Senescence has been considered a programmed cellular response, parallel to apoptosis, that is turned on when a cell reaches Hayflick's limit. Once cells enter the senescence program, they cease to proliferate and undergo a series of morphological and functional changes. Studies support a central role for Rb protein in controlling this process after it receives senescent signals from the p53 and p16 pathways. Cellular senescence is considered an essential contributor to the aging process and has been shown to be an important tumor suppression mechanism. In addition, emerging evidence suggests that senescence may also be involved in the pathogenesis of stem cell dysfunction and chronic human diseases. Under these circumstances cells undergo stress-induced premature senecence, which has several specific features. Focusing on endothelial cells, we discuss recent advances in our understanding of the stresses and their pathways that prompt the premature senescence response, evaluate their correlation with the apoptotic response, and examine their links to the development of chronic diseases and the impaired function of endothelial progenitor cells, with the emphasis on vasculopathy. Emerging novel therapeutic interventions based on recent experimental findings are also reviewed.

Brain natriuretic peptide (BNP) produced by cardiac myocytes has antifibrotic and antigrowth properties and is a marker of cardiac hypertrophy. We previously showed that prostaglandin E₂ (PGE₂) is the main prostaglandin produced in myocytes treated with proinflammatory stimuli and stimulates protein synthesis by binding to its EP₄ receptor. We hypothesized that PGE₂, acting through EP₄, also regulates BNP gene expression. We transfected neonatal ventricular myocytes with a plasmid encoding the human BNP (hBNP) promoter driving expression of a luciferase reporter gene. PGE₂ increased hBNP promoter activity 3.5-fold. An EP₄ antagonist reduced the stimulatory effect of PGE₂ but not an EP₁ antagonist. Because EP₄ signaling can involve adenylate cyclase, cAMP, and protein kinase A (PKA), we tested the effect of H-89, a PKA inhibitor, on PGE₂ stimulation of the hBNP promoter. H-89 at 5 μM decreased PGE₂ stimulation of BNP promoter activity by 100%. Because p42/44 MAPK mediates the effect of PGE₂ on protein synthesis, we also examined the role of MAPKs in the regulation of BNP promoter activity. PGE₂ stimulation of the hBNP promoter was inhibited by a MEK1/2 inhibitor and a dominant-negative mutant of Raf, indicating that p42/44 MAPK was involved. In contrast, neither a p38 MAPK inhibitor nor a JNK inhibitor reduced the stimulatory effect of PGE₂. Involvement of small GTPases was also studied. Dominant-negative Rap inhibited PGE₂ stimulation of the hBNP promoter, but dominant-negative Ras did not. We concluded that PGE₂ stimulates the BNP promoter mainly via EP₄, PKA, Rap, and p42/44 MAPK.

l-Arginine, the sole substrate for the nitric oxide (NO) synthase (NOS) enzyme in producing NO, is also a substrate for arginase. We examined normal feline hearts and hearts with compensated left ventricular (LV) hypertrophy (LVH) produced by ascending aorta banding. Using Western blot analysis, we examined the abundance of arginase isozymes in crude homogenates and isolated cardiac myocytes obtained from the LVs of normal and LVH hearts. We examined the functional significance of myocyte arginase via measurement of shortening and intracellular calcium in isolated myocytes in the presence and absence of boronoethyl chloride (BEC), a specific pharmacological inhibitor of arginase. Both arginase I and II were detected in crude myocardial homogenates, but only arginase I was present in isolated cardiac myocytes. Arginase I was downregulated in LVH compared with normal. Inhibition of arginase with BEC reduced fractional shortening, maximal rate of shortening (+dL/dt) and relengthening (−dL/dt), and the peak of the free cytosolic calcium transient in normal myocytes but did not affect these parameters in LVH myocytes. These negative inotropic actions of arginase inhibition were associated with increases in cGMP generation. These studies indicate that only arginase I is present in cardiac myocytes where it tends to limit NO and cGMP production with the effect of supporting basal contractility. In experimental LVH induced by pressure overload, our studies demonstrate reduced arginase I expression and reduced functional significance, allowing greater arginine substrate availability for NO/cGMP signaling.

Diabetic (db/db) mice provide an animal model of Type 2 diabetes characterized by marked in vivo insulin resistance. The effect of insulin on myocardial metabolism has not been fully elucidated in this diabetic model. In the present study we tested the hypothesis that the metabolic response to insulin in db/db hearts will be diminished due to cardiac insulin resistance. Insulin-induced changes in glucose oxidation (GLUox) and fatty acid (FA) oxidation (FAox) were measured in isolated hearts from control and diabetic mice, perfused with both low as well as high concentration of glucose and FA: 10 mM glucose/0.5 mM palmitate and 28 mM glucose/1.1 mM palmitate. Both in the absence and presence of insulin, diabetic hearts showed decreased rates of GLUox and elevated rates of FAox. However, the insulin-induced increment in GLUox, as well as the insulin-induced decrement in FAox, was similar or even more pronounced in diabetic that in control hearts. During elevated FA and glucose supply, however, the effect of insulin was blunted in db/db hearts with respect to both FAox and GLUox. Finally, insulin-stimulated deoxyglucose uptake was markedly reduced in isolated cardiomyocytes from db/db mice, whereas glucose uptake in isolated perfused db/db hearts was clearly responsive to insulin. These results show that, despite reduced insulin-stimulated glucose uptake in isolated cardiomyocytes, isolated perfused db/db hearts are responsive to metabolic actions of insulin. These results should advocate the use of insulin therapy (glucose-insulin-potassium) in diabetic patients undergoing cardiac surgery or during reperfusion after an ischemic insult.

Activation of the mitochondrial ATP-sensitive K⁺ channel (mitoK_ATP) and its regulation by PKC are critical events in preconditioning induced by ischemia or pharmaceutical agents in animals and humans. The properties of the human cardiac mitoK_ATP channel are unknown. Furthermore, there is no evidence that cytosolic PKC can directly regulate the mitoK_ATP channel located in the inner mitochondrial membrane (IMM) due to the physical barrier of the outer mitochondrial membrane. In the present study, we characterized the human cardiac mitoK_ATP channel and its potential regulation by PKC associated with the IMM. IMM fractions isolated from human left ventricles were fused into lipid bilayers in symmetrical potassium glutamate (150 mM). The conductance of native mitoK_ATP channels was usually below 80 pS (∼70%), which was reduced by ATP and 5-hydroxydecanoic acid (5-HD) in a dose- and time-dependent manner. The native mitoK_ATP channel is activated by diazoxide and inhibited by ATP and 5-HD. The PKC activator phorbol 12-myristate 13-acetate (2 μM) increased the cumulative open probability of the mitoK_ATP channel previously inhibited by ATP (P < 0.05), but its inactive analog 4α-phorbol 12,13-didecanoate had no effect. Western blot analysis detected an inward rectifying K⁺ channel (Kir6.2) immunoreactive protein at 56 kDa and PKC-δ in the IMM. These data provide the first characterization of the human cardiac mitoK_ATP channel and its regulation by PKC(s) in IMM. This local PKC control mechanism may represent an alternative pathway to that proposed previously for cytosolic PKC during ischemic/pharmacological preconditioning.

Electrophilic lipids, such as 4-hydroxynonenal (HNE), and the cyclopentenones 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) and 15-J₂-isoprostane induce both reactive oxygen species (ROS) formation and cellular antioxidant defenses, such as heme oxygenase-1 (HO-1) and glutathione (GSH). When we compared the ability of these distinct electrophiles to stimulate GSH and HO-1 production, the cyclopentenone electrophiles were somewhat more potent than HNE. Over the concentration range required to observe equivalent induction of GSH, dichlorofluorescein fluorescence was used to determine both the location and amounts of electrophilic lipid-dependent ROS formation in endothelial cells. The origin of the ROS on exposure to these compounds was largely mitochondrial. To investigate the possibility that the increased ROS formation was due to mitochondrial localization of the lipids, we prepared a novel fluorescently labeled form of the electrophilic lipid 15d-PGJ₂. The lipid demonstrated strong colocalization with the mitochondria, an effect which was not observed by using a fluorescently labeled nonelectrophilic lipid. The role of mitochondria was confirmed by using cells deficient in functional mitochondria. On the basis of these data, we propose that ROS formation in endothelial cells is due to the direct interaction of these lipids with the organelle.

We have recently shown that estrogen causes vessel dilation through receptor-mediated stimulation of nitric oxide (NO) production. Here, we hypothesize that estrogen modulates the mechanical homeostasis in the blood vessel wall through NO production. The mechanical properties of female ovariectomized (ovx) mice, female mice lacking the gene for endothelial NO synthase (eNOS^−/−), and control female and male mice were studied to test the hypothesis. The femoral and carotid arteries and aorta were cannulated in situ and mechanically distended. The stress, strain, elastic modulus, and wall thickness of vessels in ovx and eNOS^−/− mice, as well as intact female and male mice, were determined. Western blot and immunohistochemistry were used to assess eNOS protein expression in the aorta. Moreover, NO by-products of the femoral and carotid artery were determined by measuring the levels of nitrite and nitrate. Our results show that ovariectomy and eNOS^−/− significantly decrease the strain in all arteries. Furthermore, the eNOS protein was significantly reduced in ovx mice. Finally, the NO metabolites were significantly decreased both in ovx and eNOS^−/− mice. We found statistically significant correlations between the structural (wall thickness), mechanical (stress, strain, and elastic modulus), and biochemical parameters (NO by-products). These novel results connect NO to the structural and mechanical properties of the vessel wall. Hence, the effect of endogenous estrogen on the arterial mechanical properties is mediated by the regulation of NO derived from eNOS.

