Replacement Of cTnI Research Articles

Influence of a constitutive increase in myofilament Ca2+-sensitivity on Ca2+-fluxes and contraction of mouse heart ventricular myocytes

Chronic increases in myofilament Ca2+-sensitivity in the heart are known to alter gene expression potentially modifying Ca2+-homeostasis and inducing arrhythmias. We tested age-dependent effects of a chronic increase in myofilament Ca2+-sensitivity on induction of altered alter gene expression and activity of Ca2+ transport systems in cardiac myocytes. Our approach was to determine the relative contributions of the major mechanisms responsible for restoring Ca2+ to basal levels in field stimulated ventricular myocytes. Comparisons were made from ventricular myocytes isolated from non-transgenic (NTG) controls and transgenic mice expressing the fetal, slow skeletal troponin I (TG-ssTnI) in place of cardiac TnI (cTnI). Replacement of cTnI by ssTnI induces an increase in myofilament Ca2+-sensitivity. Comparisons included myocytes from relatively young (5–7months) and older mice (11–13months). Employing application of caffeine in normal Tyrode and in 0Na+ 0Ca2+ solution, we were able to dissect the contribution of the sarcoplasmic reticulum Ca2+ pump (SR Ca2+-ATPase), the Na+/Ca2+ exchanger (NCX), and “slow mechanisms” representing the activity of the sarcolemmal Ca2+ pump and the mitochondrial Ca2+ uniporter. The relative contribution of the SR Ca2+-ATPase to restoration of basal Ca2+ levels in younger TG-ssTnI myocytes was lower than in NTG (81.12±2.8% vs 92.70±1.02%), but the same in the older myocytes. Younger and older NTG myocytes demonstrated similar contributions from the SR Ca2+-ATPase and NCX to restoration of basal Ca2+. However, the slow mechanisms for Ca2+ removal were increased in the older NTG (3.4±0.3%) vs the younger NTG myocytes (1.4±0.1%). Compared to NTG, younger TG-ssTnI myocytes demonstrated a significantly bigger contribution of the NCX (16±2.7% in TG vs 6.9±0.9% in NTG) and slow mechanisms (3.3±0.4% in TG vs 1.4±0.1% in NTG). In older TG-ssTnI myocytes the contributions were not significantly different from NTG (NCX: 4.9±0.6% in TG vs 5.5±0.7% in NTG; slow mechanisms: 2.5±0.3% in TG vs 3.4±0.3% in NTG). Our data indicate that constitutive increases in myofilament Ca2+-sensitivity alter the relative significance of the NCX transport system involved in Ca2+-homeostasis only in a younger group of mice. This modification may be of significance in early changes in altered gene expression and electrical stability hearts with increased myofilament Ca-sensitivity.

Effect of Hypertrophic Cardiomyopathy Linked Troponin C Mutations on the Calcium Sensitivity of Acto-Myosin ATPase under Different Phosphorylation States of Troponin I

The objective of this work was to investigate the effect of hypertrophic cardiomyopathy linked A8V and E134D cardiac troponin C (cTnC) mutations on the calcium sensitivity of acto-myosin ATPase under different phosphorylation states of cardiac troponin I (cTnI). Consistent with its effect on the calcium sensitivity of cTnC reconstituted into the thin filament, the A8V mutation led to substantially higher calcium sensitivity of acto-myosin ATPase. The effect of the A8V mutation on the calcium sensitivity of acto-myosin ATPase was also evaluated after cTnI was replaced by the PKA phosphorylation mimetic of cTnI (S22D/S23DcTnI). Replacement of cTnI by the PKA phosphorylation mimetic of cTnI led to a larger decrease in the calcium sensitivity of acto-myosin ATPase for the A8VcTnC compared to wild-type cTnC. In fact, the ability of the A8V mutation to sensitize the acto-myosin ATPase to calcium was almost completely eliminated after cTnI was replaced by the PKA phosphorylation mimetic of cTnI. The PKA phosphorylation mimetic of cTnI also had a bigger effect on the rate of calcium dissociation from the A8VcTnC reconstituted into the thin filament than on that from wild-type cTnC. The E134D mutation exerted no effect on the calcium sensitivity of the acto-myosin ATPase regardless of cTnI phosphorylation status. Thus, the ability of hypertrophic cardiomyopathy linked cTnC mutations to affect the calcium sensitivity of acto-myosin ATPase varies depending on phosphorylation status of cTnI.

