Systematic in vivo candidate evaluation uncovers therapeutic targets for LMNA dilated cardiomyopathy and risk of Lamin A toxicity.

BACKGROUND: Dilated cardiomyopathy (DCM) is a severe, non-ischemic heart disease which ultimately results in heart failure (HF). Decades of research on DCM have revealed diverse aetiologies. Among them, familial DCM is the major form of DCM, with pathogenic variants in LMNA being the second most common form of autosomal dominant DCM. LMNA DCM is a multifactorial and complex disease with no specific treatment thus far. Many studies have demonstrated that perturbing candidates related to various dysregulated pathways ameliorate LMNA DCM. However, it is unknown whether these candidates could serve as potential therapeutic targets especially in long term efficacy. METHODS: We evaluated 14 potential candidates including Lmna gene products (Lamin A and Lamin C), key signaling pathways (Tgfß/Smad, mTor and Fgf/Mapk), calcium handling, proliferation regulators and modifiers of LINC complex function in a cardiac specific Lmna DCM model. Positive candidates for improved cardiac function were further assessed by survival analysis. Suppressive roles and mechanisms of these candidates in ameliorating Lmna DCM were dissected by comparing marker gene expression, Tgfß signaling pathway activation, fibrosis, inflammation, proliferation and DNA damage. Furthermore, transcriptome profiling compared the differences between Lamin A and Lamin C treatment. RESULTS: Cardiac function was restored by several positive candidates (Smad3, Yy1, Bmp7, Ctgf, aYAP1, Sun1, Lamin A, and Lamin C), which significantly correlated with suppression of HF/fibrosis marker expression and cardiac fibrosis in Lmna DCM. Lamin C or Sun1 shRNA administration achieved consistent, prolonged survival which highly correlated with reduced heart inflammation and DNA damage. Importantly, Lamin A treatment improved but could not reproduce long term survival, and Lamin A administration to healthy hearts itself induced DCM. Mechanistically, we identified this lapse as caused by a dose-dependent toxicity of Lamin A, which was independent from its maturation. CONCLUSIONS: In vivo candidate evaluation revealed that supplementation of Lamin C or knockdown of Sun1 significantly suppressed Lmna DCM and achieve prolonged survival. Conversely, Lamin A supplementation did not rescue long term survival and may impart detrimental cardiotoxicity risk. This study highlights a potential of advancing Lamin C and Sun1 as therapeutic targets for the treatment of LMNA DCM.

Tissue factor cytoplasmic domain exacerbates post-infarct left ventricular remodeling via orchestrating cardiac inflammation and angiogenesis.

The coagulation protein tissue factor (TF) regulates inflammation and angiogenesis via its cytoplasmic domain in infection, cancer and diabetes. While TF is highly abundant in the heart and is implicated in cardiac pathology, the contribution of its cytoplasmic domain to post-infarct myocardial injury and adverse left ventricular (LV) remodeling remains unknown. Methods: Myocardial infarction was induced in wild-type mice or mice lacking the TF cytoplasmic domain (TF∆CT) by occlusion of the left anterior descending coronary artery. Heart function was monitored with echocardiography. Heart tissue was collected at different time-points for histological, molecular and flow cytometry analysis. Results: Compared with wild-type mice, TF∆CT had a higher survival rate during a 28-day follow-up after myocardial infarction. Among surviving mice, TF∆CT mice had better cardiac function and less LV remodeling than wild-type mice. The overall improvement of post-infarct cardiac performance in TF∆CT mice, as revealed by speckle-tracking strain analysis, was attributed to reduced myocardial deformation in the peri-infarct region. Histological analysis demonstrated that TF∆CT hearts had in the infarct area greater proliferation of myofibroblasts and better scar formation. Compared with wild-type hearts, infarcted TF∆CT hearts showed less infiltration of proinflammatory cells with concomitant lower expression of protease-activated receptor-1 (PAR1) - Rac1 axis. In particular, infarcted TF∆CT hearts displayed markedly lower ratios of inflammatory M1 macrophages and reparative M2 macrophages (M1/M2). In vitro experiment with primary macrophages demonstrated that deletion of the TF cytoplasmic domain inhibited macrophage polarization toward the M1 phenotype. Furthermore, infarcted TF∆CT hearts presented markedly higher peri-infarct vessel density associated with enhanced endothelial cell proliferation and higher expression of PAR2 and PAR2-associated pro-angiogenic pathway factors. Finally, the overall cardioprotective effects observed in TF∆CT mice could be abolished by subcutaneously infusing a cocktail of PAR1-activating peptide and PAR2-inhibiting peptide via osmotic minipumps. Conclusions: Our findings demonstrate that the TF cytoplasmic domain exacerbates post-infarct cardiac injury and adverse LV remodeling via differential regulation of inflammation and angiogenesis. Targeted inhibition of the TF cytoplasmic domain-mediated intracellular signaling may ameliorate post-infarct LV remodeling without perturbing coagulation.

