Inherited cardiomyopathies are life-threatening disorders that generally emerge in the teenage years or later in life. Cardiomyopathies involve profound changes to the structure and function of heart tissue, all stemming from one or more gene mutations carried by the patient. Our lab is working to piece together the events that link specific gene mutations to altered muscle function and ultimately diseased tissue. In building accurate biophysical and biomechanical descriptions of the disease, we will ultimately be able to improve diagnosis and design novel therapeutic approaches. Our recent work is leveraging a combination of multi-scale computer modeling, cardiac tissue engineering, and clinical genetics to study cardiomyopathy.
Cardiac muscle physiology is a classical field of study that still holds many unanswered questions. These knowledge gaps are fascinating scientifically and, in many cases, critical to human health. Our approach to cardiac muscle physiology makes use of engineering tools such as computational modeling and biomechanical measurements to investigate fundamental muscle phenomena. Ongoing areas of interest include the basis for the Frank-Starling relationship, the roles of various proteins in the regulation of muscle contraction, and the impact of myofilament protein mutations on cardiac muscle behavior.
Cardiomyocyte heterogeneity is readily apparent simply by looking at isolated cardiac cells under low magnification. Their sizes and shapes differ drastically - even when cells are obtained from the same small sample of heart tissue. There is also substantial cell-to-cell variation in the speed of contraction and relaxation, something we showed in recent work. We were able to demonstrate that these functional differences between cells are the result of apparently stochastic variation in levels of protein phosphorylation. Ongoing work aims to understand how this cellular heterogeneity contributes to behavior of the intact heart.
Cardiac mechanobiology is the study of how cardiac cells and tissues respond to mechanical cues. These cues include the properties of the extracellular matrix and applied mechanical loads. Apart from key roles in cardiac development and tissue homeostasis, the way heart cells sense and respond to their mechanical environment is fundamental to many cardiac diseases. In our group, we are using engineered heart tissue in combination with custom mechanical bioreactors to probe relationships between mechanical loads, extracellular matrix, and cardiomyocyte behavior.
Our approaches and tools
Computational models are often used in our work. Through collaborations with computational chemists and structural biologists, we use molecular dynamics simulations to predict the impact of missense mutations on protein behavior. We also use meso-scale models to transfer molecular-scale information to higher scales where their effects on key kinetic steps in muscle contraction can be predicted. We frequently use sarcomere-level models to fit our experimental data and refine hypotheses. Finally, we are actively developing models of cardiomyocyte mechanotransduction to aid in the design and analysis of experiments in engineered heart tissues.
Our lab has developed a novel technique for generating engineered heart tissues (EHTs). Much of the fundamental research on cardiac muscle physiology has been performed on small linear segments of muscle that can be dissected from the inner surface of the right or left ventricles. These are known as trabeculae. The trabecular preparation is convenient for biomechanical study because it can be mounted between a force transducer and a length controller, thereby enabling measurements of muscle stiffness and force production at different muscle lengths and shortening velocities. In designing our EHT system, we sought to replicate the thin, ribbon-like structure of trabeculae while also allowing the user to select the desired cell source (neonatal rat or mouse cardiomyocytes, or human induced pluripotent stem cell derived cardiomyocytes). We also wanted our tissue platform to include secure mechanical attachments to enable precisely controlled loading conditions and accurate force measurements. To meet these requirements, we developed a process for creating EHTs that starts with cryosectioning porcine heart tissue and laser cutting these thin sheets into desired shapes. The native cells are removed through a series of chemical treatments, leaving the extracellular matrix behind. This delicate tissue scaffold is fixed securely to custom-designed retainer clips and seeded with heart muscle cells of our choosing. The seeded cells combine with the matrix in a matter of days to form a beating cardiac tissue construct suitable for a range of physiological experiments.
Human induced pluripotent stem cells (iPSC) have become an experimental mainstay of our research program, providing the means of studying our questions of interest in a human context. iPSC-derived cardiomyocytes can be seeded into our EHT system to produce tissue constructs that mimic many of the key properties of the adult human myocardium. We are leveraging these human EHTs to study fundamental physiology, the effects of gene mutations on muscle function (using CRISPR/Cas9), and the behavior of tissue derived from cardiomyopathy patients.
Custom devices to facilitate our research aims are constantly under development in our group. Examples include:
Stretch and pacing bioreactors: EHTs can be stretched both statically and dynamically during culture via a voice coil actuator while being simultaneously paced by field stimulation through carbon electrodes. We can impose custom work loops onto the EHTs to study the effects of exercise, stretch and relax tissues to gain insight into tissue remodeling, and promote maturation through pacing regimens.
Biomechanics test rig: Our custom mechanics testing apparatus allows for micromanipulation of EHTs within a temperature-controlled, continuously superfused bath. One end of the EHT is supported by a force transducer, while the other end can be controlled by a high-speed motor for dynamic length control. The system is mounted on a microscope to allow for ratiometric quantification of Fura-2 fluorescent dye loaded into the tissues to measure intracellular Ca2+ transients alongside force and length records.
Single cell microfluidic device: We have developed a custom microwell testing device for prolonged measurements of sarcomere shortening and Ca2+ transients. PDMS microwells allow for consistent positioning and alignment of cardiomyocytes, sustained cardiac drug superfusion, and aspiration via an automated single-cell pipettor for proteomic and transcriptomic analysis.
