Circulating histones released from damaged cells cause secondary impairment to organs, including the heart. Here, we investigate their effects and the underlying pathological mechanisms on isolated ventricular cardiomyocytes. Exogenous H3 or H4 (0.01–1 µg ml−1) induced progressive membrane depolarization, action potential prolongation, membrane potential oscillations resembling early afterdepolarizations, and Ca2+ dysregulation. These changes resulted from a histone-induced nonspecific depolarizing current with a linear current voltage relationship, consistent with the formation of transient pores in the sarcolemma. These effects were replicated by the addition of endogenous H3 isolated from media surrounding myocytes where necrosis has been induced. Acetylation by histone deacetylase inhibitors improved cell viability, reduced and delayed the electrophysiological changes observed with nonacetylated H3. Recombinant pan-acetyl H3 eliminated lethal depolarization. Complexes of histone and histone antibodies produced smaller depolarizations after longer exposures and delayed electrophysiological response. This is consistent with neutralization of the positive charges associated with the histone and suggest a novel therapeutic strategy to limit pathology resulting from histone toxicity.

Carbon monoxide (CO) exposure can result in cardiovascular complications including arrhythmias. Previous work has shown that CO induces early afterdepolarization arrhythmias (EADs) in rat or guinea pig ventricular myocytes. However, in rat, the EADs were caused by activation of the late Na current (INalate) while in guinea pig, the dominant effect was inhibition of the ERG K+ channel. Here, we examined CO effects on iCell2 human induced pluripotent stem cell derived cardiomyocytes (hiPSC). CO was applied as CORM-2 (a CO releasing molecule; 10 μM) and its inactive form (iCORM; 10 μM) served as control.

iCell2 cardiomyocytes action potentials (AP) can be classified into atrial and ventricular like-phenotypes, both of which can exhibit endogenous activity. Here we characterise the variability of ICaL in these phenotypes. Voltage clamp and action potential recording were obtained from iCell2 prepared as instructed by the manufacturer (FujiFilm cellular dynamics) at room temperature. APs were modelled by modified O'Hara-Rudy (ORd) model with 5% variability in maximal conductance values. Currents obtained from rectangular voltage clamp, and computed by AP clamp of ORd model. Atrial shaped APs were paced at 1 Hz show stationary APDs, with APD50 and APD90 of 102 ± 6.4 and 257 ± 10.5 ms (N = 25) and ventricular-like AP 90.9 ± 0.62 and ± 143.9 0.64 ms (N = 28) When AP clamped by these recorded APs the mean modified ORd model produces local maxima of ICaL of −7.23 ± 0.36 during the upswing of the action potential, and of −5.99 ± 0.23 μA/μF during the plateau for ventricular-like APs; and −10.88 ± 3.78 and −2.64 ± 0.16 μA/μF, for atrial-like AP (N = 10). Accumulative peak value for the ventricular cell population is −10.45 ±1.87 during the upswing and −3.79 ±1.97 (n = 6) during the plateau. In the atrial like cells, accumulative peak during the upswing −12.02 ± 5.7 and plateau −3.73 ± 1.08 μA/μF (n = 4). There are no significant differences between the atrial and ventricular-like magnitudes of these ICaL. Rectangular voltage clamp experiments give a peak ICaL with maxima of −13.22 ± 7.84 pA/pF, (n = 7), at a holding potential of 0mV. The experimental and computed magnitudes of ICaL are similar. However, the ICaL has local maxima during the upswing and repolarising phase of the AP; in the ventricular-like cells the second local maximum has moved towards the spike phase of the AP, and decays slowly the plateau and more rapidly during repolarisation.

