M. Benjamin Perryman

Use of a Rapid Assay of Subforms of Creatine Kinase MB to Diagnose or Rule Out Acute Myocardial Infarction

BACKGROUND Ruling out myocardial infarction in patients coming to the emergency room with chest pain is hindered by the lack of a specific early diagnostic marker. Less than 30 percent of patients admitted to coronary care units have infarction, resulting in substantial unnecessary expenditures. We developed a rapid assay of the subforms of creatine kinase MB (CK-MB) and prospectively analyzed its sensitivity and specificity in diagnosing myocardial infarction in the first six hours after the onset of chest pain. METHODS In 1110 consecutive patients who came to the emergency room with chest pain, blood samples were collected every 30 to 60 minutes until at least 6 hours after the onset of symptoms; in patients who were then admitted to the hospital, samples were collected every 4 hours for up to 48 hours. The samples were analyzed for CK-MB subforms, and the diagnosis of myocardial infarction was confirmed by conventional CK-MB analysis. RESULTS Of the 1110 patients evaluated, 121 had myocardial infarction. The sensitivity of the assay of CK-MB subforms to detect myocardial infarction in the first six hours after the onset of symptoms was 95.7 per cent, as compared with only 48 percent for the conventional CK-MB assay; the specificity was 93.9 percent among patients hospitalized without myocardial infarction and 96.2 percent among those sent home. Among the patients with myocardial infarction, definitive results of the subform assay were available a mean (+/- SD) of 1.22 +/- 1.17 hours after their arrival in the emergency room. CONCLUSIONS The assay of CK-MB subforms reliably detected myocardial infarction within the first six hours after the onset of symptoms, and its use could reduce admission to the coronary care unit by 50 to 70 percent, thereby reducing costs.

NONINVASIVE DETECTION OF REPERFUSION IN ACUTE MYOCARDIAL INFARCTION BASED ON PLASMA ACTIVITY OF CREATINE KINASE MB SUBFORMS

Successful thrombolytic therapy is associated with an accelerated release of creatine kinase (CK) MB from necrotic myocardium. With use of a previously validated assay, the plasma kinetics of the myocardial subform (MB2) and the plasma-modified subform (MB1) were determined in blood samples obtained from 56 patients with acute Q wave myocardial infarction: 33 patients who received thrombolytic therapy (group A) and 23 patients managed conservatively (group B). Plasma MB2 activity increased more rapidly in the group A patients, but there was substantial overlap with group B. Plasma MB1 activity did not differ significantly between the two groups. The MB2/MB1 ratio was significantly higher in group A patients than in group B patients between 2 and 10 h after the onset of infarction. Among group A patients, the ratio increased from 2.4 +/- 1.6 to 4.6 +/- 2.0 in the 1st h after therapy (p less than 0.001). The peak ratio was 6.3 +/- 2.5 in group A patients and 3.1 +/- 1.2 in group B patients. Twenty-seven of the 33 group A patients had a peak ratio greater than 3.8 versus 5 of the 23 group B patients (p less than 0.001). In seven group A patients, the ratio was greater than 3.8 before plasma CK MB activity was out of the normal range. Angiography was performed at 5.0 +/- 3.5 days in 39 patients. Eighteen (90%) of 20 patients with a patent infarct-related artery had a peak ratio greater than 3.8; 17 (89.5%) of 19 patients with an occluded infarct-related artery had a ratio less than 3.8 (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

The Ku Protein Complex Interacts with YY1, Is Up-Regulated in Human Heart Failure, and Represses α Myosin Heavy-Chain Gene Expression

