RNA Helicase DDX5 Maintains Cardiac Function by Regulating CamkIIδ Alternative Splicing.

BACKGROUND: Heart failure (HF) is a leading cause of morbidity and mortality worldwide. RNA-binding proteins are identified as regulators of cardiac disease; DDX5 (dead-box helicase 5) is a master regulator of many RNA processes, although its function in heart physiology remains unclear. METHODS: We assessed DDX5 expression in human failing hearts and a mouse HF model. To study the function of DDX5 in heart, we engineered cardiomyocyte-specific Ddx5 knockout mice. We overexpressed DDX5 in cardiomyocytes using adeno-associated virus serotype 9 and performed transverse aortic constriction to establish the murine HF model. The mechanisms underlined were subsequently investigated using immunoprecipitation-mass spectrometry, RNA-sequencing, alternative splicing analysis, and RNA immunoprecipitation sequencing. RESULTS: We screened transcriptome databases of murine HF and human dilated cardiomyopathy samples and found that DDX5 was significantly downregulated in both. Cardiomyocyte-specific deletion of Ddx5 resulted in HF with reduced cardiac function, an enlarged heart chamber, and increased fibrosis in mice. DDX5 overexpression improved cardiac function and protected against adverse cardiac remodeling in mice with transverse aortic constriction-induced HF. Furthermore, proteomics revealed that DDX5 is involved in RNA splicing in cardiomyocytes. We found that DDX5 regulated the aberrant splicing of Ca2+/calmodulin-dependent protein kinase IIδ (CamkIIδ), thus preventing the production of CaMKIIδA, which phosphorylates L-type calcium channel by serine residues of Cacna1c, leading to impaired Ca2+ homeostasis. In line with this, we found increased intracellular Ca2+ transients and increased sarcoplasmic reticulum Ca2+ content in DDX5-depleted cardiomyocytes. Using adeno-associated virus serotype 9 knockdown of CaMKIIδA partially rescued the cardiac dysfunction and HF in Ddx5 knockout mice. CONCLUSIONS: These findings reveal a role for DDX5 in maintaining calcium homeostasis and cardiac function by regulating alternative splicing in cardiomyocytes, identifying the DDX5 as a potential target for therapeutic intervention in HF.

Low-Dose Prasugrel vs. Standard-Dose Clopidogrel for Patients Undergoing Percutaneous Coronary Intervention.

BACKGROUND: Low-dose prasugrel (3.75 mg) is used as maintenance therapy for percutaneous coronary intervention; however, data on long-term outcomes are scarce.Methods and Results: We analyzed 5,392 participants in the KiCS-PCI registry who were administered low-dose prasugrel or clopidogrel at discharge between 2008 and 2018 and for whom 2-year follow-up data were available. We adjusted for confounders using matching weight analyses and multiple imputations. Similarly, we used inverse probability- and propensity score-weighted analyses. We also performed instrumental variable analyses. The primary outcomes were acute coronary syndrome (ACS) and bleeding requiring readmission. Secondary outcomes were all-cause death and a composite outcome of ACS, bleeding, heart failure, stroke, coronary bypass requiring admission, and all-cause death. In this cohort, 12.2% of patients were discharged with low-dose prasugrel. Compared with clopidogrel, low-dose prasugrel was associated with a reduced risk of ACS (hazard ratio [HR] 0.58; 95% confidence interval [CI] 0.39-0.85), bleeding (HR 0.62; 95% CI 0.40-0.97), and the composite outcome (HR 0.71; 95% CI 0.59-0.86). Inverse probability-weighted analysis yielded similar results; however, matching weight analysis without multiple imputations and propensity score-matched analyses showed similar outcomes in both groups. Instrumental variable analyses showed reduced risks of ACS and composite outcome for those on low-dose prasugrel. All-cause mortality did not differ in all analyses. CONCLUSIONS: Low-dose prasugrel demonstrates comparable outcomes to clopidogrel in terms of ACS and bleeding.

A deep learning-based automated diagnosis system for SPECT myocardial perfusion imaging.

