Biology subjects

Caldwell, J. L.

Publications and source records attributed to Caldwell, J. L..

6 recordsLinked to original sources

bioRxiv2025

Multi-parametric imaging of spatio-temporal cAMP signaling, transmembrane potential, and intracellular calcium in the Langendorff-perfused heart

The spatiotemporal dynamics of intracellular second messengers and signaling molecules, including cyclic adenosine monophosphate (cAMP), have been studied extensively in isolated cardiomyocytes using Forster resonance energy transfer (FRET)-based reporters. However, little is known about how molecular signaling events affect tissue-level function. Optical mapping of transmembrane potential (Vm) and intracellular calcium (Ca2+i) is frequently performed in isolated intact hearts to reveal tissue-level electrophysiological function but cannot reveal molecular underpinnings. Here, we developed a novel multi-parametric optical imaging system that enables multi-parametric recording of four wavelengths to visualize cAMP and real-time electrophysiological responses (Vm and Ca2+i) by utilizing concurrent FRET imaging and dual optical mapping (FRE-DOM) in the intact heart from a cardiac-specific FRET-based cAMP reporter mouse. We showed that cAMP is strongly and heterogeneously activated throughout the heart (atria and ventricles) in response to {beta}-AR stimulation, the time course of which matches heart rate, action potential, and Ca2+ responses. This novel imaging system will provide insight into the relationship between cAMP signaling, electrophysiology, and arrhythmogenesis at high spatio-temporal resolution.

bioengineering↗

bioRxiv2024

Sympathetic structural and electrophysiological remodeling in a rabbit model of reperfused myocardial infarction

Chondroitin sulfate proteoglycans (CSPGs) inhibit sympathetic reinnervation in rodent hearts post myocardial infarction (MI), causing regional hypo-innervation that is associated with supersensitivity of {beta}-adrenergic receptors and increased arrhythmia susceptibility. To investigate the role of CSPGs and hypo-innervation in the heart of larger mammals, we used a rabbit model of reperfused MI and tested electrophysiological responses to sympathetic nerve stimulation (SNS). Innervated hearts from MI and sham rabbits were optically mapped using voltage and Ca2+-sensitive dyes. SNS was performed with electrical stimulation of the spinal cord and {beta}-adrenergic responsiveness was tested using isoproterenol. Sympathetic nerve density and CSPG expression were evaluated using immunohistochemistry. CSPGs were robustly expressed in the infarct and border zone of all MI hearts, and the presence of CSPGs was associated with reduced sympathetic nerve density in the infarct vs. remote region. Action potential duration (APD) dispersion and susceptibility to ventricular tachycardia/fibrillation (VT/VF) were increased with SNS in MI hearts but not in sham. SNS decreased APD80 in MI but not sham hearts, while isoproterenol decreased APD80 in both groups. Isoproterenol also shortened Ca2+ transient duration (CaTD80) in both groups but to a greater extent in MI hearts. Our data suggest sympathetic remodeling post-MI is similar between species, with CSPGs associated with sympathetic hypo-innervation. Despite a reduction in sympathetic nerve density, the infarct region of MI hearts remained responsive to both physiological SNS and isoproterenol, potentially through preserved or elevated {beta}-adrenergic responsiveness, which may underly increased APD dispersion and susceptibility for VT/VF. NEW & NOTEWORTHYHere we show that CSPGs are present in the infarcts of rabbit hearts with reperfused MI, where they are associated with reduced sympathetic nerve density. Despite hypo-innervation, sympathetic responsiveness is maintained or enhanced in MI rabbit hearts, which also demonstrate increased APD dispersion and tendency for arrhythmias following sympathetic modulation. Together, this study indicates that the mechanisms of sympathetic remodeling post-MI are similar between species, with hypo-innervation likely associated with enhanced {beta}-adrenergic sensitivity.

physiology↗

bioRxiv2023

Chronic nicotine exposure is associated with electrophysiological and sympathetic remodeling in the intact rabbit heart

Nicotine is the primary addictive component in tobacco products. Through its actions on the heart and autonomic nervous system, nicotine exposure is associated with electrophysiological changes and increased arrhythmia susceptibility. However, the underlying mechanisms are unclear. To address this, we treated rabbits with transdermal nicotine (NIC, 21 mg/day) or control (CT) patches for 28 days prior to performing dual optical mapping of transmembrane potential (RH237) and intracellular Ca2+ (Rhod-2 AM) in isolated hearts with intact sympathetic innervation. Sympathetic nerve stimulation (SNS) was performed at the 1st - 3rd thoracic vertebrae, and {beta}-adrenergic responsiveness was additionally evaluated as changes in heart rate (HR) following norepinephrine (NE) perfusion. Baseline ex vivo HR and SNS stimulation threshold were increased in NIC vs. CT (P = 0.004 and P = 0.003 respectively). Action potential duration alternans emerged at longer pacing cycle lengths (PCL) in NIC vs. CT at baseline (P = 0.002) and during SNS (P = 0.0003), with similar results obtained for Ca2+ transient alternans. SNS reduced the PCL at which alternans emerged in CT but not NIC hearts. NIC exposed hearts also tended to have slower and reduced HR responses to NE perfusion. While fibrosis was unaltered, NIC hearts had lower sympathetic nerve density (P = 0.03) but no difference in NE content vs. CT. These results suggest both sympathetic hypo-innervation of the myocardium and diminished {beta}-adrenergic responsiveness with NIC. This autonomic remodeling may underlie the increased risk of arrhythmias associated with nicotine exposure, which may be further exacerbated with continued long-term usage. NEW & NOTEWORTHYHere we show that chronic nicotine exposure was associated with increased heart rate, lower threshold for alternans and reduced sympathetic electrophysiological responses in the intact rabbit heart. We suggest that this was due to the sympathetic hypo-innervation of the myocardium and diminished {beta}- adrenergic responsiveness observed following nicotine treatment. Though these differences did not result in increased arrhythmia propensity in our study, we hypothesize that prolonged nicotine exposure may exacerbate this pro-arrhythmic remodeling.

