p38 MAPK and the compromised regenerative ...

Cardiovascular Regenerative Medicine 2017; 4: e1508. doi: 10.14800/crm.1508; © 2017 by Angelino Calderone http://www.smartscitech.com/index.php/crm

REVIEW

p38 MAPK and the compromised regenerative response of the infarcted adult heart Angelino Calderone Département de Pharmacologie et Physiologie, Université de Montréal and Montreal Heart Institute, Montréal, Québec, Canada Correspondence: Angelino Calderone E-mail: [email protected] Received: January 17, 2017 Published online: February 27, 2017

Mononucleated cardiomyocytes promote the hyperplasic growth of the embryonic mammalian heart and the proliferative phenotype was retained by neonatal cardiomyocytes for a short duration after birth. Despite the cell cycle re-entry of pre-existing cardiomyocytes, regeneration of the damaged neonatal rat heart required the coordinated effort of reparative embryonic macrophages. During postnatal development, the majority of pre-existing rodent cardiomyocytes underwent karyokinesis in the absence of cytokinesis and the ability to proliferate was lost. Nonetheless, a paucity of mononucleated cardiomyocytes persisted in the adult heart and after an ischemic insult re-entered the cell cycle thereby providing a semblance of hope that a cardiac regenerative response was possible. However, the already compromised regenerative response of the ischemically damaged adult mammalian heart may be further hindered by the expansion of pro-inflammatory monocyte-derived macrophages. The serine/threonine kinase p38 MAPK was identified as a seminal target of pro-inflammatory cytokines and p38 MAPK inhibition reduced infarct size. The smaller infarct was attributed in part to cardiac regeneration as p38 MAPK suppressed the cell cycle re-entry of neonatal/adult cardiomyocytes in response to peptide growth factors. p38 MAPK-mediated inhibition of cell cycle re-entry may be related in part to the attenuation of nestin expression by pre-existing cardiomyocytes as the intermediate filament protein was directly implicated in the cell proliferation of normal and tumorigenic cells. Targeting the inflammatory response via p38 MAPK inhibition may represent a rational approach to unmask the proliferative potential of pre-existing mononucleated cardiomyocytes and initiate a partial regenerative response of the infarcted adult mammalian heart. Keywords: cardiac regeneration; inflammation; cardiomyocytes; p38 MAPK; nestin To cite this article: Angelino Calderone. p38 MAPK and the compromised regenerative response of the infarcted adult heart. Cardiovasc Regen Med 2017; 4: e1508. doi: 10.14800/crm.1508. Copyright: © 2017 The Authors. Licensed under a Creative Commons Attribution 4.0 International License which allows users including authors of articles to copy and redistribute the material in any medium or format, in addition to remix, transform, and build upon the material for any purpose, even commercially, as long as the author and original source are properly cited or credited.

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Introduction Hyperplasic growth of the embryonic mammalian heart was facilitated by mononucleated ventricular cardiomyocytes that possessed the inherent capacity to re-enter the cycle and proliferate in response to locally released stimuli (Figure 1) [1,

. Following birth, the majority of neonatal pre-existing cardiomyocytes were mononucleated and still retained the ability to re-enter the cell cycle [1, 3]. The latter paradigm was confirmed as injury to the neonatal mouse heart led to cardiac regeneration by pre-existing ventricular cardiomyocytes that re-entered the cell cycle and underwent

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Figure 1. Embryonic and postnatal myocardial growth and adaptation to pathological stimuli. Hyperplasic growth of the embryonic rodent heart was mediated by the proliferation of mononucleated cardiomyocytes. Following birth, the majority of neonatal ventricular cardiomyocytes were mononucleated and retained a proliferative phenotype for a short duration whereas a modest population underwent karyokinesis in the absence of cytokinesis leading to binucleated/multinucleated cardiomyocytes. Postnatal development led to the preferential accumulation of binucleated/multinucleated cardiomyocytes and the significant loss of mononucleated cardiomyocytes. However, a residual population of mononucleated cardiomyocytes persisted during postnatal development. The increase of cardiac mass during postnatal development was attributed predominantly to a physiological hypertrophic response of binucleated/multinucleated cardiomyocytes supported by a modest proliferative response of mononucleated cardiomyocytes. The imposition of a hemodynamic stress (e.g. pressure/volume overload, myocardial infarction) on the adult heart further increased cardiac mass via a predominant hypertrophic response (e.g. concentric or eccentric) of binucleated/multinucleated cardiomyocytes. However, at least after an ischemic insult, a paucity of mononucleated cardiomyocytes identified predominantly at the peri-infarct region re-entered the cell cycle and underwent cytokinesis.

