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Gene remodeling in type 2 diabetic cardiomyopathy and its phenotypic rescue with SERCA2a.

Karakikes I, Kim M, Hadri L, Sakata S, Sun Y, Zhang W, Chemaly ER, Hajjar RJ, Lebeche D - PLoS ONE (2009)

Bottom Line: Diabetes-associated myocardial dysfunction results in altered gene expression in the heart.Diabetic cardiomyopathic hearts have reduced levels of SERCA2a.In isolated cardiomyocytes in vitro, SERCA2a overexpression significantly modified the expression of a number of transcripts known to be involved in insulin signaling, glucose metabolism and cardiac remodeling.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Center, Mount Sinai School of Medicine, New York, New York, United States of America.

ABSTRACT

Background/aim: Diabetes-associated myocardial dysfunction results in altered gene expression in the heart. We aimed to investigate the changes in gene expression profiles accompanying diabetes-induced cardiomyopathy and its phenotypic rescue by restoration of SERCA2a expression.

Methods/results: Using the Otsuka Long-Evans Tokushima Fatty rat model of type 2 diabetes and the Agilent rat microarray chip, we analyzed gene expression by comparing differential transcriptional changes in age-matched control versus diabetic hearts and diabetic hearts that received gene transfer of SERCA2a. Microarray expression profiles of selected genes were verified with real-time qPCR and immunoblotting. Our analysis indicates that diabetic cardiomyopathy is associated with a downregulation of transcripts. Diabetic cardiomyopathic hearts have reduced levels of SERCA2a. SERCA2a gene transfer in these hearts reduced diabetes-associated hypertrophy, and differentially modulated the expression of 76 genes and reversed the transcriptional profile induced by diabetes. In isolated cardiomyocytes in vitro, SERCA2a overexpression significantly modified the expression of a number of transcripts known to be involved in insulin signaling, glucose metabolism and cardiac remodeling.

Conclusion: This investigation provided insight into the pathophysiology of cardiac remodeling and the potential role of SERCA2a normalization in multiple pathways in diabetic cardiomyopathy.

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Related in: MedlinePlus

Validation of microarray data by Immunoblotting.Immunoblotting analysis of selected proteins was performed on samples from control, DM and Serca2a-transduced hearts. A representative blot from 2–3 experiments is shown. sFRP4, secreted frizzled-related protein 4; DSC2, desmocollin 2. GAPDH is also shown to verify protein loading. Cont, control non-diabetic; DM, hearts with diabetes mellitus; Serca2a, diabetic hearts infected with Ad.Serca2a; MW, molecular weight.
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pone-0006474-g004: Validation of microarray data by Immunoblotting.Immunoblotting analysis of selected proteins was performed on samples from control, DM and Serca2a-transduced hearts. A representative blot from 2–3 experiments is shown. sFRP4, secreted frizzled-related protein 4; DSC2, desmocollin 2. GAPDH is also shown to verify protein loading. Cont, control non-diabetic; DM, hearts with diabetes mellitus; Serca2a, diabetic hearts infected with Ad.Serca2a; MW, molecular weight.

Mentions: qRT-PCR was used to verify the oligonucleotide microarray expression data. The differential expressions of 19 randomly selected genes identified by microarray were validated. Relative transcript levels were determined in control non-diabetic and diabetic hearts compared to diabetic hearts transduced with SERCA2a. qRT-PCR analysis confirmed that the majority of genes were statistically differentially regulated (P<0.05) in DM+SERCA2a samples (Table 4). Although the pattern of fold-change of many of the genes, as determined by qRT-PCR analysis, correlated with fold-change reported by microarray analysis, there were some discrepancies in at least 5 cases. The greatest discrepancy was seen in the expression of ryanodine receptor 2 (RyR2), which was reported as 11.4-fold up-regulated in DM hearts by microarray analysis compared with a modest 1.19-fold by qRT-PCR (Table 4).The second major discrepancy was observed in the expression levels of GLUT4, which was reported as 1.30-fold up-regulated by microarray analysis compared with 0.21-fold down-regulation by qRT-PCR (Table 4) in DM samples. GLUT4 expression is widely reported to be down-regulated in the diabetic heart, consistent with our qRT-PCR data. Immunoblotting of GLUT4 protein expression confirmed this observation (Fig. 4) in deference to the microarray data. The other discrepancy between qRT-PCR and microarray was seen with the relative expression of MMP7, 7.08-fold versus 1.63-fold, respectively (Table 4). FGF4 and Cacnb3, although displayed on the microarray, were not amplified by qRT-PCR. Nevertheless, the qRT-PCR results are in general concordance with the microarray data and serve to verify the results. The variations observed could be due to the differences in the techniques and/or the sequences of oligonucleotide probes used in the two approaches.


