Single Mutation (A162H) in Human Cardiac Troponin I Corrects Acid pH Sensitivity of Ca2+-regulated Actomyosin S1 ATPase

In contrast to skeletal muscle, the efficiency of the contractile apparatus of cardiac tissue has long been known to be severely compromised by acid pH as in the ischemia of myocardial infarction and other cardiac myopathies. Recent reports (Westfall, M. V., and Metzger, J. M. (2001) News Physiol. Sci.16, 278–281; Li, G., Martin, A. F., and Solaro, R. J. (2001) J. Mol. Cell. Cardiol.33, 1309–1320) have indicated that the reduced Ca2+ sensitivity of cardiac contractility at low pH (≤pH 6.5) is attributable to structural difference(s) in the cardiac and skeletal inhibitory components (TnIs) of their troponins. Here, using a reconstituted Ca2+-regulated human cardiac troponin-tropomyosin actomyosin S1 ATPase assay, we report that a single TnI mutation, A162H, restores Ca2+ sensitivity at pH 6.5 to that at pH 7.0. Levels of inhibition (pCa 7.0), activation (pCa 4.0), and cooperativity of ATPase activity were minimally affected. Two other mutations (Q155R and E164V) also previously suggested by us (Pearlstone, J. R., Sykes, B. D., and Smillie, L. B. (1997) Biochemistry36, 7601–7606) and involving charged residues showed no such effects. With fast skeletal muscle troponin, a single TnI H130A mutation reduced Ca2+sensitivity at pH 6.5 to levels approaching the cardiac system at pH 6.5. These observations provide structural insight into long-standing physiological and clinical phenomena and are of potential relevance to therapeutic treatments of heart disease by gene transfer, stem cell, and cell transplantation approaches. In contrast to skeletal muscle, the efficiency of the contractile apparatus of cardiac tissue has long been known to be severely compromised by acid pH as in the ischemia of myocardial infarction and other cardiac myopathies. Recent reports (Westfall, M. V., and Metzger, J. M. (2001) News Physiol. Sci.16, 278–281; Li, G., Martin, A. F., and Solaro, R. J. (2001) J. Mol. Cell. Cardiol.33, 1309–1320) have indicated that the reduced Ca2+ sensitivity of cardiac contractility at low pH (≤pH 6.5) is attributable to structural difference(s) in the cardiac and skeletal inhibitory components (TnIs) of their troponins. Here, using a reconstituted Ca2+-regulated human cardiac troponin-tropomyosin actomyosin S1 ATPase assay, we report that a single TnI mutation, A162H, restores Ca2+ sensitivity at pH 6.5 to that at pH 7.0. Levels of inhibition (pCa 7.0), activation (pCa 4.0), and cooperativity of ATPase activity were minimally affected. Two other mutations (Q155R and E164V) also previously suggested by us (Pearlstone, J. R., Sykes, B. D., and Smillie, L. B. (1997) Biochemistry36, 7601–7606) and involving charged residues showed no such effects. With fast skeletal muscle troponin, a single TnI H130A mutation reduced Ca2+sensitivity at pH 6.5 to levels approaching the cardiac system at pH 6.5. These observations provide structural insight into long-standing physiological and clinical phenomena and are of potential relevance to therapeutic treatments of heart disease by gene transfer, stem cell, and cell transplantation approaches. In both cardiac and skeletal muscles, the generation of force through the interaction of thick and thin filaments of the sarcomere is under the control of Ca2+ concentration in the sarcoplasm. The heterotrimeric troponin (Tn) 1The abbreviations used are: Tn, troponin; A, F-actin; TM, tropomyosin; S1, subfragment 1 of myosin; TnC, Ca2+ binding component of Tn; TnI, inhibitory component of Tn; TnT, TM binding component of Tn; fsTnI, chicken fast