Author response:
The following is the authors’ response to the original reviews
Public Reviews:
Reviewer #1 (Public Review):
Summary:
In the manuscript submission by Zhao et al. entitled, "Cardiac neurons expressing a glucagon-like receptor mediate cardiac arrhythmia induced by high-fat diet in Drosophila" the authors assert that cardiac arrhythmias in Drosophila on a high-fat diet are due in part to adipokinetic hormone (Akh) signaling activation. High-fat diet induces Akh secretion from activated endocrine neurons, which activate AkhR in posterior cardiac neurons. Silencing or deletion of Akh or AkhR blocks arrhythmia in Drosophila on a high-fat diet. Elimination of one of two AkhR-expressing cardiac neurons results in arrhythmia similar to a high-fat diet.
Strengths:
The authors propose a novel mechanism for high-fat diet-induced arrhythmia utilizing the Akh signaling pathway that signals to cardiac neurons.
Weaknesses:
Major comments:
(1) The authors state, "Arrhythmic pathology is rooted in the cardiac conduction system." This assertion is incorrect as a blanket statement on arrhythmias. There are certain arrhythmias that have been attributable to the conduction system, such as bradycardic rhythms, heart block, sinus node reentry, inappropriate sinus tachycardia, AV nodal reentrant tachycardia, bundle branch reentry, fascicular ventricular tachycardia, or idiopathic ventricular fibrillation to name a few. However the etiological mechanism of many atrial and ventricular arrhythmias, such as atrial fibrillation or substrate-based ventricular tachycardia, are not rooted in the conduction system. The introduction should be revised to reflect a clear focus (away from?) on atrial fibrillation (AF). In addition, AF susceptibility is known to be modulated by autonomic tone, which is topically relevant (irrelevant?) to this manuscript.
Thank you for the helpful comment. We rephrased the sentence as “Arrhythmic pathology is often rooted in the cardiac conduction system”.
(2) The authors state that "HFD led to increased heartbeat and an irregular rhythm." In representative examples shown, HFD resulted in pauses, slower heart rate, and increased irregularity in rhythm but not consistently increased heart rate (Figures 1B, 3A, and 4C). Based on the cited work by Ocorr et al (https://doi.org/10.1073/pnas.0609278104), Drosophila heart rate is highly variable with periods of fast and slow rates, which the authors attributed to neuronal and hormonal inputs. Ocorr et al then describe the use of "semi-intact" flies to remove autonomic input to normalize heart rate. Were semi-intact flies used? If not, how was heart rate variability controlled? And how was heart rate "increase" quantified in high-fat diet compared to normal-fat diet? Lastly, how does one measure "arrhythmia" when there is so much heart rate variability in normal intact flies?
We also observed that fly heart rate is highly variable with periods of fast and slow rates. To control heart rate variability, Ocorr et al. used semi-intact flies to record the heartbeat (https://doi.org/10.1073/pnas.0609278104). We consider it a rigorous method to get highly consistent results with high quality videos/images. Since our work has a focus on the neuronal inputs to the heart, we did not use the semi-intact method. Our concern is that it is likely to disrupt the neuronal processes during the dissection. Using OCT, we recorded the heartbeat of intact flies in an 8 s time window, when the heartbeat was relatively stable. The different groups of flies, which were fed on a high-fat diet or a normal-fat diet, were recorded using the same method. Thus, we could compare the differences in heart rate.
(3) The authors state, "to test whether the HFD-induced increase in Akh in the APC affects APC neuron activity, we used CaLexA (https://doi.org/10.3109/01677063.2011.642910)." According to the reference, CaLexA is a tool to map active neurons and would not indicate, as the authors state, whether Akh affects APC neuron activity specifically. It is equally possible that APC neurons may be activated by HFD and produce more Akh. Please clarify this language.
Thank you for clarifying the calcium reporter, CaLexA. We rephrased this sentence to “to test whether HFD affects APC neuron activity, we used CaLexA”.
(4) Are the AkhR+ neurons parasympathetic or sympathetic? Please provide additional experimentation that characterizes these neurons. The AkhR+ neurons appear to be anti-arrhythmic. Please expand the discussion to include a working hypothesis of the overall findings on Akh, AkhR, and AkhR+ neurons.