Gene remodeling in type 2 diabetic cardiomyopathy and its phenotypic rescue with SERCA2a.

Karakikes I, Kim M, Hadri L, Sakata S, Sun Y, Zhang W, Chemaly ER, Hajjar RJ, Lebeche D - PLoS ONE (2009)

Validation of microarray data by Immunoblotting.Immunoblotting analysis of selected proteins was performed on samples from control, DM and Serca2a-transduced hearts. A representative blot from 2–3 experiments is shown. sFRP4, secreted frizzled-related protein 4; DSC2, desmocollin 2. GAPDH is also shown to verify protein loading. Cont, control non-diabetic; DM, hearts with diabetes mellitus; Serca2a, diabetic hearts infected with Ad.Serca2a; MW, molecular weight.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2714457&req=5

pone-0006474-g004: Validation of microarray data by Immunoblotting.Immunoblotting analysis of selected proteins was performed on samples from control, DM and Serca2a-transduced hearts. A representative blot from 2–3 experiments is shown. sFRP4, secreted frizzled-related protein 4; DSC2, desmocollin 2. GAPDH is also shown to verify protein loading. Cont, control non-diabetic; DM, hearts with diabetes mellitus; Serca2a, diabetic hearts infected with Ad.Serca2a; MW, molecular weight.
Mentions: qRT-PCR was used to verify the oligonucleotide microarray expression data. The differential expressions of 19 randomly selected genes identified by microarray were validated. Relative transcript levels were determined in control non-diabetic and diabetic hearts compared to diabetic hearts transduced with SERCA2a. qRT-PCR analysis confirmed that the majority of genes were statistically differentially regulated (P<0.05) in DM+SERCA2a samples (Table 4). Although the pattern of fold-change of many of the genes, as determined by qRT-PCR analysis, correlated with fold-change reported by microarray analysis, there were some discrepancies in at least 5 cases. The greatest discrepancy was seen in the expression of ryanodine receptor 2 (RyR2), which was reported as 11.4-fold up-regulated in DM hearts by microarray analysis compared with a modest 1.19-fold by qRT-PCR (Table 4).The second major discrepancy was observed in the expression levels of GLUT4, which was reported as 1.30-fold up-regulated by microarray analysis compared with 0.21-fold down-regulation by qRT-PCR (Table 4) in DM samples. GLUT4 expression is widely reported to be down-regulated in the diabetic heart, consistent with our qRT-PCR data. Immunoblotting of GLUT4 protein expression confirmed this observation (Fig. 4) in deference to the microarray data. The other discrepancy between qRT-PCR and microarray was seen with the relative expression of MMP7, 7.08-fold versus 1.63-fold, respectively (Table 4). FGF4 and Cacnb3, although displayed on the microarray, were not amplified by qRT-PCR. Nevertheless, the qRT-PCR results are in general concordance with the microarray data and serve to verify the results. The variations observed could be due to the differences in the techniques and/or the sequences of oligonucleotide probes used in the two approaches.

Bottom Line: Diabetes-associated myocardial dysfunction results in altered gene expression in the heart.Diabetic cardiomyopathic hearts have reduced levels of SERCA2a.In isolated cardiomyocytes in vitro, SERCA2a overexpression significantly modified the expression of a number of transcripts known to be involved in insulin signaling, glucose metabolism and cardiac remodeling.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Center, Mount Sinai School of Medicine, New York, New York, United States of America.

ABSTRACT

Background/aim: Diabetes-associated myocardial dysfunction results in altered gene expression in the heart. We aimed to investigate the changes in gene expression profiles accompanying diabetes-induced cardiomyopathy and its phenotypic rescue by restoration of SERCA2a expression.

Methods/results: Using the Otsuka Long-Evans Tokushima Fatty rat model of type 2 diabetes and the Agilent rat microarray chip, we analyzed gene expression by comparing differential transcriptional changes in age-matched control versus diabetic hearts and diabetic hearts that received gene transfer of SERCA2a. Microarray expression profiles of selected genes were verified with real-time qPCR and immunoblotting. Our analysis indicates that diabetic cardiomyopathy is associated with a downregulation of transcripts. Diabetic cardiomyopathic hearts have reduced levels of SERCA2a. SERCA2a gene transfer in these hearts reduced diabetes-associated hypertrophy, and differentially modulated the expression of 76 genes and reversed the transcriptional profile induced by diabetes. In isolated cardiomyocytes in vitro, SERCA2a overexpression significantly modified the expression of a number of transcripts known to be involved in insulin signaling, glucose metabolism and cardiac remodeling.

Conclusion: This investigation provided insight into the pathophysiology of cardiac remodeling and the potential role of SERCA2a normalization in multiple pathways in diabetic cardiomyopathy.

Show MeSH
Related in: MedlinePlus