A novel technique for isolating functional mast cells from the heart.

OBJECTIVE AND DESIGN: The purpose of this study was to determine the feasibility of adapting peritoneal and pleural mast cell isolation techniques to recover cardiac mast cells that retain their functional response to the secretagogue, compound 48/80. METHODS: Using a novel protocol in rats, viable epicardial mast cells were recovered by aspiration of HBSS injected into the pericardial space. Functionality of these cells was determined by ELISA quantification of histamine release in response to compound 48/80, calcium ionophore A23187 and substance P. Mast cell phenotype was determined based on the presence of chymase and tryptase demonstrated by immunofluorescence, alcian blue-safranin staining, and Western blotting. RESULTS: Mast cells isolated in this manner have low basal rates of histamine release and are highly responsive to these secretagogues. These epicardial mast cells were of the connective tissue type, which is consistent with previous reports characterizing cardiac mast cells isolated from the heart by enzymatic dispersion techniques. CONCLUSIONS: This novel pericardial aspiration technique facilitates the straightforward characterization of isolated epicardial mast cell functionality in a controlled in vitro environment, furthering our understanding of their contribution to myocardial disease.

Rat cardiac mast cell maturation and differentiation following acute ventricular volume overload.

OBJECTIVE: Cardiac mast cell numbers increase significantly within 12 h following the creation of an aortocaval (AV) fistula in rats and play a central role in mediating adverse left ventricular remodeling. We studied whether this increase was related to maturation of resident immature mast cells. METHODS: We measured percentages of immature and mature cardiac mast cells at 1, 2 and 7 days following AV-fistula or sham surgery and in non-surgical control rats using the alcian-blue safranin reaction. RESULTS: Relative to sham-operated and control rats, there was a significant shift from immature to a greater percentage of mature cardiac mast cells at 1 day and 2 days post-fistula that returned to a normal distribution by 7 days. CONCLUSIONS: We conclude that the acute increase in mast cell density following volume overload is due to a paracrine response in the heart that stimulates the maturation and differentiation of resident immature cardiac mast cells.

Spontaneous histamine secretion during isolation of rat cardiac mast cells.

OBJECTIVE: To test the hypothesis that spontaneous release of histamine occurring during an isolation protocol may modify responses of rat cardiac mast cells (connective tissue-type mast cells) to secretagogues. METHODS: We assessed two protocols for enzymatic dispersion utilizing collagenase, hyaluronidase, and deoxyribonuclease; with protease (Protocol 1, n = 8) or without protease (Protocol 2, n = 3). Spontaneous release of histamine was quantified following mechanical and enzymatic dispersion of the whole heart. RESULTS: Total histamine loss (Mean +/- SEM) was 963+/-92 and 833+/-60 ng/g of tissue weight following Protocols 1 and 2. Percentages of histamine release from cell isolates following Protocol 1 were 40+/-5%, 41+/-6%, and 51+/-7% at 0, 30, and 300 microg/mL of compound 48/80. CONCLUSIONS: Enzymatic dispersion of cardiac mast cells affects their response to secretagogues.

Effect of a myocardial volume overload on lactate transport in skeletal muscle sarcolemmal vesicles.

This study sought to determine the effect of a myocardial volume overload (MVO) on sarcolemmal (SL) lactate (La(-)) transport and the aerobic profile of skeletal muscle. SL vesicles were obtained from female rats 10 wk after either a MVO was induced by creation of an infrarenal fistula (n = 10), or sham surgeries were performed (n = 11). Influx of (14)C-labeled L(+)-La(-) was measured at various unlabeled La(-) concentrations under zero-trans conditions. La(-) transport kinetics were determined using a Michaelis-Menten equation with an added linear component to discriminate between carrier-mediated and diffusional transport. Although heart and lung weights were significantly increased (P < 0.0001) in the MVO group, left ventricular function was only modestly altered (P < 0.05). A significant reduction in type I myosin heavy chain (MHC) in the soleus and a strong trend (P = 0.06) for a reduced type IIx MHC in the plantaris were observed in MVO rats, but no differences in citrate synthase activity or monocarboxylate transporter proteins (MCT)-1 expression were noted in any muscle. Carrier-mediated La(-) influx into SL vesicles was similar between sham and MVO (K(m) = 12 +/- 1 and 18 +/- 3 mM; apparent V(max) = 772 +/- 99 and 827 +/- 80 nmol. mg(-1). min(-1), respectively). Total influx at 100 mM was lower in MVO, and this was due to a 30% reduction in membrane diffusion. In conclusion, a 10-wk MVO did not alter MCT-mediated La(-) transport or protein expression but was associated with modest changes in myofibrillar proteins and impaired SL diffusive properties.

Contribution of ventricular remodeling to pathogenesis of heart failure in rats.

We previously reported an approximately 50% incidence of rats with symptoms of congestive heart failure (CHF) at 8 wk postinfrarenal aorto-caval fistula. However, it was not clear whether compensatory ventricular remodeling could continue beyond 8 wk or whether the remaining animals would have developed CHF or died. Therefore, the intent of this study was to complete the characterization of this model of sustained volume overload by determining the morbidity and mortality and the temporal response of left ventricular (LV) remodeling and function beyond 8 wk. The findings demonstrate an upper limit to LV hypertrophy and substantial increases in LV volume and compliance, matrix metalloproteinase activity, and collagen volume fraction associated with the development of CHF. There was an 80% incidence of morbidity and mortality following 21 wk of chronic volume overload. These findings indicate that the development of CHF is triggered by marked ventricular dilatation and increased compliance occurring once the myocardial hypertrophic response is exhausted.

