Innate immune signaling induces expression and shedding of the heparan sulfate proteoglycan syndecan-4 in cardiac fibroblasts and myocytes, affecting inflammation in the pressure-overloaded heart.

Sustained pressure overload induces heart failure, the main cause of mortality in the Western world. Increased understanding of the underlying molecular mechanisms is essential to improve heart failure treatment. Despite important functions in other tissues, cardiac proteoglycans have received little attention. Syndecan-4, a transmembrane heparan sulfate proteoglycan, is essential for pathological remodeling, and we here investigated its expression and shedding during heart failure. Pressure overload induced by aortic banding for 24 h and 1 week in mice increased syndecan-4 mRNA, which correlated with mRNA of inflammatory cytokines. In cardiac myocytes and fibroblasts, tumor necrosis factor-α, interleukin-1ß and lipopolysaccharide through the toll-like receptor-4, induced syndecan-4 mRNA. Bioinformatical and mutational analyses in HEK293 cells identified a functional site for the proinflammatory nuclear factor-κB transcription factor in the syndecan-4 promoter, and nuclear factor-κB regulated syndecan-4 mRNA in cardiac cells. Interestingly, tumor necrosis factor-α, interleukin-1ß and lipopolysaccharide induced nuclear factor-κB-dependent shedding of the syndecan-4 ectodomain from cardiac cells. Overexpression of syndecan-4 with mutated enzyme-interacting domains suggested enzyme-dependent heparan sulfate chains to regulate shedding. In cardiac fibroblasts, lipopolysaccharide reduced focal adhesion assembly, shown by immunohistochemistry, suggesting that inflammation-induced shedding affects function. After aortic banding, a time-dependent cardiac recruitment of T lymphocytes was observed by measuring CD3, CD4 and CD8 mRNA, which was reduced in syndecan-4 knockout hearts. Finally, syndecan-4 mRNA and shedding were upregulated in failing human hearts. Conclusively, our data suggest that syndecan-4 plays an important role in the immune response of the heart to increased pressure, influencing cardiac remodeling and failure progression.

Hypoxia inducible factor 1 links fast-patterned muscle activity and fast muscle phenotype in rats.

Exercise influences muscle phenotype by the specific pattern of action potentials delivered to the muscle, triggering intracellular signalling pathways. PO2 can be reduced by an order of magnitude in working muscle. In humans, carriers of a hyperactive polymorphism of the transcription factor hypoxia inducible factor 1α (HIF-1α) have 50% more fast fibres, and this polymorphism is prevalent among strength athletes. We have investigated the putative role of HIF-1α in mediating activity changes in muscle.When rat muscles were stimulated with short high frequency bursts of action potentials known to induce a fast muscle phenotype, HIF-1α increased by about 80%. In contrast, a pattern consisting of long low frequency trains known to make fast muscles slow reduced the HIF-1α level of the fast extensor digitorum longus (EDL) muscle by 44%. Nuclear protein extracts from normal EDL contained 2.3-fold more HIF-1α and 4-fold more HIF-1ß than the slow soleus muscle, while von-Hippel-Lindau protein was 4.8-fold higher in slow muscles. mRNA displayed a reciprocal pattern; thus FIH-1 mRNA was almost 2-fold higher in fast muscle, while the HIF-1α level was half, and consequently protein/mRNA ratio for HIF-1α was more than 4-fold higher in the fast muscle, suggesting that HIF-1α is strongly suppressed post-transcriptionally in slow muscles.When HIF-1α was overexpressed for 14 days after somatic gene transfer in adult rats, a slow-to-fast transformation was observed, encompassing an increase in fibre cross sectional area, oxidative enzyme activity and myosin heavy chain. The latter was shown to be regulated at the mRNA level in C2C12 myotubes.

The ups and downs of gene regulation by electrical activity in skeletal muscles.

Adult skeletal muscles retain an adaptive capacity to switch between slow- and fast-twitch properties that are largely dependent on motoneuron activity. Our studies on the transcriptional regulation of the Troponin I slow (TnIs) and fast (TnIf) genes uncovered a dual mechanism of transcriptional enhancement and repression by a single activity pattern, that promotes the phenotypic differences among myofibers while preserving their adaptive capacity. Using the Tnf Fast Intronic Regulatory Element (FIRE), we initially demonstrated that fast-patterned activity (infrequent, high frequency depolarization) is necessary to up-regulate FIRE-dependent transcription and that its effect differs dramatically from muscle denervation. Hence, the "fast muscle program" is not a default state mimicked simply by denervation or muscle inactivity. Next, we found that slow-patterned activity (tonic, slow frequency stimulation) selectively represses FIRE-dependent transcription while enhancing transcription from the TnIs Slow Upstream Regulatory Element. Unexpectedly, repression of the TnIf FIRE by slow-patterned activity is mediated by an NFAT element that directly binds NFATc1, a transcription factor that translocates to the nucleus selectively by slow-pattern depolarization and has been implicated in the up-regulation of the slow muscle program. Transfection of siRNAs targeting NFATc1 or mutation of the TnIFIRE NFAT site result in the upregulation of FIRE-dependent transcription in slow muscle, but have no effect in fast muscle. These findings demonstrate a novel function of NFAT as a repressor of transcription of fast contractile genes in slow muscles and, more importantly, they illustrate how specific activity patterns can enhance the phenotypic differences among fibre-types by differentially regulating transcription in a use-dependent manner while retaining the adaptive properties of adult muscles.

