Abstinence Restores Cardiac Function in Mice with Established Alcohol-Induced Cardiomyopathy.

Alcohol-induced cardiomyopathy (ACM) has a poor prognosis with up to a 50% chance of death within four years of diagnosis. There are limited studies investigating the potential of abstinence for promoting repair after alcohol-induced cardiac damage, particularly in a controlled preclinical study design. Here, we developed an exposure protocol that led to significant decreases in cardiac function in C57BL6/J mice within 30 days; dP/dt max decreased in the mice fed alcohol for 30 days (8054 ± 664.5 mmHg/s compared to control mice: 11,188 ± 724.2 mmHg/s, p < 0.01), and the dP/dt min decreased, as well (-7711 ± 561 mmHg/s compared to control mice: -10,147 ± 448.2 mmHg/s, p < 0.01). Quantitative PCR was used to investigate inflammatory and fibrotic biomarkers, while histology was used to depict overt changes in cardiac fibrosis. We observed a complete recovery of function after abstinence (dP/dt max increased from 8054 ± 664 mmHg/s at 30 days to 11,967 ± 449 mmHg/s after abstinence, p < 0.01); further, both inflammatory and fibrotic biomarkers decreased after abstinence. These results lay the groundwork for future investigation of the molecular mechanisms underlying recovery from alcohol-induced damage in the heart.

H2S Prodrug, SG-1002, Protects against Myocardial Oxidative Damage and Hypertrophy In Vitro via Induction of Cystathionine ß-Synthase and Antioxidant Proteins.

Endogenously produced hydrogen sulfide (H2S) is critical for cardiovascular homeostasis. Therapeutic strategies aimed at increasing H2S levels have proven cardioprotective in models of acute myocardial infarction (MI) and heart failure (HF). The present study was undertaken to investigate the effects of a novel H2S prodrug, SG-1002, on stress induced hypertrophic signaling in murine HL-1 cardiac muscle cells. Treatment of HL-1 cells with SG-1002 under serum starvation without or with H2O2 increased the levels of H2S, H2S producing enzyme, and cystathionine ß-synthase (CBS), as well as antioxidant protein levels, such as super oxide dismutase1 (SOD1) and catalase, and additionally decreased oxidative stress. SG-1002 also decreased the expression of hypertrophic/HF protein markers such as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), galectin-3, TIMP1, collagen type III, and TGF-ß1 in stressed HL-1 cells. Treatment with SG-1002 caused a significant induction of cell viability and a marked reduction of cellular cytotoxicity in HL-1 cells under serum starvation incubated without or with H2O2. Experimental results of this study suggest that SG-1002 attenuates myocardial cellular oxidative damage and/or hypertrophic signaling via increasing H2S levels or H2S producing enzymes, CBS, and antioxidant proteins.

Ovarian hormones do not mediate protection against pulmonary hypertension and right ventricular remodeling in female mice exposed to chronic, inhaled nicotine.

Electronic cigarette use has increased globally prompting calls for improved understanding of nicotine's cardiovascular health effects. Our group has previously demonstrated that chronic, inhaled nicotine induces pulmonary hypertension and right ventricular (RV) remodeling in male mice, but not female mice, suggesting sex differences in nicotine-related pathology. Clinically, biological females develop pulmonary hypertension more often but have less severe disease than biological males, likely because of the cardiopulmonary protective effects of estrogen. Nicotine is also metabolized more rapidly in biological females because of differences in cytochrome-P450 activity, which are thought to be mediated by female sex hormones. These findings led us to hypothesize that female mice are protected against nicotine-induced pulmonary hypertension by an ovarian hormone-dependent mechanism. In this study, intact and ovariectomized (OVX) female mice were exposed to chronic, inhaled nicotine or room air for 12 h/day for 10-12 wk. We report no differences in serum cotinine levels between intact and OVX mice. In addition, we found no structural (RV or left ventricular dimensions and Fulton index) or functional (RV systolic pressure, pulmonary vascular resistance, cardiac output, ejection fraction, and fractional shortening) evidence of cardiopulmonary dysfunction in intact or OVX mice. We conclude that ovarian hormones do not mediate cardiopulmonary protection against nicotine-induced pulmonary hypertension. Due to profound sex differences in clinical pulmonary hypertension pathogenesis and nicotine metabolism, further studies are necessary to elucidate mechanisms underlying protection from nicotine-induced pathology in female mice.NEW & NOTEWORTHY The emergence of electronic cigarettes poses a threat to cardiovascular and pulmonary health, but the direct contribution of nicotine to these disease processes is largely unknown. Our laboratory has previously shown that chronic, inhaled nicotine induces pulmonary hypertension and right ventricular remodeling in male mice, but not female mice. This study using a bilateral ovariectomy model suggests that the cardiopulmonary protection observed in nicotine-exposed female mice may be independent of ovarian hormones.

