TREATMENT OF TACHYCARDIA

Abstract

The invention provides compounds which are selective PDE2 inhibitors for use in the treatment of tachycardia or tachyarrhythmia. Such compounds are particularly suitable for use in the treatment of any of the following conditions: atrial tachycardia, atrial fibrillation, atrial flutter, paroxysmal supraventricular tachycardia, premature ventricular contractions (PVCs), ventricular fibrillation and ventricular tachycardia, and may be used alone or in combination therapy with other conventional cardiovascular drugs, e.g. beta-blockers. In particular, the invention provides compounds which are selective PDE2 inhibitors for use in the treatment of ventricular tachycardia in patients who are suffering from, or who are at risk of suffering from heart failure, CPVT or long QT syndrome.

Claims

1. A compound which is a selective PDE2 inhibitor for use in the treatment or prevention of ventricular tachycardia in a subject.

2. A compound for use as claimed in claim 1, wherein said compound has a selectivity for inhibiting the activity of PDE2 (e.g. human PDE2) which is at least 10-fold compared to at least one other PDE type (e.g. at least one other human PDE type).

3. A compound for use as claimed in claim 2, wherein said compound has a selectivity for inhibiting the activity of PDE2 (e.g. human PDE2) which is at least 10-fold compared to all other PDE types (e.g. compared to all other human PDE types).

4. A compound for use as claimed in claim 2 or claim 3, wherein said selectivity is at least 20-fold, preferably at least 30-fold, more preferably at least 50-fold, e.g. at least 100-fold.

5. A compound for use as claimed in any one of the preceding claims, wherein said compound inhibits PDE2, e.g. human PDE2, with an IC.sub.50 value of less than about 100 nM, preferably less than about 50 nM, e.g. less than about 10 nM.

6. A compound for use as claimed in any one of the preceding claims, wherein said compound is selected from any of the following, their pharmaceutically acceptable salts or prodrugs thereof: TABLE-US-00002 Compound Name BAY 60-7550 2-(3,4- dimethoxybenzyl)-7- [(2R,3R)-2-hydroxy- 6-phenylhexan-3-yl]- 5-methylimidazo[5,1- f][1,2,4]triazin-4(3H)- one Lu AF64280 PF-05180999 TAK-915 N-((1S)-1-(3-fluoro-4- (trifluoromethoxy) phenyl)-2- methoxyethyl)-7- methoxy-2-oxo-2,3- dihydropyrido[2,3- b]pyrazine-4-(1H)- carboxamide N-(1S)-2-hydroxy-2- methyl-1-(4- (trifluoromethoxy)- phenyl)propyl)-5-(1H- pyrazol-1-yl) pyrazolo[1,5- a]pyrimidine-3- carboxamide N-(1S)-2-hydroxy-2- methyl-1-(4- (trifluoromethoxy)- phenyl)propyl)-5-(4- methyl-1H-1,2,3- triazol-1- yl)pyrazolo[1,5- a]pyrimidine-3- carboxamide N-(1S)-2-hydroxy-2- methyl-1-(4- (trifluoromethoxy)- phenyl)propyl)-5-(3- methyl-1H-1,2,4- triazol-1- yl)pyrazolo[1,5- a]pyrimidine-3- carboxamide ND-7001 3-(8-methoxy-1- methyl-2-oxo-7- phenyl-2,3-dihydro- 1H-benzo[e]- [1,4]diazepin-5- yl)benzamide PDM-631 ((S)-3-cyclopropyl-6- methyl-1-(1-(4- (trifluoromethoxy) phenyl)propan-2-yl)- 1,5-dihydro-4H- pyrazolo[3,4-d] pyrimidin-4-one DNS-8254 (5S)-1-[(3-bromo-4- fluorophenyl) carbonyl]-3,3- difluoro-5-{5-methyl- [1,2,4]triazolo[1,5- a]pyrimidin-7- yl}piperidine 1-[2,3-dihydro-1- benzofuran-5- yl)carbonyl]-3-{5- methyl-[1,2,4]- triazolo[1,5- a]pyrimidin-7- yl}piperidine 6-[(3-{5-methyl- [1,2,4]triazolo[1,5- a]pyrimidin-7- yl}piperidin-1- yl)carbonyl]quinolone EHNA (erythro-9-(2- hydroxy-3- nonyl)adenine)

7. A compound for use as claimed in claim 1, wherein said compound is selected from TAK-915 (N-((1S)-1-(3-fluoro-4-(trifluoromethoxy)phenyl)-2-methoxyethyl)-7-methoxy-2-oxo-2,3-dihydropyrido[2,3-b]pyrazine-4-(1H)-carboxamide); ND-7001 (3-(8-methoxy-1-methyl-2-oxo-7-phenyl-2,3-dihydro-1H-benzo[e]-[1,4]diazepin-5-yl)benzamide); PF-05180999 or Lu AF64280 as defined in claim 6; and their pharmaceutically acceptable salts and prodrugs thereof.

8. A compound for use as claimed in claim 1, wherein said compound is TAK-915 (N-((1S)-1-(3-fluoro-4-(trifluoromethoxy)phenyl)-2-methoxyethyl)-7-methoxy-2-oxo-2,3-dihydropyrido[2,3-b]pyrazine-4-(1H)-carboxamide), or PF-05180999 as defined in claim 6, or a pharmaceutically acceptable salt or prodrug thereof.

9. A compound for use as claimed in claim 1, wherein said compound is BAY 60-7550 (2-(3,4-dimethoxybenzyl)-7-[(2R,3R)-2-hydroxy-6-phenylhexan-3-yl]-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one), ND-7001 (3-(8-methoxy-1-methyl-2-oxo-7-phenyl-2,3-dihydro-1H-benzo[e]-[1,4]diazepin-5-yl)benzamide), PF-05180999 as defined in claim 6, or Lu AF64280 as defined in claim 6, or a pharmaceutically acceptable salt or prodrug thereof.

10. A compound for use as claimed in any one of the preceding claims in the treatment of a subject who has previously suffered a myocardial infarction, a subject who has heart failure, or a subject predisposed to tachycardia, e.g. a subject having a genetic predisposition to catecholaminergic polymorphic ventricular tachycardia (CPVT).

11. A compound for use as claimed in any one of claims 1 to 9 in the treatment of a subject suffering from, or at risk of suffering from, heart failure, CPVT or long QT syndrome,

12. A compound for use as claimed in claim 10 or claim 11, wherein said subject is selected from any of the following: a subject previously diagnosed with, and/or treated for, a cardiac arrhythmia, e.g. a subject undergoing treatment with an anti-arrhythmic drug such as a beta-blocker; a subject who has an implanted cardiac defibrillator (ICD); and a subject undergoing long term treatment for a cardiac arrhythmia, e.g. a subject that has been undergoing treatment with a beta-blocker for at least 6 months.

13. A compound for use as claimed in 12, wherein said subject has an implanted cardiac defibrillator (ICD) and is undergoing treatment with an anti-arrhythmic drug, e.g. a beta-blocker.

14. A compound for use as claimed in claim 13, wherein said beta-blocker is a β.sub.1-selective beta-blocker, e.g. Metoprolol.

15. A compound for use as claimed in any one of the preceding claims, wherein said subject is a mammalian subject, preferably a human.

16. A compound for use as claimed in any one of the preceding claims in a combination therapy with one or more cardiovascular drugs, preferably a drug for the treatment of hypertension, heart failure, arrhythmia and/or post infarction myocardial reperfusion syndrome, e.g. wherein said drug is selected from any of the following: beta-blockers, calcium antagonists, ACE-inhibitors, ATII/-blockers and anti-arrhythmic drugs.

17. A compound for use as claimed in claim 16, wherein the beta-blocker is a β.sub.1-selective beta-blocker, e.g. Metoprolol.

18. A pharmaceutical composition comprising a selective PDE2 inhibitor as defined in any one of claims 1 to 9, together with one or more cardiovascular drugs, e.g. an anti-arrhythmic drug, and optionally in combination with at least one pharmaceutically acceptable carrier or excipient.

19. A composition as claimed in claim 18, wherein the anti-arrhythmic drug is a beta-blocker, preferably a β.sub.1-selective beta-blocker, e.g. Metoprolol.

20. A method of treatment of ventricular tachycardia in a subject in need of such treatment (preferably a mammalian subject, e.g. a human), said method comprising the step of administering to said subject a selective PDE2 inhibitor as defined in any one of claims 1 to 9, or a pharmaceutical composition as claimed in claim 18 or claim 19.

21. A method of treatment as claimed in claim 20, wherein said subject has previously suffered a myocardial infarction, has heart failure, or is predisposed to tachycardia, e.g. wherein said subject has a genetic predisposition to catecholaminergic polymorphic ventricular tachycardia (CPVT).

22. A method of treatment as claimed in claim 20, wherein said subject is suffering from, or is at risk of suffering from, heart failure, CPVT or long QT syndrome.

23. A method of treatment as claimed in any one of claims 20 to 22, wherein said subject is selected from any of the following: a subject previously diagnosed with, and/or treated for, a cardiac arrhythmia, e.g. a subject undergoing treatment with an anti-arrhythmic drug such as a beta-blocker; a subject who has an implanted cardiac defibrillator (ICD); and a subject undergoing long term treatment for a cardiac arrhythmia, e.g. a subject that has been undergoing treatment with a beta-blocker for at least 6 months.

24. A method of treatment as claimed in 23, wherein said subject has an implanted cardiac defibrillator (ICD) and is undergoing treatment with an anti-arrhythmic drug, preferably a beta-blocker, e.g. a β.sub.1-selective beta-blocker such as Metoprolol.

25. A method of treatment as claimed in any one of claims 20 to 24, wherein said method additionally comprises the step of administering to said subject (e.g. simultaneously, separately or sequentially) one or more cardiovascular drugs, preferably a drug for the treatment of hypertension, heart failure, arrhythmia and/or post infarction myocardial reperfusion syndrome, e.g. wherein said drug is selected from any of the following: beta-blockers, calcium antagonists, ACE-inhibitors, ATII/-blockers and anti-arrhythmic drugs.

26. A method of treatment as claimed in claim 25, wherein the beta-blocker is a β.sub.1-selective beta-blocker, e.g. Metoprolol.

27. Use of a selective PDE2 inhibitor as defined in any one of claims 1 to 9 or a pharmaceutical composition as claimed in claim 18 or claim 19 in the manufacture of a medicament for use in the treatment of ventricular tachycardia in a subject.

