Leonardo Sacconi

Understanding the three‐dimensional organisation of the myocardium requires imaging approaches that can resolve cellular structures across the entire heart. Existing methods are limited by trade‐offs between spatial resolution, imaging depth and throughput. Here we present a high‐throughput imaging pipeline for high‐fidelity reconstruction of whole mouse hearts. The approach combines a CUBIC tissue clearing protocol optimized for cardiac tissue, a dual‐camera selective plane‐illumination microscope (dual‐mesoSPIM) and an automated fusion algorithm that selects the highest‐contrast information from the opposing detection views. Whole hearts ( n = 9) were imaged label free (using myocardial autofluorescence) at a voxel size of 3.25 × 3.25 × 3 µm ³ in approximately 25 min per sample. Dual‐view fusion significantly reduces depth‐dependent information loss compared to single‐camera acquisitions, enabling robust three‐dimensional reconstruction and morphometry of cardiomyocyte orientations and laminar tissue organisation across the ventricular walls. Quantitative morphometry of fibre and sheet orientations confirmed the expected transmural organisation of the myocardium, including the progressive rotation of the helix angle and the regional variation in sheet architecture. The protocol is compatible with endogenous fluorescent reporters and whole‐mount staining, allowing, for example, the reconstruction of the cardiac sympathetic nervous system throughout the intact mouse organ. Our new pipeline provides a rapid and scalable strategy for mesoscale mapping of cardiac cytoarchitecture in physiological and pathological models. image Key points: Understanding cardiac function requires mapping the three‐dimensional arrangement of cardiomyocytes throughout the whole organ, yet reconstructing this cytoarchitecture at cellular resolution across an intact heart remains technically demanding. We combined CUBIC tissue clearing with a dual‐camera light‐sheet microscope (dual‐mesoSPIM) to reconstruct the entire adult mouse heart in three dimensions at cellular scale, rapidly and with high fidelity. Acquiring and fusing images from two opposing cameras substantially reduced the loss of information at depth, allowing the local orientation of cardiomyocytes and myocardial sheetlets to be quantified throughout the ventricular walls, including the expected transmural rotation of the fibre helix angle. The approach preserves endogenous fluorescent reporters and is compatible with whole‐heart immunostaining, which we used to reconstruct the cardiac sympathetic innervation across the entire organ, opening the way to mapping cellular organization and its remodelling in health and disease.

This study explores the use of polarized second-harmonic generation (pSHG) to investigate myosin conformation in the relaxed state, differentiating between the actin-available, disordered (ON) state and the energy-conserving, ordered (OFF) state. By shifting the ON/OFF equilibrium using both physical and chemical manipulations, we demonstrate the sensitivity of pSHG in quantifying the ON/OFF ratio in skeletal and cardiac tissues. Comparisons with X-ray diffraction measurements further validate our findings. Applying this approach to a sarcomeric mutation associated with hypertrophic cardiomyopathy, we show that R403Q/MYH7-mutated minipig ventricle tissue exhibits a higher ON fraction compared to controls. This difference is abolished under high concentrations of a myosin activator (2-deoxyATP) and an inhibitor (Mavacamten), indicating structural similarity between R403Q and controls in these two states. ATPase assays reveal increased resting ATPase activity in R403Q samples, which persists even in the presence of 2-deoxyATP, suggesting that the elevated energy consumption in the R403Q mutation is driven by both a population shift toward the ON state and enhanced myosin ATPase activity per motor head.

This study explores the use of polarized second-harmonic generation (pSHG) to investigate myosin conformation in the relaxed state, differentiating between the actin-available, disordered (ON) state and the energy-conserving, ordered (OFF) state. By shifting the ON/OFF equilibrium using both physical and chemical manipulations, we demonstrate the sensitivity of pSHG in quantifying the ON/OFF ratio in skeletal and cardiac tissues. Comparisons with X-ray diffraction measurements further validate our findings. Applying this approach to a sarcomeric mutation associated with hypertrophic cardiomyopathy, we show that R403Q/MYH7-mutated minipig ventricle tissue exhibits a higher ON fraction compared to controls. This difference is abolished under high concentrations of a myosin activator (2-deoxyATP) and an inhibitor (Mavacamten), indicating structural similarity between R403Q and controls in these two states. ATPase assays reveal increased resting ATPase activity in R403Q samples, which persists even in the presence of 2-deoxyATP, suggesting that the elevated energy consumption in the R403Q mutation is driven by both a population shift toward the ON state and enhanced myosin ATPase activity per motor head.

