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Sustained hyperglycemia results in excess protein O-GlcNAcylation, leading to vascular complications in diabetes. This study aims to investigate the role of O-GlcNAcylation in the progression of coronary microvascular disease (CMD) in inducible type 2 diabetic (T2D) mice generated by a high-fat diet with a single injection of low-dose streptozotocin. Inducible T2D mice exhibited an increase in protein O-GlcNAcylation in cardiac endothelial cells (CECs) and decreases in coronary flow velocity reserve (CFVR, an indicator of coronary microvascular function) and capillary density accompanied by increased endothelial apoptosis in the heart. Endothelial-specific O-GlcNAcase (OGA) overexpression significantly lowered protein O-GlcNAcylation in CECs, increased CFVR and capillary density, and decreased endothelial apoptosis in T2D mice. OGA overexpression also improved cardiac contractility in T2D mice. OGA gene transduction augmented angiogenic capacity in high-glucose treated CECs. PCR array analysis revealed that seven out of 92 genes show significant differences among control, T2D, and T2D + OGA mice, and Sp1 might be a great target for future study, the level of which was significantly increased by OGA in T2D mice. Our data suggest that reducing protein O-GlcNAcylation in CECs has a beneficial effect on coronary microvascular function, and OGA is a promising therapeutic target for CMD in diabetic patients.

Background Obesity is an established risk factor for hypertension. Although obesity-induced gut barrier breach leads to the leakage of various microbiota-derived products into host circulation and distal organs, the roles of microbiota in mediating the development of obesity-associated adrenomedullary disorders and hypertension have not been elucidated. We seek to explore the impacts of microbial DNA enrichment on inducing obesity-related adrenomedullary abnormalities and hypertension. Methods and Results Obesity was accompanied by remarkable bacterial DNA accumulation and elevated inflammation in the adrenal glands. Gut microbial DNA containing extracellular vesicles (mEVs) were readily leaked into the bloodstream and infiltrated into the adrenal glands in obese mice, causing microbial DNA enrichment. In lean wild-type mice, adrenal macrophages expressed CRIg (complement receptor of the immunoglobulin superfamily) that efficiently blocks the infiltration of gut mEVs. In contrast, the adrenal CRIg+ cell population was greatly decreased in obese mice. In lean CRIg-/- or C3-/- (complement component 3) mice intravenously injected with gut mEVs, adrenal microbial DNA accumulation elevated adrenal inflammation and norepinephrine secretion, concomitant with hypertension. In addition, microbial DNA promoted inflammatory responses and norepinephrine production in rat pheochromocytoma PC12 cells treated with gut mEVs. Depletion of microbial DNA cargo markedly blunted the effects of gut mEVs. We also validated that activation of cGAS (cyclic GMP-AMP synthase)/STING (cyclic GMP-AMP receptor stimulator of interferon genes) signaling is required for the ability of microbial DNA to trigger adrenomedullary dysfunctions in both in vivo and in vitro experiments. Restoring CRIg+ cells in obese mice decreased microbial DNA abundance, inflammation, and hypertension. Conclusions The leakage of gut mEVs leads to adrenal enrichment of microbial DNA that are pathogenic to induce obesity-associated adrenomedullary abnormalities and hypertension. Recovering the CRIg+ macrophage population attenuates obesity-induced adrenomedullary disorders.

The obesity epidemic has increased type II diabetes mellitus (T2DM) across developed countries. Cardiac T2DM risks include ischemic heart disease, heart failure with preserved ejection fraction, intolerance to ischemia-reperfusion (I-R) injury, and refractoriness to cardioprotection. While opioids are cardioprotective, T2DM causes opioid receptor signaling dysfunction. We tested the hypothesis that sustained opioid receptor stimulus may overcome diabetes mellitus-induced cardiac dysfunction via membrane/mitochondrial-dependent protection. In a murine T2DM model, we investigated effects of morphine on cardiac function, I-R tolerance, ultrastructure, subcellular cholesterol expression, mitochondrial protein abundance, and mitochondrial function. T2DM induced 25% weight gain, hyperglycemia, glucose intolerance, cardiac hypertrophy, moderate cardiac depression, exaggerated postischemic myocardial dysfunction, abnormalities in mitochondrial respiration, ultrastructure and Ca2+ -induced swelling, and cell death were all evident. Morphine administration for 5 days: (1) improved glucose homeostasis; (2) reversed cardiac depression; (3) enhanced I-R tolerance; (4) restored mitochondrial ultrastructure; (5) improved mitochondrial function; (6) upregulated Stat3 protein; and (7) preserved membrane cholesterol homeostasis. These data show that morphine treatment restores contractile function, ischemic tolerance, mitochondrial structure and function, and membrane dynamics in type II diabetic hearts. These findings suggest potential translational value for short-term, but high-dose morphine administration in diabetic patients undergoing or recovering from acute ischemic cardiovascular events.

