Acute Β-adrenergic Stimulation Research Articles

Hepato-renal oxidative disturbances following acute β-adrenergic stimulation by isoprenaline in male Wistar rat: Attenuative role of taurine, a β-amino acid

Hepato-renal oxidative disturbances following acute β-adrenergic stimulation by isoprenaline in male Wistar rat: Attenuative role of taurine, a β-amino acid

Live-cell photoactivated localization microscopy correlates nanoscale ryanodine receptor configuration to calcium sparks in cardiomyocytes

Ca2+ sparks constitute the fundamental units of Ca2+ release in cardiomyocytes. Here we investigate how ryanodine receptors (RyRs) collectively generate these events by employing a transgenic mouse with a photoactivated label on RyR2. This allowed correlative imaging of RyR localization, by super-resolution photoactivated localization microscopy, and Ca2+ sparks, by high-speed imaging. Two populations of Ca2+ sparks were observed: stationary events and ‘traveling’ events that spread between neighboring RyR clusters. Traveling sparks exhibited up to eight distinct releases, sourced from local or distal junctional sarcoplasmic reticulum. Quantitative analyses showed that sparks may be triggered by any number of RyRs within a cluster, and that acute β-adrenergic stimulation augments intracluster RyR recruitment to generate larger events. In contrast, RyR ‘dispersion’ during heart failure facilitates the generation of traveling sparks. Thus, RyRs cooperatively generate Ca2+ sparks in a complex, malleable fashion, and channel organization regulates the propensity for local propagation of Ca2+ release.

Mitochondrial ROS-Mediated Metabolic and Cytotoxic Effects of Isoproterenol on Cardiomyocytes Are p53-Dependent and Reversed by Curcumin.

Acute β-adrenergic stimulation contributes to heart failure. Here, we investigated the role of p53 in isoproterenol (ISO)-mediated metabolic and oxidative stress effects on cardiomyocytes and explored the direct protective effects offered by the antioxidant nutraceutical curcumin. Differentiated H9C2 rat cardiomyocytes treated with ISO were assayed for glucose uptake, lactate release, and mitochondrial reactive oxygen species (ROS) generation. Survival was assessed by sulforhodamine B assays. Cardiomyocytes showed significantly decreased glucose uptake and lactate release, as well as increased cellular toxicity by ISO treatment. This was accompanied by marked dose-dependent increases of mitochondria-derived ROS. Scavenging with N-acetyl-L-cysteine (NAC) effectively lowered ROS levels, which completely recovered glycolytic metabolism and survival suppressed by ISO. Mechanistically, ISO reduced extracellular-signal-regulated kinase (ERK) activation, whereas it upregulated p53 expression in an ROS-dependent manner. Silencing of p53 with siRNA blocked the ability of ISO to stimulate mitochondrial ROS and suppress glucose uptake, and partially recovered cell survival. Finally, curcumin completely reversed the metabolic and ROS-stimulating effects of ISO. Furthermore, curcumin improved survival of cardiomyocytes exposed to ISO. Thus, ISO suppresses cardiomyocyte glycolytic metabolism and survival by stimulating mitochondrial ROS in a p53-dependent manner. Furthermore, curcumin can efficiently rescue cardiomyocytes from these adverse effects.

新生児マウス心筋細胞におけるβアドレナリン受容体刺激による好気的解糖系の亢進

新生児マウス心筋細胞におけるβアドレナリン受容体刺激による好気的解糖系の亢進

Adaptive versus maladaptive cardiac remodelling in response to sustained β-adrenergic stimulation in a new 'ISO on/off model'.

