Exercise triggers CAPN1-mediated AIF truncation, inducing myocyte cell death in arrhythmogenic cardiomyopathy.

Myocyte death occurs in many inherited and acquired cardiomyopathies, including arrhythmogenic cardiomyopathy (ACM), a genetic heart disease plagued by the prevalence of sudden cardiac death. Individuals with ACM and harboring pathogenic desmosomal variants, such as desmoglein-2 (DSG2), often show myocyte necrosis with progression to exercise-associated heart failure. Here, we showed that homozygous Dsg2 mutant mice (Dsg2 mut/mut), a model of ACM, die prematurely during swimming and display myocardial dysfunction and necrosis. We detected calcium (Ca2+) overload in Dsg2 mut/mut hearts, which induced calpain-1 (CAPN1) activation, association of CAPN1 with mitochondria, and CAPN1-induced cleavage of mitochondrial-bound apoptosis-inducing factor (AIF). Cleaved AIF translocated to the myocyte nucleus triggering large-scale DNA fragmentation and cell death, an effect potentiated by mitochondrial-driven AIF oxidation. Posttranslational oxidation of AIF cysteine residues was due, in part, to a depleted mitochondrial thioredoxin-2 redox system. Hearts from exercised Dsg2 mut/mut mice were depleted of calpastatin (CAST), an endogenous CAPN1 inhibitor, and overexpressing CAST in myocytes protected against Ca2+ overload-induced necrosis. When cardiomyocytes differentiated from Dsg2 mut/mut embryonic stem cells (ES-CMs) were challenged with ß-adrenergic stimulation, CAPN1 inhibition attenuated CAPN1-induced AIF truncation. In addition, pretreatment of Dsg2 mut/mut ES-CMs with an AIF-mimetic peptide, mirroring the cyclophilin-A (PPIA) binding site of AIF, blocked PPIA-mediated AIF-nuclear translocation, and reduced both apoptosis and necrosis. Thus, preventing CAPN1-induced AIF-truncation or barring binding of AIF to the nuclear chaperone, PPIA, may avert myocyte death and, ultimately, disease progression to heart failure in ACM and likely other forms of cardiomyopathies.

CaMKII exacerbates heart failure progression by activating class I HDACs.

BACKGROUND: Persistent cardiac Ca2+/calmodulin dependent Kinase II (CaMKII) activation plays an essential role in heart failure development. However, the molecular mechanisms underlying CaMKII induced heart failure progression remains incompletely understood. Histone deacetylases (HDACs) are critical for transcriptional responses to stress, and contribute to expression of pathological genes causing adverse ventricular remodeling. Class I HDACs, including HDAC1, HDAC2 and HDAC3, promote pathological cardiac hypertrophy, whereas class IIa HDACs suppress cardiac hypertrophy. While it is known that CaMKII deactivates class IIa HDACs to enhance cardiac hypertrophy, the role of CaMKII in regulating class I HDACs during heart failure progression is unclear. METHODS AND RESULTS: CaMKII increases the deacetylase activity of recombinant HDAC1, HDAC2 and HDAC3 via in vitro phosphorylation assays. Phosphorylation sites on HDAC1 and HDAC3 are identified with mass spectrometry. HDAC1 activity is also increased in cardiac-specific CaMKIIδC transgenic mice (CaMKIIδC-tg). Beyond post-translational modifications, CaMKII induces HDAC1 and HDAC3 expression. HDAC1 and HDAC3 expression are significantly increased in CaMKIIδC-tg mice. Inhibition of CaMKII by overexpression of the inhibitory peptide AC3-I in the heart attenuates the upregulation of HDAC1 after myocardial infarction surgery. Importantly, a potent HDAC1 inhibitor Quisinostat improves downregulated autophagy genes and cardiac dysfunction in CaMKIIδC-tg mice. In addition to Quisinostat, selective class I HDACs inhibitors, Apicidin and Entinostat, HDAC3 specific inhibitor RGFP966, as well as HDAC1 and HDAC3 siRNA prevent CaMKII overexpression induced cardiac myocyte hypertrophy. CONCLUSION: CaMKII activates class I HDACs in heart failure, which may be a central mechanism for heart failure progression. Selective class I HDACs inhibition may be a novel therapeutic avenue to alleviate CaMKII hyperactivity induced cardiac dysfunction.

