Myosin Heavy Chain Promoter Research Articles

Abstract 15568: Selective Deletion of Smad4 in Smooth Muscle Cells Causes Thoracic Aortic Aneurysm in Mice

TGFβ signaling have been implicated in the onset of thoracic aortic aneurysms (TAAs). Recently, it has been described that mutations in SMAD4, an intracellular transducer required in the TGFβ canonical pathway, present aortic disease in humans. Although transgenic models recapitulating mutations of TGFβ pathway have been studied, the role of Smad4 in the onset of TAA has never been explored. Smad4 homozygotes mice are embryonically lethal, thus, we postnatally targeted the gene, using a tamoxifen (TAM)-inducible Cre recombinase under the smooth muscle myosin heavy chain promoter. Activation by TAM suppressed Smad4 in aorta SMCs of Smadf/f;Myh11-CreERT2 mice. Most of these mice died 4/6 months later from hemothorax due to rupture of thoracic aorta, while untreated animals survived. Echocardiography showed a progressive dilatation of aorta and TAA formation. Histological analysis revealed progressive fragmentation of elastic lamellae with increased inflammatory infiltrates at sites of disarrangement, rich in macrophages. At the molecular level, we found that lack of Smad4 was followed by increased levels of P-Smad2 and P-Smad1/5/8 early after TAM. This finding suggested an enhancement of TGFβ receptors activity, a condition that could increase non-canonical TGFβ signaling, the canonical one being certainly depressed. We actually found a significant increase in one of the non-canonical TGFβ signaling, i.e. the p65 subunit of NFkB, a protein complex involved in regulating responses to different stress stimuli, including cytokines. Given the particular relevance that the latter molecules may have in TAA, we tested expression of some of them, finding a selective IL-1β upregulation, produced and stored as an inactive precursor and released in its active form by caspase-1 cleavage. In turn, caspase-1 requires the function of NLRP3 inflammasome, a multiprotein platform that assembles in response to infection and tissue damage and is responsible for activation of inflammatory caspases. Therefore, we also tested NLRP3 mRNA levels and found them to be significantly upregulated. Overall, we demonstrated that selective Smad4 deletion in the SMC of adult mice is sufficient to induce ultrastructural damage and immune reaction culminating in the onset of TAA.

Abstract 400: Regulated Levels of Sarcolemmal Membrane Protein Isoform 3 Impact Cardiac Function in Mice

The sarcolemmal membrane-associated proteins (SLMAPs) are a family of tail-anchored membrane proteins generated by alternative splicing of the SLMAP gene. A ubiquitously expressed SLMAP isoform 3 encompasses an N-terminal FHA domain with extended coiled-coil structure and has been implicated in cell cycle control. Heart function in transgenic mice with cardiac-specific overexpression of SLMAP3 cDNA driven by α myosin heavy chain promoter was evaluated by echocardiography. qPCR and western blot were used to analyze gene and protein expression respectively. Structure and fibrosis was analyzed by H&E and Masson’s Trichrome staining. Function analysis showed a 15% (p<0.05) decrease in ejection fraction and 19% (p<0.05) decrease in fractional shortening in transgenic mice as early as 5 weeks and persisted into old age at 44 weeks. Transgenic mice presented a mild systolic dysfunction and a trend towards dilated cardiomyopathy without any premature death. Natriuretic peptide ANP and BNP levels were not changed and there was no difference in left ventricular mass or activation of the hypertrophic factor Akt1 in SLMAP3 expressing myocardium. However, significant changes in calcium handling proteins with a significant decrease in phosphorylation of phospholamban ser16 (p<0.05) along with a down-regulation of sarco-endoplasmic reticulum Ca2+ ATPase protein and increased ryanodine receptor 2 phosphorylation ser2808 (p<0.05) were noted at 5 weeks of age in transgenic hearts. These data indicate that increased SLMAP3 levels did not influence cardiac remodeling or hypertrophic growth but did impact membrane biology of calcium transport systems in myocardium leading to depressed contractility. Thus regulated levels of SLMAP3 are important to support normal heart function.

Hypoxia fate mapping identifies cycling cardiomyocytes in the adult heart.

