Targeting miRNAs to Facilitate Cardiac Regenerative Pathways

A limited number of cardiac cells are able or regain the potential to reenter cell cycle (Bergmann et al. 2009). However, this cell renewal cannot compensate for cardiomyocyte loss after acute myocardial infarction or in the progression of chronic heart failure. Therefore, therapeutic strategies to induce cardiac regeneration are currently intensely investigated. As miRNAs are crucially involved in cardiac development, lineage commitment, differentiation, and maturation of car- diomyocytes, miRNAs were recently investigated for their potential to regenerate the heart, either by direct cardiac reprogramming or induction of cardiomyocyte proliferation (Fig. 4.2).

Direct cardiac reprogramming describes a process in which resident cardiac host cells are directly trans-differentiated into cardiomyocytes. Direct reprogramming therefore circumvents the step of dedifferentiation into pluripotent stem cells but

Induction or inhibition of cardiomyocyte proliferation is regulated by miRNAs

Fig. 4.2 Induction or inhibition of cardiomyocyte proliferation is regulated by miRNAs. Whereas neonatal mice show a robust cardiac repair response after cardiac injury, proliferative capacity of cardiomyocytes is (almost) lost in the adult heart of human and mice. Therapeutic targeting of miRNAs that suppresses genes involved in cell cycle reentry and mitosis results in an increased proliferation of cardiomyocytes. Chekl checkpoint kinase 1, Fntb beta subunit of farnesyltransfer- ase, Smarca5 SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily a, member 5, Btg3 B-cell translocation gene 3, Mpsl monopolar spindle 1, Cdc37cell division cycle 37, PA2G4 proliferation associated protein, mstl mammalian STE20-like protein kinase 1, Lats2 large tumor suppressor 2, Mob1b Mps one binder kinase activator 1B, Hopx HOP homeo- box, PTEN phosphatase and tensin homolog pursues reprogramming directly from endogenous non-cardiomyocytes (e.g., cardiac fibroblasts) toward functional cardiomyocytes. Ieda et al. (2010) reported recently reprogramming of mouse fibroblasts into cardiomyocytes. Delivery of three cardiac transcription factors (Gata4, Mef2c, Tbx5, or GMT) into fibroblasts derived from mouse hearts and skin resulted in cardiomyocyte-like cells with expression of cardiomyocyte-specific promoters and structures. The induction was also observed in a murine myocardial infarction model by using viral transfection of cardiac transcription factors (Qian et al. 2012; Song et al. 2012). Interestingly, the addition of miR-133a to GMT increased the number of directly reprogrammed car- diomyocytes and accelerated onset of beating cells by suppression of fibroblast signatures through SNAI1, a master gene of epithelial-to-mesenchymal transition (Muraoka et al. 2014). Jayawardena et al. (2012) extended these observations by using miRNAs involved in cardiac muscle development. Overexpression of miR-1, miR-133, miR-208, and miR-499 in mouse cardiac fibroblasts drives these cells toward cardiomyocytes with expression of cardiomyocyte markers and functions in vitro. Moreover, genetic tracing methods showed that intramyocardial injection of this set of miRNAs after experimental myocardial infarction converted cardiac fibroblast into cardiomyocytes-like cells (Jayawardena et al. 2012). In a follow-up study (Jayawardena et al. 2015), serial echocardiography in mice undergoing MI and injected with miR-1, miR-133, miR-208, and miR-499 showed an improvement in cardiac function over 3 months as compared to controls. Furthermore, reprogrammed rod-shaped cells exhibited similar physiological properties as mature adult ventricular cardiomyocytes. Fibroblasts from humans are more resistant to reprogramming techniques, as cardiac transcription factors (Gata4, Mef2c, Tbx5, and Hand2) used in mouse fibroblasts (Song et al. 2012) failed to reprogram human fibroblasts (Nam et al. 2013). However, addition of MyocD effectively induced cardiac gene expression. Interestingly, miR-1 and miR-133 together with four transcription factors (Gata4, Hand2, Tbx5, and MyocD) further enhanced reprogramming efficiency toward a cardiomyocyte phenotype (Nam et al. 2013). Though 19% of reprogrammed cells were cardiac troponin T-positive, functional characteristics of mature cardiomyocytes such as upregulation of cardiac genes, calcium transients, and beating cells were rarely observed.

