Skeletal troponin C reduces contractile sensitivity to acidosis in cardiac myocytes from transgenic mice.

Abstract

Depressed contractile function plays a primary role in the pathophysiology of acute myocardial ischemia. Intracellular acidification is an important factor underlying the inhibition offorce production in the ischemic myocardium. The effect of acidosis to depress contractiliy is markedly greater in cardiac as compared to skeletal muscle; however, the molecular basis of this difference in sensitivity to acidosis is not clearly understood. In this report, we describe transgenic mice that express the fast skeletal isoform oftroponin C (sTnC) in cardiac muscle. In permeabilized single cardiac myocytes the shift in the midpoint of the tension-pCa relationship (i.e., pCaso, where pCa is -log[Ca2+]) due to lowering pH from 7.00 to 6.20 was 1.27 ± 0.03 (n = 7) pCa units in control cardiac TnC (cTnC) expressing myocytes and 0.96 0.04 (n = 11) pCa unit in transgenic cardiac myocytes (P < 0.001). The effect of pH to alter maximum Ca2+-activated tension was unchanged by TnC isoforms in these cardiac myocytes. In a reciprocal experiment, contractile sensitivity to acidosis was increased in fast skeletal muscle fibers following extraction of endogenous sTnC and reconstitution with purified cTnC in vitro. Our findings demonstrate that TnC plays an important role in determining the profound sensitivity of cardiac muscle to acidosis and identify cTnC as a target for therapeutic interventions designed to modify ischemia-induced myocardial contractile dysfunction.It is well known that force generation diminishes markedly during ischemia in the heart (1-3). In contrast, studies on isolated whole heart or isolated papillary muscles using NMR, fluorescent indicators, or the photoprotein aequorin reveal that the Ca2+ transient of contraction is generally little changed during the time period of ischemia when force is declining markedly (2). Thus, the basis of contractile dysfunction in the ischemic heart appears to involve events in the excitation-contraction coupling scheme distal to Ca2+ release from the sarcoplasmic reticulum. During ischemia in the heart, metabolic byproducts of ATP hydrolysis and the glycolytic energy pathway, including H+, inorganic phosphate, and ADP, accumulate within the myoplasm and these byproducts have been implicated in mediating the observed altered contractile function ofthe failing heart in ischemia (2). For example, it has been shown that intracellular pH declines from about 7.00 to 6.20 during ischemia in the heart (2, 4). Studies using the permeabilized muscle preparation, in which the sarcolemma is chemically or mechanically disrupted to allow access and direct control of the intracellular solution bathing the otherwise intact contractile apparatus, provide evidence that low pH has a direct effect to depress Ca2+-activated contraction in cardiac and skeletal muscle, with the effect being greater in cardiac muscle (refs. 5 and 6; present study). Taken together these results suggest that the basis of pH-induced inhibition of force generation resides in desen-The publication costs of this artic...

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

“…4 Left) demonstrated normal sarcomeric structure in AdCMVssTnI-treated myocytes, indicating that gene transfer did not alter the highly ordered myofibrillar lattice. Taken together, these results provide strong evidence that the cardiac myocyte contractile apparatus is preserved after adenovirus-mediated transfer of the ssTnI cDNA, in agreement with earlier transgenic studies on other myofilament genes (21,22). This point is important because myofibrillar disarray is associated with overexpression of a normal or mutant myofilament protein in the indirect flight muscle of Drosophila melanogaster (23).…”

Sequence mutations in teleost cardiac troponin C that are permissive of high Ca2+ affinity of site II

“…We have demonstrated the effectiveness of Trp at residue 27 in reporting Ca 2ϩ binding to site II in McTnC and ScTnC without significantly affecting the ␣-helical content of the protein, which reflects the general structure of the molecule, using far UV circular dichroism spectra (27). Additionally, the K1⁄2 values of Ca 2ϩ binding to the F27W cNTnC mutants and full-length IFcTnC in the present study are within the range previously reported for that of Ca 2ϩ -triggered tension generation in cardiac myocytes under similar conditions (16,18,23,24,28,39). We therefore maintain that the F27W mutation effec-tively reports on the Ca 2ϩ -induced conformational response of the cTnC isoforms without significantly altering the functional characteristics of the proteins.…”

“…Ca 2ϩ affinity is very sensitive to pH in cardiac muscle but much less so in skeletal muscle. Studies using transgenic mice (24) and TnC replacement in skinned fibers (7,25) have demonstrated that TnC isoforms influence the pH sensitivity of striated muscle. Cardiac muscle demonstrates important differences in the length-tension relationship, the basis of the Starling relationship, that have been attributed, at least in part, to cTnC (2,13), though there is also compelling evidence to the contrary (22,26).…”

A Serum Response Factor-Dependent Transcriptional Regulatory Program Identifies Distinct Smooth Muscle Cell Sublineages

“…Transgenic mice harboring the Ϫ5000SM22-lacZ, Ϫ441SM22-lacZ, Ϫ441SM22CArG-lacZ, Ϫ441SM22SME-3-lacZ, Ϫ441SM22CArG-lacZ, and Ϫ280SM22-lacZ transgenes were produced according to standard techniques as previously described (34). To identify transgenic founder mice, Southern blot analyses were performed with the radiolabeled lacZ probe and high-molecularweight DNA prepared from tail biopsies of each potential founder.…”