28. Use as claimed in claim 27, wherein said subject is as defined in any one of claims 21 to 24.

29. Use as claimed in claim 27 or claim 28, wherein said treatment additionally comprises administration to said subject (e.g. simultaneously, separately or sequentially) of one or more cardiovascular drugs, preferably a drug for the treatment of hypertension, heart failure, arrhythmia and/or post infarction myocardial reperfusion syndrome, e.g. wherein said drug is selected from any of the following: beta-blockers, calcium antagonists, ACE-inhibitors, ATII/-blockers and anti-arrhythmic drugs.

30. Use as claimed in claim 29, wherein the beta-blocker is a β.sub.1-selective beta-blocker, e.g. Metoprolol.

Description

[0070] The following Examples are given by way of illustration only and with reference to the accompanying figures in which:

[0071] FIG. 1: NKA currents are regulated by cAMP and local AKAP-bound PKA. A) Outline of protocol for NKA current measurements. Isolated cardiomyocytes were voltage clamped at −20 mV and externally superfused and internally dialyzed with solutions with symmetrical Na.sub.+ concentrations (left panel). NKA currents were measured by removing K.sup.+ from the superfusate (right panel). B) Effect of increasing concentrations of cAMP on NKA currents. *=p<0.05 to 0 cAMP. 6-13 ARVMs from 2-5 rats. C) Effect of 20 μM superAKAP on the NKA current. 6-7 ARVMs from 2 rats. *=p<0.05 to 100 μM cAMP.

[0072] FIG. 2: PDE2 regulates NKA activity. A) Effect of three different PDE2 inhibitors on the NKA current. 5-8 ARVMs from 3 rats. NKA currents from control vs Bay 60-7550 and control vs PF05180999 are paired. *=p<0.05 to control. B) NKA currents in PDE2KO vs WT mice. 7-8 myocytes from 3 mice. *=p<0.05 to WT. C) Effect of PDE3 and PDE4 inhibition on the NKA current. D) Phosphorylation at ser68 on phospholemman (PLM) after treatment with isoprenaline and PDE inhibitors.

[0073] FIG. 3: NKA and PDE2 colocalize and interact. A-B) Proximity ligation assay of NKA and PDE2 in ARVMs. *=p<0.05 to experiments with no or single antibody. C) Co-immunoprecipitation of NKA and PDE2 in HEK293 cells.

[0074] FIG. 4: PDE2 inhibition reduces Ca.sup.2+ transient amplitude and SR Ca.sup.2+ load. A) Effect of Bay 60-7550 on Ca.sup.2+ transient amplitude (left) (12 ARVMs from 3 rats), Ca.sup.2+ extrusion rate (middle) (12 ARVMs from 3 rats) and SR Ca.sup.2+ load (right) (11 ARVMs from 3 rats) in ARVMs. *=p<0.05 to control. B-C) Representative tracings of Bay 60-7550 effect on Ca.sup.2+ transients (B) and tracing showing typical caffeine response (C). D) Ca.sup.2+ transient amplitude (left) (14-16 myocytes from 3 mice), Ca.sup.2+ extrusion rate (middle) (14-16 myocytes from 3 mice) and SR Ca.sup.2+ load (right) (11-14 myocytes from 3 mice) in PDE2KO vs WT. *=p<0.05 to WT.

[0075] FIG. 5: PDE2 inhibition increases NCX-dependent Ca.sup.2+ extrusion. A) Representative tracings of showing protocol for measurements of the activity of the main Ca.sup.2+ extruding proteins. B) Effect of Bay 60-7550 on SERCA2 activity (left) (10 ARVMs from 3 rats), NCX activity (middle) (11 ARVMs from 3 rats) and non-SERCA2/non-NCX Ca.sup.2+ extrusion (right) (5 ARVMs from 2 rats). *=p<0.05 to control. C) SERCA2 activity (left) (10-14 myocytes from 3 mice), NCX activity (middle) (10-14 myocytes from 3 mice) and non-SERCA2/non-NCX Ca.sup.2+ extrusion (right) (4-5 myocytes from 3 mice) in PDE2KO vs WT. *=p<0.05 to WT. D) Intracellular Na.sup.+ measured with SBFI. Representative tracings (left) and average data (right). 6 ARVMs from 3 rats.

[0076] FIG. 6: No effect of PDE2 inhibition on LTCC and RyR activity. A-B) Representative tracings (A) and average data (B) of LTCC with Bay 60-7550 treatment. 5 ARVMs from 3 rats. C-D) Representative tracings (C) and average data (D) of Ca.sup.2+ sparks frequency with Bay 60-7550 treatment. 6 ARVMs from 2 rats. E) Average data of Na.sup.+ current with Bay 60-7550. 6-7 ARVMs from 3 rats.

[0077] FIG. 7: PDE2 inhibition reduces cellular arrhythmias in post-banding HF and AnkB.sup.+/−. A) NKA current with Bay 60-7550 in myocytes from post-banding HF (6 myocytes from 3 mice) and SHAM (2 myocytes from 1 mouse). *=p<0.05 to control. B) Effect of Bay 60-7550 on Ca.sup.2+ transient amplitude (left) (11-12 myocytes from 3 mice) and Ca.sup.2+ extrusion rate (right) (10-12 myocytes from 3 mice) in post-banding HF myocytes. *=p<0.05 to control. C) Representative tracing of protocol for detecting Ca.sup.2+ waves from post-banding HF myocytes. D) Ca.sup.2+ wave frequency in isolated myocytes from post-banding HF mice (11-12 myocytes from 3 mice), AnkB.sup.+/− mice (18 myocytes from 3 mice) and WT (AnkB.sup.+/+) mice (11-13 myocytes from 3 mice). *=p<0.05 to control.

[0078] FIG. 8: PDE2 inhibition protects against ventricular tachycardia and death in post-banding HF mice. A) Protocol for in vivo arrhythmias in post-banding HF mice. B) Representative ECG tracings from HF mice showing examples of VTs (bidirectional VT and multifocal VT) and sinus rhythm. C) Ventricular tachycardia and death (left) and QT time (right) with Bay 60-7550 in HF mice (n=5 in both groups). *=p<0.05 to vehicle. D) Ventricular tachycardia (left) and QT interval (right) with PF05180999 in HF mice (n=5 in both groups).

[0079] FIG. 9: PDE2 inhibition protects against ventricular tachycardia in AnkB.sup.+/− mice. A) Protocol for in vivo arrhythmias in AnkB.sup.+/− mice. B) Ventricular tachycardia (left) and QT interval (right) with Bay 60-7550 in AnkB.sup.+/− mice (n=8 in both groups). *=p<0.05 to vehicle. D) Ventricular tachycardia (left) and QT interval (right) with PF05180999 in AnkB.sup.+/− mice (n=5 in both groups). *=p<0.05 to vehicle.

[0080] FIG. 10: PDE2 inhibition increases NKA current and prevent cellular arrhythmias through local pools of cAMP. A) Average data (left) and representative tracing (right) showing global intracellular cAMP levels in ventricular myocytes from mice after treatment with Bay 60-7550 (2 mice, 2 repeats per mouse). Isoprenaline was used as a positive control. B) Co-immunoprecipitation between NKA and PKA catalytic site and PKA RII. C) NKA currents with EHNA after treatment with superAKAP/RIAD (5-8 ARVMs from 3 rats). *=p<0.05 to RIAD. D) Ca.sup.2+ waves in myocytes with Bay 60-7550 from post-banding HF mice (10-14 myocytes from 3 mice) and AnkB.sup.+/− mice (10-13 myocytes from 2 mice) after pre-treatment with superAKAP. *=p<0.05 to superAKAP.

[0081] FIG. 11: PDE2 inhibition no longer protects against ventricular tachycardia in post-banding HF mice and AnkB.sup.+/− mice after pre-treatment with superAKAP. A) Protocol for in vivo arrhythmias in post-banding HF mice and AnkB.sup.+/− mice with Bay 60-7550 and superAKAP/scramble. B) Ventricular tachycardia (left) and QT interval (right) with Bay 60-7550 and superAKAP/scramble in HF mice (n=6 in both groups). *=p<0.05 to vehicle. D) Ventricular tachycardia (left) and QT interval (right) with with Bay 60-7550 and superAKAP/scramble in AnkB.sup.+/− mice (n=5 in both groups).

[0082] FIG. 12: Proposed mechanism for local PDE2 regulation of NKA. A) We propose that PDE2 and NKA assemble in a common domain, where PDE2 locally regulates cAMP levels and AKAP-bound PKA-RII activity. PKA-RII phosphorylates phospholemman, which regulates NKA activity. B) With superAKAP, which disrupts PKA-RII from AKAPs, PDE2 is no longer able to regulate NKA.

[0083] FIG. 13: Increased PDE2-mRNA expression in human cardiac hypertrophy and ageing and in rat post-banding HF. Data from mRNA sequencing from isolated and sorted cardiomyocyte nuclei.

[0084] FIG. 14: PDE2 inhibition protects against ventricular tachycardia in post-banding HF mice. Ventricular tachycardia with historical controls (n=15), ND-7001 (n=5) and LuAF64280 (n=3) in HF mice. *=p<0.05 to control.

[0085] FIG. 15: PDE2 inhibition is superior to beta-blockers in preventing Ca2+/− induced ventricular arrhythmias A) Protocol for chronic benchmarking study in AnkB+/− mice. B) Representative tracings of ECG recordings in the four treatment groups. C) Average data showing presence of ventricular tachyarrhythmias and ventricular tachycardias in the four treatment groups. Control 11 mice, Metoprolol 15 mice, Bay 60-7550 13 mice, Bay 60-7550+ Metoprolol 15 mice. D) QT time in the four treatment groups with injection of treatment (left) or caffeine (right). *=p<0.05. #=p<0.01.

[0086] FIG. 16: No effect of PDE2 inhibition RyR activity. A) Representative tracings and average data of PDE2 inhibition on Ca.sup.2+ spark frequency, Ca.sup.2+ transient amplitude and SR Ca.sup.2+ load in field stimulated ARVMs. 18 ARVMs from 3 rats. *=p<0.05 to control. B) Representative tracings and average data of PDE2 inhibition on Ca.sup.2+ spark frequency and SR Ca.sup.2+ load in non-stimulated ARVMs. 19 (Bay 60-7550) and 22 (control) ARVMs from 3 rats. C) Representative tracings and average data of PDE2 inhibition on Ca.sup.2+ spark frequency in saponin-permeabilized ARVMs. 25 (Bay 60-7550) and 24 (control) ARVMs from 3 rats.

