• Aucun résultat trouvé

Dysregulation of vascular TRPM7 and annexin-1 is associated with endothelial dysfunction in inherited hypomagnesemia

N/A
N/A
Protected

Academic year: 2021

Partager "Dysregulation of vascular TRPM7 and annexin-1 is associated with endothelial dysfunction in inherited hypomagnesemia"

Copied!
7
0
0

Texte intégral

(1)

Associated With Endothelial Dysfunction in

Inherited Hypomagnesemia

Tamara M. Paravicini, Alvaro Yogi, Andrzej Mazur, Rhian M. Touyz

Abstract—Inadequate magnesium intake and hypomagnesemia may contribute to chronic diseases, such as hypertension.

The novel magnesium transporter TRPM7 is a critical regulator of magnesium homeostasis in vascular cells, but its role in pathophysiology is unclear. In a model of hypomagnesemia, we examined microvascular structure and function, TRPM7 expression, and vascular inflammatory status using inbred mice selected for normal-high intracellular magnesium levels or low intracellular magnesium levels (MgLs). Blood pressure was significantly increased in MgLs compared with normal-high intracellular magnesium levels. Pressurized myography of mesenteric resistance arteries showed that MgLs had significantly impaired endothelial function together with decreased plasma nitrate levels and endothelial NO synthase expression when compared with normal-high intracellular magnesium levels. Significant differences in vascular structure were also evident in both mesenteric arteries and aortas from MgLs. Aortas from MgLs had increased medial cross-sectional areas, whereas mesenteric arteries from MgLs had increased lumen diameters with increased medial cross-sectional areas, indicating outward hypertrophic remodeling. Expression of the magnesium transporter TRPM7 was significantly elevated in the vasculature of MgLs, whereas expression of a TRPM7 downstream target, the anti-inflammatory molecule annexin-1, was reduced. MgLs had increased expression of vascular cell adhesion molecule-1 and plasminogen activator inhibitor-1, indicating vascular inflammation. Taken together, these data demonstrate that the inherited magnesium status of MgLs and normal-high intracellular magnesium levels mice affects magnesium transporter expression, endothelial function, vascular structure, and inflammation. Our findings suggest a potential regulatory role for TRPM7 signaling in the maintenance of vascular integrity. Alterations in magnesium status and/or TRPM7 signaling may contribute to vascular injury in conditions associated with hypomagnesemia.

(Hypertension. 2009;53[part 2]:423-429.)

Key Words: magnesium 䡲 TRPM7 䡲 endothelial function 䡲 remodeling 䡲 hypertrophy

M

agnesium, the second most abundant intracellular cat-ion, is involved in many physiological processes reg-ulating cardiovascular function. Magnesium influences vas-cular tone, vasvas-cular smooth muscle cell growth, inflammation, ion channel activity, and the production of vasoactive agents.1 Under normal physiological conditions, magnesium levels in serum are maintained within a narrow range (0.7 to 1.1 mmol/L), and whole body magnesium balance is tightly controlled by regulating gastrointestinal absorption and renal excretion. On the other hand, in patho-logical conditions, hypomagnesemia and decreased tissue content of magnesium have been reported in various chronic diseases, such as type 2 diabetes mellitus and hypertension.2 Several studies demonstrated that, in various experimental models of hypertension, magnesium supplementation attenu-ates the increase in blood pressure and ameliorattenu-ates vascular

damage.3,4Epidemiological data indicate an inverse associa-tion between dietary magnesium intake and blood pressure in humans,5,6whereas data from the National Health and Nutri-tion ExaminaNutri-tion study suggests that many American adults have an inadequate dietary magnesium intake.7

Although magnesium is an abundant cytosolic cation important in many biological processes, little is known about the transport mechanisms that regulate its homeostasis, espe-cially in vascular cells. Transporters and exchangers that have been implicated include the Na⫹/Mg2⫹exchanger, Mg2⫹/Ca2⫹ exchanger, and, more recently, 2 novel ion channels of the transient receptor potential (TRP) cation channel superfamily, namely, TRPM6 and TRPM7, were identified as critical regulators of vertebrate intracellular magnesium levels.1 TRPM6 and TRPM7 are members of the newly described “chanzyme” family, composed of both a

magnesium-Received October 3, 2008; first decision October 21, 2008; revision accepted December 1, 2008.

From the Kidney Research Centre (T.M.P., A.Y., R.M.T.), Ottawa Health Research Institute, University of Ottawa, Ottawa, Ontario, Canada; and Institut National de la Recherche Agronomique (A.M.), Clermont Ferrand/Theix, Centre de Recherche en Nutrition Humaine, d’Auvergne, Unite´ de Nutrition Humaine, Equipe Stress Me´tabolique et Micronutriments, Saint-Gene`s-Champanelle, France.

Correspondence to Rhian M. Touyz, Kidney Research Centre, University of Ottawa/Ottawa Health Research Institute, 451 Smyth Rd, Ottawa, ON, KIH 8M5 Canada. E-mail rtouyz@uottawa.ca

© 2009 American Heart Association, Inc.

Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.108.124651

423

(2)

permeable ion channel pore and an unique carboxy-terminal ␣-kinase domain, which may signal independently of the ion channel through its recently identified substrates annexin-1, calpain, and myosin II heavy chain.8 –10TRPM6 and TRPM7 are functionally nonredundant11 and have differing tissue distribution patterns. TRPM6 is expressed primarily in the cecum and kidney, specifically, the apical membrane of distal convoluted tubule cells, where it regulates transepithelial magnesium reabsorption.12TRPM6 is considered to be cru-cial for magnesium homeostasis, with mutations in TRPM6 being the causative mutations in patients with autosomal recessive hypomagnesemia with secondary hypocalcemia.13 In contrast, TRPM7 is ubiquitously expressed, and targeted deletion of TRPM7 is lethal,14indicating its vital physiolog-ical role.

We showed that, in vascular cells, magnesium influx is driven mainly through TRPM7-sensitive pathways and that an altered cellular magnesium homeostasis and abnormal vascular smooth muscle cell function in hypertension may be, in part, related to defective TRPM7 expression/activity.15,16 To further explore the pathophysiological significance of these findings, we used a model of inherited hypomag-nesemia to investigate the effects of chronic magnesium deficiency on microvascular structure and function, magne-sium transporter (TPRM7) expression, and vascular inflam-matory status. Using bidirectional selective breeding, mice from a heterogenous population were selected for low (MgL) and normal-high (MgH) levels of erythrocyte magnesium. The MgL mice demonstrate inherited hypomagnesemia, with significant reductions in plasma, bone, and kidney magne-sium levels.17

Methods

For detailed methodology, please see the online data supplement, available at http://hyper.ahajournals.org.

Animals

Experiments in this study were approved by the University of Ottawa Animal Ethics Committee and performed according to the recom-mendations of the Canadian Council for Animal Care. Mouse colonies were selectively bred for low and normal-high erythrocyte magnesium concentrations.17 Colonies were subsequently

main-tained at the University of Ottawa and allowed standard rodent chow (Teklad Global 18% protein diet, Harland) and tap water ad libitum. Animals from matched age ranges (18⫾2 weeks) were used for this study.

Blood Pressure

Systolic blood pressure was measured via tail cuff plethysmography (BP-2000, Visitech Systems). Animals were trained to the system for 7 consecutive days, and measurements were recorded for 3 days after the acclimatization period.

Plasma and Urine Analysis

To measure electrolyte levels, spot urine and blood samples (via cardiac puncture) were collected immediately before sacrifice. Lev-els of electrolytes, Mg2⫹, Ca2⫹, and creatinine were determined using an automated analyzer (Synchron CX5 PRO, Beckman). To measure plasma nitrate, samples were filtered through 10 000 mo-lecular weight cutoff filters before analyzing with a commercially available kit (Total Nitric Oxide, Assay Designs). Albuminuria (albumin:creatinine ratio) was measured with commercially avail-able kits (Albuwell M and Creatinine Companion, Exocell).

Myography

Pressurized myography was used to measure microvascular function (contractility and endothelial function), structure (lumen diameter and medial cross-sectional area), and mechanics (distensibility). Second-order branches of mesenteric arteries (corresponding with resistance arteries) from MgLs and MgHs were cleaned of connec-tive tissue and mounted in a pressurized myograph at 45 mm Hg.

Vessel contractility was assessed by cumulative concentrationresponse curves to norepinephrine. Endotheliumdependent and -independent relaxations were assessed using acetylcholine and sodium nitroprusside, respectively. To measure microvascular struc-ture and mechanics, vessels were superfused with Ca2⫹-free physi-ological salt solution containing 1 mmol/L of EGTA to remove intrinsic tone. In response to increasing intraluminal pressure (3 to 140 mm Hg), lumen diameter and vessel thickness were measured at 3 points along the vessel and medial cross-sectional area (CSA), and distensibility, circumferential stress, and strain were calculated as described previously.18

Histology

Medial CSA, media:lumen ratio, and collagen deposition were examined in aortas from MgLs and MgHs. Aortic segments were fixed in methacarn (60% methanol, 30% chloroform, and 10% acetic acid) for 6 hours before paraffin embedding and sectioning (5␮m). Slides were stained with Sirius red and hematoxylin for the deter-mination of collagen content and measurement of the media:lumen ratio and CSA.

Analysis of TRPM7 Expression

Quantitative real-time PCR (TaqMan, Applied Biosystems) was used to measure the expression of TRPM7 mRNA in aortas from MgLs and MgHs. The expression of TRPM7 in the samples was interpo-lated from a standard curve (constructed from an independent sample of mouse kidney cDNA) and expressed relative to the housekeeping gene 18S.

