Objective: This study used a novel multiscale electromechanical model of the rabbit ventricles to investigate the origin of and the substrate for spontaneous arrhythmias arising from ischemia-induced electrophysiological and mechanical changes.

Methods and Results: Two stages of ischemia were simulated. Dynamic mechanoelectrical feedback was modeled as spatially and temporally nonuniform membrane currents through mechanosensitive channels, the conductances of which depended on local strain rate. Our results reveal that both strains and strain rates were significantly larger in the central ischemic zone than in the border zone. However, in both ischemia stages, a VPB originated from the ischemic border in the left ventricular apical endocardium because of mechanically induced suprathreshold depolarizations. It then traveled fully intramurally until emerging from the ischemic border on the anterior epicardium. Reentry was formed only in the advanced ischemia stage as the result of a widened temporal excitable gap. Mechanically induced delayed afterdepolarization-like events contributed to the formation of reentry by further decreasing the already reduced-by-hyperkalemia local excitability, causing extended conduction block lines and slowed conduction in the ischemic region.

Conclusions: Mechanically induced membrane depolarizations in the ischemic region are the mechanism by which mechanical activity contributes to both the origin of and substrate for spontaneous arrhythmias under the conditions of acute regional ischemia.

Objective: Our goal was to determine the role of resident intimal DCs in the initiation of atherosclerosis.

Methods and Results: En face immunostaining of nascent atherosclerotic lesions in low-density lipoprotein receptor–deficient (Ldlr^-/-) mice fed a cholesterol-rich diet for 5 or 10 days demonstrated that foam cells expressed DC markers CD11c, 33D1, and major histocompatibility complex class II. Transmission electron microscopy revealed that the majority of intimal lipid was intracellular. The role of resident intimal DCs in lesion formation was verified by their conditional depletion using transgenic mice expressing the simian diphtheria toxin receptor in CD11c⁺ cells. A single injection of diphtheria toxin depleted intimal CD11c⁺ DCs by >98% within 24 hours, with 25% and 75% recovery at 1 and 3 weeks, respectively. When bred onto the Ldlr^-/- background, intimal DC depletion with diphtheria toxin during 5 days of lesion formation reduced the intimal lipid area by 55% relative to undepleted controls. Transmission electron microscopy revealed few foam cells in DC-depleted mice and abundant accumulation of subendothelial extracellular lipid.

Conclusions: Induction of hypercholesterolemia in mice triggers rapid ingestion of lipid by resident intimal DCs, which initiate nascent foam cell lesion formation.

Objective: We aimed to examine the effect of class II HDACs on post–myocardial infarction remodeling in male and female mice.

Methods and Results: Here we show that the absence of HDAC5 or -9 in female mice protects against maladaptive remodeling following myocardial infarction, which coincides with upregulation of estrogen-responsive genes in the heart. This genetic reprogramming coincides with a pronounced increase in expression of the estrogen receptor (ER) gene, which we show to be a direct MEF2 target gene. ER also directly interacts with class II HDACs. Cardioprotection resulting from the absence of HDAC5 or -9 in female mice can be attributed, at least in part, to enhanced neoangiogenesis in the infarcted region via upregulation of the ER target gene vascular endothelial growth factor-a.

Conclusions: Our results reveal a novel gender-specific pathway of cardioprotection mediated by ER and its regulation by MEF2 and class II HDACs.

Objective: We tested the hypothesis that the structural heterogeneity of the ventricular wall modulates globally applied stretches to create heterogeneous strain distributions that lead to the initiation of arrhythmias.

Methods and Results: We applied global stretches to arterially perfused rabbit right ventricular tissue preparations. The distribution of strain (determined by marker tracking) and the transmembrane potential (measured by optical mapping) were simultaneously recorded while accounting for motion artifacts. The 3D structure of the preparations was also examined using a laser displacement meter. To examine whether such observations can be translated to the physiological condition, we performed similar measurements in whole heart preparations while applying volume pulses to the right ventricle. At the tissue level, larger stretches (≥20%) caused synchronous excitation of the entire preparation, whereas medium stretches (10% and 15%) induced focal excitation. We found a significant correlation between the local strain and the local thickness, and the probability for focal excitation was highest for medium stretches. In the whole heart preparations, we observed that such focal excitations developed into reentrant arrhythmias.

