Cysteine And Glycine-rich Protein 3 Research Articles

A novel gene-trap line reveals the dynamic patterns and essential roles of cysteine and glycine-rich protein 3 in zebrafish heart development and regeneration

Mutations in cysteine and glycine-rich protein 3 (CSRP3)/muscle LIM protein (MLP), a key regulator of striated muscle function, have been linked to hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) in patients. However, the roles of CSRP3 in heart development and regeneration are not completely understood. In this study, we characterized a novel zebrafish gene-trap line, gSAIzGFFM218A, which harbors an insertion in the csrp3 genomic locus, heterozygous fish served as a csrp3 expression reporter line and homozygous fish served as a csrp3 mutant line. We discovered that csrp3 is specifically expressed in larval ventricular cardiomyocytes (CMs) and that csrp3 deficiency leads to excessive trabeculation, a common feature of CSRP3-related HCM and DCM. We further revealed that csrp3 expression increased in response to different cardiac injuries and was regulated by several signaling pathways vital for heart regeneration. Csrp3 deficiency impeded zebrafish heart regeneration by impairing CM dedifferentiation, hindering sarcomere reassembly, and reducing CM proliferation while aggravating apoptosis. Csrp3 overexpression promoted CM proliferation after injury and ameliorated the impairment of ventricle regeneration caused by pharmacological inhibition of multiple signaling pathways. Our study highlights the critical role of Csrp3 in both zebrafish heart development and regeneration, and provides a valuable animal model for further functional exploration that will shed light on the molecular pathogenesis of CSRP3-related human cardiac diseases.

LIM domain-wide comprehensive virtual mutagenesis provides structural rationale for cardiomyopathy mutations in CSRP3

Cardiomyopathies are a severe and chronic cardiovascular burden worldwide, affecting a large cohort in the general population. Cysteine and glycine-rich protein 3 (CSRP3) is one of key proteins implicated in dominant dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). In this study, we device a rapid in silico screening protocol that creates a mutational landscape map for all possible allowed and disallowed substitutions in the protein of interest. This map provides the structural and functional insights on the stability of LIM domains of CSRP3. Further, the sequence analysis delineates the eukaryotic CSRP3 protein orthologs which complements the mutational map, but provide limited information of amino acid exchanges. Next, we also evaluated the effect of HCM/DCM mutations on these domains. One of highly destabilising mutations—L44P (also disease causing) and a neutral mutation—L44M were further subjected to molecular dynamics (MD) simulations. The results establish that L44P substitution affects the LIM domain structure by altering secondary structure and due to loss of hydrophobic interaction with Phenylananine 35. The present study provides a useful perspective to our understanding of the role of mutations in the CSRP3 LIM domains and their evolution. This study provides a novel computational screening method for quick identification of key mutation sites for specific protein structures that can reduce the burden on experimental research.

CSRP3, p.Arg122*, is responsible for hypertrophic cardiomyopathy in a Chinese family

Hypertrophic cardiomyopathy (HCM) is a hereditary disease manifested by a thickened ventricular wall. Cysteine and glycine-rich protein 3 (CSRP3), the gene encoding muscle LIM protein, is important for initiating hypertrophic gene expression. The mutation of CSRP3 causes dilated cardiomyopathy or HCM. In the present study, we enrolled a Chinese family with HCM across three generations. Whole-exome sequencing (WES) was performed in the proband to detect the candidate genes of the family. Sanger sequencing was performed for mutational analysis and confirmation of cosegregation. Through histopathological and imaging examinations, an obvious left ventricular hypertrophy was found in the proband. After WES data filtering, bioinformatic prediction and co-segregation analysis, a nonsense mutation (NM_003476.5:c.364C>T; NP_003467.1:p.Arg122*) of CSRP3 was identified in this family. This variant was predicted to be disease-causing and resulted in a truncated protein. This is the first HCM family case of CSRP3 (p.Arg122*) variation in Asia. The finding here not only contributes to the genetic diagnosis and counseling of the family, but also provides a new case with detailed phenotypes that may be caused by the CSRP3 variant.

