Karen G. Green

Tissue-specific determinants of anisotropic conduction velocity in canine atrial and ventricular myocardium.

Electrical conduction is very rapid and highly anisotropic in atrial fiber bundles, such as the crista terminalis. In contrast to left ventricular myocardium in which the ratio of longitudinal to transverse conduction velocities is approximately 3, propagation velocity in the crista terminalis is approximately 10 times greater in the longitudinal than in the transverse direction. To elucidate potential determinants of these distinct conduction properties, we characterized structural and molecular features of intercellular coupling in the crista terminalis and left ventricular myocardium of the canine heart. Analysis of the number and spatial orientation of myocyte interconnections at gap junctions revealed that a typical left ventricular myocyte was connected to 11.3 +/- 2.2 other myocytes. Approximately equal numbers of connections occurred between ventricular myocytes juxtaposed in side-to-side and end-to-end orientation. In contrast, a typical myocyte of the crista terminalis was connected to only 6.4 +/- 1.7 other cells (P < .05), but nearly 80% of these connections occurred between cells oriented in an end-to-end configuration. In comparison with the ventricular pattern, this spatial distribution of connections would limit intercellular current transfer between laterally apposed cells and thereby enhance anisotropy of conduction velocity in the longitudinal and transverse directions. Ultrastructural analysis showed that crista terminalis myocytes were connected by numerous small gap junctions that occurred in relatively simple, straight intercalated disks. Northern blot analysis showed approximately equivalent amounts of mRNAs encoding the gap junction channel proteins connexin43 and connexin45 but approximately four times more connexin40 mRNA in crista terminalis than in the left ventricle.(ABSTRACT TRUNCATED AT 250 WORDS)

Gap Junction Protein Phenotypes of the Human Heart and Conduction System

Connexin Phenotypes in the Human Heart. Introduction: Gap junction channels are major determinants of intercellular resistance to current flow between cardiac myocytes. Alterations in gap junctions may contribute to development of arrhythmia substrates in patients. However, there is significant interspecies variation in the types and amounts of gap junction subunit proteins (connexins) expressed in disparate regions of mammalian hearts. To elucidate determinants of conduction properties in the human heart, we characterized connexin phenotypes of specific human cardiac tissues with different conduction properties.

Multiple connexins colocalize in canine ventricular myocyte gap junctions.

We have recently shown that adult canine ventricular myocytes express three distinct gap junction channel proteins, connexin40 (Cx40), connexin43 (Cx43), and connexin45 (Cx45). These proteins have unique cytoplasmic domains that likely confer connexin-specific physiological properties. To determine whether the three distinct channel proteins are distributed in identical or different populations of gap junctions, we performed double-label immunofluorescence on disaggregated canine ventricular myocytes incubated simultaneously with a mouse monoclonal anti-Cx43 and affinity-purified polyclonal rabbit antibodies against Cx40 or Cx45. Analysis of double-labeled cardiac myocytes using laser scanning confocal microscopy revealed virtually identical patterns of immunoreactivity for both the Cx43/Cx40 and Cx43/Cx45 pairs. Double-label immunoelectron microscopy confirmed that ultrastructurally identified cardiac myocyte gap junctions contain multiple channel proteins. Thus, three channel proteins colocalize in canine cardiac myocyte gap junctions. The presence of multiple functionally distinct connexins suggests complex possibilities regarding the composition of individual channels and the regulation of intercellular coupling.

