A POLYPEPTIDE AND RELATED PRODUCTS, METHODS AND USES

Abstract

The invention relates to an isolated polypeptide encoded by Sghrt and related uses. In addition, methods of assessing a heart function, treating impaired heart function by inhibiting Sghrt, identifying a potential drug for treating impaired heart function, and dedifferentiating and/or proliferating a heart cell by inhibiting Sghrt are claimed.

Claims

1. An isolated polypeptide encoded by Sghrt.

2. The polypeptide according to claim 1, wherein the polypeptide comprises a mitochondrial polypeptide.

3. The polypeptide according to claim 1, wherein the polypeptide shares at least 75% sequence identity with any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25.

4. The polypeptide according to claim 1, wherein the polypeptide comprises a micropeptide.

5. The polypeptide according to claim 1, wherein the polypeptide comprises a homo-oligomer.

6. A method of assessing heart function in a subject, the method comprising: determining an expression level of the polypeptide according to claim 1 in a sample obtained from the subject; comparing the expression level to a reference expression level, wherein if the expression level exceeds the reference expression level, the subject is considered to have impaired or deteriorated heart function.

7. The method according to claim 6, wherein the reference expression level comprises an expression level of the polypeptide in a healthy population.

8. The method according to claim 6, wherein the reference expression level comprises an expression level of the polypeptide in an earlier sample obtained from the subject.

9. The method according to claim 6, the method further comprising administering to the subject an inhibitor of the polypeptide.

10. A method of treating impaired heart function in a subject, the method comprising: inhibiting an expression of the polypeptide according to claim 1 in the subject.

11. A method of identifying a potential drug for treating impaired heart function, the method comprising: determining a first expression level of the polypeptide according to claim 1 in a cell; exposing the cell to a drug candidate; and determining a second expression level of the polypeptide after the exposure, wherein if the second expression level is lower than the first expression level, then the drug candidate is identified as a potential drug for treating impaired heart function.

12. An inhibitor of the polypeptide according to claim 1.

13. (canceled)

14. (canceled)

15. (canceled)

16. The method of claim 6, wherein the impaired heart function is selected from the group consisting of myocardial infarction, heart failure, coronary artery disease, narrowing of the arteries, heart attack, abnormal heart rhythms, arrhythmias, heart failure, heart valve disease, congenital heart disease, heart muscle disease, cardiomyopathy, pericardial disease, aorta disease, marfan syndrome, genetic cardiomyopathy, non-genetic cardiomyopathy, heart hypertrophy, pressure overload-induced heart dysfunction, and damaged heart tissue.

17. A pharmaceutical composition comprising the inhibitor according to claim 12, and a suitable carrier, adjuvant, diluent and/or excipient.

18. A vector comprising a polynucleotide sequence encoding for the polypeptide according to claim 1.

19. A host cell transfected with the vector according to claim 18.

20. A transgenic non-human subject comprising a polynucleotide construct encoding for the polypeptide according to claim 1.

21. The polypeptide according to claim 1 coupled to a detectable label.

22. A method of dedifferentiating and/or proliferating a heart cell, the method comprising: inhibiting the expression of the polypeptide according to claim 1 in the heart cell.

23. (canceled)

24. An isolated polynucleotide encoding for the polypeptide according to claim 1.

Description

BRIEF DESCRIPTION OF FIGURES

[0126] FIG. 1 is a mass spectrum result of the membrane proteins extracted from the mitochondria of a rat heart tissue.

[0127] FIG. 2 is a deconvolution of the mass spectrum of FIG. 1.

[0128] FIG. 3 is a sequencing result of the peptide, with a peak at 886.48 m/z, using tandem mass spectrometry (ms/ms).

[0129] FIG. 4. Peptide mapping and coverage of STMP1 in mammalian mitochondria samples.

[0130] FIG. 5. Preliminary evidence indicates SGHRT micropeptide oligomerisation. A, 1 ug and 3 ug of SGHRT synthesised peptides were boiled for 10 mins and run in 16.5% Tricine gel and the Gel was stained with Coomassie Blue solution. B, 3 ug of SGHRT synthesised peptides were treated with 1 mM and 10 mM EDTA for 30 mins and then run in 16.5% Tricine gel and the Gel was stained with Coomassie Blue solution. C, Analytical Ultracentrifugation (AUC) analysis of SGHRT synthesised peptides suggested that molecular weight of SGHRT peptides is in the range of 15-20 KDa, and the highest peak is at ˜17 KDa. D, Western blot with FLAG antibody showing a 12 KDa band in protein lysates of HEK cells transfected with SGHRT-FLAG vector in which SGHRT was C-terminal FLAG tagged, but not in lysates from cells transfected with FLAG-SGHRT vector in which SGHRT was N-terminal FLAG-tagged or with ATG-mutated SGHRT-FLAG.

[0131] FIG. 6. Upregulation of SGHRT in human hearts with End-stage HF. A, RNA-seq tracks showing higher expression of SGHRT in Dilated Cardiomyocpathy (DCM), Hypertrophic cardiomyocpathy (HCM) and Ischemic Cardiomyopathy (IHD) patients compared to heathy hearts. B, RT-qPCR validation of SGHRT upregulation in cardiac tissue from patients with end-stage HF.

[0132] FIG. 7. SGHRT knockout in hESC-CMs results in increased DAB2+ CMs. A, RNA-seq data showing increased mRNA expression of DAB2 in D58 SGHRT KO hESC-CMs. B, Western blot result showing increased DAB2 protein expression in D58 SGHRT KO hESC-CMs. GAPDH was used as loading control. C, Representative microscope images showing an increase in DAB2+ hESC-CMs and a decrease in cTnT+ hESC-CMs after SGHRT knockout at D58. White boxed areas are magnified to 40×. D, SGHRT knockout resulted in increased DAB2+ CMs at D58 time points.

