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.

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.

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.

Thyroid hormone analog, diiodothyropropionic acid (DITPA), exerts beneficial effects on chamber and cellular remodeling in cardiomyopathic hamsters.

Diiodothyropropionic acid (DITPA) is a thyroid hormone analog that is currently in phase II clinical trials. However, there have not been any studies to comprehensively analyze its effect on myocyte morphology. In addition, long-term studies with DITPA have not been done. This study compares the effects of DITPA with L-thyroxine (T4) on chamber remodeling, cardiac function, cellular morphology, cardiac blood flow, and protein expression. Normal and cardiomyopathic hamsters were treated with T4 or DITPA for 2 months. At the end of the treatment, echos, hemodynamics, coronary blood flow, cell morphology, and protein expression data were collected. Both T4 and DITPA treatment reduced chamber diameter during diastole, suggesting attenuated chamber dilatation in cardiomyopathic hamsters. Wall thickness also tended to increase, which was supported by cell morphology data in which DITPA significantly increased cross-sectional growth of myocytes specifically in the minor dimension, which is oriented transmurally. T4 and DITPA also increased myocardial blood flow both at baseline and after maximal dilation. This suggests there was increased angiogenesis or reduced loss of arterioles. Both T4 and DITPA had beneficial effects on chamber remodeling, which was most likely due to beneficial changes in cell shape and improved vascular supply.

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.

A method to collect isolated myocytes and whole tissue from the same heart.

A technique for isolation of cardiac myocytes and collection of whole heart tissue from individual hearts of adult rats is described in this study. After excision of the apical half of the left ventricle (LV) and cauterization of the cut edge, aortas were cannulated and high-quality isolated cardiac myocytes were collected after collagenase perfusion of the basal portion. Myocyte dimensions from the basal portion of cauterized and noncauterized hearts from matching rats were identical. Additionally, myocyte dimensions from the basal and apical halves of the LV were compared with the use of whole heart-isolated myocyte preps. No regional differences between basal and apical LV myocyte size were found. Therefore, this cauterization method can be used to collect isolated myocytes from the basal half and whole heart tissue from the apical half, with each half being representative of the other with respect to myocyte dimensions.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection spreads by cell-to-cell transfer in cultured MARC-145 cells, is dependent on an intact cytoskeleton, and is suppressed by drug-targeting of cell permissiveness to virus infection.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of PRRS, causing widespread chronic infections which are largely uncontrolled by currently available vaccines or other antiviral measures. Cultured monkey kidney (MARC-145) cells provide an important tool for the study of PRRSV replication. For the present study, flow cytometric and fluorescence antibody (FA) analyses of PRRSV infection of cultured MARC-145 cells were carried out in experiments designed to clarify viral dynamics and the mechanism of viral spread. The roles of viral permissiveness and the cytoskeleton in PRRSV infection and transmission were examined in conjunction with antiviral and cytotoxic drugs. RESULTS: Flow cytometric and FA analyses of PRRSV antigen expression revealed distinct primary and secondary phases of MARC-145 cell infection. PRRSV antigen was randomly expressed in a few percent of cells during the primary phase of infection (up to about 20-22 h p.i.), but the logarithmic infection phase (days 2-3 p.i.), was characterized by secondary spread to clusters of infected cells. The formation of secondary clusters of PRRSV-infected cells preceded the development of CPE in MARC-145 cells, and both primary and secondary PRRSV infection were inhibited by colchicine and cytochalasin D, demonstrating a critical role of the cytoskeleton in viral permissiveness as well as cell-to-cell transmission from a subpopulation of cells permissive for free virus to secondary targets. Cellular expression of actin also appeared to correlate with PRRSV resistance, suggesting a second role of the actin cytoskeleton as a potential barrier to cell-to-cell transmission. PRRSV infection and cell-to-cell transmission were efficiently suppressed by interferon-gamma (IFN-gamma), as well as the more-potent experimental antiviral agent AK-2. CONCLUSION: The results demonstrate two distinct mechanisms of PRRSV infection: primary infection of a relatively small subpopulation of innately PRRSV-permissive cells, and secondary cell-to-cell transmission to contiguous cells which appear non-permissive to free virus. The results also indicate that an intact cytoskeleton is critical for PRRSV infection, and that viral permissiveness is a highly efficient drug target to control PRRSV infection. The data from this experimental system have important implications for the mechanisms of PRRSV persistence and pathology, as well as for a better understanding of arterivirus regulation.

Low thyroid function leads to cardiac atrophy with chamber dilatation, impaired myocardial blood flow, loss of arterioles, and severe systolic dysfunction.

