Autophagy guided interventions to modify the cardiac phenotype of Danon disease.

Danon disease is a lethal X-linked genetic syndrome resulting from radical mutations in the LAMP2 gene. LAMP2 protein deficiency results in defective lysosomal function, autophagy arrest and a multisystem disorder primarily involving the heart, skeletal muscle and the central nervous system. Cardiomyopathy is the main cause of morbidity and mortality. To investigate the mechanisms of and develop therapies for cardiac Danon disease we engineered a mouse model carrying an exon 6 deletion human mutation in LAMP2, which recapitulates the human cardiac disease phenotype. Mice develop cardiac hypertrophy followed by left ventricular dilatation and systolic dysfunction, in association with progressive fibrosis, oxidative stress, accumulation of autophagosomes and activation of proteasome. Stimulation of autophagy in Danon mice (by exercise training, caloric restriction, and rapamycin) aggravate the disease phenotype, promoting dilated cardiomyopathy. Inhibiting autophagy (by high fat diet or hydroxychloroquine) is better tolerated by Danon mice compared to wild type but is not curative. Inhibiting proteasome by Velcade was found to be highly toxic to Danon mice, suggesting that proteasome is activated to compensate for defective autophagy. In conclusion, activation of autophagy should be avoided in Danon patients. Since Danon's is a lifelong disease, we suggest that lifestyle interventions to decrease cardiac stress may be useful to slow progression of Danon's cardiomyopathy. While Danon mice better tolerate high fat diet and sedentary lifestyle, the benefit regarding cardiomyopathy in humans needs to be balanced against other health consequences of such interventions.

Leptin modulates gene expression in the heart, cardiomyocytes and the adipose tissue thus mitigating LPS-induced damage.

Leptin is an adipokine of pleiotropic effects linked to energy metabolism, satiety, the immune response, and cardioprotection. We have recently shown that leptin causally conferred resistance to myocardial infarction-induced damage in transgenic αMUPA mice overexpressing leptin compared to their wild type (WT) ancestral mice FVB/N. Prompted by these findings, we have investigated here if leptin can counteract the inflammatory response triggered after LPS administration in tissues in vivo and in cardiomyocytes in culture. The results have shown that LPS upregulated in vivo and in vitro all genes examined here, both pro-inflammatory and antioxidant, as well as the leptin gene. Pretreating mice with leptin neutralizing antibodies further upregulated the expression of TNFα and IL-1ß in the adipose tissue of both mouse types, and in the αMUPA heart. The antibodies also increased the levels of serum markers for cell toxicity in both mouse types. These results indicate that under LPS, leptin actually reduced the levels of these inflammatory-related parameters. In addition, pretreatment with leptin antibodies reduced the levels of HIF-1α and VEGF mRNAs in the heart, indicating that under LPS leptin increased the levels of these mRNAs. In cardiomyocytes, pretreatment with exogenous leptin prior to LPS reduced the expression of both pro-inflammatory genes, enhanced the expression of the antioxidant genes HO-1, SOD2 and HIF-1α, and lowered ROS staining. In addition, results obtained with leptin antibodies and the SMLA leptin antagonist indicated that endogenous and exogenous leptin can inhibit leptin gene expression. Together, these findings have indicated that under LPS, leptin concomitantly downregulated pro-inflammatory genes, upregulated antioxidant genes, and lowered ROS levels. These results suggest that leptin can counteract inflammation in the heart and adipose tissue by modulating gene expression.

Weak Electromagnetic Fields Accelerate Fusion of Myoblasts.

Weak electromagnetic fields (WEF) alter Ca2+ handling in skeletal muscle myotubes. Owing to the involvement of Ca2+ in muscle development, we investigated whether WEF affects fusion of myoblasts in culture. Rat primary myoblast cultures were exposed to WEF (1.75 µT, 16 Hz) for up to six days. Under control conditions, cell fusion and creatine kinase (CK) activity increased in parallel and peaked at 4-6 days. WEF enhanced the extent of fusion after one and two days (by ~40%) vs. control, but not thereafter. Exposure to WEF also enhanced CK activity after two days (almost four-fold), but not afterwards. Incorporation of 3H-thymidine into DNA was enhanced by one-day exposure to WEF (~40%), indicating increased cell replication. Using the potentiometric fluorescent dye di-8-ANEPPS, we found that exposure of cells to 150 mM KCl resulted in depolarization of the cell membrane. However, prior exposure of cells to WEF for one day followed by addition of KCl resulted in hyperpolarization of the cell membrane. Acute exposure of cells to WEF also resulted in hyperpolarization of the cell membrane. Twenty-four hour incubation of myoblasts with gambogic acid, an inhibitor of the inward rectifying K+ channel 2.1 (Kir2.1), did not affect cell fusion, WEF-mediated acceleration of fusion or hyperpolarization. These data demonstrate that WEF accelerates fusion of myoblasts, resulting in myotube formation. The WEF effect is associated with hyperpolarization but WEF does not appear to mediate its effects on fusion by activating Kir2.1 channels.

