MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures.

Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming.

Identification of Left Atrial Appendage Thrombi in Patients With Persistent and Long-Standing Persistent Atrial Fibrillation Using Intra-Cardiac Echocardiography and Cardiac Computed Tomography.

BACKGROUND: Intracardiac echocardiography (ICE) and cardiac computed tomography (CCT), in addition to standard transesophageal echocardiography (TEE), have been used to identify left atrial (LA) thrombi prior to ablation for atrial fibrillation (AF). The clinical advantages of this, however, remain unclear. This study therefore investigated the advantages of additional pre-procedural LA appendage (LAA) thrombus evaluation using ICE and the clinical value of CCT in persistent and long-standing persistent AF.Methods and Results:We analyzed data from 108 consecutive patients with persistent and long-standing persistent AF who were scheduled to undergo AF ablation. TEE was performed within 24 h prior to ablation. ICE was performed for 97 patients in whom a thrombus was not detected on TEE. CCT was performed in 95 patients. Thrombus or sludge was detected on TEE in 11 patients (10.3%), for whom ablation was cancelled. Four additional patients were diagnosed with LAA thrombus on ICE. When TEE and ICE were used as the reference for thrombus detection, the sensitivity, specificity, positive predictive value, and negative predictive value of CCT for identifying contrast defects in the LAA were 100%, 81.0%, 40.7%, and 100%, respectively. CONCLUSIONS: ICE combined with TEE increased the detection rate of LAA thrombi in patients with persistent and long-standing persistent AF. Moreover, CCT had high sensitivity and negative predictive value for LAA thrombus detection.

Induction of human cardiomyocyte-like cells from fibroblasts by defined factors.

Heart disease remains a leading cause of death worldwide. Owing to the limited regenerative capacity of heart tissue, cardiac regenerative therapy has emerged as an attractive approach. Direct reprogramming of human cardiac fibroblasts (HCFs) into cardiomyocytes may hold great potential for this purpose. We reported previously that induced cardiomyocyte-like cells (iCMs) can be directly generated from mouse cardiac fibroblasts in vitro and vivo by transduction of three transcription factors: Gata4, Mef2c, and Tbx5, collectively termed GMT. In the present study, we sought to determine whether human fibroblasts also could be converted to iCMs by defined factors. Our initial finding that GMT was not sufficient for cardiac induction in HCFs prompted us to screen for additional factors to promote cardiac reprogramming by analyzing multiple cardiac-specific gene induction with quantitative RT-PCR. The addition of Mesp1 and Myocd to GMT up-regulated a broader spectrum of cardiac genes in HCFs more efficiently compared with GMT alone. The HCFs and human dermal fibroblasts transduced with GMT, Mesp1, and Myocd (GMTMM) changed the cell morphology from a spindle shape to a rod-like or polygonal shape, expressed multiple cardiac-specific proteins, increased a broad range of cardiac genes and concomitantly suppressed fibroblast genes, and exhibited spontaneous Ca(2+) oscillations. Moreover, the cells matured to exhibit action potentials and contract synchronously in coculture with murine cardiomyocytes. A 5-ethynyl-2'-deoxyuridine assay revealed that the iCMs thus generated do not pass through a mitotic cell state. These findings demonstrate that human fibroblasts can be directly converted to iCMs by defined factors, which may facilitate future applications in regenerative medicine.

Visualization of the left atrial appendage by phased-array intracardiac echocardiography from the pulmonary artery in patients with atrial fibrillation.

AIMS: The left atrial appendage (LAA) represents the major source of cardiac thrombus formation in patients with atrial fibrillation (AF). Phased-array intracardiac echocardiography (ICE) has become available and frequently used during catheter ablation of AF. We attempted to study the feasibility of using ICE for the visualization and evaluation of the LAA from the pulmonary artery (PA) in patients with AF. METHODS AND RESULTS: Eighty patients with AF undergoing catheter ablation (70 males, 57.5 ± 9.1 years) were included. Transoesophageal echocardiography was performed on the prior day before the catheter ablation, and ICE was performed just before the transseptal puncture during the catheter ablation. The ICE catheter was advanced up into the PA from the femoral vein, where the LAA was clearly and entirely visualized by manipulating the ICE catheter. We compared the degree of spontaneous echo contrast, and the correlation was obtained between the ICE and TEE (κ = 0.534, P < 0.001). Furthermore, the LAA flow velocity (LAA emptying and filling velocities) measured by ICE had a good correlation to that measured by TEE (R = 0.872, P < 0.01 and R = 0.753, P < 0.01, respectively). No patients developed any complications. CONCLUSION: The utilization of ICE in the PA is feasible for the observation and evaluation of the LAA.

