text:
Doxorubicin induces caspase-mediated proteolysis of KV7.1

Kv7.1 (KCNQ1) coassembles with KCNE1 to generate the cardiac IKs-channel. Gain- and loss-of-function mutations in KCNQ1 are associated with cardiac arrhthymias, highlighting the importance of modulating IKs activity for cardiac function. Here, we report proteolysis of Kv7.1 as an irreversible posttranslational modification. The identification of two C-terminal fragments of Kv7.1 led us to identify an aspartate critical for the generation of one of the fragments and caspases as responsible for mediating proteolysis. Activating caspases reduces Kv7.1/KCNE1 currents, which is abrogated in cells expressing caspase-resistant channels. Enhanced cleavage of Kv7.1 can be detected for the LQT mutation G460S, which is located adjacent to the cleavage site, whereas a calmodulin-binding-deficient mutation impairs cleavage. Application of apoptotic stimuli or doxorubicin-induced cardiotoxicity provokes caspase-mediated cleavage of endogenous IKs in human cardiomyocytes. In summary, caspases are novel regulatory components of IKs channels that may have important implications for the molecular mechanism of doxorubicin-induced cardiotoxicity.

redox activity of dexrazoxane on Fe 2+ ions is much lower, so that the Fe 2+ -scavenging activity and the consequently reduced ROS production by dexrazoxane have been suggested to underlie the alleviating effect on cardiotoxicity 26 .
Caspases are cysteine proteases, which specifically cleave their substrates at the C-terminal side of an aspartic acid and play important roles in numerous aspects of physiology such as apoptosis, aging, development, and inflammation 27 . Caspases are well known for their executive role in apoptosis and can be grouped into initiator (caspase 2, [8][9][10] and effector caspases (caspase 3, 6 and 7) 28 . Activation of caspases is triggered either by the extrinsic pathway, mediated by ligand binding to death receptors and activation of the initiator caspase 8, or by the intrinsic pathway 28 . In the latter case, mitochondrial membranes are permeabilized by the proapoptotic proteins BCL-2 and BAX, leading to a loss of mitochondrial transmembrane potentials and to the release of other proapoptotic proteins such as cytochrome C into the cytosol, which results in the activation of the initiator caspase 9. Activated caspases 8 and 9 specifically cleave effector caspases, which finally execute apoptosis 28 . With the exception of caspase 14, all other caspases in humans have been implicated in inflammation 28 . Moreover, altered caspase expression levels have been correlated with ageing 29 and heart failure 30,31 . Recently, it has become evident that caspases also have nonapoptotic and noninflammatory functions, such as regulation of long-term depression 32 or organelle removal during terminal differentiation 33 .
By using K v 7.1-specific antibodies directed against an epitope on the channel's C-terminus, we detected two c-terminal fragments, leading us to the hypothesis that K v 7.1 is processed by unknown proteases. In the present study, we identify K v 7.1 as a novel substrate for caspases, which may have important implications for understanding the role of K v 7.1 in cardiac arrhythmias and its function as a tumor suppressor. Our data suggest that caspase-mediated proteolysis of K v 7.1 leads to decreased K v 7.1-mediated currents, representing a novel regulatory mechanism for modulating K v 7.1 channel activity. Furthermore, we show that K v 7.1 cleavage is induced upon administration of doxorubicin, which efficiently activates caspase 3 in human cardiomyocytes. To our knowledge, K v 7.1 is the first example of a voltage-gated potassium channel that acts as a substrate for caspases.

