Integrated proteomics reveals alterations in sarcomere composition and developmental processes during postnatal swine heart development.

The neonatal swine heart possesses an endogenous ability to regenerate injured myocardium through the proliferation of pre-existing cardiomyocyte (CM) populations. However, this regenerative capacity is lost shortly after birth. Normal postnatal developmental processes and the regenerative capacity of mammalian hearts are tightly linked, but not much is known about how the swine cardiac proteome changes throughout postnatal development. Herein, we integrated robust and quantitative targeted "top-down" and global "bottom-up" proteomic workflows to comprehensively define the dynamic landscape of the swine cardiac proteome throughout postnatal maturation. Using targeted top-down proteomics, we were able to identify significant alterations in sarcomere composition, providing new insight into the proteoform landscape of sarcomeres that can disassemble, a process necessary for productive CM proliferation. Furthermore, we quantified global changes in protein abundance using bottom-up proteomics, identified over 700 differentially expressed proteins throughout postnatal development, and mapped these proteins to changes in developmental and metabolic processes. We envision these results will help guide future investigations to comprehensively understand endogenous cardiac regeneration toward the development of novel therapeutic strategies for heart failure.

Turning back the clock: A concise viewpoint of cardiomyocyte cell cycle activation for myocardial regeneration and repair.

Patients with acute myocardial infarction (MI) could progress to end-stage congestive heart failure, which is one of the most significant problems in public health. From the molecular and cellular perspective, heart failure often results from the loss of cardiomyocytes-the fundamental contractile unit of the heart-and the damage caused by myocardial injury in adult mammals cannot be repaired, in part because mammalian cardiomyocytes undergo cell-cycle arrest during the early perinatal period. However, recent studies in the hearts of neonatal small and large mammals suggest that the onset of cardiomyocyte cell-cycle arrest can be reversed, which may lead to the development of entirely new strategies for the treatment of heart failure. In this Viewpoint, we summarize these and other provocative findings about the cellular and molecular mechanisms that regulate cardiomyocyte proliferation and how they may be targeted to turn back the clock of cardiomyocyte cell-cycle arrest and improve recovery from cardiac injury and disease.

Cyclin D2 Overexpression Enhances the Efficacy of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Myocardial Repair in a Swine Model of Myocardial Infarction.

BACKGROUND: Human induced pluripotent stem cells with normal (wild-type) or upregulated (overexpressed) levels of CCND2 (cyclin D2) expression were differentiated into cardiomyocytes (CCND2WTCMs or CCND2OECMs, respectively) and injected into infarcted pig hearts. METHODS: Acute myocardial infarction was induced by a 60-minute occlusion of the left anterior descending coronary artery. Immediately after reperfusion, CCND2WTCMs or CCND2OECMs (3×107 cells each) or an equivalent volume of the delivery vehicle was injected around the infarct border zone area. RESULTS: The number of the engrafted CCND2OECMs exceeded that of the engrafted CCND2WTCMs from 6- to 8-fold, rising from 1 week to 4 weeks after implantation. In contrast to the treatment with the CCND2WTCMs or the delivery vehicle, the administration of CCND2OECM was associated with significantly improved left ventricular function, as revealed by magnetic resonance imaging. This correlated with reduction of infarct size, fibrosis, ventricular hypertrophy, and cardiomyocyte apoptosis, and increase of vascular density and arterial density, as per histologic analysis of the treated hearts. Expression of cell proliferation markers (eg, Ki67, phosphorylated histone 3, and Aurora B kinase) was also significantly upregulated in the recipient cardiomyocytes from the CCND2OECM-treated than from the CCND2WTCM-treated pigs. The cell proliferation rate and the hypoxia tolerance measured in cultured human induced pluripotent stem cell cardiomyocytes were significantly greater after treatment with exosomes isolated from the CCND2OECMs (CCND2OEExos) than from the CCND2WTCMs (CCND2WTExos). As demonstrated by our study, CCND2OEExos can also promote the proliferation activity of postnatal rat and adult mouse cardiomyocytes. A bulk miRNA sequencing analysis of CCND2OEExos versus CCND2WTExos identified 206 and 91 miRNAs that were significantly upregulated and downregulated, respectively. Gene ontology enrichment analysis identified significant differences in the expression profiles of miRNAs from various functional categories and pathways, including miRNAs implicated in cell-cycle checkpoints (G2/M and G1/S transitions), or the mechanism of cytokinesis. CONCLUSIONS: We demonstrated that enhanced potency of CCND2OECMs promoted myocyte proliferation in both grafts and recipient tissue in a large mammal acute myocardial infarction model. These results suggest that CCND2OECMs transplantation may be a potential therapeutic strategy for the repair of infarcted hearts.

