cAMP-dependent regulation of HCN4 controls the tonic entrainment process in sinoatrial node pacemaker cells

It is highly debated how cyclic adenosine monophosphate-dependent regulation (CDR) of the major pacemaker channel HCN4 in the sinoatrial node (SAN) is involved in heart rate regulation by the autonomic nervous system. We addressed this question using a knockin mouse line expressing cyclic adenosine monophosphate-insensitive HCN4 channels. This mouse line displayed a complex cardiac phenotype characterized by sinus dysrhythmia, severe sinus bradycardia, sinus pauses and chronotropic incompetence. Furthermore, the absence of CDR leads to inappropriately enhanced heart rate responses of the SAN to vagal nerve activity in vivo. The mechanism underlying these symptoms can be explained by the presence of nonfiring pacemaker cells. We provide evidence that a tonic and mutual interaction process (tonic entrainment) between firing and nonfiring cells slows down the overall rhythm of the SAN. Most importantly, we show that the proportion of firing cells can be increased by CDR of HCN4 to efficiently oppose enhanced responses to vagal activity. In conclusion, we provide evidence for a novel role of CDR of HCN4 for the central pacemaker process in the sinoatrial node.

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Software and code
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Data analysis
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub Reporting for specific materials, systems and methods The Microarray dataset generated and analysed during the current study is available in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) repository and is accessible through the GEO Series accession number GSE138086.
The source data underlying Any other datasets generated during and/or analysed during the current study are available from the corresponding author upon reasonable request.
The Figure of the C-terminus structure was prepared using PyMOL. The structure is based on sequence 521 -723 of the human HCN4 structure in complex with cAMP, downloaded from the protein data base (accession code: 3U11 At least triplicates were used to meet the minimal requirements for statistical analysis. Effort was taken to keep the number of animals at a minimum.
Animals with unsufficient quality of the ECG and/or blood pressure signal were excluded from the study. Possible technical problems during in-vivo EPS that lead to data exclusion were measurements in which the stimulus did not always lead to a capture/response, in which an animal's spontaneous heart rate was temporarily or permanently faster than the S1S1 stimulus interval, or premature termination of the experiment due to an uncontrollable stage of anaesthesia. All exclusion criteria were pre-established All animal studies and in vitro electrophysiology with mouse tissue were performed with littermates from multiple litters over time. Histology and immunohistology were performed at least three times independently. Western blot analysis from SAN tissue and microarray was performed once. All attempts at replication were successful.
All animals and cells were randomly assigned to the experimental group.
In vivo telemetry data analysis was not blinded because the ECG phenotype of the HCN4FEA animals was obvious. Analysis of all other in vivo experiments was blinded. Microscopy data analysis was performed in a blinded way, as this method is very sensitive to experimenter's biases but sample preparation, histological and biochemical experiments were not performed in a blinded way. qPCR values were normalized to the internal control, thus blinded experiment is not necessary. Data analysis of voltage-clamp electrophysiology was not blinded but performed independently by two researchers. Data analysis of current-clamp electrophysiology was not blinded but in some cases performed independently by two researchers. Optical imaging data analysis was mainly performed blinded but sample preparation and measurements were not blinded.
All other experiments were not performed or analysed in a blinded way.

October 2018
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Guinea pig polyclonal anti-HCN4, supplier: Alomone Labs, catalog number: AGP-004, polyclonal antibody, Lot AGP004AN0202 Validation: HCN4 expression was only detected in the sinoatrial node of explants and was absent in surrounding atrial tissue as shown in Supplemental Figure 1F,G. Additionally the antibody was validated by the manufacturer.
Rabbit polyclonal anti-HCN1, supplier: Alomone Labs, catalog number: APC-056, polyclonal antibody, Lot APC056AN1402 Validation: HCN1 expression was only detected in the sinoatrial node of explants and was absent in surrounding atrial tissue as shown in Supplemental Figure 1F,G. Additionally the antibody was validated by the manufacturer.
Mouse anti-HCN1, supplier: Abcam, catalog number: ab176304, polyclonal antibody, Lot n/a. Validation: The antibody was validated by the manufacturer by western blot analysis using lysates from murine tissues and several cell lines.
Rat anti-HCN4, supplier: Santa Cruz Biotechnology, catalog number: Sc-58622, clone SHG 1E5, Lot n/a. The antibody was validated by the manufacturer by western blot analysis using whole cell lysates from several cell lines.  HEK293: ATCC states that they comprehensively perform authentication (Short Tandem Repeat profiling) and quality-control tests on all distribution lots of cell lines. In cell culture the HEK cells had shown typical morphology and cell growth. Flip-In™-293: cell lines were not authenticated Cell lines were not tested for mycoplasma contamination.
No commonly misidentified cell lines were used.