On the one hand, sustained β-adrenergic stress is a hallmark of heart failure (HF) and exerts maladaptive cardiac remodelling. On the other hand, acute β-adrenergic stimulation maintains cardiac function under physiological stress. However, it is still incompletely understood to what extent the adaptive component of β-adrenergic signaling contributes to the maintenance of cardiac function during chronic β-adrenergic stress. We developed an experimental catecholamine-based protocol to distinguish adaptive from maladaptive effects. Mice were for 28 days infused with 30 mg/kg body weight/day isoproterenol (ISO) by subcutaneously implanted osmotic minipumps (‘ISO on’). In a second and third group, ISO infusion was stopped after 26 days and the mice were observed for additional two or seven days without further ISO infusion (‘ISO off short’, ‘ISO off long’). In this setup, ‘ISO on’ led to cardiac hypertrophy and slightly improved cardiac contractility. In stark contrast, ‘ISO off’ mice displayed progressive worsening of left ventricular ejection fraction that dropped down below 40%. While fetal and pathological gene expression (increase in Nppa, decrease in Myh6/Myh7 ratios, increase in Xirp2) was not induced in ‘ISO on’, it was activated in ‘ISO off’ mice. After ISO withdrawal, phosphorylation of phospholamban (PLN) at the protein kinase A (PKA) phosphorylation site Ser-16 dropped down to 20% as compared to only 50% at the Ca2+/Calmodulin-dependent kinase II (CaMKII) phosphorylation site Thr-17 in ‘ISO off’ mice. PKA-dependent cardioprotective production of the N-terminal proteolytic product of histone deacetylase 4 (HDAC4-NT) was reduced in ‘ISO off’ as compared to ‘ISO on’. Taken together, these data indicate that chronic ISO infusion induces besides maladaptive remodelling also adaptive PKA signalling to maintain cardiac function. The use of the ‘ISO on/off’ model will further enable the separation of the underlying adaptive from maladaptive components of β-adrenergic signalling and may help to better define and test therapeutic targets downstream of β-adrenergic receptors.

Differentiating the effects of β-adrenergic stimulation and stretch on calcium and force dynamics using a novel electromechanical cardiomyocyte model.

Cardiac electrophysiology and mechanics are strongly interconnected. Calcium is crucial in this complex interplay through its role in cellular electrophysiology and sarcomere contraction. We aim to differentiate the effects of acute β-adrenergic stimulation (β-ARS) and cardiomyocyte stretch (increased sarcomere length) on calcium-transient dynamics and force generation, using a novel computational model of cardiac electromechanics. We implemented a bidirectional coupling between the O'Hara-Rudy model of human ventricular electrophysiology and the MechChem model of sarcomere mechanics through the buffering of calcium by troponin. The coupled model was validated using experimental data from large mammals or human samples. Calcium transient and force were simulated for various degrees of β-ARS and initial sarcomere lengths. The model reproduced force-frequency, quick-release, and isotonic contraction experiments, validating the bidirectional electromechanical interactions. An increase in β-ARS increased the amplitudes of force (augmented inotropy) and calcium transient, and shortened both force and calcium-transient duration (lusitropy). An increase in sarcomere length increased force amplitude even more, but decreased calcium-transient amplitude and increased both force and calcium-transient duration. Finally, a gradient in relaxation along the thin filament may explain the nonmonotonic decay in cytosolic calcium observed with high tension. Using a novel coupled human electromechanical model, we identified differential effects of β-ARS and stretch on calcium and force. Stretch mostly contributed to increased force amplitude and β-ARS to the reduction of calcium and force duration. We showed that their combination, rather than individual contributions, is key to ensure force generation, rapid relaxation, and low diastolic calcium levels.NEW & NOTEWORTHY This work identifies the contribution of electrical and mechanical alterations to regulation of calcium and force under exercise-like conditions using a novel human electromechanical model integrating ventricular electrophysiology and sarcomere mechanics. By better understanding their individual and combined effects, this can uncover arrhythmogenic mechanisms in exercise-like situations. This publicly available model is a crucial step toward understanding the complex interplay between cardiac electrophysiology and mechanics to improve arrhythmia risk prediction and treatment.

S-nitrosylation of connexin43 hemichannels elicits cardiac stress-induced arrhythmias in Duchenne muscular dystrophy mice.

