ABSTRACT
Inositol trisphosphate (IP3) is a Ca2+-mobilizing second messenger shown to modulate atrial muscle contraction and is thought to contribute to atrial fibrillation. Cellular pathways underlying IP3 actions in cardiac tissue remain poorly understood, and the work presented here addresses the question whether IP3-mediated Ca2+ release from the sarcoplasmic reticulum is linked to adenylyl cyclase activity including Ca2+-stimulated adenylyl cyclases (AC1 and AC8) that are selectively expressed in atria and sinoatrial node (SAN). Immunocytochemistry in guinea pig atrial myocytes identified colocalization of type 2 IP3 receptors with AC8, while AC1 was located in close vicinity. Intracellular photorelease of IP3 by UV light significantly enhanced the amplitude of the Ca2+ transient (CaT) evoked by electrical stimulation of atrial myocytes (31 ± 6% increase 60 s after photorelease, n = 16). The increase in CaT amplitude was abolished by inhibitors of adenylyl cyclases (MDL-12,330) or protein kinase A (H89), showing that cAMP signaling is required for this effect of photoreleased IP3. In mouse, spontaneously beating right atrial preparations, phenylephrine, an α-adrenoceptor agonist with effects that depend on IP3-mediated Ca2+ release, increased the maximum beating rate by 14.7 ± 0.5%, n = 10. This effect was substantially reduced by 2.5 µmol/L 2-aminoethyl diphenylborinate and abolished by a low dose of MDL-12,330, observations which are again consistent with a functional interaction between IP3 and cAMP signaling involving Ca2+ stimulation of adenylyl cyclases in the SAN pacemaker. Understanding the interaction between IP3 receptor pathways and Ca2+-stimulated adenylyl cyclases provides important insights concerning acute mechanisms for initiation of atrial arrhythmias.NEW & NOTEWORTHY This study provides evidence supporting the proposal that IP3 signaling in cardiac atria and sinoatrial node involves stimulation of Ca2+-activated adenylyl cyclases (AC1 and AC8) by IP3-evoked Ca2+ release from junctional sarcoplasmic reticulum. AC8 and IP3 receptors are shown to be located close together, while AC1 is nearby. Greater understanding of these novel aspects of the IP3 signal transduction mechanism is important for future study in atrial physiology and pathophysiology, particularly atrial fibrillation.
Subject(s)
Adenylyl Cyclases/metabolism , Biological Clocks , Calcium Signaling , Heart Atria/enzymology , Heart Rate , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Inositol 1,4,5-Trisphosphate/metabolism , Myocytes, Cardiac/enzymology , Sinoatrial Node/enzymology , Action Potentials , Animals , Cyclic AMP-Dependent Protein Kinases/metabolism , Guinea Pigs , Heart Atria/cytology , Isoenzymes , Male , Mice , Sarcoplasmic Reticulum/enzymology , Time FactorsABSTRACT
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.
Subject(s)
Biological Clocks , Cyclic AMP-Dependent Protein Kinases/metabolism , Heart Rate , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism , Models, Cardiovascular , Phosphorylation , Second Messenger Systems , Sinoatrial Node/enzymology , Tachycardia/enzymology , Action Potentials , Adenylyl Cyclases/metabolism , Animals , Calcium-Binding Proteins/metabolism , Computer Simulation , Cyclic AMP/metabolism , Genetic Predisposition to Disease , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/genetics , Kinetics , Mutation , Numerical Analysis, Computer-Assisted , Phenotype , Rabbits , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Sinoatrial Node/cytology , Tachycardia/genetics , Tachycardia/physiopathologyABSTRACT
One of the main strategies for cancer therapy is to use tyrosine kinase inhibitors for inhibiting tumor proliferation. Increasing evidence has demonstrated the potential risks of cardiac arrhythmias (such as prolonged QT interval) of these drugs. We report here that a widely used selective inhibitor of Src tyrosine kinases, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), can inhibit and prevent ß-adrenergic stimulation of cardiac pacemaker activity. First, in dissected rat sinus node, PP2 inhibited and prevented the isoproterenol-induced increase of spontaneous beating rate. Second, in isolated rat sinus node myocytes, PP2 suppressed the hyperpolarization-activated "funny" current (traditionally called cardiac pacemaker current, I(f)) by negatively shifting the activation curve and decelerating activation kinetics. Third, in isolated rat sinus node myocytes, PP2 decreased the Src kinase activity, the cell surface expression, and tyrosine phosphorylation of hyperpolarization-activated, cyclic nucleotide-modulated channel 4 (HCN4) channel proteins. Finally, in human embryonic kidney 293 cells overexpressing recombinant human HCN4 channels, PP2 reversed the enhancement of HCN4 channels by isoproterenol and inhibited 573x, a cyclic adenosine momophosphate-insensitive human HCN4 mutant. These results demonstrated that inhibition of Src kinase activity in heart by PP2 decreased and prevented ß-adrenergic stimulation of cardiac pacemaker activity. These effects are mediated, at least partially, by a cAMP-independent attenuation of channel activity and cell surface expression of HCN4, the main channel protein that controls the heart rate.
