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Despite emerging data indicating a role for T cells in profibrotic cardiac repair and healing after ischemia, little is known about whether T cells directly impact cardiac fibroblasts (CFBs) to promote cardiac fibrosis (CF) in nonischemic heart failure (HF). Recently, we reported increased T cell infiltration in the fibrotic myocardium of nonischemic HF patients, as well as the protection from CF and HF in TCR-α−/− mice. Here, we report that T cells activated in such a context are mainly IFN-γ+, adhere to CFB, and induce their transition into myofibroblasts. Th1 effector cells selectively drive CF both in vitro and in vivo, whereas adoptive transfer of Th1 cells, opposite to activated IFN-γ−/− Th cells, partially reconstituted CF and HF in TCR-α−/− recipient mice. Mechanistically, Th1 cells use integrin α4 to adhere to and induce TGF-β in CFB in an IFN-γ–dependent manner. Our findings identify a previously unrecognized role for Th1 cells as integrators of perivascular CF and cardiac dysfunction in nonischemic HF.

Clinical trial and epidemiological data support that the cardiovascular effects of estrogen are complex, including a mixture of both potentially beneficial and harmful effects. In animal models, estrogen protects females from vascular injury and inhibits atherosclerosis. These effects are mediated by estrogen receptors (ERs), which, when bound to estrogen, can bind to DNA to directly regulate transcription. ERs can also activate several cellular kinases by inducing a rapid nonnuclear signaling cascade. However, the biological significance of this rapid signaling pathway has been unclear.

Heart failure is a major cause of morbidity and mortality worldwide. The ubiquitously expressed cytokine transforming growth factor-1 (TGF1) promotes cardiac fibrosis, an important component of progressive heart failure. Membrane-associated endoglin is a coreceptor for TGF1 signaling and has been studied in vascular remodeling and preeclampsia. We hypothesized that reduced endoglin expression may limit cardiac fibrosis in heart failure.

Autophagy is a cytoprotective pathway used to degrade and recycle cytoplasmic content. Dysfunctional autophagy has been linked to both cancer and cardiomyopathies. Here, we show a role for the transcriptional regulator p8 in autophagy. p8 RNA interference (RNAi) increases basal autophagy markers in primary cardiomyocytes, in H9C2 and U2OS cells, and decreases cellular viability after autophagy induction. This autophagy is associated with caspase activation and is blocked by atg5 silencing and by pharmacological inhibitors. FoxO3 transcription factor was reported to activate autophagy by enhancing the expression of autophagy-related genes. P8 expression represses FoxO3 transcriptional activity, and p8 knockdown affects FoxO3 nuclear localization. Thus, p8 RNAi increases FoxO3 association with bnip3 promoter, a known proautophagic FoxO3 target, resulting in higher bnip3 RNA and protein levels. Accordingly, bnip3 knockdown restores cell viability and blocks apoptosis of p8-deficient cells. In vivo, p8 –/– mice have higher autophagy and express higher cardiac bnip3 levels. These mice develop left ventricular wall thinning and chamber dilation, with consequent impaired cardiac function. Our studies provide evidence of a p8-dependent mechanism regulating autophagy by acting as FoxO3 corepressor, which may be relevant for diseases associated with dysregulated autophagy, as cardiovascular pathologies and cancer.

In this study, we investigate a role for p8 in autophagy in vitro and in vivo, by using p8−/− mice. In both settings, silencing of p8 is associated with basal up-regulation of autophagy and apoptosis. In vivo, the hearts of p8knockout mice develop features that provoke a decreased left ventricular functionality.

