Revitalizing the heart: cutting-edge nucleic acid therapies in cardiac regeneration

Cardiovascular disease (CVD) is a leading global cause of death. Heart failure (HF) results from cardiac damage and subsequent loss of contractile function following injury. Some organisms, like zebrafish or neonatal mice, can regenerate the heart via proliferation of pre-existing cardiomyocytes. On the opposite, the mammalian adult heart is largely a post-mitotic organ, cardiomyocyte replication stops at birth and their endogenous regenerative capacity is not sufficient to compensate the loss of myocardium. Despite extensive research on regenerative medicine, cell or gene therapies adopted in clinical settings have not yet achieved significant improvements in patient’s heart function.

In my PhD research, I aim to better understand the biology of pro-proliferative miRNAs. One key challenge lies in finding a master gene regulator that can stimulate cardiomyocyte proliferation. miRNAs are small pieces of genetic information that can bind many different targets, which offer a promising opportunity in terms of boosting cardiac proliferation (Figure 1). Professor Mauro Giacca’s laboratory-unique approach was to perform a large unbiased functional screening. In their seminal study, they identified various human-specific miRNAs that can enhance cardiomyocyte proliferation including miR-199a-3p, miR-1825 and miR-33b-3p. miRNAs can be delivered to the heart either upon expression from adeno-associated viral (AAV) vectors or as synthetic molecules. Through gene transfer of pri-miRNA DNA using AAVs, miR-199a was found to induce cardiac regeneration and improve cardiac function in mouse and pig hearts after infarction. These improvements were assessed using echocardiography for mice and cardiac magnetic resonance imaging (MRI) for pigs. These studies constitute the first evidence that miRNA therapy delivered via AAV can stimulate cardiac regeneration in vivo in large mammals. Nonetheless, the clinical application of miRNA-based therapy must overcome the problems associated with uncontrolled expression of miRNAs, such as cardiomyocyte dedifferentiation followed by sudden arrhythmic death. Therefore, it is imperative to understand the function of pro-proliferative miRNAs and to study safe and effective methods of delivery to the heart.

In terms of delivery, we are comparing a viral delivery approach with the SNALP (Stable Nucleic Acid Lipid Particle) technology. In the latter, the miRNAs are encapsulated into lipid nanoparticles, a technology that has taken off following the success of mRNA-based therapies against COVID-19. I also employ novel AAV-based molecular tools to genetically label mitotic cells, in particular, the CycleTrack system (characterized and developed by Ilaria Secco, PhD). In this system, a small (312 bp) fragment of cyclin B2 promoter drives Cre recombinase expression in the nucleus of all cells that proliferate during G2/M phases of the cell cycle. This is monitored by the cumulative expression of EGFP fluorescence upon Cre-mediated excision. In this way, all cardiomyocytes that are green are those in which at least one round of replication has occurred. From a translational perspective, the most effective miRNAs and delivery strategies are being tested in both rat and human living myocardial slices.

Myocardial slices consist of sections of ultrathin (100–400 μm) living cardiac tissue with over 95% viable cardiomyocytes and robust electro-physiological activity. The thinness of these slices ensures the preservation of cardiomyocyte viability during culture, allowing for efficient diffusion of oxygen and essential nutrients without the requirement for coronary perfusion in vitro. Thanks to the development of novel culture methods and biomimetic culture devices, human myocardial slices can now be kept in culture for several weeks in vitro while preserving the function of the intact myocardium. This approach creates a much-needed bridge in translational research between in vitro and in vivo systems as it now allows chronic investigations in a cardiac multicellular preparation.

Going back to Sir Hans A. Krebs, I found it surprising when he wrote the following commentary on the history of the tricarboxylic acid cycle that made use of rat liver slices to study synthetic metabolic reactions: “The use of tissue slices, which I had learned in Otto Warburg’s laboratory (…) opened up an entirely new kind of approach to many problems of metabolism.” This highlights the relevance of taking a novel approach, which in this case led to the 1932 discovery of the ornithine cycle of urea formation, which provided a pattern of metabolic organization that set the precedent for the tricarboxylic acid cycle, that was ultimately awarded the Nobel Prize in 1953. In parallel to my own research, I have started using myocardial slices which are amenable to genetic manipulation (with AAV-based reporters that label proliferation or genetically encoded calcium indicators, etc.) to test many different conditions (such as a variety of miRNAs delivered via lipid nanoparticles). This system overcomes many of the drawbacks of studying cells in monolayer, such as maintaining the mechanical and electrical stimulation that prevents cellular dedifferentiation. I believe that we are equipped with the tools to answer the long-sought question of what orchestrates changes in cardiomyocyte proliferation.

In terms of framework, molecular biology research is grounded in the belief that biochemical events follow well-ordered causal chains. Studying pathways of proliferation, such as the precise targets of miR-199a-3p, yields valuable information on biological processes that are mechanical and susceptible to control and design. Conversely, physiology and cells can also be viewed as fractals, where cells behave as self-similar and self-referential units of life across different timescales, provided the external environment remains consistent (as in cardiac slices derived from different organisms) (Figure 2). This fractal perspective allows us to investigate whether the proliferation mechanisms or functional changes in the electrophysiology of cells observed across lower vertebrates and neonatal mammals can be mimicked in quiescent adult hearts when delivering pro-proliferative miRNAs.

I obtained my BSc in Biochemistry with Professional Experience at the University of Manchester. During my studies, I completed a placement year at the University of California San Francisco at Guo Huang's laboratory to study the regenerative potential of the heart. Upon completing my undergraduate studies, I joined the Wellcome Trust ‘Advanced Therapies for Regenerative Medicine’ MRes and PhD programme at King’s College London. Currently, I am pursuing a PhD project at Professor Mauro Giacca's laboratory, where my focus lies in understanding and testing the efficacy of pro-proliferative microRNAs in primary mouse cardiomyocytes and human myocardial slices. Email: [email protected]. Twitter: @@Sara_go2000