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: 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: 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 Ends) 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.

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: 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: 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 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: 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.

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 in 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: 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.