CNPase, a 2’,3’-Cyclic-nucleotide 3’-phosphodiesterase, as a Therapeutic Target to Attenuate Cardiac Hypertrophy by Enhancing Mitochondrial Energy Production

Heart failure is the end-stage of all cardiovascular diseases with a ~25% 5-year survival rate, and insufficient mitochondrial energy production to meet myocardial demand is the hallmark of heart failure. Mitochondrial components involved in the regulation of ATP production remain to be fully elucidated. Recently, roles of 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) in the pathophysiological processes of heart diseases have emerged, implicated by evidence that mitochondrial CNPase proteins are associated with mitochondrial integrity under metabolic stress. In this study, a zebrafish heart failure model was established, by employing antisense morpholino oligonucleotides and the CRISPR-Cas9 gene-editing system, which recapitulates heart failure phenotypes including heart dysfunction, pericardial edema, ventricular enlargement, bradycardia, and premature death. The translational implications of CNPase in the pathophysiological process of heart failure were tested in a pressure overload-induced heart hypertrophy model, which was carried out in rats through transverse abdominal aorta constriction (TAAC). AAV9-mediated myocardial delivery of CNPase mitigated the hypertrophic response through the specific hydrolysis of 2′-3′-cyclic nucleotides, supported by the decrease of cardiac hypertrophy and fibrosis, the integrity of mitochondrial ultrastructure, and indicators of heart contractility in the AAV9-TAAC group. Finally, the biometrics of a mitochondrial respiration assay carried out on a Seahorse cellular energy analyzer demonstrated that CNPase protects mitochondrial respiration and ATP production from AngII-induced metabolic stress. In summary, this study provides mechanistic insights into CNPase-2′,3′-cyclic nucleotide metabolism that protects the heart from energy starvation and suggests novel therapeutic approaches to treat heart failure by targeting CNPase activity.

Mitochondrial ATP production is required for endothelial cell control of vascular tone

Arteries and veins are lined by non-proliferating endothelial cells that play a critical role in regulating blood flow. Endothelial cells also regulate tissue perfusion, metabolite exchange, and thrombosis. It is thought that endothelial cells rely on ATP generated via glycolysis to fuel each of these energy-demanding processes. However, endothelial metabolism has mainly been studied in the context of proliferative cells in angiogenesis, and little is known about energy production in endothelial cells within the fully-formed vascular wall. Using intact arteries isolated from rats and mice, we show that inhibiting mitochondrial oxidative phosphorylation disrupts endothelial control of vascular tone. The role for endothelial cell energy production is independent of species, sex, or vascular bed. Basal, mechanically-activated, and agonist-evoked calcium activity in intact artery endothelial cells are each prevented by inhibiting mitochondrial ATP synthesis. This effect is mimicked by blocking the transport of pyruvate, the master fuel for mitochondrial energy production, through the mitochondrial pyruvate carrier. These data show that mitochondrial ATP is necessary for calcium-dependent, nitric oxide mediated endothelial control of vascular tone, and identifies the critical role of endothelial mitochondrial energy production in fueling perfused blood vessel function.

Mitochondrial energy dysfunction induces remodeling of the cardiac mitochondrial protein acylome

ABSTRACTMitochondria are increasingly recognized as signaling organelles because, under conditions of stress, mitochondria can trigger various signaling pathways to coordinate the cell’s response. The specific pathway(s) engaged by mitochondria in response to defects in mitochondrial energy production in vivo and in high-energy tissues like the heart are not fully understood. Here, we investigated cardiac pathways activated in response to mitochondrial energy dysfunction by studying mice with cardiomyocyte-specific loss of the mitochondrial phosphate carrier (SLC25A3), an established model that develops cardiomyopathy as a result of defective mitochondrial ATP synthesis. In heart tissue from these mice, mitochondrial energy dysfunction induced a striking pattern of acylome remodeling, with significantly increased post-translational acetylation and malonylation. Mass spectrometry-based proteomics further revealed that energy dysfunction-induced remodeling of the acetylome and malonylome preferentially impacts mitochondrial proteins. Acetylation and malonylation modified a highly interconnected interactome of mitochondrial proteins, and both modifications were present on the enzyme isocitrate dehydrogenase 2 (IDH2). Intriguingly, IDH2 activity was enhanced in SLC25A3-deleted mitochondria, and further study of IDH2 sites targeted by both acetylation and malonylation revealed that these modifications can have site-specific and distinct functional effects. Finally, we uncovered a novel crosstalk between the two modifications, whereby mitochondrial energy dysfunction-induced acetylation of sirtuin 5 (SIRT5), inhibited its function. Because SIRT5 is a mitochondrial deacylase with demalonylase activity, this finding suggests that acetylation can modulate the malonylome. Together, our results position acylations as an arm of the mitochondrial response to energy dysfunction and suggest a mechanism by which focal disruption to the mitochondrial energy production machinery can have an expanded impact on global mitochondrial function.

Methylpiperidinopyrazole Attenuates Estrogen-Induced Mitochondrial Energy Production and Subsequent Osteoblast Maturation via an Estrogen Receptor Alpha-Dependent Mechanism

An estrogen deficiency is the main cause of osteoporosis in postmenopausal women. In bone remodeling, estrogen receptors (ERs) can mediate estrogen-transducing signals. Methylpiperidinopyrazole (MPP) is a highly specific antagonist of ER-alpha (ERα). This study was designed to evaluate the effects of MPP on estrogen-induced energy production, subsequent osteoblast maturation, and the possible mechanisms. Exposure of primary osteoblasts isolated from neonatal rat calvarias to MPP did not affect cell morphology or survival. Estradiol can induce translocation of ERα into mitochondria from the cytoplasm. Interestingly, pretreatment of rat calvarial osteoblasts with MPP lowered estrogen-induced ERα translocation. Sequentially, estrogen-triggered expressions of mitochondrial energy production-linked cytochrome c oxidase (COX) I and COX II messenger (m)RNAs were inhibited following pretreatment with MPP. Consequently, MPP caused decreases in estrogen-triggered augmentation of the activities of mitochondrial respiratory complex enzymes and levels of cellular adenosine phosphate (ATP). During progression of osteoblast maturation, estrogen induced bone morphogenetic protein (BMP)-6 and type I collagen mRNA expressions, but MPP treatment inhibited such induction. Consequently, estrogen-induced osteoblast activation and mineralization were attenuated after exposure to MPP. Taken together, MPP suppressed estrogen-induced osteoblast maturation through decreasing chromosomal osteogenesis-related BMP-6 and type I collagen mRNA expressions and mitochondrial ATP synthesis due to inhibiting energy production-linked COX I and II mRNA expressions. MPP can appropriately be applied to evaluate estrogen-involved bioenergetics and osteoblast maturation.

Serial cryoFIB/SEM reveals profound cytoarchitectural disruptions caused by a pathogenic mutation in Leigh syndrome patient cells

The advancement of serial cryo-FIB/SEM offers a new opportunity to study large volumes of near-native, fully hydrated frozen cells and tissues at voxel sizes of 10 nm and below. We explored this capability for pathologic characterization of vitrified human patient cells. We demonstrate profound disruption of subcellular architecture in primary fibroblasts from a Leigh syndrome patient harboring a disease-causing mutation in USMG5 protein responsible for impaired mitochondrial energy production.