c-kit Haploinsufficiency impairs adult cardiac stem cell growth, myogenicity and myocardial regeneration

An overdose of Isoproterenol (ISO) causes acute cardiomyocyte (CM) dropout and activates the resident cardiac c-kitpos stem/progenitor cells (CSCs) generating a burst of new CM formation that replaces those lost to ISO. Recently, unsuccessful attempts to reproduce these findings using c-kitCre knock-in (KI) mouse models were reported. We tested whether c-kit haploinsufficiency in c-kitCreKI mice was the cause of the discrepant results in response to ISO. Male C57BL/6J wild-type (wt) mice and c-kitCreKI mice were given a single dose of ISO (200 and/or 400 mg/Kg s.c.). CM formation was measured with different doses and duration of BrdU or EdU. We compared the myogenic and regenerative potential of the c-kitCreCSCs with wtCSCs. Acute ISO overdose causes LV dysfunction with dose-dependent CM death by necrosis and apoptosis, whose intensity follows a basal-apical and epicardium to sub-endocardium gradient, with the most severe damage confined to the apical sub-endocardium. The damage triggers significant new CM formation mainly in the apical sub-endocardial layer. c-kit haploinsufficiency caused by c-kitCreKIs severely affects CSCs myogenic potential. c-kitCreKI mice post-ISO fail to respond with CSC activation and show reduced CM formation and suffer chronic cardiac dysfunction. Transplantation of wtCSCs rescued the defective regenerative cardiac phenotype of c-kitCreKI mice. Furthermore, BAC-mediated transgenesis of a single c-kit gene copy normalized the functional diploid c-kit content of c-kitCreKI CSCs and fully restored their regenerative competence. Overall, these data show that c-kit haploinsufficiency impairs the endogenous cardioregenerative response after injury affecting CSC activation and CM replacement. Repopulation of c-kit haploinsufficient myocardial tissue with wtCSCs as well c-kit gene deficit correction of haploinsufficient CSCs restores CM replacement and functional cardiac repair. Thus, adult neo-cardiomyogenesis depends on and requires a diploid level of c-kit.


Introduction
The lack of curative therapies for heart failure (HF) 1 has made the search for an effective cellular replacement of lost/damaged cardiomyocytes (CMs) a priority in the war against HF epidemics 1,2 . Ex-vivo expanded resident cardiac stem cells (CSCs) and progenitor cells have been reproducibly shown to repair extensive myocardial damage and regenerate lost CMs [3][4][5][6] . Nevertheless, it is argued 7-9 that the myocardium lacks a bona fide endogenous CM-generating progenitor cell population of biological significance. This controversy has been mainly nurtured by Cre-lox mouse lines with Cre knocked-in (KI) to the c-kit locus (c-kit Cre KI) [10][11][12][13] . c-kit Cre KI lines show that myocardial c-kit-expressing (c-kit pos ) cells only minimally, if not negligibly, contribute CMs [10][11][12][13][14] . However, the very low number of endogenous c-kit pos CSC-generated CMs detected in the c-kit Cre mice, simply reflects the failure of the c-kit Cre -null allele produced by Cre insertion to recombine the CSCs and to track their progeny together with the severe defect in CSCs myogenesis produced by the c-kit hemizygosity [15][16][17][18] .
Recently, the deleterious effects of excessive catecholamines have been linked to the development of Takotsubo syndrome, a stress-related form of acute severe, generally transient, left ventricular (LV) dysfunction 19 . In rats and mice, a single excessive dose injection (subcutaneous (s.c.)) of the synthetic catecholamine, isoproterenol (ISO), causes CM death 20,21 , which leads to the development of acute HF, fully reversible by 28 days. Using this acute ISOdamage model, by means of several genetic tests and selective cell ablation/repopulation experiments, we provided the proof-of-concept evidence that resident CSCs are necessary and sufficient for the regeneration and repair of myocardial damage 21 .
In contrast to our data, Wallner et al. 22 reported that ISO caused minimal myocardial necrosis, which recovered without any meaningful contribution of "c-Kit (+)-CSC-derived CM regeneration." These results obtained using the c-kit Cre KI mutant mice 10,22 have raised further scepticism about the role of CSCs in the adult heart.
Here we show that c-kit haploinsufficiency in c-kit Cre KI mice impairs the endogenous cardioregenerative response after ISO affecting CSC activation and CM replacement. Repopulation of c-kit haploinsufficient myocardial tissue with wild-type CSCs, as well c-kit gene deficit correction of haploinsufficient CSCs, restores CM replacement and cardiac function after ISO. Thus, these data show that adult cardiomyogenesis depends on a diploid level of c-kit in CSCs.

Animals
All animal experimental procedures were approved by Magna Graecia Institutional Review Boards on Animal Use and Welfare and performed according to the Guide for the Care and Use of Laboratory Animals from directive 2010/63/EU of the European Parliament. All animals received human care and all efforts were made to minimize animal suffering.
To assess CM replenishment by CSCs after ISO, Tg (Myh6-Cre/Esr1)1Jmk/J male mice (abbreviated as Tg-myh6 MCM , Jackson Labs, stock number 005650) were crossed with homozygous Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo /J reporter mice (abbreviated as R26 mT/mG or R26 mt-mg , Jackson Labs, stock number 007576), to generate double heterozygous animals. These double mutant mice carry a TAM-inducible Cre Recombinase driven by the cardiac α-myosin heavy chain (α-MHC or myh6) promoter and a double fluorescent (dT/mGFP) gene reporter in the ROSA26 locus where, upon TAM-induced Cre-dependent recombination, a membrane-bound dimeric tomato (dT) is replaced by a membrane-bound green fluorescent protein (mGFP). Eight-week-old double heterozygous Tg-myh6 MCM :R26 mT/mG male mice were fed for 4 weeks with 40 mg/Kg bw/d of TAM citrate chow (400 mg/Kg diet, ENVIGO, TD.130860) or alternatively were i.p. injected in alternate days with (Z)-4-Hydroxytamoxifen (Sigma, H7904) at the dose of 40 mg/Kg for 2 weeks and then used 2 weeks later. For each experimental procedure with TAM diet/injection, double heterozygous Tg-myh6 MCM :R26 mT/mG , Tg-myh6 MCM and R26 mT/mG heterozygous littermates mice, were fed with standard normal mouse diet as controls.
The final n-value for each experimental group is specified in the relative figure legends.

ISO administration
ISO (Sigma-Aldrich #I6504; St. Louis, MO) was prepared by dissolving a desired amount of powder in NaCl 0.9%. The solution obtained was protected from the light and kept on ice until the injections. Before any ISO/Saline injection, the body weight of animals was determined, mice were anaesthetised using isoflurane and baseline echocardiography obtained. Then, mice were randomly divided in the different groups and on awakening prepared to receive saline or ISO at dose of 200 mg/Kg or 400 mg/Kg. The solutions were injected s. c. under the inter-scapular skin. All ISO injections were administered to male, 12-week-old mice of the specified strains.