Patients with Duchenne muscular dystrophy (DMD) commonly present with severe ventricular arrhythmias that contribute to heart failure. Arrhythmias and lethality are also consistently observed in adult Dmdmdx mice, a mouse model of DMD, after acute β-adrenergic stimulation. These pathological features were previously linked to aberrant expression and remodeling of the cardiac gap junction protein connexin43 (Cx43). Here, we report that remodeled Cx43 protein forms Cx43 hemichannels in the lateral membrane of Dmdmdx cardiomyocytes and that the β-adrenergic agonist isoproterenol (Iso) aberrantly activates these hemichannels. Block of Cx43 hemichannels or a reduction in Cx43 levels (using Dmdmdx Cx43+/- mice) prevents the abnormal increase in membrane permeability, plasma membrane depolarization, and Iso-evoked electrical activity in these cells. Additionally, Iso treatment promotes nitric oxide (NO) production and S-nitrosylation of Cx43 hemichannels in Dmdmdx heart. Importantly, inhibition of NO production prevents arrhythmias evoked by Iso. We found that NO directly activates Cx43 hemichannels by S-nitrosylation of cysteine at position 271. Our results demonstrate that opening of remodeled and S-nitrosylated Cx43 hemichannels plays a key role in the development of arrhythmias in DMD mice and that these channels may serve as therapeutic targets to prevent fatal arrhythmias in patients with DMD .

Assessing structural and functional response of murine vasculature to acute β-adrenergic stimulation in vivo during hypothermic and hyperthermic conditions

Background: Because of the importance of adrenoreceptors in regulating the cardiovascular (CV) system and the role of the CV system in thermoregulation, understanding the response to these two stressors is of interest. The purpose of this study was to assess changes of arterial geometry and function in vivo during thermal and β-adrenergic stress induced in mice and quantified by MRI.Methods: Male mice were anesthetized and imaged at 7 T. Anatomical and functional data were acquired from the neck (carotid artery), torso (suprarenal and infrarenal aorta and iliac artery) and periphery (femoral artery). Intravenous dobutamine (tail vein catheter, 40 µg/kg/min, 0.12 mL/h) was used as β-adrenergic stressor. Baseline and dobutamine data were acquired at minimally hypothermic (35 °C) and minimally hyperthermic (38 °C) core temperatures. Cross-sectional vessel area and maximum cyclic strain were measured across the cardiac cycle.Results: Vascular response varied by location and by core temperature. For minimally hypothermic conditions (35 °C), average, maximum and minimum areas decreased with dobutamine only at the suprarenal aorta (avg: −17.9%, max: −13.5%, min: −21.4%). For minimally hyperthermic conditions (38 °C), vessel areas decreased between baseline and dobutamine at the carotid (avg: −19.6%, max: −15.5%, min: −19.3%) and suprarenal aorta (avg: −24.2%, max: −17.4%, min: −17.3%); whereas, only the minimum vessel area decreased for the iliac artery (min: −14.4%). Maximum cyclic strain increased between baseline and dobutamine at the iliac artery for both conditions and at the suprarenal aorta at hyperthermic conditions.Conclusions: At hypothermic conditions, the vessel area response to dobutamine is diminished compared to hyperthermic conditions where the vessel area response mimics normothermic dobutamine conditions. The varied response emphasizes the need to monitor and control body temperature during medical conditions or treatments that may be accompanied by hypothermia, especially when vasoactive agents are used.

Abstract 277: Enhanced Mitochondrial Calcium Uptake Promotes Deleterious Remodeling and Impaired Left Ventricular Function During Chronic Adrenergic Stimulation

The mitochondrial calcium uniporter (MCU) constitutes the pore-forming unit of the mitochondrial calcium uniporter channel (mtCU) and is necessary for mitochondrial Ca 2+ ( m Ca 2+ ) uptake. We previously reported that MCU is required to increase cardiac energetics and contractility in response to acute β-adrenergic stimulation. However, deletion of MCU has no detrimental effect on basal cardiac function or metabolism. While these findings support a role for MCU-dependent Ca 2+ flux in functional adaptations to acute stress, little is known about the involvement of MCU in cardiac responses to sustained stress. We compared mice with adult, cardiomyocyte (ACM)-specific loss ( Mcu fl/fl ; Mcu -cKO) or gain (CAG-CAG-MCU; MCU-Tg) of MCU function to explore the role of m Ca 2+ uptake in a model of chronic catecholamine overload. Biophysical characterization confirmed that MCU overexpression enhanced and MCU deletion inhibited ACM m Ca 2+ uptake. Neither loss nor gain of MCU had a significant impact on baseline contractile function. In αMHC-MCM control mice, fractional shortening was transiently increased after 2 days of isoproterenol infusion (Fig 1). This early increase in contractility was attenuated in Mcu -cKO mice. MCU-Tg mice exhibited decreased fractional shortening at 7 and 14 days of isoproterenol infusion. This detrimental effect on LV function was associated with increased LV dilation, HW/BW ratio, and lung edema as compared to αMHC-MCM controls. These data suggest that although mtCU-dependent m Ca 2+ uptake is essential for early energetic adaptations to high adrenergic load, under conditions of chronic adrenergic stress it is maladaptive and predisposes to heart failure.

