Targeting cardiac arrhythmia by enhancing mitochondrial calcium uptake

Cardiovascular diseases remain the number one cause for morbidity and mortality worldwide with an estimated half of cardiovascular disease-related deaths being attributed to cardiac arrhythmia. Despite this enormous importance for public health, existing antiarrhythmic drugs are still far from being ideal as they display perilous side effects and can not be administered over extended time periods. It is thus a major endeavor of cardiovascular research to identify novel safer drug targets and therapeutic strategies for the treatment of cardiac arrhythmia. Since cardiac rhythmicity is directly dependent on a tight regulation of intracellular Ca2+ and cardiac arrhythmia is often associated with disturbances in Ca2+ homeostasis, we used an unbiased approach to identify novel regulators of cardiac Ca2+ handling and modulators thereof. A library of newly synthesized, organic-like compounds was screened for their ability to restore rhythmic cardiac contractions in a zebrafish model for cardiac fibrillation. From this screen we identified the small ester compound efsevin, which binds to the voltage-dependent anion channel 2 (VDAC2) in the outer mitochondrial membrane. We demonstrated that treatment with efsevin enhances mitochondrial Ca2+ uptake and thereby prevents propagation of spontaneous intracellular Ca2+ release events in cardiomyocytes, the triggers for ectopic excitations and arrhythmia. Since this work presented a novel role for VDAC2 in cardiomyocytes we analyzed the structure of VDAC2 by crystallography to identify elements that promote specificity of this isoform over VDAC1 and VDAC3. Though we could not detect large structural differences, we identified moieties that interact with regulatory proteins, which differ between the isoforms, and could thus explain the distinct role of VDAC2 in cardiomyocytes. We then used the crystal structure of VDAC2 to identify the binding site of efsevin by computational modeling and identified a binding pocket located between the wall of the VDAC2 pore and the pore-lining α helix, that was previously suggested to promote channel gating. In planar lipid bilayers we demonstrated that efsevin promotes gating of the channel from an anion-selective high conductance state into a more cation-selective low conductance state, thereby explaining the enhanced mitochondrial Ca2+ uptake induced by efsevin. To analyze the translational potential of efsevin, we tested efsevin in experimental models for the human cardiac arrhythmia catecholaminergic polymorphic ventricular tachycardia (CPVT). Efsevin reduced spontaneous diastolic Ca2+ signals and action potentials in cardiomyocytes isolated from CPVT mice and significantly reduced episodes of ventricular tachycardia in in vivo. Furthermore, efsevin reduced spontaneous, diastolic Ca2+ signals in induced pluripotent stem cell derived cardiomyocytes from a CPVT patient. Because efsevin lacks several features essential for druggability like e.g. a nanomolar affinity to the target and key pharmacokinetic properties like oral bioavailability, we then screened a library of clinically approved compounds for additional mitochondrial Ca2+ uptake enhancers. We identified increased uptake of Ca2+ into mitochondria of cardiomyocytes upon treatment with either the cholesterol uptake inhibitor ezetimbe or disulfiram, used for the treatment of alcohol abuse. Both were active at significantly lower concentrations compared to efsevin and showed efficacy experimental models for cardiac arrhythmia. Taken together, this thesis (i) establishes the outer mitochondrial membrane as a regulated barrier for Ca2+, (ii) establishes mitochondrial Ca2+ uptake as a novel regulator of cardiac rhythmicity and (iii) provides a novel candidate structure and lead substances for the development of a treatment for human cardiac arrhythmia

TRPM6 and TRPM7 differentially contribute to the relief of heteromeric TRPM6/7 channels from inhibition by cytosolic Mg2+ and Mg center dot ATP

