Journal of Physiology-London最新文献

Ryanodine receptor 2 (RyR2) hyperactivity is frequently observed in structural heart disease (SHD), commonly caused by ischemic heart disease. This aberrant Ca²⁺ release promotes irregular electrical activity and life-threatening arrhythmias. Our previous work demonstrated that the pan-ryanodine receptor (RyR) inhibitor dantrolene can reverse arrhythmogenic substrates, but its lack of RyR2 selectivity limits its therapeutic potential. The unnatural verticilide enantiomer (ent-verticilide) was identified as a selective RyR2 inhibitor, showing promising specificity without activating skeletal muscle RyR1. This study evaluated the antiarrhythmic potential of a selective RyR2 antagonist, ent-(+)-verticilide (ent-Vert), in human ventricular slices. Right and left ventricular slices were prepared from human hearts not designated for transplantation. Pseudo-electrocardiograms (ECG) tracked premature ventricular contraction (PVC) incidence, and optical mapping identified arrhythmogenic substrates. Baseline optical recordings were obtained before treatment with isoproterenol (Iso) (250 nM) and caffeine (200 µM) to induce Ca^{2 +} leak and arrhythmogenic activity, followed by sequential ent-Vert application (1 and 3 µM). The effects of ent-Vert alone were tested with baseline recordings followed by a single 3 µM dose. Iso and caffeine increased PVC incidence in both right (RV) and left ventricular (LV) slices, but ent-Vert (1 and 3 µM) significantly reduced it. Iso and caffeine also shortened action potential duration (APD) in both slice types, but ent-Vert, after Iso and caffeine or alone (under control conditions), did not significantly change APD. In conclusion, ent-Vert suppressed arrhythmic triggers and reduced arrhythmogenic substrates in human ventricular slices, highlighting its potential as a targeted antiarrhythmic therapy for SHD. KEY POINTS: RyR2 hyperactivity is a major driver of arrhythmias in structural heart disease, producing abnormal Ca^{2 +} release and premature ventricular contractions. The RyR2-selective inhibitor ent-Vert was evaluated in human RV and LV slices to determine its antiarrhythmic potential. β-Adrenergic and RyR2 activation with Iso + caffeine induced Ca^{2 +} leak, ectopic beats and shortened APD. Sequential ent-Vert application (1 and 3 µM) significantly suppressed ectopy incidence in a dose-dependent manner. ent-Vert did not alter APD or conduction velocity under control or stimulated conditions, demonstrating that it suppresses arrhythmic triggers without disrupting normal cardiac electrophysiology.

Ion channels are often depicted as autonomous membrane switches, yet their function depends on upstream vascular and mitochondrial processes that deliver ATP to maintain the ionic gradients essential for cellular excitability. Here, we propose a unifying framework, in the form of a capillary-mitochondria-ion channel (CMIC) axis, that links microvascular architecture to beat-to-beat performance in the heart and spike-to-spike behaviour in the brain. In this formulation, capillaries define the spatial resolution of oxygen and energy substrate delivery, while mitochondria serve as the critical intermediary that couples vascular supply to ion channel performance. CMIC coupling is critical in both cardiac function, where each cycle initiates with an electrical spike in pacemaking cells, and neural activity, where electrical spikes encode language, memories and other cognitive processes. Both neurons and cardiomyocytes have limited metabolic reserves, making them vulnerable to microvascular changes. Accordingly, these shared energetic demands, the brain and heart, exhibit similar microvascular topologies where capillary density scales with local metabolic demand. The myocardium is far more densely vascularized than the cerebral cortex, consistent with the higher energetic cost of pacemaking and contraction relative to individual neuronal spikes. Within this axis, mitochondria shape ATP waveforms to power rapid ionic gradients and Ca²⁺ cycling. Changes in any CMIC component, such as capillary rarefaction or mitochondrial dysfunction, alter energetics and thus cellular excitability, with effects ranging from adaptive to pathological depending on severity. Recognizing excitability as a product of vascular-initiated processes shifts the therapeutic focus toward preserving microvasculature function, retuning mitochondria-channel coupling and restoring capillary signalling.