Conclusions: Global stretches of intermediate strength, rather than intense stretches, created heterogeneous strain (excitation) distributions in the ventricular wall, which can trigger fatal arrhythmias.

Objective: To prevent the "normal" downregulation of miR-133a induced by an acute hypertrophic stimulus in the adult heart.

Methods and Results: miR-133a is downregulated in transverse aortic constriction (TAC) and isoproterenol-induced hypertrophy, but not in 2 genetic hypertrophy models. Using MYH6 promoter-directed expression of a miR-133a genomic precursor, increased cardiomyocyte miR-133a had no effect on postnatal cardiac development assessed by measures of structure, function, and mRNA profile. However, increased miR-133a levels increased QT intervals in surface electrocardiographic recordings and action potential durations in isolated ventricular myocytes, with a decrease in the fast component of the transient outward K⁺ current, I_to,f, at baseline. Transgenic expression of miR-133a prevented TAC-associated miR-133a downregulation and improved myocardial fibrosis and diastolic function without affecting the extent of hypertrophy. I_to,f downregulation normally observed post-TAC was prevented in miR-133a transgenic mice, although action potential duration and QT intervals did not reflect this benefit. miR-133a transgenic hearts had no significant alterations of basal or post-TAC mRNA expression profiles, although decreased mRNA and protein levels were observed for the I_to,f auxiliary KChIP2 subunit, which is not a predicted target.

Conclusions: These results reveal striking differences between in vitro and in vivo phenotypes of miR expression, and further suggest that mRNA signatures do not reliably predict either direct miR targets or major miR effects.

Objective: We hypothesized that extracellular HSP60 (exHSP60) would mediate apoptosis via TLR-4.

Methods and Results: Adult rat cardiac myocytes were treated with HSP60, either recombinant human or with HSP60 purified from the media of injured rat cardiac myocytes. ExHSP60 induced apoptosis in cardiac myocytes, as detected by increased caspase 3 activity and increased DNA fragmentation. Apoptosis could be reduced by blocking antibodies to TLR-4 and by nuclear factor B binding decoys, but not completely inhibited, even though similar treatment blocked lipopolysaccharide-induced apoptosis. Three distinct controls showed no evidence for involvement of a ligand other than exHSP60 in the mediation of apoptosis.

Conclusions: This is the first report of HSP60-induced apoptosis via the TLRs. HSP60-mediated activation of TLR-4 may be a mechanism of myocyte loss in heart failure, where HSP60 has been detected in the plasma.

Objective: To test the role of S100A12 in vascular inflammation, we generated and analyzed mice expressing human S100A12 in vascular smooth muscle under control of the smooth muscle 22 promoter because S100A12 is not present in mice.

Methods and Results: Transgenic mice displayed pathological vascular remodeling with aberrant thickening of the aortic media, disarray of elastic fibers, and increased collagen deposition, together with increased latent matrix metalloproteinase-2 protein and reduction in smooth muscle stress fibers leading to a progressive dilatation of the aorta. In primary aortic smooth muscle cell cultures, we found that S100A12 mediates increased interleukin-6 production, activation of transforming growth factor β pathways and increased metabolic activity with enhanced oxidative stress. To correlate our findings to human aortic aneurysmal disease, we examined S100A12 expression in aortic tissue from patients with thoracic aortic aneurysm and found increased S100A12 expression in vascular smooth muscle cells.

Conclusions: S100A12 expression is sufficient to activate pathogenic pathways through the modulation of oxidative stress, inflammation and vascular remodeling in vivo.

Objective: To characterize the role of Inpp5f in regulating cardiac hypertrophy.

Methods and Results: We generated homozygous Inpp5f knockout mice and cardiac specific Inpp5f overexpression transgenic mice. We evaluated their hearts for biochemical, structural and functional changes. Inpp5f knockout mice have augmented hypertrophy and reactivation of the fetal gene program in response to stress when compared to wild-type littermates. Furthermore, cardiac overexpression of Inpp5f in transgenic mice reduces hypertrophic responsiveness.

Conclusions: Our results suggest that Inpp5f is a functionally important endogenous modulator of cardiac myocyte size and of the cardiac response to stress.

Objective: To explore the mechanism underlying the roles of 14-3-33 and filamin A in regulating the VWF binding function of GPIb-IX.

