Medical Foundation Building K25, The University of Sydney, NSW 2006 Australia
ccam8612@uni.sydney.edu.au
I have recently completed a PhD in the laboratory of Exocrine Physiology and Biophysics under the supervision of Prof David Cook and A/Prof Anuwat Dinudom at the University of Sydney. In my time as a PhD student, I have learned to apply electrophysiological techniques to investigate a broad range of physiological questions. I am interested in furthering my use of these and other cellular techniques in a research environment.
Abstract: We investigated the relationship between gut health, visceral fat dysfunction and metabolic disorders in diet-induced obesity. C57BL/6J mice were fed control or high saturated fat diet (HFD). Circulating glucose, insulin and inflammatory markers were measured. Proximal colon barrier function was assessed by measuring transepithelial resistance and mRNA expression of tight-junction proteins. Gut microbiota profile was determined by 16S rDNA pyrosequencing. Tumor necrosis factor (TNF)-α and interleukin (IL)-6 mRNA levels were measured in proximal colon, adipose tissue and liver using RT-qPCR. Adipose macrophage infiltration (F4/80(+)) was assessed using immunohistochemical staining. HFD mice had a higher insulin/glucose ratio (P = 0.020) and serum levels of serum amyloid A3 (131%; P = 0.008) but reduced circulating adiponectin (64%; P = 0.011). In proximal colon of HFD mice compared to mice fed the control diet, transepithelial resistance and mRNA expression of zona occludens 1 were reduced by 38% (P<0.001) and 40% (P = 0.025) respectively and TNF-α mRNA level was 6.6-fold higher (P = 0.037). HFD reduced Lactobacillus (75%; P<0.001) but increased Oscillibacter (279%; P = 0.004) in fecal microbiota. Correlations were found between abundances of Lactobacillus (r = 0.52; P = 0.013) and Oscillibacter (r = -0.55; P = 0.007) with transepithelial resistance of the proximal colon. HFD increased macrophage infiltration (58%; P = 0.020), TNF-α (2.5-fold, P<0.001) and IL-6 mRNA levels (2.5-fold; P = 0.008) in mesenteric fat. Increased macrophage infiltration in epididymal fat was also observed with HFD feeding (71%; P = 0.006) but neither TNF-α nor IL-6 was altered. Perirenal and subcutaneous adipose tissue showed no signs of inflammation in HFD mice. The current results implicate gut dysfunction, and attendant inflammation of contiguous adipose, as salient features of the metabolic dysregulation of diet-induced obesity.
Abstract: The G protein-coupled receptor kinase (GRK2) belongs to a family of protein kinases that phosphorylates agonist-activated G protein-coupled receptors, leading to G protein-receptor uncoupling and termination of G protein signaling. GRK2 also contains a regulator of G protein signaling homology (RH) domain, which selectively interacts with α-subunits of the Gq/11 family that are released during G protein-coupled receptor activation. We have previously reported that kinase activity of GRK2 up-regulates activity of the epithelial sodium channel (ENaC) in a Na(+) absorptive epithelium by blocking Nedd4-2-dependent inhibition of ENaC. In the present study, we report that GRK2 also regulates ENaC by a mechanism that does not depend on its kinase activity. We show that a wild-type GRK2 (wtGRK2) and a kinase-dead GRK2 mutant ((K220R)GRK2), but not a GRK2 mutant that lacks the C-terminal RH domain (ΔRH-GRK2) or a GRK2 mutant that cannot interact with Gαq/11/14 ((D110A)GRK2), increase activity of ENaC. GRK2 up-regulates the basal activity of the channel as a consequence of its RH domain binding the α-subunits of Gq/11. We further found that expression of constitutively active Gαq/11 mutants significantly inhibits activity of ENaC. Conversely, co-expression of siRNA against Gαq/11 increases ENaC activity. The effect of Gαq on ENaC activity is not due to change in ENaC membrane expression and is independent of Nedd4-2. These findings reveal a novel mechanism by which GRK2 and Gq/11 α-subunits regulate the activity ENaC.
