Date: 2nd July 2021
Heart disease has remained the leading cause of death at the global level for the last 20 years. However, it is now killing more people than ever before, in the main because after infarction the heart muscle cells, cardiomyocytes, have a poor renewal capacity. The best current treatment for heart failure is a transplant, but availability of organs is a massive hinderance to this being a widespread and only option, meaning there is an urgent need for alternative solutions. Now, researchers have used gene therapy to knock down a signalling pathway that represses cardiomyocyte proliferation and renewal, showing it can improve heart function and myocardial regeneration after infarction.
After myocardial infarction in humans and other large animals, millions of cardiomyocytes fail to regenerate and die. However, this lack of regenerative capabilities is not the case for all animals, and those such as zebrafish can fully regenerate after amputations of up to 20% of the ventricle.
Other animals that cannot regenerate in adulthood, are able to for a short time window just after birth, such as mice and pigs, and there is also some anecdotal evidence that the human heart may also be able to do this postnatally. These data have led to ongoing efforts to manipulate adult cardiomyocytes to regenerate the heart either by re-activating signalling pathways present in the neonatal or embryonic heart that may be responsible for this ability or to inhibit signals that prevent regeneration in the mature heart.
Hippo signalling is an evolutionarily conserved pathway that controls organ size by regulating cell proliferation, apoptosis, and stem cell self renewal. Previous work has shown that the Hippo signalling pathway is upregulated in patients with heart failure. Furthermore, inhibiting the pathway in a mouse model that mimics advanced human heart failure results in the murine hearts with recovered pumping function. The Hippo pathway consists of a core kinase cascade in which Hpo, called MST1/2 in mammals, phosphorylates the protein kinase Warts (Wts), facilitated in part by the adapter protein, Salvador, or SAV1 in mammals.
Now, researchers at Baylor College of Medicine, US, led by James Martin have developed a gene therapy using adeno-associated virus (AAV) to locally knock down the Hippo pathway gene Sav in a myocardial infarction model in pigs, after which an improvement in ejection fraction, increase in cardiomycocyte division, and reduced scar size was observed.
The team started by generating a AAV vector encoding short hairpin RNAs (shRNAs) against Sav, called AAV9-Sav-shRNA which also encoded a green fluorescent protein (GFP) reporter to visualise infected cardiomyocytes. These were locally administered into the border zone of the myocardial wall of adult, 3-month-old pigs via subendocardial injection. One month after viral injection into the uninjured hearts the team observed a reduction in Hippo pathway activity and an induction of cardiomyocyte proliferation over non-treated animals.
To determine whether AAV9-Sav-shRNA promoted heart regeneration after myocardial infarction, the team induced damage via ischemia/reperfusion (I/R) injury in the pig hearts and then administered the therapy two weeks later. At this stage all groups showed a reduction in left ventricular ejection fraction to below 40%. Whilst, the left ventricular function steadily worsened in the control treated group, those that had received the therapy saw a progressive improvement over time, along with an improved stroke volume and reduced scarring.
To delve into the mechanism of repair, the team examined the hearts yet further. They found that viral vector delivery of Sav-shRNA increased cardiomyocyte cell cycle entry and division after treatment, and the number of mononucleated cardiomyocytes, indicative of division, in the GFP-positive areas were higher than in the control hearts. Furthermore, sarcomere breakdown, which occurs as the cardiomyocytes de-differentiate, was also transiently observed. Finally, the therapy encouraged vascularity, inducing more capillaries.
Conclusions and future applications
The team here have revealed a new strategy for tissue renewal by inhibiting an endogenous pathway in a poorly regenerative organ, the heart. The AAV-based gene therapy inhibited the Hippo pathway, providing an effective and safe treatment option, by inducing cardiomyocyte regeneration, accompanied by improved function in a large-animal model after myocardial infarction.
The cardiovascular system of pigs has many important similarities to humans and has long been one of the closest models for human hearts, with pig heart valves often transplanted into patients. Therefore, the team see this work as an important step towards translation into humans, and one they will be pursuing in the near future as they hope to move on to clinical trials. Although safety and efficacy will be foremost, the initial study here showed the therapy was well tolerated, there were no deleterious effects of Hippo pathway loss in non-cardiomyocytes, and there were no tumours detected in other organs in the 3 month monitoring period. The next step will be to determine how human cardiomyocytes respond to Sav knockdown with shRNAs, as pig and human cardiomyocytes differ somewhat, for example in the number of nuclei they possess.
The lack of transplantable organs is an ongoing problem and one that is deteriorating as demand outstrips availability. Scientists around the world are trying to overcome this shortage by seeking alternative treatments and therapies. One recent promising breakthrough was the development of artificial paediatric heart valves, from a hybrid of tissue engineering and regenerative medicine, that created a tri-tube heart valve that can grow with the patient. Others are investigating biomimetic hydrogels that mimic natural elastin, and have also been shown to lead to functional recovery of ejection fraction after myocardial infarction. Cell-free therapies such as exosomes can induce cardiac regeneration, whilst base editing therapies to treat coronary disease may also provide an alternative treatment in the future. The work here complements these discoveries, adding valuable targets to restore heart function, and offering hope to the millions of patients that are diagnosed with cardiovascular disease a year.
For more information please see the press release from Baylor College of Medicine
Liu, S., Li, K., Wagner Florencio, L., Tang, L., Heallen, T.R., Leach, J.P., Wang, Y., Grisanti, F., Willerson, J.T., Perin, E.C., et al. (2021). Gene therapy knockdown of Hippo signaling induces cardiomyocyte renewal in pigs after myocardial infarction. Science Translational Medicine 13, eabd6892.
https://doi.org/10.1126/scitranslmed.abd6892
