Beneficial effect of voluntary physical exercise in Plakophilin2 transgenic mice.

Arrhythmogenic right ventricular cardiomyopathy is a hereditary, rare disease with an increased risk for sudden cardiac death. The disease-causing mutations are located within the desmosomal complex and the highest incidence is found in plakophilin2. However, there are other factors playing a role for the disease progression unrelated to the genotype such as inflammation or exercise. Competitive sports have been identified as risk factor, but the type and extend of physical activity as cofactor for arrhythmogenesis remains under debate. We thus studied the effect of light voluntary exercise on cardiac health in a mouse model. Mice with a heterozygous PKP2 loss-of-function mutation were given the option to exercise in a running wheel which was monitored 24 h/d. We analyzed structural and functional development in vivo by echocardiography which revealed that neither the genotype nor the exercise caused any significant structural changes. Ejection fraction and fractional shortening were not influenced by the genotype itself, but exercise did cause a drop in both parameters after 8 weeks, which returned to normal after 16 weeks of training. The electrophysiological analysis revealed that the arrhythmogenic potential was slightly higher in heterozygous animals (50% vs 18% in wt littermates) and that an additional stressor (isoprenaline) did not lead to an increase of arrhythmogenic events pre run or after 8 weeks of running but the vulnerability was increased after 16 weeks. Exercise-induced alterations in Ca handling and contractility of isolated myocytes were mostly abolished in heterozygous animals. No fibrofatty replacements or rearrangement of gap junctions could be observed. Taken together we could show that light voluntary exercise can cause a transient aggravation of the mutation-induced phenotype which is abolished after long term exercise indicating a beneficial effect of long term light exercise.

Hypertonicity counteracts MCL-1 and renders BCL-XL a synthetic lethal target in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive and difficult-to-treat cancer entity. Current therapies ultimately aim to activate the mitochondria-controlled (intrinsic) apoptosis pathway, but complex alterations in intracellular signaling cascades and the extracellular microenvironment hamper treatment response. On the one hand, proteins of the BCL-2 family set the threshold for cell death induction and prevent accidental cellular suicide. On the other hand, controlling a cell's readiness to die also determines whether malignant cells are sensitive or resistant to anticancer treatments. Here, we show that HNSCC cells upregulate the proapoptotic BH3-only protein NOXA in response to hyperosmotic stress. Induction of NOXA is sufficient to counteract the antiapoptotic properties of MCL-1 and switches HNSCC cells from dual BCL-XL/MCL-1 protection to exclusive BCL-XL addiction. Hypertonicity-induced functional loss of MCL-1 renders BCL-XL a synthetically lethal target in HNSCC, and inhibition of BCL-XL efficiently kills HNSCC cells that poorly respond to conventional therapies. We identify hypertonicity-induced upregulation of NOXA as link between osmotic pressure in the tumor environment and mitochondrial priming, which could perspectively be exploited to boost efficacy of anticancer drugs.

Long-term effects of empagliflozin on excitation-contraction-coupling in human induced pluripotent stem cell cardiomyocytes.