Echocardiography
Prior to echocardiography, mice were anaesthetised with isoflurane. Unconscious mice were weighed and secured in a supine position on a temperature-controlled restraining board. Anesthesia was maintained with 1-2% isoflurane in oxygen delivered through a nose cone. Fourlimb lead electrocardiograms (Vevo 3100 and MP150, Biopac, Goleta, CA, USA) were simultaneously recorded. All hair in the thoracic region was removed using a depilatory agent and the area was cleaned with water. Ultrasound gel was applied to the thoracic region to improve sound wave transmission. All mice were maintained at heart rates > 400 b.p.m., while images were recorded. Echocardiographic images were obtained with a Vevo 3100 system (Visualsonics, Inc., Toronto, Canada) equipped with a MX550D ultra-high frequency linear array transducer . The transducer was positioned in a stationary stand perpendicular to the mouse (in some cases, manual adaptations were needed for optimal imaging). In brief, a frame rate of > 200 frames per minute was maintained for all B-mode and M-mode images. B-mode long-axis parasternal images were recorded when optimal views of the aorta, papillary muscle, and endocardium were visible. M-mode shortaxis images were recorded at the level of the papillary muscles and the LV was bisected to obtain the optimal Mmode selection. Conventional echocardiographic measurements of the LV included ejection fraction (EF), fractional shortening (FS), end-diastolic dimension, endsystolic dimension, anterior and posterior wall thickness, and mass were obtained. For long-axis B-mode measurements, the endocardium was traced semiautomatically beginning from the mitral valve and excluding the papillary muscle. EF and FS were calculated by software using standard computational methods. Advanced cardiac analysis (regional and global cardiac measurements) were assessed by speckle-tracking echocardiography (Vevo LAB analysis software; VisualSonics). Cardiac cycles were acquired digitally from the parasternal long-axis and mid-ventricular short-axis views for the assessment of radial, circumferential, and longitudinal systolic strain/velocity (in accordance with myocardial fibre orientation at varying levels of the LV wall) and time-to-peak systolic strain/velocity. Images selected for strain analysis had well-defined endocardium and epicardium borders and no substantial image artefacts. Image analysis was performed according to the manufacturer's instructions. The endocardium and epicardium were traced semi-automatically using VevoStrain software. The traces were manually adjusted to ensure adequate tracking of endocardium and epicardium borders.
Velocity, displacement, strain, and strain rate were calculated for radial and longitudinal planes. In long axis, the basal anterior-septum, mid-anterior-septum, apical anterior-septum, basal posterior wall, mid-posterior wall, and apical posterior segments were defined. In midventricular short axis, the anterior, anterior-septum, inferior-septum, inferior, posterior, and anterior-lateral segments were further delineated. Tissue contraction patterns were expressed as negative strain values for longitudinal and circumferential motion, and positive values for radial strain. In each segment, peak systolic strain (%) and time-to-peak systolic strain (ms) were analysed. Global average peak values for circumferential and longitudinal strain are reported.
The n-value for each experimental group is specified in the relative figure legends.

Myocyte necrosis analysis
To assess CM necrosis, 12-week-old male mice were used. Mice (strains are specified in the main text and figure legends) received i.p. injection of 100 µg/100 µl of a monoclonal antibody against cardiac myosin (MF-20, ID: AB_2147781, DSHB) 2 h after ISO injection. Alternatively, mice received i.p. injections of 50 µl/10 g of the body weight of a 20 mg/ml stock solution of Evan's blue dye (EBD, Sigma-Aldrich) dissolved in NaCl 0.9% administrated 24 h before ISO/Saline injections. All animals were sacrified at 1 day after ISO/Saline injection and the heart fixed with 4% paraformaldehyde (PFA).
The n-value for each experimental group is specified in the relative figure legends.

Cardiac troponin analysis
To assess the presence of myocyte necrosis induced by a single dose of ISO, the levels of high-sensitive cardiac Troponin T (cTnT) in the blood serum were analysed 1 day after ISO/Saline. Animals were anaesthetized (ketamine 100 mg/Kg and xylazine 5 mg/Kg) and 1 ml of blood was taken from the orbital sinus inserting the tip of a fine-glass Pasteur pipette into the corner of the eye underneath the eyeball, directing the tip at a 45°angle towards the middle of the eye. The blood was collected in a vacutainer (Vacuette Tube 3 ml K3E K3EDTA, # 454086). The threshold for cTnT positivity in mice was established at the 99th percentile of cTnT values in blood samples from 15 consecutive control mice, using a commercially available and clinically validated blood cTnT kit. The n-value for each experimental group is specified in the relative figure legends.

BrdU and Edu incorporation in vivo
To detect in vivo cell proliferation after ISO administration, 12-week-old male C57BL/6J mice were injected at 12 pm with saline or ISO at dose of 200 mg/Kg. Six hours later, mice received an i.p. injection of Bromodeoxyuridine (BrdU) or 5-Ethynyl-2´-deoxyuridine (EdU) at a concentration of 50 mg/Kg in 100 µl of a 50% de-ionized water and 50% dimethylsulfoxide (DMSO) solution (Brdu, Sigma B9285; EdU, Life Technologies E10187). The morning after, all mice were anaesthetized using isoflurane and implanted subcutaneously (between the two scapulae) with mini-osmotic pumps (ALZET) 21 to systemically release BrdU or EdU (50 mg/Kg/Day both) for 7 or 28 days prepared by dissolving the powders in 50% deionized water and 50% DMSO. Mice from each group were sacrified at 28 days after ISO/Saline administration and the hearts were fixed in formalin for immunohistochemistry analysis.
To assess the regenerative potential of c-kit Cre mice, heterozygous c-kit Cre/+ mice, heterozygous R26 dT/+ mice, and double heterozygous c-kit Cre/+ :R26 dT/+ mice were treated with ISO/Saline and implanted for 28 days with BrdU pumps as described above. In addition,~35% of available male c-kit Cre/+ :R26 dT/+ exhibited spontaneous alterations of cardiac function at baseline pre-ISO injections and had to be excluded. To assess the effects of ISO administration on activation of quiescent resident CSCs, R26 dT/+ and c-kit Cre/+ :R26 dT/+ mice were divided in saline-injected and ISO-injected groups as above. BrdU, at a concentration of 50 mg/Kg/Day, was i.p. administered in vivo for 3 days in adult mice every 12 h before sacrifice. The mice were sacrified at 1 day or 3 days after ISO injection and hearts were dissociated to obtain a myocytedepleted cardiac cell preparation for fluorescenceactivated cell sorting (FACS) analysis as described 18,21,23 . To further assess the cardiac generation process, myocardial nuclei were isolated 28 days after ISO or Saline from mouse hearts continuously labelled with BrdU and evaluated by FACS analysis for BrdU detection as previously reported 24 .
The n-value for each experimental group is specified in the relative figure legends.

ISO + 5-FU-induced cardiomyopathy
Chronic HF was induced in 12-week-old male C57BL/6J mice, in heterozygous Tg-myh6 MCM mice, and in Tg-myh6 MCM : R26 mT-mG/+ double heterozygous mice by a subcutaneous single-dose administration of ISO 200 mg/Kg followed at 72 h by systemic administration of 5-Fluorouracil (5-FU,15 mg/Kg/daily) for 25 days through subcutaneously implantation of mini-osmotic pumps. Before pumps implantation the mice were anaesthetized using isoflurane. Twenty-eight days after ISO injection, 5-FU-realizing mini-pumps were removed. Tg-myh6 MCM : R26 mT-mG/+ double heterozygous mice were killed and the hearts were fixed in 4% PFA for immunohistochemistry analysis. C57BL/6J mice were randomly divided to receive tail vein CSCs/Saline injections as indicated. The animals were sacrified 28 days after CSCs/Saline injections and the hearts were fixed in formalin for immunohistochemistry analysis or dissociated to obtain a cardiac cell preparation for FACS analysis.
The n-value for each experimental group is specified in the relative figure legends.

