Objectives Vitamin D deficiency has been associated with incident cardiovascular disease and cardiovascular mortality. Vitamin D receptor (VDR) activation through vitamin D or its analogues attenuates cardiomyocyte hypertrophy and cardiac dysfunction. Whether VDR status influences cardiomyocyte fate in the setting of myocardial ischaemia/reperfusion (MI/R) injury remains unknown. Our aim was to determine effects of cardiac VDR activation on MI/R injury.
Methods The mice were randomly assigned to seven groups: sham, vehicle, paricalcitol (PC), In vivo VDR-siRNA + PC, In vivo VDR-siRNA + vehicle, In vivo control siRNA + PC, and In vivo control siRNA + vehicle. PC was administered through intraperitoneal injection at 20 ng/mouse/day for 2 days before MI/R. The last dose was administered at 15 min before reperfusion. The vehicle groups received vehicle injections using the same schedule. In vivo siRNA-mediated cardiac-specific VDR gene silencing was performed 48 h before MI/R. Following 30 minutes of ischaemia, the myocardium was reperfused. The mice were reperfused for 15 min (for protein phosphorylation levels), 3 h (for apoptosis, oxidation, nitration,and inflammation assays), 24 h (for cardiac function and infarct size assays), or 4 weeks (for left ventricular remodelling). The myocardial infarct size was determined using Evans blue-TTC double staining methods. Cardiac function was measured using an echocardiographic imaging system. DHE was used to detect ROS generation within the myocardium in situ. The nitrotyrosine levels were measured using immunostaining and ELISA.caspase-9, caspase-12, cleaved-caspase-8,gp91phox, iNOS, phospho-JNK, and phospho-p38 MAPK were detected using western blot analysis. ANP, β-MHC, TNFα, IL1β, and IL6 expression were determined using real-time quantitative PCR.
Results Low VDR expression was detected in the mouse heart, and MI/R upregulated VDR expression. The activation of VDR through PC reduced myocardial infarct size (23% vs. 44%, p < 0.01), improved cardiac function[left ventricular ejection fraction (61% vs. 48%, p < 0.01) and left ventricular fractional shortening (32% vs. 24%, p < 0.01)], and prevented adverse cardiac remodelling [ANP(-40%, p < 0.05) and β-MHC (-44%, p < 0.01)] after MI/R injury compared with the vehicle group. Mechanistic studies demonstrated that VDR activation reduced reactive oxygen species (ROS) and nitrotyrosine production through the inhibition of gp91phox and iNOS expression. Moreover, VDR activation suppressed the activation of proinflammatory kinases (p38 and JNK) and inflammatory cytokines [TNFα(-34%, p < 0.01), IL6(-73%, p < 0.05), and IL1β(-86% p < 0.05)]. VDR activation also attenuated the intrinsic mitochondrial-caspase 9, endoplasmic reticulum (ER) stress-caspase 12, and extrinsic death receptor-caspase 8 pathways apoptosis. TUNEL staining showed PC treatment attenuated the amount of apoptosis by 37% compared with vehicle administration, p < 0.01. Furthermore, an In vivo cardiac-specific VDR knockdown experiment demonstrated that intact VDR signalling was required for the anti-oxidative/anti-nitrative and anti-inflammatory effect of PC.
Conclusions The results obtained in this study demonstrate VDR is a potent cardioprotective receptor against MI/R injury through the inhibition of oxidative/nitrative stress and inflammation and subsequent intrinsic and extrinsic caspase-mediated apoptosis. VDR represents a potential molecular therapeutic target for the treatment of ischaemic heart disease.