Upregulation of Yy1 Suppresses Dilated Cardiomyopathy caused by Ttn insufficiency.

Truncating variants in TTN (TTNtv), coding for the largest structural protein in the sarcomere, contribute to the largest portion of familial and ambulatory dilated cardiomyopathy (DCM). TTN haploinsufficiency caused by TTNtv is suggested as the disease mechanism. However, it is unclear whether TTN insufficiency causes DCM. Moreover, it is unknown whether modulation of downstream pathways serves as a therapeutic strategy for DCM caused by TTN insufficiency. Here, we show that reduction of cardiac Ttn expression by adeno-associated virus mediated shRNA (Ttn shRNA) generated DCM in mouse, demonstrating impaired cardiac performance, enlarged left ventricle (LV) and reduced LV wall thickness. A screen of 10 dysregulated and selected genes identified that Yin Yang 1 (Yy1) significantly suppressed DCM caused by Ttn shRNA. Gene profiling by RNAseq showed Yy1 modulated cell growth related genes. Ttn insufficiency activated cardiomyocyte cell cycle reentry by upregulating of Ccnd1 and Ccnd2. Cardiomyocytes activated by Ttn insufficiency did not advance to S phase by EdU incorporation assay. Yy1 promoted cardiomyocyte cell cycle by further enhancing Ccnd1 and Ccnd2 and increasing DNA replication without undergoing cell division. Importantly, upregulation of Ccnd1 and Ccnd2 suppressed DCM caused by Ttn insufficiency. Our findings demonstrate that DCM caused by Ttn insufficiency can be treated by therapeutically promoting cardiac cell cycle.

Yin Yang 1 Suppresses Dilated Cardiomyopathy and Cardiac Fibrosis Through Regulation of Bmp7 and Ctgf.

RATIONALE: Pathogenic variations in the lamin gene (LMNA) cause familial dilated cardiomyopathy (DCM). LMNA insufficiency caused by LMNA pathogenic variants is believed to be the basic mechanism underpinning LMNA-related DCM. OBJECTIVE: To assess whether silencing of cardiac Lmna causes DCM and investigate the role of Yin Yang 1 (Yy1) in suppressing Lmna DCM. METHODS AND RESULTS: We developed a Lmna DCM mouse model induced by cardiac-specific Lmna short hairpin RNA. Silencing of cardiac Lmna induced DCM with associated cardiac fibrosis and inflammation. We demonstrated that upregulation of Yy1 suppressed Lmna DCM and cardiac fibrosis by inducing Bmp7 expression and preventing upregulation of Ctgf. Knockdown of upregulated Bmp7 attenuated the suppressive effect of Yy1 on DCM and cardiac fibrosis. However, upregulation of Bmp7 alone was not sufficient to suppress DCM and cardiac fibrosis. Importantly, upregulation of Bmp7 together with Ctgf silencing significantly suppressed DCM and cardiac fibrosis. Mechanistically, upregulation of Yy1 regulated Bmp7 and Ctgf reporter activities and modulated Bmp7 and Ctgf gene expression in cardiomyocytes. Downregulation of Ctgf inhibited TGF-ß (transforming growth factor-ß)/Smad signaling in DCM hearts. Regulation of both Bmp7 and Ctgf further suppressed TGFß/Smad signaling. In addition, co-modulation of Bmp7 and Ctgf reduced CD3+ T cell numbers in DCM hearts. CONCLUSIONS: Our findings demonstrate that upregulation of Yy1 or co-modulation of Bmp7 and Ctgf offer novel therapeutic strategies for the treatment of DCM caused by LMNA insufficiency.

mTORC1 dependent regulation of REDD1 protein stability.