Co-culture hydrogel micro-chamber array-based plate for anti-tumor drug development at single-element resolution.

In response to the need for reliable cellular models that reflect complex tumor microenvironmental properties, and enable more precise testing of anti-cancer therapeutics effects on humans, a co-culture platform for in-vitro model that enhances the physiology of breast cancer (BC) microenvironment is presented. A six well imaging plate wherein each macro-well contains several separate compartments was designed. Three-dimensional (3D) cancer spheroids are generated and cultured in the inner compartment which is embossed with an array of nano-liter micro-chambers made of hydrogel. Stromal cells are cultured in the outer chambers. The two cell types are cultured side-by-side, sharing a common space, thus enabling extra-cellular communication via secreted molecules. As proof of concept, a model of BC tumor microenvironment was recapitulated by co-cultivating 3D MCF7 spheroids in the presence of tumor-associated macrophages (TAMs). The presence of TAMs induced an aggressive phenotype by promoting spheroid growth, enhancing survivin expression levels and enabling invasive behavior. Moreover, TAMs influenced the response of BC spheroids to cytotoxic treatment as well as hormonal drug therapy, and enhanced the effects of nitric oxide donor. The platform enables time-lapse imaging and treatment without losing spatial location of the measured spheroids, thereby allowing measurements and analysis at individual-object resolution in an easy and efficient manner.

Leptin modulates gene expression in the heart and cardiomyocytes towards mitigating ischemia-induced damage.

Leptin, an adipocyte-derived satiety hormone, has been previously linked to cardioprotection. We have shown before that leptin conferred resistance to ischemic damage in the heart in long-lived transgenic αMUPA mice overexpressing leptin compared to the wild type (WT) FVB/N control mice. To better understand the contribution of leptin to the ischemic heart, we measured here the expression of genes encoding leptin and ischemia-related proteins in αMUPA and WT mice in the heart vs adipose tissue after MI. In addition, we investigated gene expression in neonatal rat cardiomyocytes under hypoxia in the absence and presence of exogenously added leptin or a leptin antagonist. We used real time RT-PCR and ELISA or Western blot assays to measure, respectively, mRNA and protein levels. The results have shown that circulating leptin levels and mRNA levels of leptin and heme oxygenase-1 (HO-1) in the heart were elevated in both mouse genotypes after 24 h myocardial infarction (MI), reaching higher values in αMUPA mice. In contrast, leptin gene expression in the adipose tissue was significantly increased only in WT mice, but reaching lower levels compared to the heart. Expression of the proinflammatory genes encoding TNFα and IL-1ß was also largely increased after MI in the heart in both mouse types, however reaching considerably lower levels in αMUPA mice indicating a mitigated inflammatory state. In cardiomyocytes, mRNA levels of all aforementioned genes as well as HIF-1α and SOD2 genes were elevated after hypoxia. Pretreatment with exogenous leptin largely reduced the mRNA levels of TNFα and IL-1ß after hypoxia, while enhancing expression of all other genes and reducing ROS levels. Pretreating the cells with a leptin antagonist increased solely the levels of leptin mRNA, suggesting a negative regulation of the hormone on the expression of its own gene. Overall, the results have shown that leptin affects expression of genes in cardiomyocytes under hypoxia in a manner that could mitigate inflammation and oxidative stress, suggesting a similar influence by endogenous leptin in αMUPA mice. Furthermore, leptin is likely to function in the ischemic murine heart more effectively in an autocrine compared to paracrine manner. These results suggest that leptin can reduce ischemic damage by modulating gene expression in the heart.

Farewell to Professor David Yaffe - A pillar of the myogenesis field.

