Adult Rat Ventricular Myocytes Research Articles

ENDOGENOUS β 3 -ADRENERGIC RECEPTOR ACTIVATION ALLEVIATES SEPSIS-INDUCED CARDIOMYOCYTE APOPTOSIS VIA PI3K/AKT SIGNALING PATHWAY.

β 3 -adrenergic receptor (β 3 -AR) has been proposed as a new therapy for several myocardial diseases. However, the effect of β 3 -AR activation on sepsis-induced myocardial apoptosis is unclear. Here, we investigated the effect of β 3 -AR activation on the cardiomyocyte apoptosis and cardiac dysfunction in cecal ligation and puncture (CLP)-operated rats and lipopolysaccharide (LPS)-treated cardiomyocytes. We found that β 3 -AR existed both in adult rat ventricular myocytes (ARVMs) and H9c2 cells. The expression of β 3 -AR was upregulated in LPS-treated ARVMs and the heart of CLP rats. Pretreatment with β 3 -AR agonist, BRL37344, inhibited LPS-induced cardiomyocyte apoptosis and caspase-3, -8, and -9 activation in ARVMs. BRL37344 also reduced apoptosis and increased the protein levels of PI3K, p-Akt Ser473 and p-eNOS Ser1177 in LPS-treated H9c2 cells. Inhibition of PI3K using LY294002 abolished the inhibitory effect of BRL37344 on LPS-induced caspase-3, -8, and -9 activation in H9c2 cells. Furthermore, administration of β 3 -AR antagonist, SR59230A (5 mg/kg), significantly decreased the maximum rate of left ventricular pressure rise (+dP/dt) in CLP-induced septic rats. SR59230A not only increased myocardial apoptosis, reduced p-Akt Ser473 and Bcl-2 contents, but also increased mitochondrial Bax, cytoplasm cytochrome c, cleaved caspase-9, and cleaved caspase-3 levels of the myocardium in septic rats. These results suggest that endogenous β 3 -AR activation alleviates sepsis-induced cardiomyocyte apoptosis via PI3K/Akt signaling pathway and maintains intrinsic myocardial systolic function in sepsis.

Persistent PKA activation redistributes NaV1.5 to the cell surface of adult rat ventricular myocytes.

During chronic stress, persistent activation of cAMP-dependent protein kinase (PKA) occurs, which can contribute to protective or maladaptive changes in the heart. We sought to understand the effect of persistent PKA activation on NaV1.5 channel distribution and function in cardiomyocytes using adult rat ventricular myocytes as the main model. PKA activation with 8CPT-cAMP and okadaic acid (phosphatase inhibitor) caused an increase in Na+ current amplitude without altering the total NaV1.5 protein level, suggesting a redistribution of NaV1.5 to the myocytes' surface. Biotinylation experiments in HEK293 cells showed that inhibiting protein trafficking from intracellular compartments to the plasma membrane prevented the PKA-induced increase in cell surface NaV1.5. Additionally, PKA activation induced a time-dependent increase in microtubule plus-end binding protein 1 (EB1) and clustering of EB1 at myocytes' peripheral surface and intercalated discs (ICDs). This was accompanied by a decrease in stable interfibrillar microtubules but an increase in dynamic microtubules along the myocyte surface. Imaging and coimmunoprecipitation experiments revealed that NaV1.5 interacted with EB1 and β-tubulin, and both interactions were enhanced by PKA activation. We propose that persistent PKA activation promotes NaV1.5 trafficking to the peripheral surface of myocytes and ICDs by providing dynamic microtubule tracks and enhanced guidance by EB1. Our proposal is consistent with an increase in the correlative distribution of NaV1.5, EB1, and β-tubulin at these subcellular domains in PKA-activated myocytes. Our study suggests that persistent PKA activation, at least during the initial phase, can protect impulse propagation in a chronically stressed heart by increasing NaV1.5 at ICDs.

Abstract 16997: Cardiomyocyte-Autonomous and Microtubule Dependent Mechanisms Are Sufficient to Mediate Load-Dependent T-Tubule Remodeling

