RESUMO
Nanometre-scale cellular information obtained through super-resolution microscopies are often unaccompanied by functional information, particularly transient and diffusible signals through which life is orchestrated in the nano-micrometre spatial scale. We describe a correlative imaging protocol which allows the ubiquitous intracellular second messenger, calcium (Ca2+), to be directly visualised against nanoscale patterns of the ryanodine receptor (RyR) Ca2+ channels which give rise to these Ca2+ signals in wildtype primary cells. This was achieved by combining total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals, with the subsequent DNA-PAINT imaging of the RyRs. We report a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets and demonstrate how such data can be used to visually identify the ensembles of Ca2+ channels that are locally activated during the genesis of cytoplasmic Ca2+ signals.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Citosol/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Fatores de TempoRESUMO
Right heart failure is the major cause of death in Pulmonary Artery Hypertension (PAH) patients but is not a current, specific therapeutic target. Pre-clinical studies have shown that adrenoceptor blockade can improve cardiac function but the mechanisms of action within right ventricular (RV) myocytes are unknown. We tested whether the ß1-adrenoceptor blocker metoprolol could improve RV myocyte function in an animal model of PAH, by attenuating adverse excitation-contraction coupling remodeling. PAH with RV failure was induced in rats by monocrotaline injection. When PAH was established, animals were given 10â¯mg/kg/day metoprolol (MCTâ¯+â¯BB) or vehicle (MCT). The median time to the onset of heart failure signs was delayed from 23â¯days (MCT), to 31â¯days (MCTâ¯+â¯BB). At 23⯱â¯1â¯days post-injection, MCTâ¯+â¯BB showed improved in vivo cardiac function, measured by echocardiography. RV hypertrophy was reduced despite persistent elevated afterload. RV myocyte contractility during field stimulation was improved at higher pacing frequencies in MCTâ¯+â¯BB. Preserved t-tubule structure, more uniform evoked Ca2+ release, increased SERCA2a expression and faster ventricular repolarization (measured in vivo by telemetry) may account for the improved contractile function. Sarcoplasmic reticulum Ca2+ overload was prevented in MCTâ¯+â¯BB myocytes resulting in fewer spontaneous Ca2+ waves, with a lower pro-arrhythmic potential. Our novel finding of attenuation of defects in excitation contraction coupling by ß1-adrenoceptor blockade with delays in the onset of HF, identifies the RV as a promising therapeutic target in PAH. Moreover, our data suggest existing therapies for left ventricular failure may also be beneficial in PAH induced RV failure.
Assuntos
Antagonistas de Receptores Adrenérgicos beta 1/uso terapêutico , Cálcio/metabolismo , Hipertensão Pulmonar/tratamento farmacológico , Metoprolol/uso terapêutico , Miócitos Cardíacos/metabolismo , Artéria Pulmonar/fisiopatologia , Disfunção Ventricular Direita/tratamento farmacológico , Antagonistas de Receptores Adrenérgicos beta 1/administração & dosagem , Análise de Variância , Animais , Modelos Animais de Doenças , Ecocardiografia , Eletrocardiografia , Insuficiência Cardíaca/metabolismo , Hipertensão Pulmonar/diagnóstico por imagem , Hipertrofia Ventricular Direita/tratamento farmacológico , Masculino , Metoprolol/administração & dosagem , Ratos , Ratos Wistar , Volume Sistólico/efeitos dos fármacos , Disfunção Ventricular Direita/diagnóstico por imagemRESUMO
Pulmonary hypertension (PH) increases the work of the right ventricle (RV) and causes right-sided heart failure. This study examined RV mitochondrial function and ADP transfer in PH animals advancing to right heart failure, and investigated a potential therapy with the specific ß1-adrenergic-blocker metoprolol. Adult Wistar rats (317 ± 4 g) were injected either with monocrotaline (MCT, 60 mg kg-1) to induce PH, or with an equivalent volume of saline for controls (CON). At three weeks post-injection the MCT rats began oral metoprolol (10 mg kg-1 day-1-) or placebo treatment until heart failure was observed in the MCT group. Mitochondrial function was then measured using high-resolution respirometry from permeabilised RV fibres. Relative to controls, MCT animals had impaired mitochondrial function but maintained coupling between myofibrillar ATPases and mitochondria, despite an increase in ADP diffusion distances. Cardiomyocytes from the RV of MCT rats were enlarged, primarily due to an increase in myofibrillar protein. The ratio of mitochondria per myofilament area was decreased in both MCT groups (p ≤ 0.05) in comparison to control (CON: 1.03 ± 0.04; MCT: 0.74 ± 0.04; MCT + BB: 0.74 ± 0.03). This not only implicates impaired energy production in PH, but also increases the diffusion distance for metabolites within the MCT cardiomyocytes, adding an additional hindrance to energy supply. Together, these changes may limit energy supply in MCT rat hearts, particularly at high cardiac workloads. Metoprolol treatment did not delay the onset of heart failure symptoms, improve mitochondrial function, or regress RV hypertrophy.
