Calcium signaling in endothelial and vascular smooth muscle cells: sex differences and the influence of estrogens and androgens.

Calcium signaling in vascular endothelial cells (ECs) and smooth muscle cells (VSMCs) is essential for the regulation of vascular tone. However, the changes to intracellular Ca2+ concentrations are often influenced by sex differences. Furthermore, a large body of evidence shows that sex hormone imbalance leads to dysregulation of Ca2+ signaling and this is a key factor in the pathogenesis of cardiovascular diseases. In this review, the effects of estrogens and androgens on vascular calcium-handling proteins are discussed, with emphasis on the associated genomic or nongenomic molecular mechanisms. The experimental models from which data were collected were also considered. The review highlights 1) in female ECs, transient receptor potential vanilloid 4 (TRPV4) and mitochondrial Ca2+ uniporter (MCU) enhance Ca2+-dependent nitric oxide (NO) generation. In males, only transient receptor potential canonical 3 (TRPC3) plays a fundamental role in this effect. 2) Female VSMCs have lower cytosolic Ca2+ levels than males due to differences in the activity and expression of stromal interaction molecule 1 (STIM1), calcium release-activated calcium modulator 1 (Orai1), calcium voltage-gated channel subunit-α1C (CaV1.2), Na+-K+-2Cl- symporter (NKCC1), and the Na+/K+-ATPase. 3) When compared with androgens, the influence of estrogens on Ca2+ homeostasis, vascular tone, and incidence of vascular disease is better documented. 4) Many studies use supraphysiological concentrations of sex hormones, which may limit the physiological relevance of outcomes. 5) Sex-dependent differences in Ca2+ signaling mean both sexes ought to be included in experimental design.

A hydroalcoholic extract of Senecio nutans SCh. Bip (Asteraceae); its effects on cardiac function and chemical characterization.

ETHNOPHARMACOLOGY RELEVANCE: The plant Senecio nutans SCh. Bip. is used by Andean communities to treat altitude sickness. Recent evidence suggests it may produce vasodilation and negative cardiac inotropy, though the cellular mechanisms have not been elucidated. PURPOSE: To determinate the mechanisms action of S. nutans on cardiovascular function in normotensive animals. METHODS: The effect of the extract on rat blood pressure was measured with a transducer in the carotid artery and intraventricular pressure by a Langendorff system. The effects on sheep ventricular intracellular calcium handling and contractility were evaluated using photometry. Ultra-high-performance liquid-chromatography with diode array detection coupled with heated electrospray-ionization quadrupole-orbitrap mass spectrometric detection (UHPLC-DAD-ESI-Q-OT-MSn) was used for extract chemical characterization. RESULTS: In normotensive rats, S. nutans (10 mg/kg) reduced mean arterial pressure (MAP) by 40% (p < 0.05), causing a dose-dependent coronary artery dilation and decreased left ventricular pressure. In isolated cells, S. nutans extract (1 µg/ml) rapidly reduced the [Ca2+]i transient amplitude and sarcomere shorting by 40 and 49% (p < 0.001), respectively. The amplitude of the caffeine evoked [Ca2+]i transient was reduced by 24% (p < 0.001), indicating reduced sarcoplasmic reticulum (SR) Ca2+ content. Sodium-calcium exchanger (NCX) activity increased by 17% (p < 0.05), while sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) activity was decreased by 21% (p < 0.05). LC-MS results showed the presence of vitamin C, malic acid, and several antioxidant phenolic acids reported for the first time. Dihydroeuparin and 4-hydroxy-3-(3-methylbut-2-enyl) acetophenone were abundant in the extract. CONCLUSION: In normotensive animals, S. nutans partially reduces MAP by decreasing heart rate and cardiac contractility. This negative inotropy is accounted for by decreased SERCA activity and increased NCX activity which reduces SR Ca2+ content. These results highlight the plant's potential as a source of novel cardio-active phytopharmaceuticals or nutraceuticals.

Systolic [Ca2+ ]i regulates diastolic levels in rat ventricular myocytes.

