Importantly, we present here a direct experimental validation of differential mRNA targeting by 59 isomiRs using the example of miR-133a. Interestingly, alteration to the 39 end of miR-133a mimics did not affect the level of mRNA repression, suggesting that in this instance the 39 end is not essential for efficient target binding. Our work further provides information on expression of novel miR*, a new candidate for AGO2-mediated processing and antisense-miRNAs, as well as documenting a substantial repertoire of entirely novel miRNA candidates. Remarkably, we also described numerous examples of extreme miRNA variants, whose existence may often be explained by the primiRNA adopting an alternate secondary structure that differ from the MK-1775 Wee1 inhibitor predicted structures deposited in miRBase. We therefore suggest that many novel e-miRs may be produced by canonical Drosha and Dicer processing of alternate miRNA harpin structures. While novel as a concept in this context, it is in general well established that long-range interactions within RNA molecules or interactions with cellular proteins can affect local RNA secondary structure and the current CUDC-907 supply catalog of known miRNA hairpin interacting proteins is rapidly expanding. The HL-1 cell line is a popular cell culture model of cardiomyocyte biology and, as shown previously for transcriptomic and phenotypic aspects, we have demonstrated here that they express an adult cardiomyocyte-like miRNA profile. Furthermore, a small number of miRNAs with prominent roles in cardiac biology represented the bulk of the HL-1 cell tag count, as is typically seen in differentiated cells. Nevertheless, the miRNA expression profile of HL-1 cells did deviate from a previously described whole heart miRNA expression profile and our own left ventricle dataset in some respects, e.g. exhibiting higher miR-145 and lower miR-1 expression. This may partly be due to the transformed nature and cell culture environment of HL-1 cells, or their atrial origin as there is some evidence that expression of miRNAs differs between atrium and ventricle. Importantly, our observations of the many miRNA processing variants were remarkably consistent between HL-1 cells and our ventrical biopsy. It was interesting to test whether the characteristic change from a non-beating to a beating state mimicked a distinct step in cardiomyocyte differentiation, however, the relative lack of differential miRNA expression we found argues against this notion. Instead, we saw a bulk up-regulation in miRNA level, consistent with observations in other cells types reaching confluency. It remains to be tested whether this merely correlates with the beating state, or directly contributes to it in some way. The importance of miRNAs for cardiac development and cardiomyocyte function is well described.