Apex 英雄M APK: A Licensed and Co-Developed Mobile Version of Apex Legends

During the acute phase of healing after apex resection of the neonatal mouse heart, the protease thrombin converts fibrinogen to fibrin leading to the formation of a thrombus sealing the exposed left ventricular chamber3,5. In addition to fibrin clot formation; thrombin was reported to play a seminal role in scar expansion of the infarcted adult heart as pharmacological inhibition of thrombin synthesis or inactivation of the protease significantly reduced scar size8,9. The smaller infarct may be related in part to the attenuation of thrombin mediated recruitment of the inflammatory response post-myocardial infarction10. In addition, the protease directly influences cardiac remodelling as thrombin stimulation of ventricular cardiomyocytes via activation of the protease activated receptor-1 (PAR-1) induced a hypertrophic response11,12. Although not examined, thrombin may further prevent the cell cycle re-entry of mononucleated ventricular cardiomyocytes via recruitment of the serine/threonine kinase p38α MAPK13,14. Numerous studies have unequivocally revealed that pharmacological inhibition or inactivation of p38α MAPK in response to various stimuli (e.g. acidic fibroblast growth factor, interleukin-1β, neuroregulin-1 and protein kinase C) inhibits the cell cycle re-entry of neonatal and adult ventricular rodent cardiomyocytes15,16,17,18. Moreover, work from my lab has reported that phorbol 12,13-dibutyrate (PDBu) activation of p38α MAPK in neonatal rat ventricular cardiomyocytes prevented de novo expression of the intermediate filament protein nestin15,16. The intermediate filament protein facilitated cell cycle re-entry as shRNA-mediated depletion of constitutive nestin expression in embryonic rat ventricular cardiomyocytes or preventing induction in neonatal rat ventricular cardiomyocytes co-treated with PDBu and the p38α/β MAPK inhibitor SB203580 attenuated bromodeoxyuridine incorporation15,16. Based on these data, it is tempting to speculate that the local accumulation of thrombin during the acute phase of fibrin clot formation after ventricular apex resection of the neonatal heart may partially suppress cell cycle re-entry of ventricular cardiomyocytes and prevent de novo nestin expression via recruitment of p38α MAPK-dependent signalling events. However, directly examining the latter premise is not possible as thrombin inactivation after ventricular apex resection will prevent fibrin clot formation leading to exsanguination and death. In this regard, two complementary approaches will address the potential relationship between thrombin, p38α MAPK, cell cycle re-entry and nestin in neonatal ventricular cardiomyocytes. The first series of experiments will test the hypothesis that thrombin treatment of 1-day old neonatal rat ventricular cardiomyocytes prevents cell cycle re-entry and de novo nestin expression via p38α MAPK signalling. A second series of experiments will test the hypothesis that administration of the p38α/β MAPK inhibitor SB203580 during the acute phase of fibrin clot formation after ventricular apex resection of the neonatal rat heart increases the density of ventricular cardiomyocytes and subpopulation of nestin(+)-ventricular cardiomyocytes that re-enter the cell cycle translating to a partial cardiac regenerative response.

The predominant appearance of nestin(+)-cells along the border of the fibrin clot. (A) In a vehicle-treated apex-resected heart, a large fibrin clot sealing the exposed left ventricular chamber was evident. Furthermore, a population of nestin(+)-cells (red fluorescence) was detected bordering the entire length of the fibrin clot region. (B) In a SB203580-treated apex-resected heart, the resected left ventricle was completely sealed with myocardial tissue characterized by the appearance of cardiac troponin-T(+)-NNVMs. Furthermore, an important population of nestin(+)-cells (red fluorescence) was still prevalent juxtaposed to the fibrin clot. DAPI staining identifies the nucleus (blue fluorescence).

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Nestin/cardiac troponin-T(+)-ventricular cardiomyocytes were detected exclusively adjacent to the fibrin clot region and a subpopulation re-entered the cell cycle. (A) The majority of nestin(+)-cells (red fluorescence) identified adjacent to the fibrin clot region were neonatal ventricular cardiomyocytes characterized by the co-expression of cardiac troponin-T (green fluorescence). (B) In SB203580-treated apex-resected hearts, a subpopulation of cardiac troponin-T/nestin(+)-neonatal ventricular cardiomyocytes bordering the fibrin clot region re-entered the cell cycle (indicated by arrow) characterized by bromodeoxyuridine (Brdu; grey fluorescence) incorporation. Cell cycle re-entry was also observed in cardiac troponin-T/nestin(+)-neonatal ventricular cardiomyocytes along the border of the vehicle-treated apex-resected heart (see Fig. 11). DAPI staining identified the nucleus (blue fluorescence).

