Claudia Crocini

Defects in T-tubular electrical activity underlie local alterations of calcium release in heart failure

Significance The plasma membrane of cardiac myocytes contains complex invaginations known as transverse tubules (T-tubules). In heart failure, T-tubule loss is a major contributor to Ca2+ transient abnormalities, leading to weaker and slower contraction. Current therapeutic strategies are often based on attempts to accelerate Ca2+ transients. Here, we demonstrate that T-tubular loss represents just one way by which T-tubule dysfunction leads to asynchronous Ca2+ release across the myocyte. In fact, we report that defects in T-tubular electrical activity may contribute to Ca2+-mediated arrhythmogenesis not only by favoring asynchronous Ca2+ release, but also by generating voltage-associated Ca2+ sparks. This work provides the first description to our knowledge of these novel proarrhythmogenic events that could help guide future therapeutic strategies. 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 previously documented the presence of TATS elements which failed to propagate AP and displayed spontaneous electrical activity; the consequence for Ca2+ release remained, however, unsolved. Here, 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.

The transverse-axial tubular system of cardiomyocytes

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 Ca2+ 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 Ca2+-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.

Optogenetics design of mechanistically-based stimulation patterns for cardiac defibrillation

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 was developed to optically map action potential propagation with a red-shifted 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 sub-millisecond temporal resolution allowing precise epicardial activation of Channelrhodopsin2 (ChR2). We employed this optical system in the setting of ventricular tachycardia to optimize mechanistic, multi-barrier cardioversion/defibrillation patterns. Multiple regions of conduction block were created with a very high cardioversion efficiency but with lower energy requirements as compared to whole ventricle interventions to interrupt arrhythmias. This work demonstrates that defibrillation energies can be substantially reduced by applying discrete stimulation patterns and promotes the progress of current anti-arrhythmic strategies.

Novel insights on the relationship between T-tubular defects and contractile dysfunction in a mouse model of hypertrophic cardiomyopathy

Abnormalities of cardiomyocyte Ca2 + homeostasis and excitation–contraction (E–C) coupling are early events in the pathogenesis of hypertrophic cardiomyopathy (HCM) and concomitant determinants of the diastolic dysfunction and arrhythmias typical of the disease. T-tubule remodelling has been reported to occur in HCM but little is known about its role in the E–C coupling alterations of HCM. Here, the role of T-tubule remodelling in the electro-mechanical dysfunction associated to HCM is investigated in the Δ160E cTnT mouse model that expresses a clinically-relevant HCM mutation. Contractile function of intact ventricular trabeculae is assessed in Δ160E mice and wild-type siblings. As compared with wild-type, Δ160E trabeculae show prolonged kinetics of force development and relaxation, blunted force-frequency response with reduced active tension at high stimulation frequency, and increased occurrence of spontaneous contractions. Consistently, prolonged Ca2 + transient in terms of rise and duration are also observed in Δ160E trabeculae and isolated cardiomyocytes. Confocal imaging in cells isolated from Δ160E mice reveals significant, though modest, remodelling of T-tubular architecture. A two-photon random access microscope is employed to dissect the spatio-temporal relationship between T-tubular electrical activity and local Ca2 + release in isolated cardiomyocytes. In Δ160E cardiomyocytes, a significant number of T-tubules (> 20%) fails to propagate action potentials, with consequent delay of local Ca2 + release. At variance with wild-type, we also observe significantly increased variability of local Ca2 + transient rise as well as higher Ca2 +-spark frequency. Although T-tubule structural remodelling in Δ160E myocytes is modest, T-tubule functional defects determine non-homogeneous Ca2 + release and delayed myofilament activation that significantly contribute to mechanical dysfunction.

Ranolazine Prevents Phenotype Development in a Mouse Model of Hypertrophic Cardiomyopathy.

Background— Current therapies are ineffective in preventing the development of cardiac phenotype in young carriers of mutations associated with hypertrophic cardiomyopathy (HCM). Ranolazine, a late Na+ current blocker, reduced the electromechanical dysfunction of human HCM myocardium in vitro. Methods and Results— To test whether long-term treatment prevents cardiomyopathy in vivo, transgenic mice harboring the R92Q troponin-T mutation and wild-type littermates received an oral lifelong treatment with ranolazine and were compared with age-matched vehicle-treated animals. In 12-months-old male R92Q mice, ranolazine at therapeutic plasma concentrations prevented the development of HCM-related cardiac phenotype, including thickening of the interventricular septum, left ventricular volume reduction, left ventricular hypercontractility, diastolic dysfunction, left-atrial enlargement and left ventricular fibrosis, as evaluated in vivo using echocardiography and magnetic resonance. Left ventricular cardiomyocytes from vehicle-treated R92Q mice showed marked excitation–contraction coupling abnormalities, including increased diastolic [Ca2+] and Ca2+ waves, whereas cells from treated mutants were undistinguishable from those from wild-type mice. Intact trabeculae from vehicle-treated mutants displayed inotropic insufficiency, increased diastolic tension, and premature contractions; ranolazine treatment counteracted the development of myocardial mechanical abnormalities. In mutant myocytes, ranolazine inhibited the enhanced late Na+ current and reduced intracellular [Na+] and diastolic [Ca2+], ultimately preventing the pathological increase of calmodulin kinase activity in treated mice. Conclusions— Owing to the sustained reduction of intracellular Ca2+ and calmodulin kinase activity, ranolazine prevented the development of morphological and functional cardiac phenotype in mice carrying a clinically relevant HCM-related mutation. Pharmacological inhibitors of late Na+ current are promising candidates for an early preventive therapy in young phenotype-negative subjects carrying high-risk HCM-related mutations.