The contribution of altered mitochondrial Ca2+ handling to metabolic and functional defects in type 2 diabetic (T2D) mouse hearts is not well understood. In this study, we show that the T2D heart is metabolically inflexible and almost exclusively dependent on mitochondrial fatty acid oxidation as a consequence of mitochondrial calcium uniporter complex (MCUC) inhibitory subunit MCUb overexpression. Using a recombinant endonuclease-deficient Cas9-based gene promoter pulldown approach coupled with mass spectrometry, we found that MCUb is upregulated in the T2D heart due to loss of glucose homeostasis regulator nuclear receptor corepressor 2 repression, and chromatin immunoprecipitation assays identified peroxisome proliferator-activated receptor α as a mediator of MCUb gene expression in T2D cardiomyocytes. Upregulation of MCUb limits mitochondrial matrix Ca2+ uptake and impairs mitochondrial energy production via glucose oxidation by depressing pyruvate dehydrogenase complex activity. Gene therapy displacement of endogenous MCUb with a dominant-negative MCUb transgene (MCUbW246R/V251E) in vivo rescued T2D cardiomyocytes from metabolic inflexibility and stimulated cardiac contractile function and adrenergic responsiveness by enhancing phospholamban phosphorylation via protein kinase A. We conclude that MCUb represents one newly discovered molecular effector at the interface of metabolism and cardiac function, and its repression improves the outcome of the chronically stressed diabetic heart.

AimsWe previously reported that increased protein O-GlcNAcylation in diabetic mice led to vascular rarefaction in the heart. In this study, we aimed to investigate whether and how coronary endothelial cell (EC) apoptosis is enhanced by protein O-GlcNAcylation and thus induces coronary microvascular disease (CMD) and subsequent cardiac dysfunction in diabetes. We hypothesize that excessive protein O-GlcNAcylation increases p53 that leads to CMD and reduced cardiac contractility.Methods and resultsWe conducted in vivo functional experiments in control mice, TALLYHO/Jng (TH) mice, a polygenic type 2 diabetic (T2D) model, and EC-specific O-GlcNAcase (OGA, an enzyme that catalyzes the removal of O-GlcNAc from proteins)-overexpressing TH mice, as well as in vitro experiments in isolated ECs from these mice. TH mice exhibited a significant increase in coronary EC apoptosis and reduction of coronary flow velocity reserve (CFVR), an assessment of coronary microvascular function, in comparison to wild-type mice. The decreased CFVR, due at least partially to EC apoptosis, was associated with decreased cardiac contractility in TH mice. Western blot experiments showed that p53 protein level was significantly higher in coronary ECs from TH mice and T2D patients than in control ECs. High glucose treatment also increased p53 protein level in control ECs. Furthermore, overexpression of OGA decreased protein O-GlcNAcylation and down-regulated p53 in coronary ECs, and conferred a protective effect on cardiac function in TH mice. Inhibition of p53 with pifithrin-α attenuated coronary EC apoptosis and restored CFVR and cardiac contractility in TH mice.ConclusionsThe data from this study indicate that inhibition of p53 or down-regulation of p53 by OGA overexpression attenuates coronary EC apoptosis and improves CFVR and cardiac function in diabetes. Lowering coronary endothelial p53 levels via OGA overexpression could be a potential therapeutic approach for CMD in diabetes.

Ischemic damage is recognized to cause cardiomyocyte (CM) death and myocardial dysfunction, but the role of cell-matrix interactions and integrins in this process has not been extensively studied. Expression of α7β1D integrin, the dominant integrin in normal adult CMs, increases during ischemia/reperfusion (I/R), while deficiency of β1 integrins increases ischemic damage. We hypothesized that the forced overexpression of integrins on the CM would offer protection from I/R injury. Tg mice with CM-specific overexpression of integrin α7β1D exposed to I/R had a substantial reduction in infarct size compared with that of α5β1D-overexpressing mice and WT littermate controls. Using isolated CMs, we found that α7β1D preserved mitochondrial membrane potential during hypoxia/reoxygenation (H/R) injury via inhibition of mitochondrial Ca2+ overload but did not alter H/R effects on oxidative stress. Therefore, we assessed Ca2+ handling proteins in the CM and found that β1D integrin colocalized with ryanodine receptor 2 (RyR2) in CM T-tubules, complexed with RyR2 in human and rat heart, and specifically bound to RyR2 amino acids 165-175. Integrins stabilized the RyR2 interdomain interaction, and this stabilization required integrin receptor binding to its ECM ligand. These data suggest that α7β1D integrin modifies Ca2+ regulatory pathways and offers a means to protect the myocardium from ischemic injury.