TRPM6 and its homologue TRPM7 are alpha-kinase-coupled divalent cation-selective channels activated upon reduction of cytosolic levels of Mg2+ and Mg center dot ATP. TRPM6 is vital for organismal Mg2+ balance. However, mechanistically the cellular role and functional nonredundancy of TRPM6 remain incompletely understood. Comparative analysis of native currents in primary cells from TRPM6-versus TRPM7-deficient mice supported the concept that native TRPM6 primarily functions as a constituent of heteromeric TRPM6/7 channels. However, heterologous expression of the human TRPM6 protein engendered controversial results with respect to channel characteristics including its regulation by Mg2+ and Mg center dot ATP. To resolve this issue, we cloned the mouse TRPM6 (mTRPM6) cDNA and compared its functional characteristics to mouse TRPM7 (mTRPM7) after heterologous expression. Notably, we observed that mTRPM6 and mTRPM7 differentially regulate properties of heteromeric mTRPM6/7 channels: In the presence of mTRPM7, the extreme sensitivity of functionally expressed homomeric mTRPM6 to Mg2+ is tuned to higher concentrations, whereas mTRPM6 relieves mTRPM7 from the tight inhibition by Mg center dot ATP. Consequently, the association of mTRPM6 with mTRPM7 allows for high constitutive activity of mTRPM6/7 in the presence of physiological levels of Mg2+ and Mg center dot ATP, thus laying the mechanistic foundation for constant vectorial Mg2+ transport specifically into epithelial cells

The antiarrhythmic compound efsevin directly modulates voltage‐dependent anion channel 2 by binding to its inner wall and enhancing mitochondrial Ca2+ uptake

Background and Purpose The synthetic compound efsevin was recently identified to suppress arrhythmogenesis in models of cardiac arrhythmia, making it a promising candidate for antiarrhythmic therapy. Its activity was shown to be dependent on the voltage‐dependent anion channel 2 (VDAC2) in the outer mitochondrial membrane. Here, we investigated the molecular mechanism of the efsevin–VDAC2 interaction. Experimental Approach To evaluate the functional interaction of efsevin and VDAC2, we measured currents through recombinant VDAC2 in planar lipid bilayers. Using molecular ligand‐protein docking and mutational analysis, we identified the efsevin binding site on VDAC2. Finally, physiological consequences of the efsevin‐induced modulation of VDAC2 were analysed in HL‐1 cardiomyocytes. Key Results In lipid bilayers, efsevin reduced VDAC2 conductance and shifted the channel's open probability towards less anion‐selective closed states. Efsevin binds to a binding pocket formed by the inner channel wall and the pore‐lining N‐terminal α‐helix. Exchange of amino acids N207, K236 and N238 within this pocket for alanines abolished the channel's efsevin‐responsiveness. Upon heterologous expression in HL‐1 cardiomyocytes, both channels, wild‐type VDAC2 and the efsevin‐insensitive VDAC2AAA restored mitochondrial Ca2+ uptake, but only wild‐type VDAC2 was sensitive to efsevin. Conclusion and Implications In summary, our data indicate a direct interaction of efsevin with VDAC2 inside the channel pore that leads to modified gating and results in enhanced SR‐mitochondria Ca2+ transfer. This study sheds new light on the function of VDAC2 and provides a basis for structure‐aided chemical optimization of efsevin

Mitochondrial Ca(2+) uptake by the voltage-dependent anion channel 2 regulates cardiac rhythmicity.

Tightly regulated Ca(2+) homeostasis is a prerequisite for proper cardiac function. To dissect the regulatory network of cardiac Ca(2+) handling, we performed a chemical suppressor screen on zebrafish tremblor embryos, which suffer from Ca(2+) extrusion defects. Efsevin was identified based on its potent activity to restore coordinated contractions in tremblor. We show that efsevin binds to VDAC2, potentiates mitochondrial Ca(2+) uptake and accelerates the transfer of Ca(2+) from intracellular stores into mitochondria. In cardiomyocytes, efsevin restricts the temporal and spatial boundaries of Ca(2+) sparks and thereby inhibits Ca(2+) overload-induced erratic Ca(2+) waves and irregular contractions. We further show that overexpression of VDAC2 recapitulates the suppressive effect of efsevin on tremblor embryos whereas VDAC2 deficiency attenuates efsevin\u27s rescue effect and that VDAC2 functions synergistically with MCU to suppress cardiac fibrillation in tremblor. Together, these findings demonstrate a critical modulatory role for VDAC2-dependent mitochondrial Ca(2+) uptake in the regulation of cardiac rhythmicity

