A case of atrial septal defect with right-to-left shunting without pulmonary hypertension.

We report a case of a 54-year-old man with atrial septal defect who presented with oxygen desaturation on pulse oximetry. Cardiac magnetic resonance imaging and transesophageal echocardiography showed right-to-left shunting through an atrial septal defect, which was confirmed by superior vena cavography and suggested Eisenmenger syndrome. However, cardiac catheterization revealed a normal pulmonary arterial pressure. Simultaneous measurement of interatrial pressure identified two transient interatrial pressure gradient points, where the right atrial pressure was higher than the left atrial pressure. The patient was finally diagnosed with atrial septal defect without pulmonary hypertension. Right-to-left shunting was primarily caused by a transient interatrial pressure gradient due to a time delay in both initial atrial contraction and completion of passive ventricular filling between the right and left sides of the heart. Surgical closure of the atrial septal defect was performed, and hypoxemia improved. This is the first report of right-to-left shunting without pulmonary hypertension caused by a transient interatrial pressure gradient due to a time delay between the right and left cardiac cycles. Precise assessment of the simultaneous interatrial pressure in addition to diagnostic imaging played a pivotal role in clarifying the etiology of this rare condition. Learning objective: Atrial septal defect with right-to-left shunting without Eisenmenger syndrome is a rare condition. We identified transient interatrial pressure gradients associated with a time delay in both initial atrial contraction and completion of the passive ventricular filling phase, which we considered as the primary mechanism underpinning right-to-left shunting. Simultaneous measurement of interatrial pressure played a pivotal role in elucidating the hemodynamics and abnormal shunt flow mechanism.

MEF2C/p300-mediated epigenetic remodeling promotes the maturation of induced cardiomyocytes.

Cardiac transcription factors (TFs) directly reprogram fibroblasts into induced cardiomyocytes (iCMs), where MEF2C acts as a pioneer factor with GATA4 and TBX5 (GT). However, the generation of functional and mature iCMs is inefficient, and the molecular mechanisms underlying this process remain largely unknown. Here, we found that the overexpression of transcriptionally activated MEF2C via fusion of the powerful MYOD transactivation domain combined with GT increased the generation of beating iCMs by 30-fold. Activated MEF2C with GT generated iCMs that were transcriptionally, structurally, and functionally more mature than those generated by native MEF2C with GT. Mechanistically, activated MEF2C recruited p300 and multiple cardiogenic TFs to cardiac loci to induce chromatin remodeling. In contrast, p300 inhibition suppressed cardiac gene expression, inhibited iCM maturation, and decreased the beating iCM numbers. Splicing isoforms of MEF2C with similar transcriptional activities did not promote functional iCM generation. Thus, MEF2C/p300-mediated epigenetic remodeling promotes iCM maturation.

Inhibition of microRNA-33b in humanized mice ameliorates nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) can lead to cirrhosis and hepatocellular carcinoma in their advanced stages; however, there are currently no approved therapies. Here, we show that microRNA (miR)-33b in hepatocytes is critical for the development of NASH. miR-33b is located in the intron of sterol regulatory element-binding transcription factor 1 and is abundantly expressed in humans, but absent in rodents. miR-33b knock-in (KI) mice, which have a miR-33b sequence in the same intron of sterol regulatory element-binding transcription factor 1 as humans and express miR-33b similar to humans, exhibit NASH under high-fat diet feeding. This condition is ameliorated by hepatocyte-specific miR-33b deficiency but unaffected by macrophage-specific miR-33b deficiency. Anti-miR-33b oligonucleotide improves the phenotype of NASH in miR-33b KI mice fed a Gubra Amylin NASH diet, which induces miR-33b and worsens NASH more than a high-fat diet. Anti-miR-33b treatment reduces hepatic free cholesterol and triglyceride accumulation through up-regulation of the lipid metabolism-related target genes. Furthermore, it decreases the expression of fibrosis marker genes in cultured hepatic stellate cells. Thus, inhibition of miR-33b using nucleic acid medicine is a promising treatment for NASH.

Essential thrombocythemia complicated with simultaneous two-vessel acute myocardial infarction in the subacute phase of takotsubo cardiomyopathy: A case report.

