Β-adrenergic Receptor Stimulation Research Articles

Quantitative Cross-Species Prediction of β-Adrenergic Response in Ventricular Myocytes

Despite the large body of research on neural control of the heart, our quantitative understanding of the role of β-adrenergic receptor (β-AR) stimulation in human cardiac electrophysiology and arrhythmogenesis remains poor. One possible reason is in the fact that our knowledge is mostly based on experimental animal models (e.g., mouse and rabbit) characterized by quite different cellular electrophysiology. Indeed, we have shown that well conserved mammalian responses to β-AR stimulation (fight or flight) are mediated by different sub-cellular processes in mouse vs.

Interplay Between β-Adrenergic Stimulation and CaMKII Signaling Favors Human Atrial Arrhythmogenesis: Insights from Populations of Models

Excessive β-adrenergic receptor (βAR) stimulation and hyperactivation of the multifunctional Ca2+-calmodulin-dependent protein kinase II (CaMKII) play important roles in the initiation and maintenance of atrial fibrillation (AF), the world's most common cardiac arrhythmia. It has been postulated that the interplay of these two pathways with intracellular Ca2+ signaling drives a vicious cycle that promotes arrhythmogenesis. Here, we coupled our well-established computational model of human atrial cellular electrophysiology (extended to incorporate two recently-characterized atrial-predominant K+ currents) and Ca2+ handling (Grandi et al., 2011) with detailed descriptions of βAR/cAMP/PKA and CaMKII pathways (Soltis and Saucerman 2010; Morotti et al., 2014) to investigate their respective roles and potential synergy in promoting AF.

Improved workflow for mass spectrometry–based metabolomics analysis of the heart

MS-based metabolomics methods are powerful techniques to map the complex and interconnected metabolic pathways of the heart; however, normalization of metabolite abundance to sample input in heart tissues remains a technical challenge. Herein, we describe an improved GC-MS-based metabolomics workflow that uses insoluble protein-derived glutamate for the normalization of metabolites within each sample and includes normalization to protein-derived amino acids to reduce biological variation and detect small metabolic changes. Moreover, glycogen is measured within the metabolomics workflow. We applied this workflow to study heart metabolism by first comparing two different methods of heart removal: the Langendorff heart method (reverse aortic perfusion) and in situ freezing of mouse heart with a modified tissue freeze-clamp approach. We then used the in situ freezing method to study the effects of acute β-adrenergic receptor stimulation (through isoproterenol (ISO) treatment) on heart metabolism. Using our workflow and within minutes, ISO reduced the levels of metabolites involved in glycogen metabolism, glycolysis, and the Krebs cycle, but the levels of pentose phosphate pathway metabolites and of many free amino acids remained unchanged. This observation was coupled to a 6-fold increase in phosphorylated adenosine nucleotide abundance. These results support the notion that ISO acutely accelerates oxidative metabolism of glucose to meet the ATP demand required to support increased heart rate and cardiac output. In summary, our MS-based metabolomics workflow enables improved quantification of cardiac metabolites and may also be compatible with other methods such as LC or capillary electrophoresis.

Obesity Affects β2 Adrenergic Regulation of the Inflammatory Profile and Phenotype of Circulating Monocytes from Exercised Animals.

Anomalous immune/inflammatory responses in obesity take place along with alterations in the neuroendocrine responses and dysregulation in the immune/stress feedback mechanisms. Exercise is a potential anti-inflammatory strategy in this context, but the influence of exercise on the β2 adrenergic regulation of the monocyte-mediated inflammatory response in obesity remains completely unknown. The first objective of this study was to analyze the effect of exercise on the inflammatory profile and phenotype of monocytes from obese and lean animals, and the second aim was to determine whether obesity could affect monocytes’ inflammatory response to β2 adrenergic activation in exercised animals. C57BL/6J mice were allocated to different lean or obese groups: sedentary, with acute exercise, or with regular exercise. The inflammatory profile and phenotype of their circulating monocytes were evaluated by flow cytometry in the presence or absence of the selective β2 adrenergic receptor agonist terbutaline. Exercise caused an anti-inflammatory effect in obese individuals and a pro-inflammatory effect in lean individuals. β2 adrenergic receptor stimulation exerted a global pro-inflammatory effect in monocytes from exercised obese animals and an anti-inflammatory effect in monocytes from exercised lean animals. Thus, β2 adrenergic regulation of inflammation in monocytes from exercised animals seems to depend on the inflammatory basal set-point.

