Molecular mechanisms of exercise as novel therapies for heart failure
Molecular mechanisms of exercise as novel therapies for heart failure.
The cardiac benefits of exercise have been vastly documented, however, part of the patient population is unable to exercise sufficiently or respond poorly to exercise. This bottleneck calls for research to develop therapeutic alternatives. In this project we use the molecular mechanisms of exercise as inspiration to develop novel drug therapies.
In 2016 we used silencing RNA (siRNA) technology to knock down 16 molecular targets of exercise targets in human iPSC-derived cardiomyocytes, as a screening model to verify whether perturbation of these molecules has adverse effects in cardiomyocytes. As screening readout we adopted the expression of BNP, a validated biomarker of cardiac failure. These experiments identified 3 targets whose knockdown increased BNP expression significantly, indicating that inhibition of these targets (as found in our rat heart failure model, oppositely to exercise) triggers a pathological state. We also assessed whether the expression of the 16 exercise targets was disturbed in biopsies from heart failure patients, using RNA sequencing datasets. Altogether, these analyses singled out the protein Proline dehydrogenase (PRODH), which became our priority for further analyses. PRODH was reduced by 60% in rat failing hearts and rescued to completely normal levels by aerobic exercise. Moreover, the expression of PRODH is highly enriched in rat cardiomyocytes, as compared to cardiac fibroblasts from the same hearts. PRODH expression is disrupted by >70% in human failing hearts, with similar patterns observed between ischemic and non-ischemic heart failure. We generated adenovirus carrying human PRODH cDNA, to overexpress PRODH in human iPSC-cardiomyocytes. We initially used qPCR to quantify expression of selected markers of cardiac remodeling, metabolism and failure, in response to PRODH overexpression. The findings from these experiments confirmed that PRODH overexpression positively regulates cardiac genes related to enhanced cardiac function. Built on these findings, we decided to use RNA sequencing technology to assess, in an unbiased manner, whether PRODH overexpression (as seen in exercise failing hearts) would affect cardiomyocyte gene expression at genome-wide scale. Gene Set Enrichment Analysis revealed that PRODH overexpression upregulates transcripts related to oxidative phosphorylation in the mitochondria, and downregulates a gene signature related to endoplasmic reticulum stress. These findings gave us confidence to conduct further analyses of mitochondrial function, because PRODH is located in the mitochondria and due to our GSEA results. We used the Seahorse extracellular flux analyzer to assess mitochondrial oxygen consumption and extracellular acidification rates (ie. glycolysis) in human iPSC- cardiomyocytes with and without manipulation of PRODH expression. We observed that PRODH knockdown switched cardiomyocyte metabolism towards glycolysis, which is a hallmark metabolic characteristic observed in failing hearts. All these experiments were extensively reproduced and validated, and many of them were repeated in cultured adult cardiomyocytes from rats. The immediate next step is to conduct the in vivo follow-up of heart failure rats receiving AAV- PRODH or control AAV, to verify whether rescuing PRODH expression with a relevant therapeutic manipulation improves cardiovascular outcomes in the animal model. We are also conducting mechanistic studies both in adult rat cardiomyocytes and human iPSC-cardiomyocytes, to further characterize the function of PRODH, particularly in regard to regulation of mitochondrial function. We stress that PRODH has never been studied in the heart or in any other therapeutic context. These facts make our studies unique and highly original, thereby enhancing the potential for publication in prestigious journals.
Molecular mechanisms of exercise as novel therapies for heart failure
Heart failure is the main cause of death worldwide, which calls for development of improved therapeutics. Here we hypothesize that molecular mechanisms recruited by exercise are valuable drug targets for heart failure, and can bring forward the possibility to “deliver” the benefits of exercise to patients that are not able to exercise.
The original goals described in the application for the period were achieved with success, including: • Identification of circulating proteins induced by exercise: the proteomics screenings in human blood were concluded in the first semester of 2015. The initial data was used for two master theses, successfully defended and approved earlier this year • We started pilot experiments to identify the source (organ) of circulating molecules induced by exercise. Isolated organ baths are being used for this task. • We have validated the cardiac gene targets discovered in the rat model, using a separate cohort of animals. A group of 20 genes demonstrated robust modulation after exercise training in both cohorts. We have also verified whether they are expressed in cardiac myocytes and/or fibroblasts and identified 16 genes with cardiomyocyte expression. These genes form the initial list to be manipulated in vitro. • Since we concluded these activities faster than originally planned, we have started in the next step of the project. Therefore, the project is in fact ahead of schedule. We have initiated cell cultures of human cardiomyocytes derived from induced-pluripotent stem cells, and established optimized protocols for transfection and gene transfer. The cardiac genes identified in the animal models are currently being manipulated and the results will be available in February 2016 (several months in advance). • In addition to two cohorts of ischemic heart failure rats (myocardial infarction), we included data from an animal model of heart failure with preserved ejection fraction (Dahl salt-sensitive rats), to verify whether exercise-induced genes were induced exclusively in the ischemic model or not. Some of the targets were also similarly regulated in the animal model of heart failure with preserved ejection fraction. Concluded master theses related to the project: Inger Ane Hole (MSc in Chemistry, NTNU). Proteomic profiling of human blood after aerobic exercise training. Thesis approved on 08/April/2015 with grade “A”. Lars Einar Garnvik (MSc in Exercise Physiology). Identification of Circulating Biomolecules Induced by Endurance Exercise Training - First unbiased evidence in humans. Thesis approved on 16/06/2015 with grade “B”.
Role of KATP Channels in Beneficial Effects of Exercise in Ischemic Heart Failure.
Med Sci Sports Exerc 2015 Dec;47(12):2504-12.
Deletion of Kinin B2 Receptor Alters Muscle Metabolism and Exercise Performance.
PLoS One 2015;10(8):e0134844. Epub 2015 aug 24