Noyes et al. showed that Akh treatment increases heartbeat (Noyes, B. E., F. N. Katz, and M. H. Schaffer. 1995. “Identification and Expression of the Drosophila Adipokinetic Hormone Gene.” Molecular and Cellular Endocrinology 109 (2): 133–41.), suggesting that AkhR+ neurons are sympathetic. We showed that high-fat diet induced Akh expression and secretion, which led to stimulation of AkhR+ neuron and increased heart rate, supporting the sympathetic role of the AkhR+ neurons. Additional explanation on the sympathetic & anti-arrhythmic role of the Akh, AkhR, and AkhR+ neurons were added to the discussion.
(5) The authors state, "Heart function is dependent on glucose as an energy source." However, the heart's main energy source is fatty acids with minimal use of glucose (doi: 10.1016/j.cbpa.2006.09.014). Glucose becomes more utilized by cardiomyocytes under heart failure conditions. Please amend/revise this statement.
Thank you for pointing this out and providing the reference. We rephrased this sentence “Heart function is dependent on continuous ATP production. Cardiac ATP in Drosophila might come from fatty acids, glucose, and lactate (Kodde et al., 2007), as well as trehalose.”
Reviewer #2 (Public Review):
This manuscript explores mechanisms underlying heart contractility problems in metabolic disease using Drosophila as a model. They confirm, as others have demonstrated, that a high-fat diet (HFD) induces cardiac problems in flies. They showed that a high-fat diet increased Akh mRNA levels and calcium levels in the Akh-producing cells (APC), suggesting there is increased production and release of this hormone in a HFD context. When they knock down Akh production in the APCs using RNAi they see that cardiac contractility problems are abolished. They similarly show that levels of the Akh receptor (Akhr) are increased on a HFD and that loss of Akhr also rescues contractility problems on a HFD.
One highlight of the paper was the identification of a pair of neurons that express a receptor for the metabolic hormone Akh, and showing initial data that these neurons innervate the cardiac muscle. They then overexpress cell death gene reaper (rpr) in all Akhr-positive cells with Akhr-GAL4 and see that cardiac contractility becomes abnormal.
However, this paper contains several findings that have been reported elsewhere and it contains key flaws in both experimental design and data interpretation. There is some rationale for doing the experiments, and the data and images are of good quality. However, others have shown that HFD induces cardiac contractility problems (Birse 2010), that Akh mRNA levels are changed with HFD (Liao 2021) that Akh modulates cardiac rhythms (Noyes 1995), so Figures 1-4 are largely a confirmation of what is already known. This limits the overall magnitude of the advances presented in these figures. Overall, the stated concerns limit the impact of the manuscript in advancing our understanding of heart contractility.
We thank the reviewer for the positive comments and appreciate the reviewer for the instructive suggestions. Birse 2010 (PMID: 21035763) was cited in our manuscript. Liao 2021 showed that Akh mRNA levels are changed with HFD. We added the reference to the revised manuscript and modified the text as: “In consistent with a previous work (Liao et al., 2020), we showed that the expression of Akh was significantly up-regulated in the flies fed a HFD, compared to NFD-fed flies (Figure 2B)”. Our qPCR verified Liao’s results. On top of this, we investigated the calcium levels in the Akh producing cells (APCs) and showed elevated calcium levels in the APC in HFD fed flies. In the revised version, we added more data to show that Akh protein levels were increased with HFD (Figure 2E-F). In line with Noyes' discovery, which showed that Akh injection caused cardioaccelation in prepupae, we showed that genetic manipulation of Akh expression affected heartbeat in the adults.
