[Treatment with cardiac electronic implantable devices]. / Therapie mit kardialen elektronischen implantierbaren Devices.

Cardiac device therapy provides not only treatment options for bradyarrhythmia but also advanced treatment for heart failure and preventive measures against sudden cardiac death. In heart failure treatment it enables synergistic reverse remodelling and reduces pharmacological side effects. Cardiac resynchronization therapy (CRT) has revolutionized the treatment of reduced left ventricular ejection fraction (LVEF) and left bundle branch block by decreasing the mortality and morbidity with improvement of the quality of life and resilience. Conduction system pacing (CSP) as an alternative method of physiological stimulation can improve heart function and reduce the risk of pacemaker-induced cardiomyopathy. Leadless pacers and subcutaneous/extravascular defibrillators offer less invasive options with lower complication rates. The prevention of infections through preoperative and postoperative strategies enhances the safety of these therapies.

PCSK9 Activity Is Potentiated Through HDL Binding.

Rationale: Proprotein convertase subtilisin/kexin type 9 (PCSK9) circulates in a free and lipoprotein-bound form, yet the functional consequence of the association between PCSK9 and high-density lipoprotein (HDL) remains unexplored. Objective: This study sought to interrogate the novel relationship between PCSK9 and HDL in humans. Methods and Results: Comparing lipoprotein and apolipoprotein profiles by nuclear magnetic resonance and targeted mass spectrometry measurements with PCSK9 levels in the community-based Bruneck (n=656) study revealed a positive association of plasma PCSK9 with small HDL, alongside a highly significant positive correlation between plasma levels of PCSK9 and apolipoprotein-C3, an inhibitor of lipoprotein lipase. The latter association was replicated in an independent cohort, the SAPHIR study (n=270). Thus, PCSK9-HDL association was determined during the postprandial response in two dietary studies (n=20 participants each, 8 times points). Peak triglyceride levels coincided with an attenuation of the PCSK9-HDL association, a loss of apolipoprotein-C3 from HDL and lower levels of small HDL as measured by nuclear magnetic resonance. Crosslinking mass spectrometry (XLMS) upon isolated HDL identified PCSK9 as a potential HDL-binding partner. PCSK9 association with HDL was confirmed through size-exclusion chromatography and immuno-isolation. Quantitative proteomics upon HDL isolated from patients with coronary artery disease (n=172) returned PCSK9 as a core member of the HDL proteome. Combined interrogation of the HDL proteome and lipidome revealed a distinct cluster of PCSK9, phospholipid transfer protein, clusterin and apolipoprotein-E within the HDL proteome, that was altered by sex and positively correlated with sphingomyelin content. Mechanistically, HDL facilitated PCSK9-mediated low-density lipoprotein receptor degradation and reduced low-density lipoprotein uptake through the modulation of PCSK9 internalisation and multimerisation. Conclusions: This study reports HDL as a binder of PCSK9 and regulator of its function. The combination of -omic technologies revealed postprandial lipaemia as a driver of PCSK9 and apolipoprotein-C3 release from HDL.

Flagellin-Induced Immune Response in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Pathogen-associated molecular patterns (PAMPs) are involved in the pathogenesis of septic cardiomyopathy through a toll-like receptor (TLR)-mediated immune response. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can reflect the innate immune abilities of cardiomyocytes. Therefore, hiPSC-CMs may provide an attractive tool with which to study PAMP-induced alterations in cardiomyocytes. HiPSC-CMs from two different healthy donors were exposed to the PAMP flagellin (FLA) at different doses and exposure times. Alterations in the expression levels of distinct inflammation-associated cytokines, intracellular inflammation pathways including TLR5 downstream signaling, reactive oxygen species levels and surface antigen composition were assessed using PCR, ELISA and FACS techniques. Higher doses of flagellin increased the expression levels of inflammation-associated cytokines like TNFα (p < 0.01) and downstream signaling molecules like caspase-8 (p < 0.05). TLR5 expression (p < 0.01) and TLR5 fluorescence proportion (p < 0.05) increased in hiPSC-CMs after prolonged FLA exposure. FLA-induced innate immune response processes in cardiomyocytes might be detectable with an hiPSC-CMs-based in vitro model.

