Vascular calcium signalling and ageing

Abstract

Changes in cellular Ca²⁺ levels have major influences on vascular function and blood pressure regulation. Vascular smooth muscle cells (SMCs) and endothelial cells (ECs) orchestrate vascular activity in distinct ways, often involving highly specific fluctuations in Ca²⁺ signalling. Ageing is a major risk factor for cardiovascular diseases, but the impact of ageing perse on vascular Ca²⁺ signalling has received insufficient attention. We reviewed the literature for age-related changes in Ca²⁺ signalling in relation to vascular structure and function. Vascular tone dysregulation in several vascular beds has been linked to abnormal expression or activity of SMC voltage-gated Ca²⁺ channels, Ca²⁺-activated K⁺ channels or TRPC6 channels. Some of these effects were linked to altered caveolae density, microRNA expression or 20-HETE abundance. Intracellular store Ca²⁺ handling was suppressed in ageing mainly via reduced expression of intracellular Ca²⁺ release channels, and Ca²⁺ reuptake or efflux pumps. An increase in mitochondrial Ca²⁺ uptake, leading to oxidative stress, could also play a role in SMC hypercontractility and structural remodelling in ageing. In ECs, ageing entailed diverse effects on spontaneous and evoked Ca²⁺ transients, as well as structural changes at the EC-SMC interface. The concerted effects of altered Ca²⁺ signalling on myogenic tone, endothelium-dependent vasodilatation, and vascular structure are likely to contribute to blood pressure dysregulation and blood flow distribution deficits in critical organs. With the increase in the world’s ageing population, future studies should be directed at solving specific ageing-induced Ca²⁺ signalling deficits to combat the imminent accelerated vascular ageing and increased risk of cardiovascular diseases.

Graphical Abstract

Ageing is often associated with a change in vascular function. Resistance vessels – which control peripheral resistance, and therefore blood pressure – demonstrate altered vascular tone and impaired vascular conduction. These changes can be traced back to defective Ca²⁺ signalling in the building blocks of resistance vessels, smooth muscle cells (SMCs) and endothelial cells (ECs). Alterations in the activity of ion channels that permeate or respond to Ca²⁺ signals, intracellular handling of Ca²⁺ and mitochondrial dysfunction have been implicated in ageing-associated vascular dysfunction.

Introduction

Calcium signalling and muscle contraction

Ca²⁺ is the most abundant and versatile second messenger in signal transduction pathways. A deviation in Ca²⁺ concentration ([Ca²⁺]) from baseline levels is a crucial step in a vast majority of biological processes, such as muscle contraction, neurotransmitter release and egg fertilization (Berridge et al. 2003). Disruption of Ca²⁺ homeostasis is a hallmark of ageing and disease. A multitude of proteins and cellular pathways are involved in steady-state Ca²⁺ handling and in the control of [Ca²⁺] dynamics in response to different triggers. The four primary mechanisms involved in determining cytosolic [Ca²⁺] are Ca²⁺ influx, efflux, release and reuptake. Flux of Ca²⁺ into (influx) and out of (efflux) the cytoplasm is respectively enabled by Ca²⁺-permeable ion channels and active transporters that allow Ca²⁺ movement across a concentration gradient. Proteins localized in the membranes of intracellular Ca²⁺ stores permit Ca²⁺ release (to the cytoplasm) and reuptake (into the Ca²⁺ store). The endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR) are the main intracellular Ca²⁺ stores, and the mitochondria also play a role in Ca²⁺ storage. The concerted action of several cellular mechanisms (Ca²⁺ entry channels, efflux transporters, release channels and reuptake transporters) is indispensable for Ca²⁺ homeostasis (Clapham 2007).

One of the earliest demonstrations of the universal nature of Ca²⁺ signalling was pioneered by Ringer who showed that isolated hearts only contract in the presence of Ca²⁺ (Ringer 1883). Vascular smooth muscle similarly contracts in a Ca²⁺-dependent process classically known as excitation-contraction coupling (Sandow 1952). The vasculature comprises arteries, arterioles, capillaries, venules and veins. Smaller arteries (<500 μm) and arterioles (<100 μm) together form resistance vessels since they represent the site of vascular resistance that is crucial in the regulation of blood pressure. Resistance arteries control tissue perfusion via vasomotor responses to a range of neurohormonal stimuli and local factors, such as sympathetic vasoconstrictors and perturbations in arterial pressure (wall stress) and flow (shear stress). Arteries are made up of layers of smooth muscle cells (SMCs) that are circumferentially arranged and a single layer of endothelial cells (ECs) (Fig. 1A). The ability of arteries to constrict to elevated intravascular pressure, known as the ‘myogenic response’, is intrinsic to vascular SMCs and it establishes the basal tone (myogenic tone) (Fig. 1B) from which other vasoconstrictor or vasodilator mechanisms can adjust the vascular diameter to regulate blood pressure and organ blood flow. The myogenic response is also crucial for autoregulation of organ blood flow, such as in the brain, to protect downstream capillaries from excess flow and rupture. Canonically, elevated intravascular pressure leads to a rise in [Ca²⁺]_i in SMCs which ultimately facilitates myosin-actin cross-bridging and SMC contraction (Frearson et al. 1976; Dabrowska et al. 1977; Olson et al. 1990; Webb 2003). Importantly, the fact that the myogenic response is driven by changes in [Ca²⁺]_i makes it vulnerable in disease and during ageing (Fig. 1C).

Figure 1. Properties of small arteries and arterioles in relation to ageing.

A, general vessel structure with a single endothelial cell layer (a.k.a. intima) towards the lumen and a smooth muscle layer (a.k.a. media) consisting of one cell layer (in arterioles) to several layers of smooth muscle cells in small arteries. Small arteries (<500 μm lumen diameter) and arterioles (<100 μm lumen diameter) are generally termed resistance vessels. For clarity, we have not shown an outer adventitia layer with peripheral nerves and fibroblasts. B, the concept of vascular tone is illustrated by a vessel with 50% reduction in active diameter compared with the fully relaxed state (i.e. 50% tone). C, schematic showing the possible effects of ageing in resistance vessels, and their likely causal relationship with age-related diseases.