BACKGROUND: Although thyroid dysfunction has been linked to heart failure, it is not clear whether hypothyroidism alone can cause heart failure. METHODS AND RESULTS: Hypothyroidism was induced in adult rats by treatment with 0.025% propylthiouracil (PTU) for 6 weeks (PTU-S) and 1 year (PTU-L). Echocardiographic measurements, left ventricular (LV) hemodynamics, isolated myocyte length (KOH method), myocardial blood flow (fluorescent microspheres), arteriolar morphometry, and gene expression (Western blot) were determined. Heart weight, heart rate, LV systolic blood pressure, LV ejection fraction, LV fractional shortening, and systolic wall thickness were reduced in PTU-S and PTU-L rats. LV internal diameter in systole increased by 40% in PTU-S and 86% in PTU-L. LV internal dimension in diastole was increased in PTU-S and PTU-L rats, but only PTU-L rats showed a significant increase in myocyte length due to series sarcomere addition. Resting and maximum (adenosine) myocardial blood flow were reduced in both PTU-S and PTU-L rats. Impaired blood flow was due to a large reduction in arteriolar length density and small arterioles in PTU-S and PTU-L (P<0.05 or greater for all of the above comparisons). Expression of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)-2a and alpha-myosin heavy chain were reduced in hypothyroidism, whereas phospholamban and beta-myosin heavy chain were increased. CONCLUSIONS: Hypothyroidism led to severe, progressive systolic dysfunction and increased chamber diameter/wall thickness ratio despite a reduction in cardiac mass. Chamber dilatation in PTU-L rats was due to series sarcomere addition, typical of heart failure. Hypothyroidism resulted in impaired myocardial blood flow due to a dramatic loss of arterioles. Thus, we have identified 2 important new mechanisms by which low thyroid function may lead to heart failure.

Treatment of subclinical hypothyroidism reverses ischemia and prevents myocyte loss and progressive LV dysfunction in hamsters with dilated cardiomyopathy.

Growing evidence suggests that thyroid dysfunction may contribute to progression of cardiac disease to heart failure. We investigated the effects of a therapeutic dose of thyroid hormones (TH) on cardiomyopathic (CM) hamsters from 4 to 6 mo of age. CM hamsters had subclinical hypothyroidism (normal thyroxine, elevated TSH). Left ventricular (LV) function was determined by echocardiography and hemodynamics. Whole tissue pathology and isolated myocyte size and number were assessed. TH treatment prevented the decline in heart rate and rate of LV pressure increase and improved LV ejection fraction. The percentage of fibrosis/necrosis in untreated 4-mo-old CM (4CM; 15.5 +/- 2.2%) and 6-mo-old CM (6CM; 21.5 +/- 2.4%) hamsters was pronounced and was reversed in treated CM (TCM; 11.9 +/- 0.9%) hamsters. Total ventricular myocyte number was the same between 4- and 6-mo-old controls but was reduced by 30% in 4CM and 43% in 6CM hamsters. TH treatment completely prevented further loss of myocytes in TCM hamsters. Compared with age-matched controls, resting and maximum coronary blood flow was impaired in 4CM and 6CM hamsters. Blood flow was completely normalized by TH treatment. We conclude that TH treatment of CM hamsters with subclinical hypothyroidism normalized impaired coronary blood flow, which prevented the decline in LV function and loss of myocytes.

Effects of induced hyperthyroidism in normal and cardiomyopathic hamsters.

Thyroid hormones (TH) enhance cardiac function and reverse gene changes typical of pathological hypertrophy. However, reports in humans, but not animals, indicate that excess TH can cause heart failure. Also, the effects of TH on normal and cardiomyopathic hearts are likely to be different. The goal of this study was to characterize the effects of prolonged hyperthyroidism on cardiac function, chamber and cellular remodeling, and protein expression in both normal and cardiomyopathic hearts. Hyperthyroidism was induced in 3-mo-old normal BIO F1B and dilated cardiomyopathic BIO TO2 hamsters. After TH treatment for 10 days and 2 mo, hemodynamics, echos, myocyte length, histology, and protein expression were assessed. After 10 days and 2 mo, there were no differences between TO2-treated (Tx) and TO2-untreated (Untx) hamsters in chamber diameters or left ventricular function. After 2 mo of treatment, however, F1B-Tx showed evidence of dilated heart failure vs. F1B-Untx. Chamber diameters were increased, and ejection fraction and positive and negative changes in pressure over time were reduced. In F1B-Tx and TO2-Tx hamsters, beta-myosin isoform expression was reduced, whereas alpha-myosin increased significantly in F1B-Tx only. In TO2-Tx hamsters, the percent of viable myocardium was increased, and percent fibronecrosis was reduced vs. TO2-Untx. Myocyte length increased with TH treatment in both hamster strains. We conclude that 1) excess TH can induce heart failure in normal animals as observed in humans, 2) reversal of myosin heavy chain expression does not necessarily improve heart function, and 3) excess TH altered cellular remodeling but did not adversely affect chamber function or dimensions in TO2 hamsters.

Protein kinase C isozymes in hypertension and hypertrophy: insight from SHHF rat hearts.