[0087] FIG. 17: PDE2 inhibition has no effect on other anti-arrhythmic targets. A) Effect of PDE2 inhibition on LTCC. 8 (control) and 10 (Bay 60-7550) ARVMs from 3 rats (both groups). B) Effect of PDE2 inhibition on background K+ currents 7 (both groups) ARVMs from 3 rats. C) Effect of PDE2 inhibition on Na.sup.+ currents. 7 (control) and 8 (Bay 60-7550) ARVMs from 3 rats. D) Effect of PDE2 inhibition on action potential duration. *=p<0.05 vs control.

[0088] FIG. 18: PDE2 inhibition protects against ventricular tachycardia in CPVT mice. Ventricular tachycardia with controls (n=6) and Bay 60-7550 (n=7) in CPVT mice. *=p<0.05 vs control.

EXAMPLES

Example 1

[0089] The following methods were carried out to demonstrate that phosphodiesterase 2A inhibition activates the Na.sup.+/K.sup.+-ATPase and prevents ventricular tachycardias.

[0090] Methods:

[0091] Animal Models

[0092] Animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Project approval was granted by the Norwegian National Animal Research Committee (FDU 2146, 7016 and 7040). Male Wistar rats with ˜300 g body weight (Møllegaard, Denmark) were stored two per cage in a temperature-regulated room on a 12:12 h day/night cycle and given access to food and water ad libitum. Mice were stored under similar conditions, and maximally six mice per cage were allowed. Aorta banding was performed in C57BL6/J mice by a standardized constriction of the ascending aorta, and the mice were followed for 14-16 weeks. Development of congestive heart failure was verified by echocardiography and post-mortem examination as previously described (Aronsen, J. M., et al., Noninvasive stratification of postinfarction rats based on the degree of cardiac dysfunction using magnetic resonance imaging and echocardiography. American Journal of Physiology-Heart and Circulatory Physiology, 2017. 312(5): H932-H942). Ankyrin B.sup.+/− mice were bred as previously described (Mohler, P. J., et al., Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death. Nature, 2003. 421(6923): 634-9). Floxed PDE2A mice were crossed with alpha-MHC MerCreMer mice and were used to test the cardiomyocyte specific role of PDE2A with methods as previously described (Hougen, K., et al., Cre-loxP DNA recombination is possible with only minimal unspecific transcriptional changes and without cardiomyopathy in Tg(alpha MHC-MerCreMer) mice. American Journal of Physiology-Heart and Circulatory Physiology, 2010. 299(5): H1671-H1678).

[0093] Cell Isolation

[0094] Male Wistar rats were anaesthetized in 4% isoflurane, 65% N.sub.2O and 31% O.sub.2, and intubated and ventilated with 2% isoflurane, 66% N.sub.2O and 32% O.sub.2. Deep surgical anesthesia was confirmed by abolished pain reflexes. 150 IE heparin was administrated intravenously for post-excision thrombosis prophylaxis. The heart was excised and immediately cooled in 0.9% NaCl at 4° C. Aorta was cannulated and the coronary arteries retrogradely perfused in a modified Langendorff setup with buffer A (in mM: Hepes 25, NaCl 130, KCl 5.4, NaH.sub.2PO.sub.4 0.4, MgCl.sub.2 0.5, D-glucose 22, pH 7.4) at 37° C. for 2-4 min, then with buffer A containing 0.8 g/L collagenase II (Worthington Biochemical Corporation, USA) and 6.7 μM CaCl.sub.2 for 18-22 min. Atria and the right ventricular free wall were removed before the LV was cut into small pieces in 8-10 mL of the perfusate added 500 μL 2% BSA, and mechanically isolated by careful pipetting with a Pasteur pipette for 1 min. The cardiomyocyte suspension was filtered through a nylon mesh (200 μm, Burmeister, Lørenskog, Norway), and left at room temperature for sedimentation. Immediately following sedimentation (˜5 min), the supernatant was removed. For single-cell experiments and generation of primary cultures for cAMP measurements, the cardiomyocytes were washed three times in buffer A containing 1) 0.1% BSA and 0.1 mM CaCl.sub.2, 2) 0.1% BSA and 0.2 mM CaCl.sub.2, and 3) 0.05% BSA and 0.5 mM CaCl.sub.2. For generation of primary cultures for proximity ligation assay, the cardiomyocytes were washed three times in buffer A with decreasing concentration of BSA (0.1%, 0.05% and 0%).

[0095] Left ventricular myocytes from PDE2-deficient mice, Ankyrin.sup.+/− mice and C57BL6/J mice after sham or AB operation were isolated based on a similar protocol as recently described (Ackers-Johnson, M., et al., A Simplified, Langendorff-Free Method for Concomitant Isolation of Viable Cardiac Myocytes and Nonmyocytes From the Adult Mouse Heart. Circ Res, 2016. 119(8): 909-20). Mice were anesthetized in a combination of 5% isoflurane and 95% O.sub.2, and mask ventilated by a combination of 5% isoflurane and 95% O.sub.2. Deep surgical anesthesia was confirmed by abolished pain reflexes. The chest was opened, before the descending aorta and inferior caval vein was cut. 7 mL of buffer A with 5 mM EDTA was injected into the right ventricle. Thereafter, the aorta was clamped and the heart was excised. 10 mL of the buffer solution and thereafter 3 ml of the buffer solution without EDTA was injected into the left ventricle over 2-5 min. Then preheated solution A containing 0.8 mg/mL collagenase II was injected into the left ventricle over ˜20 min. The atria and right ventricle was removed, and the remaining procedure was similar the procedure described for the Langendorff-based isolation above.

[0096] Proximity Ligation Assay

[0097] Isolated cardiomyocytes were washed twice in PBS at room temperature, transferred to 4% paraformaldehyde (PFA) and gently shaken for 30 min, then washed twice again in PBS. The cardiomyocyte suspension was next transferred to 0.8 cm.sup.2 wells, each coated with 8 μg laminin (Invitrogen), and incubated at 37° C. for 2 h. PBS was replaced by 0.1% Triton X100 in PBS, and incubated for 10 min at 37° C. Proximity ligation assay was then performed with the Duolink II proprietary system (Olink Bioscience, Uppsala, Sweden), according to the manufacturer's protocol (Soderberg, O., et al., Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods, 2006. 3(12): 995-1000).

[0098] Cardiomyocytes were scanned on a Zeiss LSM 710 confocal microscope (excitation 543 nm HeNe laser, through a MBS 488/543 dichroic mirror, emission collected at 565-589 nm). ImageJ 1.44p software (http://imagej.nih.gov/ij) was used for analysis of single-cell intracellular fluorescence intensity by measuring whole cell mean gray value. Results were corrected for background fluorescence signal.

[0099] Whole-Cell Voltage Clamp Experiments

[0100] Whole-cell continuous voltage clamp was performed in isolated cardiomyocytes, using an Axoclamp 2B or 2A amplifier and pCLAMP software (both Axon Instruments, Foster City, Calif., USA). The signal was sampled at 10 kHz and filtered with a low-pass filter before analysis. All amplifier and program settings were held constant during and between experiments. The cells were superfused at 37° C., and the superfusion system was arranged to allow rapid switch of solution.

[0101] Nka Currents:

[0102] Wide tipped patch pipettes (1.5-2.5 MΩ) were filled with internal solution (in mM, modified from Despa, S. and D. M. Bers, Na/K pump current and [Na](i) in rabbit ventricular myocytes: local [Na](i) depletion and Na buffering. Biophys J, 2003. 84(6): 4157-66: NaCl 17, KCl 13, K-Aspartate 85, TEA-CI 20, HEPES 10, MgATP 5, MgCl.sub.2 0.7 (free Mg.sup.2+ −1.0 mM using Maxchelator, Stanford), BAPTA 3, CaCl.sub.2 1.15 (free Ca.sup.2+150 nM), pH=7.2 (adjusted with KOH). After reaching whole-cell access, the cells were dialyzed for at least 4 minutes at −20 mV. Holding potential in the remaining experiment was −20 mV. The series resistance was 3-6 MΩ in most cells and any cell with a series resistance >9 MΩ was discarded.

[0103] The cells were patched in solution A (mM): NaCl 140, Hepes 5, KCl 5.4, CaCl.sub.2 1, MgCl.sub.2 0.5, D-glucose 5.5 and NaH.sub.2PO.sub.4 0.4. pH was adjusted to 7.4. After reaching whole-cell access, cells were superfused with solution B (in mM): N-methyl-D-glucamine 108, NaCl 17, D-glucose 10, HEPES 5, KCl 15, NiCl.sub.2 5, BaCl.sub.2 2, MgCl.sub.2 1, pH adjusted to 7.4 HCl. 5 μM cAMP and peptides (1 or 20 μM superAKAP or 1 μM RIAD) were added to the internal solution on experiment day. NKA currents were elicited by rapidly removing extracellular KCl (replaced with equal amounts of TrisCl). Solutions with symmetrical Na.sub.+ concentrations were used (i.e. the same concentration of Na.sup.+ in both the superfusate and the internal solution) in order to reduce the influence of intracellular Na.sup.+ gradients on the NKA currents. 100 nM Bay 60-7550, 100 nM PF05180999 or 10 μM EHNA were added to the superfusate on the day of experiments to measure the effect of PDE2 inhibition on NKA currents. Both paired and unpaired recordings were performed, but with consistency within one data set. In the paired recordings, NKA currents during control conditions and with PDE2 inhibitor were measured in the same cell, but with at least 5 minutes between the recordings. We alternated whether the first recorded NKA current in one cell was with or without PDE2 inhibitor to minimize any unwanted time-dependent effects. NKA currents were related to cell capacitance to account for differences in cell size.

[0104] L-Type Ca.sup.2+ Currents (LTCC):

[0105] Wide tipped patch pipettes (1.4-1.8 MΩ) were filled with internal solution (in mM, modified from Leroy, J., et al., Phosphodiesterase 48 in the cardiac L-type Ca(2)(+) channel complex regulates Ca(2)(+) current and protects against ventricular arrhythmias in mice. J Clin Invest, 2011. 121(7): 2651-61: CsCl 122, HEPES 10, MgATP 10, MgCl.sub.2 0.7 (free Mg.sup.2+ −0.6 mM), Na.sub.2Phosphodicreatinine 5, EGTA 10, CaCl.sub.2 0.2 (free Ca.sup.2+3 nM), pH 7.2 with CsOH. Series resistance was between 4-8 MΩ in all recordings. The holding potential was −45 mV, and Ca.sup.2+ transients were triggered by a 100 ms square voltage step from −45 to 0 mV at 0.125 Hz. The cells were patched in solution A, but after whole-cell access was reached, solution C was applied (in mM, modified from Leroy, J., et al., Phosphodiesterase 48 in the cardiac L-type Ca(2)(+) channel complex regulates Ca(2)(+) current and protects against ventricular arrhythmias in mice. J Clin Invest, 2011. 121(7): 2651-61: NaCl 118, CsCl 20, D-glucose 5, MgCl.sub.2 1.8, HEPES 10, NaH.sub.2PO.sub.4 0.8, CaCl.sub.2 1.8, pH 7.4 with NaOH. The internal solution was allowed to equilibrate for at least 4 minutes before recordings were started. No recordings were initiated before the LTCC were completely stable. Control recordings and recordings with Bay 60-7550 were performed in the same cell.