Hepatocyte Nuclear Factor 1A Is a Cell-Intrinsic Transcription Factor Required for B Cell Differentiation and Development in Mice.

The hepatocyte NF (HNF) family of transcription factors regulates the complex gene networks involved in lipid, carbohydrate, and protein metabolism. In humans, HNF1A mutations cause maturity onset of diabetes in the young type 3, whereas murine HNF6 participates in fetal liver B lymphopoiesis. In this study, we have identified a crucial role for the prototypical member of the family HNF1A in adult bone marrow B lymphopoiesis. HNF1A(-/-) mice exhibited a clear reduction in total blood and splenic B cells and a further pronounced one in transitional B cells. In HNF1A(-/-) bone marrow, all B cell progenitors-from pre-pro-/early pro-B cells to immature B cells-were dramatically reduced and their proliferation rate suppressed. IL-7 administration in vivo failed to boost B cell development in HNF1A(-/-) mice, whereas IL-7 stimulation of HNF1A(-/-) B cell progenitors in vitro revealed a marked impairment in STAT5 phosphorylation. The B cell differentiation potential of HNF1A(-/-) common lymphoid progenitors was severely impaired in vitro, and the expression of the B lymphopoiesis-promoting transcription factors E2A, EBF1, Pax5, and Bach2 was reduced in B cell progenitors in vivo. HNF1A(-/-) bone marrow chimera featured a dramatic defect in B lymphopoiesis recapitulating that of global HNF1A deficiency. The HNF1A(-/-) lymphopoiesis defect was confined to B cells as T lymphopoiesis was unaffected, and bone marrow common lymphoid progenitors and hematopoietic stem cells were even increased. Our data demonstrate that HNF1A is an important cell-intrinsic transcription factor in adult B lymphopoiesis and suggest the IL-7R/STAT5 module to be causally involved in mediating its function.

Clinical outcomes associated with catecholamine use in patients diagnosed with Takotsubo cardiomyopathy.

BACKGROUND: Recent hypotheses have suggested the pathophysiological role of catecholamines in the evolution of the Takotsubo syndrome (TTS). The extent of cardiac and circulatory compromise dictates the use of some form of supportive therapy. This study was designed to investigate the clinical outcomes associated with catecholamine use in TTS patients. METHODS: Our institutional database constituted a collective of 114 patients diagnosed with TTS between 2003 and 2015. The study-patients were subsequently classified into two groups based on the need for catecholamine support during hospital stay (catecholamine group n = 93; 81%, non-catecholamine group = 21; 19%). The primary end-point of our study was all-cause mortality. RESULTS: Patients receiving catecholamine support showed higher grades of circulatory and cardiac compromise (left ventricular ejection fraction (LVEF) 39.6% vs. 32.7%, p-value < 0.01) and the course of disease was often complicated by the occurrence of different TTS-associated complications. The in-hospital mortality (3.2% vs. 28.5%, p < 0.01), 30-day mortality (17.2% vs. 51.4%, p < 0.01) as well as long-term mortality (38.7% vs. 80.9%, p < 0.01) was significantly higher in the group of patients receiving catecholamine support. A multivariate Cox regression analysis attributed EF ≤ 35% (HR 3.6, 95% CI 1.6-8.1; p < 0.01) and use of positive inotropic agents (HR 2.2, 95% CI 1.0-4.8; p 0.04) as independent predictors of the adverse outcome. CONCLUSION: Rates of in-hospital events and short- as well as long-term mortality were significantly higher in TTS patients receiving catecholamine support as compared to the other study-patients. These results need further evaluation in pre-clinical and clinical trials to determine if external catecholamines contribute to an adverse clinical outcome already compromised by the initial insult.

Interventional Left Atrial Appendage Closure Affects the Metabolism of Acylcarnitines.

