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Author Correction: Connexin43 expression in bone marrow derived cells contributes to the electrophysiological properties of cardiac scar tissue

Vasquez, Carolina; Mezzano, Valeria; Kessler, Newman; Swardh, Freja; Ernestad, Desiree; Mahoney, Vanessa M; Hanna, John; Morley, Gregory E
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
PMID: 32632225
ISSN: 2045-2322
CID: 4545862

Connexin43 expression in bone marrow derived cells contributes to the electrophysiological properties of cardiac scar tissue

Vasquez, Carolina; Mezzano, Valeria; Kessler, Newman; Swardh, Freja; Ernestad, Desiree; Mahoney, Vanessa M; Hanna, John; Morley, Gregory E
Cardiac pathologies associated with arrhythmic activity are often accompanied by inflammation. The contribution of inflammatory cells to the electrophysiological properties of injured myocardium is unknown. Myocardial scar cell types and intercellular contacts were analyzed using a three-dimensional reconstruction from serial blockface scanning electron microscopy data. Three distinct cell populations were identified: inflammatory, fibroblastic and endocardial cells. While individual fibroblastic cells interface with a greater number of cells, inflammatory cells have the largest contact area suggesting a role in establishing intercellular electrical connections in scar tissue. Optical mapping was used to study the electrophysiological properties of scars in fetal liver chimeric mice generated using connexin43 knockout donors (bmpKO). Voltage changes were elicited in response to applied current pulses. Isopotential maps showed a steeper pattern of decay with distance from the electrode in scars compared with uninjured regions, suggesting reduced electrical coupling. The tissue decay constant, defined as the distance voltage reaches 37% of the amplitude at the edge of the scar, was 0.48 ± 0.04 mm (n = 11) in the scar of the bmpCTL group and decreased 37.5% in the bmpKO group (n = 10). Together these data demonstrate inflammatory cells significantly contribute to scar electrophysiology through coupling mediated at least partially by connexin43 expression.
PMID: 32054938
ISSN: 2045-2322
CID: 4304592

Disruption of Ca2+i Homeostasis and Cx43 Hemichannel Function in the Right Ventricle Precedes Overt Arrhythmogenic Cardiomyopathy in PKP2-Deficient Mice

Kim, Joon-Chul; Pérez-Hernández Duran, Marta; Alvarado, Francisco J; Maurya, Svetlana R; Montnach, Jerome; Yin, Yandong; Zhang, Mingliang; Lin, Xianming; Vasquez, Carolina; Heguy, Adriana; Liang, Feng-Xia; Woo, Sun-Hee; Morley, Gregory E; Rothenberg, Eli; Lundby, Alicia; Valdivia, Hector H; Cerrone, Marina; Delmar, Mario
BACKGROUND:Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy (ARVC). A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. METHODS:We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice ("PKP2cKO") 14 days post-tamoxifen (post-TAM) injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. RESULTS:homeostasis. Similarly, PKC inhibition normalized spark frequency at comparable SR load levels. CONCLUSIONS:handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.
PMID: 31315456
ISSN: 1524-4539
CID: 3977952

Mechanistic investigation of Ca2+ alternans in human heart failure and its modulation by fibroblasts

Mora, Maria T; Gomez, Juan F; Morley, Gregory; Ferrero, Jose M; Trenor, Beatriz
BACKGROUND:Heart failure (HF) is characterized, among other factors, by a progressive loss of contractile function and by the formation of an arrhythmogenic substrate, both aspects partially related to intracellular Ca2+ cycling disorders. In failing hearts both electrophysiological and structural remodeling, including fibroblast proliferation, contribute to changes in Ca2+ handling which promote the appearance of Ca2+ alternans (Ca-alt). Ca-alt in turn give rise to repolarization alternans, which promote dispersion of repolarization and contribute to reentrant activity. The computational analysis of the incidence of Ca2+ and/or repolarization alternans under HF conditions in the presence of fibroblasts could provide a better understanding of the mechanisms leading to HF arrhythmias and contractile function disorders. METHODS AND FINDINGS/RESULTS:The goal of the present study was to investigate in silico the mechanisms leading to the formation of Ca-alt in failing human ventricular myocytes and tissues with disperse fibroblast distributions. The contribution of ionic currents variability to alternans formation at the cellular level was analyzed and the results show that in normal ventricular tissue, altered Ca2+ dynamics lead to Ca-alt, which precede APD alternans and can be aggravated by the presence of fibroblasts. Electrophysiological remodeling of failing tissue alone is sufficient to develop alternans. The incidence of alternans is reduced when fibroblasts are present in failing tissue due to significantly depressed Ca2+ transients. The analysis of the underlying ionic mechanisms suggests that Ca-alt are driven by Ca2+-handling protein and Ca2+ cycling dysfunctions in the junctional sarcoplasmic reticulum and that their contribution to alternans occurrence depends on the cardiac remodeling conditions and on myocyte-fibroblast interactions. CONCLUSION/CONCLUSIONS:It can thus be concluded that fibroblasts modulate the formation of Ca-alt in human ventricular tissue subjected to heart failure-related electrophysiological remodeling. Pharmacological therapies should thus consider the extent of both the electrophysiological and structural remodeling present in the failing heart.
PMID: 31211790
ISSN: 1932-6203
CID: 3939102

