RESUMEN
In the heart, genetic or acquired mishandling of diastolic [Ca2+] by ryanodine receptor type 2 (RyR2) overactivity correlates with risks of arrhythmia and sudden cardiac death. Strategies to avoid these risks include decrease of Ca2+ release by drugs modulating RyR2 activity or increase in Ca2+ uptake by drugs modulating SR Ca2+ ATPase (SERCA2a) activity. Here, we combine these strategies by developing experimental compounds that act simultaneously on both processes. Our screening efforts identified the new 1,4-benzothiazepine derivative GM1869 as a promising compound. Consequently, we comparatively studied the effects of the known RyR2 modulators Dantrolene and S36 together with GM1869 on RyR2 and SERCA2a activity in cardiomyocytes from wild type and arrhythmia-susceptible RyR2R2474S/+ mice by confocal live-cell imaging. All drugs reduced RyR2-mediated Ca2+ spark frequency but only GM1869 accelerated SERCA2a-mediated decay of Ca2+ transients in murine and human cardiomyocytes. Our data indicate that S36 and GM1869 are more suitable than dantrolene to directly modulate RyR2 activity, especially in RyR2R2474S/+ mice. Remarkably, GM1869 may represent a new dual-acting lead compound for maintenance of diastolic [Ca2+].
Asunto(s)
Dantroleno , Canal Liberador de Calcio Receptor de Rianodina , Animales , Humanos , Ratones , Arritmias Cardíacas/metabolismo , Transporte Biológico , Dantroleno/farmacología , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
To discover new multifunctional agents for the treatment of cardiovascular diseases, we designed and synthesized a series of compounds with a cyclopropyl alcohol moiety and evaluated them in biochemical assays. Biological screening identified derivatives with dual activity: preventing Ca2+ leak through ryanodine receptor 2 (RyR2) and enhancing cardiac sarco-endoplasmic reticulum (SR) Ca2+ load by activation of Ca2+-dependent ATPase 2a (SERCA2a). The compounds that stabilize RyR2 at micro- and nanomolar concentrations are either structurally related to RyR-stabilizing drugs or Rycals or have structures similar to them. The novel compounds also demonstrate a good ability to increase ATP hydrolysis mediated by SERCA2a activity in cardiac microsomes, e.g., the half-maximal effective concentration (EC50) was as low as 383 nM for compound 12a, which is 1,4-benzothiazepine with two cyclopropanol groups. Our findings indicate that these derivatives can be considered as new lead compounds to improve cardiac function in heart failure.
Asunto(s)
Canal Liberador de Calcio Receptor de Rianodina , Retículo Sarcoplasmático , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Miocitos Cardíacos , Canal Liberador de Calcio Receptor de Rianodina/farmacología , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Tiazepinas/química , Tiazepinas/farmacologíaRESUMEN
Amlodipine, a unique long-lasting calcium channel antagonist and antihypertensive drug, has weak fluorescence in aqueous solutions. In the current paper, we show that direct visualization of amlodipine in live cells is possible due to the enhanced emission in cellular environment. We examined the impact of pH, polarity and viscosity of the environment as well as protein binding on the spectral properties of amlodipine in vitro, and used quantum chemical calculations for assessing the mechanism of fluorescence quenching in aqueous solutions. The confocal fluorescence microscopy shows that the drug readily penetrates the plasma membrane and accumulates in the intracellular vesicles. Visible emission and photostability of amlodipine allow confocal time-lapse imaging and the drug uptake monitoring.