Genetic Disruption of Guanylyl Cyclase/Natriuretic Peptide Receptor-A Triggers Differential Cardiac Fibrosis and Disorders in Male and Female Mutant Mice: Role of TGF-ß1/SMAD Signaling Pathway.

The global targeted disruption of the natriuretic peptide receptor-A (NPRA) gene (Npr1) in mice provokes hypertension and cardiovascular dysfunction. The objective of this study was to determine the mechanisms regulating the development of cardiac fibrosis and dysfunction in Npr1 mutant mice. Npr1 knockout (Npr1-/-, 0-copy), heterozygous (Npr1+/-, 1-copy), and wild-type (Npr1+/+, 2-copy) mice were treated with the transforming growth factor (TGF)-ß1 receptor (TGF-ß1R) antagonist GW788388 (2 µg/g body weight/day; ip) for 28 days. Hearts were isolated and used for real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot, and immunohistochemical analyses. The Npr1-/- (0-copy) mice showed a 6-fold induction of cardiac fibrosis and dysfunction with markedly induced expressions of collagen-1α (3.8-fold), monocyte chemoattractant protein (3.7-fold), connective tissue growth factor (CTGF, 5.3-fold), α-smooth muscle actin (α-SMA, 6.1-fold), TGF-ßRI (4.3-fold), TGF-ßRII (4.7-fold), and phosphorylated small mothers against decapentaplegic (pSMAD) proteins, including pSMAD-2 (3.2-fold) and pSMAD-3 (3.7-fold), compared with wild-type mice. The expressions of phosphorylated extracellular-regulated kinase ERK1/2 (pERK1/2), matrix metalloproteinases-2, -9, (MMP-2, -9), and proliferating cell nuclear antigen (PCNA) were also significantly upregulated in Npr1 0-copy mice. The treatment of mutant mice with GW788388 significantly blocked the expression of fibrotic markers, SMAD proteins, MMPs, and PCNA compared with the vehicle-treated control mice. The treatment with GW788388 significantly prevented cardiac dysfunctions in a sex-dependent manner in Npr1 0-copy and 1-copy mutant mice. The results suggest that the development of cardiac fibrosis and dysfunction in mutant mice is predominantly regulated through the TGF-ß1-mediated SMAD-dependent pathway.

Nicotine and novel tobacco products drive adverse cardiac remodeling and dysfunction in preclinical studies.

Background: The heart undergoes structural and functional changes in response to injury and hemodynamic stress known as cardiac remodeling. Cardiac remodeling often decompensates causing dysfunction and heart failure (HF). Cardiac remodeling and dysfunction are significantly associated with cigarette smoking. Although cigarette smoking has declined, the roles of nicotine and novel tobacco products (including electronic cigarettes and heat-not-burn tobacco) in cardiac remodeling are unclear. In this perspective, we present evidence demonstrating maladaptive cardiac remodeling in nicotine-exposed mice undergoing hemodynamic stress with angiotensin (Ang)-II infusion and review preclinical literature linking nicotine and novel tobacco products with cardiac remodeling and dysfunction. Methods: Adult, male C57BL/6J mice were exposed to room air or chronic, inhaled nicotine for 8 weeks. A subset of mice was infused with Ang-II via subcutaneous osmotic mini-pumps during the final 4 weeks of exposure. Left ventricular structure and function were assessed with echocardiography. Results: Chronic, inhaled nicotine abrogated Ang-II-induced thickening of the left ventricular posterior wall, leading to reduced relative wall thickness. Ang-II infusion was associated with increased left ventricular mass index in both air- and nicotine-exposed mice. Conclusions: These changes suggest a phenotypic shift from concentric hypertrophy to eccentric hypertrophy in nicotine-exposed, hemodynamically-stressed mice which could drive HF pathogenesis. These findings join a growing body of animal studies demonstrating cardiac remodeling and dysfunction following nicotine and electronic cigarette exposure. Further exploration is necessary; however, clinicians and researchers should not overlook these emerging products as potential risk factors in the pathogenesis of cardiac remodeling and associated diseases including HF.

