Recombinant Atrial Natriuretic Peptide Prevents Aberrant Ca²⁺ Leakage through the Ryanodine Receptor by Suppressing Mitochondrial Reactive Oxygen Species Production Induced by Isoproterenol in Failing Cardiomyocytes

<div><p>Catecholamines induce intracellular reactive oxygen species (ROS), thus enhancing diastolic Ca²⁺ leakage through the ryanodine receptor during heart failure (HF). However, little is known regarding the effect of atrial natriuretic peptide (ANP) on ROS generation and Ca²⁺ handling in failing cardiomyocytes. The aim of the present study was to clarify the mechanism by which an exogenous ANP exerts cardioprotective effects during HF. Cardiomyocytes were isolated from the left ventricles of a canine tachycardia-induced HF model and sham-operated vehicle controls. The degree of mitochondrial oxidized DNA was evaluated by double immunohistochemical (IHC) staining using an anti-VDAC antibody for the mitochondria and an anti-8-hydroxy-2′-deoxyguanosine antibody for oxidized DNA. The effect of ANP on ROS was investigated using 2,7-dichlorofluorescin diacetate, diastolic Ca²⁺ sparks assessed by confocal microscopy using Fluo 4-AM, and the survival rate of myocytes after 48 h. The double IHC study revealed that isoproterenol (ISO) markedly increased oxidized DNA in the mitochondria in HF and that the ISO-induced DNA damage was markedly inhibited by the co-presence of ANP. ROS production and Ca²⁺ spark frequency (CaSF) were increased in HF compared to normal controls, and were further increased in the presence of ISO. Notably, ANP significantly suppressed both ISO-induced ROS and CaSF without changing sarcoplasmic reticulum Ca²⁺ content in HF (p<0.01, respectively). The survival rate after 48 h in HF was significantly decreased in the presence of ISO compared with baseline (p<0.01), whereas it was significantly improved by the co-presence of ANP (p<0.01). Together, our results suggest that ANP strongly suppresses ISO-induced mitochondrial ROS generation, which might correct aberrant diastolic Ca²⁺ sparks, eventually contributing to the improvement of cardiomyocyte survival in HF.</p></div

A low-dose β1-blocker in combination with milrinone improves intracellular Ca2+ handling in failing cardiomyocytes by inhibition of milrinone-induced diastolic Ca2+ leakage from the sarcoplasmic reticulum.

OBJECTIVES:The purpose of this study was to investigate whether adding a low-dose β1-blocker to milrinone improves cardiac function in failing cardiomyocytes and the underlying cardioprotective mechanism. BACKGROUND:The molecular mechanism underlying how the combination of low-dose β1-blocker and milrinone affects intracellular Ca(2+) handling in heart failure remains unclear. METHODS:We investigated the effect of milrinone plus landiolol on intracellular Ca(2+) transient (CaT), cell shortening (CS), the frequency of diastolic Ca(2+) sparks (CaSF), and sarcoplasmic reticulum Ca(2+) concentration ({Ca(2+)}SR) in normal and failing canine cardiomyocytes and used immunoblotting to determine the phosphorylation level of ryanodine receptor (RyR2) and phospholamban (PLB). RESULTS:In failing cardiomyocytes, CaSF significantly increased, and peak CaT and CS markedly decreased compared with normal myocytes. Administration of milrinone alone slightly increased peak CaT and CS, while CaSF greatly increased with a slight increase in {Ca(2+)}SR. Co-administration of β1-blocker landiolol to failing cardiomyocytes at a dose that does not inhibit cardiomyocyte function significantly decreased CaSF with a further increase in {Ca(2+)}SR, and peak CaT and CS improved compared with milrinone alone. Landiolol suppressed the hyperphosphorylation of RyR2 (Ser2808) in failing cardiomyocytes but had no effect on levels of phosphorylated PLB (Ser16 and Thr17). Low-dose landiolol significantly inhibited the alternans of CaT and CS under a fixed pacing rate (0.5 Hz) in failing cardiomyocytes. CONCLUSION:A low-dose β1-blocker in combination with milrinone improved cardiac function in failing cardiomyocytes, apparently by inhibiting the phosphorylation of RyR2, not PLB, and subsequent diastolic Ca(2+) leak

SR Ca²⁺ content in sham and failing cardiomyocytes.