Myocyte-depleted cardiac cell isolation
CSCs were isolated from the relative adult mouse hearts by enzymatic dissociation using a Langerdoff-modified apparatus 23 or using gentle MACS Dissociator (Miltenyi Biotec) 17,18 . Briefly, for the Langerdoff-modified apparatus, the heart was excised, the aorta cannulated, and hung on a retrograde perfusion system. Heart was perfused with collagenase type II dissolved HEPES-MEM (Sigma) (Worthington) at 37°C with 85% O 2 and 15% N 2 , then the heart was removed from the apparatus, cut into small pieces, and the fragments shaken in re-suspension medium at 37°C. CMs and myocyte-depleted small cardiac cells were separated by centrifugation. For gentle MACSisolation, manufacturer instructions were followed to obtain myocyte-depleted cardiac small cells for FACS analysis. To obtain CSC-enriched CD45 neg CD31 neg ckit pos cells, the MACS technology was used with direct CD45-and CD31-negative and then c-kit-positive specific anti-mouse microbeads sorting (Miltenyi Biotec) 17,18,23 .
The n-value of biological replicates is specified in the relative figure legends.

BAC c-kit vector generation and transfection in W Cre CSCs
A purified bacterial artificial chromosome (BAC), BAC c-kit DNA was generated and then transfected in a dT-W Cre CSCs clone obtained from c-kit CreER(T2)/+ :R26 mT-mG/+ mice as previously described 18,25 . A single-cellderived sub-clone of dT-W Cre CSCs containing a single BAC c-kit copy was obtained as previously described and used for the in vivo cell transplantation experiments 18 .

MethoCult assay in vitro
Colony Forming Unit (CFU) formation from cloned wtCSCs vs. W Cre CSCs was assessed by plating relative cells in 1.2 ml of MethoCult medium (STEMCELL Technologies, Vancouver, Canada) (1% methylcellulose in CSC growth medium). Colonies were counted at 14 days after plating. The n-value for the biological replicates is specified in the relative figure legends.

Bone marrow cell isolation
Bone marrow (BM) cells were isolated from mice by flushing both femurs and tibias with phosphate-buffered saline (w/o Ca, w/o Mg) using a 26-gauge needle syringe. Cells were pelleted, washed, and re-suspended for FACS analysis. For BM lineage-negative FACS analysis, a Lineage Negative Depletion Kit was used (Miltenyi Biotec). The n-value of biological replicates is specified in the relative figure legends.

Tissue harvesting, histology, and immunohistochemistry
For immunohistochemistry analysis, the abdominal aorta was cannulated and the heart arrested in diastole using cadmium chloride/potassium solution. The animals were perfused through the cannulated aorta and fixed with 10% buffered formalin or with 4% PFA. The hearts were cut into apical, mid, and basal regions, and the right and left atria. After being weighed, the LV was sectioned embedded in paraffin or in Optimal Cutting Temperature Compound. Tissues were cut in 5 µm or 10 µm crosssections, respectively. Apoptotic cells on cross-sections were detected using the primary antibody caspase-3 (1:100 dilution: Abcam) and horseradish peroxidaseconjugated secondary antibody, and visualized by 3,3′-Diaminobenzidine (DAB) substrate histochemistry. Sections were stained with hematoxylin and eosin (H&E) following standard procedures. Formalin-fixed sections were stained with Masson's Trichome (PolySciences, Inc.) for bioquantification of fibrosis 26 . CM cross-sectional area was measured through immunostaining with Wheat Germ Agglutinin (WGA) Alexa Fluor 647 conjugate (1:200 dilution; Invitrogen) and digital analysis of acquired cardiac cross-section images (Leica, 1128 LAS AF Software). CM diameter was measured across the nucleus on three transverse sections (~500 myocytes/animal were sampled). For immunostaining and BrdU detection, antigen retrieval was achieved using Target Retrieval Solution, Citrate pH 6 (DAKO). For EdU detection, the Click-iT EdU Imaging Kit (Life Technologies) was used. The following primary antibodies were used: anti-BrdU (1:50 dilution; Sigma), anti-EdU (1:500, Click-iT EdU Imaging Kit, Life Technologies), monoclonal antibody against cardiac myosin (MF-20, ID: AB_2147781, DSHB), anti-Actinin (1:50 dilution; Santa Cruz), anti-Cardiac Troponin I (1:50 dilution; Abcam), anti-GFP (for YFP detection) (1:50 dilution; Rockland), anti-RFP (for dTomato detection) (1:50 dilution; Rockland), anti-c-kit (1:50 dilution; R&D Systems), and anti-pericentriolar material 1 (PCM-1) (1:200 dilution; Atlas Antibodies). The primary antibody was revealed by respective anti-mouse IgG, antirabbit IgG, or anti-goat IgG secondary antibody (1:100 dilution; Jackson Immunoresearch). The nuclei were counterstained with the DNA binding dye, DAPI (4, 6diamidino-2-phenylindole, Sigma) at 1 µg/ml. BrdU and EdU fluorescence quantification has been obtained through manual counting of respective histologic samples and the number of BrdU pos or EdU pos CMs was expressed as a percent fraction of the total CM nuclei 17,21 . To evaluate the CM progeny of the YFP pos or dTomato pos CSCs in vivo, the number of double positive YFP pos / actinin pos (or YFP pos /cTnI pos ) and dTomato pos /actinin pos (or dTomato pos /cTnI pos ) CMs were counted in cardiac cross-sections derived from mice for each power field using a ×63 objective for a total of 20 fields 17,21 . The number of c-kit pos CD45 neg CD31 neg cells in all experimental group was expressed per mm 2 . The number of YFP pos or dTomato pos CMs in all experimental group was expressed as a percent fraction of the total CM number per mm 2 . The number of necrotic/dead MF-20 pos and EBD pos (the latter fluoresces in red) CMs was manually counted in cardiac cross-sections for each power field using a ×63 objective for a total of 20 fields 17,21 , and the number of MF-20 pos and EBD pos CMs was expressed as a percent fraction of the total CM number per mm 2 . All stainings were acquired and analysed using confocal microscopy (LEICA TCS SP5 and SP8).

Flow cytometry
Cell analysis on total freshly isolated c-kit pos cardiac cells, freshly isolated CD45 neg CD31 neg c-kit pos CSCs, and BM cells was performed using a FACSCanto II (BD Biosciences) or a BD FACSAria™ III (BD Biosciences). FlowJo software (Treestar) was used to identify the percentage of cells expressing the different cell surface markers of interest. Voltages for cytometry acquisition were determined using single stain. Specific antibodies used are shown in Supplementary Table 1 (bottom of this file). Appropriate labelled isotype controls were used to define the specific gates. The n-value for each group is specified in the relative figure legends.

