Soluble complement receptor-1 protects heart, lung, and cardiac myofilament function from cardiopulmonary bypass damage.

BACKGROUND: Host defense system activation occurs with cardiopulmonary bypass (CPB) and is thought to contribute to the pathophysiological consequences of CPB. Complement inhibition effects on the post-CPB syndrome were tested with soluble complement receptor-1 (sCR1). METHODS AND RESULTS: Twenty neonatal pigs (weight 1.8 to 2.8 kg) were randomized to control and sCR1-treated groups. LV pressure and volume, left atrial pressure, pulmonary artery pressure and flow, and respiratory system compliance and resistance were measured. Preload recruitable stroke work, isovolumic diastolic relaxation time constant (tau), and pulmonary vascular resistance were determined. Pre-CPB measures were not statistically significantly different between the 2 groups. After CPB, preload recruitable stroke work was significantly higher in the sCR1 group (n=5, 46.8+/-3.2x10(3) vs n=6, 34.3+/-3.7x10(3) erg/cm(3), P=0.042); tau was significantly lower in the sCR1 group (26.4+/-1.5, 42.4+/-6. 6 ms, P=0.003); pulmonary vascular resistance was significantly lower in the sCR1 group (5860+/-1360 vs 12 170+/-1200 dyn. s/cm(5), P=0.009); arterial PO(2) in 100% FIO(2) was significantly higher in the sCR1 group (406+/-63 vs 148+/-33 mm Hg, P=0.01); lung compliance and airway resistance did not differ significantly. The post-CPB Hill coefficient of atrial myocardium was higher in the sCR1 group (2.88+/-0.29 vs 1.88+/-0.16, P=0.023). CONCLUSIONS: sCR1 meaningfully moderates the post-CPB syndrome, supporting the hypothesis that complement activation contributes to this syndrome.

Immunological identification of five troponin T isoforms reveals an elaborate maturational troponin T profile in rabbit myocardium.

Myocardium is generally thought to express no more than two isoforms of troponin T (TnT). We have recently reported that TnT purified from rabbit myocardium is resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into five proteins (TnT1, TnT2, TnT3, TnT4, and TnT5). In this study, these proteins are characterized immunologically and a novel elaborate maturational profile is described. Myocardium was obtained from 23 days of gestation fetal rabbits and 2-day, 6-week, 3-month, and 6-month postnatal rabbits. The major species in the adult myocardium, TnT4, was identified on sodium dodecyl sulfate-polyacrylamide gels and excised. The protein was electroeluted and purified. An amino acid microsequence of a cleaved fragment of this protein was found to be virtually identical to residues 86-99 from adult rabbit cardiac TnT. The protein, TnT4, was used to raise a polyclonal antibody. This antibody recognized all five isoforms from purified cardiac TnT, but none of the TnT isoforms from fast skeletal muscle. A monoclonal antibody, Mab JLT-12, raised against a highly conserved epitope of rabbit fast skeletal muscle, recognized all five cardiac as well as five skeletal muscle isoforms. Western blots performed on intact myocardial preparations demonstrated that TnT1, the cardiac isoform with the slowest electrophoretic mobility, was expressed prominently in the immature hearts, in addition to TnT2, TnT3, and TnT4, but TnT1 was not evident in the 3-month and 6-month postnatal hearts. The expression of TnT2 also decreased with maturation. Thus, the number of TnT isoforms expressed in the rabbit decreases with maturation.(ABSTRACT TRUNCATED AT 250 WORDS)

In utero right ventricular output in the fetal lamb: the effect of heart rate.

