Comprehensive analyses of the inotropic compound omecamtiv mecarbil in rat and human cardiac preparations

Omecamtiv mecarbil (OM), a myosin activator, was reported to induce complex concentration- and species-dependent effects on contractile function and clinical studies indicated a low therapeutic index with diastolic dysfunction at concentrations above 1 µM. To further characterize effects of OM in a human context and under different preload conditions, we constructed a setup that allows isometric contractility analyses of human induced pluripotent stem cell (hiPSC)-derived engineered heart tissues (EHTs). The results were compared to effects of OM on the very same EHTs measured under auxotonic conditions. OM induced a sustained, concentration-dependent increase in time-to-peak under all conditions (maximally 2-3 fold). Peak force, in contrast, was increased by OM only in human, but not rat EHTs and only under isometric conditions, varied between hiPSC lines and showed a biphasic concentration-dependency with maximal effects at 1 µM. Relaxation time tended to fall under auxotonic and strongly increase under isometric conditions, again with biphasic concentration-dependency. Diastolic tension concentration-dependently increased under all conditions. The latter was reduced by an inhibitor of the mitochondrial sodium calcium exchanger (CGP-37157). OM induced increases in mitochondrial oxidation in isolated cardiomyocytes, indicating that OM, an inotrope that does not increase intracellular and mitochondrial Ca2+, can induce mismatch between an increase in ATP and ROS production and unstimulated mitochondrial redox capacity. Taken together, we developed a novel setup well suitable for isometric measurements of EHTs. The effects of OM on contractility and diastolic tension are complex with concentration-, time-, species- and loading-dependent differences. Effects on mitochondrial function require further studies.

Mutant D96V calmodulin induces unexpected remodeling of cardiac nanostructure and physiology

Calmodulin (CaM) prevents proarrhythmic late sodium current (INa) by facilitating normal inactivation of sodium channels (NaV). Since dysfunction of NaV1.6 has been implicated in late INa-mediated arrhythmias, we investigated its role in arrhythmias promoted by CaM mutant D96V. Super-resolution STED microscopy revealed enlarged NaV1.6 clusters in NaV1.6-expressing Chinese hamster ovary cells transfected with D96V-CaM relative to those transfected with WT-CaM. Therefore, we examined NaV1.6 clustering in transgenic mice with cardiac-specific expression of D96V-CaM (cD96V) with a C-terminal FLAG tag. Confocal microscopy confirmed expression of NaV1.6 and FLAG-tagged D96V-CaM in a striated pattern along with RYR2 in cD96V hearts, consistent with T-tubular localization. In both WT and cD96V hearts, STORM single molecule localization microscopy revealed that ∼50% of NaV1.6 clusters localized <100 nm from RYR2. However, NaV1.6 density within these regions was 67% greater in cD96V relative to WT. Consistent with this result, SICM-guided “smart” patch clamp recording of NaV activity from T-tubule openings revealed more frequent late-burst openings involving larger NaV clusters in cD96V myocytes relative to WT. Previous work identifies the sodium-calcium exchanger (NCX) as a key link between aberrant late NaV1.6 activity and proarrhythmic Ca2+ mishandling. Therefore, we explored the spatial organization of NaV1.6 and NCX using STORM. Consistent with their close association, 89% of NaV1.6 clusters localized <100 nm from NCX in cD96V hearts, compared with 77% in WT. Notably, density of both NaV1.6 and NCX was increased at these sites by 48% and 31%, respectively, in cD96V relative to WT. Consistent with these data, cD96V myocytes displayed larger, more frequent Ca2+ sparks relative to WT. These proarrhythmic functional effects were abrogated by cardiac-specific knockout of NaV1.6. To our knowledge, this is the first demonstration of proarrhythmic cardiac structural remodeling secondary to a defect in calmodulin, offering novel mechanistic insight into calmodulinopathy.

The fungicide Tebuconazole induces electromechanical cardiotoxicity in murine hearts

Tebuconazole (TEB) is an important fungicide that belongs to the triazole family. It is largely applied in agriculture and its use has increased in the last decade. Since TEB is stable in water and soil, long-term exposure of humans to this pesticide is a real threat. Acute toxicological studies to uncover the toxicity of TEB are limited, and there is evidence of an association between long-term exposure to TEB and damage of several biological systems, including hepatotoxicity and cardiotoxicity. In this paper, the effects of acute exposure of cardiomyocytes and murine hearts to TEB were addressed to elucidate its impact on electromechanical properties of the cardiac tissue. In whole-cell patch-clamp records, TEB inhibited both the total outward potassium current (IC50=5.7±1.5 μmol.l−1) and the L-type calcium current (IC50=33.2±7.4 μmol.l−1). Acute exposure to TEB at 30 μmol.l−1 prolonged the action potential duration as well as an induced out-of-pace action potential, and increased the sodium/calcium exchanger current in its forward and reverse modes. Moreover, sarcomere shortening and calcium transient in isolated cardiomyocytes was enhanced when cells were exposed to TEB at 30 μmol.l−1. In ex vivo experiments, TEB 30 μmol.l−1 caused significant electrocardiogram remodeling with prolonged PR, QRS, and QT interval duration. Accordingly, TEB exposure was prone to the appearance of arrhythmias. Combined, our results demonstrate that acute TEB exposure affects the cardiomyocyte's electro-contractile properties and triggers the appearance of ECG abnormalities, including conduction defects and arrhythmias.