Statistical analysis
Statistical analysis was performed with GraphPad Prism version 6.00 for Macintosh (GraphPad Software). Quantitative data are reported as mean ± SD and binary data by counts. Significance between two groups was determined by Student's t-test or paired t-test as appropriate. For comparison between multiple groups, analysis of variance was used. A P-value < 0.05 was considered significant. Tukey's post-hoc method was used to locate the differences. In these cases, the Type 1 error (α = 0.05) was corrected by the number of statistical comparisons performed. For the in vitro cell and molecular biology experiments, the Kruskal-Wallis test (for multiple-group comparison) and the Mann-Whitney U-test (for comparison between two groups) were also performed.

Results
Acute ISO overdose causes CM necrosis and apoptosis in a dose-dependent manner in the LV sub-endocardial apex ISO at 200 mg/Kg 21 (n = 14) or at 400 mg/Kg (lethal dose in the study by Wallner et al. 22 ) (n = 14) caused diffuse CM necrosis in 12-week-old C57BL/6J male mice as revealed by in vivo myosin antibody labelling 27 1 day after ISO injection ( Fig. 1). Myosin-labelled CMs showed clear morphologic features of necrosis with loss of cell membrane integrity and architectural disarray (Fig. 1). The damage was distributed in an intensity/density progressive gradient from epicardium to sub-endocardium and from base to apex (Fig. 1a-c). CM death mostly concentrated to the sub-endocardial apex where myosinlabelled necrotic CMs reached up to~8% and~12% after ISO 200 or 400 mg/Kg ISO, respectively ( Fig. 1a-d). Only very rare (~0,001% of total) myosin-labelled CMs were detected in the saline-injected mice; however, these antimyosin-labelled CMs were always normally shaped and without additional signs of necrosis (Fig. 1e). Increased cTnT blood levels independently confirmed CM necrosis directly proportional to the ISO dose (Fig. 1f). The normal cTnT values baseline was established at <0.01 ng/ml in 15 consecutive control mice. Blood cTnT levels increased to 0.25 ± 0.14 and 0.58 ± 0.21 ng/ml 1 day after 200 or 400 mg/Kg ISO, respectively (Fig. 1f).
The assessment of EBD 28 -positive CMs at 1 day after ISO closely reproduced the CM necrosis data obtained with myosin antibody labelling ( Supplementary Fig. 1). In addition, CM necrotic death after ISO was also evident by H&E histochemistry (Supplementary Fig. 1B). Finally, ISO exposure caused apoptotic CM death in a dose-dependent manner as identified by caspase-3 labelling (Supplementary Fig. 1C).

High dose of ISO transiently impairs LV regional performance
To assess the functional consequences of ISO-induced CM damage and death, additional mice were injected with either 200 mg/Kg or 400 mg/Kg ISO and analysed by echocardiography (ECHO). Saline-injected mice were used as sham controls. Two mice in the 200 mg/Kg and 3 mice in the 400 mg/Kg ISO group died during the first week.
Only ISO at 400 mg/Kg significantly decreased LV EF and FS after 1 day compared with baseline. There was a nonsignificant decrease in EF in mice treated with 200 mg/Kg ISO (Fig. 2a-d, Supplementary Fig. 2 Table 2). EF 2 days after 200 mg/Kg ISO showed a significant depression compared with baseline. The latter could be explained by the evidence that a detectable reduction in EF is often a late phenomenon 29 (Fig. 2d and Supplementary Table 2). At the same time point, LV function was also consistently depressed in mice treated with 400 mg/Kg ISO (Fig. 2a-d and Supplementary Table 2). Segmental dysfunction was more prominent at mid-apical regions (see below) and evident apical ballooning was present in approximately a third of the injected mice.
Diastolic dysfunction was evident at 1 and 2 days after ISO, both at 200 or 400 mg/Kg, when compared with base ( Fig. 2e, f, Supplementary Fig. 2, and Supplementary Table 2). Specifically, the diastolic function shows that the E'-value decreases (indicating a reduction of LV longitudinal myocardial relaxation) (Fig. 2f and Supplementary Table 2), whereas E/E' (the ratio of transmitral Doppler early-filling velocity to tissue Doppler early-diastolic mitral annular velocity, an index of LV end-diastolic filling pressure)  Table  2). All these parameters had returned to baseline at 28 days after ISO (Fig. 2a-f and Supplementary Table 2). Global strain analysis 29 was able to detect subtle changes in cardiac performance earlier than conventional standard echocardiographic measures in 200 mg/ Kg ISO-treated mice (Fig. 2g, h and Supplementary Table 2). ISO, both at 200 and 400 mg/Kg, significantly decreased the value of global longitudinal strain at 1 day compared with baseline. Global longitudinal strain remained consistently depressed at 2 days ( Fig. 2g and Supplementary Table 2). Interestingly, only in mice treated with 400 mg/ Kg ISO, global circumferential strain was significantly  Table 2). All strain values were normalized at 28 days (Fig. 2g, h and Supplementary Table 2). Finally, regional speckle-tracking strain analysis throughout four segments at 1 day after ISO 200 mg/Kg showed an increase in time-to peak value in apical segments (even in mice with preserved EF) when compared with the uninjured segments ( Fig. 2i and Supplementary Table 2). Also, segments analysis of wall motion abnormalities and wall synchronicity detected early cardiac damage (Fig. 2i and Supplementary Table 2).

CM loss by a single high dose of ISO triggers their replacement with new CMs by the CSCs and not through division of pre-existing CMs
We assessed new CM formation 28 days after ISO by comparing head-to-head BrdU vs. EdU continuous labelling (through mini-pump implants releasing BrdU or EdU 50 mg/Kg daily) for 7 or 28 days after ISO (200 mg/Kg). We detected new CM formation both in EdU-and BrdU-continuously labelled mice, which was more intense and mainly localized to the subendocardial apex (Fig. 3a). The fraction of BrdU pos and EdU pos CMs was~7% and~3% of the CMs in the subendocardial apical layer at 28 days, respectively, compared with~0,1% in saline-treated mice. Newly formed CMs were significantly less abundant in the mid-and basal myocardial regions (Fig. 3b), in agreement with less CM damage in these areas (Fig. 1). Also, BrdU pos and EdU pos CMs were significantly less abundant with only 7 days compared with 28 days continuous thymidine analogues' administration after ISO (Fig. 3b). In all cases, however, EdU-labelled less (< 50%) newly formed CMs than BrdU either after 7-days or 28-days of continuous administration (Fig. 3b).
CM nuclei-specific identification with PCM-1 antibody 24 show that the number of BrdU pos PCM-1 pos cTnI pos CMs (5.6 ± 1%, Fig. 3c) 28 days after ISO in the subendocardial layer of the apical section was practically undistinguishable from the number of BrdU pos CM nuclei identified and counted by morphological features (Fig. 3b). Isolated CM nuclei were evaluated for PCM-1 and BrdU detection by FACS (Fig. 3d), allowing for unambiguous direct evaluation of BrdU pos CMs. These data well reproduced the immunohistochemistry data in Fig. 3b.
We also analysed new CM formation using double transgenic myh6-mER-Cre-mER//R26R mT-mG mice (abbreviated as Tg-myh6 MCM: R26 mT-mG mice). Dilution of the GFP pos CMs/dTomato pos CMs ratio with an increase of dTomato pos CMs after ISO ( Supplementary Fig. 3) in these mice show that the new CM formation was not due to division of pre-existing CMs. Also, these data implicate that increased number of dTomato pos CMs had to have arisen from non-myocyte myogenic progenitor/precursor cells [30][31][32] .

