Phospholamban Mutation Research Articles

The phenotypic characteristic observed by cardiac magnetic resonance in a PLN-R14del family

Phospholamban (PLN) is an important regulator for sarcoendoplasmic reticulum (SR) calcium transport ATPase (SERCA), which uptakes Ca2+ to SR during the diastolic phase of cardiomyocytes to maintain intracellular calcium homeostasis. Mutations on PLN result in intracellular calcium disorder, myocardial contraction defect, and eventually heart failure and/or malignant ventricular arrhythmia. Since 2003, several kinds of PLN mutations have been identified in familial dilated cardiomyopathy (DCM) patients, illustrating a few clinical characteristics that differs from classical DCM patients. Herein, we report a large PLN-R14del family with typical clinical characteristics reported including relatively late-onset clinical symptoms, low-voltage in ECG, as well as frequent ventricular arrythmias. Moreover, members underwent cardiac magnetic resonance (CMR) examination showed a strikingly similar pattern of late gadolinium enhancement (LGE)—Sub-epicardial involvement in the left ventricular (LV) lateral wall with or without linear mid-wall enhancement in the interventricular septum. The former one can also present in younger PLN-R14del carriers despite completely normal LV structure and function. Meanwhile, T1 mapping also found significantly increased extracellular volume (ECV) in PLN-R14del carriers. These findings highlight the special role of CMR to phenotyping PLN-induced cardiomyopathy patients and distinguish them from other types of cardiomyopathy.

Computer versus cardiologist: Is a machine learning algorithm able to outperform an expert in diagnosing a phospholamban p.Arg14del mutation on the electrocardiogram?

Phospholamban (PLN) p.Arg14del mutation carriers are known to develop dilated and/or arrhythmogenic cardiomyopathy, and typical electrocardiographic (ECG) features have been identified for diagnosis. Machine learning is a powerful tool used in ECG analysis and has shown to outperform cardiologists. We aimed to develop machine learning and deep learning models to diagnose PLN p.Arg14del cardiomyopathy using ECGs and evaluate their accuracy compared to an expert cardiologist. We included 155 adult PLN mutation carriers and 155 age- and sex-matched control subjects. Twenty-one PLN mutation carriers (13.4%) were classified as symptomatic (symptoms of heart failure or malignant ventricular arrhythmias). The data set was split into training and testing sets using 4-fold cross-validation. Multiple models were developed to discriminate between PLN mutation carriers and control subjects. For comparison, expert cardiologists classified the same data set. The best performing models were validated using an external PLN p.Arg14del mutation carrier data set from Murcia, Spain (n = 50). We applied occlusion maps to visualize the most contributing ECG regions. In terms of specificity, expert cardiologists (0.99) outperformed all models (range 0.53-0.81). In terms of accuracy and sensitivity, experts (0.28 and 0.64) were outperformed by all models (sensitivity range 0.65-0.81). T-wave morphology was most important for classification of PLN p.Arg14del carriers. External validation showed comparable results, with the best model outperforming experts. This study shows that machine learning can outperform experienced cardiologists in the diagnosis of PLN p.Arg14del cardiomyopathy and suggests that the shape of the T wave is of added importance to this diagnosis.

Phosphoproteomic analysis identifies phospho-Threonine-17 site of phospholamban important in low molecular weight isoform of fibroblast growth factor 2-induced protection against post-ischemic cardiac dysfunction

