Transient Receptor Potential Melastatin 7 Cation Channel Kinase: New Player in Angiotensin II-Induced Hypertension

Abstract

Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein comprising a magnesium (Mg(2+))/cation channel and a kinase domain. We previously demonstrated that vasoactive agents regulate vascular TRPM7. Whether TRPM7 plays a role in the pathophysiology of hypertension and associated cardiovascular dysfunction is unknown. We studied TRPM7 kinase-deficient mice (TRPM7Δkinase; heterozygous for TRPM7 kinase) and wild-type (WT) mice infused with angiotensin II (Ang II; 400 ng/kg per minute, 4 weeks). TRPM7 kinase expression was lower in heart and aorta from TRPM7Δkinase versus WT mice, effects that were further reduced by Ang II infusion. Plasma Mg(2+) was lower in TRPM7Δkinase versus WT mice in basal and stimulated conditions. Ang II increased blood pressure in both strains with exaggerated responses in TRPM7Δkinase versus WT groups (P<0.05). Acetylcholine-induced vasorelaxation was reduced in Ang II-infused TRPM7Δkinase mice, an effect associated with Akt and endothelial nitric oxide synthase downregulation. Vascular cell adhesion molecule-1 expression was increased in Ang II-infused TRPM7 kinase-deficient mice. TRPM7 kinase targets, calpain, and annexin-1, were activated by Ang II in WT but not in TRPM7Δkinase mice. Echocardiographic and histopathologic analysis demonstrated cardiac hypertrophy and left ventricular dysfunction in Ang II-treated groups. In TRPM7 kinase-deficient mice, Ang II-induced cardiac functional and structural effects were amplified compared with WT counterparts. Our data demonstrate that in TRPM7Δkinase mice, Ang II-induced hypertension is exaggerated, cardiac remodeling and left ventricular dysfunction are amplified, and endothelial function is impaired. These processes are associated with hypomagnesemia, blunted TRPM7 kinase expression/signaling, endothelial nitric oxide synthase downregulation, and proinflammatory vascular responses. Our findings identify TRPM7 kinase as a novel player in Ang II-induced hypertension and associated vascular and target organ damage.

Keywords: blood pressure; downregulation; hypertension; magnesium; signal transduction.

Figure 1. TRPM7kinase expression in heart and aorta of TRPM7+/+ and TRPM7Δkinase mice

A. Validation of TRPM7kinase antibody (Epitomics; # 3828-1). TRPM7 kinase expression in HEK cells expressing TRPM7 full length protein (WT) and TRPM7 lacking the kinase domain (Δ kinase) under the control of tetracycline. Cells deficient in TRPM7kinase failed to express the protein. B. Hearts were isolated from TRPM7^+/+ and TRPM7Δkinase mice infused with vehicle or Ang II. Protein expression of TRPM7kinase was analyzed as a ratio to β-actin. Upper panels, representative immunoblots of TRPM7 and β-actin. Bar graphs represent mean ±SEM of 9–11 mice per group. *p<0.05 vs vehicle-infused TRPM7^+/+. C. TRPM7kinase expression was analyzed in aortas from vehicle and Ang II-infused mice. Panels are representative photomicrographs of paraffin-embedded aortic sections processed for immunohistochemistry of TRPM7kinase, with original magnification of ×100. Data were obtained from 5 mice/group. Stars (*) and arrows (↑) indicate the presence of TRPM7kinase in vascular media and endothelium respectively.

Figure 2. Systolic blood pressure (BP) in TRPM7^+/+ and TRPM7^+/− mice infused with Ang II

TRPM7^+/+ and TRPM7^+/− mice were infused with vehicle or Ang II (400 ng/Kg/min., osmotic minipumps; 4 weeks). BP was measured weekly for 4 weeks (W) by tail-cuff plethysmography. Line graphs represent mean ± SEM of 10–19 mice per group. *p<0.05 vs vehicle-infused counterparts: ⁺p<0.05 vs Ang II-infused TRPM7^+/+.