PKG1α Cysteine-42 Redox State Controls mTORC1 Activation in Pathological Cardiac Hypertrophy.

RATIONALE: Stimulated PKG1α (protein kinase G-1α) phosphorylates TSC2 (tuberous sclerosis complex 2) at serine 1365, potently suppressing mTORC1 (mechanistic [mammalian] target of rapamycin complex 1) activation by neurohormonal and hemodynamic stress. This reduces pathological hypertrophy and dysfunction and increases autophagy. PKG1α oxidation at cysteine-42 is also induced by these stressors, which blunts its cardioprotective effects. OBJECTIVE: We tested the dependence of mTORC1 activation on PKG1α C42 oxidation and its capacity to suppress such activation by soluble GC-1 (guanylyl cyclase 1) activation. METHODS AND RESULTS: Cardiomyocytes expressing wild-type (WT) PKG1α (PKG1αWT) or cysteine-42 to serine mutation redox-dead (PKG1αCS/CS) were exposed to ET-1 (endothelin 1). Cells expressing PKG1αWT exhibited substantial mTORC1 activation (p70 S6K [p70 S6 kinase], 4EBP1 [elF4E binding protein-1], and Ulk1 [Unc-51-like kinase 1] phosphorylation), reduced autophagy/autophagic flux, and abnormal protein aggregation; all were markedly reversed by PKG1αCS/CS expression. Mice with global knock-in of PKG1αCS/CS subjected to pressure overload (PO) also displayed markedly reduced mTORC1 activation, protein aggregation, hypertrophy, and ventricular dysfunction versus PO in PKG1αWT mice. Cardioprotection against PO was equalized between groups by co-treatment with the mTORC1 inhibitor everolimus. TSC2-S1365 phosphorylation increased in PKG1αCS/CS more than PKG1αWT myocardium following PO. TSC2S1365A/S1365A (TSC2 S1365 phospho-null, created by a serine to alanine mutation) knock-in mice lack TSC2 phosphorylation by PKG1α, and when genetically crossed with PKG1αCS/CS mice, protection against PO-induced mTORC1 activation, cardiodepression, and mortality in PKG1αCS/CS mice was lost. Direct stimulation of GC-1 (BAY-602770) offset disparate mTORC1 activation between PKG1αWT and PKG1αCS/CS after PO and blocked ET-1 stimulated mTORC1 in TSC2S1365A-expressing myocytes. CONCLUSIONS: Oxidation of PKG1α at C42 reduces its phosphorylation of TSC2, resulting in amplified PO-stimulated mTORC1 activity and associated hypertrophy, dysfunction, and depressed autophagy. This is ameliorated by direct GC-1 stimulation.

Cardiac regeneration using human-induced pluripotent stem cell-derived biomaterial-free 3D-bioprinted cardiac patch in vivo.

One of the leading causes of death worldwide is heart failure. Despite advances in the treatment and prevention of heart failure, the number of affected patients continues to increase. We have recently developed 3D-bioprinted biomaterial-free cardiac tissue that has the potential to improve cardiac function. This study aims to evaluate the in vivo regenerative potential of these 3D-bioprinted cardiac patches. The cardiac patches were generated using 3D-bioprinting technology in conjunction with cellular spheroids created from a coculture of human-induced pluripotent stem cell-derived cardiomyocytes, fibroblasts, and endothelial cells. Once printed and cultured, the cardiac patches were implanted into a rat myocardial infarction model (n = 6). A control group (n = 6) without the implantation of cardiac tissue patches was used for comparison. The potential for regeneration was measured 4 weeks after the surgery with histology and echocardiography. 4 weeks after surgery, the survival rates were 100% and 83% in the experimental and the control group, respectively. In the cardiac patch group, the average vessel counts within the infarcted area were higher than those within the control group. The scar area in the cardiac patch group was significantly smaller than that in the control group. (Figure S1) Echocardiography showed a trend of improvement of cardiac function for the experimental group, and this trend correlated with increased patch production of extracellular vesicles. 3D-bioprinted cardiac patches have the potential to improve the regeneration of cardiac tissue and promote angiogenesis in the infarcted tissues and reduce the scar tissue formation.

Calpain 9 as a therapeutic target in TGFß-induced mesenchymal transition and fibrosis.