RationaleAmong its many biological roles, fibroblast growth factor 2 (FGF2) protects the heart from dysfunction and damage associated with an ischemic attack. Our laboratory demonstrated that its protection against myocardial dysfunction occurs by the low molecular weight (LMW) isoform of FGF2, while the high molecular weight (HMW) isoforms are associated with a worsening in post-ischemic recovery of cardiac function. LMW FGF2-mediated cardioprotection is facilitated by activation of multiple kinases, including PKCalpha, PKCepsilon, and ERK, and inhibition of p38 and JNK. ObjectiveYet, the substrates of those kinases associated with LMW FGF2-induced cardioprotection against myocardial dysfunction remain to be elucidated. Methods and resultsTo identify substrates in LMW FGF2 improvement of post-ischemic cardiac function, mouse hearts expressing only LMW FGF2 were subjected to ischemia-reperfusion (I/R) injury and analyzed by a mass spectrometry (MS)-based quantitative phosphoproteomic strategy. MS analysis identified 50 phosphorylation sites from 7 sarcoendoplasmic reticulum (SR) proteins that were significantly altered in I/R-treated hearts only expressing LMW FGF2 compared to those hearts lacking FGF2. One of those phosphorylated SR proteins identified was phospholamban (PLB), which exhibited rapid, increased phosphorylation at Threonine-17 (Thr17) after I/R in hearts expressing only LMW FGF2; this was further validated using Selected Reaction Monitoring-based MS workflow. To demonstrate a mechanistic role of phospho-Thr17 PLB in LMW FGF2-mediated cardioprotection, hearts only expressing LMW FGF2 and those expressing only LMW FGF2 with a mutant PLB lacking phosphorylatable Thr17 (Thr17Ala PLB) were subjected to I/R. Hearts only expressing LMW FGF2 showed significantly improved recovery of cardiac function following I/R (p < 0.05), and this functional improvement was significantly abrogated in hearts expressing LMW FGF2 and Thr17Ala PLB (p < 0.05). ConclusionThe findings indicate that LMW FGF2 modulates intracellular calcium handling/cycling via regulatory changes in SR proteins essential for recovery from I/R injury, and thereby protects the heart from post-ischemic cardiac dysfunction.

Abstract 530: Mechanisms Underlying Phospholamban L39 Stop (PLN L39X) Cardiomyopathy

Background: Defective calcium (Ca++) handling is a hallmark of HF across species. Together with the Sarco/Endoplasmic Reticulum Ca-ATPase (SERCA2a), Phospholamban (PLN) has emerged as a critical regulator of Ca++homeostasis. Worldwide, PLN mutations are identified with increasing frequency in patients with dilated, hypertrophic and arrhythmogenic cardiomyopathy (CMPs) but the causative defects leading to the CMP remain incompletely understood. While preclinical studies have unequivocally shown that absence of PLN (PLNKO) is therapeutic in rodent HF models, the discovery of a human pathogenic mutation (L39X) presumed to be the human equivalent of the PLNKO lead to the conclusion that PLN ablation was lethal in human, mitigating any enthusiasm in targeting PLN inhibition in PLN-associated disease or HF treatment. The objective of this proposal is to levarge the use of “induced-pluripotent” stem cells (iPSCs) derived cardiomyocytes (CM) from homozygote L39X carriers to elucidate the role of PLN (L39) in human pathophysiology. Methodology and Results: We obtained mononuclear cells from Homozygotes (Hom) L39 carriers and generated 11 iPSC clones. To derive CMs, we used the direct differentiation method temporally modulating the Wnt/β-catenin signaling. Immunocytostaining revealed positive expression of cardiac troponin T as well as PLN. In Hom L39 derived CMs, PLN showed an abnormal cytoplasmic distribution, formed intracellular aggregates and there was loss of perinuclear localization when compared to matched WT iPSC-CMs. Using fura-2AM, we observed decreased calcium transient amplitude in iPSC-CMs from L39 compared to WT with prolongation of the time constant of relaxation and early after depolarization (EAD). Lastly, we saw a 70% and 50% reduction in the protein and mRNA expression of PLN and SERCA2a respectively. Conclusions: Our data suggest that the L39 PLN mutant is expressed but mis-located within the cardiomyocytes. The mis-location of PLN was associated to decreased SERCA2a expression impaired Ca++ handling and increased arrhythmogenicity. Further studies will be required to fully elucidate the impact of the mutation in HF pathophysiology

Predicting sustained ventricular arrhythmias in dilated cardiomyopathy: a meta-analysis and systematic review.