BACKGROUND: Left atrial appendage closure (LAAC) represents the interventional alternative to oral anticoagulation for stroke prevention in atrial fibrillation (AF). The metabolism of acylcarnitines was shown to affect cardiovascular diseases. This study evaluates the influence of successful LAAC on the metabolism of acylcarnitines. METHODS: Patients undergoing successful LAAC were enrolled prospectively. Peripheral blood samples for metabolomics measurements were collected immediately before (i.e., index) and six months after LAAC (i.e., mid-term). A targeted metabolomics analysis based on electrospray ionization-liquid chromatography-mass spectrometry (ESI-LC-MS/MS) and MS/MS measurements was performed. RESULTS: 44 patients with non-valvular AF (median CHA2DS2-VASc score 4, median HAS-BLED score 4) and successful LAAC were included. Significant changes in acylcarnitine levels were found in the total cohort, which were mainly attributed to patients with impaired left ventricular and renal function, elevated amino-terminal pro-brain natriuretic peptide (NT-proBNP) and diabetes mellitus. Adjusted multivariable regression models revealed significant changes of five metabolites over mid-term follow-up: C2, C14:1, C16, and C18:1 decreased significantly (each p < 0.05); short-chain C5 acylcarnitine plasma levels increased significantly (p < 0.05). CONCLUSION: This study demonstrates that successful LAAC affects the metabolism of acylcarnitines at mid-term follow-up. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02985463.

HDL-bound sphingosine 1-phosphate (S1P) predicts the severity of coronary artery atherosclerosis.

BACKGROUND: We have recently demonstrated a reduction in HDL-bound sphingosine 1-phosphate (S1P) in patients with stable coronary artery disease (CAD). In the current study, we tested whether HDL-associated S1P is predictive for the degree of coronary stenosis, restenosis and overall CAD severity on follow up in patients undergoing elective percutaneous coronary intervention (PCI). METHODS: Coronary angiography of patients with CAD (n=59) undergoing elective PCI and presenting for a follow up after 6 months (n=48) was graded for disease severity defined clinically as 1- or multi-vessel disease. Target lesion stenosis was quantified by quantitative coronary angiography (QCA). S1P in plasma and isolated HDL were measured by mass spectrometry in the initial samples and in 32 available follow up samples. RESULTS: HDL-bound S1P levels remained stable over time and correlated closely at first visit and follow up. While not associated with the extent of target lesion stenosis or restenosis, HDL-bound S1P correlated negatively with the overall severity of CAD and discriminated 1-vessel-disease from multi-vessel disease. Furthermore, low HDL-bound S1P was predictive for CAD extent. CONCLUSION: In stable CAD, HDL-bound S1P does not predict the degree of stenosis or restenosis of the target lesion but constitutes a marker of clinically defined disease burden.

Catecholamine induces endothelial dysfunction via Angiotensin II and intermediate conductance calcium activated potassium channel.

Endothelial dysfunction contributes to the pathogenesis of Takotsubo syndrome (TTS). However, the exact mechanism underlying endothelial dysfunction in the setting of TTS has not been completely clarified. This study aims to investigate the roles of angiotensin II (Ang II) and intermediate-conductance Ca2+-activated K+ (SK4) channels in catecholamine-induced endothelial dysfunction. Human cardiac microvascular endothelial cells (HCMECs) were exposed to 100 µM epinephrine (Epi), mimicking the setting of TTS. Epi treatment increased the ET-1 concentration and reduced NO levels in HCMECs. Importantly, the effects of Epi were found to be mitigated in the presence of Ang II receptor blockers. Furthermore, Ang II mimicked Epi effects on ET-1 and NO production. Additionally, Ang II inhibited tube formation and increased cell apoptosis. The effects of Ang II could be reversed by an SK4 activator NS309 and mimicked by an SK4 channel blocker TRAM-34. Ang II also inhibited the SK4 channel current (ISK4) without affecting its expression level. Ang II could depolarize the cell membrane potential. Ang II promoted ROS release and reduced protein kinase A (PKA) expression. A ROS blocker prevented Ang II effect on ISK4. The PKA activator Sp-8-Br-cAMPS increased SK4 channel currents. Epinephrine enhanced the activity of ACE by activating the α1 receptor/Gq/PKC signal pathway, thereby promoting the secretion of Ang II. The study suggested that high-level catecholamine can increase Ang II release from endothelial cells by α1 receptors/Gq/PKC signal pathway. Ang II can inhibit SK4 channel current by increasing ROS generation and reducing PKA expression, thereby contributing to endothelial dysfunction.

