The cardiac Pi3k signaling cascade – New drug target for the prevention and treatment of cardiovascular disease?
Cardiac diseases ware by far the main cause of death in humans and the prognosis for cardiac patients remains unacceptably poor, which calls for development of novel pharmacological therapies. In this regard, previous studies demonstrated that transgenic animals with overactivation of cardiac PI3K-AKT cascade display reduced infarct size after ischemia and genetic disruption of PI3K-AKT signaling accelerates the development of heart failure and impedes the cardiac benefits promoted by exercise. While evidence from transgenic animals suggests an important role for PI3K-AKT in the heart, it is unknown whether targeted activation of PI3K-AKT signaling with therapeutically relevant strategies evokes cardioprotection after ischemia or mimics the benefits of exercise in the heart. Therefore, the translation of this knowledge into the clinic requires further investigation on molecular targeting approaches with clear therapeutic potential. Considering these points, the goals of the studies comprising this project were to: (Paper I) verify whether protein kinase AKT is activated in the human heart by a cardioprotective intervention, remote ischemic preconditioning (RIPC), in a randomized controlled trial; Published in the International Journal of Cardiology. Remote ischemic preconditioning preserves mitochondrial function and activates pro- survival protein kinase AKT in the left ventricle during cardiac surgery: A randomized trial. Katrine H. Slagsvold*, Jose B.N. Moreira*, Øivind Rognmo, Morten Høydal, Anja Bye, Ulrik Wisløff, Alexander Wahba. *Shared first authors (Paper II) test whether a small molecule activator of AKT reduces infarct size after ischemia; Under 2nd revision in the Journal of Translational Medicine. A small molecule activator of AKT does not reduce ischemic injury of the heart. Jose B.N. Moreira, Martin Wohlwend, Marcia NM Alves, Ulrik Wisløff, Anja Bye (Paper III) evaluate the effects of cardiac-specific PI3K gene therapy on mitochondrial function, cardiac function and aerobic capacity (VO2max) in a rat model artificially selected for low aerobic running capacity. Manuscript submitted. Cardiac PI3K gene transfer in a rat model of low aerobic capacity. Jose B.N. Moreira, Martin Wohlwend, Lauren G. Koch, Steven L. Britton, Ulrik Wisløff, Anja Bye In study I we demonstrated that RIPC activates protein kinase AKT in the left ventricle of patients undergoing coronary artery bypass graft surgery. AKT was substantially activated by RIPC before the onset of ischemia (aortic clamping), which induced phosphorylation of AKT-specific targets at reperfusion, suggesting that AKT is relevant for cardioprotection in humans. AKT activation was also associated with preserved mitochondrial function in RIPC group, as compared to control. In study II we showed that the small molecule SC79 activates cardiac AKT within 30min. Surprisingly, activation of AKT by SC79 either before or after ischemia, in low or high doses, was not sufficient to rescue myocardium ATP levels or reduce infarct size. Finally, in study III we found that PI3K-AKT signaling is deactivated in the heart of rats with inherited low aerobic capacity, which was accompanied by mitochondrial dysfunction and impaired cardiomyocyte function. We used clinical trial-like gene therapy with a cardiotropic adeno-associated virus (AAV9) to transfect PI3K in this animal model, and assessed mitochondrial function, cardiac function and VO2max. Despite inducing an efficient transfection, AAV9 carrying PI3K did not improve cardiac function, mitochondrial function or VO2max in our animal model. We conclude that although PI3K-AKT signaling is affected by RIPC in the human heart and is disrupted in an animal of low aerobic capacity, targeted activation of cardiac PI3K-AKT signaling with the selected therapeutic strategies did not induce significant benefits. PhD thesis related to this project was delivered on the 16.December.2014. Preliminary date for the defense: 09.April.2015
In Paper I we provided the first evidence in humans that remote ischemic preconditioning activates pro-survival protein kinase AKT in the heart. We followed-up this finding in Paper II by answering a research question that intrigued the literature for decades: whether acute activation of AKT reduces the size of infarction in an animal model. In Paper III we provided evidence that PI3K-AKT signaling is deactivated in the heart of rats with inherited low aerobic capacity, which was accompanied by mitochondrial dysfunction and impaired cardiomyocyte function. However, gene therapy to restore Pi3k-Akt signalling in the heart did not rescues cardiac function and VO2max in the animal model.
