Beta-adrenergic Stimulation Research Articles

Correcting Inter-domain Interaction of Cardiac Ryanodine Receptor Inhibits Aberrant Ca2+ Release in Cardiac Troponin T-related Familial Hypertrophic Cardiomyopathy Mouse Model

Cardiac troponin T (cTnT) mutations causes Familial Hypertrophic Cardiomyopathy (FHC), which leads to sudden cardiac death in young age. However, the underlying mechanism by which the cTnT mutations leads to lethal arrhythmia remains elusive. Here, we hypothesized that aberrant Ca2+ release could be altered arrhythmogenesis in FHC. To verify this hypothesis, we investigated the pathogenic role of aberrant Ca2+ release via the cardiac ryanodine receptor (RyR2) in transgenic mouse (TG) model with FHC-related cTnT mutation (δ160E). There was no appreciable structural difference of the hearts between wild-type (WT) mice and TG in 6th month of age. In response to isoproterenol (ISO; 10 nmol/L), the Ca2+ spark frequency was much higher in TG cardiomyocytes than in WT (TG (n=5): 7.8±0.9; WT (n=5): 3.6±0.4; p<0.01), whereas it was largely reversed by dantrolene (1μM), which was found to correct the defective inter-domain interaction in failing hearts (TG(n=5): 5.0±0.3; p<0.05). ISO-treated TG cardiomyocytes (but not ISO-treated WT: 0/14; n=14) showed spontaneous Ca2+ transient (sCaT) after 5 Hz pacing (8/13; n=13). The sCaT was again attenuated by dantrolene (4/14; n=14). In FHC-linked cTnT mutated hearts, aberrant local Ca2+ release through defective RyR2 induced by beta-adrenergic stimulation could be attenuated by correcting the inter-domain interaction of RyR2.

Heterogeneous response of J-wave syndromes to beta-adrenergic stimulation

Inferolateral early repolarization (ER) and Brugada syndrome manifest with J waves. Isoproterenol suppresses recurrent ventricular arrhythmias while reducing J waves in both disorders. To characterize the effect of isoproterenol on J waves. We analyzed the impact of isoproterenol on J waves in 20 patients with Brugada-type electrocardiogram (Br group) and 38 patients with ER (ER group). In the ER group, J waves were present in inferior leads in 32 patients (84%) and in lateral leads in 23 patients (61%). Isoproterenol increased the heart rate by 75 beats/min in the ER group and by 71 beats/min in the Br group (P = .20). The incidences of persistent (≤ 0.05-mV decrease), decreased, and normalized J waves (residual J wave ≤ 0.05 mV) were 20%, 80%, and 0% for Br group patients and 29%, 8%, and 63% for ER group patients, respectively (P <.001). Within the ER group, inferior J waves persisted in 34% of the cases, decreased in 9%, and normalized in 56% whereas lateral J waves always normalized (P <.001). Baseline QRS width was broader in ER group patients with persistent J waves (90 ms vs 80 ms; P = .003) and was unchanged with isoproterenol (90 ms; P = .19), whereas it decreased in the remaining patients (75 ms; P <.001). J-wave syndromes have distinct regional sensitivity to beta-adrenergic stimulation. J waves may persist in a subset of patients with right precordial and inferior J waves but never in lateral location. This heterogeneous response to isoproterenol may indicate distinctive mechanisms for Brugada and ER patterns, including depolarization abnormalities or ion channel sensitivity.

Abstract 227: Cardiomyocytes from Human Pluripotent Stem Cells Predict Chemotherapy-Induced Cardiotoxicity

The potential of human pluripotent stem cells-derived cardiomyocytes (hPSC-CM) to mimic human cardiac toxicity tests is largely unknown. Here we investigate the interaction between the chemotherapeutic agents Doxorubicin (DOX) and Lapatinib (LAP) and the protective effect of beta2 adrenergic signalling in hPSC-CM and murine myocytes. We hypothesized that death and survival pathways in hPSC-CM differ from those in animal-derived myocytes and thereby may predict better clinical outcome of chemotherapeutics. Human induced pluripotent or embryonic stem cell H7 cell lines were differentiated into cardiomyocytes (hiPSC-CM and hESC-CM, respectively), treated with drugs and stained with fluorescent dyes (Hoechst 33342, mitochondrial probes, effector caspases activation and impermeant nuclear dyes). Cell death profile was generated from high content screening and analysis (Cellomics). In isolated hPSC-CM, the anthracycline DOX induced apoptosis in a concentration-dependent manner (1-100µM), whereas LAP did not produce any toxicity. However, the combination of the two chemotherapeutic agents worsened the toxicity profile compared to DOX alone (p&lt;0.001, n=12), consistent with clinical observations. In contrast, rat neonates were sensitive to LAP and showed a different cell death profile in DOX and drug combination experiments. Activation of apoptotic caspases in hESC-CM clusters was coincident with the dissipation of the mitochondrial membrane potential, while the potential was preserved in isolated cells during this phase. Beta2 adrenergic stimulation did not reverse DOX pro-apoptotic effect but modified toxicity-related nuclear events. In conclusion, we have shown that hPSC-CM reproduce the clinical adverse cardiac effects after treatment with anti-cancer drugs, making these cells a more relevant system for future drug development than animal-derived cells. However, our data suggest that cell death pathways involved may depend on culture condition for cardiomyocytes.

