RELAXIN RECEPTOR 1 FOR USE IN TREATMENT AND PREVENTION OF HEART FAILURE

Abstract

The present invention relates to a polynucleotide comprising an expressible nucleic acid sequence encoding a relaxin family peptide receptor (RXFP) polypeptide for use in treatment and/or prevention of heart failure in a subject. The present invention further relates to a vector comprising the polynucleotide of the present invention for use in treatment and/or prevention of heart failure, as well as to host cells, RXFP agonists, kits and devices related thereto.

Claims

1. A method for treating and/or preventing heart failure in a subject, comprising: (i) contacting the subject with a polynucleotide comprising an expressible nucleic acid sequence encoding a relaxin family peptide receptor (RXFP) polypeptide, and (ii) thereby treating heart failure.

2. The method of claim 1, wherein said heart failure is chronic heart failure.

3. The method of claim 1, wherein said polynucleotide comprises the sequence of SEQ ID NO:1, 3, or 5.

4. The method of claim 1, wherein said RXFP polypeptide comprises at least the amino acids corresponding to amino acids 1 to 766 of human RXFP1.

5. The method of claim 1, wherein said RXFP is RXFP1.

6. The method of claim 1, wherein said polynucleotide comprises, preferably consists of, the sequence of SEQ ID NO:3.

7. The method of claim 1, wherein said polynucleotide is comprised in a vector, preferably a viral vector.

8. The method of claim 1, wherein said method comprises intravenous and/or intracardial administration of said polynucleotide to said subject.

9. The method of claim 1, wherein said method further comprises administration of an RXFP agonist.

10. The method of claim 9, wherein said RXFP agonist is relaxin or a derivative thereof.

11. (canceled)

12. The method of claim 1, wherein the polynucleotide is comprised in a host cell.

13. The method of claim 1, wherein the subject is a mammal.

14. A kit comprising a polynucleotide as specified in claim 1, and a pharmaceutical preparation comprising an RXFP agonist.

15. A device comprising a polynucleotide as specified in claim 1.

16. The method of claim 1, wherein the subject is a human.

17. The method of claim 5, wherein the RXFP (i) comprises the amino acid sequence of SEQ ID NO:2 and/or 4; or (ii) comprises an amino acid sequence at least 70% identical to SEQ ID NO:2 and/or 4.

18. The method of claim 7, wherein the viral vector is an adeno-associated virus (AAV) vector.

19. The method of claim 18, wherein the AAV vector is selected from the group consisting of AAV1, AAV2, AAV5, AAV6, and an AAV9 vector.

20. The method of claim 19, wherein the AAV vector is an AAV9 vector.

Description

FIGURE LEGENDS

[0079] FIG. 1: RXFP1 gene therapy with chronic administration of RLN (RXFP1/RLN) improves cardiac function in TAC-induced heart failure in mice. (A) Experimental design. (B) Representative echocardiography recordings from midventricular M-Mode used for assessment of cardiac function. (C) Ejection Fraction (EF) and (D) fractional shortening (FS) of sham mice treated with and without recombinant RLN and TAC mice treated with control Luc virus and RXFP1 virus with and without recombinant RLN treatment (Sham+NaCl, Sham+RLN, TAC+LUC+NaCl, TAC+LUC+RLN, TAC+RXFP1+NaCl and TAC+RXFP1+RLN, n=8-9 in sham groups and n=14-15 in TAC groups). TAC animals were injected with 1×10.sup.12 vg/animal and implanted with either NaCl pumps or RLN pumps 4 weeks after TAC operation. (E) RXFP1 mRNA expression in the ventricle detected 8 weeks after RXFP1 gene therapy treatment. RXFP1 mRNA expression from the treated samples were compared to the native RXFP1 expression in the atria. (F) BNP expression quantification by qPCR, HPRT1 was used as a reference gene. (G) cardiac P-PLB(S16)/PLB quantification by immunoblot; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) immunodetection was used as an internal control. (H) Relaxin plasma concentrations were determined using an Elisa kit. For (C) and (D) *FL NaCl vs. FR RLN, #FL RLN vs. FR RLN § FR NaCl vs. FR RLN, */#/§ P<0.05, for (E) to (H) *P<0.05; **P<0.01; ***P<0.001

[0080] FIG. 2: RXFP1 gene therapy with chronic administration of RLN (RXFP1/RLN) maintains left ventricular internal diameter, but does not affect left ventricular mass and heart rate in TAC-induced heart failure in mice. TAC animals were injected with 1×10.sup.12 vg/animal and implanted with either NaCl pumps or RLN pumps 4 weeks after TAC operation. *FL NaCl vs. FR RLN; #FL RLN vs. FR RLN; § FR NaCl vs. FR RLN (A) Left ventricular internal diameter (LVIdD) of sham mice treated with and without recombinant RLN and TAC mice treated with control Luc virus and RXFP1 virus with and without recombinant RLN (Sham+NaCl, Sham+RLN, TAC+LUC+NaCl, TAC+LUC+RLN, TAC+RXFP1+NaCl and TAC+RXFP1+RLN, n=8-9 in sham groups and n=14-15 in TAC groups); (B) Left ventricular (LV) mass of sham mice treated with and without recombinant RLN and TAC mice treated with control Luc virus and RXFP1 virus with and without recombinant RLN (Sham+NaCl, Sham+RLN, TAC+LUC+NaCl, TAC+LUC+RLN, TAC+RXFP1+NaCl and TAC+RXFP1+RLN, n=8-9 in sham groups and n=14-15 in TAC groups); (C) Heart rate of sham mice treated with and without recombinant RLN and TAC mice treated with control Luc virus and RXFP1 virus with and without recombinant RLN (Sham+NaCl, Sham+RLN, TAC+LUC+NaCl, TAC+LUC+RLN, TAC+RXFP1+NaCl and TAC+RXFP1+RLN, n=8-9 in sham groups and n=14-15 in TAC groups). */#/§ P<0.05

[0081] FIG. 3: Effect of RXFP1 gene therapy with chronic administration of RLN (RXFP1/RLN) on cardiac gene expression in TAC-induced heart failure in mice. Cardiac remodeling genes (A) ANP (B) β-MHC and collagen regulation genes (C) Col1a1 (D) Col3a1 (E) Postn mRNA expression in TAC animals receiving RXFP1 gene therapy with and without RLN quantified by qPCR with HPRT1 as reference gene. (n=8-15). *P<0.05; **P<0.01; ***P<0.001; B)

[0082] FIG. 4: Overexpression of RXFP1 in combination with RLN stimulation induces cAMP-dependent signaling in vitro; (A) RXFP1 mRNA expression in transduced NRVMs, n=3 per condition. *P<0.05; **P<0.01; ***P<0.001; (B) cAMP production in transduced NRVMs, 100 nM of RLN was used for RXFP1 stimulation under all conditions, n=3. *P<0.05; **P<0.01; ***P<0.001; (C) RXFP1 mRNA expression in transduced NRVMs quantified by qPCR with HPRT1 as reference gene (n=6), control virus and RXFP1 virus were deployed in NRVMs, non-treated neonatal rat atrial cardiomyocytes (NRAM) were used as endogenous reference, *P<0.05; **P<0.01; ***P<0.001; (D) RXFP1-RLN dose response as assessed by cAMP production, NRVMs were transduced with RXFP1 or control virus and stimulated with different dosages of RLN ranging from 1 pM to 100 nM and changes in cAMP production were measured, n=4-6, *P<0.05; **P<0.01; ***P<0.001; (E-F) Characterization of cAMP-dependent downstream signaling, NRVMs were transduced with RXFP1 or Luc control virus and stimulated with 100 nM RLN or NaCl as control, phospholamban phosphorylation at serine 16 in relation to total phosholamban expression (P-PLB(S16)/PLB) was quantified by immunobloting, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, (E) quantitative results, (F) representative immunoblots, n=4, *P<0.05; **P<0.01; ***P<0.001.

