Rudolf J. Wiesner

A Variant Form of the Nuclear Triiodothyronine Receptor c-ErbAα1 Plays a Direct Role in Regulation of Mitochondrial RNA Synthesis

ABSTRACT In earlier research, we identified a 43-kDa c-ErbAα1 protein (p43) in the mitochondrial matrix of rat liver. In the present work, binding experiments indicate that p43 displays an affinity for triiodothyronine (T3) similar to that of the T3 nuclear receptor. Using in organello import experiments, we found that p43 is targeted to the organelle by an unusual process similar to that previously reported for MTF1, a yeast mitochondrial transcription factor. DNA-binding experiments demonstrated that p43 specifically binds to four mitochondrial DNA sequences with a high similarity to nuclear T3 response elements (mt-T3REs). Using in organello transcription experiments, we observed that p43 increases the levels of both precursor and mature mitochondrial transcripts and the ratio of mRNA to rRNA in a T3-dependent manner. These events lead to stimulation of mitochondrial protein synthesis. In transient-transfection assays with reporter genes driven by the mitochondrial D loop or two mt-T3REs located in the D loop, p43 stimulated reporter gene activity only in the presence of T3. All these effects were abolished by deletion of the DNA-binding domain of p43. Finally, p43 overexpression in QM7 cells increased the levels of mitochondrial mRNAs, thus indicating that the in organello influence of p43 was physiologically relevant. These data reveal a novel hormonal pathway functioning within the mitochondrion, involving a truncated form of a nuclear receptor acting as a potent mitochondrial T3-dependent transcription factor.

Cytochrome Oxidase Activity and Mitochondrial Gene Expression in Skeletal Muscle of Patients with Chronic Obstructive Pulmonary Disease

Several recent studies have suggested that skeletal muscle bioenergetics are abnormal in patients with chronic obstructive pulmonary disease (COPD). This study investigates the activity of cytochrome oxidase (COX), the terminal enzyme in the mitochondrial electron transport chain, and the expression of two mitochondrial DNA genes related to COX (mRNA of subunit I of COX [COX-I] and the RNA component of the 12S ribosomal subunit [12S rRNA]), in quadriceps femoris muscle biopsies obtained from COPD patients with various degrees of arterial hypoxemia, and from healthy sedentary control subjects of similar age. The activity of COX was measured spectrophotometrically in fresh tissue at 37 degrees C with excess substrate. RNA transcripts were measured using reverse transcription and polymerase chain reaction. The measurements of mRNA COX-I and 12S rRNA were normalized to the mRNA of actin, which is a housekeeping gene not influenced by hypoxia. We found that, compared with control subjects, COPD patients with chronic respiratory failure (PaO2 < 60 mm Hg) showed increased COX activity (p < 0.05). Further, the activity of COX was inversely related to arterial PO2 value (Rho -0.59, p < 0.01). The COX-I mRNA content was not different between patients and control subjects but patients with chronic respiratory failure had higher levels of 12S rRNA (p < 0.05), which were again inversely related to PaO2 (Rho -0.49, p < 0.05). These results indicate that the activity of COX is increased in skeletal muscle of patients with COPD and chronic respiratory failure, and they suggest that this is likely regulated at the translational level by increasing the number of mitochondrial ribosomes.

Counting target molecules by exponential polymerase chain reaction: copy number of mitochondrial DNA in rat tissues.

In this report, we show that the actual number of target molecules of the polymerase chain reaction can be determined by measuring the concentration of product accumulating in consecutive cycles. The equation describing product accumulation, log Nn = log eff x n + log N0, can be analyzed by linear regression and the molar concentration of target at cycle zero, N0, is obtained. Using this new approach, the actual content of mitochondrial DNA was determined in rat tissues and ranged from 116 x 10(9) molecules/g in fast-twitch skeletal muscle to 743 x 10(9) molecules/g in liver. Using morphometric data from the literature, mitochondria were found to contain 1 to 3 DNA molecules. There was no relation between the oxidative capacity of a tissue and its content of mitochondrial DNA, indicating that transcriptional and posttranscriptional mechanisms rather than gene dosage, as postulated by others, determine to what extent the mitochondrial genome is expressed.

Chronic ETA Receptor Blockade Attenuates Cardiac Hypertrophy Independently of Blood Pressure Effects in Renovascular Hypertensive Rats