Methods and Results: A truncation mutant of GPIb (565) deleting the C-terminal 14-3-33 binding sites retained 14-3-33 binding function, in contrast, deletion of the C-terminal residues 551 to 610 of GPIb totally abolished 14-3-33 binding, indicating that the residues 551 to 564 of GPIb are important in the interaction between 14-3-33 and GPIb-IX. An antibody recognizing phosphorylated R557GpSLP561 sequence reacted with GPIb suggesting phosphorylation of a population of GPIb molecules at Ser559, and a membrane permeable phosphopeptide (MP-P), R557GpSLP561 corresponding to residues 557 to 561 of GPIb eliminated the association of 14-3-33 with 565. MP-P also promoted GPIb-IX association with the membrane skeleton, and inhibited ristocetin-induced platelet agglutination, VWF binding to platelets and platelet adhesion to immobilized VWF. Furthermore, a GPIb-IX mutant replacing Ser559 of GPIb with alanine showed an enhanced association with the membrane skeleton, reduced ristocetin-induced VWF binding, and diminished ability to mediate cell adhesion to VWF under flow conditions.

Conclusions: These data suggest a phosphorylation-dependent binding of 14-3-33 to central filamin A binding site of GPIb, and the dimeric 14-3-33 binding to both the C-terminal site and central RGpSLP site inhibits GPIb-IX association with the membrane skeleton and promotes the VWF binding function of GPIb-IX.

Objective: We investigated the net effects of acute hypoxia and catecholamines on the cardiac action potential.

Methods and Results: We incorporated all published data on the effects of hypoxia on the late Na⁺ current (I_Na-L), the fast Na⁺ current (I_Na), the basal L-type Ca²⁺ channel current (I_Ca-L), and the slow (I_Ks) and rapid components of the delayed rectifier K⁺-current (I_Kr) in the absence and presence of β-adrenergic receptor (β-AR) stimulation into the Luo–Rudy model of the action potential. Hypoxia alone had little effect on the action potential configuration or action potential duration. However in the presence of β-AR stimulation, hypoxia caused a prolongation of the action potential and early afterdepolarizations (EADs) and spontaneous tachycardia were induced. Experiments performed in guinea pig ventricular myocytes confirmed the modeling results.

Conclusions: EADs occur predominantly because of the increased sensitivity of I_Ca-L to β-AR stimulation during hypoxia. β-AR stimulation is necessary to induce EADs as EADs are never observed during hypoxia in the absence of β-AR stimulation.

Objective: To analyze connexin changes and their potential contribution to the atrial fibrillation (AF) substrate during the development and reversal of CHF.

Methods and Results: Three groups of dogs were studied: CHF induced by 2-week ventricular tachypacing (240 bpm, n=15); CHF dogs allowed a 4-week nonpaced recovery interval after 2-week tachypacing (n=16); and nonpaced sham controls (n=19). Left ventricular (LV) end-diastolic pressure and atrial refractory periods increased with CHF and normalized on CHF recovery. CHF caused abnormalities in atrial conduction indices and increased the duration of burst pacing-induced AF (DAF, from 22±7 seconds in control to 1100±171 seconds, P<0.001). CHF did not significantly alter overall atrial connexin (Cx)40 and Cx43 mRNA and protein expression levels, but produced Cx43 dephosphorylation, increased Cx40/Cx43 protein expression ratio and caused Cx43 redistribution toward transverse cell-boundaries. All of the connexin-alterations reversed on CHF recovery, but CHF-induced conduction abnormalities and increased DAF (884±220 seconds, P<0.001 versus control) remained. The atrial fibrous tissue content increased from 3.6±0.7% in control to 14.7±1.5% and 13.3±2.3% in CHF and CHF recovery, respectively (both P<0.01 versus control), with transversely running zones of fibrosis physically separating longitudinally directed muscle bundles. In an ionically based action potential/tissue model, fibrosis was able to account for conduction abnormalities associated with CHF and recovery.

Conclusions: CHF causes atrial connexin changes, but these are not essential for CHF-related conduction disturbances and AF promotion, which are rather related primarily to fibrotic interruption of muscle bundle continuity.

Objective: Our objective in the present study was to characterize the signal transduction mechanism of phosphatidylinositol 3-kinase (PI3K) involvement in Ang II–induced stimulation of central neuronal activity in cultured neurons and Ang II–induced inhibition of baroreflex in spontaneously hypertensive rats (SHR) versus WKY rats.