Subject(s)
Adrenergic beta-Agonists/pharmacology , Pyrimidines/pharmacology , Sinoatrial Node/drug effects , src-Family Kinases/antagonists & inhibitors , Animals , Cells, Cultured , HEK293 Cells , Humans , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/biosynthesis , Muscle Proteins/biosynthesis , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/enzymology , Potassium Channels/biosynthesis , Rats , Rats, Sprague-Dawley , Sinoatrial Node/enzymology , src-Family Kinases/metabolismABSTRACT
RATIONALE: Sinoatrial node cells (SANCs) generate local, subsarcolemmal Ca(2+) releases (LCRs) from sarcoplasmic reticulum (SR) during late diastolic depolarization. LCRs activate an inward Na(+)-Ca(2+) exchange current (I(NCX)), which accelerates diastolic depolarization rate, prompting the next action potential (AP). The LCR period, ie, a delay between AP-induced Ca(2+) transient and LCR appearance, defines the time of late diastolic depolarization I(NCX) activation. Mechanisms that control the LCR period, however, are still unidentified. OBJECTIVE: To determine dependence of the LCR period on SR Ca(2+) refilling kinetics and establish links between regulation of SR Ca(2+) replenishment, LCR period, and spontaneous cycle length. METHODS AND RESULTS: Spontaneous APs and SR luminal or cytosolic Ca(2+) were recorded using perforated patch and confocal microscopy, respectively. Time to 90% replenishment of SR Ca(2+) following AP-induced Ca(2+) transient was highly correlated with the time to 90% decay of cytosolic Ca(2+) transient (T-90(C)). Local SR Ca(2+) depletions mirror their cytosolic counterparts, LCRs, and occur following SR Ca(2+) refilling. Inhibition of SR Ca(2+) pump by cyclopiazonic acid dose-dependently suppressed spontaneous SANCs firing up to ≈50%. Cyclopiazonic acid and graded changes in phospholamban phosphorylation produced by ß-adrenergic receptor stimulation, phosphodiesterase or protein kinase A inhibition shifted T-90(C) and proportionally shifted the LCR period and spontaneous cycle length (R(2)=0.98). CONCLUSIONS: The LCR period, a critical determinant of the spontaneous SANC cycle length, is defined by the rate of SR Ca(2+) replenishment, which is critically dependent on SR pumping rate, Ca(2+) available for pumping, supplied by L-type Ca(2+) channel, and ryanodine receptor Ca(2+) release flux, each of which is modulated by cAMP-mediated protein kinase A-dependent phosphorylation.
Subject(s)
Biological Clocks/physiology , Calcium Signaling/physiology , Heart Rate/physiology , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Sarcoplasmic Reticulum/enzymology , Sinoatrial Node/enzymology , Action Potentials/drug effects , Animals , Biological Clocks/drug effects , Calcium Signaling/drug effects , Heart Rate/drug effects , Rabbits , Sarcoplasmic Reticulum/metabolism , Sarcoplasmic Reticulum/physiology , Sarcoplasmic Reticulum Calcium-Transporting ATPases/antagonists & inhibitors , Sarcoplasmic Reticulum Calcium-Transporting ATPases/physiology , Sinoatrial Node/cytology , Sinoatrial Node/metabolism , Sinoatrial Node/physiology , Time FactorsABSTRACT
Decades of intensive research of primary cardiac pacemaker, the sinoatrial node, have established potential roles of specific membrane channels in the generation of the diastolic depolarization, the major mechanism allowing sinoatrial node cells to generate spontaneous beating. During the last three decades, multiple studies made either in the isolated sinoatrial node or sinoatrial node cells have demonstrated a pivotal role of Ca(2+) and, specifically Ca(2+) release from sarcoplasmic reticulum, for spontaneous beating of cardiac pacemaker. Recently, spontaneous, rhythmic local subsarcolemmal Ca(2+) releases from ryanodine receptors during late half of the diastolic depolarization have been implicated as a vital factor in the generation of sinoatrial node cell spontaneous firing. Local Ca(2+) releases are driven by a unique combination of high basal cAMP production by adenylyl cyclases, high basal cAMP degradation by phosphodiesterases and a high level of cAMP-mediated PKA-dependent phosphorylation. These local Ca(2+) releases activate an inward Na(+)-Ca(2+) exchange current which accelerates the terminal diastolic depolarization rate and, thus, controls the spontaneous pacemaker firing. Both the basal primary pacemaker beating rate and its modulation via beta-adrenergic receptor stimulation appear to be critically dependent upon intact RyR function and local subsarcolemmal sarcoplasmic reticulum generated Ca(2+) releases. This review aspires to integrate the traditional viewpoint that has emphasized the supremacy of the ensemble of surface membrane ion channels in spontaneous firing of the primary cardiac pacemaker, and these novel perspectives of cAMP-mediated PKA-dependent Ca(2+) cycling in regulation of the heart pacemaker clock, both in the basal state and during beta-adrenergic receptor stimulation.