We report the case of a 79-year-old woman with essential thrombocythemia who presented with simultaneous two-vessel acute myocardial infarction (AMI) in the subacute phase of takotsubo cardiomyopathy. Despite sufficient anticoagulation therapy with warfarin to prevent thrombus formation in the left ventricle, the patient developed simultaneous two-vessel AMI in the right and left circumflex coronary arteries 16 days after the onset of takotsubo cardiomyopathy. Thromboembolism from the left ventricle associated with takotsubo cardiomyopathy was considered a potential cause of this event. However, macroscopic and pathological findings of the aspirated thrombi revealed that the primary cause of AMI was non-organized white platelet thrombi associated with essential thrombocythemia. In addition to oral anticoagulation therapy with warfarin, low-dose aspirin was started. The patient was discharged without any symptoms, and the clinical course has been uneventful for >5 years. This case highlights the potential risk of fatal complications associated with essential thrombocythemia, including simultaneous multivessel AMI. Additionally, pathological findings of the thrombi may play a crucial role in clarifying the etiology in such complicated cases. Appropriate antithrombotic therapy should be selected according to the pathogenesis of the condition. Learning objective: We describe a 79-year-old woman with essential thrombocythemia complicated with simultaneous two-vessel acute myocardial infarction (AMI) in the subacute phase of takotsubo cardiomyopathy. Although patients with essential thrombocythemia are highly predisposed to thrombotic events including AMI, the appropriate antithrombotic regimen remains controversial. The macroscopic and pathological findings of the thrombi play a pivotal role in clarifying the etiology, which may lead to the appropriate antithrombotic therapy.

Prevalence of the Academic Research Consortium high bleeding risk criteria in patients undergoing endovascular therapy for peripheral artery disease in lower extremities.

The Academic Research Consortium (ARC) recently published a definition of patients at high bleeding risk (HBR) undergoing percutaneous coronary intervention. However, the prevalence of the ARC-HBR criteria in patients undergoing endovascular therapy (EVT) for peripheral artery disease in lower extremities has not been thoroughly investigated. This study sought to investigate the prevalence and impact of the ARC-HBR criteria in patients undergoing EVT. We analyzed 277 consecutive patients who underwent their first EVT from July 2011 to September 2019. We applied the full ARC-HBR criteria to the study population. The primary end point was a composite outcome of all-cause mortality, Bleeding Academic Research Consortium 3 or 5 bleeding, and lower limb amputation within 12 months of EVT. Among the 277 patients, 193 (69.7%) met the ARC-HBR criteria. HBR patients had worse clinical outcomes compared with non-HBR patients at 12 months after EVT, including a higher incidence of the composite primary outcome (19.2% vs. 3.6%, p < 0.001) and all-cause death (7.8% vs. 0%, p = 0.007). In a multivariate Cox proportional hazards regression analysis, presence of the ARC-HBR criteria [hazard ratio (HR) 4.15, 95% confidence interval (CI) 1.25-13.80, p = 0.020], body mass index (HR 1.13, 95% CI 1.01-1.27, p = 0.042), diabetes mellitus (HR 2.70, 95% CI 1.28-5.69, p = 0.009), hyperlipidemia (HR 0.41, 95% CI 0.21-0.80, p = 0.009), and infrapopliteal lesions (HR 3.51, 95% CI 1.63-7.56, p = 0.001) were independent predictors of the primary composite outcome. Approximately 70% of Japanese patients undergoing EVT met the ARC-HBR criteria, and its presence was strongly associated with adverse outcomes within 12 months of EVT.

A woman complicated by sudden cardiac arrest owing to spontaneous coronary artery dissection after stillbirth.

Spontaneous coronary artery dissection (SCAD) is the most important cause of acute coronary syndrome in pregnant women. Pregnancy-associated SCAD frequently occurs in the third trimester or postpartum period. However, little is known regarding the relationship between the occurrence of SCAD and stillbirth. We describe here a 41-year-old woman complicated by sudden cardiac arrest owing to SCAD in the distal segment of the right coronary artery 13 days after stillbirth. After contacting emergency medical services, she was resuscitated by an automated external defibrillator because the initial electrocardiographic waveform was ventricular fibrillation. After cardiopulmonary resuscitation, the diagnosis of SCAD was confirmed by coronary angiography and intracoronary imaging, including intravascular ultrasound and optical coherence tomography. The patient was managed with conservative medical therapy because the culprit lesion was present in the distal segment of the right coronary artery and coronary blood flow was preserved. No major adverse cardiovascular events, including recurrent ventricular arrhythmia, were observed during hospitalization. Our findings indicate that pregnancy-associated SCAD leading to sudden cardiac arrest may occur in the postpartum period, even after stillbirth. Intravascular imaging plays a pivotal role in diagnosing SCAD. .

Successful Transcatheter Aortic Valve Implantation in a Patient with Radiation-induced Aortic Stenosis for Mediastinal Hodgkin Lymphoma.

