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
Optogenetics has provided new insights into cardiovascular research, leading to new methods for cardiac pacing, resynchronization therapy and cardioversion. Although these interventions have clearly demonstrated the feasibility of cardiac manipulation, current optical stimulation strategies do not take into account cardiac wave dynamics in real time. Here, we developed an all-optical platform complemented by integrated, newly developed software to monitor and control electrical activity in intact mouse hearts. The system combined a wide-field mesoscope with a digital projector for optogenetic activation. Cardiac functionality could be manipulated either in free-run mode with sub-millisecond temporal resolution or in a closed-loop fashion: a tailored hardware and software platform allowed real-time intervention capable of reacting within 2 ms. The methodology was applied to restore normal electrical activity after atrioventricular block, by triggering the ventricle in response to optically mapped atrial activity with appropriate timing. Real-time intra-ventricular manipulation of the propagating electrical wavefront was also demonstrated, opening the prospect for real-time resynchronization therapy and cardiac defibrillation. Furthermore, the closed-loop approach can be applied to simulate a re-entrant circuit across ventricle demonstrating the capability of our system to manipulate heart conduction with high versatility even towards arrhythmogenic conditions. The development of this innovative optical methodology provides the first proof-of-concept that a real-time optical-based stimulation can control cardiac rhythm in normal and abnormal conditions, promising a new approach for the investigation of the (patho)physiology of the heart.
Whole heart cytoarchitecture at micron-scale resolution
Remodelling processes, associated with genetic and non-genetic cardiac diseases, can cause alterations of the electrical conduction and mechanical dysfunction. Current models employed to predict functional alterations caused by structural remodeling commonly do not draw upon comprehensive functional and structural data and furthermore are often based on low-resolution and non-integrated information. Here, we present a multi-modal optical approach to correlate electrophysiological dysfunction found in a Hypertrophic Cardiomyopathy mouse model with its structural alterations. In detail, we first employed an optical mapping system to characterize action potential propagation in diseased and control whole-heart preparations. To gain the structural data on the same intact hearts, we combined advances in tissue clearing, staining and high-resolution light-sheet microscopy to reconstruct the three-dimensional organization of the cardiac conduction system on a cellular level. In particular, we optimized a passive Clarity protocol for clearing the whole heart and for achieving homogeneously fluorescent probe penetration into the entire tissue. Moreover, we developed a cytoarchitectonic analysis software to identify cells and to map fibers alignment in three-dimensions. The structural reconstruction was directly used to generate a high-resolution image-based computational model to simulate the conduction pathway of action potential propagation across the whole organ with the aim to elucidate the role of cellular disorganization in the electrical dysfunctions mapped previously through optical mapping. We believe that this innovative experimental approach will pave the way for a unifying model which integrates functional and structural data and enable a comprehensive investigation of the morphological causes that lead to electrical alterations after structural remodelling.