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. You have full access to this article via your institution. Download PDF SIMILAR CONTENT BEING VIEWED BY OTHERS SOLUBLE KLOTHO PROTECTS AGAINST

GLOMERULAR INJURY THROUGH REGULATION OF ER STRESS RESPONSE Article Open access 22 February 2023 KLF9 IS ESSENTIAL FOR CARDIAC MITOCHONDRIAL HOMEOSTASIS Article 08 November 2024 TDAG51

INDUCES RENAL INTERSTITIAL FIBROSIS THROUGH MODULATION OF TGF-Β RECEPTOR 1 IN CHRONIC KIDNEY DISEASE Article Open access 08 October 2021 INTRODUCTION Klotho is an antiageing protein

predominantly produced in the kidney and several other tissues including parathyroid glands and epithelial cells of the choroids plexus1. Mice homozygous for a hypomorphic _Klotho_ allele

(_kl_/_kl_) manifest multiple ageing-related phenotypes including skin and muscle atrophy, hyperphosphatemia, osteoporosis and vascular calcification, and die prematurely at around 2–3

months of age. The full-length Klotho protein is a type-1 membrane protein with a large extracellular domain of 952 amino acids in human, a membrane-spanning segment, and a short 11 amino

acids intracellular carboxyl terminus1. Membranous Klotho associates with fibroblast growth factor (FGF) receptors to form co-receptors for the ligand FGF23, a bone-derived circulating

hormone that lowers serum phosphate levels by increasing renal phosphate excretion, suppressing 1,25-dihyroxyvitamin D synthesis, and decreasing gastrointestinal phosphate absorption2,3,4,5.

Klotho-deficient mice have severe hyperphosphatemia due to defects in the Klotho-FGF23-vitamin D regulatory axis5,6,7. This phosphate retention is pivotal for growth retardation and

premature death of Klotho-deficient mice. Dietary phosphate restriction rescues growth defects and premature death of the mice5,6,7. The notion that FGF receptor and Klotho form obligatory

co-receptors for FGF23 is supported by the demonstration that systemic injection of bioactive FGF23 decreases serum levels of phosphate in wild–type (WT) mice, but not in Klotho-deficient

mice8. The extracellular domain of Klotho is composed of two internal repeats, KL1 and KL2, each sharing amino-acid sequence homology to family 1 glycosidases1. The extracellular domain of

Klotho is shed into the systemic circulation, urine and cerebrospinal fluid9. In urine, soluble Klotho regulates several ion transporters in the apical membrane of kidney tubules10,11,12.

The physiological function of soluble Klotho present in the systemic circulation is mostly unknown. The heart responds to injury and stress signals by pathological growth and remodelling

that often progresses to heart failure and sudden death13. One key regulatory step in the development of pathological cardiac growth and remodelling is activation of calmodulin-dependent

serine–threonine protein phosphatase calcineurin by abnormal calcium signalling14. Once activated by increases in intracellular calcium, calcineurin dephosphorylates and causes nuclear

translocation of nuclear factor of activated T cells (NFAT) transcription factors, which bind the regulator regions of cardiac genes and in conjunction with other transcription factors

induce gene expression and promote hypertrophic growth and remodelling. Extracellular stimuli increase intracellular Ca2+ levels by either promoting its release from intracellular organelles

or its entry across the plasma membrane. The TRPC family channels are Ca2+-permeable cation channels expressed in the plasma membrane of many tissues including the heart15. The TRPC family

includes seven members, and is divided into two groups based on structural and functional similarities: TRPC1/4/5, which are not sensitive to diacylglycerol (DAG), and TRPC3/6/7, which are

activated by DAG. TRPC2 is not expressed in humans. Evidence indicates that Ca2+ influx through cardiac TRPC channels—including TRPC1, 3, 4, 5 and 6—is important in calcineurin signalling

and hypertrophic growth of hearts16,17,18,19,20,21,22. The expression of TRPC1, 3, 4, 5 and/or 6 is increased in hypertrophic hearts stimulated by various types or forms of stresses and

their downregulation protects against cardiac hypertrophy. Some members of TRPC family channels, such as TRPC6, contain NFAT-responsive elements in their promoters, which have a pivotal role

in amplifying and sustaining gene expression through a feed-forward circuit16. Thus, TRPC6 is an important modulator of cardiac hypertrophy and a potential target for treatment. However,

physiological function of TRPC6 in the heart and its regulation remain poorly understood, limiting therapeutic strategies for targeting the pathway. Here we show that soluble Klotho inhibits

cardiac TRPC6 channels and protects the heart against stress-induced pathological hypertrophy and remodelling. RESULTS KLOTHO DEFICIENCY AGGRAVATES PATHOLOGICAL HEART GROWTH Klotho

expression is decreased in aging, a condition associated with increased risk for heart failure23,24. We examined the role of Klotho in protecting the heart using _Klotho_-hypomorphic mice

rescued by dietary phosphate restriction. To avoid potential variations caused by strain and gender differences, we studied male mice congenic for the 129/SvJ background by backcrossing for