The SGLT2 inhibitor empagliflozin improved cardiovascular outcomes in patients with diabetes. As the cardiac mechanisms remain elusive, we investigated the long-term effects (up to 2 months) of empagliflozin on excitation-contraction (EC)-coupling in human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CM) in a blinded manner. IPSC from 3 donors, differentiated into pure iPSC-CM (4 differentiations), were treated with a clinically relevant concentration of empagliflozin (0.5 µmol/l) or vehicle control. Treatment, data acquisition, and analysis were conducted externally blinded. Epifluorescence microscopy measurements in iPSC-CM showed that empagliflozin has neutral effects on Ca2+ transient amplitude, diastolic Ca2+ levels, Ca2+ transient kinetics, or sarcoplasmic Ca2+ load after 2 weeks or 8 weeks of treatment. Confocal microscopy determining possible effects on proarrhythmogenic diastolic Ca2+ release events showed that in iPSC-CM, Ca2+ spark frequency and leak was not altered after chronic treatment with empagliflozin. Finally, in patch-clamp experiments, empagliflozin did not change action potential duration, amplitude, or resting membrane potential compared with vehicle control after long-term treatment. Next-generation RNA sequencing (NGS) and mapped transcriptome profiles of iPSC-CMs untreated and treated with empagliflozin for 8 weeks showed no differentially expressed EC-coupling genes. In line with NGS data, Western blots indicate that empagliflozin has negligible effects on key EC-coupling proteins. In this blinded study, direct treatment of iPSC-CM with empagliflozin for a clinically relevant duration of 2 months did not influence cardiomyocyte EC-coupling and electrophysiology. Therefore, it is likely that other mechanisms independent of cardiomyocyte EC-coupling are responsible for the beneficial treatment effect of empagliflozin. KEY MESSAGES: This blinded study investigated the clinically relevant long-term effects (up to 2 months) of empagliflozin on cardiomyocyte excitation-contraction (EC)-coupling. Human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CM) were used to study a human model including a high repetition number of experiments. Empagliflozin has neutral effects on cardiomyocyte Ca2+ transients, sarcoplasmic Ca2+ load, and diastolic sarcoplasmic Ca2+ leak. In patch-clamp experiments, empagliflozin did not change the action potential. Next-generation RNA sequencing, mapped transcriptome profiles, and Western blots of iPSC-CM untreated and treated with empagliflozin showed no differentially expressed EC-coupling candidates.

Ca2+/calmodulin-dependent protein kinase II is essential in hyperacute pressure overload.

BACKGROUND: Activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) is established as a central intracellular trigger for various cardiac pathologies such as hypertrophy, heart failure or arrhythmias in animals and humans suggesting CaMKII as a promising target protein for future medical treatments. However, the physiological role of CaMKII is scarcely well defined. AIM & METHODS: To investigate the role of CaMKII in hyperacute pressure overload, we evaluated the effects of pressure overload induced by transverse aortic constriction (TAC) on survival, cardiac function, protein expression and excitation-contraction coupling (ECC) in female WT littermate vs. AC3-I mice 2 days after TAC (2d post TAC). AC3-I mice express the CaMKII inhibitor autocamtide-3 related inhibitory peptide (AiP) under the control of the α-myosin heavy chain promotor in the heart. RESULTS: CaMKII activation is significantly increased in WT TAC vs. sham mice 2d post TAC. Interestingly, survival is significantly reduced in AC3-I animals within the first five days after TAC compared to WT TAC littermates, while systolic cardiac function is markedly reduced in AC3-I TAC vs. AC3-I sham mice, but preserved in WT TAC vs. WT sham mice. Proteins regulating ECC such as ryanodine receptors (RyR2) and phospholamban (PLB) are hypophosphorylated at their CaMKII phosphorylation site in AC3-I TAC mice, but hyperphosphorylated in WT TAC mice compared to controls. In isolated cardiomyocytes fractional shortening is significantly impaired in AC3-I compared to WT mice 2d post TAC, and CaMKII incubation with AiP mimics the AC3-I phenotype in cardiomyocytes from WT TAC mice in vitro. In summary, this suggests cardiac dysfunction due to CaMKII inhibition as a potential cause of increased mortality in AC3-I TAC mice. However, proarrhythmic spontaneous Ca2+ release events (SCR) appear less frequent in cardiomyocytes from AC3-I TAC mice than in WT TAC mice. CONCLUSIONS: Our data indicate that excessive CaMKII inhibition as present in AC3-I transgenic mice leads to an impaired adaptation of ECC to hyperacute pressure overload resulting in diminished cardiac contractility and increased death. Thus, our data suggest that in pressure overload the activation of CaMKII is a pivotal, but previously unknown part of hyperacute stress physiology in the heart, while CaMKII inhibition, albeit potentially antiarrhythmic, can be detrimental. This should be taken into account for future studies with CaMKII inhibitors as therapeutic agents.

Empagliflozin reduces Ca/calmodulin-dependent kinase II activity in isolated ventricular cardiomyocytes.

