VSMCs respond to changes in the local environment by adjusting their phenotype from contractile to synthetic, a phenomenon known as phenotypic modulation or switching. Failure of VSMCs to acquire and maintain the contractile phenotype plays a key role in a number of major human diseases, including arteriosclerosis. Although several regulatory circuits that control differentiation of SMCs have been identified, the decisive mechanisms that govern phenotypic modulation remain unknown. Here, we demonstrate that the mouse miR-143/145 cluster, expression of which is confined to SMCs during development, is required for VSMC acquisition of the contractile phenotype. VSMCs from miR-143/145-deficient mice were locked in the synthetic state, which incapacitated their contractile abilities and favored neointimal lesion development. Unbiased high-throughput, quantitative, mass spectrometry-based proteomics using reference mice labeled with stable isotopes allowed identification of miR-143/145 targets; these included angiotensin-converting enzyme (ACE), which might affect both the synthetic phenotype and contractile functions of VSMCs. Pharmacological inhibition of either ACE or the AT 1 receptor partially reversed vascular dysfunction and normalized gene expression in miR-143/145-deficient mice. We conclude that manipulation of miR-143/145 expression may offer a new approach for influencing vascular repair and attenuating arteriosclerotic pathogenesis.

Apelin constitutes a novel endogenous peptide system suggested to be involved in a broad range of physiological functions, including cardiovascular function, heart development, control of fluid homeostasis, and obesity. Apelin is also a catalytic substrate for angiotensin-converting enzyme 2, the key severe acute respiratory syndrome receptor. The in vivo physiological role of Apelin is still elusive. Here we report the generation of Apelin gene-targeted mice. Apelin mutant mice are viable and fertile, appear healthy, and exhibit normal body weight, water and food intake, heart rates, and heart morphology. Intriguingly, aged Apelin knockout mice developed progressive impairment of cardiac contractility associated with systolic dysfunction in the absence of histological abnormalities. We also report that pressure overload induces upregulation of Apelin expression in the heart. Importantly, in pressure overload-induced heart failure, loss of Apelin did not significantly affect the hypertrophy response, but Apelin mutant mice developed progressive heart failure. Global gene expression arrays and hierarchical clustering of differentially expressed genes in hearts of banded Apelin(-/y) and Apelin(+/y) mice showed concerted upregulation of genes involved in extracellular matrix remodeling and muscle contraction. These genetic data show that the endogenous peptide Apelin is crucial to maintain cardiac contractility in pressure overload and aging.

Nemaline myopathy (NM) is a congenital myopathy that can result in lethal muscle dysfunction and is thought to be a disease of the sarcomere thin filament. Recently, several proteins of unknown function have been implicated in NM, but the mechanistic basis of their contribution to disease remains unresolved. Here, we demonstrated that loss of a muscle-specific protein, kelch-like family member 40 (KLHL40), results in a nemaline-like myopathy in mice that closely phenocopies muscle abnormalities observed in KLHL40-deficient patients. We determined that KLHL40 localizes to the sarcomere I band and A band and binds to nebulin (NEB), a protein frequently implicated in NM, as well as a putative thin filament protein, leiomodin 3 (LMOD3). KLHL40 belongs to the BTB-BACK-kelch (BBK) family of proteins, some of which have been shown to promote degradation of their substrates. In contrast, we found that KLHL40 promotes stability of NEB and LMOD3 and blocks LMOD3 ubiquitination. Accordingly, NEB and LMOD3 were reduced in skeletal muscle of both Klhl40 -/-mice and KLHL40-deficient patients. Loss of sarcomere thin filament proteins is a frequent cause of NM; therefore, our data that KLHL40 stabilizes NEB and LMOD3 provide a potential basis for the development of NM in KLHL40-deficient patients.

We report that mice lacking the heterogeneous nuclear ribonucleoprotein U (hnRNP U) in the heart develop lethal dilated cardiomyopathy and display numerous defects in cardiac pre-mRNA splicing. Mutant hearts have disorganized cardiomyocytes, impaired contractility, and abnormal excitation-contraction coupling activities. RNA-seq analyses of Hnrnpu mutant hearts revealed extensive defects in alternative splicing of pre-mRNAs encoding proteins known to be critical for normal heart development and function, including Titin and calcium/calmodulin-dependent protein kinase II delta (Camk2d). Loss of hnRNP U expression in cardiomyocytes also leads to aberrant splicing of the pre-mRNA encoding the excitation-contraction coupling component Junctin. We found that the protein product of an alternatively spliced Junctin isoform is N-glycosylated at a specific asparagine site that is required for interactions with specific protein partners. Our findings provide conclusive evidence for the essential role of hnRNP U in heart development and function and in the regulation of alternative splicing.T he expression of more than 95% of human genes is affected by alternative pre-mRNA splicing (AS) (1, 2). Differentially spliced isoforms play distinct roles in a temporally and spatially specific manner (3), and mutations that lead to aberrant splicing are the cause of many human genetic diseases (4). RNA-binding proteins (RBPs) play a central role in the regulation of alternative splicing during development and disease. They function primarily by positively or negatively regulating splice-site recognition by the spliceosome (1). Many RBPs are expressed in specific tissues, and AS is regulated by the combinatorial activities of these factors on specific pre-mRNAs through their interactions with distinct regulatory sequences in premRNA that function as splicing enhancers or silencers (5).The developing heart is one of the best studied systems where splicing changes occur during normal development, and mutations affecting specific splicing outcomes contribute to cardiomyopathy (6, 7). Although these mutations can either disrupt splicing elements or affect the expression of specific splicing factors, the latter mechanism is clearly responsible for the distinct splicing profiles at different developmental stages. For example, the dynamics of alternative splicing during postnatal heart development correlate with expression changes of many RBPs, including CUG-BP, Elavlike family member 1 (CELF1), Muscleblind-like 1 (MBNL1), and FOX proteins (8). Detailed biochemical studies have elucidated the mechanisms by which these splicing factors regulate splicing in a position-and context-dependent manner (9, 10). The function of other RBPs during heart development has also been studied. For example, two of the muscle-specific splicing factors, RBM20 and RBM24, play distinct roles in splicing regulation. RBM20 mainly acts as a splicing repressor, as its absence leads to multiple exon inclusion events in the heart. For example, the Titin gene is one of...