Images obtained from single-photon emission computed tomography for myocardial perfusion imaging (MPI SPECT) contain noises and artifacts, making cardiovascular disease diagnosis difficult. We developed a deep learning-based diagnosis support system using MPI SPECT images. Single-center datasets of MPI SPECT images (n = 5443) were obtained and labeled as healthy or coronary artery disease based on diagnosis reports. Three axes of four-dimensional datasets, resting, and stress conditions of three-dimensional reconstruction data, were reconstructed, and an AI model was trained to classify them. The trained convolutional neural network showed high performance [area under the curve (AUC) of the ROC curve: approximately 0.91; area under the recall precision curve: 0.87]. Additionally, using unsupervised learning and the Grad-CAM method, diseased lesions were successfully visualized. The AI-based automated diagnosis system had the highest performance (88%), followed by cardiologists with AI-guided diagnosis (80%) and cardiologists alone (65%). Furthermore, diagnosis time was shorter for AI-guided diagnosis (12 min) than for cardiologists alone (31 min). Our high-quality deep learning-based diagnosis support system may benefit cardiologists by improving diagnostic accuracy and reducing working hours.

A Novel Risk Score for Major Bleeding in Japanese Patients with Non-Valvular Atrial Fibrillation: The J-RISK AF Study.

AIM: Oral anticoagulants (OACs) reduce the risk of ischemic stroke but may increase the risk of major bleeding in patients with non-valvular atrial fibrillation (NVAF). Various risk scores, such as HAS-BLED, ATRIA, ORBIT, and DOAC, have been proposed to assess the risk of major bleeding in patients with NVAF receiving OACs. However, limited data are available regarding bleeding risk stratification in Japanese patients with NVAF. METHODS: Of the 16,098 NVAF patients from the J-RISK AF study, the combined data of the five major AF registries in Japan (J-RHYTHM Registry, Fushimi AF Registry, Shinken Database, Keio interhospital Cardiovascular Studies, and Hokuriku-Plus AF Registry), we analyzed 11,539 patients receiving OACs (median age, 71 years old; women, 29.6%; median CHA2DS2-VASc score, 3). RESULTS: During the 2-year follow-up period, major bleeding occurred in 274 patients (1.3% per patient-year). In a multivariate Cox proportional hazards analysis, an advanced age, hypertension (systolic blood pressure ≥ 150 mmHg), bleeding history, anemia, thrombocytopenia, and concomitant antiplatelet agents were significantly associated with a higher incidence of major bleeding. We developed a novel risk stratification system, HED-[EPA]2-B3 score, which had a better predictive performance for major bleeding (C-statistics 0.67, [95% confidence interval, 0.63-0.70]) than the HAS-BLED (0.64, [0.60-0.67], P for difference 0.02) and ATRIA (0.63, [0.60-0.66], P for difference ＜0.01) scores. Furthermore, it was non-significantly higher than the ORBIT (0.65, [0.62-0.68], P for difference 0.07) and DOAC (0.65, [0.62-0.68], P for difference 0.17) scores. CONCLUSION: Our novel risk stratification system, the HED-[EPA]2-B3 score, may be useful for identifying Japanese patients receiving OACs at a risk of major bleeding.

H1FOO-DD promotes efficiency and uniformity in reprogramming to naive pluripotency.

Heterogeneity among both primed and naive pluripotent stem cell lines remains a major unresolved problem. Here we show that expressing the maternal-specific linker histone H1FOO fused to a destabilizing domain (H1FOO-DD), together with OCT4, SOX2, KLF4, and LMYC, in human somatic cells improves the quality of reprogramming to both primed and naive pluripotency. H1FOO-DD expression was associated with altered chromatin accessibility around pluripotency genes and with suppression of the innate immune response. Notably, H1FOO-DD generates naive induced pluripotent stem cells with lower variation in transcriptome and methylome among clones and a more uniform and superior differentiation potency. Furthermore, we elucidated that upregulation of FKBP1A, driven by these five factors, plays a key role in H1FOO-DD-mediated reprogramming.

Regeneration of Nonhuman Primate Hearts With Human Induced Pluripotent Stem Cell-Derived Cardiac Spheroids.