skeletal isoform of TnI; ssTnI, slow skeletal isoform of TnI; cTnI, human cardiac isoform of TnI; cTnC, human cardiac isoform of TnC; fsTnC, chicken fast skeletal isoform of TnC; cTnT, human cardiac isoform of TnT; fsTnT, chicken fast skeletal isoform of TnT; Ip, inhibitory peptide. complex through its interaction with the thin filament proteins F-actin (A) and tropomyosin (TM) is the dominant player in this control. Conformational changes associated with Ca2+ binding to the regulatory N domain of troponin C (TnC) alter its interactions with both of the other Tn subunits, troponin I (TnI, inhibitory subunit) and troponin T (TnT, TM binding subunit), and of TnI and TnT with TM and A. In consequence, TM moves from its steric blocking position, facilitating interaction of thick filament myosin heads with the thin filament and generation of ATPase activity and contraction. Sequestration of Ca2+ by the sarcoplasma reticulum reverses the process with ensuing relaxation (for reviews, see Refs. 1Geaves M.A. Holmes K.C. Annu. Rev. Biochem. 1999; 68: 687-728Crossref PubMed Scopus (640) Google Scholar and 2Gordon A.M. Homsher E. Regnier M. Physiol. Rev. 2000; 80: 853-924Crossref PubMed Scopus (1341) Google Scholar). As key components of this system, the functional and structural relationships of TnC and TnI and their interaction in the presence and absence of Ca2+ have been the subject of intensive investigation over several decades (for reviews, see Refs. 2Gordon A.M. Homsher E. Regnier M. Physiol. Rev. 2000; 80: 853-924Crossref PubMed Scopus (1341) Google Scholar, 3Farah C.S. Reinach F.C. FASEB J. 1995; 9: 755-767Crossref PubMed Scopus (476) Google Scholar, 4Tobacman L.S. Annu. Rev. Physiol. 1996; 58: 447-481Crossref PubMed Scopus (461) Google Scholar, 5Perry S.V. Mol. Cell. Biochem. 1999; 190: 9-32Crossref PubMed Google Scholar). While high resolution structures of the binary or ternary (with TnT) complexes by x-ray crystallographic or NMR approaches have remained elusive, considerable progress has been made in the mapping of interaction sites of the TnC and TnI components. Two such TnI regions, first described by S. V. Perry and colleagues (for review, see Ref. 5Perry S.V. Mol. Cell. Biochem. 1999; 190: 9-32Crossref PubMed Google Scholar) (see Fig. 1), involve the NH2-terminal segment of fast skeletal TnI (fsTnI) (residues ∼1–30) and the so-called inhibitory peptide or Ip region (residues 96–116). While the high affinity interaction involving residues ∼1–30 likely persists throughout the contraction-relaxation cycle, the weaker Ca2+-dependent interaction involving Ip permits toggling between TnC (+Ca2+) and TM·A (−Ca2+). As such it accounts for a significant proportion of the inhibitory capacity of TnI and the Tn complex (−Ca2+). More recently several studies have shown that for full functional competence additional TnI segments that are COOH-terminal to the Ip region are operative. Thus, for example, Tripetet al. (6Tripet B. Van Eyk J.E. Hodges R.S. J. Mol. Biol. 1997; 271: 728-750Crossref PubMed Scopus (184) Google Scholar) identified an additional Ca2+-sensitive TnC binding region (fsTnI-(116–131)) and a second TM·A binding segment (fsTnI-(140–148)). In a report from our laboratory (7Pearlstone J.R. Sykes B.D. Smillie L.B. Biochemistry. 1997; 36: 7601-7606Crossref PubMed Scopus (56) Google Scholar), we described a sequence motif repeated 3-fold, once in the Ip region (residues ∼101–114, designated α) and twice more in the region of residues ∼121–132 (β) and ∼135–146 (γ). The latter two correspond approximately to those described by Tripetet al. (6Tripet B. Van Eyk J.E. Hodges R.S. J. Mol. Biol. 1997; 271: 728-750Crossref PubMed Scopus (184) Google Scholar). Based on binding studies to TnC and its isolated domains we concluded that while residues 96–116 (Ip) largely interact with the structural C domain, residues 117–148 (β and γ) bind to the regulatory N domain (7Pearlstone J.R. Sykes B.D. Smillie L.B. Biochemistry. 