Subject(s)
Calcium Channels/metabolism , Calcium Signaling , Cell Membrane/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Sinoatrial Node/cytology , Sinoatrial Node/enzymology , Animals , Humans , Receptors, Adrenergic, beta/metabolismABSTRACT
Ablation of the enzyme phosphoinositide 3-kinase (PI3K)gamma (PI3Kgamma(-/-)) in mice increases cardiac contractility by elevating intracellular cAMP and enhancing sarcoplasmic reticulum Ca(2+) handling. Because cAMP is a critical determinant of heart rate, we investigated whether heart rate is altered in mice lacking PI3Kgamma. Heart rate was similar in anesthetized PI3Kgamma(-/-) and wild-type (PI3Kgamma(+/+)) mice. However, IP injection of atropine (1 mg/kg), propranolol (1 mg/kg), or both drugs in combination unmasked elevated heart rates in PI3Kgamma(-/-) mice, suggesting altered sinoatrial node (SAN) function. Indeed, spontaneous action potential frequency was approximately 35% greater in SAN myocytes isolated from PI3Kgamma(-/-) mice compared with PI3Kgamma(+/+) mice. These differences in action potential frequency were abolished by intracellular dialysis with the cAMP/protein kinase A antagonist Rp-cAMP but were unaffected by treatment with ryanodine to inhibit sarcoplasmic reticulum Ca(2+) release. Voltage-clamp experiments demonstrated that elevated action potential frequencies in PI3Kgamma(-/-) SAN myocytes were more strongly associated with cAMP-dependent increases in L-type Ca(2+) current (I(Ca,L)) than elevated hyperpolarization-activated current (I(f)). In contrast, I(Ca,L) was not increased in working atrial myocytes, suggesting distinct subcellular regulation of L-type Ca(2+) channels by PI3Kgamma in the SAN compared with the working myocardium. In summary, PI3Kgamma regulates heart rate by the cAMP-dependent modulation of SAN function. The effects of PI3Kgamma ablation in the SAN are unique from those in the working myocardium.
Subject(s)
Cyclic AMP/physiology , Heart Rate/genetics , Phosphatidylinositol 3-Kinases/deficiency , Sinoatrial Node/enzymology , Sinoatrial Node/physiopathology , Action Potentials/genetics , Animals , Class Ib Phosphatidylinositol 3-Kinase , Isoenzymes/biosynthesis , Isoenzymes/deficiency , Isoenzymes/genetics , Male , Mice , Mice, Knockout , Myocytes, Cardiac/enzymology , Myocytes, Cardiac/pathology , Myocytes, Cardiac/physiology , Phosphatidylinositol 3-Kinases/biosynthesis , Phosphatidylinositol 3-Kinases/geneticsABSTRACT
BACKGROUND: Spontaneous firing of sinoatrial node cells (SANCs) is regulated by cAMP-mediated, PKA (protein kinase A)-dependent (cAMP/PKA) local subsarcolemmal Ca2+ releases (LCRs) from RyRs (ryanodine receptors). LCRs occur during diastolic depolarization and activate an inward Na+/Ca2+ exchange current that accelerates diastolic depolarization rate prompting the next action potential. PDEs (phosphodiesterases) regulate cAMP-mediated signaling; PDE3/PDE4 represent major PDE activities in SANC, but how they modulate LCRs and basal spontaneous SANC firing remains unknown. METHODS: Real-time polymerase chain reaction, Western blot, immunostaining, cellular perforated patch clamping, and confocal microscopy were used to elucidate mechanisms of PDE-dependent regulation of cardiac pacemaking. RESULTS: PDE3A, PDE4B, and PDE4D were the major PDE subtypes expressed in rabbit SANC, and PDE3A was colocalized with α-actinin, PDE4D, SERCA (sarcoplasmic reticulum Ca2+ ATP-ase), and PLB (phospholamban) in Z-lines. Inhibition of PDE3 (cilostamide) or PDE4 (rolipram) alone increased spontaneous SANC firing by ≈20% (P<0.05) and ≈5% (P>0.05), respectively, but concurrent PDE3+PDE4 inhibition increased spontaneous firing by ≈45% (P<0.01), indicating synergistic effect. Inhibition of PDE3 or PDE4 alone increased L-type Ca2+ current (ICa,L) by ≈60% (P<0.01) or ≈5% (P>0.05), respectively, and PLB phosphorylation by ≈20% (P>0.05) each, but dual PDE3+PDE4 inhibition increased ICa,L by ≈100% (P<0.01) and PLB phosphorylation by ≈110% (P<0.05). Dual PDE3+PDE4 inhibition increased the LCR number and size (P<0.01) and reduced the SR (sarcoplasmic reticulum) Ca2+ refilling time (P<0.01) and the LCR period (time from action potential-induced Ca2+ transient to subsequent LCR; P<0.01), leading to decrease in spontaneous SANC cycle length (P<0.01). When RyRs were disabled by ryanodine and LCRs ceased, dual PDE3+PDE4 inhibition failed to increase spontaneous SANC firing. CONCLUSIONS: Basal cardiac pacemaker function is regulated by concurrent PDE3+PDE4 activation which operates in a synergistic manner via decrease in cAMP/PKA phosphorylation, suppression of LCR parameters, and prolongation of the LCR period and spontaneous SANC cycle length.
Subject(s)
Action Potentials , Biological Clocks , Cyclic Nucleotide Phosphodiesterases, Type 3/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Heart Rate , Sinoatrial Node/enzymology , Action Potentials/drug effects , Animals , Calcium Signaling , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 3/genetics , Cyclic Nucleotide Phosphodiesterases, Type 4/genetics , Enzyme Activation , Heart Rate/drug effects , Kinetics , Phosphodiesterase 3 Inhibitors/pharmacology , Phosphodiesterase 4 Inhibitors/pharmacology , Rabbits , Ryanodine Receptor Calcium Release Channel/metabolism , Sinoatrial Node/cytology , Sinoatrial Node/drug effectsABSTRACT
AIMS: RHOA-ROCK signalling regulates cell migration, proliferation, differentiation, and transcription. RHOA is expressed in the developing cardiac conduction system in chicken and mice. In early development, the entire sinus venosus myocardium, including both the transient left-sided and the definitive sinoatrial node (SAN), has pacemaker potential. Later, pacemaker potential is restricted to the right-sided SAN. Disruption of RHOA expression in adult mice causes arrhythmias including bradycardia and atrial fibrillation, the mechanism of which is unknown but presumed to affect the SAN. The aim of this study is to assess the role of RHOA-ROCK signalling in SAN development in the chicken heart. METHODS AND RESULTS: ROCK signalling was inhibited chemically in embryonic chicken hearts using Y-27632. This prolonged the immature state of the sinus venosus myocardium, evidenced by up-regulation of the transcription factor ISL1, wide distribution of pacemaker potential, and significantly reduced heart rate. Furthermore ROCK inhibition caused aberrant expression of typical SAN genes: ROCK1, ROCK2, SHOX2, TBX3, TBX5, ISL1, HCN4, CX40, CAV3.1, and NKX2.5 and left-right asymmetry genes: PITX2C and NODAL. Anatomical abnormalities in pulmonary vein development were also observed. Patch clamp electrophysiology confirmed the immature phenotype of the SAN cells and a residual left-sided sinus venosus myocardium pacemaker-like potential. CONCLUSIONS: RHOA-ROCK signalling is involved in establishing the right-sided SAN as the definitive pacemaker of the heart and restricts typical pacemaker gene expression to the right side of the sinus venosus myocardium.