Reviewer #3 (Public Review):
Zhao et al. provide new insights into the mechanism by which a high-fat diet (HFD) induces cardiac arrhythmia employing Drosophila as a model. HFD induces cardiac arrhythmia in both mammals and Drosophila. Both glucagon and its functional equivalent in Drosophila Akh are known to induce arrhythmia. The study demonstrates that Akh mRNA levels are increased by HFD and both Akh and its receptor are necessary for high-fat diet-induced cardiac arrhythmia, elucidating a novel link. Notably, Zhao et al. identify a pair of AKH receptor-expressing neurons located at the posterior of the heart tube. Interestingly, these neurons innervate the heart muscle and form synaptic connections, implying their roles in controlling the heart muscle. The study presented by Zhao et al. is intriguing, and the rigorous characterization of the AKH receptor-expressing neurons would significantly enhance our understanding of the molecular mechanism underlying HFD-induced cardiac arrhythmia.
Many experiments presented in the manuscript are appropriate for supporting the conclusions while additional controls and precise quantifications should help strengthen the authors' augments. The key results obtained by loss of Akh (or AkhR) and genetic elimination of the identified AkhR-expressing cardiac neurons do not reconcile, complicating the overall interpretation.
It is intriguing to see an increase in Akh mRNA levels in HFD-fed animals. This is a key result for linking HFD-induced arrhythmia to Akh. Thus, demonstrating that HFD also increases the Akh protein levels and Akh is secreted more should significantly strengthen the manuscript.
Thank you for the positive comments and the instructive suggestions. We performed immunostaining to show that Akh protein levels increased, which is consistent with elevated Akh mRNA expression in HFD-fed flies. The data was added to Figure 2, panels E and F. Akh secretion from the APCs is regulated by APC activity (https://doi.org/10.1038/s41586-019-1675-4). We used a calcium reporter CaLexA (https://doi.org/10.3109/01677063.2011.642910) to monitor APC activity and showed that HFD increased APC activity (Figure 2, C-D).
The experiments employing an AkhR null allele nicely demonstrate its requirement for HFD-induced cardiac arrhythmia. Depletion of Akh in Akh-expressing cells recapitulates the consequence of AkhR knockout, supporting that both Akh and its receptor are required for HFD-induced cardiac arrhythmia. Given that RNAi is associated with off-target effects and some RNAi reagents do not work, testing multiple independent RNAi lines is the standard procedure. It is also important to show the on-target effect of the RNAi reagents used in the study.
Indeed, RNAi approaches can suffer from off-target effects. For Akh experiments, we used an RNAi line BL_34960, which was generated using artificial microRNAs shRNA (DOI: 10.1038/nmeth.1592). In comparison to long-hairpin constructs, shRNA constructs are expected to be advantageous, e.g., more efficient and minimized off-target. We performed immunostaining to determine Akh-Gal4>UAS-Akh-RNAi efficiency. We showed that anti-Akh fluorescence diminished in Akh-Gal4>UAS-Akh-RNAi APCs. The data was added to Figure 3-figure supplement 1.
The most exciting result is the identification of AkhR-expressing neurons located at the posterior part of the heart tube (ACNs). The authors attempted to determine the function of ACNs by expressing rpr with AkhR-GAL4, which would induce cell death in all AkhR-expressing cells, including ACNs. The experiments presented in Figure 6 are not straightforward to interpret. Moreover, the conclusion contradicts the main hypothesis that elevated Akh is the basis of HFD-induced arrhythmia. The results suggest the importance of AkhR-expressing cells for normal heartbeat. However, elimination of Akh or AkhR restores normal rhythm in HFD-fed animals, suggesting that Akh and AkhR are not important for maintaining normal rhythms. If Akh signaling in ACNs is key for HFD-induced arrhythmia, genetic elimination of ACNs should unalter rhythm and rescue the HFD-induced arrhythmia. An important caveat is that the experiments do not test the specific role of ACNs. ACNs should be just a small part of the cells expressing AkhR. The experiments presented in Figure 6 cannot justify the authors' conclusion. Specific manipulation of ACNs will significantly improve the study. Moreover, the main hypothesis suggests that HFD may alter the activity of ACNs in a manner dependent on Akh and AkhR. Testing how HFD changes calcium, possibly by CaLexA (Figure 2) and/or GCaMP, in wild-type and AkhR mutants could be a way to connect ACNs to HFD-induced arrhythmia. Moreover, optogenetic manipulation of ACNs will allow for specific manipulation of ACNs, which is crucial for studying the specific role of ACNs in controlling cardiac rhythms.