<p>A. Measurement of SR Ca²⁺ content by caffeine application. After isolation of cardiomyocytes, cardiomyocytes were loaded with 20 μM Fluo-4 AM (Molecular Probes) for 30 min at room temperature in the dark. Then, these cardiomyocytes were washed with Tyrode solution containing final concentration of 2 mM Ca²⁺. The cardiomyocytes were electrically stimulated by a field stimulator (IonOptix, MA) at a frequency of 0.5 Hz for 30 sec, and then final concentration of 20 mM caffeine were added. An arrow shows a point of addition of caffeine to the dish. B. Bar graph representation of the data in Fig 4A. Each group included 20–30 cells. At least 4 cells were evaluated for each preparation. The bars indicate the means ± SE.</p

Proposed ANP cardioprotective mechanism in heart failure.

<p>A. Leaky RyR channel in failing cardiomyocytes. B. ISO enhanced diastolic SR Ca²⁺ leak. C. ANP inhibited ISO-induced mitochondrial ROS, leading to the decrease of diastolic Ca²⁺ leak through RyR. ANP, atrial natriuretic peptide; GCA-R, membrane guanylate coupled A receptor; ISO, isoproterenol; β-AR, β adrenal receptor; PKA, protein kinase A; CaMKII, Ca²⁺/calmodulin-dependent protein kinase II; RyR, ryanodine receptor; ROS, reactive oxygen species; SR, sarcoplasmic reticulum.</p

Effect of ISO or ANP on Ca²⁺ sparks in sham and failing cardiomyocytes.

<p>A. Representative data for diastolic Ca²⁺ sparks in sham and failing cardiomyocytes. B. Bar graph representation of the data in Fig 3A. The bars indicate the means ± SE. Each group included 20–30 cells. At least 4 cells were evaluated for each preparation. Notably, ISO-induced aberrant diastolic Ca²⁺ sparks were inhibited by 10 nM ANP, but upon the addition of H₂O₂ (25 μM), aberrant diastolic Ca²⁺ sparks reappeared in the failing cardiomyocytes.</p

Effects of ISO and/or ANP on ROS production in sham and failing cardiomyocytes.

<p>A. Representative images of echocardiography in a sham operated dog and a HF dog. Left ventricular ejection fraction (LVEF) in a sham operated dog was 79%, while LVEF in a HF dog was 22%. RV, right ventricle; IVS, interventricular septum; LV, left ventricle; Sham, sham operated control. B. Representative images depicting intracellular ROS production in sham and failing cardiomyocytes corresponding to Fig A. Cardiomyocytes were subjected to immunofluorescence staining with a ROS-sensitive fluorescent dye (DCFH-DA) after electrical pacing at 0.5 Hz. Upper panels: sham cardiomyocytes. Bottom panels: failing cardiomyocytes. C. Bar graph representation of the data in Fig 1B. The bars indicate the means ± SE. Each group included 20–30 cells. At least 4 cells were evaluated for each preparation. D. Representative images depicting the antioxidant effect of the free radical scavenger edaravone (100 μM), ANP (10 nM) and Mito-tempo (100 μM) after exposure to H₂O₂ (25 μM) in sham cardiomyocytes. E. Bar graph representation of the data in Fig 1D. The bars indicate the means ± SE. Changes in the fluorescence intensities of DCFH-DA were compared among cell treatment with edaravone (100 μM), ANP (10 nM) and Mito-tempo (100 μM) Each group included 20–30 cells. At least 4 cells were evaluated for each preparation.</p