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Expression of microRNA-208 is Associated With Adverse Clinical Outcomes in Human Dilated Cardiomyopathy
Journal of Cardiac Failure Vol. 16 No. 5 2010
Translational Science
Expression of microRNA-208 is Associated With Adverse Clinical Outcomes in Human Dilated Cardiomyopathy MAMORU SATOH, MD, YOSHITAKA MINAMI, MD, YUJI TAKAHASHI, MD, TSUYOSHI TABUCHI, MD, AND MOTOYUKI NAKAMURA, MD Iwate Japan
ABSTRACT Background: Recently, microRNA-208 (miR-208) encoded by the a-myosin heavy chain (MHC) gene, has been shown to be involved in pathological cardiac growth, fibrosis, and up-regulation of b-MHC expression. A recent study has also reported 2 additional myosin-expressed miRNAs (miR-208b and miR-499). The aim of this study was to determine whether miR-208, miR-208b, and miR-499 are expressed with MHC mRNA in human dilated cardiomyopathy (DCM), and whether these levels are related to left ventricular (LV) function and to clinical outcomes. Methods and Results: Endomyocardial biopsy tissues were obtained from 82 patients with DCM and 21 subjects without LV dysfunction as controls. Levels of miR-208, miR-208b, and miR-499 were higher in DCM patients than in controls. Levels of a-MHC mRNA were lower in patients with DCM than in controls, whereas b-MHC mRNA levels were higher in patients with DCM compared with controls. Levels of miR-208 were correlated with b-MHC mRNA levels and myocardial collagen volume, whereas levels of miR-208b and miR-499 showed no correlation. After a mean follow-up of 517 days, an increase in miR-208 levels was shown to be a strong predictor of clinical outcomes (RR 3.4, 95% CI 1.1e11.2). Conclusions: This study suggests that myocardial expression of miR-208 is associated with MHC mRNA expression and with poor clinical outcomes in patients with DCM. We conclude that miR-208 may therefore be involved in the progression of human DCM. (J Cardiac Fail 2010;16:404e410) Key Words: Biopsy, collagen volume, heart failure, myosin heavy chains.
The myosin heavy chains (MHCs) are molecule motors of muscle and have a potential functional significance for cardiac performance.1 Two isoforms of MHC consisting of a-MHC and b-MHC are expressed in the mammalian heart.2 The myosin of a-MHC has higher adenosine triphosphatase (ATPase) activity than myosin of b-MHC, and contractile velocity correlates with the balance of MHC isoforms.2
The MHC composition of normal human ventricular myocardium has been reported to have b-MHC as the predominant isoform (O95%) among MHC isoforms.3 Impairment of MHC composition, such as a decrease in a-MHC mRNA and its protein levels, has been shown in failing human heart compared to non-failing human heart.4 In addition, improvement in left ventricular ejection fraction (LVEF) through b-blocker therapy is associated with normalization of a-MHC and b-MHC expression resulting from upregulation of a-MHC and downregulation of b-MHC in patients with dilated cardiomyopathy (DCM).5 MHC expression may therefore be critical for maintaining normal cardiac function, which suggests that impairment of MHC composition, such as a subtle shift in the balance between a-MHC and b-MHC, may play a significant role in cardiac failure. However, the precise mechanism underlying the change in MHC composition in human DCM is unclear. It has recently been reported that microRNAs (miRs) are small noncoding RNAs that negatively regulate protein expression at the posttranscription level.6 Through specific targeting of the 30 untranslated regions of multicellular
From the Division of Cardiology, Department of Internal Medicine and Memorial Heart Center, Iwate Medical University School of Medicine, Iwate, Japan. Manuscript received November 17, 2009; revised manuscript received January 8, 2010; revised manuscript accepted January 15, 2010. Reprint requests: Mamoru Satoh, Division of Cardiology, Department of Internal Medicine and Memorial Heart Center, Iwate Medical University School of Medicine, Uchimaru 19-1, Morioka, 020-8505, Iwate, Japan. Tel: þ81-19-651-5111; Fax: þ81-19-651-0401. E-mail: m_satoh@imu. ncvc.go.jp Supported by a Grant-in-Aid for General Scientific Research from the Japanese Ministry of Education, Science, Sports and Culture (No. 20590886), a grant from the Keiryokai Research Foundation (No. 98). 1071-9164/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cardfail.2010.01.002
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miR-208 and Clinical Outcomes in DCM
eukaryotic mRNAs, miRs downregulate gene expression either by inducing degradation of target mRNA or by impairing its translation.7,8 It has been recently reported that miR-208, a cardiac-specific miR, encodes within intron 27 of the gene encoding a-MHC and is essential for expression of the genes involved in cardiac contractility and fibrosis.9 In response to cardiac stress such as pressure overload, miR-208 knockdown mice showed virtually no cardiomyocytic hypertrophy and fibrosis and no impact on the balance between a-MHC and b-MHC expression compared with wild-type mice.9 A more recent study has also reported 2 myosin-expressed microRNAs, miR-208b and miR-499, which are encoded by b-MHC genes.10 This report provides important new information about the pathogenesis of failing human heart and points the way toward new therapies to combat this condition. However, there have been no reports on the relationship between myosin-expressed microRNAs and MHC expression in human DCM. The aim of this study was to determine whether the expression of miR-208, miR-208b, and miR-499 is related to MHC expression in the myocardium of patients with DCM, and whether miR-208 levels are related to LV geometry and function and to clinical outcomes in human DCM. Methods and Materials Subjects Endomyocardial tissues were obtained from 82 consecutive patients with DCM by right ventricular endomyocardial biopsy. The clinical diagnosis of DCM was made according to the World Health Organization/International Society and Federation of Cardiology Task Force criteria.11 Left ventricular angiography was performed to determine LVEF and left ventricular end-systolic volume index (LVESVI) at the time of biopsy. Control myocardial samples were obtained by endomyocardial biopsy from 21 subjects with suspected cardiac disorders such as myocardial deposition disease, hypertensive heart disease, and hypertrophic cardiomyopathy on the basis of arrhythmia and echocardiographic changes such as premature beats and a slight increase in ventricular wall thickness. Five biopsy samples were taken from each subject from the right ventricular myocardium. Three of the 5 samples were fixed in formalin and paraffin-embedded for histopathology and morphometry. The remaining 2 samples were immediately frozen for subsequent investigation by real-time reverse transcriptase polymerase chain reaction (PCR). The resulting pathologic findings and close clinical examination failed to show any evidence of myocardial disease or functional abnormality, and these subjects were thus designated as controls. This study protocol was approved by our hospital ethics committee, and written informed consent was obtained from all subjects.
