Matriglycan maintains t-tubule structural integrity in cardiac muscle.

Maintaining the structure of cardiac membranes and membrane organelles is essential for heart function. A critical cardiac membrane organelle is the transverse tubule system (called the t-tubule system) which is an invagination of the surface membrane. A unique structural characteristic of the cardiac muscle t-tubule system is the extension of the extracellular matrix (ECM) from the surface membrane into the t-tubule lumen. However, the importance of the ECM extending into the cardiac t-tubule lumen is not well understood. Dystroglycan (DG) is an ECM receptor in the surface membrane of many cells, and it is also expressed in t-tubules in cardiac muscle. Extensive posttranslational processing and O-glycosylation are required for DG to bind ECM proteins and the binding is mediated by a glycan structure known as matriglycan. Genetic disruption resulting in defective O-glycosylation of DG results in muscular dystrophy with cardiorespiratory pathophysiology. Here, we show that DG is essential for maintaining cardiac t-tubule structural integrity. Mice with defects in O-glycosylation of DG developed normal t-tubules but were susceptible to stress-induced t-tubule loss or severing that contributed to cardiac dysfunction and disease progression. Finally, we observed similar stress-induced cardiac t-tubule disruption in a cohort of mice that solely lacked matriglycan. Collectively, our data indicate that DG in t-tubules anchors the luminal ECM to the t-tubule membrane via the polysaccharide matriglycan, which is critical to transmitting structural strength of the ECM to the t-tubules and provides resistance to mechanical stress, ultimately preventing disruptions in cardiac t-tubule integrity.

Hypersensitivity of Zebrafish htr2b Mutant Embryos to Sertraline Indicates a Role for Serotonin Signaling in Cardiac Development.

ABSTRACT: Selective serotonin reuptake inhibitors (SSRIs) are antidepressants prescribed in 10% of pregnancies in the United States. Maternal use of SSRIs has been linked to an elevated rate of congenital heart defects, but the exact mechanism of pathogenesis is unknown. Previously, we have shown a decrease in cardiomyocyte proliferation, left ventricle size, and reduced cardiac expression of the serotonin receptor 5-HT 2B in offspring of mice exposed to the SSRI sertraline during pregnancy, relative to offspring of untreated mice. These results suggest that disruption of serotonin signaling leads to heart defects. Supporting this conclusion, we show here that zebrafish embryos exposed to sertraline develop with a smaller ventricle, reduced cardiomyocyte number, and lower cardiac expression of htr2b relative to untreated embryos. Moreover, zebrafish embryos homozygous for a nonsense mutation of htr2b ( htr2bsa16649 ) were sensitized to sertraline treatment relative to wild-type embryos. Specifically, the ventricle area was reduced in the homozygous htr2b mutants treated with sertraline compared with wild-type embryos treated with sertraline and homozygous htr2b mutants treated with vehicle control. Whereas long-term effects on left ventricle shortening fraction and stroke volume were observed by echocardiography in adult mice exposed to sertraline in utero, echocardiograms of adult zebrafish exposed to sertraline as embryos were normal. These results implicate the 5-HT 2B receptor functions in heart development and suggest zebrafish are a relevant animal model that can be used to investigate the connection between maternal SSRI use and elevated risk of congenital heart defects.

Functional resilience of C57BL/6J mouse heart to dietary fat overload.

