Using mouse and cellular models to investigate the ACTN2 M228T variant in the heart and its link to cardiac disease

The Z-disc acts as a protein-rich structure to tether thin filament in the contractile units also known as sarcomeres, of striated muscle cells. Numerous proteins interact in the Z-disc and are integral for maintaining the architecture of the sarcomere, while facilitating force transduction and intracellular signalling in both cardiac and skeletal muscle. Pathogenic variants in Z-disc proteins can cause diseases, e.g. cardiomyopathies. These are diseases of the heart muscle, affecting its structure and function which ultimately impede the hearts ability to pump blood around the body. They can also be associated with a high risk of sudden cardiac death. This project focused on the Z-disc sarcomeric α-actinin 2 (ACTN2) and its link to cardiomyopathies. Pathogenic variants in the gene coding for ACTN2 have been identified and linked to cardiomyopathies, although there is limited understanding about the mechanisms involved. Actn2 M228T mouse and cellular models were used to test the impact of this pathogenic variant on cardiac hypertrophy and to explore underlying disease mechanisms. Morphometrics of wild type (WT) and heterozygous (Het) adult mice were used to test for hypertrophic differences between mice of different ages and sexes. Western blots were performed on adult mouse hearts to investigate protein expression of hypertrophic markers. While increased protein expression of hypertrophic markers was observed suggesting molecular changes occurred, this was not translated into pathological signs of hypertrophy such as changes in heart weight. The homozygous (Hom) variant resulted in embryonic lethality, however no developmental delay was detected by Theiler staging at embryonic day 15.5 embryos. Both gene and protein expression were explored in hearts from homozygous embryos. An increase in Actn2 transcript expression saw a down-regulation of the corresponding Actn2 protein, suggesting degradation of the aberrant protein. Increased expression of Actn3 was not translated at the protein level and suggests Actn3 cannot compensate for reduction of Actn2. Increased expression was seen for genes involved in the foetal gene programme and supports enhancement of this programme in the presence of this genetic variant. Reduced colocalisation of Actn2 and titin seen in homozygous embryos further suggests disorganisation of sarcomeres, a feature seen in some instances of hypertrophy. Preliminary results in an induced pluripotent stem cell (iPSC) line homozygous for the variant, further suggest less mature sarcomeres in the homozygous ACTN2 M228T variant compared to the KOLFC2 parental WT. Future work should aim to phenotype this line further. Overall, these data support the pathogenic role of ACTN2 M228T in the development of cardiomyopathy and cardiac disease

Long QT syndrome-associated calmodulin variants disrupt the activity of the slowly activating delayed rectifier potassium channel.

Calmodulin (CaM) is a highly conserved mediator of calcium (Ca2+ )-dependent signalling and modulates various cardiac ion channels. Genotyping has revealed several CaM mutations associated with long QT syndrome (LQTS). LQTS patients display prolonged ventricular recovery times (QT interval), increasing their risk of incurring life-threatening arrhythmic events. Loss-of-function mutations to Kv7.1 (which drives the slow delayed rectifier potassium current, IKs, a key ventricular repolarising current) are the largest contributor to congenital LQTS (>50% of cases). CaM modulates Kv7.1 to produce a Ca2+ -sensitive IKs, but little is known about the consequences of LQTS-associated CaM mutations on Kv7.1 function. Here, we present novel data characterising the biophysical and modulatory properties of three LQTS-associated CaM variants (D95V, N97I and D131H). We showed that mutations induced structural alterations in CaM and reduced affinity for Kv7.1, when compared with wild-type (WT). Using HEK293T cells expressing Kv7.1 channel subunits (KCNQ1/KCNE1) and patch-clamp electrophysiology, we demonstrated that LQTS-associated CaM variants reduced current density at systolic Ca2+ concentrations (1 μm), revealing a direct QT-prolonging modulatory effect. Our data highlight for the first time that LQTS-associated perturbations to CaM's structure impede complex formation with Kv7.1 and subsequently result in reduced IKs. This provides a novel mechanistic insight into how the perturbed structure-function relationship of CaM variants contributes to the LQTS phenotype. KEY POINTS: Calmodulin (CaM) is a ubiquitous, highly conserved calcium (Ca2+ ) sensor playing a key role in cardiac muscle contraction. Genotyping has revealed several CaM mutations associated with long QT syndrome (LQTS), a life-threatening cardiac arrhythmia syndrome. LQTS-associated CaM variants (D95V, N97I and D131H) induced structural alterations, altered binding to Kv7.1 and reduced IKs. Our data provide a novel mechanistic insight into how the perturbed structure-function relationship of CaM variants contributes to the LQTS phenotype

Insights into the Role of a Cardiomyopathy-Causing Genetic Variant in ACTN2

Pathogenic variants in ACTN2, coding for alpha-actinin 2, are known to be rare causes of Hyper-trophic Cardiomyopathy. However, little is known about the underlying disease mechanisms. Adult heterozygous mice carrying the Actn2 p.Met228Thr variant were phenotyped by echocar-diography. For homozygous mice, viable E15.5 embryonic hearts were analysed by High Reso-lution Episcopic Microscopy and wholemount staining, complemented by unbiased proteomics, qPCR and Western blotting. Heterozygous Actn2 p.Met228Thr mice have no overt phenotype. Only mature males show molecular parameters indicative of cardiomyopathy. By contrast, the variant is embryonically lethal in the homozygous setting and E15.5 hearts show multiple morphological abnormalities. Molecular analyses, including unbiased proteomics, identified quantitative abnormalities in sarcomeric parameters, cell cycle defects and mitochondrial dys-function. The mutant alpha-actinin protein is found to be destabilised, associated with increased activity of the ubiquitin-proteosomal system. This missense variant in alpha-actinin renders the protein less stable. In response, the ubiquitin-proteosomal system is activated; a mechanism which has been implicated in cardiomyopathies previously. In parallel, lack of functional al-pha-actinin is thought to cause energetic defects through mitochondrial dysfunction. This seems, together with cell cycle defects, the likely cause of death of the embryos. The defects also have wide-ranging morphological consequences