Targeted ablation of PINCH1 and PINCH2 from murine myocardium results in dilated cardiomyopathy and early postnatal lethality - PubMed (original) (raw)

. 2009 Aug 18;120(7):568-76.

doi: 10.1161/CIRCULATIONAHA.109.864686. Epub 2009 Aug 3.

Yunfu Sun, Maoqing Ye, Maria C Scimia, Hongqiang Cheng, Jody Martin, Gang Wang, Ann Rearden, Chuanyue Wu, Kirk L Peterson, Henry C Powell, Sylvia M Evans, Ju Chen

Affiliations

Targeted ablation of PINCH1 and PINCH2 from murine myocardium results in dilated cardiomyopathy and early postnatal lethality

Xingqun Liang et al. Circulation. 2009.

Abstract

Background: PINCH proteins are 5 LIM domain-only adaptor proteins that function as key components of the integrin signaling pathway and play crucial roles in multiple cellular processes. Two PINCH proteins, PINCH1 and PINCH2, have been described in mammals and share high homology. Both PINCH1 and PINCH2 are ubiquitously expressed in most tissues and organs, including myocardium. Cardiac-specific PINCH1 knockout or global PINCH2 knockout mice exhibit no basal cardiac phenotype, which may reflect a redundant role for these 2 PINCH proteins in myocardium. A potential role for PINCH proteins in myocardium remains unknown.

Methods and results: To define the role of PINCH in myocardium, we generated mice that were doubly homozygous null for PINCH1 and PINCH2 in myocardium. Resulting mutants were viable at birth but developed dilated cardiomyopathy and died of heart failure within 4 weeks. Mutant hearts exhibited disruptions of intercalated disks and costameres accompanied by fibrosis. Furthermore, multiple cell adhesion proteins exhibited reduced expression and were mislocalized. Mutant cardiomyocytes were significantly smaller and irregular in size. In addition, we observed that the absence of either PINCH1 or PINCH2 in myocardium leads to exacerbated cardiac injury and deterioration in cardiac function after myocardial infarction.

Conclusions: These results demonstrate essential roles for PINCHs in myocardial growth, maturation, remodeling, and function and highlight the importance of studying the role of PINCHs in human cardiac injury and cardiomyopathy.

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Conflict of interest statement

Disclosures

The authors report no conflicts.

Figures

Fig. 1

Fig. 1. The expression pattern of PINCH1 and PINCH2

A: In Situ Hybridization detects PINCH1 in the Embryonic day (E) 9.5. B: Northern blotting (quantification shown in lower panel) reveals highest expression of PINCH1 in the adult mouse heart. C: In Situ Hybridization detects PINCH2 at E14.5. D: Northern blotting (quantification shown in lower panel) reveals highest expression of PINCH2 in the adult mouse heart. E, G: Immunstaining shows expression of PINCH1 in cardiomyocytes isolated from the new-born (E) and adult mice (G) and control section without PINCH1 antibody added shows negative staining. F, H: Immunstaining shows expression of PINCH2 in cardiomyocytes isolated from the new born (F) and adult mice (H) and control section without PINCH2 antibody added shows negative staining. α-actinin marks the myocytes. H: heart; B: brain; Sp: spleen; Lu: lung; Li: liver; Sk: skeletal muscle; K: kidney; T: thymus. RA: right atria; RV: right ventricle; LA: left atria; LV: left ventricle; VS: ventricular septum.

Fig. 2

Fig. 2. General analyses of PINCH CDKO

A: Growth retardation of the double knockouts. B: The survival curve shows that the double knockouts died within one month. C: Morphological and histological analyses show a dilated heart of the new born double knockouts compared with the control littermate. D: Morphological and histological analyses show cardiomyopathy with extensive fibrosis in the double knockouts at P20 compared with the wild type littermate (WT). Arrows indicate fibrosis; arrowheads demonstrate thrombosis.

