Going full circle - optogenetic control of Ca2+ release from and reuptake into the endoplasmic reticulum
Participants:
1) Alexander Gottschalk (PI; University of Frankfurt), coworker: Frank Becker
2) Stefan Lehnart (PI; University of Göttingen), coworker: Miroslav Dura
3) Philipp Sasse (PI; University of Bonn), coworker: Wanchana Jangsangthong
Abstract:
In excitable cells, intracellular Ca2+ release and uptake from and into the endo/sarcoplasmic reticulum, via the Ryanodine Receptor (RyR) and SERCA-Ca2+-ATPase, respectively, are functionally coupled. This allows for tight spatiotemporal control of local Ca2+ signals (Ca2+ sparks) and safeguards cell viability through low resting Ca2+ concentrations. Despite the eminent role of RyR Ca2+ release channels and SERCA Ca2+-pumps in the brain, heart, pancreas and skeletal muscle, both in health and disease, their molecularly targeted manipulation remains challenging and complex. For example, patient mutations cause dangerous arrhythmias via RyR2 channels in the heart through increased Ca2+ leak. However, in experimental cellular models Ca2+ leak is commonly induced by non-physiological, typically pharmacological interventions at the cost of significant off-target effects. Importantly, the lack of RyR- and SERCA-specific tools compromises and delays the development of drug compounds for a large number of diseases, including arrhythmias, cancer, cognitive dysfunction, diabetes, epilepsy, heart failure, and muscle fatigue. While optogenetic manipulation of RyR channels and SERCA pumps addresses a challenging area, our collaborative efforts to develop the first OptoRyR tool are not only promising, but can open major opportunities for faster and combinatory strategies, with the vision to facilitate drug-safety testing.
In the first funding period we have developed de novo strategies for light-induced Ca2+ release. The most successful strategy used a Ca2+-conducting Channelrhodopsin2 variant in fusion to the RyR2. This OptoRyR2 relies on an endogenous amplification mechanism of Ca2+-induced Ca2+-release through cytosolic Ca2+-activation of RyR2 channels.
For the second funding period we will significantly extend the planned work through three major directions: In Aim 1 we will apply the mechanistic concept of OptoRyR2 to C. elegans and human stem cell-derived cardiomyocytes using CRISPR/Cas9 mediated gene editing to address fundamental biological questions in intact animals and to develop a novel optogenetic platform for drug-safety testing of RyR2-targeted chemical compounds. Aim 2 broadens the utility concept through a new mechanistic approach of direct opto-mechanical light-gating of RyR2 through insertion of LOV domains in the channel shell structure. This became possible through recent high-resolution CryoEM structures of RyR channels. Aim 3, literally closing the circle, extends the optogenetic toolbox to control Ca2+ uptake through two parallel strategies: light-activation of cAMP production molecularly linked to phospholamban and opto-mechanical control of SERCA will both modulate Ca2+ uptake. Thus, we will develop a toolbox for subcellular Ca2+ control and apply this in vitro and in vivo to enable exploring basic questions of Ca2+ homeostasis, as well as a new concept of drug-safety testing.
Logic of interactions:
OptoRyR and optoSERCA constructs will be generated for C. elegans and tested by the Gottschalk group, and in parallel, by the Sasse group, for the mouse. The Lehnart group will provide essential biophysical experiments (single-channel measurements) to characterize the mechanism of action of the generated constructs.
Publications Gottschalk:
Azimi Hashemi N, Bergs ACF, Schüler C, Scheiwe AR, Steuer Costa W, Bach M, Liewald JF, Gottschalk A (2019) Rhodopsin-based voltage imaging tools for use in muscles and neurons of Caenorhabditis elegans. PNAS 116: 17051-60 https://doi.org/10.1073/pnas.1902443116
Brown J, Behnam R, Coddington L, Tervo DGR, Martin K, Proskurin M, Kuleshova E, Park J, Phillips J, Bergs ACF, Gottschalk A, Dudman JT, Karpova AY. (2018) Expanding the Optogenetics Toolkit by Topological Inversion of Rhodopsins. Cell 175: 1131-1140. https://doi.org/10.1016/j.cell.2018.09.026
Bergs ACF, Schultheis C, Fischer E, Tsunoda SP, Erbguth K, Husson SJ, Govorunova E, Spudich JL, Nagel G, Gottschalk A*, Liewald JF*. (2018) Rhodopsin optogenetic toolbox v2.0 for light-sensitive excitation and inhibition in Caenorhabditis elegans. PLoS ONE 13(2): e0191802. https://doi.org/10.1371/journal.pone.0191802
Fischer E, Gottschalk A*, Schueler C* (2017) An optogenetic arrhythmia model to study catecholaminergic polymorphic ventricular tachycardia mutations. Sci. Rep. 7: 17514
Schüler C, Fischer E, Shaltiel L, Steuer Costa W, Gottschalk A. (2015) Arrhythmogenic effects of mutated L-type Ca2+-channels on an optogenetically paced muscular pump in Caenorhabditis elegans. Scientific Reports 5:14427.
