(A) NRVM over-expressing precursors for miR-34b/c were collected for mRNA transcript levels. Results (normalized to non-targeting miR) show down-regulation of SCN5A, and SCN1B, but unchanged levels for KCND3 (n = 7–8). (B) Protein levels from NRVM with over-expressed miR-34b showing reduced protein expression for Kv4.3 (KCND3). Multiple bands for Kv4.3 represent different glycosylation states of the protein. (C) Summary data of the immunblot for Kv4.3 (n = 4). (D) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c, with mutations made to the seed regions (highlighted in red) to disrupt interaction at the seed region. (E) Reporter assay with the 3’-UTR cloned into pmiRGlo reporter construct. Luciferase activity in HEK cells transfected with WT or mutant 3’-UTRs. Results are presented as a percent change from a non-targeting miR precursor (n = 5) normalized to renillin activity. (F) I/V curves for INa measured in NRVM over-expressing precursors for control (n = 24), miR-34b (n = 24), or miR-34c (n = 18). (G) I/V curves for Ito,total measured in NRVM over-expressing precursors for control (n = 15), miR-34b (n = 12), or miR-34c (n = 11). (H) Ito,f was also assessed in NRVM through kinetic subtraction of Ito,s. Resulting I/V curves now reveal a significant reduction in current density in miR-34b (n = 14) and miR-34c (n = 15) precursor treated cells compared to control (n = 16). (I) The same experiments conducted in human derived cardiomyocytes (iCells) expressing either control or miR-34b/c precursor together, measuring INa (control, n = 24; miR-34b/c, n = 21) and (J) Ito,total (control, n = 24; miR-34b/c, n = 25). Data presented as mean ± SEM. *p<0.05; **p<0.01, as indicated or compared to control. See also Figure 3—figure supplement 1.