Histones are highly basic proteins which are a major component of nuclear chromatin. In sepsis, or following blunt trauma, histones released from damaged cells have been implicated in secondary damage to organs including the heart. We investigated the mechanism of H3 histone toxicity in ventricular myocytes from Wistar rats. Animals were sacrificed in accordance with the UK Home Office Guidance on the Operation of Animals Act of 1986. "n" = cells, number of hearts is indicated in parentheses. Data are mean ± SEM, significance was determined using Student's t-test. H3 (5μg ml−1) induced membrane damage and cell necrosis. After 10 min exposure, topographical images obtained using scanning ion conductance microscopy showed fine dark lines consistent with "micro-tears" in the membrane. H3 effects on membrane potential (Em) were assessed using whole-cell current clamp. H3 application (1, 0.1, or 0.01 μg ml−1) induced dose and time dependent depolarisation of Em from control (in mV; −79.3 ± 0.79; n = 31(15)) to −42.36 ± 2.88 in 127.15 ± 27.52 sec (n = 14 (9)); −38.51 ± 4.98 in 218 ± 64.06 sec (n = 9 (7)) and to −43.84 ± 5.33 in 236.57 ± 68.89 sec (n = 8 (4)) respectively. Spontaneous action potentials (APs) and the development of early afterdepolarisations were detected at all H3 concentrations. Low levels of H3 (0.1–0.01 μg ml−1) increased AP duration at 90% of peak amplitude (APD0.9) from (ms) 66.55 ± 13.71 to 85.2 ± 14.91 (n = 9 (7); p < 0.05) and from 77.78 ± 17.56 to 102.83 ± 24.98 (n = 8 (4); p < 0.05). These data show that H3 induces membrane damage, myocyte depolarisation and pro-arrhythmic changes in myocyte APs. These effects may contribute to cardiac dysfunction a rise in circulating histones. This work was funded by the British Heart Foundation.

Sunitinib malate is known to cause cardiotoxicity in a sub-population of patients, with heart failure seen in more severe cases. Cardiac progenitor cells (CPCs) have been identified in adult human myocardium and contribute to overall tissue maintenance, with previous work identifying negative impacts of sunitinib on these cells. This study aimed to characterise the toxic effects of sunitinib in human CPCs, applying sunitinib concentrations equivalent to clinical plasma levels to these cells in vitro. Cell viability was reduced by 26.5 ± 6.6 % by 2 μM sunitinib for 24 h (p < 0.01); this concentration also induced fold-change increases in gene expression of: calpain (3.1 ± 0.73, p < 0.05), FAS (2.3 ± 0.8, p < 0.05) and BAX (1.9 ± 0.2, p < 0.05), and a decrease in BCL-2 (3.5 ± 0.0, p < 0.001), vs. control (1.0 ± 0.0). This was affirmed by sunitinib inducing fold changes in protein expression of: calpain-1 (2.5 ± 0.5, p < 0.05); FAS receptor (2.1 ± 0.2, p < 0.05) and BAX (2.1 ± 0.2, p < 0.05) vs. control (1.0 ± 0.0). These results indicated that sunitinib induced apoptosis in CPCs, but negative annexin V staining and lack of protection by caspase inhibitors indicated this was not the cell death pathway activated. Further investigation found sunitinib was concentrated in the lysosomes and autophagosomes within CPCs, but did not induce accumulation of acidic organelles. In conclusion, these data confirm that cell death is caused by sunitinib in CPCs at concentrations equivalent to clinical plasma levels, inducing cell death pathway signals that lead to non-apoptotic cell death.

Rapid release of calcium from internal stores via ryanodine receptors (RyRs) is one of the fastest types of cytoplasmic second messenger signalling in excitable cells. In the heart, rapid summation of the elementary events of calcium release, 'calcium sparks', determine the contraction of the myocardium. We adapted a correlative super-resolution microscopy protocol to correlate sub-plasmalemmal spontaneous calcium sparks in rat right ventricular myocytes with the local nanoscale RyR2 positions. This revealed a steep relationship between the integral of a calcium spark and the sum of the local RyR2s. Segmentation of recurring spark sites showed evidence of repeated and triggered saltatory activation of multiple local RyR2 clusters. In myocytes taken from failing right ventricles, RyR2 clusters themselves showed a dissipated morphology and fragmented (smaller) clusters. They also featured greater heterogeneity in both the spark properties and the relationship between the integral of the calcium spark and the local ensemble of RyR2s. While fragmented (smaller) RyR2 clusters were rarely observed directly underlying the larger sparks or the recurring spark sites, local interrogation of the channel-to-channel distances confirmed a clear link between the positions of each calcium spark and the tight, non-random clustering of the local RyR2 in both healthy and failing ventricles.

