MiR-133a Mimic Alleviates T1DM-Induced Systolic Dysfunction in Akita: An MRI-Based Study

Diabetic cardiomyopathy is a leading cause of heart failure. Developing a novel therapeutic strategy for diabetic cardiomyopathy and characterizing animal models used for diabetes mellitus (DM) are important. Insulin 2 mutant (Ins2+/-) Akita is a spontaneous, genetic, mouse model for T1DM, which is relevant to humans. There are contrasting reports on systolic dysfunction and pathological remodeling (hypertrophy and fibrosis) in Akita heart. Here, we used magnetic resonance imaging (MRI) approach, a gold standard reference for evaluating cardiac function, to measure ejection fraction (indicator of systolic dysfunction) in Akita. Moreover, we performed Wheat Germ Agglutinin (WGA) and hematoxylin and Eosin stainings to determine cardiac hypertrophy, and Masson\u27s Trichrome and picrosirius red stainings to determine cardiac fibrosis in Akita. MiR-133a, an anti-hypertrophy and anti-fibrosis miRNA, is downregulated in Akita heart. We determined if miR-133a mimic treatment could mitigate systolic dysfunction and remodeling in Akita heart. Our MRI results revealed decreased ejection fraction in Akita as compared to WT and increased ejection fraction in miR-133a mimic-treated Akita. We also found that miR-133a mimic treatment mitigates T1DM-induced cardiac hypertrophy and fibrosis in Akita. We conclude that Akita shows cardiac hypertrophy, fibrosis and systolic dysfunction and miR-133a mimic treatment to Akita could ameliorate them

Induction of autophagy markers is associated with attenuation of miR-133a in diabetic heart failure patients undergoing mechanical unloading.

Autophagy is ubiquitous in all forms of heart failure and cardioprotective miR-133a is attenuated in human heart failure. Previous reports from heart failure patients undergoing left ventricular assist device (LVAD) implantation demonstrated that autophagy is upregulated in the LV of the failing human heart. Studies in the murine model show that diabetes downregulates miR-133a. However, the role of miR-133a in the regulation of autophagy in diabetic hearts is unclear. We tested the hypothesis that diabetes exacerbates cardiac autophagy by inhibiting miR-133a in heart failure patients undergoing LVAD implantation. The miRNA assay was performed on the LV of 15 diabetic (D) and 6 non-diabetic (ND) heart failure patients undergoing LVAD implantation. Four ND with highly upregulated and 5 D with highly downregulated miR-133a were analyzed for autophagy markers (Beclin1, LC3B, ATG3) and their upstream regulators (mTOR and AMPK), and hypertrophy marker (beta-myosin heavy chain) by RT-qPCR, Western blotting and immunofluorescence. Our results demonstrate that attenuation of miR-133a in diabetic hearts is associated with the induction of autophagy and hypertrophy, and suppression of mTOR without appreciable difference in AMPK activity. In conclusion, attenuation of miR-133a contributes to the exacerbation of diabetes mediated cardiac autophagy and hypertrophy in heart failure patients undergoing LVAD implantation

Transgenic Expression of miR-133a in the Diabetic Akita Heart Prevents Cardiac Remodeling and Cardiomyopathy