Left ventricular (LV) remodelling following myocardial infarction (MI) is associated with increased morbidity and mortality. Previous data suggest that angiotensin II (Ang II) plays a central role in the molecular events contributing to LV remodelling. We explored the effects of angiotensin-converting-enzyme (ACE) inhibition versus Ang II (AT1) receptor blockade on LV remodelling in mice post-MI. Mice underwent sham procedure or left coronary artery ligation, and received placebo, the AT1 receptor antagonist, losartan or the ACE inhibitor, enalapril. At 6 weeks, echocardiography and haemodynamic studies were performed. Infarct size and interstitial collagen content were determined. Expression of genes encoding atrial natriuretic peptide (ANP), collagen type I, AT1a and AT1b receptors were measured. The placebo MI group showed increased LV end-diastolic diameter, LV end-systolic diameter with depressed fractional shortening (P<0.01 versus shams), increased LV mass and volume (both P<0.01 versus shams). The placebo MI group also exhibited increased non-infarct zone collagen content (P<0.01), ANP (P<0.01) and collagen type 1 (P<0.01) gene expression, with a non-significant rise in AT1a receptor gene expression. Neither losartan or enalapril prevented LV dilation or improved fractional shortening. Both similarly lowered systolic blood pressure (P<0.01 for each versus placebo). Losartan and enalapril inhibited LV hypertrophy (P<0.01), and decreased ANP (P<0.01) and collagen type 1 gene expression (P<0.05). Levels of AT1a receptor gene expression were higher than shams (P<0.05 for both), but similar to placebo. AT1b receptor gene expression was much lower than that for AT1a receptor and similar in all groups. Thus, in this model, AT1 receptor antagonism and ACE inhibition have equivalent inhibitory effects on myocardial hypertrophy and fibrosis. These results serve as an important basis for planned investigations to evaluate the anti-remodelling effects of these agents on mice in which genetic manipulations are used to disrupt components of the Ang II signalling system.

Transgenic mice are increasingly being utilized for understanding cardiac electrophysiologic abnormalities. However, little is known about the normal atrioventricular nodal and infraHisian physiology in the mouse because of the prior inability to record a His-bundle deflection. We present the first comprehensive examination of the murine atrioventricular nodal and His-Purkinje systems employing His-bundle recordings. Normal, healthy, male C57BL/6J mice (n = 48) underwent an in vivo electrophysiology study using a 2 F octapolar electrode catheter. Effective refractory periods were determined during premature atrial and ventricular stimulation. The PR interval measured 44 ± 6 ms with a mean sinus cycle length of 185 ± 42 ms. Baseline AH intervals were 36 ± 5 ms and HV intervals were 10 ± 2 ms. At a pacing cycle length of 140 ms the atrioventricular nodal effective refractory period (AVNERP) and atrial effective refractory period (AERP) were 86 ± 19 ms and 57 ± 17 ms, respectively. The mean AV Wenckebach and 2:1 paced cycle length were 103 ± 14 ms and 84 ± 13 ms, respectively. Premature atrial stimulation curves were asymptotic without discontinuity. A subset of nine mice was studied after administration of isoproterenol. The sinus cycle length, AVNERP and AERP decreased significantly from baseline measurements. This method establishes a practical and feasible technique to record in vivo His-bundle electrograms in the mouse to assess atrioventricular nodal and infraHisian physiology. Use of this model will allow for the examination of abnormalities of atrioventricular nodal and infraHisian conduction in transgenic murine models.

ABSTRACT. This report analyzes the effect on pulmonary vascular structure and function in the newborn guinea pig of chronic fetal hypoxemia, induced by maternal hypobaric hypoxia (380-420 torr) without direct interference with the utero-placental blood flow. Six pregnant guinea pigs were maintained in hypobaric chambers, 10 newborns from their litters being the hypoxic group. Twenty newborns from the litters of 10 pregnant guinea pigs maintained in air were the control group. After spontaneous delivery catheters were placed in the left carotid and the main pulmonary arteries of the newborns. Oxygen consumption was measured, pressures recorded from the pulmonary artery, right ventricle, and right atrium, oxygen saturation and hemoglobin concentration estimated on blood from systemic arteries, pulmonary artery, and right atrium, and cardiac output calculated (Fick principle). All data were normalized per kilogram body weight. Morphometric analysis of the pulmonary vasculature after injection of the pulmonary arteries showed no difference between the groups. Contrary to expectation chronic hypoxemia in utero did not cause pulmonary hypertension or the precocious muscularization of the precapillary unit found in human cases of persistent pulmonary hypertension: it caused growth retardation, a feature not typical of the human syndrome.

