Mechanisms of Electrical Remodeling in Lipotoxic Guinea Pig Heart.
Objectives: To develop an adult guinea pig model of lipotoxicity and explore the underlying mechanisms associated with changes in the expression of the delayed rectifier potassium current (IK). Background: Lipotoxicity may represent a common link among metabolic disorders and a higher vulnerability to arrhythmias. Methods: Whole-cell patch clamp, and palmitic acid (PA, a potent inducer of lipotoxicity), were used to assess mechanisms of short-term (~50 days) high-fat diet (HFD) feeding on atrial electrophysiology in guinea pig hearts and myocytes. Results: HFD fed guinea pigs were significantly heavier, displayed hypertriglyceridemia and hypercholesterolemia; but no signs of hyperglycemia or inflammation compared to low-fat diet fed controls. Increasing cardiac PA levels, resulted in shortened atrial action potential duration, and increased IK density. Inhibition of phosphoinositide 3-kinase (PI3K) prevented increases in IK due to PA. Acute (\xe2\x89\xa5 1hr) exposure of atrial myocytes to exogenous PA (1 mM) increased the density of the rapid delayed potassium current IKr, while it was decreased with the unsaturated oleic acid (OA, 1 mM). Serine-threonine protein phosphatase-2 (PP2A) inhibition with cantharidin reversed the effect of OA on IKr. Conclusion and Implications: Our data provide evidence of a novel lipotoxic guinea pig model with signs of vulnerability to arrhythmias. Inhibition of PA/PI3K/IK and/or activation of the OA/PP2A/IKr pathways may be therapeutically beneficial for lipotoxic arrhythmias.
Introduction<!>Methods<!>Electrophysiology<!>High-fat diet feeding in guinea pig model<!>Intramyocardial injection of palmitic acid in guinea pigs<!>Isolation of guinea pig atrial myocytes<!>Preparation of bovine serum albumin conjugated FFA solutions<!>Data and statistical analyses<!>Effects of HFD feeding in guinea pig<!>Short-term HFD feeding separates hyperlipidemia from hyperglycemia and inflammation in guinea pig<!>Determination of intramyocardial palmitic acid effects on atrial myocyte electrophysiology<!>Effects of intramyocardial palmitic acid on atrial IK in guinea pig heart<!>Effects of LY294002 on palmitic acid-induced increases in IK density in atrial myocytes<!>Effects of exogenous palmitic acid and oleic acid on IKr in native atrial myocytes<!>Effects of Cantharidin on oleic acid-induced depression of IKr density in atrial myocytes<!>Discussion<!>Therapeutic effects of PI3K inhibition on short-term HFD-induced modulation of IK in guinea pig myocytes<!>Beneficial effect of unsaturated oleic acid in limiting atrial arrhythmogenesis due to short-term HFD feeding in guinea pig heart
<p>Obesity is a major contributor to the increasing prevalence of cardiovascular diseases worldwide [1], with significant implications for supraventricular arrhythmias [2]. Obesity mechanisms may have a direct impact on the electrical activity of the atria (Figure 1A). Lipotoxicity or abnormal accumulation of serum free fatty acids (FFAs) [3], looms as a key mechanism underlying these interlinked pathologies [4].</p><p>Lipotoxicity is associated with changes in protein kinases (phosphatidylinositide 3-kinase (PI3K)) [5], and protein phosphatases (serine-threonine protein phosphatase-2, or PP2A) [6], PI3K upregulates IKr function in HEK293 cells, while dominant negative mutants reduced the current [7]. Reduced PP2A activity is associated with arrythmias including atrial fibrillation (AF) [8], and there is growing interest in PP2A activators in treating arrhythmias [8]. Thus, PP2A and PI3K are strong candidates for modulation of IK function in lipotoxic heart.</p><p>The study presents a novel guinea pig model of lipotoxicity with early signs of atrial electrical remodeling (shortened action potential duration (APD) and increased IK density), which may be useful for investigating mechanisms that initiate arrhythmias. Furthermore, PI3K inhibition prevented increases of IK due to palmitic acid (PA), while PP2A inhibition prevented oleic acid (OA)-mediated depression of IKr. The data suggest that individual or multiple combinations of dietary (OA) and therapeutic interventions (potassium channel blockers, PI3K inhibitors, and PP2A activators) may be beneficial as early treatment options in obese patients with atrial arrhythmias.