Inhibition of β1-AR/Gαs signaling promotes cardiomyocyte proliferation in juvenile mice through activation of RhoA-YAP axis
- Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, United States
- Department of Pediatrics, College of Medicine, University of Cincinnati, United States
Figures
Figure 1 with 1 supplement
β-blocker (Metoprolol) treatment promotes neonatal-juvenile cardiomyocyte proliferation.
(a) Schematic of experimental timeline. (b) Heart rate of the saline (control) and β-blocker-treated mice at P7 and P14. Data are as mean ± SD, Student’s t-test, P7, n≥5; P14, n≥5. (c) H&E staining …
Figure 1—figure supplement 1
β-blocker treatment promotes cardiomyocyte proliferation after the cardiac regeneration window.
(a) Heart weight (HW) to body weight (BW) ratio of β-blocker treated heart at P7 (Student’s t-test, n≥3). (b) Ki67 and cardiac α-actinin staining of heart sections from control and β-blocker treated …
Figure 2 with 2 supplements
β-blocker treatment promotes cardiac regeneration and cardiomyocyte proliferation following injury in juvenile hearts.
(a) Schematic of experimental timeline. (b) Masson’s trichrome staining of heart sections from control and β-blocker-treated mice 3 weeks post-MI (left panel). Scale bar: 500μm. Quantification of …
Figure 2—figure supplement 1
Scar area is reduced in β-blocker treated hearts 3 weeks post MI.
Hearts were subjected to LAD ligation at P7 and treated with β-blocker from P8 for 3 weeks. Scar area was analyzed by sirius red staining of transverse sections at P28. Serial sections were cut at …
Figure 2—figure supplement 2
β-blocker doesn’t have cardioprotective effect at juvenile period.
2,3,5-Tripherylterazolium chloride (TTC) staining of control and β-blocker treated hearts 1 day post-MI. Right panel showed the % of ischemic area of each heart. Student t-test. N.S., not significant.
Figure 3 with 1 supplement
Deletion of Gnas promotes cardiomyocyte proliferation.
(a) Hematoxylin and eosin staining of P7 control and Gnas cKO heart sections. Scale bar: 500μm. (b) Heart weight (HW) to body weight (BW) ratio of P7 Gnas cKO mice. Data are as mean ± SD, Student’s …
Figure 3—figure supplement 1
Gnas cKO hearts exhibit enlarged phenotype but do not show cardiac hypertrophy.
(a) H&E staining of control and Gnas cKO hearts at P1 (upper panels) and P14 (lower panels) and quantification of heart weight-to-body weight ratio of control and Gnas cKO neonates at P1 and P14, …
Figure 4 with 2 supplements
Inhibition of βAR-Gαs signaling leads to elevation of YAP activity in the cardiomyocytes.
(a) Functional enrichment of GO terms for the common down-regulated genes in Gnas cKO and β-blocker treated hearts at P7 (fold change ≤0.8). (b) GSEA plot shows that fatty acid metabolic genes are …
Figure 4—figure supplement 1
Differential gene expression in Gnas cKO and β-blocker-treated hearts.
(a) The Venn diagram shows the down-regulated genes in Gnas cKO and β-blocker treated hearts at P7 (fold change ≤0.8). The total and overlay numbers of down-regulated genes are 1700 and 109, …
Figure 4—figure supplement 2
YAP activity is regulated by Gαs.
(a) Western blot analysis of lysate from P4, P7, and P9 hearts using phospho-YAP (P-YAP) or total YAP antibodies. (b) YAP and cardiac α-actinin immunostaining of P4 and P7 control and Gnas cKO heart …
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Figure 4—figure supplement 2—source data 1
Raw data of Western Blots.
- https://cdn.elifesciences.org/articles/74576/elife-74576-fig4-figsupp2-data1-v2.zip
Figure 5 with 2 supplements
GαS regulates cardiomyocyte proliferation through RhoA mediated YAP activation.
(a) Functional enrichment of GO terms for the common up-regulated genes. (b) Active-RhoA pull-down assay and western blot analysis of P7 control and Gnas cKO hearts. (c) Active-RhoA pull-down assay …
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Figure 5—source data 1
Raw data of Western Blots.
- https://cdn.elifesciences.org/articles/74576/elife-74576-fig5-data1-v2.zip
Figure 5—figure supplement 1
RhoA activity is only detected in the embryonic and postnatal hearts.
Active-RhoA pull-down assay of wild type hearts at several time points. The online version of this article includes the following source data for Figure 5—figure supplement 1.
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Figure 5—figure supplement 1—source data 1
Raw data of Western Blots.
- https://cdn.elifesciences.org/articles/74576/elife-74576-fig5-figsupp1-data1-v2.zip
Figure 5—figure supplement 2
Comparison of heart rate at different ages.
Heart rate at P0, P7, and P14 in C57BL6 mice. Data are as mean ± SD, n≥5.