Abstract 26: Myocardial Phosphodiesterase-2A Is Upregulated in Human and Experimental Heart Failure and Blunts Cardiac β-Adrenergic Inotropic Responsiveness

Augmented cGMP- and diminished cAMP-signaling within cardiomyocytes is characteristic for failing hearts. Cyclic nucleotide phosphodiesterases (PDEs) comprise a family of cyclic-nucleotide hydrolyzing enzymes, controlling cAMP and cGMP levels. Among them the PDE-2A isoform has the unique property to be stimulated by cGMP but primarily hydrolyzing cAMP. This appears to mediate a negative cross-talk between both signaling pathways. However, a potential role for PDE-2A in the failing heart has not been addressed yet. Here we show that PDE-2A protein levels were ∼2-fold higher in failing human hearts as well as in a large animal heart failure model from dog hearts subjected to rapid-pacing (n≥6, p&lt;0.05). Intriguingly, PDE-2A protein levels were normal in hypertrophied hearts from patients with preserved cardiac function who underwent aortic valve replacement. Chronic beta-adrenergic stimulation by catecholamine infusions enhanced cAMP hydrolyzing activity of PDE-2A by four-fold (n≥6, p&lt;0.05) in rat hearts in vivo and in isolated cardiomyocytes (measured by radioimmunoassay and FRET-based sensors, respectively) and correlated with blunted beta-adrenergic responsiveness. Consistent with this observation, overexpressed PDE-2A, which localized to the sarcomeric Z-line, blunted the rise in cAMP by 70% (n≥6, p&lt;0.05) and abolished the positive inotropic effect after acute beta-adrenergic stimulation by 70% (n≥6, p&lt;0.05) in isolated cardiomyocytes. Notably, those cardiomyocytes also showed marked protection from norepinephrine-induced hypertrophic responses, e. g. 40% less increase in cell surface area (n≥10, p&lt;0.05). In summary, PDE-2A is markedly upregulated in human and experimental failing hearts. This may constitute an important defense mechanism during cardiac stress, by antagonizing the cAMP-mediated toxic effects. Thus, activating myocardial PDE-2A may represent a new intracellular anti-adrenergic therapeutic strategy in heart failure.

Chromogranin-A: A Multifaceted Cardiovascular Role in Health and Disease

Chromogranin A (CgA), a major component of the chromaffin granules, is co-stored and co-released with catecholamines. It is also expressed in extra-adrenal sites, including the heart. In the rat, CgA localizes in atrial myoendocrine cells, associated with Atrial Natriuretic Peptide (ANP), and in the conduction system. In the human heart it is present in the ventricular myocardium, co-localized with B-type NP (BNP). CgA is the precursor of several biologically active peptides generated by proteolytic processing also in the heart. Two of them, vasostatin-1 (VS-1) and catestatin (Cst), inhibit cardiac contraction and relaxation, counter-regulate beta-adrenergic and endothelinergic stimulation, and protect the heart against ischemia/reperfusion damages. Recently, clinical studies have suggested CgA to be involved also in cardiovascular pathologies. High plasma CgA levels were found in hypertension, chronic and acute heart failure, myocardial infarction, decompensated and hypertrophic heart, and acute coronary syndromes. These alterations correlate with those of conventional cardiovascular biomarkers, such as NP and endothelin-1 (ET-1), and have prognostic relevance, being indicative of both severity of the disease and mortality. Accordingly, the current knowledge indicates CgA as a multifaceted peptide in cardiovascular homeostasis. Whether the influence elicited by the protein on both normal and failing heart is beneficial and/or detrimental, as well as its implication in the cardiac neuroendocrine scenario is under intense investigation. This review will focus on: i) the involvement of CgA and its derived peptides in the mechanisms which sustain cardiac function and compensation, ii) CgA clinical relevance, and iii) its putative value as a clinical biomarker.