Rodent studies suggest that peroxisome proliferator-activated receptor-α (PPAR-α) activation reduces myocardial ischemia-reperfusion (I/R) injury and infarct size; however, effects of PPAR-α activation in large animal models of myocardial I/R are unknown. We determined whether chronic treatment with the PPAR-α activator fenofibrate affects myocardial I/R injury in pigs. Domestic farm pigs were assigned to treatment with fenofibrate 50 mg·kg⁻¹·day⁻¹ orally or no drug treatment, and either a low-fat (4% by weight) or a high-fat (20% by weight) diet. After 4 wk, 66 pigs underwent 90 min low-flow regional myocardial ischemia and 120 min reperfusion under anesthetized open-chest conditions, resulting in myocardial stunning. The high-fat group received an infusion of triglyceride emulsion and heparin during this terminal experiment to maintain elevated arterial free fatty acid (FFA) levels. An additional 21 pigs underwent 60 min no-flow ischemia and 180 min reperfusion, resulting in myocardial infarction. Plasma concentration of fenofibric acid was similar to the EC₅₀ for activation of PPAR-α in vitro and to maximal concentrations achieved in clinical use. Myocardial expression of PPAR-α mRNA was prominent but unaffected by fenofibrate treatment. Fenofibrate increased expression of carnitine palmitoyltransferase (CPT)-I mRNA in liver and decreased arterial FFA and lactate concentrations (each P < 0.01). However, fenofibrate did not affect myocardial CPT-I expression, substrate uptake, lipid accumulation, or contractile function during low-flow I/R in either the low- or high-fat group, nor did it affect myocardial infarct size. Despite expression of PPAR-α in porcine myocardium and effects of fenofibrate on systemic metabolism, treatment with this PPAR-α activator does not alter myocardial metabolic or contractile responses to I/R in pigs.

Nitric oxide (NO) plays an important role in anoxic preconditioning to protect the heart against ischemia-reperfusion injuries. The present work was performed to study better the NO-cGMP-protein kinase G (PKG) signaling pathway in the activation of both sarcolemmal and mitochondrial ATP-sensitive K⁺ (K_ATP) channels during anoxic preconditioning (APC) and final influence on reducing anoxia-reperfusion (A/R)-induced cardiac damage in rat hearts. The upstream regulating elements controlling NO-cGMP-PKG signal-induced K_ATP channel opening that leads to cardioprotection were investigated. The involvement of both inducible and endothelial NO synthases (iNOS and eNOS) in the progression of this signaling pathway was followed. Final cellular outcomes of ischemia-induced injury after different preconditioning in the form of lactate dehydrogenase release, DNA strand breaks, and malondialdehyde formation as indexes of cell injury and lipid peroxidation, respectively, were investigated. The lactate dehydrogenase and malondialdehyde values decreased in the groups that underwent preconditioning periods with specific mitochondrial K_ATP channels opener diazoxide (100 μM), nonspecific mitochondrial K_ATP channels opener pinacidil (50 μM), S-nitroso-N-acetylpenicillamine (SNAP, 300 μM), or β-phenyl-1,N²-etheno-8-bromoguanosine-3′,5′-cyclicmonophosphorothioate, Sp-isomer (10 μM) before the A/R period. Preconditioning with SNAP significantly reduced the DNA damage. The effect was blocked by glibenclamide (50 μM), 5-hydroxydecanoate (100 μM), N^G-nitro-l-arginine methyl ester (200 μM), and β-phenyl-1,N²-etheno-8-bromoguanosine-3′,5′-cyclic monophosphorothioate, Rp-isomer (1 μM). The results suggest iNOS, rather than eNOS, as the major contributing NO synthase during APC treatment. Moreover, the PKG shows priority over NO as the upstream regulator of NO-cGMP-PKG signal-induced K_ATP channel opening that leads to cardioprotection during APC treatment.

Using high-performance liquid chromatography techniques with fluorescence and electrochemical detection, we found that β-nicotinamide adenine dinucleotide (β-NAD) is released in response to electrical field stimulation (4–16 Hz, 0.3 ms, 15 V, 120 s) along with ATP and norepinephrine (NE) in the canine isolated mesenteric arteries. The release of β-NAD increases with number of pulses/stimulation frequencies. Immunohistochemistry analysis showed dense distribution of tyrosine hydroxylase-like immunoreactivity (TH-LI) and sparse distribution of TH-LI-negative nerve processes, suggesting that these blood vessels are primarily under sympathetic nervous system control with some contribution of other (e.g., sensory) neurons. Exogenous NE (3 μmol/l), α,β-methylene ATP (1 μmol/l), neuropeptide Y (NPY, 0.1 μmol/l), CGRP (0.1 μmol/l), vasoactive intestinal peptide (VIP, 0.1 μmol/l), and substance P (SP, 0.1 μmol/l) had no effect on the basal release of β-NAD, suggesting that the overflow of β-NAD is evoked by neither the sympathetic neurotransmitters NE, ATP, and NPY, nor the neuropeptides CGRP, VIP, and SP. Botulinum neurotoxin A (BoNTA, 0.1 μmol/l) abolished the evoked release of NE, ATP, and β-NAD at 4 Hz, suggesting that at low levels of neural activity, release of these neurotransmitters results from N-ethylmaleimide-sensitive factor attachment protein receptor/synaptosomal-associated protein of 25 kDa-mediated exocytosis. At 16 Hz, however, the evoked release of NE, ATP, and β-NAD was reduced by BoNTA by ∼90, 60, and 80%, respectively, suggesting that at higher levels of neural activity, β-NAD is likely to be released from different populations of synaptic vesicles or different populations of nerve terminals (i.e., sympathetic and sensory terminals).

Inhaled nitric oxide (NO) is a highly selective pulmonary vasodilator. It was recently reported that inhaled NO causes peripheral vasodilatation after treatment with a NO synthase (NOS) inhibitor. These findings suggested the possibility that inhibition of endogenous NOS uncovered the systemic vasodilating effect of NO or NO adducts absorbed via the lungs during NO inhalation. To learn whether inhaled NO reduces systemic vascular resistance in the absence of endothelial NOS, we studied the systemic vascular effects of NO breathing in wild-type mice treated without and with the NOS inhibitor N^ω-nitro-l-arginine methyl ester and in NOS3-deficient (NOS3^−/−) mice. During general anesthesia, the cardiac output, left ventricular function, and systemic vascular resistance were not altered by NO breathing at 80 parts/million in both genotypes. Breathing NO in air did not alter blood pressure and heart rate, as measured by tail-cuff and telemetric methods, in either awake wild-type mice (whether or not they were treated with N^ω-nitro-l-arginine methyl ester), or in awake NOS3^−/− mice. Our findings suggest that absorption of NO or adducts during NO breathing is insufficient to cause systemic vasodilation in mice, even when endogenous endothelial NO production is congenitally absent.

Feedback control of total peripheral resistance (TPR) by the arterial and cardiopulmonary baroreflex systems is an important mechanism for short-term blood pressure regulation. Existing methods for measuring this TPR baroreflex mechanism typically aim to quantify only the gain value of one baroreflex system as it operates in open-loop conditions. As a result, the normal, integrated functioning of the arterial and cardiopulmonary baroreflex control of TPR remains to be fully elucidated. To this end, the laboratory of Mukkamala et al. (Mukkamala R, Toska K, and Cohen RJ. Am J Physiol Heart Circ Physiol 284: H947–H959, 2003) previously proposed a potentially noninvasive technique for estimating the closed-loop (dimensionless) gain values of the arterial TPR baroreflex (G_A) and the cardiopulmonary TPR baroreflex (G_C) by mathematical analysis of the subtle, beat-to-beat fluctuations in arterial blood pressure, cardiac output, and stroke volume. Here, we review the technique with additional details and describe its experimental evaluation with respect to spontaneous hemodynamic variability measured from seven conscious dogs, before and after chronic arterial baroreceptor denervation. The technique was able to correctly predict the group-average changes in G_A and G_C that have previously been shown to occur following chronic arterial baroreceptor denervation. That is, reflex control by the arterial TPR baroreflex was virtually abolished (G_A = −2.1 ± 0.6 to 0.3 ± 0.2; P < 0.05), while reflex control by the cardiopulmonary TPR baroreflex more than doubled (G_C = −0.7 ± 0.4 to −1.8 ± 0.2; P < 0.05). With further successful experimental testing, the technique may ultimately be employed to advance the basic understanding of TPR baroreflex functioning in both humans and animals in health and disease.

Pial arterioles do not express N-methyl-d-aspartate (NMDA) receptors but dilate in response to topical NMDA application. We explored the mechanism underlying NMDA-mediated responses in murine pial arterioles (11–31 μm), using a closed cranial window preparation, and found that arteriolar dilation was not concentration dependent. Pial arteriolar diameter abruptly increased within 3 min of superfusing 50 or 100 μM NMDA. Dilation reached a peak within 1 min (46 ± 14%) and then declined to a plateau (28 ± 13%) for the duration of superfusion. Whereas a higher concentration (200 μM) did not produce further dilation, lower concentrations (1–10 μM) did not dilate the arterioles at all. MK-801 (10 μM) abrogated the dilation response, whereas N^ω-nitro-l-arginine (1 mM) attenuated the peak and abolished the sustained dilation during NMDA superfusion. We determined that NMDA-induced pial arteriolar responses were evoked by cortical spreading depression, because abrupt vasodilation during 50 or 100 μM NMDA superfusion was associated with a large negative slow potential shift and electrocorticogram suppression that spread from the superfusion window to distant cortical areas. Our data suggest that the responses of pial arterioles to NMDA are caused in part by neurovascular coupling due to cortical spreading depression.