Alpha7 nicotinic acetylcholine receptor mediates chronic nicotine inhalation-induced cardiopulmonary dysfunction.

Cigarette smoking remains the leading modifiable risk factor for cardiopulmonary diseases; however, the effects of nicotine alone on cardiopulmonary function remain largely unknown. Previously, we have shown that chronic nicotine vapor inhalation in mice leads to the development of pulmonary hypertension (PH) with right ventricular (RV) remodeling. The present study aims to further examine the cardiopulmonary effects of nicotine and the role of the α7 nicotinic acetylcholine receptor (α7-nAChR), which is widely expressed in the cardiovascular system. Wild-type (WT) and α7-nAChR knockout (α7-nAChR-/-) mice were exposed to room air (control) or nicotine vapor daily for 12 weeks. Consistent with our previous study, echocardiography and RV catheterization reveal that male WT mice developed increased RV systolic pressure with RV hypertrophy and dilatation following 12-week nicotine vapor exposure; in contrast, these changes were not observed in male α7-nAChR-/- mice. In addition, chronic nicotine inhalation failed to induce PH and RV remodeling in female mice regardless of genotype. The effects of nicotine on the vasculature were further examined in male mice. Our results show that chronic nicotine inhalation led to impaired acetylcholine-mediated vasodilatory response in both thoracic aortas and pulmonary arteries, and these effects were accompanied by altered endothelial nitric oxide synthase phosphorylation (enhanced inhibitory phosphorylation at threonine 495) and reduced plasma nitrite levels in WT but not α7-nAChR-/- mice. Finally, RNA sequencing revealed up-regulation of multiple inflammatory pathways in thoracic aortas from WT but not α7-nAChR-/- mice. We conclude that the α7-nAChR mediates chronic nicotine inhalation-induced PH, RV remodeling and vascular dysfunction.

Angiotensin II type 1 receptor mediates pulmonary hypertension and right ventricular remodeling induced by inhaled nicotine.

Use of electronic cigarettes is rapidly increasing among youth and young adults, but little is known regarding the long-term cardiopulmonary health impacts of these nicotine-containing devices. Our group has previously demonstrated that chronic, inhaled nicotine induces pulmonary hypertension (PH) and right ventricular (RV) remodeling in mice. These changes were associated with upregulated RV angiotensin-converting enzyme (ACE). Angiotensin II receptor blockers (ARBs) have been shown to reverse cigarette smoking-induced PH in rats. ACE inhibitor and ARB use in a large retrospective cohort of patients with PH is associated with improved survival. Here, we utilized losartan (an ARB specific for angiotensin II type 1 receptor) to further explore nicotine-induced PH. Male C57BL/6 mice received nicotine vapor for 12 h/day, and exposure was assessed using serum cotinine to achieve levels comparable to human smokers or electronic cigarette users. Mice were exposed to nicotine for 8 wk and a subset was treated with losartan via an osmotic minipump. Cardiac function was assessed using echocardiography and catheterization. Although nicotine exposure increased angiotensin II in the RV and lung, this finding was nonsignificant. Chronic, inhaled nicotine significantly increased RV systolic pressure and RV free wall thickness versus air control. These parameters were significantly lower in mice receiving both nicotine and losartan. Nicotine significantly increased RV internal diameter, with no differences seen between the nicotine and nicotine-losartan group. Neither nicotine nor losartan affected left ventricular structure or function. These findings provide the first evidence that antagonism of the angiotensin II type 1 receptor can ameliorate chronic, inhaled nicotine-induced PH and RV remodeling.NEW & NOTEWORTHY Chronic, inhaled nicotine causes pulmonary hypertension and right ventricular remodeling in mice. Treatment with losartan, an angiotensin II type 1 receptor antagonist, ameliorates nicotine-induced pulmonary hypertension and right ventricular remodeling. This novel finding provides preclinical evidence for the use of renin-angiotensin system-based therapies in the treatment of pulmonary hypertension, particularly in patients with a history of tobacco-product use.

