Mono- and Biallelic Protein-Truncating Variants in Alpha-Actinin 2 Cause Cardiomyopathy Through Distinct Mechanisms

Background: ACTN2 (alpha-actinin 2) anchors actin within cardiac sarcomeres. The mechanisms linking ACTN2 mutations to myocardial disease phenotypes are unknown. Here, we characterize patients with novel ACTN2 mutations to reveal insights into the physiological function of ACTN2. Methods: Patients harboring ACTN2 protein-truncating variants were identified using a custom mutation pipeline. In patient-derived iPSC-cardiomyocytes, we investigated transcriptional profiles using RNA sequencing, contractile properties using video-based edge detection, and cellular hypertrophy using immunohistochemistry. Structural changes were analyzed through electron microscopy. For mechanistic studies, we used coimmunoprecipitation for ACTN2, followed by mass-spectrometry to investigate protein-protein interaction, and protein tagging followed by confocal microscopy to investigate introduction of truncated ACTN2 into the sarcomeres. Results: Patient-derived iPSC-cardiomyocytes were hypertrophic, displayed sarcomeric structural disarray, impaired contractility, and aberrant Ca 2+ -signaling. In heterozygous indel cells, the truncated protein incorporates into cardiac sarcomeres, leading to aberrant Z-disc ultrastructure. In homozygous stop-gain cells, affinity-purification mass-spectrometry reveals an intricate ACTN2 interactome with sarcomere and sarcolemma-associated proteins. Loss of the C-terminus of ACTN2 disrupts interaction with ACTN1 and GJA1, 2 sarcolemma-associated proteins, which may contribute to the clinical arrhythmic and relaxation defects. The causality of the stop-gain mutation was verified using CRISPR-Cas9 gene editing. Conclusions: Together, these data advance our understanding of the role of ACTN2 in the human heart and establish recessive inheritance of ACTN2 truncation as causative of disease.

Transcriptome analysis of conditionally immortalized atrial myocytes: identification of a novel atrium-enriched protein involved in sarcomere assembly and maintenance

Background Heart development relies on the tight spatiotemporal control of cardiac gene expression. Genes involved in these processes have been identified using mainly (transgenic) animals models and pluripotent stem cell-derived cardiomyocytes (CMs). Recently, the repertoire of cardiomyocyte differentiation models has been expanded with iAM-1, a monoclonal cell line of conditionally immortalized neonatal rat atrial myocytes (NRAMs) which allows toggling between proliferative and differentiated (i.e. excitable and contractile) phenotypes in a synchronized and homogenous manner. Purpose To identify and characterize (lowly expressed) genes with an as-of-yet uncharacterized role in cardiomyocyte differentiation, dedifferentiation and proliferation by exploiting the unique properties of conditionally immortalized NRAMs (iAMs). Methods and results RNA sequencing was performed during a full cycle of iAM-1 differentiation and subsequent dedifferentiation, identifying ±13,000 transcripts, of which the dynamic expressional changes during cardiomyogenic differentiation in most cases opposed those during dedifferentiation. Among the genes whose expression increased during differentiation and decreased during dedifferentiation were many genes with a known (lineage-specific) role in cardiac muscle formation, thereby confirming the relevance of iAMs as cardiomyogenic differentiation model. Filtering for cardiomyocyte-enriched low abundancy transcripts, resulted in the identification of an uncharacterized protein, which is highly conserved among Nephrozoa and up- and downregulated during cardiomyocyte differentiation and dedifferentiation, respectively. In neonatal and adult rats, this protein is muscle-specific, highly atrium-enriched and localized around the C-zone of cardiac sarcomeres. Lentiviral shRNA-mediated knockdown resulted in loss of sarcomeric organization in both NRAMs and iAMs. Neither knockdown nor overexpression of this protein affected the electrophysiological properties of differentiated iAM monolayers. Conclusions iAM-1 cells offer a relevant model to identify and characterize novel (low abundancy) genes involved in cardiomyocyte (de)differentiation as exemplified by the identification a novel uncharacterized protein that is muscle-specific, highly atrium-enriched, localized around the C-zone of cardiac sarcomeres and plays a specific role in atrial sarcomerigenesis. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Netherlands Organisation for Health Research and Development (ZonMw) Leiden Regenerative Medicine Platform Holding project with number (LRMPH) Figure 1. (A) Experimental setup. At the indicated timepoints iAM-1 cells were fixed for immunostaining and RNA extraction for transcriptome analysis. (B) Immunochemical staining of iAM-1 cells for the proliferation marker Ki-67 and the Z-line marker sarcomeric α-actinin. (C & D) Immunohistological double stainings of longitudinal sections of neonatal rat hearts for the uncharacterized protein (GOI 1) and the sarcomeric protein cardiac troponin I (TNNI3). LA, left atrium; RA, right atrium; LV, left ventricle; RV, right ventricle. Scale bar, 250 μm.