Adrenomedullin (ADM) acts as an autocrine or a paracrine factor in the regulation of cardiac function. The intracellular mechanisms involved in the direct effect of ADM on adult rat ventricular myocytes (ARVMs) are still to be elucidated. In ARVMs from normal rats, ADM produced an initial (<30 min) increase in cell shortening and Ca²⁺ transients and a marked decrease in both on prolonged incubation (>1 h). Both effects were sensitive to ADM antagonist ADM-(22–52). Treatment with SQ-22536, an inhibitor of adenylate cyclase, blocked the positive inotropic effect of ADM and potentiated its negative inotropic effect. The negative inotropic effect was sensitive to inhibition by pertussis toxin (PTX), an inhibitor of G_i proteins and KT-5720, an inhibitor of PKA. The observations suggest a switch from G_s-coupled to PTX-sensitive, PKA-dependent G_i coupling by ADM in ARVMs. The ADM-mediated G_i-signaling system involves cAMP-dependent pathways because SQ-22536 further increased the negative inotropic actions of ADM. Also, because ADM is overproduced by ARVMs in our rat model of septic shock, ARVMs from LPS-treated rats were subjected to treatment with ADM-(22–52) and PTX. The decrease in cell shortening and Ca²⁺ transients in LPS-treated ARVMs could be reversed back with ADM-(22–52) and PTX. This indicates that ADM plays a role in mediating the negative inotropic effect in LPS-treated ARVM through the activation of G_i signaling. This study delineates the intracellular pathways involved in ADM-mediated direct inotropic effects on ARVMs and also suggests a role of ADM in sepsis.

In its role as an endothelial cell proliferation and migration factor, vascular endothelial growth factor (VEGF) can affect peripheral circulation and therefore impact maximal oxygen consumption (V̇o_{2 max}). Because of the role of VEGF, and because variation in the VEGF gene has the ability to alter VEGF gene expression and VEGF protein level, we hypothesized that VEGF gene polymorphisms are related to VEGF gene expression in human myoblasts and V̇o_{2 max} before and after aerobic exercise training. We analyzed the effects of the VEGF −2578/−1154/−634 promoter region haplotype on VEGF gene expression by using a luciferase reporter assay in cultured human myoblasts and found that the AAG and CGC haplotypes resulted in significantly higher hypoxia-stimulated VEGF gene expression than the AGG and CGG haplotypes. Consistent with these results, we found that individuals with at least one copy of the AAG or CGC haplotype had higher V̇o_{2 max} before and after aerobic exercise training than did subjects with only the AGG and/or CGG haplotype. In conclusion, we found that VEGF −2578/−1154/−634 haplotype impacts VEGF gene expression in human myoblasts and is associated with V̇o_{2 max}. These results have potential implications for aerobic exercise training and may prove relevant in the study of pathological conditions that can be affected by angiogenesis, such as coronary artery disease and peripheral artery disease.

We used a longitudinal study design (gestational weeks 8, 15, 22, 29, and 36 and 12 wk postpartum ) to investigate the effect of normal pregnancy on cerebral autoregulation and pressor response. Blood flow velocities in the right internal carotid artery, end-tidal CO₂, and mean arterial pressure (MAP) were simultaneously and continuously recorded in 16 healthy pregnant women during standardized hyperventilation and handgrip. Blood flow velocities were recorded using Doppler ultrasound sampled beat by beat using the ECG signal. The results demonstrate that the vasoconstrictor response to hyperventilation is unchanged during pregnancy. During standardized handgrip, MAP showed a statistically significant increase during pregnancy that did not affect cerebral blood flow. A statistically significant reduction in the MAP response to handgrip was seen in week 36. In conclusion, pregnancy has no impact on cerebral autoregulation. There is an impact on the pressor response resulting in a blunted reaction at week 36, probably caused by a fall in the baroreflex set point.

The antioxidant response element (ARE) is a transcriptional control element that mediates expression of a set of antioxidant proteins. NF-E2-related factor 2 (Nrf2) is a transcription factor that activates ARE-containing genes. In endothelial cells, the ARE-mediated genes are upregulated by atheroprotective laminar flow through a Nrf2-dependent mechanism. We tested the hypothesis that activation of ARE-regulated genes via adenovirus-mediated expression of Nrf2 may suppress redox-sensitive inflammatory gene expression. Expression of Nrf2 in human aortic endothelial cells (HAECs) resulted in a marked increase in ARE-driven transcriptional activity and protected HAECs from H₂O₂-mediated cytotoxicity. Nrf2 suppressed TNF-α-induced monocyte chemoattractant protein (MCP)-1 and VCAM-1 mRNA and protein expression in a dose-dependent manner and inhibited TNF-α-induced monocytic U937 cell adhesion to HAECs. Nrf2 also inhibited IL-1β-induced MCP-1 gene expression in human mesangial cells. Expression of Nrf2 inhibited TNF-α-induced activation of p38 MAP kinase. Furthermore, expression of a constitutively active form of MKK6 (an upstream kinase for p38 MAP kinase) partially reversed Nrf2-mediated inhibition of VCAM-1 expression, suggesting that p38 MAP kinase, at least in part, mediates Nrf2's anti-inflammatory action. In contrast, Nrf2 did not inhibit TNF-α-induced NF-κB activation. These data identify the Nrf2/ARE pathway as an endogenous atheroprotective system for antioxidant protection and suppression of redox-sensitive inflammatory genes, suggesting that targeting the Nrf2/ARE pathway may represent a novel therapeutic approach for the treatment of inflammatory diseases such as atherosclerosis.

The zero-stress state of a blood vessel has been extensively studied because it is the reference state for which all calculations of intramural stress and strain must be based. It has also been found to reflect nonuniformity in growth and remodeling in response to chemical or physical changes. The zero-stress state can be characterized by an opening angle, defined as the angle subtended by two radii connecting the midpoint of the inner wall. All prior studies documented the zero-stress state or opening angle with no regard to duration of the no-load state. Our hypotheses were that, given the viscoelastic properties of blood vessels, the zero-stress state may have “memory” of prior circumferential and axial loading, i.e., duration of the no-load state influences opening angle. To test these hypotheses, we considered ring pairs of porcine coronary arteries to examine the effect of duration in the no-load state after circumferential distension. Our results show a significant reduction in opening angle as duration of the no-load state increases, i.e., vessels that are reduced to the zero-stress state directly from the loaded state attain much larger opening angles at 30 min after the radial cut than rings that are in the no-load state for various durations. To examine the effect of axial loading, we found similar reductions in opening angle with duration in the no-load from the in situ state, albeit the effect was significantly smaller than that of circumferential loading. Hence, we found that the zero-stress state has memory of both circumferential and axial loading. These results are important for understanding viscoelastic properties of coronary arteries, interpretation of the enormous data on the opening angle and strain in the literature, and standardization of future measurements on the zero-stress state.

The autocrine modulation of cardiac K⁺ currents was compared in ventricular and atrial cells (V and A cells, respectively) from Type 1 diabetic rats. K⁺ currents were measured by using whole cell voltage clamp. ANG II was measured by ELISA and immunofluorescent labeling. Oxidative stress was assessed by immunofluorescent labeling with dihydroethidium, a measure of superoxide ions. In V cells, K⁺ currents are attenuated after activation of the renin-angiotensin system (RAS) and the resulting ANG II-mediated oxidative stress. In striking contrast, these currents are not attenuated in A cells. Inhibition of the angiotensin-converting enzyme (ACE) also has no effect, in contrast to current augmentation in V cells. ANG II levels are enhanced in V, but not in A, cells. However, the high basal ANG II levels in A cells suggest that in these cells, ANG II-mediated pathways are suppressed, rather than ANG II formation. Concordantly, superoxide ion levels are lower in diabetic A than in V cells. Several findings indicate that high atrial natriuretic peptide (ANP) levels in A cells inhibit RAS activation. In male diabetic V cells, in vitro ANP (300 nM–1 μM, >5 h) decreases oxidative stress and augments K⁺ currents, but not when excess ANG II is present. ANP has no effect on ventricular K⁺ currents when the RAS is not activated, as in control males, in diabetic males treated with ACE inhibitor and in diabetic females. In conclusion, the modulation of K⁺ currents and oxidative stress is significantly different in A and V cells in diabetic rat hearts. The evidence suggests that this is largely due to inhibition of RAS activation and/or action by ANP in A cells. These results may underlie chamber-specific arrhythmogenic mechanisms.

Activation of adenosine A_2a receptors in the nucleus of the solitary tract (NTS) decreases mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA), whereas increases in preganglionic adrenal sympathetic nerve activity (pre-ASNA) occur, a pattern similar to that observed during hypotensive hemorrhage. Central vasopressin V₁ receptors may contribute to posthemorrhagic hypotension and bradycardia. Both V₁ and A_2a receptors are densely expressed in the NTS, and both of these receptors are involved in cardiovascular control; thus they may interact. The responses elicited by NTS A_2a receptors are mediated mostly via nonglutamatergic mechanisms, possibly via release of vasopressin. Therefore, we investigated whether blockade of NTS V₁ receptors alters the autonomic response patterns evoked by stimulation of NTS A_2a receptors (CGS-21680, 20 pmol/50 nl) in α-chloralose-urethane anesthetized male Sprague-Dawley rats. In addition, we compared the regional sympathetic responses to microinjections of vasopressin (0.1–100 ng/50 nl) into the NTS. Blockade of V₁ receptors reversed the normal decreases in MAP into increases (−95.6 ± 28.3 vs. 51.4 ± 15.7 ∫Δ%), virtually abolished the decreases in HR (−258.3 ± 54.0 vs. 18.9 ± 57.8 ∫Δbeats/min) and RSNA (−239.3 ± 47.4 vs. 15.9 ± 36.1 ∫Δ%), and did not affect the increases in pre-ASNA (279.7 ± 48.3 vs. 233.1 ± 54.1 ∫Δ%) evoked by A_2a receptor stimulation. The responses partially returned toward normal values ∼90 min after the blockade. Microinjections of vasopressin into the NTS evoked dose-dependent decreases in HR and RSNA and variable MAP and pre-ASNA responses with a tendency toward increases. We conclude that the decreases in MAP, HR, and RSNA in response to NTS A_2a receptor stimulation may be mediated via release of vasopressin from neural terminals in the NTS. The differential effects of NTS V₁ and A_2a receptors on RSNA versus pre-ASNA support the hypothesis that these receptor subtypes are differentially located/expressed on NTS neurons/neural terminals controlling different sympathetic outputs.

