Dynamin-2 mutations linked to neonatal-onset centronuclear myopathy impair exocytosis and endocytosis in adrenal chromaffin cells.

Dynamins are large GTPases whose primary function is not only to catalyze membrane scission during endocytosis but also to modulate other cellular processes, such as actin polymerization and vesicle trafficking. Recently, we reported that centronuclear myopathy associated dynamin-2 mutations, p.A618T, and p.S619L, impair Ca2+-induced exocytosis of the glucose transporter GLUT4 containing vesicles in immortalized human myoblasts. As exocytosis and endocytosis occur within rapid timescales, here we applied high-temporal resolution techniques, such as patch-clamp capacitance measurements and carbon-fiber amperometry to assess the effects of these mutations on these two cellular processes, using bovine chromaffin cells as a study model. We found that the expression of any of these dynamin-2 mutants inhibits a dynamin and F-actin-dependent form of fast endocytosis triggered by single action potential stimulus, as well as inhibits a slow compensatory endocytosis induced by 500 ms square depolarization. Both dynamin-2 mutants further reduced the exocytosis induced by 500 ms depolarizations, and the frequency of release events and the recruitment of neuropeptide Y (NPY)-labeled vesicles to the cell cortex after stimulation of nicotinic acetylcholine receptors with 1,1-dimethyl-4-phenyl piperazine iodide (DMPP). They also provoked a significant decrease in the Ca2+-induced formation of new actin filaments in permeabilized chromaffin cells. In summary, our results indicate that the centronuclear myopathy (CNM)-linked p.A618T and p.S619L mutations in dynamin-2 affect exocytosis and endocytosis, being the disruption of F-actin dynamics a possible explanation for these results. These impaired cellular processes might underlie the pathogenic mechanisms associated with these mutations.

Autocrine activation of P2X7 receptors mediates catecholamine secretion in chromaffin cells.

BACKGROUND AND PURPOSE: ATP is highly accumulated in secretory vesicles and secreted upon exocytosis from neurons and endocrine cells. In adrenal chromaffin granules, intraluminal ATP reaches concentrations over 100 mM. However, how these large amounts of ATP contribute to exocytosis has not been investigated. EXPERIMENTAL APPROACH: Exocytotic events in bovine and mouse adrenal chromaffin cells were measured with single cell amperometry. Cytosolic Ca2+ measurements were carried out in Fluo-4 loaded cells. Submembrane Ca2+ was examined in PC12 cells transfected with a membrane-tethered Ca2+ indicator Lck-GCaMP3. ATP release was measured using the luciferin/luciferase assay. Knockdown of P2X7 receptors was induced with short interfering RNA (siRNA). Direct Ca2+ influx through this receptor was measured using a P2X7 receptor-GCamp6 construct. KEY RESULTS: ATP induced exocytosis in chromaffin cells, whereas the ectonucleotidase apyrase reduced the release events induced by the nicotinic agonist dimethylphenylpiperazinium (DMPP), high KCl, or ionomycin. The purinergic agonist BzATP also promoted a secretory response that was dependent on extracellular Ca2+. A740003, a P2X7 receptor antagonist, abolished secretory responses of these secretagogues. Exocytosis was also diminished in chromaffin cells when P2X7 receptors were silenced using siRNAs and in cells of P2X7 receptor knockout mice. In PC12 cells, DMPP induced ATP release, triggering Ca2+ influx through P2X7 receptors. Furthermore, BzATP, DMPP, and KCl allowed the formation of submembrane Ca2+ microdomains inhibited by A740003. CONCLUSION AND IMPLICATIONS: Autocrine activation of P2X7 receptors constitutes a crucial feedback system that amplifies the secretion of catecholamines in chromaffin cells by favouring submembrane Ca2+ microdomains.

Membrane retrieval after Immediately Releasable Pool (IRP) exocytosis is produced by dynamin-dependent and dynamin-independent/protein kinase C-dependent mechanisms.

