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. 2010 Jan 8;106(1):166-75.
doi: 10.1161/CIRCRESAHA.109.202176. Epub 2009 Nov 5.

MicroRNA-133a protects against myocardial fibrosis and modulates electrical repolarization without affecting hypertrophy in pressure-overloaded adult hearts

Scot J Matkovich  1 , Wei Wang, Yizheng Tu, William H Eschenbacher, Lisa E Dorn, Gianluigi Condorelli, Abhinav Diwan, Jeanne M Nerbonne, Gerald W Dorn 2nd
Affiliations

MicroRNA-133a protects against myocardial fibrosis and modulates electrical repolarization without affecting hypertrophy in pressure-overloaded adult hearts

Scot J Matkovich et al. Circ Res. .

Abstract

Rationale: MicroRNA (miR)-133a regulates cardiac and skeletal muscle differentiation and plays an important role in cardiac development. Because miR-133a levels decrease during reactive cardiac hypertrophy, some have considered that restoring miR-133a levels could suppress hypertrophic remodeling.

Objective: To prevent the "normal" downregulation of miR-133a induced by an acute hypertrophic stimulus in the adult heart.

Methods and results: miR-133a is downregulated in transverse aortic constriction (TAC) and isoproterenol-induced hypertrophy, but not in 2 genetic hypertrophy models. Using MYH6 promoter-directed expression of a miR-133a genomic precursor, increased cardiomyocyte miR-133a had no effect on postnatal cardiac development assessed by measures of structure, function, and mRNA profile. However, increased miR-133a levels increased QT intervals in surface electrocardiographic recordings and action potential durations in isolated ventricular myocytes, with a decrease in the fast component of the transient outward K+ current, I(to,f), at baseline. Transgenic expression of miR-133a prevented TAC-associated miR-133a downregulation and improved myocardial fibrosis and diastolic function without affecting the extent of hypertrophy. I(to,f) downregulation normally observed post-TAC was prevented in miR-133a transgenic mice, although action potential duration and QT intervals did not reflect this benefit. miR-133a transgenic hearts had no significant alterations of basal or post-TAC mRNA expression profiles, although decreased mRNA and protein levels were observed for the I(to,f) auxiliary KChIP2 subunit, which is not a predicted target.

Conclusions: These results reveal striking differences between in vitro and in vivo phenotypes of miR expression, and further suggest that mRNA signatures do not reliably predict either direct miR targets or major miR effects.

