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1.  Genomic Oncology Education: An Urgent Need, A New Approach 
Genomic testing has entered oncology practice. With reduced cost and faster turn-around-times, clinical applications for next-generation sequencing-based assays will only continue to increase. As such, there is an urgent need for health professional education to allow implementation of these new diagnostic tools. However, current medical school, residency and fellowship training has had limited success in educating physicians in the fundamentals of single gene testing let alone genomic methods. In this review, we describe the novel approach the pathology community has taken in genomic education and the potential for application to oncology trainees.
PMCID: PMC3924419  PMID: 24445771
Genomics; next-generation sequencing; gene panels; oncology; cancer; molecular pathology; medical education; residency training
2.  Role of the intercalated disc in cardiac propagation and arrhythmogenesis 
This review article discusses mechanisms underlying impulse propagation in cardiac muscle with specific emphasis on the role of the cardiac cell-to-cell junction, called the “intercalated disc.”The first part of this review deals with the role of gap junction channels, formed by connexin proteins, as a determinant of impulse propagation. It is shown that, depending on the underlying structure of the cellular network, decreasing the conductance of gap junction channels (so-called “electrical uncoupling”) may either only slow, or additionally stabilize propagation and reverse unidirectional propagation block to bidirectional propagation. This is because the safety factor for propagation increases with decreasing intercellular electrical conductance. The role of heterogeneous connexin expression, which may be present in disease states, is also discussed. The hypothesis that so-called ephaptic impulse transmission plays a role in heart and can substitute for electrical coupling has been revived recently. Whereas ephaptic transmission can be demonstrated in theoretical simulations, direct experimental evidence has not yet been presented. The second part of this review deals with the interaction of three protein complexes at the intercalated disc: (1) desmosomal and adherens junction proteins, (2) ion channel proteins, and (3) gap junction channels consisting of connexins. Recent work has revealed multiple interactions between these three protein complexes which occur, at least in part, at the level of protein trafficking. Such interactions are likely to play an important role in the pathogenesis of arrhythmogenic cardiomyopathy, and may reveal new therapeutic concepts and targets.
PMCID: PMC4201087  PMID: 25368581
propagation velocity in heart; electrical-cell-to-cell coupling; intercalated disc; cardiac connexins; discontinuous propagation
3.  Gap Junctions and Arrhythmogenic Cardiomyopathy 
Heart Rhythm  2011;9(6):992-995.
PMCID: PMC3358518  PMID: 22100709
4.  Altered Desmosomal Proteins in Granulomatous Myocarditis and Potential Pathogenic Links to Arrhythmogenic Right Ventricular Cardiomyopathy 
Immunoreactive signal for the desmosomal protein plakoglobin (γ-catenin) is reduced at cardiac intercalated disks in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC), a highly arrhythmogenic condition caused by mutations in genes encoding desmosomal proteins. Previously, we observed a “false positive” case in which plakoglobin signal was reduced in a patient initially thought to have ARVC but who actually had cardiac sarcoidosis. Sarcoidosis can masquerade clinically as ARVC, but has not previously been associated with altered desmosomal proteins.
Methods and Results
We observed marked reduction in immunoreactive signal for plakoglobin at cardiac myocyte junctions in patients with sarcoidosis and giant cell myocarditis, both highly arrhythmogenic forms of myocarditis associated with granulomatous inflammation. In contrast, plakoglobin signal was not depressed in lymphocytic (non-granulomatous) myocarditis. To determine whether cytokines might promote dislocation of plakoglobin from desmosomes, we incubated cultures of neonatal rat ventricular myocytes with selected inflammatory mediators. Brief exposure to low concentrations of IL-17, TNFα and IL-6, cytokines implicated in granulomatous myocarditis, caused translocation of plakoglobin from cell-cell junctions to intracellular sites, whereas other potent cytokines implicated in non-granulomatous myocarditis had no effect, even at much high concentrations. We also observed myocardial expression of IL-17 and TNFα, and elevated serum levels of inflammatory mediators including IL-6R, IL-8, MCP1 and MIP1β in ARVC patients (all p<0.0001 compared with controls).
These results suggest novel disease mechanisms involving desmosomal proteins in granulomatous myocarditis and implicate cytokines, perhaps derived in part from the myocardium, in disruption of desmosomal proteins and arrhythmogenesis in ARVC.
PMCID: PMC3203520  PMID: 21859801
plakoglobin; desmosome; sarcoidosis; giant cell myocarditis; cytokines
5.  The Role of Endomyocardial Biopsy in ARVC: Looking Beyond Histology in Search of New Diagnostic Markers 
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by a high incidence of ventricular tachyarrhythmias and sudden cardiac death that appear early in the natural history of the disease and may precede significant ventricular remodeling. The classical pathology of ARVC is degeneration of right ventricular free wall myocardium and its replacement by fat and fibrous tissue. The clinical presentation may be highly variable, however, and genetic penetrance is typically low which makes definitive diagnosis difficult, especially in early stages of the disease. Endomyocardial biopsy (EMB) has not been used widely in the diagnosis of ARVC, in part because pathological changes are usually most conspicuous in the epicardium of the right ventricular free wall and tend to spare the endocardium and interventricular septum. Thus, diagnostic pathological features of ARVC are often not seen in conventional septal biopsies. Diagnostic yield may be increased by sampling the RV free wall or by using 3-dimensional electroanatomic voltage mapping to identify affected areas, but these approaches can carry increased risk and require specialized equipment and experience. Moreover, diagnostic pathological changes may not be apparent in early disease despite an increased risk of arrhythmias and sudden death. This review considers the role of EMB in the diagnosis of ARVC and highlights recent advances in identifying potential tissue biomarkers that arise early in the disease process and occur diffusely throughout the myocardium. Analysis of conventional EMB using such biomarkers could improve diagnostic sensitivity and accuracy but widespread clinical application of this approach requires further validation.
