Sumoylation-deficient phosphoglycerate mutase 2 impairs myogenic differentiation.

Phosphoglycerate mutase 2 (PGAM2) is a critical glycolytic enzyme that is highly expressed in skeletal muscle. In humans, naturally occurring mutations in Phosphoglycerate mutase 2 have been etiologically linked to glycogen storage disease X (GSDX). Phosphoglycerate mutase 2 activity is regulated by several posttranslational modifications such as ubiquitination and acetylation. Here, we report that Phosphoglycerate mutase 2 activity is regulated by sumoylation-a covalent conjugation involved in a wide spectrum of cellular events. We found that Phosphoglycerate mutase 2 contains two primary SUMO acceptor sites, lysine (K)49 and K176, and that the mutation of either K to arginine (R) abolished Phosphoglycerate mutase 2 sumoylation. Given that K176 is more highly evolutionarily conserved across paralogs and orthologs than K49 is, we used the CRISPR-mediated homologous recombination technique in myogenic C2C12 cells to generate homozygous K176R knock-in cells (PGAM2K176R/K176R). Compared with wild-type (WT) C2C12 cells, PGAM2K176R/K176R C2C12 cells exhibited impaired myogenic differentiation, as indicated by decreased differentiation and fusion indexes. Furthermore, the results of glycolytic and mitochondrial stress assays with the XF96 Extracellular Flux analyzer revealed a reduced proton efflux rate (PER), glycolytic PER (glycoPER), extracellular acidification rate (ECAR), and oxygen consumption rate (OCR) in PGAM2K176R/K176R C2C12 cells, both at baseline and in response to stress. Impaired mitochondrial function was also observed in PGAM2K176R/K176R P19 cells, a carcinoma cell line. These findings indicate that the PGAM2-K176R mutation impaired glycolysis and mitochondrial function. Gene ontology term analysis of RNA sequencing data further revealed that several downregulated genes in PGAM2K176R/K176R C2C12 cells were associated with muscle differentiation/development/contraction programs. Finally, PGAM2 with either of two naturally occurring missense mutations linked to GSDX, E89A (conversion of glutamic acid 89 to alanine) or R90W (conversion of arginine 90 to tryptophan), exhibited reduced Phosphoglycerate mutase 2 sumoylation. Thus, sumoylation is an important mechanism that mediates Phosphoglycerate mutase 2 activity and is potentially implicated in Phosphoglycerate mutase 2 mutation-linked disease in humans.

Contribution of Increased Expression of Yin Yang 2 to Development of Cardiomyopathy.

Yin Yang 2 (YY2) is a member of the Yin Yang family of transcription factors. Although the bioactivity of YY2 has been previously studied, its role in cardiovascular diseases is not known. We observed the increased expression of YY2 in failing human hearts compared with control hearts, raising the question of whether YY2 is involved in the pathogenesis of cardiomyopathy. To investigate the potential contribution of YY2 to the development of cardiomyopathy, we crossed two independent transgenic (Tg) mouse lines, pCAG-YY2-Tg+and alpha-myosin heavy chain-cre (α-MHC-Cre), to generate two independent double transgenic (dTg) mouse lines in which the conditional cardiomyocyte-specific expression of YY2 driven by the α-MHC promoter was mediated by Cre recombinase, starting at embryonic day 9.0. In dTg mice, we observed partial embryonic lethality and hearts with defective cardiomyocyte proliferation. Surviving dTg mice from both lines developed cardiomyopathy and heart failure that occurred with aging, showing different degrees of severity that were associated with the level of transgene expression. The development of cardiomyopathy was accompanied by increased levels of cardiac disease markers, apoptosis, and cardiac fibrosis. Our studies further revealed that the Cre-mediated cardiomyocyte-specific increase in YY2 expression led to increased levels of Beclin 1 and LC3II, indicating that YY2 is involved in mediating autophagic activity in mouse hearts in vivo. Also, compared with control hearts, dTg mouse hearts showed increased JNK activity. Because autophagy and JNK activity are important for maintaining cardiac homeostasis, the dysregulation of these signaling pathways may contribute to YY2-induced cardiomyopathy and heart failure in vivo.

Colon adenocarcinoma after jejunoileal bypass for morbid obesity.

Jejunoileal bypass (JIB) was developed as a surgical treatment for morbid obesity in the early 1950s. However, this procedure is now known to be associated with multiple metabolic complications and has subsequently been abandoned as a viable bariatric procedure. Some of these known complications include renal stone formation, liver failure, migratory arthritis, fat-soluble deficiencies, blind-loop syndrome and severe diarrhea. Additionally, there have been animal models suggesting colon dysplasia after JIB. To our knowledge however, in humans, no colon cancers have been attributed to JIB in the literature. Here we report a 63-year-old morbidly obese female who had a JIB surgery in 1973 and subsequently was found to have numerous sessile colonic polyps throughout her colon and adenocarcinoma of the ascending colon without any family history of colonic polyposis syndromes or colon cancer.