Fibrosis is a common pathologic outcome of chronic disease resulting in the replacement of normal tissue parenchyma with a collagen-rich extracellular matrix produced by myofibroblasts. Although the progenitor cell types and cellular programs giving rise to myofibroblasts through mesenchymal transition can vary between tissues and diseases, their contribution to fibrosis initiation, maintenance, and progression is thought to be pervasive. Here, we showed that the ability of transforming growth factor-ß (TGFß) to efficiently induce myofibroblast differentiation of cultured epithelial cells, endothelial cells, or quiescent fibroblasts is dependent on the induced expression and activity of dimeric calpains, a family of non-lysosomal cysteine proteases that regulate a variety of cellular events through posttranslational modification of diverse substrates. siRNA-based gene silencing demonstrated that TGFß-induced mesenchymal transition of a murine breast epithelial cell line was dependent on induction of expression of calpain 9 (CAPN9), an isoform previously thought to be restricted to the gastrointestinal tract. Mice lacking functional CAPN9 owing to biallelic targeting of Capn9 were viable and fertile but showed overt protection from bleomycin-induced lung fibrosis, carbon tetrachloride-induced liver fibrosis, and angiotensin II-induced cardiac fibrosis and dysfunction. A predicted loss-of-function allele of CAPN9 is common in Southeast Asia, with the frequency of homozygosity matching the prediction of Hardy-Weinberg equilibrium. Together with the highly spatially restricted pattern of CAPN9 expression under physiologic circumstances and the heartiness of the murine knockout, these data provide a strong signature for tolerance of therapeutic strategies for fibrosis aimed at CAPN9 antagonism.

Regenerative and durable small-diameter graft as an arterial conduit.

Despite significant research efforts, clinical practice for arterial bypass surgery has been stagnant, and engineered grafts continue to face postimplantation challenges. Here, we describe the development and application of a durable small-diameter vascular graft with tailored regenerative capacity. We fabricated small-diameter vascular grafts by electrospinning fibrin tubes and poly(ε-caprolactone) fibrous sheaths, which improved suture retention strength and enabled long-term survival. Using surface topography in a hollow fibrin microfiber tube, we enable immediate, controlled perfusion and formation of a confluent endothelium within 3-4 days in vitro with human endothelial colony-forming cells, but a stable endothelium is noticeable at 4 weeks in vivo. Implantation of acellular or endothelialized fibrin grafts with an external ultrathin poly(ε-caprolactone) sheath as an interposition graft in the abdominal aorta of a severe combined immunodeficient Beige mouse model supports normal blood flow and vessel patency for 24 weeks. Mechanical properties of the implanted grafts closely approximate the native abdominal aorta properties after just 1 week in vivo. Fibrin mediated cellular remodeling, stable tunica intima and media formation, and abundant matrix deposition with organized collagen layers and wavy elastin lamellae. Endothelialized grafts evidenced controlled healthy remodeling with delayed and reduced macrophage infiltration alongside neo vasa vasorum-like structure formation, reduced calcification, and accelerated tunica media formation. Our studies establish a small-diameter graft that is fabricated in less than 1 week, mediates neotissue formation and incorporation into the native tissue, and matches the native vessel size and mechanical properties, overcoming main challenges in arterial bypass surgery.

Inorganic arsenic exposure induces sex-disparate effects and exacerbates ischemia-reperfusion injury in the female heart.