Methods and Results: Application of Ang II to neurons produced a 42% greater increase in neuronal firing in cells from the SHR than the WKY rat. Although the Ang II–mediated increase in firing rate was abolished entirely by the protein kinase (PK)C inhibitor GF109230 in the WKY, blockade of both PKC and PI3K activity was necessary in the SHR. This was associated with an increased ability of Ang II to stimulate NADPH oxidase–reactive oxygen species (ROS)–mediated signaling involving phosphorylation of the p47phox subunit of the NADPH oxidase and was dependent on the activation of PI3K in the SHR. Inhibition of PI3K resulted in the reduction of levels of p47phox phosphorylation, NADPH oxidase activity, ROS levels, and ultimately neuronal activity in cells from the SHR but not the WKY rat. In addition, in working heart–brainstem preparations, inhibition of PKC activity in the nucleus of the solitary tract in situ abolished the Ang II–mediated depression of cardiac and sympathetic baroreceptor reflex gain in the WKY. In contrast, PKC inhibition in the nucleus of the solitary tract of SHR only partially reduced the effect of Ang II on the baroreceptor reflex gain.

Conclusions: These observations demonstrate that PI3K in the cardiovascular brainstem regions of the SHR may be selectively involved in Ang II–mediated signaling that includes a reduction in baroreceptor reflex function, presumably via a NADPH-ROS mediated pathway.

Objective: Characterize functional effects of increased PKC-induced cTnI phosphorylation and identify underlying mechanisms using a transgenic mouse model (cTnI_PKC-P) expressing mutant cTnI (S43E, S45E, T144E).

Methods and Results: Two-dimensional gel analysis showed 7.2±0.5% replacement of endogenous cTnI with the mutant form. Experiments included: mechanical measurements (perfused isolated hearts, isolated papillary muscles, and skinned fiber preparations), biochemical and molecular biological measurements, and a mathematical model–based analysis for integrative interpretation. Compared to wild-type mice, cTnI_PKC-P mice exhibited negative inotropy in isolated hearts (14% decrease in peak developed pressure), papillary muscles (53% decrease in maximum developed force), and skinned fibers (14% decrease in maximally activated force, F_max). Additionally, cTnI_PKC-P mice exhibited slowed relaxation in both isolated hearts and intact papillary muscles. The cTnI_PKC-P mice showed no differences in calcium sensitivity, cooperativity, steady-state force-MgATPase relationship, calcium transient (amplitude and relaxation), or baseline phosphorylation of other myofilamental proteins. The model-based analysis revealed that experimental observations in cTnI_PKC-P mice could be reproduced by 2 simultaneous perturbations: a decrease in the rate of cross-bridge formation and an increase in calcium-independent persistence of the myofilament active state.

Conclusions: A modest increase in PKC-induced cTnI phosphorylation (7%) can significantly alter cardiac muscle contraction: negative inotropy via decreased cross-bridge formation and negative lusitropy via persistence of myofilament active state. Based on our data and data from the literature we speculate that effects of PKC-mediated cTnI phosphorylation are site-specific (S43/S45 versus T144).

Objective: To investigate in cultured myocytes from adult cat and rat, 2 species in which Ang II has opposite inotropic effects, the signaling cascade involved in Ang II–induced apoptosis.

Methods and Results: Ang II (1 µmol/L) reduced cat/rat myocytes viability by 40%, in part, because of apoptosis (TUNEL/caspase-3 activity). In both species, apoptosis was associated with reactive oxygen species (ROS) production, Ca²⁺/calmodulin–dependent protein kinase (CaMK)II, and p38 mitogen-activated protein kinase (p38MAPK) activation and was prevented by the ROS scavenger MPG (2-mercaptopropionylglycine) or the NADPH oxidase inhibitor DPI (diphenyleneiodonium) by CaMKII inhibitors (KN-93 and AIP [aryl hydrocarbon receptor–interacting protein]) or in transgenic mice expressing a CaMKII inhibitory peptide and by the p38MAPK inhibitor, SB202190. Furthermore, p38MAPK overexpression exacerbated Ang II–induced cell mortality. Moreover, although KN-93 did not affect Ang II–induced ROS production, it prevented p38MAPK activation. Results further show that CaMKII can be activated by Ang II or H₂O₂, even in the presence of the Ca²⁺ chelator BAPTA-AM, in myocytes and in EGTA-Ca²⁺–free solutions in the presence of the calmodulin inhibitor W-7 in in vitro experiments.