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Extracted RNA from endomyocardial biopsy tissues and human myocardial mRNA (Applied Biosystem, Foster City, CA) as a standard control were initially reverse transcribed using a high-capacity cDNA Archive Kit (Applied Biosystem, Foster City, CA) and then amplified in a 10 mL PCR reaction and primer set for amplification of human miR-208 (Assay ID: 000511), miR-208b (Assay ID: 002290), miR-499 (Assay ID: 002427), and U6 (Assay ID: 001093) using TaqMan microRNA assays according to the manufacturer’s recommended protocol (Applied Biosystem).12 Levels of a-MHC (Assay ID: Hs00411908) and b-MHC (Assay ID: Hs01110632) mRNAs were amplified using TaqMan Gene Expression Assays (Applied Biosystem). For all myocardial specimens, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Assay ID: Hs99999905) mRNA was amplified using TaqMan Gene Expression Assays as an endogenous control (Applied Biosystem). The amplification steps consisted of denaturation at 95 C, followed by 40 cycles of denaturation at 95 C for 15 seconds and then annealing at 60 C for 1 minute. All reactions were carried out on the 7500 real-time PCR system (Applied Biosystem) using the TaqMan Universal PCR master Mix and Assays on demand (Applied Biosystem). Relative quantitation was carried out using the DD threshold cycle (Ct) method for recurrent versus primary with U6 or GAPDH as an endogenous control and fold changes were calculated for each gene.13 Replicates with a Ct O40 were excluded. The assay was run in triplicate for each case to allow for assessment of technical variability. To account for PCR amplification of contaminating genomic DNA, a control without reverse transcription was included. To improve the accuracy of real-time reverse transcriptase PCR for quantification, amplifications were performed in triplicate for each RNA sample. Collagen Morphometry Serial paraffin sections (5 mm thick) were stained with Masson trichrome. Myocardial collagen volume was estimated from each specimen by quantitation of fibrillar collagen accumulation stained by Masson trichrome omitting perivascular fibrosis using a computer-associated morphometric program (Olympus, Tokyo, Japan) with WinROOF image analysis software (version 6.01, Mitani Corporation, Tokyo, Japan). Ten randomly selected different areas per specimen were analyzed (magnification 62.5). Myocardial collagen volume fraction (CVF) was calculated as the percentage of myocardial collagen volume in the total area of the specimen. Follow-up Study We planned a minimum follow-up of 12 months in all patients with DCM. This was achieved by outpatient assessments and telephone contact with the patient or his or her local physician. Clinical outcomes were defined as total mortality and development of heart failure (HF). Occurrence of HF required the presence of rest or effort dyspnea and $1 of pulmonary congestion at chest x-ray, new appearance of peripheral edema, or use of diuretics. All deaths were considered to be of cardiac origin unless a noncardiac origin was established clinically or at autopsy. Statistical Analysis
Real-time Reverse Transcriptase PCR for miR-208, miR-208b, miR-499, a-MHC and b-MHC mRNAs Total RNA including the small RNA fraction was extracted from endomyocardial biopsy tissues using a mirVana Paris miRNA isolation kit (Ambion, Inc, Austin, TX) in accordance with the manufacturer’s instructions.
All values are presented as mean 6 standard deviation. Kolmogorov-Smirnov analysis was performed to assess data distribution. Unpaired Student t-test was performed for normally distributed data, and nonparametric Mann-Whitney test was performed where this was not appropriate. Statistical analysis of categorical variables was also carried out using chi-square analysis and Fisher exact
406 Journal of Cardiac Failure Vol. 16 No. 5 May 2010 analysis. Spearman correlation coefficients were used to examine the relationship between miR-208 levels, MHC mRNA levels, CVF, and left ventricular geometry and function. An event-free survival curve was generated by the Kaplan-Meier method, and event-free survival among the groups was compared by use of the log-rank test. Multivariate association between miR-208 levels and clinical outcomes was evaluated with logistic regression models. Odds ratios are reported with logistic regression models that adjust for factors (sex, age, LVEF, LVESVI, New York Heart Association, and medications) independently associated with the outcome variable. A value of P ! .05 was considered statistically significant.
Results Baseline Characteristics
Baseline and clinical characteristics of patients with DCM and controls are shown in Table 1. There were no differences in age and sex between patients with DCM and controls. Data on New York Heart Association functional class, left ventricular geometry, and pressure studies showed significant differences between DCM patients and controls. Levels of miR-208, miR-208b, miR-499, a-MHC, and ß-MHC mRNAs
Histopathology and CVF
There was no difference in average Ct of U6 between patients with DCM and controls (20.6 6 5.1 vs. 19.9 6 4.9; not significant). Levels of miR-208, miR-208b, and miR-499 in the myocardium were higher in patients with DCM than in controls (miR-208: 0.94 6 0.78 vs. 0.21 6 0.19; miR-208b: 0.20 6 0.12 vs. 0.13 6 0.10; miR-499: 0.53 6 0.27 vs. 0.20 6 0.25; Table 1. Baseline and Clinical Characteristics of Study Populations Characteristics
DCM (n 5 82)
Age (y) 55.8 6 12.1 Sex (male/female) 59/23 NYHA functional class at the time of biopsy I or II, n (%) 51 (62)* III or IV, n (%) 31 (38)* LVG at time of biopsy LVEF (%) 34.1 6 10.3* 57.4 6 29.3* LVESVI (mL/mm2 BSA) Pressure study at time of biopsy Systolic BP (mm Hg) 122 6 31 Diastolic BP (mm Hg) 71 6 16 LVEDP (mm Hg) 11.5 6 7.1* 2.9 6 0.9* CI (L$min$m2 BSA) Mean PC (mm Hg) 11.6 6 7.1* Medication, n (%) Loop diuretics 56 (68)* Digitalis 28 (34)* Spironolactone 45 (55)* ACEIs 44 (54)* ARBs 42 (51)* b-blocker 49 (60)*
DCM group vs. controls, all P ! .01) (Fig. 1A). Average Ct of miR-208 was lower in patients with DCM compared with controls (23.8 6 6.4 vs. 28.5 6 7.2; DCM group vs. control group, P ! .01). Levels of a-MHC mRNAwere lower in patients with DCM than in controls (0.07 6 0.02 vs. 0.42 6 0.10, P ! .001) (Fig. 1B). In contrast, b-MHC mRNA levels were higher in patients with DCM compared with controls (1.63 6 1.25 vs. 1.04 6 0.43, P 5 .03) (Fig. 1B). Average Ct of GAPDH did not differ between patients with DCM and controls (20.2 6 4.3 vs. 20.8 6 4.4; not significant). Levels of miR-208 were negatively correlated with a-MHC mRNA levels in patients with DCM (r 5 0.65, P ! .001) (Fig. 2A). There was a positive correlation between miR-208 and b-MHC mRNA levels in patients with DCM (r 5 0.80, P ! .001) (Fig. 2B). Levels of miR-208b and miR-499 were not statistically correlated with b-MHC mRNA levels in patients with DCM (miR208b vs. b-MHC: r 5 0.11, P 5 .35; miR-499 vs. b-MHC: r 5 0.19, P 5 .09).
Controls (n 5 21) 54.2 6 9.6 15/6 21 (100) 0 (0) 69.5 6 7.1 17.2 6 5.9 127 70 5.4 3.9 7.9
6 6 6 6 6
19 11 1.1 0.9 2.9
0 0 0 5 (24) 4 (19) 3 (14)
ACEI, angiotensin-converting enzyme inhibitors; ARB, angiotensin II type 1 receptor blockers; BP, blood pressure; CI, cardiac index; DCM, dilated cardiomyopathy; LVEDP, left ventricular end-diastolic pressure; LVEF, left ventricular ejection fraction; LVESVI, left ventricular end-systolic volume index; LVG, left ventricular angiography; N /A, not available; NYHA, New York Heart Association; PC, pulmonary capillary pressure. *P ! .05 vs. controls.
