Advantages:
-It requires very small amounts of cardiac tissue
-Frozen tissue samples can be used as a source of cardiomyocytes
-Tissue samples can be transported at <-20 degrees of Celsius
-A wide range of species can be tested, including mice, rat, pig, dog, guinea pig and human (latter requires ethical approval, depending on the project)
-Pathophysiological conditions (e.g. ischemia) can easily be modelled
Cardiomyocyte contractile parameters to be determined:
-Maximal Ca-activated (active) force (Fo) in kN/m2
-Ca-independent (passive) force (Fpassive) in kN/m2
-Ca-sensitivity of isometric force (pCa50) in pCa units
-Cooperativity (nHill)
-Actin-myosin turnover rate (ktr) in 1/sec units
-[Ca2+] dependencies of F, nHill and ktr
-Sarcomere length dependencies of Fo, Fpassive, pCa50, nHill, ktr
The cardiomyocyte force measurement system consists of an electromagnetic motor and a force transducer. The motor is used to adjust cardyomyocyte length, while the force transducer is to measure isometric cardiomyocyte contraction. A permeabilized ("skinned") cardyomyocyte is mounted between needles attached to the motor and the force transducer. A specially developed optical system is used to determine cardiomyocyte morphology from both the horizontal and the vertical directions. The experimental protocol usually consists of a series of force measurements upon Ca2+ stimuli (using Ca2+ buffer solutions), the determination of actin-myosin crossbridge kinetics and the measurement of the passive tension of the mounted cardiomyocytes at pre-defined sarcomer lengths (Fig. 1).
Fig. 1 Cardiomyocyte force measurement system
A number of parameters can be determined using the single cardiomyocyte force measurement system. The first set of such data is related to the Ca-dependence of force production. A set of Ca-buffers are used to establish a Ca-force relationship (Fig. 2). When the measured force is plotted as the function of the Ca-concentration a sigmoidal relationship between these variables is approximated. Fitting the peak force values to the Hill equation reveals the Ca-sensitivity of force production with parameters like the Ca-concentration resulting in half maximal contraction, the level of maximal Ca-activated force production and the cooperativity (nHill) in the contractile system (Fig. 2). These parameters can be useful to characterize myofibrillar pathologies of various origin (e.g. Molnar et al.) and drug effects.
Fig. 2 Determination of Ca-sensitvity, cooperativity and maximal Ca-activated force.
Kinetics of the contractile responses can be also measured (Fig. 3A and C) besides to the steady-state chracteristics (Fig. 3B). When the kinetics of force recoveries during rapid length changes are fitted to a single exponential, the time constant (ktr) of the fit reveals the actin-myosin turnover kinetics (see Edes et al. for further details).
Fig. 3 Determination of actin-myosin turnover rate
Some of the drug candidates are developed to directly improve cardiac contractility (e.g. positive inotropic drugs), while others may have side effects affecting cardiac contractility. Drugs may affect several parameters of the Ca-sensitivity curve. E.g.: EMD 53998 increased passive force, Ca-sensitivity and maximal Ca-activated force simultaneously (Fig. 4).
Fig. 4 Effects on Ca-activated force production by 10 microM EMD 53998
The Frank-Starling law of the heart (also known as Starling's law or the Frank-Starling mechanism) states that the greater the volume of blood entering the heart during diastole (end-diastolic volume), the greater the volume of blood ejected during systolic contraction (stroke volume) and vice-versa (as quated from Wikipedia). There is still a debate about the exact molecular mechanisms behind this physiological observation, but it is widely accepted, that changes in Ca-sensitivity of the contractile system dominantly contributes to this phenomenon (Edes et al.). Accordingly, the Ca-sensitivity of force production in a single, permeabilized cardiomyocyte increases with the sarcomere length (SL) (Fig. 5). An increase in passive force can be also observed in addition to the changes in active force with potential pathophysiological significance ( e.g. in heart failure with preserved ejection fraction (Borbely et. al., Fig. 5).
Fig. 5 Measurement of stretch-dependence of Ca-sensitivity and passive force