AIMS: The EMPA-REG OUTCOME study showed reduced mortality and hospitalization due to heart failure (HF) in diabetic patients treated with empagliflozin. Overexpression and Ca2+ -dependent activation of Ca2+ /calmodulin-dependent kinase II (CaMKII) are hallmarks of HF, leading to contractile dysfunction and arrhythmias. We tested whether empagliflozin reduces CaMKII- activity and improves Ca2+ -handling in human and murine ventricular myocytes. METHODS AND RESULTS: Myocytes from wild-type mice, mice with transverse aortic constriction (TAC) as a model of HF, and human failing ventricular myocytes were exposed to empagliflozin (1 µmol/L) or vehicle. CaMKII activity was assessed by CaMKII-histone deacetylase pulldown assay. Ca2+ spark frequency (CaSpF) as a measure of sarcoplasmic reticulum (SR) Ca2+ leak was investigated by confocal microscopy. [Na+ ]i was measured using Na+ /Ca2+ -exchanger (NCX) currents (whole-cell patch clamp). Compared with vehicle, 24 h empagliflozin exposure of murine myocytes reduced CaMKII activity (1.6 ± 0.7 vs. 4.2 ± 0.9, P < 0.05, n = 10 mice), and also CaMKII-dependent ryanodine receptor phosphorylation (0.8 ± 0.1 vs. 1.0 ± 0.1, P < 0.05, n = 11 mice), with similar results upon TAC. In murine myocytes, empagliflozin reduced CaSpF (TAC: 1.7 ± 0.3 vs. 2.5 ± 0.4 1/100 µm-1  s-1 , P < 0.05, n = 4 mice) but increased SR Ca2+ load and Ca2+ transient amplitude. Importantly, empagliflozin also significantly reduced CaSpF in human failing ventricular myocytes (1 ± 0.2 vs. 3.3 ± 0.9, P < 0.05, n = 4 patients), while Ca2+ transient amplitude was increased (F/F0 : 0.53 ± 0.05 vs. 0.36 ± 0.02, P < 0.05, n = 3 patients). In contrast, 30 min exposure with empagliflozin did not affect CaMKII activity nor Ca2+ -handling but significantly reduced [Na+ ]i . CONCLUSIONS: We show for the first time that empagliflozin reduces CaMKII activity and CaMKII-dependent SR Ca2+ leak. Reduced Ca2+ leak and improved Ca2+ transients may contribute to the beneficial effects of empagliflozin in HF.

Cardiac myocyte alternans in intact heart: Influence of cell-cell coupling and ß-adrenergic stimulation.

BACKGROUND: Cardiac alternans are proarrhythmic and mechanistically link cardiac mechanical dysfunction and sudden cardiac death. Beat-to-beat alternans occur when beats with large Ca(2+) transients and long action potential duration (APD) alternate with the converse. APD alternans are typically driven by Ca(2+) alternans and sarcoplasmic reticulum (SR) Ca(2+) release alternans. But the effect of intercellular communication via gap junctions (GJ) on alternans in the intact heart remains unknown. OBJECTIVE: We assessed the effects of cell-to-cell coupling on local alternans in intact Langendorff-perfused mouse hearts, measuring single myocyte [Ca(2+)] alternans synchronization among neighboring cells, and effects of ß-adrenergic receptor (ß-AR) activation and reduced GJ coupling. METHODS AND RESULTS: Mouse hearts (C57BL/6) were retrogradely perfused and loaded with Fluo8-AM to record cardiac myocyte [Ca(2+)] in situ with confocal microscopy. Single cell resolution allowed analysis of alternans within the intact organ during alternans induction. Carbenoxolone (25 µM), a GJ inhibitor, significantly increased the occurrence and amplitude of alternans in single cells within the intact heart. Alternans were concordant between neighboring cells throughout the field of view, except transiently during onset. ß-AR stimulation only reduced Ca(2+) alternans in tissue that had reduced GJ coupling, matching effects seen in isolated myocytes. CONCLUSIONS: Ca(2+) alternans among neighboring myocytes is predominantly concordant, likely because of electrical coupling between cells. Consistent with this, partial GJ uncoupling increased propensity and amplitude of Ca(2+) alternans, and made them more sensitive to reversal by ß-AR activation, as in isolated myocytes. Electrical coupling between myocytes may thus limit the alternans initiation, but also allow alternans to be more stable once established.

