Milica Vukmirovic

Withaferin-A Reduces Type I Collagen Expression In Vitro and Inhibits Development of Myocardial Fibrosis In Vivo

Type I collagen is the most abundant protein in the human body. Its excessive synthesis results in fibrosis of various organs. Fibrosis is a major medical problem without an existing cure. Excessive synthesis of type I collagen in fibrosis is primarily due to stabilization of collagen mRNAs. We recently reported that intermediate filaments composed of vimentin regulate collagen synthesis by stabilizing collagen mRNAs. Vimentin is a primary target of Withaferin-A (WF-A). Therefore, we hypothesized that WF-A may reduce type I collagen production by disrupting vimentin filaments and decreasing the stability of collagen mRNAs. This study is to determine if WF-A exhibits anti-fibrotic properties in vitro and in vivo and to elucidate the molecular mechanisms of its action. In lung, skin and heart fibroblasts WF-A disrupted vimentin filaments at concentrations of 0.5–1.5 µM and reduced 3 fold the half-lives of collagen α1(I) and α2(I) mRNAs and protein expression. In addition, WF-A inhibited TGF-β1 induced phosphorylation of TGF-β1 receptor I, Smad3 phosphorylation and transcription of collagen genes. WF-A also inhibited in vitro activation of primary hepatic stellate cells and decreased their type I collagen expression. In mice, administration of 4 mg/kg WF-A daily for 2 weeks reduced isoproterenol-induced myocardial fibrosis by 50%. Our findings provide strong evidence that Withaferin-A could act as an anti-fibrotic compound against fibroproliferative diseases, including, but not limited to, cardiac interstitial fibrosis.

Serine-threonine kinase receptor associated protein (STRAP) regulates translation of type I collagen mRNAs

ABSTRACT Type I collagen is the most abundant protein in the human body and is composed of two α1(I) and one α2(I) polypeptides which assemble into a triple helix. For the proper assembly of the collagen triple helix, the individual polypeptides must be translated in coordination. Here, we show that serine-threonine kinase receptor-associated protein (STRAP) is tethered to collagen mRNAs by interaction with LARP6. LARP6 is a protein which directly binds the 5′ stem-loop (5′SL) present in collagen α1(I) and α2(I) mRNAs, but it interacts with STRAP with its C-terminal domain, which is not involved in binding 5′SL. Being tethered to collagen mRNAs, STRAP prevents unrestricted translation, primarily that of collagen α2(I) mRNAs, by interacting with eukaryotic translation initiation factor 4A (eIF4A). In the absence of STRAP, more collagen α2(I) mRNA can be pulled down with eIF4A, and collagen α2(I) mRNA is unrestrictedly loaded onto the polysomes. This results in an imbalance of synthesis of α1(I) and α2(I) polypeptides, in hypermodifications of α1(I) polypeptide, and in inefficient assembly of the polypeptides into a collagen trimer and their secretion as monomers. These defects can be partially restored by supplementing STRAP. Thus, we discovered STRAP as a novel regulator of the coordinated translation of collagen mRNAs.

In Vivo Analysis of Troponin C Knock-In (A8V) Mice: Evidence that TNNC1 Is a Hypertrophic Cardiomyopathy Susceptibility Gene

