Adult Mouse Ventricular Myocytes Research Articles

Abstract 105: Tuning EC-Coupling: β -Adrenergic Receptor Activation Stimulates Dynamic Augmentation of Ca V 1.2 Channel Sarcolemmal Abundance and Cooperativity

Voltage-gated L-type Ca V 1.2 channels play an essential role in cardiac excitation-contraction coupling. These channels decorate the z-lines of ventricular myocytes where they form discrete clusters on the t-tubule membrane. We have previously reported that groups of channels within these clusters are capable of influencing each other’s gating behavior and undergo dynamic, cooperative interactions. This occurs via Ca 2+ -calmodulin-dependent, physical interactions between the C-terminal tails of adjacent channels. The interactions increase the open probability ( P o ) of adjoined channels, and thus amplify Ca 2+ influx into the myocytes. It has long been known that activation of β -adrenergic receptors during the ‘fight or flight’ response, leads to increased mean open time and P o of Ca V 1.2 channels, and thus enhanced Ca 2+ influx. Given our previous finding that Ca V 1.2 channel cooperativity is triggered by Ca 2+ -influx, we predicted that β -AR stimulation could affect Ca V 1.2 channel cooperativity in ventricular myocytes. To test this hypothesis, we investigated the effect of the β -agonist isoproterenol (ISO) on Ca V 1.2 channel clustering and cooperativity using a combination of optical and electrophysiological approaches. Ground State Depletion super-resolution nanoscopy revealed ‘super-clustering’ of Ca V 1.2 channels in ISO-stimulated adult mouse ventricular myocytes. Channels within these super-clusters display enhanced physical interactions compared to control unstimulated cells as revealed by Bimolecular fluorescence complementation. To visualize this process, we performed live cardiomyocyte imaging and observed a robust, dynamic increase in sarcolemmal Ca V 1.2 channel abundance in response to ISO. Functionally, optical and electrophysiologicaI recordings of Ca V 1.2 channel activity revealed that ISO-stimulated cells display enhanced Ca V 1.2 channel cooperativity, and amplified Ca 2+ influx. On the basis of these data, we propose that stimulation of β ARs initiates dynamic augmentation of Ca V 1.2 channel abundance, and enhanced cooperative gating of Ca V 1.2 channels, in the sarcolemma of ventricular myocytes, to amplify Ca 2+ influx into these cells and tune EC-coupling in times of high metabolic or hemodynamic demand.

Cartilage intermediate layer protein-1 alleviates pressure overload-induced cardiac fibrosis via interfering TGF-β1 signaling

Cardiac fibrosis is characterized by excessive deposition of extracellular matrix (ECM) proteins in the myocardium and results in decreased ventricular compliance and diastolic dysfunction. Cartilage intermediate layer protein-1 (CILP-1), a novel identified cardiac matricellular protein, is upregulated in most conditions associated with cardiac remodeling, however, whether CILP-1 is involved in pressure overload-induced fibrotic response is unknown. Here, we investigated whether CILP-1 was critically involved in the fibrotic remodeling induced by pressure overload. Western blot analysis and immunofluorescence staining showed that CILP-1 was predominantly detected in cardiac myocytes and to a less extent in the interstitium. In isolated adult mouse ventricular myocytes and nonmyocytes, CILP-1 was found to be mainly synthesized by myocytes. CILP-1 expression in left ventricles was upregulated in C57BL/6 mice undergoing transverse aortic constriction (TAC). Myocardial CILP-1 knockdown aggravated whereas CILP-1 overexpression attenuated TAC-induced ventricular remodeling and dysfunction, as measured by echocardiography test, morphological examination, and gene expressions of fibrotic molecules. Incubation of cardiac fibroblasts with the conditioned medium containing full-length, N-terminal, or C-terminal CILP-1 inhibited transforming growth factor (TGF)-β1-induced Smad3 phosphorylation and the subsequent profibrotic events. We first demonstrated that C-terminal CILP-1 increased Akt phosphorylation, promoted the interaction between Akt and Smad3, and suppressed Smad3 phosphorylation. Blockade of PI3K-Akt pathway attenuated the inhibitory effect of C-CILP-1 on TGF-β1-induced Smad3 activation. We conclude that CILP-1 is a novel ECM protein possessing anti-fibrotic ability in pressure overload-induced fibrotic remodeling. This anti-fibrotic effect of CILP-1 attributes to interfering TGF-β1 signaling through its N- and C- terminal fragments.

