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.

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.

Mesenteric artery smooth muscle cells from hypertensive rats have increased microtubule acetylation.

The dynamic nature of the microtubule network is dependent in part by post-translational modifications (PTMs) - particularly through acetylation, which stabilizes the microtubule network. Whether PTMs of the microtubule network in vascular smooth muscle cells (VSMCs) contribute to the pathophysiology of hypertension is unknown. The aim of this study was to determine the acetylated state of the microtubule network in the mesenteric arteries of spontaneously hypertensive rats (SHR). Experiments were performed on male normotensive rats and SHR mesenteric arteries. Western blotting and mass spectrometry determined changes in tubulin acetylation. Wire myography was used to investigate the effect of tubacin on isoprenaline-mediated vasorelaxations. Isolated cells from normotensive rats were used for scanning ion conductance microscopy (SICM). Mass spectrometry and Western blotting showed that tubulin acetylation is increased in the mesenteric arteries of the SHR compared with normotensive rats. Tubacin enhanced the ß-adrenoceptor-mediated vasodilatation by isoprenaline when the endothelium was intact, but attenuated relaxations when the endothelium was denuded or nitric oxide production was inhibited. By pre-treating vessels with colchicine to disrupt the microtubule network, we were able to confirm that the effects of tubacin were microtubule-dependent. Using SICM, we examined the cell surface Young's modulus of VSMCs, but found no difference in control, tubacin-treated, or taxol-treated cells. Acetylation of tubulin at Lys40 is elevated in mesenteric arteries from the SHR. Furthermore, this study shows that tubacin has an endothelial-dependent bimodal effect on isoprenaline-mediated vasorelaxation.

Development and characterization of the mode-of-action of inhibitory and agonist peptides targeting the voltage-gated sodium channel SCN1B beta-subunit.

Cardiac arrhythmia treatment is a clinical challenge necessitating safer and more effective therapies. Recent studies have highlighted the role of the perinexus, an intercalated disc nanodomain enriched in voltage-gated sodium channels including both Nav1.5 and ß1 subunits, adjacent to gap junctions. These findings offer insights into action potential conduction in the heart. A 19-amino acid SCN1B (ß1/ß1B) mimetic peptide, ßadp1, disrupts VGSC beta subunit-mediated adhesion in cardiac perinexii, inducing arrhythmogenic changes. We aimed to explore ßadp1's mechanism and develop novel SCN1B mimetic peptides affecting ß1-mediated adhesion. Using patch clamp assays in neonatal rat cardiomyocytes and electric cell substrate impedance sensing (ECIS) in ß1-expressing cells, we observed ßadp1 maintained inhibitory effects for up to 5 h. A shorter peptide (LQLEED) based on the carboxyl-terminus of ßadp1 mimicked this inhibitory effect, while dimeric peptides containing repeated LQLEED sequences paradoxically promoted intercellular adhesion over longer time courses. Moreover, we found a link between these peptides and ß1-regulated intramembrane proteolysis (RIP) - a signaling pathway effecting gene transcription including that of VGSC subunits. ßadp1 increased RIP continuously over 48 h, while dimeric agonists acutely boosted RIP for up to 6 h. In the presence of DAPT, an RIP inhibitor, ßadp1's effects on ECIS-measured intercellular adhesion was reduced, suggesting a relationship between RIP and the peptide's inhibitory action. In conclusion, novel SCN1B (ß1/ß1B) mimetic peptides are reported with the potential to modulate intercellular VGSC ß1-mediated adhesion, potentially through ß1 RIP. These findings suggest a path towards the development of anti-arrhythmic drugs targeting the perinexus.

Caveolae-associated cAMP/Ca2+-mediated mechano-chemical signal transduction in mouse atrial myocytes.

