Platelet TGF-ß1 contributions to plasma TGF-ß1, cardiac fibrosis, and systolic dysfunction in a mouse model of pressure overload.

Circulating platelets contain high concentrations of TGF-ß1 in their α-granules and release it on platelet adhesion/activation. We hypothesized that uncontrolled in vitro release of platelet TGF-ß1 may confound measurement of plasma TGF-ß1 in mice and that in vivo release and activation may contribute to cardiac pathology in response to constriction of the transverse aorta, which produces both high shear and cardiac pressure overload. Plasma TGF-ß1 levels in blood collected from C57Bl/6 mice by the standard retro-bulbar technique were much higher than those obtained when prostaglandin E1 was added to inhibit release or when blood was collected percutaneously from the left ventricle under ultrasound guidance. Even with optimal blood drawing, plasma TGF-ß1 was lower in mice rendered profoundly thrombocytopenic or mice with selectively low levels of platelet TGF-ß1 because of megakaryocyte-specific disruption of their TGF-ß1 gene (Tgfb1(flox)). Tgfb1(flox) mice were also partially protected from developing cardiac hypertrophy, fibrosis, and systolic dysfunction in response to transverse aortic constriction. These studies demonstrate that plasma TGF-ß1 levels can be assessed accurately, but it requires special precautions; that platelet TGF-ß1 contributes to plasma levels of TGF-ß1; and that platelet TGF-ß1 contributes to the pathologic cardiac changes that occur in response to aortic constriction.

Phosphatase-resistant gap junctions inhibit pathological remodeling and prevent arrhythmias.

RATIONALE: Posttranslational phosphorylation of connexin43 (Cx43) has been proposed as a key regulatory event in normal cardiac gap junction expression and pathological gap junction remodeling. Nonetheless, the role of Cx43 phosphorylation in the context of the intact organism is poorly understood. OBJECTIVE: To establish whether specific Cx43 phosphorylation events influence gap junction expression and pathological remodeling. METHODS AND RESULTS: We generated Cx43 germline knock-in mice in which serines 325/328/330 were replaced with phosphomimetic glutamic acids (S3E) or nonphosphorylatable alanines (S3A). The S3E mice were resistant to acute and chronic pathological gap junction remodeling and displayed diminished susceptibility to the induction of ventricular arrhythmias. Conversely, the S3A mice showed deleterious effects on cardiac gap junction formation and function, developed electric remodeling, and were highly susceptible to inducible arrhythmias. CONCLUSIONS: These data demonstrate a mechanistic link between posttranslational phosphorylation of Cx43 and gap junction formation, remodeling, and arrhythmic susceptibility.

The sarcoplasmic reticulum luminal thiol oxidase ERO1 regulates cardiomyocyte excitation-coupled calcium release and response to hemodynamic load.

Two related ER oxidation 1 (ERO1) proteins, ERO1α and ERO1ß, dynamically regulate the redox environment in the mammalian endoplasmic reticulum (ER). Redox changes in cysteine residues on intralumenal loops of calcium release and reuptake channels have been implicated in altered calcium release and reuptake. These findings led us to hypothesize that altered ERO1 activity may affect cardiac functions that are dependent on intracellular calcium flux. We established mouse lines with loss of function insertion mutations in Ero1l and Ero1lb encoding ERO1α and ERO1ß. The peak amplitude of calcium transients in homozygous Ero1α mutant adult cardiomyocytes was reduced to 42.0 ± 2.2% (n=10, P ≤ 0.01) of values recorded in wild-type cardiomyocytes. Decreased ERO1 activity blunted cardiomyocyte inotropic response to adrenergic stimulation and sensitized mice to adrenergic blockade. Whereas all 12 wild-type mice survived challenge with 4 mg/kg esmolol, 6 of 8 compound Ero1l and Ero1lb mutant mice succumbed to this level of ß adrenergic blockade (P ≤ 0.01). In addition, mice lacking ERO1α were partially protected against progressive heart failure in a transaortic constriction model [at 10 wk postprocedure, fractional shortening was 0.31 ± 0.02 in the mutant (n=20) vs. 0.23 ± 0.03 in the wild type (n=18); P ≤ 0.01]. These findings establish a role for ERO1 in calcium homeostasis and suggest that modifying the lumenal redox environment may affect the progression of heart failure.

Remodeling of atrial ATP-sensitive K⁺ channels in a model of salt-induced elevated blood pressure.