Transporting human brain tissue blocks or slices from the operating theatre or on-site laboratory to an off-site laboratory may affect sample integrity for electrophysiological studies. In this study, we investigated how a 30-40 min transport influenced the intrinsic, synaptic and morphological properties of human cortical neurons. Electrophysiological recordings were performed on layer 2/3 (L2/3) pyramidal cells and fast-spiking (FS) interneurons from acute human cortical slices (n = 200 neurons from 32 surgeries, in which 112 neurons passed quality control for further analyses). Recordings were performed on-site at RWTH Aachen University Hospital and off-site at the Research Centre Jülich, which are approximately 40 km apart. Action potential (AP) firing patterns remained largely preserved across both recording sites, but several differences were observed. Off-site recorded pyramidal cells showed a depolarised resting membrane potential and a lowered rheobase current. In off-site recorded FS interneurons, we found a narrower AP half-width and an increased AP amplitude, suggesting altered ion channel kinetics and/or neuromodulatory environment. Additionally, a significant reduction in large rhythmic depolarisations and the amplitudes of spontaneous excitatory postsynaptic potentials in off-site recorded FS interneurons indicated an altered synaptic efficacy. Although overall dendritic architecture was preserved, the dendritic spine densities in apical oblique and apical tuft dendrites of off-site recorded pyramidal cells were also reduced. These findings emphasise the need for optimised transport conditions to preserve synaptic integrity, network activity and neuronal morphology. Standardised protocols are crucial for ensuring reliable and reproducible results in studies of human cortical function and structure. KEY POINTS: Effects of transportation on neuronal properties: Brief transportation of human brain tissue retains many key neuronal properties, while still exhibiting measurable alterations in certain intrinsic, synaptic and morphological properties. Mechanical stress and neuromodulator dysfunction may underlie alterations: These changes are likely due to the combined effects of mechanical stress and altered neuromodulator signalling during transportation. Advancing understanding of cortical function and structure: This research provides valuable insights into the impact of transportation on human brain tissue, advancing our understanding of cortical function and structure and highlighting the importance of optimising transport protocols to preserve tissue integrity and neuronal function.

In lowland mammals that ascend to high elevation, hypoxia-induced changes in the pulmonary circulation can give rise to hypoxic pulmonary hypertension (HPH) and associated right-ventricle (RV) hypertrophy. Some mammals that are native to high elevation have evolved a means of attenuating HPH, demonstrating how genetic mechanisms of hypoxia adaptation may sometimes counteract the effects of ancestral acclimatization responses. Here, we examine elevational variation in heart mass and measures of RV hypertrophy in four closely-related species of leaf-eared mice (genus Phyllotis) that are broadly co-distributed across a steep elevational gradient on the western slope of the Andes. All species exhibited a positive relationship between heart mass and elevation that reflected proportional changes in both the right and left ventricles. Thus, elevation-related increases in overall heart mass are not generally attributable to RV hypertrophy, suggesting that this group of predominantly highland species have evolved a means of avoiding HPH and/or attenuating the cardiac response to HPH. To gain insight into possible regulatory mechanisms, we examined patterns of transcriptomic variation in the right ventricles of Phyllotis vaccarum from two geographically distinct highland populations (both from elevations >5000 m) that exhibit strikingly different levels of RV hypertrophy. Suppression of RV hypertrophy is associated with differential expression of key regulatory genes related to striated muscle structure, immune processes, and the inflammatory response. Analysis of co-expression modules identified a promising set of candidate genes for mediating the development of RV hypertrophy at extremely high elevations. KEY POINTS: Hypoxic pulmonary hypertension (HPH) and associated right-ventricle hypertrophy are common maladies at high elevation. Some animal species that are native to especially high elevations appear to have evolved a means of attenuating the effects of HPH. Species of Andean leaf-eared mice (Phyllotis) that have extraordinarily broad elevational distributions exhibit elevational increases in overall heart mass. Elevation-related increases in heart mass are not generally attributable to right-ventricle hypertrophy, indicating that highland species of Phyllotis have evolved a means of avoiding HPH and/or attenuating the cardiac response to HPH. In populations of Phyllotis vaccarum from elevations >5000 m, analysis of co-expression modules in the right ventricle transcriptome identified candidate genes for mediating hypoxia-induced hypertrophy.

