Search

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disorder affecting 1 in 500 people in the general population. Characterized by asymmetric left ventricular hypertrophy, cardiomyocyte disarray, cardiac fibrosis and increased risk of sudden cardiac death, HCM is in fact a highly complex disease with heterogenous clinical presentation, onset and complication. While mutations in the sarcomere gene can account for a substantial proportion of familial cases of HCM, up to 70% of HCM patients do not carry such sarcomere variants and the causal mutations for their diseases remain elusive. Recently, we identified a novel variant of the alpha-crystallin B chain (CRYAB R123W ) in a pair of monozygotic twins who developed concordant HCM phenotypes that manifested over a nearly identical time course. Yet, how CRYAB R123W promotes HCM phenotype remains unclear. Here, we generated mice carrying the Cryab R123W -knockin allele and demonstrated that hearts from these animals exhibit increased elastance, reduced end diastolic volume and increased E/E’ consistent with increased systolic and reduced diastolic function. Upon transverse aortic constriction, mice carrying the Cryab R123W allele developed pathological left ventricular hypertrophy with substantial cardiac fibrosis and progressively reduced ejection fraction. In contrast to another well-characterized CRYAB variant (R120G) which induced Desmin aggregation, no evidence of protein aggregation was observed in hearts expressing CRYAB R123W despite its potent effect on driving cellular hypertrophy. Unexpectedly, CRYAB R123W appears to enhance calcium signaling by promoting nuclear localization of NFAT through direct interaction with calcineurin. Mice also were more susceptible to ventricular tachycardia with programmed stimulation. Single nuclei RNA-sequencing of mouse hearts is underway and preliminarily reveals reduction in oxidative phosphorylation, consistent with known effects of Cryab on mitochondrial function. Thus, our data establishes the Cryab R123W allele as a novel genetic model of HCM and unveiled additional sarcomere-independent mechanisms of cardiomyocyte hypertrophy.

Background The incidence of sudden arrhythmic death is markedly increased in diabetics. Objective The purpose of this study was to develop a mouse model for postmyocardial infarction (post-MI) ventricular tachycardia (VT) in the diabetic heart and determine the mechanism of an antiarrhythmic effect of statins. Methods ECG transmitters were implanted in wild-type (WT), placebo, and pravastatin-treated type I diabetic Akita mice. MIs were induced by coronary ligation, and Ca 2+ transients were studied by optical mapping, and Ca 2+ transients and sparks in left ventricular myocytes (VM) by the Ionoptix system and confocal microscopy. Results Burst pacing of Akita mouse hearts resulted in rate-related QRS/T-wave alternans, which was attenuated in pravastatin-treated mice. Post-MI Akita mice developed QRS/T-wave alternans and VT at 2820 ± 879 beats per mouse, which decreased to 343 ± 115 in pravastatin-treated mice (n = 13, P 2+ alternans, both attenuated in hearts of statin-treated mice. Akita VM displayed Ca 2+ alternans, and triggered activity as well as increased Ca 2+ transient decay time (Tau), Ca 2+ sparks, and cytosolic Ca 2+ and decreased SR Ca 2+ stores all of which were in part reversed in cells from statin treated mice. Homogenates of Akita ventricles demonstrated decreased SERCA2a/PLB ratio and increased ratio of protein phosphatase (PP-1) to the PP-1 inhibitor PPI-1 which were reversed in homogenates of pravastatin-treated Akita mice. Conclusion Pravastatin decreased the incidence of post-MI VT and Ca 2+ alternans in Akita mouse hearts in part by revering abnormalities of Ca 2+ handling via the PP-1/PPI-1 pathway.

The pathophysiology of pulmonary hypertension (PH) and heart failure (HF) includes fibrogenic remodeling associated with loss of pulmonary arterial (PA) and cardiac compliance. We and others have previously identified that transglutaminase 2 (TG2) as a participant in adverse fibrogenic remodeling. However, little is known about the biologic mechanisms that regulate TG2 function. We examined physiological mouse models of experimental PH, HF and type 1 diabetes that are associated with altered glucose metabolism/ glycolysis and report here that TG2 expression and activity are elevated in pulmonary and cardiac tissues under all these conditions. We additionally used PA adventitial fibroblasts to test the hypothesis that TG2 is an intermediary between enhanced tissue glycolysis and fibrogenesis. Our in vitro results show that glycolytic enzymes and TG2 are up-regulated in fibroblasts exposed to high glucose, which stimulates cellular glycolysis as measured by Seahorse analysis. We examined the relationship of TG2 to a terminal glycolytic enzyme, pyruvate kinase M2 (PKM2), and found that PKM2 regulates glucose -induced TG2 expression and activity as well as fibrogenesis. Our studies further show that TG2 inhibition blocks glucose-induced fibrogenesis and cell proliferation. Our findings support a novel role for glycolysis -mediated TG2 induction and tissue fibrosis associated with experimental PH, HF and hyperglycemia.

Type 1 diabetic Akita mice develop severe cardiac parasympathetic dysfunction that we have previously demonstrated is due at least in part to an abnormality in the response of the end organ to parasympathetic stimulation. Specifically, we had shown that hypoinsulinemia in the diabetic heart results in attenuation of the G-protein coupled inward rectifying K channel (GIRK) which mediates the negative chronotropic response to parasympathetic stimulation due at least in part to decreased expression of the GIRK1 and GIRK4 subunits of the channel. We further demonstrated that the expression of GIRK1 and GIRK4 is under the control of the Sterol Regulatory element Binding Protein (SREBP-1), which is also decreased in response to hypoinsulinemia. Finally, given that hyperactivity of Glycogen Synthase Kinase (GSK)3β, had been demonstrated in the diabetic heart, we demonstrated that treatment of Akita mice with Li+, an inhibitor of GSK3β, increased parasympathetic responsiveness and SREBP-1 levels consistent with the conclusion that GSK3β might regulate IKACh via an effect on SREBP-1. However, inhibitor studies were complicated by lack of specificity for GSK3β. Here we generated an Akita mouse with cardiac specific inducible knockout of GSK3β. Using this mouse, we demonstrate that attenuation of GSK3β expression is associated with an increase in parasympathetic responsiveness measured as an increase in the heart rate response to atropine from 17.3 ± 3.5% (n = 8) prior to 41.2 ± 5.4% (n = 8, P = 0.017), an increase in the duration of carbamylcholine mediated bradycardia from 8.43 ± 1.60 min (n = 7) to 12.71 ± 2.26 min (n = 7, P = 0.028) and an increase in HRV as measured by an increase in the high frequency fraction from 40.78 ± 3.86% to 65.04 ± 5.64 (n = 10, P = 0.005). Furthermore, patch clamp measurements demonstrated a 3-fold increase in acetylcholine stimulated peak IKACh in atrial myocytes from GSK3β deficiency mice compared with control. Finally, western blot analysis of atrial extracts from knockout mice demonstrated increased levels of SREBP-1, GIRK1 and GIRK4 compared with control. Taken together with our prior observations, these data establish a role of increased GSK3β activity in the pathogenesis of parasympathetic dysfunction in type 1 diabetes via the regulation of IKACh and GIRK1/4 expression.