The cardiac Pi3k signaling cascade – New drug target for the prevention and treatment of cardiovascular disease?
Cardiovascular disease (CVD) is a leading public health problem worldwide and recent data suggest it as the main cause of death in human specie, therefore, investigation on novel therapeutic targets are of interest. In this sense, the goal of this project is to study cardiac Pi3k-Akt signaling in different animal models of heart disease.
The activities in 2013 were:
1. We analysed the effects of Pi3k modulation in cardiac myocytes isolated from rats artificially selected for Low and High running capacity (LCR and HCR, respectively). After Pi3k activation, isolated cardiomyocytes from LCR no longer presented contractile dysfunction, neither reduced time to peak shortening or slowed relenghtening, when compared with HCR control cardiomyocytes. On the other hand, cardiomyocytes from HCR treated with Pi3k inhibitor presented a functional phenotype similar to LCR control, regarding fractional shortening and contractile kinetics (Group effect, p<0.01; Treatment effect, p<0.05). Cardiomyocyte calcium handling (calcium peak, time to peak and decay velocity) was not altered by any of the treatments (Group effect, p<0.01; Treatment effect, p=0.95). These results were presented in 2013 at the European Society of Cardiology congress (Amsterdam, Netherlands) and at the Chronic Heart Failure Symposium is Oslo.
2. One of the aims with this study is to modulate Pi3k-Akt signaling in vivo by gene transfection using adeno-associated viruses (AAV) in rats. In 2013 we conducted a pilot study using different AAV serotypes to verify if we could achieve in vivo transfesction via tail vein injections. Unfortunately, none of the AAV serotypes provided robust transgene expression at the dose used in the initial pilot study. We have started a new pilot study using a five-fold higher dose of AAV to verify whether cardiac transfection can be achieved.
3. In December 2013, Jose Bianco Moreira visited the laboratory of Prof Roger Hajjar at the Mount Sinai School of Medicine (USA), a pioneer on experimental transfection in vivo. In the visit, he learned the technique for intracardiac AAV injections in rats, which will be used in case the intravenous AAV injections do not provide robust and reproducible gene transfection in the heart.
4. Jose Bianco started his participation in the translation research project Optimex (http://www.ntnu.edu/optimex), which aims to develop training strategies as therapy for diastolic heart failure. Jose is involved in the experimental arm of the study, which tests the safety and efficacy of different training intensities in rats with diastolic heart failure. The hypothesis is that exercise training will deactivate intracellular signaling responsible for pathological cardiac hypertrophy and will activate signaling responsible for physiological cardiac hypertrophy, in particular the Pi3k-Akt cascade. The first batch of animals will be sacrificed in February 2014 and molecular experiments will start immediately after all samples have been collected. If the previously mentioned hypothesis is confirmed, we will activate Pi3k-Akt signaling in isolated cardiomyocytes from diastolic heart failure rats and verify if this activation is sufficient to improve relaxation function in the isolated cardiac myocytes.
5. Left ventricular samples were also obtained from infarcted rats exercised at moderate or high-intensity for 8 weeks, in order to test whether training can activate Pi3k-Akt cascade in a model of systolic heart failure. If so, we will also verify whether high-intensity training can provide superior Pi3k-Akt activation than moderate-intensity. These experiments are ongoing.
For 2014 we aim to finish ongoing activities (points 4 and 5 above) and to standardise the technique for in vivo cardiac gene transfection in rats.