Abstract: One unique physiological characteristic of frogs is that their main route for intake of water is across the skin. In these animals, the skin acts in concert with the kidney and urinary bladder to maintain electrolyte homeostasis. Water absorption across the skin is driven by the osmotic gradient that develops as a consequence of solute transport. Our recent study demonstrated that chytridiomycosis, an infection of amphibian skin by the fungal pathogen, Batrachochytrium dendrobatidis, inhibits epithelial Na(+) channels, attenuating Na(+) absorption through the skin. In frogs that become severely affected by this fungus, systemic depletion of Na(+), K(+) and Cl(-) is thought to cause deterioration of cardiac electrical function, leading to cardiac arrest. Here we review the ion transport mechanisms of frog skin, and discuss the effect of chytridiomycosis on these mechanisms.
Abstract: We have previously reported that P2Y(2) purinoceptors and muscarinic M(3) receptors trigger Ca(2+) responses in HT-29 cells that differ in their timecourse, the Ca(2+) response to P2Y(2) receptor activation being marked by a more rapid decline of intracellular Ca(2+) concentration ([Ca(2+)](i)) after the peak response and that this rapid decline of [Ca(2+)](i) was slowed in cells expressing heterologous beta-adrenergic receptor kinase (betaARK). In the present study, we demonstrate that, during P2Y(2) receptor activation, betaARK expression increases the rate of Gd(3+)-sensitive Mn(2+) influx, a measure of the rate of store-operated Ca(2+) entry from the extracellular space, during P2Y(2) activation and that this effect of betaARK is mimicked by exogenous alpha-subunits of G(q), G(11) and G(i2). The effect of betaARK on the rate of Mn(2+) influx is thus attributable to its ability to scavenge G protein betagamma-subunits released during activation of P2Y(2) receptor. We further find that the effect of betaARK on the rate of Mn(2+) influx during P2Y(2) receptor activation can be overcome by arachidonic acid. In addition, the UTP-induced Mn(2+) influx rate was significantly increased by inhibitors of phospholipase A(2) (PLA(2)) and an siRNA directed against PLA(2)beta, but not by an siRNA directed against PLA(2)alpha or by inhibitors of arachidonic acid metabolism. These findings provide evidence for the existence of a P2Y(2) receptor-activated signalling system that acts in parallel with depletion of intracellular Ca(2+) stores to inhibit Ca(2+) influx across the cell membrane. This signalling process is mediated via Gbetagamma and involves PLA(2)beta and arachidonic acid.
Abstract: The pathogen Batrachochytrium dendrobatidis (Bd), which causes the skin disease chytridiomycosis, is one of the few highly virulent fungi in vertebrates and has been implicated in worldwide amphibian declines. However, the mechanism by which Bd causes death has not been determined. We show that Bd infection is associated with pathophysiological changes that lead to mortality in green tree frogs (Litoria caerulea). In diseased individuals, electrolyte transport across the epidermis was inhibited by >50%, plasma sodium and potassium concentrations were respectively reduced by approximately 20% and approximately 50%, and asystolic cardiac arrest resulted in death. Because the skin is critical in maintaining amphibian homeostasis, disruption to cutaneous function may be the mechanism by which Bd produces morbidity and mortality across a wide range of phylogenetically distant amphibian taxa.