Abdominal Aortic Aneurysm (AAA) is a major cause of mortality and morbidity in men over 65 years of age. Male apolipoprotein E knockout (ApoE(-/-)) mice infused with angiotensin II (AngII) develop AAA. Although AngII stimulates both JAK/STAT and Toll-like receptor 4 (TLR4) signaling pathways, their involvement in AngII mediated AAA formation is unclear. Here we used the small molecule STAT3 inhibitor, S3I-201, the TLR4 inhibitor Eritoran and ApoE(-/-)TLR4(-/-) mice to evaluate the interaction between STAT3 and TLR4 signaling in AngII-induced AAA formation. ApoE(-/-) mice infused for 28 days with AngII developed AAAs and increased STAT3 activation and TLR4 expression. Moreover, AngII increased macrophage infiltration and the ratio of M1 (pro-inflammatory)/M2 (healing) macrophages in aneurysmal tissue as early as 7-10 days after AngII infusion. STAT3 inhibition with S3I-201 decreased the incidence and severity of AngII-induced AAA formation and decreased MMP activity and the ratio of M1/M2 macrophages. Furthermore, AngII-mediated AAA formation, MMP secretion, STAT3 phosphorylation and the ratio of M1/M2 macrophages were markedly decreased in ApoE(-/-)TLR4(-/-) mice, and in Eritoran-treated ApoE(-/-) mice. TLR4 and pSTAT3 levels were also increased in human aneurysmal tissue. These data support a role of pSTAT3 in TLR4 dependent AAA formation and possible therapeutic roles for TLR4 and/or STAT3 inhibition in AAA.

Decreased heart rate variability (HRV) is a major risk factor for sudden death and cardiovascular disease. We previously demonstrated that parasympathetic dysfunction in the heart of the Akita type 1 diabetic mouse was due to a decrease in the level of the sterol response element–binding protein (SREBP-1). Here we demonstrate that hyperactivity of glycogen synthase kinase-3β (GSK3β) in the atrium of the Akita mouse results in decreased SREBP-1, attenuation of parasympathetic modulation of heart rate, measured as a decrease in the high-frequency (HF) fraction of HRV in the presence of propranolol, and a decrease in expression of the G-protein coupled inward rectifying K+ (GIRK4) subunit of the acetylcholine (ACh)-activated inward-rectifying K+ channel (IKACh), the ion channel that mediates the heart rate response to parasympathetic stimulation. Treatment of atrial myocytes with the GSK3β inhibitor Kenpaullone increased levels of SREBP-1 and expression of GIRK4 and IKACh, whereas a dominant-active GSK3β mutant decreased SREBP-1 and GIRK4 expression. In Akita mice treated with GSK3β inhibitors Li+ and/or CHIR-99021, Li+ increased IKACh, and Li+ and CHIR-99021 both partially reversed the decrease in HF fraction while increasing GIRK4 and SREBP-1 expression. These data support the conclusion that increased GSK3β activity in the type 1 diabetic heart plays a critical role in parasympathetic dysfunction through an effect on SREBP-1, supporting GSK3β as a new therapeutic target for diabetic autonomic neuropathy.

Although a reduction in the high-frequency (HF) component of heart rate variability (HRV) is a major complication of diabetes and a risk factor for sudden death, its relationship to ventricular tachycardia (VT) is unknown. We developed a mouse model for the study of VT and its relationship to changes in HRV in the Akita type 1 diabetic mouse. Programmed ventricular stimulation of anesthetized mice demonstrated that Akita mice were more inducible for VT compared with wild-type mice: 78.6% versus 28.6% ( P = 0.007). Optical mapping of perfused hearts demonstrated multifocal breakthroughs that occasionally gave rise to short-lived rotors consistent with focal initiation and maintenance of VT. Treatment of Akita mice with pravastatin, which had been previously shown clinically to decrease ventricular ectopy and to increase HRV, decreased the inducibility of VT: 36.8% compared with 75.0% with placebo treatment ( P = 0.022). The HF fraction of HRV was reduced in Akita mice (48.6 ± 5.2% vs. 70.9 ± 4.8% in wild-type mice, P = 0.005) and was increased compared with placebo treatment in pravastatin-treated mice. Pretreatment of Akita mice with the muscarinic agonist carbamylcholine or the β-adrenergic receptor blocker propranolol decreased the inducibility of VT ( P = 0.001). In conclusion, the increased inducibility of focally initiated VT and reduced HF fraction in Akita mice were partially reversed by both pravastatin treatment and pharmacologic reversal of parasympathetic dysfunction. In this new animal model for the study of the pathogenesis of VT in type 1 diabetes, pravastatin may play a role in the prevention of VT by attenuating parasympathetic dysfunction.

Objective— Atherosclerosis encompasses a conspicuously maladaptive inflammatory response that might involve innate immunity. Here, we compared the role of Toll-like receptor 4 (TLR4) with that of TLR2 in intimal foam cell accumulation and inflammation in apolipoprotein E (ApoE) knockout (KO) mice in vivo and determined potential mechanisms of upstream activation and downstream action. Methods and Results— We measured lipid accumulation and gene expression in the lesion-prone lesser curvature of the aortic arch. TLR4 deficiency reduced intimal lipid by ≈75% in ApoE KO mice, despite unaltered total serum cholesterol and triglyceride levels, whereas TLR2 deficiency reduced it by ≈45%. TLR4 deficiency prevented the increased interleukin-1α (IL-1α) and monocyte chemoattractant protein-1 mRNA levels seen within lesional tissue, and it also lowered serum IL-1α levels. Smooth muscle cells (SMC) were present within the intima of the lesser curvature of the aortic arch at this early lesion stage, and they enveloped and permeated nascent lesions, which consisted of focal clusters of foam cells. Cholesterol enrichment of SMC in vitro stimulated acyl-coenzyme A:cholesterol acyltransferase-1 mRNA expression, cytoplasmic cholesterol ester accumulation, and monocyte chemoattractant protein-1 mRNA and protein expression in a TLR4-dependent manner. Conclusion— TLR4 contributes to early-stage intimal foam cell accumulation at lesion-prone aortic sites in ApoE KO mice, as does TLR2 to a lesser extent. Intimal SMC surround and penetrate early lesions, where TLR4 signaling within them may influence lesion progression.