It is with great sadness that we have learned about the passing of Professor David Yaffe (1929-2020, Israel). Yehi Zichro Baruch - May his memory be a blessing. David was a man of family, science and nature. A native of Israel, David grew up in the historic years that preceded the birth of the State of Israel. He was a member of the group that established Kibbutz Revivim in the Negev desert, and in 1948 participated in Israel's War of Independence. David and Ruth eventually joined Kibbutz Givat Brenner by Rehovot, permitting David to be both a kibbutz member and a life-long researcher at the Weizmann Institute of Science, where David received his PhD in 1959. David returned to the Institute after his postdoc at Stanford. Here, after several years of researching a number of tissues as models for studying the process of differentiation, David entered the myogenesis field and stayed with it to his last day. With his dedication to the field of myogenesis and his commitment to furthering the understanding of the People and the Land of Israel throughout the international scientific community, David organized the first ever myogenesis meeting that took place in Shoresh, Israel in 1975. This was followed by the 1980 myogenesis meeting at the same place and many more outstanding meetings, all of which brought together myogenesis, nature and scenery. Herein, through the preparation and publication of this current manuscript, we are meeting once again at a "David Yaffe myogenesis meeting". Some of us have been members of the Yaffe lab, some of us have known David as his national and international colleagues in the myology field. One of our contributors has also known (and communicates here) about David Yaffe's earlier years as a kibbutznick in the Negev. Our collective reflections are a tribute to Professor David Yaffe. We are fortunate that the European Journal of Translational Myology has provided us with tremendous input and a platform for holding this 2020 distance meeting "Farwell to Professor David Yaffe - A Pillar of the Myogenesis Field".

The Role of Heme Oxygenase 1 in the Protective Effect of Caloric Restriction against Diabetic Cardiomyopathy.

Type 2 diabetes mellitus (DM2) leads to cardiomyopathy characterized by cardiomyocyte hypertrophy, followed by mitochondrial dysfunction and interstitial fibrosis, all of which are exacerbated by angiotensin II (AT). SIRT1 and its transcriptional coactivator target PGC-1α (peroxisome proliferator-activated receptor-γ coactivator), and heme oxygenase-1 (HO-1) modulates mitochondrial biogenesis and antioxidant protection. We have previously shown the beneficial effect of caloric restriction (CR) on diabetic cardiomyopathy through intracellular signaling pathways involving the SIRT1-PGC-1α axis. In the current study, we examined the role of HO-1 in diabetic cardiomyopathy in mice subjected to CR. METHODS: Cardiomyopathy was induced in obese diabetic (db/db) mice by AT infusion. Mice were either fed ad libitum or subjected to CR. In an in vitro study, the reactive oxygen species (ROS) level was determined in cardiomyocytes exposed to different glucose levels (7.5-33 mM). We examined the effects of Sn(tin)-mesoporphyrin (SnMP), which is an inhibitor of HO activity, the HO-1 inducer cobalt protoporphyrin (CoPP), and the SIRT1 inhibitor (EX-527) on diabetic cardiomyopathy. RESULTS: Diabetic mice had low levels of HO-1 and elevated levels of the oxidative marker malondialdehyde (MDA). CR attenuated left ventricular hypertrophy (LVH), increased HO-1 levels, and decreased MDA levels. SnMP abolished the protective effects of CR and caused pronounced LVH and cardiac metabolic dysfunction represented by suppressed levels of adiponectin, SIRT1, PPARγ, PGC-1α, and increased MDA. High glucose (33 mM) increased ROS in cultured cardiomyocytes, while SnMP reduced SIRT1, PGC-1α levels, and HO activity. Similarly, SIRT1 inhibition led to a reduction in PGC-1α and HO-1 levels. CoPP increased HO-1 protein levels and activity, SIRT1, and PGC-1α levels, and decreased ROS production, suggesting a positive feedback between SIRT1 and HO-1. CONCLUSION: These results establish a link between SIRT1, PGC-1α, and HO-1 signaling that leads to the attenuation of ROS production and diabetic cardiomyopathy. CoPP mimicked the beneficial effect of CR, while SnMP increased oxidative stress, aggravating cardiac hypertrophy. The data suggest that increasing HO-1 levels constitutes a novel therapeutic approach to protect the diabetic heart. Brief Summary: CR attenuates cardiomyopathy, and increases HO-1, SIRT activity, and PGC-1α protein levels in diabetic mice. High glucose reduces adiponectin, SIRT1, PGC1-1α, and HO-1 levels in cardiomyocytes, resulting in oxidative stress. The pharmacological activation of HO-1 activity mimics the effect of CR, while SnMP increased oxidative stress and cardiac hypertrophy. These data suggest the critical role of HO-1 in protecting the diabetic heart.

Structurally Simple, Readily Available Peptidomimetic 1-Benzyl-5-methyl-4-( n-octylamino)pyrimidin-2(1 H)-one Exhibited Efficient Cardioprotection in a Myocardial Ischemia (MI) Mouse Model.

