Pancreatic β-cell death in response to pro-inflammatory cytokines is distinct from genuine apoptosis

A reduction in functional β-cell mass leads to both major forms of diabetes; pro-inflammatory cytokines, such as interleukin-1beta (IL-1β) and gamma-interferon (γ-IFN), activate signaling pathways that direct pancreatic β-cell death and dysfunction. However, the molecular mechanism of β-cell death in this context is not well understood. In this report, we tested the hypothesis that individual cellular death pathways display characteristic phenotypes that allow them to be distinguished by the precise biochemical and metabolic responses that occur during stimulus-specific initiation. Using 832/13 and INS-1E rat insulinoma cells and isolated rat islets, we provide evidence that apoptosis is unlikely to be the primary pathway underlying β-cell death in response to IL-1β+γ-IFN. This conclusion was reached via the experimental results of several different interdisciplinary strategies, which included: 1) tandem mass spectrometry to delineate the metabolic differences between IL-1β+γ-IFN exposure versus apoptotic induction by camptothecin and 2) pharmacological and molecular interference with either NF-κB activity or apoptosome formation. These approaches provided clear distinctions in cell death pathways initiated by pro-inflammatory cytokines and bona fide inducers of apoptosis. Collectively, the results reported herein demonstrate that pancreatic β-cells undergo apoptosis in response to camptothecin or staurosporine, but not pro-inflammatory cytokines.     

AMP-activated protein kinase (AMPK) is a tau kinase, activated in response to amyloid β-peptide exposure

Hyperphosphorylation of tau is a hallmark of Alzheimer's disease and other tauopathies. Although the mechanisms underlying hyperphosphorylation are not fully understood, cellular stresses such as impaired energy metabolism are thought to influence the signalling cascade. The AMPK (AMP-activated protein kinase)-related kinases MARK (microtubule-associated protein-regulating kinase/microtubule affinity-regulating kinase) and BRSK (brain-specific kinase) have been implicated in tau phosphorylation, but are insensitive to activation by cellular stress. In the present study, we show that AMPK itself phosphorylates tau on a number of sites, including Ser2?2 and Ser3??, altering microtubule binding of tau. In primary mouse cortical neurons, CaMKKβ (Ca2+/calmodulin-dependent protein kinase kinase β) activation of AMPK in response to Aβ (amyloid-β peptide)-(1-42) leads to increased phosphorylation of tau at Ser2?2/Ser3?? and Ser33??. Activation of AMPK by Aβ-(1-42) is inhibited by memantine, a partial antagonist of the NMDA (N-methyl-D-aspartate) receptor and currently licensed for the treatment of Alzheimer's disease. These findings identify a pathway in which Aβ-(1-42) activates CaMKKβ and AMPK via the NMDA receptor, suggesting the possibility that AMPK plays a role in the pathophysiological phosphorylation of tau.     

Threonine 32 (Thr32) of FoxO3 is critical for TGF-β-induced apoptosis via Bim in hepatocarcinoma cells

Transforming growth factor- β (TGF- β) exerts apoptotic effects on various types of malignant cells, including liver cancer cells. However, the precise mechanisms by which TGF- β induces apoptosis remain poorly known. In the present study, we have showed that threonine 32 (Thr32) residue of FoxO3 is critical for TGF- β to induce apoptosis via Bim in hepatocarcinoma Hep3B cells. Our data demonstrated that TGF- β induced FoxO3 activation through specific de-phosphorylation at Thr32. TGF- β-activated FoxO3 cooperated with Smad2/3 to mediate Bim up-regulation and apoptosis. FoxO3 (de)phosphorylation at Thr32 was regulated by casein kinase I- ? (CKI- ?). CKI inhibition by small molecule D4476 could abrogate TGF- β-induced FoxO/Smad activation, reverse Bim up-regulation, and block the sequential apoptosis. More importantly, the deregulated levels of CKI- ? and p32FoxO3 were found in human malignant liver tissues. Taken together, our findings suggest that there might be a CKI-FoxO/Smad-Bim engine in which Thr32 of FoxO3 is pivotal for TGF- β-induced apoptosis, making it a potential therapeutic target for liver cancer treatment.     

