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PhD Program in
“Translational Medicine”
Pro-regenerative microRNAs delivered during the
sub-acute phase of myocardial infarction
Candidate: Thesis Advisor:
Nikoloz Gorgodze Fabio Recchia
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LIST OF CONTENTS
ABSTRACT
………...8Chapter 1: INTRODUCTION
………...101.1 Cardiomyocyte proliferation capacity………...13
1.2 Therapeutic strategies for cardiac regeneration………...18
1.3 MicroRNAs - the sound of (gene) silence………...21
1.4 MicroRNAs in cardiovascular diseases………...22
1.5 Pioneering evidence of microRNA-induced cardiac regeneration - the beginning of the Story ………...30
1.6 Large animal models: an obligatory step towards clinical applications………...34
1.7 A step forward - Successful validation of miRNA-induced cardiac repair in a swine model of MI………...35
Chapter 2: OBJECTIVE OF THE STUDY
………...44
Chapter 3: METHODS
………...453.1 Production and purification of recombinant AAV vectors and microRNA ………...45
3.2 Closed-chest myocardial infarction model………...45
3.3 Experimental protocols………...….48
3.4 Open-chest surgery and direct intramyocardial injections of AAV6………...50
3.5 Cardiac MRI ………...51
3.6 Cardiac MRI data analyses ………...52
3.6.1 Cardiac mechanical function………...52
3.6.2 Myocardial fibrosis ………...55
3.6.3 Myocardial perfusion………...56
3.7 Electrophysiology (EP) study………...58
3.8 Invasive hemodynamic measurements and tissue sampling………...60
3.9 Tissue analysis………...…...62
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3.11 Quantification of AAV6 by real time PCR taqman assay………...63
3.12 Total RNA isolation………...63
3.13 Quantification of miRNA by Mircury LNA universal rt microRNA PCR………...63
3.14 Immunostaining for Phosphorylated histone H3 (PHH3)………...64
3.15 Statistical analysis ………...64
Chapter 4: RESULTS
………...654.1 PROTOCOL 1………...65
4.1.1 Mortality rate………...65
4.1.2 Cardiac volumes and LV global function ………...66
4.1.3 LV regional contractility……….……...68
4.1.4 Infarct scar size………...71
4.1.5 Myocardial perfusion………...74
4.2 PROTOCOL 2………...76
4.2.1 Mortality rate………...76
4.2.2 Cardiac volumes and LV global function ………...77
4.2.3 LV regional contractility………...79
4.2.4 Scar scar size………...83
4.2.5 Myocardial perfusion………...86
4.3 Histological and molecular analyses………...88
4.3.1 Cardiomyocyte proliferation ………...88
4.3.2 Quantification of hsa-miR-199a-3p expression……….…..90
4.3.3 MiR-199a target gene expression……….…….91
4.4 Electrophysiology (EP) study results………...92
Chapter 5: DISCUSSION………...94
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LIST OF ABREVIATIONS AND ACRONYMS
AAPC = Average annual percentage change AAR = Area at risk
AAV6 = Adenoassociated virus serotype 6 AD = Adenovirus
AGO = The Argonaute protein ANP = Atrial natriuretic peptide AOP = Aortic blood pressure ATP = Adenosine triphosphate
BIRC5 = Baculoviral repeat-containing 5 BNP = Brain natriuretic peptide
BRDU = Bromodeoxyuridine
CARP = Cardiac ankyrin repeat protein CLIC = Clathrin Independent Carriers
CLIC5 = Chloride intracellular channel protein 5 CMRI = Cardiac magnetic resonance imaging CO = Cardiac output
CTGF = Connective Tissue Growth Factor CX = Circumflex branch of left coronary artery CYR61= Cysteine-rich angiogenic inducer 61 DALY = Disability adjusted life years
DAPI= 4',6-diamidino-2-phenylindole
DGCR8 = Digeorge syndrome chromosomal region 8 Ea = arterial elastance
ECC = Circumferential strain
ECG = Electrocardiogram ECM = Extracellular martrix EDM = End-diastolic mass EDP = End-diastolic pressure
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EDPVR=End dystolic pressure volume relationship EDU = 5-ethynyl-2′-deoxyuridine
EDV = End-diastolic volume
EDWT = End-diastolic wall thickness EF = Ejection fraction
ELL = Longitudinal strain
ERR = Radial strain
ERα = Estrogen receptor α ESM = End-systolic mass
ESPVR=End systolic pressure volume relationship ESV = End-systolic volume
ESWT = End-systolic wall thickness FRMD6 = FERM-domain-containing-6
GEEC = GPI-anchored protein Enriched Endocytic Compartment HARP = Harmonic phase analysis
HNRNP A1 = Heterogeneous nuclear ribonucleoprotein A1 HOMER1 = Homer protein homolog 1 protein
HOPX = Homeodomain-only protein HR = Heart rate
IAP = Inhibitor of apoptosis IR = Inversion recovery IVS = Interventricular septum
KSRP = The KH-type splicing regulatory protein LAD = Left anterior descending coronary artery LATS = Large tumour suppressor
LV = Left ventricle
LVP = left ventricular pressure LVV = and left ventricular volume LVWT = Left ventricle wall thickening MHC = Myosine heavy chain
MI = Myocardial infarction MiRNA = MicroRNA
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MOB1 = Mps-one binder kinase activator-1 MRI = Magnetic resonance imaging
MRNA = Messenger RNA
MST1, MST2 = Mammalian sterile-20-like kinases type 1 and type 2 NCRNA = Small non-coding RNA
NF2 = Neurofibromatosis type II protein (Merlin) NHLBI = National Heart, Lung, and Blood Institute NPRA = Natriuretic peptide receptor A
ORF = Open reading frame
PACT = Protein kinase RNA activator PBS = Phosphate-buffered saline
PCI = percutaneous coronary intervention PET= Positron emission tomography PH3= Phospho-histone-3
Pre-miRNA = Precursor miRNA
PRSW=preload recruitable stroke work Pri-miRNA = Primary microRNA
P53 = Tumor protein 53 P1 = Postnatal day 1
Ran-GTP = GTP-binding nuclear protein Ran RCA = Right coronary artery
RISC = RNA-induced silencing complex ROS = Reactive oxygen species
RV = Right ventricle RYR = Ryanodine receptor SAV1 = Salvador homolog 1 SCD = sudden cardiac death SE = Echo sequence
SEM = Standard error of mean SI = signal intensity
SI-RNA = Short interfering RNA
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SPECT = Single-photon emission computed tomography SV = Stroke volume
TARBP = Transactivation response RNA binding protein
TEAD = TEA domain-containing sequence-specific transcription factor TIMI = thrombolysis in myocardial infarction
TNF = Tumor necrosis factor VF = Ventricular fibrillation VG = vector genomes YAP = Yes-associated protein YKI = Yorkie proten
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ABSTRACT
The acute loss of a large portion of cardiac tissue after infarction often evolves into heart failure, a syndrome characterized by very poor quality of life and high mortality rate. The incidence of heart failure is increasing dramatically, therefore the development of an effective and safe cardio-reparative therapy might represent a major milestone in cardiovascular medicine. A recent study from our laboratory demonstrated in a pig model that intramyocardial injection of miRNA-199a genes carried by adeno-associated viral vectors (AAV) soon after the induction of acute myocardial infarction (MI) resulted in almost complete recovery of cardiac morpho-functional parameters. However, 6-7 weeks after miRNA-199a administration most animals died from sudden death, likely due to fatal arrhythmias.
The overall aim of the present study was to test the hypothesis that a delayed administration of AAV6-miR199a during the sub-acute phase of MI displays therapeutic effects without causing long-term deadly arrhythmias.