Chronic hypertension results in cardiac hypertrophy and may lead to congestive heart failure. The protein kinase C (PKC) family has been identified as a signaling component promoting cardiac hypertrophy. We hypothesized that PKC activation may play a role mediating hypertrophy in the spontaneously hypertensive heart failure (SHHF) rat heart. Six-month-old SHHF and normotensive control Wistar Furth (WF) rats were used. Hypertension and cardiac hypertrophy were confirmed in SHHF rats. PKC expression and activation were analyzed by Western blots using isozyme-specific antibodies. Compared to WF, untreated SHHF rats had increased phospho-active alpha (10-fold), delta (4-fold), and epsilon (3-fold) isozyme expression. Furthermore, we analyzed the effect of an angiotensin II type 1 receptor blocker (ARB) and hydralazine (Hy) on PKC regulation in SHHF rat left ventricle (LV). Both the ARB and Hy normalized LV blood pressure, but only the ARB reduced heart mass. Neither treatment affected PKC expression or activity. Our data show differential activation of PKC in the hypertensive, hypertrophic SHHF rat heart. Regression of hypertrophy elicited by an ARB in this model occurred independently of changes in the expression and activity of the PKC isoforms examined.

Vascular supplies differ in regenerating and nonregenerating amputated rodent digits.

Bone regenerates following amputation through the level of the nail, but bone is capped following amputation through more proximal levels. Because osteogenesis requires an ample blood supply, we postulated that a restricted vascular supply might be correlated with restricted regenerative ability at proximal levels. More than 40 rats and mice were injected with ink or resin to visualize vascular supplies of intact, regenerating, and nonregenerating rat and mouse digits. Ink-injected specimens were viewed as histological sections or cleared whole mounts. Partially digested resin casts were viewed using scanning electron microscopy. Contrary to our hypothesis, prior to amputation, proximal sites are more vascular than distal sites. At both proximal and distal levels, endosteal and periosteal vascular systems are evident. However, in proximal phalanges, additional subcutaneous and dermal layers encircle the bone. Beneath the distal nail, these layers are absent, and a single layer of vessels provides both periosteal and cutaneous supplies. After amputation at both levels, new vessels sprout profusely in osteogenic areas of both endosteum and periosteum. However, at proximal levels, the additional hypodermal and dermal vessels contribute to a vascular plexus that, paradoxically, may impair bone regrowth by contributing to the formation of dermal scar rather than bone.

Actin fiber patterns detected by Alexafluor 488 phalloidin suggest similar cell migration in regenerating and nonregenerating rodent toes.

Although mammals do not regenerate most appendages, they are able to regenerate toetips if the amputation occurs through the nail bed. The reasons for different outcomes following amputation at different levels are not understood. It is possible that cells at regenerating and nonregenerating sites migrate from fundamentally different tissues. If so, different migratory pathways could be detected. To identify putative migrating cells, microscope slides were made from both regenerating and nonregenerating toes of rats and mice on successive days after amputation. Fluorescent-labeled phalloidin, which binds polymerized f-actin, was used to identify actin filaments and fibers. Cells containing prominent actin bundles were distinguishable from those containing diffuse fibrils and those in which visible fibers were absent. Phalloidin labeling was similar in regenerating and nonregenerating digits after amputation. As early as 2 days after amputation at either proximal or distal levels, many cells of the hypodermis adjacent to the wound became labeled with phalloidin. The number and intensity of labeled hypodermal cells containing stress fiber-like bundles increased rapidly with time, and at successive times cells were seen progressively further distally. By approximately 7 days, they occupied the wound site immediately distal to bone of both regenerating and nonregenerating digits. Most dermal cells were unlabeled and endosteal and marrow cells contained only fibrillar actin. Phalloidin labeling does not support the concept of migration from different tissues in regenerating and nonregenerating amputation sites.

Temporal regression of myocyte hypertrophy in hypertensive, heart failure-prone rats treated with an AT1-receptor antagonist.

BACKGROUND: A recent study showed reverse remodeling of left ventricular myocyte shape when the type 1 angiotensin II (AT1)-receptor antagonist L-158,809 was administered to spontaneously hypertensive heart failure (SHHF) rats 4 months before the onset of failure. The aim of this study was to characterize temporally early treatment-induced reverse remodeling at the organ and cellular level by echocardiography and morphometry of isolated left ventricular myocytes. METHODS AND RESULTS: L-158,809 was administered to 9-month-old SHHF rats. Blood pressure normalized shortly after initiation of treatment. Isolated myocytes were collected in terminal experiments to assess cell remodeling. L-158,809 reduced myocyte volume and cross-sectional area significantly after 1 week of treatment with maximal regression of hypertrophy, including reduced cell length, obtained by 4 weeks. Reduced wall thickness was clearly detectable by echocardiography within 4 weeks after initiation of treatment. CONCLUSIONS: Regression of left ventricular myocyte hypertrophy occurred rapidly after initiation of AT1-blocker therapy in SHHF rats and was completed within 1 month. Regression of myocyte hypertrophy was associated with reduced wall thickness, which was detected consistently by echocardiography within 1 month after initiation of treatment.