Caveolae are tiny invaginations in the sarcolemma that buffer extra membrane and contribute to mechanical regulation of cellular function. While the role of caveolae in membrane mechanosensation has been studied predominantly in non-cardiomyocyte cells, caveolae contribution to cardiac mechanotransduction remains elusive. Here, we studied the role of caveolae in the regulation of Ca2+ signaling in atrial cardiomyocytes. In Langendorff-perfused mouse hearts, atrial pressure/volume overload stretched atrial myocytes and decreased caveolae density. In isolated cells, caveolae were disrupted through hypotonic challenge that induced a temporal (<10 min) augmentation of Ca2+ transients and caused a rise in Ca2+ spark activity. Similar changes in Ca2+ signaling were observed after chemical (methyl-ß-cyclodextrin) and genetic ablation of caveolae in cardiac-specific conditional caveolin-3 knock-out mice. Acute disruption of caveolae, both mechanical and chemical, led to the elevation of cAMP level in the cell interior, and cAMP-mediated augmentation of protein kinase A (PKA)-phosphorylated ryanodine receptors (at Ser2030 and Ser2808). Caveolae-mediated stimulatory effects on Ca2+ signaling were abolished via inhibition of cAMP production by adenyl cyclase antagonists MDL12330 and SQ22536, or reduction of PKA activity by H-89. A compartmentalized mathematical model of mouse atrial myocytes linked the observed changes to a microdomain-specific decrease in phosphodiesterase activity, which disrupted cAMP signaling and augmented PKA activity. Our findings add a new dimension to cardiac mechanobiology and highlight caveolae-associated cAMP/PKA-mediated phosphorylation of Ca2+ handling proteins as a novel component of mechano-chemical feedback in atrial myocytes.

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.

Interaction of the Joining Region in Junctophilin-2 With the L-Type Ca2+ Channel Is Pivotal for Cardiac Dyad Assembly and Intracellular Ca2+ Dynamics.

RATIONALE: Ca2+-induced Ca2+ release (CICR) in normal hearts requires close approximation of L-type calcium channels (LTCCs) within the transverse tubules (T-tubules) and RyR (ryanodine receptors) within the junctional sarcoplasmic reticulum. CICR is disrupted in cardiac hypertrophy and heart failure, which is associated with loss of T-tubules and disruption of cardiac dyads. In these conditions, LTCCs are redistributed from the T-tubules to disrupt CICR. The molecular mechanism responsible for LTCCs recruitment to and from the T-tubules is not well known. JPH (junctophilin) 2 enables close association between T-tubules and the junctional sarcoplasmic reticulum to ensure efficient CICR. JPH2 has a so-called joining region that is located near domains that interact with T-tubular plasma membrane, where LTCCs are housed. The idea that this joining region directly interacts with LTCCs and contributes to LTCC recruitment to T-tubules is unknown. OBJECTIVE: To determine if the joining region in JPH2 recruits LTCCs to T-tubules through direct molecular interaction in cardiomyocytes to enable efficient CICR. METHODS AND RESULTS: Modified abundance of JPH2 and redistribution of LTCC were studied in left ventricular hypertrophy in vivo and in cultured adult feline and rat ventricular myocytes. Protein-protein interaction studies showed that the joining region in JPH2 interacts with LTCC-α1C subunit and causes LTCCs distribution to the dyads, where they colocalize with RyRs. A JPH2 with induced mutations in the joining region (mutPG1JPH2) caused T-tubule remodeling and dyad loss, showing that an interaction between LTCC and JPH2 is crucial for T-tubule stabilization. mutPG1JPH2 caused asynchronous Ca2+-release with impaired excitation-contraction coupling after ß-adrenergic stimulation. The disturbed Ca2+ regulation in mutPG1JPH2 overexpressing myocytes caused calcium/calmodulin-dependent kinase II activation and altered myocyte bioenergetics. CONCLUSIONS: The interaction between LTCC and the joining region in JPH2 facilitates dyad assembly and maintains normal CICR in cardiomyocytes.

Microtubules regulate cardiomyocyte transversal Young's modulus.

The field of cardiomyocyte mechanobiology is gaining significant attention, due to accumulating evidence concerning the significant role of cellular mechanical effects on the integrated function of the heart. To date, the protein titin has been demonstrated as a major contributor to the cardiomyocytes Young's modulus (YM). The microtubular network represents another potential regulator of cardiac mechanics. However, the contribution of microtubules (MTs) to the membrane YM is still understudied and has not been interrogated in the context of myocardial infarction (MI) or mechanical loading and unloading. Using nanoscale mechanoscanning ion conductance microscopy, we demonstrate that MTs contribute to cardiomyocyte transverse YM in healthy and pathological states with different mechanical loading. Specifically, we show that posttranslational modifications of MTs have differing effects on cardiomyocyte YM: Acetylation provides flexibility, whereas detyrosination imparts rigidity. Further studies demonstrate that there is no correlation between the total protein amount of acetylated and detyrosinated MT. Yet, in the polymerized-only populations, an increased level of acetylation results in a decline of detyrosinated MTs in an MI model.