The first specific aim was to determine whether the therapeutic efficacy of AAV6-miR-199a delivery is time-dependent. MI was induced by inserting a balloon-tipped catheter in the left anterior descending coronary artery under X-ray fluoroscopic guidance and occluding it for 90 minutes. All pigs underwent baseline cardiac MRI evaluation at day 13 (Protocol 1) or day 6 (Protocol 2) post-MI to assess baseline cardiac morpho-functional parameters and scar dimensions. On the following day, they were subjected to open-chest surgery and randomly assigned to receive 2 x 1013 of
AAV6-miR-199a or empty AAV6 (AAV-control) injected in multiple sites of the cardiac wall along the infarct border zone. One month after AAV delivery, the animals underwent a second cardiac MRI. In Protocol 1, the scar mass and the indexes of global and regional contractile performance were not significantly different between AAV6-miR-199a and AAV-control MI. No significant differences were found also in Protocol 2, although we observed a trend towards reduced scar size and improved ejection fraction as well as wall thickening and circumferential strain, two indexes of regional contractility. Post-mortem histological and molecular analysis of myocardial tissue revealed that only in Protocol 2 there was a significant increase in cardiomyocytes positive for proliferation markers (PHH3) and downregulation of 3 messenger RNAs targeted by miR-199a.
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Despite the absence of significant morpho-functional improvements, most of the pigs in both protocols died from sudden cardiac death at 6-8 weeks after AAV6-miR-199a delivery. In some cases we were able to record ECG tracings indicative of ventricular fibrillation as the cause of death. The second specific aim of our study was to determine whether infarcted hearts treated with miR-199a display a lower threshold for ventricular arrhythmias in response to abnormal electrical stimuli. Electrophysiology studies were performed at six weeks after AAV6-miR-199a or AAV6-control delivery in infarcted hearts. While ventricular arrhythmias were induced in all the animals of the AAV6-control group, AAV6-miR-199a-treated pigs surprisingly displayed relative resistance to external extrastimuli even with the most aggressive stimulation pattern.
In conclusion, our study showed that gene therapy with miR-199a for cardiac repair loses its efficacy when administered 7 or more days after MI, while the predisposition to lethal arrhythmias in treated hearts is still present. Paradoxically, infarcted hearts treated with miR-199a display a higher threshold for ventricular arrhythmias in response to abnormal electrical stimuli. These findings further support the possibility that delivery via AAV vectors, which causes long-term expression of miR-199a, is not the right approach. Future studies shall test different types of vector for short-term expression of miR-199a, for instance synthetic nanoparticles, which must be delivered only during the acute phase of MI.
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Chapter 1: INTRODUCTION
Ischemic heart disease (IHD) is the top cause of global mortality and accounts for about 9 million deaths annually 1 (Figure 1). However, there has been a substantial decrease in the mortality rate
from IHD by almost 60% over the past five decades 2 (Figure 2). Advances in preventative actions
such as reduction in cigarette smoking, improvement in hypertension control and healthy diets as well as the progress in the coronary artery revascularization therapy are the main reasons responsible for this improved outcome.
Figure 1.
Global health estimates 2016. Death by cause, world health organization (WHO)Decreased mortality from IHD automatically generates a growing number of surviving patients with compromised heart function, frequently leading to pump failure. Finding an effective therapeutic approach to recover the cardiac normal morpho-functional parameters rather than provide short-term relief has long been a holy grail for basic and clinical investigators.
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Transmural myocardial infarction (MI) affects not only the area corresponding to the culprit artery, but it might expand to the non-infarcted regions too. Loss of functional cardiomyocytes leads to cellular and interstitial alterations eventually evolving in cardiac remodeling. The latter is a complex phenomenon involving structural and functional reprogramming, such as loss of cardiac myofibers, apoptosis, and disruption of the cytoskeleton, accelerated deposition of collagen and fibroblasts, as well as changes in receptor density, signal transduction and Ca(2+) homeostasis. 3 4
Figure 2:
Age-adjusted cardiovascular mortality Rates, 1950–2014 years, Source: CDC/NCHS; National Vital Statistics System, Mortality Multiple-Cause-of-Death. CVD (cardiovascular disease); IHD (Ischemic heart disease) Figure taken from reference 2The reduction or complete loss of oxygen delivery to cardiomyocytes alters the function of mitochondrial respiratory chain enzymes and superoxide, hydrogen peroxide, peroxynitrite and hydroxyl radicals accumulate. 5 Further impairment of mitochondrial function causes the
generation of reactive oxygen species (ROS) which, in turn, accelerate electron leakage and create a vicious cycle of free radical generation. Next, permeability transition pores (PTP) in the inner mitochondrial membrane stop functioning and the mitochondrial matrix gets swollen.
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Finally, loss of the membrane potential and ATP depletion leads to cardiomyocyte dysfunction with subsequent cell death. 6
During MI, cardiomyocytes start to die approximately 30 minutes after oxygen deprivation. The death of cardiomyocytes is accompanied by the release of intracellular proteins. Myocyte death occurs through two independent pathways, i.e. myocyte apoptosis, recognizable by cell shrinkage, and myocyte necrosis characterized by swelling of the cells. Surprisingly, apoptosis occurs also in the peri-infarct and remote myocardium and the relative mechanism is not fully understood. The peak intensity of apoptosis occurs 6-8 hours after MI and represents the dominant source of myocyte loss after MI. 7 Most apoptotic cells are not eliminated by
phagocytosis and trigger a second cascade of cell necrosis from 12 h to 4 days after MI.8 This
secondary necrosis of cardiac cells induces a massive inflammatory response and a number of cytokines are released in the bloodstream. Two to 3 days after MI, myofibroblasts start producing extracellular matrix proteins to prevent cardiac rupture. The scar formation ends 3-4 weeks after MI, resulting in cell-free tissue, predominantly containing cross-linked collagen. 9
One of the characteristic hallmarks of adult mammalian cardiomyocytes is the inability to regenerate after damage. Despite the evidence of minimal spontaneous proliferation, the rate of which slightly increases after acute injury, mammalian adult hearts are not able to compensate for the loss of hundreds of millions cardiomyocytes after MI, hence to regain the normal morpho-functional parameters. Consequently, an effective and safe strategy for boosting cardiomyocyte proliferation in the long-term could be of paramount importance.
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1.1 Cardiomyocyte proliferation capacity
The main characteristic of any proliferating somatic cell is karyokinesis followed by cytokinesis yielding genetically identical cells. 10 11 Fetal cardiomyocytes in the mammalian hearts strictly
follow this biological pattern and are highly proliferative, while, a few days after birth, their potential for regeneration drastically diminishes, likely due to the poor ability to reenter the cell cycle and undergo cell division.1213 Therefore, heart growth and also any damage to the adult
mammalian heart resulting in the loss of functional myocardium is mainly compensated by increasing the size of pre-existing cardiomyocytes.1415 Unlike some lower vertebrates such as
amphibians and teleost fish with potent cardiac regenerative capacities,1617 adult mammalian
cardiomyocyte DNA might replicate with or without nuclear division (karyokinesis). In adult human cardiomyocytes, the nucleus continues to replicate its DNA, but in most cells does not divide. This process is called endoreduplication and is characterized by copying rounds of DNA eventually leading to polyploidy. 18 On the other hand, cardiomyocytes in other mammals may
divide their nuclei, but cannot proceed with cell division. (acytokinetic mitosis) 18 In the adult
human hearts, cardiomyocytes typically are mononucleated with tetraploid or higher DNA content, whereas the adult mouse hearts contain binucleated diploid cardiomyocytes. Because of interrupted cytokinesis, the number of nuclei may vary among species and might reach up to thirty-two per cardiomyocyte as in the case of pigs.1819 (Figure 3).