Diffusional and Electrical Properties of T-Tubules Are Governed by Their Constrictions and Dilations

Cardiac t-tubules (TTs) form a network of complex surface membrane invaginations that is essential for proper excitation-contraction coupling. Although electron and optical microscopy studies provided a wealth of important information about the structure of TTs, assessing their functional properties remains a challenge. In this study, we investigated the diffusional accessibility of TTs in intact isolated adult mouse ventricular myocytes using, to our knowledge, a novel fluorescence-based assay. In this approach, a small part of TTs is first locally filled with fluorescent dextran and then its diffusion out of TTs is monitored after rapid removal of extracellular dextran. In normal cells, diffusion of 3 kDa dextran is characterized by an average time constant of 3.9 ± 1.2 s with the data ranging from 1.8 to 10.5 s. The data are consistent with essentially free diffusion of dextran in TTs although measurable contribution of binding is also evident. TT fluorescence is abolished in cells treated with high concentration of formamide or after hyposmotic stress. Importantly, the assay we use allows for quantitative, repetitive measurements of subtle dynamic changes in TT structure of the same cell that are not possible to observe with other approaches. In particular, dextran diffusion rate decreases two-to-threefold during cell swelling, suggesting significant structural remodeling of TTs. Computer modeling shows that diffusional accessibility and electrical properties of TTs are primarily determined by the constrictions and dilations of individual TTs and that, from a functional perspective, TTs cannot be considered as a network of cylinders of the same average diameter. Constriction/dilation model of cardiac TTs is in a quantitative agreement with previous high-resolution microscopy studies of TT structure and alternative measurements of diffusional and electrical time constants of TTs. The data also show that the apparent electrical length constant of cardiac TTs is likely several-fold smaller than that estimated in earlier studies.

High-Throughput Screen Identifies Novel Small Molecule Stress Regulator That Confers Cardioprotection During Ischemia-Reperfusion Injury

Rationale: During ischemia-reperfusion (IR) injury undue amounts of reactive oxygen species are generated and potentiate myocyte death and cardiac dysfunction. Recently, our lab published a new role for the adaptive cardiac sensor, ATF6, as a key contributor to promoting an adaptive response to oxidative stress. These results highlight the need for the development of small molecules that preferentially activate ATF6 to ameliorate myocardial damage. Through the use of a cell-based high throughput-screen we have identified a small molecular activator of ATF6 and seek to test its efficacy in the heart. Objective/Methods: To address this question, we performed dose response curves of the activator, Compound 147, in cultured neonatal rat ventricular myocytes to examine specific activity of ATF6. We then performed viability studies with chemical stressors and simulated IR. We replicated these activity and IR viability experiments in adult mouse ventricular myocytes and in an ex vivo Langendorff model of the isolated perfused heart. Results: Treatment of cultured ventricular myocytes with Compound 147 specifically and acutely activated ATF6 and primed cells to mount an adaptive response when treated with pharmacological cytotoxic stressors. Treatment of both neonatal and adult ventricular myocytes with Compound 147 increased survivability when subjected to simulated IR injury in vitro. The specificity and activation potential of Compound 147 was confirmed in vivo, by injecting mice intravenously. Furthermore, hearts from mice receiving Compound 147 injections displayed better recovery in response to IR ex vivo. Conclusions: ATF6 is an adaptive cardiac sensor responsible for promoting cardioprotection through the induction of protective targets (e.g. catalase) in response to oxidative stress. As such, it was pertinent to find a small molecule activator of this pathway and examine its efficacy in a clinically relevant scenario of ischemia-reperfusion injury. Compound 147 specifically activates ATF6 and confers cardioprotection during IR in vitro and ex vivo.

An Alpha-1A Adrenergic Receptor Agonist Prevents Acute Doxorubicin Cardiomyopathy in Male Mice.

Alpha-1 adrenergic receptors mediate adaptive effects in the heart and cardiac myocytes, and a myocyte survival pathway involving the alpha-1A receptor subtype and ERK activation exists in vitro. However, data in vivo are limited. Here we tested A61603 (N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide), a selective imidazoline agonist for the alpha-1A. A61603 was the most potent alpha-1-agonist in activating ERK in neonatal rat ventricular myocytes. A61603 activated ERK in adult mouse ventricular myocytes and protected the cells from death caused by the anthracycline doxorubicin. A low dose of A61603 (10 ng/kg/d) activated ERK in the mouse heart in vivo, but did not change blood pressure. In male mice, concurrent subcutaneous A61603 infusion at 10 ng/kg/d for 7 days after a single intraperitoneal dose of doxorubicin (25 mg/kg) increased survival, improved cardiac function, heart rate, and cardiac output by echocardiography, and reduced cardiac cell necrosis and apoptosis and myocardial fibrosis. All protective effects were lost in alpha-1A-knockout mice. In female mice, doxorubicin at doses higher than in males (35–40 mg/kg) caused less cardiac toxicity than in males. We conclude that the alpha-1A-selective agonist A61603, via the alpha-1A adrenergic receptor, prevents doxorubicin cardiomyopathy in male mice, supporting the theory that alpha-1A adrenergic receptor agonists have potential as novel heart failure therapies.

