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.

Objective: To establish the transfection method of vascular endothelial growth factor (VEGF) gene into mesenchymal stem cells (MSCs), to investigate the effect of this gene-transfected MSCs for heart function restoration and angiogenesis after myocardial infarction, and to compare the therapeutic differences among cell therapy, gene therapy, and combined therapy. Methods: Ischemic heart models were constructed in inbred Wistar rats by ligation of the left anterior descending coronary artery. MSCs of Wistar rats were isolated by density gradient centrifugation and purified on the basis of their ability to adhere to plastic, and identified by checking the surface markers and their differentiation capacity, and then followed by transfection of pcDNA_3.1-hVEGF₁₆₅ using the liposome-mediated method. The expression of hVEGF₁₆₅ in the transfected cells was detected by Enzyme-Linked Immunosorbent Assay, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Western Blot Analysis. The ligated animals were randomly divided into four groups (12 in each) and, after 2 weeks, were injected at the heart infarct zone with hVEGF₁₆₅-transfected MSCs (Combo group), MSCs (Cell group), liposome-hVEGF gene plasmid (Gene group), or medium (Control group). And other six ligated rats (without any injection) were used as Model-assessment group for the baseline heart infarcted size evaluation, and other 12 non-ligated rats (Non-ischemic group) were used as the normal control. Four weeks after the injection, the rats’ cardiac function was measured by the Buxco system. Brdu and Troponin-T double labeling and factor VIII were identified by immunohistochemical staining to demonstrate the survival and differentiation of engrafted cells or to evaluate the angiogenesis in the injured heart area; heart infarcted size was calculated by Evan’s blue staining. VEGF expression was evaluated by RT-PCR. Results: MSCs can be successfully isolated and cultured by density gradient centrifugation followed by adherence-separation. The cultured MSCs were CD34–, CD45–, CD44+ and SH+. They can differentiate into osteoblasts and adipocytes successfully. The expression of hVEGF₁₆₅ in the transfected MSCs was demonstrated with Enzyme-Linked Immunosorbent Assay, RT-PCR and Western Blot Assay. Four weeks after the cells were transplanted, among all groups but the Non-ischemic group, the Combo group had the smallest heart infarcted size and the best heart function. The capillary density of the Combo group was significantly greater than those of both Cell and Control groups. The heart infarcted size, heart function and capillary density of both Cell and Gene groups were similar with each other and smaller, better and greater than those of the Control group, respectively. Brdu and Troponin-T double staining detected a varied increase in the number of survived cardiomyoctyes at the heart infarcted area, some of which were double stain positive. RT-PCR showed that the hVEGF₁₆₅ gene was expressed in the...

Ezh2 is a histone trimethyltransferase that silences genes mainly via catalyzing trimethylation of histone 3 lysine 27 (H3K27Me3). The role of Ezh2 as a regulator of gene silencing and cell proliferation in cancer development has been extensively investigated; however, its function in heart development during embryonic cardiogenesis has not been well studied. In the present study, we used a genetically modified mouse system in which Ezh2 was specifically ablated in the mouse heart. We identified a wide spectrum of cardiovascular malformations in the Ezh2 mutant mice, which collectively led to perinatal death. In the Ezh2 mutant heart, the endocardial cushions (ECs) were hypoplastic and the endothelial-to-mesenchymal transition (EMT) process was impaired. The hearts of Ezh2 mutant mice also exhibited decreased cardiomyocyte proliferation and increased apoptosis. We further identified that the Hey2 gene, which is important for cardiomyocyte proliferation and cardiac morphogenesis, is a downstream target of Ezh2. The regulation of Hey2 expression by Ezh2 may be independent of Notch signaling activity. Our work defines an indispensible role of the chromatin remodeling factor Ezh2 in normal cardiovascular development.