>6 generations. As reported previously5,6,7, dietary phosphate restriction lowered serum phosphate levels and rescued growth defects and premature death of Klotho-deficient mice

(Supplementary Figs S1 and S2a). Serum levels of sodium, potassium, chloride, calcium, magnesium and urea nitrogen were not different between WT and _kl_/_kl_ mice on a phosphate-restricted

diet (Supplementary Table S1). Phosphate restriction did not affect the growth of WT mice (Supplementary Fig. S1). _Klotho_-hypomorphic mice on low-phosphate diet remained markedly

Klotho-deficient (Supplementary Fig. S2b). To investigate the potential cardioprotective effect of Klotho, we measured heart weight indices (heart weight normalized to body weight or tibia

length) as well as the overall heart size in WT and Klotho-deficient mice. Heart weight indices (Fig. 1a) and the overall heart size measured using magnetic resonance imaging (MRI) (Fig. 1c)

were not different between WT and Klotho-deficient mice at baseline. Overstimulation by isoproterenol (ISO) induced pathological hypertrophy in WT mice as reflected by increase in heart

weight indices and the overall heart size, and these ISO-induced changes were aggravated in Klotho-deficient mice (Fig. 1a–c). ISO overstimulation is a well-accepted experimental model of

stress-induced cardiac hypertrophy25,26. Phosphate restriction itself did not alter cardiac responses to stress, as baseline and ISO-induced increases in heart mass were not different

between WT mice fed normal and phosphate-restricted diets (Supplementary Fig. S2c). Pathological cardiac hypertrophy and remodelling are also characterized by increased (re)expression of

fetal genes that are normally quiescent in adult hearts, including brain natriuretic peptide (_BNP_), atrial natriuretic peptide (_ANP_) and β-myosin heavy chain (_β_-_MHC_)13,14. Consistent

with the notion that Klotho deficiency accelerates ISO-induced pathological cardiac remodelling, the expression of cardiac fetal genes was increased by ISO in WT mice, and such increase in

gene expression was augmented in Klotho-deficient mice (Fig.1d–f). Increased expression of these cardiac fetal genes is mediated by activation of the calcineurin-NFAT pathway14. The _Trpc6_

gene contains NFAT-responsive elements in the promoter and its expression is upregulated in several human and rodent models of heart failure16,17,20. We therefore measured the expression of

_Trpc6_ in ISO-treated WT and Klotho-deficient hearts. _Trpc6_ mRNA levels were increased in WT hearts after ISO treatment (Supplementary Fig. S3a). For comparison, ISO treatment did not

alter the expression of _Trpc6_ in other tissues including the blood vessels, lung, kidney and liver. As was observed for cardiac fetal genes, ISO-induced increases in _Trpc6_ mRNA were

enhanced in Klotho-deficient relative to WT mice (Supplementary Fig. S3b). Interstitial fibrosis is another consequence of pathological cardiac hypertrophy and remodelling16. Trichrome

staining of heart sections revealed fibrosis in WT hearts after ISO treatment, and Klotho deficiency worsened ISO-induced cardiac fibrosis (Fig. 1g). In support of these results from

morphometric and gene expression studies, functional analysis of hearts using MRI showed that ISO treatment decreased the ejection fraction of WT hearts, and Klotho-deficiency markedly

aggravated the ISO-induced decline in the ejection fraction (Fig. 1h). Left ventricular end-systolic and end-diastolic volumes were markedly increased, and stroke volumes were decreased in

Klotho-deficient mice after ISO treatment (Supplementary Fig. S4a,b), indicating chamber dilatation as well as impaired contractility of the left ventricle (LV). Severe heart failure with

lung oedema developed in some Klotho-deficient mice after ISO treatment (Supplementary Fig. S4c,d). Thus, Klotho deficiency does not cause baseline cardiac abnormalities but renders the

heart more susceptible to stress-induced pathological cardiac remodelling. KLOTHO ATTENUATES STRESS-INDUCED CARDIAC HYPERTROPHY To further corroborate the above experimental data indicating

that Klotho protects the heart against stress-induced cardiac remodelling, we examined ISO-induced cardiac changes in transgenic mice that overexpress Klotho (KL-Tg). These mice live ~20–30%

longer than WT littermates, and the circulating level of soluble Klotho in transgenic mice is ~100% higher than WT (~200 pM in transgenic mice versus ~100 pM in WT mice)27. Klotho

overexpression in mice did not cause detectable changes in heart mass index and the heart size at baseline (Fig. 2a), and nor did it alter the systemic blood pressure (systolic BP: 103±7 mm 