</p><!><p>This study was performed in accordance with the guidelines of the Columbia University and the Veterans Healthcare System Animal Care and Use Committees.</p><!><p>Whole-cell current recordings in adult guinea pig atria myocytes were performed as previously described [9, 10].</p><!><p>Male and female guinea pigs (200-250 g or 2-3 weeks of age) were purchased from Charles River Laboratories (Wilmington, MA). Guinea pigs were fed ad libitum a low-fat diet (LFD), and high-fat diet (HFD) as previously described [11].</p><!><p>Bovine serum albumin-conjugated palmitic acid (PA-BSA, 1 mM, or 50 mg/kg) or vehicle (albumin alone, 50 mg/kg) was introduced into slightly anaesthetized [12] guinea pig hearts through the cranial vena cava, over 10 days. 12.5 mg/kg of PA-albumin or albumin alone were injected at day 0, 2, 8, and 10 (Figure 2A), to achieve a cumulative concentration of 50 mg/kg. All animals recovered within 2-5 minutes of the injection of PA-BSA or control albumin.</p><!><p>Primary atrial myocytes were isolated by enzymatic dissociation as previously described [9].</p><!><p>Palmitic acid (PA) stock solution was prepared as previously described [9].</p><!><p>Electrophysiological data were analyzed off-line using built in functions in Fitmaster (HEKA), Clampfit and Origin software. Current densities are expressed as current amplitudes (in pA) relative to cell size (in pF) or pA/pF. Data are reported as means±S.E.M. Statistical differences were determined using one-way ANOVA with Bonferroni post-hoc analysis or two-tailed unpaired t test for comparisons between groups and considered significant at P < 0.05.</p><!><p>The effects of short-term (~50 days), LFD and HFD feeding were investigated in male and female guinea pigs. As illustrated in Figure 1 switching from normal chow to the respective LFD or HFD led to progressive weight gain relative to time (Figure 1C). After ~50 days, HFD guinea pigs (Open triangles) were heavier (733±48 g, n=5, or a 19% increase) when compared to LFD-fed controls (589±10g, n=3, Open circles), in line with our previous report [9].</p><!><p>The Table shows the blood cell counts and glucose values before and after LFD or HFD feeding. We found that both complete blood cell (white blood cells, red blood cells, platelets, hemoglobin) count analyses and serum glucose measurements revealed that no significant changes were associated with HFD feeding, compared to LFD-fed guinea pigs.</p><!><p>Next the effect of lipotoxicity directly on cardiac function was evaluated using intracardiac injection of saturated PA (a potent inducer of lipotoxicity) [13]. After 40 days, electrophysiological assays were conducted (Figure 2A). Figure 2B shows representative superimposed AP traces recorded in myocytes isolated from guinea pigs injected with PA-BSA and vehicle control (BSA alone). On average atrial myocytes from PA-BSA guinea pigs displayed significantly shorter APD90 (107.9±13.3 ms, n=3 or 33%, *P<0.05, Figure 2C) compared to BSA alone control (151.3±3.42 ms, n=4, Figure 2C). Average APD30 was reduced to 41.3±2.67ms, (or by 68%, n=3, *P<0.05, Figure. 2C) from a control value of 92.5±10ms (n=4). Resting membrane potential (RMP) was more depolarized in guinea pigs injected with PA-BSA (−47±2 mV, n=3, *P<0.05, Figure. 2C), compared to control (−70 ±1.44 mV, n=4), suggesting that a reduced function of other major atrial ion channels (TWIK-related acid sensitive potassium channel or TASK-2) could depolarize RMP [14] in our lipotoxic guinea pig model and contribute to the vulnerability to atrial arrhythmogenic events.</p><!