Physiological cholesterol concentration is a neuroprotective factor against β-amyloid and β-amyloid-metal complexes toxicity

Alzheimer's disease is one of the most common forms of dementia in the elderly. One of its hallmarks is the abnormal aggregation and deposition of β-amyloid (Aβ). Endogenous and exogenous metal ions seem to influence β-amyloid folding process, aggregation and deposition. Besides these variables other elements appear to affect β-amyloid behavior, such as cholesterol. The physiological concentration of cholesterol in the cerebrospinal fluid (CSF) was used in order to determine the extent in which Aβ and Aβ-metal complexes in vitro aggregation and their toxicity on human neuroblastoma cell cultures is affected. Cholesterol did not appear to influence Aβ and Aβ-metal complexes aggregation, but it was effective in protecting neuroblastoma cells against Aβ complexes' toxicity. The Aβ-Al complex seemed to be the most effective in disrupting and damaging membrane external layer, and simultaneously it appears to increase its toxicity on cell cultures; both of these effects are preventable by cholesterol. The presence in physiological concentrations of cholesterol seemed to compensate membrane damage that occurred to neuroblastoma cells. These findings appear to contradict some data reported in literature. We believe that our results might shed some light on the role played by cholesterol at physiological concentrations in both cellular balance and membrane protection.     

PKR downregulation prevents neurodegeneration and β-amyloid production in a thiamine-deficient model

Brain thiamine homeostasis has an important role in energy metabolism and displays reduced activity in Alzheimer''s disease (AD). Thiamine deficiency (TD) induces regionally specific neuronal death in the animal and human brains associated with a mild chronic impairment of oxidative metabolism. These features make the TD model amenable to investigate the cellular mechanisms of neurodegeneration. Once activated by various cellular stresses, including oxidative stress, PKR acts as a pro-apoptotic kinase and negatively controls the protein translation leading to an increase of BACE1 translation. In this study, we used a mouse TD model to assess the involvement of PKR in neuronal death and the molecular mechanisms of AD. Our results showed that the TD model activates the PKR-eIF2α pathway, increases the BACE1 expression levels of Aβ in specific thalamus nuclei and induces motor deficits and neurodegeneration. These effects are reversed by PKR downregulation (using a specific inhibitor or in PKR knockout mice).Thiamine (vitamin B1) deficiency (TD) induces regionally selective neurodegeneration in the mammal''s brains, particularly in specific thalamus nuclei (submedial thalamus nuclei (SmTN) and ventral lateral nuclei (VLN)) and cerebellum.^{1, 2} TD-induced neuronal loss is associated with a chronic impairment of oxidative metabolism and neuroinflammation associated with glial activation.^{3, 4, 5} Diminished thiamine-dependent processes in humans is not only associated with Wernicke–Korsakoff syndrome but also accompany other neurodegenerative disorders, such as Alzheimer''s disease (AD).⁶ Experimental TD has been used to model the pathogenesis and investigate the cellular mechanisms of neurodegenerative disorders. In mice, TD-induced oxidative stress enhances Aβ accumulation by regulating β-site APP-cleaving enzyme 1 (BACE1) maturation. These effects are amplified in AD mouse model.^{7, 8}The double-stranded RNA-dependent protein kinase (PKR) is one of the four mammalian serine–threonine kinases—the others being HRI (heme-regulated eukaryotic translation initiation factor-2α (eIF2α) kinase), GCN2 (general control nonderepressible 2) and PERK (protein kinase RNA-like endoplasmic reticulum kinase)—that catalyzes the phosphorylation of the α subunit of eIF2 in response to stress signals, leading to an inhibition of protein synthesis.^{9, 10} Activation of PKR is induced by various triggers such as viral infection and endoplasmic reticulum or oxidative stresses^{11, 12} and could be controlled by an interaction with its specific activator PACT (PKR activator), also named RAX in rodents. PKR phosphorylation is known to have a significant role in AD,¹³ and cerebrospinal fluid (CSF) PKR levels could be used as a potential diagnostic biomarker in AD patients.^{14, 15} PKR is a proapoptotic kinase¹⁶ and can exert a number of toxic effects on neurons that could contribute to the functional and pathological alterations in AD brains. PKR also contributes to neurodegeneration and to the pathological molecular mechanisms observed in AD. PKR can mediate Tau phosphorylation induced by Aβ exposure in cell cultures.¹⁷ Additionally, several investigators have demonstrated that eIF2α phosphorylation, via PKR-induced cellular stress, leads to increased BACE1 mRNA translation when, paradoxically, global protein translation is inhibited.^{18, 19, 20, 21} These alterations of BACE1 translational control could be explained by a stress-dependent phenomenon of translation initiation.^{22, 23, 24} Moreover, PKR and eIF2α are highly phosphorylated in SmTN and the cerebellum of TD mouse model. Analyses performed on cerebellar granule neurons exposed to a thiamine metabolic antagonist suggest that TD-induced neuronal death could be partially mediated by PKR activation.²⁵To date, all studies that have explored the deleterious role of PKR activation in neurodegeneration indicate that inhibition of PKR is a promising approach to inhibit pathological mechanisms. Moreover, recent studies have shown that the genetic lack of PKR enhances learning and memory in several behavioral tasks while increasing network excitability.²⁶ The goal of this study was to assess the role of PKR downregulation on neurodegeneration and Aβ production in a mouse model of neurodegeneration.     

