Julia Münch

FHL2 expression and variants in hypertrophic cardiomyopathy

Based on evidence that FHL2 (four and a half LIM domains protein 2) negatively regulates cardiac hypertrophy we tested whether FHL2 altered expression or variants could be associated with hypertrophic cardiomyopathy (HCM). HCM is a myocardial disease characterized by left ventricular hypertrophy, diastolic dysfunction and increased interstitial fibrosis and is mainly caused by mutations in genes coding for sarcomeric proteins. FHL2 mRNA level, FHL2 protein level and I-band-binding density were lower in HCM patients than control individuals. Screening of 121 HCM patients without mutations in established disease genes identified 2 novel (T171M, V187L) and 4 known (R177Q, N226N, D268D, P273P) FHL2 variants in unrelated HCM families. We assessed the structural and functional consequences of the nonsynonymous substitutions after adeno-associated viral-mediated gene transfer in cardiac myocytes and in 3D-engineered heart tissue (EHT). Overexpression of FHL2 wild type or nonsynonymous substitutions in cardiac myocytes markedly down-regulated α-skeletal actin and partially blunted hypertrophy induced by phenylephrine or endothelin-1. After gene transfer in EHTs, force and velocity of both contraction and relaxation were higher with T171M and V187L FHL2 variants than wild type under basal conditions. Finally, chronic phenylephrine stimulation depressed EHT function in all groups, but to a lower extent in T171M-transduced EHTs. These data suggest that (1) FHL2 is down-regulated in HCM, (2) both FHL2 wild type and variants partially protected phenylephrine- or endothelin-1-induced hypertrophy in cardiac myocytes, and (3) FHL2 T171M and V187L nonsynonymous variants induced altered EHT contractility. These findings provide evidence that the 2 novel FHL2 variants could increase cardiac function in HCM.

The E3 ubiquitin ligase Asb2β is downregulated in a mouse model of hypertrophic cardiomyopathy and targets desmin for proteasomal degradation

BACKGROUND Hypertrophic cardiomyopathy (HCM) is an autosomal-dominant disease with mutations in genes encoding sarcomeric proteins. Previous findings suggest deregulation of the ubiquitin proteasome system (UPS) in HCM in humans and in a mouse model of HCM (Mybpc3-targeted knock-in (KI) mice). In this study we investigated transcript levels of several muscle-specific E3 ubiquitin ligases in KI mice and aimed at identifying novel protein targets. METHODS AND RESULTS Out of 9 muscle-specific E3 ligases, Asb2β was found with the lowest mRNA level in KI compared to wild-type (WT) mice. After adenoviral-mediated Asb2β transduction of WT neonatal mouse cardiomyocytes with either a WT or inactive Asb2β mutant, desmin was identified as a new target of Asb2β by mass spectrometry, co-immunoprecipitation and immunoblotting. Immunofluorescence analysis revealed a co-localization of desmin with Asb2β at the Z-disk of the sarcomere. Knock-down of Asb2β in cardiomyocytes resulted in higher desmin protein levels. Furthermore, desmin levels were higher in ventricular samples of HCM mice and patients than controls. CONCLUSIONS This study identifies desmin as a new Asb2β target for proteasomal degradation in cardiomyocytes and suggests that accumulation of desmin could contribute to UPS impairment in HCM mice and patients.

Evaluation of MYBPC3 trans-Splicing and Gene Replacement as Therapeutic Options in Human iPSC-Derived Cardiomyocytes