Permanent and Transient Electrophysiological Effects During Cardiac Cryoablation Documented by Optical Activation Mapping and Thermal Imaging

Morley, Greg; Bernstein, Scott; Kuznekoff, Laura; Vasquez, Carolina; Saul, Phil; Haemmerich, Dieter
OBJECTIVE:Cardiac catheter cryoablation is a safer alternative to radiofrequency ablation for arrhythmia treatment, but electrophysiological (EP) effects during and after freezing are not adequately characterized. The goal of this study was to determine transient and permanent temperature induced EP effects, during and after localized tissue freezing. METHODS:Conduction in right (RV) and left ventricles (LV) was studied by optical activation mapping during and after cryoablation in paced, isolated Langendorff-perfused porcine hearts. Cryoablation was performed endocardially (n=4) or epicardially (n=4) by a cryoprobe cooled to -120 °C for 8 minutes. Epicardial surface temperature was imaged with an infrared camera. Viability staining was performed after ablation. Motion compensation and co-registration was performed between optical mapping data, temperature image data, and lesion images. RESULTS:Cryoablation produced lesions 14.9 +/- 3.1 mm in diameter and 5.8 +/- 1.7 mm deep. A permanent lesion was formed in tissue cooled below -5 +/- 4 °C. Transient EP changes observed at temperatures between 17 and 37 °C during cryoablation surrounding the frozen tissue region directly correlated with local temperature, and include action potential (AP) duration prolongation, decrease in AP magnitude, and slowing in conduction velocity (Q10=2.0). Transient conduction block was observed when epicardial temperature reached <17 °C, but completely resolved upon tissue rewarming, within 5 minutes. CONCLUSION/CONCLUSIONS:Transient EP changes were observed surrounding the permanent cryo lesion (<-5 °C), including conduction block (-5 to 17 °C), and reduced conduction velocity (>17 °C). SIGNIFICANCE/CONCLUSIONS:The observed changes explain effects observed during clinical cryoablation, including transient increases in effective refractory period, transient conduction block, and transient slowing of conduction. The presented quantitative data on temperature dependence of EP effects may enable the prediction of the effects of clinical cryoablation devices.
PMID: 30418875
ISSN: 1558-2531
CID: 3657812

Plakophilin-2 is required for transcription of genes that control calcium cycling and cardiac rhythm

Cerrone, Marina; Montnach, Jerome; Lin, Xianming; Zhao, Yan-Ting; Zhang, Mingliang; Agullo-Pascual, Esperanza; Leo-Macias, Alejandra; Alvarado, Francisco J; Dolgalev, Igor; Karathanos, Thomas V; Malkani, Kabir; Van Opbergen, Chantal J M; van Bavel, Joanne J A; Yang, Hua-Qian; Vasquez, Carolina; Tester, David; Fowler, Steven; Liang, Fengxia; Rothenberg, Eli; Heguy, Adriana; Morley, Gregory E; Coetzee, William A; Trayanova, Natalia A; Ackerman, Michael J; van Veen, Toon A B; Valdivia, Hector H; Delmar, Mario
Plakophilin-2 (PKP2) is a component of the desmosome and known for its role in cell-cell adhesion. Mutations in human PKP2 associate with a life-threatening arrhythmogenic cardiomyopathy, often of right ventricular predominance. Here, we use a range of state-of-the-art methods and a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mouse to demonstrate that in addition to its role in cell adhesion, PKP2 is necessary to maintain transcription of genes that control intracellular calcium cycling. Lack of PKP2 reduces expression of Ryr2 (coding for Ryanodine Receptor 2), Ank2 (coding for Ankyrin-B), Cacna1c (coding for CaV1.2) and Trdn (coding for triadin), and protein levels of calsequestrin-2 (Casq2). These factors combined lead to disruption of intracellular calcium homeostasis and isoproterenol-induced arrhythmias that are prevented by flecainide treatment. We propose a previously unrecognized arrhythmogenic mechanism related to PKP2 expression and suggest that mutations in PKP2 in humans may cause life-threatening arrhythmias even in the absence of structural disease.It is believed that mutations in desmosomal adhesion complex protein plakophilin 2 (PKP2) cause arrhythmia due to loss of cell-cell communication. Here the authors show that PKP2 controls the expression of proteins involved in calcium cycling in adult mouse hearts, and that lack of PKP2 can cause arrhythmia in a structurally normal heart.
PMID: 28740174
ISSN: 2041-1723
CID: 2653852