Background— Mutations in thin-filament proteins have been linked to hypertrophic cardiomyopathy, but it has never been demonstrated that variants identified in the TNNC1 (gene encoding troponin C) can evoke cardiac remodeling in vivo. The goal of this study was to determine whether TNNC1 can be categorized as an hypertrophic cardiomyopathy susceptibility gene, such that a mouse model can recapitulate the clinical presentation of the proband. Methods and Results— The TNNC1-A8V proband diagnosed with severe obstructive hypertrophic cardiomyopathy at 34 years of age exhibited mild-to-moderate thickening in left and right ventricular walls, decreased left ventricular dimensions, left atrial enlargement, and hyperdynamic left ventricular systolic function. Genetically engineered knock-in (KI) mice containing the A8V mutation (heterozygote=KI-TnC-A8V+/−; homozygote=KI-TnC-A8V+/+) were characterized by echocardiography and pressure–volume studies. Three-month-old KI-TnC-A8V+/+ mice displayed decreased ventricular dimensions, mild diastolic dysfunction, and enhanced systolic function, whereas KI-TnC-A8V+/− mice displayed cardiac restriction at 14 months of age. KI hearts exhibited atrial enlargement, papillary muscle hypertrophy, and fibrosis. Liquid chromatography–mass spectroscopy was used to determine incorporation of mutant cardiac troponin C (≈21%) into the KI-TnC-A8V+/− cardiac myofilament. Reduced diastolic sarcomeric length, increased shortening, and prolonged Ca2+ and contractile transients were recorded in intact KI-TnC-A8V+/− and KI-TnC-A8V+/+ cardiomyocytes. Ca2+ sensitivity of contraction in skinned fibers increased with mutant gene dose: KI-TnC-A8V+/+>KI-TnC-A8V+/−>wild-type, whereas KI-TnC-A8V+/+ relaxed more slowly on flash photolysis of diazo-2. Conclusions— The TNNC1-A8V mutant increases the Ca2+-binding affinity of the thin filament and elicits changes in Ca2+ homeostasis and cellular remodeling, which leads to diastolic dysfunction. These in vivo alterations further implicate the role of TNNC1 mutations in the development of cardiomyopathy.Background—Mutations in thin-filament proteins have been linked to hypertrophic cardiomyopathy, but it has never been demonstrated that variants identified in the TNNC1 (gene encoding troponin C) can evoke cardiac remodeling in vivo. The goal of this study was to determine whether TNNC1 can be categorized as an hypertrophic cardiomyopathy susceptibility gene, such that a mouse model can recapitulate the clinical presentation of the proband. Methods and Results—The TNNC1-A8V proband diagnosed with severe obstructive hypertrophic cardiomyopathy at 34 years of age exhibited mild-to-moderate thickening in left and right ventricular walls, decreased left ventricular dimensions, left atrial enlargement, and hyperdynamic left ventricular systolic function. Genetically engineered knock-in (KI) mice containing the A8V mutation (heterozygote=KI-TnC-A8V+/−; homozygote=KI-TnC-A8V+/+) were characterized by echocardiography and pressure–volume studies. Three-month-old KI-TnC-A8V+/+ mice displayed decreased ventricular dimensions, mild diastolic dysfunction, and enhanced systolic function, whereas KI-TnC-A8V+/− mice displayed cardiac restriction at 14 months of age. KI hearts exhibited atrial enlargement, papillary muscle hypertrophy, and fibrosis. Liquid chromatography–mass spectroscopy was used to determine incorporation of mutant cardiac troponin C (≈21%) into the KI-TnC-A8V+/− cardiac myofilament. Reduced diastolic sarcomeric length, increased shortening, and prolonged Ca2+ and contractile transients were recorded in intact KI-TnC-A8V+/− and KI-TnC-A8V+/+ cardiomyocytes. Ca2+ sensitivity of contraction in skinned fibers increased with mutant gene dose: KI-TnC-A8V+/+>KI-TnC-A8V+/−>wild-type, whereas KI-TnC-A8V+/+ relaxed more slowly on flash photolysis of diazo-2. Conclusions—The TNNC1-A8V mutant increases the Ca2+-binding affinity of the thin filament and elicits changes in Ca2+ homeostasis and cellular remodeling, which leads to diastolic dysfunction. These in vivo alterations further implicate the role of TNNC1 mutations in the development of cardiomyopathy.

Identification and validation of differentially expressed transcripts by RNA-sequencing of formalin-fixed, paraffin-embedded (FFPE) lung tissue from patients with Idiopathic Pulmonary Fibrosis

BackgroundIdiopathic Pulmonary Fibrosis (IPF) is a lethal lung disease of unknown etiology. A major limitation in transcriptomic profiling of lung tissue in IPF has been a dependence on snap-frozen fresh tissues (FF). In this project we sought to determine whether genome scale transcript profiling using RNA Sequencing (RNA-Seq) could be applied to archived Formalin-Fixed Paraffin-Embedded (FFPE) IPF tissues.ResultsWe isolated total RNA from 7 IPF and 5 control FFPE lung tissues and performed 50 base pair paired-end sequencing on Illumina 2000 HiSeq. TopHat2 was used to map sequencing reads to the human genome. On average ~62 million reads (53.4% of ~116 million reads) were mapped per sample. 4,131 genes were differentially expressed between IPF and controls (1,920 increased and 2,211 decreased (FDR < 0.05). We compared our results to differentially expressed genes calculated from a previously published dataset generated from FF tissues analyzed on Agilent microarrays (GSE47460). The overlap of differentially expressed genes was very high (760 increased and 1,413 decreased, FDR < 0.05). Only 92 differentially expressed genes changed in opposite directions. Pathway enrichment analysis performed using MetaCore confirmed numerous IPF relevant genes and pathways including extracellular remodeling, TGF-beta, and WNT. Gene network analysis of MMP7, a highly differentially expressed gene in both datasets, revealed the same canonical pathways and gene network candidates in RNA-Seq and microarray data. For validation by NanoString nCounter® we selected 35 genes that had a fold change of 2 in at least one dataset (10 discordant, 10 significantly differentially expressed in one dataset only and 15 concordant genes). High concordance of fold change and FDR was observed for each type of the samples (FF vs FFPE) with both microarrays (r = 0.92) and RNA-Seq (r = 0.90) and the number of discordant genes was reduced to four.ConclusionsOur results demonstrate that RNA sequencing of RNA obtained from archived FFPE lung tissues is feasible. The results obtained from FFPE tissue are highly comparable to FF tissues. The ability to perform RNA-Seq on archived FFPE IPF tissues should greatly enhance the availability of tissue biopsies for research in IPF.