AimsPatients with non‐ischaemic dilated cardiomyopathy (DCM) are at increased risk of sudden cardiac death. Identification of patients that may benefit from implantable cardioverter‐defibrillator implantation remains challenging. In this study, we aimed to determine predictors of sustained ventricular arrhythmias in patients with DCM.Methods and resultsWe searched MEDLINE/Embase for studies describing predictors of sustained ventricular arrhythmias in patients with DCM. Quality and bias were assessed using the Quality in Prognostic Studies tool, articles with high risk of bias in ≥2 areas were excluded. Unadjusted hazard ratios (HRs) of uniformly defined predictors were pooled, while all other predictors were evaluated in a systematic review. We included 55 studies (11 451 patients and 3.7 ± 2.3 years follow‐up). Crude annual event rate was 4.5%. Younger age [HR 0.82; 95% CI (0.74–1.00)], hypertension [HR 1.95; 95% CI (1.26–3.00)], prior sustained ventricular arrhythmia [HR 4.15; 95% CI (1.32–13.02)], left ventricular ejection fraction on ultrasound [HR 1.45; 95% CI (1.19–1.78)], left ventricular dilatation (HR 1.10), and presence of late gadolinium enhancement [HR 5.55; 95% CI (4.02–7.67)] were associated with arrhythmic outcome in pooled analyses. Prior non‐sustained ventricular arrhythmia and several genotypes [mutations in Phospholamban (PLN), Lamin A/C (LMNA), and Filamin‐C (FLNC)] were associated with arrhythmic outcome in non‐pooled analyses. Quality of evidence was moderate, and heterogeneity among studies was moderate to high.ConclusionsIn patients with DCM, the annual event rate of sustained ventricular arrhythmias is approximately 4.5%. This risk is considerably higher in younger patients with hypertension, prior (non‐)sustained ventricular arrhythmia, decreased left ventricular ejection fraction, left ventricular dilatation, late gadolinium enhancement, and genetic mutations (PLN, LMNA, and FLNC). These results may help determine appropriate candidates for implantable cardioverter‐defibrillator implantation.

1041 Early detection of biventricular mechanical dysfunction in PLN R14del mutation carriers

Abstract Funding Acknowledgements PLN Genetic Heart Disease Foundation Background Carriers of the phospholamban (PLN) R14del founder mutation may develop an arrhythmogenic and/or dilated cardiomyopathy. Overt disease is preceded by a pre-symptomatic phase of variable length in which structural abnormalities seem to be absent. Purpose We aimed to explore echocardiographic characteristics of PLN R14del mutation carriers, particularly in early disease stages. Methods We included 120 PLN R14del mutation carriers and classified them to the pre-symptomatic stage (no symptoms and no structural disease, n = 60), the arrhythmic stage (arrhythmic symptoms and left ventricular ejection fraction (LVEF) ≥50%, n = 30) or the structural stage (LVEF &amp;lt;50%, n = 30). We included 60 healthy control subjects who were age- and gender matched with pre-symptomatic mutation carriers. All subjects underwent comprehensive echocardiographic analysis, including deformation imaging. Results Values are provided in the abstract table. Patients in the structural stage had significantly impaired left/right ventricular (LV/RV) function and increased LV/RV size when compared to the other mutation carriers (p &amp;lt; 0.001). In the pre-symptomatic and arrhythmic stage, LV function and volumes did not differ significantly from controls by conventional measurements. However, LV global longitudinal strain (GLS) and LV mechanical dispersion (MD) were already significantly impaired in the pre-symptomatic and arrhythmic stage when compared to controls (p &amp;lt; 0.001). RV function by conventional measurements was lower in arrhythmic subjects than in controls (p = 0.016). A strong linear correlation was found between LV GLS and RV GLS (r = 0.8, p &amp;lt; 0.001). Conclusion Echocardiographic deformation imaging reveals biventricular mechanical alterations in PLN R14del mutation carriers before arrhythmic symptoms and overt structural disease. Longitudinal studies are needed to determine the incremental prognostic value of these findings. Control subjects (n = 60) Pre-symptomatic (n = 60) Arrhythmic (n = 30) Strucutral (n = 30) LVEF(%) 60.3 ± 4.2 58.6 ± 4.2 57.1 ± 5.3 38.8 ± 10.6* LVEDV (ml/m2) 54.6 ± 10.0 53.7 ± 9.5 56.1 ± 11.6 70.1 ± 25.8* LV GLS (%) 21.5 ± 1.8 19.5 ± 1.5* 19.1 ± 1.7* 12.7 ± 3.8* LV MD (msec) 24.4 ± 5.9 33.1 ± 9.5* 48.2 ± 13.3* 60.8 ± 17.1* RV FAC (%) 46.4 ± 4.9 44.7 ± 5.0 42.6 ± 7.4* 33.2 ± 8.0* RV GLS (%) 26.5 ± 3.7 24.2 ± 2.9* 22.5 ± 4.2* 15.1 ± 5.7* FAC = Fractional area change; GLS = Global longitudinal strain; LV/RV = Left/right ventricular; LVEDV = Left ventricular end-diastolic volume; LVEF = Left ventricular ejection fraction; MD = Mechanical dispersion. *p &amp;lt; 0.05 when compared to control subjects. Abstract 1041 Figure. Clinical stages in PLN mutation carriers

