Is myocardial Na+/Ca2+ exchanger transcription a marker for different stages of myocardial dysfunction? Quantitative polymerase chain reaction of the messenger RNA in endomyocardial biopsies of patients with heart failure.

OBJECTIVES: This study was designed to determine the stage of myocardial dysfunction at which an upregulation of the Na+/Ca2+ exchanger (EXCH) transcription takes place. BACKGROUND: Because EXCH is an important regulator of intracellular calcium homeostasis, alterations in EXCH expression may occur before the onset of end-stage heart failure (HF) to maintain normal intracellular Ca2+ concentrations. We analyzed whether the EXCH transcription level is correlated to the degree of myocardial dysfunction and whether it can be a suitable molecular marker to define the transition to myocardial decompensation early on. METHODS: By quantitative polymerase chain reaction technique, the level of EXCH transcription was analyzed in myocardial biopsies from 40 patients with various degrees of myocardial dysfunction due to valvular heart disease (VHD; n = 22) or dilated cardiomyopathy (DCM; n = 18). Additionally, biopsies from 7 individuals with excluded heart disease and explanted heart tissue from 13 patients with end-stage HF were investigated. RESULTS: The level of EXCH transcription of controls (2.6 +/- 1.2 attomoles [amol]/ng total RNA) did not differ from that of patients with DCM (2.3 +/- 1.5 amol/ng) or VHD (2.1 +/- 1.5 amol/ng). No alteration in the EXCH transcription was found in VHD and DCM patients with respect to the severity of myocardial dysfunction. However, patients with end-stage HF showed a four-fold increase in EXCH transcription, amounting to 8.9 +/- 1.9 amol/ng (p < 0.05). CONCLUSIONS: The upregulation in EXCH transcription either occurs very late in human heart failure or is a phenomenon of heart transplantation in end-stage HF. Consequently, myocardial EXCH transcription cannot be used as a marker for early myocardial decompensation.

Enteroviral and immune mediated myocarditis in SCID mice.

Severe combined immune deficiency (SCID) mice have been used as an animal model to study both the direct cytopathic effect of enteroviruses on the heart in the absence of an effective immune system and to investigate the role of immune mediated processes in the pathogenesis of human myocarditis. The infection of SCID mice with coxsackievirus B3 resulted in severe myocarditis with very high titers of the virus in the myocardium and severe necrosis of myocytes. This direct cytopathic effect caused an impairment of the myocardial function and resulted in a high mortality rate of the infected animals. For the study of the immune mechanisms in human myocarditis, peripheral blood leukocytes of patients with myocarditis, having an impaired left ventricular function without viral persistence in the myocardium, were transferred into SCID mice. As controls peripheral blood leukocytes of normal donors were used. At 60 days after transfer, human immunoglobulines could be demonstrated in the peripheral blood of the SCID mice, however, human autoantibodies against the adenine nucleotide translocator, a myocardial autoantigen, were only present in the animals receiving peripheral blood leukocytes from patients with myocarditis. Cellular infiltrates of human leukocytes in the myocardium and an impaired left ventricular function were also only observed in animals reconstituted with peripheral blood leukocytes from patients. These effects were T cell dependent as shown by differential transfer. These results are of interest for the treatment of human myocarditis, suggesting the avoidance of an immunosuppressive therapy in acute or chronic myocarditis with viral persistence to prevent a direct cytopathic effect in the absence of an effective immune system. However, in the setting of a chronic, (auto-)immunological myocarditis with the proven absence of entero- or adenoviral sequences an immunomodulatory therapy seems to be effective and safe.

Dilated cardiomyopathy is associated with significant changes in collagen type I/III ratio.

BACKGROUND: It is controversial whether myocardial fibrosis in end-stage dilated cardiomyopathy (DCM) is associated with altered collagen type I/type III (Col I/Col III) ratio. METHODS AND RESULTS: Patients with DCM (ejection fraction [EF] <50%, n=12) and with mild global left ventricular dysfunction (EF >50%, n=18) were examined. Col I, Col III, and transforming growth factors-beta1 (TGF-beta1) and -beta2 (TGF-beta2) gene expression in endomyocardial biopsies was evaluated by quantitative competitive reverse transcriptase-polymerase chain reaction (qRT-PCR). Collagen content was quantified after picrosirius red and immunohistological staining and by hydroxyproline assay. In patients with EF <50%, there was a pronounced 2- to 6-fold increase of myocardial Col I mRNA abundance (P<0.01), with a corresponding 1.6-fold increase at the protein level versus that found in patients with EF >50%. The Col III mRNA abundance showed a 2.0-fold increase (P<0.04). There was a relevant shift in the Col I/Col III mRNA ratio for DCM patients (Col I/Col III, 8.2) compared with patients with an EF >50% (Col I/Col III, 6. 4). In addition, total collagen content was increased in patients with EF <50% (n=3) (4.3+/-0.1%) compared with patients with EF >50% (n=8) (2.7+/-0.9%) (P<0.004). The biochemically determined ratio of hydroxyproline/total protein (n=12) was correlated to the Col I mRNA abundance (P<0.05, r=0.77). TGF-beta1 and TGF-beta2 showed elevated myocardial mRNA abundances (1- to 7-fold and 4- to 5-fold, respectively) in DCM patients. CONCLUSIONS: Differential increase of Col I and Col III leads to an increased Col I/Col III ratio in DCM myocardium. Because Col I provides substantial tensile strength and stiffness, this may contribute to systolic and in particular diastolic dysfunction in DCM.