Apart from direct reprogramming, induction of cardiomyocyte proliferation is an alternative different strategy to enhance cardiac function in injured hearts that likely underlies the capacity of zebra fish and postnatal mice to regenerate after cardiac injury. Cardiomyocyte proliferation is recognized for decades to be a prerequisite in embryogenesis and for lower vertebrates. However, Bergmann et al. showed that in adults, a low but relevant count of cardiomyocytes still proliferates (approx. 1% turnover rate/year) (Bergmann et al. 2009), which raises the possibility to enhance cell cycle in mature cardiomyocytes. In experimental studies, proliferation of cardiomyocytes after surgical injury in neonatal mice has been reported (Porrello et al. 2011b). These observations indicate postnatal regeneration of the heart. MiRNAs are required for modulation of proliferative and apoptotic processes in cardiomyocytes, as cardiac deletion of enzymes required in the biogenesis of miRNAs resulted in dilatation of the heart and premature lethality (Chen et al. 2008; Rao et al. 2009). MiR-1 and miR-133 have been shown to regulate mitotic processes. MiR-1 is specifically expressed in the skeletal and cardiac muscle and consists of two miRNAs, miR-1-1 and miR-1-2 (Zhao et al. 2007). Mice lacking miR-1-2 die early due to ventricular septal defects (Zhao et al. 2007). Adult mice lacking miR-1 will result to overt cardiomyocyte hyperplasia. Molecular studies showed an increased expression of proteins involved in cardiac morphogenesis and development, such as Hand2 (Zhao et al. 2007). In contrast cardiac- specific overexpression of miR-1 leads to decreased ventricular cardiomyocyte proliferation (Zhao et al. 2005). MiR-133a is co-transcribed as a bicistronic construct with miR-1 and involved in cardiac development. Deletion of miR-133a-1/ miR-133a-2 causes lethal ventricular septal defects in embryonic and neonatal stages and dilated cardiomyopathy in surviving adult mice (Liu et al. 2008). In these double-mutant mice, a disorganization of sarcomeres and an increased proliferation and apoptosis of cardiomyocytes were detected. Consistently, cell cycle genes were upregulated in double knockout mice. In zebra fish, downregulation of miR-133 was observed after resection of the cardiac apex. Transgenic overexpression of miR-133 suppresses cell cycle genes btg3, cdc37, PA2G4 and mps1, and connexin-43, a gap junction protein required for intercellular communication (Yin et al. 2012), as it was shown for miR-499 (Hosoda et al. 2011). MiR-133a therefore suppresses cardiomyocyte cell cycle and guides differentiation into cardiomyo- cytes. As changes in spatiotemporal expression of miRNA are observed, a study linked the transient regenerative capacity in postnatal murine hearts (Porrello et al. 2011b) to detect up- and downregulated miRNAs using a microarray approach (Porrello et al. 2011a). MiR-195, a member of the miR-15 family, is highly upregu- lated in mouse hearts between day 1 and 10 after birth. Delivery of anti-miRs targeting miR-15 family members in neonatal mice increased cardiomyocyte proliferation by de-inhibition of cell cycle genes (Porrello et al. 2011a). Porrello et al. (2013) further investigated the impact of miR-15 on cardiac regeneration after cardiac injury in postnatal mice. Postnatal MI at day 1 resulted in an extensive infarcted area. However, at day 21, a functional recovery can be observed (Porrello et al. 2013). Cardiac-specific overexpression of miR-195 (a member of the miR-15 family) in these mice impaired cardiac regenerative capacity with extensive fibrosis in the infarcted area and decreased proliferating cardiomyocytes (Porrello et al. 2013). Furthermore, pretreatment of postnatal mice with anti-miR-15 improved cardiac function after induction of myocardial infarction in adult mice (Porrello et al. 2013). Of note, transgenic overexpression of miR-195 results in cardiac growth and disassembly of cardiomyocytes (van Rooij et al. 2006), leading to dilated cardiomyopathy. However, inhibition of miR-195 was recently shown to increase elastin deposition in the aorta of mice (Zampetaki et al. 2014). Therefore, the role of miR-195 in cardiac extracellular matrix deposition has to be determined in future studies. Similar to miR-195, miR-29a is upregulated when comparing miRNA array expression data from cardiomyocytes derived from rats at postnatal day 2 when compared to postnatal day 28 (Cao et al. 2013). MiR-29a targets cell cycle genes (CCND2). In vitro, inhibition of miR-29a in neonatal cardiomyocytes enhances cardiomyocyte proliferation (Cao et al. 2013).

