Activated expression of cardiac adenylyl cyclase 6 reduces dilation and dysfunction of the pressure-overloaded heart.

BACKGROUND AND OBJECTIVE: Cardiac-directed adenylyl cyclase 6 (AC6) expression attenuates left ventricular (LV) hypertrophy and dysfunction in cardiomyopathy, but its effects in the pressure-overloaded heart are unknown. METHODS: Mice with cardiac-directed and regulated expression of AC6 underwent transaortic constriction (TAC) to induce LV pressure overload. Ten days prior to TAC, and for the duration of the 4 week study, cardiac myocyte AC6 expression was activated in one group (AC-On) but not the other (AC-Off). Multiple measures of LV systolic and diastolic function were obtained 4 weeks after TAC, and LV samples assessed for alterations in Ca2+ signaling. RESULTS: LV contractility, as reflected in the end-systolic pressure-volume relationship (Emax), was increased (p=0.01) by activation of AC6 expression. In addition, diastolic function was improved (p<0.05) and LV dilation was reduced (p<0.05). LV samples from AC-On mice showed reduced protein expression of sodium/calcium exchanger (NCX1) (p<0.05), protein phosphatase 1 (PP1) (p<0.01), and increased phosphorylation of phospholamban (PLN) at Ser16 (p<0.05). Finally, sarcoplasmic reticulum (SR) Ca2+ content was increased in cardiac myocytes isolated from AC-On mice (p<0.05). CONCLUSIONS: Activation of cardiac AC6 expression improves function of the pressure-overloaded and failing heart. The predominant mechanism for this favorable adaptation is improved Ca2+ handling, a consequence of increased PLN phosphorylation, reduced NCX1, reduced PP1 expression, and increased SR Ca2+ content.

Increased cardiac adenylyl cyclase expression is associated with increased survival after myocardial infarction.

BACKGROUND: Cardiac-directed expression of adenylyl cyclase type VI (AC(VI)) in mice results in structurally normal hearts with normal basal heart rate and function but increased responses to catecholamine stimulation. We tested the hypothesis that increased left ventricular (LV) AC(VI) content would increase mortality after acute myocardial infarction (MI). METHODS AND RESULTS: Transgenic mice with cardiac-directed AC(VI) expression and their transgene-negative littermates (control) underwent coronary ligation, and survival, infarct size, and LV size and function were assessed 1 to 7 days after MI. Mice with increased AC(VI) expression had increased survival (control 41%, AC(VI) 74%; P = 0.004). Infarct size and myocardial apoptotic rates were similar in AC(VI) and control mice; however, AC(VI) mice had less LV dilation (P < 0.001) and increased ejection fractions (P < 0.03). Three days after MI, studies in isolated perfused hearts showed that basal LV +dP/dt was similar, but graded dobutamine infusion was associated with a more robust LV contractile response in AC(VI) mice (P < 0.05). Increased LV function was associated with increases in cAMP generation (P = 0.0002), phospholamban phosphorylation (P < 0.04), sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) affinity for calcium (P < 0.015), and reduced AV block (P = 0.04). CONCLUSIONS: In acute MI, increased cardiac AC(VI) content attenuates adverse LV remodeling, preserves LV contractile function, and reduces mortality.

Nicotine inhibits cardiac apoptosis induced by lipopolysaccharide in rats.