Urocortin-2 (UCn2) peptide infusion increases cardiac function in patients with heart failure, but chronic peptide infusion is cumbersome, costly, and provides only short-term benefits. Gene transfer would circumvent these shortcomings. Here we ask whether a single intravenous injection of adeno-associated virus type 8 encoding murine urocortin-2 (AAV8.UCn2) could provide long-term elevation in plasma UCn2 levels and increased left ventricular (LV) function. Normal mice received AAV8.UCn2 (5×10¹¹ genome copies, intravenous). Plasma UCn2 increased 15-fold 6 weeks and >11-fold 7 months after delivery. AAV8 DNA and UCn2 mRNA expression was persistent in LV and liver up to 7 months after a single intravenous injection of AAV8.UCn2. Physiological studies conducted both in situ and ex vivo showed increases in LV +dP/dt and in LV -dP/dt, findings that endured unchanged for 7 months. SERCA2a mRNA and protein expression was increased in LV samples and Ca²⁺ transient studies showed an increased rate of Ca²⁺ decline in cardiac myocytes from mice that had received UCn2 gene transfer. We conclude that a single intravenous injection of AAV8.UCn2 increases plasma UCn2 and increases LV systolic and diastolic function for at least 7 months. The simplicity of intravenous injection of a long-term expression vector encoding a gene with paracrine activity to increase cardiac function is a potentially attractive strategy in clinical settings. Future studies will determine the usefulness of this approach in the treatment of heart failure.

Emerging evidence indicates the critical roles of microbiota in mediating host cardiac functions in ageing, however, the mechanisms underlying the communications between microbiota and cardiac cells during the ageing process have not been fully elucidated. Bacterial DNAwas enriched in the cardiomyocytes of both ageing humans and mice. Antibiotic treatment remarkably reduced bacterial DNA abundance in ageingmice. Gutmicrobial DNA containing extracellular vesicles (mEVs) were readily leaked into the bloodstream and infiltrated into cardiomyocytes in ageing mice, causing cardiac microbial DNA enrichment. Vsig4+ macrophages efficiently block the spread of gut mEVs whereas Vsig4+ cell population was greatly decreased in ageingmice. GutmEV treatment resulted in cardiac inflammation and a reduction in cardiac contractility in young Vsig4-/- mice. Microbial DNA depletion attenuated the pathogenic effects of gut mEVs. cGAS/STING signaling is critical for the effects of microbial DNA. Restoring Vsig4+ macrophage population in ageingWT mice reduced cardiac microbial DNA abundance and inflammation and improved heart contractility. [ABSTRACT FROM AUTHOR]

Pressure overload-induced cardiac hypertrophy results in a pathological type of hypertrophy with activation of signaling cascades like the extracellular signal-regulated kinase (ERK) pathway, which promotes negative cardiac remodeling and decreased contractile function. In contrast, thyroid hormone mediates a physiological type of hypertrophy resulting in enhanced contractile function. In addition, thyroid hormone action is diminished in pressure overload-induced cardiac hypertrophy. We hypothesized that thyroid hormone status modulates ERK activity and that administration of thyroid hormone could alter the activity of this kinase in cardiac hypertrophy induced by pressure overload. ERK is activated by phosphorylation; accordingly, we investigated phosphorylation of ERK in hearts of control, hypothyroid, and hyperthyroid mice. In addition, the effect of T3 treatment on ERK phosphorylation in hypertrophied hearts from transverse aortic-constricted (TAC) mice was investigated. Results showed that phosphorylated ERK (p-ERK) was decreased by 25% in hyperthyroid mice. In contrast, hypothyroid mice presented increased p-ERK by 80%. TAC mice presented a greater than fourfold increase of p-ERK compared with control mice. Interestingly, T3 administration dramatically canceled TAC-induced ERK phosphorylation (36% lower compared with control). Raf-1 is upstream of the ERK pathway. TAC mice presented a 45% increase in phospho-Raf-1 (Ser338). T3 treatment inhibited this effect of pressure overload and further decreased p-Raf-1 (Ser338) by 37%, compared with control. Overexpression of thyroid hormone receptor-[alpha] in cultured cardiomyocytes potentiated the inhibitory effect of T3 on ERK phosphorylation. We concluded that thyroid hormone has an inhibitory effect on the Raf-1/ERK pathway. Furthermore, treatment of TAC mice with T3 inhibited Raf-1/ERK pathway by a thyroid hormone receptor-dependent mechanism. MAPK; thyroid hormone receptors doi: 10.1152/ajpcell.00168.2010.