PMCID: PMC3058333  PMID: 21235662
arrhythmogenic right ventricular cardiomyopathy; endomyocardial biopsy; intercalated disks; desmosomes; plakoglobin
6.  Remodeling of the cardiac sodium channel, Connexin43 and Plakoglobin at the intercalated disk in patients with arrhythmogenic cardiomyopathy 
Arrhythmogenic cardiomyopathy (AC) is tightly associated with desmosomal mutations in the majority of patients. Arrhythmogenesis in AC patients is likely related to remodeling of cardiac gap junctions and increased levels of fibrosis. Recently, using experimental models, we also identified sodium channel dysfunction secondary to desmosomal dysfunction. The aim of the present study was to assess the immunoreactive signal levels of the sodium channel protein NaV1.5, as well as Connexin43 and Plakoglobin, in myocardial specimens obtained from AC patients.
Left and right ventricular free wall (LVFW/RVFW) post-mortem material was obtained from 5 AC patients and 5 age and sex-matched controls. RV septal biopsies (RVSB) were taken from another 15 AC patients. All patients fulfilled the 2010 revised Task Force Criteria for AC diagnosis. Immunohistochemical analyses were performed using antibodies against Connexin43 (Cx43), Plakoglobin, NaV1.5, Plakophilin-2 and N-Cadherin.
N-Cadherin and Desmoplakin immunoreactive signals and distribution were normal in AC patients compared to control. Plakophilin-2 signals were unaffected unless a PKP2 mutation predicting haploinsufficiency was present. Distribution was unchanged compared to control. Immunoreactive signal levels of PKG, Cx43 and NaV1.5 were disturbed in 74%, 70% and 65% of the patients, respectively.
Reduced immunoreactive signal of PKG, Cx43 and NaV1.5 at the intercalated disks can be observed in a large majority of the patients. Decreased levels of Nav1.5 might contribute to arrhythmia vulnerability and, in the future, potentially could serve as a new clinically relevant tool for risk assessment strategies.
PMCID: PMC3608196  PMID: 23178689
7.  Phospholamban R14del mutation in patients diagnosed with dilated cardiomyopathy or arrhythmogenic right ventricular cardiomyopathy: evidence supporting the concept of arrhythmogenic cardiomyopathy 
European Journal of Heart Failure  2012;14(11):1199-1207.
To investigate whether phospholamban gene (PLN) mutations underlie patients diagnosed with either arrhythmogenic right ventricular cardiomyopathy (ARVC) or idiopathic dilated cardiomyopathy (DCM).
Methods and results
We screened a cohort of 97 ARVC and 257 DCM unrelated index patients for PLN mutations and evaluated their clinical characteristics. PLN mutation R14del was identified in 12 (12 % ) ARVC patients and in 39 (15 % ) DCM patients. Haplotype analysis revealed a common founder, estimated to be between 575 and 825 years old. A low voltage electrocardiogram was present in 46 % of R14del carriers. Compared with R14del– DCM patients, R14del+ DCM patients more often demonstrated appropriate implantable cardioverter defibrillator discharge (47 % vs. 10 % , P < 0.001), cardiac transplantation (18 % vs. 2 % , P < 0.001), and a family history for sudden cardiac death (SCD) at < 50 years (36 % vs. 16 % , P = 0.007). We observed a similar pattern in the ARVC patients although this was not statistically significant. The average age of 26 family members who died of SCD was 37.7 years. Immunohistochemistry in available myocardial samples revealed absent/depressed plakoglobin levels at intercalated disks in five of seven (71 % ) R14del+ ARVC samples, but in only one of nine (11 % ) R14del+ DCM samples (P = 0.03).
The PLN R14del founder mutation is present in a substantial number of patients clinically diagnosed with DCM or ARVC. R14del+ patients diagnosed with DCM showed an arrhythmogenic phenotype, and SCD at young age can be the presenting symptom. These findings support the concept of ‘arrhythmogenic cardiomyopathy’.
PMCID: PMC3475434  PMID: 22820313
Arrhythmia; Arrhythmogenic cardiomyopathy; Arrhythmogenic right ventricular cardiomyopathy; Dilated cardiomyopathy; Genetics
8.  Protein Kinase Cε Mediates Salutary Effects on Electrical Coupling Induced by Ischemic Preconditioning 
Ischemic preconditioning delays the onset of electrical uncoupling and prevents loss of the primary ventricular gap junction protein connexin43 (Cx43) from gap junctions during subsequent ischemia.
