METHODS FOR PREDICTING AND TREATING CARDIAC DYSFUNCTION

Abstract

Ageing myocardium undergoes structural and functional changes characterized by progressive cardiomyocyte hypertrophy, interstitial fibrosis and inflammation ultimately leading to diastolic and systolic dysfunction. Whilst most focus has been placed on established risk factors such as dyslipidaemia, hypertension and obesity in accelerating cardiac ageing, a potential role for amino acids has received little attention. Here the inventors show that increased phenylalanine (PA) levels induced in vitro cytosolic oxidative stress and senescence whilst in vivo led to senile-like cardiac deterioration in young mice. Moreover, they demonstrated that hepatic PA catabolism declined with age in a p21-dependent manner, whilst p21 deficiency prevented age-related cardiac dysfunction. Finally, the inventors found that Pah cofactor BH4 reversed the age-related rise in plasma PA levels and senile cardiac alterations. These observations have immediate implications for promoting cardiac health and healthspan and suggest that phenylalanine can be used as a biomarker and biotarget of cardiac dysfunction.

Claims

1. A method of predicting whether a subject has or is at risk of having a cardiac dysfunction comprising determining the level of phenylalanine in a sample obtained from the subject wherein said level indicates whether the subject has or is at risk of having a cardiac dysfunction.

2. The method of claim 1 wherein the subject exhibits one or more risk factors for cardiac dysfunction or is a subject who does not exhibit risk factors, or is a subject who is asymptomatic for cardiac dysfunction.

3. The method of claim 1 wherein the subject is an elderly subject.

4. The method of claim 1 wherein the subject is obese.

5. The method of claim 1 wherein the subject suffers from a form of acquired and hereditary hyperphenylalaninemia.

6. The method of claim 1 which comprises a step of comparing the determined level of phenylalanine with a predetermined reference value.

7. The method of claim 6 wherein when the determined level of phenylalanine is higher than the predetermined reference value it is concluded that the subject has or is at risk of having a cardiac dysfunction.

8. The method of claim 1, wherein the cardiac dysfunction is cardiac senescence.

9. The method of claim 8, wherein the cardiac senescence is premature cardiac senescence.

10. (canceled)

11. (canceled)

12. A method of determining whether a patient achieves a response with a drug that is used for the treatment of cardiac dysfunction in a patient, comprising determining the level of phenylalanine in a sample obtained from the patient during the course of the treatment wherein an increase in said level indicates that the patient does not achieve a response or wherein a stable level or a decreased level indicates that the patient achieves a response.

13. A method of preventing or treating cardiac dysfunction in a patient in need thereof comprising administering to the patient a therapeutically effective agent that is capable of increasing the catabolism of phenylalanine, thereby lowering phenylalanine levels.

14. The method of claim 13 wherein the method is performed prophylactically to prevent cardiac dysfunction in elderly patients, and/or obese patients and/or patients who exhibit one or more risk factors for cardiac dysfunction and/or patients suffering from acquired and hereditary hyperphenylalaninemia.

15. The method of claim 11 wherein the agent is BH4.

16. A method of preventing or treating cardiac dysfunction in a subject in need thereof, comprising determining the level of phenylalanine in a sample obtained from the subject, and, when the level is higher than a previously determined reference value, administering to the subject a therapeutically effective amount of an agent that increases the catabolism of phenylalanine.

17. The method of claim 16, wherein the agent is BH4.

18. The method of claim 2 wherein the one or more risk factors include age, alcohol consumption, cigarette smoking, metabolic syndrome, obesity, diabetes/insulin resistance, hypertension, dyslipidaemia, liver disease and chronic kidney disease.

19. The method of claim 5 wherein the subject suffers from phenylketonuria (PKU).

20. The method of claim 14 wherein the one or more risk factors include age, alcohol consumption, cigarette smoking, metabolic syndrome, obesity, diabetes/insulin resistance, hypertension, dyslipidaemia, liver disease or chronic kidney disease.

21. The method of claim 14 wherein the subject suffers from phenylketonuria (PKU).

Description

FIGURES

[0050] FIG. 1. Phenylalanine (PA) induces senescence. a, Immunoblot of indicated markers of senescence in C2C12 cells treated with PA (5 mM) or vehicle with quantification (n=6/group). b, Immunoblot of p21 as a function of PA concentration with quantification (n=4/concentration). c, p21 protein levels after treatment with PA (5 mM), BH4 (10 μM) and 4-CPA (1.5 mM) with quantification as indicated (n=4/condition). d, Quantification of EdU incorporation in C2C12 cells after treatment with PA, BH4 and 4-CPA as indicated (n=4/condition). e, Cytosolic superoxide levels (with CellROX probe) in C2C12 cells as a function of PA concentration (n=4/concentration). f, Cytosolic superoxide levels as treated with PA, BH4 and 4-CPA as indicated (n=4/condition). g, Immunoblot of p21 in primary adult rat cardiomyocytes treated with PA (5 mM) and/or BH4 (10 μM) vs. vehicle. Data are presented as original immunoblot images (a-c) and mean±SEM analysed with ANOVA with Bonferroni post-hoc test (a-f); ns: non-significant, *p<0.05, **p<0.01, ***p<0.001 as indicated.