Vascular smooth muscle calcium signalling and vascular function

The ability of vascular SMCs to contract and relax – and therefore mediate vasoconstriction and vasodilation, respectively – is intimately tied to [Ca²⁺]_i. Even though spontaneous action potentials are not characteristic of vascular SMCs as primary drivers of changes in [Ca²⁺]_i, active depolarization and repolarization of membrane potential (V_M) do occur in vascular SMCs. Arterial SMC V_M sits around −40 mV in vessels pressurized to physiological levels. Maximum vasodilation occurs at ~−60 mV and maximum constriction at ~−30 mV, leading to a steep relationship between SMC V_M and arterial diameter (Knot & Nelson 1998). In fact, the primary driver of a global increase in SMC [Ca²⁺]_i is V_M depolarization and the subsequent activation of Ca²⁺ channels that are gated by voltage. A physiological stimulus that depolarizes SMC V_M will typically facilitate voltage-gated Ca²⁺ fluxes, increase [Ca²⁺]_i, and activate downstream signalling pathways including those mediating contraction (Knot & Nelson 1998). SMC V_M hyperpolarization, on the other hand, deactivates voltage-gated Ca²⁺ flux, lowers [Ca²⁺]_i and leads to SMC relaxation and vasodilation. Local, microdomain increases in SMC [Ca²⁺]_i can couple to signalling cascades leading to vasodilation (see later sections).

Ca²⁺ influx pathways in vascular smooth muscle

Vascular SMCs of resistance vessels have the ability to contract in response to increases in intravascular pressure. When pressure increases, the vascular wall stress increases, leading to vascular SMC depolarization and the graded opening of Ca²⁺ channels located at the plasma membrane (Knot & Nelson 1998; Welsh et al. 2002). The global [Ca²⁺]_i in SMCs is primarily set by steady-state changes in V_M and the graded entry of Ca²⁺ from the extracellular space through voltage-gated Ca²⁺ channels (VGCC) to establish the arterial tone (Fig. 1B). The primary Ca²⁺ influx pathway in vascular SMC is the high voltage-activated, L-type Cav1.2 channel (Knot & Nelson 1998). Consistent with the crucial role of the Cav1.2 channel in setting arterial tone through mediating Ca²⁺ influx, pharmacological blockers of Cav1.2 channels dilate arteries and lower arterial resistance therapeutically.

In addition to Cav1.2, vascular SMCs express other voltage-activated Ca²⁺ channels. In particular, the low-voltage-activated, T-type Cav3.x channels are expressed in rodent and human arteries (Braunstein et al. 2009; Kuo et al. 2010; Abd El-Rahman et al. 2013; Björling et al. 2013; Harraz, Visser, et al. 2015). As their name implies, T-type Ca²⁺ channels are activated by more modest depolarizations (i.e. at a V_M more hyperpolarized than that required to activate L-type channels). Cav3 channels are also characterized by fast activation/inactivation and low unitary conductance, when compared with Cav1.2 channels (Perez-Reyes et al. 1998). Intriguingly, the activation of Cav3.2 channel in vascular SMCs does not lead to a global rise in [Ca²⁺]_i, but rather mediates a Ca²⁺-induced Ca²⁺ release-like mechanism in which Ca²⁺ influx triggers localized Ca²⁺ release from the SR, activates a K⁺ channel, hyperpolarizes SMCs and counterbalances arterial tone (contractility) (Harraz et al. 2014). Cav3.1 and Cav3.3 channels demonstrate mutually exclusive expression in vascular SMC from rodents and humans (Harraz, Visser, et al. 2015). Studies have shown that Cav3.1 channels activate at lower intravascular pressures (40–80 mmHg) – where V_M is more hyperpolarized – and they facilitate myogenic tone development (Abd El-Rahman et al. 2013; Björling et al. 2013).

Transient receptor potential (TRP) channels comprise a family of non-selective cation channels. TRP channels are distinct from voltage-gated Ca²⁺ channels and they represent the primary non-voltage-activated Ca²⁺ influx pathway in vascular SMCs. Several TRP channels participate in the regulation of SMC contraction by either altering V_M and therefore affecting voltage-gated channels, or by directly (via Ca²⁺ influx) or indirectly (via facilitating Ca²⁺ release) triggering a global or local change in [Ca²⁺]_i. Vascular SMCs express several functional TRP channels that are permeable to Ca²⁺ (Inoue et al. 2006; Earley & Brayden 2015), such as canonical TRP (TRPC) channels. Additionally, vasoactive substances acting through G-protein-coupled receptors or receptor tyrosine kinases have been shown to activate TRPC channels, which in turn leads to rises in [Ca²⁺]_i (for review, see Inoue et al. 2006; Earley & Brayden 2015). When functionally coupled to Ca²⁺-activated channels, TRP channel-mediated localized changes in [Ca²⁺]_i can lead to V_M depolarization (e.g. TRPC6 activation→Ca²⁺ release→TRPM4 activation) or hyperpolarization (e.g. TRPV4 activation→Ca²⁺ release Ca²⁺-activated K⁺ channel activation) (Welsh et al. 2002; Earley et al. 2005; Gonzales et al. 2014).

Ca²⁺ release pathways in vascular smooth muscle

The SR in vascular SMC is equipped with transmembrane ion channels – ryanodine receptors (RyRs) and IP₃ receptors (IP₃R) – that mediate Ca²⁺ release down its concentration gradient to the cytosol (Clapham 2007). While RyR-mediated Ca²⁺ release is an essential step in the contraction of cardiac and skeletal muscle, its role in the vasculature is quite distinct. RyRs are activated by Ca²⁺ acting on the RyR cytosolic domain. Vascular RyRs, encoded by RYR2 in humans, mediate Ca²⁺ release from the SR in the form of Ca²⁺ sparks. The latter activate the large-conductance, Ca²⁺-activated K⁺ channel (BK_Ca), thus leading to SMC hyperpolarization and vasodilation – rather than vasoconstriction (Nelson et al. 1995). On the other hand, IP₃ evokes Ca²⁺ release from the SR by sensitizing its cognate receptor IP₃R to stimulatory Ca²⁺. IP₃ is typically formed downstream of G_q protein-coupled receptor activation as a metabolite of phosphatidylinositol-4,5-bisphosphate (PIP₂) hydrolysis. Vascular SMC IP₃-mediated Ca²⁺ release is a major signalling pathway that has significant effects, such as the activation of depolarizing TRP channels (e.g. TRPM4) that further facilitate voltage-gated Ca²⁺ influx (Gonzales et al. 2010).