cytokinesis [3]. However, several additional studies have in part contradicted the latter finding and demonstrated that significant fibrosis rather than ventricular regeneration was the prevalent response following injury to the neonatal mouse heart [4, 5]. It was determined that the extent and type of injury (e.g. apical resection, myocardial infarction versus cryoinjury) as well as the timing (e.g. age of neonatal mice) influenced the cardiac regenerative response [6]. Regardless the underlying reason, pre-existing mononucleated neonatal

cardiomyocytes still represented a suitable model to ascertain the underlying biological events implicated in cell cycle re-entry and cytokinesis [1, 6]. During postnatal development, the regenerative capacity of the adult heart was lost and attributed to the decreased density of mononucleated ventricular cardiomyocytes and concomitant appearance of binucleated/multinucleated post-mitotic terminally differentiated cardiomyocytes (Figure 1) [1, 6]. Following the imposition of a hemodynamic overload or after myocardial

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infarction of the adult mammalian heart, concentric or eccentric hypertrophy facilitated by binucleated/multinucleated ventricular cardiomyocytes represented the primary adaptive growth response (Figure 1) [7, 8] . However, a residual population of pre-existing mononucleated cardiomyocytes that retained an embryonic phenotype persisted and via a proliferative response contributed in part to the increase of cardiac mass during postnatal development (Figure 1) [9-12]. Moreover, ischemic injury represented an additional cue that induced the cell cycle re-entry and cytokinesis of pre-existing mononucleated cardiomyocytes (Figure 1) [12]. Interestingly, the newly formed diploid mononucleated cardiomyocytes that appeared secondary to an ischemic insult were detected predominantly at the peri-infarct region, albeit the response was modest (Figure 1) [12]. Collectively, these data provided a semblance of hope that a cardiac regenerative response of the infarcted mammalian adult heart driven by pre-existing mononucleated cardiomyocytes was possible. Therefore, identifying and targeting biological events that may selectively limit the proliferative capacity of mononucleated ventricular cardiomyocytes that have retained an embryonic phenotype may represent a viable approach to initiate a significant regenerative response of the infarcted adult mammalian heart.

deletion of the transcriptional factor led to myocardial hypoplasia and lethality at embryonic day 10,5 [14]. Work from Martin’s group further revealed that Yap-1 phosphorylation in the 2 day postnatal (p2) mouse heart which still retained the inherent ability to regenerate cardiac tissue was significantly lower than that observed in p7 and p21 mouse hearts which mounted a predominant reactive fibrotic response following cardiac injury [13]. These data supported the premise that HIPPO-dependent phosphorylation and subsequent inhibition of Yap-1 signaling during postnatal development may represent a seminal underlying event that repressed the regenerative capacity of the adult heart. Indeed, the selective loss of Hippo signalling (e.g. Salvador depletion) in pre-existing ventricular cardiomyocytes or the constitutive activation of Yap-1 (lacking the serine127 phosphorylation site) led to an exaggerated growth response of the normal heart and promoted ventricular regeneration after myocardial infarction associated with normal contractile function [13, 14]. Thus, the cardiac regenerative response attributed to dephosphorylated Yap-1 apparently rekindled an embryonic proliferative phenotype of pre-existing adult ventricular cardiomyocytes.

HIPPO Signalling Pathway

It was originally assumed that the steady state level of macrophages identified in normal adult tissue was derived primarily from circulating monocytes [16]. However, several studies have reported that a subpopulation of resident tissue macrophages were of embryonic lineage and possessed the ability to repopulate in situ without a concomitant recruitment of circulating monocytes [16-18]. Moreover, the monocyte/macrophage population recruited after an ischemic insult dictated in part whether the subsequent response led to partial cardiac regeneration or scar formation [16-18]. The adult mouse heart contained two discrete and distinct populations of macrophage. A resident population of macrophages was characterized as Ly6clow and further identified as CD11clow/MHCIIlow and CD11clow/MHCIIhigh. Lineage tracing experiments revealed that CD11clow/MHCIIlow and CD11clow/MHCIIhigh macrophages represented a mixed population of definitive hematopoietic-derived cells and embryonically derived from the yolk sac (15-25%) [16, 17]. The second distinct population of macrophages identified in the adult heart originated from circulating monocytes, were Ly6chi and further characterized as CD11chigh/MHCIIhigh. An additional distinguishing feature of Ly6chi/CD11chigh/MHCIIhigh macrophages as compared to Ly6clow macrophages was expression of CCR2, the cognate receptor for the chemotactic stimulus monocyte chemoattractant protein-1 (MCP-1 or CCL2) [17]. Work from Lavine and colleagues further revealed that the normal adult