Cardiac Pi3k signaling cascade in cardiovascular diseases
Cardiovascular disease (CVD) is a leading public health problem worldwide and recent data suggest it as the main cause of death in human specie, therefore, investigation on novel therapeutic targets are of interest. In this sense, the goal of this project is to study cardiac Pi3k-Akt signaling in a rat model.
Prospective studies suggested that low aerobic fitness (as measured by maximal oxygen uptake) is the single best predictor of all-cause and cardiovascular mortality in healthy individuals and patients with CVD.
More recently, unique rat lineages were developed as an attempt to demonstrate a causal relationship between aerobic fitness, complex diseases and mortality, resulting in rats with Low or High inherited Running Capacity (LCR/HCR rats, respectively). These contrasting strains are born with a large difference in aerobic fitness level, and LCR rats accumulate risk factors of CVD and have shorter lifespan compared to HCR. Therefore, our study is currently using these animals as models to investigate the proposed molecular mechanisms involved in the cardioprotection provided by high aerobic fitness.
A previous study of our group demonstrated by a high-throughput screening that hearts from HCR presented higher expression of genes related to defense response against pathological stimuli and that LCR’s hearts showed an up-regulation of embryonic growth factors, along with pathological cardiac growth, which led us to look for intracellular mechanisms that could modulate both the physiological cardiac cellular response to pathological stimuli and inhibit pathological cardiac growth, leading to healthier cardiac cells. Therefore, we hypothesized that the Pi3k (Phosphoinositide-3 kinase)-Akt signalling cascade would be up-regulated in HCR hearts, since this pathway was extensively shown as a positive modulator of physiological cardiac response, an inhibitor of pathological cardiac remodeling and required for several exercise-induced cardiac adaptations. If the proposed hypothesis is confirmed, this pathway might turn into a novel pharmaceutical target to prevent and/or treat cardiovascular diseases.
The project started in March 2012. Besides assessing physiological parameters and cardiomyocyte function, we have measured mitochondrial respiration in permeabilized cardiac fibers and our results show that maximal mitochondrial respiration (as induced by a cocktail of different mitochondrial substrates) was 40% reduced in LCR, while non-stimulated respiration was unchanged.
As a starting strategy to verify differential regulation of Pi3k-Akt signaling, we isolated total RNA from left ventricles of HCR and LCR and performed real-time polymerase chain reaction (PCR) to detect messenger RNA levels of several genes known to encode important components of Pi3k-Akt cascade or cross-talking actors. We identified that eight out of the 84 tested targets were significantly overexpressed in HCR, while none of them were overexpressed in LCR. The overexpressed genes in HCR hearts were Adenosine deaminase, RNA-specific (Adar), Cell division cycle 42 (CDC42), Beta Catenin, FK506 binding protein 1a (FKBP1), Connexin 43, Mitogen activated protein kinase 3 (MAPK-3 or ERK1), Protein Kinase C alpha (PKC alpha) and SOS1.
Considering our findings, we went back to the related literature to check the current state of knowledge regarding those genes and gene products, in order to adjust the next steps of the project and take available evidence as support for future experiments. Our next step is to follow-up on investing the role played CDC42, Connexin 43, MAPK-3, Beta Catenin and FKBP1 in our animal model.
The study is in good pace and perfectly on schedule. Pi3k modulation in cardiac HCR and LCR cells was already performed and the data is being analyzed.
Remote ischemic preconditioning preserves mitochondrial function and activates pro-survival protein kinase Akt in the left ventricle during cardiac surgery: a randomized trial.
Int J Cardiol 2014 Dec 15;177(2):409-17. Epub 2014 okt 14
PMID: 25456576 - Inngår i doktorgradsavhandlingen
A small molecule activator of AKT does not reduce ischemic injury of the heart
Under review in the Journal of Translational Medicine (January 2015)
Cardiac PI3K(p110a) gene transfer in a rat model of low aerobic capacity
Conference abstract: Pi3k modulates cardiomyocyte phenotype in rats selected for low aerobic capacity
Eur Heart J (2013) 34 (supplement 1): doi: 10.1093/eurheartj/eht309.P4202