Arsenic is a common contaminant in drinking water throughout the world, and recent studies support a link between inorganic arsenic (iAS) exposure and ischemic heart disease in men and women. Female hearts exhibit an estrogen-dependent reduction in susceptibility to myocardial ischemic injury compared with males, and as such, female hearts may be more susceptible to the endocrine-disrupting effects of iAS exposure. However, iAS exposure and susceptibility to ischemic heart injury have not been examined in mechanistic studies. Male and female mice (8 wk) were exposed to environmentally relevant concentrations of sodium arsenite (0, 10, 100, and 1,000 parts/billion) via drinking water for 4 wk. Pre- and postexposure echocardiography was performed, and postexposure plasma was collected for 17ß-estradiol measurement. Hearts were excised and subjected to ischemia-reperfusion (I/R) injury via Langendorff perfusion. Exposure to 1,000 parts/billion iAS led to sex-disparate effects, such that I/R injury was exacerbated in female hearts but unexpectedly attenuated in males. Assessment of echocardiographic parameters revealed statistically significant structural remodeling in iAS-treated female hearts with no change in function; males showed no change. Plasma 17ß-estradiol levels were not significantly altered by iAS in male or female mice versus nontreated controls. Although total eNOS protein levels did not change in whole heart homogenates from iAS-treated male or female mice, eNOS phosphorylation (Ser1177) was significantly elevated in iAS-treated male hearts. These results suggest that iAS exposure can induce sex-disparate effects and modulate susceptibility to ischemic heart injury by targeting distinct sex-dependent pathways. NEW & NOTEWORTHY This is the first mechanistic study examining iAS exposure on myocardial ischemia-reperfusion injury in male and female mice. Following iAS exposure, ischemia-reperfusion injury was exacerbated in female hearts but attenuated in males. iAS treatment induced statistically significant cardiac remodeling in females, with no change in males. iAS treatment also enhanced phosphorylated eNOS levels at Ser1177, but only in male hearts. These results suggest that iAS alters susceptibility to myocardial I/R injury through distinct sex-dependent pathways.

Inhibition of glycosphingolipid synthesis reverses skin inflammation and hair loss in ApoE-/- mice fed western diet.

Sphingolipids have been accorded numerous biological functions however, the effects of feeding a western diet (diet rich in cholesterol and fat) on skin phenotypes, and color is not known. Here, we observed that chronic high-fat and high-cholesterol diet intake in a mouse model of atherosclerosis (ApoE-/-) decreases the level of ceramides and glucosylceramide. At the expense of increased levels of lactosylceramide due to an increase in the expression of lactosylceramide synthase (GalT-V). This is accompanied with neutrophil infiltration into dermis, and enrichment of tumor necrosis factor-stimulated gene-6 (TSG-6) protein. This causes skin inflammation, hair discoloration and loss, in ApoE-/- mice. Conversely, inhibition of glycosphingolipid synthesis, by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), unbound or encapsulated in a biodegradable polymer (BPD) reversed these phenotypes. Thus, inhibition of glycosphingolipid synthesis represents a unique therapeutic approach relevant to human skin and hair Biology.

Transient receptor potential channel 6 regulates abnormal cardiac S-nitrosylation in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked disorder with dystrophin loss that results in skeletal and cardiac muscle weakening and early death. Loss of the dystrophin-sarcoglycan complex delocalizes nitric oxide synthase (NOS) to alter its signaling, and augments mechanosensitive intracellular Ca2+ influx. The latter has been coupled to hyperactivation of the nonselective cation channel, transient receptor potential canonical channel 6 (Trpc6), in isolated myocytes. As Ca2+ also activates NOS, we hypothesized that Trpc6 would help to mediate nitric oxide (NO) dysregulation and that this would be manifest in increased myocardial S-nitrosylation, a posttranslational modification increasingly implicated in neurodegenerative, inflammatory, and muscle disease. Using a recently developed dual-labeling proteomic strategy, we identified 1,276 S-nitrosylated cysteine residues [S-nitrosothiol (SNO)] on 491 proteins in resting hearts from a mouse model of DMD (dmdmdx:utrn+/-). These largely consisted of mitochondrial proteins, metabolic regulators, and sarcomeric proteins, with 80% of them also modified in wild type (WT). S-nitrosylation levels, however, were increased in DMD. Genetic deletion of Trpc6 in this model (dmdmdx:utrn+/-:trpc6-/-) reversed ∼70% of these changes. Trpc6 deletion also ameliorated left ventricular dilation, improved cardiac function, and tended to reduce fibrosis. Furthermore, under catecholamine stimulation, which also increases NO synthesis and intracellular Ca2+ along with cardiac workload, the hypernitrosylated state remained as it did at baseline. However, the impact of Trpc6 deletion on the SNO proteome became less marked. These findings reveal a role for Trpc6-mediated hypernitrosylation in dmdmdx:utrn+/- mice and support accumulating evidence that implicates nitrosative stress in cardiac and muscle disease.

Divergent Effects of miR-181 Family Members on Myocardial Function Through Protective Cytosolic and Detrimental Mitochondrial microRNA Targets.

