TITLE: Distribution and Morphology of Calcitonin Gene-Related Peptide (CGRP) Innervation in Flat Mounts of Whole Rat Atria and Ventricles
AUTHORS: Jin Chen, Kohlton T Bendowski, Ariege Bizanti, Yuanyuan Zhang, Jichao Ma, Donald Hoover, David Gozal, Kalyanam Shivkumar, Zixi (Jack) Cheng

[DESCRIPTION]
Calcitonin gene-related peptide (CGRP) is widely used as a marker for nociceptive afferent axons. However, the distribution of CGRP-IR axons has not been fully determined in the whole rat heart. Immunohistochemically labeled flat-mounts of the right and left atria and ventricles, and the interventricular septum in rats for CGRP were assessed with a Zeiss imager to generate complete montages of the entire atria, ventricles, and septum, and a confocal microscope was used to acquire detailed images of selected regions. We found that 1) CGRP-IR axons extensively innervated all regions of the atrial walls and the walls of the great vessels including the sinoatrial node region, auricles, atrioventricular node region, superior/inferior vena cava, left pre-caval vein, and pulmonary veins. 2) CGRP-IR axons formed varicose terminals around individual neurons in some cardiac ganglia but passed through other ganglia without making appositions with cardiac neurons. 3) Varicose CGRP-IR axons innervated the walls of blood vessels. 4) CGRP-IR axons extensively innervated the right/left ventricular walls and interventricular septum. Our data shows the rather ubiquitous distribution of CGRP-IR axons in the whole rat heart at single-cell/axon/varicosity resolution for the first time. This study lays the foundation for future studies to quantify the differences in CGRP-IR axon innervation between sexes, disease models, and species.


[STEPS]
SECTION: Whole heart dissection and flat-mount preparation
1. Rats were first deeply anesthetized with isoflurane (5%; 5-10 minutes), with an oxygen flow rate of 1 liter per minute. When the animals were not responsive to the hind-toe pinch withdrawal reflex, the chest was opened and an injection of heparin (100 units) was made in the apex of the left ventricle. After the inferior vena cava was cut to drain the blood and perfusate, a needle was inserted into the left ventricle and the perfusion began. The rats were first perfused with 500 ml of 37-40 °C phosphate-buffered saline (0.1 M PBS, pH = 7.4) followed by 300 ml paraformaldehyde (4%; 4 °C).
SECTION: Whole heart dissection and flat-mount preparation
2. The tissues were separated in the same manner that is shown in Figure 1 of Bizanti et al., 2023 [1] (see the figure below). Note: The following figure uses an FVB mouse as an example, however the current protocol uses Sprague-Dawley rats. The heart, lungs, and trachea were removed immediately after perfusion and stored in 4 % paraformaldehyde for at least 24 hours at 4 ºC. The atria and ventricles were separated at the atrial-ventricular groove, and the left and right atria and ventricles were separated. The left atrium was prepared with the entrance portions of all pulmonary veins (PVs) attached and the right atrium was prepared with the superior vena cava (SVC), inferior vena cava (IVC) and left precaval vein (LPCV) attached.
 