[0083] FIG. 5: Comparison of human and rat RXFP1 receptor activation by recombinant RLN; A) P-PLB(S16)/PLB and (B) representative western blot of NRVMs transduced with different versions of RXFP1 receptor virus and stimulated by recombinant RLN (n=3), transduced NRVMs was stimulated with 100 nM recombinant RLN and phospholamban phosphorylation at serine 16 was quantified by immunoblot; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) immunodetection was used as an internal control. *P<0.05; **P<0.01; ***P<0.001.

[0084] FIG. 6: Activation of human and rat RXFP1 receptor by a chemical agonist (ML290); (A) P-PLB(S16)/PLB in NRVMs transduced with different versions of RXFP1 receptor virus and stimulated by ML290 (n=3), transduced NRVMs were stimulated with 1 μM ML290 and phospholamban phosphorylation at serine 16 was quantified by immunoblot; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) immunodetection was used as internal control. *P<0.05; **P<0.01; ***P<0.001 (B) Western blot of P-PLB(S16)/PLB in NRVMs transduced with different versions of RXFP1 receptor virus and stimulated by ML290 (n=3), transduced NRVMs were stimulated with 1 μM ML290 and phospholamban phosphorylation at serine 16 was quantified by immunoblot; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) immunodetection was used as an internal control. *P<0.05; **P<0.01; ***P<0.001.

[0085] FIG. 7: Functional and molecular improvements after huRXFP1 gene therapy treatment with RLN: (A) Timeline of huRXFP1 study. (B) Ejection fraction (EF), of sham mice treated with and without recombinant RLN treatment and TAC mice treated with and without huRXFP1 gene therapy and RLN (Sham+NaCl, Sham+RLN, TAC+LUC+NaCl, TAC+LUC+RLN, TAC+huRXFP1+NaCl and TAC+huRXFP1+RLN, n=15 in sham groups and n=15-22 in TAC groups). TAC animals were injected with 1×10.sup.12 vg/animal and implanted with either NaCl pumps or RLN pumps 4 weeks after TAC operation. (C) BNP and (D) huRXFP1 mRNA expression in the ventricle detected 8 weeks after huRXFP1 gene therapy treatment (n=15 in sham groups and n=15-22 in TAC groups). mRNA expression was quantified using qPCR, with HPRT1 as a reference gene. For (B) *FL NaCl vs. huRXFP1 RLN, #LUC RLN vs. huRXFP1 RLN § huRXFP1 NaCl vs. huRXFP1 RLN, for (C) and (D) *P<0.05; **P<0.01; ***P<0.001. Error bars depict in SEM.

[0086] FIG. 8: Functional and molecular improvements in stress model with high expression of huRXFP1. (A) Timeline of the experiment. (B) Ejection fraction (EF) of TAC transgenic animals (n=10-20). After TAC operation, the animals were periodically followed using echocardiography. (C) huRXFP1, (D) ANP, (E) BNP, (F) Collagen1A1, and (G) Collagen1A3 mRNA expression in the ventricle detected 6 weeks after the TAC operation (n=10-20). mRNA expression was quantified using qPCR, with HPRT1 as a reference gene. For (B) *TAC TG vs TAC WT, for (C-G) *P<0.05; **P<0.01; ***P<0.001. Error bars depict in SEM.

[0087] FIG. 9: Calcium transients measurement in RXFP1 transduced NRVCMs: (A), (B) Transients amplitude and representative Ca.sup.2+ transients and (C) Diastolic Ca.sup.2+ after 5 minutes of NaCl, RLN, and ISO treatments (n=3-8). NRVCMs were transduced with RXFP1 and LUC virus for 5 days. Ca.sup.2+ transients were measured with 1 Hz electrical stimulation. (D) RXFP1 mRNA expression after the experiment. RXFP1 mRNA expression was analyzed using HPRT1 as a reference gene. For figure A only the first 4 groups were compared. *P<0.05; **P<0.01; ***P<0.001. Error bars depict in SEM.

[0088] FIG. 10: Adult cardiomyocyte Ca.sup.2+ transient: (A) Ca.sup.2+ peak height, (B) Departure velocity, (C) Return velocity after RLN treatment (n=2). Adult cardiomyocytes were isolated from transgenic mice that expressed RXFP1 specifically in the heart. Baseline Ca.sup.2+ was measured. The cells were treated with NaCl, RLN (100 nM), or ISO (10 nM) for 1 minute and the transients were measured again. Alteration of Ca.sup.2+ signal was calculated using automatic software and normalized against baseline value. *P<0.05; **P<0.01; ***P<0.001. Error bars depict in SEM.

[0089] FIG. 11: RXFP1 expression in heart failure: (A) BNP mRNA expression. (B) RXFP1 mRNA expression. mRNA expression profile of fetal gene and RXFP1 from explanted hearts and cardiac biopsies were performed. mRNA expression was quantified using qPCR, with HPRT1 as a reference gene. *P<0.05; **P<0.01; ***P<0.001. Error bars depict in SEM.

[0090] FIG. 12: FIG. 12: Hemodynamics changes in transgenic animals after RLN administration (Example 10). (A) Representative PV loops recording, (B) Heart Rate (HR), and (C) dP/dt maximum. Continuous PV loops were measured from WT and huRXFP1 TG animals. 10 μg in 100 μL of RLN was administered to stimulate huRXFP1. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Error bars depict in SEM.

[0091] The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

EXAMPLE 1: RXFP1 GENE THERAPY IN COMBINATION WITH RLN ADMINISTRATION INDUCES POSITIVE INOTROPIC EFFECTS AND RESCUES HEART FAILURE

Virus Production

[0092] For all in vivo experiments adeno-associated viral vectors serotype 9 (AAV9) were generated. Recombinant AAV vectors expressing rat (Rattus norwegicus) and human (Homo sapiens) relaxin receptor 1 (RXFP1) were generated: AAV9-CMV-MLC260-FLAG-Rel1.sup.Rattus (AAV9-RXFP1) and AAV9-CMV-MLC260-FLAG-Rel1.sup.human (AAV9-huRXFP1). Both optimized rat and human Rel1 cDNA (NM_201417.1 and NM_021634.3, National Center for Biotechnology Information (NCBI)) were synthesized and cloned into a pCDNA3.1 plasmid with N-terminal FLAG-TAG. After extensive in vitro testing, the transgene cassette was subcloned into an AAV transfer plasmid downstream of a cardiac myocyte-specific cytomegalovirus (CMV) enhancer coupled with a short 260 bp myosin light chain promoter (MLC260) and the whole construct was flanked by two ITRs. Recombinant vectors were generated by packaging of AAV-inverted terminal repeat recombinant genomes into AAV9 capsids using the two plasmids transfection protocol (Grimm et al., 2003, Mol. Ther. 7: 839-850). High titer vectors were produced in cell stacks (Corning) with polyethylenimine transfection. After 48 hours the vectors were harvested and purified by density filtration in an iodixanol gradient (Jungmann et al., 2017, Hum. Gene. Ther. Methods 28: 235-246). Using the same method, a control vector expressing firefly luciferase was generated: AAV9-CMV-MLC260-FLAG-Luc.sup.Firefly (AAV9-LUC). Viral titers from all viral vectors were quantified at the same time using a SYBR-green real time PCR assay (Bio-Rad) and expressed as viral genomes per milliliter (vg/mL).

Heart Failure Model and Experimental Set Up

[0093] Transverse aortic constriction (TAC) operation was previously described (Rockman et al., 1991, Proc. Natl. Acad. Sci. 88: 8277-8281). TAC model was used as heart failure model. 8 weeks old C57BL/6 mice were weighted prior to the operation. TAC operation was performed on day 0 using 26-gauge blunt needle effectively reducing the diameter of the transverse aorta to approximately 0.46 mm (Rockman et al., 1994, Proc. Natl. Acad. Sci. 91(7): 2694-2698). Cardiac function was assessed by echocardiography (Vevo 2100 VisualSonics) before TAC operation (day −2), before virus infusion (day 7), before pump implantation (day 28), and at 2-month follow up (day 55) (FIG. 1A and B). At day 7, animals were randomized to systemic injection of luciferase (LUC) control virus or RXFP1 virus. Osmotic pumps (Alzet 2004) were implanted on day 28 with animals randomized to get either saline or recombinant relaxin pumps. Follow up assessments were performed on day 55 and the animals were sacrificed 3 days later. In addition, 17 mice served as control animals which underwent sham operation, followed by either saline or recombinant relaxin pump implantation.