This report analyzes the effect on pulmonary vascular structure and function in the newborn guinea pig of chronic fetal hypoxemia, induced by maternal hypobaric hypoxia (380-420 torr) without direct interference with the utero-placental blood flow. Six pregnant guinea pigs were maintained in hypobaric chambers, 10 newborns from their litters being the hypoxic group. Twenty newborns from the litters of 10 pregnant guinea pigs maintained in air were the control group. After spontaneous delivery catheters were placed in the left carotid and the main pulmonary arteries of the newborns. Oxygen consumption was measured, pressures recorded from the pulmonary artery, right ventricle, and right atrium, oxygen saturation and hemoglobin concentration estimated on blood from systemic arteries, pulmonary artery, and right atrium, and cardiac output calculated (Fick principle). All data were normalized per kilogram body weight. Morphometric analysis of the pulmonary vasculature after injection of the pulmonary arteries showed no difference between the groups. Contrary to expectation chronic hypoxemia in uterodid not cause pulmonary hypertension or the precocious muscularization of the precapillary unit found in human cases of persistent pulmonary hypertension it caused growth retardation, a feature not typical of the human syndrome.

The clinical syndrome of persistent pulmonary hypertension of the newborn results from failure of the normal perinatal vascular adaptation, and functionally is characterized by persistent right to left shunting of blood through the foramen ovale and ductus arteriosus. Exposure of the fetus to drugs that inhibit prostaglandin synthesis and cause closure of the ductus arteriosus has been suggested as one cause of persistent pulmonary hypertension of the newborn. We attempted to produce a functional and structural model of persistent pulmonary hypertension of the newborn by administration of indomethacin, a cyclooxygenase inhibitor, to pregnant guinea pigs. Five pregnant guinea pigs received 3.5 mg/kg indomethacin intravenously twice each day for the 12 to 19 days before delivery and seven controls received saline. Hemodynamie studies were performed in eight “treated” newborns and in 12 controls. After sacrifice, the ductus was ligated and, for morphomelrie studies, the pulmonary arteries were distended with barium/gelatin. The treated animals did not show the intraacinar structural or hemodynamie changes of persistent pulmonary hypertension of the newborn. It seems that the indomethacin did cross the placenta because lung structure was modified. The radial alveolar count and alveolar/artery ratio were increased and the preacinar arteries dilated, with more increase in muscle mass. This could be explained by increased pulmonary blood flow because of ductal constriction but direct effect of indomethacin cannot be excluded.

Left ventricular hypertrophy produced by supravalvar aortic banding in infant rats was studied by proton magnetic resonance spectroscopy. Weight gain at 11 weeks of age in the 11 male Sprague-Dawley rats with aortic bands placed at three weeks was similar to that of the 14 controls. The left ventricle of banded rats hypertrophied, increasing the ratio of left ventricle plus septum to body weight (LVSBW) by more than 50 (P<.00001). Right ventricular weight (RVBW) increased slightly (P<.03). T1 and T2 relaxation times of LVS, RV, and thigh muscle (Th) from the banded and control rats were compared. The T2 value distinguished hypertrophied from control LVS (P< .003), but not between RV or Th from the two groups. For banded rats only, the T2 value distinguished each muscle type LVS from RV, LVS from Th, and RV from Th (P<.00001 for each). For control rats, cardiac muscle was distinguished from Th (P<.00001), but LVS and RV were similar. The T1 value did not distinguish either the banded from the control group or any of the muscle types. Percent water content was similar for all tissues. Any correlation between water content and T1 or T2 was inconsistent or weak.