Ca²⁺ reuptake and efflux (SERCA, Ca²⁺-ATPase)

While Ca²⁺ influx from the extracellular space or release from intracellular stores lead to Ca²⁺ flux along the concentration gradient – and therefore a rise in [Ca²⁺]_i – several mechanisms are able to move Ca²⁺ in the opposite direction (i.e. against the concentration gradient). These mechanisms utilize energy in the form of ATP to lower [Ca²⁺]_i, by mediating Ca²⁺ reuptake into the SR or Ca²⁺ efflux to the extracellular space. In particular, Ca²⁺-ATPase pumps are expressed in vascular SMCs on the SR membrane (SERCA pump: the sarco-endoplasmic reticulum Ca²⁺-ATPase) and on the plasma membrane (PMCA pump: the plasma membrane Ca²⁺-ATPase) (Ottolini & Sonkusare 2021). It is noteworthy that altered expression and/or function of Ca²⁺-ATPase pumps might be implicated in the disruption of Ca²⁺ homeostasis during ageing (see later sections).

Endothelial calcium signalling and vascular function

The endothelium is composed of ECs that line all blood vessels (Fig. 1A). The luminal side of ECs is continuously in contact with blood cells and blood-borne factors, both of which represent important triggers of endothelial Ca²⁺ signalling. On the other hand, the abluminal side of arterial and arteriolar ECs faces the internal elastic lamina that separates ECs from SMCs. Projections from ECs make close contacts with SMCs and form myoendothelial junctions, which are important connections that facilitate vascular communication between ECs and SMCs (Straub et al. 2014; Sandow and Hill 2000; Moore & Ruska 1957).

Impact of EC Ca²⁺signalling on vascular function

Cation influx (Ca²⁺ or Na⁺) shifts the V_M toward the equilibrium potential of these cations, leading to membrane depolarization. While Ca²⁺ influx in vascular SMC typically associates with muscle contraction, EC Ca²⁺ signals are unique in the essence that not only are they capable of altering V_M, these EC Ca²⁺ transients can affect downstream targets that counteract vascular contraction. Endothelial Ca²⁺ signals are crucial in the activation of endothelial nitric oxide synthase (eNOS) and the production of the potent vasodilator NO (Fleming & Busse 1999; Cohen & Adachi 2006). Furthermore, studies have shown the coupling between endothelial Ca²⁺ signalling and EC hyperpolarization via microdomain signalling. In particular, Ca²⁺ influx (TRPV4 channels) or release (IP₃Rs) is coupled to the activation of Ca²⁺-activated K⁺ channels leading to hyperpolarization and ultimately vasodilation (Fig. 2) (Ledoux et al. 2008; Sonkusare et al. 2012). Additionally, EC Ca²⁺ transients can activate a number of protein kinases such as protein kinase C (PKC) and Ca²⁺-calmodulin kinase (CaMK) leading to the modulation of several EC processes (Förstermann et al. 1991) (Fig. 3). Notably, ECs form a unique barrier between the vessel lumen and the vascular wall. Ca²⁺ transients in ECs play a crucial role in determining barrier activity and intercellular junctions between ECs (Dalal et al. 2020).

Figure 2. The prominent molecular players of endothelial cell Ca²⁺ handling and the impact of ageing.

Top: The primary Ca²⁺ influx pathways in endothelial cells (ECs) include: TRP channels such as TRPV4 and TRPA1; the mechanosensitive Piezo1 channel; the ionotropic purinergic receptor P2X; and the Na⁺/Ca²⁺ exchanger (NCX) in the reverse mode. Ca²⁺ release from the endoplasmic reticulum (ER) is facilitated by IP₃R and can activate a Ca²⁺-sensitive target (K_Ca channels). Ca²⁺ influx by TRPV4 channel has also been linked to downstream activation of Ca²⁺-activated K⁺ channels. The efflux of Ca²⁺ across the plasma membrane and the reuptake into the ER are facilitated by the active transporters PMCA and SERCA, respectively. Bottom: EC Ca²⁺ handling and morphology are altered during ageing. Inset box summarizes key findings in studies that investigated the impact of ageing on Ca²⁺ transients (see text for details). A putative change (shown by ‘?’) should be further investigated.

Figure 3. Targets of endothelial cell Ca²⁺ signals.

Ca²⁺ is a crucial second messenger in endothelial cells (ECs) that affects several downstream signalling pathways. Cation influx depolarizes the membrane potential of ECs. Activation of the endothelial NO synthase (eNOS) leads to the generation of the potent vasodilator NO that dilates the neighbouring smooth muscle cells (SMCs). Microdomain Ca²⁺ signals (either through Ca²⁺ influx or release) can activate K_Ca channels leading to EC hyperpolarization that is transmitted to SMCs via gap junctions which ultimately induces vasodilation. Several protein kinases in ECs are Ca²⁺-activated such as PKC and CaM Kinase.

Mechanisms driving EC Ca²⁺ signals

The influx of Ca²⁺ into ECs is mediated by a number of channels that are not gated by voltage. Among these, several polymodal TRP channels have been shown to mediate endothelial Ca²⁺ influx and vascular tone regulation. For instance, these TRP channels have been reported: vanilloid TRPV4, TRPV3 and TRPV1 channels, ankyrin TRPA1, polycystic TRPP1 (Polycystin-2 or PKD2), canonical TRPC1, TRPC3, TRPC4, TRPC5, TRPC6 and melastatin TRPM2 (Earley & Brayden 2015; Thakore & Earley 2019) (Fig. 2). Recent evidence further unravelled a role for the Ca²⁺-permeable Piezo1 channel in sensing mechanical forces associated with blood flow (Wang et al. 2016). Ionotropic purinergic receptors (P2X) and reverse-mode Na⁺/Ca²⁺ exchangers (NCX) are additional proposed Ca²⁺ influx mechanisms in ECs (Harrington & Mitchell, 2004; Lillo et al. 2018). Endothelial Ca²⁺ release from and reuptake into the ER is driven by IP3R and SERCA, respectively (Ottolini & Sonkusare 2021) (Fig. 2).