Induction of cardiomyocyte-like cells in infarct hearts by gene transfer of Gata4, Mef2c, and Tbx5.

RATIONALE: After myocardial infarction (MI), massive cell death in the myocardium initiates fibrosis and scar formation, leading to heart failure. We recently found that a combination of 3 cardiac transcription factors, Gata4, Mef2c, and Tbx5 (GMT), reprograms fibroblasts directly into functional cardiomyocytes in vitro. OBJECTIVE: To investigate whether viral gene transfer of GMT into infarcted hearts induces cardiomyocyte generation. METHODS AND RESULTS: Coronary artery ligation was used to generate MI in the mouse. In vitro transduction of GMT retrovirus converted cardiac fibroblasts from the infarct region into cardiomyocyte-like cells with cardiac-specific gene expression and sarcomeric structures. Injection of the green fluorescent protein (GFP) retrovirus into mouse hearts, immediately after MI, infected only proliferating noncardiomyocytes, mainly fibroblasts, in the infarct region. The GFP expression diminished after 2 weeks in immunocompetent mice but remained stable for 3 months in immunosuppressed mice, in which cardiac induction did not occur. In contrast, injection of GMT retrovirus into α-myosin heavy chain (αMHC)-GFP transgenic mouse hearts induced the expression of αMHC-GFP, a marker of cardiomyocytes, in 3% of virus-infected cells after 1 week. A pooled GMT injection into the immunosuppressed mouse hearts induced cardiac marker expression in retrovirus-infected cells within 2 weeks, although few cells showed striated muscle structures. To transduce GMT efficiently in vivo, we generated a polycistronic retrovirus expressing GMT separated by 2A "self-cleaving" peptides (3F2A). The 3F2A-induced cardiomyocyte-like cells in fibrotic tissue expressed sarcomeric α-actinin and cardiac troponin T and had clear cross striations. Quantitative RT-PCR also demonstrated that FACS-sorted 3F2A-transduced cells expressed cardiac-specific genes. CONCLUSIONS: GMT gene transfer induced cardiomyocyte-like cells in infarcted hearts.

Serum inflammation markers predicting successful initial catheter ablation for atrial fibrillation.

BACKGROUND: We investigated various serum inflammatory markers to predict ablation responders who have no atrial fibrillation (AF) relapse after the initial ablation. METHODS: Forty-four consecutive AF patients (age: 59 ± 8 years, paroxysmal: 31, CHADS2: 1.1 ± 1.1) who underwent an initial pulmonary vein isolation were investigated. Various serum inflammatory markers, such as adiponectin, ANP, BNP, 1CTP, F1+2, hs-CRP, IL-6, intact P1NP, MDA-LDL, MMP-2, TGF-ß, TIMP-2, and TNF-α, were evaluated prior to ablation. AF relapse was defined as AF documented in telemonitoring electrocardiograms twice a day during 9.7 ± 2.4 months of follow-up with three months of a blanking-period. RESULTS: A total of 29 patients (paroxysmal: 21) maintained sinus rhythm after the initial catheter ablation. These ablation responders had significantly lower MMP-2 (Sinus vs. Relapsed: 748 ± 132.7 vs. 841.2 ± 152.4 ng/mL, P=0.042) and TNF-α (1.1 ± 0.4 vs. 1.8 ± 1.7 pg/mL, P=0.046) levels prior to ablation. A BNP-adjusted Cox multivariate regression analysis revealed that the independent predictive factor for AF recurrence was high MMP-2 levels (>766 ng/mL) accompanied by high TNF-α levels (>1.2 pg/mL). CONCLUSIONS: The levels of MMP-2 and TNF-α might be useful for predicting initial AF catheter ablation responders.

Storms of ventricular fibrillation responsive to isoproterenol in an idiopathic ventricular fibrillation patient demonstrating complete right bundle branch block.

