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Angiocrine IGFBP3 Spatially Coordinates IGF Signaling During Neonatal Cardiac Regeneration

Shah R. Ali, Waleed Elhelaly, Ngoc Uyen Nhi Nguyen, Shujuan Li, Ivan Menendez-Montes, Zhaoning Wang, Miao Cui, Abdallah Elnwasany, Feng Xiao, Suwannee Thet, Nicholas T. Lam, Alisson Cardoso, Ana Helena Pereira, Mohammad Goodarzi, Michael T. Kinter, Andrew Lemoff, Luke I. Szweda, John Shelton, Wataru Kimura, Hesham A. Sadek

Preprint posted on 16 September 2021 https://www.biorxiv.org/content/10.1101/2021.09.16.460522v1

When endothelial-derived IGFBP3 meets cardiomyocytes: fanning the flames for heart regeneration.

Selected by Laura Alonso-Herranz

Categories: cell biology, pathology

Background

The adult mammalian heart presents a minor cardiomyocyte turnover that is, however, not sufficient to replace the millions of cells lost upon myocardial infarction (MI) injury (Bergmann et al., 2009; Mollova et al., 2013). The underlying cause of this is the postnatal withdrawal of cardiomyocytes from the cell cycle (Ali et al., 2020). Nevertheless, the mammalian heart does retain the capacity for regeneration during a short period after birth (Porrello et al., 2011). Neonatal murine cardiac regeneration relies on the proliferation of pre-existing cardiomyocytes with negligible contributions from any type of stem cell population (Porrello et al., 2013; Senyo et al., 2013). Therefore, the finding of pro-mitogen effectors to extend the regenerative temporal window into adulthood is the focus of many laboratories.

In this preprint, Ali et al. aim to identify non-cell-autonomous mechanisms driving cardiomyocyte mitosis by looking for secreted molecules that differ in expression between regenerating versus non-regenerating neonatal hearts subjected to MI.

Key findings

IGFBP3 is expressed in vascular cells in the heart after neonatal MI

Regenerating (postnatal day 1, P1) and non-regenerating hearts (P14) underwent MI injury (or sham intervention) and then microarray was performed. The authors focused their study on secreted proteins whose expression was upregulated upon injury in the regenerating heart. The candidate list was shortened by in situ hybridization to Igfbp3, which was the only transcript that recapitulated the microarray pattern and was expressed only in the area surrounding the injury, more precisely associated with endothelial cells. Interestingly, immunostaining of IGFBP3 revealed the presence of the protein in the injury zone instead, suggesting IFGBP3 synthesis and release from endothelial cells in the border zone and diffusion into the injury.

IGFBP3 deletion impairs neonatal regeneration

Next, Ali et al. used a global Igfbp3-/- mouse to assess the role of IGFBP3 in cardiomyocyte proliferation and regeneration (Figure 1). The lack of Igfbp3 had no baseline effects on cardiac development and function in adult mice. However, Igfbp3-/- mice presented diminished cardiomyocyte mitosis and cytokinesis, and a significant, although subtle, worsened post-MI cardiac function (Figure 1), suggesting the contribution of IGFBP3 to cardiomyocyte division and ultimately heart regeneration.

Figure 1. Igfbp3 global deletion impairs neonatal heart regeneration. A) Genetic construct of Igfbp3-KOLacZ model. B) Western blot of pH3-S10 as a hallmark for mitosis from cardiac ventricular tissue 3 days after described operation and genotype. C-D) Indicators of systolic function C) ejection fraction (%) and D) fractional shortening (%) 4 weeks post-MI in P1 neonates. Data represent mean ± SEM. Unpaired t-test. S: sham; MI: myocardial infarction injury. [Figure 2A, 2G-J, & 2K from the preprint. Adapted with permission from Ali et al.]
Endothelial overexpression of IGFBP3 increased cardiomyocyte proliferation

The authors generated two mouse lines with endothelial-specific overexpression of murine IGFBP3. Overexpression of IGFBP3 during the regenerative time window (Tie2CreERT2Igfbp3OE) resulted in hyperplasia of the myocardium, indicating that ectopic endothelial-derived IGFBP3 can increase the number of cycling myocytes in the absence of injury. Furthermore, endothelial overexpression of IGFBP3 in adulthood (Cdh5CreERT2Igfbp3OE) augmented cardiomyocyte numbers ex vivo, an indirect measurement of cell proliferation. This data may indicate a pro-mitotic effect of IGFBP3 on cardiomyocytes.

In addition, coimmunoprecipitation experiments with cultured neonatal rat cardiomyocytes showed IGFBP3 binding to IGF2, suggesting a regulatory role of IGFBP3 in the canonical IGF pathway.

