Yohei Ohno

Nongenetic method for purifying stem cell-derived cardiomyocytes.

Several applications of pluripotent stem cell (PSC)-derived cardiomyocytes require elimination of undifferentiated cells. A major limitation for cardiomyocyte purification is the lack of easy and specific cell marking techniques. We found that a fluorescent dye that labels mitochondria, tetramethylrhodamine methyl ester perchlorate, could be used to selectively mark embryonic and neonatal rat cardiomyocytes, as well as mouse, marmoset and human PSC-derived cardiomyocytes, and that the cells could subsequently be enriched (>99% purity) by fluorescence-activated cell sorting. Purified cardiomyocytes transplanted into testes did not induce teratoma formation. Moreover, aggregate formation of PSC-derived cardiomyocytes through homophilic cell-cell adhesion improved their survival in the immunodeficient mouse heart. Our approaches will aid in the future success of using PSC-derived cardiomyocytes for basic and clinical applications.

In vitro pharmacologic testing using human induced pluripotent stem cell-derived cardiomyocytes

The lethal ventricular arrhythmia Torsade de pointes (TdP) is the most common reason for the withdrawal or restricted use of many cardiovascular and non-cardiovascular drugs. The lack of an in vitro model to detect pro-arrhythmic effects on human heart cells hinders the development of new drugs. We hypothesized that recently established human induced pluripotent stem (hiPS) cells could be used in an in vitro drug screening model. In this study, hiPS cells were driven to differentiate into functional cardiomyocytes, which expressed cardiac markers including Nkx2.5, GATA4, and atrial natriuretic peptide. The hiPS-derived cardiomyocytes (hiPS-CMs) were analyzed using a multi electrode assay. The application of ion channel inhibitors resulted in dose-dependent changes to the field potential waveform, and these changes were identical to those induced in the native cardiomyocytes. This study shows that hiPS-CMs represent a promising in vitro model for cardiac electrophysiologic studies and drug screening.

Disease characterization using LQTS-specific induced pluripotent stem cells

AIMS Long QT syndrome (LQTS) is an inheritable and life-threatening disease; however, it is often difficult to determine disease characteristics in sporadic cases with novel mutations, and more precise analysis is necessary for the successful development of evidence-based clinical therapies. This study thus sought to better characterize ion channel cardiac disorders using induced pluripotent stem cells (iPSCs). METHODS AND RESULTS We reprogrammed somatic cells from a patient with sporadic LQTS and from controls, and differentiated them into cardiomyocytes through embryoid body (EB) formation. Electrophysiological analysis of the LQTS-iPSC-derived EBs using a multi-electrode array (MEA) system revealed a markedly prolonged field potential duration (FPD). The IKr blocker E4031 significantly prolonged FPD in control- and LQTS-iPSC-derived EBs and induced frequent severe arrhythmia only in LQTS-iPSC-derived EBs. The IKs blocker chromanol 293B did not prolong FPD in the LQTS-iPSC-derived EBs, but significantly prolonged FPD in the control EBs, suggesting the involvement of IKs disturbance in the patient. Patch-clamp analysis and immunostaining confirmed a dominant-negative role for 1893delC in IKs channels due to a trafficking deficiency in iPSC-derived cardiomyocytes and human embryonic kidney (HEK) cells. CONCLUSIONS This study demonstrated that iPSCs could be useful to characterize LQTS disease as well as drug responses in the LQTS patient with a novel mutation. Such analyses may in turn lead to future progress in personalized medicine.

G-CSF Promotes the Proliferation of Developing Cardiomyocytes In Vivo and in Derivation from ESCs and iPSCs

During a screen for humoral factors that promote cardiomyocyte differentiation from embryonic stem cells (ESCs), we found marked elevation of granulocyte colony-stimulating factor receptor (G-CSFR) mRNA in developing cardiomyocytes. We confirmed that both G-CSFR and G-CSF were specifically expressed in embryonic mouse heart at the midgestational stage, and expression levels were maintained throughout embryogenesis. Intrauterine G-CSF administration induced embryonic cardiomyocyte proliferation and caused hyperplasia. In contrast, approximately 50% of csf3r(-/-) mice died during late embryogenesis because of the thinning of atrioventricular walls. ESC-derived developing cardiomyocytes also strongly expressed G-CSFR. When extrinsic G-CSF was administered to the ESC- and human iPSC-derived cardiomyocytes, it markedly augmented their proliferation. Moreover, G-CSF-neutralizing antibody inhibited their proliferation. These findings indicated that G-CSF is critically involved in cardiomyocyte proliferation during development, and may be used to boost the yield of cardiomyocytes from ESCs for their potential application to regenerative medicine.

5-Year Outcomes After Transcatheter Aortic Valve Implantation With CoreValve Prosthesis.