Although the adult mammalian heart is incapable of meaningful functional recovery following substantial cardiomyocyte loss, it is now clear that modest cardiomyocyte turnover occurs in adult mouse and human hearts, mediated primarily by proliferation of pre-existing cardiomyocytes. However, fate mapping of these cycling cardiomyocytes has not been possible thus far owing to the lack of identifiable genetic markers. In several organs, stem or progenitor cells reside in relatively hypoxic microenvironments where the stabilization of the hypoxia-inducible factor 1 alpha (Hif-1α) subunit is critical for their maintenance and function. Here we report fate mapping of hypoxic cells and their progenies by generating a transgenic mouse expressing a chimaeric protein in which the oxygen-dependent degradation (ODD) domain of Hif-1α is fused to the tamoxifen-inducible CreERT2 recombinase. In mice bearing the creERT2-ODD transgene driven by either the ubiquitous CAG promoter or the cardiomyocyte-specific α myosin heavy chain promoter, we identify a rare population of hypoxic cardiomyocytes that display characteristics of proliferative neonatal cardiomyocytes, such as smaller size, mononucleation and lower oxidative DNA damage. Notably, these hypoxic cardiomyocytes contributed widely to new cardiomyocyte formation in the adult heart. These results indicate that hypoxia signalling is an important hallmark of cycling cardiomyocytes, and suggest that hypoxia fate mapping can be a powerful tool for identifying cycling cells in adult mammals.

Abstract 251: Selective Deletion of Smad4 in Smooth Muscle Cells Causes Thoracic Aortic Aneurysm in Mice

TGFβ signaling have been implicated in the onset of thoracic aortic aneurysms (TAA). Recently, it has been described that mutations in SMAD4, an intracellular transducer required in the TGFβ canonical pathway, present aortic disease in humans. Although transgenic models recapitulating mutations of TGFβ pathway have been studied, Smad4 role in the onset of TAA has never been explored. Smad4 homozygotes mice are embryonically lethal, thus, we postnatally targeted the gene, using a tamoxifen (TAM)-inducible Cre recombinase under the smooth muscle myosin heavy chain promoter. Activation by TAM suppressed Smad4 in aorta SMCs of Smadf/f;Myh11-CreERT2 mice. We found that, most of these mice died 4/6 months later from hemothorax due to rupture of thoracic aorta, while untreated mice survived. Echocardiography showed a progressive dilation of aorta and TAA formation. Histology revealed progressive fragmentation of elastic lamellae with increased inflammatory infiltrates at sites of disarrangement, rich in macrophages. At the molecular level, we found that lack of Smad4 was followed by increased levels of PSmad2 and PSmad1/5/8 early after TAM, suggesting an enhancement of TGFβ receptors activity, a condition that could increase non-canonical TGFβ signaling, the canonical one being depressed. We actually found a significant increase in one of the non-canonical TGFβ signaling, i.e. the p65 subunit of NFkB, a protein complex controlling DNA transcription and involved in regulating responses to different stress stimuli, including cytokines. Given the particular relevance that the latter molecules may have in TAA, we tested expression of some of them, finding a selective IL-1β upregulation, produced and stored in cells as an inactive precursor and released in its active form by caspase-1 cleavage. In turn, caspase-1 requires NLRP3 inflammasome, a multiprotein platform that assembles in response to infection and tissue damage and is responsible for activation of inflammatory caspases. Therefore, we also tested NLRP3 mRNA levels and found them to be significantly upregulated. We demonstrated for the first time that selective Smad4 deletion in the SMC of adult mice is sufficient to induce ultrastructural damage and immune reaction culminating in the onset of TAA.

Involvement of activated SUMO-2 conjugation in cardiomyopathy

Sumoylation is a posttranslational modification that regulates a wide spectrum of cellular activities. Cardiomyopathy is the leading cause of heart failure. Whether sumoylation, particularly SUMO-2/3 conjugation, is involved in cardiomyopathy has not been investigated. We report here that SUMO-2/3 conjugation was elevated in the human failing hearts, and we investigated the impact of increased SUMO-2 conjugation on heart function by using the gain-of-function approach in mice, in which cardiac specific expression of constitutively active SUMO-2 was governed by alpha myosin heavy chain promoter (MHC-SUMO-2 transgenic, SUMO-2-Tg). Four of five independent SUMO-2-Tg mouse lines exhibited cardiomyopathy with various severities, ranging from acute heart failure leading to early death to the development of chronic cardiomyopathy with aging. We further revealed that SUMO-2 directly regulated apoptotic process by at least partially targeting calpain 2 and its natural inhibitor calpastatin. SUMO conjugation to calpain 2 promoted its enzymatic activity, and SUMO attachment to calpastatin mainly promoted its turnover and altered its subcellular distribution. Thus, enhanced SUMO-2 conjugation led to increased apoptosis and played a pathogenic role in the development of cardiomyopathy and heart failure.

