Lysine crotonylation of SERCA2a correlates to cardiac dysfunction and arrhythmia in Sirt1 cardiac-specific knockout mice.

Protein post-translational modifications (PTMs) are important regulators of protein functions and produce proteome complexity. SIRT1 has NAD+-dependent deacylation of acyl-lysine residues. The present study aimed to explore the correlation between lysine crotonylation (Kcr) on cardiac function and rhythm in Sirt1 cardiac-specific knockout (ScKO) mice and related mechanism. Quantitative proteomics and bioinformatics analysis of Kcr were performed in the heart tissue of ScKO mice established with a tamoxifen-inducible Cre-loxP system. The expression and enzyme activity of crotonylated protein were assessed by western blot, co-immunoprecipitation, and cell biology experiment. Echocardiography and electrophysiology were performed to investigate the influence of decrotonylation on cardiac function and rhythm in ScKO mice. The Kcr of SERCA2a was significantly increased on Lys120 (1.973 folds). The activity of SERCA2a decreased due to lower binding energy of crotonylated SERCA2a and ATP. Changes in expression of PPAR-related proteins suggest abnormal energy metabolism in the heart. ScKO mice had cardiac hypertrophy, impaired cardiac function, and abnormal ultrastructure and electrophysiological activities. We conclude that knockout of SIRT1 alters the ultrastructure of cardiac myocytes, induces cardiac hypertrophy and dysfunction, causes arrhythmia, and changes energy metabolism by regulating Kcr of SERCA2a. These findings provide new insight into the role of PTMs in heart diseases.

Soluble epoxide hydrolase and TRPC3 channels jointly contribute to homocysteine-induced cardiac hypertrophy: Interrelation and regulation by C/EBPß.

OBJECTIVES: Studies in certain cardiac hypertrophy models suggested the individual role of soluble epoxide hydrolase (sEH) and canonical transient receptor potential 3 (TRPC3) channels, however, whether they jointly mediate hypertrophic process remains unexplored. Hyperhomocysteinemia promotes cardiac hypertrophy while the involvement of sEH and TRPC3 channels remains unknown. This study aimed to explore the role of, and interrelation between sEH and TRPC3 channels in homocysteine-induced cardiac hypertrophy. METHODS: Rats were fed methionine-enriched diet to induce hyperhomocysteinemia. H9c2 cells and neonatal rat cardiomyocytes were incubated with homocysteine. Cardiac hypertrophy was evaluated by echocardiography, histological examination, immunofluorescence imaging, and expressions of hypertrophic markers. Epoxyeicosatrienoic acids (EETs) were determined by ELISA. TRPC3 current was recorded by patch-clamp. Gene promotor activity was measured using dual-luciferase reporter assay. RESULTS: Inhibition of sEH by 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) reduced ventricular mass, lowered the expression of hypertrophic markers, decreased interstitial collagen deposition, and improved cardiac function in hyperhomocysteinemic rats, associated with restoration of EETs levels in myocardium. TPPU or knockdown of sEH suppressed TRPC3 transcription and translation as well as TRPC3 current that were enhanced by homocysteine. Exogenous 11,12-EET inhibited homocysteine-induced TRPC3 expression and cellular hypertrophy. Silencing C/EBPß attenuated, while overexpressing C/EBPß promoted homocysteine-induced hypertrophy and expressions of sEH and TRPC3, resulting respectively from inhibition or activation of sEH and TRPC3 gene promoters. CONCLUSIONS: sEH and TRPC3 channels jointly contribute to homocysteine-induced cardiac hypertrophy. Homocysteine transcriptionally activates sEH and TRPC3 genes through a common regulatory element C/EBPß. sEH activation leads to an upregulation of TRPC3 channels via a 11,12-EET-dependent manner.

Inhibition of the INa/K and the activation of peak INa contribute to the arrhythmogenic effects of aconitine and mesaconitine in guinea pigs.

