Reference genes for the developing mouse lung under consideration of biological, technical and experimental confounders.

For gene expression analysis, the raw data obtained from RT-qPCR are preferably normalized to reference genes, which should be constantly expressed regardless of experimental conditions. Selection of reference genes is particularly challenging for the developing lung because of the complex transcriptional and epigenetic regulation of genes during organ maturation and injury repair. To date, there are only limited experimental data addressing reliable reference genes for this biological circumstance. In this study, we evaluated reference genes for the lung in neonatal C57BL/6 mice under consideration of biological, technical and experimental conditions. For that, we thoroughly selected candidates from commonly used reference genes side-by-side with novel ones by analyzing publicly available microarray datasets. We performed RT-qPCR of the selected candidate genes and analyzed their expression variability using GeNorm and Normfinder. Cell-specific expression of the candidate genes was analyzed using our own single-cell RNA-sequencing data from the developing mouse lung. Depending on the investigated conditions, i.e., developmental stages, sex, RNA quality, experimental condition (hyperoxia) and cell types, distinct candidate genes demonstrated stable expression confirming their eligibility as reliable reference genes. Our results provide valuable information for the selection of proper reference genes in studies investigating the neonatal mouse lung.

Clinical Impact of the Polypill for Cardiovascular Prevention in Latin America: A Consensus Statement of the Inter-American Society of Cardiology.

The burden of cardiovascular diseases (CVD) is increasing, particularly in low-middle-income countries such as most of Latin America. This region presents specific socioeconomic characteristics, generating a high incidence of CVD despite efforts to control the problem. A consensus statement has been developed by Inter-American Society of Cardiology with the aim of answering some important questions related to CVD in this region and the role of the polypill in cardiovascular (CV) prevention as an intervention to address these issues. A multidisciplinary team composed of Latin American experts in the prevention of CVD was convened by the Inter-American Society of Cardiology and participated in the process and the formulation of statements. To characterize the prevailing situation in Latin American countries, we describe the most significant CV risk factors in the region. The barriers that impair the use of CV essential medications are also reviewed. The role of therapeutic adherence in CV prevention and how the polypill emerges as an effective strategy for optimizing adherence, accessibility, and affordability in the treatment of CVDs are discussed in detail. Clinical scenarios in which the polypill could represent an effective intervention in primary and secondary CV prevention are described. This initiative is expected to help professionals involved in the management of CVD and public health policymakers develop optimal strategies for the management of CVDs.

Protein kinase A is activated by the n-3 polyunsaturated fatty acid eicosapentaenoic acid in rat ventricular muscle.

During cardiac ischaemia antiarrhythmic n-3 polyunsaturated fatty acids (PUFAs) are released following activation of phospholipase A2, if they are in the diet prior to ischaemia. Here we show a positive lusitropic effect of one such PUFA, eicosapentaenoic acid (EPA) in the antiarrhythmic concentration range in Langendorff hearts and isolated rat ventricular myocytes due to activation of protein kinase A (PKA). Several different approaches indicated activation of PKA by EPA (5-10 micromol l(-1)): the time constant of decay of the systolic Ca2+ transient decreased to 65.3 +/- 5.0% of control, Western blot analysis showed a fourfold increase in phospholamban phosphorylation, and PKA activity increased by 21.0 +/- 7.3%. In addition myofilament Ca2+ sensitivity was reduced in EPA; this too may have resulted from PKA activation. We also found that EPA inhibited L-type Ca2+ current by 38.7 +/- 3.9% but this increased to 63.3 +/- 3.4% in 10 micromol l(-1) H89 (to inhibit PKA), providing further evidence of activation of PKA by EPA. PKA inhibition also prevented the lusitropic effect of EPA on the systolic Ca2+ transient and contraction. Our measurements show, however, PKA activation in EPA cannot be explained by increased cAMP levels and alternative mechanisms for PKA activation are discussed. The combined lusitropic effect and inhibition of contraction by EPA may, respectively, combat diastolic dysfunction in ischaemic cardiac muscle and promote cell survival by preserving ATP. This is a further level of protection for the heart in addition to the well-documented antiarrhythmic qualities of these fatty acids.

