Functional analysis of a gene-edited mouse model to gain insights into the disease mechanisms of a titin missense variant.

Titin truncating variants are a well-established cause of cardiomyopathy; however, the role of titin missense variants is less well understood. Here we describe the generation of a mouse model to investigate the underlying disease mechanism of a previously reported titin A178D missense variant identified in a family with non-compaction and dilated cardiomyopathy. Heterozygous and homozygous mice carrying the titin A178D missense variant were characterised in vivo by echocardiography. Heterozygous mice had no detectable phenotype at any time point investigated (up to 1 year). By contrast, homozygous mice developed dilated cardiomyopathy from 3 months. Chronic adrenergic stimulation aggravated the phenotype. Targeted transcript profiling revealed induction of the foetal gene programme and hypertrophic signalling pathways in homozygous mice, and these were confirmed at the protein level. Unsupervised proteomics identified downregulation of telethonin and four-and-a-half LIM domain 2, as well as the upregulation of heat shock proteins and myeloid leukaemia factor 1. Loss of telethonin from the cardiac Z-disc was accompanied by proteasomal degradation; however, unfolded telethonin accumulated in the cytoplasm, leading to a proteo-toxic response in the mice.We show that the titin A178D missense variant is pathogenic in homozygous mice, resulting in cardiomyopathy. We also provide evidence of the disease mechanism: because the titin A178D variant abolishes binding of telethonin, this leads to its abnormal cytoplasmic accumulation. Subsequent degradation of telethonin by the proteasome results in proteasomal overload, and activation of a proteo-toxic response. The latter appears to be a driving factor for the cardiomyopathy observed in the mouse model.

Mutant Muscle LIM Protein C58G causes cardiomyopathy through protein depletion.

Cysteine and glycine rich protein 3 (CSRP3) encodes Muscle LIM Protein (MLP), a well-established disease gene for Hypertrophic Cardiomyopathy (HCM). MLP, in contrast to the proteins encoded by the other recognised HCM disease genes, is non-sarcomeric, and has important signalling functions in cardiomyocytes. To gain insight into the disease mechanisms involved, we generated a knock-in mouse (KI) model, carrying the well documented HCM-causing CSRP3 mutation C58G. In vivo phenotyping of homozygous KI/KI mice revealed a robust cardiomyopathy phenotype with diastolic and systolic left ventricular dysfunction, which was supported by increased heart weight measurements. Transcriptome analysis by RNA-seq identified activation of pro-fibrotic signalling, induction of the fetal gene programme and activation of markers of hypertrophic signalling in these hearts. Further ex vivo analyses validated the activation of these pathways at transcript and protein level. Intriguingly, the abundance of MLP decreased in KI/KI mice by 80% and in KI/+ mice by 50%. Protein depletion was also observed in cellular studies for two further HCM-causing CSRP3 mutations (L44P and S54R/E55G). We show that MLP depletion is caused by proteasome action. Moreover, MLP C58G interacts with Bag3 and results in a proteotoxic response in the homozygous knock-in mice, as shown by induction of Bag3 and associated heat shock proteins. In conclusion, the newly generated mouse model provides insights into the underlying disease mechanisms of cardiomyopathy caused by mutations in the non-sarcomeric protein MLP. Furthermore, our cellular experiments suggest that protein depletion and proteasomal overload also play a role in other HCM-causing CSPR3 mutations that we investigated, indicating that reduced levels of functional MLP may be a common mechanism for HCM-causing CSPR3 mutations.

When signalling goes wrong: pathogenic variants in structural and signalling proteins causing cardiomyopathies.

Cardiomyopathies are a diverse group of cardiac disorders with distinct phenotypes, depending on the proteins and pathways affected. A substantial proportion of cardiomyopathies are inherited and those will be the focus of this review article. With the wide application of high-throughput sequencing in the practice of clinical genetics, the roles of novel genes in cardiomyopathies are recognised. Here, we focus on a subgroup of cardiomyopathy genes [TTN, FHL1, CSRP3, FLNC and PLN, coding for Titin, Four and a Half LIM domain 1, Muscle LIM Protein, Filamin C and Phospholamban, respectively], which, despite their diverse biological functions, all have important signalling functions in the heart, suggesting that disturbances in signalling networks can contribute to cardiomyopathies.

The essential autophagy gene ATG7 modulates organ fibrosis via regulation of endothelial-to-mesenchymal transition.

Pulmonary fibrosis is a progressive disease characterized by fibroblast proliferation and excess deposition of collagen and other extracellular matrix components. Although the origin of fibroblasts is multifactorial, recent data implicate endothelial-to-mesenchymal transition as an important source of fibroblasts. We report herein that loss of the essential autophagy gene ATG7 in endothelial cells (ECs) leads to impaired autophagic flux accompanied by marked changes in EC architecture, loss of endothelial, and gain of mesenchymal markers consistent with endothelial-to-mesenchymal transition. Loss of ATG7 also up-regulates TGFß signaling and key pro-fibrotic genes in vitro. In vivo, EC-specific ATG7 knock-out mice exhibit a basal reduction in endothelial-specific markers and demonstrate an increased susceptibility to bleomycin-induced pulmonary fibrosis and collagen accumulation. Our findings help define the role of endothelial autophagy as a potential therapeutic target to limit organ fibrosis, a condition for which presently there are no effective available treatments.