Variations in local calcium signaling in adjacent cardiac myocytes of the intact mouse heart detected with two-dimensional confocal microscopy.

Dyssynchronous local Ca release within individual cardiac myocytes has been linked to cellular contractile dysfunction. Differences in Ca kinetics in adjacent cells may also provide a substrate for inefficient contraction and arrhythmias. In a new approach we quantify variation in local Ca transients between adjacent myocytes in the whole heart. Langendorff-perfused mouse hearts were loaded with Fluo-8 AM to detect Ca and Di-4-ANEPPS to visualize cell membranes. A spinning disc confocal microscope with a fast camera allowed us to record Ca signals within an area of 465 µm by 315 µm with an acquisition speed of 55 fps. Images from multiple transients recorded at steady state were registered to their time point in the cardiac cycle to restore averaged local Ca transients with a higher temporal resolution. Local Ca transients within and between adjacent myocytes were compared with regard to amplitude, time to peak and decay at steady state stimulation (250 ms cycle length). Image registration from multiple sequential Ca transients allowed reconstruction of high temporal resolution (2.4 ± 1.3 ms) local CaT in 2D image sets (N = 4 hearts, n = 8 regions). During steady state stimulation, spatial Ca gradients were homogeneous within cells in both directions and independent of distance between measured points. Variation in CaT amplitudes was similar across the short and the long side of neighboring cells. Variations in TAU and TTP were similar in both directions. Isoproterenol enhanced the CaT but not the overall pattern of spatial heterogeneities. Here we detected and analyzed local Ca signals in intact mouse hearts with high temporal and spatial resolution, taking into account 2D arrangement of the cells. We observed significant differences in the variation of CaT amplitude along the long and short axis of cardiac myocytes. Variations of Ca signals between neighboring cells may contribute to the substrate of cardiac remodeling.

Relationship between oral fluid and blood concentrations of drugs of abuse in drivers suspected of driving under the influence of drugs.

In recent years, the interest in the use of oral fluid as a biological matrix has increased significantly, particularly for detecting driving under the influence of drugs (DUID). In this study, the relationship between the oral fluid and the blood concentrations of drugs of abuse in drivers suspected of DUID is discussed. Blood and oral fluid samples were collected from drivers suspected of DUID or stopped during random controls by the police in Belgium, Germany, Finland, and Norway for the ROSITA-2 project. The blood samples were analyzed by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS), sometimes preceded by immunoassay screening of blood or urine samples. The oral fluid samples were analyzed by GC-MS or LC-MS(/MS). Scatter plots and trend lines of the blood and oral fluid concentrations and the median, mean, range, and SD of the oral fluid to blood (OF:B) ratios were calculated for amphetamines, benzodiazepines, cocaine, opiates, and Delta(9)-2 tetrahydrocannabinol. The ratios found in this study were comparable with those that were published previously, but the range was wider. The OF:B ratios of basic drugs such as amphetamines, cocaine, and opiates were >1 [amphetamine: median (range) 13 (0.5-182), methylenedioxyamphetamine: 4 (1-15), methylenedioxymethamphetamine: 6 (0.9-88), methamphetamine: 5 (2-23), cocaine: 22 (4-119), benzoylecgonine: 1 (0.2-11), morphine: 2 (0.8-6), and codeine: 10 (0.8-39)]. The ratios for benzodiazepines were very low, as could be expected as they are highly protein bound and weakly acidic, leading to low oral fluid concentrations [diazepam: 0.02 (0.01-0.15), nordiazepam: 0.04 (0.01-0.23), oxazepam: 0.05 (0.03-0.14), and temazepam: 0.1 (0.06-0.54)]. For tetrahydrocannabinol, an OF:B ratio of 15 was found (range 0.01-569). In this study, the time of last administration, the dose, and the route of administration were unknown. Nevertheless, the data reflect the variability of the OF:B ratios in drivers thought to be under the influence of drugs. The wide range of the ratios, however, does not allow reliable calculation of the blood concentrations from oral fluid concentrations.