The Protein Acyl Transferase ZDHHC21 Modulates α1 Adrenergic Receptor Function and Regulates Hemodynamics.

OBJECTIVE: Palmitoylation, the reversible addition of the lipid palmitate to a cysteine, can alter protein localization, stability, and function. The ZDHHC family of protein acyl transferases catalyzes palmitoylation of numerous proteins. The role of ZDHHC enzymes in intact tissue and in vivo is largely unknown. Herein, we characterize vascular functions in a mouse that expresses a nonfunctional ZDHHC21 (F233Δ). APPROACH AND RESULTS: Physiological studies of isolated aortae and mesenteric arteries from F233Δ mice revealed an unexpected defect in responsiveness to phenylephrine, an α1 adrenergic receptor agonist. In vivo, F233Δ mice displayed a blunted response to infusion of phenylephrine, and they were found to have elevated catecholamine levels and elevated vascular α1 adrenergic receptor gene expression. Telemetry studies showed that the F233Δ mice were tachycardic and hypotensive at baseline, consistent with diminished vascular tone. In biochemical studies, ZDHHC21 was shown to palmitoylate the α1D adrenoceptor and to interact with it in a molecular complex, thus suggesting a possible molecular mechanism by which the receptor can be regulated by ZDHHC21. CONCLUSIONS: Together, the data support a model in which ZDHHC21 F233Δ diminishes the function of vascular α1 adrenergic receptors, leading to reduced vascular tone, which manifests in vivo as hypotension and tachycardia. This is to our knowledge the first demonstration of a ZDHHC isoform affecting vascular function in vivo and identifies a novel molecular mode of regulation of vascular tone and blood pressure.

Dynamics of PKA phosphorylation and gain of function in cardiac pacemaker cells: a computational model analysis.

Cardiac pacemaker cell function is regulated by a coupled-clock system that integrates molecular cues on the cell-membrane surface (i.e., membrane clock) and on the sarcoplasmic reticulum (SR) (i.e., Ca(2+) clock). A recent study has shown that cotransfection of spontaneous beating cells (HEK293 cells and neonatal rat myocytes) with R524Q-mutant human hyperpolarization-activated cyclic nucleotide-gated molecules (the dominant component of funny channels) increases the funny channel's sensitivity to cAMP and leads to a decrease in spontaneous action potential (AP) cycle length (i.e., tachycardia). We hypothesize that in rabbit pacemaker cells, the same behavior is expected, and because of the coupled-clock system, the resultant steady-state decrease in AP cycle length will embody contributions from both clocks: the initial decrease in the spontaneous AP beating interval, arising from increased sensitivity of the f-channel to cAMP, will be accompanied by an increase in the adenylyl cyclase (AC)-cAMP-PKA-dependent phosphorylation activity, which will further decrease this interval. To test our hypothesis, we used the recently developed Yaniv-Lakatta pacemaker cell numerical model. This model predicts the cAMP signaling dynamics, as well as the kinetics and magnitude of protein phosphorylation in both normal and mutant pacemaker cells. We found that R524Q-mutant pacemaker cells have a shorter AP firing rate than that of wild-type cells and that gain in pacemaker function is the net effect of the R514Q mutation on the functioning of the coupled-clock system. Specifically, our results directly support the hypothesis that changes in Ca(2+)-activated AC-cAMP-PKA signaling are involved in the development of tachycardia in R524Q-mutant pacemaker cells.

Modulation by Central MAPKs/PI3K/sGc of the TNF-α/iNOS-dependent Hypotension and Compromised Cardiac Autonomic Control in Endotoxic Rats.