B7-H3 drives immunosuppression and Co-targeting with CD47 is a new therapeutic strategy in β-catenin activated melanomas

In melanoma, immune cell infiltration into the tumor is associated with better patient outcomes and response to immunotherapy. T-cell non-inflamed tumors (cold tumors) are associated with tumor cell-intrinsic Wnt/β-catenin activation, and are typically resistant to anti-PD-1 alone or in combination with anti-CTLA-4 therapy. Reversal of the ‘cold tumor’ phenotype and identifying new effective immunotherapies are challenges. We sought to investigate the role of a newer immunotherapy agent, B7-H3, in this setting. RNA sequencing was used to identify co-targeting strategies upon B7-H3 inhibition in a well-defined preclinical melanoma model driven by β-catenin. We found that immune checkpoint molecule B7-H3 confers a suppressive tumor microenvironment by modulating antiviral signals and innate immunity. B7-H3 inhibition led to an inflamed microenvironment, up-regulation of CD47/SIRPa signaling, and together with blockade of the macrophage checkpoint CD47 resulted in additive antitumor responses. We found that the antitumor effects of the B7-H3/CD47 antibody combination were dependent on cytokine signaling pathways (CCR5/CCL5 and IL4).     

Effect of Melatonin and Melatonylvalpromide on β-amyloid and Neurofilaments in N2a Cells

In the present study, we have studied the effect of melatonin (Mt) and melatonin derivative, i.e., melatonylvalpromide (Mtv), on cell viability, β-amyloid (Aβ) production, cell morphology, and expression and phosphorylation of neurofilament proteins in wild-type murine neuroblastoma N2a (N2a/wt) and N2a stably transfected with amyloid precursor protein (N2a/APP) cell lines. The study used MTT assay, Sandwich ELISA, immunocytochemistry and Western blots techniques. The results showed that both Mt and Mtv could increase cell viability, but Mtv did so more effectively. The N2a/APP showed shorter and less amount of cell processes than N2a/wt, and Mtv but not Mt slightly improved the morphological changes in N2A/APP. Both Mt and Mtv suppressed the Aβ level in cell lysates, but the effect of Mtv was stronger than Mt. The immunoreaction to the non-phosphorylated neurofilament proteins probed by SMI32 and SMI33 were remarkably weaker in N2a/APP than N2a/wt, while the immunoreaction to the phosphorylated neurofilament proteins at SMI34 epitopes was slightly stronger in N2a/APP than N2a/wt, suggesting higher phosphorylation level of neurofilament proteins in N2a/APP. Treatment of the cells with Mt and Mtv increased the immunoreaction at SMI32 and SMI33 epitopes, while only Mtv but not Mt decreased the staining at SMI34 epitope, suggesting both Mt and Mtv promote dephosphorylation of neurofilament at SMI32 and SMI33 epitopes, while Mtv stimulates dephosphorylation of neurofilament at SMI34 epitope. These results suggest that Mtv may be a better candidate in arresting the intracellular accumulation of Aβ and protecting the cells from Aβ-related toxicity. Xiao-Chuan Wang and Yin-Chun Zhang equally contributed to the work.     

