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Key points: Although optogenetics has clearly demonstrated the feasibility of cardiac manipulation, current optical stimulation strategies lack the capability to react acutely to ongoing cardiac wave dynamics. Here, we developed an all‐optical platform to monitor and control electrical activity in real‐time. The methodology was applied to restore normal electrical activity after atrioventricular block and to manipulate the intraventricular propagation of the electrical wavefront. The closed‐loop approach was also applied to simulate a re‐entrant circuit across the ventricle. The development of this innovative optical methodology provides the first proof‐of‐concept that a real‐time all‐optical stimulation can control cardiac rhythm in normal and abnormal conditions. Abstract: Optogenetics has provided new insights in 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 submillisecond 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 intraventricular 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 was applied to simulate a re‐entrant circuit across the ventricle demonstrating the capability of our system to manipulate heart conduction with high versatility even in arrhythmogenic conditions. The development of this innovative optical methodology provides the first proof‐of‐concept that a real‐time optically 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. [ABSTRACT FROM AUTHOR]

Well-coordinated activation of all cardiomyocytes must occur on every heartbeat. At the cell level, a complex network of sarcolemmal invaginations, called the transverse-axial tubular system (TATS), propagates membrane potential changes to the cell core, ensuring synchronous and uniformexcitation-contraction coupling. Although myocardial conduction of excitation has been widely described, the electrical properties of the 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 photobleaching (FRAP) microscopy is used to probe the diffusion properties of TATS in isolated rat cardiomyocytes: A fluorescent dextran inside TATS lumen is photobleached, 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. [ABSTRACT FROM AUTHOR]

A characteristic histological feature of striated muscle cells is the presence of deep invaginations of the plasma membrane (sarcolemma), most commonly referred to as T-tubules or the transverse-axial tubular system (TATS). TATS mediates the rapid spread of the electrical signal (action potential) to the cell core triggering Ca 2+ release from the sarcoplasmic reticulum, ultimately inducing myofilament contraction (excitation–contraction coupling). T-tubules, first described in vertebrate skeletal muscle cells, have also been recognized for a long time in mammalian cardiac ventricular myocytes, with a structure and a function that in recent years have been shown to be far more complex and pivotal for cardiac function than initially thought. Renewed interest in T-tubule function stems from the loss and disorganization of T-tubules found in a number of pathological conditions including human heart failure (HF) and dilated and hypertrophic cardiomyopathies, as well as in animal models of HF, chronic ischemia and atrial fibrillation. Disease-related remodeling of the TATS leads to asynchronous and inhomogeneous Ca 2+-release, due to the presence of orphan ryanodine receptors that have lost their coupling with the dihydropyridine receptors and are either not activated or activated with a delay. Here, we review the physiology of the TATS, focusing first on the relationship between function and structure, and then describing T-tubular remodeling and its reversal in disease settings and following effective therapeutic approaches. [ABSTRACT FROM AUTHOR]

Action potential (AP), via the transverse axial tubular system (TATS), synchronously triggers uniform Ca2+-release throughout the cardiomyocyte. Cardiac diseases associated with TATS structural remodeling preclude a uniform Ca2+-release across the myocyte, contributing to contractile dysfunction. Here, we combined the advantage of an ultrafast random access multi-photon (RAMP) microscope with a double staining approach to optically record AP in several TATS elements and, simultaneously, the corresponding local Ca2+-transient.In rat control cardiomyocytes, although AP was uniform between surface sarcolemma (SS) and t-tubules (TT) at steady-state stimulation, we observed a non-negligible beat-to-beat variability of local Ca2+-transient amplitude and kinetics. This variability was significantly reduced applying 0.1 μM Isoproterenol, which increases the open probability of Ca2+-release units. Ca2+ sparks, detected at times throughout the TATS, did not induce any membrane potential variation in the surrounding tubular elements.In a rat model of post-ischemic heart failure (HF), we previously demonstrated that some tubular elements fail to propagate AP (AP-; Sacconi PNAS 2012). Here, we found that those AP- tubules displayed a slower and reduced local Ca2+-transient compared to electrically coupled tubules (AP+). Consistently, in a model of acute detubulation, tubules that do not conduct AP showed a local reduction and delay of Ca2+-transient rise. In addition, variability of Ca2+-transient kinetics was increased in HF. Finally, AP- tubular elements, occasionally exhibited spontaneous depolarisations of various amplitude and kinetics. These events were never accompanied by local Ca2+-release in the absence of pro-arrhythmogenic stimuli, while they could induce local Ca2+-release in the presence of 0.1 μM Isoproterenol. Simultaneous recording of AP and Ca2+-transient allows us to probe the spatio-temporal variability of Ca2+-release, whereas the investigation of Ca2+-transient in HF discloses an unexpected uncoupling between t-tubular depolarization and Ca2+-release in remodelled tubules. This work was supported by the European Union 7th Framework Program (FP7/2007- 2013) under grant agreement n° 284464, 241526, by the Italian Ministry of University and Research (NANOMAX), and by Telethon-Italy (GGP13162). [ABSTRACT FROM AUTHOR]