Histological analysis showed no active or borderline myocarditis in any of the DCM samples analyzed. CVF was higher in patients with DCM than in controls (6.02 6 2.41% vs. 0.63 6 0.69%, P ! .001) (Fig. 3A). There was a moderate positive correlation between CVF and miR-208 levels in patients with DCM (r 5 0.44, P ! .001) (Fig. 3B), whereas miR-208b and miR-499 levels were not correlated with CVF in patients with DCM (miR-208b vs. CVF: r 5 0.36, P 5 .11; miR-499 vs. CVF: r 5 0.12, P 5 .30). Relationship between Clinical Data and miR-208 Levels
There was a moderate negative correlation between miR208 levels and LVEF in patients with DCM (r 5 0.52, P ! .001) (Fig. 4A). LVESVI was positively correlated with miR-208 levels in patients with DCM (r 5 0.54, P ! .001) (Fig. 4B). Relationship between miR-208 Levels and Clinical Outcomes in Patients with DCM
After a mean follow-up of 517 days (range, 48 to 983 days), 7 (8.5%) DCM patients had died from a cardiac cause and 11 (13.4%) had developed HF. There were no significant differences in baseline characteristics of DCM patients between event-free patients and patients with events (Table 2). Figure 5 shows differences in clinical outcomes when patients with DCM were divided into tertiles according to miR-208 levels (high, moderate, and low miR-208 groups). Log-rank analysis showed that the DCM subgroup with high miR-208 levels was associated with poor clinical outcomes. In the multivariate model, after adjustment for baseline characteristics (sex, age, LVEF, LVESVI, New York Heart Association, and medications), high levels of miR-208 (miR-208 levels 5 1.15 to 2.79) were a strong independent
miR-208 and Clinical Outcomes in DCM 2
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Fig. 1. Levels of microRNA-208 (miR-208), miR-206b, miR-499, a-myosin heavy chain (MHC), and ß-MHC mRNA in patients with dilated cardiomyopathy (DCM) and controls. (A) Comparison of miR-208, miR-208b, and miR-499 levels between patients with DCM and controls. (B) Comparison of a-MHC and ß-MHC mRNA levels between patients with DCM and controls. *P ! .05 vs. controls.
predictor of cardiac death and HF (RR 3.4, 95% CI 1.111.2). Discussion Relative expression levels of MHC isoforms including a-MHC and b-MHC can be altered in disease states such as cardiac failure or hypertrophy.1 In the present study, both upregulation of b-MHC levels and downregulation of a-MHC levels were shown in myocardium from patients with DCM compared with subjects without LV dysfunction. In agreement with these findings, a shift from a-MHC toward b-MHC is often observed in failing human heart.14 In addition, a b-MHC-transgenic mouse model has shown that a shift from a-MHC to b-MHC isoform reduced left ventricular contractility and dilated left ventricular volume.15 Myocardial expression of a-MHC has more rapid contractile velocity than myocardial expression of b-MHC, because the tension-time integral for force per cross-bridge cycle is greater in a-MHC than in b-MHC.16,17 Indeed, the proportion of a-MHC mRNA relative to total MHC mRNA levels is reduced in myocardium obtained from patients undergoing cardiac transplantation
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for chronic end-stage HF from several forms of heart disease, including DCM.4,14 From these observations, it has been suggested that alternation in MHC isoform ratio which represses a-MHC and reexpresses b-MHC in the heart may be involved in LV dysfunction and dilatation in human DCM. However, the mechanisms underlying this alternation in MHC isoforms in patients with DCM are poorly understood. MicroRNAs, a newly discovered class of small ribonucleotide-based regulators of gene expression, modulate gene expression at the posttranscriptional level.6,7 Several recent studies have demonstrated that expression of numerous microRNAs, such as miR-21, miR-133, miR195, and miR-208, have key roles in the growth, development, function, and stress responsiveness of the heart.9,18-21 In particular, it has been reported that myocardial expression of miR-208 regulates the balance between a-MHC and b-MHC isoforms and induces adverse cardiac remodeling resulting from a reduction in cardiac contractility and an increase in cardiac fibrosis.9 A more recent report has also shown that miR-208b and miR-499 were encoded by b-MHC genes as myosin-expressed microRNAs.10 These reports provide important new information about the
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Fig. 2. Correlation plot between microRNA-208 (miR-208) levels and a-myosin heavy chain (MHC) and ß-MHC mRNA levels in dilated cardiomyopathy (DCM) patients. (A) miR-208 vs. a-MHC mRNA, r 5 0.65, P ! .001. (B) miR-208 vs. ß-MHC mRNA, r 5 0.80, P ! .001.
408 Journal of Cardiac Failure Vol. 16 No. 5 May 2010
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Fig. 3. (A) Comparison of collagen volume fraction (CVF) between patients with dilated cardiomyopathy (DCM) and controls. *P ! .01 vs. controls. (B) Correlation between microRNA-208 (miR-208) levels and CVF in patients with DCM (r 5 0.44, P ! .001).
A3
B3 miR-208 levels
miR-208) in the heart dramatically increased b-MHC protein levels and then induced adverse cardiac remodeling.24 In contrast, deletion of miR-208a resulted in decreased b-MHC expression in the adult heart, providing genetic evidence that miR-208a is required for b-MHC expression.24 Although the present study could not confirm the mechanism by which miR-208 levels were increased in the myocardium in DCM, these observations have therefore suggested a critical role for miR-208 transcripts in the induction of b-MHC and cardiac remodeling during stress conditions. The present study has shown a positive correlation between miR-208 levels and CVF in myocardium obtained from patients with DCM. MiR-208 knockdown mice demonstrated diminished collagen accumulation and upregulation of b-MHC expression in response to pressure overload.9 A relationship between b-MHC gene expression and cardiac fibrosis has also been found in animal heart.25 From these observations, it has been speculated that miR-208 expression could affect myocardial collagen accumulation via upregulation of b-MHC expression in human DCM. Using right ventricular septal biopsy specimens for measurement of miR-208 expression, the present study found a correlation between miR-208 levels and LV function and volume in human DCM. Although it could not be confirmed whether miR-208 expression in right ventricular
miR-208 levels
pathogenesis of failing human heart and points the way toward new therapies to combat this condition. The present study has shown that miR-208, miR-208b, and miR-499 levels were higher in myocardium obtained from patients with DCM compared with that from subjects without LV dysfunction. There was a negative correlation between miR-208 and a-MHC mRNA levels in DCM. These observations have suggested that myocardial expression of miR208 may downregulate a-MHC mRNA levels resulting from inducing degradation of a-MHC mRNA. In addition, miR-208 had a long half-life and may be elevated in the myocardium in DCM, although a-MHC levels had declined.9 It has therefore been speculated that a closed relationship exists between miR-208 expression and a change in MHC isoform composition in human DCM. The present study has shown a positive correlation between miR-208 and b-MHC levels in patients with DCM, whereas both miR-208b and miR-499 levels were not correlated with b-MHC levels. It has been reported that both normal and dilated cardiomyopathic human ventricles express predominantly b-MHC and very low levels of a-MHC, and a-MHC levels are lower in failing ventricles comparing with normal ventricles, suggesting a shift toward b-MHC in failing ventricles.22 In addition, increased b-MHC expression is a feature of HF in human hearts.23 It has recently been reported that transgenic overexpression of miR-208a (also known as
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Fig. 4. Correlation between LV geometry and microRNA-208 (miR-208) levels in patients with dilated cardiomyopathy (DCM). (A) LVEF vs. miR-208 levels, r 5 0.52, P ! .001. (B) LVESVI vs. miR-208 levels, r 5 0.54, P ! .001. LVEF, left ventricular ejection fraction; LVESVI, left ventricular end-systolic volume index.