1. The effect of heart rate on right ventricular output was examined in six lambs during a period extending from 126 to 139 days of gestation. The fetuses had been surgically instrumented at least four days previously with a main pulmonary artery flow probe, right ventricular dimension transducers and left and right atrial pacing electrodes. 2. During spontaneous variations in heart rate, rate was correlated positively with right ventricular output (P less than 0.0001) and end-diastolic dimension (P less than 0.0001) among the lambs considered as a group, but no significant effect of rate on stroke volume was found. When individual responses were examined, output increased significantly with rate in sixteen out of seventeen observations. 3. With left atrial pacing, heart rate did not affect output. With right atrial pacing, rate correlated negatively with output (P less than 0.0001). With pacing from either site, rate correlated negatively with end-diastolic dimension (P less than 0.0001) and stroke volume (P less than 0.0001). 4. The introduction of a longer period interval during each pacing rate inhibited the rate-related decrease in dimension and allowed the ventricle to fill to the same end-diastolic dimension. The systole following these longer intervals had a greater stroke volume than did the preceding systoles with smaller end-diastolic dimension. The faster the preceding paced rate, the greater was the increase in stroke volume (P less than 0.001). 5. Right ventricular dimensions and volumes were measured in vitro, and the relationship was found to be linear using regression analysis. 6. This study demonstrates that experimentally induced variations in heart rate produce changes in end-diastolic volume and contractility which prominently affect right ventricular stroke volume. As a consequence, rate has, over a broad range, either no significant effect on output or a negative one. With spontaneous variations in rate, additional changes in contractility and venous return occur which affect stroke volume and end-diastolic volume and enhance right ventricular output. These relationships are similar to those in the adult heart, and demonstrate the absence of a maturational change in the effects of rate on ventricular function from the fetus to the adult.

A cardiac troponin T epitope conserved across phyla.

Troponin T is a thin filament protein that is important in regulating striated muscle contraction. We have raised a monoclonal antibody against rabbit cardiac troponin T, monoclonal (mAb) 13-11, that recognizes its epitope in cardiac troponin T isoforms from fish, bird, and mammal but not from frog. The number of these isoforms expressed in cardiac muscle varies among species and during development. Cardiac troponin T isoforms were not found in adult skeletal muscle, while they were expressed transiently in immature skeletal muscle. We have mapped the epitope recognized by mAb 13-11 using rabbit cardiac troponin T isoforms. Analysis of stepwise cyanogen bromide digestion, which allowed association of the epitope to regions spanning methionine residues, coupled with immunoactivity of synthetic peptides, corresponding to sequences containing methionine residues, indicated that mAb 13-11 recognized its epitope in a 17-residue sequence containing the methionine at position 68, SKPKPRPFMPNLVPPKI. Comparison of skeletal and cardiac troponin T sequences suggested that the epitope was contained within the sequence FMPNLVPPKI. Synthetic peptides PFMPNLVPPKI and FMPNLVPPKI were recognized by mAb 13-11 on slot-blots. Enzyme-linked immunosorbent assay demonstrated mAb 13-11 recognized, in order of descending affinity, the 17-, 11-, and 10-residue sequence. Preabsorption of mAb 13-11 with each of these sequences blocked the recognition of the 17-residue peptide by mAb 13-11. The domain, PFMPNLVPPKI is encoded by the 5' region of the cardiac gene exon 10 and is present in hearts across a broad range of phyla. These findings suggest that this cardiac troponin T-specific sequence confers onto myofilaments structural and functional properties unique to the heart.

Force-pCa relation and troponin T isoforms of rabbit myocardium.

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.

Cardiac troponin T isoform expression correlates with pathophysiological descriptors in patients who underwent corrective surgery for congenital heart disease.

BACKGROUND: This study examined cardiac troponin T (cTnT) isoform expression in patients who had undergone surgery at Duke University Medical Center (Durham, NC) between December 1, 1993, and January 31, 1995, to correct congenital heart defects. The human heart expresses four cTnT isoforms (cTnT1 through cTnT4) whose sequence differences result from combinatorial alternative splicing of two exons. We have previously shown that cTnT4 is expressed at higher levels in severely failing hearts from transplant patients. In this study, we tested the hypothesis that congenital heart defects that have a more negative effect on myocardial function increase cTnT4 expression. We used the presence or absence of drug treatment for heart failure or congested circulation before surgery and the duration of inotropic support after corrective surgery as indicators of the pathophysiological state of the heart just before surgery. METHODS AND RESULTS: Right atrial appendage tissue was collected from 34 patients, 6 days to 35 years old (median age, 3.4 months). The amounts of the cTnT1 through cTnT4 isoforms, measured as a percentage of total cTnT, were determined from Western blots probed with MAb13-11, a cTnT-specific monoclonal antibody. We found that cTnT4 expression correlated positively with the duration of inotropic support and was higher in patients who received drug treatment before surgery than in those who did not. Furthermore, we found that the percent of cTnT4 was significantly higher in hearts with congenital defects that caused congestive failure than in hearts with tetralogy of Fallot. CONCLUSIONS: 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.

