Functional LTCC-β₂AR complex needs Caveolin-3 and is disrupted in heart failure

Background: Beta-2 adrenergic receptors (β2ARs) but not beta-2 adrenergic receptors (β1ARs) form a functional complex with L-type Ca2+ channels (LTCCs) on the cardiomyocyte membrane. However, how microdomain localization in the plasma membrane affects the function of these complexes is unknown. We aim to study the coupling between LTCC and β adrenergic receptors in different cardiomyocyte microdomains, the distinct involvement of PKA and CAMKII (Ca2+/calmodulin-dependent protein kinase II) and explore how this functional complex is disrupted in heart failure. Methods: Global signaling between LTCCs and β adrenergic receptors was assessed with whole-cell current recordings and western blot analysis. Super-resolution scanning patch-clamp was used to explore the local coupling between single LTCCs and β1AR or β2AR in different membrane microdomains in control and failing cardiomyocytes. Results: LTCC open probability (Po) showed an increase from 0.054±0.003 to 0.092±0.008 when β2AR was locally stimulated in the proximity of the channel (<350 nm) in the transverse tubule microdomain. In failing cardiomyocytes, from both rodents and humans, this transverse tubule coupling between LTCC and β2AR was lost. Interestingly, local stimulation of β1AR did not elicit any change in the Po of LTCCs, indicating a lack of proximal functional interaction between the two, but we confirmed a general activation of LTCC via β1AR. By using blockers of PKA and CaMKII and a Caveolin-3-knockout mouse model, we conclude that the β2AR-LTCC regulation requires the presence of caveolin-3 and the activation of the CaMKII pathway. By contrast, at a cellular “global” level PKA plays a major role downstream β1AR and results in an increase in LTCC current. Conclusions: Regulation of the LTCC activity by proximity coupling mechanisms occurs only via β2AR, but not β1AR. This may explain how β2ARs tune the response of LTCCs to adrenergic stimulation in healthy conditions. This coupling is lost in heart failure; restoring it could improve the adrenergic response of failing cardiomyocytes

Supernatants from lymphocytes stimulated with Bacillus Calmette-Guerin can modify the antigenicity of tumours and stimulate allogeneic T-cell responses

BACKGROUND: Reduced expression of class 1 human leucocyte antigens (HLA1) is often a mechanism by which tumours evade surveillance by the host immune system. This is often associated with an immune function that is unable to mount appropriate responses against disease, which can result in a state that favours carcinogenesis. METHODS: In the current study, we have explored the effects of Bacillus Calmette-Guerin (BCG) on the cytokine output of leucocytes, which is a key determinant in generating antitumour action, and have also assessed the effect of these cytokine cocktails on HLA1 expression in solid tumour cell lines. RESULTS: BCG potently activated a broad range of leucocytes, and also enhanced the production of cytokines that were Th(1)-predominant. Supernatants from BCG-treated leucocytes significantly increased the expression of HLA1 on the surface of cancer cell lines, which correlated with increased cytolytic T-cell activity. We also showed that the increased HLA1 expression was associated with activation of intracellular signalling pathways, which was triggered by the increases in the Th(1)-cytokines interferon-γ and tumour necrosis factor-α, as counteracting their effects negated the enhancement. CONCLUSION: These studies reaffirm the role of BCG as a putative immunotherapy through their cytokine-modifying effects on leucocytes and their capacity to enhance tumour visibility

Reclassification of a likely pathogenic Dutch founder variant in KCNH2; implications of reduced penetrance

Background: Variants in KCNH2, encoding the human ether a-go-go (hERG) channel that is responsible for the rapid component of the cardiac delayed rectifier K+ current (IKr), are causal to long QT syndrome type 2 (LQTS2). We identified eight index patients with a new variant of unknown significance (VUS), KCNH2:c.2717C > T:p.(Ser906Leu). We aimed to elucidate the biophysiological effect of this variant, to enable reclassification and consequent clinical decision-making. Methods: A genotype–phenotype overview of the patients and relatives was created. The biophysiological effects were assessed independently by manual-, and automated calibrated patch clamp. HEK293a cells expressing (i) wild-type (WT) KCNH2, (ii) KCNH2-p.S906L alone (homozygous, Hm) or (iii) KCNH2-p.S906L in combination with WT (1:1) (heterozygous, Hz) were used for manual patching. Automated patch clamp measured the variants function against known benign and pathogenic variants, using Flp-In T-rex HEK293 KCNH2-variant cell lines. Results: Incomplete penetrance of LQTS2 in KCNH2:p.(Ser906Leu) carriers was observed. In addition, some patients were heterozygous for other VUSs in CACNA1C, PKP2, RYR2 or AKAP9. The phenotype of carriers of KCNH2:p.(Ser906Leu) ranged from asymptomatic to life-threatening arrhythmic events. Manual patch clamp showed a reduced current density by 69.8 and 60.4% in KCNH2-p.S906L-Hm and KCNH2-p.S906L-Hz, respectively. The time constant of activation was significantly increased with 80.1% in KCNH2-p.S906L-Hm compared with KCNH2-WT. Assessment of KCNH2-p.S906L-Hz by calibrated automatic patch clamp assay showed a reduction in current density by 35.6%. Conclusion: The reduced current density in the KCNH2-p.S906L-Hz indicates a moderate loss-of-function. Combined with the reduced penetrance and variable phenotype, we conclude that KCNH2:p.(Ser906Leu) is a low penetrant likely pathogenic variant for LQTS2