Peer review of: Cardiac dopamine D1 receptor triggers ventricular arrhythmia in chronic heart failure (2020)

Reviewers' comments: 

 

Reviewer #1 (Remarks to the Author): 

 

General comments: This report deals with the role of the cardiac dopamine D1 receptor (D1R) in the 
pathogenesis of cardiac arrhythmia in heart failure. The combination of studies in rodents and 
humans is a strength of this report. There is a decrease in premature ventricular contractions (PVCs) 
in mice with cardiomyocyte-specific deletion of Drd1 subjected to thoracic aorta constriction (TAC) 
(Figure 1g). However, the PVCs induced by dopamine and caffeine do not seem to be decreased by 
cardiomyocyte-specific deletion of Drd1 (Figure 2a). The blood pressures of the mice were not 
measured (Card Electrophysiol Clin. 2015 Jun;7(2):207-20). 

Specific concerns: 

A. Major: 

1. The statement in (line 14) “Increased expression of Drd1, but no other dopamine receptor genes 
was [sic] confirmed by quantitative PCR (qPCR)” needs proof. The D1R, D3R, and D4R have been 
reported to be expressed in the heart (Pharmacol Ther. 2019 Jul 9:107392), as well as the D2R and 
D5R in the human (Heart Vessels. 2010 Sep;25(5):432-7 (reference 17)) and rat hearts (Circulation. 
2012 Dec 11;126(24):2859-69). 

2. Figure 2b is cited to indicate that the prevention of the cardiomyocyte-specific deletion of D1R 
decreases the number of PVCs. However, it seems that the number of PVCs with doxycycline 

-tTA. 

3. Supplementary Fig. 5b. The failure to find a significant difference in cardiac DRD1 mRNA in 
patients with history of sustained ventricular tachycardia and/or with an implantable cardioverter 
defibrillator device than patients without ventricular tachycardia weakens the linkage between 
increased cardiac DRD1 expression and ventricular tachycardia. 

3. Supplementary Table. The average expression of DRD1 mRNA in patients with no tachycardia or 
nonsustained tachycardia can be higher (44 years old, 40 years old#2 and #3, and 38 years old) than 
several patients with sustained ventricular tachycardia). 

B. Minor: 

1. References: 

a. Reference 5 deals with ibopamine which is and agonist at both α-adrenergic and dopamine 
receptor (Clin Cardiol. 1995 Mar;18(3 Suppl I):I17-21). 

b. Reference 6 deals with the treatment of shock, not heart failure. 

2. Grammar. The manuscript needs editing for grammar and syntax. 

3. Formatting: 

a. LacZ is upside down in Figure 1f. Sometimes, there is a space between Lac and Z. 

 

 

 

Reviewer #2 (Remarks to the Author): 

 

This manuscript investigates the role of role of dopamine receptor D1 (D1R)-expressing 
cardiomyocytes in triggering heart failure-associated ventricular arrhythmias. The authors show that 
D1R-expressing cardiomyocytes are found in mice subject to TAC induced heart failure as well as 
heart failure patients with sustained ventricular arrhythmias. Overexpression and deletion of D1R is 
generated in mouse models and correlates directly with the incidence of ventricular arrhythmias. 
The authors conclude that abnormal calcium handling in cardiomyocytes results from D1R 
expression during heart failure and predisposes to ventricular arrhythmia.This study reveals a 
potentially interesting role for the dopamine receptor in heart failure associated ventricular 
arrhythmias. The data does present significant data that expression levels of D1R rise during heart 
failure and correlates with an increased frequency of mild ventricular arrhythmias. Concerns are 
listed below. 

 

Specific Points: 

1. Of the 18 genes that qualified as significantly changed in the RNA sequencing why was the DR1 
chosen and why were others excluded. Is currently very arbitrary. 

2. Data is presented regarding mouse models of heart failure. Criteria is listed for excluding animals 
but none regarding those included for data analysis. Were hearts analyzed for structural changes 
consistent with TAC, were other parameters such as ejection fractions, fractional shortening and 
chamber dimensions monitored to determine the success and progression of heart failure in 
experimental animals. 

3. In accordance with the above issue (ques.2) does the overexpression of DR1 accelerate TAC in 
αMHC2 tTA/TRE-D1R mice and slow in the DR1 knockdown model? Could the increased incidence of 
arrhythmias observed be related to the extent of HF rather than altered calcium dynamics? 

4. It appears that DR1 expression is localized to a small percentage of myocytes with even fewer cells 
showing significantly elevated levels at 8 wks post-TAC. Although DR1 levels correlate with increased 
PVCs there is not any evidence that demonstrates that these few DR1 positive cells are the initiating 
source of these PVCs. Although one can draw the conclusion that DR1 overexpression results in 
more calcium dysfunction (sparks) there is no direct evidence in the WT TAC model that this is the 
case. 

5. No data is presented regarding the mechanism of increased sparks in the DR1 overexpression 
model. IS the level of overexpression similar,more,less to what is seen in the average ventricular 
myocytes after 8 wks TAC. Is the arrhythmia burden significantly higher in the overexpression model 
assuming that all and not only a fraction of cells are now overexpressing DR1 or less because of the 
now more homogeneous (since heterogeneous distribution has been proposed as mechanism for 
arrhythmia) nature of the substrate. 