P4373Post systolic shortening in the apex of the left ventricular is a typical finding in patients with PLN mutation

Abstract Introduction The p.Arg14del founder mutation in the gene encoding phospholamban (PLN) is associated with an increased risk of malignant ventricular arrhythmia and heart failure. Ejection fraction (EF) is the gold standard of systolic left ventricular (LV) function, however new technics using global longitudinal strain (GLS) are able to detect LV dysfunction early, e.g. in patients with chemotherapy. Moreover, strain analysis on post systolic shortening (PSS) can show distinct pattern in various diseases, e.g. patients with myocardial fibrosis and amyloid. The goal of this study was to investigate whether speckle tracking (GLS and PSS) of the LV can detect LV dysfunction in patients with PLN mutations and whether these patients have a specific PSS pattern. Method 72 Patients (49 female, mean age 32 years) diagnosed with the genetic PLN mutation p.Arg14del and 21 controls (10 female, mean age 27 years) underwent complete Echocardiographic exam including LVEF, GLS and PSI of all LV segments. LVEF &lt;50% was considered as LV dysfunction. The diagnostic characteristics of GLS and PSI were determined. Results Pearson correlation is in PLN patients between PSI apex and EF 0.677 (P &lt;.0001) PSI apex and GLS 0.692 (P &lt;.0001) PLN patients appeared to have a typical pattern with a high PSI in the apical segments. An apical PSI &gt;20 was the strongest diagnostic parameter indicating LV dysfunction. Apical PSI &gt;20 and LVEF &lt;50% have a sensitivity and specificity of 78% and 85%. Diagnostic characteristics PLN Controls P-value LVEF, % 55.2 59.3 0.033 GLS, % 15.5 17.3 0.163 PSI basal 8.5 10.1 0.326 PSI mid 5.6 2.9 0.070 PSI apical 15.6 2.1 0.01 LVEF, left ventricular ejection fraction; GLS, Global longitudinal strain; PSI, post systolic index. PSI Conclusion PLN patients have a typical pattern of high PSI in the apex of the left ventricular which can be used as a diagnostic test to detect LV dysfunction.

Delineating the Link Between Dilated Cardiomyopathy and Arrhythmogenic Symptoms

Dilated cardiomyopathy (DCM) is a common cause of heart failure and frequently involves life‐threatening ventricular arrhythmia and sudden cardiac death. In addition, DCM patients often show a heightened susceptibility to drug‐induced arrhythmia. The mechanisms that link DCM to ventricular arrhythmia are not well understood but are thought to involve structural alterations in the heart wall and in the ion channel makeup of individual cardiomyocytes. About 50 genetic mutations are known to cause congenital forms of DCM. Induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs) generated from patient tissue recapitulate many pathophysiological phenotypes characteristic of congenital heart disease and offer an unprecedented tool to study disease mechanisms. To model DCM, we used iPSC‐CMs harboring a DCM‐causing mutation in phospholamban (PLN), a protein involved in calcium regulation. Using the PLN mutant cells, we tested whether the disease would increase arrhythmic susceptibility to drugs that target certain ion channels. Initial experiments from my internship found that both Sodium and human Ether‐à‐go‐go Related channel agonists elicited an exacerbated proarrhythmic response from mutant cells. Action potential durations were disproportionately increased in the DCM‐afflicted mutants. If these data are sustained throughout the experiment, they support the model that the PLN mutant DCM involves a channelopathy that increases proarrhythmic sensitivity to drugs.Support or Funding InformationTM acknowledges support from the American Heart Association and the Stanford Cardiovascular Institute.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