Ca2+/calmodulin-dependent kinase II-dependent regulation of atrial myocyte late Na+ current, Ca2+ cycling, and excitability: a mathematical modeling study.

Atrial fibrillation (AF) affects more than three million people per year in the United States and is associated with high morbidity and mortality. Both electrical and structural remodeling contribute to AF, but the molecular pathways underlying AF pathogenesis are not well understood. Recently, a role for Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the regulation of persistent "late" Na+ current ( INa,L) has been identified. Although INa,L inhibition is emerging as a potential antiarrhythmic strategy in patients with AF, little is known about the mechanism linking INa,L to atrial arrhythmogenesis. A computational approach was used to test the hypothesis that increased CaMKII-activated INa,L in atrial myocytes disrupts Ca2+ homeostasis, promoting arrhythmogenic afterdepolarizations. Dynamic CaMKII activity and regulation of multiple downstream targets [ INa,L, L-type Ca2+ current, phospholamban, and the ryanodine receptor sarcoplasmic reticulum Ca2+-release channel (RyR2)] were incorporated into an existing well-validated computational model of the human atrial action potential. Model simulations showed that constitutive CaMKII-dependent phosphorylation of Nav1.5 and the subsequent increase in INa,L effectively disrupt intracellular atrial myocyte ion homeostasis and CaMKII signaling. Specifically, increased INa,L promotes intracellular Ca2+ overload via forward-mode Na+/Ca2+ exchange activity, which greatly increases RyR2 open probability beyond that observed for CaMKII-dependent phosphorylation of RyR2 alone. Increased INa,L promotes atrial myocyte repolarization defects (afterdepolarizations and alternans) in the setting of acute β-adrenergic stimulation. We anticipate that our modeling efforts will help identify new mechanisms for atrial NaV1.5 regulation with direct relevance for human AF. NEW & NOTEWORTHY Here, we present a novel computational model to study the effects of late Na+ current ( INa,L) in human atrial myocytes. Simulations predict that INa,L promotes intracellular accumulation of Ca2+, with subsequent dysregulation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling and ryanodine receptor 2-mediated Ca2+ release. Although INa,L plays a small role in regulating atrial myocyte excitability at baseline, CaMKII-dependent enhancement of the current promoted arrhythmogenic dynamics. Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/camkii-dependent-regulation-of-atrial-late-sodium-current-and-excitability/ .

Reduced Binding of the M-Domain of Cardiac Myosin Binding Protein C to Actin Impairs the Ability of the Heart to Respond to Chronic Stress

Cardiac myosin binding protein C (cMyBP-C) is a sarcomeric protein that regulates cross bridge cycling to control timing and strength of cardiac contraction. However, the molecular mechanisms by which cMyBP-C influences cross bridge cycling are incompletely understood. The M-domain of cMyBP-C can interact with actin and myosin, and phosphorylation of cMyBP-C in response to β-adrenergic stimulation during cardiac stress diminishes its affinity for both.In this study we determined the functional relevance of cMyBP-C binding to actin on cardiac function. Binding of cMyBP-C to actin can activate the thin filament and we hypothesize that dissociation of cMyBP-C from actin is essential for cardiac relaxation. To test our hypothesis, we created a transgenic mouse model with a mutation in the M-domain of cMyBP-C (E330K) that reduced binding affinity for actin in vitro. We then used echocardiography and pressure-volume recordings to assess cardiac function under sedentary and stress conditions.Both systolic function (e.g. stroke volume, ejection fraction and maximal rate of pressure development (+dP/dt)) and diastolic function (e.g. –dP/dt, isovolumetric relaxation time, E/A) were normal in E330K-Tg mice under sedentary conditions. Acute β-adrenergic stimulation with isoprenaline also led to normal increases in both contractility and relaxation in E330K-Tg mice. However, chronic cardiac stress by increasing preload to the heart by aortocaval fistula was fatal to all E330K-Tg mice within 10 days after surgery.Our results demonstrate that reduced interaction between the M-domain of cMyBP-C and actin did not impair cardiac function in E330K-Tg mice under sedentary conditions, but that this interaction may be essential for the heart to cope with chronic volume overload.This work is supported by NIH HL-080367 and AHA 15POST25700403.

