4,072 research outputs found
RGS4 Expression is Associated with Spontaneously Beating Cardiomyocytes during Sinoatrial Node Development
No full textRGS4 is highly expressed in the SAN and is an important regulator of parasympathetic signaling in those cells. Our study set out to determine the utility of RGS4 expression as a marker of SAN myocyte progenitors during cardiac development. In the intact RGS4-LacZ mouse, RGS4 expression was observed in the SAN region starting at E11.5 and its expression remains strong in the SAN throughout cardiac maturation. In mESC-derived cardiomyocytes, RGS4 expression was observed to be associated in the contractile regions of the monolayer. When a RGS4-GFP transgenic mESC line was used to select for differentiated cardiomyocytes with high RGS4 expression, this cell population showed many similarities to SAN myocytes. Specifically, they expressed the pacemaker channel, HCN4, and showed spontaneous action potentials that were characteristic of SAN myocytes. Taken together these data suggest that RGS4 reporter expression may be used to select for SAN myocyte-like cells during cardiomyocyte differentiation from mESCs.MAS
The Functional Characterization of Two Regulators of G-protein Signaling Proteins Abundantly Expressed in Vascular Smooth Muscle Cells
No full textPrecise regulation of heterotrimeric G-protein signaling is important for maintaining proper cardiovascular system function. Indeed, G-protein signaling is frequently upregulated during cardiovascular disease suggesting that identifying mechanisms for inhibiting G-protein signaling may be an effective therapeutic strategy for the treatment and prevention of disease. The work presented in this thesis is directed at two RGS proteins, RGS2 and RGS5, the two highest expressing RGS proteins in VSMCs. Despite the large number of studies published on them, there is still much to be learned about the specific G-protein pathways that each RGS protein controls. Using genetic and molecular models, we set out to identify novel regulatory pathways controlling RGS2 and RGS5 function. We hypothesize that characterizing the determinants and regulation of RGS protein function will provide a better understanding of the signaling that occurs within VSMCs under both physiologic and pathophysiologic conditions.
Our work presented in the first three studies of this thesis, describes novel regulatory pathways that are involved in regulating RGS2 protein function. We describe the production of RGS2 protein isoforms that are the result of alternative translational start site usage. Interestingly, the expression pattern of these proteins is controlled by the signaling status of the cell. In the second two studies, we identify a functional consequence of RGS2-interaction with the plasma membrane. We show that this is dependent on the interaction between the amphipathic α-helix and anionic phospholipids present in the plasma membrane. We further show that disruptions in this interaction, as occurs in the human population, can lead to reduced RGS2 function and thus potentially hypertension.
Finally, our last study focuses on the function and regulation of RGS5, the single highest expressing RGS protein in VSMCs. We show that the regulation of RGS5 is dependent, similar to other VSMC-specific genes, on the activity of SRF and myocardin. However, interestingly, RGS5 expression is further controlled by the extent of DNA methylation that occurs in its proximal promoter. We show that this is an important regulator of RGS5 expression both in development as well as during disease, specifically in-stent restenosis.Ph
Characterizing the Role of Regulator of G-protein Signalling 4 as a Mediator of Sinoatrial Node and Atrial Cardiomyocyte Function
No full textHeart rate is modulated by the opposing activities of sympathetic and parasympathetic inputs to pacemaker cardiomyocytes in the sinoatrial (SA) node. Parasympathetic activity on nodal myocytes is mediated by acetylcholine-dependent stimulation of M2 muscarinic receptors and activation of Gαi/o signalling. Although, regulators of G-protein signalling (RGS) proteins are potent inhibitors of Gαi/o signalling in many tissues, the RGS protein(s) that regulate parasympathetic tone in the SA node are unknown. Our results demonstrate that RGS4 mRNA levels are higher in the SA node compared to right atrium. Conscious freely moving RGS4-null mice showed a greater extent of bradycardia in response to parasympathetic agonists compared to wild-type animals. Moreover, anaesthetized rgs4-null mice had lower baseline heart rates and greater heart rate increases following atropine administration. Retrograde-perfused hearts from rgs4-null mice also showed enhanced negative chronotropic responses to carbachol, while isolated SA node myocytes showed greater sensitivity to carbachol-mediated reduction in the action potential firing rate. Finally, rgs4-null SA node cells showed decreased levels of G-protein-coupled inward rectifying potassium (GIRK) channel desensitization, and altered modulation of acetylcholine-sensitive potassium current (IKACh) kinetics following carbachol stimulation. Taken together, our studies establish that RGS4 plays an important role in regulating sinus rhythm by inhibiting parasympathetic signalling and IKACh activity. Following these results, we predicted that loss of RGS4 expression and function will result in increased levels of parasympathetic effector activity leading to increased susceptibility to atrial fibrillation.
