Mirjam Keßler

Recent progress in the use of zebrafish for novel cardiac drug discovery

Introduction: Cardiovascular disease is the leading cause of morbidity and mortality worldwide, thereby putting a large burden on our healthcare costs. Using both human genetic approaches, as well as forward and reverse genetic strategies in animal models, significant progress has been made to unravel the genetic and molecular etiology of human cardiovascular disease that is crucial to define novel therapeutic targets. In this context, the zebrafish has emerged as an important in vivo vertebrate animal system to study and to model human cardiovascular diseases as well as for in vivo cardiovascular drug discovery. Areas covered: This review describes the rationale for using the in vivo model system zebrafish in whole-organism-based drug discovery strategies. It also highlights recent developments in the fields of drug target identification, disease modeling, and automation of high-throughput small compound screening. Expert opinion: Novel genome-editing techniques such as the clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) and transcription activator-like effector nuclease (TALEN) technologies allow highly efficient and reliable disease modeling in the in vivo system zebrafish. The ambition of developing personalized therapeutic options will clearly be fostered by the establishment of animal disease models that accurately simulate the patient’s situation and the use of these disease models in ‘next-generation’ high-throughput small compound screens to define treatment options tailored to individual needs. To define suitable targets for therapeutic modulation, systems biology approaches that study complex biological systems as an integrated whole will pave the way to successful in vivo disease modeling and future drug discovery.

Ion Flux Dependent and Independent Functions of Ion Channels in the Vertebrate Heart: Lessons Learned from Zebrafish

Ion channels orchestrate directed flux of ions through membranes and are essential for a wide range of physiological processes including depolarization and repolarization of biomechanical activity of cells. Besides their electrophysiological functions in the heart, recent findings have demonstrated that ion channels also feature ion flux independent functions during heart development and morphogenesis. The zebrafish is a well-established animal model to decipher the genetics of cardiovascular development and disease of vertebrates. In large scale forward genetics screens, hundreds of mutant lines have been isolated with defects in cardiovascular structure and function. Detailed phenotyping of these lines and identification of the causative genetic defects revealed new insights into ion flux dependent and independent functions of various cardiac ion channels.

Loss of dihydrolipoyl succinyltransferase (DLST) leads to reduced resting heart rate in the zebrafish

The genetic underpinnings of heart rate regulation are only poorly understood. In search for genetic regulators of cardiac pacemaker activity, we isolated in a large-scale mutagenesis screen the embryonic lethal, recessive zebrafish mutant schneckentempo (ste). Homozygous ste mutants exhibit a severely reduced resting heart rate with normal atrio-ventricular conduction and contractile function. External electrical pacing reveals that defective excitation generation in cardiac pacemaker cells underlies bradycardia in ste−/− mutants. By positional cloning and gene knock-down analysis we find that loss of dihydrolipoyl succinyltransferase (DLST) function causes the ste phenotype. The mitochondrial enzyme DLST is an essential player in the citric acid cycle that warrants proper adenosine-tri-phosphate (ATP) production. Accordingly, ATP levels are significantly diminished in ste−/− mutant embryos, suggesting that limited energy supply accounts for reduced cardiac pacemaker activity in ste−/− mutants. We demonstrate here for the first time that the mitochondrial enzyme DLST plays an essential role in the modulation of the vertebrate heart rate by controlling ATP production in the heart.

Predictors of permanent pacemaker implantation after transfemoral aortic valve implantation with the Lotus valve

BACKGROUND Permanent pacemaker implantation (PPMI) after transcatheter aortic valve implantation is of high clinical relevance, but PPMI rates differ widely between valve types. Although the Lotus valve can be repositioned, reported rates for PPMI are high. The predictors of PPMI after Lotus valve implantation have not been defined yet. METHODS We analyzed the impact of preexisting conduction disturbances, depth of implantation, oversizing, and amount of calcification on PPMI in 216 patients with severe symptomatic aortic stenosis underdoing Lotus valve implantation. RESULTS PPMI was required in 39.8% of patients. Patients with need for PPMI compared with patients without need for PPMI had more often the following criteria: male gender (P=.035); preprocedural right bundle-branch block (RBBB) (16.3% vs 0, P<.001); atrioventricular (AV) block first degree (26.7% vs 10.1%, P=.004); higher calcium volume of the left coronary cusp (63.1±87.5 mm3 vs 42.8±49.3 mm3, P=.05); and deeper valve implantation at right coronary (P=.011), noncoronary (P=.026), and left coronary (P=.012) position. Oversizing in relation to annulus and left ventricular outflow tract did not have an impact on need for PPMI. By multiple regression analysis, preprocedural AV block first degree (P=.005), RBBB (P<.001), and depth of implantation (P=.006) were independent risk factors for need of PPMI. CONCLUSIONS In patients with severe aortic stenosis receiving transfemoral Lotus valve, preexisting AV block first degree, RBBB, and implantation depth are independent predictors of PPMI, highlighting the importance of careful valve positioning.

Tbx20 Is an Essential Regulator of Embryonic Heart Growth in Zebrafish.