The Role of Reactive Oxygen Species in β-Adrenergic Signaling in Cardiomyocytes from Mice with the Metabolic Syndrome.

The metabolic syndrome is associated with prolonged stress and hyperactivity of the sympathetic nervous system and afflicted subjects are prone to develop cardiovascular disease. Under normal conditions, the cardiomyocyte response to acute β-adrenergic stimulation partly depends on increased production of reactive oxygen species (ROS). Here we investigated the interplay between beta-adrenergic signaling, ROS and cardiac contractility using freshly isolated cardiomyocytes and whole hearts from two mouse models with the metabolic syndrome (high-fat diet and ob/ob mice). We hypothesized that cardiomyocytes of mice with the metabolic syndrome would experience excessive ROS levels that trigger cellular dysfunctions. Fluorescent dyes and confocal microscopy were used to assess mitochondrial ROS production, cellular Ca2+ handling and contractile function in freshly isolated adult cardiomyocytes. Immunofluorescence, western blot and enzyme assay were used to study protein biochemistry. Unexpectedly, our results point towards decreased cardiac ROS signaling in a stable, chronic phase of the metabolic syndrome because: β-adrenergic-induced increases in the amplitude of intracellular Ca2+ signals were insensitive to antioxidant treatment; mitochondrial ROS production showed decreased basal rate and smaller response to β-adrenergic stimulation. Moreover, control hearts and hearts with the metabolic syndrome showed similar basal levels of ROS-mediated protein modification, but only control hearts showed increases after β-adrenergic stimulation. In conclusion, in contrast to the situation in control hearts, the cardiomyocyte response to acute β-adrenergic stimulation does not involve increased mitochondrial ROS production in a stable, chronic phase of the metabolic syndrome. This can be seen as a beneficial adaptation to prevent excessive ROS levels.

Assessing structural and functional responses of murine hearts to acute and sustained β-adrenergic stimulation in vivo

IntroductionGiven the importance of β-adrenoceptor signalling in regulating cardiac structure and function, robust protocols are required to assess potential alterations in such regulation in murine models in vivo. MethodsEchocardiography was performed in naïve and stressed (isoprenaline; 30μg/g/day s.c. for up to 14days) mice, in the absence or presence of acute β-adrenergic stimulation (dobutamine 0.75μg/g, i.p.). Controls received saline infusion and/or injection. Hearts were additionally analysed gravimetrically, histologically and biochemically. ResultsIn naïve mice, acute β-adrenoceptor stimulation with dobutamine increased heart rate, left ventricular (LV) fractional shortening (LVFS), ejection fraction (LVEF) and wall thickness and decreased LV diameter (p<0.05). In stressed mice, dobutamine failed to induce further inotropic and chronotropic responses. Furthermore, following dobutamine injection, these mice exhibited lower LVEF and LVFS at identical heart rates, relative to corresponding controls. Sustained isoprenaline infusion induced LV hypertrophy (increased heart weight, heart weight/body weight ratio, heart weight/tibia length ratio and LV wall thickness (p<0.05)) by 3days, with little further change at 14days. In contrast, increases in LVEF and LVFS were seen only at 14days (p<0.05). DiscussionWe describe protocols for and illustrative data from the assessment of murine cardiac responses to acute and sustained β-adrenergic stimulation in vivo, which would be of value in determining the impact of genetic or pharmacological interventions on such responses. Additionally, our data indicate that acute dobutamine stimulation unmasks early signs of LV dysfunction in the remodelled heart, even at a stage when basal function is enhanced.

