Sandra Ruppenthal

Dantrolene rescues arrhythmogenic RYR2 defect in a patient-specific stem cell model of catecholaminergic polymorphic ventricular tachycardia

Coordinated release of calcium (Ca2+) from the sarcoplasmic reticulum (SR) through cardiac ryanodine receptor (RYR2) channels is essential for cardiomyocyte function. In catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited disease characterized by stress‐induced ventricular arrhythmias in young patients with structurally normal hearts, autosomal dominant mutations in RYR2 or recessive mutations in calsequestrin lead to aberrant diastolic Ca2+ release from the SR causing arrhythmogenic delayed after depolarizations (DADs). Here, we report the generation of induced pluripotent stem cells (iPSCs) from a CPVT patient carrying a novel RYR2 S406L mutation. In patient iPSC‐derived cardiomyocytes, catecholaminergic stress led to elevated diastolic Ca2+ concentrations, a reduced SR Ca2+ content and an increased susceptibility to DADs and arrhythmia as compared to control myocytes. This was due to increased frequency and duration of elementary Ca2+ release events (Ca2+ sparks). Dantrolene, a drug effective on malignant hyperthermia, restored normal Ca2+ spark properties and rescued the arrhythmogenic phenotype. This suggests defective inter‐domain interactions within the RYR2 channel as the pathomechanism of the S406L mutation. Our work provides a new in vitro model to study the pathogenesis of human cardiac arrhythmias and develop novel therapies for CPVT.

Irisin and exercise training in humans - results from a randomized controlled training trial.

BackgroundThe recent discovery of a new myokine (irisin) potentially involved in health-related training effects has gained great attention, but evidence for a training-induced increase in irisin remains preliminary. Therefore, the present study aimed to determine whether irisin concentration is increased after regular exercise training in humans.MethodsIn a randomized controlled design, two guideline conforming training interventions were studied. Inclusion criteria were age 30 to 60 years, <1 hour/week regular activity, non-smoker, and absence of major diseases. 102 participants could be included in the analysis. Subjects in the training groups exercised 3 times per week for 26 weeks. The minimum compliance was defined at 70%. Aerobic endurance training (AET) consisted of 45 minutes of walking/running at 60% heart rate reserve. Strength endurance training (SET) consisted of 8 machine-based exercises (2 sets of 15 repetitions with 100% of the 20 repetition maximum). Serum irisin concentrations in frozen serum samples were determined in a single blinded measurement immediately after the end of the training study. Physical performance provided positive control for the overall efficacy of training. Differences between groups were tested for significance using analysis of variance. For post hoc comparisons with the control group, Dunnett’s test was used.ResultsMaximum performance increased significantly in the training groups compared with controls (controls: ±0.0 ± 0.7 km/h; AET: 1.1 ± 0.6 km/h, P < 0.01; SET: +0.5 ± 0.7 km/h, P = 0.01). Changes in irisin did not differ between groups (controls: 101 ± 81 ng/ml; AET: 44 ± 93 ng/ml; SET: 60 ± 92 ng/ml; in both cases: P = 0.99 (one-tailed testing), 1−β error probability = 0.7). The general upward trend was mainly accounted for by a negative association of irisin concentration with the storage duration of frozen serum samples (P < 0.01, β = −0.33). After arithmetically eliminating this confounder, the differences between groups remained non-significant.ConclusionsA training-induced increase in circulating irisin could not be confirmed, calling into question its proposed involvement in health-related training effects. Because frozen samples are prone to irisin degradation over time, positive results from uncontrolled trials might exclusively reflect the longer storage of samples from initial tests.Trial registrationClinicaltrials.gov. Identifier: NCT01263522.

Functional and morphological preservation of adult ventricular myocytes in culture by sub-micromolar cytochalasin D supplement

In cardiac myocytes, cytochalasin D (CytoD) was reported to act as an actin disruptor and mechanical uncoupler. Using confocal and super-resolution STED microscopy, we show that CytoD preserves the actin filament architecture of adult rat ventricular myocytes in culture. Five hundred nanomolar CytoD was the optimal concentration to achieve both preservation of the T-tubular structure during culture periods of 3 days and conservation of major functional characteristics such as action potentials, calcium transients and, importantly, the contractile properties of single myocytes. Therefore, we conclude that the addition of CytoD to the culture of adult cardiac myocytes can indeed be used to generate a solid single-cell model that preserves both morphology and function of freshly isolated cells. Moreover, we reveal a putative link between cytoskeletal and T-tubular remodeling. In the absence of CytoD, we observed a loss of T-tubules that led to significant dyssynchronous Ca(2+)-induced Ca(2+) release (CICR), while in the presence of 0.5 μM CytoD, T-tubules and homogeneous CICR were majorly preserved. Such data suggested a possible link between the actin cytoskeleton, T-tubules and synchronous, reliable excitation-contraction-coupling. Thus, T-tubular re-organization in cell culture sheds some additional light onto similar processes found during many cardiac diseases and might link cytoskeletal alterations to changes in subcellular Ca(2+) signaling revealed under such pathophysiological conditions.