[0133] FIG. 8. SGHRT KO EHT show poorly formed EHT profile and increased dedifferentiated DAB2+ CMs. A, Representative pictures of EHT generated from wildtype and SGHRT knockout hESC-CMs at D16. B, Contractility profile of SGHRT WT and KO of D16 EHT. C, Representative microscope images showing an increase in DAB2+ CMs in SGHRT KO D14 EHT.

[0134] FIG. 9. Sghrt knockdown resulted in CM dedifferentiation in neonatal (P7) mouse. A, Expression of Sghrt in whole hearts from P1 to P56 mice. B, Injection of AAV9-cTNT-GFP-RNAi to knockdown Sghrt in P7 WT mice. C, Representative pictures of a typical DAB2+ and cTNI+ cardiomyocytes, confocal images with z-stacking showing co-localization of DAB2 and cTNI signals. D, Quantification of DAB2+ CMs per heart section from control mice and mice with Sghrt KD (2 sections from each heart were analysed). E, Cell sizes of DAB2+ CMs and their neighbouring DAB2− CMs. F, Representative pictures of a binucleated DAB2+ CM (left, the two nuclei are indicated by the white arrows) and a mononucleated DAB2+ CM (right). G, Graph showing the percentage of binucleated and mononucleated DAB2+ CMs. ****P<0.0001 (Student's t-test).

[0135] FIG. 10. KD of Sghrt rescued cardiac function and induced CM dedifferentiation in TAC mouse model within 6 weeks. A, Single nuclear RNA-seq show an upregulation of Sghrt mRNA expression in single nucleus isolated from TAC CMs compared to those from Sham CMs. Each dot represents a single CM nucleus. B, Diagram showing the timeline of TAC surgery on 8-week-old WT mice, followed by AAV9 injection and harvesting. C, Graphs showing EF and FS of Sham mice, control mice with TAC and Sghrt KD mice with TAC, 6 weeks after AAV9 injection. D, Representative pictures of a typical DAB2+ and cTNI+ cell. E, Quantification of DAB2+ CMs per heart section (3 sections from each heart were analysed). F, The cell size of DAB2+ CMs and their neighbouring DAB2− CMs. G, Representative pictures of a binucleated DAB2+ CM (top panel) and a mononucleated pH3+ CM (bottom panel). (I) Graph showing the percentage of binucleated and mononucleated DAB2+ CMs. n.s. P>0.05, **P<0.01, ***P<0.0001 (Student's t-test).

[0136] FIG. 11. A, Representative DNA sequencing result confirmed that 2 A-to-G (BE19-4 and BE19-10) and 2 C-to-T (BE20-1 and BE20-2) based editing ES clones were successfully generated. B, Q-PCR results showed no change in SGHRT RNA expression in BE ES clones. C, Quantification of ATP production in WT, KO and BE ES-derived CM clones with and without Fatty Acid treatment.

EXAMPLES

[0137] Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following discussions and if applicable, in conjunction with the figures. It should be appreciated that other modifications related to structural, electrical and optical changes may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new exemplary embodiments.

Key Regulators of the Cardiomyocytes Dedifferentiation and Proliferation

[0138] The neonatal mammalian heart possesses a robust but transient regenerative ability that is rapidly lost after the first few days of life. The cardiac regeneration is likely due to dedifferentiation and subsequent proliferation of pre-existing CMs. Dedifferentiation of cardiomyocytes is characterized by the disassembly of sarcomeric structure, extrusion of mitochondria, electrical uncoupling and expression of regulators of cell cycle progression. Understanding the biology of CM dedifferentiation and proliferation could be of great clinical significance for treating heart failure.

[0139] Signaling pathways, transcription factors, cell cycle regulators, epigenetic modifiers, environmental factors and extracellular matrix have been identified to control mammalian CM dedifferentiation and proliferation. Manipulating these pathways reactivates CM proliferation and regenerates the injured adult mammalian hearts. In human HF, the limited functional recovery clearly demonstrates insufficient regeneration of human adult CMs. The evidence of human CM renewal suggests that the development of pharmacological strategies to stimulate this process may be a rational alternative or complement to cell transplantation strategies for CM replacement. Therefore, it is important to understand the biology and uncover novel gene regulatory pathways/drivers mediating CM dedifferentiation and proliferation in normal and failing hearts.

Long Non-Coding RNA-Derived Micropeptides in Cardiomyopathy

[0140] Long noncoding RNAs (lncRNAs) has been recently identified as a new layer of complexity to the regulation of the biological processes underlying normal cardiac development and myocardial remodeling during disease. Dysregulation in lncRNA regulatory circuits have been associated with cardiac pathological hypertrophy and heart failure. LncRNAs represent potential targets for novel therapeutic strategies for cardiovascular diseases. Interestingly, recent emerging evidence indicates that several LncRNAs have been mis-annotated as noncoding and in fact contains short open reading frames (sORFs) that encode functional peptides. sORF-encoded peptides (SEPs), or micropeptides, have been shown to have important roles in fundamental biological processes and in the maintenance of cellular homeostasis. These small proteins can act independently as ligands or signaling molecules, or they can exert their biological functions by engaging with and modulating larger regulatory proteins. Biological roles have been discovered to a small fraction of the total putative micropeptides and a huge amount of work remains to be done to prove their existence and elucidate their functions.

[0141] Single nuclear RNA-seq was previously performed on left ventricular (LV) CMs isolated from healthy and failing adult mouse and human hearts. This was followed by a mapping out of gene regulatory networks that contained key nodal lincRNAs (Sghrt and Gas5) that regulate dedifferentiation and cell cycle gene expression in CM subpopulations. This was also described in International Application No. PCT/SG2017/050620, the contents of which are fully incorporated herein by reference.

