Eugene Morkin

Control of cardiac myosin heavy chain gene expression.

The α‐ and β‐myosin genes extend over 51 kb on chromosome 14 in human and 11 in mouse separated by about 4.5 kb of intergenic sequence. They are located in tandem in the order of their expression during development. Transcription of each gene is independently controlled but coordinately regulated. During each embryogenesis, the β‐MHC gene is expressed as part of the cardiac myogenic program under the control of NKX‐2.5, MEF‐2C, and GATA‐4/5/6. After birth, thyroid hormone induces expression of α‐MHC mRNA and inhibits expression of the β‐MHC gene. While a large number of physiological stimuli are capable of modifying this basic paradigm, thyroid hormone is required for expression of α‐MHC in ventricular muscle. The positive TRE for T3‐stimulation of α‐MHC is an imperfect direct repeat located in the proximal promoter of the gene. The negative TRE for the β‐MHC gene is probably a binding half‐site that is located adjacent to the TATA box. Binding of TEF‐1 to a strong positive element in the proximal promoter is important in basal expression of β‐MHC gene and in the response to α1‐adrenergic stimulation. The β‐MHC gene also is induced together with several other “fetal” genes during cardiac hypertrophy by a mechanism involving Ca2+‐mediated activation of calcineurin and NF‐AT3. Upon activation, NF‐AT3 translocates to the nucleus and interacts with GATA‐4 to stimulate β‐MHC expression. Changes in chromatin structure mediated by the association of histone acetylases and deacetylases with transcription factors are essential in regulating cell‐specific expression of MHC genes. Microsc. Res. Tech. 50:522–531, 2000.

Biochemical and physiologic effects of thyroid hormone on cardiac performance

Abstract Recognition of the existence of ventricular myosin isozymes and their regulation by thyroid hormone may provide a biochemical basis for some of the effects of the hormone on myocardial performance. The isozymes, which are referred to as V 3 , in order of decreasing electrophoretic mobility and ATPase activity, contain the same light chain components, but differ in their heavy chain composition. V 1 contains two A heavy chains, V 3 two B heavy chains, and V 2 one heavy chain of each type. Rabbit and several other mammalian species, including man, have a predominance of the low activity V 3 form. The relative proportions of these myosin forms seems consistent with a random association of the heavy chain types. Thyroid hormone administration leads to a predominance of the high activity V 1 form of the enzyme, indicating that A chain synthesis has been stimulated and B chain synthesis suppressed. By contrast, the V 1 form normally is predominant in rat ventricle; thyroid ablation procedures lead to expression of the V 3 form, indicating stimulation of B chain synthesis and suppression of A chain synthesis. Since the ATPase activity of myosin seems closely related to the intrinsic speed of cardiac contraction, a change in myosin isozyme composition from V 3 to the V 1 may explain the increase in velocity of shortening observed in papillary muscles from thyrotoxic cat and rabbit hearts. Similarly, the switch from V 1 to V 3 may account for the decrease in performance of hypothyroid rat cardiac muscle. However, in man and other species which normally have a predominance of the V 3 form, the reported changes in myocardial performance in hypothyroidism may be related to other factors, including alterations in electro-mechanical coupling, myxedematous infiltration and coronary atherosclerosis. Assessments of LV performance in thyrotoxic subjects and in conscious animal models have indicated that LV wall velocity is not usually greater than normal because of the increase in afterload. Although LV systolic and diastolic dimensions are often increased, ventricular filling pressures remain normal, and there is no evidence of sarcomere stretching or disruption. Acute B-adrenergic blockade has very little influence on any of these alterations in LV performance. Furthermore, animal experiments and computer simulation of the thyrotoxic circulatory system indicate that there are important changes in the peripheral vascular compartments as well as in the heart. These alterations, which include a decrease in peripheral arterial resistance and an increase in P ms , should be regarded as essential features of the high-output state associated with thyrotoxicosis. A change from the V 1 to the V 3 form also has been shown to occur in the rat ventricle during development, aging, chemically-induced diabetes, and in hemodynamic overload. In some cases, these changes seem to be associated with alterations in plasma thyroid hormone concentration. Also, in diabetes and pressure overload, the changes in myosin isozyme composition can be reversed by thyroxine treatment. Taken together, the experimental data suggest that thyroid hormone may play at least a permissive role in regulating ventricular myosin isozyme expression in many nonthyroidal cardiac conditions. The potential to alter myosin isozyme composition constitutes an important area for future research.