Ageing disrupts vascular function

Ageing is a major risk factor for cardiovascular disease (Abete et al. 2014; Berridge 2016). In humans, ageing is an important contributor to the risk of developing hypertension (Buford, 2016; Hay et al. 2020), and a large clinical trial showed that mean arterial pressure increases with age until around age 70 for both sexes (Cheng et al. 2012). Ageing can entail both healthy ageing and senescence. Here, we review situations where vascular Ca²⁺ signalling was associated with loss of normal function in senescent cells, as opposed to the default healthy ageing. Nearly every organ will be affected by ageing, but the most well-known loss-of-function effects are observed in skeletal muscles and the brain. The ‘Ca²⁺ hypothesis of brain ageing’ poses that long-lasting small or short-lasting large increases in intracellular Ca²⁺ levels in the brain increase the risk of neuronal damage and neurodegenerative diseases (Berridge 2010). Ageing is indeed an important risk factor in Alzheimer’s disease (AD), and studies suggest that one of the earliest events in AD is a decrease in cerebral blood flow (Korte et al. 2020). Along these lines, a role for Ca²⁺ and VGCC in AD has been suggested based on the efficacy of a blood-brain barrier-permeable VGCC inhibitor in restoring cerebral blood flow to normal levels in a mouse model of AD (Paris et al. 2004). In support of this, two independent epidemiological studies showed that the use of Ca²⁺ channel blockers was associated with a lower risk of dementia in elderly hypertensive patients (Lovell et al. 2015; Wu & Wen 2016). Furthermore, the induction of cell senescence with various stressors leads to an increase in [Ca²⁺]_i in several cell types through either Ca²⁺ influx or intracellular Ca²⁺ release pathways (Martin & Bernard 2018). Prolonged increases in [Ca²⁺]_i may additionally result in mitochondrial Ca²⁺ accumulation and dysfunction, which can lead to oxidative stress and inflammatory signalling (Peng & Jou, 2010; Chaudhari et al. 2014; Görlach et al. 2015; Martin & Bernard 2018). This may in turn have a deleterious impact on gene expression programmes and ion channel or membrane transporter function, ultimately leading to altered vascular function.

The impact of ageing on microvascular reactivity in skeletal muscle, for example, depends on the size of the vessels and type of skeletal muscle fibres nourished, and it is noteworthy that endothelial vs. smooth muscle effects sometimes oppose one another (Muller-Delp, 2006; Sinkler & Segal, 2014; Muller-Delp, 2016). Myogenic tone is reported to be increased in middle-aged mice and rats (Mikkelsen et al. 2016; Björling et al. 2018; Wang et al. 2020; Young et al. 2021), while others have found that advanced age leads to a loss of myogenic tone (Gros et al. 2002). The myogenic tone in cerebral arteries in particular seems to be reduced with advanced age as shown in endothelium-denuded preparations and under pulsatile pressure (Geary & Buchholz 2003; Springo et al. 2015), thereby potentially impairing cerebral autoregulation and leading to excess flow-induced impairment of downstream capillaries. Due to the large and often diverse impact that ageing can have on microvascular structure and function, it is important to shed more light on the role of Ca²⁺ in the ageing microvasculature (Fig. 1C). For a definition of the age of mice and rats corresponding to different age groups in this review, please see Table 1. For an overview of the major findings concerning effects of ageing on calcium signaling in the vasculature, please see Table 2.

Table 1.

Defining the relevant age range for ageing studies in mice and rats

	Mouse1	Rat2
Young (mature adult)	3–6 months	7–10 months
Middle-aged	10–14 months	12–18 months
Old	18–24 months	22–30 months

¹According to Flurkey et al. 2007 [https://mouseion.jax.org/stfb2000_2009/1685].

²As suggested by Harraz & Jensen, 2020.

Table 2.

Summary of key findings in vascular smooth muscle cells and endothelial cells in relation to age, sex, species and vascular bed

Mechanism	Key finding	Species	Age (sex)	Vessel (cell type)	Reference
Myogenic tone	Increased myogenic tone in ageing with intact endothelium, but reduced in endothelial-denuded preparations. Intracellular [Ca2+] in VSMCs increased at low pressures, but not at high pressures	Rat	Old (M)	Middle cerebral artery (SMC)	Geary & Bucholz, 2003
Myogenic tone	Loss of negative feedback on myogenic tone via CaV3.2 channels, caveolae and BKCa channels in ageing. Increased myogenic tone in ageing	Mouse	Middle-aged (M)	Small mesenteric artery (SMC)	Mikkelsen et al. 2016; Fan et al. 2020
Intracellular Ca2+ handling	Decreased Ca2+ extrusion and Ca2+ fluxes across the SR membrane. Reduced expression of Ca2+-release channels and Ca2+ ATPases	Mouse	Old (M)	Small mesenteric artery; cerebral artery (SMC)	Del Corsso et al. 2006; Goyal et al. 2009; Georgeon-Chartier et al. 2013
Structural remodelling	Increased VSMC cell size; vascular wall hypertrophia; and inward remodelling in ageing	Mouse (WT) Mouse (WT) Heterozygous PMCA1+/− mouse	Old (M) Middle-aged (M) Middle-aged and Old (M)	Small mesenteric artery (SMC)	Del Corsso et al. 2006; Mikkelsen et al. 2016; Little et al. 2017
microRNA expression	Altered miR-328 and miR-155 expression in ageing causes opposite changes in L-type Ca2+ channel expression	Rat Mouse (WT and MR-KO)	Middle-aged (M)	Small mesenteric artery (SMC)	DuPont et al. 2016; Liao et al. 2017
Local Ca2+ transients	Reduced frequency of spontaneous local Ca2+ signals. Reduced number of holes of internal elastic lamina.	Mouse (WT, genetically encoded Ca2+ indicator)	Old (M and F)	Small mesenteric artery (EC)	Boerman et al. 2016
Ca2+ transients	EC sensitivity to ACh increased with age. Frequency of Ca2+ oscillations were unchanged with age, but the latency of events was increased. Pressure-dependent inhibition of Ca2+ signalling was reduced with age.	Rat	Middle-aged (M)	Carotid artery (EC)	Wilson et al. 2017
Muscarinic-mediated Ca2+ transients	Minimally altered with ageing. Enhanced with ageing.	Mouse	Middle-aged and Old (M and F) Old (M)	Cerebral artery (EC tubes) Skeletal muscle artery (EC tubes)	Socha et al. 2015; Hakim et al. 2020
Hydrogen peroxide-mediated Ca2+ transients	Reduced with ageing.	Mouse	Old (M)	Skeletal muscle artery (EC tubes)	Socha et al. 2015
Electrical conduction	Reduced with ageing due to enhanced Ca2+-activated K+ channel activation.	Mouse	Middle-aged and Old (M)	Skeletal muscle artery (EC tubes)	Behringer et al. 2013