Noradrenergic signaling in the wakeful state inhibits microglial surveillance and synaptic plasticity in the mouse visual cortex.

Microglia are the brain’s resident innate immune cells and also have a role in synaptic plasticity. Microglial processes continuously survey the brain parenchyma, interact with synaptic elements and maintain tissue homeostasis. However, the mechanisms that control surveillance and its role in synaptic plasticity are poorly understood. Microglial dynamics in vivo have been primarily studied in anesthetized animals. Here we report that microglial surveillance and injury response are reduced in awake mice compared to anesthetized mice, suggesting that arousal state modulates microglial function. Pharmacological stimulation of β2-adrenergic receptors recapitulated these observations and disrupted experience-dependent plasticity, and these effects required the presence of β2-adrenergic receptors in microglia. These results indicate that microglial roles in surveillance and synaptic plasticity in the mouse brain are modulated by noradrenergic tone fluctuations between arousal states and emphasize the need to understand the effect of disruptions of adrenergic signaling in neurodevelopment and neuropathology.

Artificial taste avoidance memory induced by coactivation of NMDA and β-adrenergic receptors in the amygdala

The association between a taste and gastric malaise allows animals to avoid the ingestion of potentially toxic food. This association has been termed conditioned taste aversion (CTA) and relies on the activity of key brain structures such as the amygdala and the insular cortex.The establishment of this gustatory-avoidance memory is related to glutamatergic and noradrenergic activity within the amygdala during two crucial events: gastric malaise (unconditioned stimulus, US) and the post-acquisition spontaneous activity related to the association of both stimuli. To understand the functional implications of these neurochemical changes on avoidance memory formation, we assessed the effects of pharmacological stimulation of β-adrenergic and glutamatergic NMDA receptors through the administration of a mixture of L-homocysteic acid and isoproterenol into the amygdala after saccharin exposure on specific times to emulate the US and post-acquisition local signals that would be occurring naturally under CTA training. Our results show that activation of NMDA and β-adrenergic receptors generated a long-term avoidance response to saccharin, like a naturally induced rejection with LiCl. Moreover, the behavioral outcome was accompanied by changes in glutamate, norepinephrine and dopamine levels within the insular cortex, analogous to those displayed during memory retrieval of taste aversion memory. Therefore, we suggest that taste avoidance memory can be induced artificially through the emulation of specific amygdalar neurochemical signals, promoting changes in the amygdala-insular cortex circuit enabling memory establishment.

Effects of adrenaline on contractility and endurance of isolated mammalian soleus with different calcium concentrations.

The β-adrenergic receptor stimulation improves endurance in fast twitch muscles and these effects are sensitive to extracellular Ca2+ influx. Present study is aimed to determine the effects of adrenaline, with different concentrations of extracellular Ca2+[Formula: see text], on the contractility and endurance of slow twitch muscles during high frequency stimulations (HFS). Isolated soleus of rabbit was electrically stimulated (strength; 50Hz, duration; 0.5ms) in the presence (Test) of adrenaline (1 × 10-7mM) or without adrenaline (CTL). Fatigue was induced with HFS (80Hz) for the duration of 20s. Contractions were recorded through isometric transducer connected with Powerlab. Kreb's buffer was used with three compositions: standard with 2.5mM Ca2+ (Ca-S), Ca2+ free buffer (Ca-F) and buffer with raised Ca2+ i.e., 10mM (Ca-R). Muscles endurance was assessed by measuring the decline in tetanic tension in the terms of percentage (%Pmax) and rate of decline in tetanic tension (dP/dt). During 20s, %Pmax showed reduction of only 10% in Ca-S. This decline was enhanced in Ca-F (50%) and reduced in Ca-R (6%). Effect of adrenaline was observed only in Ca-F where %Pmax was about 20% greater in Test than CTL. These effects were not observed in both Ca-S and Ca-R during 20s. However, when duration of stimulation was increased to 120 or 150s in Ca-S and Ca-R respectively, decline in %Pmax was less in Test as compared to CTL. Thus, [Formula: see text] plays protective role against fatigue during continuous HFS in slow twitch muscles. In addition, adrenaline improves the muscles endurance during fatiguing contraction but these effects are not mediated through [Formula: see text] influx.