The importance of the immediately releasable pool (IRP) of vesicles was proposed to reside in the maintenance of chromaffin cell secretion during the firing of action potentials at basal physiological frequencies. To accomplish this duty, IRP should be replenished as a function of time. We have previously reported that an action potential-like stimulus (APls) triggers the release of ~50% IRP, followed by a fast dynamin-dependent endocytosis and an associated rapid replenishment process. In this work, we investigated the endocytosis and IRP replenishment produced after the exocytosis of variable IRP fractions in mice primary chromaffin cell cultures. Exocytosis and endocytosis were estimated by membrane capacitance measurements obtained in patch-clamped cells. In addition to the dynamin-dependent fast endocytosis activated after the application of APls or 5 ms squared depolarizations, we found that depolarizations lasting 25-50 ms, which release >80% of IRP, are related with a fast dynamin-independent, Ca2+ - and protein kinase C (PKC)-dependent endocytosis (time constant <1 s). PKC inhibitors, such as staurosporine, bisindolylmaleimide XI, PKC 19-31 peptide, and prolonged treatments with high concentrations of phorbol esters, reduced and decelerated this endocytosis. Additionally, we found that the inhibition of PKC also abolished a slow component of replenishment (time constant ~8 s) observed after total IRP exocytosis. Therefore, our results suggest that PKC contributes to the coordination of membrane retrieval and vesicle replenishment mechanisms that occur after the complete exocytosis of IRP.

Gain-of-Function Dynamin-2 Mutations Linked to Centronuclear Myopathy Impair Ca2+-Induced Exocytosis in Human Myoblasts.

Gain-of-function mutations of dynamin-2, a mechano-GTPase that remodels membrane and actin filaments, cause centronuclear myopathy (CNM), a congenital disease that mainly affects skeletal muscle tissue. Among these mutations, the variants p.A618T and p.S619L lead to a gain of function and cause a severe neonatal phenotype. By using total internal reflection fluorescence microscopy (TIRFM) in immortalized human myoblasts expressing the pH-sensitive fluorescent protein (pHluorin) fused to the insulin-responsive aminopeptidase IRAP as a reporter of the GLUT4 vesicle trafficking, we measured single pHluorin signals to investigate how p.A618T and p.S619L mutations influence exocytosis. We show here that both dynamin-2 mutations significantly reduced the number and durations of pHluorin signals induced by 10 µM ionomycin, indicating that in addition to impairing exocytosis, they also affect the fusion pore dynamics. These mutations also disrupt the formation of actin filaments, a process that reportedly favors exocytosis. This altered exocytosis might importantly disturb the plasmalemma expression of functional proteins such as the glucose transporter GLUT4 in skeletal muscle cells, impacting the physiology of the skeletal muscle tissue and contributing to the CNM disease.

Rapid vesicle replenishment after the immediately releasable pool exocytosis is tightly linked to fast endocytosis, and depends on basal calcium and cortical actin in chromaffin cells.

The maintenance of the secretory response requires a continuous replenishment of releasable vesicles. It was proposed that the immediately releasable pool (IRP) is important in chromaffin cell secretion during action potentials applied at basal physiological frequencies, because of the proximity of IRP vesicles to voltage-dependent Ca2+ channels. However, previous reports showed that IRP replenishment after depletion is too slow to manage such a situation. In this work, we used patch-clamp measurements of membrane capacitance, confocal imaging of F-actin distribution, and cytosolic Ca2+ measurements with Fura-2 to re-analyze this problem in primary cultures of mouse chromaffin cells. We provide evidence that IRP replenishment has one slow (time constant between 5 and 10 s) and one rapid component (time constant between 0.5 and 1.5 s) linked to a dynamin-dependent fast endocytosis. Both, the fast endocytosis and the rapid replenishment component were eliminated when 500 nM Ca2+ was added to the internal solution during patch-clamp experiments, but they became dominant and accelerated when the cytosolic Ca2+ buffer capacity was increased. In addition, both rapid replenishment and fast endocytosis were retarded when cortical F-actin cytoskeleton was disrupted with cytochalasin D. Finally, in permeabilized chromaffin cells stained with rhodamine-phalloidin, the cortical F-actin density was reduced when the Ca2+ concentration was increased in a range of 10-1000 nM. We conclude that low cytosolic Ca2+ concentrations, which favor cortical F-actin stabilization, allow the activation of a fast endocytosis mechanism linked to a rapid replenishment component of IRP.

Ca2+ -independent and voltage-dependent exocytosis in mouse chromaffin cells.

