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. 2012 Mar;52(3):638-49.
doi: 10.1016/j.yjmcc.2011.11.011. Epub 2011 Dec 1.

Enhanced desumoylation in murine hearts by overexpressed SENP2 leads to congenital heart defects and cardiac dysfunction

Eun Young Kim  1 , Li Chen, Yanlin Ma, Wei Yu, Jiang Chang, Ivan P Moskowitz, Jun Wang
Affiliations

Enhanced desumoylation in murine hearts by overexpressed SENP2 leads to congenital heart defects and cardiac dysfunction

Eun Young Kim et al. J Mol Cell Cardiol. 2012 Mar.

Abstract

Sumoylation is a posttranslational modification implicated in a variety of cellular activities, and its role in a number of human pathogeneses such as cleft lip/palate has been well documented. However, the importance of the SUMO conjugation pathway in cardiac development and functional disorders is newly emerging. We previously reported that knockout of SUMO-1 in mice led to congenital heart diseases (CHDs). To further investigate the effects of imbalanced SUMO conjugation on heart development and function and its underlying mechanisms, we generated transgenic (Tg) mice with cardiac-specific expression of SENP2, a SUMO-specific protease that deconjugates sumoylated proteins, to evaluate the impact of desumoylation on heart development and function. Overexpression of SENP2 resulted in premature death of mice with CHDs-atrial septal defects (ASDs) and/or ventricular septal defects (VSDs). Immunobiochemistry revealed diminished cardiomyocyte proliferation in SENP2-Tg mouse hearts compared with that in wild type (WT) hearts. Surviving SENP2-Tg mice showed growth retardation, and developed cardiomyopathy with impaired cardiac function with aging. Cardiac-specific overexpression of the SUMO-1 transgene reduced the incidence of cardiac structural phenotypes in the sumoylation defective mice. Moreover, cardiac overexpression of SENP2 in the mice with Nkx2.5 haploinsufficiency promoted embryonic lethality and severity of CHDs, indicating the functional interaction between SENP2 and Nkx2.5 in vivo. Our findings indicate the indispensability of a balanced SUMO pathway for proper cardiac development and function. This article is part of a Special Issue entitled 'Post-translational Modification SI'.