Spatiotemporal expression of IGFBP3 and its regulators modulate IGF signaling after neonatal MI

Data obtained with the Igfbp3-/- mouse suggest that IGFBP3 participates in the activation of the IGFR-1/ERK/AKT pathway in the regenerating heart upon injury. The authors found upregulation of the IGFBP3 protease PAPPA2 and curtailed expression of its inhibitor STC2 in the border and infarct zone of the regenerating heart, therefore suggesting that the PAPPA2/STC2/IGFBP3 axis coordinates the spatial release of IGF2 in the infarcted myocardium and activates myocyte proliferation via ERK/AKT pathway.

What I liked about this preprint:

Nowadays, it is widely accepted that heart regeneration relies on the proliferation of surviving cardiomyocytes to replace the lost tissue without compromising heart function. Despite being the most prominent cell type, myocytes are not the only cells that make up the heart (Pinto et al., 2016). Endothelial cells constitute the majority of non-myocyte cells and are likely to play a greater role in physiological function and response to injury than previously appreciated. I am deeply fascinated by intercellular communication in the heart, with a special interest in the mechanisms underlying the regenerative capacity of the neonatal heart.

Ali et al. used microarray to identify IGFBP3 as an angiocrine produced by the regenerating heart, and combining loss- and gain-of-function models, the authors proposed an elegant mechanism of intercellular communication that led to myocyte proliferation. As a summary, endothelial IGFBP3 would guide the pro-mitotic IGF2 into the injured region and the protease PAPPA2 would fragment IGFBP3 yielding free IGF2. Thus, IGFP3 would act as a link with the IGF signaling pathway, known to play a pivotal role in heart regeneration.

Future directions

  1. You used a global Igfbp3 KO mouse model arguing that Igfbp3 is at least expressed by fibroblasts and endothelial cells. Nonetheless, your working model describes intercellular crosstalk between endothelial cells (secreting IGFBP3 that binds and protects IGF2 from its degradation) and cardiomyocytes (binding IGF2 and activating ERK/AKT proliferating pathway). Have you considered creating an endothelial-specific Igfbp3 KO mouse model to support it?
  2. Although significant, changes in EF and FS are very subtle between WT and global Igfbp3-/- mouse. Would you expect these changes to be still significant in an endothelial-specific Igfbp3 KO mouse? On the other hand, did you observe a positive effect on post-MI cardiac function (EF and/or FS) with the gain-of-function mouse models Tie2OE or Cdh5OE?
  3. In a previous study, this lab showed that cardiomyocyte proliferation in the regenerating heart is not limited to the injured area but takes place in the whole myocardium (infarct, border, and healthy/remote myocardium) (Porrello et al., 2013). In which area of the myocardium was pH3+ cardiomyocyte quantification performed in the current study (Figure 2E-F)? Did you see blunted proliferation in the whole myocardium of Igfbp3-/- mice or only in the injured zone? Since IGFBP3 expression is restricted to the border zone, which alternative mechanisms do you hypothesize are driving cardiomyocyte division in the remote areas of the myocardium?
  4. Both injection of recombinant IGFBP3 or ectopic-endothelial overexpression of IGFBP3 (Tie2-CreERT2) increased cardiomyocyte division in baseline conditions (Figures 3I-J and 4F-G). What happens if IGFBP3 is injected in the non-regenerating heart (from P7 on) subjected to MI?
  5. Figure 3I: not only pH3+TnT+ cells raised in IGFBP3 treated mice but also pH3+TnT- seemed to be increased. Have you checked if the vasculature network, a key element to sustain cardiac regeneration (Aurora et al., 2014), was enhanced in IGFBP3 injected mice?

References

Ali, S.R., Lam, N.T., and Sadek, H.A. (2020). 3 – Cellular Basis for Myocardial Regeneration and Repair. In Heart Failure: a Companion to Braunwald’s Heart Disease (Fourth Edition), G.M. Felker, and D.L. Mann, eds. (Philadelphia: Elsevier), pp. 43-61.e43.

Aurora, A.B., Porrello, E.R., Tan, W., Mahmoud, A.I., Hill, J.A., Bassel-Duby, R., Sadek, H.A., and Olson, E.N. (2014). Macrophages are required for neonatal heart regeneration. The Journal of clinical investigation 124, 1382-1392.

Bergmann, O., Bhardwaj, R.D., Bernard, S., Zdunek, S., Barnabé-Heider, F., Walsh, S., Zupicich, J., Alkass, K., Buchholz, B.A., Druid, H., et al. (2009). Evidence for cardiomyocyte renewal in humans. Science (New York, NY) 324, 98-102.

Mollova, M., Bersell, K., Walsh, S., Savla, J., Das, L.T., Park, S.-Y., Silberstein, L.E., dos Remedios, C.G., Graham, D., Colan, S., et al. (2013). Cardiomyocyte proliferation contributes to heart growth in young humans.  110, 1446-1451.