Quantitative assessment of passive electrical properties of the cardiac T-tubular system by FRAP microscopy

Significance The homogenous propagation of the action potential in cardiac cells is guaranteed by a complex network of membrane invaginations called the T-tubular system. In cardiac diseases, T-tubules may show electrical defects that can compromise cell function. Here, we investigate the diffusional properties of fluorescent probes inside T-tubules to predict electrical conductivity of the tubular network. We apply this method to detecting alterations of T-tubule conductivity in a pathological setting characterized by compromised T-tubule integrity. We found that in heart failure, T-tubule conductivity is significantly reduced compared with healthy cardiac cells. A reduction in conductivity can impair the propagation of action potential across the network and may explain the presence of conduction defects found at the single tubular level. 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 uniform excitation–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.

Functional cardiac imaging by random access microscopy

Advances in the development of voltage sensitive dyes and Ca2+ sensors in combination with innovative microscopy techniques allowed researchers to perform functional measurements with an unprecedented spatial and temporal resolution. At the moment, one of the shortcomings of available technologies is their incapability of imaging multiple fast phenomena while controlling the biological determinants involved. In the near future, ultrafast deflectors can be used to rapidly scan laser beams across the sample, performing optical measurements of action potential and Ca2+ release from multiple sites within cardiac cells and tissues. The same scanning modality could also be used to control local Ca2+ release and membrane electrical activity by activation of caged compounds and light-gated ion channels. With this approach, local Ca2+ or voltage perturbations could be induced, simulating arrhythmogenic events, and their impact on physiological cell activity could be explored. The development of this optical methodology will provide fundamental insights in cardiac disease, boosting new therapeutic strategies, and, more generally, it will represent a new approach for the investigation of the physiology of excitable cells.

Optogenetics gets to the heart: A guiding light beyond defibrillation

Optogenetics provides a tool for controlling the electrical activity of excitable cells by means of the interaction of light with light-gated ion channels. Despite the fact that optogenetics has been intensively utilized in the neurosciences, it has been more rarely employed as an instrument for studying cardiac pathophysiology. However, the advantages of optical approaches to perturb cardiac electrical activity are numerous, especially when the spatio-temporal qualities of light are utterly exploited. Here, we review the main breakthroughs employing optogenetics to perturb cardiac pathophysiology and attempt a comparison of methods and procedures that have employed optogenetics in the heart. We particularly focus on light-based defibrillation strategies that represent one of the latest achievements in this field. We highlight the important role of advanced optical methods for detecting and stimulating electrical activity for optimizing defibrillation strategies and, more generally, for dissecting novel insights in cardiac physiology. Finally, we discuss the main future perspectives that we envision for optogenetics in the heart, both in terms of translational applications and for addressing fundamental questions of cardiac function.

T-Tubular Electrical Defects Contribute to Blunted β-Adrenergic Response in Heart Failure

Alterations of the β-adrenergic signalling, structural remodelling, and electrical failure of T-tubules are hallmarks of heart failure (HF). Here, we assess the effect of β-adrenoceptor activation on local Ca2+ release in electrically coupled and uncoupled T-tubules in ventricular myocytes from HF rats. We employ an ultrafast random access multi-photon (RAMP) microscope to simultaneously record action potentials and Ca2+ transients from multiple T-tubules in ventricular cardiomyocytes from a HF rat model of coronary ligation compared to sham-operated rats as a control. We confirmed that β-adrenergic stimulation increases the frequency of Ca2+ sparks, reduces Ca2+ transient variability, and hastens the decay of Ca2+ transients: all these effects are similarly exerted by β-adrenergic stimulation in control and HF cardiomyocytes. Conversely, β-adrenergic stimulation in HF cells accelerates a Ca2+ rise exclusively in the proximity of T-tubules that regularly conduct the action potential. The delayed Ca2+ rise found at T-tubules that fail to conduct the action potential is instead not affected by β-adrenergic signalling. Taken together, these findings indicate that HF cells globally respond to β-adrenergic stimulation, except at T-tubules that fail to conduct action potentials, where the blunted effect of the β-adrenergic signalling may be directly caused by the lack of electrical activity.

Electrical defects of the transverse‐axial tubular system in cardiac diseases

Electrical excitability is an essential feature of cardiomyocytes and the homogenous propagation of the action potential is guaranteed by a complex network of membrane invaginations called the transverse‐axial tubular system (TATS). TATS structural remodelling is a hallmark of cardiac diseases and we demonstrated that this can be accompanied by electrical defects at single T‐tubular level. Using a random‐access multi‐photon (RAMP) microscope, we found that pathological T‐tubules can fail to conduct action potentials, which delays local Ca2+ release. Although the underlying causes for T‐tubular electrical failure are still unknown, our findings suggest that they are likely to be related to local ultrastructural alterations. Here, we first review the experimental approach that allowed us to observe and dissect the consequences of TATS electrical dysfunction and then propose two different strategies to unveil the reasons for T‐tubular electrical failures. The first strategy consists in a correlative approach, in which the failing T‐tubule identified with the RAMP microscope is then imaged with electron microscopy. The second approach exploits the diffusion of molecules within TATS to gain insights into the local TATS structure, even without a thorough reconstruction of the tubular network. Although challenging, the local electrical failure occurring at single T‐tubules is a fundamental question that needs to be addressed and could provide novel insights in cardiac pathophysiology.