Hg and 103±4 mm Hg, WT versus KL-Tg, _n_=4 each). Klotho overexpression yet blunted the ISO-induced cardiac hypertrophic responses (Fig. 2a). Consistent with the notion that Klotho protects

against stress-induced cardiac remodelling, Klotho overexpression did not alter _BNP_ and _Trpc6_ mRNA levels at baseline, but attenuated ISO-induced increases in _BNP_ and _Trpc6_ mRNA

expression (Fig. 2c). It has been reported that elevated serum FGF23 promotes cardiac hypertrophy28. Because Klotho and FGF23 work in the same pathway to regulate phosphate metabolism, we

measured serum phosphate and FGF23 levels. Klotho overexpression in mice did not alter serum phosphate or FGF23 levels (Fig. 2e), indicating that the cardioprotective effect of Klotho was

not mediated by serum FGF23. As Klotho overexpression mice and control WT littermates were fed normal phosphate diets, these studies also exclude the role of dietary phosphate restriction in

cardioprotection by Klotho. KLOTHO PROTECTS THE HEART BY DOWNREGULATION OF TRPC6 Next, we investigated the mechanism by which Klotho protects against stress-induced cardiac hypertrophy and

remodelling. We have observed that Klotho-deficiency aggravated ISO-induced increases in _Trpc6_ expression, and conversely Klotho overexpression attenuated the ISO-induced increases in

cardiac _Trpc6_ mRNA expression (Supplementary Fig. S3b and Fig. 2d). Inhibition of cardiac TRPC6 by gene silencing or by dominant-negative expression of mutant channels confers

cardioprotection17,20. We examined whether Klotho may protect the heart by inhibiting TRPC6 by crossing Klotho-deficient mice with global _Trpc6_-knockout mice29. Mice with global deletion

of _Trpc6_ grow normally and have no apparent defects in major organ systems at baseline30. Consistently, we found that the baseline heart mass index was not different between mice with

global deletion of _Trpc6_ and control WT littermates (Fig. 3a). Deletion of _Trpc6_ partially protected against ISO-induced cardiac remodelling, but completely prevented the exaggerated

ISO-induced cardiac hypertrophy in Klotho-deficient mice. Consistent with these results, deletion of _Trpc6_ attenuated ISO-induced increases in _ANP_ and _BNP_ mRNA, and abolished the

exaggerated ISO-induced increases in the mRNA in Klotho-deficient mice (Fig. 3b). We further investigated cardioprotection by Klotho using transgenic mice that overexpress TRPC6 in hearts.

Mice with cardiac-specific overexpression of TRPC6 develop spontaneous cardiac hypertrophy and also have heightened sensitivity to stress-induced hypertrophy16. We crossed mice with cardiac

TRPC6 overexpression with transgenic mice overexpressing Klotho to create double transgenic mice, and compared the survival rate, heart mass index and cardiac fetal gene expression of double

transgenic mice with those having cardiac overexpression of TRPC6 and with WT littermates. Compared with WT littermates at 24 months of age, cardiac TRPC6-overexpressing mice had decreased

survival and increased heart mass index and cardiac fetal gene expression without ISO treatment (Fig. 3d–f). Klotho overexpression tended to improve the survival of cardiac

TRPC6-overexpressing mice, prevented the increase in heart mass, and markedly diminished the increase in fetal gene expression induced by overexpression of TRPC6 in hearts. These studies

also support the notion that Klotho protects the heart by inhibiting cardiac TRPC6. SOLUBLE KLOTHO INHIBITS TRPC6 IN ISOLATED CARDIAC MYOCYTES Klotho is not expressed in the heart. We tested

the hypothesis that soluble Klotho present in the systemic circulation mediates the inhibition of cardiac TRPC6. TRPC6 is activated by DAG15. We examined TRPC6 channel activity in freshly

isolated ventricular myocytes by whole-cell patch-clamp recording using stimulation by endothelin-1 (ET1) to release DAG (Fig. 4a). Ventricular myocytes from WT mice without ISO treatment

showed baseline ET-1-activated TRPC-like currents presumably mediated by non-TRPC6 channels, as currents were not different between _Trpc6_-knockout mice (C6−/−) and WT littermates (Fig.

4b). ISO treatment increased TRPC6-mediated currents in WT hearts (Fig. 4a); the role of TRPC6 is supported by the facts that the increase was eliminated in _Trpc6_-knockout mice (Fig. 4b),

and identical currents were seen in TRPC6-overexpressing hearts (Fig. 4a and Supplementary Fig. S5). Cell capacitance (a measurement of surface area of cells) of ventricular myocytes was

increased in WT hearts after ISO and in TRPC6-overexpressing hearts (Fig. 4c), supporting the conclusion that myocyte hypertrophy occurred under these conditions. Klotho overexpression in

mice prevented the ISO-induced increases in currents (Fig. 4b), and acute addition of soluble Klotho to culture media decreased TRPC6-mediated currents in myocytes isolated from WT mice

after ISO treatment (Fig. 4d). Similarly, direct addition of soluble Klotho inhibited the currents in myocytes isolated from TRPC6-overexpressing mice (Fig. 4e). It is theoretically possible

that Klotho inhibits TRPC6 by decreasing the production of DAG. However, soluble Klotho decreased TRPC6 currents in cardiomyocytes directly activated by membrane-permeant DAG (Fig. 4f),

