Fotis Kolokathis

Human phospholamban null results in lethal dilated cardiomyopathy revealing a critical difference between mouse and human

In human disease and experimental animal models, depressed Ca(2+) handling in failing cardiomyocytes is widely attributed to impaired sarcoplasmic reticulum (SR) function. In mice, disruption of the PLN gene encoding phospholamban (PLN) or expression of dominant-negative PLN mutants enhances SR and cardiac function, but effects of PLN mutations in humans are unknown. Here, a T116G point mutation, substituting a termination codon for Leu-39 (L39stop), was identified in two families with hereditary heart failure. The heterozygous individuals exhibited hypertrophy without diminished contractile performance. Strikingly, both individuals homozygous for L39stop developed dilated cardiomyopathy and heart failure, requiring cardiac transplantation at ages 16 and 27. An over 50% reduction in PLN mRNA and no detectable PLN protein were noted in one explanted heart. The expression of recombinant PLN-L39stop in human embryonic kidney (HEK) 293 cells and adult rat cardiomyocytes showed no PLN inhibition of SR Ca(2+)-ATPase and the virtual absence of stable PLN expression; where PLN was expressed, it was misrouted to the cytosol or plasma membrane. These findings describe a naturally-occurring loss-of-function human PLN mutation (PLN null). In contrast to reported benefits of PLN ablation in mouse heart failure, humans lacking PLN develop lethal dilated cardiomyopathy.

A mutation in the human phospholamban gene, deleting arginine 14, results in lethal, hereditary cardiomyopathy

The sarcoplasmic reticulum Ca2+-cycling proteins are key regulators of cardiac contractility, and alterations in sarcoplasmic reticulum Ca2+-cycling properties have been shown to be causal of familial cardiomyopathies. Through genetic screening of dilated cardiomyopathy patients, we identified a previously uncharacterized deletion of arginine 14 (PLN-R14Del) in the coding region of the phospholamban (PLN) gene in a large family with hereditary heart failure. No homozygous individuals were identified. By middle age, heterozygous individuals developed left ventricular dilation, contractile dysfunction, and episodic ventricular arrhythmias, with overt heart failure in some cases. Transgenic mice overexpressing the mutant PLN-R14Del recapitulated human cardiomyopathy exhibiting similar histopathologic abnormalities and premature death. Coexpression of the normal and mutant-PLN in HEK-293 cells resulted in sarcoplasmic reticulum Ca2+-ATPase superinhibition. The dominant effect of the PLN-R14Del mutation could not be fully removed, even upon phosphorylation by protein kinase A. Thus, by chronic suppression of sarcoplasmic reticulum Ca2+-ATPase activity, the nonreversible superinhibitory function of mutant PLN-R14Del may lead to inherited dilated cardiomyopathy and premature death in both humans and mice.

Correction of human phospholamban R14del mutation associated with cardiomyopathy using targeted nucleases and combination therapy

A number of genetic mutations is associated with cardiomyopathies. A mutation in the coding region of the phospholamban (PLN) gene (R14del) is identified in families with hereditary heart failure. Heterozygous patients exhibit left ventricular dilation and ventricular arrhythmias. Here we generate induced pluripotent stem cells (iPSCs) from a patient harbouring the PLN R14del mutation and differentiate them into cardiomyocytes (iPSC-CMs). We find that the PLN R14del mutation induces Ca2+ handling abnormalities, electrical instability, abnormal cytoplasmic distribution of PLN protein and increases expression of molecular markers of cardiac hypertrophy in iPSC-CMs. Gene correction using transcription activator-like effector nucleases (TALENs) ameliorates the R14del-associated disease phenotypes in iPSC-CMs. In addition, we show that knocking down the endogenous PLN and simultaneously expressing a codon-optimized PLN gene reverses the disease phenotype in vitro. Our findings offer novel strategies for targeting the pathogenic mutations associated with cardiomyopathies.

The Ser96Ala variant in histidine-rich calcium-binding protein is associated with life-threatening ventricular arrhythmias in idiopathic dilated cardiomyopathy.