Discriminating aspects of global metabolism of neonatal cardiomyocytes from wild type and KO-CSRP3 rats using proton magnetic resonance spectroscopy of culture media samples.

Knockout of multifunction gene cysteine- and glycine-rich protein 3 (CSRP3) in cardiomyocytes (CMs) of mice leads to heart dilation, severely affecting its functions. In humans, CSRP3 mutations are associated with hypertrophic (HCM) and dilated cardiomyopathy (DCM). The absence of the CSRP3 expression produces unknown effects on in vitro neonatal CMs' metabolism. The metabolome changes in culture media conditioned by CSRP3 knockout (KO-CSRP3), and wild type (WT) neonatal cardiomyocytes were investigated under untreated or after metabolic challenging conditions produced by isoproterenol (ISO) stimulation, by in vitro high-resolution proton magnetic resonance spectroscopy (1H-MRS)-based metabolomics. Metabolic differences between neonatal KO-CSRP3 and WT rats' CMs were identified. After 72h of culture, ISO administration was associated with increased CMs' energy requirements and increased levels of threonine, alanine, and 3-hydroxybutyrate in both neonatal KO-CSRP3 and WT CMs conditioned media. When compared with KO-CSRP3, culture media derived from WT cells presented higher lactate concentrations either under basal or ISO-stimulated conditions. The higher activity of ketogenic biochemical pathways met the elevated energy requirements of the contractile cells. Both cells are considered phenotypically indistinguishable in the neonatal period of animal lives, but the observed metabolic stress responses of KO-CSRP3 and WT CMs to ISO were different. KO-CSRP3 CMs produced less lactate than WT CMs in both basal and stimulated conditions. Mainly, ISO-stimulated conditions produced evidence for lactate overload within KO-CSRP3 CMs, while WT CMs succeeded to manage the metabolic stress. Thus, 1H-MRS-based metabolomics was suitable to identify early inefficient energetic metabolism in neonatal KO-CSRP3 CMs. These results may reflect an apparent lower lactate transport and consumption, in association with protein catabolism.

Correcting an instance of synthetic lethality with a pro-survival sequence

A human cDNA encoding the LIM domain containing 194 amino acid cysteine and glycine rich protein 3 (CSRP3) was identified as a BAX suppressor in yeast and a pro-survival sequence that abrogated copper mediated regulated cell death (RCD). Yeast lacks a CSRP3 orthologue but it has four LIM sequences, namely RGA1, RGA2, LRG1 and PXL1. These are known regulators of stress responses yet their roles in RCD remain unknown. Given that LIMs interact with other LIMs, we ruled out the possibility that overexpressed yeast LIMs alone could prevent RCD and that CSRP3 functions by acting as a dominant regulator of yeast LIMs. Of interest was the discovery that even though yeast cells lacking the LIM encoding PXL1 had no overt growth defect, it was nevertheless supersensitive to the effects of sublethal levels of copper. Heterologous expression of human CSPR3 as well as the pro-survival 14-3-3 sequence corrected this copper supersensitivity. These results show that the pxl1∆-copper synthetic lethality is likely due to the induction of RCD. This differs from the prevailing model in which synthetic lethality occurs because of specific defects generated by the combined loss of two overlapping but non-essential functions.

Zebrafish cysteine and glycine-rich protein 3 is essential for mechanical stability in skeletal muscles

Cysteine and glycine-rich protein 3 (CSRP3) is a striated muscle-specific cytoskeleton protein which participates in cardiac stretch sensing. Mutations in CSRP3 gene cause cardiomyopathies and deregulation of CSRP3 has been found in patients with heart failure and several skeletal muscle diseases. However, the mechanism underneath these disorders still remains poorly understood. Here we generated the first csrp3 knockout zebrafish. csrp3−/− embryos showed no gross morphological defects but csrp3 deficient skeletal muscle fibers were prone to lesions upon prolonged stretching force. Further studies revealed csrp3 cooperatively interacted with ilk to maintain skeletal muscle mechanical stability and regulated tcap activation. Thus, our work has established a zebrafish model to investigate the function of csrp3 gene, and provides novel insights towards how csrp3 defects may lead to skeletal myopathies by a mechanistic link between Csrp3 and force stimuli.