TLR4, a member of the Toll-like receptor (TLR) family, serves as a pattern recognition receptor in the innate immune response to microbial pathogens. TLR4 also regulates the inflammatory reaction to ischemic injury in the heart. The TRIF-related adaptor molecule (TRAM) is an adapter that recruits the Toll/interleukin 1 receptor (TIR) domain, which contains adapter-inducing IFN-ß (TRIF), to activate TLR4, following TRIF-dependent cytokine gene transcription. On the basis of a known TRAM-derived decoy peptide, 10 of its peptidomimetics were synthesized. One of them, 1-benzyl-5-methyl-4-( n-octylamino)pyrimidin-2(1 H)-one (21), exhibited high potency and efficacy in vitro. In vitro results and in silico analysis provided evidence for the possible direct interaction of 21 with the TLR4 complex. Administered in mice, 21 was able to block the pathophysiological manifestation of MI, restoring the concomitant tissue damage, with a 100% survival rate. Thus, inhibition of TLR4-mediated inflammation in postischemic myocardium could be used as an approach for developing cardioprotective drugs.

PARP-1 inhibition protects the diabetic heart through activation of SIRT1-PGC-1α axis.

Type 2 diabetes mellitus (DM2) follows impaired glucose tolerance in obesity and is frequently associated with hypertension, causing adverse myocardial remodelling and leading to heart failure. The DNA bound protein PARP (poly ADP ribose) polymerase catalyses a post translational modification (polymerization of negatively charged ADP-ribose chains) of nuclear proteins. PARP-1 activation is NAD+ dependent and takes part in DNA repair and in chromatin remodelling and has a function in transcriptional regulation, intracellular trafficking and energy metabolism. PARP-1 is activated in diabetic cardiomyopathy. We hypothesized that PARP-1 inhibition in diabetic mice may protect cardiomyocytes from inflammation and ROS production. METHODS: Obese Leptin resistant (db/db) mice suffering from DM2, were treated with angiotensin II (AT) for 4 weeks to enhance the development of cardiomyopathy. Mice were concomitantly treated with the PARP-1 inhibitor INO1001. Neonatal cardiomyocytes exposed to high levels of glucose (33 mM) with or without AT were treated with INO1001. or with SIRT inhibitor (EX-527) in the presence of INO1001 were tested in-vitro. RESULTS: The in-vivo tests show that hearts from AT treated DM2 mice exhibited cardiac hypertrophy, fibrosis and an increase in the inflammatory marker TNFα. DM2 mice had an increased oxidative stress, concomitant with elevated PARP-1 activity and reduced Sirtuin-1 (SIRT1) expression. PARP-1 inhibition led to increased SIRT1 and Peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) levels, attenuating oxidative stress, inflammation and fibrosis. In-vitro experiments demonstrated that inhibition of PARP-1 in cardiomyocytes exposed to high levels of glucose and AT led to a significant reduction in ROS (P < 0.01), which was abolished in the presence of the SIRT1 inhibitor together with increased protein expression of SIRT1 and PGC-1α. CONCLUSION: PARP1 inhibitor INO1001 attenuated cardiomyopathic features in diabetic mice through the activation of SIRT1 and its downstream antioxidant defence mechanisms. The results of this study suggest a pivotal role of PARP-1 inhibition in treating diabetic and AT-induced cardiomyopathy.

Muscle Response to Complete Peripheral Nerve Injury: Changes of Acetylcholine Receptor and Creatine Kinase Activity over Time.

Background This study was designed to assess the changes of acetylcholine receptor (AChR) and creatine kinase (CK) levels, which are important biochemical markers for muscle viability in cases of long-term muscle denervation. Scientists and peripheral nerve surgeons may find these data important regarding maximal range of muscle viability applicable for timing of effective peripheral nerve reconstructive surgery. Methods The study was conducted on 48 rats (96 gastrocnemius muscles), whose right legs were denervated by removing a 10-mm segment of sciatic nerve, while their left legs remained intact. Under general anesthesia, the rats were euthanized at seven points in time, on days 7, 14, 21, 30, 60, 120, and 210. In both legs, AChR was quantified by 125I-α-bungarotoxin, whereas CK activity was measured using a spectrophotometric method. Results CK levels in the denervated limb reached a minimal level of 34% on day 30 in comparison to the intact limb and remained at this level up to 210 days after operation. AChR levels in the denervated limb reached a minimal level of 38% on day 120 in comparison to the intact limb and remained at this level up to 210 days after operation. Conclusion The present study shows that AChR and CK levels in rat denervated muscles remain constant at about third of its intact condition for a period of at least a third of rat's lifetime postinjury.