Aims To investigate whether genetic variants of the histidine-rich calcium (HRC)-binding protein are associated with idiopathic dilated cardiomyopathy (DCM) and its progression. Methods and results We screened 123 idiopathic DCM patients and 96 healthy individuals by single-strand conformation polymorphism analysis and direct sequencing for genetic variants in HRC. Six polymorphisms were detected: Leu35Leu (A/G), Ser43Asn (G/A), Ser96Ala (T/G), Glu202_Glu203insGlu (−/GAG), Asp261del (GAT/−), and an in-frame insertion of 51 amino acids at His321. The analysis of their frequencies did not reveal any significant correlation with DCM development. However, the Ser96Ala polymorphism exhibited a statistically significant correlation with the occurrence of life-threatening ventricular arrhythmias. During a follow-up of 4.02 ± 2.4 years, the risk for ventricular arrhythmias was higher (HR, 9.620; 95% CI, 2.183–42.394; P = 0.003) in the Ala/Ala patients, compared with Ser/Ser homozygous patients. On multivariable Cox regression analysis, the Ser96Ala polymorphism was the only significant genetic arrythmogenesis predictor in DCM patients (HR, 4.191; 95% CI, 0.838–20.967; P = 0.018). Conclusion The Ser96Ala genetic variant of HRC is associated with life-threatening ventricular arrhythmias in idiopathic DCM and may serve as an independent predictor of susceptibility to arrhythmogenesis in the setting of DCM.

Antiinflammatory Effects of Cardiac Resynchronization Therapy in Patients with Chronic Heart Failure

Background: Cardiac resynchronization therapy (CRT) pacing has been proposed as an additional treatment to medical therapy to improve heart failure patients with left ventricular asynchrony. The aim of this study was to evaluate the influence of CRT treatment on proinflammatory cytokines in patients with heart failure.

Serum markers of deranged myocardial collagen turnover: their relation to malignant ventricular arrhythmias in cardioverter-defibrillator recipients with heart failure.

BACKGROUND Pathologic collagen remodeling has been involved in the occurrence of ventricular arrhythmias and sudden cardiac death in heart failure. The aim of the study was to investigate the relationship between malignant ventricular arrhythmias and cardiac collagen turnover indexes, expressing specific types of derangement in collagen physiology, in stable patients with an implantable cardioverter-defibrillator (ICD). METHODS Seventy-four patients with an ICD and heart failure were studied. They had coronary artery disease (n = 42) or dilated cardiomyopathy, New York Heart Association classes I and II, and left ventricular ejection fraction 29% ± 1%. An ICD had been implanted for secondary (n = 36) or primary prevention of sudden cardiac death. We assessed (1) markers of collagen types I and III synthesis and their ratio: procollagen type I carboxyterminal peptide (PICP), procollagen type III aminoterminal peptide (PIIINP), and PICP/PIIINP; (2) markers of collagen degradation, degradation inhibition, and their ratio: matrix metalloproteinase 9 (MMP-9), tissue inhibitor of matrix metalloproteinase (TIMP) 1 (TIMP-1), and MMP-9/TIMP-1. Patients were prospectively followed up for 1 year. The number of episodes necessitating appropriate interventions for ventricular tachyarrhythmias (>170 beat/min) was related to the assessed parameters. RESULTS Multivariate analysis revealed a significant relation between the number of tachyarrhythmic episodes and MMP-9/TIMP-1 (P = .007), PICP/PIIINP (P = .007), and ejection fraction (P = .04). No other significant relation was observed between arrhythmias and the remaining parameters. CONCLUSION In heart failure, biochemical markers indicative of a deranged equilirium in myocardial collagen deposition/degradation and collagen I/III synthesis are related to ventricular arrhythmogenesis. Further studies are needed to investigate their predictive ability.

A human phospholamban promoter polymorphism in dilated cardiomyopathy alters transcriptional regulation by glucocorticoids

