Faculty Bibliography

The neurovascular unit (NVU) consists of cells intrinsic to the vessel wall, the endothelial cells and pericytes, and astrocyte endfeet that surround the vessel but are separated from it by basement membrane. Endothelial cells are primarily responsible for creating and maintaining blood-brain-barrier (BBB) tightness, but astrocytes contribute to the barrier through paracrine signaling to the endothelial cells and by forming the glia limitans. Gap junctions (GJs) between astrocyte endfeet are composed of connexin 43 (Cx43) and Cx30, which form plaques between cells. GJ plaques formed of Cx43 do not diffuse laterally in the plasma membrane and thus potentially provide stable organizational features to the endfoot domain, whereas GJ plaques formed of other connexins and of Cx43 lacking a large portion of its cytoplasmic carboxyl terminus are quite mobile. In order to examine the organizational features that immobile GJs impose on the endfoot, we have used super-resolution confocal microscopy to map number and sizes of GJ plaques and aquaporin (AQP)-4 channel clusters in the perivascular endfeet of mice in which astrocyte GJs (Cx30, Cx43) were deleted or the carboxyl terminus of Cx43 was truncated. To determine if BBB integrity was compromised in these transgenic mice, we conducted perfusion studies under elevated hydrostatic pressure using horseradish peroxide as a molecular probe enabling detection of micro-hemorrhages in brain sections. These studies revealed that microhemorrhages were more numerous in mice lacking Cx43 or its carboxyl terminus. In perivascular domains of cerebral vessels, we found that density of Cx43 GJs was higher in the truncation mutant, while GJ size was smaller. Density of perivascular particles formed by AQP4 and its extended isoform AQP4ex was inversely related to the presence of full length Cx43, whereas the ratio of sizes of the particles of the AQP4ex isoform to total AQP4 was directly related to the presence of full length Cx43. Confocal analysis showed that Cx43 and Cx30 were substantially colocalized in astrocyte domains near vasculature of truncation mutant mice. These results showing altered distribution of some astrocyte nexus components (AQP4 and Cx30) in Cx43 null mice and in a truncation mutant, together with leakier cerebral vasculature, support the hypothesis that localization and mobility of gap junction proteins and their binding partners influences organization of astrocyte endfeet which in turn impacts BBB integrity of the NVU.

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.

Dysfunction of the specialized cardiac conduction system (CCS) is associated with life-threatening arrhythmias. Strategies to derive CCS cells, including rare Purkinje cells (PCs), would facilitate models for mechanistic studies and drug discovery and also provide new cellular materials for regenerative therapies. A high-throughput chemical screen using CCS:lacz and Contactin2:egfp (Cntn2:egfp) reporter embryonic stem cell (ESC) lines was used to discover a small molecule, sodium nitroprusside (SN), that efficiently promotes the generation of cardiac cells that express gene profiles and generate action potentials of PC-like cells. Imaging and mechanistic studies suggest that SN promotes the generation of PCs from cardiac progenitors initially expressing cardiac myosin heavy chain and that it does so by activating cyclic AMP signaling. These findings provide a strategy to derive scalable PCs, along with insight into the ontogeny of CCS development.

The cardiac Purkinje fiber network is comprised of highly specialized cardiomyocytes responsible for the synchronous excitation and contraction of the ventricles. Computational modeling, experimental animal studies and intracardiac electrical recordings from patients with heritable and acquired forms of heart disease suggest that Purkinje cells (PC) may also serve as critical triggers of life-threatening arrhythmias. Nonetheless, owing to the difficulty in isolating and studying this rare population of cells, the precise role of PC in arrhythmogenesis and the underlying molecular mechanisms responsible for their pro-arrhythmic behavior are not fully characterized. Conceptually, a stem cell-based model system might facilitate studies of PC-dependent arrhythmia mechanisms and serve as a platform to test novel therapeutics. Here, we describe the generation of murine embryonic stem cells (ESC) harboring pan-cardiomyocyte and PC-specific reporter genes. We demonstrate that the dual reporter gene strategy may be used to identify and isolate the rare ESC-derived PC (ESC-PC) from a mixed population of cardiogenic cells. ESC-PC display transcriptional signatures and functional properties, including action potentials, intracellular calcium cycling and chronotropic behavior comparable to endogenous PC. Our results suggest that stem-cell derived PC are a feasible new platform for studies of developmental biology, disease pathogenesis and screening for novel anti-arrhythmic therapies