BACKGROUND: MicroRNA (miRNA) is a type of noncoding RNA that can repress the expression of target genes through posttranscriptional regulation. In addition to numerous physiologic roles for miRNAs, they play an important role in pathophysiologic processes affecting cardiovascular health. Previously, we reported that nuclear encoded microRNA (miR-181c) is present in heart mitochondria, and importantly, its overexpression affects mitochondrial function by regulating mitochondrial gene expression. METHODS AND RESULTS: To investigate further how the miR-181 family affects the heart, we suppressed miR-181 using a miR-181-sponge containing 10 repeated complementary miR-181 "seed" sequences and generated a set of H9c2 cells, a cell line derived from rat myoblast, by stably expressing either a scrambled or miR-181-sponge sequence. Sponge-H9c2 cells showed a decrease in reactive oxygen species production and reduced basal mitochondrial respiration and protection against doxorubicin-induced oxidative stress. We also found that miR-181a/b targets phosphatase and tensin homolog (PTEN), and the sponge-expressing stable cells had increased PTEN activity and decreased PI3K signaling. In addition, we have used miR-181a/b-/- and miR-181c/d-/- knockout mice and subjected them to ischemia-reperfusion injury. Our results suggest divergent effects of different miR-181 family members: miR-181a/b targets PTEN in the cytosol, resulting in an increase in infarct size in miR-181a/b-/- mice due to increased PTEN signaling, whereas miR-181c targets mt-COX1 in the mitochondria, resulting in decreased infarct size in miR-181c/d-/- mice. CONCLUSIONS: The miR-181 family alters the myocardial response to oxidative stress, notably with detrimental effects by targeting mt-COX1 (miR-181c) or with protection by targeting PTEN (miR-181a/b).

Echocardiographic Characterization of a Murine Model of Hypertrophic Obstructive Cardiomyopathy Induced by Cardiac-specific Overexpression of Epidermal Growth Factor Receptor 2.

Although rare, hypertrophic cardiomyopathy (HCM) with midventricular obstruction is often associated with severe symptoms and complications. None of the existing HCM animal models display this particular phenotype. Our group developed a mouse line that overexpresses the ErbB2 receptor (ErbB2(tg)) in cardiomyocytes; we previously showed that the ErbB2 receptor induces cardiomyocyte hypertrophy, myocyte disarray, and fibrosis compatible with HCM. In the current study, we sought to further echocardiographically characterize the ErbB2(tg) mouse line as a model of HCM. Compared with their wild-type littermates, ErbB2(tg) mice show increased left ventricular (LV) mass, concentric LV hypertrophy, and papillary muscle hypertrophy. This hypertrophy was accompanied by diastolic dysfunction, expressed as reduced E:A ratio, prolonged deceleration time, and elevated E:e' ratio. In addition, ErbB2(tg) mice consistently showed midcavity obstruction with elevated LV gradients, and the flow profile revealed a prolonged pressure increase and a delayed peak, indicating dynamic obstruction. The ejection fraction was increased in ErbB2(tg) mice, due to reduced end-diastolic and end-systolic LV volumes. Furthermore, systolic radial strain and systolic radial strain rate but not systolic circumferential strain and longitudinal strain were decreased in ErbB2(tg) compared with wild-type mice. In conclusion, the phenotype of the ErbB2(tg) mouse model is consistent with midventricular HCM in many important aspects, including massive LV hypertrophy, diastolic dysfunction, and midcavity obstruction. This pattern is unique for a small animal model, suggesting that ErbB2(tg) mice may be well suited for research into the hemodynamics and treatment of this rare form of HCM.

Tetrahydrobiopterin Protects Against Hypertrophic Heart Disease Independent of Myocardial Nitric Oxide Synthase Coupling.