Hypertension is associated with the development of atrial fibrillation; however, the electrophysiological consequences of this condition remain poorly understood. ATP-sensitive K(+) (K(ATP)) channels, which contribute to ventricular arrhythmias, are also expressed in the atria. We hypothesized that salt-induced elevated blood pressure (BP) leads to atrial K(ATP) channel activation and increased arrhythmia inducibility. Elevated BP was induced in mice with a high-salt diet (HS) for 4 wk. High-resolution optical mapping was used to measure atrial arrhythmia inducibility, effective refractory period (ERP), and action potential duration at 90% repolarization (APD(90)). Excised patch clamping was performed to quantify K(ATP) channel properties and density. K(ATP) channel protein expression was also evaluated. Atrial arrhythmia inducibility was 22% higher in HS hearts compared with control hearts. ERP and APD(90) were significantly shorter in the right atrial appendage and left atrial appendage of HS hearts compared with control hearts. Perfusion with 1 µM glibenclamide or 300 µM tolbutamide significantly decreased arrhythmia inducibility and prolonged APD(90) in HS hearts compared with untreated HS hearts. K(ATP) channel density was 156% higher in myocytes isolated from HS animals compared with control animals. Sulfonylurea receptor 1 protein expression was increased in the left atrial appendage and right atrial appendage of HS animals (415% and 372% of NS animals, respectively). In conclusion, K(ATP) channel activation provides a mechanistic link between salt-induced elevated BP and increased atrial arrhythmia inducibility. The findings of this study have important implications for the treatment and prevention of atrial arrhythmias in the setting of hypertensive heart disease and may lead to new therapeutic approaches.

Gap junction remodeling and spironolactone-dependent reverse remodeling in the hypertrophied heart.

Pressure overload is a common pathological insult to the heart and the resulting hypertrophy is an independent risk factor for sudden cardiac death. Gap junction remodeling (GJR) has been described in hypertrophied hearts; however, a detailed understanding of the remodeling process and its effects on impulse propagation is lacking. Moreover, there has been little progress developing therapeutic strategies to diminish GJR. Accordingly, transverse aortic banding (TAC) was performed in mice to determine the effects of progressive pathological hypertrophy on connexin (Cx)43 expression, posttranslational phosphorylation, gap junction assembly, and impulse propagation. Within 2 weeks after TAC, total and phospho-Cx43 abundance was reduced and incorporation of Cx43 into gap junctional plaques was markedly diminished. These molecular changes were associated with progressive slowing of impulse propagation, as determined by optical mapping with voltage-sensitive dyes. Treatment with the aldosterone receptor antagonist spironolactone, which has been shown to diminish sudden arrhythmic death in clinical trials, was examined for its effects on GJR. We found that spironolactone blunted the development of GJR and also potently reversed established GJR, both at the molecular and functional levels, without diminishing the extent of hypertrophy. These data suggest a potential mechanism for some of the salutary electrophysiological and clinical effects of mineralocorticoid antagonists in myopathic hearts.

Gap junction remodeling and cardiac arrhythmogenesis in a murine model of oculodentodigital dysplasia.

Gap junction channels are required for normal cardiac impulse propagation, and gap junction remodeling is associated with enhanced arrhythmic risk. Oculodentodigital dysplasia (ODDD) is a multisystem syndrome due to mutations in the connexin43 (Cx43) gap junction channel gene. To determine the effects of a human connexin channelopathy on cardiac electrophysiology and arrhythmogenesis, we generated a murine model of ODDD by introducing the disease-causing I130T mutant allele into the mouse genome. Cx43 abundance was markedly reduced in mutant hearts with preferential loss of phosphorylated forms that interfered with trafficking and assembly of gap junctions in the junctional membrane. Dual whole-cell patch-clamp studies showed significantly lower junctional conductance between neonatal cell pairs from mutant hearts, and optical mapping of isolated-perfused hearts with voltage-sensitive dyes demonstrated significant slowing of conduction velocity. Programmed electrical stimulation revealed a markedly increased susceptibility to spontaneous and inducible ventricular tachyarrhythmias. In summary, our data demonstrate that the I130T mutation interferes with Cx43 posttranslational processing, resulting in diminished cell-cell coupling, slowing of impulse propagation, and a proarrhythmic substrate.

Common variants in 22 loci are associated with QRS duration and cardiac ventricular conduction.

The QRS interval, from the beginning of the Q wave to the end of the S wave on an electrocardiogram, reflects ventricular depolarization and conduction time and is a risk factor for mortality, sudden death and heart failure. We performed a genome-wide association meta-analysis in 40,407 individuals of European descent from 14 studies, with further genotyping in 7,170 additional Europeans, and we identified 22 loci associated with QRS duration (P < 5 × 10(-8)). These loci map in or near genes in pathways with established roles in ventricular conduction such as sodium channels, transcription factors and calcium-handling proteins, but also point to previously unidentified biologic processes, such as kinase inhibitors and genes related to tumorigenesis. We demonstrate that SCN10A, a candidate gene at the most significantly associated locus in this study, is expressed in the mouse ventricular conduction system, and treatment with a selective SCN10A blocker prolongs QRS duration. These findings extend our current knowledge of ventricular depolarization and conduction.

Upregulation of gamma-catenin compensates for the loss of beta-catenin in adult cardiomyocytes.