Pinto, A.R., Ilinykh, A., Ivey, M.J., Kuwabara, J.T., D’Antoni, M.L., Debuque, R., Chandran, A., Wang, L., Arora, K., Rosenthal, N.A., et al. (2016). Revisiting Cardiac Cellular Composition. Circulation research 118, 400-409.

Porrello, E.R., Mahmoud, A.I., Simpson, E., Hill, J.A., Richardson, J.A., Olson, E.N., and Sadek, H.A. (2011). Transient regenerative potential of the neonatal mouse heart. Science (New York, NY) 331, 1078-1080.

Porrello, E.R., Mahmoud, A.I., Simpson, E., Johnson, B.A., Grinsfelder, D., Canseco, D., Mammen, P.P., Rothermel, B.A., Olson, E.N., and Sadek, H.A. (2013). Regulation of neonatal and adult mammalian heart regeneration by the miR-15 family. Proceedings of the National Academy of Sciences of the United States of America 110, 187-192.

Senyo, S.E., Steinhauser, M.L., Pizzimenti, C.L., Yang, V.K., Cai, L., Wang, M., Wu, T.D., Guerquin-Kern, J.L., Lechene, C.P., and Lee, R.T. (2013). Mammalian heart renewal by pre-existing cardiomyocytes. Nature 493, 433-436.

 

Tags: cardiomyocyte proliferation, heart regeneration, igfbp3, intercellular crosstalk

Posted on: 7 January 2022

doi: https://doi.org/10.1242/prelights.31227

Read preprint (No Ratings Yet)

Author's response

Hesham Sadek shared

  1. You used a global Igfbp3 KO mouse model arguing that Igfbp3 is at least expressed by fibroblasts and endothelial cells. Nonetheless, your working model describes intercellular crosstalk between endothelial cells (secreting IGFBP3 that binds and protects IGF2 from its degradation) and cardiomyocytes (binding IGF2 and activating ERK/AKT proliferating pathway). Have you considered creating an endothelial-specific Igfbp3 KO mouse model to support it?

Yes, we have these mice and we are conducting these studies. We chose the global KO initially given that expression does not seem to be restricted to endothelial cells based on scRNA seq data

  1. Although significant, changes in EF and FS are very subtle between WT and global Igfbp3-/- mouse. Would you expect these changes to be still significant in an endothelial-specific Igfbp3 KO mouse? On the other hand, did you observe a positive effect on post-MI cardiac function (EF and/or FS) with the gain-of-function mouse models Tie2OE or Cdh5OE?

This is an important point. I can only speculate that what IGFBP3 is doing is guiding proliferation in the injury zone. As you mention later, post injury response appears to be global at least in the neonatal models that we have used. So we don’t think that loss of IGFBP3 completely abolishes proliferation, but rather abolishes or decreases proliferation that occurs in the injury zone. 

The manuscript is currently under review, and the experiments you suggest are included!

  1. In a previous study, this lab showed that cardiomyocyte proliferation in the regenerating heart is not limited to the injured area but takes place in the whole myocardium (infarct, border, and healthy/remote myocardium) (Porrello et al., 2013). In which area of the myocardium was pH3+ cardiomyocyte quantification performed in the current study (Figure 2E-F)? Did you see blunted proliferation in the whole myocardium of Igfbp3-/- mice or only in the injured zone? Since IGFBP3 expression is restricted to the border zone, which alternative mechanisms do you hypothesize are driving cardiomyocyte division in the remote areas of the myocardium?

See response to the previous point. An additional point is that we do see epicardial activation in these models and we are not sure whether this epicardial activation mediates the global proliferative response or not. There is certainly no paucity of epicardial factors that have been shown to impact cardiomyocyte proliferation, and any of them may play a role here. An important point to mention here is that when we injure the heart in these models we go through the epicardium (in either apical resection or LAD ligation). So it is unclear whether epicardial activation is because we injure the epicardium, or whether this would also happen if you for example have a classic plaque rupture inside a coronary as in case of myocardial infarctions in humans. 

  1. Both injection of recombinant IGFBP3 or ectopic-endothelial overexpression of IGFBP3 (Tie2-CreERT2) increased cardiomyocyte division in baseline conditions (Figures 3I-J and 4F-G). What happens if IGFBP3 is injected in the non-regenerating heart (from P7 on) subjected to MI?

Good experiment. We haven’t tried that.

  1. Figure 3I: not only pH3+TnT+ cells raised in IGFBP3 treated mice but also pH3+TnT- seemed to be increased. Have you checked if the vasculature network, a key element to sustain cardiac regeneration (Aurora et al., 2014), was enhanced in IGFBP3 injected mice?

Another good point. No, we have not.

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