Gene therapy is a promising option for severe forms of genetic diseases. We previously provided evidence for the feasibility of trans-splicing, exon skipping, and gene replacement in a mouse model of hypertrophic cardiomyopathy (HCM) carrying a mutation in MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C). Here we used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from an HCM patient carrying a heterozygous c.1358-1359insC MYBPC3 mutation and from a healthy donor. HCM hiPSC-CMs exhibited ∼50% lower MYBPC3 mRNA and cMyBP-C protein levels than control, no truncated cMyBP-C, larger cell size, and altered gene expression, thus reproducing human HCM features. We evaluated RNA trans-splicing and gene replacement after transducing hiPSC-CMs with adeno-associated virus. trans-splicing with 5′ or 3′ pre-trans-splicing molecules represented ∼1% of total MYBPC3 transcripts in healthy hiPSC-CMs. In contrast, gene replacement with the full-length MYBPC3 cDNA resulted in ∼2.5-fold higher MYBPC3 mRNA levels in HCM and control hiPSC-CMs. This restored the cMyBP-C level to 81% of the control level, suppressed hypertrophy, and partially restored gene expression to control level in HCM cells. This study provides evidence for (1) the feasibility of trans-splicing, although with low efficiency, and (2) efficient gene replacement in hiPSC-CMs with a MYBPC3 mutation.

Serum Matrix Metalloproteinases as Quantitative Biomarkers for Myocardial Fibrosis and Sudden Cardiac Death Risk Stratification in Patients With Hypertrophic Cardiomyopathy

BACKGROUND Hypertrophic cardiomyopathy (HCM) is associated with an increased risk of sudden cardiac death due to ventricular tachycardia (VT), and myocardial fibrosis reflects an important risk factor. Several matrix metalloproteinases (MMPs) and procollagen N-terminal propeptides (PNPs) are involved in collagen turnover and discussed as fibrosis biomarkers. We aimed to identify biomarkers that correlate with myocardial fibrosis in late-gadolinium-enhancement cardiac magnetic resonance imaging (LGE-CMR) and/or cardiac events (syncope, VT) in HCM patients. METHODS AND RESULTS In 54 HCM patients (age 55.9 ± 14.3 y, 50% female) fibrosis was quantified by LGE-CMR. Serum concentrations of MMP-1, -2, -3, -9, and tissue inhibitor of MMP (TIMP) 1 were analyzed by means of enzyme-linked immunosorbent assay and PINP, PIIINP, and type I collagen C-terminal telopeptide (ICTP) concentrations by radioimmunoassay. MMP-9 was associated with fibrosis in LGE-CMR (mean increase 0.66 g/unit MMP9 [95% confidence interval [CI] 0.50-0.82]; P < .001) and with cardiac events in women (odds ratio [OR] 1.07 [1.01-1.12], P = .01) but not in men. Increased MMP-2 levels in women were associated with lower fibrosis (0.05 [-0.09 to -0.01]; P = .015). MMP-3 levels were positively associated with cardiac events (OR 1.13 [1.05-1.22]; P = .001) independently from fibrosis and sex. No association was detected for MMP-1, TIMP-1, PNPs, and ICTP. CONCLUSIONS These data suggest that MMP-9 is a useful biomarker for fibrosis and cardiac events in female HCM patients, whereas MMP-3 is associated with a higher event rate independent from fibrosis and sex.

Prediction of the estimated 5-year risk of sudden cardiac death and syncope or non-sustained ventricular tachycardia in patients with hypertrophic cardiomyopathy using late gadolinium enhancement and extracellular volume CMR

AbstractObjectivesTo evaluate the ability of late gadolinium enhancement (LGE) and mapping cardiac magnetic resonance (CMR) including native T1 and global extracellular volume (ECV) to identify hypertrophic cardiomyopathy (HCM) patients at risk for sudden cardiac death (SCD) and to predict syncope or non-sustained ventricular tachycardia (VT).MethodsA 1.5-T CMR was performed in 73 HCM patients and 16 controls. LGE size was quantified using the 3SD, 5SD and full width at half maximum (FWHM) method. T1 and ECV maps were generated by a 3(3)5 modified Look-Locker inversion recovery sequence. Receiver-operating curve analysis evaluated the best parameter to identify patients with increased SCD risk ≥4% and patients with syncope or non-sustained VT.ResultsGlobal ECV was the best predictor of SCD risk with an area under the curve (AUC) of 0.83. LGE size was significantly inferior to global ECV with an AUC of 0.68, 0.70 and 0.70 (all P < 0.05) for 3SD-, 5SD- and FWHM-LGE, respectively. Combined use of the SCD risk score and global ECV significantly improved the diagnostic accuracy to identify HCM patients with syncope or non-sustained VT.ConclusionsCombined use of the SCD risk score and global ECV has the potential to improve HCM patient selection, benefiting most implantable cardioverter defibrillators.Key Points• Global ECV identified the best HCM patients with increased SCD risk. • Global ECV performed equally well compared to a SCD risk score. • Combined use of the SCD risk score and global ECV improved test accuracy. • Combined use potentially improves selection of HCM patients for ICD implantation.

