Haider Mehdi

Mutation in Glycerol-3-Phosphate Dehydrogenase 1–Like Gene (GPD1-L) Decreases Cardiac Na+ Current and Causes Inherited Arrhythmias

Background— Brugada syndrome is a rare, autosomal-dominant, male-predominant form of idiopathic ventricular fibrillation characterized by a right bundle-branch block and ST elevation in the right precordial leads of the surface ECG. Mutations in the cardiac Na+ channel SCN5A on chromosome 3p21 cause ≈20% of the cases of Brugada syndrome; most mutations decrease inward Na+ current, some by preventing trafficking of the channels to the surface membrane. We previously used positional cloning to identify a new locus on chromosome 3p24 in a large family with Brugada syndrome and excluded SCN5A as a candidate gene. Methods and Results— We used direct sequencing to identify a mutation (A280V) in a conserved amino acid of the glycerol-3-phosphate dehydrogenase 1–like (GPD1-L) gene. The mutation was present in all affected individuals and absent in >500 control subjects. GPD1-L RNA and protein are abundant in the heart. Compared with wild-type GPD1-L, coexpression of A280V GPD1-L with SCN5A in HEK cells reduced inward Na+ currents by ≈50% (P<0.005). Wild-type GPD1-L localized near the cell surface to a greater extent than A280V GPD1-L. Coexpression of A280V GPD1-L with SCN5A reduced SCN5A cell surface expression by 31±5% (P=0.01). Conclusions— GPD1-L is a novel gene that may affect trafficking of the cardiac Na+ channel to the cell surface. A GPD1-L mutation decreases SCN5A surface membrane expression, reduces inward Na+ current, and causes Brugada syndrome.

Genetic variation in the alternative splicing regulator RBM20 is associated with dilated cardiomyopathy

BACKGROUND Dilated cardiomyopathy (DCM) is a leading cause of heart failure and death. The etiology of DCM is genetically heterogeneous. OBJECTIVES We sought to define the prevalence of mutations in the RNA splicing protein RBM20 in a large cohort with DCM and to determine whether genetic variation in RBM20 is associated with clinical outcomes. METHODS Subjects included in the Genetic Risk Assessment of Defibrillator Events (GRADE) study were aged at least 18 years, had an ejection fraction of ≤30%, and an implantable cardioverter-defibrillator (ICD). The coding region and splice junctions of RBM20 were screened in subjects with DCM; 2 common polymorphisms in RBM20, rs942077 and rs35141404, were genotyped in all GRADE subjects. RESULTS A total of 1465 subjects were enrolled in the GRADE study, and 283 with DCM were screened for RBM20 mutations. The mean age of subjects with DCM was 58 ± 13 years, 64% were males, and the mean follow-up time was 24.2 ± 17.1 months after ICD placement. RBM20 mutations were identified in 8 subjects with DCM (2.8%). Mutation carriers had a similar survival, transplantation rate, and frequency of ICD therapy compared with nonmutation carriers. Three of 8 subjects with RBM20 mutations (37.5%) had atrial fibrillation (AF), whereas 19 subjects without mutations (7.4%) had AF (P = .02). Among all GRADE subjects, rs35141404 was associated with AF (minor allele odds ratio = 0.62; 95% confidence interval = 0.44-0.86; P = .006). In the subset of GRADE subjects with DCM, rs35141404 was associated with AF (minor allele odds ratio = 0.58; P = .047). CONCLUSIONS Mutations in RBM20 were observed in approximately 3% of subjects with DCM. There were no differences in survival, transplantation rate, and frequency of ICD therapy in mutation carriers.

Genetic variation in apolipoprotein H (β2-glycoprotein I) affects the occurrence of antiphospholipid antibodies and apolipoprotein H concentrations in systemic lupus erythematosus