[0106] Na.sup.+ Currents:

[0107] Low resistance pipettes (1.4-2.5 MΩ) were filled with internal solution (in mM, modified from Leroy, J., et al., Phosphodiesterase 48 in the cardiac L-type Ca(2)(+) channel complex regulates Ca(2)(+) current and protects against ventricular arrhythmias in mice. J Clin Invest, 2011. 121(7): 2651-61): CsCl 122, HEPES 10, MgATP 5, MgCl.sub.2 0.7 (free Mg.sup.2+ −0.6 mM), Na.sub.2Phosphodicreatinine 5, EGTA 10, CaCl.sub.2 0.2 (free Ca.sup.2+3 nM), pH 7.2 with CsOH. Series resistance was between 4-7.5 MΩ in all recordings. The cells were patched in solution A, but after whole-cell access was reached, solution D was applied: N-methyl-D-glucamine 125, NaCl 10, CsCl 5, D-glucose 5, MgCl.sub.2 1.2, HEPES 10, NiCl.sub.2 5, pH 7.4 with CsOH. 20 μM Nifedipine was added on experiment day to inhibit L-type Ca.sup.2+ channels. Holding potential was −80 mV. Na.sup.+ currents were measured in discontinuous mode (switching rate 9 kHz) by applying a 50 ms square voltage step from the holding potential of −80 mV to −10 mV at 1 Hz. Good voltage control was maintained by symmetrical Na.sup.+ solutions and a low series resistance. A multistep protocol with −10 mV incremental steps ranging from −70 mV to +50 mV (all from the −80 mV holding potential) was run prior to these experiments to determine the test potential that yielded the largest peak current, with no difference between control and Bay 60-7550. The internal solution was allowed to equilibrate for at least 4 minutes before recordings were started. No recordings were initiated before the Na.sup.+ currents were completely stable. Control recordings and recordings with Bay 60-7550 were mostly performed in separate cells. However, in a subset of cells, both control and Bay 60-7550 were recorded in the same cell.

[0108] Field Stimulation Experiments

[0109] Whole-Cell Ca.sup.2+ Transients:

[0110] Ca.sup.2+ transients were recorded in field stimulated myocytes loaded with 5 μM Fluo4-AM for 10-15 minutes (Molecular Probes, Eugene, USA), followed by 5 minutes de-esterification. Experiments were either performed with and without 100 nM Bay 60-7550 in the same cell (rat ventricular myocytes) or in PDE2KO or WT ventricular myocytes. In experiments with peptides, myocytes were incubated with either 1 μM TAT-conjugated peptides (superAKAP or corresponding scrambled peptide) for 20 minutes. During the recordings the myocytes were then superfused with solution A containing the same peptide.

[0111] Cellular fluorescence was obtained with Cairn Research Optoscan Monochromator (excitation 485 nm, emission 515 nm long pass) (Cairn Research Ltd., Faverham, UK). Cell-free fluorescence was obtained after each experiment and subtracted from the tracing to correct for background fluorescence. Cells were stimulated at 0.5 Hz for at least 3 minutes or until the Ca.sup.2+ transients stabilized before recordings were initiated. Cells without stable Ca.sup.2+ transients (both baseline and peak Ca.sup.2+ levels) were discarded. Field stimulation was then stopped and a brief pulse of 10 mM caffeine was added. The SR Ca.sup.2+ content was recorded as the peak of the caffeine-evoked Ca.sup.2+ transient. The same experiment was performed after addition of 10 mM Ni.sup.2+ in the superfusate to block NCX activity. Tau values were obtained by monoexponential fitting of the Ca.sup.2+ extrusion phase from regular transients (T), caffeine transients (T.sub.caff) and caffeine transients with Ni.sup.2+ (T.sub.Ni). SERCA2 rate constant was calculated as the difference between the rate constant for field stimulated Ca.sup.2+ transients and the caffeine-evoked Ca.sup.2+ transient (Trafford, A. W., M. E. Diaz, and D. A. Eisner, Measurement of sarcoplasmic reticulum Ca content and sarcolemmal fluxes during the transient stimulation of the systolic Ca transient produced by caffeine. Ann N Y Acad Sci, 1998. 853: 368-71) while T.sub.caff was interpreted as Ca.sup.2+ extrusion through NCX in absence of any difference in T.sub.Ni.

[0112] Protocol for Detecting Cellular Arrhythmias:

[0113] Isolated ventricular myocytes from post-banding HF mice, AnkB.sup.+/− mice and WT (AnkB.sup.+/+) were pre-conditioned for 3 min at 0.5 Hz and 1 minute at 1 Hz, before stimulation was stopped for 15 seconds after each stimulation frequency. Ca.sup.2+ waves and/or spontaneous contractions were detected during the pauses as previously described (Aronsen, J. M., et al., Hypokalaemia induces Ca(2+) overload and Ca(2+) waves in ventricular myocytes by reducing Na(+),K(+)-ATPase alpha2 activity. J Physiol, 2015. 593(6): 1509-21). Inclusion criteria were rod-shaped and striated cardiomyocytes and absence of Ca.sup.2+ waves upon visual inspection 10 seconds prior to electrical stimulation. Cellular arrhythmias were recorded with and without 100 nM Bay 60-7550. In a subset of experiments, cellular arrhythmias were detected with 100 nM Bay 60-7550 with 1 μM TAT-conjugated scrambled peptide or superAKAP. In experiments with peptides, cells were incubated with the peptides for 20 minutes before the start of the protocol.

[0114] Whole-Cell Na.sup.+ Measurements:

[0115] To measure the cytosolic Na.sup.+ concentration, isolated rat ventricular myocytes were loaded at room temperature in 10 μM SBFI for 120 min, in the presence of 0.05% Pluronic F-127, followed by 20 minutes of de-esterification. SBFI ratios were detected with a photomultiplier (Photon Technology International, NJ, USA) in myocytes superfused with solution A and field stimulated at 0.5 Hz. Single excitation (340 nm) and dual ratiometric emission (410 nm/590 nm) were used as previously described (Baartscheer, A., C. A. Schumacher, and J. W. Fiolet, Small changes of cytosolic sodium in rat ventricular myocytes measured with SBFI in emission ratio mode. J Mol Cell Cardiol, 1997. 29(12): 3375-83). The signal was sampled at 1 Hz and allowed to stabilize before recordings started (typically 10 minutes). 100 nM Bay 60-7550 was applied to study the effect of PDE2 inhibition on cytosolic Na.sup.+.

[0116] Each cell was calibrated by superfusing the cell with a solution containing 0 and 20 mM Na.sup.+. In this range, the SBFI signal was assumed to be linear with the intracellular Na.sup.+ levels, as previously described (Despa, S., et al., Intracellular [Na+] and Na+ pump rate in rat and rabbit ventricular myocytes. J Physiol, 2002. 539(Pt 1): 133-43). Two different calibration solutions were made, and they were mixed to achieve the desired Na.sup.+ concentrations. Both calibration solutions contained (in mM) gramicidin 0.01, ouabain 0.1, Hepes 5, Glucose 5.5, EGTA 2, adjusted to pH 7.2 with TrisBase. Calibration solution with 145 Na.sup.+ contained also (in mM): Na-gluconate 115, NaCl 30, KCl 0. Calibration solution with 145 contained (in mM): K-gluconate 115, KCl 30, NaCl 0.

[0117] Confocal Ca.sup.2+ Measurements:

[0118] Ca.sup.2+ sparks were recorded in line-scan mode with a confocal microscope (Zeiss LSM Live7), as described previously (Louch, W. E., et al., T-tubule disorganization and reduced synchrony of Ca2+ release in murine cardiomyocytes following myocardial infarction. J Physiol, 2006. 574(Pt 2): 519-33). In short, a 512 pixel line was drawn longitudinally across the cell and scan time was 1.5 ms. Rat ventricular myocytes were field stimulated at 1 Hz for 3 minutes, before Ca.sup.2+ sparks were recorded immediately after cessation of stimulation. After a few seconds, 10 mM caffeine was applied to measure the SR Ca.sup.2+ load. The Ca.sup.2+ sparks frequency was related to the SR Ca.sup.2+ load, to prevent load-dependent effects on spark frequency. Ca.sup.2+ sparks were measured with and without Bay 60-7550 in the same cell. Ca.sup.2+ sparks were detected and analyzed using SparkMaster in ImageJ (NIH) (Picht, E., et al., SparkMaster: automated calcium spark analysis with ImageJ. Am J Physiol Cell Physiol, 2007. 293(3): C1073-81).

[0119] In Vivo Recordings of Arrhythmias

[0120] Post-banding HF mice, AnkB.sup.+/− mice and WT (AnkB.sup.+/+) mice were anaesthetized in 4% isoflurane, 65% N.sub.2O and 31% O.sub.2, and ventilated with 2% isoflurane, 66% N.sub.2O and 32% O.sub.2. Deep surgical anesthesia was confirmed by abolished pain reflexes. Every other mouse was randomized to one of the treatment groups, while the next one was assigned to the other group. Efforts were made to assure that littermate controls were used within one comparison (for instance vehicle vs. Bay 60-7550). One-lead ECG recordings was performed by attaching the mouse limbs to incorporated ECG electrodes on the operating table, which was pre-heated to 40° C. to maintain a stable body temperature. The ECG was continuously recorded by VEVO2100 software (Visualsonics, Toronto, Canada) during the entire protocol, and the signal was not filtered. A baseline ECG was recorded for 3-5 minute before starting the protocol, and in the rare event of mice having ventricular extrasystoles during this period, they were excluded and the protocol was stopped prior to drug injection. The mice were first injected intraperitoneally (i.p.) with either Bay 60-7550 or vehicle (3 mg/kg) or PF05180999 (1 mg/kg), which was allowed to work for 10 minutes (Vettel, C., et al., Phosphodiesterase 2 Protects Against Catecholamine-Induced Arrhythmia and Preserves Contractile Function After Myocardial Infarction. Circ Res, 2017. 120(1): 120-132) before the first injection of caffeine (120 mg/kg i.p.). In experiments with peptides, 5 mM (calculated by assuming free distribution of peptides in the liquid phase of the body, which was assumed to be 0.7 of the total body weight) of TAT-conjugated superAKAP or scrambled peptide were injected i.p. 5 minutes prior to caffeine injection. In experiments with AnkB.sup.+/− mice, caffeine was injected i.p. (120 mg/kg i.p.) fora second time 10 minutes after the first injection. The animals were sacrificed by excision of the heart after the end of the protocol, the lung and heart weight were obtained and the left ventricle was transferred to an Eppendorf tube and immediately moved to liquid nitrogen and stored at −80° C. ECG was recorded during the entire protocol. ECG was analyzed using VEVO software.