In isolated cardiac myocytes, the direct effects of angiotensin II on cellular growth and gene expression were shown to be mediated by endothelin via the endothelin subtype A (ETA) receptor. To determine whether this pathway is also involved in the cardiovascular adaptations to a chronic activation of the renin-angiotensin system in vivo, the effects of a selective ETA receptor antagonist (LU 127043) were investigated in adult rats with renal artery stenosis. Four groups of rats (n=107) were studied over a period of 10 days after surgery: (1) sham-operated animals with saline administration, (2) rats subjected to left renal artery clipping with saline administration, (3) sham-operated rats with LU 127043 administration, and (4) rats subjected to left renal artery clipping with LU 127043 administration. LU 127043 (50 mg/kg) or saline was given by gavage twice daily starting 1 day before the operation. In clipped rats with saline administration, plasma renin activity, the ratio of left ventricular weight to body weight, and mRNAs for beta-myosin heavy chain and atrial natriuretic peptide were significantly elevated as early as 2 days after surgery. Blood pressure started to rise on the third postoperative day and attained a steady state hypertensive level by day 6. Blockade of ETA receptors had no effects on plasma renin activity or the time course of hypertension in clipped animals but completely prevented left ventricular hypertrophy and the re-expression of the beta-myosin heavy chain and atrial natriuretic peptide genes on day 2. While the expressions of the beta-myosin heavy chain and atrial natriuretic peptide genes were not different from saline-treated, clipped animals after day 4, the development of left ventricular hypertrophy remained markedly blunted (-50%) during ETA receptor blockade until day 10. These results show that a continuous blockade of ETA receptors significantly attenuates the development of left ventricular hypertrophy and, more transiently, fetal gene expression in the early phase of renovascular hypertension. Since neither blood pressure nor the increase in plasma renin activity was significantly altered by ETA receptor blockade, the inhibitory influences of the ETA receptor antagonist on left ventricular hypertrophy and gene expression were mediated most likely through a direct blockade of myocardial ETA receptors.

Regulation of mitochondrial transcription by mitochondrial transcription factor A

In order to test the hypothesis that mitochondrial transcription factor A (mtTFA) regulates mitochondrial transcription in vivo, mtTFA was overexpressed in HeLa cells and imported into isolated rat liver mitochondria. Five hours after transfection with an eukaryotic expression vector, mitochondrial transcripts for cytochrome-c-oxidase subunit I and 12 S rRNA were increased over controls. In the presence of rat liver mitochondria, the 29 kDa mtTFA, generated by in vitro translation, was processed to a 24 kDa protein which was protected from protease digestion. This demonstrates that mtTFA was imported into the matrix. Incorporation of 32P-UTP into mitochondrial transcripts was stimulated following import of mtTFA. We conclude that the intracellular and intramitochondrial concentration of mtTFA, respectively, indeed regulates mitochondrial transcription. (Mol Cell Biochem 174: 227–230, 1997)

Dissociation of Left Ventricular Hypertrophy, β-Myosin Heavy Chain Gene Expression, and Myosin Isoform Switch in Rats After Ascending Aortic Stenosis

BACKGROUNDnReexpression of the fetal beta-myosin heavy chain (beta-MHC) gene was reported to be a marker for phenotypic reprogramming and cardiac hypertrophy in rats. Recent in vitro studies strongly suggested a role of angiotensin II for phenotypic reprogramming. In the present investigation, beta-MHC gene expression was studied in an experimental model of pressure-over-load hypertrophy that is not associated with a concurrent activation of the circulating renin-angiotensin system.nnnMETHODS AND RESULTSnHypertrophy was induced in rats by ascending aortic banding (n = 40). After 7 days, myosin contained 31% (P < .05) of the beta-MHC isoform in banded but < 5% in sham-operated animals. However, no specific elevation of beta-MHC mRNA levels was found in banded animals. In contrast, hearts of rats with abdominal aortic banding displayed a marked increase in beta-MHC mRNA levels (3-fold to 5-fold, P < .05). Both the left ventricular weight and left ventricular peak systolic pressure were significantly elevated compared with sham-operated animals (abdominal aortic banding, +13% and 164 +/- 7 mm Hg; ascending aortic banding, +27% and 191 +/- 9 mm Hg). Plasma renin activity was elevated in rats with abdominal aortic banding (2.5-fold, P < .05) but not in rats with ascending aortic banding.nnnCONCLUSIONSnThe results of the present work do not support the concept that increased beta-MHC gene expression is a general stable late marker of myocardial hypertrophy in rats. Our results suggest that the stimulation of the renin-angiotensin system is crucial for the activation of the beta-MHC gene.

Stimulation of Mitochondrial Gene Expression and Proliferation of Mitochondria Following Impairment of Cellular Energy Transfer by Inhibition of the Phosphocreatine Circuit in Rat Hearts

Mitochondria proliferate when cellular energy demand increases. However, the pathwaysleading to enhanced expression of mitochondrial genes are largely unknown. We tested thehypothesis that an altered flux through energy metabolism is the key regulatory event bydecreasing mitochondrial energy supply to rat heart cells by creatine depletion. Electronmicroscopy showed that the density of mitochondria increased by 75% in such hearts(p < 0.01). Levels of representative mRNAs encoded on mitochondrial DNA (mtDNA) or on nuclearchromosomes were elevated 1.5 to 2-fold (p < 0.05), while the mtDNA content was unchanged.The mRNA for the nuclear encoded mitochondrial transcription factor A (mtTFA) was increasedafter GPA feeding (p < 0.05). Thus, we have shown that an impairment of mitochondrialenergy supply causes stimulation of gene expression resulting in mitochondrial proliferation,probably as a compensatory mechanism. The observed activation of the mtTFA genecorroborates the important function of this protein in nuclear—mitochondrial communication.

