Ca2+-Driven Selectivity of the Effect of the Cardiotonic Steroid Marinobufagenin on Rabbit Sinoatrial Node Function.

The synergy between Na+-K+ pumps, Na+-Ca2+ exchangers, membrane currents and the sarcoplasmic reticulum (SR) generates the coupled-clock system, which governs the spontaneous electrical activity of heart sinoatrial node cells (SANCs). Ca2+ mediates the degree of clock coupling via local Ca2+ release (LCR) from the SR and activation of cAMP/PKA signaling. Marinobufagenin (MBG) is a natural Na+-K+ pump inhibitor whose effect on SANCs has not been measured before. The following two hypotheses were tested to determine if and how MBG mediates between the Na+-K+ pump and spontaneous SAN activity: (i) MBG has a distinct effect on beat interval (BI) due to variable effects on LCR characteristics, and (ii) Ca2+ is an important mediator between MBG and SANC activity. Ca2+ transients were measured by confocal microscopy during application of increasing concentrations of MBG. To further support the hypothesis that Ca2+ mediates between MBG and SANC activity, Ca2+ was chelated by the addition of BAPTA. Dose response tests found that 100 nM MBG led to no change in BI in 6 SANCs (no BI change group), and to BI prolongation in 10 SANCs (BI change group). At the same concentration, the LCR period was prolonged in both groups, but more significantly in the BI change group. BAPTA-AM prolonged the BI in 12 SANCs. In the presence of BAPTA, 100 nM MBG had no effect on SANC BI or on the LCR period. In conclusion, the MBG effects on SANC function are mediated by the coupled clock system, and Ca2+ is an important regulator of these effects.

cAMP signaling affects age-associated deterioration of pacemaker beating interval dynamics.

Sinoatrial node (SAN) beating interval variability (BIV) and the average beating interval (BI) are regulated by a coupled-clock system, driven by Ca2+-calmodulin activated adenylyl cyclase, cAMP, and downstream PKA signaling. Reduced responsiveness of the BI and BIV to submaximal, [X]50, ß-adrenergic receptor (ß-AR) stimulation, and phosphodiesterase inhibition (PDEI) have been documented in aged SAN tissue, whereas the maximal responses, [X]max, do not differ by age. To determine whether age-associated dysfunction in cAMP signaling leads to altered responsiveness of BI and BIV, we measured cAMP levels and BI in adult (2-4 months n = 27) and aged (22-26 months n = 25) C57/BL6 mouse SAN tissue in control and in response to ß-AR or PDEI at X50 and [X]max. Both cAMP and average BI in adult SAN were reduced at X50, whereas cAMP and BI at Xmax did not differ by age. cAMP levels and average BI were correlated both within and between adult and aged SAN. BIV parameters in long- and short-range terms were correlated with cAMP levels for adult SAN. However, due to reduced cAMP within aged tissues at [X]50, these correlations were diminished in advanced age. Thus, cAMP level generated by the coupled clock mechanisms is tightly linked to average BI. Reduced cAMP level at X50 in aged SAN explains the reduced responsiveness of the BI and BIV to ß-AR stimulation and PDEI.

Changes in cAMP signaling are associated with age-related downregulation of spontaneously beating atrial tissue energetic indices.

The prevalence of atria-related diseases increases exponentially with age and is associated with ATP supply-to-demand imbalances. Because evidence suggests that cAMP regulates ATP supply-to-demand, we explored aged-associated alterations in atrial ATP supply-to-demand balance and its correlation with cAMP levels. Right atrial tissues driven by spontaneous sinoatrial node impulses were isolated from aged (22-26 months) and adult (3-4 months) C57/BL6 mice. ATP demand increased by addition of isoproterenol or 3-Isobutyl-1-methylxanthine (IBMX) and decreased by application of carbachol. Each drug was administrated at the dose that led to a maximal change in beating rate (Xmax) and to 50% of that maximal change in adult tissue (X50). cAMP, NADH, NAD + NADH, and ATP levels were measured in the same tissue. The tight correlation between cAMP levels and the beating rate (i.e., the ATP demand) demonstrated in adult atria was altered in aged atria. cAMP levels were lower in aged compared to adult atrial tissue exposed to X50 of ISO or IBMX, but this difference narrowed at Xmax. Neither ATP nor NADH levels correlated with ATP demand in either adult or aged atria. Baseline NADH levels were lower in aged as compared to adult atria, but were restored by drug perturbations that increased cAMP levels. Reduction in Ca2+-activated adenylyl cyclase-induced decreased cAMP and prolongation of the spontaneous beat interval of adult atrial tissue to their baseline levels in aged tissue, brought energetics indices to baseline levels in aged tissue. Thus, cAMP regulates right atrial ATP supply-to-demand matching and can restore age-associated ATP supply-to-demand imbalance.