Reduced blood pressure (BP) and cardiac autonomic activity are early manifestations of endotoxemia. We investigated whether these effects are modulated by central mitogen-activated protein kinases (MAPKs) and related phosphoinositide-3-kinase (PI3K)/soluble guanylate cyclase (sGC) signaling in conscious rats. The effect of pharmacologic inhibition of these molecular substrates on BP, heart rate (HR), and heart rate variability (HRV) responses evoked by intravascular lipopolysaccharide (LPS) (10 mg/kg) were assessed. LPS (1) lowered BP (2) increased HR, (3) reduced time [SD of beat-to-beat intervals (SDNN), and root mean square of successive differences in R-R intervals (rMSSD)], and frequency domain indices of HRV (total power and spectral bands of low and high-frequency), and (4) elevated serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels. The inhibition of TNF-α (pentoxifylline) or inducible nitric oxide synthase (iNOS, aminoguanidine) abolished hemodynamic, HRV, and inflammatory actions of LPS. Intracisternal (i.c.) injection of ODQ (sGC inhibitor), wortmannin (PI3K inhibitor), and SP600125 (MAPKJNK inhibitor) mitigated the hypotensive and tachycardic actions of LPS but failed to affect associated decreases in HRV. MAPKp38 inhibition by i.c. SB203580 produced exactly opposite effects. None of the LPS effects was altered after i.c. PD98059 (MAPKERK1/2 inhibitor). Overall, central MAPKs/PI3K/sGC pathways variably contribute to the TNF-α/iNOS-dependent reductions in BP and HRV seen during endotoxic shock.

Impact of citrate pretreatment on ventricular arrhythmia and myocardial capase-3 expression in ischemia/reperfusion injury.

Ischemia/reperfusion (I/R) injury often triggers ventricular arrhythmia. Citrate binds calcium ions, forming a soluble calcium citrate complex that may reduce I/R injury by affecting calcium ion concentration. We tested the effects of citrate pretreatment on ventricular heart rate and related factors in a rat I/R model. Fifty male Sprague Dawley rats weighing 350-400 g were randomly divided into equally sized control (A), model (B), and 0.1 M (C), 0.05 M (D), and 0.025 M (E) citrate groups. An I/R model was established by ligating the left anterior descending coronary artery. Serum calcium ion concentration was measured before and after citrate treatment. Triphenyltetrazolium chloride staining and spectrophotometry were used to determine infarction area and caspase-3 protein levels in myocardial tissue, respectively. Polymerase chain reaction was performed to test myocardial calmodulin (CAM) expression. The frequency of ventricular arrhythmia in group B was significantly higher than in the sham surgery group (P < 0.05). Citrate pretreatment resulted in lower and higher frequencies than those observed in the model and control groups, respectively, in a dose-independent manner. The most obvious reduction in ventricular arrhythmia was seen in Group D. Serum calcium ion concentration decreased markedly after citrate treatment (P < 0.05), with a specific pattern emerging over time. Infarction area and caspase-3 and CAM levels were significantly lower in the citrate groups compared with the model group (P < 0.05). Citrate can reduce myocardial cell apoptosis, alleviating ventricular arrhythmia and protecting the myocardium by reducing serum calcium ion concentration and downregulating caspase-3 and CAM expression.

Estimating sympathetic tone by recording subcutaneous nerve activity in ambulatory dogs.

INTRODUCTION: We tested the hypothesis that subcutaneous nerve activity (SCNA) of the thorax correlates with the stellate ganglion nerve activity (SGNA) and can be used to estimate the sympathetic tone. METHODS AND RESULTS: We implanted radio transmitters in 11 ambulatory dogs to record left SGNA, left thoracic vagal nerve activity (VNA), and left thoracic SCNA, including 3 with simultaneous video monitoring and nerve recording. Two additional dogs were studied under general anesthesia with apamin injected into the right stellate ganglion while the right SGNA and the right SCNA were recorded. There was a significant positive correlation between integrated SGNA (iSGNA) and integrated SCNA (iSCNA) in the first 7 ambulatory dogs, with correlation coefficient of 0.70 (95% confidence interval [CI] 0.61-0.84, P < 0.05 for each dog). Tachycardia episodes (heart rate exceeding 150 bpm for ≥3 seconds) were invariably preceded by SGNA and SCNA. There was circadian variation of both SCNA and SGNA. Crosstalk was ruled out because SGNA, VNA, and SCNA bursts had different timing and activation patterns. In an eighth dog, closely spaced bipolar subcutaneous electrodes also recorded SCNA, but with reduced signal to noise ratio. Video monitoring in additional 3 dogs showed that movement was not a cause of high frequency SCNA. The right SGNA correlated strongly with right SCNA and heart rate in 2 anesthetized dogs after apamin injection into the right stellate ganglion. CONCLUSIONS: SCNA recorded by bipolar subcutaneous electrodes correlates with the SGNA and can be used to estimate the sympathetic tone.