[0142] Mass spectrometry to profile small peptides in mitochondrial extract of bovine hearts identified that Sghrt is also potentially a micropeptide that has 100% homology with “Short transmembrane mitochondrial protein 1 precursor” (STMP1) and was co-purified with subunit 9 of mitochondrial respiratory complex III. Multiple sequence alignment demonstrated that the STMP1 micropeptide sequence is also highly conserved across vertebrates. Because there is no experimental evidence yet confirming the presence of Sghrt micropeptide in human and other mammals, it was proposed to perform Mass Spectrometry analysis on mitochondrial membrane proteins isolated from mouse, rat, pig and human heart as well as human pluripotent stem cell-derived cardiomyocytes.

Identification Sqhrt-Encoded Micropeptides in Mitochondria Membrane Proteins Isolated from Rat Hearts

[0143] Predicted mass-to-charge ratios (m/z) of peptides of interest were generated using an online tool (http://prospector.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msisotope) (see sequences in Zhang D et al., Functional prediction and physiological characterization of a novel short trans-membrane protein 1 as a subunit of mitochondrial respiratory complexes. Physiol Genomics. 2012; 44: 1133-1140. doi: 10.1152/physiolgenomics.00079.2012.). The m/z ratios allow for identification of the right peak of mass in a mass spectrum and consequently, the identification of the peptide sequence of that peak. The results are shown in Table 2 below.

TABLE-US-00003 TABLE 2 Predicted mass and m/z of the membrane peptide SGHRT in various animal species SEQ ID Species Predicted Sequence Mass M + 7 M + 6 M + 5 NO: Homo sapien MLQFLLGFTLGNVVGMYLAQ 5264.87 753.13 878.49 1053.98 1 (Human) NYDIPNLAKKLEEIKKDLDA KKKPPSA Pan troglodytes Conserved (Chimpanzee) Pan paniscus Conserved (Bonobo) Sus scrofa (pig) MLQFLLGFTLGNVVGMYLAQ 5266.85 753.41 878.82 1054.38 2 NYDIPNLAKKLEDIKKDLDA KKKPPSS Bos taurus (cow) MLQFLLGFTLGNVVGMYLAQ 5282.83 755.70 881.48 1057.57 3 NYDIPNLAKKLEEIKKDLDA KKKPPSC Rattus norvegicus MLQFLLGFTLGNVVGMYLAQ 5312.84 759.98 886.48 1063.57 4 (rat) NYDMPNLAKKLEEIKKDLDA KKKPPSS Mus musculus MLQFLLGFTWGNVVGMYLAQ 5399.85 772.41 900.98 1080.98 5 (mouse) NYEMPNLAKKLEEIKKDLEA KKKPPSS Gallus gallus MLQFVLGFTLGNVVGMYLAQ 5529.94 791.00 922.66 1106.99 6 (chicken) NYDIPNIAKKLEDFKKDVEA KKKPPSDKS Danio rerio MLQFILGFTLGNVVGMYLAQ 5245.78 750.40 875.30 1050.16 7 (zebrafish) NYEVPNISKKIEAFKKDVEA KKKPPE

[0144] Mitochondrial membrane proteins were extracted from rat heart tissue and subjected to liquid chromatography-mass spectrometry (LC-MS). Presence of the predicted m/z was determined by using the extracted ion chromatogram (FIG) function of Agilent Mass Hunter B08 software. The mass spectrum obtained is shown in FIG. 1. A prominent molecular feature 886.48 m/z eluting at around 9 minutes was identified.

[0145] The mass spectrum was then deconvoluted to obtain estimates of original molecular mass (FIG. 2). The prominent molecular feature 886.48 m/z was estimated to be of mass 5321.84, which is suggestive of the rat STMP1 peptide.

[0146] For confirmation of peptide identity, amino acid sequencing was done using tandem mass spectrometry (ms/ins). The sample was rerun on the LC-MS, with the collision-induced dissociation (ClD) cell configured to fragment the molecular feature at various fragmentation energies, and the resulting mass spectrum was collected. Amino acid sequences were then confirmed by manually matching the ms/ms spectrum to predicted a, b, and y-series ions obtained from http://prospector.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msproduct (FIG. 3). Several different fragments were identified, including for example, a fragment comprising the sequence “MLQFLLGFTL”. Based on the identified fragments, the full sequence of Sghrt is predicted to be “MLQFLLGFTLGNWGMYLAQNYDIPNLAKKLEEIKKDLDAKKKPPSA”. The ms/ms spectrum is in agreement with b ions generated from the N-terminus of rat Sghrt (see predicted N-terminal amino acids of rat Sghrt in Table 2). Strong signal of a2 and y4 ions were also noted.

[0147] Mass spectrometry analysis was performed in a further experiment to confirm and resolve the protein sequence for SGHRT in mouse, rat, pig and human. Samples were prepared for proteomics analysis using a gel-assisted sample preparation (GASP) strategy (Fischer, R. & Kessler, B. M. Gel-aided sample preparation (GASP)—a simplified method for gel-assisted proteomic sample generation from protein extracts and intact cells. Proteomics 15, 1224-1229, doi:10.1002/pmic.201400436 (2015)). Peptide mapping was done in mitochondria enriched heart samples and hES-derived CM. To increase protein coverage, LC-MS/MS was done in undigested, trypsin digested, as well as chymotrypsin digested samples. Fragments of human SGHRT peptides were detected and confirmed in limited human heart cadaver samples available, and full coverage was achieved in rat and mouse samples (FIG. 4).