><p>The potential mechanisms of adverse atrial electrical remodeling in PA+BSA injected hearts were assessed by measuring IK, because of its role in repolarization and that dysfunction of IK [15, 16] contributes to arrhythmias [17]. Figure 2D shows typical whole-cell atrial IK current traces measured in guinea pig atrial myocytes from PA+BSA- and BSA alone-injected hearts (controls). Compared to controls PA-BSA injection increased IK densities at all potentials positive to +10 mV (Figure 2E). At +50 mV, IK peak density was increased from 4.79±0.41 pA/pF (n=6) in control to 6.88±1.13 pA/pF, (n=6, or by 30.4%, P>0.05, Figure. 2E) with PA-BSA. At +100 mV averaged IK density was significantly increased from 11.3±3.42 pA/pF (n=6, control) to 22.1±2.34 pA/pF (or by 48.7% n=6, *P<0.05) with PA+BSA.</p><!><p>To interpret the mechanistic bases of the adverse effects by lipotoxicity on atrial electrical properties, effects of exogenous pretreatment with inhibitors of critical metabolic pathways on IK density were determined in atrial myocytes. PI3K, is a serine/threonine kinase which is sensitive to lipotoxicity [5] and a key regulator of cardiac ion channels [18]. Figure 3A shows typical IK currents measured in basal conditions (i.e. untreated, BSA alone). Exogenous pretreatment with PA+BSA (1 mM) significantly increased IK density (Figure 3B), while pre-exposure to the PI3K inhibitor, LY294002 (10μM) [19], completely reversed this effect (Figure 3C) consistent with a role for PI3K in the facilitatory effect of PA on IK current density in atrial myocytes. On average PA+BSA significantly increased Imax, or maximal current at +30 mV from 2.59±0.44 (n=10, control) to 5.27±0.22 pA/pF (n=9, PA+BSA) (or by ~51%, *P<0.05; Figure 3D). IK current density measured in PA+BSA-treated cells that were pre-exposed to LY294002 was 2.91±0.42 pA/pF (n=9, P>0.05) compared to 2.59±0.44 pA/pF (BSA alone, n=10, Figure 3D) measured in basal conditions.</p><!><p>Previously we demonstrated that OA prolonged atrial APD and severely depressed IKr density in HEK293 cells but had no effect on IKs, while PA shortened APD and increased both IKr and IKs densities [9]. Next we determined the effects the lipids on IKr currents measured in native atrial cardiomyocytes. We used the whole-cell patch clamp technique to assess the functional properties of IKr in atrial myocytes incubated ≥1hr with either 1 mM BSA control, PA, or OA. (Figure 3E). Compared to control or basal IKr (Figure 3E, Top), PA augmented (Figure 3E, Middle), whereas OA+BSA severely depressed IKr (Figure 3E, Bottom). PA+BSA significantly increased Imax, or maximal current at +30 mV from 0.29±0.03 (BSA alone, n=7 separate myocytes) to 0.91±0.12 (n=8 separate myocytes) (or by ~68%, *P<0.05; Figure 3F), whereas OA+BSA significantly reduced Imax by ~45% (0.16±0.01, n=5 separate myocytes; *P<0.05; Figure 3F). The data suggest that alteration of cellular lipids, either by disease, diet, or metabolic state, can potentially affect the expression of IK channels with significant implications for cardiac function.</p><!><p>Our results presented in Figure 3E and Figure 3F suggests that OA through inhibition of IKr, may be a key anti-arrhythmic mechanism in lipotoxicity by antagonizing the shortening of APD. Further there are reports that reduced PP2A activity is associated with AF through modulation of cardiac ion channels [8]. To elucidate the mechanism of APD prolongation and reduced IKr density by OA, the effect of PP2A inhibition with cantharidin (30 μM) on IKr was examined. Pretreatment (10-mins) of atrial myocytes with cantharidin prevented OLA+BSA-mediated depression of IKr (Figure 3G). At +30 mV IKr density was significantly (*P<0.05) increased from 0.16±0.01 pA/pF (n=9) in the presence of OA+BSA to 0.49±0.17 pA/pF (or increased by ~67%, n=3, Figure 3G,H) with cantharidin. The data suggests that activation of the OA/PP2A/IKr pathway may prevent or lower threshold of vulnerability to supraventricular arrhythmias.</p><!