Cardioprotection by Klotho through downregulation of TRPC6 channels in the mouse heart

Abstract

Klotho is a membrane protein predominantly produced in the kidney that exerts some antiageing effects. Ageing is associated with an increased risk of heart failure; whether Klotho is cardioprotective is unknown. Here we show that Klotho-deficient mice have no baseline cardiac abnormalities but develop exaggerated pathological cardiac hypertrophy and remodelling in response to stress. Cardioprotection by Klotho in normal mice is mediated by downregulation of TRPC6 channels in the heart. We demonstrate that deletion of Trpc6 prevents stress-induced exaggerated cardiac remodelling in Klotho-deficient mice. Furthermore, mice with heart-specific overexpression of TRPC6 develop spontaneous cardiac hypertrophy and remodelling. Klotho overexpression ameliorates cardiac pathologies in these mice and improves their long-term survival. Soluble Klotho present in the systemic circulation inhibits TRPC6 currents in cardiomyocytes by blocking phosphoinositide-3-kinase-dependent exocytosis of TRPC6 channels. These results provide a new perspective on the pathogenesis of cardiomyopathies and open new avenues for treatment of the disease.

Figure 1. Klotho-deficient mice display exaggerated ISO-induced cardiac hypertrophy

(a and b) Heart weight/body weight (HW/BW) (a) and heart weight/tibia length (HW/TL) (b) ratios of wild-type (WT) and homozygous klotho-hypomorphic mice (kl/kl) treated with ISO or vehicle (PBS). Mice were fed a low-phosphate diet after weaning. At the time of study (~3 months of age), body weight of WT and kl/kl mice were not different (25.1 ± 0.76 g vs 24.2 ± 1.22 g). Also, systemic blood pressure of WT and kl/kl mice were not different (systolic BP: 122 ± 4 mmHg vs 119 ± 5 mmHg). Data were mean ± s.e.m.; n = 6 for each group. * P < 0.01 vs no ISO; # P < 0.02 between indicated groups. (c) Magnetic resonance images of WT (n = 5) and kl/kl (n = 4) mice (along the long axis of hearts) before and after ISO treatment. Scale bar, 1 cm. (d-f) Expression of ANP (d), BNP (e), andβ-MHC (f) in hearts of WT and kl/kl mice described in panel a, measured by qRT-PCR, and normalized to GAPDH. Shown are mRNA levels relative to wild-type mice without ISO treatment (which is assigned the value 1). Data were mean ± s.e.m.; n = 6 for each group. * P < 0.01 vs no ISO; # P < 0.01 between indicated groups. (g) Heart sections of mice (from panel a) were stained with Masson’s trichrome (blue is collagen). Magnification was ×200 and ×25 (insets), respectively. Yellow scale bar, 50 μm; white scale bar, 250 μm. (h) Ejection fraction of WT and kl/kl mice (from panel a) calculated based on left ventricular stroke and end-diastolic volumes measured by MRI (see Supplementary Fig. S4 for measurements). * P < 0.01 vs no ISO; # P < 0.01 between indicated groups.

Figure 2. Klotho overexpression in mice attenuates cardiac hypertrophic responses to ISO

(a) HW/BW ratios of WT (n = 9) and Klotho-overexpressing transgenic mice (KL-Tg, n = 8 each) ± ISO treatment. Mice were fed a normal phosphate diet after weaning, and studied at 3 months of age. * P < 0.01 vs no ISO; # P < 0.02 between indicated groups. (b) Representative H&E-stained heart sections of WT and KL-Tg mice described in panel a. Scale bar, 2 mm. (c and d) Expression of BNP (c) and Trpc6 (d) in hearts of WT and KL-Tg mice described above. n = 8 each group.* P < 0.01 vs no ISO; # P < 0.02 between indicated groups. * P < 0.01 vs no ISO; # P < 0.01 between indicated groups. (e and f) Serum phosphate (e) and FGF23 (f) levels of WT and KL-Tg mice before ISO treatment. n = 8 each. ns, not significant. All data are expressed as mean ± s.e.m.

