Research ArticlePEDIATRIC CARDIOLOGY

Neuregulin stimulation of cardiomyocyte regeneration in mice and human myocardium reveals a therapeutic window

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Science Translational Medicine  01 Apr 2015:
Vol. 7, Issue 281, pp. 281ra45
DOI: 10.1126/scitranslmed.aaa5171
  • Fig. 1. Cryoinjury induces cell death, myocardial dysfunction, and decreased cardiomyocyte cell cycle activity in neonatal mice.

    Mice underwent sham surgery or cryoinjury on P1. (A) Hematoma at the injury site. (B) Vital staining with TTC shows the injury zone indicated by yellow arrowhead. (C and D) Myocardial cell death visualized by TUNEL staining (red) and DNA staining with Hoechst (blue) (C) and quantification (D). (E) Cryoinjury induces a sustained decrease in EF. (F) Acid fuchsin–orange G (AFOG)–stained sections show scar (blue) formation (within 7 dpi and present 30 days later). (G) Cryoinjury-induced scars, visualized on two sections of the same heart (500 μm apart) by Masson trichrome staining, persist to 7 months after injury. (H) Quantification of scar size. (I) Two cardiomyocytes in M phase visualized with antibodies against H3P (green/yellow), α-actinin (red), and Hoecsht (blue). The position of orthogonal reconstructions (along the XZ and YZ axes) of the cardiomyocyte in the center are indicated by yellow arrowheads. (J) Quantification of M-phase cardiomyocytes in the region around the injury zone shows significant and sustained reduction after cryoinjury. Scar region is indicated by black arrowheads (F and G). Scale bars, 1 mm (A, B, F, and G) and 20 μm (C and I). Statistical analysis by Student’s t test (D) and analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test (E, H, and J). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Cryo, cryoinjury.

  • Fig. 2. Early administration of rNRG1 improves myocardial function and structure.

    (A) Experimental design of mouse preclinical trials. (B to N) Mice underwent cryoinjury on P1 and were treated with BSA or rNRG1 from P0 (early administration; B to D) or from P5 (late administration; E to G). Prolonged improvement in myocardial function after early administration shown by echocardiography (B) and cMRI at 64 dpi (C). Late administration of rNRG1 resulted in transient improvement of myocardial function measured by echocardiography (E) and cMRI at 64 dpi (F). (D and G) Indexed heart weights showed that early rNRG1 administration reduced cardiac hypertrophy at 64 dpi. (H) Time series of AFOG-stained section shows that scar (blue) is formed within 10 dpi and is still present at 64 dpi. Note transmural scars after cryoinjury in BSA and late-administration rNRG1 treatment groups. Quantification of scar size after AFOG staining shows transient and significant scar reduction after early rNRG1 administration (I) when compared to late administration (J). (K) Early administration reduces the percentage of transmural scars at 34 and 64 dpi. (L) Nontransmural injury site thickens in systole (64 dpi, early administration). (M) Relative thickening of nontransmural scars is similar to remote LV free wall myocardium. (N) Transmural and nontransmural scars were identified by AFOG sections (left panels). Black rectangles indicate photomicrographs shown in the middle panels. Nontransmural scars have cardiomyocytes connected by gap junctions visualized with connexin 43 staining (64 dpi, early administration; middle and right panels). Yellow squares indicate zoomed-in areas of scar region (right panels). Scale bars, 1 mm (H), 500 μm (N, center panel), and 50 μm (N, far right). SC, subcutaneous injection. Statistical significance was tested with Student’s t test (C, F, and M), ANOVA followed by Bonferroni’s multiple comparison test (B, D, E, G, I, and J), and Fisher’s exact test (K). *P< 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.

  • Fig. 3. Early administration of rNRG1 reduces myocardial death and stimulates cardiomyocyte proliferation.