Conclusions: (1) The Ang II–induced apoptotic cascade converges in both species, in a common pathway mediated by ROS-dependent CaMKII activation which results in p38MAPK activation and apoptosis. (2) In the presence of Ang II or ROS, CaMKII may be activated at subdiastolic Ca²⁺ concentrations, suggesting a new mechanism by which ROS reset the Ca²⁺ dependence of CaMKII to extremely low Ca²⁺ levels.

Objective: The goal of the present study was therefore to gain insights into the role of NF-B pathway specifically in mouse cardiomyocytes by conditional deletion of the NF-B essential modulator (NEMO).

Methods and Results: Using a Cre/loxP system, we disrupted the Nemo gene in a cardiomyocyte-specific manner in the heart, which simulated gene expression changes underlying human heart failure and caused adult-onset dilated cardiomyopathy accompanied by inflammation and apoptosis. Pressure overload challenges of NEMO-deficient young hearts precociously induced the functional decrements that develop spontaneously in older knockout animals. Moreover, oxidative stress in NEMO-deficient cardiomyocytes is a critical pathological component that can be attenuated with antioxidant diet in vivo.

Conclusions: These results reveal an essential physiological role for NEMO-mediated signaling in the adult heart to maintain cardiac function in response to age-related or mechanical challenges, in part through modulation of oxidative stress.

Objective: Herein, we generated a cardiac-specific overexpression model, carrying nonphosphorylatable Hsp20, where serine 16 was substituted with alanine (Hsp20^S16A). By subjecting this model to ischemia/reperfusion, we addressed whether: (1) the cardioprotective effects of Hsp20 are associated with serine 16 phosphorylation; (2) blockade of Hsp20 phosphorylation influences the balance between autophagy and cell death; and (3) the aggregation pattern of Hsp20 is altered by its phosphorylation.

Methods and Results: Our results demonstrated that Hsp20^S16A hearts were more sensitive to ischemia/reperfusion injury, evidenced by lower recovery of contractile function and increased necrosis and apoptosis, compared with non-TG hearts. Interestingly, autophagy was activated in non-TG hearts but significantly inhibited in Hsp20^S16A hearts following ischemia/reperfusion. Accordingly, pretreatment of Hsp20^S16A hearts with rapamycin, an activator of autophagy, resulted in improvement of functional recovery, compared with saline-treated Hsp20^S16A hearts. Furthermore, on ischemia/reperfusion, the oligomerization pattern of Hsp20 appeared to shift to higher aggregates in Hsp20^S16A hearts.

Conclusions: Collectively, these data indicate that blockade of Ser16-Hsp20 phosphorylation attenuates the cardioprotective effects of Hsp20 against ischemia/reperfusion injury, which may be attributable to suppressed autophagy and increased cell death. Therefore, phosphorylation of Hsp20 at serine 16 may represent a potential therapeutic target in ischemic heart disease.

Objective: We studied the role of hypertrophy signal-mediated IL-1β/insulin-like growth factor (IGF)-1 production in regulating the progression from compensative pressure-mediated hypertrophy to heart failure.

Methods and Results: Pressure overload was performed by aortic banding in IL-1β–deficient mice. Primarily cultured cardiac fibroblasts (CFs) and cardiac myocytes (CMs) were exposed to cyclic stretch. Heart weight, myocyte size, and left ventricular ejection fraction were significantly lower in IL-1β–deficient mice (20%, 23% and 27%, respectively) than in the wild type 30 days after aortic banding, whereas interstitial fibrosis was markedly augmented. DNA microarray analysis revealed that IGF-1 mRNA level was markedly (50%) decreased in the IL-1β–deficient hypertrophied heart. Stretch of CFs, rather than CMs, abundantly induced the generation of IL-1β and IGF-1, whereas such IGF-1 induction was markedly decreased in IL-1β–deficient CFs. IL-1β released by stretch is at a low level unable to induce IL-6 but sufficient to stimulate IGF-1 production. Promoter analysis showed that stretch-mediated IL-1β activates JAK/STAT to transcriptionally regulate the IGF-1 gene. IL-1β deficiency markedly increased c-Jun N-terminal kinase (JNK) and caspase-3 activities and enhanced myocyte apoptosis and fibrosis, whereas replacement of IGF-1 or JNK inhibitor restored them.