REDD1 is known to be transcriptionally upregulated in hypoxia. During hypoxic stress, REDD1 plays an important role as a mediator of mTORC1 inhibition. REDD1 is also subject to highly dynamic transcriptional regulation in response to a variety of other stress signals. In addition, the REDD1 protein is highly unstable. However, it is currently not well understood how REDD1 protein stability is regulated. In this study, we discovered that mTORC1 regulates REDD1 protein stability in a 26S proteasome dependent manner. Inhibition of mTORC1 resulted in reduced REDD1 protein stability and a consequent decrease in REDD1 expression. Conversely, activation of the mTORC1 pathway increases REDD1 protein levels. We show that REDD1 degradation is not regulated by HUWE1, Cul4a or other Cullin E3 ubiquitin ligases. Our study shows that mTORC1 increases REDD1 protein stability and reveals a novel mTORC1-REDD1 feedback loop. This feedback mechanism may limit the inhibitory action of REDD1 on mTORC1.

Destabilization of CDC6 upon DNA damage is dependent on neddylation but independent of Cullin E3 ligases.

CDC6 is an important component of the pre-replication complex and plays an essential role in the regulation of DNA replication in eukaryotic cells. Deregulation of CDC6 protein levels results in rereplication and genomic instability. CDC6 expression is tightly regulated during the cell cycle. One major mechanism of cell cycle dependent regulation of CDC6 is APC(Cdh1) mediated protein ubiquitination and degradation during G1 phase. In addition to APC(Cdh1) dependent degradation, alternative, Cullin RING E3 ubiquitin ligase dependent degradation pathways have been characterized in yeast. Here we studied whether Cullin RING E3 ligases also play a role in the turnover of CDC6 protein in mammalian cells. To this end, we used the Nedd8 E1 inhibitor MLN4924, which blocks the activity of all Cullin E3 ligases. We observed that treatment with MLN4924 increased CDC6 protein expression. However, this effect was due to a delay in cell cycle progression from G1 to S phase, resulting in accumulation of cells with high CDC6 protein levels. Therefore, our results indicate that Cullin E3 ligases are not involved in the basal turnover of CDC6 in mammalian cells. Interestingly, we also found that the DNA cross-linker mitomycin C induces marked CDC6 protein degradation. Mitomycin C induced CDC6 degradation is not mediated by APC(Cdh1), Cullin or HUWE1 E3 ubiquitin ligases. Notably, mitomycin C mediated CDC6 degradation requires the neddylation pathway. Our results provide evidence for a novel, cullin independent mechanism of CDC6 posttranslational regulation upon DNA damage that involves the neddylation pathway.

Post-translational regulation of mTOR complex 1 in hypoxia and reoxygenation.

The mechanistic target of rapamycin complex 1 (mTORC1) is a key regulator of cell growth and proliferation in response to various upstream signals. Hypoxia has been shown to exert a strong inhibitory effect on mTORC1 activity. Various mechanisms involving gene transcription have been proposed to mediate the effect of hypoxia on mTORC1 activity. Here we show that oxygen concentrations regulate mTORC1 activity in a highly dynamic manner. The rapid response of mTORC1 to changes in oxygen concentrations was not mediated by the HIF transcription factor or its transcriptional targets, REDD1 and BNIP3. Interestingly, we observed that the rapid response of mTORC1 activity to changes in oxygen concentrations is independent of transcription and new protein synthesis. This suggests a post-translational regulation mTORC1 activity in hypoxia and reoxygenation. We also provide evidence that hypoxia does not regulate mTORC1 via the TSC1/2 or Ragulator pathways but directly at the level of mTORC1. In conclusion, our results suggest that mTORC1 can respond rapidly to changes in oxygen concentrations via a post-translational mechanism that may involve a heme containing protein.

Biochemical and cellular effects of inhibiting Nedd8 conjugation.

