EPA, not DHA, prevents fibrosis in pressure overload-induced heart failure: potential role of free fatty acid receptor 4.

Heart failure with preserved ejection fraction (HFpEF) is half of all HF, but standard HF therapies are ineffective. Diastolic dysfunction, often secondary to interstitial fibrosis, is common in HFpEF. Previously, we found that supra-physiologic levels of ω3-PUFAs produced by 12 weeks of ω3-dietary supplementation prevented fibrosis and contractile dysfunction following pressure overload [transverse aortic constriction (TAC)], a model that resembles aspects of remodeling in HFpEF. This raised several questions regarding ω3-concentration-dependent cardioprotection, the specific role of EPA and DHA, and the relationship between prevention of fibrosis and contractile dysfunction. To achieve more clinically relevant ω3-levels and test individual ω3-PUFAs, we shortened the ω3-diet regimen and used EPA- and DHA-specific diets to examine remodeling following TAC. The shorter diet regimen produced ω3-PUFA levels closer to Western clinics. Further, EPA, but not DHA, prevented fibrosis following TAC. However, neither ω3-PUFA prevented contractile dysfunction, perhaps due to reduced uptake of ω3-PUFA. Interestingly, EPA did not accumulate in cardiac fibroblasts. However, FFA receptor 4, a G protein-coupled receptor for ω3-PUFAs, was sufficient and required to block transforming growth factor ß1-fibrotic signaling in cultured cardiac fibroblasts, suggesting a novel mechanism for EPA. In summary, EPA-mediated prevention of fibrosis could represent a novel therapy for HFpEF.

Longstanding hyperthyroidism is associated with normal or enhanced intrinsic cardiomyocyte function despite decline in global cardiac function.

Thyroid hormones (THs) play a pivotal role in cardiac homeostasis. TH imbalances alter cardiac performance and ultimately cause cardiac dysfunction. Although short-term hyperthyroidism typically leads to heightened left ventricular (LV) contractility and improved hemodynamic parameters, chronic hyperthyroidism is associated with deleterious cardiac consequences including increased risk of arrhythmia, impaired cardiac reserve and exercise capacity, myocardial remodeling, and occasionally heart failure. To evaluate the long-term consequences of chronic hyperthyroidism on LV remodeling and function, we examined LV isolated myocyte function, chamber function, and whole tissue remodeling in a hamster model. Three-month-old F1b hamsters were randomized to control or 10 months TH treatment (0.1% grade I desiccated TH). LV chamber remodeling and function was assessed by echocardiography at 1, 2, 4, 6, 8, and 10 months of treatment. After 10 months, terminal cardiac function was assessed by echocardiography and LV hemodynamics. Hyperthyroid hamsters exhibited significant cardiac hypertrophy and deleterious cardiac remodeling characterized by myocyte lengthening, chamber dilatation, decreased relative wall thickness, increased wall stress, and increased LV interstitial fibrotic deposition. Importantly, hyperthyroid hamsters demonstrated significant LV systolic and diastolic dysfunction. Despite the aforementioned remodeling and global cardiac decline, individual isolated cardiac myocytes from chronically hyperthyroid hamsters had enhanced function when compared with myocytes from untreated age-matched controls. Thus, it appears that long-term hyperthyroidism may impair global LV function, at least in part by increasing interstitial ventricular fibrosis, in spite of normal or enhanced intrinsic cardiomyocyte function.

Post-myocardial infarction left ventricular myocyte remodeling: are there gender differences in rats?

BACKGROUND: Previous studies have shown gender differences in left ventricular remodeling after myocardial infarction. Results are varied, however, and reliable, comprehensive data for changes in cardiac myocyte shape are not available. METHODS: Young adult female and male Sprague-Dawley rats were used in this study and randomly assigned to the myocardial infarction and sham myocardial infarction groups. Myocardial infarction was produced by ligation of the left descending coronary artery. Four weeks after surgery, left ventricular echocardiography and hemodynamics were performed before isolating myocytes for size determination. RESULTS: In general, left ventricular functional changes after myocardial infarction were comparable. Females developed slightly, but significantly, more left ventricular hypertrophy than males, and this was reflected by the relative increases in left ventricular myocyte volume. In both males and females, however, myocyte hypertrophy was due exclusively to lengthening of myocytes with no change in myocyte cross-sectional area. CONCLUSIONS: This study demonstrates that post-myocardial infarction changes in LV function and myocyte remodeling are remarkably similar in young adult male and female rats.