Left Ventricular Aneurysm Repair with Endoaneurysmorrhaphy Technique: An Assessment of Two Different Ventriculotomy Closure Methods.

BACKGROUND: Left ventricular aneurysm is a serious mechanical complication of myocardial infarction and has an incidence of 10-35% after myocardial infarction. Ventricular aneurysm in patients with angina, heart failure, and ventricular arrhythmia should be surgically treated. Endoaneurysmorrhaphy is one of the repair techniques that results in better left ventricular geometry and function. After this surgical procedure the ventriculotomy is repaired either with Teflon felt strips or by direct suture of the epicardium. METHODS: In this study, we described the postoperative early outcomes of two ventriculotomy closing techniques such as Teflon felt versus direct closure after aneurysm repair. This retrospective study included a total of 73 patients (mean age > 70 years) with left ventricular aneurysm, who underwent endoaneurysmorrhaphy repair between 1997 and 2009. All selected patients were divided into two groups according to the ventriculotomy closure technique either by Teflon felt or direct by epicardial closure. The pre-, intra-, and postoperative results of these patients were analyzed accordingly. RESULTS: The postoperative early mortality rate and postoperative bleeding were not significantly different between the Teflon felt and primary closure groups (P = .246 and P = .371 respectively), but postoperative arrhythmias were significantly higher in the Teflon felt repair group (P = .049). CONCLUSION: Endoaneurysmorrhaphy is a better surgical technique in left ventricle aneurysm to restore the internal contour and preserve the surface anatomy of the ventricle. The ventriculotomy closure can be performed with two different approaches, including Teflon felt strips or by direct suture of the epicardium. Based on this study's findings, two repair techniques have similar impact on the early outcomes. However, with overall outcomes with respect to Teflon felt repair, direct closure of the ventriculotomy after endoaneurysmorrhaphy was superior.

cis-Regulatory control of Mesp1 expression by YY1 and SP1 during mouse embryogenesis.

BACKGROUND: Mesp1 is critical for early cardiomyocyte differentiation and heart development. We previously observed down-regulation of Mesp1 expression in YY1-ablated mouse embryonic hearts. However, how Mesp1 expression is mediated by YY1 is not well understood. RESULTS: We excised YY1 in the murine embryos using Sox2-cre and found that Mesp1 was down-regulated in the embryonic day (E) 7.5 mutant embryos. Also, YY1 activated the 6 kb Mesp1 regulatory element fused to a luciferase reporter. We identified two putative YY1 binding sites in the proximal promoter region of Mesp1 gene, and found that mutation of these sites significantly reduced YY1-induced activation of the Mesp1 promoter. We also uncovered one cognitive site for SP1, one of the earliest binding partners of YY1 identified. Mutation of this SP1 site repressed SP1-induced activation of the Mesp1 promoter. Moreover, YY1 and SP1 synergistically activated the Mesp1 promoter. Consistently, while Lacz expression driven by the wild-type 6 kb regulatory element of Mesp1 gene was robust in E7.5 mouse embryos, the mutation of these binding sites in the context of this 6 kb sequence substantially reduced the LacZ expression during embryogenesis. CONCLUSIONS: YY1 and SP1 independently and cooperatively govern the Mesp1 expression during embryogenesis.

Involvement of activated SUMO-2 conjugation in cardiomyopathy.

Sumoylation is a posttranslational modification that regulates a wide spectrum of cellular activities. Cardiomyopathy is the leading cause of heart failure. Whether sumoylation, particularly SUMO-2/3 conjugation, is involved in cardiomyopathy has not been investigated. We report here that SUMO-2/3 conjugation was elevated in the human failing hearts, and we investigated the impact of increased SUMO-2 conjugation on heart function by using the gain-of-function approach in mice, in which cardiac specific expression of constitutively active SUMO-2 was governed by alpha myosin heavy chain promoter (MHC-SUMO-2 transgenic, SUMO-2-Tg). Four of five independent SUMO-2-Tg mouse lines exhibited cardiomyopathy with various severities, ranging from acute heart failure leading to early death to the development of chronic cardiomyopathy with aging. We further revealed that SUMO-2 directly regulated apoptotic process by at least partially targeting calpain 2 and its natural inhibitor calpastatin. SUMO conjugation to calpain 2 promoted its enzymatic activity, and SUMO attachment to calpastatin mainly promoted its turnover and altered its subcellular distribution. Thus, enhanced SUMO-2 conjugation led to increased apoptosis and played a pathogenic role in the development of cardiomyopathy and heart failure.