BACKGROUND: The clinical application of human induced pluripotent stem cell-derived cardiomyocytes (CMs) for cardiac repair commenced with the epicardial delivery of engineered cardiac tissue; however, the feasibility of the direct delivery of human induced pluripotent stem cell-derived CMs into the cardiac muscle layer, which has reportedly induced electrical integration, is unclear because of concerns about poor engraftment of CMs and posttransplant arrhythmias. Thus, in this study, we prepared purified human induced pluripotent stem cell-derived cardiac spheroids (hiPSC-CSs) and investigated whether their direct injection could regenerate infarcted nonhuman primate hearts. METHODS: We performed 2 separate experiments to explore the appropriate number of human induced pluripotent stem cell-derived CMs. In the first experiment, 10 cynomolgus monkeys were subjected to myocardial infarction 2 weeks before transplantation and were designated as recipients of hiPSC-CSs containing 2×107 CMs or the vehicle. The animals were euthanized 12 weeks after transplantation for histological analysis, and cardiac function and arrhythmia were monitored during the observational period. In the second study, we repeated the equivalent transplantation study using more CMs (6×107 CMs). RESULTS: Recipients of hiPSC-CSs containing 2×107 CMs showed limited CM grafts and transient increases in fractional shortening compared with those of the vehicle (fractional shortening at 4 weeks after transplantation [mean ± SD]: 26.2±2.1%; 19.3±1.8%; P<0.05), with a low incidence of posttransplant arrhythmia. Transplantation of increased dose of CMs resulted in significantly greater engraftment and long-term contractile benefits (fractional shortening at 12 weeks after transplantation: 22.5±1.0%; 16.6±1.1%; P<0.01, left ventricular ejection fraction at 12 weeks after transplantation: 49.0±1.4%; 36.3±2.9%; P<0.01). The incidence of posttransplant arrhythmia slightly increased in recipients of hiPSC-CSs containing 6×107 CMs. CONCLUSIONS: We demonstrated that direct injection of hiPSC-CSs restores the contractile functions of injured primate hearts with an acceptable risk of posttransplant arrhythmia. Although the mechanism for the functional benefits is not fully elucidated, these findings provide a strong rationale for conducting clinical trials using the equivalent CM products.

Pre-Existing Left Bundle Branch Block and Clinical Outcomes After Transcatheter Aortic Valve Replacement.

Background: Few reports on pre-existing left bundle branch block (LBBB) in patients undergoing transcatheter aortic valve replacement (TAVR) are currently available. Further, no present studies compare patients with new onset LBBB with those with pre-existing LBBB. Objectives: This study aimed to investigate the association between pre-existing or new onset LBBB and clinical outcomes after TAVR. Methods: Using data from the Japanese multicenter registry, 5,996 patients who underwent TAVR between October 2013 and December 2019 were included. Patients were classified into 3 groups: no LBBB, pre-existing LBBB, and new onset LBBB. The 2-year clinical outcomes were compared between 3 groups using Cox proportional hazards models and propensity score analysis to adjust the differences in baseline characteristics. Results: Of 5,996 patients who underwent TAVR, 280 (4.6%) had pre-existing LBBB, while 1,658 (27.6%) experienced new onset LBBB. Compared with the no LBBB group, multivariable Cox regression analysis showed that pre-existing LBBB was associated not only with a higher 2-year all-cause (adjusted HR: 1.39; 95% CI: 1.06-1.82; P = 0.015) and cardiovascular (adjusted HR: 1.60; 95% CI: 1.04-2.48; P = 0.031) mortality, but also with higher all-cause (adjusted HR: 1.43, 95% CI: 1.07-1.91; P = 0.016) and cardiovascular (adjusted HR: 1.81, 95% CI:1.12-2.93; P = 0.014) mortality than the new onset LBBB group. Heart failure was the most common cause of cardiovascular death, with more heart failure deaths in the pre-existing LBBB group. Conclusions: Pre-existing LBBB was independently associated with poor clinical outcomes, reflecting an increased risk of cardiovascular mortality after TAVR. Patients with pre-existing LBBB should be carefully monitored.

Orphan Nuclear Receptor NR4A3 Promotes Vascular Calcification via Histone Lactylation.