Distinct PKA Signaling in Cytosolic and Mitochondrial Compartments in Electrically Paced Atrial Myocytes.

Protein kinase A (PKA) is a key nodal signaling molecule that regulates a wide range of cellular functions in the cytosol and mitochondria. The distribution of A-kinase anchoring proteins that tether PKA, the local interaction with degradation molecules, and regulation by Ca2+, may lead to distinct spatiotemporal cAMP/PKA signaling in these compartments. In this work, FRET-based sensors were used to investigate PKA signaling in the cytosol, outer mitochondrial membrane (OMM), and mitochondrial matrix (MM) and its crosstalk with Ca2+ in response to electrical stimulation of cultured rabbit atrial cells. A gradual decrease in PKA activity eliminating the ability of the atrial cells to respond to physiological electrical stimulation, was observed upon treatment of cells with H-89. Chelation of intracellular Ca2+ by BAPTA reduced PKA activity and diminished its response to forskolin, an AC stimulator. Under basal conditions, PKA activity in response to forskolin was lower in the OMM compared to the cytosol and MM. In response to electrical stimulation in the presence of ISO, distinct compartmentalization of PKA activity was observed, with higher activity in the cytosol and MM than in the OMM. Thus, distinct Ca2+-dependent spatiotemporal cAMP/PKA signaling exists in atrial cells, likely mediating its excitation and mitochondrial function.

Increase in Ca2+-Activated cAMP/PKA Signaling Prevents Hydroxychloroquine-Induced Bradycardia of the Cardiac Pacemaker.

Bradycardia or tachycardia are known side effects of drugs that limit their clinical use. The heart pacemaker function which control the heart rate under normal conditions is determined by coupled clock system. Thus, interfering with specific clock mechanism will affect other clock mechanisms through changes in interconnected signaling and can lead to rhythm disturbance. However, upregulation of a different clock components can compensate for this change. We focus here on hydroxychloroquine (HCQ), which has been shown effective in treating COVID-19 patients, however its bradycardic side effect limits its clinical use. We aim to decipher the mechanisms underlying the effect of HCQ on pacemaker automaticity, to identify a potential drug that will eliminate the bradycardia. We used isolated rabbit sinoatrial node (SAN) cells, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and mouse SAN cells residing in SAN tissue. Further, we employed SAN cell computational model to suggest mechanistic insights of the effect of HCQ on pacemaker function. HCQ increased mean spontaneous beat interval and variability in all three models in parallel to slower intracellular kinetics. The computational model suggested that HCQ affects the pacemaker (funny) current (If), L-type Ca2+ current (ICa,L), transient outward potassium (Ito) and due to changes in Ca2+ kinetics, the sodium-calcium exchanger current (INCX). Co-application of 3'-isobutylmethylxanthine (IBMX) and HCQ prevented the increase in beat interval and variability in all three experimental models. The HCQ-induced increase in rabbit and mice SAN cell and hiPSC-CM spontaneous beat interval, can be prevented by a phosphodiester inhibitor that restores automaticity due to slower intracellular Ca2+ kinetics.

Functional capacity and cognitive performance of adults from Argentina in cardiopulmonary rehabilitation / Capacidad funcional y rendimiento cognitivo de adultos argentinos en rehabilitación cardiopulmonar.

