Regulation of the Muscarinic M3 Receptor by Myocardin-Related Transcription Factors.

Myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MRTFA, and MRTF-B/MRTFB) are co-factors of serum response factor (SRF) that activate the smooth muscle cell (SMC) gene program and that play roles in cardiovascular development and mechanobiology. Gain and loss of function experiments have defined the SMC gene program under control of MRTFs, yet full understanding of their impact is lacking. In the present study, we tested the hypothesis that the muscarinic M3 receptor (CHRM3) is regulated by MRTFs together with SRF. Forced expression of MYOCD (8d) in human coronary artery (SMC) followed by RNA-sequencing showed increased levels of M2, M3, and M5 receptors (CHRM2: 2-fold, CHRM3: 16-fold, and CHRM5: 2-fold). The effect of MYOCD on M3 was confirmed by RT-qPCR using both coronary artery and urinary bladder SMCs, and correlation analyses using human transcriptomic datasets suggested that M3 may also be regulated by MRTF-B. Head-to-head comparisons of MYOCD, MRTF-A and MRTF-B, argued that while all MRTFs are effective, MRTF-B is the most powerful transactivator of CHRM3, causing a 600-fold increase at 120h. Accordingly, MRTF-B conferred responsiveness to the muscarinic agonist carbachol in Ca2+ imaging experiments. M3 was suppressed on treatment with the MRTF-SRF inhibitor CCG-1423 using SMCs transduced with either MRTF-A or MRTF-B and using intact mouse esophagus in culture (by 92±2%). Moreover, silencing of SRF with a short hairpin reduced CHRM3 (by >60%) in parallel with α-actin (ACTA2). Tamoxifen inducible knockout of Srf in smooth muscle reduced Srf (by 54±4%) and Chrm3 (by 41±6%) in the urinary bladder at 10days, but Srf was much less reduced or unchanged in aorta, ileum, colon, trachea, and esophagus. Longer induction (21d) further accentuated the reduction of Chrm3 in the bladder and ileum, but no change was seen in the aorta. Single cell RNA-sequencing revealed that Mrtfb dominates in ECs, while Myocd dominates in SMCs, raising the possibility that Chrm3 may be driven by Mrtfb-Srf in the endothelium and by Myocd-Srf in SMCs. These findings define a novel transcriptional control mechanism for muscarinic M3 receptors in human cells, and in mice, that could be targeted for therapy.

MRTFA overexpression promotes conversion of human coronary artery smooth muscle cells into lipid-laden foam cells.

OBJECTIVE: Smooth muscle cells contribute significantly to lipid-laden foam cells in atherosclerotic plaques. However, the underlying mechanisms transforming smooth muscle cells into foam cells are poorly understood. The purpose of this study was to gain insight into the molecular mechanisms regulating smooth muscle foam cell formation. APPROACH AND RESULTS: Using human coronary artery smooth muscle cells we found that the transcriptional co-activator MRTFA promotes lipid accumulation via several mechanisms, including direct transcriptional control of LDL receptor, enhanced fluid-phase pinocytosis and reduced lipid efflux. Inhibition of MRTF activity with CCG1423 and CCG203971 significantly reduced lipid accumulation. Furthermore, we demonstrate enhanced MRTFA expression in vascular remodeling of human vessels. CONCLUSIONS: This study demonstrates a novel role for MRTFA as an important regulator of lipid homeostasis in vascular smooth muscle cells. Thus, MRTFA could potentially be a new therapeutic target for inhibition of vascular lipid accumulation.

Array profiling reveals contribution of Cthrc1 to growth of the denervated rat urinary bladder.