Metabolic changes in adipose tissues in response to β3-adrenergic receptor activation in mice

Brown and beige adipocytes dissipate energy as heat. Thus, the activation of brown adipocytes and the emergence of beige adipocytes in white adipose tissue (WAT) are suggested to be useful for preventing and treating obesity. Although β₃-adrenergic receptor activation is known to stimulate lipolysis and activation of brown and beige adipocytes, fat depot–dependent changes in metabolite concentrations are not fully elucidated. The current study examined the effect of treatment with CL-316,243, a β₃-adrenergic receptor agonist, on the relative abundance of metabolites in interscapular brown adipose tissue (iBAT), inguinal WAT (ingWAT), and epididymal WAT (epiWAT). Intraperitoneal injection of CL-316,243 (1 mg/kg) for 3 consecutive days increased the relative abundance of several glycolysis-related metabolites in all examined fat depots. The cellular concentrations of metabolites involved in the citric acid cycle and of free amino acids were also increased in epiWAT by CL-316,243. CL-316,243 increased the expression levels of several enzymes and transporters related to glucose metabolism and amino acid catabolism in ingWAT and iBAT but not in epiWAT. CL-316,243 also induced the emergence of more beige adipocytes in ingWAT than in epiWAT. Furthermore, adipocytes surrounded by macrophages were detected in the epiWAT of mice given CL-316,243. The current study reveals the fat depot–dependent modulation of cellular metabolites in CL-316,243-treated mice, presumably resulting from differential regulation of cell metabolism in different cell populations.     

Hypotension in the chronically hypoxic chicken embryo is related to the β-adrenergic response of chorioallantoic and femoral arteries and not to bradycardia

Prolonged fetal hypoxia leads to growth restriction and can cause detrimental prenatal and postnatal alterations. The embryonic chicken is a valuable model to study the effects of prenatal hypoxia, but little is known about its long-term effects on cardiovascular regulation. We hypothesized that chicken embryos incubated under chronic hypoxia would be hypotensive due to bradycardia and βAR-mediated relaxation of the systemic and/or the chorioallantoic (CA) arteries. We investigated heart rate, blood pressure, and plasma catecholamine levels in 19-day chicken embryos (total incubation 21 days) incubated from day 0 in normoxia or hypoxia (14-15% O(2)). Additionally, we studied α-adrenoceptor (αAR)-mediated contraction, relaxation to the β-adrenoceptor (βAR) agonist isoproterenol, and relaxation to the adenylate cyclase activator forskolin in systemic (femoral) and CA arteries (by wire myography). Arterial pressure showed a trend toward hypotension in embryos incubated under chronic hypoxic conditions compared with the controls (mean arterial pressure 3.19 ± 0.18 vs. 2.59 ± 0.13 kPa, normoxia vs. hypoxia, respectively. P = 0.056), without an accompanied bradycardia and elevation in plasma norepinephrine and lactate levels. All vessels relaxed in response to βAR stimulation with isoproterenol, but the CA arteries completely lacked an αAR response. Furthermore, hypoxia increased the sensitivity of femoral arteries (but not CA arteries) to isoproterenol. Hypoxia also increased the responsiveness of femoral arteries to forskolin. In conclusion, we suggest that hypotension in chronic hypoxic chicken embryos is the consequence of elevated levels of circulating catecholamines acting in vascular beds with exclusive (CA arteries) or exacerbated (femoral arteries) βAR-mediated relaxation, and not a consequence of bradycardia.     

FoxO1 as a double-edged sword in the pancreas: analysis of pancreas- and β-cell-specific FoxO1 knockout mice

Diabetes is characterized by an absolute or relative deficiency of pancreatic β-cells. New strategies to accelerate β-cell neogenesis or maintain existing β-cells are desired for future therapies against diabetes. We previously reported that forkhead box O1 (FoxO1) inhibits β-cell growth through a Pdx1-mediated mechanism. However, we also reported that FoxO1 protects against β-cell failure via the induction of NeuroD and MafA. Here, we investigate the physiological roles of FoxO1 in the pancreas by generating the mice with deletion of FoxO1 in the domains of the Pdx1 promoter (P-FoxO1-KO) or the insulin 2 promoter (β-FoxO1-KO) and analyzing the metabolic parameters and pancreatic morphology under two different conditions of increased metabolic demand: high-fat high-sucrose diet (HFHSD) and db/db background. P-FoxO1-KO, but not β-FoxO1-KO, showed improved glucose tolerance with HFHSD. Immunohistochemical analysis revealed that P-FoxO1-KO had increased β-cell mass due to increased islet number rather than islet size, indicating accelerated β-cell neogenesis. Furthermore, insulin-positive pancreatic duct cells were increased in P-FoxO1-KO but not β-FoxO1-KO. In contrast, db/db mice crossed with P-FoxO1-KO or β-FoxO1-KO showed more severe glucose intolerance than control db/db mice due to decreased glucose-responsive insulin secretion. Electron microscope analysis revealed fewer insulin granules in FoxO1 knockout db/db mice. We conclude that FoxO1 functions as a double-edged sword in the pancreas; FoxO1 essentially inhibits β-cell neogenesis from pancreatic duct cells but is required for the maintenance of insulin secretion under metabolic stress.     