Skeletal muscle atrophy and weakness are major contributors to frailty and impact significantly on quality of life of older people. Muscle aging is characterized by a loss of maximum tetanic force ([P.sub.o]) generation, primarily due to muscle atrophy, to which mitochondrial dysfunction is hypothesized to contribute. We hypothesized that lifelong overexpression of the mitochondrial heat shock protein (HSP) HSP10 in muscle of mice would protect against development of these deficits. [P.sub.o] generation by extensor digitorum longus muscles of adult and old wild-type and HSP10-overexpressing mice was determined in situ. Muscles were subjected to damaging lengthening contractions, and force generation was remeasured at 3 h or 28 days to examine susceptibility to, and recovery from, damage, respectively. Muscles of old wild-type mice had a 23% deficit in [P.sub.o] generation and a 10% deficit in muscle cross-sectional area compared with muscles of adult wild-type mice. Overexpression of HSP10 prevented this age-related fall in [P.sub.o] generation and reduction in cross-sectional area observed in muscles of old wild-type mice. Additionally, overexpression of HSP10 protected against contraction-induced damage independent of age but did not improve recovery if damage occurred. Preservation of muscle force generation and CSA by HSP10 overexpression was associated with protection against the age-related accumulation of protein carbonyls. Data demonstrate that development of age-related muscle weakness may not be inevitable and show, for the first time, that lifelong overexpression of an HSP prevents the age-related loss of [P.sub.o] generation. These findings support the hypothesis that mitochondrial dysfunction is involved in the development of age-related muscle deficits. aging; heat shock protein; Cpnl0; Hspel; atrophy doi: 10.1152/ajpregu.00334.2009.

Downregulation of the sarcoplasmic reticulum calcium ATPase (SERCA2) is associated with diastolic dysfunction in the failing heart. Elevated plasma endothelin-1 (ET) levels are correlated with congestive heart failure suggesting that ET may play a pathophysiological role. We have investigated the ability of ET to regulate SERCA2 gene expression in isolated adult rat ventricular myocytes. We find that ET enhances net protein synthesis by ~40% but significantly downregulates SERCA2 mRNA expression, time dependently, by ~30-50%, and the expression of SERCA2 protein by ~ 50%. In myoyctes, ET binds to ETA receptor that couples to [G.sub.q] and [G.sub.i] proteins. Inhibition of [G.sub.q]-PLC-induced phosphoinositide (PI) hydrolysis with U73122 (1 [micro]M) or inhibition of [G.sub.i] protein with pertussis toxin (PTX) abolishes the ability of ET to downregulate SERCA2 mRNA gene expression. Further investigation suggests that ET coupling to PTX-sensitive [G.sub.i] with consequent lowering of cAMP is required for downregulation of SERCA2 mRNA levels. Increasing intracellular cAMP quantity using cAMP-specific PDE inhibitor Ro20-1724 or cAMP analog dibutyryl-cAMP reverses ET-induced downregulation of SERCA2 mRNA levels. The data indicate that, in adult myocytes, ET downregulates SERCA2 mRNA and protein levels, and the effect requires cross-talk between [G.sub.q] and PTX-sensitive [G.sub.i] pathways. hypertrophy; sarcoplasmic reticulum calcium ATPase; [G.sub.i]; [G.sub.q]

Mitochondrial transcription factor A (TFAM) is essential for mitochondrial DNA transcription and replication. TFAM transcriptional activity is decreased in diabetic cardiomyopathy; however, the functional implications are unknown. We hypothesized that a reduced TFAM activity may be responsible for some of the alterations caused by hyperglycemia. Therefore, we investigated the effect of TFAM overexpression on hyperglycemia-induced cytosolic calcium handling and mitochondrial abnormalities. Neonatal rat cardiomyocytes were exposed to high glucose (30 mM) for 48 h, and we examined whether TFAM overexpression, by protecting mitochondrial DNA, could reestablish calcium fluxes and mitochondrial alterations toward normal. Our results shown that TFAM overexpression increased to more than twofold mitochondria copy number in cells treated either with normal (5.5 mM) or high glucose. ATP content was reduced by 30% and mitochondrial calcium decreased by 40% after high glucose. TFAM overexpression returned these parameters to even higher than control values. Calcium transients were prolonged by 70% after high glucose, which was associated with diminished sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase 2a and cytochrome-c oxidase subunit 1 expression. These parameters were returned to control values after TFAM overexpression. High glucose-induced protein oxidation was reduced by TFAM overexpression, indicating a reduction of the high glucose-induced oxidative stress. In addition, we found that TFAM activity can be modulated by O-linked [[beta]-N-acetylglucosamine glycosylation. In conclusion, TFAM overexpression protected cell function against the damage induced by high glucose in cardiomyocytes. mitochondria; free radicals; SERCA2a

