Dilated cardiomyopathy (DCM) is characterized by dilation of left ventricular cavity with systolic dysfunction. Clinical symptom of DCM is heart failure, often associated with cardiac sudden death. About 20-35% of DCM patients have apparent family histories and it has been revealed that mutations in genes for sarcomere proteins cause DCM. However, the disease-causing mutations can be found only in about 17% of Japanese patients with familial DCM. Bcl-2-associated athanogene 3 (BAG3) is a co-chaperone protein with antiapoptotic function, which localizes at Z-disc in the striated muscles. Recently, BAG3 gene mutations in DCM patients were reported, but the functional abnormalities caused by the mutations are not fully unraveled. In this study, we analyzed 72 Japanese familial DCM patients for mutations in BAG3 and found two mutations, p.Arg218Trp and p.Leu462Pro, in two cases of adult-onset DCM without skeletal myopathy, which were absent from 400 control subjects. Functional studies at the cellular level revealed that the DCM-associated BAG3 mutations impaired the Z-disc assembly and increased the sensitivities to stress-induced apoptosis. These observations suggested that BAG3 mutations present in 2.8% of Japanese familial DCM patients caused DCM possibly by interfering with Z-disc assembly and inducing apoptotic cell death under the metabolic stress.
Abstract Bradyarrhythmia is a common heart rhythm abnormality comprising number of diseases and is associated with decreased heart rate due to the failure of action potential generation and propagation at the sinus node. Permanent pacemaker implantation is often used therapeutically to compensate for decreased heart rate and cardiac output. The vast majority of bradyarrhythmia cases are attributable either to aging or to structural abnormalities of the cardiac conduction system, caused by underlying structural heart disease. However, there is a subset of bradyarrhythmia primarily caused by genetic defects in the absence of aging or underlying structural heart disease. These include several genes that play principal roles in cardiac electrophysiology, heart development, cardioprotection, and the structural integrity of the membrane and sarcomere. Recent advances in the functional analysis of mutations using a heterologous expression system and genetically engineered animal models have provided significant insights into the underlying molecular mechanisms responsible for inherited arrhythmia. In this review, current understandings of the genetic and molecular basis of inherited bradyarrhythmia are presented.
Although shortening of the corrected QT interval (QTc) is a key finding in the diagnosis of short QT syndrome (SQTS), there may be overlap of the QTc between SQTS patients and normal subjects in childhood and adolescence. We aimed to investigate electrocardiographic findings for differentiation of SQTS patients.The SQTS group comprised 34 SQTS patients <20 years old, including 9 from our institutions and 25 from previous reports. The control group comprised 61 apparently healthy subjects with an QTc of <360 ms who were selected from 13 314 participants in a school-based screening programme. We compared electrocardiographic findings, including QT and Jpoint-Tpeak intervals (QT and J-Tpeak, respectively), those corrected by using the Bazett's and Fridericia's formulae (cB and cF, respectively) and early repolarization (ER) between the groups. QT, QTc by using Bazett's formula (QTcB), QTc by using Fridericia's formula (QTcF), J-Tpeak, J-Tpeak cB, and J-Tpeak cF were significantly shorter in the SQTS group than in the control group. On receiver operating characteristic curve analysis, the area under the curve (AUC) was largest for QTcB (0.888) among QT, QTcB, and QTcF, with a cut-off value of 316 ms (sensitivity: 79.4% and specificity: 96.7%). The AUC was largest for J-Tpeak cB (0.848) among J-Tpeak, J-Tpeak cB, and J-Tpeak cF, with a cut-off value of 181 ms (sensitivity: 80.8% and specificity: 91.8%). Early repolarization was found more frequently in the SQTS group than in the control group (67% vs. 23%, P = 0.001).A QTcB <316 ms, J-Tpeak cB < 181 ms, and the presence of ER may indicate SQTS patients in childhood and adolescence.
