Growth differentiation factor 11 is a circulating factor that reverses age-related cardiac hypertrophy.

The most common form of heart failure occurs with normal systolic function and often involves cardiac hypertrophy in the elderly. To clarify the biological mechanisms that drive cardiac hypertrophy in aging, we tested the influence of circulating factors using heterochronic parabiosis, a surgical technique in which joining of animals of different ages leads to a shared circulation. After 4 weeks of exposure to the circulation of young mice, cardiac hypertrophy in old mice dramatically regressed, accompanied by reduced cardiomyocyte size and molecular remodeling. Reversal of age-related hypertrophy was not attributable to hemodynamic or behavioral effects of parabiosis, implicating a blood-borne factor. Using modified aptamer-based proteomics, we identified the TGF-ß superfamily member GDF11 as a circulating factor in young mice that declines with age. Treatment of old mice to restore GDF11 to youthful levels recapitulated the effects of parabiosis and reversed age-related hypertrophy, revealing a therapeutic opportunity for cardiac aging.

Myocardial pressure overload induces systemic inflammation through endothelial cell IL-33.

Hypertension increases the pressure load on the heart and is associated with a poorly understood chronic systemic inflammatory state. Interleukin 33 (IL-33) binds to membrane-bound ST2 (ST2L) and has antihypertrophic and antifibrotic effects in the myocardium. In contrast, soluble ST2 appears to act as a decoy receptor for IL-33, blocking myocardial and vascular benefits, and is a prognostic biomarker in patients with cardiovascular diseases. Here we report that a highly local intramyocardial IL-33/ST2 conversation regulates the heart's response to pressure overload. Either endothelial-specific deletion of IL33 or cardiomyocyte-specific deletion of ST2 exacerbated cardiac hypertrophy with pressure overload. Furthermore, pressure overload induced systemic circulating IL-33 as well as systemic circulating IL-13 and TGF-beta1; this was abolished by endothelial-specific deletion of IL33 but not by cardiomyocyte-specific deletion of IL33. Our study reveals that endothelial cell secretion of IL-33 is crucial for translating myocardial pressure overload into a selective systemic inflammatory response.

Transcriptional reversion of cardiac myocyte fate during mammalian cardiac regeneration.

RATIONALE: Neonatal mice have the capacity to regenerate their hearts in response to injury, but this potential is lost after the first week of life. The transcriptional changes that underpin mammalian cardiac regeneration have not been fully characterized at the molecular level. OBJECTIVE: The objectives of our study were to determine whether myocytes revert the transcriptional phenotype to a less differentiated state during regeneration and to systematically interrogate the transcriptional data to identify and validate potential regulators of this process. METHODS AND RESULTS: We derived a core transcriptional signature of injury-induced cardiac myocyte (CM) regeneration in mouse by comparing global transcriptional programs in a dynamic model of in vitro and in vivo CM differentiation, in vitro CM explant model, as well as a neonatal heart resection model. The regenerating mouse heart revealed a transcriptional reversion of CM differentiation processes, including reactivation of latent developmental programs similar to those observed during destabilization of a mature CM phenotype in the explant model. We identified potential upstream regulators of the core network, including interleukin 13, which induced CM cell cycle entry and STAT6/STAT3 signaling in vitro. We demonstrate that STAT3/periostin and STAT6 signaling are critical mediators of interleukin 13 signaling in CMs. These downstream signaling molecules are also modulated in the regenerating mouse heart. CONCLUSIONS: Our work reveals new insights into the transcriptional regulation of mammalian cardiac regeneration and provides the founding circuitry for identifying potential regulators for stimulating heart regeneration.

A systematic analysis of neonatal mouse heart regeneration after apical resection.