During organogenesis, while remaining highly proliferative, mammalian hearts undergo periodic (intermittent) proliferation, which plays a key role in growth and shaping of the developing hearts. 20 21 For instance, during gastrulation, myocardial progenitor cells derived from the
primitive streak maintain vigorous proliferative activity until the cardiac tube, a well-defined paired structure on the ventral surface of the developing embryo, takes shape. 2022 Once the
tubular heart is formed, cardiomyocytes exit the G1 phase and enter the quiescent stage. As cardiogenesis progresses and looping process starts, the venous end, the atrioventricular canal, the inner curvature and the outflow tract remain poorly proliferative, while the outer layer continues extensive mitotic activity to form heart chambers. 23 This inside‐to‐outside
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conduction system, which during cardiogenesis might also function as a substitute for sphincters to prevent blood back flow until the heart valves develop. 2425 On the other hand,the highly
proliferative outer layer (ventricular compact layer) generates most part of contractile myocardial tissue.
Figure 3:
A; B; C; Mammalian and rodent hearts stop proliferating shortly after birth, but the DNA continues to duplicate with or without karyokinesis. Consequently, cardiomyocytes might remain mononucleated with polyploid genome as in humans or become polynucleated with diploid genome. D; Teleost fish cardiomyocytes undergo complete karyokinesis followed by cell division even after birth and, as a result, their hearts maintain elevated proliferative activity throughout their life course. Figure taken and modified from reference 10Several studies demonstrated that, despite the minimal regenerative capacity after acute injury, adult mammalian cardiomyocytes undergo spontaneous proliferation throughout life. 26 27
Based on the mathematical modeling of radiocarbon (14C) data, the annual cardiomyocyte
renewal in humans gradually declines from 1% at the age of 25 to 0.45% at the age of 75. To infer from the aforementioned data, more than 45 % of cardiomyocytes are replenished during
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the entire lifespan. 26 Unfortunately, this low rate of cardiomyocyte turnover is far too small to
compensate for the acute loss of millions of cardiomyocytes after MI.
The exact mechanisms prompting the cardiomyocytes to stop proliferation and mature is not fully understood. Nonetheless, it has been almost two decades since the discovery of the Hippo signaling pathway.28 This pathway is responsible for the cell cycle arrest in adult hearts,
regulates organ size and growth and plays an important role in cell proliferation, apoptosis, and differentiation. 29
Figure 4:
Schematic representation of the Hippo pathway components and their interaction is shown here A. The activated Hippo pathway sets a cascade of phosphorylation, which prevents the YAP/TAZ complex from nuclear transposition; consequently, the target genes for cell proliferation are silenced. B. when the Hippo pathway is off, the YAP/TAZ complex accumulates in a nucleus, displaces VGL4, binds to TEADs and activates gene expression. Figure taken from reference 20The Hippo pathway consists of several kinases and its activation triggers a phosphorylation cascade leading to the inhibition of cell proliferation (Figure 4). When the pathway is on, two
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potent kinases, namely MST1 and/or MST2 (mammalian STE20-like protein kinase 1 and 2), phosphorylate Salvador homolog 1 (SAV1). MST1/MST2, together with SAV1, phosphorylate and activate the downstream factors MOB1A and MOB1B (MOB kinase activator 1A and 1B). MOB1A and MOB1B, in turn, phosphorylate LATS1/LATS2 kinases (large tumor suppressor homolog 1 and 2 kinases), which then phosphorylate the Yes-associated protein (YAP), a master regulator of cell proliferation, together with the transcriptional co-activator TAZ. Phosphorylated YAP/TAZ complex is then sequestrated and subjected to proteasomal degradation in the cytoplasm. As a consequence, the TEA domain-containing sequence-specific transcription factors (TEADs) bind the transcription cofactor vestigial-like protein 4 (VGL4) in the nucleus and suppress target gene expression. Conversely, when the Hippo pathway is off, the kinases MST1, MST2, LATS1, and LATS2 are inactive, the YAP/TAZ complex is not phosphorylated and instead translocates into the nucleus: here, the YAP/TAZ complex displaces VGL4 and binds to TEADs, which promotes the expression of target genes responsible for cell proliferation. 30 Apart from the canonical
pathway of the YAP regulation, many other molecules can facilitate or hamper the nuclear translocation of YAP, resulting in the target gene expression or repression, respectively. (Some of these molecules are discussed in the following paragraphs)
A number of alternative pathways have been proposed to explain the mechanisms of postnatal cardiomyocyte cell cycle withdrawal. Research in rodents showed that neonatal mice at postnatal day 1 (P1) possess a vigorous regenerative capacity, the rate of which gradually lessens and becomes almost nulled at postnatal day 7 (P7). 31 Several morphological and metabolic
alterations, as well as the activation of transcriptional factors responsible for cell proliferation inhibition were observed in the first post-natal week (P1-P7 period). With the aim to precisely characterize the changes in the composition of the extracellular matrix (ECM) occurring in the P1-P7 period, Bassat et al.32 performed mass spectrometry analysis and identified
ECM-associated Glico-protein Agrin. The latter, which is normally minimally expressed in adult mammalian hearts, proved to induce cardiac proliferation with concomitant attenuation of cardiomyocyte maturation. Indeed, the genetic ablation of Agrin in P1 neonatal mice halted cardiac repair after apex resection whereas its upregulation in both neonatal and adult mice significantly reduced the scar size and improved heart function. Further research demonstrated that Agrin interacts with the DG complex (Dystrophin-glycoprotein complex) and disrupts the integrity of the cytoskeleton and myofibrils, - a process necessary for cardiomyocyte dedifferentiation. Additionally, Agrin-DG interaction enables the YAP protein to dissociate from
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the DG complex and translocate in the nucleus to activate gene expression required for cell proliferation. 33 (Figure 5). In line with the morphological changes such as the cytoskeleton
stabilization and contractile proteins shift from fetal to adult isoforms, metabolic reprogramming is one of the major differences between proliferating and quiescent cardiomyocytes. The fructose-induced glycolysis was the main energy source for the heart in P1 mice, whereas switch to β-oxidation was revealed in cardiomyocytes of P7 mice. 34 Moreover,
the cessation of the cell division in cultured neonatal cardiomyocytes coincided with the same process in neonatal mice, indicating the existence of an intrinsic timer responsible for the proliferation schedule. 35 Indeed, activation of the pocket proteins (p21, p27, and p57), as well
as some transcriptional factors (Meis-1 and HIF-1), occurred simultaneously in both cultured neonatal cardiomyocytes and P7 mice hearts. En masse, these molecular changes inhibited Cyclins and Cyclin-dependent kinases (CDKs), key factors of the cell cycle progress, and contributed to the cell-cycle withdrawal.36
Apart from the abovementioned intrinsic stimuli and multiple intracellular alterations, environmental factors could also play an important role in directing the proliferating cardiomyocytes to the quiescent stage. For instance, oxygen tension in fetal cardiomyocytes ranges from 18 to 28 mmHg, but, after birth, it quickly rises and reaches 100 mmHg. 37 This
rapid increase in oxygenation leads to the accumulation of reactive oxygen species (ROS), which, in turn, might stop cardiomyocyte proliferation and induce their terminal differentiation. 35
Another major environmental event that happens at birth is the abrupt increase in the left ventricular workload o. Due to the greater strain, the cells at the inner surface are the first to exit the cell cycle compared to those at the outer layer. 38
Despite a large number of the possible mechanisms mediating the cell cycle arrest in cardiomyocytes, further research is required to elucidate the main determinants for the fate of adult cardiomyocytes. This will contribute to establishing new and hopefully more effective therapeutic strategies in the field of cardiac regeneration.
