Unknown,Transcriptomics,Genomics,Proteomics

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Therapy with bone marrow cells recovers gene expression alterations in hearts of mice with chronic chagasic cardiomyopathy

ABSTRACT: Chronic chagasic cardiomyopathy is one of the leading causes of heart failure in Latin American countries, being associated with intense inflammatory response and fibrosis. We have previously shown that bone marrow mononuclear cell (BMC) transplantation improves inflammation, fibrosis and ventricular diameter in hearts of mice with chronic Chagas’ disease. Here we investigated alterations of gene expression in the hearts of chronic chagasic mice submitted or not to BMC therapy. C57Bl/6 mice chronically infected with T. cruzi (6 months) were transplanted with BMC or saline i.v. and sacrificed 2 months later. RNA was extracted from the hearts of normal controls, chagasic and BMC transplanted mice and microarray analysis was performed using MO30k oligonucleotide arrays. Out of the 9390 unigenes quantified in all samples, 1702 had their expression altered in chronic chagasic hearts compared to those of normal mice. Major categories of significantly upregulated genes were related to inflammation, fibrosis and immune responses, while genes involved in mitochondrion function were downregulated. When BMC-treated chagasic hearts were compared to infected mice, 1631 (96%) of the alterations detected in infected hearts were not found, although an additional 109 genes were altered by treatment, indicating a remarkable 84% transcriptomic recovery. Immunofluorescence and morphometric analyses confirmed the effects of BMC therapy in the pattern of inflammatory-immune response and expression of adhesion molecules. Our results demonstrate important immunomodulatory effects of BMC therapy in chagasic cardiomyopathy and indicate potentially relevant factors involved in the pathogenesis of the disease that may provide new therapeutic targets. We compared RNA samples extracted from whole hearts of 4 control, 4 chagasic and 4 BMC-treated chagasic mice by analyzing hybridization to microarrays printed by Duke University (http://www.ncbi.nlm.nih.gove/geo/query/acc.cgi?acc=GPL8938) spotted with MO30k mouse Operon version 3.0 70-mer oligonucleotides. The hybridization protocol (see Soares et al, 2010), the slide type and the scanner settings were uniform throughout the entire experiment to minimize the technical noise. Briefly, 20 ug total RNA extracted in Trizol from each of the twelve samples (individual hearts) was reverse transcribed in the presence of fluorescent Alexa Fluor® 555- and Alexa Fluor®647-aha-dUTPs (Invitrogen, Carlsbad, CA) to obtain labeled cDNA. Red and green labeled samples of biological replicas were then co-hybridized (“multiple yellow” strategy, 22) overnight at 50° C. After washing (0.1% SDS and 1% SSC) to remove the non-hybridized cDNA, each array was scanned at 630V (635 nm) and 580V (532 nm) with GenePix 4100B scanner (Axon Instruments, Union City, CA) and images were primarily analyzed with GenePixPro 6.0 (Molecular Devices, Sunnyvale, CA). Microarray data were processed as described previously (Soares et al, 2010). A gene was considered as significantly up- or down-regulated when comparing four hearts from one condition to those from another if the absolute fold change was >1.5x and the p-vlaue of the Sutdent”s heteroscedastic t-test of equality of the means of the distributions with a Bonferroni-type adjustment for each redundancy group (set of spots probing the same gene) was <0.05.

ORGANISM(S): Mus musculus

SUBMITTER: Dumitru Iacobas

PROVIDER: E-GEOD-24088 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Although bone marrow-derived mononuclear cells (BMNC) have been extensively used in cell therapy for cardiac diseases, little mechanistic information is available to support reports of their efficacy. To address this shortcoming, we compared structural and functional recovery and associated global gene expression profiles in post-ischaemic myocardium treated with BMNC transplantation. BMNC suspensions were injected into cardiac scar tissue 10 days after experimental myocardial infarction. Six weeks later, mice undergoing BMNC therapy were found to have normalized antibody repertoire and improved cardiac performance measured by ECG, treadmill exercise time and echocardiography. After functional testing, gene expression profiles in cardiac tissue were evaluated using high-density oligonucleotide arrays. Expression of more than 18% of the 11981 quantified unigenes was significantly altered in the infarcted hearts. BMNC therapy restored expression of 2099 (96.2%) of the genes that were altered by infarction but led to altered expression of 286 other genes, considered to be a side effect of the treatment. Transcriptional therapeutic efficacy, a metric calculated using a formula that incorporates both recovery and side effect of treatment, was 73%. In conclusion, our results confirm a beneficial role for bone marrow-derived cell therapy and provide new information on molecular mechanisms operating after BMNC transplantation on post ischemic heart failure in mice. We compared RNA samples extracted from whole hearts of infarcted mouse myocardium treated with bone marrow mononuclear cells control with untreated infarcted and control mice samples by analyzing hybridization to AECOM 32k mouse microarrays (http://microarray1k.aecom.yu.edu/) spotted with Operon version 3.0 70-mer oligonucleotides. The hybridization protocol and the slide type were uniform throughout the entire experiment to minimize the technical noise. Treated, control (sham) and infarcted red-labeled heart samples were hybridized against an in-house prepared green-labeled universal mouse reference.