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Figures

Figure 1
Figure 1. microRNA and mRNA regulation in hypertrophy models
A. miR-133a expression determined by RT-qPCR: left panel, baseline (n = 10) and 7 days after TAC (n = 6 for 1 week, n = 8 for 3 weeks); center panel, at baseline (n = 5) and in response to 60 mg/kg/d isoproterenol (ISO, n = 5) for 14 days; right panel, nontransgenic (ntg, n=4), Gαq transgenic (Gq, n = 4) and constitutively active PI3K transgenic (caPI3K, n = 4). B. Microarray analysis of microRNAs in nontransgenic, Gq and caPI3K hearts (n=4 each). Blue denotes low expression, red denotes high expression. C. Microarray analysis of mRNAs in ntg nonoperated hearts, ntg hearts 7 days after TAC, Gq, and caPI3K hearts (n=4 each). D. Venn diagram of regulated mRNAs.
Figure 2
Figure 2. αMHC-miR-133a transgenic mice have normal basal cardiac characteristics
A. Schematic diagram of αMHC-miR-133a transgene construct depicting the encoded stem-loop structure. a and b correspond to PCR primers. B. Top: miR-133a genomic precursor (transgene) expression by Northern blot. Middle: RT-PCR of miR-133a genomic precursor expression. Bottom: RT-PCR in the absence of reverse transcriptase. ntg = non-transgenic, miR = αMHC-miR-133a transgenic mice. C. Mature miR-133a expression in ntg and miR hearts measured by RT-qPCR (n = 6 pairs). D. Gross structure of ntg and miR hearts at 12 weeks of age (top); wheat-germ agglutinin (WGA) staining (bottom). E. Cardiac performance as a function of infused dobutamine dose. n=6 mice per group. F. Representative signal-averaged ECGs. ntg = non-transgenic; miR = αMHC-miR-133a.
Figure 3
Figure 3. Functional and molecular responses to surgical pressure overload are similar in αMHC-miR-133a (miR) transgenic mice
A. Morphometric data. Left panel; gravimetric heart weight (hw) vs tibial length (tb) in non-operated (white bars) and 3 week TAC (black bars) mice. n=6 non-transgenic (ntg) non-operated; n=6 miR non-operated; n=9 ntg TAC; n=8 miR TAC. Center panel; echocardiographic left ventricular mass (LVM). Right panel; myocyte cross-sectional area (CSA) measured by WGA stain. B. Functional data. Left panel; echocardiographic fractional shortening (%FS). Center panel; maximal rate of increase in left ventricular pressure (dP/dt-max). Right panel; maximal rate of decrease of left ventricular pressure (dP/dt-min). C. Fetal gene expression at baseline and 1 week after TAC. White bars = ntg nonoperated (n=5), black bars = ntg TAC (n=4), gray bars = miR nonoperated (n=6), striped bars = miR TAC (n=4). D. Volcano plot of cardiac-expressed mRNAs by microarray analyses (~7200 genes), with –log(p-value) plotted on the y-axis vs fold-change (TAC vs nonoperated) on the x-axis. Red squares are mRNAs upregulated at least 2-fold (P≤0.001) by TAC in ntg. Green triangles are mRNAs downregulated at least 2-fold (P≤0.001) by TAC in miR.
Figure 4
Figure 4. Preventing miR-133a downregulation by TAC decreases fibrosis and apoptosis and improves diastolic function in pressure-overloaded hearts
A. left panels, picrosirius red staining of ntg and miR myocardium, nonoperated (nonop) and 3 weeks after TAC. right graphs, quantitative collagen content data (n=6 per group). B. Apoptosis in non-operated (white bars) and 1-week TAC hearts (black bars) measured by TUNEL; n=6 per group. (right) Representative histological sections showing caspase 3 activation. C. Pressure volume loops and expanded end diastolic parameters. D. Quantitative data from hemodynamic analyses. Left is end-systolic pressure volume relationship; middle is left ventricular end diastolic pressure; right is left ventricular stiffness constant.
Figure 5
Figure 5. Repolarization and repolarizing Kv currents in αMHC-miR-133a ventricular myocytes
A. Representative whole-cell action potential waveforms. ntg = non-transgenic; miR = αMHC-miR-133a; TAC = 1 week after TAC. B. Action potential durations at 20% (APD20) 50% (APD50), and 90% (APD90) repolarization. White bars are non-operated. Black bars are 1 week after TAC. C. Corrected QT intervals by signal-averaged ECG, bar codes are same as Figure 5b. D. Resting membrane potentials (Vm) and action potential amplitudes (APA). ntg, n = 12; ntg-TAC, n = 13; miR, n = 16; miR-TAC, n = 11 myocytes. E. Representative whole-cell Kv currents, evoked during 4.5 sec depolarizing voltage steps to test potentials from −40 to +40 mV (in 10 mV increments) from a holding potential (HP) of −70. F–I. Group mean measures of peak current density (F), Iss density (G), IK, slow density (H), and Ito,f density (I). White bars are non-operated. Black bars are 1 week after TAC.
Figure 6
Figure 6. Kv channel subunit expression in miR-133a-expressing and hypertrophied ventricular myocytes
A. Kv channel transcripts were examined using RT-qPCR (mean ± SEM, n=3 hearts per group). B. Kv4.2 and KChIP2 protein expression (mean ± SEM, n=4 hearts per group, representative blots shown). For A and B, white bars are non-transgenic, black bars are αMHC-miR-133a transgenic hearts. C. Kv channel transcripts and D. immunoblot analysis in non-transgenic and αMHC-miR-133a transgenic hearts (mean ± SEM, n=5 non-operated per group, n=6 TAC per group). Representative immunoblots (2 samples per group) are shown. White bars are non-operated, black bars are 1 week after TAC.
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