ABSTRACT Human heart failure is accompanied by repression of genes such as α myosin heavy chain (αMyHC) and SERCA2A and the induction of fetal genes such as βMyHC and atrial natriuretic factor. It seems likely that changes in MyHC isoforms contribute to the poor contractility seen in heart failure, because small changes in isoform composition can have a major effect on the contractility of cardiac myocytes and the heart. Our laboratory has recently shown that YY1 protein levels are increased in human heart failure and that YY1 represses the activity of the human αMyHC promoter. We have now identified a region of the αMyHC promoter that binds a factor whose expression is increased sixfold in failing human hearts. Through peptide mass spectrometry, we identified this binding activity to be a heterodimer of Ku70 and Ku80. Expression of Ku represses the human αMyHC promoter in neonatal rat ventricular myocytes. Moreover, overexpression of Ku70/80 decreases αMyHC mRNA expression and increases skeletal α-actin. Interestingly, YY1 interacts with Ku70 and Ku80 in HeLa cells. Together, YY1, Ku70, and Ku80 repress the αMyHC promoter to an extent that is greater than that with YY1 or Ku70/80 alone. Our results suggest that Ku is an important factor in the repression of the human αMyHC promoter during heart failure.

Molecular heterogeneity of creatine kinase isoenzymes.

Cytoplasmic creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) is a dimeric enzyme exhibiting three isoenzymes (MM, MB and BB). The two subunits have been reported to have identical molecular weights (Mr) of 41 000. We have demonstrated that the M subunits from human, canine, rabbit, mouse and bovine tissue have similar apparent Mr values of 43 000 as determined by SDS-polyacrylamide gel electrophoresis. In contrast, the Mr of the B subunits was different from that of the M subunit and varied with each species (human Mr 44 500; canine Mr 46 000; rabbit Mr 44 000 and mouse Mr 49 000). Cyanogen bromide cleavage showed all M subunits to have identical fragments, while B subunits exhibited cleavage products with patterns unique for each species. Despite the differences in Mr and cyanogen bromide fragment patterns, all B subunits were capable of producing enzymatically active hybrid (MB) molecules in combination with M subunits from any species tested. Mitochondrial creatine kinase subunits exhibited identical molecular weights and were similar to the M subunits and failed to combine with either the cytosolic M or B subunits. Thus, B subunits appear less conserved during evolution compared to M subunits, but have retained the amino acid sequences essential for subunit interaction and enzymatic activity.

Metabolic and diagnostic significance of creatine kinase isoenzymes.

Creatine kinase isoenzyme content is frequently used to assess the state of differentiation of muscle and neural tissue and following release into plasma as diagnostic markers for acute myocardial infarction, skeletal muscle disease, and neurologic injury. The establishment of thrombolytic therapy as the standard of care for acute myocardial infarction and new information on the tissue distribution of creatine kinase isoenzymes has necessitated the development of more rapid assays for the diagnosis of infarction and expanded the potential use of these isoenzymes as markers for other disease states.

Developmental expression of creatine kinase isozymes in mammalian lens

Four different isoforms are thought to comprise the creatine kinase of enzymes which regulate energy metabolism through the interconversion of ADP and creatine phosphate. In addition to these well characterized isoforms, MM, MB, BB and mitochondrial creatine kinase, several uncharacterized variants with atypical electrophoretic mobility have been described. In mammalian lens, creatine kinase isoforms exhibit both a regional and developmental pattern of expression. In neonatal rat and human lens, the only isoform expressed is a variant cathodic creatine kinase. Near the time of sexual maturation (11-13 yr) there is a dramatic increase in the expression of BB creatine kinase in human lens. In rat lens, a similar pattern of isoenzyme expression is also seen near the time of sexual maturation (5-6 weeks). In the mature rat lens, in addition to the cathodic variant, there is expression of BB and, to a lesser extent, MM creatine kinase. Using a polyclonal antisera, we have localized BB creatine kinase to the cuboidal epithelial cells of the adult rat lens. This unique pattern of isoenzyme expression and developmental regulation suggests a more complex scheme for the regulation of creatine kinase gene expression than previously postulated.