Bladder denervation and bladder outlet obstruction are urological conditions that cause bladder growth. Transcriptomic surveys in outlet obstruction have identified differentially expressed genes, but similar studies following denervation have not been done. This was addressed using a rat model in which the pelvic ganglia were cryo-ablated followed by bladder microarray analyses. At 10 days following denervation, bladder weight had increased 5.6-fold, and 2,890 mRNAs and 135 micro-RNAs (miRNAs) were differentially expressed. Comparison with array data from obstructed bladders demonstrated overlap between the conditions, and 10% of mRNAs changed significantly and in the same direction. Many mRNAs, including collagen triple helix repeat containing 1 ( Cthrc1), Prc1, Plod2, and Dkk3, and miRNAs, such as miR-212 and miR-29, resided in the shared signature. Discordantly regulated transcripts in the two models were rare, making up for <0.07% of all changes, and the gene products in this category localized to the urothelium of normal bladders. These transcripts may potentially be used to diagnose sensory denervation. Western blotting demonstrated directionally consistent changes at the protein level, with increases of, e.g., Cthrc1, Prc1, Plod2, and Dkk3. We chose Cthrc1 for further studies and found that Cthrc1 was induced in the smooth muscle cell (SMC) layer following denervation. TGF-ß1 stimulation and miR-30d-5p inhibition increased Cthrc1 in bladder SMCs, and knockdown and overexpression of Cthrc1 reduced and increased SMC proliferation. This work defines common and distinguishing features of bladder denervation and obstruction and suggests a role for Cthrc1 in bladder growth following denervation.

Loss of Vascular Myogenic Tone in miR-143/145 Knockout Mice Is Associated With Hypertension-Induced Vascular Lesions in Small Mesenteric Arteries.

OBJECTIVE: Pressure-induced myogenic tone is involved in autoregulation of local blood flow and confers protection against excessive pressure levels in small arteries and capillaries. Myogenic tone is dependent on smooth muscle microRNAs (miRNAs), but the identity of these miRNAs is unclear. Furthermore, the consequences of altered myogenic tone for hypertension-induced damage to small arteries are not well understood. APPROACH AND RESULTS: The importance of smooth muscle-enriched microRNAs, miR-143/145, for myogenic tone was evaluated in miR-143/145 knockout mice. Furthermore, hypertension-induced vascular injury was evaluated in mesenteric arteries in vivo after angiotensin II infusion. Myogenic tone was abolished in miR-143/145 knockout mesenteric arteries, whereas contraction in response to calyculin A and potassium chloride was reduced by ≈30%. Furthermore, myogenic responsiveness was potentiated by angiotensin II in wild-type but not in knockout mice. Angiotensin II administration in vivo elevated systemic blood pressure in both genotypes. Hypertensive knockout mice developed severe vascular lesions characterized by vascular inflammation, adventitial fibrosis, and neointimal hyperplasia in small mesenteric arteries. This was associated with depolymerization of actin filaments and fragmentation of the elastic laminae at the sites of vascular lesions. CONCLUSIONS: This study demonstrates that miR-143/145 expression is essential for myogenic responsiveness. During hypertension, loss of myogenic tone results in potentially damaging levels of mechanical stress and detrimental effects on small arteries. The results presented herein provide novel insights into the pathogenesis of vascular disease and emphasize the importance of controlling mechanical factors to maintain structural integrity of the vascular wall.

MicroRNA-Dependent Control of Serotonin-Induced Pulmonary Arterial Contraction.

BACKGROUND: Serotonin (5-HT) is considered to play a role in pulmonary arterial hypertension by regulating vascular remodeling and smooth muscle contractility. Here, arteries from mice with inducible and smooth muscle-specific deletion of Dicer were used to address mechanisms by which microRNAs control 5-HT-induced contraction. METHODS: Mice were used 5 weeks after Dicer deletion, and pulmonary artery contractility was analyzed by wire myography. RESULTS: No change was seen in right ventricular systolic pressure following dicer deletion, but systemic blood pressure was reduced. Enhanced 5-HT-induced contraction in Dicer KO pulmonary arteries was associated with increased 5-HT2A receptor mRNA expression whereas 5-HT1B and 5-HT2B receptor mRNAs were unchanged. Contraction by the 5-HT2A agonist TCB-2 was increased in Dicer KO as was the response to the 5-HT2B agonist BW723C86. Effects of Src and protein kinase C inhibition were similar in control and KO arteries, but the effect of inhibition of Rho kinase was reduced. We identified miR-30c as a potential candidate for 5-HT2A receptor regulation as it repressed 5-HT2A mRNA and protein. CONCLUSION: Our findings show that 5-HT receptor signaling in the arterial wall is subject to regulation by microRNAs and that this entails altered 5-HT2A receptor expression and signaling.