physiology↗

bioRxiv2023

Atrial t-tubules adopt a specialist developmental state while alterations to Ca2+ buffering maintain systolic Ca2+ during postnatal development

Transverse (t)-tubules ensure a uniform rise in calcium (Ca2+) and thus contraction in cardiac cells. Though more extensively studied in the ventricle, t-tubules also play a key role in the atria of large mammals, such as human, and their loss in heart failure is associated with impaired Ca2+ release and thus contractility. T-tubule restoration is therefore an ideal therapeutic target but the process of t-tubule formation is not understood. The aim of this study was to determine how t-tubules develop in the healthy atria and the impact this has on Ca2+ handling. Postnatal development was assessed in sheep from newborn through to adulthood. Atrial t-tubules were present at birth in the sheep atria and increased in density up until 3 months of age. In the latter part of development (3 months to adult) a lack of t-tubule growth but increase in cell width results in t-tubule density decreasing. In the newborn, despite reduced t-tubule density, we found the amplitude of the Ca2+ transient was maintained and this was associated with increases in the L-type Ca2+ current (ICa-L) and the Ca2+ content of the sarcoplasmic reticulum (SR). We suggest these changes are sufficient to overcome the elevated cytosolic Ca2+ buffering in the newborn and the decreased t-tubule density. We have shown the neonate atria is highly specialised to negate reduced central Ca2+ release through enhanced surface ICa-L and SR load. This maintains atrial function despite immature t-tubules highlighting important differences in Ca2+ handling in the newborn and heart failure atria where t-tubules are sparse.

physiology↗

bioRxiv2021

Disordered yet functional atrial t-tubules on recovery from heart failure

Transverse (t)-tubules drive the rapid and synchronous Ca2+ rise in cardiac myocytes. The virtual complete loss of atrial t-tubules in heart failure (HF) decreases Ca2+ release. It is unknown if or how atrial t-tubules can be restored and if restored t-tubules are functional. Sheep were tachypaced to induce HF and recovered when pacing was stopped. Serial block face Scanning Electron Microscopy and confocal imaging were used to understand t-tubule ultrastructure and function. Candidate proteins involved in atrial t-tubule recovery were identified by western blot and causality determined using expression studies. Sheep atrial t-tubules reappeared following recovery from HF. Despite being disordered (branched, longer and longitudinally arranged) recovered t-tubules triggered Ca2+ release and were associated with restoration of systolic Ca2+. Telethonin and myotubularin abundance correlated with t-tubule density and altered the density and structure of BIN1-driven tubules in neonatal myocytes. Myotubularin had a greater effect, increasing tubule length and branching, replicating that seen in the recovery atria. Recovery from HF restores atrial t-tubules and systolic Ca2+ and myotubularin facilitates this process. Atrial t-tubule restoration could present a new and viable therapeutic strategy. Brief SummaryThe loss of atrial transverse (t)-tubules and the associated dysfunction in heart failure is reversible and the protein myotubularin plays an important role.

physiology↗

bioRxiv2018

PDE5 inhibition improves symptom-free survival and restores transverse tubule loss and catecholamine responsiveness in heart failure

Heart failure is characterized by poor survival, a loss of catecholamine reserve and cellular structural remodeling in the form of disorganization and loss of the transverse tubule network. Indeed, survival rates for heart failure are worse than many common cancers and have not improved over time. Tadalafil is a clinically relevant drug that blocks phosphodiesterase 5 with high specificity and is used to treat erectile dysfunction. Using a sheep model of advanced heart failure, we show that tadalafil treatment results in a marked improvement in symptom-free survival, reverses transverse tubule loss and restore cardiac myocyte systolic calcium transient amplitude and the hearts chronotropic and contractile response to catecholamines. These effects are independent of changes in myocardial cGMP content and are associated with upregulation of both monomeric and dimerized forms of protein kinase G and of the cGMP hydrolyzing phosphodiesterase 2 and 3. We propose that the molecular switch for the loss of transverse tubules in heart failure and their restoration following tadalafil treatment involves the BAR domain protein Amphiphysin II (BIN1) and the restoration of catecholamine sensitivity is through reductions in G-protein receptor kinase 2, protein phosphatase 1 and protein phosphatase 2A abundance following phosphodiesterase 5 inhibition.

physiology↗