Heat-not-burn tobacco products: an emerging threat to cardiovascular health.

Cigarette smoking is at all-time lows globally, but the use of electronic cigarettes has increased profoundly. Recent reports of electronic cigarette or vaping use-associated lung injury may lead individuals to explore novel methods of nicotine consumption, such as heat-not-burn devices. IQOS from Philip Morris, a heat-not-burn device, became available for purchase in the United States in October 2019. Philip Morris claims that 8.8 million people have abandoned traditional cigarettes in favor of IQOS; however, evidence suggests that it may act as a gateway or complement to cigarette smoking, rather than a replacement. Surveys indicate that 96% of Korean IQOS users also smoke cigarettes, and 45% of Italian users of IQOS had never smoked cigarettes. In the United States, Canada, and England, susceptibility of youth to trying IQOS was slightly lower than electronic cigarettes, but higher than cigarette smoking. Heat-not-burn products produce mainstream and second-hand emissions of harmful chemicals, including nicotine, particulate matter, benzene, acrolein, and tobacco-specific nitrosamines. The levels of these emissions, despite being less than those of traditional cigarettes, are potentially harmful to cardiovascular health. A study of current smokers showed similar acute effects of heat-not-burn tobacco products and traditional cigarettes on heart rate, blood pressure, and arterial stiffness. Rats exposed to IQOS had similar vascular endothelial function impairment to those exposed to cigarettes. Heat-not-burn aerosol exposure of cultured macrophages elicited increased oxidative stress, although less than that induced by cigarette smoke. Further studies are needed to better understand the cardiovascular effects of heat-not-burn tobacco products.

Effects of Chronic Nicotine Inhalation on Systemic and Pulmonary Blood Pressure and Right Ventricular Remodeling in Mice.

Cigarette smoking is the single most important risk factor for the development of cardiovascular and pulmonary diseases; however, the role of nicotine in the pathogenesis of these diseases is incompletely understood. The purpose of this study was to examine the effects of chronic nicotine inhalation on the development of cardiovascular and pulmonary disease with a focus on blood pressure and cardiac remodeling. Male C57BL6/J mice were exposed to air (control) or nicotine vapor (daily, 12 hour on/12 hour off) for 8 weeks. Systemic blood pressure was recorded weekly by radio-telemetry, and cardiac remodeling was monitored by echocardiography. At the end of the 8 weeks, mice were subjected to right heart catheterization to measure right ventricular systolic pressure. Nicotine-exposed mice exhibited elevated systemic blood pressure from weeks 1 to 3, which then returned to baseline from weeks 4 to 8, indicating development of tolerance to nicotine. At 8 weeks, significantly increased right ventricular systolic pressure was detected in nicotine-exposed mice compared with the air controls. Echocardiography showed that 8-week nicotine inhalation resulted in right ventricular (RV) hypertrophy with increased RV free wall thickness and a trend of increase in RV internal diameter. In contrast, there were no significant structural or functional changes in the left ventricle following nicotine exposure. Mechanistically, we observed increased expression of angiotensin-converting enzyme and enhanced activation of mitogen-activated protein kinase pathways in the RV but not in the left ventricle. We conclude that chronic nicotine inhalation alters both systemic and pulmonary blood pressure with the latter accompanied by RV remodeling, possibly leading to progressive and persistent pulmonary hypertension.