The finding that neonatal mice are able to regenerate myocardium after apical resection has recently been questioned. We determined if heart regeneration is influenced by the size of cardiac resection and whether surgical retraction of the ventricular apex results in an increase in cardiomyocyte cell cycle activity. We performed moderate or large apical ventricular resections on neonatal mice and quantified scar infiltration into the left ventricular wall at 21 days post-surgery. Moderately resected hearts had 15±2% of the wall infiltrated by a collagen scar; significantly greater scar infiltration (23±4%) was observed in hearts with large resections. Resected hearts had higher levels of cardiomyocyte cell cycle activity relative to sham hearts. Surgically retracting the ventricle often resulted in fibrosis and induced cardiomyocyte cell cycle activity that were comparable to that of resected hearts. We conclude that apical resection in neonatal mice induces cardiomyocyte cell cycle activity and neomyogenesis, although scarring can occur. Surgical technique and definition of approach to assessing the extent of regeneration are both critical when using the neonatal mouse apical resection model.

An engineered bivalent neuregulin protects against doxorubicin-induced cardiotoxicity with reduced proneoplastic potential.

BACKGROUND: Doxorubicin (DOXO) is an effective anthracycline chemotherapeutic, but its use is limited by cumulative dose-dependent cardiotoxicity. Neuregulin-1ß is an ErbB receptor family ligand that is effective against DOXO-induced cardiomyopathy in experimental models but is also proneoplastic. We previously showed that an engineered bivalent neuregulin-1ß (NN) has reduced proneoplastic potential in comparison with the epidermal growth factor-like domain of neuregulin-1ß (NRG), an effect mediated by receptor biasing toward ErbB3 homotypic interactions uncommonly formed by native neuregulin-1ß. Here, we hypothesized that a newly formulated, covalent NN would be cardioprotective with reduced proneoplastic effects in comparison with NRG. METHODS AND RESULTS: NN was expressed as a maltose-binding protein fusion in Escherichia coli. As established previously, NN stimulated antineoplastic or cytostatic signaling and phenotype in cancer cells, whereas NRG stimulated proneoplastic signaling and phenotype. In neonatal rat cardiomyocytes, NN and NRG induced similar downstream signaling. NN, like NRG, attenuated the double-stranded DNA breaks associated with DOXO exposure in neonatal rat cardiomyocytes and human cardiomyocytes derived from induced pluripotent stem cells. NN treatment significantly attenuated DOXO-induced decrease in fractional shortening as measured by blinded echocardiography in mice in a chronic cardiomyopathy model (57.7±0.6% versus 50.9±2.6%, P=0.004), whereas native NRG had no significant effect (49.4±3.7% versus 50.9±2.6%, P=0.813). CONCLUSIONS: NN is a cardioprotective agent that promotes cardiomyocyte survival and improves cardiac function in DOXO-induced cardiotoxicity. Given the reduced proneoplastic potential of NN versus NRG, NN has translational potential for cardioprotection in patients with cancer receiving anthracyclines.

Evidence from a genetic fate-mapping study that stem cells refresh adult mammalian cardiomyocytes after injury.

An emerging concept is that the mammalian myocardium has the potential to regenerate, but that regeneration might be too inefficient to repair the extensive myocardial injury that is typical of human disease. However, the degree to which stem cells or precursor cells contribute to the renewal of adult mammalian cardiomyocytes remains controversial. Here we report evidence that stem cells or precursor cells contribute to the replacement of adult mammalian cardiomyocytes after injury but do not contribute significantly to cardiomyocyte renewal during normal aging. We generated double-transgenic mice to track the fate of adult cardiomyocytes in a 'pulse-chase' fashion: after a 4-OH-tamoxifen pulse, green fluorescent protein (GFP) expression was induced only in cardiomyocytes, with 82.7% of cardiomyocytes expressing GFP. During normal aging up to one year, the percentage of GFP+ cardiomyocytes remained unchanged, indicating that stem or precursor cells did not refresh uninjured cardiomyocytes at a significant rate during this period of time. By contrast, after myocardial infarction or pressure overload, the percentage of GFP+ cardiomyocytes decreased from 82.8% in heart tissue from sham-treated mice to 67.5% in areas bordering a myocardial infarction, 76.6% in areas away from a myocardial infarction, and 75.7% in hearts subjected to pressure overload, indicating that stem cells or precursor cells had refreshed the cardiomyocytes.

Identification of targeting peptides for ischemic myocardium by in vivo phage display.

