The E3 ubiquitin ligase HectD3 attenuates cardiac hypertrophy and inflammation in mice.

Myocardial inflammation has recently been recognized as a distinct feature of cardiac hypertrophy and heart failure. HectD3, a HECT domain containing E3 ubiquitin ligase has previously been investigated in the host defense against infections as well as neuroinflammation; its cardiac function however is still unknown. Here we show that HectD3 simultaneously attenuates Calcineurin-NFAT driven cardiomyocyte hypertrophy and the pro-inflammatory actions of LPS/interferon-γ via its cardiac substrates SUMO2 and Stat1, respectively. AAV9-mediated overexpression of HectD3 in mice in vivo not only reduced cardiac SUMO2/Stat1 levels and pathological hypertrophy but also largely abolished macrophage infiltration and fibrosis induced by pressure overload. Taken together, we describe a novel cardioprotective mechanism involving the ubiquitin ligase HectD3, which links anti-hypertrophic and anti-inflammatory effects via dual regulation of SUMO2 and Stat1. In a broader perspective, these findings support the notion that cardiomyocyte growth and inflammation are more intertwined than previously anticipated.

The A818-6 system as an in-vitro model for studying the role of the transportome in pancreatic cancer.

BACKGROUND: The human pancreatic cancer cell line A818-6 can be grown in vitro either as a highly malignant, undifferentiated monolayer (ML) or as three-dimensional (3D) single layer hollow spheres (HS) simulating a benign, highly differentiated, duct-like pancreatic epithelial structure. This characteristic allowing A818-6 cells to switch from one phenotype to another makes these cells a unique system to characterize the cellular and molecular modifications during differentiation on one hand and malignant transformation on the other hand. Ion channels and transport proteins (transportome) have been implicated in malignant transformation. Therefore, the current study aimed to analyse the transportome gene expression profile in the A818-6 cells growing as a monolayer or as hollow spheres. METHODS & RESULTS: The study identified the differentially expressed transportome genes in both cellular states of A818-6 using Agilent and Nanostring arrays and some targets were validated via immunoblotting. Additionally, these results were compared to a tissue Affymetrix microarray analysis of pancreatic adenocarcinoma patients' tissues. The overall transcriptional profile of the ML and HS cells confirmed the formerly described mesenchymal features of ML and epithelial nature of HS which was further verified via high expression of E-cadherin and low expression of vimentin found in HS in comparison to ML. Among the predicted features between HS and ML was the involvement of miRNA-9 in this switch. Importantly, the bioinformatics analysis also revealed substantial number (n = 126) of altered transportome genes. Interestingly, three genes upregulated in PDAC tissue samples (GJB2, GJB5 and SLC38A6) were found to be also upregulated in ML and 3 down-regulated transportome genes (KCNQ1, TRPV6 and SLC4A) were also reduced in ML. CONCLUSION: This reversible HS/ML in vitro system might help in understanding the pathophysiological impact of the transportome in the dedifferentiation process in pancreatic carcinogenesis. Furthermore, the HS/ML model represents a novel system for studying the role of the transportome during the switch from a more benign, differentiated (HS) to a highly malignant, undifferentiated (ML) phenotype.

Cardiac α-Actin (ACTC1) Gene Mutation Causes Atrial-Septal Defects Associated With Late-Onset Dilated Cardiomyopathy.

BACKGROUND: Familial atrial septal defect (ASD) has previously been attributed primarily to mutations in cardiac transcription factors. Here, we report a large, multi-generational family (78 members) with ASD combined with a late-onset dilated cardiomyopathy and further characterize the consequences of mutant α-actin. METHODS: We combined a genome-wide linkage analysis with cell biology, microscopy, and molecular biology tools to characterize a novel ACTC1 (cardiac α-actin) mutation identified in association with ASD and late-onset dilated cardiomyopathy in a large, multi-generational family. RESULTS: Using a genome-wide linkage analysis, the ASD disease locus was mapped to chromosome 15q14 harboring the ACTC1 gene. In 15 affected family members, a heterozygous, nonsynonymous, and fully penetrant mutation (p. Gly247Asp) was identified in exon 5 of ACTC1 that was absent in all healthy family members (n=63). In silico tools predicted deleterious consequences of this variant that was found absent in control databases. Ultrastructural analysis of myocardial tissue of one of the mutation carriers showed sarcomeric disarray, myofibrillar degeneration, and increased apoptosis, while cardiac proteomics revealed a significant increase in extracellular matrix proteins. Consistently, structural defects and increased apoptosis were also observed in neonatal rat ventricular cardiomyocytes overexpressing the mutant, but not native human ACTC1. Molecular dynamics studies and additional mechanistic analyses in cardiomyocytes confirmed actin polymerization/turnover defects, thereby affecting contractility. CONCLUSIONS: A combined phenotype of ASD and late-onset heart failure was caused by a heterozygous, nonsynonymous ACTC1 mutation. Mechanistically, we found a shared molecular mechanism of defective actin signaling and polymerization in both cardiac development and contractile function. Detection of ACTC1 mutations in patients with ASD may thus have further clinical implications with regard to monitoring for (late-onset) dilated cardiomyopathy.