We have previously found that in failing human hearts, Rho-associated coiled-coil protein kinase 1 (ROCK1) is processed by caspase-3 into an active isoform, ROCKΔ1. The purpose of the current investigation was to elucidate the pathological consequences of truncated ROCK1 accumulation in the heart, the associated molecular mechanism of ROCKΔ1-mediated cardiac phenotype, and the molecular signaling between Rho kinase activation in cardiomyocytes and extracellular matrix response. We generated transgenic mice expressing ROCKΔ1 in cardiomyocytes to mimic the situation observed in human heart disease, whereas an additional kinase-deficient mouse was generated as a control. The ROCKΔ1 transgenic mice developed fibrotic cardiomyopathy with diastolic dysfunction. Transgenic hearts displayed activated TGFβ1 and NF-κB signaling and a release of a subset of cytokines and were susceptible to angiotensin II stress. Treatment with a Rho kinase inhibitor attenuated the fibrotic phenotype. Cardiac fibroblasts differentiated into myofibroblasts when cocultured with transgenic cardiomyocytes but not with wild-type cardiomyocytes. Inhibitors of Rho kinase as well as TGFβR1 and NF-κB decreased these effects. The serum response factor-dependent TGFβ1 regulation was shown to be responsible for the Rho kinase-mediated activation of TGFβ1 signaling. We conclude that ROCKΔ1 is a novel fibrotic factor. Activation of TGFβ1 and NF-κB signaling contributes to the Rho kinase-mediated pathological fibrosis.

Nkx2.5 is a cardiac specific homeobox gene critical for normal heart development. We previously identified Nkx2.5 as a target of sumoylation, a posttranslational modification implicated in a variety of cellular activities. Sumoylation enhanced Nkx2.5 activity via covalent attachment to the lysine residue 51, the primary SUMO acceptor site. However, how sumoylation regulates the activity of Nkx2.5 in vivo remains unknown. We generated transgenic mice overexpressing sumoylation deficient mutant K51R (conversion of lysine 51 to arginine) specifically in mouse hearts under the control of cardiac α-myosin heavy chain (α-MHC) promoter (K51R-Tg). Expression of the Nkx2.5 mutant transgene in the wild type murine hearts did not result in any overt cardiac phenotype. However, in the presence of Nkx2.5 haploinsufficiency, cardiomyocyte-specific expression of the Nkx2.5 K51R mutant led to congenital heart diseases (CHDs), accompanied with decreased cardiomyocyte proliferation. Also, a number of human CHDs-associated Nkx2.5 mutants exhibited aberrant sumoylation. Our work demonstrates that altered sumoylation status may underlie the development of human CHDs associated with Nkx2.5 mutants.

ZIC3, an X-linked zinc finger transcription factor, was the first identified gene involved in establishing normal left-right patterning in humans. Mutations in the Zic3 gene in patients cause heterotaxy, which includes congenital heart defects. However, very little is known about how the function of the ZIC3 protein is regulated. Sumoylation is a posttranslational modification process in which a group of small ubiquitin-like modifier (SUMO) proteins is covalently attached to targets via a series of enzymatic reactions. Here, we report for the first time that sumoylation targets human ZIC3 primarily on the consensus lysine residue K248, which is critical for the nuclear retention of ZIC3. Consequently, SUMO modification potentiates the repressive activity of ZIC3 on the promoter of its target gene cardiac α-actin, and the mutation of lysine 248 to arginine (K248R) abolishes its repressive function. We further revealed that ZIC3 variants with mutations found in human patients with congenital anomalies exhibit aberrant sumoylation activity, which at least partially accounts for their cytoplasmic diffusion. Improved sumoylation of human disease-associated ZIC3 variants reestablishes their nuclear occupancy in the presence of SUMO E3 ligase and SUMO-1. Thus, the altered sumoylation status of ZIC3 underpins the developmental abnormalities associated with these ZIC3 mutants. The SUMO targeting consensus sequence in ZIC3 is highly conserved in its paralogs and orthologs, pointing to sumoylation as a general mechanism underlying the functional control of ZIC proteins. This study provides a potential therapeutic strategy to regain the normal subcellular distribution and function of ZIC3 mutants by restoring SUMO conjugation.

