Ca2+-regulatory function of the inhibitory peptide region of cardiac troponin I is aided by the C-terminus of cardiac troponin T: Effects of familial hypertrophic cardiomyopathy mutations cTnI R145G and cTnT R278C, alone and in combination, on filament sliding

Abstract

Investigations of cardiomyopathy mutations in Ca2+ regulatory proteins troponin and tropomyosin provide crucial information about cardiac disease mechanisms, and also provide insights into functional domains in the affected polypeptides. Hypertrophic cardiomyopathy-associated mutations TnI R145G, located within the inhibitory peptide (Ip) of human cardiac troponin I (hcTnI), and TnT R278C, located immediately C-terminal to the IT arm in human cardiac troponin T (hcTnT), share some remarkable features: structurally, biochemically, and pathologically. Using bioinformatics, we find compelling evidence that TnI and TnT, and more specifically the affected regions of hcTnI and hcTnT, may be related not just structurally but also evolutionarily. To test for functional interactions of these mutations on Ca2+-regulation, we generated and characterized Tn complexes containing either mutation alone, or both mutations simultaneously. The most important results from in vitro motility assays (varying [Ca2+], temperature or HMM density) show that the TnT mutant “rescued” some deleterious effects of the TnI mutant at high Ca2+, but exacerbated the loss of function, i.e., switching off the actomyosin interaction, at low Ca2+. Taken together, our experimental results suggest that the C-terminus of cTnT aids Ca2+-regulatory function of cTnI Ip within the troponin complex.

Order–Disorder Transitions in the Cardiac Troponin Complex

“…However, different laboratories and techniques give conflicting results, with determinations of increased calcium sensitivity compared with wild-type, decreased calcium sensitivity, and no or statistically insignificant change. 72; 73; 74; 75; 76 Even in cases without conflicting results, the effects of HCM mutations within TnI C in vitro are highly dependent on method and system complexity.…”

The functional significance of the last 5 residues of the C-terminus of cardiac troponin I

“…Flow cells for motility assays were constructed as described [25, 33-36]. Solutions for assays with regulated thin filaments was calculated to have a composition of 2 mM MgATP, 1 mM Mg 2+ , 10 mM ethylene glycol tetraacetic acid (EGTA) total, sufficient Ca(CH 3 COO) 2 to achieve the desired pCa (pCa 9 – 4), 50 mM K + , 15 mM Na + , 20 mM 3-(N-morpholino) propanesulfonic acid (MOPS) total, pH 7.00 at 30°C, 0.3% methyl cellulose, and ionic strength was adjusted to 0.085 mM with TrisOH/CH 3 COOH) [18, 32, 37]; stock motility buffers were made at pCa 9, 8, 7, 6, 5 and 4, and intermediate pCa solutions were obtained by mixing appropriate, calculated volumes of the two stock solutions at the nearest, whole pCa values.…”

“…Solutions for assays with regulated thin filaments was calculated to have a composition of 2 mM MgATP, 1 mM Mg 2+ , 10 mM ethylene glycol tetraacetic acid (EGTA) total, sufficient Ca(CH 3 COO) 2 to achieve the desired pCa (pCa 9 – 4), 50 mM K + , 15 mM Na + , 20 mM 3-(N-morpholino) propanesulfonic acid (MOPS) total, pH 7.00 at 30°C, 0.3% methyl cellulose, and ionic strength was adjusted to 0.085 mM with TrisOH/CH 3 COOH) [18, 32, 37]; stock motility buffers were made at pCa 9, 8, 7, 6, 5 and 4, and intermediate pCa solutions were obtained by mixing appropriate, calculated volumes of the two stock solutions at the nearest, whole pCa values. To obtain regulated thin filaments, equimolar concentrations (25 nM each) of cTn and cTm were applied to RhPh-labeled F-actin in the flow cell immediately preceding addition of the motility buffer, as described [18, 19, 24, 31, 32, 36]; cTn and cTm (25 nM each) were also added to motility buffers for assays with regulated thin filaments. To minimize photo-oxidative damage, motility buffers additionally contained 3 mg ml −1 glucose, 100 mg ml −1 glucose oxidase, 18 mg ml −1 catalase and 40 mM DTT.…”

Role of cardiac troponin I carboxy terminal mobile domain and linker sequence in regulating cardiac contraction

“…Relevant protein purification procedures have been described previously [29–40]. Skeletal muscle myosin and actin were isolated from the muscle of adult New Zealand White rabbits.…”

“…Rabbit procedures were approved by Florida State University’s Animal Care and Use Committee and rabbits were handled in accordance with the current National Institutes of Health/National Research Council Guide for the Care and Use of Laboratory Animals. Heavy meromyosin (HMM) was prepared according to Kron et al [41] and stored at 4°C for up to three days [29–33]. ATP-insensitive heads were removed by ultracentrifugation [41] and competent HMM diluted to the final working concentration (typically 250 μg/ml).…”