Comparison of the prognosis and outcome of heart failure with reduced ejection fraction patients treated with sacubitril/valsartan according to age.

The treatment with sacubitril/valsartan in patients suffering from chronic heart failure with reduced ejection fraction increases left ventricular ejection fraction and decreases the risk of sudden cardiac death. We conducted a retrospective analysis regarding the impact of age differences on the treatment outcome of sacubitril/valsartan in patients with chronic heart failure with reduced ejection fraction. Patients were defined as adults if ≤65 years (n = 51) and older if >65 years of age (n = 76). The incidence of ventricular arrhythmias at 1-year follow-up was comparable in both groups (30.8 vs 26.5%; p = 0.71). The mortality rate in adult patients is significantly lower as compared with older patients (2 vs 14.5%; log-rank = 0.04). Older patients may suffer remarkably more side effects than adult patients (21.1 vs 11.8%; p = 0.03).

Lay abstract Many patients still die from heart failure (HF). Better drugs for this condition were developed over the past 20 years. The drug combination sacubitril/valsartan improves heart function and might lower the risk of sudden cardiac death from a fast heart rate (ventricular arrythmia). More research is needed on the effect of sacubitril/valsartan on older patients with HF. We wanted to see how sacubitril/valsartan affects older patients with HF. Seventy-six patients above 65 years of age were compared with 51 patients below 65 years. A year later, the number of ventricular arrhythmias was similar in both groups. Side effects were more common in older patients. At 2 years, there were more deaths in the older group. In summary, side effects of sacubitril/valsartan could be an obstacle to treating older patients. Finding better concepts to treat older patients with a weak heart remains necessary.

TRPV1 activation and internalization is part of the LPS-induced inflammation in human iPSC-derived cardiomyocytes.

The non-selective cation channel transient receptor potential vanilloid 1 (TRPV1) is expressed throughout the cardiovascular system. Recent evidence shows a role for TRPV1 in inflammatory processes. The role of TRPV1 for myocardial inflammation has not been established yet. Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (hiPSC-CM) from 4 healthy donors were incubated with lipopolysaccharides (LPS, 6 h), TRPV1 agonist capsaicin (CAP, 20 min) or the antagonist capsazepine (CPZ, 20 min). TRPV1 expression was studied by PCR and western blotting. TRPV1 internalization was analyzed by immunofluorescence. Interleukin-6 (IL-6) secretion and phosphorylation of JNK, p38 and ERK were determined by ELISA. TRPV1-associated ion channel current was measured by patch clamp. TRPV1-mRNA and -protein were expressed in hiPSC-CM. TRPV1 was localized in the plasma membrane. LPS significantly increased secretion of IL-6 by 2.3-fold, which was prevented by pre-incubation with CPZ. LPS induced TRPV1 internalization. Phosphorylation levels of ERK, p38 or JNK were not altered by TRPV1 stimulation or inhibition. LPS and IL-6 significantly lowered TRPV1-mediated ion channel current. TRPV1 mediates the LPS-induced inflammation in cardiomyocytes, associated with changes of cellular electrophysiology. LPS-induced inflammation results in TRPV1 internalization. Further studies have to examine the underlying pathways and the clinical relevance of these findings.

Sphingosine 1-phosphate levels in plasma and HDL are altered in coronary artery disease.