c-kit receptor hemizygosity impairs the adult cardiac regenerative response after ISO
Wallner et al. 22 used a constitutive and TAM-inducible c-kit Cre KI mice 10 to track the fate of the "c-kit+cells" and did not find any increase in "c-kit+cell-derived CMs" after ISO. Their work was based on the assumption that the ckit Cre KI allele in their mouse models efficiently recombines the marker gene in the c-kit pos CSCs. However, we have recently shown that all the c-kit Cre KI alleles so far used 10-12 are very inefficient in driving recombination of the marker gene in many c-kit+cells, particularly the CSCs 18 . This is so because the efficiency of Cre recombination in these mice is directly proportional to the level of c-kit expression in the cell 15,16 , which is high in mast (see figure on previous page) Fig. 2 Isoproterenol transiently impairs cardiac function. a Representative M-mode tracing at Base, 1 day and 2 days after a single s.c. injection of Isoproterenol (ISO) at 200 or 400 mg/Kg. b-d Cumulative data of cardiac dimensions and function at the different time points. LVESD = left ventricular end-systolic diameter (b), FS = fractional shortening (c), and EF = ejection fraction (d). b, d *p < 0.05 vs. Base, 1d and 28d in ISO 200 mg; *p < 0.05 vs. Base and 28d in ISO 400 mg (one-way ANOVA analysis with Tukey's multiple comparison test). c *p < 0.05 vs. Base and 28d in ISO 200 mg and *p < 0.05 vs. Base and 28d in ISO 400 mg (one-way ANOVA analysis with Tukey's multiple comparison test). e Representative pulsed wave Doppler Mitral Velocity tracing (PW MV, left) and cumulative transmitral Doppler early filling velocity (MV E data, right) at Base, 1d and 2d after a single s.c. injection of ISO at 200 or 400 mg/Kg. *p < 0.05 vs. Base, 1d, and 28d in ISO 400 mg (one-way ANOVA analysis with Tukey's multiple comparison test). f Representative pulsed wave tissue Doppler imaging velocity tracing (PW TDI, left) and cumulative early diastolic mitral annular velocity value (E', mid) and E/E' ratio (right) data at Base, 1d and 2d after a single s.c. injection of ISO at 200 or 400 mg/Kg. *p < 0.05 vs. Base and 28d in ISO 200 mg and *p < 0.05 vs. Base and 28d in ISO 400 mg (one-way ANOVA analysis with Tukey's multiple comparison test). g, h Cumulative data of global longitudinal and circumferential strain (GLS and GCS, respectively) at the different time points at Base, 1d and 2d after a single s.c. injection of ISO at 200 or 400 mg/Kg. g *p < 0.05 vs. Base and 28d in ISO 200 mg and *p < 0.05 vs. Base and 28d in ISO 400 mg (one-way ANOVA analysis with Tukey's multiple comparison test). h *p < 0.05 vs. Base, 1d, and 28d in ISO 400 mg (one-way ANOVA analysis with Tukey's multiple comparison test). i Representative images of regional speckle-tracking strain analysis throughout four segments (apical, anterior and inferior segments wall, and midanterior and inferior wall) at Base, 1d and 2d after a single s.c. injection of ISO at 200 mg/Kg. n= 15 (ISO 200 mg/Kg, BASE, 1d, and 2d); n = 13 (ISO 200 mg/Kg, 28d); n = 15 (ISO 400 mg/Kg, BASE and 1d); n = 12 (ISO 400 mg/Kg, 2d, and 28d). All data are mean ± SD and endothelial (progenitor) cells but many fold lower in the CSCs 18 . Both constitutive c-kit CreGFPnls/+ and TAMinducible c-kit mERCremER/+ (c-kit MCM/+ ) mouse lines have a null c-kit allele and thus they are phenotypically similar to W/+mice [33][34][35] , show the typical white spotting in the coat (Fig. 4a), have a 50% decrease of c-kit expression 18 , and are incompatible with late fetal/early post-natal life in homozygosis 10,12 . The W-mutation phenotype of the c-kit Cre KIs mice is confirmed by the size of a prototypical c-kit-dependent organ, such as the testis, from 2-monthold c-kit Cre mice that shows a significant size reduction (Fig. 4b), which, in part, explains the low fertility of the ckit Cre KI lines. Most relevant, the c-kit haploinsufficiency severely impairs the c-kit pos CSCs growth, clonal expansion (Fig. 4c, d), and myogenic differentiation 18 . In addition, in constitutive c-kit CreGFPnls mice, the GFP nuclear expression (GFP nls ) has a surprisingly low coincidence with c-kit expression. Only~25% of the c-kit pos BM cells expressed detectable GFP nls (Fig. 4e), while the vast majority of c-kit pos cardiac cells were GFP nls negative by FACS and/or tissue immunohistochemistry (Fig. 4f).
ISO (200 mg/Kg) was injected into 12-week-old male double mutant mice, c-kit CreGFPnls : R26 floxed-stop-dTomato/+ and in control R26 floxed-stop-dTomato/+ littermates 36 , all implanted with subcutaneous mini-pumps to release BrdU for 28 days. In the Cre-constitutive double transgenic mice, all the cells, which have undergone Credependent recombination (in theory, all cells expressing c-kit) should become dTomato pos . However, at baseline before ISO,~70% of Lineage-committed (CD45 pos / CD31 pos ) c-kit pos cardiac cells (mainly blood and endothelial lineage-derived cells) are dTomato pos (Fig. 4g). On the other hand, only 10 ± 3% of the Lin neg (CD45 neg / CD31 neg ) c-kit pos cells (which include all the CSCs) are dTomato pos (Fig. 4g). Thus, this system is very inefficient and nor reliable to track the resident CSCs and their progeny 15,16,18 .
In a separate experiment with c-kit CreGFPnls/+ (abbreviated also as c-kit CRE/+ ) mice, c-kit Cre hearts show a similar CM necrosis as the control hearts from wt c-kit +/+ littermates at 1 day after ISO (Fig. 5a, b). At 3 days after ISO, control R26 floxed-stop-dTomato/+ (abbreviated also as R26 dT/+ ) hearts show a significant increase in the number of resident CD45 neg CD31 neg c-kit pos CSCs, whereas the c-kit CRE/+ : R26 dT/+ hearts only show a minimal increase when compared with the respective uninjured controls (Fig. 5c). In the uninjured control R26 dT/+ mice, the majority of freshly isolated CD45 neg CD31 neg c-kit pos/low CSCs (a representative scheme of their identification and isolation is presented in Fig. 5d) are quiescent with only 5 ± 1% in the cell cycle, as measured by BrdU incorporation (Fig. 5e). In these mice, ISO injury caused a rapid and significant egress of the CSC pool from the quiescent state with 80 ± 8% of activated BrdU pos CSCs at 3 days (Fig. 5e). In addition, transient amplifying and myogenic-committed c-kit pos Gata-4 pos cardiac progenitors significantly increased after ISO compared with vehicle-treated mice (Fig. 5f). In contrast, when compared with control R26 dT/+ mice, both the percentage of BrdU pos CSCs and myogenic-committed c-kit pos Gata pos cardiac progenitors were much lower in c-kit CRE/+ :R26 dT/+ mice after ISO (Fig. 5e, f).
At 28 days after ISO, we detected only a slight increase of c-kit pos cell-derived dTomato pos CMs (0.55 ± 0.07% vs. 0.21 ± 0.04%, p < 0.05) in the sub-endocardial apex of ISOinjured vs. saline-injected c-kit CRE/+ :R26 dT/+ mice (Fig. 6a). The number of BrdU pos CMs was also significantly lower in the c-kit CRE/+ :R26 dT/+ mice than in the R26 dT/+ control littermates (0.75 ± 0.17% vs. 6.52 ± 1.04%, p < 0.05) (Fig. 6b). The severe deficit of CM regeneration in c-kit CRE/+ :R26 dT/+ mice was accompanied by hypertrophy of the surviving pre-existing CMs, which was absent in R26 dT/+ control mice (Fig. 6c). Also, although the hearts of R26 dT/+ control mice at 28 days after ISO did not show evidence of fibrosis, there was multiple areas (see figure on previous page) Fig. 3 Cardiomyocyte Replenishment after ISO-induced CM loss. a Representative confocal microscopy images of BrdU (left) and EdU (middle) incorporation in the apical endocardium 28 days after ISO in wt C57BL/6J mice implanted with subcutaneous mini-pumps to systemically release BrdU or EdU for 28 days. Right, representative confocal microscopy of BrdU incorporation from epicardium to endocardium of the LV Apex 28 days after ISO in wt mice implanted with subcutaneous mini-pumps to systemically release BrdU for 28 days. It is evident the intensity gradient of BrdU incorporation from the epicardium to the endocardium. Scale bar = 50 μm. b Number of newly generated BrdU pos or EdU pos CMs at 7 and 28 days in saline or ISO-treated wt mice implanted with subcutaneous mini-pumps to systemically release EDU or BrdU for 7 or 28 days, respectively. n = 7 per group, *p < 0.05 vs. Saline, #p < 0.05 vs. Epi, §p < 0.05 vs. Myo, and †p < 0.05 vs. EdU (one-way ANOVA analysis with Tukey's multiple comparison test). c Representative reconstruction of three overlapping confocal microscopy images (left) and a high magnification field (×3 zoom from a ×63 microscopy objective, right) showing newly formed cardiomyocytes (arrowheads) whose nuclei are unambiguously identified by PCM-1 (white fluorescence) while being BrdU-positive (green) 28 days after ISO injury in the apical sub-endocardium. DAPI (Blue) depicts cell nuclei. d Cardiac nuclei are identified by FACS analysis based on DAPI labelling (left panel). Fluorescent gating allows the separation of cardiomyocyte nuclei (PCM-1positive) and non-cardiomyocyte (PCM-1-negative) nuclei in cardiac nuclei preparations isolated from digested heart tissue (left-mid panel). Representative plots show the number of BrdU-labelled cardiomyocyte nuclei (gated on PCM-1-positive nuclei) 28 after saline (right-mid panel) or Isoproterenol (ISO, right panel) injection. It should be noted that the number of BrdU pos cardiomyocyte nuclei should be corrected when extrapolating it to number of cardiomyocytes considering that by the vast majority of adult cardiomyocytes in the adult murine hearts are binucleated. Scale bars = 50 µm. All data are mean ± SD of fibrosis in the sub-endocardial apex of the c-kit CRE/+ : R26 dT/+ mice, in agreement with the decreased CM replacement (Fig. 6d). Twenty-eight days after ISO, the lack of robust CM regeneration was accompanied by persistence of LV functional impairment in c-kit CRE/+ :R26 dT/+ , which was absent in R26 dT/+ control mice (Fig. 6e).