Introduction: Functional capacity is decreased in people with Cardiovascular Diseases and Chronic Respiratory Diseases. These diseases have also been associated with cognitive dysfunction. The study examines the efficacy of a cardiopulmonary rehabilitation program in the recovery of functional capacity and analyzes whether subjects with cardiopulmonary diseases suffer from cognitive dysfunction. Materials and methods: Participated 50 adults with medium-high education who completed a cardiopulmonary rehabilitation program of between 3 and 6 months based on physical education, nutritional education, promotion of healthy habits and medication management. Functional capacity was evaluated with the Duke index at the beginning and end of the program. Memory and language tests were also administered, for the only time, at the beginning of the program, comparing the values obtained with Argentine normative studies. The data was analyzed with the Wilcoxon test, bivariate correlations, and linear regression. Results: Functional capacity increased significantly at the end of the program. In any case, the post-program Duke value suggests that the functional capacity of the patients continues to be affected. On the other hand, a memory test explained 10,8% of the variance in the Duke index, and there are no findings of cognitive dysfunction. Conclusion: The functional capacity of cardiopulmonary patients improved with the rehabilitation program, although this improvement is clinically insufficient. Better memory performance predicted greater functional capacity, which is why it is suggested to add cognitive stimulation workshops to cardiopulmonary rehabilitation programs. This sample with cardiopulmonary disease does not present cognitive dysfunction, probably due to its high cognitive reserve. Introduction: Functional capacity is decreased in people with Cardiovascular Diseases and Chronic Respiratory Diseases. These diseases have also been associated with cognitive dysfunction. The study examines the efficacy of a cardiopulmonary rehabilitation program in the recovery of functional capacity and analyzes whether subjects with cardiopulmonary diseases suffer from cognitive dysfunction. Materials and methods: Participated 50 adults with medium-high education who completed a cardiopulmonary rehabilitation program of between 3 and 6 months based on physical education, nutritional education, promotion of healthy habits and medication management. Functional capacity was evaluated with the Duke index at the beginning and end of the program. Memory and language tests were also administered, for the only time, at the beginning of the program, comparing the values obtained with Argentine normative studies. The data was analyzed with the Wilcoxon test, bivariate correlations, and linear regression. Results: Functional capacity increased significantly at the end of the program. In any case, the post-program Duke value suggests that the functional capacity of the patients continues to be affected. On the other hand, a memory test explained 10,8% of the variance in the Duke index, and there are no findings of cognitive dysfunction. Conclusion: The functional capacity of cardiopulmonary patients improved with the rehabilitation program, although this improvement is clinically insufficient. Better memory performance predicted greater functional capacity, which is why it is suggested to add cognitive stimulation workshops to cardiopulmonary rehabilitation programs. This sample with cardiopulmonary disease does not present cognitive dysfunction, probably due to its high cognitive reserve.

Introducción: La capacidad funcional está disminuida en personas con Enfermedades Cardiovasculares y Enfermedades Respiratorias Crónicas. Estas enfermedades también han sido asociadas a disfunción cognitiva. El estudio examina la eficacia de un programa de rehabilitación cardiopulmonar en la recuperación de la capacidad funcional, y analiza si sujetos con enfermedades cardiopulmonares sufren disfunción cognitiva. Materiales y métodos: Participaron 50 personas adultas con instrucción media-alta que completaron un programa de rehabilitación cardiopulmonar de entre 3 y 6 meses basado en educación física, educación nutricional, promoción de hábitos saludables y manejo de medicación. Se evaluó la capacidad funcional con el índice Duke al iniciar y finalizar el programa. Se administró también, por única vez, al iniciar el programa, pruebas de memoria y de lenguaje, comparando los valores obtenidos con estudios normativos argentinos. Se analizó los datos con test Wilcoxon, correlaciones bivariadas y regresión lineal. Resultados: La capacidad funcional aumentó significativamente al finalizar el programa. De todos modos el valor Duke pos- programa sugiere que la capacidad funcional de los pacientes continúa afectada. Por otro lado, una prueba de memoria explicó el 10,8% de la varianza en el índice Duke, y no hay hallazgos de disfunción cognitiva. Conclusión: La capacidad funcional de los pacientes cardiopulmonares mejoró con el programa de rehabilitación, aunque dicha mejora, es clínicamente insuficiente. Mejor rendimiento de memoria predijo mayor capacidad funcional, por lo que se sugiere añadir talleres de estimulación cognitiva a los programas de rehabilitación cardiopulmonares. Esta muestra con enfermedad cardiopulmonar no presenta disfunción cognitiva, probablemente por su elevada reserva cognitiva.

May the Force Not Be With You During Culture: Eliminating Mechano-Associated Feedback During Culture Preserves Cultured Atrial and Pacemaker Cell Functions.

Cultured cardiomyocytes have been shown to possess significant potential as a model for characterization of mechano-Ca2+, mechano-electric, and mechano-metabolic feedbacks in the heart. However, the majority of cultured cardiomyocytes exhibit impaired electrical, mechanical, biochemical, and metabolic functions. More specifically, the cells do not beat spontaneously (pacemaker cells) or beat at a rate far lower than their physiological counterparts and self-oscillate (atrial and ventricular cells) in culture. Thus, efforts are being invested in ensuring that cultured cardiomyocytes maintain the shape and function of freshly isolated cells. Elimination of contraction during culture has been shown to preserve the mechano-Ca2+, mechano-electric, and mechano-metabolic feedback loops of cultured cells. This review focuses on pacemaker cells, which reside in the sinoatrial node (SAN) and generate regular heartbeat through the initiation of the heart's electrical, metabolic, and biochemical activities. In parallel, it places emphasis on atrial cells, which are responsible for bridging the electrical conductance from the SAN to the ventricle. The review provides a summary of the main mechanisms responsible for mechano-electrical, Ca2+, and metabolic feedback in pacemaker and atrial cells and of culture methods existing for both cell types. The work concludes with an explanation of how the elimination of mechano-electrical, mechano-Ca2+, and mechano-metabolic feedbacks during culture results in sustained cultured cell function.