Results
Proteolysis of K v 7.1 produces C-terminal fragments. We and others 34,35 have noted the occurrence of K v 7.1 C-terminal fragments in transfected cells, when antibodies directed against epitopes on the K v 7.1 C-terminus were used, which prompted us to further analyze the specificity of these fragments. We found C-terminal fragments of the full-length K v 7.1 channel in lysates derived from transiently transfected cells with human or murine K v 7.1 cDNA constructs (Fig. 1a). Two fragments with a molecular mass of about~40 and~28 kDa can be detected in addition to the full-length form of K v 7.1 at~70 kDa, when an antibody directed against a C-terminally derived peptide of K v 7.1 was used ( Fig. 1a and Supplementary Fig. 1A). Next, we demonstrated the specificity of the K v 7.1 antibody by the absence of signals in various tissues derived from K v 7.1-deficient mice, which do not show detectable K v 7.1 in immunoblots ( Supplementary Fig. 1B). As expected, we found the highest expression of Kv7.1 in murine heart, when compared to kidney and pancreas, further supporting the specificity of the antibody (Supplementary Fig. 1B). C-terminal fragments of K v 7.1 were also detectable in cells transfected with a cDNA construct of human K v 7.1 tagged at the C-terminus with an MYC epitope (Fig. 1b). Immunoblots with either the MYC or the K v 7.1 antibodies resulted in the same pattern. Next, we analyzed neonatal rat ventricular cardiomyocytes (NRVMs) for endogenous expression of K v 7.1. Again, we found immunoreactive bands resembling full-length K v 7.1 (~70 kDa) and bands at~40 and~28 kDa, albeit with low intensity (Supplementary Fig. 1C). We concluded that K v 7.1 is cleaved twice at its C-terminus, resulting in three fragments: The Nterminal fragment comprising the cytoplasmic N-terminus and the membrane-embedded part of the protein and two C-terminal fragments showing immunoreactivity with the K v 7.1 antibody, which we termed CTF1 (~40 kDa) and CTF2 (~28 kDa) (Supplementary Fig. 1A).
To determine the cleavage sites, we generated two K v 7.1 constructs: one comprising the complete C-terminus (CT, starting with glycine at position 348), and the other beginning at helix B (CT1 beginning with amino acid 505) ( Supplementary  Fig. 1A). After expression of these fragments in HeLa cells, we compared the size of the resulting bands of CT and CT1 with the C-terminal fragments CTF1 and CTF2 (Fig. 1c). CT and CTF1 migrate at the same molecular weight, whereas CTF2 runs slightly slower than the CT1 construct. From these data, we conclude that the cleavage site for the generation of CTF1 resides between the end of the transmembrane domain S6 and helix A, while the a 28  Since CTF2 is the most abundant C-terminal fragment (Fig. 1a, b, Supplementary Fig. 1C), we focused our analysis on the region between helices A and B. We generated five K v 7.1 constructs with varying deletions within this linker region (Fig. 1c). Immunoblot analysis of lysates derived from cells expressing these constructs revealed that a stretch of six amino acids (S 457 VDGYD 462 ) was essential for the occurrence of CTF2 (Fig. 1c). Next, we performed an alanine scan over the SVDGYD region to define the precise cleavage site. Mutating the aspartate at position 459 to an alanine resulted in a complete loss of CTF2 generation, whereas mutating the other five residues (including Asp-462) appeared to have no significant effect (Fig. 1d). These data demonstrate that K v 7.1 is cleaved within the SVDGYD motif at position D459 by a protease, which requires an aspartate. However, K v 7.1 is the only member within the Kv7 family carrying an aspartate at this position ( Supplementary Fig. 1D). This aspartate is highly conserved within K v 7.1 protein sequences derived from over 20 different species ( Fig. 1e and Supplementary  Fig. 2).
K v 7.1 is cleaved by caspases upon apoptosis. To identify the protease responsible for the generation of CTF2, we searched the Merops Database for proteases that require an aspartate in the cleavage site 36 . Since caspases critically depend on an aspartate at the P1 position 27 , we used two caspase inhibitors (Q-VD-OPH and Z-VAD(OMe)-FMK) to determine whether caspases are responsible for the generation of CTF2. Both compounds effectively inhibited the generation of CTF2 but not CTF1 ( Fig. 2a and Supplementary Fig. 3). To activate caspases, we induced apoptosis in cells overexpressing wild-type K v 7.1 and the D459A mutant by applying staurosporine, a nonselective protein kinase inhibitor widely used as a proapoptotic stimulus. Whereas wild-type K v 7.1 was efficiently proteolysed, the D459A mutant appeared resistant to staurosporine treatment (Fig. 2b), demonstrating that the D459A mutant is insensitive to staurosporine-induced caspase activation and cleavage. Next, we asked whether the generation of the CTF2 was dependent on the full-length K v 7.1 α-subunit. We therefore expressed the CT construct ( Supplementary Fig. 1A) under control and apoptotic conditions. Again, an increase in CTF2 generation could be observed upon staurosporine treatment ( Fig. 2c) indicating that cleavage can occur independent from the membrane-embedded part of the Kv7.1 protein. To gain deeper insight into the involvement of caspases in K v 7.1 proteolysis, we applied staurosporine and the specific caspase 8 inhibitor II to cells expressing K v 7.1. As shown in Fig. 3a, inhibition of caspase 8 efficiently blocked activation of downstream caspase 3 and the generation of CTF2 dose-dependently, demonstrating that the staurosporine-induced CTF2 generation is mediated by caspases.
To address the question whether caspase 3, one of the major effector caspases, is solely responsible for K v 7.1 cleavage at position D459, we used a human breast carcinoma MCF-7 cell line, which is deficient for caspase 3 37 . Overexpression of K v 7.1 in MCF-7 cells still resulted in the generation of CTF2 albeit to a lower extent (Fig. 3b, eGFP labeled lane). Whereas cotransfection of K v 7.1 together with wild-type caspase-3 restored CTF2 production, the overexpression of an inactive form of caspase-3 led to a reduction of CTF2 levels ( Fig. 3b) likely by protecting K v 7.1 from endogenous caspases. To determine whether all caspases cleave K v 7.1 to the same extent, we coexpressed K v 7.1 with at least one caspase of each group, namely caspases 1, 2, 3, 7, and 8. Immunoblots revealed that all tested caspases are able to generate CTF2 (Fig. 3c). Stronger cleavage could be observed by overexpression of the initiator caspase 1, 2 and 8, which might be due to an activation of downstream effector caspases. Nevertheless, these data suggest that K v 7.1 is a substrate of all analyzed caspases.