Abstract: BACKGROUND: Myocardial refractoriness and repolarization is an important electrophysiological property that, when altered, increases the risk of arrhythmogenesis. These electrophysiological changes associated with chronic myocardial infarction (MI) have not been studied in detail. We assessed the influence of left ventricular (LV) scarring on local refractoriness, repolarization, and electrogram characteristics. METHODS: MI was induced in five sheep by percutaneous left anterior descending artery occlusion for 3 hours. Mapping was performed at 19 +/- 6 weeks post-MI. A total of 20 quadripolar transmural needles were deployed at thoracotomy in the LV within and surrounding scar. Bipolar pacing was performed from each needle to assess the effective refractory period (ERP) of the subendocardium and subepicardium. The activation (AT) and repolarization (RT) times, and modified activation recovery interval (ARI(m)) were determined from endocardial unipolar electrograms recorded in sinus rhythm simultaneously from all needles. Scarring was quantified histologically and compared with electrophysiological characteristics. RESULTS: Increased scarring corresponded with increased ERP (P < 0.01), decreased subendocardial electrogram amplitude (P < 0.001), and slope (P < 0.001). ERP did not differ between endocardium and epicardium (P > 0.05). The ARI(m) and RT were prolonged during early myocardial activation (P < 0.001). After adjusting for AT, the RT and ARI(m) were prolonged in areas of scarring (P < 0.001). After adjusting for electrogram amplitude, the ARI(m) was prolonged in dense scar (P < 0.05). CONCLUSIONS: We confirmed histologically that scarring contributes to prolongation of repolarization, increased refractoriness, and reductions in conduction and voltage post-MI. Prolongation of repolarization may be further augmented when local activation is earliest or electrogram voltage is decreased within scar.
Abstract: It has recently been shown that the epithelial Na(+) channel (ENaC) is compartmentalized in caveolin-rich lipid rafts and that pharmacological depletion of membrane cholesterol, which disrupts lipid raft formation, decreases the activity of ENaC. Here we show, for the first time, that a signature protein of caveolae, caveolin-1 (Cav-1), down-regulates the activity and membrane surface expression of ENaC. Physical interaction between ENaC and Cav-1 was also confirmed in a coimmunoprecipitation assay. We found that the effect of Cav-1 on ENaC requires the activity of Nedd4-2, a ubiquitin protein ligase of the Nedd4 family, which is known to induce ubiquitination and internalization of ENaC. The effect of Cav-1 on ENaC requires the proline-rich motifs at the C termini of the beta- and gamma-subunits of ENaC, the binding motifs that mediate interaction with Nedd4-2. Taken together, our data suggest that Cav-1 inhibits the activity of ENaC by decreasing expression of ENaC at the cell membrane via a mechanism that involves the promotion of Nedd4-2-dependent internalization of the channel.
Abstract: 1. The epithelial sodium channel (ENaC) is tightly regulated by hormonal and humoral factors, including cytosolic ion concentration and glucocorticoid and mineralocorticoid hormones. Many of these regulators of ENaC control its activity by regulating its surface expression via neural precursor cell-expressed developmentally downregulated (gene 4) protein (Nedd4-2). 2. During the early phase of aldosterone action, Nedd4-2-dependent downregulation of ENaC is inhibited by the serum- and glucocorticoid-induced kinase 1 (Sgk1). 3. Sgk1 phosphorylates Nedd4-2. Subsequently, phosphorylated Nedd4-2 binds to the 14-3-3 protein and, hence, reduces binding of Nedd4-2 to ENaC. 4. Nedd4-2 is also phosphorylated by protein kinase B (Akt1). Both Sgk1 and Akt1 are part of the insulin signalling pathway that increases transepithelial Na(+) absorption by inhibiting Nedd4-2 and activating ENaC.
Abstract: Endocardial radiofrequency ablation of the left ventricle does not create transmural lesions reliably even with active electrode cooling. The authors developed a prototype catheter with an internally cooled needle electrode that could be advanced an adjustable distance into the myocardium. Freshly excised hearts from eight male sheep were perfused and superfused using oxygenated ovine blood. Ablations were performed for 2 minutes using the prototype catheter and a conventional endocardial 5-mm irrigated tip ablation catheter at target temperatures of 80 degrees C and 50 degrees C, respectively. The prototype catheter needle was inserted 12 mm deep for all ablations. The maximal power and irrigation rate was 50 W, 20 mL/min for the irrigated tip catheter and 20 W, 10 mL/min for the intramural needle catheter. Intramural needle lesions were significantly deeper (13.5 +/- 2.3 vs 9.1 +/- 1.3 mm, P < 0.01) but less wide (8.7 +/- 1.5 vs 12.7 +/- 1.9 mm, P < 0.01) than irrigated tip lesions. Popping occurred during 12 (37%) of the 32 irrigated tip ablations. Popping did not occur during intramural needle ablation. The cooled intramural needle ablation catheter creates lesions that are significantly deeper than irrigated tip catheters with less tissue boiling. In contrast to irrigated tip ablation, electrode temperature monitoring can be used to determine if a lesion has been created during intramural needle ablation. The cooled intramural needle ablation lesions were of a clinically useful width, addressing one of the main recognized deficiencies of intramural needle ablation.