Differential Expression of Gap Junction Proteins in the Canine Sinus Node

Electrical coupling of pacemaker cells at gap junctions appears to play an important role in sinus node function. Although the major cardiac gap junction protein, connexin43 (Cx43), is expressed abundantly in atrial and ventricular muscle, its expression in the sinus node has been a subject of controversy. The objectives of the present study were to determine whether Cx43 is expressed by sinus node myocytes, to characterize the spectrum of connexin expression phenotypes in sinus node pacemaker cells, and to define the spatial distribution of different connexin phenotypes in the intact sinus node. To fulfill these objectives, we performed high-resolution immunohistochemical analysis of disaggregated adult canine sinus node preparations. Using enhanced tissue preservation and antigen retrieval techniques, we also performed immunohistochemical studies on sections of intact canine sinus node tissue. Analysis of disaggregated sinus node preparations revealed three populations of pacemaker cells distinguished on the basis of connexin immunohistochemical phenotype: approximately 55% of cells expressed only connexin40 (Cx40); 30% to 35% of cells expressed Cx43, connexin45 (Cx45), and Cx40; and the remaining cells had no detectable connexin expression. In immunostained sections of intact sinus node, Cx43- and Cx45-positive cells were limited in their distribution and were observed in discrete bundles that appeared to abut atrial myocytes. In contrast, Cx40 immunoreactive signal was widely distributed in the sinus node region. These results indicate that subsets of pacemaker cells express distinct connexin phenotypes. Differential expression of connexins could create regions within the sinus node with different conduction properties, thereby contributing to the nonuniform conduction properties seen in this tissue.

Electrical Propagation in Synthetic Ventricular Myocyte Strands From Germline Connexin43 Knockout Mice

To characterize the role of connexin43 (Cx43) as a determinant of cardiac propagation, we synthesized strands and pairs of ventricular myocytes from germline Cx43−/− mice. The amount of Cx43, Cx45, and Cx40 in gap junctions was analyzed by immunohistochemistry and confocal microscopy. Intercellular electrical conductance, gj, was measured by the dual-voltage clamp technique (DVC), and electrical propagation was assessed by multisite optical mapping of transmembrane potential using a voltage-sensitive dye. Compared with wild-type (Cx43+/+) strands, immunoreactive signal for Cx43 was reduced by 46% in Cx43+/− strands and was absent in Cx43−/− strands. Cx45 signal was reduced by 46% in Cx43+/− strands and to the limit of detection in Cx43−/− strands, but total Cx45 protein levels measured in immunoblots of whole cell homogenates were equivalent in all genotypes. Cx40 was detected in ≈ 2% of myocytes. Intercellular conductance, gj, was reduced by 32% in Cx43+/− cell pairs and by 96% in Cx43−/− cell pairs. The symmetrical dependence of gj on transjunctional voltage and properties of single-channel recordings indicated that Cx45 was the only remaining connexin in Cx43−/− cells. Propagation in Cx43−/− strands was very slow (2.1 cm/s versus 52 cm/s in Cx43+/+) and highly discontinuous, with simultaneous excitation within and long conduction delays (2 to 3 ms) between individual cells. Propagation was abolished by 1 mmol/L heptanol, indicating residual junctional coupling. In summary, knockout of Cx43 in ventricular myocytes leads to very slow conduction dependent on the presence of Cx45. Electrical field effect transmission does not contribute to propagation in synthetic strands.

Autophagy regulates pancreatic beta cell death in response to Pdx1 deficiency and nutrient deprivation

There are three types of cell death; apoptosis, necrosis, and autophagy. The possibility that activation of the macroautophagy (autophagy) pathway may increase beta cell death is addressed in this study. Increased autophagy was present in pancreatic islets from Pdx1+/− mice with reduced insulin secretion and beta cell mass. Pdx1 expression was reduced in mouse insulinoma 6 (MIN6) cells by delivering small hairpin RNAs using a lentiviral vector. The MIN6 cells died after 7 days of Pdx1 deficiency, and autophagy was evident prior to the onset of cell death. Inhibition of autophagy prolonged cell survival and delayed cell death. Nutrient deprivation increased autophagy in MIN6 cells and mouse and human islets after starvation. Autophagy inhibition partly prevented amino acid starvation-induced MIN6 cell death. The in vivo effects of reduced autophagy were studied by crossing Pdx1+/− mice to Becn1+/− mice. After 1 week on a high fat diet, 4-week-old Pdx1+/− Becn1+/− mice showed normal glucose tolerance, preserved beta cell function, and increased beta cell mass compared with Pdx1+/− mice. This protective effect of reduced autophagy had worn off after 7 weeks on a high fat diet. Increased autophagy contributes to pancreatic beta cell death in Pdx1 deficiency and following nutrient deprivation. The role of autophagy should be considered in studies of pancreatic beta cell death and diabetes and as a target for novel therapeutic intervention.