Agonist-mediated switching of ion selectivity in TPC2 differentially promotes lysosomal function

Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009;Guo et al., 2017;Jha et al., 2014;Ruas et al., 2015;Wang et al., 2012), depends on the activating ligand. A high-throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca2+-signals and non-selective cation currents, the other weaker Ca2+-signals and Na+-selective currents. These properties were mirrored by the Ca2+ mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P-2, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic signatures on demand

E as in Enigma: The Mysterious Role of the Voltage-Dependent Anion Channel Glutamate E73

The voltage-dependent anion channel (VDAC) is the main passageway for ions and metabolites over the outer mitochondrial membrane. It was associated with many physiological processes, including apoptosis and modulation of intracellular Ca2+ signaling. The protein is formed by a barrel of 19 beta-sheets with an N-terminal helix lining the inner pore. Despite its large diameter, the channel can change its selectivity for ions and metabolites based on its open state to regulate transport into and out of mitochondria. VDAC was shown to be regulated by a variety of cellular factors and molecular partners including proteins, lipids and ions. Although the physiological importance of many of these modulatory effects are well described, the binding sites for molecular partners are still largely unknown. The highly symmetrical and sleek structure of the channel makes predictions of functional moieties difficult. However, one residue repeatedly sticks out when reviewing VDAC literature. A glutamate at position 73 (E73) located on the outside of the channel facing the hydrophobic membrane environment was repeatedly proposed to be involved in channel regulation on multiple levels. Here, we review the distinct hypothesized roles of E73 and summarize the open questions around this mysterious residue

Glutamate 73 Promotes Anti-arrhythmic Effects of Voltage-Dependent Anion Channel Through Regulation of Mitochondrial Ca2+ Uptake

Mitochondria critically regulate a range of cellular processes including bioenergetics, cellular metabolism, apoptosis, and cellular Ca2+ signaling. The voltage-dependent anion channel (VDAC) functions as a passageway for the exchange of ions, including Ca2+, across the outer mitochondrial membrane. In cardiomyocytes, genetic or pharmacological activation of isoform 2 of VDAC (VDAC2) effectively potentiates mitochondrial Ca2+ uptake and suppresses Ca2+ overload-induced arrhythmogenic events. However, molecular mechanisms by which VDAC2 controls mitochondrial Ca2+ transport and thereby influences cardiac rhythmicity remain elusive. Vertebrates express three highly homologous VDAC isoforms. Here, we used the zebrafish tremblor/ncx1h mutant to dissect the isoform-specific roles of VDAC proteins in Ca2+ handling. We found that overexpression of VDAC1 or VDAC2, but not VDAC3, suppresses the fibrillation-like phenotype in zebrafish tremblor/ncx1h mutants. A chimeric approach showed that moieties in the N-terminal half of VDAC are responsible for their divergent functions in cardiac biology. Phylogenetic analysis further revealed that a glutamate at position 73, which was previously described to be an important regulator of VDAC function, is sevolutionarily conserved in VDAC1 and VDAC2, whereas a glutamine occupies position 73 (Q73) of VDAC3. To investigate whether E73/Q73 determines VDAC isoform-specific anti-arrhythmic effect, we mutated E73 to Q in VDAC2 (VDAC2E73Q) and Q73 to E in VDAC3 (VDAC3Q73E). Interestingly, VDAC2E73Q failed to restore rhythmic cardiac contractions in ncx1 deficient hearts, while the Q73E conversion induced a gain of function in VDAC3. In HL-1 cardiomyocytes, VDAC2 knockdown diminished the transfer of Ca2+ from the SR into mitochondria and overexpression of VDAC2 or VDAC3Q73E restored SR-mitochondrial Ca2+ transfer in VDAC2 deficient HL-1 cells, whereas this rescue effect was absent for VDAC3 and drastically compromised for VDAC2E73Q. Collectively, our findings demonstrate a critical role for the evolutionary conserved E73 in determining the anti-arrhythmic effect of VDAC isoforms through modulating Ca2+ cross-talk between the SR and mitochondria in cardiomyocytes