AIM: It is widely accepted that the exocytosis of synaptic and secretory vesicles is triggered by Ca2+ entry through voltage-dependent Ca2+ channels. However, there is evidence of an alternative mode of exocytosis induced by membrane depolarization but lacking Ca2+ current and intracellular Ca2+ increase. In this work we investigated if such a mechanism contributes to secretory vesicle exocytosis in mouse chromaffin cells. METHODS: Exocytosis was evaluated by patch-clamp membrane capacitance measurements, carbon fibre amperometry and TIRF. Cytosolic Ca2+ was estimated using epifluorescence microscopy and fluo-8 (salt form). RESULTS: Cells stimulated by brief depolatizations in absence of extracellular Ca+2 show moderate but consistent exocytosis, even in presence of high cytosolic BAPTA concentration and pharmacological inhibition of Ca+2 release from intracellular stores. This exocytosis is tightly dependent on membrane potential, is inhibited by neurotoxin Bont-B (cleaves the v-SNARE synaptobrevin), is very fast (saturates with time constant <10 ms), it is followed by a fast endocytosis sensitive to the application of an anti-dynamin monoclonal antibody, and recovers after depletion in <5 s. Finally, this exocytosis was inhibited by: (i) ω-agatoxin IVA (blocks P/Q-type Ca2+ channel gating), (ii) in cells from knock-out P/Q-type Ca2+ channel mice, and (iii) transfection of free synprint peptide (interferes in P/Q channel-exocytic proteins association). CONCLUSION: We demonstrated that Ca2+ -independent and voltage-dependent exocytosis is present in chromaffin cells. This process is tightly coupled to membrane depolarization, and is able to support secretion during action potentials at low basal rates. P/Q-type Ca2+ channels can operate as voltage sensors of this process.

How does the stimulus define exocytosis in adrenal chromaffin cells?

The extent and type of hormones and active peptides secreted by the chromaffin cells of the adrenal medulla have to be adjusted to physiological requirements. The chromaffin cell secretory activity is controlled by the splanchnic nerve firing frequency, which goes from approximately 0.5 Hz in basal conditions to more than 15 Hz in stress. Thus, these neuroendocrine cells maintain a tonic release of catecholamines under resting conditions, massively discharge intravesicular transmitters in response to stress, or adequately respond to moderate stimuli. In order to adjust the secretory response to the stimulus, the adrenal chromaffin cells have an appropriate organization of Ca2+ channels, secretory granules pools, and sets of proteins dedicated to selectively control different steps of the secretion process, such as the traffic, docking, priming and fusion of the chromaffin granules. Among the molecules implicated in such events are the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, Ca2+ sensors like Munc13 and synaptotagmin-1, chaperon proteins such as Munc18, and the actomyosin complex. In the present review, we discuss how these different actors contribute to the extent and maintenance of the stimulus-dependent exocytosis in the adrenal chromaffin cells.

Single particle tracking of internalized metallic nanoparticles reveals heterogeneous directed motion after clathrin dependent endocytosis in mouse chromaffin cells.

Most accepted single particle tracking methods are able to obtain high-resolution trajectories for relatively short periods of time. In this work we apply a straightforward combination of single-particle tracking microscopy and metallic nanoparticles internalization on mouse chromaffin cells to unveil the intracellular trafficking mechanism of metallic-nanoparticle-loaded vesicles (MNP-V) complexes after clathrin dependent endocytosis. We found that directed transport is the major route of MNP-Vs intracellular trafficking after stimulation (92.6% of the trajectories measured). We then studied the MNP-V speed at each point along the trajectory, and found that the application of a second depolarization stimulus during the tracking provokes an increase in the percentage of low-speed trajectory points in parallel with a decrease in the number of high-speed trajectory points. This result suggests that stimulation may facilitate the compartmentalization of internalized MNPs in a more restricted location such as was already demonstrated in neuronal and neuroendocrine cells (Bronfman et al 2003 J. Neurosci. 23 3209-20). Although further experiments will be required to address the mechanisms underlying this transport dynamics, our studies provide quantitative evidence of the heterogeneous behavior of vesicles mobility after endocytosis in chromaffin cells highlighting the potential of MNPs as alternative labels in optical microscopy to provide new insights into the vesicles dynamics in a wide variety of cellular environments.

Coronary microvascular dysfunction in women with nonobstructive ischemic heart disease as assessed by positron emission tomography.

