The Unfolded Protein Response in Amelogenesis and Enamel Pathologies

During the secretory phase of their life-cycle, ameloblasts are highly specialized secretory cells whose role is to elaborate an extracellular matrix that ultimately confers both form and function to dental enamel, the most highly mineralized of all mammalian tissues. In common with many other “professional” secretory cells, ameloblasts employ the unfolded protein response (UPR) to help them cope with the large secretory cargo of extracellular matrix proteins transiting their ER (endoplasmic reticulum)/Golgi complex and so minimize ER stress. However, the UPR is a double-edged sword, and, in cases where ER stress is severe and prolonged, the UPR switches from pro-survival to pro-apoptotic mode. The purpose of this review is to consider the role of the ameloblast UPR in the biology and pathology of amelogenesis; specifically in respect of amelogenesis imperfecta (AI) and fluorosis. Some forms of AI appear to correspond to classic proteopathies, where pathological intra-cellular accumulations of protein tip the UPR toward apoptosis. Fluorosis also involves the UPR and, while not of itself a classic proteopathic disease, shares some common elements through the involvement of the UPR. The possibility of therapeutic intervention by pharmacological modulation of the UPR in AI and fluorosis is also discussed

Targeting the mitochondrial inner membrane to improve bioenergetics in the diseased heart

Cardiovascular diseases continue to exact unparalleled economic and humanitarian costs across the globe. Manifestations of cardiovascular diseases include acute coronary syndromes and heart failure, both of which are exacerbated in diabetic patients. Although the underlying cellular culprits responsible for these cardiomyopathies are multi-factorial, aberrant cellular bioenergetics is emerging as a central component. Decrements in mitochondrial function impair cardiac function, and accordingly the development of novel therapies that improve cardiac function by targeting mitochondria has enormous therapeutic potential. In the work presented herein, we studied two diseases where impaired bioenergetics comprises a central component: diabetes, and ischemia/reperfusion injury. In diabetic heart studies, we determined the mechanisms responsible for the decline in mitochondrial bioenergetics of the diabetic heart. Comprehensive mitochondrial functional assays coupled with molecular techniques were employed. Our results showed that mitochondrial respiration and reactive oxygen species buffering capacity were significantly decreased in diabetic hearts. Diabetic mitochondria displayed aberrant mitochondrial calcium handling, post-translational oxidative modification of the adenine nucleotide translocase, increased sensitivity to permeability transition pore opening, and lowered overall expression of proteins involved in the electron transport system. These effects led to inefficient energy supply-demand matching and heightened reperfusion injury in intact heart studies. Treatment with several novel therapies that target the mitochondrial inner membrane reduced the extent of injury and restored mitochondrial function in the diabetic heart. In ischemia/reperfusion studies, we tested the hypothesis that aberrant respiration in the post-ischemic heart was due to impaired molecular organization along the inner mitochondrial membrane. Specifically, we used a novel respiratory substrate-inhibitor titration protocol to determine complex-specific changes, in the electron transport system, that lead to poor respiration. These respiratory studies were coupled with experiments using native gel electrophoresis, allowing us to link changes in respiration to altered expression of native respiratory "supercomplex" clusters. The decrease in mitochondrial respiration after ischemia/reperfusion was observed along several different sites of the electron transport system. These changes were associated with lower supercomplex expression, and altered levels of several native respiratory complexes. Post-ischemic treatment with a mitochondria-targeting peptide restored supercomplex assembly and was associated with improved respiration and a decreased extent of injury. Taken together, the results presented herein provide new insight into the molecular and functional alterations that occur along the mitochondrial inner membrane in diabetic and post-ischemic hearts. These data provide a basis for novel therapies targeting the inner mitochondrial membrane as viable pharmacological approaches to improving bioenergetics in diseased myocardium.  Ph.D

Targeting the mitochondrial inner membrane to improve bioenergetics in the diseased heart

Cardiovascular diseases continue to exact unparalleled economic and humanitarian costs across the globe. Manifestations of cardiovascular diseases include acute coronary syndromes and heart failure, both of which are exacerbated in diabetic patients. Although the underlying cellular culprits responsible for these cardiomyopathies are multi-factorial, aberrant cellular bioenergetics is emerging as a central component. Decrements in mitochondrial function impair cardiac function, and accordingly the development of novel therapies that improve cardiac function by targeting mitochondria has enormous therapeutic potential. In the work presented herein, we studied two diseases where impaired bioenergetics comprises a central component: diabetes, and ischemia/reperfusion injury. In diabetic heart studies, we determined the mechanisms responsible for the decline in mitochondrial bioenergetics of the diabetic heart. Comprehensive mitochondrial functional assays coupled with molecular techniques were employed. Our results showed that mitochondrial respiration and reactive oxygen species buffering capacity were significantly decreased in diabetic hearts. Diabetic mitochondria displayed aberrant mitochondrial calcium handling, post-translational oxidative modification of the adenine nucleotide translocase, increased sensitivity to permeability transition pore opening, and lowered overall expression of proteins involved in the electron transport system. These effects led to inefficient energy supply-demand matching and heightened reperfusion injury in intact heart studies. Treatment with several novel therapies that target the mitochondrial inner membrane reduced the extent of injury and restored mitochondrial function in the diabetic heart. In ischemia/reperfusion studies, we tested the hypothesis that aberrant respiration in the post-ischemic heart was due to impaired molecular organization along the inner mitochondrial membrane. Specifically, we used a novel respiratory substrate-inhibitor titration protocol to determine complex-specific changes, in the electron transport system, that lead to poor respiration. These respiratory studies were coupled with experiments using native gel electrophoresis, allowing us to link changes in respiration to altered expression of native respiratory "supercomplex" clusters. The decrease in mitochondrial respiration after ischemia/reperfusion was observed along several different sites of the electron transport system. These changes were associated with lower supercomplex expression, and altered levels of several native respiratory complexes. Post-ischemic treatment with a mitochondria-targeting peptide restored supercomplex assembly and was associated with improved respiration and a decreased extent of injury. Taken together, the results presented herein provide new insight into the molecular and functional alterations that occur along the mitochondrial inner membrane in diabetic and post-ischemic hearts. These data provide a basis for novel therapies targeting the inner mitochondrial membrane as viable pharmacological approaches to improving bioenergetics in diseased myocardium