High-density lipoproteins (HDL) are the major plasma carriers for sphingosine 1-phosphate (S1P) in healthy individuals, but their S1P content is unknown for patients with coronary artery disease (CAD). The aim of the study was to determine whether the S1P levels in plasma and HDL are altered in coronary artery disease. S1P was determined in plasma and HDL isolated by ultracentrifugation from patients with myocardial infarction (MI, n = 83), stable CAD (sCAD, n = 95), and controls (n = 85). In our study, total plasma S1P levels were lower in sCAD than in controls (305 vs. 350 pmol/mL). However, normalization to HDL-cholesterol (a known determinant of plasma S1P) revealed higher normalized plasma S1P levels in sCAD than in controls (725 vs. 542 pmol/mg) and even higher ones in MI (902 pmol/mg). The S1P amount contained in isolated HDL from these individuals was lower in sCAD than in controls (S1P per protein in HDL: 132 vs. 153 pmol/mg). The amount of total plasma S1P bound to HDL was lower in sCAD and MI than in controls (sCAD: 204, MI: 222, controls: 335 pmol/mL), while the non-HDL-bound S1P was, accordingly, higher (sCAD: 84, MI: 81, controls: 10 pmol/mL). HDL-bound plasma S1P was dependent on the plasma HDL-C in all groups, but normalization to HDL-C still yielded lower HDL-bound plasma S1P in patients with sCAD than in controls (465 vs. 523 pmol/mg). The ratio of non-HDL-bound plasma S1P to HDL-C-normalized HDL-bound S1P was also higher in both sCAD (0.18 mg/mL) and MI (0.15 mg/mL) than in controls (0.02 mg/mL). Remarkably, levels of non-HDL-bound plasma S1P correlated with the severity of CAD symptoms as graded by Canadian Cardiovascular Score, and discriminated patients with MI and sCAD from controls. Furthermore, a negative association was present between non-HDL-bound plasma S1P and the S1P content of isolated HDL in controls, but was absent in sCAD and MI. Finally, MI patients with symptom duration of less than 12 h had the highest levels of total and normalized plasma S1P, as well as the highest levels of S1P in isolated HDL. The HDL-C-normalized plasma level of S1P is increased in sCAD and even further in MI. This may be caused by an uptake defect of HDL for plasma S1P in CAD, and may represent a novel marker of HDL dysfunction.

High high-density lipoprotein-cholesterol reduces risk and extent of percutaneous coronary intervention-related myocardial infarction and improves long-term outcome in patients undergoing elective percutaneous coronary intervention.

AIMS: The study tested whether high-density lipoprotein-cholesterol (HDL-C) has an effect on percutaneous coronary intervention (PCI)-induced myocardial infarction and its prognosis. Elevation of cardiac troponin I (cTnI) > 3x upper normal limit after PCI is defined as PCI-related myocardial infarction (PMI) and is associated with a negative prognosis. No data exist on the relationship of HDL-C to PMI and PMI-related outcome. METHODS AND RESULTS: Pre-procedural HDL-C levels and post-procedural peak cTnI levels were collected in 350 patients undergoing PCI. Data were analysed for PMI and for acute myocardial infarction (AMI) during follow-up. Patients with PMI (n = 115) had lower HDL-C levels than patients without PMI [n = 235; 1.17 mmol/L (0.75-2.51) vs. 1.27 mmol/L (0.70-2.87), P < 0.001]. Pre-procedural HDL-C levels were inversely related to the occurrence of PMI [odds ratio for PMI: 0.884, 95% CI: 0.80, 0.98; P = 0.02 for an HDL-C-increment of 5 mg/dL (0.13 mmol/L)] and to AMI during follow-up [hazard ratio (HR): 0.697, 95% CI: 0.54, 0.90; P = 0.005]. The occurrence of PMI was associated with an elevated HR for AMI (4.702, 95% CI: 1.79, 12.37; P = 0.002). Low-risk levels of pre-procedural HDL-C [men >or=40 mg/dL (>or=1.03 mmol/L), women >or=45 mg/dL (>or=1.16 mmol/L)] did not influence the negative effects of PMI on outcome (HR: 5.510, 95% CI: 1.43, 21.31; P = 0.013) and reduction of AMI-free survival [mean AMI-free survival time with PMI: 1167.5 days (95% CI: 1098.27, 1236.67) vs. 1240.7 days (95% CI: 1220.94, 1290.49) without PMI; log-rank P = 0.005]. Conclusion Small increases in HDL-C in patients undergoing elective PCI convert into a substantial reduction of risk for PMI, which has adverse effects on the long-term prognosis. Patients with PMI are at a high risk for AMI at any HDL-C level and therefore should receive particular monitoring by the treating physician over a long period after PCI.

Improved Outcome of Cardiogenic Shock Triggered by Takotsubo Syndrome Compared With Myocardial Infarction.