SECTION: Immunohistochemistry
3. Dissected tissues were washed 3 times for 15 min in 0.01 M PBS (pH = 7.4) in a 6-well plate on an orbital shaker to remove the remaining fixative.
SECTION: Immunohistochemistry
4. To prevent non-specific binding and enhance antibody penetration, the atria were fully submerged in a blocking solution (2% bovine serum albumin, 10% normal donkey serum, 2% Triton X-100, 0.08% NaN3 (in 0.1 M PBS, pH = 7.4)) for 5 days (atria) at 4 ºC.
SECTION: Immunohistochemistry
5. Anti-CGRP primary antibody (Cell Signaling, Danvers, MA; Cat # 14959, RRID: AB_2798662; 1:200) was mixed with the primary solution (2% bovine serum albumin, 4% normal donkey serum, 0.5% Triton X-100, 0.08% NaN3 in 0.1 M PBS, pH = 7.4) and incubated with the tissues for 5 days (for atria) or 7 days (for ventricles) at 4 ºC.
SECTION: Immunohistochemistry
6. Tissues were thoroughly washed 6 times for 10 min in PBST (phosphate buffered saline with Triton X-100; 0.5% Triton X-100 in 0.01 M PBS) to remove unbound primary antibodies.
SECTION: Immunohistochemistry
7. Tissues were then kept in the dark and incubated in a fluorescent secondary antibody solution (Alexa Fluor 488; Thermo Fisher, Waltham, MA, Cat# A21202, RRID:AB_141607; 1:100) overnight at room temperature. Unbound secondary antibodies were removed by washing 6 times for 10 minutes in PBS at room temperature.
SECTION: Immunohistochemistry
8. Tissues were then mounted onto glass slides and flattened with lead weights for either 2 days (20 lbs for atria) or 3 weeks (30 lbs for ventricles) at 4 ºC.
SECTION: Immunohistochemistry
9. Slides were dehydrated in four ascending concentrations of ethanol (75%, 95%, 100%, and 100%) for 2 min in each concentration, followed by 2 × 10 min washes in 100% xylene.
SECTION: Immunohistochemistry
10. Coverslips were then attached using DEPEX mounting medium (Electron Microscopy Sciences #13514) and allowed to dry overnight.
SECTION: Immunohistochemistry
11. Negative controls, which excluded primary antibodies, showed no immunoreactivity, confirming that nonspecific binding of secondary antibodies did not occur. Antibody specificity was tested by Cell Signaling Technology using Western Blot and immunofluorescence (https://www.cellsignal.com/products/primary-antibodies/cgrp-d5r8f-rabbitmab/14959). Western Blot analysis shows a single band at the correct molecular weight of CGRP in TT cells but not in HeLa cells. Moreover, confocal immunofluorescent analysis of TT and HeLa cells confirms that CGRP were in TT cells but not in HeLa cells.
SECTION: Image acquisition
13. Hundreds of overlapping maximum projection images from stacks of optical sections (z-step: 1.5 μm for atria and 2 μm for the ventricles) from each tissue were captured using a Zeiss M2 Imager (20x lens; NA 0.8) and stitched back together seamlessly to yield full photo montages of the right and left atria and ventricles. An LED light source with a 488 nm wavelength was used to visualize the CGRP-IR axons in the tissues.
SECTION: Image acquisition
14. A Leica TCS SP5 laser-scanning confocal microscope (40x oil immersion lens; NA 1.25; z-step: 1 μm) was used to capture detailed images of CGRP-IR axons and their targets in selected locations of the heart. An argon–krypton laser (488 nm) was used to visualize CGRP-IR axons in the tissue, and a helium-neon laser (543 nm) was used to detect the autofluorescence of the tissues in the background (e.g., cardiac muscles, ganglionic cells, and blood vessels).
SECTION: Image acquisition
15. Modifications, including brightness and contrast adjustments, and scale bar additions, were performed using Photoshop or FIJI ImageJ software [3]. Of the 6 rat hearts that were used in this study, we showed the representative cases from each tissue in our figures (RA, LA, RV, LV, and IVS).
SECTION: Immunohistochemistry
7.1. Rat ventricles were pretreated with methanol (see the following sub-steps) and the immunolabeling procedures outlined in [2] Note: the clearing step was omitted entirely from the procedures outlined in the referenced publication.
SECTION: Immunohistochemistry
7.2. Tissues were dehydrated in methanol/H2O in the following series of concentrations: 20%, 40%, 60%, 80%, 100% for 1 hour each.
SECTION: Immunohistochemistry
7.3. Tissues were further washed in 100% methanol for 1 hour and then chilled in 4o C.
SECTION: Immunohistochemistry
7.4. Tissues were incubated overnight on a shaker in 66% DCM/33% methanol at room temperature.
SECTION: Immunohistochemistry
7.5. Tissues were washed twice in 100% methanol at room temperature and then they were chilled at 4o C.
SECTION: Immunohistochemistry
7.6. Tissues were bleached in chilled fresh 5% H2O2 in methanol (1 volume 30% H2O2 to 5 volumes methanol) overnight at 4o C.
SECTION: Immunohistochemistry
7.7. Tissues were rehydrated in the following series of methanol/H2O solutions: 80%, 60%, 40%, 20%, PBS for 1 hour each at room temperature.
SECTION: Immunohistochemistry
7.8. Tissues were washed twice in room temperature PBST (PTx.2) for 1 hour at room temperature.
SECTION: Immunohistochemistry
12.