Hitoo Nishi

MicroRNA-33 encoded by an intron of sterol regulatory element-binding protein 2 (Srebp2) regulates HDL in vivo

Sterol regulatory element-binding protein 2 (SREBP-2) transcription factor has been identified as a key protein in cholesterol metabolism through the transactivation of the LDL receptor and cholesterol biosynthesis genes. Here, we generated mice lacking microRNA (miR)-33, encoded by an intron of the Srebp2, and showed that miR-33 repressed the expression of ATP-binding cassette transporter A1 (ABCA1) protein, a key regulator of HDL synthesis by mediating cholesterol efflux from cells to apolipoprotein A (apoA)-I. In fact, peritoneal macrophages derived from miR-33–deficient mice showed a marked increase in ABCA1 levels and higher apoA-I–dependent cholesterol efflux than those from WT mice. ABCA1 protein levels in liver were also higher in miR-33–deficient mice than in WT mice. Moreover, miR-33–deficient mice had significantly higher serum HDL cholesterol levels than WT mice. These data establish a critical role for miR-33 in the regulation of ABCA1 expression and HDL biogenesis in vivo.

Acute doxorubicin cardiotoxicity is associated with miR-146a-induced inhibition of the neuregulin-ErbB pathway

Aims A significant increase in congestive heart failure (CHF) was reported when the anti-ErbB2 antibody trastuzumab was used in combination with the chemotherapy drug doxorubicin (Dox). The aim of the present study was to investigate the role(s) of miRNAs in acute Dox-induced cardiotoxicity. Methods and results Neuregulin-1-ErbB signalling is essential for maintaining adult cardiac function. We found a significant reduction in ErbB4 expression in the hearts of mice after Dox treatment. Because the proteasome pathway was only partially involved in the reduction of ErbB4 expression, we examined the involvement of microRNAs (miRs) in the reduction of ErbB4 expression. miR-146a was shown to be up-regulated by Dox in neonatal rat cardiac myocytes. Using a luciferase reporter assay and overexpression of miR-146a, we confirmed that miR-146a targets the ErbB4 3′UTR. After Dox treatment, overexpression of miR-146a, as well as that of siRNA against ErbB4, induced cell death in cardiomyocytes. Re-expression of ErbB4 in miR-146a-overexpressing cardiomyocytes ameliorated Dox-induced cell death. To examine the loss of miR-146a function, we constructed ‘decoy’ genes that had tandem complementary sequences for miR-146a in the 3′UTR of a luciferase gene. When miR-146a ‘decoy’ genes were introduced into cardiomyocytes, ErbB4 expression was up-regulated and Dox-induced cell death was reduced. Conclusion These findings suggested that the up-regulation of miR-146a after Dox treatment is involved in acute Dox-induced cardiotoxicity by targeting ErbB4. Inhibition of both ErbB2 and ErbB4 signalling may be one of the reasons why those patients who receive concurrent therapy with Dox and trastuzumab suffer from CHF.

MicroRNA-15b Modulates Cellular ATP Levels and Degenerates Mitochondria via Arl2 in Neonatal Rat Cardiac Myocytes