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Figure 5:
Agrin binds DGC and disrupts the cytoskeleton and myofibrils. Agrin-DGC association helps the YAP to dissociate from DGC and translocate into the nucleus to activate the target genes. Figure taken from reference 231.2 Therapeutic strategies for cardiac regeneration
For many years, a plethora of studies have been performed seeking an effective approach to stimulate cardiomyocyte regeneration and recover cardiac function after MI. Despite some encouraging findings of some translational value, to date none of them has really impacted the clinical practice. Two basic methodologies are employed in the field of cardiac regeneration, one based on cell therapies and the other based on cell-free approaches. 39
Cell-based therapy
The first attempt to implement cell-based therapy for cardiac repair dates back to 1992, when Marelli and colleagues 40 tested non-cardiac, skeletal myoblasts (SM) in canine models of MI.
Surprisingly, autologous transplants survived in recipient myocardium and continued to differentiate into a myogenic lineage, leading to improved ejection fraction after MI. A number of short-term clinical trials have demonstrated this method to be effective for human use as well, 4142 but subsequent follow-up studies have failed to confirm permanent curative effects.
Furthermore, SM therapy contributed to arrhythmogenesis, most likely due to the poor ability of skeletal myoblasts to electromechanically integrate with the surrounding myocardium. 43
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pro-proliferative potential in infarcted hearts. Next, bone marrow-derived mononuclear cells (MNCs) 44 and mesenchymal stem cells (MSCs) showed the capacity for self-renewal and
differentiation into cardiomyocytes during their co-culture with primary cardiomyocytes or after the addition methyltransferase inhibitors. 4546 Unfortunately, none of these cell lines proved to
have substantial cardiomyogenic potential in vivo. 4748 The failure of non-cardiac cell transplants
to provide durable and safe cardiac regeneration prompted investigators to change strategy and shift their interests towards harvesting, expanding and re-implanting cardiac stem cells (CSCs). CADUCEUS- the largest clinical trial 49 has then tested whether intracoronary infusion of
autologous cardiac stem cells is therapeutically effective in human hearts with post-MI failure. This treatment considerably reduced the scar size, increased the amount of viable myocardium, but, surprisingly, did not affect the ejection fraction, as shown by cardiac MRI. 49 While the
treatment with CSCs still needs further research to determine its therapeutic efficacy, an alternative option based on pluripotent embryonic stem cells (ESCs) transplantation was found to be promising, despite the low engraftment rate of these cells. 5051 However, apart from being
tumorigenic, the use of ESCs even for therapeutic purposes raised ethical issues, since the primary source of these cells is the inner cell mass of blastocysts taken from human embryos.
In 2006, Yamanaka and colleagues were the first to generate ESCs by forced expression of some transcription factors in mouse and human fibroblasts. These cells were named “induced pluripotent stem cells (iPSCs)”. This groundbreaking discovery has not only solved the ethical concerns and offered a novel cell-based approach for heart repair, but was the first successful precedent of cell reprogramming in this area of research. 5253 In 2016, an immunosuppressed
macaque model of MI demonstrated that allogeneic iPSC-derived cardiomyocytes retained in the recipient’s heart improved cardiac function and did not contribute to the tumor formation.
54 However, the majority of experimental animals receiving iPSC transplants suffered from an
increased incidence of ventricular tachycardia, most probably due to the immaturity of the iPSC-derived cardiomyocytes. To summarize, before the cell-based cardiac therapy reaches the patient’s bedside, some fundamental issues must be addressed, such as the minimization of the risk for arrhythmias and tumorigenesis as well as the engraftment rate enhancement and the differentiation into functional cardiac cells.
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The cell-free approach includes any strategy that does not employ cell transplant and works through targeting several intrinsic pathways to promote cardiac regeneration. Such approaches include a set of technologies aimed at inducing direct reprogramming into functional cardiomyocytes. For instance, retrovirus-mediated delivery of well-established reprograming cocktails (GMT and GHMT) forced the cardiac fibroblasts to express several genes necessary to acquire cardiomyocyte characteristics.55 Though effective in mice, such treatment in humans
did not provide any clinical importance, most likely attributable to epigenetic barriers between mouse and human fibroblasts. 56 Another cell-free method utilizes secretory factors to boost
cardiomyocyte proliferation and several pilot studies exploring exosomes and growth factors revealed these molecules to be encouraging potential targets. 57 58 The stimulation of
endogenous cardiac repair is considered as one of the most promising cell-free approaches, which targets universal cell cycle regulators and transcriptional factors responsible for the post-natal cell-cycle arrest.39 For instance, the concomitant overexpression of cyclins and
cyclin-dependent kinases (CDKs) contributed to cardiomyocyte proliferation and improved cardiac contractile function after MI, as reported recently. 59 Next, the well-known transcriptional factor
homeobox protein MEIS1 was shown to activate a number of CDK inhibitors, thus inducing the withdrawal from cell cycle. 60 Consequently, cardiac-specific overexpression of MEIS1 in
neonatal mice hampered cardiomyocyte proliferation and abrogated heart regeneration. Conversely, the selective deletion of MEIS1 in transgenic mice resulted in the widened time window of postnatal cardiomyocyte proliferation and caused the cardiomyocytes to enter the cell cycle, even in adult mice. 60 Manipulation of the Hippo signaling pathway (see Hippo
pathway in a previous paragraph), a potent regulator of cell proliferation and organ size, 61 is an
additional target for endogenous cardiac repair. For instance, studies in mutant mice lacking the Hippo pathway key components showed a massive proliferation of adult cardiomyocytes. 62
Similar data were obtained by the activation of the Yes-associated protein 1 (YAP1), a central transcriptional co-factor of the Hippo pathway, which is normally deactivated in quiescent cardiomyocytes. 63 In 2012, Eulalio et al. reported unique findings and provided novel insights
into the mechanisms of cardiac repair. 64 The authors identified a number of microRNAs able to
target YAP1 and display a potent pro-proliferative capacity, both in vitro and in vivo. The best performing exemplars of such microRNAs prevented YAP1 from phosphorylation and promoted its transport into the nucleus, eventually leading the adult cardiomyocytes to enter the cell cycle, as evidenced by proliferation markers 6566 Apart from being the direct modulators of the Hippo
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pathway, microRNAs are also conceivably involved in the paracrine signaling. They represent important secretory factors produced by several cells contributing to cardiac repair. 39 Hence,
the mounting interest in microRNAs as excellent therapeutic targets for cardiac regeneration.
