Microtubule-Mediated Regulation of ß2AR Translation and Function in Failing Hearts.

BACKGROUND: ß1AR (beta-1 adrenergic receptor) and ß2AR (beta-2 adrenergic receptor)-mediated cyclic adenosine monophosphate signaling has distinct effects on cardiac function and heart failure progression. However, the mechanism regulating spatial localization and functional compartmentation of cardiac ß-ARs remains elusive. Emerging evidence suggests that microtubule-dependent trafficking of mRNP (messenger ribonucleoprotein) and localized protein translation modulates protein compartmentation in cardiomyocytes. We hypothesized that ß-AR compartmentation in cardiomyocytes is accomplished by selective trafficking of its mRNAs and localized translation. METHODS: The localization pattern of ß-AR mRNA was investigated using single molecule fluorescence in situ hybridization and subcellular nanobiopsy in rat cardiomyocytes. The role of microtubule on ß-AR mRNA localization was studied using vinblastine, and its effect on receptor localization and function was evaluated with immunofluorescent and high-throughput Förster resonance energy transfer microscopy. An mRNA protein co-detection assay identified plausible ß-AR translation sites in cardiomyocytes. The mechanism by which ß-AR mRNA is redistributed post-heart failure was elucidated by single molecule fluorescence in situ hybridization, nanobiopsy, and high-throughput Förster resonance energy transfer microscopy on 16 weeks post-myocardial infarction and detubulated cardiomyocytes. RESULTS: ß1AR and ß2AR mRNAs show differential localization in cardiomyocytes, with ß1AR found in the perinuclear region and ß2AR showing diffuse distribution throughout the cell. Disruption of microtubules induces a shift of ß2AR transcripts toward the perinuclear region. The close proximity between ß2AR transcripts and translated proteins suggests that the translation process occurs in specialized, precisely defined cellular compartments. Redistribution of ß2AR transcripts is microtubule-dependent, as microtubule depolymerization markedly reduces the number of functional receptors on the membrane. In failing hearts, both ß1AR and ß2AR mRNAs are redistributed toward the cell periphery, similar to what is seen in cardiomyocytes undergoing drug-induced detubulation. This suggests that t-tubule remodeling contributes to ß-AR mRNA redistribution and impaired ß2AR function in failing hearts. CONCLUSIONS: Asymmetrical microtubule-dependent trafficking dictates differential ß1AR and ß2AR localization in healthy cardiomyocyte microtubules, underlying the distinctive compartmentation of the 2 ß-ARs on the plasma membrane. The localization pattern is altered post-myocardial infarction, resulting from transverse tubule remodeling, leading to distorted ß2AR-mediated cyclic adenosine monophosphate signaling.

Functional LTCC-ß2AR Complex Needs Caveolin-3 and Is Disrupted in Heart Failure.

BACKGROUND: Beta-2 adrenergic receptors (ß2ARs) but not beta-2 adrenergic receptors (ß1ARs) form a functional complex with L-type Ca2+ channels (LTCCs) on the cardiomyocyte membrane. However, how microdomain localization in the plasma membrane affects the function of these complexes is unknown. We aim to study the coupling between LTCC and ß adrenergic receptors in different cardiomyocyte microdomains, the distinct involvement of PKA and CAMKII (Ca2+/calmodulin-dependent protein kinase II) and explore how this functional complex is disrupted in heart failure. METHODS: Global signaling between LTCCs and ß adrenergic receptors was assessed with whole-cell current recordings and western blot analysis. Super-resolution scanning patch-clamp was used to explore the local coupling between single LTCCs and ß1AR or ß2AR in different membrane microdomains in control and failing cardiomyocytes. RESULTS: LTCC open probability (Po) showed an increase from 0.054±0.003 to 0.092±0.008 when ß2AR was locally stimulated in the proximity of the channel (<350 nm) in the transverse tubule microdomain. In failing cardiomyocytes, from both rodents and humans, this transverse tubule coupling between LTCC and ß2AR was lost. Interestingly, local stimulation of ß1AR did not elicit any change in the Po of LTCCs, indicating a lack of proximal functional interaction between the two, but we confirmed a general activation of LTCC via ß1AR. By using blockers of PKA and CaMKII and a Caveolin-3-knockout mouse model, we conclude that the ß2AR-LTCC regulation requires the presence of caveolin-3 and the activation of the CaMKII pathway. By contrast, at a cellular "global" level PKA plays a major role downstream ß1AR and results in an increase in LTCC current. CONCLUSIONS: Regulation of the LTCC activity by proximity coupling mechanisms occurs only via ß2AR, but not ß1AR. This may explain how ß2ARs tune the response of LTCCs to adrenergic stimulation in healthy conditions. This coupling is lost in heart failure; restoring it could improve the adrenergic response of failing cardiomyocytes.