The voltage-gated K+ channel plays a key role in atrial excitability, conducting the ultra-rapid rectifier K+ current (IKur) and contributing to the repolarization of the atrial action potential. In this study, we examine its regulation by hydrogen sulfide (H2S) in HL-1 cardiomyocytes and in HEK293 cells expressing human Kv1.5. Pacing induced remodeling resulted in shorting action potential duration, enhanced both Kv1.5 channel and H2S producing enzymes protein expression in HL-1 cardiomyocytes. H2S supplementation reduced these remodeling changes and restored action potential duration through inhibition of Kv1.5 channel. H2S also inhibited recombinant hKv1.5, lead to nitric oxide (NO) mediated S-nitrosylation and activated endothelial nitric oxide synthase (eNOS) by increased phosphorylation of Ser1177, prevention of NO formation precluded these effects. Regulation of Ikur by H2S has important cardiovascular implications and represents a novel and potential therapeutic target.

Background and purpose: While flecainide is now an accepted treatment for arrhythmias associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), its mechanism of action remains controversial. In studies on myocytes from CPVT mice, inhibition of proarrhythmic Ca2+ waves was initially attributed to a novel action on the type-2 ryanodine receptor (RyR2). However, subsequent work on wild type (WT) myocytes questioned the conclusion that flecainide has a direct action on RyR2. In the present study, the effects of flecainide were compared in intact and permeabilized WT myocytes.Experimental approach: Intracellular Ca2+ was measured using confocal microscopy in intact or saponin permeabilized adult rat ventricular myocytes (ARVM). In some experiments on permeabilized cells, flecainide was studied following partial inhibition of the sarcoplasmic reticulum (SR) counter-current.Key results: Flecainide induced sustained changes Ca2+ sparks and waves in permeabilized ARVM, which were comparable to those reported in intact or permeabilized myocytes from CPVT mice. However, a relatively high level of flecainide (25 μM) was required to induce these effects. Inhibition of the SR counter-current potentiated the effects of flecainide on SR Ca2+ waves. In intact field stimulated ARVM, prolonged exposure to 15 μM flecainide decreased wave frequency but RyR2 dependent effects on Ca2+ sparks were absent; higher drug concentrations blocked field stimulation, consistent with inhibition of Nav1.5.Conclusions and implications: In intact ARVM, the absence of effects on Ca2+ sparks suggests that the intracellular flecainide concentration was insufficient to influence RyR2. Wave inhibition in intact ARVM may reflect secondary effects of Nav1.5 inhibition. Potentiation of flecainide’s action by counter-current inhibition can be explained if transient polarization of the SR membrane during SR Ca2+ release facilitates its action on RyR2.

The heart is a biomechanical pump at the centre of our cir-culatory system. It contracts rhythmically from approxi-mately 6 weeks of gestational age until death.1 Contractions are initiated by action potentials, arising from pacemaker cells, transmitted to individual cardiomyocytes via specialised conduction pathways, through intercalated discs and gap junctions between cells.2 The arrival of this electrical signal results in excitation-contraction coupling, sarcomere and cellular shortening and ejection of blood.3 In this article we describe the cellular processes involved in the activation and mechanics of cardiac muscle contraction and the metabolism fuelling this activity.

Coordinated events of calcium (Ca2+) released from the endoplasmic reticulum (ER) are key second messengers in excitable cells. In pain-sensing dorsal root ganglion (DRG) neurons, these events can be observed as Ca2+ sparks, produced by a combination of ryanodine receptors (RyR) and inositol 1,4,5-triphosphate receptors (IP3R1). These microscopic signals offer the neuronal cells with a possible means of modulating the subplasmalemmal Ca2+ handling, initiating vesicular exocytosis. With super-resolution dSTORM and expansion microscopies, we visualised the nanoscale distributions of both RyR and IP3R1 that featured loosely organised clusters in the subplasmalemmal regions of cultured rat DRG somata. We adapted a novel correlative microscopy protocol to examine the nanoscale patterns of RyR and IP3R1 in the locality of each Ca2+ spark. We found that most subplasmalemmal sparks correlated with relatively small groups of RyR whilst larger sparks were often associated with larger groups of IP3R1. These data also showed spontaneous Ca2+ sparks in <30% of the subplasmalemmal cell area but consisted of both these channel species at a 3.8–5 times higher density than in nonactive regions of the cell. Taken together, these observations reveal distinct patterns and length scales of RyR and IP3R1 co-clustering at contact sites between the ER and the surface plasmalemma that encode the positions and the quantity of Ca2+ released at each Ca2+ spark.