[0148] Sghrt locus is therefore found to harbour a sORF and encode for a 47-amino acid mitochondrial micropeptide (derived from a mitochondrial long noncoding RNA) that may account for Sghrt therapeutic and biomarker potential/value. The micropeptide is named Short transmembrane mitochondrial protein 1 precursor, or STMP1. A peptide sequence with 100% homology has been isolated in a proteome screen of bovine cardiac mitochondrial fractions, and co-purified with subunit 9 of mitochondrial respiratory complex III. Moreover, multiple sequence alignment demonstrates that the STMP1 micropeptide sequence is highly conserved across vertebrates and targeting this sequence by morpholinos in zebrafish produced a phenotype including cardiac edema. In the present disclosure, Sghrt peptide sequence and fragments thereof were identified from the heart tissue extracts of mouse, rat, pig and humans. The results showed that Sghrt peptide sequence is highly conserved among different species, including among mammalian species.

Evidence Indicates Oligomerization of SGHRT Micropeptides

[0149] The expected size of SGHRT micropeptide is ˜5.8 KDa. However, when SGHRT synthesized peptides were run in 16.5% Tricine SDS gel, three bands were observed at ˜5.8 KDa, ˜8 KDa and ˜12 KDa, with the 12 KDa band being most abundant (FIG. 5A). This result suggests that SGHRT presents in an oligomer, which could be a dimer or trimer. The SGHRT oligomer was also resistant to heat (FIG. 5A) and EDTA treatment (FIG. 5B), suggesting that a covalent bond is not responsible for the oligomerisation. To analyze the molecular weight of SGHRT peptides quantitatively in solution, Analytical Ultracentrifugation (AUC) analysis of SGHRT synthetic peptides was performed. The result indicated that the molecular weight of SGHRT peptide is in the range of 15-20 KDa and the highest peak was again at ˜17 KDa (FIG. 5C). To validate that SGHRT oligomerisation also occurs in vivo, HEK cells were transfected with SGHRT-FLAG or FLAG-SGHRT vectors in which SGHRT was FLAG-tagged at the C- or the N-terminus, respectively. Interestingly, a strong band was consistently identified at ˜12 KDa when SGHRT-FLAG was C-terminal tagged, but not N-terminal. As additional control, this band was absent in HEK cells transfected with mutated SGHRT in which the ATG was replaced by GGG (FIG. 5D). Taken together, the results were consistent with the conclusion that SGHRT micropeptides form oligomers, which could be dimers or trimers.

SGHRT mRNA Expression is Upregulated in the Left Ventricles of End-Stage HF Patients

[0150] Human LV samples harvested with an ongoing protocol approved by the Papworth (Cambridge) Hospital Tissue Bank Review Board and the Cambridgeshire Research Ethics Committee (UK) were available. These samples were from patients undergoing cardiac transplantation for end-stage ischemic and idiopathic cardiomyopathy. Based on >50 of these samples, and a continuous constant supply every month, a genome-wide mapping of differential DNA methylation and H3K36me3 enrichment profiles, a landscape of DNA repeats and circular RNAs in human end-stage HF was done. Relevant to this Gap proposal, RNA-seq and RT-qPCR validation using the human LV samples showed that SGHRT mRNA levels were upregulated in a range of diseased HF hearts (FIG. 6, DCM: dilated cardiomyopathy; HCM: hypertrophic cardiomyopathy; IHD: ischaemic cardiomyopathy).

Increased Dedifferentiated CMs in SGHRT-KO hES Differentiation and Engineered Heart Tissue (EHT)

[0151] To validate whether SGHRT regulates differentiation of human CM, a SGHRT knockout hES cell-line was generated using CRISPR/CAS9 technology to specifically delete the promoter and first exon of the SGHRT gene. The efficient and precise genome edited in SGHRT-KO hESCs, with a complete loss of SGHRT mRNA expression, was validated. By RT-qPCR, immunostaining, and Western blot, it was proven that that there was no change to OCT4 mRNA/protein and NANOG mRNA, showing that SGHRT-KO hESCs maintained their pluripotency, and stemness was not affected by SGHRT KO.

[0152] Next, CM differentiation was performed using SGHRT-KO hESCs and harvested CM cultures at D58. SGHRT-KO CMs (MYH6-GFP reporter positive) appeared rounder, smaller, and with higher nucleus/cytoplasm ratio compared to the WT CMs (data not shown), suggesting that these cells may be acquiring cardiac progenitor-like characteristics. DAB2 mRNA and protein increase was also proven with RNA-seq and Western blot (FIG. 7A-B). There was significant increase in DAB2.sup.+ cells in SGHRT-KO (<1% in the WT compared to 11.2% DAB2.sup.+ CMs in D58 SGHRT KO, FIG. 1C-D). Consistently, there was also a significant decrease in cTnT.sup.+ (96.0% in WT cultures compared to 71.3% in D58 SGHRT-KO). These results lend further suggestion that dedifferentiated CMs lose CM characteristics and take on more progenitor-like characteristics. Moreover, using an EHT system that the lab has now robustly established (http://foo-lab.com/video/EHT.html) in collaboration with Thomas Eschenhagen (Hamburg), constructed EHT using WT and KO D14 hES-CM showed a loss in tissue structure (FIG. 8A-B) and increased DAB2.sup.+ CMs (FIG. 8C). Taken all together, the results thus far showed that the loss of SGHRT increases dedifferentiation of mature CMs both in vivo and in vitro.