Golgi localized β1-adrenergic receptors stimulate Golgi PI4P hydrolysis by PLCε to regulate cardiac hypertrophy.

Increased adrenergic tone resulting from cardiovascular stress leads to development of heart failure, in part, through chronic stimulation of β1 adrenergic receptors (βARs) on cardiac myocytes. Blocking these receptors is part of the basis for β-blocker therapy for heart failure. Recent data demonstrate that G protein-coupled receptors (GPCRs), including βARs, are activated intracellularly, although the biological significance is unclear. Here we investigated the functional role of Golgi βARs in rat cardiac myocytes and found they activate Golgi localized, prohypertrophic, phosphoinositide hydrolysis, that is not accessed by cell surface βAR stimulation. This pathway is accessed by the physiological neurotransmitter norepinephrine (NE) via an Oct3 organic cation transporter. Blockade of Oct3 or specific blockade of Golgi resident β1ARs prevents NE dependent cardiac myocyte hypertrophy. This clearly defines a pathway activated by internal GPCRs in a biologically relevant cell type and has implications for development of more efficacious β-blocker therapies.

Abstract 123: Osteopontin Regulates Adult Cardiomyocyte Division in a Mouse Model of Pressure Overload Induced Heart Failure

Background: Our previous work showed that pharmacological blockade of Osteopontin (OPN) signaling can prevent and reverse heart failure induced by pressure overload in a transverse aortic construction (TAC) mouse model. Surprisingly, OPN Knockout (KO) mice subjected to 3 month TAC had worse cardiac function and bigger hearts than wild type (WT) TAC mice, despite lack of cardiomyocyte hypertrophy. We hypothesized that OPN KO increased adult cardiomyocyte proliferation in TAC-induced heart failure. Methods: Male C57Bl/6 (n=17) or OPN KO (n=11) mice were subject to TAC for 3 months. The protein levels of the mitosis marker H3P was quantified using immunofluorescence in paraffin-embedded myocardial sections. Myocytes were co-stained with WGA and MLC2 to count the number of myocytes per field. Adult primary cardiomyocytes from WT hearts were isolated and analyzed with co-imunoprecipitation (Co-IP) to study the interation between the regeneration factor YAP1, OPN and OPN-regulated proteins such as LDLR. For validation and mechanistic studies, more Co-IP experiments were performed in proliferative human liver HEPG2 cells. To study the effect of OPN blockade on YAP1 nuclear translocation, HEPG2 cells and human iPSC- derived cardiomyocytes (hiPS-CMs) were treated with an IgG or OPN blocking antibody for 24 hours followed by immunostaining for YAP1 and PITX2. Results: Nuclear H3P normalized to myocyte count was significantly increased in OPN KO relative to WT TAC hearts (Fold Change = 1.4; p=0,04). Co-IP results revealed a novel interaction between OPN, LDLR and YAP1. Stimulation of β2 adrenergic receptor increased the formation of this multi-molecular complex in a time-dependent manner. Blockade of OPN by a monocolonal antibody for 24 hours caused nuclear localization of YAP1 and PITX2 in HEPG2 cells and hiPS-CMs. Conclusion: OPN regulates the mitotic program in adult cardiomyocytes. Furthermore, the interaction of OPN with LDLR and YAP1 to form a new multi-molecular protein complex is regulated by β2-cAMP signaling pathway. Importantly, OPN regulates nuclear translocation of the regeneration factors YAP1 and PITX2, suggesting that OPN signaling may be important for adult cardiomyocyte division in TAC and potentially myocardial infarction and aging.