OBJECTIVES: Apoptosis develops in several heart diseases, but the therapeutic options are limited. It was hypothesized that nicotine, which inhibits apoptosis in several cells, inhibits cardiac apoptosis induced by lipopolysaccharide (LPS). BACKGROUND: Over-the-counter nicotine produces sustained levels (10 to 25 ng/ml) that may be antiapoptotic. Low levels of LPS induce apoptosis by activating tissue renin-angiotensin to stimulate angiotensin II, type 1 (AT(1)) receptors in cardiac myocytes. METHODS: Adult Sprague Dawley rats were pretreated with nicotine (6 mg/kg/day) or saline for seven to ten days (miniosmotic pumps). The LPS (1 mg/kg) was injected intravenously. Toll-like receptor 4 (TLR4) and angiotensinogen messenger ribonucleic acid (mRNA) were measured in the heart after 0, 4, 8, 16, and 24 h. Cardiac apoptosis was measured by terminal deoxy-nucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining after 24 h. In vitro effects of LPS (10 ng/ml, 24 h) were studied in cardiac myocytes isolated from rats pretreated with nicotine for 7 to 10 days, or after pre-exposing myocytes to nicotine (15 ng/ml) for 1, 4, 16, or 24 h. RESULTS: Neither nicotine nor LPS affected systolic blood pressure. The LPS increased cardiac apoptosis after 24 h in saline-treated, but not nicotine-treated rats, despite similar increases in cardiac TLR4 and angiotensinogen mRNA over 8 to 16 h. The LPS-induced apoptosis was blocked by pre-exposing myocytes to nicotine for 4 to 24 h (partial inhibition after 1 h). Nicotine did not inhibit apoptosis induced by angiotensin II (100 nM, 24 h). CONCLUSIONS: Therapeutic levels of nicotine inhibit LPS-induced cardiac apoptosis. This occurs after LPS increases TLR4 and angiotensinogen mRNA, but proximal to AT(1) receptor activation. Nicotine may be a novel inhibitor of cardiac apoptosis in conditions associated with circulating LPS (e.g., decompensated heart failure, acute and chronic infections).

Myocardial fibrosis induced by exposure to subclinical lipopolysaccharide is associated with decreased miR-29c and enhanced NOX2 expression in mice.

BACKGROUND: Exposure to subclinical levels of lipopolysaccharide (LPS) occurs commonly and is seemingly well tolerated. However, recurrent LPS exposure induces cardiac fibrosis over 2 to 3 months in a murine model, not mediated by the renin-angiotensin system. Subclinical LPS induces cardiac fibrosis by unique mechanisms. METHODS: In C57/Bl6 mice, LPS (10 mg/kg) or saline (control) were injected intraperitoneally once a week for 1-4 weeks. Mice showed no signs of distress, change in activity, appetite, or weight loss. Mice were euthanized after 3 days, 1, 2, or 4 weeks to measure cardiac expression of fibrosis-related genes and potential mediators (measured by QRT-PCR), including micro-RNA (miR) and NADPH oxidase (NOX). Collagen fraction area of the left ventricle was measured with picrosirius red staining. Cardiac fibroblasts isolated from adult mouse hearts were incubated with 0, 0.1, 1.0 or 10 ng/ml LPS for 48 hours. RESULTS: Cardiac miR expression profiling demonstrated decreased miR-29c after 3 and 7 days following LPS, which were confirmed by QRT-PCR. The earliest changes in fibrosis-related genes and mediators that occurred 3 days after LPS were increased cardiac expression of TIMP-1 and NOX-2 (but not of NOX-4). This persisted at 1 and 2 weeks, with additional increases in collagen Iα1, collagen IIIα1, MMP2, MMP9, TIMP1, TIMP2, and periostin. There was no change in TGF-ß or connective tissue growth factor. Collagen fraction area of the left ventricle increased after 2 and 4 weeks of LPS. LPS decreased miR-29c and increased NOX-2 in isolated cardiac fibroblasts. CONCLUSIONS: Recurrent exposure to subclinical LPS induces cardiac fibrosis after 2-4 weeks. Early changes 3 days after LPS were decreased miR-29c and increased NOX2 and TIMP1, which persisted at 1 and 2 weeks, along with widespread activation of fibrosis-related genes. Decreased miR-29c and increased NOX2, which induce cardiac fibrosis in other conditions, may uniquely mediate LPS-induced cardiac fibrosis.

Recurrent exposure to subclinical lipopolysaccharide increases mortality and induces cardiac fibrosis in mice.