[0121] Preparation of Cardiomyocyte Lysate for Immunoblotting

[0122] Non-sterile solution A with 10 mM BDM was pre-heated to 37° C., and plastic wells were coated with 4% laminin in solution A/BDM for 1 hour. Rat ventricular myocytes were isolated as described above, re-suspended in solution A/BDM, plated in the laminin-coated wells and incubated for 1 hour at 37° C. The cells were then gently washed in solution A/BDM, and again incubated for 1 hour at 37° C. Peptides, Isoprenaline or inhibitors were then added as indicated, and allowed to equilibrate for 10 minutes. Finally, cells were harvested in hot (90-100° C.) lysis buffer (1% SDS, 2 mM Na.sub.3VO.sub.4, 10 mM Tris-HCl, 10 mM NaF, dH.sub.2O, pH 7,4), transferred to liquid nitrogen and stored at −80° C.

[0123] Immunoprecipitation

[0124] Lysates from HEK293 transfected cells were incubated with antibodies for 2 h, thereafter 50 μL protein NG PLUS agarose beads (sc-2003, Santa Cruz Biotechnology) were added overnight at 4° C. Immunocomplexes were washed three times in cold IP-buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 Triton X-100), centrifuged at 3000 g for 1 min at 4° C., boiled in SDS loading buffer and analysed by immunoblotting. HEK293 cells not transfected with FLAG-PDE2 were used for control.

[0125] Immunoblotting

[0126] Immunoprecipitates were analysed on 4-15% or 15% SDS-PAGE before blotting onto PVDF membranes. The PVDF membranes and peptide membranes were blocked in 1% casein or 5% milk in TBST for 60 min at room temperature, incubated overnight at 4° C. with primary antibodies, washed three times 10 min in TBS-T and incubated with a HRP-conjugated primary or secondary antibody. Blots were incubated in ECL Prime (GE Healthcare, RPN2232) and chemiluminescence signals were detected by LAS-4000 (Fujifilm, Tokyo, Japan).

[0127] Antibodies

[0128] Ser68-PLM was a gift from William Fuller. Anti-FLAG and Anti-GFP were used to blot FLAG-PDE2 and GFP-NKA in the immunoprecipitations from HEK293 cells. PDE2 and NKA.sub.a1 antibodies were used in proximity ligation assay experiments.

[0129] cAMP Measurements

[0130] Adult mice ventricular myocytes were transfected with adenovirus type 5 containing GloSensor (Promega, United States) and allowed to incubate for 48 hours before cAMP measurements. The sensor binds cAMP and, when bound to cAMP, emits a light signal that is proportional to cAMP levels.

[0131] Nuclear Isolation and Sorting/mRNA Sequencing

[0132] Nuclear isolation/sorting and mRNA sequencing were performed as previously described (Thienpont, B., et al., The H3K9 dimethyltransferases EHMT1/2 protect against pathological cardiac hypertrophy. J Clin Invest, 2017. 127(1): 335-348).

[0133] Statistics

[0134] Data are presented as mean values ±S.E. For voltage clamp and field stimulation experiments we used two tailed student's t-test, while Fisher's exact test was employed in all experiments on cellular and in vivo arrhythmias. p<0.05 was considered significant.

[0135] Results

[0136] PDE2 Regulates NKA Activity

[0137] We performed a voltage clamp protocol where the isolated rat ventricular myocytes were exposed to symmetrical concentrations of Na.sup.+ (i.e. similar Na.sub.+ concentrations in the superfusate and the internal solution) to reduce the unwanted effects of intracellular Na.sup.+ gradients. The NKA current was measured as the K.sup.+-sensitive current after removal of a saturating concentration of extracellular K.sup.+ (from 15 mM to 0 mM [K.sup.+.sub.e]) (see FIG. 1A). The NKA current increased in a concentration-response-dependent manner (FIG. 1B), while a high dose of superAKAP, disrupting both PKA-RI and PKA-RII from AKAPs (Gold et al, Molecular basis of AKAP specificity for PKA regulatory subunits, Mol. Cell. 2006 Nov. 3; 24(3): 383-95) reduced the NKA current (FIG. 1C). This suggests that the NKA activity is regulated by local regulation of cAMP, and that increasing cAMP by inhibiting the cAMP-degrading phosphodiesterases PDE2-4 might increase NKA current, which we next wanted to test.

[0138] PDE2 inhibition with three different pharmacological inhibitors (EHNA, PF05180999 and Bay 60-7550) robustly increased the NKA current (FIG. 2A), while neither PDE3 inhibition with Cilostamide or PDE4 inhibition with Rolipram had any detectable effects (FIG. 2C). We also found increased NKA current in a PDE2-deficient mice model (FIG. 2B) further strengthening the finding of PDE2 regulating NKA activity.

[0139] In line with the functional recordings, we also found increased phosphorylation of phospholemman (PLM) at its main site for PKA phosphorylations, serine 68, after inhibition of PDE2. We also found increased PLM ser-68 phosphorylation with PDE4 inhibition, while there was no effect of PDE3 inhibition (FIG. 2D).

[0140] PDE2 and NKA Colocalize and Interact

[0141] PDE2 appears to be the main cAMP-PDE regulating NKA activity. If PDE2 regulates NKA through a local regulatory effect, then this interaction could be targeted specifically for therapeutically purposes. To investigate whether PDE2 and NKA reside in the same intracellular compartment, we performed a proximity ligation assay (Duolink®) which is used to detect intracellular colocalization between proteins with 30-40 nm resolution (Soderberg, O., et al., Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods, 2006. 3(12): 995-1000). The light dots in FIG. 3A (image to the right) suggest colocalization in intact rat ventricular myocytes between PDE2 and NKA, while the other images are negative controls. FIG. 3B shows a quantification of the light dots, with the three bars to the right showing much higher levels of light dots when both NKA and PDE2 antibodies were present. We then co-expressed FLAG-tagged PDE2 and GFP-tagged NKA or GFP-NKA alone in HEK293 cells, and immunoprecipitated FLAG. The levels of GFP-tagged NKA was higher when co-expressed with FLAG-PDE2, showing that PDE2 and NKA co-immunoprecipitates (FIG. 3C). These results indicate that PDE2 and NKA co-localizes in cells, providing a structural basis for the proposed local regulation of NKA activity by PDE2.

[0142] PDE2 Inhibition Reduces Ca.sup.2+ Transient Amplitude and SR Ca.sup.2+ Load

[0143] Having shown that PDE2 regulates NKA activity and colocalizes with NKA, we next wanted to investigate whether PDE2 influenced the Ca.sup.2+ homeostasis in ventricular myocytes. First, we measured Ca.sup.2+ transients in Fluo4-AM-loaded, field-stimulated rat ventricular myocytes, and we observed a decrease in Ca.sup.2+ transient amplitude without alterations on the Ca.sup.2+ extrusion rate (FIG. 4A-B) after application of Bay 60-7550. SR Ca.sup.2+ load was measured with rapid pulses of caffeine (FIG. 4C), and we found reduced SR Ca.sup.2+ load after PDE2 inhibition (FIG. 4A, right panel), in accordance with the observed reduction in Ca.sup.2+ transient amplitude. We also performed the same experiments in isolated ventricular myocytes from PDE2KO mice, and we found similar results with reduced Ca.sup.2+ transient amplitude and SR Ca.sup.2+ load with no effect on the Ca.sup.2+ extrusion rate (FIG. 4D).

[0144] PDE2 Inhibition Increases NCX-Mediated Ca.sup.2+ Extrusion

[0145] A reduction in Ca.sup.2+ transient amplitude and SR Ca.sup.2+ load after PDE2 inhibition could be explained by altered activity in the main Ca.sup.2+ handling proteins in the ventricular myocyte, i.e. L-type Ca.sup.2+ channel, RyR, NCX, SERCA or non-SERCA/non-NCX Ca.sup.2+ extruding proteins (PMCA and mitochondrial uniporter). To elucidate the role of the various Ca.sup.2+ handling proteins in the Ca.sup.2+ homeostasis following PDE2 inhibition, we did a series of experiments where we measured their activity after application of Bay 60-7550. We did not find any effect on SERCA function or the non-SERCA/non-NCX activity in rat ventricular myocytes after PDE2 inhibition, while the Ca.sup.2+ extrusion through NCX was significantly increased (FIG. 5A-B). This same pattern was also found in experiments conducted in PDE2KO mice, with increased Ca.sup.2+ extrusion through NCX and unaltered activity of SERCA and non-SERCA/non-NCX Ca.sup.2+ handling proteins (FIG. 5C). Altered NKA activity could affect NCX activity through alterations in local or global Na.sup.+ (Despa, S., J. B. Lingrel, and D. M. Bers, Na(+)/K)+)-ATPase alpha2-isoform preferentially modulates Ca2(+) transients and sarcoplasmic reticulum Ca2(+) release in cardiac myocytes. Cardiovasc Res, 2012. 95(4): 480-6), but we were not able to detect any differences in global intracellular Na.sup.+ using SBFI (FIG. 5D), suggesting that local Na.sup.+ gradients are involved.

[0146] In voltage clamped rat ventricular myocytes, we did not find any effect of PDE2 inhibition on the L-type Ca.sup.2+ current (FIG. 6A-B) or Na.sup.+ current (FIG. 6E). We also measured Ca.sup.2+ sparks in Fluo4-AM-loaded rat ventricular myocytes using line-scan mode on a confocal microscopy, and this revealed no difference in the Ca.sup.2+ sparks frequency between control and PDE2 inhibition, suggesting unaltered RyR activity (FIG. 6C-D).