OBJECTIVES The purpose of this analysis was to assess 5-year outcomes of transcatheter aortic valve implantation (TAVI) using the current technology of the self-expanding CoreValve prosthesis (Medtronic Inc., Minneapolis, Minnesota). BACKGROUND There is a paucity of evidence on long-term durability of currently available transcatheter heart valves. METHODS Starting in June 2007, all consecutive patients with severe aortic stenosis undergoing TAVI with the third-generation 18-F CoreValve device in 8 Italian centers were prospectively included in the ClinicalService Project. For the purposes of this study, we included only consecutive patients with 5-year follow-up data available (n = 353) treated from June 2007 to August 2009. All outcomes were reported according to VARC (Valve Academic Research Consortium)-1 criteria. RESULTS All-cause mortality rates at 1, 2, 3, 4, and 5 years were 21%, 29%, 38%, 48%, and 55.0%, respectively. Cardiovascular mortality rates at 1, 2, 3, 4, and 5 years were 10%, 14%, 19%, 23%, and 28.0%, respectively. The overall neurological event rate at 5 years was 7.5%, of which more than two-thirds occurred early after the procedure. During follow-up, there were 241 rehospitalizations for cardiovascular reasons in 164 (46%) patients. Among all rehospitalizations, acute heart failure was the most frequently reported (42.7%), followed by requirement of permanent pacemaker implantation (17.4%). On echocardiography, mean transaortic gradients decreased from 55.6 ± 16.8 mm Hg (pre-TAVI) to 12.8 ± 10.9 mm Hg (5-year post-TAVI) (p < 0.001). Late prosthesis failure occurred in 5 cases (1.4%); among these, redo TAVI was successfully carried out in 2 patients (0.6%) presenting with symptomatic prosthesis restenosis. The remaining 3 cases of prosthesis failure did not undergo further invasive interventions. Ten patients (2.8%) showed late mild stenosis with a mean transaortic gradient ranging from 20 to 40 mm Hg. No other cases of structural or nonstructural valvular deterioration were observed. Valve thrombosis or late valve embolization were not reported. CONCLUSIONS TAVI with the currently adopted CoreValve generation was associated with sustained clinical outcomes up to 5-year follow-up, with a low rate (1.4%) of significant prosthetic valve degeneration. The procedure appears to be an adequate and lasting resolution of aortic stenosis in selected high-risk patients.

Impact of periprocedural bleeding on incidence of contrast-induced acute kidney injury in patients treated with percutaneous coronary intervention.

OBJECTIVES This study sought to evaluate the association between contrast-induced acute kidney injury (CI-AKI) after percutaneous coronary intervention and severity of bleeding estimated from periprocedural hemoglobin (Hb) measurement. BACKGROUND The relationship between CI-AKI and bleeding in contemporary practice remains controversial. METHODS In a retrospective analysis of the prospectively maintained Japan Cardiovascular Database-Keio Interhospital Cardiovascular Studies (JCD-KICS) multicenter registry, we divided 2,646 consecutive patients into 5 groups according to the change of Hb level after compared with before percutaneous coronary intervention: patients without a decrease in Hb level (group A) and patients with a decreased Hb level: <1 g/dl (group B); 1 to <2 g/dl (group C); 2 to <3g/dl (group D); and >3 g/dl (group E). CI-AKI was defined as an increase in serum creatinine level ≥ 0.5 mg/dl or ≥ 25% above baseline values at 48 h after administration of contrast media. Procedure and outcome variables were compared. RESULTS The mean patient age was 67 ± 11 years. Of the 2,646 patients, CI-AKI developed in 315 (11.9%). The CI-AKI incidence was 6.2%, 7.5%, 10.7%, 17.0%, and 26.2%, in groups A through E, respectively (p < 0.01), whereas the incidence of major bleeding was 0.7%, 1.3%, 2.0%, 4.1%, and 28.3%, respectively (p < 0.01). CI-AKI was associated with higher rates of mortality (5.4% vs. 0.6%, p < 0.01) and of composite of heart failure, cardiogenic shock, and death (16.5% vs. 2.8%, p < 0.01). CONCLUSIONS Periprocedural bleeding was significantly associated with CI-AKI, with CI-AKI incidence correlating with bleeding severity.

Mechanisms, Pathophysiology, and Clinical Aspects of Incomplete Stent Apposition

Incomplete stent apposition (ISA) is characterized by the lack of contact of at least 1 stent strut with the vessel wall in a segment not overlying a side branch; it is more commonly found in drug-eluting stents than bare-metal stents. The accurate diagnosis of ISA, initially only possible with intravascular ultrasound, can currently be performed with higher accuracy by optical coherence tomography, which also enables strut-level assessment due to its higher axial resolution. Different circumstances related both to the index procedure and to vascular healing might influence ISA occurrence. Although several histopathology and clinical studies linked ISA to stent thrombosis, potential selection bias precluded definitive conclusions. Initial studies usually performed single time point assessments comparing overall ISA percentage and magnitude in different groups (i.e., stent type), thus hampering a comprehensive understanding of its relationship with vascular healing. Serial intravascular imaging studies that evaluated vascular response heterogeneity recently helped fill this gap. Some particular clinical scenarios such as acute coronary syndromes, bifurcations, tapered vessels, overlapping stents, and chronic total occlusions might predispose to ISA. Interventional cardiologists should be committed to optimal stent choices and techniques of implantation and use intravascular imaging guidance when appropriate to aim at minimizing acute ISA. In addition, the active search for new stent platforms that could accommodate vessel remodeling over time (i.e., self-expandable stents) and for new polymers and/or eluting drugs that could induce less inflammation (hence, less positive remodeling) could ultimately reduce the occurrence of ISA and its potentially harmful consequences.