Changes in left ventricular function and remodeling after myocardial infarction in hypothyroid rats.

It has been shown that hypothyroidism may lead to delayed wound healing after experimental myocardial infarction (MI) in rats and increased infarct size in dogs. However, the long-term effect of hypothyroidism on left ventricular (LV) remodeling after MI has not been determined. Adult female Sprague-Dawley rats with and without surgical thyroidectomy (TX) were used in the study. Four weeks after TX, MI or sham MI was performed on TX and non-TX rats. Rats from all groups were examined 4 wk later. Four weeks after TX, hypothyroid-induced LV dysfunction was confirmed by echocardiography. In terminal experiments 4 wk after MI, TX sham-MI rats showed smaller hearts and impaired LV function compared with non-TX sham-MI controls. TX + MI rats showed smaller hearts with bigger infarct areas, higher LV end-diastolic pressures, and greater impairment of relaxation (-dP/dt) compared with non-TX MI rats. Relative changes after MI between TX and non-TX rats for most other hemodynamic and echocardiographic indexes were similar. These results suggest that preexisting hypothyroidism exaggerates post-MI remodeling and worsens LV function, particularly diastolic function.

Regulation of arteriolar density in adult myocardium during low thyroid conditions.

Low thyroid function induced by either propylthiouracil (PTU) treatment or thyroidectomy surgery led to a reduction of arteriolar density in adult rat myocardium, which can be prevented by treatment with thyroxine or the thyroid hormone analogue 3,5-diiodothyropropionic acid (DITPA). However, many questions related to pathophysiological changes and the regulation of arteriolar density in the heart due to hypothyroidism remain unanswered. Sprague-Dawley rats were treated with PTU in drinking water for 1, 3, and 6weeks, or co-treated with the vasodilator dipyridamole and PTU for 6weeks, or treated with PTU for 6weeks and treatment discontinued for 2 or 4weeks. Heart mass, body mass, cardiac function and myocardial arteriolar density were determined. Arteriolar loss in hypothyroidism induced by PTU treatment progressed gradually with a 22% reduction after 3weeks treatment and 34% by 6weeks which was largely reversed after discontinuing PTU treatment for only 2weeks. Combined treatment with the vasodilator dipyridamole during the 6-week PTU treatment period prevented vessel loss indicating the mechanism of arteriolar loss from hypothyroidism may result from vasoconstriction. These results suggest that thyroid hormone is a powerful regulator of vasculature in adult myocardium, particularly in low thyroid states.

Thyroid hormone analog 3,5-diiodothyropropionic acid promotes healthy vasculature in the adult myocardium independent of thyroid effects on cardiac function.

Patients with hypothyroidism are at a higher risk for coronary vascular disease. Patients with diabetes and related vascular complications also have an increased incidence of low thyroid function. While thyroid hormones (THs) may be key regulators of a healthy vasculature, potential undesirable side effects hinder their use in the treatment of vascular disorders. TH analogs such as 3,5-diiodothyropropionic acid (DITPA) may provide a safer treatment option. However, the relative potency of DITPA on vascular growth, cardiac function, and metabolism is poorly understood. We hypothesized that the vascular growth-promoting effects of DITPA can be obtained with a minimum effect on cardiac function. Thyroidectomized Sprague-Dawley rats were given slow-release pellets with either thyroxine (T4, 2.7 or 5.2 mg) or DITPA (80 mg) for 6 wk and were compared with placebo. Heart mass, body mass, body temperature, serum THs, cardiac function (echocardiograms and hemodynamics), and myocardial arteriolar density were determined. Hypothyroidism led to reductions in cardiac function, heart mass, body temperature, and myocardial arterioles. High-dose T4 prevented arteriolar loss and the development of hypothyroidism. Low-dose T4 partially prevented the reduction in cardiac function but had minimal effects on arteriolar loss. In contrast, DITPA treatment prevented myocardial arteriolar loss but not the progression of hypothyroid-induced changes in cardiac function. The results suggested that DITPA can promote a healthy vasculature independently from its thyroid-related metabolic effects. Drugs in this class may provide new therapeutic options for patients with vascular disease.