Molecular mechanisms underlying cardiac dysfunction and subsequent heart failure in diabetic cardiomyopathy are incompletely understood. Initially we intended to test the role of G protein-coupled receptor kinase 2 (GRK2), a potential mediator of cardiac dysfunction in diabetic cardiomyopathy, but found that control animals on HFD did not develop cardiomyopathy. Cardiac function was preserved in both wild-type and GRK2 knockout animals fed high-fat diet as indicated by preserved left ventricular ejection fraction (LVEF) although heart mass was increased. The absence of cardiac dysfunction led us to rigorously evaluate the utility of diet-induced obesity to model diabetic cardiomyopathy in mice. Using pure C57BL/6J animals and various diets formulated with different sources of fat-lard (32% saturated fat, 68% unsaturated fat) or hydrogenated coconut oil (95% saturated fat), we consistently observed left ventricular hypertrophy, preserved LVEF, and preserved contractility measured by invasive hemodynamics in animals fed high-fat diet. Gene expression patterns that characterize pathological hypertrophy were not induced, but a modest induction of various collagen isoforms and matrix metalloproteinases was observed in heart with high-fat diet feeding. PPARα-target genes that enhance lipid utilization such as Pdk4, CD36, AcadL, and Cpt1b were induced, but mitochondrial energetics was not impaired. These results suggest that although long-term fat feeding in mice induces cardiac hypertrophy and increases cardiac fatty acid metabolism, it may not be sufficient to activate pathological hypertrophic mechanisms that impair cardiac function or induce cardiac fibrosis. Thus, additional factors that are currently not understood may contribute to the cardiac abnormalities previously reported by many groups.NEW & NOTEWORTHY Dietary fat overload (DFO) is widely used to model diabetic cardiomyopathy but the utility of this model is controversial. We comprehensively characterized cardiac contractile and mitochondrial function in C57BL6/J mice fed with lard-based or saturated fat-enriched diets initiated at two ages. Despite cardiac hypertrophy, contractile and mitochondrial function is preserved, and molecular adaptations likely limit lipotoxicity. The resilience of these hearts to DFO underscores the need to develop robust alternative models of diabetic cardiomyopathy.

Mitochondrial pyruvate carriers are required for myocardial stress adaptation.

In addition to fatty acids, glucose and lactate are important myocardial substrates under physiologic and stress conditions. They are metabolized to pyruvate, which enters mitochondria via the mitochondrial pyruvate carrier (MPC) for citric acid cycle metabolism. In the present study, we show that MPC-mediated mitochondrial pyruvate utilization is essential for the partitioning of glucose-derived cytosolic metabolic intermediates, which modulate myocardial stress adaptation. Mice with cardiomyocyte-restricted deletion of subunit 1 of MPC (cMPC1-/-) developed age-dependent pathologic cardiac hypertrophy, transitioning to a dilated cardiomyopathy and premature death. Hypertrophied hearts accumulated lactate, pyruvate and glycogen, and displayed increased protein O-linked N-acetylglucosamine, which was prevented by increasing availability of non-glucose substrates in vivo by a ketogenic diet (KD) or a high-fat diet, which reversed the structural, metabolic and functional remodelling of non-stressed cMPC1-/- hearts. Although concurrent short-term KDs did not rescue cMPC1-/- hearts from rapid decompensation and early mortality after pressure overload, 3 weeks of a KD before transverse aortic constriction was sufficient to rescue this phenotype. Together, our results highlight the centrality of pyruvate metabolism to myocardial metabolism and function.

Arginine vasopressin infusion is sufficient to model clinical features of preeclampsia in mice.

Copeptin, a marker of arginine vasopressin (AVP) secretion, is elevated throughout human pregnancies complicated by preeclampsia (PE), and AVP infusion throughout gestation is sufficient to induce the major phenotypes of PE in mice. Thus, we hypothesized a role for AVP in the pathogenesis of PE. AVP infusion into pregnant C57BL/6J mice resulted in hypertension, renal glomerular endotheliosis, intrauterine growth restriction, decreased placental growth factor (PGF), altered placental morphology, placental oxidative stress, and placental gene expression consistent with human PE. Interestingly, these changes occurred despite a lack of placental hypoxia or elevations in placental fms-like tyrosine kinase-1 (FLT1). Coinfusion of AVP receptor antagonists and time-restricted infusion of AVP uncovered a mid-gestational role for the AVPR1A receptor in the observed renal pathologies, versus mid- and late-gestational roles for the AVPR2 receptor in the blood pressure and fetal phenotypes. These findings demonstrate that AVP is sufficient to initiate phenotypes of PE in the absence of placental hypoxia, and indicate that AVP may mechanistically (independently, and possibly synergistically with hypoxia) contribute to the development of clinical signs of PE in specific subtypes of human PE. Additionally, they identify divergent and gestational time-specific signaling mechanisms that mediate the development of PE phenotypes in response to AVP.

Discovery of an Experimental Model of Unicuspid Aortic Valve.