miR-208 and Clinical Outcomes in DCM Table 2. Baseline and Clinical Characteristics of Event-free Patients and Patients with Events Patients with Event-free Patients Events (n 5 18) (n 5 64)
Characteristics
Age (y) Sex (male/female) NYHA functional class at time of I or II, n (%) III or IV, n (%) LVG at time of biopsy LVEF (%) LVESVI (mL/mm2 BSA) Pressure study at time of biopsy Systolic BP (mm Hg) Diastolic BP (mm Hg) LVEDP (mm Hg) CI (L$min$m2 BSA) Mean PC (mm Hg) Medication, n (%) Loop diuretics Digitalis Spironolactone ACEIs ARBs b-blocker
56.3 6 13.1 15/3 biopsy 11 (61) 7 (39)
55.1 6 12.5 44/20
34.1 6 11.4 70.1 6 23.4
34.1 6 10.2 67.1 6 21.3
117 69 15.8 3.0 12.4 14 6 10 10 9 11
6 6 6 6 6
40 (65) 24 (38)
26 17 9.1 0.9 9.6
132 72 10.4 2.9 10.7
(78) (34) (56) (56) (50) (61)
42 22 35 34 33 38
6 6 6 6 6
29 18 6.6 0.7 6.8
(66) (34) (55) (53) (52) (59)
ACEI, angiotensin-converting enzyme inhibitors; ARB, angiotensin II type 1 receptor blockers; BP, blood pressure; CI, cardiac index; DCM, dilated cardiomyopathy; LVEDP, left ventricular end-diastolic pressure; LVEF, left ventricular ejection fraction; LVESVI, left ventricular end-systolic volume index; LVG, left ventricular angiography; N /A, not available; NYHA, New York Heart Association; PC, pulmonary capillary pressure.
septum directly reflects LV function in human DCM, it has been reported that changes in expression of several genes in the septum reflect those in both right and left ventricular free walls in human DCM.26 Another important finding of the present study was high levels of miR-208 expression in association with poor clinical outcomes in DCM, showing miR-208 to be a predictor of cardiac death and progression of HF in DCM patients. It has been suggested that miR-208 is required for the development of cardiac hypertrophy and myocardial fibrosis.9 These events may lead to
Event-free survival
1 Group 1: low miR-208 Group 2: moderate miR-208
0.8 0.6 0.4 0.2
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Group 3 vs. 1 or 2, P = 0.02
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adverse left ventricular remodeling and the development of cardiac events. On the basis of these observations, we propose that myocardial expression of miR-208 may give rise to a HF phenotype that consists of reexpression of an embryonic gene pattern (eg, upregulation of b-MHC) and an increase in myocardial collagen accumulation, leading to irreversible myocardial injury in human DCM. The results of the present study therefore provide a pathogenic mechanism for human DCM based on the relationship between cardiac miR-208 expression and this HF phenotype. Myocardial expression of miR-208 may therefore represent a novel therapeutic opportunity. Limitations
A limitation of this study is the small size of the study population. In addition, no miR-208 functional analysis of cardiac myocytes was carried out, such as that in these microRNAs upregulated or downregulated in vitro models. Further studies will therefore be needed to determine the mechanism by which miR-208 expression regulates a HF phenotype, such as upregulation of b-MHC, in human DCM. In addition, recent reports have identified myocardial expression of microRNAs (eg, miR-1, miR-133) in addition to miR-208.27,28 Further studies will thus be needed to characterize the expression profile of myocardial microRNAs in the myocardium in DCM. The present study could not confirm miR-208 as a marker of HF, because the characteristics of the DCM and control materials used differed significantly, including LV geometry and medication. We will therefore need to examine miR-208 levels in HF from DCM in comparison to that arising from various other origins, such as secondary cardiomyopathy, ischemic heart, and valvular disease. The present study has also not confirmed whether miR-208 levels provide incremental information beyond other clinical parameters (eg, brain natriuretic peptide, high-sensitivity C-reactive protein) that are already known to be prognostic factors. Followup studies will be needed to compare miR-208 with other prognostic parameters. Conclusions In conclusion, the present study suggests that myocardial expression of miR-208 is associated with reexpression of bMHC mRNA and cardiac collagen accumulation and may be involved in adverse clinical outcomes in human DCM.
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References
Time (days) Group 1:n = 27; miR-208 levels, 0.03 to 0.35 Group 2:n = 28; miR-208 levels, 0.36 to 1.14 Group 3:n = 27; miR-208 levels, 1.15 to 2.79
Fig. 5. Kaplan-Meier analysis. Event-free (P 5 .02, log-rank test) for group 3 (high microRNA-208 [miR-208] levels). HRs were 5.8 (95% CI, 1.4 to 23.8) for group 1 vs. group 3 and 4.0 (95% CI, 1.1 to 14.7) for group 2 vs. group 3. Group 1: n 5 27, miR-208 levels 5 0.03 to 0.35; Group 2: n 5 28, miR-208 5 0.36 to 1.14; Group 3: n 5 27, miR-208 5 1.15 to 2.79.