Intracardiac accumulation of lipid and related intermediates (e.g., ceramide) is associated with cardiac dysfunction and may contribute to the progression of heart failure (HF). Overexpression of nuclear receptor peroxisome proliferator-activated receptor-α (PPARα) increases intramyocellular ceramide and left ventricular (LV) dysfunction. We tested the hypothesis that activation of fatty acid metabolism with fat feeding or a PPARα agonist increases myocardial triglyceride and/or ceramide and exacerbates LV dysfunction in HF. Rats with infarct-induced HF (n = 38) or sham-operated rats (n = 10) were either untreated (INF, n = 10), fed a high-fat diet (45% kcal fat, INF + Fat, n = 15), or fed the PPARα agonist fenofibrate (150 mg·kg⁻¹·day⁻¹, INF + Feno, n = 13) for 12 wk. LV ejection fraction was significantly reduced with HF (49 ± 6%) compared with sham operated (86 ± 2%) with no significant differences in ejection fraction (or other functional or hemodynamic measures) among the three infarcted groups. Treatment with the PPARα agonist resulted in LV hypertrophy (24% increase in LV/body mass ratio) and induced mRNAs encoding for PPARα-regulated genes, as well as protein expression and activity of medium chain acyl-CoA dehydrogenase (compared with INF and INF + Fat groups). Myocardial ceramide content was elevated in the INF group compared with sham-operated rats, with no further change in the INF + Fat or INF + Feno groups. Myocardial triglyceride was unaffected by infarction but increased in the INF + Fat group. In conclusion, LV dysfunction and dilation are not worsened despite upregulation of the fatty acid metabolic pathway and LV hypertrophy or accumulation of myocardial triglyceride in the rat infarct model of HF.

Extracellular acidic pH was found to induce an outwardly rectifying Cl⁻ current (I_Cl,acid) in mouse ventricular cells, with a half-maximal activation at pH 5.9. The current showed the permeability sequence for anions to be SCN⁻ > Br⁻ > I⁻ > Cl⁻ > F⁻ > aspartate, while it exhibited a time-dependent activation at large positive potentials. Similar currents were also observed in mouse atrial cells and in atrial and ventricular cells from guinea pig. Some Cl⁻ channel blockers (DIDS, niflumic acid, and glibenclamide) inhibited I_Cl,acid, whereas tamoxifen had little effect on it. Unlike volume-regulated Cl⁻ current (I_Cl,vol) and CFTR Cl⁻ current (I_Cl,CFTR), I_Cl,acid was independent of the presence of intracellular ATP. Activation of I_Cl,acid appeared to be also independent of intracellular Ca²⁺ and G protein. I_Cl,acid and I_Cl,vol could develop in an additive fashion in acidic hypotonic solutions. Isoprenaline-induced I_Cl,CFTR was inhibited by acidification in a pH-dependent manner in guinea pig ventricular cells. Our results support the view that I_Cl,acid and I_Cl,vol stem from two distinct populations of anion channels and that the I_Cl,acid channels are present in cardiac cells. I_Cl,acid may play a role in the control of action potential duration or cell volume under pathological conditions, such as ischemia-related cardiac acidosis.

The ability of arterial smooth muscle to respond to vasoconstrictor stimuli is reduced in chronic portal hypertension (PHT). Additional evidence supports the existence of a postreceptor defect in vascular smooth muscle excitation contraction coupling. However, the nature of this defect is unclear. Recent studies have shown that vasoconstrictor stimuli induce actin polymerization in smooth muscle and that the associated increase in F-actin is necessary for force development. In the present study we have tested the hypothesis that impaired actin polymerization contributes to reduced vasoconstrictor function in small mesenteric arteries derived from rats with chronic prehepatic PHT. In vitro studies were conducted on small mesenteric artery vessel rings isolated from normal and PHT rats. Isometric tension responses to incremental concentrations of phenylephrine were significantly reduced in PHT arteries. The ability to polymerize actin in portal hypertensive mesenteric arteries stimulated by phenylephrine was attenuated compared with control. Inhibition of cAMP-dependent protein kinase (PKA) restored agonist-induced actin polymerization of arteries from PHT rats to normal levels. Depolymerization of actin in arteries from normal rats reduced maximal contractile force but not myosin phosphorylation, suggesting a key role for the dynamic regulation of actin polymerization in the maintenance of vascular smooth muscle contraction. We conclude that reductions in agonist-induced maximal force development of PHT vascular smooth muscle is due, in part, to impaired actin polymerization, and prolonged PKA activation may underlie these changes.

Several congenital heart defects require surgery that acutely increases pulmonary blood flow (PBF). This can lead to dynamic alterations in postoperative pulmonary vascular resistance (PVR) and can contribute to morbidity and mortality. Thus the objective of this study was to determine the role of nitric oxide (NO), endothelin (ET)-1, and their interactions in the alterations of PVR after surgically induced increases in PBF. Twenty lambs underwent placement of an aortopulmonary vascular graft. Lambs were instrumented to measure vascular pressures and PBF and studied for 4 h. Before and after shunt opening, lambs received an infusion of saline (n = 9), tezosentan, an ET_A- and ET_B-receptor antagonist (n = 6), or N^ω-nitro-l-arginine (l-NNA), a NO synthase (NOS) inhibitor (n = 5). In control lambs, shunt opening increased PBF by 117.8% and decreased PVR by 40.7% (P < 0.05) by 15 min, without further changes thereafter. Plasma ET-1 levels increased 17.6% (P < 0.05), and total NOS activity decreased 61.1% (P < 0.05) at 4 h. ET-receptor blockade (tezosentan) prevented the plateau of PBF and PVR, such that PBF was increased and PVR was decreased compared with controls at 3 and 4 h (P < 0.05). These changes were associated with an increase in total NOS activity (+61.4%; P < 0.05) at 4 h. NOS inhibition (l-NNA) after shunt placement prevented the sustained decrease in PVR seen in control lambs. In these lambs, PVR decreased by 15 min (P < 0.05) but returned to baseline by 2 h. Together, these data suggest that surgically induced increases in PBF are limited by vasoconstriction, at least in part by an ET-receptor-mediated decrease in lung NOS activity. Thus NO appears to be important in maintaining a reduction in PVR after acutely increased PBF.

Several essential components of NADPH oxidase, including p22^phox, gp91^phox (nox2) and its homologs nox1 and nox4, p47^phox, p67^phox, and rac1, are present in the vasculature. We previously reported that p67^phox is essential for adventitial fibroblast NADPH oxidase O₂⁻ production. Thus we postulated that inhibition of adventitial p67^phox activity would attenuate angioplasty-induced hyperplasia. To test this hypothesis, we treated the adventitia of carotid arteries with a control adenovirus (Ad-control), a virus expressing dominant-negative p67^phox (Ad-p67dn), or a virus expressing a competitive peptide (gp91ds) targeting the p47^phox-gp91^phox interaction (Ad-gp91ds). Common carotid arteries (CCAs) from male Sprague-Dawley rats were transfected with Ad-control, Ad-p67dn, or Ad-gp91ds in pluronic gel. After 2 days, a 2-F (Fogarty) catheter was used to injure CCAs in vivo. After 14 days, CCAs were perfusion-fixed and analyzed. In 13 experiments, digital morphometry suggested a reduction of neointimal hyperplasia with Ad-p67dn compared with Ad-control; however, the reduction did not reach statistical significance (P = 0.058). In contrast, a significant reduction was achieved with Ad-gp91ds (P = 0.006). No changes in medial area or remodeling were observed with either treatment. Moreover, adventitial fibroblast proliferation in vitro was inhibited by Ad-gp91ds but not by Ad-p67dn, despite confirmation that Ad-p67dn inhibits NADPH oxidase in fibroblasts. These data appear to suggest that a multicomponent vascular NADPH oxidase plays a role in neointimal hyperplasia. However, inhibition of p47^phox may be more effective than inhibition of p67^phox at attenuating neointimal growth.

AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that plays a key role in the regulation of energy metabolism. In humans, mutations in the γ2-subunit of AMPK cause cardiac hypertrophy associated with Wolff-Parkinson-White syndrome, characterized by ventricular preexcitation. The effect of these mutations on AMPK activity and in development of the disease is enigmatic. Here we report that transgenic mice with cardiac-specific expression of γ2 harboring a mutation of arginine residue 531 to glycine (RG-TG) develop a striking cardiac phenotype by 4 wk of age, including hypertrophy, impaired contractile function, electrical conduction abnormalities, and marked glycogen accumulation. At this stage, AMPK activity isolated from hearts of RG-TG mice was almost completely abolished but could be restored after phosphorylation by an upstream AMPK kinase. At 1 wk of age, there was no detectable evidence of a cardiac phenotype, and AMPK activity in RG-TG hearts was similar to that in nontransgenic, control mice. We propose that mutations in γ2 lead to suppression of total cardiac AMPK activity secondary to increased glycogen accumulation. The subsequent decrease in AMPK activity provides a mechanism that may explain the development of cardiac hypertrophy in this model.