Cardiac Function Measurements

[0094] Echocardiography was performed using Vevo 2100, VisualSonics. The mice were shaved and held in prone position. Warm ultrasound coupling gel (37° C.) was placed on the shaved chest area, and the MS400 transducer was positioned to obtain 2D B-mode parasternal long and short axis views and M-mode short axis view. The ejection fraction (EF), fractional shortening (FS), left ventricular internal diameter (LVIDd) and heart rate (HR) were calculated from 6 consecutive heartbeats in the M-mode using LV trace function.

[0095] EF and FS of all TAC animals gradually decreased starting from day 7 post TAC operation (EF: 62.25±10.41% and FS: 33.17±7.38). Further reduction was detected in all TAC operated animals on day 28 post operation before pump implantation (EF: 54.09±12.65 and FS: 28.07±7.63). The follow up assessments at day 55 showed further reduction in EF and FS in control groups treated with AAV9.LUC receiving either NaCl or RLN (LUC NaCl EF: 48.78±18.61; FS: 25.11±10.69 and LUC RLN EF: 47.79±13.38; FS: 23.98±7.64). An attenuated however non-significant decrease in EF and FS was observed in RXFP1 groups treated with NaCl (EF: 53.99±13.75 and FS: 27.95±8.40) and a significant increase in EF and FS was detected in RXFP1 groups treated with RLN (EF: 65.11±14.39 and FS: 35.74±9.92) (FIG. 1C and D). Other parameters such as left ventricular internal diameter (LVIdD), left ventricular mass corrected, and heart rate were also measured, but no significant difference between TAC operated groups could be observed (FIG. 2A, B, and C).

Molecular Analysis

RNA Expression

[0096] RNA was extracted using TRlzol® reagent (Ambion) from snap frozen tissue (10 mg) or cell lysate (2×10.sup.6 cells) according to the manufacturer's protocols. 1 μg of total the RNA was reverse transcribed into cDNA using the iScript cDNA-Synthesis Kit (Bio-Rad). iQ SYBR

[0097] Green Supermix was used according to the manufacturer's protocol. A final volume of 15 μL per reaction consisted of 7.5 μL iQ SYBR Green Supermix, 1 μL of forward and reverse primers mix (final concentration of 300 nM each), and 6.5 μL diluted cDNA prepared (final concentration of 3.25 ng per reaction). The quantitative real-time PCR was performed in duplicate on Bio-Rad CFX96 real-time PCR detection system (Bio-Rad). The 2.sup.−ΔΔCT method was used to quantify relative gene expression levels between samples. Specificity of PCR products was confirmed by gel electrophoresis. Primers listed in table 1 were used for mRNA expression quantification.

TABLE-US-00001 TABLE 1 Primers for RT-PCR SEQ Genes Primers Template ID NO ANP Forward primer: NM_008725.3 14 5′-TGCCGGTAGAAGATGAGGTC-3′ Reverse primer: 15 5′-TGCTTTTCAAGAGGGCAGAT-3′ β-MHC Forward primer: NM_080728.3 16 5′-GCCAACACCAACCTGTCCAAGTTC-3′ Reverse primer: 17 5′-TGCAAAGGCTCCAGGTCTGAGGGC-3′ BNP Forward primer: NM_008726.5 18 5′-CTGAAGGTGCTGTCCCAGAT-3′ Reverse primer: 19 5′-CCTTGGTCCTTCAAGAGCTG-3′ Collα1 Forward primer: NM_007742.4 20 5′-GTGTTCCCTACTCAGCCGTC-3′ Reverse primer: 21 5′-ACTCGAACGGGAATCCATCG-3′ Col3α1 Forward primer: NM_009930.2 22 5′-TGACTGTCCCACGTAAGCAC-3′ Reverse primer: 23 5′-GAGGGCCATAGCTGAACTGA-3′ Hprt1 Forward primer: NM_013556.2 24 5′-GAGGAGTCCTGTTGATGTTGCCAG-3′ Reverse primer: 25 5′-GGCTGGCCTATAGGCTCATAGTGC-3′ Postn Forward primer: NM_009071.2 26 5′-ACAAAAGGGTTCAAGGGCCTA-3′ Reverse primer: 27 5′-TTGGCTTCTGTTGGTTGTCA-3′ Rattus Forward primer: NM_201417.1 28 Norvegicus 5′-CCATGCATTGTTTGTGCCGA-3′ Rel1 Reverse primer: 29 5′-TTTGCAGGCACAGCTTTTGG-3′ Homo Forward primer: NM_021634.3 30 sapiens 5′-CTACAAGGACGACGATGACAAG-3′ Rel1 Reverse primer: 31 5′-GAAATAGCCAAGGGAGCACTTG-3′

TABLE-US-00002 TABLE 2 RT-PCR program Number of Step Temperature Time Cycles Denaturation 95° C. 3:00  1x Denaturation 95° C. 0:10 Annealing Vary annealing Temperature 0:10 40x Elongation and reading 72° C. 0:30 Termination 95° C. 0:10  1x Melting Curve 65 .fwdarw. 95° C. with 0.5° C. 0:05 60x increment

[0098] Post-mortem mRNA expression analysis using RTPCR revealed a significant increase in RXFP1 mRNA expression within the ventricle of TAC animals treated with RXFP1 virus (FIG. 1E). Compared to a TAC group treated with control virus alone, a significant decrease in BNP mRNA expression was observed in RXFPI and RLN treated mice (FIG. 1F).

[0099] Furthermore, a trend towards reduced activation of fetal genes like ANP or β-MHC was detected in the TAC group receiving both RXFP1 gene therapy and RLN treatment (FIG. 3A and B). Interestingly, a trend toward decreased collagen mRNA expression (Col1a1, Col3a1 and Postn) post TAC was detected in both RXFP1 groups (with and without RLN treatment) (FIG. 3C, D and E).

Protein Expression

[0100]

TABLE-US-00003 TABLE 3 List of antibodies used for immunoblotting Protein Manufacturer Type Species Reactivity Dilution Phospholamban Sigma Aldrich Monoclonal Mouse Mouse, Rat, 1:7500 (PLB) Human Phospho Upstate Polyclonal Rabbit Mouse, Rat, 1:5000 (serine16)- Human phospholamban [P-PLB(S16)] Phosphor Badrilla Polyclonal Rabbit Mouse, Rat, 1:5000 (threonine17)- Human phospholamban [P-PLB(T17)] GAPDH Merck- Monoclonal Mouse Mouse and Rat  1:10000 Millipore

[0101] Tissue samples were cut into 10 mg pieces prior to freezing. Proteins from tissues were extracted using 1% SDS buffer (1% SDS, 1 mM EDTA, 1 mM EGTA supplemented with protease inhibitor and phosphatase inhibitor cocktail 2 and 3). Extracted proteins were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting as previously described (Towbin et al., 1979, Proc. Natl. Acad. Sci. 76: 4350-4354). Membranes were probed with antibodies listed in table 3. Mouse secondary antibody coupled to Alexa Flour 680 (Invitrogen) and rabbit secondary antibody coupled to Dylight™ 800 (Cell Signaling) were used to detect the signals from the immunoblots utilizing an Odyssey infrared imager (LI-COR) detection system. Images were processed using Odyssey imaging software. A higher ratio of phospholamban phosphorylation at serine 16 [P-PLB(S16)] was detected in TAC animals that received both RXFP1 gene therapy and RLN (FIG. 1G).

RLN Measurements

[0102] Circulating plasma levels of recombinant relaxin H2 were measured using a ready to use relaxin H2 Quantikine ELISA kit from R&D Systems (DLR200). Plasma samples were diluted 1:200 to stay within the detection range of the assay. 50 μL of negative control, positive control, standard and diluted samples were used for the measurement.

[0103] Measurements of RLN plasma levels revealed elevated plasma levels of RLN in all animals receiving RLN. RLN plasma concentrations were equal in all treated groups (FIG. 1H).