Viral delivered gene therapy to treat catecholaminergic polymorphic ventricular tachycardia (CPVT2) in mouse models.

BACKGROUND: The recessive form of catecholaminergic polymorphic ventricular tachycardia 2 (CPVT2) is caused by mutations in cardiac calsequestrin (CASQ2), leading to protein deficiency. OBJECTIVES: The aims of this study were to develop a viral-delivered gene therapy for CPVT2 and to determine the relationship between CASQ2 expression and antiarrhythmic efficacy in a murine model. METHODS: We used a murine model of CPVT2 caused by the D307H human mutation (CASQ2D307H) or CASQ2 knockout (CASQ2Δ/Δ). Adeno-associated virus (AAV) particles containing the CASQ2 gene (AAVCASQ2) were injected into the heart or intraperitoneally to 12-week-old mice. A telemetry device was implanted, and mice underwent provocation testing 7-8 weeks after gene therapy. RESULTS: CASQ2Δ/Δ mice injected intracardiacally with AAVCASQ2 expressed 40% ± 25% of the normal CASQ2 protein level, which was increased compared to untreated CASQ2Δ/Δ mice (n = 10; P < .05). Intraperitoneal therapy led to a significantly elevated expression of the CASQ2 protein, which was comparable in CASQ2D307H (n = 12) and CASQ2Δ/Δ (n = 4) mice. All control mice with CPVT2 had nonsustained ventricular tachycardia (VT) and 8 of 13 had sustained VT on provocation. Expressing ≥33% of the normal CASQ2 level was needed to protect from nonsustained VT as well as stress-induced premature ventricular contractions. Lower levels of expression prevented sustained VT in AAVCASQ2-treated mice (0 of 26; P < .001 vs controls). CONCLUSION: AAVCASQ2 displays a long-lasting capacity to attenuate and potentially cure CPVT2. Systemic delivery is feasible and convenient, reproducibly providing adequate levels of transgene expression. Antiarrhythmic efficacy depends on the CASQ2 level: ≥33% of the normal CASQ2 level is needed to prevent arrhythmia. However, even lower levels of protein protect from sustained VT, thereby potentially reducing the risk of sudden death.

Nitric oxide is cytoprotective to breast cancer spheroids vulnerable to estrogen-induced apoptosis.

Estrogen-induced apoptosis has become a successful treatment for postmenopausal metastatic, estrogen receptor-positive breast cancer. Nitric oxide involvement in the response to this endocrine treatment and its influence upon estrogen receptor-positive breast cancer progression is still unclear. Nitric oxide impact on the MCF7 breast cancer line, before and after estrogen-induced apoptosis, was investigated in 3D culture systems using unique live-cell imaging methodologies. Spheroids were established from MCF7 cells vulnerable to estrogen-induced apoptosis, before and after exposure to estrogen. Spheroids derived from estrogen-treated cells exhibited extensive apoptosis levels with downregulation of estrogen receptor expression, low proliferation rate and reduced metabolic activity, unlike spheroids derived from non-treated cells. In addition to basic phenotypic differences, these two cell cluster types are diverse in their reactions to exogenous nitric oxide. A dual effect of nitric oxide was observed in the breast cancer phenotype sensitive to estrogen-induced apoptosis. Nitric oxide, at the nanomolar level, induced cell proliferation, high metabolic activity, downregulation of estrogen receptor and enhanced collective invasion, contributing to a more aggressive phenotype. Following hormone supplementation, breast cancer 3D clusters were rescued from estrogen-induced apoptosis by these low nitric oxide-donor concentrations, since nitric oxide attenuates cell death levels, upregulates survivin expression and increases metabolic activity. Higher nitric oxide concentrations (100nM) inhibited cell growth, metabolism and promoted apoptosis. These results suggest that nitric oxide, in nanomolar concentrations, may inhibit estrogen-induced apoptosis, playing a major role in hormonal therapy. Inhibiting nitric oxide activity may benefit breast cancer patients and ultimately reduce tumor recurrence.

Silencing cardiomyocyte TLR4 reduces injury following hypoxia.