To demonstrate that endogenous K v 7.1, embedded in the I Ks channel complex undergoes caspase-mediated proteolysis, we treated murine cardiac muscle cells (HL-1 cells 38 ) with increasing concentrations of staurosporine, confirming a dose-dependent occurrence of CTF2 (Fig. 3d). Notably, full-length K v 7.1 channel was efficiently cleaved at higher staurosporine concentrations. In summary, our data strongly suggest that K v 7.1 is cleaved by caspases at an aspartate at position 459.
Functional impact of proteolysis on K v 7.1/KCNE1 channels. To analyze the functional impact of caspase-mediated cleavage of K v 7.1 upon induction of apoptosis, we cotransfected HEK 293T cells with wild-type Kv7.1 and the D459A mutant together with KCNE1 and measured whole-cell currents under staurosporine treatment and control conditions (Fig. 4a). Drug treatment produced a small but significant reduction of K v 7.1/KCNE1 currents, whereas currents generated by K v 7.1 D459A/KCNE1 channels remained unaffected (Fig. 4b). Cells were harvested after patch-clamp measurements and were subjected to immunoblot analysis to probe for CTF2. Again, we were able to detect CTF2 in cells expressing wild-type K v 7.1 but not the D459A mutant ( Supplementary Fig. 3B). One possible explanation for the small functional effect of caspase-mediated cleavage on Kv7.1/KCNE1mediated currents could be a protective effect of KCNE1 on K v 7.1. Since the majority of our analysis so far was done using cells expressing homomeric Kv7.1 channels, we therefore compared the generation of CTF2 in presence and absence of KCNE1. As shown in Fig. 4c, Kv7.1 is efficiently cleaved upon caspase activation even in the presence of KCNE1 indicating that the β-subunit is not shielding the heteromeric Kv7.1/KCNE1 complex from proteolysis. Subsequently, we performed surface biotinylation experiments to prove that the Kv7.1/KCNE1 heteromeric channels can be processed at the plasma membrane by caspases as demonstrated by the presence of CTF2 in the isolated fraction of cell surface proteins (Fig. 4d). Furthermore, these data suggest that CTF2 is, under this condition, still associated with the apoprotein complex. In summary, these data strongly suggest that the activity of heteromeric K v 7.1/KCNE1 channels can be modified by caspase-mediated cleavage in the C-terminus of Kv7.1.
Identifying long-QT mutations modulating K v 7.1 proteolysis. Next, we tested if disease-causing mutations in K v 7.1 can interfere with the generation of CTF2 and focused on the LQT1 mutation G460S, which is located just one amino acid downstream of the aspartate residue important for Kv7.1 proteolysis (Fig. 4d). Immunoblot analysis of the G460S mutant protein demonstrated significantly increased CTF2 levels when compared to wild-type K v 7.1, indicating that this LQT1 mutation renders Kv7.1 more susceptible to caspase-mediated cleavage even under nonstaurosporine treatment conditions (Fig. 4d). This observation is in line with the finding that this mutation causes a decrease in I Ks -like currents 39 .
The caspase cleavage site resides in an approximately 80 amino acid-long intervening loop between helices A and B (Fig. 4d). Both helices contain an IQ motif, which is important for calmodulin binding (Fig. 4d). Recently, it has been suggested by crystallography, molecular modeling, biochemical, and functional analyses that one bifunctional calmodulin molecule embraces both helices from one K v 7.1 subunit 34 . Given the important role of calmodulin for K v 7.1 function, we asked whether LQT1associated mutations within the calmodulin binding sites interfere with CTF2 generation. Indeed, the analyses of the K v 7.1 A372D mutation, which is located close to the calmodulin binding motif in helix A, revealed no detectable CTF2 in immunoblots (Fig. 4d). Coimmunoprecipitations proved that the A372D mutation impaired the interaction of endogenous calmodulin with K v 7.1 (Fig. 4e), suggesting that calmodulin binding is necessary for caspase-mediated proteolysis of K v 7.1.
Doxorubicin induces K v 7.1 proteolysis in cardiomyocytes. It is well known that cancer treatment by the common antineoplastic doxorubicin is hindered by severe cardiotoxic side effects, and there is strong evidence in the literature that doxorubicin leads to caspase activation in cardiomyocytes 40 . In order to determine whether interference with cardiac function by doxorubicin also involves caspase-mediated cleavage of Kv7.1, we treated humaninduced pluripotent stem cell-derived cardiomyocytes with staurosporine and doxorubicin. In total lysates derived from untreated human-induced pluripotent stem cell-derived cardiomyocytes, we could only detect trace amounts of CTF2, when we used the C-terminal K v 7.1 antibody to precipitate K v 7.1 (compare Fig. 5a and Supplementary Fig. 3C). We were also unable to detect CTF2 in murine cardiomyocytes (Fig. 3d) and tissue ( Supplementary Fig. 1B), suggesting that baseline levels of CTF2 and likely K v 7.1 proteolysis are rather low. However, staurosporine and doxorubicin activated caspase 3 as indicated by the occurrence of cleaved-active forms and a reduction of the inactive zymogen of the protease (Fig. 5a). Doxorubicin treatment at higher concentration appeared to be more efficient in producing active caspase 3, which correlated with a higher abundance of CTF2 and a strong reduction of monomeric and tetrameric forms of Kv7.1 (Fig. 5a). Interestingly, we detected a potentially dimeric form of Kv7.1 at 150 kDa in staurosporine-and doxorubicintreated cells, suggesting that the caspase-mediated destruction of tetramers results in Kv.7.1 dimers in human-induced pluripotent stem cell-derived cardiomyocytes (Fig. 5a). Nevertheless, lower doses of doxorubicin failed to produce similar levels of CTF2, which correlated with a decrease in caspase 3 activation (Fig. 5b).