Rho-family GTPase 1 (Rnd1) is a biomechanical stress-sensitive activator of cardiomyocyte hypertrophy.

Cardiac remodeling is induced by mechanical or humoral stress causing pathological changes to the heart. Here, we aimed at identifying the role of differentially regulated genes upon dynamic mechanical stretch. Microarray of dynamic stretch induced neonatal rat ventricular cardiomyocytes (NRVCMs) discovered Rho family GTPase 1 (Rnd1) as one of the significantly upregulated genes, a cardiac role of which is not known yet. Rnd1 was consistently upregulated in NRVCMs after dynamic stretch or phenylephrine (PE) stimulation, and in a mouse model of pressure overload. Overexpression of Rnd1 in NRVCMs activated the fetal gene program (including nppa and nppb) effected into a significant increase in cell surface area in untreated, stretched or PE-treated cells. Furthermore, Rnd1 overexpression showed a positive effect on cell proliferation as detected by significant increase in Ki67, Phosphohistone H3, and EdU positive NRVCMs. Through a Yeast two-hybrid screen and immunoprecipitation analysis, we identified Myozap, an intercalated disc protein, as novel interaction partner of Rnd1. Importantly, functional analysis of this interaction revealed the importance of RND1 in the RhoA and Myozap protein network that activates serum-response factor (SRF) signaling. In summary, we identified Rnd1 as a novel stretch-sensitive gene which influences cell proliferation and cellular hypertrophy via activation of RhoA-mediated SRF dependent and independent signaling pathways.

TRIM24 protein promotes and TRIM32 protein inhibits cardiomyocyte hypertrophy via regulation of dysbindin protein levels.

We have previously shown that dysbindin is a potent inducer of cardiomyocyte hypertrophy via activation of Rho-dependent serum-response factor (SRF) signaling. We have now performed a yeast two-hybrid screen using dysbindin as bait against a cardiac cDNA library to identify the cardiac dysbindin interactome. Among several putative binding proteins, we identified tripartite motif-containing protein 24 (TRIM24) and confirmed this interaction by co-immunoprecipitation and co-immunostaining. Another tripartite motif (TRIM) family protein, TRIM32, has been reported earlier as an E3 ubiquitin ligase for dysbindin in skeletal muscle. Consistently, we found that TRIM32 also degraded dysbindin in neonatal rat ventricular cardiomyocytes as well. Surprisingly, however, TRIM24 did not promote dysbindin decay but rather protected dysbindin against degradation by TRIM32. Correspondingly, TRIM32 attenuated the activation of SRF signaling and hypertrophy due to dysbindin, whereas TRIM24 promoted these effects in neonatal rat ventricular cardiomyocytes. This study also implies that TRIM32 is a key regulator of cell viability and apoptosis in cardiomyocytes via simultaneous activation of p53 and caspase-3/-7 and inhibition of X-linked inhibitor of apoptosis. In conclusion, we provide here a novel mechanism of post-translational regulation of dysbindin and hypertrophy via TRIM24 and TRIM32 and show the importance of TRIM32 in cardiomyocyte apoptosis in vitro.

Sumoylation-independent activation of Calcineurin-NFAT-signaling via SUMO2 mediates cardiomyocyte hypertrophy.