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liberation in normal and hypertrophied right ventricular papillary muscles. Circ Res 1982;50:491e500. Holubarsch C, Goulette RP, Litten RZ, Martin BJ, Mulieri LA, Alpert NR. The economy of isometric force development, myosin isoenzyme pattern and myofibrillar ATPase activity in normal and hypothyroid rat myocardium. Circ Res 1985;56:78e86. Cheng Y, Ji R, Yue J, Yang J, Liu X, Chen H, et al. MicroRNAs are aberrantly expressed in hypertrophic heart: do they play a role in cardiac hypertrophy? Am J Pathol 2007;170:1831e40. Care` A, Catalucci D, Felicetti F, Bonci D, Addario A, Gallo P, et al. MicroRNA-133 controls cardiac hypertrophy. Nat Med 2007;13:613e8. Van Rooij E, Sutherland LB, Liu N, Williams AH, McAnally J, Gerard RD, et al. A signature pattern of stress-responsive microRNAs that can evoke cardiac hypertrophy and heart failure. Proc Natl Acad Sci USA 2006;103:18255e60. Ambros V, Lee RC, Lavanway A, Williams PT, Jewell D. MicroRNAs and other tiny endogenous RNAs in C. elegans. Curr Biol 2003;13: 807e18. Reiser PJ, Kline WO. Electrophoretic separation and quantitation of cardiac myosin heavy chain isoforms in eight mammalian species. Am J Physiol 1998;274:H1048e53. Chien KR. Genomic circuits and the integrative biology of cardiac diseases. Nature 2000;407: 227e2. Callis TE, Pandya K, Seok HY, Tang RH, Tatsuguchi M, Huang ZP, et al. MicroRNA-208a is a regulator of cardiac hypertrophy and conduction in mice. J Clin Invest 2009;119:2772e86. Pandya K, Kim HS, Smithies O. Fibrosis, not cell size, delineates betamyosin heavy chain reexpression during cardiac hypertrophy and normal aging in vivo. Proc Natl Acad Sci USA 2006;103:16864e9. Lowes BD, Minobe W, Abraham WT, Rizeq MN, Bohlmeyer TJ, Quaife RA, et al. Changes in gene expression in the intact human heart. Downregulation of alpha-myosin heavy chain in hypertrophied, failing ventricular myocardium. J Clin Invest 1997;100:2315e24. Chen JF, Mandel EM, Thomson JM, Wu Q, Callis TE, Hammond SM, et al. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet 2006;38:228e33. Liu N, Williams AH, Kim Y, McAnally J, Bezprozvannaya S, Sutherland LB, et al. An intragenic MEF2-dependent enhancer directs muscle-specific expression of microRNAs 1 and 133. Proc Natl Acad Sci USA 2007;104:20844e9.
Translational Science
Expression of microRNA-208 is Associated With Adverse Clinical Outcomes in Human Dilated Cardiomyopathy MAMORU SATOH, MD, YOSHITAKA MINAMI, MD, YUJI TAKAHASHI, MD, TSUYOSHI TABUCHI, MD, AND MOTOYUKI NAKAMURA, MD Iwate Japan
ABSTRACT Background: Recently, microRNA-208 (miR-208) encoded by the a-myosin heavy chain (MHC) gene, has been shown to be involved in pathological cardiac growth, fibrosis, and up-regulation of b-MHC expression. A recent study has also reported 2 additional myosin-expressed miRNAs (miR-208b and miR-499). The aim of this study was to determine whether miR-208, miR-208b, and miR-499 are expressed with MHC mRNA in human dilated cardiomyopathy (DCM), and whether these levels are related to left ventricular (LV) function and to clinical outcomes. Methods and Results: Endomyocardial biopsy tissues were obtained from 82 patients with DCM and 21 subjects without LV dysfunction as controls. Levels of miR-208, miR-208b, and miR-499 were higher in DCM patients than in controls. Levels of a-MHC mRNA were lower in patients with DCM than in controls, whereas b-MHC mRNA levels were higher in patients with DCM compared with controls. Levels of miR-208 were correlated with b-MHC mRNA levels and myocardial collagen volume, whereas levels of miR-208b and miR-499 showed no correlation. After a mean follow-up of 517 days, an increase in miR-208 levels was shown to be a strong predictor of clinical outcomes (RR 3.4, 95% CI 1.1e11.2). Conclusions: This study suggests that myocardial expression of miR-208 is associated with MHC mRNA expression and with poor clinical outcomes in patients with DCM. We conclude that miR-208 may therefore be involved in the progression of human DCM. (J Cardiac Fail 2010;16:404e410) Key Words: Biopsy, collagen volume, heart failure, myosin heavy chains.
The myosin heavy chains (MHCs) are molecule motors of muscle and have a potential functional significance for cardiac performance.1 Two isoforms of MHC consisting of a-MHC and b-MHC are expressed in the mammalian heart.2 The myosin of a-MHC has higher adenosine triphosphatase (ATPase) activity than myosin of b-MHC, and contractile velocity correlates with the balance of MHC isoforms.2
The MHC composition of normal human ventricular myocardium has been reported to have b-MHC as the predominant isoform (O95%) among MHC isoforms.3 Impairment of MHC composition, such as a decrease in a-MHC mRNA and its protein levels, has been shown in failing human heart compared to non-failing human heart.4 In addition, improvement in left ventricular ejection fraction (LVEF) through b-blocker therapy is associated with normalization of a-MHC and b-MHC expression resulting from upregulation of a-MHC and downregulation of b-MHC in patients with dilated cardiomyopathy (DCM).5 MHC expression may therefore be critical for maintaining normal cardiac function, which suggests that impairment of MHC composition, such as a subtle shift in the balance between a-MHC and b-MHC, may play a significant role in cardiac failure. However, the precise mechanism underlying the change in MHC composition in human DCM is unclear. It has recently been reported that microRNAs (miRs) are small noncoding RNAs that negatively regulate protein expression at the posttranscription level.6 Through specific targeting of the 30 untranslated regions of multicellular
From the Division of Cardiology, Department of Internal Medicine and Memorial Heart Center, Iwate Medical University School of Medicine, Iwate, Japan. Manuscript received November 17, 2009; revised manuscript received January 8, 2010; revised manuscript accepted January 15, 2010. Reprint requests: Mamoru Satoh, Division of Cardiology, Department of Internal Medicine and Memorial Heart Center, Iwate Medical University School of Medicine, Uchimaru 19-1, Morioka, 020-8505, Iwate, Japan. Tel: þ81-19-651-5111; Fax: þ81-19-651-0401. E-mail: m_satoh@imu. ncvc.go.jp Supported by a Grant-in-Aid for General Scientific Research from the Japanese Ministry of Education, Science, Sports and Culture (No. 20590886), a grant from the Keiryokai Research Foundation (No. 98). 1071-9164/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cardfail.2010.01.002
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eukaryotic mRNAs, miRs downregulate gene expression either by inducing degradation of target mRNA or by impairing its translation.7,8 It has been recently reported that miR-208, a cardiac-specific miR, encodes within intron 27 of the gene encoding a-MHC and is essential for expression of the genes involved in cardiac contractility and fibrosis.9 In response to cardiac stress such as pressure overload, miR-208 knockdown mice showed virtually no cardiomyocytic hypertrophy and fibrosis and no impact on the balance between a-MHC and b-MHC expression compared with wild-type mice.9 A more recent study has also reported 2 myosin-expressed microRNAs, miR-208b and miR-499, which are encoded by b-MHC genes.10 This report provides important new information about the pathogenesis of failing human heart and points the way toward new therapies to combat this condition. However, there have been no reports on the relationship between myosin-expressed microRNAs and MHC expression in human DCM. The aim of this study was to determine whether the expression of miR-208, miR-208b, and miR-499 is related to MHC expression in the myocardium of patients with DCM, and whether miR-208 levels are related to LV geometry and function and to clinical outcomes in human DCM. Methods and Materials Subjects Endomyocardial tissues were obtained from 82 consecutive patients with DCM by right ventricular endomyocardial biopsy. The clinical diagnosis of DCM was made according to the World Health Organization/International Society and Federation of Cardiology Task Force criteria.11 Left ventricular angiography was performed to determine LVEF and left ventricular end-systolic volume index (LVESVI) at the time of biopsy. Control myocardial samples were obtained by endomyocardial biopsy from 21 subjects with suspected cardiac disorders such as myocardial deposition disease, hypertensive heart disease, and hypertrophic cardiomyopathy on the basis of arrhythmia and echocardiographic changes such as premature beats and a slight increase in ventricular wall thickness. Five biopsy samples were taken from each subject from the right ventricular myocardium. Three of the 5 samples were fixed in formalin and paraffin-embedded for histopathology and morphometry. The remaining 2 samples were immediately frozen for subsequent investigation by real-time reverse transcriptase polymerase chain reaction (PCR). The resulting pathologic findings and close clinical examination failed to show any evidence of myocardial disease or functional abnormality, and these subjects were thus designated as controls. This study protocol was approved by our hospital ethics committee, and written informed consent was obtained from all subjects.