Thank you for the insightful comments. We have been trying to find a way to only target the AkhR neurons using split-Gal4. Up to now, it’s not successful. Akh/AkhR signaling shall play a key role in the ACNs, however, we cannot rule out the possibility that ACNs also receive signals other than Akh in the modulation of heartbeat.
Interestingly, expressing rpr with AkhR-GAL4 was insufficient to eliminate both ACNs. It is not clear why it didn't eliminate both ACNs. Given the incomplete penetrance, appropriate quantifications should be helpful. Additionally, the impact on other AhkR-expressing cells should be assessed. Adding more copies of UAS-rpr, AkhR-GAL4, or both may eliminate all ACNs and other AkhR-expressing cells. The authors could also try UAS-hid instead of UAS-rpr.
We added more data to show that AkhR+ neurons are positive in anti-Akh staining, indicating the AkhR+ neurons indeed receive Akh.
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
Typo in line 765: "increased Akh section into the circulation." Section should be secretion.
Thank you for finding the typo. We changed section to secretion.
Reviewer #2 (Recommendations For The Authors):
One interesting extension to our knowledge in Figures 3 & 4 is that loss of Akhr and loss of Akh both block the cardiac contractility defects that accompany a HFD. The main concern I have with the Akh finding is that the authors use only a GAL4 control and no UAS alone control. Metabolic phenotypes often show strain-specific effects, so to make conclusions it is essential that the authors include a UAS alone control alongside the other genotypes to be sure it does not rescue the cardiac contractility defects that accompany a HFD by itself.
I am interested in the authors' identification of a pair of Akhr-positive neurons that innervate the cardiac muscle. I am not aware of any other studies identifying these neurons, or revealing their function. The contents of Figure 5 therefore represent the largest advance in the study. However, the characterization of these neurons is very superficial, and a lot more work to understand their regulation and function in a HFD context is needed to make conclusions about their role in any HFD-induced cardiac contractility problems. Or to determine how Akh influences the function of these specific neurons in an HFD context.
The reason I say this is that the authors ablate all Akhr-positive cells in Figure 6 and show that this disturbs normal cardiac contractility. While studies on the one pair of Akhr-positive neurons would be really interesting, ablating all Akhr-positive cells, which includes the fat and many other cell types in the fly, is not a scientifically rigorous approach to answering this question. As a result, the authors are only able to make the claim that ablating many cell types throughout the animal disrupts cardiac contractility, which does not advance our understanding of mechanisms underlying heart contractility problems. In addition, because the experiments they designed did not test whether it was Akh binding to Akhr on those neurons that regulate cardiac contractility problems in a HFD context, their experiments do not support their model in Figure 7.
The authors also make conclusions that are fairly speculative around Line 231 when describing their model in Figure 7. These claims are simply not supported by the data they present and must be removed. For example, the authors have not identified an endocrine-heart axis, they simply showed that changes in Akh can influence the heart, but this is not necessarily a direct effect on a specific cell type. They do not show data that Akh binds the newly identified Akhr-positive neuron pair to mediate the effects of HFD-induced contractility defects - they just ablate all Akhr-positive cells (fat, neurons, and other types) and show cardiac defects. If those neurons did mediate the abnormal cardiac rhythm promoted by Akh, then ablating those neurons (and not a large number of additional tissues) should rescue HFD-induced heart defects just like reducing Akhr or Akh did (but this is the opposite of what they see). Overall, concerns with experimental design, data interpretation, and relatively few findings that aren't reported elsewhere reduce the impact of this paper.
We appreciate the positive comments and helpful suggestions. Indeed, it is important to get clean genetic access to the cardiac neurons. We intended to use split Gal4 system to target the AkhR cardiac neurons. We have tried to build a split Gal4 driver AkhR-p65.AD. Two rounds of injection were carried out. However, we did not recover a transgenic line.
In the revised version, we performed immunostaining using Akh antibodies to show that anti-Akh fluorescence was observed in AkhR neurons (Figure 5-figure supplement 1), indicating an endocrine-heart axis.
