Identification of KIAA0196 as a novel susceptibility gene for myofibril structural disorganization in cardiac development

Highlights

• We identified KIAA0196 may as a susceptibility gene of cardiac development.

• Loss-of-function of KIAA0196 in zebrafish and mouse model leads to cardiac dysfunction.

• KIAA0196 insufficiency induced sarcomere disorganization and myofibril disruption.

• Transcriptomic and proteomic show KIAA0196 deficiency affects sarcomere proteins/RNAs.

Abstract

Background

Congenital heart disease is one of the most common cardiac malformation-related diseases worldwide. Some causative genes have been identified but can explain only a small proportion of all cases; therefore, the discovery of novel susceptibility genes and/or modifier genes for abnormal cardiac development remains a major challenge.

Methods

We used a single nucleotide polymorphism (SNP) array, and next-generation sequencing (NGS) was conducted to screen and quickly identify candidate genes. KIAA0196 knockout zebrafish and mice were generated by CRISPR/Cas9 to detect whether or how KIAA0196 deficiency would influence cardiac development.

Results

Homozygous, but not heterozygous, zebrafish and mice showed early embryonic lethality. At the embryonic stage, microscopic examination and dissection revealed pericardial edema and ventricle enlargement in homozygous zebrafish and obviously delayed cardiac development in heterozygous mice, while echocardiography and tissue staining showed that significantly decreased cardiac function, ventricle enlargement, myofibril loss, and significantly reduced trabecular muscle density were observed in adult heterozygous zebrafish and mice. Most importantly, immunostaining and electron microscopy showed that there was a significant increase in sarcomere structural disorganization and myofibril structural integrity loss in KIAA0196 mutants. Furthermore, substantial downregulation in other sarcomeric genes and proteins was detected and verified in a mouse model via transcriptome and proteomics analyses; these changes especially affected the myosin heavy or light chain (MYH or MYL) family genes.

Conclusion

We identified KIAA0196 for the first time as a susceptibility gene for abnormal cardiac development. KIAA0196 deficiency may cause abnormal heart development by influencing the structural integrity of myofibrils.

Introduction

Congenital heart diseases refer to anatomical abnormalities caused by developmental disorders of the heart and blood vessels during embryonic development; these diseases remain the leading cause of infant mortality [1] and are a major public health problem. Although some cardiac-development-associated susceptibility genes have been identified, including GATA-binding protein 4 (GATA4) [2], myosin heavy chain-6 and -7 (MYH6 [3], MYH7 [4]), T-box transcription factor 5 (TBX5) [5], and NK2 homeobox 5 (NKX2.5) [6], these genes account for a minority of patients, and the etiology of most cases remains genetically elusive. Therefore, the discovery of novel susceptibility genes and/or modifier genes for cardiac development remains a major challenge.

The sarcomere is the basic contractile unit of the cardiac muscles; it consists of central bipolar thick myosin filaments surrounded by parallel thin actin filaments, which are anchored to the Z-disc. The normal structure and function of sarcomeres are critical for the development of the cardiac, especially in the embryonic stage. Moreover, the underlying pathogenesis in some cardiac diseases is primarily attributed to cardiac sarcomere gene mutations, leading to blurred Z-disc, sparse myofilament, and rupture or disorder of the myofilaments, which results in myocardial contractile dysfunction [4]. In addition to well-known calcium homeostasis and sarcomere maintenance, cardiac contractility is regulated by signaling pathways. Accumulating evidence suggests that the Wnt/β-catenin signaling pathway plays an important role in myocardial and early cardiac development.

The KIAA0196 gene, also called Wiskott–Aldrich syndrome protein and scar homologue complex subunit 5 (WASHC5), is situated at 8q24.13 of chromosome 8, and the protein encoded by this gene is known as strumpellin. It is ubiquitously expressed in multiple systems and muscles at high levels and is the core component of the WASH complex (an actin-regulating complex) [7,8]. Mutations in KIAA0196 have been examined through functional studies in human hereditary spastic paraplegia (HSP) [[9], [10], [11]], but the complexities of their overlapping phenotypes, especially in studies of cardiac development or the role of KIAA0196 in cardiac sarcomere development, have rarely been investigated. A recent study [12] showed that patients with KIAA0196 gene mutations exhibited cardiac defects and the expression of strumpellin protein was also reduced by 60%. Decreased levels of KIAA0196 or strumpellin in zebrafish and mice have also resulted in cardiac edema or an enlarged heart [11,13], and homozygous mutations lead to early lethality [10]. However, none of these studies has elucidated whether it has an effect on cardiac function and the reasons for the abnormal cardiac phenotype. In this study, we identified the KIAA0196 gene as a novel genetic modifier and/or susceptibility gene for abnormal cardiac development by establishing two knockout mutant animal models, one vertebrate and one mammalian.