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Conserved Chamber-Specific Polyploidy Maintains Heart Function in Drosophila

Archan Chakraborty, Nora G. Peterson, Juliet S. King, Ryan T. Gross, Michelle Mendiola Pla, Aatish Thennavan, Kevin C. Zhou, Sophia DeLuca, Nenad Bursac, Dawn E. Bowles, Matthew J. Wolf, Donald T. Fox

Posted on: 3 March 2023

Preprint posted on 12 February 2023

The importance of being in the right place, with the right ploidy, at the right time- and how not to mend broken hearts.

Selected by Anastasia Moraiti

The more the merrier? Polyploidy in the heart

The heart is primarily made up of polyploid cells; that is either cells with more than one nucleus, or cells with one nucleus bearing more than  two copies of the genome.

The presence of polyploid cells in the heart has been linked to its low regeneration potential. Most polyploid cells in the human heart do not regenerate after, for example, a myocardial infarction, making heart disease a significant health burden. Other species are much better than us at regenerating their hearts. Newts, zebrafish, and neonatal mice have highly regenerative myocardia, but their hearts are composed mainly of diploid cardiomyocytes (CMs). There is an increased amount of evidence (González-Rosa et al., 2018) that polyploidisation might pose a barrier to proliferation. This has led to a new tempting idea in the field of heart regeneration: What if CMs were not polyploid? (Derks and Bergmann, 2020)

Before jumping in and manipulating developmentally regulated polyploidization as a therapeutic option, Chakraborty et al. studied its emergence in the Drosophila cardiac organ. They highlight the importance of ploidy for the maintenance of cardiac structural functionality, identify a potential regulatory mechanism for polyploidy, and hint that this is conserved in humans too. Their work acts as a word of caution against blocking CM polyploidisation as a means of boosting the heart’s regenerative capacity.

 

Temporal and molecular regulation of polyploidy emergence in Drosophila cardiomyocytes

Before we dive deeper, here’s some background: flies have hearts, they do. The cardiac organ of Drosophila is called the dorsal vessel. It is a tubular organ, spanning the anteroposterior axis of the animal, and can be divided further into the more anterior aorta and the more posterior heart. Its main function is to contract in order to disperse lymph (think of it as the fly’s blood) with immune cells, from the heart, through the aorta and then to the rest of the body (Figure 1).

 

Figure 1. The Drosophila cardiac organ (dorsal vessel). The dorsal vessel spans the AP axis of the animal. The anterior part, spanning segments thoracic 2- abdominal 4, is the aorta, and the posterior, segments abdominal 5-7, is the heart. The ostia between the segments are formed by specialised cardiomyocytes and act as inflow openings for the hemolymph.

 

Adult CMs in Drosophila are known to be polyploid, but how this emerges during development was so far not understood. Chakraborty et al. set out to investigate this, starting from the embryonic mesoderm, but found no evidence of polyploidisation there. Comparing early and late larval stages though, they observed an increase in CM nuclear size without a change in their number, suggesting incomplete cell cycle, presumably alternating only between G and S phases. They confirmed this by feeding the larvae BrdU up to late stages and observing consistently BrdU-negative heterochromatin in otherwise positive nuclei, a hallmark of cells initiating but not completing S phase (as heterochromatin tends to replicate in late S phase). Such early-S-phase-only cycles have previously been observed in other Drosophila tissues that are known to be polyploid, such as the salivary gland (Fig. 2A).

Interestingly, the authors observed two populations of polyploid CMs: the higher ploidy cells were found in the heart chamber, and the lower ploidy ones mostly in the aorta. They found that this corresponded with having larger CMs in the heart. To investigate this further, the authors used optical coherence tomography imaging (OCT) and found that differences in cell size were reflected in the overall organ structure. The heart showed a thicker muscle wall and larger lumen compared to the aorta, which argues that ploidy can play a role in establishing the architecture of each cardiac chamber (Fig.2B-D).

 

Figure 2. A. BrdU assay showing late larval (WL3) CMs with BrdU positive nuclei, excluding the heterochromatin (marked with the dotted circle), indicating that larval CMs undergo incomplete divisions, stopping at early S phase. B, C, D. Chamber specific differences in area of CMs (B), wall thickness (C) and end diastolic dimension area (D).

 

A BrdU pulse-chase experiment showed that endocycles start immediately after larval hatching (similar to the early transition to polyploidy in mammals), but stop after 48 h. Interestingly, although this is true for the heart and aorta, the higher final ploidy of the former indicates that heart cells endocycle more quickly. The early period of life when polyploidy is established coincides with the time when the developing fly is feeding, and indeed starvation hampers endocyclying.

Given the link between diet, PI3K/insulin signalling and growth, the next step was to ask whether Insulin Receptor (InR) signalling regulates CM polyploidisation during larval life. CM-specific RNAi of InR did not affect CM number but led to cells with smaller nuclei and decreased DNA content. Interestingly, although the driver used was both expressed in the heart and aorta, the effect of InR RNAi was different in the two chambers. That was also the case when they impacted ploidy using fzr RNAi (to manipulate ploidy without interfering with insulin signalling), showcasing another chamber-specific difference in polyploidy, suggesting a differential role of ploidy in each compartment.