Abstract: BACKGROUND: We assessed the hypothesis that "virtual electrograms" from a noncontact mapping system (EnSite 3000) could be used to localize myocardial scar. METHODS AND RESULTS: Myocardial infarctions were induced in sheep by inflating an angioplasty balloon in the left anterior descending coronary artery for 3 hours. Scar mapping was performed on 8 sheep without inducible ventricular tachycardia by use of the noncontact mapping system and a 256-channel contact mapping system. Transmural mapping needles were inserted into myocardial regions that were (1) scarred, (2) peripheral to the scar, and (3) distant from the scar. Unipolar electrograms were exported from both systems and analyzed on a personal computer workstation. The percentage of myocardial scarring at each needle site was assessed histologically. Pearson's correlation was used to assess the degree of association between various electrogram characteristics and the presence of myocardial scarring. The only noncontact electrogram characteristic that showed any association with the presence of myocardial scarring was the negative slope duration (contact, r=0.62, P<0.001; noncontact, r=0.23, P=0.004). The other electrogram characteristics studied were electrogram maximal deflection (contact, r=0.38, P<0.001; noncontact, r=0.03, P=0.75) and minimal slope (contact, r=0.42, P<0.001; noncontact, r=0.05, P=0.54). CONCLUSIONS: Noncontact electrograms do not reliably identify ventricular scar. Alternative strategies such as importing computed tomography images into the geometry should be used when scar localization is important.
Abstract: The aim of this study was to evaluate intramural temperature-controlled radiofrequency ablation by determining the intramural temperature profile during ablation and by correlating lesion geometry with intramural electrode size and temperature. Intramural ablation might be useful to create deeper lesions for ventricular tachycardia secondary to underlying heart disease. Intramural radiofrequency ablation was performed in 17 greyhounds at thoracotomy, from an epicardial approach, using a 21-gauge needle electrode. Sixty-eight lesions were created in 11 dogs at electrode temperatures of 70 degrees C, 80 degrees C, 90 degrees C, and 100 degrees C for 60 seconds. Intramural thermocouples at 1-, 2-, 3-, 4-, and 5-mm distances were used to identify simultaneous intramural temperature profile. An epicardial approach was used to ensure accurate positioning of the ablating and temperature monitoring needles within the myocardium with fixed interneedle distances. Ninety-nine radiofrequency ablations were performed in six greyhounds using three different intramural electrode lengths (1 mm, 2.5 mm, and 5.5 mm). Lesions were created at 70 degrees C, 80 degrees C, and 90 degrees C for 60 seconds. All lesions were measured after staining with Gomori Trichrome. Lesion dimensions increased in a highly predictable manner with increasing electrode temperature or length. There was no popping or charring, even with target electrode temperature of 100 degrees C. There was significant correlation between intramural temperature 4 mm from the ablating electrode and lesion width (P < 0.001, R2= 0.45) and depth (P = 0.02, R2= 0.08). Feedback control of electrode temperature enables reliable intramural radiofrequency ablation without impedance rise even with target electrode temperature of 100 degrees C. Increasing the length of the intramural ablating electrode to > or = 5.5 mm and increasing temperatures to 90 degrees C-100 degrees C creates the largest lesions.