Nerve growth factor transfer from cardiomyocytes to innervating sympathetic neurons activates TrkA receptors at the neuro-cardiac junction.

Sympathetic neurons densely innervate the myocardium with non-random topology and establish structured contacts (i.e. neuro-cardiac junctions, NCJ) with cardiomyocytes, allowing synaptic intercellular communication. Establishment of heart innervation is regulated by molecular mediators released by myocardial cells. The mechanisms underlying maintenance of cardiac innervation in the fully developed heart, are, however, less clear. Notably, several cardiac diseases, primarily affecting cardiomyocytes, are associated with sympathetic denervation, supporting the hypothesis that retrograde 'cardiomyocyte-to-sympathetic neuron' communication is essential for heart cellular homeostasis. We aimed to determine whether cardiomyocytes provide nerve growth factor (NGF) to sympathetic neurons, and the role of the NCJ in supporting such retrograde neurotrophic signalling. Immunofluorescence on murine and human heart slices shows that NGF and its receptor, tropomyosin-receptor-kinase-A, accumulate, respectively, in the pre- and post-junctional sides of the NCJ. Confocal immunofluorescence, scanning ion conductance microscopy and molecular analyses, in co-cultures, demonstrate that cardiomyocytes feed NGF to sympathetic neurons, and that this mechanism requires a stable intercellular contact at the NCJ. Consistently, cardiac fibroblasts, devoid of NCJ, are unable to sustain SN viability. ELISA assay and competition binding experiments suggest that this depends on the NCJ being an insulated microenvironment, characterized by high [NGF]. In further support, real-time imaging of tropomyosin-receptor-kinase-A vesicle movements demonstrate that efficiency of neurotrophic signalling parallels the maturation of such structured intercellular contacts. Altogether, our results demonstrate the mechanisms which link sympathetic neuron survival to neurotrophin release by directly innervated cardiomyocytes, conceptualizing sympathetic neurons as cardiomyocyte-driven heart drivers. KEY POINTS: CMs are the cell source of nerve growth factor (NGF), required to sustain innervating cardiac SNs; NCJ is the place of the intimate liaison, between SNs and CMs, allowing on the one hand neurons to peremptorily control CM activity, and on the other, CMs to adequately sustain the contacting, ever-changing, neuronal actuators; alterations in NCJ integrity may compromise the efficiency of 'CM-to-SN' signalling, thus representing a potentially novel mechanism of sympathetic denervation in cardiac diseases.

Fetal cardiac dysfunction in intrahepatic cholestasis of pregnancy is associated with elevated serum bile acid concentrations.

BACKGROUND & AIMS: Intrahepatic cholestasis of pregnancy (ICP) is associated with an increased risk of stillbirth. This study aimed to assess the relationship between bile acid concentrations and fetal cardiac dysfunction in patients with ICP who were or were not treated with ursodeoxycholic acid (UDCA). METHODS: Bile acid profiles and NT-proBNP, a marker of ventricular dysfunction, were assayed in umbilical venous serum from 15 controls and 76 ICP cases (36 untreated, 40 UDCA-treated). Fetal electrocardiogram traces were obtained from 43 controls and 48 ICP cases (26 untreated, 22 UDCA-treated). PR interval length and heart rate variability (HRV) parameters were measured in 2 behavioral states (quiet and active sleep). RESULTS: In untreated ICP, fetal total serum bile acid (TSBA) concentrations (r = 0.49, p = 0.019), hydrophobicity index (r = 0.20, p = 0.039), glycocholate concentrations (r = 0.56, p = 0.007) and taurocholate concentrations (r = 0.44, p = 0.039) positively correlated with fetal NT-proBNP. Maternal TSBA (r = 0.40, p = 0.026) and alanine aminotransferase (r = 0.40, p = 0.046) also positively correlated with fetal NT-proBNP. There were no significant correlations between maternal or fetal serum bile acid concentrations and fetal HRV parameters or NT-proBNP concentrations in the UDCA-treated cohort. Fetal PR interval length positively correlated with maternal TSBA in untreated (r = 0.46, p = 0.027) and UDCA-treated ICP (r = 0.54, p = 0.026). Measures of HRV in active sleep and quiet sleep were significantly higher in untreated ICP cases than controls. HRV values in UDCA-treated cases did not differ from controls. CONCLUSIONS: Elevated fetal and maternal serum bile acid concentrations in untreated ICP are associated with an abnormal fetal cardiac phenotype characterized by increased NT-proBNP concentration, PR interval length and HRV. UDCA treatment partially attenuates this phenotype. LAY SUMMARY: The risk of stillbirth in intrahepatic cholestasis of pregnancy (ICP) is linked to the level of bile acids in the mother which are thought to disrupt the baby's heart rhythm. We found that babies of women with untreated ICP have abnormally functioning hearts compared to those without ICP, and the degree of abnormality is closely linked to the level of harmful bile acids in the mother and baby's blood. Babies of women with ICP who received treatment with the drug UDCA do not have the same level of abnormality in their hearts, suggesting that UDCA could be a beneficial treatment in some ICP cases, although further clinical trials are needed to confirm this.