Aconitine (ACO), a main active ingredient of Aconitum, is well-known for its cardiotoxicity. However, the mechanisms of toxic action of ACO remain unclear. In the current study, we investigated the cardiac effects of ACO and mesaconitine (MACO), a structurally related analog of ACO identified in Aconitum with undocumented cardiotoxicity in guinea pigs. We showed that intravenous administration of ACO or MACO (25 µg/kg) to guinea pigs caused various types of arrhythmias in electrocardiogram (ECG) recording, including ventricular premature beats (VPB), atrioventricular blockade (AVB), ventricular tachycardia (VT), and ventricular fibrillation (VF). MACO displayed more potent arrhythmogenic effect than ACO. We conducted whole-cell patch-clamp recording in isolated guinea pig ventricular myocytes, and observed that treatment with ACO (0.3, 3 µM) or MACO (0.1, 0.3 µM) depolarized the resting membrane potential (RMP) and reduced the action potential amplitude (APA) and durations (APDs) in a concentration-dependent manner. The ACO- and MACO-induced AP remodeling was largely abolished by an INa blocker tetrodotoxin (2 µM) and partly abolished by a specific Na+/K+ pump (NKP) blocker ouabain (0.1 µM). Furthermore, we observed that treatment with ACO or MACO attenuated NKP current (INa/K) and increased peak INa by accelerating the sodium channel activation with the EC50 of 8.36 ± 1.89 and 1.33 ± 0.16 µM, respectively. Incubation of ventricular myocytes with ACO or MACO concentration-dependently increased intracellular Na+ and Ca2+ concentrations. In conclusion, the current study demonstrates strong arrhythmogenic effects of ACO and MACO resulted from increasing the peak INa via accelerating sodium channel activation and inhibiting the INa/K. These results may help to improve our understanding of cardiotoxic mechanisms of ACO and MACO, and identify potential novel therapeutic targets for Aconitum poisoning.

Protection of dilator function of coronary arteries from homocysteine by tetramethylpyrazine: Role of ER stress in modulation of BKCa channels.

OBJECTIVES: We recently reported the involvement of ER stress-mediated BKCa channel inhibition in homocysteine-induced coronary dilator dysfunction. In another study, we demonstrated that tetramethylpyrazine (TMP), an active ingredient of the Chinese herb Chuanxiong, possesses potent anti-ER stress capacity. The present study investigated whether TMP protects BKCa channels from homocysteine-induced inhibition and whether suppression of ER stress is a mechanism contributing to the protection. Furthermore, we explored the signaling transduction involved in TMP-conferred protection on BKCa channels. METHODS: BKCa channel-mediated relaxation was studied in porcine small coronary arteries. Expressions of BKCa channel subunits, ER stress molecules, and E3 ubiquitin ligases, as well as BKCa ubiquitination were determined in porcine coronary arterial smooth muscle cells (PCASMCs). Whole-cell BKCa currents were recorded. RESULTS: Exposure of PCASMCs to homocysteine or the chemical ER stressor tunicamycin increased the expression of ER stress molecules, which was significantly inhibited by TMP. Suppression of ER stress by TMP preserved the BKCa ß1 protein level and restored the BKCa current in PCASMCs, concomitant with an improved BKCa-mediated dilatation in coronary arteries. TMP attenuated homocysteine-induced BKCa ß1 protein ubiquitination, in which inhibition of ER stress-mediated FoxO3a activation and FoxO3a-dependent atrogin-1 and Murf-1 was involved. CONCLUSIONS: Reversal of BKCa channel inhibition via suppressing ER stress-mediated loss of ß1 subunits contributes to the protective effect of TMP against homocysteine on coronary dilator function. Inhibition of FoxO3a-dependent ubiquitin ligases is involved in TMP-conferred normalization of BKCa ß1 protein level. These results provide new mechanistic insights into the cardiovascular benefits of TMP.

Activation of PERK branch of ER stress mediates homocysteine-induced BKCa channel dysfunction in coronary artery via FoxO3a-dependent regulation of atrogin-1.