BACKGROUND: The epithelial growth factor receptor family of tyrosine kinases modulates embryonic formation of semilunar valves. We hypothesized that mice heterozygous for a dominant loss-of-function mutation in epithelial growth factor receptor, which are EgfrVel/+ mice, would develop anomalous aortic valves, valve dysfunction, and valvular cardiomyopathy. METHODS AND RESULTS: Aortic valves from EgfrVel/+ mice and control mice were examined by light microscopy at 2.5 to 4 months of age. Additional EgfrVel/+ and control mice underwent echocardiography at 2.5, 4.5, 8, and 12 months of age, followed by histologic examination. In young mice, microscopy revealed anatomic anomalies in 79% of EgfrVel/+ aortic valves, which resembled human unicuspid aortic valves. Anomalies were not observed in control mice. At 12 months of age, histologic architecture was grossly distorted in EgfrVel/+ aortic valves. Echocardiography detected moderate or severe aortic regurgitation, or aortic stenosis was present in 38% of EgfrVel/+ mice at 2.5 months of age (N=24) and in 74% by 8 months of age. Left ventricular enlargement, hypertrophy, and reversion to a fetal myocardial gene expression program occurred in EgfrVel/+ mice with aortic valve dysfunction, but not in EgfrVel/+ mice with near-normal aortic valve function. Myocardial fibrosis was minimal or absent in all groups. CONCLUSIONS: A new mouse model uniquely recapitulates salient functional, structural, and histologic features of human unicuspid aortic valve disease, which are phenotypically distinct from other forms of congenital aortic valve disease. The new model may be useful for elucidating mechanisms by which congenitally anomalous aortic valves become critically dysfunctional.

Lipopolysaccharide-induced maternal inflammation induces direct placental injury without alteration in placental blood flow and induces a secondary fetal intestinal injury that persists into adulthood.

PROBLEM: Premature birth complicates 10%-12% of deliveries. Infection and inflammation are the most common etiologies and are associated with increased offspring morbidity and mortality. We hypothesize that lipopolysaccharide (LPS)-induced maternal inflammation causes direct placenta injury and subsequent injury to the fetal intestine. METHOD OF STUDY: Pregnant C57Bl6 mice were injected intraperitoneally on day 15.5 with 100 µg/kg LPS or saline. Maternal serum, amniotic fluid, placental samples, and ileal samples of offspring were obtained assessed for inflammation and/or injury. Maternal placental ultrasounds were performed. Placental DNA was isolated for microbiome analysis. RESULTS: Maternal injection with LPS caused elevated IL-1ß, IL-10, IL-6, KC-GRO, and TNF. Placental tissue showed increased IL-1ß, IL-6, and KC-GRO and decreased IL-10, but no changes were observed in amniotic fluid. Placental histology demonstrated LPS-induced increases in mineralization and necrosis, but no difference in placental blood flow. Most placentas had no detectable microbiome. Exposure to maternal LPS induced significant injury to the ilea of the offspring. CONCLUSION: Lipopolysaccharide causes a maternal inflammatory response that is mirrored in the placenta. Placental histology demonstrates structural changes; however, placental blood flow is preserved. LPS also induces an indirect intestinal injury in the offspring that lasts beyond the neonatal period.

MG53 is dispensable for T-tubule maturation but critical for maintaining T-tubule integrity following cardiac stress.

The cardiac transverse (T)-tubule membrane system is the safeguard for cardiac function and undergoes dramatic remodeling in response to cardiac stress. However, the mechanism by which cardiomyocytes repair damaged T-tubule network remains unclear. In the present study, we tested the hypothesis that MG53, a muscle-specific membrane repair protein, antagonizes T-tubule damage to protect against maladaptive remodeling and thereby loss of excitation-contraction coupling and cardiac function. Using MG53-knockout (MG53-KO) mice, we first established that deficiency of MG53 had no impact on maturation of the T-tubule network in developing hearts. Additionally, MG53 ablation did not influence T-tubule integrity in unstressed adult hearts as late as 10months of age. Following left ventricular pressure overload-induced cardiac stress, MG53 protein levels were increased by approximately three-fold in wild-type mice, indicating that pathological stress induces a significant upregulation of MG53. MG53-deficient mice had worsened T-tubule disruption and pronounced dysregulation of Ca2+ handling properties, including decreased Ca2+ transient amplitude and prolonged time to peak and decay. Moreover, MG53 deficiency exacerbated cardiac hypertrophy and dysfunction and decreased survival following cardiac stress. Our data suggest MG53 is not required for T-tubule development and maintenance in normal physiology. However, MG53 is essential to preserve T-tubule integrity and thereby Ca2+ handling properties and cardiac function under pathological cardiac stress.

