Background— Atrial fibrosis is an important component of the arrhythmogenic substrate in patients with atrial fibrillation (AF). We studied the effect of interstitial fibrosis on conduction velocity (CV) in the left atrial appendage of patients with AF. Methods and Results— Thirty-five left atrial appendages were obtained during AF surgery. Preparations were superfused and stimulated at 100 beats per minute. Activation was recorded with optical mapping. Longitudinal CV (CV L ), transverse CV (CV T ), and activation times (>2 mm distance) were measured. Interstitial collagen was quantified and graded qualitatively. The presence of fibroblasts and myofibroblasts was assessed immunohistochemically. Mean CV L was 0.55±0.22 m/s, mean CV T was 0.25±0.15 m/s, and the mean activation time was 9.31±5.45 ms. The amount of fibrosis was unrelated to CV or patient characteristics. CV L was higher in left atrial appendages with thick compared with thin interstitial collagen strands (0.77±0.22 versus 0.48±0.19 m/s; P =0.012), which were more frequently present in persistent patients with AF. CV T was not significantly different ( P =0.47), but activation time was 14.93±4.12 versus 7.95±4.12 ms in patients with thick versus thin interstitial collagen strands, respectively ( P =0.004). Fibroblasts were abundantly present and were associated with the presence of thick interstitial collagen strands ( P =0.008). Myofibroblasts were not detected in the left atrial appendage. Conclusions— In patients with AF, thick interstitial collagen strands are associated with higher CV L and increased activation time. Our observations demonstrate that the severity and structure of local interstitial fibrosis is associated with atrial conduction abnormalities, presenting an arrhythmogenic substrate for atrial re-entry.

Stem cell therapy is a promising therapeutic option to treat patients after myocardial infarction. However, the intramyocardial administration of large amounts of stem cells might generate a proarrhythmic substrate. Proarrhythmic effects can be explained by electrotonic and/or paracrine mechanisms. The narrow therapeutic time window for cell therapy and the presence of comorbidities limit the application of autologous cell therapy. The use of allogeneic or xenogeneic stem cells is a potential alternative to autologous cells, but differences in the proarrhythmic effects of adipose‐derived stromal cells (ADSCs) across species are unknown. Using microelectrode arrays and microelectrode recordings, we obtained local unipolar electrograms and action potentials from monolayers of neonatal rat ventricular myocytes (NRVMs) that were cocultured with rat, human, or pig ADSCs (rADSCs, hADSCs, pADSCs, respectively). Monolayers of NRVMs were cultured in the respective conditioned medium to investigate paracrine effects. We observed significant conduction slowing in all cardiomyocyte cultures containing ADSCs, independent of species used (p < .01). All cocultures were depolarized compared with controls (p < .01). Only conditioned medium taken from cocultures with pADSCs and applied to NRVM monolayers demonstrated similar electrophysiological changes as the corresponding cocultures. We have shown that independent of species used, ADSCs cause conduction slowing in monolayers of NRVMs. In addition, pADSCs exert conduction slowing mainly by a paracrine effect, whereas the influence on conduction by hADSCs and rADSCs is preferentially by electrotonic interaction. Stem Cells Translational Medicine 2017;6:22–30

Stem cell therapy has been suggested to be a promising option for regeneration of injured myocardium, for example following a myocardial infarction. For clinical use cell-based therapies have to be safe and applicable and are aimed to renovate the architecture of the heart. Yet for functional and coordinated activity synchronized with the host myocardium stem cells have to be capable of forming electrical connections with resident cardiomyocytes. In this paper we discuss whether stem cells are capable of establishing functional electrotonic connections with cardiomyocytes and whether these may generate a risk for arrhythmias. Application of stem cells in the clinical setting with outcomes concerning arrhythmogenic safety and future perspectives will also briefly be touched upon.

Heart failure is still a major cause of hospitalization and mortality in developed countries. Many clinical trials have tested the use of multipotent stem cells as a cardiac regenerative medicine. The benefit for the patients of this therapeutic intervention has remained limited. Herein, we review the pluripotent stem cells as a cell source for cardiac regeneration. We more specifically address the various challenges of this cell therapy approach. We question the cell delivery systems, the immune tolerance of allogenic cells, the potential proarrhythmic effects, various drug mediated interventions to facilitate cell grafting and, finally, we describe the pathological conditions that may benefit from such an innovative approach. As members of a transatlantic consortium of excellence of basic science researchers and clinicians, we propose some guidelines to be applied to cell types and modes of delivery in order to translate pluripotent stem cell cardiac derivatives into safe and effective clinical trials. STEM CELLS 2016;34:34-43 SIGNIFICANCE STATEMENTCell therapy of heart failure has emerged as a promising approach for the last decade. However, the use of non cardiogenic stem cells in clinical trials has lead to disapointing results. Here we challenge the use of pluripotent stem cell derived cardiac progenitors as a mean to regnerate diseased hearts.

Background: Cardiomyocyte progenitor cells (CMPCs) are a promising cell source for regenerative cell therapy to improve cardiac function after myocardial infarction. However, it is unknown whether undifferentiated CMPCs have arrhythmogenic risks. We investigate whether undifferentiated, regionally applied, human fetal CMPCs form a pro-arrhythmic substrate in co-culture with neonatal rat ventricular myocytes (NRVMs).Method: Unipolar extracellular electrograms, derived from micro-electrode arrays (8 × 8 electrodes) containing monolayers of NRVMs (control), or co-cultures of NRVMs and locally seeded CMPCs were used to determine conduction velocity and the incidence of tachy-arrhythmias. Micro-electrodes were used to record action potentials. Conditioned medium (Cme) of CMPCs was used to distinguish between coupling or paracrine effects.Results: Co-cultures demonstrated conduction slowing (5.6 ± 0.3 cm/s, n = 50) compared to control monolayers (13.4 ± 0.4 cm/s, n = 26) and monolayers subjected to Cme (13.7 ± 0.6 cm/s, n = 11, all p < 0.001). Furthermore, co-cultures had a more depolarized resting membrane than control monolayers (−47.3 ± 17.4 vs. −64.8 ± 7.7 mV, p < 0.001) and monolayers subjected to Cme (−64.4 ± 8.1 mV, p < 0.001). Upstroke velocity was significantly decreased in co-cultures and action potential duration was prolonged. The CMPC region was characterized by local ST-elevation in the recorded electrograms. The spontaneous rhythm was faster and tachy-arrhythmias occurred more often in co-cultured monolayers than in control monolayers (42.0 vs. 5.4%, p < 0.001).Conclusion: CMPCs form a pro-arrhythmic substrate when co-cultured with neonatal cardiomyocytes. Electrical coupling between both cell types leads to current flow between a, slowly conducting, depolarized and the normal region leading to local ST-elevations and the occurrence of tachy-arrhythmias originating from the non-depolarized zone.

BackgroundStem cell therapy to improve cardiac function after myocardial infarction is hampered by poor cell retention, while it may also increase the risk of arrhythmias by providing an arrhythmogenic substrate. We previously showed that porcine adipose tissue-derived-stromal cells (pASC) induce conduction slowing through paracrine actions, whereas rat ASC (rASC) and human ASC (hASC) induce conduction slowing by direct coupling. We postulate that biomaterial microspheres mitigate the conduction slowing influence of pASC by interacting with paracrine signaling.AimTo investigate the modulation of ASC-loaded recombinant human collagen-based microspheres, on the electrophysiological behavior of neonatal rat ventricular myocytes (NRVM).MethodUnipolar extracellular electrograms, derived from microelectrode arrays (8x8 electrodes) containing NRVM, co-cultured with ASC or ASC loaded microspheres, were used to determine conduction velocity (CV) and conduction heterogeneity. Conditioned medium (Cme) of (co)cultures was used to assess paracrine mechanisms.ResultsMicrospheres did not affect CV in control (NRVM) monolayers. In co-cultures of NRVM and rASC, hASC or pASC, CV was lower than in controls (14.4±1.0, 13.0±0.6 and 9.0± 1.0 vs. 19.5±0.5 cm/s respectively, p<0.001). Microspheres loaded with either rASC or hASC still induced conduction slowing compared to controls (13.5±0.4 and 12.6±0.5 cm/s respectively, p<0.001). However, pASC loaded microspheres increased CV of NRVM compared to pASC and NRMV co-cultures (16.3±1.3 cm/s, p< 0.001) and did not differ from controls (p = NS). Cme of pASC reduced CV in control monolayers of NRVM (10.3±1.1 cm/s, p<0.001), similar to Cme derived from pASC-loaded microspheres (11.1±1.7 cm/s, p = 1.0). The presence of microspheres in monolayers of NRVM abolished the CV slowing influence of Cme pASC (15.9±1.0 cm/s, p = NS vs. control).ConclusionThe application of recombinant human collagen-based microspheres mitigates indirect paracrine conduction slowing through interference with a secondary autocrine myocardial factor.

We thank Dr Patanè 1 for his congratulations regarding our recent article in this journal 2 and for bringing forward 2 issues for discussion. His first comment relates to the observation that the amount of fibrosis was unrelated to conduction velocity. In our study, we found that longitudinal conduction velocity is increased in patients with more thick strands of collagen (>0.01 mm), which, in combination with unchanged transversal conduction velocity confers increased vulnerability for reentry. In addition, activation time was longer in specimens with thick strands of collagen, indicating more discontinuous conduction. Thick strands of collagen were predominantly found in patients with more advanced atrial fibrillation (AF), as 7 of 8 patients with thick strands of collagen had persistent AF. Interestingly, the mere percentage of collagen, that is, the area of the histological section being occupied by picrosirius red staining, did not correlate with conduction velocity, nor with the clinical severity of AF. Thus, the quality rather than the quantity of fibrosis seems to cause an arrhythmogenic substrate in the setting of human AF. This is consistent with earlier work of our group, that showed that dense, long and thick strands of fibrosis, but not diffuse fibrosis was associated with conduction slowing on premature stimulation in ventricular tissue of patients with end stadium heart failure.4 Taken together, these observations do not contradict, but fit perfectly with the comment of Dr Patanè 1 that older patients have an increase in longitudinal conduction velocity and that age is one of the determinants of fibrosis.The second question of Dr Patanè 1 relates to our histochemical analysis of myofibroblasts. The differentiation of fibroblasts into myofibroblasts is characterized by the expression of α-smooth muscle actin. With a combination of α-smooth muscle actin and CD31 staining, we were able to detect a significant amount of smooth muscle cells and pericytes in the microvasculature. No isolated α-smooth muscle actin positive cells were found, indicating the absence of myofibroblasts. Hence, the cytokines and messenger molecules that may have contributed to the differentiation of fibroblasts into myofibroblasts seemed of lesser relevance to this study. Although there may be a important role of myofibroblasts in the early development of the fibrotic substrate for AF, their role in clinically symptomatic AF seems to be limited. The observation that there were many fibroblasts in all specimens with thick strands of collagen remains intriguing. This could either point at dedifferentiation of myofibroblasts, or otherwise at their disappearance through a process of apoptotic cell death after maturation of the collagen deposits. Meanwhile other cell types, probably fibroblasts, might be of importance for the maintenance of the fibrotic substrate for AF.

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