KEY POINTS: For the heart to function as a pump, intracellular calcium concentration ([Ca2+ ]i ) must increase during systole to activate contraction and then fall, during diastole, to allow the myofilaments to relax and the heart to refill with blood. The present study investigates the control of diastolic [Ca2+ ]i in rat ventricular myocytes. We show that diastolic [Ca2+ ]i is increased by manoeuvres that decrease sarcoplasmic reticulum function. This is accompanied by a decrease of systolic [Ca2+ ]i such that the time-averaged [Ca2+ ]i remains constant. We report that diastolic [Ca2+ ]i is controlled by the balance between Ca2+ entry and Ca2+ efflux during systole. The results of the present study identify a novel mechanism by which changes of the amplitude of the systolic Ca transient control diastolic [Ca2+ ]i . ABSTRACT: The intracellular Ca concentration ([Ca2+ ]i ) must be sufficently low in diastole so that the ventricle is relaxed and can refill with blood. Interference with this will impair relaxation. The factors responsible for regulation of diastolic [Ca2+ ]i , in particular the relative roles of the sarcoplasmic reticulum (SR) and surface membrane, are unclear. We investigated the effects on diastolic [Ca2+ ]i that result from the changes of Ca cycling known to occur in heart failure. Experiments were performed using Fluo-3 in voltage clamped rat ventricular myocytes. Increasing stimulation frequency increased diastolic [Ca2+ ]i . This increase of [Ca2+ ]i was larger when SR function was impaired either by making the ryanodine receptor leaky (with caffeine or ryanodine) or by decreasing sarco/endoplasmic reticulum Ca-ATPase activity with thapsigargin. The increase of diastolic [Ca2+ ]i produced by interfering with the SR was accompanied by a decrease of the amplitude of the systolic Ca transient, such that there was no change of time-averaged [Ca2+ ]i . Time-averaged [Ca2+ ]i was increased by ß-adrenergic stimulation with isoprenaline and increased in a saturating manner with increased stimulation frequency; average [Ca2+ ]i was a linear function of Ca entry per unit time. Diastolic and time-averaged [Ca2+ ]i were decreased by decreasing the L-type Ca current (with 50 µm cadmium chloride). We conclude that diastolic [Ca2+ ]i is controlled by the balance between Ca entry and efflux during systole. Furthermore, manoeuvres that decrease the amplitude of the Ca transient (without decreasing Ca influx) will therefore increase diastolic [Ca2+ ]i . This identifies a novel mechanism by which changes of the amplitude of the systolic Ca transient control diastolic [Ca2+ ]i .

Disulfide-activated protein kinase G Iα regulates cardiac diastolic relaxation and fine-tunes the Frank-Starling response.

The Frank-Starling mechanism allows the amount of blood entering the heart from the veins to be precisely matched with the amount pumped out to the arterial circulation. As the heart fills with blood during diastole, the myocardium is stretched and oxidants are produced. Here we show that protein kinase G Iα (PKGIα) is oxidant-activated during stretch and this form of the kinase selectively phosphorylates cardiac phospholamban Ser16-a site important for diastolic relaxation. We find that hearts of Cys42Ser PKGIα knock-in (KI) mice, which are resistant to PKGIα oxidation, have diastolic dysfunction and a diminished ability to couple ventricular filling with cardiac output on a beat-to-beat basis. Intracellular calcium dynamics of ventricular myocytes isolated from KI hearts are altered in a manner consistent with impaired relaxation and contractile function. We conclude that oxidation of PKGIα during myocardial stretch is crucial for diastolic relaxation and fine-tunes the Frank-Starling response.

Biphasic decay of the Ca transient results from increased sarcoplasmic reticulum Ca leak.

KEY POINTS: Ca leak from the sarcoplasmic reticulum through the ryanodine receptor (RyR) reduces the amplitude of the Ca transient and slows its rate of decay. In the presence of ß-adrenergic stimulation, RyR-mediated Ca leak produces a biphasic decay of the Ca transient with a fast early phase and a slow late phase. Two forms of Ca leak have been studied, Ca-sensitising (induced by caffeine) and non-sensitising (induced by ryanodine) and both induce biphasic decay of the Ca transient. Only Ca-sensitising leak can be reversed by traditional RyR inhibitors such as tetracaine. Ca leak can also induce Ca waves. At low levels of leak, waves occur. As leak is increased, first biphasic decay and then slowed monophasic decay is seen. The level of leak has major effects on the shape of the Ca transient. In heart failure, a reduction in Ca transient amplitude and contractile dysfunction can by caused by Ca leak through the sarcoplasmic reticulum (SR) Ca channel (ryanodine receptor, RyR) and/or decreased activity of the SR Ca ATPase (SERCA). We have characterised the effects of two forms of Ca leak (Ca-sensitising and non-sensitising) on calcium cycling and compared with those of SERCA inhibition. We measured [Ca(2+)]i with fluo-3 in voltage-clamped rat ventricular myocytes. Increasing SR leak with either caffeine (to sensitise the RyR to Ca activation) or ryanodine (non-sensitising) had similar effects to SERCA inhibition: decreased systolic [Ca(2+)]i , increased diastolic [Ca(2+)]i and slowed decay. However, in the presence of isoproterenol, leak produced a biphasic decay of the Ca transient in the majority of cells while SERCA inhibition produced monophasic decay. Tetracaine reversed the effects of caffeine but not of ryanodine. When caffeine (1 mmol l(-1)) was added to a cell which displayed Ca waves, the wave frequency initially increased before waves disappeared and biphasic decay developed. Eventually (at higher caffeine concentrations), the biphasic decay was replaced by slow decay. We conclude that, in the presence of adrenergic stimulation, Ca leak can produce biphasic decay; the slow phase results from the leak opposing Ca uptake by SERCA. The degree of leak determines whether decay of Ca waves, biphasic or monophasic, occurs.