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Extracted RNA from endomyocardial biopsy tissues and human myocardial mRNA (Applied Biosystem, Foster City, CA) as a standard control were initially reverse transcribed using a high-capacity cDNA Archive Kit (Applied Biosystem, Foster City, CA) and then amplified in a 10 mL PCR reaction and primer set for amplification of human miR-208 (Assay ID: 000511), miR-208b (Assay ID: 002290), miR-499 (Assay ID: 002427), and U6 (Assay ID: 001093) using TaqMan microRNA assays according to the manufacturer’s recommended protocol (Applied Biosystem).12 Levels of a-MHC (Assay ID: Hs00411908) and b-MHC (Assay ID: Hs01110632) mRNAs were amplified using TaqMan Gene Expression Assays (Applied Biosystem). For all myocardial specimens, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Assay ID: Hs99999905) mRNA was amplified using TaqMan Gene Expression Assays as an endogenous control (Applied Biosystem). The amplification steps consisted of denaturation at 95 C, followed by 40 cycles of denaturation at 95 C for 15 seconds and then annealing at 60 C for 1 minute. All reactions were carried out on the 7500 real-time PCR system (Applied Biosystem) using the TaqMan Universal PCR master Mix and Assays on demand (Applied Biosystem). Relative quantitation was carried out using the DD threshold cycle (Ct) method for recurrent versus primary with U6 or GAPDH as an endogenous control and fold changes were calculated for each gene.13 Replicates with a Ct O40 were excluded. The assay was run in triplicate for each case to allow for assessment of technical variability. To account for PCR amplification of contaminating genomic DNA, a control without reverse transcription was included. To improve the accuracy of real-time reverse transcriptase PCR for quantification, amplifications were performed in triplicate for each RNA sample. Collagen Morphometry Serial paraffin sections (5 mm thick) were stained with Masson trichrome. Myocardial collagen volume was estimated from each specimen by quantitation of fibrillar collagen accumulation stained by Masson trichrome omitting perivascular fibrosis using a computer-associated morphometric program (Olympus, Tokyo, Japan) with WinROOF image analysis software (version 6.01, Mitani Corporation, Tokyo, Japan). Ten randomly selected different areas per specimen were analyzed (magnification 62.5). Myocardial collagen volume fraction (CVF) was calculated as the percentage of myocardial collagen volume in the total area of the specimen. Follow-up Study We planned a minimum follow-up of 12 months in all patients with DCM. This was achieved by outpatient assessments and telephone contact with the patient or his or her local physician. Clinical outcomes were defined as total mortality and development of heart failure (HF). Occurrence of HF required the presence of rest or effort dyspnea and $1 of pulmonary congestion at chest x-ray, new appearance of peripheral edema, or use of diuretics. All deaths were considered to be of cardiac origin unless a noncardiac origin was established clinically or at autopsy. Statistical Analysis
Real-time Reverse Transcriptase PCR for miR-208, miR-208b, miR-499, a-MHC and b-MHC mRNAs Total RNA including the small RNA fraction was extracted from endomyocardial biopsy tissues using a mirVana Paris miRNA isolation kit (Ambion, Inc, Austin, TX) in accordance with the manufacturer’s instructions.
All values are presented as mean 6 standard deviation. Kolmogorov-Smirnov analysis was performed to assess data distribution. Unpaired Student t-test was performed for normally distributed data, and nonparametric Mann-Whitney test was performed where this was not appropriate. Statistical analysis of categorical variables was also carried out using chi-square analysis and Fisher exact
406 Journal of Cardiac Failure Vol. 16 No. 5 May 2010 analysis. Spearman correlation coefficients were used to examine the relationship between miR-208 levels, MHC mRNA levels, CVF, and left ventricular geometry and function. An event-free survival curve was generated by the Kaplan-Meier method, and event-free survival among the groups was compared by use of the log-rank test. Multivariate association between miR-208 levels and clinical outcomes was evaluated with logistic regression models. Odds ratios are reported with logistic regression models that adjust for factors (sex, age, LVEF, LVESVI, New York Heart Association, and medications) independently associated with the outcome variable. A value of P ! .05 was considered statistically significant.
Results Baseline Characteristics
Baseline and clinical characteristics of patients with DCM and controls are shown in Table 1. There were no differences in age and sex between patients with DCM and controls. Data on New York Heart Association functional class, left ventricular geometry, and pressure studies showed significant differences between DCM patients and controls. Levels of miR-208, miR-208b, miR-499, a-MHC, and ß-MHC mRNAs
Histopathology and CVF
There was no difference in average Ct of U6 between patients with DCM and controls (20.6 6 5.1 vs. 19.9 6 4.9; not significant). Levels of miR-208, miR-208b, and miR-499 in the myocardium were higher in patients with DCM than in controls (miR-208: 0.94 6 0.78 vs. 0.21 6 0.19; miR-208b: 0.20 6 0.12 vs. 0.13 6 0.10; miR-499: 0.53 6 0.27 vs. 0.20 6 0.25; Table 1. Baseline and Clinical Characteristics of Study Populations Characteristics
DCM (n 5 82)
Age (y) 55.8 6 12.1 Sex (male/female) 59/23 NYHA functional class at the time of biopsy I or II, n (%) 51 (62)* III or IV, n (%) 31 (38)* LVG at time of biopsy LVEF (%) 34.1 6 10.3* 57.4 6 29.3* LVESVI (mL/mm2 BSA) Pressure study at time of biopsy Systolic BP (mm Hg) 122 6 31 Diastolic BP (mm Hg) 71 6 16 LVEDP (mm Hg) 11.5 6 7.1* 2.9 6 0.9* CI (L$min$m2 BSA) Mean PC (mm Hg) 11.6 6 7.1* Medication, n (%) Loop diuretics 56 (68)* Digitalis 28 (34)* Spironolactone 45 (55)* ACEIs 44 (54)* ARBs 42 (51)* b-blocker 49 (60)*
DCM group vs. controls, all P ! .01) (Fig. 1A). Average Ct of miR-208 was lower in patients with DCM compared with controls (23.8 6 6.4 vs. 28.5 6 7.2; DCM group vs. control group, P ! .01). Levels of a-MHC mRNAwere lower in patients with DCM than in controls (0.07 6 0.02 vs. 0.42 6 0.10, P ! .001) (Fig. 1B). In contrast, b-MHC mRNA levels were higher in patients with DCM compared with controls (1.63 6 1.25 vs. 1.04 6 0.43, P 5 .03) (Fig. 1B). Average Ct of GAPDH did not differ between patients with DCM and controls (20.2 6 4.3 vs. 20.8 6 4.4; not significant). Levels of miR-208 were negatively correlated with a-MHC mRNA levels in patients with DCM (r 5 0.65, P ! .001) (Fig. 2A). There was a positive correlation between miR-208 and b-MHC mRNA levels in patients with DCM (r 5 0.80, P ! .001) (Fig. 2B). Levels of miR-208b and miR-499 were not statistically correlated with b-MHC mRNA levels in patients with DCM (miR208b vs. b-MHC: r 5 0.11, P 5 .35; miR-499 vs. b-MHC: r 5 0.19, P 5 .09).
