PCSK9 inhibition with alirocumab reduces lipoprotein(a) levels in nonhuman primates by lowering apolipoprotein(a) production rate.

Therapeutic antibodies targeting proprotein convertase subtilisin kexin type 9 (PCSK9) (e.g. alirocumab) lower low-density lipoprotein cholesterol (LDL-C) and lipoprotein (a) [Lp(a)] levels in clinical trials. We recently showed that PCSK9 enhances apolipoprotein(a) [apo(a)] secretion from primary human hepatocytes but does not affect Lp(a) cellular uptake. Here, we aimed to determine how PCSK9 neutralization modulates Lp(a) levels in vivoSix nonhuman primates (NHP) were treated with alirocumab or a control antibody (IgG1) in a crossover protocol. After the lowering of lipids reached steady state, NHP received an intravenous injection of [2H3]-leucine, and blood samples were collected sequentially over 48 h. Enrichment of apolipoproteins in [2H3]-leucine was assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Kinetic parameters were calculated using numerical models with the SAAMII software. Compared with IgG1, alirocumab significantly reduced total cholesterol (TC) (-28%), LDL-C (-67%), Lp(a) (-56%), apolipoprotein B100 (apoB100) (-53%), and apo(a) (-53%). Alirocumab significantly increased the fractional catabolic rate of apoB100 (+29%) but not that of apo(a). Conversely, alirocumab sharply and significantly reduced the production rate (PR) of apo(a) (-42%), but not significantly that of apoB100, compared with IgG1, respectively.In line with the observations made in human hepatocytes, the present kinetic study establishes that PCSK9 neutralization with alirocumab efficiently reduces circulating apoB100 and apo(a) levels by distinct mechanisms: apoB primarily by enhancing its catabolism and apo(a) primarily by lowering its production.

Exploring the fate of inhaled monoclonal antibody in the lung parenchyma by microdialysis.

Therapeutic antibodies (Abs) are emerging as major drugs to treat respiratory diseases, and inhalation may provide substantial benefits for their delivery. Understanding the behavior of Abs after pulmonary deposition is critical for their development. We investigated the pharmacokinetics of a nebulized Ab by continuous sampling in lung parenchyma using microdialysis in non-human primates. We defined the optimal conditions for microdialysis of Ab and demonstrated that lung microdialysis of Ab is feasible over a period of several days. The concentration-profile indicated a two-phase non-linear elimination and/or distribution of inhaled mAbX. Lung exposition was higher than the systemic one over a period of 33 hours and above MabX affinity for its target. The microdialysis results were supported by an excellent relationship with dosages from lung extracts.

Cardiac sodium channel Nav1.5 is regulated by a multiprotein complex composed of syntrophins and dystrophin.

The cardiac sodium channel Na(v)1.5 plays a key role in cardiac excitability and conduction. The purpose of this study was to elucidate the role of the PDZ domain-binding motif formed by the last three residues (Ser-Ile-Val) of the Na(v)1.5 C-terminus. Pull-down experiments were performed using Na(v)1.5 C-terminus fusion proteins and human or mouse heart protein extracts, combined with mass spectrometry analysis. These experiments revealed that the C-terminus associates with dystrophin, and that this interaction was mediated by alpha- and beta-syntrophin proteins. Truncation of the PDZ domain-binding motif abolished the interaction. We used dystrophin-deficient mdx(5cv) mice to study the role of this protein complex in Na(v)1.5 function. Western blot experiments revealed a 50% decrease in the Na(v)1.5 protein levels in mdx(5cv) hearts, whereas Na(v)1.5 mRNA levels were unchanged. Patch-clamp experiments showed a 29% decrease of sodium current in isolated mdx(5cv) cardiomyocytes. Finally, ECG measurements of the mdx(5cv) mice exhibited a 19% reduction in the P wave amplitude, and an 18% increase of the QRS complex duration, compared with controls. These results indicate that the dystrophin protein complex is required for the proper expression and function of Na(v)1.5. In the absence of dystrophin, decreased sodium current may explain the alterations in cardiac conduction observed in patients with dystrophinopathies.

Long-term amiodarone administration remodels expression of ion channel transcripts in the mouse heart.