Angiotensin-(1-7) attenuates atrial tachycardia-induced sympathetic nerve remodeling.

INTRODUCTION: The effect of Angiotensin-(1-7) (Ang-(1-7)) on atrial autonomic remodeling is still unknown. We hypothesized that Ang-(1-7) could inhibit sympathetic nerve remodeling in a canine model of chronic atrial tachycardia. MATERIALS AND METHODS: Eighteen dogs were randomly assigned to sham group, pacing group and Ang-(1-7) group. Rapid atrial pacing was maintained for 14 days in the pacing and Ang-(1-7) groups. Ang-(1-7) was administered intravenously in the Ang-(1-7) group. The atrial effective refractory period and atrial fibrillation inducibility level were measured at baseline and under sympathetic nerve stimulation after 14 days of measurement. The atrial sympathetic nerves labeled with tyrosine hydroxylase were detected using immunohistochemistry and Western blotting, and tyrosine hydroxylase and nerve growth factor mRNA levels were measured by reverse transcription polymerase chain reaction. RESULTS: Pacing shortened the atrial effective refractory period and increased the atrial fibrillation inducibility level at baseline and under sympathetic nerve stimulation. Ang-(1-7) treatment attenuated the shortening of the atrial effective refractory period and the increase in the atrial fibrillation inducibility level. Immunohistochemistry and Western blotting showed sympathetic nerve hyperinnervation in the pacing group, while Ang-(1-7) attenuated sympathetic nerve proliferation. Ang-(1-7) alleviated the pacing-induced increases in tyrosine hydroxylase and nerve growth factor mRNA expression levels. CONCLUSION: Ang-(1-7) can attenuate pacing-induced atrial sympathetic hyperinnervation.

Prognostic significance of isolated sinus tachycardia during first three days of acute myocardial infarction.

Sinus tachycardia often accompanies other indicators of poor prognosis in acute myocardial infarction. This study was performed to evaluate the prognostic significance of early (Days 1 to 3) in-hospital sinus tachycardia (isolated sinus tachycardia) in the absence of other common indicators of poor prognosis. All patients consecutively admitted directly to the cardiac care unit during a six-year period were evaluated. Patients who had confirmed acute myocardial infarction and no urgent complications during Days 1 to 3 with isolated sinus tachycardia (99 patients) or without isolated sinus tachycardia (159 patients) were included in the study. Both groups were followed for subsequent in-hospital outcome and long-term survival. Univariable and multivariable analysis of historical and demographic characteristics showed no significant differences between the two groups. When clinical descriptors of the infarct were evaluated, the group with isolated sinus tachycardia had a significantly higher mean peak creatine kinase level (p = 0.0007), a larger proportion of anterior infarcts and multiple infarct sites (p less than 0.001) by electrocardiography, a higher incidence of peri-infarction pericarditis (p = 0.007), and a higher incidence of recurrent chest pain (p = 0.03). Twenty-five patients (25 percent) in the group with isolated sinus tachycardia had subsequent urgent complications during the hospitalization compared with 11 patients (7 percent) in the control group (p = 0.00005). In multivariable analysis, isolated sinus tachycardia was an independent predictor of subsequent urgent complications (p = 0.0009) and mortality (p = 0.05).

Cardiac and regional haemodynamics, inducible nitric oxide synthase (NOS) activity, and the effects of NOS inhibitors in conscious, endotoxaemic rats.