Subject(s)
Biological Clocks , Cell Differentiation , Signal Transduction , Sinoatrial Node/enzymology , rho-Associated Kinases/metabolism , rhoA GTP-Binding Protein/metabolism , Action Potentials , Animals , Arrhythmias, Cardiac/enzymology , Arrhythmias, Cardiac/genetics , Arrhythmias, Cardiac/physiopathology , Biological Clocks/drug effects , Cell Differentiation/drug effects , Cells, Cultured , Chick Embryo , Gene Expression Regulation, Developmental , Heart Defects, Congenital/enzymology , Heart Defects, Congenital/genetics , Heart Defects, Congenital/physiopathology , Heart Rate , Morphogenesis , Myocytes, Cardiac/enzymology , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effects , Sinoatrial Node/drug effects , Sinoatrial Node/embryology , Sinoatrial Node/physiopathology , Time Factors , rho-Associated Kinases/antagonists & inhibitors , rho-Associated Kinases/genetics , rhoA GTP-Binding Protein/geneticsABSTRACT
The expression of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel isoforms varies among species, cardiac tissues, developmental stages, and disease generation. However, alterations in the HCN channels during aging remain unclear. We investigated the protein expressions of HCN channel isoforms, HCN1-HCN4, in the sinoatrial nodes (SANs) from young (1-month-old), adult (4-month-old), and aged (30-month-old) rats. We found that HCN2 and HCN4 proteins were present in rat SAN using immunohistochemistry; therefore, we quantitatively analyzed their expression by Western blot. Aim to correlate protein expression and pacemaking function, specific blockade of HCN channels with 3 µmol/L ivabradine prolonged the cycle length in the intact rat heart. During the senescent process, the HCN2 and HCN4 protein levels declined, which was accompanied with a decreased effect of ivabradine on rat SAN automaticity. These results indicated the age-associated expression and relative function of HCN channel isoforms.
Subject(s)
Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/biosynthesis , Sinoatrial Node/enzymology , Age Factors , Animals , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/chemistry , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/genetics , Male , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Rats, Sprague-DawleyABSTRACT
1. We have characterized alpha 1-adrenoceptor in the conduction systems of the rat heart by quantitative autoradiography. 2. Consecutive 20 micron thick sections from a single rat heart containing the sinoatrial (SA) node and atrioventricular (AV) node were incubated with increasing concentrations of [3H]-prazosin with or without 10 microM phentolamine. After exposure to 3H-Ultrofilm, optical densities corresponding to the SA node and AV node were determined by computerized densitometry after comparison with 3H standards. 3. The SA node and AV node were stained heavily for cholinesterase and they contained a higher concentration of alpha 1-adrenoceptors than the adjacent myocardium without a significant change in the affinity. 4. These results support the hypothesis that alpha 1-adrenoceptors may play an important role not only in inotropism but also in chronotropism of rat hearts.
Subject(s)
Heart Conduction System/metabolism , Receptors, Adrenergic, alpha-1/metabolism , Animals , Atrioventricular Node/drug effects , Atrioventricular Node/enzymology , Atrioventricular Node/metabolism , Autoradiography , Cholinesterases/metabolism , Heart Conduction System/drug effects , Heart Conduction System/enzymology , Image Processing, Computer-Assisted , In Vitro Techniques , Male , Prazosin/pharmacokinetics , Rats , Rats, Wistar , Receptors, Adrenergic, alpha-1/drug effects , Sinoatrial Node/drug effects , Sinoatrial Node/enzymology , Sinoatrial Node/metabolismSubject(s)
Catecholamines/physiology , Sinoatrial Node/physiology , 3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , 3',5'-Cyclic-AMP Phosphodiesterases/metabolism , Adenosine Triphosphate/metabolism , Adrenergic beta-Antagonists/pharmacology , Animals , Biological Transport , Calcium/metabolism , Catecholamines/metabolism , Cyclic AMP/metabolism , Cyclic GMP/metabolism , Exocytosis , Heart/embryology , Heart Rate/drug effects , Mechanoreceptors/physiology , Membrane Potentials , Methyltyrosines/pharmacology , Models, Biological , Myocardial Contraction/drug effects , Reserpine/pharmacology , Sarcolemma/metabolism , Sinoatrial Node/enzymology , Sinoatrial Node/innervation , Sinoatrial Node/ultrastructureABSTRACT