BACKGROUND: Medial arterial calcification is a chronic systemic vascular disorder distinct from atherosclerosis and is commonly observed in patients with chronic kidney disease, diabetes, and aging individuals. We previously showed that NR4A3 (nuclear receptor subfamily 4 group A member 3), an orphan nuclear receptor, is a key regulator in apo (apolipoprotein) A-IV-induced atherosclerosis progression; however, its role in vascular calcification is poorly understood. METHODS: We generated NR4A3-/- mice and 2 different types of medial arterial calcification models to investigate the biological roles of NR4A3 in vascular calcification. RNA-seq was performed to determine the transcriptional profile of NR4A3-/- vascular smooth muscle cells under ß-glycerophosphate treatment. We integrated Cleavage Under Targets and Tagmentation analysis and RNA-seq data to further investigate the gene regulatory mechanisms of NR4A3 in arterial calcification and target genes regulated by histone lactylation. RESULTS: NR4A3 expression was upregulated in calcified aortic tissues from chronic kidney disease mice, 1,25(OH)2VitD3 overload-induced mice, and human calcified aorta. NR4A3 deficiency preserved the vascular smooth muscle cell contractile phenotype, inhibited osteoblast differentiation-related gene expression, and reduced calcium deposition in the vasculature. Further, NR4A3 deficiency lowered the glycolytic rate and lactate production during the calcification process and decreased histone lactylation. Mechanistic studies further showed that NR4A3 enhanced glycolysis activity by directly binding to the promoter regions of the 2 glycolysis genes ALDOA and PFKL and driving their transcriptional initiation. Furthermore, histone lactylation promoted medial calcification both in vivo and in vitro. NR4A3 deficiency inhibited the transcription activation and expression of Phospho1 (phosphatase orphan 1). Consistently, pharmacological inhibition of Phospho1 attenuated calcium deposition in NR4A3-overexpressed vascular smooth muscle cells, whereas overexpression of Phospho1 reversed the anticalcific effect of NR4A3 deficiency in vascular smooth muscle cells. CONCLUSIONS: Taken together, our findings reveal that NR4A3-mediated histone lactylation is a novel metabolome-epigenome signaling cascade mechanism that participates in the pathogenesis of medial arterial calcification.

Pluripotent stem cell-based cardiac regenerative therapy for heart failure.

Cardiac regenerative therapy using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is expected to become an alternative to heart transplantation for severe heart failure. It is now possible to produce large numbers of human pluripotent stem cells (hPSCs) and eliminate non-cardiomyocytes, including residual undifferentiated hPSCs, which can cause teratoma formation after transplantation. There are two main strategies for transplanting hPSC-CMs: injection of hPSC-CMs into the myocardium from the epicardial side, and implantation of hPSC-CM patches or engineered heart tissues onto the epicardium. Transplantation of hPSC-CMs into the myocardium of large animals in a myocardial infarction model improved cardiac function. The engrafted hPSC-CMs matured, and microvessels derived from the host entered the graft abundantly. Furthermore, as less invasive methods using catheters, injection into the coronary artery and injection into the myocardium from the endocardium side have recently been investigated. Since transplantation of hPSC-CMs alone has a low engraftment rate, various methods such as transplantation with the extracellular matrix or non-cardiomyocytes and aggregation of hPSC-CMs have been developed. Post-transplant arrhythmias, imaging of engrafted hPSC-CMs, and immune rejection are the remaining major issues, and research is being conducted to address them. The clinical application of cardiac regenerative therapy using hPSC-CMs has just begun and is expected to spread widely if its safety and efficacy are proven in the near future.

Right Ventricular Dysfunction in Patients With Concomitant Tricuspid Regurgitation Undergoing Transcatheter Aortic Valve Implantation.

BACKGROUND: This study investigated the impact and predictive factors of concomitant significant tricuspid regurgitation (TR) and evaluated the roles of right ventricle (RV) function and the etiology of TR in the clinical outcomes of patients with severe aortic stenosis undergoing transcatheter aortic valve implantation (TAVI).Methods and Results: We assessed grading of TR severity, TR etiology, and RV function in pre- and post-TAVI transthoracic echocardiograms for 678 patients at Keio University School of Medicine. TR etiology was divided into 3 groups: primary TR, ventricular functional TR (FTR), and atrial FTR. The primary outcomes were all-cause and cardiovascular death. At baseline, moderate or greater TR was found in 55 (8%) patients and, after adjustment for comorbidities, was associated with increased all-cause death (hazard ratio [HR] 2.11; 95% confidence interval [CI] 1.19-3.77; P=0.011) and cardiovascular death (HR 2.29; 95% CI 1.06-4.99; P=0.036). RV dysfunction (RVD) also remained an independent predictor of cardiovascular death (HR 2.06; 95% CI 1.03-4.14; P=0.042). Among the TR etiology groups, patients with ventricular FTR had the lowest survival rate (P<0.001). Patients with persistent RVD after TAVI had a higher risk of cardiovascular death than those with a normal or recovered RV function (P<0.001). CONCLUSIONS: The etiology of TR and RV function play an important role in predicting outcomes in concomitant TR patients undergoing TAVI.