1.3 MicroRNAs - the sound of (gene) silence
MicroRNAs are small, non-coding RNAs, about 20-22 nucleotides in length, and play an important role in the regulation of gene expression at the posttranscriptional level. These multi-target molecules control mRNA translation and their cytosolic stability. 67 MicroRNAs together
with other double-stranded RNAs (siRNAs and piRNAs) take part in RNA silencing - the evolutionary conserved gene-inactivation system. According to microRNA target databases, one microRNA might modulate hundreds of genes, 6869 since microRNAs do not require precise base
pairing for their target recognition. On the other hand, a one-to-one relationship between microRNA and its target gene has also been documented. 70
Figure 6
: Schematic representation of the canonical pathway of microRNA biogenesis and microRNA mediated gene silencing. Figure taken from reference 7222
The canonical pathway (Figure 6) of microRNA biogenesis starts with the generation of pri-miRNA transcripts by RNA polymerase II (Pol II) in the nucleus. 7172 Pri-miRNAs are then cleaved by the
microprocessor enzymes DROSHA and DGCR8 (DiGeorge syndrome critical region 8) in conjunction with p68 and p72, yielding the 60–70-nucleotide long precursor miRNAs (Pre-miRNAs). The pre-miRNAs are subjected to cytosolic transportation by exportin 5 (XPO5) and are further processed by DICER1 producing mature miRNAs. 73 Finally, only the guide strand of the mature miRNA duplex
(either the 5p or 3p strands) is incorporated into the Argonaute (AGO) proteins and form a miRNA-induced silencing complex (miRISC). MiRISC recognizes its target mRNA seed region by sequence complementary binding and induces mRNA translational repression and/or mRNA degradation in processing bodies. (P-bodies) Most microRNAs bind specific sequences at the 3′ UTR sites of their target mRNAs, but 5’ UTR sites, coding sequences and promoter regions may also be targeted. While binding to 3’UTR and 5’UTR sites as well as coding sequences induce gene silence, interaction with promoters results in gene activation. 74 75 76 Based on the extent of miRNA-mRNA
complementarity, target mRNAs can be cleaved and irreversibly sequestrated from the cytosol as in the case of perfect base pairing. Alternatively, poormiRNA-mRNA complementarity causes mRNAs to temporarily detach from the ribosomes, leading to translation repression. This latter is not a permanent inhibitory mechanism of gene expression and holds the potential for translation reactivation. 77 Studies in microRNA mutant mice demonstrated that most miRNA-mRNA
interactions lacked perfect complementarity; therefore, 48% of target genes were regulated by translation repression, 29% by mRNA cleavage with their subsequent degradation, and 23% by both.
78 However, further research is warranted to reveal the precise mechanisms responsible for the
dominant mode of microRNA-mediated gene silencing.
1.4 MicroRNAs in cardiovascular diseases
MicroRNAs have first been described almost three decades ago in the worm Caenorhabditis
elegans by Lee et al. 79 80 Later on, in 1998, Fire et al. discovered and described microRNA
involvement in gene silencing. 81 The first two microRNAs to be identified and meticulously
studied were lin-4 and let-7 controlling developmental timing by repressing their target mRNA products of the lin-14 and lin-41 genes. 7982 Thanks to the extensive and continuous exploration
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field of biomedicine and genetics elucidating their biogenesis and biological functions. MicroRNAs, evolutionarily highly conserved master regulators of gene expression, represent primary players in cell differentiation, proliferation, apoptosis, immune responses, fat metabolism and neuronal patterning. 83 84 85 86 Apart from being involved in normal
physiological processes, dozens of recent studies have demonstrated their contribution to pathological cellular mechanisms. 87 88 For instance, in tumors some microRNAs function as
oncogenes (oncomiRs), whereas others can suppress abnormal cell proliferation and act as tumor suppressors. 8789
The first well-studied microRNAs that suggested their potential implication in cardiovascular diseases were miR-1 and miR-133. Their discovery dates back to 2005, when Zhao et al., and a year later in 2006 Chen et al. , demonstrated that these two molecules, deriving from the same precursor transcript, were highly expressed in cardiac and skeletal-muscles of rodents and amphibians. 90 91 MiR-1 regulates cardiac differentiation by targeting the transcription factor
Hand2 and the importance of this microRNA in cardiogenesis was revealed by its selective
deletion in mouse models. 92 Mice lacking miR-1 showed a wide array of abnormalities, which
included heart structural defects (ventricular septal defect), heart rhythm disturbances and pathological cardiac hypertrophy causing early postnatal mortality. 93 Furthermore, prominent
cell-cycle anomalies were observed, resulting in continuous nuclear and cellular division that promote pathological cardiomyocyte hyperplasia, even postnatally. One of the miR-1 targets is the cytoskeleton regulatory protein twinfilin-1 (Twf1), which acts through binding actin monomers and thus hampering their assembly into filaments. 94 The expression of Twf1 is
relatively low in the adult heart and inversely correlates with miR-1. For instance, hypertrophic stimuli in rats, such as aortic banding or α-adrenergic stimulation, can induce the downregulation of miR-1 with concomitant overexpression of Twf1, eventually leading to cardiac hypertrophy. 95 The fact that Twf1 overexpression is sufficient to cause experimental
cardiac hypertrophy renders the abovementioned protein a valuable therapeutic target for the future studies.
The implication of miR-1 in the genesis of cardiac arrhythmia has long been documented,9697
however several studies produced conflicting data on its pro-arrhythmogenic effects. For instance, accelerated atrioventricular conduction developed in mice with a targeted deletion of miR-1.92 Continuous ECG monitoring revealed delayed intraventricular conduction converting
24
into ventricular arrhythmia that killed the majority of mutant mice.92 K+ channel defects,
responsible for transient outward K+ current (Ito), were manifested as the main cause for arrhythmia.98 Paradoxically, the upregulation of miR-1 yielded similar results, as reported by
Terentyev et al. in 2009.99 Utilizing quantitative immunoblotting, Ca2+ imaging and
electrophysiology studies, the authors investigated excitation-contraction coupling and Ca2+ cycling in rat cardiomyocytes previously transduced with adenoviral vectors expressing miR-1. In the presence of β adrenoreceptor agonist, while rhythmically paced, miR-1–overexpressing myocytes displayed persistent spontaneous arrhythmogenic oscillations of intracellular Ca2+, compared to control myocytes under the same conditions.
Su et al. continued to explore the underling pro-arrhythmogenic mechanisms of miR-1 in vivo.100
Those authors carried out microarray analyses of the transcriptomes in transgenic mice in which miR-1 was over-expressed (miR-1 Tg). They demonstrated that in such mice a number of intracellular trafficking-related genes were downregulated, including Stx6, which is minimally expressed also in ischemic cardiomyocytes during infarction.101 Underexpression of Stx6 in
miR-1 Tg mice resulted in the impairment of L-type calcium channels that eventually led to the increased level of resting ([Ca2+]i) and thus contributed to cardiac arrhythmia. Though contradictory, the existing data indicate that any pathological expression of miR-1 might be related to cardiac arrhythmia.
Regarding miR-133, double mutant-mice with deletion of both miR-133 subtypes (miR-133a-1 and miR-133a-2), developed heart structural defects that killed approximately half of the embryos or neonates. The surviving ones suffered from dilated cardiomyopathy and subsequent heart failure. 102 Dysregulation of the genes involved in cell cycle control was also displayed,
most likely attributable to the overexpression of the miR-133 mRNA targets, such as Cyclin D2 and serum response factor (SRF).103
In 2008, Morton et al. reported the first data on miR-138, an important regulator of species-specific cardiac morphogenesis . This microRNA was species-specifically expressed in certain domains of the teleost fish heart and was essential to establish normal temporal and spatial chamber-specific gene expression .104 The authors demonstrated that time-specific knockdown of
miR-138 by antagomiRs in zebrafish led to atrioventricular canal (AVC) gene expansion into the ventricular chambers. The disrupted gene expression eventually resulted in a failure of ventricular cardiomyocytes to thoroughly mature and acquire the normal morphology,
25
highlighting the importance of the abovementioned microRNA in cardiac morphogenesis and maturation. Being a part of the transcriptional networks controlling chamber-specific gene expression, miR-138 is highly conserved across species, varying from zebrafish to humans. However, its role in the shaping of the mammalian four-chambered heart is not fully understood.105
The same year novel evidence was given about microRNAs involved in cardiac fibrosis after MI and miR-29 family was revealed as an important suppressor of fibrotic response to myocardial ischemia. 106 Experiments carried out in rodents and humans revealed that the inhibition of the
miR-29 family with anti-miRs resulted in upregulation of the proteins enhancing collagen and elastin deposition. On the other hand, over-expression of miR-29 in fibroblasts drastically lowered collagen expression and the extent of cardiac fibrosis.