BACKGROUND: Nitric oxide synthase uncoupling occurs under conditions of oxidative stress modifying the enzyme's function so it generates superoxide rather than nitric oxide. Nitric oxide synthase uncoupling occurs with chronic pressure overload, and both are ameliorated by exogenous tetrahydrobiopterin (BH4)-a cofactor required for normal nitric oxide synthase function-supporting a pathophysiological link. Genetically augmenting BH4 synthesis in endothelial cells fails to replicate this benefit, indicating that other cell types dominate the effects of exogenous BH4 administration. We tested whether the primary cellular target of BH4 is the cardiomyocyte or whether other novel mechanisms are invoked. METHODS AND RESULTS: Mice with cardiomyocyte-specific overexpression of GTP cyclohydrolase 1 (mGCH1) and wild-type littermates underwent transverse aortic constriction. The mGCH1 mice had markedly increased myocardial BH4 and, unlike wild type, maintained nitric oxide synthase coupling after transverse aortic constriction; however, the transverse aortic constriction-induced abnormalities in cardiac morphology and function were similar in both groups. In contrast, exogenous BH4 supplementation improved transverse aortic constricted hearts in both groups, suppressed multiple inflammatory cytokines, and attenuated infiltration of inflammatory macrophages into the heart early after transverse aortic constriction. CONCLUSIONS: BH4 protection against adverse remodeling in hypertrophic cardiac disease is not driven by its prevention of myocardial nitric oxide synthase uncoupling, as presumed previously. Instead, benefits from exogenous BH4 are mediated by a protective effect coupled to suppression of inflammatory pathways and myocardial macrophage infiltration.

Bidirectional cross-regulation between ErbB2 and ß-adrenergic signalling pathways.

AIMS: Despite the observation that ErbB2 regulates sensitivity of the heart to doxorubicin or ErbB2-targeted cancer therapies, mechanisms that regulate ErbB2 expression and activity have not been studied. Since isoproterenol up-regulates ErbB2 in kidney and salivary glands and ß2AR and ErbB2 complex in brain and heart, we hypothesized that ß-adrenergic receptors (AR) modulate ErbB2 signalling status. METHODS AND RESULTS: ErbB2 transfection of HEK293 cells up-regulates ß2AR, and ß2AR transfection of HEK293 up-regulates ErbB2. Interestingly, cardiomyocytes isolated from myocyte-specific ErbB2-overexpressing (ErbB2(tg)) mice have amplified response to selective ß2-agonist zinterol, and right ventricular trabeculae baseline force generation is markedly reduced with ß2-antagonist ICI-118 551. Consistently, receptor binding assays and western blotting demonstrate that ß2ARs levels are markedly increased in ErbB2(tg) myocardium and reduced by EGFR/ErbB2 inhibitor, lapatinib. Intriguingly, acute treatment of mice with ß1- and ß2-AR agonist isoproterenol resulted in myocardial ErbB2 increase, while inhibition with either ß1- or ß2-AR antagonist did not completely prevent isoproterenol-induced ErbB2 expression. Furthermore, inhibition of ErbB2 kinase predisposed mice hearts to injury from chronic isoproterenol treatment while significantly reducing isoproterenol-induced pAKT and pERK levels, suggesting ErbB2's role in transactivation in the heart. CONCLUSION: Our studies show that myocardial ErbB2 and ßAR signalling are linked in a feedback loop with ßAR activation leading to increased ErbB2 expression and activity, and increased ErbB2 activity regulating ß2AR expression. Most importantly, ErbB2 kinase activity is crucial for cardioprotection in the setting of ß-adrenergic stress, suggesting that this mechanism is important in the pathophysiology and treatment of cardiomyopathy induced by ErbB2-targeting antineoplastic drugs.

A deleterious gene-by-environment interaction imposed by calcium channel blockers in Marfan syndrome.

Calcium channel blockers (CCBs) are prescribed to patients with Marfan syndrome for prophylaxis against aortic aneurysm progression, despite limited evidence for their efficacy and safety in the disorder. Unexpectedly, Marfan mice treated with CCBs show accelerated aneurysm expansion, rupture, and premature lethality. This effect is both extracellular signal-regulated kinase (ERK1/2) dependent and angiotensin-II type 1 receptor (AT1R) dependent. We have identified protein kinase C beta (PKCß) as a critical mediator of this pathway and demonstrate that the PKCß inhibitor enzastaurin, and the clinically available anti-hypertensive agent hydralazine, both normalize aortic growth in Marfan mice, in association with reduced PKCß and ERK1/2 activation. Furthermore, patients with Marfan syndrome and other forms of inherited thoracic aortic aneurysm taking CCBs display increased risk of aortic dissection and need for aortic surgery, compared to patients on other antihypertensive agents.

Molecular imaging of inflammation in the ApoE -/- mouse model of atherosclerosis with IodoDPA.