BACKGROUND: Cardiogenic shock (CS) is a severe complication of myocardial infarction (MI) or of takotsubo syndrome (TTS). For both diseases, CS is related to a worse long-term outcome. The outcome of CS has not been studied in a direct comparison of patients with MI and patients with TTS. METHODS: Mortality and cardiovascular complications were compared in patients presenting with CS based on MI or TTS between 2003 and 2017 during a follow-up of 5 years. A total of 138 patients with TTS and 532 patients with MI were included. Of these, 66 patients with MI and 25 patients with TTS developed CS (12% vs 18%, P = 0.08). RESULTS: Patients with MI and CS had more often malignant arrhythmias (74% vs 28%, P < 0.01), and need for resuscitation (80% vs 24%, P < 0.01) or death (71% vs 24%, P < 0.01) than patients with TTS and CS during the first 30 days. Although the overall rate of death remained higher in MI than in TTS (75.8% vs 52%, log rank, P < 0.01), deaths occurred in TTS constantly throughout the follow-up time, but not in MI. The incidence of heart failure increased in MI but not in TTS (31.8% vs 4%, P < 0.01) during follow-up. CONCLUSIONS: Patients with MI and CS have a worse prognosis than patients with TTS and CS. This is driven by cardiovascular events or death during the first 30 days after the index event. However, patients with TTS and CS show high mortality as well, especially during long-term follow-up.

Serum of patients with acute myocardial infarction prevents inflammation in iPSC-cardiomyocytes.

Acute myocardial infarction (MI) evokes a systemic inflammatory response and locally the degradation of the necrotic tissue, followed by scar formation. The mechanisms for containment of the infarct zone are not studied well. The study aimed to examine the response of healthy cardiomyocytes to serum of patients with myocardial infarction. Human iPSC-cardiomyocytes (iPSC-CM) generated from two healthy donors were incubated with serum of patients with MI with and without ventricular fibrillation (VF) or of healthy controls. Different cell adhesion molecules were studied by flow cytometry and immunostaining. Cellular electrophysiology was studied by patch clamp. The cell adhesion molecules CD54/ICAM-1, CD58/LFA-3 and CD321/JAM-A were expressed on iPSC-CM within the plasma membrane. Incubation with serum of MI patients reduced the levels of expression of CD54/ICAM-1 and CD321/JAM-A by 15-20%. VF serum was less effective than serum of MI patients without VF. MI serum or VF serum did not affect resting potential, action potential duration or maximum depolarization velocity. Myocardial infarction serum exerts anti-inflammatory effects on healthy cardiomyocytes without affecting their electrical activity, thus helping to contain the infarct zone and to protect healthy tissue. Ventricular fibrillation during MI drives healthy cardiomyocytes towards a pro-inflammatory phenotype.

Ion Channel Expression and Characterization in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

BACKGROUND: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are providing new possibilities for the biological study, cell therapies, and drug discovery. However, the ion channel expression and functions as well as regulations in hiPSC-CMs still need to be fully characterized. METHODS: Cardiomyocytes were derived from hiPS cells that were generated from two healthy donors. qPCR and patch clamp techniques were used for the study. RESULTS: In addition to the reported ion channels, INa, ICa-L, ICa-T, If, INCX, IK1, Ito, IKr, IKs IKATP, IK-pH, ISK1-3, and ISK4, we detected both the expression and currents of ACh-activated (KACh) and Na+-activated (KNa) K+, volume-regulated and calcium-activated (Cl-Ca) Cl-, and TRPV channels. All the detected ion currents except IK1, IKACh, ISK, IKNa, and TRPV1 currents contribute to AP duration. Isoprenaline increased ICa-L, If, and IKs but reduced INa and INCX, without an effect on Ito, IK1, ISK1-3, IKATP, IKr, ISK4, IKNa, ICl-Ca, and ITRPV1. Carbachol alone showed no effect on the tested ion channel currents. CONCLUSION: Our data demonstrate that most ion channels, which are present in healthy or diseased cardiomyocytes, exist in hiPSC-CMs. Some of them contribute to action potential performance and are regulated by adrenergic stimulation.