Diabetic cardiomyopathy is characterized by reduced cardiac contractility independent of vascular disease. A contributor to contractile dysfunction in the diabetic heart is impaired sarcoplasmic reticulum function with reduced sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase (SERCA2a) pump activity, leading to disturbed intracellular calcium handling. It is currently unclear whether increasing SERCA2a activity in hearts with existing diabetic cardiomyopathy could still improve calcium flux and contractile performance. To test this hypothesis, we generated a cardiac-specific tetracycline-inducible double transgenic mouse, which allows for doxycycline (DOX)-based inducible SERCA2a expression in which DOX exposure turns on SERCA2a expression. Isolated cardiomyocytes and Langendorff perfused hearts from streptozotocin-induced diabetic mice were studied. Our results show that total SERCA2a protein levels were decreased in the diabetic mice by 60% compared with control. SERCA2a increased above control values in the diabetic mice after DOX. Dysfunctional contractility in the diabetic cardiomyocyte was restored to normal by induction of SERCA2a expression. Calcium transients from diabetic cardiomyocytes showed a delayed rate of diastolic calcium decay of 66%, which was reverted toward normal after SERCA2a expression induced by DOX. Global cardiac function assessed in the diabetic perfused heart showed diminished left ventricular pressure, rate of contraction, and relaxation. These parameters were returned to control values by SERCA2a expression. In conclusion, we have used mice allowing for inducible expression of SERCA2a and could demonstrate that increased expression of SERCA2a leads to improved cardiac function in mice with an already established diabetic cardiomyopathy in absence of detrimental effects. transgenic mice; doxycycline; cardiac myocytes

A member of the TNF receptor family, the p75 neurotrophin receptor ([p75.sup.NTR]) has been previously shown to play a role in the regulation of fibrin deposition in the lung. However, the role of [p75.sup.NTR] in the regulation of pulmonary vascular tone in the lung is unknown. In the present study, we evaluated the expression of [p75.sup.NTR] in mouse pulmonary arteries and the putative role of [p75.sup.NTR] in modulating pulmonary vascular tone and agonist responsiveness using wild-type (WT) and [p75.sup.NTR] knockout ([p75.sup.-/-]) mice. Our data indicated that [p75.sup.NTR] is expressed in both smooth muscle and endothelial cells within the pulmonary vascular wall in WT mice. Pulmonary artery tings from [p75.sup.-/-] mice exhibited significantly elevated active tension due to endothelin-1-mediated [Ca.sup.2+] influx. Furthermore, the contraction due to capacitative [Ca.sup.2+] entry (CCE) in response to phenylephrine-mediated active depletion of intracellular [Ca.sup.2+] stores was significantly enhanced compared with WT rings. The contraction due to CCE induced by passive store depletion, however, was comparable between WT and [p75.sup.-/-] rings. Active tension induced by serotonin, U-46619 (a thromboxane [A.sub.2] analog), thrombin, 4-aminopyridine (a [K.sup.+] channel blocker), and high extracellular [K.sup.+] in [p75.sup.-/-] rings was similar to that in WT rings. Deletion of [p75.sup.NTR] did not alter pulmonary vasodilation to sodium nitroprusside (a nitric oxide donor). These data suggest that intact [p75.sup.NTR] signaling may play a role in modulating pulmonary vasoconstriction induced by endothelin-1 and by active store depletion. mouse; pulmonary artery; pharmacology; vasoconstriction; store depletion

Vascular endothelial cells (ECs) play a major role in regulating vascular tone and in revascularization. There is increasing evidence showing endothelial dysfunction in diabetes, although little is known about the contribution of connexins (Cxs) to vascular complications in the diabetic heart. This study was designed to investigate the role of Cxs in coronary endothelial dysfunction in diabetic mice. Coronary ECs isolated from diabetic mice exhibit lowered protein levels of Cx37 and Cx40 (but not Cx43) and a loss of gap junction intercellular communication (GJIC). Vasodilatation induced by the assumed contribution of EC-dependent hyperpolarization was significantly reduced in the diabetic coronary artery (CA). Cx40-specific inhibitory peptide [sup.40]GAP27 strongly attenuated endothelium-dependent relaxation in diabetic CA at the concentration that does not affect the relaxation in control CA, suggesting that the total amount of Cx40 is lower in diabetic CA than in control CA. In diabetic mice, coronary capillary density was significantly decreased in vivo. In vitro, GJIC inhibitor attenuated the ability of EC capillary network formation. High-glucose treatment caused a decrease in Cx40 protein expression in ECs and impaired endothelial capillary network formation, which was restored by Cx40 overexpression. Furthermore, we found that the hyperglycemia-induced decrease in Cx40 was associated with inhibited protein expression of Spl, a transcriptional factor that regulates Cx40 expression. These data suggest that downregulation of Cx40 protein expression and resultant inhibition of GJIC contribute to coronary vascular dysfunction in diabetes. gap junction; endothelium-dependent relaxation; microvascular rarefaction; coronary vascular complications; Spl