Akerboom, J, Carreras Calderon N, Tian L, Wabnig S, Prigge M, Tolö J, Gordus A, Orger MB, Severi KE, Macklin JJ, Patel R, Pulver SR, Wardill TJ, Fischer E, Schüler C, Chen T-W, Sarkisyan, KS, Marvin JS, Bargmann, CI, Kim DK, Kügler S, Lagnado L, Hegemann P, Gottschalk A, Schreiter ER and Looger LL. (2013) Genetically encoded calcium indicators for multi-color neural activity imaging in combination with optogenetics. Frontiers in Molecular Neuroscience 6: 2. doi.org/10.3389/fnmol.2013.00002
Erbguth K, Prigge M, Schneider F, Hegemann P, Gottschalk A. (2012) Bimodal activation of different neuron classes with the spectrally red-shifted Channelrhodopsin chimera C1V1 in Caenorhabditis elegans. PLoS ONE 7: e46827. doi.org/10.1371/journal.pone.0046827
Schultheis C, Liewald JF, Bamberg, E, Nagel G, Gottschalk A. Optogenetic long-term manipulation of behavior and animal development. PLoS One 2011 April 20; 6(4): e18766.
Liewald JF, Brauner M, Stephens GJ, Bouhours M, Schultheis C, Zhen M, Gottschalk A. (2008) Optogenetic analysis of synaptic function. Nat Methods 5: 895-902.
Gottschalk A*, Almedom RB, Schedletzky T, Anderson SD, Yates III JR, Schafer WR.* (2005) Identification and characterization of novel nicotinic receptor-associated proteins in Caenorhabditis elegans. EMBO J 24: 2566-78. (* corresponding authors)
Publications Lehnart:
Zaglia T, Pianca N, Borile G, Da Broi F, Richter C, Campione M, Lehnart SE, Luther S, Corrado D, Miquerol L, Mongillo M. (2015) Optogenetic determination of the myocardial requirements for extrasystoles by cell type-specific targeting of ChannelRhodopsin-2. PNAS 112: E4495-504.
Walker MA, Williams GSB, Kohl T, Lehnart SE, Jafri MS, Greenstein J, Lederer WJ, Winslow RL. (2014) Super-resolution modeling of calcium release in heart. Biophys J 107: 3009-20.
Wagner E, Lauterbach MA, Kohl T, Westphal V, Williams GSB, Steinbrecher JH, Streich JH, Korff B, Tuan HTM, Hagen B, Luther S, Hasenfuss G, Parlitz U, Jafri MS, Hell SW, Lederer WJ, Lehnart SE. (2012) STED live cell super-resolution imaging shows proliferative remodeling of T-tubule membrane structures after myocardial infarction. Circ Res 111: 402-414.
Wehrens XH1, Lehnart SE1, Huang F, Vest JA, Reiken SR, Mohler PJ, Sun J, Guatimosim S, Song LS, Rosemblit N, D’Armiento JM, Napolitano C, Memmi M, Priori SG, Lederer WJ, Marks AR. (2003) FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death. Cell 113: 829-840. (1 first authors)
Publications Sasse:
Roell W*, Lewalter T*, Sasse P* (*: shared authorships), Tallini YN, Choi BR, Breitbach M, Doran R, Becher U, Hwang SM, Bostani T, von Maltzahn J, Hofmann A, Reining S, Eiberger B, Gabris B, Pfeifer A, Welz A, Willecke K, Salama G, Schrickel JW, Kotlikoff MI, Fleischmann BK: Engraftment of connexin 43-expressing cells prevents post -infarct arrhythmia. Nature 2007; 450:819-24
Bruegmann T, Malan D, Hesse M, Beiert T, Fuegemann CJ, Fleischmann BK, Sasse P: Optogenetic control of heart muscle in vitro and in vivo. Nature Methods 2010;7:897-900.
Beiert T, Bruegmann T, Sasse P: Optogenetic activation of Gq-signaling in cardiomyocytes modulates pacemaker activity. Cardiovasc Res. 2014; 102:507-516
Vogt CC, Bruegmann T, Malan D, Ottersbach A, Roell W, Fleischmann BK, Sasse P: Systemic gene transfer enables optogenetic pacing of mouse hearts. Cardiovasc. Res. 2015, 106:338-43
Bruegmann T, van Bremen T, Vogt C, Send T, Fleischmann BK, Sasse P: Optogenetic control of contractile function in skeletal muscle. Nat. Commun. 2015; 6:7153