Caveolin-3 and Caveolae regulate ventricular repolarization.

RATIONALE: Flask-shaped invaginations of the cardiomyocyte sarcolemma called caveolae require the structural protein caveolin-3 (Cav-3) and host a variety of ion channels, transporters, and signaling molecules. Reduced Cav-3 expression has been reported in models of heart failure, and variants in CAV3 have been associated with the inherited long-QT arrhythmia syndrome. Yet, it remains unclear whether alterations in Cav-3 levels alone are sufficient to drive aberrant repolarization and increased arrhythmia risk. OBJECTIVE: To determine the impact of cardiac-specific Cav-3 ablation on the electrophysiological properties of the adult mouse heart. METHODS AND RESULTS: Cardiac-specific, inducible Cav3 homozygous knockout (Cav-3KO) mice demonstrated a marked reduction in Cav-3 expression by Western blot and loss of caveolae by electron microscopy. However, there was no change in macroscopic cardiac structure or contractile function. The QTc interval was increased in Cav-3KO mice, and there was an increased propensity for ventricular arrhythmias. Ventricular myocytes isolated from Cav-3KO mice exhibited a prolonged action potential duration (APD) that was due to reductions in outward potassium currents (Ito, Iss) and changes in inward currents including slowed inactivation of ICa,L and increased INa,L. Mathematical modeling demonstrated that the changes in the studied ionic currents were adequate to explain the prolongation of the mouse ventricular action potential. Results from human iPSC-derived cardiomyocytes showed that shRNA knockdown of Cav-3 similarly prolonged APD. CONCLUSION: We demonstrate that Cav-3 and caveolae regulate cardiac repolarization and arrhythmia risk via the integrated modulation of multiple ionic currents.

Localization of stomatal lineage proteins reveals contrasting planar polarity patterns in Arabidopsis cotyledons.

Many plant cells exhibit polarity, revealed by asymmetric localization of specific proteins within each cell.1,2,3,4,5,6 Polarity is typically coordinated between cells across a tissue, raising the question of how coordination is achieved. One hypothesis is that mechanical stresses provide cues.7 This idea gains support from experiments in which cotyledons were mechanically stretched transversely to their midline.8 These previously published results showed that without applied tension, the stomatal lineage cell polarity marker, BREVIS RADIX-LIKE 2 (BRXL2), exhibited no significant excess in the transverse orientation. By contrast, 7 h after stretching, BRXL2 polarity distribution exhibited transverse excess, aligned with the stretch direction. These stretching experiments involved statistical comparisons between snapshots of stretched and unstretched cotyledons, with different specimens being imaged in each case.8 Here, we image the same cotyledon before and after stretching and find no evidence for reorientation of polarity. Instead, statistical analysis shows that cotyledons contain a pre-existing transverse excess in BRXL2 polarity orientation that is not significantly modified by applied tension. The transverse excess reflects BRLX2 being preferentially localized toward the medial side of the cell, nearer to the cotyledon midline, creating a weak medial bias. A second polarity marker, BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), also exhibits weak medial bias in stomatal lineages, whereas ectopic expression of BASL in non-stomatal cells exhibits strong proximal bias, as previously observed in rosette leaves. This proximal bias is also unperturbed by applied tension. Our findings therefore show that cotyledons contain two near-orthogonal coordinated biases in planar polarity: mediolateral and proximodistal.