Several studies have revealed that repression of the HIPPO signalling pathway in pre-existing cardiomyocytes provided the requisite phenotype to initiate ventricular regeneration following myocardial infarction [13, 14]. The Hippo pathway originally identified in Drosophila plays a seminal role in organ growth [15]. The core Hippo pathway consists of a cascade that signals from the kinase Mst1/2 (mammalian STE20-like protein kinase 1) to the kinase Lats1/2 (large tumour suppressor kinase) to limit the activity of Yap-1 (Yes-associated protein-1), a transcriptional coactivator that binds to the TEA domain (TEAD)-containing transcriptional factors to induce expression of cell cycle regulators implicated in cell growth [15] . Hippo signal transduction requires the subcellular compartmentalization of the kinases at the plasma membrane, which is dependent on interactions between Mst1/2 and Lats1/2 with the scaffold proteins Salvador and Merlin, respectively. Mst1/2 binding to the scaffold protein Salvador is required to phosphorylate and activate LATS1/2 which in turn directly phosphorylates Yap-1 on serine127 leading to the cytoplasmic retention of Yap-1 thereby inhibiting the transcription of target genes implicated in cell growth [15]. During embryogenesis, dephosphorylated Yap-1 was identified as an important mediator of ventricular cardiomyocyte proliferation as cardiomyocyte specific

Macrophages, scar formation and cardiac regeneration following myocardial injury

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Figure 2. Neonatal heart regeneration. Ischemic damage to the neonatal rodent heart led to the recruitment of reparative embryonic CCR2(-) macrophages and via the release of a panel of anti-inflammatory/pro-angiogenic peptide growth factors initiated a robust angiogenic response in the infarcted region. The de novo synthesis of new blood vessels and the coordinated recruitment and proliferation of pre-existing mononucleated cardiomyocytes led to the regeneration of the damaged neonatal rodent heart.

mouse heart contained an additional monocyte subset characterized as MHCIIlow/CCR2(+) [17]. These distinct populations were further defined by unique biological characteristics as resident CD11clow/MHCIIhigh/low/CCR2(-) macrophages displayed superior phagocytic properties, promoted angiogenesis and participated in tissue regeneration, whereas CD11chigh/MHCIIhigh/low/CCR2(+) macrophages were primarily pro-inflammatory in nature leading to scar formation, adverse cardiac remodelling and diminished contractile function [17]. Indeed, the mRNA levels of a panel of pro-inflammatory cytokines including MCP-1, MCP-3, IL-6, IL1-β, and TNF-α were significantly greater in CCR2(+) versus CCR2(-) macrophages [17]. These distinct properties explained in part the predominant reparative fibrotic response [e.g. scar formation and healing] of the adult heart following cardiomyocyte injury, as monocyte-derived macrophage populations CD11chigh/MHCIIhigh/low/CCR2(+) were selectively expanded whereas resident CD11clow/MHCIIhigh/low/CCR2(-) macrophage levels were significantly reduced [17]. In the infarcted adult heart, activation of the sympathetic system played an instrumental role in hematopoiesis and monocyte

production in the spleen [19]. After release from the spleen, two sequential phases of recruitment of Ly6chi/CCR2(+) monocyte/macrophages were identified in the infarcted adult heart [19]. The first phase involved the transformation of monocytes to macrophages that removed tissue debris by phagocytosis and released proteolytic enzymes [19]. In the second phase, Ly6chi macrophages were converted to Ly6clow macrophages and facilitated wound healing by promoting myofibroblast accumulation leading to collagen deposition [19, 20] . To reaffirm the role of CCR2, the receptor was pharmacologically inhibited or depleted via a transgenic approach. Pharmacological inhibition of CCR2 following cardiomyocyte ablation of the adult mouse heart which induced cardiomyocyte cell death without systemic inflammation and cardiac fibrosis, selectively prevented the expansion of CD11chigh/MHCIIhigh/low/CCR2(+) macrophage populations leading to a reduced pro-inflammatory response and robust angiogenesis [17]. An analogous paradigm was reported in CCR2-/- mice as recruitment of bone marrow-derived monocytes to the infarcted mouse heart was significantly reduced whereas neutrophil levels remained unchanged [21]. The adaptive pattern of ventricular

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remodeling following the pharmacological inhibition of CCR2 in the infarcted adult mouse heart following cardiomyocyte ablation was attributed in part to the unopposed reparative phenotype of resident CD11clow/MHCIIhigh/low/CCR2(-) macrophages of which 15-25% were of embryonic origin [17].

macrophages in the injured adult heart led to scar formation and maladaptive ventricular remodeling.