Background: Mechanical overload is an established contributor to T-tubule (TT) remodeling and associated dyssynchronous Ca 2+ release leading to abnormal excitation-contraction coupling in cardiomyopathies. We examined the induction of pathological TT remodeling in isolated cardiomyocytes and the involvement of microtubules (MT) in this process. Methods: Adult rat ventricular myocytes (aRVMs) were cultured with applied variable in vitro load for 48hrs at either physiological (10kPa) or pathological (50kPa) stiffness, using custom magnetorheological elastomers with tunable stiffness. Cells were stained with Di-8-ANEPPS and Fluo-4-AM and imaged using confocal microscopy. Field stimulation of cells allowed for Ca 2+ transient acquisition via line scans. Protein expression changes were determined via Western blotting. IF staining allowed for visualization of the MT network and relevant TT proteins (TCAP, JPH2, BIN1). MT and tyrosination state dependency of remodeling was investigated by 1μM nocodazole (MT depolymerizer), taxol (MT stabilizer), and tubulin tyrosine ligase (TTL) treatments to alter the MT network accordingly. Results: Compared to the physiological stiffness group, aRVMs subjected to pathologically increased stiffness exhibited a 14.7% decrease in density, 18.9% decrease in regularity, and a 26.6% increase in luminal dimension of the TT system, along with an increase in longitudinal elements (p<0.05). The changes in both Ca 2+ handling parameters and protein expression resembled those typical of the in vivo myopathic phenotype. This included a 21.7% decrease in amplitude, along with a 33.7%, 29.8% and 21.1% increase in time to peak (TTP), variance in TTP, and the signal decay tau, respectively (p<0.05). Treatment of pathologically loaded cells with taxol worsened TT remodeling, specifically with respect to TT density which decreased by 12.5% compared to nontreatment cells, and longitudinal elements which increased by 24%. Increased MT tyrosination through TTL treatment showed a 37% recovery in TT density in cells cultured at pathological stiffness compared to nontreatment cells (p<0.05). Conclusion: The results suggest that load-induced TT remodeling occurs via a cardiomyocyte autonomous, MT-dependent mechanism.

Ionic current changes underlying action potential repolarization responses to physiological pacing and adrenergic stimulation in adult rat ventricular myocytes.

This study aimed to simulate ventricular responses to elevations in myocyte pacing and adrenergic stimulation using a novel electrophysiological rat model and investigate ion channel responses underlying action potential (AP) modulations. Peak ion currents and AP repolarization to 50% and 90% of full repolarization (APD50-90 ) were recorded during simulations at 1-10 Hz pacing under control and adrenergic stimulation conditions. Further simulations were performed with incremental ion current block (L-type calcium current, ICa ; transient outward current, Ito ; slow delayed rectifier potassium current, IKs ; rapid delayed rectifier potassium current, IKr ; inward rectifier potassium current, IK1 ) to identify current influence on AP response to exercise. Simulated APD50-90 closely resembled experimental findings. Rate-dependent increases in IKs (6%-101%), IKr (141%-1339%), and ICa (0%-15%) and reductions in Ito (11%-57%) and IK1 (1%-9%) were observed. Meanwhile, adrenergic stimulation triggered moderate increases in all currents (23%-67%) except IK1 . Further analyses suggest AP plateau is most sensitive to modulations in Ito and ICa while late repolarization is most sensitive to IK1 , ICa , and IKs , with alterations in IKs predominantly stimulating the greatest magnitude of influence on late repolarization (35%-846% APD90 prolongation). The modified Leeds rat model (mLR) is capable of accurately modeling APs during physiological stress. This study highlights the importance of ICa , Ito , IK1, and IKs in controlling electrophysiological responses to exercise. This work will benefit the study of cardiac dysfunction, arrythmia, and disease, though future physiologically relevant experimental studies and model development are required.

Flecainide induces a sustained countercurrent dependent effect on RyR2 in permeabilized WT ventricular myocytes but not in intact cells.

Background and purpose: While flecainide is now an accepted treatment for arrhythmias associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), its mechanism of action remains controversial. In studies on myocytes from CPVT mice, inhibition of proarrhythmic Ca2+ waves was initially attributed to a novel action on the type-2 ryanodine receptor (RyR2). However, subsequent work on wild type (WT) myocytes questioned the conclusion that flecainide has a direct action on RyR2. In the present study, the effects of flecainide were compared in intact and permeabilized WT myocytes. Experimental approach: Intracellular Ca2+ was measured using confocal microscopy in intact or saponin permeabilized adult rat ventricular myocytes (ARVM). In some experiments on permeabilized cells, flecainide was studied following partial inhibition of the sarcoplasmic reticulum (SR) counter-current. Key results: Flecainide induced sustained changes Ca2+ sparks and waves in permeabilized ARVM, which were comparable to those reported in intact or permeabilized myocytes from CPVT mice. However, a relatively high level of flecainide (25μM) was required to induce these effects. Inhibition of the SR counter-current potentiated the effects of flecainide on SR Ca2+ waves. In intact field stimulated ARVM, prolonged exposure to 15μM flecainide decreased wave frequency but RyR2 dependent effects on Ca2+ sparks were absent; higher drug concentrations blocked field stimulation, consistent with inhibition of Nav1.5. Conclusions and implications: In intact ARVM, the absence of effects on Ca2+ sparks suggests that the intracellular flecainide concentration was insufficient to influence RyR2. Wave inhibition in intact ARVM may reflect secondary effects of Nav1.5 inhibition. Potentiation of flecainide's action by counter-current inhibition can be explained if transient polarization of the SR membrane during SR Ca2+ release facilitates its action on RyR2.