Sghrt KD Induces Dedifferentiation of Neonatal Mouse Cardiomyocytes in Vivo

[0153] Mouse CMs enter cell cycle arrest at the end of their proliferation window at the 7th postnatal day (P7). Hence, Sghrt transcript levels during normal mouse heart development across this proliferation time-course window were assessed. It was found that Sghrt expression increased progressively from P10 onwards with age (FIG. 9A). The end of the proliferation window at P7 coincided with a small but statistically significant expression spike in Sghrt. To test if Sghrt regulates CM dedifferentiation in vivo and assess the validity of the in vitro KD results, CM-specific in vivo KD in P7 mice was targeted (FIG. 9B) using the AAV9-TNNT2-eGFP RNAi delivery system. Following injection of either AAV9-TNNT2-eGFP-Sghrt KD in P7 mice, hearts were harvested at P14. Sghrt-KD hearts indeed bore significantly more dedifferentiated CMs (DAB2.sup.+) (FIG. 9C,D).

[0154] Next, it was confirmed that DAB2+ CMs possessed the typical properties of dedifferentiated CMs with cell size and number of nuclei. DAB2+ CMs (n=15) were smaller than their neighbouring DAB2− CMs (n=45) (FIG. 9E). Among all 478 DAB2.sup.+ CMs, 94.98% were mononucleated (FIG. 9F, right panel and FIG. 9G) and only very few of them were binucleated (FIG. 9F, left panel). These results indicated that KD of Sghrt resulted in CM dedifferentiation in neonatal mouse.

Sghrt Knockdown Results in Increased Dedifferentiation of Adult Mouse Cardiomyocytes and Functional Recovery after TAC Pressure Overload

[0155] Single nuclear RNA-seq on cardiomyocytes isolated from healthy and failing adult mouse left ventricles revealed Sghrt to be highly upregulated in a subpopulation of cardiomyocytes (FIG. 10A). This subpopulation of cardiomyocytes was unique because of their expression pattern in the genes encoding for cell cycle activators and inhibitors. Through gene network analysis, Sghrt stood out as a putative key regulator for the cardiomyocyte stress gene programme. Therefore, it was decided to carry out Sghrt knockdown in the TAC pressure overload HF model.

[0156] Four weeks after TAC surgery, mice were injected with AAV9 to deliver 2 unique Sghrt RNAi reagents of either Sghrt-RNAi-#1, Sghrt-RNAi-#2, or the control LacZ sequence. Six weeks after AAV9 injection, mice were assessed by echocardiography before their hearts were harvested and assessed for CM dedifferentiation by immunofluorescence staining (FIG. 10B). Mice receiving LacZ as control (n=4) showed the expected significant deterioration in cardiac function (EF % and FS %) from TAC-surgery, compared to Sham-operated mice (n=3). Cardiac function of mice from Sghrt-RNAi-#2 group was significantly rescued. No significant difference was found between the EF % and FS % of Sghrt-RNAi-#1 and control groups (FIG. 10C), possibly due to the larger variation within groups and small sample size.

[0157] Importantly, more DAB2.sup.+ cardiomyocytes (FIG. 10D) per heart section were found, 6 weeks after Sghrt KD, compared to control mice (FIG. 10E), where DAB2 has been proposed as a bona fide marker of cardiomyocyte dedifferentiation. Consistent with the hallmark of cardiomyocyte dedifferentiation in DAB2-positive cells, DAB2+ CMs (n=36 CMs) were overall smaller than the DAB2− CMs (n=108 CMs) (FIG. 10F), and they were also predominantly mononucleated (FIG. 10G-H). The results thus suggest that Sghrt KD improves heart function of failing mouse heart, associated with enhanced dedifferentiation of adult mouse cardiomyocytes in Sghrt-inhibited hearts.

SGHRT Micropeptide is Required for Fatty Acid Utilization in Cardiomyocytes

[0158] To study function of SGHRT micropeptide in cardiomyocytes, ATG (TCG in reverse strand)-mutated human embryonic stem (ES) cell clones were generated using CRISPR-CAS based editing technology to change A or C into G or T (Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420-424, doi:10.1038/nature17946 (2016); Gaudelli, N. M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464-471, doi:10.1038/nature24644 (2017).) DNA sequencing confirmed that 2 A-to-G (BE19-4 and BE19-10) and 2 C-to-T (BE20-1 and BE20-2) based editing ES clones were successfully generated (FIG. 11A). The Q-PCR data indicated there was no significant change in SGHRT RNAs in BE ES clones compared to WT ES (FIG. 11B), suggesting that changing ATG site in SGHRT DNA did not affect RNA expression. These BE ES clones were then differentiated into cardiomyoctes (CMs) and ATP measurement was performed on these cardiomyocytes treated with or without Fatty Acid (FA). It was found that WT CMs produce more ATP when treated with FA compared to non-treated condition whereas KO and BE CM clones did not produce more ATP in response to FA treatment (FIG. 11C). Taken together, these results suggested that SGHRT is a mitochondria micropeptide required for FA utilization in cardiomyocytes.

Experimental Procedures

Tissue and Cell Samples

[0159] Human heart (Left Ventricle) samples were collected with an ongoing protocol approved by the Papworth (Cambridge) Hospital Tissue Bank Review Board and the Cambridgeshire Research Ethics Committee (UK). Samples were from patients undergoing cardiac transplantation for end-stage ischemic and idiopathic cardiomyopathy. The tissues were frozen and kept in −80° C. freezer until being used for mitochondria extraction.

[0160] Pig, rat and mouse heart (Left Ventricles) samples were collected from pig, rat and mouse respectively, snapped frozen and kept in −80° C. freezer until being used for mitochondria extraction.

[0161] Human ES-derived cardiomyocytes were collected from cell culture, snapped frozen and kept in −80° C. freezer until being used for extraction.