    (A) Hematomas are present at the zone of injury at 1 dpi. (B) Hematoma size quantification shows no change after early administration of rNRG1. (C and D) Photomicrographs (C) and quantification (D) of myocardial cell death visualized by TUNEL staining at 1 dpi after early administration. (E) Cardiomyocytes in M phase were visualized with an antibody against H3P. (F and G) H3P-positive cardiomyocytes were quantified around the injury zone after early (F) and late (G) administration of rNRG1. Treatment with rNRG1 increases cardiomyocyte cell cycle activity, and early administration captures the regenerative phase (F). (H and I) Cardiomyocytes in cytokinesis were visualized with an antibody against Aurora B kinase (H) and quantified around the injury zone after early administration at 1 dpi (I). (J) Cardiomyocyte nuclear density is increased after early administration of rNRG1 (34 dpi). (K) Early administration of rNRG1 increases the cardiomyocyte density by ~62,000 cardiomyocytes/mm3 within the first 8 days, compared to BSA controls. CM, cardiomyocytes. Scale bars, 1 mm (A) and 20 μm (C, E, and H). Statistical significance was tested with Student’s t test (B, D, I, and J) and ANOVA followed by Bonferroni’s multiple comparison test (F and G). *P < 0.05, ***P < 0.001, ****P < 0.0001; ns, not significant.

  • Fig. 4. rNRG1 acts through ErbB4 on cardiomyocytes in neonatal mouse hearts in vivo.

    (A to C) Experiments were performed in αMHC-MerCreMer+/+; ErbB4F/WT (control) and αMHC-MerCreMer+/+; ErbB4F/F (test) mice. rNRG1 or BSA was administered from P0 until P12. ErbB4 inactivation was induced with tamoxifen administration from days P1 to P3, which caused a significant down-regulation of ErbB4 mRNA levels (A). Representative example of a cardiomyocyte in M phase with orthogonal reconstructions along the XZ and YZ axes (shown by staining of heart sections with an antibody against H3P, B). Quantification of these sections showed that rNRG1 increased cardiomyocyte cell cycle activity in ErbB4F/WT but not in ErbB4F/F mice (C). Statistical test by Student’s t test (A) and ANOVA followed by Bonferroni’s multiple comparison test (C). *P < 0.05, ****P < 0.0001. Scale bar, 50 μm.

  • Fig. 5. Cryoinjury and rNRG1 administration induce gene regulation patterns that are consistent with structural and functional changes.

    Mice underwent cryoinjury on P1 and were treated with BSA or rNRG1 according to the early administration protocol. Expression profiling was performed at 10 dpi with five mice per group and were normalized to sham (n = 5). (A) Heat map shows 622 genes whose expression was significantly different (P < 0.05, Cuffdiff) in BSA- and rNRG1-treated mice relative to sham-operated mice. Selected genes discussed in the text are indicated. The color chart indicates fold change of expression using a log2 scale. (B) Functional annotation clustering of differentially expressed genes shows significant differences in the expression of components of multiple biological pathways by rNRG1. GO, Gene Ontology, a major bioinformatics initiative to unify the representation of gene and gene product attributes enabling functional interpretation of experimental data through enrichment analysis. P values were generated by DAVID bioinformatics tools. Fisher’s exact test was adopted to measure gene enrichment in annotation terms.

  • Fig. 6. Pediatric patients with heart disease show decreased cardiomyocyte cell cycle activity.

    Cardiomyocytes from patients were isolated, stained, and analyzed by flow cytometry. (A) Isolated human cardiomyocytes were intact as evidenced by staining with antibodies to pan-cadherin and α-actinin. Intact desmosomes are indicated by white arrowheads. (B) Representative double-marker plot of a 3-month-old patient showing flow cytometric analysis of cardiomyocyte cell cycle activity using cardiomyocyte (α-actinin) and cell cycle (H3P) markers. (C) Summary graph showing that patients with heart disease exhibited decreased cell cycling compared to age-matched controls without heart disease. Red dots connected with solid lines indicate results from patients with heart disease, and numbers of corresponding patients are indicated in red. Each donor heart control without heart disease is indicated with an open black symbol connected with dotted lines. Circles represent right ventricular and triangles represent LV samples. Scale bar, 50 μm.

  • Fig. 7. rNRG1 stimulates cardiomyocyte cycling in myocardium from infants with heart disease (younger than 6 months of age).

    For organotypic culture, chunks of myocardium were maintained in the presence of 1% FCS or rNRG1 for 3 days, fixed, and analyzed by immunofluorescence microscopy. (A and B) Preculture is fresh myocardium. Post–72 hour culture is after 72 hours of organotypic culture. Incubation of cells in organotypic culture for 3 days did not change microscopic architecture (A). Gap junctions and electromechanical connections were identified by connexin 43 staining and were present after 72 hours of organotypic culture (B). (C and D) rNRG1 stimulates cardiomyocytes to enter M phase in a 2-month-old patient with Tetralogy of Fallot. The position of orthogonal reconstructions of the cardiomyocyte in the center are indicated by yellow arrowheads (along the XZ and YZ axes) (C). Quantitative analysis showed that rNRG1 increased M-phase cardiomyocytes in an age-dependent manner (D). Numbers of patients per data point are indicated (D). Scale bars, 20 μm (A and C) and 50 μm (B).