Controls (n 5 21) 54.2 6 9.6 15/6 21 (100) 0 (0) 69.5 6 7.1 17.2 6 5.9 127 70 5.4 3.9 7.9
6 6 6 6 6
19 11 1.1 0.9 2.9
0 0 0 5 (24) 4 (19) 3 (14)
ACEI, angiotensin-converting enzyme inhibitors; ARB, angiotensin II type 1 receptor blockers; BP, blood pressure; CI, cardiac index; DCM, dilated cardiomyopathy; LVEDP, left ventricular end-diastolic pressure; LVEF, left ventricular ejection fraction; LVESVI, left ventricular end-systolic volume index; LVG, left ventricular angiography; N /A, not available; NYHA, New York Heart Association; PC, pulmonary capillary pressure. *P ! .05 vs. controls.
Histological analysis showed no active or borderline myocarditis in any of the DCM samples analyzed. CVF was higher in patients with DCM than in controls (6.02 6 2.41% vs. 0.63 6 0.69%, P ! .001) (Fig. 3A). There was a moderate positive correlation between CVF and miR-208 levels in patients with DCM (r 5 0.44, P ! .001) (Fig. 3B), whereas miR-208b and miR-499 levels were not correlated with CVF in patients with DCM (miR-208b vs. CVF: r 5 0.36, P 5 .11; miR-499 vs. CVF: r 5 0.12, P 5 .30). Relationship between Clinical Data and miR-208 Levels
There was a moderate negative correlation between miR208 levels and LVEF in patients with DCM (r 5 0.52, P ! .001) (Fig. 4A). LVESVI was positively correlated with miR-208 levels in patients with DCM (r 5 0.54, P ! .001) (Fig. 4B). Relationship between miR-208 Levels and Clinical Outcomes in Patients with DCM
After a mean follow-up of 517 days (range, 48 to 983 days), 7 (8.5%) DCM patients had died from a cardiac cause and 11 (13.4%) had developed HF. There were no significant differences in baseline characteristics of DCM patients between event-free patients and patients with events (Table 2). Figure 5 shows differences in clinical outcomes when patients with DCM were divided into tertiles according to miR-208 levels (high, moderate, and low miR-208 groups). Log-rank analysis showed that the DCM subgroup with high miR-208 levels was associated with poor clinical outcomes. In the multivariate model, after adjustment for baseline characteristics (sex, age, LVEF, LVESVI, New York Heart Association, and medications), high levels of miR-208 (miR-208 levels 5 1.15 to 2.79) were a strong independent
miR-208 and Clinical Outcomes in DCM 2
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Fig. 1. Levels of microRNA-208 (miR-208), miR-206b, miR-499, a-myosin heavy chain (MHC), and ß-MHC mRNA in patients with dilated cardiomyopathy (DCM) and controls. (A) Comparison of miR-208, miR-208b, and miR-499 levels between patients with DCM and controls. (B) Comparison of a-MHC and ß-MHC mRNA levels between patients with DCM and controls. *P ! .05 vs. controls.
predictor of cardiac death and HF (RR 3.4, 95% CI 1.111.2). Discussion Relative expression levels of MHC isoforms including a-MHC and b-MHC can be altered in disease states such as cardiac failure or hypertrophy.1 In the present study, both upregulation of b-MHC levels and downregulation of a-MHC levels were shown in myocardium from patients with DCM compared with subjects without LV dysfunction. In agreement with these findings, a shift from a-MHC toward b-MHC is often observed in failing human heart.14 In addition, a b-MHC-transgenic mouse model has shown that a shift from a-MHC to b-MHC isoform reduced left ventricular contractility and dilated left ventricular volume.15 Myocardial expression of a-MHC has more rapid contractile velocity than myocardial expression of b-MHC, because the tension-time integral for force per cross-bridge cycle is greater in a-MHC than in b-MHC.16,17 Indeed, the proportion of a-MHC mRNA relative to total MHC mRNA levels is reduced in myocardium obtained from patients undergoing cardiac transplantation
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for chronic end-stage HF from several forms of heart disease, including DCM.4,14 From these observations, it has been suggested that alternation in MHC isoform ratio which represses a-MHC and reexpresses b-MHC in the heart may be involved in LV dysfunction and dilatation in human DCM. However, the mechanisms underlying this alternation in MHC isoforms in patients with DCM are poorly understood. MicroRNAs, a newly discovered class of small ribonucleotide-based regulators of gene expression, modulate gene expression at the posttranscriptional level.6,7 Several recent studies have demonstrated that expression of numerous microRNAs, such as miR-21, miR-133, miR195, and miR-208, have key roles in the growth, development, function, and stress responsiveness of the heart.9,18-21 In particular, it has been reported that myocardial expression of miR-208 regulates the balance between a-MHC and b-MHC isoforms and induces adverse cardiac remodeling resulting from a reduction in cardiac contractility and an increase in cardiac fibrosis.9 A more recent report has also shown that miR-208b and miR-499 were encoded by b-MHC genes as myosin-expressed microRNAs.10 These reports provide important new information about the
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Fig. 2. Correlation plot between microRNA-208 (miR-208) levels and a-myosin heavy chain (MHC) and ß-MHC mRNA levels in dilated cardiomyopathy (DCM) patients. (A) miR-208 vs. a-MHC mRNA, r 5 0.65, P ! .001. (B) miR-208 vs. ß-MHC mRNA, r 5 0.80, P ! .001.
408 Journal of Cardiac Failure Vol. 16 No. 5 May 2010
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Fig. 3. (A) Comparison of collagen volume fraction (CVF) between patients with dilated cardiomyopathy (DCM) and controls. *P ! .01 vs. controls. (B) Correlation between microRNA-208 (miR-208) levels and CVF in patients with DCM (r 5 0.44, P ! .001).