Aurora and colleagues were the first to highlight that macrophage recruitment played a seminal role in promoting the regenerative response by pre-existing cardiomyocytes following ischemic injury to the heart of neonatal mice (day 1) [18]. This study further demonstrated that two sequential phases of recruitment akin to that described in the ischemically damaged adult heart was evident as an initial increase of Ly6chigh monocytes was observed throughout the injured neonatal heart followed by a second phase characterized by the increased appearance of Ly6clow monocytes and concomitant reduction of Ly6chigh monocytes [18] . To reaffirm the role of macrophages in cardiac regeneration, neonatal mice were injected with liposomes containing clodronate. The cardiac regenerative response of the ischemically damaged heart of clodronate-treated neonatal mice was significantly compromised, albeit macrophage depletion did not inhibit cardiomyocyte proliferation (Figure 2) [18]. The major biological effect attributed to macrophage depletion was impaired vascularisation leading to significant scar formation and depressed contractile function (Figure 2) [18]. The importance of macrophages in the cardiac regenerative response of the injured neonatal heart was reaffirmed by Lavine and colleagues as the study further highlighted a seminal reparative role of cardiac resident macrophages that originated from an embryonic lineage (Figure 2) [17]. The neonatal mouse heart contained a resident macrophage [MHCIIlow/CCR2(-)] and monocyte [MHCIIlow/CCR2(+)] population. Following cardiomyocyte ablation, a selective expansion of MHCIIlow/CCR2(-) macrophages was observed whereas MHCIIlow/CCR2(+) levels remained unchanged [17]. The selective expansion of MHCIIlow/CCR2(-) macrophages coincided with a cardiac regenerative response of the neonatal heart following cardiomyocyte injury (Figure 2) [17]. Consistent with the latter finding, the media released from CCR2(-) macrophages stimulated angiogenesis as reflected by capillary-like tubule formation by endothelial cells plated in matrigel and the cell cycle re-entry of neonatal ventricular cardiomyocytes, whereas neither biological response was observed with media secreted by CCR2(+) macrophages [17]. Collectively, these data have demonstrated that anti-inflammatory/reparative embryonic CCR2(-) macrophages played a pivotal role in the cardiac regenerative response of the injured neonatal heart whereas the predominant expansion of pro-inflammatory CCR2(+)

During the acute phase of scar formation following myocardial infarction of the adult heart, the expansion of CCR2(+)-macrophages led to the overt expression and release of a panel of pro-inflammatory cytokines including TNF-α and IL-1β [17, 22]. Despite the adaptive physiological role of scar formation of the infarcted adult heart in the absence of a significant cardiac regenerative response, inflammatory cytokines also contribute to maladaptive remodeling by exerting a potent and direct negative inotropic effect and exacerbating the apoptotic loss of cardiomyocytes [23-25]. Activation of the serine/threonine p38 mitogen activated protein kinase (MAPK) was identified as a seminal signaling event driving a plethora of biological actions attributed to pro-inflammatory cytokines [26]. Four isoforms [α, β, γ and δ] of p38 MAPK were identified and each isoform was encoded by a distinct gene. p38α and p38β MAPKs shared high sequence homology and both isoforms were sensitive to pyridinyl imidazole molecules (e.g. SB203580) [27, 28]. By contrast, p38γ and p38δ shared only 60% homology with p38α/β and were resistant to SB203580 inhibition. Recruitment of p38 MAPK required the dual phosphorylation of the threonine180 (Thr180) and tyrosine182 (Tyr182) residues located in the Thr-Gly-Tyr motif by putative upstream MAPK kinases MKK3 and MKK6 [27]. An alternative non-canonical pathway that selectively targeted p38α MAPK was reported during myocardial ischemia and involved the physical interaction of TAB-1 (TAK-1 binding protein) with p38α and induced the autophosphorylation of the Thr-Gly-Tyr motif [29]. It has been well established that adult ventricular cardiomyocytes expressed p38α/p38β and p38α was identified as the predominant isoform implicated in a variety of biological actions [26, 27]. p38 MAPK phosphorylation and activity were increased in the rodent and human infarcted heart and the reported pro-apoptotic action of the serine/threonine kinase may have directly contributed to scar expansion by exacerbating the loss of cardiomyocytes bordering the ischemic region [26,27]. The latter effect was reaffirmed as SB203580 treatment inhibited cardiomyocyte apoptosis following exposure to pro-apoptotic stimuli and facilitated by p38α MAPK as overexpression of the isoform led to programmed cell death [30-32]. The depressed inotropic state of the ischemically damaged heart secondary to the loss of cardiac tissue was further compromised by activated p38α MAPK via the dysregulation of calcium homeostasis and reduced myofilament sensitivity to calcium of surviving cardiomyocytes [26, 33, 34]. Moreover, an important positive