EC: endothelial cell; mineralocorticoid receptors knockout mice: MR-KO; SMC: smooth muscle cell; SR: sarcoplasmic reticulum; VSMC: vascular smooth muscle cell; WT: wild-type.

Ageing and smooth muscle Ca²⁺ handling

Role of voltage-gated Ca²⁺ (L-, T-type) channels

Several studies have investigated the potential role of altered expression and function of L-type Ca²⁺ channels (Ca_V1.2) in the ageing vasculature. Studies in rats revealed a discrepancy between different vascular beds of the effects of ageing on L-type channels. In mesenteric resistance arteries, a loss of L-type channel expression and function was seen in middle-aged and old male rats (Albarwani et al. 2016; Liao et al. 2017), whereas no change was observed in coronary resistance arteries from old rats (Korzick et al. 2005). An enhanced myogenic tone without a change in SMC [Ca²⁺]_i at high intravascular pressure suggested an increase in SMC Ca²⁺ sensitivity in middle cerebral arteries with intact endothelium from old rats (Geary & Buchholz 2003). Later studies in mouse and rat mesenteric artery implicated increased Rho kinase activity in the age-dependent increase in vascular SMC Ca²⁺ sensitivity resulting in an augmentation of myogenic (Björling et al. 2018) and α₁-adrenoceptor-induced (Wei et al. 2021) arterial tone. Similar to the discrepancy in rat studies, investigations of mouse small mesenteric arteries demonstrated an inconsistent impact of ageing on L-type channel expression and function (del Corsso et al. 2006; DuPont et al. 2016; Mikkelsen et al. 2016). Discrepancies possibly reflect methodological differences and the use of different mouse ages. Notably, reports of reduced vascular tone in aged cerebral arteries are consistent with the loss of L-type channel expression (Georgeon-Chartier et al. 2013; Springo et al. 2015; Toth et al. 2017).

T-type Ca²⁺ channels (Ca_V3.1/Ca_V3.2) are widely expressed in the cerebral, mesenteric, renal and pulmonary vasculature where they are robustly detected in vascular SMCs, despite a number of studies suggesting endothelial expression (Hansen et al. 2001; Rodman et al. 2005; Zhou & Wu 2006; Braunstein et al. 2009; Kuo et al. 2010; Abd El-Rahman et al. 2013). The observation that the myogenic tone was enhanced and [Ca²⁺]_i was elevated at low intravascular pressures (20–40 mmHg) in old rats (Geary & Buchholz 2003) is consistent with data showing that Ca_V3.1 T-type Ca²⁺ channels sustain myogenic tone at these lower pressures (Abd El-Rahman et al. 2013; Björling et al. 2013). On the other hand, mRNA expression of Cacna1g (encoding Ca_V3.1) is diminished by ageing in mouse small mesenteric arteries (Mikkelsen et al. 2016) and mouse aortae (Thuesen et al. 2018), consistent with a report of reduced Ca_V3.1 mRNA and protein expression in certain brain areas of old humans and mice (Rice et al. 2014). This warrants further investigation into the effects of ageing on vascular Ca_V3.1 T-type channel.

A more distinct role has been elucidated for Ca_V3.2 T-type channels in ageing (Fig. 4). Arterial Ca_V3.2 channels located in caveolar membrane subdomains are involved in the activation of RyRs to trigger local Ca²⁺ sparks from adjacent SR and elicit outward K⁺ currents through BK_Ca channels (Harraz et al. 2014). In turn this leads to vasodilatation and negative feedback on myogenic tone in cerebral and mesenteric arteries (Harraz et al. 2014; 2015; Mikkelsen et al. 2016). This Ca_V3.2-mediated negative feedback control of myogenic tone was absent in middle-aged wild-type mice, and this absence was mimicked by the genetic deletion of Ca_V3.2 channels in mesenteric arteries from young mice (Mikkelsen et al. 2016). A recent study showed that a disruption of caveolae could explain the loss of the Ca_V3.2-RyR-BK_Ca signalling axis in middle-aged mice (Fan et al. 2020). Further, it is possible that the loss of Ca_V3.2/BK_Ca axis could be caused by a shift in the intracellular location of SR from a sub-membranous, peripheral location in young vascular SMCs to a deeper, less peripheral location in aged SMCs (Harraz & Jensen, 2020). Interestingly, another Ca²⁺ influx pathway, which was Gd³⁺-sensitive, seems to substitute for the loss of Ca_V3.2 signalling in middle-aged mice, suggesting a role for vascular TRP channels in triggering BK_Ca activity (Hashad et al. 2017; Fan et al. 2020).

Figure 4. Vascular smooth muscle model for Ca²⁺ signalling in young (top) vs. middle-aged or old (bottom) subjects.