><p>Lipotoxicity is an independent risk factor for arrhythmias in obese patients; however the mechanisms are poorly understood. The goal of the present study is to determine whether we could examine the effects of HFD-induced lipotoxicity on atrial electrophysiology in guinea pig independent of other obesity co-morbidities. Short-term HFD diet feeding in guinea pig separates hyperlipidemia from hyperglycemia and inflammation. Lipotoxic hearts also displayed atrial electrical dysfunction (Figure 4A). The data suggests that short-term HFD diet feeding in guinea pig permits investigation of lipotoxic mechanisms and atrial electrical remodeling prior to supraventricular arrhythmias.</p><!><p>Shortening of atrial APD contributes to mechanisms (refractory periods, reentry circuits) [20], that lead to arrhythmias. Therefore, enhanced IK density will be expected to accelerate repolarization and contribute to supraventricular arrythmias. The relevance of IK mechanisms is highlighted by recent studies in AF patients [21, 22]. Mechanistically, increased IK may be due to altered posttranslational modifications [22, 23]. As shown in this study, inhibition of the lipid kinase PI3K [24], inhibited IK density and suggests that myocardial PI3K inhibition has the potential to be anti-arrhythmic by prolonging APD and reducing vulnerability to supraventricular arrhythmias (Figure 4B). This is supported by a previous report in mouse hearts lacking PI3K p110α catalytic subunit, demonstrating prolonged APD and QT interval [25]. Diabetic mice which lacks the PI3K p110γ or expressing the catalytically inactive p110γ prevented systolic and diastolic dysfunction [26]. Therefore, it would also be valuable to explore the link between PI3K, IK and atrial arrhythmias.</p><!><p>Our finding that exogenous OA selectively inhibited IKr density in atrial myocytes, is in-line with its role in atrial APD prolongation in obese myocytes [9]. Electrophysiological studies have shown that fish oil diets prevented cardiac hypertrophy in a rat model of pressure overload [27] and a rabbit model of heart failure [28] through modulation of potassium channels [28]. Therefore, dietary supplementation of OA may be a valuable anti-arrhythmic mechanism in lipotoxicity by antagonizing the shortening of APD and raising the threshold for inducibility of atrial tachycardia (Figure 4C).</p><p>One of the well-accepted mechanisms underlying initiation and maintenance of supraventricular arrythmias is impaired posttranslational regulation due to reversible phosphorylation by protein phosphatases [29]. Furthermore, reduced PP2A activity is associated with AF [8], suggesting therapeutic potential of PP2A activators. Evidence shows that decreased ICa,L current density associated with AF [22] is due to increased calcium channel dephosphorylation by increased activity of PP2A [8]. The findings of this study further show that PP2A inhibition prevented decreases in IKr current density due to OA and suggests that PP2A activation contributed to OA-induced APD.</p><p>These results suggest that in lipotoxic atrial myocytes: 1) activation of endogenous of PI3K may underlie increased densities of IKr and IKs; and 2) activation of native PP2A may mediate the selective down regulation of atrial IKr seen in the presence OA. Overall, the DATA preview the new mechanistic insights likely to be gathered from future studies (Figure 4).</p><p>Taking together these results and the findings of Aromolaran et al [9], short-term HFD feeding in guinea pig leads to significant weight gain, lipotoxicity and adverse remodeling of atrial IK. These findings have potential implications for atria electrical activities, possibly through significant elevations in intramyocardial saturated palmitic acid. It is intriguing to speculate that timely interventions that either prevent: 1) ectopic lipid accumulation (anti-lipotoxic drugs that divert lipid to adipose stores or decrease systemic lipid levels); 2) increased IKs function (dietary unsaturated FFAs) may be anti-arrhythmic, and therefore beneficial to obese patients with cardiac events (Figure 4A).</p>