Troponin I isoforms and differential effects of acidic pH on soleus and cardiac myofilaments

Abstract

Differences in pH sensitivity of tension generation between developing and adult cardiac myofilaments, which contain the same isoform of troponin C (TnC), have been proposed to be due to troponin I (TnI) isoform switching from the slow skeletal (ss) to cardiac (c) TnI isoforms (21). We investigated the effects of acidic pH on Ca(2+)-activation of force in chemically skinned preparations of adult rat trabeculae and single soleus fibers that also share the same TnC isoform. Compared with the soleus fibers, trabeculae demonstrated a greater suppression of tension and a rightward shift in pCa50 (-log half-maximally activating molar Ca2+ concentration) when pH was decreased from 7.0 to 6.2. The pH-induced shift in pCa50 in soleus fibers did not change with sarcomere length. Troponin subunit interactions were also investigated, using cardiac troponin C (cTnCIA) labeled with a fluorescent probe, 2-(4'-iodoacetamidoanilino)-naphthalene-6-sulfonic acid. Under acidic conditions, cTnCIA demonstrated a decrease in Ca(2+)-affinity. This decrease was amplified both in the binary complex cTnCIA-cTnI and in the complex cTnCIA-cTnI-cTnT-tropomyosin to the same extent. In contrast, substitution of ssTnI for cTnI in these complexes produced the same decrease in Ca2+ affinity in response to acidic pH as cTnCIA alone. These results support our hypothesis that differential effects of pH on tension generation and Ca2+ sensitivity between soleus fibers and trabeculae are due to the presence of different isoforms of TnI.

Slow skeletal troponin I gene transfer, expression, and myofilament incorporation enhances adult cardiac myocyte contractile function

“…Studies using permeabilized muscle preparations also have implicated TnI in this pH effect. For example, the acidosis-induced shift in myofilament Ca 2ϩ sensitivity is greater in cTnI-expressing adult myocardium than in slow skeletal muscle fibers (9,10) or fetal͞neonatal myocardium (3,5), which express ssTnI (1,2). Together, these results suggest that TnI influences the myofilament Ca 2ϩ sensitivity response to acidosis in an isoformdependent manner.…”

Effects of exercise training on contractile function in myocardial trabeculae after ischemia-reperfusion

“…Transmural samples from the left and right ventricular free walls were obtained by dissection after 60 min of perfusion for the control hearts and after reperfusion in the ischemia-reperfusion hearts and stored at Ϫ80°C for later analysis of MHC compositions. The rest of the myocardium was placed in glycerinating solution (32) for skinning and stored at Ϫ22°C until analyzed for measurements of trabecula contractile properties. The frozen transmural samples were thawed and prepared for analysis of MHC isoform composition as described in Blough et al (2), including the homogenization step.…”

Regulatory Domain Conformational Exchange and Linker Region Flexibility in Cardiac Troponin C Bound to Cardiac Troponin I

“…Spectral widths in the t 1 and t 2 dimensions were 3.3 kHz and 12 kHz respectively. Spectra were collected with Carr-Purcell-Meiboom-Gill relaxation periods of 0, 8,16,24,32,40,48, and 64 ms. Double points were collected with relaxation periods of 8, 16, 32, and 40 ms for error analysis.…”

“…Fluorescence Assay-Calcium binding to cTnC was measured as described previously (24). Binding measurements were made with the aid of the fluorescent probe, 2-(4Ј-iodoacetamidoanilino)-naphthalene-6-sulfonic acid (IAANS), which reports Ca 2ϩ binding to the regulatory site.…”

“…Surprisingly, binding of both the cardiac specific N terminus and the C-domain of cTnI altered the cTnC regulatory domain conformation. The absence of the cardiac specific N terminus (cTnI-(33-211)), mutation of Ser 23 and Ser 24 to Asp (cTnI-(1-80)DD) or phosphorylation at Ser residues 23 and 24 (cTnI-(1-80)pp), shifts the conformational equilibrium toward that observed in free Ca 2ϩ -saturated cTnC. This provides the first evidence that the cardiac specific N terminus modulates the function of cTnC by altering the conformational equilibria within the regulatory domain.…”