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Parasympathetic stimulation of the heart, which provides protection from arrhythmias and sudden death, involves activation of the G protein--coupled inward rectifying [K.sup.+] channel GIRK1/4 and results in an acetylcholine-sensitive [K.sup.+] current, [I.sub.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.sub.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.sub.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.sub.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., Introduction The balance between the response of the heart to sympathetic and parasympathetic stimuli not only determines the rate and force of contraction but also plays a role in regulating [...]

The comparison of cell development in medium supplemented with fetal calf serum (FCS) and lipoprotein-depleted serum (LPDS) showed that the capacity of Ca2+ and the Ca2+ channel agonist, BAY K 8644, to increase the amplitude of contraction was lowered by 25 and 50% respectively. Incubation with mevinolin reversed this influence of development in LPDS. The control of the contractile reaction to Ca2+ channel agonists and to exogenous Ca2+ may be influenced by the variations in the comparative amounts of alpha-o and alpha-s in cells developed in LPDS.

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. [ABSTRACT FROM AUTHOR]

Transforming growth factor-[Beta] (TGF[Beta]) signaling has been shown to play a role in cardiac development as well as in the pathogenesis of cardiovascular disease. Prior studies have suggested a relationship between cholesterol metabolism and TGF[Beta] signaling. Here we demonstrate that induction of the cholesterol metabolic pathway by growth of embryonic chicken atrial cells in medium supplemented with lipoprotein-depleted serum coordinately decreased the expression of the TGF[Beta] type II receptor (TGF[Beta]RII), TGF[[Beta].sub.1], and TGF[Beta] signaling as measured by plasminogen activator inhibitor-1 (PAI-1) promoter activity. Inhibition of the cholesterol metabolic pathway by the hydrophobic 3-hydroxy-3-methylglutaryl CoA (HMGCoA) reductase inhibitors, simvastatin and atorvastatin, reversed the effect of lipoprotein-depleted serum and up-regulated TGF[Beta]RII expression, whereas the hydrophilic HMGCoA reductase inhibitor, pravastatin, had no effect. Simvastatin stimulated the expression of TGF[Beta]RII, TGF[[Beta].sub.1], and PAI-1 at the level of transcription. Experiments using specific inhibitors of different branches of the cholesterol metabolic pathway demonstrated that simvastatin exerted its effect on TGF[Beta] signaling by inhibition of the geranylgeranylation pathway. C3 exotoxin, which specifically inactivates geranylgeranylated Rho GTPases, mimicked the effect of simvastatin on PAI-1 promoter activity. Cotransfection of cells with a PAI-1 promoter-reporter and a dominant-negative RhoA mutant increased PAI-1 promoter activity, whereas cotransfection with a dominant-active RhoA mutant decreased PAI-1 promoter activity. These data support the conclusion that TGF[Beta] signaling is regulated by RhoA GTPase and demonstrate a relationship between cholesterol metabolism and TGF[Beta] signaling. Our data suggest that in patients treated with HMGCoA reductase inhibitors, these agents may exert effects independent of cholesterol lowering on TGF[Beta] signaling in the heart.

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. [ABSTRACT FROM AUTHOR]