[0051] FIG. 2. p21 deficiency protects against senile molecular, structural and functional changes in the myocardium. a, Plasma PA levels in 2 and 15 month-old WT and p21-/- mice (n=9-10/group). b, Immunoblot analysis of hearts of 2- and 15-month-old WT mice (n=3/age). c, p21 colocalisation with cardiomyocyte marker troponin I and fibroblast marker vimentin, but not CD31 in aged WT hearts. d, Percentage of 4-HNE-positive area in hearts of WT and p21-/- mice at indicated ages (n=4/condition). e, Representative immunofluorescent images of Pah and 2-SC (and merge) in hearts of WT and p21-/- mice at indicated ages (n=3/condition). f, Representative immunofluorescent images of Pah and 2-SC (and merge) in human hearts of indicated ages. g, Heart weight to tibia length (HW/TL) of WT and p21-/- mice at indicated ages (n=8-11/condition). h, Quantification of myocardial interstitial fibrosis (Sirius red) of WT and p21-/- mice at indicated ages (n=4/condition). i-k, Systolic strain rate (i), dP/dt.sub.max (j) & dP/dt.sub.min (k) of WT and p21-/- mice at indicated ages (n=8-11/condition). For microscopic images in panel c, magnification: 400×, scale-bar: 50 μm, in panels e-f: 200× scale-bar: 100 μm. Data are presented as original images (b-c, e-f) or mean±SEM analysed with ANOVA with Bonferroni post-hoc test (a, d, g-k); ns: non-significant, *p<0.05, **p<0.01, ***p<0.001 as indicated.

[0052] FIG. 3. Phenylalanine (PA) is a driver of cardiac ageing. a, Schematic of treatment and evaluation protocol. b, Plasma PA levels in vehicle- or PA-treated WT mice of 6 and 12 months of age (n=8-11/condition). c, Heart weight to tibia length (HW/TL) for the same groups (n=8-11/condition). d, Representative images of hearts stained with wheat-germ agglutinin (WGA) Sirius red, vimentin for the same groups (n=4/condition). e-g, Quantification of cardiomyocyte cross-sectional area (CSA; e; n=4/group), interstitial fibrosis (Sirius red, f; n=4/group) and vimentin-positive areas (g; n=4/group) in hearts of the same animals as above. h-k, LV ejection fraction (h), systolic strain rate (i), dP/dt.sub.max (j) & dP/dt.sub.min (k; n=8-11/condition) in the same animals as above. l, Representative microscopic images of myocardial p21, Pah, Gch1, 2-SC & 4-HNE in the same animals as above (n=3-4/condition). For all microscopic images, magnification: 200×, scale-bar: 50 μm. Data are presented either as original images (d & l) or as mean±SEM analysed with ANOVA with Bonferroni post-hoc test (b-c, e-k); ns: non-significant, *p<0.05, **p<0.01, ***p<0.001 as indicated.

[0053] FIG. 4. BH4 rescues plasma PA levels and senile cardiac deterioration by enhancing hepatic PA catabolism. a, Plasma PA levels in 12.5 month-old WT mice treated with intraperitoneal BH4 or vehicle (n=8-10/group). b-f, Heart weight to tibia length (b; HW/TL), systolic strain rate (c), dP/dt.sub.max (d) & dP/dt.sub.min (e) and arterial pressures (f; n=9-10/group) in mice treated as above. g-h, Quantification of myocardial Sirius red (g) and vimentin staining (h) (n=4/group). i, Myocardial nitric oxide synthase activity in 12.5 month-old WT mice treated with intraperitoneal BH4 or vehicle (NOS; n=9-10/group). j, Immunofluorescence of p21, Pah, Gch1 and 2-SC as above (n=4/group). k, Representative Pah and Gch1 immunofluorescent images of livers in 2 and 15 month-old WT and p21-/- mice (n=3/group). l, Immunoblots of senescence markers 2- and 15-month-old WT as indicated (n=.sup.3/.sub.age). m, Hepatic phenylalanine content after subcutaneous PA administration or with increased age (n=9-10/group). n, Immunoblot of p21 from AML-12 hepatocytes as a function of PA (0-10 mM) added. o, Immunoblots of vehicle (VEH)- and Nutlin3a (NU)-treated AML-12 hepatocytes with p21 or scrambled siRNA (scr; n=3/condition). p-q, Tyrosine levels in media of AML-12 cells treated with vehicle or Nutlin3a and p21 or scrambled siRNA (p) and treatment with Nutlin3a without or with BH4 (q; n=5-6/group). r, Pah immunoblot in AML-12 cells treated with Nutlin3a+/−BH4 (n=3/group). s, Representative immunofluorescent images of liver Pah in vehicle- and BH4-treated 12.5-month-old WT mice (n=3/group). t, Hepatic PA content in 12.5-month-old WT mice treated with BH4 or vehicle (n=9-10/group). u, Standardized expression of PAH against age in biopsies from liver donors (n=33). For microscopic images, magnification: 200×, scale-bar: 50 μm. Data are presented as original images (j-l, n-o, r-s) or mean±SEM and analysed with two-tailed unpaired t-test (a-i, q, t), ANOVA with Bonferroni post-hoc test (m and p) or linear regression analysis (u); ns: non-significant, *p<0.05, **p<0.01, ***p<0.001.

