Miguel Fernandez-Tenorio

NO-dependent CaMKII activation during β-adrenergic stimulation of cardiac muscle.

AIMS During β-adrenergic receptor (β-AR) stimulation, phosphorylation of cardiomyocyte ryanodine receptors by protein kinases may contribute to an increased diastolic Ca(2+) spark frequency. Regardless of prompt activation of protein kinase A during β-AR stimulation, this appears to rely more on activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), by a not yet identified signalling pathway. The goal of the present study was to identify and characterize the mechanisms which lead to CaMKII activation and elevated Ca(2+) spark frequencies during β-AR stimulation in single cardiomyocytes in diastolic conditions. METHODS AND RESULTS Confocal imaging revealed that β-AR stimulation increases endogenous NO production in cardiomyocytes, resulting in NO-dependent activation of CaMKII and a subsequent increase in diastolic Ca(2+) spark frequency. These changes of spark frequency could be mimicked by exposure to the NO donor GSNO and were sensitive to the CaMKII inhibitors KN-93 and AIP. In vitro, CaMKII became nitrosated and its activity remained increased independent of Ca(2+) in the presence of GSNO, as assessed with biochemical assays. CONCLUSIONS β-AR stimulation of cardiomyocytes may activate CaMKII by a novel direct pathway involving NO, without requiring Ca(2+) transients. This crosstalk between two established signalling pathways may contribute to arrhythmogenic diastolic Ca(2+) release and Ca(2+) waves during adrenergic stress, particularly in combination with cardiac diseases. In addition, NO-dependent activation of CaMKII is likely to have repercussions in many cellular signalling systems and cell types.

Stabilization of Ca 2+ signaling in cardiac muscle by stimulation of SERCA

AIMS In cardiac muscle, phosphorylation of the RyRs is proposed to increase their Ca2+ sensitivity. This mechanism could be arrhythmogenic via facilitation of spontaneous Ca2+ waves. Surprisingly, the level of Ca2+ inside the SR needed to initiate such waves has been reported to increase upon β-adrenergic stimulation, an observation which cannot be easily reconciled with elevated Ca2+ sensitivity of the RyRs. We tested the hypothesis that this change of Ca2+ wave threshold could occur indirectly, subsequent to SERCA stimulation. METHODS AND RESULTS Cytosolic and intra-SR Ca2+ waves were simultaneously recorded with confocal line-scan imaging in intact and permeabilized mouse cardiomyocytes using Rhod-2 and Fluo-5-N, respectively. We analyzed changes of several Ca2+ signaling parameters during specific SERCA stimulation by ochratoxin A (OTA), jasmonate or the Fab fragment of a phospholamban antibody. SERCA stimulation resulted in a substantial increase of the threshold for Ca2+ wave initiation. Faster Ca2+ transient decay and SR refilling confirmed SERCA acceleration. CONCLUSIONS These results suggest that isolated SERCA stimulation can elevate the intra-SR threshold for the generation of Ca2+ waves, independently of RyR phosphorylation. Simultaneously, fractional Ca2+ release and wave amplitudes are reduced. Thus, SERCA stimulation appears to exert a negative feed-back on the Ca2+-induced Ca2+ release mechanisms sustaining the waves. Thereby, it may be profoundly antiarrhythmic. This may be clinically relevant when therapies are applied to stimulate the SERCA activity (e.g. SERCA overexpression with gene therapy, future small molecule SERCA stimulators).

Cardiomyocyte Lineage Specification in Adult Human Cardiac Precursor Cells Via Modulation of Enhancer-Associated Long Noncoding RNA Expression

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Reassessment of the Transport Mechanism of the Human Zinc Transporter SLC39A2

The human zinc transporter SLC39A2, also known as ZIP2, was shown to mediate zinc transport that could be inhibited at pH <7.0 and stimulated by HCO3-, suggesting a Zn2+/HCO3- cotransport mechanism [Gaither, L. A., and Eide, D. J. (2000) J. Biol. Chem. 275, 5560-5564]. In contrast, recent experiments in our laboratory indicated that the functional activity of ZIP2 increases at acidic pH [Franz, M. C., et al. (2014) J. Biomol. Screening 19, 909-916]. The study presented here was therefore designed to reexamine the findings about the pH dependence and to extend the functional characterization of ZIP2. Our current results show that ZIP2-mediated transport is modulated by extracellular pH but independent of the H+ driving force. Also, in our experiments, ZIP2-mediated transport is not modulated by extracellular HCO3-. Moreover, a high extracellular [K+], which induces depolarization, inhibited ZIP2-mediated transport, indicating that the transport mechanism is voltage-dependent. We also show that ZIP2 mediates the uptake of Cd2+ ( Km ∼ 1.57 μM) in a pH-dependent manner ( KH+ ∼ 66 nM). Cd2+ transport is inhibited by extracellular [Zn2+] (IC50 ∼ 0.32 μM), [Cu2+] (IC50 ∼ 1.81 μM), and to a lesser extent [Co2+], but not by [Mn2+] or [Ba2+]. Fe2+ is not transported by ZIP2. Accordingly, the substrate selectivity of ZIP2 decreases in the following order: Zn2+ > Cd2+ ≥ Cu2+ > Co2+. Altogether, we propose that ZIP2 is a facilitated divalent metal ion transporter that can be modulated by extracellular pH and membrane potential. Given that ZIP2 expression has been reported in acidic environments [Desouki, M. M., et al. (2007) Mol. Cancer 6, 37; Inoue, Y., et al. (2014) J. Biol. Chem. 289, 21451-21462; Tao, Y. T., et al. (2013) Mol. Biol. Rep. 40, 4979-4984], we suggest that the herein described H+-mediated regulatory mechanism might be important for determining the velocity and direction of the transport process.

Real-time intra-store confocal Ca(2+) imaging in isolated mouse cardiomyocytes.

To initiate the contraction of cardiomyocytes, Ca2+ is released from the SR to the cytosol via ryanodine receptors (RyRs), which are activated by the Ca2+-induced Ca2+ release mechanism (CICR). The activity of RyRs is regulated by both, cytosolic and SR luminal Ca2+. Deregulation of the CICR, by dysfunctional SR Ca2+ release or uptake, is frequently associated with cardiac pathologies (e.g. arrhythmias, CPVT, heart failure). Recently, the interest to directly measure changes of the free Ca2+ concentration within the SR ([Ca2+]SR) has led to the application of low affinity Ca2+ indicators (mag-fluo-4, Fluo-5N) to follow changes of [Ca2+]SR in cardiomyocytes from some species. However, direct measurement of Ca2+ signals from the SR have not been possible in freshly isolated mouse cardiomyocytes. Here, we show a new protocol optimized to measure changes of [Ca2+]SR in mouse cardiomyocytes using fluorescent Ca2+ indicators and confocal microscopy. The application of this protocol permits the design of experimental studies with direct evaluation of SR Ca2+ in real time in various mouse models of cardiac disease, including transgenic animals harboring mutants of RyRs or other Ca2+ signaling proteins. The technique, in combination with these models, will help to understand how these diseases and mutations affect Ca2+ signals within the SR and the Ca2+ sensitivity of the RyRs for cytosolic and SR luminal Ca2+, thereby contributing to arrhythmias or weak heart beat.