Extraction of Mitochondria from Tissue

[0162] Tissue was cut into small pieces using tweezer and scissors, then homogenised with a mechanical blender in hypotonic buffer (NaCl 10 mM, MgCl.sub.2 1.5 mM, Tris 10 mM, adjusted with HCl to pH 7.5). Cells were then ruptured by douncing until approximately 80% of cell nucleus was naked. The cell lysate was then rendered isotonic by adding a concentrated mannitol solution to final concentration of 210 mM mannitol, 70 mM sucrose, 5 mM Tris, 1 mM EDTA, adjusted with HCl to pH 7.5.

[0163] Intact cells and nucleus were then depleted from the lysate by centrifuging at 1300 g for 5 minutes at 4° C. and discarding the pellet for three cycles. Mitochondria were then harvested from the supernatant as a pellet by centrifuging at 8000 g for 15 minutes at 4° C., discarding the supernatant, and then resuspending in isotonic mannitol solution for two cycles.

Extraction of Membrane Protein from Mitochondria

[0164] Organic cell lysis buffer [2-propanol:acetonitrile:hexafluoro-isopropanol:water (70:25:0.56:4.44, by vol) containing 20 mM ammonium formate, pH 3.7] was added to the mitochondria pellet at approximately 9:1 volumetric ratio. The lysate was then vortexed for 1 minute and sonicated for 15 minutes in ice water bath. The supernatant obtained after centrifuging at 21,000 g for 10 minutes at 4° C. was then used for LC-MS.

LC-MS

[0165] An Agilent Infinity II 6550B UPLC-QTOF system was used for LC-MS. Mobile phases for UPLC chromatography were water with 0.1% formic acid and acetonitrile with 0.1% formic acid. Reverse phase separation was performed using a Phenomenex Luna Omega 1.6 um Polar C18 100 A LC Column 100×2.1 mm, with linear gradient of 20% to 80% over 20 minutes. The eluent was connected online to the mass spectrometer to acquire mass spectrum over time.

[0166] It will be appreciated by a person skilled in the art that other variations and/or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.

Stem Cell Maintenance and Differentiation

[0167] Human embryonic stem cell line H1 was maintained using mTeSR medium (Stemcell Technologies, 85850) on 1:200 growth factor-reduced Geltrex (Thermo fisher, A1413202) coated tissue culture plates and passaged regularly as cell aggregates every 4-5 days using ReLeSR (Stemcell Technologies, 05872), an enzyme-free dissociation reagent specific for human pluripotent stem cells). Two days prior to starting differentiation, cells were dissociated using Accutase (Stemcell Technologies, 07922) and seeded as single cells in Geltrex-coated 12-well plates (passage ratio 1:2, between 500′000-600′000 cells). Differentiation was performed following the published protocol by Lian et al. (Lian et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/beta-catenin signaling under fully defined conditions. Nature protocols 8, 162-175, doi:10.1038/nprot.2012.150 (2013)) with modifications as follows. 10 μM of CHIR99021 (Stemcell Technologies, 72054) was added on day 0 and left for 24 hours followed by medium change. On day 3, 5 uM IWP2 (Sigma Aldrich, 10536) was added using 50/50 mix of new fresh medium and conditioned medium collected from each well and left for 48 hours. Culture medium from day 0 until day 7 was RPM11640 (HyClone, SH30027.01) plus B-27 serum-free supplement without insulin (Gibco, A1895601). From day 7 and onwards, RPM11640 with B-27 serum free supplement with insulin (Gibco, 17504044) was used and changed every 2-3 days.

Generation of MYH6-GFP Reporter Line

[0168] EGFP cassette with kanamycin selection was inserted into BACs for MYH6 (RP11-834J17, BacPac) immediately before the initiating Methionine (ATG) using recombineering (Quick & Easy BAC Modification Kit, KD-001, Gene Bridges GmbH). The Tol2 transposon cassette with Ampicilin selection mark was inserted into the loxp site of the BAC in the backbone using recombineering. Ten million H1 cells were cultured in CF1 conditioned medium (20% KO serum replacement, 1 mMI-glutamine, 1% non-essential amino acids, 0.1 mM 2-mercaptoethanol and 8 ng ml-1 of basic fibroblast growth factor in DMEM:F12) for 6 days and dissociated into single cells with TrypLE™ Express (Life Technologies) and electroporated with 20 micrograms of Tol2 transposes and 100 micrograms of Tol2/EGFP modified Transposon-BACs. After electroporation, cells were re-suspended in conditioned medium with 10 μM ROCK inhibitor Y276329 (Y27632 (Stemcell technology, 72302). ROCK inhibitor was added for the first 48 hours after electroporation. Fifty μg/ml geneticin (Gibco, 10131035) was added for selection of positive clones 72 hours post-electroporation. Fourteen days later after drug selection, single colonies were picked into 24 well plates for expansion. Fluorescent in situ hybridyzation (FISH) using non-modified BACs as probes was carried out to validate the incorporation of BAC construct into genome of ES cells (Cytogenetics Services, Genome Institute of Singapore). Karyotyping was performed to confirm a normal chromosome pattern.