MicroRNAs (miRNAs or miRs) are small, non-coding RNAs that modulate mRNA stability and post-transcriptional translation. A growing body of evidence indicates that specific miRNAs can affect the cellular function of cardiomyocytes. In the present study, miRNAs that are highly expressed in the heart were overexpressed in neonatal rat ventricular myocytes, and cellular ATP levels were assessed. As a result, miR-15b, -16, -195, and -424, which have the same seed sequence, the most critical determinant of miRNA targeting, decreased cellular ATP levels. These results suggest that these miRNAs could specifically down-regulate the same target genes and consequently decrease cellular ATP levels. Through a bioinformatics approach, ADP-ribosylation factor-like 2 (Arl2) was identified as a potential target of miR-15b. It has already been shown that Arl2 localizes to adenine nucleotide transporter 1, the exchanger of ADP/ATP in mitochondria. Overexpression of miR-15b, -16, -195, and -424 suppressed the activity of a luciferase reporter construct fused with the 3′-untranslated region of Arl2. In addition, miR-15b overexpression decreased Arl2 mRNA and protein expression levels. The effects of Arl2 siRNA on cellular ATP levels were the same as those of miR-15b, and the expression of Arl2 could restore ATP levels reduced by miR-15b. A loss-of-function study of miR-15b resulted in increased Arl2 protein and cellular ATP levels. Electron microscopic analysis revealed that mitochondria became degenerated in cardiomyocytes that had been transduced with miR-15b and Arl2 siRNA. The present results suggest that miR-15b may decrease mitochondrial integrity by targeting Arl2 in the heart.

Oxidative stress induces GLUT4 translocation by activation of PI3-K/Akt and dual AMPK kinase in cardiac myocytes

In response to metabolic stress, GLUT4, the most abundant glucose transporter, translocates from intracellular vesicles to the plasma membrane. This appears to play an important role in protecting cardiac myocytes from ischemic injury. To investigate the precise mechanisms of GLUT4 translocation in cardiomyocytes, we have established a method for quantifying the relative proportion of sarcolemmal GLUT4 to total GLUT4 in these cells. Stimulation with H2O2 resulted in a concentration‐dependent increase in GLUT4 translocation, which peaked at 15 min after stimulation. The dominant‐negative form (DN) of AMP‐activated protein kinase (AMPK) α2 inhibited the H2O2‐induced translocation of GLUT4. We further examined the role of two known AMPK kinases (AMPKKs), calmodulin‐dependent protein kinase kinase (CaMKK)β and LKB1. The DN of CaMKKβ or LKB1 alone inhibited H2O2‐induced GLUT4 translocation only partially compared to the inhibition produced by the DN of AMPKα2. However, the combination of DN‐LKB1 and DN‐CaMKKβ inhibited translocation to an extent similar to with DN‐AMPKα2. Stimulation with H2O2 also activated Akt and the inhibition of PI3‐K/Akt prevented GLUT4 translocation to the same extent as with AMPK inhibition. When the DN of AMPKα2 was applied with DN‐PI3‐K, there was a complete reduction in the GLUT4 membrane level similar to that seen at the 0 time‐point. These results demonstrate that AMPK and PI3‐K/Akt have an additive effect on oxidative stress‐mediated GLUT4 translocation. J. Cell. Physiol. 215: 733–742, 2008.

Cholesterol Loading Reprograms the MicroRNA-143/145–Myocardin Axis to Convert Aortic Smooth Muscle Cells to a Dysfunctional Macrophage-Like Phenotype

Objective— We previously showed that cholesterol loading in vitro converts mouse aortic vascular smooth muscle cells (VSMC) from a contractile state to one resembling macrophages. In human and mouse atherosclerotic plaques, it has become appreciated that ≈40% of cells classified as macrophages by histological markers may be of VSMC origin. Therefore, we sought to gain insight into the molecular regulation of this clinically relevant process. Approach and Results— VSMC of mouse (or human) origin were incubated with cyclodextrin–cholesterol complexes for 72 hours, at which time the expression at the protein and mRNA levels of contractile-related proteins was reduced and of macrophage markers increased. Concurrent was downregulation of miR-143/145, which positively regulate the master VSMC differentiation transcription factor myocardin. Mechanisms were further probed in mouse VSMC. Maintaining the expression of myocardin or miR-143/145 prevented and reversed phenotypic changes caused by cholesterol loading. Reversal was also seen when cholesterol efflux was stimulated after loading. Notably, despite expression of macrophage markers, bioinformatic analyses showed that cholesterol-loaded cells remained closer to the VSMC state, consistent with impairment in classical macrophage functions of phagocytosis and efferocytosis. In apoE-deficient atherosclerotic plaques, cells positive for VSMC and macrophage markers were found lining the cholesterol-rich necrotic core. Conclusions— Cholesterol loading of VSMC converts them to a macrophage-appearing state by downregulating the miR-143/145–myocardin axis. Although these cells would be classified by immunohistochemistry as macrophages in human and mouse plaques, their transcriptome and functional properties imply that their contributions to atherogenesis would not be those of classical macrophages.