To test the hypothesis that these effects are mediated by protein kinase C epsilon (PKCε), we studied isolated Langendorff-perfused hearts from mice with homozygous germline deletion of PKCε (PKCε-KO). Cx43 phosphorylation and distribution were measured by quantitative immunoblotting and confocal microscopy. Changes in electrical coupling were monitored using the 4-electrode technique to measure whole-tissue resistivity.
The amount of Cx43 located in gap junctions, measured by confocal microscopy under basal conditions, was significantly greater in PKCε-KO hearts compared to wildtype but total Cx43 content measured by immunoblotting was not different. These unanticipated results indicate that PKCε regulates subcellular distribution of Cx43 under normal conditions. Preconditioning prevented loss of Cx43 from gap junctions during ischemia in wildtype but not PKCε-KO hearts. Specific activation of PKCε, but not PKCδ, also prevented ischemia-induced loss of Cx43 from gap junctions. Preconditioning delayed the onset of uncoupling in wildtype but hastened uncoupling in PKCε-KO hearts. Cx43 phosphorylation at the PKC site Ser368 increased 5-fold after ischemia in wildtype hearts and, surprisingly, by nearly 10-fold in PKCε-KO hearts. Preconditioning prevented phosphorylation of Cx43 in gap junction plaques at Ser368 in wildtype but not PKCε-KO hearts.
Taken together, these results indicate that PKCε plays a critical role in preconditioning to preserve Cx43 signal in gap junctions and delay electrical uncoupling during ischemia.
PMCID: PMC2711555  PMID: 17765619
preconditioning; gap junctions; connexin43; coupling; protein kinase C
Circulation Research  2012;110(11):1445-1453.
Spatial heterogeneity in connexin (Cx) expression has been implicated in arrhythmogenesis.
This study was carried out to quantify the relation between the degree of heterogeneity in Cx43 expression and disturbances in electrical propagation.
Methods and Results
Cell pairs and strands composed of mixtures of Cx43-/- (Cx43KO) or GFP-expressing Cx43+/+ (WTGFP) murine ventricular myocytes were patterned using microlithographic techniques. At the interface between pairs of WTGFP and Cx43KO cells, dual voltage clamp showed a marked decrease in electrical coupling (~5% of wildtype) and voltage gating suggested the presence of mixed Cx43/Cx45 channels. Cx43 and Cx45 immunofluorescence signals were not detectable at this interface, probably because of markedly reduced gap junction size. Macroscopic propagation velocity, measured by multisite high-resolution optical mapping of transmembrane potential in strands of cells of mixed Cx43 genotype, decreased with an increasing proportion of Cx43KO cells in the strand. A marked decrease in conduction velocity was observed in strands composed of <50% wildtype cells. Propagation at the microscopic scale showed a high degree of dissociation between WTGFP and Cx43KO cells, but consistent excitation without development of propagation block.
Heterogeneous ablation of Cx43 leads to a marked decrease in propagation velocity in tissue strands composed of <50% cells with wildtype Cx43 expression and marked dissociation of excitation at the cellular level. However, the small residual electrical conductance between Cx43 and WTGFP myocytes assures excitation of Cx43-/- cells. This explains the previously reported undisturbed contractility in tissues with spatially heterogeneous downregulation of Cx43 expression.
PMCID: PMC3381798  PMID: 22518032
Cx43 genotypes; Myocardium; Cellular Coupling; Propagation
10.  Connexin43 Mutation Causes Heterogeneous Gap Junction Loss and Sudden Infant Death 
Circulation  2011;125(3):474-481.
An estimated 10-15% of sudden infant death syndrome (SIDS) may stem from channelopathy-mediated lethal arrhythmias. Loss of the GJA1-encoded gap junction channel protein connexin43 (Cx43) is known to underlie formation of lethal arrhythmias. GJA1 mutations have been associated with cardiac diseases including atrial fibrillation. Therefore, GJA1 is a plausible candidate gene for premature sudden death.
Methods and Results
GJA1 open reading frame mutational analysis was performed using PCR, DHPLC, and direct DNA sequencing on DNA from 292 SIDS cases. Immunofluorescence and dual whole cell patch-clamp studies were performed to determine functionality of mutant gap junctions. Immunostaining for gap junction proteins was performed on SIDS-associated paraffin-embedded cardiac tissue. Two rare, novel missense mutations, E42K and S272P, were detected in 2 of 292 SIDS cases, a 2-month-old white male and a 3-month-old white female, respectively. Analysis of the E42K victim’s parental DNA demonstrated a de novo mutation. Both mutations involved highly conserved residues and were absent in over 1000 ethnic-matched reference alleles. Immunofluorescence demonstrated no trafficking abnormalities for either mutation and S272P demonstrated wildtype junctional conductance. However, junctional conductance measurements for the E42K mutation demonstrated a loss-of-function not rescued by wildtype. Moreover, the E42K victim cardiac tissue demonstrated a mosaic immunostaining pattern for Cx43 protein.