N-Acetylcysteine prevents the decreases in cardiac collagen I/III ratio and systolic function in neonatal mice with prenatal alcohol exposure.

Prenatal alcohol exposure (PAE) is often associated with congenital heart defects, most commonly septal, valvular, and great vessel defects. However, there have been no known studies on whether PAE affects the resulting fibroblast population after development, and whether this has any consequences in the postnatal period. Our previous study focused on the effects of PAE on the postnatal fibroblast population, which translated into changes in cardiac extracellular matrix (ECM) composition and cardiac function in the neonatal heart. Moreover, our lab has previously demonstrated that alcohol-induced fibrosis is mediated by oxidative stress mechanisms in adult rat hearts following chronic alcohol exposure. Thus, we hypothesize that PAE alters cardiac ECM composition that persists into the postnatal period, leading to cardiac dysfunction, and these effects are prevented by antioxidant treatment. To investigate these effects, pregnant mice were intraperitoneally injected with 2.9 g EtOH/kg body weight on gestation days 6.75 and 7.25. Controls were injected with vehicle saline. Randomly selected dams in both groups were then treated with 100 mg/kg body weight of the antioxidant N-acetylcysteine (NAC) immediately after EtOH or vehicle administration. Left ventricular (LV) chamber dimension and function were assessed in sedated animals on neonatal day 5 using echocardiography. Ejection fraction decreased in the PAE group. NAC treatment prevented this depression of systolic function in PAE neonates. Hearts were analyzed for expression of fibroblast activation markers. Alpha smooth muscle actin (α-SMA) increased in PAE neonatal hearts, and this increase was prevented by NAC treatment. In PAE pups, collagen I decreased, but collagen III expression increased compared to saline animals; the overall collagen I/III ratio significantly decreased. When PAE mice were treated with NAC, collagen I/III ratio did not change. Overall, our data demonstrate that prenatal alcohol exposure produces changes in collagen subtype in neonatal cardiac ECM and a decline in systolic function, and these adverse effects were prevented by NAC treatment.

The prevalence of cardio-metabolic risk factors is differentially elevated in obesity-prone Osborne-Mendel and obesity-resistant S5B/Pl rats.

AIMS: Individual susceptibility to develop obesity may impact the development of cardio-metabolic risk factors that lead to obesity-related comorbid conditions. Obesity-prone Osborne-Mendel (OM) rats expressed higher levels of visceral adipose inflammation than obesity-resistant, S5B/Pl (S5B) rats. However, the consumption of a high fat diet (HFD) differentially affected OM and S5B rats and induced an increase in visceral adipose inflammation in S5B rats. The current study examined the effects of HFD consumption on cardio-metabolic risk factors in OM and S5B rats. MATERIALS & METHODS: Glucose regulation and circulating levels of lipids, adiponectin and C-reactive protein were assessed following 8 weeks of HFD or low fat diet (LFD) consumption. Left ventricle hypertrophy and mRNA expression of cardiovascular disease biomarkers were also quantified in OM and S5B rats. KEY FINDINGS: Circulating levels of triglycerides were higher, while HDL cholesterol, adiponectin and glycemic control were lower in OM rats, compared to S5B rats. In the left ventricle, BNP and CTGF mRNA expression were higher in OM rats and IL-6, IL-1ß, VEGF, and iNOS mRNA expression were higher in S5B rats. SIGNIFICANCE: These findings support the hypothesis that cardio-metabolic risk factors are increased in obesity-prone individuals, which may increase the risk for the development of obesity-related comorbidities. In the current models, obesity-resistant S5B rats also exhibited cardiovascular risk factors supporting the importance of monitoring cardiovascular health in individuals characterized as obesity-resistant.

Prenatal Alcohol Exposure Causes Adverse Cardiac Extracellular Matrix Changes and Dysfunction in Neonatal Mice.