A 45-year-old male was admitted to our hospital after successful resuscitation of cardiac arrest. Ventricular fibrillation (VF) had occurred during breakfast and was defibrillated by an automated external defibrillator operated by emergency medical service staff. On admission, his ECG demonstrated complete right bundle branch block as the sole abnormality. Intensive examination could not detect any structural disease leading to a diagnosis of idiopathic VF and implantation of an ICD. VF storm occurred one month after hospital discharge and beta-blocker, amiodarone, and sedative administration had no effect on VF. Likewise, catheter ablation for triggering premature ventricular beats failed to control the VF storm. The VF storm then subsided in the following weeks and the patient was discharged on amiodarone. A half month later VF storm recurred and the patient was admitted again. This time, isoproterenol infusion was effective in suppressing VF, and thereafter the patient was administered bepridil and followed up without recurrence of VF for 1.5 years. From these beneficial effects, the VF of the patient was suggested to share common arrhythmogenic characteristics to those of Brugada syndrome or J-wave associated VF.

Downregulation of KIF23 suppresses glioma proliferation.

To identify therapeutic molecular targets for glioma, we performed modified serological identification of antigens by recombinant complementary DNA (cDNA) expression cloning using sera from a mouse glioma model. Two clones, kinesin family member 23 (Kif23) and structural maintenance of chromosomes 4 (Smc4), were identified as antigens through immunological reaction with sera from mice harboring synergic GL261 mouse glioma and intratumoral inoculation with a mutant herpes simplex virus. The human Kif23 homolog KIF23 is a nuclear protein that localizes to the interzone of mitotic spindles, acting as a plus-end-directed motor enzyme that moves antiparallel microtubules in vitro. Expression analysis revealed a higher level of KIF23 expression in glioma tissues than in normal brain tissue. The introduction of small interfering RNA (siRNA) targeting KIF23 into two different glioma cell lines, U87MG and SF126, downregulated KIF23 expression, which significantly suppressed glioma cell proliferation in vitro. KIF23 siRNA-treated glioma cells exhibited larger cell bodies with two or more nuclei compared with control cells. In vivo analysis using mouse xenograft showed that KIF23 siRNA/DNA chimera-treated tumors were significantly smaller than tumors treated with control siRNA/DNA chimera. Taken together, our results indicate that downregulation of KIF23 decreases proliferation of glioma cells and that KIF23 may be a novel therapeutic target in malignant glioma.

A Cluster Analysis of the Japanese Multicenter Outpatient Registry of Patients With Atrial Fibrillation.

Recently, cluster analysis was used to identify unique clinically relevant phenotypes of atrial fibrillation (AF) in a cohort from the United States (US) and classified clusters according to the presence of comorbid behavioral disorders, those with conduction disorders, or atherosclerotic comorbidities. Whether these phenotypes are consistent in AF cohorts outside the US remains unknown. Thus, we sought to conduct a cluster analysis in a cohort of Japanese AF patients. We conducted a cluster analysis of phenotypic data (46 variables) in an AF patient cohort recruited from 11 Japanese sites participating in the KiCS-AF Registry. Overall, 2,458 AF patients (median [IQR] age, 68.0 [60.0 to 76.0]; 30.3% female; median [IQR] CHA2DS2-Vasc, 2 [1, 3]) were analyzed. Similar to the US cohort, atherosclerotic comorbidities were identified as distinguishing factors to characterize clusters. Distribution of AF type and left atrial (LA) size substantially varied and was the key feature for cluster formation. CHA2DS2-Vasc score also contributed to cluster formation, although behavioral disorders and/or conduction disorders did not readily characterize clusters. Subsequently, the cohort was classified into 3 clusters: (1) Younger paroxysmal AF (n = 1,190); (2) Persistent/permanent AF with LA enlargement (n = 1,143); and (3) Atherosclerotic comorbid AF in elderly patients (N = 125). In conclusion, conventional classifications, such as atherosclerotic risk factors and CHA2DS2-Vasc score contributed to cluster formation in mutually, whereas in nonatherosclerotic clusters, AF type or LA size rather than the presence or absence of behavior risk factors or sinus node dysfunction (tachy-brady syndrome) seemed to contribute to cluster formation in the Japanese cohort.

Asymptomatic Cerebral Infarction During Catheter Ablation for Atrial Fibrillation: Comparing Uninterrupted Rivaroxaban and Warfarin (ASCERTAIN).