Fhf2 gene deletion causes temperature-sensitive cardiac conduction failure

Park, David S; Shekhar, Akshay; Marra, Christopher; Lin, Xianming; Vasquez, Carolina; Solinas, Sergio; Kelley, Kevin; Morley, Gregory; Goldfarb, Mitchell; Fishman, Glenn I
Fever is a highly conserved systemic response to infection dating back over 600 million years. Although conferring a survival benefit, fever can negatively impact the function of excitable tissues, such as the heart, producing cardiac arrhythmias. Here we show that mice lacking fibroblast growth factor homologous factor 2 (FHF2) have normal cardiac rhythm at baseline, but increasing core body temperature by as little as 3 degrees C causes coved-type ST elevations and progressive conduction failure that is fully reversible upon return to normothermia. FHF2-deficient cardiomyocytes generate action potentials upon current injection at 25 degrees C but are unexcitable at 40 degrees C. The absence of FHF2 accelerates the rate of closed-state and open-state sodium channel inactivation, which synergizes with temperature-dependent enhancement of inactivation rate to severely suppress cardiac sodium currents at elevated temperatures. Our experimental and computational results identify an essential role for FHF2 in dictating myocardial excitability and conduction that safeguards against temperature-sensitive conduction failure.
PMID: 27701382
ISSN: 2041-1723
CID: 2273672

Connexin43 contributes to electrotonic conduction across scar tissue in the intact heart

Mahoney, Vanessa M; Mezzano, Valeria; Mirams, Gary R; Maass, Karen; Li, Zhen; Cerrone, Marina; Vasquez, Carolina; Bapat, Aneesh; Delmar, Mario; Morley, Gregory E
Studies have demonstrated non-myocytes, including fibroblasts, can electrically couple to myocytes in culture. However, evidence demonstrating current can passively spread across scar tissue in the intact heart remains elusive. We hypothesize electrotonic conduction occurs across non-myocyte gaps in the heart and is partly mediated by Connexin43 (Cx43). We investigated whether non-myocytes in ventricular scar tissue are electrically connected to surrounding myocardial tissue in wild type and fibroblast-specific protein-1 driven conditional Cx43 knock-out mice (Cx43fsp1KO). Electrical coupling between the scar and uninjured myocardium was demonstrated by injecting current into the myocardium and recording depolarization in the scar through optical mapping. Coupling was significantly reduced in Cx43fsp1KO hearts. Voltage signals were recorded using microelectrodes from control scars but no signals were obtained from Cx43fsp1KO hearts. Recordings showed significantly decreased amplitude, depolarized resting membrane potential, increased duration and reduced upstroke velocity compared to surrounding myocytes, suggesting that the non-excitable cells in the scar closely follow myocyte action potentials. These results were further validated by mathematical simulations. Optical mapping demonstrated that current delivered within the scar could induce activation of the surrounding myocardium. These data demonstrate non-myocytes in the scar are electrically coupled to myocytes, and coupling depends on Cx43 expression.
PMID: 27244564
ISSN: 2045-2322
CID: 2124772

Bone marrow derived cells populate post-ablation scar tissue and couple to surrounding myocardium [Meeting Abstract]

Mezzano, V; Kessler, N; Mahoney, V M; Morley, G E
Introduction: Post-ablation scarring is used as a method to uncouple and/or silence pro-arrhythmic circuits. It has been previously suggested that circulating bone marrow derived cells (BMDC) are capable of homing into myocardial infarction scars. It is possible that intercellular junctions form between myocytes and BMDCs and may contribute to ablation failure and recurrence of arrhythmias. Methods: We tested whether BMDCs populate an ablation scars and contribute to functional coupling between the scar and surrounding myocardium. Wild type C57BI/6 mice (n=17) underwent radiation-induced myeloablation and subsequent transplantation with bone marrow progenitors obtained from fetal Cx43 WT (bmcWT) or Cx43 deficient (bmcKO) mice. Results: All donor cells constitutively expressed mCherry protein. Right ventricular ablation was carried out thirty days post transplantation and hearts were studied 30 day post ablation. Cells expressing mCherry and vimentin were observed throughout the scar suggesting donor cells differentiated into a mesenchymal lineage. Coupling between the uninjured myocardium and the scar was assayed with optical mapping. Suction electrode was placed on uninjured myocardium next to the scar to deliver current pulses. Conclusions: Changes in membrane voltage were measured optically at three different sites: Uninjured myocardium, Scar and Remote area (see figure). These data demonstrate that BMDCs can couple to the surrounding myocardium and contribute to the electrophysiological properties of ablation scar tissue. Delivery of modified BMDCs could be used to modifythe post-ablation scar electrophysiological properties. (Figure Presented)
ISSN: 1556-3871
CID: 2150962

A review of the literature on cardiac electrical activity between fibroblasts and myocytes

Mahoney, Vanessa; Mezzano, Valeria; Morley, Gregory E
Myocardial injuries often lead to fibrotic deposition. This review presents evidence supporting the concept that fibroblasts in the heart electrically couple to myocytes.
PMID: 26713556
ISSN: 1873-1732
CID: 1895142