Effect of ovariectomy on blood pressure and venous tone in female spontaneously hypertensive rats.

BACKGROUND: Venous capacitance plays an important role in circulatory homeostasis. A number of reports have suggested an effect of estrogen on venous function. This study tested the hypothesis that ovariectomy would increase venous tone in the female spontaneously hypertensive rat (SHR) via autonomic mechanisms. METHODS: Five-week-old female SHR were subjected to sham operation (Sham) or ovariectomy (OVX). At 10 weeks of age, the rats were instrumented for the measurement of arterial and venous pressure. A balloon catheter was advanced into the right atrium. Mean circulatory filling pressure (MCFP), an index of venous tone, was calculated. Mean arterial pressure (MAP), heart rate (HR), and MCFP were recorded from conscious rats. Postsynaptic adrenergic responsiveness was assessed by constructing cumulative dose-response curves to norepinephrine (NE). RESULTS: MAP was not significantly affected by ovariectomy (Sham 127 +/- 6 mm Hg vs. OVX 130 +/- 3 mm Hg). HR also was not different between groups (Sham 409 +/- 11 bpm vs. OVX 399 +/- 12 bpm). Conversely, MCFP was significantly, but moderately, increased in OVX SHR (Sham 5.2 +/- 0.2 mm Hg vs. OVX 5.9 +/- 0.2 mm Hg). Ganglionic blockade produced marked decreases in MAP, HR, and MCFP in both groups; however, the responses were not different between groups. Infusion of NE caused dose-dependent increases in MAP and MCFP. There were no statistically significant differences in these responses between Sham and OVX SHR. CONCLUSION: Endogenous ovarian hormones effect a small reduction in MCFP. This effect does not appear to be mediated by adrenergic mechanisms.

Effects of thyroidectomy, T4, and DITPA replacement on brain blood vessel density in adult rats.

In hypothyroid patients, altered microvascular structure and function may affect mood and cognitive function. We hypothesized that adult male hypothyroid rats will have significantly lower forebrain blood vessel densities (BVD) than euthyroid rats and that treatment with 3,5-diiothyroprionic acid (DITPA) (a thyroid hormone analog) or thyroxine (T(4)) will normalize BVDs. The euthyroid group received no thyroidectomy or treatment. The other three groups received thyroidectomies and pellets. The hypothyroid group received a placebo pellet, the DITPA group received an 80-mg DITPA-containing pellet, and the T(4) group received a 5.2-mg T(4) slow-release pellet for 6 wk. Body weights, cardiac function, and body temperatures were measured. A monoclonal antiplatelet endothelial cell adhesion antibody was used to visualize blood vessels. The euthyroid group averaged body weights of 548 +/- 54 g, while the hypothyroid group averaged a body weight of 332 +/- 19 g (P value < 0.001). Relative to the euthyroid group, the DITPA-treated group was significantly lighter (P value < 0.05), while the T(4)-treated group was comparable in body weight to the euthyroid group. The same trends were seen with body temperature and cardiac function with the largest difference between the euthyroid and hypothyroid groups. BVD in the euthyroid group was 147 +/- 12 blood vessels/mm(2) and in hypothyroid group 69 +/- 5 blood vessels/mm(2) (P = 0.013) but similar among the euthyroid, DITPA, and T(4) groups. These results show that hypothyroidism decreased BVD in adult rat forebrain regions. Moreover, DITPA and T(4) were efficacious in preventing effects of hypothyroidism on cardiac function and BVD.

Serum thyroid hormone levels may not accurately reflect thyroid tissue levels and cardiac function in mild hypothyroidism.