Maladaptive remodelling of the ischemically damaged heart secondary to the inflammatory response was mediated in part by p38 MAPK

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feedback paradigm was identified between inflammation and p38 MAPK as the co-injection of adenoviruses containing constitutively active MKK3B and wildtype p38α directly into the LV of normal mice led to a massive infiltration of inflammatory cells and the activated serine/threonine kinase induced the expression of a panel of pro-inflammatory cytokines [26, 35]. p38 MAPK and cell cycle re-entry of pre-existing cardiomyocytes Although a disparate regenerative response was apparent between the neonatal and adult mammalian heart following injury, a paucity of pre-existing mononucleated adult ventricular cardiomyocytes still retained the capacity to re-enter the cell cycle and were detected predominantly bordering the peri-infarct region [12]. It is tempting to speculate that the selective expansion of CCR2(+)-macrophages and release of pro-inflammatory cytokines may have further compromised the already modest regenerative response of the infarcted adult heart. It is noteworthy to reiterate that in the absence of significant cardiac regeneration, the inflammatory response post-myocardial infarction represents an adaptive physiological response facilitating scar formation and healing [17-19] . The importance of the latter paradigm was supported by the observation that global suppression of the pro-inflammatory response following myocardial infarction led to maladaptive scar thinning [inadequate reparative fibrotic response], excessive left ventricular dilatation and in some cases rupture of the infarcted myocardium.[36-38] However, despite an adaptive seminal role on scar formation, the inflammatory response contributed to infarct expansion and remains equivocal as to whether the effect was mediated predominantly via cardiomyocyte apoptosis and/or occurred in part by inhibiting the cell cycle re-entry of a residual population of mononucleated cardiomyocytes [24, 25]. Thus, targeting the inflammatory response following ischemic damage to the adult heart remains a conundrum as adaptive and maladaptive biological effects are prevalent. Nonetheless, identifying the signalling events that may preferentially influence survival and/or cell cycle re-entry of pre-existing cardiomyocytes with a minimal impact on scar formation and healing represents a plausible option. Based on the available literature, targeting p38 MAPK may represent a viable approach to resolve the aforementioned conundrum. As previously discussed, p38 MAPK was recruited by pro-inflammatory cytokines and in addition to initiating programmed cell death [30-32, 39], may likewise play a pivotal role in suppressing the cell cycle re-entry of pre-existing cardiomyocytes. Engel and colleagues revealed that p38 MAPK expression inversely correlated with cardiac proliferation during embryonic development and

overexpression of p38α MAPK in fetal mouse cardiomyocytes inhibited DNA synthesis following a 24 hour exposure to FGF1 [40]. An analogous paradigm was reported in neonatal mouse cardiomyocytes as treatment with the p38 MAPK inhibitor SB203580 or overexpression of dominant negative p38α MAPK further increased DNA synthesis in response to a panel of peptide growth factors whereas dominant negative p38β MAPK was without effect [40]. Moreover, a subpopulation of neonatal cardiomyocytes that re-entered the cell cycle following the overexpression of dominant negative p38α MAPK in presence of peptide growth factor stimulation underwent cytokinesis as reflected by the appearance of the G2-M phase marker phosphohistone-3 [40]. Phosphorylation of the serine 10 residue of histone 3 was identified as a distinctive marker of dividing cells, highly phosphorylated in late G2 phase and during chromatin condensation in mitosis [41]. Furthermore, FGF1-mediated DNA synthesis in cardiomyocytes in the presence of p38 MAPK inhibition was suppressed by LY294002 supporting a seminal role of phosphatidylinositol 3-kinase-dependent pathway [40]. An analogous relationship between p38 MAPK, FGF1, cell cycle re-entry and cytokinesis was observed in adult mouse ventricular cardiomyocytes, albeit a longer period of exposure was required and the magnitude of the response was markedly smaller as compared to fetal and neonatal ventricular cardiomyocytes [40]. To examine the paradigm in an in vivo setting, FGF1 and SB203580 were co-administered to 3 month post-infarcted adult mice for a period 4 weeks [42]. The study revealed that FGF1/SB203580 co-administration reduced infarct size and improved contractile function attributed in part to the cell cycle re-entry of pre-existing cardiomyocytes and subsequent cytokinesis [42]. Moreover, FGF1/SB203580 co-administration did not adversely impact scar remodelling [42]. The authors further demonstrated that p38 MAPK inhibition alone was sufficient to promote the cell cycle re-entry of pre-existing cardiomyocytes but the overall improvement of cardiac function and remodelling was dependent on the concomitant angiogenic response by FGF1 [42]. An analogous pattern was observed following the administration of the p38α/β MAPK inhibitor RWJ-67657 to 1-week post myocardial infarcted rats as infarct size was reduced and improved contractile function [43]. By contrast, minimal impact on scar remodelling was observed, as collagen type I and III accumulation in the infarct region was modestly reduced by RWJ-67657 treatment as compared to untreated post-myocardial infarcted rats [43]. A similar antiproliferative role of p38α MAPK was identified during the cardiac regenerative response of the ischemically damaged zebrafish heart [44, 45]. Natural tissue regeneration was reported in amphibians and fish as these lower vertebrates have the greatest capacity for replacing removed and/or injured body parts and this property extended to the