Therapies selectively targeting ischemic myocardium could be applied by intravenous injection. Here, we report an approach for ischemic tissue-selective targeting based on in vivo screening of random peptide sequences using phage display. We performed in vivo biopanning using a phage library in a rat model of ischemia-reperfusion and identified three peptide motifs, CSTSMLKAC, CKPGTSSYC, and CPDRSVNNC, that exhibited preferential binding to ischemic heart tissue compared to normal heart as well as other control organs. The CSTSMLKAC sequence was capable of mediating selective homing of phage to ischemic heart tissue. The CSTSMLKAC peptide was then made as a fusion protein with Sumo-mCherry and injected intravenously in a mouse model of myocardial ischemia-reperfusion injury; subsequently, bio-distribution of Sumo-mCherry-CSTSMLKAC was measured with quantitative ELISA. The targeting peptide led to a significant increase in homing to ischemic left ventricle compared to tissues from non-ischemic left ventricle, the right ventricle, lung, liver, spleen, skeletal muscle, and brain (all p<0.001). These results indicate that the peptide sequence CSTSMLKAC represents a novel molecular tool that may be useful in targeting ischemic tissue and delivering bioengineered proteins into the injured myocardium by systemic intravenous administration.

Attenuated hypertrophic response to pressure overload in a lamin A/C haploinsufficiency mouse.

Inherited mutations cause approximately 30% of all dilated cardiomyopathy cases, with autosomal dominant mutations in the LMNA gene accounting for more than one third of these. The LMNA gene encodes the nuclear envelope proteins lamins A and C, which provide structural support to the nucleus and also play critical roles in transcriptional regulation. Functional deletion of a single allele is sufficient to trigger dilated cardiomyopathy in humans and mice. However, whereas Lmna(-/-) mice develop severe muscular dystrophy and dilated cardiomyopathy and die by 8 weeks of age, heterozygous Lmna(+/-) mice have a much milder phenotype, with changes in ventricular function and morphology only becoming apparent at 1 year of age. Here, we studied 8- to 20-week-old Lmna(+/-) mice and wild-type littermates in a pressure overload model to examine whether increased mechanical load can accelerate or exacerbate myocardial dysfunction in the heterozygotes. While overall survival was similar between genotypes, Lmna(+/-) animals had a significantly attenuated hypertrophic response to pressure overload as evidenced by reduced ventricular mass and myocyte size. Analysis of pressure overload-induced transcriptional changes suggested that the reduced hypertrophy in the Lmna(+/-) mice was accompanied by impaired activation of the mechanosensitive gene Egr-1. In conclusion, our findings provide further support for a critical role of lamins A and C in regulating the cellular response to mechanical stress in cardiomyocytes and demonstrate that haploinsufficiency of lamins A and C alone is sufficient to alter hypertrophic responses and cardiac function in the face of pressure overload in the heart.

Controlled delivery of PDGF-BB for myocardial protection using injectable self-assembling peptide nanofibers.

Endothelial cells can protect cardiomyocytes from injury, but the mechanism of this protection is incompletely described. Here we demonstrate that protection of cardiomyocytes by endothelial cells occurs through PDGF-BB signaling. PDGF-BB induced cardiomyocyte Akt phosphorylation in a time- and dose-dependent manner and prevented apoptosis via PI3K/Akt signaling. Using injectable self-assembling peptide nanofibers, which bound PDGF-BB in vitro, sustained delivery of PDGF-BB to the myocardium at the injected sites for 14 days was achieved. A blinded and randomized study in 96 rats showed that injecting nanofibers with PDGF-BB, but not nanofibers or PDGF-BB alone, decreased cardiomyocyte death and preserved systolic function after myocardial infarction. A separate blinded and randomized study in 52 rats showed that PDGF-BB delivered with nanofibers decreased infarct size after ischemia/reperfusion. PDGF-BB with nanofibers induced PDGFR-beta and Akt phosphorylation in cardiomyocytes in vivo. These data demonstrate that endothelial cells protect cardiomyocytes via PDGF-BB signaling and that this in vitro finding can be translated into an effective in vivo method of protecting myocardium after infarction. Furthermore, this study shows that injectable nanofibers allow precise and sustained delivery of proteins to the myocardium with potential therapeutic benefits.