indicating that it inhibits cardiac TRPC6 channel function acting downstream of DAG. KLOTHO BLOCKS IGF AND PI3K-DEPENDENT EXOCYTOSIS OF TRPC6 Because isolated mouse cardiomyocytes cannot be

cultured continuously and the low abundance of endogenous TRPC6 in hearts, we further investigated the mechanism of regulation by soluble Klotho using HEK cells expressing recombinant TRPC6

as well as isolated cardiac myocytes. As in cardiomyocytes, soluble Klotho decreased DAG-activated TRPC6 channels in HEK cells (Fig. 5a). Soluble Klotho treatment decreased cell surface

abundance of TRPC6 measured by biotinylation assays (Fig. 5b). Soluble Klotho exhibits sialidase (Sial) activity and increases cell surface abundance of TRPV5 channels by cleaving sialic

acids in the N-glycans of channels11. The Sial activity of Klotho is not responsible for the regulation of TRPC6, as purified Sial had no effect on TRPC6 whereas it stimulated TRPV5 (Fig.

5c). The above results also indicate that soluble Klotho decreases cell surface expression of TRPC6 via a mechanism not restricted to cardiomyocytes. The decrease in cell-surface abundance

of TRPC6 by soluble Klotho may be caused by decreased exocytosis and/or increased endocytosis of the channel. Blocking exocytosis by v-SNARE inhibitor tetanus toxin31 decreased TRPC6

currents, and prevented further inhibition by soluble Klotho (Fig. 5e). Because tetanus toxin completely prevented the effect by Klotho, the major (if not the sole) action of Klotho on TRPC6

is by blocking exocytosis. Consistent with this notion, we found that blocking endocytosis using a dominant-negative dynamin did not affect the ability of Klotho to inhibit TRPC6:

Coexpression with dominant-negative dynamin increased the basal (that is, without KL) TRPC6 currents, indicating inhibition of endocytosis of channels, but soluble Klotho decreased TRPC6

currents similarly in cells expressing dominant-negative dynamin and in cell expressing the control WT dynamin (Fig. 5f). Phosphoinositide-3-kinase (PI3K)-activating growth factors increase

cell surface abundance of TRPC channels by stimulating exocytosis32. Soluble Klotho inhibits PI3K signalling by insulin and insulin-like growth factors (IGF), which contributes to the

antiaging and tumor suppression effects of Klotho27,33,34,35. We therefore tested the hypothesis that soluble Klotho inhibits TRPC6 by interfering with IGF1 activation of PI3K to promote

exocytosis of channels. To allow for studying the effect of IGF1, we first examined the effect of serum deprivation. Serum deprivation lowered TRPC6 cell surface abundance and activities,

and prevented the inhibition by soluble Klotho (Fig. 6a). The role of IGF1 is demonstrated by findings showing that physiological concentrations of IGF1 (10 nM) reproduced the effect of

serum to promote TRPC6 currents and that soluble Klotho inhibited IGF1-stimulated TRPC6 currents (Fig. 6c). Moreover, PI3K inhibitor wortmannin decreased TRPC6 currents stimulated by IGF1,

and prevented a further decrease of TRPC6 by soluble Klotho (Fig. 6d). These results support the notion that soluble Klotho inhibits IGF1 and PI3K-dependent exocytosis of TRPC6. Because

wortmannin abrogates its effect, soluble Klotho acts upstream of PI3K. Finally, we examined whether soluble Klotho regulates TRPC6 in hearts via the same mechanism. Wortmannin decreased

TRPC6 currents and prevented the inhibition by soluble Klotho in isolated cardiomyocytes (Fig. 6e). Furthermore, the effect of wortmannin to decrease cardiac TRPC6 currents and to prevent

further inhibition by soluble Klotho was reproduced by tetanus toxin, and the effects of wortmannin and tetanus toxin were not additive (Fig. 6f). Thus, tetanus toxin and wortmannin inhibit

cardiac TRPC6 via the same mechanism; that is, by blocking exocytosis of channels. Collectively, these data strongly support the hypothesis that soluble Klotho inhibits TRPC6 by blocking

PI3K-dependent exocytosis of channels (Fig. 7). DISCUSSION The data presented in this study provide compelling evidence indicating that soluble Klotho protects the heart against

stress-induced cardiac hypertrophy and remodelling. Klotho expression is decreased in aging23, thus decline in circulating soluble Klotho may contribute to age-related cardiomyopathy in

humans. One consequence of cardiac aging is increased sensitivity to stress-induced heart failure36 similar to the changes in Klotho-deficient mice we observed here. Many Klotho-mediated

aging phenotypes, such as vascular calcification, growth defects and premature death, are attributed to defects in the function of membrane Klotho as co-receptors for FGF23 and phosphate

retention3,4,5,6,7,8. Our results indicate that the cardioprotective effect of soluble Klotho is independent of FGF23 and phosphate metabolism. First, Klotho overexpression in mice confers