Mutant Muscle LIM Protein C58G causes cardiomyopathy through protein depletion

Cysteine and glycine rich protein 3 (CSRP3) encodes Muscle LIM Protein (MLP), a well-established disease gene for Hypertrophic Cardiomyopathy (HCM). MLP, in contrast to the proteins encoded by the other recognised HCM disease genes, is non-sarcomeric, and has important signalling functions in cardiomyocytes. To gain insight into the disease mechanisms involved, we generated a knock-in mouse (KI) model, carrying the well documented HCM-causing CSRP3 mutation C58G.In vivo phenotyping of homozygous KI/KI mice revealed a robust cardiomyopathy phenotype with diastolic and systolic left ventricular dysfunction, which was supported by increased heart weight measurements. Transcriptome analysis by RNA-seq identified activation of pro-fibrotic signalling, induction of the fetal gene programme and activation of markers of hypertrophic signalling in these hearts. Further ex vivo analyses validated the activation of these pathways at transcript and protein level. Intriguingly, the abundance of MLP decreased in KI/KI mice by 80% and in KI/+ mice by 50%. Protein depletion was also observed in cellular studies for two further HCM-causing CSRP3 mutations (L44P and S54R/E55G). We show that MLP depletion is caused by proteasome action. Moreover, MLP C58G interacts with Bag3 and results in a proteotoxic response in the homozygous knock-in mice, as shown by induction of Bag3 and associated heat shock proteins.In conclusion, the newly generated mouse model provides insights into the underlying disease mechanisms of cardiomyopathy caused by mutations in the non-sarcomeric protein MLP. Furthermore, our cellular experiments suggest that protein depletion and proteasomal overload also play a role in other HCM-causing CSPR3 mutations that we investigated, indicating that reduced levels of functional MLP may be a common mechanism for HCM-causing CSPR3 mutations.

Molecular cloning, characterization, and expression analysis of the three cysteine and glycine-rich protein genes in the Chinese fire-bellied newt Cynops orientalis

The cysteine- and glycine-rich protein (CRP) family members, including the cysteine- and glycine-rich protein 1 (CSRP1), cysteine- and glycine-rich protein 2 (CSRP2), and the cysteine- and glycine-rich protein 3 (CSRP3), have exhibited various cellular functions during cell development and differentiation. However, the sequences of the three CSRP genes and their functions are still poorly understood in newts. In this study, we cloned the complete open reading frame (ORF) sequences of the three CSRP genes from the Chinese fire-bellied newt, Cynops orientalis (C. orientalis). The complete ORF sequences of Co-CSRP1, Co-CSRP2, and Co-CSRP3 were 582, 582, and 576bp, respectively, and encoded 193, 193, and 191 amino acids, respectively. The deduced amino acid sequences of the three CRP members showed high similarities with that of other species, particularly, with amphibians. Co-CSRP1 was highly expressed in the kidney, limb, and stomach, however, the expression was low in the spleen, heart, intestine, liver, and tail (P<0.05). The mRNA expression of Co-CSRP2 was higher in the kidney and heart than that in other organs (P<0.05). It was observed that Co-CSRP3 was only expressed in the heart, limb, and tail. The mRNA expression of Co-CSRP1 and Co-CSRP3 was lower in the digits in comparison to other limb segments. However, there was no significant difference of Co-CSRP2 mRNA expression in the four limb segments. The Co-CSRP1 and Co-CSRP2 mRNA expressions were significantly increased, whereas the expression of Co-CSRP3 was remarkably decreased during the limb regeneration. This study will provide useful information for further elucidating the role of Co-CSRP genes during newt limb regeneration.

Clinical features, spectrum of causal genetic mutations and outcome of hypertrophic cardiomyopathy in South Africans.