Abstract: It is not clear whether the noncontact electrograms obtained using the EnSite system in the left ventricle resemble most closely endocardial, intramural, or epicardial contact electrograms or a summation of transmural electrograms. This study compared unipolar virtual electrograms from the EnSite system with unipolar contact electrograms from transmural plunge needle electrodes using a 256-channel mapping system. The study also evaluated the effects of differing activation sites (endocardial, intramural, or epicardial). A grid of 50-60 plunge needles was positioned in the left ventricles of eight male sheep. Each needle had four electrodes to record from the endocardium, two intramural sites, and the epicardium. Correlations between contact and noncontact electrograms were calculated on 32,242 electrograms. Noncontact electrograms correlated equally well in morphology and accuracy of timing with endocardial (0.88 +/- 0.15), intramural (0.87 +/- 0.15), epicardial (0.88 +/- 0.15), and transmural summation contact electrograms (0.89 +/- 0.14) during sinus rhythm, endocardial pacing, and epicardial pacing. There was a nonlinear relationship between noncontact electrogram accuracy as measured by correlation with the contact electrogram and distance from the multielectrode array (MEA): beyond 40 mm accuracy decreased rapidly. The accuracy of noncontact electrograms also decreased with increasing distance from the equator of the MEA. Virtual electrograms from noncontact mapping of normal left ventricles probably represent a summation of transmural activation. Noncontact mapping has similar accuracy with either endocardial or epicardial sites of origin of electrical activity provided the MEA is within 40 mm of the recording site.
Abstract: Transmural recordings using plunge needle electrodes are useful in mapping ventricular tachyarrhythmia, but they interfere with activation sequences or damage the myocardium. This study evaluated the effects of insertion of 66 transmural needles on myocardial activation, structure, and function. Epicardial maps were performed at thoracotomy using a 40-electrode plaque in five mongrel dogs. Sixty-six transmural plunge needles were introduced into the anterior aspect of the septum and left ventricle. Transmural maps of unipolar electrograms were recorded every 15 minutes via 124 electrodes over a 2-hour period. Epicardial maps were repeated after the needles were removed. All recordings were performed during sinus rhythm and ventricular pacing at 300- and 200-ms cycle lengths. Gated heart pool studies were performed preoperatively and 2 weeks after thoracotomy. Programmed ventricular stimulation was performed 2 weeks after thoracotomy. In total, 15,996 electrograms were analyzed. Maximum negative dV/dt of each electrogram and the activation time at each electrode did not change significantly over the 2 hours of needle insertion. After removal of the needles, epicardial maps were unchanged compared to before needle insertion. Mean left ventricular ejection fraction 2 weeks after needle insertion was 59% versus 58% before needle insertion (P=0.9). No dogs had inducible ventricular tachycardia. Histology showed contraction bands of 0.8-mm diameter adjacent to the needle tracks but no scarring. Insertion of 66 closely spaced plunge needles did not distort epicardial or transmural maps. Multiple needles did not result in myocardial scarring, left ventricular dysfunction, or predispose to ventricular tachycardia.
Abstract: Radiofrequency ablation of the left ventricle using an endocardially placed electrode is unable to reliably create transmural lesions even with active electrode cooling. To produce deeper radiofrequency lesions, the authors developed and tested a prototype intramural needle ablation catheter that had a distal 1.1-mm diameter straight needle that could be advanced 12 mm into the myocardium. Freshly excised hearts from eight male sheep were perfused and superfused with oxygenated ovine blood. Ablations were performed for 60 seconds with the prototype catheter and a conventional 5-mm irrigated tip ablation catheter at target temperatures of 90 degrees C and 50 degrees C, respectively. The ablation lesions were bisected and stained with blue tetrazolium to assess lesion geometry. The irrigated tip ablation catheter required significantly more power than the intramural needle ablation catheter (37.7 +/- 7.3 vs 6.4 +/- 2.1 W, P < 0.01). Intramural needle lesions were significantly deeper (12.5 +/- 3.0 mm vs 8.3 +/- 2.1 mm, P < 0.01) but less wide (3.9 +/- 1.1 mm vs 11.5 +/- 2.0 mm, P < 0.01) than irrigated tip lesions. There was a high incidence of crater formation (74%), popping (45%), and myocardial charring (29%) during irrigated tip ablation; these phenomena were not observed during intramural needle ablation. The intramural needle ablation catheter creates significantly deeper but narrower lesions without evidence of tissue boiling. This technology may be particularly useful for ablation of ventricular tachycardia originating from regions where tissue depth is increased, like the ventricular septum.