Perturbed atrial calcium handling in an ovine model of heart failure: potential roles for reductions in the L-type calcium current.

Heart failure (HF) is commonly associated with reduced cardiac output and an increased risk of atrial arrhythmias particularly during ß-adrenergic stimulation. The aim of the present study was to determine how HF alters systolic Ca(2+) and the response to ß-adrenergic (ß-AR) stimulation in atrial myocytes. HF was induced in sheep by ventricular tachypacing and changes in intracellular Ca(2+) concentration studied in single left atrial myocytes under voltage and current clamp conditions. The following were all reduced in HF atrial myocytes; Ca(2+) transient amplitude (by 46% in current clamped and 28% in voltage clamped cells), SR dependent rate of Ca(2+) removal (kSR, by 32%), L-type Ca(2+) current density (by 36%) and action potential duration (APD90 by 22%). However, in HF SR Ca(2+) content was increased (by 19%) when measured under voltage-clamp stimulation. Inhibiting the L-type Ca(2+) current (ICa-L) in control cells reproduced both the decrease in Ca(2+) transient amplitude and increase of SR Ca(2+) content observed in voltage-clamped HF cells. During ß-AR stimulation Ca(2+) transient amplitude was the same in control and HF cells. However, ICa-L remained less in HF than control cells whilst SR Ca(2+) content was highest in HF cells during ß-AR stimulation. The decrease in ICa-L that occurs in HF atrial myocytes appears to underpin the decreased Ca(2+) transient amplitude and increased SR Ca(2+) content observed in voltage-clamped cells.

Direct measurements of SR free Ca reveal the mechanism underlying the transient effects of RyR potentiation under physiological conditions.

AIMS: Most of the calcium that activates contraction is released from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR). It is controversial whether activators of the RyR produce a maintained increase in the amplitude of the systolic Ca transient. We therefore aimed to examine the effects of activation of the RyR in large animals under conditions designed to be as physiological as possible while simultaneously measuring SR and cytoplasmic Ca. METHODS AND RESULTS: Experiments were performed on ventricular myocytes from canine and ovine hearts. Cytoplasmic Ca was measured with fluo-3 and SR Ca with mag-fura-2. Application of caffeine resulted in a brief increase in the amplitude of the systolic Ca transient accompanied by an increase of action potential duration. These effects disappeared with a rate constant of ∼3 s(-1). Similar effects were seen in cells taken from sheep in which heart failure had been induced by rapid pacing. The decrease of Ca transient amplitude was accompanied by a decrease of SR Ca content. During this phase, the maximum (end-diastolic) SR Ca content fell while the minimum systolic increased. CONCLUSIONS: This study shows that, under conditions designed to be as physiological as possible, potentiation of RyR opening has no maintained effect on the systolic Ca transient. This result makes it unlikely that potentiation of the RyR has a maintained role in positive inotropy.

Ca analysis: an Excel based program for the analysis of intracellular calcium transients including multiple, simultaneous regression analysis.

Here I present an Excel based program for the analysis of intracellular Ca transients recorded using fluorescent indicators. The program can perform all the necessary steps which convert recorded raw voltage changes into meaningful physiological information. The program performs two fundamental processes. (1) It can prepare the raw signal by several methods. (2) It can then be used to analyze the prepared data to provide information such as absolute intracellular Ca levels. Also, the rates of change of Ca can be measured using multiple, simultaneous regression analysis. I demonstrate that this program performs equally well as commercially available software, but has numerous advantages, namely creating a simplified, self-contained analysis workflow.