Studying signal compartmentation in adult cardiomyocytes.

Multiple intra-cellular signalling pathways rely on calcium and 3'-5' cyclic adenosine monophosphate (cAMP) to act as secondary messengers. This is especially true in cardiomyocytes which act as the force-producing units of the cardiac muscle and are required to react rapidly to environmental stimuli. The specificity of functional responses within cardiomyocytes and other cell types is produced by the organellar compartmentation of both calcium and cAMP. In this review, we assess the role of molecular localisation and relative contribution of active and passive processes in producing compartmentation. Active processes comprise the creation and destruction of signals, whereas passive processes comprise the release or sequestration of signals. Cardiomyocytes display a highly articulated membrane structure which displays significant cell-to-cell variability. Special attention is paid to the way in which cell membrane caveolae and the transverse-axial tubule system allow molecular localisation. We explore the effects of cell maturation, pathology and regional differences in the organisation of these processes. The subject of signal compartmentation has had a significant amount of attention within the cardiovascular field and has undergone a revolution over the past two decades. Advances in the area have been driven by molecular imaging using fluorescent dyes and genetically encoded constructs based upon fluorescent proteins. We also explore the use of scanning probe microscopy in the area. These techniques allow the analysis of molecular compartmentation within specific organellar compartments which gives researchers an entirely new perspective.

Correlative SICM-FCM reveals changes in morphology and kinetics of endocytic pits induced by disease-associated mutations in dynamin.

Dynamin 2 (DNM2) is a GTP-binding protein that controls endocytic vesicle scission and defines a whole class of dynamin-dependent endocytosis, including clathrin-mediated endocytosis by caveoli. It has been suggested that mutations in the DNM2 gene, associated with 3 inherited diseases, disrupt endocytosis. However, how exactly mutations affect the nanoscale morphology of endocytic machinery has never been studied. In this paper, we used live correlative scanning ion conductance microscopy (SICM) and fluorescence confocal microscopy (FCM) to study how disease-associated mutations affect the morphology and kinetics of clathrin-coated pits (CCPs) by directly following their dynamics of formation, maturation, and internalization in skin fibroblasts from patients with centronuclear myopathy (CNM) and in Cos-7 cells expressing corresponding dynamin mutants. Using SICM-FCM, which we have developed, we show how p.R465W mutation disrupts pit structure, preventing its maturation and internalization, and significantly increases the lifetime of CCPs. Differently, p.R522H slows down the formation of CCPs without affecting their internalization. We also found that CNM mutations in DNM2 affect the distribution of caveoli and reduce dorsal ruffling in human skin fibroblasts. Collectively, our SICM-FCM findings at single CCP level, backed up by electron microscopy data, argue for the impairment of several forms of endocytosis in DNM2-linked CNM.-Ali, T., Bednarska, J., Vassilopoulos, S., Tran, M., Diakonov, I. A., Ziyadeh-Isleem, A., Guicheney, P., Gorelik, J., Korchev, Y. E., Reilly, M. M., Bitoun, M., Shevchuk, A. Correlative SICM-FCM reveals changes in morphology and kinetics of endocytic pits induced by disease-associated mutations in dynamin.