1997; 36: 7601-7606Crossref PubMed Scopus (56) Google Scholar). In contrast to skeletal muscle, the efficiency of the contractile apparatus of cardiac tissue has long been known to be compromised by acid pH (≤6.5) as in the ischemia of myocardial infarction and other cardiac myopathies (8Gaskell W.H. J. Physiol. (Lond.). 1880; 3: 48-75Crossref Scopus (133) Google Scholar, 9Fabiato A. Fabiato F. J. Physiol. (Lond.). 1978; 276: 233-255Crossref Scopus (838) Google Scholar, 10Orchard C.H. Kentish J.C. Am. J. Physiol. 1990; 258: C967-C981Crossref PubMed Google Scholar). Recent reports (11Westfall M.V. Metzger J.M. News Physiol. Sci. 2001; 16: 278-281PubMed Google Scholar, 12Li G. Martin A.F. Solaro R.J. J. Mol. Cell. Cardiol. 2001; 33: 1309-1320Abstract Full Text PDF PubMed Scopus (26) Google Scholar, 13Westfall M.V. Albayya F.P. Turner I.I. Metzger J.M. Circ. Res. 2000; 86: 470-477Crossref PubMed Scopus (50) Google Scholar, 14Wolska B.M. Vijayan K. Arteaga G.M. Konhilas J.P. Phillips R.M. Kim R. Naya T. Leiden J.M. Martin A.F. de Tombe P.P. Solaro R.J. J. Physiol. 2001; 536: 863-870Crossref PubMed Scopus (63) Google Scholar) have indicated that this differential effect can be ascribed to differences in their TnI components and in particular to the COOH-terminal regions distal to their Ip segments. Consistent with these observations and from a comparison of the cardiac and skeletal TnI α, β, and γ sequence motifs, we previously identified several highly conserved charge differences, especially in the β motif, and suggested (7Pearlstone J.R. Sykes B.D. Smillie L.B. Biochemistry. 1997; 36: 7601-7606Crossref PubMed Scopus (56) Google Scholar) these as potential candidates responsible for the differential acid pH effect on the Ca2+ sensitivities of the two systems (see Fig.1). Here we describe experiments to test this proposal. The pET3a·cTnC and pET3a·fsTnC constructs have been described previously (15Pearlstone J.R. Chandra M. Sorenson M.M. Smillie L.B. J. Biol. Chem. 2000; 275: 35106-35115Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 16Slupsky C.M. Kay C.M. Reinach F.C. Smillie L.B. Sykes B.D. Biochemistry. 1995; 34: 7365-7375Crossref PubMed Scopus (62) Google Scholar). For the present study, the Cys-34 and Cys-83 codons (TGC) of pET3a·cTnC were mutated to Ser codons (AGC) using paired 31- and 37-mer oligonucleotides, respectively (Stratagene). The 37-mer oligonucleotide also incorporated a base change for the Arg-84 codon from CGG (rare in Escherichia coli) to CGC (common). This construct has been designated herein as pET3a·cTnC“WT”. Protocols for transformation, expression in BL21 (DE3) pLysS cells, and purification were as described previously (7Pearlstone J.R. Sykes B.D. Smillie L.B. Biochemistry. 1997; 36: 7601-7606Crossref PubMed Scopus (56) Google Scholar). The cDNA encoding human cardiac TnI (cTnI) was kindly provided as clone pCT1-2 by P. J. R. Barton of Imperial College School of Medicine, London, UK (17Vallins W.J. Brand N.J. Dabhade N. Butler-Browne G. Yacoub M.H. Barton P.J.R. FEBS Lett. 1990; 270: 57-61Crossref PubMed Scopus (127) Google Scholar). It was engineered into theNdeI/BamHI sites of pET3a using standard PCR technology. These procedures also involved the mutation of Ala-1 and Gly-3 codons to GCC and GGT, respectively, as recommended (18Al-Hillawi E. Minchin S.D. Trayer I.P. Eur. J. Biochem. 