Author response image 1
Author response image 2
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| gene (Mus musculus) | Gnas | NCBI | Gene:14683 | |
| strain, strain background (Mus musculus) | C57BL/6 J | The Jackson Laboratory | IMSR_JAX:000664 | |
| genetic reagent (Mus musculus) | Gnasflox/flox | The Jackson Laboratory | IMSR_JAX:035239 | |
| genetic reagent (Mus musculus) | Yapflox/flox | Xin et al., 2011 | ||
| genetic reagent (Mus musculus) | Myh6-Cre | Sanbe et al., 2003 | ||
| genetic reagent (Mus musculus) | Myh6-MerCreMer | Sohal et al., 2001 | ||
| biological sample (Rattus norvegicus) | Primary neonatal rat cardiomyocytes | In this paper | Freshly isolated from neonatal rats | |
| antibody | PCM1 (rabbit polyclonal) | Sigma | Car#: HPA023370 | IHC (1:1000) |
| antibody | Phspho-histon H3 (PH3) (mouse monoclonal) | Millipore | Car#: 05–806 | IHC (1:100) |
| antibody | Sarcomeric a-actinin (mouse monoclonal) | Sigma | Car#: A7811 | IHC (1:200) |
| antibody | Cardiac Troponin T (mouse monoclonal) | Thermo | Car#: MA295-P1 | IHC (1:200) |
| antibody | YAP (Rabbit monoclonal) | Cell Signaling | Cat#: 14074 | IHC (1:100) |
| antibody | YAP (Rabbit polyclonal) | Novus | Cat#: NB110-58358 | WB (1:1000) |
| antibody | Phospho-YAP (Rabbit monoclonal) | Cell Signaling | Cat#: 13008 | WB (1:1000) |
| antibody | RhoA (Rabbit monoclonal) | Cell Signaling | Cat#: 2117 | WB (1:1000) |
| antibody | GAPDH (Rabbit monoclonal) | Cell Signaling | Cat#: 2118 | WB (1:1000) |
| antibody | Ki67 (Rabbit monoclonal) | Thermo | Cat#: RM9106 | IHC (1:200) |
| antibody | Aurora B (AurkB) (Rabbit polyclonal) | Abcam | Cat#: ab2254 | IHC (1:100) |
| antibody | Histon H3 (Rabbit polyclonal) | Abcam | Cat#: ab1791 | WB (1:3000) |
| sequence- based reagent | Hmgcs2-F | This paper | qPCR primer | GAAGAGAGCGA TGCAGGAAAC |
| sequence- based reagent | Hmgcs2-R | This paper | qPCR primer | GTCCACATATT GGGCTGGAAA |
| sequence- based reagent | Nqo1-F | This paper | qPCR primer | AGGATGGGAG GTACTCGAATC |
| sequence- based reagent | Nqo1-R | This paper | qPCR primer | TGCTAGAGATG ACTCGGAAGG |
| sequence- based reagent | Pla2g4e-F | This paper | qPCR primer | AGGTGGAGTTC CTACTCGAAG |
| sequence- based reagent | Pla2g4e-R | This paper | qPCR primer | TGTTCTCGAAGG AGTCTGTCA |
| sequence- based reagent | Pla2g5-F | This paper | qPCR primer | CCAGGGGGCT TGCTAGAA |
| sequence- based reagent | Pla2g5-R | This paper | qPCR primer | AGCACCAATC AGTGCCATCC |
| sequence- based reagent | mtDN1-F | This paper | qPCR primer | CTCTTATCCACG CTTCCGTTACG |
| sequence- based reagent | mtDN1-R | This paper | qPCR primer | GATGGTGGTAC TCCCGCTGTA |
| sequence- based reagent | mtDN2-F | This paper | qPCR primer | CCCATTCCACT TCTGATTACC |
| sequence- based reagent | mtDN2-R | This paper | qPCR primer | ATGATAGTAGAG TTGAGTAGCG |
| sequence- based reagent | CTGF-F | This paper | qPCR primer | GGGCCTCTT CTGCGATTTC |
| sequence- based reagent | CTGF-R | This paper | qPCR primer | ATCCAGGCAAG TGCATTGGTA |
| sequence- based reagent | Ankrd1-F | This paper | qPCR primer | GGATGTGCCGA GGTTTCTGAA |
| sequence- based reagent | Ankrd1-R | This paper | qPCR primer | GTCCGTTTATAC TCATCGCAGAC |
| commercial assay or kit | Neonatal cardiomyocyte isolation kit | Cellutron | NC-6031 | |
| commercial assay or kit | RNeasy mini kit | Qiagen | 74104 | |
| chemical compound, drug | Epinephrine | Sigma | E4375 | 100 mM |
| chemical compound, drug | C3 | Cytoskeleton | CT04 | 1 mg/ml |
| chemical compound, drug | GO4 | Provided by Dr. Yi Zheng | 100 mM | |
| chemical compound, drug | Tamoxifen | Sigma | T5648 | 50 mg/kg, IP injection |
| chemical compound, drug | Metoprolol (b-blocker) | Sigma | M5391 | 2 mg/kg, IP injection |
| chemical compound, drug | 5-ethynyl-2- deoxyuridine (EdU) | Thermo | Cat#: C10337 | 5 mg/kg, IP injection |
| software, algorithm | ImageJ | National Institutes of Health (NIH) | ||
| other | 4',6-diamidino-2- phenylindole (DAPI) | Invitrogen | D1306 | For nuclear staining |
| other | Wheat germ agglutinin (WGA) | Invitrogen | W11261 | For plasma membrane staining |
| other | Collagenase B | Roche | 11088815001 | 1.8 mg/ml For heart digestion |
| other | Collagenase D | Roche | 11088866001 | 2.4 mg/ml For heart digestion |
| other | 2,3,5- Tripherylterazolium chloride (TTC) | Sigma | T8877 | For staining of ischemic region |