Regulation of cardiac myocyte cyclic AMP production by intracellular α1-adrenergic receptors.

Sympathetic regulation of cardiac function involves β1‑adrenergic receptor (β1AR) production of cAMP. However, the effects are typically mediated by the endogenous neurotransmitter norepinephrine (NE), which is also a potent α1-adrenergic receptor (α1AR) agonist. Furthermore, activation of a mitogen-activated protein kinase (MAPK) signaling pathway by nuclear α1ARs is thought to be cardioprotective, although the exact mechanism has yet to be elucidated. Because long-term stimulation of cAMP production by β1ARs contributes to hypertrophy and apoptosis, we tested the hypothesis that the cardioprotective α1AR signaling pathway inhibits β1AR cAMP production. Changes in cAMP activity were measured in adult rat ventricular myocytes (ARVMs) using FRET-based biosensors. We found that the increase in cAMP produced by the βAR agonist isoproterenol could be antagonized by the selective α1AR agonist methoxamine (METH), and the response to METH could be antagonized by the selective α1AR antagonist prazosin (PRAZ). The effects of METH could also be antagonized by blocking the MAPK signaling pathway with non-selective tyrosine kinase inhibitors genistein and lavendustin A as well as the selective MEK inhibitors U0126 and PD98059. Furthermore, NE produced increases in cAMP activity that was enhanced in the presence of PRAZ, demonstrating α1AR inhibition of β1AR cAMP production contributes to the net response. NE stimulation of cAMP production was also significantly enhanced when the organic cation transporter OCT3 was inhibited with cortisol, and addition of PRAZ caused no further increase, suggesting the α1ARs responsible for inhibiting cAMP production are intracellular. Confocal imaging of ARVMs treated with BODIPY-labeled PRAZ confirmed α1ARs are primarily perinuclear in these cells, a result confirmed by the labeling of α1ARs on isolated nuclei. These results suggest the cardioprotective effect of α1AR stimulation may involve nuclear receptor activation of a MAPK signaling mechanism that inhibits β1AR production of cAMP.

Abstract 11506: Roles of Mechanical Overload and the Microtubule Network in Mediating T-Tubule Remodeling

Background: Mechanical overload is an established contributor to the remodeling of T-tubules (TT) and associated dyssynchronous Ca 2+ release leading to abnormal excitation-contraction coupling in cardiomyopathies. We examined the induction of pathologic TT remodeling in isolated cardiomyocytes and the involvement of microtubules in this process. Methods: Normal adult rat ventricular myocytes (aRVMs) were cultured for 48hrs on MREs at either physiological (10kPa) or pathological (50kPa) stiffness. Variable in vitro load application was achieved using custom magnetorheologic elastomers (MREs) with tunable stiffness. Cells were stained with Di-8-ANEPPS and Fluo-4-AM and imaged using Airyscan confocal microscopy. Microtubule dependency was investigated by treating cells with 1μM nocodazole, a microtubule depolymerization agent.Structural changes in TT networks were quantified in terms of regularity, luminal dimensions, density, and directionality. Field stimulation of cells allowed for acquisition of [Ca 2+ ] i transients via line scans and subsequent analysis of key [Ca 2+ ] i transient parameters. Changes in protein expression were determined via Western blotting. Results: Compared to the physiological stiffness group, aRVMs subjected to pathologically increased stiffness exhibited decreased density and regularity of the TT system, and increased luminal dimension. Direction-specific density analysis showed that pathologically loaded cells have more longitudinal tubule elements and fewer transverse tubule elements. This remodeling was attenuated in aRVMs treated with nocodazole in terms of regularity, luminal diameter, and direction-specific density. The observed changes in both Ca 2+ handling parameters and key protein expression resembled those typical of the in vivo myopathic phenotype. Conclusion: The results suggest that load-induced TT remodeling occurs via a cardiomyocyte autonomous, microtubule-dependent mechanism.