Traditional approaches for risk assessment of ischemic heart disease (IHD) are based on the physiological consequences of an epicardial coronary stenosis. Of note, normal coronary arteries or nonobstructive coronary artery disease (CAD) is a common finding in women with signs and symptoms of ischemia. Therefore, assessment of risk based on a coronary stenosis approach may fail in women. Positron emission tomography (PET) quantifies absolute myocardial blood flow (MBF) which may help to elucidate other mechanisms involved such as endothelial dysfunction and alterations in the smooth muscle cell relaxation responsible for IHD in women. The objective of the present review is to describe the current state of the art of PET imaging in assessing IHD in women with nonobstructive CAD.

Sustained Exocytosis after Action Potential-Like Stimulation at Low Frequencies in Mouse Chromaffin Cells Depends on a Dynamin-Dependent Fast Endocytotic Process.

Under basal conditions the action potential firing rate of adrenal chromaffin cells is lower than 0.5 Hz. The maintenance of the secretory response at such frequencies requires a continuous replenishment of releasable vesicles. However, the mechanism that allows such vesicle replenishment remains unclear. Here, using membrane capacitance measurements on mouse chromaffin cells, we studied the mechanism of replenishment of a group of vesicles released by a single action potential-like stimulus (APls). The exocytosis triggered by APls (ETAP) represents a fraction (40%) of the immediately releasable pool, a group of vesicles highly coupled to voltage dependent calcium channels. ETAP was replenished with a time constant of 0.73 ± 0.11 s, fast enough to maintain synchronous exocytosis at 0.2-0.5 Hz stimulation. Regarding the mechanism involved in rapid ETAP replenishment, we found that it depends on the ready releasable pool; indeed depletion of this vesicle pool significantly delays ETAP replenishment. On the other hand, ETAP replenishment also correlates with a dynamin-dependent fast endocytosis process (τ = 0.53 ± 0.01 s). In this regard, disruption of dynamin function markedly inhibits the fast endocytosis and delays ETAP replenishment, but also significantly decreases the synchronous exocytosis during repetitive APls stimulation at low frequencies (0.2 and 0.5 Hz). Considering these findings, we propose a model in where both the transfer of vesicles from ready releasable pool and fast endocytosis allow rapid ETAP replenishment during low stimulation frequencies.

How the stimulus defines the dynamics of vesicle pool recruitment, fusion mode, and vesicle recycling in neuroendocrine cells.

The pattern of stimulation defines important characteristics of the secretory process in neurons and neuroendocrine cells, including the pool of secretory vesicles being recruited, the type and amount of transmitters released, the mode of membrane retrieval, and the mechanisms associated with vesicle replenishment. This review analyzes the mechanisms that regulate these processes in chromaffin cells, as well as in other neuroendocrine and neuronal models. A common factor in these mechanisms is the spatial and temporal distribution of the Ca(2+) signal generated during cell stimulation. For instance, neurosecretory cells and neurons have pools of vesicles with different locations with respect to Ca(2+) channels, and those pools are therefore differentially recruited following different patterns of stimulation. In this regard, a brief stimulus will induce the exocytosis of a small pool of vesicles that is highly coupled to voltage-dependent Ca(2+) channels, whereas longer or more intense stimulation will provoke a global Ca(2+) increase, promoting exocytosis irrespective of vesicle location. The pattern of stimulation, and therefore the characteristics of the Ca(2+) signal generated by the stimulus also influence the mode of exocytosis and the type of endocytosis. Indeed, low-frequency stimulation favors kiss-and-run exocytosis and clathrin-independent fast endocytosis, whereas higher frequencies promote full fusion and clathrin-dependent endocytosis. This latter type of endocytosis is accelerated at high-frequency stimulation. Synaptotagmins, calcineurin, dynamin, complexin, and actin remodeling, appear to be involved in the mechanisms that determine the response of these processes to Ca(2+) . In chromaffin cells, a brief stimulus induces the exocytosis of a small pool of vesicles that is highly coupled to voltage-dependent Ca(2+) channels (A), whereas longer or high-frequency stimulation provokes a global Ca(2+) increase, promoting exocytosis irrespective of vesicle location (B). Furthermore, low-frequency stimulation favors kiss-and-run exocytosis (A), whereas higher frequencies promote full fusion (B). In this review, we analyze the mechanisms by which a given stimulation pattern defines the mode of exocytosis, and recruitment and recycling of neurosecretory vesicles. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015).