Toll-like receptor 4 (TLR4), the receptor for lipopolysaccharide (LPS) of gram-negative pathogens expressed in the heart, is activated by several endogenous ligands associated with tissue injury in response to myocardial infarction (MI). The aim of this study was to investigate the involvement of TLR4 signaling in cardiomyocytes dysfunction following hypoxia (90min) using multiple methodologies such as knocking down TLR4 and small interfering RNA (siTLR4). Cardiomyocytes of C57Bl/6 mice (WT) subjected to hypoxic stress showed increased cardiac release of LDH, HMGB1, IκB, TNF-α and myocardial apoptotic and necrotic markers (BAX, PI) compared to TLR4 knock out mice (TLR4KO). Treating these cardiomyocytes with siRNA against TLR4 decreased the damage markers (LDH, IκB, TNF-α). TLR4 silencing during hypoxic stress resulted in the activation of the p-AKT and p-GSK3ß (by ∼25%). The latter is an indicator that there is a reduction of mitochondrial permeability transition pore (mPTP) opening following hypoxic myocardial induced injury leading to preserved mitochondrial membrane potential. Silencing TLR4 in cardiomyocytes improved cell survival following hypoxic injury through activation of the AKT/GSK3ß pathway, reduced inflammatory and apoptotic signals. These findings suggest that TLR4 may serve as a potential target in the treatment of ischemic myocardial injury. Moreover, RNA interfering targeting TLR4 expression represents a therapeutic strategy.

Weak electromagnetic fields alter Ca(2+) handling and protect against hypoxia-mediated damage in primary newborn rat myotube cultures.

Weak electromagnetic fields (WEF) enhance Ca(2+) entry into cells via voltage-gated Ca(2+) channels and affect various aspects of metabolism, structure, and function. However, little information is available on the effect of WEF on skeletal muscle, which depends primarily on intracellular Ca(2+) stores for function and metabolism. Here, we examine the effects of 30 min exposure of rat primary myotube cultures to WEF (1.75 µT, 16 Hz) on Ca(2+) handling and creatine kinase (CK) release. Free myoplasmic Ca(2+) concentration ([Ca(2+) i]) was measured with the ratiometric dye indo-1. WEF did not affect basal [Ca(2+)]i but decreased the twitch [Ca(2+)]i transient in a time-dependent manner, and the twitch amplitude was decreased to ∼30 % after 30 min. WEF completely abolished the increase in [Ca(2+)]i induced by potassium chloride (∼60 mM) but had no effect on the increase induced by caffeine (∼6 mM). Hypoxia (2 h exposure to 100 % argon) resulted in a marked loss of CK into the medium (400 % of normoxic value), as well as a rapid (within 20 min) and sustained increase in basal [Ca(2+)]i (∼20 % above baseline). However, during exposure to WEF, basal [Ca(2+)]i remained constant during the initial 60 min of hypoxia and, thereafter, increased to levels similar to those observed in the absence of WEF. Finally, WEF blocked about 80 % of hypoxia-mediated CK release (P < 0.05). These data demonstrate that WEF inhibits increases in [Ca(2+)]i by interfering with muscle excitation and protects against muscle damage induced by hypoxia. Thus, WEF may have therapeutic/protective effects on skeletal muscle.

Long-Lived αMUPA Mice Show Attenuation of Cardiac Aging and Leptin-Dependent Cardioprotection.

αMUPA transgenic mice spontaneously consume less food compared with their wild type (WT) ancestors due to endogenously increased levels of the satiety hormone leptin. αMUPA mice share many benefits with mice under caloric restriction (CR) including an extended life span. To understand mechanisms linked to cardiac aging, we explored the response of αMUPA hearts to ischemic conditions at the age of 6, 18, or 24 months. Mice were subjected to myocardial infarction (MI) in vivo and to ischemia/reperfusion ex vivo. Compared to WT mice, αMUPA showed functional and histological advantages under all experimental conditions. At 24 months, none of the WT mice survived the first ischemic day while αMUPA mice demonstrated 50% survival after 7 ischemic days. Leptin, an adipokine decreasing under CR, was consistently ~60% higher in αMUPA sera at baseline. Leptin levels gradually increased in both genotypes 24h post MI but were doubled in αMUPA. Pretreatment with leptin neutralizing antibodies or with inhibitors of leptin signaling (AG-490 and Wortmannin) abrogated the αMUPA benefits. The antibodies also reduced phosphorylation of the leptin signaling components STAT3 and AKT specifically in the αMUPA myocardium. αMUPA mice did not show elevation in adiponectin, an adipokine previously implicated in CR-induced cardioprotection. WT mice treated for short-term CR exhibited cardioprotection similar to that of αMUPA, however, along with increased adiponectin at baseline. Collectively, the results demonstrate a life-long increased ischemic tolerance in αMUPA mice, indicating the attenuation of cardiac aging. αMUPA cardioprotection is mediated through endogenous leptin, suggesting a protective pathway distinct from that elicited under CR.