A perinuclear calcium compartment regulates cardiac myocyte hypertrophy

The pleiotropic Ca2+/calmodulin-dependent phosphatase calcineurin is a key regulator of pathological cardiac myocyte hypertrophy. The selective activation of hypertrophic calcineurin signaling under stress conditions has been attributed to compartmentation of Ca2+ signaling in cardiac myocytes. Here, perinuclear signalosomes organized by the scaffold protein muscle A-Kinase Anchoring Protein β (mAKAPβ/AKAP6β) are shown to orchestrate local Ca2+ transients, inducing calcineurin-dependent NFATc nuclear localization and myocyte hypertrophy in response to β-adrenergic receptor activation. Fluorescent biosensors for Ca2+ and calcineurin and protein kinase A (PKA) activity, both diffusely expressed and localized by nesprin-1α to the nuclear envelope, are used to define an autonomous mAKAPβ signaling compartment in adult and neonatal rat ventricular myocytes. Notably, β-adrenergic-stimulated perinuclear Ca2+ and PKA and CaN activity transients depended upon mAKAPβ expression, while Ca2+ elevation and PKA and CaN activity in the cytosol were mAKAPβ independent. Buffering perinuclear cAMP and Ca2+ prevented calcineurin-dependent NFATc nuclear translocation and myocyte hypertrophy, without affecting cardiac myocyte contractility. Additional findings suggest that the perinuclear Ca2+ transients were mediated by signalosome-associated ryanodine receptors regulated by local PKA phosphorylation. These results demonstrate the existence of a functionally independent Ca2+ signaling compartment in the cardiac myocyte regulating hypertrophy and provide a premise for targeting mAKAPβ signalosomes to prevent selectively cardiac hypertrophy in disease.

Tetramisole is a new IK1 channel agonist and exerts IK1 -dependent cardioprotective effects in rats.

Cardiac ischemia, hypoxia, arrhythmias, and heart failure share the common electrophysiological changes featured by the elevation of intracellular Ca2+ (Ca2+ overload) and inhibition of the inward rectifier potassium (IK1) channel. IK1 channel agonists have been considered a new type of anti‐arrhythmia and cardioprotective agents. We predicted using a drug repurposing strategy that tetramisole (Tet), a known anthelminthic agent, was a new IK1 channel agonist. The present study aimed to experimentally identify the above prediction and further demonstrate that Tet has cardioprotective effects. Results of the whole‐cell patch clamp technique showed that Tet at 1–100 μmol/L enhanced IK1 current, hyperpolarized resting potential (RP), and shortened action potential duration (APD) in isolated rat cardiomyocytes, while without effects on other ion channels or transporters. In adult Sprague–Dawley (SD) rats in vivo, Tet showed anti‐arrhythmia and anticardiac remodeling effects, respectively, in the coronary ligation‐induced myocardial infarction model and isoproterenol (Iso, i.p., 3 mg/kg/day, 10 days) infusion‐induced cardiac remodeling model. Tet also showed anticardiomyocyte remodeling effect in Iso (1 μmol/L) infused adult rat ventricular myocytes or cultured H9c2 (2‐1) cardiomyocytes. Tet at 0.54 mg/kg in vivo or 30 μmol/L in vitro showed promising protections on acute ischemic arrhythmias, myocardial hypertrophy, and fibrosis. Molecular docking was performed and identified the selective binding of Tet with Kir2.1. The cardioprotection of Tet was associated with the facilitation of IK1 channel forward trafficking, deactivation of PKA signaling, and inhibition of intracellular calcium overload. Enhancing IK1 may play dual roles in anti‐arrhythmia and antiventricular remodeling mediated by restoration of Ca2+ homeostasis.

B-adrenergic/cAMP signaling in doxorubicin-induced cardiotoxicity

Doxorubicin (Dox) is one of the most commonly used chemotherapy but also one of the most cardiotoxic. As of today, only the iron chelator Dexrazoxane has been FDA and EMA approved as a cardioprotective agent for Dox but its usage is still controversial. Thus, there is an urgent need to identify new targets to develop an effective protection against Dox-induced cardiotoxicity (DIC) without compromising its anti-tumor action. Well-characterized DIC mechanisms include topoisomerase II inhibition, DNA damages, induction of ROS and bioenergetic distress that lead to cardiomyocyte death. Alternative mechanisms are emerging such as an alteration of the β-adrenergic system. Our objective here was to study DIC mechanisms with a specific focus on how Dox treatment affects the β-adrenergic/cAMP signaling cascade. We used FRET-based sensors for cAMP targeted to two different cellular compartments, cytosol and nucleus, and measured the cAMP response to the β-adrenergic agonist isoprenaline (Iso, 10 nM) in adult rat ventricular myocytes after 24 h treatment with 1 or 10 μM Dox. Basal cAMP concentration was measured by Elisa. Adenylyl cyclase (AC5 and AC6 isoforms) mRNA was measured by RT-qPCR and phosphodiesterase (PDE) isoform expression and activity by western blot and radioenzymatic assay, respectively. cAMP response to Iso was decreased by 25% at 1 μM and > 50% at 10 μM Dox in both cytosol and nucleus. The maximal response to 20 μM L858051 (a forskolin analog) + 200 μM IBMX (a PDE inhibitor) on top of Iso was also decreased by Dox. Dox treatment did not change basal cAMP level nor PDE activity, but decreased AC5 and AC6 mRNA and PDE4 protein expression. Doxorubicin treatment downregulates the β-adrenergic/cAMP cascade in ventricular myocytes by decreasing adenylyl cyclase expression and maximal activity. This may contribute to the Dox-induced cardiotoxicity mechanisms.