[0147] PDE2 inhibition thus leads to increased NKA current, increased Ca.sup.2+ extrusion through NCX and reduced Ca.sup.2+ transient amplitude and SR Ca.sup.2+ load, and these results harmonize with the following reasoning: 1) Increased NKA activity leads to reduced local intracellular Na.sup.+, 2) and the reduced intracellular Na.sup.+ increases the driving force for Ca.sup.2+ efflux through NCX, meaning that more Ca.sup.2+ is extruded through NCX out of the myocyte, 3) Finally, more sarcolemmal Ca.sup.2+ extrusion through NCX means that less Ca.sup.2+ is available for intracellular Ca.sup.2+ cycling through SERCA, with the net effect being that Ca.sup.2+ is removed out of the cell, giving a smaller Ca.sup.2+ transient and a smaller SR Ca.sup.2+ load.

[0148] PDE2 Inhibition Prevents Cellular Tachycardias

[0149] Ca.sup.2+ overloading is a well-established cause of arrhythmias (Pogwizd, S. M. and D. M. Bers, Cellular basis of triggered arrhythmias in heart failure. Trends Cardiovasc Med, 2004. 14(2): 61-6; and Kranias, E. G. and D. M. Bers, Calcium and cardiomyopathies. Subcell Biochem, 2007. 45: 523-37) leading to Ca.sup.2+ waves (Aronsen, J. M., et al., Hypokalaemia induces Ca(2+) overload and Ca(2+) waves in ventricular myocytes by reducing Na(+),K(+)-ATPase alpha2 activity. J Physiol, 2015. 593(6): 1509-21) and deleterious secondary effects (Pezhouman, A., et al., Molecular Basis of Hypokalemia-Induced Ventricular Fibrillation. Circulation, 2015. 132(16): 1528-1537), and conversely, agents that reduces intracellular Ca.sup.2+, such as Ca.sup.2+ channel blockers, might protect against arrhythmias. Heart failure is a disease with a huge risk of ventricular arrhythmias (Pogwizd, S. M. and D. M. Bers, Cellular basis of triggered arrhythmias in heart failure. Trends Cardiovasc Med, 2004. 14(2): 61-6), while Ankyrin B syndrome is a genetic disease which causes type 4 long-QT syndrome (Mohler, P. J., et al., Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death. Nature, 2003. 421(6923): 634-9). Since PDE2 inhibition reduced Ca.sup.2+ transient amplitude, we hypothesized that PDE2 inhibition would prevent Ca.sup.2+ waves in ventricular myocytes from heart failure mice and Ankyrin B.sup.+/− mice (long QT syndrome). First, we confirmed that PDE2 inhibition increased NKA currents and reduced Ca.sup.2+ transients in ventricular myocytes from post-banding mice with heart failure (FIG. 7A-B). We detected a considerable amount of Ca.sup.2+ waves in myocytes both from heart failure mice and Ankyrin B.sup.+/− mice (long QT syndrome), but the Ca.sup.2+ waves were largely reduced or abolished with PDE2 inhibition (FIG. 7C-D). In WT mice (Ankyrin B.sup.+/− mice littermates), there was no difference in the Ca.sup.2+ wave frequency, which is as expected considering the low frequency of Ca.sup.2+ waves during control conditions (FIG. 7D, right panel).

[0150] In conclusion, PDE2 inhibition prevents cellular tachycardias in known models of cardiac arrhythmias.

[0151] PDE2 Inhibition Prevents Ventricular Tachycardia in Mice with Heart Failure and AnkB.sup.+/− Syndrome (Long QT Syndrome)

[0152] To test whether PDE2 inhibition protects against ventricular tachycardias in vivo, we performed a protocol where we acutely injected anesthetized post-banding HF mice and AnkB.sup.+/− mice with 120 mg/kg caffeine (Kannankeril, P. J., et al., Mice with the R176Q cardiac ryanodine receptor mutation exhibit catecholamine-induced ventricular tachycardia and cardiomyopathy. Proc Natl Acad Sci USA, 2006. 103(32): 12179-84) and monitored the heart rhythm with a one-lead ECG (protocols outlined in FIGS. 8A and 9A). The protocol was also tested in WT animals, where one caffeine injection was not sufficient to elicit arrhythmias (data not shown), while both post-banding HF mice and AnkB.sup.+/− mice exhibited ventricular arrhythmias. Examples of a normal sinus rhythm and ventricular tachycardia are shown in FIG. 8B, and the typical ventricular tachycardias that we observed were bidirectional ventricular tachycardias and multifocal ventricular tachycardias. In experiments with Bay 60-7550, 4 out of 5 (80%) of the heart failure mice that received vehicle injection (control) developed ventricular tachycardia and cardiac arrest, while this outcome was seen in 0 out of 5 (0%) of the heart failure mice that received an injection with 3 mg/kg Bay 60-7550 (Vettel, C., et al., Phosphodiesterase 2 Protects Against Catecholamine-Induced Arrhythmia and Preserves Contractile Function After Myocardial Infarction. Circ Res, 2017. 120(1): 120-132) (FIG. 8C). 1 mouse in the Bay 60-7550 group developed ventricular arrhythmias, but this did notably not develop into cardiac arrest. In experiments with PF05180999, 4 out of 5 (80%) of the heart failure mice that received vehicle injection (control) developed ventricular tachycardia, while this was seen in only 1 out of 5 (20%) of the heart failure mice that received an injection with 1 mg/kg PF05180999 (FIG. 8D).

[0153] In the experiments with Bay 60-7550 on Ankyrin B.sup.+/− mice (long QT syndrome), 5 out of 8 (63%) in the control group (vehicle injection) developed ventricular tachycardia (FIG. 8D). In addition, 2 mice developed supraventricular tachycardia (data not shown). In contrast, 0 out of 8 (0%) in the intervention group (Bay 60-7550 injection) developed ventricular tachycardia (FIG. 9C) or supraventricular tachycardia (data not shown). In the experiments with PF05180999, 5 out of 5 (100%) developed ventricular tachycardia in the control group (vehicle injection), while 1 out of 5 (20%) developed ventricular tachycardia with 1 mg/kg PF05180999 (FIG. 9D).

[0154] QT prolongation is a known risk factor for ventricular tachycardias (Osadchii, O. E., Impact of hypokalemia on electromechanical window, excitation wavelength and repolarization gradients in guinea-pig and rabbit hearts. PLoS One, 2014. 9(8): e105599). We observed an increased QT interval from baseline ECG to post-caffeine injection in the vehicle group in both post-banding HF and AnkB.sup.+/− mice, while there was no difference in the QT interval in the Bay 60-7550 and PF05180999 groups (FIGS. 8C-D and 9C-D, right panels).

[0155] In summary, we find that PDE2 inhibition prevents deleterious ventricular tachycardias and QT prolongation in established mice models of cardiac arrhythmias. This indicates that PDE2 inhibition is a novel target for treatment of cardiac tachycardias in a variety of clinical settings, for instance in heart failure patients.

[0156] PDE2 Regulation of NKA is Dependent on Local PKA-RII Activity

[0157] Since PDE2 and NKA colocalize in intact ventricular myocytes, an intriguing possibility is that PDE2 regulation of NKA is dependent on local cAMP gradients and local PKA activity. In line with this idea, we did not detect any global increase or decrease in cAMP levels after treatment with Bay 60-7550. We used Isoprenaline as a positive control, and application of 20 nM Isoprenaline largely increased cAMP levels (FIG. 10A). Low doses of RIAD and superAKAP (1 μM) are established, highly specific disruptors of AKAP-bound PKA-RI (RIAD) and PKA-RII (superAKAP), while a higher concentration of superAKAP (20 μM) disrupts both PKA-RI and PKA-RII (Gold et al, Molecular basis of AKAP specificity for PKA regulatory subunits, Mol. Cell. 2006 Nov. 3; 24(3): 383-95). Following dialysis of superAKAP (both high and low dose) into the voltage clamped myocytes, PDE2 inhibition did not alter NKA current. However, PDE2 inhibition still increased the NKA current in the presence of RIAD (PKA-RI disruptor), suggesting that PDE2 regulation of NKA is exclusively dependent on AKAP-bound, local PKA-RII activity, not PKA-RI (FIG. 10C). We also found positive co-IP between NKAα2 and both the catalytic site on PKA and PKA RIIα (FIG. 10B), which supports the idea of a local, regulatory complex consisting of NKA, PDE2 and an AKAP-bound PKA-RII (see FIG. 12 for proposed model).

[0158] The Anti-Arrhythmic Effect of PDE2 Inhibition is Dependent on AKAP-Bound PKA-RII

[0159] We propose that PDE2 inhibition activates NKA through regulation of local cAMP pools and prevents ventricular arrhythmias, so we hypothesized that both the cellular and in vivo arrhythmias that were prevented with PDE2 inhibition would reappear after application of superAKAP. As shown in FIG. 10C, there is an increased frequency of Ca.sup.2+ waves with Bay 60-7550 present when cells were incubated and superfused with TAT-superAKAP compared to TAT-scrambled peptide. The increased amount of Ca.sup.2+ waves was seen both in post-banding HF and AnkB.sup.+/−, and at both frequencies tested (FIG. 10D).

[0160] We also tested the ability of superAKAP to reverse the anti-arrhythmic effect of PDE2 inhibition in vivo as outlined in FIG. 11A. Both in post-banding HF mice (4 out of 6) and in AnkB.sup.+/− mice (3 out of 5) did ventricular arrhythmias appear during Bay 60-7550 treatment when the mice were injected with TAT-superAKAP. However, there were no observed arrhythmias in post-banding HF mice (0 out of 6) and in AnkB.sup.+/− mice (0 out of 5) during Bay 60-7550 treatment when the mice were injected with TAT-scramble. These results confirm that PDE2 inhibition prevents cellular and in vivo tachycardias through regulation of a local signaling domain that activates NKA.

[0161] PDE2 is Up-Regulated in Cardiomyocytes in Human Hypertrophy and Ageing

[0162] In order for PDE2 inhibition to be a future anti-arrhythmic treatment option, it needs to be established that PDE2 is present in human cardiac tissue and in disease. Although it previously has been reported that PDE2 is up-regulated in human heart failure (Mehel, H., et al., Phosphodiesterase-2 is up-regulated in human failing hearts and blunts beta-adrenergic responses in cardiomyocytes. J Am Coll Cardiol, 2013. 62(17): 1596-606), these analysis were performed on left ventricular tissue, which also contains other cell types (Thienpont, B., et al., The H3K9 dimethyltransferases EHMT1/2 protect against pathological cardiac hypertrophy. J Clin Invest, 2017. 127(1): 335-348). We did mRNA sequencing on isolated and sorted cardiomyocyte nuclei, and find that PDE2A-mRNA expression is up-regulated in human left ventricular hypertrophy and in elderly individuals. PDE2A-mRNA expression was also increased in cardiomyocyte nuclei from rats with post-banding HF (FIG. 13). Thus, it is found that PDE2A is up-regulated in cardiomyocytes in relevant disease models, meaning that PDE2A can be targeted for anti-arrhythmic purposes in humans.

