Abstract
One of the earliest maturation steps in cardiomyocytes (CMs) is the sarcomere protein isoform switch between TNNI1 and TNNI3 (fetal and neonatal/adult troponin I). Here, we generate human ...induced pluripotent stem cells (hiPSCs) carrying a TNNI1
EmGFP
and TNNI3
mCherry
double reporter to monitor and isolate mature sub-populations during cardiac differentiation. Extensive drug screening identifies two compounds, an estrogen-related receptor gamma (ERRγ) agonist and an S-phase kinase-associated protein 2 inhibitor, that enhances cardiac maturation and a significant change to TNNI3 expression. Expression, morphological, functional, and molecular analyses indicate that hiPSC-CMs treated with the ERRγ agonist show a larger cell size, longer sarcomere length, the presence of transverse tubules, and enhanced metabolic function and contractile and electrical properties. Here, we show that ERRγ-treated hiPSC-CMs have a mature cellular property consistent with neonatal CMs and are useful for disease modeling and regenerative medicine.
Human pluripotent stem cell-derived cardiomyocytes (CMs) are a promising tool for cardiac cell therapy. Although transplantation of induced pluripotent stem cell (iPSC)-derived CMs have been reported ...in several animal models, the treatment effect was limited, probably due to poor optimization of the injected cells. To optimize graft cells for cardiac reconstruction, we compared the engraftment efficiency of intramyocardially-injected undifferentiated-iPSCs, day 4 mesodermal cells, and day 8, day 20, and day 30 purified iPSC-CMs after initial differentiation by tracing the engraftment ratio (ER) using in vivo bioluminescence imaging. This analysis revealed the ER of day 20 CMs was significantly higher compared to other cells. Transplantation of day 20 CMs into the infarcted hearts of immunodeficient mice showed good engraftment, and echocardiography showed significant functional improvement by cell therapy. Moreover, the imaging signal and ratio of Ki67-positive CMs at 3 months post injection indicated engrafted CMs proliferated in the host heart. Although this graft growth reached a plateau at 3 months, histological analysis confirmed progressive maturation from 3 to 6 months. These results suggested that day 20 CMs had very high engraftment, proliferation, and therapeutic potential in host mouse hearts. They also demonstrate this model can be used to track the fate of transplanted cells over a long time.
Many studies have shown the feasibility of in vivo cardiac transplantation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in animal experiments. However, nano-structural ...confirmation of the successful incorporation of the engrafted iPSC-CMs including electron microscopy (EM) has not been accomplished, partly because identification of graft cells in EM has proven to be difficult. Using APEX2, an engineered ascorbate peroxidase imaging tag, we successfully localized and analyzed the fine structure of sarcomeres and the excitation contraction machinery of iPSC-CMs 6 months after their engraftment in infarcted mouse hearts. APEX2 made iPSC-CMs visible in multiple imaging modalities including light microscopy, X-ray microscopic tomography, transmission EM, and scanning EM. EM tomography allowed assessment of the differentiation state of APEX2-positive iPSC-CMs and analysis of the fine structure of the sarcomeres including T-tubules and dyads.
Here, we find that human-induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM)-fated progenitors (CFPs) that express a tetraspanin family glycoprotein, CD82, almost exclusively ...differentiate into CMs both in vitro and in vivo. CD82 is transiently expressed in late-stage mesoderm cells during hiPSC differentiation. Purified CD82+ cells gave rise to CMs under nonspecific in vitro culture conditions with serum, as well as in vivo after transplantation to the subrenal space or injured hearts in mice, indicating that CD82 successfully marks CFPs. CD82 overexpression in mesoderm cells as well as in undifferentiated hiPSCs increased the secretion of exosomes containing β-catenin and reduced nuclear β-catenin protein, suggesting that CD82 is involved in fated restriction to CMs through Wnt signaling inhibition. This study may contribute to the understanding of CM differentiation mechanisms and to cardiac regeneration strategies.
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•Cardiomyocyte (CM)-fated progenitors (CFPs) from human iPSCs•CD82 as a specific cell-surface CFP marker•Specific differentiation of CD82+ cells to CMs in vitro and in vivo•CD82 in CM-fate restriction through exosome-mediated Wnt inhibition
Takeda et al. find that CD82+ is a cell-surface marker on cardiomyocyte-fated progenitors made from human iPSCs.