Fibrotic Aortic Valve Stenosis in Hypercholesterolemic/Hypertensive Mice.

OBJECTIVE: Hypercholesterolemia and hypertension are associated with aortic valve stenosis (AVS) in humans. We have examined aortic valve function, structure, and gene expression in hypercholesterolemic/hypertensive mice. APPROACH AND RESULTS: Control, hypertensive, hypercholesterolemic (Apoe(-/-)), and hypercholesterolemic/hypertensive mice were studied. Severe aortic stenosis (echocardiography) occurred only in hypercholesterolemic/hypertensive mice. There was minimal calcification of the aortic valve. Several structural changes were identified at the base of the valve. The intercusp raphe (or seam between leaflets) was longer in hypercholesterolemic/hypertensive mice than in other mice, and collagen fibers at the base of the leaflets were reoriented to form a mesh. In hypercholesterolemic/hypertensive mice, the cusps were asymmetrical, which may contribute to changes that produce AVS. RNA sequencing was used to identify molecular targets during the developmental phase of stenosis. Genes related to the structure of the valve were identified, which differentially expressed before fibrotic AVS developed. Both RNA and protein of a profibrotic molecule, plasminogen activator inhibitor 1, were increased greatly in hypercholesterolemic/hypertensive mice. CONCLUSIONS: Hypercholesterolemic/hypertensive mice are the first model of fibrotic AVS. Hypercholesterolemic/hypertensive mice develop severe AVS in the absence of significant calcification, a feature that resembles AVS in children and some adults. Structural changes at the base of the valve leaflets include lengthening of the raphe, remodeling of collagen, and asymmetry of the leaflets. Genes were identified that may contribute to the development of fibrotic AVS.

Spontaneous Aortic Regurgitation and Valvular Cardiomyopathy in Mice.

OBJECTIVE: We studied the mechanistic links between fibrocalcific changes in the aortic valve and aortic valve function in mice homozygous for a hypomorphic epidermal growth factor receptor mutation (Wave mice). We also studied myocardial responses to aortic valve dysfunction in Wave mice. APPROACH AND RESULTS: At 1.5 months of age, before development of valve fibrosis and calcification, aortic regurgitation, but not aortic stenosis, was common in Wave mice. Aortic valve fibrosis, profibrotic signaling, calcification, osteogenic markers, lipid deposition, and apoptosis increased dramatically by 6 and 12 months of age in Wave mice. Aortic regurgitation remained prevalent, however, and aortic stenosis was rare, at all ages. Proteoglycan content was abnormally increased in aortic valves of Wave mice at all ages. Treatment with pioglitazone prevented abnormal valve calcification, but did not protect valve function. There was significant left ventricular volume overload, hypertrophy, and fetal gene expression, at all ages in Wave mice with aortic regurgitation. Left ventricular systolic function was normal until 6 months of age in Wave mice, but became impaired by 12 months of age. Myocardial transverse tubules were normal in the presence of left ventricular hypertrophy at 1.5 and 3 months of age, but became disrupted by 12 months of age. CONCLUSIONS: We present the first comprehensive phenotypic and molecular characterization of spontaneous aortic regurgitation and volume-overload cardiomyopathy in an experimental model. In Wave mice, fibrocalcific changes are not linked to valve dysfunction and are epiphenomena arising from structurally incompetent myxomatous valves.

Angiotensin-dependent autonomic dysregulation precedes dilated cardiomyopathy in a mouse model of muscular dystrophy.