Calcitriol (1,25-dihydroxyvitamin D3) increases L-type calcium current via protein kinase A signaling and modulates calcium cycling and contractility in isolated mouse ventricular myocytes

Calcitriol, the bioactive metabolite of vitamin D, exerts its effects through interaction with the nuclear vitamin D receptor (VDR) to induce genomic responses. Calcitriol may also induce rapid responses via plasma membrane-associated VDR, involving the activation of second messengers and modulation of voltage-dependent channels. VDR is expressed in cardiomyocytes, but the molecular and cellular mechanisms involved in the rapid responses of calcitriol in the heart are poorly understood. The aim of the present study was to analyze the rapid nongenomic effect of calcitriol on L-type calcium channels, intracellular Ca2+ ([Ca2+]i) transients, and cell contractility in ventricular myocytes. We used the whole-cell patch-clamp technique to record L-type calcium current (ICaL) and confocal microscopy to study global [Ca2+]i transients evoked by electrical stimulation and cell shortening in adult mouse ventricular myocytes treated with vehicle or with calcitriol. In some experiments, ICaL was recorded using the perforated patch-clamp technique. Calcitriol treatment of cardiomyocytes induced a concentration-dependent increase in ICaL density (Half maximal effective concentration (EC50) = 0.23 nM) and a significant increase in peak [Ca2+]i transients and cell contraction. The effect of calcitriol on ICaL was prevented by pretreatment of cardiomyocytes with the protein kinase A (PKA) inhibitor KT-5720 but not with the β-adrenergic blocker propranolol. The effect of calcitriol on ICaL was absent in myocytes isolated from VDR knockout mice. Calcitriol induces a rapid response in mouse ventricular myocytes that involves a VDR-PKA-dependent increase in ICaL density, enhancing [Ca2+]i transients and contraction.

BAG3 regulates contractility and Ca2 + homeostasis in adult mouse ventricular myocytes

Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations, including mutations leading to loss of protein, are associated with familial cardiomyopathy. Furthermore, BAG3 levels have been found to be reduced in end-stage non-familial failing myocardium. In contrast to neonatal myocytes in which BAG3 is found in the cytoplasm and involved in protein quality control and apoptosis, in adult mouse left ventricular (LV) myocytes BAG3 co-localized with Na+–K+–ATPase and L-type Ca2+ channels in the sarcolemma and t-tubules. BAG3 co-immunoprecipitated with β1-adrenergic receptor, L-type Ca2+ channels and phospholemman. To simulate decreased BAG3 protein levels observed in human heart failure, we targeted BAG3 by shRNA (shBAG3) in adult LV myocytes. Reducing BAG3 by 55% resulted in reduced contraction and [Ca2+]i transient amplitudes in LV myocytes stimulated with isoproterenol. L-type Ca2+ current (ICa) and sarcoplasmic reticulum (SR) Ca2+ content but not Na+/Ca2+ exchange current (INaCa) or SR Ca2+ uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyryl cAMP restored ICa amplitude in shBAG3 myocytes to that observed in WT myocytes, consistent with BAG3 having effects upstream and at the level of the receptor. Resting membrane potential and action potential amplitude were unaffected but APD50 and APD90 were prolonged in shBAG3 myocytes. Protein levels of Ca2+ entry molecules and other important excitation–contraction proteins were unchanged in myocytes with lower BAG3. Our findings that BAG3 is localized at the sarcolemma and t-tubules while modulating myocyte contraction and action potential duration through specific interaction with the β1-adrenergic receptor and L-type Ca2+ channel provide novel insight into the role of BAG3 in cardiomyopathies and increased arrhythmia risks in heart failure.

Higenamine protects ischemia/reperfusion induced cardiac injury and myocyte apoptosis through activation of β2-AR/PI3K/AKT signaling pathway

Cardiomyocyte apoptosis contributes to ischemic cardiac injury and the development of heart failure. Higenamine is a key component of the Chinese herb aconite root that has been prescribed for treating symptoms of heart failure for thousands of years in the oriental Asian countries. It has been shown that higenamine has anti-apoptotic effects in a few cell types including cardiomyocytes. However, the pharmacological target and molecular mechanism of higenamine in the heart are still not fully illustrated. Herein, we report that higenamine protected myocyte apoptosis and ischemia/reperfusion (I/R) injury through selective activation of beta2-adrenergic receptor (β2-AR). In particular, we show that higenamine significantly reduced I/R-induced myocardial infarction in mice. In both primary neonatal rat and adult mouse ventricular myocytes, we show higenamine inhibited cell apoptosis and also reduced biochemical markers of apoptosis such as cleaved caspase 3 and 9. More importantly, we show that the anti-apoptotic effects of higenamine in cardiomyocytes were completely abolished by β2-AR but not β1-AR antagonism. Furthermore, we confirmed that higenamine attenuated I/R-induced myocardial injury and reduced cleaved caspases in a β2-AR dependent manner in intact mouse hearts. Higenamine stimulated AKT phosphorylation and required PI3K activation for the anti-apoptotic effect in cardiomyocytes. These findings together suggest that anti-apoptotic and cardiac protective effects of higenamine are mediated by the β2-AR/PI3K/AKT cascade.

