Rutger J. Hassink

Increased cardiomyocyte differentiation from human embryonic stem cells in serum-free cultures.

Human embryonic stem cells (hESCs) can differentiate into cardiomyocytes, but the efficiency of this process is low. We routinely induce cardiomyocyte differentiation of the HES‐2 cell line by coculture with a visceral endoderm‐like cell line, END‐2, in the presence of 20% fetal calf serum (FCS). In this study, we demonstrate a striking inverse relationship between cardiomyocyte differentiation and the concentration of FCS during HES‐2‐END‐2 coculture. The number of beating areas in the cocultures was increased 24‐fold in the absence of FCS compared with the presence of 20% FCS. An additional 40% increase in the number of beating areas was observed when ascorbic acid was added to serum‐free cocultures. The increase in serum‐free cocultures was accompanied by increased mRNA and protein expression of cardiac markers and of Isl1, a marker of cardiac progenitor cells. The number of beating areas increased up to 12 days after initiation of coculture of HES‐2 with END‐2 cells. However, the number of α‐actinin–positive cardiomyocytes per beating area did not differ significantly between serum‐free cocultures (503 ± 179; mean ± standard error of the mean) and 20% FCS cocultures (312 ± 227). The stimulating effect of serum‐free coculture on cardiomyocyte differentiation was observed not only in HES‐2 but also in the HES‐3 and HES‐4 cell lines. To produce sufficient cardiomyocytes for cell replacement therapy in the future, upscaling cardiomyocyte formation from hESCs is essential. The present data provide a step in this direction and represent an improved in vitro model, without interfering factors in serum, for testing other factors that might promote cardiomyocyte differentiation.

The human adult cardiomyocyte phenotype

AIMnDetermination of the phenotype of adult human atrial and ventricular myocytes based on gene expression and morphology.nnnMETHODSnAtrial and ventricular cardiomyocytes were obtained from patients undergoing cardiac surgery using a modified isolation procedure. Myocytes were isolated and cultured with or without serum. The relative cell attachment promoting efficiency of several reagents was evaluated and compared. Morphological changes during long-term culture were assessed with phase contrast microscopy, morphometric analysis and immunocytochemistry or RT-PCR of sarcomeric markers including alpha-actinin, myosin light chain-2 (MLC-2) and the adhesion molecule, cadherin.nnnRESULTSnThe isolation method produced viable rod-shaped atrial (16.6+/-6.0%, mean+/-S.E.; n=5) and ventricular cells (22.4+/-8.0%, mean+/-S.E.; n=5) in addition to significant numbers of apoptotic and necrotic cells. Cell dedifferentiation was characterized by the loss of sarcomeric structure, condensation and extrusion of sarcomeric proteins. Cells cultured with low serum recovered and assumed a flattened, spread form with two distinct morphologies apparent. Type I cells were large, had extensive sarcolemmal spreading, with stress fibers and nascent myofibrils, whilst type II cells appeared smaller, with more mature myofibril organisation and focal adhesions.nnnCONCLUSIONnCharacterization of the redifferentiation capabilities of cultured adult cardiac myocytes in culture, provides an important system for comparing cardiomyocytes differentiating from human stem cells and provides the basis for an in vitro transplantation model to study interaction and communication between primary adult and stem cell-derived cardiomyocytes.

Heart repair and stem cells.

Of the medical conditions currently being discussed in the context of possible treatments based on cell transplantation therapy, few have received more attention than the heart. Much focus has been on the potential application of bone marrow‐derived cell preparations, which have already been introduced into double‐blind, placebo‐controlled clinical trials. The consensus is that bone marrow may have therapeutic benefit but that this is not based on the ability of bone marrow cells to transdifferentiate into cardiac myocytes. Are there potential stem cell sources of cardiac myocytes that may be useful in replacing those lost or dysfunctional after myocardial infarction? Here, this question is addressed with a review of the recent literature.