Purpose: Current understanding of ventricular action potential adaptation to physiological stress is generally based on protocols using non-physiological rates and conditions isolating rate effects from escalating adrenergic stimulation. To permit refined understanding, ventricular action potentials were assessed across physiological pacing frequencies in the presence and absence of adrenergic stimuli. Isolated and combined effects were analyzed to assess their ability to replicate in-vivo responses. Methods: Steady-state action potentials from ventricular myocytes isolated from male Wistar rats (3 months; N = 8 animals) were recorded at 37 degrees C with steady-state pacing at 1, 2, 4, 6, 8 and 10 Hz using whole-cell patch-clamp. Action potential repolarization to 25, 50, 75, 90 and 100% of full repolarization (APD(25-100)) was compared before and after 5 nM, 100 nM and 1 mu M isoproterenol doses. Results: A Repeated measures ANOVA found APD(50-90) shortened with 5 nM isoproterenol infusion by 6-25% (but comparable across doses) (p <= 0.03). Pacing frequencies emulating a normal rat heart rate (6 Hz) prolonged APD(50) 23% compared with 1 Hz pacing. Frequencies emulating exercise or stress (10 Hz) shortened APD(90) (29%). Conclusion: These results demonstrate modest action potential shortening in response to adrenergic stimulation and elevations in pacing beyond physiological resting rates. Our findings indicate changes in action potential plateau and late repolarization predominantly underlie simulated exercise responses in the rat heart. This work provides novel action potential reference data and will help model cardiac responses to physiological stimuli in the rat heart via computational techniques.

The receptor tyrosine kinase inhibitor imatinib improves patient cancer survival but is linked to cardiotoxicity. This study investigated imatinib’s effects on cell viability, apoptosis, autophagy, and necroptosis in human cardiac progenitor cells in vitro. Imatinib reduced cell viability (75.9 ± 2.7% vs. 100.0 ± 0.0%) at concentrations comparable to peak plasma levels (10 µM). Imatinib reduced cells’ TMRM fluorescence (74.6 ± 6.5% vs. 100.0 ± 0.0%), consistent with mitochondrial depolarisation. Imatinib increased lysosome and autophagosome content as indicated by LAMP2 expression (2.4 ± 0.3-fold) and acridine orange fluorescence (46.0 ± 5.4% vs. 9.0 ± 3.0), respectively. Although imatinib increased expression of autophagy-associated proteins and also impaired autophagic flux, shown by proximity ligation assay staining for LAMP2 and LC3II (autophagosome marker): 48 h of imatinib treatment reduced visible puncta to 2.7 ± 0.7/cell from 11.3 ± 2.1 puncta/cell in the control. Cell viability was partially recovered by autophagosome inhibition by wortmannin, with the viability increasing 91.8 ± 8.2% after imatinib-wortmannin co-treatment (84 ± 1.5% after imatinib). Imatinib-induced necroptosis was associated with an 8.5 ± 2.5-fold increase in mixed lineage kinase domain-like pseudokinase activation. Imatinib-induced toxicity was rescued by RIP1 inhibition: 88.6 ± 3.0% vs. 100.0 ± 0.0% in the control. Imatinib applied to human cardiac progenitor cells depolarises mitochondria and induces cell death through necroptosis, recoverable by RIP1 inhibition, with a partial role for autophagy.

Hydrogen sulfide (H2S) is gaining interest as a mammalian signalling molecule with wide ranging effects. S-sulfhydration is one mechanism that is emerging as a key post translational modification through which H2S acts. Ion channels and neuronal receptors are key target proteins for S-sulfhydration and this can influence a range of neuronal functions. Voltage-gated K+ channels, including Kv2.1, are fundamental components of neuronal excitability. Here, we show that both recombinant and native rat Kv2.1 channels are inhibited by the H2S donors, NaHS and GYY4137. Biochemical investigations revealed that NaHS treatment leads to S-sulfhydration of the full length wild type Kv2.1 protein which was absent (as was functional regulation by H2S) in the C73A mutant form of the channel. Functional experiments utilising primary rat hippocampal neurons indicated that NaHS augments action potential firing and thereby increases neuronal excitability. These studies highlight an important role for H2S in shaping cellular excitability through S-sulfhydration of Kv2.1 at C73 within the central nervous system.