Effects of angiotensin II on expression of the gap junction channel protein connexin43 in neonatal rat ventricular myocytes.

OBJECTIVES To elucidate signal transduction pathways regulating expression of myocardial gap junction channel proteins (connexins) and to determine whether mediators of cardiac hypertrophy might promote remodeling of gap junctions, we characterized the effects of angiotensin II on expression of the major cardiac gap junction protein connexin43 (Cx43) in cultured neonatal rat ventricular myocytes. BACKGROUND Remodeling of the distribution of myocardial gap junctions appears to be an important feature of anatomic substrates of ventricular arrhythmias in patients with heart disease. Remodeling of intercellular connections may be initiated by changes in connexin expression caused by chemical mediators of the hypertrophic response. METHODS Cultures were exposed to 0.1 micromol/liter angiotensin II for 6 or 24 h, and Cx43 expression was characterized by immunoblotting, confocal microscopy and electron microscopy. RESULTS Immunoblot analysis revealed a twofold increase in Cx43 content in cells treated for 24 h with angiotensin II (n=4, p < 0.05). This response was inhibited by the presence of 1.0 micromol/liter losartan, an AT1-receptor blocker. Confocal and electron microscopy demonstrated enhanced Cx43 immunoreactivity and increases in the number and size of gap junction profiles in cells exposed to angiotensin II for 24 h. These effects were also blocked by losartan. Immunoprecipitation of Cx43 from cells metabolically labeled with [35S]methionine demonstrated 2.4- and 2.9-fold increases in Cx43 radioactivity after 6 and 24 h exposure to angiotensin II, respectively (p < 0.03 at each time point). CONCLUSIONS Angiotensin II up-regulates gap junctions in cultured neonatal rat ventricular myocytes by increasing Cx43 synthesis. Signal transduction pathways activated by angiotensin II under pathophysiologic conditions could initiate remodeling of conduction pathways, leading to the development of anatomic substrates of arrhythmias.

Proteolysis of connexin43-containing gap junctions in normal and heat-stressed cardiac myocytes.

OBJECTIVE The present studies were performed to examine the degradation of connexin43-containing gap junctions by the lysosome or the proteasome in normal and heat-stressed cultures of neonatal rat ventricular myocytes. METHODS Primary cultures were prepared from neonatal rat ventricular myocytes. Connexin43 was detected by immunoblotting, immunofluorescence, or immunoprecipitation. Gap junction profiles were detected by transmission electron microscopy. RESULTS Immunoblots of whole cell lysates demonstrated increased levels of connexin43 in cultures treated with lysosomal inhibitors (chloroquine, leupeptin, E-64, or ammonium chloride) or proteasomal inhibitors (lactacystin or ALLN). Pulse-chase experiments showed that the half-life of connexin43 was 1.4 h in control cultures, but was prolonged to 2.0 or 2.8 h in cultures treated with chloroquine or lactacystin, respectively. Immunofluorescence and electron microscopy showed a significant increase in the number of gap junction profiles in myocytes treated with either chloroquine or lactacystin. Heat treatment of cultures (43.5 degrees C for 30 min) produced a rapid loss of connexin43 as detected by immunoblotting or immunofluorescence. Heat-induced connexin43 degradation was prevented by simultaneous treatment with lactacystin, ALLN, or chloroquine. Connexin43 levels and distribution returned to normal by 3 h following a heat shock and were resistant to a subsequent repeat heat stress. The heat shock also led to production of HSP70 as detected by immunoblotting. CONCLUSIONS These data suggest that Cx43 gap junctions in myocytes are degraded by the proteasome and the lysosome, that this proteolysis can be augmented by heat stress, and that inducible factors such as HSP70 may protect against Cx43 degradation.