EXAMPLE 2 POSITIVE INOTROPY FROM RXFP1 GENE THERAPY AND RECOMBINANT RLN STIMULATION IN VITRO

Virus Production

[0104] For all in vitro experiments AAV6 serotype vectors were deployed. The recombinant adeno-associated viral vectors AAV6-CMV-MLC260-FLAG-Rel1.sup.Rattus (AAV6-RXFP1) and AAV6-CMV-MLC260-FLAG-Rel1.sup.human (AAV6-huRXFP1) were generated as follows. Both optimized Rattus norvegicus and Homo sapiens RXFP1 cDNA [NM_201417.1 and NM_021634.3, National Center for Biotechnology Information (NCBI)] were synthesized and cloned into a pCDNA3.1 plasmid with N-terminal FLAG-TAG. After extensive in vitro testing, the transgene cassette was subcloned into an AAV transfer plasmid downstream of a cardiac myocyte-specific cytomegalovirus (CMV) enhancer coupled with a short 260 bp myosin light chain promoter (MLC260) and the whole construct was flanked by two ITRs. Recombinant vectors were generated by packaging of AAV-inverted terminal repeat recombinant genomes into AAV6 capsids using the two plasmids transfection protocol (Grimm et al., 2003, Mol. Ther. 7: 839-850). High titer vectors were produced in cell stacks (Corning) with polyethylenimine transfection. After 48 hours the vectors were harvested and purified by density filtration in an iodixanol gradient (Jungmann et al., 2017, Hum. Gene. Ther. Methods 28: 235-246). Using the same method, the control vector AAV6-CMV-MLC260-FLAG-Luc.sup.Firefly (AAV6-LUC) was generated. Viral titers from all viral vectors were quantified at the same time using a SYBR-green real time PCR assay (Bio-Rad) and expressed as viral genomes per milliliter (vg/mL).

Isolation of Neonatal Rat Cardiomyocytes

[0105] Primary cultures of neonatal rat ventricular cardiac myocytes (NRVMs) were prepared from 1-2 days old Wistar rats (Charles River). The neonatal rats were euthanatized by decapitation and the heart dissected and placed in 1× cold ADS solution. The atria and other vessels were removed, and the hearts were transferred into a new 1× cold ADS solution. The ADS solution was removed, and the ventricles were washed with 3 mL of cold ADS before being minced. Tissue fragments were digested in a T75 bottle with 30 mL of a digestion solution containing collagenase and pancreatin for 5 minutes at 37° C. The tissue fragments were allowed to settle on one side of the bottle and the solution removed and discarded. Fresh digestion solution was added, and the tissue fragments were digested for 20 minutes at 37° C. After 20 minutes, the tissue fragments were pipetted up and down 5 times at maximum power and the solution removed and filtered through a 40 μm cell strainer into a 50 mL falcon. 10 mL of FCS was added through the cell strainer and the solution was centrifuged at 1000 rpm for 5 minutes at room temperature. After centrifugation, the supernatant was removed, and 7 mL of FCS was added. The cell solution was placed in a 37° C. incubator awaiting further processing. The tissue fragments were digested at least 5 times in order to completely digest all the tissue pieces. After the digestion, all cell suspensions were combined and centrifuged at 1000 rpm for 5 minutes at room temperature. The supernatant was removed, and the cells were resuspended in 12 mL of cold 1×ADS. The fibroblasts and cardiac myocytes were separated using percoll density gradient. To obtain cardiomyocytes with high purity, a two layers Percoll density gradient was performed. The gradient consisted of 63% red Percoll solution, and a 40.5% clear Percoll solution. 4 mL of red Percoll solution was added into each 15 mL falcon. Then 3 mL of clear percoll solution was added using underlaying technique. 2 mL of cell solution containing 1× cold ADS was layered at a 45 degrees angle onto of the gradient. The falcons were centrifuged at 2400 rpm for 30 minutes at 37° C. deceleration speed of 0. The upper stromal cells band including fibroblasts was removed by aspiration. The lower cardiomyocytes band was kept and washed twice with cold 1×ADS before being resuspended in a warm Medium 199 supplemented with 10% v/v fetal calf serum, 100 U/mL penicillin, 100 μg/streptomycin, 2 mM L-glutamine, and 1 mM calcium chloride. The cells were counted manually in a Neubauer chamber and plated accordingly (Table 4). The NRVCM were cultured at 37° C. and 5% CO.sub.2 humidified atmosphere. After 48 hours fetal calf serum was reduced to 0.5% and cells were cultured for 2-5 days. Two days after the cells were plated, the medium was removed, and the cells were washed once with warm PBS before starving medium M199 supplemented with 0.5% FCS, 1% penicillin/streptomycin, 1% L-glutamine, 1 mM CaCl.sub.2 was added. In the following, the medium was changed every two days until the cells were harvested. Cells were used for the maximum of 7 days before being discarded.

TABLE-US-00004 TABLE 4 Optimal plating density for NRVMs Area per Number of Volume per well (cm.sup.2) cells seeded well (mL)  6-well plate 10 2 × 10.sup.6 2 12-well plate 4 5 × 10.sup.5 1 96-well plate 0.32 2 × 10.sup.4 0.1

Optimization of Neonatal Rat Ventricular Cardiomyocyte Transfection for In Vitro Testing

[0106] of RXFP1 Gene Therapy After the first medium change NRVMs seeded in 6-well plates were transduced with different amounts of RXFP1 virus (ranging from viral MOI of 5,000 to 50,000 vg/cell) along with Luc control virus with the MOI of 10,000 vg/cell. The virus was allowed to transduce the cells for 48 hours before the medium was changed. After 5 days the transduced NRVMs were harvested and RXFP1 mRNA expression was quantified by qPCR using primers from table 1. Significant increase in RXFP1 mRNA expression could be detected in a dose dependent manner (FIG. 4A). To further confirm the optimal MOI, cAMP measurements were performed using a cAMP Glo™ assay kit from Promega. Manufacture's manual was followed. First NRVMs were seeded into a 96-well according to table 4. The cells were transduced with different MOI of RXFP1 virus ranging from MOI of 5,000 to 50,000 vg/cell and a fixed MOI of Luc control virus at 10,000 vg/cell. The virus was allowed to transduce the cell for 48 hours before the medium was changed. After 5 days the transduced NRVMs were treated with 100 nM of recombinant RLN for 30 minutes. To stimulate the traduced NRVMs, the medium was removed and 20 μL of RLN dissolved in induction buffer (1×PBS supplemented with 500 μM IBMX and 100 μM Ro 20-1724) was added to the cells to stimulate cAMP production. After 30 minutes, 20 μL of lysis buffer was added to all wells. The cells were lysed at room temperature for 15 minutes. 40 μL of detection buffer (Reaction buffer supplemented with Protein Kinase A) was added to all wells, and the plate was mixed by shaking for 1 minute and incubated at room temperature for 20 minutes. 80 μL of Kinase Glo® Reagent was added to all wells and the plate was mixed by shaking for 1 minute and incubated at room temperature for 10 minutes. The luminescence was measured with a luminometer. A significant increase in cAMP production was observed in samples that received at least 10,000 vg/cell RXFP1, but a decrease in cAMP production was detected with a MOI of 50,000 vg/cell (FIG. 2B). Thus, lowest viral MOI with both RXFP1 mRNA expression and significant increase in intracellular cAMP production following RLN stimulation was 10,000 vg/cell.

Ectopic RXFP1 Expression Vs Native RXFP1 Expression

[0107] NRVMs seeded in 6-well plates were transduced with RXFP1 virus and Luc control virus with the viral MOI of 10,000 vg/cell. The virus was allowed to transduce the cells for 48 hours before the medium was changed. After 5 days the transduced NRVMs were harvested and RXFP1 mRNA expression was quantified by qPCR using primers listed table 1. Significant increase in RXFP1 mRNA expression could be detected in RXFP1 virus treated cells compared to Luc control treated cells and untreated cells with native RXFP1 expression from the atria (neonatal rat atrial cardiomyocytes, NRAM) (FIG. 4C).