In recent years, transgenic mouse models have been developed to examine the underlying cellular and molecular mechanisms of lung disease and pulmonary vascular disease, such as asthma, pulmonary thromboembolic disease, and pulmonary hypertension. However, there has not been systematic characterization of the basic physiological pulmonary vascular reactivity in normal and transgenic mice. This represents an intellectual 'gap', since it is important to characterize basic murine pulmonary vascular reactivity in response to various contractile and relaxant factors to which the pulmonary vasculature is exposed under physiological conditions. The present study evaluates excitation- and pharmacomechanical-contraction coupling in pulmonary arteries (PA) isolated from wild-type BALB/c mice. We demonstrate that both pharmaco-and electromechanical coupling mechanisms exist in mice PA. These arteries are also reactive to stimulation by [[alpha].sub.1]-adrenergic agonists, serotonin, endothelin-1, vasopressin, and U-46619 (a thromboxane [A.sub.2] analog). We conclude that the basic vascular responsiveness of mouse PA is similar to those observed in PA of other species, including rat, pig, and human, albeit on a different scale and to varying amplitudes. pharmacology; store depletion; excitation-contraction coupling; G protein-coupled receptors

The majority of mitochondrial proteins are encoded by nuclear genes and synthesized in the cytosol as preproteins containing a mitochondria import sequence. Preproteins traverse the outer mitochondrial membrane in an unfolded state and then translocate through the inner membrane into the matrix via import machinery that includes mitochondrial heat shock protein 70 (mtHSP70). Neonatal rat cardiac myocytes (NCM) infected with an adenoviral vector expressing mtHSP70 or an empty control ([Adv.sup.-]) for 48 h were submitted to 8 h of simulated ischemia (hypoxia) followed by 16 h of reperfusion (reoxygenation). Infection with mtHSP70 virus yielded an increase in mtHSP70 protein in NCM mitochondria compared with [Adv.sup.-] (P < 0.05). Cell viability after simulated ischemia/reperfusion (I/R) was decreased in both [Adv.sup.-] and mtHSP70 groups, relative to control (P < 0.05), but mtHSP70-infected NCM had enhanced viability after I/R relative to Adv-infected NCM (P < 0.05). Simulated I/R caused an increase in reactive oxygen species generation and lipid peroxidation in Adv-infected NCM (P < 0.05, for both) that was not observed in mtHSP70-infected NCM. Mitochondrial complex III and IV activities were greater in mtHSP70-infected NCM after simulated I/R compared with [Adv.sup.-] (P < 0.05 for both). After simulated I/R, ATP content increased in mtHSP70-infected NCM, compared with [Adv.sup.-] (P < 0.05). Apoptotic markers were decreased in mtHSP70-infected NCM compared with [Adv.sup.-] after simulated I/R (P < 0.05). These results indicate that overexpression of mtHSP70 protects the mitochondria against damage from simulated I/R that may be due to a decrease in reactive oxygen species leading to preservation of mitochondrial complex function activities and ATP formation. ischemia; reperfusion; free radical scavenger

While several transgenic mouse models exhibit improved contractile characteristics in the heart, less is known about how these changes influence energy metabolism, specifically the balance between carbohydrate and fatty acid oxidation. In the present study we examine glucose and fatty acid oxidation in transgenic mice, generated to overexpress sarco(endo) plasmic reticulum calcium-ATPase (SERCA), which have an enhanced contractile phenotype. Energy substrate metabolism was measured in isolated working hearts using radiolabeled glucose and palmitate. We also examined oxygen consumption to see whether SERCA overexpression is associated with increased oxygen utilization. Since SERCA is important in calcium handling within the cardiac myocyte, we examined cytosolic calcium transients in isolated myocytes using indo-1, and mitochondrial calcium levels using pericam, an adenovirally expressed, mitochondrially targeted ratiometric calcium indicator. Oxygen consumption did not differ between wildtype and SERCA groups; however, we were able to show an increased utilization of glucose for oxidative metabolism and a corresponding decreased utilization of fatty acids in the SERCA group. Cytosolic calcium transients were increased in myocytes isolated from SERCA mice, and they show a faster rate of decay of the calcium transient. With these observations we noted increased levels of mitochondrial calcium in the SERCA group, which was associated with an increase in the active form of the pyruvate dehydrogenase complex. Since an increase in mitochondrial calcium levels leads to activation of the pyruvate dehydrogenase complex (the rate-limiting step for carbohydrate oxidation), the increased glucose utilization observed in isolated perfused hearts in the SERCA group may reflect a higher level of mitochondrial calcium. mitochondrial calcium; pyruvate dehydrogenase; cardiac energetics; sarco(endo)plasmic reticulum calcium-adenosine 5'-triphosphatase