Distinct Pathways Regulate Expression of Cardiac Electrical and Mechanical Junction Proteins in Response to Stretch

To define mechanisms regulating expression of cell–cell junction proteins, we have developed an in vitro system in which neonatal rat ventricular myocytes were subjected to pulsatile stretch. Previously, we showed that expression of the gap junction protein, connexin (Cx) 43, is increased by ≈2-fold after 1 hour of stretch, and this response is mediated by stretch-induced secretion of vascular endothelial growth factor (VEGF). Here, we report that the mechanical junction proteins plakoglobin, desmoplakin, and N-cadherin are also upregulated by pulsatile stretch but by a mechanism independent of VEGF or other secreted chemical signals. Stretch-induced upregulation of mechanical junction proteins was blocked by anti–β1 and anti–β3 integrin antibodies. Transfection of cells with adenovirus expressing GFP-FRNK, a dominant-negative inhibitor of focal adhesion kinase (FAK)-dependent signaling, blocked stretch-induced upregulation of Cx43 and mechanical junction proteins but did not block the ability of exogenous VEGF to upregulate Cx43 expression. Conditioned medium removed from uninfected cells after stretch increased Cx43 expression when added to nonstretched cells, and this effect was blocked by anti-VEGF antibodies, but stretch-conditioned medium from GFP-FRNK cells had no effect on Cx43 expression. The src kinase inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine blocked stretch-induced upregulation of mechanical junction proteins but not Cx43. Thus, stretch upregulates expression of both electrical and mechanical junction proteins via integrin-dependent activation of FAK. Stretch-induced upregulation of Cx43 expression is mediated by FAK-dependent secretion of VEGF. In contrast, stretch-induced upregulation of adhesion junction proteins involves intracellular mechanotransduction pathways initiated via integrin signaling and acting downstream of src kinase.

Relative Contributions of Connexins 40 and 43 to Atrial Impulse Propagation in Synthetic Strands of Neonatal and Fetal Murine Cardiomyocytes

Atrial tissue expresses both connexin 40 (Cx40) and 43 (Cx43) proteins. To assess the relative roles of Cx40 and Cx43 in atrial electrical propagation, we synthesized cultured strands of atrial myocytes derived from mice with genetic deficiency in Cx40 or Cx43 expression and measured propagation velocity (PV) by high-resolution optical mapping of voltage-sensitive dye fluorescence. The amount of Cx40 and/or Cx43 in gap junctions was measured by immunohistochemistry and total or sarcolemmal Cx43 or Cx40 protein by immunoblotting. Progressive genetic reduction in Cx43 expression decreased PV from 34±6 cm/sec in Cx43+/+ to 30±8 cm/sec in Cx43+/− and 19±11 cm/sec in Cx43−/− cultures. Concomitantly, the cell area occupied by Cx40 immunosignal in gap junctions decreased from 2.0±1.6% in Cx43+/+ to 1.7±0.5% in Cx43+/− and 1.0±0.2% in Cx43−/− strands. In contrast, progressive genetic reduction in Cx40 expression increased PV from 30±2 cm/sec in Cx40+/+ to 40±7 cm/sec in Cx40+/− and 45±10 cm/sec in Cx40−/− cultures. Concomitantly, the cell area occupied by Cx43 immunosignal in gap junctions increased from 1.2±0.9% in Cx40+/+ to 2.8±1.4% in Cx40+/− and 3.1±0.6% in Cx40−/− cultures. In accordance with the immunostaining results, immunoblots of the Triton X-100–insoluble fraction revealed an increase of Cx43 in gap junctions in extracts from Cx40-ablated atria, whereas total cellular Cx43 remained unchanged. Our results suggest that the relative abundance of Cx43 and Cx40 is an important determinant of atrial impulse propagation in neonatal hearts, whereby dominance of Cx40 decreases and dominance of Cx43 increases local propagation velocity.