1994; 225: 1195-1201Crossref PubMed Scopus (32) Google Scholar) for high yield expression. Using this construct (pET3a·cTnI) as template, the Cys-79 and Cys-96 codons were mutated to Ser (AGC) in two rounds of mutagenesis using paired 24- and 27-mer primers, respectively. In the case of the Cys-96 to Ser mutation the oligonucleotides used also introduced a codon mutation for Arg-97 from CGA (rare) to CGC (common in E. coli). The final construct is designated pET3a·cTnI“WT.” The pET3a·cTnI(Q155R/A162H/E164V), pET3a·cTnI(Q155R/E164V), and pET3a·cTnI(A162H) were initially constructed using pET3a·cTnI as template for sequential rounds of mutagenesis using paired 25-, 27-, or 29-mer oligonucleotides. Codon changes were to CGT for Arg, GTG for Val, and CAT for His. The C79S and C96S mutants were then introduced into these constructs as for pET3a·cTnC“WT.” The fsTnI construct in pET3a was a gift from F. C. Reinach (19Quaggio R.B. Ferro J.A. Monteiro P.B. Reinach F.C. Protein Sci. 1993; 2: 1053-1056Crossref PubMed Scopus (24) Google Scholar). Sequence analysis of this construct, which expressed poorly in our experiments, showed that the codons for Arg-13 and Arg-14 were both CGG (rare in E. coli). Mutation of these to CGT (common) by the PCR chain extension method markedly improved expression. The codons for Cys-48 and Cys-65 were mutated to AGC (Ser) by two cycles of mutagenesis using paired oligonucleotides. This construct is herein designated pET3a·fsTnI“WT”. Using the latter as template, mutation of the His-130 codon from GAC to that for Ala (GCC) was by the PCR chain extension method to produce pET3a·fsTnI(H130A). Transformation and protein expression of all TnI constructs were the same as for the TnCs. Following cell lysis in a French press at pH 9.0 and centrifugation, the pellets were extracted with 8 murea, pH 9.0 and fractionated on a CM-Sepharose column in 8m urea, pH 7.0 with a 0–0.3 m NaCl gradient. Final purities were assessed by SDS-polyacrylamide gel electrophoresis and amino acid analyses. A clone (pSBET·cTnT) for expression of cTnT was a gift from Dr. S. Hitchcock-DeGregori (20Mukherjea P. Tong L. Seidman J.G. Seidman C.E. Hitchcock-DeGregori S.E. Biochemistry. 1999; 38: 13296-13301Crossref PubMed Scopus (23) Google Scholar). Expression was the same as for the TnCs except that BL21 (DE3) cells and kanamycin for antibiotic selection were used. Purification of cTnT from a cell acetone powder extract (pH 8.0, 6 m urea) was on Q-Sepharose (pH 8.0) and CM-Sepharose columns (pH 6.0), both in 6 m urea. Chicken muscle fsTnT was prepared as described for the rabbit protein (21Mak A.S. Golosinska K. Smillie L.B. J. Biol. Chem. 1983; 258: 14330-14334Abstract Full Text PDF PubMed Google Scholar). A from rabbit skeletal muscle was prepared as described previously (22Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar). Final dialysis was against pH 7.0 or 6.5 assay buffer (plus 1 mm ATP). Final stock concentration was 90–115 μm. Assays were carried out within 2 weeks. Rabbit skeletal myosin subfragment 1 (S1) with associated light chains (A1 and A2) was prepared as described previously (23Weeds A.G. Taylor R.S. Nature. 1975; 257: 54-56Crossref PubMed Scopus (931) Google Scholar) and dialyzed against pH 7.0 or 6.5 assay buffer. Final stock concentration was 22–26 μm. Rabbit skeletal TM, prepared as freeze-dried powder (24Smillie L.B. Colowich S.P. Kaplan N.B. Methods in Enzymology. 