In 2009, soon after identifying the role of the miR-29 family, Rane and her team made a striking discovery. The authors observed that the total mature miR-199a was decreased to undetectable levels in both cultured neonatal cardiomyocytes under hypoxia and infarcted mice hearts, 107
whereas the direct targets of miR-199a -Hypoxia induced factor 1α (Hif-1α) and Sirtuin 1 (Sirt1) were overexpressed. Overexpression of Hif-1α induced p53 stabilization and triggered a cascade of hypoxia-induced apoptosis. Conversely, replenishing miR-199a diminished Hif-1 expression, blocked the Hif-1-p53 pathway and markedly decreased apoptotic activity in vitro. This finding was of paramount importance, since therapeutic upregulation of miR-199a could decrease the apoptotic death of hypoxic cardiomyocytes (Table 1).
During the following years, a number of microRNAs have been found to exhibit important regulatory effects. For instance, in 2010, miR-499 was recognized to decrease the proliferation of human fetal cardiomyocyte progenitor cells (CMPCs) along with miR-1. 108 Transient
transfection of CMPCs with miR-1 and miR-499 declined cell proliferation rate by 25% and 15%, respectively, and significantly increased their differentiation into cardiomyocytes, attributable to the repression of histone deacetylase 4 and Sox6. This was the pioneering demonstration that modifications of the levels of specific microRNAs could direct progenitor cell fate towards either proliferation or differentiation in vitro.
Subsequent research by Fiedler et al. Identified miR-24, a key player of endothelial apoptosis in ischemic hearts. 109 Overexpression of miR-24 or silencing of its target genes- GATA2 and PAK4
miR-26
24 in endothelial cells limited myocardial infarct size in mice and increased their survival through the prevention of endothelial apoptosis and increase of vascular density.
Figure 7:
A. Relative expression level of miR-135a and miR-147 in peripheral blood mononuclear cells (PBMCs) from coronary arteries of disease patients (CAD; black bars) and healthy controls (Control; white bars). ∗∗P < 0.01, ∗∗∗P < 0.001 B. Calculated ratio of microRNA-135a and microRNA-147 expression levels in PBMCs from CAD patients (black bar) and healthy controls (white bar). ∗∗P < 0.01 C. Relative expression levels of miR-198 in PBMCs from patients diagnosed with unstable (UAP; black bars) or stable angina pectoris (SAP; gray bars) ∗∗P < 0.01. Figure is taken and modified from reference 50Significant headway was made in 2010, when Hoekstra M. et al. first considered the microRNAs as potential biomarkers for coronary artery disease (CAD) and acute myocardial infarction (AMI).
110 111 Furthermore, those authors were the first to put forward the idea that circulating
microRNAs in the bloodstream and extracellular space could not only carry diagnostic value but also be a robust tool for making prognosis and disease stratification for several pathologies. They screened the microRNA profile of peripheral blood mononuclear cells (PBMCs) from stable and unstable CAD patients along with healthy controls and observed a 5-fold increase in the relative level of miR-135 in both CAD groups compared to controls (Figure 7). In line with overexpressed 135, a 4-fold decrease in 147 level was detected, resulting in a 19-fold higher miR-135a/miR-147 ratio in the CAD groups. Additionally, miR-198 was shown to be 12-fold upregulated in patients with recent ischemic pain at rest (Braunwald’s class III-B) compared to those having stable angina diagnosed for >6 months (Figure 7). This difference between the CAD groups might suggest that the overexpression of miR-198 can help distinguishing between
27
patients at high risk for acute coronary events and those previously diagnosed with stable angina.
Figure 8:
Time course of TnI and microRNAs plasma levels in patients with documented myocardial infarction. In all patients, the first plasma sample was taken 156 ± 72 min after the MI symptoms. (T0) miR-1, (A) miR-133a, (B) and miR -133b (C) and reached their peak concentration before troponin I, while miR-499-5p (D) displayed a slower time course. Figure taken from reference 52The same year D’Alessandra et al. and other groups found that the levels of some microRNAs, namely mir-1, miR-133a/miR-133b, miR-208 and miR-499, were significantly elevated in plasma obtained from rodents with experimental MI as well as in patients diagnosed with acute MI. 112 113114 The first three from the abovementioned microRNAs were overexpressed as early as 156
minutes after the first symptoms of MI, similarly to Troponin-I (TnI) - an established marker of myocardial necrosis, but unlike TnI their plasma level reached the peak at T0, slightly before the peak increase in TnI (Figure 8).
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Figure 9:
Schematic representation of MI-related release of microRNAs and their further entry into the bloodstream A. Myocardial necrosis due to acute ischemia and release of some muscle enriched (1, miR-133a/b and miR-499) and cardiac-specific (miR-208a) microRNAs are shown. B. MicroRNA uptake by apoptotic bodies, membrane-derived vesicles and protein complexes. C. Active microRNAs enter the circulation and become detectable as early as 156 minutes after the first symptoms of myocardial infarctionThe mechanisms by which microRNAs enter the circulation and become detectable in the serum are not completely understood, however mounting evidence indicates that they are resistant to RNase-dependent degradation. 113115116 So far, at least three possible explanations have been
proposed to mediate the protection of circulating microRNAs from being deactivated into the bloodstream. These include the transportation of microRNAs by membrane-derived vesicles (exosomes, microvesicles), 117 118 119 through apoptotic bodies 120 or by forming the protein–
miRNA complexes. 121 (Figure 9) Exosomes are produced by intercellular endosomes, have a
relatively small size (40 ~ 100 nm in diameter) and the ceramide dependent pathway was shown to be one of the mechanisms responsible for exosome-dependent microRNA transfer. 122
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their larger dimensions (50 ~ 1,000 nm in diameter) enable them to play a role in cell-to-cell communication. 123 Apoptotic bodies are even larger particles (1 ~ 5 μm in diameter) and include
a portion of cytoplasm with microRNA, both taken from apoptotic cells. They display a wide array of inhibitory effects on recipient cells. For instance, endothelial cell-derived apoptotic bodies expressing miR-126 can hamper atheroma formation by activating CXCL12 chemokine in recipient vascular cells. 120 Several RNA-binding proteins, such as Argonaute family (mainly
AGO2) and nucleophosmin 1 can also prevent their cargo from degradation by RNase, thus contributing to protection and delivery of microRNAs, as confirmed by various studies. 121124
One of the surprising findings that broadened the knowledge of microRNAs biology was the evidence of cross-kingdom regulation provided in 2012. Zhang et al were the first to demonstrate that plant-derived, exogenous microRNAs could regulate mammalian gene expression. 125 The authors performed both in vivo and in vitro experiments and found that
ingested rice-derived miR-168a maintained its original chemical structure, passed through the mammalian small intestine in association with microvesicles (MV), entered the circulation and by suppressing the expression of human/mouse LDL receptor adaptor protein 1 (LDLRAP1) eventually lowered hepatic LDL uptake. These findings prompted numerous experiments exploring a “cross-kingdom” platform by which plant microRNAs could modulate mammalian gene expression. Chin et al. detected broccoli-derived miR-159 in human sera, the level of which was inversely correlated with breast cancer (BC) progression: TCF7, a mammalian target gene for miR-159, was thus identified. 126 However, the data obtained from such studies have not
always been promising and several others have failed to show any substantial uptake of plant microRNAs. 127128129130 Consequently, further and rigorous research is required in this regard
to confirm the uptake of extrinsic, diet-derived miRNAs through the digestive system in the biologically relevant context.