The effects of tumor necrosis factor-alpha on systolic and diastolic function in rat ventricular myocytes.

The proinflammatory cytokine tumor necrosis factor-alpha (TNF-α) is associated with myocardial dysfunction observed in sepsis and septic shock. There are two fundamental components to this dysfunction. (1) systolic dysfunction; and (2) diastolic dysfunction. The aim of these experiments was to determine if any aspect of whole-heart dysfunction could be explained by alterations to global intracellular calcium ([Ca(2+)]i), contractility, and [Ca(2+)]i handling, by TNF-α, at the level of the individual rat myocyte. We took an integrative approach to simultaneously measure [Ca(2+)]i, contractility and sarcolemmal Ca fluxes using the Ca indicator fluo-3, video edge detection, and the perforated patch technique, respectively. All experiments were performed at 37°C. The effects of 50 ng/mL TNF-α were immediate and sustained. The amplitude of systolic [Ca(2+)]i was reduced by 31% and systolic shortening by 19%. Diastolic [Ca(2+)]i, myocyte length and relaxation rate were not affected, nor were the activity of the [Ca(2+)]i removal mechanisms. The reduction in systolic [Ca(2+)]i was associated with a 14% reduction in sarcoplasmic reticulum (SR) content and a 11% decrease in peak L-type Ca current (IC a-L). Ca influx was decreased by 7% associated with a more rapid IC a-L inactivation. These data show that at the level of the myocyte, TNF-α reduces SR Ca which underlies a reduction in systolic [Ca(2+)]i and thence shortening. Although these findings correlate well with aspects of systolic myocardial dysfunction seen in sepsis, in this model, acutely, TNF-α does not appear to provide a cellular mechanism for sepsis-related diastolic myocardial dysfunction.

Diastolic spontaneous calcium release from the sarcoplasmic reticulum increases beat-to-beat variability of repolarization in canine ventricular myocytes after ß-adrenergic stimulation.

RATIONALE: Spontaneous Ca(2+) release (SCR) from the sarcoplasmic reticulum can cause delayed afterdepolarizations and triggered activity, contributing to arrhythmogenesis during ß-adrenergic stimulation. Excessive beat-to-beat variability of repolarization duration (BVR) is a proarrhythmic marker. Previous research has shown that BVR is increased during intense ß-adrenergic stimulation, leading to SCR. OBJECTIVE: We aimed to determine ionic mechanisms controlling BVR under these conditions. METHODS AND RESULTS: Membrane potentials and cell shortening or Ca(2+) transients were recorded from isolated canine left ventricular myocytes in the presence of isoproterenol. Action-potential (AP) durations after delayed afterdepolarizations were significantly prolonged. Addition of slowly activating delayed rectifier K(+) current (I(Ks)) blockade led to further AP prolongation after SCR, and this strongly correlated with exaggerated BVR. Suppressing SCR via inhibition of ryanodine receptors, Ca(2+)/calmodulin-dependent protein kinase II inhibition, or by using Mg(2+) or flecainide eliminated delayed afterdepolarizations and decreased BVR independent of effects on AP duration. Computational analyses and voltage-clamp experiments measuring L-type Ca(2+) current (I(CaL)) with and without previous SCR indicated that I(CaL) was increased during Ca(2+)-induced Ca(2+) release after SCR, and this contributes to AP prolongation. Prolongation of QT, T(peak)-T(end) intervals, and left ventricular monophasic AP duration of beats after aftercontractions occurred before torsades de pointes in an in vivo dog model of drug-induced long-QT1 syndrome. CONCLUSIONS: SCR contributes to increased BVR by interspersed prolongation of AP duration, which is exacerbated during I(Ks) blockade. Attenuation of Ca(2+)-induced Ca(2+) release by SCR underlies AP prolongation via increased I(CaL.) These data provide novel insights into arrhythmogenic mechanisms during ß-adrenergic stimulation besides triggered activity and illustrate the importance of I(Ks) function in preventing excessive BVR.

Age-related divergent remodeling of the cardiac extracellular matrix in heart failure: collagen accumulation in the young and loss in the aged.