Discussion
To date, over 1500 caspase cleavage sites and substrates have been identified 41 . The vast majority of caspase-mediated cleavage occurs during apoptosis, but caspase function has also been demonstrated in nonapoptotic cellular responses 42 , suggesting that caspases cleave a specific subset of substrates independent of apoptosis. Here, we report K v 7.1 as a novel substrate for caspases, representing to our knowledge the first example of a voltage-gated cation channel undergoing caspase-mediated proteolysis. However, the transient receptor potential melastin-like 7 (TRMP7) has been also identified as a caspase substrate, which appears to be critical for Fas-induced apoptosis 43 . Cleavage of human K v 7.1 by caspases occurs after an aspartate at position 459, which is located within the intervening loop between helices A and B in the channel's large cytoplasmic C-terminal domain that serves as a scaffold for numerous protein −protein interactions involved in cellular signaling cascades 15 . Both helices appear to form a two-helical bundle, which is embraced by a calmodulin molecule as revealed by X-ray crystallography of recombinantly expressed calmodulin and the proximal C-terminus of K v 7.1 34 . In this study, the intervening loop, in which the caspase-mediated proteolysis of K v 7.1 occurs, was deleted. Thus, structural information about the caspase cleavage site is missing so far. However, our finding that the calmodulin binding-deficient Kv7.1 A372D mutant is not processed by caspases strongly suggest that calmodulin either helps to recruit caspases to the channel complex or determines the structure of the intervening loop necessary for proper caspase recognition.
Our functional analysis using patch-clamp suggests that caspase cleavage interferes with the function of K v 7.1. This finding is supported by numerous reports showing that the intracellular C-terminal domain of K v 7.1 is responsible for channel tetramerization, trafficking and modulating the biophysical properties of the channel 15 . The rather small effect of Kv7.1 cleavage on whole-cell currents could be explained by the continued association of CTF2 with the apoprotein complex. For example, calmodulin could, by binding to helices A and B, act as a bridging molecule. Alternatively, the interaction of CTF2 via helices C and D with uncleaved Kv7.1 subunits could keep CTF2 in the channel complex. However, our cell surface biotinylation data strongly suggest that CTF2 is still present in heteromeric Kv7.1/KCNE1 localized at the plasmalemma.
Many pathogenic LQT1 mutations have been mapped to the C-terminus of K v 7.1 emphasizing the functional importance of this particular channel region 44 . Our finding that the LQT1 mutation G460S, which is adjacent to the aspartate 459 residue, is more susceptible to caspase cleavage suggest a potential novel pathophysiologic mechanism for LQT1 mutations located in the C-terminus of K v 7.1.
Based on the analysis of a number of cleavage sites, a general consensus motif of DXED-A/G/S/T has been proposed for apoptosis executioner caspases such as caspases 3 and 7, whereas caspases 2, 8, 9, and 10 and caspases 1, 4, 5, 6, and 14, prefer isoleucine/ leucine or tryptophan/tyrosine/valine instead of an aspartate at the first position, respectively 41 . Due to the overlapping specificity of caspases and the significantly different K v 7.1 cleavage site (amino acid sequence: FSVD-G), it is difficult to predict which individual caspase cleaves K v 7.1. Our coexpression data suggest that K v 7.1 can be cleaved by all tested caspases including caspases 1, 2, 3, 7 and 8. In this group, caspase 1 was more efficient in K v 7.1 processing, which is in agreement with the larger similarity of the preferred cleavage site 41 .
Caspases have well-established functions in the execution of apoptosis, as well as inflammation 28 . However, transiently active caspases have also been detected in nonapoptotic cells. For example, in neurons, caspases 3 and 9 are critical for long-term depression and AMPA receptor internalization 32 . In the heart, increased expression of caspase 1 was found in murine heart failure models and in patients with end-stage heart failure 31 . Analysis of mice with heart-targeted overexpression of caspases 1 and 3 further supported the notion that caspases contribute to heart diseases, likely based on an overlap of apoptotic and nonapoptotic functions 30,31 . Our finding that I Ks is sensitive to caspase-mediated cleavage uncovers a novel molecular mechanism that may contribute to cardiac arrhythmias, which is strongly supported by an increased susceptibility of the LQT1 G460S mutant to proteolytic processing by caspases. Thus, it is likely that the reported 40% smaller current density of the G460S mutant, when compared to wild-type I Ks , is at least partially due to an increased cleavage of the mutant 39 . Although several C-terminally located LQT1 mutations have been identified which interfere with channel function by modulating calmodulin 16,45 or phosphatidylinositol 4,5-bisphosphate 46 binding and/or disrupt assembly of functional I Ks 15 , for most of the C-terminal LQT mutations, the pathophysiological mechanism that leads to disease is unknown. Our data strongly suggest that susceptibility to caspase-mediated degradation should also be considered when analyzing these mutations.