3213 Unraveling the role of Phospholamban (PLN) in humans via the characterization of Induced Pluripotent Stem Cell (iPSC) Cardiomyocytes (CM) derived from carriers of a lethal PLN mutation

OBJECTIVES/SPECIFIC AIMS: To study the biology of Phosholamban (PLN) in a human relevant model. METHODS/STUDY POPULATION: State of the art stem-cell technologies using iPSC-CMs derived from carriers of a lethal PLN mutation. RESULTS/ANTICIPATED RESULTS: Our preliminary data demonstrate that this particular PLN mutation (L39) results in reduced expression and mis-localization of PLN as well as increased incidence of early after depolarization in isolated iPSC-CMs. DISCUSSION/SIGNIFICANCE OF IMPACT: Phospholamban (PLN) is a critical regulator of Ca++ homeostasis yet many uncertainties still remain regarding its role in humans. Our study will provide unique insights into the pathophysiology of this protein in HF.

The R9H phospholamban mutation is associated with highly penetrant dilated cardiomyopathy and sudden death in a spontaneous canine model

Causative mutations for familial dilated cardiomyopathy (DCM) have been identified in the phospholamban gene. There are many poorly understood aspects about familial DCM (variable penetrance, expression) which may be studied in natural animal models.We characterized genetic aspects of familial DCM in a canine model with a high incidence of sudden death. A missense G > A mutation in exon 1 of the phospholamban gene that changed an amino acid from arginine to histidine was identified in affected dogs. This variant was predicted to be deleterious.We describe a spontaneous canine model of familial DCM and sudden death with the R9H mutation. In comparison to a reported human family, the variant was highly penetrant and resulted in sudden death. Genetic penetrance of this mutation may be influenced by genetic or environmental modifiers. The dog provides an excellent model in which to study complex aspects of familial DCM.

Utilizing Empirical Data and Structural Dynamics Prediction to Optimize Rational Design of Therapeutic Phospholamban Mutations to Tune SERCA Function

Utilizing Empirical Data and Structural Dynamics Prediction to Optimize Rational Design of Therapeutic Phospholamban Mutations to Tune SERCA Function

Prevalence and cardiac phenotype of patients with aphospholamban mutation.

Pathogenic mutations in the phospholamban (PLN) gene may give rise to inherited cardiomyopathies due to its role in calcium homeostasis. Several PLN mutations have been identified, with the R14del mutation being the most prevalent cardiomyopathy-related mutation in the Netherlands. It is present in patients diagnosed with arrhythmogenic cardiomyopathy as well as dilated cardiomyopathy. Awareness of the phenotype of this PLN mutation is of great importance, since many carriers remain to be identified. Patients with the R14del mutation are characterised by older age at onset, low-voltage electrocardiograms and a high frequency of ventricular arrhythmias. Additionally, these patients have a poor prognosis often with left ventricular dysfunction and early-onset heart failure. Therefore, when there is a suspicion of a PLN mutation, cardiac and genetic screening is strongly recommended.