H-89 decreases the gain of excitation–contraction coupling and attenuates calcium sparks in the absence of beta-adrenergic stimulation

This study used the selective protein kinase A (PKA) inhibitor H-89 (N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) to determine the role of basal PKA activity in modulating cardiac excitation–contraction coupling in the absence of β-adrenergic stimulation. Basal intracellular cyclic AMP (cAMP) levels measured in isolated murine ventricular myocytes with an enzyme immunoassay were increased upon adenylyl cyclase activation (forskolin; 1 and 10μM) or phosphodiesterase inhibition (3-isobutyl-1-methylxanthine, IBMX; 300μM). Forskolin and IBMX also caused concentration-dependent increases in peak Ca2+ transients (fura-2) and cell shortening (edge-detector) measured simultaneously in field-stimulated myocytes (37°C). Similar effects were seen upon application of dibutyryl cAMP. In voltage-clamped myocytes, H-89 (2μM) decreased basal Ca2+ transients, contractions and underlying Ca2+ currents. H-89 also decreased diastolic Ca2+ and the gain of excitation–contraction coupling (Ca2+ release/Ca2+ current), especially at negative membrane potentials. This was independent of alterations in sarcoplasmic reticulum (SR) Ca2+ loading, as SR stores were unchanged by PKA inhibition. H-89 also decreased the frequency, amplitude and width of spontaneous Ca2+ sparks measured in quiescent myocytes (loaded with fluo-4), but increased time-to-peak. Thus, H-89 suppressed SR Ca2+ release by decreasing Ca2+ current and by reducing the gain of excitation–contraction coupling, in part by decreasing the size of individual Ca2+ release units. These data suggest that basal PKA activity enhances SR Ca2+ release in the absence of ß-adrenergic stimulation. This may depress contractile function in models such as aging, where the cAMP/PKA pathway is altered due to low basal cAMP levels.

Myokardschädigung und Paroxysmale Ventrikuläre Tachykardie bei einem Hund nach Albuterolintoxikation

Intoxication with the beta2-agonist Albuterol may lead to immediate signs of beta-adrenergic stimulation like excitation, tachypnea and tachycardia. Furthermore, it typically causes severe hypokalemia, which then leads to muscle weakness and which predisposes to ventricular arrhythmias. We describe a dog where albuterol intoxication caused runs of fast paroxysmal ventricular tachycardia that persisted after normalization of the hypokalemia. Based on a markedly elevated serum troponin I level acute myocardial damage was identified as cause of the tachyarrhythmia. Repeated Troponin I measurements and Holter-ECGs were the means to document complete cure.

The C57BL/6 genetic background confers cardioprotection in iron-overloaded mice.

Chronic transfusion therapy causes a progressive iron overload that damages many organs including the heart. Recent evidence suggests that L-type calcium channels play an important role in iron uptake by cardiomyocytes under conditions of iron overload. Given that beta-adrenergic stimulation significantly enhances L-type calcium current, we hypothesised that beta-adrenergic blocking drugs could reduce the deleterious effects of iron overload on the heart. Iron overload was generated by intraperitoneal injections of iron dextran (1g/kg) administered once a week for 8 weeks in male C57bl/6 mice, while propranolol was administered in drinking water at the dose of 40 mg/kg/day. Cardiac function and ventricular remodelling were evaluated by echocardiography and histological methods. As compared to placebo, iron injection caused cardiac iron deposition. Surprisingly, despite iron overload, myocardial function and ventricular geometry in the iron-treated mice resulted unchanged as compared to those in the placebo-treated mice. Administration of propranolol increased cardiac performance in iron-overloaded mice. Specifically, as compared to the values in the iron-overloaded group, in iron-overloaded animals treated with propranolol left ventricular fractional shortening increased (from 31.6% to 44.2%, P =0.01) whereas left ventricular end-diastolic diameter decreased (from 4.1 ± 0.1 mm to 3.5 ± 0.1 mm, P =0.03). Propranolol did not alter cardiac systolic function or left ventricular sizes in the placebo group. These results demonstrate that C57bl/6 mice are resistant to iron overload-induced myocardial injury and that treatment with propranolol is able to increase cardiac performance in iron-overloaded mice. However, since C57bl/6 mice were resistant to iron-induced injury, it remains to be evaluated further whether propranolol could prevent iron-overload cardiomyopathy.