Ultra Low Dose Delta 9-Tetrahydrocannabinol Protects Mouse Liver from Ischemia Reperfusion Injury.

BACKGROUND/AIMS: Ischemia/reperfusion (I/R) injury is the main cause of both primary graft dysfunction and primary non-function of liver allografts. Cannabinoids has been reported to attenuate myocardial, cerebral and hepatic I/R oxidative injury. Delta-9-tetrahydrocannabinol (THC), a cannabinoid agonist, is the active components of marijuana. In this study we examined the role of ultralow dose THC (0.002mg/kg) in the protection of livers from I/R injury. This extremely low dose of THC was previously found by us to protect the mice brain and heart from a variety of insults. METHODS: C57Bl Mice were studied in in vivo model of hepatic segmental (70%) ischemia for 60min followed by reperfusion for 6 hours. RESULTS: THC administration 2h prior to the induction of hepatic I/R was associated with significant attenuated elevations of: serum liver transaminases ALT and AST, the hepatic oxidative stress (activation of the intracellular signaling CREB pathway), the acute proinflammatory response (TNF-α, IL-1α, IL-10 and c-FOS hepatic mRNA levels, and ERK signaling pathway activation). This was followed by cell death (the cleavage of the pro-apoptotic caspase 3, DNA fragmentation and TUNEL) after 6 hours of reperfusion. Significantly less hepatic injury was detected in the THC treated I/R mice and fewer apoptotic hepatocytes cells were identified by morphological criteria compared with untreated mice. CONCLUSION: A single ultralow dose THC can reduce the apoptotic, oxidative and inflammatory injury induced by hepatic I/R injury. THC may serve as a potential target for therapeutic intervention in hepatic I/R injury during liver transplantation, liver resection and trauma.

TLR4 Expression Is Associated with Left Ventricular Dysfunction in Patients Undergoing Coronary Artery Bypass Surgery.

INTRODUCTION: Toll-like receptor 4 (TLR4) is an innate immune receptor expressed in immune cells and the heart. Activation of the immune system following myocardial ischemia causes the release of proinflammatory mediators that may negatively influence heart function. AIM: The aim of this study is to determine whether TLR4 is activated in peripheral monocytes and heart tissue taken from patients with varying degrees of myocardial dysfunction caused by coronary artery diseases and scheduled for coronary artery bypass graft (CABG) surgery before 12 months following operation. METHODS AND RESULTS: Patients (n = 44) undergoing CABG surgery having left ventricular ejection fraction ≤ 45% ('reduced EF', n = 20) were compared to patients with preserved EF >45% ('preserved EF' group, n = 24). 'Reduced EF' patients exhibited increased TLR4 expression in monocytes (2.78±0.49 vs. 1.76±0.07 rMFI, p = 0.03). Plasma levels of C-reactive protein, microRNA miR-320a, brain natriuretic peptide (pro BNP) and NADPH oxidase (NOX4) were also significantly different between the 'preserved EF' and 'reduced EF'groups. Elevated TLR4 gene expression levels in the right auricle correlated with those of EF (p<0.008), NOX4 (p<0.008) and miR320, (p<0.04). In contrast, no differences were observed in peripheral monocyte TLR2 expression. After CABG surgery, monocyte TLR4 expression decreased in all patients, reaching statistical significance in the 'reduced EF' group. CONCLUSION: TLR4 is activated in peripheral monocytes and heart tissue obtained from patients with ischemic heart disease and reduced left ventricular function. Coronary revascularization decreases TLR4 expression. We therefore propose that TLR4 plays a pathogenic role and may serve as an additional marker of ischemic myocardial dysfunction.

Sirtuin 6 protects the heart from hypoxic damage.