Irisin does not mediate resistance training-induced alterations in resting metabolic rate.

PURPOSE This study aimed to investigate the effects of a 6-month preventive resistance training program on resting metabolic rate (RMR) and its associations with fat-free mass (FFM) and the newly described myokine irisin as two potential mechanistic links between exercise training and RMR. METHODS In a randomized controlled trial, 74 sedentary healthy male and female participants either completed 6 months of high-repetition resistance training 3 d·wk in accordance with the American College of Sports Medicine recommendations (RT: n = 37; 47 ± 7 yr; body mass index, 25.0 ± 3.4 kg·m) or served as controls (CO: n = 37; 50 ± 7 yr; body mass index, 24.2 ± 3.2 kg·m). Strength (one-repetition maximum), RMR (indirect calorimetry), body fat (caliper method), and serum irisin concentration (enzyme-linked immunosorbent assay) were measured before and after 6 months of training. RESULTS Training led to an increase in strength (one-repetition maximum leg press, 16% ± 7%; P < 0.001). RMR increased in RT (1671 ± 356 vs 1843 ± 385 kcal·d, P < 0.001) but not in CO (1587 ± 285 vs 1602 ± 294 kcal·d, P = 0.97; group-time interaction, P < 0.01). Body weight (RT, -0.5 ± 2.4 kg; CO, 0.1 ± 2.3 kg), body fat percentage (RT, -1.1% ± 2.5%; CO, -0.7% ± 2.9%), and FFM (RT, 0.4 ± 2.1 kg; CO, 0.6 ± 1.9 kg) did not develop differently between groups (group-time interaction: P = 0.29, P = 0.54, and P = 0.59, respectively). Serum irisin concentration increased in CO (70.8 ± 83.4 ng·mL, P < 0.001) but not in RT (22.4 ± 92.6 ng·mL, P = 0.67; group-time interaction, P < 0.01). The change in RMR was not associated with the change in FFM (r = -0.11, P = 0.36) or irisin (r = -0.004, P = 0.97). CONCLUSIONS Preventive resistance training elicits an increase in RMR. However, in contrast to currently discussed hypotheses, this increase does not seem to be mediated by training-induced changes in FFM or circulating irisin concentration, which casts doubt in the meaning of irisin for human energy balance.

Moderate Calcium Channel Dysfunction in Adult Mice with Inducible Cardiomyocyte-specific Excision of the cacnb2 Gene

The major L-type voltage-gated calcium channels in heart consist of an α1C (CaV1.2) subunit usually associated with an auxiliary β subunit (CaVβ2). In embryonic cardiomyocytes, both the complete and the cardiac myocyte-specific null mutant of CaVβ2 resulted in reduction of L-type calcium currents by up to 75%, compromising heart function and causing defective remodeling of intra- and extra-embryonic blood vessels followed by embryonic death. Here we conditionally excised the CaVβ2 gene (cacnb2) specifically in cardiac myocytes of adult mice (KO). Upon gene deletion, CaVβ2 protein expression declined by >96% in isolated cardiac myocytes and by >74% in protein fractions from heart. These latter protein fractions include CaVβ2 proteins expressed in cardiac fibroblasts. Surprisingly, mice did not show any obvious impairment, although cacnb2 excision was not compensated by expression of other CaVβ proteins or changes of CaV1.2 protein levels. Calcium currents were still dihydropyridine-sensitive, but current density at 0 mV was reduced by <29%. The voltage for half-maximal activation was slightly shifted to more depolarized potentials in KO cardiomyocytes when compared with control cells, but the difference was not significant. In summary, CaVβ2 appears to be a much stronger modulator of L-type calcium currents in embryonic than in adult cardiomyocytes. Although essential for embryonic survival, CaVβ2 down-regulation in cardiomyocytes is well tolerated by the adult mice.

Isolation and genetic manipulation of adult cardiac myocytes for confocal imaging.

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Genetically Encoded Ca2+ Indicators in Cardiac Myocytes

Genetically encoded Ca(2+) indicators constitute a powerful set of tools to investigate functional aspects of Ca(2+) signaling in isolated cardiomyocytes, cardiac tissue, and whole hearts. Here, we provide an overview of the concepts, experiences, state of the art, and ongoing developments in the use of genetically encoded Ca(2+) indicators for cardiac cells and heart tissue. This review is supplemented with in vivo viral gene transfer experiments and comparisons of available genetically encoded Ca(2+) indicators with each other and with the small molecule dye Fura-2. In the context of cardiac myocytes, we provide guidelines for selecting a genetically encoded Ca(2+) indicator. For future developments, we discuss improvements of a broad range of properties, including photophysical properties such as spectral spread and biocompatibility, as well as cellular and in vivo applications.

