Thus far, clock genes in the heart have been described only in rodents, and alterations of these genes have been associated with various myocardial malfunctions. In this study, we analyzed the expression of clock genes in human hearts. Left papillary muscles of 16 patients with coronary heart disease, 39 subjects with cardiomyopathy, and 9 healthy donors (52 males and 12 females, mean age 55.7+/-11.2; 16-70 yrs) were obtained during orthotopic heart transplantation. We assessed the mRNA levels of PER1, PER2, BMAL1, and CRY1 by real time PCR and analyzed their rhythmic expression by sliding means and Cosinor functions. Furthermore, we sought for differences between the three groups (by ANOVAs) for both the total 24 h period and separate time bins. All four clock genes were expressed in human hearts. The acrophases (circadian rhythm peak time) of the PER mRNAs occurred in the morning (PER1: 07:44 h [peak level 187% higher than trough, p = .008]; PER2: 09:42 h [peak 254% higher than trough, p < .0001], and BMAL1 mRNA in the evening at 21:44 h [peak 438% higher than trough; p < .0001]. No differences were found in the rhythmic patterns between the three groups. No circadian rhythm was detected in CRY1 mRNA in any group. PER1, PER2, and BMAL1 mRNAs revealed clear circadian rhythms in the human heart, with their staging being in antiphase to those in rodents. The circadian amplitudes of the mRNA clock gene levels in heart tissue are more distinct than in any other human tissue so far investigated. The acrophase of the myocardial PER mRNAs and the trough of the myocardial BMAL1 coincide to the time of day of most frequent myocardial incidents.

We showed that the melatonin receptor subtype, MT1, is expressed in healthy and diseased human coronary arteries. As studies in experimental animals suggest that the MT2 melatonin receptor subtype is also present in the vasculature, we investigated whether the MT2 is expressed in human aorta and coronary arteries. Additionally, MT2 expression in human ventricular specimens was analysed, as melatonin was shown to affect myocyte function. Expression of the MT2-receptor was studied in sections of isolated coronary arteries, aorta and left ventricular specimens from healthy heart donors (control) and patients with dilated or ischemic cardiomyopathy. MT2 expression was found by reverse transcriptase (RT)-nested-polymerase chain reaction (PCR) in all of the specimens (aorta, left ventricle and coronary arteries) derived from controls. Also, visible evidence for receptor expression was found in 12 of 15 samples from cardiomyopathy patients and 10 of 15 of coronary heart disease patients. Additionally, the expression of MT2-receptor between aorta, left ventricle and coronary arteries varied among the individuals, some of them showing highest expression in the aorta while in others principal expression sites were coronary arteries or left ventricles. In conclusion, the MT2-receptor subtype is present in human arteries and left ventricles and it is suggested that in coronary heart disease MT2-receptor expression is altered. Furthermore, there is evidence for heterogeneous MT2 expression patterns in individual patients.

: Based on the fact that human bile and, particularly gallbladder bile, contains high physiological levels of the antioxidant melatonin, the aim of this study was to investigate whether the melatonin receptor MT1 is present in human gallbladder. Expression and localization of MT1 was assessed by RT‐PCR, Western blotting and immunofluorescence analysis in gallbladder samples from patients with cholelithiasis and with advanced gallbladder carcinoma. Additionally, we monitored mRNA expression of the two key enzymes of melatonin synthesis, i.e. arylalkylamine‐N‐acetyltransferase (AANAT) and hydroxyindole‐O‐methyltransferase (HIOMT). MT1 mRNA and protein were present in all cholelithiasis (n = 10) and gallbladder carcinoma (n = 5) samples. As indicated from RT‐PCR and Western blot studies, MT1 is located in gallbladder epithelia. Epithelial expression was further proven by immunofluorescence staining of MT1 in paraffin‐embedded cholelithiasis and gallbladder carcinoma sections. Analysis of AANAT and HIOMT mRNA expression showed that HIOMT mRNA is present in gallbladder. Surprisingly, AANAT was not detectable under conditions where it was found in a human colon specimen. The absence of AANAT suggests that in human gallbladder, HIOMT might be involved in the formation of 5‐hydroxytryptamine products other than melatonin. In summary, our results provide the first evidence for the presence of MT1 in human gallbladder epithelia. Therefore, in addition to its profound antioxidative effects in the biliary system, melatonin might also act through MT1‐mediated signal transduction pathways. Thereby, it might be involved in the regulation of gallbladder function.

PPARg, a nuclear transcription factor, is expressed in various cells within the vasculature and in cardiomyocytes. It has been suggested that PPARg is involved in atherogenesis and in cardiac hypertrophy. Therefore, we sought to quantify PPARg mRNA in coronary arteries, the aorta and left ventricular specimens from patients with ischaemic (CHD) and dilated cardiomyopathy (CMP). Using real-time PCR, we were able to demonstrate the expression of PPARg in all of the human specimens. The lowest expression of PPARg was detected in the aorta specimens of both groups (this was set to one). In comparison, the expression in coronary arteries was 2.32-fold in CHD-and 3.78-fold in CMP specimens and in the left ventricle specimens, 2.12-fold in CHDand 3.51-fold in CMP. Samples from CHD patients showed a higher expression of PPARg in all of the samples compared to those from CMP patients (aorta: 1.99-fold; coronary arteries: 1.35; left ventricles: 1.23). PPARg levels were not significantly correlated to CD 36 expression values in any group, suggesting that higher levels of PPARg are not principally due to increased PPARg expression in macrophages. This was confirmed by immunohistochemical analysis, which showed that PPARg is also located in the smooth muscle layer and in cardiomyocytes. In conclusion, our observations of increased PPAR mRNA expression in the coronary arteries and left ventricles from CHD and CMP patients suggest an important function of this nuclear receptor in the pathogenesis of heart disease.

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