Figure 3. Functional responses in mesenteric arteries from Ang II-infused TRPM7^+/+ and TRPM7^+/− mice

Mesenteric arteries were isolated from TRPM7^+/+ and TRPM7^+/− mice infused with vehicle or Ang II. Concentration-response curves to cumulative concentrations of acetylcholine (Ach, 10⁻⁹- 3 × 10⁻⁵ M) (A), diethylamine NONOate (DEA-NO, 10⁻⁹- 3 × 10⁻⁵ M) (B), and norepinephrine (NE, 10⁻⁹- 3 × 10⁻⁵ M) (C) were analyzed in vessels mounted on wire myographs. Line graphs represent mean ± SEM of 7- 17 mice per group. Inset (A) shows the sensitivity to Ach expressed by pD2 values. *p<0.05 vs vehicle-infused counterparts: ⁺p<0.05 vs Ang II-infused TRPM7^+/+.

Figure 4. Mechanical properties in mesenteric resistance arteries from Ang II-infused mice

Mesenteric resistance arteries were isolated from TRPM7^+/+ and TRPM7^+/− mice infused with vehicle or Ang II. Strain (A), Stress (B), and Stress-strain relationship curves (C) were analyzed by pressure myography. Mesenteric arteries were incubated in 0 Ca²⁺⁻physiological salt solution containing 10 mmol/L EGTA and were subjected to increasing levels of intraluminal pressure. Line graphs represent mean ± SEM of 5 mice per group. *p<0.05 vs vehicle-infused counterparts; ^†p<0.05 vehicle-infused TRPM7^+/−

Figure 5. Ang II effects on expression and phosphorylation of eNOS and Akt in aorta from TRPM7^+/+ and TRPM7^+/− mice

Representative immunoblots and bar graphs of eNOS content (A), eNOS (B) and Akt (C) phosphorylation in vessels from vehicle- and Ang II-infused TRPM7^+/+ and TRPM7^+/− mice. Levels of total eNOS, phospho eNOS and phospho Akt are expressed relative to expression of β-actin. Results are presented as mean ±SEM of 8–9 mice per group. *p<0.05 vs vehicle-treated groups; + p<0.05 vs Ang II-infused TRPM7^+/+ mice.

Figure 6. Expression of TRPM7 kinase targets calpain and annexin-1 in membrane-enriched fractions of mesenteric arteries from Ang II-infused TRPM7^+/+ and TRPM7Δkinase mice

Mesenteric arteries were isolated from TRPM7^+/+ and TRPM7Δkinase mice infused with vehicle or Ang II. Protein content of calpain (A) and annexin-1 (B) was measured in membrane-enriched fractions. Data are presented as the expression of calpain or annexin-1 relative to the expression of β-actin. Upper panels, representative immunoblots of calpain, annexin-1 and α actin. Results are presented as mean ±SEM of 6–9 mice per group. *p<0.05 vs vehicle-infused TRPM7^+/+.

Figure 7. Expression of VCAM-1 and ERK1/2 in vessels from Ang II-infused TRPM7^+/+ and TRPM7Δkinase mice

Aortas were isolated from TRPM7^+/+ and TRPM7Δkinase mice infused with vehicle or Ang II. (A) Upper panels, representative immunoblots of VCAM-1 and β-actin. Data are presented as the expression of VCAM-1 relative to the expression of β-actin. (B) Phosphorylation levels of ERK1/2 are presented relative to expression of β-actin. Upper panels, representative immunoblots of phospho-ERK1/2 and β-actin. Results are presented as mean ±SEM of 5–9 mice per group. ^*p<0.05 vs vehicle infused counterparts; ⁺p<0.05 vs Ang II-infused TRPM7^+/+.

Figure 8. Possible molecular mechanisms whereby TRPM7 kinase deficiency amplifies Ang II cardiovascular and blood pressure (BP) effects

Blunted TRPM7 kinase signaling by Ang II through its G protein-coupled receptor is associated with reduced activation of TRPM7 kinase downstream targets, including Akt, calpain and annexin-1, leading to vascular and cardiac dysfunction and amplified increase in BP. These processes may also be influenced by reduced intracellular Mg²⁺, possibly due to decreased transmembrane Mg²⁺ transport as a consequence of TRPM7 kinase downregulation (as we previously demonstrated (1)). (−) denotes inhibitory effect. Dashed lines are possible pathways.