NEW FINDINGS: What is the central question of this study? Is autonomic dysregulation in a mouse model of muscular dystrophy dependent on left ventricular systolic dysfunction and/or activation of the renin-angiotensin system (RAS) and does it predict development of dilated cardiomyopathy (DCM)? What is the main finding and its importance? The results demonstrate that autonomic dysregulation precedes and predicts left ventricular dysfunction and DCM in sarcoglycan-δ-deficient (Sgcd-/-) mice. The autonomic dysregulation is prevented by treatment of young Sgcd-/- mice with the angiotensin II type 1 receptor blocker losartan. Measurements of RAS activation and autonomic dysregulation may predict risk of DCM, and therapies targeting the RAS and autonomic dysregulation at a young age may slow disease progression in patients. Sarcoglycan mutations cause muscular dystrophy. Patients with muscular dystrophy develop autonomic dysregulation and dilated cardiomyopathy (DCM), but the temporal relationship and mechanism of autonomic dysregulation are not well understood. We hypothesized that activation of the renin-angiotensin system (RAS) causes autonomic dysregulation prior to development of DCM in sarcoglycan-δ-deficient (Sgcd-/-) mice and that the severity of autonomic dysfunction at a young age predicts the severity of DCM at older ages. At 10-12 weeks of age, when left ventricular function assessed by echocardiography remained normal, Sgcd-/- mice exhibited decreases in arterial pressure, locomotor activity, baroreflex sensitivity and cardiovagal tone and increased sympathetic tone compared with age-matched C57BL/6 control mice (P < 0.05). Systemic and skeletal muscle RAS were activated, and angiotensin II type 1 receptor (AT1 R) expression, superoxide and fibrosis were increased in dystrophic skeletal muscle (P < 0.05). Treatment with the AT1 R blocker losartan for 7-9 weeks beginning at 3 weeks of age prevented or strongly attenuated the abnormalities in Sgcd-/- mice (P < 0.05). Repeated assessment of phenotypes between 10 and 75 weeks of age demonstrated worsening of autonomic function, progressive cardiac dysfunction and DCM and increased mortality in Sgcd-/- mice. High sympathetic tone predicted subsequent left ventricular dysfunction. We conclude that activation of the RAS causes severe autonomic dysregulation in young Sgcd-/- mice, which portends a worse long-term prognosis. Therapeutic targeting of the RAS at a young age may improve autonomic function and slow disease progression in muscular dystrophy.

Aortic valve sclerosis in mice deficient in endothelial nitric oxide synthase.

Risk factors for fibrocalcific aortic valve disease (FCAVD) are associated with systemic decreases in bioavailability of endothelium-derived nitric oxide (EDNO). In patients with bicuspid aortic valve (BAV), vascular expression of endothelial nitric oxide synthase (eNOS) is decreased, and eNOS(-/-) mice have increased prevalence of BAV. The goal of this study was to test the hypotheses that EDNO attenuates profibrotic actions of valve interstitial cells (VICs) in vitro and that EDNO deficiency accelerates development of FCAVD in vivo. As a result of the study, coculture of VICs with aortic valve endothelial cells (vlvECs) significantly decreased VIC activation, a critical early phase of FCAVD. Inhibition of VIC activation by vlvECs was attenuated by N(G)-nitro-l-arginine methyl ester or indomethacin. Coculture with vlvECs attenuated VIC expression of matrix metalloproteinase-9, which depended on stiffness of the culture matrix. Coculture with vlvECs preferentially inhibited collagen-3, compared with collagen-1, gene expression. BAV occurred in 30% of eNOS(-/-) mice. At age 6 mo, collagen was increased in both bicuspid and trileaflet eNOS(-/-) aortic valves, compared with wild-type valves. At 18 mo, total collagen was similar in eNOS(-/-) and wild-type mice, but collagen-3 was preferentially increased in eNOS(-/-) mice. Calcification and apoptosis were significantly increased in BAV of eNOS(-/-) mice at ages 6 and 18 mo. Remarkably, these histological changes were not accompanied by physiologically significant valve stenosis or regurgitation. In conclusion, coculture with vlvECs inhibits specific profibrotic VIC processes. In vivo, eNOS deficiency produces fibrosis in both trileaflet and BAVs but produces calcification only in BAVs.

Microtubule-mediated defects in junctophilin-2 trafficking contribute to myocyte transverse-tubule remodeling and Ca2+ handling dysfunction in heart failure.

