Exploring cardiac biophysical properties

Abstract

The heart is subject to multiple sources of stress. To maintain its normal function, and successfully overcome these stresses, heart muscle is equipped with fine-tuned regulatory mechanisms. Some of these mechanisms are inherent within the myocardium itself and are known as intrinsic mechanisms. Over a century ago, Otto Frank and Ernest Starling described an intrinsic mechanism by which the heart, even ex vivo, regulates its function on a beat-to-beat basis. According to this phenomenon, the higher the ventricular filling is, the bigger the stroke volume. Thus, the Frank-Starling law establishes a direct relationship between the diastolic and systolic function of the heart. To observe this biophysical phenomenon and to investigate it, technologic development has been a pre-requisite to scientific knowledge. It allowed for example to observe, at the cellular level, a Frank-Starling like mechanism and has been termed: Length Dependent Activation (LDA). In this review, we summarize some experimental systems that have been developed and are currently still in use to investigate cardiac biophysical properties from the whole heart down to the single myofibril. As a scientific support, investigation of the Frank-Starling mechanism will be used as a case study.

Figure 1.

Frank-Starling Law as observed on a typical pressure-volume loop. Solid curve; control pressure-volume loop shape at rest. Dashed curve; pressure-volume loop shape following increased diastolic volume. Dashed double arrow; increased stroke volume in response to increased ventricular volume (Frank-Starling Law). The ESPVR (End Systolic Pressure-Volume Relationship) is often used to estimate systolic function of the heart. The EDPVR (End Diastolic Pressure-Volume Relationship) is an indication diastolic function of the heart.

Figure 2.

Myocardium multicellular preparation. Single trabeculae and papillary muscles are isolated from the right ventricle (A). Using aluminum T-clips, the samples are mounted between a force transducer and a piezo actuator (B and C). The muscle quality can be estimated based on the overall muscle shape (10 ×  magnification; C) and presence of clear and uniform striations pattern (40 ×  magnification; D).

Figure 3.

A typical attached intact cardiomyocyte with carbon fibers. The black box defines a ROI (Region of Interest) that is used by the FFT (Fast Fourier Transform) algorithm to compute sarcomere length. The edge detection algorithm is used to follow the compliant carbon fiber's displacement (right tip on the picture). The optical displacement is then converted to force.

Figure 4.

Single skinned cardiac cell attachment apparatus. Typical experimental system employed to investigate single skinned cardiac mechanical properties. The system consists of an inverted microscope surrounded by two attachment stainless iron needles. A, one needle is connected to a tiny force transducer (AE801 from Kronex), where the other is connected to a piezo actuator. In some cases, a rapid change (2 milliseconds) is needed to evaluate contraction kinetics, hence, the use of a piezo actuator. B, a perfusion system is used to expose the attached permeabilized cell to different calcium containing solutions. The inset image shows a typical attached cardiac cell at rest (∼ 1.9 microns).

Figure 5.

Force-Calcium relationship. Force is measured at slack (1.9 mm) and after stretch (2.3 mm) at different calcium concentration. At a given calcium concentration, the attached cardiac cell (insets) generates a corresponding active force. When this active force is potted against calcium concentration, a typical Tension-pCa curve is obtained. This curve can be fitted with a Hill equation and both pCa50 (calcium concentration generating 50 % maximal force) and nH (hill coefficient) are estimated. Stretch induces a leftward shift of this relationship (dashed curve; horizontal arrow). This shift reflects an increased myofilament sensitivity to calcium. DpCa50 is used to estimate LDA, the myofilament basis of Frank-Starling law of the heart. It is obtained by computing the difference between pCa50 at long and short sarcomere length (double horizontal arrow).

Figure 6.

Transmural contractile heterogeneity across the mammalian left ventricular free wall. A, DpCa50: index of myofilament sensitization to calcium with stretch. Solid line shows the correlation between DpCa50 and passive tension in healthy conditions. Ischemic heart failure (HF) eliminates this gradient (dashed red line) by mainly affecting the Endocardium contractility (downward red arrow). Physiological (exercise) and pharmacological (SR33805) treatment both reverse HF effect by restoring the altered Endocardium cellular contractility (upward green arrow and dashed green line).

Figure 7.

Sarcomere structure. A) Electron microscope image revealing the contractile machinery organization in a cardiomyocyte. B) Schematic of the contractile apparatus (i.e. sarcomere). The contractile machinery is composed of thick (myosin) and thin (actin) filament. During contraction, thin filament slide over thick filament to produce cellular shortening. The shown dimensions are in micrometers (according to).

Figure 8.

Single cardiac myofibril force measurement system. To measure force generated by a single myofibril, the sample is attached to two tips with distinguish compliance. A steep tips (A; Tip 1) is connected to a piezo motor where the more compliant tip (A; Tip 2) is used to report developed active and passive forces. A double barrel pipette is used to expose the myofibril to relax (low [Ca2+]; blue flow on panel A) and active (higher [Ca2+]; red flow on panel A) solutions. Perfusion solutions are quickly changed by rapidly moving the pipette's position back and forth between the two solutions. A typical resulted force record is shown on panel B. the horizontal bare shows current perfusion solution; white: relaxing solution and black: activating solution. Due to the sample's size and solution rapid switching, parameter such as activation and relaxation kinetics can be measured (B; zoom trace).