β₁-adrenoceptor stimulation promotes LPS-induced cardiomyocyte apoptosis through activating PKA and enhancing CaMKII and IκBα phosphorylation.

IntroductionCaspase activation and cardiomyocyte apoptosis have been implicated in lipopolysaccharide (LPS)-induced cardiac contractile dysfunction. We have recently demonstrated that β1-adrenoceptor (AR) activation by endogenous norepinephrine contributes to cardiomyocyte apoptosis in endotoxemic mice. Here, we further investigated the molecular mechanisms for the enhancing effect of β1-AR activation on LPS-induced cardiomyocyte apoptosis.MethodsThe adult mouse ventricular myocytes were exposed to LPS, dobutamine, protein kinase A (PKA) inhibitor or/and nifedipine, an L-type Ca2+ channel blocker. Male BALB/c mice were treated with LPS or/ and β1-AR antagonist, atenolol. Cardiomyocyte apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assay and apoptosis-associated molecules were detected.ResultsLPS induced apoptosis in adult mouse ventricular myocytes, dobutamine (DOB), a β1-AR agonist, promoted apoptosis, caspase-8, 9 and 3 activation and increased cytosolic Ca2+ concentration in LPS-challenged cardiomyocytes. DOB also up-regulated TNF-α expression, decreased Bcl-2 levels, promoted Bax translocation to mitochondria, mitochondrial membrane potential loss and cytochrome c release as well as IκBα, p38 MAPK, JNK and Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation in LPS-treated cardiomyocytes. PKA inhibitor abolished the effects of DOB on caspase-9 activation, Bcl-2 levels as well as JNK and p38 MAPK phosphorylation, but not on IκBα phosphorylation, TNF-α expression and caspase-8 activation in LPS-stimulated cardiomyocytes. Pretreatment with nifedipine not only significantly blocked the enhancing effects of DOB on LPS-induced elevation in cytosolic Ca2+ concentration and CaMKII phosphorylation in cardiomyocytes, but also partly reversed the effects of DOB on caspase-9 and caspase-3/7 activities in LPS-treated cardiomyocytes. Furthermore, atenolol suppressed TNF-α expression, JNK, p38 MAPK and CaMKII phosphorylation, increased Bcl-2 expression, and inhibited cytochrome c release and cardiomyocyte apoptosis in the myocardium of endotoxemic mice.Conclusionsβ1-AR activation promotes LPS-induced apoptosis through activating PKA, increasing CaMKII phosphorylation as well as enhancing IκBα phosphorylation and TNF-α expression in cardiomyocytes.

Abstract 11016: Bag3 Regulates Contractility and Ca Homeostasis in Adult Mouse Ventricular Myocytes

Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations are associated with familial dilated cardiomyopathy and reduced BAG3 expression was observed in murine transverse aortic constriction and porcine myocardial infarction models. BAG3 is expressed in the sarcolemma and t-tubules in adult myocytes. To simulate reduced BAG3 expression in disease states, we downregulated BAG3 in adult myocytes by shRNA (shBAG3). Despite ~50% reduction in BAG3 by shRNA, expression of α-subunit of L-type Ca channel, SR Ca-ATPase, Na/Ca exchanger, α1- and α2-subunits of Na-K-ATPase, and ryanodine receptor was unchanged. At baseline, BAG3 downregulation had no effect on myocyte contraction and [Ca]i dynamics. After isoproterenol, BAG3 downregulation resulted in reduced myocyte contraction amplitudes, shortening and re-lengthening velocities, lower systolic [Ca]i and [Ca]i transient amplitudes, and prolongation in both APD50 and APD90. L-type Ca current (ICa) and sarcoplasmic reticulum (SR) Ca content but not Na/Ca exchange current or SR Ca uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyrl cAMP restored ICa amplitude in shBAG3 myocytes to that observed in WT myocytes. On the other hand, BAG3 overexpression resulted in enhanced myocyte contractility after isoproterenol. In addition, BAG3 overexpression rescued the depressed contraction amplitudes in myocytes overexpressing the HIV protein Tat, and improved myocardial contractility in Tg26 (HIV-1 transgenic) mice. We conclude: (1) BAG3 modulates myocyte contraction, Ca dynamics and action potential duration by regulating ion channels and provides a novel paradigm for contractile dysfunction and increased arrhythmia risks in familial dilated cardiomyopathy; (2) BAG3 is a therapeutic target for cardiomyopathy including HIV-associated cardiomyopathy.