Neurite outgrowth in cultured mouse pelvic ganglia - Effects of neurotrophins and bladder tissue.

Neurotrophic factors regulate survival and growth of neurons. The urinary bladder is innervated via both sympathetic and parasympathetic neurons located in the major pelvic ganglion. The aim of the present study was to characterize the effects of the neurotrophins nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3) on the sprouting rate of sympathetic and parasympathetic neurites from the female mouse ganglion. The pelvic ganglion was dissected out and attached to a petri dish and cultured in vitro. All three factors (BDNF, NT-3 and NGF) stimulated neurite outgrowth of both sympathetic and parasympathetic neurites although BDNF and NT-3 had a higher stimulatory effect on parasympathetic ganglion cells. The neurotrophin receptors TrkA, TrkB and TrkC were all expressed in neurons of the ganglia. Co-culture of ganglia with urinary bladder tissue, but not diaphragm tissue, increased the sprouting rate of neurites. Active forms of BDNF and NT-3 were detected in urinary bladder tissue using western blotting whereas tissue from the diaphragm expressed NGF. Neurite outgrowth from the pelvic ganglion was inhibited by a TrkB receptor antagonist. We therefore suggest that the urinary bladder releases trophic factors, including BDNF and NT-3, which regulate neurite outgrowth via activation of neuronal Trk-receptors. These findings could influence future strategies for developing pharmaceuticals to improve re-innervation due to bladder pathologies.

Cavin-3 (PRKCDBP) deficiency reduces the density of caveolae in smooth muscle.

Cavins belong to a family of proteins that contribute to the formation of caveolae, which are membrane organelles with functional roles in muscle and fat. Here, we investigate the effect of cavin-3 ablation on vascular and urinary bladder structure and function. Arteries and urinary bladders from mice lacking cavin-3 (knockout: KO) and from controls (wild type: WT) were examined. Our studies revealed that the loss of cavin-3 resulted in ∼40% reduction of the caveolae protein cavin-1 in vascular and bladder smooth muscle. Electron microscopy demonstrated that the loss of cavin-3 was accompanied by a reduction of caveolae abundance by 40-45% in smooth muscle, whereas the density of caveolae in endothelial cells was unchanged. Vascular contraction in response to an α1-adrenergic agonist was normal but nitric-oxide-dependent relaxation was enhanced, in parallel with an increased relaxation on direct activation of soluble guanylyl cyclase (sGC). This was associated with an elevated expression of sGC, although blood pressure was similar in WT and KO mice. Contraction of the urinary bladder was not affected by the loss of cavin-3. The proteomic response to outlet obstruction, including STAT3 phosphorylation, the induction of synthetic markers and the repression of contractile markers were identical in WT and KO mice, the only exception being a curtailed induction of the Golgi protein GM130. Loss of cavin-3 thus reduces the number of caveolae in smooth muscle and partly destabilizes cavin-1 but the functional consequences are modest and include an elevated vascular sensitivity to nitric oxide and slightly disturbed Golgi homeostasis in situations of severe cellular stress.

Assessing the contribution of thrombospondin-4 induction and ATF6α activation to endoplasmic reticulum expansion and phenotypic modulation in bladder outlet obstruction.