Cell Culture

Endothelial cells (ECs) were isolated and cultured from MgL and MgH aortas to measure the expression of endothelial NO synthase (eNOS).

Western Blotting

Expression of eNOS, annexin-1, calpain, plasminogen activator inhibitor (PAI-1), and vascular cell adhesion molecule (VCAM) was measured by Western immunoblotting. Proteins extracted from frozen aortic tissue (20␮g) and ECs (30 ␮g) were separated using polyacrylamide gel electrophoresis (10%) and transferred to nitro-cellulose membranes. Nonspecific binding sites were blocked by incubating in 5% skim milk in Tris-buffered saline solution with Tween before incubating at 4°C overnight with primary antibodies to eNOS, calpain, annexin-1, PAI-1, or VCAM-1 (1:500, Santa Cruz) diluted in Tris-buffered saline solution with Tween with 3% BSA. After washing, membranes were incubated for 1 hour with secondary antibody (1:1000) diluted in 5% milk in Tris-buffered saline solution with Tween before the development with chemiluminescence (Pi-coSignal West, Pierce). Membranes were stripped before reprobing with an antibody for GAPDH (ECs, 1:10 000, Chemicon) or␤-actin (aortic homogenates, 1:20 000, Sigma) as an internal control. Den-sitometric analysis of the resulting bands was performed using ScionImage (National Institutes of Health).

Statistics

All of the data are presented as means⫾SEMs. Groups were compared using the unpaired t test or 2-way ANOVA as appropriate, with significance taken at P⬍0.05.

Results

Blood Pressure, Plasma, and Urine Analysis Systolic blood pressure, measured by tail-cuff plethysmogra-phy, was increased in MgLs compared with MgHs (116⫾2

(3)

versus 104⫾1 mm Hg; n⫽11 to 14; P⬍0.05). No significant differences between MgLs and MgHs were found in plasma electrolytes. In contrast, urinary magnesium (MgH: 3.46⫾0.39 versus MgL: 6.00⫾0.72 mmol Mg2⫹/mmol of creatinine; n⫽11 to 13; P⬍0.05) and potassium (MgH: 53.2⫾3.8 versus MgL: 80.4⫾5.4 mmol K⫹/mmol of creati-nine; n⫽11 to 13; P⬍0.05) concentrations were significantly increased in MgL mice compared with MgH mice. MgL mice exhibited some microalbuminuria as evidenced by the in-creased albumin:creatinine ratio (MgH: 27.2⫾6.8 versus MgL 97.9⫾22.9␮g of albumin per milligram of creatinine; n⫽6; P⬍0.05). Urinary creatinine was slightly lower in MgL mice (2.62⫾0.23 mmol/L) than in MgH mice (4.44⫾ 0.61 mmol/L).

Microvascular Function

In mesenteric arteries, maximum contractile responses and sensitivity to norepinephrine were similar in MgL and MgH mice (pEC50⫽7.10⫾0.15 in MgH versus 6.78⫾0.24 in MgL; maximum response⫽97.2⫾2.0% in MgH and 99.9⫾1.9% in MgL; n⫽6; Figure 1A). Similarly, no differences in the magnitudes of the maximal responses to high-potassium physiological salt solution were observed between MgL and MgH mice. However, arteries from MgLs showed significant impairment of endothelial function, as determined by re-sponses to the vasodilator acetylcholine (pEC50⫽6.80⫾0.12

in MgH versus 6.89⫾0.41 in MgL; maximal relax-ation⫽78.6⫾8.9% in MgH versus 37.4⫾16.2 in MgL; n⫽6; P⬍0.05; Figure 1B). This impairment was specific for endothelium-dependent relaxation, because arteries from MgL and MgH mice showed similar responsiveness to the NO donor sodium nitroprusside (pEC50⫽6.85⫾0.30 in MgH versus 6.67⫾0.46 in MgL; maximal relaxation⫽88.9⫾3.3% in MgH versus 79.0⫾7.3% in MgL; n⫽6; Figure 1C).

To further investigate the underlying mechanisms of this endothelial dysfunction, we examined plasma nitrate levels and eNOS expression. MgLs had reduced plasma nitrate levels when compared with MgHs (7.30⫾0.59 and 5.66⫾0.43 ␮mol/L in MgH and MgL, respectively; n⫽6; P⬍0.05). eNOS expression was significantly reduced in both cultured ECs and aortic homogenates from MgL mice (P⬍0.05; Figure 2).