Objective— Abdominal aortic aneurysm (AAA) is a life-threatening disease affecting almost 10% of the population over age 65. Generation of AAAs by infusion of angiotensin (Ang) II in apolipoprotein E-knockout (ApoE −/− ) mice is an animal model which supports an imbalance of the renin-angiotensin system in the pathogenesis of AAA. The effect of statins on AngII-mediated AAA formation and the associated neovascularization is not known. Here we determined the effect of simvastatin and the ERK inhibitor, CI1040, on AngII-stimulated AAA formation. Methods and Results— ApoE −/− mice infused for 28 days with AngII using osmotic minipumps were treated with placebo, 10 mg/kg/d simvastatin, or 100 mg/kg/d CI1040. 95% of AngII-treated mice developed AAA with neovascularization of the lesion, increased ERK phosphorylation, MCP-1 secretion, and MMP activity. These effects were markedly reversed by simvastatin and in part by CI1040. Furthermore, simvastatin and the ERK inhibitor U0126 reversed AngII-stimulated angiogenesis and MMP secretion by human umbilical vein endothelial cells. Conclusions— These data support the conclusion that simvastatin interferes with AAA formation induced by AngII in ApoE −/− mice at least in part via ERK inhibition.

Parasympathetic stimulation of the heart, which provides protection from arrhythmias and sudden death, involves activation of the G protein-coupled inward rectifying K+ channel GIRK1/4 and results in an acetylcholine-sensitive K+ current, I KACh. We describe a unique relationship between lipid homeostasis, the lipid-sensitive transcription factor SREBP-1, regulation of the cardiac parasympathetic response, and the development of ventricular arrhythmia. In embryonic chick atrial myocytes, lipid lowering by culture in lipoprotein-depleted serum increased SREBP-1 levels, GIRK1 expression, and I KACh activation. Regulation of the GIRK1 promoter by SREBP-1 and lipid lowering was dependent on interaction with 2 tandem sterol response elements and an upstream E-box motif. Expression of dominant negative SREBP-1 (DN-SREBP-1) reversed the effect of lipid lowering on I KACh and GIRK1. In SREBP-1 knockout mice, both the response of the heart to parasympathetic stimulation and the expression of GIRK1 were reduced compared with WT. I KACh, attenuated in atrial myocytes from SREBP-1 knockout mice, was stimulated by SREBP-1 expression. Following myocardial infarction, SREBP-1 knockout mice were twice as likely as WT mice to develop ventricular tachycardia in response to programmed ventricular stimulation. These results demonstrate a relationship between lipid metabolism and parasympathetic response that may play a role in arrhythmogenesis.

HMG-CoA reductase inhibitors (statins) are widely used in the treatment and prevention of atherosclerosis. Here we demonstrate that the HMG-CoA reductase inhibitor simvastatin potentiates TNFalpha-mediated apoptosis and TNFalpha signaling in human umbilical vein endothelial cells (HUVECs). While 2.5 microM simvastatin or 40 ng/ml TNFalpha alone had only a small effect on apoptosis in HUVECs, co-incubation with simvastatin and TNFalpha markedly increased apoptosis in a time- and dose-dependent manner as measured by FACS analysis of propidium iodide-stained cells. Geranylgeraniol, which serves as a substrate for the geranylgeranylation of small GTP binding proteins such as RhoA, which is required for the function and membrane localization of Rho, reversed the effect of simvastatin on apoptosis. GGTI, an inhibitor of protein geranylgeranylation, mimicked the effect of simvastatin on apoptosis and interfered with the membrane localization of RhoA. Furthermore, simvastatin increased the expression of the TNFalpha type I receptor (TNFalphaRI) with a dose dependence and a dependence on geranylgeranylation similar to that demonstrated for the potentiation of TNFalpha-mediated apoptosis. Adenoviral expression of a dominant-negative RhoA mimicked the effect of simvastatin on the expression of TNFalphaRI, while adenoviral expression of a dominant-activating RhoA mutant reversed the effect of simvastatin on the expression of TNFalphaRI. Simvastatin also potentiated TNFalpha signaling as determined by increased TNFalpha-mediated E-selectin expression. These data support the conclusion that TNFalpha signaling is under the negative control of RhoA and that statins potentiate TNFalpha signaling at least in part via interference with RhoA inhibition of TNFalpha type I receptor expression.

Transforming growth factor beta (TGFbeta) signaling is involved in the development and regulation of multiple organ systems and cellular signaling pathways. We recently demonstrated that TGFbeta regulates the response of atrial myocytes to parasympathetic stimulation. Here, TGFbeta(1) is shown to inhibit expression of the M(2) muscarinic receptor (M(2)), which plays a critical role in the parasympathetic response of the heart. This effect is mimicked by overexpression of a dominant negative mutant of RhoA and by the RhoA kinase inhibitor Y27632, whereas adenoviral expression of a dominant activating-RhoA reverses TGFbeta inhibition of M(2) expression. TGFbeta(1) also mediates a decrease in GTP-bound RhoA and a reciprocal increase in the expression of the RhoA GTPase-activating protein, p190RhoGAP, whereas total RhoA is unchanged. Inhibition of M(2) promoter activity by TGFbeta(1) is mimicked by overexpression of p190RhoGAP, whereas a dominant negative mutant of p190RhoGAP reverses this effect of TGFbeta(1). In contrast to atrial myocytes, in mink lung epithelial cells, in which TGFbeta signaling through activation of RhoA has been previously identified, TGFbeta(1) stimulated an increase in GTP-bound RhoA in association with a reciprocal decrease in the expression of p190RhoGAP. Both effects demonstrated a similar dose dependence on TGFbeta(1). Thus TGFbeta regulation of M(2) muscarinic receptor expression is dependent on RhoA, and TGFbeta regulation of p190RhoGAP expression may be a cell type-specific mechanism for TGFbeta signaling through RhoA.

Human umbilical vein endothelial cells (HUVECs) have played a major role as a model system for the study of the regulation of endothelial cell function and the role of the endothelium in the response of the blood vessel wall to stretch, shear forces, and the development of atherosclerotic plaques and angiogenesis. Here, we use HUVECs and human microvascular endothelial cells to study the role of the HMG-CoA reductase inhibitor, simvastatin, and the small GTP-binding protein Rho in the regulation of angiogenesis. Simvastatin inhibited angiogenesis in response to FGF-2 in the corneal pocket assay of the mouse and in vascular endothelial growth factor (VEGF)-stimulated angiogenesis in the chick chorioallontoic membrane. Furthermore, simvastatin inhibited VEGF-stimulated tube formation by human dermal microvascular endothelial cells and the formation of honeycomb-like structures by HUVECs. The effect was dose-dependent and was not secondary to apoptosis. Geranylgeranyl-pyrophosphate (GGPP), a product of the cholesterol metabolic pathway that serves as a substrate for the posttranslational lipidation of RhoA, was required for membrane localization, but not farnesylpyrophosphate (FPP), the substrate for the lipidation of Ras. Furthermore, GGTI, a specific inhibitor of GGPP, mimicked the effect of simvastatin of tube formation and the formation of honeycombs whereas FTI, a specific inhibitor of the farnesylation of Ras, had no effect. Adenoviral expression of a DN-RhoA mutant mimicked the effect of simvastatin on tube formation and the formation of honeycombs, whereas a dominant activating mutant of RhoA reversed the effect of simvastatin on tube formation. Finally, simvastatin interfered with the membrane localization of RhoA with a dose-dependence similar to that for the inhibition of tube formation. Simvastatin also inhibited the VEGF-stimulated phosphorylation of the VEGF receptor KDR, and the tyrosine kinase FAK, which plays a role in cell migration. These data demonstrate that simvastatin interfered with angiogenesis via the inhibition of RhoA. Data supporting a role for angiogenesis in the development and growth of atherosclerotic plaques suggest that this antiangiogenic effect of Statins might prevent the progression of atherosclerosis via the inhibition of plaque angiogenesis.