Enteroviral RNA replication in the myocardium of patients with left ventricular dysfunction and clinically suspected myocarditis.

BACKGROUND: Previous studies dealing with the detection of enteroviral RNA in human endomyocardial biopsies have not differentiated between latent persistence of the enteroviral genome and active viral replication. Enteroviruses that are considered important factors for the development of myocarditis have a single-strand RNA genome of positive polarity that is transcribed by a virus-encoded RNA polymerase into a minus-strand mRNA during active viral replication. The synthesis of multiple copies of minus-strand enteroviral RNA therefore occurs only at sites of active viral replication but not in tissues with mere persistence of the viral genome. METHODS AND RESULTS: We investigated enteroviral RNA replication versus enteroviral RNA persistence in endomyocardial biopsies of 45 patients with left ventricular dysfunction and clinically suspected myocarditis. Using reverse-transcriptase polymerase chain reaction in conjunction with Southern blot hybridization, we established a highly sensitive assay to specifically detect plus-strand versus minus-strand enteroviral RNA in the biopsies. Plus-strand enteroviral RNA was detected in endomyocardial biopsies of 18 (40%) of 45 patients, whereas minus-strand RNA as an indication of active enteroviral RNA replication was detected in only 10 (56%) of these 18 plus-strand-positive patients. Enteroviral RNA was not found in biopsies of the control group (n=26). CONCLUSIONS: These data demonstrate that a significant fraction of patients with left ventricular dysfunction and clinically suspected myocarditis had active enteroviral RNA replication in their myocardium (22%). Differentiation between patients with active viral replication and latent viral persistence should be particularly important in future studies evaluating different therapeutic strategies. In addition, molecular genetic detection of enteroviral genome and differentiation between replicating versus persistent viruses is possible in a single endomyocardial biopsy.

Differential myocardial abundance of collagen type I and type III mRNA in dilated cardiomyopathy: effects of myocardial inflammation.

OBJECTIVE: The collagen subtypes I (Col I) and III (Col III) are essential components of the cardiac extracellular matrix (ECM) maintaining the functional integrity of the heart. Histological, immunohistological, and biochemical studies, however, demonstrate characteristical changes of the ECM in dilated cardiomyopathy, myocarditis, ischemic cardiomyopathy, and hypertensive heart disease. METHODS: In order to investigate possible effects of inflammatory processes on mRNA abundance of Col I and Col III, we examined 24 patients with the presumptive clinical diagnosis of dilated cardiomyopathy (EF = 30 +/- 11%). 12 Patients were classified as idiopathic dilated cardiomyopathy without any evidence of myocardial inflammation; the remaining 12 patients were classified as inflammatory cardiomyopathy due to the immunohistologically documented inflammatory myocardial process. RESULTS: Quantification of reverse transcription polymerase chain reaction (RT-PCR) products revealed significant differences as to the mRNA abundance ratio Col III/Col I between subgroups of patients with inflammatory cardiomyopathy (1.16 +/- 0.18) and idiopathic dilated cardiomyopathy (2.77 +/- 0.65) regardless of left ventricular dysfunction (p < or = 0.05). CONCLUSION: It is not yet known, whether different Col III/Col I ratios differentially influence diastolic compliance. Our data suggest that inflammatory mechanisms seen in inflammatory cardiomyopathy influence the mRNA abundance of collagen subtypes I and III.

Tissue-specific transcription pattern of the adenine nucleotide translocase isoforms in humans.

Three adenine nucleotide translocase isoforms (ANT1, ANT2 and ANT3) are coded by different genes. The relative amounts of the three ANT isoform mRNAs were determined in detail in various human tissues. ANT isoforms were co-expressed in all tested tissues revealing tissue-specific transcription patterns. The highest ANT1 mRNA proportions were found in terminally differentiated tissues like skeletal muscle, heart and brain, whereas ANT2 was mainly expressed in tissues capable of proliferation and regeneration as in the kidneys, spleen, liver, fibroblasts and lymphocytes. The ANT3 mRNA proportion was not prominently expressed in any of the tissues tested. In conclusion, tissue-specific expression of ANT isoforms is strongly related to the state of cellular differentiation.

Study of the demethylation of [1,3,7-Me-13C] caffeine in man using respiratory exchange measurements.