Apolipoprotein H (apoH, protein; APOH, gene) is a required cofactor for the production of antiphospholipid antibodies (APA). In this study we have examined whether genetic variation in the APOH gene affects variation in risk for systemic lupus erythematosus (SLE), occurrence of antiphospholipid antibodies (APA), anti-apoH, and plasma apoH concentrations. A total of 222 white SLE women were screened for four APOHpolymorphisms (codons 88, 247, 306, and 316) by polymerase chain reaction, and for plasma apoH concentrations by ELISA. Of these, 29.3% were positive for APA (APA-positive group) and 31.1% for anti-apoH. None of the four APOH polymorphisms were significantly associated with variation in risk for SLE. The codons 306 and 316 polymorphisms showed significant, gene-dosage effects on plasma apoH concentrations (P<0.0001) and explained 30% and 13%, respectively, of the residual variation in apoH concentrations. No significant association was observed between anti-apoH status and APOH polymorphisms or plasma apoH levels. However, plasma apoH concentrations were significantly higher in patients positive for APA than in patients negative for APA (18.5±4.0 mg/dl vs 17.1+3.8 mg/dl; P=0.02). The distribution of the Trp3l6Ser polymorphism was significantly different between the APA-positive and APA-negative groups. The frequency of the mutant allele (Ser316) was significantly lower in the APA-positive group than the APA-negative group (3.1% vs 12.1% P < 0.04), indicating that the Ser316 mutation is protective against the production of phospholipid-apoH dependent APA. Our data indicate that common genetic variation in the APOH gene is a significant determinant of plasma apoH variation in SLE patients, and the Trp3l6Ser polymorphism appears to provide protection against the production of APA in SLE patients.

Single nucleotide polymorphisms in the coding region of the apolipoprotein H (beta2-glycoprotein I) gene and their correlation with the protein polymorphism, anti-beta2glycoprotein I antibodies and cardiolipin binding: description of novel haplotypes and their evolution.

Apolipoprotein H (APOH), also known as β2‐glycoprotein I, is a major autoantigen for the production of antiphospholipid antibodies (APA) in autoimmune diseases. APA is also recognized by a cryptic epitope generated following the interaction of APOH with anionic phospholipids (PL). The prevalence of APA in the general U.S. white population is about 10%, but it ranges from 30–70% in patients with lupus and antiphospholipid syndrome. Since the structural characterization of APOH from different mammalian species is important to identify the evolutionary conserved regions that may be critical for its function, we have previously determined the chimpanzee APOH gene structure and the prevalence of APA. There are only two amino acid differences between the chimpanzee and human wild type APOH proteins. Chimpanzees have an unusually high prevalence (64%) of APA. There is a common protein polymorphism in the human APOH gene, with the occurrence of four alleles APOH*1, APOH*2, APOH*3 and APOH*4, the latter being present only in blacks. Based on its differential reactivity with an APOH monoclonal antibody, the APOH*3 allele is further divided into APOH*3W (present only in whites) and APOH*3B (present only in blacks). In this study we have screened a large African population (n = 755) to determine the prevalence of APA and the molecular basis of the protein polymorphism. Almost 50% of the Africans were found to be positive for APA. The APOH*3B allele was found to be identical to the chimpanzees wild type APOH. Novel two‐site or three‐site haplotypes, encoded in the third domain of APOH, explained the molecular basis of the APOH*3B, APOH*3W and APOH*4 alleles. Based on the comparison of the human and chimpanzee APOH DNA sequences, we suggest that the APOH*3W and APOH*4 alleles arose on the ancestral APOH*3B haplotype after the split of human races. We also found that these haplotypes are associated with the occurrence of APA. Recombinant APOH haplotypes, expressed in COS‐1 cells, showed that these mutations also affect the binding of APOH to anionic PL.

Effect of Right Ventricular versus Biventricular Pacing on Electrical Remodeling in the Normal Heart

Background—Biventricular (BIV) pacing can improve cardiac function in heart failure by altering the mechanical and electric substrates. We investigated the effect of BIV versus right ventricular (RV) pacing on the normal heart. Methods and Results—Male New Zealand White rabbits (n=33) were divided into 3 groups: sham-operated (control), RV pacing, and BIV pacing groups. Four weeks after surgery, the native QT (P=0.004) interval was significantly shorter in the BIV group compared with the RV or sham-operated groups. Also, compared with rabbits in the RV group, rabbits in the BIV group had shorter RV effective refractory period at all cycle lengths and shorter LV paced QT interval during the drive train of stimuli and close to refractoriness (P<0.001 for all comparisons). Protein expression of the KVLQT1 was significantly increased in the BIV group compared with the RV and control groups, whereas protein expression of SCN5A and connexin43 was significantly decreased in the RV compared with the other study groups. Erg protein expression was significantly increased in both pacing groups compared with the controls. Conclusions—In this rabbit model, we demonstrate a direct effect of BIV but not RV pacing on shortening the native QT interval as well as the paced QT interval during burst pacing and close to the ventricular effective refractory period. These findings underscore the fact that the effect of BIV pacing is partially mediated through direct electric remodeling and may have implications as to the effect of BIV pacing on arrhythmia incidence and burden.