Protein tyrosine phosphatase-like A (PTPLa) has been implicated in skeletal myogenesis and cardiogenesis. Mutations in PTPLa correlated with arrhythmogenic right ventricular dysplasia in humans and congenital centronuclear myopathy with severe hypotonia in dogs. The molecular mechanisms of PTPLa in myogenesis are unknown. In this report, we demonstrate that PTPLa is required for myoblast growth and differentiation. The cells lacking PTPLa remained immature and failed to differentiate into mature myotubes. The repressed MyoG expression was responsible for the impaired myoblast differentiation. Meanwhile, impeded cell growth, with an obvious S-phase arrest and compromised G 2 /M transition, was observed in PTPLa-deficient myoblasts. Further study demonstrated that the upregulation of cyclin D1 and cyclin E2 complexes, along with a compromised G 2 /M transition due to the decreased CDK1 (cyclin-dependent kinase 1) activity and upregulated p21, contributed to the mutant cell S-phase arrest and eventually led to the retarded cell growth. Finally, the transcriptional regulation of the PTPLa gene was explored. We identified PTPLa as a new target gene of the serum response factor (SRF). Skeletal-and cardiac-muscle-specific SRF knockouts resulted in significant decreases in PTPLa expression, suggesting a conserved transcriptional regulation of the PTPLa gene in mice. Skeletal myogenesis involves multiple processes in which undifferentiated myoblasts proliferate, withdraw from the cell cycle, and differentiate into mononucleated myocytes followed by a subsequent fusion of myocytes into multinucleated myotubes. The latter are assembled into mature muscle fibers along with the expression of muscle-specific proteins. The multistep process is tightly regulated in order to secure normal myogenesis development. Extensive studies that have focused on myogenic transcriptional regulation revealed four essential myogenic regulatory factors (MRFs), MyoD (17), MyoG (myogenin) (20, 65), Myf5 (muscle regulatory factor 5) (11), and MRF4 (muscle regulatory factor 4) (10,47,55). These factors function coordinately at different stages of muscle cell fate during development and play crucial roles in myogenesis. In comparison with myogenic transcriptional regulation, there have been far fewer studies of posttranslational regulation of myogenesis. Accumulating evidence has begun to reveal that tyrosyl phosphorylation and its opposite, dephosphorylation, are important regulatory components during myogenic progression. Several representative studies have examined focal adhesion kinase (FAK), a nonreceptor tyrosine kinase also known as protein tyrosine kinase 2 (53, 54), phosphatidylinositol 3=-kinase (PI3K) (16, 30), phosphoinositide phosphatase myotubularin, and protein tyrosine phosphatase 32,33).Protein tyrosine phosphatase-like A (PTPLa) is a protein tyrosine phosphatase in which the active motif (I/V)HCXXGXXP (S/T) contains an arginine-to-proline replacement (indicated by boldface) (61). While the significance of this substitution remains ...

Acylglycerol kinase (AGK) had been shown to contribute to cancer progression and unfavorable clinical outcomes of patients. Our study aimed to investigate the expression pattern and clinical significance of AGK in patients with early-stage cervical squamous cell cancer (CSCC). The protein and messenger RNA (mRNA) expression of AGK was analyzed in six cervical cancer cell lines and four paired early-stage CSCC specimens and normal cervical tissues (NCT), using Western blotting and real-time PCR (RT-PCR). And we investigated the AGK protein expression in paraffin-embedded specimens from 140 patients with early-stage CSCC and 30 cases of NCT by immunohistochemistry (IHC). Statistical analyses were performed to evaluate the clinicopathological significance of AGK expression. The expressions of AGK protein and mRNA were significantly up-regulated in cervical cancer cell lines and cancer tissues. IHC analyses revealed that AGK was highly expressed in 93 (66.4 %) of 140 early-stage CSCC specimens, but in none of the NCT. Moreover, AGK expression in early-stage CSCC was significantly correlated with tumor stage (P < 0.001), tumor size (P < 0.001), and tumor type (P < 0.001). Early-stage CSCC patients with high AGK expression level had shorter progress-free survival (PFS) and overall survival (OS) time compared with patients with low AGK expression levels. Univariate and multivariate analyses identified AGK expression level as an independent prognostic factor for survival of early-stage CSCC patients. We showed that AGK was over-expressed in cervical cancer cell lines and clinical tissues, and over-expression of AGK was associated with poor survival outcomes of early-stage CSCC patients. AGK can be used as an independent prognostic marker for early-stage CSCC.