ABSTRACT. The clinical syndrome of persistent pulmonary hypertension of the newborn results from failure of the normal perinatal vascular adaptation, and functionally is characterized by persistent right to left shunting of blood through the foramen ovale and ductus arteriosus. Exposure of the fetus to drugs that inhibit prostaglandin synthesis and cause closure of the ductus arteriosus has been suggested as one cause of persistent pulmonary hypertension of the newborn. We attempted to produce a functional and structural model of persistent pulmonary hypertension of the newborn by administration of indomethacin, a cyclooxygenase inhibitor, to pregnant guinea pigs. Five pregnant guinea pigs received 3.5 mg/kg indomethacin intravenously twice each day for the 12 to 19 days before delivery and seven controls received saline. Hemodynamie studies were performed in eight “treated” newborns and in 12 controls. After sacrifice, the ductus was ligated and, for morphomelrie studies, the pulmonary arteries were distended with barium/gelatin. The treated animals did not show the intraacinar structural or hemodynamie changes of persistent pulmonary hypertension of the newborn. It seems that the indomethacin did cross the placenta because lung structure was modified. The radial alveolar count and alveolar/artery ratio were increased and the preacinar arteries dilated, with more increase in muscle mass. This could be explained by increased pulmonary blood flow because of ductal constriction but direct effect of indomethacin cannot be excluded.

Objectives. This study was designed to describe the hemodynamic abnormalities associated with the appearance of left ventricular diastolic collapse in the setting of regional left heart cardiac tamponade.

The atheroprotective effects of estrogen in women are well recognized1, but the underlying mechanisms responsible are not well understood. Blood vessel cells express the classic estrogen receptor, ERα (ref. 2—6), and are directly affected by estrogen, which inhibits the development of atherosclerotic and injury-induced vascular lesions78. We have generated mice in which the ERα gene is disrupted9and have used a mouse model of carotid arterial injury to compare the effects of estrogen on wild-type and estrogen receptor-deficient mice. Increases in vascular medial area and smooth muscle cell proliferation were quantified following vascular injury in ovariectomized mice treated with vehicle or with physiologic levels of 17β-estradiol. Suprisingly, in both wild-type and estrogen receptor-deficient mice, 17β-estradiol markedly inhibited to the same degree all measures of vascular injury. These data demonstrate that estrogen inhibits vascular injury by a novel mechanism that is independent of the classic estrogen receptor, ERα.

The extensive homology between apolipoprotein(a) and plasminogen has led to the hypothesis that the increased risk for atherosclerosis, cardiac disease and stroke associated with elevated levels of apolipoprotein(a) may reflect modulation of fibrinolysis. We have investigated the role of apolipoprotein(a) on clot lysis in transgenic mice expressing the human apolipoprotein(a) gene. These mice develop fatty streak lesions resembling early lesions of human atherosclerosis. Pulmonary emboli were generated in mice by injection, through the right jugular vein, of a human platelet-rich plasma clot radiolabelled with technetium-99m-labelled antifibrin antibodies. Tissue plasminogen activator was introduced continuously via the right jugular vein. Clot lysis, determined by ex vivoimaging, was depressed in mice carrying the apolipoprotein(a) transgene relative to their sex-matched normal littermates. These results directly demonstrate an in vivoeffect of apolipoprotein(a) on fibrinolysis, an effect that may contribute to the pathology associated with elevated levels of this protein.

Genetically-manipulated mice harboring an α-myosin heavy chain Arg403Gln missense mutation (α-MHC 403/+ ) display a phenotype characteristic of familial hypertrophic cardiomyopathy (FHC). Male and female (30±8 week old) heterozygous α-MHC 403/+ mice and littermate controls were evaluated using a surface electrocardiogram (ECG) and an in vivo cardiac electrophysiology study (EPS). Wild type animals had normal intracardiac electrophysiology, with no significant differences between male and female control mice during EPS. The female wild-type mice did have slower heart rates and longer ECG intervals than their male wild-type counterparts. The female α-MHC 403/+ mice had similar ECG's, cardiac conduction times, and refractory periods compared with female wild-type mice. In contrast, male FHC mice had distinctive ECG and electrophysiologic abnormalities including right axis deviation, prolonged ventricular repolarization and prolonged sinus node recovery times. During programmed ventricular stimulation, 62% of male α-MHC 403/+ mice and 28% of female α-MHC 403/+ mice had inducible ventricular tachycardia. These studies identify gender-specific electrophysiologic abnormalities in α-MHC 403/+ FHC mice, concordant with the histological and hemodynamic derangements previously reported.