cardioprotection without altering serum phosphate and FGF23 levels. Second, dietary phosphate restriction normalizes serum phosphate levels of Klotho-deficient mice to the level of WT mice,

excluding hyperphosphatemia as the culprit of cardiac dysfunction in these mice. Moreover, deletion of _Trpc6_ completely prevents exaggerated stress-induced cardiac hypertrophy and

remodelling in Klotho-deficient mice, indicating that cardioprotection by Klotho is mediated by downregulation of TRPC6. TRPC6 is broadly expressed in tissues15,37. Although it is possible

that the effect of Klotho on TRPC6 in other tissues also contributes to cardioprotection, the following results indicate that Klotho inhibition of cardiac TRPC6 has a critical role in the

process. First, Klotho ameliorates cardiac hypertrophy and remodelling induced by heart-specific overexpression of TRPC6, and Klotho inhibits TRPC6 in isolated cardiomyocytes. Second,

Klotho-deficient mice have no cardiac dysfunction at baseline, but develop exaggerated cardiomyopathy in response to ISO treatment. ISO treatment causes upregulation of _Trpc6_ mRNA in the

heart, but not in other tissues that influence cardiac function, such as the blood vessels, lung, and kidney. The effect of _Trpc6_ deletion to prevent ISO-induced exaggerated cardiomyopathy

in Klotho-deficient mice is therefore most likely due to abolition of ISO-induced increases of cardiac TRPC6. Our findings also have important implications in chronic kidney disease (CKD),

a disease that affects ~10% of the general population38,39. The prevalence of cardiac hypertrophy in patients of advanced stages of CKD is estimated as high as 90%, and cardiac dysfunction

is the main cause of death for the patients39,40,41. Klotho is predominantly produced in the kidney, and circulating levels of soluble Klotho are reportedly decreased in CKD42,43. Our study

supports that decreased levels of soluble Klotho contribute to the pathogenesis of cardiac hypertrophy in CKD. Recently, Faul _et al._ reported that FGF23 stimulates cardiomyocyte growth and

increased serum FGF23 contributes to cardiac hypertrophy in CKD28. Interestingly, FGF23 appears to induce cardiac hypertrophy independently of stress factors, whereas Klotho deficiency

predisposes the heart to stress-induced pathological hypertrophy. Thus, increased FGF23 and Klotho deficiency may synergistically contribute to cardiac hypertrophy in CKD by participating at

different stages of pathogenesis. The physiological role of TRPC6 in hearts is elusive. Its function appears to be dispensable, as mice with deletion of _Trpc6_ have no apparent cardiac

dysfunction. Consistent with this observation, we found that TRPC6 channel activity is undetectable in hearts at baseline. Overstimulation by ISO leads to increase in _Trpc6_ mRNA levels and

functional TRPC6 currents in mouse hearts. Increased expression of cardiac TRPC6 has also been reported in mouse models of cardiac hypertrophy induced by calcineurin gene overexpression, by

overstimulation by neuroendocrine hormones including ET-1, phenylephrine and angiotensin II, by thoracic aortic banding pressure overload, and in human failing hearts16,17,20. Thus, soluble

Klotho protects the heart by acting on a molecule that is normally quiescent but activated during stresses. Mechanistically, we propose that IGFs such as IGF1 provide a tonic stimulation

for exocytosis of TRPC6 via PI3K, and soluble Klotho exerts a tonic inhibition to the system (model in Fig. 7). Cardiac stresses increase the intracellular Ca2+ concentration from multiple

mechanisms44. The abnormal intracellular calcium signalling in the heart activates calcineurin and NFAT to initiate fetal gene expression and pathological cardiac hypertrophy and

remodelling. TRPC6 contains NFAT-responsive elements in its promoter and is also upregulated by stress. The increased Ca2+ influx through TRPC6 causes a feed-forward cycle and further

amplifies and sustains the process. By placing a brake on the system, Klotho protects the heart. Conversely, Klotho deficiency accelerates stress-induced cardiac remodelling. Without stress

signals to upregulate TRPC6, neither Klotho deficiency nor overexpression in mice affects cardiac function at baseline. Multiple studies have reported that soluble Klotho inhibits

intracellular signalling by insulin and IGF127,33,34,35. Kurosu _et al._27 first reported that soluble Klotho inhibits insulin and IGF-mediated activation of PI3K pathway by inhibiting

activation of receptors and repressing activated receptors. This antiinsulin/IGF effect contributes to aging suppression by Klotho in mice. Wolf _et al._ further found that soluble Klotho

suppresses the growth of human breast and pancreatic cancer cells33,34. They also found that soluble Klotho coimmunoprecipitated with IGF1 receptors, and suggested that soluble Klotho

inhibits the intracellular signalling by IGF1 at least partly by direct interactions with receptors33. It was also reported that Klotho prolongs lifespan and stress resistance in _C.

elegans_ by blocking insulin and IGF-like signalling in worms35. Our results support these previous reports and extend the antiinsulin/IGF role of Klotho to cardioprotection.