Extended use of percutaneous edge-to-edge mitral valve repair beyond EVEREST (Endovascular Valve Edge-to-Edge Repair) criteria: 30-day and 12-month clinical and echocardiographic outcomes from the GRASP (Getting Reduction of Mitral Insufficiency by Percutaneous Clip Implantation) registry.

OBJECTIVES This study sought to compare, in high-risk patients with 3+ to 4+ mitral regurgitation (MR) dichotomized by baseline echocardiographic features, acute, 30-day, and 12-month outcomes following percutaneous mitral valve repair using the MitraClip. BACKGROUND The feasibility and mid-term outcomes after MitraClip implantation in patients with echocardiographic features different from the EVEREST (Endovascular Valve Edge-to-Edge Repair) I and II trials have been scarcely studied. METHODS Clinical and echocardiographic outcomes through 12-month follow-up of consecutive patients who underwent MitraClip implantation were obtained from an ongoing prospective registry. Two different groups, divided according to baseline echocardiographic criteria (investigational group [EVERESTOFF] and control group [EVERESTON]), were compared. RESULTS Seventy-eight patients were included in EVERESTOFF and 93 patients in EVERESTON groups. Important and comparable acute reductions in MR and no clip-related complications were revealed. The primary safety endpoint at 30 days was comparable between groups (2.6% vs. 6.5%, respectively, p = 0.204); in addition, MR reduction was mostly sustained, whereas equivalent improvement in New York Heart Association functional class were demonstrated. Kaplan-Meier freedom from death, surgery for mitral valve dysfunction, or grade ≥3+ MR at 12 months was demonstrated in 71.4% and 76.2%, respectively, in the EVERESTOFF and EVERESTON groups (log rank p = 0.378). Significant improvements in ejection fraction and reduction in left ventricle volumes were demonstrated in both groups over time, but the baseline between-group differences were sustained. CONCLUSIONS MitraClip implantation in patients with expanded baseline echocardiographic features, compared with the control group, was associated with similar rates of safety and efficacy through 12-month follow-up. Further validation of our findings is warranted.

G-CSF influences mouse skeletal muscle development and regeneration by stimulating myoblast proliferation

Granulocyte colony-stimulating factor and its receptor are needed for skeletal muscle development and injury-induced regeneration in mice.

Association of tricuspid regurgitation with clinical and echocardiographic outcomes after percutaneous mitral valve repair with the MitraClip System: 30-day and 12-month follow-up from the GRASP Registry

AIM The aim of this study was to evaluate the association of baseline tricuspid regurgitation (TR) on the outcomes after percutaneous mitral valve repair (PMVR) with the MitraClip system. METHODS AND RESULTS Data from 146 consecutive patients with functional mitral regurgitation (MR) were obtained. Two different groups, dichotomized according to the degree of pre-procedural TR (moderate/severe, n = 47 and none/mild, n = 99), had their clinical and echocardiographic outcomes through 12-month compared. At 30-day, the primary safety endpoint was significantly higher in moderate/severe TR compared with none/mild TR (10.6 vs. 2.0%, P = 0.035). Marked reduction in MR grades observed post-procedure were maintained through 12 months. Although NYHA functional class significantly improved in both groups compared with baseline, it was impaired in moderate/severe TR compared with the none/mild TR group (NYHA > II at 30 day: 33.3 vs. 9.2%, P < 0.001; at 1 year: 38.5 vs. 12.3%, respectively, P = 0.006). Left ventricle reverse remodelling and ejection fraction improvement were revealed in both groups. The primary efficacy endpoint at 12-month determined by freedom from death, surgery for mitral valve dysfunction, or grade ≥ 3+ MR was comparable between groups, but combined death and re-hospitalization for heart failure rates were higher in the moderate/severe TR group. Multivariable Cox regression analysis demonstrated that baseline moderate/severe TR and chronic kidney disease were independent predictors of this combined endpoint. CONCLUSIONS Although PMVR with MitraClip led to improvement in MR, TR, and NYHA functional class in patients with baseline moderate/severe TR, the primary safety endpoint at 30-day was impaired, while moderate/severe TR independently predicted death and re-hospitalization for heart failure at 12-month.