Functional and transcriptomic insights into pathogenesis of R9C phospholamban mutation using human induced pluripotent stem cell-derived cardiomyocytes

Dilated cardiomyopathy (DCM) can be caused by mutations in the cardiac protein phospholamban (PLN). We used CRISPR/Cas9 to insert the R9C PLN mutation at its endogenous locus into a human induced pluripotent stem cell (hiPSC) line from an individual with no cardiovascular disease. R9C PLN hiPSC-CMs display a blunted β-agonist response and defective calcium handling. In 3D human engineered cardiac tissues (hECTs), a blunted lusitropic response to β-adrenergic stimulation was observed with R9C PLN. hiPSC-CMs harboring the R9C PLN mutation showed activation of a hypertrophic phenotype, as evidenced by expression of hypertrophic markers and increased cell size and capacitance of cardiomyocytes. RNA-seq suggests that R9C PLN results in an altered metabolic state and profibrotic signaling, which was confirmed by gene expression analysis and picrosirius staining of R9C PLN hECTs. The expression of several miRNAs involved in fibrosis, hypertrophy, and cardiac metabolism were also perturbed in R9C PLN hiPSC-CMs. This study contributes to better understanding of the pathogenic mechanisms of the hereditary R9C PLN mutation in the context of human cardiomyocytes.

Effects of the Arg9Cys and Arg25Cys mutations on phospholamban's conformational equilibrium in membrane bilayers

Approximately, 70% of the Ca2+ ion transport into the sarcoplasmic reticulum is catalyzed by the sarcoplasmic reticulum Ca2+-ATPase (SERCA), whose activity is endogenously regulated by phospholamban (PLN). PLN comprises a TM inhibitory region and a cytoplasmic regulatory region that harbors a consensus sequence for cAMP-dependent protein kinase (PKA). The inhibitory region binds the ATPase, reducing its apparent Ca2+ binding affinity. β-adrenergic stimulation activates PKA, which phosphorylates PLN at Ser 16, reversing its inhibitory function. Mutations and post-translational modifications of PLN may lead to dilated cardiomyopathy (DCM) and heart failure. PLN's cytoplasmic region interconverts between a membrane-associated T state and a membrane-detached R state. The importance of these structural transitions on SERCA regulation is emerging, but the effects of natural occurring mutations and their relevance to the progression of heart disease are unclear. Here we use solid-state NMR spectroscopy to investigate the structural dynamics of two lethal PLN mutations, R9C and R25C, which lead to DCM. We found that the R25C mutant enhances the dynamics of PLN and shifts the conformational equilibrium toward the R state confirmation, whereas the R9C mutant drives the amphipathic cytoplasmic domain toward the membrane-associate state, enriching the T state population. The changes in membrane interactions caused by these mutations may explain the aberrant regulation of SERCA.

Abstract 185: Genome Editing of Isogenic Human Induced Pluripotent Stem Cells Allows for Functional and Transcriptomic Insights Into Hereditary Dilated Cardiomyopathy Caused by Phospholamban Mutations

Dilated cardiomyopathy (DCM) can be caused by genetic mutations in numerous cardiac proteins, including phospholamban (PLN). PLN mutations are quite rare and obtaining patient samples for mechanistic insights can be challenging. We used genome editing with CRISPR/Cas9 to successfully insert R14del and R9C PLN mutations into a human induced pluripotent stem cell (hiPSC) line from an individual with no cardiovascular disease. HiPSC-cardiomyocytes (hiPSC-CMs) with the inserted R14del PLN mutation recapitulate the phenotype observed in patient-derived R14del hiPSC-CMs, characterized by abnormal intracellular calcium cycling and arrhythmogenicity. Insertion of R9C PLN results in hiPSC-CMs displaying an abnormal response to β-agonists, defective calcium handling, and a hypertrophic phenotype. In human engineered cardiac tissues (hECTs) created from hiPSC-CMs in a 3D matrix, R14del results in a progressive worsening of developed force and R9C PLN demonstrates an abnormal lusitropic response following β-adrenergic stimulation. Further, transcriptional profiling using RNAseq suggests a role for lipid metabolism in R14del. DNA methylation studies showed that differentially expressed genes were enriched for lipoprotein metabolism and chylomicron-mediated lipid transport pathways in R14del PLN. This was also confirmed with the observation of lipid deposition in R14del hECTs and human myocardial tissues from explanted hearts of affected patients. Furthermore, small RNAseq identified 2 miRNAs that were differentially regulated in R14del hiPSC-CMs (miR-449c-5p and miR-483-3p). For R9C PLN, RNAseq suggests that the mutation results in profibrotic signaling, activation of autophagy, and an altered metabolic state. Our findings demonstrate that gene editing of hiPSCs can be used to successfully create models and delineate molecular mechanisms of human PLN mutations associated with DCM.