Vascular Structure

Pressurized mesenteric arteries from MgLs had significant increases in both lumen diameter (Figure 3A) and medial CSA (Figure 3B) when compared with arteries from MgH. Both strains showed similar media:lumen ratios (Figure 3C). In response to increases in intraluminal pressure, arteries from MgL and MgH mice showed similar deformation (Figure 4A), stress-pressure (Figure 4B), and stress-strain relationships (Figure 4C). -10 -9 -8 -7 -6 -5 -4 -3 0 25 50 75 100 MgH MgL Log [ACh] (M) % r e la x a ti o n -10 -9 -8 -7 -6 -5 -4 -3 0 25 50 75 100 MgH MgL Log [SNP] (M) % r e la x a ti o n -10 -9 -8 -7 -6 -5 -4 0 25 50 75 100 MgH MgL Log [NA] (M) % K P S Sma x

A

B

C

*

Figure 1. Functional responses of pressurized mesenteric arteries from MgL and MgH mice in response to cumulative additions of (A)

norepinephrine, (B) acetylcholine, and (C) sodium nitroprusside. Data in A are expressed as a percentage of the maximum contraction to high-potassium physiological salt solution. In B and C, relaxations were measured in vessels precontracted to⬇70% of maximum with norepinephrine and responses to the vasodilator agonist expressed as a percentage of that precontraction. N⫽6; *P⬍0.05.

MgL MgH 0.00 0.25 0.50 0.75 1.00 eNOS:GAPDH

*

MgL MgH eNOS GAPDH

A

B

0.0 0.1 0.2 0.3 0.4

*

eNOS: β actin MgL MgH eNOS β actin MgL MgH

Figure 2. eNOS expression in (A)

cul-tured ECs and (B) aortic homogenates from MgL and MgH mice. Top, Repre-sentative immunoblots and (bottom) the corresponding bar graphs of the above data expressed as relative optical den-sity of the target protein normalized to GAPDH (A) and␤-actin (B). N⫽4 to 5; *P⬍0.05.

(4)

Measurement of fixed aortic sections from MgL and MgH mice showed that MgL had increased medial CSA (5.37⫾0.10 versus 6.89⫾0.61 arbitrary units squared in MgH and MgL, respectively; n⫽4 to 6; P⬍0.05; Figure 5A), whereas media:lumen ratios and luminal CSA were not significantly different between the 2 groups (media:lumen ratio: 0.60⫾0.06 and 0.68⫾0.09; luminal CSA: 8.6⫾1.1 and 10.1⫾1.2 arbitrary units squared in MgH and MgL, respec-tively; n⫽5 to 6; Figure 5B). There were no differences in aortic collagen content between MgL and MgH mice (Figure 5C and 5D).

Alterations in TRPM7, Annexin-1, PAI-1, and VCAM-1 Expression

In aortas from MgL mice, TRPM7 mRNA was significantly increased (⬇18-fold) when compared with MgH (Figure 6A). We also found that annexin-1, a TRPM7 downstream target, expression was reduced in aortas from MgL mice (expressed relative to a ␤-actin control, 0.78⫾0.09 in MgH versus 0.42⫾0.08 in MgL; n⫽4 to 5; P⬍0.05; Figure 6B), and this was associated with an increased expression of the proinflam-matory markers VCAM-1 and PAI-1 (Figure 7A and 7B). Expression of the TRPM7 target calpain also tended to be lower in MgL aortas, but this did not reach statistical significance (Figure 6C).

Discussion

Major findings from the present study demonstrate that inbred mice with intracellular magnesium deficiency have the

following: (1) increased systolic blood pressure; (2) reduced endothelial function associated with low plasma nitrate levels and eNOS expression; (3) increased lumen diameter and medial CSA, indicating outward hypertrophic remodeling; (4) increased vascular expression of the magnesium transporter TRPM7 with decreased expression of the TRPM7 substrate annexin-1; and (5) increased expression of proinflammatory markers (VCAM-1 and PAI-1). Taken together, these data identify a vascular phenotype associated with magnesium deficiency and highlight an important role for magnesium in the maintenance of vascular integrity and function.

We observed a small but significantly higher systolic blood pressure in the MgL mice. This is consistent with previous reports indicating that chronic magnesium deficiency is associated with increased blood pressure in normotensive animals.19,20It has been demonstrated that magnesium sup-plementation attenuates blood pressure increases in various models of hypertension, including the stroke-prone spontane-ously hypertensive rat,21 angiotensin II–induced hyperten-sion,22 and mineralocorticoid salt hypertension.3,23,24 Al-though the effects of magnesium on blood pressure seem to be more rapid and pronounced in disease states such as hypertension,21 our data indicate that chronic magnesium deficiency also influences blood pressure in normotensive animals.

Our data that mesenteric resistance arteries from MgL exhibit increased lumen diameter and medial CSA indicate an outward hypertrophic remodeling in these animals. This is

A

B

C

0 25 50 75 100 125 150 0.00 0.05 0.10 0.15 0.20 0.25 MgH MgL Intraluminal Pressure (mmHg) M e di a: Lum e n 0 25 50 75 100 125 150 0 25 50 75 100 125 150 175 200 225 250 275 MgH MgL

*

Intraluminal Pressure (mmHg) µ m 0 25 50 75 100 125 150 0 MgH MgL 5000 7500 10000 12500 15000

*

Pressure (mmHg) Me d ia CS A ( µ m 2 )

Figure 3. Lumen diameter (A), medial CSA (B), and media:lumen ratio (C) in response to increasing intraluminal pressure in deactivated

mesenteric arteries from MgL and MgH mice. N⫽6 to 8; *P⬍0.05.