The objective of this study was to identify unknown modulators of Calcineurin (Cn)-NFAT signaling. Measurement of NFAT reporter driven luciferase activity was therefore utilized to screen a human cardiac cDNA-library (~107 primary clones) in C2C12 cells through serial dilutions until single clones could be identified. This extensive screening strategy culminated in the identification of SUMO2 as a most efficient Cn-NFAT activator. SUMO2-mediated activation of Cn-NFAT signaling in cardiomyocytes translated into a hypertrophic phenotype. Prohypertrophic effects were also observed in mice expressing SUMO2 in the heart using AAV9 (Adeno-associated virus), complementing the in vitro findings. In addition, increased SUMO2-mediated sumoylation in human cardiomyopathy patients and in mouse models of cardiomyopathy were observed. To decipher the underlying mechanism, we generated a sumoylation-deficient SUMO2 mutant (ΔGG). Surprisingly, ΔGG replicated Cn-NFAT-activation and the prohypertrophic effects of native SUMO2, both in vitro and in vivo, suggesting a sumoylation-independent mechanism. Finally, we discerned a direct interaction between SUMO2 and CnA, which promotes CnA nuclear localization. In conclusion, we identified SUMO2 as a novel activator of Cn-NFAT signaling in cardiomyocytes. In broader terms, these findings reveal an unexpected role for SUMO2 in cardiac hypertrophy and cardiomyopathy, which may open the possibility for therapeutic manipulation of this pathway.

Myoscape controls cardiac calcium cycling and contractility via regulation of L-type calcium channel surface expression.

Calcium signalling plays a critical role in the pathogenesis of heart failure. Here we describe a cardiac protein named Myoscape/FAM40B/STRIP2, which directly interacts with the L-type calcium channel. Knockdown of Myoscape in cardiomyocytes decreases calcium transients associated with smaller Ca(2+) amplitudes and a lower diastolic Ca(2+) content. Likewise, L-type calcium channel currents are significantly diminished on Myoscape ablation, and downregulation of Myoscape significantly reduces contractility of cardiomyocytes. Conversely, overexpression of Myoscape increases global Ca(2+) transients and enhances L-type Ca(2+) channel currents, and is sufficient to restore decreased currents in failing cardiomyocytes. In vivo, both Myoscape-depleted morphant zebrafish and Myoscape knockout (KO) mice display impairment of cardiac function progressing to advanced heart failure. Mechanistically, Myoscape-deficient mice show reduced L-type Ca(2+)currents, cell capacity and calcium current densities as a result of diminished LTCC surface expression. Finally, Myoscape expression is reduced in hearts from patients suffering of terminal heart failure, implying a role in human disease.

Myozap Deficiency Promotes Adverse Cardiac Remodeling via Differential Regulation of Mitogen-activated Protein Kinase/Serum-response Factor and ß-Catenin/GSK-3ß Protein Signaling.

The intercalated disc (ID) is a "hot spot" for heart disease, as several ID proteins have been found mutated in cardiomyopathy. Myozap is a recent addition to the list of ID proteins and has been implicated in serum-response factor signaling. To elucidate the cardiac consequences of targeted deletion of myozap in vivo, we generated myozap-null mutant (Mzp(-/-)) mice. Although Mzp(-/-) mice did not exhibit a baseline phenotype, increased biomechanical stress due to pressure overload led to accelerated cardiac hypertrophy, accompanied by "super"-induction of fetal genes, including natriuretic peptides A and B (Nppa/Nppb). Moreover, Mzp(-/-) mice manifested a severe reduction of contractile function, signs of heart failure, and increased mortality. Expression of other ID proteins like N-cadherin, desmoplakin, connexin-43, and ZO-1 was significantly perturbed upon pressure overload, underscored by disorganization of the IDs in Mzp(-/-) mice. Exploration of the molecular causes of enhanced cardiac hypertrophy revealed significant activation of ß-catenin/GSK-3ß signaling, whereas MAPK and MKL1/serum-response factor pathways were inhibited. In summary, myozap is required for proper adaptation to increased biomechanical stress. In broader terms, our data imply an essential function of the ID in cardiac remodeling beyond a mere structural role and emphasize the need for a better understanding of this molecular structure in the context of heart disease.

Mice with cardiac-restricted overexpression of Myozap are sensitized to biomechanical stress and develop a protein-aggregate-associated cardiomyopathy.

