A study led by German scientists has developed a new therapeutic to treat a currently untreatable form of heart failure by targeting a splice regulator in stiffened cardiac muscle cells to render them more pliable.

Heart failure is often preceded by shortness of breath, tiredness, water retention, heart palpitations, dizziness, increased blood pressure, diabetes, kidney disease, or infections. Although heart failure is a leading cause of death in older people, especially in older women, not all heart failures stem from the same immediate cause.

In some heart failures, the pumping activity of the heart is compromised. Currently, there are medications available to treat this type of heart failure called “heart failure with reduced ejection fraction” or HFrEF.

In some heart failures, however, the larger chambers of the heart—the ventricles—fail to fill adequately even when the heart continues to pump normally. This is usually due to the stiffening and thickening of the muscles in the walls of the ventricles of the heart. At present, there is no effective therapy for this form of heart failure called “heart failure with preserved ejection fraction” or HFpEF.

A team of scientists including Michael Gotthardt, PhD, professor at the Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), and his colleagues from Heidelberg University and Ionis Pharmaceuticals, a California-based company, has developed new therapeutic approach for HFpEF.

The new therapeutic approach is reported in an article published in the journal Science Translational Medicine titled, “Therapeutic inhibition of RBM20 improves diastolic function in a murine heart failure model and human engineered heart tissue.

Victor Badillo Lisakowski (left) and Michael Radke, PhD (right) from Michael Gotthardt’s lab, view ultrasound data from the mouse heart with and without ASO treatment. [Source: M. Gotthardt, MDC]
Michael Radke, PhD, postdoctoral researcher at the MDC and a co-first author of the paper said, “We provide a therapeutic approach that targets cardiac splicing to improve cardiac filling. We show how to accommodate species differences in evaluating antisense oligonucleotide treatment by employing complementary model systems—the mouse and engineered heart tissue.”

A gigantic protein aptly called titin, controls the elasticity of muscles in the ventricular wall. It is produced by cardiac muscle cells in different variants called isoforms that differ in their degree of flexibility. Growing infants produce highly elastic isoform of titin. Once growth has plateaued, the body produces stiffer titin isoforms that increase the heart’s pumping efficiency. However, stiffer titin filaments may lead to impaired filling of the ventricles and heart failure.

In adults, heart muscle cells are virtually unable to renew themselves. Yet the constant pumping action puts severe strain on titin proteins. Worn-out proteins are degraded and replaced every three to four days.

“The mechanical properties of titin proteins are difficult to adjust,” said Gotthardt. “But we can now intervene in the process preceding protein synthesis—alternative splicing.”

Alternative splicing is an ingenious biological ploy that allows the production of several protein isoforms from a single gene. The process is controlled by splicing factors that include some protein-coding fragments of genes called exons while excluding others from the processed messenger RNA produced from a gene.

“One of these [splicing factors], the master regulator RBM20, is a suitable target that we can address therapeutically,” said Gotthardt.

Earlier studies from the group have shown RBM20 determines the elastic, contractile, and electrical properties of the walls of the heart chambers. Mutant mice that produce only half as much RBM20 as normal mice produce more of the elastic titin isoforms. Animal models that replicate phenotypic features of HFpEF, such as increased stiffness of the cardiac ventricle, respond to a 50% reduction in RBM20 expression with improved diastolic filling and cardiac function.

However, pharmacological methods used in earlier studies by the team to reduce RBM20 expression cause cellular toxicity and cannot be used as therapeutics. Therefore, in the current study the team explored the use of antisense oligonucleotides (ASOs) optimized for delivery in vivo to the heart, to downregulate RBM20.

“We were surprised at how easily this could be done,” said Gotthardt.

ASOs are synthetic short chains of single-stranded nucleic acids. They bind specifically to the complementary messenger RNA sequences, the blueprint of the targeted protein and block its synthesis.

The researchers showed in adult mice with stiffened cardiac walls, that weekly application of ASOs over two months increased the expression of elastic titin isoforms that improved cardiac function. The team measured cardiac function using echocardiography and conductance catheter. The present study is the first to use ASOs to successfully influence alternative splicing in cardiac disease in an animal model.

The authors also sequenced RNA transcripts to confirm RBM20-dependent isoform changes of titin. This also pointed out potential side effects which were largely limited to genes related to the immune response.

To assess the human relevance and the potential for translation, the authors generated human RBM20 ASOs and evaluated the molecular and mechanical effects in human engineered heart tissue (hEHT) using expression analysis and video microscopy.

The authors noted, “We validated our approach in human engineered heart tissue, showing down-regulation of RBM20 to less than 50% within three weeks of treatment with ASOs, resulting in adapted relaxation kinetics in the absence of cardiac pathology. Our data suggest anti-RBM20 ASOs as powerful cardiac splicing regulators for the causal treatment of human HFpEF.”

Victor Badillo-Lisakowski, PhD student at the MDC and co-lead author on the study, grew heart muscle cells from human stem cells into artificial heart tissue. The tiny 3D heart tissue structures can be stimulated to contract and relax enabling them to mimic the pumping action of the heart. The researchers demonstrated that ASO molecules penetrate the cells and trigger the desired response.

“These tests on artificial heart tissue were an important step, because the primary sequences for titin are not identical in mice and humans,” said Radke, co-lead author of the study.

Researchers from Ionis Pharmaceuticals stabilized the sensitive ASO molecule so that most of the injected ASOs reached the striated muscles of the heart in the mouse model without being degraded in the blood, liver, or eliminated by the kidneys, although some did enter skeletal muscles.

“In the mouse model, however, we observed that it has no disruptive effect if increased amounts of elastic titin are formed in skeletal muscle,” stressed Radke.

The authors also conducted long-term treatment of mice with anti-RBM20 ASOs to model chronic cardiac disease that requires prolonged treatment.

“We treated our mice over a longer period of time and were able to see lasting treatment effects,” said Gotthardt. The therapeutic approach still needs some work, he said, adding, “An improvement over a weekly injection, which many patients are already familiar with from insulin or heparin, would be oral administration.”

Radke said, “Next steps are the evaluation of the treatment in different HFpEF-like animal models and modifying ASOs for improved targeting and fewer side effects as well as optimizing delivery. After that a large animal model would be important to evaluate scalability.”

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