In the infarcted adult rabbit heart, increased expression of the transmembrane receptor tissue factor in ventricular cardiomyocytes bordering the ischemically damaged region initiated VII/VIIa mediated accumulation of the protease thrombin at the site of injury9. The latter study established a biological role as pharmacological inhibition of tissue factor or hirudin inhibition of activated thrombin reduced infarct size9. A significant reduction of infarct size was also reported in apolipoprotein E-deficient/LDL receptor-deficient adult male mice treated with the activated thrombin inhibitor melagatran after exposure of the heart to acute hypoxic stress22. Consistent with the latter paradigm, pharmacological inhibition of thrombin activation of the protease-activated receptor-1 (PAR-1) limited scar expansion of the adult rodent heart after ischemia/reperfusion injury23,24. In contrast to the established pharmacological data, PAR-1 deficiency failed to reduce infarct size and administration of PAR-1 agonists did not further increase the scar region after ischemic damage24,25. It has been suggested that the disparate pharmacological and transgenic effect on infarct size may be attributed in part to off-target actions of PAR-1 antagonists24. Alternatively, protease-activated receptor-4 (PAR-4) was reported to compensate for PAR-1 deficiency as pharmacological inhibition or transgenic depletion of PAR-4 reduced infarct size in the ischemically damaged adult heart24. Based on these observations, the local accumulation of thrombin during the acute phase of fibrin clot formation after apex resection of the left ventricle of the neonatal rat heart may directly influence cardiomyocyte responsiveness after injury. Indeed, the reported recruitment of p38α MAPK signalling after thrombin stimulation of neonatal ventricular cardiomyocytes may prevent cell cycle re-entry after apex resection11,12. Engel and colleagues demonstrated that p38α MAPK activity was significantly repressed during the embryonic proliferative phase of heart development and expression of a dominant-negative form of the serine/threonine kinase increased cell cycle re-entry and cytokinesis of fetal cardiomyocytes delineated by nuclear phosphohistone-3 staining17. In neonatal rodent ventricular cardiomyocytes, pharmacological suppression of p38α MAPK with the inhibitor SB203580 potentiated cell cycle re-entry and cytokinesis in response to diverse stimuli including acidic fibroblast growth factor, IL-1β, neuroregulin-1 and phorbol ester recruitment of protein kinase C-dependent pathways15,16,17. The latter paradigm was preserved in the infarcted adult mouse heart as treatment with SB203580 alone was sufficient to increase the density of ventricular cardiomyocytes that re-entered the cell cycle18. An analogous mechanism is prevalent during regeneration of the injured adult zebrafish heart as p38α MAPK inactivation was identified as a seminal prerequisite event initiating the migration of mononucleated cardiomyocytes to the damaged region and subsequent cell cycle re-entry1,2. In the present study, the absence of an increase in cell cycle re-entry of cardiac troponin-T(+)-neonatal ventricular cardiomyocytes after thrombin treatment was associated with the acute recruitment of p38α MAPK signalling delineated by the increased phosphorylation of the serine/threonine kinase and an enhanced phosphorylated state of the putative downstream target HSP27. Pre-treatment with the p38α/β MAPK inhibitor SB203580 abrogated thrombin stimulated HSP27 phosphorylation and concomitantly increased the number of cardiac troponin-T(+)-neonatal ventricular cardiomyocytes that incorporated bromodeoxyuridine. Cell cycle re-entry of fetal chicken cardiomyocytes cultured in three-dimensional myocardial tissue was also reported after thrombin activation of PAR-126. However, the latter study did not examine the downstream signalling events coupling PAR-1 activation to cell cycle re-entry26. Thus, the in vitro data highlight a novel antiproliferative role of thrombin and further suggests that the local accumulation of the protease in the apex-resected neonatal heart or infarcted adult heart may in part suppress the cell cycle re-entry of mononucleated ventricular cardiomyocytes6,7,15.

The study by Porello and colleagues reported that following ventricular apex resection of the 1-day old neonatal mouse heart, a complete regenerative was observed 28 days post-surgery5. By contrast, Andersen and colleagues revealed that apex resection was associated with a modest number of cardiomyocytes that re-entered the cell cycle translating to significant scarring in the absence of a regenerative cardiac response33. The study by Bryant et al. suggested that apex resection of a large region of the neonatal mouse heart may in part compromise the cardiac regenerative response34. Despite these discordant findings, a salient feature of healing after apex resection of the neonatal heart or the adult zebrafish heart is the formation of a thrombus that seals the exposed left ventricular chamber1,3,5,34. In the present study, successful ventricular apex resection of 1-day old neonatal rat hearts was confirmed by the appearance of minute bleeding immediately after surgery leading to the formation of a fibrin clot sealing the exposed chamber. The latter paradigm may further represent an essential prerequisite biological event initiating a cardiac regenerative response as fibrin is an important natural scaffold providing the necessary matrix for cell migration, adhesion and proliferation35. Morphologically, fibrin clot formation after apex resection was associated with a significant reduction of left ventricular free wall thickness and septal wall thickness in vehicle-treated apex-resected heart as compared to neonatal sham hearts. Within the remaining viable myocardium of the apex region and at the border of the fibrin clot, a subpopulation of cardiac troponin-T(+)-ventricular cardiomyocytes re-entered S-phase and progressed to the G2-M phase characterized by nuclear staining of exogenously administered bromodeoxyuridine and phosphistone-3, respectively. Furthermore, a subpopulation of ventricular cardiomyocytes identified in vehicle-treated apex-resected hearts that expressed nuclear phosphohistone-3 was characterized by the marginalization of cardiac troponin-T to the periphery5. The acute phase of healing after apex resection was further highlighted by the novel appearance of a population of cycling ventricular cardiomyocytes along the entire length of the fibrin clot that expressed the intermediate filament protein nestin. The latter response was analogous to that observed in the infarcted adult rodent heart21,27. However, in contrast to the infarcted adult heart, the response was robust and nestin(+)-ventricular cardiomyocytes bordering the fibrin clot of the vehicle-treated apex resected neonatal heart re-entered the cell cycle15. Collectively, these data are consistent with the preponderance of previously published studies demonstrating that fibrin clot formation secondary to apex resection of the neonatal rodent heart was associated with the cell cycle re-entry of ventricular cardiomyocytes3,5,34,36.