Targeting exosome-associated human antigen R attenuates fibrosis and inflammation in diabetic heart.

RNA-binding proteins like human antigen R (HuR) are key regulators in post-transcriptional control of gene expression in several pathophysiological conditions. Diabetes adversely affects monocyte/macrophage biology and function. It is not known whether diabetic milieu affects cellular/exosome-HuR and its implications on cardiac inflammation and fibrosis. Here, we evaluate in vitro and in vivo effects of diabetic milieu on macrophage cellular/exosome-HuR, alterations in intercellular cross talk with fibroblasts, and its impact on cardiac remodeling. Human failing hearts show higher HuR levels. Diabetic milieu activates HuR expression in cardiac- and cultured bone marrow-derived macrophages (BMMØ) and stimulates HuR nuclear-to-cytoplasmic translocation and exosome transfer. Exosomes from macrophages exposed to diabetic milieu (high glucose or db/db mice) significantly increase inflammatory and profibrogenic responses in fibroblast (in vitro) and cardiac fibrosis in mice. Intriguingly, Exo-HuR deficiency (HuR knockdown in macrophage) abrogates the above effects. In diabetic mice, macrophage depletion followed by reconstitution with BMMØ-derived HuR-deficient exosomes inhibits angiotensin II-induced cardiac fibrosis response and preserves left ventricle function as compared to control-exosome administration. To the best of our knowledge, this is the first study to demonstrate that diabetes activates BMMØ HuR expression and its transfer into exosome. The data suggest that HuR might be targeted to alleviate macrophage dysfunction and pathological fibrosis in diabetes.

Convergences of Life Sciences and Engineering in Understanding and Treating Heart Failure.

On March 1 and 2, 2018, the National Institutes of Health 2018 Progenitor Cell Translational Consortium, Cardiovascular Bioengineering Symposium, was held at the University of Alabama at Birmingham. Convergence of life sciences and engineering to advance the understanding and treatment of heart failure was the theme of the meeting. Over 150 attendees were present, and >40 scientists presented their latest work on engineering human functional myocardium for disease modeling, drug development, and heart failure research. The scientists, engineers, and physicians in the field of cardiovascular sciences met and discussed the most recent advances in their work and proposed future strategies for overcoming the major roadblocks of cardiovascular bioengineering and therapy. Particular emphasis was given for manipulation and using of stem/progenitor cells, biomaterials, and methods to provide molecular, chemical, and mechanical cues to cells to influence their identity and fate in vitro and in vivo. Collectively, these works are profoundly impacting and progressing toward deciphering the mechanisms and developing novel treatments for left ventricular dysfunction of failing hearts. Here, we present some important perspectives that emerged from this meeting.

CHIR99021 and fibroblast growth factor 1 enhance the regenerative potency of human cardiac muscle patch after myocardial infarction in mice.