The incidence of heart failure (HF) increases with age. This study sought to determine whether aging exacerbates structural and functional remodeling of the myocardium in HF. HF was induced in young (~18 months) and aged sheep (>8 years) by right ventricular tachypacing. In non-paced animals, aging was associated with increased left ventricular (LV) end diastolic internal dimensions (EDID, P<0.001), reduced fractional shortening (P<0.01) and an increase in myocardial collagen content (P<0.01). HF increased EDID and reduced fractional shortening in both young and aged animals, although these changes were more pronounced in the aged (P<0.05). Age-associated differences in cardiac extracellular matrix (ECM) remodeling occurred in HF with collagen accumulation in young HF (P<0.001) and depletion in aged HF (P<0.05). MMP-2 activity increased in the aged control and young HF groups (P<0.05). Reduced tissue inhibitor of metalloproteinase (TIMP) expression (TIMPs 3 and 4, P<0.05) was present only in the aged HF group. Secreted protein acidic and rich in cysteine (SPARC) was increased in aged hearts compared to young controls (P<0.05) while serum procollagen type I C-pro peptide (PICP) was increased in both young failing (P<0.05) and aged failing (P<0.01) animals. In conclusion, collagen content of the cardiac ECM changes in both aging and HF although; whether collagen accumulation or depletion occurs depends on age. Changes in TIMP expression in aged failing hearts alongside augmented collagen synthesis in HF provide a potential mechanism for the age-dependent ECM remodeling. Aging should therefore be considered an important factor when elucidating cardiac disease mechanisms.

Impaired ß-adrenergic responsiveness accentuates dysfunctional excitation-contraction coupling in an ovine model of tachypacing-induced heart failure.

Reduced inotropic responsiveness is characteristic of heart failure (HF). This study determined the cellular Ca2+ homeostatic and molecular mechanisms causing the blunted ß-adrenergic (ß-AR) response in HF.We induced HF by tachypacing in sheep; intracellular Ca2+ concentration was measured in voltage-clamped ventricular myocytes. In HF, Ca2+ transient amplitude and peak L-type Ca2+ current (ICa-L) were reduced (to 70 ± 11% and 50 ± 3.7% of control, respectively, P <0.05) whereas sarcoplasmic reticulum (SR) Ca2+ content was unchanged. ß-AR stimulation with isoprenaline (ISO) increased Ca2+ transient amplitude, ICa-L and SRCa2+ content in both cell types; however, the response of HF cells was markedly diminished (P <0.05).Western blotting revealed an increase in protein phosphatase levels (PP1, 158 ± 17% and PP2A, 188 ± 34% of control, P <0.05) and reduced phosphorylation of phospholamban in HF (Ser16, 30 ± 10% and Thr17, 41 ± 15% of control, P <0.05). The ß-AR receptor kinase GRK-2 was also increased in HF (173 ± 38% of control, P <0.05). In HF, activation of adenylyl cyclase with forskolin rescued the Ca2+ transient, SR Ca2+ content and SR Ca2+ uptake rate to the same levels as control cells in ISO. In conclusion, the reduced responsiveness of the myocardium to ß-AR agonists in HF probably arises as a consequence of impaired phosphorylation of key intracellular proteins responsible for regulating the SR Ca2+ content and therefore failure of the systolic Ca2+ transient to increase appropriately during ß-AR stimulation.

The effects of hydrogen peroxide on intracellular calcium handling and contractility in the rat ventricular myocyte.

Elevations in reactive oxygen species are implicated in many disease states and cause systolic and diastolic myocardial dysfunction. To understand the underlying cellular dysfunction, we characterised the effects of H2O2 on [Ca(2+)](i) handling and contractility in the rat ventricular myocyte. This was achieved using patch clamping, [Ca(2+)](i) measurement using Fluo-3, video edge detection and confocal microscopy. All experiments were performed at 37°C. 200 µM H2O2 resulted in a 44% decrease in the [Ca(2+)](i) transient amplitude, a 30% increase in diastolic [Ca(2+)](i) and an 18% decrease in the rate of systolic Ca(2+) removal. This was associated with a 61% reduction in systolic shortening, a contracture of 3 µm and a 42% increase in relaxation time respectively. The decrease in the [Ca(2+)](i) transient amplitude could be explained by a 27% decrease in SR Ca(2+) content. This, in turn results from a 22% decrease of SERCA activity. The decreased SR Ca(2+) content also provides a mechanism for a reduction in [Ca(2+)](i) spark frequency with no evidence for a Ca(2+) independent modification of ryanodine receptor open probability. We conclude that decreased SERCA activity is the major factor responsible for the changes of the systolic [Ca(2+)](i) transient.