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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.

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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.

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Although, myocardial conduction of excitation has been widely described, the electrical properties of the transverse-axial tubular system (TATS) remain mostly unknown. Here, we exploit the formal analogy between diffusion and electrical conductivity to link the latter with the diffusional properties of TATS. Fluorescence recovery after photo-bleaching (FRAP) microscopy is used to probe the diffusion properties of TATS in isolated rat cardiomyocytes: a fluorescent dextran inside TATS lumen is photo-bleached and signal recovery by diffusion of unbleached dextran from the extracellular space is monitored. We designed a mathematical model to correlate the time constant of fluorescence recovery with the apparent diffusion coefficient of the fluorescent molecules. Then, apparent diffusion is linked to electrical conductivity and used to evaluate the efficiency of the passive spread of membrane depolarization along TATS. The method is first validated in cells where most TATS elements are acutely detached by osmotic shock and then applied to probe TATS electrical conductivity in failing heart cells. We find that acute and pathological tubular remodeling significantly affect TATS electrical conductivity. This may explain the occurrence of defects in action potential propagation at the level of single T-tubules, recently observed in diseased cardiomyocytes.

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NEW RESEARCH ACTIVITY!
Both genetic and non-genetic cardiac diseases are often characterized by cardiac remodeling processes that cause alterations in electrical conduction and electro-mechanical dysfunction that lead to the development of arrhythmias. Predictive models of these alterations are based on non-integrated and low-resolution information. In this project, we combine advances in high-resolution optical microscopy, tissue clearing and immunostaining to reconstruct the three-dimensional organization of the cardiac conduction system in the whole mouse heart. We exploit the advantages of two-photon microscopy in terms of high contrast and resolution deep within tissue. A custom-made software for cytoarchitectonic analysis is used to identify cells and the alignment of myofilaments in three dimensions defining the conduction pathway of action potential propagation at intercellular level. This innovative experimental approach will allow to dissect the morphological causes leading to electro-mechanical dysfunction.