A

B

0 25 50 75 100 125 150 0.00 0.25 0.50 0.75 1.00 MgH MgL Intraluminal Pressure (mmHg) St ra in (D/ D o ) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 0.0 0.5 1.0 1.5 2.0 2.5 MgH MgL Strain (∆D/Do) S tr ess ( x 10 6 dyn es/ cm 2)

C

0 25 50 75 100 125 150 0.0 0.5 1.0 1.5 2.0 2.5 MgH MgL Intraluminal Pressure (mmHg) St re s s ( x 1 0 6 dy nes /c m 2)

Figure 4. Distensibility of deactivated mesenteric arteries from MgL and MgH mice. A and B, Respectively, deformation (relative to

original diameter) and wall stress in response to increasing intraluminal pressure. C, Stress-strain relationship in the same ves-sels. N⫽6 to 8.

(5)

supported in part by findings that long-term magnesium deprivation is associated with increased CSA in various blood vessels (mesenteric resistance artery, common carotid artery, and aorta), although no changes in lumen diameter were observed in these studies.19,25,26Reasons for these discrepan-cies are still unclear, but one may argue that differences in the model being used (inherited hypomagnesemia versus dietary magnesium deprivation of adult animals) are likely to be a contributing factor. Alternatively, the remodeling seen in MgL mice may be a secondary response, because outward hypertrophic remodeling of the resistance vasculature has been shown to occur in both aging27 and in response to increased blood flow.28,29 Arterial remodeling is often con-sidered an adaptive process to normalize vascular wall stress and maintain tissue perfusion and is associated with structural changes in the vessel wall in response to various pathophys-iological conditions. The outward hypertrophic remodeling seen in the MgL mice may be such an adaptive response, because the stress-pressure and stress-strain response curves are similar in MgL and MgH mice. Furthermore, in our study, there were no differences in either arterial distensibility or aortic collagen deposition between MgL and MgH mice, indicating that the remodeling is occurring without overt alterations in vascular stiffness. Although we did not inves-tigate the molecular mechanisms underlying the outward hypertrophic remodeling in MgL mice we speculate that mitogen-activated protein kinases may be involved.

Mitogen-activated protein kinase signaling pathways are involved in many of the processes that contribute to vascular remodeling (eg, vascular smooth muscle cell hypertrophy, hyperplasia, and migration), and we have demonstrated previously that activation of mitogen-activated protein kinases in hyperten-sion is enhanced by magnesium deficiency.21

An intriguing finding is that endothelial function is signif-icantly impaired in MgL mice, as indicated by the impaired endothelium-dependent vasodilatation. This seems to be me-diated by a reduction in eNOS expression, although at this point we cannot rule out a role for increased reactive oxygen species production, because magnesium deficiency is known to increase oxidative stress.21,30 Studies from cultured cells support a role for magnesium in modulating the NO pathway, because it has been demonstrated that magnesium supple-mentation increases both eNOS expression and NO produc-tion in cultured cells.31Conversely, magnesium deprivation in cultured ECs inhibits cell proliferation, increases the expression of the proinflammatory markers VCAM-1 and PAI-1, and induces a senescent phenotype.31,32

Whole body magnesium balance is determined by the combination of gastrointestinal absorption and renal excre-tion, and both processes are tightly regulated to maintain magnesium homeostasis. In recent years, there has been significant progress in our understanding of the molecular mechanisms underlying magnesium transport with the dis-covery that TRPM6 and TRPM7 are critical magnesium

MgL MgH 0.0 3.5 7.0

*

Me d ia C S A (a rb it ra ry u n it s ) MgL MgH 0.00 0.25 0.50 0.75 M e di a :Lu m e n

A

B

C

D

Figure 5. Medial CSAs (A) and media:lumen ratio (B) measured in aortic sections from MgL and MgH mice. N⫽4 to 5; *P⬍0.05.

Bottom, Representative sections of MgL (C) and MgH (D) aortas stained for collagen with Sirius red.

Figure 6. Expression of aortic TRPM7 mRNA (A), annexin-1 protein (B), and calpain protein (C) in MgL and MgH mice. A, Data are

expressed as relative levels of TRPM7:18S; N⫽5. B and C, Top, Representative immunoblots and (bottom) corresponding bar graphs of the above data normalized to␤-actin. N⫽4 to 5; *P⬍0.05.

(6)

transporters. The primary mechanism underlying hypomag-nesemia in the MgL mouse is unclear but may relate to increased renal magnesium wasting.17This is supported by findings from the present study where we observed higher levels of magnesium in the urine of MgL mice. The molecular mechanisms underlying this magnesium wasting in MgL are yet to be fully elucidated; however, dysregulation of TRPM6 in both kidneys and cecum may be important.33 It is also possible that magnesium wasting and blood pressure eleva-tion may be attributed, at least in part, to renal dysfunceleva-tion in MgL, because these mice exhibited microalbuminuria (as evidenced by higher urinary albumin:creatinine ratios in MgL compared with MgH mice).