Abstract 335: Exercise Protects the Heart by Preserving Mitochondrial Membrane Potential During Early Reperfusion

Exercise evokes adaptations intrinsic to the myocardium that protect against ventricular arrhythmia, yet the underlying mechanisms are not completely understood. We have previously shown that the transition to arrhythmia occurs concomitant with a collapse in mitochondrial membrane potential (ΔΨm). As our previous studies indicated that exercise preserves intracellular redox homeostasis, which directly influences mitochondrial energetics, we hypothesized that rats exposed to exercise (Ex, 10 d of treadmill running) would be protected against reperfusion arrhythmia via better maintenance of ΔΨm. To fully understand the temporal relationship between ΔΨm and cardiac electrical activity, two-photon microscopy images (using the fluorescent probe TMRM) and volume-conducted electrocardiogram were simultaneously recorded. Langendorff-perfused hearts underwent 40/30 min of ischemia/reperfusion. Exercise lowered the incidence of arrhythmia, with 3 of 8 Ex hearts experiencing tachycardia or fibrillation compared to 7 of 8 sedentary (Sed) hearts. Ex prevented the collapse of ΔΨm during the first 10 min of reperfusion (74±6.4% v 57±1.5% of baseline fluorescence intensity; P&lt;0.05). To gain a more comprehensive understanding of energetics throughout the heterogeneous mitochondrial population, we then measured mean TMRM fluorescence intensity in isolated ventricular mitochondria harvested after reperfusion using flow cytometry (n=100,000 events per group). Interestingly, mean fluorescence intensity for ΔΨm was similar in Ex and Sed mitochondria (278±33 v 309 ±44 AU, respectively). Mitochondrial respiratory control ratios were also similar in Ex and Sed (9.03±0.70 v 9.00±0.92, respectively). Taken together, the isolated mitochondrial assessment did not reflect what was observed in vivo. This suggests that either intracellular factors influenced in vivo mitochondrial energetics, or our isolated mitochondria may have been enriched with predominantly healthy mitochondria. Our studies demonstrate for the first time that exercise prevents electrical dysfunction following an ischemic insult through better preservation of mitochondrial energetics, and that this preservation is only observed in the intact organ.</jats:p

Abstract 225: The Mitochondria-localizing Peptide Bendavia Reduces Cardiac Injury By Targeting Cardiolipin

Bendavia is a cardioprotective mitochondria-targeting peptide that is currently being utilized in the EMBRACE-STEMI clinical trial for acute coronary syndromes (ACS). Across a variety of pre-clinical models examining ACS and heart failure, administration of Bendavia reduces infarction and coronary “no-reflow”, improves function, preserves mitochondrial energetics, and lowers ROS-dependent cell death. Despite the clear effects in mitigating reperfusion injury, Bendavia’s molecular targets and underlying mechanisms are not fully understood. The objective of these studies was to determine Bendavia’s molecular mechanism of action and pharmacokinetic profile. In diabetic animals, Bendavia treatment reversed the decline in 18:2 cardiolipin composition observed in diabetic heart. In vivo (i.v.) administration of 3H-Bendavia showed tissue distribution that correlated with tissue mitochondrial volume density. 3H-Bendavia levels in heart mitochondria peaked 1 hour after treatment, and by 24 hours there was no discernible 3H-Bendavia in the myocardium. Biologic processing of Bendavia results in two major metabolites, and neither of these metabolites showed cardioprotective efficacy, suggesting that the cardioprotection is unique to the intact, tetra-peptide. In isolated heart cells, 3H-Bendavia uptake was found to be independent of mitochondrial membrane potential, and respiratory studies revealed no discernible uncoupling activity. We then tested the hypothesis that Bendavia selectively targeted cardiolipin biophysical organization by synthesizing lipid vesicles of differing phospholipid composition. Fluorescence quenching and electrostatically sensitive probes revealed Bendavia preferentially associated with cardiolipin-enriched lipid vesicles. We modeled changes in inner membrane lipids during cardiac ischemia/reperfusion. Bendavia augmented membrane microviscosity in proportion to the cardiolipin content. As a number of disease states (heart failure, diabetes, ischemia/reperfusion) are characterized by low membrane microviscosity, Bendavia’s putative ability to restore the lipid microenvironment represents a novel paradigm for preserving tissue bioenergetics.</jats:p