This study provides the first molecular and functional evidence implicating a GJA1 mutation as a novel pathogenic substrate for SIDS. E42K-Cx43 demonstrated a trafficking-independent reduction in junctional coupling in vitro as well as demonstrating a mosaic pattern of mutational DNA distribution in deceased cardiac tissue, suggesting a novel mechanism of Cx43-associated sudden death.
PMCID: PMC3332541  PMID: 22179534
arrhythmia; connexins; death; sudden; electrophysiology; genetics
11.  Electrophysiological abnormalities precede overt structural changes in arrhythmogenic right ventricular cardiomyopathy due to mutations in desmoplakin-A combined murine and human study 
European Heart Journal  2012;33(15):1942-1953.
Anecdotal observations suggest that sub-clinical electrophysiological manifestations of arrhythmogenic right ventricular cardiomyopathy (ARVC) develop before detectable structural changes ensue on cardiac imaging. To test this hypothesis, we investigated a murine model with conditional cardiac genetic deletion of one desmoplakin allele (DSP ±) and compared the findings to patients with non-diagnostic features of ARVC who carried mutations in desmoplakin.
Methods and results
Murine: the DSP (±) mice underwent electrophysiological, echocardiographic, and immunohistochemical studies. They had normal echocardiograms but delayed conduction and inducible ventricular tachycardia associated with mislocalization and reduced intercalated disc expression of Cx43. Sodium current density and myocardial histology were normal at 2 months of age. Human: ten patients with heterozygous mutations in DSP without overt structural heart disease (DSP+) and 12 controls with supraventricular tachycardia were studied by high-density electrophysiological mapping of the right ventricle. Using a standard S1–S2 protocol, restitution curves of local conduction and repolarization parameters were constructed. Significantly greater mean increases in delay were identified particularly in the outflow tract vs. controls (P< 0.01) coupled with more uniform wavefront progression. The odds of a segment with a maximal activation–repolarization interval restitution slope >1 was 99% higher (95% CI: 13%; 351%, P= 0.017) in DSP+ vs. controls. Immunostaining revealed Cx43 mislocalization and variable Na channel distribution.
Desmoplakin disease causes connexin mislocalization in the mouse and man preceding any overt histological abnormalities resulting in significant alterations in conduction–repolarization kinetics prior to morphological changes detectable on conventional cardiac imaging. Haploinsufficiency of desmoplakin is sufficient to cause significant Cx43 mislocalization. Changes in sodium current density and histological abnormalities may contribute to a worsening phenotype or disease but are not necessary to generate an arrhythmogenic substrate. This has important implications for the earlier diagnosis of ARVC and risk stratification.
PMCID: PMC3409421  PMID: 22240500
Arrhythmia; Conduction; ARVC; Repolarization; Desmosome; Desmoplakin
12.  A novel desmocollin-2 mutation reveals insights into the molecular link between desmosomes and gap junctions 
Heart Rhythm  2011;8(5):711-718.
Cellular adhesion mediated by cardiac desmosomes is a prerequisite for proper electric propagation mediated by gap junctions in the myocardium. However, the molecular principles underlying this interdependence are not fully understood.
The purpose of this study was to determine potential causes of right ventricular conduction abnormalities in a patient with borderline diagnosis of arrhythmogenic right ventricular cardiomyopathy.
To assess molecular changes, the patient's myocardial tissue was analyzed for altered desmosomal and gap junction (connexin43) protein levels and localization. In vitro functional studies were performed to characterize the consequences of the desmosomal mutations.
Loss of plakoglobin signal was evident at the cell junctions despite expression of the protein at control levels. Although the distribution of connexin43 was not altered, total protein levels were reduced and changes in phosphorylation were observed. The truncation mutant in desmocollin-2a is deficient in binding plakoglobin. Moreover, the ability of desmocollin-2a to directly interact with connexin43 was abolished by the mutation. No pathogenic potential of the desmoglein-2 missense change was identified.
The observed abnormalities in gap junction protein expression and phosphorylation, which precede an overt cardiac phenotype, likely are responsible for slow myocardial conduction in this patient. At the molecular level, altered binding properties of the desmocollin-2a mutant may contribute to the changes in connexin43. In particular, the newly identified interaction between the desmocollin-2a isoform and connexin43 provides novel insights into the molecular link between desmosomes and gap junctions.
PMCID: PMC3085091  PMID: 21220045
Cardiomyopathy; Conduction; Connexin43; Desmocollin-2; Desmoglein-2; Desmosome; Functional studies; Gap junction; Mutation; Plakoglobin; ARVC, arrhythmogenic right ventricular cardiomyopathy; Cx43, connexin43; DAPI, 4′,6-diamidino-2-phenylindole; DSC2, desmocollin-2; DSG2, desmoglein-2; DSP, desmoplakin; GFP, green fluorescent protein; GST, glutathione-S-transferase; ICS, intracellular cadherin segment; PG, plakoglobin; PKP2, plakophilin-2; RV, right ventricle; YFP, yellow fluorescent protein
13.  Diagnosis of Arrhythmogenic Right Ventricular Cardiomyopathy/Dysplasia (ARVC/D) 
Circulation  2010;121(13):1533-1541.
