Targeting miR-423-5p Reverses Exercise Training–Induced HCN4 Channel Remodeling and Sinus Bradycardia

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  Figure 1.. Evidence of a role for HCN4 (hyperpolarization-activated cyclic nucleotide gated channel 4) in the resting bradycardia in human athletes. A, VO2max of sedentary human subjects and human athletes (n=7/8). B, Heart rates measured under baseline conditions and after complete autonomic blockade of sedentary human subjects and human athletes (n=10/8). C, Complete autonomic blockade abolishes heart rate variability. Left, Examples traces of RR interval under baseline conditions and after complete autonomic blockade. Right, Spectral analyses of corresponding RR interval plots demonstrating near-abolition of high-frequency (HF) heart rate variability after autonomic blockade (note difference in y axis scale). D, Relationship between the intrinsic heart rate measured after complete autonomic blockade and VO2max in sedentary human subjects and human athletes (n=7/8). E, Relationship between ivabradine-induced decrease in heart rate and the intrinsic heart rate measured after complete autonomic blockade in sedentary human subjects and human athletes (n=10/8). In D and E, data fit by linear regression; best fit line, 95% confidence limits, and R2 and P values shown. PSD indicates power…

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  Figure 2.. If remodeling in training-induced bradycardia is accompanied by dysregulation of miRs. A, Heart rates of sedentary and trained mice measured in vivo in conscious animals (n=5/9) and in vitro in isolated sinus node preparations (n=6/6). B, Expression of HCN4 (hyperpolarization-activated cyclic nucleotide gated channel 4) mRNA in sinus node of sedentary and trained mice (n=5/5). C, Current–voltage relationships for If recorded from single sinus node cells from sedentary (n=47 cells/5 mice) and trained (n=58 cells/4 mice) mice. D, Relationship between ivabradine-induced decrease in heart rate and the intrinsic heart rate measured in vivo after complete autonomic blockade in sedentary and trained mice (n=8/10). Data fit by linear regression; best fit line, 95% confidence limits, and R2 and P values are shown. E, Significant (as determined by DeSeq) training-induced changes in miR expression (measured by next generation sequencing) in sinus node of mice. Ratio of miR expression in trained mice to expression in sedentary mice shown on logarithmic scale. Hatched bars indicate significant differences after Benjamini–Hochberg false discovery rate (FDR) correction (P<0.05). Data obtained…

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  Figure 3.. HCN4 (hyperpolarization-activated cyclic nucleotide gated channel 4) is a target gene for miR-423-5p. A, Luciferase reporter assay showing post-transcriptional repression of HCN4 by miRs. H9C2 cells were cotransfected with precursor miR and 3′-UTR of HCN4 cloned into expression vector downstream of luciferase gene. Luciferase activity is shown 24 h after cotransfection with different miRs, including a control (scrambled) miR. n=3 batches of cells with 4 to 5 replicates/batch. B, Predicted miR-423-5p binding sites in HCN4 3′-UTR and corresponding sequence of mutant HCN4 3′-UTR tested. C, Luciferase reporter assay showing dose-dependent repression of HCN4 by miR-423-5p and loss of repression by mutation of HCN4 3′-UTR. Luciferase activity is shown 24 h after cotransfection with different amounts of wild-type or mutant miR-423-5p. n=3 batches of cells with 4 replicates/batch. D, Relationship between HCN4 mRNA and log miR-423-5p in sedentary and trained mice (n=5/5). E, Relationship between the heart rate measured in vitro in the isolated sinus node and log miR-423-5p in sedentary and trained mice (n=6/7). In D and E, data fit by linear regression; best fit line, 95% confidence…

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  Figure 4.. Anti-miR to miR-423-5p reverses training-induced bradycardia and blunts HCN4 (hyperpolarization-activated cyclic nucleotide gated channel 4) channel remodeling. A, Time course of exercise training and anti-miR administration. B, Anti-miR abolishes training-induced upregulation of miR-423-5p in sinus node. miR-423-5p (determined by quantitative real-time reverse transcription polymerase chain reaction [qPCR]) in sinus node of vehicle- or anti–miR-treated sedentary and trained mice shown (n=6/5/5/5). C, Anti-miR reverses training-induced bradycardia. Representative ECG traces (recorded from conscious animals) from a vehicle-treated sedentary mouse, vehicle-treated trained mouse, and anti–miR-treated trained mouse shown on left and mean heart rates (measured in vivo and in isolated sinus node) on right (n=10/12/12 and 9/12/12). D, Anti-miR reverses training-induced downregulation of HCN4. Western blots using antibodies recognizing HCN4 and actin (housekeeper) proteins for sinus node from vehicle-treated sedentary, vehicle-treated trained, and anti-miR-treated trained mice shown as well as mean expression level of HCN4 protein (normalized to actin) in the 3 groups (n=5/4/5 with 3…

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  Figure 5.. Nkx2.5 regulation of miR-423-5p. A, map of NSRP1 gene (solid blocks show exons with introns in between) showing location of Nkx2.5 binding sites and intronic location of the miR-423 gene. B, Expression of NSRP1 mRNA in sinus node of sedentary and trained mice (n=8/8). C, Significant (P<0.05) training-induced changes in expression of transcription factor transcripts (measured by quantitative real-time reverse transcription polymerase chain reaction [qPCR]) in sinus node of mice. Ratio of mRNA expression in trained mice to expression in sedentary mice shown (n=6/8). D, Expression of Nkx2.5 mRNA in sinus node of sedentary and trained mice (n=8/8). E, Luciferase reporter assay showing activation of NSRP1 transcription by Nkx2.5. H9c2 cells were transfected with 2.1 kb of the 5′ flanking region of NSRP1 cloned into expression vector downstream of gene for luciferase. The cells were cotransfected with Nkx2.5; control cells were not cotransfected with Nkx2.5. Luciferase activity is shown 48 h after transfection. n=3 independent batches of cells with 4 replicates/batch. F, Upregulation of miR-423-5p by Nkx2.5. miR-423-5p expression is shown in H9c2 cells not transfected (control) or…