Isolated Permeablized Cardiomyocyte Functional Analysis
The myofilament is activated by calcium cycling inside the cell. We isolate individual cardiomyocytes and remove the cell membrane so we can control the precise level of calcium the myofilament is exposed to. We attach the cell to a sensitive force transducer and piezo length controller so we can measure the force generated by the cell at various calcium concentrations and length. This allows us to determine in what ways the myofilament is altered during disease and in response to various stimuli and possible therapeutic
Furthermore, cells can be isolated from very small pieces of frozen tissue. This allows us the ability to assay myofilament function in cells from biopsies, previously frozen samples from tissue banks, or previously completed experiments which now suggest a myofilament role.
Intact Cell Shortening and Calcium Transient Analysis
By enzymatic isolation from a heart, we can collect individual cardiomyocytes that are still intact and beating in response to electrical stimulation. These cells actively cycle calcium with each beat, and this intracellular calcium level can be determined using fluorescent dyes loaded inside each cell, such as indo-1 or fura-2. Our Ionoptix-based system can simultaneously record cell shortening and calcium transients in cardiomyocytes. This gives us important information regarding how well the myocytes can contract and relax with each beat, as well as whether there are any changes in the excitation-contraction network within the cell.
The myofilament is regulated by protein phosphorylation and other post-translational modifications (PTMs). Mass spectrometry provides an un-biased snapshot at a huge number of proteins and PTMs in a sample. Combined with sample fractionation and enrichment, this allows us to observe even very rare events that can have very significant affects in the heart.
The Proteomics Resource Center is Directed by Dr. Kirk. The current instrumentation includes an Orbitrap XL w/ UltiMate 3000 nanoHPLC, Vantage Triple Quad w/ UltiMate 3000 nanoHPLC, Shimadzu Axima-CFR+ MALDI-TOF MS, and a Typhoon scanner,
Animal Surgery and In Vivo Function
Our in vivo work is supported by the Department of Cell of Molecular Physiology Small Animal Core Facility.
Bioengineering and Prototyping
We have our own protoyping bench, but work closely with the Department's Machine Shop Core facility to generate new experimental systems and improve our existing instruments. The machine shop is fully equipped, including a computer-aided mill, a lathe, and 3D printer.