Early segmental relaxation abnormalities in hypertrophic cardiomyopathy for differential diagnostic of patients with left ventricular hypertrophy

Hypertrophic cardiomyopathy (HCM) is characterized by asymmetric left ventricular hypertrophy (LVH). However, clinical signs can be subtle and differentiation from other causes of LVH is challenging.

Comparison of global extracellular volume (ECV) and late gadolinium enhancement (LGE) to predict the estimated 5 year risk of sudden cardiac death (SCD) in patients with hypertrophic cardiomyopathy (HCM)

University Medical Center Hamburg-Eppendorf, Hamburg, Germany Full list of author information is available at the end of the article Figure 1 Correlation of global ECV (R = 0.68, p < 0.01) and global LGE with the SCD Risk Score (R = 0.39, p = 0.08). Dashed lines define a 95% confidence interval. Continuous line represents the fitting line. Avanesov et al. Journal of Cardiovascular Magnetic Resonance 2016, 18(Suppl 1):P127 http://www.jcmr-online.com/content/18/S1/P127

Orthostatic Blood Pressure Test for Risk Stratification in Patients with Hypertrophic Cardiomyopathy

Background Hypertrophic cardiomyopathy (HCM) is the most common cause of sudden cardiac death (SCD) in young adults, mainly ascribed to ventricular tachycardia (VT). Assuming that VT is the major cause of (pre-) syncope in HCM patients, its occurrence is essential for SCD risk stratification and primarily preventive ICD-implantation. However, evidence of VT during syncope is often missing. As the differentiation of potential lethal causes for syncope such as VT from more harmless reasons is crucial, HCM patients were screened for orthostatic dysregulation by using a simple orthostatic blood pressure test. Methods Over 15 months (IQR [9;20]) 100 HCM patients (55.8±16.2 yrs, 61% male) were evaluated for (pre-)syncope and VT (24h-ECGs, device-memories) within the last five years. Eighty patients underwent an orthostatic blood pressure test. Logistic regression models were used for statistical analysis. Results In older patients (>40 yrs) a positive orthostatic test result increased the chance of (pre-) syncope by a factor of 63 (95%-CI [8.8; 447.9], p<0.001; 93% sensitivity, 95%-CI [76; 99]; 74% specificity, 95%-CI [58; 86]). No correlation with VT was shown. A prolonged QTc interval also increased the chance of (pre-) syncope by a factor of 6.6 (95%-CI [2.0; 21.7]; p=0.002). Conclusions The orthostatic blood pressure test is highly valuable for evaluation of syncope and presyncope especially in older HCM patients, suggesting that orthostatic syncope might be more relevant than previously assumed. Considering the high complication rates due to ICD therapies, this test may provide useful information for the evaluation of syncope in individual risk stratification and may help to prevent unnecessary device implantations, especially in older HCM patients.

Extent of late gadolinium enhancement in patients with hypertrophic cardiomyopathy in correlation with serum MMP9 as an indicator of myocardial fibrosis

Results Nine out of 50 patients with HCM (18 %) showed no fibrosis on LGE-CMR. In the remaining 41 patients, mean size of fibrosis was 13,3 ± 10,3% on LGE-CMR. In all patients, the mean MMP9 level was 54,4 ± 35,2 ng/ ml. Size of fibrosis on LGE-CMR strongly correlated with MMP9 levels (R2 = 0,557, Pearson’s r = 0,75, p < 0,01). In the 9 patients with no LGE, MMP9 was with 29,6 ± 14,2 ng/ml significantly lower compared to the 41 patients with LGE and levels of 59,8 ± 36,2 ng/ml (p = 0,01).