c-kit hemizygosity supresses CSC myogenicity and regenerative potential
We tested whether transplantation of exogenous wtCSCs would rescue the defective cardioregenerative phenotype of c-kit Cre KI mice. To this end, c-kit mERCremER/+ mice (hereafter c-kit MCM/+ mice) 10 were ISO-injured (200 mg/Kg). Twelve hours later, they were administered through the tail vein with saline or 4 × 10 5 each of either wt YFP + CSCs (CSC YFP ) or wt YFP + cFBs (cFBs YFP ) (Fig. 6f, g). To further assess c-kit gene function in CSC regenerative potential in vivo, additional ISO-injured mice were similarly transplanted with CSC YFP transfected with a KIT siRNA knocking down c-kit expression (Supplementary Fig. 4). At 28 days after ISO, the transplanted exogenous wtCSC YFP had differentiated into new YFP + CMs in the sub-endocardial layer where they reached 4 ± 1% of total CMs (Fig. 6g, i), which were clearly absent in cFBs transplanted mice (Fig. 6f, i). c-kit knockdown significantly reduced wtCSC myogenic potential, as indeed CSC YFP -KIT KD did not generate significant new CMs (Fig.  6h, i). New CM formation, derived from the transplanted wtCSC YFP , prevented cardiac fibrosis when compared with the recipients of either cFBs YFP , CSC YFP -KIT KD , or saline (Fig. 6j). wt CSC YFP transplantation in c-kit MCM/+ mice also normalized cardiac function, whereas the saline-, cFBs-, and CSC YFP -KIT KD -injected mice remained in HF (Fig. 6k).