BACKGROUND: The basis for the unique effectiveness of long-term amiodarone treatment on cardiac arrhythmias is incompletely understood. The present study investigated the pharmacogenomic profile of amiodarone on genes encoding ion-channel subunits. METHODS AND RESULTS: Adult male mice were treated for 6 weeks with vehicle or oral amiodarone at 30, 90, or 180 mg x kg(-1) x d(-1). Plasma and myocardial levels of amiodarone and N-desethylamiodarone increased dose-dependently, reaching therapeutic ranges observed in human. Plasma triiodothyronine levels decreased, whereas reverse triiodothyronine levels increased in amiodarone-treated animals. In ECG recordings, amiodarone dose-dependently prolonged the RR, PR, QRS, and corrected QT intervals. Specific microarrays containing probes for the complete ion-channel repertoire (IonChips) and real-time reverse transcription-polymerase chain reaction experiments demonstrated that amiodarone induced a dose-dependent remodeling in multiple ion-channel subunits. Genes encoding Na+ (SCN4A, SCN5A, SCN1B), connexin (GJA1), Ca2+ (CaCNA1C), and K+ channels (KCNA5, KCNB1, KCND2) were downregulated. In patch-clamp experiments, lower expression of K+ and Na+ channel genes was associated with decreased I(to,f), I(K,slow), and I(Na) currents. Inversely, other K+ channel alpha- and beta-subunits, such as KCNA4, KCNK1, KCNAB1, and KCNE3, were upregulated. CONCLUSIONS: Long-term amiodarone treatment induces a dose-dependent remodeling of ion-channel expression that is correlated with the cardiac electrophysiologic effects of the drug. This profile cannot be attributed solely to the amiodarone-induced cardiac hypothyroidism syndrome. Thus, in addition to the direct effect of the drug on membrane proteins, part of the therapeutic action of long-term amiodarone treatment is likely related to its effect on ion-channel transcripts.

Moderate and chronic hemodynamic overload of sheep atria induces reversible cellular electrophysiologic abnormalities and atrial vulnerability.

OBJECTIVES: The aim of this study was to evaluate the myocardial consequences of a chronic volume overload of the left atrium (LA). BACKGROUND: Atrial dilation is a major risk factor for atrial fibrillation (AF), but the underlying mechanisms are poorly understood. METHODS: A left-right aorto-pulmonary artery shunt (APS) was created in sheep. The cardiopathy was characterized by echocardiography, electrophysiologic testing, and histologic analysis. Cellular action potential (AP) and calcium current (I(Ca)) were recorded by means of microelectrode and patch clamp techniques. RESULTS: Three to four months after surgery, all animals in the APS state had a dilated LA (146.2 +/- 35.4 cm(2)/m(2) vs. 91.7 +/- 10.4 cm(2)/m(2) in the control state; p = 0.0024) but remained in sinus rhythm. Repetitive atrial firing was triggered by a single extra beat in five of six animals in the APS state and in two of six animals in the control state. Moreover, in two animals in the APS state, a single extra beat triggered sustained AF. Myocytes were enlarged and 39.8% showed some degree of myolysis. In animals in the APS state, the AP had no plateau phase or small amplitude and numerous myocytes were unexcitable. The I(Ca) density was 45.2% lower in APS animals than in control animals. Beta-adrenergic stimulation normalized I(Ca) and restored the plateau phase of the AP. After shunt suppression, the electrophysiologic properties of the atria returned to normal. CONCLUSIONS: The APS induced moderate, isolated LA dilation, which was sufficient to cause major changes in cellular electrophysiologic properties and to render the atria vulnerable to fibrillation. These effects were reversed by shunt suppression.

Fibrosis of the left atria during progression of heart failure is associated with increased matrix metalloproteinases in the rat.

OBJECTIVES: The purpose of this study was to determine the pathogenic factors and molecular mechanisms involved in fibrosis of the atria. BACKGROUND: Fibrosis is an important component of the pathophysiology of atrial fibrillation, especially when the arrhythmia is associated with heart failure (HF) or atrial dilation. METHODS: We used a rat model of myocardial infarction (MI) complicated by various degrees of left ventricular dysfunction and atrial dilation to study fibrosis and matrix metalloproteinase (MMP) activity in the left atrial (LA) myocardium by means of histologic, Western blot, zymographic, and immunohistologic techniques. RESULTS: Three months after surgical ligature of the left coronary artery, 27 rats had a large MI, 12 were in mild HF, and 15 in severe HF. Both groups had LA enlargement at the echocardiography. Masson's trichrome and picrosirius staining of tissue sections revealed marked fibrosis at the periphery of trabeculae and also surrounding myolytic myocytes, in both mild and severe HF. In mild HF, the activity and expression of the matrilysin MMP-7 were increased (122%), whereas in severe HF, both MMP-7 (211%) and the gelatinase MMP-2 (187%) were up-regulated. There were no changes in the expression or activity of MMP inhibitors, TIMP-1, -2, and -4. Immunostaining of cryosections showed that MMP-2 was present in the interstitial spaces, whereas MMP-7 accumulated in myolytic myocytes. CONCLUSIONS: Hemodynamic overload of the atria is an important pathogenic factor of fibrosis; MMP-7 appears to be involved in the early stage of this tissue remodeling process.

