Metabolic Maturation Exaggerates Abnormal Calcium Handling in a Lamp2 Knockout Human Pluripotent Stem Cell-Derived Cardiomyocyte Model of Danon Disease.

Danon disease (DD) is caused by mutations of the gene encoding lysosomal-associated membrane protein type 2 (LAMP2), which lead to impaired autophagy, glycogen accumulation, and cardiac hypertrophy. However, it is not well understood why a large portion of DD patients develop arrhythmia and sudden cardiac death. In the current study, we generated LAMP2 knockout (KO) human iPSC-derived cardiomyocytes (CM), which mimic the LAMP2 dysfunction in DD heart. Morphologic analysis demonstrated the sarcomere disarrangement in LAMP2 KO CMs. In functional studies, LAMP2 KO CMs showed near-normal calcium handling at base level. However, treatment of pro-maturation medium (MM) exaggerated the disease phenotype in the KO cells as they exhibited impaired calcium recycling and increased irregular beating events, which recapitulates the pro-arrhythmia phenotypes of DD patients. Further mechanistic study confirmed that MM treatment significantly enhanced the autophagic stress in the LAMP2 KO CMs, which was accompanied by an increase of both cellular and mitochondrial reactive oxygen species (ROS) levels. Excess ROS accumulation in LAMP2 KO CMs resulted in the over-activation of calcium/calmodulin dependent protein kinase IIδ (CaMKIIδ) and arrhythmogenesis, which was partially rescued by the treatment of ROS scavenger. In summary, our study has revealed ROS induced CaMKIIδ overactivation as a key mechanism that promotes cardiac arrhythmia in DD patients.

Ultrafine particles from diesel vehicle emissions at different driving cycles induce differential vascular pro-inflammatory responses: implication of chemical components and NF-kappaB signaling.

BACKGROUND: Epidemiological evidence supports the association between exposure to ambient particulate matter (PM) and cardiovascular diseases. Chronic exposure to ultrafine particles (UFP; Dp <100 nm) is reported to promote atherosclerosis in ApoE knockout mice. Atherogenesis-prone factors induce endothelial dysfunction that contributes to the initiation and progression of atherosclerosis. We previously demonstrated that UFP induced oxidative stress via c-Jun N-terminal Kinases (JNK) activation in endothelial cells. In this study, we investigated pro-inflammatory responses of human aortic endothelial cells (HAEC) exposed to UFP emitted from a diesel truck under an idling mode (UFP1) and an urban dynamometer driving schedule (UFP2), respectively. We hypothesize that UFP1 and UFP2 with distinct chemical compositions induce differential pro-inflammatory responses in endothelial cells. RESULTS: UFP2 contained a higher level of redox active organic compounds and metals on a per PM mass basis than UFP1. While both UFP1 and UFP2 induced superoxide production and up-regulated stress response genes such as heme oxygenease-1 (HO-1), OKL38, and tissue factor (TF), only UFP2 induced the expression of pro-inflammatory genes such as IL-8 (2.8 +/- 0.3-fold), MCP-1 (3.9 +/- 0.4-fold), and VCAM (6.5 +/- 1.1-fold) (n = 3, P < 0.05). UFP2-exposed HAEC also bound to a higher number of monocytes than UFP1-exposed HAEC (Control = 70 +/- 7.5, UFP1 = 106.7 +/- 12.5, UFP2 = 137.0 +/- 8.0, n = 3, P < 0.05). Adenovirus NF-kappaB Luciferase reporter assays revealed that UFP2, but not UFP1, significantly induced NF-kappaB activities. NF-kappaB inhibitor, CAY10512, significantly abrogated UFP2-induced pro-inflammatory gene expression and monocyte binding. CONCLUSION: While UFP1 induced higher level of oxidative stress and stress response gene expression, only UFP2, with higher levels of redox active organic compounds and metals, induced pro-inflammatory responses via NF-kappaB signaling. Thus, UFP with distinct chemical compositions caused differential response patterns in endothelial cells.

Ultrafine particles from diesel engines induce vascular oxidative stress via JNK activation.

Exposure to particulate air pollution is linked to increased incidences of cardiovascular diseases. Ambient ultrafine particles (UFP) from diesel vehicle engines have been shown to be proatherogenic in ApoE knockout mice and may constitute a major cardiovascular risk in humans. We posited that circulating nano-sized particles from traffic pollution sources induce vascular oxidative stress via JNK activation in endothelial cells. Diesel UFP were collected from a 1998 Kenworth truck. Intracellular superoxide assay revealed that these UFP dose-dependently induced superoxide (O(2)(-)) production in human aortic endothelial cells (HAEC). Flow cytometry showed that UFP increased MitoSOX red intensity specific for mitochondrial superoxide. Protein carbonyl content was increased by UFP as an indication of vascular oxidative stress. UFP also up-regulated heme oxygenase-1 (HO-1) and tissue factor (TF) mRNA expression, and pretreatment with the antioxidant N-acetylcysteine significantly decreased their expression. Furthermore, UFP transiently activated JNK in HAEC. Treatment with the JNK inhibitor SP600125 and silencing of both JNK1 and JNK2 with siRNA inhibited UFP-stimulated O(2)(-) production and mRNA expression of HO-1 and TF. Our findings suggest that JNK activation plays an important role in UFP-induced oxidative stress and stress response gene expression.