OCT2013, an ischaemia-activated antiarrhythmic prodrug, devoid of the systemic side effects of lidocaine.

Sudden cardiac death (SCD) caused by acute myocardial ischaemia and ventricular fibrillation (VF) is an unmet therapeutic need. Lidocaine suppresses ischaemia-induced VF, but its utility is limited by side effects and a narrow therapeutic index. Here, we characterise OCT2013, a putative ischaemia-activated prodrug of lidocaine. The rat Langendorff-perfused isolated heart, anaesthetised rat and rat ventricular myocyte preparations were utilised in a series of blinded and randomised studies to investigate the antiarrhythmic effectiveness, adverse effects and mechanism of action of OCT2013, compared with lidocaine. In isolated hearts, OCT2013 and lidocaine prevented ischaemia-induced VF equi-effectively, but OCT2013 did not share lidocaine's adverse effects (PR widening, bradycardia and negative inotropy). In anaesthetised rats, i.v. OCT2013 and lidocaine suppressed VF and increased survival equi-effectively; OCT2013 had no effect on cardiac output even at 64 mg·kg-1 i.v., whereas lidocaine reduced it even at 1 mg·kg-1 . In adult rat ventricular myocytes, OCT2013 had no effect on Ca2+ handling, whereas lidocaine impaired it. In paced isolated hearts, lidocaine caused rate-dependent conduction slowing and block, whereas OCT2013 was inactive. However, during regional ischaemia, OCT2013 and lidocaine equi-effectively hastened conduction block. Chromatography and MS analysis revealed that OCT2013, detectable in normoxic OCT2013-perfused hearts, became undetectable during global ischaemia, with lidocaine becoming detectable. OCT2013 is inactive but is bio-reduced locally in ischaemic myocardium to lidocaine, acting as an ischaemia-activated and ischaemia-selective antiarrhythmic prodrug with a large therapeutic index, mimicking lidocaine's benefit without adversity.

Chrysosplenol-C Increases Contraction by Augmentation of Sarcoplasmic Reticulum Ca2+ Loading and Release via Protein Kinase C in Rat Ventricular Myocytes.

Naturally found chrysosplenol-C (4',5,6-trihydroxy-3,3',7-trimethoxyflavone) increases the contractility of cardiac myocytes independent of β-adrenergic signaling. We investigated the cellular mechanism for chrysosplenol-C-induced positive inotropy. Global and local Ca2+ signals, L-type Ca2+ current (ICa), and contraction were measured from adult rat ventricular myocytes using two-dimensional confocal Ca2+ imaging, the whole-cell patch-clamp technique, and video-edge detection, respectively. Application of chrysosplenol-C reversibly increased Ca2+ transient magnitude with a maximal increase of ∼55% within 2- to 3-minute exposures (EC50 ≅ 21 μM). This chemical did not alter ICa and slightly increased diastolic Ca2+ level. The frequency and size of resting Ca2+ sparks were increased by chrysosplenol-C. Chrysosplenol-C significantly increased sarcoplasmic reticulum (SR) Ca2+ content but not fractional release. Pretreatment of protein kinase C (PKC) inhibitor but not Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor abolished the stimulatory effects of chrysosplenol-C on Ca2+ transients and Ca2+ sparks. Chrysosplenol-C-induced positive inotropy was removed by the inhibition of PKC but not CaMKII or phospholipase C. Western blotting assessment revealed that PKC-δ protein level in the membrane fractions significantly increase within 2 minutes after chrysosplenol-C exposure with a delayed (5-minute) increase in PKC-α levels in insoluble membrane. These results suggest that chrysosplenol-C enhances contractility via PKC (most likely PKC-δ)-dependent enhancement of SR Ca2+ releases in ventricular myocytes. SIGNIFICANCE STATEMENT: Study shows that chrysosplenol-C, a natural flavone showing a positive inotropic effect, increases SR Ca2+ releases on depolarizations and Ca2+ sparks with an increase of SR Ca2+ loading but not L-type Ca2+ current in ventricular myocytes. Chrysosplenol-C-induced enhancement in contraction is eliminated by PKC inhibition, and it is associated with redistributions of PKC to the membrane. These indicate that chrysosplenol-C enhances contraction via PKC-dependent augmentations of SR Ca2+ release and Ca2+ loading during action potentials.