Distinct fibrosis pattern in desmosomal and phospholamban mutation carriers in hereditary cardiomyopathies

Desmosomal and phospholamban (PLN) mutations are associated with arrhythmogenic cardiomyopathy. Ultimately, most cardiomyopathic hearts develop significant cardiac fibrosis. To compare the fibrosis patterns of desmosomal and p. Arg14del PLN-associated cardiomyopathies with the pattern in hearts with other hereditary cardiomyopathies. A midventricular transversal slice was obtained from hearts of 30 patients with a cardiomyopathy with a known underlying mutation and from 8 controls. Fibrosis and fatty changes were quantitatively analyzed using digital microscopy. Hearts from patients with desmosomal mutations (n=6) showed fibrosis and fibrofatty replacement in the left ventricular (LV) outer myocardium, mainly in the posterolateral wall, and in the right ventricle. A similar phenotype, but with significantly more severe fibrotic changes in the LV, was found in the PLN mutation group (n = 8). Cardiomyopathies associated with lamin A/C (n= 5), sarcomeric (n = 8), and desmin (n = 3) mutations all showed a different pattern from that of the desmosomal and PLN mutation carriers. The posterolateral LV wall appeared to be the most discriminative area with fibrosis andfatty changes predominantly at the outer compact myocardium in 13 of 14 hearts with desmosomal and PLN mutations (93%), in 0 of 13 hearts with lamin A/C and sarcomeric mutations (0%), and in 1 of 3 desminopathic hearts (33%) (P<.001). Desmosomal- and PLN-associated cardiomyopathies have a fibrosis pattern distinct from the patterns in other hereditary cardiomyopathies. The posterolateral LV wall appeared to be the most discriminative region between mutation groups. These results may provide a roadmap for cardiac imaging interpretation and may help in further unraveling disease mechanisms.

NMR Resolves Phospholamban's Allosteric Regulation of the Sarcoplasmic Reticulum Ca2+-ATPase

Disruption of cardiac calcium cycling due to dysregulation of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) by the small transmembrane protein, phospholamban (PLN) leads to severe phenotypes including arrhythmia and dilated cardiomyopathy (DCM). In order to understand how inherited mutations in PLN lead to these severe phenotypes it is essential to map PLN's binding interaction with SERCA and how this binding interaction is modulated by post-translational modifications in PLN.Here we have applied both magic angle spinning (MAS) and oriented solid-state NMR techniques to characterize the structure, dynamics, and topology of monomeric constructs of PLN in complex with SERCA to better understand how post-translational modifications and mutations in PLN alter SERCA's activity. By combining chemical shift perturbations, dipolar couplings, and other structural restraints with molecular dynamics simulations, we have built a model of the PLN-SERCA complex that outlines a mechanism for PLN's allosteric regulation of SERCA via its cytoplasmic domain.