When we examined the expression of the Mg transporter TRPM7 in vascular tissue, we observed that aortas from MgL mice have a striking increase in TRPM7 mRNA levels compared with MgH mice. Previously, studies from our laboratory have demonstrated that TRPM7 is a critical regu-lator of magnesium influx and intracellular magnesium levels in vascular smooth muscle cells.15 Furthermore, TRPM7 expression can be modulated by vasoactive agents such as angiotensin II and is involved in mediating angiotensin II–induced growth of vascular smooth muscle cells.15 The increased TRPM7 expression shown in the present study may be a compensatory mechanism in response to hypo-magnesemia and may contribute to the associated vascular hypertrophy.

To date, 3 substrates for the␣-kinase domain of TRPM7 have been identified: annexin-1, calpain, and myosin II heavy chain.8 –10Annexin-1 is an endogenous modulator of inflam-mation that was originally identified as a mediator of glu-cocorticoid signaling. Anti-inflammatory actions of annexin-1 include the inhibition of the phospholipase A2/ arachadonic acid cascade and reduced neutrophil-endotheli-um interactions.34 Angiotensin II activates annexin-1 in vascular smooth muscle cells, an effect that is blunted in cells obtained from hypertensive animals.16In the present study, we demonstrated reduced expression of annexin-1 in aortas from MgL mice together with increased expression of the proinflammatory molecules VCAM-1 and PAI-1.

Interest-ingly, the expression of annexin-1 does not parallel the changes seen in TRPM7 levels, as we observed previously in the kidneys of aldosterone-infused mice.35 This disconnect may relate to the tissues under examination (renal versus vascular) or may reflect the complicated relationship between the ion channel and the kinase domain. This relationship is yet to be fully characterized, although it has been shown that magnesium is required for kinase activity,36which may be a contributing factor to the reduced levels of annexin-1 seen in the MgL mice.

In conclusion, data from the present study demonstrate that, in inherited hypomagnesemia, there is an increase in blood pressure, impaired endothelial function, outward hy-pertrophic remodeling of arteries, and vascular inflammation. We also report increased vascular TRPM7 expression and reduced annexin-1 expression. These novel findings suggest a potential regulatory role for TRPM7 in the vasculature, together with a protective effect of magnesium on vascular function and structure.

Perspectives

Inadequate magnesium intake and hypomagnesemia are widely prevalent in the community and may contribute to the pathophysiology of chronic diseases, such as hypertension. Our data demonstrate that magnesium plays an important role in the maintenance of vascular integrity, with hypomag-nesemia exerting detrimental effects on endothelial function, vascular structure, and inflammation. We also demonstrate that the magnesium transporter TRPM7 is upregulated in hypomagnesemia, which may represent a compensatory re-sponse. Additional work is required to fully understand the relationships between the complex systems responsible for the regulation of magnesium homeostasis, the role of TRPM7 in vascular signaling, and their impact on the cardiovascular system. Such research has important consequences for under-standing and estimating the clinical implications of hypomag-nesemia for cardiovascular disease.

Sources of Funding

This study was supported by grants from the Canadian Institute of Health Research and Heart and Stroke Foundation of Canada.

MgL MgH 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

*

VCA M -1 -a c ti n

A

MgL MgH VCAM-1 β-actin MgL MgH 0.0 0.1 0.2 0.3 0.4 0.5 0.6

*

PA I-1 -a c ti n

B

MgL MgH PAI-1 β-actin

Figure 7. Expression of VCAM-1 (A) and PAI-1

(B) in MgL and MgH aortas. Top, Representa-tive immunoblots and (bottom) corresponding bar graphs of the above data normalized to ␤-actin. N⫽4 to 5; *P⬍0.05.

(7)

R.M.T. is supported through a Canada Research Chair/Canadian Foundation for Innovation Award. T.M.P. was supported by post-doctoral fellowships from the Heart and Stroke Foundation of Canada and the National Heart Foundation of Australia (O 04M 1727).

Disclosures

None.

References

1. Touyz RM. Transient receptor potential melastatin 6 and 7 channels, magnesium transport, and vascular biology: implications in hypertension. Am J Physiol Heart Circ Physiol. 2008;294:H1103–H1118.

2. Barbagallo M, Dominguez LJ, Resnick LM. Magnesium metabolism in hypertension and type 2 diabetes mellitus. Am J Ther. 2007;14:375–385. 3. Laurant P, Kantelip JP, Berthelot A. Dietary magnesium supplemen-tation modifies blood pressure and cardiovascular function in mineralocorticoid-salt hypertensive rats but not in normotensive rats. J Nutr. 1995;125:830 – 841.