Furthermore, our results demonstrate that doxorubicin treatment of human-induced pluripotent stem cell-derived cardiomyocytes efficiently induced caspase-mediated cleavage of K v 7.1, suggesting that this pathway might contribute to doxorubicininduced cardiotoxicity. Interestingly, doxorubicin has also been shown to induce electrocardiogram abnormalities such as QT interval prolongations, which are often observed within the first day after chemotherapy 25 . These results have been confirmed in animal studies, showing that doxorubicin prolongs the cardiac action potential duration by specifically inactivating I Ks but not I Kr , which both compose the delayed rectifier potassium current I K 23 . In cardiomyocytes, I Ks is mediated by a macromolecular complex formed by assembly of the pore-forming subunits K v 7.1 with KCNE1 β-subunits, which are linked to the scaffolding protein yotiao/A-kinase anchoring protein 9 (AKAP-9) 20 . Yotiao binds to the distal part of the C-terminus of K v 7.1 and recruits PKA, protein phosphatase 1 (PP1), adenylate cyclase 9 (AC9) and phosphodiesterase PDE4D3 to the complex, allowing the control of the phosphorylation state of K v 7.1, which is the molecular basis Fig. 4 Cleavage of K v 7.1 in physiology and pathophysiology. a Representative current traces for K v 7.1-MYC and K v 7.1-D459A-MYC, both coexpressed with KCNE1. b Mean currents amplitude was plotted versus voltage to obtain current−voltage (I−V) relationships in cells expressing K v 7.1-MYC (n = 39 for vehicle, n = 16 for staurosporine treatment) or K v 7.1-D459A-MYC (n = 27 for vehicle, n = 17 for staurosporine treatment) and KCNE1 treated with 500 nmol per L staurosporine for 10-12 h. Statistics were tested with two-way ANOVA followed by Bonferroni post-tests. c Immunoblot analysis of HeLa cells coexpressing Kv7.1 with KCNE1-MYC treated with 1 µM staurosporine for 4.5 h. Untransfected (Ø) and vehicle-treated cells served as negative controls. d Biotinylating study analyzed by immunoblots of Hek 293 cells coexpressing K v 7.1 and KCNE1-MYC treated with 1 µM staurosporine for 3 h. Untransfected (Ø) cells as well as cells not treated with biotin served as negative controls. IP Immunoprecipitation. TL total lysate. e Schematic illustration to highlight the position of G460 and A372 and calmodulin binding site in helix A. Western blot analysis of HeLa cell lysates overexpressing indicated constructs. Untransfected (Ø) and K v 7.1-D459A-transfected cells served as negative controls. Densitometric analysis of four independent experiments of CTF2 band intensity normalized to K v 7.1 full-length band intensity. Statistics were tested with one-way-ANOVA followed by Bonferroni's Multiple Comparison test. f Coimmunoprecipitation study analyzed by immunoblots of HeLa cells overexpressing wild-type K v 7.1 and the A372D mutant with endogenous calmodulin. IP Immunoprecipitation with anti-K v 7.1 antibody, IB Immunoblot, TL total lysate. Untransfected cells (Ø) served as negative control. c Anti-KCNE1 antibody, anti-caspase 3 antibody. c, d Anti-K v 7.1 antibody, anti-GAPDH antibody. e Anti-β-actin antibody. e, f Anti-MYC antibody. f Anti-calmodulin antibody. All graphs are shown as mean and error bars as SEM for the β-adrenergic regulation of I Ks 20,47 . In silico sequence analyses of yotiao predict several potential caspase cleavage sites, suggesting that this scaffold protein is also processed by caspases 41 . Thus, it is conceivable that caspase-mediated processing of K v 7.1 and likely yotiao contributes to the prolongation of the QT interval mediated by doxorubicin. It will be important to determine the pathway by which doxorubicin treatment leads to elevated caspase activity and K v 7.1 cleavage. While it is widely accepted that doxorubicin-induced cardiotoxicity is due to the induction of mitochondrial dysfunction, resulting in an increased production of ROS in the cytoplasm and consequent activation of extrinsic and intrinsic apoptotic pathways, a more direct effect on ROS-mediated signaling by the oxidizing activity of doxorubicin on Fe 2+ ions, as suggested by the alleviating effect of dexrazoxane, must also be considered. This will also clarify the issue of whether regulated C-terminal cleavage of K v 7.1 is more generally involved in ROS-mediated cardiac responses.
In summary, the present study demonstrates caspase-mediated proteolysis of K v 7.1. Posttranslational modifications such as phosphorylation, ubiquitination, sumoylation, palmitoylation, and glycosylation have been reported for potassium channels 48 . According to our data, proteolysis of K v 7.1 mediated by caspases is another important mechanism of posttranslational modification of K v 7.1, and we hypothesize that analysis of this regulation will advance understanding of the molecular mechanism of doxorubicin-induced cardiotoxicity.