β‐Adrenergic stimulation increases the intra‐sarcoplasmic reticulum Ca2+ threshold for Ca2+ wave generation

β-Adrenergic signalling induces positive inotropic effects on the heart that associate with pro-arrhythmic spontaneous Ca(2+) waves. A threshold level of sarcoplasmic reticulum (SR) Ca(2+) ([Ca(2+)](SR)) is necessary to trigger Ca(2+) waves, and whether the increased incidence of Ca(2+) waves during β-adrenergic stimulation is due to an alteration in this threshold remains controversial. Using the low-affinity Ca(2+) indicator fluo-5N entrapped within the SR of rabbit ventricular myocytes, we addressed this controversy by directly monitoring [Ca(2+)](SR) and Ca(2+) waves during β-adrenergic stimulation. Electrical pacing in elevated extracellular Ca(2+) ([Ca(2+)](o) = 7 mM) was used to increase [Ca(2+)](SR) to the threshold where Ca(2+) waves were consistently observed. The β-adrenergic agonist isoproterenol (ISO; 1 μM) increased [Ca(2+)](SR) well above the control threshold and consistently triggered Ca(2+) waves. However, when [Ca(2+)](SR) was subsequently lowered in the presence of ISO (by lowering [Ca(2+)](o) to 1 mM and partially inhibiting sarcoplasmic/endoplasmic reticulum calcium ATPase with cyclopiazonic acid or thapsigargin), Ca(2+) waves ceased to occur at a [Ca(2+)](SR) that was higher than the control threshold. Furthermore, for a set [Ca(2+)](SR) level the refractoriness of wave occurrence (Ca(2+) wave latency) was prolonged during β-adrenergic stimulation, and was highly dependent on the extent that [Ca](SR) exceeded the wave threshold. These data show that acute β-adrenergic stimulation increases the [Ca(2+)](SR) threshold for Ca(2+) waves, and therefore the primary cause of Ca(2+) waves is the robust increase in [Ca(2+)](SR) above this higher threshold level. Elevation of the [Ca(2+)](SR) wave threshold and prolongation of wave latency represent potentially protective mechanisms against pro-arrhythmogenic Ca(2+) release during β-adrenergic stimulation.

Res-Erection of Viagra as a Heart Drug

In 1998, the world was formally introduced to Viagra (sildenafil), the first oral agent to be approved in the United States for the treatment of erectile dysfunction. Developed by Pfizer chemists, sildenafil inhibited phosphodiesterase type 5 (PDE5), one of the 11-member superfamily of enzymes that hydrolyze cyclic nucleotide monophosphates and among the first discovered that was selective for cGMP. PDE5 had been identified more than 2 decades earlier, first as a protein that bound cGMP and later as one that hydrolyzed it (the binding function turning out to be a mechanism to regulate activity).1 Sildenafil induces vasorelaxation by blocking PDE5-cGMP hydrolysis, raising cGMP levels in smooth muscle to activate protein kinase G (also known as cGK). In platelets, this blunts thrombosis; together, the effects suggested a potential use for coronary vascular disease and hypertension. Pfizer initiated trials targeting coronary ischemia, but, although antianginal effects were disappointing, the study gave a whole new meaning to the term “side effect,” effectively hijacking sildenafil from cardiovascular development to a rather different indication. Article see p 8 There were still some cardiovascular studies performed, but essentially all were limited assessments of volunteer populations, mostly normal and at rest and some with cardiovascular disease; the results were interpreted to support safety for patients taking the drug. In particular, blood pressure changes were slight if present at all, and cardiac function appeared unaltered. Well into the mid 2000s, PDE5 was thought to be expressed in selective vascular beds, lung and corpus cavernosum being dominant, and to have a minor cardiovascular profile elsewhere, with a negligible role in the heart. Sildenafil reemergence as …

The new HNO donor, 1-nitrosocyclohexyl acetate, increases contractile force in normal and β-adrenergically desensitized ventricular myocytes