Advanced diabetes mellitus (DM) may have both insulin resistance and deficiency (double DM) that accelerates diabetic cardiomyopathy (DMCM), a cardiac muscle disorder. Reduced cardiac miR-133a, a cardioprotective miRNA, is associated with DMCM. However, it is unclear whether increasing miR-133a levels in the double DM heart could prevent DMCM. We hypothesized that increasing cardiac levels of miR-133a could prevent DMCM in Akita, a mouse model of double DM. To test the hypothesis, we created Akita/miR-133aTg mice, a new strain of Akita where miR-133a is overexpressed in the heart, by crossbreeding male Akita with female cardiac-specific miR-133a transgenic mice. We validated Akita/miR-133aTg mice by genotyping and phenotyping (miR-133a levels in the heart). To determine whether miR-133a overexpression could prevent cardiac remodeling and cardiomyopathy, we evaluated cardiac fibrosis, hypertrophy, and dysfunction (P-V loop) in 13–15 week male WT, Akita, Akita/miR-133aTg, and miR-133aTg mice. Our results revealed that miR-133a overexpression in the Akita heart prevents DM-induced cardiac fibrosis (reduced collagen deposition), hypertrophy (decreased beta-myosin heavy chain), and impaired contractility (downregulated calcium handling protein sarco-endoplasmic reticulum-ATPase-2a). These results demonstrate that increased levels of miR-133a in the DM heart could prevent cardiac remodeling. Our P-V loop analysis showed a trend of decreased cardiac output, stroke volume, and ± dp/dt in Akita, which were blunted in Akita/miR-133aTg heart. These findings suggest that 13–15 week Akita heart undergoes adverse remodeling toward cardiomyopathy, which is prevented by miR-133a overexpression. In addition, increased cardiac miR-133a in the Akita heart did not change blood glucose levels but decreased lipid accumulation in the heart, suggesting inhibition of metabolic remodeling in the heart. Thus, miR-133a could be a promising therapeutic candidate to prevent DMCM

Cardiac transcriptome profiling of diabetic Akita mice using microarray and next generation sequencing.

Although diabetes mellitus (DM) causes cardiomyopathy and exacerbates heart failure, the underlying molecular mechanisms for diabetic cardiomyopathy/heart failure are poorly understood. Insulin2 mutant (Ins2+/-) Akita is a mouse model of T1DM, which manifests cardiac dysfunction. However, molecular changes at cardiac transcriptome level that lead to cardiomyopathy remain unclear. To understand the molecular changes in the heart of diabetic Akita mice, we profiled cardiac transcriptome of Ins2+/- Akita and Ins2+/+ control mice using next generation sequencing (NGS) and microarray, and determined the implications of differentially expressed genes on various heart failure signaling pathways using Ingenuity pathway (IPA) analysis. First, we validated hyperglycemia, increased cardiac fibrosis, and cardiac dysfunction in twelve-week male diabetic Akita. Then, we analyzed the transcriptome levels in the heart. NGS analyses on Akita heart revealed 137 differentially expressed transcripts, where Bone Morphogenic Protein-10 (BMP10) was the most upregulated and hairy and enhancer of split-related (HELT) was the most downregulated gene. Moreover, twelve long non-coding RNAs (lncRNAs) were upregulated. The microarray analyses on Akita heart showed 351 differentially expressed transcripts, where vomeronasal-1 receptor-180 (Vmn1r180) was the most upregulated and WD Repeat Domain 83 Opposite Strand (WDR83OS) was the most downregulated gene. Further, miR-101c and H19 lncRNA were upregulated but Neat1 lncRNA was downregulated in Akita heart. Eleven common genes were upregulated in Akita heart in both NGS and microarray analyses. IPA analyses revealed the role of these differentially expressed genes in key signaling pathways involved in diabetic cardiomyopathy. Our results provide a platform to initiate focused future studies by targeting these genes and/or non-coding RNAs, which are differentially expressed in Akita hearts and are involved in diabetic cardiomyopathy

Metabolites and Genes behind Cardiac Metabolic Remodeling in Mice with Type 1 Diabetes Mellitus

Metabolic remodeling is at the heart of diabetic cardiomyopathy. High glycemic fluctuations increase metabolic stress in the type 1 diabetes mellitus (T1DM) heart. There is a lack of understanding on how metabolites and genes affect metabolic remodeling in the T1DM heart. We hypothesize that differential expression of metabolic genes and metabolites synergistically influence metabolic remodeling preceding T1DM cardiomyopathy. To test our hypothesis, we conducted high throughput analysis of hearts from adult male hyperglycemic Ins2+/− (Akita) and littermate normoglycemic Ins2+/+ (WT) mice. The Akita mouse is a spontaneous, genetic model of T1DM that develops increased levels of consistent glycemic variability without the off-target cardiotoxic effects present in chemically- induced models of T1DM. After validating the presence of a T1DM phenotype, we conducted metabolomics via LC-MS analysis and genomics via next-generation sequencing in left ventricle tissue from the Akita heart. Ingenuity Pathway Analyses revealed that 108 and 30 metabolic pathways were disrupted within the metabolomics and genomics datasets, respectively. Notably, a comparison between the two analyses showed 15 commonly disrupted pathways, including ketogenesis, ketolysis, cholesterol biosynthesis, acetyl CoA hydrolysis, and fatty acid biosynthesis and beta-oxidation. These identified metabolic pathways predicted by the differential expression of metabolites and genes provide the foundation for understanding metabolic remodeling in the T1DM heart. By limited experiment, we revealed a predicted disruption in the metabolites and genes behind T1DM cardiac metabolic derangement. Future studies targeting these genes and metabolites will unravel novel therapies to prevent/improve metabolic remodeling in the T1DM heart