A Receptor of the Immunoglobulin Superfamily Regulates Adaptive Thermogenesis

SUMMARYExquisite regulation of energy homeostasis protects from nutrient deprivation but causes metabolic dysfunction upon nutrient excess. In human and murine adipose tissue, the accumulation of ligands of the receptor for advanced glycation end products (RAGE) accompanies obesity, implicating this receptor in energy metabolism. Here, we demonstrate that mice bearing global- or adipocyte-specific deletion of Ager, the gene encoding RAGE, display superior metabolic recovery after fasting, a cold challenge, or high-fat feeding. The RAGE-dependent mechanisms were traced to suppression of protein kinase A (PKA)-mediated phosphorylation of its key targets, hormone-sensitive lipase and p38 mitogen-activated protein kinase, upon β-adrenergic receptor stimulation—processes that dampen the expression and activity of uncoupling protein 1 (UCP1) and thermogenic programs. This work identifies the innate role of RAGE as a key node in the immunometabolic networks that control responses to nutrient supply and cold challenges, and it unveils opportunities to harness energy expenditure in environmental and metabolic stress.

Overexpression of a Neuronal Type Adenylyl Cyclase (Type 8) in Sinoatrial Node Markedly Impacts Heart Rate and Rhythm

Heart rate (HR) and HR variability (HRV), predictors of over-all organism health, are widely believed to be driven by autonomic input to the sinoatrial node (SAN), with sympathetic input increasing HR and reducing HRV. However, variability in spontaneous beating intervals in isolated SAN tissue and single SAN cells, devoid of autonomic neural input, suggests that clocks intrinsic to SAN cells may also contribute to HR and HRV in vivo. We assessed contributions of both intrinsic and autonomic neuronal input mechanisms of SAN cell function on HR and HRV via in vivo, telemetric EKG recordings. This was done in both wild type (WT) mice, and those in which adenylyl cyclase type 8 (ADCY8), a main driver of intrinsic cAMP-PKA-Ca2+ mediated pacemaker function, was overexpressed exclusively in the heart (TGAC8). We hypothesized that TGAC8 mice would: (1) manifest a more coherent pattern of HRV in vivo, i.e., a reduced HRV driven by mechanisms intrinsic to SAN cells, and less so to modulation by autonomic input and (2) utilize unique adaptations to limit sympathetic input to a heart with high levels of intrinsic cAMP-Ca2+ signaling. Increased adenylyl cyclase (AC) activity in TGAC8 SAN tissue was accompanied by a marked increase in HR and a concurrent marked reduction in HRV, both in the absence or presence of dual autonomic blockade. The marked increase in intrinsic HR and coherence of HRV in TGAC8 mice occurred in the context of: (1) reduced HR and HRV responses to β-adrenergic receptor (β-AR) stimulation; (2) increased transcription of genes and expression of proteins [β-Arrestin, G Protein-Coupled Receptor Kinase 5 (GRK5) and Clathrin Adaptor Protein (Dab2)] that desensitize β-AR signaling within SAN tissue, (3) reduced transcripts or protein levels of enzymes [dopamine beta-hydorxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT)] required for catecholamine production in intrinsic cardiac adrenergic cells, and (4) substantially reduced plasma catecholamine levels. Thus, mechanisms driven by cAMP-PKA-Ca2+ signaling intrinsic to SAN cells underlie the marked coherence of TGAC8 mice HRV. Adaptations to limit additional activation of AC signaling, via decreased neuronal sympathetic input, are utilized to ensure the hearts survival and prevent Ca2+ overload.

Imipramine as an alternative to formamide to detubulate rat ventricular cardiomyocytes