The cytoplasmic C-terminus of the gap junction forming protein Cx43 regulates channel function and binding to other proteins through posttranslational modification to serine, tyrosine, and other residues. Live high-resolution and super-resolution microscopy allowed us to show that the cysteine residues of the Cx43 C-terminus are required for formation of the normally stable gap junction macromolecular complex. In addition we used two-color live imaging to show that the Cx43 C-terminus also determines the mobility of other components of the specialized astrocyte end-foot compartment. Nitric oxide and associated enzymes are an important signaling pathway in astrocyte endfoot regulation of blood brain barrier maintenance and vascular- two processes partly governed by astrocyte connexins. We found that pharmacological treatment with nitrosylation modifying enzymes leads to changes in mobility of gap junction Nexus components, dependent on the presence of cysteine residues in the C-terminus of Cx43. Using mice expressing truncated Cx43 lacking the C-terminus (including the cysteine residues) we have found compromised blood brain barrier. We hypothesize that nitrosylation of cysteine residues within the C-terminus of Cx43 regulates astrocyte endfoot macromolecular organization and communication between astrocyte endfeet that is required for formation of a blood brain barrier adequately robust to resist heightened blood pressure. Since nitrosylation and connexin expression are altered in conditions of inflammation and brain trauma these new insights into gap junction biology will help us to understand why blood brain barrier weakness occurs and provide targets for therapeutic intervention

BACKGROUND: We previously demonstrated that the cell adhesion protein contactin2 (Cntn2) is enriched in Purkinje cells of the cardiac conduction system (CCS). The objective of this study was generation of a mouse embryonic stem cell (mESC) reporter line that allows identification of Purkinje-like cardiomyocytes in vitro. METHODS AND RESULTS: mESC were generated from transgenic mice carrying a BAC Cntn2-eGFP reporter gene and were subsequently transduced with lentivirus coding for a MHCalpha-mCherry cardiomyocyte reporter gene. Immunostaining analysis confirmed that mESC expressed markers of pluripotency (Oct3/4; Klf4) and spontaneously differentiated into cells of all three germ layers in the absence of LIF (alpha-smooth muscle actin, beta-tubulin, alpha-fetoprotein). Spontaneous or serum-free directed cardiac differentiation resulted in generation of spontaneously beating cardiomyocytes, of which single positive MHCalpha-mCherry cells comprised 57.76% +/- 3.7% and MHCalpha-mCherry/Cntn2-eGFP cells comprised 1.9% +/- 0.9% of the total population as determined by fluorescence-activated cell sorting (FACS) (n = 5 differentiation cultures). Quantitative real-time PCR analysis of FACS isolated double-positive cells verified cardiomyocyte-specific transcript expression (Mlc2v: 33-fold, P <.01; Nkx2.5: 178-fold, P <.001) compared to MHCalpha-mCherry/Cntn2-eGFP-negative noncardiomyocytes. Moreover, double-positive cells expressed significantly elevated levels of CCS-specific transcripts compared to mCherry single-positive cardiomyocytes (Cntn2: 31-fold, P <.001; Cx40: 8-fold, P <.01). Action potential recordings of double-positive cells demonstrated distinct plateau phase and elongated action potential duration (APD50 = 79.9 +/- 10.4 ms, APD90 = 170.2 +/- 17.5 ms, n = 11) compared with eGFP-negative cardiomyocytes (APD50 = 53.4 +/- 9.4 ms, APD90 = 120.6 +/- 17.3 ms, n = 15), consistent with their assignment as Purkinje-like derivatives. CONCLUSIONS: We established an mESC reporter line that allows for the identification and enrichment of ventricular CCS derivatives. This model should be useful for downstream studies of CCS development and pathology, including the role of Purkinje cells as arrhythmogenic triggers. Cntn2 may also be a useful marker of CCS-like cells derived from human embryonic stem and/or induced pluripotent stem cells.