Abstract Background Aortic valve replacement (AVR) is currently the standard therapy for severe aortic stenosis (AS), and regression of left ventricular (LV) hypertrophy after AVR has been reported. ...However, data regarding a temporal relation between LV mass and left atrial (LA) volume are limited, and their prognostic impacts have not been fully elucidated. We aimed to clarify the temporal patterns of LA and LV reverse remodeling and their associations with clinical outcomes. Methods We retrospectively reviewed 198 consecutive patients who underwent AVR for severe AS. After excluding patients with prior cardiac surgery, atrial fibrillation, concomitant moderate to severe aortic regurgitation, or concurrent mitral valve surgery, 83 patients with echocardiographic LV mass index (LVMI) and LA volume index (LAVI) data before and 1 year after AVR were eligible for the outcome analysis and 29 patients with these 2 measures before surgery, 1 month, 1 year, and 3 years after surgery were eligible for the analysis of time-dependent change of LVMI and LAVI. Results Significant reductions in LVMI and LAVI (both p < 0.001) after surgery were observed over time. LA dilatation improved and reached a plateau 1 month after surgery, whereas LV hypertrophy improved more gradually and reached a plateau at 1 year. The presence of both LV hypertrophy and LA dilatation 1 year after surgery was associated with significantly higher mortality (patients with both conditions vs. patients with neither or one condition = 22.6% vs. 7.3% at 3 years; p = 0.031) and major adverse cardiac and cerebrovascular events (38.9% vs. 12.6% at 3 years; p = 0.021). Conclusions LA reverse remodeling occurred rapidly after AVR for severe AS, and regression of LV hypertrophy was more gradual. The presence of both residual LV hypertrophy and LA dilatation 1 year after AVR was associated with poor long-term outcomes.
Recent high-throughput approaches have revealed a vast number of transcripts with unknown functions. Many of these transcripts are long noncoding RNAs (lncRNAs), and intergenic region-derived lncRNAs ...are classified as long intergenic noncoding RNAs (lincRNAs). Although Myosin heavy chain 6 (Myh6) encoding primary contractile protein is down-regulated in stressed hearts, the underlying mechanisms are not fully clarified especially in terms of lincRNAs. Here, we screen upregulated lincRNAs in pressure overloaded hearts and identify a muscle-abundant lincRNA termed Lionheart. Compared with controls, deletion of the Lionheart in mice leads to decreased systolic function and a reduction in MYH6 protein levels following pressure overload. We reveal decreased MYH6 results from an interaction between Lionheart and Purine-rich element-binding protein A after pressure overload. Furthermore, human LIONHEART levels in left ventricular biopsy specimens positively correlate with cardiac systolic function. Our results demonstrate Lionheart plays a pivotal role in cardiac remodeling via regulation of MYH6.
ABSTRACT
Background and objective: Septic pulmonary embolism due to periodontal disease (SPE‐PD) is rarely reported and little is known about its clinical features. The purpose of this study was to ...evaluate the clinical and radiological features, as well as outcome, in SPE‐PD.
Methods: Patients' records were retrospectively reviewed and 12 patients with SPE‐PD were identified (10 men, mean age 60.5 years). The patients' demographic features, laboratory data, physical and radiological findings, and clinical outcomes were evaluated.
Results: All but one patient were smokers. Eight of the 12 patients had comorbidities including hypertension (58%) and/or diabetes mellitus (17%). Prevalent symptoms were fever (67%) and chest pain (58%). Only two patients fulfilled the criteria of systemic inflammatory response syndrome; most of the subjects were not clinically severely ill. Blood cultures were negative in all cases. Contrast‐enhanced chest computed tomography (CT) showed multiple peripheral nodules in all 12 patients, wedge‐shaped peripheral lesions abutting on the pleura in 10 (83%) and a feeding‐vessel sign in 9 (75%). All patients recovered from their illness after antimicrobial therapy concomitant with tooth extraction or periodontal care. The median duration of antibiotic administration was 51 days.
Conclusions: Most patients with SPE‐PD were not seriously ill. Contrast‐enhanced chest CT appeared to be useful to diagnose SPE‐PD.
SPE‐PD is rarely reported. We retrospectively reviewed 12 patients with SPE‐PD. Most of the cases were not seriously ill. With early diagnosis by contrast‐enhanced chest CT, appropriate antimicrobial therapy and periodontal surgery, resolution of the illness can be expected in most patients.