Eliminating contraction during culture maintains global and local Ca2+ dynamics in cultured rabbit pacemaker cells.

Pacemaker cells residing in the sinoatrial node generate the regular heartbeat. Ca2+ signaling controls the heartbeat rate-directly, through the effect on membrane molecules (NCX exchange, K+ channel), and indirectly, through activation of calmodulin-AC-cAMP-PKA signaling. Thus, the physiological role of signaling in pacemaker cells can only be assessed if the Ca2+ dynamics are in the physiological range. Cultured cells that can be genetically manipulated and/or virally infected with probes are required for this purpose. Because rabbit pacemaker cells in culture experience a decrease in their spontaneous action potential (AP) firing rate below the physiological range, Ca2+ dynamics are expected to be affected. However, Ca2+ dynamics in cultured pacemaker cells have not been reported before. We aim to a develop a modified culture method that sustains the global and local Ca2+ kinetics along with the AP firing rate of rabbit pacemaker cells. We used experimental and computational tools to test the viability of rabbit pacemaker cells in culture under various conditions. We tested the effect of culture dish coating, pH, phosphorylation, and energy balance on cultured rabbit pacemaker cells function. The cells were maintained in culture for 48 h in two types of culture media: one without the addition of a contraction uncoupler and one enriched with either 10 mM BDM (2,3-Butanedione 2-monoxime) or 25 µM blebbistatin. The uncoupler was washed out from the medium prior to the experiments. Cells were successfully infected with a GFP adenovirus cultured with either BDM or blebbistatin. Using either uncoupler during culture led to the cell surface area being maintained at the same level as fresh cells. Moreover, the phospholamban and ryanodine receptor densities and their phosphorylation level remained intact in culture when either blebbistatin or BDM were present. Spontaneous AP firing rate, spontaneous Ca2+ kinetics, and spontaneous local Ca2+ release parameters were similar in the cultured cells with blebbistatin as in fresh cells. However, BDM affects these parameters. Using experimental and a computational model, we showed that by eliminating contraction, phosphorylation activity is preserved and energy is reduced. However, the side-effects of BDM render it less effective than blebbistatin.

A Method Sustaining the Bioelectric, Biophysical, and Bioenergetic Function of Cultured Rabbit Atrial Cells.

Culturing atrial cells leads to a loss in their ability to be externally paced at physiological rates and to maintain their shape. We aim to develop a culture method that sustains the shape of atrial cells along with their biophysical and bioenergetic properties in response to physiological pacing. We hypothesize that adding 2,3-Butanedione 2-monoxime (BDM), which inhibits contraction during the culture period, will preserve these biophysical and bioenergetic properties. Rabbit atrial cells were maintained in culture for 24 h in a medium enriched with a myofilament contraction inhibitor, BDM. The morphology and volume of the cells, including their ability to contract in response to 1-3 Hz electrical pacing, was maintained at the same level as fresh cells. Importantly, the cells could be successfully infected with a GFP adenovirus. Action potentials, Ca2+ transients, and local Ca2+ spark parameters were similar in the cultured and in fresh cells. Finally, these cultured cells' flavoprotein autofluorescence was maintained at a constant level in response to electrical pacing, a response similar to that of fresh cells. Thus, eliminating contraction during the culture period preserves the bioelectric, biophysical and bioenergetic properties of rabbit atrial myocytes. This method therefore has the potential to further improve our understanding of energetic and biochemical regulation in the atria.

Semi-automated program for analysis of local Ca2+ spark release with application for classification of heart cell type.