OBJECTIVES: This randomized study compared uninterrupted rivaroxaban therapy with warfarin therapy as prophylaxis against catheter ablation (CA)-induced asymptomatic cerebral infarction (ACI) and identified the risk factors of rivaroxaban. BACKGROUND: The reported incidence of ACI during CA for atrial fibrillation (AF) remains at 10% to 30%, and periprocedural oral anticoagulation could affect this incidence. METHODS: Patients with nonvalvular AF undergoing radiofrequency CA were randomly assigned to receive either uninterrupted rivaroxaban or warfarin as periprocedural anticoagulation therapy. CA was performed after at least 1 month of adequate anticoagulation. Cerebral magnetic resonance imaging (MRI) was performed within 2 weeks before and 1 day after CA to detect ACI. RESULTS: A total 132 patients were enrolled; 127 (median: 60.0 years of age; 83.5% males; 64.6% incidence of paroxysmal AF) complied with the study protocol and were analyzed; 64 patients received rivaroxaban, and 63 patients received warfarin. The rates of CA-induced ACI in the rivaroxaban group (15.6% [10 of 64 patients]) were similar to those in the warfarin group (15.9% [10 of 63 patients]; p = 1.000). No thromboembolic events developed; no differences in major or nonmajor bleeding rates were observed between the 2 drug groups (3.1% vs. 1.6%, respectively, or 18.8% vs. 19.0%, respectively). Multiple regression analysis indicated that the presence of deep and subcortical white matter hyperintensity (p = 0.002; odds ratio [OR]: 5.323) and the frequency of cardioversions (p = 0.016; OR: 1.250) were associated with the incidence of ACI. CONCLUSIONS: No notable differences were found between the incidence of CA-induced ACI in the rivaroxaban group and that in the warfarin group in this randomized study.

Direct reprogramming of mouse fibroblasts into cardiac myocytes.

The potency of specific transcription factors as cell fate determinants was first demonstrated by the discovery of MyoD, a master gene for skeletal muscle transdifferentiation. More recently, the induction of pluripotency in somatic cells using a combination of stem cell-specific transcription factors has been reported. That elegant study altered the approach to regenerative medicine and inspired new strategies for generating specific cell types by introducing combinations of lineage-specific transcription factors. A diverse range of cell types, such as pancreatic ß-cells, neurons, chondrocytes, and hepatocytes, can be induced from heterologous cells using lineage-specific reprogramming factors. Furthermore, functional cardiomyocytes can be generated directly from differentiated somatic cells using several combinations of cardiac-enriched defined factors in the mouse. The present article reviews the pioneering and recent studies in cellular reprogramming and discusses the perspectives and challenges of direct cardiac reprogramming in regenerative therapy.

Anatomical characteristics of the left atrial appendage in cardiogenic stroke with low CHADS2 scores.

BACKGROUND: Strokes develop even in patients with low CHADS2 scores, and the left atrial appendage (LAA) is the embolic source 90% of the time. We focused on the LAA morphology as a new predictor of strokes. OBJECTIVE: To clarify the anatomical characteristics of the LAA for risk stratification of strokes in patients with nonvalvular atrial fibrillation (AF) who have low CHADS2 scores. METHODS: Among 80 patients who underwent catheter ablation of AF with contrast-enhanced computed tomography, the LAA characteristics were compared between 30 patients with histories of strokes and 50 age-matched controls. The LAA anatomy was classified into 4 types--"cactus," "cauliflower," "chicken wing," and "windsock"--discriminated by the computed tomography measurements of the length, angle, and number of lobes of the LAA. RESULTS: The average CHADS2 score did not differ significantly between patients with stroke and controls (0.8 ± 0.8 vs 0.6 ± 0.7; P = .277). Eight (26.7%) patients with stroke had CHA2DS2-VASc scores of 0. The left atrial size, LAA flow velocity, left ventricular function, and serum brain natriuretic peptide level were also unable to predict strokes. However, a "cauliflower" LAA, defined as a main lobe of less than 4 cm long without forked lobes, was significantly more common in patients with stroke (odds ratio 3.857; 95% confidence interval 1.482-10.037; P = .005). The CHA2DS2-VASc score-adjusted logistic regression analysis revealed the cauliflower LAA as an independent predictor of a stroke (odds ratio 3.355; 95% confidence interval 1.243-9.055; P = .017). CONCLUSIONS: The LAA anatomy might be useful for predicting strokes in patients with nonvalvular AF who have low CHADS2 scores.