EXAMPLE

Methods

Animal Husbandry

[0054] Procedures involving animals were approved by the Institutional Animal Care and Use Committee of the French National Institute of Health and Medical Research (INSERM)-Unit 955, Créteil, France (ComEth 15-001). Global p21-/- mice backcrossed to C57BL6 background for at least 10 generations (Jackson) as well as wild-type (WT) littermates were kept in individually ventilated cages in a high-health facility with 12-hour light-dark cycle, controlled temperature (20-22° C.) and humidity. Water and chow were provided ad libitum.

Cardiac Phenotyping

[0055] Male WT and p21-/- mice were followed from the age of 2 to 15 months of age. These mice were sequentially evaluated for myocardial structure and function. Animals were euthanized and tissues harvested for histology and molecular biology (at ages 2, 6, 10 and 15 months). A separate group of p21-/- mice (n=17) were allowed to age further. These mice displayed an inconspicuous phenotype with no mortality until at least 24 months (not shown).

In Vivo Drug Treatment

[0056] PA in a dose of 200 mg/kg twice a day or vehicle (1×PBS) was subcutaneously administered to 11 month-old WT mice (Janvier Labs, France) in vivo for a month. BH4 in a dose of 10 mg/kg/die 2×/die or vehicle (1×PBS with 10 mM sodium ascorbate and citric acid to pH 4.5) was intraperitoneally administered to 11 month-old WT mice in vivo over six weeks. General state of the mice (body weight & wellbeing) was closely monitored. In both cases drug treatment was completed as scheduled without incidents.

2D Transthoracic Echocardiography in Conscious Mice

[0057] Mice were trained to be grasped for transthoracic echocardiography (TTE) that was performed in non-sedated mice to avoid the cardiac depressor effect of anesthetic agents, as previously reported..sup.1 Heart rates at recordings were typically above 600 beats per minute (bpm).

[0058] Data acquisition for a single cohort was performed by a single operator (JT or ER). Images were acquired from a parasternal position at the level of the papillary muscles using a 13-MHz linear-array transducer with a digital ultrasound system (Vivid 7, GE Medical System, Horton, Norway). Left ventricular dimensions and ejection fraction, anterior and posterior wall thicknesses were serially obtained from M-mode acquisition. Relative LV wall thickness (RWT) was defined as the sum of septal and posterior wall thickness over LV end-diastolic diameter, and LV mass was determined using the uncorrected cube assumption formula (LV mass=(AWTd+LVEDD+PWTd).sup.3−(LVEDD).sup.3). Peak systolic values of radial strain rate of the anterior and posterior wall were obtained using Tissue Doppler Imaging (TDI) as previously described..sup.2 TDI loops were acquired from parasternal view with a careful alignment with the radial component of the deformation' at a mean frame rate of 514 fps and a depth of 1 cm. The Nyquist velocity limit was set at 12 cm/s. Radial strain rate analysis was performed offline using the EchoPac Software (GE Medical Systems) blindly by a single operator (GD). Peak systolic of radial strain rate was computed from a region of interest positioned in the mid anterior wall and was measured over an axial distance of 0.6 mm. The temporal smoothing filters were turned off for all measurements. Because of the inevitable respiratory variability, we averaged peak systolic of radial strain rate on 8 consecutive cardiac cycles.

Invasive In Vivo Haemodynamic Assessment of Left Ventricular Function

[0059] In vivo haemodynamic measurements were performed just before mice of indicated ages were sacrificed.' Haemodynamic evaluation was performed in mice placed on a homeothermic operating table in supine position under 1.5% isoflurane anesthesia with spontaneous breathing. A 1.4-Fr microcatheter (Millar Instruments, Houston, USA) was calibrated manually before each experiment, inserted via the right carotid artery into the aorta and subsequently advanced to the left ventricle. Data were collected after at least 10 min of baseline, using the lowest isoflurane concentration tolerated to ensure minimal cardiodepression during measurement of peak rates of isovolumetric pressure development (dP/dt.sub.max) and pressure decay (dP/dt.sub.min). The microcatheter was then withdrawn to the aorta for measurement of systolic and diastolic pressure. Data were analyzed using the IOX software (EMKA, France).

Tissue Harvesting and Processing

[0060] All mice were weighed, euthanized by cervical dislocation, followed by rapid excision of the organs of interest. The heart was cannulated through the aorta for perfusion with ice-cold 1×PBS, then blotted and weighed. The heart was cut in half perpendicular to its axis: the apical two-third was snap-frozen in liquid nitrogen, whilst the basic one-third was recannulated through the aorta, perfused with 10% formalin for histology. Hearts were kept in formalin at 4° C. for at least 24-48 hours before embedding. Snap-frozen tissues were kept at −80° C., until they were powdered using a mortar and pestle cooled with liquid nitrogen and collected as aliquots. Livers and kidneys were processed in a similar manner.