Local hyperactivation of L-type Ca2+ channels increases spontaneous Ca2+ release activity and cellular hypertrophy in right ventricular myocytes from heart failure rats.

Right ventricle (RV) dysfunction is an independent predictor of patient survival in heart failure (HF). However, the mechanisms of RV progression towards failing are not well understood. We studied cellular mechanisms of RV remodelling in a rat model of left ventricle myocardial infarction (MI)-caused HF. RV myocytes from HF rats show significant cellular hypertrophy accompanied with a disruption of transverse-axial tubular network and surface flattening. Functionally these cells exhibit higher contractility with lower Ca2+ transients. The structural changes in HF RV myocytes correlate with more frequent spontaneous Ca2+ release activity than in control RV myocytes. This is accompanied by hyperactivated L-type Ca2+ channels (LTCCs) located specifically in the T-tubules of HF RV myocytes. The increased open probability of tubular LTCCs and Ca2+ sparks activation is linked to protein kinase A-mediated channel phosphorylation that occurs locally in T-tubules. Thus, our approach revealed that alterations in RV myocytes in heart failure are specifically localized in microdomains. Our findings may indicate the development of compensatory, though potentially arrhythmogenic, RV remodelling in the setting of LV failure. These data will foster better understanding of mechanisms of heart failure and it could promote an optimized treatment of patients.

Ventricular fibrillation mechanism and global fibrillatory organization are determined by gap junction coupling and fibrosis pattern.

AIMS: Conflicting data exist supporting differing mechanisms for sustaining ventricular fibrillation (VF), ranging from disorganized multiple-wavelet activation to organized rotational activities (RAs). Abnormal gap junction (GJ) coupling and fibrosis are important in initiation and maintenance of VF. We investigated whether differing ventricular fibrosis patterns and the degree of GJ coupling affected the underlying VF mechanism. METHODS AND RESULTS: Optical mapping of 65 Langendorff-perfused rat hearts was performed to study VF mechanisms in control hearts with acute GJ modulation, and separately in three differing chronic ventricular fibrosis models; compact fibrosis (CF), diffuse fibrosis (DiF), and patchy fibrosis (PF). VF dynamics were quantified with phase mapping and frequency dominance index (FDI) analysis, a power ratio of the highest amplitude dominant frequency in the cardiac frequency spectrum. Enhanced GJ coupling with rotigaptide (n = 10) progressively organized fibrillation in a concentration-dependent manner; increasing FDI (0 nM: 0.53 ± 0.04, 80 nM: 0.78 ± 0.03, P < 0.001), increasing RA-sustained VF time (0 nM: 44 ± 6%, 80 nM: 94 ± 2%, P < 0.001), and stabilized RAs (maximum rotations for an RA; 0 nM: 5.4 ± 0.5, 80 nM: 48.2 ± 12.3, P < 0.001). GJ uncoupling with carbenoxolone progressively disorganized VF; the FDI decreased (0 µM: 0.60 ± 0.05, 50 µM: 0.17 ± 0.03, P < 0.001) and RA-sustained VF time decreased (0 µM: 61 ± 9%, 50 µM: 3 ± 2%, P < 0.001). In CF, VF activity was disorganized and the RA-sustained VF time was the lowest (CF: 27 ± 7% vs. PF: 75 ± 5%, P < 0.001). Global fibrillatory organization measured by FDI was highest in PF (PF: 0.67 ± 0.05 vs. CF: 0.33 ± 0.03, P < 0.001). PF harboured the longest duration and most spatially stable RAs (patchy: 1411 ± 266 ms vs. compact: 354 ± 38 ms, P < 0.001). DiF (n = 11) exhibited an intermediately organized VF pattern, sustained by a combination of multiple-wavelets and short-lived RAs. CONCLUSION: The degree of GJ coupling and pattern of fibrosis influences the mechanism sustaining VF. There is a continuous spectrum of organization in VF, ranging between globally organized fibrillation sustained by stable RAs and disorganized, possibly multiple-wavelet driven fibrillation with no RAs.