Modeling Short QT Syndrome Using Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

BACKGROUND: Short QT syndrome (SQTS), a disorder associated with characteristic ECG QT-segment abbreviation, predisposes affected patients to sudden cardiac death. Despite some progress in assessing the organ-level pathophysiology and genetic changes of the disorder, the understanding of the human cellular phenotype and discovering of an optimal therapy has lagged because of a lack of appropriate human cellular models of the disorder. The objective of this study was to establish a cellular model of SQTS using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). METHODS AND RESULTS: This study recruited 1 patient with short QT syndrome type 1 carrying a mutation (N588K) in KCNH2 as well as 2 healthy control subjects. We generated hiPSCs from their skin fibroblasts, and differentiated hiPSCs into cardiomyocytes (hiPSC-CMs) for physiological and pharmacological studies. The hiPSC-CMs from the patient showed increased rapidly activating delayed rectifier potassium channel current (IKr) density and shortened action potential duration compared with healthy control hiPSC-CMs. Furthermore, they demonstrated abnormal calcium transients and rhythmic activities. Carbachol increased the arrhythmic events in SQTS but not in control cells. Gene and protein expression profiling showed increased KCNH2 expression in SQTS cells. Quinidine but not sotalol or metoprolol prolonged the action potential duration and abolished arrhythmic activity induced by carbachol. CONCLUSIONS: Patient-specific hiPSC-CMs are able to recapitulate single-cell phenotype features of SQTS and provide novel opportunities to further elucidate the cellular disease mechanism and test drug effects.

Estradiol protection against toxic effects of catecholamine on electrical properties in human-induced pluripotent stem cell derived cardiomyocytes.

BACKGROUND AND PURPOSE: Previous studies revealed that Takotsubo cardiomyopathy (TTC), a transient disorder of ventricular dysfunction affecting predominantly postmenopausal women, is associated with acquired long QT syndrome and arrhythmias, but the exact pathophysiologic mechanism is unknown. Our aim is to investigate the electrophysiological mechanism for QT-prolongation in TTC-patients by using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). METHODS: hiPSC-CMs, which were generated from human skin fibroblasts of three healthy donors, were treated by estradiol (10µM for one week) and a toxic concentration of isoprenaline (Iso, 1mM for 2h). Patch clamp techniques, qPCR and fluorescence-activated cell sorting (FACS) were employed for the study. KEY RESULTS: Iso enhanced late INa and suppressed Ito and thus prolonged the action potential duration (APD), suggesting possible reasons for arrhythmias in TTC. Iso elevated the production of reactive oxygen species (ROS). N-acetylcystein (1mM), a ROS-blocker, abolished the effects of Iso on late INa and Ito. H2O2 (100µM) mimicked Iso effects on late INa and Ito. These data indicate that the effects of Iso were mediated by ROS. Metoprolol (1mM), a beta-blocker, prevented the effects of Iso on late INa and APD, confirming the adrenoceptor-dependent effects of Iso. Estradiol treatment prevented the APD-prolongation, attenuated the enhancement of INa, diminished the reduction of Ito, suppressed ROS-production induced by Iso and reduced the expression levels of adrenoceptors, suggesting protective effects of estragon against toxic effects of catecholamine. CONCLUSIONS: Estradiol has protective effects against catecholamine excess and hence reduction in estrogen level may increase the risk of acquired long QT syndrome in TTC.

Ion Channel Dysfunctions in Dilated Cardiomyopathy in Limb-Girdle Muscular Dystrophy.