BACKGROUND: Circulating subclinical lipopolysaccharide (LPS) occurs in health and disease. Ingesting high fatty meals increases LPS that cause metabolic endotoxemia. Subclinical LPS in periodontal disease may impair endothelial function. The heart may be targeted as cardiac cells express TLR4, the LPS receptor. It was hypothesized that recurrent exposure to subclinical LPS increases mortality and causes cardiac fibrosis. METHODS: C57Bl/6 mice were injected with intraperitoneal saline (control), low dose LPS (0.1 or 1 mg/kg), or moderate dose LPS (10 or 20 mg/kg), once a week for 3 months. Left ventricular (LV) function (echocardiography), hemodynamics (tail cuff pressure) and electrocardiograms (telemetry) were measured. Cardiac fibrosis was assessed by picrosirius red staining and LV expression of fibrosis related genes (QRT-PCR). Adult cardiac fibroblasts were isolated and exposed to LPS. RESULTS: LPS injections transiently increased heart rate and blood pressure (<6 hours) and mildly decreased LV function with full recovery by 24 hours. Mice tolerated weekly LPS for 2-3 months with no change in activity, appearance, appetite, weight, blood pressure, LV function, oximetry, or blood chemistries. Mortality increased after 60-90 days with moderate, but not low dose LPS. Arrhythmias occurred a few hours before death. LV collagen fraction area increased dose-dependently from 3.0±0.5% (SEM) in the saline control group, to 5.6±0.5% with low dose LPS and 9.7±0.9% with moderate dose LPS (P<0.05 moderate vs low dose LPS, and each LPS dose vs control). LPS increased LV expression of collagen Iα1, collagen IIIα1, MMP2, MMP9, TIMP1, periostin and IL-6 (P<0.05 moderate vs low dose LPS and vs control). LPS increased α-SMA immunostaining of myofibroblasts. LPS dose-dependently increased IL-6 in isolated adult cardiac fibroblasts. CONCLUSIONS: Recurrent exposure to subclinical LPS increases mortality and induces cardiac fibrosis.

Lipopolysaccharide activates calcineurin in ventricular myocytes.

OBJECTIVES: We investigated whether lipopolysaccharide (LPS), a proximate cause of inflammation, activates calcineurin in cardiac myocytes and if calcineurin regulates apoptosis in this setting. BACKGROUND: Calcineurin regulates myocardial growth and hypertrophy, but its role in inflammation is unknown. Calcineurin has proapoptotic or antiapoptotic effects depending on the stimuli. METHODS: Calcineurin activity was measured in left ventricular myocytes from adult Sprague Dawley rats. Cardiac apoptosis was measured by terminal deoxy-nucleotidyl transferase-mediated dUTP nick end-labeling staining and caspase-3 activity after in vitro and in vivo exposure to LPS. RESULTS: Lipopolysaccharide increased calcineurin activity in myocytes over 1 to 24 h (t 1/2 = 4.8 h) with an EC(50) of 0.80 ng/ml LPS (p < 0.05, n = 4). The LPS (10 ng/ml) effects were mimicked by angiotensin II (Ang II) (100 nmol/l); both increased calcineurin activity and induced apoptosis without additive effects (p < 0.05, n = 5 to 9). Lipopolysaccharide and/or Ang II effects were prevented by 1 h pre-treatment with an Ang II type 1 receptor blocker (losartan, 1 micromol/l), calcineurin inhibitor (cyclosporin A, 0.5 micromol/l), calcium chelator (1,2-Bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester, 0.1 micromol/l), or by inhibiting sarcoplasmic reticulum (SR) calcium (Ca)-ATPase (thapsigargin, 1 micromol/l) or SR calcium release channel (ryanodine, 1 micromol/l). Left ventricular apoptosis increased from 4 to 24 h after LPS (1 mg/kg intravenously) in vivo, but not in rats pre-treated with cyclosporin A (20 mg/kg/day subcutaneously) for 3 days (p < 0.05, n = 5). CONCLUSIONS: In cardiac myocytes, LPS activates calcineurin in association with apoptosis by Ang II and SR calcium-dependent mechanisms. This expands the paradigm for cardiac calcineurin to be activated by low levels of LPS in inflammation and chronic conditions (e.g., infections, smoking, and heart failure).