Caffeine was shown to be demethylated both in the rat and in man into dimethyl and monomethyl derivatives. There are no quantitative data on the demethylation process because in man the identified metabolites have not all been quantified, and in addition unidentified polar metabolites have been reported recently both in the rat and in man. To quantify the total demethylation process and to study the use of caffeine as a clinical test to analyse hepatic functions, [1,3,7-Me-13C]caffeine was synthesized and 200 mg was administered orally to volunteers. Expired carbon dioxide was continuously measured and collected in a liquid nitrogen trap and then analysed on a double inlet mass spectrometer. A significant increase of the 13CO2 over the basal value was already observed in the first sample collected 15 minutes after the administration. The 13CO2 enrichment reached a maximum within one hour, exhibited a plateau and after 5 hours decreased slowly to return near the basal value after 24 hours. From 21 to 26% of total 13C administration was recovered in expired CO2 over 24 hours. These percentages corresponded also to a mean rate of demethylation for each methyl group. Thus, [1,3,7-Me-13C]caffeine is a molecule suitable for a breath test. However, it remains to show whether or not P-448 induction stimulates a specific demethylation. From these data a specific enrichment of a methyl group could be decided. The use of a physiological dose of caffeine and the use of stable isotopes constitute a non-invasive and safe technique to study human liver functions.

Carbohydrate utilization in obese subjects after an oral load of 100 g naturally-labelled [13C] glucose.

1. Total carbohydrate (CHO) and ingested glucose oxidation was measured in five obese subjects with normal glucose tolerance after an oral load of 100 g naturally-labelled [13C]glucose using indirect calorimetry and mass spectrometry respectively. 2. CHO utilization rate (107 +/- 14 mg/min in the post-absorptive state) increased 30 min after the glucose load to reach a plateau (245 +/- 25 mg/min) between 90 and 120 min. It then decreased to basal values at 330 min. Cumulative CHO oxidation over 480 min was 66 +/- 7 g and the CHO oxidized above basal levels was 26 +/- 7 g. 3. Enrichment of expired carbon dioxide with 13C began at 45 min and maximum values were observed between 210 and 300 min. At 480 min, cumulative oxidation of the ingested glucose was 24 +/- 2 g. 4. Compared with controls, the obese subjects exhibit an impairment of CHO utilization which precedes glucose intolerance. This impairment can be explained by an increased availability of free fatty acids which favours lipid oxidation at the expense of ingested [13C]glucose oxidation.

Substrate utilization during prolonged exercise after ingestion of 13C-glucose in obese and control subjects.

This study was performed to investigate whether the difference in substrate utilization observed between obese and control individuals at rest still exists during prolonged exercise. Using a combination of respiratory exchange and tracer techniques, six obese and six control subjects were investigated while exercising for 2 h on a bicycle ergometer, 1 h after ingesting 100 g naturally-enriched 13C-glucose. Oxidation rates of protein, lipid and carbohydrate (CHO) were measured by indirect calorimetry and that of exogenous glucose by mass spectrometry (13CO2). Before exercise the obese subjects presented a lower rate of CHO utilization with a mean respiratory quotient of .803 compared to .858 for the controls. This impairement of CHO utilization disappeared during exercise where total CHO oxidation was found to be comparable for the obese (94.0 +/- 8.4 g) and the control group (94.3 +/- 6.1g). Exogenous-glucose oxidation was even slightly more elevated in the obese subjects (33.6 +/- 2.5 g compared with 28.1 +/- 2.3 g). Two hypotheses have been proposed to explain the improvement in CHO utilization during exercise after a glucose load: (1) The fall in FFA is sufficient to suppress the inhibition of CHO uptake and oxidation; (2) The insulin resistance decreases during exercise. In conclusion, this study supports the concept that exercise performed by obese individuals stimulates CHO utilization with a concomitant improvement in glucose tolerance.

Comparison of carbohydrate utilization in man using indirect calorimetry and mass spectrometry after an oral load of 100 g naturally-labelled [13C]glucose.

1. Carbohydrate (CHO) oxidation was measured simultaneously in a group of five normal subjects after an oral load of 100 g naturally-labelled [13C]glucose, using indirect calorimetry and mass spectrometry. 2. CHO utilization, calculated from the results of indirect calorimetry, increased 30 min after the glucose load to reach a peak at 90 min. It then decreased to reach basal values at 380 min. Cumulative total CHO oxidation at 480 min was 83 +/- 8 g, and CHO oxidized above basal levels, 37 +/- 3 g. 3. Enrichment of expired carbon dioxide with 13C began at 60 min and maximum values were observed at 270 min. At 480 min, cumulative CHO oxidation measured by use of [13C]glucose was 29 g. The difference from calorimetric values can be attributed in part to the slow isotopic dilution in the glucose and bicarbonate pools. 4. Thus, approximately 30% of the glucose load was oxidized during the 8 h after its ingestion and this accounts for a significant part of the increased CHO oxidation (37 g), as measured by indirect calorimetry.