Fetal alcohol syndrome (FAS) is the most severe condition of fetal alcohol spectrum disorders (FASD) and is associated with congenital heart defects. However, more subtle defects such as ventricular wall thinning and cardiac compliance may be overlooked in FASD. Our studies focus on the role of cardiac fibroblasts in the neonatal heart, and how they are affected by prenatal alcohol exposure (PAE). We hypothesize that PAE affects fibroblast function contributing to dysregulated collagen synthesis, which leads to cardiac dysfunction. To investigate these effects, pregnant C57/BL6 mice were intraperitoneally injected with 2.9 g EtOH/kg dose to achieve a blood alcohol content of approximately 0.35 on gestation days 6.75 and 7.25. Pups were sacrificed on neonatal day 5 following echocardiography measurements of left ventricular (LV) chamber dimension and function. Hearts were used for primary cardiac fibroblast isolation or protein expression analysis. PAE animals had thinner ventricular walls than saline exposed animals, which was associated with increased LV wall stress and decreased ejection fraction. In isolated fibroblasts, PAE decreased collagen I/III ratio and increased gene expression of profibrotic markers, including α-smooth muscle actin and lysyl oxidase. Notch1 signaling was assessed as a possible mechanism for fibroblast activation, and indicated that gene expression of Notch1 receptor and downstream Hey1 transcription factor were increased. Cardiac tissue analysis revealed decreased collagen I/III ratio and increased protein expression of α-smooth muscle actin and lysyl oxidase. However, Notch1 signaling components decreased in whole heart tissue. Our study demonstrates that PAE caused adverse changes in the cardiac collagen profile and a decline in cardiac function in the neonatal heart.

Nicotine and the renin-angiotensin system.

Cigarette smoking is the single most important risk factor for the development of cardiovascular and pulmonary diseases (CVPD). Although cigarette smoking has been in constant decline since the 1950s, the introduction of e-cigarettes or electronic nicotine delivery systems 10 yr ago has attracted former smokers as well as a new generation of consumers. Nicotine is a highly addictive substance, and it is currently unclear whether e-cigarettes are "safer" than regular cigarettes or whether they have the potential to reverse the health benefits, notably on the cardiopulmonary system, acquired with the decline of tobacco smoking. Of great concern, nicotine inhalation devices are becoming popular among young adults and youths, emphasizing the need for awareness and further study of the potential cardiopulmonary risks of nicotine and associated products. This review focuses on the interaction between nicotine and the renin-angiotensin system (RAS), one of the most important regulatory systems on autonomic, cardiovascular, and pulmonary functions in both health and disease. The literature presented in this review strongly suggests that nicotine alters the homeostasis of the RAS by upregulating the detrimental angiotensin-converting enzyme (ACE)/angiotensin (ANG)-II/ANG II type 1 receptor axis and downregulating the compensatory ACE2/ANG-(1-7)/Mas receptor axis, contributing to the development of CVPD.

Chronic Ethanol Administration Prevents Compensatory Cardiac Hypertrophy in Pressure Overload.

BACKGROUND: Alcohol is among the most commonly abused drugs worldwide and affects many organ systems, including the heart. Alcoholic cardiomyopathy is characterized by a dilated cardiac phenotype with extensive hypertrophy and extracellular matrix (ECM) remodeling. We have previously shown that chronic ethanol (EtOH) administration accelerates the progression to heart failure in a rat model of volume overload. However, the mechanism by which this decompensation occurs is unknown. For this study, we hypothesized that chronic EtOH administration would prevent compensatory hypertrophy and cardiac remodeling in a rodent model of pressure overload (PO). METHODS: Abdominal aortic constriction was used to create PO in 8-week-old male Wistar rats. Alcohol administration was performed via chronic intermittent EtOH vapor inhalation for 2 weeks prior to surgery and for the duration of the 8-week study. Echocardiography measurements were taken to assess ventricular functional and structural changes. RESULTS: PO increased posterior wall thickness and the hypertrophic markers, atrial and B-type natriuretic peptides (ANP and BNP). With the added stressor of EtOH, wall thickness, ANP, and BNP decreased in PO animals. The combination of PO and EtOH resulted in increased wall stress compared to PO alone. PO also caused increased expression of collagen I and III, whereas EtOH alone only increased collagen III. The combined stresses of PO and EtOH led to an increase in collagen I expression, but collagen III did not change, resulting in an increased collagen I/III ratio in the PO rats treated with EtOH. Lastly, Notch1 expression was significantly increased only in the PO rats treated with EtOH. CONCLUSIONS: Our data indicate that chronic EtOH may limit the cardiac hypertrophy induced by PO which may be associated with a Notch1 mechanism, resulting in increased wall stress and altered ECM profile.