CSC activation and ensuing CM formation depends on a proper level of c-kit function
To devoid the hearts of functional resident CSCs, ISO injury was followed by 5-FU treatment 21 . This model invariably leads to HF in rats and mice 21 . 5-FU regime ablated proliferating CSCs and resulted in a severe CSC deficit, absent CM replacement, continued CM loss, hypertrophy of surviving CMs and dilated decompensated cardiomyopathy ( Supplementary Fig. 5A-F). The lack of CSC-derived CM regeneration in ISO + 5-FU cardiomyopathy was confirmed using double transgenic Tg-myh6 MCM :R26 mT/mG mice after TAM treatment ( Supplementary Fig. 5G).
Next, we addressed whether the CSCs are sufficient to regenerate the cardiac tissue lost and to restore myocardial function after ISO injury. If so, whether the CSC regenerative properties are dependent on a normal diploid c-kit level. We compared in vivo the regenerative properties of cloned W Cre CSCs (so called for the W phenotype of the c-kit CRE mice) obtained from c-kit CreER(T2)/+ :R26 mT-mG/+ mice (hereafter dT-W Cre CSCs) with cloned wtCSCs from R26 mT-mG/+ littermates (dT-wtCSCs). A total of 4 × 10 5 cloned cells of each type (all of them the progeny of a single cell) were transplanted through the systemic circulation by tail vein injection, into 16-week-old syngeneic male C57BL/ 6J mice with ISO + 5-FU failing cardiomyopathy 21 (Fig. 7a). Control ISO + 5-FU mice were injected an equal volume of saline. One month after treatment, all saline-injected mice and those transplanted with dT-W Cre CSCs were still in overt cardiac failure (Fig. 7b,  Supplementary Fig. 6A). In contrast, those treated with dT-wtCSCs had completely recovered from cardiac dysfunction (Fig. 7b, Supplementary Fig. 6A). In addition, although the dT-wtCSCs reconstituted the host's myocardial c-kit pos CSC pool previously ablated by the ISO + 5-FU regime, the dT-W Cre CSCs did not and the myocardium of the relative recipient animals was practically devoid of CSCs (Fig. 7c, d). Concordantly, dTomato pos newly formed CMs (6.08 ± 1.16%) were detected (see figure on previous page) Fig. 4 c-kit Cre mice fail to label resident CSCs. a Photographs of representative adult c-kit MCM and c-kit CreGFPnls/+ mice showing the typical W-mutation-determined piebald and white belly spot as compared with c-kit +/+ C57BL/6J mouse. b The testis from 2-month-old kit MCM and c-kit CreGFPnls/+ show a significant size reduction compared to c-kit +/+ mice. c Cell growth curve of freshly isolated (P1) W Cre CSCs vs. wtCSCs over 96 h. n = 7 biological replicates, *p < 0.05 vs. wtCSCs (Kruskal-Wallis test). d Colony number of 100 cloned wtCSCs vs. W Cre CSCs in Methocult. n = 7 biological replicates, *p < 0.05 vs. wtCSCs (Student's t-test). On the right, representative light microscopy images of a colony from wtCSCs and W Cre CSCs, respectively. n = 6 plots biological replicates. Scale bars = 1000 µm. e GFP expression (from the c-kit CreGFPnls allele) in the monocyte-lymphocyte gate of total bone marrow (BM) cells population and in total c-kit pos BM cells from 2-month-old c-kit CreGFPnls/+ mice. Less than 25% of the total c-kit pos BM cells express GFP in c-kit CreGFPnls/+ mice. Concurrently, <35% of the lineage-negative (Lin neg ) c-kit pos BM cells (including HSCs) express GFP in c-kit CreGFPnls/+ mice. f GFP expression (from the c-kit CreGFPnls allele) in cardiomyocyte-depleted cardiac cells (left) and in cardiac c-kit pos cardiac cells (right) from 2-month-old c-kit CreGFPnls/+ mice. Less than 10% of these cells express GFP. Right, confocal microscopy representative images of cardiac cross-sections showing that GFP nuclear expression is expressed only in some (20 ± 3%) of the c-kit-expressing cardiac cells. Scale bar = 20 µm. (e, f representative of n = 5 BM/Hearts). g Mice KI-ed within the first exon of the c-kit locus to express Cre recombinase and GFP with a nuclear localization sequence (eGFPnls) behind an internal ribosome entry site (IRES) (c-kit CreGFPnls or c-kit Cre ) were crossed with B6.129S6-Gt(ROSA) 26Sortm9(CAG-tdTomato)Hze/J (abbreviated as R26 floxed-dTomato ) Cre-reporter mice, which harbour a targeted mutation of the Gt(ROSA)26Sor locus with a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (tdTomato), which is expressed following Cre-mediated recombination. Approximately 20% of total cardiac cells and~60% of c-kit pos cardiac cells from these mice are dTomato pos . Approximately 70% of lineage-committed endothelial/mast cell (CD45 pos /CD31 pos c-kit pos ) cardiac cells are recombined to express dTomato, whereas <10% of the CSC-enriched CD45 neg CD31 neg /c-kit pos were recombined to become dTomato pos (g representative of n = 5 hearts). All data are mean ± SD in the mice injected with dT-wtCSCs (Fig. 7e), whereas the dT-W Cre CSCs minimally contributed dTomato pos CMs (0.18 ± 0.08%) in the ISO + 5-FU failing hearts (Fig. 7e). None of these effects was due to differential tissue-homing and engraftment of dT-wtCSCs and dT-W Cre CSCs, as similar fractional numbers of each were detected in the myocardium 24 h after cell transplantation ( Supplementary Fig. 6B). were similarly increased in wild-type c-kit +/+ and heterozygous c-kit +/Cre mice at 1 day after ISO (200) in the Apex sub-endocardium. n = 5 mice per group, *p < 0.05 vs. saline (one-way ANOVA analysis with Tukey's multiple comparison test). Scale bars = 50 µm. c c-kit CreGFPnls :R26 floxed-dTomato/+ (abbreviated as c-kit Cre/+ :R26 dT/+ ) mice show a blunt increase of resident CD45 neg c-kit pos CSCs when compared with control R26 floxed-dTomato/+ (abbreviated hereafter as R26 dT/+ ) mice 3 days after ISO. n = 5 mice per group; *p < 0.05 vs. Saline; # p < 0.05 vs. R26 dT/+ mice (one-way ANOVA analysis with Tukey's multiple comparison test). d Representative FACS detection and isolation of CSCs from cardiomyocyte-depleted cardiac cell preparations. Left, representative gating strategy using isotype antibodies. Pre-sorting, flow cytometry dot plots show that the majority of total c-kit pos cardiac cells are CD45 or CD31 positive (red box), whereas only a minority are CD45 and CD31 negative (green box). Post sorting, flow cytometry dot plots show that CD45 pos CD31 pos (lin pos ) cells are efficiently removed from cardiac cells by CD45 − CD31 sorting and CD45 neg CD31 neg c-kit pos -sorted cells uniformly express low levels of c-kit. e Flow cytometry dot plots (representative of n = 3) show BrdU incorporation in CD45 neg c-kit pos cardiac cells from saline and ISO-treated R26 dT/+ and c-kit Cre/+ :R26 dT/+ mice. BrdU-35mg/Kg bid-was intraperitoneally administered in vivo for 3 days in adult mice every 12 h before killing. f Flow cytometry dot plots (representative of n = 3) show the fraction of myogenic-committed Gata-4 pos CD45 neg c-kit pos CSCs isolated from saline and ISO-treated R26 dT/+ and c-kit Cre/+ :R26 dT/+ mice. All data are mean ± SD The regenerative phenotype of W Cre CSCs was further tested in a competitive reconstitution assay by coinjecting YFP-positive (YFP pos ) wtCSCs (obtained from R26 stopYFP reporter mice 21 ) and dT-W Cre CSCs in a 1:3 ratio (a total of 4 × 10 5 cells injected) into mice with ISO +5-FU cardiomyopathy. At 28 days, the YFP pos wtCSC +W Cre CSC combination had partially reverted the cardiac dysfunction and CSC pool in all the animals treated (Fig. 7a-c and Supplementary Fig. 6A). However, in these hearts-although the YFP pos wtCSC were only 25% of the CSCs transplanted-the Lin neg c-kit pos CSC pool was constituted exclusively by YFP pos wtCSCs with no detectable persistence of dT-W Cre CSCs (Fig. 7d). Also, the newly formed CMs were exclusively YFP pos (4.85 ± 1.03%) with only an occasional dTomato pos CM (< 0.01%) (Fig. 7e, f).
To test whether restoring the diploid level of c-kit expression in the dT-W Cre CSCs would correct their myogenic and regenerative defects in vivo, we transfected a BAC construct spanning the entire c-kit gene locus 25 into cloned dT-W Cre CSCs, as recently reported 18 . This clone carries a single BAC/c-kit copy (hereafter dT-BAC ckit W Cre CSCs) and expresses c-kit mRNA and protein levels similar to those of dT-wtCSCs and doubled those of the untransfected parent c-kit/haploinsufficient dT-W Cre CSCs 18 . Adult C57BL/6J male mice with ISO + 5-FU-induced cardiomyopathy were injected as above with either dT-BAC c-kit W Cre CSCs, unedited BAC-naïve dT-W Cre CSCs, or saline. At 56 days, mice injected with dT-W Cre CSCs remained in overt HF similar to saline-injected mice (Fig. 7g). In striking contrast and comparable to dT-wtCSCs ( Fig. 7b and Supplementary Fig. 6A), the dT-BAC c-kit W Cre CSCs (n = 5) had fully restored cardiac function (Fig. 7g). The dT-BAC c-kit W Cre CSCs produced new dTomato pos CMs (~5%) in an amount similar to the dT-wtCSCs (Fig. 7h, i). In addition, dT-BAC ckit W Cre CSCs had restored the resident CSC cohort in the host myocardium (Fig. 7j).
Lastly, dT-wtCSCs from R26 mT/mG mice were injected into Tg-myh6 MCM mice with ISO + 5-FU cardiomyopathy, fed with a TAM diet for 28 days. In this genetic arrangement, all dTomato pos CMs (6.5 ± 2%) were invariably only dTomato pos , with none of them coexpressing GFP (Supplementary Fig. 7). These data rule out cell fusion as a relevant mechanism for new CMs contributed by the c-kit pos CSCs.