MioLab: Simulator for cardiac myocyte contractile force of rat based on the dynamics of calcium

BackgroundThe calcium ion (Ca2+) is essential in cardiac electrical activity since it is the direct activator of myofilaments, which cause contraction. For this reason, irregular Ca2+ flow in the cardiac myocyte is one of the main causes of cardiac arrhythmias and contractile dysfunction. In this sense, the formulation of mathematical and computational models of the mammalian ventricular myocyte has played an important role in understanding cardiac physiology. This paper proposes a biophysical model for the rat cardiac myocyte in order to reduce the number of degrees of freedom and provides a mathematical framework that makes computation simpler for our understanding of the complex process of excitation–contraction–relaxation (ECR), uses low-order lumped parameter of the myofilaments and Ca2+handling. The model allows the calculation of contractile force mainly based on the dynamics of Ca2+, provides data on transient Ca2+andenables the analysis of the effects of drugs used in the treatment of cardiac arrhythmias. MethodsThe mathematical model which describes the kinetics of cytosolic Ca2+ and dynamic cross-bridges, was implemented through nine differential equations, auxiliary equations, three 47 biophysical parameters. Each was implemented using C++ programming and its user-friendly interface in the programming language Delphi. The results of each simulation were stored in a text file and its analysis was shown in a graphical interface using executable programs in Matlab®. Additionally, the validation model was performed by comparing both the experimental data of Ca2+ transient and several drug tests. ResultsThe model satisfactorily reproduced the Ca2+ transient, as well as the effects of drugs that cause beta-adrenergic stimulation and the inhibition of the Ca2+ uptake mechanism (SERCA). Changes in the parameters regulating Ca2+ entry through L-type channels produced the oscillation amplitude of the Ca2+ transient known as the syndrome of pulses alternans. Changes in parameters related to the Na+–Ca2+ exchange has already stabilized the transient Ca2+ and produced a decrease in amplitude of the contractile force. ConclusionThe simulator proved to be a tool to study and understand the mechanisms that involve the kinetics of Ca2+ and the dynamics of cross-bridges in the unit heart muscle, as well as a tool to analyze the possible factors that may cause arrhythmias and study the effects of drugs that are used in the treatment of cardiovascular diseases in general.

Interleukin 6 is not necessary for STAT3 phosphorylation and myocardial hypertrophy following short term beta-adrenergic stimulation

In recent years several reports have suggested involvement of interleukin 6 (IL-6) in beta-adrenergic effects on myocardium, particularly in enhancement of STAT3 phosphorylation (downstream signal transducer of IL-6). Here we present a study of isoproterenol effects on hearts of IL-6 deficient mice. Male 12 week old C57Bl6/J mice and age and sex matched mice from IL-6 knockout strain (C57Bl6/J(IL6-/-)) received a single intraperitoneal bolus of either isoproterenol (15 mg/kg) or placebo (0.9% NaCl) and were sacrificed after 1 or 24 hours (n=8 in each group). Another group of mice from both genotypes received a three-day isoproterenol treatment (20 mg/kg every 8h). Activation of STAT3 and MEK/ERK pathways were assessed after a single dose of isoproterenol by means of western blotting. After injection of placebo a significantly lower level of STAT3 phosphorylation was observed in IL-6 KO animals. This difference was abolished after isoproterenol both at 1 and 24-hour time points. Isoproterenol produced potent and rapid activation of both STAT3 and MEK/ERK pathways that returned to the levels of placebo treated controls after 24 hours. Lack of IL-6 did not affect phosphorylation of ERKs. Three-day treatment with isoproterenol caused significant increase of indices of RV and LV hypertrophy in both WT and IL-6 KO animals with no significant differences between genotypes. IL-6 is not necessary for isoproterenol induced STAT3 phosphorylation, but may affect activation of this pathway by mild non-specific stimuli. Lack of IL-6 does not affect activation of MEK/ERK pathway nor cardiac hypertrophy by beta-adrenergic agonists.