BACKGROUND: Cardiac dysfunction in failing hearts of human patients and animal models is associated with both microtubule densification and transverse-tubule (T-tubule) remodeling. Our objective was to investigate whether microtubule densification contributes to T-tubule remodeling and excitation-contraction coupling dysfunction in heart disease. METHODS AND RESULTS: In a mouse model of pressure overload-induced cardiomyopathy by transaortic banding, colchicine, a microtubule depolymerizer, significantly ameliorated T-tubule remodeling and cardiac dysfunction. In cultured cardiomyocytes, microtubule depolymerization with nocodazole or colchicine profoundly attenuated T-tubule impairment, whereas microtubule polymerization/stabilization with taxol accelerated T-tubule remodeling. In situ immunofluorescence of heart tissue sections demonstrated significant disorganization of junctophilin-2 (JP2), a protein that bridges the T-tubule and sarcoplasmic reticulum membranes, in transaortic banded hearts as well as in human failing hearts, whereas colchicine injection significantly preserved the distribution of JP2 in transaortic banded hearts. In isolated mouse cardiomyocytes, prolonged culture or treatment with taxol resulted in pronounced redistribution of JP2 from T-tubules to the peripheral plasma membrane, without changing total JP2 expression. Nocodazole treatment antagonized JP2 redistribution. Moreover, overexpression of a dominant-negative mutant of kinesin 1, a microtubule motor protein responsible for anterograde trafficking of proteins, protected against JP2 redistribution and T-tubule remodeling in culture. Finally, nocodazole treatment improved Ca(2+) handling in cultured myocytes by increasing the amplitude of Ca(2+) transients and reducing the frequency of Ca(2+) sparks. CONCLUSION: Our data identify a mechanistic link between microtubule densification and T-tubule remodeling and reveal microtubule-mediated JP2 redistribution as a novel mechanism for T-tubule disruption, loss of excitation-contraction coupling, and heart failure.

Nox1 NADPH oxidase is necessary for late but not early myocardial ischaemic preconditioning.

AIMS: Ischaemic preconditioning (IPC) is an adaptive mechanism that renders the myocardium resistant to injury from subsequent hypoxia. Although reactive oxygen species (ROS) contribute to both the early and late phases of IPC, their enzymatic source and associated signalling events have not yet been understood completely. Our objective was to investigate the role of the Nox1 NADPH oxidase in cardioprotection provided by IPC. METHODS AND RESULTS: Wild-type (WT) and Nox1-deficient mice were treated with three cycles of brief coronary occlusion and reperfusion, followed by prolonged occlusion either immediately (early IPC) or after 24 h (late IPC). Nox1 deficiency had no impact on the cardioprotection afforded by early IPC. In contrast, deficiency of Nox1 during late IPC resulted in a larger infarct size, cardiac remodelling, and increased myocardial apoptosis compared with WT hearts. Furthermore, expression of Nox1 in WT hearts increased in response to late IPC. Deficiency of Nox1 abrogated late IPC-mediated activation of cardiac nuclear factor-κB (NF-κB) and induction of tumour necrosis factor-α (TNF-α) in the heart and circulation. Finally, knockdown of Nox1 in cultured cardiomyocytes prevented TNF-α induction of NF-κB and the protective effect of IPC on hypoxia-induced apoptosis. CONCLUSIONS: Our data identify a critical role for Nox1 in late IPC and define a previously unrecognized link between TNF-α and NF-κB in mediating tolerance to myocardial injury. These findings have clinical significance considering the emergence of Nox1 inhibitors for the treatment of cardiovascular disease.

Critical roles of junctophilin-2 in T-tubule and excitation-contraction coupling maturation during postnatal development.