SummaryBackgroundLittle is known about the clinical characteristics, spectrum of causal genetic mutations and outcome of hypertrophic cardiomyopathy (HCM) in Africans. The objective of this study was to delineate the clinical and genetic features and outcome of HCM in African patients.MethodsInformation on clinical presentation, electrocardiographic and echocardiographic findings, and outcome of cases with HCM was collected from the Cardiac Clinic at Groote Schuur Hospital over a mean duration of follow up of 9.1 ± 3.4 years. Genomic DNA was screened for mutations in 15 genes that cause HCM, i.e. cardiac myosinbinding protein C (MYBPC3), cardiac β-myosin heavy chain (MYH7), cardiac troponin T2 (TNNT2), cardiac troponin I (TNNI3), regulatory light chain of myosin (MYL2), essential light chain of myosin (MYL3), tropomyosin 1 (TPM1), phospholamban (PLN), α-actin (ACTC1), cysteine and glycine-rich protein 3 (CSRP3), AMP-activated protein kinase (PRKAG2), α-galactosidase (GLA), four-and-a-half LIM domains 1 (FHL1), lamin A/C (LMNA) and lysosomeassociated membrane protein 2 (LAMP2). Survival and its predictors were analysed using the Kaplan–Meier and Cox proportional hazards regression methods, respectively.ResultsForty-three consecutive patients [mean age 38.5 ± 14.3 years; 25 (58.1%) male; and 13 (30.2%) black African] were prospectively enrolled in the study from January 1996 to December 2012. Clinical presentation was similar to that reported in other studies. The South African founder mutations that cause HCM were not found in the 42 probands. Ten of 35 index cases (28.6%) tested for mutations in 15 genes had disease-causing mutations in MYH7 (six cases or 60%) and MYBPC3 (four cases or 40%). No disease-causing mutation was found in the other 13 genes screened. The annual mortality rate was 2.9% per annum and overall survival was 74% at 10 years, which was similar to the general South African population. Cox’s proportional hazards regression showed that survival was predicted by New York Heart Association (NYHA) functional class at last visit (p = 0.026), but not by the presence of a disease-causing mutation (p = 0.474).ConclusionsComprehensive genetic screening was associated with a 29% yield of causal genetic mutations in South African HCM cases, all in MYH7 and MBPC3 genes. A quarter of the patients had died after a decade of follow up, with NYHA functional class serving as a predictor of survival.

Molecular cloning, characterization and tissue specificity of the expression of the ovine CSRP2 and CSRP3 genes from Small-tail Han sheep (Ovis aries)

Two genes, cysteine- and glycine-rich protein 2 (CSRP2) and cysteine- and glycine-rich protein 3 (CSRP3), play important roles in tissue-specific cell growth and development. However, few CSRP2 and CSRP3 genes have been functionally characterized in sheep. In this study, the full-length cDNAs of the CSRP2 and CSRP3 genes were cloned from Small-tail Han sheep by rapid amplification of cDNA ends-PCR. The GenBank accession numbers of the full-length CSRP2 and CSRP3 cDNA sequences are KJ743957 and KJ743958, respectively. The full-length cDNA of ovine CSRP2 was 917bp, with a 582-bp open reading frame encoding 193 amino acids. The complete ovine CSRP3 cDNA was 917bp, with a 585-bp open reading frame encoding 194 amino acids. Alignment and phylogenetic analyses revealed that their amino acid sequences are highly similar to those of other vertebrates, all of which contain two conserved LIM-only domains and a relatively conserved nuclear targeting sequence. To further validate the functions of the two genes, their mRNA expression patterns were evaluated in various Small-tail Han and Dorper sheep tissues using qRT-PCR analyses. CSRP2 was mainly detected in the aorta, whereas CSRP3 was highly concentrated in the heart and the muscle. CSRP3 was expressed to a higher level in the hearts of Small-tail Han sheep than in Dorper sheep (P<0.05). However, the opposite was found in the muscle (the longissimus dorsi and biceps femoris); CSRP3 was expressed to a higher level in Dorper sheep than in Small-tail Han sheep (P<0.05). We quantified the CRP3 protein (coded by the CSRP3 gene) levels in different tissues in Small-tail Han and Dorper sheep. We also detected a putative isoform of the CRP3 protein in sheep, which was significantly different in the heart tissue of the two breeds (P<0.05). The expression patterns of the two genes' mRNAs and CRP3 protein showed clear tissue specificity in both sheep breeds.