Using 111In-labeled autologous platelets, we studied the kinetics of pulmonary platelet deposition and clearance in relation to hemodynamic and structural events during thrombin-induced pulmonary microembolism in rabbits. Autologous platelets were radiolabeled and returned to animals prior to infusion of thrombin (100 units/kg over 15 min) (n = 20) or saline (n = 6). All animals were pretreated with tranexamic add, an inhibitor of fibrinolysis. Thrombin-treated animals manifested progressive increases in mean pulmonary platelet activity, reaching a maximum of 38% above baseline (p < .0001), whereas no change was observed in saline-treated controls. Animals that died during, or immediately following, thrombin infusion manifested significantly greater increases in pulmonary platelet uptake (mean 1.55 ± 0.47 times baseline), compared to surviving animals (1.14 ± 0.16; p < .05 survivors vs. nonsurvivors). In surviving animals, following cessation of thrombin, pulmonary platelet activity cleared gradually, with a half-time of approximately 12 min. Thrombin reduced circulating platelet counts (p < .001), increased mean pulmonary artery pressure (13 ± 3 mm Hg to 18 ± 6 mm Hg; p < .0001), and reduced mean systemic arterial pressure (55 ± 10 mm Hg to 44 ± 7 mm Hg; p < .001). The time courses of these events approximated that of thrombin-induced pulmonary platelet uptake. Furthermore, the increase in pulmonary artery pressure occurred predominantly in the group of animals in which the increase in pulmonary radiolabeled platelet activity exceeded the median value of 20%. Postmortem histology showed extensive pulmonary thrombus extending from small arterial to capillary levels in animals that died during, or immediately following, thrombin infusion, but not in surviving animals. Our findings suggest that platelet aggregation

There is mechanistic rationale to suggest differential effects of angiotensin-converting enzyme (ACE) inhibition and angiotensin II type 1 (AT1)-receptor antagonism on ventricular remodeling after myocardial infarction (MI). We compared the effects of ACE inhibition, AT1-receptor antagonism, and their combination on post-MI ventricular remodeling in rats. We induced MI in 62 rats, which then received one of four treatments: (a) placebo; (b) the ACE inhibitor, enalapril; (c) the AT1-receptor antagonist, losartan; and (d) enalapril and losartan in combination. Two weeks after MI, we examined: (a) heart weight (HW)/body weight (BW) ratio; (b) nonmyocyte cellular proliferation in the noninfarct zone by using proliferating cell nuclear antigen staining; and (c) collagen content within the noninfarct zone. Placebo-treated, infarcted rats developed significant increases in HW/BW ratio (p < 0.001), left ventricular (LV) volume (p < 0.01), nonmyocyte cellular proliferation (p < 0.04), and collagen content (p < 0.01) compared with noninfarcted controls. Enalapril, losartan, and combination therapy limited the increase in HW/BW ratio (all p values <0.01 vs. placebo). Enalapril inhibited nonmyocyte proliferation (p < 0.01 vs. placebo), whereas losartan had a smaller effect (p = NS vs. placebo; p < 0.03 vs. enalapril); combined treatment also reduced nonmyocyte cellular proliferation but did not reach statistical significance (p = 0.08 vs. placebo). Enalapril and combination treatment significantly diminished collagen content (both p values <0.01 vs. placebo), whereas losartan did not. Thus, ACE inhibition and AT1-receptor antagonism equally limited myocardial hypertrophy after MI in rats, but ACE inhibition more effectively prevented nonmyocyte cellular proliferation and collagen deposition in the noninfarcted myocardium. Combination therapy was no more effective than was ACE inhibition alone. These data suggest that the myocyte hypertrophic response after MI is strongly influenced by activation of the AT1receptor, whereas nonmyocyte cellular proliferation and collagen deposition result, in part, from mechanisms separate from AT1-receptor activation.