Troponin T isoform expression in the normal and failing human left ventricle: a correlation with myofibrillar ATPase activity.

The expression of troponin T, a thin filament regulatory protein, was examined in normal and failing left ventricles. The samples were obtained from the hearts of patients with severe heart failure who were undergoing cardiac transplantation, and from normal adult hearts that could not be used for transplantation. Western blots of the myofibrillar proteins demonstrated two isoforms, troponin T 1 (TnT1) and troponin T 2 (TnT2). TnT2 is expressed at significantly higher levels in failing hearts (p less than 0.004). Western blots of two-dimension SDS-PAGE gels resolved two dominant spots of TnT1 and of TnT2 and several minor troponin T species. Alkaline phosphatase treatment markedly decreased the sizes of the two acidic spots while increasing the two more basic spots by a comparable amount. Myofibrillar ATPase activity had an inverse and negative linear relationship (r = 0.7, p less than 0.02) with the myofibrillar percentage of total troponin T comprised of TnT2. In that heart failure in these transplant patients had multiple bases, we propose that rather than a cause of heart failure, the disease-associated changes in troponin T isoform expression are an adaptation to abnormal myocardial function.

Molecular basis of cardiac troponin T isoform heterogeneity in rabbit heart.

In the rabbit heart, multiple isoforms of cardiac troponin T (cTnT1 through cTnT5, from largest in size to smallest), a protein essential for calcium-regulated myofibrillar ATPase activity, have been identified, and a correlation has been found between these isoforms and myofilament sensitivity to calcium. We have sought to establish the molecular basis of this diversity. Restriction-digest analysis of genomic DNA has indicated that the rabbit cTnT gene is a single-copy gene. cTnT cDNA clones were isolated from cDNA libraries, yielding a consensus sequence for the protein. Newborn rabbit heart cDNAs, obtained using the reverse-transcriptase polymerase chain reaction (RT-PCR), were amplified using primers derived from this cDNA. Three full-length cDNAs that differed by the inclusion or exclusion of three short nucleotide sequences within the cDNAs were obtained. Amplification in the 5' half of the cDNAs confirmed that multiple cTnT products arose because of the variable inclusion of an 18- and a 30-nt sequence. The 30-nt sequence has homology with previously described alternatively spliced exons in rat and chicken cTnT, whereas the 18-nt sequence has not been described previously. RT-PCR in the 3' half of the cDNAs confirmed an additional region of heterogeneity: the presence, in part or in full, or absence of a 9-nt region, which matches the alternatively spliced exon 12 described for rat cTnT. In vitro transcription and translation of four cDNA clones containing both the 18- and 30-nt sequences, the 30-nt sequence, the 18-nt sequence, or neither generated protein isoforms that comigrated with cTnT1, cTnT2, cTnT3, and cTnT4, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Troponin T isoform expression in humans. A comparison among normal and failing adult heart, fetal heart, and adult and fetal skeletal muscle.