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heart [45]. Cardiomyocytes of lower vertebrates were predominantly mononucleated, smaller in size, contained less myofibrils than adult mammalian cardiomyocytes and maintained a proliferative phenotype throughout life [45]. The study by Jopling and colleagues demonstrated that the natural regenerative response of pre-existing zebrafish cardiomyocytes following cardiac damage was suppressed via the overexpression of p38α MAPK whereas a dominant negative kinase was without effect [44]. Interestingly, pre-existing zebrafish cardiomyocytes expressed p38α MAPK but the serine/threonine kinase was inactivated prior to entry into the G2-M phase [44]. Collectively, these data have demonstrated that whereas adult zebrafish cardiomyocytes have the inherent ability to overcome the anti-proliferative action of p38 MAPK, the overt proinflammatory response following ischemic damage to the adult mammalian heart provides an unfavourable environment as sustained activation of the serine/threonine kinase may further compromise the already modest regenerative response on the infarcted heart. The relationship between p38 MAPK, nestin and cell cycle re-entry of pre-existing cardiomyocytes; recapitulating an embryonic phenotype Nestin, a member of the class VI family of intermediate filament proteins, was first identified in CNS-derived neural progenitor/stem cells and expression in normal and tumour cells promoted proliferation, provided an anti-apoptotic phenotype and enhanced migration [46-48]. In the normal adult rodent heart, a resident population of neural progenitor/stem cells that expressed nestin and derived from the neural crest were identified [49, 50]. Following ischemic damage, a subpopulation of nestin(+)-neural progenitor/stem cells migrated to the scar and contributed to neurogenic innervation of the infarct region and differentiated to a vascular phenotype [e.g. endothelial cells] providing a novel cellular substrate of angiogenesis [51, 52]. However, nestin expression was not exclusive to the neural progenitor/stem cell population as the intermediate filament protein was also detected in ventricular and lung fibroblasts, endothelial cells of newly formed blood vessels and vascular smooth muscle cells that re-entered the cell cycle [49, 53-55]. In ventricular fibroblasts and vascular smooth muscle cells, nestin depletion via a lentiviral shRNA approach inhibited basal and peptide growth factor stimulated DNA synthesis, thereby reaffirming the role of the intermediate filament protein in cell cycle re-entry [53, 56]. Work from our lab and confirmed by others have also identified a modest population of troponin-T(+)-cardiomyocytes bordering the peri-infarct region of the infarcted rodent heart that were morphologically and structurally incomplete, exhibited an aberrant pattern of connexin-43 expression and/or

organization and selectively expressed the intermediate filament protein nestin in a striated pattern (Figure 3) [57-60]. The appearance of nestin(+)-cardiomyocytes was not an acute transient response to ischemic damage as a modest number persisted in the heart of 9-month post-myocardial infarcted rats [57]. These findings were not limited to the rodent heart, as a modest population of cardiomyocytes identified in close proximity to the peri-infarct region of the infarcted human heart were likewise nestin immunoreactive [50, 51, 58, 59]. By contrast, nestin staining was not detected in adult ventricular cardiomyocytes residing in the normal heart or non-infarcted region of the ischemically damaged rodent heart.[49,50] A transgenic lineage tracing approach revealed that nestin was expressed by pre-existing troponin-T(+)-cardiomyocytes exclusively bordering the peri-infarct region of the infarcted mouse heart, albeit cell cycle re-entry was not observed [60]. These observations provided the impetus to assess whether p38 MAPK may have influenced the appearance of nestin(+)-cardiomyocytes by directly inhibiting the expression of the intermediate filament protein and concomitantly suppressing cell cycle re-entry. To examine the latter paradigm, in vitro experiments were performed on neonatal rat ventricular cardiomyocytes (NVCMs) [3-6]. In an attempt to mimic the hypertrophic response of cardiomyocytes and concomitant recruitment of p38 MAPK reported in vivo following myocardial infarction, NVCMs were treated for 24 hours with the protein kinase C (PKC) activator phorbol 12, 13 dibutyrate (PDBu). Previous studies have reported that PKC contributed in part to cardiomyocyte hypertrophy, PKC activation directly phosphorylated and increased p38 MAPK activity and pro-inflammatory cytokines recruited PKC-dependent signalling events (Figure 4) [61-65]. Moreover, the robust production of reactive oxygen species after ischemic damage secondary to the increased expression of NADPH oxidase 4 (Nox4) in cardiomyocytes likewise recruited p38 MAPK (Figure 4) [66-68]. Pro-inflammatory cytokines may have further contributed to the production of reactive oxygen species via PKC-dependent expression of Nox4 (Figure 4) [63, 64, 67, 69]. Consistent with previous studies, 24 hour stimulation with PDBu induced a hypertrophic response characterized by a significant increase of the surface area of NVCMs [60-62]. In unstimulated conditions, a paucity of NVCMs expressed nestin and incorporated bromodexoyuridine (BrdU) and the percentage remained unchanged following the induction of a hypertrophic response after PDBu treatment alone [60]. These data provided the impetus to elucidate whether the lack of effect of PDBu on nestin expression and cell cycle re-entry was attributed in part to a concomitant recruitment of p38 MAPK. Indeed, PDBu treatment of NVCMs rapidly increased the phosphorylation of p38 MAPK and downstream target heat shock protein 27 (HSP27). The pre-treatment with a p38α/β inhibitor SB203580 abrogated PDBu-mediated p38 MAPK