Five-year efficacy of pulmonary vein antrum isolation as a primary ablation strategy for atrial fibrillation: a single-centre cohort study

AIMSnPulmonary vein antrum isolation (PVAI) is the cornerstone of atrial fibrillation (AF) ablation. There is an ongoing discussion on whether and when to add substrate modification to PVAI. This study evaluates (1) long-term efficacy of PVAI as a primary ablation strategy in all patients independently from AF type and (2) predictors of arrhythmia recurrence.nnnMETHODS AND RESULTSnA total of 509 consecutive patients (mean age 57 years, 38.9% non-paroxysmal AF) with AF underwent PVAI. In redo procedures, ablation was restricted to re-pulmonary vein (PV) isolation in case of PV reconnection. If the PVs were found to be isolated, substrate modification was performed. In total, 774 procedures were performed. Mean follow-up duration after the first and last ablation was, respectively, 66 ± 23 and 55 ± 25 months. A single PVAI was sufficient in restoring and maintaining long-term sinus rhythm in 41.3% (n = 210) of patients. Multiple procedures (mean 1.5) with re-PV isolation increased long-term success to 58.3% (n = 297). Additional substrate modification (n = 70) increased success to 62.5% (n = 318). After the last ablation, 87.5% of patients experienced success or significant clinical improvement on or off antiarrhythmic drugs. The incidence of left-sided atrial flutter or atrial tachycardia was 5% after PVAI and increased to 32% after additional substrate modification. Independent predictors for arrhythmia recurrence after the last ablation were non-paroxysmal AF, female sex, body mass index, hypertension, and AF duration.nnnCONCLUSIONnFive-year freedom of atrial tachyarrhythmia could be achieved by PVAI as primary ablation strategy in 58.3% of patients. Additional substrate modification only moderately increased overall success.

Role of the autonomic nervous system in vagal atrial fibrillation

Based on the seminal work of Coumel, the autonomic nervous system is considered to play a pathophysiological role in a subset of patients with atrial fibrillation (AF).1 In particular, Coumel put forward the concept of so-called vagal AF. Yet, the precise role of the autonomic nervous system in vagal AF is unclear. Is vagal AF caused by abnormal autonomic function or abnormal autonomic tone, or is there perhaps an increased sensitivity of the heart to vagal influences? In previous studies,2,3 both autonomic function and autonomic tone were shown to be normal in patients with AF, suggesting that increased sensitivity of the heart to vagal influences plays a role. However, these studies were limited by small sample size. In the present study we sought to collect additional data to define the role of the autonomic nervous system in vagal AF.nnThe study group comprised 73 patients with paroxysmal AF. None of the patients had congestive heart failure. On average, patients had a three year history of one paroxysm per week lasting two hours. Patients were considered to have vagal AF if they met the following clinical criteria: (1) most attacks occurred at rest or during sleep, and generally terminated with exercise or in the morning; and (2) if available, typical electrocardiographic findings (preceding sinus bradycardia and a slow ventricular response during AF) were present. Ten patients fulfilled these clinical criteria (vagal AF group); eight men, mean (SD) age 59 (12) years. One patient had hypertension, one patient had mild aortic valve disease, and one patient had angina pectoris, but the other seven patients had lone AF. Mean left …

Impaired autonomic function predicts dizziness at onset of paroxysmal atrial fibrillation

BACKGROUNDnParoxysmal atrial fibrillation is associated with various symptoms, including dizziness, which presumably reflects hemodynamic deterioration. Given the importance of the autonomic nervous system in mitigating the hemodynamic effect of atrial fibrillation, we hypothesized that autonomic function would be predictive of the severity of dizziness.nnnMETHODSnThe study group comprised 73 patients with paroxysmal atrial fibrillation (mean age 54.1 years, 51 males). Forty-three (59%) patients had lone atrial fibrillation. Mean ventricular rate during atrial fibrillation was 99+/-16 beats/min. On average, patients had a 3-year history of one paroxysm per week lasting 2 h. Autonomic function was assessed using autonomic function tests, including noninvasive measurement of baroreflex sensitivity. Head up tilting was used to test vasovagal reactivity. Severity of dizziness at onset of atrial fibrillation was quantified by the patients using a five-point scale (1=none; 2=light; 3=mild; 4=moderate; and 5=severe). Multivariate analysis was performed to identify the independent predictors of the severity of dizziness.nnnRESULTSnMean severity of dizziness was 3.36+/-1.65. Multivariate predictors of moderate-to-severe dizziness as opposed to none-to-mild dizziness were a low 30-15 ratio after standing up and low baroreflex sensitivity. Though syncope was never reported nine patients showed a full vasovagal response during head up tilting.nnnCONCLUSIONSnIt is concluded that dizziness in patients with treated atrial fibrillation in the setting of none to mild structural heart disease is predicted by impaired autonomic function. Vasovagal reactivity appears not to be involved in this connection.

Cell-cycle-based strategies to drive myocardial repair.