␣ 2 -Adrenoceptors mediate diverse functions of the sympathetic system and are targets for the treatment of cardiovascular disease, depression, pain, glaucoma, and sympathetic activation during opioid withdrawal. To determine whether ␣ 2 -adrenoceptors on adrenergic neurons or ␣ 2 -adrenoceptors on nonadrenergic neurons mediate the physiological and pharmacological responses of ␣ 2 -agonists, we used the dopamine ␤-hydroxylase (Dbh) promoter to drive expression of ␣ 2A -adrenoceptors exclusively in noradrenergic and adrenergic cells of transgenic mice. Dbh-␣ 2A transgenic mice were crossed with double knockout mice lacking both ␣ 2A -and ␣ 2C -receptors to generate lines with selective expression of ␣ 2A -autoreceptors in adrenergic cells. These mice were subjected to a comprehensive phenotype analysis and compared with wild-type mice, which express ␣ 2A -and ␣ 2C -receptors in both adrenergic and nonadrenergic cells, and ␣ 2A /␣ 2C double-knockout mice, which do not express these receptors in any cell type. We were surprised to find that only a few functions previously ascribed to ␣ 2 -adrenoceptors were mediated by receptors on adrenergic neurons, including feedback inhibition of norepinephrine release from sympathetic nerves and spontaneous locomotor activity. Other agonist effects, including analgesia, hypothermia, sedation, and anesthetic-sparing, were mediated by ␣ 2 -receptors in nonadrenergic cells. In dopamine ␤-hydroxylase knockout mice lacking norepinephrine, the ␣ 2 -agonist medetomidine still induced a loss of the righting reflex, confirming that the sedative effect of ␣ 2 -adrenoceptor stimulation is not mediated via autoreceptor-mediated inhibition of norepinephrine release. The present study paves the way for a revision of the current view of the ␣ 2 -adrenergic receptors, and it provides important new considerations for future drug development.Adrenergic receptors are important targets for the treatment of human diseases and conditions including hypertension and heart failure, psychiatric and neurological diseases, asthma, and pain (Westfall and Westfall, 2006). To date, nine different adrenergic receptor subtypes have been cloned and grouped into three receptor groups, including ␣ 1A,B,D , ␣ 2A,B,C , and ␤ 1,2,3 (Bylund et al., 1994). However, the therapeutic potential of these subtypes has not been fully explored because of the lack of ligands with sufficient subtype-selectivity. At present, only four of the nine possible subtype distinctions (i.e., ␣ 1 , ␣ 2 , ␤ 1 , and ␤ 2 ) have achieved clinical relevance (Westfall and Westfall, 2006). Especially within the ␣ 1 -and ␣ 2 -receptor subgroups, the physiological significance of individual receptor subtypes has remained unclear until recently. For the ␣ 2 -adrenoceptors, mouse models with targeted deletions of the individual subtypes have greatly advanced our understanding of the physiological role and the therapeutic potential of these receptors (Gilsbach and Hein, 2008). Activation of ␣ 2A -receptors could be linked with bradycardia ...

No abstract

Hypertension and its complications represent leading causes of morbidity and mortality. Although the cause of hypertension is unknown in most patients, genetic factors are recognized as contributing significantly to an individual's lifetime risk of developing the condition. Here, we investigated the role of the G protein regulator phosducin (Pdc) in hypertension. Mice with a targeted deletion of the gene encoding Pdc (Pdc -/-mice) had increased blood pressure despite normal cardiac function and vascular reactivity, and displayed elevated catecholamine turnover in the peripheral sympathetic system. Isolated postganglionic sympathetic neurons from Pdc -/-mice showed prolonged action potential firing after stimulation with acetylcholine and increased firing frequencies during membrane depolarization. Furthermore, Pdc -/-mice displayed exaggerated increases in blood pressure in response to post-operative stress. Candidate gene-based association studies in 2 different human populations revealed several SNPs in the PDC gene to be associated with stress-dependent blood pressure phenotypes. Individuals homozygous for the G allele of an intronic PDC SNP (rs12402521) had 12-15 mmHg higher blood pressure than those carrying the A allele. These findings demonstrate that PDC is an important modulator of sympathetic activity and blood pressure and may thus represent a promising target for treatment of stress-dependent hypertension.

Adrenal alpha(2)-mediated feedback regulation of epinephrine secretion differs fundamentally from sympathetic feedback control. A single adrenoceptor subtype, alpha(2C), operates without a significant receptor reserve to prevent elevation of circulating epinephrine levels. This genetic model may provide an experimental basis to study the pathophysiology of alpha(2C)-adrenoceptor dysfunction in humans.