  • Fig. 8. rNRG1 stimulates cardiomyocyte proliferation in myocardium from infants with heart disease (younger than 6 months).

    Organotypic cultures of human myocardium were metabolically labeled with CFSE and then maintained in the presence of 1% FBS or rNRG1 for 3 days. Cardiomyocytes were analyzed and isolated by fluorescence-activated cell sorting (FACS). (A) FACS strategy for enrichment by size (left panel), doublet discrimination (middle panel), and viability (right panel). (B) Flow cytometric analysis of a 3-month-old infant reveals a CFSElo population of 4.1%. (C and D) After fixation, the CFSElo population was stained with isotype control (C, left panel) and antibodies against cTnT (C, right panel). Analysis by flow cytometry shows that 94.7% of the population were cardiomyocytes (C, right panel) with forward and side scatter characteristics (D) similar to (A). (E and F) RT-PCR showed that CFSElo cardiomyocytes expressed markers of mature differentiated cardiomyocytes (E) and cell cycle–associated genes (F). (G) The graph of proportion of CFSElo populations shows that stimulation of cardiomyocyte proliferation in patients with heart disease is age-dependent. Numbers of patients per data point are indicated. (H and I) Laser scanning cytometry shows that administration of rNRG1 in organotypic culture did not change the overall percentage of mononucleated cardiomyocytes (H) or the ploidy pattern of mononucleated cardiomyocytes (I). Red boxes indicate the gating parameters used for data acquisition and analysis (A to D). FSC, forward scatter; SSC, side scatter; FSC-H, forward scatter height; SSC-W, side scatter width; FL4, detector for 488-nm laser with a 695/40 bandpass filter; FITC, fluorescein isothiocyanate; Ctrl, control.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/281/281ra45/DC1

    Materials and Methods

    Results

    Fig. S1. Contribution of cardiomyocyte proliferation to regeneration.

    Fig. S2. Cryoinjured hearts showed loss of sarcomeric organization.

    Fig. S3. Characterization of myocardial repair after cryoinjury.

    Fig. S4. Transmural scar persists even after 7 months after cryoinjury.

    Fig. S5. Visualization of scar and quantification of cardiac function by MRI from early administration group.

    Fig. S6. Visualization of scar and quantification of cardiac function by MRI for late administration group.

    Fig. S7. Time course of myocardial repair after cryoinjury from early rNRG1 administration group.

    Fig. S8. Time course of myocardial repair after cryoinjury from late rNRG1 administration group.

    Fig. S9. Failure to visualize transmural scars with late gadolinium enhancement due to low spatial resolution of cMRI in mice.

    Fig. S10. Schematic representation illustrating the nongenetic labeling technique with CFSE.

    Fig. S11. rNRG1-stimulated cardiomyocyte proliferation in infants is age-dependent (2-month-, 6-month-, 1.5- to 5-year-, and 10-year-old patients).

    Table S1. List of all differentially expressed genes between the BSA and rNRG1 treatment groups relative to sham mice (P < 0.05).

    Table S2. Clinical information of patients with heart disease analyzed for Fig. 6C (H3P activity over age).

    Table S3. Clinical information for normal hearts analyzed for Fig. 6C (H3P activity over age).

    Table S4. Clinical information of patients with heart disease analyzed for Fig. 7D (rNRG1 stimulation).

    Table S5. Clinical information of patients with heart disease analyzed for Fig. 8G (CFSE assay).

    Table S6. Comparison of tissue response after cryoinjury in mice and myocardial disease in human infants (myocardial dysfunction, scar formation, and decreased cardiomyocyte cycling).

    Table S7. Antibody manufacturers and dilutions.

    Table S8. Image acquisition hardware and settings.

    Table S9. Quantification of numeric data.

    Table S10. Human primers for quantitative RT-PCR for calculation of fold change in expression levels.

    Table S11. Mouse primers for quantitative PCR for calculation of fold change in expression levels.

    Movie S1. BSA-treated mouse from early administration.