1. A reproducible model of the hyperdynamic circulatory sequelae of endotoxaemia in conscious, chronically-instrumented Long Evans rats, was achieved with a continuous infusion of lipopolysaccharide (LPS, 150 micro g kg(-1) h(-1)) for 32 h. Over the first 2 h of LPS infusion, there was a transient hypotension and tachycardia, accompanied by a marked increase in renal flow and vascular conductance, although there were reductions in cardiac and stroke index. Between 4-8 after the start of LPS infusion, there was slight hypotension and tachycardia, and a transient rise in mesenteric flow and conductance, but reductions in the hindquarters vascular bed; the hyperaemic vasodilatation in the renal vascular bed was maintained. At this stage, all cardiac haemodynamic variables were not different from baseline. At this stage, cardiac and stroke index were substantially elevated, in association with marked increases in peak aortic flow, dF/dtmax and total peripheral conductance; these changes were well-maintained over the following 8 h of LPS infusion. 2. By 2 h after the start of LPS infusion, only lung inducible nitric oxide synthase (iNOS) activity was increased, but at 6 h there were significant increases in iNOS activity in lung, liver, spleen, heart and aorta. (43.3 +/- 7.8, 28.8 +/- 3.3, 50.8 +/- 7.2, 3.04 +/- 0.29, 3.76 +/- 0.94 pmol min(-1) mg(-1) protein, respectively). However, by 24 h after the start of LPS infusion, iNOS activity was not elevated significantly in any tissue examined, and kidney iNOS activity did not change significantly during LPS infusion. Plasma nitrite/nitrate levels were increased after 2 h infusion of LPS (from 6.07 +/- 1.23 to 29.44 +/- 7.08 micromol l(-1)), and further by 6 h (228.10 +/- 29.20 micromol l(-1)), but were less 24 h after onset of LPS infusion (74.96 +/- 11.34 micromol l(-1)). Hence, the progressive hypotension, increasing cardiac function and developing hyperaemic vasodilatation in renal and hindquarters vascular beds between 8-24 h after the onset of LPS infusion, occurred when tissue iNOS activity and plasma nitrite/nitrate levels were falling. 3. Pretreatment with NG-monomethyl-L-arginine (L-NMMA, 30 mg kg(-1) bolus, 30 mg kg(-1) h(-1) infusion) 1 h before LPS infusion did not prevent the early hypotension, but abolished the initial renal vasodilatation and the later (6-8 h) fall in mean arterial pressure (MAP), and the additional renal vasodilatation.

Serum dopamine beta-hydroxylase activity in acute cardiac disease.

The activity of dopamine beta-hydroxylase in the serum was measured in patients with acute cardiac disorders who were admitted to a coronary care unit. The activity of the enzyme on admission was similar in patients with acute myocardial infarction and in those with other conditions; on discharge, the activity was reduced significantly. Patients with myocardial infarction had the greatest fall in the activity of dopamine beta-hydroxylase. Patients with multiple signs of sympathetic hyperactivity had significantly higher values for the activity of dopamine beta-hydroxylase on admission. Pain was found to significantly increase the activity of the enzyme. These data confirm findings that the activity of dopamine beta-hydroxylase in the serum reflects the intensity of sympathetic activity. The factors contributing to the elevated sympathetic activity in some patients with acute myocardial infarction seem to be nonspecific, but because of the ominous nature of increased sympathetic tone in this condition, it could be advantageous to identify these patients.

Association of angiotensin converting enzyme gene polymorphism with tachycardia cardiomyopathy.

Despite incessant tachycardia, not all patients develop tachycardia-mediated cardiomyopathy. The cardiac renin-angiotensin system may be involved in cardiac remodelling and fibrosis. The level of angiotension-converting enzyme (ACE) in the serum is associated with a 287 bp insertion (I)/deletion (D) polymorphism in intron 16 of the ACE gene. The DD genotype is associated with increased serum ACE levels and a higher incidence of idiopathic dilated and ischemic cardiomyopathy. The objective of this study was to assess whether the ACE gene I/D polymorphism is responsible for development of tachycardia-mediated cardiomyopathy. We identified 20 consecutive patients with persistent tachycardia and cardiomyopathy who showed significant improvement in ejection fraction after rate control (group A, tachycardia cardiomyopathy group). We compared the I/D genotype frequency of group A with the gene frequency of a separate group of 20 patents who, despite rapid atrial arrhythmias had preserved left ventricular ejection fraction (group B, tachycardia without cardiomyopathy group). These two groups were then compared with 24 healthy normal volunteers (group C). After a mean follow-up of 30 months, group A patients showed improvement in ejection fraction from 20+/-7 to 43+/-9% (p<0.001). Group A had a significantly higher frequency of the DD genotype than groups B and C (p-value <0.035 and <0.009 respectively). The profile of group B patients was intermediate between normal and patients with tachycardia-mediated cardiomyopathy. I/D polymorphism of the ACE gene may account for cardiomyopathy secondary to tachycardia.