Contractile dysfunction and diminished response to β-adrenergic agonists are characteristics for failing hearts. Chemically donated nitroxyl (HNO) improves contractility in failing hearts and thus may have therapeutic potential. Yet, there is a need for pharmacologically suitable donors. In this study we tested whether the pure and long acting HNO donor, 1-nitrosocyclohexyl acetate (NCA), affects contractile force in normal and pathological ventricular myocytes (VMs) as well as in isolated hearts. VMs were isolated from mice either subjected to isoprenaline-infusion (ISO; 30 μg/g per day) or to vehicle (0.9% NaCl) for 5 days. Sarcomere shortening and Ca 2+ transients were simultaneously measured using the IonOptix system. Force of contraction of isolated hearts was measured by a Langendorff-perfusion system. NCA increased peak sarcomere shortening by + 40–200% in a concentration-dependent manner (EC 50 ∼55 μM). Efficacy and potency did not differ between normal and chronic ISO VMs, despite the fact that the latter displayed a markedly diminished inotropic response to acute β-adrenergic stimulation with ISO (1 μM). NCA (60 μM) increased peak sarcomere shortening and Ca 2+ transient amplitude by ∼200% and ∼120%, respectively, suggesting effects on both myofilament Ca 2+ sensitivity and sarcoplasmic reticulum (SR) Ca 2+ cycling. Importantly, NCA did not affect diastolic Ca 2+ or SR Ca 2+ content, as assessed by rapid caffeine application. NCA (45 μM) increased force of contraction by 30% in isolated hearts. In conclusion, NCA increased contractile force in normal and β-adrenergically desensitized VMs as well as in isolated mouse hearts. This profile warrants further investigations of this HNO donor in the context of heart failure.

Acute β-adrenergic stimulation does not alter mitochondrial protein synthesis or markers of mitochondrial biogenesis in adult men

Exercise-induced expression of peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) is dramatically inhibited in mice pretreated with a beta-adrenergic receptor (beta-AR) antagonist, suggesting that beta-ARs play an important role in the regulation of skeletal muscle PGC-1alpha expression, and potentially, mitochondrial biogenesis. Accordingly, we hypothesized that acute beta-AR stimulation would induce transcriptional pathways involved in skeletal muscle mitochondrial biogenesis in humans. Whole body protein turnover (WBPT), myofibrillar protein synthesis (MyPS), skeletal muscle mitochondrial protein synthesis (MiPS), and mitochondrial biogenic signaling were determined in samples of vastus lateralis obtained on two separate occasions in 10 young adult males following 1 h of continuous intravenous administration of saline (CON) or a nonspecific beta-AR agonist [isoproterenol (ISO): 12 ng.kg fat free mass(-1).min(-1)], combined with coinfusion of [1,2](13)C-leucine. beta-AR stimulation induced appreciable increases in heart rate and systolic blood pressure (both P < 0.001) but did not affect mitochondrial biogenic signaling (no change in PGC-1alpha, TFAM, NRF-1, NRF-2, COX, or NADHox expression via RT-PCR; P > 0.05). Additionally, MiPS [CON: 0.099 +/- 0.028, ISO: 0.074 +/- 0.046 (mean +/- SD); P > 0.05] and MyPS (CON: 0.059 +/- 0.008, ISO: 0.055 +/- 0.009; P > 0.05), as well as measures of WBPT were unaffected. On the basis of this investigation, we conclude that acute intravenous beta-AR stimulation does not increase mitochondrial protein synthesis or biogenesis signals in skeletal muscle.

Is Phospholamban or Troponin I the “Prima Donna” in β-Adrenergic Induced Lusitropy?

See related articles, pages 377–386 In normal myocardium, acute β-adrenergic stimulation augments both systolic and diastolic performance via protein kinase A (PKA)–mediated phosphorylation of key proteins governing Ca2+ handling and the contractile machinery. Twenty-five years ago Kranias and Solaro identified troponin I (TnI) and phospholamban (PLN) as the 2 major cardiac proteins which were nearly simultaneously phosphorylated by β-adrenergic stimulation of the beating heart in synchrony with the agonist effect of augmenting contractility (inotropy) and rate of relaxation (lusitropy).1 Numerous subsequent studies have mechanistically examined the role of these proteins in inotropy and lusitropy. It is well established that enhanced Ca2+ availability during systole is the major, though perhaps not the only, driver of enhanced inotropy with β- adrenergic stimulation.2–4 The effect on Ca2+ dynamics is primarily mediated by phosphorylation of PLN, though increased ionic current through the L-type calcium channel contributes to Ca2+ loading. When PLN is phosphorylated by PKA, the “brake” imposed by PLN on sarcoplasmic reticulum (SR) Ca2+ ATPase is relieved, resulting in an increase in the activity of the latter that leads to a faster sequestration of Ca2+ into the SR, enhancing cardiac relaxation and re-loading the SR with Ca2+ to increase Ca2+ release in subsequent beats. Yet, PLN phosphorylation by PKA is not the sole mechanism implicated in enhancing relaxation. In particular, phosphorylation of TnI by PKA has long been proposed to have a role in diastole because it desensitizes the myofilament to Ca2+, increases the off rate of Ca …