BACKGROUND: We have shown that genetic overexpression of cell cycle proteins can increase the proliferation of transplanted cardiomyocytes derived from human induced-pluripotent stem cells (hiPSC-CMs) in animal models of myocardial infarction (MI). Here, we introduce a new, non-genetic approach to promote hiPSC-CM cell cycle activity and proliferation in transplanted human cardiomyocyte patches (hCMPs). METHODS: Mice were randomly distributed into 5 experimental groups (n = 10 per group). One group underwent Sham surgery, and the other 4 groups underwent MI induction surgery followed by treatment with hCMPs composed of hiPSC-CMs and nanoparticles that contained CHIR99021 and FGF1 (the NPCF-hCMP group), with hCMPs composed of hiPSC-CMs and empty nanoparticles (the NPE-hCMP group); with patches containing the CHIR99021/FGF-loaded nanoparticles but lacking hiPSC-CMs (the NPCF-Patch group), or patches lacking both the nanoparticles and cells (the E-Patch group). Cell cycle activity was evaluated via Ki67 and Aurora B expression, bromodeoxyuridine incorporation, and phosphorylated histone 3 levels (immunofluorescence); engraftment via human cardiac troponin T or human nuclear antigen expression (immunofluorescence) and bioluminescence imaging; cardiac function via echocardiography; infarct size and wall thickness via histology; angiogenesis via isolectin B4 expression (immunofluorescence); and apoptosis via TUNEL and caspace 3 expression (immunofluorescence). RESULTS: Combined CHIR99021- and FGF1-treatment significantly increased hiPSC-CM cell cycle activity both in cultured cells (by 4- to 6-fold) and in transplanted hCMPs, and compared to treatment with NPE-hCMPs, NPCF-hCMP transplantation increased hiPSC-CM engraftment by ~4-fold and was associated with significantly better measurements of cardiac function, infarct size, wall thickness, angiogenesis, and hiPSC-CM apoptosis four weeks after MI induction. CONCLUSIONS: Nanoparticle-mediated CHIR99021 and FGF1 delivery promotes hiPSC-CM cell cycle activity and proliferation, as well as the engraftment and regenerative potency of transplanted hCMPs, in a mouse MI model.

Fluorescent indicators for continuous and lineage-specific reporting of cell-cycle phases in human pluripotent stem cells.

Proper cell-cycle progression is essential for the self-renewal and differentiation of human pluripotent stem cells (hPSCs). The fluorescent ubiquitination-based cell-cycle indicator (FUCCI) has allowed the dual-color visualization of the G1 and S/G2 /M phases in various dynamic models, but its application in hPSCs is not widely reported. In addition, lineage-specific FUCCI reporters have not yet been developed to analyze complex tissue-specific cell-cycle progression during hPSC differentiation. Desiring a robust tool for spatiotemporal reporting of cell-cycle events in hPSCs, we employed the CRISPR/Cas9 genome editing tool and successfully knocked the FUCCI reporter into the AAVS1 safe harbor locus of hPSCs for stable and constitutive FUCCI expression, exhibiting reliable cell-cycle-dependent fluorescence in both hPSCs and their differentiated progeny. We also established a cardiac-specific TNNT2-FUCCI reporter for lineage-specific cell-cycle monitoring of cardiomyocyte differentiation from hPSCs. This powerful and modular FUCCI system should provide numerous opportunities for studying human cell-cycle activity, and enable the identification and investigation of novel regulators for adult tissue regeneration.

Can We Engineer a Human Cardiac Patch for Therapy?

Some of the most significant leaps in the history of modern civilization-the development of article in China, the steam engine, which led to the European industrial revolution, and the era of computers-have occurred when science converged with engineering. Recently, the convergence of human pluripotent stem cell technology with biomaterials and bioengineering have launched a new medical innovation: functional human engineered tissue, which promises to revolutionize the treatment of failing organs including most critically, the heart. This compendium covers recent, state-of-the-art developments in the fields of cardiovascular tissue engineering, as well as the needs and challenges associated with the clinical use of these technologies. We have not attempted to provide an exhaustive review in stem cell biology and cardiac cell therapy; many other important and influential reports are certainly merit but already been discussed in several recent reviews. Our scope is limited to the engineered tissues that have been fabricated to repair or replace components of the heart (eg, valves, vessels, contractile tissue) that have been functionally compromised by diseases or developmental abnormalities. In particular, we have focused on using an engineered myocardial tissue to mitigate deficiencies in contractile function.

CCND2 Overexpression Enhances the Regenerative Potency of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Remuscularization of Injured Ventricle.