Asunto(s)
Amlodipino/farmacología , Microscopía Fluorescente , Amlodipino/química , Supervivencia Celular/efectos de los fármacos , Células HEK293 , Humanos , Indoles/metabolismo , Microscopía Confocal , Modelos Biológicos , Conformación Molecular , SolucionesRESUMEN
Atrial dysfunction is highly prevalent and associated with increased severity of heart failure. While rapid excitation-contraction coupling depends on axial junctions in atrial myocytes, the molecular basis of atrial loss of function remains unclear. We identified approximately 5-fold lower junctophilin-2 levels in atrial compared with ventricular tissue in mouse and human hearts. In atrial myocytes, this resulted in subcellular expression of large junctophilin-2 clusters at axial junctions, together with highly phosphorylated ryanodine receptor (RyR2) channels. To investigate the contribution of junctophilin-2 to atrial pathology in adult hearts, we developed a cardiomyocyte-selective junctophilin-2-knockdown model with 0 mortality. Junctophilin-2 knockdown in mice disrupted atrial RyR2 clustering and contractility without hypertrophy or interstitial fibrosis. In contrast, aortic pressure overload resulted in left atrial hypertrophy with decreased junctophilin-2 and RyR2 expression, disrupted axial junctions, and atrial fibrosis. Whereas pressure overload accrued atrial dysfunction and heart failure with 40% mortality, additional junctophilin-2 knockdown greatly exacerbated atrial dysfunction with 100% mortality. Strikingly, transgenic junctophilin-2 overexpression restored atrial contractility and survival through de novo biogenesis of polyadic junctional membrane complexes maintained after pressure overload. Our data show a central role of junctophilin-2 cluster disruption in atrial hypertrophy and identify transgenic augmentation of junctophilin-2 as a disease-mitigating rationale to improve atrial dysfunction and prevent heart failure deterioration.
Asunto(s)
Fibrilación Atrial/metabolismo , Insuficiencia Cardíaca/fisiopatología , Proteínas de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Miocardio/metabolismo , Animales , Fibrilación Atrial/mortalidad , Femenino , Técnicas de Silenciamiento del Gen , Atrios Cardíacos/metabolismo , Atrios Cardíacos/fisiopatología , Insuficiencia Cardíaca/metabolismo , Humanos , Uniones Intercelulares/metabolismo , Masculino , Proteínas de la Membrana/genética , Ratones , Proteínas Musculares/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
Rationale: Recently, abundant axial tubule (AT) membrane structures were identified deep inside atrial myocytes (AMs). Upon excitation, ATs rapidly activate intracellular Ca2+ release and sarcomeric contraction through extensive AT junctions, a cell-specific atrial mechanism. While AT junctions with the sarcoplasmic reticulum contain unusually large clusters of ryanodine receptor 2 (RyR2) Ca2+ release channels in mouse AMs, it remains unclear if similar protein networks and membrane structures exist across species, particularly those relevant for atrial disease modeling. Objective: To examine and quantitatively analyze the architecture of AT membrane structures and associated Ca2+ signaling proteins across species from mouse to human. Methods and Results: We developed superresolution microscopy (nanoscopy) strategies for intact live AMs based on a new custom-made photostable cholesterol dye and immunofluorescence imaging of membraneous structures and membrane proteins in fixed tissue sections from human, porcine, and rodent atria. Consistently, in mouse, rat, and rabbit AMs, intact cell-wide tubule networks continuous with the surface membrane were observed, mainly composed of ATs. Moreover, co-immunofluorescence nanoscopy showed L-type Ca2+ channel clusters adjacent to extensive junctional RyR2 clusters at ATs. However, only junctional RyR2 clusters were highly phosphorylated and may thus prime Ca2+ release at ATs, locally for rapid signal amplification. While the density of the integrated L-type Ca2+ current was similar in human and mouse AMs, the intracellular Ca2+ transient showed quantitative differences. Importantly, local intracellular Ca2+ release from AT junctions occurred through instantaneous action potential propagation via transverse tubules (TTs) from the surface membrane. Hence, sparse TTs were sufficient as electrical conduits for rapid activation of Ca2+ release through ATs. Nanoscopy of atrial tissue sections confirmed abundant ATs as the major network component of AMs, particularly in human atrial tissue sections. Conclusion: AT junctions represent a conserved, cell-specific membrane structure for rapid excitation-contraction coupling throughout a representative spectrum of species including human. Since ATs provide the major excitable membrane network component in AMs, a new model of atrial "super-hub" Ca2+ signaling may apply across biomedically relevant species, opening avenues for future investigations about atrial disease mechanisms and therapeutic targeting.
