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Induction of the cell cycle is emerging as an intervention to treat heart failure. Here, we tested the hypothesis that enhanced cardiomyocyte renewal in transgenic mice expressing cyclin D2 would be beneficial during hemodynamic overload. We induced pressure overload by transthoracic aortic constriction (TAC) or volume overload by aortocaval shunt in cyclin D2expressing and WT mice. Although cyclin D2 expression dramatically improved survival following TAC, it did not confer a survival advantage to mice following aortocaval shunt. Cardiac function decreased following TAC in WT mice, but was preserved in cyclin D2-expressing mice. On the other hand, cardiac structure and function were compromised in response to aortocaval shunt in both WT and cyclin D2-expressing mice. The preserved function and improved survival in cyclin D2expressing mice after TAC was associated with an approximately 50% increase in cardiomyocyte number and exaggerated cardiac hypertrophy, as indicated by increased septum thickness. Aortocaval shunt did not further impact cardiomyocyte number in mice expressing cyclin D2. Following TAC, cyclin D2 expression attenuated cardiomyocyte hypertrophy, reduced cardiomyocyte apoptosis, fibrosis, calcium/calmodulin-dependent protein kinase IIS phosphorylation, brain natriuretic peptide expression, and sustained capillarization. Thus, we show that cyclin D2-induced cardiomyocyte renewal reduced myocardial remodeling and dysfunction after pressure overload but not after volume overload., Introduction In structural heart diseases, increased ventricular load is a key trigger mechanism for altered gene expression and cardiac remodeling (1). Increased ventricular load translates into increased wall stress for [...]

Background: Identifying genetic variants which impact the level of cell cycle reentry and establishing the degree of cell cycle progression in those variants could help guide development of therapeutic interventions aimed at effecting cardiac regeneration. We observed that C57Bl6/NCR (B6N) mice have a marked increase in cardiomyocyte S-phase activity following permanent coronary artery ligation as compared to infarcted DBA/2J (D2J) mice.Methods: Cardiomyocyte cell cycle activity post-infarction was monitored in D2J, (D2J x B6N)-F1 and [(D2J x B6N)-F1 x D2J] backcross mice via bromodeoxyuridine or 5-ethynyl-2' -deoxyuridine incorporation, using a nuclear-localized transgenic reporter to identify cardiomyocyte nuclei. Genome-wide quantitative trait locus (QTL) analysis, fine scale genetic mapping, whole exome sequencing and RNA-seq analyses of the backcross mice were performed to identify the gene responsible for the elevated cardiomyocyte S-phase phenotype.Results: (D2J x B6N)-F1 mice exhibited a 14-fold increase in cardiomyocyte S-phase activity in ventricular regions remote from infarct scar as compared to D2J mice (0.798 ± 0.09% vs. 0.056 ± 0.004%; p < 0.001). QTL analysis of [(D2J x B6N)-F1 x D2J] backcross mice revealed that the gene responsible for differential S-phase activity was located on the distal arm of Chromosome 3 (LOD score = 6.38; p < 0.001). Additional genetic and molecular analyses identified 3 potential candidates. Of these, troponin I-interacting kinase (Tnni3k) is expressed in B6N hearts but not in D2J hearts. Transgenic expression of Tnni3k in a D2J genetic background results in elevated cardiomyocyte S-phase activity post-injury. Cardiomyocyte S-phase activity in both TNNI3K-expressing and TNNI3K-nonexpressing mice results in the formation of polyploid nuclei.Conclusions: These data indicate that TNNI3K expression increases the level of cardiomyocyte S-phase activity following injury. [ABSTRACT FROM AUTHOR]