What is the central question of this study? Can imipramine, an antidepressant agent that is a cationic amphiphilic drug that interferes with the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) interactions with proteins maintaining the tubular system, be validated as a new detubulating tool? What is the main finding and its importance? Imipramine was validated as a more efficient and less toxic detubulating agent of cardiomyocytes than formamide. New insights are provided on how PI(4,5)P2 is crucial to maintaining T-tubule attachment to the cell surface and on the cardiotoxic effects of imipramine overdoses. Cardiac T-tubules are membrane invaginations essential for excitation-contraction coupling (ECC). Imipramine, like other cationic amphiphilic drugs, interferes with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) interactions with proteins maintaining the tubular system connected to the cell surface. Our main purpose was to validate imipramine as a new detubulating agent in cardiomyocytes. Staining adult rat ventricular myocytes (ARVMs) with di-4-ANEPPS, we showed that unlike formamide, imipramine induces a complete detubulation with no impact on cell viability. Using the patch-clamp technique, we observed a ∼40% decrease in cell capacitance after imipramine pretreatment and a reduction of ICa,L amplitude by ∼72%. These parameters were not affected in atrial cells, excluding direct side effects of imipramine. β-Adrenergic receptor (β-AR) stimulation of the remaining ICa,L with isoproterenol (Iso) was still effective. ECC was investigated in ARVMs loaded with Fura-2 and paced at 1Hz, allowing simultaneous measurement of the Ca2+ transient (CaT) and sarcomere shortening (SS). Amplitude of both CaT and SS was decreased by imipramine and partially restored by Iso. Furthermore, detubulated cells exhibited Ca2+ homeostasis perturbations. Real-time cAMP variations induced by Iso using a Förster resonance energy transfer biosensor revealed ∼27% decreased cAMP elevation upon β-AR stimulation. To conclude, we validated a new cardiomyocyte detubulation method using imipramine, which is more efficient and less toxic than formamide. This antidepressant agent induces the hallmark effects of detubulation on ECC and its β-AR stimulation. Besides, we provide new insights on how an imipramine overdose may affect cardiac function and suggest that PI(4,5)P2 is crucial for maintaining T-tubule structure.

Synergic PDE3 and PDE4 control intracellular cAMP and cardiac excitation-contraction coupling in a porcine model

AimsCyclic AMP phosphodiesterases (PDEs) are important modulators of the cardiac response to β-adrenergic receptor (β-AR) stimulation. PDE3 is classically considered as the major cardiac PDE in large mammals and human, while PDE4 is preponderant in rodents. However, it remains unclear whether PDE4 also plays a functional role in large mammals. Our purpose was to understand the role of PDE4 in cAMP hydrolysis and excitation-contraction coupling (ECC) in the pig heart, a relevant pre-clinical model. Methods and resultsReal-time cAMP variations were measured in isolated adult pig right ventricular myocytes (APVMs) using a Förster resonance energy transfer (FRET) biosensor. ECC was investigated in APVMs loaded with Fura-2 and paced at 1 Hz allowing simultaneous measurement of intracellular Ca2+ and sarcomere shortening. The expression of the different PDE4 subfamilies was assessed by Western blot in pig right ventricles and APVMs. Similarly to PDE3 inhibition with cilostamide (Cil), PDE4 inhibition with Ro 20-1724 (Ro) increased cAMP levels and inotropy under basal conditions. PDE4 inhibition enhanced the effects of the non-selective β-AR agonist isoprenaline (Iso) and the effects of Cil, and increased spontaneous diastolic Ca2+ waves (SCWs) in these conditions. PDE3A, PDE4A, PDE4B and PDE4D subfamilies are expressed in pig ventricles. In APVMs isolated from a porcine model of repaired tetralogy of Fallot which leads to right ventricular failure, PDE4 inhibition also exerts inotropic and pro-arrhythmic effects. ConclusionsOur results show that PDE4 controls ECC in APVMs and suggest that PDE4 inhibitors exert inotropic and pro-arrhythmic effects upon PDE3 inhibition or β-AR stimulation in our pre-clinical model. Thus, PDE4 inhibitors should be used with caution in clinics as they may lead to arrhythmogenic events upon stress.

Circulating messenger for neuroprotection induced by molecular hydrogen.

Molecular hydrogen (H2) showed protection against various kinds of oxidative-stress-related diseases. First, it was reported that the mechanism of therapeutic effects of H2 was antioxidative effect due to inhibition of the most cytotoxic reactive oxygen species, hydroxy radical (•OH). However, after chronic administration of H2 in drinking water, oxidative-stress-induced nerve injury is significantly attenuated even in the absence of H2. It suggests indirect signaling of H2 and gastrointestinal tract is involved. Indirect effects of H2 could be tested by giving H2 water only before nerve injury, as preconditioning. For example, preconditioning of H2 for certain a period (∼7 days) in Parkinson's disease model mice shows significant neuroprotection. As the mechanism of indirect effect, H2 in drinking water induces ghrelin production and release from the stomach via β1-adrenergic receptor stimulation. Released ghrelin circulates in the body, being transported across the blood-brain barrier, activates its receptor, growth-hormone secretagogue receptor. H2-induced upregulation of ghrelin mRNA is also shown in ghrelin-producing cell line, SG-1. These observations help with understanding the chronic effects of H2 and raise intriguing preventive and therapeutic options using H2.