Optimal Concentration of RLN Stimulation Determined by cAMP Accumulation

[0108] cAMP measurement was performed using an cAMP Glo™ assay kit from Promega. Manufacture's manual was followed. First NRVCMs were seeded on a 96-well plate according to table 4. The cells were transduced with RXFP1 virus and Luc control virus with a viral MOI of 10,000 vg/cell. The virus was allowed to transduce the cell for 48 hours before the medium was changed. After 5 days the transduced NRVMs were treated with different amounts of recombinant RLN ranging from 1 pM to 10 nM for 30 minutes. To stimulate the transduced NRVMs, the medium was removed and 20 μL of RLN in induction buffer (1×PBS supplemented with 500 μM IBMX and 100 μM Ro 20-1724) were added to the cells to stimulate cAMP production. After 30 minutes, 20 μL of lysis buffer was added to all wells. The cells were lysed at room temperature for 15 minutes. 40 μL of detection buffer (Reaction buffer supplemented with Protein Kinase A) was added to all wells, and the plate was mixed by shaking for 1 minute and incubated at room temperature for 20 minutes. 80 μL of Kinase Glo® Reagent was added to all wells and the plate was mixed by shaking for 1 minute and incubated at room temperature for 10 minutes. The luminescence was measured with a luminometer. The results confirmed that a substantial increase in cAMP production was observed in RXFP1 virus and recombinant RLN treated cells compared to cells treated with Luc control virus and recombinant RLN (FIG. 4D). Peak stimulation of the RXFP1 receptor was observed at a recombinant RLN concentration of 10 nM. Interestingly, no further increase in cAMP accumulation with higher recombinant RLN concentrations was found.

Positive Inotropy from RXFP1 Gene Therapy with Recombinant RLN Treatment

[0109] NRVMs seeded in 6-well plates were transduced with RXFP1 virus and Luc control virus at a viral MOI of 10,000 vg/cell. The virus was allowed to transduce the cell for 48 hours before the medium was changed. After 5 days the transduced NRVMs were stimulated with 100 nM recombinant RLN for 5 hours before being harvested for immunoblotting using 1% SDS buffer (1% SDS, 1 mM EDTA, 1 mM EGTA supplemented with protease inhibitor and phosphatase inhibitor cocktail 2 and 3). Proteins were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting as previously described (Towbin et al., 1979, Proc. Natl. Acad. Sci. 76: 4350-4354). Membranes were probed with antibodies listed in table 3. Mouse secondary antibody coupled to Alexa Flour 680 (Invitrogen) and rabbit secondary antibody coupled to Dylight™ 800 (Cell Signaling) were used to detect the signals from the immunoblots utilizing Odyssey infrared imager (LI-COR) detection system. Images were processed using Odyssey imaging software. A significant increase in P-PLB(S16) was observed in the NRVM groups transduced with RXFP1 virus and treated with recombinant RLN (FIG. 4E-F). In line with in vivo experiments, molecular evidences from the in vitro experiments suggest that positive inotropy is a result of increased cAMP production and increased phospholamban phosphorylation at serine 16.

EXAMPLE 3 COMPARISON OF HUMAN AND RAT RXFP1 GENE THERAPY

Virus Production

[0110] For all in vitro experiments AAV6 serotype vectors were deployed. AAV6-CMV-MLC260-Rel1.sup.Rattus (designated AAV6-naRXFP1) was generated as follows. Optimized Rattus norvegicus Rel1 cDNA [NM_201417.1, National Center for Biotechnology Information (NCBI)] were synthesized and cloned into a pCDNA3.1 plasmid. After extensive in vitro testing, the transgene cassette was subcloned into an AAV transfer plasmid downstream of a cardiac myocyte-specific cytomegalovirus (CMV) enhancer coupled with a short 260 bp myosin light chain promoter (MLC260) and the whole construct was flanked by two ITRs. Recombinant vectors were generated by packaging of AAV-inverted terminal repeat recombinant genomes into AAV6 capsids using the two plasmids transfection protocol (Grimm et al., 2003, Mol. Ther. 7: 839-850). High titer vectors were produced in cell stacks (Corning) with polyethylenimine transfection. After 48 hours the vectors were harvested and purified by density filtration in an iodixanol gradient (Jungmann et al., 2017, Hum. Gene. Ther. Methods 28: 235-246). Viral titers from all viral vectors were quantified at the same time using a SYBR-green real time PCR assay (Bio-Rad) and expressed as viral genomes per milliliter (vg/mL).

Receptor Function Measurements

[0111] NRVMs seeded in 6-well plates were transduced with virus containing human and rat RXFP1 virus and Luc control virus at a viral MOI of 10,000 vg/cell. The virus was allowed to transduce the cell for 48 hours before the medium was changed. After 5 days the transduced NRVMs were stimulated with 100 nM recombinant RLN for 5 hours before being harvested for immunoblotting using 1% SDS buffer (1% SDS, 1 mM EDTA, 1 mM EGTA supplemented with protease inhibitor and phosphatase inhibitor cocktail 2 and 3). Proteins were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting as previously described (Towbin et al., 1979, Proc. Natl. Acad. Sci. 76: 4350-4354). Membranes were probed with antibodies listed in table 3 overnight. Mouse secondary antibody coupled to Alexa Flour 680 (Invitrogen) and rabbit secondary antibody coupled to Dylight™ 800 (Cell Signaling) were used to detect the signals from the immunoblots utilizing Odyssey infrared imager (LI-COR) detection system. Images were processed using Odyssey imaging software. Significant increase in P-PLB(S16) was observed in the NRVM groups transduced with human or rat RXFP1 virus and both treated with recombinant RLN (FIG. 5A and B), with no significant difference between human and rat RXFP1 virus.

EXAMPLE 4 ACTIVATION OF RXFP1 BY ML290 (HUMAN RXFP1 CHEMICAL AGONIST)

RXFP1 Activation by ML290 In Vitro

[0112] NRVMs seeded in 6-well plates were transduced with virus containing human RXFP1, rat RXFP1 and Luc control virus at the viral MOI of 10,000 vg/cell. The virus was allowed to transduce the cell for 48 hours before the medium was changed. After 5 days the transduced NRVMs were stimulated with 1 μM of ML290 for 30 minutes before being harvested for immunoblotting using 1% SDS buffer (1% SDS, 1 mM EDTA, 1 mM EGTA supplemented with protease inhibitor and phosphatase inhibitor cocktail 2 and 3). Proteins were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting as previously described (Towbin et al., 1979, Proc. Natl. Acad. Sci. 76: 4350-4354). Membranes were probed with antibodies listed in table 3 overnight. Mouse secondary antibody coupled to Alexa Flour 680 (Invitrogen) and rabbit secondary antibody coupled to Dylight™ 800 (Cell Signaling) were used to detect the signals from the immunoblots utilizing Odyssey infrared imager (LI-COR) detection system. Images were processed using Odyssey imaging software. Significant increase in P-PLB(S16) was detected in the NRVMs group transduced with human RXFP1 virus and treated with 1 μM ML290 (FIG. 6A and B). Marked increase in P-PLB(S16) was also observed in the NRVMs group transduced with rat RXFP1 virus and treated with 1 μM ML290. No difference in P-PLB(S16) was observed between human RXFP1 virus and treated with 1 μM ML290 and human RXFP1 virus and treated with 100 nM recombinant RLN.