The Tei index is clinically useful to quantify left ventricular (LV) function, but it requires sequential Doppler recordings from two different views. A related myocardial performance index (MPI) using tissue Doppler (TD) can be rapidly calculated from a single beat; however, its ability to quantify contractility and the effects of acute changes in loading have not been determined. Our aim was to test the hypothesis that TD MPI can quantify contractile state but is affected by acute alterations in loading, using LV pressure-volume relations in an animal model. Eight dogs were studied by using mitral annular TD, high-fidelity pressure, and conductance catheters. TD MPI was calculated as (a′ − b′)/b′, where a′ was the duration of mitral annular velocity during diastole and b′ was the duration of the systolic wave. End-systolic elastance (E_es), the time constant of isovolumic relaxation (τ), and peak positive and negative first derivative of pressure (dP/dt_max and dP/dt_min, respectively) were used as measures of LV function. Data were obtained at baseline, at dobutamine and esmolol infusion to alter contractile state, and at inferior vena cava and aortic occlusion to alter preload and afterload. TD MPI decreased from 0.83 (SD 0.19) to 0.62 (SD 0.20) with dobutamine and increased to 1.19 (SD 0.26) with esmolol. TD MPI significantly correlated with dP/dt_max (r = −0.76), E_es (r = −0.68), dP/dt_min (r = 0.82), and τ (r = 0.78); however, it was affected by acute decreases in preload [from 0.83 (SD 0.19) to 1.09 (SD 0.36)] and acute increases in afterload [to 1.23 (SD 0.17)]. All the above increases and decreases and r values were significant (P < 0.05 vs. baseline). In conclusion, TD MPI can rapidly quantify alterations in LV contractile state but is affected by acute alterations in preload and afterload.

We determined the effects of cyclooxygenase-1 (COX-1; SC-560), COX-2 (SC-58125), and inducible nitric oxide synthase (iNOS; 1400W) inhibitors on atorvastatin (ATV)-induced myocardial protection and whether iNOS mediates the ATV-induced increases in COX-2. Sprague-Dawley rats received 10 mg ATV·kg⁻¹·day⁻¹ added to drinking water or water alone for 3 days and received intravenous SC-58125, SC-560, 1400W, or vehicle alone. Anesthesia was induced with ketamine and xylazine and maintained with isoflurane. Fifteen minutes after intravenous injection rats underwent 30-min myocardial ischemia followed by 4-h reperfusion [infarct size (IS) protocol], or the hearts were explanted for biochemical analysis and immunoblotting. Left ventricular weight and area at risk (AR) were comparable among groups. ATV reduced IS to 12.7% (SD 3.1) of AR, a reduction of 64% vs. 35.1% (SD 7.6) in the sham-treated group (P < 0.001). SC-58125 and 1400W attenuated the protective effect without affecting IS in the non-ATV-treated rats. ATV increased calcium-independent NOS (iNOS) [11.9 (SD 0.8) vs. 3.9 (SD 0.1) × 1,000 counts/min; P < 0.001] and COX-2 [46.7 (SD 1.1) vs. 6.5 (SD 1.4) pg/ml of 6-keto-PGF_1α; P < 0.001] activity. Both SC-58125 and 1400W attenuated this increase. SC-58125 did not affect iNOS activity, whereas 1400W blocked iNOS activity. COX-2 was S-nitrosylated in ATV-treated but not sham-treated rats or rats pretreated with 1400W. COX-2 immunoprecipitated with iNOS but not with endothelial nitric oxide synthase. We conclude that ATV reduced IS by increasing the activity of iNOS and COX-2, iNOS is upstream to COX-2, and iNOS activates COX-2 by S-nitrosylation. These results are consistent with the hypothesis that preconditioning effects are mediated via PG.

The aim of this study was to investigate acute vasodilator responses to phytoestrogens and selective estrogen receptor-α (ERα) agonist in isolated small arteries from men with established coronary heart disease (CHD) and with a history of myocardial infarction versus healthy male control subjects. As to methodology, small arteries obtained from subcutaneous fat biopsies and mounted on a wire myograph were preconstricted with norepinephrine, and dilator responses to increasing nanomolar-micromolar concentrations of the phytoestrogens resveratrol and genistein (predominantly ERβ agonists) and to propyl-[1H]-pyrazole-1,3,5-triyl-trisplenol (PPT, a selective ERα agonist) were determined. These were compared with responses to reference compound 17β-estradiol (17β-E₂). Concentration-response curves were constructed before and after nitric oxide (NO) synthase inhibition with N^ω-nitro-l-arginine methyl ester. As a result, relaxation induced by the investigated compounds was similar in men with CHD and control men, but in both groups PPT and genistein-induced relaxation was greater than that of resveratrol and 17β-E₂. NO contributed to both phytoestrogens and PPT-induced relaxation but not to 17β-E₂ responses in arteries from control men. This NO-mediated component of relaxation was absent in arteries from men with established CHD. In conclusion, phytoestrogens, at concentrations achievable by ingestion of phytoestrogen-rich food products, evoke dilatation ex vivo of small peripheral arteries from normal men and those with established CHD. The contribution of NO to dilatory responses by these compounds is pertinent to arteries from control males, whereas other NO-independent dilatory mechanism(s) are involved in arteries from CHD.

We analyzed central interstitial potential differences during multisite stimulation to assess the feasibility of using those recordings to measure cardiac microimpedances in multidimensional preparations. Because interstitial current injected and removed using electrodes with different proximities allows modulation of the portion of current crossing the membrane, we hypothesized that multisite interstitial stimulation would give rise to central interstitial potential differences that depend on intracellular and interstitial microimpedances, allowing measurement of those microimpedances. Simulations of multisite stimulation with fine and wide spacing in two-dimensional models that included dynamic membrane equations for guinea pig ventricular myocytes were performed to generate test data (∂φ_o). Isotropic interstitial and intracellular microimpedances were prescribed for one set of simulations, and anisotropic microimpedances with unequal ratios (intracellular to interstitial) along and across fibers were prescribed for another set of simulations. Microimpedance measurements were then obtained by making statistical comparisons between ∂φ_o values and interstitial potential differences from passive bidomain simulations (Δφ_o) in which a wide range of possible microimpedances were considered. Possible microimpedances were selected at 25% increments. After demonstrating the effectiveness of the overall method with microimpedance measurements using one-dimensional test data, we showed microimpedance measurements within 25% of prescribed values in isotropic and anisotropic models. Our findings suggest that development of microfabricated devices to implement the procedure would facilitate routine measurement as a component of cardiac electrophysiological study.

Aortic smooth muscle cell release of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) has been implicated in aortic aneurysm pathogenesis, but proximal modulation of release is poorly understood. Extracellular nucleotides regulate vascular smooth muscle cell metabolism in response to physiochemical stresses, but nucleotide modulation of MMP and/or TIMP release has not been reported. We hypothesized that nucleotides modulate MMP-2 and TIMP-2 release from human aortic smooth muscle cells (HASMCs) via distinct purinergic receptors and signaling pathways. We exposed HASMCs to exogenous ATP and other nucleotides with and without interleukin-1β (IL-1β). HASMCs were pretreated in some experiments with apyrase, which degrades ATP, and inhibitors of ERK1/2, JNK, and p38 MAPK. MMP-2 and TIMP-2 released into supernatant were assessed using ELISA and Western blotting. ATP, adenosine, and UTP significantly stimulated MMP-2 release in the presence of IL-1β (300 nM ATP: 181 ± 22%, P = 0.003; 30 μm adenosine: 244 ± 150%, P = 0.001; and 200 μm UTP: 153 ± 40%, P = 0.015; vs. 100% constitutive). ATP also stimulated MMP-2 release in the absence of IL-1β (100 μm ATP: 148 ± 38% vs. 100% constitutive). Apyrase significantly reduced ATP-stimulated MMP-2 release (apyrase + 500 nM ATP: 59 ± 3% vs. 124 ± 7% with 500 nM ATP). Rank-order agonist potency for MMP-2 release was consistent with ATP activation of PAY and PAY receptors. ATP induced phosphorylation of intracellular JNK, and inhibition of the JNK pathway blocked ATP-stimulated MMP-2 release, indicating signaling via this pathway. Nucleotides are thus novel stimulants of MMP-2 release from HASMCs and may provide a mechanistic link between physiochemical stress in the aorta and aneurysms, especially in the context of inflammation.

Pathological cardiac hypertrophy is considered a precursor to clinical heart failure. Understanding the transcriptional regulators that suppress the hypertrophic response may have profound implications for the treatment of heart disease. We report the generation of transgenic mice that overexpress the transcription factor CHF1/Hey2 in the myocardium. In response to the α-adrenergic agonist phenylephrine, they show marked attenuation in the hypertrophic response compared with wild-type controls, even though blood pressure is similar in both groups. Isolated myocytes from transgenic mice demonstrate a similar resistance to phenylephrine-induced hypertrophy in vitro, providing further evidence that the protective effect of CHF1/Hey2 is mediated at the myocyte level. Induction of the hypertrophy marker genes ANF, BNP, and β-MHC in the transgenic cells is concurrently suppressed in vivo and in vitro, demonstrating that the induction of hypertrophy-associated genes is repressed by CHF1/Hey2. Transfection of CHF1/Hey2 into neonatal cardiomyocytes suppresses activation of an ANF reporter plasmid by the transcription factor GATA4, which has previously been shown to activate a hypertrophic transcriptional program. Furthermore, CHF1/Hey2 binds GATA4 directly in coimmunoprecipitation assays and inhibits the binding of GATA4 to its recognition sequence within the ANF promoter. Our findings demonstrate that CHF1/Hey2 functions as an antihypertrophic gene, possibly through inhibition of a GATA4-dependent hypertrophic program.