Normal Ca²⁺ signalling components in young vascular SMCs comprise voltage-gated Ca²⁺ channels (Ca_V1.2 L-type; Ca_V3.1, and Ca_V3.2 T-types), Transient Receptor Potential C6 channels (TRPC6), large-conductance Ca²⁺-activated K⁺ channels (BK_Ca), ryanodine receptor (RyR) and inositol-triphosphate (IP₃) receptor release channels, Ca²⁺-ATPase reuptake pump (SERCA) in the sarcoplasmic reticulum (SR) membrane, plasma membrane Ca²⁺-ATPase (PMCA) and Gα_q/11-coupled receptors (GPCR). Caveolae are shown as membrane invaginations in a limited area of the plasma membrane, for clarity. Mitochondria can release reactive oxygen species (ROS) under basal conditions. In middle-aged or old vascular smooth muscle cells (SMCs) the following mechanisms are changed, or a putative change (shown by ‘?’) should be investigated in further studies: the number of and area covered by caveolae are severely reduced, Ca²⁺ influx via Ca_V3.2 channels therefore causes less activation of RyR-mediated Ca²⁺ release and BK_Ca activation. Proximity of the SR to BK_Ca channels may be impaired via reduced function of junctophilin (JP) and microtubular networks (MTs). The SR is in general found at a deeper location in the cell, thereby preventing Ca_V3.2 channels from activating RyRs. Ca_V1.2 L-type channel expression is negatively affected by miR-155 and miR-328 expression. Putative regulation of TRPC6, T-type channels and BK_Ca channels by miRs should be investigated. Increased myogenic tone and intracellular [Ca²⁺] at relatively lower intravascular pressures (40–80 mm Hg) might be due to increased Ca_V3.1 activity (Björling et al. 2013). Loss of TRPC6 function in aged hypertensive animals is attributed to decreased Cyp4a activity and 20-HETE production. Ca²⁺ loading of mitochondria may lead to increased ROS release, causing increased activity of inflammatory mediators (INFL) (such as IL-1β, IL-6, and TNFα), growth factors (GFs) (such as IGF-1 and TGF-β1), and matrix metalloproteinases (MMPs) (such as MMP2 and MMP9). Reduced Ca²⁺ efflux and Ca²⁺ reuptake into the SR may be caused by crippled expression and/or activity of the key transporters involved.

Role of intracellular Ca²⁺ stores

Studies in mice have shown an effect of age on intracellular Ca²⁺ handling via channels and pumps controlling release and uptake of Ca²⁺ in the SR. In aged mouse mesenteric arteries, the size of individual vascular SMCs was increased, but there were no major changes in basal Ca²⁺ levels (del Corsso et al. 2006; Goyal et al. 2009). RyR- and IP₃R-mediated Ca²⁺ release and the refilling of SR were suppressed in mesenteric and cerebral artery SMCs from old mice (Fig. 4) (del Corsso et al. 2006; Georgeon-Chartier et al. 2013). This was consistent with the reduced expression of channels and pumps involved in intracellular Ca²⁺ homeostasis (Georgeon-Chartier et al. 2013), and the loss of PKD2 (Polycystin-2) channel functional role in facilitating Ca²⁺ release in aged mouse cerebral arteries (Abdi et al. 2015). Interestingly, SR Ca²⁺ storage was enhanced while Ca²⁺ extrusion and SR Ca²⁺ reuptake were unchanged in mesenteric SMCs from old mice (Goyal et al. 2009). Taken together, different mouse studies suggest that intracellular Ca²⁺ handling is altered with ageing, which may lead to changes in vascular tone or phenotypic modulation of old arteries. Given the discrepant findings of investigations carried out only in mouse arteries, further studies are needed to resolve the matter (Fig. 5).

Figure 5. The impact of ageing on vascular Ca²⁺ signalling.

Ageing impairs vascular function and myogenic tone. These changes are attributed partly to altered Ca²⁺ handling in vascular smooth muscle cells and endothelial cells. See text for further explanations and additional mechanisms. ‘?’ indicates future directions and unanswered questions.

Role of TRPC6 channels

TRPC6 channels are non-selective cation channels with a Ca²⁺/Na⁺ permeability ratio of about 5:1 (Owsianik et al. 2006). They were shown to constitute the essential component of the α₁-adrenoceptor activated cation current causing sympathetic Ca²⁺ entry and vasoconstriction in blood vessels via activation of the G_αq-protein/PLC/DAG cascade (Inoue et al. 2001; 2006). In addition, smooth muscle TRPC6 channels are purported to be involved in vascular mechanosensitivity – either directly or indirectly via local 20-hydroxyeicosatetraenoic acid (20-HETE) production – and to contribute to myogenic responsiveness in small arteries (Welsh et al. 2002; Inoue et al. 2009). TRPC6 channels are upregulated in hypertension and contribute to an enhanced sustained myogenic constriction in cerebral arteries, thus protecting against blood-brain barrier disruption, oedema formation and haemorrhages (Toth et al. 2020). This protective upregulation of TRPC6 is, however, lost in ageing hypertensive animals (Fig. 4) due to reduced local levels of 20-HETE and reduced circulating levels of insulin-like growth factor 1 (Toth et al. 2014; 2020).

Role of BK_Ca channels

Smooth muscle K⁺ channels are crucial for setting the resting V_M, and the BK_Ca channel is particularly prominent for its role as a negative feedback regulator of vascular tone (Nelson et al. 1995). As explained earlier, altered Ca²⁺ influx through VGCCs during ageing can drive changes in BK_Ca activity and the subsequent effects on vascular tone. Furthermore, reduced functional expression of BK_Ca α- and β₁-subunits was reported in aged rat and human coronary arteries, thus leading to reduced relaxation to nitric oxide donors (Marijic et al. 2001; Nishimaru et al. 2004). Similar functional effects were observed in small mesenteric arteries from aged rats, but in that study the downregulation of BK_Ca β₁-subunits predominated over that of α-subunits (Shi et al. 2013). In contrast, a recent study (Reed et al. 2021) demonstrated an increased expression of BK_Ca channel in aged male rat cerebral arteries leading to an attenuated myogenic reactivity. The opposite effects were observed in aged female rats, where a loss of female sex hormones by ageing caused a loss of K⁺ channel expression and exaggerated vascular tone (Reed et al. 2021), highlighting a possible role for sexual dimorphism in the role of vascular BK_Ca channels during ageing. It is noteworthy, however, that an earlier study found no change in BK_Ca expression during ageing in male rat cerebral artery SMCs (Nishimaru et al. 2004).