Little is known regarding factors that induce parasympathetic responsiveness during cardiac development. We demonstrated previously that in atrial cells cultured from chicks 14 days in ovo, transforming growth factor β (TGFβ) decreased parasympathetic inhibition of beat rate by the muscarinic agonist, carbamylcholine, by 5-fold and decreased expression of Gαi2. Here in atrial cells 5 days in ovo,TGFβ increased carbamylcholine inhibition of beat rate 2.5-fold and increased expression of Gαi2. TGFβ also stimulated Gαi2 mRNA expression and promoter activity at day 5 while inhibiting them at day 14 in ovo. Over the same time course expression of type I TGFβ receptors, chick activin receptor-like kinase 2 and 5 increased with a 2.3-fold higher increase in activin receptor-like kinase 2. Constitutively active activin receptor-like kinase 2 inhibited Gαi2 promoter activity, whereas constitutively active activin receptor-like kinase 5 stimulated Gαi2 promoter activity independent of embryonic age. In 5-day atrial cells, TGFβ stimulated the p3TP-lux reporter, which is downstream of activin receptor-like kinase 5 and had no effect on the activity of the pVent reporter, which is downstream of activin receptor-like kinase 2. In 14-day cells, TGFβ stimulated both pVent and p3TP-lux. Thus TGFβ exerts opposing effects on parasympathetic response and Gαi2 expression by activating different type I TGFβ receptors at distinct stages during cardiac development.

S. M. Ward, A. P. Gadbut, D. Tang, A. G. Papageorge, L. Wu, G. LI, J. V. Barnett and J. B. Galper. TGFβ Regulates the Expression of Gαi2 via an Effect on the Localization of Ras. Journal of Molecular and Cellular Cardiology (2002) 34, 1217–1226. The negative chronotropic response of the heart to parasympathetic stimulation is mediated via the interaction of M2 muscarinic receptors, Gαi2 and the G-protein coupled inward rectifying K+ channel, GIRK1. Here TGFβ1 is shown to decrease the expression of Gαi2 in cultured chick atrial cells in parallel with attenuation of the negative chronotropic response to parasympathetic stimulation. The response to the acetylcholine analogue, carbamylcholine, decreased from a 95±2% (±SEM, n=8) inhibition of beat rate in control cells to 18±2% (±SEM,n =8) in TGFβ1 treated cells. Data support the conclusion that TGFβ regulation of Gαi2 expression was mediated via an effect on Ras. TGFβ1 inhibited Gαi2 promoter activity by 56±6% (±SEM, n=4) compared to control. A dominant activating Ras mutant reversed the effect of TGFβ on Gαi2 expression and stimulated Gαi2 promoter activity 1.7 fold above control. A dominant negative Ras mutant mimicked the effect of TGFβ1 on Gαi2 promoter activity. TGFβ had no effect on the ratio of GDP/GTP bound Ras, but markedly decreased the level of membrane associated Ras and increased the level of cytoplasmic Ras compared to control. Furthermore, farnesol, a precursor to farnesylpyrophosphate, the substrate for the farnesylation of Ras, not only reversed TGFβ1 inhibition of Ras localization to the membrane, but also reversed TGFβ1 inhibition of Gαi2promoter activity. FTI-277, a specific inhibitor of the farnesylation of Ras, mimicked the effect of TGFβ1 on Ras localization and Gαi2 promoter activity. These data suggest a novel relationship between TGFβ signaling, regulation of Ras function and the autonomic response of the heart.

We have previously demonstrated that growth of embryonic chick atrial cells in medium supplemented with lipoprotein-depleted serum (LPDS) resulted in a coordinate increase in the expression of genes involved in the parasympathetic response of the heart (the M 2 muscarinic receptor; the α-subunit of the heterotrimeric G protein, Gα i2 ; and the inward rectifying K + channel protein, GIRK1) and a marked increase in the negative chronotropic response of atrial cells to muscarinic stimulation. In the present study, we demonstrate that regulation of Gα i2 promoter activity by LPDS is mediated by the binding of a sterol regulatory element binding protein (SREBP) to a sterol regulatory element (SRE) in the Gα i2 promoter. Deletion and point mutation of this putative SRE interfered with the regulation of the Gα i2 promoter by SREBP and LPDS. Furthermore gel shift assays demonstrated that point mutations in the putative Gα i2 SRE markedly inhibited the binding of purified SREBP to oligonucleotides containing the Gα i2 SRE sequence. The expression of a dominant-negative SREBP mutant interfered with LPDS stimulation of Gα i2 promoter activity. Finally, we demonstrate that SREBP-1 is markedly more potent than SREBP-2 for the stimulation of Gα i2 promoter activity, suggesting that SREBP1 may play a role in the regulation of Gα i2 expression. These are the first data to demonstrate SREBP regulation of a protein not involved in lipid homeostasis and suggest a new relationship between lipid metabolism and the parasympathetic response of the heart.

Abdominal aortic aneurysm (AAAs) is a life threatening disease and currently the only treatment is surgical or endovascular repair. Using an animal model for AngiotensinII (AngII) mediated AAA formation in the ApoE-/- mouse, we demonstrate that AAA formation is dependent on the activation of the JAK/STAT pathway. STAT3 activity was increased in the abdominal aortas of Ang II treated ApoE-/- mice. Treatment of ApoE-/- mice with AngII and the p-STAT3 inhibitor S3I-201 decreased the incidence of AAA formation and aortic diameter by greater than 50% compared to placebo. While AngII treatment increased activity of MMP2 and MMP9 in aortas of ApoE-/- mice, S3I-201 treatment inhibited p-STAT3 stimulation and decreased MMP2 and MMP9 activity to levels similar to those in control aortas. The increase in p-STAT3 levels and the secretion of MMP2 and MMP9 in AngII treated bone marrow derived macrophages (BDMs) from ApoE-/- mice was inhibited by pretreatment with S3I-201. Interestingly, AngII stimulated TLR4 expression in AAAs and in BDMs from ApoE-/- mice. Based on these findings we determined the role of TLR4 on AAA formation and p-STAT3 stimulation in ApoE-/-TLR4-/- mice. The incidence of AAA formation, increase in aortic diameter and MMP2 and MMP9 activity were markedly decreased in AngII treated ApoE-/-TLR4-/- mice compared to ApoE-/- controls. Importantly, AngII stimulation of p-STAT3 was also decreased in ApoE-/-TLR4-/- mice compared to control. Similar effects on AngII stimulated p-STAT3 and activity of MMP2 and MMP9 were demonstrated in BMDs from ApoE-/-TLR4-/- mice. Treatment of ApoE-/- mice with the TLR4 inhibitor Eritoran (Eisai, Inc) also decreased the incidence and severity of AAAs, MMPs secretion and p-STAT3 activity. Finally, we determined that S3I-201 decreased AngII stimulated TLR4 expression in AngII treated ApoE-/- mice and in BDM from ApoE-/- mice compared to placebo. These data supported the conclusion that AngII stimulation of AAAs is mediated via a TLR4 dependent activation of p-STAT3 and that both p-STAT3 and TLR4 might be potential therapeutic targets for the treatment of AAAs.