Local Ca2+ spark releases are essential to the Ca2+ cycling process. Thus, they play an important role in ventricular and atrial cell contraction, as well as in sinoatrial cell automaticity. Characterizing their properties in healthy cells from different regions in the heart can reveal the basic biophysical differences among these regions. We designed a semi-automatic Matlab Graphical User Interface (called Sparkalyzer) to characterize parameters of Ca2+ spark release from any major cardiac tissue, as recorded in line-scan mode with a confocal laser-scanning microscope. We validated the algorithm on experimental images from rabbit sinoatrial, atrial, and ventricular cells loaded with Fluo-4 AM. The program characterizes general image parameters of Ca2+ transients and sparks: spark duration, which indicates for how long the spark provides Ca2+ to the closed intracellular mechanisms (typical value: 25±1, 23±1, 26±1ms for sinoatrial, atrial, and ventricular cells, respectively); spark amplitude, which indicates the amount of Ca2+ released by a single spark (1.6±0.1, 1.6±0.2, 1.4±0.1F/F0 for sinoatrial, atrial, and ventricular cells, respectively); spark length, which is the length of the Ca2+ wavelets fired out of a row of ryanodine receptors (5±0.1, 5±0.2, 3.4±0.3µm for sinoatrial, atrial, or ventricular cells, respectively) and number of sparks (0.14±0.02, 0.025±0.01, 0.02±0.01 for 1µm in 1s for sinoatrial, atrial, and ventricular cells, respectively). This method is reliable for Ca2+ spark analysis of sinoatrial, atrial, or ventricular cells. Moreover, by examining the average value of Ca2+ spark characteristics and their scattering around the mean, atrial, ventricular and sinoatrial cells can be differentiated.

Adipose-derived endothelial and mesenchymal stem cells enhance vascular network formation on three-dimensional constructs in vitro.

BACKGROUND: Adipose-derived mesenchymal stem cells (MSCs) have been gaining fame mainly due to their vast clinical potential, simple isolation methods and minimal donor site morbidity. Adipose-derived MSCs and microvascular endothelial cells have been shown to bear angiogenic and vasculogenic capabilities. We hypothesized that co-culture of human adipose-derived MSCs with human adipose-derived microvascular endothelial cells (HAMECs) will serve as an effective cell pair to induce angiogenesis and vessel-like network formation in three-dimensional scaffolds in vitro. METHODS: HAMECs or human umbilical vein endothelial cells (HUVECs) were co-cultured on scaffolds with either MSCs or human neonatal dermal fibroblasts. Cells were immunofluorescently stained within the scaffolds at different time points post-seeding. Various analyses were performed to determine vessel length, complexity and degree of maturity. RESULTS: The HAMEC:MSC combination yielded the most organized and complex vascular elements within scaffolds, and in the shortest period of time, when compared to the other tested cell combinations. These differences were manifested by higher network complexity, more tube alignment and higher α-smooth muscle actin expression. Moreover, these generated microvessels further matured and developed during the 14-day incubation period within the three-dimensional microenvironment. CONCLUSIONS: These data demonstrate optimal vascular network formation upon co-culture of microvascular endothelial cells and adipose-derived MSCs in vitro and constitute a significant step in appreciation of the potential of microvascular endothelial cells and MSCs in different tissue engineering applications that can also be advantageous in in vivo studies.

Resistance of Helicobacter pylori to tetracycline, amoxicillin, clarithromycin and metronidazole in Israeli children and adults.

The aim of this study was to examine Helicobacter pylori-resistance rate to different antibiotics: tetracycline, amoxicillin, clarithromycin and metronidazole, and to compare eradication rates in adults and children in Israel. The study was based on the hypothesis of high-resistance rates to clarithromycin and metronidazole especially in adults and overall low-resistance rates to tetracycline and amoxicillin. One seventy six biopsies from patients with dyspeptic symptoms were cultured of which 100 were from adults (19-79 years) and 76 from children (7-17 years). All positive cultures were examined by Epsilometer test for MIC determination against tetracycline, amoxicillin, clarithromycin and metronidazole. 48.3% (85 out of 176) were H. pylori positive, of which 44% were from adults and 54% from children. Antibiotic resistance was seen in 31 out of 44 (70.5%) for metronidazole, 1 out of 44 (2.3%) for amoxicillin, 10 out of 44 (22.3%) for clarithromycin and 1 out of 44 (2.3%) for tetracycline among adults. Antibiotic resistance was seen in 10 out of 41 (24.4%) for metronidazole, 5 out of 41 (12.2%) for amoxicillin, 10 out of 41 (24.4%) for clarithromycin and 1 out of 41 (2.4%) for tetracycline among children. High rates of H. pylori resistance to metronidazole and clarithromycin was found especially among adults. Therefore, to increase the success rate of anti-H. pylori treatment, other classes of antibiotics need to be considered.