Intrinsic Cell Polarity Coupled to Growth Axis Formation in Tobacco BY-2 Cells.

Several plant proteins are preferentially localized to one end of a cell, allowing a polarity to be assigned to the cell. These cell polarity proteins often exhibit coordinated patterns between neighboring cells, termed tissue cell polarity. Tissue cell polarity is widespread in plants and can influence how cells grow, divide, and differentiate [1-5]. However, it is unclear whether cell polarity is established through cell-intrinsic or -extrinsic mechanisms and how polarity is coupled to growth. To address these issues, we analyzed the behavior of a tissue cell polarity protein BASL (BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE) in the simplifying context of cultured cell filaments and in protoplasts before and during regeneration. We show that BASL is polarly localized when ectopically expressed in tobacco BY-2 cell cultures. Ectopic BASL is found preferentially at the developing tips of cell filaments, likely marking a polarized molecular address. Polarity can shift during the cell cycle and is resistant to treatment with microtubule, actin or auxin transport inhibitors. BASL also exhibits polar localization in spherical protoplasts, in contrast to other polarity proteins so far tested. BASL polarity within protoplasts is dynamic and resistant to auxin transport inhibitors. As protoplasts regenerate, polarity remains dynamic in isotropically growing cells but becomes fixed in anisotropic cells and aligns with the axis of cell growth. Our findings suggest that plant cells have an intrinsic ability to polarize and that environmental or developmental cues may act by biasing the direction of this polarity and thus the orientation of anisotropic growth.

Prolonged ursodeoxycholic acid administration reduces acute ischaemia-induced arrhythmias in adult rat hearts.

Acute myocardial ischaemia and reperfusion (I-R) are major causes of ventricular arrhythmias in patients with a history of coronary artery disease. Ursodeoxycholic acid (UDCA) has previously been shown to be antiarrhythmic in fetal hearts. This study was performed to investigate if UDCA protects against ischaemia-induced and reperfusion-induced arrhythmias in the adult myocardium, and compares the effect of acute (perfusion only) versus prolonged (2 weeks pre-treatment plus perfusion) UDCA administration. Langendorff-perfused adult Sprague-Dawley rat hearts were subjected to acute regional ischaemia by ligation of the left anterior descending artery (10 min), followed by reperfusion (2 min), and arrhythmia incidence quantified. Prolonged UDCA administration reduced the incidence of acute ischaemia-induced arrhythmias (p = 0.028), with a reduction in number of ventricular ectopic beats during the ischaemic phase compared with acute treatment (10 ± 3 vs 58 ± 15, p = 0.036). No antiarrhythmic effect was observed in the acute UDCA administration group. Neither acute nor prolonged UDCA treatment altered the incidence of reperfusion arrhythmias. The antiarrhythmic effect of UDCA may be partially mediated by an increase in cardiac wavelength, due to the attenuation of conduction velocity slowing (p = 0.03), and the preservation of Connexin43 phosphorylation during acute ischaemia (p = 0.0027). The potential antiarrhythmic effects of prolonged UDCA administration merit further investigation.

Mediastinal Lymphadenopathy, Class-Switched Auto-Antibodies and Myocardial Immune-Complexes During Heart Failure in Rodents and Humans.