85. Academic Press, New York1982: 234-241Google Scholar) without fractionation into α and β chains, was dissolved and dialyzed against pH 7.0 or 6.5 assay buffer to give a 12–14 μm stock solution. Precise concentrations of all three stock solutions were determined by amino acid analyses. The stock solution of TnC (∼5 mg/ml) was in assay buffer; TnI (∼5 mg/ml) was in H2O at pH 4.0–4.5; and TnT (∼5 mg/ml) was in 4 m guanidine·HCl, 1 m KCl, 100 mm imidazole, 4.5 mmMgCl2, 1 mm dithiothreitol, pH 7.0. Concentrations of each stock solution were determined by amino acid analyses, and then all three were combined to give a molar ratio of TnI:TnT:TnC of 1:1:2. Following dialysis against pH 7.0 or 6.5 assay buffer (−EGTA, +50 μm Ca2+) the precise Tn stock concentration (16–24 μm), expressed as TnI, was determined by amino acid analyses. This simplified reconstitution protocol gave equivalent ATPase assay data as native Tn in control experiments. At pH 7.0 the assay buffer was 30 mm KCl, 20 mm imidazole, 4.5 mmMgCl2, 0.15 mm NaN3, 0.5 mm 1 mm At pH 6.5 the was reduced to The protein molar for were for were and for were Final was μm in all The of to each assay was pH 7.0 or 6.5 assay S1, A, TM, of 2 in pH 7.0 or 6.5 assay and at was by final of of 20 mm in pH 7.0 or 6.5 assay buffer to a final of Final was with an and expressed as were and was determined by the method of and R.J. Biochem. PubMed Scopus Google Scholar). as we human cardiac and chicken fast skeletal reconstituted from their isolated components. In all experiments the two residues on each of and fsTnI been previously mutated to These are designated In the two residues in were also mutated to These changes have previously been shown to alter ATPase or Ca2+ sensitivity in reconstituted actomyosin S1 ATPase J.A. P. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, L. K. FEBS Lett. 1993; PubMed Scopus Google Scholar) and in our experiments assay A comparison of Ca2+ sensitivities of the cardiac and skeletal at pH 7.0 and 6.5 is shown in Fig. In with reports the skeletal reconstituted system showed a in Ca2+ sensitivity pH was to 6.5. This can be as an to as in The Ca2+ sensitivity of the cardiac system, was from a of to of Ca2+-regulated troponin-tropomyosin actomyosin S1 ATPase reconstituted with cardiac or skeletal or mutated TnI at pH 7.0 and of ATPase in of F-actin from to pH 7.0 and from to at pH 6.5 with the ratio and buffer used. are from paired experiments in which and at pH 7.0 and 6.5 were with the same F-actin is at ATPase of ATPase in of F-actin from to pH 7.0 and from to at pH 6.5 with the ratio and buffer used. are from paired experiments in which and at pH 7.0 and 6.5 were with the same F-actin is at ATPase in a test the of the β motif charge differences between the cardiac and skeletal (see Fig. we first mutated all three residues in human to the residues in the β motif of fsTnI to produce Following reconstitution with human cardiac TnC and TnT and of ATPase activity as a of at pH 7.0 and 6.5 (see A and it was that the mutation of all three residues markedly the acid sensitivity of the human cardiac the of two of the residues to produce ATPase at pH 7.0 and 6.5 as a showed that this a Ca2+sensitivity to these mutations no effect on the acid sensitivity of the cardiac this indicated the of the mutation in this we carried out on the single In comparison with A and it was that this single mutation largely the acid pH sensitivity of the while the the was reduced from at pH 7.0 to at pH the for were and respectively (see This latter in is to that with the as shown in Fig. 2 and of Ca2+ of and (A) and and fsTnI A, pH pH pH pH pH pH pH pH have also the of the single mutation in the reconstituted fast skeletal muscle Tn system 4 and The data that in contrast to this single mutation the acid pH sensitivity of the Ca2+-regulated the in from pH 7.0 to 6.5 are for and for The data an for His-130 in the of Ca2+ sensitivity against the of pH in the fast skeletal muscle The ATPase data of I provide additional into the of the cardiac and skeletal Tn components and of their TnI the of both the cardiac and skeletal Tn systems showed activity levels that of as studies C.S. Reinach F.