Cardioprotection by Klotho may contribute to the antiaging effect of Klotho in mice. Activation of PI3K and downstream Akt signalling cascade in the heart is important for physiological

cardiac growth, but it can also lead to pathological cardiac hypertrophy13,45. Inhibition of TRPC6 by soluble Klotho may be a mechanism for preventing PI3K to cause pathological cardiac

hypertrophy in the normal heart. Interestingly, shedding of soluble Klotho from membranous Klotho is mediated by metalloproteinases ADAM 10 and 17, and insulin stimulates the shedding

through the PI3K pathway46. Whether the regulation of shedding of soluble Klotho by insulin/IGF1 has any roles in the control of cardiac functions awaits future investigation. Other TRPC

channels including TRPC1, 3, 4 and 5 are also present in the heart. Increased expression of these channels is also associated with cardiac hypertrophy induced by pathological stimuli and

downregulation confers the protection17,18,19,20. Cardiac TRPC channels are likely heteromultimers of different TRPC members15,22,23, which may partly explain why inhibition of different

TRPC channels can confer cardioprotection. The exact molecular composition and stoichiometry of TRPC channels that form heteromultimers with TRPC6 in the heart, however, remains unknown. In

this study, deletion of _Trpc6_ totally abolishes exaggerated ISO-induced cardiac hypertrophy and remodelling in Klotho-deficient mice, indicating that inhibition of TRPC6 alone is

sufficient for cardioprotection. Our study yet does not exclude the possibility that soluble Klotho also exerts inhibition on other TRPC channels that form multimers with TRPC6. Of note,

_Trpc6_ deletion only partially blunts ISO-induced hypertrophy, and other factors besides TRPC6 (such as other TRPC channels) are also involved in the hypertrophic response to ISO treatment.

Pharmacological TRPC antagonism is in development as a potential treatment of cardiac hypertrophy21,22,47. As an endogenous hormone that may extend human lifespan, soluble Klotho or its

analogues or activators48 may prove to be important therapeutic agents. The involvement of TRPC6 in multiple models of cardiac hypertrophy and heart failure16,17,20 and the ability of Klotho

to protect against cardiac hypertrophy induced by heart-specific overexpression of TRPC6 suggest that Klotho-based therapeutic strategies may be applicable to diverse cardiac diseases.

TRPC6 is also expressed in the kidney, and systemic and pulmonary vasculature, and increased TRPC6 function in these tissues leads to pathologies15,37,49,50. Klotho-based therapeutics may

also be valuable in treating TRPC6-related pathologies in other organs. One major unanswered question in the pathogenesis of cardiac hypertrophy and remodelling is how the heart

distinguishes between overwhelming intracellular calcium transients during each normal cardiac cycle and the abnormal calcium signalling induced by stress signals44. Mice with Klotho

deficiency or overexpression have no apparent cardiac abnormalities at baseline. The selective targeting to the stress-induced abnormal calcium signalling by Klotho may provide clues to

answer this question in the future. METHODS GENERAL EXPERIMENTAL PROCEDURES OF MICE _Klotho_-hypomorphic, KL-Tg, _Trpc6_-knockout, and TRPC6-Tg (line L16) mice have been described1,16,27,29.

Each mouse line was backcrossed to 129/SvJ mice for >6 generations to achieve congenic background. For dietary phosphate restriction, mice were fed a purified diet containing with 0.2%

(wt/wt) inorganic phosphate (TD-09073, Harlan Teklad, Madison, WI) from weaning at ~3 weeks of age. Normal phosphate diets contain 0.35% inorganic phosphate. All mice subjected to

experiments were males at ~3 months of age, unless otherwise specified. Blood pressure was measured in WT, _Klotho_-hypomorphic and KL-Tg mice using a tail-cuff sphygmomanometer as

previously described51. For induction of cardiac hypertrophy, ISO (2 mg kg−1 per day diluted in PBS) was injected subcutaneously to mice once per day for 10 consecutive days52. Control mice

received PBS injection. At day 11, mice were euthanized and hearts were isolated. After measurement of weight, a portion of the hearts was snap-frozen in liquid N2 and saved for RNA

isolation, and the remainder fixed and stored for histology. All animal protocols were approved by the University of Texas Southwestern Institutional Animal Care and Use Committee. REAL-TIME

QUANTITATIVE RT–PCR ANALYSIS OF MRNA RNA was extracted from heart samples with trizol (Invitrogen), reverse-transcribed into cDNA (Taqman reverse transcription reagents, Applied

Biosystems-Roche), and mRNA abundance was analysed by real-time PCR with SYBR-green (iTaq or iQ SYBR-green Supermix, BioRad). Primers: _GAPDH_, 5′-tgcaccaccaactgcttagc-3′,

5′-ggcatggactgtggtcatgag-3′; _ANP_, 5′-gccatattggagcaaatcct-3′, 5′-gcaggttcttgaaatccatca-3′; _BNP_, 5′-ccaaggcctcacaaaagaac-3′, 5′-agacccaggcagagtcagaa-3′; _β-MHC_,