Dynamic changes of the actin cytoskeleton are instrumental in morphogenetic processes including changes in cell shape and adhesion. Formin proteins regulate actin microfilament assembly and can specifically influence adherens junction formation. Previous studies in our lab have demonstrated remarkable plasticity of formin isoforms during heart development and in vitro cardiomyocyte differentiation. As gap junction stability is dependent on the presences of mechanical junctions we were interested if modulation of cardiac formins influences expression of Cx43 protein and gap junction function. Objective: To investigate the effect of cardiac formin knockdown (KD) on cell-cell contact formation and functional coupling of cardiomyocytes. Methods: Cardiomyocytes were isolated from neonatal rat hearts (NRCM) and cultured as monolayers (d0); NRCM were treated with transfection agent only (TF), control siRNA (Ctr) or formin specific rat siRNAs (Daam1; Fhod1; Fhod3; Dharmacon) (d1); cultures were subjected to high resolution optical mapping or processed for immunofluorescence analysis (d4). Results: KD of Fhod1 or Fhod3 lead to disruption of sarcomers, cell rounding and ultimately resulted in complete dissociation of NRCM. In contrast, Daam1 KD resulted in significant cell elongation without loss of cell-cell contacts (mean cell areas in mum²: 681.8 + 99.1 (Daam1) vs. 594.9 + 67.6 (TF), 564 + 53.3 (Ctr), 455.9 + 47.4 (Fhod1), 339.3 + 14.3 (Fhod3); P: 0.01, ANOVA). As expected, optical mapping data for discontinuous Fhod1 and Fhod3 monolayers were very variable due to areas of complete block of conduction. Optical mapping analysis of Daam1 silenced NRCM demonstrated significant increase in conduction velocity (0.241 + 0.004 m/s; n=4) compared to NRCM treated with TF only (0.197 + 0.010 m/s, n=3) or Ctr (0.207 + 0.005 m/s, n=3; P: 0.003, ANOVA). Average gap junction diameter (0.24 + 0.03 mum (n=489; Daam1) vs. 0.41 + 0.03 mum (n=550; DF), 0.38 + 0.04 mum (n=574; Ctr) P: 0.009, ANOVA), and total !

Background: We have previously demonstrated that the cell adhesion protein contactin2 (Cntn2) is enriched in Purkinje cells of the cardiac conduction system (CCS). Objective: Generation of a mouse embryonic stem cell (mESC) reporter line that allows identification of Purkinje-like cardiomyocytes in vitro. Methods and Results: mESC were generated from transgenic mice carrying a BAC Cntn2-eGFP reporter gene and were subsequently transduced with lentivirus coding for a selectable MHCalpha-mCherry cardiomyocyte reporter gene. Immunostaining analysis confirmed that mESC expressed markers of pluripotency (Oct3/4; Klf4) and spontaneously differentiated into cells of all three germ layers in the absence of LIF (alpha- smooth muscle actin; beta-tubulin; alpha-fetoprotein). Spontaneous or serum-free directed cardiac differentiation resulted in generation of double positive, spontaneously beating cardiomyocytes after three weeks. Yield of double positive cells could be increased by adding endocardialderived factors (Nrg1; ET-1). FACS isolated double positive cells were enriched in transcripts of cardiomyocytes (Tbx5; Nkx2.5) and the CCS (Cntn2; Cx40). Action potential recordings of eGFP positive cardiomyocytes demonstrated distinct plateau phase and elongated action potential duration (APD50=79.9+10.4ms, APD90=170.2+17.5ms; n=11) compared with eGFP negative cardiomyocytes (APD50=53.4+9.4ms, APD90=120.6+17.3ms; n=15). Conclusion: We have established a mESC reporter line for the identification of CCS-like cells. This model should be useful for downstream studies of CCS development and pathology. Cntn2 may also be a useful marker of CCS-like cells derived from human ES and/or iPS cells

Background: Hypertension is associated with the development of atrial fibrillation, however the electrophysiological consequences of this condition remain poorly understood. K(ATP) channels, which contribute to ventricular arrhythmias, are also expressed in the atria. We hypothesized that salt-induced elevated blood pressure leads to atrial K(ATP) channel activation and increased arrhythmia inducibility. Methods and Results: Elevated blood pressure was induced in mice with a high salt diet (HS) for four weeks. High resolution optical mapping was used to measure atrial arrhythmia inducibility, effective refractory period (ERP) and action potential duration (APD(90)). Excised patch clamping was performed to quantify K(ATP) channel properties and density. K(ATP) channel protein expression was also evaluated. Atrial arrhythmia inducibility was 22% higher in HS compared to control hearts. ERP and APD(90) were significantly shorter in the RAA and LAA of HS compared to control hearts. Perfusion with 1 muM glibenclamide or 300 muM tolbutamide significantly decreased arrhythmia inducibility and prolonged APD(90) in HS hearts compared to untreated HS hearts. K(ATP) channel density was 156% higher in myocytes isolated from HS compared to control animals. SUR1 protein expression was increased in the HS LAA (415% of NS) and RAA (372% of NS). Conclusion: K(ATP) channel activation provides a mechanistic link between salt-induced elevated BP and increased atrial arrhythmia inducibility. The findings of this study have important implications for the treatment and prevention of atrial arrhythmias in the setting of hypertensive heart disease and may lead to new therapeutic approaches