RATIONALE: The effectiveness of transplanted, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for treatment of ischemic myocardial injury is limited by the exceptionally low engraftment rate. OBJECTIVE: To determine whether overexpression of the cell cycle activator CCND2 (cyclin D2) in hiPSC-CMs can increase the graft size and improve myocardial recovery in a mouse model of myocardial infarction by increasing the proliferation of grafted cells. METHODS AND RESULTS: Human CCND2 was delivered to hiPSCs via lentiviral-mediated gene transfection. In cultured cells, markers for cell cycle activation and proliferation were ≈3- to 7-folds higher in CCND2-overexpressing hiPSC-CMs (hiPSC-CCND2OECMs) than in hiPSC-CMs with normal levels of CCND2 (hiPSC-CCND2WTCMs; P<0.01). The pluripotent genes (Oct 4, Sox2, and Nanog) decrease to minimal levels and undetectable levels at day 1 and 10 after differentiating to CMs. In the mouse myocardial infarction model, cardiac function, infarct size, and the number of engrafted cells were similar at week 1 after treatment with hiPSC-CCND2OECMs or hiPSC-CCND2WTCMs but was about tripled in hiPSC-CCND2OECM-treated than in hiPSC-CCND2WTCM-treated animals at week 4 (P<0.01). The cardiac function and infarct size were significantly better in both cell treatment groups' hearts than in control hearts, which was most prominent in hiPSC-CCND2OECM-treated animals (P<0.05, each). No tumor formation was observed in any hearts. CONCLUSIONS: CCND2 overexpression activates cell cycle progression in hiPSC-CMs that results in a significant enhanced potency for myocardial repair as evidenced by remuscularization of injured myocardium. This left ventricular muscle regeneration and increased angiogenesis in border zone are accompanied by a significant improvement of left ventricular chamber function.

Cardiomyocytes from CCND2-overexpressing human induced-pluripotent stem cells repopulate the myocardial scar in mice: A 6-month study.

BACKGROUND: Cardiomyocytes that have been differentiated from CCND2-overexpressing human induced-pluripotent stem cells (hiPSC-CCND2OE CMs) can proliferate when transplanted into mouse hearts after myocardial infarction (MI). However, it is unknown whether remuscularization can replace the thin LV scar and if the large muscle graft can electrophysiologically synchronize to the recipient myocardium. Our objectives are to evaluate the structural and functional potential of hiPSC-CCND2OE CMs in replacing the LV thin scar. METHODS: NOD/SCID mice were treated with hiPSC-CCND2OE CMs (i.e., the CCND2OE group), hiPSC-CCND2WT CMs (the CCND2WT group), or an equal volume of PBS immediately after experimentally-induced myocardial infarction. The treatments were administered to one site in the infarcted zone (IZ), two sites in the border zone (BZ), and a fourth group of animals underwent Sham surgery. RESULTS: Six months later, engrafted cells occupied >50% of the scarred region in CCND2OE animals, and exceeded the number of engrafted cells in CCND2WT animals by ~8-fold. Engrafted cells were also more common in the IZ than in the BZ for both cell-treatment groups. Measurements of cardiac function, infarct size, wall thickness, and cardiomyocyte hypertrophy were significantly improved in CCND2OE animals compared to animals from the CCND2WT or PBS-treatment groups. Measurements in the CCND2WT and PBS groups were similar, and markers for cell cycle activation and proliferation were significantly higher in hiPSC-CCND2OE CMs than in hiPSC-CCND2WT CMs. Optical mapping of action potential propagation indicated that the engrafted hiPSC-CCND2OE CMs were electrically coupled to each other and to the cells of the native myocardium. No evidence of tumor formation was observed in any animals. CONCLUSIONS: Six months after the transplantation, CCND2-overexpressing hiPSC-CMs proliferated and replaced >50% of the myocardial scar tissue. The large graft hiPSC-CCND2OE CMs also electrically integrated with the host myocardium, which was accompanied by a significant improvement in LV function.

Protein phosphatase 5 and the tumor suppressor p53 down-regulate each other's activities in mice.