Abstract 355: β-Adrenergic Stimulation Impact on Cerebrovascular Contractility Through Actin Cytoskeleton Disorganization in Rabbits

Beta-adrenoceptor stimulations have been well-known to initiate cardiac hypertrophy accompanied by extracellular remodeling that predisposes heart failure and cardiac dysarrhythmia. However, less is known concerning cerebrovascular dysfunction induced by beta-adrenergic stimulation. We investigated whether cerebrovascular functions and related biochemical signal pathways were altered during beta-adrenergic stimulations in rabbits. New Zealand white rabbits have been injected with isoproterenol (ISO, 0.3 mg/kg/day) for 7 days. We have studied the alteration of protein expression in cerebral arteries (CAs) using 2DE proteomics and western blot analysis and further analyzed those data using bioinformatics software. Reactive oxygen species generation and followed DNA damage were assessed deteriorative effect of ISO on CAs. Vasocontractility response to angiotensin II (Ang II) and related biochemical signal pathways were assessed to evaluate functional alteration of CAs. Proteomic analysis revealed remarkably decreased expression of cytoskeleton organizing proteins, (e.g. actin related protein 1A and 2, α-actin, capping protein Z beta, and vimentin) and anti-oxidative stress proteins (e.g. heat shock protein 9A and stress-induced-phosphoprotein 1) in ISO-stimulated CAs. As a cause of dysregulation of actin cytoskeleton organization, we found decreased level of RhoA/ROCK1, major regulators of actin cytoskeleton organization. As functional consequences of proteomic alteration, we found the decreased transient Ca 2+ efflux and contraction response to Ang II and high K + in ISO-stimulated CAs. We suggested that ISO stimulations reduced RhoA/ROCK activities and modified key actin based cytoskeletal and antioxidative proteins in CAs, which consequently lead to impair cerebrovascular contractility response to Ang II in rabbits.

Contractile Properties of Early Human Embryonic Stem Cell-Derived Cardiomyocytes: Beta-Adrenergic Stimulation Induces Positive Chronotropy and Lusitropy but Not Inotropy

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) provide the unique opportunity to study the very early development of the human heart. The aim of this study was to investigate the effect of calcium and beta-adrenergic stimulation on the contractile properties of early hESC-CMs. Beating clusters containing hESC-CMs were co-cultured in vitro with noncontractile slices of neonatal murine ventricles. After 5-7 days, when beating clusters had integrated morphologically into the damaged tissue, isometric force measurements were performed during spontaneous beating as well as during electrical field stimulation. Spontaneous beating stopped when extracellular calcium ([Ca²⁺](ec)) was removed or after administration of the Ca²⁺ channel blocker nifedipine. During field stimulation at a constant rate, the developed force increased with incremental concentrations of [Ca²⁺](ec). During spontaneous beating, rising [Ca²⁺](ec) increased beating rate and developed force up to a [Ca²⁺](ec) of 2.5 mM. When [Ca²⁺](ec) was increased further, spontaneous beating rate decreased, whereas the developed force continued to increase. The beta-adrenergic agonist isoproterenol induced a dose-dependent increase of the frequency of spontaneous beating; however, it did not significantly change the developed force during spontaneous contractions or during electrical stimulation at a constant rate. Force developed by early hESC-CMs depends on [Ca²⁺](ec) and on the L-type Ca²⁺ channel. The lack of an inotropic reaction despite a pronounced chronotropic response after beta-adrenergic stimulation most likely indicates immaturity of the sarcoplasmic reticulum. For cell-replacement strategies, further maturation of cardiac cells has to be achieved either in vitro before or in vivo after transplantation.

Critical role of cardiac t-tubule system for the maintenance of contractile function revealed by a 3D integrated model of cardiomyocytes

T-tubules in mammalian ventricular myocytes constitute an elaborate system for coupling membrane depolarization with intracellular Ca2+ signaling to control cardiac contraction. Deletion of t-tubules (detubulation) has been reported in heart diseases, although the complex nature of the cardiac excitation–contraction (E–C) coupling process makes it difficult to experimentally establish causal relationships between detubulation and cardiac dysfunction. Alternatively, numerical simulations incorporating the t-tubule system have been proposed to elucidate its functional role. However, the majority of models treat the subcellular spaces as lumped compartments, and are thus unable to dissect the impact of morphological changes in t-tubules. We developed a 3D finite element model of cardiomyocytes in which subcellular components including t-tubules, myofibrils, sarcoplasmic reticulum, and mitochondria were modeled and realistically arranged. Based on this framework, physiological E–C coupling was simulated by simultaneously solving the reaction-diffusion equation and the mechanical equilibrium for the mathematical models of electrophysiology and contraction distributed among these subcellular components. We then examined the effect of detubulation in this model by comparing with and without the t-tubule system. This model reproduced the Ca2+ transients and contraction observed in experimental studies, including the response to beta-adrenergic stimulation, and provided detailed information beyond the limits of experimental approaches. In particular, the analysis of sarcomere dynamics revealed that the asynchronous contraction caused by a large detubulated region can lead to impairment of myocyte contractile efficiency. These data clearly demonstrate the importance of the t-tubule system for the maintenance of contractile function.