Phosphodiesterases reduce spontaneous sinoatrial beating but not the 'fight or flight' tachycardia elicited by agonists through Gs-protein-coupled receptors.

Cyclic AMP (cAMP) steers the generation of basal heart beat in the sinoatrial node. It also induces sinoatrial tachycardia and increased cardiac force, elicited through activation of Gs-protein-coupled receptors (GsPCRs). Phosphodiesterases (PDEs) hydrolyse cAMP. In the heart mainly PDE3 and PDE4 would be expected to limit those functions, and the PDE isoenzymes do indeed reduce basal sinoatrial beating rate and blunt the positive inotropic effects of agonists, mediated by GsPCRs. By contrast, recent evidence shows that GsPCR-mediated sinoatrial tachycardia is not controlled by PDE1-5. A PDE-resistant cAMP pool in sinoatrial cells, generated through activation of GsPCRs, including ß(1)- and ß(2)-adrenoceptors, appears to guarantee unrestrained tachycardia during fight or flight stress.

HSPB1, HSPB6, HSPB7 and HSPB8 protect against RhoA GTPase-induced remodeling in tachypaced atrial myocytes.

BACKGROUND: We previously demonstrated the small heat shock protein, HSPB1, to prevent tachycardia remodeling in in vitro and in vivo models for Atrial Fibrillation (AF). To gain insight into its mechanism of action, we examined the protective effect of all 10 members of the HSPB family on tachycardia remodeling. Furthermore, modulating effects of HSPB on RhoA GTPase activity and F-actin stress fiber formation were examined, as this pathway was found of prime importance in tachycardia remodeling events and the initiation of AF. METHODS AND RESULTS: Tachypacing (4 Hz) of HL-1 atrial myocytes significantly and progressively reduced the amplitude of Ca²âº transients (CaT). In addition to HSPB1, also overexpression of HSPB6, HSPB7 and HSPB8 protected against tachypacing-induced CaT reduction. The protective effect was independent of HSPB1. Moreover, tachypacing induced RhoA GTPase activity and caused F-actin stress fiber formation. The ROCK inhibitor Y27632 significantly prevented tachypacing-induced F-actin formation and CaT reductions, showing that RhoA activation is required for remodeling. Although all protective HSPB members prevented the formation of F-actin stress fibers, their mode of action differs. Whilst HSPB1, HSPB6 and HSPB7 acted via direct prevention of F-actin formation, HSPB8-protection was mediated via inhibition of RhoA GTPase activity. CONCLUSION: Overexpression of HSPB1, as well as HSPB6, HSPB7 and HSPB8 independently protect against tachycardia remodeling by attenuation of the RhoA GTPase pathway at different levels. The cardioprotective role for multiple HSPB members indicate a possible therapeutic benefit of compounds able to boost the expression of single or multiple members of the HSPB family.

Development of porcine model of chronic tachycardia-induced cardiomyopathy.