Transgenic Over-Expression of Carbonic Anhydrase III in Cardiac Muscle Demonstrates a Mechanism to Resist Acidosis

Carbonic anhydrase III (CAIII) is an abundant protein in skeletal muscle, liver and adipose cells. A cytosolic enzyme that catalyzes conversions between CO2 and HCO3- in regulating intracellular pH, its physiological function in muscle is unclear. Mice lacking CAIII showed lower than wild type intracellular pH in skeletal muscle cells during fatigue treatment. To further understand the role of CAIII in muscle functions and stress adaptation, we developed transgenic mice overexpressing CAIII in the heart under the control of a cloned myosin heavy chain promoter for phenotype comparisons with wild type mouse hearts that are CAIII negative. Three months old transgenic mice showed normal cardiac phenotypes under non-stress conditions. Cardiac function was examined using ex vivo working heart preparations under normal and low pH conditions to investigate CAIII function in pH regulation of cardiac muscle. With equilibration of 5% CO2 generating pH 7.4 in normal Kreb's perfusion buffer, 10% CO2 was used to lower pH to 7.0. Functional data showed that transgenic and wild type hearts had similar pumping functions under normal pH. Perfused with low pH buffer, heart functions of both groups were decreased. In comparison with wild type controls at low pH, CAIII transgenic mouse hearts showed higher left ventricular pressure development and systolic and diastolic velocities under both baseline conditions and increased afterload stress, indicating a better tolerance to acidosis. The results suggest that CAIII may function in compensating for intracellular pH under acidotic conditions, a tractive novel approach to develop new treatment of chronic congestive heart failure.

Role of peroxisome proliferator-activated receptor-γ in vascular muscle in the cerebral circulation.

Although peroxisome proliferator-activated receptor-γ (PPARγ) is thought to play a protective role in the vasculature, its cell-specific effect, particularly in resistance vessels, is poorly defined. Nitric oxide (NO) plays a major role in vascular biology in the brain. We examined the hypothesis that selective interference with PPARγ in vascular muscle would impair NO-dependent responses and augment vasoconstrictor responses in the cerebral circulation. We studied mice expressing a dominant negative mutation in human PPARγ (P467L) under the control of the smooth muscle myosin heavy chain promoter (S-P467L). In S-P467L mice, dilator responses to exogenously applied or endogenously produced NO were greatly impaired in cerebral arteries in vitro and in small cerebral arterioles in vivo. Select NO-independent responses, including vasodilation to low concentrations of potassium, were also impaired in S-P467L mice. In contrast, increased expression of wild-type PPARγ in smooth muscle had little effect on vasomotor responses. Mechanisms underlying impairment of both NO-dependent and NO-independent vasodilator responses after interference with PPARγ involved Rho kinase with no apparent contribution by oxidative stress-related mechanisms. These findings support the concept that via effects on Rho kinase-dependent signaling, PPARγ in vascular muscle is a major determinant of vascular tone in resistance vessels and, in particular, NO-mediated signaling in cerebral arteries and brain microvessels. Considering the importance of NO and Rho kinase, these findings have implications for regulation of cerebral blood flow and the pathogenesis of large and small vessel disease in brain.

DNA Methylation of a GC Repressor Element in the Smooth Muscle Myosin Heavy Chain Promoter Facilitates Binding of the Notch-Associated Transcription Factor, RBPJ/CSL1

The goal of the present study was to identify novel mechanisms that regulate smooth muscle cell (SMC) differentiation marker gene expression. We demonstrate that the CArG-containing regions of many SMC-specific promoters are imbedded within CpG islands. A previously identified GC repressor element in the SM myosin heavy chain (MHC) promoter was highly methylated in cultured aortic SMC but not in the aorta, and this difference was inversely correlated with SM MHC expression. Using an affinity chromatography/mass spectroscopy-based approach, we identified the multifunctional Notch transcription factor, recombination signal binding protein for immunoglobulin κ J region (RBPJ), as a methylated GC repressor-binding protein. RBPJ protein levels and binding to the endogenous SM MHC GC repressor were enhanced by platelet-derived growth factor-BB treatment. A methylation mimetic mutation to the GC repressor that facilitated RBPJ binding inhibited SM MHC promoter activity as did overexpression of RBPJ. Consistent with this, knockdown of RBPJ in phenotypically modulated human aortic SMC enhanced endogenous SMC marker gene expression, an effect likely mediated by increased recruitment of serum response factor and Pol II to the SMC-specific promoters. In contrast, the depletion of RBPJ in differentiated transforming growth factor-β-treated SMC inhibited SMC-specific gene activation, supporting the idea that the effects of RBPJ/Notch signaling are context dependent. Our results indicate that methylation-dependent binding of RBPJ to a GC repressor element can negatively regulate SM MHC promoter activity and that RBPJ can inhibit SMC marker gene expression in phenotypically modulated SMC. These results will have important implications on the regulation of SMC phenotype and on Notch-dependent transcription.