5′-ttggatgagcgactcaaaaa-3′, 5′-gctccttgagcttcttctgc-3′; _Trpc6_, 5′-cgctgccaccgtatgg-3′, 5′-ccgccggtgagtcagt-3′. HISTOLOGICAL ANALYSIS Dissected hearts were rinsed in PBS and incubated in

Krebs–Henseleit solution lacking Ca2+ for 30 min, and were then fixed in 4% paraformaldehyde overnight at room temperature. Samples were dehydrated and stored in 50% ethanol, mounted in

paraffin and sectioned. Sections were then stained with haematoxylin and eosin or with Masson’s Trichrome stain. SERUM COLLECTION AND MEASUREMENT Blood was drawn from mice using

retro-orbital bleeding method. Samples were immediately centrifuged, and supernatant collected and stored. Serum phosphate and FGF23 levels were measured using a phosphate assay kit (Stanbio

labs, San Antonio, TX) and FGF23 ELISA kit (Kainos lab, Japan), respectively. CARDIAC MRI Cardiac MRI of mice was performed in the Mouse MRI Core Facility of UT Southwestern Medical Center

as previously described53. To determine the LV volume, multiple parallel slices of 1-mm thickness perpendicular to the long heart axis were imaged. The area of LV of each slice at both

end-diastolic and end-systolic phases was measured using ImageJ software. LV volume was calculated as the sum of area of all slices at either phase. Stroke volume is the difference between

end-diastolic and end-systolic volumes. Ejection fraction is the percentage of stroke volume over end-diastolic volume. ISOLATION OF CARDIAC VENTRICULAR MYOCYTE Isolation of mouse

ventricular myocytes was performed per established procedure54. Briefly, mice received heparin (100 U per mouse) and anaesthesia. Hearts were quickly removed and perfused retrograde via the

aorta with a solution containing (in mM) 113 NaCl, 4.7 KCl, 1.2 MgSO4, 0.6 KH2PO4, 0.6 Na2HPO4, 10 NaHCO3, 30 taurine, 5.5 glucose, 10 2,3-butanedione monoxime, 10 HEPES (at pH 7.4) and

followed by a solution containing in addition 1 mg ml−1 type 2 collagenase (Worthinton) and 0.1 mg ml−1 protease XIV (Sigma). The perfusion solution was maintained at 37 °C and equilibrated

with 100% O2. Thereafter, the ventricle was removed, chopped into small pieces, and further digested in the enzyme solution. After stopping enzyme digestion by adding 2.5% bovine serum

albumin and 0.1 mM CaCl2, the tissue-cell suspension was filtered through a sterilized-gauze sponge, centrifuged using a tabletop centrifuge at 50_g_ for 1 min. The resulting cell pellet was

resuspended in the stopping buffer and [Ca2+] titrated to 0.5 mM by addition of 100 mM CaCl2 stock solution in four steps over 20 min. Isolated myocytes were stored at room temperature

until use. WHOLE-CELL RECORDING For recording of isolated myocytes, cells were transferred into a perfusion chamber mounted on an inverted microscope and continually perfused at the rate of

1 ml min−1 with bath solution. Whole-cell currents were recorded under voltage-clamp using an Axopatch 200B patch-clamp amplifier (Axon instruments Inc., Foster City, CA, USA)55. Voltage

protocol consists of holding at −40 mV and repetitive descending ramp pulses from +120 to −120 mV for 500 ms applied every 10 sec. The pipette solution contained (in mM) 9.4 NaCl, 120 CsCl,

1 MgCl2, 3.5 CaCl2, 10 BAPTA, 10 HEPES, 0.2 NaGTP (pH 7.2) (calculated ionized [Ca2+]~80 nM) and the bath solution contained 140 NaCl, 5 CsCl, 1 MgCl2, 1.2 CaCl2, 10 glucose, 10 HEPES (pH

7.4). Bath solution also contained 1 μM of nifedipine and 3 mM of NiCl2 to block current flow through L-type Ca2+ channel and Na+/Ca2+ exchanger, respectively. The pipette resistance was

~2–3 MΩ when filled with the pipette solution. Whole-cell access resistance was <10 MΩ. ET-1 (20 nM) was administrated using focal application method. The distance between the tip of the

applicator and myocyte was <50 μm. Currents were low-pass filtered at 2 kHz and sampled every 0.1 ms. Data acquisition was performed using pClamp9.2 program (Axon Instrument, Inc.) and

analysis using Prism (V3.0) software (GraphPad Software, San Diego, CA, USA). For whole-cell recording of recombinant TRPC6 channels in HEK cells, the pipette and bath solution contained (in

mM) 120 Cs-aspartate (Cs-Asp), 10 CsCl, 1 MgCl2, 2 MgATP, 5 EGTA, 1.5 CaCl2 (free [Ca2+]=70 nM) and 10 CsHEPES (pH 7.2) and 140 NaCl, 5 KCl, 0.5 EGTA and 10 NaHEPES (pH 7.4), respectively.

