In situ three-dimensional reconstruction of mouse heart sympathetic innervation by two-photon excitation fluorescence imaging

Background Sympathetic nerve wiring in the mammalian heart has remained largely unexplored. Resolving the wiring diagram of the cardiac sympathetic network would help establish the structural underpinnings of neurocardiac coupling. New Method We used two-photon excitation fluorescence microscopy, combined with a computer-assisted 3-D tracking algorithm, to map the local sympathetic circuits in living hearts from adult transgenic mice expressing enhanced green fluorescent protein (EGFP) in peripheral adrenergic neurons. Results Quantitative co-localization analyses confirmed that the intramyocardial EGFP distribution recapitulated the anatomy of the sympathetic arbor. In the left ventricular subepicardium of the uninjured heart, the sympathetic network was composed of multiple subarbors, exhibiting variable branching and looping topology. Axonal branches did not overlap with each other within their respective parental subarbor nor with neurites of annexed subarbors. The sympathetic network in the border zone of a 2-week-old myocardial infarction was characterized by substantive rewiring, which included spatially heterogeneous loss and gain of sympathetic fibers and formation of multiple, predominately nested, axon loops of widely variable circumference and geometry. Comparison with Existing Methods In contrast to mechanical tissue sectioning methods that may involve deformation of tissue and uncertainty in registration across sections, our approach preserves continuity of structure, which allows tracing of neurites over distances, and thus enables derivation of the three-dimensional and topological morphology of cardiac sympathetic nerves. Conclusions Our assay should be of general utility to unravel the mechanisms governing sympathetic axon spacing during development and disease

Electrical coupling between ventricular myocytes and myofibroblasts in the infarcted mouse heart

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

Cardiac engraftment of genetically-selected parthenogenetic stem cell-derived cardiomyocytes

Parthenogenetic stem cells (PSCs) are a promising candidate donor for cell therapy applications. Similar to embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), PSCs exhibit self-renewing capacity and clonogenic proliferation in vitro. PSCs exhibit largely haploidentical genotype, and as such may constitute an attractive population for allogenic applications. In this study, PSCs isolated from transgenic mice carrying a cardiomyocyte-restricted reporter transgene to permit tracking of donor cells were genetically modified to carry a cardiomyocyte-restricted aminoglycoside phosphotransferase expression cassette (MHC-neor/pGK-hygror) to permit the generation of highly enriched cardiomyocyte cultures from spontaneously differentiating PSCs by simple selection with the neomycin analogue G148. Following engraftment into isogenic recipient hearts, the selected cardiomyocytes formed a functional syncytium with the host myocardium as evidenced by the presence of entrained intracellular calcium transients. These cells thus constitute a potential source of therapeutic donor cells

Myocardial polyploidization creates a barrier to heart regeneration in zebrafish

Correlative evidence suggests that polyploidization of heart muscle, which occurs naturally in post-natal mammals, creates a barrier to heart regeneration. Here, we move beyond a correlation by demonstrating that experimental polyploidization of zebrafish cardiomyocytes is sufficient to suppress their proliferative potential during regeneration. Initially, we determined that zebrafish myocardium becomes susceptible to polyploidization upon transient cytokinesis inhibition mediated by dominant-negative Ect2. Using a transgenic strategy, we generated adult animals containing mosaic hearts composed of differentially labeled diploid and polyploid-enriched cardiomyocyte populations. Diploid cardiomyocytes outcompeted their polyploid neighbors in producing regenerated heart muscle. Moreover, hearts composed of equivalent proportions of diploid and polyploid cardiomyocytes failed to regenerate altogether, demonstrating that a critical percentage of diploid cardiomyocytes is required to achieve heart regeneration. Our data identify cardiomyocyte polyploidization as a barrier to heart regeneration and suggest that mobilizing rare diploid cardiomyocytes in the human heart will improve its regenerative capacity

Lineage Tracing of Cardiac Explant Derived Cells

AIMS: Cultured cardiac explants produce a heterogeneous population of cells including a distinctive population of refractile cells described here as small round cardiac explant derived cells (EDCs). The aim of this study was to explore the source, morphology and cardiogenic potential of EDCs. METHODS: Transgenic MLC2v-Cre/ZEG, and actin-eGFP mice were used for lineage-tracing of EDCs in vitro and in vivo. C57B16 mice were used as cell transplant recipients of EDCs from transgenic hearts, as well as for the general characterisation of EDCs. The activation of cardiac-specific markers were analysed by: immunohistochemistry with bright field and immunofluorescent microscopy, electron microscopy, PCR and RT-PCR. Functional engraftment of transplanted cells was further investigated with calcium transient studies. RESULTS: Production of EDCs was highly dependent on the retention of blood-derived cells or factors in the cultured explants. These cells shared some characteristics of cardiac myocytes in vitro and survived engraftment in the adult heart in vivo. However, EDCs failed to differentiate into functional cardiac myocytes in vivo as demonstrated by the absence of stimulation-evoked intracellular calcium transients following transplantation into the peri-infarct zone. CONCLUSIONS: This study highlights that positive identification based upon one parameter alone such as morphology or immunofluorescene is not adequate to identify the source, fate and function of adult cardiac explant derived cells