Nanometre-scale cellular information obtained through super-resolution microscopies are often unaccompanied by functional information, particularly transient and diffusible signals through which life is orchestrated in the nano-micrometre spatial scale. We describe a correlative imaging protocol which allows the ubiquitous intracellular second messenger, calcium (Ca2+), to be directly visualised against nanoscale patterns of the ryanodine receptor (RyR) Ca2+ channels which give rise to these Ca2+ signals in wildtype primary cells. This was achieved by combining total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals, with the subsequent DNA-PAINT imaging of the RyRs. We report a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets and demonstrate how such data can be used to visually identify the ensembles of Ca2+ channels that are locally activated during the genesis of cytoplasmic Ca2+ signals.

Chronic exposure to low levels of Carbon Monoxide is associated with an increased risk of cardiac arrhythmia. Microelectrode recordings from rat and guinea pig single isolated ventricular myocytes exposed to CO releasing molecule CORM-2 and excited at 0.2/s show repolarisation changes that develop over hundreds of seconds: action potential prolongation by delayed repolarisation, EADs, multiple EADs and oscillations around the plateau, leading to irreversible repolarisation failure. The measured direct effects of CO on currents in these cells, and ion channels expressed in mammalian systems showed an increase in prolonged late Na+, and a decrease in the maximal T- and L-type Ca++. peak and late Na+, ultra-rapid delayed, delayed rectifier, and the inward rectifier K+ currents. Incorporation of these CO induced changes in maximal currents in ventricular cell models; (Gattoni et al., J. Physiol., 2016, 594, 4193–4224) (rat) and (Luo and Rudy, Circ. Res., 1994, 74, 1071–1096) (guinea-pig) and human endo-, mid-myo- and epi-cardial (O’Hara et al., PLoS Comput. Biol., 2011, 7, e1002061) models, by changes in maximal ionic conductance reproduces these repolarisation abnormalities. Simulations of cell populations with Gaussian distributions of maximal conductance parameters predict a CO induced increase in APD and its variability. Incorporation of these predicted CO induced conductance changes in human ventricular cell electrophysiology into ventricular tissue and wall models give changes in indices for the probability of the initiation of re-entrant arrhythmia.

Voltage-gated Kv7 (or KCNQ) channels control activity of excitable cells, including vascular smooth muscle cells (VSMCs), by setting their resting membrane potential and controlling other excitability parameters. Excitation -contraction coupling in muscle cells is mediated by Ca2+ but until now, the exact role of Kv7 channels in cytosolic Ca2+ dynamics in VSMCs has not been fully elucidated. We utilised microfluorimetry to investigate the impact of Kv7 channel activity on intracellular Ca2+ levels and electrical activity of rat A7r5 VSMCs and primary human internal mammary artery (IMA) SMCs. Both, direct (XE991) and G protein coupled receptor mediated (vasopressin, AVP) Kv7 channel inhibition induced robust Ca2+ oscillations, which were significantly reduced in the presence of Kv7 channel activator, retigabine, L-type Ca2+ channel inhibitor, nifedipine, or T-type Ca2+ channel inhibitor, NNC 55-0396, in A7r5 cells. Membrane potential measured using FluoVolt exhibited a slow depolarisation followed by a burst of sharp spikes in response to XE991; spikes were temporally correlated with Ca2+ oscillations. Phospholipase C inhibitor (edelfosine) reduced AVP-induced, but not XE991-induced Ca2+ oscillations. AVP and XE991 induced a large increase of [Ca2+]i in human IMA, which was also attenuated with retigabine, nifedipine and NNC 55-0396. RT-PCR, immunohistochemistry and electrophysiology suggested that Kv7.5 was the predominant Kv7 subunit in both rat and human arterial SMCs; CACNA1C (Cav1.2; L-type) and CACNA1 G (Cav3.1; T-type) were the most abundant voltage-gated Ca2+ channel gene transcripts in both types of VSMCs. This study establishes Kv7 channels as key regulators of Ca2+ signalling in VSMCs with Kv7.5 playing a dominant role.