Mediastinal lymphadenopathy and auto-antibodies are clinical phenomena during ischemic heart failure pointing to an autoimmune response against the heart. T and B cells have been convincingly demonstrated to be activated after myocardial infarction, a prerequisite for the generation of mature auto-antibodies. Yet, little is known about the immunoglobulin isotype repertoire thus pathological potential of anti-heart auto-antibodies during heart failure. We obtained human myocardial tissue from ischemic heart failure patients and induced experimental MI in rats. We found that anti-heart autoimmunity persists during heart failure. Rat mediastinal lymph nodes are enlarged and contain active secondary follicles with mature isotype-switched IgG2a B cells. Mature IgG2a auto-antibodies specific for cardiac antigens are present in rat heart failure serum, and IgG and complement C3 deposits are evident in heart failure tissue of both rats and human patients. Previously established myocardial inflammation, and the herein provided proof of B cell maturation in lymph nodes and myocardial deposition of mature auto-antibodies, provide all the hallmark signs of an established autoimmune response in chronic heart failure.

Short-term angiotensin II treatment regulates cardiac nanomechanics via microtubule modifications.

Mechanical properties of single myocytes contribute to the whole heart performance, but the measurement of mechanics in living cells at high resolution with minimal force interaction remains challenging. Angiotensin II (AngII) is a peptide hormone that regulates a number of physiological functions, including heart performance. It has also been shown to contribute to cell mechanics by inducing cell stiffening. Using non-contact high-resolution Scanning Ion Conductance Microscopy (SICM), we determine simultaneously cell topography and membrane transverse Young's modulus (YM) by a constant pressure application through a nanopipette. While applying pressure, the vertical position is recorded and a deformation map is generated from which YM can be calculated and corrected for the uneven geometry. High resolution of this method also allows studying specific membrane subdomains, such as Z-grooves and crests. We found that short-term AngII treatment reduces the transversal YM in isolated adult rat cardiomyocytes acting via an AT1 receptor. Blocking either a TGF-ß1 receptor or Rho kinase abolishes this effect. Analysis of the cytoskeleton showed that AngII depletes microtubules by decreasing long-lived detyrosinated and acetylated microtubule populations. Interestingly, in the failing cardiomyocytes, which are stiffer than controls, the short-term AngII treatment also reduces the YM, thus normalizing the mechanical state of cells. This suggests that the short-term softening effect of AngII on cardiac cells is opposite to the well-characterized long-term hypertrophic effect. In conclusion, we generate a precise nanoscale indication map of location-specific transverse cortical YM within the cell and this can substantially advance our understanding of cellular mechanics in a physiological environment, for example in isolated cardiac myocytes.

ß3-Adrenoceptor redistribution impairs NO/cGMP/PDE2 signalling in failing cardiomyocytes.

Cardiomyocyte ß3-adrenoceptors (ß3-ARs) coupled to soluble guanylyl cyclase (sGC)-dependent production of the second messenger 3',5'-cyclic guanosine monophosphate (cGMP) have been shown to protect from heart failure. However, the exact localization of these receptors to fine membrane structures and subcellular compartmentation of ß3-AR/cGMP signals underpinning this protection in health and disease remain elusive. Here, we used a Förster Resonance Energy Transfer (FRET)-based cGMP biosensor combined with scanning ion conductance microscopy (SICM) to show that functional ß3-ARs are mostly confined to the T-tubules of healthy rat cardiomyocytes. Heart failure, induced via myocardial infarction, causes a decrease of the cGMP levels generated by these receptors and a change of subcellular cGMP compartmentation. Furthermore, attenuated cGMP signals led to impaired phosphodiesterase two dependent negative cGMP-to-cAMP cross-talk. In conclusion, topographic and functional reorganization of the ß3-AR/cGMP signalosome happens in heart failure and should be considered when designing new therapies acting via this receptor.

Interventricular Differences in Action Potential Duration Restitution Contribute to Dissimilar Ventricular Rhythms in ex vivo Perfused Hearts.