Effects of eicosapentaenoic acid on cardiac SR Ca(2+)-release and ryanodine receptor function.

n-3 polyunsaturated fatty acids (PUFAs) can prevent life-threatening arrhythmias but the mechanisms responsible have not been established. There is strong evidence that part of the antiarrhythmic action of PUFAs is mediated through inhibition of the Ca(2+)-release mechanism of the sarcoplasmic reticulum (SR). It has also been shown that PUFAs activate protein kinase A (PKA) and produce effects in the cardiac cell similar to beta-adrenergic stimulation. We have investigated whether the inhibitory effect of PUFAs on the Ca(2+)-release mechanism is caused by direct inhibition of the SR Ca(2+)-release channel/ryanodine receptor (RyR) or requires activation of PKA. Experiments in intact cells under voltage-clamp show that the n-3 PUFA eicosapentaenoic acid (EPA) is able to reduce the frequency of spontaneous waves of Ca(2+)-release while increasing SR Ca(2+) content even when PKA activity is inhibited with H-89. This suggests that the EPA-induced inhibition of SR Ca(2+)-release is not dependent on activation of PKA. Consistent with this, single-channel studies demonstrate that EPA (10-100 microM), but not saturated fatty acids, reduce the open probability (Po) of the cardiac RyR incorporated into phospholipid bilayers. EPA also inhibited the binding of [3H]ryanodine to isolated heavy SR. Our results indicate that direct inhibition of RyR channel gating by PUFAs play an important role in the overall antiarrhythmic properties of these compounds.

Direct and indirect modulation of rat cardiac sarcoplasmic reticulum function by n-3 polyunsaturated fatty acids.

Measurements were made of trans-sarcolemmal Ca(2+) fluxes and intracellular [Ca(2+)](i) in rat ventricular myocytes loaded with Indo-1 to determine how the n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) suppresses spontaneous waves of Ca(2+) release. We report that in 10 microM EPA, the Ca(2+) efflux generated by individual waves increased by 11.3 +/- 4.9 % over control levels. However, wave-generated efflux per unit time fell overall by 19 +/- 5.3 %. On removal of EPA, wave frequency increased transiently such that Ca(2+) efflux was greater than normal and the cell lost 28.0 +/- 10.6 micromol l(-1) Ca(2+). This probably represents the loss of extra Ca(2+) accumulated by the sarcoplasmic reticulum (SR), while Ca(2+) release was inhibited. These results are evidence of inhibition of the SR Ca(2+)-release mechanism and reduced availability of Ca(2+) to the SR. From the relationship between average intracellular Ca(2+) and the frequency of spontaneous waves, we have calculated the relative contributions of these different mechanisms to the lower frequency of waves. In EPA, the frequency of spontaneous waves fell by 37.5 +/- 8.1 %, the majority of this (29.2 +/- 8.8 %) is due to inhibition of the Ca(2+)-release mechanism. In EPA, the rate of fall of Ca(2+) in the caffeine response (an indicator of surface membrane Ca(2+) efflux pathway activity) was not altered. We conclude, therefore, that the lower resting level of Ca(2+) observed in EPA is due to a lower influx of Ca(2+) across the surface membrane rather than increased activation of efflux pathways. How these effects might contribute to the anti-arrhythmic actions of EPA is discussed.

Inhibition of sarcoplasmic reticulum function by polyunsaturated fatty acids in intact, isolated myocytes from rat ventricular muscle.

1. We have studied the effects of two polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on spontaneous and electrically stimulated contractions in single, isolated ventricular myocytes from rat hearts. 2. The frequency of spontaneous waves of calcium release and contraction (induced by elevation of the bathing calcium concentration) is reduced in the presence of EPA. At the same time the resting level of intracellular calcium falls, the resting cell length increases and the amplitude of shortening decreases. All these effects are reversed on removal of EPA. 3. Imaging of the waves of calcium release shows that the amplitude and the rate of propagation of the wave is increased in EPA. Consistent with the increased amplitude, integration of the caffeine-induced Na+-Ca2+ exchange current (a measure of the sarcoplasmic reticulum (SR) calcium content) is increased by both EPA and DHA. 4. EPA has a maintained negative inotropic effect on voltage clamped myocytes. This seems to be entirely due to inhibition of the L-type calcium current. Smaller depolarising pulses in control conditions that elicit the same calcium current as in EPA also activate the same level of contraction. This is in spite of the increased SR calcium content in EPA. 5. It is concluded that PUFAs have two effects on the SR; they reduce the availability of calcium for uptake and they inhibit the release mechanism. Both of these effects should lower the frequency of spontaneous waves of calcium release. As spontaneous release of calcium can initiate arrhythmias, some of the anti-arrhythmic action of PUFAs must be exerted at the level of the SR.

Enhanced Ca2+ current and decreased Ca2+ efflux restore sarcoplasmic reticulum Ca2+ content after depletion.