Rho-dependent kinases serve as downstream effectors of several vasoconstrictor systems, the activities of which are upregulated in congestive heart failure (CHF). We evaluated renal and cardiac effects of Y-27632, a highly selective Rho kinase inhibitor, in an experimental model of volume-overload CHF. Effects of acute administration of Y-27632 (0.3 mg/kg) on renal hemodynamic and clearance parameters and effects of chronic treatment (10.0 mg·kg⁻¹·day⁻¹ for 7 days via osmotic minipumps) on cardiac hypertrophy and cumulative Na⁺ excretion were studied in male Wistar rats with aortocaval fistula and control rats. The Y-27632-induced decrease in renal vascular resistance (from 40.4 ± 4.6 to 26.0 ± 3.1 resistance units, P < 0.01) in CHF rats was associated with a significant increase in total renal blood flow (+34%) and cortical and medullary blood flow (approx +37 and +27%, respectively). These values were significantly higher than those in control rats and occurred despite a decrease in mean arterial pressure (−15 mmHg). Despite the marked renal vasodilatory effect, Y-27632 did not alter glomerular filtration rate and renal Na⁺ excretion. Chronic administration of Y-27632 did not alter daily or cumulative renal Na⁺ excretion in CHF rats but was associated with a significant decrease in heart-to-body weight ratio, an index of cardiac hypertrophy: 0.32 ± 0.007, 0.46 ± 0.017, and 0.37 ± 0.006% in control, CHF, and CHF + Y-27632 rats, respectively. The findings suggest that Rho kinase-dependent pathways are involved in the mechanisms of renal vasoconstriction and cardiac hypertrophy in rats with volume-overload heart failure. Selective blockade of these signaling pathways may be considered an additional tool to improve renal perfusion and attenuate cardiac hypertrophy in heart failure.

Direct cell-to-cell transfer of ions and small signaling molecules via gap junctions plays a key role in vessel wall homeostasis. Vascular endothelial gap junctional channels are formed by the connexin (Cx) proteins Cx37, Cx40, and Cx43. The mechanisms regulating connexin expression and assembly into functional channels have not been fully identified. We investigated the dynamic regulation of endothelial gap junctional intercellular communication (GJIC) by fluid flow and the participation of each vascular connexin in functional human endothelial gap junctions in vitro. Human aortic endothelial cells (HAEC) were exposed for 5, 16, and 24 h to physiological flows in a parallel-plate flow chamber. Connexin protein expression and localization were evaluated by immunocytochemistry, and functional GJIC was evaluated by dye injection. Connexin-mimetic peptide inhibitors were used to assess the specific connexin composition of functional channels. HAEC monolayers in culture exhibited baseline functional communication at a striking low level despite abundant expression of Cx43 and Cx40 localized at cell-to-cell appositions. Upon exposure to flow, GJIC by dye spread demonstrated a significant time-dependent increase from baseline levels, reaching 7.5-fold in 24 h. Inhibition studies revealed that this response was mediated primarily by Cx40, with lesser contributions of the other two vascular connexins assembled into functional homotypic and/or heterotypic channels. This is the first study to demonstrate that flow simultaneously and differentially regulates expression of the Cx37, Cx40, and Cx43 proteins and their involvement in the augmentation of intercellular communication by dye transfer in human endothelial cells in vitro.

We investigated the role of pH, reactive oxygen species (ROS), Ca²⁺, and the mitochondrial permeability transition (MPT) in pH-dependent ischemia-reperfusion injury to adult rat myocytes. Myocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH 6.2 for 3 h to simulate ischemia. To simulate reperfusion, myocytes were reoxygenated at pH 6.2 or 7.4 for 2 h. Some myocytes were treated with MPT blockers (cyclosporin A and N-methyl-4-isoleucine cyclosporin) and antioxidants (desferal, diphenylphenylene diamine, and 2-mercaptopropionyl glycine). Mitochondrial membrane potential, inner membrane permeabilization, and ROS formation were imaged with tetramethylrhodamine methyl ester, calcein, and chloromethyldichlorofluorescein diacetate, respectively. For Ca²⁺ imaging, myocytes were coloaded with rhod-2 and fluo-4 to evaluate mitochondrial and cytosolic Ca²⁺, respectively. After 10 min of reperfusion at pH 7.4, calcein redistributed across the mitochondrial inner membrane, an event preceded by mitochondrial ROS formation and accompanied by hypercontracture, mitochondrial depolarization, and then cell death. Acidotic reperfusion, antioxidants, and MPT blockers each prevented the MPT, depolarization, hypercontraction, and cell killing. Antioxidants, but neither MPT blockers nor acidotic reperfusion, inhibited ROS formation after reperfusion. Furthermore, anoxic reperfusion at pH 7.4 prevented cell death. Both mitochondrial and cytosolic Ca²⁺ increased during ischemia but recovered in the first minutes of reperfusion. Mitochondrial and cytosolic Ca²⁺ overloading again occurred late after reperfusion. This late Ca²⁺ overloading was blocked by MPT inhibition. Intramitochondrial Ca²⁺ chelation by cold loading/warm incubation of BAPTA did not prevent cell death after reperfusion. In conclusion, mitochondrial ROS, together with normalization of pH, promote MPT onset and subsequent myocyte death after reperfusion. In contrast, Ca²⁺ overloading appears to be the consequence of bioenergetic failure after the MPT and is not a factor promoting MPT onset.

The intracellular protease calpain, abundant in endothelial cells (EC), is assumed to be inactive under physiological conditions but may account for Ca²⁺-linked pathophysiological events. However, nonstimulated EC contained autolyzed, activated calpain. Adding 12–48 μM calpain inhibitor I (CI) or 0.5–1 μM of the novel, membrane-permeable conjugate of calpastatin peptide-penetratin (CPP) caused rapid rounding and retraction of cultured EC (phase contrast, capacitance) and translocation of Syk, Rac, and Rho to the membrane, signifying activation upon inhibition of calpain. Isolated hearts (guinea pig) perfused with 12 μM CI or 0.5 μM CPP developed coronary leak. We conclude that calpain is constitutively active in EC and regulates vascular permeability by governing cell-cell attachment.

Cardiac rupture can be fatal after myocardial infarction (MI). Experiments in animals revealed gender differences in rupture rate; however, patient data are controversial. We found a significantly higher rupture rate in testosterone-treated female mice within 1 wk after MI, whereas castration in males significantly reduced rupture. We hypothesized that testosterone may adversely affect remodeling after MI, exaggerating the inflammatory response and increasing cardiac rupture, whereas estrogen may be cardioprotective, attenuating early remodeling and reducing rupture rate. We studied the effect of gender and hormone manipulation on morphological and histological changes during early remodeling after MI in 4-wk-old male and female C57BL/6J mice and how these events could affect cardiac function. Females were randomly divided into 1) sham ovariectomy + placebo (s-ovx + P), 2) s-ovx + testosterone (T), 3) ovx + P, and 4) ovx + T; males were divided into 1) sham castration + P (s-cas + P), 2) s-cas + 17β-estradiol (E), 3) cas + P, and 4) cas + E. At 6 wk after gonadectomy and hormone manipulation, MI was induced. Mice were randomly killed 1, 2, 4, 7, and 14 days after MI. The left ventricle was weighed and sectioned for evaluation of MI size, infarct expansion index (IEI), and neutrophil infiltration. Transthoracic echocardiography was performed in conscious mice in the 14-day group before organ harvest. Cardiac rupture rate and IEI were significantly higher in testosterone-treated females and noncastrated males than in controls; these effects were accompanied by enhanced neutrophil infiltration and pronounced deterioration of cardiac function and left ventricular dilatation. Ovariectomy in females and estrogen supplementation in males did not confer significant protection from cardiac rupture, IEI, or neutrophil infiltration. We concluded that, in mice, high testosterone levels enhance acute myocardial inflammation, adversely affecting myocardial healing and early remodeling, as indicated by increased cardiac rupture, and possibly causing deterioration of cardiac function after MI, and, conversely, estrogen seems to have no significant protective effect in the acute phase after MI.

Peripheral polymorphonuclear leukocytes (PMNL) in hemodialysis (HD) patients are primed, continually releasing and exposing the vascular endothelium to soluble factors such as reactive oxygen species and inflammatory mediators. To mimic the close proximity between PMNL and the endothelial monolayer and to monitor and characterize the influence of soluble mediators released from PMNL, we developed a novel cocultivation system using primary human umbilical vein endothelial cell (HUVEC) cultures and PMNL, with a sieve separating the two cell types to prevent direct adhesive effects. PMNL (10⁶) from HD patients or from healthy normal controls were cocultivated with HUVEC (10⁵) for 15 min, and endothelial cell injury was assessed by HUVEC morphology, cell detachment, and apoptosis. Proinflammatory changes were estimated by expression of HUVEC adhesion molecule P-selectin and by endothelial IL-8 and endothelial nitric oxide synthase mRNA. The levels of intracellular tissue factor reflected the procoagulant state, whereas NADPH oxidase activity served as an indicator for prooxidative changes in HUVEC. Mediators released from the primed PMNL triggered activation/dysfunction of endothelial cells, causing 1) an increase in endothelial cell detachment and apoptosis, 2) a proinflammatory state manifested by increased IL-8 mRNA expression and P-selectin on the endothelial surface, 3) activation of endothelial NADPH oxidase, 4) an increase in endothelial cell tissue factor that directly correlated with PMNL priming index, and 5) a decrease in endothelial nitric oxide synthase mRNA. Our data support a pathogenic link between PMNL priming and endothelial dysfunction, suggesting that PMNL priming is a potential new nontraditional risk factor for the development of atherosclerosis.

