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This study investigated the influence of chronic beta(3)-adrenoceptor deficiency on myocardial function. Therefore, we investigated Ca(2+)-regulatory proteins, SERCA 2a activity, and myofibrillar and mitochondrial function in hearts of wild-type (WT, n=7) and beta(3)-adrenoceptor knockout mice (beta(3)-KNO, n=7). Morphometric heart analysis showed no difference between WT and beta(3)-KNO. No alterations were observed for the protein expression of the ryanodine receptor or phospholamban. However, in beta(3)-KNO mice, protein expression of SERCA 2a and phospholamban phosphorylation were significantly increased. These changes were accompanied by an increased SERCA 2a activity in beta(3)-KNO. Alterations in phospholamban phosphorylation were independent of alterations in beta(1)/beta(2)-adrenoceptor distribution and protein expression of G proteins in beta(3)-KNO. Measurement of myofibrillar Ca(2+) sensitivity showed no difference in the Ca(2+)/force relation for WT and beta(3)-KNO. The same seems to hold true for mitochondrial function since the protein expressions of cytochrome c, uncoupling protein 3 and cytochrome c oxidase subunit IV were similar in WT and beta(3)-KNO. The conclusion is that depression of beta(3)-adrenergic stimulation may modulate the protein expression of SERCA 2a and phospholamban phosphorylation, thereby improving sarcoplasmic reticulum Ca(2+) uptake. Thus, beta(3)-adrenergic depression may be a therapeutic aim in situations of impaired SERCA 2a activity, e.g. for the treatment of heart failure.

Mitochondrial proliferation was studied in chronically stimulated rabbit skeletal muscle over a period of 50 days. After this time, subunits of COX had increased about fourfold. Corresponding mRNAs, encoded on mitochondrial DNA as well as on nuclear genes, were unchanged when related to total tissue RNA, however, they were elevated two- to fivefold when the massive increase of ribosomes per unit mass of muscle was taken into account. The same was true for the mRNA encoding mitochondrial transcription factor A. Surprisingly, tissue levels of mtTFA protein were reduced about twofold, together with mitochondrial DNA. In conclusion, mitochondria are able to maintain high rates of mitochondrial transcription even in the presence of reduced mtTFA protein and mtDNA levels. Therefore, stimulated mtTFA gene expression accompanies stimulated mitochondrial transcription, as in other models, but it is not sufficient for an increase of mtDNA copy number and other, yet unknown, factors have to be postulated.

Mitochondria proliferate when cellular energy demand increases. However, the pathways leading to enhanced expression of mitochondrial genes are largely unknown. We tested the hypothesis that an altered flux through energy metabolism is the key regulatory event by decreasing mitochondrial energy supply to rat heart cells by creatine depletion. Electron microscopy 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 nuclear chromosomes 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 increased after GPA feeding (p < 0.05). Thus, we have shown that an impairment of mitochondrial energy supply causes stimulation of gene expression resulting in mitochondrial proliferation, probably as a compensatory mechanism. The observed activation of the mtTFA gene corroborates the important function of this protein in nuclear-mitochondrial communication.

Left ventricular hypertrophy (LVH) is often associated with an impaired maximal coronary blood flow and increases the vulnerability of the heart tissue to ischaemia. In this study, the correlation between coronary blood flow and expression of the vascular endothelial growth factor (VEGF) mRNA was investigated. Using both haemodynamic measurements and analysis of mRNA, we have demonstrated that during development of LVH, in spontaneously hypertensive rats (SHR), an impaired maximal coronary flow at 12 weeks of age is associated with low levels of VEGF mRNA. However, in older SHR (32 weeks) with stabilised hypertrophy and a normal maximal coronary flow response, VEGF mRNA levels are increased 3-fold. These results suggest that the mechanism for the impaired flow, observed in some types of cardiac hypertrophy, might involve an inadequate growth of the coronary vessels due to insufficient activation of the VEGF gene.

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 mTFA. We conclude that the intracellular and intramitochondrial concentration of mtTFA, respectively, indeed regulates mitochondrial transcription.

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 Ca2+, 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 "mu"M), 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 "mu"M and 150 "mu"M, 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 in Escherichia 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-S1 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 when gly110sTnI, thr110sTnI or gly111sTnI was used for reconstitution. The mutant gly105sTnI, on the other hand, reduced the ability of skinned fibers to relax at low Ca2+ concentrations and it caused an increase in Ca2+ sensitivity.

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 Ca(2+)-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 microM). The Ca(2+)-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.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome c oxidase (COX), like other multi-subunit components of the respiratory chain, is controlled by both the nuclear and the mitochondrial genome. In order to find wether there is a close relationship between mRNAs encoded by the nucleus and by the mitochondrion, and between these mRNAs and enzyme activity, we compared six rat tissues (ventricle, liver, m. soleus, m. plantaris, and the white and red portions of m. gastrocnemius). We found a tenfold range for COX activity, a tenfold range for the contents of mRNA III (mitochondrial) and mRNA VIc (nuclear), a threefold range for total [poly(A)+] mRNA content and a sevenfold range for total RNA content in these tissues. The ratio of mRNA III to mRNA VIc was equal in each tissue, indicating the presence of a mechanism that coordinates the two genomes. There was a good correlation between mRNA content and COX activity (r = 0.78 for VIc, r = 0.77 for III; p less than 0.0001), demonstrating that the expression of this enzyme is mainly under pretranslational control.