Up-regulation of micro-RNA765 in human failing hearts is associated with post-transcriptional regulation of protein phosphatase inhibitor-1 and depressed contractility.

Impaired sarcoplasmic reticulum (SR) Ca(2+) cycling and depressed contractility, a hallmark of human and experimental heart failure, has been partially attributed to increased protein phosphatase 1 (PP-1) activity, associated with down-regulation of its endogenous inhibitor-1. The levels and activity of inhibitor-1 are reduced in failing hearts, contributing to dephosphorylation and inactivation of key calcium cycling proteins. Therefore, we investigated the mechanisms that mediate decreases in inhibitor-1 by post-transcriptional modification. Bioinformatics revealed that 17 human microRNAs may serve as modulators of inhibitor-1. However, real-time PCR analysis identified only one of these microRNAs, miR-765, as being increased in human failing hearts concomitant with decreased inhibitor-1 levels. Expression of miR-765 in HEK293 cells or mouse ventricular myocytes confirmed suppression of inhibitor-1 levels through binding of this miR-765 to the 3'-untranslated region of inhibitor-1 mRNA. To determine the functional significance of miR-765 in Ca(2+) cycling, pri-miR-765 as well as a non-translated nucleotide sequence (miR-Ctrl) were expressed in adult mouse ventricular myocytes. The inhibitor-1 expression levels were decreased, accompanied by enhanced PP-1 activity in the miR-765 cardiomyocytes, and these reflected depressed contractile mechanics and Ca(2+) transients, compared with the miR-Ctrl group. The depressive effects were associated with decreases in the phosphorylation of phospholamban and SR Ca(2+) load. These miR-765 negative inotropic effects were abrogated in inhibitor-1-deficient cardiomyocytes, suggesting its apparent specificity for inhibitor-1. miR-765 levels are increased in human failing hearts. Such increases may contribute to depressed cardiac function through reduced inhibitor-1 expression and enhanced PP-1 activity, associated with reduced SR Ca(2+) load.

Cx43 phosphorylation on S279/282 and intercellular communication are regulated by IP3/IP3 receptor signaling.

BackgroundInositol 1,4,5-trisphosphate receptor (IP3R) plays a pivotal role in the Ca2+ release process in a variety of cell types. Additionally, IP3R is distributed in ventricular intercalated discs, but its function(s) in this particular site remains unknown. Connexin (Cx43), the predominant gap junction (GJ) protein in ventricular myocardium, is linked to several signaling pathways that regulate Cx43 properties by (de)phosphorylation on multiple residues. Here, we investigated the regulatory role of IP3R in cell-cell communication and the mechanism(s) underlying this effect.ResultsIn neonatal rat and adult mouse ventricular myocytes IP3R co-localized and co-immunoprecipitated with Cx43 in GJ plaques detected by immunostaining and western blot assays. Blocking IP3R with antagonists or silencing pan-IP3R expression with shRNA hindered the 6-carboxyfluorescein (6-CFDA) diffusion through GJs and desynchronized Ca2+ transients among confluent neonatal myocytes in culture, whereas stimulation of IP3R with IP3 ester or ATP exerted the opposite effect. Likewise, 6-CFDA propagation through GJs was modulated by IP3R activation or inhibition in cell pairs of isolated adult cardiomyocytes. Furthermore, IP3R activation or IP3R suppression promoted or suppressed, respectively, Cx43 phosphorylation on S279/282. Site-directed mutagenesis indicated that expression of a mutant Cx43-S282A (alanine) inhibited S279/282 phosphorylation and GJ permeability, while the S279A mutant showed the opposite effect in ventricular myocytes. Expression of these mutants in HEK293 cells revealed that cells with a dual S279/282 mutation failed to express exogenous Cx43, whereas cells with a single S279 or S282 mutation displayed Cx43 overexpression with increased phosphorylation of S279/282 and promotion of intercellular communication.ConclusionsThese results demonstrated, for the first time, that IP3R physically interacts with Cx43 and participates in the regulation of Cx43 phosphorylation on S279/282, thereby affecting GJ intercellular communication in ventricular myocytes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-014-0058-6) contains supplementary material, which is available to authorized users.