Figure 3. Klotho and TPRC6 exert opposite effects on cardiac hypertrophy

(a) HW/BW ratios of WT, kl/kl, homozygous Trpc6-knockout (C6^−/−), and C6^−/−; kl/kl double mutant mice ± ISO treatment. All mice were fed a low-Pi diet after weaning and ~3 months of age (n = 6–8 for each group). * P < 0.01 vs no ISO; # P < 0.02 between indicated groups. (b and c) ISO-induced increases of expression of ANP (b) and BNP (c) in hearts of above mice. Abundance of mRNA determined by real time RT-PCR was plotted as fold-increase in ISO-treated group vs sham (ISO-untreated) group. n = 6–8 for each group. * P < 0.01 vs WT; # P < 0.01 between indicated groups. (d) Kaplan-Meier survival analysis of WT, cardiac-specific TRPC6-overexpressing transgenic mice (C6-Tg), and double transgenic mice (C6-Tg; KL-Tg) mice at ~24 months. n = 5 each group. * P < 0.01 vs WT; φ, not significant vs. either WT or C6-Tg. (e) HW/BW ratios of WT, C6-Tg, and C6-Tg; KL-Tg living mice at ~24 months. n = 5 each group. * P < 0.01 between indicated groups. (f) Changes of β-MHC, ANP and BNP expression in hearts of C6-Tg and double Tg mice vs. WT mice. mRNA abundance in the hearts of the groups in panel e were plotted as fold changes vs. WT group. n = 5 each group. * P < 0.01 vs C6-Tg. Data in a-c, e-f are expressed as mean ± s.e.m.

Figure 4. Soluble Klotho inhibits TRPC6 currents in isolated ventricular myocytes

(a) Top panel shows recording conditions. Currents were recorded in ruptured whole-cell mode. Voltage protocol consists of holding at −40 mV and repetitive descending ramp pulses (500 ms duration) from +120 mV to −120 mV. This protocol diminishes the activation of Na_v. Bath solution contains inhibitors for L-type Ca_v channel (Nifedipine, 1μM) and Na⁺-Ca²⁺ exchanger (NiCl, 3 mM). Bottom panel shows representative current-voltage (I–V) relationships of currents activated by endothelin-1 (ET1, 20 nM) in myocytes isolated from WT mice, WT mice after ISO treatment, and from C6-Tg mice. n = 7–9 each group. (b) Currents were recorded from myocytes isolated from WT, C6^−/−, and KL-Tg mice treated with or without ISO. Inward current density (current at −100 mV divided by cell capacitance; pA/pF) is shown. n = 7–9 for each group. * P < 0.01 vs. no ISO. (c) Cell capacitance of myocytes recorded described in panel a. n = 7–9 for each group. (d) Myocytes were isolated from WT mice treated with ISO and incubated with or without purified recombinant soluble Klotho (200 pM for 2h) before ruptured whole-cell recording. n = 6 each. * P < 0.01 vs. no KL. (e) Myocytes were isolated from C6-Tg mice and incubated with or without purified recombinant soluble Klotho before ruptured whole-cell recording. n = 7 each. * P < 0.01 vs. no KL. (f) Myocytes were isolated from WT mice treated with ISO and incubated with or without purified recombinant soluble Klotho before ruptured whole-cell recording. TRPC6 currents were activated by extracellular addition of a membrane-permeant DAG (1-oleoyl-2-acetyl glycerol, 50 μM). Activation of TRPC6 occurred within 1–2 min. n = 8 each. * P < 0.01 vs. no KL. Data in b-f are expressed as mean ± s.e.m.