The immediately releasable pool of mouse chromaffin cell vesicles is coupled to P/Q-type calcium channels via the synaptic protein interaction site.

It is generally accepted that the immediately releasable pool is a group of readily releasable vesicles that are closely associated with voltage dependent Ca(2+) channels. We have previously shown that exocytosis of this pool is specifically coupled to P/Q Ca(2+) current. Accordingly, in the present work we found that the Ca(2+) current flowing through P/Q-type Ca(2+) channels is 8 times more effective at inducing exocytosis in response to short stimuli than the current carried by L-type channels. To investigate the mechanism that underlies the coupling between the immediately releasable pool and P/Q-type channels we transiently expressed in mouse chromaffin cells peptides corresponding to the synaptic protein interaction site of Cav2.2 to competitively uncouple P/Q-type channels from the secretory vesicle release complex. This treatment reduced the efficiency of Ca(2+) current to induce exocytosis to similar values as direct inhibition of P/Q-type channels via ω-agatoxin-IVA. In addition, the same treatment markedly reduced immediately releasable pool exocytosis, but did not affect the exocytosis provoked by sustained electric or high K(+) stimulation. Together, our results indicate that the synaptic protein interaction site is a crucial factor for the establishment of the functional coupling between immediately releasable pool vesicles and P/Q-type Ca(2+) channels.

The immediately releasable vesicle pool: highly coupled secretion in chromaffin and other neuroendocrine cells.

In neuroendocrine cells, such as adrenal chromaffin cells, the exocytosis of hormone-filled vesicles is triggered by a localized Ca(2+) increase that develops after the activation of voltage-dependent Ca(2+) channels. To reach the fusion competent state, vesicles have to go through a series of maturation steps that involve the detachment from cytoskeletal proteins, docking and priming. However, the fusion readiness of vesicles will also depend on their proximity to the calcium source. The immediately releasable pool is a small group of ready-to-fuse vesicles, whose fusion is tightly coupled to Ca(2+) entry through channels. Recent work indicates that such coupling is not produced by a random distribution between vesicles and channels, but would be the result of a specific interaction of immediately releasable vesicles with particular Ca(2+) channel subtypes. The immediately releasable pool is able to sustain, with high efficiency, the secretion triggered by the small and localized Ca(2+) gradients produced by brief depolarizations at low frequencies, like action potentials at basal conditions in adrenal chromaffin cells.

Rapid endocytosis and vesicle recycling in neuroendocrine cells.

Endocytosis is a crucial process for neuroendocrine cells that ensures membrane homeostasis, vesicle recycling, and hormone release reliability. Different endocytic mechanisms have been described in chromaffin cells, such as clathrin-dependent slow endocytosis and clathrin-independent rapid endocytosis. Rapid endocytosis, classically measured in terms of a fast decrease in membrane capacitance, exhibits two different forms, "rapid compensatory endocytosis" and "excess retrieval." While excess retrieval seems to be associated with formation of long-lasting endosomes, rapid compensatory endocytosis is well correlated with exocytotic activity, and it is regarded as a mechanism associated to rapid vesicle recycling during normal secretory activity. It has been suggested that rapid compensatory endocytosis may be related to the prevalence of a transient fusion mode of exo-endocytosis. In the latter mode, the fusion pore, a nanometric-sized channel formed at the onset of exocytosis, remains open for a few hundred milliseconds and later abruptly closes, releasing a small amount of transmitters. By this mechanism, endocrine cell selectively releases low molecular weight transmitters, and rapidly recycles the secretory vesicles. In this article, we discuss the cellular and molecular mechanisms that define the different forms of exocytosis and endocytosis and their impact on vesicle recycling pathways.

P/Q Ca2+ channels are functionally coupled to exocytosis of the immediately releasable pool in mouse chromaffin cells.