Urinary neutrophil gelatinase-associated lipocalin (NGAL) can potentially predict vascular complications and reliably risk stratify patients with peripheral arterial disease.

Neutrophil gelatinase-associated lipocalin (NGAL) is expressed in atherosclerotic plaques and implicated in the development of cardiovascular diseases. Peripheral arterial disease (PAD) is an atherosclerotic disease that often results in major cardiovascular events. This study aimed to prospectively examine the potential of urine NGAL (uNGAL) in predicting worsening PAD status and major adverse limb events (MALE). Baseline urine NGAL (uNGAL) and urine creatinine (uCr) concentrations were measured in PAD (n = 121) and non-PAD (n = 77) patients. Levels of uNGAL were normalized for urine creatinine (uNGAL/uCr). Outcomes included worsening PAD status, which was defined as a drop in ankle brachial index (ABI) > 0.15, and major adverse limb events (MALE), which was defined as a need for surgical revascularization or amputations. PAD patients had 2.30-fold higher levels of uNGAL/uCr [median (IQR) 31.8 (17.0-62.5) µg/g] in comparison to non-PAD patients [median (IQR) 73.3 (37.5-154.7) µg/g] (P = 0.011). Multivariate cox analysis showed that uNGAL/uCr levels were independently associated with predicting worsening PAD status and MALE outcomes. Cumulative survival analysis, over follow up period, demonstrated a direct correlation between elevated uNGAL/uCr levels and PAD disease progression and MALE outcomes. These data demonstrate an association between elevated uNGAL/uCr levels and worsening PAD disease status and MALE outcomes, indicating its potential for risk-stratification of PAD patients.

Pravastatin-induced changes in expression of long non-coding and coding RNAs in endothelial cells.

OBJECTIVE: Atherosclerosis is the main cause of the cardiovascular disease (CVD). Elevated blood cholesterol and inflammation of the endothelium are two major mechanisms contributing to the establishment of atherosclerotic plaques. Statins, such as pravastatin, are blood-cholesterol lowering drugs commonly prescribed for patients with or at risk for CVDs. In addition to lowering blood cholesterols, statins have recently been shown to improve endothelial function in both hyper- and normocholesterolemic patients with atherosclerosis. To understand the molecular mechanisms underlying the endothelial function improvement by statins, we assessed the RNA profile of pravastatin-treated endothelial cells, particularly their mRNAs and long non-coding RNAs (lncRNAs). METHODS: Human umbilical vein endothelial cells (HUVECs) treated with pravastatin (10 µM) for 24 hr were profiled for lncRNAs and mRNAs using the Arraystar Human lncRNA Expression Microarray V3.0. RESULTS: Of the 30,584 different lncRNAs screened, 95 were significantly upregulated, while 86 were downregulated in HUVECs responding to pravastatin. LINC00281 and BC045663 were the most upregulated (~8-fold) and downregulated (~3.5-fold) lncRNAs, respectively. Of the 26,106 different mRNAs screened in the pravastatin-treated HUVEC samples, 190 were significantly upregulated, while 90 were downregulated. Assigning the differentially expressed genes by bioinformatics into functional groups revealed their molecular signaling involvement in the following physiological processes: osteoclast differentiation, Rap1 signaling pathway, hematopoiesis, immunity, and neurotrophin signaling pathway. CONCLUSIONS: This is the first lncRNA and mRNA expression profiling of pravastatin-mediated changes in human endothelial cells. Our results reveal potential novel targets and mechanisms for pravastatin-mediated vascular protection in atherosclerosis.

BReast CAncer susceptibility gene 2 deficiency exacerbates oxidized LDL-induced DNA damage and endothelial apoptosis.