Targeted deletion of thioredoxin-interacting protein regulates cardiac dysfunction in response to pressure overload.

Biomechanical overload induces cardiac hypertrophy and heart failure, and reactive oxygen species (ROS) play a role in both processes. Thioredoxin-Interacting Protein (Txnip) is encoded by a mechanically-regulated gene that controls cell growth and apoptosis in part through interaction with the endogenous dithiol antioxidant thioredoxin. Here we show that Txnip is a critical regulator of the cardiac response to pressure overload. We generated inducible cardiomyocyte-specific and systemic Txnip-null mice (Txnip-KO) using Flp/frt and Cre/loxP technologies. Compared with littermate controls, Txnip-KO hearts had attenuated cardiac hypertrophy and preserved left ventricular (LV) contractile reserve through 4 weeks of pressure overload; however, the beneficial effects were not sustained and Txnip deletion ultimately led to maladaptive LV remodeling at 8 weeks of pressure overload. Interestingly, these effects of Txnip deletion on cardiac performance were not accompanied by global changes in thioredoxin activity or ROS; instead, Txnip-KO hearts had a robust increase in myocardial glucose uptake. Thus, deletion of Txnip plays an unanticipated role in myocardial energy homeostasis rather than redox regulation. These results support the emerging concept that the function of Txnip is not as a simple thioredoxin inhibitor but as a metabolic control protein.

Local delivery of protease-resistant stromal cell derived factor-1 for stem cell recruitment after myocardial infarction.

BACKGROUND: Local delivery of chemotactic factors represents a novel approach to tissue regeneration. However, successful chemokine protein delivery is challenged by barriers including the rapid diffusion of chemokines and cleavage of chemokines by proteases that are activated in injured tissues. Stromal cell-derived factor-1 (SDF-1) is a well-characterized chemokine for attracting stem cells and thus a strong candidate for promoting regeneration. However, SDF-1 is cleaved by exopeptidases and matrix metalloproteinase-2, generating a neurotoxin implicated in some forms of dementia. METHODS AND RESULTS: We designed a new chemokine called S-SDF-1(S4V) that is resistant to matrix metalloproteinase-2 and exopeptidase cleavage but retains chemotactic bioactivity, reducing the neurotoxic potential of native SDF-1. To deliver S-SDF-1(S4V), we expressed and purified fusion proteins to tether the chemokine to self-assembling peptides, which form nanofibers and allow local delivery. Intramyocardial delivery of S-SDF-1(S4V) after myocardial infarction recruited CXCR4+/c-Kit+ stem cells (46+/-7 to 119+/-18 cells per section) and increased capillary density (from 169+/-42 to 283+/-27 per 1 mm2). Furthermore, in a randomized, blinded study of 176 rats with myocardial infarction, nanofiber delivery of the protease-resistant S-SDF-1(S4V) improved cardiac function (ejection fraction increased from 34.0+/-2.5% to 50.7+/-3.1%), whereas native SDF-1 had no beneficial effects. CONCLUSIONS: The combined advances of a new, protease-resistant SDF-1 and nanofiber-mediated delivery promoted recruitment of stem cells and improved cardiac function after myocardial infarction. These data demonstrate that driving chemotaxis of stem cells by local chemokine delivery is a promising new strategy for tissue regeneration.

Local controlled intramyocardial delivery of platelet-derived growth factor improves postinfarction ventricular function without pulmonary toxicity.

