The Cardiac Imaging Group is developing innovative imaging methodologies to increase the understanding of cardiac physiology. For instance, we recentlly developed an ultrafast random access multi-photon (RAMP) microscope that, in combination with a custom synthesized voltage-sensitive dye, is used to simultaneously measure action potentials and intracellular calcium transients at multiple sites within the sarcolemma of cardiac cells with submillisecond temporal and submicrometer spatial resolution. An ultrafast wide- field macroscope was also developed to optically map action potential propagation with a redshifted voltage sensitive dye in whole mouse hearts. The macroscope was implemented with a random-access scanning head capable of drawing arbitrarily-chosen stimulation patterns with submillisecond temporal resolution allowing precise epicardial activation of Channelrhodopsin2 (ChR2). We employed this optical system in the setting of ventricular tachycardia to optimize mechanistic-based, multi-barrier defibrillation patterns.
Cardiac imaging by random access microscopy
Action potentials (APs), via the transverse axial tubular system (TATS), synchronously trigger uniform Ca2+ release throughout the cardiomyocyte. In heart failure (HF), TATS structural remodeling occurs, leading to asynchronous Ca2+ release across the myocyte and contributing to contractile dysfunction. In cardiomyocytes from failing rat hearts, we documented the presence of TATS elements which failed to propagate AP and displayed spontaneous electrical activity; the consequence for Ca2+ release remained, however, unsolved.
Recentlly, we develop an imaging method to simultaneously assess TATS electrical activity and local Ca2+ release. In HF cardiomyocytes, sites where T-tubules fail to conduct AP show a slower and reduced local Ca2+ transient compared with regions with electrically coupled elements. It is concluded that TATS electrical remodeling is a major determinant of altered kinetics, amplitude, and homogeneity of Ca2+ release in HF. Moreover, spontaneous depolarization events occurring in failing T-tubules can trigger local Ca2+ release, resulting in Ca2+ sparks. The occurrence of tubule-driven depolarizations and Ca2+ sparks may contribute to the arrhythmic burden in heart failure. This research provides the first description to our knowledge of these novel proarrhythmogenic events that could help guide future therapeutic strategies.
Optical control of the whole heart activity
Current rescue therapies for life-threatening arrhythmias ignore the pathological electro-anatomical substrate and base their efficacy on a generalized electrical discharge. Here, we developed an all-optical platform to examine less invasive defibrillation strategies. An ultrafast wide-field macroscope operating at 2 KHz (100 x 100 pixel) was developed to optically map action potential propagation with a red-shifted voltage sensitive dye (di-4-ANBDQPQ) in whole mouse hearts. Control of the electrical activity was achieved by employing transgenic mouse hearts expressing Channel Rhodopsin-2 (ChR2). In order to draw arbitrarily-chosen ChR2 stimulation patterns with sub-millisecond temporal resolution, the macroscope was implemented with a random-access scanning head based on acousto-optic deflectors (AODs).
AODs rapidly scan the laser beam across the whole field of view exciting different volume with a commutation time of few μs. At the end of one cycle the AODs return to the initial position and repeat the stimulation cycle. Alternatively, a simpler optical solution based on digital micromirror device (DMD) in combination with a high power LED was used to manipulate light positioning in a real simultaneous manner. We employed the macroscope to study the mechanistic features of ventricular tachycardia and we designed mechanistically-based cardioversion/defibrillation patterns exploiting the transient refractoriness of myocardium produced by the ChR2 stimulation. Multiple regions of conduction block revealed to efficiently defibrillate arrhythmic hearts but with lower energy requirements as compared to whole ventricle interventions.
Diffusion properties of cardiac T-tubular system
T-tubular structural remodelling that is generally associated with pathological settings may increase the tubular electrical resistance, augmenting the probability that the depolarization wavefront fails in reaching the AP threshold at the tubular membrane. T-tubular structural changes can modify the diffusion properties of T-tubule lumen. Here, we used fluorescence recovery after photo-bleaching (FRAP) microscopy to probe the diffusion properties of TATS lumen in cardiomyocytes.
T-tubules of isolated cardiomyocytes from rodent models of cardiac diseases are labelled using fluorescent dextran that diffuses from extracellular space to TATS lumen. The fluorescent dextran present inside TATS lumen is photo-bleached and the diffusion of unbleached dextran from extracellular space to TATS is monitored using confocal imaging. We designed a mathematical model that correlate the apparent diffusion of dextran inside T-tubules with the geometrical factor of the network. Then, exploiting the analogy between diffusion and electrical conductivity we link the diffusional properties of TATS with its electrical resistance.
• Cecilia Ferrantini, MD, PhD, University of Florence, Italy.
• Corrado Poggesi, MD, PhD, University of Florence, Italy.
• Raffaele Coppini, MD, PhD, University of Florence, Italy.
• Elisabetta Cerbai, PhD, University of Florence, Italy.
• Leonardo Bocchi, University of Florence, Italy.
• Leslie M. Loew, Ph.D., University of Connecticut Health Center, Connecticut, US.
Selected Recent Publications
M. Scardigli, C. Crocini, C. Ferrantini, T. Gabbrielli, L. Silvestri, R. Coppini, C. Tesi, E. A. Rog-Zielinska, P. Kohl, E. Cerbai, C. Poggesi, F. S. Pavone, L. Sacconi, Quantitative assessment of passive electrical properties of the cardiac T-tubular system by FRAP microscopy, Proc Natl Acad Sci U S A vol. 114 pp. 5737-5742 (2017).
C. Crocini, C. Ferrantini, R. Coppini, L. Sacconi, Electrical defects of the transverse-axial tubular system in cardiac diseases. J Physiol. Epub ahead of print (2016).
C. Crocini, C. Ferrantini, R. Coppini, M. Scardigli, P. Yan, L. M. Loew, G. Smith, E. Cerbai, C. Poggesi, F. S. Pavone, L. Sacconi, Optogenetics design of mechanistically-based stimulation patterns for cardiac defibrillation. Sci Rep. vol. 6 pp. 35628 (2016).
C. Crocini, R. Coppini, C. Ferrantini, F. S. Pavone, L. Sacconi, Functional cardiac imaging by random access microscopy. Front. Physiol. vol. 5 (2014).
C. Crocini, R. Coppini, C. Ferrantini, P. Yan, L. M. Loew, C. Tesi, E. Cerbai, C. Poggesi, F. S. Pavone, L. Sacconi, Defects in T-tubular electrical activity underlie local alterations of calcium release in heart failure. Proc Natl Acad Sci U S A vol. 42 pp. 15196-15201 (2014).
C. Ferrantini, C. Crocini, R. Coppini, F. Vanzi, C. Tesi, E. Cerbai, C. Poggesi, F. S. Pavone, L. Sacconi, The transverse-axial tubular system of cardiomyocytes, Cell. Mol. Life Sci. vol. 70 pp 4695-4710 (2013).