Myotonic Dystrophy Protein Kinase Is Critical for Nuclear Envelope Integrity

Background: Triplet repeat expansions in myotonic dystrophy reduce muscle expression of myotonic dystrophy protein kinase (DMPK). Results: DMPK localizes to the nuclear envelope, and DMPK depletion disrupts the nuclear envelope. Conclusion: DMPK is critical to maintain nuclear envelope integrity. Significance: Reduced DMPK expression in myotonic dystrophy could contribute to nuclear instability, a common mechanism of muscle wasting in muscular dystrophies. Myotonic dystrophy 1 (DM1) is a multisystemic disease caused by a triplet nucleotide repeat expansion in the 3′ untranslated region of the gene coding for myotonic dystrophy protein kinase (DMPK). DMPK is a nuclear envelope (NE) protein that promotes myogenic gene expression in skeletal myoblasts. Muscular dystrophy research has revealed the NE to be a key determinant of nuclear structure, gene regulation, and muscle function. To investigate the role of DMPK in NE stability, we analyzed DMPK expression in epithelial and myoblast cells. We found that DMPK localizes to the NE and coimmunoprecipitates with Lamin-A/C. Overexpression of DMPK in HeLa cells or C2C12 myoblasts disrupts Lamin-A/C and Lamin-B1 localization and causes nuclear fragmentation. Depletion of DMPK also disrupts NE lamina, showing that DMPK is required for NE stability. Our data demonstrate for the first time that DMPK is a critical component of the NE. These novel findings suggest that reduced DMPK may contribute to NE instability, a common mechanism of skeletal muscle wasting in muscular dystrophies.

Serial Alu sequence transposition interrupting a human B creatine kinase pseudogene

We have isolated, sequenced, and characterized a single-copy B creatine kinase pseudogene. The chromosomal assignment of this gene is 16p13 and a unique sequence probe from this locus detects EcoRI restriction fragment length polymorphisms of 7.8 and 5.4 kb. In 26 unrelated individuals, the frequencies for the 7.8- and 5.4-kb B creatine kinase pseudogene alleles were calculated to be 17.3 and 82.7%, respectively. The B creatine kinase pseudogene is interrupted by a 904-bp DNA insertion composed of three Alu repeat sequences in tandem flanked by an 18-bp direct repeat, derived from the pseudogene sequence. Nucleotide sequence analysis of the Alu elements suggests that the Alu sequences were incorporated into this locus in three separate integration events. Several complex clustered Alu repeat sequences without defined integration borders have been previously identified at different genomic loci. This is the first evidence that complex tandem Alu elements can integrate in an apparently serial manner in the human genome and supports the contention that Alu repeats integrate nonrandomly into the human genome.

Design of a multicenter, double-blind study to assess the effects of prophylactic diltiazem on early reinfarction after non-Q-wave acute myocardial infarction: Diltiazem reinfarction study

This protocol describes the design of an international study to examine the potential prophylactic effect of 14 days of diltiazem therapy to prevent early reinfarction in patients initially presenting with non-Q-wave acute myocardial infarction. Reinfarction was defined by the detection of a secondary elevation of plasma MB-creatine kinase. The results of clinical trials, instrumental in this protocol design, that established the increased propensity for early reinfarction after initial non-Q-wave infarction are described and the implications of the present study are discussed.

In vitro translation of canine mitochondrial creatine kinase messenger RNA.

The cell-free translation products of mRNA from canine myocardium were immunoprecipitated using antiserum specific for either the MM or mitochondrial creatine kinase subunit. The two subunits were shown to be encoded by the nuclear genome and translated from separate mRNAs. The mitochondrial subunit was translated as a polypeptide with a molecular weight approximately 6,000 greater than the mature form of the enzyme. In contrast, the M-subunit was translated as a polypeptide having a molecular weight identical to that of the mature cytosolic M-subunit. It is assumed that the mitochondrial subunit precursor must be proteolytically processed during translocation from the cytoplasm into mitochondria.