Critical role of YY1 in cardiac morphogenesis.

BACKGROUND: Yin Yang 1 (YY1), the only DNA binding polycomb group protein, was reported to regulate cardiomyocyte differentiation during early cardiac mesoderm development. However, whether it contributes to cardiac morphogenesis at later developmental stage(s) during embryogenesis is unknown. RESULTS: We excised YY1 in murine hearts during embryogenesis using two temporal-spatially controlled cre activation approaches, and revealed critical roles of YY1 in cardiac structural formation. Alpha-myosin heavy chain-cre (α-MHC-cre)-mediated cardiomyocyte-specific ablation of YY1 (MHC-YY1) resulted in perinatal death of mutant mice, and Nkx2.5-cre-mediated YY1 null embryos (Nkx2.5-YY1) died embryonically. In the Nkx2.5-YY1 mutants, the endocardial cushions (ECs) of both atrioventricular canal (AVC) and outflow tract (OFT) were hypoplastic due to decreased proliferation and increased apoptosis. Endothelial-to-mesenchymal transition (EMT) progress was also compromised in ECs. Nkx2.5-YY1 mutant hearts had normal formation of extracellular matrix, suggesting that the impaired EMT resulted from the direct loss of YY1. We further uncovered that a number of factors that are involved in normal cardiogenesis were downstream targets of YY1. CONCLUSIONS: YY1 plays a critical role in cardiac development and occupies a high-level position within the hierarchy of the cardiac transcriptional network that governs normal cardiogenesis.

SENP5, a SUMO isopeptidase, induces apoptosis and cardiomyopathy.

Cardiomyopathy presents a major health issue and is a leading cause of heart failure. Although a subset of familial cardiomyopathy is associated with genetic mutations, over 50% of cardiomyopathy is defined as idiopathic, the mechanisms underlying which are under intensive investigation. SUMO conjugation is a dynamic posttranslational modification that can be readily reversed by the activity of sentrin-specific proteases (SENPs). However, whether SENPs are implicated in heart disease pathophysiology remains unexplored. We observed a significant increase in the level of SENP5, a SUMO isopeptidase, in human idiopathic failing hearts. To reveal whether it plays a role in the pathogenesis of cardiac muscle disorders, we used a gain-of-function approach to overexpress SENP5 in murine cardiomyocytes (SENP5 transgenic, SENP5-Tg). Overexpression of SENP5 led to cardiac dysfunction, accompanied by decreased cardiomyocyte proliferation and elevated apoptosis. The increase in apoptosis preceded other detectable pathological changes, suggesting its causal link to cardiomyopathy. Further examination of SENP5-Tg hearts unveiled a decrease in SUMO attachment to dynamin related protein (Drp1), a factor critical for mitochondrial fission. Correspondingly, the mitochondria of SENP5-Tg hearts at an early developmental stage were significantly larger compared with those in the control hearts, suggesting that desumoylation of Drp1 at least partially accounts for the cardiac phenotypes observed in the SENP5-Tg mice. Finally, overexpression of Bcl2 in SENP5-Tg hearts improved cardiac function of SENP5-Tg mice, further supporting the notion that SENP5 mainly targets mitochondrial function in vivo. Our findings demonstrate an important role of the desumoylation enzyme SENP5 in the development of cardiac muscle disorders, and point to the SUMO conjugation pathway as a potential target in the prevention/treatment of cardiomyopathy. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".

Potentiation of Tbx5-mediated transactivation by SUMO conjugation and protein inhibitor of activated STAT 1 (PIAS1).

The role of the T-box transcription factor Tbx5 in heart and limb development has been well documented; however, how posttranslational modification is involved in mediating its activity is unknown. Here we report that Tbx5 is a novel target by SUMO conjugation, a posttranslational modification that is involved in a variety of cellular events. Sumoylation potentiated the transcriptional activity of Tbx5, and PIAS family members, a group of SUMO E3 ligase, differentially mediated sumoylation and function of Tbx5. PIAS1 potently stimulated SUMO conjugation to Tbx5, and the physical association of Tbx5 with PIAS1 was required for its full sumoylation. PIAS1 also enhanced the functional cooperation between Tbx5 and its interaction partners. Overlapping expression pattern and colocalization of PIAS1 and Tbx5 in the mouse embryonic hearts and on the native target gene promoter were observed, pointing to a potential functional interaction of these two factors in vivo. These findings provide novel insights into how the transcriptional activity of a cardiac-specific factor, Tbx5, is regulated both directly and indirectly via posttranslational modification by a non-tissue-specific factor, PIAS1.