Yasuhide Kuwabara

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.

MicroRNA-33 Deficiency Reduces the Progression of Atherosclerotic Plaque in ApoE−/− Mice

Background Cholesterol efflux from cells to apolipoprotein A-I (apoA-I) acceptors via the ATP-binding cassette transporters ABCA1 and ABCG1 is thought to be central in the antiatherogenic mechanism. MicroRNA (miR)-33 is known to target ABCA1 and ABCG1 in vivo. Methods and Results We assessed the impact of the genetic loss of miR-33 in a mouse model of atherosclerosis. MiR-33 and apoE double-knockout mice (miR-33−/−Apoe−/−) showed an increase in circulating HDL-C levels with enhanced cholesterol efflux capacity compared with miR-33+/+Apoe−/− mice. Peritoneal macrophages from miR-33−/−Apoe−/− mice showed enhanced cholesterol efflux to apoA-I and HDL-C compared with miR-33+/+Apoe−/− macrophages. Consistent with these results, miR-33−/−Apoe−/− mice showed reductions in plaque size and lipid content. To elucidate the roles of miR-33 in blood cells, bone marrow transplantation was performed in these mice. Mice transplanted with miR-33−/−Apoe−/− bone marrow showed a significant reduction in lipid content in atherosclerotic plaque compared with mice transplanted with miR-33+/+Apoe−/− bone marrow, without an elevation of HDL-C. Some of the validated targets of miR-33 such as RIP140 (NRIP1) and CROT were upregulated in miR-33−/−Apoe−/− mice compared with miR-33+/+Apoe−/− mice, whereas CPT1a and AMPKα were not. Conclusions These data demonstrate that miR-33 deficiency serves to raise HDL-C, increase cholesterol efflux from macrophages via ABCA1 and ABCG1, and prevent the progression of atherosclerosis. Many genes are altered in miR-33-deficient mice, and detailed experiments are required to establish miR-33 targeting therapy in humans.

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.

MicroRNAs and cardiovascular diseases.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that have gained status as important regulators of gene expression. Recent studies have demonstrated that miRNAs are aberrantly expressed in the cardiovascular system under some pathological conditions. Gain‐ and loss‐of‐function studies using in vitro and in vivo models have revealed distinct roles for specific miRNAs in cardiovascular development and physiological function. The implications of miRNAs in cardiovascular disease have recently been recognized, representing the most rapidly evolving research field. In the present minireview, the current relevant findings on the role of miRNAs in cardiac diseases are updated and the target genes of these miRNAs are summarized.

MicroRNA-33 regulates sterol regulatory element-binding protein 1 expression in mice

MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports have indicated that miR-33, which is located within the intron of sterol regulatory element-binding protein (SREBP) 2, controls cholesterol homoeostasis and may be a potential therapeutic target for the treatment of atherosclerosis. Here we show that deletion of miR-33 results in marked worsening of high-fat diet-induced obesity and liver steatosis. Using miR-33−/−Srebf1+/− mice, we demonstrate that SREBP-1 is a target of miR-33 and that the mechanisms leading to obesity and liver steatosis in miR-33−/− mice involve enhanced expression of SREBP-1. These results elucidate a novel interaction between SREBP-1 and SREBP-2 mediated by miR-33 in vivo.

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.

MicroRNA-451 Exacerbates Lipotoxicity in Cardiac Myocytes and High-Fat Diet-Induced Cardiac Hypertrophy in Mice Through Suppression of the LKB1/AMPK Pathway

Rationale: In some patients with type 2 diabetes mellitus (DM) without hypertension, cardiac hypertrophy and attenuated cardiac function are observed, and this insult is termed diabetic cardiomyopathy. To date, microRNA (miRNAs or miR) functions in diabetic cardiomyopathy remain to be elucidated. Objective: To clarify the functions of miRNAs involved in diabetic cardiomyopathy caused by type 2 DM. Methods and Results: C57BL/6 mice were fed a high-fat diet (HFD) for 20 weeks, which induced obesity and type 2 DM. miRNA microarray analyses and real-time polymerase chain reaction revealed that miR-451 levels were significantly increased in the type 2 DM mouse hearts. Because excess supply of saturated fatty acids is a cause of diabetic cardiomyopathy, we stimulated neonatal rat cardiac myocytes with palmitic acid and confirmed that miR-451 expression was increased in a dose- and time-dependent manner. Loss of miR-451 function ameliorated palmitate-induced lipotoxicity in neonatal rat cardiac myocytes. Calcium-binding protein 39 (Cab39) is a scaffold protein of liver kinase B1 (LKB1), an upstream kinase of AMP-activated protein kinase (AMPK). Cab39 was a direct target of miR-451 in neonatal rat cardiac myocytes and Cab39 overexpression rescued the lipotoxicity. To clarify miR-451 functions in vivo, we generated cardiomyocyte-specific miR-451 knockout mice. HFD-induced cardiac hypertrophy and contractile reserves were ameliorated in cardiomyocyte-specific miR-451 knockout mice compared with control mice. Protein levels of Cab39 and phosphorylated AMPK were increased and phosphorylated mammalian target of rapamycin (mTOR) was reduced in cardiomyocyte-specific miR-451 knockout mouse hearts compared with control mouse hearts. Conclusions: Our results demonstrate that miR-451 is involved in diabetic cardiomyopathy through suppression of the LKB1/AMPK pathway.

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.

MicroRNA-33b knock-in mice for an intron of sterol regulatory element-binding factor 1 (Srebf1) exhibit reduced HDL-C in vivo

MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports, including ours, indicated that miR-33a located within the intron of sterol regulatory element-binding protein (SREBP) 2 controls cholesterol homeostasis and can be a possible therapeutic target for treating atherosclerosis. Primates, but not rodents, express miR-33b from an intron of SREBF1. Therefore, humanized mice, in which a miR-33b transgene is inserted within a Srebf1 intron, are required to address its function in vivo. We successfully established miR-33b knock-in (KI) mice and found that protein levels of known miR-33a target genes, such as ABCA1, ABCG1, and SREBP-1, were reduced compared with those in wild-type mice. As a consequence, macrophages from the miR-33b KI mice had a reduced cholesterol efflux capacity via apoA-I and HDL-C. Moreover, HDL-C levels were reduced by almost 35% even in miR-33b KI hetero mice compared with the control mice. These results indicate that miR-33b may account for lower HDL-C levels in humans than those in mice and that miR-33b is possibly utilized for a feedback mechanism to regulate its host gene SREBF1. Our mice will also aid in elucidating the roles of miR-33a/b in different genetic disease models.

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.