AIMS: Emerging evidence indicates a critical role for junctophilin-2 (JP2) in T-tubule integrity and assembly of cardiac dyads in adult ventricular myocytes. In the postnatal stage, one of the critical features of myocyte maturation is development of the T-tubule system, though the mechanisms remain poorly understood. In this study, we aim to determine whether JP2 is required for normal cardiac T-tubule maturation. METHODS AND RESULTS: Using in situ confocal imaging of intact murine hearts, we found T-tubules were absent in both left- and right-ventricular myocytes at postnatal Day 8 and did not appear until Day 10. Quantification of T-tubule structural integrity using the T-tubule power (TT(power)) index revealed a progressive increase in TT(power) between postnatal Days 10 and 19. By postnatal Day 19, TT(power) was similar to that in adult murine cardiomyocytes, indicative of a nearly matured T-tubule network. JP2 levels increased dramatically during development, reaching levels observed in adult hearts by postnatal Day 14. Deficiency of JP2, using a mouse model in which a JP2-specific shRNA is expressed during embryonic development, severely impaired T-tubule maturation, with equivalent decreases in the left- and right-ventricular TT(power). We also detected a gradual increase in the density of transverse but not longitudinal tubules during development, and JP2 deficiency abolished the increase in the density of transverse elements. Alterations in T-tubules caused significant reduction in Ca(2+) transient amplitude and marked increase in Ca(2+) release dyssynchrony, Ca(2+) alternans, and spontaneous Ca(2+) waves, leading to contractile failure. CONCLUSION: Our data identify a critical role for JP2 in T-tubule and excitation-contraction coupling maturation during development.

Osteoprotegerin inhibits aortic valve calcification and preserves valve function in hypercholesterolemic mice.

BACKGROUND: There are no rigorously confirmed effective medical therapies for calcific aortic stenosis. Hypercholesterolemic Ldlr (-/-) Apob (100/100) mice develop calcific aortic stenosis and valvular cardiomyopathy in old age. Osteoprotegerin (OPG) modulates calcification in bone and blood vessels, but its effect on valve calcification and valve function is not known. OBJECTIVES: To determine the impact of pharmacologic treatment with OPG upon aortic valve calcification and valve function in aortic stenosis-prone hypercholesterolemic Ldlr (-/-) Apob (100/100) mice. METHODS: Young Ldlr (-/-) Apob (100/100) mice (age 2 months) were fed a Western diet and received exogenous OPG or vehicle (N = 12 each) 3 times per week, until age 8 months. After echocardiographic evaluation of valve function, the aortic valve was evaluated histologically. Older Ldlr (-/-) Apob (100/100) mice were fed a Western diet beginning at age 2 months. OPG or vehicle (N = 12 each) was administered from 6 to 12 months of age, followed by echocardiographic evaluation of valve function, followed by histologic evaluation. RESULTS: In Young Ldlr (-/-) Apob (100/100) mice, OPG significantly attenuated osteogenic transformation in the aortic valve, but did not affect lipid accumulation. In Older Ldlr (-/-) Apob (100/100) mice, OPG attenuated accumulation of the osteoblast-specific matrix protein osteocalcin by ∼80%, and attenuated aortic valve calcification by ∼ 70%. OPG also attenuated impairment of aortic valve function. CONCLUSIONS: OPG attenuates pro-calcific processes in the aortic valve, and protects against impairment of aortic valve function in hypercholesterolemic aortic stenosis-prone Ldlr (-/-) Apob (100/100) mice.

Genetic inhibition of Na+-Ca2+ exchanger current disables fight or flight sinoatrial node activity without affecting resting heart rate.

RATIONALE: The sodium-calcium exchanger 1 (NCX1) is predominantly expressed in the heart and is implicated in controlling automaticity in isolated sinoatrial node (SAN) pacemaker cells, but the potential role of NCX1 in determining heart rate in vivo is unknown. OBJECTIVE: To determine the role of Ncx1 in heart rate. METHODS AND RESULTS: We used global myocardial and SAN-targeted conditional Ncx1 knockout (Ncx1(-/-)) mice to measure the effect of the NCX current on pacemaking activity in vivo, ex vivo, and in isolated SAN cells. We induced conditional Ncx1(-/-) using a Cre/loxP system. Unexpectedly, in vivo and ex vivo hearts and isolated SAN cells showed that basal rates in Ncx1(-/-) (retaining ≈20% of control level NCX current) and control mice were similar, suggesting that physiological NCX1 expression is not required for determining resting heart rate. However, increases in heart rate and SAN cell automaticity in response to isoproterenol or the dihydropyridine Ca(2+) channel agonist BayK8644 were significantly blunted or eliminated in Ncx1(-/-) mice, indicating that NCX1 is important for fight or flight heart rate responses. In contrast, the pacemaker current and L-type Ca(2+) currents were equivalent in control and Ncx1(-/-) SAN cells under resting and isoproterenol-stimulated conditions. Ivabradine, a pacemaker current antagonist with clinical efficacy, reduced basal SAN cell automaticity similarly in control and Ncx1(-/-) mice. However, ivabradine decreased automaticity in SAN cells isolated from Ncx1(-/-) mice more effectively than in control SAN cells after isoproterenol, suggesting that the importance of NCX current in fight or flight rate increases is enhanced after pacemaker current inhibition. CONCLUSIONS: Physiological Ncx1 expression is required for increasing sinus rates in vivo, ex vivo, and in isolated SAN cells, but not for maintaining resting heart rate.