Discontinuation of Oral Anticoagulants in Atrial Fibrillation Patients: Impact of Treatment Strategy and on Patients' Health Status.

AIMS: The discontinuation of oral anticoagulants (OACs) remains as a significant concern in the management of atrial fibrillation (AF). The discontinuation rate may vary depending on management strategy, and physicians may also discontinue OACs due to concerns about patient satisfaction with their care. We aimed to assess the incidence of OAC discontinuation and its relationship to patients' health in an outpatient AF registry. METHODS AND RESULTS: From a multicenter registry for newly recognized AF patients (n = 3313), we extracted 1647 (49.7%) patients with OACs and a CHA2DS2-Vasc score of ≥2. Discontinuation was defined as sustained cessation of OACs within a 1-year follow-up. We examined predictors associated with discontinuation and its relations to health status defined by the AFEQT questionnaire. Of the 1647 patients, 385 (23.6%) discontinued OACs after 1 year, with discontinuation rates varying across treatment strategies (15.3% for catheter ablation, 4.9% for rhythm control with antiarrhythmic drugs, and 3.0% for rate control). Successful rhythm control was associated with discontinuation in the catheter ablation (OR 6.61, 95% CI 3.00-14.6, p < 0.001) and antiarrhythmic drugs (OR 6.47, 95% CI 2.62-15.9, p < 0.001) groups, whereas the incidence of bleeding events within 1 year was associated with discontinuation in the rate control group. One-year AFEQT scores did not significantly differ between patients who discontinued OACs and those who did not in each treatment strategy group. CONCLUSIONS: OAC discontinuation was common among AF patients with significant stroke risk but varied depending on the chosen treatment strategy. This study also found no significant association between OAC discontinuation and patients' health status.

Anaerobic threshold using sweat lactate sensor under hypoxia.

We aimed to investigate the reliability and validity of sweat lactate threshold (sLT) measurement based on the real-time monitoring of the transition in sweat lactate levels (sLA) under hypoxic exercise. In this cross-sectional study, 20 healthy participants who underwent exercise tests using respiratory gas analysis under hypoxia (fraction of inspired oxygen [FiO2], 15.4 ± 0.8%) in addition to normoxia (FiO2, 20.9%) were included; we simultaneously monitored sLA transition using a wearable lactate sensor. The initial significant elevation in sLA over the baseline was defined as sLT. Under hypoxia, real-time dynamic changes in sLA were successfully visualized, including a rapid, continual rise until volitionary exhaustion and a progressive reduction in the recovery phase. High intra- and inter-evaluator reliability was demonstrated for sLT's repeat determinations (0.782 [0.607-0.898] and 0.933 [0.841-0.973]) as intraclass correlation coefficients [95% confidence interval]. sLT correlated with ventilatory threshold (VT) (r = 0.70, p < 0.01). A strong agreement was found in the Bland-Altman plot (mean difference/mean average time: - 15.5/550.8 s) under hypoxia. Our wearable device enabled continuous and real-time lactate assessment in sweat under hypoxic conditions in healthy participants with high reliability and validity, providing additional information to detect anaerobic thresholds in hypoxic conditions.

Scalable production of homogeneous cardiac organoids derived from human pluripotent stem cells.

Three-dimensional (3D) cultures are known to more closely mimic in vivo conditions compared with 2D cultures. Cardiac spheroids (CSs) and organoids (COs) are useful for 3D tissue engineering and are advantageous for their simplicity and mass production for regenerative therapy and drug discovery. Herein, we describe a large-scale method for producing homogeneous human induced pluripotent stem cell (hiPSC)-derived CSs (hiPSC-CSs) and COs without scaffolds using a porous 3D microwell substratum with a suction system. Our method has many advantages, such as increased efficiency and improved functionality, homogeneity, and sphericity of hiPSC-CSs. Moreover, we have developed a substratum on a clinically relevant large scale for regenerative therapy and have succeeded in producing approximately 40,000 hiPSC-CSs with high sphericity at once. Furthermore, we efficiently produced a fused CO model consisting of hiPSC-derived atrial and ventricular cardiomyocytes localized on opposite sides of one organoid. This method will facilitate progress toward hiPSC-based clinical applications.