Background: Dissimilar ventricular rhythms refer to the occurrence of different ventricular tachyarrhythmias in the right and left ventricles or different rates of the same tachyarrhythmia in the two ventricles. Objective: We investigated the inducibility of dissimilar ventricular rhythms, their underlying mechanisms, and the impact of anti-arrhythmic drugs (lidocaine and amiodarone) on their occurrence. Methods: Ventricular tachyarrhythmias were induced with burst pacing in 28 Langendorff-perfused Sprague Dawley rat hearts (14 control, 8 lidocaine, 6 amiodarone) and bipolar electrograms recorded from the right and left ventricles. Fourteen (6 control, 4 lidocaine, 4 amiodarone) further hearts underwent optical mapping of transmembrane voltage to study interventricular electrophysiological differences and mechanisms of dissimilar rhythms. Results: In control hearts, dissimilar ventricular rhythms developed in 8/14 hearts (57%). In lidocaine treated hearts, there was a lower cycle length threshold for developing dissimilar rhythms, with 8/8 (100%) hearts developing dissimilar rhythms in comparison to 0/6 in the amiodarone group. Dissimilar ventricular tachycardia (VT) rates occurred at longer cycle lengths with lidocaine vs. control (57.1 ± 7.9 vs. 36.6 ± 8.4 ms, p < 0.001). The ratio of LV:RV VT rate was greater in the lidocaine group than control (1.91 ± 0.30 vs. 1.76 ± 0.36, p < 0.001). The gradient of the action potential duration (APD) restitution curve was shallower in the RV compared with LV (Control - LV: 0.12 ± 0.03 vs RV: 0.002 ± 0.03, p = 0.015), leading to LV-to-RV conduction block during VT. Conclusion: Interventricular differences in APD restitution properties likely contribute to the occurrence of dissimilar rhythms. Sodium channel blockade with lidocaine increases the likelihood of dissimilar ventricular rhythms.

Ectopic BASL Reveals Tissue Cell Polarity throughout Leaf Development in Arabidopsis thaliana.

Tissue-wide polarity fields, in which cell polarity is coordinated across the tissue, have been described for planar organs such as the Drosophila wing and are considered important for coordinating growth and differentiation [1]. In planar plant organs, such as leaves, polarity fields have been identified for subgroups of cells, such as stomatal lineages [2], trichomes [3, 4], serrations [5], or early developmental stages [6]. Here, we show that ectopic induction of the stomatal protein BASL (BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE) reveals a tissue-wide epidermal polarity field in leaves throughout development. Ectopic GFP-BASL is typically localized toward the proximal end of cells and to one lobe of mature pavement cells, revealing a polarity field that aligns with the proximodistal axis of the leaf (base to tip). The polarity field is largely parallel to the midline of the leaf but diverges in more lateral positions, particularly at later stages in development, suggesting it may be deformed during growth. The polarity field is observed in the speechless mutant, showing that it is independent of stomatal lineages, and is observed in isotropic cells, showing that cell shape anisotropy is not required for orienting polarity. Ectopic BASL forms convergence and divergence points at serrations, mirroring epidermal PIN polarity patterns, suggesting a common underlying polarity mechanism. Thus, we show that similar to the situation in animals, planar plant organs have a tissue-wide cell polarity field, and this may provide a general cellular mechanism for guiding growth and differentiation.

Auxetic Cardiac Patches with Tunable Mechanical and Conductive Properties toward Treating Myocardial Infarction.