Accurate nuclear identification is crucial for distinguishing the role of cardiac myocytes in intrinsic and experimentally induced regenerative growth of the myocardium. Conventional histologic analysis of myocyte nuclei relies on the optical sectioning capabilities of confocal microscopy in conjunction with immunofluorescent labeling of cytoplasmic proteins such as troponin T, and dyes that bind to double-strand DNA to identify nuclei. Using heart sections from transgenic mice in which the cardiomyocyte-restricted [alpha]-cardiac myosin heavy chain promoter targeted the expression of nuclear localized [beta]-galactosidase reporter in >99% of myocytes, we systematically compared the fidelity of conventional myocyte nuclear identification using confocal microscopy, with and without the aid of a membrane marker. The values obtained with these assays were then compared with those obtained with anti-[beta]-galactosidase immune reactivity in the same samples. In addition, we also studied the accuracy of anti-GATA4 immunoreactivity for myocyte nuclear identification. Our results demonstrate that, although these strategies are capable of identifying myocyte nuclei, the level of interobserver agreement and margin of error can compromise accurate identification of rare events, such as cardiomyocyte apoptosis and proliferation. Thus these data indicate that morphometric approaches based on segmentation are justified only if the margin of error for measuring the event in question has been predetermined and deemed to be small and uniform. We also illustrate the value of a transgene-based approach to overcome these intrinsic limitations of identifying myocyte nuclei. This latter approach should prove quite useful when measuring rare events. myocyte nuclear identification; cardiac myocyte-restricted [alpha]-cardiac myosin heavy chain promoter [beta]-galactosidase reporter mice; cell membrane marker; cardiomyogenic transcription factor; confocal microscopy doi: 10.1152/ajpcell.00435.2009.

Aims Doxorubicin (DOX) is a widely used and effective anti-cancer therapeutic. DOX treatment is associated with both acute and late onset cardiotoxicity, limiting its overall efficacy. Here, the impact of cardiomyocyte cell cycle activation was examined in a juvenile model featuring aspects of acute and late onset DOX cardiotoxicity. Methods and results Two-week old MHC-cycD2 transgenic mice (which express cyclin D2 in postnatal cardiomyocytes and exhibit sustained cardiomyocyte cell cycle activity; D2 mice) and their wild type (WT) littermates received weekly DOX injections for 5 weeks (25 mg/kg cumulative dose). One week after the last DOX treatment (acute stage), cardiac function was suppressed in both groups. Acute DOX cardiotoxicity in D2 and WT mice was associated with similar increases in the levels of cardiomyocyte apoptosis and Ku70/Ku80 expression (markers of DNA damage and oxidative stress), as well as similar reductions in hypertrophic cardiomyocyte growth. Cardiac dysfunction persisted in WT mice for 13 weeks following the last DOX treatment (late stage) and was accompanied by increased levels of cardiomyocyte apoptosis, Ku expression, and myocardial fibrosis. In contrast, D2 mice exhibited a progressive recovery in cardiac function, which was indistinguishable from saline-treated animals by 9 weeks following the last DOX treatment. Improved cardiac function was accompanied by reductions in the levels of late stage cardiomyocyte apoptosis, Ku expression, and myocardial fibrosis. Conclusion These data suggest that cardiomyocyte cell cycle activity can promote recovery of cardiac function and preserve cardiac structure following DOX treatment. [ABSTRACT FROM AUTHOR]

Aims: Recent studies have demonstrated electrotonic coupling between scar tissue and the surrounding myocardium in cryoinjured hearts. However, the electrical dynamics occurring at the myocyte-nonmyocyte interface in the fibrotic heart remain undefined. Here, we sought to develop an assay to interrogate the nonmyocyte cell type contributing to heterocellular coupling and to characterize, on a cellular scale, its voltage response in the infarct border zone of living hearts. Methods And Results: We used two-photon laser scanning microscopy in conjunction with a voltage-sensitive dye to record transmembrane voltage changes simultaneously from cardiomyocytes and adjoined nonmyocytes in Langendorff-perfused mouse hearts with healing myocardial infarction. Transgenic mice with cardiomyocyte-restricted expression of a green fluorescent reporter protein underwent permanent coronary artery ligation and their hearts were subjected to voltage imaging 7-10 days later. Reporter-negative cells, i.e. nonmyocytes, in the infarct border zone exhibited depolarizing transients at a 1:1 coupling ratio with action potentials recorded simultaneously from adjacent, reporter-positive ventricular myocytes. The electrotonic responses in the nonmyocytes exhibited slower rates of de- and repolarization compared to the action potential waveform of juxtaposed myocytes. Voltage imaging in infarcted hearts expressing a fluorescent reporter specifically in myofibroblasts revealed that the latter were electrically coupled to border zone myocytes. Their voltage transient properties were indistinguishable from those of nonmyocytes in hearts with cardiomyocyte-restricted reporter expression. The density of connexin43 expression at myofibroblast-cardiomyocyte junctions was -5% of that in the intercalated disc regions of paired ventricular myocytes in the remote, uninjured myocardium, whereas the ratio of connexin45 to connexin43 expression levels at heterocellular contacts was -1%. Conclusion: Myofibroblasts contribute to the population of electrically coupled nonmyocytes in the infarct border zone. The slower kinetics of myofibroblast voltage responses may reflect low electrical conductivity across heterocellular junctions, in accordance with the paucity of connexin expression at myofibroblast-cardiomyocyte contacts. [ABSTRACT FROM AUTHOR]