In 1994, an International Task Force proposed criteria for the clinical diagnosis of ARVC/D which facilitated recognition and interpretation of the frequently non-specific clinical features of ARVC/D. This enabled confirmatory clinical diagnosis in index cases through exclusion of phenocopies, and provided a standard upon which clinical research and genetic studies could be based. Structural, histological, electrocardiographic, arrhythmic, and familial features of the disease were incorporated into the criteria, subdivided into major and minor according to the specificity of their association with ARVC/D. At that time, clinical experience with ARVC/D was dominated by symptomatic index cases and sudden cardiac death victims: the overt and/or severe end of the disease spectrum. Consequently, the 1994 criteria were highly specific but lacked sensitivity for early and familial disease.
Methods and Results
Revision of the diagnostic criteria provides guidance on the role of emerging diagnostic modalities and advances in the genetics of ARVC/D. The criteria have been modified to incorporate new knowledge and technology to improve diagnostic sensitivity, but with the important requisite of maintaining diagnostic specificity. The approach classifying structural, histological, electrocardiographic, arrhythmic, and genetic features of the disease as major and minor criteria has been maintained. In this modification of the Task Force Criteria, quantitative criteria are proposed and abnormalities are defined based on comparison with normal subject data.
The diagnosis of ARVC/D based on modification of the original Task Force criteria is a working framework to improve the diagnosis and management of this condition.
PMCID: PMC2860804  PMID: 20172911
cardiomyopathy; diagnosis; echocardiography; electrocardiography; magnetic resonance imaging
14.  Diagnosis of arrhythmogenic right ventricular cardiomyopathy/dysplasia 
European Heart Journal  2010;31(7):806-814.
In 1994, an International Task Force proposed criteria for the clinical diagnosis of arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) that facilitated recognition and interpretation of the frequently nonspecific clinical features of ARVC/D. This enabled confirmatory clinical diagnosis in index cases through exclusion of phenocopies and provided a standard on which clinical research and genetic studies could be based. Structural, histological, electrocardiographic, arrhythmic, and familial features of the disease were incorporated into the criteria, subdivided into major and minor categories according to the specificity of their association with ARVC/D. At that time, clinical experience with ARVC/D was dominated by symptomatic index cases and sudden cardiac death victims–the overt or severe end of the disease spectrum. Consequently, the 1994 criteria were highly specific but lacked sensitivity for early and familial disease.
Methods and Results
Revision of the diagnostic criteria provides guidance on the role of emerging diagnostic modalities and advances in the genetics of ARVC/D. The criteria have been modified to incorporate new knowledge and technology to improve diagnostic sensitivity, but with the important requisite of maintaining diagnostic specificity. The approach of classifying structural, histological, electrocardiographic, arrhythmic, and genetic features of the disease as major and minor criteria has been maintained. In this modification of the Task Force criteria, quantitative criteria are proposed and abnormalities are defined on the basis of comparison with normal subject data.
The present modifications of the Task Force Criteria represent a working framework to improve the diagnosis and management of this condition.
Clinical Trial Registration Identifier: NCT00024505.
PMCID: PMC2848326  PMID: 20172912
Arrhythmias, cardiac; Arrhythmogenic right ventricular cardiomyopathy/dysplasia; Death, sudden, cardiac; Diagnosis; Echocardiography; Electrocardiography; Magnetic resonance imaging
15.  Mechanistic insights into arrhythmogenic right ventricular cardiomyopathy caused by desmocollin-2 mutations 
Cardiovascular Research  2010;90(1):77-87.
Recent immunohistochemical studies observed the loss of plakoglobin (PG) from the intercalated disc (ID) as a hallmark of arrhythmogenic right ventricular cardiomyopathy (ARVC), suggesting a final common pathway for this disease. However, the underlying molecular processes are poorly understood.
Methods and results
We have identified novel mutations in the desmosomal cadherin desmocollin 2 (DSC2 R203C, L229X, T275M, and G371fsX378). The two missense mutations (DSC2 R203C and T275M) have been functionally characterized, together with a previously reported frameshift variant (DSC2 A897fsX900), to examine their pathogenic potential towards PG's functions at the ID. The three mutant proteins were transiently expressed in various cellular systems and assayed for expression, processing, localization, and binding to other desmosomal components in comparison to wild-type DSC2a protein. The two missense mutations showed defects in proteolytic cleavage, a process which is required for the functional activation of mature cadherins. In both cases, this is thought to cause a reduction of functional DSC2 at the desmosomes in cardiac cells. In contrast, the frameshift variant was incorporated into cardiac desmosomes; however, it showed reduced binding to PG.
Despite different modes of action, for all three variants, the reduced ability to provide a ligand for PG at the desmosomes was observed. This is in agreement with the reduced intensity of PG at these structures observed in ARVC patients.
PMCID: PMC3058729  PMID: 21062920
Arrhythmogenic right ventricular cardiomyopathy; Desmocollin-2; Desmosome; Functional studies; Mutation
16.  Novel missense mutations in exon 15 of desmoglein-2: Role of the intracellular cadherin segment in arrhythmogenic right ventricular cardiomyopathy? 
Heart Rhythm  2010;7(10):1446-1453.