BACKGROUND: Local delivery methods can target therapies to specific tissues and potentially avoid toxicity to other organs. Platelet-derived growth factor can protect the myocardium, but it also plays an important role in promoting pulmonary hypertension. It is not known whether local myocardial delivery of platelet-derived growth factor during myocardial infarction (MI) can lead to sustained cardiac benefit without causing pulmonary hypertension. METHODS AND RESULTS: We performed a randomized and blinded experiment of 127 rats that survived experimental MI or sham surgery. We delivered platelet-derived growth factor (PDGF)-BB with self-assembling peptide nanofibers (NFs) to provide controlled release within the myocardium. There were 6 groups with n > or = 20 in each group: sham, sham+NF, sham+NF/PDGF, MI, MI+NF, and MI+NF/PDGF. Serial echocardiography from 1 day to 3 months showed significant improvement of ventricular fractional shortening, end-systolic dimension, and end-diastolic dimension with local PDGF delivery (P < 0.05 for MI+NF/PDGF versus MI or MI+NF). Catheterization at 4 months revealed improved ventricular function in the controlled delivery group (left ventricular end-diastolic pressure, cardiac index, +dP/dt, -dP/dt, and time constant of exponential decay all P < 0.05 for MI+NF/P versus MI or MI+NF). Infarcted myocardial volume was reduced by NF/PDGF therapy (34.0 +/- 13.3% in MI, 28.9 +/- 12.9% in MI+NF, and 12.0 +/- 5.8% in MI+NF/PDGF; P < 0.001). There was no evidence of pulmonary toxicity from the therapy, with no differences in right ventricular end-systolic pressure, right ventricular dP/dt, bromodeoxyuridine staining, or pulmonary artery medial wall thickness. CONCLUSIONS: Intramyocardial delivery of PDGF by self-assembling peptide NFs leads to long-term improvement in cardiac performance after experimental infarction without apparent pulmonary toxicity. Local myocardial protection may allow prevention of heart failure without systemic toxicity.

Effect of a cleavage-resistant collagen mutation on left ventricular remodeling.

Matrix metalloproteinase-mediated degradation of type I collagen may play a role in cardiac remodeling after strain or injury. To explore this hypothesis, we used mice homozygous (r/r) for a targeted mutation in Col1a1; these mice synthesize collagen I that resists collagenase cleavage at Gly975-Leu976. A total of 64 r/r and 84 littermate wild-type mice (WT) underwent experimental pressure overload by transverse aortic constriction (TAC) or myocardial infarction (MI). Echocardiographic, hemodynamic, and histological parameters were evaluated up to 12 weeks after TAC or 21 days after MI. At 4 weeks after TAC, collagen levels, wall thickness, and echocardiographic parameters were similar in the 2 groups. At 12 weeks after TAC, r/r mice had smaller LV dimensions (ESD: 2.7+/-0.2 mm WT versus 1.7+/-0.2 mm r/r, P<0.013; EDD: 3.8+/-0.2 mm WT versus 3.1+/-0.1 mm r/r, P<0.013); better fractional shortening (30+/-2% WT versus 46+/-4% r/r; P<0.013); and lower LV/body weight ratios (7.3+/-0.6 WT and 5.1+/-0.5 r/r; P<0.013). Surprisingly, these differences were not accompanied by differences in collagen accumulation, myocyte cross-sectional areas, wall thickness, or microvessel densities. Furthermore, no differences in LV remodeling assessed by echocardiography, fibrosis, or hemodynamic parameters were found between r/r and WT mice after MI. Thus, a mutation that encodes a collagenase cleavage-resistant collagen I does not affect early LV remodeling after TAC or MI, suggesting that collagen cleavage at this site is not the mechanism by which metalloproteinases mediate LV remodeling. Collagen cleavage could, however, have a role in preservation of cardiac function in late remodeling by mechanisms independent of collagen accumulation. We were not able to detect collagen cleavage fragments, and could not, therefore, rule out the possibility of collagen cleavage at additional sites.

Thioredoxin-interacting protein controls cardiac hypertrophy through regulation of thioredoxin activity.