EXAMPLE 5: INOTROPIC EFFECTS OF HURXFP1 GENE THERAPY WITH RLN ADMINISTRATION RESCUE HEART FAILURE

Virus Production

[0113] For all in vivo experiments AAV9 serotype vectors were deployed. The recombinant adeno-associated viral vectors AAV9-CMV-MLC260-FLAG-Rel1.sup.Rattus (designated AAV9-RXFP1) and AAV9-CMV-MLC260-FLAG-Rel1.sup.human (designated AAV9-huRXFP1) were generated as follows. Both optimized Rattus norvegicus and Homo sapiens Rel1 cDNA [NM_201417.1 and NM_021634.3, National Center for Biotechnology Information (NCBI)] were synthesized and cloned into a pCDNA3.1 plasmid with N-terminal FLAG-TAG. After extensive in vitro testing, the transgene cassette was subcloned into an AAV transfer plasmid downstream of a cardiac myocyte-specific cytomegalovirus (CMV) enhancer coupled with a short 260 bp myosin light chain promoter (MLC260) and the whole construct was flanked by two ITRs. Recombinant vectors were generated by packaging of AAV-inverted terminal repeat recombinant genomes into AAV9 capsids using the two plasmids transfection protocol (Grimm et al., 2003, Mol. Ther. 7: 839-850). High titer vectors were produced in cell stacks (Corning) with polyethylenimine transfection. After 48 hours the vectors were harvested and purified by density filtration in an iodixanol gradient (Jungmann et al., 2017, Hum. Gene. Ther. Methods 28: 235-246). Using the same method, the control vector AAV9-CMV-MLC260-FLAG-Luc.sup.Firefly (designated AAV9-LUC) was generated. Viral titers from all viral vectors were quantified at the same time using a SYBR-green real time PCR assay (Bio-Rad) and expressed as viral genomes per milliliter (vg/mL).

Heart Failure Model Generation and Experimental Set Up

[0114] Heart failure model was generated by transverse aortic constriction operation (TAC) previously described (Rockman et al., 1991, Proc. Natl. Acad. Sci. 88: 8277-8281). 8 weeks old C57BL/6 mice were ordered and weighted prior to the operation. TAC operation was performed on day 0 using 26-gauge blunt needle effectively reducing the diameter of the transverse aorta to approximately 0.46 mm (Rockman et al., 1994, Proc. Natl. Acad. Sci. 91(7): 2694-2698). Cardiac function measurements were performed before Transverse aortic Constriction (TAC) operation (day −2) before virus infusion (day 7), before pump implantation (day 28), and at 2-month follow up time point (day 55) (FIG. 7A). At day 7, animals were randomized to systemic injection of Luciferase control virus or RXFP1 virus. Osmotic pumps were implanted on day 28 with animals randomized to get either saline or recombinant relaxin pumps. Follow up assessments were performed on day 55 and the animals were sacrificed 3 days later. In addition, 17 mice served as control animals that were sham operated followed by either saline or recombinant relaxin pumps implantation.

Cardiac Function Measurements

[0115] Echocardiography was performed using Vevo 2100, VisualSonics. The mice were shaved on the chest and held in prone position. Warm ultrasound coupling gel (37° C.) was placed on the shaved chest area, and the MS400 transducer was positioned to obtain 2D B-mode parasternal long and short axis views and 1D M-mode short axis view. The ejection fraction (EF) was calculated from 6 consecutive heartbeats in the M-mode using LV trace function.

[0116] The EF of all TAC animals were gradually reduced starting from day 7 post operation (EF: 61.49±8.48%). Further reduction was detected in all TAC operated animals on day 28 post operation before the pump implantation (EF: 61.16±13.47). The follow up assessments at day 55 showed further reduction in EF in control groups treated with AAV9.LUC receiving either NaCl or RLN (LUC NaCl EF: 51.54±14.17 and LUC RLN EF: 50.02±18.31). Sight increase in EF was observed in RXFP1 groups treated with NaCl (EF: 60.25±16.57) and significant increase in EF was detected in RXFP1 groups treated with RLN (EF: 64.97±12.78) (FIG. 7B). Other parameters such as left ventricular internal diameter (LVIdD), Left ventricular mass corrected, and heart rate were also measured, but no significant difference was observed.

Molecular Analysis

RNA Expression

[0117] RNA was extracted using TRlzol® reagent (Ambion) from snap frozen tissue (10 mg) or cell lysate (2×10.sup.6 cells) according to the manufacturer's protocols. 1 μg of total the RNA was reverse transcribed into cDNA using the iScript cDNA-Synthesis Kit (Bio-Rad). iQ SYBR Green Supermix was used according to the manufacturer's protocol. A final volume of 15 μL per reaction consisted of 7.5 μL iQ SYBR Green Supermix, 1 μL of forward and reverse primers mix (final concentration of 300 nM each), and 6.5 μL diluted cDNA prepared (final concentration of 3.25 ng per reaction). The quantitative real-time PCR was performed in duplicate on Bio-Rad CFX96 real-time PCR detection system (Bio-Rad) The 2.sup.−ΔΔCT method was used to quantify relative gene expression levels between samples. Specificity of PCR products were confirmed by gel electrophoresis. Primers listed in Table 1 above were used for mRNA expression quantification and general RT-PCR program in Table 5 was used for all primers with only adjustment in annealing temperatures being made.

TABLE-US-00005 TABLE 5 RT-PCR program Number of Step Temperature Time Cycles Denaturation 95° C. 3:00  1x Denaturation 95° C. 0:10 Annealing Vary annealing Temperature 0:10 40x Elongation and reading 72° C. 0:30 Termination 95° C. 0:10  1x Melting Curve 65 .fwdarw. 95° C. with 0.5° C. 0:05 60x increment

[0118] Post-mortem mRNA expression analysis using RTPCR revealed a significant increase in RXFP1 mRNA expression within the ventricle of TAC animals treated with huRXFP1 virus (FIG. 7C). Compared to a TAC group treated with control virus alone, a significant decrease in BNP mRNA expression was observed in huRXFPI and RLN treated mice (FIG. 7D).

[0119] Other pathogenic cardiac remodeling genes expression was quantified. A trend toward decreasing cardiac remodeling genes ANP and β-MHC expression was detected in a TAC group that received both huRXFP1 gene therapy and RLN. Furthermore, a trend toward a decrease in collagen production proteins (Col1a1, Col3a1 and Postn) was also detected in both huRXFP1 gene therapy with and without RLN treated groups.

EXAMPLE 6: PROTECTIVE EFFECTS OF HURXFP1 WHEN HIGHLY EXPRESSED

Transgenic Animal Generation

[0120] FLAG huRXFP1 was cloned onto a place holder plasmid containing a full length α-MHC promoter flanked by a restriction site. After the correct clone was identified, the plasmid was propagated and the construct including the gene of interest and the promoter were excised from the plasmid using a restriction digest. The correct DNA fragment was identified and purified before given to the Animal facility at the University of Heidelberg for nuclear injection. The transgenic (TG) animal generation protocol was roughly based on Davis et al. (Davis et al., 2012, Circ. Res. 111(6): 761-777).

Heart Failure Model Generation and Experimental Set Up

[0121] Heart failure model was generated by transverse aortic constriction operation (TAC) previously described (Rockman et al., 1991, Proc. Natl. Acad. Sci. 88: 8277-8281). 8 weeks old C57BL/6 mice were ordered and weighted prior to the operation. TAC operation was performed on day 0 using a 27-gauge blunt needle effectively reducing the diameter of the transverse aorta to approximately 0.44 mm (Rockman et al., 1994, Proc. Natl. Acad. Sci. 91(7): 2694-2698). Cardiac function measurements were performed before Transverse Aortic Constriction (TAC) operation (day −2) 7 days after the operation (day 7), 28 days after the operation (day 28), and 42 days after the operation (day 42) (FIG. 8A). The animals were sacrificed 2 days after the last assessment. In addition, 20 mice served as control animals that were sham operated.

Cardiac Function Measurements

[0122] Echocardiography was performed using Vevo 2100, VisualSonics. The mice were shaved on the chest and held in prone position. Warm ultrasound coupling gel (37° C.) was placed on the shaved chest area, and the MS400 transducer was positioned to obtain 2D B-mode parasternal long and short axis views and 1D M-mode short axis view. The ejection fraction (EF) was calculated from 6 consecutive heartbeats in the M-mode using LV trace function.

[0123] The EF of all TAC animals were gradually reduced starting from day 7 post operation (EF: 62.80±12.31%). A further and significant reduction in cardiac function was detected in WT TAC operated animals on day 28 (EF: 58.59±11.70) compared to TG TAC operated animals (EF: 66.53±7.33). The follow up assessments at day 42 showed even further reduction in EF in WT TAC animals (EF: 53.79±14.97) compared to TG TAC animals (EF: 67.96±8.50) (FIG. 8B). Other parameters such as left ventricular internal diameter (LVIdD), Left ventricular mass corrected, and heart rate were also measured, but no significant different was observed.