The link between thyroid dysfunction and cardiovascular diseases has been recognized for more than 100 years. Although overt hypothyroidism leads to impaired cardiac function and possibly heart failure, the cardiovascular consequences of borderline low thyroid function are not clear. Establishment of a suitable animal model would be helpful. In this study, we characterized a rat model to study the relationship between cardiovascular function and graded levels of thyroid activity. We used rats with surgical thyroidectomy and subcutaneous implantation of slow release pellets with three different T(4) doses for 3 wk. In terminal experiments, cardiac function was evaluated by echocardiograms and hemodynamics. Myocardial arteriolar density was also quantified morphometrically. Thyroid hormone levels in serum and heart tissue were determined by RIA assays. Thyroidectomy alone led to cardiac atrophy, severe cardiac dysfunction, and a dramatic loss of arterioles. The low T(4) dose normalized serum T(3) and T(4) levels, but cardiac tissue T(3) and T(4) remained below normal. Low-dose T(4) failed to prevent cardiac atrophy or restore cardiac function and arteriolar density to normal values. All cardiac function parameters and myocardial arteriolar density were normalized with the middle dose of T(4), whereas the high dose produced hyperthyroidism. Our results show that thyroid hormones are important regulators of cardiac function and myocardial arteriolar density. This animal model will be useful in studying the pathophysiological consequences of mild thyroid dysfunction. Results also suggest that cardiac function may provide valuable supplemental information in proper diagnosis of mild thyroid conditions.

Effect of low thyroid function on cardiac structure and function in spontaneously hypertensive heart failure rats.

BACKGROUND: Although low thyroid function is known to have detrimental effects on the cardiovascular system, including microvascular impairment, little is known about the pathophysiologic consequences of hypothyroidism in the background of hypertension. METHODS AND RESULTS: Hypothyroidism was induced in female spontaneously hypertensive heart failure (SHHF) rats by treatment with propylthiouracil (PTU) for 6 months. Untreated SHHF and normotensive Wistar Furth (WF) rats served as controls. In terminal experiments, heart weight, echocardiographic measurements, hemodynamics, and arteriolar morphometry were performed. Left ventricular internal diameter in systole and diastole were increased and wall thickness, ejection fraction, heart rate, systolic blood pressure, and +/-dP/dt were significantly decreased in the treatment group. Surprisingly, there were no observed differences in arteriolar density among the 3 groups. CONCLUSIONS: As expected, PTU treatment of SHHF rats led to systolic dysfunction and chamber dilation. However, PTU treatment did not lead to arteriolar loss as previously observed in normotensive rats treated with PTU. These finding suggest that induced hypothyroidism leads to detrimental changes in SHHF rats, but the overall effects were no worse than those previously observed in normotensive rats treated with PTU.

Short term triiodo-L-thyronine treatment inhibits cardiac myocyte apoptosis in border area after myocardial infarction in rats.

Thyroid hormone (TH) levels decline after a myocardial infarction (MI). Treatment with TH has been shown to improve left ventricular (LV) function in MI and other cardiovascular diseases, but the mechanisms are not clear. We have previously shown that TH can prevent myocyte apoptosis via Akt signaling in cultured neonatal rat cardiomyocytes. In this study, the effects of triiodo-L-thyronine (T3) on LV function and myocyte apoptosis after MI was examined in rats. After surgery, MI rats were treated with T3 for 3 days. Compared with sham-operated rats, MI rats showed significantly increased LV chamber dimension during systole and decreased LV function. T3 treatment increased LV +/-dP/dt but did not change LV chamber dimensions. MI rats also showed significantly increased myocyte apoptosis in the border area as assessed by DNA laddering and TUNEL assay. T3 treatment decreased the amount of DNA laddering, and reduced TUNEL positive myocytes in the border area, which was associated with phosphorylation of Akt at serine 473. These results suggest that T3 can protect myocytes against ischemia-induced apoptosis, which may be mediated by Akt signaling.

Effects of excessive long-term exercise on cardiac function and myocyte remodeling in hypertensive heart failure rats.