The diagnosis of arrhythmogenic right ventricular cardiomyopathy can be challenging. Disease-causing mutations in desmosomal genes have been identified. A novel diagnostic feature, loss of immunoreactivity for plakoglobin from the intercalated disks, recently was proposed.
The purpose of this study was to identify two novel mutations in the intracellular cadherin segment of desmoglein-2 (G812S and C813R in exon 15). Co-segregation of the G812S mutation with disease expression was established in a large Caucasian family. Endomyocardial biopsies of two individuals showed reduced plakoglobin signal at the intercalated disk.
To understand the pathologic changes occurring in the diseased myocardium, functional studies on three mutations in exon 15 of desmoglein-2 (G812C, G812S, C813R) were performed.
Localization studies failed to detect any differences in targeting or stability of the mutant proteins, suggesting that they act via a dominant negative mechanism. Binding assays were performed to probe for altered binding affinities toward other desmosomal proteins, such as plakoglobin and plakophilin-2. Although no differences were observed for the mutated proteins in comparison to wild-type desmoglein-2, binding to plakophilin-2 depended on the expression system (i.e., bacterial vs mammalian protein expression). In addition, abnormal migration of the C813R mutant protein was observed in gel electrophoresis.
Loss of plakoglobin immunoreactivity from the intercalated disks appears to be the endpoint of complex pathologic changes, and our functional data suggest that yet unknown posttranslational modifications of desmoglein-2 might be involved.
PMCID: PMC2994644  PMID: 20708101
Arrhythmogenic right ventricular cardiomyopathy; Desmoglein-2; Desmosome; Genetics; Missense mutation; Plakophilin-2; ARVC, arrhythmogenic right ventricular cardiomyopathy; Cx43, connexin43; DSC2, desmocollin-2; DSG2, desmoglein-2; DSP, desmoplakin; GFP, green fluorescent protein; GST, glutathione-S-transferase; ICS, intracellular cadherin segment; PG, plakoglobin; PKP2, plakophilin-2; RV, right ventricle
17.  Prolonged RV Endocardial Activation Duration: A Novel Marker of Arrhythmogenic Right Ventricular Dysplasia/ /Cardiomyopathy 
Parietal block, defined as intra right ventricular (RV) conduction slowing is a major diagnostic criterion for ARVD/C.
We evaluated the utility of total RV endocardial activation duration (EAD) measured by 3D electroanatomic mapping during sinus rhythm in the diagnosis of ARVD/C.
25 consecutive patients with frequent LBBB morphology PVCs who underwent electroanatomic mapping as a part of the evaluation for ARVD/C were included in the study. All patients were evaluated using standard protocol that included, ECG, signal averaged ECG, Holter, Echocardiography, and MRI. Invasive testing was performed as indicated. Total RV EAD was measured as the time interval between the onset of RV activation to the latest activated region in the RV.
Mean age of the study subjects was 38±11 and 32% were men. 14 subjects were diagnosed as ARVD/C using task force criteria and the remainder had idiopathic VT. While the surface QRS durations were similar, the total RV EAD was significantly prolonged in ARVD/C compared with idiopathic VT (83.9±10 msec vs. 50.8±7 msec, p<0.001). None of the idiopathic VT subjects had RV EAD of > 65 msec. RV EAD also showed significant negative correlation with RV ejection fraction.
Total RV EAD obtained by 3D electroanatomic mapping is a sensitive marker of intra RV conduction delay in ARVD/C and a total RV EAD of >65 msec accurately differentiates ARVD/C from idiopathic VT.
PMCID: PMC2778730  PMID: 19419906
18.  Impulse Propagation in Synthetic Strands of Neonatal Cardiac Myocytes With Genetically Reduced Levels of Connexin43 
Circulation research  2003;92(11):1209-1216.
Connexin43 (Cx43) is a major determinant of the electrical properties of the myocardium. Closure of gap junctions causes rapid slowing of propagation velocity (θ), but the precise effect of a reduction in Cx43 levels due to genetic manipulation has only partially been clarified. In this study, morphological and electrical properties of synthetic strands of cultured neonatal ventricular myocytes from Cx43+/+ (wild type, WT) and Cx+/− (heterozygote, HZ) mice were compared. Quantitative immunofluorescence analysis of Cx43 demonstrated a 43% reduction of Cx43 expression in the HZ versus WT mice. Cell dimensions, connectivity, and alignment were independent of genotype. Measurement of electrical properties by microelectrodes and optical mapping showed no differences in action potential amplitude or minimum diastolic potential between WT and HZ. However, maximal upstroke velocity of the transmembrane action potential, dV/dtmax, was increased and action potential duration was reduced in HZ versus WT. θ was similar in the two genotypes. Computer simulation of propagation and dV/dtmax showed a relatively small dependence of θ on gap junction coupling, thus explaining the lack of observed differences in θ between WT and HZ. Importantly, the simulations suggested that the difference in dV/dtmax is due to an upregulation of INa in HZ versus WT. Thus, heterozygote-null mutation of Cx43 produces a complex electrical phenotype in synthetic strands that is characterized by both changes in ion channel function and cell-to-cell coupling. The lack of changes in θ in this tissue is explained by the dominating role of myoplasmic resistance and the compensatory increase of dV/dtmax.