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Figure 3. Nestin expression in adult and neonatal cardiomyocytes. (Panel A) In the infarcted heart of 3-week post-MI mice, striated nestin staining was detected in numerous pre-existing adult ventricular cardiomyocytes [several indicated by arrow] bordering the peri-infarct region. Furthermore, the majority of nestin(+)-cardiomyocytes were mononucleated. The morphologically small nestin(+)-cells detected in the peri-infarct and infarct region may represent neural/progenitor stem cells or myofibroblasts. (Panel B) The treatment of neonatal rat ventricular cardiomyocytes with PDBu and SB203580 for 24 hours increased the percentage that expressed nestin (red fluorescence; indicated by arrow) and incorporated BrdU (purple fluorescence). Nestin(+)-cardiomyocytes that incorporated BrdU were predominantly mononucleated. Neonatal ventricular fibroblasts were identified by collagen type I immunostaining (green fluorescence). Lastly, a subpopulation of cardiomyocytes [indicated by asterisk and lacked collagen staining] failed to express nestin or re-enter the cell cycle following exposure to PDBu/SB203580. The nucleus was identified by To-PRO-3 staining (blue fluorescence).

activity as reflected by the complete inhibition of HSP27 phosphorylation [60]. Moreover, SB20350 treatment prior to the addition of PDBu significantly increased the percentage of neonatal cardiomyocytes that expressed nestin and the percentage of nestin(+)-neonatal cardiomyocytes that incorporated BrdU (Figure 3). Thus, the in vitro data reaffirmed the premise that the appearance of nestin(+)-cardiomyocytes in the infarcted adult mouse heart was attributed in part to the de novo synthesis of the intermediate filament by pre-existing cardiomyocytes [60]. Based on the established proliferative role, nestin expression represented an identifiable phenotypic marker of pre-existing cardiomyocytes that may have the untapped potential to re-enter the cell cycle. However, recruitment of the p38 MAPK pathway post-myocardial infarction (Figure 4) may negatively regulate nestin expression by pre-existing cardiomyocytes and concomitantly inhibit cell cycle re-entry [40, 42] .

Based on the aforementioned data, additional studies were performed to assess whether nestin expression by pre-existing neonatal and adult cardiomyocytes of the infarcted mammalian heart recapitulated an embryonic phenotype. Kachinsky and colleagues revealed that nestin mRNA and protein were expressed in the mouse heart during mid-embryonic development (E9.0-E11) and nestin immunoreactivity was detected in embryonic cardiomyocytes [70] . A recent study by Liu and colleagues further revealed that nestin overexpression increased heart size during embryogenesis attributed in part via cardiomyocyte proliferation [71]. Work from our lab extended these observations and detected striated and/or cytoplasmic nestin staining of troponin-T(+)-cardiomyocytes of embryonic 10,5 day mice and further revealed that this population was cycling as depicted by nuclear Ki67 immunoreactivity.[60] Filamentous nestin was also identified in proliferating H9c2 embryonic rat ventricular cardiomyocytes and lentiviral shRNA-mediated depletion of the intermediate filament

