Cardiac Slice Contraction-Relaxation Function: The experimental model system is the excitable cardiac slice prepared from mammalian left ventricle. Unlike other excitable preparations, sarcomere length can be established in this model system. Characteristics of isometric force transients (max force, max rate of force development, max rate of force decline, etc.) are quantified as measures of contraction- relaxation function and their dependence on sarcomere length, preload, and afterload including variable preload and afterload. In addition, cardiac slices will be used to generate force-length work loops, which mimic the pressure-volume work loops of a heart in vivo, at multiple sarcomere lengths. The dependence of contractility on sarcomere length, i.e., length-dependent activation also known as the Frank-Starling response in heart muscle, provides a measure of intrinsic contractility of the heart.

Cardiac Slice Intracellular Calcium Regulation: The experimental model system is the excitable cardiac slice prepared from mammalian left ventricle, loaded with CalRed AM, and exposed to excitation light at 490 nm. The ratio of emissions at 525 nm / 650 nm provides a measure of intracellular calcium concentration. Characteristics of intracellular calcium transients (max ratio, time to peak ratio, max rate of ratio development, max rate of ratio decline, etc.) quantify intracellular calcium regulation and can be used to distinguish the effects of disease, sex, intervention by drugs, intervention by known signaling agonists (e.g., isoproterenol), or gene therapies. This assay is often performed in conjunction with contraction-relaxation function.

Myofilament Force Production and Calcium Sensitivity: The experimental model system is demembranated mammalian myocardium, i.e., not excitable, that is ~120 um in diameter and 1 mm long. Sarcomere length can be visualized and used to establish starting muscle length, and isometric tension is recorded over multiple calcium concentrations (tension-pCa relationship). The result indicates the maximum force per cross-sectional area of the muscle sample and the calcium sensitivity of the myofilaments.

Myosin Crossbridge Kinetics: Myosin crossbridge kinetics can be quantified in demembranated myocardium using sinusoidal analysis and a force-response to a quick stretch. Myosin crossbridge detachment rate and rates of force decline and redevelopment can be measured and used to distinguish the effects of disease, sex, intervention or gene therapy.

Cardiac Myocyte Contraction-Relaxation Function: The experimental model system is the excitable cardiac myocyte prepared from left ventricle. Characteristics of contraction-relaxation function include (max shortening, max rate of shortening, max rate of relaxation, etc.). Cardiac myocytes are especially useful for rapid detection of a drug candidate’s dose response and cardiotoxicity with high sample counts, i.e., n=50-500.

Cardiac Myocyte Intracellular Calcium Regulation: The experimental model system is the excitable cardiac myocyte prepared from left ventricle, loaded with CalRed AM, and exposed to excitation light at 490 nm. The ratio of emissions at 525 nm / 650 nm provides a measure of intracellular calcium concentration. Characteristics of intracellular calcium transients (max ratio, time to peak ratio, max rate of ratio development, max rate of ratio decline, etc.) quantify intracellular calcium regulation and can be used to distinguish the effects of disease, sex, intervention by drugs, intervention by known signaling agonists (e.g., isoproterenol), or gene therapies. This assay is often performed in conjunction with contraction-relaxation function.

Long-Term Incubation:The experimental model system is the excitable cardiac slice prepared from mammalian left ventricle. Long-term incubation maintains functional tissue for days to weeks, allowing for detection of compound responses that require extended time and remodeling after gene therapy or other treatments.

Measurement of Myofilament Spacing using X-ray Diffraction: The experimental model system is the excitable cardiac slice prepared from mammalian left ventricle. X-ray video crystallography simultaneously detects the recovery of both functional and structural dynamics in cardiac tissue, providing a unique tool for evaluating the effects of therapies.

PrincipaI Investigator:

Brad Palmer, PhD
Sarcometrics, LLC – Burlington, VT
[email protected]
(802) 324-3764 (cell, text)

Contractor

Sarcometrics, LLC
(802) 324-3764 (cell, text)
[email protected]
[email protected]

Deliverables

1. Raw data in tab-delimited text format readable in Excel.
2. Detailed notes related to experimental conditions.
3. Representative raw data traces of isometric force transient, force-length work loops, and pressure transients for visual inspection of the effects of each condition.
4. Calculated parameter values provided in tab-delimited text format readable in Excel.
5. Results of statistical analyses.
6. Presentation by Principal Investigator of results and interpretations.