Years Finding Involved microRNAs Authors
2005 MicroRNA is essential for proper muscle development
miR-1 miR-133a/b
Sokol et al. Kwon C et al.
30
2006/2007 A. Muscle microRNAs regulate myoblast
proliferation and differentiation in skeletal muscle
B. MicroRNAs expression regulated in
response to cardiac muscle hypertrophy
miR-1 miR-133a/b miR-195 miR-21 miR-1 Chen JF et al. KIM HK et al. Van Rooij E et al. Sayed D et al Cheng Y et al.
2008/2009 A. MicroRNA regulates species-dependent
cardiac patterning
B. microRNAs are involved in cardiac
conduction, cardiomyocyte hyperplasia, fibrosis and apoptosis
miR-138 miR-1 , miR-133 miR-29 family miR-199a S. U. Morton et al. Rane S et al. Terentyev D et al. Van Rooij E et al.
2010/2011 A. MicroRNAs regulate cardiac stem cell
fate
B. Targeting a miRNA promotes revascularization after myocardial infarction
C. Circulating microRNAs may be novel
biomarkers for coronary artery disease and acute myocardial infarction
miR-499 miR-24 miR-135,↑ miR-198 ↑ miR-147 ↓ miR-1,↑ miR133a/b, ↑ miR-499,↑ miR-208a ↑ Sluijter JP et al. Fiedler J et al. Hoekstra M et al. V. Di Stefano et al
Table1:
Breakthrough microRNA discoveries in the field of cardiovascular diseases from 2005 to 2011. ↑-upregulated and ↓-downregulated microRNAs in different pathologies. The table is taken from the LC Sciences database1.5 Pioneering evidence of microRNA-induced cardiac regeneration -
the beginning of the story
Decades after intensive research in the field of cardiac regeneration, a study published in 2012 by Eulalio et al.64 provided the first experimental demonstration that some microRNAs could
significantly boost adult cardiomyocyte proliferation. Based on previous findings showing that some microRNAs could direct cardiomyocytes to the quiescent stage, 90 102 131 those authors
Fluorescence-microscopy-31
based, high-throughput screening technique was utilized to screen 875 miRNA mimics for their pro-proliferative capacity. In rat and mouse cardiomyocytes, more than 200 miRNA mimics were identified as able to stimulate cell proliferation, but only 40 of them could induce both of DNA replication and subsequent entry into mitosis. Therefore, hsa-miR-199a-3p and hsa-miR-590-3p, the most potent pro-regenerative microRNAs, were selected to further test them in vivo. First the authors examined whether cardiac delivery of the aforementioned microRNAs via cardiotropic adeno-associated viruses (AAV) could stimulate cardiomyocyte proliferation in healthy hearts (Figure 9). An S phase proliferation marker, namely 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, revealed a pronounced increase in EdU positive cells, indicating that the hearts treated with hsa-miR-199a-3p and hsa-miR-590-3p underwent intensive DNA replication (Figure 9 B). In line with these findings, the same treatment given to infarcted mice hearts yielded striking results.In addition to the EdU+ cells, the heart samples from the treated animals were positive for Ki 67-and PHH3 markers as well, proving the cell-cycle progress from the G2 phase to mitosis. Consistent with the immunostaining data, AAV-mediated delivery of hsa-miR-199a-3p and hsa-miR-590-3p in the peri-infarct zone, shortly after LAD ligation, substantially decreased the scar size and preserved the LV contractile function compared to hearts receiving a control cel-miR-67 (Figure 10).
With the scope of identifying the global transcriptome change consequent to overexpression of the selected microRNAs, Eulalio et al. applied the deep sequencing technology to measure the mRNA dynamics in cardiomyocytes receiving hsa-miR-590-3p or hsa-miR-199a-3p. This analysis revealed 1,056 upregulated transcripts (773 for hsa-miR-199a-3p; 283 for hsa-miR-590-3p) and 697 downregulated transcripts (95 for hsa -miR-590-3p; 602 for hsa-miR-199a-3p).
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Figure 9:
a. The experimental protocol for non-infarcted neonatal rat hearts b. Immunostaining for the detection of EdU+ cardiomyocytes. Scale bars, 1 mm (top), 100 mm (bottom); c. Bars showing the difference of EdU+ cells (%) between the groups. mean ± s.e.m.; **P < 0.01, ***P < 0.001 relative to control d. Confocal microscopy of rat hearts receiving hsa-miR-590-3p and hsa-miR-199a-3p. Scale bar, 10 µm. Taken from reference 64Most of the downregulated genes, were characterized as genes necessary for skeletal and muscular system development, cellular assembly and organization, whereas the upregulated ones belonged to the cell cycle and cellular growth categories. In other words, downregulated genes contributed to the cell-cycle progression towards mitosis, at least to some extent, while upregulated ones directed cell fate to terminal maturation.
To determine the direct targets specifically involved in cardiomyocyte proliferation, the authors performed siRNA mediated knockdown of the downregulated transcripts and found 45 genes that prevent neonatal cardiomyocytes entry into the cell cycle. Of these, five siRNAs targeted transcripts were downregulated by hsa-miR-590-3p, 43 by hsa-miR-199a-3p and three by both. None of the target genes’ selective knockdown revealed to be as effective as miRNA overexpression in boosting cell proliferation, indicating that the identified miRNAs most likely function by modulating multiple targets. From the downregulated genes Homer1, Hopx, and Clic5 were common targets for hsa-miR-590-3p and hsa-miR-199a-3p. Homer1 protein regulates the ryanodine receptor mediated Ca2+ signaling, necessary for Ca2+ release during the excitation-contraction coupling process in skeletal and cardiac muscles. 132 Moreover,
33
Figure 10:
A. The experimental protocol of MI in adult mice. B. LVEF (%) measured by echocardiography, dashed line, non-infarcted animals injected with AAV9-control. N=10–16 per group. C. Infarct size. n = 6–10 per group D. Masson trichrome staining of heart cross E. Confocal microscopy at the border zone of the infarct, 60 days post-MI. Scale bar, 10 µm. All panels, mean ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001 relative to control. Taken from reference 64responsible for premature termination of gene transcription. 133 Hopx is the smallest protein
from homeodomain-containing family, which together with histone deacetylase 2 (Hdac2) deactivates GATA4 and blocks cardiac myocyte proliferation.134 Clic5 is present in the inner
mitochondrial membrane and regulates chloride ion homeostasis in cardiomyocytes. It also controls mitochondrial reactive oxygen species (ROS) production 135 and is shown to inhibit
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The present study was the first of its kind to test a novel approach for cardiac regeneration utilizing microRNAs for therapeutic purposes. Despite the fact that the exact origin of the replicating cells in the treated animals was not clear, the improved morpho-functional parameters after MI, as well as the expression of well-established proliferation markers, proved the authors’ concept. AAV mediated delivery of hsa-miR-590-3p and hsa-miR-199a-3p displayed effectiveness for the stimulation of cardiac regeneration. With this study and others
64136 the new course of strategies for cardiac regeneration based on delivery of molecules rather
than cells has begun.
1.6 Large animal models: an obligatory step towards clinical
applications
While significant insights into cellular and molecular basis of cardiovascular biology have derived from experiments in vitro and in small animal models of myocardial infarction, promising findings must be further verified in more clinically relevant animal models before they are translated into medical practice. Besides the fact that hemodynamic and physiological parameters such as heart rate, oxygen consumption and adrenergic receptor ratios differ considerably between rodents and large mammals, the expression of contractile proteins that are critically involved in the excitation-contraction coupling process seems to be different too.