The effects of cGMP analogues and phosphodiesterase inhibitors were investigated on cAMP and cGMP hydrolysis by cGMP-stimulated phosphodiesterase (cGS-PDE), isolated from a canine heart sinoatrial node-enriched preparation and from the left ventricle. There was no significant difference between the effects of drugs and cGMP analogues on cGS-PDE from the cardiac ventricle and from the sinoatrial node, suggesting that cGS-PDE has similar characteristics in the two tissues, cGMP itself, 8-bromo-cGMP and 2'-deoxy-cGMP had dual effects: at low concentrations, cAMP hydrolysis was stimulated (maximal effect at 10 microM, 100 microM and 100 microM respectively), while at higher concentrations these compounds inhibited cAMP hydrolysis. Monobutyryl-cGMP and dibutyryl-cGMP had only an inhibitory effect on cAMP hydrolysis. Inhibitors of cAMP- or cGMP-selective PDEs, including the cardiotonic drugs rolipram and zaprinast, were not effective inhibitors of cGS-PDE. Cilostamide (a selective inhibitor of cGMP-inhibited PDE). IBMX (nonspecific inhibitor of PDEs) and dipyridamole inhibited basal cGS-PDE hydrolysis of cAMP and cGMP, and their apparent Ki for cAMP hydrolysis was decreased by 5 microM cGMP (from 30, 14 and 18 to 15.7 and 2.6 microM, respectively, for the ventricular enzyme).(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
2',3'-Cyclic-Nucleotide Phosphodiesterases/metabolism , Cyclic GMP/analogs & derivatives , Cyclic GMP/pharmacology , Myocardium/metabolism , 2',3'-Cyclic-Nucleotide Phosphodiesterases/antagonists & inhibitors , Animals , Cardiotonic Agents/pharmacology , Chromatography, DEAE-Cellulose , Chromatography, High Pressure Liquid , Cyclic AMP/metabolism , Dogs , Heart Ventricles/enzymology , Hydrolysis , In Vitro Techniques , Kinetics , Phosphodiesterase Inhibitors/pharmacology , Sinoatrial Node/enzymology , Stimulation, ChemicalABSTRACT
Recently, we observed that atrial natriuretic peptide (ANP) immunoreactivity (IR) was present not only in the Purkinje fibres, but also in nerve fibre varicosities in the conduction system of the bovine heart. These findings and previous observations that ANP is able to influence autonomic neurotransmission in the heart, lead us to elucidate the possible occurrence of ANP in the sympathetic and/or parasympathetic nervous systems and/or in various types of peptidergic innervation in the conduction system. The different parts of the conduction system of bovine hearts were dissected out and processed for immunohistochemistry including double-staining, using antisera against ANP, tyrosine hydroxylase and different neuropeptides. We observed that some of the nerve fibre varicosities exhibiting ANP-IR showed substance P-IR and that ANP was present as scattered immunoreactive granules in intracardial, presumably parasympathetic, ganglionic cells. The study shows that ANP is likely to be present in parasympathetic innervation and in afferent nerve endings in the bovine heart conduction system.
Subject(s)
Atrial Natriuretic Factor/metabolism , Autonomic Nervous System/metabolism , Heart Conduction System/metabolism , Substance P/metabolism , Animals , Atrioventricular Node/enzymology , Atrioventricular Node/metabolism , Autonomic Nervous System/enzymology , Cattle , Ganglia, Autonomic/cytology , Ganglia, Autonomic/enzymology , Heart Conduction System/enzymology , Immunohistochemistry , In Vitro Techniques , NADH Tetrazolium Reductase/metabolism , Nerve Fibers/enzymology , Neurons, Afferent/enzymology , Neurons, Afferent/physiology , Neuropeptides/metabolism , Sinoatrial Node/enzymology , Sinoatrial Node/metabolismABSTRACT
Effects of phorbol esters on spontaneously beating rabbit sino-atrial (SA) node cells were investigated by means of voltage clamp technique. In a small SA node specimen, 12-O-tetradecanoylphorbol-13-acetate (TPA) 10(-7) mol/l lengthened the cycle length (CL) and at over 3 X 10(-7) mol/l prolonged the action potential duration (APD). Action potential amplitude (APA), maximum diastolic potential (MDP) and maximum rate of rise (Vmax) were unaffected. Amiloride 10(-3) mol/l, an inhibitor of Na+-H+ exchange, did not reverse the phorbol ester-induced effects. In voltage-clamp experiments, TPA 1-10 X 10(-7) mol/l slightly increased the slow inward current (Isi) and the time-dependent inward current (Ih) which activates during hyperpolarization. The outward current and the tail current were reduced, although the activation curve was not shifted along the voltage axis. In the presence of 10(-7) mol/l isoprenaline, TPA produced dysrhythmia and a transient inward current in voltage-clamp experiments. In the presence of 5 X 10(-5) mol/l phenylephrine or 2 X 10(-6) mol/l acetylcholine, TPA also elicited dysrhythmia. 4-beta-phorbol-12,13-dibutyrate (PBD) induced similar electrophysiological effects as TPA, but 4-alpha-phorbol-12,13-didecanoate (PDD) never did so even in the presence of isoprenaline. These results suggest that TPA and PDB might mobilize intracellular Ca2+ via protein kinase C activation in the presence of isoprenaline, phenylephrine or acetylcholine, resulting in dysrhythmia due to delayed afterdepolarization.
