Scientists have revealed genetic changes that may weaken a key protein in the heart muscle, offering a new explanation for how inherited mutations contribute to hypertrophic cardiomyopathy, which in some cases leads to serious heart rhythm disturbances or sudden cardiac death.

Researchers from the Universities of Birmingham, Oxford, Nottingham, and the Harwell Research Centre in the UK analyzed 17 genetic changes in a protein called alpha-actinin-2, or ACTN2, and found that these changes may reduce the protein's stability, cause it to clump, or alter its ability to bind to actin filaments inside heart cells.

The researchers said the findings, published in the journal Nature Communications, show that disease-associated changes do not all work in the same way, but may disrupt protein structure and function through several different molecular mechanisms.

Hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy is a genetic disease in which the heart muscle, especially the left ventricular wall, becomes thicker than normal. This thickening can impede the filling of the heart with blood or its ejection, and can be associated with heart rhythm disturbances.

Many affected individuals have no obvious symptoms, while others may experience symptoms such as shortness of breath, chest pain, fainting, and palpitations.

In a limited proportion of cases, an electrical disturbance of the heart can lead to sudden cardiac arrest, making the disease a known cause of sudden death in some young people and athletes.

Protein anchoring the heart's internal structure

The ACTN2 protein plays a key role in the sarcomere, the basic unit responsible for contraction of heart muscle cells. The protein is localized in a region known as the Z-disc, where it anchors thin actin filaments and links them together, maintaining the mechanical organization of the heart muscle during repeated contraction.

Previous studies have linked changes in the gene responsible for producing ACTN2 to cardiomyopathies, but researchers did not know precisely how each mutation affects the protein's shape, stability, and interaction with other components inside the cell.

The team selected 17 changes of the 'missense mutation' type, which are DNA changes that replace one amino acid with another within the protein. The changes were distributed across different regions of ACTN2, including five mutations in the actin-binding domain, ten mutations in the central rod-like region, and two mutations in the C-terminal region.

The researchers used a range of biochemical and structural biology techniques, including measuring protein melting and thermal stability, X-ray crystallography, molecular structure modeling, actin-binding assays, as well as techniques to study protein size and aggregation propensity.

The results showed that all tested variant forms exhibited reduced solubility compared to the normal protein, with a clear tendency for some to form protein aggregates.

Highly sensitive region

The actin-binding domain emerged as the most vulnerable part of the protein; mutations in this region showed greater thermal instability, meaning their structure may begin to unfold under conditions that do not affect the normal protein to the same extent.

Molecular models predicted that some changes could weaken ACTN2's binding to actin, while others might lock the protein in an open position that increases its adhesion to actin filaments excessively.

Experiments confirmed that a mutation designated T247M increased binding strength and capacity to actin, indicating that disease does not necessarily result only from loss of protein function, but may also arise when some functions become excessive or dysregulated.

Computational predictions and lab experiments

The researchers also predicted that other changes in the central region might disrupt the ability of two ACTN2 molecules to bind and form the dimeric structure required for protein function. However, crystal structure analysis of two of these mutations showed that the molecules remained capable of forming stable pairs.

The research team said this result highlights the importance of combining computational predictions with laboratory experiments, because models alone may not reveal the true effect of a mutation within the full-length protein.

Co-author Dr. Katja Gehmlich, Professor of Molecular Cardiology at the University of Birmingham, said that hypertrophic cardiomyopathy can affect people who appear healthy and have good physical fitness, and its effects can be devastating.

She added that the findings will help researchers understand how these proteins reshape the hearts of affected individuals, and may in the future support the search for ways to address the molecular weaknesses resulting from genetic changes.

Interpreting genetic tests

Interpreting the results of genetic tests is one of the major challenges in inherited heart diseases, as analysis may reveal a change in a gene without it being clear whether this change actually causes disease or represents a harmless genetic variation.

Some of the changes included in the study were classified in databases as having 'uncertain clinical significance,' while others had no clear classification, and interpretations conflicted over whether other changes were benign or disease-causing.

Co-author Dr. Fayad Mohammed, a lecturer at the University of Birmingham, said the methods used can be replicated in other labs and may provide a framework for improving the interpretation of genetic tests related to ACTN2.

He added that the same approach can be adapted to study genetic changes affecting other cardiac proteins, which may help doctors distinguish between dangerous mutations and harmless changes.

Lead author Dr. Maya Nour-Eldin, a researcher at the University of Birmingham, said the inability to determine whether a specific genetic change is responsible for the disease makes it more difficult to provide a clear explanation to patients and their families.

She added that the systematic combination of structural and functional tests helped the team identify changes likely to affect the protein, and may later support the development of treatments for hypertrophic cardiomyopathy and other heart diseases.

However, the study did not test a new treatment, nor did it prove that targeting ACTN2 pharmacologically can prevent sudden death. Moreover, most tests were performed on isolated proteins and in laboratory systems, not in a clinical trial on patients.

The researchers added that further studies on heart muscle cells, animal models, and clinical data from mutation carriers are needed to understand how each mutation affects the heart over time, and whether the mechanisms observed are amenable to therapeutic targeting.