We examined the myofibril biochemical, structural, and biophysical properties of C2C12, a mouse skeletal muscle cell line (American Type Culture Collection), to assess whether force development and the sensitivity of the myofilaments to calcium could be measured in C2C12 myotubes and whether a cardiac contractile protein, troponin T, is expressed and incorporated into C2C12 myofibrils. When myoblasts fused and differentiated into myotubes, expression of myofilament proteins was initiated. Multiple cardiac and skeletal muscle troponin T isoforms were coexpressed. Cardiac troponin T expression increased and then decreased with time. Fluorescence immunocytochemistry demonstrated incorporation of cardiac troponin T isoforms into the myofibrils. At the time of the biophysical studies, mean myotube diameter was 12 microns (range 5-25 microns), and mean length was 290 microns (range 130-520 microns). The estimated maximum force developed by chemically skinned myotubes at 6-7 days poststarvation, 0.88 +/- 0.12 microN (mean +/- 95% confidence interval, n = 5), was significantly less (P < 0.05) than that at 10-13 days poststarvation, 1.12 +/- 0.12 microN (n = 7). The force-pCa relation yielded a Hill coefficient of 2.9 +/- 0.6 (n = 7) and half-maximal activation at pCa of 5.77 +/- 0.20. The demonstration that the biophysical properties of C2C12 cells can be measured and that cardiac and skeletal muscle troponin T isoforms are incorporated and colocalized into myofibrils suggest that these cells could be a useful model to assess the effects of exogenous native and mutated cardiac and skeletal contractile protein isoforms on myofilament function.

Sarcomere shortening and ultrastructure of intact isolated myocytes from ventricles of three-week-old and adult rabbits were examined. Cells were fixed and embedded, and after measuring their sarcomere shortening in response to electrical stimulation, they were examined in serial thin sections by electron microscopy. This structure-function analysis showed that adult cells were significantly larger, had longer rest sarcomere lengths, greater amount and velocity of sarcomere shortening, greater velocity of reextension, and shorter contraction duration than immature cells. In immature myocytes, a thin outer shell of myofibrils enveloped a central mass of mitochondria and nuclei, but in adult cells, the cytoskeleton divided the cell into compartments with the mitochondria arranged around and interspersed among the myofibrils. The different arrangement of the organelles and the cytoskeleton at the two ages may account for the shorter rest sarcomere length in the young myocytes and may confer differing internal loads that contribute to their smaller amount and velocity of sarcomere shortening. The corbular and longitudinal sarcoplasmic reticulum were less demarcated in immature than in adult cells. Myocytes from both ages showed postextrasystolic potentiation, suggesting that the sarcoplasmic reticulum modulates calcium at both ages. Restitution of contractility between contractions, obtained by measuring sarcomere shortening of interpolated extrasystoles, was faster in immature than in adult cells and may reflect the structural differences in the sarcoplasmic reticulum. The developmental differentiation in the sarcoplasmic reticulum suggests that changes in compartmentalization of calcium and in the distribution of putative calcium-release sites contribute to the increased contractility of adult myocytes.

We have previously reported the existence of at least four troponin T isoforms in rabbit ventricular muscle and described the changes in their distribution with development. In this report we test whether the proportions of the troponin T isoforms are related to the sensitivity of the myofilaments to calcium. We measured the force-pCa relations in 12 detergent-skinned ventricular strands of cardiac muscle from newborn (2-5-day-old) rabbits. We determined from each strand the amount of each troponin T isoform relative to the total amount of troponin T by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometric scans of Western blots probed with a cardiac-specific troponin T monoclonal antibody, MAb 13-11. To assess the presence of different relative amounts of cardiac and slow skeletal troponin I among the strands, we determined the amount of cardiac troponin I relative to tropomyosin. We determined the Hill coefficient and the pCa for half-maximal force, pCa50, for each strand. pCa50 was related directly to the relative amount of troponin T2 (pslope = 0.037). Our results do not indicate a relation between the Hill coefficient and troponin T2. We also did not find a relation between pCa50 and the cardiac troponin I/tropomyosin ratio, which suggests that the correlation between pCa50 and troponin T2 was not a result of changes in the relative amounts of cardiac and slow skeletal muscle troponin I. Our findings indicate that a relation exists between the force-pCa characteristics of rabbit myocardium and the troponin T isoforms that it expresses, suggesting a role for troponin T in modulating the sensitivity of cardiac myofilaments to calcium.

These findings suggest that in patients with congenital cardiac defects, cTnT4 expression is modulated by heart failure and is increased in hearts that are more hemodynamically stressed.