However, important regulators of regenerative processes can be missed in mammals when evolutionary conserved mechanisms are not activated upon heart injury. In zebra fish, heart amputation results in a downregulation of miR-99/100 and let-- 7a/c, which is not observed in mice after MI (Aguirre et al. 2014). However, intramyocardial delivery of an adenovirus encoding for anti-miR-99/100 and anti-let-7a/c in mice undergoing MI improved cardiac function and decreased scar formation. These effects were triggered by an increase in dedifferentiated and proliferation of cardiomyocytes and resembled the regenerative mechanisms observed in zebra fish (Aguirre et al. 2014).

These studies investigated miRNAs with antiproliferative effects on cardiomyo- cytes. In contrast, a recent study reported that miRNAs can also induce proliferation of cardiomyocytes (Eulalio et al. 2012). A functional high-throughput screening was performed to detect miRNAs involved in cardiomyocyte proliferation. Neonatal cardiomyocytes were transfected with a miRNA library consisting of 875 miRNAs (Eulalio et al. 2012). Remarkably, 204 miRNAs increased neonatal cardiomyocyte proliferation in vitro. Two pro-proliferative miRNAs - miR-199a and miR-590 - were further used for in vivo experiments. Overexpression of these miRNAs in neonatal rats revealed a thicker myocardium and increased cardiomyocyte proliferation. Moreover, intramyocardial overexpression of miR-199a and miR-590 in adult mice undergoing myocardial infarction induced cardiomyocyte proliferation in the peri- infarct area, reduced infarct size, and improved cardiac function (Eulalio et al. 2012).

The role of the miRNA cluster miR-17-92 for cardiac proliferative processes was also investigated. Cardiac-specific deletion of miR-17-92 leads to decreased cardiomyocyte proliferation in postnatal hearts (Chen et al. 2013a). Consistently, overexpression of miR-17-92 in embryonic and postnatal cardiomyocytes increased their proliferative capacity with a thickened myocardium due to hyperplasia. Intriguingly, induced cardiac expression of miR-17-92 in adult mice, where proliferative capacity of cardiomyocytes is almost lost, resulted in an increased cardiomyocyte proliferation. In addition, cardiac overexpression of miR-17-92 preserved cardiac function after myocardial infarction (Chen et al. 2013a). Another study investigated the miR- 302-367 cluster in hearts due to its contribution in lung development. Cardiac- specific knockout resulted in decreased embryonic cell proliferation associated with a decreased cardiomyocyte differentiation (Tian et al. 2015). Target analysis after overexpression of miR-302-367 showed suppression of Mst1, Lats2, and Mob1b, which are all acting as contributors of the Hippo signaling pathway upstream of the Yes-associated protein (YAP). Phosphorylation of the transcriptional co-activator YAP results in suppression of cell proliferation. Consistently, transgenic cardiac or systemic transient overexpression of the miR-302-367 enhanced cardiomyocyte proliferation and improved cardiac function in a mouse myocardial infarction model. However, long-term overexpression of miR-302-367, consistent with the role of the Hippo pathway in the regulation of organ growth, leads to dilatation of the left ventricle, which favors a transient application of this miR cluster (Tian et al.

2015). These studies indicate that cell cycle reentry of cardiomyocytes can be induced by administration of pro-proliferative miRNAs.

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