Mono- and bi-allelic protein truncating variants in alpha-actinin 2 cause cardiomyopathy through distinct mechanisms

Alpha-actinin 2 (ACTN2) anchors actin within cardiac sarcomeres. The mechanisms linking ACTN2 mutations to myocardial disease phenotypes are unknown. Here, we characterize patients with novel ACTN2 mutations to reveal insights into the physiological function of ACTN2. Patient-derived iPSC-cardiomyocytes harboring ACTN2 protein-truncating variants were hypertrophic, displayed sarcomeric structural disarray, impaired contractility, and aberrant Ca2+-signaling. In heterozygous indel cells, the truncated protein incorporates into cardiac sarcomeres, leading to aberrant Z-disc structure. In homozygous stop-gain cells, affinity-purification mass spectrometry reveals an intricate ACTN2 interactome with sarcomere and sarcolemma-associated proteins. Loss of the C-terminus of ACTN2 disrupts interaction with ACTN1 and GJA1, two sarcolemma-associated proteins, that may lead to the clinical arrhythmic and relaxation defects. The causality of the stop-gain mutation was verified using CRISPR-Cas9 gene editing. Together, these data advance our understanding of the role of ACTN2 in the human heart and establish recessive inheritance of ACTN2 truncation as causative of disease.

Abstract 14355: Mono- and Biallelic Structural Variants in Alpha-actinin 2 Cause Contractile Dysfunction and Cardiomyopathy in Humans

Background and Aim: Cardiomyopathy is a common cause of sudden cardiac death in children that often requires heart transplantation. Alpha-actinins are critical cytoskeletal proteins that anchor actin filaments within the cardiac sarcomere. Mutations in ACTN2 have been associated with cardiomyopathy, but the mechanisms behind how those lead to cardiac dysfunction remain poorly understood. The aim of the present study was to investigate the effects of two novel structural ACTN2 mutations in human cardiac tissue and patient-specific iPSC-derived cardiomyocytes. Methods and Results: We identified a patient homozygous for a stop-gain mutation (p.Q860X) and a family heterozygous for a large exon 8-10 deletion with a 41bp insertion (indel) in ACTN2 , using a custom mutation pipeline optimized for rare variant discovery. In the explanted cardiac tissue of the homozygous patient, we observed mild hypertrophy and interstitial fibrosis. Patient-specific hiPSC-CMs from both families were hypertrophic, displayed sarcomeric structural disarray on transmission electron microscopy, and had slower contractile velocity compared to control hiPSC-CMs. The ACTN2indel protein was expressed, with subsequent incorporation into cardiac sarcomeres. In the homozygous stop-gain cells and normal controls, we used Co-IP followed by mass-spectrometry to identify C-terminal interacting proteins that are disrupted with C terminal loss. We found an intricate ACTN2 interactome with many sarcolemma-associated proteins and showed lack of interaction of the truncated ACTN2 with ACTN1 and GJA1 (Fig. 1). ACTN1 association was verified using colocalization and Co-IP followed by Western blot. Conclusion: We provide evidence that two loss of function genetic variants in ACTN2 are associated with contractile dysfunction and lead to cardiac abnormalities through distinct mechanisms within cardiac sarcomeres and through lack of critical protein-protein interactions.

Potential roles in cardiac physiology and pathology of the cation channel TRPV2 expressed in cardiac cells and cardiac macrophages: a mini-review

TRPV2 is a well-conserved channel protein expressed in almost all tissues. Cardiomyocyte TRPV2 is expressed in the intercalated disks of the cardiac sarcomeres, where it is involved in maintaining the proper mechanoelectric coupling and structure. It is also abundantly expressed in the intracellular pools, mainly the endoplasmic reticulum. Under pathological conditions, TRPV2 is translocated to the sarcolemma, where it mediates an abnormal [Ca]2+ entry that may contribute to disease progression. In addition, an intracellularly diffused TRPV2 expression is present in resident cardiac macrophages. Upon infection or inflammation, TRPV2 is engaged in early phagosomes and is, therefore, potentially involved in protecting the cardiac tissue. Following acute myocardial infarction, a profound elevated expression of TRPV2 is observed on the cell membrane of the peri-infarct macrophages. The macrophage TRPV2 may harbor a detrimental effect in cardiac recovery by increasing unfavorable migration and phagocytosis processes in the injured heart. Most reports suggest that while cardiac TRPV2 activation may be beneficial under specific physiological conditions, both cardiac- and macrophage-related TRPV2 blocking can significantly ameliorate disease progression in various pathological states. To verify this possibility, the time frame of TRPV2 overexpression and its mediated signaling need to be fully characterized in both cardiomyocyte and cardiac macrophage populations.

Cardiac actin changes in the actomyosin interface have different effects on myosin duty ratio

Hypertrophic cardiomyopathy (HCM) is an inherited cardiovascular disease (CD) that commonly causes an increased size of cardiomyocytes in the left ventricle. The proteins myosin and actin interact in the myocardium to produce contraction through the actomyosin ATPase cycle. The duty ratio (r) of myosin is the proportion of the actomyosin ATPase cycle that myosin is bound to actin and does work. A common hypothesis is that HCM mutations increase contraction in cardiac sarcomeres; however, the available data are not clear on this connection. Based on previous work with human α-cardiac actin (ACTC), we hypothesize that HCM-linked ACTC variants with alterations near the myosin binding site have an increased r, producing more force. Myosin duty ratios using human ACTC variant proteins were calculated with myosin ATPase activity and in-vitro motility data. We found no consistent changes in the duty ratio of the ACTC variants, suggesting that other factors are involved in the development of HCM when ACTC variants are present.