Phenotypic modulation of smooth muscle cells is a hallmark of disease. The associated expansion of endoplasmic reticulum (ER) volume remains unexplained. Thrombospondin-4 was recently found to promote ATF6α activation leading to ER expansion. Using bladder outlet obstruction as a paradigm for phenotypic modulation, we tested if thrombospondin-4 is induced in association with ATF6α activation and ER expansion. Thrombospondin-4 was induced and ATF6α was activated after outlet obstruction in rodents. Increased abundance of spliced of Xbp1, another ER-stress sensor, and induction of Atf4 and Creb3l2 was also seen. Downstream of ATF6α, Calr, Manf, Sdf2l1 and Pdi increased as did ER size, whereas contractile markers were reduced. Overexpression of ATF6α, but not of thrombospondin-4, increased Calr, Manf, Sdf2l1 and Pdi and caused ER expansion, but the contractile markers were inert. Knockout of thrombospondin-4 neither affected bladder growth nor expression of ATF6α target genes, and repression of contractile markers was the same, even if ATF6α activation was curtailed. Increases of Xbp1s, Atf4 and Creb3l2 were similar. Our findings demonstrate reciprocal regulation of the unfolded protein response, including ATF6α activation and ER expansion, and reduced contractile differentiation in bladder outlet obstruction occurring independently of thrombospondin-4, which however is a sensitive indicator of obstruction.

MicroRNAs in Bladder Outlet Obstruction: Relationship to Growth and Matrix Remodelling.

The discovery of microRNAs (miRNAs), which are ~22 nucleotide RNAs that inhibit protein synthesis in a sequence-specific manner and are present in a range of species, has born hope of new therapeutic strategies. miRNAs play important roles in development and disease, but they remain poorly studied in uropathologies beyond cancer. Here, we discuss biological functions of miRNAs in the lower urogenital tract. A special focus is on miRNAs that change in bladder outlet obstruction (BOO). This is a condition that affects nearly one third of all men over 60 years and that involves growth and fibrosis of the urinary bladder. Animal models of BOO, such as that in rat, have been developed, and they feature a massive 6-fold bladder growth over 6 weeks. Using microarrays, we have charted the miRNAs that change during the time course of this process and identified several with important modulatory roles. We discuss known and predicted functions of miR-1, miR-29, miR-30, miR-132/212, miR-204 and miR-221, all of which change in BOO. The majority of the miRNA-mediated influences in BOO are expected to favour growth. We also outline evidence that miR-29 represents a key effector molecule in a generic response to mechanical distension that is designed to counteract exaggerated organ deformation via effects on matrix deposition and stiffness. We conclude that miRNAs play important roles in bladder remodelling and growth and that they may be targeted pharmacologically to combat diseases of the lower urinary tract.

Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization.

Both type 1 and type 2 diabetes are associated with increased risk of cardiovascular disease. This is in part attributed to the effects of hyperglycemia on vascular endothelial and smooth muscle cells, but the underlying mechanisms are not fully understood. In diabetic animal models, hyperglycemia results in hypercontractility of vascular smooth muscle possibly due to increased activation of Rho-kinase. The aim of the present study was to investigate the regulation of contractile smooth muscle markers by glucose and to determine the signaling pathways that are activated by hyperglycemia in smooth muscle cells. Microarray, quantitative PCR, and Western blot analyses revealed that both mRNA and protein expression of contractile smooth muscle markers were increased in isolated smooth muscle cells cultured under high compared with low glucose conditions. This effect was also observed in hyperglycemic Akita mice and in diabetic patients. Elevated glucose activated the protein kinase C and Rho/Rho-kinase signaling pathways and stimulated actin polymerization. Glucose-induced expression of contractile smooth muscle markers in cultured cells could be partially or completely repressed by inhibitors of advanced glycation end products, L-type calcium channels, protein kinase C, Rho-kinase, actin polymerization, and myocardin-related transcription factors. Furthermore, genetic ablation of the miR-143/145 cluster prevented the effects of glucose on smooth muscle marker expression. In conclusion, these data demonstrate a possible link between hyperglycemia and vascular disease states associated with smooth muscle contractility.