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Project description:After myocardial infarction (MI) activation of the immune system and inflammatory mechanisms, among others, can lead to ventricular remodeling and heart failure (HF). Interaction between these systemic alterations and corresponding changes in the heart has not been extensively examined in the setting of chronic ischemia. The main purpose of this study was to investigate alterations in cardiac gene and systemic cytokine profile in mice with post-ischemic HF. Plasma was tested for IgM and IgG anti-heart reactive repertoire and inflammatory cytokines. Heart samples were assayed for gene expression. Ischemic HF significantly increased the levels of serum IgM (by 5.2 fold) and IgG (by 3.6 fold) associated with remarkable content of anti-heart specificity. Comparable increase was observed in levels of circulating pro-inflammatory cytokines, such as IL-1Î² (3.8x) and TNF-Î± (6.0x). IFN-gamma was also increased in the MI group by 3.1x. However, IL-4 and IL-10 showed no significant difference between MI and sham groups. Chemokines such as MCP-1 and IL-8 were enhanced in the plasma of infarcted mice. We identified 2079 well annotated unigenes that were significantly regulated by the post-ischemic HF. Complement activation and immune response was among the most up-regulated processes. Interestingly, 21 out of the 101 quantified unigenes involved in inflammatory response were significantly up-regulated and none were down-regulated. These data indicate that post-ischemic heart remodeling is accompanied by immune mediated mechanisms that act both systemically and locally. We compared RNA samples extracted from whole hearts of control and infarcted mice samples by analyzing hybridization to AECOM 32k mouse microarrays (http://microarray1k.aecom.yu.edu/) spotted with Operon version 3.0 70-mer oligonucleotides. The hybridization protocol, the slide type and the scanner settings were uniform throughout the entire experiment to minimize the technical noise. Control (sham) and infarcted red-labeled heart samples were hybridized against an in-house prepared green-labeled universal mouse reference.

Project description:Expression level, control and intercoordination of 66 selected heart rhythm determinant (HRD) genes were compared in atria and ventricles of 4 male and 4 female adult mice. We found that genes encoding various adrenergic receptors, ankyrins, ion channels and transporters, connexins and other components of the intercalated discs form a complex network that is chamber dependent and differs between the two sexes. In addition, most HRD genes in atria had higher expression in males than in females, while in ventricles expression levels were mostly higher in females than in males. Moreover, significant chamber-differences were observed between the sexes, with higher expression in atria than ventricles for males and higher expression in ventricles than atria for females. We have ranked the selected genes according to their prominence in controlling the HRD gene web through expression coordination with the other web genes and protecting the web though their own expression stability. Interestingly, the prominence hierarchy was substantially different between the two sexes. Taken together these findings indicate that the organizational principles of the heart rhythm transcriptome are sex-dependent, with the newly introduced prominence analysis allowing identification of genes that are pivotal for the sexual dichotomy. Four adult male (M) and 4 adult female (F) mice were decapitated, the hearts removed and atria (A) and ventricles (V) collected in separate tubes. 20 Âµg total RNA extracted in Trizol from each of the 16 samples was reverse transcribed in the presence of fluorescent Alexa FluorÂ®_647 and Alexa FluorÂ®_555-aha-dUTPs (Invitrogen, CA) to obtain labeled cDNAs Red and green-labeled samples of biological replicas were then co-hybridized (âmultiple yellowâ strategy) overnight at 50Â°C with mouse MO36k oligonucleotide arrays printed by Duke University (http://microarray.genome.duke.edu/spotted-arrays) with Operon Mouse Oligo Set, version 4.0. After washing (0.1% SDS and 1% SSC) to remove the non-hybridized cDNA, each array was scanned with GenePix 4000B scanner (MDS, Toronto, Canada) and images were primarily analyzed with GenePixPro 6.0 (Axon Instruments, CA).

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Therapy with bone marrow cells recovers gene expression alterations in hearts of mice with chronic chagasic cardiomyopathy

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