An auxetic conductive cardiac patch (AuxCP) for the treatment of myocardial infarction (MI) is introduced. The auxetic design gives the patch a negative Poisson's ratio, providing it with the ability to conform to the demanding mechanics of the heart. The conductivity allows the patch to interface with electroresponsive tissues such as the heart. Excimer laser microablation is used to micropattern a re-entrant honeycomb (bow-tie) design into a chitosan-polyaniline composite. It is shown that the bow-tie design can produce patches with a wide range in mechanical strength and anisotropy, which can be tuned to match native heart tissue. Further, the auxetic patches are conductive and cytocompatible with murine neonatal cardiomyocytes in vitro. Ex vivo studies demonstrate that the auxetic patches have no detrimental effect on the electrophysiology of both healthy and MI rat hearts and conform better to native heart movements than unpatterned patches of the same material. Finally, the AuxCP applied in a rat MI model results in no detrimental effect on cardiac function and negligible fibrotic response after two weeks in vivo. This approach represents a versatile and robust platform for cardiac biomaterial design and could therefore lead to a promising treatment for MI.

Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Encapsulating Bioactive Hydrogels Improve Rat Heart Function Post Myocardial Infarction.

Tissue engineering offers an exciting possibility for cardiac repair post myocardial infarction. We assessed the effects of combined polyethylene glycol hydrogel (PEG), human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM), and erythropoietin (EPO) therapy in a rat model of myocardial infarction. PEG with/out iPSC-CMs and EPO; iPSC-CMs in saline; or saline alone was injected into infarcted hearts shortly after infarction. Injection of almost any combination of the therapeutics limited acute elevations in chamber volumes. After 10 weeks, attenuation of ventricular remodeling was identified in all groups that received PEG injections, while ejection fractions were significantly increased in the gel-EPO, cell, and gel-cell-EPO groups. In all treatment groups, infarct thickness was increased and regions of muscle were identified within the scar. However, no grafted cells were detected. Hence, iPSC-CM-encapsulating bioactive hydrogel therapy can improve cardiac function post myocardial infarction and increase infarct thickness and muscle content despite a lack of sustained donor-cell engraftment.

T-tubule remodelling disturbs localized ß2-adrenergic signalling in rat ventricular myocytes during the progression of heart failure.

AIMS: Cardiomyocyte ß2-adrenergic receptor (ß2AR) cyclic adenosine monophosphate (cAMP) signalling is regulated by the receptors' subcellular location within transverse tubules (T-tubules), via interaction with structural and regulatory proteins, which form a signalosome. In chronic heart failure (HF), ß2ARs redistribute from T-tubules to the cell surface, which disrupts functional signalosomes and leads to diffuse cAMP signalling. However, the functional consequences of structural changes upon ß2AR-cAMP signalling during progression from hypertrophy to advanced HF are unknown. METHODS AND RESULTS: Rat left ventricular myocytes were isolated at 4-, 8-, and 16-week post-myocardial infarction (MI), ß2ARs were stimulated either via whole-cell perfusion or locally through the nanopipette of the scanning ion conductance microscope. cAMP release was measured via a Förster Resonance Energy Transfer-based sensor Epac2-camps. Confocal imaging of di-8-ANNEPS-stained cells and immunoblotting were used to determine structural alterations. At 4-week post-MI, T-tubule regularity, density and junctophilin-2 (JPH2) expression were significantly decreased. The amplitude of local ß2AR-mediated cAMP in T-tubules was reduced and cAMP diffused throughout the cytosol instead of being locally confined. This was accompanied by partial caveolin-3 (Cav-3) dissociation from the membrane. At 8-week post-MI, the ß2AR-mediated cAMP response was observed at the T-tubules and the sarcolemma (crest). Finally, at 16-week post-MI, the whole cell ß2AR-mediated cAMP signal was depressed due to adenylate cyclase dysfunction, while overall Cav-3 levels were significantly increased and a substantial portion of Cav-3 dissociated into the cytosol. Overexpression of JPH2 in failing cells in vitro or AAV9.SERCA2a gene therapy in vivo did not improve ß2AR-mediated signal compartmentation or reduce cAMP diffusion. CONCLUSION: Although changes in T-tubule structure and ß2AR-mediated cAMP signalling are significant even at 4-week post-MI, progression to the HF phenotype is not linear. At 8-week post-MI the loss of ß2AR-mediated cAMP is temporarily reversed. Complete disorganization of ß2AR-mediated cAMP signalling due to changes in functional receptor localization and cellular structure occurs at 16-week post-MI.