Autophagy in microglia degrades extracellular β-amyloid fibrils and regulates the NLRP3 inflammasome

Accumulation of β-amyloid (Aβ) and resultant inflammation are critical pathological features of Alzheimer disease (AD). Microglia, a primary immune cell in brain, ingests and degrades extracellular Aβ fibrils via the lysosomal system. Autophagy is a catabolic process that degrades native cellular components, however, the role of autophagy in Aβ degradation by microglia and its effects on AD are unknown. Here we demonstrate a novel role for autophagy in the clearance of extracellular Aβ fibrils by microglia and in the regulation of the Aβ-induced NLRP3 (NLR family, pyrin domain containing 3) inflammasome using microglia specific atg7 knockout mice and cell cultures. We found in microglial cultures that Aβ interacts with MAP1LC3B-II via OPTN/optineurin and is degraded by an autophagic process mediated by the PRKAA1 pathway. We anticipate that enhancing microglial autophagy may be a promising new therapeutic strategy for AD.     

Autophagy in microglia degrades extracellular β-amyloid fibrils and regulates the NLRP3 inflammasome

Accumulation of β-amyloid (Aβ) and resultant inflammation are critical pathological features of Alzheimer disease (AD). Microglia, a primary immune cell in brain, ingests and degrades extracellular Aβ fibrils via the lysosomal system. Autophagy is a catabolic process that degrades native cellular components, however, the role of autophagy in Aβ degradation by microglia and its effects on AD are unknown. Here we demonstrate a novel role for autophagy in the clearance of extracellular Aβ fibrils by microglia and in the regulation of the Aβ-induced NLRP3 (NLR family, pyrin domain containing 3) inflammasome using microglia specific atg7 knockout mice and cell cultures. We found in microglial cultures that Aβ interacts with MAP1LC3B-II via OPTN/optineurin and is degraded by an autophagic process mediated by the PRKAA1 pathway. We anticipate that enhancing microglial autophagy may be a promising new therapeutic strategy for AD.     

A 3-hydroxy β-end group in xanthophylls is preferentially oxidized to a 3-oxo ε-end group in mammals

We previously found that mice fed lutein accumulated its oxidative metabolites (3′-hydroxy-ε,ε-caroten-3-one and ε,ε-carotene-3,3′-dione) as major carotenoids, suggesting that mammals can convert xanthophylls to keto-carotenoids by the oxidation of hydroxyl groups. Here we elucidated the metabolic activities of mouse liver for several xanthophylls. When lutein was incubated with liver postmitochondrial fraction in the presence of NAD⁺, (3′R,6′R)-3′-hydroxy-β,ε-caroten-3-one and (6RS,3′R,6′R)-3′-hydroxy-ε,ε-caroten-3-one were produced as major oxidation products. The former accumulated only at the early stage and was assumed to be an intermediate, followed by isomerization to the latter. The configuration at the C3′ and C6′ of the ε-end group in lutein was retained in the two oxidation products. These results indicate that the 3-hydroxy β-end group in lutein was preferentially oxidized to a 3-oxo ε-end group via a 3-oxo β-end group. Other xanthophylls such as β-cryptoxanthin and zeaxanthin, which have a 3-hydroxy β-end group, were also oxidized in the same manner as lutein. These keto-carotenoids, derived from dietary xanthophylls, were confirmed to be present in plasma of normal human subjects, and β,ε-caroten-3′-one was significantly increased by the ingestion of β-cryptoxanthin. Thus, humans as well as mice have oxidative activity to convert the 3-hydroxy β-end group of xanthophylls to a 3-oxo ε-end group.     