Aortic stenosis (AS), a late complication of thoracic radiation therapy for chest lesions, is often coincident with porcelain aorta or hostile thorax. We herein report a 59-year-old man with a history of mediastinal Hodgkin lymphoma treated with radiation therapy but later presenting with heart failure caused by severe AS. Severe calcification in the mediastinum and around the ascending aorta made it difficult to perform surgical aortic valve replacement. The patient therefore underwent transcatheter aortic valve implantation (TAVI). It is important to recognize radiation-induced AS early, now that TAVI is a well-established treatment required by increasing numbers of successfully treated cancer patients.

Soft Matrix Promotes Cardiac Reprogramming via Inhibition of YAP/TAZ and Suppression of Fibroblast Signatures.

Direct cardiac reprogramming holds great potential for regenerative medicine. However, it remains inefficient, and induced cardiomyocytes (iCMs) generated in vitro are less mature than those in vivo, suggesting that undefined extrinsic factors may regulate cardiac reprogramming. Previous in vitro studies mainly used hard polystyrene dishes, yet the effect of substrate rigidity on cardiac reprogramming remains unclear. Thus, we developed a Matrigel-based hydrogel culture system to determine the roles of matrix stiffness and mechanotransduction in cardiac reprogramming. We found that soft matrix comparable with native myocardium promoted the efficiency and quality of cardiac reprogramming. Mechanistically, soft matrix enhanced cardiac reprogramming via inhibition of integrin, Rho/ROCK, actomyosin, and YAP/TAZ signaling and suppression of fibroblast programs, which were activated on rigid substrates. Soft substrate further enhanced cardiac reprogramming with Sendai virus vectors via YAP/TAZ suppression, increasing the reprogramming efficiency up to ∼15%. Thus, mechanotransduction could provide new targets for improving cardiac reprogramming.

Tbx6 induces cardiomyocyte proliferation in postnatal and adult mouse hearts.

Cardiovascular disease is a leading cause of death worldwide. Mammalian cardiomyocytes (CMs) proliferate during embryonic development, whereas they largely lose their regenerative capacity after birth. Defined factors expressed in cardiac progenitors or embryonic CMs may activate the cell cycle and induce CM proliferation in postnatal and adult hearts. Here, we report that the overexpression of Tbx6, enriched in the cardiac mesoderm (progenitor cells), induces CM proliferation in postnatal and adult mouse hearts. By screening 24 factors enriched in cardiac progenitors or embryonic CMs, we found that only Tbx6 could induce CM proliferation in primary cultured postnatal rat CMs. Intriguingly, it did not induce the proliferation of cardiac fibroblasts. We next generated a recombinant adeno-associated virus serotype 9 vector encoding Tbx6 (AAV9-Tbx6) for transduction into mouse CMs in vivo. The subcutaneous injection of AAV9-Tbx6 into neonatal mice induced CM proliferation in postnatal and adult mouse hearts. Mechanistically, Tbx6 overexpression upregulated multiple cell cycle activators including Aurkb, Mki67, Ccna1, and Ccnb2 and suppressed the tumor suppressor Rb1. Thus, Tbx6 promotes CM proliferation in postnatal and adult mouse hearts by modifying the expression of cell cycle regulators.

Role of cyclooxygenase-2-mediated prostaglandin E2-prostaglandin E receptor 4 signaling in cardiac reprogramming.

Direct cardiac reprogramming from fibroblasts can be a promising approach for disease modeling, drug screening, and cardiac regeneration in pediatric and adult patients. However, postnatal and adult fibroblasts are less efficient for reprogramming compared with embryonic fibroblasts, and barriers to cardiac reprogramming associated with aging remain undetermined. In this study, we screened 8400 chemical compounds and found that diclofenac sodium (diclofenac), a non-steroidal anti-inflammatory drug, greatly enhanced cardiac reprogramming in combination with Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2. Intriguingly, diclofenac promoted cardiac reprogramming in mouse postnatal and adult tail-tip fibroblasts (TTFs), but not in mouse embryonic fibroblasts (MEFs). Mechanistically, diclofenac enhanced cardiac reprogramming by inhibiting cyclooxygenase-2, prostaglandin E2/prostaglandin E receptor 4, cyclic AMP/protein kinase A, and interleukin 1ß signaling and by silencing inflammatory and fibroblast programs, which were activated in postnatal and adult TTFs. Thus, anti-inflammation represents a new target for cardiac reprogramming associated with aging.

A tale of two sisters with hypertrophic cardiomyopathy and recurrent embolism: When is the optimal timing of the intervention for left atrial appendage?

