HSA+ immature cardiomyocytes persist in the adult heart and expand after ischemic injury

The assessment of the regenerative capacity of the heart has been compromised by the lack of surface signatures to characterize cardiomyocytes. Here, combined multiparametric surface marker analysis with single cell transcriptional profiling and in vivo transplantation, identify the main fetal cardiac populations and their progenitors. We found that cardiomyocytes at different stages of differentiation co-exist during development. We identified a population of immature HSA/CD24+ cardiomyocytes that persists throughout life and that, unlike other cardiomyocyte subsets, actively proliferates up to one week of age and engraft cardiac tissue upon transplantation. In adult heart HSA/CD24+ cardiomyocytes appear as mononucleated cells that cycle and increase in frequency after infarction. Our work identified cell surface signatures that allow the prospective isolation of cardiomyocytes at any developmental stage and the detection of adult cardiomyocytes poised for activation in response to ischemic stimuli. This work opens new perspectives in the understanding and treatment of heart pathologies.


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The cell types that form the mammalian heart have diverse developmental origins 46 and temporal differentiation 1 . In the mouse, cardiomyocytes (CMs) are initially 47 specified by embryonic day (E) 7.5 from the first set of cardiogenic progenitors (first 48 heart field) 2 followed by the incoming cells from the second heart field (SHF) 3 . At E 49 9.5 (looping-heart stage), the heart is divided into primitive (P) atria (At), ventricle Vt

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To probe the homogeneity of the cardiac subsets here defined we carried out

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The ensemble of these results validated a phenotyping strategy ( Fig. 1; 150 Supplementary Table 1) that allowed the identification (Fig. 2a-b) and the prospective 151 isolation of the major cardiac cell subsets in the fetal heart (Fig. 3g).    and Vt 78% in G 0 , Supplementary Fig. 3c). The stromal subsets were highly 184 proliferative (At: 36.6% and 11.2%; and Vt: 27.6% and 11.3%, respectively in G 1 and

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Because HSA is the last surface protein to be lost before the acquisition of Cav3,

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we used these two markers to discriminate immature and mature CMs, respectively.

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The detection of an immature CM subset in the adult prompted us to investigate 275 its frequency in the diseased heart. We found HSA expression largely circumscribed 276 to the non-CM compartment in sham-operated hearts, although rare 277 HSA + Actinin + CMs were also detected (outlined by laminin expression, Fig. 5c). Seven

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We identified HSA, so far never associated to heart development or maturation,

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Although HSA + immature CMs do not proliferate sufficiently to regenerate the 360 myocardium, they might account for the low CM turnover rate previously described in 361 the adult 21,22,39 and be more amenable than binucleated CMs to respond to mitotic 362 stimuli. Importantly, using the strategy herein described, CMs at different maturation 363 stages can now be prospectively isolated as viable cells from the adult heart, 364 enabling further mechanistic studies.

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Mice. C57BL/6 mice (Charles River) 6 -8 week-old or timed pregnant females were 367 used. Timed-pregnancies were generated after overnight mating. The following 368 morning, females with vaginal plug were considered to be at E0.5.

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All animal manipulations at i3S were approved by the Animal Ethics Committee and           Fig. 1 and in Supplementary Fig. 1, was used to define each population.

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Map clustering of the flow cytometry data was performed using custom R scripts from R

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Statistical Analysis. All results are shown as mean ± standard deviation (SD).

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Statistical significance was determined using the t-Student test, except when comparing 508 the frequency of HSA + CMs form E13.5 to P7 (one-way ANOVA followed by Tukey    Supplementary Fig. 1

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Color-code as in Supplementary Fig. 1