Background It is not clear whether the increase in the myocardial guanylyl nucleotide inhibitory protein (G i ), frequently observed in heart failure, is associated with any functional effects. Methods and Results Eight sham-operated dogs and 10 dogs were studied with pacing-induced heart failure (240 bpm for 4 to 7 weeks), characterized by reduced ( P P i α 2 was increased by 55% in heart failure. Dose-response curves for carbachol (10 − 8 to 10 − 3 mol/L) inhibition of isoproterenol-stimulated adenylyl cyclase demonstrated significantly greater ( P 3 H-quinuclidinyl benzilate, in heart failure (153±6.2 fmol/mg protein) compared with sham-operated dogs (124±7.4 fmol/mg protein). Agonist binding with carbachol also revealed an increase in total muscarinic receptors in heart failure without a change in fraction of high- and low-affinity receptors. Conclusions These data, in the aggregate, provide physiological and biochemical evidence to support the concept that the coordinate increases in muscarinic receptor number and G i levels in heart failure are coupled to increased inhibition of adenylyl cyclase activity and an increased inhibition of myocardial contractility.

The G-protein-gated inward-rectifying K+ channel GIRK1 has been demonstrated in heart and brain. These tissues also both express the M2, M3, and M4, muscarinic acetylcholine receptors (mAChR) (Gadbut, A.P., and Galper, J.B. (1994),J. Biol. Chem. 269,25823-25829). Only the M2 mAChR has been demonstrated to couple to GIRK1 (Kubo, Y., Reuveny, E., Slesinger, P. A., Jan, Y. N., and Jan, L. Y. (1993) Nature 264, 802-806). In this study we determined the specificity of coupling of the M3 and M4 mAChR to a new GIRK1 cloned from a chick brain cDNA library. This clone codes for a 492-amino acid protein that is 93% identical to rat GIRK1 and is expressed in brain, atrium, and ventricle, but not skeletal muscle. In Xenopus laetis oocytes co-expression of GIRK1 with either the chick M2 or M4 mAChR gave carbamylcholine (10 microm)-stimulated K+ currents of 308 +/-26 nA and 298 +/-29 nA, respectively, which were both Ba2+- and pertussis toxin-sensitive. Activation of the M3 receptor produced 2382 +/-478 nA of current which was insensitive to Ba2+ and pertussis toxin, but was 85% inhabitable by the Cl channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (10-20 microm) consistent with coupling to an endogenous Ca2+-activated Cl- channel via a phosphatidylinositol-dependent mechanism. Co-expression of the cardiac inward rectifier CIR with chick M2 or M4 mAChR and GIRK1 increased currents more than 10-fold, but had no effect on specificity of coupling. These data demonstrate a new function for the M4 mAChR and a high degree of specificity for coupling of each receptor subtype to GIRK1.

SummaryBackground A test dose is used to detect intravascular injection during neuraxial block in pediatrics. Accidental intravascular epidural local anesthetic injection might be unrecognized in anesthetized children leading to potential life-threatening complications. In children, sevoflurane anesthesia blunts the hemodynamic response when intravascular cathecolamines are administered. No studies have explored the hemodynamics and the criteria for a positive test dose result following phenylephrine in sevoflurane anesthetized children. Methods Healthy children undergoing minor procedures were randomly assigned to receive intravenous placebo, or 5 μg∙kg−1 phenylephrine (n = 11/group) during sevoflurane anesthesia. Hemodynamic response was assessed using electrocardiography, pulse oxymetry and non-invasive blood pressure monitoring for 5 min following drug administration in anesthetized patients. Results All patients receiving phenylephrine showed a decreased heart rate (HR) but not all of them met the positive criterion for test dose response. Overall, at 1 min, patients receiving phenylephrine showed a 25% decrease in HR from the baseline while an increase in blood pressure was noticed in 54% of patients receiving phenylephrine. Discussion Phenylephrine might be a future indicator of positive intravascular test dose. Further investigation is needed to find out the phenylephrine dose that elicits a reliable hemodynamic response and whether phenylephrine needs to be dose age-adjusted in order to appreciate relevant hemodynamic changes in children receiving neuraxial blocks undergoing general anesthesia.

Abstract We studied several aspects of guanine nucleotide–stimulated adenylate cyclase function in patients after orthotopic cardiac transplantation. In 28 patients, adenylate cyclase activity was measured in endomyocardial biopsy samples obtained just before and at monthly intervals after cardiac transplantation. In biopsies obtained ≥6 months after transplantation, basal adenylate cyclase activity was decreased by 67% (n=12; P P +2 +forskolin–stimulated adenylate cyclase activity was decreased by 80% (n=8; P +2 -stimulated adenylate cyclase activity (a measure of activity of the catalytic subunit of adenylate cyclase) was decreased by 83% (n=8, P sα protein was decreased by 61±12% (n=8; P iα as assessed by pertussis toxin–catalyzed ADP-ribosylation (n=4; P =NS). With the use of the quantitative polymerase chain reaction, a 50±10% (n=6; P sα mRNA was observed. There was no change in the level of mRNA for G i-3α . Thus, after orthotopic cardiac transplantation in humans, guanine nucleotide–stimulated adenylate cyclase activity is decreased in parallel with decreased levels of G sα protein and mRNA.