Transcriptome profiles in sarcoidosis and their potential role in disease prediction

Purpose of review Sarcoidosis is a systemic disease defined by the presence of nonnecrotizing granuloma in the absence of any known cause. Although the heterogeneity of sarcoidosis is well characterized clinically, the transcriptome of sarcoidosis and underlying molecular mechanisms are not. The signal of all transcripts, small and long noncoding RNAs, can be detected using microarrays or RNA-Sequencing. Analyzing the transcriptome of tissues that are directly affected by granulomas is of great importance to understand biology of the disease and may be predictive of disease and treatment outcome. Recent findings Multiple genome wide expression studies performed on sarcoidosis affected tissues were published in the last 11 years. Published studies focused on differences in gene expression between sarcoidosis vs. control tissues, stable vs. progressive sarcoidosis, as well as sarcoidosis vs. other diseases. Strikingly, all these transcriptomics data confirm the key role of TH1 immune response in sarcoidosis and particularly of interferon-&ggr; (IFN-&ggr;) and type I IFN-driven signaling pathways. Summary The steps toward transcriptomics of sarcoidosis in precision medicine highlight the potentials of this approach. Large prospective follow-up studies are required to identify signatures predictive of disease progression and outcome.

Impact of Transcriptomics on Our Understanding of Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease characterized by aberrant remodeling of the lung parenchyma with extensive changes to the phenotypes of all lung resident cells. The introduction of transcriptomics, genome scale profiling of thousands of RNA transcripts, caused a significant inversion in IPF research. Instead of generating hypotheses based on animal models of disease, or biological plausibility, with limited validation in humans, investigators were able to generate hypotheses based on unbiased molecular analysis of human samples and then use animal models of disease to test their hypotheses. In this review, we describe the insights made from transcriptomic analysis of human IPF samples. We describe how transcriptomic studies led to identification of novel genes and pathways involved in the human IPF lung such as: matrix metalloproteinases, WNT pathway, epithelial genes, role of microRNAs among others, as well as conceptual insights such as the involvement of developmental pathways and deep shifts in epithelial and fibroblast phenotypes. The impact of lung and transcriptomic studies on disease classification, endotype discovery, and reproducible biomarkers is also described in detail. Despite these impressive achievements, the impact of transcriptomic studies has been limited because they analyzed bulk tissue and did not address the cellular and spatial heterogeneity of the IPF lung. We discuss new emerging technologies and applications, such as single-cell RNAseq and microenvironment analysis that may address cellular and spatial heterogeneity. We end by making the point that most current tissue collections and resources are not amenable to analysis using the novel technologies. To take advantage of the new opportunities, we need new efforts of sample collections, this time focused on access to all the microenvironments and cells in the IPF lung.

Data challenges of biomedical researchers in the age of omics

Background High-throughput technologies are rapidly generating large amounts of diverse omics data. Although this offers a great opportunity, it also poses great challenges as data analysis becomes more complex. The purpose of this study was to identify the main challenges researchers face in analyzing data, and how academic libraries can support them in this endeavor. Methods A multimodal needs assessment analysis combined an online survey sent to 860 Yale-affiliated researchers (176 responded) and 15 in-depth one-on-one semi-structured interviews. Interviews were recorded, transcribed, and analyzed using NVivo 10 software according to the thematic analysis approach. Results The survey response rate was 20%. Most respondents (78%) identified lack of adequate data analysis training (e.g., R, Python) as a main challenge, in addition to not having the proper database or software (54%) to expedite analysis. Two main themes emerged from the interviews: personnel and training needs. Researchers feel they could improve data analyses practices by having better access to the appropriate bioinformatics expertise, and/or training in data analyses tools. They also reported lack of time to acquire expertise in using bioinformatics tools and poor understanding of the resources available to facilitate analysis. Conclusions The main challenges identified by our study are: lack of adequate training for data analysis (including need to learn scripting language), need for more personnel at the University to provide data analysis and training, and inadequate communication between bioinformaticians and researchers. The authors identified the positive impact of medical and/or science libraries by establishing bioinformatics support to researchers.