 

Location, location, location

All this evidence was pointing at the importance of differences in ploidy between the cardiac chambers, and Chakraborty et al. went on to examine the geometry and physiology of ploidy-manipulated organs. Indeed, in fzrRNAi and InRRNAi hearts, they observed reduced chamber length and end diastolic dimension area and they computed the stroked volume to also be decreased. This was not the case for the aorta, or when they induced a constitutively active Insulin Receptor in both chambers. Furthermore, while they found heart rate not to be affected by ploidy manipulation, there was a difference in cardiac output with the heart chamber output being lower than the aorta output in ploidy-compromised animals. Confirming the functional consequences of reduced ploidy and the associated chamber defects in the heart, hemocyte velocity was also affected: in wildtype animals hemocytes move in an anterior direction from the posterior end of the heart and accelerate as they move toward the aorta. In InRRNAi animals though, this did not occur, with the hemocytes travelling at approximately the same velocity in both chambers.

 

Figure 3. A and B. Cardiac output between the aorta (A) and heart (B) in ploidy compromised tissue (InRRNAi and fzrRNAi). Ploidy reduction has a significant effect on the heart, but not on the aorta. D. Hemocytes (marked with Hml-DsRed) travelling in an anterior direction from the heart to the aorta. E and F. Stills from hemocyte tracking, anterior to the right of the image. Normal ploidy in E, ploidy compromised in F.

 

Of flies and men

Finally, given the crucial role of ploidy in shaping heart form and, as a result, heart function, the authors wondered whether that might also be the case in humans. They studied tissue sections to look for any differences between the left atrium (LA) and left ventricle (LV), as the left side of the human heart is the one that pumps blood through the body, and the LV chamber has thicker walls. They found that LV CMs have a higher volume than LA ones suggesting an asymmetry in nuclear DNA content between the chambers, as was seen in the fly. Finally, they performed pathway analysis on publicly available transcriptomic datasets and found that insulin signalling is upregulated in the ventricular compared to atrial CMs, which they also confirmed by staining for the human insulin receptor (INSR) in LV and LA sections.

 

How (not) to mend a broken heart

In this preprint, Chakraborty et al. provide a description of the temporal and molecular regulation of polyploidisation of Drosophila CMs. They report a functional role for polyploidy in sculpting the form of the organ, so that the form can support proper function. The compromised physiology that resulted from their manipulations of ploidy goes to show that developmentally regulated polyploidy plays a critical role in proper heart functioning. This is very important to notice as human heart regenerative therapies are potentially angling towards blocking polyploidisation as a means of boosting regenerative capacity through a diploid myocardium. Despite some compelling evidence showing that manipulating ploidy has an effect on regenerative potential and certain associations (Patterson et al., 2017), it is not established that polyploidy itself blocks regeneration, as it is well known that other polyploid organs can regenerate. The authors argue that developmentally regulated polyploidy should be disentangled from other changes that might occur while CMs transition to a higher ploidy state, and note that blocking polyploidisation to boost regenerative capacity might indeed compromise heart function instead.

My questions to the authors

  • When you used the constitutively active InR and saw the increased ploidy, was this still a case of incomplete cell/nuclear divisions? Did you also drive an fzrCA and if so, what was the result of that?
  • One assumption on the role of polyploidy in the heart has been that it is actually preferred over sequential cell cycles, as that would require disrupting the sarcomere network at every cycle, hampering the muscle ability to contract (Ovrebo and Edgar, 2018). I was wondering if it is technically possible to perform your beautiful quantifications of the heartbeat and/or output before the endocycles start, or if you can speculate on the effect of a full cytokinesis on contractility.
  • I’d be very curious to know whether late larval or even pupal dorsal vessels have any regenerative capacity in line with the low renewal rates that have observed for example in humans. Do you have any evidence, or can you speculate whether the different ploidy levels of the heart and aorta might affect the response to mechanical injury or genetic ablation?

Tags: drosophila, heart physiology, heart regeneration, polyploidy

doi: https://doi.org/10.1242/prelights.33981

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Author's response

Donald Fox and and Archan Chakraborty shared

When you used the constitutively active InR and saw the increased ploidy, was this still a case of incomplete cell/nuclear divisions? Did you also drive an fzrCA and if so, what was the result of that?

We don’t see any change in numbers of nuclei in InRCAanimals, and this indicates that the increase in ploidy is due to increased endocycles, and not by incomplete cell division. We haven’t tried fzrCA, but we predict that it would show similar results to InRCA.

 

One assumption on the role of polyploidy in the heart has been that it is actually preferred over sequential cell cycles, as that would require disrupting the sarcomere network at every cycle, hampering the muscle ability to contract (Ovrebo and Edgar, 2018). I was wondering if it is technically possible to perform your beautiful quantifications of the heartbeat and/or output before the endocycles start, or if you can speculate on the effect of a full cytokinesis on contractility.

We haven’t measured the heartbeat in any embryonic stages. However, we preliminarily found that newly hatched larvae (L1 stage-during the endocycles) have a heartrate that is twice as fast as the heartrate of mature larvae (WL3 stage-after the endocycles). This may suggest that ploidy levels inversely correlate with heartrate.

 

I’d be very curious to know whether late larval or even pupal dorsal vessels have any regenerative capacity in line with the low renewal rates that have observed for example in humans. Do you have any evidence, or can you speculate whether the different ploidy levels of the heart and aorta might affect the response to mechanical injury or genetic ablation?

We are quite interested in the question of whether Drosophila cardiomyocytes have regenerative potential. We are currently investigating this question- hopefully this will be the next paper!

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