Subject(s)
Phorbol Esters/pharmacology , Protein Kinase C/metabolism , Sinoatrial Node/enzymology , Acetylcholine/pharmacology , Action Potentials/drug effects , Animals , Female , In Vitro Techniques , Isoproterenol/pharmacology , Male , Membrane Potentials/drug effects , Phenylephrine/pharmacology , Rabbits , Sinoatrial Node/cytology , Time FactorsABSTRACT
Choline acetyltransferase activity, a biochemical indication of parasympathetic innervation, is increased in the hypertrophied right ventricle of guinea pigs after pulmonary artery constriction (PAC). The increase appears to be dependent on the severity and the duration of hypertrophy. This change in choline acetyltransferase activity suggests compensatory changes occur in the parasympathetic innervation of PAC guinea pigs which allows the right ventricle to maintain its level of parasympathetic innervation despite marked hypertrophy. Unlike the right ventricle, the SA node of PAC guinea pigs does not have detectable changes in choline acetyltransferase activity. This model of right ventricular hypertrophy also does not have detectable changes in baroreflex control of heart rate.
Subject(s)
Cardiomegaly/enzymology , Choline O-Acetyltransferase/metabolism , Myocardium/enzymology , Animals , Atrioventricular Node/enzymology , Constriction , Guinea Pigs , Heart Rate , Male , Pulmonary Artery , Sinoatrial Node/enzymologyABSTRACT
The activity of acetylcholinesterase (Ache) and butyrilcholinesterase (Bche) in the blood and the heart of 3 and 13-month old control male rats is considerably lower than in female rats. In old rats (25 months) no sex differences in the Ache and Bche were revealed in the heart. In 3 and 13-month old male and female rats, under conditions of muscular exercises, the Ache and Bche activity is lower, and in hypokinetic male rats--higher than that in respective control animals. In all the rats, irrespective of sex, age and motor conditions, the Ache and Bche activity tended to decrease, in direction from the sino-atrial node to the heart apex.
Subject(s)
Acetylcholinesterase/metabolism , Butyrylcholinesterase/metabolism , Cholinesterases/metabolism , Myocardium/enzymology , Physical Exertion , Acetylcholinesterase/blood , Age Factors , Animals , Atrioventricular Node/enzymology , Butyrylcholinesterase/blood , Female , Male , Rats , Restraint, Physical , Sex Factors , Sinoatrial Node/enzymologyABSTRACT
UNLABELLED: : Ca(2+)-activated basal adenylate cyclase (AC) in rabbit sinoatrial node cells (SANC) guarantees, via basal cAMP/PKA-calmodulin/CaMKII-dependent protein phosphorylation, the occurrence of rhythmic, sarcoplasmic-reticulum generated, sub-membrane Ca(2+) releases that prompt rhythmic, spontaneous action potentials (APs). This high-throughput signaling consumes ATP. AIMS: We have previously demonstrated that basal AC-cAMP/PKA signaling directly, and Ca(2+) indirectly, regulate mitochondrial ATP production. While, clearly, Ca(2+)-calmodulin-CaMKII activity regulates ATP consumption, whether it has a role in the control of ATP production is unknown. METHODS AND RESULTS: We superfused single, isolated rabbit SANC at 37°C with physiological saline containing CaMKII inhibitors, (KN-93 or autocamtide-2 Related Inhibitory Peptide (AIP)), or a calmodulin inhibitor (W-7) and measured cytosolic Ca(2+), flavoprotein fluorescence and spontaneous AP firing rate. We measured cAMP, ATP and O2 consumption in cell suspensions. Graded reductions in basal CaMKII activity by KN-93 (0.5-3 µmol/L) or AIP (2-10 µmol/L) markedly slow the kinetics of intracellular Ca(2+) cycling, decrease the spontaneous AP firing rate, decrease cAMP, and reduce O2 consumption and flavoprotein fluorescence. In this context of graded reductions in ATP demand, however, ATP also becomes depleted, indicating reduced ATP production. CONCLUSIONS: CaMKII signaling, a crucial element of normal automaticity in rabbit SANC, is also involved in SANC bioenergetics.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Calcium/metabolism , Energy Metabolism , Sinoatrial Node/cytology , Sinoatrial Node/enzymology , Action Potentials/drug effects , Adenosine Triphosphate/metabolism , Animals , Benzylamines/pharmacology , Calcium Signaling/drug effects , Calcium-Calmodulin-Dependent Protein Kinase Type 2/antagonists & inhibitors , Cyclic AMP/metabolism , Energy Metabolism/drug effects , Isoquinolines/pharmacology , Mitochondria/drug effects , Mitochondria/metabolism , Models, Biological , Protein Kinase Inhibitors/pharmacology , Rabbits , Sulfonamides/pharmacologyABSTRACT