[Ca2+]i was measured using the fluorescent indicator indo 1 in voltage-clamped ferret and rat ventricular myocytes. The Ca2+ content of the sarcoplasmic reticulum (SR) was estimated from the integral of the Na(+)-Ca2+ exchange current activated by caffeine. Refilling of the SR after caffeine removal was enhanced by stimulation. As the systolic Ca2+ transient recovered, the integral of the L-type Ca2+ current decreased and that of the Na(+)-Ca2+ exchange tail current increased. For the early pulses, the gain of Ca2+ via the Ca2+ current is greater than the loss via the exchanger, and during steady state stimulation, the fluxes are equal. The difference in the integrals gives a measure of the net gain of cell Ca2+ with each pulse. When these are summed, the calculated gain of cell Ca2+ agrees well with the increase of SR Ca2+ produced by stimulation, as measured from the caffeine-evoked currents. There was a nonlinear relationship between SR Ca2+ content and the magnitude of the systolic Ca2+ transient such that at high SR Ca2+ content a given increase of content had a greater effect on the Ca2+ transient than did an increase at low SR content. In conclusion, the effects of systolic Ca2+ on the Ca2+ current and Na(+)-Ca2+ exchange current provide a means to regulate SR Ca2+ content and thence the systolic Ca2+ transient.

Estimate of net calcium fluxes and sarcoplasmic reticulum calcium content during systole in rat ventricular myocytes.

1. The experiments were performed on voltage-clamped cells in which intracellular calcium concentration ([Ca2+]i) was measured with the fluorescent indicator indo-1 (acetoxymethyl ester (AM) loading). When cells were stimulated with a short (100 ms) depolarizing pulse, following a rest, the magnitude of the first systolic calcium transient was greater than that in the steady state (rest potentiation) and decayed to its steady level over a few stimuli. If a longer pulse (800 ms) was used then the systolic calcium transient was either unaffected or increased in magnitude following a rest. During constant stimulation, if the length of the pulse is decreased, then the magnitude of the calcium transient decreased reversibly over several beats. 2. The calcium entry into the cell was measured from the integral of the inward calcium current and the efflux from the Na(+)-Ca2+ exchange current on repolarization. During the negative staircase the calcium current was approximately constant whilst the Na(+)-Ca2+ exchange current decayed in parallel with the systolic calcium transient. A net loss of calcium from the cell can be calculated from the extra Na(+)-Ca2+ exchange current following the initial pulses. 3. The application of caffeine produces a transient increase of both [Ca2+]i and an inward Na(+)-Ca2+ exchange current. The integral of this current can be used to estimate the caffeine-releasable calcium content of the sarcoplasmic reticulum (SR), which decreases following stimulation with short compared to long pulses. This difference in SR calcium content is quantitatively similar to that estimated from the sarcolemmal currents. 4. At a given membrane potential, the relationship between [Ca2+]i and current during the caffeine exposure can be used to estimate the Na(+)-Ca2+ exchange flux from the measured [Ca2+]i and thence the Na(+)-Ca2+ exchange flux during depolarization. 5. For a long depolarizing pulse the extrusion of calcium from the cell on Na(+)-Ca2+ exchange is comparable to the entry on the calcium current. In contrast, for short pulses the extrusion of calcium on the Na(+)-Ca2+ exchange immediately after the pulse is greater than the entry during the pulse on the calcium current. 6. These results show that rest potentiation can be correlated with changes in the amount of calcium stored in the SR and this, in turn, can be accounted for by sarcolemmal fluxes.

Comparison of the effects of caffeine and other methylxanthines on [Ca2+]i in rat ventricular myocytes.

1. The effects of caffeine and other methylxanthines were investigated on intracellular calcium concentration ([Ca2+]i) and contraction in rat isolated ventricular myocytes. The use of the fluorescent indicator, Indo-1, allowed simultaneous measurement of [Ca2+]i and the intracellular concentration of the methylxanthines. 2. Rapid application of caffeine (10 mM) produced a transient rise of [Ca2+]i which decayed to resting levels. This was accompanied by a transient contraction which decayed to a level above baseline. The addition of theophylline also produced a transient increase of [Ca2+]i. However, following the initial transient, contraction decayed before redeveloping to a maintained level. 3. Direct measurements showed that [caffeine]i rose more quickly than did [theophylline]i. The slower rise of [theophylline]i was associated with a delay in the increase of [Ca2+]i. At lower concentrations of the methylxanthines, theophylline was less effective than caffeine at initiating Ca release. The rate of entry of theobromine was similar to that of theophylline. 4. Isocaffeine did not produce a rise of [Ca2+]i. The rate of rise of [isocaffeine]i was much slower than that of either caffeine or theophylline. 5. Measurements of the oil:water partition coefficient showed that the order of relative partitioning into oil was: caffeine > theophylline > theobromine > isocaffeine. This is similar to the order of rate of entry into the cell. 6. We conclude that many of the differences in the effects of these methylxanthines can be attributed to differences in membrane permeability due to differences in oil:water partition.