Abdominal aortic aneurysm (AAA) is a potentially life-threatening degenerative vascular disease that affects 6-9% of men over 65. Although Toll-like receptors (TLRs) mediate the innate immune response resulting in the recruitment of inflammatory cells to the site of a range of pathogen-associated molecular patterns, they have also been implicated in immune tolerance. To determine the role of TLR2 signaling in AAA formation we compared AAA formation in ApoE -/- and ApoE-/-TLR2 -/- DKO mice using a mouse model for angiotensin II (ANGII) mediated AAA formation. Treatment of ApoE -/- mice with Alzet pumps releasing 750 ng/kg/min ANG II resulted in formation of AAAs in 75% of mice (n=23) and a mean increase in aortic diameter from 1.0 mm in saline treated controls to 2.15±0.15 mm, p-/- DKO mice compared to ApoE -/- alone was unchanged, of the 12 ApoE-/-TLR2-/-DKO who developed AAAs, half demonstrated severe aneurismal involvement of the aortic arch and thoracic aorta compared to the ApoE -/- group consistent with a protective effect of TLR2. To test this hypothesis, mice were pretreated with PAM3, 50ug/ mouse/week. The incidence of AAA formation decreased to 32% in PAM 3 treated mice and aortic diameter decreased to 1.17±0.08 (n=18, p

Growth of chick atrial cells in medium supplemented with lipoprotein-depleted serum has been shown to result in an increase in total cell cholesterol, and an increase in the negative chronotropic response to muscarinic stimulation in parallel with an increase in levels of muscarinic receptors and the G-protein alpha-subunits alpha i and alpha o (Haigh, L. S., Leatherman, G. F., O'Hara, D. S., Smith, T. W., and Galper, J. B. (1988) J. Biol. Chem. 263, 15608-15618). In this study we determined whether growth of chick ventricular cells in medium supplemented with lipoprotein depleted serum could alter levels of muscarinic receptors and G-protein alpha-subunits and induce a negative chronotropic response to muscarinic stimulation. We further determined whether levels of mRNA coding for muscarinic receptors, G-proteins, and the acetylcholine-sensitive K+ channel were coordinately regulated. Growth of embryonic chick ventricular cells from hearts 14 days in ovo in medium supplemented with lipoprotein depleted serum resulted in a 21 +/- 5% (n = 3, +/- S.E.) increase in muscarinic receptor number as demonstrated by [3H]quinuclidinyl benzilate binding and a 4.7 +/- 1.0 (+/- S.E., n = 4)-fold increase in G alpha i2 as demonstrated by Western blot analysis. These changes in receptor and G-protein were associated with a coordinate increase in levels of mRNA coding for the M2 muscarinic receptor, G alpha i2 and the acetylcholine sensitive K+ channel as determined by RNase protection. These increases were reversed by addition of 30 microM mevinolin, an inhibitor of HMG-CoA reductase activity. Carbamylcholine (0.1 mM) had no effect on beat rate in ventricular cells grown in medium supplemented with fetal calf serum. Cells grown in medium supplemented with lipoprotein depleted serum demonstrated a 40 +/- 8% (+/- S.E., n = 10, p < 0.0001) decrease in beat rate in response to 0.1 mM carbamylcholine which was reversed by the addition of 30 microM mevinolin. These data suggest that, during growth in medium supplemented with lipoprotein depleted serum, a component of the cholesterol biosynthetic pathway plays a role in the coordinate induction of mRNAs coding for receptors, G-proteins, and an effector (ion channel) that results in the induction of a parasympathetic response in the ventricular cell characteristic of the atrial phenotype.

Prior studies have suggested that heart expresses only the M2 isoform of the muscarinic receptor (Peralta, E.G., Ashkenazi, A., Winslow, J.W., Smith, D.H., Ramachandran, J., and Capon, D.J. (1987) EMBO J. 6, 3923-3929). Tietje and Nathanson (Tietje, K.M., and Nathanson, N. M. (1991) J. Biol. Chem. 266, 17382-17387) have recently demonstrated that the chick heart may be unique since it expresses both the M2 and M4 isoforms of the muscarinic receptor. In this study, in order to determine whether other isoforms of the muscarinic receptor were present in the chick heart, a chick M3 muscarinic receptor receptor was cloned, characterized, and its expression in chick tissues determined. Using a human M3 muscarinic receptor cDNA as a probe, a 2.4-kilobase pair cDNA was isolated from a chick brain cDNA library which contained an open reading frame coding for a 639 amino acid protein. This protein demonstrated an 87 and 86% homology to the human and rat M3 muscarinic receptor, respectively. Chinese hamster ovary (CHO-GRA) cells were stably transfected with the chick M3 muscarinic receptor and one clone (CHO-CM3) expressed the M3 receptor, as measured by the binding of quinuclidinly benzilate at 116 +/- 14 (+/- S.E., n = 3) fmol/mg protein with a Kd of 76 +/- 17 pM. This receptor demonstrated a rank order of potency for muscarinic antagonist binding characteristic for the M3 receptor: with high affinity binding for hexahydrosiladifenidol, Kd: 16 +/- 2 nM (+/- S.E., n = 3); intermediate affinity for pirenzepine, Kd: 383 +/- 47 nM, and low affinity for methoctramine, Kd: 533 +/- 185 nM (+/- S.E., n = 3). Carbamylcholine stimulation of CHO-CM3 cells resulted in a 1.6-fold increase in cyclic AMP accumulation and a 3.5-fold increase in a pertussis toxin-insensitive inositol phosphate release. These data demonstrate that the chick M3 muscarinic receptor has the properties characteristic of M3 receptors from other species. RNase protection studies demonstrated the presence of M3 muscarinic receptor mRNA in the brain, atria, and ventricle of chicks 17 days in ovo. Hence, the chick heart appears to have the unique capacity to express mRNAs coding not only for the M2 and M4 muscarinic receptors but also for the M3 muscarinic receptor.

We have cloned cDNAs coding for G-protein alpha subunits from a chick brain cDNA library. Based on sequence similarity to G-protein alpha subunits from other eukaryotes, one clone was designated G alpha i3. A second clone, G alpha i3-o, was identical to the G alpha i3 clone over 932 bases on the 3′ end. The 5′ end of G alpha i3-o, however, contained an alternative sequence in which the first 45 amino acids coded for are 100% identical to the conserved N-terminus of G alpha o from species such as rat, mouse, human, bovine and hamster. Both clones were found to be expressed in all tissues studied. The unusual alpha o-alpha i3-like G-protein chimera, G alpha i3-o, was found to be expressed at significantly lower levels than G alpha i3. In vitro transcription and translation of the G alpha i3-o cDNA clone gave a protein of approx. 41 kDa which stably bound guanosine 5′-[gamma-thio]triphosphate. G alpha i3-o appears to be the first G-protein alpha subunit cloned which contains ends that are homologous to two different alpha subunit isoforms, G alpha o and G alpha i3.

We have cloned and characterized several cDNAs coding for G-protein inhibitory α subunits (Gai) from a chick brain cDNA library. Based on homology to Ga subunits from other eukaryotes, these clones were designated chick Gαil and Gαi2 . On the deduced amino-acid level, Gαi1 and Gαi2 were found to be 98 and 95% identical to rat Goal and Gαi2 , respectively. Using RNase protection analysis, the Gαi1 and Gαi2 mRNAs were found to be expressed in chick atria, ventricle, lung, liver, brain and kidney.