Abstract 051: The Myogenic Tone Of Resistance Arteries Is Controlled By A Molecular Pathway Driven By Tgfβ Through Emilin1 And Converging On The Regulation Of Trp Channels.

Emilin1 (E1) is a protein of the extracellular matrix that regulates TGFβ bioavailability through proteolysis of the proTGFβ. E1 KO mice are hypertensive, with increased TGFβ activation. As E1 is expressed in blood vessels starting from embryonic life to adulthood, is still unknown whether the E1 KO phenotype results from a developmental defect or lack of a homeostatic role in the adult. To dissect this issue, we used a conditional gene targeting approach to inactivate E1 in smooth muscle cells (VSMCs) of adult mice, by the use of floxed E1 and CreERT2 [tamoxifen (TAM) inducible Cre recombinase] under the control of the smooth muscle myosin heavy chain (Smmhc) promoter. When E1fl/fl mice carrying the Smmhc-CreERT2 were given TAM blood pressure significantly increased (124±1 vs basal condition 106±1 mmHg) as well as myogenic response in resistance arteries (16.3±0.7 vs basal condition 11.4±0.1 % at 125 mmHg). In order to understand how E1 ablation in VSMCs could functionally determine the increase in myogenic tone, we used an ex vivo approach in resistance arteries, to target the molecular pathway that could regulate intracellular calcium fluxes, the main regulator of myogenic contraction. Experiments performed in isolated VSMCs from E1 KO as compared to WT arteries, suggested the involvement of a ROCE (receptor operated calcium entry) mechanism, whose main final molecular determinant is represented by the TRP channels, known to be regulated by a canonical PLC-DAG pathway, but also by a parallel one controlled by the growth factors signaling cascade. By exploring this pathway, we found that E1 regulates the activity of TRPC3 channels and thus of the myogenic response. The molecular pathway delineated by our studies started from the transactivation of EGFR through metalloproteinases and converged on the Src kinases that increased TRPC3 function and thus calcium entry. In particular, with an in vivo targeting of TRPC3 in E1 KO with an adenovirus carrying a dominant negative mutation, we demonstrated a rescue of the increased myogenic response of E1 KO resistance arteries. Overall our results suggest that this pathway in VSMC, driven by TGFβ and regulated by E1, is crucial for the myogenic tone of resistance arteries and is linked to the regulation of blood pressure.

Abstract 511: Emilin1 Controls Myogenic Response of Resistance Arteries by Modulation of TRPC6 Through the TGFß/ALK5/ADAM17/EGFR Pathway

Emilin1 (E1), a protein of the extracellular matrix expressed in the cardiovascular system, regulates TGFβ bioavailability through proteolysis of the proTGFβ, that releases the growth factor. E1 null mice are hypertensive, with increased TGFβ activation. As E1 is expressed in blood vessels starting from embryonic life to adulthood, is still unknown whether the E1 null phenotype results from a developmental defect or lack of a homeostatic role exerted during the adult life. To dissect this issue, we used a conditional gene targeting approach to inactivate E1 in smooth muscle cells (SMCs) of adult mice, by the use of floxed E1 and CreER T2 [a tamoxifen (TAM) inducible Cre recombinase] under the control of the smooth muscle myosin heavy chain (Smmhc) promoter. TAM induced Cre activity selectively in SMCs, as shown in mice with the Rosa26R mutation. When E1 fl/fl mice carrying the Smmhc-CreER T2 (Smmhc-CreER T2 E1 fl/fl ) were given TAM, E1 protein completely disappeared and blood pressure significantly increased, as compared to E1 fl/fl . While no structural changes of arteries were detected, we found that mesenteric arteries of Smmhc-CreER T2 E1 fl/fl displayed an increased myogenic response to pressure as compared to E1 fl/fl . Since the hypertensive phenotype of Smmhc-CreER T2 E1 fl/fl mice was rescued by a treatment with a TGFβ neutralizing antibody, we investigated the molecular mechanism in SMCs that, downstream to this pathway, could lead to an increased Ca 2+ -mediated contractility. In particular, also the increased myogenic response was rescued by exposure of resistance arteries to a TGFβ neutralizing antibody and, more important, by the inhibition of TGFβ receptor ALK5, obtained by in vivo vessels transfection with an adenovirus carrying a dominant negative ALK5 mutant. Moreover, we found that the increased TGFβ signaling, by phosphorylating the ADAM17/EGFR cascade, converged on overactivation of the Transient receptor potential cation channel C6 (TRPC6), rescued by in vivo vessels transfection with an adenovirus carrying a dominant negative TRPC6 mutant. Our results demonstrate that E1 exerts a homeostatic control of myogenic response by regulating the TRPC6 Ca 2+ channel, through a modulation of the TGFβ/ALK5/ADAM17/EGFR signaling pathway in SMCs.