The relative contributions of different intracellular and sarcolemmal systems to relaxation in rat ventricular myocytes.

OBJECTIVE: The aim was to estimate the relative contributions of the various intracellular and sarcolemmal systems to the relaxation of the systolic calcium transient. METHODS: The experiments were performed on isolated rat ventricular myocytes. The cells were loaded with the fluorescent indicator indo-1 in order to measure [Ca2+]i. RESULTS: The application of caffeine to release calcium from the sarcoplasmic reticulum produced a rise of [Ca2+]i which decayed about 7-8 times more slowly than the electrically stimulated calcium transient. This suggests that the sarcoplasmic reticulum accounts for about 87% of the calcium removal. The rate of decay of the caffeine response was decreased to about 33% of the control by inhibiting the Na-Ca exchange with Ni2+. In the presence of Ni2+ the rate could be inhibited further by inhibiting either the sarcolemmal Ca-ATPase (by increasing extracellular calcium concentration, [Ca2+]o) or the mitochondria (with FCCP and oligomycin). The relative contributions of the various processes were estimated to be: sarcoplasmic reticulum 87%, mitochondria 1.7%, Na-Ca 8.7%, sarcolemmal Ca-ATPase 2.6%. CONCLUSIONS: These experiments show that the Na-Ca exchange accounts for 67% of the calcium removal not mediated by the sarcoplasmic reticulum. This is a smaller fraction than in rabbit cardiac cells and highlights the importance of the Ca-ATPase in the rat heart.

The effects of inhibitors of sarcoplasmic reticulum function on the systolic Ca2+ transient in rat ventricular myocytes.

1. The effects of thapsigargin, ryanodine and caffeine were examined on systolic Ca2+ transients in indo-1-loaded rat ventricular myocytes. 2. Thapsigargin (1-10 microM) decreased the magnitude of the Ca2+ transient. This was accompanied by a decrease of the rate constant of decay of the transient. 3. Ryanodine (1-10 microM) decreased the magnitude of the Ca2+ transient. Initially there was no change in the rate of decay but further reduction of the magnitude was accompanied by a slowing. 4. Caffeine (0.5-10 mM) decreased the magnitude of the Ca2+ transient and its rate of decay. These effects were graded with caffeine concentration. 5. For a given submaximal reduction of the magnitude of the Ca2+ transient, the effect on the rate of decay was greatest for thapsigargin, least for ryanodine and intermediate for caffeine. 6. The above data are reproduced by a model in which all three agents decrease the magnitude of the Ca2+ transient by decreasing the calcium content of the sarcoplasmic reticulum (SR) (thapsigargin by inhibiting the Ca2+ pump and ryanodine and caffeine by increasing the leak of Ca2+ from the SR). The decreased contribution of the SR will thereby slow relaxation. The fact that thapsigargin inhibits the SR Ca2+ pump accounts for the observation that, for a given decrease of amplitude, it has more effect than the other agents on the rate of decay. The difference between caffeine and ryanodine is suggested to arise because caffeine potentiates Ca2+ release from the SR and thereby attenuates the effect of the decreased SR calcium content on the magnitude of the Ca2+ transient.

An estimate of the calcium content of the sarcoplasmic reticulum in rat ventricular myocytes.

The aim of this paper was to estimate the Ca content of the sarcoplasmic reticulum (s.r.) and to compare this with the amount of Ca which enters the cell via the calcium current in systole. The s.r. Ca content was measured electrophysiologically in voltage-clamped rat ventricular myocytes. Rapid application of caffeine produced a transient increase of [Ca2+]i which was accompanied by a transient inward Na-Ca exchange current. The integral of this current gives a measure of the Ca2+ pumped out of the cell by Na-Ca exchange. Ni2+ (5 mM) inhibited the current and decreased the rate of fall of [Ca2+]i to 32% of the control suggesting that Na-Ca exchange is responsible for 68% of Ca removal from the cytoplasm following the addition of caffeine. Correcting for the Na-Ca independent Ca removal suggests that the s.r. Ca content is equivalent to about 120 mumol per litre cell. Furthermore we estimate that, during systole, Ca entry into the cell via the sarcolemmal calcium current is equal to about 6% of the Ca content of the s.r.