To address the role of peptide growth factors in chick organogenesis, we have focused on TGF beta 2 and have cloned the chick Type II and Type III TGF beta receptors. The chick Type II receptor is a serine/threonine kinase with a ligand binding profile identical to the human receptor and a divergent N-terminus when compared to the mammalian receptors. The chick Type III receptor is a beta-glycan that demonstrates a binding profile identical to the rat receptor and contains a single transmembrane spanning domain and short cytoplasmic tail that are highly conserved when compared to the mammalian receptors. Both the Type II and Type III TGF beta receptors are coexpressed during chick embryogenesis in the developing heart, lung, and eye, and are developmentally upregulated in parallel in the heart and lung. Levels of both receptor proteins and mRNAs also increase in cardiocytes cultured from different developmental stages, in agreement with the increase in Type II and Type III receptor mRNA levels observed in the developing heart. Although exhibiting different temporal or spatial profiles from the receptors, TGF beta 2 is also expressed in the developing heart, lung, and eye. These findings are consistent with recent data indicating that co-expression of both the Type II and Type III TGF beta receptors is required for high affinity binding of TGF beta 2 by the Type II receptor and suggest that TGF beta 2 and the Type II and Type III TGF beta receptors participate in heart, lung, and eye development.

We have developed a system for the co-culture of embryonic chick heart cells obtained from embryos at 3.5 days in ovo with ciliary ganglia from chick embryos at 7 days in vivo. After 3 days of co-culture, removal of the ciliary ganglia resulted in complete degeneration of axons within 6-8 h, leaving the post-innervated heart cell culture devoid of neurons. Embryonic chick heart cells at 3.5 days in ovo are unresponsive to muscarinic stimulation. However, following 3 days of co-culture with ciliary ganglia, the heart cells developed a negative chronotropic response to muscarinic stimulation (paired t test, P < 0.02) which persisted for at least 24 h after removal of the ciliary ganglion. The development of muscarinic responsiveness was associated with an increase in the levels of specific alpha-subunits of the guanine nucleotide binding proteins (G-proteins), with a 3-fold increase in the level of alpha 39 (39 kDa subunit) and a 2.5-fold increase in the level of alpha 41. The level of the G-protein subunit alpha s remained unchanged. Culture of embryonic chick heart cells at 3.5 days in ovo with medium conditioned by the growth of embryonic chick heart cells and ciliary ganglia had an effect on the chronotropic response to muscarinic stimulation and on alpha 39 and alpha 41 levels identical to that of co-culture. These data suggest that a soluble factor released during the co-culture of embryonic chick heart cells and ciliary ganglia is capable of inducing muscarinic responsiveness. These studies suggest that innervation of the heart may induce parasympathetic responsiveness by increasing the availability of G-proteins which couple the muscarinic receptor to a physiological response.

Retrospective studies suggest that statins might exert an antiarrhythmic effect on the heart. The mechanism of this effect is unclear. Parasympathetic stimulation of the heart has been shown to protect against ventricular arrhythmias. The goal of this study was to determine the effect of statins on ventricular arrhythmias and its correlation with changes in parasympathetic responsiveness and Galpha(i2) expression. Patients were randomized to pravastatin and simvastatin in a double-blind crossover design. Ventricular arrhythmias were determined by analysis of 24-hour Holter recordings. Spectral RR interval analysis of Holter studies determined peak high-frequency power fraction, which reflects parasympathetic modulation of heart rate. Expression of Galpha(i2), a molecular component of the parasympathetic response pathway, was determined by Western blots of patients' lymphocytes. Pravastatin treatment decreased the incidence of ventricular premature complexes by 22.5 + or - 3.4% (n = 20, p

Rationale: Diabetic autonomic neuropathy (DAN), a major complication of diabetes mellitus, is characterized, in part, by impaired cardiac parasympathetic responsiveness. Parasympathetic stimulation of the heart involves activation of an acetylcholine-gated K + current, I KAch , via a (GIRK1) 2 /(GIRK4) 2 K + channel. Sterol regulatory element binding protein-1 (SREBP-1) is a lipid-sensitive transcription factor. Objective: We describe a unique SREBP-1–dependent mechanism for insulin regulation of cardiac parasympathetic response in a mouse model for DAN. Methods and Results: Using implantable EKG transmitters, we demonstrated that compared with wild-type, Ins2 Akita type I diabetic mice demonstrated a decrease in the negative chronotropic response to carbamylcholine characterized by a 2.4-fold decrease in the duration of bradycardia, a 52±8% decrease in atrial expression of GIRK1 ( P P KAch with a peak value of −181±31 pA/pF compared with −451±62 pA/pF ( P KAch activity in atrial myocytes from these mice to levels in wild-type mice. Insulin treatment of cultured atrial myocytes stimulated GIRK1 expression 2.68±0.12-fold ( P KAch to levels in cells from wild-type mice. Conclusions: These results support a unique molecular mechanism for insulin regulation of GIRK1 expression and parasympathetic response via SREBP-1, which might play a role in the pathogenesis of DAN in response to insulin deficiency in the diabetic heart.

We have demonstrated that muscarinic stimulation of inositol phosphate production in cultured atrial cells from chicks at 14 days in ovo is partially sensitive to inhibition by pertussis toxin. In these cells, muscarinic agonist binding is coupled to phospholipase C activity via at least two guanine-nucleotide-binding proteins (G-proteins), one sensitive to pertussis toxin and the other (Gp) insensitive to pertussis toxin [Barnett, Shamah, Lassegue, Griendling & Galper (1990) Biochem. J. 271, 437-442]. In the current study we demonstrate that during embryonic development of the chick heart, muscarinic stimulation of inositol phosphate production decreases by 50% between days 5 and 14 in ovo in cells cultured from both atrium and ventricle. In atrial cells, however, pertussis toxin-sensitive muscarinic stimulation of inositol phosphate production increased from undetectable levels at day 5 in ovo to 40% of total stimulation at day 12 in ovo. Muscarinic stimulation of inositol phosphate production in the ventricle did not become sensitive to pertussis toxin at any age studied. In permeabilized atrial cells from embryonic chicks at 5 days in ovo, guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulated InsP1 levels by 40 +/- 10% (mean +/- S.E.M., n = 3), InsP2 levels by 117 +/- 18% and InsP3 levels by 51 +/- 8%, suggesting that at day 5 in ovo all of the muscarinic-stimulated inositol phosphate production was coupled to phospholipase C via Gp. H.p.l.c. analysis demonstrated that, in spite of these changes in coupling of phospholipase C to different G-proteins, no changes could be demonstrated in the isomers of InsP3 produced in response to carbamylcholine at both days 5 and 14 in ovo. These data demonstrate that embryonic development of the chick atrium is associated with a switch in coupling of muscarinic receptors to phospholipase C from Gp to a pertussis toxin substrate. This developmental switch in coupling of G-proteins may be related to possible developmental switches in levels of muscarinic receptor isoforms or switches in the subtype of phospholipase C.