Conclusions: We demonstrate for the first time that pressure-mediated hypertrophy and mechanical stretch generates a subinflammatory low level of IL-1β, which constitutively causes IGF-1 production to maintain adaptable compensation hypertrophy and inhibit interstitial fibrosis.

Methods: In the present study, we performed sex-mismatched bone marrow transplantation using Ddr1^+/+;Ldlr^-/- and Ddr1^-/-;Ldlr^-/- mice to investigate the role of macrophage Ddr1 during atherogenesis. Chimeric mice with deficiency of Ddr1 in bone marrow–derived cells (Ddr1^-/-->+/+) or control chimeric mice that received Ddr1^+/+;Ldlr^-/- marrow (Ddr1^+/+->+/+) were fed an atherogenic diet for 12 weeks.

Results: We observed a 66% reduction in atherosclerosis in the descending aorta and a 44% reduction in plaque area in the aortic sinus in Ddr1^-/-->+/+ mice compared to Ddr1^+/+->+/+ mice. Furthermore, we observed a specific reduction in the number of donor-derived macrophages in Ddr1^-/-->+/+ plaques, suggesting that bone marrow deficiency of Ddr1 attenuated atherogenesis by limiting macrophage accumulation to the plaque. We have also demonstrated that the effects of Ddr1 on macrophage infiltration and accumulation can occur at the earliest stage of atherogenesis, the formation of the fatty streak. Deficiency of Ddr1 limited the appearance of 5-bromodeoxyuridine–labeled monocytes/macrophages in the fatty streak and resulted in reduced lesion size in Ldlr^-/- mice fed a high fat diet for 2 weeks. In vitro studies to investigate the mechanisms involved revealed that macrophages from Ddr1^-/- mice had decreased adhesion to type IV collagen and decreased chemotactic invasion of type IV collagen in response to monocyte chemoattractant protein-1.

Conclusions: Taken together, our data support an independent and critical role for Ddr1 in macrophage accumulation at early and late stages of atherogenesis.

Objective: To determine whether there is defective efferocytosis in a mouse model of obesity and atherosclerosis.

Methods and Results: We quantified efferocytosis in peritoneal macrophages and in atherosclerotic lesions of obese ob/ob or ob/ob;Ldlr^-/- mice and littermate controls. Peritoneal macrophages from ob/ob and ob/ob;Ldlr^-/- mice showed impaired efferocytosis, reflecting defective phosphatidylinositol 3-kinase activation during uptake of apoptotic cells. Membrane lipid composition of ob/ob and ob/ob;Ldlr^-/- macrophages showed an increased content of saturated fatty acids (FAs) and decreased -3 FAs (eicosapentaenoic acid and docosahexaenoic acid) compared to controls. A similar defect in efferocytosis was induced by treating control macrophages with saturated free FA/BSA complexes, whereas the defect in ob/ob macrophages was reversed by treatment with eicosapentaenoic acid/BSA or by feeding ob/ob mice a fish oil diet rich in -3 FAs. There was also defective macrophage efferocytosis in atherosclerotic lesions of ob/ob;Ldlr^-/- mice and this was reversed by a fish oil–rich diet.

Conclusions: The findings suggest that in obesity and type 2 diabetes elevated levels of saturated FAs and/or decreased levels of -3 FAs contribute to decreased macrophage efferocytosis. Beneficial effects of fish oil diets in atherosclerotic cardiovascular disease may involve improvements in macrophage function related to reversal of defective efferocytosis and could be particularly important in type 2 diabetes and obesity.

Objective: Here we tested whether the pathological manifestations of the transplanted heart can be corrected partly by a strategy that implements the use of cardiac progenitor cells from the recipient to repopulate the donor heart with immunocompatible cardiomyocytes and coronary vessels.

Methods and Results: A large number of cardiomyocytes and coronary vessels were created in a rather short period of time from the delivery, engraftment, and differentiation of cardiac progenitor cells from the recipient. A proportion of newly formed cardiomyocytes acquired adult characteristics and was integrated structurally and functionally within the transplant. Similarly, the regenerated arteries, arterioles, and capillaries were operative and contributed to the oxygenation of the chimeric myocardium. Attenuation in the extent of acute damage by repopulating cardiomyocytes and vessels decreased significantly the magnitude of myocardial scarring preserving partly the integrity of the donor heart.

Conclusions: Our data suggest that tissue regeneration by differentiation of recipient cardiac progenitor cells restored a significant portion of the rejected donor myocardium. Ultimately, immunosuppressive therapy may be only partially required improving quality of life and lifespan of patients with cardiac transplantation.