Human Heart Biopsies

[0061] Human heart biopsies (right atrial appendage) were obtained from patients undergoing elective coronary artery bypass grafting. Biopsies were obtained after approval of the ethical committee (Comité de Protection des Personnes Ile-de France VI) of Pitié-Salpêtrière Hospital, Paris and informed consent was acquired from each patient prior to the procedure..sup.4

Human Liver Data

[0062] BioMart (https://www.ncbi.nlm.nih.gov/pubmed/14707178) was used to map microarray probesets to the human assembly in Ensembl release 96. The available human liver transcriptomic data was generated in 33 non-diseased, beating heart liver donors with Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays (https://www.ncbi.nlm.nih.gov/pubmed/29554203), which had 3 probesets that mapped to PAH..sup.5 The annotations and normalized data were downloaded from NCBI Gene Expression Omnibus (accession number GSE107039). Principal components analysis in IBM SPSS Statistics v25 was used to reduce the data to a standardized expression of PAH, which was regressed as a dependent variable against donor age in GraphPad Prism v8.

Expression Profiling Across Human Tissues

[0063] The expression data from the Genotype-Tissue Expression Project (http://gtexportal.org/, GTEx) (https://www.ncbi.nlm.nih.gov/pubmed/23715323) reflects 16,000 samples taken from multiple tissues across 752 donors (65% male, age 20-79) within 24 hours of death and quantified by RNAseq. The median TPM by tissue analysis V7 was downloaded, underwent log 10 transformation, and visualized with Java TreeView (https://www.ncbi.nlm.nih.gov/pubmed/15180930)..sup.6,7

Senescence-Associated β-galactosidase Staining

[0064] Senescence-associated β-galactosidase activity was used to estimate global cardiac senescence. Briefly, a section of freshly harvested hearts was incubated for 1 hour at 37° C. in β-galactosidase staining solution containing 1 mg/ml X-Gal (Sigma), 40 mM citric acid, 150 mM NaCl, 2 mM MgCl.sub.2, 5 mM potassium ferrocyanide and 5 mM potassium ferricyanide with the pH adjusted to 6.0. Stained sections were then scanned.

Isolation and Culture of Ventricular Primary Adult Rat Cardiomyocytes

[0065] Male Spargue Dawley rats (9 weeks, 300-350 g) were anesthetised with ketamine and xylazine (100 and 10 mg/kg, respectively) with heparin added (100 UI/kg). Hearts were excised and retrogradely perfused with an oxygenated (95% CO.sub.2, 5% O.sub.2) perfusion buffer consisting of NaCl 113 mM, KCl 4.7 mM, KH.sub.2PO.sub.4 0.6 mM, Na.sub.2HPO.sub.4 0.6 mM, MgSO.sub.4-7H.sub.2O 1.2 mM, NaHCO.sub.3 12 mM, KHCO.sub.3 10 mM, HEPES 10 mM, Taurine 30 mM, phenol red 0.032 mM, D-glucose 5.5 mM, 2,3-butanedionemonoxime 10 mM, pH 7.4) for 2 minutes to wash out the blood from the coronary arteries. Then hearts were perfused with a digestion buffer (perfusion buffer supplemented with 0.1 mg/mL liberase, 0.14 mg/mL trypsine-EDTA and 12.5 μM Ca.sup.2+) for 10-12 minutes. Hearts were then placed in a stopping buffer (perfusion buffer supplemented with 10% NBCS and 12.5 μM Ca.sup.2+). Atria and right ventricles were removed. Left ventricles were dissected into small fragments, then subjected to successive aspirations-reflux. Digested ventricles were filtered through a 250 μm cell strainer. After 10 minutes of incubation at 37° C., supernatants were discarded and cells were resuspended with a calcium buffer (perfusion buffer supplemented with 5% NBCS, 12.5 μM Ca.sup.2+). Extracellular calcium was added incrementally up to 1 mM. Finally, cells were suspended in culture medium M199 (supplemented with 1% ITS), seeded on wells pre-coated with 10 μg/mL laminin and allowed to attach for 2 h before starting treatments. Cardiomyocytes were subsequently treated with 5 mM PA with or without BH4 against vehicle. After a 4-hour incubation cardiomyocytes were snap-frozen and transferred to −80° C. for protein work conducted a few days later. At the time of harvesting rod shape, the presence of cross-striations and the absence of vesicles on their surface were visually confirmed to insure viable status of cardiomyocytes.

RNA Work and Quantitative RT-PCR

[0066] Total RNA from cells or powdered tissue was extracted using the RNeasy mini kit or RNeasy mini fibrous tissue kit (QIAGEN), respectively, as previously described..sup.4 RNA yield was measured with Nanodrop ND-1000, a >1.9 260/280 ratio was accepted. For reverse transcription the High Capacity cDNA kit was used according to the manufacturer's instructions (Applied Biosystems). Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to quantify relative levels of genes of interest. Briefly, amplification reactions were carried out using Fast Universal Master Mix on a StepOnePlus system (Applied Biosystems) in a multiplexed design (FAM: gene of interest; VIC: β-actin as endogenous control) in a reaction volume of 10 μl. All Taqman oligos used were inventoried Taqman-MGB oligos from Applied Biosystems. Relative expression was quantified using the ΔC.sub.T method with the formula RQ=2.sup.−ΔΔCT (User Bulletin #2; Applied Biosystems)..sup.4