These studies demonstrate a novel mechanism for the coupling of the muscarinic receptor to phospholipase C activity in embryonic chick atrial cells. In monolayer cultures of atrial cells from hearts of embryonic chicks at 14 days in ovo, carbamylcholine stimulated the sequential appearance of InsP3, InsP2 and InsP1 with an EC50 (concn. causing 50% of maximal stimulation) of 30 microM. In the presence of 15 mM-Li, a 5 min exposure to carbamylcholine (0.1 mM) increased InsP3 levels to a maximum of 47 +/- 12% over basal, InsP2 to 108 +/- 13% over basal and InsP1 to 42 +/- 5% over basal. This effect was blocked by 5 microM-atropine. Incubation of these cells with pertussis toxin (15 h; 0.5 ng/ml) inhibited carbamylcholine-stimulated InsP3, InsP2 and InsP1 formation by 42 +/- 7%, 30 +/- 3% and 48 +/- 7% respectively. The IC50 (concn. causing 50% inhibition) for pertussis toxin inhibition of all three inositol phosphates was 0.01 ng/ml, with a half-time of 6 h at 0.5 ng/ml. This partial sensitivity to pertussis toxin was not due to incomplete ADP-ribosylation of the guanine-nucleotide-binding protein (G-protein), since autoradiography of polyacrylamide gels of cell homogenates incubated with [32P]NAD+ in the presence of pertussis toxin demonstrated that incubation of cells with 0.5 ng of pertussis toxin/ml for 15 h resulted in complete ADP-ribosylation of pertussis toxin substrates by endogenous NAD+. In cells permeabilized with saponin (10 micrograms/ml), 0.1 mM-GTP[S] (guanosine 5′-[gamma-thio]triphosphate) stimulated InsP1 by 102 +/- 15% (mean +/- S.E.M., n = 4), InsP2 by 421 +/- 67% and InsP3 by 124 +/- 33% above basal. Incubation of cells for 15 h with 0.5 ng of pertussis toxin/ml decreased GTP[S]-stimulated InsP1 production in saponin-treated cells by 30 +/- 10% (n = 3), InsP2 production by 45 +/- 7% (n = 4) and InsP3 production by 49 +/- 6% (n = 4). These data demonstrate that in embryonic chick atrial cells at least two independent G-proteins, a pertussis toxin-sensitive G-protein and a pertussis toxin-insensitive G-protein, play a role in coupling muscarinic agonist binding to phospholipase C activation and to inositol phosphate production.

Studies of the development of parasympathetic responsiveness in embryonic chick hearts have demonstrated that between days 2.5 and 10 in ovo the ability of muscarinic agonists to inhibit adenylate cyclase activity increases 10-fold in parallel with a 2.7-fold increase in the level of alpha i and alpha o. Thus, muscarinic inhibition of adenylate cyclase increases in parallel with an increase in alpha o and alpha i. These data suggest that changes in levels of guanine nucleotide regulatory proteins control, at least in part, the appearance of a parasympathetic response in the heart during embryonic development of the chick.

to cardiac myocyte hypertrophy, although exogenously administered BNP shows poor clinical efficacy in heart failure and hypertension. Here we tested whether PDE2A was directly involved in modulating cardiac neurotransmitter release from pre-hypertensive spontaneously hypertensive rats (SHRs) that show an enhanced Ca2+ phenotype. Methods and Results: [3H] labelled noradrenaline (NA) release was measured from isolated spontaneously beating atrial in response to 5Hz field stimulation for 1 minute at the 16th (S1, control) and 40th (S2, with 250nM BNP) minutes. PDE2A activity in the SHR (n = 8) was higher (~60%) than in WKY stellate ganglia tissue (n= 8). BNP significantly reduced [3H]-NA release (S1:+0.92 ± 0.09%, S2: +0.59 ± 0.09% n= 9,) in the WKY, whereas it failed in the SHR (S1: +1.34 ± 0.34% vs S2:+1.16 ± 0.23%, n = 9). Overexpression of PDE2A in the right atria abrogated the response to BNP in the WKY and mimicked the neurotransmission seen in the SHR neuron. This response was rescued by inhibition of PDE2A by BAY60-7550 or the dominant negative of PDE2A. Conclusion: These data support the hypothesis that higher PDE2A expression in the SHR abolishes the capacity of BNP to reduce neurotransmitters release. They also suggest that inhibiting neuronal PDE2 may be important in restoring the efficacy of BNP to decrease sympathetic neurotransmission in cases of dysautonomia.

Background— We have previously demonstrated in an in vitro model for lipid lowering that lipoprotein depletion resulted in a marked increase in the negative chronotropic response to the acetylcholine analogue carbamylcholine. In this study we used heart rate variability analysis to determine the effect of lipid lowering by statins on the response of the heart to parasympathetic stimulation. In parallel, we examined whether changes in parasympathetic responsiveness correlated with changes in the expression of Gα i2 , a molecular component of the parasympathetic signaling pathway in the heart. Methods and Results— Patients were randomized in a crossover study of pravastatin and simvastatin. R-R interval analysis of Holter monitor studies demonstrated that in patients treated initially with pravastatin, the peak high-frequency power fraction during sleep, which reflects parasympathetic modulation of heart rate, increased by 24.0±5.02% (SEM, n=13, P P =0.009) increase in Gα i2 expression, whereas simvastatin had no effect. Relative changes in Gα i2 correlated significantly with the changes in the fraction of high-frequency power (ρ=0.574, P =0.016). Conclusions— Taken together with our in vitro data, these data are the first to suggest that cholesterol lowering by pravastatin might increase the response of the heart to parasympathetic stimulation and that changes in Gα i2 expression might serve as a molecular marker for this effect.

The negative chronotropic response of the heart to parasympathetic stimulation is mediated via the interaction of M(2) muscarinic receptors, Galpha(i2) and the G-protein coupled inward rectifying K(+) channel, GIRK1. Here TGFbeta(1) is shown to decrease the expression of Galpha(i2) in cultured chick atrial cells in parallel with attenuation of the negative chronotropic response to parasympathetic stimulation. The response to the acetylcholine analogue, carbamylcholine, decreased from a 95+/-2% (+/-SEM, n=8) inhibition of beat rate in control cells to 18+/-2% (+/-SEM,n =8) in TGFbeta(1) treated cells. Data support the conclusion that TGFbeta regulation of Galpha(i2) expression was mediated via an effect on Ras. TGFbeta(1) inhibited Galpha(i2) promoter activity by 56+/-6% (+/-SEM, n=4) compared to control. A dominant activating Ras mutant reversed the effect of TGFbeta on Galpha(i2) expression and stimulated Galpha(i2) promoter activity 1.7 fold above control. A dominant negative Ras mutant mimicked the effect of TGFbeta(1) on Galpha(i2) promoter activity. TGFbeta had no effect on the ratio of GDP/GTP bound Ras, but markedly decreased the level of membrane associated Ras and increased the level of cytoplasmic Ras compared to control. Furthermore, farnesol, a precursor to farnesylpyrophosphate, the substrate for the farnesylation of Ras, not only reversed TGFbeta(1) inhibition of Ras localization to the membrane, but also reversed TGFbeta(1) inhibition of Galpha(i2)promoter activity. FTI-277, a specific inhibitor of the farnesylation of Ras, mimicked the effect of TGFbeta(1) on Ras localization and Galpha(i2) promoter activity. These data suggest a novel relationship between TGFbeta signaling, regulation of Ras function and the autonomic response of the heart.