Ageing and endothelial Ca²⁺ handling

A key indicator of ageing-related vascular pathology is endothelial dysfunction that is well manifested in ageing subjects (Bugiardini et al. 2004; James et al. 2006). Ageing may lead to endothelial cell senescence and vascular dysfunction through abnormal Ca²⁺ signalling (Jia et al. 2019), and a multitude of endothelial signalling pathways are altered with ageing. The impact of ageing on EC Ca²⁺ handling is quite complex, and further complicated by the fact that ageing ECs become morphologically thinner which could dramatically affect Ca²⁺ dynamics (Kalaria 1996; Altschul 1955; Alba et al. 2004). In vivo studies revealed that advanced age profoundly decreases the frequency of spontaneous local EC Ca²⁺ transients in mesenteric arteries (Boerman et al. 2016) and suggested that diminished signalling between the endothelium and SMCs could contribute to age-dependent vascular dysfunction. Along these lines, Wilson and colleagues demonstrated that ageing negatively impacts the intravascular pressure-mediated suppression of EC Ca²⁺ signalling (Wilson et al. 2017), which could in part be attributed to the reduced density of holes in the internal elastic lamina that facilitate EC-SMC communication (Boerman et al. 2016) (Fig. 2).

Elderly human subjects show diminished responses to the endothelium-dependent vasodilator acetylcholine (ACh) (Taddei et al. 2001; Kirby et al. 2009). However, experimental evidence demonstrated that muscarinic receptor-mediated Ca²⁺ signals in arterial ECs were minimally altered (Prendergast et al. 2014; Hakim et al. 2020) or even enhanced (Socha et al. 2015) during ageing. Ca²⁺ imaging in carotid arteries showed that EC sensitivity to ACh increased with age but the latency of Ca²⁺ responses significantly increased (Wilson et al. 2017). These observations collectively indicate the complexity of age-related effects on EC Ca²⁺ signals. A key target of EC Ca²⁺ signalling is the activation of the small- and intermediate-conductance Ca²⁺-activated K⁺ (SK/IK) channels. Behringer, Segal and colleagues (Behringer et al. 2013) investigated whether ageing affects SK/IK channels given their essential involvement in electrical signal transmission (Behringer & Segal 2012). Despite intact EC hyperpolarization to ACh, ageing impaired intercellular vascular electrical conduction due to an enhanced current loss (leaky conduction) attributed to enhanced SK/IK channel activation (Fig. 2) (Behringer et al. 2013). These findings highlight how ageing could negatively impact vascular function and blood flow control, despite a seemingly unaltered Ca²⁺ signalling in response to muscarinic receptor activation.

Even though ageing-triggered oxidative stress is deleterious, an intriguing finding from the Segal group indicated that advanced age was protective of ECs from the damaging Ca²⁺ signals associated with oxidative stress (Socha et al. 2015). In particular, aged ECs from skeletal muscle arteries were less responsive to hydrogen peroxide-mediated aberrant Ca²⁺ signals – while maintaining an intact or slightly enhanced Ca²⁺ response to Ach – and therefore were protected from cell death associated with oxidative stress. The authors further proposed that this protection attributed to the attenuation of EC Ca²⁺ influx, possibly via TRP channels, during ageing (Fig. 2) (Socha et al. 2015). A later study, however, did not demonstrate a clear effect of ageing on hydrogen peroxide-mediated EC Ca²⁺ signalling in cerebral arteries (Hakim et al. 2020), a clear distinction from skeletal muscle arteries (Socha et al. 2015; Norton et al. 2019). The latter observations highlight the vascular bed-specific responsiveness of endothelial Ca²⁺ signalling during ageing. Further investigations into possible alterations in Ca²⁺ influx pathways, like TRP channels, during ageing are needed. Impaired EC TRPV4-mediated Ca²⁺ signalling during ageing likely contributes to endothelial dysfunction and vascular complications (Fig. 2) (Du et al. 2016).

In summary, endothelial dysfunction is a hallmark of ageing and the associated vascular challenges. However, the impact of advanced age on EC Ca²⁺ signalling and its downstream effectors is complex and might at times appear to be subtle or vascular bed-dependent (Fig. 2, Fig. 5).

Ageing, Ca²⁺ and cell senescence

Accumulation of reactive oxygen species leads to oxidative stress and triggers cell senescence. Oxidative stress is driven by Ca²⁺ release from the ER/SR leading to the build-up of mitochondrial [Ca²⁺] and subsequent mitochondrial dysfunction (Martin & Bernard 2018). Further, cellular stressors increase Ca²⁺ entry, activate the calmodulin/calcineurin/NFAT transcription factor pathway, and lead to the expression of senescence-related genes (Martin & Bernard 2018). Ca²⁺ also activates the calpain protease leading to vascular wall fibrosis, remodelling and senescence through activation of pro-inflammatory mediators and matrix metalloproteinases (Jiang et al. 2012; Martin & Bernard 2018). Interestingly, vascular SMC senescence induced by hydrogen peroxide can be prevented by inhibiting Ca²⁺ influx using a VGCC blocker (Kim et al. 2020). In summary, aberrant vascular Ca²⁺ signals are crucially implicated in cellular senescence and may present a potential therapeutic target to slow down ageing-associated detrimental impacts of senescence on vascular function.

Ageing, epigenetic regulation and post-translational modifications

MicroRNAs (miR) are short strands (about 22 base pairs) of non-coding RNA molecules involved in epigenetic modifications and gene expression regulation via post-transcriptional silencing in many organisms and tissues (Gebert & MacRae, 2019). For instance, miR-328 regulates Ca_V1.2 channel expression in vascular SMCs. In rat mesenteric arteries, miR-328 expression increased in middle-aged rats compared with young rats along with decreased L-type channel expression. Furthermore, transient overexpression of miRNA-328 in cultured vascular SMCs suppressed L-type channel expression (Liao et al. 2017). On the other hand, miR-155 expression was reduced in the mesenteric arteries of ageing mice, a reduction concomitant with increased expression of Ca_V1.2 channels, mineralocorticoid receptors (MR), and angiotensin II type-1 receptors (AT₁-R). Restoring miR-155 expression in vascular SMCs from old mice decreased AT₁-R and Ca_V1.2 AngII- and Ca_V1.2-mediated vasoconstriction (DuPont et al. 2016). Using smooth muscle-specific MR knockout mice, it was shown that MR controlled the promotor activity of the miR-155 gene, thus establishing increased MR expression in ageing as the culprit behind epigenetic regulation of Ca²⁺ signalling downstream to increased AT₁-R and L-type channel activity in aged mouse mesenteric arteries (DuPont et al. 2016). Epigenetic regulation of smooth muscle ion channel expression – such as TRP, T-type or BK_Ca channels – therefore seems to be worth exploring using various animal models at different age stages (Fig. 4, Fig. 5). Furthermore, numerous epigenetic factors, such as histone acetylation and methylation, are involved in vascular calcification associated with ageing and VSMC senescence (Kwon et al. 2020).