Protein Extraction and Western Blotting

[0067] Cells were washed briefly with ice-cold 1×PBS, scraped in 400 μL T-PER lysis buffer (ThermoFisher) supplemented with 0.1 mM phenylmethylsulphonyl fluoride (PMSF) and protease/phosphatase inhibitor cocktail tablet (ThermoFisher). Pulverised tissue samples (20-30 mg) were lysed in the same buffer accelerated by sonication and passing through a 21 G needle. To pellet any debris, lysates were centrifuged at 10,000 g for 10 minutes at 4° C., with the supernatant transferred to fresh tubes. Protein concentrations were determined with Bradford assay (Biorad) and lysates were diluted to equal concentration with lysis buffer. Protein lysates were then mixed with Laemmli buffer, vortexed and heated to 95° C. for 5 min. Equal amounts of protein lysates in parallel with protein molecular weight marker (ThermoFisher) were loaded onto pre-cast polyacrylamide gels (Nupage 10 or 12% Bis Tris gel, Novex, Invitrogen) and separated by electrophoresis at 200V for 1 hour. Proteins were transferred onto polyvinylidene difluoride (PVDF) membrane (Invitrogen) using an electrophoretic transfer cell (Mini Trans-Blot, Bio-Rad) in ice-cold transfer buffer (48 mM Tris base, 390 mM glycine, 0.1% SDS, 20% methanol (v/v)) at 300 mA for 2 hours. Membranes were blocked in 1× skimmed milk (ThermoFisher) for 1 hour at room temperature, followed by overnight incubation with primary antibody diluted in blocking buffer at 4° C. The antibodies and concentrations used are shown in Table 2. Next day membranes were washed, followed by 60 minutes incubation in the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody diluted 1:2000-10000 in blocking buffer (all Abcam). Blots were developed using enhanced chemiluminescence (ECL) reagents (normal, Prime & Select) and digital images were acquired using an imaging system (Azure Biosystems). Membranes were usually stripped of antibodies and reprobed with an antibody targeting a protein serving as loading control (α-actinin for hearts & β-actin for all others). For this procedure, membranes were placed in Guanidine-HCl-based stripping solution (6M Guanidine-HCl, 0.2% Nonidet P-40, 0.1M β-mercaptoethanol, 20 mM Tris-HCl, pH 7.5 and 100 mM 2-mercaptoethanol) and incubated for 10 minutes at room temperature with gentle agitation, followed by extensive washing and re-blocking..sup.5

TABLE-US-00001 TABLE 1 Primary antibodies used in the study. Target Application/ protein Producer/Cat# Species concentration p21 Abcam: ab188224 rabbit WB: 1:1000 p21 Abcam: ab80633 mouse IF: 1:200 p21 Santa Cruz: sc-397 rabbit or IF: 1:50 goat p-p53 Cell Signaling: 9284S rabbit WB: 1:1000 p53 Cell Signaling: 2524S mouse IF: 1:200 p16 Abbiotec: 250804 rabbit IF: 1:100, WB: 1:100 β-actin Abcam: ab49900 mouse- WB: 1:10000 HRP Pah Sigma: SAB250434 goat IF: 100, WB: 1:200 Pah Abcam: ab148430 rabbit IHC: 1:200, IF: 1:100, WB: 1:200-1000 Gch1 Bioss antibodies: rabbit IF: 100, WB: 1:200- bs-0136R 1000 2-SC Cambridge BioSciences: rabbit IHC/IF: 1:100, WB: crb2005017e 1:200 Nrf2 Genetex: GTX103322 rabbit WB: 1:500 4-HNE Millipore: AB5605 goat IHC: 1:200 vimentin Abcam: ab92547 rabbit IF: 1:200 α-actinin Abcam: ab68167 rabbit WB: 1:5000 CD31 Santa Cruz: sc-18916 rat IF: 1:200 cardiac Abcam: ab47003 rabbit IF: 1:200 troponin I IHC: immunohistochemistry, IF: immunofluorescence, WB: Western blot.

Histology

[0068] Formalin-fixed organs were embedded in paraffin. Cross-sections were cut into 7 μm thickness using a rotary microtome (Leica). To assess cardiac fibrosis, Sirius red staining was performed, followed by dehydration and mounting with Eukitt quick-hardening mounting medium (Sigma). To visualise cardiomyocyte hypertrophy, sections were incubated in 2 μg/mL Texas red-conjugated wheat germ agglutinin (WGA; Invitrogen) in 1×PBS at room temperature for 45 minutes. Slides were then mounted with fluorescent mounting media (Abcam). Images were acquired using a Zeiss fluorescent microscope. Whole-mount preparations were made of hearts and livers of 2- and 15-month-old p21-mCherry reporter mice. Briefly, organs were quickly removed and 1-mm-thick sections were cut with a custom-made chamber equipped with razor blades. Subsequently, sections were laid out on a slide, soaked in 1×PBS and mounted with a cover slip for fluorescent microscopic evaluation.