Allopregnanolone promotes regeneration and reduces β-amyloid burden in a preclinical model of Alzheimer's disease

Previously, we demonstrated that allopregnanolone (APα) promoted proliferation of rodent and human neural progenitor cells in vitro. Further, we demonstrated that APα promoted neurogenesis in the hippocampal subgranular zone (SGZ) and reversed learning and memory deficits in the male triple transgenic mouse model of Alzheimer's (3xTgAD). In the current study, we determined the efficacy of APα to promote the survival of newly generated neural cells while simultaneously reducing Alzheimer's disease (AD) pathology in the 3xTgAD male mouse model. Comparative analyses between three different APα treatment regimens indicated that APα administered 1/week for 6 months was maximally efficacious for simultaneous promotion of neurogenesis and survival of newly generated cells and reduction of AD pathology. We further investigated the efficacy of APα to impact Aβ burden. Treatment was initiated either prior to or post intraneuronal Aβ accumulation. Results indicated that APα administered 1/week for 6 months significantly increased survival of newly generated neurons and simultaneously reduced Aβ pathology with greatest efficacy in the pre-pathology treatment group. APα significantly reduced Aβ generation in hippocampus, cortex, and amygdala, which was paralleled by decreased expression of Aβ-binding-alcohol-dehydrogenase. In addition, APα significantly reduced microglia activation as indicated by reduced expression of OX42 while increasing CNPase, an oligodendrocyte myelin marker. Mechanistic analyses indicated that pre-pathology treatment with APα increased expression of liver-X-receptor, pregnane-X-receptor, and 3-hydroxy-3-methyl-glutaryl-CoA-reductase (HMG-CoA-R), three proteins that regulate cholesterol homeostasis and clearance from brain. Together these findings provide preclinical evidence for the optimal treatment regimen of APα to achieve efficacy as a disease modifying therapeutic to promote regeneration while simultaneously decreasing the pathology associated with Alzheimer's disease.     

Dermal β-catenin activity in response to epidermal Wnt ligands is required for fibroblast proliferation and hair follicle initiation

Dermal fibroblasts are required for structural integrity of the skin and for hair follicle development. Uniform Wnt signaling activity is present in dermal fibroblast precursors preceding hair follicle initiation, but the functional requirement of dermal Wnt signaling at early stages of skin differentiation and patterning remains largely uncharacterized. We show in mice that epidermal Wnt ligands are required for uniform dermal Wnt signaling/β-catenin activity and regulate fibroblast cell proliferation and initiation of hair follicle placodes. In the absence of dermal Wnt signaling/β-catenin activity, patterned upregulation of epidermal β-catenin activity and Edar expression are absent. Conversely, forced activation of β-catenin signaling leads to the formation of thickened dermis, enlarged epidermal placodes and dermal condensates that result in prematurely differentiated enlarged hair follicles. These data reveal functional roles for dermal Wnt signaling/β-catenin in fibroblast proliferation and in the epidermal hair follicle initiation program.     

Phospholamban phosphorylation in ischemia-reperfused heart. Effect of pacing during ischemia and response to a β-adrenergic challenge

The status of phospholamban (PLB) phosphorylation in the ischemia-reperfused hearts remains controversial. Although a decrease in the phosphorylation of both PLB residues (Ser¹⁶, PKA site, and Thr¹⁷, CaMKII site) was previously reported, experiments from our laboratory failed to detect this decrease. In an attempt to elucidate the cause for this discrepancy, experiments were performed in Langendorff-perfused rat hearts with two main goals: (1) To determine whether keeping pacing during ischemia, a protocol followed in other ischemia-reperfusion models, decreases the phosphorylation of PLB residues, below pre-ischemic values; (2) To investigate whether a maximal -adrenergic challenge allows to detect a decrease in the ability of PLB to be phosphorylated in ischemia-reperfused hearts. Hearts were submitted to a global ischemia/reperfusion protocol (20/30 min) with (P) or without (NP) pacing during ischemia, and phosphorylation of PLB residues was assessed by immunodetection. The recovery of contractility upon reperfusion was lower in P vs. NP hearts. Ser¹⁶ of PLB, was phosphorylated at the end of ischemia in NP hearts. This increase appeared earlier in P hearts and was significantly diminished by catecholamine depletion and -blockade. Thr¹⁷ site was phosphorylated at the beginning of ischemia and the onset of reperfusion. The ischemia-induced phosphorylation of Thr¹⁷ was higher and more sustained in P vs. NP hearts, and inhibited by the calcium channel blocker, nifedipine, whereas the reperfusion-induced increase in Thr¹⁷ phosphorylation was similar in P and NP hearts and was significantly diminished by the Na⁺/Ca²⁺ exchanger inhibitor KB-R7943. Phosphorylation of PLB residues did not decrease below basal levels at any time during ischemia and reperfusion. However, the phosphorylation, inotropic and lusitropic response to -adrenergic stimulation was significantly decreased both in P and NP hearts.