Myofilament and Myocyte Functional Assays
                                                
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 that 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 therapeutics. 

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. 




Proteomics Facility

    The myofilament is regulated by protein phosphorylation and other post-translational modifications (PTMs). While antibody-based approaches are useful (and utilized in our lab), they come with a variety of drawbacks including a bias - they can only discover changes that have been looked for. 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 Loyola Proteomics Resource Center is Directed by Dr. Jonathan Kirk, and is located on the 1st floor of the Center for Translational Research & Education. Our current instrumentation includes 2-dimensional gel equipment, Typhoon scanner, Shimadzu Axima-CFR+ MALDI-TOF MS, a Thermo Fisher TSQ Vantage Triple Quadrupole MS with Agilent 1200 HPLC, a Thermo Fisher TSQ Vantage Triple Quadrupole MS with ekspert nanoLC, and a Thermo Fisher Orbitrap XL with Dionex UltiMate 3000. If you are interested in working with the Proteomics Resource Center, please email Jonathan Kirk



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.