Hypertrophic cardiomyopathy (HCM) is an extremely heterogeneous genetic disease that affects the left ventricle (LV) and has a varied clinical course and phenotypic expression. Here, we report a case of two sisters with HCM who developed a massive refractory left atrial appendage (LAA) thrombus and recurrent embolism. The older sister, who was at a high surgical risk due to progressive LV systolic dysfunction with an ejection fraction of 19%, underwent LAA plication in combination with implantation of an LV assist device after progression to treatment-refractory heart failure at the age of 49. The younger sister underwent surgical thrombectomy, LAA plication, and Maze surgery before deterioration of heart failure at the age of 47. She was free from embolism and atrial fibrillation for 2years after surgery. Individualized therapeutic approaches targeting the LAA at a relatively early stage are required in the subgroups of HCM patients with left atrial dysfunction.

Tbx6 Induces Nascent Mesoderm from Pluripotent Stem Cells and Temporally Controls Cardiac versus Somite Lineage Diversification.

The mesoderm arises from pluripotent epiblasts and differentiates into multiple lineages; however, the underlying molecular mechanisms are unclear. Tbx6 is enriched in the paraxial mesoderm and is implicated in somite formation, but its function in other mesoderms remains elusive. Here, using direct reprogramming-based screening, single-cell RNA-seq in mouse embryos, and directed cardiac differentiation in pluripotent stem cells (PSCs), we demonstrated that Tbx6 induces nascent mesoderm from PSCs and determines cardiovascular and somite lineage specification via its temporal expression. Tbx6 knockout in mouse PSCs using CRISPR/Cas9 technology inhibited mesoderm and cardiovascular differentiation, whereas transient Tbx6 expression induced mesoderm and cardiovascular specification from mouse and human PSCs via direct upregulation of Mesp1, repression of Sox2, and activation of BMP/Nodal/Wnt signaling. Notably, prolonged Tbx6 expression suppressed cardiac differentiation and induced somite lineages, including skeletal muscle and chondrocytes. Thus, Tbx6 is critical for mesoderm induction and subsequent lineage diversification.

Direct In Vivo Reprogramming with Sendai Virus Vectors Improves Cardiac Function after Myocardial Infarction.

Direct cardiac reprogramming holds great promise for regenerative medicine. We previously generated directly reprogrammed induced cardiomyocyte-like cells (iCMs) by overexpression of Gata4, Mef2c, and Tbx5 (GMT) using retrovirus vectors. However, integrating vectors pose risks associated with insertional mutagenesis and disruption of gene expression and are inefficient. Here, we show that Sendai virus (SeV) vectors expressing cardiac reprogramming factors efficiently and rapidly reprogram both mouse and human fibroblasts into integration-free iCMs via robust transgene expression. SeV-GMT generated 100-fold more beating iCMs than retroviral-GMT and shortened the duration to induce beating cells from 30 to 10 days in mouse fibroblasts. In vivo lineage tracing revealed that the gene transfer of SeV-GMT was more efficient than retroviral-GMT in reprogramming resident cardiac fibroblasts into iCMs in mouse infarct hearts. Moreover, SeV-GMT improved cardiac function and reduced fibrosis after myocardial infarction. Thus, efficient, non-integrating SeV vectors may serve as a powerful system for cardiac regeneration.

Distinct expression patterns of Flk1 and Flt1 in the coronary vascular system during development and after myocardial infarction.

The coronary vascular system is critical for myocardial growth and cardiomyocyte survival. However, the molecular mechanism regulating coronary angiogenesis remains elusive. Vascular endothelial growth factor (VEGF) regulates angiogenesis by binding to the specific receptors Flk1 and Flt1, which results in different functions. Despite the importance of Flk1 and Flt1, their expression in the coronary vasculature remains largely unknown due to the lack of appropriate antibodies for immunostaining. Here, we analyzed multiple reporter mice including Flk1-GFP BAC transgenic (Tg), Flk1-LacZ knock-in, Flt1-DsRed BAC Tg, and Flk1-GFP/Flt1-DsRed double Tg animals to determine expression patterns in mouse hearts during cardiac growth and after myocardial infarction (MI). We found that Flk1 was expressed in endothelial cells (ECs) with a pattern of epicardial-to-endocardial transmural gradients in the neonatal mouse ventricle, which was downregulated in adult coronary vessels with development. In contrast, Flt1 was homogeneously expressed in the ECs of neonatal mouse hearts and expression was maintained until adulthood. After MI, expression of both Flk1 and Flt1 was induced in the regenerating coronary vessels at day 7. Intriguingly, Flk1 expression was downregulated thereafter, whereas Flt1 expression was maintained in the newly formed coronary vessels until 30 days post-MI, recapitulating their expression kinetics during development. This is the first report demonstrating the spatiotemporal expression patterns of Flk1 and Flt1 in the coronary vascular system during development and after MI; thus, this study suggests that these factors have distinct and important functions in coronary angiogenesis.