The conjugation of proteins with the ubiquitin-like protein Nedd8 is an essential cellular process and an important anti-cancer therapeutic target. The major known role of Nedd8 is the attachment to and activation of Cullin RING E3 ubiquitin ligases (CRL). The attachment of Nedd8 to its substrates occurs via a process analogous to ubiquitin transfer, involving a Nedd8 E1 activating enzyme and a Nedd8 E2 conjugating enzyme, Ubc12, which transfers Nedd8 onto lysine residues of target proteins. In this study, we utilize dominant-negative Ubc12 (dnUbc12) and the Nedd8 E1 inhibitor MLN4924 to inhibit cellular neddylation. We demonstrate that dnUbc12 functions by depleting cellular Nedd8 concentrations. Inhibition of cellular neddylation leads to rapid accumulation of CRL substrates and an enlarged and flattened morphology in HEK293 cells. Inhibiting Nedd8 conjugation also causes abnormalities in the actin cytoskeleton. This is likely at least partially mediated via accumulation of the small GTPase RhoA, a recently identified CRL substrate. We indeed found that siRNA mediated knockdown of RhoA can reverse the morphological changes observed upon inhibition of cellular neddylation. In conclusion, the Nedd8 pathway plays an important role in regulating the actin cytoskeleton and cellular morphology. Dysfunction of the actin cytoskeleton may contribute to the anti-cancer effect of Nedd8 inhibition.

Possible gene-gene interaction of KIR2DL4 with its cognate ligand HLA-G in modulating risk for preeclampsia.

Preeclampsia (PE) is a leading cause of maternal and fetal mortality and morbidity that occurs only during pregnancy. Pregnancy is the only physiological situation where killer-cell immunoglobulin-like receptors (KIRs) may meet cognate nonself variants of human leukocyte antigen (HLA) allotypes. We previously reported that presence of fetal HLA-G*0106 was significantly associated with risk for PE in multigravid pregnancies. We have now tested the KIR2DL4 receptor gene for association with PE, as well as for its interaction with HLA-G in modulating disease risk, in a case-control study of 83 PE and 240 normotensive pregnancies. No significant association was observed between alleles of KIR2DL4 and PE in both maternal and fetal groups, either among primigravid or multigravid pregnancies. Alleles of KIR2DL4 and HLA-G were then analyzed together to determine whether particular variant ligand-receptor combinations were associated with an increased risk for PE. Gene-gene interaction analyses suggest that the presence of fetal HLA-G*0106 in combination with maternal KIR2DL4*006 is significantly associated with PE risk in multigravid pregnancies (P < .001). These data provide the first preliminary evidence suggesting that although KIR2DL4 itself is not associated with PE, it may modulate the effect of HLA-G*0106 on risk for PE.

Paternal contribution of HLA-G*0106 significantly increases risk for pre-eclampsia in multigravid pregnancies.

Pre-eclampsia (PE) is a leading cause of maternal and fetal mortality and morbidity. Structural or functional alterations of human leukocyte antigen (HLA)-G present at the maternal-fetal interface may predispose women to PE. We tested the HLA-G gene for association with PE in a case-control study of 83 PE and 240 normotensive Malay women. HLA-G was amplified in a single-tube multiplex-PCR reaction and genotyped for 18 single nucleotide polymorphisms (SNPs) by multiplex-minisequencing. Case-control comparisons were performed, and associations with disease were expressed as odds ratios (ORs). Risk for PE was significantly associated with fetal allele G*0106 only in multigravid pregnancies (P = 0.002, OR = 5.0, 95% CI = 1.8-13.8). Among multigravid pregnancies, the frequency of PE babies heterozygous or homozygous for G*0106 was also significantly higher compared with normal control babies (P = 0.002, OR = 5.4, 95% CI = 1.9-15.4). Multivariate analyses with adjustment for factors associated with PE revealed similar results (P = 0.003, OR = 10.1, 95% CI = 2.2-46.8). Additionally, a significantly higher frequency of fetal-maternal G*0106 genotype mismatch was observed in PE compared with normal multigravid pregnancies (P = 0.001, OR = 9.6, 95% CI = 2.4-38.7). Thus, paternal HLA-G G*0106 contribution significantly increases risk for PE in multigravidas who do not carry this allele, potentially mediated by a gradual maternal alloimmune response to repeated exposure to the paternal HLA-G variant.