In a commonly occurring Ca²⁺-dependent post-translational modification known as citrullination, Ca²⁺ is essential for the activation of peptidylarginine deiminase, an enzyme that converts basic arginine to neutral citrulline residues (Ishigami & Maruyama, 2010). Nitric oxide is synthesized in vascular ECs in a reaction catalysed by eNOS as L-arginine is being converted to L-citrulline (Palmer & Moncada, 1989). Citrullination has been implicated in cardiovascular diseases, such as atherosclerosis and heart failure (Gallart-Palau et al. 2019), and in the progression of AD in the ageing human brain (Ishigami & Maruyama, 2010). Furthermore, the high levels of serum antibodies to citrullinated proteins observed in patients with coronary artery disease warrants further study to define whether citrullination is a risk factor for atherosclerosis and heart failure (Cambridge et al. 2013). Thus, it is presumably worth exploring whether Ca²⁺-dependent citrullination is implicated in age-related deficits.

Cellular oxidative damage in the form of protein nitration, frequently seen as formation of 3-nitrotyrosine residues, is another commonly occurring post-translational modification in ageing (Tohgi et al. 1999; Beal, 2002). In relation to cellular Ca²⁺ homeostasis and signalling, nitration of the SERCA Ca²⁺ pump in the SR membrane of skeletal, cardiac and SMCs is increased by ageing (Schöneich et al. 1999; Squier & Bigelow, 2000; Bigelow 2009), and this enhanced nitration may prevent cyclic guanosine monophosphate-independent vasodilatation to nitric oxide in aortae (Cohen & Adachi 2006). Glycation of elastin residues in aortae leads to age-dependent accumulation of advanced glycation end products in the extracellular matrix, vascular calcification and increased vessel stiffness, which is the primary cause of systolic hypertension and increased pulse pressure in the elderly population (Sawabe, 2010; Ott et al. 2014; Lakatta 2015). Clearly, however, a general understanding of the specific consequences of post-translational modifications of Ca²⁺ signalling molecular players in ageing resistance vessels has not yet emerged.

Ageing and calcium-dependent structural remodelling

The Ca²⁺ extruding plasma membrane Ca²⁺ ATPase (PMCA1) has been linked to hypertension in genome-wide association studies (Levy et al. 2009; Tabara et al. 2010). Intriguingly, PMCA1 heterozygous knockout mice exhibited normal blood pressure as young and mature adults, but developed elevated blood pressure compared with age-matched wild-type mice at ≥12 months of age (Little et al. 2017). Blood pressure elevation in heterozygous PMCA1 knockout mice was preceded by an inward eutrophic remodelling with increased vascular wall thickness and an increased wall:lumen ratio from age 9 months and older (Little et al. 2017), suggesting a role for PMCA1-mediated structural remodelling in age-dependent hypertension. Along these lines, small mesenteric arteries displayed hypertrophic remodelling in middle-aged mice (Mikkelsen et al. 2016), consistent with the increased vascular SMC size noted earlier (del Corsso et al. 2006). In summary, while these observations suggest a role for PMCA and intracellular Ca²⁺ handling in structural remodelling in old arteries, this subject deserves to be addressed in various animal models of ageing and vascular beds before generalizing conclusions on the link between ageing and structural remodelling (Fig. 5).

Concluding remarks

With the ageing demographics of the population worldwide, it is of great socioeconomic value to better understand vascular ageing, which is one of the most important risk factors of cardiovascular and neurodegenerative diseases. In fact, accelerated vascular ageing is an important manifestation in several lifestyle-related diseases threatening human health. Ca²⁺ is perhaps the most important intracellular signalling molecule in vertebrate cells, and the Ca²⁺ ion is implicated in a multitude of cellular functions at all life stages. However, vascular Ca²⁺ signalling and ageing has so far received inadequate attention. Here, we have provided an overview of studies on the ageing process of small arteries and arterioles involved in blood pressure and organ blood flow regulation. Endothelial Ca²⁺ signalling in ageing is quite complex and given its central importance in blood flow control, more studies are needed at all levels of integration in order to understand the impact of ageing. In vascular SMCs, distinct changes in the expression and function of voltage-gated L- and T-type Ca²⁺ channels, TRPC6 channels and BK_Ca channels have emerged. Furthermore, a suppression of the function of transporters involved in intracellular Ca²⁺ handling is evident. The exact causes of the altered functions of plasma membrane ion channels and intracellular Ca²⁺ transporters are not well understood. However, based on recent studies we propose that epigenetic changes, post-translational modifications and Ca²⁺-induced cell senescence are major contributors to the altered vascular Ca²⁺ signalling and function in the ageing resistance vasculature (Fig. 5). Given the complexity of the signalling and the vascular changes occurring, an ‘omics’ and ‘big data’ approach to studying age-related vascular changes is recommended. One thing is for sure, we are all getting older and the sooner we understand the vascular consequences of ageing, the better we can withstand the health and economic impacts of ageing on tomorrow’s society.

Biography

Osama Harraz, PhD, is an Assistant Professor of Pharmacology at the University of Vermont. The research in his laboratory focuses on vascular ion channels and signal transduction pathways. In order to investigate how ion channels impact blood flow regulation in the brain, he employs different electrophysiological approaches, genetically engineered animal models and vascular imaging. The major goal of his research is to understand vascular signalling and the changes associated with small-vessel and neurodegenerative diseases. Lars Jørn Jensen’s laboratory studies arteriolar function in health and disease. He investigates fundamental mechanisms in the regulation of arteriolar tone involved in the control of blood pressure and organ/brain perfusion. He specializes in myogenic tone, flow-mediated vasodilatation and structural remodelling, which are key determinants of vascular function in vivo. He aims to describe new molecular mechanisms, while applying his research using rodent models of hypertension, ageing and obesity/diabetes, as well as porcine biomedical models.