4. Touyz RM, Milne FJ. Magnesium supplementation attenuates, but does not prevent, development of hypertension in spontaneously hypertensive rats. Am J Hypertens. 1999;12:757–765.

5. Geleijnse JM, Witteman JC, den Breeijen JH, Hofman A, de Jong PT, Pols HA, Grobbee DE. Dietary electrolyte intake and blood pressure in older subjects: the Rotterdam Study. J Hypertens. 1996;14:737–741. 6. Joffres MR, Reed DM, Yano K. Relationship of magnesium intake and

other dietary factors to blood pressure: the Honolulu Heart Study. Am J Clin Nutr. 1987;45:469 – 475.

7. Ford ES, Mokdad AH. Dietary magnesium intake in a national sample of US adults. J Nutr. 2003;133:2879 –2882.

8. Clark K, Langeslag M, van LB, Ran L, Ryazanov AG, Figdor CG, Moolenaar WH, Jalink K, van Leeuwen FN. TRPM7, a novel regulator of actomyosin contractility and cell adhesion. EMBO J. 2006;25:290 –301. 9. Dorovkov MV, Ryazanov AG. Phosphorylation of annexin I by TRPM7

channel-kinase. J Biol Chem. 2004;279:50643–50646.

10. Su LT, Agapito MA, Li M, Simonson WT, Huttenlocher A, Habas R, Yue L, Runnels LW. TRPM7 regulates cell adhesion by controlling the calcium-dependent protease calpain. J Biol Chem. 2006;281: 11260 –11270.

11. Schmitz C, Dorovkov MV, Zhao X, Davenport BJ, Ryazanov AG, Perraud AL. The channel kinases TRPM6 and TRPM7 are functionally nonredundant. J Biol Chem. 2005;280:37763–37771.

12. Hoenderop JG, Bindels RJ. Calciotropic and magnesiotropic TRP channels. Physiology. 2008;23:32– 40.

13. Schlingmann KP, Weber S, Peters M, Niemann NL, Vitzthum H, Klingel K, Kratz M, Haddad E, Ristoff E, Dinour D, Syrrou M, Nielsen S, Sassen M, Waldegger S, Seyberth HW, Konrad M. Hypomagnesemia with sec-ondary hypocalcemia is caused by mutations in TRPM6, a new member of the TRPM gene family. Nat Genet. 2002;31:166 –170.

14. Nadler MJ, Hermosura MC, Inabe K, Perraud AL, Zhu Q, Stokes AJ, Kurosaki T, Kinet JP, Penner R, Scharenberg AM, Fleig A. LTRPC7 is a Mg.ATP-regulated divalent cation channel required for cell viability. Nature. 2001;411:590 –595.

15. He Y, Yao G, Savoia C, Touyz RM. Transient receptor potential melastatin 7 ion channels regulate magnesium homeostasis in vascular smooth muscle cells: role of angiotensin II. Circ Res. 2005;96:207–215. 16. Touyz RM, He Y, Montezano AC, Yao G, Chubanov V, Gudermann T, Callera GE. Differential regulation of transient receptor potential melastatin 6 and 7 cation channels by ANG II in vascular smooth muscle cells from spontaneously hypertensive rats. Am J Physiol Regul Integr Comp Physiol. 2006;290:R73–R78.

17. Henrotte JG, Franck G, Santarromana M, Frances H, Mouton D, Motta R. Mice selected for low and high blood magnesium levels: a new model for stress studies. Physiol Behav. 1997;61:653– 658.

18. Iglarz M, Touyz RM, Amiri F, Lavoie MF, Diep QN, Schiffrin EL. Effect of peroxisome proliferator-activated receptor-alpha and -gamma activa-tors on vascular remodeling in endothelin-dependent hypertension. Arte-rioscler Thromb Vasc Biol. 2003;23:45–51.

19. Adrian M, Chanut E, Laurant P, Gaume V, Berthelot A. A long-term moderate magnesium-deficient diet aggravates cardiovascular risks asso-ciated with aging and increases mortality in rats. J Hypertens. 2008;26: 44 –52.

20. Laurant P, Dalle M, Berthelot A, Rayssiguier Y. Time-course of the change in blood pressure level in magnesium-deficient Wistar rats. Br J Nutr. 1999;82:243–251.

21. Touyz RM, Pu Q, He G, Chen X, Yao G, Neves MF, Viel E. Effects of low dietary magnesium intake on development of hypertension in stroke-prone spontaneously hypertensive rats: role of reactive oxygen species. J Hypertens. 2002;20:2221–2232.

22. Finckenberg P, Merasto S, Louhelainen M, Lindgren L, Vapaatalo H, Muller DN, Luft FC, Mervaala EM. Magnesium supplementation prevents angiotensin II-induced myocardial damage and CTGF overex-pression. J Hypertens. 2005;23:375–380.

23. Berthon N, Laurant P, Fellmann D, Berthelot A. Effect of magnesium on mRNA expression and production of endothelin-1 in DOCA-salt hyper-tensive rats. J Cardiovasc Pharmacol. 2003;42:24 –31.