Immunohistochemistry/Immunofluorescence

[0069] Whenever a dispersed pattern was expected, immunohistochemistry was performed on paraffin-embedded sections. Briefly, after rehydration, citrate buffer-assisted, heat-mediated antigen retrieval and blocking, sections were incubated overnight at +4° C. with primary antibodies raised against the following targets: 4-HNE (Millipore; goat, 1:200), Pah (Abcam; rabbit, 1:200) or 2-SC (Cambridge Biosciences; rabbit, 1:100; also see Table 1). Next day sections were washed, then incubated with the corresponding HRP-conjugated secondary antibodies (Abcam; 1:200) for 30 minutes at room temperature, washed again, followed by incubation with 3,3′-diaminobenzidine (DAB; Sigma) under visual observation until the signal appeared. Then slides were either counterstained with haematoxylin or not, dehydrated and mounted.

[0070] Immunofluorescence was used for higher sensitivity and co-localisation of proteins of interest. Briefly, paraffin-embedded sections were rehydrated, followed by antigen retrieval with citrate-assisted heat. Sections were then blocked with 30% goat serum (using antibody dilution solution with background reducing components; Dako) or Bloxall™ artificial blocking agent (Dako) and incubated overnight at +4° C. with primary antibodies raised against target proteins (see Table 1 for details). The following day sections were washed in 1×TBST, incubated with a mixture of the corresponding Alexa Fluor 555-labeled secondary antibodies (Invitrogen) and Alexa-488-conjugated Phalloidin (to counterstain cardiomyocytes) for 30 minutes at room temperature, and washed again. In case of co-labelling Alexa Fluor 555-labelled and Alexa Fluor 647-labeled secondary antibodies were used (Invitrogen), occasionally in combination with the avidin/biotin enhancement system (Dako). Specific protocols are available upon request. Slides were mounted with fluorescent mounting media with DAPI (4′,6-diamidino-2-phenylindole; Abcam). Images were obtained with a Zeiss confocal microscope.

Cell Culture

[0071] C2C12 (ATCC® CRL-1772™) myoblasts maintained in DMEM supplemented with L-glutamine (2 mM), penicillin/streptomycin (1%) and 5% fetal bovine serum (FBS) were seeded either at a density of 5×10.sup.4 cells (<50%) per well in 24-well plates or 10.sup.4 cells in 4-well chamber slides. Cells were with L-phenylalanine (1-10 mM)+BH4 (10 μM) and incubation with increasing concentrations of N-acetylcysteine (0.5, 2.5, 5 mM) vs. vehicle overnight as indicated. Cells were analysed for oxidative stress with MitoSOX™ (mitochondrial superoxide; 5 μM, ThermoFisher Scientific), CellROX™ (cytosolic superoxide; 5 μM, ThermoFisher Scientific), gene or protein expression, or various metabolites (see below).

[0072] AML12 cells (ATCC® CRL-2254™) were maintained in DMEM/F-12 (1:1) culture media supplemented with 10% FBS, 10 μg/ml insulin, 5.5 μg/ml transferrin, 5 ng/ml selenium, 40 ng/ml dexamethasone and 1% penicillin/streptomycin. Cells were sub-cultivated from subconfluent flasks with a ratio from 1:4 to 1:8. For WB analysis, cells were plated on 6-well plates and harvested at 80-90% of confluence. AML12 cells were transfected with indicated siRNA (Table 2) at 80% confluence using Lipofectamine RNAiMAX Transfection Reagent (ThermoFischer Scientific 13778030) following the manufacturer's instructions. Transfection efficiency was constantly checked using BLOCK-IT Alexa Fluor Red Fluorescent Control (ThermoFischer Scientific 14750100).

[0073] All cell culture experiments were performed in biological triplicates at the least.

TABLE-US-00002 TABLE 2 siRNA used for transfection experiments. Target mRNA Producer/Cat# Cdkn1a (p21) ThermoFischer Scientific: 60538 Scrambled ThermoFischer Scientific: 12935112

Determination of Myocardial Nitric Oxide Synthase Activity

[0074] Myocardial nitric oxide synthase (NOS) activity was determined in hearts of 12.5-month-old WT mice treated with BH4 or vehicle using a commercial kit (Biovision). Briefly, pulverized heart aliquots were processed according to the manufacturer's instructions and fluorometric readouts (excitation: 360, emission: 450) were normalised to protein concentration determined by Bradford assay. Activity of recombinant NOS protein served as positive control.

Determination of Metabolite Levels

[0075] Levels of reduced glutathione (GSH; ThermoFisher), phenylalanine (BioVision) and tyrosine (BioVision) were determined from cells or plasma, with respective agents/kits following the manufacturer's recommendations. Specifically, GSH levels were estimated with the aid of ThiolTracker™ Violet fluorescent probe (ThermoFisher). Plasma and liver phenylalanine levels were determined using an enzyme-coupled, fluorometric method (BioVision). Briefly, samples were deproteinized via trichloroacetate precipitation, neutralised, tyrosinase-treated (to remove tyrosine that may interfere) and diluted to fit in the standard curve. Fluorescence reading took place at 587 nm after excitation at 535 nm. Levels of tyrosine in cells or conditioned media were determined after deproteinization using 10 kDa cut-off columns with an enzyme-coupled, colorimetric method with reading at 491 nm (BioVision).

