The inotropic and arrhythmogenic effects of acutely increased late INa are associated with elevated ROS but not oxidation of PKARIα.

Background: Acute stimulation of the late sodium current (INaL) as pharmacologically induced by Anemonia toxin II (ATX-II) results in Na+-dependent Ca2+ overload and enhanced formation of reactive oxygen species (ROS). This is accompanied by an acute increase in the amplitude of the systolic Ca2+ transient. Ca2+ transient amplitude is determined by L-type Ca2+-mediated transsarcolemmal Ca2+ influx (ICa) into the cytosol and by systolic Ca2+ release from the sarcoplasmic reticulum (SR). Type-1 protein kinase A (PKARIα) becomes activated upon increased ROS and is capable of stimulating ICa, thereby sustaining the amplitude of the systolic Ca2+ transient upon oxidative stress. Objectives: We aimed to investigate whether the increase of the systolic Ca2+ transient as acutely induced by INaL (by ATX-II) may involve stimulation of ICa through oxidized PKARIα. Methods: We used a transgenic mouse model in which PKARIα was made resistant to oxidative activation by homozygous knock-in replacement of redox-sensitive Cysteine 17 with Serine within the regulatory subunits of PKARIα (KI). ATX-II (at 1 nmol/L) was used to acutely enhance INaL in freshly isolated ventricular myocytes from KI and wild-type (WT) control mice. Epifluorescence and confocal imaging were used to assess intracellular Ca2+ handling and ROS formation. A ruptured-patch whole-cell voltage-clamp was used to measure INaL and ICa. The impact of acutely enhanced INaL on RIα dimer formation and PKA target structures was studied using Western blot analysis. Results: ATX-II increased INaL to a similar extent in KI and WT cells, which was associated with significant cytosolic and mitochondrial ROS formation in both genotypes. Acutely activated Ca2+ handling in terms of increased Ca2+ transient amplitudes and elevated SR Ca2+ load was equally present in KI and WT cells. Likewise, cellular arrhythmias as approximated by non-triggered Ca2+ elevations during Ca2+ transient decay and by diastolic SR Ca2+-spark frequency occurred in a comparable manner in both genotypes. Most importantly and in contrast to our initial hypothesis, ATX-II did not alter the magnitude or inactivation kinetics of ICa in neither WT nor KI cells and did not result in PKARIα dimerization (i.e., oxidation) despite a clear prooxidant intracellular environment. Conclusions: The inotropic and arrhythmogenic effects of acutely increased INaL are associated with elevated ROS, but do not involve oxidation of PKARIα.

Potency and durability of T and B cell immune responses after homologous and heterologous vector delivery of a trimer-stabilized, membrane-displayed HIV-1 clade ConC Env protein.

Introduction: The generation of an HIV-1 vaccine able to induce long-lasting protective immunity remains a main challenge. Here, we aimed to modify next-generation soluble, prefusion-stabilized, close-to-native, glycan-engineered clade C gp140 envelope (Env) trimers (sC23v4 KIKO and ConCv5 KIKO) for optimal display on the cell surface following homologous or heterologous vector delivery. Methods: A combination of the following modifications scored best regarding the preservation of closed, native-like Env trimer conformation and antigenicity when using a panel of selected broadly neutralizing (bnAb) and non-neutralizing (nnAb) monoclonal antibodies for flow cytometry: i) replacing the natural cleavage site with a native flexible linker and introducing a single amino acid substitution to prevent CD4 binding (*), ii) fusing a heterologous VSV-G-derived transmembrane moiety to the gp140 C-terminus, and iii) deleting six residues proximal to the membrane. Results: When delivering membrane-tethered sC23v4 KIKO* and ConCv5 KIKO* via DNA, VSV-GP, and NYVAC vectors, the two native-like Env trimers provide differential antigenicity profiles. Whereas such patterns were largely consistent among the different vectors for either Env trimer, the membrane-tethered ConCv5 KIKO* trimer adopted a more closed and native-like structure than sC23v4 KIKO*. In immunized mice, VSV-GP and NYVAC vectors expressing the membrane-tethered ConCv5 KIKO* administered in prime/boost combination were the most effective regimens for the priming of Env-specific CD4 T cells among all tested combinations. The subsequent booster administration of trimeric ConCv5 KIKO* Env protein preserved the T cell activation levels between groups. The evaluation of the HIV-1-specific humoral responses induced in the different immunization groups after protein boosts showed that the various prime/boost protocols elicited broad and potent antibody responses, preferentially of a Th1-associated IgG2a subclass, and that the obtained antibody levels remained high at the memory phase. Discussion: In summary, we provide a feasible strategy to display multiple copies of native-like Env trimers on the cell surface, which translates into efficient priming of sustained CD4+ T cell responses after vector delivery as well as broad, potent, and sustained antibody responses following booster immunizations with the homologous, prefusion-stabilized, close-to-native ConCv5 KIKO* gp140 Env trimer.

Ibrutinib impairs IGF-1-dependent activation of intracellular Ca handling in isolated mouse ventricular myocytes.

Background: The Bruton tyrosine kinase (BTK) inhibitor Ibrutinib is associated with a higher incidence of cardiotoxic side effects including heart failure (HF). Objectives: Ibrutinib is capable of inhibiting PI3K/Akt signaling in neonatal rat ventricular cardiomyocytes when stimulated with insulin-like growth factor 1 (IGF-1). We therefore hypothesized that Ibrutinib might disrupt IGF-1-mediated activation of intracellular Ca handling in adult mouse cardiomyocytes by inhibiting PI3K/Akt signaling. Methods: Isolated ventricular myocytes (C57BL6/J) were exposed to IGF-1 at 10 nmol/L in the presence or absence of Ibrutinib (1 µmol/L) or Acalabrutinib (10 µmol/L; cell culture for 24 ± 2 h). Intracellular Ca handling was measured by epifluorescence (Fura-2 AM) and confocal microscopy (Fluo-4 AM). Ruptured-patch whole-cell voltage-clamp was used to measure ICa. Levels of key cardiac Ca handling proteins were investigated by immunoblots. Results: IGF-1 significantly increased Ca transient amplitudes by ∼83% as compared to vehicle treated control cells. This was associated with unaffected diastolic Ca, enhanced SR Ca loading and increased ICa. Co-treatment with Ibrutinib attenuated both the IGF-1-mediated increase in SR Ca content and in ICa. IGF-1 treated cardiomyocytes had significantly increased levels of pS473Akt/Akt and SERCA2a expression as compared to cells concomitantly treated with IGF-1 and Ibrutinib. SR Ca release (as assessed by Ca spark frequency) was unaffected by either treatment. In order to test for potential off-target effects, second generation BTK inhibitor Acalabrutinib with greater BTK selectivity and lower cardiovascular toxicity was tested for IGF1-mediated activation of intracellular Ca handling. Acalabrutinib induced similar effects on Ca handling in IGF-1 treated cultured myocytes as Ibrutinib in regard to decreased Ca transient amplitude and slowed Ca transient decay, hence implying a functional class effect of BTK inhibitors in cardiac myocytes. Conclusions: Inhibition of BTK by Ibrutinib impairs IGF-1-dependent activation of intracellular Ca handling in adult ventricular mouse myocytes in the face of disrupted Akt signaling and absent SERCA2a upregulation.