The expression of troponin (Tn) T, a thin-filament regulatory protein, was examined in left ventricular myocardium from normal and from failing adult human hearts. The differences in isoform expression between normal and failing myocardium led us to examine the ontogenic expression of TnT in human striated muscle. Left ventricular samples were obtained from patients with severe heart failure undergoing cardiac transplantation and normal adult organ donors. Fetal muscle was obtained from aborted fetuses after 14-15 weeks of gestation, and adult skeletal muscle was obtained from surgical biopsies. Western blots of normal and failing adult heart proteins demonstrated that two isoforms, TnT1 and TnT2, are expressed in different amounts, with TnT2 being significantly greater in failing hearts (p less than 0.004). Western blots of two-dimensional gels of these proteins resolved two predominant spots of both TnT1 and TnT2 and several minor TnT species. Alkaline phosphatase treatment converted the two major spots of each isoform into the single more basic spots. A comparison of the ATPase activities and the TnT2 percentage of total TnT in individual failing and normal adult hearts demonstrated an inverse and negative relation (r = 0.7, p less than 0.02). In the fetal heart, four TnT isoforms were found, two of which had the same electrophoretic mobilities as the adult cardiac isoforms TnT1 and TnT2. Fetal skeletal muscle expressed two of the four fetal cardiac TnT isoforms, one of which comigrated with adult cardiac TnT1. These cardiac isoforms were expressed in low abundance in fetal skeletal muscle relative to seven fast skeletal muscle TnT isoforms. No cardiac isoforms were present in adult skeletal muscle. Because many etiologies caused heart failure in the transplant patients, we propose that the disease-associated increased expression of the TnT isoform TnT2 is an adaptation to the heart failure state and a partial recapitulation of the fetal expression of cardiac TnT isoforms.

Troponin I phosphorylation in the normal and failing adult human heart.

BACKGROUND: In the failing human heart myofibrillar calcium sensitivity of tension development is greater and maximal myofibrillar ATPase activity is less than in the normal heart. Phosphorylation of the cardiac troponin I (cTnI)-specific NH2-terminus decreases myofilament sensitivity to calcium, while phosphorylation of other cTnI sites decreases maximal myofibrillar ATPase activity. METHODS AND RESULTS: We examined cTnI phosphorylation in left ventricular myocardium collected from failing hearts at the time of transplant (n=20) and normal hearts from trauma victims (n=24). The relative amounts of actin, tropomyosin, and TnI did not differ between failing and normal myocardium. Using Western blot analysis with a monoclonal antibody (MAb) that recognizes the striated muscle TnI isoforms, we confirmed that the adult human heart expresses only cTnI. A cTnI-specific MAb recognized two bands of cTnI, designated cTnI1 and cTnI2, while a MAb whose epitope is located in the cTnI-specific NH2-terminus recognized only cTnI1. Alkaline phosphatase decreased the relative amount of cTnl1, while protein kinase A and protein kinase C increased cTnI1. The percentage of cTnI made up of cTnI1, the phosphorylated form of TnI, is greater in the normal than the failing human heart (P<.00). CONCLUSIONS: This phosphorylation difference could underlie the reported greater myofibrillar calcium sensitivity of failing myocardium. The functional consequence of this difference may be an adaptive or maladaptive response to the lower and longer calcium concentration transient of the failing heart, eg, enhancing force development or producing ventricular diastolic dysfunction.

C2C12 cells: biophysical, biochemical, and immunocytochemical properties.

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.

Molecular basis of human cardiac troponin T isoforms expressed in the developing, adult, and failing heart.

Cardiac troponin T (cTnT), a protein essential for calcium-regulated myofibrillar ATPase activity, is expressed in the human heart as four isoforms (cTnT1 through cTnT4, numbered in the order of decreasing molecular size). The expression of these isoforms at the protein level has previously been found by us to differ in the normal and failing adult and fetal human heart. In the present study, we have cloned and sequenced four full-length cDNAs corresponding to the four native cTnT protein isoforms and have expressed these cDNAs in an in vitro transcription and translation system. The cDNAs differ by the variable inclusion of a 15- and a 30-nt exon in the 5' half of the coding region. These cDNAs yielded proteins that comigrate with the native isoforms, cTnT1 through cTnT4. Polyclonal antisera, raised against a synthetic peptide corresponding to the 10-residue peptide encoded by the 30-nt exon, reacted with the two human isoforms largest in molecular size (cTnT1 and cTnT2) and the two largest cTnT isoforms of the rabbit and rat. The isoforms cTnT1 and cTnT2, containing either both peptides encoded by the 30- and 15-nt exons or the peptide encoded by the 30-nt exon alone, are expressed in the fetal heart, with cTnT2 being expressed at a very low level. cTnT4, lacking both of these sequences, is expressed in the fetal heart and is reexpressed in the failing adult heart, whereas cTnT3, containing the 5-residue peptide, is the dominant isoform in the adult heart.(ABSTRACT TRUNCATED AT 250 WORDS)