Transgenic Mice for Real-Time Visualization of cGMP in Intact Adult Cardiomyocytes

3',5'-Cyclic guanosine monophosphate (cGMP) is an important second messenger that regulates cardiac contractility and protects the heart from hypertrophy. However, because of the lack of real-time imaging techniques, specific subcellular mechanisms and spatiotemporal dynamics of cGMP in adult cardiomyocytes are not well understood. Our aim was to generate and characterize a novel cGMP sensor model to measure cGMP with nanomolar sensitivity in adult cardiomyocytes. We generated transgenic mice with cardiomyocyte-specific expression of the highly sensitive cytosolic Förster resonance energy transfer-based cGMP biosensor red cGES-DE5 and performed the first Förster resonance energy transfer measurements of cGMP in intact adult mouse ventricular myocytes. We found very low (≈10 nmol/L) basal cytosolic cGMP levels, which can be markedly increased by natriuretic peptides (C-type natriuretic peptide >> atrial natriuretic peptide) and, to a much smaller extent, by the direct stimulation of soluble guanylyl cyclase. Constitutive activity of this cyclase contributes to basal cGMP production, which is balanced by the activity of clinically established phosphodiesterase (PDE) families. The PDE3 inhibitor, cilostamide, showed especially strong cGMP responses. In a mild model of cardiac hypertrophy after transverse aortic constriction, PDE3 effects were not affected, whereas the contribution of PDE5 was increased. In addition, after natriuretic peptide stimulation, PDE3 was also involved in cGMP/cAMP crosstalk. The new sensor model allows visualization of real-time cGMP dynamics and pharmacology in intact adult cardiomyocytes. Förster resonance energy transfer imaging suggests the importance of well-established and potentially novel PDE-dependent mechanisms that regulate cGMP under physiological and pathophysiological conditions.

Cx43 phosphorylation on S279/282 and intercellular communication are regulated by IP 3 /IP 3 receptor signaling

Inositol 1,4,5-trisphosphate receptor (IP3R) plays a pivotal role in the Ca2+ release process in a variety of cell types. Additionally, IP3R is distributed in ventricular intercalated discs, but its function(s) in this particular site remains unknown. Connexin (Cx43), the predominant gap junction (GJ) protein in ventricular myocardium, is linked to several signaling pathways that regulate Cx43 properties by (de)phosphorylation on multiple residues. Here, we investigated the regulatory role of IP3R in cell-cell communication and the mechanism(s) underlying this effect. In neonatal rat and adult mouse ventricular myocytes IP3R co-localized and co-immunoprecipitated with Cx43 in GJ plaques detected by immunostaining and western blot assays. Blocking IP3R with antagonists or silencing pan-IP3R expression with shRNA hindered the 6-carboxyfluorescein (6-CFDA) diffusion through GJs and desynchronized Ca2+ transients among confluent neonatal myocytes in culture, whereas stimulation of IP3R with IP3 ester or ATP exerted the opposite effect. Likewise, 6-CFDA propagation through GJs was modulated by IP3R activation or inhibition in cell pairs of isolated adult cardiomyocytes. Furthermore, IP3R activation or IP3R suppression promoted or suppressed, respectively, Cx43 phosphorylation on S279/282. Site-directed mutagenesis indicated that expression of a mutant Cx43-S282A (alanine) inhibited S279/282 phosphorylation and GJ permeability, while the S279A mutant showed the opposite effect in ventricular myocytes. Expression of these mutants in HEK293 cells revealed that cells with a dual S279/282 mutation failed to express exogenous Cx43, whereas cells with a single S279 or S282 mutation displayed Cx43 overexpression with increased phosphorylation of S279/282 and promotion of intercellular communication. These results demonstrated, for the first time, that IP3R physically interacts with Cx43 and participates in the regulation of Cx43 phosphorylation on S279/282, thereby affecting GJ intercellular communication in ventricular myocytes.

Nitroxyl (HNO)

Background— The nitroxyl (HNO) donor, Angeli’s salt, exerts positive inotropic, lusitropic, and vasodilator effects in vivo that are cAMP independent. Its clinical usefulness is limited by chemical instability and cogeneration of nitrite which itself has vascular effects. Here, we report on effects of a novel, stable, pure HNO donor (CXL-1020) in isolated myoctyes and intact hearts in experimental models and in patients with heart failure (HF). Methods and Results— CXL-1020 converts solely to HNO and inactive CXL-1051 with a t 1/2 of 2 minutes. In adult mouse ventricular myocytes, it dose dependently increased sarcomere shortening by 75% to 210% (50–500 μmol/L), with a ≈30% rise in the peak Ca 2+ transient only at higher doses. Neither inhibition of protein kinase A nor soluble guanylate cyclase altered this contractile response. Unlike isoproterenol, CXL-1020 was equally effective in myocytes from normal or failing hearts. In anesthetized dogs with coronary microembolization-induced HF, CXL-1020 reduced left ventricular end-diastolic pressure and myocardial oxygen consumption while increasing ejection fraction from 27% to 40% and maximal ventricular power index by 42% (both P <0.05). In conscious dogs with tachypacing-induced HF, CXL-1020 increased contractility assessed by end-systolic elastance and provided venoarterial dilation. Heart rate was minimally altered. In patients with systolic HF, CXL-1020 reduced both left and right heart filling pressures and systemic vascular resistance, while increasing cardiac and stroke volume index. Heart rate was unchanged, and arterial pressure declined modestly. Conclusions— These data show the functional efficacy of a novel pure HNO donor to enhance myocardial function and present first-in-man evidence for its potential usefulness in HF. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifiers: NCT01096043, NCT01092325.