Depressed calcium handling by the sarcoplasmic reticulum (SR) Ca‐ATPase and its regulator phospholamban (PLN) is a key characteristic of human and experimental heart failure. Accumulating evidence indicates that increases in the relative levels of PLN to Ca‐ATPase in failing hearts and resulting inhibition of Ca sequestration during diastole, impairs contractility. Here, we identified a genetic variant in the PLN promoter region, which increases its expression and may serve as a genetic modifier in dilated cardiomyopathy (DCM). The variant AF177763.1:g.203A>C (at position –36 bp relative to the PLN transcriptional start site) was found only in the heterozygous form in 1 out of 296 normal subjects and in 22 out of 381 cardiomyopathy patients (heart failure at age of 18–44 years, ejection fraction=22±9%). In vitro analysis, using luciferase as a reporter gene in rat neonatal cardiomyocytes, indicated that the PLN‐variant increased activity by 24% compared to the wild type. Furthermore, the g.203A>C substitution altered the specific sequence of the steroid receptor for the glucocorticoid nuclear receptor (GR)/transcription factor in the PLN promoter, resulting in enhanced binding to the mutated DNA site. These findings suggest that the g.203A>C genetic variant in the human PLN promoter may contribute to depressed contractility and accelerate functional deterioration in heart failure. Hum Mutat 29(5), 640–647, 2008.

Relationship between plasma osteopontin and oxidative stress in patients with coronary artery disease

Background: It is known that oxidative stress plays an important role in the pathogenesis of atherosclerosis and that an association exists between osteopontin (OPN) and atherosclerosis. Objectives: It was proposed that malondialdehyde (MDA), a biomarker of lipid peroxidation and oxidative stress, would be related to plasma OPN levels in patients with coronary artery disease (CAD). Methods/results: Plasma OPN and MDA levels were measured in 71 patients (60 males and 11 females; mean age 61.7 ± 10 years). Fifty-eight patients had significant CAD (group I) and 13 patients were free of CAD as defined angiographically (group II). Plasma OPN was measured by enzyme-linked immunosorbent assay (ELISA), while MDA was determined spectrophotometrically. Multivariate regression analysis revealed that ln-transformed OPN levels were independently associated with MDA after adjustment for age, hypertension and diabetes mellitus (R2 = 0.278, p = 0.0004 and β regression coefficient = 0.252 [standard error = 0.0958], p = 0.011). OPN and MDA levels were higher in patients with diabetes (73.6 ± 36.2 ng/ml versus 56.1 ± 30.9 ng/ml, p = 0.02 and 2.5 ± 0.5 μM versus 2.0 ± 0.5 μM, p = 0.002, respectively). Conclusions: The association between OPN and MDA levels in patients with CAD suggests an interaction between OPN and oxidative stress. This interaction may play a role in the pathogenesis of atherosclerosis.

Effect of biventricular pacing on heart rate variability in patients with chronic heart failure

Biventricular pacing is emerging as a long‐term therapy for symptomatic heart failure. Analysis of heart rate variability (HRV) has become an important predictive tool in this syndrome.

Cytokine gene polymorphisms are associated with markers of disease severity and prognosis in patients with idiopathic dilated cardiomyopathy

AIMS To identify potential genetic associations of five cytokine gene polymorphisms with disease severity and prognosis in patients with idiopathic dilated cardiomyopathy (DCM). METHODS AND RESULTS Eighty patients with DCM were genotyped for transforming growth factor beta1 (TGF-β1)+869 T/C (codon10 Leu→Pro), TGF-β1 +915 G/C (codon25 Arg→Pro), interleukin (IL)-6 -174G/C, tumor necrosis factor-alpha (TNF-α) -308A/G, interferon-gamma (IFN-γ) +874T/A, IL-10 -1082A/G, IL-10 -819T/C and IL-10 -592A/C gene polymorphisms. In homozygous TT patients for TGF-β1 +869 T/C polymorphism mean VO(2) max was significantly higher than in CC homozygous patients (25.67±6.73ml/kg/min vs. 20.29±6.35 ml/kg/min, p = 0.046), which remained significant only for patients younger than 39 years old after adjusting for age and sex (p = 0.009). C carriers of TGF-β1 +915 G/C polymorphism are 4.2 times more likely to be in a worse NYHA stage (III-IV) than non C carriers [OR: 4.25, 95% CI (1.53-11.80), p = 0.006]. Patients GG homozygous for IL-6 -174G/C polymorphism presented greater left ventricle end-systolic (p = 0.018) and end-diastolic (p = 0.04) diameters in comparison to the CC homozygous. The AA homozygote for IFN-γ +874T/A polymorphism (p = 0.02) and the combination of the TGF-β1 +869 T/C and TGF-β1 +915 G/C genotypes were associated with adverse outcome (p = 0.014). CONCLUSION Specific cytokine gene polymorphisms seem to be associated with worse prognosis as well as with measures of disease severity in DCM.