Endothelium‐targeted deletion of Nox4 attenuates hypoxia‐induced increases in right ventricular pressure (1089.19)

The complex pathobiology of pulmonary hypertension (PH) involves reactive oxygen species (ROS) and increased pulmonary vascular resistance. NADPH oxidase 4 (Nox4) has been shown to be an important source of ROS in PH, and systemic administration of a Nox4 pharmacological inhibitor attenuated hypoxia‐induced PH. To further explore the role of pulmonary vascular Nox4 in hypoxia‐induced PH, a floxed Nox4 mouse model was crossed with mice that: 1) express Cre recombinase driven by the tie‐2 promoter to target deletion of endothelial Nox4 (eNox4), or 2) express Cre recombinase driven by the smooth muscle myosin heavy chain promoter to target deletion of smooth muscle Nox4 (smNox4). Littermate controls, eNox4 and smNox4 mice were exposed to normoxia (21% O2) or hypoxia (10% O2) for 3 weeks. PH development was assessed by measuring right ventricular systolic pressures (RVSP) and right ventricular hypertrophy (RVH). Hypoxia‐induced increases in RVSP were attenuated in the eNox4 model (N=5; p<0.05) whereas the RVSP in smNox4 knockout mice was not different than controls. In contrast, hypoxia‐induced RVH was no different than controls in either the eNox4 or smNox4 mice. These studies suggest that endothelial Nox4 promotes pulmonary vascular derangements involved in PH pathogenesis.Grant Funding Source: Supported by VA Merit Review 1I01BX001910 (CMH) and NIH grant R01 HL102167 (CMH and RLS).

Smooth muscle‐targeted overexpression of peroxisome proliferator‐activated receptor gamma disrupts vascular wall structure and function (866.4)

Objective: To explore the role of PPARγ in vascular regulation, smooth muscle VP16‐PPARγ‐overexpressing mice were subjected to studies of systemic vascular function, biomechanical testing, histological analysis, and molecular characterization. Methods: Mice with a flox‐controlled VP16‐PPARγ gene were crossed with mice containing a tamoxifen‐inducible Cre recombinase driven by a smooth muscle myosin heavy chain promoter. All mice received tamoxifen injections at 6 weeks of age. Experiments were performed at 10‐16 weeks of age. Results: Compared to littermate controls, transgenic mice displayed: 1) aortic dilation (by histology, echocardiography, and microCT), 2) increased aortic compliance, 3) reduced blood pressure, 4) 80% reduction in aortic ring contractile forces in response to KCl or phenylephrine, 5) lipid accumulation in the aortic media (Oil Red O staining), and 6) reduction in aortic contractile markers and extra‐cellular matrix components with increased expression of adipose markers and selected mitochondrial proteins (by qPCR).Conclusion: These results demonstrate that targeted overexpression of activated PPARγ in smooth muscle disrupts vascular wall structure and function in part by driving medial smooth muscle towards an adipocyte phenotype.Grant Funding Source: Supported by grants from NIH (R01 HL102167) and VA (Merit Review Award 1I01BX001910)

Pluripotent stem cells – from physiology to cell therapy, disease models and test systems

Owing to their ability to reproduce the embryonic, fetal and neonatal differentiation of all different organotypic cellular phenotypes, pluripotent stem cells represent an ideal tool to study physiological processes of embryogenesis under in vitro conditions, as well as provide the basis of cellular therapeutics to develop novel disease models and to build up test assay systems for drug discovery and toxicology. In particular, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells can reproduce all organotypic electrophysiology, signalling cascades and genes involved in development (functional genomics). This spontaneously occurs within three-dimensional cell aggregates – embryoid bodies – which were developed 25 years ago. To select only one lineage (e.g. the cardiac lineage) and to allow the identification of the transplanted cells, transgenic ES and iPS cells were used. They contained a vector with two cloning sites for enhanced green fluorescent protein and a puromycin resistance gene for selection under the α -MHC (myosin heavy chain) promoter. We aimed to generate iPS cell-derived cardiomyocytes and their molecular and functional characterization in comparison with cardiomyocytes derived from established ES cells on a transcriptomic and electrophysiological level. To demonstrate the ability of ES cells for regenerative medicine and tissue repair, cardiomyocytes differentiated from ES cells were injected into the cryoinfarcted left ventricular wall of adult wild-type mice. Translation from the laboratory into the clinic is one of the remaining key issues for applied stem cell research.