Methods
Plasmids and antibodies. Human K v 7.1 and caspase cDNAs were subcloned in the expression vectors pFrog and pcDNA4/TO (Invitrogen, Waltham, USA), respectively. Mutations, deletions and tags for antibodies were constructed/introduced by recombinant PCR and verified by sequencing. The C-terminal anti-K v 7.1 antibody was directed against the following peptide sequence TVPRRGPDEGS. Protein extraction, immunoprecipitation, and immunoblotting. Cells were washed twice with phosphate-buffered saline (PBS) and harvested in PBS, containing a protease inhibitor cocktail (Complete, Roche, Basel, Switzerland). After centrifugation, cell pellets were lysed in PBS/Complete (1% Triton X-100, Carl Roth) by sonication. After 1 h incubation on ice, samples were centrifuged, and supernatants were analyzed by SDS-PAGE. For coimmunoprecipitations and immunoprecipitations, cells were lysed in EBC buffer/Complete (120 mM NaCl, 50 mM Tris-HCl, 0.5% NP-40, pH 7.4 (all from Carl Roth)) and treated as described above. Lysates were incubated with mouse anti-MYC antibody (coimmunoprecipitation) or treated with K v 7.1-myc antibody (immunoprecipitation) at 4°C overnight. For precipitation, protein G agarose beads (coimmunoprecipitation) or protein G dynabeads (immunoprecipitation) were used. After thorough washing of the beads, protein complexes were released by denaturation. Samples were subjected to SDS-PAGE and transferred onto nitrocellulose membranes by tank blotting. Membranes were blocked and incubated overnight at 4°C in primary antibody solution followed by incubation with the appropriate secondary antibodies conjugated to horseradish peroxidase. After thorough washing, bound antibodies were detected by chemiluminescence using a luminescent imager (LAS-4000, Fujifilm, GE Healthcare, Little Chalfont, UK). For quantifications, ImageJ software was used.
Biotinylating assay. After washing cells twice with PBS/CM (0.1 mM CaCl 2 , 1 mM MgCl 2 ), cells were incubated with 0.5 mg NHS biotin ester (Thermo Fisher) in PBS/CM for 10 min. By adding 50 mM Glycin in PBS/CM biotinylation was stopped. After two washing steps with PBS/CM, cells were lysed as described for immunoprecipitation studies above. Incubation with Streptavidin beads for 1 h at 4°C was used to precipitate biotinylated proteins. After through washing proteins were released by denaturation and subjected to SDS-PAGE and immunoblotting as described above.
Isolation of neonatal rat ventricular cardiomyocytes. Hearts of 1-2-day-old Wistar rats were harvested and minced in buffer (120 mmol NaCl, 20 mmol HEPES, 8 mmol NaH 2 PO 4 , 6 mmol glucose, 5 mmol KCl, 0.8 mmol MgSO 4 , pH = 7.4). Subsequently, up to six digestion steps were carried out with 0.6 mg per mL pancreatin (Sigma-Aldrich) and 0.5 mg per mL collagenase type II (Worthington, Lakewood, USA) in sterile ADS buffer. Cardiomyocytes were purified from contaminating fibroblasts using a Percoll gradient centrifugation step. Finally, b Densitometric analysis of 3-6 independent experiments of band intensities of CTF2 normalized to GAPDH band intensity. Statistics were tested with one-way-ANOVA followed by Bonferroni's Multiple Comparison test. a Anti-K v 7.1 antibody, anti-caspase-3 antibody, anti-GAPDH antibody. All dot blots are shown as mean and error bars as SEM