Postischemic reperfusion of rat or mouse hearts causes generation of inositol (,,)trisphosphate [Ins(,,)P₃] and the initiation of arrhythmias. In the current study we investigated the possibility that the enhanced Ins(,,)P₃ generation in postischemic reperfusion was associated with an increased availability of the precursor lipid phosphatidylinositol(,)bisphosphate (PIP₂) for α₁-adrenergic receptor-activated phospholipase C (PLC). Isolated-perfused rat hearts were labeled with [³H]inositol and subjected to ischemia-reperfusion or stimulation with norepinephrine under normoxic conditions. Caveolar fractions were prepared by buoyant density sucrose gradient centrifugation. [³H]PIP₂ was concentrated in caveolae, along with Gαq and PLCβ1b. Caveolae contained only 27.3 ± 6.9% (means ± SE, n = 6) of the total α₁-adrenergic receptor complement of the heart. These did not migrate to PIP₂-containing caveolar fractions with norepinephrine stimulation under normoxic conditions, even though caveolar PIP₂ was depleted. In contrast, [³H]PIP₂ in caveolae increased during 2 min of reperfusion, independently of norepinephrine release and thus of α₁-adrenergic receptor activation. The increased PIP₂ in the caveolar fractions where signaling proteins are concentrated may be critical for the heightened generation of Ins(,,)P₃ in early reperfusion.

Previous studies have demonstrated that skeletal muscle perfusion is impaired in obese Zucker rats (OZR) under control conditions and with elevated metabolic demand versus responses in lean Zucker rats (LZR). To further our understanding of processes contributing to impaired perfusion, we determined whether hyperemic responses following periods of occlusion were altered in skeletal muscle of OZR versus LZR. In isolated hindlimbs, basal blood flow in OZR was less than in LZR, and total perfusion responses after 30, 90, and 180 s of occlusion were reduced. Treatment of animals with an antioxidant (polythethylene glycol-superoxide dismutase) had no effect on reactive hyperemia, although blockade of α-adrenoreceptors (α₁ > α₂) improved responses to 30 and 90 s of occlusion; responses to 180 s of occlusion were unaltered. Pump perfusion of a dilated distal hindlimb demonstrated that increased volume flow elicited a greater increase in perfusion pressure in OZR versus LZR, suggesting structural contributions to an increased vascular resistance. Responses were comparable for in situ cremaster muscle because reactive hyperemia following serial arteriolar occlusion was attenuated in OZR versus LZR, treatment with polythethylene glycol-superoxide dismutase was ineffective, and hyperemic responses were improved following inhibition of α-adrenoreceptors (α₁ > α₂). Treatment of cremaster muscle with adenosine (10⁻³ M) caused flow to increase to a level comparable to that following 180 s of occlusion in both strains, although this level was reduced in OZR versus LZR. These results suggest that increased adrenergic tone may constrain reactive hyperemia in OZR with brief occlusion, although structural increases in vascular resistance can contribute to constrained perfusion after longer periods of occlusion.

Studies have shown that hypertrophied hearts are unusually vulnerable to ischemia. Compromised O₂ supply has been postulated as a possible explanation for this phenomenon on the basis of elongated O₂ diffusion distance and altered coronary vasculature found in hypertrophied myocardium. To examine the postulate, perfused heart experiments followed the metabolic and functional responses of hypertrophic myocardium to ischemia. ¹H/³¹P NMR was used to measure cellular oxygenation and energy level during ischemia-reperfusion. The left ventricles from spontaneously hypertensive rats (SHR) were enlarged by 48%. With this moderate degree of hypertrophy, cellular O₂ and energy levels were normal during baseline perfusion. After an ischemic episode, however, cellular O₂ was severely deprived in the SHR hearts compared with the normal hearts. Depressed postischemic O₂ reperfusion correlated well with depressed energetic and functional recovery. The results from the current study thus demonstrate a critical relationship between reperfused O₂ level and functional recovery in hypertrophic myocardium. The role of reperfused O₂, however, is time dependent. During early reperfusion, factor(s) other than O₂ appear to limit functional recovery. It is when the mechanical function of the heart approaches a new steady state that O₂ becomes a dominant factor. Meanwhile, the finding of a normal O₂ level in preischemic SHR hearts defies the notion of preexisting hypoxia as a primer of ischemic damage.

Mechanisms underlying coronary spasm are still poorly understood. The aim of the study was to assess the hypothesis that fluctuations in the development of coronary spasm might reflect inputs from the adjacent esophageal system. We enrolled patients admitted to the coronary care unit for episodes of nocturnal angina. Seven patients with variant angina and five with coronary artery disease (CAD) had concurrent ECG and esophageal manometric monitoring. ECG monitoring documented 28 episodes of ST elevation in variant angina patients and 16 episodes of ST depression in CAD patients. Manometric analysis showed that esophageal spasms resulted remarkably more frequently in variant angina patients (143 total spasms; individual range 9–31) than in CAD patients (20 total spasms; individual range 0–9; P < 0.01). Time series analysis was used to assess fluctuations in the occurrence of abnormal esophageal waves and its relationship with spontaneous episodes of ST shift. Episodes of esophageal spasm in CAD were sporadic (<1 in 30 min) and not related to ECG-recorded ischemia. In the variant angina group, esophageal spasms were time related to ischemia (>1 into 5 min before ECG-recorded ischemia) (P < 0.05). A bidirectional analysis of causal effects showed that the influence processes between esophageal and coronary spasms were mutual and reciprocal (transfer function model, P < 0.05) in variant angina. We concluded that in variant angina patients, episodes of esophageal spasms and myocardial ischemia influenced each other. Mechanisms that cause esophageal spasm can feed back to produce coronary spasm. Coronary spasm may feed forward to produce additional episodes of esophageal spasm.

Cardiac contraction-relaxation coupling is determined by both the free intracellular calcium concentration ([Ca²⁺]_i) and myofilament properties. We set out to develop a technique where we could assess these parameters (twitch and steady-state force [Ca²⁺]_i) under near physiological conditions. Bis-fura-2 was iontophorically introduced into ultrathin rat trabeculae preparations to monitor the [Ca²⁺]_i, and steady-state contractures were achieved by using a modified Krebs-Henseleit solution containing high K⁺. During K⁺ contractures, the very slow changes in [Ca²⁺]_i and force development were in equilibrium and allowed for the construction of a steady-state, force-[Ca²⁺]_i relationship. Twitch contractions before and after this myofilament calcium sensitivity assessment were unaltered, and this protocol could be repeated several times. For the first time, this novel protocol allows us to measure myofilament calcium sensitivity under physiological temperature. Not only do the data so obtained allow us to assess myofilament calcium sensitivity, the data also will allow us, in the same preparation under nearly identical conditions, to compare the dynamic to the steady-state, force-calcium relationship. To test whether the steady-state relationship between force and calcium in our novel protocol reproduces expected changes, we determined this relationship in the presence of isoproterenol and under acidosis and alkalosis. As expected, β-adrenergic stimulation resulted in an increase of calcium amplitude and twitch force and a desensitization of the myofilaments as indicated by a rightward shift of the obtained steady-state, force-calcium relationship. An increase in pH shifted the curve leftward, whereas a decrease in pH resulted in the expected rightward shift.

Mice that overexpress the inflammatory cytokine tumor necrosis factor-α in the heart (TNF mice) develop heart failure characterized by atrial and ventricular dilatation, decreased ejection fraction, atrial and ventricular arrhythmias, and increased mortality (males > females). Abnormalities in Ca²⁺ handling, prolonged action potential duration (APD), calcium alternans, and reentrant atrial and ventricular arrhythmias were previously observed with the use of optical mapping of perfused hearts from TNF mice. We therefore tested whether altered voltage-gated outward K⁺ and/or inward Ca²⁺ currents contribute to the altered action potential characteristics and the increased vulnerability to arrhythmias. Whole cell voltage-clamp recordings of K⁺ currents from left ventricular myocytes of TNF mice revealed an ∼50% decrease in the rapidly activating, rapidly inactivating transient outward K⁺ current I_to and in the rapidly activating, slowly inactivating delayed rectifier current I_K,slow1, an ∼25% decrease in the rapidly activating, slowly inactivating delayed rectifier current I_K,slow2, and no significant change in the steady-state current I_ss compared with controls. Peak amplitudes and inactivation kinetics of the L-type Ca²⁺ current I_Ca,L were not altered. Western blot analyses revealed a reduction in the proteins underlying Kv4.2, Kv4.3, and Kv1.5. Thus decreased K⁺ channel expression is largely responsible for the prolonged APD in the TNF mice and may, along with abnormalities in Ca²⁺ handling, contribute to arrhythmias.