Susceptibility to atrial fibrillation (AF) depends strongly on parasympathetic activity. Since RGS4 inhibits parasympathetic / M2-dependent Gαi/o signalling in the SA node, we explored whether changes in RGS4 levels altered the susceptibility of atrial fibrillation. We found that, RGS4 levels were decreased in atria of tachypaced dogs prior to their development of chronic AF. Moreover, in vivo ECG recordings of anaesthetized mice showed greater susceptibility to AF while optical mapping of isolated atrial preparations using a voltage-sensitive dye revealed greatly increased susceptibility to rotor formation when RGS4 was ablated. Consistent with altered parasympathetic signalling in the myocardium of rgs4-null mice, IKACh evoked by carbachol application were greater in isolated atrial myocytes from rgs4-null mice. These IKACh changes were, as expected, associated with marked action potential duration shortening in response to parasympathetic activation, but not to slower conduction velocities. Together, our findings establish that RGS4 protects atrial tissues from excess parasympathetic signalling that predispose to atrial fibrillation.Ph
Defining the Mechanisms by which Palmitoylation Regulates the Localization and Function of RGS4
No full textRegulator of G-protein signalling 4 (RGS4) modulates Gq and Gi signalling at the plasma membrane (PM). It has been demonstrated that the addition of palmitate to cysteine residues is an important regulator of RGS protein localization and function. The family of palmitate transferase enzymes shares a conserved Asp-His-His-Cys (DHHC) motif. We set out to establish the DHHC isoform(s) that affect RGS4 activity in HEK201 cells. Confocal microscopy revealed that overexpression of DHHCs 3 and 7 mobilized RGS4 to the Golgi. Knockdown of either DHHC3 or DHHC7 attenuated RGS4 inhibition of Gαq-coupled Ca2+ release and reduced RGS4 PM localization. Consistent with a role in promoting RGS4 lipid bilayer targeting, dominant negative mutants of the five most highly expressed DHHCs in HEK201 cells also diminished RGS4 PM association. Together, these data suggest that members of the mammalian DHHC family regulate RGS4 localization and function, likely through palmitoylation of its target cysteine residues.MAS
The Functional Characterization of Two Regulators of G-protein Signaling Proteins Abundantly Expressed in Vascular Smooth Muscle Cells
No full textPrecise regulation of heterotrimeric G-protein signaling is important for maintaining proper cardiovascular system function. Indeed, G-protein signaling is frequently upregulated during cardiovascular disease suggesting that identifying mechanisms for inhibiting G-protein signaling may be an effective therapeutic strategy for the treatment and prevention of disease. The work presented in this thesis is directed at two RGS proteins, RGS2 and RGS5, the two highest expressing RGS proteins in VSMCs. Despite the large number of studies published on them, there is still much to be learned about the specific G-protein pathways that each RGS protein controls. Using genetic and molecular models, we set out to identify novel regulatory pathways controlling RGS2 and RGS5 function. We hypothesize that characterizing the determinants and regulation of RGS protein function will provide a better understanding of the signaling that occurs within VSMCs under both physiologic and pathophysiologic conditions.