Calmodulin is a ubiquitous Ca2+ sensor molecule encoded by three distinct calmodulin genes, CALM1-3. Recently, mutations in CALM1-3 have been reported to be associated with severe early-onset long-QT syndrome (LQTS). However, the underlying mechanism through which heterozygous calmodulin mutations lead to severe LQTS remains unknown, particularly in human cardiomyocytes. We aimed to establish an LQTS disease model associated with a CALM2 mutation (LQT15) using human induced pluripotent stem cells (hiPSCs) and to assess mutant allele-specific ablation by genome editing for the treatment of LQT15. We generated LQT15-hiPSCs from a 12-year-old boy with LQTS carrying a CALM2-N98S mutation and differentiated these hiPSCs into cardiomyocytes (LQT15-hiPSC-CMs). Action potentials (APs) and L-type Ca2+ channel (LTCC) currents in hiPSC-CMs were analyzed by the patch-clamp technique and compared with those of healthy controls. Furthermore, we performed mutant allele-specific knockout using a CRISPR-Cas9 system and analyzed electrophysiological properties. Electrophysiological analyses revealed that LQT15-hiPSC-CMs exhibited significantly lower beating rates, prolonged AP durations, and impaired inactivation of LTCC currents compared with control cells, consistent with clinical phenotypes. Notably, ablation of the mutant allele rescued the electrophysiological abnormalities of LQT15-hiPSC-CMs, indicating that the mutant allele caused dominant-negative suppression of LTCC inactivation, resulting in prolonged AP duration. We successfully recapitulated the disease phenotypes of LQT15 and revealed that inactivation of LTCC currents was impaired in CALM2-N98S hiPSC model. Additionally, allele-specific ablation using the latest genome-editing technology provided important insights into a promising therapeutic approach for inherited cardiac diseases.
In higher plants, four types of mRNA variants, one form (tAPX-I) encoding thylakoid-bound APX (tAPX) and three forms (sAPX-I, -II, -III) encoding stromal APX (sAPX), are produced by the alternative splicing of the 3?-terminal region [Yoshimura et al. (1999) Biochem. J., 338, 41-48]. The same observation is also true in the cases of chloroplastic ascorbate peroxidases (chlAPX) isoenzymes in pumpkin, M. crystallinum, and tobacco, indicating that the alternative splicing of chlAPX isoenzymes is a universal regulation mechanism in higher plants. To explore the regulation mechanism of alternative splicing, the expression of chlAPX isoenzymes in the leaf, stem, and root of spinach was analyzed. The expression ratio of four types of chlAPX mRNA variants was different in a tissue-specific manner, whereas the transcript levels of total chlAPX were almost equal in each tissue. The expression ratio of sAPX mRNAs (sAPX-I, -II, -III) to tAPX mRNA was almost equal in leaf; in root, however, the ratio of sAPX mRNAs to tAPX mRNA was greatly enhanced due to the decrease in tAPX-I mRNA and the increase in sAPX-III mRNA. A putative splicing regulatory cis-element (SRE), which was highly conserved in the sequences of chlAPX genes from higher plants, was identified. In fact, gel-shift analysis showed a tissue-specific interaction between SRE and a nuclear protein prepared from each tissue. These results indicate that the tissue-specific alternative splicing of chlAPX isoenzymes is regulated by SRE as a splicing enhancer. We are progressing in the identification of a trans-acting factor to regulate the alternative splicing mechanism.
Mutations in the human calmodulin genes (CALM1, CALM2, and CALM3) are associated with life-threatening conditions in childhood, such as idiopathic ventricular fibrillation (VF) and long QT syndrome (LQTS).1–3 Furthermore, CALM1 mutations were described in a catecholaminergic polymorphic ventricular tachycardia (CPVT)-like phenotype.4 Sudden unexplained death in the young can be the first clinical manifestation of an underlying arrhythmogenic disorder such as idiopathic VF.5,6 After an aborted cardiac arrest, determining the diagnosis begins with a systematic clinical evaluation.