Protein phosphatase 5 (PP5), a serine/threonine phosphatase, has a wide range of biological functions and exhibits elevated expression in tumor cells. We previously reported that pp5-deficient mice have altered ataxia-telangiectasia mutated (ATM)-mediated signaling and function. However, this regulation was likely indirect, as ATM is not a known PP5 substrate. In the current study, we found that pp5-deficient mice are hypersensitive to genotoxic stress. This hypersensitivity was associated with the marked up-regulation of the tumor suppressor tumor protein p53 and its downstream targets cyclin-dependent kinase inhibitor 1A (p21), MDM2 proto-oncogene (MDM2), and phosphatase and tensin homolog (PTEN) in pp5-deficient tissues and cells. These observations suggested that PP5 plays a role in regulating p53 stability and function. Experiments conducted with p53+/-pp5+/- or p53+/-pp5-/- mice revealed that complete loss of PP5 reduces tumorigenesis in the p53+/- mice. Biochemical analyses further revealed that PP5 directly interacts with and dephosphorylates p53 at multiple serine/threonine residues, resulting in inhibition of p53-mediated transcriptional activity. Interestingly, PP5 expression was significantly up-regulated in p53-deficient cells, and further analysis of pp5 promoter activity revealed that p53 strongly represses PP5 transcription. Our results suggest a reciprocal regulatory interplay between PP5 and p53, providing an important feedback mechanism for the cellular response to genotoxic stress.

Regenerative Potential of Neonatal Porcine Hearts.

BACKGROUND: Rodent hearts can regenerate myocardium lost to apical resection or myocardial infarction for up to 7 days after birth, but whether a similar window for myocardial regeneration also exists in large mammals is unknown. METHODS: Acute myocardial infarction (AMI) was surgically induced in neonatal pigs on postnatal days 1, 2, 3, 7, and 14 (ie, the P1, P2, P3, P7, and P14 groups, respectively). Cardiac systolic function was evaluated before AMI and at 30 days post-AMI via transthoracic echocardiography. Cardiomyocyte cell cycle activity was assessed via immunostaining for proliferation and mitosis markers, infarct size was evaluated histologically, and telomerase activity was measured by quantitative polymerase chain reaction. RESULTS: Systolic function at day 30 post-AMI was largely restored in P1 animals and partially restored in P2 animals, but significantly impaired when AMI was induced on postnatal day 3 or later. Hearts of P1 animals showed little evidence of scar formation or wall thinning on day 30 after AMI, with increased measures of cell-cycle activity seen 6 days after AMI (ie, postnatal day 7) compared with postnatal day 7 in noninfarcted hearts. CONCLUSIONS: The neonatal porcine heart is capable of regeneration after AMI during the first 2 days of life. This phenomenon is associated with induction of cardiomyocyte proliferation and is lost when cardiomyocytes exit the cell cycle shortly after birth.

Large Cardiac Muscle Patches Engineered From Human Induced-Pluripotent Stem Cell-Derived Cardiac Cells Improve Recovery From Myocardial Infarction in Swine.

BACKGROUND: Here, we generated human cardiac muscle patches (hCMPs) of clinically relevant dimensions (4 cm × 2 cm × 1.25 mm) by suspending cardiomyocytes, smooth muscle cells, and endothelial cells that had been differentiated from human induced-pluripotent stem cells in a fibrin scaffold and then culturing the construct on a dynamic (rocking) platform. METHODS: In vitro assessments of hCMPs suggest maturation in response to dynamic culture stimulation. In vivo assessments were conducted in a porcine model of myocardial infarction (MI). Animal groups included: MI hearts treated with 2 hCMPs (MI+hCMP, n=13), MI hearts treated with 2 cell-free open fibrin patches (n=14), or MI hearts with neither experimental patch (n=15); a fourth group of animals underwent sham surgery (Sham, n=8). Cardiac function and infarct size were evaluated by MRI, arrhythmia incidence by implanted loop recorders, and the engraftment rate by calculation of quantitative polymerase chain reaction measurements of expression of the human Y chromosome. Additional studies examined the myocardial protein expression profile changes and potential mechanisms of action that related to exosomes from the cell patch. RESULTS: The hCMPs began to beat synchronously within 1 day of fabrication, and after 7 days of dynamic culture stimulation, in vitro assessments indicated the mechanisms related to the improvements in electronic mechanical coupling, calcium-handling, and force generation, suggesting a maturation process during the dynamic culture. The engraftment rate was 10.9±1.8% at 4 weeks after the transplantation. The hCMP transplantation was associated with significant improvements in left ventricular function, infarct size, myocardial wall stress, myocardial hypertrophy, and reduced apoptosis in the periscar boarder zone myocardium. hCMP transplantation also reversed some MI-associated changes in sarcomeric regulatory protein phosphorylation. The exosomes released from the hCMP appeared to have cytoprotective properties that improved cardiomyocyte survival. CONCLUSIONS: We have fabricated a clinically relevant size of hCMP with trilineage cardiac cells derived from human induced-pluripotent stem cells. The hCMP matures in vitro during 7 days of dynamic culture. Transplantation of this type of hCMP results in significantly reduced infarct size and improvements in cardiac function that are associated with reduction in left ventricular wall stress. The hCMP treatment is not associated with significant changes in arrhythmogenicity.