Fig. 3

Fig. 3. Ultrastructural analysis of the CDKO

Representative electron micrographs from the ventricular myocardium of WT and CDKO at day 1 and day 10. A and B: At day 1, in a few focal regions, the gaps of intercalated discs (arrows) are observed to be widened with disarrayed sarcomeric structure and distorted Z-line. C–F: At day 10, wild type mice show preserved intercalated discs structures (arrows), and well-aligned arrays and insertions of myofibrils at Z lines (C and E); whereas CDKO mice show impaired intercalated discs (D, arrows), sarcomere and sarcolemmal connection and increased intercellular spaces (F arrowheads and double-head arrows). Bar: 2µm

Fig. 4

Fig. 4. Disrupted Integrin-ILK signaling in PINCH CDKO hearts

A and B: Deletion of PINCH1 and PINCH2 suppresses ILK and parvin expression in new-born cardiomyocytes (A) and in myocardium at day 10 (B), and reduction of integrinβ1 at day 10. Expression of Talin is unchanged (B), Quantifications of protein expression level are shown in lower panels. C and D: ILK expression is significantly reduced in the large area of CDKO myocardium (D) compared with WT (C). F-actin mainly stains the myocytes. E and F: The diffused and disorganized fibronectin expression with disarrayed F-actin is observed in the area close to aorta and pulmonary artery of myocardium in CDKO (F) compared with that of WT (E). G: Inactivation of PINCH1 and PINCH2 inhibits phosphorylation of Akt (Ser473). Quantifications are shown in right panels. H and I: Caspase 3 activation is significantly increase in myocardium of CDKO (I) compared with WT (H). Casp3: activated Caspase 3. In each case of the Western Blotting, each expression is normalized to GAPDH

Fig. 5

Fig. 5. Alteration of intercalated disc proteins in myocardium of CDKO at day 10

A: Western blot: The expression of Connexin43 (CNX43), α-E-cadherin (α-E-CAT) and ZO-1 are reduced in myocardium of CDKO at day 10 compared with WT (**p<0.01; *p<0.05), but expression levels of Cadherin (CAD), β-catenin (β-CAT) and vinculin (VIN) are not significantly changed Each expression is normalized to GAPDH expression and quantified in lower panel. B: Immunostaining: CNX43 (a, a’) and α-E-Catenin (b, b’) are reduced and mislocalized to the lateral side of CDKO cardiomyocytes. Mislocalized Cadherin (c, c’) and β-Catenin (d, d’) expression are seen in CDKO myocardium. The ZO-1 expression is reduced and absent in the intercalated discs of CDKO (e’) compared with WT (e). The Vinculin expression is absent in the intercalated discs of CDKO (f’) compared with WT (f).

Fig. 6

Fig. 6. Impaired spreading, focal adhesion and attachment in the cardiomyocytes of CDKO

A–F: Immunostaining on isolated cardiomyocytes from WT (A–C) and CDKO (D–F) newborn hearts shows that impaired spreading and focal adhesion (indication by arrows) in the CDKO myocytes. G: Attachment of the isolated myocytes from the newborn heart of CDKO is significantly (p<0.01) reduced compared with WT. H: The size of the isolated myocytes from the newborn heart of CDKO is significantly (p<0.01) smaller compared with WT. I and J: WGA staining on myocardium of WT (I) and CDKO (J) at day 10 shows that disarrayed myocytes with reduced various sizes is observed in CDKO. K: Quantitative analysis on the WGA staining myocardium shows that the size of CDKO myocytes is significantly smaller (p<0.01).

Fig. 7

Fig. 7. Either cardiac specific PINCH1 mutants or global PINCH2 knockouts show susceptibility to injury after myocardial infarction (MI)

A: MI in cardiac specific PINCH1 mutants (PINCH1 KO) (n=6) and controls (WT) (n=6) at 20 weeks. a and b: Macroscopic appearance of representative hearts of WT (a) and MUT (b) at 12 days post-MI. c and d: Representative trichrome stain of transverse heart sections at comparable levels 12 days after MI in WT(c) and PINCH1 KO (d). e: Echocardiogram of left ventricles 12 days after MI in WT and MUT. Left ventricular fractional shortening (FS) of PINCH1 KO hearts is significantly (p<0.01) decreased compared with WT. B: MI global PINCH2 knockouts (PINCH2 KO) (n=6) and WT (n=6) at 20 weeks. f and g: Macroscopic appearance of representative mouse hearts of WT (f) and PINCH2 KO (g). h and i: Representative trichrome stain of transverse heart sections in comparable levels at 12 days after MI in WT (h) and PINCH2 KO (i). j: Echocardiogram of left ventricles 12 day after MI in WT and PINCH1 KO. Left ventricular fractional shortening (FS) of PINCH2 KO hearts is significantly decreased compared with WT (p<0.01). Arrows indicate the damaged area of the control heart; arrowheads indicate the damaged area of the PINCH1 or PINCH2 mutant heart.

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