Diabetes increases oxidant stress and doubles the risk of dying after myocardial infarction, but the mechanisms underlying increased mortality are unknown. Mice with streptozotocin-induced diabetes developed profound heart rate slowing and doubled mortality compared with controls after myocardial infarction. Oxidized Ca(2+)/calmodulin-dependent protein kinase II (ox-CaMKII) was significantly increased in pacemaker tissues from diabetic patients compared with that in nondiabetic patients after myocardial infarction. Streptozotocin-treated mice had increased pacemaker cell ox-CaMKII and apoptosis, which were further enhanced by myocardial infarction. We developed a knockin mouse model of oxidation-resistant CaMKIIδ (MM-VV), the isoform associated with cardiovascular disease. Streptozotocin-treated MM-VV mice and WT mice infused with MitoTEMPO, a mitochondrial targeted antioxidant, expressed significantly less ox-CaMKII, exhibited increased pacemaker cell survival, maintained normal heart rates, and were resistant to diabetes-attributable mortality after myocardial infarction. Our findings suggest that activation of a mitochondrial/ox-CaMKII pathway contributes to increased sudden death in diabetic patients after myocardial infarction.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Diabetes Mellitus, Experimental/enzymology , Myocardial Infarction/enzymology , Sinoatrial Node/enzymology , Animals , Apoptosis , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Cardiac Output , Cells, Cultured , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/mortality , Female , Fibrosis , Heart Rate , Humans , In Vitro Techniques , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mitochondria, Heart/metabolism , Myocardial Infarction/etiology , Myocardial Infarction/mortality , Myocardium/enzymology , Myocardium/pathology , Oxidation-Reduction , Oxidative Stress , Peptides/pharmacology , Reactive Oxygen Species/metabolism , Sinoatrial Node/pathology , Sinoatrial Node/physiopathologySubject(s)
3',5'-Cyclic-AMP Phosphodiesterases/metabolism , Sinoatrial Node/enzymology , Animals , Calcium/pharmacology , Cyclic AMP/metabolism , Cyclic GMP/metabolism , Enzyme Activation , Female , Heart Atria/enzymology , Heart Ventricles/enzymology , Kinetics , Macromolecular Substances , Male , RabbitsABSTRACT
The sympathetic nervous system increases heart rate by activating beta adrenergic receptors and increasing cAMP levels in myocytes in the sinoatrial node. The molecular basis for this response is not well understood; however, the cardiac funny current (I(f)) is thought to be among the end effectors for cAMP signaling in sinoatrial myocytes. I(f) is produced by hyperpolarization-activated cyclic nucleotide-sensitive (HCN4) channels, which can be potentiated by direct binding of cAMP to a conserved cyclic nucleotide binding domain in the C terminus of the channels. beta Adrenergic regulation of I(f) in the sinoatrial node is thought to occur via this direct binding mechanism, independent of phosphorylation. Here, we have investigated whether the cAMP-activated protein kinase (PKA) can also regulate sinoatrial HCN4 channels. We found that inhibition of PKA significantly reduced the ability of beta adrenergic agonists to shift the voltage dependence of I(f) in isolated sinoatrial myocytes from mice. PKA also shifted the voltage dependence of activation to more positive potentials for heterologously expressed HCN4 channels. In vitro phosphorylation assays and mass spectrometry revealed that PKA can directly phosphorylate at least 13 sites on HCN4, including at least three residues in the N terminus and at least 10 in the C terminus. Functional analysis of truncated and alanine-substituted HCN4 channels identified a PKA regulatory site in the distal C terminus of HCN4, which is required for PKA modulation of I(f). Collectively, these data show that native and expressed HCN4 channels can be regulated by PKA, and raise the possibility that this mechanism could contribute to sympathetic regulation of heart rate.