Mitochondrial A-kinase anchoring proteins in cardiac ventricular myocytes.

Compartmentation of cAMP signaling is a critical factor for maintaining the integrity of receptor‐specific responses in cardiac myocytes. This phenomenon relies on various factors limiting cAMP diffusion. Our previous work in adult rat ventricular myocytes (ARVMs) indicates that PKA regulatory subunits anchored to the outer membrane of mitochondria play a key role in buffering the movement of cytosolic cAMP. PKA can be targeted to discrete subcellular locations through the interaction of both type I and type II regulatory subunits with A‐kinase anchoring proteins (AKAPs). The purpose of this study is to identify which AKAPs and PKA regulatory subunit isoforms are associated with mitochondria in ARVMs. Quantitative PCR data demonstrate that mRNA for dual specific AKAP1 and 2 (D‐AKAP1 & D‐AKAP2), acyl‐CoA‐binding domain‐containing 3 (ACBD3), optic atrophy 1 (OPA1) are most abundant, while Rab32, WAVE‐1, and sphingosine kinase type 1 interacting protein (SPHKAP) were barely detectable. Biochemical and immunocytochemical analysis suggests that D‐AKAP1, D‐AKAP2, and ACBD3 are the predominant mitochondrial AKAPs exposed to the cytosolic compartment in these cells. Furthermore, we show that both type I and type II regulatory subunits of PKA are associated with mitochondria. Taken together, these data suggest that D‐AKAP1, D‐AKAP2, and ACBD3 may be responsible for tethering both type I and type II PKA regulatory subunits to the outer mitochondrial membrane in ARVMs. In addition to regulating PKA‐dependent mitochondrial function, these AKAPs may play an important role by buffering the movement of cAMP necessary for compartmentation.

Human BIN1 isoforms grow, maintain, and regenerate excitation-contraction couplons in adult rat and human stem cell-derived cardiomyocytes.

In ventricular myocytes, transverse-tubules (T-tubules) are instrumental for excitation-contraction (EC)coupling and their disarray is a hallmark of cardiac diseases. BIN1 is a key contributor to their biogenesis. Our study set out to investigate the role of human BIN1 splice variants in the maintenance and regeneration of EC-coupling in rat adult ventricular myocytes and human-induced pluripotent stem cell-derived cardiac myocytes (hiPS-CMs). In heart samples from healthy human donors expression patterns of five BIN1 splice variants were identified. Following viral transduction of human BIN1 splice variants in cellular models of T-tubular disarray, we employed high-speed confocal calcium imaging and CaCLEAN analysis to identify functional EC-coupling sites (couplons) and T-tubular architecture. Adult rat ventricular myocytes were used to investigate the regeneration after loss and maintenance of EC-coupling while we studied the enhancement of EC-coupling in hiPS-CMs. All five human BIN1 splice variants induced de-novo generation of T-tubules in both cell types. Isoforms with the phosphoinositide-binding motif (PI) were most potent in maintenance and regeneration of T-tubules and functional EC-coupling in adult rat myocytes. In hiPSC-CMs, BIN1 variants with PI-motif-induced de novo generation of T-tubules, functional couplons and enhanced calcium handling. BIN1 is essential for the maintenance, regeneration, and de novo generation of functional T-tubules. Isoforms with PI-motifs appeared as particulalrly potent. These T-tubules trigger the development of functional couplons resulting in enhanced calcium handling.