Background: Mavacamten, a first-in-class allosteric myosin inhibitor, has demonstrated efficacy and safety in obstructive hypertrophic cardiomyopathy (oHCM), notably reducing symptoms, left ventricular outflow obstruction, and wall thickness over 30 weeks. We recently reported that the MYBPC3 :c.772G>A variant causes HCM through cMyBP-C haploinsufficiency, leading to accelerated sarcomere kinetics and higher energy consumption in patient myocardium and hiPSC- derived cardiomyocytes (hiPSC-CMs). These effects are counterbalanced by prolonged action potentials and slower Ca²⁺ transients, which preserve twitch duration but may increase arrhythmic risk. Mavacamten may reduce myocardial energetic defects in HCM. Objectives: To investigate the long-term effects of Mavacamten on sarcomere structure, contractility, and transcriptional remodeling using patient-specific and CRISPR-corrected isogenic hiPSC-derived cardiomyocyte models of HCM. Methods: HiPSC-CMs and engineered heart tissues (EHTs) derived from a MYBPC3:c.772G>A patient and its CRISPR-corrected line were first exposed to increasing concentrations of Mavacamten to assess acute dose–response relationships and determine IC ₅₀ values. Based on these data, chronic treatments (0.3– 0.75 μM for 20 days) were performed mechanical, structural, electrophysiological, and transcriptomic adaptations. Results: Acute exposure produced a rapid and fully reversible reduction in active force, while chronic treatment for 20 days induced a sustained decrease in contractility with incomplete recovery after 4 days of washout, indicating a two-phase mechanism of action. Long-term force reduction was paralleled by decreased cell area and sarcomere density, indicating that structural disassembly contributes to sustained functional depression and re-assembly after washout. Electrophysiological analysis confirmed the specific alterations of the MYBPC3 :c.772G>A mutation previously observed, with no detectable effects following treatment with Mavacamten. In addition, transcriptome analysis was used to study the molecular mechanisms underlying the long-term effect. Conclusions: Mavacamten induces a biphasic, persistent-to-reversible, reduction of sarcomeric force associated with structural remodeling, providing mechanistic insight into its capacity to promote favorable cardiac remodeling in oHCM.

Cardiac fibrosis contributes to electrical conduction disturbances, yet its specific impact on conduction remains unclear, hindering predictive insight into cardiac electrophysiology and arrhythmogenesis. Arrhythmogenic cardiomyopathy is associated with fibrotic remodeling, and it accounts for most cases of stress-related arrhythmic sudden death. Here we develop a correlative imaging approach to integrate macroscale cardiac electrophysiology with three-dimensional microscale reconstructions of the ventricles. We apply this tool to a desmoglein-2 mutant mouse model to characterize the dynamics of conduction wavefronts and relate them to the underlying structural substrate. We observed that conduction through fibrotic tissue areas shows a frequency-dependent behavior, where conduction fails at high stimulation frequencies; this promotes reentrant arrhythmias, even in regions that were electrophysiologically inconspicuous at lower stimulation rates. We found that fibrotic areas undergo electrophysiological remodeling that acts as a low-pass filter for conduction, quantitatively explained by computational models informed by structural data. Collectively, our study provides a structure–function mapping pipeline and describes a pro-arrhythmogenic mechanism in arrhythmogenic cardiomyopathy.

Obscurin is a large muscle protein whose multiple functions include providing mechanical strength to the M-band and linking the sarcomere to the sarcoplasmic reticulum. Mutations in obscurin are linked to various forms of muscle diseases. This study compares cardiac function in a murine model of obscurin deletion (KO) with wild-type (WT) in vivo and ex vivo. Echocardiography showed that KO hearts had larger (+20%) end-diastolic and end-systolic volumes, reduced fractional shortening, and impaired ejection fraction, consistent with dilated cardiomyopathy. However, stroke volume and cardiac output were preserved due to increased end-diastolic volume. Morphological analyses revealed reduced sarcoplasmic reticulum volume, with preserved T-tubule network. While myofilament function was preserved in isolated myofibrils and skinned trabeculae, experiments in intact trabeculae revealed that Obscn KO hearts compared with WT displayed (1) reduced active tension at high frequencies and during resting-state contractions, (2) impaired positive inotropic and lusitropic response to β-adrenergic stimulation (isoproterenol 0.1 μM), and (3) faster mechanical restitution, suggesting reduced sarcoplasmic reticulum refractoriness. Intracellular [Ca2+]i measurements showed reduced peak systolic and increased diastolic levels in KO versus WT cardiomyocytes. Western blot experiments revealed lower SERCA and phospholamban (PLB) expression and reduced PLB phosphorylation in KO mice. While action potential parameters and conduction velocity were unchanged, β-adrenergic stimulation induced more frequent spontaneous Ca2+ waves and increased arrhythmia susceptibility in KO compared with WT. Taken together, these findings suggest that obscurin deletion, in adult mice, is linked to compensated dilated cardiomyopathy, altered E-C coupling, impaired response to inotropic agents, and increased propensity to arrhythmias.