Ontogeny of phosphoinositide 3-kinase signaling in developing heart: effect of acute β-adrenergic stimulation

Signaling pathways underlying transition of cardiomyocyte growth from hyperplasia in fetal/newborn to hypertrophy in postnatal/adult hearts are not well understood. We have shown that beta-adrenergic receptor (beta-AR)-mediated regulation of neonatal cardiomyocyte proliferation involves p70 ribosomal protein S6 kinase (p70S6K). Here we examined the ontogeny of phosphoinositide 3-kinase (PI3K)/p70S6K signaling pathway in rat hearts and investigated the influence of beta-AR on this pathway during development. Cardiac PI3K and p70S6K1 activities were high in the embryonic day 20 fetus, decreased gradually postnatally, and were low in the adult. In contrast, p70S6K2 was barely detectable. Phosphorylation of p70S6K1, Akt, and phosphoinositide-dependent protein kinase 1 were markedly increased in late gestation and early postnatal life but not in adult hearts. Phosphatase and tensin homolog on chromosome 10 (PTEN), a negative regulator of PI3K, was highly expressed in adult hearts but only at low levels and mostly in the phosphorylated (inactivated) form in the fetus. Beta-AR stimulation resulted in increased cardiac p70S6K1 activity only in animals > or = 2 wk old, whereas Akt level was increased in all developmental stages tested. These increases were accompanied by increased Bcl-2 associated death promoter (Ser136) phosphorylation without changes in PTEN level. Thus there is globally high input of cardiac PI3K signaling during the fetal-neonatal transition period. Inactivation of PTEN may in part contribute to the high activity of PI3K signaling, which coincides with the period of high cardiomyocyte proliferation. Beta-AR stimulation activates cardiac p70S6K1 and Akt in postnatal animals and may activate cardiac survival signals. These data provide further evidence for the importance of beta-AR and PI3K signaling in the regulation of cardiac growth during development.

Characterization of a Novel PKA Phosphorylation Site, Serine-2030, Reveals No PKA Hyperphosphorylation of the Cardiac Ryanodine Receptor in Canine Heart Failure

Hyperphosphorylation of the cardiac Ca2+ release channel (ryanodine receptor, RyR2) by protein kinase A (PKA) at serine-2808 has been proposed to be a key mechanism responsible for cardiac dysfunction in heart failure (HF). However, the sites of PKA phosphorylation in RyR2 and their phosphorylation status in HF are not well defined. Here we used various approaches to investigate the phosphorylation of RyR2 by PKA. Mutating serine-2808, which was thought to be the only PKA phosphorylation site in RyR2, did not abolish the phosphorylation of RyR2 by PKA. Two-dimensional phosphopeptide mapping revealed two major PKA phosphopeptides, one of which corresponded to the known serine-2808 site. Another, novel, PKA phosphorylation site, serine 2030, was identified by Edman sequencing. Using phospho-specific antibodies, we showed that the novel serine-2030 site was phosphorylated in rat cardiac myocytes stimulated with isoproterenol, but not in unstimulated cells, whereas serine-2808 was considerably phosphorylated before and after isoproterenol treatment. We further showed that serine-2030 was stoichiometrically phosphorylated by PKA, but not by CaMKII, and that mutations of serine-2030 altered neither the FKBP12.6-RyR2 interaction nor the Ca2+ dependence of [3H]ryanodine binding. Moreover, the levels of phosphorylation of RyR2 at serine-2030 and serine-2808 in both failing and non-failing canine hearts were similar. Together, our data indicate that serine-2030 is a major PKA phosphorylation site in RyR2 responding to acute beta-adrenergic stimulation, and that RyR2 is not hyperphosphorylated by PKA in canine HF.