BACKGROUND: Although cellular redox balance plays an important role in mechanically induced cardiac hypertrophy, the mechanisms of regulation are incompletely defined. Because thioredoxin is a major intracellular antioxidant and can also regulate redox-dependent transcription, we explored the role of thioredoxin activity in mechanically overloaded cardiomyocytes in vitro and in vivo. METHODS AND RESULTS: Overexpression of thioredoxin induced protein synthesis in cardiomyocytes (127+/-5% of controls, P<0.01). Overexpression of thioredoxin-interacting protein (Txnip), an endogenous thioredoxin inhibitor, reduced protein synthesis in response to mechanical strain (89+/-5% reduction, P<0.01), phenylephrine (80+/-3% reduction, P<0.01), or angiotensin II (80+/-4% reduction, P<0.01). In vivo, myocardial thioredoxin activity increased 3.5-fold compared with sham controls after transverse aortic constriction (P<0.01). Aortic constriction did not change thioredoxin expression but reduced Txnip expression by 40% (P<0.05). Gene transfer studies showed that cells that overexpress Txnip develop less hypertrophy after aortic constriction than control cells in the same animals (28.1+/-5.2% reduction versus noninfected cells, P<0.01). CONCLUSIONS: Thus, even though thioredoxin is an antioxidant, activation of thioredoxin participates in the development of pressure-overload cardiac hypertrophy, demonstrating the dual function of thioredoxin as both an antioxidant and a signaling protein. These results also support the emerging concept that the thioredoxin inhibitor Txnip is a critical regulator of biomechanical signaling.

Consequences of pressure overload on sarcomere protein mutation-induced hypertrophic cardiomyopathy.

BACKGROUND: Whether ventricular remodeling from hypertrophic cardiomyopathy (HCM), systemic hypertension, or other pathologies arises through a common signaling pathway or through independent molecular mechanisms is unknown. To study this, we assessed cardiac hypertrophy in a mouse model of HCM subjected to increased left ventricular (LV) load. METHODS AND RESULTS: Transverse aortic banding of mice with or without an Arg403Gln cardiac myosin heavy chain mutation (alphaMHC403/+) produced similarly elevated LV pressures (120+/-30 versus 112+/-14 mm Hg; P=NS). No mice developed heart failure, and mortality (26% alphaMHC403/+, 35% wild-type) was comparable. Load-induced hypertrophy was identical in banded 129SvEv alphaMHC403/+ mice (LV anterior wall [LVAW]=1.28+/-0.11) and 129SvEv wild-type mice (LVAW=1.29+/-0.11 mm; P=NS). Genetically outbred Black Swiss (BS) alphaMHC403/+ mice showed only mildly exaggerated hypertrophy in response to aortic banding (BS alphaMHC403/+ LVAW=1.30+/-0.13 mm; BS wild-type LVAW=1.17+/-0.15 mm; P=0.03), suggesting some effect from a BS genetic locus that modifies hypertrophy induced by the cardiac MHC Arg403Gln mutation. Histopathology and molecular markers of hypertrophy were comparable in all banded 129SvEv or BS mice. Banded alphaMHC403/+ mice had potential for greater hypertrophy, because cyclosporin A treatment markedly augmented hypertrophy. CONCLUSIONS: The uniform hypertrophic response to increased ventricular load in wild-type and alphaMHC403/+ mice indicates independent cardiac remodeling pathways and predicts that coexistent hypertension and HCM should not profoundly exacerbate cardiac hypertrophy. In contrast, sarcomere mutation and cyclosporin A-mediated calcineurin inhibition stimulate a shared hypertrophic signaling pathway. Defining distinct signaling pathways that trigger myocyte growth should help to tailor therapies for cardiac hypertrophy.

Selective matrix metalloproteinase inhibition reduces left ventricular remodeling but does not inhibit angiogenesis after myocardial infarction.