The molecular mechanisms that regulate cardiomyocyte proliferation during embryonic heart growth are not completely deciphered yet. In a forward genetic N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we identified the recessive embryonic-lethal zebrafish mutant line weiches herz (whz). Homozygous mutant whz embryos display impaired heart growth due to diminished embryonic cardiomyocyte proliferation resulting in cardiac hypoplasia and weak cardiac contraction. By positional cloning, we found in whz mutant zebrafish a missense mutation within the T-box 20 (Tbx20) transcription factor gene leading to destabilization of Tbx20 protein. Morpholino-mediated knock-down of Tbx20 in wild-type zebrafish embryos phenocopies whz, indicating that the whz phenotype is due to loss of Tbx20 function, thereby leading to significantly reduced cardiomyocyte numbers by impaired proliferation of heart muscle cells. Ectopic overexpression of wild-type Tbx20 in whz mutant embryos restored cardiomyocyte proliferation and heart growth. Interestingly, ectopic overexpression of Tbx20 in wild-type zebrafish embryos resulted, similar to the situation in the embryonic mouse heart, in significantly reduced proliferation rates of ventricular cardiomyocytes, suggesting that Tbx20 activity needs to be tightly fine-tuned to guarantee regular cardiomyocyte proliferation and embryonic heart growth in vivo.

Mutation of the Na + /K + -ATPase Atp1a1a.1 causes QT interval prolongation and bradycardia in zebrafish

The genetic underpinnings that orchestrate the vertebrate heart rate are not fully understood yet, but of high clinical importance, since diseases of cardiac impulse formation and propagation are common and severe human arrhythmias. To identify novel regulators of the vertebrate heart rate, we deciphered the pathogenesis of the bradycardia in the homozygous zebrafish mutant hiphop (hip) and identified a missense-mutation (N851K) in Na+/K+-ATPase α1-subunit (atp1a1a.1). N851K affects zebrafish Na+/K+-ATPase ion transport capacity, as revealed by in vitro pump current measurements. Inhibition of the Na+/K+-ATPase in vivo indicates that hip rather acts as a hypomorph than being a null allele. Consequently, reduced Na+/K+-ATPase function leads to prolonged QT interval and refractoriness in the hip mutant heart, as shown by electrocardiogram and in vivo electrical stimulation experiments. We here demonstrate for the first time that Na+/K+-ATPase plays an essential role in heart rate regulation by prolonging myocardial repolarization.

Procedural and Clinical Results of the New MitraClip® NT after Percutaneous Edge to Edge Repair of Mitral Valve Regurgitation

Objective: The MitraClip® NT (MC-NT) as an updated version aim to facilitate improved maneuvering of the guide catheter and more efficient leaflet capture due to technical improvements of the delivery system and clip material. We evaluate procedural results and 12-month clinical outcomes of the new MC-NT device and compare them with the previous MitraClip® MC. Methods: We analyzed a total population of 231 patients from our Ulm - Transcatheter Mitral Valve Repair registry. To adjust for differences of baseline characteristics a propensity-score matching was performed (N=142). 30-day endpoints were analyzed in accordance with the Mitral Valve Academic Research Consortium (MVARC). For both devices 12-month clinical results (MACCE, mortality, and heart failure rehospitalization) were evaluated. Results: Acute technical success was achieved in 100% with both device groups. Device, procedure and fluoroscopy time were comparable in both treatment groups. In-hospital mortality was 2.8% in the MC-NT group and 4.2% in the MC group (p=0.65). After 30 days single-leaflet clip detachment was observed in 2.8% in the MC-NT group compared to 1.4% (p=0.56). Mitral regurgitation was reduced to the same extent in both treatment groups (1.6 ± 0.6 vs. 1.6 ± 0.5 in MC-NT group, p=0.57) with comparable rates of recurrent moderate to severe mitral regurgitation after 30 days and during 12-month follow-up. Clinical results up to 12 months and improvement of New York Heart Association functional class were comparable in both device groups (3.1 ± 0.7 to 2.1 ± 0.9 vs. 3.2 ± 0.6 to 2.1 ± 0.9, p=0.75). Conclusion: The new MitraClip® NT as an updated version presents itself as an efficacious and safe device, but all in all failed to prove superiority compared to the original MitraClip® in terms of procedural and clinical results.

Predictors of rehospitalization after percutaneous edge-to-edge mitral valve repair by MitraClip implantation: Predictors of rehospitalization after MitraClip

In patients at increased surgical risk, transcatheter edge‐to‐edge mitral valve repair by MitraClip implantation for severe mitral regurgitation (MR) has proven to relieve symptoms of MR, reduce New York Heart Association (NYHA) functional class and improve quality of life. Rehospitalization for decompensated heart failure occurs frequently after MitraClip implantation, negatively impacting quality of life. We aimed here to determine predictors of 1‐year rehospitalization for decompensated heart failure.

Intra-aortic balloon counterpulsation pump in heart failure patients during MitraClip implantation-A propensity-score matched analysis

To analyze 30‐day and 6‐month results after percutaneous mitral valve repair using an IABP as circulatory support.

Long-term clinical outcome of persistent left bundle branch block after transfemoral aortic valve implantation

The impact of persistent left bundle‐branch block (pLBBB) on long‐term clinical outcome remains to be determined.