BACKGROUND: Atherosclerosis is a common and serious vascular disease predisposing individuals to myocardial infarction and stroke. Intravascular plaques, the pathologic lesions of atherosclerosis, are largely composed of cholesterol-laden luminal macrophage-rich infiltrates within a fibrous cap. The ability to detect those macrophages non-invasively within the aorta, carotid artery and other vessels would allow physicians to determine plaque burden, aiding management of patients with atherosclerosis. METHODS AND RESULTS: We previously developed a low-molecular-weight imaging agent, [(125)I]iodo-DPA-713 (iodoDPA), which selectively targets macrophages. Here we use it to detect both intravascular macrophages and macrophage infiltrates within the myocardium in the ApoE -/- mouse model of atherosclerosis using single photon emission computed tomography (SPECT). SPECT data were confirmed by echocardiography, near-infrared fluorescence imaging and histology. SPECT images showed focal uptake of radiotracer at the aortic root in all ApoE -/- mice, while the age-matched controls were nearly devoid of radiotracer uptake. Focal radiotracer uptake along the descending aorta and within the myocardium was also observed in affected animals. CONCLUSIONS: IodoDPA is a promising new imaging agent for atherosclerosis, with specificity for the macrophage component of the lesions involved.

Cardiac fibroblasts mediate IL-17A-driven inflammatory dilated cardiomyopathy.

Inflammatory dilated cardiomyopathy (DCMi) is a major cause of heart failure in individuals below the age of 40. We recently reported that IL-17A is required for the development of DCMi. We show a novel pathway connecting IL-17A, cardiac fibroblasts (CFs), GM-CSF, and heart-infiltrating myeloid cells with the pathogenesis of DCMi. Il17ra(-/-) mice were protected from DCMi, and this was associated with significantly diminished neutrophil and Ly6Chi monocyte/macrophage (MO/MΦ) cardiac infiltrates. Depletion of Ly6Chi MO/MΦ also protected mice from DCMi. Mechanistically, IL-17A stimulated CFs to produce key chemokines and cytokines that are critical downstream effectors in the recruitment and differentiation of myeloid cells. Moreover, IL-17A directs Ly6Chi MO/MΦ in trans toward a more proinflammatory phenotype via CF-derived GM-CSF. Collectively, this IL-17A-fibroblast-GM-CSF-MO/MΦ axis could provide a novel target for the treatment of DCMi and related inflammatory cardiac diseases.

PDE5 inhibitor efficacy is estrogen dependent in female heart disease.

Inhibition of cGMP-specific phosphodiesterase 5 (PDE5) ameliorates pathological cardiac remodeling and has been gaining attention as a potential therapy for heart failure. Despite promising results in males, the efficacy of the PDE5 inhibitor sildenafil in female cardiac pathologies has not been determined and might be affected by estrogen levels, given the hormone's involvement in cGMP synthesis. Here, we determined that the heart-protective effect of sildenafil in female mice depends on the presence of estrogen via a mechanism that involves myocyte eNOS-dependent cGMP synthesis and the cGMP-dependent protein kinase Iα (PKGIα). Sildenafil treatment failed to exert antiremodeling properties in female pathological hearts from Gαq-overexpressing or pressure-overloaded mice after ovary removal; however, estrogen replacement restored the effectiveness of sildenafil in these animals. In females, sildenafil-elicited myocardial PKG activity required estrogen, which stimulated tonic cardiomyocyte cGMP synthesis via an eNOS/soluble guanylate cyclase pathway. In contrast, eNOS activation, cGMP synthesis, and sildenafil efficacy were not estrogen dependent in male hearts. Estrogen and sildenafil had no impact on pressure-overloaded hearts from animals expressing dysfunctional PKGIα, indicating that PKGIα mediates antiremodeling effects. These results support the importance of sex differences in the use of PDE5 inhibitors for treating heart disease and the critical role of estrogen status when these agents are used in females.

Geranylgeranylacetone blocks doxorubicin-induced cardiac toxicity and reduces cancer cell growth and invasion through RHO pathway inhibition.