Cardiovascular and Renal Actions of the PDZ protein SAP97

Synapse Associated Protein-97 (SAP97) is a PDZ protein that is expressed in the CNS as well as in peripheral organs. SAP97 is a major compartmentalizing protein involved in organizing signaling complexes. We investigated the role of SAP97 in anterograde translocation of beta1-adrenergic receptors (beta1-AR) in adult rat ventricular myocytes (ARVM) and in translocation of aquaporin-2 forming channels in kidney cells. Interactions between SAP97 and other proteins are mediated by binding of one of its three PDZ domains with type-1 PDZ binding motifs in target proteins. We determined that SAP97 forms a ternary complex with the beta1-AR and with A-kinase anchoring protein 79/150 (AKAP5)-PKA, which targets the beta1-AR to AKAP-PKA. Activation of the beta1-AR by agonists promotes induces PKA phosphorylation of the beta1-AR at serine312 in the third intracellular loop and its internalization to endosomes. This phospho-serine along with the type- PDZ binding motif in the C-tail of the beta1-AR serve as trafficking barcodes which promote the recycling of the agonist-internalized beta1-AR back to the membrane. Similarly, SAP97 binds to AQP2 via its type1-PDZ binding motif and to an unidentified AKAP-PKA complex. Exposing renal collecting cells to vasopressin, promotes the phosphorylation of AQP2 on serine256 in the C-tail, which is a primary signal for translocating cytoplasmic AQP2 storage vesicles to the apical membrane. These two pathways were dependent on targeting these trafficking proteins to an AKAP-PKA complex via SAP97. In this regard we verified that anterograde translocation of beta1-AR and AQP2 was dependent on their type-1 PDZs, and on the SAP97-anchored complex to faithfully phosphorylate a specific trafficking serine. In addition, we determined that SAP97 co-localized with beta1-AR in ARVM and with AQP2 in the kidneys, and knockdown of SAP97 inhibited anterograde translocation of these proteins, without affecting their retrograde internalization. Here in we describe these pathways and will show that SAP97 provides a novel paradigm to decipher trafficking events localized around cis-acting barcodes and their trans-acting proteins that might ultimately be used for therapeutic advantage.

Mitochondrial A Kinase Anchoring Proteins in Cylic AMP compartmentation

Cyclic AMP is the basis of sympathetic control of cardiac contractility. Upon activation of the beta-adrenergic receptors numerous proteins in the excitation-contraction machinery are phosphorylated by the cAMP effector protein kinase A (PKA). In addition to this, cAMP is also utilized by other systems to modulate gene transcription, apoptosis, and cell survival. To preserve the fidelity of receptor-specific responses, precise spatial and temporal control of cAMP occurs and this has been experimentally observed. This phenomenon of cAMP compartmentation relies on various factors limiting cAMP mobility. One of these involves the buffering effect of PKA. Binding and unbinding to the regulatory subunits of PKA slow cAMP as it attempts to move throughout the cytoplasm. Previous work indicates that regulatory subunits anchored to the outer membrane of mitochondria by A-kinase anchoring proteins (AKAPs) play a particularly important role in this process. At least seven different mitochondrial AKAPs have been identified in various cell types. These include AKAPs capable of interacting with type I and/or type II PKA regulatory subunits. The purpose of this study was to determine which of those AKAPs are associated with mitochondria in adult rat ventricular myocytes. Quantitative PCR detected evidence for expression of OPA1, D‑AKAP1, D‑AKAP2, and ACBD3, while message levels for Rab32, SKIP, and WAVE1 were barely measurable. Western blot analysis determined that D‑AKAP1, D‑AKAP2, ACBD3 are expressed in mitochondrial fractions of these cells, along with type I and type II PKA regulatory subunits. The mitochondrial association of these proteins was confirmed by immunocytochemistry. Our results also show low expression levels of OPA1 and Rab32 in the mitochondrial fraction. These results suggest that D-AKAP1, D‑AKAP2, and ACBD3 are involved in anchoring type I and type II PKA regulatory subunits to mitochondria in adult ventricular myocytes. In addition to playing a role in PKA-dependent regulation of mitochondrial function, they may play an essential role in cAMP compartmentation by buffering its movement throughout the cell.

A modification of cAMP compartmentation is involved in the doxorubicin-induced cardiotoxicity

Doxorubicin (Dox) is one of the most commonly used chemotherapy but also one of the most cardiotoxic. As of today, only the Iron chelator Dexrazoxane has been FDA and EMA approved as a cardioprotective agent for Dox but its usage is still controversial. Thus, there is an urgent need to identify new targets to develop an effective protection against Dox-induced cardiotoxicity (DIC) without compromising its anti-tumor action. Well-known and studied DIC mechanisms include Topoisomerase II inhibition, DNA damages, induction of ROS and bioenergetic distress that lead to the death of the cardiomyocyte. Alternative mechanisms are emerging such as the alteration of the ß adrenergic system. cAMP is one of the most important second messengers in the heart and is found compartmentalized in specific subcellular domain modulating downstream effectors and thus many physiological processes (contraction/relaxation). Our objective is to study DIC mechanisms with a specific focus on Dox effect on the ß adrenergic system and especially on the cAMP compartmentation. We used FRET-based sensors for cAMP targeted to different compartment of the cell (cytosol and nucleus) in order to study cAMP production in adult rat ventricular myocyte under Isoprenalin stimulation after Dox treatment. Involvement of the main molecular actors of the cAMP compartmentation system has been studied. Under β adrenergic stimulation, cAMP production decreases in the Dox treated cells compare to the control, in the cytosol as well as in the nucleus. Furthermore, the maximal level of cAMP production reached by the Dox treated myocytes is also lower than the control. Based on this data we looked at the expression of AC5 and 6, the two most expressed in myocytes. AC5 but not AC6 mRNA expression is decreased by Dox treatment indicating a potential implication of AC5 in the DIC. Moreover, PDEs involvement cannot be exclude yet. Here, we show for the first time the involvement of cAMP compartmentation in the DIC.