PMCID: PMC2242733  PMID: 12730095
synthetic cardiac strands; neonatal mouse cardiomyocytes; connexin43 expression; conduction velocity
19.  Quantifying Effects of Plaque Structure and Material Properties on Stress Distributions in Human Atherosclerotic Plaques Using 3D FSI Models 
Journal of biomechanical engineering  2005;127(7):1185-1194.
Atherosclerotic plaques may rupture without warning and cause acute cardiovascular syndromes such as heart attack and stroke. Methods to assess plaque vulnerability noninvasively and predict possible plaque rupture are urgently needed.
MRI-based three-dimensional unsteady models for human atherosclerotic plaques with multi-component plaque structure and fluid-structure interactions are introduced to perform mechanical analysis for human atherosclerotic plaques.
Stress variations on critical sites such as a thin cap in the plaque can be 300% higher than that at other normal sites. Large calcification block considerably changes stress/strain distributions. Stiffness variations of plaque components (50% reduction or 100% increase) may affect maximal stress values by 20–50 %. Plaque cap erosion causes almost no change on maximal stress level at the cap, but leads to 50% increase in maximal strain value.
Effects caused by atherosclerotic plaque structure, cap thickness and erosion, material properties, and pulsating pressure conditions on stress/strain distributions in the plaque are quantified by extensive computational case studies and parameter evaluations. Computational mechanical analysis has good potential to improve accuracy of plaque vulnerability assessment.
PMCID: PMC1474006  PMID: 16502661
20.  Local Maximal Stress Hypothesis and Computational Plaque Vulnerability Index for Atherosclerotic Plaque Assessment 
Annals of biomedical engineering  2005;33(12):1789-1801.
It is believed that atherosclerotic plaque rupture may be related to maximal stress conditions in the plaque. More careful examination of stress distributions in plaques reveals that it may be the local stress/strain behaviors at critical sites such as very thin plaque cap and locations with plaque cap weakness that are more closely related to plaque rupture risk. A “local maximal stress hypothesis” and a stress-based computational plaque vulnerability index (CPVI) are proposed to assess plaque vulnerability. A critical site selection (CSS) method is proposed to identify critical sites in the plaque and critical stress conditions which are be used to determine CPVI values. Our initial results based on 34 2D MRI slices from 14 human coronary plaque samples indicate that CPVI plaque assessment has an 85% agreement rate (91% if the square root of stress values is used) with assessment given by histopathological analysis. Large-scale and long-term patient studies are needed to further validate our findings for more accurate quantitative plaque vulnerability assessment.
PMCID: PMC1474005  PMID: 16389527
Stroke; Heart attack; Plaque cap rupture; Fluid-structure interaction; Carotid artery; Coronary; Blood flow; Cardiovascular diseases
21.  Quantitative Assessment of Coronary Artery Plaque Vulnerability by High-Resolution Magnetic Resonance Imaging and Computational Biomechanics: A Pilot Study ex Vivo 
The risk of atherosclerotic plaque disruption is thought to be closely related to plaque composition and rupture triggers such as external mechanical forces. The purpose of this study was to integrate MR imaging and computational techniques for the quantification of plaque vulnerability with morphologic data and biomechanical stress/strain distributions that were all based on high-resolution MR images of coronary artery plaque specimens ex vivo. Twenty-two coronary artery plaque specimens were selectively collected from 10 cadavers. Multislice T2-weighted spin echo images were acquired with a resolution of 100 × 100 μm2. Histopathological images were used as the gold standard for the identification of plaque components and vulnerability. Plaque components were classified on MR images, and the stress/strain components were calculated with a two-dimensional computational model with fluid–structure interactions. As expected, vulnerable plaques appeared to result from a large lipid pool, a thin fibrous cap, and some critical stress/strain conditions. An empiric vulnerability marker was derived and was closely related to the vulnerability score that was determined through pathologic examination. Noninvasive quantification of the MR contrast and mechanical properties of plaque may provide a comprehensive biomarker for the assessment of vulnerability of atherosclerotic plaques.
PMCID: PMC1474004  PMID: 16265643
MRI; atherosclerosis; coronary disease; biomechanics; vulnerability
22.  A novel mouse model of lipotoxic cardiomyopathy 
Journal of Clinical Investigation  2001;107(7):813-822.
Inherited and acquired cardiomyopathies are associated with marked intracellular lipid accumulation in the heart. To test the hypothesis that mismatch between myocardial fatty acid uptake and utilization leads to the accumulation of cardiotoxic lipid species, and to establish a mouse model of metabolic cardiomyopathy, we generated transgenic mouse lines that overexpress long-chain acyl-CoA synthetase in the heart (MHC-ACS). This protein plays an important role in vectorial fatty acid transport across the plasma membrane. MHC-ACS mice demonstrate cardiac-restricted expression of the transgene and marked cardiac myocyte triglyceride accumulation. Lipid accumulation is associated with initial cardiac hypertrophy, followed by the development of left-ventricular dysfunction and premature death. Terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling staining and cytochrome c release in transgenic hearts suggest that cardiac myocyte death occurs, in part, by lipid-induced programmed cell death. Taken together, our data demonstrate that fatty acid uptake/utilization mismatch in the heart leads to accumulation of lipid species toxic to cardiac myocytes. This novel mouse model will provide insight into the role of perturbations in myocardial lipid metabolism in the pathogenesis of inherited and acquired forms of heart failure.
