MicroRNA-1 and -133 increase arrhythmogenesis in heart failure by dissociating phosphatase activity from RyR2 complex

Abstract

In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca(2+) release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca(2+) waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca(2+)/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca(2+) waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca(2+) waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca(2+) cycling, and increased propensity to arrhythmogenesis.

Figure 1. Inhibition of phosphatase activity is sufficient to promote arrhythmogenic spontaneous Ca²⁺ waves in normal paced myocytes during beta-adrenergic stimulation.

A. Representative confocal Ca²⁺ images, with corresponding time dependent profiles (bottom) and membrane potential recordings (top) in control myocytes at basal conditions, preincubated with 100 nM Calyculin A for 30 min, incubated with 100 nM isoproterenol for 3 min (Iso) and challenged with Iso after 30 min incubation with Calyculin A. B. Pooled data for a number of delayed after-depolarizations due to spontaneous Ca²⁺ waves per cycle. Basal SCW frequency was 0 out of 9 cells. Statistically significant at p<0.05, vs. * Calyculin A and † vs Iso, n = 5.

Figure 2. Heart failure animals have increased LV chamber size and reduced fractional shortening.

A. LV internal diameter- end diastole is increased in the 4 month HF dogs. B. LV internal diameter- end systole is increased in the 4 month HF dogs. C. LV fractional shortening is reduced in the 4 month HF dogs. *p<0.05, n = 5–6.

Figure 3. Changes in Expression Levels of miR-1 and miR-133 and their targets in canine HF Myocytes.

A, B. Normalized levels of miR-1 and -133 assessed with qRT-PCR. *p<0.05, n = 5. C. Representative Western blots of putative targets of miR-1 and -133, regulatory subunits B56α, B56δ and catalytic subunit of PP2A respectively. D,E,F. Normalized optical density (OD) for B56α, B65δ and catalytic subunit of PP2A respectively; *p<0.05, n = 8. Note that anti-PP2AC antibody recognizes both α and β isoforms. G. Total PP2A activity is samples prepared from LV tissues of normal and failing hearts, *p<0.05, n = 4. H,I,J. Decrease in levels of PP2A scaffolded to RyR2 tested using coimmunoprecipitation with anti-RyR2 antibodies; (H) representative Western blots and pooled data for B56α (I) and PP2AC (J), *significantly different vs. control, p<0.05, n = 6. K. Local CaMKII activity measured in samples with immunoprecipitated RyR2s (n = 4). Local CaMKII activity levels and levels of PP2AC and B56α were normalized to the levels of RyR2s.

Figure 4. MiR-133 targets both catalytic subunits of the PP2A phosphatase.

A. CHO cells were transfected with either the psiCHECK-PP2ACα or psiCHECK-PP2ACα-mut (i.e. the putative miR-133 binding has been mutated) luciferase reporter construct and miR-133, or negative control miRNA mimics at the concentrations indicated. B. Alternatively, CHO cells were transfected with either the psiCHECK-PP2ACβ or psiCHECK-PP2ACβ-mut luciferase reporter construct and miR-133, or negative control miRNA mimics at the concentrations indicated. Twenty-four hours following transfection, luciferase activities were measured. Renilla luciferase activity was normalized to firefly luciferase activity and mean activities ± S.E. from three-five independent experiments are shown (*p<0.05 vs. 0 nM).

Figure 5. Inhibition of PP2A promotes spontaneous Ca²⁺ release under condition of β-adrenergic stimulation.

A, Representative line-scan images and temporal profiles of Rhod-2 fluorescence recorded in control myocyte treated with 100 nM Iso alone, 300 nM fostriecin (30 min preincubation), a specific PP2A inhibitor, alone, and 300 nM fostriecin plus 100 nM Iso, respectively. Cells were field-stimulated at 0.3 Hz. B, Frequency of diastolic SCWs was calculated for myocytes treated with Iso alone (n = 12), fostriecin (Fost) alone (n = 11), and Fost plus Iso (n = 11). *, p<0.05 Fost plus Iso vs. Iso alone; †, p<0.05 Fost plus Iso vs. Fost alone.

Figure 6. Enhanced phosphorylation of RyR2 at sites S-2814 and S-2030 in HF.

A, B. Representative Western blots (A) and pooled data (B) for normalized RyR2 phosphorylation at sites - S-2030 and S-2808 and S-2814 to total RyR2 content measured in gels run in parallel. Levels of RyR2s in HF and control cells were compared using GAPDH as loading control. *p<0.05, n = 8. C.D, Representative Western blots (C) and pooled data (D) for β-adrenergic agonist Isoproterenol dose-dependence of S-2030. Signals obtained with anti pS-2030 ab were normalized to the levels of RyR2s assessed in gels run in parallel and normalized to maximum level of phosphorylation achieved by 30 min incubation of myocytes isolated from normal hearts with PP1 and PP2A inhibitor Calyculin A (100 nM) and exposed to 1 µM Iso for 3 min. *†,p<0.05 vs baseline and Iso+Calyculin respectively, n = 6. E,F. Representative Western blots and pooled data illustrating sensitivity of S-2030 to phosphorylation by CaMKII in myocytes isolated from failing hearts. Incubation with CaMKII inhibitor KN93 (1 µM, 15 min) significantly reduced S-2030 phosphorylation at basal conditions and after myocytes treatment with Iso and Calyculin A. *p<0.05 vs no KN93, n = 4.

Figure 7. Overexpression of miR-1 and miR-133 increases the frequency of pro-arrhythmic spontaneous Ca²⁺ waves (SCW) in rat myocytes.

A, Time dependent profiles of electrically- (field stimulation at 0.5 Hz) and 10 mM caffeine-evoked Ca²⁺ transients in myocytes infected with scrambled and miR-133 carrying Advs (MOI 100) after 48 hrs in primary culture. B,C,D. Pooled data for electrically and caffeine evoked Ca²⁺ transient amplitude and fractional release in miR-133 overexpressing myocytes vs. controls. E. Representative confocal Ca²⁺ images with corresponding time dependent profiles recorded in field-stimulated (0.2 Hz) myocytes in the presence of 100 nM isoproterenol infected with miR-1 and/or miR-133 Adv. F. Pooled data for the number of SCWs per second. *Statistically significant at p<0.05 vs. scrambled, n = 15–25. G. Representative Western Blots (upper panel) and pooled data (lower panel) of PP2AC in myocytes infected with control Adv (scrambled) or miR-1, or miR-133 or both miR-1 and miR-133; *<0.05 vs control, n = 6. H. Levels of miR-1 and miR-133 in myocytes assessed by qRT-PCR. *<0.05 vs control, n = 3. I. Infection of non-muscle cells (HEK) with miR-1 does not result in increase in expression levels of miR-133 and vise versa, *p<0.05 vs control, n = 3.

Figure 8. Inhibition of CaMKII activity attenuates pro-arrhythmic spontaneous Ca²⁺ waves in HF myocytes.

A. Representative confocal Ca²⁺ images, with corresponding time dependent profiles (bottom) and membrane potential recordings (top) in myocytes isolated from normal and failing hearts in the presence of 100 nM Iso. B. Pooled data for a number of spontaneous Ca²⁺ waves per cycle. *p<0.05, n = 5–10.