Prevention of adverse electrical and mechanical remodeling with biventricular pacing in a rabbit model of myocardial infarction

BACKGROUND Biventricular (BIV) pacing can improve cardiac function in heart failure (HF). OBJECTIVE This study sought to investigate the mechanisms of benefit of BIV pacing using a rabbit model of myocardial infarction (MI). METHODS New Zealand White rabbits were divided into 4 groups (sham-operated [C], MI with no pacing [MI], MI with right ventricular pacing [MI+RV], and MI with BIV pacing [MI+BIV]) and underwent serial electrocardiograms and echocardiograms. At 4 weeks, hearts were excised and tissue was extracted from various areas of the left ventricle (LV). RESULTS Four weeks after coronary ligation, BIV pacing prevented systolic and diastolic dilation of the LV as well as the reduction in its fractional shortening, restored the QRS width and the rate-dependent QT intervals to their baseline values, and prevented the decline of the ether-a-go-go (Erg) protein levels. This prevention of remodeling was not documented in the MI+RV groups. CONCLUSION In this rabbit model of BIV pacing and MI, we show prevention of adverse mechanical and electrical remodeling of the heart. These changes may underlie some of the benefits seen with BIV pacing in HF patients with more severe LV dysfunction.

Recombinant hepatitis B surface antigen and anionic phospholipids share a binding region in the fifth domain of β2-glycoprotein I (apolipoprotein H)

Human beta2-glycoprotein I (beta 2GPI) binds to recombinant hepatitis B surface antigen (rHBsAg), but the location of the binding domain on beta 2GPI is unknown. It has been suggested that the lipid rather than the protein moiety of rHBsAg binds to beta 2GPI. Since beta 2GPI binds to anionic phospholipids (PL) through its lipid-binding region in the fifth domain of beta 2GPI, we predicted that this lipid-binding region may also be involved in binding rHBsAg. In this study, we examined rHBsAg binding to two naturally occurring mutants of beta 2GPI, Cys306Gly and Trp316Ser, or evolutionarily conserved hydrophobic amino acid sequence, Leu313-Ala314-Phe315 in the fifth domain of beta 2GPI. The two naturally occurring mutations and two mutagenized amino acids, Leu313Gly or Phe315Ser, disrupted the binding of recombinant beta 2GPI (rbeta 2GPI) to both rHBsAg and cardiolipin (CL), an anionic PL. These results suggest that rHBsAg and CL share the same region in the fifth domain of beta2GPI. Credence to this conclusion was further provided by competitive ELISA, where CL-bound rbeta 2GPI was incubated with increasing amounts of rHBsAg. As expected, pre-incubation of rbeta 2GPI with CL precluded binding to rHBsAg, indicating that CL and rHBsAg bind to the same region on beta 2GPI. Our data provide evidence that the lipid (PL) rather than the protein moiety of rHBsAg binds to beta 2GPI and that this binding region is located in the fifth domain of beta 2GPI, which also binds to anionic PL.

Sirtuin 1 regulates cardiac electrical activity by deacetylating the cardiac sodium channel