Objective: We investigated how the heart compensates for the accelerated accumulation of aldehydes.

Methods and Results: Aldehyde dehydrogenase 2 (ALDH2) has a major role in aldehyde detoxification in the mitochondria, a major source of aldehydes. Transgenic (Tg) mice carrying an Aldh2 gene with a single nucleotide polymorphism (Aldh2*2) were developed. This polymorphism has a dominant-negative effect and the Tg mice exhibited impaired ALDH activity against a broad range of aldehydes. Despite a shift toward the oxidative state in mitochondrial matrices, Aldh2*2 Tg hearts displayed normal left ventricular function by echocardiography and, because of metabolic remodeling, an unexpected tolerance to oxidative stress induced by ischemia/reperfusion injury. Mitochondrial aldehyde stress stimulated eukaryotic translation initiation factor 2 phosphorylation. Subsequent translational and transcriptional activation of activating transcription factor-4 promoted the expression of enzymes involved in amino acid biosynthesis and transport, ultimately providing precursor amino acids for glutathione biosynthesis. Intracellular glutathione levels were increased 1.37-fold in Aldh2*2 Tg hearts compared with wild-type controls. Heterozygous knockout of Atf4 blunted the increase in intracellular glutathione levels in Aldh2*2 Tg hearts, thereby attenuating the oxidative stress–resistant phenotype. Furthermore, glycolysis and NADPH generation via the pentose phosphate pathway were activated in Aldh2*2 Tg hearts. (NADPH is required for the recycling of oxidized glutathione.)

Conclusions: The findings of the present study indicate that mitochondrial aldehyde stress in the heart induces metabolic remodeling, leading to activation of the glutathione–redox cycle, which confers resistance against acute oxidative stress induced by ischemia/reperfusion.

Objective: However, the role that ASK1 plays in regulating the cardiac hypertrophic response in vivo remains controversial.

Methods and Results: Here, we generated mice with cardiac-specific and inducible overexpression of ASK1 in the heart to assess its gain-of-function effect. ASK1 transgenic mice exhibited no induction of cardiac hypertrophy or pathology at 3 and 12 months of age, and these mice showed an identical hypertrophic response to controls following 2 weeks of pressure-overload stimulation or isoproterenol infusion. Although ASK1 overexpression did not alter the cardiac hypertrophic response, it promoted cardiomyopathy and greater TUNEL following pressure-overload stimulation and myocardial infarction. Indeed, ASK1 transgenic mice showed a greater than 2-fold increase in ischemia reperfusion-induced injury to the heart compared with controls. Examination of downstream signaling showed a prominent activation of mitogen-activated protein kinase kinase 4/6 and c-Jun NH₂-terminal kinase (JNK)1/2 (but not p38 or extracellular signal-regulated kinases [ERKs]), inhibition of calcineurin-NFAT (nuclear factor of activated T cells), and induction of Bax in the hearts of ASK1 transgenic mice following 1 and 8 weeks of pressure-overload stimulation. Mechanistically, cardiomyopathy associated with ASK1 overexpression after 8 weeks of pressure overload was significantly reduced in the calcineurin Aβ–null (CnAβ^-/-) background.

Conclusions: These results indicate that ASK1 does not directly regulate the cardiac hypertrophic response in vivo, but it does alter cell death and propensity to cardiomyopathy, in part, through a calcineurin-dependent mechanism.

Objective: We sought to understand the function of NMII-B in adult mouse hearts and to see whether the brain defects found in germline-ablated mice influence cardiac development.

Methods and Results: We used a loxP/Cre recombinase strategy to specifically ablate NMII-B in the brains or hearts of mice. Mice ablated for NMII-B in neural tissues die between postnatal day 12 and 22 without showing cardiac defects. Mice deficient in NMII-B only in cardiac myocytes (B^MHC/B^MHC mice) do not show brain defects. However, B^MHC/B^MHC mice display novel cardiac defects not seen in NMII-B germline-ablated mice. Most of the B^MHC/B^MHC mice are born with enlarged cardiac myocytes, some of which are multinucleated, reflecting a defect in cytokinesis. Between 6 to 10 months, they develop a cardiomyopathy that includes interstitial fibrosis and infiltration of the myocardium and pericardium with inflammatory cells. Four of 5 B^MHC/B^MHC hearts develop marked widening of intercalated discs.