BACKGROUND: There are few experimental models of heart failure (HF) in large animals, despite structural and functional similarities to human myocardium. We have developed a porcine model of chronic tachycardia-induced cardiomyopathy. METHODS: Homogenous siblings of White Large breed swine (n=6) underwent continuous right ventricular (RV) pacing at 170 bpm; 2 subjects served as controls. In the course of RV pacing, animals developed a clinical picture of HF and were presented for euthanasia at subsequent stages: mild, moderate and end-stage HF. Left ventricle (LV) sections were analyzed histologically and relative ANP, BNP, phospholamban and sarcoplasmic reticulum calcium ATPase 2a transcript levels in LV were quantified by real time RT-PCR. RESULTS: In the course of RV pacing, animals demonstrated reduced exercise capacity (time of running until being dyspnoeic: 6.6 ± 0.5 vs. 2.4 ± 1.4 min), LV dilatation (LVEDD: 4.9 ± 0.4 vs. 6.7 ± 0.4 cm), impaired LV systolic function (LVEF: 69 ± 8 vs. 32 ± 7 %), (all baseline vs. before euthanasia, all p<0.001). LV tissues from animals with moderate and end-stage HF demonstrated local foci of interstitial fibrosis, congestion, cardiomyocyte hypertrophy and atrophy, which was not detected in controls and mild HF animals. The up-regulation of ANP and BNP and a reduction in a ratio of sarcoplasmic reticulum calcium ATPase 2a and phospholamban in failing myocardium were observed as compared to controls. CONCLUSIONS: In pigs, chronic RV pacing at relatively low rate can be used as an experimental model of HF, as it results in a gradual deterioration of exercise tolerance accompanied by myocardial remodeling confirmed at subcellular level.

Propafenone for the prevention of atrial tachyarrhythmias after cardiac surgery: a randomized, double-blind placebo-controlled trial.

We studied the efficacy of propafenone in preventing atrial tachyarrhythmias after cardiac surgery, and the possible relationships between CYP2D6 polymorphism and the efficacy, pharmacokinetics, and tolerability of propafenone. One hundred and sixty patients were randomized (double blind) to receive propafenone (n= 78) or placebo (n= 82) for 1 week after cardiac surgery. The patients who were assigned to the propafenone group received 1 mg/kg infused in 1 h, followed by a continuous infusion at a rate of 4 mg/kg/24 h until the following morning, and subsequently 450 mg/day orally until the sixth postoperative day. Thirty-seven patients completed the trial in the propafenone group and 45 in the placebo group. The frequency of occurrence of atrial tachyarrhythmia was lower in the propafenone group than in the placebo group (29.7% vs. 53.3%, P< 0.05; relative risk, 0.56). Plasma propafenone concentrations were markedly influenced by CYP2D6 genotype-derived phenotype.

Ultrastructural abnormalities of mitochondria and deficiency of myocardial cytochrome c oxidase in a patient with ventricular tachycardia.

A 30-year-old woman presented with life-threatening ventricular tachycardia without overt heart disease. Ultrastructural investigation of endomyocardial biopsy disclosed abnormally structured and often enlarged mitochondria. Morphometry revealed the ratio of volume density of mitochondria to myofibrils to be markedly increased to 0.667 as compared with five controls (mean: 0.46; range: 0.445-0.479). Investigation of mitochondrial respiratory chain enzymes revealed a 90% reduction in activity of cytochrome c oxidase. Our data suggest that mitochondrial cardiomyopathy may induce malignant ventricular arrhythmias.

5'-Nucleotidase and adenosine deaminase activities in hypertrophied rat heart. The effect of tachycardia.

Activity of 5'-nucleotidase (5NT) and adenosine deaminase (ADA) in the left heart ventricle was examined at normal heart rate and after atrial pacing in six weeks after constriction of the aorta above or below the renal vessels and in sham operated rats. The aorta constriction above the vessels reduced activity of 5NT by 29% and increased activity of ADA by 33%, whereas the constriction below the vessels resulted in elevated activity of both enzymes: 5NT by 16% and ADA by 145% at normal heart rate. The atrial pacing reduced 5NT activity and did not affect ADA activity in control rats and in those with constriction of the aorta below the vessels. The activity of both enzymes increased after constriction of the aorta above the renal vessels. It is concluded that adenosine metabolism in the hypertrophied heart may be changed due to changes in its production and degradation. Renin-angiotensin-aldosterone system seems to be involved in regulation of the activities of adenosine metabolizing enzymes in the hypertrophied heart.