 data to mathematical modelers to create calcium homeostasis models, which will help reduce the use of experimental animals.
Reduction: The number of required animals has been minimized. 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 timepoints examined after myocardial infarction or aortic banding are chosen based on previous findings which have indicated that these represent distinct, progressive stages of HF development. Trained surgical staff reproducibly induce HF with minimum animal suffering.