Cardiomyocyte-myofibroblast contact dynamism is modulated by connexin-43.

Healthy cardiomyocytes are electrically coupled at the intercalated discs by gap junctions. In infarcted hearts, adverse gap-junctional remodeling occurs in the border zone, where cardiomyocytes are chemically and electrically influenced by myofibroblasts. The physical movement of these contacts remains unquantified. Using scanning ion conductance microscopy, we show that intercellular contacts between cardiomyocytes and myofibroblasts are highly dynamic, mainly owing to the edge dynamics (lamellipodia) of the myofibroblasts. Decreasing the amount of functional connexin-43 (Cx43) at the membrane through Cx43 silencing, suppression of Cx43 trafficking, or hypoxia-induced Cx43 internalization attenuates heterocellular contact dynamism. However, we found decreased dynamism and stabilized membrane contacts when cellular coupling was strengthened using 4-phenylbutyrate (4PB). Fluorescent-dye transfer between cells showed that the extent of functional coupling between the 2 cell types correlated with contact dynamism. Intercellular calcein transfer from myofibroblasts to cardiomyocytes is reduced after myofibroblast-specific Cx43 down-regulation. Conversely, 4PB-treated myofibroblasts increased their functional coupling to cardiomyocytes. Consistent with lamellipodia-mediated contacts, latrunculin-B decreases dynamism, lowers physical communication between heterocellular pairs, and reduces Cx43 intensity in contact regions. Our data show that heterocellular cardiomyocyte-myofibroblast contacts exhibit high dynamism. Therefore, Cx43 is a potential target for prevention of aberrant cardiomyocyte coupling and myofibroblast proliferation in the infarct border zone.-Schultz, F., Swiatlowska, P., Alvarez-Laviada, A., Sanchez-Alonso, J. L., Song, Q., de Vries, A. A. F., Pijnappels, D. A., Ongstad, E., Braga, V. M. M., Entcheva, E., Gourdie, R. G., Miragoli, M., Gorelik, J. Cardiomyocyte-myofibroblast contact dynamism is modulated by connexin-43.

Heart and bile acids - Clinical consequences of altered bile acid metabolism.

Cardiac dysfunction has an increased prevalence in diseases complicated by liver cirrhosis such as primary biliary cholangitis and primary sclerosing cholangitis. This observation has led to research into the association between abnormalities in bile acid metabolism and cardiac pathology. Approximately 50% of liver cirrhosis cases develop cirrhotic cardiomyopathy. Bile acids are directly implicated in this, causing QT interval prolongation, cardiac hypertrophy, cardiomyocyte apoptosis and abnormal haemodynamics of the heart. Elevated maternal serum bile acids in intrahepatic cholestasis of pregnancy, a disorder which causes an impaired feto-maternal bile acid gradient, have been associated with fatal fetal arrhythmias. The hydrophobicity of individual bile acids in the serum bile acid pool is of relevance, with relatively lipophilic bile acids having a more harmful effect on the heart. Ursodeoxycholic acid can reverse or protect against these detrimental cardiac effects of elevated bile acids.

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.

Nanoscale, Voltage-Driven Application of Bioactive Substances onto Cells with Organized Topography.

With scanning ion conductance microscopy (SICM), a noncontact scanning probe technique, it is possible both to obtain information about the surface topography of live cells and to apply molecules onto specific nanoscale structures. The technique is therefore widely used to apply chemical compounds and to study the properties of molecules on the surfaces of various cell types. The heart muscle cells, i.e., the cardiomyocytes, possess a highly elaborate, unique surface topography including transverse-tubule (T-tubule) openings leading into a cell internal system that exclusively harbors many proteins necessary for the cell's physiological function. Here, we applied isoproterenol into these surface openings by changing the applied voltage over the SICM nanopipette. To determine the grade of precision of our application we used finite-element simulations to investigate how the concentration profile varies over the cell surface. We first obtained topography scans of the cardiomyocytes using SICM and then determined the electrophoretic mobility of isoproterenol in a high ion solution to be -7 × 10(-9) m(2)/V s. The simulations showed that the delivery to the T-tubule opening is highly confined to the underlying Z-groove, and especially to the first T-tubule opening, where the concentration is ∼6.5 times higher compared to on a flat surface under the same delivery settings. Delivery to the crest, instead of the T-tubule opening, resulted in a much lower concentration, emphasizing the importance of topography in agonist delivery. In conclusion, SICM, unlike other techniques, can reliably deliver precise quantities of compounds to the T-tubules of cardiomyocytes.