Activity-dependent repression of muscle genes by NFAT.

Adult skeletal muscles retain an adaptive capacity to switch between slow- and fast-twitch properties that largely depend on motoneuron activity. The NFAT (nuclear factor of activated T cells) family of calcium-dependent transcription factors has been implicated in the up-regulation of genes encoding slow contractile proteins in response to slow-patterned motoneuron depolarization. Here, we demonstrate an unexpected, novel function of NFATc1 in slow-twitch muscles. Using the troponin I fast (TnIf) intronic regulatory element (FIRE), we identified sequences that down-regulate its function selectively in response to patterns of electrical activity that mimic slow motoneuron firing. A bona fide NFAT binding site in the TnIf FIRE was identified by site-directed mutations and by electrophoretic mobility and supershift assays. The activity-dependent transcriptional repression of FIRE is mediated through this NFAT site and, importantly, its mutation did not alter the up-regulation of TnIf transcription by fast-patterned activity. siRNA-mediated knockdown of NFATc1 in adult muscles resulted in ectopic activation of the FIRE in the slow soleus, without affecting enhancer activity in the fast extensor digitorum longus muscle. These findings demonstrate that NFAT can function as a repressor of fast contractile genes in slow muscles and they exemplify how an activity pattern can increase or decrease the expression of distinct contractile genes in a use-dependent manner as to enhance phenotypic differences among fiber types or induce adaptive changes in adult muscles.

PPARdelta expression is influenced by muscle activity and induces slow muscle properties in adult rat muscles after somatic gene transfer.

The effects of exercise on skeletal muscle are mediated by a coupling between muscle electrical activity and gene expression. Several activity correlates, such as intracellular Ca(2+), hypoxia and metabolites like free fatty acids (FFAs), might initiate signalling pathways regulating fibre-type-specific genes. FFAs can be sensed by lipid-dependent transcription factors of the peroxisome proliferator-activated receptor (PPAR) family. We found that the mRNA for the predominant muscle isoform, PPARdelta, was three-fold higher in the slow/oxidative soleus compared to the fast/glycolytic extensor digitorum longus (EDL) muscle. In histological sections of the soleus, the most oxidative fibres display the highest levels of PPARdelta protein. When the soleus muscle was stimulated electrically by a pattern mimicking fast/glycolytic IIb motor units, the mRNA level of PPARdelta was reduced to less than half within 24 h. In the EDL, a three-fold increase was observed after slow type I-like electrical stimulation. When a constitutively active form of PPARdelta was overexpressed for 14 days in normally active adult fibres after somatic gene transfer, the number of I/IIa hybrids in the EDL more than tripled, IIa fibres increased from 14% to 25%, and IIb fibres decreased from 55% to 45%. The level of succinate dehydrogenase activity increased and size decreased, also when compared to normal fibres of the same type. Thus PPARdelta can change myosin heavy chain, oxidative enzymes and size locally in muscle cells in the absence of general exercise. Previous studies on PPARdelta in muscle have been performed in transgenic animals where the transgene has been present during muscle development. Our data suggest that PPARdelta can mediate activity effects acutely in pre-existing adult fibres, and thus is an important link in excitation-transcription coupling.

Imaging transcription in vivo: distinct regulatory effects of fast and slow activity patterns on promoter elements from vertebrate troponin I isoform genes.

Firing patterns typical of slow motor units activate genes for slow isoforms of contractile proteins, but it remains unclear if there is a distinct pathway for fast isoforms or if their expression simply occurs in the absence of slow activity. Here we first show that denervation in adult soleus and EDL muscles reverses the postnatal increase in expression of troponin I (TnI) isoforms, suggesting that high-level transcription of both genes in mature muscles is under neural control. We then use a combination of in vivo transfection, live muscle imaging and fluorescence quantification to investigate the role of patterned electrical activity in the transcriptional control of troponin I slow (TnIs) and fast (TnIf) regulatory sequences by directly stimulating denervated muscles with pattern that mimic fast and slow motor units. Rat soleus muscles were electroporated with green fluorescent protein (GFP) reporter constructs harbouring 2.7 and 2.1 kb of TnIs and TnIf regulatory sequences, respectively. One week later, electrodes were implanted and muscles stimulated for 12 days. The change in GFP fluorescence of individual muscle fibres before and after the stimulation was used as a measure for transcriptional responses to different patterns of action potentials. Our results indicate that the response of TnI promoter sequences to electrical stimulation is consistent with the regulation of the endogenous genes. The TnIf and TnIs enhancers were activated by matching fast and slow activity patterns, respectively. Removal of nerve-evoked activity by denervation, or stimulation with a mismatching pattern reduced transcriptional activity of both enhancers. These results strongly suggest that distinct signalling pathways couple both fast and slow patterns of activity to enhancers that regulate transcription from the fast and slow troponin I isoforms.