Effects of β-Adrenergic Antagonists on Chemoradiation Therapy for Locally Advanced Non-Small Cell Lung Cancer.

Introduction: Locally advanced non-small cell lung cancer (NSCLC) is highly resistant to chemoradiotherapy, and many cancer patients experience chronic stress. Studies that suggest stimulation of β-adrenergic receptors (β-AR) promotes tumor invasion and therapy resistance. We investigated whether β-AR inhibition with beta-blockers acts as a chemotherapy and radiation sensitizer in vitro and in patients treated with chemoradiation for locally advanced NSCLC. Methods: We investigated the effects of the non-selective beta-blocker propranolol on two human lung adenocarcinoma cell lines (PC9, A549) treated with radiation or cisplatin. We retrospectively evaluated 77 patients with Stage IIIA NSCLC who received induction chemoradiation followed by surgery. Pathological and imaging response, metastatic rate, and survival were analyzed using SPSS v22.0 and PrismGraphpad6. Results: Propranolol combined with radiation or cisplatin decreased clonogenic survival of PC9 and A549 cells in vitro (p < 0.05). Furthermore, propranolol decreased expression of phospho-protein kinase A (p-PKA), a β-adrenergic pathway downstream activation target, in both cell lines compared to irradiation or cisplatin alone (p < 0.05). In patients treated for Stage IIIA NSCLC, 16 took beta-blockers, and 61 did not. Beta-blockade is associated with a trend to improved overall survival (OS) at 1 year (81.3% vs 57.4%, p = 0.08) and distant metastasis-free survival (DMFS) (2.6 years vs. 1.3 years, p = 0.16). Although beta-blocker use was associated with decreased distant metastases (risk ratio (RR) 0.19; p = 0.03), it did not affect primary tumor pathological response (p = 0.40) or imaging response (p = 0.36). Conclusions: β-AR blockade enhanced radiation and cisplatin sensitivity of human lung cancer cells in vitro. Use of beta-blockers is associated with decreased distant metastases and potentially improved OS and DMFS. Additional studies are warranted to evaluate the role of beta-blockers as a chemoradiation sensitizer in locally advanced NSCLC.

β-Adrenergic Receptor Stimulation and Alternans in the Border Zone of a Healed Infarct: An ex vivo Study and Computational Investigation of Arrhythmogenesis.

Background: Following myocardial infarction (MI), the myocardium is prone to calcium-driven alternans, which typically precedes ventricular tachycardia and fibrillation. MI is also associated with remodeling of the sympathetic innervation in the infarct border zone, although how this influences arrhythmogenesis is controversial. We hypothesize that the border zone is most vulnerable to alternans, that β-adrenergic receptor stimulation can suppresses this, and investigate the consequences in terms of arrhythmogenic mechanisms.Methods and Results: Anterior MI was induced in Sprague-Dawley rats (n = 8) and allowed to heal over 2 months. This resulted in scar formation, significant (p < 0.05) dilation of the left ventricle, and reduction in ejection fraction compared to sham operated rats (n = 4) on 7 T cardiac magnetic resonance imaging. Dual voltage/calcium optical mapping of post-MI Langendorff perfused hearts (using RH-237 and Rhod2) demonstrated that the border zone was significantly more prone to alternans than the surrounding myocardium at longer cycle lengths, predisposing to spatially heterogeneous alternans. β-Adrenergic receptor stimulation with norepinephrine (1 μmol/L) attenuated alternans by 60 [52–65]% [interquartile range] and this was reversed with metoprolol (10 μmol/L, p = 0.008). These results could be reproduced by computer modeling of the border zone based on our knowledge of β-adrenergic receptor signaling pathways and their influence on intracellular calcium handling and ion channels. Simulations also demonstrated that β-adrenergic receptor stimulation in this specific region reduced the formation of conduction block and the probability of premature ventricular activation propagation.Conclusion: While high levels of overall cardiac sympathetic drive are a negative prognostic indicator of mortality following MI and during heart failure, β-adrenergic receptor stimulation in the infarct border zone reduced spatially heterogeneous alternans, and prevented conduction block and propagation of extrasystoles. This may help explain recent clinical imaging studies using meta-iodobenzylguanidine (MIBG) and 11C-meta-hydroxyephedrine positron emission tomography (PET) which demonstrate that border zone denervation is strongly associated with a high risk of future arrhythmia.