Myocardin Family Members Drive Formation of Caveolae.

Caveolae are membrane organelles that play roles in glucose and lipid metabolism and in vascular function. Formation of caveolae requires caveolins and cavins. The make-up of caveolae and their density is considered to reflect cell-specific transcriptional control mechanisms for caveolins and cavins, but knowledge regarding regulation of caveolae genes is incomplete. Myocardin (MYOCD) and its relative MRTF-A (MKL1) are transcriptional coactivators that control genes which promote smooth muscle differentiation. MRTF-A communicates changes in actin polymerization to nuclear gene transcription. Here we tested if myocardin family proteins control biogenesis of caveolae via activation of caveolin and cavin transcription. Using human coronary artery smooth muscle cells we found that jasplakinolide and latrunculin B (LatB), substances that promote and inhibit actin polymerization, increased and decreased protein levels of caveolins and cavins, respectively. The effect of LatB was associated with reduced mRNA levels for these genes and this was replicated by the MRTF inhibitor CCG-1423 which was non-additive with LatB. Overexpression of myocardin and MRTF-A caused 5-10-fold induction of caveolins whereas cavin-1 and cavin-2 were induced 2-3-fold. PACSIN2 also increased, establishing positive regulation of caveolae genes from three families. Full regulation of CAV1 was retained in its proximal promoter. Knock down of the serum response factor (SRF), which mediates many of the effects of myocardin, decreased cavin-1 but increased caveolin-1 and -2 mRNAs. Viral transduction of myocardin increased the density of caveolae 5-fold in vitro. A decrease of CAV1 was observed concomitant with a decrease of the smooth muscle marker calponin in aortic aneurysms from mice (C57Bl/6) infused with angiotensin II. Human expression data disclosed correlations of MYOCD with CAV1 in a majority of human tissues and in the heart, correlation with MKL2 (MRTF-B) was observed. The myocardin family of transcriptional coactivators therefore drives formation of caveolae and this effect is largely independent of SRF.

Non-uniform changes in membrane receptors in the rat urinary bladder following outlet obstruction.

The aim of the present study was to investigate the expression and distribution of membrane receptors after bladder outlet obstruction (BOO). Partial bladder outlet obstruction (BOO) was induced in female rats and bladders were harvested after either 10 days or 6 weeks of BOO. The expression of different receptors was surveyed by microarrays and corroborated by immunohistochemistry and western blotting. A microarray experiment identified 10 membrane receptors that were differentially expressed compared to sham-operated rats including both upregulated and downregulated receptors. Four of these were selected for functional experiments on the basis of magnitude of change and relevance to bladder physiology. At 6 weeks of BOO, maximal contraction was reduced for neuromedin B and vasopressin (AVP), consistent with reductions of receptor mRNA levels. Glycine receptor-induced contraction on the other hand was increased and receptor mRNA expression was accordingly upregulated. Maximal relaxation by the ß3-adrenergic receptor agonist CL316243 was reduced as was the receptor mRNA level. Immunohistochemistry supported reduced expression of neuromedin B receptors, V1a receptors and ß3-adrenergic receptors, but glycine receptor expression appeared unchanged. Western blotting confirmed repression of V1a receptors and induction of glycine receptors in BOO. mRNA for vasopressin was detectable in the bladder, suggesting local AVP production. We conclude that changes in receptor expression following bladder outlet obstruction are non-uniform. Some receptors are upregulated, conferring increased responsiveness to agonist, whereas others are downregulated, leading to decreased agonist-induced responses. This study might help to select pharmacological agents that are effective in modulating lower urinary tract symptoms in BOO.

Regulation of smooth muscle dystrophin and synaptopodin 2 expression by actin polymerization and vascular injury.