24. Kh R, Khullar M, Kashyap M, Pandhi P, Uppal R. Effect of oral mag-nesium supplementation on blood pressure, platelet aggregation and calcium handling in deoxycorticosterone acetate induced hypertension in rats. J Hypertens. 2000;18:919 –926.

25. Laurant P, Hayoz D, Brunner HR, Berthelot A. Effect of magnesium deficiency on blood pressure and mechanical properties of rat carotid artery. Hypertension. 1999;33:1105–1110.

26. Laurant P, Hayoz D, Brunner H, Berthelot A. Dietary magnesium intake can affect mechanical properties of rat carotid artery. Br J Nutr. 2000; 84:757–764.

27. Laurant P, Adrian M, Berthelot A. Effect of age on mechanical properties of rat mesenteric small arteries. Can J Physiol Pharmacol. 2004;82: 269 –275.

28. Buus CL, Pourageaud F, Fazzi GE, Janssen G, Mulvany MJ, De Mey JG. Smooth muscle cell changes during flow-related remodeling of rat mes-enteric resistance arteries. Circ Res. 2001;89:180 –186.

29. Ceiler DL, De Mey JG. Chronic N(G)-nitro-L-arginine methyl ester treatment does not prevent flow-induced remodeling in mesenteric feed arteries and arcading arterioles. Arterioscler Thromb Vasc Biol. 2000;20: 2057–2063.

30. Blache D, Devaux S, Joubert O, Loreau N, Schneider M, Durand P, Prost M, Gaume V, Adrian M, Laurant P, Berthelot A. Long-term moderate magnesium-deficient diet shows relationships between blood pressure, inflammation and oxidant stress defense in aging rats. Free Radic Biol Med. 2006;41:277–284.

31. Maier JA, Malpuech-Brugere C, Zimowska W, Rayssiguier Y, Mazur A. Low magnesium promotes endothelial cell dysfunction: implications for atherosclerosis, inflammation and thrombosis. Biochim Biophys Acta. 2004;1689:13–21.

32. Ferre S, Mazur A, Maier JA. Low-magnesium induces senescent features in cultured human endothelial cells. Magnes Res. 2007;20:66 –71. 33. Rondon LJ, Groenestege WM, Rayssiguier Y, Mazur A. Relationship

between low magnesium status and TRPM6 expression in the kidney and large intestine. Am J Physiol Regul Integr Comp Physiol. 2008;294: R2001–R2007.

34. Parente L, Solito E. Annexin 1: more than an anti-phospholipase protein. Inflamm Res. 2004;53:125–132.

35. Sontia B, Montezano AC, Paravicini T, Tabet F, Touyz RM. Downregu-lation of renal TRPM7 and increased inflammation and fibrosis in aldo-sterone-infused mice: effects of magnesium. Hypertension. 2008;51: 915–921.

36. Ryazanova LV, Dorovkov MV, Ansari A, Ryazanov AG. Character-ization of the protein kinase activity of TRPM7/ChaK1, a protein kinase fused to the transient receptor potential ion channel. J Biol Chem. 2004; 279:3708 –3716.

Figure

Figure 1. Functional responses of pressurized mesenteric arteries from MgL and MgH mice in response to cumulative additions of (A) norepinephrine, (B) acetylcholine, and (C) sodium nitroprusside
Figure 3. Lumen diameter (A), medial CSA (B), and media:lumen ratio (C) in response to increasing intraluminal pressure in deactivated mesenteric arteries from MgL and MgH mice
Figure 5. Medial CSAs (A) and media:lumen ratio (B) measured in aortic sections from MgL and MgH mice
Figure 7. Expression of VCAM-1 (A) and PAI-1 (B) in MgL and MgH aortas. Top,  Representa-tive immunoblots and (bottom) corresponding bar graphs of the above data normalized to

Références

Documents relatifs

To conclude, we hypothesise that an increased Plin2 expression in the muscle has different effects according to the age and the level of activity of the subjects: while

Although prostacyclin and EDHF may also participate in the bradykinin effects, the fact that indomethacin did not significantly affect the relaxation response to

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des

The patients with liver stiffness of ≥ 12.5 kPa, presumably having cirrhosis, had significantly higher plasma VAP-1 concentrations as compared to the patients with liver

An additional sensitivity analysis, in which participants with factors potentially associated with a BP non-dipping profile (cardiovascular drug intake, clinical hypertension,

Tryptase level (panel A), Tryptophan level (panel B), Kynurenine level (panel C) and IDO activity (panel D) in blood of mastocytosis patients with or without

neurotransmitter levels and anxiety were assessed in 6 months old PLTP − / − mice born from vitamin E-supplemented parents (‘‘E’’ group); and (ii)

The levels of most of the EET and DHET regioisomers and subsequently total EETs + DHETs levels increased during heating in the healthy subjects but did not change in