Accurate nuclear identification is crucial for distinguishing the role of cardiac myocytes in intrinsic and experimentally induced regenerative growth of the myocardium. Conventional histologic analysis of myocyte nuclei relies on the optical sectioning capabilities of confocal microscopy in conjunction with immunofluorescent labeling of cytoplasmic proteins such as troponin T, and dyes that bind to double-strand DNA to identify nuclei. Using heart sections from transgenic mice in which the cardiomyocyte-restricted α-cardiac myosin heavy chain promoter targeted the expression of nuclear localized β-galactosidase reporter in >99% of myocytes, we systematically compared the fidelity of conventional myocyte nuclear identification using confocal microscopy, with and without the aid of a membrane marker. The values obtained with these assays were then compared with those obtained with anti-β-galactosidase immune reactivity in the same samples. In addition, we also studied the accuracy of anti-GATA4 immunoreactivity for myocyte nuclear identification. Our results demonstrate that, although these strategies are capable of identifying myocyte nuclei, the level of interobserver agreement and margin of error can compromise accurate identification of rare events, such as cardiomyocyte apoptosis and proliferation. Thus these data indicate that morphometric approaches based on segmentation are justified only if the margin of error for measuring the event in question has been predetermined and deemed to be small and uniform. We also illustrate the value of a transgene-based approach to overcome these intrinsic limitations of identifying myocyte nuclei. This latter approach should prove quite useful when measuring rare events. [ABSTRACT FROM AUTHOR]

Induction of the cell cycle is emerging as an intervention to treat heart failure. Here, we tested the hypothesis that enhanced cardiomyocyte renewal in transgenic mice expressing cyclin D2 would be beneficial during hemodynamic overload. We induced pressure overload by transthoracic aortic constriction (TAC) or volume overload by aortocaval shunt in cyclin D2-expressing and WT mice. Although cyclin D2 expression dramatically improved survival following TAC, it did not confer a survival advantage to mice following aortocaval shunt. Cardiac function decreased following TAC in WT mice, but was preserved in cyclin D2-expressing mice. On the other hand, cardiac structure and function were compromised in response to aortocaval shunt in both WT and cyclin D2-expressing mice. The preserved function and improved survival in cyclin D2-expressing mice after TAC was associated with an approximately 50% increase in cardiomyocyte number and exaggerated cardiac hypertrophy, as indicated by increased septum thickness. Aortocaval shunt did not further impact cardiomyocyte number in mice expressing cyclin D2. Following TAC, cyclin D2 expression attenuated cardiomyocyte hypertrophy, reduced cardiomyocyte apoptosis, fibrosis, calcium/calmodulin-dependent protein kinase IIδ phosphorylation, brain natriuretic peptide expression, and sustained capillarization. Thus, we show that cyclin D2-induced cardiomyocyte renewal reduced myocardial remodeling and dysfunction after pressure overload but not after volume overload. [ABSTRACT FROM AUTHOR]

The paucity of mammalian adult cardiac myocytes (CM) proliferation following myocardial infarction (MI) and the remodeling of the necrotic tissue that ensues, result in non-regenerative repair. In contrast, zebrafish (ZF) can regenerate after an apical resection or cryoinjury of the heart. There is considerable interest in models where regeneration proceeds in the presence of necrotic tissue. We have developed and characterized a cautery injury model in the giant danio (GD), a species closely related to ZF, where necrotic tissue remains part of the ventricle, yet regeneration occurs. By light and transmission electron microscopy (TEM), we have documented four temporally overlapping processes: (1) a robust inflammatory response analogous to that observed in MI, (2) concomitant proliferation of epicardial cells leading to wound closure, (3) resorption of necrotic tissue and its replacement by granulation tissue, and (4) regeneration of the myocardial tissue driven by 5-EDU and [(3) H]thymidine incorporating CMs. In conclusion, our data suggest that the GD possesses robust repair mechanisms in the ventricle and can serve as an important model of cardiac inflammation, remodeling and regeneration., (Copyright © 2011 Wiley-Liss, Inc.)