Inducible heat shock protein 70 (HSP70i) has been shown to exert a protective effect in hearts subjected to ischemia-reperfusion. Although studied in heat-shocked animals and in transgenic mice that constitutively overexpress the protein, the therapeutic application of the protein in the form of a viral vector-mediated HSP70i expression has not been widely examined. Accordingly, we have examined the effects of HSP70i delivered in vivo to the left ventricular free wall of the heart via viral gene therapy in mice. The affect of virally mediated HSP70i expression in preserving cardiac function following ischemia-reperfusion was examined after short-term expression (5-day adenovirus mediated) and long-term expression (8-too adeno-associated virus mediated) in mice by subjecting ex vivo Langendorff perfused hearts to a regime of ischemia-reperfusion. Both vectors were capable of increasing HSP70i expression in the heart, and neither vector had any effect on cardiac function during aerobic (preischemic) perfusion when compared with corresponding controls. In contrast, both adenovirus-mediated and adeno-associated virus-mediated expression of HSP70i improved the contractile recovery of the heart after 120 min of reperfusion following ischemia. This study demonstrates the feasibility of using both short- and long-term expression of virally mediated HSP70i as a therapeutic intervention against cardiac ischemia-reperfusion injury. inducible heat shock protein 70; left ventricle

Hyperthyroidism has been associated with atrial fibrillation (AF); however, hyperthyroidism-induced ion channel changes that may predispose to AF have not been fully elucidated. To understand the electrophysiological changes that occur in left and right atria with hyperthyroidism, the patch-clamp technique was used to compare action potential duration (APD) and whole cell currents in myocytes from left and right atria from both control and hyperthyroid mice. Additionally, RNase protection assays and immunoblotting were performed to evaluate the mRNA and protein expression levels of [K.sup.+] channel [alpha]-subunits in left and right atria. The results showed that 1) in control mice, the APD was shorter and the ultra-rapid delayed rectifier [K.sup.+] conductance ([I.sub.Kur]) and the sustained delayed rectifier [K.sup.+] conductance ([I.sub.ss]) were larger in the left than in the right atrium; also, mRNA and protein expression levels of K v 1.5.and K v 2.1 were higher in the left atrium; 2) in hyperthyroid mice, the APD was shortened and [I.sub.Kur] and [I.sub.ss] were increased in both left and right atrial myocytes, and the protein expression levels of K v 1.5 and K v 2.1 were increased significantly in both atria; and 3) the influence of hyperthyroidism on APD and delayed rectifier [K.sup.+] currents was more prominent in right than in left atrium, which minimized the interatrial APD difference. In conclusion, hyperthyroidism resulted in more significant APD shortening and greater delayed rectifier [K.sup.+] current increases in the right vs. the left atrium, which can contribute to the propensity for atrial arrhythmia in hyperthyroid heart. action potential duration; atrial fibrillation

Angiotensin II (ANG II) plays important roles in cardiac extracellular matrix remodeling via its type 1A (A[T.sub.1A]) receptor. The cytokines tumor necrosis factor-[alpha] and interleukin-1[beta] (IL-1[beta] were shown previously to upregulate A[T.sub.1A] receptor mRNA and protein, thereby increasing the profibrotic response to ANG II in cardiac fibroblasts. The present experiments implicate increased nuclear factor-[kappa]B (NF-[kappa]B)-dependent transcription and also, to a lesser extent, altered mRNA splicing in the mechanism of receptor upregulation. Cytokine stimulation was found to increase A[T.sub.1A] heterogeneous nuclear RNA levels, which strongly suggests that mRNA upregulation occurs transcriptionally. The transcription factor NF-[kappa]B was previously deemed necessary for cytokine-induced A[T.sub.1A] receptor mRNA upregulation. Computer analysis of upstream DNA sequences revealed putative NF-[kappa]B elements at -365 and -2540 bp. Both isolated elements were shown to bind NF-KB (using gel-shift assays) and to transactivate a minimal promoter (using reporter assays), although the element at -365 bp appeared stronger. Three splice variants of A[T.sub.1A] receptor mRNA that have different 5' untranslated regions were detected in rat tissues, namely, exons 1-2-3 (predominant), 1-2-3+6, and 1-3. Cytokine treatment of fibroblasts upregulated all splice variants, but exon 1-3 increased more than the others. This differential upregulation, albeit of modest magnitude, was statistically significant with IL-1[beta] treatment. Exon 2 contains an inhibitory minicistron and a predicted inhibitory hairpin structure. Luciferase reporter assays indicated that each splice variant translates at a different efficiency, with exon 1-2-3+6 (both minicistron and hairpin) < exon 1-2-3 (minicistron only) < exon 1-3 (neither minicistron or hairpin). These results provide evidence that cytokines increase A[T.sub.1] protein levels by altering both transcription and splicing. messenger RNA; heart; nuclear factor-[kappa]B; tumor necrosis factor-[alpha]; interleukin-1[beta]

