The Stelzer Lab - Techniques

Molecular Biology

Our lab employs a suite of advanced molecular biology techniques to investigate the molecular mechanisms of the cardiac sarcomere. We produce and purify proteins and peptides to study their roles within the sarcomere and assess their biochemical properties.

Myosin ATPase assays – Measure the enzymatic activity of myosin, providing insights into sarcomere function and energy transduction in health and disease.
High Throughput Screens (HTS) – Enables the rapid identification of small molecules that modulate sarcomeric protein function, aiding therapeutic development.
Isothermal Titration Calorimetry (ITC) – Quantifies protein-protein and protein-ligand interactions, helping to characterize molecular mechanisms governing sarcomere function.
Rapid kinetic measurements using stopped-flow techniques – Reveal transient biochemical states in sarcomeric proteins, crucial for understanding dynamic contraction and relaxation processes.
Immunohistochemistry – Allows visualization and localization of sarcomeric proteins in tissue samples, validating molecular findings in biological contexts.
Gel Electrophoresis/Western Blot – Enables the detection and quantification of sarcomeric protein expression and post-translational modifications in disease models.
Molecular Dynamics – Simulates the behavior of sarcomeric proteins at atomic resolution, uncovering mechanisms of structural changes and drug interactions.
Peptide Drug Design – Develops targeted therapeutic peptides to modulate sarcomeric function, offering novel treatment avenues for cardiomyopathies.
Protein Structure/Function – Investigates how structural changes in sarcomeric proteins impact their biochemical and mechanical roles in contraction.
Protein-Protein Interactions – Defines the molecular interactions that regulate sarcomere assembly and function, identifying potential therapeutic targets.
Protein Crystallization – Facilitates high-resolution structural analysis of sarcomeric proteins, providing insights into their function and potential drug binding sites.

Animal Models

Our lab utilizes a diverse array of animal models, including mice and pigs, to advance our understanding of cardiovascular physiology and disease while evaluating novel therapeutic strategies.

In-vivo Animal Models – Provide a physiological context to study cardiac disease mechanisms and test therapeutic interventions in a controlled system.
Sophisticated in vivo measurements such as 4D echocardiography, 4D strain analysis, Doppler imaging, and ECG analysis – Enable precise, real-time assessment of cardiac structure and function, capturing disease progression and treatment effects.
Gene therapy treatments – Target sarcomeric dysfunction at the genetic level, offering potential long-term or curative strategies for inherited cardiomyopathies.
Physiological Phenotyping – Integrates molecular, cellular, and whole-organ function assessments to comprehensively characterize disease models and therapeutic outcomes.

Human Tissue

Human tissue is invaluable in cardiovascular research because it provides a direct link between experimental findings and clinical relevance. Unlike animal models, human tissue reflects the unique genetic, molecular, and physiological characteristics of human biology, offering unparalleled accuracy in studying disease mechanisms and responses to treatment.

Muscle Fiber Mechanics

Heart muscle force generation relies on the interaction between actin and myosin filaments within muscle fibers. By attaching the cells to a sensitive force transducer and length controller, we can measure the force generated at different calcium concentrations and lengths. This approach helps identify how myofilament function is altered in disease, in response to various stimuli, and during potential treatments.

Calcium Sensitivity – Determines how changes in intracellular calcium levels regulate myofilament activation and force generation, providing insights into altered contractility in disease states.
Stretch Activation – Examines the response of muscle fibers to mechanical stretch, revealing how sarcomere proteins affect force generation
Force-Velocity – Measures the relationship between force production and contraction speed, helping to characterize muscle performance and the effects of genetic mutations or therapeutic interventions.

Metabolism Studies

Indirect Calorimetry (IDC)- Mice are placed in an air-tight chamber for up to 72 hours to measure changes in oxygen consumption and the release of carbon dioxide during natural feeding and fasting periods to provide insight into systemic metabolic flux.
Exercise Training & Challenges – We have access to a six-lane mouse treadmill for exercise training and challenges. We monitor systemic metabolic flux either before and after exercise using handheld blood glucose/ketone meters, or in real-time by measuring the Respiratory Exchange Ratio (RER) via IDC.
High-Resolution Respirometry – By measuring the rate of oxygen uptake in permeabilized muscle fibers or isolated mitochondria in response to different substrates, we can study changes in the TCA cycle and its activity linked to the Electron Transport Chain.
Seahorse Extracellular Flux Analysis – Primary cells isolated from mouse heart and liver are plated and subject to a series of substrate and drug injections to test mitochondrial capacity, cellular flux, and glycolytic capacity.
Substrate Utilization – With a chromatogram generated from mass spectrometry, we can identify circulating or stored substrates in mouse models of heart failure.

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