Abstract 194: Phosphodiesterase 3 Controls Basal cGMP Levels And Regulates cGMP/cAMP Cross-talk In Adult Mouse Ventricular Cardiomyocytes

PURPOSE: cGMP is an important second messenger which is involved in the regulation of cardiac contractility and pathological hypertrophy. In cardiomyocytes, signaling by cGMP is organized in microdomains and is considered cardioprotective. Especially in adult cardiac myocytes, measurements of cGMP have been challenging and little is known about the spatio-temporal dynamics of cGMP. Here we developed a transgenic mouse model to visualize cGMP dynamis in adult cardiac myocytes. Methods: We generated transgenic mice with cardiomyocyte-specific expression of a highly sensitive fluorescence resonance energy transfer (FRET)-based cGMP biosensor red cGES-DE5 and performed FRET measurements in freshly isolated adult mouse ventricular myocytes. To analyze cGMP/cAMP crosstalk, FRET experiments were performed in cardiomyocytes isolated from mice transgenically expressing the cAMP sensor Epac1-camps. Results: Basal cytosolic cGMP levels were very low (~10 nM), but could be markedly increased by stimulation with natriuretic peptides (CNP>>ANP). In contrast, direct stimulation of the soluble guanylyl cyclase (sGC) with NO-donors such as SNAP showed no effect. However, constitutive activity of this cyclase contributes to basal cGMP production, since a clear decrease of basal cGMP levels was observed after stimulation with the sGC inhibitor ODQ. This basal cGMP production is regulated by phosphodiesterase (PDE) activity. Unexpectedly, PDE3 is most important in controlling basal cGMP levels, whereas PDE2 and PDE5 are much less active. We could also show that cGMP pools produced by GC-B after CNP stimulation are mainly regulated by PDE3, so that the receptor and this PDE form one functional unit important for the regulation of cGMP/cAMP cross-talk. Conclusion: In summary, we performed the first FRET-based measurements of cGMP in adult cardiomyocytes and we could highlight the key role of PDE3 in the regulation of basal cGMP levels and cGMP/cAMP cross-talk.

PDE3 is the major cGMP-PDE in adult mouse ventricular cardiomyocytes

Materials and methods We generated a transgenic mouse model with cardiomyocyte-specific expression of the highly sensitive fluorescence resonance energy transfer (FRET)-based cGMP biosensor red cGES-DE5. This sensor mouse allows us to performed FRET measurements of cGMP in freshly isolated adult mouse ventricular myocytes, to visulaize realtime dynamics of cGMP. To analyze cAMP and cGMP interactions (cGMP/cAMP signaling crosstalk), FRET experiments were performed in cardiomyocytes isolated from mice, transgenically expressing the FRET-based cAMP sensor Epac1-camps.

Abstract 19629: Expression and Role of TRPC Channels in Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells

Store-operated Ca 2+ entry (SOCE) is a mechanism for increasing cytosolic Ca 2+ (Ca i 2+ ) load after the SR/ER Ca 2+ depletion in many cell types. Our previous study has implicated SOCE, which is at least partially mediated by transient receptor potential canonical (TRPC) channels, exists in adult mouse ventricular myocytes, and contributes to the generation of spontaneous SR Ca 2+ release and triggered arrhythmias. However, it is unclear if this mechanism also exists in human cardiac myocytes. In the present study, we investigate the expression and role of TRPC channels in cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) (purchased from Cellular Dynamics). Abundant distributions of TRPC3 and 6 channels were detected in the plasma membrane of hiPSC-CMs using immunocytochemical staining. Ca i 2+ was imaged in fluo-4-AM loaded cells. A SOCE (F/F 0 = 1.75 ± 0.08, n = 47) was observed in hiPSC-CMs when the external Ca 2+ concentration ([Ca 2+ ] o ) was switched from 0 to 1 mM after SR Ca 2+ was completely depleted by caffeine (20 mM) and thapsigargin (10 µM). The SOCE was inhibited by non-selective TRPC blockers gadolinium (F/F 0 = 1.05 ± 0.03, n = 13) and SKF-96265 (F/F 0 = 1.04 ± 0.03, n = 17), respectively, compared to control (p<0.05). Whereas, the SOCE in hiPSC-CMs was significantly reduced only by pretreatment with anti-TRPC6 antibody (F/F 0 = 1.38 ± 0.06, n = 15, p<0.05), but not with anti-TRPC1 (F/F 0 = 1.93 ± 0.19, n = 8) or anti-TRPC3 antibodies (F/F 0 = 1.49 ± 0.05, n = 14), indicating TRPC6 channels are essential for SOCE in hiPSC-CMs. In addition, the hiPSC-CMs exhibit a property of spontaneous rhythmic beating. After SR Ca 2+ depletion, spontaneous Ca 2+ waves reappeared via refilling from the extracellular Ca 2+ source, suggesting the involvement of SOCE in the generation of spontaneous Ca 2+ waves. This idea was confirmed by the application of TRPC blockers, including gadolinium, SKF-96265 and Pyr3, all of which showed mild to potent inhibitory effects on the spontaneous Ca 2+ waves. In conclusion, we demonstrated the existence of SOCE in hiPSC-CMs. SOCE mediated by TRPC channels, especially the TRPC6, may act as an important mechanism of controlling Ca 2+ handling and arrhythmogenesis in the developing human heart.