Myocardial Rad Deletion Modulates L-Type Calcium Channel Current

Rad inhibits L-type calcium channel current (ICa,L), and in vivo deletion of Rad results in elevated ICa,L density. The aim of this study is to test the hypothesis that Rad-deletion modulates L-type calcium channel current (ICa,L). We engineered a cardiac-restricted inducible Rad knockout mouse (cRadKO). We examined ICa,L using the whole cell configuration and the cell attached configuration of the patch clamp technique. We assessed global and local cellular calcium handling using Fura-2 AM and Fluo-4 AM, respectively. cRadKO displayed an increase in ICa,L compared to WT (maximal conductance: cRadKO = 254 ± 19 pS/pF, n=15; WT= 144 ± 12 pS/pF, n=18; p<0.0001). ICa,L activated at more negative voltages (activation midpoint: cRadKO = −18.3 ± 1.0 mV, n=15; WT= −8.1 ± 1.9 mV, n=18; p<10−4). WT cells treated with forskolin and IBMX demonstrated a ceiling effect of modulation in WT ICa,L. ICa,L kinetics are accelerated in cRadKO concordant with the upstroke velocity of Ca2+ (transient velocity 48.7 ± 2.4 AU/s, n=49; WT=38.1 ± 3.5 AU/s, n=37; p=.014). cRadKO ICa,L was no different when treated with isoproterenol, consistent with saturable modulation of the LTCC. Myocardial Rad deletion increases ICa,L and activates ICa,L at more negative potentials that is beyond PKA induced modulation of the L-type calcium channel; cRadKO ICa,L remains unchanged when PKA is activated. This unmasks a new form of modulation of ICa,L, demonstrating that Rad plays a critical role in how the L-type calcium channel responds to β-Adrenergic Receptor stimulation.

Exogenous ubiquitin reduces inflammatory response and preserves myocardial function 3 days post-ischemia-reperfusion injury.

β-Adrenergic receptor (β-AR) stimulation increases extracellular levels of ubiquitin (UB) in myocytes, and exogenous UB decreases β-AR-stimulated myocyte apoptosis and myocardial fibrosis. Here, we hypothesized that exogenous UB modulates the inflammatory response, thereby playing a protective role in cardiac remodeling after ischemia-reperfusion (I/R) injury. C57BL/6 mice infused with vehicle or UB (1 μg·g-1·h-1) were subjected to myocardial I/R injury. Functional and biochemical parameters of the heart were examined 3 days post-I/R. Heart weight-to-body weight ratios were similarly increased in I/R and UB + I/R groups. The area at risk and infarct size were significantly lower in UB + I/R versus I/R groups. Measurement of heart function using echocardiography revealed that I/R decreases percent fractional shortening and percent ejection fraction. However, the decrease in fractional shortening and ejection fraction was significantly lower in the UB + I/R group. The UB + I/R group displayed a significant decrease in inflammatory infiltrates, neutrophils, and macrophages versus the I/R group. Neutrophil activity was significantly lower in the UB + I/R group. Analysis of the concentration of a panel of 23 cytokines/chemokines in the serum using a Bio-Plex assay revealed a significantly lower concentration of IL-12 subunit p40 in the UB + I/R versus I/R group. The concentration of monocyte chemotactic protein-1 was lower, whereas the concentration of macrophage inflammatory protein-1α was significantly higher, in the UB+I/R group versus the sham group. Expression of matrix metalloproteinase (MMP)-2 and activity of MMP-9 were higher in the UB + I/R group versus the I/R group. Levels of ubiquitinated proteins and tissue inhibitor of metalloproteinase 2 expression were increased to a similar extent in both I/R groups. Thus, exogenous UB plays a protective role in myocardial remodeling post-I/R with effects on cardiac function, area at risk/infarct size, the inflammatory response, levels of serum cytokines/chemokines, and MMP expression and activity. NEW & NOTEWORTHY Stimulation of β-adrenergic receptors increases extracellular levels of ubiquitin (UB) in myocytes, and exogenous UB decreases β-adrenergic receptor-stimulated myocyte apoptosis and myocardial fibrosis. Here, we provide evidence that exogenous UB decreases the inflammatory response and preserves heart function 3 days after myocardial ischemia-reperfusion injury. Further identification of the molecular events involved in the anti-inflammatory role of exogenous UB may provide therapeutic targets for patients with ischemic heart disease.