The intercalated disc (ID) is a major component of the cell-cell contact structures of cardiomyocytes and has been recognized as a hot spot for cardiomyopathy. We have previously identified Myozap as a novel cardiac-enriched ID protein, which interacts with several other ID proteins and is involved in RhoA/SRF signaling in vitro. To now study its potential role in vivo we generated a mouse model with cardiac overexpression of Myozap. Transgenic (Tg) mice developed cardiomyopathy with hypertrophy and LV dilation. Consistently, these mice displayed upregulation of the hypertrophy-associated and SRF-dependent gene expression. Pressure overload (transverse aortic constriction, TAC) caused exaggerated cardiac hypertrophy, further loss of contractility and LV dilation. Similarly, a physiological stimulus (voluntary running) also led to significant LV dysfunction. On the ultrastructural level, Myozap-Tg mouse hearts exhibited massive protein aggregates composed of Myozap, desmoplakin and other ID proteins. This aggregate-associated pathology closely resembled the alterations observed in desmin-related cardiomyopathy. Interestingly, desmin was not detectable in the aggregates, yet was largely displaced from the ID. Molecular analyses revealed induction of autophagy and dysregulation of the unfolded protein response (UPR), associated with apoptosis. Taken together, cardiac overexpression of Myozap leads to cardiomyopathy, mediated, at least in part by induction of Rho-dependent SRF signaling in vivo. Surprisingly, this phenotype was also accompanied by protein aggregates in cardiomyocytes, UPR alteration, accelerated autophagy and apoptosis. Thus, this mouse model may also offer additional insight into the pathogenesis of protein-aggregate-associated cardiomyopathies and represents a new candidate gene itself.

Nuclear death receptor TRAIL-R2 inhibits maturation of let-7 and promotes proliferation of pancreatic and other tumor cells.

BACKGROUND & AIMS: Tumor necrosis factor-related apoptosis inducing ligand (TRAIL-R1) (TNFRSF10A) and TRAIL-R2 (TNFRSF10B) on the plasma membrane bind ligands that activate apoptotic and other signaling pathways. Cancer cells also might have TRAIL-R2 in the cytoplasm or nucleus, although little is known about its activities in these locations. We investigated the functions of nuclear TRAIL-R2 in cancer cell lines. METHODS: Proteins that interact with TRAIL-R2 initially were identified in pancreatic cancer cells by immunoprecipitation, mass spectrometry, and immunofluorescence analyses. Findings were validated in colon, renal, lung, and breast cancer cells. Functions of TRAIL-R2 were determined from small interfering RNA knockdown, real-time polymerase chain reaction, Drosha-activity, microRNA array, proliferation, differentiation, and immunoblot experiments. We assessed the effects of TRAIL-R2 overexpression or knockdown in human pancreatic ductal adenocarcinoma (PDAC) cells and their ability to form tumors in mice. We also analyzed levels of TRAIL-R2 in sections of PDACs and non-neoplastic peritumoral ducts from patients. RESULTS: TRAIL-R2 was found to interact with the core microprocessor components Drosha and DGCR8 and the associated regulatory proteins p68, hnRNPA1, NF45, and NF90 in nuclei of PDAC and other tumor cells. Knockdown of TRAIL-R2 increased Drosha-mediated processing of the let-7 microRNA precursor primary let-7 (resulting in increased levels of mature let-7), reduced levels of the let-7 targets (LIN28B and HMGA2), and inhibited cell proliferation. PDAC tissues from patients had higher levels of nuclear TRAIL-R2 than non-neoplastic pancreatic tissue, which correlated with increased nuclear levels of HMGA2 and poor outcomes. Knockdown of TRAIL-R2 in PDAC cells slowed their growth as orthotopic tumors in mice. Reduced nuclear levels of TRAIL-R2 in cultured pancreatic epithelial cells promoted their differentiation. CONCLUSIONS: Nuclear TRAIL-R2 inhibits maturation of the microRNA let-7 in pancreatic cancer cell lines and increases their proliferation. Pancreatic tumor samples have increased levels of nuclear TRAIL-R2, which correlate with poor outcome of patients. These findings indicate that in the nucleus, death receptors can function as tumor promoters and might be therapeutic targets.

Dysbindin is a potent inducer of RhoA-SRF-mediated cardiomyocyte hypertrophy.

Dysbindin is an established schizophrenia susceptibility gene thoroughly studied in the context of the brain. We have previously shown through a yeast two-hybrid screen that it is also a cardiac binding partner of the intercalated disc protein Myozap. Because Dysbindin is highly expressed in the heart, we aimed here at deciphering its cardiac function. Using a serum response factor (SRF) response element reporter-driven luciferase assay, we identified a robust activation of SRF signaling by Dysbindin overexpression that was associated with significant up-regulation of SRF gene targets, such as Acta1 and Actc1. Concurrently, we identified RhoA as a novel binding partner of Dysbindin. Further phenotypic and mechanistic characterization revealed that Dysbindin induced cardiac hypertrophy via RhoA-SRF and MEK1-ERK1 signaling pathways. In conclusion, we show a novel cardiac role of Dysbindin in the activation of RhoA-SRF and MEK1-ERK1 signaling pathways and in the induction of cardiac hypertrophy. Future in vivo studies should examine the significance of Dysbindin in cardiomyopathy.