Data Analysis and Statistics

[0076] Mice were randomly assigned to experimental groups and data were acquired and analysed blind to genotype, age or treatment. Statistical analyses were performed using GraphPad Prism Software (version 6). In all cases n numbers were raised to obtain Gaussian distribution and parametric tests were used. Accordingly, Student's t-test was used to compare two groups, whilst more than two groups were compared using one-way analysis of variance (ANOVA), with Bonferroni post-hoc test for multiple comparisons. Two-way ANOVA was used to compare groups with time-dependent evolution of readouts, with Bonferroni post-hoc test for more than two groups. Data are presented as mean±standard error of the mean. Annotations used: *p<0.05, **p<0.01, ***p<0.001 compared to groups indicated. A p value of <0.05 was considered significant.

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Results and Discussion

[0086] To assess the role of PA in cellular senescence in vitro, we treated C2C12 myoblasts with PA.sup.9. PA selectively induced cyclin-dependent kinase 1a (Cdkn1a=p21) and suppressed EdU incorporation, whilst other markers of senescence (Cdkn2a=p16 and phospho-p53=p-p53) remained unaltered (FIG. 1a-d). BH4 antagonised PA-dependent senescence, which was reversed by PA analogue 4-chlorophenylalanine (4-CPA; FIG. 1c-d). BH4 stimulated PA catabolism, as evidenced by increased cellular levels of Pah, tyrosine and 2-succinylcysteine (2-SC; data not shown). BH4-dependent succination, a spontaneous posttranslational modification between PA catabolite fumarate and cysteine, came along with activation of the succination-sensitive Nrf2-antioxidant pathway (data not shown)..sup.10 Interestingly, PA incrementally increased cytosolic, but not mitochondrial superoxide levels (FIG. 1e & data not shown). BH4 normalized superoxide levels, a rescue prevented by 4-CPA (FIG. 1f). This raised the question whether PA induces senescence through oxidative stress..sup.11 To resolve this matter, we used the antioxidant N-acetylcysteine (NAC), which like BH4, restored normal cytosolic superoxide and reduced glutathione (GSH; data not shown) levels, yet failed to prevent p21 induction (data not shown), suggesting an oxidative stress-independent role for PA in triggering cellular senescence. Finally, we confirmed the selective induction of p21 protein (but not that of p53 and p16) and its rescue by BH4 in primary adult rat cardiomyocytes (FIG. 1g). Collectively, our experiments establish the link between PA and cellular senescence by p21 induction, motivating in vivo studies.

[0087] Based on our in vitro findings showing PA activating p21 together with reports indicating that p21 deficiency improves lifespan.sup.12, we focused our efforts on p21-/- mice. As reported in humans,.sup.13, 14 plasma PA levels increased in wild-type mice (WT) with age (2 vs. 15 months, an age with senile cardiac alterations),.sup.2, 3 whilst p21-/- mice were protected from senile rise in plasma PA levels (FIG. 2a).

[0088] With chronological ageing, p21 and senescence-associated β-galactosidase, but not p53 and p16 expression increased in WT myocardium (FIG. 2b & data not shown). Induction of p21 protein colocalised with cardiomyocyte marker troponin I and fibroblast marker vimentin, but not endothelial cell marker CD31 in aged WT hearts (FIG. 2c). In addition, 4-hydroxynonenal (4-HNE), a marker of lipid peroxidation with cytosolic presence'.sup.s also increased with age in hearts of WT but not p21-/- mice (FIG. 2d & data not shown). Ageing progressively upregulated and co-localized Pah and 2SC in WT hearts, but p21 deficiency prevented this (FIG. 2e & data not shown). Notably, human hearts presented with a comparable induction and colocalization of Pah and 2SC with age (FIG. 2f). Similarly, the rate-limiting enzyme of de novo BH4 biosynthesis GTP cyclohydrolase 1 (Geh1) was upregulated in old WT but not in p21-/- hearts, as were transcriptional targets of Nrf2, a sensitive responder to succination.sup.10 (data not shown).

[0089] The observed increase in plasma PA and cardiac Pah levels were associated with cardiac hypertrophy and myocardial interstitial fibrosis in aged WT mice (FIG. 2g-h & data not shown). By contrast, aged p21-/- mice did not show any cardiac remodeling in line with normal plasma PA levels. Furthermore, whilst left ventricular ejection fraction remained normal (data not shown), diastolic (dP/dt.sub.min) and systolic dysfunction (dP/dt.sub.max, systolic strain rate) were unmasked by in vivo haemodynamics and advanced echocardiographic deformation imaging in aged WT but not p21-/- mice (FIG. 2i-k). Collectively, p21 deficiency protected against molecular, structural and functional changes of chronologically ageing myocardium while preventing increase in plasma PA levels.