MicroRNA-27a regulates beta cardiac myosin heavy chain gene expression by targeting thyroid hormone receptor beta1 in neonatal rat ventricular myocytes.

ABSTRACT MicroRNAs (miRNAs), small noncoding RNAs, are negative regulators of gene expression and play important roles in gene regulation in the heart. To examine the role of miRNAs in the expression of the two isoforms of the cardiac myosin heavy chain (MHC) gene, α- and β-MHC, which regulate cardiac contractility, endogenous miRNAs were downregulated in neonatal rat ventricular myocytes (NRVMs) using lentivirus-mediated small interfering RNA (siRNA) against Dicer, an essential enzyme for miRNA biosynthesis, and MHC expression levels were examined. As a result, Dicer siRNA could downregulate endogenous miRNAs simultaneously and the β-MHC gene but not α-MHC, which implied that specific miRNAs could upregulate the β-MHC gene. Among 19 selected miRNAs, miR-27a was found to most strongly upregulate the β-MHC gene but not α-MHC. Moreover, β-MHC protein was downregulated by silencing of endogenous miR-27a. Through a bioinformatics screening using TargetScan, we identified thyroid hormone receptor β1 (TRβ1), which negatively regulates β-MHC transcription, as a target of miR-27a. Moreover, miR-27a was demonstrated to modulate β-MHC gene regulation via thyroid hormone signaling and to be upregulated during the differentiation of mouse embryonic stem (ES) cells or in hypertrophic hearts in association with β-MHC gene upregulation. These findings suggested that miR-27a regulates β-MHC gene expression by targeting TRβ1 in cardiomyocytes.

TG-interacting factor is required for the differentiation of preadipocytes

The accumulation of visceral adipose tissue is closely associated with insulin resistance and metabolic syndrome. Therefore, it is important to identify genes that are required for adipocyte differentiation. To identify genes that are required for the differentiation of 3T3-L1 preadipocytes into mature adipocytes, we used retrovirus insertion-mediated random mutagenesis to generate 3T3-L1 cell lines that lose their ability to differentiate into mature adipocytes. One of the genes identified was TG-interacting factor (TGIF), a DNA binding homeodomain protein that has been demonstrated to suppress Smad-mediated activation of transforming growth factor β (TGF-β)-regulated transcription. In the TGIF-disrupted clone of 3T3-L1 preadipocytes, the rate of differentiation into mature adipocytes was clearly reduced compared with that in the wild-type clone. Suppression of TGIF by lentivirus-mediated RNAi also inhibited the differentiation of 3T3-L1 cells. Insulin specifically increased the abundance of TGIF protein, primarily by enhancing its stability. In addition, insulin caused the rapid accumulation of TGIF in the nuclei. Forced expression of exogenous TGIF repressed both endogenous and overexpressed Smad2/3-mediated promoter activity in 3T3-L1. These findings suggest that insulin specifically antagonizes TGF-β signaling in preadipocytes by stabilizing the putative Smad transcriptional corepressor TGIF and regulates adipocyte differentiation.