GlyH-101 is a commonly used pore blocker of the cystic fibrosis transmembrane conductance regulator (CFTR) to confirm the functional role of CFTR in model systems. Unlike most other anionic CFTR blockers GlyH-101 blocks CFTR from the extracellular side, albeit previous studies suggesting an additional internal binding site. To explore the detailed mechanism of GlyH-101 block we first examined GlyH-101's effects on a hydrolysis-deficient CFTR mutant (E1371S) whose open probability approaches unity. Whole-cell recordings with extracellularly applied GlyH-101 revealed two phases of current reduction: a rapid initial drop within seconds and a slower decay over tens of seconds. The fast phase blockade exhibited voltage dependence, consistent with previously reported external pore blocking. Single-channel recordings in excised inside-out patches with GlyH-101 in the pipette solution showed a voltage-dependent blockade with |zδ| of 0.38, close to that reported previously. However intracellular application of GlyH-101 also induced a similar voltage-dependent block, suggesting membrane permeation and subsequent development of external block. To decrease the membrane permeability of GlyH-101 we synthesised a hydrophilic analogue GlyH-101-1. Application of GlyH-101-1 from the cytoplasmic side of the membrane induced voltage-independent CFTR current inhibition. Single-channel recordings revealed two distinct shut states in the presence of cytoplasmic GlyH-101-1, consistent with the two-step blocking kinetics previously described for CFTRinh-172. Single-channel kinetics with a hydrophilic CFTRinh-172 analogue further confirmed this inhibitory mechanism. Taken together our findings establish two distinct inhibitory mechanisms for GlyH-101: a voltage-dependent on-off block through the external entrance and a two-step voltage-independent inhibition through the internal entrance. KEY POINTS: GlyH-101, a hydrophobic cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor, can diffuse across cell membranes and plug CFTR's pore from both external and internal ends. Although the external block is voltage dependent, the internal block is not. A hydrophilic GlyH-101 derivative is synthesized and shown to block CFTR's pore from its internal entrance in a voltage-independent manner. At the single-channel level our data support a two-step inhibitory mechanism: a fast binding of the blocker in the pore and a slow conformational change step following binding - a mechanism first proposed for CFTRinh-172. Although CFTRinh-172 itself did not show two-step inhibitory action, leveraging the cryo-EM structure of CFTR/CFTRinh-172 complex, we synthesized an analogue that indeed blocks CFTR through the same two-step mechanism. Our studies uncovered a common mechanism for CFTR inhibitors and underscore the potential role of structure-based drug design in developing drugs for diseases caused by hyperactive CFTR.

Accurate morphometric measurements are essential for estimating body size and condition in animals. These characteristics are, in turn, key to eco-physiological studies, wildlife management and conservation. For free-ranging cetaceans, however, collecting non-invasive morphometric data is challenging. Unoccupied aerial vehicle (UAV) photogrammetry offers a promising solution but requires ground-truthing to assess accuracy and precision. Similarly, morphometric-based indices of body condition must be validated against the animals' true body condition. Here we validated UAV-derived estimates of body size and condition in bottlenose dolphins (Tursiops spp.) under human care by comparing photogrammetry-based measurements of body length, width, height and girth from both stationary and swimming individuals with manual measurements. The two methods showed negligible differences, with UAV-based data yielding lower variability, confirming both high measurement accuracy and precision. Using UAV-derived measurements we calculated a volume-based body condition index (BCI) and compared it with a mass-based BCI, a standard metric in ecological research. The two indices showed a near-perfect fit, demonstrating that volume-based metrics reliably reflect true body condition in small cetaceans. Body density decreased with increasing body condition, consistent with higher fat-to-muscle ratios. By combining UAV-derived body volume with predicted density, based on their body condition, we accurately estimated individual body mass (mean error = 6.4%). This study provides a comprehensive validation of UAV-based photogrammetry to estimate body size, condition and mass in small cetaceans, highlighting its value as a non-invasive and cost-effective tool for ecological and conservation research. KEY POINTS: Measuring body size and condition in free-ranging dolphins is difficult, yet essential to understand their physiology, energy reserves and health. We used unoccupied aerial vehicles (UAV) to obtain accurate, non-invasive body measurements of bottlenose dolphins and compared them with direct manual measurements. UAV-based photogrammetry produced highly precise and accurate estimates of body length, girth and overall body volume, even for freely swimming animals. A UAV-derived, volume-based body condition index matched traditional mass-based indices and enabled accurate estimation of body mass. These results validate UAV photogrammetry as a reliable, ethical and cost-effective method for assessing body size, condition and mass in small cetaceans, thereby advancing ecological and physiological research in the wild.