Microdomain-Specific Modulation of L-Type Calcium Channels Leads to Triggered Ventricular Arrhythmia in Heart Failure.

RATIONALE: Disruption in subcellular targeting of Ca(2+) signaling complexes secondary to changes in cardiac myocyte structure may contribute to the pathophysiology of a variety of cardiac diseases, including heart failure (HF) and certain arrhythmias. OBJECTIVE: To explore microdomain-targeted remodeling of ventricular L-type Ca(2+) channels (LTCCs) in HF. METHODS AND RESULTS: Super-resolution scanning patch-clamp, confocal and fluorescence microscopy were used to explore the distribution of single LTCCs in different membrane microdomains of nonfailing and failing human and rat ventricular myocytes. Disruption of membrane structure in both species led to the redistribution of functional LTCCs from their canonical location in transversal tubules (T-tubules) to the non-native crest of the sarcolemma, where their open probability was dramatically increased (0.034±0.011 versus 0.154±0.027, P<0.001). High open probability was linked to enhance calcium-calmodulin kinase II-mediated phosphorylation in non-native microdomains and resulted in an elevated ICa,L window current, which contributed to the development of early afterdepolarizations. A novel model of LTCC function in HF was developed; after its validation with experimental data, the model was used to ascertain how HF-induced T-tubule loss led to altered LTCC function and early afterdepolarizations. The HF myocyte model was then implemented in a 3-dimensional left ventricle model, demonstrating that such early afterdepolarizations can propagate and initiate reentrant arrhythmias. CONCLUSIONS: Microdomain-targeted remodeling of LTCC properties is an important event in pathways that may contribute to ventricular arrhythmogenesis in the settings of HF-associated remodeling. This extends beyond the classical concept of electric remodeling in HF and adds a new dimension to cardiovascular disease.

A conducting polymer with enhanced electronic stability applied in cardiac models.

Electrically active constructs can have a beneficial effect on electroresponsive tissues, such as the brain, heart, and nervous system. Conducting polymers (CPs) are being considered as components of these constructs because of their intrinsic electroactive and flexible nature. However, their clinical application has been largely hampered by their short operational time due to a decrease in their electronic properties. We show that, by immobilizing the dopant in the conductive scaffold, we can prevent its electric deterioration. We grew polyaniline (PANI) doped with phytic acid on the surface of a chitosan film. The strong chelation between phytic acid and chitosan led to a conductive patch with retained electroactivity, low surface resistivity (35.85 ± 9.40 kilohms per square), and oxidized form after 2 weeks of incubation in physiological medium. Ex vivo experiments revealed that the conductive nature of the patch has an immediate effect on the electrophysiology of the heart. Preliminary in vivo experiments showed that the conductive patch does not induce proarrhythmogenic activities in the heart. Our findings set the foundation for the design of electronically stable CP-based scaffolds. This provides a robust conductive system that could be used at the interface with electroresponsive tissue to better understand the interaction and effect of these materials on the electrophysiology of these tissues.

Rice perception of symbiotic arbuscular mycorrhizal fungi requires the karrikin receptor complex.

In terrestrial ecosystems, plants take up phosphate predominantly via association with arbuscular mycorrhizal fungi (AMF). We identified loss of responsiveness to AMF in the rice (Oryza sativa) mutant hebiba, reflected by the absence of physical contact and of characteristic transcriptional responses to fungal signals. Among the 26 genes deleted in hebiba, DWARF 14 LIKE is, the one responsible for loss of symbiosis . It encodes an alpha/beta-fold hydrolase, that is a component of an intracellular receptor complex involved in the detection of the smoke compound karrikin. Our finding reveals an unexpected plant recognition strategy for AMF and a previously unknown signaling link between symbiosis and plant development.