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The adult mammalian heart is non-regenerative owing to the post-mitotic nature of cardiomyocytes. The neonatal mouse heart can regenerate, but only during the first week of life. Here we show that changes in the composition of the extracellular matrix during this week can affect cardiomyocyte growth and differentiation in mice. We identify agrin, a component of neonatal extracellular matrix, as required for the full regenerative capacity of neonatal mouse hearts. In vitro, recombinant agrin promotes the division of cardiomyocytes that are derived from mouse and human induced pluripotent stem cells through a mechanism that involves the disassembly of the dystrophin–glycoprotein complex, and Yap- and ERK-mediated signalling. In vivo, a single administration of agrin promotes cardiac regeneration in adult mice after myocardial infarction, although the degree of cardiomyocyte proliferation observed in this model suggests that there are additional therapeutic mechanisms. Together, our results uncover a new inducer of mammalian heart regeneration and highlight fundamental roles of the extracellular matrix in cardiac repair.

The only stoichiometric proteasomal subunit found to reside outside the proteasome is the ubiquitin receptor S5a. S5a-dependent binding of substrates and shuttle factors is restricted to occur only on the proteasome, thus increasing efficiency of substrate degradation by the 26S proteasome.

In this study, we explored the possibility that PKR, a dsRNA-activated regulatory protein, is an essential component in the differentiation program of myogenic cells in vitro.For this purpose, we used a retroviral expression vector pMV7, harboring the PKR dominant-negative mutant PKRΔ6 (pMV7-p68Δ6). Murine C2C12 myogenic cells were transfected either with pMV7 or with pMV7-p68Δ6. Neomycin-resistant clones from both types were isolated and expanded and the results obtained with the representative clones C2-NEO (transfected with pMV7) and clone 17 and clone 22 (both transfected with pMV7-p68Δ6) are presented. In clone 17 and 22 cells, regardless of IFN treatment, a similar level of the transfected human p68 PKR mutant was detected. This protein was absent in C2-NEO cells. In parallel, in all types of cells, a low basal level of the endogenous murine p65 PKR protein was observed, which was further induced by IFN. However, PKR enzymatic activity was significantly induced by IFN only in C2-NEO cells, while it was hardly detected in both clones 17 and 22, even after IFN treatment. Furthermore, in contrast to C2-NEO cells, only a slight to moderate increase in enzymatic activity was observed in clone 17 and 22 differentiating cells. Next, cells were grown either in growth medium (GM) or differentiation medium (DM), and the progression of the myogenic program was studied. An inhibition in myotube formation in clone 17 versus C2-NEO cells cultivated in DM was clearly observed. Furthermore, while the growth rate and thymidine incorporation were reduced in C2-NEO cells grown in DM, both clone 17 and 22 cells were less affected under the same conditions. Similarly, a delay in the accumulation of the transcription factors MyoD and myogenin, as well as in creatine kinase activity and accumulation of troponin T, was detected in DM-cultivated clone 17 and clone 22 cells. Moreover, a delay in the induction of p21 (WAF1), in down-regulation of cyclin D1 and c-myc, and in the accumulation of the underphosphorylated form of pRb was also observed in clone 17 cells. We conclude that inhibition of endogenous PKR activity by a PKR dominant-negative mutant interferes with the myogenic program of murine C2C12 myogenic cells.