PMCID: PMC199569  PMID: 11285300
23.  Peroxisome proliferator–activated receptor γ coactivator-1 promotes cardiac mitochondrial biogenesis 
Journal of Clinical Investigation  2000;106(7):847-856.
Cardiac mitochondrial function is altered in a variety of inherited and acquired cardiovascular diseases. Recent studies have identified the transcriptional coactivator peroxisome proliferator–activated receptor γ coactivator-1 (PGC-1) as a regulator of mitochondrial function in tissues specialized for thermogenesis, such as brown adipose. We sought to determine whether PGC-1 controlled mitochondrial biogenesis and energy-producing capacity in the heart, a tissue specialized for high-capacity ATP production. We found that PGC-1 gene expression is induced in the mouse heart after birth and in response to short-term fasting, conditions known to increase cardiac mitochondrial energy production. Forced expression of PGC-1 in cardiac myocytes in culture induced the expression of nuclear and mitochondrial genes involved in multiple mitochondrial energy-transduction/energy-production pathways, increased cellular mitochondrial number, and stimulated coupled respiration. Cardiac-specific overexpression of PGC-1 in transgenic mice resulted in uncontrolled mitochondrial proliferation in cardiac myocytes leading to loss of sarcomeric structure and a dilated cardiomyopathy. These results identify PGC-1 as a critical regulatory molecule in the control of cardiac mitochondrial number and function in response to energy demands.
PMCID: PMC517815  PMID: 11018072
24.  Truncated apo B-70.5–containing lipoproteins bind to megalin but not the LDL receptor 
Journal of Clinical Investigation  1999;103(10):1419-1430.
Apo B-100 of LDL can bind to both the LDL receptor and megalin, but the molecular interactions of apo B-100 with these 2 receptors are not completely understood. Naturally occurring mutant forms of apo B may be a source of valuable information on these interactions. Apo B-70.5 is uniquely useful because it contains the NH2-terminal portion of apo B-100, that includes only one of the two putative LDL receptor–binding sites (site A). The lipoprotein containing apo B-70.5 (Lp B-70.5) was purified from apo B-100/apo B-70.5 heterozygotes by sequential ultracentrifugation combined with immunoaffinity chromatography. Cell culture experiments, ligand blot analysis, and in vivo studies all consistently showed that Lp B-70.5 is not recognized by the LDL receptor. The kidney was identified as a major organ in catabolism of Lp B-70.5 in New Zealand white rabbits. Autoradiographic analysis revealed that renal proximal tubular cells selectively removed Lp B-70.5. On ligand blotting of renal cortical membranes, Lp B-70.5 bound only to megalin. The ability of megalin to mediate cellular endocytosis of Lp B-70.5 was confirmed using retinoic acid/dibutyryl cAMP–treated F9 cells. This study suggests that the putative LDL receptor–binding site A on apo B-100 might not by itself be a functional binding domain and that the apo B–binding sites recognized by the LDL receptor and by megalin may be different. Moreover, megalin may play an important role in renal catabolism of apo B truncations, including apo B-70.5.
PMCID: PMC408451  PMID: 10330424
25.  PGC-1α Deficiency Causes Multi-System Energy Metabolic Derangements: Muscle Dysfunction, Abnormal Weight Control and Hepatic Steatosis 
PLoS Biology  2005;3(4):e101.
The gene encoding the transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) was targeted in mice. PGC-1α null (PGC-1α−/−) mice were viable. However, extensive phenotyping revealed multi-system abnormalities indicative of an abnormal energy metabolic phenotype. The postnatal growth of heart and slow-twitch skeletal muscle, organs with high mitochondrial energy demands, is blunted in PGC-1α−/− mice. With age, the PGC-1α−/− mice develop abnormally increased body fat, a phenotype that is more severe in females. Mitochondrial number and respiratory capacity is diminished in slow-twitch skeletal muscle of PGC-1α−/− mice, leading to reduced muscle performance and exercise capacity. PGC-1α−/− mice exhibit a modest diminution in cardiac function related largely to abnormal control of heart rate. The PGC-1α−/− mice were unable to maintain core body temperature following exposure to cold, consistent with an altered thermogenic response. Following short-term starvation, PGC-1α−/− mice develop hepatic steatosis due to a combination of reduced mitochondrial respiratory capacity and an increased expression of lipogenic genes. Surprisingly, PGC-1α−/− mice were less susceptible to diet-induced insulin resistance than wild-type controls. Lastly, vacuolar lesions were detected in the central nervous system of PGC-1α−/− mice. These results demonstrate that PGC-1α is necessary for appropriate adaptation to the metabolic and physiologic stressors of postnatal life.
Eliminating the activity of the gene PGC-1 α in mice reveals its role in post-natal metabolism and provides a link to obesity and some intriguing differences with another report of this knockout
PMCID: PMC1064854  PMID: 15760270

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