Inhibitor of lysyl oxidase improves cardiac function and the collagen/MMP profile in response to volume overload.

The cardiac extracellular matrix is a complex architectural network that serves many functions, including providing structural and biochemical support to surrounding cells and regulating intercellular signaling pathways. Cardiac function is directly affected by extracellular matrix (ECM) composition, and alterations of the ECM contribute to the progression of heart failure. Initially, collagen deposition is an adaptive response that aims to preserve tissue integrity and maintain normal ventricular function. However, the synergistic effects of proinflammatory and profibrotic responses induce a vicious cycle, which causes excess activation of myofibroblasts, significantly increasing collagen deposition and accumulation in the matrix. Furthermore, excess synthesis and activation of the enzyme lysyl oxidase (LOX) during disease increases collagen cross-linking, which significantly increases collagen resistance to degradation by matrix metalloproteinases (MMPs). In the present study, the aortocaval fistula model of volume overload (VO) was used to determine whether LOX inhibition could prevent adverse changes in the ECM and subsequent cardiac dysfunction. The major findings from this study were that LOX inhibition 1) prevented VO-induced increases in left ventricular wall stress; 2) partially attenuated VO-induced ventricular hypertrophy; 3) completely blocked the increases in fibrotic proteins, including collagens, MMPs, and their tissue inhibitors; and 4) prevented the VO-induced decline in cardiac function. It remains unclear whether a direct interaction between LOX and MMPs exists; however, our experiments suggest a potential link between the two because LOX inhibition completely attenuated VO-induced increases in MMPs. Overall, our study demonstrated key cardioprotective effects of LOX inhibition against adverse cardiac remodeling due to chronic VO. NEW & NOTEWORTHY Although the primary role of lysyl oxidase (LOX) is to cross-link collagens, we found that elevated LOX during cardiac disease plays a key role in the progression of heart failure. Here, we show that inhibition of LOX in volume-overloaded rats prevented the development of cardiac dysfunction and improved ventricular collagen and matrix metalloproteinase/tissue inhibitor of metalloproteinase profiles.

A novel experimental model of erectile dysfunction in rats with heart failure using volume overload.

BACKGROUND: Patients with heart failure (HF) display erectile dysfunction (ED). However, the pathophysiology of ED during HF remains poorly investigated. OBJECTIVE: This study aimed to characterize the aortocaval fistula (ACF) rat model associated with HF as a novel experimental model of ED. We have undertaken molecular and functional studies to evaluate the alterations of the nitric oxide (NO) pathway, autonomic nervous system and oxidative stress in the penis. METHODS: Male rats were submitted to ACF for HF induction. Intracavernosal pressure in anesthetized rats was evaluated. Concentration-response curves to contractile (phenylephrine) and relaxant agents (sodium nitroprusside; SNP), as well as to electrical field stimulation (EFS), were obtained in the cavernosal smooth muscle (CSM) strips from sham and HF rats. Protein expression of endothelial NO synthase (eNOS) and neuronal NO synthase (nNOS) and phosphodiestarese-5 in CSM were evaluated, as well as NOX2 (gp91phox) and superoxide dismutase (SOD) mRNA expression. SOD activity and thiobarbituric acid reactive substances (TBARs) were also performed in plasma. RESULTS: HF rats display erectile dysfunction represented by decreased ICP responses compared to sham rats. The neurogenic contractile responses elicited by EFS were greater in CSM from the HF group. Likewise, phenylephrine-induced contractions were greater in CSM from HF rats. Nitrergic response induced by EFS were decreased in the cavernosal tissue, along with lower eNOS, nNOS and phosphodiestarese-5 protein expressions. An increase of NOX2 and SOD mRNA expression in CSM and plasma TBARs of HF group were detected. Plasma SOD activity was decreased in HF rats. CONCLUSION: ED in HF rats is associated with decreased NO bioavailability in erectile tissue due to eNOS/nNOS dowregulation and NOX2 upregulation, as well as hypercontractility of the penis. This rat model of ACF could be a useful tool to evaluate the molecular alterations of ED associated with HF.