Single-Construct Polycistronic Doxycycline-Inducible Vectors Improve Direct Cardiac Reprogramming and Can Be Used to Identify the Critical Timing of Transgene Expression.

Direct reprogramming is a promising approach in regenerative medicine. Overexpression of the cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2 (GHMT) directly reprogram fibroblasts into cardiomyocyte-like cells (iCMs). However, the critical timing of transgene expression and the molecular mechanisms for cardiac reprogramming remain unclear. The conventional doxycycline (Dox)-inducible temporal transgene expression systems require simultaneous transduction of two vectors (pLVX-rtTA/pLVX-cDNA) harboring the reverse tetracycline transactivator (rtTA) and the tetracycline response element (TRE)-controlled transgene, respectively, leading to inefficient cardiac reprogramming. Herein, we developed a single-construct-based polycistronic Dox-inducible vector (pDox-cDNA) expressing both the rtTA and TRE-controlled transgenes. Fluorescence activated cell sorting (FACS) analyses, quantitative RT-PCR, and immunostaining revealed that pDox-GMT increased cardiac reprogramming three-fold compared to the conventional pLVX-rtTA/pLVX-GMT. After four weeks, pDox-GMT-induced iCMs expressed multiple cardiac genes, produced sarcomeric structures, and beat spontaneously. Co-transduction of pDox-Hand2 with retroviral pMX-GMT increased cardiac reprogramming three-fold compared to pMX-GMT alone. Temporal Dox administration revealed that Hand2 transgene expression is critical during the first two weeks of cardiac reprogramming. Microarray analyses demonstrated that Hand2 represses cell cycle-promoting genes and enhances cardiac reprogramming. Thus, we have developed an efficient temporal transgene expression system, which could be invaluable in the study of cardiac reprogramming.

Discovery and progress of direct cardiac reprogramming.

Cardiac disease remains a major cause of death worldwide. Direct cardiac reprogramming has emerged as a promising approach for cardiac regenerative therapy. After the discovery of MyoD, a master regulator for skeletal muscle, other single cardiac reprogramming factors (master regulators) have been sought. Discovery of cardiac reprogramming factors was inspired by the finding that multiple, but not single, transcription factors were needed to generate induced pluripotent stem cells (iPSCs) from fibroblasts. We first reported a combination of cardiac-specific transcription factors, Gata4, Mef2c, and Tbx5 (GMT), that could convert mouse fibroblasts into cardiomyocyte-like cells, which were designated as induced cardiomyocyte-like cells (iCMs). Following our first report of cardiac reprogramming, many researchers, including ourselves, demonstrated an improvement in cardiac reprogramming efficiency, in vivo direct cardiac reprogramming for heart regeneration, and cardiac reprogramming in human cells. However, cardiac reprogramming in human cells and adult fibroblasts remains inefficient, and further efforts are needed. We believe that future research elucidating epigenetic barriers and molecular mechanisms of direct cardiac reprogramming will improve the reprogramming efficiency, and that this new technology has great potential for clinical applications.

Fibroblast Growth Factors and Vascular Endothelial Growth Factor Promote Cardiac Reprogramming under Defined Conditions.

Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors, including Gata4, Mef2c, and Tbx5; however, this process is inefficient under serum-based culture conditions, in which conversion of partially reprogrammed cells into fully reprogrammed functional iCMs has been a major hurdle. Here, we report that a combination of fibroblast growth factor (FGF) 2, FGF10, and vascular endothelial growth factor (VEGF), termed FFV, promoted cardiac reprogramming under defined serum-free conditions, increasing spontaneously beating iCMs by 100-fold compared with those under conventional serum-based conditions. Mechanistically, FFV activated multiple cardiac transcriptional regulators and converted partially reprogrammed cells into functional iCMs through the p38 mitogen-activated protein kinase and phosphoinositol 3-kinase/AKT pathways. Moreover, FFV enabled cardiac reprogramming with only Mef2c and Tbx5 through the induction of cardiac reprogramming factors, including Gata4. Thus, defined culture conditions promoted the quality of cardiac reprogramming, and this finding provides new insight into the mechanism of cardiac reprogramming.