β1 Integrin Gene Excision in the Adult Murine Cardiac Myocyte Causes Defective Mechanical and Signaling Responses

How mechanical signals are transmitted in the cardiac myocyte is poorly understood. In this study, we produced a tamoxifen-inducible mouse model in which β1 integrin could be reduced specifically in the adult cardiomyocyte, so that the function of this integrin could be assessed in the postnatal and mechanically stressed heart. The expression of β1 integrin was reduced to 35% of control levels, but function remained normal at baseline. With aortic constriction, the knockout mice survived but had a blunted hypertrophic response. Integrin knockout myocytes, in contrast to controls, showed reduced integrin-linked kinase expression both at baseline and after hemodynamic stress; focal adhesion kinase expression was reduced after stress. Alterations in multiple signaling pathways were detected in the integrin knockout group after acute and chronic hemodynamic stress. Most remarkably, when we challenged the knockout mice with short-term loading, the robust responses of several kinases (extracellular signal-regulated kinase 1/2, p38, and Akt) evident in control mice were essentially abolished in the knockout mice. We also found that reduction of myocyte β1 integrin expression modified adrenergic-mediated signaling through extracellular signal-regulated kinase, p38, and Akt. Reduction of β1 integrin expression in the mature cardiac myocyte leads to a varied response compared with when this protein is reduced during either the embryonic or perinatal period. These results show that β1 integrin expression is required for proper mechanotransductive and adrenergic responses of the adult heart.

3D Imaging of Nanoscopic Membrane Systems Regulating Cardiac Excitation-Contraction Coupling: Multi-Color Optical Super-Resolution and Serial Block-Face Scanning Electron Microscopy

Cardiac excitation contraction (E-C) coupling is controlled by ionic fluxes in couplons, nano-scale junctional structures between sarcolemma and sarcoplasmic reticulum (SR) membranes. The 3-dimensional (3D) geometry of cardiac couplons and their relationship to the transverse-tubule system (t-system) is complex with little 3D data available. Using super-resolution microscopy, we successfully imaged couplons in normal adult mouse ventricular myocytes with a resolution of about 30nm. A fluorescence microscope equipped with an insertable cylindrical lens and an EMCCD camera enabled multi-color 3-dimensional dSTORM imaging and recorded fluorophore “blinking”: rapid transitions between a photon-emitting “on”-state and laser-induced “dark”-state. This data was analyzed with custom-written software to localize individual fluorophores and render super-resolution images. We used ryanodine receptor (RyR) to identify couplons and the sarcolemmal protein Caveolin-3 (CAV3) to visualize the t-system. Separately, we applied an advanced 3D electron microscopic technology, Serial Block-face Scanning Electron microscopy (SBFSEM), to cardiac muscle for the first time to image, segment, and annotate couplons and the t-system in mouse cardiac myocytes. Expectedly, the majority of RyR clusters aggregated near the t-system. The individual morphology and size of couplons visualized with dSTORM and SBFSEM were both highly variable, in agreement with our recent publications. Furthermore, both microscopies strongly suggested a significant enlargement in diameter of the t-system at loci where couplons accumulated. This enlargement may contribute to minimizing ion accumulation or depletion. Alternatively, it could reflect a geometrical requirement for forming larger junctional complexes. Collectively, the study revealed the strength of integrating new advanced 3D light and electron microscopies. Knowledge of nanoscale anatomy may be critical both to mechanistic understanding of E-C coupling and as a resource to generate realistic computational models that can simulate E-C coupling and other muscle biology.