ABSTRACTDespite the existing association of gut dysbiosis and T cell inflammation in heart failure (HF), whether and how gut microbes contribute to T cell immune responses, cardiac fibrosis and dysfunction in HF remains largely unexplored. Our objective was to investigate whether gut dysbiosis is induced by cardiac pressure overload, and its effect in T cell activation, adverse cardiac remodeling, and cardiac dysfunction.We used 16S rRNA sequencing of fecal samples and discovered that cardiac pressure overload-induced by transverse aortic constriction (TAC) results in gut dysbiosis, characterized by a reduction of tryptophan and short-chain fatty acids producing bacteria in WT mice, but not in T cell-deficient mice (Tcra−/-) mice. These changes did not result in T cell activation in the gut or gut barrier disruption. Strikingly, microbiota depletion in WT mice resulted in decreased heart T cell infiltration, decreased cardiac fibrosis, and protection from systolic dysfunction in response to TAC. Spontaneous reconstitution of the microbiota partially reversed these effects. We observed decreased cardiac expression of the Aryl hydrocarbon receptor (AhR) and enzymes associated with tryptophan metabolism in WT mice, but not in Tcra−/-mice, or in mice depleted of the microbiota.These findings demonstrate that cardiac pressure overload induced gut dysbiosis and T cell immune responses contribute to adverse cardiac remodeling, and identify the potential contribution of tryptophan metabolites and the AhR to protection from adverse cardiac remodeling and systolic dysfunction in HF.

To determine the effect of age and sex on the rise in pulmonary artery(PA)pressure(P)and on morphologic changes in the PV bed in response to chronic hypoxia, 12 Sprague-Dawley infant rats (I-rats)and 11 adult rats(A-rats)were placed in hypobaria(air at 380 mmHg)for 1 month. The I-rats, 8 male(M)and 3 female(F)were 8 days old(mean weight=10 grams); the A-rats, 7M and 4F, 3 months old (mean weight=280 grams). After exposure, indwelling PA and aortic(Ao)catheters were inserted under pentobarbitol anesthesia and the next day unanesthetized pressure measurements were recorded. The rats were sacrificed and their lungs analysed(after injecting the PA tree with barium-gelatin and the parenchyma with formalin)by 3 structural features: 1)extension of muscle into small arteries(EMSA), 2)percentage wall thickness of peripheral arteries(%WT)and 3)alveolar artery ratio(A/A). A rise in mean PAP to 51±5.1(compared with 16.6±1.1 in C-rats)observed in I-rats was similar in AM-rats, but mean PAP in AF-rats was lower(29.6± 2.1, p < 0.03); AoP was unchanged in all. Increased EMSA, %WT and A/A were found in I-rats and A-rats compared with C-rats(p<0.005); however AF-rats compared with AM-rats showed less increase in %WT(p<0.05). Thus, after chronic hypoxic exposure, adult female rats had developed milder PA hypertension associated with less medial hypertrophy.

Secause microwave (MW) energy may achieve high temperatures (TEMP), TEMP monitoring may be important to prevent excessive endocardial damage. Since MW delivery is radial, we examined the value of TEMP monitoring using 3 circumferentially placed thermocouples (THERMO) rather than a single TEMP monitoring. During 15 energy deliveries of 20–40 Watts for 30sec at separate left ventricular sites in 5 dogs, there was a significant difference in maximum TEMP recorded among the 3 THERMO: 93.6±25.32 vs. 68.73±18.28 degrees C (p&lt;0.001). Mean TEMP curves are shown: