Recombinant human collagen-based microspheres mitigate cardiac conduction slowing induced by adipose tissue-derived stromal cells

Background Stem 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. Aim To investigate the modulation of ASC-loaded recombinant human collagen-based microspheres, on the electrophysiological behavior of neonatal rat ventricular myocytes (NRVM). Method Unipolar 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. Results Microspheres 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 Conclusion The application of recombinant human collagen-based microspheres mitigates indirect paracrine conduction slowing through interference with a secondary autocrine myocardial factor

Increased amount of atrial fibrosis in patients with atrial fibrillation secondary to mitral valve disease

Objective: Atrial fibrosis is related to atrial fibrillation but may differ in patients with mitral valve disease or lone atrial fibrillation. Therefore, we studied atrial fibrosis in patients with atrial fibrillation + mitral valve disease or with lone atrial fibrillation and compared it with controls. Methods: Left and right atrial appendages amputated during Maze III surgery for lone atrial fibrillation (n = 85) or atrial fibrillation + mitral valve disease (n = 26) were embedded in paraffin, sectioned, and stained with picrosirius red. Atria from 10 deceased patients without a cardiovascular history served as controls. A total of 1048 images (4-mu m sections, 10-fold magnification, 4 images per appendage) were obtained and digitized. The percentage of fibrous tissue was calculated by quantitative morphometry. Results: Irrespective of the presence or absence of atrial fibrillation or mitral valve disease, more fibrous tissue was present in right atrial appendages than in left atrial appendages (12.7% +/- 5.7% vs 8.2% +/- 3.9%; P <.0001). The mean amount of fibrous tissue in the atria was significantly larger in patients with atrial fibrillation + mitral valve disease than in patients with lone AF and controls (13.6% +/- 5.8%, 9.7% +/- 3.2%, and 8.8% +/- 2.4%, respectively; P <.01). No significant differences existed between patients with lone atrial fibrillation and patients without a cardiovascular history (controls). Conclusions: Atria of patients with atrial fibrillation and mitral valve disease have more fibrosis than atria of patients with lone atrial fibrillation. However, patients with lone atrial fibrillation have an equal amount of atrial fibrosis compared with controls. These findings support the notion that fibrosis plays a more important role in the pathogenesis of atrial fibrillation secondary to mitral valve disease than in lone atrial fibrillation and potentially explains the relatively poor success of antiarrhythmic surgery in patients with mitral valve disease. (J Thorac Cardiovasc Surg 2012;144:327-33

Profibrillatory Structural and Functional Properties of the Atrial-Pulmonary Junction in the Absence of Remodeling

Background: Sole pulmonary vein (PV) isolation by ablation therapy prevents atrial fibrillation (AF) in patients with short episodes of AF and without comorbidities. Since incomplete PV isolation can be curative, we tested the hypothesis that the PV in the absence of remodeling and comorbidities contains structural and functional properties that are proarrhythmic for AF initiation by reentry. Methods: We performed percutaneous transvenous in vivo endocardial electrophysiological studies and quantitative histological analysis of PV from healthy sheep. Results: The proximal PV contained more myocytes than the distal PV and a higher percentage of collagen and fat tissue relative to myocytes than the left atrium. Local fractionated electrograms occurred in both the distal and proximal PVs, but a large local activation (>0.75 mV) was more often present in the proximal PV than in the distal PV (86 vs. 50% of electrograms, respectively, p = 0.017). Atrial arrhythmias (run of premature atrial complexes) occurred more often following the premature stimulation in the proximal PV than in the distal PV (p = 0.004). The diastolic stimulation threshold was higher in the proximal PV than in the distal PV (0.7 [0.3] vs. 0.4 [0.2] mA, (median [interquartile range]), p = 0.004). The refractory period was shorter in the proximal PV than in the distal PV (170 [50] vs. 248 [52] ms, p < 0.001). A linear relation existed between the gradient in refractoriness (distal-proximal) and atrial arrhythmia inducibility in the proximal PV. Conclusion: The structural and functional properties of the native atrial-PV junction differ from those of the distal PV. Atrial arrhythmias in the absence of arrhythmia-induced remodeling are caused by reentry in the atrial-PV junction. Ablative treatment of early paroxysmal AF, rather than complete isolation of focal arrhythmia, may be limited to inhibition of reentry

AAV9-mediated Rbm24 overexpression induces fibrosis in the mouse heart

The RNA-binding protein Rbm24 has recently been identified as a pivotal splicing factor in the developing heart. Loss of Rbm24 in mice disrupts cardiac development by governing a large number of muscle-specific splicing events. Since Rbm24 knockout mice are embryonically lethal, the role of Rbm24 in the adult heart remained unexplored. Here, we used adeno-associated viruses (AAV9) to investigate the effect of increased Rbm24 levels in adult mouse heart. Using high-resolution microarrays, we found 893 differentially expressed genes and 1102 differential splicing events in 714 genes in hearts overexpressing Rbm24. We found splicing differences in cardiac genes, such as PDZ and Lim domain 5, Phospholamban, and Titin, but did not find splicing differences in previously identified embryonic splicing targets of Rbm24, such as skNAC, αNAC, and Coro6. Gene ontology enrichment analysis demonstrated increased expression of extracellular matrix (ECM)-related and immune response genes. Moreover, we found increased expression of Tgfβ-signaling genes, suggesting enhanced Tgfβ-signaling in these hearts. Ultimately, this increased activation of cardiac fibroblasts, as evidenced by robust expression of Periostin in the heart, and induced extensive cardiac fibrosis. These results indicate that Rbm24 may function as a regulator of cardiac fibrosis, potentially through the regulation of TgfβR1 and TgfβR2 expression

Differential Mechanisms of Myocardial Conduction Slowing by Adipose Tissue-Derived Stromal Cells Derived From Different Species

: 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. Cell-based therapy is a promising option to treat patients after myocardial infarction. Although cell-based therapy may help replace infarcted heart tissue by functional tissue, it has some limitations. First, it may cause life-threatening arrhythmias. Slow conduction facilitates arrhythmias induction. Second, cells derived from and administered to the same patients may be affected by age and disease. Therefore, cells from other patients or other species may be used. This study shows that application of stromal cells caused conduction slowing in cardiomyocyte monolayers, irrespective of the specific origin of the cells, but that the conduction slowing is conferred through soluble factors or through coupling between fat-derived cells and cardiac myocytes in a species-dependent manne

Differential Mechanisms of Myocardial Conduction Slowing by Adipose Tissue-Derived Stromal Cells Derived From Different Species

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. SIGNIFICANCE: Cell-based therapy is a promising option to treat patients after myocardial infarction. Although cell-based therapy may help replace infarcted heart tissue by functional tissue, it has some limitations. First, it may cause life-threatening arrhythmias. Slow conduction facilitates arrhythmias induction. Second, cells derived from and administered to the same patients may be affected by age and disease. Therefore, cells from other patients or other species may be used. This study shows that application of stromal cells caused conduction slowing in cardiomyocyte monolayers, irrespective of the specific origin of the cells, but that the conduction slowing is conferred through soluble factors or through coupling between fat-derived cells and cardiac myocytes in a species-dependent manner

Spatial Heterogeneity of Cx43 is an Arrhythmogenic Substrate of Polymorphic Ventricular Tachycardias during Compensated Cardiac Hypertrophy in Rats

BACKGROUND: Ventricular remodeling increases the propensity of ventricular tachyarrhythmias and sudden death in patients. We studied the mechanism underlying these fatal arrhythmias, electrical and structural cardiac remodeling, as well as arrhythmogeneity during early, compensated hypertrophy in a rat model of chronic pressure overload. METHODS: Twenty-six Wistar rats were subjected to transverse aortic constriction (TAC) (n = 13) or sham operation (n = 13). Four weeks postoperative, echo- and electrocardiography was performed. Epicardial (208 or 455 sites) and transmural (30 sites) ventricular activation mapping was performed on Langendorff perfused hearts. Subsequently, hearts were processed for (immuno)histological and molecular analyses. RESULTS: TAC rats showed significant hypertrophy with preserved left ventricular (LV) function. Epicardial conduction velocity (CV) was similar, but more dispersed in TAC. Transmural CV was slowed in TAC (37.6 ± 2.9 cm s(-1)) compared to sham (58.5 ± 3.9 cm s(-1); P < 0.01). Sustained polymorphic ventricular tachycardias were induced from LV in 8/13 TAC and in 0/13 sham rats. During VT, electrical activation patterns showed variable sites of earliest epicardial activation and altering sites of functional conduction block. Wandering epicardial reentrant activation was sporadically observed. Collagen deposition was significantly higher in TAC compared to sham, but not different between arrhythmogenic and non-arrhythmogenic TAC animals. Connexin43 (Cx43) expression was heterogeneous with a higher prevalence of non-phosphorylated Cx43 in arrhythmogenic TAC animals. CONCLUSION: In TAC rats with compensated cardiac hypertrophy, dispersion of conduction correlated to arrhythmogenesis, an increased heterogeneity of Cx43, and a partial substitution with non-phosphorylated Cx43. These alterations may result in the increased vulnerability to polymorphic VTs

Atrial Fibrosis and Conduction Slowing in the Left Atrial Appendage of Patients Undergoing Thoracoscopic Surgical Pulmonary Vein Isolation for Atrial Fibrillation

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 (CVL), transverse CV (CVT), 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 CVL was 0.55+/-0.22 m/s, mean CVT 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. CVL 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. CVT 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 CVL 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-entr

Pulsed-field gel electrophoresis analysis of Bordetella pertussis populations in various European countries with different vaccine policies.

The increasing incidence of pertussis in a number of countries, despite good vaccination coverage, is a cause for concern. We used pulsed-field gel electrophoresis (PFGE) typing to examine the genetic diversity of 101 clinical isolates of Bordetella pertussis, recovered during 1999-2001, and circulating in five different European countries to evaluate temporal and geographical distribution. This DNA fingerprinting approach seems to be a more discriminative epidemiological tool than sequencing of individual genes. Despite differences in vaccination policies in the five countries, these European isolates were found to be very similar and fell into the same major PFGE profile groups, with a predominance of one profile group. There was no evidence of geographic clustering, except that one new profile subgroup was predominantly found in one country. This study provides a baseline for continued surveillance of the B. pertussis population in Europe

Genetic variation in GNB5 causes bradycardia by augmenting the cholinergic response via increased acetylcholine-activated potassium current (I K,ACh)

Mutations in GNB5, encoding the G-protein β5 subunit (Gβ5), have recently been linked to a multisystem disorder that includes severe bradycardia. Here, we investigated the mechanism underlying bradycardia caused by the recessive p.S81L Gβ5 variant. Using CRISPR/Cas9-based targeting, we generated an isogenic series of human induced pluripotent stem cell (hiPSC) lines that were either wild type, heterozygous or homozygous for the GNB5 p.S81L variant. These were differentiated into cardiomyocytes (hiPSC-CMs) that robustly expressed the acetylcholine-activated potassium channel [I(KACh); also known as I K,ACh]. Baseline electrophysiological properties of the lines did not differ. Upon application of carbachol (CCh), homozygous p.S81L hiPSC-CMs displayed an increased acetylcholine-activated potassium current (I K,ACh) density and a more pronounced decrease of spontaneous activity as compared to wild-type and heterozygous p.S81L hiPSC-CMs, explaining the bradycardia in homozygous carriers. Application of the specific I(KACh) blocker XEN-R0703 resulted in near-complete reversal of the phenotype. Our results provide mechanistic insights and proof of principle for potential therapy in patients carrying GNB5 mutations