This study explores the use of polarized second-harmonic generation (pSHG) to investigate myosin conformation in the relaxed state, differentiating between the actin-available, disordered (ON) state and the energy-conserving, ordered (OFF) state. By shifting the ON/OFF equilibrium using both physical and chemical manipulations, we demonstrate the sensitivity of pSHG in quantifying the ON/OFF ratio in skeletal and cardiac tissues. Comparisons with X-ray diffraction measurements further validate our findings. Applying this approach to a sarcomeric mutation associated with hypertrophic cardiomyopathy, we show that R403Q/MYH7-mutated minipig ventricle tissue exhibits a higher ON fraction compared to controls. This difference is abolished under high concentrations of a myosin activator (2-deoxyATP) and an inhibitor (Mavacamten), indicating structural similarity between R403Q and controls in these two states. ATPase assays reveal increased resting ATPase activity in R403Q samples, which persists even in the presence of 2-deoxyATP, suggesting that the elevated energy consumption in the R403Q mutation is driven by both a population shift toward the ON state and enhanced myosin ATPase activity per motor head.

Cardiac fibrosis is a key factor in electrical conduction disturbances, yet its specific impact on conduction remains unclear, hindering predictive insight of cardiac electrophysiology and arrhythmogenesis. Among the different cardiac disorders, arrhythmogenic cardiomyopathy (ACM) is known to be associated with massive fibrotic remodelling of the myocardium, and it accounts for most cases of stress-related arrhythmic sudden death. To explore ACM further, we employed a Desmoglein-2-mutant mouse model and developed a correlative imaging approach to integrate macro-scale cardiac electrophysiology with 3D micro-scale reconstructions of the ventricles, to characterise the dynamics of conduction wavefronts and relate them to the underlying structural substrate. Our findings confirm that this ACM model shows localised replacement of cardiomyocytes with collagen and non-myocytes, contributing to electrical dysfunction. Moreover, we observed that conduction through fibrotic tissue areas shows a frequency-dependent behaviour, where conduction fails at high stimulation frequencies, promoting re-entrant arrhythmias, even in regions that were electrophysiologically inconspicuous at lower stimulation rates. Using a computational model, informed by high-resolution structural data, we found that frequency-dependent conduction through fibrotic tissue cannot be explained solely by collagen deposition or cardiomyocyte re-organisation. Indeed, fibrotic areas feature electrophysiological remodelling which acts as a low-pass filter for conduction, which can be quantitatively explained by electrotonic coupling of cardiomyocytes with non-myocytes. Collectively, our study provides a novel structure-function mapping pipeline and describes a previously unrecognised pro-arrhythmogenic mechanism in ACM, underscoring the need for dynamic assessment of functional conduction block in fibrotic myocardium using multiple diagnostic pacing protocols.

Inflammatory cytokines, including interleukin 6 (IL6), are associated with ion channel remodeling and enhance the propensity to alterations in cardiac rhythm generation and propagation, in which the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a crucial role. Hence, we investigated the consequences of exposure to IL6 on HCN channels in cell models and human atrial biopsies. In murine atrial HL1 cells and in cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CMs), IL6 elicited STAT3 phosphorylation, a receptor-mediated downstream signaling. Downregulation of HCN1,2,4 by IL6 was observed after 24–48 h; in hiPS-CMs, this effect was reverted by 24 h of application of tocilizumab, a human IL6 receptor antagonist. In parallel, hiPS-CM action potentials (APs) showed a reduced spontaneous frequency. Moreover, we assessed IL6 and HCN expression in dilated left atrial samples from patients with mitral valve disease, an AF-prone condition. IL6 levels were increased in dilated atria compared to controls and positively correlated with echocardiographic atrial dimensions. Interestingly, the highest IL6 transcript levels and the lowest HCN4 and HCN2 expression were in these samples. In conclusion, our data uncovered a novel link between IL6 and cardiac HCN channels, potentially contributing to atrial electrical disturbances and a higher risk of dysrhythmias in conditions with elevated IL6 levels.