Pathologic Stimulus Determines Lineage Commitment of Cardiac C-kit+ Cells.

BACKGROUND: Although cardiac c-kit+ cells are being tested in clinical trials, the circumstances that determine lineage differentiation of c-kit+ cells in vivo are unknown. Recent findings suggest that endogenous cardiac c-kit+ cells rarely contribute cardiomyocytes to the adult heart. We assessed whether various pathological stimuli differentially affect the eventual cell fates of c-kit+ cells. METHODS: We used single-cell sequencing and genetic lineage tracing of c-kit+ cells to determine whether various pathological stimuli would result in different fates of c-kit+ cells. RESULTS: Single-cell sequencing of cardiac CD45-c-kit+ cells showed innate heterogeneity, indicative of the existence of vascular and mesenchymal c-kit+ cells in normal hearts. Cardiac pressure overload resulted in a modest increase in c-kit-derived cardiomyocytes, with significant increases in the numbers of endothelial cells and fibroblasts. Doxorubicin-induced acute cardiotoxicity did not increase c-kit-derived endothelial cell fates but instead induced cardiomyocyte differentiation. Mechanistically, doxorubicin-induced DNA damage in c-kit+ cells resulted in expression of p53. Inhibition of p53 blocked cardiomyocyte differentiation in response to doxorubicin, whereas stabilization of p53 was sufficient to increase c-kit-derived cardiomyocyte differentiation. CONCLUSIONS: These results demonstrate that different pathological stimuli induce different cell fates of c-kit+ cells in vivo. Although the overall rate of cardiomyocyte formation from c-kit+ cells is still below clinically relevant levels, we show that p53 is central to the ability of c-kit+ cells to adopt cardiomyocyte fates, which could lead to the development of strategies to preferentially generate cardiomyocytes from c-kit+ cells.

VEGF nanoparticles repair the heart after myocardial infarction.

Vascular endothelial growth factor (VEGF) is a well-characterized proangiogenic cytokine that has been shown to promote neovascularization in hearts of patients with ischemic heart disease but can also lead to adverse effects depending on the dose and mode of delivery. We investigated whether prolonged exposure to a low dose of VEGF could be achieved by encapsulating VEGF in polylactic coglycolic acid nanoparticles and whether treatment with VEGF-containing nanoparticles improved cardiac function and protected against left ventricular remodeling in the hearts of mice with experimentally induced myocardial infarction. Polylactic coglycolic acid nanoparticles with a mean diameter of ~113 nm were generated via double emulsion and loaded with VEGF; the encapsulation efficiency was 53.5 ± 1.7% (107.1 ± 3.3 ng VEGF/mg nanoparticles). In culture, VEGF nanoparticles released VEGF continuously for at least 31 days, and in a murine myocardial infarction model, VEGF nanoparticle administration was associated with significantly greater vascular density in the peri-infarct region, reductions in infarct size, and improvements in left ventricular contractile function 4 wk after treatment. Thus, our study provides proof of principle that nanoparticle-mediated delivery increases the angiogenic and therapeutic potency of VEGF for the treatment of ischemic heart disease. NEW & NOTEWORTHY Vascular endothelial growth factor (VEGF) is a well-characterized proangiogenic cytokine but has a short half-life and a rapid clearance rate. When encapsulated in nanoparticles, VEGF was released for 31 days and improved left ventricular function in infarcted mouse hearts. These observations indicate that our new platform increases the therapeutic potency of VEGF.