BACKGROUND: Limb-Girdle muscular dystrophies (LGMD) are a heritable group of genetically determined disorders with a primary involvement of the pelvic or shoulder girdle musculature with partially cardiac manifestation, such as dilated cardiomyopathy (DCM) and life-threatening tachyarrhythmia. We report here that human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes from a patient with LGMD2I and DCM associated with recurrent ventricular tachycardia displayed ion channel dysfunction and abnormality of calcium homeostasis. METHODS: Dermal fibroblasts obtained from a patient with LGMD2I harboring a fukutin-related protein gene mutation (826C>A; Leu276Ile) and 3 healthy donors were reprogrammed to hiPSCs. The hiPSCs were differentiated into cardiomyocytes and used for biological and electrophysiological studies. RESULTS: Compared with hiPSC cardiomyocytes from the healthy donors, the hiPSC cardiomyocytes from the patient exhibited abnormal action potentials characterized by reduced amplitude and upstroke velocity. The peak and late Na channel currents (INa) as well as the peak L-type calcium channel currents were significantly reduced. The expression of SCN5A and CACNA1C was reduced in DCM cardiomyocytes, consistent with reduction of INa and L-type calcium channel currents. In addition, the rapidly activating delayed rectifier potassium current (IKr) was reduced, whereas the transient outward current (Ito) and slowly activating delayed rectifier potassium current (IKs) were similar in DCM and control cardiomyocytes. Finally, a significant reduction of systolic and diastolic intracellular Ca2+ concentrations was detected in DCM cardiomyocytes. CONCLUSIONS: This study demonstrates that patient-specific hiPSC cardiomyocytes can recapitulate some phenotypic properties of LGMD2I with DCM and provide a platform for studies on the cardiac events in LGMD.

Dysregulation of the ubiquitin-proteasome system in human carotid atherosclerosis.

OBJECTIVE: The ubiquitin-proteasome system is the principal degradation route of intracellular and oxidized proteins, thus regulating many cellular processes conceivably important for atherosclerosis. The aim of this study was to evaluate the activity of ubiquitin-proteasome system in human carotid artery plaques in relation to oxidative stress and clinical manifestation. METHODS AND RESULTS: In carotid endarterectomy specimens from 83 asymptomatic and 94 symptomatic patients, content of ubiquitin, ubiquitin conjugates, matrix metalloproteases, and NADPH-oxidase-p67 was evaluated by immunoblotting; proteolytic proteasome activity by fluorometric assay; single and double immunostaining for ubiquitin conjugates, 3-nitrotyrosine, apoptosis, smooth muscle alpha-actin, and macrophage CD-68, as well as Sirius Red staining for collagen were performed. Compared with asymptomatic patients, symptomatic patients showed a more unstable plaque phenotype, an increased degree of apoptosis, a significantly higher ubiquitin conjugates content (17.72+/-1.36 versus 10.99+/-1.04; P<0.001), and lower proteasome activity (5.01+/-0.70 versus 9.41+/-1.19 nmol AMC/mg protein/min; P<0.01). Ubiquitin conjugates content was directly correlated to NADPH-p67 and degree of apoptosis. Immunostaining revealed colocalization of ubiquitin conjugates and 3-nitrotyrosine, and accumulation of ubiquitin conjugates in smooth muscle cells and macrophages. CONCLUSIONS: In human carotid plaques increased oxidative stress is associated with inhibition of the proteasome activity and accumulation of ubiquitin conjugates, particularly in symptomatic patients. These results suggest a possible role of the ubiquitin-proteasome system in influencing plaque stability.

Incidence and Prognostic Relevance of Cardiopulmonary Failure in Takotsubo Cardiomyopathy.

Recent studies have indicated that patients with takotsubo cardiomyopathy (TTC) have a higher mortality rate than the general population. There is a distinct possibility that TTC could be associated with adverse life-threatening complications like cardiopulmonary failure. Our institutional database constituted a collective of 114 patients diagnosed with TTC. The frequency, determinants and predictors of cardiopulmonary failure were assessed. The patients were subsequently classified into two groups based on the presence (n = 44, 38.6%) or absence (n = 70, 61.4%) of cardiopulmonary failure. Multivariable logistic-regression analysis identified impaired left ventricular function defined as ≤35% at presentation and life-threatening arrhythmia as a positive significant independent predictor of cardiopulmonary failure. A majority of the patients with cardiopulmonary failure were treated with either non-invasive or invasive ventilator support (88%), while 48% of the patients required treatment with catecholamine. The in-hospital mortality rate was greater in the cardiopulmonary failure group. Cardiopulmonary failure patients were at ongoing increased risk of death with a higher mortality at 30-day, 1-year and at 5 years of follow-up. Cardiopulmonary failure is a frequent complication in TTC with an increased short- and long-term mortality. Patient susceptible to this condition could be identified by a reduced ejection fraction and life-threatening arrhythmia.