Molecular Analysis

[0124] All RNA was analyzed using primers listed in table 1 above. Post-mortem mRNA expression analysis using RTPCR revealed a significant increase in huRXFP1 mRNA expression within the ventricle of all huRXFP1 transgenic animals (FIG. 8C). Compared to a WT TAC group, a significant decrease in ANP and BNP mRNA expression was observed in TAC operated huRXFPI transgenic animals (FIG. 8C and D).

[0125] Other pathogenic cardiac remodeling genes expression was quantified. Furthermore, a significant decrease in collagen production proteins (Col1a1 and Col3a1) was detected in TAC operated huRXFP1 TG animals (FIG. 8E and F).

EXAMPLE 7: POSITIVE INOTROPIC EFFECTS OF RXFP1 IMPROVES INTRACELLULAR CALCIUM HANDLING IN NEONATAL RAT VENTRICULAR CARDIOMYOCYTES

Isolation of Neonatal Rat Ventricular Cardiomyocytes (NRVCM)

[0126] Primary cultures of neonatal rat ventricular cardiac myocytes (NRVCM) were prepared from 1-2 days old Wistar rats (Charles River). The neonatal rats were euthanatized by decapitation and the heart removed and placed in 1× cold ADS solution. The atria and other vessels were removed, and the hearts were transferred into a new 1× cold ADS solution. The ADS solution was removed, and the ventricles were washed with 3 mL of cold ADS before being minced. Tissue fragments were digested in a T75 bottle with 30 mL of a digestion solution containing collagenase and pancreatin for 5 minutes at 37° C. The tissue fragments were allowed to settle on one side of the bottle and the solution removed and discarded. Fresh digestion solution was added, and the tissue fragments were digested for 20 minutes at 37° C. After 20 minutes, the tissue fragments were pipetted up and down 5 times at maximum power and the solution removed and filtered through a 40 μm cell strainer into a 50 mL falcon. 10 mL of FCS was added through the cell strainer and the solution was centrifuged at 1000 rpm for 5 minutes at room temperature. After centrifugation, the supernatant was removed, and 7 mL of FCS was added. The cell solution was placed in a 37° C. incubator awaiting further processing. The tissue fragments were digested at least 5 times in order to completely digest all the tissue pieces. After the digestion, all cell suspensions were combined and centrifuged at 1000 rpm for 5 minutes at room temperature. The supernatant was removed, and the cells were resuspended in 12 mL of cold 1×ADS. The fibroblasts and cardiac myocytes were separated using percoll density gradient. To obtain cardiomyocytes with high purity, two layers Percoll density gradient was performed. The gradient consisted of 63% red Percoll solution, and a 40.5% clear Percoll solution. 4 mL of red Percoll solution was added into each 15 mL falcon. Then 3 ml, of clear percoll solution was added using underlaying technique. 2 mL of cell solution containing 1× cold ADS was layered at a 45 degrees angle onto of the gradient. The falcons were centrifuged at 2400 rpm for 30 minutes at 37° C. deceleration speed of 0. The upper stromal cells band including fibroblasts was removed by aspiration. The lower cardiomyocytes band was kept and washed twice with cold 1×ADS before being resuspended in a warm Medium 199 supplemented with 10% v/v fetal calf serum, 100 U/mL penicillin, 100 μg/streptomycin, 2 mM L-glutamine, and 1 mM calcium chloride. The cells were counted manually in a Neubauer chamber and plated accordingly (Table 6). The NRVCM were cultured at 37° C. and 5% CO.sub.2 humidified atmosphere. After 48 hours fetal calf serum was reduced to 0.5% and cells were cultured for 2-5 days. Two days after the cells were plated, the medium was removed, and the cells were washed once with warm PBS before starving medium M199 supplemented with 0.5% FCS, 1% penicillin/streptomycin, 1% L-glutamine, 1 mM CaCl.sub.2 was added. In the following, the medium was changed every two days until the cells were harvested. Cells were used for the maximum of 7 days before being discarded.

TABLE-US-00006 TABLE 6 Optimal plating density for NRVCMs calcium transient Table 6: Optimal plating density for NRVCMs calcium transient Area per Number of Volume per well (cm.sup.2) cells seeded well (mL) Glass-bottom 24 2 × 10.sup.4 3 dish

Ectopic RXFP1 Expression

[0127] NRVCMs seeded in glass-bottom dishes were transduced with RXFP1 virus and LUC control virus with the viral MOI of 10,000 vg/cell. The virus was allowed to transduce the cells for 48 hours before the medium was changed.

Ca.SUP.2+ Transient Measurement in NRVCMS

[0128] Intracellular Ca.sup.2+ transients were measured according to Yu et al. with modifications to fit available apparatus (Yu et al., 2013, J. Biol. Chem. 288(31): 22481-22492). NRVCMs were transduced with RXFP1 or LUC AAV vectors for 5 days. The medium was changed every two days. On the day of the experiment, the transfected NRVCMs were loaded with 1 μM Fura 2 AM in M199 medium at 37° C. for 15 minutes protected from light. After loading, the cells were washed with warm M199 medium for 15 minutes at 37° C. protected from light and connected to an electrode system on the table of an inverted fluorescence microscope (IX70 Olympus). The cells were electrically stimulated with 1 Hz bipolar. Using a monochromator, the Fura 2 AM loaded cells were excited at 380 nm. The alternative measurement of the bispectral emission (340 nm/380 nm) was performed with the fluorescence microscope for a period of 5 minutes and the emission ratio was calculated with the software TILL vision. The data were exported to an online program written by Dr. Martin Bush. The following parameters were analyzed: a) the amplitude of the transient (Δ[340 nm/380 nm]) and b) the diastolic Ca.sup.2+ ([340 nm/380 nm]).

[0129] A significant increase in calcium amplitude was observed in a group of NRVCMs treated with both RXFP1 and RLN compared to LUC treated with both NaCl and RLN (FIG. 9A and B). Additionally, there was no difference in diastolic calcium detected (FIG. 9C). Lastly, addition mRNA quantification using rat RXFP1 primer from table 1 above was performed and significant rat RXFP1 mRNA expression was observed in NRVCMs that received RXFP1 treatment (FIG. 9D).

EXAMPLE 8: POSITIVE INOTROPIC EFFECTS OF RXFP1 IMPROVE INTRACELLULAR CALCIUM HANDLING IN ADULT CARDIOMYOCYTES

Isolation of Adult Mouse Cardiomyocytes

[0130] Adult mouse cardiac cells were isolated a modified protocol from Harding et al. (Harding et al., 1990, Cardioscience. 1: 49-54). The mouse heart was perfused with digestion buffer in the Langendorff technique, thus enabling dissociation of single cardiac cells. An adult male mouse (30-35 g) was quickly euthanatized by cervical dislocation. Incision areas on the abdomen and thorax were sterilized with 70% ethanol and opened with lateral cuts by scissors. The abdominal Vena Cava was exposed, and heparin was given I.V. Then the heart was exposed and cut out with a large portion of the aorta and quickly placed into a 6 cm Petri dish with 10 mL of room temperature perfusion buffer. The aorta was slid onto the cannula of the Langendorff apparatus and fixed with a double knots surgical suture. Perfusion was started immediately with a drip rate of ˜3.5 mL/min. Filling of the coronary arteries was checked and evaluated in order to determine, if the heart was correctly positioned on the cannula. In order to remove residual serum traces and free Ca2+, the heart was washed with perfusion buffer for 1 minute. After that the heart was perfused with digestion buffer containing a mixture of liberase (mixture of collagenases) and trypsin solution for 10 minutes. The perfusion was stopped, when the heart became flaccid and easily penetrable by fine tip forceps. The heart was quickly removed from the cannula and the atria were cut away using surgical scissor. The heart was minced into 1 mm pieces and 2.5 mL stop solution 1 containing 1% BSA and 50 μM CaCl.sub.2) was added to the solution to stop the digestion process. The heart was pipetted up and down using a cut 1 mL pipette tip for 3 minutes. The solution was filtered into a 50 mL falcon and left to precipitate for 15 minutes at room temperature. After the pellet formed, all but 1.5 mL of supernatant was removed and 5 mL of stop solution 2 containing 0.5% BSA and 38 μM CaCl.sub.2) was added to the cells. Calcium treatment was performed after every 4 minutes by adjusting calcium concentration within the solution starting from 62 μM CaCl.sub.2) at 4 minutes, 114 μM CaCl.sub.2) at 8 minutes, 191 μM CaCl.sub.2) at 12 minutes, 498 μM CaCl.sub.2) at 16 minutes, 960 μM CaCl.sub.2) at 20 minutes. After the last adjustment the cell was left to precipitate in the incubator for 12 minutes, and the cells were plated onto 4 laminin coated calcium transient glass bottom dishes. The cells were allowed to attach for 1 hour.