CONCLUSION

[0163] We have shown that PDE2 inhibition strongly prevents ventricular tachycardias in post-banding HF mice and heterozygous Ankyrin B.sup.+/− mice (long QT syndrome) through a novel anti-arrhythmic mechanism, where PDE2 inhibition increases NKA activity through regulation of local PKA activity, ultimately leading to reduced Ca.sup.t′ loading of the myocytes and reduced cellular and in vivo arrhythmogenecity. Ca.sup.2+ extrusion through NCX was increased after PDE2 inhibition, offering a mechanism for the reduced Ca.sup.2+ loading of the myocytes, whereas no other Ca.sup.2+ handling proteins was regulated by PDE2 inhibition in our hands, including LTCC, RyR, SERCA2 and non-SERCA2-non-NCX Ca.sup.2+ extruding proteins. The increased Ca.sup.2+ extrusion through NCX could either be explained by a direct PDE2-mediated effect on NCX or downstream to alterations in intracellular Na.sup.t. It has previously been shown that activation of beta-adrenergic signaling and subsequent phosphorylation of PLM regulates NCX negatively (Cheung, J. Y., et al., Regulation of cardiac Na+/Ca2+ exchanger by phospholemman. Ann N Y Acad Sci, 2007. 1099: 119-34; and Wanichawan, P., et al., Development of a high-affinity peptide that prevents phospholemman (PLM) inhibition of the sodium/calcium exchanger 1 (NCX1). Biochem J, 2016. 473(15): 2413-23). Thus, it is unlikely that PDE2 inhibition would increase Ca.sup.2+ extrusion through NCX through an effect on PLM. NKA activation leads to reduced intracellular Na.sup.+, which could have downstream effects on NCX, as shown previously by our group and others (Aronsen, J. M., et al., Hypokalaemia induces Ca(2+) overload and Ca(2+) waves in ventricular myocytes by reducing Na(+),K(+)-ATPase alpha2 activity. J Physiol, 2015. 593(6): 1509-21; and Despa, S., J. B. Lingrel, and D. M. Bers, Na(+)/K)+)-ATPase alpha2-isoform preferentially modulates Ca2(+) transients and sarcoplasmic reticulum Ca2(+) release in cardiac myocytes. Cardiovasc Res, 2012. 95(4): 480-6). NKA and NCX have been shown to interact in a local Na.sup.+ domain, where downstream effects on Ca.sup.2+ homeostasis and contractility are poorly predicted by changes in global intracellular Na.sup.+ (Despa, S., J. B. Lingrel, and D. M. Bers, Na(+)/K)+)-ATPase alpha2-isoform preferentially modulates Ca2(+) transients and sarcoplasmic reticulum Ca2(+) release in cardiac myocytes. Cardiovasc Res, 2012. 95(4): 480-6). In the present study, we did not find global changes in intracellular Na.sup.t, although the sensitivity of intracellular Na.sup.+ measurements using SBFI is low (Baartscheer, A., C. A. Schumacher, and J. W. Fiolet, Small changes of cytosolic sodium in rat ventricular myocytes measured with SBFI in emission ratio mode. J Mol Cell Cardiol, 1997. 29(12): 3375-83; and Swift, F., et al., The Na+/K+-ATPase alpha2-isoform regulates cardiac contractility in rat cardiomyocytes. Cardiovasc Res, 2007. 75(1): 109-17), suggesting that the observed effect on NCX is due to NKA regulation of local Na.sup.+ pools in a restricted domain.

[0164] Reduced NKA activity is an emerging pro-arrhythmic pathway, evident by the classical digitalis-induced arrhythmias, but reduced NKA activity has also been highlighted more recently, by our group (Aronsen, J. M., et al., Hypokalaemia induces Ca(2+) overload and Ca(2+) waves in ventricular myocytes by reducing Na(+),K(+)-ATPase alpha2 activity. J Physiol, 2015. 593(6): 1509-21) and Pezhouman et al. (Pezhouman, A., et al., Molecular Basis of Hypokalemia-Induced Ventricular Fibrillation. Circulation, 2015. 132(16): 1528-1537) to have a role in hypokalemia-induced arrhythmias (Faggioni, M. and B. C. Knollmann, Arrhythmia Protection in Hypokalemia: A Novel Role of Ca2+-Activated K+ Currents in the Ventricle. Circulation, 2015. 132(15): 1371-3). Although this implies that increased NKA activity could have an anti-arrhythmic effect, no such NKA activators exist, leaving this opportunity unexplored. We report here that PDE2 inhibition increases NKA activity with 30-50%. We and others have previously reported that a similar reduction in NKA activity has strong pro-arrhythmic effects. It has also been shown previously that small changes in total NKA activity could translate into large downstream effects in contractility (Despa, S., J. B. Lingrel, and D. M. Bers, Na(+)/K)+)-ATPase alpha2-isoform preferentially modulates Ca2(+) transients and sarcoplasmic reticulum Ca2(+) release in cardiac myocytes. Cardiovasc Res, 2012. 95(4): 480-6), depending on NKA isoform and co-localization with NCX.

[0165] A previous study found that overexpression of PDE2 protects against catecholamine-induced arrhythmias (Vettel, C., et al., Phosphodiesterase 2 Protects Against Catecholamine-Induced Arrhythmia and Preserves Contractile Function After Myocardial Infarction. Circ Res, 2017. 120(1): 120-132), which apparently contrasts with our findings. PDE2 is a dual-specific PDE, degrading both cGMP and cAMP, but with different affinities and maximal velocity rate (Bender, A. T. and J. A. Beavo, Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use. Pharmacol Rev, 2006. 58(3): 488-520). Importantly, PDE2 activity has been shown to be highly compartmentalized, regulating cAMP levels in specific domains, suggesting that PDE2 could have a highly differentiated role in various domains (Zaccolo, M. and M. A. Movsesian, cAMP and cGMP signaling cross-talk: role of phosphodiesterases and implications for cardiac pathophysiology. Circ Res, 2007. 100(11): 1569-78). Following catecholamine-induced activation of beta-adrenergic receptors, overexpression of PDE2 is expected to limit the intracellular rise in cAMP (Vettel, C., et al., Phosphodiesterase 2 Protects Against Catecholamine-Induced Arrhythmia and Preserves Contractile Function After Myocardial Infarction. Circ Res, 2017. 120(1): 120-132). However, it is not clear whether cAMP is altered in specific compartments or whether the observed beneficial effect is due to a global reduction in cAMP levels. In the present study, we report that PDE2 inhibition increases NKA current, reduces intracellular Ca.sup.2+ loading and prevents cellular and in vivo tachycardias without prior activation of the beta-adrenergic receptors. We find no global increase (or decrease) in cAMP levels after PDE2 inhibition without activation of beta-adrenergic receptors, suggesting that PDE2 inhibition is beneficial by regulating cAMP levels specific domains. We propose that PDE2 inhibition specifically activates NKA with few or no other effects on Ca.sup.2+ handling proteins. Our approach differs from the previous study in two important areas: 1) Activation of beta-adrenergic receptors leads to a global increase in intracellular cAMP; 2) The overexpression of PDE2 does not necessarily alter cAMP in specific domains, but might reflect a general, global reduction in cAMP levels. A previous study found beneficial effects on cardiac hypertrophy after PDE2 inhibition, but the effect was abrogated after disruption of local PKA activity (Zoccarato, A., et al., Cardiac Hypertrophy Is Inhibited by a Local Pool of cAMP Regulated by Phosphodiesterase 2. Circ Res, 2015. 117(8): 707-19). Together with our findings in the present study, this suggests that PDE2 inhibition is beneficial due to increase of cAMP in specific domains, while the proposed beneficial effect of PDE2 overexpression might be due to an unspecific effect with global cAMP degradation.

Example 2

[0166] The methods of Example 1 were repeated with other known PDE2 inhibitors to demonstrate their activity in the prevention of ventricular tachycardias in mice.

[0167] In Vivo Recordings of Arrhythmias

[0168] ND-7001 (10 mg/kg) and LuAF64280 (20 mg/kg), were tested in HF mice 1 week after AB using the same experimental set-up as described in Example 1. DMSO was used for control injections. Marked cardiac remodeling was verified in all mice before injection of the PDE2 inhibitor and subsequent caffeine.

[0169] Results

[0170] PDE2 Inhibition Prevents Ventricular Tachycardia in Mice with Heart Failure

[0171] Both ND-7001 and LuAF64280 protected against VTs in HF mice: ND-7001 0/5 (0%) developed VT; LuAF64280 0/3 (0%) developed VT (FIG. 14). In total, four PDE2 inhibitors, Bay 60-7550, PF05180999, ND-7001, and LuAF64280, protected against VT in HF mice.

CONCLUSION

[0172] It has been shown that two further PDE2 inhibitors prevent ventricular tachycardia in established models.

Example 3

[0173] The anti-arrhythmic effect of the PDE2 inhibitor Bay 60-7550 was tested against Metoprolol and in combination with Metoprolol, a commonly used anti-arrhythmic drug.

[0174] In Vivo Recordings of Arrhythmias

[0175] The anti-arrhythmic effect of the PDE2 inhibitor Bay 60-7550 was tested against Metoprolol in the same way as described in Example 1. We also included groups that received control injections and the combination of Bay 60-7550 and Metoprolol. Mice were randomly assigned to treatment groups, but with pre-determined group sizes. All groups were treated for five days; 5.5 mg/kg Metoprolol (see Zhou, Q., et al., Carvedilol and its new analogs suppress arrhythmogenic store overload-induced Ca2+ release. Nat. Med. 2011. 17(8): 1003-9) was injected once every day (in the morning), while 3 mg/kg Bay 60-7550 and control were injected twice every day (morning and evening). For chronic injections Bay 60-7550 was dissolved in 5% ethanol and 95% sunflower oil, and the control group received the same vehicle. 5% ethanol was added to the injection in the Metoprolol-only group to minimize ethanol-dependent effects. On the fifth day, we recorded baseline ECGs of all the animals before receiving their final injection. The final injections were given 10-20 min prior to the arrhythmia protocol. Final injections, anesthesia, arrhythmia provocation, ECG recordings and ECG analysis were performed as described in Example 1.

