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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<0.01), and a 31.3+/-2.1% decrease in SREBP-1 (P<0.05). Whole-cell patch-clamp studies of atrial myocytes from Akita mice exhibited a markedly decreased carbamylcholine stimulation of I(KAch) with a peak value of -181+/-31 pA/pF compared with -451+/-62 pA/pF (P<0.01) in cells from wild-type mice. Western blot analysis of extracts of Akita mice demonstrated that insulin treatment increased the expression of GIRK1, SREBP-1, and I(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<0.01), which was reversed by overexpression of dominant negative SREBP-1. Finally, adenoviral expression of SREBP-1 in Akita atrial myocytes reversed the impaired I(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.

Angiogenesis is implicated in the pathogenesis of cancer, rheumatoid arthritis, and atherosclerosis and in the treatment of coronary artery and peripheral vascular disease. Here, cholesterol-lowering agents, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are shown to interfere with angiogenesis. In vivo, the HMG-CoA reductase inhibitor simvastatin dose-dependently inhibited capillary growth in both vascular endothelial growth factor-stimulated chick chorioallantoic membranes and basic fibroblast growth factor-stimulated mouse corneas. In vitro, the development of tubelike structures by human microvascular endothelial cells cultured on 3D collagen gels was inhibited at simvastatin concentrations similar to those found in the serum of patients on therapeutic doses of this agent. HMG-CoA reductase inhibitors interfered with angiogenesis via inhibition of the geranylgeranylation and membrane localization of RhoA. Simvastatin inhibited membrane localization of RhoA with a concentration dependence similar to that for the inhibition of tube formation, whereas geranylgeranyl pyrophosphate, the substrate for the geranylgeranylation of Rho, reversed the effect of simvastatin on tube formation and on the membrane localization of RhoA. Furthermore, tube formation was inhibited by GGTI, a specific inhibitor of the geranylgeranylation of Rho; by C3 exotoxin, which inactivates Rho; and by the adenoviral expression of a dominant-negative RhoA mutant. The expression of a dominant-activating RhoA mutant reversed the effect of simvastatin on tube formation. Finally, HMG-CoA reductase inhibitors inhibited signaling by vascular endothelial growth factor, Akt, and focal adhesion kinase, three RhoA-dependent pathways known to be involved in angiogenesis. This study demonstrates a new relationship between lipid metabolism and angiogenesis and an antiangiogenic effect of HMG-CoA reductase inhibitors with possible important therapeutic implications.

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 M2 muscarinic receptor; the alpha-subunit of the heterotrimeric G protein, Galpha(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 Galpha(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 Galpha(i2) promoter. Deletion and point mutation of this putative SRE interfered with the regulation of the Galpha(i2) promoter by SREBP and LPDS. Furthermore gel shift assays demonstrated that point mutations in the putative Galpha(i2) SRE markedly inhibited the binding of purified SREBP to oligonucleotides containing the Galpha(i2) SRE sequence. The expression of a dominant-negative SREBP mutant interfered with LPDS stimulation of Galpha(i2) promoter activity. Finally, we demonstrate that SREBP-1 is markedly more potent than SREBP-2 for the stimulation of Galpha(i2) promoter activity, suggesting that SREBP1 may play a role in the regulation of Galpha(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.

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 > or = 6 months after transplantation, basal adenylate cyclase activity was decreased by 67% (n = 12; P < .05), GTP gamma S-stimulated adenylate cyclase activity was decreased by 78% (n = 12; P < .05), Mn+2+forskolin-stimulated adenylate cyclase activity was decreased by 80% (n = 8; P < .05), and Mn+2-stimulated adenylate cyclase activity (a measure of activity of the catalytic subunit of adenylate cyclase) was decreased by 83% (n = 8, P < .05). Western blot analysis demonstrated that 6 months after cardiac transplantation, the level of Gs alpha protein was decreased by 61 +/- 12% (n = 8; P < .001). There was no change in the level of Gi alpha 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 < .001) reduction in the steady-state level of Gs alpha mRNA was observed. There was no change in the level of mRNA for Gi-3 alpha. Thus, after orthotopic cardiac transplantation in humans, guanine nucleotide-stimulated adenylate cyclase activity is decreased in parallel with decreased levels of Gs alpha protein and mRNA.