Assuntos
Antagonistas de Receptores Adrenérgicos beta 1/farmacologia , Metoprolol/farmacologia , Mitocôndrias/efeitos dos fármacos , Função Ventricular Direita/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Administração Oral , Antagonistas de Receptores Adrenérgicos beta 1/uso terapêutico , Animais , Modelos Animais de Doenças , Metabolismo Energético/efeitos dos fármacos , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/prevenção & controle , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/tratamento farmacológico , Masculino , Metoprolol/uso terapêutico , Mitocôndrias/metabolismo , Monocrotalina/toxicidade , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miofibrilas/metabolismo , Miofibrilas/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Efeito Placebo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
Nanodomains are intracellular foci which transduce signals between major cellular compartments. One of the most ubiquitous signal transducers, the ryanodine receptor (RyR) calcium channel, is tightly clustered within these nanodomains. Super-resolution microscopy has previously been used to visualize RyR clusters near the cell surface. A majority of nanodomains located deeper within cells have remained unresolved due to limited imaging depths and axial resolution of these modalities. A series of enhancements made to expansion microscopy allowed individual RyRs to be resolved within planar nanodomains at the cell periphery and the curved nanodomains located deeper within the interiors of cardiomyocytes. With a resolution of â¼ 15 nm, we localized both the position of RyRs and their individual phosphorylation for the residue Ser2808. With a three-dimensional imaging protocol, we observed disturbances to the RyR arrays in the nanometer scale which accompanied right-heart failure caused by pulmonary hypertension. The disease coincided with a distinct gradient of RyR hyperphosphorylation from the edge of the nanodomain toward the center, not seen in healthy cells. This spatial profile appeared to contrast distinctly from that sustained by the cells during acute, physiological hyperphosphorylation when they were stimulated with a ß-adrenergic agonist. Simulations of RyR arrays based on the experimentally determined channel positions and phosphorylation signatures showed how the nanoscale dispersal of the RyRs during pathology diminishes its intrinsic likelihood to ignite a calcium signal. It also revealed that the natural topography of RyR phosphorylation could offset potential heterogeneity in nanodomain excitability which may arise from such RyR reorganization.
Assuntos
Canais de Cálcio/metabolismo , Nanoestruturas/química , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Transdução de Sinais , Agonistas Adrenérgicos beta/farmacologia , Cálcio/metabolismo , Humanos , Microscopia , Fosforilação , Transdução de Sinais/efeitos dos fármacosRESUMO
Atrial remodeling due to elevated arterial pressure predisposes the heart to atrial fibrillation (AF). Although abnormal sarcoplasmic reticulum (SR) function has been associated with AF, there is little information on the effects of elevated afterload on atrial Ca2+-handling. We investigated the effects of ascending aortic banding (AoB) on Ca2+-handling in rat isolated atrial myocytes in comparison to age-matched sham-operated animals (Sham). Myocytes were either labelled for ryanodine receptor (RyR) or loaded with fluo-3-AM and imaged by confocal microscopy. AoB myocytes were hypertrophied in comparison to Sham controls (P<0.0001). RyR labeling was localized to the z-lines and to the cell edge. There were no differences between AoB and Sham in the intensity or pattern of RyR-staining. In both AoB and Sham, electrical stimulation evoked robust SR Ca2+-release at the cell edge whereas Ca2+ transients at the cell center were much smaller. Western blotting showed a decreased L-type Ca channel expression but no significant changes in RyR or RyR phosphorylation or in expression of Na+/Ca2+ exchanger, SR Ca2+ ATPase or phospholamban. Mathematical modeling indicated that [Ca2+]i transients at the cell center were accounted for by simple centripetal diffusion of Ca2+ released at the cell edge. In contrast, caffeine (10 mM) induced Ca2+ release was uniform across the cell. The caffeine-induced transient was smaller in AoB than in Sham, suggesting a reduced SR Ca2+-load in hypertrophied cells. There were no significant differences between AoB and Sham cells in the rate of Ca2+ extrusion during recovery of electrically-stimulated or caffeine-induced transients. The incidence and frequency of spontaneous Ca2+-transients following rapid-pacing (4 Hz) was greater in AoB than in Sham myocytes. In conclusion, elevated afterload causes cellular hypertrophy and remodeling of atrial SR Ca2+-release.