Downregulation of the calcium current in human right atrial myocytes from patients in sinus rhythm but with a high risk of atrial fibrillation.

AIMS: A decrease in L-type calcium current (ICaL) is an important mechanism favouring atrial fibrillation (AF). Here, we aimed to identify pathogenic factors associated with ICaL downregulation. METHODS AND RESULTS: Atrial myocytes were isolated from right atrial appendages obtained from 86 adult patients in sinus rhythm with coronary artery disease, aortic valve disease, or mitral valve disease (MVD). Current was recorded in isolated myocytes using the whole-cell patch-clamp technique. The ICaL recorded in the 172 myocytes studied showed a marked variability of peak density ranging from 0.1 to 9.0 pA/pF. The ICaL peak density did not correlate with membrane capacitance or changes in current biophysical properties. The ICaL peak density was homogeneous for a given sample. Small ICaL values were recorded in patients with MVD or with a low left ventricular ejection fraction (<45%). Small ICaL values were more sensitive to the beta-adrenergic agonist, isoproterenol (1 microM), and to the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (10 microM). CONCLUSION: In human atrial myocytes, the variability of ICaL is related to the clinical history of the donors. The downregulation of ICaL is already observed in patients in sinus rhythm with a high risk of AF and is associated with the greatest response to beta-adrenergic agonist.

Chronic angiotensin II stimulation in the heart produces an acquired long QT syndrome associated with IK1 potassium current downregulation.

Cardiac hypertrophy is an independent predictor of cardiovascular morbidity and mortality. It predisposes patients to heart failure, QT interval prolongation and ventricular arrhythmias. Angiotensin II (Ang II) exerts direct actions on cardiac tissue inducing cardiomyocyte hypertrophy and electro-mechanical dysfunction. However, a direct association between Ang II and cardiomyocyte electrical remodeling has yet to be demonstrated. Transgenic TG1306/1R (TG) mice with cardiac-specific Ang II overproduction demonstrate blood pressure-independent cardiac hypertrophy and exhibit significant increase in sudden death associated with mechanical dysfunction. The present study makes use of TG mice to evaluate the direct effects of high levels of intracardiac Ang II on cardiac electrophysiology. Surface-limb ECG measurements were recorded on 50- to 60-week-old TG and wild-type (WT) mice. QT interval was significantly prolonged (+20%) in TG mice relative to WT. TG mice also showed an increased incidence of ventricular arrhythmias. QT prolongation was associated with prolongation of cardiomyocyte action potential at 90% repolarization (APD90). The change in APD90 correlated with a reduction in IK1 potassium current density in TG vs. WT cardiomyocytes (at -70 mV: 0.3+/-0.1 pA/pF vs. 0.8+/-0.2 pA/pF, P<0.05). In TG mice, reduction in IK1 was associated with a significant reduction (-50%) of the mRNA encoding Kir2.1 and Kir2.2 subunits of IK1-related KCNJ2 and KCNJ12 potassium channels. These data suggest that cardiac Ang II overproduction leads to the emergence of a long QT syndrome resulting from an IK1-dependent prolongation of the action potential duration through modulation of channel subunit expression.

Aldosterone increases voltage-gated sodium current in ventricular myocytes.

The role of aldosterone in the pathogenesis of heart failure (HF) is still poorly understood. Recently, aldosterone has been shown to modulate the function of cardiac Ca(2+) and K(+) channels, thus playing a role in the electrical remodeling process. The goal of this work was to investigate the role of aldosterone on the cardiac Na(+) current (I(Na)). We analyzed the effects of aldosterone on I(Na) in isolated adult mouse ventricular myocytes, using the whole cell patch-clamp technique. After 24 h incubation with 1 microM aldosterone, the I(Na) density was significantly increased (+55%), without alteration of the biophysical properties and the cell membrane capacitance. Aldosterone (10 nM) increased the I(Na) by 23%. In 24-h coincubation experiments, with the use of actinomycin D, cycloheximide, or brefeldin A, the effect of aldosterone on I(Na) was abolished. Spironolactone (mineralocorticoid receptor antagonist, 10 microM) prevented the 1 microM aldosterone-dependent I(Na) increase, whereas RU-38486 (glucocorticoid receptor antagonist, 10 microM) did not. The action potential duration (APD) was longer in aldosterone-treated (APD(90): +53%) than in control myocytes. In addition, the L-type Ca(2+) current was also upregulated (+48%). We performed quantitative RT-PCR measurements and Western blots to quantify the mRNA and protein levels of Na(v)1.5 and Ca(v)1.2 (main channels mediating cardiac I(Na) and I(Ca)), but no significant difference was found. In conclusion, this study shows that aldosterone upregulates the cardiac I(Na) and suggest that this phenomenon may contribute to the HF-induced electrical remodeling process that may be reversed by spironolactone.