Differential expression of natriuretic peptides and their receptors in volume overload cardiac hypertrophy in the rat.

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) upregulation are genetic markers for the chronic hypertrophic phenotype but also have important acute physiologic effects on salt and water balance and blood pressure control. The presence of a dual NP-system led us to hypothesize a differential expression of ANP and BNP in response to an acute hemodynamic stress of volume overload in the left ventricle (LV) and right ventricle (RV). Accordingly, we examined the temporal relationship between the RV and LV expression of ANP and BNP mRNA and NP receptor mRNA levels on days 1, 2, 3, and 7 after induction of aortocaval fistula in the rat. LV end-diastolic pressure was increased 1.5-fold by day 3 and 2.0-fold by day 7 compared to control (P<0.05). LV weight increased by day 7 compared to control (2.34+/-0.04 vs 3.07+/-0.10 mg/g, P<0.05) while RV weight did not change over the 7 days. There was a 7-fold increase of ANP mRNA in LV at day 1, which was sustained through day 7, while LV BNP mRNA levels did not differ from controls over the 7 days. In contrast, RV mRNA transcript levels for ANP and BNP were increased >2-fold by day 2 and this increase was sustained throughout 7 days. NP clearance receptor was decreased by 75% by day 7 in the LV but did not change in the RV. Thus, LV ANP mRNA levels increased before the onset of LV hypertrophy and RV BNP mRNA levels increased in the absence of RV hypertrophy. The disparate response of BNP and the NP clearance receptor transcript levels in the LV and RV may be related to differences in load and/or differential expression of the NP system in the LV and RV in response to acute haemodynamic stress.

Temporal evaluation of left ventricular remodeling and function in rats with chronic volume overload.

The left ventricle (LV) significantly dilates and hypertrophies in response to chronic volume overload. However, the temporal responses in LV mass, volume, and systolic/diastolic function secondary to chronic volume overload induced by an infrarenal arteriovenous (A-V) fistula in rats have not been well characterized. To this end, LV end-diastolic pressure, size, and function (i.e., isovolumetric pressure-volume relationships in the blood-perfused isolated heart) were assessed at 1, 2, 3, 5, and 8 wk post-A-V fistula and compared with age-matched control animals. Progressive hypertrophy (192% at 8 wk), ventricular dilatation (172% at 8 wk), and a decrease in ventricular stiffness (257% at 8 wk) occurred in the fistula groups. LV end-diastolic pressure increased from a control value of 4.2 +/- 3.1 mmHg to a peak value of 15.7 +/- 3.6 mmHg after 3 wk of volume overload. A subsequent decline in LVEDP to 11.0 +/- 6.0 mmHg together with further LV dilation (169%) corresponded to a significant decrease in LV stiffness (222%) at 5 wk post-A-V fistula. Myocardial contractility, as assessed by the isovolumetric pressure-volume relationship, was significantly reduced in all A-V fistula groups; however, the compensatory remodeling induced by 8 wk of chronic biventricular volume overload tended to preserve systolic function.

Catecholamine response to chronic ANG II infusion and its role in myocyte and coronary vascular damage.

Acute elevations in circulating angiotensin II (ANG II) are known to increase circulating norepinephrine (NE) levels. However, the time course of catecholamine release relative to chronic ANG II infusion is not known. Furthermore, it is unknown if this ANG II-induced catecholamine release is ANG II type 1 (AT1) receptor mediated or whether the increase in serum catecholamines is responsible for the myocyte and coronary vascular damage seen within the first 3 days of chronic ANG II infusion. Therefore, we examined the influence of chronic ANG II stimulation on serum catecholamine levels with and without AT1 blockade and the effect of beta-blockade on ANG II-induced myocyte and coronary vascular damage. The results indicate that NE release is AT1 mediated, but NE is not significantly elevated until day 4 of ANG II infusion after which it remains elevated. beta-Blockade prevented most ANG II-related myocyte necrosis and coronary vascular damage. Therefore, myocyte and coronary vascular damage do not appear to be related to increased serum NE levels, but instead may be due to the release of neural catecholamines within the heart.

Ventricular remodeling in heart failure: the role of myocardial collagen.

Collagen which is present in the myocardium in relatively small amounts is the most abundant structural protein of the connective tissue network. Its structural organization consists of a complex weave of collagen fibers that surrounds and interconnects myocytes, groups of myocytes, muscle fibers and muscle bundles. The conformation of interstitial fibrillar collagen makes it highly resistant to degradation by all proteinases other than specific collagenases. In hearts with myocardial damage secondary to myocardial infarction, chronic ischemia, inflammation, or cardiomyopathy, a complex sequence of compensatory events occur that eventually result in an adverse left ventricular remodeling. This continual state of remodeling is characterized by persistent collagenase activity, fibrillar collagen degradation, and progressive myocyte loss. The net effect is a shift in the balance between collagen synthesis and degradation which leads to an inadequate fibrillar collagen matrix, progressive ventricular dilatation and sphericalization with wall thinning and eventual congestive heart failure.