Recent progresses in signal transduction have revealed that beta-catenin signaling controls embryonic development, tumorigenesis, cell shape, and polarity. The role of this pathway in myocyte shape regulation during cardiac hypertrophy and failure is, however, not clearly defined. Since homozygous knockout of beta-catenin is embryonically lethal, we have deleted beta-catenin genes specifically in the heart of adult mice by crossing loxP-flanked beta-catenin mice with transgenic mice expressing tamoxifen-activated MerCreMer protein (MCM) driven by the alpha-myosin heavy chain promoter. Administration of tamoxifen to homozygous loxP-flanked beta-catenin mice positive for MCM induces the deletion of beta-catenin only in cardiomyocytes. Immunolabeling with beta-catenin antibody demonstrates that 90% of cardiomyocytes completely lose their beta-catenin expression but maintain normal rod-shaped morphology. The intercalated disk of cardiomyocytes lacking beta-catenin is morphologically unremarkable with normal distribution of vinculin, N-cadherin, desmoplakin, ZO-1, connexin43, and alpha-, gamma-, and p120 catenins. The expression level of these proteins, except that of gamma-catenin, is also similar in tamoxifen-treated and control mice with both homozygous loxP-flanked beta-catenin genes and the MCM transgene. Western blot analyses reveal that gamma-catenin increases in the heart of beta-catenin knockout mice compared with controls. Confocal microscopy also demonstrates that gamma-catenin has significantly increased in the intercalated disk of cardiomyocytes lacking beta-catenin. Echocardiographic data indicate that the knockout mice maintain normal ventricular geometry and cardiac function. The results suggest that upregulation of gamma-catenin can compensate for the loss of beta-catenin in cardiomyocytes to maintain normal cardiac structure and function.

Cardiac-specific haploinsufficiency of beta-catenin attenuates cardiac hypertrophy but enhances fetal gene expression in response to aortic constriction.

In addition to its role in cell adhesion, beta-catenin is an important signaling molecule in the Wnt/Wingless signaling pathway. Recent studies have indicated that beta-catenin is stabilized by hypertrophic stimuli and may regulate cardiac hypertrophic responses. To explore the role and requirement of beta-catenin in cardiac development and hypertrophy, we deleted the beta-catenin gene specifically in cardiac myocytes by crossing loxP-floxed beta-catenin mice with transgenic mice expressing a Cre recombinase under the control of the alpha-myosin heavy chain promoter. No homozygous beta-catenin-deleted mice were born alive and died before embryonic day 14.5, indicating significant and irreplaceable roles of beta-catenin in embryonic heart development. Heterozygous beta-catenin-deleted mice, however, demonstrated no structural and functional abnormality. The response of heterozygous beta-catenin-deleted mice to transverse aortic constriction, however, was significantly attenuated with decreased heart weight and heart weight/body weight ratio compared to controls with intact beta-catenin genes. Hemodynamic analysis revealed that there was no difference in cardiac function between wild-type and heterozygous beta-catenin-deleted mice. On the other hand, the expression of fetal genes, beta-myosin heavy chain, atrial and brain natriuretic peptides was significantly higher in heterozygous beta-catenin-deleted mice when compared to wild-type beta-catenin mice. These results suggest that the cytoplasmic level of beta-catenin modulates hypertrophic response and fetal gene reprogramming after pressure overload.

Nuclear compartmentalization of FAK and FRNK in cardiac myocytes.

Focal adhesion kinase (FAK) and FAK-related non-kinase (FRNK) accumulate in the nucleus of cardiac myocytes during hypertensive hypertrophy. Nuclear FAK and FRNK are phosphorylated on different serines and form distinct bright spots. The subnuclear distribution of serine-phosphorylated FAK and FRNK was examined in this study by double labeling with fibrillarin, a component of nucleoli, and Sam68, a constituent of Sam68 nuclear bodies. We also investigated the role of protein kinase C (PKC)-mediated phosphorylation of FAK and FRNK on nuclear translocation. PKC activation by 12-O-tetradecanoylphorbol 13-acetate treatment increased serine phosphorylation of FAK and FRNK. Specifically, FAK was phosphorylated on serine 722 but not serine 910. On the other hand, FRNK was phosphorylated on serine 217, the equivalent site of FAK serine 910, but not serine 30, the homologous site of FAK serine 722. Serine-phosphorylated FAK and FRNK redistributed into the nucleus and formed distinct patterns. FAK with phosphorylation on serine 722 colocalized with Sam68 but not fibrillarin. On the contrary, FRNK phosphorylated on 217 coexisted with fibrillarin but not Sam68. Immunoprecipitation also confirmed that FAK associated with Sam68 and FRNK interacted with fibrillarin, respectively. These results suggest that FAK and FRNK target different nuclear subdomains by their association with distinct nuclear proteins.