BACKGROUND: Broad inhibition of matrix metalloproteinases (MMPs) attenuates left ventricular remodeling after myocardial infarction (MI). However, it is not clear if selective MMP inhibition strategies will be effective or if MMP inhibition will impair angiogenesis after MI. METHODS AND RESULTS: We used a selective MMP inhibitor (MMPi) that does not inhibit MMP-1 in rabbits, which, like humans but unlike rodents, express MMP-1 as a major collagenase. On day 1 after MI, rabbits were randomized to receive either inhibitor (n=10) or vehicle (n=8). At 4 weeks after MI, there were no differences in infarct size or collagen fractional area. However, MMPi reduced ventricular dilation. The increase in end-diastolic dimension from day 1 to week 4 was 3.1+/-0.5 mm for vehicle versus 1.3+/-0.3 mm for MMPi (P<0.01). The increase in end-systolic dimension was 2.8+/-0.5 mm for vehicle and 1.3+/-0.4 mm for MMPi (P<0.05). Furthermore, MMPi reduced infarct wall thinning; the minimal infarct thickness was 0.8+/-0.1 mm for vehicle and 1.6+/-0.3 mm for MMPi (P<0.05). Interestingly, the MMPi group had increased numbers of vessels in the subendocardial layer of the infarct; the number of capillaries was increased in the subendocardial layer (46+/-4 vessels/field versus 17+/-3 vessels/field for vehicle; P<0.001), and the number of arterioles was also increased (4.0+/-0.8 vessels/field versus 2.0+/-0.4 vessels/field for vehicle; P<0.05). CONCLUSIONS: MMP inhibition attenuates left ventricular remodeling even when the dominant collagenase MMP-1 is not inhibited; furthermore, this selective MMP inhibition appears to increase rather than decrease neovascularization in the subendocardium.

Nerves Regulate Cardiomyocyte Proliferation and Heart Regeneration.

Some organisms, such as adult zebrafish and newborn mice, have the capacity to regenerate heart tissue following injury. Unraveling the mechanisms of heart regeneration is fundamental to understanding why regeneration fails in adult humans. Numerous studies have revealed that nerves are crucial for organ regeneration, thus we aimed to determine whether nerves guide heart regeneration. Here, we show using transgenic zebrafish that inhibition of cardiac innervation leads to reduction of myocyte proliferation following injury. Specifically, pharmacological inhibition of cholinergic nerve function reduces cardiomyocyte proliferation in the injured hearts of both zebrafish and neonatal mice. Direct mechanical denervation impairs heart regeneration in neonatal mice, which was rescued by the administration of neuregulin 1 (NRG1) and nerve growth factor (NGF) recombinant proteins. Transcriptional analysis of mechanically denervated hearts revealed a blunted inflammatory and immune response following injury. These findings demonstrate that nerve function is required for both zebrafish and mouse heart regeneration.

Sex dependence and temporal dependence of the left ventricular genomic response to pressure overload.

To characterize responses of the left ventricle (LV) to pressure overload at the genomic level, we performed high-density microarray analysis on individual mouse LVs. Male and female mice underwent transverse aortic constriction. At 1 day and 30 wk, the LV free wall was harvested and RNA isolated from 27 individual ventricles was analyzed on Mu74Av2 GeneChips, which contain approximately 12,483 distinct genes. Interestingly, a greater number of genes was regulated in response to acute overload than in response to chronic overload. Hierarchical cluster analysis revealed the presence of several distinct expression profiles. Of these clusters, the majority contained genes that were regulated either in response to acute overload or both acute and chronic overload. In addition, clusters revealing sex-specific responses to overload were detected. In summary, the acute and chronic genomic responses to pressure overload are distinct. Moreover, sex modifies these responses. Furthermore, these studies have uncovered several novel and potentially important genes that are regulated in response to overload and may open unrecognized avenues for further functional analysis.

Bone marrow-derived cell therapy stimulates endogenous cardiomyocyte progenitors and promotes cardiac repair.

Cell therapy can improve cardiac function in animals and humans after injury, but the mechanism is unclear. We performed cell therapy experiments in genetically engineered mice that permanently express green fluorescent protein (GFP) only in cardiomyocytes after a pulse of 4-OH-tamoxifen. Myocardial infarction diluted the GFP(+) cardiomyocyte pool, indicating refreshment by non-GFP(+) progenitors. Cell therapy with bone marrow-derived c-kit(+) cells, but not mesenchymal stem cells, further diluted the GFP(+) pool, consistent with c-kit(+) cell-mediated augmentation of cardiomyocyte progenitor activity. This effect could not be explained by transdifferentiation to cardiomyocytes by exogenously delivered c-kit(+) cells or by cell fusion. Therapy with c-kit(+) cells but not mesenchymal stem cells improved cardiac function. These findings suggest that stimulation of endogenous cardiogenic progenitor activity is a critical mechanism of cardiac cell therapy.