Functional analysis of titin/connectin N2-B mutations found in cardiomyopathy

Abstract

Hypertrophic cardiomyopathy and dilated cardiomyopathy are two major clinical phenotypes of "idiopathic" cardiomyopathy. Recent molecular genetic analyses have now revealed that "idiopathic" cardiomyopathy is caused by mutations in genes for sarcomere components. We have recently reported several mutations in titin/connectin gene found in patients with hypertrophic cardiomyopathy or dilated cardiomyopathy. A hypertrophic cardiomyopathy-associated titin/connectin mutation (Arg740Leu) was found to increase the binding to actinin, while other dilated cardiomyopathy-associated titin/connectin mutations (Ala743Val and Val54Met) decreased the binding to actinin and Tcap/telethonin, respectively. We also reported several other mutations in the N2-B region of titin/connectin found in hypertrophic cardiomyopathy and dilated cardiomyopathy. Since the N2-B region expresses only in the heart, it was speculated that functional alterations due to the mutations cause cardiomyopathies. In this study, we investigated the functional changes caused by the N2-B region mutations by using yeast-two-hybrid assays. It was revealed that a hypertrophic cardiomyopathy-associated mutation (Ser3799Tyr) increased the binding to FHL2 protein, whereas a dilated cardiomyopathy-associated mutation (Gln4053ter) decreased the binding. In addition, another TTN mutation (Arg25618Gln) at the is2 region was found in familial DCM. Because FHL2 protein is known to tether metabolic enzymes to N2-B and is2 regions of titin/connectin, these observations suggest that altered recruitment of metabolic enzymes to the sarcomere may play a role in the pathogenesis of cardiomyopathies.

Molecular basis of hereditary cardiomyopathy: abnormalities in calcium sensitivity, stretch response, stress response and beyond

“…It is not clear how the aggregated aB-crystalline resulted in cardiac hypertrophy, but impaired stress response may exaggerate hypertrophic response. 46 It should be noted here that an HCM-associated TTN mutation in N2B region increased binding to FHL2 protein 47 and decreased binding to aB-crystalline. 48 As for the function of caveolin-3 in cardiac function, it was reported that cell surface expression of caveolin-3 was associated with cardiac hypertrophy.…”

“…91 The third group is composed of mutations in genes for titin-N2B-interacting proteins, four and half LIM protein (FHL2) 92 and aBcrystallin (CRYAB). 48 As a titin-N2B region mutation found in DCM reduced binding to FHL2 47 and an FHL2 mutation reduced binding to titin-N2B, 92 impaired interaction between titin and FHL2 appeared as a result in DCM. Molecular mechanisms underlying this phenomenon may be that FHL2 function as a tethering molecule of adenyl kinase, phosphofructokinase and muscle creatine kinase; that is, proper recruitment of metabolic enzymes was impaired, although abnormality in other functions of FHL2 93 could not be neglected.…”

“…The DCMassociated CRYAB mutation decreased binding to titin-N2B region and a DCM-associated titin-N2B region mutation decreased binding to aB-crystallin, 48 suggesting that impaired interaction between titin-N2B and aB-crystallin resulted in DCM. However, an HCM-associated titin-N2B mutation also reduced the binding to aB-crystallin, 47 and it is not clear why the impaired binding of titin-N2B and aBcrystallin could express as both HCM and DCM. There may be additional factors involved in the phenotypic expression of titin-N2B mutations, such that binding to FHL2 was different between the HCM-and DCM-associated mutations and that the DCMassociated titin-N2B mutation was a truncation mutation, whereas the HCM-associated mutation was a missense mutation.…”

Novel mutations in the sarcomeric protein myopalladin in patients with dilated cardiomyopathy

“…As a number of missense mutations in genes encoding for titin and titin-associated proteins have recently been reported to be responsible for DCM, 3,[12][13][14][15][19][20][21][22][23][24][25] we searched in a well-characterized sample of DCM patients for unknown SNPs and/or mutations in the human MYPN and ANKRD1 genes. In order to extend our knowledge on disease-causing mutations in these two proteins expressed in heart tissue, we screened the coding sequences and corresponding intron flanks of the two genes and found two novel non-synonymous mutations in the MYPN gene and only one rare synonymous SNP in the coding region of the ANKRD1 gene.…”

Gigantic Business

“…90 The majority of the TTN mutations found in DCM patients are truncating mutations within A-band titin, but I-band mutations and a few Z-disk and M-band (including TK) mutations have also been reported ( Figure 4). 83,84,[90][91][92][93][94][95][96] These mutations include mostly nonsense and frameshift as well as splicing variants. Some patients with a titin mutation present with both cardiac and skeletal muscle disease, 91,96 but often it is unknown whether in patients with a cardiac phenotype the skeletal muscles are affected as well.…”