Doxorubicin is a widely used chemotherapy for solid tumors and hematologic malignancies, but its use is limited due to cardiotoxicity. Geranylgeranylacetone (GGA), an antiulcer agent used in Japan for 30 years, has no significant adverse effects, and unexpectedly reduces ovarian cancer progression in mice. Because GGA reduces oxidative stress in brain and heart, we hypothesized that GGA would prevent oxidative stress of doxorubicin cardiac toxicity and improve doxorubicin's chemotherapeutic effects. Nude mice implanted with MDA-MB-231 breast cancer cells were studied after chronic treatment with doxorubicin, doxorubicin/GGA, GGA, or saline. Transthoracic echocardiography was used to monitor systolic heart function and xenografts evaluated. Mice were euthanized and cardiac tissue evaluated for reactive oxygen species generation, TUNEL assay, and RHO/ROCK pathway analysis. Tumor metastases were evaluated in lung sections. In vitro studies using Boyden chambers were performed to evaluate GGA effects on RHO pathway activator lysophosphatidic acid (LPA)-induced motility and invasion. We found that GGA reduced doxorubicin cardiac toxicity, preserved cardiac function, prevented TUNEL-positive cardiac cell death, and reduced doxorubicin-induced oxidant production in a nitric oxide synthase-dependent and independent manner. GGA also reduced heart doxorubicin-induced ROCK1 cleavage. Remarkably, in xenograft-implanted mice, combined GGA/doxorubicin treatment decreased tumor growth more effectively than doxorubicin treatment alone. As evidence of antitumor effect, GGA inhibited LPA-induced motility and invasion by MDA-MB-231 cells. These anti-invasive effects of GGA were suppressed by geranylgeraniol suggesting GGA inhibits RHO pathway through blocking geranylation. Thus, GGA protects the heart from doxorubicin chemotherapy-induced injury and improves anticancer efficacy of doxorubicin in breast cancer.

Beneficial cardiac effects of caloric restriction are lost with age in a murine model of obesity.

Obesity is associated with increased diastolic stiffness and myocardial steatosis and dysfunction. The impact of aging on the protective effects of caloric restriction (CR) is not clear. We studied 2-month (younger) and 6-7-month (older)-old ob/ob mice and age-matched C57BL/6J controls (WT). Ob/ob mice were assigned to diet ad libitum or CR for 4 weeks. We performed echocardiograms, myocardial triglyceride assays, Oil Red O staining, and measured free fatty acids, superoxide, NOS activity, ceramide levels, and Western blots. In younger mice, CR restored diastolic function, reversed myocardial steatosis, and upregulated Akt phosphorylation. None of these changes was observed in the older mice; however, CR decreased oxidative stress and normalized NOS activity in these animals. Interestingly, myocardial steatosis was not associated with increased ceramide, but CR altered the composition of ceramides. In this model of obesity, aging attenuates the benefits of CR on myocardial structure and function.

TLR3 deficiency induces chronic inflammatory cardiomyopathy in resistant mice following coxsackievirus B3 infection: role for IL-4.

Recent findings indicate that TLR3 polymorphisms increase susceptibility to enteroviral myocarditis and inflammatory dilated cardiomyopathy (iDCM) in patients. TLR3 signaling has been found to inhibit coxsackievirus B3 (CVB3) replication and acute myocarditis in mouse models, but its role in the progression from myocarditis to iDCM has not been previously investigated. In this study we found that TLR3 deficiency increased acute (P = 5.9 × 10(-9)) and chronic (P = 6.0 × 10(-7)) myocarditis compared with WT B6.129, a mouse strain that is resistant to chronic myocarditis and iDCM. Using left ventricular in vivo hemodynamic assessment, we found that TLR3-deficient mice developed progressively worse chronic cardiomyopathy. TLR3 deficiency significantly increased viral replication in the heart during acute myocarditis from day 3 through day 12 after infection, but infectious virus was not detected in the heart during chronic disease. TLR3 deficiency increased cytokines associated with a T helper (Th)2 response, including IL-4 (P = 0.03), IL-10 (P = 0.008), IL-13 (P = 0.002), and TGF-ß(1) (P = 0.005), and induced a shift to an immunoregulatory phenotype in the heart. However, IL-4-deficient mice had improved heart function during acute CVB3 myocarditis by echocardiography and in vivo hemodynamic assessment compared with wild-type mice, indicating that IL-4 impairs cardiac function during myocarditis. IL-4 deficiency increased regulatory T-cell and macrophage populations, including FoxP3(+) T cells (P = 0.005) and Tim-3(+) macrophages (P = 0.004). Thus, TLR3 prevents the progression from myocarditis to iDCM following CVB3 infection by reducing acute viral replication and IL-4 levels in the heart.