MicroRNA 26b encoded by the intron of small CTD phosphatase (SCP) 1 has an antagonistic effect on its host gene

Tissue‐specific patterns of gene expression play an important role in the distinctive features of each organ. Small CTD phosphatases (SCPs) 1–3 are recruited by repressor element 1 (RE‐1)‐silencing transcription factor/neuron‐restrictive silencer factor (REST/NRSF) to neuronal genes that contain RE‐1 elements, leading to neuronal gene silencing in non‐neuronal cells. SCPs are highly expressed in the heart and contain microRNAs (miR)‐26b, 26a‐2, and 26a‐1 with the same seed sequence in their introns. Therefore, we tried to investigate the roles of miR‐26b and its host gene in neonatal rat cardiomyocytes. Overexpression of miR‐26b suppressed the mRNA expression levels of ANF, βMHC, and ACTA1 and reduced the cell surface area in cardiomyocytes. We confirmed that miR‐26b targets the 3′ untranslated region (3′UTR) of GATA4 and canonical transient receptor potential channel (TRPC) 3. Conversely, silencing of the endogenous miR‐26b family enhanced the expression levels of TRPC3 and GATA4. On the other hand, overexpression of SCP1 induced the mRNA expression of ANF and βMHC and increased the cell surface area in cardiomyocytes. Next, we compared the effect of overexpression of SCP1 with its introns and SCP1 cDNA to observe the net function of SCP1 expression on cardiac hypertrophy. When the expression levels of SCP1 were the same, the overexpression of SCP1 cDNA had a greater effect at inducing cardiac hypertrophy than SCP1 cDNA with its intron. In conclusion, SCP1 itself has the potential to induce cardiac hypertrophy; however, the effect is suppressed by intronic miR‐26b in cardiomyocytes. miR‐26b has an antagonistic effect on its host gene SCP1. J. Cell. Biochem. 113: 3455–3465, 2012.

Roles of microRNAs and myocardial cell differentiation.

As drug therapy is of limited efficacy in the treatment of heart diseases related to loss of cardiomyocytes, which have very poor division potential, regenerative medicine is expected to be a new strategy to address regenerative treatment in cardiac diseases. To achieve myocardial regeneration, elucidation of the mechanism of myocardial differentiation from stem cells is essential. Myocardial differentiation from embryonic pluripotent stem cells has been investigated worldwide, and remarkable developments such as establishment of induced pluripotent stem cells and transformation of somatic cells to cardiomyocytes have recently been made, markedly changing the strategy of regenerative medicine. At the same time, the close involvement of microRNA in the maintenance, proliferation, differentiation, and reprogramming of these stem cells has been revealed. In this report, microRNA is outlined, focusing on its role in myocardial differentiation.

Neural cell adhesion molecule is a cardioprotective factor up-regulated by metabolic stress

Screening for cell surface proteins up-regulated under stress conditions may lead to the identification of new therapeutic targets. To search for genes whose expression was enhanced by treatment with oligomycin, a mitochondrial-F(0)F(1) ATP synthase inhibitor, signal sequence trapping was performed in H9C2 rat cardiac myoblasts. One of the genes identified was that for neural cell adhesion molecule (NCAM, CD56), a major regulator of development, cell survival, migration, and neurite outgrowth in the nervous system. Immunohistochemical analyses in a mouse myocardial infarction model revealed that NCAM was strongly expressed in residual cardiac myocytes in the infarcted region. Increased expression of NCAM was also found during the remodeling period in a rat model of hypertension-induced heart failure. Lentivirus-mediated knockdown of NCAM decreased the cell growth and survival following oligomycin treatment in H9C2 cells. In primary rat neonatal cardiac myocytes, NCAM was also found to be up-regulated and played a protective role following oligomycin treatment. Analyses of downstream signaling revealed that knockdown of NCAM significantly decreased the basal AKT phosphorylation level. In contrast, NCAM mimetic peptide P2d activated AKT and significantly reduced oligomycin-induced cardiomyocyte death, which was abolished by treatment with the PI3K inhibitor LY-294002 as well as overexpression of the dominant-negative AKT mutant. These findings demonstrate that NCAM is a cardioprotective factor up-regulated under metabolic stress in cardiomyocytes and augmentation of this signal improved survival.