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Engineering

text:
A novel high-throughput method for kinetic characterisation of anaerobic bioproduction strains, applied to Clostridium kluyveri

Hexanoic acid (HA), also called caproic acid, can be used as an antimicrobial agent and as a precursor to various chemicals, such as fuels, solvents and fragrances. HA can be produced from ethanol and acetate by the mesophilic anaerobic bacterium Clostridium kluyveri, via two successive elongation steps over butyrate. A high-throughput anaerobic growth curve technique was coupled to a data analysis framework to assess growth kinetics for a range of substrate and product concentrations. Using this method, growth rates and several kinetic parameters were determined for C. kluyveri. A maximum growth rate (µmax) of 0.24 ± 0.01 h−1 was found, with a half-saturation index for acetic acid (KS,AA) of 3.8 ± 0.9 mM. Inhibition by butyric acid occurred at of 124.7 ± 5.7 mM (KI,BA), while the final product, HA, linearly inhibited growth with complete inhibition above 91.3 ± 10.8 mM (KHA of 10.9*10−3 ± 1.3*10−3 mM−1) at pH = 7, indicating that the hexanoate anion also exerts toxicity. These parameters were used to create a dynamic mass-balance model for bioproduction of HA. By coupling data collection and analysis to this modelling framework, we have produced a powerful tool to assess the kinetics of anaerobic micro-organisms, demonstrated here with C. kluyveri, in order further explore the potential of micro-organisms for chemicals production.

S.1.2. Production kinetics 44
A randomized sampling scheme (Figure S.1) was constructed using R 1 . One scheme ( Figure  45 S.1) was made for 3 rows and 10 columns, and applied to both conditions in the 96-WP. This 46 experiment was performed in triplicate, i.e. 3 96-WP, of which one was in the platereader for 47 continuous readings (620 nm) every 15 min. Actual time into the experiment for each timepoint 48 is given in Table S   Quantifying biomass based on OD can give valuable information, considering the large amount 85 of OD data available in the high-throughput growth curve experiments. To link these 86 parameters, a culture grown on standard DSM52 medium in 200 mL batches was taken after 2 87 and 4 days. These cultures were centrifuged for 8 min at 8610g, supernatans was removed and 88 filtered over 0.20 µm filters to obtain cell-free spent medium. This spent medium was first used 89 to resuspend the centrifuged biomass in 1/5 th of the original volume and subsequently make a 90 six-fold serial dilution (1:1 cell suspension:spent medium). For each dilution, total cell count, 91 VSS and OD were determined. Total cell counts were determined by flow cytometry 2 . This 92 experiment was performed in triplicate, i.e. a dilution series was made from 3 independently 93  was put at 35°C for one minute, after which it increased from 35°C to 230°C at a rate of 10°C 111 min -1 where it was kept for 2 minutes. FID temperature was set at 250°C, carrier gas was 112 nitrogen gas at a flow rate of 1.09 mL min -1 . The GC was externally calibrated with a minimum 113 detection limit of 25 mg/L. 114