Prevalence and risk factors of hepatitis B and C viruses among hemodialysis patients in Isfahan, Iran

Background: The aim of this study was to assess the prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) and their associated risk factors among hemodialysis patients in Isfahan, Iran in 2011. Materials and Methods : In this cross-sectional study, a total of 499 chronic hemodialysis patients from eight governmental hemodialysis centers were tested. Demographic information, time duration on hemodialysis, history of blood transfusion, and the number of transfused blood units as risk factors to HBV and HCV infections were calculated. The status of HBsAg and anti-HCV was assessed by serological testing. Results: The overall prevalence of HBV-positive and HCV-positive among study population was 1.2% and 5.2%, respectively. Age, sex, and time duration on hemodialysis were not statistically significant in HBV- and HCV-patients compared with other hemodialysis patients (P0 > 0.05). History of blood transfusion and the number of transfused blood units in HBV infected patients were similar to other patients (P > 0.05). The main risk factors in hemodialysis patients infected by HCV were history of blood transfusion and the number of transfused blood units (P < 0.0001). Conclusion: In conclusion, the prevalence of HBV and HCV infections in hemodialysis centers in Isfahan is lower than in some other cities of Iran. History of blood transfusion and the number of blood units transfused might be a risk factor for HCV gaining. However, further studies are needed to assess the role of other demographic and clinical risk factors in these patients

Ingenuity Pathway Analyses (IPA) for key signaling network in Akita heart using differentially expressed genes from NGS analyses.

<p>(A-C) The differentially expressed genes obtained from NGS analyses are involved in several key signaling networks in Akita heart.</p

Ingenuity Pathway Analyses (IPA) for key upstream regulators of diabetic heart failure signaling cascade in Akita heart using differentially expressed genes from microarray analyses.

<p>(A) Upstream regulators activated in Akita heart. (B) Upstream regulators suppressed in Akita heart.</p

Transcriptome profiling of WT and Akita hearts using next generation sequencing (NGS) and microarray analyses.

<p>(A) Top-ten upregulated transcriptome in Akita by NGS analyses. (B) Top-ten downregulated transcriptome in Akita heart by NGS analyses. (C) Top-ten upregulated transcriptome in Akita heart by microarray analyses. (D) Top-ten downregulated transcriptome in Akita heart by microarray analyses. (E) Top-ten upregulated genes in Akita heart common in both NGS and microarray analyses (fold change ≤ 2 or 2 ≥ and <i>p-</i>value < 0.05). All the data are represented as fold change of WT hearts. Values are mean ± SD. N = 3. (F-H) Validation of three upregulated genes in Akita heart using an independent experiment and qPCR analyses. Values are represented as mean ± SEM. N = 3.</p

Differentially expressed long-non-coding RNAs (lncRNAs) in Akita heart.

<p>(A) NGS analyses showing different lncRNAs upregulated in Akita hearts. (B) Microarray analyses of lncRNAs in Akita hearts. Values are mean ± S. D. N = 3. (C) Representative Western blots and densitometric analyses of bands for Nebulin in WT and Akita hearts. Values are mean ± S. E. N = 5. (D) Representative Western blots and densitometric analyses of bands for GABA (A) receptor associated protein like-1 (GABARAPLK1) in WT and Akita hearts. Values are mean ± S. E. N = 5.</p