Mutations in the tumor suppressor gene BRCA2 (BReast CAncer susceptibility gene 2) predispose carriers to breast, ovarian, and other cancers. In response to DNA damage, BRCA2 participates in homology-directed DNA damage repair to maintain genome stability. Genome-wide association studies have identified an association between BRCA2 single nucleotide polymorphisms and plasma-lipid levels and lipid deregulation in humans. To date, DNA damage, apoptosis, and lipid deregulation are recognized as central pathways for endothelial dysfunction and atherosclerosis; however, the role of BRCA2 in endothelial dysfunction remains to be elucidated. To determine the role of BRCA2 in endothelial dysfunction, BRCA2 was silenced in human umbilical vein endothelial cells (ECs) and assessed for markers of DNA damage, apoptosis, and endothelial function following oxidized low-density lipoprotein (oxLDL) treatment. OxLDL was found to induce significant reactive oxygen species (ROS) production in BRCA2-silenced ECs. This increase in ROS production was associated with exacerbated DNA damage evidenced by increased expression and activation of DNA double-stranded break (DSB) marker γH2AX and reduced RAD51-foci formation-an essential regulator of DSB repair. Increased DSBs were associated with enhanced expression and activation of pro-apoptotic p53 and significant apoptosis in oxLDL-treated BRCA2-silenced ECs. Loss of BRCA2 in ECs was further associated with oxLDL-induced impaired tube-forming potential and eNOS expression. Collectively, the data reveals, for the first time, a novel role of BRCA2 as a regulator of EC survival and function in the setting of oxLDL treatment in vitro. Additionally, the data provide important clues regarding the potential susceptibility of BRCA2 mutation carriers to endothelial dysfunction, atherosclerosis, and other cardiovascular diseases.

Endothelial-specific Loss of IFT88 Promotes Endothelial-to-Mesenchymal Transition and Exacerbates Bleomycin-induced Pulmonary Fibrosis.

Intraflagellar transport protein 88 (Ift88) is required for ciliogenesis and shear stress-induced dissolution of cilia in embryonic endothelial cells coincides with endothelial-to-mesenchymal transition (EndMT) in the developing heart. EndMT is also suggested to underlie heart and lung fibrosis, however, the mechanism linking endothelial Ift88, its effect on EndMT and organ fibrosis remains mainly unexplored. We silenced Ift88 in endothelial cells (ECs) in vitro and generated endothelial cell-specific Ift88-knockout mice (Ift88endo) in vivo to evaluate EndMT and its contribution towards organ fibrosis, respectively. Ift88-silencing in ECs led to mesenchymal cells-like changes in endothelial cells. The expression level of the endothelial markers (CD31, Tie-2 and VE-cadherin) were significantly reduced with a concomitant increase in the expression level of mesenchymal markers (αSMA, N-Cadherin and FSP-1) in Ift88-silenced ECs. Increased EndMT was associated with increased expression of profibrotic Collagen I expression and increased proliferation in Ift88-silenced ECs. Loss of Ift88 in ECs was further associated with increased expression of Sonic Hedgehog signaling effectors. In vivo, endothelial cells isolated from the heart and lung of Ift88endo mice demonstrated loss of Ift88 expression in the endothelium. The Ift88endo mice were born in expected Mendelian ratios without any adverse cardiac phenotypes at baseline. Cardiac and pulmonary endothelial cells isolated from the Ift88endo mice demonstrated signs of EndMT and bleomycin treatment exacerbated pulmonary fibrosis in Ift88endo mice. Pressure overload stress in the form of aortic banding did not reveal a significant difference in cardiac fibrosis between Ift88endo mice and control mice. Our findings demonstrate a novel association between endothelial cilia with EndMT and cell proliferation and also show that loss of endothelial cilia-associated increase in EndMT contributes specifically towards pulmonary fibrosis.

Adiponectin limits monocytic microparticle-induced endothelial activation by modulation of the AMPK, Akt and NFκB signaling pathways.

OBJECTIVE: Monocyte-derived microparticles (mono-MPs) are emerging as critical transducers of inflammatory signals, and have been suggested to link cardiovascular risk factors to vascular injury. Since adiponectin has been proposed to exert multiple anti-inflammatory and vasculoprotective effects, we hypothesized that it might serve to limit the production and/or action of mono-MPs. METHODS: Flow cytometry and western blot studies were conducted on THP-1 cells, THP-1-derived MPs, human umbilical vein endothelial cells (HUVECs), peripheral blood CD14+ monocytes and mice to evaluate the effects of adiponectin on mono-MPs. RESULTS: Adiponectin attenuated lipopolysaccharide (LPS)-evoked MP release from THP-1 monocytes (30% difference) and peripheral blood monocytes (both P < 0.05) as well as dampened LPS-induced mono-MP generation in vivo. Furthermore, peritoneal monocytes from Adipoq(-/-) mice generated significantly greater MPs than those from Adipoq(+/+) littermates in the absence (2.3 fold difference, P < 0.05) and presence (1.6 fold difference, P < 0.05) of LPS. LPS-induced MP expression of NLRP3 inflammasome and its key components, namely cleaved ASC, caspase-1 and IL-1ß (pro- and cleaved), were markedly attenuated by adiponectin. HUVECs incubated with MPs from LPS-treated THP-1 cells exhibited increased VCAM-1 levels and adhesion to THP-1 cells. Adiponectin abrogated these effects. From a mechanistic standpoint, the effects of adiponectin on MP release and molecular signaling occurred at least in part through the AMPK, Akt and NFκB pathways. CONCLUSION: Adiponectin exerts novel effects to limit the production and action of mono-MPs, underscoring yet another pleiotropic effect of this adipokine.