Recombinant troponin I substitution and calcium responsiveness in skinned cardiac muscle

Using treatment with vanadate solutions, we extracted native cardiac troponin I and troponin C (cTnI and cTnC) from skinned fibers of porcine right ventricles. These proteins were replaced by exogenously supplied TnI and TnC isoforms, thereby restoring Ca2+-dependent regulation. Force then depended on the negative logarithm of Ca2+ concentration (pCa) in a sigmoidal manner, the pCa for 50% force development, pCa50, being about 5.5. For reconstitution we used fast-twitch rabbit skeletal muscle TnI and TnC (sTnI and sTnC), bovine cTnI and cTnC or recombinant sTnIs that were altered by site-directed mutagenesis. Incubation with TnI inhibited isometric tension in TnI-extracted fibers in the absence of Ca+, but restoration of Ca2+ dependence required incubation with both TnI and TnC. Relaxation at low Ca2+ levels and the steepness of the force/pCa relation depended on the concentration of exogenously supplied TnI in the reconstitution solution (range 20–150 μM), while Ca2+ sensitivity, i.e. the pCa50, was dependent on the isoform, and also on the concentration of TnC in the reconstitution solution. At pH 6.7, skinned fibers reconstituted with optimal concentrations of sTnC and sTnI (120 μM and 150 μM, respectively) were more sensitive to Ca2+ than those reconstituted with cTnC and cTnI (difference in pCa50 approx. 0.2 units). Rabbit sTnI was cloned and expressed inEscherichia coli using a high yield expression plasmid. We introduced point mutations into the TnI inhibitory region comprising the sequence of the minimal common TnC/actin binding site (-G104-K-F-K-R-P-P-L-R-R-V-R115-). The four mutants produced by substitution of T for P110, G for P110, G for L111 and G for K105 were chosen, based on previous work with synthetic peptides showing that single amino acid substitution in this region diminished the capacity of these peptides to inhibit acto-Si, ATPase or contraction of skinned fibers. Therefore, all amino acid residues of the inhibitory region are thought to contribute to biological activity of TnI. However, each of the recombinant TnIs could substitute for endogenous TnI. In combination with exogenous TnC, Ca2+ dependence could be restored whengly110sTnI,thr110sTnI orgly111sTnI was used for reconstitution. The mutantgly105sTnI, on the other hand, reduced the ability of skinned fibers to relax at low Ca2+ concentrations and it caused an increase in Ca2+ sensitivity.

CA2+ SENSITIZING EFFECTS OF EMD 53998 AFTER TROPONIN REPLACEMENT IN SKINNED FIBRES FROM PORCINE ATRIA AND VENTRICLES

Skinned fibres from porcine ventricles exhibited a higher Ca2+ sensitivity (pCa50, i.e. -log10 Ca2+ concentration required for half-maximal activation, for force generation) than atrial fibres. The thiadiazinone derivative EMD 53998 increased Ca2+ sensitivity and Ca2+ efficacy in both preparations. The drug effect depended on the isoform of troponin (Tn). Using the vanadate method TnI and TnC could be partly extracted and replaced by foreign tropin or by the TnI subunit of added foreign troponins. We investigated the relationship between pCa and force development before and after replacement of TnI with foreign troponin (bovine ventricular troponin, cTn, or rabbit skeletal muscle troponin, sTn) in the presence and absence of EMD 53998. Substitution with bovine cTn increased Ca2+ sensitivity to a value characteristic of bovine ventricular skinned fibres (pCa50=5.4) and was further increased by EMD 53998. Substitution with sTn also increased Ca2+ sensitivity, but subsequent addition of EMD 53998 caused little further increase in Ca2+ sensitivity. Following extraction of TnI with vanadate, skinned fibres contracted in a Ca2+-independent manner and failed to relax at a pCa of 8. Relaxation could be induced, however, by bovine ventricular TnI and rabbit skeletal muscle recombinant TnI. This relaxation could be reversed by EMD 53998 (100 μM). The Ca2+-independent force of contracted fibres could also be depressed by a TnI inhibitory peptide, (cTnI 137–148) and, in addition, this effect was antagonized by EMD 53998. These results suggest that EMD 53998 antagonizes the inhibitory action of TnI, possibly by interfering with the interaction of the TnI inhibitory region with actin.

Expression of the junD proto-oncogene in the rat spinal cord and skin following noxious cutaneous ultraviolet irradiation.

Noxious peripheral stimulation induces the expression of various proto-oncogenes in rat spinal neurons. However, proto-oncogene expression seems to differ depending on the mode of the stimulus. Here, we report that noxious cutaneous ultraviolet (UV) irradiation results in a nearly 8-fold increase in junD mRNA levels in the rat lumbar spinal cord. RNA slot-blotting and hybridization techniques revealed a transcriptional activation of the junD proto-oncogene after 6 h, but not 1 h following UV exposure. These results suggest that low-frequency ongoing afferent impulse discharge is reflected by an accumulation in junD transcripts.