OBJECTIVE: Actin dynamics in vascular smooth muscle is known to regulate contractile differentiation and may play a role in the pathogenesis of vascular disease. However, the list of genes regulated by actin polymerization in smooth muscle remains incomprehensive. Thus, the objective of this study was to identify actin-regulated genes in smooth muscle and to demonstrate the role of these genes in the regulation of vascular smooth muscle phenotype. APPROACH AND RESULTS: Mouse aortic smooth muscle cells were treated with an actin-stabilizing agent, jasplakinolide, and analyzed by microarrays. Several transcripts were upregulated including both known and previously unknown actin-regulated genes. Dystrophin and synaptopodin 2 were selected for further analysis in models of phenotypic modulation and vascular disease. These genes were highly expressed in differentiated versus synthetic smooth muscle and their expression was promoted by the transcription factors myocardin and myocardin-related transcription factor A. Furthermore, the expression of both synaptopodin 2 and dystrophin was significantly reduced in balloon-injured human arteries. Finally, using a dystrophin mutant mdx mouse and synaptopodin 2 knockdown, we demonstrate that these genes are involved in the regulation of smooth muscle differentiation and function. CONCLUSIONS: This study demonstrates novel genes that are promoted by actin polymerization, that regulate smooth muscle function, and that are deregulated in models of vascular disease. Thus, targeting actin polymerization or the genes controlled in this manner can lead to novel therapeutic options against vascular pathologies that involve phenotypic modulation of smooth muscle cells.

Vasopressin-induced mouse urethral contraction is modulated by caveolin-1.

Caveolae are 50-100nm large invaginations in the cell membrane that are considered to play roles in receptor signaling. Here we aimed to investigate the expression and distribution of the arginine-vasopressin (AVP) V1a receptor and its functional dependence on caveolin-1 (Cav1) in the mouse urethra. Female Cav1 knockout (KO) and wild type (WT) mice were used, and urethral preparations were micro-dissected for mechanical experiments. Methyl-ß-cyclodextrin (mßcd) was used to deplete cholesterol and to disrupt caveolae. Protein expression and localization was determined using immunofluorescence and western blotting and transcript expression was determined by qRT-PCR. We found that Cav1 and AVP V1a receptors were expressed in urethral smooth muscle cells with apparent co-localization at the cell membrane. AVP caused urethral contraction that was inhibited by the V1a receptor antagonist SR49059. Concentration-response curves for AVP were right-shifted and maximal contraction was reduced in Cav1 KO mice and after mßcd treatment. In addition to caveolin-1 we also detected caveolin-2, cavin-1 and cavin-3 in the mouse urethra by western blotting. Caveolin-2, cavin-1 and cavin-3 as well as V1a receptor expression was reduced in KO urethra. We conclude that AVP regulates urethral contractility via the V1a receptor through a Cav1-dependent mechanism involving, in part, altered V1a receptor expression.

Detrusor induction of miR-132/212 following bladder outlet obstruction: association with MeCP2 repression and cell viability.

The microRNAs (miRNAs) miR-132 and miR-212 have been found to regulate synaptic plasticity and cholinergic signaling and recent work has demonstrated roles outside of the CNS, including in smooth muscle. Here, we examined if miR-132 and miR-212 are induced in the urinary bladder following outlet obstruction and whether this correlates with effects on gene expression and cell growth. Three to seven-fold induction of miR-132/212 was found at 10 days of obstruction and this was selective for the detrusor layer. We cross-referenced putative binding sites in the miR-132/212 promoter with transcription factors that were predicted to be active in the obstruction model. This suggested involvement of Creb and Ahr in miR-132/212 induction. Creb phosphorylation (S-133) was not increased, but the number of Ahr positive nuclei increased. Moreover, we found that serum stimulation and protein kinase C activation induced miR-132/212 in human detrusor cells. To identify miR-132/212 targets, we correlated the mRNA levels of validated targets with the miRNA levels. Significant correlations between miR-132/212 and MeCP2, Ep300, Pnkd and Jarid1a were observed, and the protein levels of MeCP2, Pnkd and Ache were reduced after obstruction. Reduction of Ache however closely matched a 90% reduction of synapse density arguing that its repression was unrelated to miR-132/212 induction. Importantly, transfection of antimirs and mimics in cultured detrusor cells increased and decreased, respectively, the number of cells and led to changes in MeCP2 expression. In all, these findings show that obstruction of the urethra increases miR-132 and miR-212 in the detrusor and suggests that this influences gene expression and limits cell growth.

