Despite the recent advances in cardiovascular therapies, cardiac diseases are still the leading cause of morbidity and mortality in developed countries and for this reason it is of pivotal importance to discover new therapeutic and prevention targets. The aim of this thesis is to implement a novel strategy to define new factors, either secreted proteins or microRNAs that protect myocardial cells from either ischemic or toxic damage. The protective activity of discovered factors will be validated in further studies in vivo in different models of myocardial damage.
The first step of the work is to clone two sub-libraries of secreted proteins and microRNAs in an adeno-associated viral vector (AAV) backbone plasmid (pZac2.1 and pAAV-MCS) containing the CMV promoter, that allows transgene expression into mammalian cells, and the ITR sequences, essential for viral genome packaging. So far many AAV serotypes have been recognized with specific capabilities to transduce different cell types. The AAV vectors are good candidates for gene therapy because of their promising features: these vectors retain less then 10% of the viral genome, do not express viral proteins, permitting a long persistence in vivo without either immunogenic or inflammatory response and are very efficient for in vivo transduction of many fully differentiated tissues, such as retina, neurons, heart and muscle. Cells are transduced at high multiplicity of infection and mixing of different rAAV preparation results in the simultaneous expression of gene combinations in vivo.
After the cloning, we obtained two different libraries to begin the selection procedure. Starting from pools of cDNAs and microRNAs cloned in an AAV backbone, we produced the corresponding pools of viruses and used the preparation to transduce cardiomyocytes. The serotype 9 was used for these experiments because it was shown that it has the highest tropism for cardiomyocytes in vitro and in vivo. Afterwards the cells were treated with a toxic chemical drug. The DNA of the surviving cells was extracted and analyzed by PCR to determine if any of the transduced genes carried by the AAV vectors was enriched; theoretically the cells that were transduced with a considerable amount of the protective factor should have a survival advantage and contain a considerable amount of the specific DNA sequence. Similarly, neonatal mice were infected with the same preparations of AAV vectors and an ischemic-like damage to the heart was induced by chemicals. Hearts were retrieved and DNA of the vectors retained by the living cells amplified in order to detect if any of the factors carried by the AAV pools had a positive effect on myocardium.
In parallel we decided to exploit also another strategy, transfecting a more permissive cardiac cell line with each interesting factor individually and inducing a toxic damage with a chemical drug. The viability and apoptotic rate of the cells treated with different factors was measured with high-throughput assays.
Two types of cells were used for the selection procedures: rat primary cardiomyocytes obtained from neonatal rats and HL-1, a murine cell line from mouse atrial cardiac myocytes that maintains the differentiated phenotype in culture, mainly used for the high-throughput screening. The mice employed for the in vivo part of this experiment belonged to the C57 strain.
To produce the toxic damage we used doxorubicin in the cells, a pro-apoptotic anthacycline drug, while isoproterenol in vivo, a synthetic catecholamine that stimulates both beta1 and beta adrenergic receptors, producing a widely accepted ischemic-like myocardial damage. The proper dose to be used in each cell type has been determined prior to the selection experiments.
1) The selection strategy was preliminarily validated in vitro in HEK 293T cells with a pool of AAV virus coding for 5 random proteins and a reporter gene, the green fluorescent protein (GFP). Cells were infected with the preparation of AAV2 virus and subsequently the ones with fluorescent phenotype were divided from the non-fluorescent cells with a fluorescence-activated cell sorter. The DNA of the nuclei of cells from the different populations was amplified by PCR and the analyses showed an enrichment in the GFP cDNA in the positive fractions.
2) Neonatay rat cardiomyocytes cell coltures were transduced with AAV serotype 9. Each viral vector preparation contained 10 different genes or 28/12 microRNAs.
The day after transduction the cells were treated with drugs for 24 hours and then stained with a fluorescent Annexin-V, that marks cells in apoptosis. With a fluorescence-activated cells sorter, the apoptotic cells are divided from the living ones. Only the DNA from the nuclei was extracted from each fraction of cells and analyzed through PCR to measure any appreciable difference in plasmid composition.
3) HL-1 cells were transfected with the most promising genes identified in step 2. The transfection was performed one plasmid at a time in a 96-well plate. The cells were subsequently treated with doxorubicin at the appropriate dose for 24 hours and analyzed in a plate reader with a luminescence-based assay that measures the viability of the cells in terms of ATP production. In this way, the protective potential of genes encoding for proteins or microRNA of unknown function can be analyzed in a short time.
4) Neonatal mice were intraperitoneally injected with the AAV9 preparations. After three weeks, the animals were treated with isoproterenol in order to induce an ischemic-like damage. Ten days later, the hearts of the mice were retrieved and DNA extracted. A PCR was used for amplifying the transduced plasmids in order to identify if one of them was preferentially retained because of its protective action on the myocardial muscle.
A single cycle of infection-damage-extraction of DNA from cardiomyocytes or hearts in most cases was not sufficient to identify a clear enrichment of one or more protective factors. In future developments of this project cDNA and microRNAs retrieved will be recloned in AAV plasmids to produce a new set of vectors carrying different proportions of the original genes. This new preparation will be used for further rounds of in vitro selection until the identification of a clear enrichment of one or more protective factors.
The final goal of this project is the in vivo and in vitro molecular characterization of the newly identified anti-apoptotic or pro-survival factors.