[0169] Generation of SGHRT Knockout ESC Lines Using CRISPR/Cas9 Knockout

[0170] Plasmid pMIA3 (Addgene plasmid #109399; http://n2t.net/addgene:109399; RRID: Addgene_109399) was used for CRISPR/Cas9 mediated KO. Two KO hESC lines were generated using dual single guide RNA (sgRNAs) to remove part of SGHRT. Single guide RNA sequence was designed using cloud based software tools for digital DNA sequence editing Benchling and CRISPOR. 10 μM sense and anti-sense oligonucleotides were ordered and annealed to generate the 20 nucleotide spacer that defines the genomic target to be modified (VHRT 5′end and 3′end). pMIA3 was digested with BsmBI and sgRNA spacers cloned after human U6 promoter with T4 DNA ligase (New England Biolabs) following manufacturer's instruction. Ligated constructs underwent transformation using RapidTrans™ Chemically Competent Cells (Active motif). Plasmid was extracted from bacterial culture, purified and Sanger sequenced to confirm successful cloning. Prior to hESC targeting, cutting efficiency of pMIA3-sgRNAs plasmids were tested on HEK293T using the EGxxFP plasmid (pCAG-EGxxFP was a gift from Masahito Ikawa, Addgene plasmid #50716). Best combination of sgRNAs was then used for final targeting of hESCs. The best pair of guides were cloned into a single pMIA3 plasmid digested with NheI & XbaI, using NEBuilder isothermal assembly (New England Biolabs), according to manufacturer's instructions to make the final pMIA3 dual sgRNA plasmid. Human ESCs were dissociated with Accutase (Stemcell Technologies, 07922) and ˜1.5×10.sup.6 cells were re-suspended in 100 μl P3 Primary Cell kit solution from Lonza (V4XP-3024) and mixed with 10 μg pMIA3dual sgRNA plasmid. To transfect hESC, nucleofection was performed using program CM-113 on the 4D-Nucleofector System (Lonza). Cells were then plated into Geltrex coated 6-well plate with mTeSR medium (Stemcell Technologies, 85850) containing 5 μM Y-27632. After 2 days in culture, cells were dissociated, FACS sorted for RFP positive cells and collected into a tube containing mTeSR medium (Stemcell Technologies, 85850) with 5 μM Y-27632. 500 to 2000 cells were plated into wells of 6-well plate containing the above media. Single cell clones were monitored and upon sizeable growth, colonies were picked and passaged. Genomic DNA was extracted for genotyping and screened for successful KO. RT-qPCR was performed to validate KD of SGHRT transcript.

Generation CRISPR/CA S9 Based Editing

[0171] Plasmid pMIA19-CBE4 and pMIA20-ABE7 were used for CRISPR/Cas9 mediated Based Editing (BE) to change from ATG to ATA and from ATG to ACG respectively. BE hESC lines were generated using single guide RNA (sgRNAs) 25 to edit from ATG to ATA site or from ATG to ACG of SGHRT. Single guide RNA sequence was designed using cloud based software tools for digital DNA sequence editing Benchling and CRISPOR. 10 μM sense and anti-sense oligonucleotides were ordered and annealed to generate the 20 nucleotide spacer that defines the genomic target to be modified (VHRT 5′end and 3′end). pMIA19-CBE4 and pMIA20-ABE7 were digested with AarI and spacers cloned after human U6 promoter with T4 DNA ligase (New England Biolabs) following manufacturer's instruction. Ligated constructs underwent transformation using RapidTrans™ Chemically Competent Cells (Active motif). Plasmid was extracted from bacterial culture, purified and Sanger sequenced to confirm successful cloning. Human ESCs were dissociated with Accutase (Stemcell Technologies, 07922) and ˜1.5×10.sup.6 cells were re-suspended in 100 μl P3 Primary Cell kit solution from Lonza (V4XP-3024) and mixed with 10 μg plasmid. To transfect hESC, nucleofection was performed using program CM-113 on the 4D-Nucleofector System (Lonza). Cells were then plated into Geltrex coated 6-well plate with mTeSR medium (Stemcell Technologies, 85850) containing 5 μM Y-27632. After 2 days in culture, cells were dissociated, FACS sorted for RFP positive cells and collected into a tube containing mTeSR medium (Stemcell Technologies, 85850) with 5 μM Y-27632. 500 to 2000 cells were plated into wells of 6-well plate containing the above media. Single cell clones were monitored and upon sizeable growth, colonies were picked and passaged. Genomic DNA was extracted for genotyping and screened for successful base editing. RT-qPCR was performed to validate if there is any change in the expression of SGHRT transcript.

Immunostaining

[0172] Cells were fixed in 3.7% formaldehyde for 15 min at room temperature and stored in DPBS. They were permeabilized in 0.2% Triton X-100 for 15 min followed by a pre-blocking step with 2% BSA for 20 min. Primary antibody incubation was performed in DPBS+10% goat serum (except for Nk×2.5 for which donkey serum was used) overnight at 4 degree and secondary antibody incubation for 2 hours at room temperature. DAPI was included during the final washing step. Antibodies used were cardiac troponin T (Lab Vision, ms-295-P0, mouse, 1:500 dilution), α-DAB2 (Santa Cruz, rabbit, 1:200 dilution), alexa fluor 594 goat-anti-mouse, alexa fluor 546 goat-anti-rabit (Life Technology, A-11071), alexa fluor 568 donkey-anti-goat (Life Technologies, A11057).

RNA and DNA Isolation

[0173] RNA was extracted using Direct-zol™ RNA MiniPrep Kit (Zymo, R2060). Cells were directly lysed using Trizol reagent (Thermo Fisher, 15596026). DNA was purified using PureLink Genomic DNA Mini Kit (Thermo Fisher, K182001). All experiments were performed following the manufacturer's instructions.

PCR and Reverse Transcription Quantitative PCR (RT-qPCR)

[0174] DNA or plasmid vector were PCR amplified using Q5 High-Fidelity 2× Master Mix (Bio Labs, M0492S) and target specific primers (IDT) following manufacturer's instructions.

[0175] RNA (50-500 ng) was reverse transcribed to cDNA using qScript Flex cDNA Kit (Quantabio, 95049-025) with a combination of random primers and oligo (dT). Subsequently, 1 ul of cDNA (1:10) was used to PCR amplify only specific SGHRT transcripts. The remaining cDNA (1:10) was mixed with PerfeCTa SYBR Green FastMix, low ROX (Quantabio, 95074-05K) and specific primers on a 384-well plate. Real time qPCR was run using ViiA 7 Real-Time PCR System (Applied biosystems). Average Cq was recorded and ΔΔCq method was used to calculate relative gene expression changes. Expression levels of genes were normalized against two housekeeping genes, GAPDH and PPIA.