In Vivo Analysis of Troponin C Knock-In (A8V) MiceCLINICAL PERSPECTIVE: Evidence that TNNC1 Is a Hypertrophic Cardiomyopathy Susceptibility Gene

Background— Mutations in thin-filament proteins have been linked to hypertrophic cardiomyopathy, but it has never been demonstrated that variants identified in the TNNC1 (gene encoding troponin C) can evoke cardiac remodeling in vivo. The goal of this study was to determine whether TNNC1 can be categorized as an hypertrophic cardiomyopathy susceptibility gene, such that a mouse model can recapitulate the clinical presentation of the proband. Methods and Results— The TNNC1-A8V proband diagnosed with severe obstructive hypertrophic cardiomyopathy at 34 years of age exhibited mild-to-moderate thickening in left and right ventricular walls, decreased left ventricular dimensions, left atrial enlargement, and hyperdynamic left ventricular systolic function. Genetically engineered knock-in (KI) mice containing the A8V mutation (heterozygote=KI-TnC-A8V+/−; homozygote=KI-TnC-A8V+/+) were characterized by echocardiography and pressure–volume studies. Three-month-old KI-TnC-A8V+/+ mice displayed decreased ventricular dimensions, mild diastolic dysfunction, and enhanced systolic function, whereas KI-TnC-A8V+/− mice displayed cardiac restriction at 14 months of age. KI hearts exhibited atrial enlargement, papillary muscle hypertrophy, and fibrosis. Liquid chromatography–mass spectroscopy was used to determine incorporation of mutant cardiac troponin C (≈21%) into the KI-TnC-A8V+/− cardiac myofilament. Reduced diastolic sarcomeric length, increased shortening, and prolonged Ca2+ and contractile transients were recorded in intact KI-TnC-A8V+/− and KI-TnC-A8V+/+ cardiomyocytes. Ca2+ sensitivity of contraction in skinned fibers increased with mutant gene dose: KI-TnC-A8V+/+>KI-TnC-A8V+/−>wild-type, whereas KI-TnC-A8V+/+ relaxed more slowly on flash photolysis of diazo-2. Conclusions— The TNNC1-A8V mutant increases the Ca2+-binding affinity of the thin filament and elicits changes in Ca2+ homeostasis and cellular remodeling, which leads to diastolic dysfunction. These in vivo alterations further implicate the role of TNNC1 mutations in the development of cardiomyopathy.Background—Mutations in thin-filament proteins have been linked to hypertrophic cardiomyopathy, but it has never been demonstrated that variants identified in the TNNC1 (gene encoding troponin C) can evoke cardiac remodeling in vivo. The goal of this study was to determine whether TNNC1 can be categorized as an hypertrophic cardiomyopathy susceptibility gene, such that a mouse model can recapitulate the clinical presentation of the proband. Methods and Results—The TNNC1-A8V proband diagnosed with severe obstructive hypertrophic cardiomyopathy at 34 years of age exhibited mild-to-moderate thickening in left and right ventricular walls, decreased left ventricular dimensions, left atrial enlargement, and hyperdynamic left ventricular systolic function. Genetically engineered knock-in (KI) mice containing the A8V mutation (heterozygote=KI-TnC-A8V+/−; homozygote=KI-TnC-A8V+/+) were characterized by echocardiography and pressure–volume studies. Three-month-old KI-TnC-A8V+/+ mice displayed decreased ventricular dimensions, mild diastolic dysfunction, and enhanced systolic function, whereas KI-TnC-A8V+/− mice displayed cardiac restriction at 14 months of age. KI hearts exhibited atrial enlargement, papillary muscle hypertrophy, and fibrosis. Liquid chromatography–mass spectroscopy was used to determine incorporation of mutant cardiac troponin C (≈21%) into the KI-TnC-A8V+/− cardiac myofilament. Reduced diastolic sarcomeric length, increased shortening, and prolonged Ca2+ and contractile transients were recorded in intact KI-TnC-A8V+/− and KI-TnC-A8V+/+ cardiomyocytes. Ca2+ sensitivity of contraction in skinned fibers increased with mutant gene dose: KI-TnC-A8V+/+>KI-TnC-A8V+/−>wild-type, whereas KI-TnC-A8V+/+ relaxed more slowly on flash photolysis of diazo-2. Conclusions—The TNNC1-A8V mutant increases the Ca2+-binding affinity of the thin filament and elicits changes in Ca2+ homeostasis and cellular remodeling, which leads to diastolic dysfunction. These in vivo alterations further implicate the role of TNNC1 mutations in the development of cardiomyopathy.