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Figures

Figure 1
Figure 1. SENP2 gene was expressed in mouse heart during embryogenesis
A. Semi-quantitative RT-PCR was performed on RNA samples purified from embryonic hearts at various developmental stages (E9.5, E10.5 and E11.5) to detect SENP2 mRNA. PCR for each sample was carried out with two cycles (30 and 35, respectively). GAPDH served as a loading control. B–D'. Immunostaining shows SENP2 expression in hearts at later developmental stages. Hearts of three embryonic stages, E14.5 (B–B'), E16.5 (C–C'), and E17.5 (D–D') were examined. Panel B, C & D show single SENP2 staining, while B', C' & D' display the merged images of three stainings: SENP2 (red), Cav3 (green, positive for cardiomyocyte), and DAPI (blue, for nucleus). The arrows in B & B' show examples of non-nuclear staining of SENP2, and the dotted staining (arrowheads) indicates blood cells. Note that in the compact zone, SENP2 was barely detected at E14.5, but was detectable at E16.5 (C & C', arrows) and became abundant at E17.5, indicating developmental and spatial regulation of SENP2 expression during cardiogenesis.
Figure 2
Figure 2. Generation of SENP2 transgenic (Tg) mice
A. Schematic representation of the construct for generation of SENP2-Tg mouse lines. B. Transcripts and protein level of transgenic flag-tagged human SENP2 were detected in the SENP2-Tg mouse hearts but not in the WT hearts. Upper panel: Reverse transcription followed by PCR was performed on total RNA purified from atria and ventricles of E16.5 WT and SENP2-Tg mouse hearts (lines #2837 and #2839) to detect mRNA of flag-tagged SENP2 and GAPDH (as a control) using specific oligos. Lower panel: Western blot was performed on lysates extracted from P14 WT and SENP2-Tg mouse hearts from both lines to detect flag-tagged SENP2 in SENP2-Tg hearts. Arrows indicate flag-tagged SENP2. C. Suppressed conjugation of SUMO-1 (upper panel) and SUMO-2/3 (middle panel) in SENP2-Tg mouse hearts by overexpression of SENP2. GAPDH served as a loading control (lower panel). HMW, high molecular weight SUMO conjugates. D. Summary of transmission at F1 and F2 generations of both SENP2-Tg lines. Note that the frequency at P1 of F1 and F2 from both lines was lower than the expected Mendelian rate of 50%.
Figure 3
Figure 3. SENP2-Tg mice exhibited congenital heart diseases
A. Global morphological view of hearts from one of the demised SENP2-Tg mice and a WT littermate control at P2. Scale bar = 1 mm. B. Histological evaluations revealed ASDs/VSDs in SENP2-Tg mice (middle and right panels) in comparison to littermate control (left panel) at P4. Arrows indicate location of ASD (middle panel) and VSD (right panel), respectively, with the following abbreviations: RA, right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle; ASD, atrial septal defect; VSD, ventricular septal defect. C. Summary of incidence of cardiac defects in the demised SENP2-Tg mice. D. RT-qPCR analysis of RNA samples isolated from E16.5 WT and SENP2-Tg hearts revealed no significant changes in the transcription levels of genes indicated between WT and SENP2-Tg mice. E. RT-qPCR revealed no significant changes in the transcription levels of SUMO factors in E16.5 hearts between the WT and SENP2-Tg mice. n=4 for each group.
Figure 4
Figure 4. SENP2-Tg mice exhibited growth retardation
A. Comparative body size of a SENP2-Tg mouse (line #2839) and a WT littermate at P31. B. Heart weight (HW) and body weight (BW) of SENP2-Tg mice (n=4) at P18 were significantly lower than those of littermates (n=5, P<0.001 and 0.00001, respectively), but cardiac index HW/BW ratio showed no significant difference (P=0.66). C. Non-invasive cardiac functional analysis of WT and SENP2-Tg mice at P40 using echocardiography showed thinner LVPW in diastole, reduced stroke volume and cardiac output in SENP2-Tg mice. Abbreviations: LVPW, left ventricular posterior wall; d, diastole; s, systole. D. RT-qPCR on RNA samples purified from both WT and SENP2-Tg mouse hearts at P18 revealed down-regulation of transcripts of a number of contractile proteins. *, P<0.05; **, P<0.01. The unpaired Student's t test was applied to determine statistical significance between groups.
Figure 5
Figure 5. Hearts of aging SENP2-Tg mice were hypertrophic and fibrotic
A–C. SENP2-Tg mice exhibited smaller cardiomyocyte size compared with WT mice at embryonic stages. Representative WGA-TRITC staining of E14.5 heart sections (A–B) and statistical analysis (C) are shown. Surface areas were measured in 10–20 randomly selected fields from each individual heart sample (n=5 per group for WT and SENP2-Tg mice) using Software ImageJ (National Institutes of Health). Bar, 500 μm. *, p<0.05. The average surface area calculated for WT cardiomyocytes was taken as 100%. Magnification, 40×. D–F. SENP2-Tg mice developed cardiac hypertrophy with aging. Sections of hearts from mice with over one year of age were WGA-TRITC stained and the surface areas were measured as described above (n=4 per group for WT and SENP2-Tg mice). Representative data are shown in D–E and statistical analysis is shown in F. Bar, 200 μm. **, p<0.001. Magnification, 20×. G–H. Aging SENP2-Tg mouse heart presented fibrosis (Masson's trichrome staining). Bar, 200 μm. Magnification, 20×.
Figure 6
Figure 6. Defect in cardiomyocyte proliferation in SENP2-Tg mice