Compartmentation of β2 -adrenoceptor stimulated cAMP responses by phosphodiesterase types 2 and 3 in cardiac ventricular myocytes.

In cardiac myocytes, cyclic AMP (cAMP) produced by both β1 - and β2 -adrenoceptors increases L-type Ca2+ channel activity and myocyte contraction. However, only cAMP produced by β1 -adrenoceptors enhances myocyte relaxation through phospholamban-dependent regulation of the sarco/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2). Here we have tested the hypothesis that stimulation of β2 -adrenoceptors produces a cAMP signal that is unable to reach SERCA2 and determine what role, if any, phosphodiesterase (PDE) activity plays in this compartmentation. The cAMP responses produced by β1 -and β2 -adrenoceptor stimulation were studied in adult rat ventricular myocytes using two different fluorescence resonance energy transfer (FRET)-based biosensors, the Epac2-camps, which is expressed uniformly throughout the cytoplasm of the entire cell and the Epac2-αKAP, which is targeted to the SERCA2 signalling complex. Selective activation of β1 - or β2 -adrenoceptors produced cAMP responses detected by Epac2-camps. However, only stimulation of β1 -adrenoceptors produced a cAMP response detected by Epac2-αKAP. Yet, stimulation of β2 -adrenoceptors was able to produce a cAMP signal detected by Epac2-αKAP in the presence of selective inhibitors of PDE2 or PDE3, but not PDE4. These results support the conclusion that cAMP produced by β2 -adrenoceptor stimulation was not able to reach subcellular locations where the SERCA2 pump is located. Furthermore, this compartmentalized response is due at least in part to PDE2 and PDE3 activity. This discovery could lead to novel PDE-based therapeutic treatments aimed at correcting cardiac relaxation defects associated with certain forms of heart failure.

Rosuvastatin Reduces L-Type Ca2+ Current and Alters Contractile Function in Cardiac Myocytes via Modulation of β-Adrenergic Receptor Signaling.

Rosuvastatin is one of the most used statins to lower plasma cholesterol levels. Although previous studies have reported remarkable cardiovascular effects of rosuvastatin (RSV), the mechanisms of these effects are largely unknown. In this study, we investigated the acute effects of RSV on L-type Ca2+ currents and contractile function of ventricular myocytes under basal conditions and during β-adrenergic stimulation. The effects of RSV were investigated in freshly isolated adult rat ventricular myocytes. L-type Ca+2 currents and myocyte contractility were recorded using patch-clamp amplifier and sarcomere length detection system. All experimental recordings were performed at 36 ± 1°C. L-type Ca+2 currents were significantly reduced with the administration of 1μM RSV (~ 24%) and this reduction in Ca2+ currents was observed at almost all potential ranges applied. Suppression of L-type Ca2+ current by RSV was prevented by adenylyl cyclase (AC) and protein kinase A (PKA) inhibitors SQ 22536 and KT5720, respectively. However, inhibition of Rho-associated kinases (ROCKs) by Y-27632 or nitric oxide synthase (NOS) by L-NAME failed to circumvent the inhibitory effect of RSV. Finally, we examined the effect of RSV during β-adrenergic receptor stimulation by isoproterenol and observed that RSV significantly suppresses the β-adrenergic responses in both L-type Ca2+ currents and contraction parameters. In conclusion, RSV modulates the β-adrenergic signaling cascade and thereby mimics the impact of β-adrenergic receptor blockers in adult ventricular myocytes through modulation of the AC-cAMP-PKA pathway.

Alpha-Adrenergic Regulation of Camp in Ventricular Cardiac Myocytes

In cardiac myocytes, α-adrenergic receptors (α-AR) mediate a wide variety of cellular responses, ranging from cell survival to contraction. Previous studies have demonstrated that α-AR activation can also inhibit β-adrenergic receptor (β-AR) induced cAMP-dependent functional responses. To investigate this phenomenon further, we studied the effects of α-AR stimulation on cAMP activity using fluorescence resonance energy transfer (FRET) based biosensors targeted to different subcellular locations in adult rat ventricular myocytes.