SURFACE BIOTINYLATION ASSAY HEK cells expressing haemagglutinin (HA)-tagged TRPC6 (in 35-mm culture dish) were incubated with or without soluble Klotho, washed with 1 ml of ice-cold PBS

three times, and incubated with 1 ml of PBS containing 1.5 mg ml−1 EZ-link-NHS-SS-biotin (Thermo Scientific) for 2 h at 4 °C. After quenching with glycine-containing PBS for 20 min, cells

were lysed in a buffer (150 mm NaCl, 50 mm Tris–HCl, 5 mm EDTA, 1% Triton X-100, 0.5% deoxycholate and 0.1% SDS) containing protease inhibitor mixture for 30 min. For detection of

biotinylated proteins, lysates were precipitated by streptavidin-agarose beads (Thermo Scientific) for 2 h at 4 °C. Beads were subsequently washed three times with TBS containing 1% Triton

X-100. Biotin-labelled proteins were eluted in sample buffer, separated by SDS–PAGE, and transferred to nitrocellulose membranes for western blotting using mouse monoclonal anti-HA antibody

(Sigma-Aldrich; 1:250 dilution) or anti-α-tubulin antibody (Sigma-Aldrich; 1:500 dilution). STATISTICAL ANALYSIS Statistical comparison was made between control and experimental groups

conducted during the same time period. Each experiment was repeated at least once at separate times and with similar results. Data are presented as means±s.e.m. Statistical comparison

between two groups of data were made using two-tailed unpaired Student’s _t_-test. Multiple comparisons were determined using one-way analysis of variance followed by Tukey's multiple

comparison tests. Statistical comparison of Kaplan–Meier cumulative survival curves was made using ‘log-rank’ analysis ( http://bioinf.wehi.edu.au/software/russell/logrank/). ADDITIONAL

INFORMATION HOW TO CITE THIS ARTICLE: Xie J. _et al._ Cardioprotection by Klotho through downregulation of TRPC6 channels in the mouse heart. _Nat. Commun._ 3:1238 doi: 10.1038/ncomms2240

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coordinate growth factor and GPCR-Gq signaling. _Curr. Biol._ 21, 1979–1987 (2011). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Eric Olson for TRPC6-Tg mice,

Jyothsna Gattineni for assistance with measurements of serum FGF23 and phosphate, Masaya Takahashi and Kim Kangasniemi for cardiac MRI, and Peter Igarashi, Orson Moe and Aylin Rodan for

discussions and comments. This work was supported by NIH (DK59530, DK85726, DK79328, DK91392), the Intramural Research Program of the NIH (ZO1-ES-101684) and by a GRIP grant from Genzyme,

Inc. C.-L.H. holds the Jacob Lemann Professorship in Calcium Transport of University of Texas Southwestern Medical Center. AUTHOR INFORMATION Author notes * Seung-Kuy Cha Present address:

Present address: Department of Physiology and Institute of Life Style Medicine, Yonsei University Wonju College of Medicine, Ilsan-Dong 162, Wonju, Kangwondo 220-701, Republic of Korea, *

Seung-Kuy Cha and Sung-Wan An: These authors contributed equally to this work AUTHORS AND AFFILIATIONS * Department of Medicine, UT Southwestern Medical Center, Dallas, 75390, Texas, USA

Jian Xie, Seung-Kuy Cha, Sung-Wan An & Chou-Long Huang * Department of Pathology, UT Southwestern Medical Center, Dallas, 75390, Texas, USA Makoto Kuro-o * Laboratory of Neurobiology,

National Institute of Environmental Health Sciences, Research Triangle Park, 27709, North Carolina, USA Lutz Birnbaumer Authors * Jian Xie View author publications You can also search for

this author inPubMed Google Scholar * Seung-Kuy Cha View author publications You can also search for this author inPubMed Google Scholar * Sung-Wan An View author publications You can also

search for this author inPubMed Google Scholar * Makoto Kuro-o View author publications You can also search for this author inPubMed Google Scholar * Lutz Birnbaumer View author publications

You can also search for this author inPubMed Google Scholar * Chou-Long Huang View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS J.X.,

S.-K.C. and S.-W.A. designed the study, conducted the experiments, analysed the data, and participated in writing the paper; M.K. contributed Klotho-deficient and overexpression mice, and

analysis of Klotho expression in Klotho-deficient mice; L.B. contributed Trpc6-deleted mice. C.-L.H. supervised the entire project and wrote the final paper. All authors read, commented and

approved the paper. CORRESPONDING AUTHOR Correspondence to Chou-Long Huang. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. SUPPLEMENTARY

INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figures S1-S5 and Supplementary Table S1 (PDF 795 kb) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE

Xie, J., Cha, SK., An, SW. _et al._ Cardioprotection by Klotho through downregulation of TRPC6 channels in the mouse heart. _Nat Commun_ 3, 1238 (2012). https://doi.org/10.1038/ncomms2240

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