Cardiomyocytes exhibit robust proliferative activity during development. After birth, cardiomyocyte proliferation is markedly reduced. Consequently, regenerative growth in the postnatal heart via cardiomyocyte proliferation (and, by inference, proliferation of stem-cell-derived cardiomyocytes) is limited and often insufficient to affect repair following injury. Here, we review studies wherein cardiomyocyte cell cycle proliferation was induced via targeted expression of cyclin D2 in postnatal hearts. Cyclin D2 expression resulted in a greater than 500-fold increase in cell cycle activity in transgenic mice as compared to their nontransgenic siblings. Induced cell cycle activity resulted in infarct regression and concomitant improvement in cardiac hemodynamics following coronary artery occlusion. These studies support the notion that cell-cycle-based strategies can be exploited to drive myocardial repair following injury.

Expression of a transgene encoding mutant p193/CUL7 preserves cardiac function and limits infarct expansion after myocardial infarction

Background: Transgenic mice expressing the dominant interfering p193 protein in cardiomyocytes (MHC-1152stop mice) exhibit an induction of cell cycle activity and altered remodelling after experimental myocardial infarction (MI). Objective: To determine whether the altered remodelling results in improved cardiac function in the MHC-1152stop mice after MI, as compared with non-transgenic mice. Methods: MHC-1152stop mice and non-transgenic littermates were subjected to experimental MI via permanent occlusion of the coronary artery. Infarct size was determined at 24 h and at 4 weeks after MI, and left ventricular pressure–volume measurements were performed at 4 weeks after MI in infarcted and sham-operated animals. Results: Infarct size in MHC-1152stop mice and non-transgenic littermates was not statistically different at 24 h after MI, as measured by tetrazolium staining. Morphometric analysis showed that infarct scar expansion at 4 weeks after MI was reduced by 10% in the MHC-1152stop mice (p<0.05). No differences in cardiac function were detected between sham-operated MHC-1152stop mice and their non-transgenic littermates. However, at 4 weeks after MI, the ventricular isovolumic relaxation time constant (τ) was decreased by 19% (p<0.05), and the slope of the dP/dtmax–EDV relationship was increased 99% (p<0.05), in infarcted MHC-1152stop mice as compared with infarcted non-transgenic littermates. Conclusion: Expression of the dominant interfering p193 transgene results in a decrease in infarct scar expansion and preservation of myocardial function at 4 weeks after MI. Antagonism of p193 activity may represent an important strategy for the treatment of MI.

Incidence of Pulmonary Vein Stenosis After Radiofrequency Catheter Ablation of Atrial Fibrillation

OBJECTIVESnThis study aimed to determine incidence of pulmonary vein stenosis (PVS) and evaluate PVS-related symptoms.nnnBACKGROUNDnThe real-life incidence of PVS after radiofrequency catheter ablation (RFCA) of atrial fibrillation (AF) is unknown.nnnMETHODSnAll patients who underwent RFCA of AF from 2005 to 2016 with routine pre- and post-ablation screening by magnetic resonance imaging or computed tomography were included. Primary ablation strategy was PV antrum isolation alone in all patients. PVS, defined as a significant reduction in the superoinferior or anteroposterior PV diameter, was classified as mild (30% to 50%), moderate (50% to 70%), or severe (>70%).nnnRESULTSnSufficient quality imaging of the PV anatomy before ablation and during follow-up (mean 6 ± 4 months) was performed in 976 patients (76.4% men, 59.1% paroxysmal AF). Of these patients, 306 (31.4%) showed mild stenosis, 42xa0(4.3%) revealed moderate stenosis, and 7 (0.7%) had a severe stenosis in at least 1 PV. Incidence of PVS fluctuated over the past decade. All severe PVS cases were likely caused by ablations being performed inside the PVs. Only 1 (0.1%) patient reported PVS-related symptoms of severe dyspnea during follow-up. Computed tomography revealed a subtotal occlusion of the left inferior PV and a severe stenosis of the left superior PV, requiring stenting.nnnCONCLUSIONSnAlthough mild PVS was frequently observed after RFCA in this large cohort, incidence of severe PVS wasxa0<1% and incidence of symptomatic PVS necessitating intervention was negligible. Based on these findings, it seems appropriate to only screen for PVS in patients with suggestive symptoms.

A Rare and Difficult Diagnosis: A Ruptured Sinus of Valsalva Aneurysm

![Figure][1] nn[![Graphic][3] ][3][![Graphic][4] ][4]nnnnA 34-year-old woman was admitted to the intensive care unit because of respiratory failure, hypotension, and tachycardia. Examination revealed increased jugular venous pressure and a rough systolic murmur loudest over the fourth