Increased Activity of Mitochondrial Complex II in Rabbit Heart Failure is Associated with Reactive Oxygen Species Generation and Impaired Excitation-Contraction Coupling

Background: In heart failure (HF), the increase in cardiac adrenergic tone, while initially beneficial, ultimately contributes to damage to the failing heart. The aim of this study was to evaluate the mechanisms responsible for cell damage during beta-adrenergic stimulation in a rabbit volume- and pressure-overload HF model.Methods and Results: Field stimulation (1 Hz) of single left ventricular HF myocytes in combination with beta-adrenergic stimulation (isoproterenol, 1 microM) was accompanied by spontaneous pro-arrhythmic Ca2+ release (Ca2+ waves), contractile dysfunction, and a robust increase in reactive oxygen species (ROS) production, eventually leading to cell death. In HF myocytes FAD/FADH2 levels remained reduced and mitochondrial complex II (succinate dehydrogenase) activity was significantly elevated (by 86%). Increased complex II activity, however did not lead to an increase in ADP-dependent respiration, indicative of an electron leak at complex II. Mitochondrial complex I-mediated state-3 respiration was decreased by 77%, while state-2 respiration remained unchanged. Supplementation of HF myocytes with substrate for complex II (10 mM dimethyl-succinate) caused a dramatic increase in rotenone-sensitive mitochondrial ROS generation compared to control cells and to HF cells treated with complex I substrates. Moreover, dimethyl-succinate itself induced spontaneous Ca2+ release in form of Ca2+ waves that was further augmented by isoproterenol and prevented by cell pre-treatment with the anti-oxidant Trolox (1 mM). The complex II inhibitor thenoyl-trifluoroacetone (100 microM) significantly decreased mitochondrial ROS generation and normalized isoproterenol-induced Ca2+-transients and cell shortening.Conclusion: Increased activity of mitochondrial complex II in rabbit HF is a major mediator of oxidative stress leading to impairment of Ca2+ handling and contractility.

Palmitate Improves Redox Balance and Enhances Contractility While Offsetting Adverse Effects of Hyperglycemia in the Diabetic Cardiomyocyte

High blood glucose and fatty acid levels constitute main hallmarks of diabetes. In heart from diabetic animals, wide variations in plasmatic glucose combined with sympathetic hyperactivation represent a challenge to mitochondria that have to supply energy and maintain redox balance. Our previous data in db/db mice unveiled a significant redox imbalance in heart cells under combined hyperglycemia (HG) and beta adrenergic stimulation (via isoproterenol, ISO), leading to cardiomyocyte dysfunction. Herein, we address the role of palmitate (Palm) upon the adverse effects of HG+ISO on diabetic cardiomyocyte contractility and intracellular redox status. The ISO (10nM) response of WT vs db/db myocytes subjected to 5mM and 30mM glucose, in the absence and presence of 0.4mM (WT) and 0.8mM (db/db) Palm, was examined comparatively with respect to: i) fractional sarcomere shortening (FS) and Ca2+ transients, and ii) the intracellular status of NADH, reduced glutathione (GSH) and reactive oxygen species (ROS). Both WT and db/db myocytes exhibited a significant increase in ISO response with Palm under 5mM glucose: FS increased by 320% and 323% in WT and db/db, p<0.01 vs 193% and 178%, respectively, no Palm. Under HG, FS was similarly enhanced in WT with Palm (372% vs 181% with no Palm, p<0.01). Diabetic myocytes showed lower contractility in HG+ISO (94%, p<0.01 vs WT) with Palm enhancing the ISO response to 380%. Under every condition, Palm strongly decreased ROS (15- to 60-fold) and significantly increased GSH (20% to 40%) in both WT and db/db. NADH was significantly increased in WT (40% to 50%) but decreased with db/db (30% to 35%). These findings reveal that Palm enhances contractility of db/db myocytes via reduction of the intracellular redox environment, thus overcoming the adverse effects of HG under ISO stimulation.