A3
B3 miR-208 levels
miR-208) in the heart dramatically increased b-MHC protein levels and then induced adverse cardiac remodeling.24 In contrast, deletion of miR-208a resulted in decreased b-MHC expression in the adult heart, providing genetic evidence that miR-208a is required for b-MHC expression.24 Although the present study could not confirm the mechanism by which miR-208 levels were increased in the myocardium in DCM, these observations have therefore suggested a critical role for miR-208 transcripts in the induction of b-MHC and cardiac remodeling during stress conditions. The present study has shown a positive correlation between miR-208 levels and CVF in myocardium obtained from patients with DCM. MiR-208 knockdown mice demonstrated diminished collagen accumulation and upregulation of b-MHC expression in response to pressure overload.9 A relationship between b-MHC gene expression and cardiac fibrosis has also been found in animal heart.25 From these observations, it has been speculated that miR-208 expression could affect myocardial collagen accumulation via upregulation of b-MHC expression in human DCM. Using right ventricular septal biopsy specimens for measurement of miR-208 expression, the present study found a correlation between miR-208 levels and LV function and volume in human DCM. Although it could not be confirmed whether miR-208 expression in right ventricular
miR-208 levels
pathogenesis of failing human heart and points the way toward new therapies to combat this condition. The present study has shown that miR-208, miR-208b, and miR-499 levels were higher in myocardium obtained from patients with DCM compared with that from subjects without LV dysfunction. There was a negative correlation between miR-208 and a-MHC mRNA levels in DCM. These observations have suggested that myocardial expression of miR208 may downregulate a-MHC mRNA levels resulting from inducing degradation of a-MHC mRNA. In addition, miR-208 had a long half-life and may be elevated in the myocardium in DCM, although a-MHC levels had declined.9 It has therefore been speculated that a closed relationship exists between miR-208 expression and a change in MHC isoform composition in human DCM. The present study has shown a positive correlation between miR-208 and b-MHC levels in patients with DCM, whereas both miR-208b and miR-499 levels were not correlated with b-MHC levels. It has been reported that both normal and dilated cardiomyopathic human ventricles express predominantly b-MHC and very low levels of a-MHC, and a-MHC levels are lower in failing ventricles comparing with normal ventricles, suggesting a shift toward b-MHC in failing ventricles.22 In addition, increased b-MHC expression is a feature of HF in human hearts.23 It has recently been reported that transgenic overexpression of miR-208a (also known as
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Fig. 4. Correlation between LV geometry and microRNA-208 (miR-208) levels in patients with dilated cardiomyopathy (DCM). (A) LVEF vs. miR-208 levels, r 5 0.52, P ! .001. (B) LVESVI vs. miR-208 levels, r 5 0.54, P ! .001. LVEF, left ventricular ejection fraction; LVESVI, left ventricular end-systolic volume index.
miR-208 and Clinical Outcomes in DCM Table 2. Baseline and Clinical Characteristics of Event-free Patients and Patients with Events Patients with Event-free Patients Events (n 5 18) (n 5 64)
Characteristics
Age (y) Sex (male/female) NYHA functional class at time of I or II, n (%) III or IV, n (%) LVG at time of biopsy LVEF (%) LVESVI (mL/mm2 BSA) Pressure study at time of biopsy Systolic BP (mm Hg) Diastolic BP (mm Hg) LVEDP (mm Hg) CI (L$min$m2 BSA) Mean PC (mm Hg) Medication, n (%) Loop diuretics Digitalis Spironolactone ACEIs ARBs b-blocker
56.3 6 13.1 15/3 biopsy 11 (61) 7 (39)
55.1 6 12.5 44/20
34.1 6 11.4 70.1 6 23.4
34.1 6 10.2 67.1 6 21.3
117 69 15.8 3.0 12.4 14 6 10 10 9 11
6 6 6 6 6
40 (65) 24 (38)
26 17 9.1 0.9 9.6
132 72 10.4 2.9 10.7
(78) (34) (56) (56) (50) (61)
42 22 35 34 33 38
6 6 6 6 6
29 18 6.6 0.7 6.8
(66) (34) (55) (53) (52) (59)
ACEI, angiotensin-converting enzyme inhibitors; ARB, angiotensin II type 1 receptor blockers; BP, blood pressure; CI, cardiac index; DCM, dilated cardiomyopathy; LVEDP, left ventricular end-diastolic pressure; LVEF, left ventricular ejection fraction; LVESVI, left ventricular end-systolic volume index; LVG, left ventricular angiography; N /A, not available; NYHA, New York Heart Association; PC, pulmonary capillary pressure.
septum directly reflects LV function in human DCM, it has been reported that changes in expression of several genes in the septum reflect those in both right and left ventricular free walls in human DCM.26 Another important finding of the present study was high levels of miR-208 expression in association with poor clinical outcomes in DCM, showing miR-208 to be a predictor of cardiac death and progression of HF in DCM patients. It has been suggested that miR-208 is required for the development of cardiac hypertrophy and myocardial fibrosis.9 These events may lead to
Event-free survival
1 Group 1: low miR-208 Group 2: moderate miR-208
0.8 0.6 0.4 0.2
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Group 3 vs. 1 or 2, P = 0.02
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adverse left ventricular remodeling and the development of cardiac events. On the basis of these observations, we propose that myocardial expression of miR-208 may give rise to a HF phenotype that consists of reexpression of an embryonic gene pattern (eg, upregulation of b-MHC) and an increase in myocardial collagen accumulation, leading to irreversible myocardial injury in human DCM. The results of the present study therefore provide a pathogenic mechanism for human DCM based on the relationship between cardiac miR-208 expression and this HF phenotype. Myocardial expression of miR-208 may therefore represent a novel therapeutic opportunity. Limitations
A limitation of this study is the small size of the study population. In addition, no miR-208 functional analysis of cardiac myocytes was carried out, such as that in these microRNAs upregulated or downregulated in vitro models. Further studies will therefore be needed to determine the mechanism by which miR-208 expression regulates a HF phenotype, such as upregulation of b-MHC, in human DCM. In addition, recent reports have identified myocardial expression of microRNAs (eg, miR-1, miR-133) in addition to miR-208.27,28 Further studies will thus be needed to characterize the expression profile of myocardial microRNAs in the myocardium in DCM. The present study could not confirm miR-208 as a marker of HF, because the characteristics of the DCM and control materials used differed significantly, including LV geometry and medication. We will therefore need to examine miR-208 levels in HF from DCM in comparison to that arising from various other origins, such as secondary cardiomyopathy, ischemic heart, and valvular disease. The present study has also not confirmed whether miR-208 levels provide incremental information beyond other clinical parameters (eg, brain natriuretic peptide, high-sensitivity C-reactive protein) that are already known to be prognostic factors. Followup studies will be needed to compare miR-208 with other prognostic parameters. Conclusions In conclusion, the present study suggests that myocardial expression of miR-208 is associated with reexpression of bMHC mRNA and cardiac collagen accumulation and may be involved in adverse clinical outcomes in human DCM.
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References
Time (days) Group 1:n = 27; miR-208 levels, 0.03 to 0.35 Group 2:n = 28; miR-208 levels, 0.36 to 1.14 Group 3:n = 27; miR-208 levels, 1.15 to 2.79
Fig. 5. Kaplan-Meier analysis. Event-free (P 5 .02, log-rank test) for group 3 (high microRNA-208 [miR-208] levels). HRs were 5.8 (95% CI, 1.4 to 23.8) for group 1 vs. group 3 and 4.0 (95% CI, 1.1 to 14.7) for group 2 vs. group 3. Group 1: n 5 27, miR-208 levels 5 0.03 to 0.35; Group 2: n 5 28, miR-208 5 0.36 to 1.14; Group 3: n 5 27, miR-208 5 1.15 to 2.79.
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