Effect of Hypertrophic Cardiomyopathy Linked Troponin C Mutations and Troponin I Phosphorylation on the Rate of Calcium Dissociation from the Thin Filament

The objective of this work was to investigate the effect of hypertrophic cardiomyopathy linked A8V, E134D and D145E mutations on the rate of calcium dissociation from cardiac troponin C (cTn)C on the thin filament under different phosphorylation states of cTnI. While the E134D mutation did not affect the rate of calcium dissociation, the A8V and D145E mutations led to significantly slower rates of calcium dissociation from cTnC on the thin filament. The effect of A8V, E134D and D145E mutations on the rate of calcium dissociation from the thin filament was also examined after cTnI was replaced by either PKA (S22D/S23DcTnI) or PKC (S41D/S43DcTnI) phosphorylation mimetic of cTnI. Replacement of cTnI by either the PKA or PKC phosphorylation mimetic of cTnI dramatically accelerated calcium dissociation from wild-type cTnC on the thin filament. The A8V mutation still led to significantly slower rate of calcium dissociation from cTnC on the thin filament after cTnI was replaced by the PKA phosphorylation mimetic of cTnI. However, in the presence of PKC phosphorylation mimetic of cTnI, the ability of A8V mutation to slow the rate of calcium dissociation from the cTnC on thin filament was abolished. The E134D mutation exerted minor to no effect on the rate of calcium dissociation from cTnC on the thin filament regardless of cTnI phosphorylation status. On the other hand, the D145E mutation led to significantly slower rates of calcium dissociation from cTnC on the thin filament after cTnI was replaced by either PKA or PKC phosphorylation mimetic of cTnI. Thus, the ability of hypertrophic cardiomyopathy linked cTnC mutations to affect the rate of calcium dissociation from cTnC on the thin filament varies depending on phosphorylation status of cTnI.

Comparison of the Binding of the Switch Regions of Cardiac Troponin-I and Skeletal Troponin-I to the Functional N-Domain of Human Cardiac Troponin-C

In the heart, there are two isoforms of troponin-I (TnI) that are developmentally regulated. The slow skeletal TnI (ssTnI) is the sole isoform expressed in the embryonic or neonatal heart, while cardiac TnI (cTnI) is expressed exclusively in the adult heart. One important distinction between ssTnI and cTnI relates to differences in myofilament Ca2+-sensitivity of force development under acidic pH conditions. Hearts expressing ssTnI show heightened Ca2+-sensitivity compared with hearts expressing cTnI under basal conditions. This isoform-specific difference is greatly accentuated during acidosis, which is often associated with ischemia of myocardial infarction. In vitro and in vivo functional studies have shown that replacement of cTnI with ssTnI results in a marked enhancement of myofilament Ca2+-sensitivity at acidic pH conditions. Recent reports have indicated that this effect can be ascribed to amino acid sequence differences in ssTnI and cTnI and in particular to a critical A162H substitution in the switch region. In this study, we have used NMR spectroscopy to examine the binding of the switch regions of ssTnI (sTnI115-131) and cTnI (cTnI147-163) to the N-domain of cardiac troponin-C (cNTnC) at physiological and acidic pH conditions. The results show that the affinity of sTnI115-131 for cNTnC•Ca2+ (KD ∼50uM) is ∼3-fold stronger than that of cTnI147-163 (KD ∼150uM), but neither are affected by a pH change from 7 to 6. The pKa of H130 in sTnI115-131 is 6.2 when free and 6.7 when bound to cNTnC•Ca2+. We have also used {1H, 15N}-HSQC NMR spectroscopy to monitor the pKa changes of cNTnC•Ca2+ from peptide free to peptide bound states. The implications of these results will be discussed in the context of structure and function of myofilament protein interactions.

Localization of Regions of Troponin I Important in Deactivation of Cardiac Myofilaments by Acidic pH

G. Li, A. F. Martin and R. J. Solaro. Localization of Regions of Troponin I Important in Deactivation of Cardiac Myofilaments by Acidic pH. Journal of Molecular and Cellular Cardiology (2001) 33, 0000–0000. Ca2+-activation of cardiac muscle myofilaments is more sensitive to depression by acidic pH than is the case with skeletal myofilaments. We tested the hypothesis that this difference is related to specific regions of the TnI (troponin I) isoforms in these muscles. We exchanged native Tn complex in detergent-extracted fiber bundles from mouse ventricles with Tn containing various combinations of fast (fsTnI) or slow skeletal (ssTnI) complexed with either cardiac TnC (cTnC) or fsTnC, and with cTnC complexed with the following chimeras: (1) fsTnI N-terminal region (fN) plus cTnI inhibitory peptide (cIp) and cTnI C-terminal region (cC); and (2) cTnI N-terminal region (cN)-cIp-fsTnI C-terminal region (fC). We determined the change in half maximal Ca2+(ΔEC50) for tension activation at pH 7.0 and pH 6.5. Similar ΔEC50values were obtained for unextracted controls (5.53±0.30 μ m), for preparations containing cTnI-cTnC (5.74±0.40μ m), and preparations exchanged with cTnI-fsTnC (5.63±0.40 μ m). However, replacement of cTnI with fsTnI significantly decreased ΔEC50to 3.95±0.17 μ m. Replacement of cTnI with ssTnI also significantly depressed ΔEC50to 2.07±0.15 μ m. Results of studies using the chimeras demonstrated that the C-terminal domains of cTnI and fsTnI are responsible for these differences. This conclusion also fits with data from experiments in which we measured Ca2+-binding to the regulatory site of cTnC in binary complexes containing cTnC with cTnI, fsTnI, or the chimeras. Our results localize a region of TnI important in effects of acidosis on cardiac myofilaments and extend our earlier data indicating that C-terminal regions of cTnI outside the Ip are critical for activation by Ca.2+