Abstract only Rationale: The only treatment for drug-resistant heart failure is heart transplantation, however, it has problems such as donor shortages and rejection. Human induced pluripotent stem ...cell-derived cardiomyocytes (hiPSC-cardiomyocytes) are promising sources of regenerative medicine that can solve these problems. The engraftment potential of hiPSC-cardiomyocytes following the transplantation into the host heart should be improved to realize this therapy. Objective: We aim to discover a compound to enhance the engraftment of hiPSC-cardiomyocytes following the cell transplantation into the damaged heart. We also aim to realize a larger graft size and recover cardiac function. Methods and Results: We established an iPSC line constitutively expressing Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI), which can show the cell cycle phase in real-time. We compared the engraftment capacity of hiPSC-cardiomyocytes in between the S/G2/M (activation) phase and G0/G1 (inactivation) phase for three months after transplantation, finding cell cycle activated hiPSC-cardiomyocytes showed significantly higher engraftment efficiency than those in inactivated. We, thus, conducted the screening to find cell-cycle activating compounds in the hiPSC-cardiomyocytes. The screening identified some candidates from more than 4000 compounds, and we focused on the most effective compound (CCA-1). An EdU assay and cell number count demonstrated the activation of proliferative capacity by CCA-1. Moreover, genetic analysis revealed a group of genes related to CCA-1-induced cell cycle activation. Finally, using in vivo bioluminescent imaging, we confirmed that CCA-1 treated hiPSC-cardiomyocytes showed enhanced graft size after transplantation into mice ischemic heart. Conclusions: This study highlights the effectiveness of the FUCCI system to analyze the cell cycle status in hiPSC-cardiomyocytes, and the cell cycle activation by CCA-1 is the efficient strategy to increase the graft size following the cell transplantation into the damaged heart.
Abstract only
Background:
For disease modeling and drug screening using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), matured iPSC-CMs are required to understand the underlying ...mechanism of diseases and to get the candidate drug’s deleterious features. We have shown that synthetic mRNAs encoding a fluorescent protein tagged with complementary sequences against specifically expressed microRNA (miRNA-switch) can be efficiently used for purification. Using miRNA-switch, we evaluated the selection method of matured iPSC-CMs from iPSC-CMs.
Methods:
We used miR-208a as a specific miRNA of CMs and miR-Matured CM (miR-MCM). We synthesized miR-208a-responsive GFP-coding mRNA (miR-208a-GFP switch), and miR-MCM-iRFP670 switch and control tagBFP mRNA and transfected them into iPSC-CMs.
Results:
After transfection of these mRNA, 25±5% of miR-208a positive cells were positive for miR-MCM. We also compared miR-208a positive and miR-MCM positive cells to miR-208a positive and miR-MCM negative cells by qPCR and electron microscopy (EM). MiR-208a-positive and miR-MCM-positive cells showed significantly higher expression of matured CM-related genes such as MYH7 and MYL2. In EM observation, miR-208a-positive and miR-MCM-positive cells showed M-bands, while miR-208a-positive and miR-MCM-negative cells did not display M-bands.
Conclusions:
We demonstrated that miR-MCM could be used for the marker of matured iPSC-CMs. The synthetic miR-switch for miR-MCM efficiently isolate matured iPSC-CMs, enabling its application in disease modeling and drug screening using differentiated cells from iPSCs.
Abstract only
Introduction:
Recently, it has also been reported that patients with ischemic heart disease have prolonged left ventricular (LV) GLS reduction after exercise.
Hypothesis:
To investigate ...the influence of prolonged LV GLS reduction after exercise on the development of cardiovascular events.
Methods:
One hundred thirty-three consecutive patients with at least one of the following coronary risk factors: hypertension, hyperlipidemia, or diabetes mellitus underwent echocardiography (ARTIDA; Canon medical, Japan) before and 30 minutes after Master double exercise and LV GLS was measured offline (Vitria; Canon medical, Japan). Patients with obvious LV wall motion abnormalities, significant valvular heart disease, or atrial fibrillation were excluded. The incidence of cardiovascular events was determined from the patients' medical records. Patients were divided into three groups based on the occurrence of cardiovascular events up to 6 months after the examination: non-events group, minor cardiovascular event group (chest pain, palpitations, other additional tests), and major cardiovascular event group (coronary revascularization, cardiac hospitalization). The LV GLSs before and 30 minutes after exercise were compared in each patient group.
Results:
Mean age was 63±11.5years (female: 25%). Patients had hypertension (53%), hyperlipidemia (47%), and diabetes (65%). Of the 133 eligible patients, non-event group (n=68), minor cardiovascular event group (n=50), and major cardiovascular event group (n=15). LV GLS before exercise was not significantly different between the groups, however LV GLS at 30 minutes after exercise were significantly lower in the cardiovascular event groups and were proportional to the severity of the event. (Figure)
Conclusions:
The prolonged reduction in LV GLS after exercise could be a useful predictive index for the development of cardiovascular events.
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