Non-coding RNA therapeutics for cardiac regeneration

Abstract

A growing body of evidence indicates that cardiac regeneration after myocardial infarction can be achieved by stimulating the endogenous capacity of cardiomyocytes (CMs) to replicate. This process is controlled, both positively and negatively, by a large set of non-coding RNAs (ncRNAs). Some of the microRNAs (miRNAs) that can stimulate CM proliferation is expressed in embryonic stem cells and is required to maintain pluripotency (e.g. the miR-302∼367 cluster). Others also govern the proliferation of different cell types, including cancer cells (e.g. the miR-17∼92 cluster). Additional miRNAs were discovered through systematic screenings (e.g. miR-199a-3p and miR-590-3p). Several miRNAs instead suppress CM proliferation and are involved in the withdrawal of CMs from the cell cycle after birth (e.g. the let-7 and miR-15 families). Similar regulatory roles on CM proliferation are also exerted by a few long ncRNAs. This body of information has obvious therapeutic implications, as miRNAs with activator function or short antisense oligonucleotides against inhibitory miRNAs or lncRNAs can be administered to stimulate cardiac regeneration. Expression of miRNAs can be achieved by gene therapy using adeno-associated vectors, which transduce CMs with high efficiency. More effective and safer for therapeutic purposes, small nucleic acid therapeutics can be obtained as chemically modified, synthetic molecules, which can be administered through lipofection or inclusion in lipid or polymer nanoparticles for efficient cardiac delivery. The notion that it is possible to reprogramme CMs into a regenerative state and that this property can be enhanced by ncRNA therapeutics remains exciting, however extensive experimentation in large mammals and rigorous assessment of safety are required to advance towards clinical application.

Keywords: Nanoparticle; AAV vectors; Cardiomyocyte; Gene therapy; Heart; Infarction; MicroRNA; Regeneration; YAP; lncRNA.

Figure 1

Extra-cellular and intra-cellular signalling regulating cardiomyocyte proliferation and cardiac regeneration. The cardiomyocyte cell cycle is under the control of several molecular regulators. Growth factors and cytokines acting on cell-membrane receptors, including IL-6, neuregulin (NRG1), Fslt1, mechanical stress signals, and modification in the extracellular matrix, mediated by the protein agrin (which acts through the dystrophin glycoprotein complex, DGC), can all stimulate entry of cardiomyocytes into the cell cycle. Three main intracellular signal transduction pathways end up in the translocation of positive transcriptional co-activators into the nucleus. These are YAP, β-catenin, and Notch intracellular domain (ICD). In particular, YAP is maintained inactive through phosphorylation by the Hippo kinase cascade and degraded through the ubiquitin-proteasome pathway. In the absence of Wnt ligands, β-catenin is also degraded, in non-proliferative conditions, by a destruction complex including GSK-3β, which can be inhibited by the small molecule BIO or by the Dishevelled (Dvl) protein. Notch is a cell-membrane receptor that, upon binding to ligands expressed by neighbouring cells (in particular Jagged1 in the heart) releases its intracellular domain that translocates into the nucleus. Similar to all cell types, the cardiomyocyte cell cycle is regulated by a number of positive activators (cyclins/CDKs, E2F transcriptional factors) and inhibitors (e.g. the cyclin-dependent kinase inhibitors p21 and p27). Several microRNAs interfere with these pathways by modulating the levels of critical regulators in the signal transduction pathways or at the level of cell cycle regulation. See text for further details.

Figure 2

Venn diagram reporting the proteins and nucleic acids shown to stimulate (black) or inhibit (blue) cardiomyocyte proliferation and cardiac repair after myocardial infarction. Factors are grouped according to their action: (i) in transgenic or knock-out mice; (ii) in cultured cardiomyocytes from embryonic stem cells, neonatal rodents, or adult hearts; (iii) after MI. A red star indicates factors that were also tested for efficacy in pigs. References are reported in the text. The effect of agrin after MI in pigs is currently reported an article in bioRxiv (https://doi.org/10.1101/854372).

Figure 3

Conserved miRNA families known to regulate cardiomyocyte proliferation. The individual miRNAs are indicated by arrowed boxes in the direction of transcription. The different colours identify specific seed sequences. (A) miRNAs active in embryonic stem cells. Human miRNAs include those in the miR-302∼367 and miR-371∼373 clusters together with several miRNAs of the miR-520 family scattered along chromosome 19. Mouse miRNAs include those in the miR-302∼367 cluster together with miRNAs belonging to the miR-290∼295 cluster, which is the functional homologue of the miR-371∼373 in humans. (B) miRNAs that also regulate proliferation of cancer cells. These belong to three clusters (the miR-17∼92 cluster and its paralogues miR-106b∼25 and miR-106a∼363 clusters). Together, these three clusters encode 15 miRNAs, which can be grouped according to their seed sequence into four families, as indicated by the different colours.

Figure 4

Effect of miRNAs stimulating cardiomyocyte proliferation on the cardiomyocyte cell cycle. Similar to other cell types, the cardiomyocyte cell cycle is governed by cyclin/CDKs, which in turn respond to a number of activator and repressor proteins (in green and red, respectively). The cartoon shows the main direct or indirect effect on these factors of the pro-proliferative miRNAs (in green) and of miRNAs inhibiting proliferation (in red). Further details are reported in the text and in Tables 1and2.

Figure 5

Effect of miRNAs on the Hippo pathway and the actin cytoskeleton. The cardiomyocyte cell cycle is controlled by the YAP transcription co-factor, which, when de-phosphorylated, translocates to the nucleus and interacts with the TEAD family of transcription factor to drive expression of pro-proliferative genes. YAP phosphorylation is controlled by various protein kinases, including TAOK1, STK38L, LATS, and MST1. These are targeted by different pro-proliferative miRNAs (in green). Proliferation is also linked to the cardiomyocyte actin cytoskeleton, which connects to cell-membrane dystrophin glycoprotein complex (DGC) and cell-to-cell junctions. Different pro-proliferative miRNAs regulate the rate of polymerized vs. globular actin (F- and G-actin, respectively) by targeting one or more of the cellular factors controlling the rate of actin polymerization (including Twinfilin, TWF, Profilin, PFN2, thymosin-b4, TMSB4X and Cofilin2, and CFL2). For further description, cf. text and references therein.