Transverse Aortic Constriction (TAC) Model

[0176] In this disclosure, transverse aortic constriction (TAC) in adult mouse was used as the model of heart failure. TAC in mouse is an experimental model for cardiac hypertrophy and heart failure induced by LV pressure overload. In this model, the constriction at aortic arch between left common carotid artery and right common carotid artery initially obstructs the blood pumped from left ventricle (LV), leading to compensated hypertrophy of the heart and a temporary enhancement of cardiac contractility. However, this response to the chronic LV overload becomes maladaptive overtime, which eventually results in cardiac dilatation and HF, accompanied by fibrosis formation within myocardium. Compared to MI model, TAC provides a more gradual time course in the development of heart failure.

Evaluating Cardiac Function in Diseased Models of Heart Failure by Echocardiography

[0177] Echocardiography (echo) detects the cavity and chamber wall of hearts by sound waves to produce live images, and is an important non-invasive experimental method to visualize the cardiovascular structures and evaluate cardiac function in mice and rats. Improved echo instrumentation can provide accurate assessment of LV systolic/diastolic function, chamber size and wall thickness in various mouse models of cardiovascular diseases. In this disclosure, echo was used to track the structural and functional changes in TAC and MI mouse models.

Quantification and Characterisation of Dedifferentiated CMs by Immunofluorescence Staining and Confocal Microscopy

[0178] The method of characterising CM dedifferentiation is not as well-established as that of CM proliferation, which can be indicated by markers of different cell-cycle stages. Based on all the existing publications related to CM dedifferentiation, the following criteria has been used to identify it: 1) loss of contractility and electrophysiological properties; 2) disassembled sarcomere or decreased expression level of sarcomeric genes; 3) expression of stem/cardiac progenitor cell markers, among which disabled homolog 2 (DAB2) is most commonly used. The first criterion is usually used to judge in vitro dedifferentiation of single CM, rather than CMs from heart tissue sections, while the last criterion is quite controversial. DAB2 is a target of GATA transcription factors and its increase may reflect increased expression of GATA4/6, a reported regulator of cardiac hypertrophy. Quantitative proteomics of human embryonic stem cells (hESCs), cardiac progenitor cells (CPCs), and cardiomyocytes also identified DAB2 as crucial cardiac developmental regulator. However, there is no direct evidence showing the change of DAB2 expression level in CMs over heart development, nor is its role in the dedifferentiation of other cell types. The first publication using DAB2 to indicate CM dedifferentiation also didn't provide detailed rationale of choosing it as a marker. Therefore, in this disclosure, immunofluorescence (IF) staining and confocal microscopy were used to identify CMs that are immune-positive of DAB2, of which the sarcomere would be judged as well-assembled or not based on IF staining of CM sarcomeric gene. Besides, the cell size of DAB2+ CMs were measured, and the number of nuclei within these CMs were counted, as dedifferentiated cells are generally smaller and have fewer organelles compared to matured cells.

In Vivo Knockdown (KD) of Sghrt in CMs Via Adeno-Associated Virus Serotype 9 (AAV9) Containing CM-Targeting RNA Interference System

[0179] RNA interference (RNAi) is an approach to silencing target genes by degrading the mRNA, achieved by introducing small double-stranded interfering RNAs (siRNA) into the cytoplasm. Adeno-associated virus (AAV) is an ideal vector for gene or siRNA delivery due to its low immunogenicity and stable gene expression, and AAV serotype 9 (AAV9) provides global cardiac gene transfer in mouse and rat, which is superior to other serotypes. To achieve knockdown (KD) of Sghrt in CMs in mouse, AAV9 containing cardiac troponin T (cTnT)-promoted and GFP-tagged siRNA targeting Sghrt (AAV9-cTNT-eGFP-RNAi) were injected into chest cavity of the mice in the dose of 5×10.sup.13 vector genomes (vg)/kg.

Studying the Effect of Sghrt KD on CM Dedifferentiation and Heart Function in TAC and MI Mouse Model

[0180] To investigate whether in vivo KD of Sghrt induces CM dedifferentiation and rescues cardiac function in diseased models of HF, the mice were given injection of AAV9-cTNT-eGFP-RNAi 4 weeks after TAC surgery and immediately after MI surgery, respectively. AAV9-cTNT-eGFP-LacZ was injected into TAC or MI mice as control. Weekly echo was applied afterwards to track the cardiac function of the mice, including the LV ejection fraction (EF) and fraction shortening (FS). 10 and 14 weeks after TAC surgery, the hearts were harvested and sectioned for histological study and immunofluorescence staining of DAB2, cardiac troponin I (cTNI) and wheat germ agglutinin (WGA) to distinguish cell membrane. The co-localization of DAB2 and cTNI signals were assessed by z-stacking function of confocal microscopy. The number of DAB2.sup.+ and cTNI.sup.+ cells in each heart section were counted, and the size of the DAB2+ CMs and 3 neighbouring DAB2− CMs were measured and analysed using ImageJ. Similarly, 4 weeks after MI surgery, the hearts were harvested for the same analysis above. Further, to study the effect of Sghrt KD on CM dedifferentiation in postnatal mouse, 7-day-old (P7) mice were given injection of AAV9-cTNT-eGFP-RNAi or AAV9-cTNT-eGFP-LacZ, followed by harvesting and downstream analysis 7 days after injection.

ATP Quantification

[0181] ATP quantification in human ES-CMs were perform using ATP Determination Kit (A22066) following instructions provided in the kit.

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