Angular Approach Scanning Ion Conductance Microscopy.

Scanning ion conductance microscopy (SICM) is a super-resolution live imaging technique that uses a glass nanopipette as an imaging probe to produce three-dimensional (3D) images of cell surface. SICM can be used to analyze cell morphology at nanoscale, follow membrane dynamics, precisely position an imaging nanopipette close to a structure of interest, and use it to obtain ion channel recordings or locally apply stimuli or drugs. Practical implementations of these SICM advantages, however, are often complicated due to the limitations of currently available SICM systems that inherited their design from other scanning probe microscopes in which the scan assembly is placed right above the specimen. Such arrangement makes the setting of optimal illumination necessary for phase contrast or the use of high magnification upright optics difficult. Here, we describe the designs that allow mounting SICM scan head on a standard patch-clamp micromanipulator and imaging the sample at an adjustable approach angle. This angle could be as shallow as the approach angle of a patch-clamp pipette between a water immersion objective and the specimen. Using this angular approach SICM, we obtained topographical images of cells grown on nontransparent nanoneedle arrays, of islets of Langerhans, and of hippocampal neurons under upright optical microscope. We also imaged previously inaccessible areas of cells such as the side surfaces of the hair cell stereocilia and the intercalated disks of isolated cardiac myocytes, and performed targeted patch-clamp recordings from the latter. Thus, our new, to our knowledge, angular approach SICM allows imaging of living cells on nontransparent substrates and a seamless integration with most patch-clamp setups on either inverted or upright microscopes, which would facilitate research in cell biophysics and physiology.

Direct Evidence for Microdomain-Specific Localization and Remodeling of Functional L-Type Calcium Channels in Rat and Human Atrial Myocytes.

BACKGROUND: Distinct subpopulations of L-type calcium channels (LTCCs) with different functional properties exist in cardiomyocytes. Disruption of cellular structure may affect LTCC in a microdomain-specific manner and contribute to the pathophysiology of cardiac diseases, especially in cells lacking organized transverse tubules (T-tubules) such as atrial myocytes (AMs). METHODS AND RESULTS: Isolated rat and human AMs were characterized by scanning ion conductance, confocal, and electron microscopy. Half of AMs possessed T-tubules and structured topography, proportional to cell width. A bigger proportion of myocytes in the left atrium had organized T-tubules and topography than in the right atrium. Super-resolution scanning patch clamp showed that LTCCs distribute equally in T-tubules and crest areas of the sarcolemma, whereas, in ventricular myocytes, LTCCs primarily cluster in T-tubules. Rat, but not human, T-tubule LTCCs had open probability similar to crest LTCCs, but exhibited ≈ 40% greater current. Optical mapping of Ca(2+) transients revealed that rat AMs presented ≈ 3-fold as many spontaneous Ca(2+) release events as ventricular myocytes. Occurrence of crest LTCCs and spontaneous Ca(2+) transients were eliminated by either a caveolae-targeted LTCC antagonist or disrupting caveolae with methyl-ß-cyclodextrin, with an associated ≈ 30% whole-cell ICa,L reduction. Heart failure (16 weeks post-myocardial infarction) in rats resulted in a T-tubule degradation (by ≈ 40%) and significant elevation of spontaneous Ca(2+) release events. Although heart failure did not affect LTCC occurrence, it led to ≈ 25% decrease in T-tubule LTCC amplitude. CONCLUSIONS: We provide the first direct evidence for the existence of 2 distinct subpopulations of functional LTCCs in rat and human AMs, with their biophysical properties modulated in heart failure in a microdomain-specific manner.

Transgenic mice for real-time visualization of cGMP in intact adult cardiomyocytes.

RATIONALE: 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. OBJECTIVE: Our aim was to generate and characterize a novel cGMP sensor model to measure cGMP with nanomolar sensitivity in adult cardiomyocytes. METHODS AND RESULTS: 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. CONCLUSIONS: 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.