The long-term effects of exercise on cardiac function and myocyte remodeling in hypertension/progression of heart failure are poorly understood. We investigated whether exercise can attenuate pathological remodeling under hypertensive conditions. Fifteen female Spontaneously Hypertensive Heart Failure rats and 10 control rats were housed with running wheels beginning at 6 months of age. At 22 months of age, heart function of the trained rats was compared with heart function of age-matched sedentary hypertensive and control rats. Heart function was measured using echocardiography and left ventricular catheterization. Cardiac myocytes were isolated to measure cellular dimensions. Fetal gene expression was determined using Western blots. Exercise did not significantly impact myocyte remodeling or ventricular function in control animals. Sedentary hypertensive rats had significant chamber dilatation and cardiac hypertrophy. In exercised hypertensive rats, however, exercise time was excessive and resulted in a 21% increase in left ventricular diastolic dimension (P<0.001), a 24% increase in heart to body weight ratio (P<0.05), a 27% increase in left ventricular myocyte volume (P<0.01), a 13% reduction in ejection fraction (P<0.001), and a 22% reduction in fractional shortening (P<0.01) compared with sedentary hypertensive rats. Exercise resulted in greater fibrosis and did not prevent activation of the fetal gene program in hypertensive rats. We conclude that excessive exercise, in the untreated hypertensive state can have deleterious effects on cardiac remodeling and may actually accelerate the progression to heart failure.

Castration reduces blood pressure and autonomic venous tone in male spontaneously hypertensive rats.

OBJECTIVE: The development of arterial hypertension is sexually dimorphic. Venous tone is elevated in the spontaneously hypertensive rat model of hypertension. This study tested the hypothesis that endogenous androgens exacerbate venous tone in the developmental stages of spontaneous hypertension. METHODS: Male spontaneously hypertensive rats (SHRs) were subjected to sham operation, castration or castration + testosterone treatment. Ten-week-old SHR rats were instrumented for the measurement of arterial and venous pressure. A balloon catheter was advanced into the right atrium. Mean circulatory filling pressure (MCFP), an index of venous tone, was calculated. Mean arterial pressure (MAP) and MCFP were recorded from conscious rats. Postsynaptic adrenergic responsiveness was assessed by constructing cumulative dose-response curves to norepinephrine (NE). Baseline values and responsiveness to NE were obtained before and after autonomic blockade. RESULTS: MAP and MCFP were significantly reduced in castrated (MAP, 130 +/- 4 mmHg; MCFP, 5.5 +/- 0.2 mmHg) versus sham-operated SHRs (MAP, 149 +/- 5 mmHg; MCFP, 6.7 +/- 0.3 mmHg) or castrated + testosterone-treated SHRs (MAP, 145 +/- 6 mmHg; MCFP, 7.1 +/- 0.4 mmHg). Ganglion blockade abolished these differences in MAP and MCFP. Infusion of NE caused dose-dependent increases in MAP and MCFP. The MAP responses in castrated SHRs were displaced to the right of those for sham and castrated + testosterone-treated SHRs. This was not evident in the venous circulation, where there were no marked differences in the NE dose-MCFP response curves. CONCLUSION: Accordingly we conclude that endogenous male sex steroids contribute to the elevated arterial and venous pressures observed in the SHR.

A method for the extraction of high-quality RNA and protein from single small samples of arteries and veins preserved in RNAlater.

INTRODUCTION: The mesenteric vasculature plays an important role in cardiovascular homeostasis, both in terms of arterial resistance and venous capacitance. However, the limited quantity of tissue available and the activity of RNases and proteases during dissection of the vessels make it difficult to study gene and protein expression in this tissue. METHODS: The mesenteric arcade was harvested from rats. The mesenteric arteries and veins were separated from each other and from contaminating adipose, nerve, and connective tissue by blunt dissection while submerged in RNAlater (Ambion). Total ribonucleic acid (RNA) and protein were extracted from the same mesenteric artery and vein samples. RESULTS: Good quality RNA and protein were obtained by this method. The method preserved the content of proteins including biglycan, adrenergic receptors, estrogen receptors, and protein kinase Cdelta, and also preserved the differential expression of estrogen receptors. DISCUSSION: A method for the isolation of high-quality RNA and protein from the arteries and veins of the same mesenteric sample is described. The content of proteins from a variety of cellular compartments and molecular weights was preserved by this method, as well as differential expression of proteins. This method will facilitate the study of gene and protein expression in the mesenteric vasculature in response to pharmacologic treatments and in disease states.