[0176] Results

[0177] PDE2 Inhibition is Superior to Beta-Blockers in Preventing Ca.sup.2+-Induced Ventricular Arrhythmias

[0178] Clinically, beta-blockers are the most commonly used anti-arrhythmic drug to prevent ventricular tachyarrhythmias, both in HF and in genetic arrhythmia syndromes (see Al-Khatib, S. M., et al., 2017 AHA/ACC/HRS Guideline for Management of Patients With Ventricular Arrhythmias and the Prevention of Sudden Cardiac Death: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines and the Heart Rhythm Society. Circulation, 2017). The anti-arrhythmic mechanism of action is mainly to block cAMP/PKA-dependent effects downstream of β-adrenergic receptors, while we propose that the anti-arrhythmic effect of PDE2 inhibition is due to specific activation of NKA and reduction in intracellular Ca2+ levels. We investigated the following questions: First, is PDE2 inhibition superior to beta-blockers in preventing Ca2+-induced ventricular arrhythmias? Second, is the anti-arrhythmic effect of PDE2 inhibition still present when combined with beta-blockers? To answer these questions, we randomized 55 AnkB+/− mice into four groups; vehicle, Metoprolol (5.5 mg/kg), Bay 60-7550 (3 mg/kg), or the combination of Bay 60-7550 (3 mg/kg) and Metoprolol (5.5 mg/kg). All mice received injections for five days, and on the final day, the mice were injected with caffeine (120 mg/kg) to induce ventricular arrhythmias (FIG. 15A). In the vehicle group, 8/11 developed VT and 11/11 developed ventricular arrhythmias (VT, ventricular bigemini, or coupled ventricular extrasystoles (VES)). In the Metoprolol group, 5/15 developed VT and 14/15 developed ventricular arrhythmias (VT, ventricular bigemini, and coupled VES). In contrast, the mice treated with Bay 60-7550 had significantly fewer ventricular arrhythmias (4/13, VT and ventricular bigemini), while 2/13 developed VT. In the group that received the combination of Metoprolol and Bay 60-7550, there were 0/15 mice with VT and 2/15 with ventricular arrhythmias (ventricular bigemini) (FIGS. 15B and C). There was a QT prolongation following Metoprolol injection in both

[0179] Metoprolol alone and in combination with Bay 60-7550. Neither of the groups that received Bay 60-7550 developed QT prolongation after caffeine injection, in contrast to Metoprolol and vehicle groups (FIG. 15D).

[0180] Beta-blockers constitute a cornerstone in modern anti-arrhythmic treatment, but our results suggest that PDE2 inhibition can complement and even provide an additive effect to this regime.

CONCLUSION

[0181] The results clearly suggest that PDE2 inhibition alone or in combination with Metoprolol prevented Ca.sup.2+-induced ventricular arrhythmias more efficiently than the control or Metoprolol alone. Many cardiac patients already use a beta-blocker, so it is an important finding that the combination of PDE2 inhibitors and beta-blockers is superior to the beta-blocker alone. This suggests that PDE2 inhibition is effective as an add-on therapy, which could be highly relevant in several clinical settings.

Example 4

[0182] In an extension to the experiments in Example 1, Ca.sup.2+ currents, Na.sup.+ currents, K.sup.+ currents and action potentials (APs) were also investigated in respect of BAY 60-7550.

[0183] L-Type Ca.sup.2+ Currents (LTCC)

[0184] Wide tipped patch pipettes (1.4-1.8 MΩ) were filled with internal solution (in mM, modified from Leroy, J. et al., Phosphodiesterase 48 in the cardiac L-type Ca(2)(+) channel complex regulates Ca(2)(+) current and protects against ventricular arrhythmias in mice. J. Clin. Invest. 2011. 121(7): 2651-61): CsCl 122, HEPES 10, MgATP 5, MgCl.sub.2 0.7 (free Mg.sup.2+0.6 mM), Na.sub.2Phosphodicreatinine 5, EGTA 10, CaCl.sub.2 0.2 (free Ca.sup.2+3 nM), cAMP 0.005, pH 7.2 with CsOH. Series resistance was between 4-8 MΩ in all recordings. 100 ms voltage steps were performed from a holding potential of −45 mV to various test potentials in the range −45 mV to 55 mV (10 mV) steps.

[0185] Na.sup.+ Currents

[0186] Low resistance pipettes (1.4-2.5 MΩ) were filled with internal solution (in mM, modified from Leroy, J. et al., Phosphodiesterase 48 in the cardiac L-type Ca(2)(+) channel complex regulates Ca(2)(+) current and protects against ventricular arrhythmias in mice. J. Clin. Invest. 2011. 121(7): 2651-61): CsCl 122, HEPES 10, MgATP 5, MgCl.sub.2 0.7 (free Mg.sup.2+0.6 mM), Na.sub.2Phosphodicreatinine 5, EGTA 10, CaCl.sub.2 0.2 (free Ca.sup.2+3 nM), cAMP 0.005, pH 7.2 with CsOH. Series resistance was between 4-7.5 MΩ in all recordings. The cells were patched in solution A, but after whole-cell access was reached, solution D was applied (in mM): N-methyl-D-glucamine 125, NaCl 10, CsCl 5, D-glucose 5, MgCl.sub.2 1.2, HEPES 10, NiCl.sub.2 5, pH 7.4 with CsOH. 20 μM Nifedipine was added on experiment day to inhibit L-type Ca.sup.2+ channels. Holding potential was −80 mV. 50 ms voltage steps were performed from a holding potential of −80 mV to various test potentials in the range −80 mV to 70 mV (15 mV) steps.

[0187] Background K.sup.+ Currents

[0188] K.sup.+ currents were measured as described previously (see Aronsen, J. M. et al., Hypokalaemia induces Ca(2+) overload and Ca(2+) waves in ventricular myocytes by reducing Na(+),K(+)-ATPase alpha2 activity. J. Physiol, 2015. 593(6): 1509-21). Briefly, 500 ms voltage steps were performed from a holding potential of −80 mV to various test potentials in the range −170 mV to 50 mV (10 mV) steps. Currents were analyzed at the stable phase towards the end of the pulse. Control recordings and recordings with Bay 60-7550 were performed in the same cells. Pipette resistance was 2-2.5 MΩ with a series resistance of 4-8 MΩ.

[0189] Action Potentials (APs)

[0190] APs were triggered by a 3-ms suprathreshold current injection. Pipette solution contained (in mM): KCl 130, NaCl 10, HEPES 10, MgATP 5, MgCl.sub.2 1, EGTA 0.5, cAMP 0.005, pH adjusted to 7.2 with KOH. The cells were superfused with solution A. Pipette resistance was 2-2.5 MΩ with a series resistance of 4.8-9.3 MΩ. APs were analyzed at 20% (APD20), 50% (APD50), 70% (APD70) and 90% (APD90), where 0% is the peak potential and 100% is the resting membrane potential, and at a given relative potential, the actual membrane potential was measured. Control recordings and recordings with Bay 60-7550 were performed in the same cells.

[0191] Results

[0192] PDE2 Inhibition has High Target Specificity

[0193] Current anti-arrhythmic treatments may be divided into class 1˜4 anti-arrhythmics, including inhibitors of Na.sup.+, K.sup.+, and Ca.sup.2+ channels. In FIG. 16c, we showed that PDE2 inhibition had no effect on Ca.sup.2+ currents. Here we also found that PDE2 inhibition had no effect on Ca.sup.2+ currents (FIG. 17a), K.sup.+ currents (FIG. 17b), or Na.sup.+ currents (FIG. 17c) in voltage clamped ARVMs. Inhibition of Na.sup.+, K.sup.+, and Ca.sup.2+ current is expected to increase action potential duration (APD). In contrast, we observed that PDE2 inhibition shortened the AP at APD50, APD70, and APD90 (FIG. 17d), consistent with the model that PDE2 inhibition selectively increases NKA activity without affecting Na.sup.+, K.sup.+, and Ca.sup.2+ currents. These results show that PDE2 inhibition activates NKA with high specificity, with no effects on other, previously known anti-arrhythmic targets.

CONCLUSION

[0194] Current pharmacological treatment strategies for ventricular arrhythmias include class I-IV anti-arrhythmic drugs. However, some anti-arrhythmic drugs are contra-indicated in patients with structural heart disease due to its pro-arrhythmic effects. New anti-arrhythmic strategies should preferably target specific arrhythmia mechanisms without too many off-target effects. Our results suggest that PDE2 inhibitors activates NKA with no effects on Ca.sup.2+ handling proteins and ion channels, including SERCA, RyR, non-SERCA-non-RyR Ca.sup.2+ extrusion proteins, Ca.sup.2+ current, Na.sup.+ current and K.sup.+ currents (inward rectifier and delayed rectifiers). We believe that this high level of specificity derives from the close interaction between NKA and PDE2.

[0195] By using a wide variety of molecular biology and imaging techniques, we show that NKA and PDE2 interacts and co-localizes in cardiomyocytes, and that PDE2 inhibition increases cAMP locally around NKA with no global increase in cAMP. Further, PDE2 regulation of NKA is blunted in the presence of superAKAP, a peptide that with high specificity displaces the RII-PKA from AKAPs. In line with this, PDE2 inhibition does not prevent VT in heart failure or AnkB.sup.+/− in the presence of superAKAP, showing that the anti-arrhythmic effect is dependent on local cAMP domains. Thus, PDE2 inhibition as an anti-arrhythmic treatment represents a novel treatment strategy in two ways: 1) As an activator of NKA and 2) by targeting cAMP levels in discrete domains.

Example 5

[0196] In Vivo Recordings of Arrhythmias

[0197] CPVT mice were bred as previously described (Lehnart S E et al., Leaky Ca2+ release channel/ryanodine receptor 2 causes seizures and sudden cardiac death in mice, Journal of Clinical Investigation 2008 June 118(6): 2230-45). Bay 60-7550 (3 mg/kg) was tested in CPVT mice. To induce ventricular tachycardia, the mice were injected with 60 mg/kg caffeine and 50 ng/kg Isoprenaline. 50% Ethanol was used for control injections.

[0198] Results

[0199] PDE2 Inhibition Prevents Ventricular Tachycardia in Mice with CPVT

[0200] Bay 60-7550 protected against VTs in CPVT mice: Bay 60-7550 2/7 (28%) developed VT; compared to 100% of controls (6/6) (FIG. 18). In total, PDE2 inhibition prevents ventricular tachycardias in three different mice models, HF, Ankyrin B.sup.+/− (long QT syndrome), and CPVT.

CONCLUSION

[0201] It has been shown that PDE2 inhibition protects against an additional cardiac disease with increased risk of ventricular tachycardias.