Docetaxel and prostaglandin E₁ (PGE₁) increase transcapillary albumin extravasation and reduce interstitial fluid pressure in the skin. In this study the microdialysate concentration (C_m) of ¹²⁵I-labeled human serum albumin (¹²⁵I-HSA) and different-sized endogenous plasma proteins (EPP) was compared to evaluate changes in transcapillary extravasation of plasma proteins. ¹²⁵I-HSA was also used to estimate changes in the specific activity of albumin. Extravasation of ¹²⁵I-HSA and EPP from plasma to interstitium in the rat skin was compared during continuous administration of docetaxel and PGE₁ by using microdialysis in anesthetized rats. Also, 20 ml of Ringer solution (RS) were injected intravenously during 10 min in a separate group. Two hollow plasmapheresis fibers (3 cm, cut off 3,000 kDa), one acting as control, were placed subcutaneously on the back skin and perfused with RS (5 μl/min, 140 min, collected every 10 min). The size of the different EPP was estimated to be 73, 65, 56, 47, and 39 Å, separated by a size-exclusion high-performance liquid chromatography column and quantified by UV detection (280 nm). Docetaxel (0.5 mg/ml, n = 5) increased C_m of ¹²⁵I-HSA and EPP of sizes 73, 65, 56, and 39 Å significantly (P < 0.05) compared with control. PGE₁ (20 μg/ml, n = 6) increased C_m of ¹²⁵I-HSA significantly (P < 0.05) but none of the different-sized EPP was increased compared with control. Intravenous RS (20 ml, n = 6) increased C_m of ¹²⁵I-HSA and increased all the different-sized EPP significantly (P < 0.05) compared with control. Although the microdialysis method is able to monitor qualitative changes in capillary permeability, a quantitative determination of the capillary reflection coefficient or permeability-surface area product was not possible, because steady state between plasma and dialysate was not achieved during the measurement period. The different pattern of extravasation of EPP and ¹²⁵I-HSA after docetaxel, PGE₁, and RS indicates increased interstitial transport rate and/or increased capillary permeability after docetaxel and RS, whereas PGE₁ seems to increase transcapillary fluid flux without altering the permeability.

In addition to well-documented vascular growth-promoting effects, ANG II exerts proapoptotic effects that are poorly understood. IGF-1 is a potent survival factor for human vascular smooth muscle cells (hVSMC), and its antiapoptotic effects are mediated via the IGF-1 receptor (IGF-1R) through a signaling pathway involving phosphatidylinositol 3-kinase and Akt. We hypothesized that there would be cross talk between ANG II proapoptotic effects and IGF-1 survival effects in hVSMC. To investigate ANG II-induced apoptosis and the potential involvement of IGF-1, we exposed quiescent and nonquiescent hVSMC to ANG II. ANG II induced apoptosis only in nonquiescent cells but stimulated hypertrophy in quiescent cells. ANG II-induced apoptosis was characterized by marked inhibition of Akt phosphorylation and stimulation of membrane Fas ligand (FasL) expression, caspase-8 activation, and a reduction in soluble FasL expression. Adenovirally mediated overexpression of Akt rescued hVSMC from ANG II-induced apoptosis. IGF-1R activation increased Akt phosphorylation and soluble FasL expression, and these effects were completely blocked by coincubating hVSMC with ANG II. In conclusion, ANG II-induced apoptosis of hVSMC is characterized by marked inhibition of Akt phosphorylation and stimulation of an extrinsic cell death signaling pathway via upregulation of membrane FasL expression, caspase-8 activation, and a reduction in soluble FasL expression. Furthermore, ANG II antagonizes the antiapoptotic effect of IGF-1 by blocking its ability to increase Akt phosphorylation and soluble FasL. These findings provide novel insights into ANG II-induced apoptotic signaling and have significant implication for understanding ANG II-induced remodeling in hypertension and atherosclerosis.

Normal pregnancy is characterized by an increased uterine blood flow due to growth and remodeling of the maternal uterine vasculature and enhanced vasodilation of the uterine arteries. The objective of the present study was to examine the role of endothelial cell Ca²⁺ signaling in augmented endothelium-mediated vasodilation of uteroplacental arteries in late pregnancy. We performed fura-2-based measurements of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the cytoplasm of endothelial cells simultaneously with diameter in pressurized uterine arteries from nonpregnant (NP) and late-pregnant (LP) rats. Basal levels of endothelial cell [Ca²⁺]_i were higher in arteries from LP rats compared with NP controls. Withdrawal of extracellular Ca²⁺ resulted in a decrease in the level of basal [Ca²⁺]_i that was significantly larger in arteries of LP than NP rats. The rate of Mn²⁺-induced quenching of fura-2 fluorescence was significantly elevated in late pregnancy, implicating augmented Ca²⁺ influx as a cause of increased basal levels of [Ca²⁺]_i in endothelial cells. Elevation of intraluminal pressure resulted in a transient increase in endothelial [Ca²⁺]_i that was markedly potentiated in late gestation. ACh-induced [Ca²⁺]_i and vasodilator responses were significantly augmented in arteries of LP compared with NP rats and were abolished by BAPTA treatment, demonstrating a critical role of [Ca²⁺]_i elevation in the production of endothelium-derived vasodilators. Together, these results indicate that late pregnancy is a state of enhanced basal and stimulated Ca²⁺ signaling in endothelial cells of uterine vessels, which may represent an important underlying mechanism for augmented vasodilation in the maternal uterine circulation.

We previously showed that C-phycocyanin (PC), an antioxidant biliprotein pigment of Spirulina platensis (a blue-green alga), effectively inhibited doxorubicin-induced oxidative stress and apoptosis in cardiomyocytes. Here we investigated the cardioprotective effect of PC against ischemia-reperfusion (I/R)-induced myocardial injury in an isolated perfused Langendorff heart model. Rat hearts were subjected to 30 min of global ischemia at 37°C followed by 45 min of reperfusion. Hearts were perfused with PC (10 μM) or Spirulina preparation (SP, 50 mg/l) for 15 min before the onset of ischemia and throughout reperfusion. After 45 min of reperfusion, untreated (control) hearts showed a significant decrease in recovery of coronary flow (44%), left ventricular developed pressure (21%), and rate-pressure product (24%), an increase in release of lactate dehydrogenase and creatine kinase in coronary effluent, significant myocardial infarction (44% of risk area), and TdT-mediated dUTP nick end label-positive apoptotic cells compared with the preischemic state. PC or SP significantly enhanced recovery of heart function and decreased infarct size, attenuated lactate dehydrogenase and creatine kinase release, and suppressed I/R-induced free radical generation. PC reversed I/R-induced activation of p38 MAPK, Bax, and caspase-3, suppression of Bcl-2, and increase in TdT-mediated dUTP nick end label-positive apoptotic cells. However, I/R also induced activation of ERK1/2, which was enhanced by PC treatment. Overall, these results for the first time showed that PC attenuated I/R-induced cardiac dysfunction through its antioxidant and antiapoptotic actions and modulation of p38 MAPK and ERK1/2.

We investigated whether benidipine, a long-acting calcium channel blocker (CCB), can normalize cardiac expression profiles of the endothelin (ET)-1 system in insulin-resistant diabetes. Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of human Type 2 diabetes, were treated for 12 wk with vehicle or benidipine (3 mg·kg⁻¹·day⁻¹). OLETF rats exhibited a significant increase in ET-1 in plasma and left ventricular (LV) tissues compared with nondiabetic controls. Expression of prepro-ET-1, ET-converting enzyme, and ET_A and ET_B receptors in LV tissues was also significantly higher in OLETF rats. The two MAPKs, JNK and p38^MAPK, both of which are activated by ET-1, were more abundantly expressed in OLETF rat LV tissues. All these alterations were reversed to nondiabetic levels when OLETF rats were treated with the subdepressor dose of benidipine. Furthermore, benidipine therapy resulted in hindering cardiomyocyte hypertrophy and cardiac perivascular fibrosis in OLETF rats. The beneficial actions of benidipine at the subdepressor dose on cardiac remodeling in insulin-resistant diabetes may involve normalization of the upregulated ET-1 system.

The Na⁺/Ca²⁺ exchanger (NCX) NCX1 exhibits tissue-specific alternative splicing. Such NCX splice variants as NCX1.1 and NCX1.3 are also differentially regulated by Na⁺ and Ca²⁺, although the physiological implications of these regulatory characteristics are unclear. On the basis of their distinct regulatory profiles, we hypothesized that cells expressing these different splice variants might exhibit unique responses to conditions promoting Ca²⁺ overload, such as during exposure to cardiac glycosides or simulated ischemia. NCX1.1 or NCX1.3 was expressed in human embryonic kidney (HEK)-293 cells or rat neonatal ventricular cardiomyocytes (NVC), and expression was confirmed by Western blotting and immunocytochemical analyses. HEK-293 cells lacked NCX1 protein before transfection. With use of adenoviral vectors, neonatal cardiomyocytes were induced to overexpress the NCX1.1 splice variant by nearly twofold, whereas the NCX1.3 isoform was expressed on the endogenous NCX1.1 background. Total expression was comparable for NCX1.1 and NCX1.3. Exposure of NVC to ouabain induced a significant increase in cellular Ca²⁺, an effect that was exaggerated in cells overexpressing NCX1.1, but not NCX1.3. The increase in intracellular Ca²⁺ was inhibited by 5 μM KB-R7943. Cardiomyocytes overexpressing NCX1.1 also exhibited a greater accumulation of intracellular Ca²⁺ in response to simulated ischemia than did cells expressing NCX1.3. Similar responses were observed in HEK-293 cells where NCX1.1 was expressed. We conclude that expression of the NCX1.3 splice variant protects against severe Ca²⁺ overload, whereas NCX1.1 promotes Ca²⁺ overload in response to cardiac glycosides and ischemic challenges. These results highlight the importance of ionic regulation in controlling NCX1 activity under conditions that promote Ca²⁺ overload.