Induction of angiotensin-converting enzyme after miR-143/145 deletion is critical for impaired smooth muscle contractility.

MicroRNAs have emerged as regulators of smooth muscle cell phenotype with a role in smooth muscle-related disease. Studies have shown that miR-143 and miR-145 are the most highly expressed microRNAs in smooth muscle cells, controlling differentiation and function. The effect of miR-143/145 knockout has been established in the vasculature but not in smooth muscle from other organs. Using knockout mice we found that maximal contraction induced by either depolarization or phosphatase inhibition was reduced in vascular and airway smooth muscle but maintained in the urinary bladder. Furthermore, a reduction of media thickness and reduced expression of differentiation markers was seen in the aorta but not in the bladder. Supporting the view that phenotype switching depends on a tissue-specific target of miR-143/145, we found induction of angiotensin-converting enzyme in the aorta but not in the bladder where angiotensin-converting enzyme was expressed at a low level. Chronic treatment with angiotensin type-1 receptor antagonist restored contractility in miR-143/145-deficient aorta while leaving bladder contractility unaffected. This shows that tissue-specific targets are critical for the effects of miR-143/145 on smooth muscle differentiation and that angiotensin converting enzyme is one such target.

HIF-mediated metabolic switching in bladder outlet obstruction mitigates the relaxing effect of mitochondrial inhibition.

Prior work demonstrated increased levels of hypoxia-inducible factor-1α (HIF-1α) in the bladder following outlet obstruction, associated with bladder growth and fibrosis. Here we hypothesized that HIF induction in outlet obstruction also switches energetic support of contraction from mitochondrial respiration to glycolysis. To address this hypothesis, we created infravesical outlet obstruction in female Sprague-Dawley rats and examined HIF induction and transcriptional activation. HIF-1α increased after 6 weeks of outlet obstruction as assessed by western blotting and yet transcription factor-binding site analysis indicated HIF activation already at 10 days of obstruction. Accumulation HIF-2α and of Arnt2 proteins were found at 10 days, providing an explanation for the lack of correlation between HIF-1α protein and transcriptional activation. HIF signature targets, including Slc2a1, Tpi1, Eno1 and Ldha increased in obstructed compared with sham-operated bladders. The autophagy markers Bnip3 and LC3B-II were also increased at 6 week of obstruction, but electron microscopy did not support mitophagy. Mitochondria were, however, remodeled with increased expression of Cox4 compared with other markers. In keeping with a switch toward glycolytic support of contraction, we found that relaxation by the mitochondrial inhibitor cyanide was reduced in obstructed bladders. This was mimicked by organ culture with the HIF-inducer dimethyloxalylglycine, which also upregulated expression of Ldha. On the basis of these findings, we conclude that HIF activation in outlet obstruction involves mechanisms beyond the accumulation of HIF-1α protein and that it results in a switch of the energetic support of contraction to anaerobic glycolysis. This metabolic adaptation encompasses increased expression of glucose transporters and glycolytic enzymes combined with mitochondrial remodeling. Together, these changes uphold contractility when mitochondrial respiration is limited.

Mir-29 repression in bladder outlet obstruction contributes to matrix remodeling and altered stiffness.