Adult Bone Marrow–derived Cells Do Not Acquire Functional Attributes of Cardiomyocytes When Transplanted into Peri-infarct Myocardium

(BM) cells after being directly transplanted into the ischemically injured heart remains a controversial issue. In this study, we investigated the ability of transplanted BM cells to develop intracellular calcium ([Ca2+] i ) transients in response to membrane depolarization in situ. Low-density mononuclear (LDM) BM cells, c-kit-enriched (c-kitenr) BM cells, and highly enriched lin– c-kit+ BM cells were obtained from adult transgenic mice ubiquitously expressing enhanced green fluorescent protein (EGFP), and injected into peri-infarct myocardiums of nontransgenic mice. After 9–10 days the mice were killed, and the hearts were removed, perfused in Langendorff mode, loaded with the calcium-sensitive fluorophore rhod-2, and subjected to two-photon laser scanning fluorescence microscopy (TPLSM) to monitor action potential–induced [Ca2+] i transients in EGFP-expressing donor-derived cells and non-expressing host cardiomyocytes. Whereas spontaneous and electrically evoked [Ca2+] i transients were found to occur synchronously in host cardiomyocytes along the graft–host border and in areas remote from the infarct, they were absent in all of the >3,000 imaged BM-derived cells that were located in clusters throughout the infarct scar or peri-infarct zone. We conclude that engrafted BM-derived cells lack attributes of functioning cardiomyocytes, calling into question the concept that adult BM cells can give rise to substantive cardiomyocyte regeneration within the infarcted heart

Absence of cardiomyocyte differentiation following transplantation of adult cardiac-resident Sca-1+ cells into infarcted mouse hearts

Although several lines of evidence suggest that the glycosyl phosphatidylinositol-anchored cell surface protein Sca-1 marks cardiac-resident stem cells, a critical analysis of the literature raises some concerns regarding their cardiomyogenic potential.1 Here, isolated adult cardiac-resident Sca-1+ cells were engrafted into infarcted hearts and monitored for cardiomyogenic differentiation. Donor cells were prepared from ACT-EGFP; MHC-nLAC double-transgenic mice ([C57/Bl6J x DBA/2J]F1 genetic background; all procedures followed were in accordance with Institutional Guidelines). The ACT-EGFP transgene targets ubiquitous expression of an enhanced green fluorescent protein reporter, and the MHC-nLAC transgene targets cardiomyocyte-restricted expression of a nuclear-localized β-galactosidase reporter. Donor cell survival was monitored via EGFP fluorescence, while cardiomyogenic differentiation was monitored by reacting with the chromogenic β-galactosidase substrate 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-GAL), which gives rise to a blue product.2 Double-transgenic hearts were dispersed with Blendzyme and the resulting cells reacted with an APC-conjugated anti-Sca-1 antibody and a PE-conjugated cocktail of antibodies recognizing hematopoietic lineage markers.3 Sca-1+, EGFP+, lineage- cells were then isolated via fluorescence-activated cell sorting (FACS; characterization of the donor cells is provided in Figure 1A), and 100,000 cells were injected into the infarct border zone of non-transgenic [C57/Bl6J x DBA/2J]F1 mice immediately following permanent coronary artery occlusion

Recombinant neuregulin 1 does not activate cardiomyocyte DNA synthesis in normal or infarcted adult mice.

Neuregulin 1 signaling plays an important role in cardiac trabecular development, and in sustaining functional integrity in adult hearts. Treatment with neuregulin 1 enhances adult cardiomyocyte differentiation, survival and/or function in vitro and in vivo. It has also been suggested that recombinant neuregulin 1β1 (NRG1β1) induces cardiomyocyte proliferation in normal and injured adult hearts. Here we further explore the impact of neuregulin 1 signaling on adult cardiomyocyte cell cycle activity.Adult mice were subjected to 9 consecutive daily injections of recombinant NRG1β1 or vehicle, and cardiomyocyte DNA synthesis was quantitated via bromodeoxyuridine (BrdU) incorporation, which was delivered using mini-osmotic pumps over the entire duration of NRG1β1 treatment. NRG1β1 treatment inhibited baseline rates of cardiomyocyte DNA synthesis in normal mice (cardiomyocyte labelling index: 0.019±0.005% vs. 0.003±0.001%, saline vs. NRG1β1, P<0.05). Acute NRG1β1 treatment did result in activation of Erk1/2 and cardiac myosin regulatory light chain (down-stream mediators of neuregulin signalling), as well as activation of DNA synthesis in non-cardiomyocytes, validating the biological activity of the recombinant protein. In other studies, mice were subjected to permanent coronary artery occlusion, and cardiomyocyte DNA synthesis was monitored via tritiated thymidine incorporation which was delivered as a single injection 7 days post-infarction. Daily NRG1β1 treatment had no impact on cardiomyocyte DNA synthesis in the infarcted myocardium (cardiomyocyte labelling index: 0.039±0.011% vs. 0.027±0.021%, saline vs. NRG1β1, P>0.05).These data indicate that NRG1β1 treatment does not increase cardiomyocyte DNA synthesis (and consequently does not increase the rate of cardiomyocyte renewal) in normal or infarcted adult mouse hearts. Thus, any improvement in cardiac structure and function observed following neuregulin treatment of injured hearts likely occurs independently of overt myocardial regeneration