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Figure 4. p38 MAPK recruitment following myocardial infarction. Following myocardial infarction (MI) of the adult mammalian heart, the population of CCR2(+) macrophages was selectively expanded and associated with the uncontrolled release of a panel of pro-inflammatory cytokines including interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α). The release of pro-inflammatory cytokines recruited p38 MAPK signalling directly or indirectly via protein kinase C (PKC)-dependent pathways. In parallel, the increased expression of NADPH oxidase 4 (Nox4) by surviving cardiomyocytes secondary to MI led to the robust accumulation of reactive oxygen species (ROS) and activation of p38 MAPK. Moreover, pro-inflammatory cytokines may have further increased Nox4 expression post-MI via a PKC-dependent pathway. Enhanced p38 MAPK signalling in adult ventricular cardiomyocytes (AVCM) led to infarct expansion via apoptosis and the concomitant inhibition of cell cycle re-entry.

protein significantly inhibited cell cycle re-entry [60]. Collectively, these data support the premise that nestin expression in pre-existing neonatal and adult ventricular cardiomyocytes recapitulated an embryonic trait and reaffirmed the concept that the intermediate filament protein may provide the requisite phenotype to re-enter the cell cycle. Conclusion The inherent ability of neonatal cardiomyocytes to re-enter the cell cycle and the concomitant recruitment of reparative embryonic CCR2(-)-macrophages provided a favourable environment to promote a regenerative cardiac response following injury to the neonatal heart (Figure 2). By contrast, a paucity of residual pre-existing cardiomyocytes re-entered the cell cycle following ischemic damage to the adult heart

and the modest response may have been further compromised by the selective expansion of pro-inflammatory CCR2(+)-macrophages that favoured scar formation and healing. The serine/threonine kinase p38 MAPK, a well-established target of pro-inflammatory cytokines and reactive oxygen species was reported to promote cardiomyocyte apoptosis and inhibit the cell cycle re-entry of neonatal and adult cardiomyocytes (Figure 4). Indeed, pharmacological inhibition of p38 MAPK reduced scar size and improved contractile function of the infarcted adult heart. Despite these data, it remains equivocal as to whether the primary response secondary to p38 MAPK inhibition was suppression of apoptosis and/or increased cell cycle re-entry and proliferation of a subpopulation of adult ventricular cardiomyocytes. Work from our lab revealed that following myocardial infarction of the adult mammalian heart, the intermediate filament protein nestin was expressed by pre-existing cardiomyocytes identified preferentially at the peri-infarct region (Figure 3). In vitro data further revealed that p38 MAPK directly inhibited the de novo synthesis of nestin by neonatal rat ventricular cardiomyocytes and the concomitant re-entry into the cell cycle (Figure 3). Nestin expression rekindled an embryonic phenotype as the intermediate filament protein was detected in proliferating troponin-T(+)-cardiomyocytes in the heart of embryonic 10,5 day mice. Furthermore, nestin depletion via a lentiviral shRNA approach inhibited the cell cycle re-entry of embryonic rat ventricular cardiomyocytes. Collectively, these data support the premise that nestin expression in pre-existing adult cardiomyocytes recapitulated an embryonic trait and may provide the requisite phenotype to re-enter the cell cycle. Therefore, the increased appearance of cycling nestin(+)-cardiomyocytes secondary to p38 MAPK inhibition would support the premise that ventricular regeneration may have contributed in part to the reported smaller infarct. However, an important corollary of our study was that nestin expression alone by a modest population of pre-existing adult cardiomyocytes identified in the infarcted adult rodent heart was insufficient to initiate cell cycle re-entry [60]. Therefore, in addition to attenuating nestin expression, activated p38 MAPK may concomitantly inhibit a downstream target of the intermediate filament protein required to initiate cell cycle re-entry. Previous studies have revealed that nestin-mediated cell proliferation involved recruitment of the phosphatidylinositol 3-kinase dependent pathway and activation of the downstream signalling event protein kinase B/AKT was reported sensitive to p38 MAPK inhibition [46, 71-73]. Thus, selectively targeting p38 MAPK in the ischemically damaged adult mammalian heart may provide a favourable environment to unmask a partial regenerative response by recapitulating an embryonic proliferative phenotype of pre-existing cardiomyocytes via

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the de novo synthesis of the intermediate filament protein nestin.

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Acknowledgements

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This work was supported by the Fondation de Recherche d’Institut de Cardiologie [FRIC] de Montreal. I would like to thank past and present members of my lab who have contributed to the work presented in this paper (Vanessa Hertig, Marc-Andre Meus, Andreanne Chabot, Kim Tardif, Robert Clement, Hugues Gosselin, Viviane El-Helou, Pauline Béguin, Jessica Drapeau and Samar Bel-Hadj). I would also like to thank Sylvie Bolduc for her assistance in the designing of the figures. I would like to also thank Vanessa Hertig and Dr. Bruce Allen for the critical reading of the manuscript. References 1.

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