Ca.SUP.2+ Transient Measurement in Adult Mouse Cardiomyocytes

[0131] Intracellular Ca.sup.2+ transients were measured according to Yu et al. with modifications to fit available apparatus (Yu et al., 2013, J. Biol. Chem. 288(31): 22481-22492). On the day of the experiment, the adult cardiomyocytes were loaded with 1 μM Fura 2 AM in BDM free medium at 37° C. for 20 minutes protected from light. After loading, the cells were washed with warm BDM free medium for 20 minutes at 37° C. Next the dish was protected from light and connected to an electrode system on the table of a high throughput inverted fluorescence microscope (Cytocypher). The cells were electrically stimulated with 1 Hz bipolar. Using a monochromator, the Fura 2 AM loaded cells were excited at 380 nm. The alternative measurement of the bispectral emission (340 nm/380 nm) was performed with the fluorescence microscope for baseline and after 1, 5, and 10 minutes stimulation with NaCl, 100 nM RLN, and 10 nM ISO. The transients were calculated using transient calculated software from the cytocypher. The following parameters were analyzed: a) Peak Height, b) Departure velocity, and c) Return velocity. A trend toward an increase in all parameters analysis was observed in the isolated TG adult cardiomyocytes after 1 minute of RLN treatment (FIG. 10A-C).

EXAMPLE 9: RXFP1 EXPRESSION ANALYSIS IN HUMAN SAMPLES

Molecular Analysis

RNA Expression

[0132] RNA was extracted using TRIzol® reagent (Ambion) from snap frozen tissue (10 mg) according to the manufacturer's protocols. 1 μg of total the RNA was reverse transcribed into cDNA using the iScript cDNA-Synthesis Kit (Bio-Rad). iQ SYBR Green Supermix was used according to the manufacturer's protocol. A final volume of 15 μL per reaction consisted of 7.5 μL iQ SYBR Green Supermix, 1 μL of forward and reverse primers mix (final concentration of 300 nM each), and 6.5 μL diluted cDNA prepared (final concentration of 3.25 ng per reaction). The quantitative real-time PCR was performed in duplicate on Bio-Rad CFX96 real-time PCR detection system (Bio-Rad) The 2.sup.−ΔΔCT method was used to quantify relative gene expression levels between samples. Specificity of PCR products were confirmed by gel electrophoresis. Listed primers in table 7 were used for mRNA expression quantification and general RT-PCR program in table 8 was used for all primers with only adjustment in annealing temperatures being made.

TABLE-US-00007 TABLE 7 Primers for human samples RT-PCR SEQ Genes Primers Template ID NO Homo Forward primer: NM_002521.3 32 sapiens 5′-CTTTCCTGGGAGGTCGTTCC-3′ BNP Reverse primer:  33 5′-GTTGCGCTGCTCCTGTAAC-3′ Homo Forward primer: NM_000194.3 34 sapiens 5′-CTCATGGACTAATTATGGACAGGAC-3′ Hprt1 Reverse primer: 35 5′-GCAGGTCAGCAAAGAATTTATAGCC-3′ Homo Forward primer2: NM_021634.4 36 sapiens 5′-GGACCTGAAGGAGCTGTCAC-3′ Rel1 Reverse primer2: 37 5′-TAGGCTGAGAGACTTGAGTTTGAC-3′

TABLE-US-00008 TABLE 8 RT-PCR program Number of Step Temperature Time Cycles Denaturation 95° C. 3:00  1x Denaturation 95° C. 0:10 Annealing Vary annealing Temperature 0:10 45x Elongation and reading 72° C. 0:30 Termination 95° C. 0:10  1x Melting Curve 65 .fwdarw. 95° C. with 0.5° C. 0:05 60x increment

[0133] Ventricular samples from 48 explanted hearts were obtained from University of Heidelberg Biobank. 10 Atria biopsy samples were obtained from AG Constanze Schmidt and 7 healthy ventricular samples were purchased from Biozol (1), Biokatz (5), and amsbio (1). mRNA expression analysis from explanted heart and cardiac atria biopsy using RTPCR revealed a significant increase in BNP mRNA expression within the ventricle of explanted heart compared to healthy control (FIG. 11A). RXFP1 mRNA was significantly expressed in the failing atria compared to non-failing ventricle (FIG. 11B). Lastly, there is a trend toward a decrease in RXFP1 mRNA in the failing ventricle compared to the atria (FIG. 11B). Thus, there is a higher expression of RXFP1 mRNA in the heart.

EXAMPLE 10: HEMODYNAMICS CHANGES IN HURXFP1 TG ANIMALS AFTER RLN ADMINISTRATION

Transgenic Animal Generation

[0134] FLAG huRXFP1 was cloned onto a place holder plasmid containing a full length α-MHC promoter flanked by restriction sites. After the correct clone was identified, the plasmid was propagated and the construct including the gene of interest and the promoter were excised from the plasmid using restriction digest. The correct DNA fragment was identified and purified before given to the Animal facility at the University of Heidelberg for nuclear injection. The transgenic (TG) animal generation protocol was based on Davis et al. (Davis et al. (2012), Circ. Res. 111(6): 761-777).

Cardiac Function Measurements

[0135] Pressure-volume loop (PV loop) measurement was performed modified from Abraham and Mao (Abraham and Mao (2015), J. Vis. Exp. (103): e52942). The animal was anesthetized according to the body weight with a cocktail of medetomidine, midazolam, and fentanyl intraperitoneally 15 minutes prior to the measurements. After the righting reflex was lost, the mouse was secured to the operating table with surgical tape. Prior to the measurement, the catheter tip was equilibrated in warm saline for 30 minutes. The neck and chest area was cleaned with ethanol and the fur was removed. An incision was made over the right carotid from mandible to sternum. The surrounding tissue was dissected to expose the right carotid, and the vagus nerve, which runs adjacent to the carotid, was cut. Sterile 6.0 silk suture was placed around the distal end (away from the chest) of the carotid artery, tied and secured. An additional suture was placed below the first suture on the carotid artery. The second suture was loosely tightened and clamped the carotid artery proximally to the second suture. After ensuring that the carotid artery was clamped both proximally and distally, a small incision was made into the carotid artery proximally to the first suture. The catheter tip was inserted into the vessel through the incision and the catheter was secured using the second suture. The catheter was gently advanced into the left ventricle through the carotid guided by the PV loop tracing to ensure correct placement. Different filter settings were applied in order to obtain calculable loops. PV loops recording was started and allowed to stabilize for 10 minutes. After 10 minutes of steady state, 100 μL of 10 μg RLN was administered intraperitoneally (I.P.). The PV loops were continuously recorded throughout the experiment until 10 minutes after administration of the tested substances.

[0136] Heart rate (HR),

[00001]$\frac{\mathrm{dP}}{\mathrm{dt}}$

maximum, and

[00002]$\frac{\mathrm{dP}}{\mathrm{dt}}$

minimum were calculated according to the manufacturer's recommendation (Transonic). The average of 20 PV loops between inhalation and exhalation was used for the calculation. A significant increase in HR and

[00003]$\frac{\mathrm{dP}}{\mathrm{dt}}$

maximum was observed in a group of RXFP1 transgenic animals receiving RLN compared to WT animals receiving RLN.

LITERATURE

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