RESUMEN
We introduce fluorogenic tubulin probes based on the recently reported fluorescent dyes (510R, 580CP, GeR and SiR) and chemotherapy agents - taxanes (docetaxel, cabazitaxel and larotaxel). The cytotoxicity of the final probe, its staining performance and specificity strongly depend on both components. We found correlation between the aggregation efficiency (related to the spirolactonization of fluorophore) and cytotoxicity. Probe optimization allowed us to reach 29 ± 11 nm resolution in stimulated emission depletion (STED) microscopy images of the microtubule network in living human fibroblasts. Application to living fruit fly (Drosophila melanogaster) tissues highlighted two distinct structures: microtubules and tracheoles. We identified 6-carboxy isomers of 580CP and SiR dyes as markers for chitin-containing taenidia, a component of tracheoles. STED microscopy revealed correlation between the taenidia periodicity and the diameter of the tracheole. Combined tubulin and taenidia STED imaging showed close interaction between the microtubules and respiratory networks in living tissues of the insect larvae.
RESUMEN
Photoactivatable rhodamine spiroamides and spirocyclic diazoketones emerged recently as synthetic markers applicable in multicolor super-resolution microscopy. However, their applicability in single molecule localization microscopy (SMLM) is often limited by aggregation, unspecific adhesion, and low reactivity caused by insufficient solubility and precipitation from aqueous solutions. We report here two synthetic modifications increasing the polarity of compact polycyclic and hydrophobic labels decorated with a reactive group: attachment of 3-sulfo-l-alanyl-beta-alanine dipeptide (a "universal hydrophilizer") or allylic hydroxylation in photosensitive rhodamine diazoketones (and spiroamides). The super-resolution images of tubulin and keratin filaments in fixed and living cells exemplify the performance of "blinking" spiroamides derived from N, N, N', N'-tetramethyl rhodamine.
RESUMEN
Visualization of the G-protein coupled receptor (GPCR) is of great importance for studying its function in a native cell. We have synthesized a series of red-emitting fluorescent probes targeting ß-adrenergic receptor (ßAR) that are compatible with confocal and Stimulated Emission Depletion (STED) microscopy as well as with Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay in living cells. The probe based on the agonist BI-167107 and fluorescent dye KK114 demonstrates nanomolar binding affinity and up to nine-fold ß2AR selectivity over ß1AR. Carazolol-derived probes are fluorogenic and allow no-wash imaging experiments. STED microscopy of ß2ARs stained at the native expression level on pancreatic CAPAN cells provides two-fold improvement in lateral optical resolution over confocal mode and reveals the formation of receptor microdomains. These probes retain their functional (agonist or antagonist) properties, allowing simultaneous modulation of cyclic adenosine monophosphate (cAMP) levels and receptor internalization as well as imaging receptor localization.
Asunto(s)
Agonistas Adrenérgicos beta/química , Imagen Molecular/métodos , Sondas Moleculares/química , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Agonistas Adrenérgicos beta/farmacología , Línea Celular Tumoral , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Células HEK293 , Humanos , Microscopía Intravital/métodos , Ligandos , Microscopía Confocal/métodos , Sondas Moleculares/farmacologíaRESUMEN
Fluorescence microscopy of the localization and the spatial and temporal dynamics of specifically labelled proteins is an indispensable tool in cell biology. Besides fluorescent proteins as tags, tag-mediated labelling utilizing self-labelling proteins as the SNAP-, CLIP-, or the Halo-tag are widely used, flexible labelling systems relying on exogenously supplied fluorophores. Unfortunately, labelling of live budding yeast cells proved to be challenging with these approaches because of the limited accessibility of the cell interior to the dyes. In this study we developed a fast and reliable electroporation-based labelling protocol for living budding yeast cells expressing SNAP-, CLIP-, or Halo-tagged fusion proteins. For the Halo-tag, we demonstrate that it is crucial to use the 6'-carboxy isomers and not the 5'-carboxy isomers of important dyes to ensure cell viability. We report on a simple rule for the analysis of ¹H NMR spectra to discriminate between 6'- and 5'-carboxy isomers of fluorescein and rhodamine derivatives. We demonstrate the usability of the labelling protocol by imaging yeast cells with STED super-resolution microscopy and dual colour live cell microscopy. The large number of available fluorophores for these self-labelling proteins and the simplicity of the protocol described here expands the available toolbox for the model organism Saccharomyces cerevisiae.