Overexpression of the SK3 channel alters vascular remodeling during pregnancy, leading to fetal demise.

The maternal cardiovascular system undergoes hemodynamic changes during pregnancy via angiogenesis and vasodilation to ensure adequate perfusion of the placenta. Improper vascularization at the maternal-fetal interface can cause pregnancy complications and poor fetal outcomes. Recent evidence indicates that small conductance Ca(2+)-activated K(+) channel subtype 3 (SK3) contributes to vascular remodeling during pregnancy, and we hypothesized that abnormal SK3 channel expression would alter the ability of the maternal cardiovascular system to adapt to pregnancy demands and lead to poor fetal outcomes. We investigated this hypothesis using transgenic Kcnn3(tm1Jpad)/Kcnn3(tm1Jpad) (SK3(T/T)) mice that overexpress the channel. Isolated pressurized uterine arteries from nonpregnant transgenic SK3(T/T) mice had larger basal diameters and decreased agonist-induced constriction than those from their wild-type counterparts; however, non-receptor-mediated depolarization remained intact. In addition to vascular changes, heart rates and ejection fraction were increased, whereas end systolic volume was reduced in SK3(T/T) mice compared with their wild-type littermates. Uterine sonography of the fetuses on pregnancy day 14 showed a significant decrease in fetal size in SK3(T/T) compared with wild-type mice; thus, SK3(T/T) mice displayed an intrauterine growth-restricted phenotype. The SK3(T/T) mice showed decreased placental thicknesses and higher incidence of fetal loss, losing over half of their complement of pups by midgestation. These results establish that the SK3 channel contributes to both maternal and fetal outcomes during pregnancy and point to the importance of SK3 channel regulation in maintaining a healthy pregnancy.

ß-Adrenergic receptor antagonists ameliorate myocyte T-tubule remodeling following myocardial infarction.

ß-Adrenergic receptor (AR) blockers provide substantial clinical benefits, including improving overall survival and left ventricular (LV) function following myocardial infarction (MI), though the mechanisms remain incompletely defined. The transverse-tubule (T-tubule) system of ventricular myocytes is an important determinant of cardiac excitation-contraction function. T-tubule remodeling occurs early during LV failure. We hypothesized that ß-AR blockers prevent T-tubule remodeling and thereby provide therapeutic benefits. A murine model of MI was utilized to examine the effect of ß-AR blockers on T-tubule remodeling following LV MI. We applied the in situ imaging of T-tubule structure from Langendorff-perfused intact hearts with laser scanning confocal microscopy. We found that MI caused remarkable T-tubule remodeling near the infarction border zone and moderate LV remodeling remote from the MI. Metoprolol and carvedilol administered 6 d after MI for 4 wk each increased the T-tubule integrity at the remote and border zones. At the molecular level, both ß-AR blockers restored border and remote zone expression of junctophilin-2 (JP-2), which is involved in T-tubule organization and formation of the T-tubule/sarcoplasmic reticulum junctions. In contrast, ß-AR blockers had no significant effects on caveolin-3 expression. In summary, our data show that ß-AR antagonists can protect against T-tubule remodeling after MI, suggesting a novel therapeutic mechanism of action for this drug class. Preservation of JP-2 expression may contribute to the beneficial effects of metoprolol and carvedilol on T-tubule remodeling.