A–C: DNA synthesis of cardiomyocyte was decreased in SENP2-Tg mice at E15.5 embryonic stage. Representative data from BrdU staining on E15.5 heart sections (A–B) and statistical analysis (C) are shown. Statistics in C represents the scores of 100 cells per section. A–B, 40× magnification. n=3 per group. **, p<0.001. D–F: SENP2-Tg mouse hearts exhibited decreased BrdU positive cardiomyocytes at age of over one year. Representative BrdU staining of heart sections from aging WT and SENP2-Tg mice (D–E) and statistical analysis (F) are shown. Scale bar, 100 μm. Statistics in F represents the scores of 5 randomly selected fields per section. D–E, 20× magnification. n=3 for each group. **, p<0.001. G–I: SENP2-Tg mouse hearts exhibited decreased Ki67 positive cardiomyocytes at E14.5 embryonic stage. Representative data from Ki67 staining on E14.5 heart sections (G–H) and statistical analysis (I) are shown. Statistical analysis in panel I represents the average scores of 100 cells per section. G–H, 40× magnification. Scale bar, 50μm. ++, p<0.001. J–L: SENP2-Tg mouse hearts exhibited decreased Ki67 positive cardiomyocytes at age of over one year. Representative data from Ki67 staining on sections of aging WT and SENP2-Tg hearts (J–K) and statistical analysis (L) are shown. Statistical results in L represent the scores from at least four randomly selected fields per section. J–K, 20× magnification. n=3 per group. ++, p<0.001. Arrows in D, E, J and K indicate BrdU (panel D–E) or Ki67 (panel J–K) positive cells.
Figure 7
Figure 7. SUMO-1 played a critical role in the development of cardiac structural malformation in SENP2-Tg mice
A. Overexpression of SENP2 in the presence of SUMO-1 haploinsufficiency led to embryonic lethality. The compound SENP2-Tg/SUMO-1+/− mice obtained from crossbreeding between SENP2-Tg mice (line #2839) and SUMO-1+/− mice were present at lower P1 frequency than the expected Mendelian rate (25%). The P1 frequency was analyzed from 7 litters comprising a total of 49 animals. The number shown above each bar indicates the P1 frequency of each corresponding group. **, P<0.01. Chi-square test was used for statistical analysis. B. The transcripts of transgene flag-tagged SUMO-1 were detected in both atria and ventricles of SUMO-1-Tg mouse hearts at E16.5 but not in the WT littermate hearts. GAPDH was used as a control. C. Mortality rate of double SUMO-1-Tg/SENP2-Tg mice was substantially decreased compared with that of single SENP2-Tg mice. Rescue data from line #2839 were compiled from 15 litters of 97 animals in total. Fisher's Exact Probability Test was used for statistical analysis. D. Improved modification of SUMO-1 in double SUMO-1-Tg/SENP2-Tg mouse heart. Western blots were performed on heart extracts from the WT, SENP2-Tg, SUMO-1-Tg and double SUMO-1-Tg/SENP2-Tg mice. Compare SUMO-1 conjugation (upper panel) in lane 2 and lane 4. GAPDH (lower panel) served as a control. E. Decreased penetrance of cardiac defects in the double SUMO-1-Tg/SENP2-Tg mice. The data were compiled from 76 available animals of 10 litters with ages ranging from E14.5 to P2. Fisher's Exact Probability Test was used for statistical analysis. The number shown above each bar indicates the incidence of cardiac defects of that corresponding group. F–G. Improved cardiomyocyte proliferation in the double SUMO-1-Tg/SENP2-Tg mouse hearts. BrdU staining was performed on sections of E14.5 embryonic hearts of the WT, SUMO-1-Tg, SENP2-Tg and double SUMO-1-Tg/SENP2-Tg mice, respectively (F) and statistical analysis was shown in G. n=4 for each group. Representative data were shown. Magnification, 20×. Bar, 200 μm.
Figure 8
Figure 8. Desumoylation of SUMO substrates in SENP2-Tg hearts
Co-immunoprecipitation (IP) was performed on the whole tissue extracts from age-matched WT and SENP2-Tg mouse hearts using agarose-conjugated anti-SUMO-1 antibody for pulldown; subsequently, immunoblot (IB) was performed using anti-RanGAP1 (A) or anti-GATA4 antibodies (B) for detection. Arrows indicate SUMO-1-conjugated RanGAP1 (A) and SUMO-1-conjugated GATA4 (B). Note the substantial decrease in the level of SUMO-1 conjugated RanGAP1 and GATA4 in SENP2-Tg heart compared with those in WT heart.
Figure 9
Figure 9. Functional interaction between the SUMO pathway component, SENP2, and Nkx2.5
A. SENP2 wt, but not the catalytic mutant, deconjugated SUMO-1-Nkx2.5 conjugates. Sumoylation assays were performed on HeLa cell extracts containing overexpressed proteins, as indicated. Upper panel: blotted with anti-Nkx2.5 antibody to detect both free and conjugated Nkx2.5 (arrows); lower panel: blotted with anti-flag antibody to detect SENP2 wt and mutant. B. SENP2 wt, but not the catalytic mutant, suppressed Nkx2.5 activity in a dose-dependent manner. Transactivation assays were performed on HeLa cells transfected with the ANF-Luc construct together with single or combined expression of vectors, as indicated. Relative activation level (fold change) was calculated based on the luciferase activity of the control (empty vector alone), which was taken as 1. The dosage for each of those expression vectors were used as follows: Nkx2.5: 0.5 μg; SUMO-1: 0.75 μg; SENP2 wt: 50 and 100 ng; SENP2 mutant: 50 and 100 ng. The number shown above each bar indicates the fold change of the luciferase activity of that particular group. C. Down-regulation of a number of Nkx2.5 target genes in SENP2-Tg hearts was shown in comparison with those of WT hearts. The microarray assays of RNA samples from E16.5 SENP2-Tg and WT hearts were performed as described in the Materials and Methods. n=3 per group, with each sample carried out in duplicate. D–E. SENP2-Tg/Nkx2.5+/− mice exhibited a lower P1 frequency (D) and a higher incidence of cardiac defects, including the dual ASD/VSD phenotype (E), compared with those of single Nkx2.5+/− and SENP2-Tg mice. Mice were obtained from crossbreeding between SENP2-Tg mice of line #2839 and Nkx2.5+/− mice. The data were then compiled from 13 litters comprising a total of 75 animals (D) and from 7 litters comprising a total of 34 embryos with developmental stages ranging from E16.5 to E17.5 (E). The total animal number (n) of each genotype group analyzed in E was shown. Chi-square test was used for statistical comparison. *, p<0.05; ++, p<0.005.
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