Synchronization of Local Calcium Releases by Beta-Adrenergic Stimulation in Cardiac Pacemaker Cells

Sympathetic heart rate acceleration is a complex orchestration of intercellular, subcellular and molecular events. According to a modern concepts, rhythmic local Ca2+releases (LCRs), generated by a subcellular “Ca-clock”, interact with a “membrane-voltage-clock”, forming a coupled-clock system that produces rhythmic action potentials (AP). We imaged LCRs using a fast-2D-camera with simultaneous AP recording via perforated-patch-clamping to explore how β-AR stimulation modulates the phase and synchronization of the ensemble of individual LCRs within a given diastole to accelerate spontaneous beating rate. In single rabbit SA-node cells (36oC) isoproterenol (100nM): (i) synchronized LCR occurrence [the standard deviation of individual LCR periods (time from Ca2+transient peak to LCR occurrence) decreased (−30%; baseline,71.5ms vs. ISO,50ms)], (ii) shifted the LCR phases to earlier time-point, (iii) shifted “Ca-nadir”, i.e. time-point when AP-induced Ca2+-transient decay ceases and [Ca2+] begins to increase due to the occurrence of LCRs (time from maximum-diastolic-potential to Ca-nadir: baseline, 103ms vs. ISO,86ms); (iv) accelerated diastolic depolarization (baseline,0.09mV/ms vs. ISO,0.15mV/ms); (v) shortened spontaneous cycle length (baseline,420ms vs. ISO,345ms). We previously had demonstrated a close relationship between the decay of the global AP-induced Ca2+ transient and the LCR phase (within a given cycle) and the AP cycle length; we now found that the decays of each LOCAL Ca2+Transient (LCaT) induced by the prior AP vary in different subcellular locations/neighborhoods and that isoproterenol not only accelerated LCaT-decays (average time constant τ: baseline, 104 vs. ISO,87ms) but also synchronized LCaT-decays among neighborhoods (τ's SD/mean: baseline,40.5% vs. ISO,33.7%). More synchronous LCaT-decay mirrors more synchronous local SR Ca2+-pumping and therefore more synchronous achievement of the threshold SR Ca load required for spontaneous LCR occurrence. Thus, earlier and more synchronized LCR occurrence under β-AR stimulation insures an earlier and larger Na+/Ca2+-exchanger current that ultimately accelerates diastolic depolarization rate.

Individual Cell Electrophysiology (ICE) of Cardiac Myocytes _ Using Innovative ‘Onion-Peeling’ Technique to Study the ICE of Excitable Cells

The action potential (AP) of a cardiac cell is a finely choreographed dance involving many ion channels and transporters interacting with one another via membrane potential and intracellular Ca2+. The ionic mechanisms that cause arrhythmogenic APs (early or delayed afterdepolarizations, short or long AP duration) are still incompletely understood. Traditional voltage-clamp experiments measured one current from one cell and in most cases without Ca2+ transients. Data averaged from many cells were then used to build a canonical AP model. However, the canonical AP may be very different from the AP of any particular cell due to cell-to-cell variations. Recently, we developed an innovative ‘Onion-Peeling’ (O-P) technique to achieve (a) a direct recording of ionic current under in situ condition (during AP, with Ca2+ cycling, in a physiological milieu, undergoing contraction) and (b) recording of multiple currents from a single cell to study how different currents interact and integrate to shape AP. Here we report the results of using O-P to investigate: (1) recording of key ionic currents (IKs, IKr, IK1, ICa,L, INCX, INa-Late) during AP in the guinea pig ventricular myocyte; (2) effects of the Ca2+ transient on modulating these currents during AP; (3) changes of multiple currents under beta-adrenergic stimulation and how concerted changes in currents integrate to reshape AP. The ability to measure many currents in a single cell has also revealed two hitherto unknown characteristics of the currents in cardiac cells: coordination of currents within a cell and large variations of currents between cells. Hence, our O-P technique presents a significant step beyond the traditional way of measuring only one current in any one cell, and provides a powerful new tool for studying the individual cell electrophysiology.

Cytosolic Calcium Wave Propagation Depends on Local Calcium Movement Inside Cardiac Sarcoplasmic Reticulum