Detrimental role of lysyl oxidase in cardiac remodeling.

A key feature of heart failure is adverse extracellular matrix (ECM) remodeling, which is associated with increases in the collagen cross-linking enzyme, lysyl oxidase (LOX). In this study, we assess the progression of cardiovascular remodeling from the compensatory to decompensatory phase, with a focus on the change in LOX expression and activity as it relates to alterations in ECM composition and changes in cardiac function. Adult male Sprague-Dawley rats were studied after 4, 14, or 21weeks of aortocaval fistula-induced volume overload (VO). Progressive increases in the left and right ventricular mass indicated biventricular hypertrophy. Echocardiography revealed significant increases in the posterior wall thickness and internal diameter of the left ventricle as early as 3weeks, which persisted until the 21week endpoint. There were also significant decreases in eccentric index and fractional shortening in VO animals. Hemodynamic measurements showed progressive decreases in contractility, indicative of systolic dysfunction. There were progressive VO-induced increases in LOX expression and activity, collagen, and collagen cross-linking during the course of these experiments. We observed a negative correlation between LOX activity and cardiac function. Additional rats were treated with an inhibitor of LOX activity starting at 2weeks post-surgery and continued to 14weeks. LOX inhibition prevented the cardiac dysfunction and collagen accumulation caused by VO. Overall these data suggest a detrimental role for the chronic increase of cardiac LOX expression and activity in the transition from compensated remodeling to decompensated failure.

Cardiac-restricted Overexpression of TRAF3 Interacting Protein 2 (TRAF3IP2) Results in Spontaneous Development of Myocardial Hypertrophy, Fibrosis, and Dysfunction.

TRAF3IP2 (TRAF3 interacting protein 2; previously known as CIKS or Act1) is a key intermediate in the normal inflammatory response and the pathogenesis of various autoimmune and inflammatory diseases. Induction of TRAF3IP2 activates IκB kinase (IKK)/NF-κB, JNK/AP-1, and c/EBPß and stimulates the expression of various inflammatory mediators with negative myocardial inotropic effects. To investigate the role of TRAF3IP2 in heart disease, we generated a transgenic mouse model with cardiomyocyte-specific TRAF3IP2 overexpression (TRAF3IP2-Tg). Echocardiography, magnetic resonance imaging, and pressure-volume conductance catheterization revealed impaired cardiac function in 2-month-old male transgenic (Tg) mice as evidenced by decreased ejection fraction, stroke volume, cardiac output, and peak ejection rate. Moreover, the male Tg mice spontaneously developed myocardial hypertrophy (increased heart/body weight ratio, cardiomyocyte cross-sectional area, GATA4 induction, and fetal gene re-expression). Furthermore, TRAF3IP2 overexpression resulted in the activation of IKK/NF-κB, JNK/AP-1, c/EBPß, and p38 MAPK and induction of proinflammatory cytokines, chemokines, and extracellular matrix proteins in the heart. Although myocardial hypertrophy decreased with age, cardiac fibrosis (increased number of myofibroblasts and enhanced expression and deposition of fibrillar collagens) increased progressively. Despite these adverse changes, TRAF3IP2 overexpression did not result in cell death at any time period. Interestingly, despite increased mRNA expression, TRAF3IP2 protein levels and activation of its downstream signaling intermediates remained unchanged in the hearts of female Tg mice. The female Tg mice also failed to develop myocardial hypertrophy. In summary, these results demonstrate that overexpression of TRAF3IP2 in male mice is sufficient to induce myocardial hypertrophy, cardiac fibrosis, and contractile dysfunction.