DNA Methylation of a GC Repressor Element in the Smooth Muscle Myosin Heavy Chain Promoter Facilitates Binding of the Notch-Associated Transcription Factor, RBPJ/CSL1

DNA Methylation of a GC Repressor Element in the Smooth Muscle Myosin Heavy Chain Promoter Facilitates Binding of the Notch-Associated Transcription Factor, RBPJ/CSL1

Abstract 9244: A Novel Phosphorylating Mechanism of STAT3 in Protection against Ischemia/reperfusion by Pharmacological Preconditioning With Tadalafil, a PDE Inhibitor

Background: Phosphodiesterase (PDE) inhibition enhances Protein Kinase G-I (PKG-I) activity leading to stem cell survival and cardioprotection against ischemia/reperfusion injury. We previously reported phosphorylation of STAT3 ser727 during late phase of protection. We aimed to investigate the specificity of STAT3 in regulation of PKG-I and cardioprotection using heart specific conditional knockout mice. Methods and Results: Heart specific conditional knockout mice were made by breeding STAT3fl/fl with tamoxifen inducible cre (MCM) transgenic mice. MCM transgene was cloned downstream to heart specific myosin heavy chain promoter (MHC). Cre positive mice (MCM:STAT3fl/fl) were selected from genotyping and tamoxifen (20mg/kg) was administrated for 10 days. KO mice were pretreated with tadalafil (1mg/kg) 1h before LAD coronary artery ligation for 30 min followed by reperfusion. Echocardiographic analysis showed abrogation of cardioprotection in STAT3 knockout mice with decreased ejection fraction and fraction shortening as compared to non-transgenic mice. Mitogen activated protein kinases ERK and P38 were known to phosphorylate STAT3 at ser727. On contrary, western blot analysis with heart tissue lysate showed reduced phosphorylation/activation of ERK both in control and tadalafil treated cells with no change in total ERK (P<0.05). Expression of pP38 and total P38 (p<0.01) were unaltered. Two fold increase in expression of PKG-I (P<0.05) during the late phase of protection was observed with tadalafil. Inhibition of PKG-I with KT5823 or antisense abolished STAT3 phosphorylation at ser727. Results were also replicated in vitro with mesenchymal stem cells isolated from adult male Fischer-344 rats by flushing the cavity of femur and cultured, purity was checked by CD44+ and CD73+ surface markers (flow cytometry). Cells were pretreated 1 h with tadalafil (100μM) then subjected to 2 h hypoxia (1% O2) and reoxygenation for 2 h/24 h. Conclusion: The study showed novel mechanism of phosphorylating STAT3 at ser727 by PKG-I leading to its entry inside nucleus and transcriptional activation of PKG-I by binding onto promoter making it a positive feedback loop. Heart specific STAT3 knockout mice showed abrogation of cardioprotection with tadalafil.

The homoeobox gene SIX1 alters myosin heavy chain isoform expression in mouse skeletal muscle

Six1 is necessary for the genesis of several tissues, but in adults, it is expressed primarily in skeletal muscle where its function is unclear. Overexpression of Six1 with a cofactor in skeletal muscle causes slow-to-fast fibre-type transition. We sought to characterize the effects of a physiologically relevant Six1 knockdown. The tibialis anterior (TA) muscles of C57BL/6 mice were electroporated with Six1 knockdown vector (siRNA) or empty vector. Muscles were collected at 2 or 14days after transfection for Six1 and myosin heavy chain (MHC) expression analysis. C2C12 mouse myoblasts were grown in standard conditions. Cells were cotransfected with MHC promoter vectors and Six1 expression vectors. Cells were harvested after 4days of differentiation. In vivo, the Six1 siRNA caused a decreased expression of Six1,1.8-fold (±0.1, P<0.05). With decreased Six1, MHC IIB expression decreased 2.7-fold (±0.7, P=0.04). Proportion of muscle fibres expressing MHC IIB decreased (45.3±4.8% vs. 65.1±7.3% in control group, P=0.04), and total area expressing MHC IIB decreased with decreased Six1 (59.6±4.3% vs. 75.2±5.4% in control group, P<0.05). Decreased Six1 increased MHC IIA expression 1.9-fold (±0.3, P=0.04). In vitro, Six1 overexpression increased promoter activation of MHC IIB 2.9-fold (±0.3, P<0.01). Six1 knockdown repressed MHC IIB promoter 2.9-fold (±0.1, P<0.05) and MHC IIX 3.7-fold (±0.08, P<0.01). Six1 knockdown caused a fast-to-slow shift in MHC isoform, and this was confirmed by promoter activity of MHC genes. Six1 may ultimately control the contractile and metabolic properties that define muscle fibre phenotype.