Chromaffin cell exocytosis is triggered by Ca(2+) entry through several voltage-dependent channel subtypes. Because it was postulated that immediately releasable vesicles are closely associated with Ca(2+) channels, we wondered what channel types are specifically coupled to the release of this pool. To study this question, cultured mouse chromaffin cell exocytosis was followed by patch-clamp membrane capacitance measurements. The immediately releasable pool was estimated using paired pulse stimulation, resulting in an upper limit of 31+/-3 fF for control conditions (I(Ca): 25+/-2 pA/pF). The N-type channel blocker omega-conotoxin-GVIA affected neither I(Ca) nor the immediately releasable pool exocytosis; although the L channel blocker nitrendipine decreased current by 50%, it did not reduce this pool significantly; and the R channel inhibitor SNX-482 significantly reduced the current but induced only a moderate decrease in the estimated IRP exocytosis. In contrast, the P/Q channel blocker omega-Agatoxin-IVA decreased I(Ca) by 37% but strongly reduced the immediately releasable pool (upper limit: 6+/-1 fF). We used alpha1A subunit knockout mice to corroborate that P/Q Ca(2+) channels were specifically linked to immediately releasable vesicles, and we found that also in this preparation the exocytosis of this pool was severely decreased (6+/-1 fF). On the other hand, application of a strong stimulus that caused the fusion of most of releasable vesicles (3 min, 50 mM K(+)) induced similar exocytosis for wild type and knockout cells. Finally, whereas application of train stimulation on chromaffin cells derived from wild type mice provoked typical early synchronous and delayed asynchronous exocytosis components, the knockout derived cells presented a strongly depressed early exocytosis but showed a prominent delayed asynchronous component. These results demonstrate that P/Q are the dominant calcium channels associated to the release of immediately releasable pool in mouse chromaffin cells.

Rapid recovery of releasable vesicles and formation of nonreleasable endosomes follow intense exocytosis in chromaffin cells.

Neurons and neuroendocrine cells must retrieve plasma membrane excess and refill vesicle pools depleted by exocytosis. To perform these tasks cells can use different endocytosis/recycling mechanisms whose selection will impact on vesicle recycling time and secretion performance. We used FM1-43 to evaluate in the same experiment exocytosis, endocytosis, and recovery of releasable vesicles on mouse chromaffin cells. Various exocytosis levels were induced by a variety of stimuli, and we discriminated the resultant endocytosis-recycling responses according to their ability to rapidly generate releasable vesicles. Exocytosis of < or =20% of plasma membrane (provoked by nicotine/acetylcholine) was followed by total recovery of releasable vesicles. If a stronger stimulus (50 mM K(+) and 2 mM Ca(2+)) provoking intense exocytosis (51 +/- 7%) was applied, endocytosis still retrieved all the fused membrane, but only a fraction (19 +/- 2%) was releasable by a second stimulus. Using ADVASEP-7 or bromophenol blue to quickly eliminate fluorescence from noninternalized FM1-43, we determined that this fraction became releasable in <2 min. The remaining nonreleasable fraction was distributed mainly as fluorescent spots ( approximately 0.7 microm) selectively labeled by 40- to 70-kDa dextrans and was suppressed by a phosphatidylinositol-3-phosphate kinase inhibitor, suggesting that it had been formed by a bulk retrieval mechanism. We concluded that chromaffin cells can rapidly recycle significant fractions of their total vesicle population, and that this pathway prevails when cholinergic agonists are used as secretagogues. When exocytosis exceeded approximately 20% of plasma membrane, an additional mechanism was activated, which was unable to produce secretory vesicles in our experimental time frame but appeared crucial to maintaining membrane surface homeostasis under extreme conditions.

Cardiovascular effects of tibolone: a selective tissue estrogenic activity regulator.

Traditionally, it was accepted that long-term hormone replacement therapy (HRT) has a cardiovascular beneficial effect in postmenopausal women with and without coronary artery disease (CAD). However, randomized trials in postmenopausal women have not shown any benefit in either primary or secondary prevention of cardiovascular events. Therefore, these findings have raised the question of whether traditional HRT (i.e., estrogen and progesterone) has a cardioprotective effect in women at risk for or with established CAD. Concerns about the use of conventional HRT have led to a search for alternatives. Tibolone is a synthetic compound with estrogenic, androgenic, and progestogenic properties that relieves climacteric symptoms and prevents postmenopausal bone loss. Tibolone possesses a tissue-selective mechanism of action that differs from that of estrogen and/or progestogen. Unlike these compounds, tibolone's metabolites play a central role in its mode of action. Tibolone is widely used for HRT. However, its clinical impact on cardiovascular disease is still under study. The current review focuses on the effects of tibolone on the cardiovascular system and discusses clinical investigations with this compound in postmenopausal women.