Initiation and Up-Titration of Guideline-Based Medications in Hospitalized Acute Heart Failure Patients　- A Report From the West Tokyo Heart Failure Registry.

BACKGROUND: Despite recommendations from clinical practice guidelines to initiate and titrate guideline-directed medical therapy (GDMT) during their hospitalization, patients with acute heart failure (AHF) are frequently undertreated. In this study we aimed to clarify GDMT implementation and titration rates, as well as the long-term outcomes, in hospitalized AHF patients.Methods and Results: Among 3,164 consecutive hospitalized AHF patients included in a Japanese multicenter registry, 1,400 (44.2%) with ejection fraction ≤40% were analyzed. We assessed GDMT dosage (ß-blockers, renin-angiotensin inhibitors, and mineralocorticoid-receptor antagonists) at admission and discharge, examined the contributing factors for up-titration, and evaluated associations between drug initiation/up-titration and 1-year post-discharge all-cause death and rehospitalization for HF via propensity score matching. The mean age of the patients was 71.5 years and 30.7% were female. Overall, 1,051 patients (75.0%) were deemed eligible for GDMT, based on their baseline vital signs, renal function, and electrolyte values. At discharge, only 180 patients (17.1%) received GDMT agents up-titrated to >50% of the maximum titrated dose. Up-titration was associated with a lower risk of 1-year clinical outcomes (adjusted hazard ratio: 0.58, 95% confidence interval: 0.35-0.96). Younger age and higher body mass index were significant predictors of drug up-titration. CONCLUSIONS: Significant evidence-practice gaps in the use and dose of GDMT remain. Considering the associated favorable outcomes, further efforts to improve its implementation seem crucial.

Long-term outcomes of periprocedural coronary dissection and perforation for patients undergoing percutaneous coronary intervention in a Japanese multicenter registry.

Long-term outcomes of iatrogenic coronary dissection and perforation in patients undergoing percutaneous coronary intervention (PCI) remains under-investigated. We analyzed 8,721 consecutive patients discharged after PCI between 2008 and 2019 from Keio Cardiovascular (KiCS) PCI multicenter prospective registry in the Tokyo metropolitan area. Significant coronary dissection was defined as persistent contrast medium extravasation or spiral or persistent filling defects with complete distal and impaired flow. The primary outcome was a composite of all-cause death, acute coronary syndrome, heart failure, bleeding, stroke requiring admission, and coronary artery bypass grafting two years after discharge. We used a multivariable Cox hazard regression model to assess the effects of these complications. Among the patients, 68 (0.78%) had significant coronary dissections, and 61 (0.70%) had coronary perforations at the index PCI. Patients with significant coronary dissection had higher rates of the primary endpoint and heart failure than those without (25.0% versus 14.3%, P = 0.02; 10.3% versus 4.2%, P = 0.03); there were no significant differences in the primary outcomes between the patients with and without coronary perforation (i.e., primary outcome: 8.2% versus 14.5%, P = 0.23) at the two-year follow-up. After adjustments, patients with coronary dissection had a significantly higher rate of the primary endpoint than those without (HR 1.70, 95% CI 1.02-2.84; P = 0.04), but there was no significant difference in the primary endpoint between the patients with and without coronary perforation (HR 0.51, 95% CI 0.21-1.23; P = 0.13). For patients undergoing PCI, significant coronary dissection was associated with poor long-term outcomes, including heart failure readmission.

Establishment and Industrialization of a New Treatment Method Using Regenerative Cardiomyocyte Transplantation for Refractory Severe Heart Failure - Secondary Publication.