Abstract 19: Blood Pressure Correlates Directly with the Expression Level of High Ouabain Affinity a2 Na+ Pumps in Arterial Smooth Muscle

High ouabain affinity α2 Na + pumps are expressed in arterial smooth muscle (ASM). They help regulate blood pressure (BP) in several forms of hypertension, but their influence on basal BP is controversial. We determined basal BP (telemetry), and the effects of subcutaneous (sc) angiotensin II (Ang II) and dietary salt on BP in: 1) α2 SM/Tg mice, which express an α2 transgene controlled by an α-actin smooth muscle (SM) promoter; 2) α2 SM/DN mice, which express a truncated α2 gene, dominant negative for native α2 Na + pumps, controlled by the SM myosin heavy chain promoter. Expression (immunoblots) of α2 was significantly up- and down-regulated in SM from α2 SM/Tg and α2 SM/DN mice, respectively. Basal 24 hr mean BP (MBP) was reduced in α2 SM/Tg mice: 103±1 ( n =15) vs 110±2 mm Hg ( n =9) in WT controls; P &lt;0.01. Ang II, 400 ng/kg/min sc x 2 wks, increased MBP in both WT and transgenic mice ( n =5 each; P &lt;0.01), but the increase (Δ) was smaller in α2 SM/Tg , Δ=20.3±3.3, than in WT mice, Δ=30.9±6.4 mm Hg . Also, a high (2% x 2 wks) NaCl diet, along with a sc 300 ng/kg/min (low dose) Ang II infusion, elevated systolic BP (SBP) in the WT mice (Δ=12.6±1.8 mm Hg; n =4; P &lt;0.05), but not in the α2 SM/Tg mice (Δ=7.2±2.0 mm Hg; n =9; NS=not significant). In contrast, in α2 SM/DN mice, basal SBP was elevated: 128±4 mm Hg ( n =11) vs 109±4 mm Hg ( n =10) in WT; P &lt;0.01. Furthermore, both high NaCl (HS, 6% x 10 days), and low dose sc Ang II infusion (240 ng/kg/min x 8 days), individually induced significantly greater increases in MBP in α2 SM/DN than in WT mice. In α2 SM/DN mice, the HS diet raised MBP by Δ=6.8±0.7 mm Hg ( n =5; P &lt;0.01); Ang II elevated MBP by Δ=17.1±3.7 mm Hg ( n =5; P &lt;0.05). In WT mice, however, HS did not elevate MBP (Δ=2.6±1.4; n =5; NS), and Ang II elevated MBP by only Δ=6.9±4.0 mm Hg; n =5; P &lt;0.05). Conclusion: ASM α2 Na + pumps help regulate basal BP and vascular reactivity. Reduced α2 expression raises basal BP and augments arterial responses to salt and Ang II; increased ASM α2 expression has the opposite effects. The α2 Na + pumps co-localize with Na/Ca exchangers in plasma membrane (PM) microdomains at MP-sarcoplasmic reticulum junctions; they are a crucial link between circulating endogenous ouabain and the control of ASM Ca 2+ homeostasis, arterial contraction and tone.

Abstract 308: Genetic Interference with Peroxisome Proliferator-Activated Receptor ? (PPAR?) in Smooth Muscle Augments Cerebrovascular Effects of Angiotensin I