Our work presented in the first three studies of this thesis, describes novel regulatory pathways that are involved in regulating RGS2 protein function. We describe the production of RGS2 protein isoforms that are the result of alternative translational start site usage. Interestingly, the expression pattern of these proteins is controlled by the signaling status of the cell. In the second two studies, we identify a functional consequence of RGS2-interaction with the plasma membrane. We show that this is dependent on the interaction between the amphipathic α-helix and anionic phospholipids present in the plasma membrane. We further show that disruptions in this interaction, as occurs in the human population, can lead to reduced RGS2 function and thus potentially hypertension.
Finally, our last study focuses on the function and regulation of RGS5, the single highest expressing RGS protein in VSMCs. We show that the regulation of RGS5 is dependent, similar to other VSMC-specific genes, on the activity of SRF and myocardin. However, interestingly, RGS5 expression is further controlled by the extent of DNA methylation that occurs in its proximal promoter. We show that this is an important regulator of RGS5 expression both in development as well as during disease, specifically in-stent restenosis.Ph
Characterizing the Role of Regulator of G-protein Signalling 4 as a Mediator of Sinoatrial Node and Atrial Cardiomyocyte Function
No full textHeart rate is modulated by the opposing activities of sympathetic and parasympathetic inputs to pacemaker cardiomyocytes in the sinoatrial (SA) node. Parasympathetic activity on nodal myocytes is mediated by acetylcholine-dependent stimulation of M2 muscarinic receptors and activation of Gαi/o signalling. Although, regulators of G-protein signalling (RGS) proteins are potent inhibitors of Gαi/o signalling in many tissues, the RGS protein(s) that regulate parasympathetic tone in the SA node are unknown. Our results demonstrate that RGS4 mRNA levels are higher in the SA node compared to right atrium. Conscious freely moving RGS4-null mice showed a greater extent of bradycardia in response to parasympathetic agonists compared to wild-type animals. Moreover, anaesthetized rgs4-null mice had lower baseline heart rates and greater heart rate increases following atropine administration. Retrograde-perfused hearts from rgs4-null mice also showed enhanced negative chronotropic responses to carbachol, while isolated SA node myocytes showed greater sensitivity to carbachol-mediated reduction in the action potential firing rate. Finally, rgs4-null SA node cells showed decreased levels of G-protein-coupled inward rectifying potassium (GIRK) channel desensitization, and altered modulation of acetylcholine-sensitive potassium current (IKACh) kinetics following carbachol stimulation. Taken together, our studies establish that RGS4 plays an important role in regulating sinus rhythm by inhibiting parasympathetic signalling and IKACh activity. Following these results, we predicted that loss of RGS4 expression and function will result in increased levels of parasympathetic effector activity leading to increased susceptibility to atrial fibrillation.
Susceptibility to atrial fibrillation (AF) depends strongly on parasympathetic activity. Since RGS4 inhibits parasympathetic / M2-dependent Gαi/o signalling in the SA node, we explored whether changes in RGS4 levels altered the susceptibility of atrial fibrillation. We found that, RGS4 levels were decreased in atria of tachypaced dogs prior to their development of chronic AF. Moreover, in vivo ECG recordings of anaesthetized mice showed greater susceptibility to AF while optical mapping of isolated atrial preparations using a voltage-sensitive dye revealed greatly increased susceptibility to rotor formation when RGS4 was ablated. Consistent with altered parasympathetic signalling in the myocardium of rgs4-null mice, IKACh evoked by carbachol application were greater in isolated atrial myocytes from rgs4-null mice. These IKACh changes were, as expected, associated with marked action potential duration shortening in response to parasympathetic activation, but not to slower conduction velocities. Together, our findings establish that RGS4 protects atrial tissues from excess parasympathetic signalling that predispose to atrial fibrillation.Ph
RGS4 Expression is Associated with Spontaneously Beating Cardiomyocytes during Sinoatrial Node Development