 

 

 

Reviewer #3 (Remarks to the Author): 

 

In this short communication, Yamaguchi, Sumida and colleagues reveal a potential role for the 
dopamine receptor D1R in triggering ventricular arrhythmia during heart failure. Evidence is 
gathered and condensed from an impressive array of techniques including single cell RNA 
sequencing of CMs in both a mouse TAC model and human heart failure patients, smFISH, loss and 
gain of function mouse genetic models and in vivo surface and wireless telemetric ECG. The work 
and findings are novel and of high clinical relevance. However, there are several areas of concern, 
outlined below. 

 

Major: 

1) The in vivo mouse experiments are incompletely described, to an extent that sometimes 
precludes interpretation of data. 

- What are the ages of mice used for all experiments? This is only specified for TAC/sham operated 
mice, described as between 8-12 weeks old. Are males and females used? 

- How were these mice allocated into experimental groups to avoid confounding factors of age (8-12 
weeks is a considerable range), gender (if applicable), and batch effects due to different litters? 

- The method for doxycycline treatment is not described. In fact, the methods section suggests 
“tetracycline” administration, via drinking water (p10 lines 15-16). Please clarify. What was the drug, 
supplier, dose and duration of treatment in this experiment? 

- Figure 1f, how many weeks post-surgery is this? 

 

2) Linked to the above, there are statistical concerns regarding the methodology used to compare 
between groups in single cell/slice comparisons, particularly because it is unclear how many actual 
mice were used per condition, and how many cells/slices came from each mouse. 

- Figure 1d, how many mice per timepoint? How many cells analysed per mouse? Why are different 
numbers of cells analysed for each timepoint, and only 680 for control? When performing the 
statistical test, should the mean values for each mouse not be calculated first, and then the 
statistical test performed on these mean values, in order to give a biologically informed comparison? 

- Figure 1e, how many mice per condition? How many cells from each mouse? 

- Figure 1f, as above for 1d. How many mice per condition? How many slices analysed per mouse? 
Were mean values per mouse used for the stats test. Also, why is a bar chart used here instead of a 
dot-plot? 

- Figure 2e, was this experiment reproduced with a second batch of neonatal CMs? Or are all data 
obtained from one biological experiment? n=9 and n=10 seem small if this refers to numbers of cells 
analysed. Were the cells randomly selected, was the experimenter blinded to the experimental 
condition? 

 

3) The study relies upon mRNA expression to infer expression of the D1R protein receptor in CMs. 
This requires validation of D1R protein expression by immunohistochemistry. There appear to be 
several antibodies available for this. Authors might co-stain with a CM-specific marker to 
demonstrate CM-specific protein expression of D1R in mouse and human control and heart failure 
tissue. 

 

4) It is claimed that figures 1d and 1f demonstrate D1R expression in CMs. However, how do the 
authors discount the possibility that some +ve stained cells are non-CMs? For example, D1R 
expression in vasculature is known. Authors should either quantify only D1R+ staining in cells that 
are co-stained with a CM-specific marker, or, clearly acknowledge the caveat that some staining here 
may arise from non-CM cells. 

 

5) Doxycycline is a known cardioprotective agent [PMIDs 24104875, 31274354]. There is therefore 
concern that reduced ventricular arrhythmia following DOX administration (Fig 2b) could be 
confounded by the effect of DOX itself, rather than reduced D1R expression. An ideal solution could 
be to repeat this experiment using a Tet-ON system (aMHC-rtTA). However, alternatively, authors 
might demonstrate that DOX administration alone does not rescue ventricular arrhythmia in wild-
type mice after TAC (i.e. figure 1g experimental setup). 

 

Minor: 

1) Were there any differences in D1R KO body weights, or heart weights? 

Also, were there differences in heart contractile function, wall thickness, heart weight or body 
weight in the D1R overexpression model? Note that dosage of the caffeine-dopamine intravenous 
drug infusion was not adjusted for body weight. 

 

2) Figure 1f: On average, how many total CMs are there per slice? 0.3 LacZ+ cells per slice (TAC 
condition) seems low. Does this correspond to the proportion of D1R+ CMs identified by scRNAseq 
(from Figure 1e, for 4 week post-TAC, this proportion is 5/73 = 6.8%)? 

The representative image in figure 1f and supplementary figure 2c suggest that fewer than 6.8% of 
CMs are LacZ+. Can the authors comment on this discrepancy? 

 

3) Supplementary Figure 3c: No error bars, was this n=1? Also, was this measured using an un-
operated mouse? If so, it’s surprising that such a clear difference exists, given that in un-operated 
mice, almost no CMs are expected to express D1R. Can the authors comment on this? 

 

4) P4 Line 14 “Increased expression of Drd1, but no other dopamine receptor genes, was confirmed 
by quantitative PCR (qPCR).” Only qPCR for D1R and D2R are shown – what about others? 

 

5) Please show primer sequences used for qPCR.