Sirtuin 6 (SIRT6) is a protein associated with prolonged life expectancy. We investigated whether life extension is associated with cardioprotection against hypoxia. The proposed study is to develop approaches to reduce hypoxic damage through the use of the sirtuin pathway and to elucidate the mechanism involved. For that purpose we subjected cardiomyocytes from transgenic mice (TG) with over-expression of SIRT6, to hypoxic stress in cell cultures. We hypothesized that cardiomyocytes from transgenic mice subjected to prolonged hypoxia may release survival factors or fewer damage markers to protect them from hypoxic stress compared with wild type (WT) mice. Lactate dehydrogenase (LDH) and creatine kinase (CK) released to the medium and propidium iodide (PI) binding, were markedly decreased following hypoxia in TG cardiomyocytes. The protective mechanism of SIRT6 over-expression includes the activation of pAMPKα pathway, the increased protein level of B-cell lymphoma 2 (Bcl2), the inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), the decrease of reactive oxygen species (ROS) and the reduction in the protein level of phospho-protein kinase B (pAkt) during hypoxia. Together, all these processes impede the necrosis/apoptosis pathways leading to the improved survival of cardiomyocytes following hypoxia, which might explain life extension.

Tumor necrosis factor alpha protects heart cultures against hypoxic damage via activation of PKA and phospholamban to prevent calcium overload.

This study aims to elucidate the mechanisms by which tumor necrosis factor alpha (TNFα) provides protection from hypoxic damage to neonatal rat cardiomyocyte cultures. We show that when intracellular Ca(2+) ([Ca(2+)]i) levels are elevated by extracellular Ca(2+) ([Ca(2+)]o) or by hypoxia, then TNFα decreased [Ca(2+)]i in individual cardiomyocytes. However, TNFα did not reduce [Ca(2+)]i after its increase by thapsigargin, (a SERCA2a inhibitor), indicating that TNFα attenuates Ca(2+) overload through Ca(2+) uptake by SERCA2a. TNFα did not reduce [Ca(2+)]i, following its elevation when [Ca(2+)]o levels were elevated in TNFα receptor knock-out mice. H-89, a protein kinase A (PKA) inhibitor, attenuated the protective effect of TNFα when the cardiomyoctyes were subjected to hypoxia, as determined by lactate dehydrogenase (LDH) and creatine kinase (CK) released and from the cardiomyocytes. Moreover, when the levels of [Ca(2+)]i were increased by hypoxia, H-89, but not KN93, (a calmodulin kinase II inhibitor), prevented the reduction in [Ca(2+)]i by TNFα. TNFα increased the phosphorylation of PKA in normoxic and hypoxic cardiomyoctes, indicating that the cardioprotective effect of TNFα against hypoxic damage was via PKA activation. Hypoxia decreased phosphorylated phospholamban levels; however, TNFα attenuated this decrease following hypoxia. It is suggested that TNFα activates phospholamban phosphorylation in hypoxic heart cultures via PKA to stimulate SERCA2a activity to limit Ca(2+) overload.

Alpha blockade potentiates CPVT therapy in calsequestrin-mutant mice.

BACKGROUND: Spontaneous calcium release evoking delayed afterdepolarization is believed to cause catecholaminergic polymorphic ventricular tachycardia (CPVT), a lethal human arrhythmia provoked by exercise or emotional stress. ß-Adrenergic blockers are the drug of choice, but fail to achieve complete arrhythmia control in some patients. These individuals often require flecainide, device implantation, and/or sympathetic denervation. OBJECTIVE: To optimize the arrhythmia therapy by pharmacological inhibition of the sympathetic nervous system in the homozygous calsequestrin knockout (CASQ2(Δ/Δ)) mouse model of CPVT2. METHODS: A heart telemetry device was implanted for continuous electrocardiographic recording at rest and during provocation testing. Calcium transients and abnormal calcium release were studied in cardiomyocytes isolated from adult mice. Adrenergic receptor expression was determined by using Western blotting and confocal microscopy. RESULTS: Adult CASQ2(Δ/Δ) mice suffer from complex ventricular arrhythmia at rest and ventricular tachycardia during treadmill exercise and after epinephrine injection. ß-Adrenergic blockers, propranolol and metoprolol, attenuated arrhythmia at rest but not after stress. Reserpine had no efficacy in controlling arrhythmia. Agents with α-blocking activity, phentolamine or labetalol, abolished both exercise- and epinephrine-induced arrhythmia. In contrast, injection of α-adrenergic agonist phenylephrine reproducibly provoked ventricular tachycardia. Isolated cardiomyocytes from CASQ2(Δ/Δ) mice had delayed calcium release waves upon exposure to sympathetic agonists, which were abolished by phentolamine. Hearts of calsequestrin-mutant mice expressed more α1-adrenergic receptor than did wild type control mice (P < .05). CONCLUSION: We identified a contribution of the α-adrenergic pathway to the pathogenesis of catecholamine-induced arrhythmia. α-Blockade emerges as an effective therapy in the murine model of CPVT2 and should be tried in humans resistant to ß-blockers.