Spheroids of cardiomyocytes derived from human-induced pluripotent stem cells improve recovery from myocardial injury in mice.

The microenvironment of native heart tissue may be better replicated when cardiomyocytes are cultured in three-dimensional clusters (i.e., spheroids) than in monolayers or as individual cells. Thus, we differentiated human cardiac lineage-induced pluripotent stem cells in cardiomyocytes (hiPSC-CMs) and allowed them to form spheroids and spheroid fusions that were characterized in vitro and evaluated in mice after experimentally induced myocardial infarction (MI). Synchronized contractions were observed within 24 h of spheroid formation, and optical mapping experiments confirmed the presence of both Ca2+ transients and propagating action potentials. In spheroid fusions, the intraspheroid conduction velocity was 7.0 ± 3.8 cm/s on days 1- 2 after formation, whereas the conduction velocity between spheroids increased significantly ( P = 0.003) from 0.8 ± 1.1 cm/s on days 1- 2 to 3.3 ± 1.4 cm/s on day 7. For the murine MI model, five-spheroid fusions (200,000 hiPSC-CMs/spheroid) were embedded in a fibrin patch and the patch was transplanted over the site of infarction. Later (4 wk), echocardiographic measurements of left ventricular ejection fraction and fractional shortening were significantly greater in patch-treated animals than in animals that recovered without the patch, and the engraftment rate was 25.6% or 30% when evaluated histologically or via bioluminescence imaging, respectively. The exosomes released from the spheroid patch seemed to increase cardiac function. In conclusion, our results established the feasibility of using hiPSC-CM spheroids and spheroid fusions for cardiac tissue engineering, and, when fibrin patches containing hiPSC-CM spheroid fusions were evaluated in a murine MI model, the engraftment rate was much higher than the rates we have achieved via the direct intramyocardial injection. NEW & NOTEWORTHY Spheroids fuse in culture to produce structures with uniformly distributed cells. Furthermore, human cardiac lineage-induced pluripotent stem cells in cardiomyocytes in adjacent fused spheroids became electromechanically coupled as the fusions matured in vitro, and when the spheroids were combined with a biological matrix and administered as a patch over the infarcted region of mouse hearts, the engraftment rate exceeded 25%, and the treatment was associated with significant improvements in cardiac function via a paracrine mechanism, where exosomes released from the spheroid patch.

Critical Roles of STAT3 in ß-Adrenergic Functions in the Heart.

BACKGROUND: ß-Adrenergic receptors (ßARs) play paradoxical roles in the heart. On one hand, ßARs augment cardiac performance to fulfill the physiological demands, but on the other hand, prolonged activations of ßARs exert deleterious effects that result in heart failure. The signal transducer and activator of transcription 3 (STAT3) plays a dynamic role in integrating multiple cytokine signaling pathways in a number of tissues. Altered activation of STAT3 has been observed in failing hearts in both human patients and animal models. Our objective is to determine the potential regulatory roles of STAT3 in cardiac ßAR-mediated signaling and function. METHODS AND RESULTS: We observed that STAT3 can be directly activated in cardiomyocytes by ß-adrenergic agonists. To follow up this finding, we analyzed ßAR function in cardiomyocyte-restricted STAT3 knockouts and discovered that the conditional loss of STAT3 in cardiomyocytes markedly reduced the cardiac contractile response to acute ßAR stimulation, and caused disengagement of calcium coupling and muscle contraction. Under chronic ß-adrenergic stimulation, Stat3cKO hearts exhibited pronounced cardiomyocyte hypertrophy, cell death, and subsequent cardiac fibrosis. Biochemical and genetic data supported that Gαs and Src kinases are required for ßAR-mediated activation of STAT3. Finally, we demonstrated that STAT3 transcriptionally regulates several key components of ßAR pathway, including ß1AR, protein kinase A, and T-type Ca(2+) channels. CONCLUSIONS: Our data demonstrate for the first time that STAT3 has a fundamental role in ßAR signaling and functions in the heart. STAT3 serves as a critical transcriptional regulator for ßAR-mediated cardiac stress adaption, pathological remodeling, and heart failure.