Muscle lim protein isoform negatively regulates striated muscle actin dynamics and differentiation

Muscle lim protein (MLP) has emerged as a critical regulator of striated muscle physiology and pathophysiology. Mutations in cysteine and glycine-rich protein 3 (CSRP3), the gene encoding MLP, have been directly associated with human cardiomyopathies, whereas aberrant expression patterns are reported in human cardiac and skeletal muscle diseases. Increasing evidence suggests that MLP has an important role in both myogenic differentiation and myocyte cytoarchitecture, although the full spectrum of its intracellular roles has not been delineated. We report the discovery of an alternative splice variant of MLP, designated as MLP-b, showing distinct expression in neuromuscular disease and direct roles in actin dynamics and muscle differentiation. This novel isoform originates by alternative splicing of exons 3 and 4. At the protein level, it contains the N-terminus first half LIM domain of MLP and a unique sequence of 22 amino acids. Physiologically, it is expressed during early differentiation, whereas its overexpression reduces C2C12 differentiation and myotube formation. This may be mediated through its inhibition of MLP/cofilin-2-mediated F-actin dynamics. In differentiated striated muscles, MLP-b localizes to the sarcomeres and binds directly to Z-disc components, including α-actinin, T-cap and MLP. The findings of the present study unveil a novel player in muscle physiology and pathophysiology that is implicated in myogenesis as a negative regulator of myotube formation, as well as in differentiated striated muscles as a contributor to sarcomeric integrity.

Expression, SNV identification, linkage disequilibrium, and combined genotype association analysis of the muscle-specific gene CSRP3 in Chinese cattle

The cysteine and glycine-rich protein 3 (CSRP3) plays an important role in the myofiber differentiation. Here, we identified five SNVs in all exon and intron regions of the CSRP3 gene using DNA sequencing, PCR-RFLP and forced-PCR-RFLP methods in 554 cattle. Four of the five SNVs were significantly associated with growth performance and carcass traits of the cattle. In addition, we evaluated haplotype frequency and linkage disequilibrium coefficient of five sequence variants. The result of haplotype analysis demonstrated 28 haplotypes present in Qinchuan and two haplotypes in Chinese Holstein. Only haplotypes 1 and 8 were being shared by two populations, haplotype 14 had the highest haplotype frequency in Qinchuan (17.4%) and haplotype 8 had the highest haplotype frequency in Chinese Holstein (94.4%). Statistical analyses of combined genotypes indicated that some combined genotypes were significantly or highly significantly associated with growth and carcass traits in the Qinchuan cattle population. qPCR analyses also showed that bovine CSRP3 gene was exclusively expressed in longissimus dorsi muscle and heart tissues. The data support the high potential of the CSRP3 as a marker gene for the improvement of growth performance and carcass traits in selection programs.

Protective role of α-actinin-3 in the response to an acute eccentric exercise bout

The ACTN3 gene encodes for the alpha-actinin-3 protein, which has an important structural function in the Z line of the sarcomere in fast muscle fibers. A premature stop codon (R577X) polymorphism in the ACTN3 gene causes a complete loss of the protein in XX homozygotes. This study investigates a possible role for the alpha-actinin-3 protein in protecting the fast fiber from eccentric damage and studies repair mechanisms after a single eccentric exercise bout. Nineteen healthy young men (10 XX, 9 RR) performed 4 series of 20 maximal eccentric knee extensions with both legs. Blood (creatine kinase; CK) and muscle biopsy samples were taken to study differential expression of several anabolic (MyoD1, myogenin, MRF4, Myf5, IGF-1), catabolic (myostatin, MAFbx, and MURF-1), and contraction-induced muscle damage marker genes [cysteine- and glycine-rich protein 3 (CSRP3), CARP, HSP70, and IL-6] as well as a calcineurin signaling pathway marker (RCAN1). Baseline mRNA content of CSRP3 and MyoD1 was 49 + or - 12 and 67 + or - 25% higher in the XX compared with the RR group (P = 0.01-0.045). However, satellite cell number was not different between XX and RR individuals. After eccentric exercise, XX individuals tended to have higher serum CK activity (P = 0.10) and had higher pain scores than RR individuals. However, CSRP3 (P = 0.058) and MyoD1 (P = 0.08) mRNA expression tended to be higher after training in RR individuals compared with XX alpha-actinin-3-deficient subjects. This study suggests a protective role of alpha-actinin-3 protein in muscle damage after eccentric training and an improved stress-sensor signaling, although effects are small.