Conclusions: By avoiding the embryonic lethality found in germline-ablated mice, we were able to study the function of NMII-B in adult mice and show that absence of NMII-B in cardiac myocytes results in cardiomyopathy in the adult heart. We also define a role for NMII-B in maintaining the integrity of intercalated discs.

Objective: We developed a novel mouse model of BD and investigated the role of complement in BD-induced myocardial inflammation and injury.

Methods and Results: BD was induced by inflation of a balloon catheter in the cranial cavity. BD in wild-type mice resulted in a significant increase in serum concentrations of the complement activation product complement component (C)3a, and immunohistochemical analysis of heart sections demonstrated C3 deposition on the vascular endothelium and surrounding myocytes. Following induction of BD in complement (C3)-deficient mice, cardiac troponin levels, and histological evidence of injury were significantly reduced compared to wild-type mice. C3 deficiency was also associated with reduced myocardial leukocyte infiltration and reduced or absent expression of P-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, tumor necrosis factor-, and interleukin-1β.

Conclusions: These data indicate an important role for complement in BD-induced inflammation and injury and suggest that a complement inhibitory strategy applied to the donor (in addition to the recipient) may provide graft protection.

Objective: To investigate reentry dynamics and the role of individual ion channels on reentry in in vitro models of the "healed" IBZ.

Methods and Results: We designed in vitro models of the healed IBZ by coculturing skeletal myotubes with neonatal rat ventricular myocytes and performed optical mapping at high temporal and spatial resolution. In culture, neonatal rat ventricular myocytes mature to form striated myocytes and electrically uncoupled skeletal myotubes simulate fibrosis seen in the healed IBZ. High resolution mapping revealed that skeletal myotubes produced localized slowing of conduction velocity (CV), increased dispersion of CV and directional-dependence of activation delay without affecting myocyte excitability. Reentry was easily induced by rapid pacing in cocultures; treatment with lidocaine, a Na⁺ channel blocker, significantly decreased reentry rate and CV, increased reentry path length and terminated 30% of reentrant arrhythmias (n=18). In contrast, nitrendipine, an L-type Ca²⁺ channel blocker terminated 100% of reentry episodes while increasing reentry cycle length and path length and decreasing reentry CV (n=16). K⁺ channel blockers increased reentry action potential duration but infrequently terminated reentry (n=12).

Conclusions: Cocultures reproduce several architectural and electrophysiological features of the healed IBZ. Reentry termination by L-type Ca²⁺ channel, but not Na⁺ channel, blockers suggests a greater Ca²⁺-dependence of propagation. These results may help explain the low efficacy of pure Na⁺ channel blockers in preventing and terminating clinical VTs late after MI.

Objective: We hypothesized that the mitoK_ATP channel contains a sulfonylurea receptor (SUR)2 regulatory subunit and aimed to identify the molecular structure.

Methods and Results: Western blot analysis in cardiac mitochondria detected a 55-kDa mitochondrial SUR2 (mitoSUR2) short form, 2 additional short forms (28 and 68 kDa), and a 130-kDa long form. RACE (rapid amplification of cDNA end products) identified a 1.5-Kb transcript, which was generated by a nonconventional intraexonic splicing (IES) event within the 4th and 29th exons of the SUR2 mRNA. The translated product matched the predicted size of the 55-kDa short form. In a knockout mouse (SUR2KO), in which the SUR2 gene was disrupted, the 130-kDa mitoSUR2 was absent, but the short forms remained expressed. Diazoxide failed to induce increased fluorescence of flavoprotein oxidation in SUR2KO cells, indicating that the diazoxide-sensitive mitoK_ATP channel activity was associated with 130-kDa–based channels. However, SUR2KO mice displayed similar infarct sizes to preconditioned wild type, suggesting a protective role for the remaining short form-based channels. Heterologous coexpression of the SUR2 IES variant and Kir6.2 in a K⁺ transport mutant Escherichia coli strain permitted improved cell growth under acidic pH conditions. The SUR2 IES variant was localized to mitochondria, and removal of a predicted mitochondrial targeting sequence allowed surface expression and detection of an ATP-sensitive current when coexpressed with Kir6.2.

Conclusions: We identify a novel SUR2 IES variant in cardiac mitochondria and provide evidence that the variant-based channel can form an ATP-sensitive conductance and may contribute to cardioprotection.