The Investigation of Combined Na+/Ca2+ Exchanger and the L-type Ca2+- Channel Inhibition in Langendorff Perfused Isolated Guinea Pig Hearts

Objective: The sodium/calcium exchanger (NCX) and the L-type Ca2+-channel (LTCC) are nowadays considered the major transmembrane transport mechanisms that control Ca2+ homeostasis. In pathophysiological conditions the altered function of these currents may influence the Ca2+ homeostasis and cardiac contractility and thereby, may enhance the development of severe tachyarrhythmias. The blockade of NCX current has been proposed as possible approach in the prevention and/or suppression of arrhythmias; however, this mechanism is not always favourable because the inhibition of both modes of NCX may induce Ca2+ overload. The decrease of the Ca2+ level by partial LTCC inhibition may be beneficial in increasing the antiarrhythmic efficacy. Therefore, the aim of our study was to investigate the antiarrhythmic effects of combined NCX and LTCC blockade in the ex vivo guinea pig arrhythmia model. Methods: We have performed Langendorff experiments in isolated guinea pig hearts. We have recorded electrocardiograms (ECG) and left ventricle pressure. We have applied 1 μM ORM-10962 (ORM), a compound that block NCX current and 30 nM nisoldipine for the inhibition of LTCC. Arrhythmias have been provoked by decreasing the activity of the sodium/potassium pump with 5 μM ouabain. Results: We found that neither LTCC nor NCX blockade alone increased, while the combined inhibition of the two currents significantly delayed (p<0.05) the mean time of appearance of ouabain-induced ventricular fibrillation. The heart f equency was affected by none of the drugs, only the left ventricular pressure (end-systolic and diastolic difference) was significantly elevated by ORM (p<0.001). Conclusion: In the Langendorff-perfused guinea pig heart, specific, combined NCX and LTCC blockade may be favourable than the inhibition of NCX or LTCC alone. However, further investigations are necessary to identify the pathological settings in which this combined cardiac drug therapy may be a potential new approach.

Reversing chemorefraction in colorectal cancer cells by controlling mucin secretion

ABSTRACT15% of colorectal cancers (CRC) cells exhibit a mucin hypersecretory phenotype, which is suggested to provide resistance to immune surveillance and chemotherapy. We now formally show that colorectal cancer cells build a barrier to chemotherapeutics by increasing mucins’ secretion. We show that low levels of KChIP3, a negative regulator of mucin secretion (Cantero-Recasens et al., 2018), is a risk factor for CRC patients’ relapse in subset of untreated tumours. Our results also reveal that cells depleted of KChIP3 are four times more resistant (measured as cell viability and DNA damage) to chemotherapeutics 5-Fluorouracil plus Irinotecan (5-FU+iri.) compared to control cells, whereas KChIP3 overexpressing cells are 10 times more sensitive to killing by chemotherapeutics. Similar increase in tumour cell death is observed upon chemical inhibition of mucin secretion by the sodium/calcium exchanger (NCX) blockers (Mitrovic et al., 2013). Finally, sensitivity of CRC patient-derived organoids to 5-FU+iri increases 40-fold upon mucin secretion inhibition. Reducing mucin secretion thus provides a means to control chemoresistance of mucinous colorectal cancer cells and other mucinous tumours.

Piezo1 is the cardiac mechanosensor that initiates the hypertrophic response to pressure overload

Pressure overload-induced cardiac hypertrophy is a maladaptive response with poor outcomes and limited treatment options. The transient receptor potential melastatin 4 (TRPM4) ion channel is key to activation of a Ca2+-calmodulin kinase II (CaMKII)-dependent hypertrophic signalling pathway after pressure overload, but TRPM4 is neither stretch-activated nor Ca2+-permeable. Here we show that Piezo1, which is both stretch-activated and Ca2+-permeable, is the mechanosensor that transduces increased myocardial forces into the chemical signal that initiates hypertrophic signalling via TRPM4. Cardiomyocyte-specific deletion of Piezo1 in adult mice prevented activation of CaMKII and inhibited the hypertrophic response: residual hypertrophy was associated with calcineurin activation in the absence of its usual inhibition by activated CaMKII. Piezo1 deletion prevented upregulation of the sodium-calcium exchanger and downregulation of the T-type calcium channel after pressure overload. These findings establish Piezo1 as the cardiomyocyte mechanosensor that instigates the maladaptive hypertrophic response to pressure overload, opening an avenue to novel therapies.

Short-term exercise affects cardiac function ex vivo partially via changes in calcium channel levels, without influencing hypoxia sensitivity

Exercise is known to improve cardiac recovery following coronary occlusion. However, whether short-term exercise can improve cardiac function and hypoxia tolerance ex vivo independent of reperfusion injury and the possible role of calcium channels in improved hypoxia tolerance remains unknown. Therefore, in the current study, heart function was measured ex vivo using the Langendorff method at different oxygen levels after a 4-week voluntary wheel-running regimen in trained and untrained male mice (C57Bl/6NCrl). The levels of cardiac Ca2+-channels: L-type Ca2+-channel (CACNA1C), ryanodine receptor (RyR-2), sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2), and sodium-calcium exchanger were measured using western blot. Trained mice displayed lower cardiac afterload pressure generation capacity (rate and amplitude), but unaltered hypoxia tolerance when compared to untrained mice with similar heart rates. The level of CACNA1C positively correlated with the pressure generation rate and amplitude. Furthermore, the CACNA1C-RYR-2 ratio also positively correlated with the pressure generation rate. While the 4-week training period was not enough to alter the intrinsic cardiac hypoxia tolerance, interestingly it decreased pressure generation capacity and slowed pressure decreasing capacity in the mouse hearts ex vivo. This reduction in pressure generation rate could be linked to the level of channel proteins in sarcolemmal Ca2+-cycling in trained mice. However, the Ca2+-channel levels did not differ significantly between the groups, and thus, the level of calcium channels cannot fully explain all the functional alterations, despite the detected correlations. Therefore, additional studies are warranted to reveal further mechanisms that contribute to the reduced intrinsic capacity for pressure production in trained mouse hearts.