Hypertrophic cardiomyopathy family with double-heterozygous mutations; does disease severity suggest doubleheterozygosity?

Background. With the improvement in genetic testing over time, double-heterozygous mutations are more often found by coincidence in families with hypertrophic cardiomyopathy (HCM). Double heterozygosity can be a cause of the wellknown clinical diversity within HCM families.Methods and results. We describe a family in which members carry either a single mutation or are double heterozygous for mutations in myosin heavy chain gene (MYH7) and cysteine and glycine-rich protein 3 (CSRP3). The described family emphasises the idea of a more severe clinical phenotype with double-heterozygous mutations. It also highlights the importance of cardiological screening where NT-proBNP may serve as an added diagnostic tool.Conclusion. With a more severe inexplicable phenotype of HCM within a family, one should consider the possibility of double-heterozygous mutations. This implies that in such families, even when one disease-causing mutation is found, all the family members still have an implication for cardiological screening parallel to extended genetic screening. (Neth Heart J 2009;17:458-63.).

소의 CSRP3, APOBEC2, Caveolin 유전자들의 단일염기다형 분석

CSRP3, APOBEC2, CAV1, CAV2 및 CAV3 유전자들은 포유동물에서 도체와 육질 형질에 중요한 역할을 하는 것으로 보고되고 있다. 따라서, 이 유전자들의 단일염기다형(Single nucleotide poly- morphism; SNP)을 8개의 다른 소의 품종에서 확인한 결과 coding region에서 caveolin family 유전자에서 9개의 SNP, CSRP3유전자에서 1개의 SNP 및 APOBEC2 유전자에서 3개의 SNP가 존재함을 확인하였다. 이 coding region의 SNP들은 PCR-RFLP 방법에 의해 재확인하였으며 이들 유전자의 intronic region에서도 9개의 SNP가 존재함을 확인할 수 있었다. 8개의 다른 품종 소에 각 유전자들의 SNP들을 이용하여 유전자 빈도를 확인한 결과 CAV2, CAV3, CSRP3 및 APOBEC2 유전자의 SNP 중에서 5개가 품종간에서 유의적으로 차이가 있음을 확인할 수 있었다. 이 SNP들은 차후 검증작업을 통하여 육질관련 형질 마커로 이용될 수 있을 것으로 사료된다. The cysteine and glycine rich protein 3 (CSRP3), apolipoprotein B mRNA editing enzyme catalytic polypeptide‐like 2(APOBEC2) and caveolin (CAV) gene family(CAV1, CAV2, CAV3) have been reported to play important roles for carcass and meat quality traits in pig, mouse, human and cattle. As an initial step, we investigated SNPs in these 5 genes among eight different cattle breeds. Eighteen primer pairs were designed from bovine sequence data of NCBI database to amplify the partial gene fragments. Sequencing results revealed 9 SNPs in the coding regions of three caveolin genes, 1 SNP in CSRP3 and 3 SNPs in APOBEC2 gene. All the identified SNPs were confirmed by PCR-RFLP. Also, 9 more intronic SNPs were detected in these genes. However, all identified mutations in the coding region do not change amino acid sequence. Allelic distributions were significantly different for 5 SNPs in CAV2, CAV3, CSRP3 and APOBEC2 genes among the eight different breeds. These results gave some clues about the polymorphisms of these genes among the cattle breeds and will be useful for further searches for identifying association between these SNPs and meat quality traits in cattle.