The voltage-gated cardiac Na+ channel (Nav1.5), encoded by the SCN5A gene, conducts the inward depolarizing cardiac Na+ current (INa) and is vital for normal cardiac electrical activity. Inherited loss-of-function mutations in SCN5A lead to defects in the generation and conduction of the cardiac electrical impulse and are associated with various arrhythmia phenotypes. Here we show that sirtuin 1 deacetylase (Sirt1) deacetylates Nav1.5 at lysine 1479 (K1479) and stimulates INa via lysine-deacetylation-mediated trafficking of Nav1.5 to the plasma membrane. Cardiac Sirt1 deficiency in mice induces hyperacetylation of K1479 in Nav1.5, decreases expression of Nav1.5 on the cardiomyocyte membrane, reduces INa and leads to cardiac conduction abnormalities and premature death owing to arrhythmia. The arrhythmic phenotype of cardiac-Sirt1-deficient mice recapitulated human cardiac arrhythmias resulting from loss of function of Nav1.5. Increased Sirt1 activity or expression results in decreased lysine acetylation of Nav1.5, which promotes the trafficking of Nav1.5 to the plasma membrane and stimulation of INa. As compared to wild-type Nav1.5, Nav1.5 with K1479 mutated to a nonacetylatable residue increases peak INa and is not regulated by Sirt1, whereas Nav1.5 with K1479 mutated to mimic acetylation decreases INa. Nav1.5 is hyperacetylated on K1479 in the hearts of patients with cardiomyopathy and clinical conduction disease. Thus, Sirt1, by deacetylating Nav1.5, plays an essential part in the regulation of INa and cardiac electrical activity.

Cardiac levels of NOS1AP RNA from right ventricular tissue recovered during lead extraction.

BACKGROUND There is a scarcity of cardiac tissue available for research. OBJECTIVE (1) To investigate the feasibility of obtaining myocardial tissue from extracted pacemaker and defibrillator leads for gene expression analysis and (2) to examine the nitric oxide 1 adaptor protein (NOS1AP) RNA expression as a function of patient genotype. METHODS Seventeen patients (age = 56 ± 20 years; 12 men; 5 pacemakers; 12 defibrillators) undergoing lead extractions for standard indications (5 device erosion; 1 vascular occlusion; 11 lead malfunction or recall) were genotyped for 2 NOS1AP single nucleotide polymorphisms-rs10494366 (T to G) and rs10918594 (C to G)-and had RNA levels measured by real-time polymerase chain reaction for collagen I, troponin I, Ca(v)1.2, Kv4.3, HERG, KvLQT1, connexin 43, NOS1AP, and sodium-calcium exchanger. Ventricular tissue obtained from 3 failing hearts at transplantation served as reference. RESULTS A high ratio of cardiac troponin I/collagen I RNA identified 9 of the 17 patient samples (muscle rich), in which the gene expression profile was similar to that of the reference ventricular samples and significantly different (P < .003) from the expression profile of samples with a low troponin I/collagen ratio (muscle poor). TT and CC polymorphisms were associated with significantly lower NOS1AP RNA levels (P < .01 compared with the GG genotype). CONCLUSIONS Performing gene expression analyses on right ventricular tissue samples extracted with pacemaker and defibrillator leads is feasible. A significant number of samples contain cardiomyocytes that express troponin I and ion channels at levels comparable to those seen in explanted hearts. Decreased NOS1AP expression in rs10494366 TT and rs10918594 CC homozygotes may underlie shorter repolarization times.

A common variant alters SCN5A–miR-24 interaction and associates with heart failure mortality

SCN5A encodes the voltage-gated Na+ channel NaV1.5 that is responsible for depolarization of the cardiac action potential and rapid intercellular conduction. Mutations disrupting the SCN5A coding sequence cause inherited arrhythmias and cardiomyopathy, and single-nucleotide polymorphisms (SNPs) linked to SCN5A splicing, localization, and function associate with heart failure–related sudden cardiac death. However, the clinical relevance of SNPs that modulate SCN5A expression levels remains understudied. We recently generated a transcriptome-wide map of microRNA (miR) binding sites in human heart, evaluated their overlap with common SNPs, and identified a synonymous SNP (rs1805126) adjacent to a miR-24 site within the SCN5A coding sequence. This SNP was previously shown to reproducibly associate with cardiac electrophysiological parameters, but was not considered to be causal. Here, we show that miR-24 potently suppresses SCN5A expression and that rs1805126 modulates this regulation. We found that the rs1805126 minor allele associates with decreased cardiac SCN5A expression and that heart failure subjects homozygous for the minor allele have decreased ejection fraction and increased mortality, but not increased ventricular tachyarrhythmias. In mice, we identified a potential basis for this in discovering that decreased Scn5a expression leads to accumulation of myocardial reactive oxygen species. Together, these data reiterate the importance of considering the mechanistic significance of synonymous SNPs as they relate to miRs and disease, and highlight a surprising link between SCN5A expression and nonarrhythmic death in heart failure.