    Movie S2. rNRG1-treated mouse from early administration.

    Movie S3. BSA-treated mouse from late administration.

    Movie S4. rNRG1-treated mouse from late administration.

    Movie S5. 3D reconstructions show myocardial syncytium adjacent to the scar after early administration (64 dpi).

    Reference (46)

  • Supplementary Material for:

    Neuregulin stimulation of cardiomyocyte regeneration in mice and human myocardium reveals a therapeutic window

    Brian D. Polizzotti, Balakrishnan Ganapathy, Stuart Walsh, Sangita Choudhury, Niyatie Ammanamanchi, David G. Bennett, Cristobal G. dos Remedios, Bernhard J. Haubner, Josef M. Penninger, Bernhard Kühn*

    *Corresponding author. E-mail: bernhard.kuhn2{at}chp.edu

    Published 1 April 2015, Sci. Transl. Med. 7, 281ra45 (2015)
    DOI: 10.1126/scitranslmed.aaa5171

    This PDF file includes:

    • Materials and Methods
    • Results
    • Fig. S1. Contribution of cardiomyocyte proliferation to regeneration.
    • Fig. S2. Cryoinjured hearts showed loss of sacromeric organization.
    • Fig. S3. Characterization of myocardial repair after cryoinjury.
    • Fig. S4. Transmural scar persists even after 7 months after cryoinjury.
    • Fig. S5. Visualization of scar and quantification of cardiac function by MRI from early administration group.
    • Fig. S6. Visualization of scar and quantification of cardiac function by MRI for late administration group.
    • Fig. S7. Time course of myocardial repair after cryoinjury from early rNRG1 administration group.
    • Fig. S8. Time course of myocardial repair after cryoinjury from late rNRG1 administration group.
    • Fig. S9. Failure to visualize transmural scars with late gadolinium enhancement due to low spatial resolution of cMRI in mice.
    • Fig. S10. Schematic representation illustrating the nongenetic labeling technique with CFSE.
    • Fig. S11. rNRG1-stimulated cardiomyocyte proliferation in infants is age-dependent (2-month-, 6-month-, 1.5- to 5-year-, and 10-year-old patients).
    • Reference (46)
    • Table S1 (Microsoft Excel format). List of all differentially expressed genes between the BSA and rNRG1 treatment groups relative to sham mice (P < 0.05).
    • Table S2. Clinical information of patients with heart disease analyzed for Fig. 6C (H3P activity over age).
    • Table S3. Clinical information for normal hearts analyzed for Fig. 6C (H3P activity over age).
    • Table S4. Clinical information of patients with heart disease analyzed for Fig. 7D (rNRG1 stimulation).
    • Table S5. Clinical information of patients with heart disease analyzed for Fig. 8G (CFSE assay).
    • Table S6. Comparison of tissue response after cryoinjury in mice and myocardial disease in human infants (myocardial dysfunction, scar formation, and decreased cardiomyocyte cycling).
    • Table S7. Antibody manufacturers and dilutions.
    • Table S8. Image acquisition hardware and settings.
    • Table S9. Quantification of numeric data.
    • Table S10. Human primers for quantitative RT-PCR for calculation of fold change in expression levels.
    • Table S11. Mouse primers for quantitative PCR for calculation of fold change in expression levels.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Fig.S3.data.pdf
    • Fig.S5.data.pdf
    • Fig.S6.data.pdf
    • Fig.S7.data.pdf
    • Fig.S8.data.pdf
    • Fig.S11.data.pdf
    • Table S1.GeneExpressionData.xlsx
    • Movie S1 (.mov format). BSA-treated mouse from early administration.
    • Movie S2 (.mov format). rNRG1-treated mouse from early administration.
    • Movie S3 (.mov format). BSA-treated mouse from late administration.
    • Movie S4 (.mov format). rNRG1-treated mouse from late administration.
    • Movie S5 (.mov format). 3D reconstructions show myocardial syncytium adjacent to the scar after early administration (64 dpi).

    [Download Table S1]

    [Download Movie S1]

    [Download Movie S2]

    [Download Movie S3]

    [Download Movie S4]

    [Download Movie S5]

    [Download Fig. S3.data.pdf]

    [Download Fig. S5.data.pdf]

    [Download Fig. S6.data.pdf]

    [Download Fig. S7.data.pdf]

    [Download Fig. S8.data.pdf]

    [Download Fig. S11.data.pdf]

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