The molecular mechanism of endoplasmic reticulum (ER) stress in vascular pathophysiology remains inadequately understood. We studied the role of ER stress in homocysteine-induced impairment of coronary dilator function, with uncovering the molecular basis of the effect of ER stress on smooth muscle large-conductance Ca2+-activated K+ (BKCa) channels. The vasodilatory function of BKCa channels was studied in a myograph using endothelium-denuded porcine small coronary arteries. Primary cultured porcine coronary artery smooth muscle cells were used for mRNA and protein measurements and current recording of BKCa channels. Homocysteine inhibited vasorelaxant response to the BKCachannel opener NS1619, lowered BKCa ß1 subunit protein level and suppressed BKCa current. Inhibition of ER stress restored BKCa ß1 protein level and NS1619-evoked vasorelaxation. Selective blockade of the PKR-like ER kinase (PERK) yielded similarly efficient restoration of BKCa ß1, preserving BKCa current and BKCa-mediated vasorelaxation. The restoration of BKCa ß1 by PERK inhibition was associated with reduced atrogin-1 expression and decreased nuclear localization of forkhead box O transcription factor 3a (FoxO3a). Silencing of atrogin-1 prevented homocysteine-induced BKCa ß1 loss and silencing of FoxO3a prevented atrogin-1 upregulation induced by homocysteine, accompanied by preservation of BKCa ß1 protein level and BKCa current. ER stress mediates homocysteine-induced BKCa channel inhibition in coronary arteries. Activation of FoxO3a by PERK branch underlies the ER stress-mediated BKCa inhibition through a mechanism involving ubiquitin ligase-enhanced degradation of the channel ß1 subunit.

Impairment of Coronary Endothelial Function by Hypoxia-Reoxygenation Involves TRPC3 Inhibition-mediated KCa Channel Dysfunction: Implication in Ischemia-Reperfusion Injury.

Despite increasing knowledge of the significance of calcium-activated potassium (KCa) and canonical transient receptor potential (TRPC) channels in endothelial physiology, no studies so far have investigated the link between these two distinct types of channels in the control of vascular tone in pathological conditions. We previously demonstrated that hypoxia-reoxygenation (H-R) inhibits endothelial KCa and TRPC3 channels in porcine coronary arteries (PCAs). The present study further investigated whether modulation of TRPC3 is involved in H-R-induced KCa channel inhibition and associated vasodilatory dysfunction using approaches of wire myography, whole-cell voltage-clamp, and coimmunoprecipitation. Pharmacological inhibition or siRNA silencing of TRPC3 significantly suppressed bradykinin-induced intermediate- and small-conductance KCa (IKCa and SKCa) currents in endothelial cells of PCAs (PCAECs). TRPC3 protein exists in physical association with neither IKCa nor SKCa. In H-R-exposed PCAECs, the response of IKCa and SKCa to bradykinin-stimulation and to TRPC3-inhibition was markedly weakened. Activation of TRPC3 channels restored H-R-suppressed KCa currents in association with an improved endothelium-derived hyperpolarizing factor (EDHF)-type vasorelaxation. We conclude that inhibition of TRPC3 channels contributes to H-R-induced suppression of KCa channel activity, which serves as a mechanism underlying coronary endothelial dysfunction in ischemia-reperfusion (I-R) injury and renders TRPC3 a potential target for endothelial protection in I-R conditions.

ER stress mediates homocysteine-induced endothelial dysfunction: Modulation of IKCa and SKCa channels.

OBJECTIVE: It remains incompletely understood how homocysteine impairs endothelial function. Whether mechanisms such as calcium-activated potassium (KCa) channels are involved is uncertain and the significance of endoplasmic reticulum (ER) stress in KCa channel-dependent endothelial function in hyperhomocysteinemia remains unexplored. We investigated the effect of homocysteine on endothelial KCa channels in coronary vasculature with further exploration of the role of ER stress. METHODS: Vasorelaxation mediated by intermediate- and small-conductance KCa (IKCa and SKCa) channels was studied in porcine coronary arteries in a myograph. IKCa and SKCa channel currents were recorded by whole-cell patch-clamp in coronary endothelial cells. Protein levels of endothelial IKCa and SKCa channels were determined for both whole-cell and surface expressions. RESULTS: Homocysteine impaired bradykinin-induced IKCa and SKCa-dependent EDHF-type relaxation and attenuated the vasorelaxant response to the channel activator. IKCa and SKCa currents were suppressed by homocysteine. Inhibition of ER stress during homocysteine exposure enhanced IKCa and SKCa currents, associated with improved EDHF-type response and channel activator-induced relaxation. Homocysteine did not alter whole-cell protein levels of IKCa and SKCa whereas lowered surface expressions of these channels, which were restored by ER stress inhibition. CONCLUSIONS: Homocysteine induces endothelial dysfunction through a mechanism involving ER stress-mediated suppression of IKCa and SKCa channels. Inhibition of cell surface expression of these channels by ER stress is, at least partially, responsible for the suppressive effect of homocysteine on the channel function. This study provides new mechanistic insights into homocysteine-induced endothelial dysfunction and advances our knowledge of the significance of ER stress in vascular disorders.