S.1.6. Model development for chemostat simulations 116
Ordinary differential equations (ODE) described in main text (Section 3.2.) were modified for 117 simulation of chemostat conditions. The modified equations for substrate consumption or 118 product formation (eq. (S1)) and biomass production (eq. (S2)) are given below. 119 = ( * 1 * µ 1 6 * + * 2 * µ 2 6 * ) * + * ( − ) (S1) For simulation of chemostat experiments 3 , the reported conditions were applied to the model 121 (see Table S.II). The concentrations of substrates (EtOH, AA) and products (BA, HA) reached 122 after simulation for 500h of chemostat operation were then compared to the reported average 123 concentration for each condition and plotted in Figure 4 of the main text. 124 H2 accumulating in the headspace can limit the metabolism of C. kluyveri by reducing the Gibbs 130 free energy (ΔG 1 ), i.e. the amount of energy liberated during the reaction. To evaluate the 131 importance of this limitation, thermodynamical calculations were performed, using data and 132 methods from literature 4 . Two situations were assessed: (1) ΔG 1 if only the first reaction step 133 is considered, and (2) ΔG 0 if only HA is an end product, i.e. the sum of both reaction steps (see 134   Table S.II.). For both situations, initial conditions were used cf. the DSM 52 medium, and ΔG 1 135 was calculated as the reaction progressed according to the stoichiometry of the reaction. The 136 limiting substrate was used to assess the reaction progression, where the first reaction was 137 limited by AA, and the second by EtOH availability.
To assess the impact of H2, this calculation was performed with and without accumulation of 139 H2 in the headspace. To calculate accumulation of H2, a Balch tube with a total volume of 26 140 mL was considered, with 11.83 mL of liquid medium. Futhermore it was assumed that: (i) no 141 substrate was used for biomass production, (ii) no H2 was lost, through diffusion or in the 142 metabolism, (iii) all H2 produced immediately dissolved from the liquid into the headspace, (iv) 143 pH does not change during the reaction, and, (v) initially a very low H2 partial pressure of 144 0.0001 atm (10.1 Pa) was present in the headspace. For the case without H2 accumulation, H2 145 partial pressure was assumed to stay constant at this same 0.0001 atm. 146 147 EtOH can be transferred through the headspace, according to concentration gradients. It appears 159 an equilibrium was established across all wells, and even in part through a physical barrier 160 (petroleum jelly). Diffusion to the environment might occur too, but 91.9% of all EtOH initially 161 present was recovered at the end of the experiment, not taking into account potential transfer to 162 the wells acting as evaporation buffer. Evaporation appears not to be troublesome for organic 163 acids, since (1) they usually behaved similarly in 96-WP and balch tubes, (2) no apparent 164 stoichiometric inconsistencies were observed as was the case with EtOH, and (3) initial pH was 165 7.85±0.14, implying all organic acids to be present in their anionic, non-volatile form. When a plastic film seal was used to physically separate each well, EtOH no longer migrated 172 between wells in an abiotic experiment. Furthermore, the seal also allowed the outer rims to be 173 used without evaporation issues. The seal used in this experiment was not sterilized, yet no 174 contamination of the medium was observed, likely due to the very restrictive medium 175 compostion. When richer media are used, or axenity is strictly necessary, sterile films could 176 also be used, but were not tested here. In a dilution series experiment, an OD was linked to VSS and total cell countsby flow 219 cytometryas well as VSS and total cell counts to each other. For a given OD, biomass 220 concentrations expressed as VSS will be lower after 4 days than at 2 days. However, the 221 opposite is true when looking at total cell counts; the same OD at 4 days implies more cells 222 present than it does at 2 days. This apparent contradiction implies the amount of cells per g VSS 223 increases between day 2 and 4confirmed by Figure

S.2.6. Model selection 233
The proposed models for each organic acid were calibrated using the data from the experiments 234 for that acid, i.e. the proposed models for AA were calibrated using data from experiments A 235 and B, BA using data from experiments E and F and HA using data from experiment G (Table  236 1, main text),. The calibrations of these models are summarised in Table S

*Bold text indicates best fit 242
Residuals are the sum of the difference between model prediction and experimental data for all data points 243

S.2.7. HA-toxicity 244
To confirm increased salinity was not the mechanism of HA-toxicity, a control experiment was 245 included in experiment G (main text, Table I that condition (n=3). 259

S.2.8. Parameter estimation 260
Fit appropriateness of the kinetic model was evaluated by a combination of 95% confidence 261 intervals (Table 3) estimated by linear approximation of the covariance matrix with the 262 inverse of the Fisher Information Matrix -, correlations between parameters (Table S.VI) and 263 normality of the model output deviation (Figure S.11). 264 The correlation matrix is shown in Table S

S.2.10. Production kinetics of Clostridium kluyveri in 96-WP 293
To validate the dynamic model for growth of and production by Clostridium kluyveri data was 294 collected of a batch growth experiment in 96-WP in the anaerobic closet. 295 A data quality control was performed by looking at replicability within a plate, as well as 296 replicability between plates. The first shows whether there is any influence due to the 297 disturbances during sampling (i.e. measuring OD, removing lid, sampling, refilling emptied 298 wells and putting back the lid), while the latter shows how replicable the experiment as a whole 299 is. 300 The replicability within a plate is excellent; average OD of the sampled wells and those

S.2.11. Assessment of buffering capacity 345
The medium used in these experiments contained less buffer capacity than the conventional 346 DSM52 medium. In Balch tubes, no CO2 was present in the headspace for buffering, while the 347 anaerobic closet contained 10% CO2 instead of the recommended 20% CO2. It was already 348 shown in the manuscript that final product concentrations in Balch tubes and 96-WP were very 349 similar, giving a first indication C. kluyveri was not majorly affected by this change. Secondly, 350 the final pH (Table S.VII), shows a similar trend. pH at the end of the experiment is slightly 351 higher in the anaerobic closet, but always within the pH range for optimum growth of C. 352 kluyveri 8 . Additionally, because the growth curve is log transformed before fitting the growth 353 curve, the early exponential phase will carry greater weight in determination of µ. In this phase 354 of growth, conditions are still close to initial conditions, and the buffering capacity is of lower 355 importance. 356