Discussion
The data presented here documents that CSCs from the c-kit Cre (also named as W Cre ) KI mice are impaired in their myogenic and cardiac regenerative properties because of their c-kit hemizygosity. These defects are fully rescued by BAC transgenesis of a single c-kit copy into the c-kit Cre CSCs to restore c-kit diploidy. On the other hand, worsening cardiac remodelling after injury in ckit Cre mice is reversed by the transplantation of wt c-kit +/ + CSCs. Therefore, the CSC's myogenic and regenerative properties are c-kit dose dependent 18 and require a diploid level of c-kit expression. For this reason, all the ckit Cre KI mouse lines, which abolish c-kit expression from the c-kit Cre KI allele [10][11][12] , cannot and should not be used to identify or to track the fate of the CSCs either in vitro or in vivo.
Acute excess catecholamines directly damage the rodent/murine heart 37-41 with resultant CM death [37][38][39][40][41] . Using ISO overdose, together with different genetic rodent models, we had previously shown that the CSCs are the main adult myocardium regenerative agents, which are necessary and sufficient for myocardial repair/ regeneration after damage 21 . Houser and colleagues 42 also reported that repair of the ISO-injured adult heart involves the generation of new CMs derived from resident progenitors 42 . This same group, however, using wild-type and c-kit Cre KI mouse models 22 , has recently reported to find no evidence of CM death or any increased CM formation after ISO, raising questions about the ISO-damage model and the role of the CSCs 22 .
The present study confirms and extends our previous results 21 , and reconciles them with those of Wallner et al. 22 . The main reasons for the discordance with Wallner et al. 22 are the different myocardial areas sampled (entire LV 22 vs. sub-endocardial apex 21 ) and the type, dose, and duration of the nucleoside analogue used to detect new cell formation. Considering cumulative EdU dose, Wallner et al. 22 underlabelled the replicating cells by about tenfold when compared with our experimental design 21 . The discrepancy in the results agrees with the well-known fact that efficiency of EdU and BrdU labelling of dividing cells is proportional to the cumulative dose used 43 .
(see figure on previous page) Fig. 6 Wild-type c-kit pos CSCs rescue the regenerative defect of c-kit Cre mice after injury in vivo. a Bar graph with cumulative data showing the number of dTomato pos myocytes in saline (n = 5) and ISO (n = 5) injected c-kit Cre/+ :R26 dT/+ mice 28 days after treatment. *p < 0.05 vs. all (one-way ANOVA analysis with Tukey's multiple comparison test). Right, representative confocal image showing dTomato pos CMs in c-kit Cre/+ :R26 dT/+ mice. b Bar graph with cumulative data shows that 28 days post-ISO, the number of newly generated BrdU pos myocytes was significantly less in c-kit Cre/+ : R26 dT/+ (n = 7) compared with R26 dT/+ mice (n = 5). p < 0,05 vs. Saline; # p < 0,05 vs. R26 dT/+ mice (one-way ANOVA analysis with Tukey's multiple comparison test). Right, representative confocal image showing BrdU pos CM in c-kit Cre/+ :R26 dT/+ mice 28 days after ISO. c Representative confocal images of cardiac cross-section showing cardiomyocyte hypertrophy in c-kit Cre/+ :R26 dT/+ mice when compared with R26 dT/+ mice (WGA, wheat germ agglutinin, Cy5 staining, and white fluorescence; cTnI, green; DAPI, blue nuclei). d Representative Masson's trichrome staining of cardiac crosssections from c-kit Cre/+ :R26 dT/+ mice 28 after ISO showing multiple areas of replacement fibrosis compared with R26 dT/+ mice. Scale bar = 250 μm. e Cardiac function 28 days after ISO as assessed by Echocardiography is depressed in c-kit Cre/+ :R26 dT/+ (n = 8) when compared with R26 dT/+ mice (n = 5). *p < 0.05 vs. all (one-way ANOVA analysis with Tukey's multiple comparison test). f Representative confocal microscopy image showing the absence of YFP pos CMs in cardiac Fibroblasts (cFb YFP )-injected c-kit MCM/+ mice (right insert, 3× zoom, shows the rare detection of YFP pos interstitial non-CM cells) in the apical sub-endocardium 28 days after ISO injury. Scale bar = 50 µm. g Robust replacement of YFP pos CMs in CSC YFP -injected ckit MCM/+ mice in the apical sub-endocardium 28 days after ISO injury. Scale bar = 50 µm. h The two representative confocal images show the very minimal number of YFP pos CMs (only visible in the right panel) in CSC YFP -KIT KD (c-kit knock-down by specific stealth RNAi siRNA transfection)-injected c-kit MCM/+ mice in the apical sub-endocardium 28 days after ISO injury where most of the engrafted cells remained as interstitial non-CM cells. Scale bar = 50 µm. i Bar graph with cumulative data showing the number of YFP pos cardiomyocytes (CMs) in injured c-kit MCM/+ mice injected with saline (n = 5), CSC YFP (n = 5), or CSC YFP -KIT KD (n = 5) injected c-kit Cre/+ :R26 dT/+ mice 28 days after ISO. *p < 0.05 vs. all (one-way ANOVA analysis with Tukey's multiple comparison test). j Representative Masson's Trichrome staining showing the presence of cardiac fibrosis in c-kit MCM/+ mice 28 days after ISO injected with cFBs YFP or CSC YFP -KIT KD that is absent in c-kit MCM/+ mice transplanted with CSC YFP . Scale bar = 100 μm. k Cumulative data showing Fractional Shortening as assessed by Echocardiography in c-kit MCM/+ mice at baseline and 28 days after ISO plus Saline or cFBs YFP or CSC YFP CSC YFP -KIT KD . n = 6 per group, p < 0.05 vs. Base and CSC YFP (one-way ANOVA analysis with Tukey's multiple comparison test). Scale bars = 50 µm unless differently specified in the panels. All data are mean ± SD The major pitfall of the reported null CSC contribution to new CMs after ISO in Wallner et al. 22 however is due to the use of a c-kit cre KI genetic mouse line 10 . The ongoing CSC controversy has been based on two assumptions: (a) that all the myocardial c-kit pos cells are expected to behave like bona fide CSCs 10 , whereas in reality they are only~1-2% of the c-kit pos cells; 17,18 (b) that the genetic tagging system used [10][11][12] does in fact tags the majority of the true c-kit pos CSCs. As we have recently shown, also the latter is incorrect 18 . All mouse lines with