Biomarkers for monitoring chemotherapy-induced cardiotoxicity.

Cardiotoxicity, including acute and late-onset cardiotoxicity, is a well-known adverse effect of many types of antitumor agents. Early identification of patients with cardiotoxicity is important to ensure prompt treatment and minimize toxic effects. The etiology of chemotherapy-induced cardiotoxicity is multifactorial. Traditional methods for assessment of chemotherapy-induced cardiotoxicity typically involve serial measurements of cardiac function via multi-modality imaging techniques. Typically, however, significant left ventricular dysfunction has already occurred when cardiotoxicity is detected by imaging techniques. Biomarkers, most importantly cardiac natriuretic peptides and troponins, are promising markers for identifying patients potentially at risk for clinical heart failure symptoms. This review summarizes the recent progress in clinical utilization of biomarkers for early diagnosis of acute cardiotoxicity and for prediction of late-onset cardiotoxicity. We also discuss the conflicting results of different studies and the association of results with study design.

Fkbp1a controls ventricular myocardium trabeculation and compaction by regulating endocardial Notch1 activity.

Trabeculation and compaction of the embryonic myocardium are morphogenetic events crucial for the formation and function of the ventricular walls. Fkbp1a (FKBP12) is a ubiquitously expressed cis-trans peptidyl-prolyl isomerase. Fkbp1a-deficient mice develop ventricular hypertrabeculation and noncompaction. To determine the physiological function of Fkbp1a in regulating the intercellular and intracellular signaling pathways involved in ventricular trabeculation and compaction, we generated a series of Fkbp1a conditional knockouts. Surprisingly, cardiomyocyte-restricted ablation of Fkbp1a did not give rise to the ventricular developmental defect, whereas endothelial cell-restricted ablation of Fkbp1a recapitulated the ventricular hypertrabeculation and noncompaction observed in Fkbp1a systemically deficient mice, suggesting an important contribution of Fkbp1a within the developing endocardia in regulating the morphogenesis of ventricular trabeculation and compaction. Further analysis demonstrated that Fkbp1a is a novel negative modulator of activated Notch1. Activated Notch1 (N1ICD) was significantly upregulated in Fkbp1a-ablated endothelial cells in vivo and in vitro. Overexpression of Fkbp1a significantly reduced the stability of N1ICD and direct inhibition of Notch signaling significantly reduced hypertrabeculation in Fkbp1a-deficient mice. Our findings suggest that Fkbp1a-mediated regulation of Notch1 plays an important role in intercellular communication between endocardium and myocardium, which is crucial in controlling the formation of the ventricular walls.

The CUL7 E3 ubiquitin ligase targets insulin receptor substrate 1 for ubiquitin-dependent degradation.

Recent genetic studies have documented a pivotal growth-regulatory role played by the Cullin 7 (CUL7) E3 ubiquitin ligase complex containing the Fbw8-substrate-targeting subunit, Skp1, and the ROC1 RING finger protein. In this report, we identified insulin receptor substrate 1 (IRS-1), a critical mediator of the insulin/insulin-like growth factor 1 signaling, as a proteolytic target of the CUL7 E3 ligase in a manner that depends on mammalian target of rapamycin and the p70 S6 kinase activities. Interestingly, while embryonic fibroblasts of Cul7-/- mice were found to accumulate IRS-1 and exhibit increased activation of IRS-1's downstream Akt and MEK/ERK pathways, these null cells grew poorly and displayed phenotypes reminiscent of those associated with oncogene-induced senescence. Taken together, our findings demonstrate a key role for the CUL7 E3 in targeting IRS-1 for degradation, a process that may contribute to the regulation of cellular senescence.