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Cell-specific ablation of Hsp47 defines the collagen-producing cells in the injured heart
Hadi Khalil, … , Kazuhiro Nagata, Jeffery D. Molkentin
Hadi Khalil, … , Kazuhiro Nagata, Jeffery D. Molkentin
Published July 2, 2019
Citation Information: JCI Insight. 2019;4(15):e128722. https://doi.org/10.1172/jci.insight.128722.
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Research Article Cardiology

Cell-specific ablation of Hsp47 defines the collagen-producing cells in the injured heart

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Abstract

Collagen production in the adult heart is thought to be regulated by the fibroblast, although cardiomyocytes and endothelial cells also express multiple collagen mRNAs. Molecular chaperones are required for procollagen biosynthesis, including heat shock protein 47 (Hsp47). To determine the cell types critically involved in cardiac injury–induced fibrosis the Hsp47 gene was deleted in cardiomyocytes, endothelial cells, or myofibroblasts. Deletion of Hsp47 from cardiomyocytes during embryonic development or adult stages, or deletion from adult endothelial cells, did not affect cardiac fibrosis after pressure overload injury. However, myofibroblast-specific ablation of Hsp47 blocked fibrosis and deposition of collagens type I, III, and V following pressure overload as well as significantly reduced cardiac hypertrophy. Fibroblast-specific Hsp47-deleted mice showed lethality after myocardial infarction injury, with ineffective scar formation and ventricular wall rupture. Similarly, only myofibroblast-specific deletion of Hsp47 reduced fibrosis and disease in skeletal muscle in a mouse model of muscular dystrophy. Mechanistically, deletion of Hsp47 from myofibroblasts reduced mRNA expression of fibrillar collagens and attenuated their proliferation in the heart without affecting paracrine secretory activity of these cells. The results show that myofibroblasts are the primary mediators of tissue fibrosis and scar formation in the injured adult heart, which unexpectedly affects cardiomyocyte hypertrophy.

Authors

Hadi Khalil, Onur Kanisicak, Ronald J. Vagnozzi, Anne Katrine Johansen, Bryan D. Maliken, Vikram Prasad, Justin G. Boyer, Matthew J. Brody, Tobias Schips, Katja K. Kilian, Robert N. Correll, Kunito Kawasaki, Kazuhiro Nagata, Jeffery D. Molkentin

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Figure 1

Cardiomyocyte-specific deletion of Hsp47 in the mouse heart does not block maladaptive fibrosis with TAC.

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Cardiomyocyte-specific deletion of Hsp47 in the mouse heart does not blo...
(A) Representative immunostaining for Hsp47 (red fluorescence) in cultured cardiac fibroblasts 72 hours after infection with Adβgal and AdCre. Nuclei are stained blue with DAPI. Scale bar: 100 μm. (B) Western blot showing levels of secreted collagen type I in the culture media of heart fibroblasts 72 hours after infection with either Adβgal or AdCre. White arrows show collagen isoforms. Molecular weight migration standard and sizes are also shown. Nonspecific (n.s.) Ponceau staining (pink) is shown as a processing and loading control. (C) Schematic representation of breeding βMHC-Cre–transgenic mice with Hsp47-loxP–targeted mice. (D) Western blot analysis for Hsp47 isolated from fractionated cardiomyocytes of the 2 genotypes of mice shown. Gapdh is shown as the loading control. (E and F) Picrosirius red–stained histological heart sections, and quantitation of the area of fibrosis (red) in hearts from the indicated genotypes of mice with the βMHC-Cre transgene after 4 weeks of TAC injury. Average fibrotic area ± SEM, n = 5–8 mice in each group, *P < 0.05 versus sham-operated βMHC-Cre mice. P values were calculated by 1-way ANOVA with Tukey’s post hoc test. Scale bar: 200 μm (G) Heart-weight-to-body-weight (HW/BW) ratio in mice after 4 weeks of TAC. n = 5–8 in each group. *P < 0.05 versus βMHC-Cre sham mice. (H) Breeding scheme of αMHC-MCM–transgenic mice with Hsp47-loxP–targeted mice. (I) Experimental regimen whereby mice were subjected to TAC injury or sham procedure for 4 weeks along with tamoxifen treatment by injection (vertical red arrows). (J) Western blot analysis for Hsp47 isolated from adult heart fractionated cardiomyocytes of the 2 genotypes shown. Gapdh is shown as a loading control. (K, L, and M) Quantitation of collagens type I, III, and V, respectively, from immunohistochemical heart images from WT αMHC-MCM mice versus Hsp47 cardiomyocyte-specific mice, as shown in Supplemental Figure 3. Ten random histological sections from each mouse heart were imaged and quantified from 5–10 mice each per group. *P < 0.05 versus αMHC-MCM Sham. P values were calculated with 1-way ANOVA with Tukey’s post hoc test.

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