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar and have shown that this of activity with the Tn complex and with The present comparison of the cardiac and fast skeletal under a in their and an to our previously The data functional differences of the two and their interactions with their TnI and TnC The data for the of the Ala to and to Ala TnI mutations on their reconstituted also the mutation showed effect on the of inhibition or on the effect (+Ca2+) in comparison with Based on the present data we that this mutation has a effect in the of acid pH on the Ca2+ sensitivity of the cardiac system without its and (+Ca2+) ATPase This is the case with the fast skeletal system in which the H130A mutation to of this effect ATPase activity from to levels to Thus, the fast skeletal TnI His-130 to a or in interactions the Tn in to acid pH also in facilitating significant in ATPase levels over those with The present data are of relevance to studies on slow skeletal muscle TnI (see Refs. M.V. Metzger J.M. News Physiol. Sci. 2001; 16: 278-281PubMed Google Scholar, 12Li G. Martin A.F. Solaro R.J. J. Mol. Cell. Cardiol. 2001; 33: 1309-1320Abstract Full Text PDF PubMed Scopus (26) Google Scholar, 13Westfall M.V. Albayya F.P. Turner I.I. Metzger J.M. Circ. Res. 2000; 86: 470-477Crossref PubMed Scopus (50) Google Scholar, 14Wolska B.M. Vijayan K. Arteaga G.M. Konhilas J.P. Phillips R.M. Kim R. Naya T. Leiden J.M. Martin A.F. de Tombe P.P. Solaro R.J. J. Physiol. 2001; 536: 863-870Crossref PubMed Scopus (63) Google Scholar and As the TnI isoform in and is by the Consistent with the and high levels of cardiac tissue Mol. Cell. Biochem. PubMed Scopus Google Scholar), its contractile have a to acid pH to that of fast skeletal Recent has the COOH-terminal region of as responsible for this (11Westfall M.V. Metzger J.M. News Physiol. Sci. 2001; 16: 278-281PubMed Google Scholar, 12Li G. Martin A.F. Solaro R.J. J. Mol. Cell. Cardiol. 2001; 33: 1309-1320Abstract Full Text PDF PubMed Scopus (26) Google Scholar, 13Westfall M.V. Albayya F.P. Turner I.I. Metzger J.M. Circ. Res. 2000; 86: 470-477Crossref PubMed Scopus (50) Google Scholar). Sequence of the several TnI that as with fsTnI the acid of can be to the equivalent His. The acid base of the chain of a of to the of in the of pH on the Tn As pH is from the charge of can be to to interaction with or more or chains in the regulatory N domain of TnC, this of such on present structural L. Sykes B.D. Biochemistry. 1999; 38: PubMed Scopus Google Scholar, Li, Sykes B.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) of the In we the observations to be of potential relevance to therapeutic treatments of heart disease by gene transfer, stem cell, and cell transplantation approaches. In to Ca2+ sensitivity at pH 6.5 to that at pH the data that ATPase (pCa inhibition (pCa is compromised by this TnI mutation as with This of be an in the of in (Westfall, A. R., F. and Metzger, J. M. J. reports that for the slow skeletal isoform of TnI, expressed in cardiac the mutations and the acid pH sensitivity of to levels to that with cardiac these are equivalent to two of the mutations in the present we that their mutation be responsible for the effects. G. and M. N. G. for