In Vivo Analysis of Troponin C Knock-In (A8V) MiceCLINICAL PERSPECTIVE

Background— Mutations in thin-filament proteins have been linked to hypertrophic cardiomyopathy, but it has never been demonstrated that variants identified in the TNNC1 (gene encoding troponin C) can evoke cardiac remodeling in vivo. The goal of this study was to determine whether TNNC1 can be categorized as an hypertrophic cardiomyopathy susceptibility gene, such that a mouse model can recapitulate the clinical presentation of the proband. Methods and Results— The TNNC1-A8V proband diagnosed with severe obstructive hypertrophic cardiomyopathy at 34 years of age exhibited mild-to-moderate thickening in left and right ventricular walls, decreased left ventricular dimensions, left atrial enlargement, and hyperdynamic left ventricular systolic function. Genetically engineered knock-in (KI) mice containing the A8V mutation (heterozygote=KI-TnC-A8V+/−; homozygote=KI-TnC-A8V+/+) were characterized by echocardiography and pressure–volume studies. Three-month-old KI-TnC-A8V+/+ mice displayed decreased ventricular dimensions, mild diastolic dysfunction, and enhanced systolic function, whereas KI-TnC-A8V+/− mice displayed cardiac restriction at 14 months of age. KI hearts exhibited atrial enlargement, papillary muscle hypertrophy, and fibrosis. Liquid chromatography–mass spectroscopy was used to determine incorporation of mutant cardiac troponin C (≈21%) into the KI-TnC-A8V+/− cardiac myofilament. Reduced diastolic sarcomeric length, increased shortening, and prolonged Ca2+ and contractile transients were recorded in intact KI-TnC-A8V+/− and KI-TnC-A8V+/+ cardiomyocytes. Ca2+ sensitivity of contraction in skinned fibers increased with mutant gene dose: KI-TnC-A8V+/+>KI-TnC-A8V+/−>wild-type, whereas KI-TnC-A8V+/+ relaxed more slowly on flash photolysis of diazo-2. Conclusions— The TNNC1-A8V mutant increases the Ca2+-binding affinity of the thin filament and elicits changes in Ca2+ homeostasis and cellular remodeling, which leads to diastolic dysfunction. These in vivo alterations further implicate the role of TNNC1 mutations in the development of cardiomyopathy.Background—Mutations in thin-filament proteins have been linked to hypertrophic cardiomyopathy, but it has never been demonstrated that variants identified in the TNNC1 (gene encoding troponin C) can evoke cardiac remodeling in vivo. The goal of this study was to determine whether TNNC1 can be categorized as an hypertrophic cardiomyopathy susceptibility gene, such that a mouse model can recapitulate the clinical presentation of the proband. Methods and Results—The TNNC1-A8V proband diagnosed with severe obstructive hypertrophic cardiomyopathy at 34 years of age exhibited mild-to-moderate thickening in left and right ventricular walls, decreased left ventricular dimensions, left atrial enlargement, and hyperdynamic left ventricular systolic function. Genetically engineered knock-in (KI) mice containing the A8V mutation (heterozygote=KI-TnC-A8V+/−; homozygote=KI-TnC-A8V+/+) were characterized by echocardiography and pressure–volume studies. Three-month-old KI-TnC-A8V+/+ mice displayed decreased ventricular dimensions, mild diastolic dysfunction, and enhanced systolic function, whereas KI-TnC-A8V+/− mice displayed cardiac restriction at 14 months of age. KI hearts exhibited atrial enlargement, papillary muscle hypertrophy, and fibrosis. Liquid chromatography–mass spectroscopy was used to determine incorporation of mutant cardiac troponin C (≈21%) into the KI-TnC-A8V+/− cardiac myofilament. Reduced diastolic sarcomeric length, increased shortening, and prolonged Ca2+ and contractile transients were recorded in intact KI-TnC-A8V+/− and KI-TnC-A8V+/+ cardiomyocytes. Ca2+ sensitivity of contraction in skinned fibers increased with mutant gene dose: KI-TnC-A8V+/+>KI-TnC-A8V+/−>wild-type, whereas KI-TnC-A8V+/+ relaxed more slowly on flash photolysis of diazo-2. Conclusions—The TNNC1-A8V mutant increases the Ca2+-binding affinity of the thin filament and elicits changes in Ca2+ homeostasis and cellular remodeling, which leads to diastolic dysfunction. These in vivo alterations further implicate the role of TNNC1 mutations in the development of cardiomyopathy.