Myofibroblast β2 adrenergic signaling amplifies cardiac hypertrophy in mice

Abnormal β-adrenergic signaling plays a central role in human heart failure. In mice, chronic β-adrenergic receptor (βAR) stimulation elicits cardiac hypertrophy. It has been reported that cultured cardiac fibroblasts express βAR; however, the functional in vivo requirement of βAR signaling in cardiac fibroblasts during the development of cardiac hypertrophy remains elusive. β2AR null mice exhibited attenuated hypertrophic responses to chronic βAR stimulation upon continuous infusion of an agonist, isoprenaline (ISO), compared to those in wildtype controls, suggesting that β2AR activation in the heart induces pro-hypertrophic effects in mice. Since β2AR signaling is protective in cardiomyocytes, we focused on β2AR signaling in cardiac myofibroblasts. To determine whether β2AR signaling in myofibroblasts affects cardiac hypertrophy, we generated myofibroblast-specific transgenic mice (TG) with the catalytic subunit of protein kinase A (PKAcα) using Cre-loxP system. Myofibroblast-specific PKAcα overexpression resulted in enhanced heart weight normalized to body weight ratio, associated with an enlargement of cardiomyocytes at 12 weeks of age, indicating that myofibroblast-specific activation of PKA mediates cardiac hypertrophy in mice. Neonatal rat cardiomyocytes stimulated with conditioned media from TG cardiac fibroblasts likewise exhibited significantly more growth than those from controls. Thus, β2AR signaling in myofibroblasts plays a substantial role in ISO-induced cardiac hypertrophy, possibly due to a paracrine effect. β2AR signaling in cardiac myofibroblasts may represent a promising target for development of novel therapies for cardiac hypertrophy.

Cardiac B55α: a Novel Regulator of β-Adrenergic Signalling and Hypertrophic Gene Expression

Introduction: Cardiac hypertrophy is an independent risk factor for heart failure. A key contributor to this maladaptation is activation of β-adrenergic signalling in cardiomyocytes. B55α, a regulatory subunit of protein phosphatase 2A, modulates hypertrophic signalling downstream of β-adrenergic receptors in vitro. To investigate the function of cardiac B55α in vivo, we generated mice with heterozygous (HET) or homozygous (HOM) deletion of the gene encoding B55α. Methods: Cardiac function, morphology, histology and molecular analyses were performed in (i) a basal cohort (n = 6–14/genotype, ∼10–12 weeks of age, both sexes) and (ii) a cohort subjected to 13 days of isoproterenol treatment administered via osmotic mini-pump (30 mg/kg/day, n = 5–6/group, male mice). Results: HOM mice were embryonically lethal. HET mice displayed a ∼50% reduction in cardiac B55α expression (p = 0.007) and showed no signs of pathology (normal systolic function, no fibrosis or induction of the cardiac stress markers Nppa and Nppb). Isoproterenol did not significantly increase heart weight/tibia length ratio of WT or HET mice, however HET mice displayed more pronounced changes in gene transcription. Specifically, expression of myosin heavy chain isoforms α and β switched in favour of the β-isoform in HET (p = 0.004) but not WT (p = 0.69) mice. This dysregulation is frequently observed in settings of cardiac hypertrophy and failure. Conclusion: Reduced B55α expression did not cause adverse remodelling in the murine heart in the absence of pathological stimuli. However, reduced B55α exacerbated hypertrophic gene transcription in response to sustained β-adrenergic receptor stimulation, identifying B55α as a potential negative regulator of hypertrophic gene expression in vivo.