The propagation of cytosolic Ca waves in cardiac myocytes is thought to occur through Ca-induced Ca release (CICR) with Ca being released from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyR) activating neighboring RyRs and propagating throughout the cell. This widely accepted paradigm has recently been challenged by the hypothesis that an intra-SR ‘sensitization’ Ca wave precedes the cytosolic Ca wave and primes RyRs for activation by CICR (Keller et al., Cardiovasc. Res., 2007). Here we tested the intra-SR sensitization wave hypothesis by direct simultaneous measurements of cytosolic ([Ca]i) and intra-SR ([Ca]SR) calcium signals during wave propagation in intact and permeabilized rabbit ventricular myocytes. [Ca]i and [Ca]SR were measured with the fluorescent probes rhod-2 and fluo-5N, respectively, using high-speed confocal imaging. Our data show that the cytosolic Ca wave front and the corresponding intra-SR Ca depletion wave is preceded by a transient elevation of [Ca]SR. This transient elevation of [Ca]SR preceded the cytosolic wave front along the path of wave propagation and could be identified at individual release junctions with high-resolution imaging. Colliding waves that originated at separate subcellular locations annihilated as expected, however the preceding elevation of [Ca]SR of each wave resulted in a further increase of [Ca]SR at the site of collision. Increasing SR Ca load (beta-adrenergic stimulation) and SR Ca buffer capacity (with the exogenous Ca buffer ADA) enhanced Ca wave amplitude and [Ca]SR elevation preceding the wave. Furthermore, the cytosolic Ca wave front was preceded by a small transient decrease of [Ca]i that coincided with the transient elevation of [Ca]SR and possibly indicates the involvement of SERCA activity. These data suggest that local Ca movement inside the SR is a prerequisite for the propagation of spontaneous Ca waves in cardiac myocytes.

Human Signaling Scaffold Protein (mAKAP) Binding Kinetics to PKA and Phosphodiesterase (PDE4DE): Implications for a Possible Role in Heart Failure

Heart failure is a leading cause of morbidity and mortality in the USA. There are several therapeutic agents available for heart failure management. In particular, agents that block beta-adrenergic receptor improve mortality rate among heart failure patients by enhancing cardiac function. Beta-adrenergic receptor stimulation signals through protein kinase A (PKA) dependent phosphorylation, partly by binding to A-kinase anchoring proteins, influencing calcium homeostasis. In particular, mAKAP (muscle-selective A-kinase anchoring protein) is targeted to specific intracellular compartments resulting in localization of PKA with its substrates as well as to bind with ryanodine receptors and phosphodiesterase-4D3 (PDE4DE). The signal transduction complex formed by the scaffold protein mAKAP at the perinuclear envelop of striated myocytes contains cAMP specific binding protein PDE4D3 which is responsible for cAMP signaling termination. Agents that modify PKA signaling would be expected to mediate an altered inotropic response. From different genomic databases, we have recently identified fifteen human mAKAP coding non-synonymous polymorphisms located within or near key protein binding sites critical to beta-adrenergic receptors signaling. Seven of these mutants were cloned for the purpose of comparing whether those substitution disrupt mAKAP binding to either the PKA binding domain R2alpha or the phosphodiesterase PDE4DE. Using surface plasmon resonance (Biacore 2000) we demonstrate specific binding of wild type mAKAP to PDE4DE. Experiments were run in triplicate and as twofold serial dilutions to explore the kinetics of the interaction and analyzed using Scrubber2 with a 1:1 Langmuir model. Comparative analysis of the binding responses of mutations to mAKAP could provide important information about how these mutations modulate signaling.

Mitochondrial ROS Production Contributes to Generation of Ca Waves During Beta-Adrenergic Receptor Stimulation in Rabbit Ventricular Myocytes

Beta-adrenergic receptor (beta-AR) activation leads to positive inotropic effect but can also increase diastolic sarcoplasmic reticulum (SR) Ca release in the form of pro-arrhythmic Ca waves. Beta-AR activation increases cellular energy demand and mitochondrial reactive oxygen species (ROS) production. We investigated the role of mitochondrial ROS in the generation of Ca waves during beta-AR stimulation. In electrically stimulated myocytes, application of ISO (0.1 microM) led to the occurrence Ca waves during diastole. The frequency of Ca waves increased over time during ISO application, with a particular steep increase after 6 minutes. Frequency of ISO-mediated Ca waves positively correlated with mitochondrial ROS production measured with Mito-SOX (a dye specifically targeted to mitochondria). The mitochondria specific antioxidant Mito-Tempo (5 microM) effectively prevented the ISO-mediated ROS production, as well as ISO-mediated Ca waves. Inhibition of the mitochondrial respiration with rotenone (0.3 microM) produced similar preventive effects on mitochondrial ROS production and Ca wave generation. Measurements of intra-SR free Ca ([Ca]SR) showed an initial increase of [Ca]SR (SR Ca load) followed by a gradual decline over time during ISO application. This decline of [Ca]SR was the result of increased SR Ca leak particularly in the form of Ca waves. Mito-Tempo did not affect the initial increase in SR Ca load, however, the ROS scavenger significantly prevented the [Ca]SR decline in the presence of ISO. Furthermore, Mito-Tempo significantly reduced ISO-mediated augmentation of SR Ca leak. These results suggest that mitochondrial ROS production play an important role in the generation of Ca waves during beta-AR stimulation.