[0090] To establish a direct role for PA in driving cardiac ageing, we decided to treat WT mice without and with senile myocardium (see FIG. 2b-k; i.e. 5 vs. 11 month-old) at a dose of PA 200 mg/kg twice a day over one month with the intent to increase plasma PA levels (FIG. 3a). Mice tolerated PA treatment well without significant weight loss (data not shown). Plasma PA levels increased in both age-groups, but were considerably higher in older animals (FIG. 3a). Young animals with PA treatment exhibited a senile-like phenotype with an increase in cardiac hypertrophy, cardiomyocyte size and myocardial interstitial fibrosis, whilst no change occurred in the older group (FIG. 3c-g). At unaltered ejection fraction, PA treatment compromised cardiac function in the young group with reduced strain rate, dP/dt.sub.max and dP/dt.sub.min (FIG. 3h-k) reaching the range of vehicle-treated old mice. PA triggered myocardial senescence with upregulation of p21, Pah, Gch1, 2-SC and 4-HNE in young hearts indistinguishable from old ones (FIG. 3l & data not shown). These findings clearly demonstrate that excess PA induces cardiac senescence in young mice, closely mimicking senile cardiac remodeling and dysfunction..sup.3

[0091] Since PA induces cardiac ageing, we speculated that PA catabolism may restore cardiac function in older mice. Accordingly, 11-month-old WT mice were intraperitoneally injected with 10 mg/kg BH4 twice a day over 6 weeks to enhance Pah activity and reduce plasma PA to young levels (FIG. 4a). BH4 ameliorated HW/TL, contractility and relaxation to young values, without lowering systemic arterial pressures (FIG. 4b-f)..sup.16, 17 Moreover, BH4 normalised interstitial fibrosis whilst suppressing senescent myocardial expression of p21, Pah, Gch1 & 2-SC without altering myocardial nitric oxide synthase activity (FIG. 4g-i & data not shown).

[0092] Restored plasma PA levels along with repressed myocardial Pah, Gch1 and 2-SC by BH4 turned our attention to natural Pah expressors, kidney and liver. Whilst no age-related downregulation of Pah and Gch1 occurred in kidney, we uncovered depressed protein levels of Pah and Gch1 in 15-month-old WT livers, which were prevented by p21 deficiency (FIG. 4k & data not shown). Since ageing induced liver senescence (by p21 expression; FIG. 4l & data not shown), we hypothesized that senescence undermines hepatic PA catabolism. To this end first we explored hepatic PA levels, finding an age-dependent increase in WT livers (FIG. 4m). Interestingly, livers of PA-treated 6-month-old WT mice had comparable hepatic PA levels to those of 12-month-old WT mice (FIG. 4m). From the findings of age-dependent increase in hepatic PA content and p21 induction we hypothesized that excess PA induces p21. To address this possibility we treated AML-12 hepatocytes with increasing concentrations of PA and found a dose-dependent induction of p21 protein (FIG. 4n). Next, we treated AML-12 hepatocytes with p53 activator Nutlin3a in order to indirectly induce p21 activity. Nutlin3a induced hepatocyte senescence (i.e. p-p53 and p21), downregulated Pah and depressed tyrosine production (FIG. 4o-p & data not shown). Knockdown with p21 siRNA in hepatocytes restored Pah levels as well as tyrosine production (FIG. 4o-p). Importantly from a translational point of view, BH4 derepressed tyrosine and Pah levels from Nutlin3a-induced senescence (FIG. 4q-r) in vitro and in vivo re-expressed Pah in liver (FIG. 4s), improved hepatic Pah activity (data not shown) and reduced hepatic PA content (FIG. 4t)..sup.18, 19 Moreover, human relevance of an age-dependent decline in hepatic Pah activity is demonstrated by an age-dependent reduction in hepatic Pah transcript levels based on reanalysis of RNAseq data of a cohort of non-diseased liver biopsies (FIG. 4u)..sup.20 In summary, our findings highlight PA toxicity in age-related cardiac decline and opens new avenues to rejuvenate aged hearts by pharmacologically restoring hepatic Pah activity.

[0093] Taken together, elevated PA levels have a previously overlooked impact in cardiac ageing. Our results suggest that myocardium takes its share in maintaining homeostasis by catabolising excess PA. Whether evolving cardiac PA catabolism along with signaling consequences,.sup.21 accumulating toxic metabolites,.sup.22, 23 or PA-fueled catecholamine biosynthesis.sup.24 accounts more for cardiac ageing needs further exploring. Our findings pointed to failing hepatic PA catabolism behind rising PA levels. Pah has by far the highest hepatic expression of the six BH4-dependent enzymes (aromatic amino acid hydroxylases & nitric oxide synthases; data not shown). Its vital dependence on BH4 is illustrated by the observation that hyperphenylalaninemia is a prominent feature in enzymatic deficiencies in de novo BH4 biosynthesis or recycling pathway BH4.sup.25. Accordingly, in naturally aged mice plasma PA levels were restored by portal BH4, consistent with revived hepatic Pah activity..sup.18, 19

[0094] Therapeutic exploitation of amino acid metabolism has been demonstrated via forced histidine catabolism to sensitise cancers to methotrexate..sup.26 According to the findings presented here, pharmacologically restored PA catabolism by BH4 or alternative means.sup.27 makes reversal of cardiac ageing a realistic aim. Further age-related states may also benefit from Pah reactivation, such as dementia.sup.28 and susceptibility to cancer..sup.29, 30 Finally, PKU patients, especially those abandoning PKU diet later in life, may be at higher cardiovascular risk..sup.31

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