Figure 5. Soluble Klotho decreases cell surface abundance of TRPC6

(a) Soluble Klotho (sKlotho) inhibits recombinant TRPC6 channels. HEK cells expressing hemagglutinin (HA)-tagged TRPC6 were incubated with or without purified recombinant soluble Klotho^, (KL 200 pM for 2h) before ruptured whole-cell recording. TRPC6 currents were activated by membrane-permeant DAG. Shown is inward current density at −100 mV (n = 7 for each). * P < 0.01 vs. control (no KL). In control cells without expression of recombinant TRPC6, inward current density after application of DAG was 21 ± 5 pA/pF (at −100 mV; n = 9). Note that I-V curves for recombinant TRPC6 currents shown here are more strongly double-rectifying than for native currents shown in Figure 4a. The differences may be partly due to formation of heteromultimers of TRPC6 with other TRPC members in native hearts. (b) Soluble Klotho decreases TRPC6 cell surface (labeled as “Surf”) abundance. Specific biotinylation of membrane TRPC6 was supported by lack of detection of α-tubulin in the membrane fraction. The abundance of α-tubulin (labeled as “α-Tub”) in lysates served as a loading control. Bar graph shows mean ± s.e.m. of four separate experiments. TRPC6 bands (detected by anti-HA antibody) were quantified by densitometry. * P < 0.05 vs. control (no KL). (c) Soluble Klotho, but not purified sialidase (Sial, 0.3 U/ml), inhibits TRPC6. n = 6 for each. * P < 0.01 vs. control (no KL). (d) Purified sialidase increases TRPV5 currents. Whole-cell TRPV5 currents were recorded as described. n = 6 each. (e) Tetanus toxin decreases TRPC6 currents, and prevent the inhibition by soluble Klotho. TRPC6-expressing cells were preincubated with tetanus toxin (50 nM for 3h) before soluble Klotho. n = 6–7 for each. * P < 0.01 vs. control (no KL). ns, not significant between indicated groups. (f) Role of endocytosis in inhibition of TRPC6 by Klotho. TRPC6 was coexpressed with a dominant-negative (K44A; lysine-44 to alanine mutation; DN Dyn II) or wild-type dynamin II (Wt DN II). n = 7–8 for each. * P < 0.01 vs. control (no KL). #, P < 0.02 between indicated groups. All data are expressed as mean ± s.e.m.

Figure 6. Soluble Klotho inhibits TRPC6 by blocking PI3K-dependent channel exocytosis

(a) Effect of serum deprivation on TRPC6 cell-surface abundance and the regulation by soluble Klotho. HEK cells expressing TRPC6 were cultured in serum-deprived or serum-containing media for 24 h and further incubated with soluble Klotho (200 pM) for 2 h before subjecting to biotinylation assay. The abundance of α-tubulin in lysates served as a loading control. Bar graph shows mean ± s.e.m. of four separate experiments. * P < 0.05 between indicated groups. (b) DAG-stimulated TRPC6 current density in HEK cells cultured in serum-deprived or serum-containing media and incubated with or without soluble Klotho for 2 h. n = 6–9 for each. * P < 0.01 vs. no KL. ns, not significant between indicated groups. (c) HEK cells were cultured in serum-containing or serum-free medium with or without IGF1 (10 nM) for 24 h, and incubated with or without soluble Klotho for 2 h before recording. n = 7–9 for each. * P < 0.01 vs. no KL. #, P < 0.01 between indicated groups. (d) HEK cells cultured with IGF1 in the serum-free medium for 24 h were preincubated with PI3K inhibitor wortmannin (Wmn; 50 nM) or vehicle (DMSO) for 2 h and further incubated with or without soluble Klotho for 2 h before recording. n = 8–9 for each. * P < 0.01 vs. no KL. ns, not significant between indicated groups. (e) Cardiomyocytes isolated from C6-Tg mice were preincubated with wortmannin (50 nM) or vehicle for 2 h and further incubated with or without soluble Klotho for 2 h before recording. Shown is ET1-activated TRPC6 current density (pA/pF at −100 mV). n = 8–10 for each. * P < 0.01 vs. no KL. ns, not significant between indicated groups. Note that cardiomyocytes were maintained in a serum-free solution after isolation, and that PI3K signaling cascade apparently remained active during our experiments (within 4 h of isolation). (f) No additive effects between inhibition of TRPC6 by tetanus toxin, by wortmannin, and by soluble Klotho in cardiomyocytes from WT mice after ISO treatment. * P < 0.01 vs control; ns, not significant between each two groups. All data are expressed as mean ± s.e.m.

Figure 7. Working model for Klotho-mediated inhibition of TRPC6

Normally, TRPC6 channel activity is undetectable in hearts. Stresses (such as ISO overstimulation in this study) cause an abnormal intracellular Ca²⁺ signaling, which activates calcineurin and NFAT, thereby inducing cardiac hypertrophy and remodeling, as well as Trpc6 gene expression. Upregulation of TRPC6 provides a feed-forward loop that amplifies and sustains the pathological cardiac responses. IGF1 activates PI3K to promote exocytosis of TRPC6. Soluble Klotho (sKlotho) inhibits IGF1 activation of PI3K, partly by direct interactions with the receptors. Inhibition of TRPC6 by soluble Klotho targeting at IGF1 and PI3K protects the heart from stress-induced cardiac hypertrophy. Without stress signal to upregulate TRPC6 expression, soluble Klotho has no effect on the heart at baseline.