Doxycycline-inducible expression of SERCA2a improves calcium handling and reverts cardiac dysfunction in pressure overload-induced cardiac hypertrophy. Ant J Physiol Heart Circ Physiol 287: H2164-H2172, 2004. First published July 15, 2004; doi:10.1152/ajpheart.00428. 2004.--Delayed cardiac relaxation in failing hearts has been attributed to reduced activity and/or expression of sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase 2a (SERCA2a). Although constitutive overexpression of SERCA2a has proven effective in preventing cardiac dysfunction, it is unclear whether increasing SERCA2a expression in hearts with preexisting hypertrophy will be therapeutic. To test this hypothesis, we generated a binary transgenic (BTG) system that allows tetracycline-inducible, cardiac-specific SERCA2a expression. In this system (tet-on SERCA2a), a FLAG-tagged SERCA2a transgene is expressed in the presence of doxycycline (Dox) but not in the absence of Dox (2.3-fold more mRNA, 45% more SERCA2a protein). Calcium transients measured in isolated cardiac myocytes from nonbanded Dox-treated BTG mice showed an accelerated calcium decline and an increased systolic [Ca.sup.2+] peak. Sarcoplasmic reticulum (SR) calcium loading was increased by 45% in BTG mice. In the presence of pressure overload (aortic banding), echocardiographic analysis revealed that expression of SERCA2a-FLAG caused an improvement in fractional shortening. SERCA2a-FLAG expression alleviated the resultant cardiac dysfunction. This was illustrated by an increase in the rate of decline of the calcium transient. Cell shortening and SR calcium loading were also improved in cardiac myocytes isolated from banded BTG mice after SERCA2a overexpression. In conclusion, we generated a novel transgenic mouse that conditionally overexpresses SERCA2a. This model is suitable for both long- and short-term studies of the effects of controlled SERCA2a expression on cardiac function. In addition, inducible overexpression of SERCA2a improved cardiac function and calcium handling in mice with established contractile dysfunction. heart failure; cardiac myocytes; transgenic mice; sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase

In many types of heart failure cardiac myocyte [Ca.sup.2+] handling is abnormal because of downregulation of key [Ca.sup.2+]-handling proteins like sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase (SERCA)2a and ryanodine receptor (RyR)2. The alteration in SERC2a and RyR2 expression results in altered cytosolic [Ca.sup.2+] transients, leading to abnormal contraction. Sorcin is an EF-hand protein that confers the property of caffeine-activated intracellular [Ca.sup.2+] release in nonmuscle cells by interacting with RyR2. To determine whether sorcin could improve the contractile function of the heart, we overexpressed sorcin in the heart of either normal or diabetic mice and in adult rat cardiomyocytes with an adenoviral gene transfer approach. Sorcin overexpression was associated with an increase in cardiac contractility of the normal heart and dramatically rescued the abnormal contractile function of the diabetic heart. These effects could be attributed to an improvement of the [Ca.sup.2+] transients found in the cardiomyocyte after sorcin overexpression. Viral vector-mediated delivery of sorcin to cardiac myocytes is beneficial, resulting in improved contractile function in diabetic cardiomyopathy. calcium handling; gene therapy

Background and Purpose: Heart failure is associated with high morbidity and mortality, and new therapeutic targets are needed. Preclinical data suggest that pharmacological activation of protein kinase G (PKG) can reduce maladaptive ventricular remodelling and cardiac dysfunction in the stressed heart. However, clinical trial results have been mixed and the effects of long‐term PKG activation in the heart are unknown. Experimental Approach We characterized the cardiac phenotype of mice carrying a heterozygous knock‐in mutation of PKG1 (Prkg1R177Q/+), which causes constitutive, cGMP‐independent activation of the kinase. We examined isolated cardiac myocytes and intact mice, the latter after stress induced by surgical transaortic constriction or angiotensin II (Ang II) infusion. Key Results: Cardiac myocytes from Prkg1R177Q/+ mice showed altered phosphorylation of sarcomeric proteins and reduced contractility in response to electrical stimulation, compared to cells from wild type mice. Under basal conditions, young PKG1R177Q/+ mice exhibited no obvious cardiac abnormalities, but aging animals developed mild increases in cardiac fibrosis. In response to angiotensin II infusion or fixed pressure overload induced by transaortic constriction, young PKGR177Q/+ mice exhibited excessive hypertrophic remodelling with increased fibrosis and myocyte apoptosis, leading to increased left ventricular dilation and dysfunction compared to wild type litter mates. Conclusion and Implications: Long‐term PKG1 activation in mice may be harmful to the heart, especially in the presence of pressure overload and neurohumoral stress. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc [ABSTRACT FROM AUTHOR]