Recent work has uncovered a role of the microRNA (miRNA) miR-29 in remodeling of the extracellular matrix. Partial bladder outlet obstruction is a prevalent condition in older men with prostate enlargement that leads to matrix synthesis in the lower urinary tract and increases bladder stiffness. Here we tested the hypothesis that miR-29 is repressed in the bladder in outlet obstruction and that this has an impact on protein synthesis and matrix remodeling leading to increased bladder stiffness. c-Myc, NF-κB and SMAD3, all of which repress miR-29, were activated in the rat detrusor following partial bladder outlet obstruction but at different times. c-Myc and NF-κB activation occurred early after obstruction, and SMAD3 phosphorylation increased later, with a significant elevation at 6 weeks. c-Myc, NF-κB and SMAD3 activation, respectively, correlated with repression of miR-29b and miR-29c at 10 days of obstruction and with repression of miR-29c at 6 weeks. An mRNA microarray analysis showed that the reduction of miR-29 following outlet obstruction was associated with increased levels of miR-29 target mRNAs, including mRNAs for tropoelastin, the matricellular protein Sparc and collagen IV. Outlet obstruction increased protein levels of eight out of eight examined miR-29 targets, including tropoelastin and Sparc. Transfection of human bladder smooth muscle cells with antimiR-29c and miR-29c mimic caused reciprocal changes in target protein levels in vitro. Tamoxifen inducible and smooth muscle-specific deletion of Dicer in mice reduced miR-29 expression and increased tropoelastin and the thickness of the basal lamina surrounding smooth muscle cells in the bladder. It also increased detrusor stiffness independent of outlet obstruction. Taken together, our study supports a model where the combined repressive influences of c-Myc, NF-κB and SMAD3 reduce miR-29 in bladder outlet obstruction, and where the resulting drop in miR-29 contributes to matrix remodeling and altered passive mechanical properties of the detrusor.

Late onset vascular dysfunction in the R6/1 model of Huntington's disease.

Huntington's disease is a neurodegenerative disorder that also gives raise to widespread changes in peripheral organs and tissues. We tested the hypothesis that vascular dysfunction may occur in Huntington's disease by studying R6/1 mice which express exon 1 of the mutant huntingtin gene. We assessed arterial function in R6/1 and wild type (WT) mice using myography. Arterial contractility was largely unaltered in R6/1 arteries at 15 and 32 weeks of age. By 40 weeks, contractility was impaired irrespective of which vasoconstrictor we tested. Endothelium-dependent relaxation was not affected, and we observed no changes in arterial geometry or expression of contractile proteins, such as myosin regulatory light chains or smooth muscle α-actin. The frequency of calcium oscillations in R6/1 arterial smooth muscle cells was higher than in WT control tissue, whereas myosin phosphorylation was unaltered. Impairment of force by the mitochondrial inhibitors cyanide and rotenone was less pronounced in R6/1 than in WT arteries and mitochondria were enlarged, in keeping with an effect related to altered mitochondrial function. Our results reveal that arteries in the R6/1 model of Huntington's disease exhibit an age-dependent impairment of contractility and that they depend less on mitochondrial function when they contract.

Impaired contractility and detrusor hypertrophy in cavin-1-deficient mice.

Caveolae are membrane invaginations present in a variety of cell types. Formation of caveolae depends on caveolins and on the more recently discovered family of proteins known as the cavins. Genetic ablation of caveolin-1 was previously shown to give rise to a number of urogenital alterations, but the effects of cavin-1 deletion on urogenital function remain unknown. Here we characterised detrusor contractility and structure in cavin-1-deficient mice. Electron microscopy demonstrated essentially complete lack of caveolae in the knock-out detrusor, and immunoblotting disclosed reduced levels of cavin-3 and of all caveolin proteins. Bladder weight was increased in male knock-out mice, and length-tension relationships demonstrated a reduction in depolarisation-induced contraction. Contractility in response to muscarinic receptor activation was similarly reduced. Despite these functional changes, micturition patterns were similar in conscious and freely moving animals and diuresis was unchanged. Our breeding additionally disclosed that the number of knock-out mice generated in heterozygous crosses was lower than expected, suggesting embryonic/perinatal lethality. In conclusion, this is the first study to show that cavin-1 is critical for detrusor caveolae and for the overall contractility and structure of the urinary bladder.