Synchronous force and calcium transient analyses in human failing myocardium reveal alterations of excitation-contraction coupling

Background: Calcium dysfunction directly impacts myocardial contractility and even arrhythmogenesis, and causes transcriptomic and proteomic modifications in heart failure. Thus, real-time synchronized visualization of calcium and force is essential to investigate the relationship between calcium events and contractility, and the alteration of excitation-contraction coupling (ECC) in human failing myocardium.

Methods: Vital myocardial tissue slices were generated from failing hearts of human transplant recipients, and were cultured in biomimetic chambers for more than 3 weeks prior to measurements. Contraction force and intracellular Ca²⁺ were simultaneously acquired in a microscopic setup which permitted both, optical analysis of intracellular Ca²⁺ using CalRed^TM R525/650 (a ratiometric calcium-sensitive indicator with visible light excitation), and direct measurement of  contraction force with the transducer integrated in preload-adjustable culture chambers. Excitation/contraction coupling was characterized with drug applications and programmed electrical stimulation sequences, which could be performed with the same slice at consecutive times.

Results: The presented technique permits simultaneous measurements of twitch force and Ca²⁺ transients in adult human myocardium, and the visualization of their kinetic relationship as a 2-dimensional force/Ca²⁺ loop. High pacing frequencies increased amplitudes of force and calcium at 1 Hz (+10.3±2.6% and +3.4±0.8%, respectively), and decreased at 2 Hz (-12.6±5.2% and -9.4±2.3%, respectively) compared to 0.5 Hz. Rate-dependent acceleration of both parameters (time to peak, decay constant tau) was maintained in a range from 0.5 Hz to 2 Hz. 12 seconds post-pause potentiation remarkably enhanced contractility (+153.6±29.3%) and the peak of intracellular calcium (+14.0±4.2%). High mechanical preload induced the enhancement of contractility immediately (+75.5±15.6%) in the absence of a notable change of intracellular Ca²⁺. Isoprenaline, a beta-selective adrenergic agonist, significantly increased amplitudes of force and calcium (+253.0±64.9%, +41.7±7.4%), and accelerated relaxation and calcium sequestration (tau, -33.4±5.4% and -34.0±4.3%, respectively). The combined inhibition of sarcoplasmic reticulum calcium-ATPase and sodium-calcium exchanger by cyclopiazonic acid and SEA0400 caused a rapid decrease of force and calcium amplitudes (-57.8±12.0% and -16.6±3.6%, respectively), and greatly increased tau of relaxation and calcium decay (+25.7±6.6% and +55.5±9.5%, respectively).

Conclusions: We present a novel approach to simultaneously and repeatedly investigate alterations of contractility and calcium signals in cultured human myocardium, which easily identifies responses to stimulation patterns and drugs. This will help to investigate the long-term effects of electrophysiological or pharmacological interventions on human myocardial ECC in heart failure.