No full textRGS4 is highly expressed in the SAN and is an important regulator of parasympathetic signaling in those cells. Our study set out to determine the utility of RGS4 expression as a marker of SAN myocyte progenitors during cardiac development. In the intact RGS4-LacZ mouse, RGS4 expression was observed in the SAN region starting at E11.5 and its expression remains strong in the SAN throughout cardiac maturation. In mESC-derived cardiomyocytes, RGS4 expression was observed to be associated in the contractile regions of the monolayer. When a RGS4-GFP transgenic mESC line was used to select for differentiated cardiomyocytes with high RGS4 expression, this cell population showed many similarities to SAN myocytes. Specifically, they expressed the pacemaker channel, HCN4, and showed spontaneous action potentials that were characteristic of SAN myocytes. Taken together these data suggest that RGS4 reporter expression may be used to select for SAN myocyte-like cells during cardiomyocyte differentiation from mESCs.MAS
Characterizing the Role of RGS5 in the Regulation of Vascular Smooth Muscle Cell Function
No full textRegulators of G-protein signaling (RGS) modulate G-protein coupled receptor (GPCR) activity in vascular smooth muscle cells (VSMCs). One such protein, RGS5, has been shown to have selective expression in VSMCs and pericytes, and can inhibit signaling from Gαq and Gαi subunits. Using an RGS5 knockout model, we assessed the functional effect of RGS5 in the constriction and dilation of resistance arterioles. Furthermore, we examined the intracellular lipid interaction of RGS proteins to identify the determinants regulating the biologic function of RGS5. Surprisingly, loss of RGS5 function in mesenteric arterioles had no effect on constriction and dilation of resistance arterioles. Cultured VSMCs showed increased basal ERK1/2 phosphorylation and increased VASP signaling in response to SNP treatment in RGS5KO VSMCs as compared to wild type controls, with no effect on cell proliferation. These data suggest RGS5 may integrate multiple intracellular pathways with competing effects on VSMC contraction.MAS
Development of a Receptor Internalization Assay for Measuring and Analysing RGS9-2 and Palmitoyl-CoA Transferase Activity in HEK Cells
No full textRegulator of G-protein signalling 9-2 (RGS9-2) complex inhibits agonist-dependent
dopamine receptor D2R activity and internalization, and plays a protective role against the
development of neuro-reward circuit dysfunction. R7 binding protein (R7BP) is necessary
for RGS9-2 protein stability and trafficking. Preliminary work from our group has shown
the palmitoyl-CoA transferase, DHHC5, can facilitate R7BP trafficking, however this
function can also enhance RGS9-2 trafficking and function. The aim of this work is to
establish an in vitro assay to verify the effects of DHHC5 and R7BP on the internalization
of D2R in HEK cells. We established a modified ELISA protocol that consistently showed
~20% internalization of surface D2R, which is not ideal, possibly due to the poor
internalization properties of D2R in HEK cells. Future efforts, therefore, will require the
development of other functional assays to examine the role of DHHC5, R7BP and RGS9-
2 as a regulator of D2R signalling and functionM.Sc.2018-05-10 00:00:0
Investigating the Putative Mechanism and Functional Role of RGS5 Upregulation in Vascular Smooth Muscle Cells Following Statin Treatment
No full textRGS5 within vascular smooth muscle cells (VSMCs) is capable of inhibiting G-protein signaling involved in VSMC recruitment. Based on the suggested ability of statin to upregulate RGS5, we investigated whether RGS5 could mediate the pleiotropic effects of statins in VSMCs. Fluvastatin treatment of isolated VSMCs significantly increased the expression of RGS5, while downregulating RGS2, RGS3, and RGS16. However, these results were not observed ex vivo or in vivo within the aorta following fluvastatin treatment. Fluvastatin also upregulated PPARδ and PPARγ, demonstrated transcriptional regulators of RGS5 expression, in VSMCs. However, PPARδ and PPARγ antagonists did not block fluvastatin-induced RGS5 upregulation and their agonists did not increase RGS5 expression. Lastly, fluvastatin did not significantly reduce neointimal hyperplasia following femoral artery injury in RGS5 WT and KO mice. These results suggest that statins robustly upregulate RGS5 only in cultured synthetic VSMCs, with the physiological role of this pleiotropic effect remaining to be elucidated.M.Sc
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