Differential Behavior of Fibroblasts and Epithelial Cells on Structured Implant Abutment Materials: A Comparison of Materials and Surface Topographies

PURPOSE The aim of this study was to compare the proliferation and attachment behavior of fibroblasts and epithelial cells on differently structured abutment materials. MATERIALS AND METHODS Three different surface topographies were prepared on zirconia and titanium alloy specimens and defined as follows: machined (as delivered without further surface modification), smooth (polished), and rough (sandblasted). Energy-dispersive X-ray spectroscopy, topographical analysis, and water contact angle measurements were used to analyze the surface properties. Fibroblasts (HGF1) and epithelial cells (HNEpC) grown on the specimens were investigated 24 hours and 72 hours after seeding and counted using fluorescence imaging. To investigate adhesion, the abundance and arrangement of the focal adhesion protein vinculin were evaluated by immunocytochemistry. RESULTS Similar surface topographies were created on both materials. Fibroblasts exhibited significant higher proliferation rates on comparable surface topographies of zirconia compared with the titanium alloy. The proliferation of fibroblasts and epithelial cells was optimal on different substrate/topography combinations. Cell spreading was generally higher on polished and machined surfaces than on sandblasted surfaces. Rough surfaces provided favorable properties in terms of cellular adhesion of fibroblasts but not of epithelial cells. CONCLUSIONS Our data support complex soft tissue cell-substrate interactions: the fibroblast and epithelial cell response is influenced by both the material and surface topography.

Mutation of the calmodulin binding motif IQ of the L-type Ca(v)1.2 Ca2+ channel to EQ induces dilated cardiomyopathy and death.

Background: Mutation of the IQ motif to EQ abolished in vitro CDI and CDF of the Cav1.2 channel. Results: Cardiac-specific expression of Cav1.2EQ prevents CDI and CDF, reduces ICa, and induces dilated cardiomyopathy. Conclusion: The cardiac-specific EQ mutation leads to premature death. Significance: Survival depends on the expression of a native Cav1.2 protein. Cardiac excitation-contraction coupling (EC coupling) links the electrical excitation of the cell membrane to the mechanical contractile machinery of the heart. Calcium channels are major players of EC coupling and are regulated by voltage and Ca2+/calmodulin (CaM). CaM binds to the IQ motif located in the C terminus of the Cav1.2 channel and induces Ca2+-dependent inactivation (CDI) and facilitation (CDF). Mutation of Ile to Glu (Ile1624Glu) in the IQ motif abolished regulation of the channel by CDI and CDF. Here, we addressed the physiological consequences of such a mutation in the heart. Murine hearts expressing the Cav1.2I1624E mutation were generated in adult heterozygous mice through inactivation of the floxed WT Cav1.2L2 allele by tamoxifen-induced cardiac-specific activation of the MerCreMer Cre recombinase. Within 10 days after the first tamoxifen injection these mice developed dilated cardiomyopathy (DCM) accompanied by apoptosis of cardiac myocytes (CM) and fibrosis. In Cav1.2I1624E hearts, the activity of phospho-CaM kinase II and phospho-MAPK was increased. CMs expressed reduced levels of Cav1.2I1624E channel protein and ICa. The Cav1.2I1624E channel showed “CDI” kinetics. Despite a lower sarcoplasmic reticulum Ca2+ content, cellular contractility and global Ca2+ transients remained unchanged because the EC coupling gain was up-regulated by an increased neuroendocrine activity. Treatment of mice with metoprolol and captopril reduced DCM in Cav1.2I1624E hearts at day 10. We conclude that mutation of the IQ motif to IE leads to dilated cardiomyopathy and death.

Optical Action Potential Screening on Adult Ventricular Myocytes as an Alternative QT-screen

Background/Aims: QT-interval screens are increasingly important for cardiac safety on all new medications. So far, investigations rely on animal experiments or cell-based screens solely probing for conductance alterations in heterologously expressed hERG-channels in cell lines allowing for a high degree of automation. Adult cardiomyocytes can not be handled by automated patch-clamp setups. Therefore optical screening of primary isolated ventricular myocytes is regarded as an alternative. Several optical voltage sensors have been reported for ratiometric measurements, but they all influenced the naïve action potential. The aim of the present study was to explore the recording conditions and define settings that allow optical QT-interval screens. Methods: Based on an improved optical design, individual action potentials could be recorded with an exceptional signal-to-noise-ratio. The sensors were validated using the patch-clamp technique, confocal microscopy and fluorescence lifetime imaging in combination with global unmixing procedures. Results: We show that the small molecule dye di-8-ANEPPS and the novel genetically encoded sensor Mermaid provide quantitative action potential information. When applying such sensors we identified distinctly different pharmacological profiles of action potentials for adult and neonatal rat cardiomyocytes. Conclusion: Optical methods can be used for QT-interval investigations based on cellular action potentials using either the small molecule dye di-8-ANEPPS or the genetically encoded sensor Mermaid. Adult cardiomyocytes are superior to neonatal cardiomyocytes for such pharmacological investigations. Optical QT-screens may replace intricate animal experiments.