137 Furthermore, there is evidence of phenotypic differences between mouse and human
cardiac cell populations and stem cells. 138 Authoritative expert panels gathered by the NIH or
endorsed by the American Heart Association (AHA) have emphasized the importance of large animal models for effective translation of new diagnostic and therapeutic strategies into clinical cardiology. 139140
Among large animal preclinical models, pigs are undoubtedly one of the best options in the cardiovascular field. Their relevance for this kind of research is due to their phylogenetic resemblance to humans, human-like coronary artery tree and non-preformed coronary collaterals, which allows to induce highly predictable infarction in terms of size and location. 141 142 Moreover, their weight and size permits to assess morpho-functional alterations utilizing the
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1.7 A step forward - Successful validation of miRNA-induced cardiac
repair in a swine model of MI
Gabisonia et al. aimed at validating the encouraging data obtained from the previous rodent study 64 in a more clinically relevant pig model of MI. 66 To induce myocardial infarction in
open-chest pigs, the left anterior descending artery (LAD) was occluded for 90 minutes distal to the first diagonal branch by applying the cross-clamp technique. At ten minutes of reperfusion, the survived animals were randomized in two groups and each of them was then assigned to 20 intramyocardial injections of2x1013 AAV6-hsa-miR-199a-3p (AAV6-miR-199a; n=13) or 2x1013
empty AAV6 (AAV6-control; n=12) along the infarct border zone (100 µl per injection). The latter was easily distinguishable from the normal myocardium by its pale color (Figure 11C) and was marked by colored epicardial stitches (Figure 11 D) to identify and sample myocardial tissue for post-mortem histological and molecular analyses. At the end of the experimental protocol (Fig 11 A) animals were euthanized, four 1-cm thick horizontal slices of the heart were obtained from the apex to the base and both histological and proteomic studies were carried out.
36
Figure 11:
A. Experimental protocol in open-chest pigs B. The LAD, its diagonal branches (arrows) and a closure point (circle) are indicated C. LAD closure with the cross-clamp technique D. Infarct border zone was visually distinguishable the healthy myocardium E. AAV-miR-199a injections in the infarct border line previously marked with stitches.Taken from reference 66To monitor and precisely characterize the changes in cardiac functional parameters and scar dimensions over time, a clinical 1.5 tesla MRI scanner (Signa excite HD; GE Medical systems) was utilized to visualize both vertical and horizontal long axis views of the entire left ventricle. Global cardiac contractility and infarct scar size was not significantly different between the AAV6-miR-199a and AAV6-control groups at baseline as evaluated by cine-CMR and late gadolinium enhancement techniques respectively. Twenty-eight days after MI, ejection fraction in the animals treated with AAV6-miR-199a improved by ~ 10 consistent with the partial normalization
of the LV end-systolic volume, while the infarct size was halved, in contrast with the AAV6-control group in which both parameters markedly worsened (Figure 12).
Figure 12:
A. LV ejection fraction differences between treated and non-treated group after myocardial infarction at four weeks. Values are mean± s.e.m. *P < 0.05 versus AAV6-control at the same time point B. LV infarct mass differences between treated and non-treated groups after myocardial infarction at four weeks. Values are mean± s.e.m. *P < 0.05 versus AAV6-control at the same time point; #P < 0.05 versus AAV6-miR-199a at day37 two after MI C. LGE-cMRI images (from apex to base) of a pig heart receiving AAV6-miR-199a are shown here. The study is performed at 2 and 28 days after MI. Taken from reference 66.
Along with global cardiac function, regional contractility was also assessed by quantifying both radial (ERR) and circumferential (CRR) strains with the tagging cMRI technique. Apical, middle and
basal short axis images were generated and divided into eight circumferential segments, to obtain curves and the area under the curve (AUC). Consistent with the data obtained from the LV systolic wall thickening analysis (Figure 13C) AAV6-miR-199a injected animals displayed considerably improved ERR and ECC valuescompared to controls (Figure 13A; 3B)
38
Figure 13:
A. Eight-segment curves corresponding to LV radial (LV Err) and circumferential (LV Ecc) strain obtained by tagging-MRI at 28 days after MI. Data are mean ± s.e.m. *P < 0.05 versus AAV6-control; #P < 0.05 versus sham; B. AUC in arbitrary units for Err and Ecc. Data are mean ± s.e.m.; the number of animals per group is indicated. *P < 0.05 versus AAV6-control; #P < 0.05 versus sham; C Eight-segment curves corresponding to LV end-systolic wall thickening (LVWT) 28 days after MI. Data are mean ± s.e.m. *P < 0.05 versus AAV6-control; #P < 0.05 versus sham. Taken from reference 66.The improved morpho-functional parameters in the treated group were in line with the increased number of cardiomyocytes expressing various proliferation markers, suggestive of regenerative repair of the infarcted hearts (Figure 14). To investigate the pro-proliferative potential of miR-199a, four well-established immunostaining methods were applied to detect non-quiescent cells at different stages of a cell cycle (Figure 16).
Figure 14:
Representative images of proliferating cells (A) Ki67 Immunostaining (B) and phosphorylated histone H3 (C) of the infarct border zone 12 days after surgery, and relative quantification. Data are mean ± s.e.m.; the39 number of animals per group is indicated. *P < 0.05; two-sided Student’s t-test. Scale bars, 100 µm. D. Aurora B kinase immunofluorescence showing localization in midbodies (arrow) in hearts from the treated pigs, 12 days after MI. Scale bars, 20 µm. Taken from reference 66.
1. BrdU incorporation assay
A synthetic nucleoside 5-bromo-2'-deoxyuridine (BrdU) replaces thymidine during DNA replication and passes to daughter cells upon division, consequently the BrdU incorporation assay detects the cells going through the S phase of cell cycle. 143144 Being BrdU an exogenous
molecular marker, continuous external injections are required in experimental animals until it becomes detectable in cells with active DNA synthesis.
2. Immunostaining for Ki 67
Ki 67 antigen is one of the commonly used markers to calculate cell proliferation rate. During the whole interphase Ki 67 is only found within the nucleus and, as the cell cycle progresses and mitosis starts, it binds the surface of chromosomes and becomes detectable in the cytoplasm too. 145 Ki 67 is present during the entire cell cycle (G
1; S; G2 and mitosis), but is absent in
quiescent cells (G0) 146 making this protein an excellent marker to accurately determine the
proliferating fraction of cell populations.
3. Immunostaining for Phosphorylated histone H3
Histone H3 is an important nuclear core protein responsible for chromosome condensation and cell cycle progression. 147 Since phosphorylation of serine residues in the histone H3 tail is a key
step to trigger mitosis, phosphorylated histone H3 (PHH3) is considered as a specific marker indicating the transition from G2 to mitosis. Phosphorylation of histone H3 reaches its peak in
metaphase and consequently finding PHH3 positive cells is only possible in mitotically active cells. 147148 On the other hand, upon exit from mitosis (also meiosis), global dephosphorylation
of H3 occurs and PHH3 becomes either minimally or no longer detectable.
4. Aurora B kinase in midbodies, a definite marker of complete cytokinesis
During early telophase, when karyokinesis is already completed and two sister nuclei are pulled towards the two opposite spindle poles, a cleavage furrow forms and initiates sister cell separation. The cleavage furrow ingression is driven by the constriction of an actomyosin contractile ring. 149 This latter tightens gradually at the cell equatorial cortex and thus separates
