Periodic Reporting for period 1 - aNtHESIS (Novel heart regeneration strategies)
Période du rapport: 2016-01-01 au 2017-06-30
Cardiovascular diseases (CVDs) are the main cause of death in Europe, accounting for over 4 million deaths each year. Worldwide, an estimated 17.3 million people died from CVDs in 2008 and by 2030 more than 23 million people are expected to die annually. Among the various CVDs, Coronary Heart Disease (CHD), known also as ischemic heart disease, is the single most common cause of death in Europe, accounting for 1.8 million deaths each year. CHD and particularly Myocardial Infarction (MI), i.e. heart attack, is the leading cause of death in the Western world today. The main cause for CHD is the incapacity of the mammalian heart to undergo regeneration after injury. Instead, a scar tissue is formed at the site of the wound, which subsequently results in decreased cardiac function and finally heart failure. The diminished regenerative potential of the heart begins shortly after birth, when most cardiomyocytes (CMs) cease to proliferate and make a transition from hyperplastic to hypertrophic growth. Cardiac regenerative medicine has focused on stem-cell therapy as a mean to replace the massive loss of CMs associated with CHD, however, the medical benefit of these studies in human patients is as yet uncertain. The lack of a real therapeutic success among these intensive clinical efforts highlights the need for novel therapeutic approaches to cure heart diseases.
We have been intensively exploring novel molecules and compounds as well as the molecular mechanisms that facilitate CM cell division in the adult heart of mammals with the hope that the insights gained from the ERC project can ultimately be translated into novel strategies for heart regeneration. Indeed, our ERC-funded studies yielded a wealth of knowledge about the processes that regulate cardiac and skeletal muscle development and disease. The exciting insights gained motivated us to employ an unbiased approach utilizing a novel state-of-the-art high-throughput screen to identify molecules that promote CM proliferation and to study their effects on cardiac regeneration in mice. As a result, we identified a novel compound, Chicago Sky Blue 6B (CSB), which significantly increased the proliferation of adult CMs. Drawing upon our recent findings, we aimed to validate the pre-clinical application of our novel compound by conducting comprehensive in-vitro and in-vivo tests in mice as well as by carrying out experiments using human CMs. Such pre-clinical studies will lead to solid data sets suitable to be undertaken by pharmaceutical companies. These studies are expected to lead to proper clinical trials in human patients towards the creation of a novel therapy for heart disease that could benefit millions of people diagnosed with heart failure each year.
As a first step, the effect of CSB was further validated in vitro. We found that 6 days incubation with CSB constitutively leads to 1.046 fold increase (4.6%) in the number of CMs; while the CM number in the untreated cultures remains unchanged. For further confirmation of CSB activity, small molecules which share structural similarity with CSB; S4, S9 and R3 were also tested by the screening system. The results demonstrated that two out of the three compounds increase the number of P7 CMs similarly to CSB. To further validate the effect of CSB on CM proliferation, P8 cardiac cells were incubated with CSB for 4 days and then stained for Ki67, a protein that is expressed in cycling cells. While in the untreated cultures 0.39% of the CMs expressed Ki67 (Ki67+), CSB treatment led to >2 fold increase, resulting in 0.89% of Ki67+ CMs. We also verified the specific effect of CSB on the CM population, as well as its induction of CM cytokinesis and not merely karyokinesis.
The next step was to further elaborate our experiments in vivo, using large numbers of mice in each group, using both juvenile and adult mice. These experiments substantiated the potential therapeutic effect of CSB treatment after MI, as it consistently induced both reduction in scar size and improvement of heart function, compared to the control treated groups. Importantly, pathological examination showed no abnormalities in the CSB treated mice. Additionally, our results demonstrate that the effect of CSB is not restricted to the cardiac system but increases proliferation of skeletal stem cells as well. Finally, we suggest that CSB promotes heart repair in a pleiotropic manner by inhibition of the Vesicular Glutamate Transporter (VGLUT), the Calcium/Calmodulin Dependent Protein Kinase II (CaMKII), and the Macrophage Migration Inhibitory Factor (MIF). Altogether, these data suggest CSB as a novel therapeutic agent for cardiac repair after MI.
We have been intensively exploring novel molecules and compounds as well as the molecular mechanisms that facilitate CM cell division in the adult heart of mammals with the hope that the insights gained from the ERC project can ultimately be translated into novel strategies for heart regeneration. Indeed, our ERC-funded studies yielded a wealth of knowledge about the processes that regulate cardiac and skeletal muscle development and disease. The exciting insights gained motivated us to employ an unbiased approach utilizing a novel state-of-the-art high-throughput screen to identify molecules that promote CM proliferation and to study their effects on cardiac regeneration in mice. As a result, we identified a novel compound, Chicago Sky Blue 6B (CSB), which significantly increased the proliferation of adult CMs. Drawing upon our recent findings, we aimed to validate the pre-clinical application of our novel compound by conducting comprehensive in-vitro and in-vivo tests in mice as well as by carrying out experiments using human CMs. Such pre-clinical studies will lead to solid data sets suitable to be undertaken by pharmaceutical companies. These studies are expected to lead to proper clinical trials in human patients towards the creation of a novel therapy for heart disease that could benefit millions of people diagnosed with heart failure each year.
As a first step, the effect of CSB was further validated in vitro. We found that 6 days incubation with CSB constitutively leads to 1.046 fold increase (4.6%) in the number of CMs; while the CM number in the untreated cultures remains unchanged. For further confirmation of CSB activity, small molecules which share structural similarity with CSB; S4, S9 and R3 were also tested by the screening system. The results demonstrated that two out of the three compounds increase the number of P7 CMs similarly to CSB. To further validate the effect of CSB on CM proliferation, P8 cardiac cells were incubated with CSB for 4 days and then stained for Ki67, a protein that is expressed in cycling cells. While in the untreated cultures 0.39% of the CMs expressed Ki67 (Ki67+), CSB treatment led to >2 fold increase, resulting in 0.89% of Ki67+ CMs. We also verified the specific effect of CSB on the CM population, as well as its induction of CM cytokinesis and not merely karyokinesis.
The next step was to further elaborate our experiments in vivo, using large numbers of mice in each group, using both juvenile and adult mice. These experiments substantiated the potential therapeutic effect of CSB treatment after MI, as it consistently induced both reduction in scar size and improvement of heart function, compared to the control treated groups. Importantly, pathological examination showed no abnormalities in the CSB treated mice. Additionally, our results demonstrate that the effect of CSB is not restricted to the cardiac system but increases proliferation of skeletal stem cells as well. Finally, we suggest that CSB promotes heart repair in a pleiotropic manner by inhibition of the Vesicular Glutamate Transporter (VGLUT), the Calcium/Calmodulin Dependent Protein Kinase II (CaMKII), and the Macrophage Migration Inhibitory Factor (MIF). Altogether, these data suggest CSB as a novel therapeutic agent for cardiac repair after MI.