Influence of Genotype on Structural Atrial Abnormalities and Atrial Fibrillation or Flutter in Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is associated with desmosomal mutations. Although desmosomal disruption affects both ventricles and atria, little is known about atrial involvement in ARVD/C. To describe the extent and clinical significance of structural atrial involvement and atrial arrhythmias (AA) in ARVD/C stratified by genotype. We included 71 patients who met ARVD/C Task Force Criteria and underwent cardiac magnetic resonance (CMR) imaging and molecular genetic analysis. Indexed atrial end-diastolic volume and area-length-ejection-fraction (ALEF) were evaluated on CMR and compared to controls with idiopathic right ventricular outflow tract tachycardia (n = 40). The primary outcome was occurrence of AA (atrial fibrillation or atrial flutter) during follow-up, recorded by 12-lead ECG, Holter monitoring or implantable cardioverter defibrillator (ICD) interrogation. Patients harbored a desmosomal plakophilin-2 (PKP2) (n = 37) or nondesmosomal phospholamban (PLN) (n = 14) mutation. In 20 subjects, no pathogenic mutation was identified. Compared to controls, right atrial (RA) volumes were reduced in PKP2 (P = 0.002) and comparable in PLN (P = 0.441) mutation carriers. In patients with no mutation identified, RA (P = 0.011) and left atrial (P = 0.034) volumes were increased. Bi-atrial ALEF showed no significant difference between the groups. AA were experienced by 27% of patients and occurred equally among PKP2 (30%) and no mutation identified patients (30%), but less among PLN mutation carriers (14%). Genotype influences atrial volume and occurrence of AA in ARVD/C. While the incidence of AA is similar in PKP2 mutation carriers and patients with no mutation identified, PKP2 mutation carriers have significantly smaller atria. This suggests a different arrhythmogenic mechanism.

Mutation analysis of the phospholamban gene in 315 South Africans with dilated, hypertrophic, peripartum and arrhythmogenic right ventricular cardiomyopathies.

Cardiomyopathy is an important cause of heart failure in Sub-Saharan Africa, accounting for up to 30% of adult heart failure hospitalisations. This high prevalence poses a challenge in societies without access to resources and interventions essential for disease management. Over 80 genes have been implicated as a cause of cardiomyopathy. Mutations in the phospholamban (PLN) gene are associated with dilated cardiomyopathy (DCM) and severe heart failure. In Africa, the prevalence of PLN mutations in cardiomyopathy patients is unknown. Our aim was to screen 315 patients with arrhythmogenic right ventricular cardiomyopathy (n = 111), DCM (n = 95), hypertrophic cardiomyopathy (n = 40) and peripartum cardiomyopathy (n = 69) for disease-causing PLN mutations by high resolution melt analysis and DNA sequencing. We detected the previously reported PLN c.25C > T (p.R9C) mutation in a South African family with severe autosomal dominant DCM. Haplotype analysis revealed that this mutation occurred against a different haplotype background to that of the original North American family and was therefore unlikely to have been inherited from a common ancestor. No other mutations in PLN were detected (mutation prevalence = 0.2%). We conclude that PLN is a rare cause of cardiomyopathy in African patients. The PLN p.R9C mutation is not well-tolerated, emphasising the importance of this gene in cardiac function.

Combining Solution and Solid State NMR Techniques in the Analysis of Arg to Cys Mutations in Phospholamban Associated with Dilated Cardiomyopathy

Dilated cardiomyopathy (DCM) is characterized by the enlargement and weakening of the left ventricle. This dilation of the ventricle prevents the heart from contracting normally and can spread to the right ventricle and atria as the disease progresses. Inherited forms of DCM have been identified in families that carry missense, deletion, or nonsense mutations in the regulatory protein, phospholamban (PLN). Two of the identified missense mutations, R9C and R25C, result in cysteine substitutions for arginine residues in PLN's cytoplasmic domain. In vitro studies with these mutants indicate that they have an altered ability to regulate SERCA, however it is unclear how these mutations lead to dysregulation of SERCA.Here we have applied both solution and solid-state NMR techniques to characterize the structure, dynamics and topology of the monomeric constructs for the R9C and R25C mutants to better understand how these mutations affect PLN's regulation of SERCA in the development of DCM. These studies were pursued under both reducing and oxidizing conditions to better reflect the physiological conditions of DCM progression and for comparison to coupled enzyme activity assays used to quantify the effect of these mutants on SERCA activity. We have utilized chemical shift perturbations as sensitive probes toward the local environment of the cytoplasmic and juxta-membrane domains of PLN which harbor the aforementioned mutations. Dynamics in lipid bilayers have been probed through cross-polarization or J-coupling enabled experiments which are exclusively sensitive toward either the rigid or mobile regions of the protein. Heteronuclear NMR was employed to probe the fast timescale dynamics of these mutants. Finally, separated local field experiments in magnetically aligned bicelles were employed to probe the topology.