Cardiomyocytes undergo cell division during the fetal period but do not divide after birth; thus, they grow into adult heart cells by enlarging their size. Therefore, heart failure occurs when a certain number of cardiomyocytes are lost owing to myocardial infarction, myocarditis, sarcoidosis, etc. Through scientific efforts, we have developed methods to safely and efficiently generate induced pluripotent stem (iPS) cells from peripheral blood T cells, generate ventricle-specific cardiomyocytes from iPS cells, and remove residual iPS cells and non-cardiomyocytes using the "metabolic selection method" and purify the cardiomyocytes from iPS cell derivatives. We have also developed the technology to mass-produce and efficiently engraft cardiomyocytes by generating cardiomyocyte spheroids and have developed devices suitable for cell transplantation. We have confirmed the safety and efficacy of these techniques by performing preclinical studies (oncogenesis, arrhythmogenicity, etc.) using immunodeficient mice, rats, pigs, and monkeys. Based on these technologies, we have successfully regenerated human ventricular muscle-specific cardiomyocytes with purity greater than 99%. We have also confirmed that the regenerated myocardium transplanted into immunodeficient mice maintained autonomic beating for more than a year without tumor formation. We are planning to conduct clinical trials to transplant iPS cell-derived cardiomyocytes into patients with heart failure associated with ischemic heart disease, which will, in the near future, enable clinical applications using HLA-deficient iPS cells and iPS cells generated from the patient's own lymphocytes to generate regenerative cardiomyocytes without rejection. It would also help establish personalized medicine for heart failure and usher in the long-awaited treatment for intractable severe heart failure using ventricular muscle supplementation.

End-of-Life Discussions and Their Timing for Patients With Cardiovascular Diseases　- From the Perspective of Bereaved Family Members.

BACKGROUND: Enhanced discussions regarding end-of-life (EOL) are crucial to provide appropriate care for seriously ill patients. However, the current status of EOL discussions, especially their timing and influencing factors, among patients with cardiovascular diseases (CVD) remains unknown.Methods and Results: We conducted a cross-sectional questionnaire survey of bereaved family members of CVD patients who died at 10 tertiary care institutes in Japan. In all, 286 bereaved family members (38.2% male; median age 66.0 [interquartile range 58.0-73.0] years) of CVD patients were enrolled; of these, 200 (69.9%) reported that their families had had EOL discussions with physicians. The major topic discussed was resuscitation (79.0%), and 21.5% discussed the place of EOL care. Most discussions were held during hospitalization of the patient (88.2%). More than half (57.1%) the discussions were initiated less than 1 month before the patient died, and 22.6% of family members felt that this timing of EOL discussions was late. Bereaved family members' perception of late EOL discussions was associated with the family members aggressive attitude towards life-prolonging treatment, less preparedness for bereavement, and less satisfaction with EOL care. CONCLUSIONS: Approximately 70% of bereaved family members of CVD patients had EOL discussions, which were often held shortly before the patient died. Further research is required to establish an ideal approach to EOL discussions at an appropriate time, which may improve the quality of EOL care.

Efficacy of exon-skipping therapy for DMD cardiomyopathy with mutations in actin binding domain 1.

Exon-skipping therapy is a promising treatment strategy for Duchenne muscular dystrophy (DMD), which is caused by loss-of-function mutations in the DMD gene encoding dystrophin, leading to progressive cardiomyopathy. In-frame deletion of exons 3-9 (Δ3-9), manifesting a very mild clinical phenotype, is a potential targeted reading frame for exon-skipping by targeting actin-binding domain 1 (ABD1); however, the efficacy of this approach for DMD cardiomyopathy remains uncertain. In this study, we compared three isogenic human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing Δ3-9, frameshifting Δ3-7, or intact DMD. RNA sequencing revealed a resemblance in the expression patterns of mechano-transduction-related genes between Δ3-9 and wild-type samples. Furthermore, we observed similar electrophysiological properties between Δ3-9 and wild-type hiPSC-CMs; Δ3-7 hiPSC-CMs showed electrophysiological alterations with accelerated CaMKII activation. Consistently, Δ3-9 hiPSC-CMs expressed substantial internally truncated dystrophin protein, resulting in maintaining F-actin binding and desmin retention. Antisense oligonucleotides targeting exon 8 efficiently induced skipping exons 8-9 to restore functional dystrophin and electrophysiological parameters in Δ3-7 hiPSC-CMs, bringing the cell characteristics closer to those of Δ3-9 hiPSC-CMs. Collectively, exon-skipping targeting ABD1 to convert the reading frame to Δ3-9 may become a promising therapy for DMD cardiomyopathy.