Angiotensin II (Ang II) plays a major role in vascular disease, including both large and small vessel disease in brain. PPARγ is a ligand-activated nuclear receptor that exerts protective effects within the vasculature. Although studies using high affinity PPARγ agonists [eg, thiazolidinediones (TZD)] suggest interactions between PPARγ and the renin-angiotensin system (RAS) exist, the importance of these interactions in the absence of TZD treatment is less clear. We examined the hypothesis that interference with PPARγ in vascular muscle would alter vascular effects of angiotensin I (Ang I) and Ang II. Ang I has little intrinsic biological activity, being functionally important only after conversion to Ang II, a key effector of the RAS. We studied pressurized cerebral arteries (resistance vessels) in vitro from transgenic mice expressing a dominant negative mutation in human PPARγ (P467L) under the control of the smooth muscle myosin heavy chain promoter (S-P467L). The approach allows cell-specific interference with normal PPARγ-dependent effects. Ang I (0.01-100 nM) produced only modest constriction in arteries from non-transgenic mice (eg, maximum reduction in diameter of 8±1% at 100 nM). In contrast, vasoconstriction to Ang I was increased ~4-fold in S-P467L mice (32±2% at 100 nM, P&lt;0.01). Augmented vasoconstrictor responses to Ang I in S-P467L mice were abolished by losartan (1 μM, an inhibitor of AT1 receptors). Captopril [10 μM, an inhibitor of angiotensin converting enzyme (ACE) which converts Ang I to Ang II] completely blocked responses to low concentrations of Ang I, but only reduced effects of 100 nM Ang I by ~40%. In contrast to effects of Ang I, vasoconstrictor responses to Ang II (0.01-10 nM) and KCl (50 mM) were similar in non-transgenic and S-P467L mice (P&gt;0.05). Vascular effects of Ang II in S-P467L mice were also abolished by losartan. These data provide the first evidence that interference with PPARγ in vascular muscle augments vascular effects of Ang I through apparent changes in both ACE-dependent and -independent metabolism of Ang I. Potent vascular effects of the precursor peptide following impairment of normal PPARγ function may exacerbate progression of Ang II-dependent vascular disease.

Post-transcriptional regulation of myotube elongation and myogenesis by Hoi Polloi

Striated muscle development requires the coordinated expression of genes involved in sarcomere formation and contractility, as well as genes that determine muscle morphology. However, relatively little is known about the molecular mechanisms that control the early stages of muscle morphogenesis. To explore this facet of myogenesis, we performed a genetic screen for regulators of somatic muscle morphology in Drosophila, and identified the putative RNA-binding protein (RBP) Hoi Polloi (Hoip). Hoip is expressed in striated muscle precursors within the muscle lineage and controls two genetically separable events: myotube elongation and sarcomeric protein expression. Myotubes fail to elongate in hoip mutant embryos, even though the known regulators of somatic muscle elongation, target recognition and muscle attachment are expressed normally. In addition, a majority of sarcomeric proteins, including Myosin Heavy Chain (MHC) and Tropomyosin, require Hoip for their expression. A transgenic MHC construct that contains the endogenous MHC promoter and a spliced open reading frame rescues MHC protein expression in hoip embryos, demonstrating the involvement of Hoip in pre-mRNA splicing, but not in transcription, of muscle structural genes. In addition, the human Hoip ortholog NHP2L1 rescues muscle defects in hoip embryos, and knockdown of endogenous nhp2l1 in zebrafish disrupts skeletal muscle development. We conclude that Hoip is a conserved, post-transcriptional regulator of muscle morphogenesis and structural gene expression.

Hypotension Due to Kir6.1 Gain‐of‐Function in Vascular Smooth Muscle

BackgroundKATP channels, assembled from pore‐forming (Kir6.1 or Kir6.2) and regulatory (SUR1 or SUR2) subunits, link metabolism to excitability. Loss of Kir6.2 results in hypoglycemia and hyperinsulinemia, whereas loss of Kir6.1 causes Prinzmetal angina–like symptoms in mice. Conversely, overactivity of Kir6.2 induces neonatal diabetes in mice and humans, but consequences of Kir6.1 overactivity are unknown.Methods and ResultsWe generated transgenic mice expressing wild‐type (WT), ATP‐insensitive Kir6.1 [Gly343Asp] (GD), and ATP‐insensitive Kir6.1 [Gly343Asp,Gln53Arg] (GD‐QR) subunits, under Cre‐recombinase control. Expression was induced in smooth muscle cells by crossing with smooth muscle myosin heavy chain promoter–driven tamoxifen‐inducible Cre‐recombinase (SMMHC‐Cre‐ER) mice. Three weeks after tamoxifen induction, we assessed blood pressure in anesthetized and conscious animals, as well as contractility of mesenteric artery smooth muscle and KATP currents in isolated mesenteric artery myocytes. Both systolic and diastolic blood pressures were significantly reduced in GD and GD‐QR mice but normal in mice expressing the WT transgene and elevated in Kir6.1 knockout mice as well as in mice expressing dominant‐negative Kir6.1 [AAA] in smooth muscle. Contractile response of isolated GD‐QR mesenteric arteries was blunted relative to WT controls, but nitroprusside relaxation was unaffected. Basal KATP conductance and pinacidil‐activated conductance were elevated in GD but not in WT myocytes.ConclusionsKATP overactivity in vascular muscle can lead directly to reduced vascular contractility and lower blood pressure. We predict that gain of vascular KATP function in humans would lead to a chronic vasodilatory phenotype, as indeed has recently been demonstrated in Cantu syndrome.