Survival after myocardial infarction in rats: captopril versus losartan

OBJECTIVES This study sought to compare the effects of angiotensin-converting enzyme inhibition versus angiotensin II receptor blockade on survival in rats with myocardial infarction. BACKGROUND The effects of specific nonpeptide angiotensin receptor blocking agents on survival after myocardial infarction are unknown. METHODS Rats with a moderate to large myocardial infarction were treated with captopril (2 g/liter drinking water, n = 87) or losartan (2 g/liter drinking water, n = 96). Therapy was initiated immediately after coronary artery ligation and continued for 1 year. RESULTS Uncensored median survival in captopril-treated rats that survived at least 48 h was 201.5 days versus 236.0 days for losartan-treated rats (p = 0.066). Median survival censored for rats with lung infections was 201.5 days in captopril-treated rats versus 243.0 days for losartan-treated rats (p = 0.028). Conscious hemodynamic measurements and remodeling data obtained at 1 year in the surviving rats (n = 5 for captopril; n = 9 for losarton) revealed no differences in heart weight, left ventricular pressure, dP/dt, cardiac index, time constant of relaxation or any variable of left ventricular remodeling. The only differences (mean +/- SD) were an increase in heart rate (293 +/- 19 vs. 266 +/- 15 beats/min, p < 0.05) and a decrease in peak developed pressure (153 +/- 21 vs. 180 +/- 16 mm Hg, p < 0.05) in the losartan-treated rats. CONCLUSIONS We conclude that in this experimental model of heart failure, there was no significant difference between survival after angiotensin II receptor blockade with losartan and with angiotensin-converting enzyme inhibition with captopril.

DITPA (3,5-diiodothyropropionic acid), a thyroid hormone analog to treat heart failure: Phase II trial veterans affairs cooperative study

Background— In animal studies and a pilot trial in patients with congestive heart failure, the thyroid hormone analog 3,5 diiodothyropropionic acid (DITPA) had beneficial hemodynamic effects. Methods and Results— This was a phase II multicenter, randomized, placebo-controlled, double-blind trial of New York Heart Association class II to IV congestive heart failure patients randomized (2:1) to DITPA or placebo and treated for 6 months. The study enrolled 86 patients (n=57 to DITPA, n=29 to placebo). The primary objective was to assess the effect of DITPA on a composite congestive heart failure end point that classifies patients as improved, worsened, or unchanged based on symptom changes and morbidity/mortality. DITPA was poorly tolerated, which obscured the interpretation of congestive heart failure–specific effects. Fatigue and gastrointestinal complaints, in particular, were more frequent in the DITPA group. DITPA increased cardiac index (by 18%) and decreased systemic vascular resistance (by 11%), serum cholesterol (−20%), low-density lipoprotein cholesterol (−30%), and body weight (−11 lb). Thyroid-stimulating hormone was suppressed in patients given DITPA, which reflects its thyromimetic effect; however, no symptoms or signs of potential hypothyroidism or thyrotoxicosis were seen. Conclusions— DITPA improved some hemodynamic and metabolic parameters, but there was no evidence for symptomatic benefit in congestive heart failure.

Clinical and experimental studies on the use of 3,5-diiodothyropropionic acid, a thyroid hormone analogue, in heart failure

Thyroid hormone has unique actions that make it a novel and possibly useful agent for treatment of heart failure. Because of potential adverse effects of thyroid hormone, however, there has been interest in developing analogues with fewer undesirable side effects. Screening of compounds structurally related to levothyroxine identified 3,5-diiodothyropropionic acid (DITPA) as an analogue with inotropic selectivity and low metabolic activity in hypothyroid rats. When DITPA was administered alone or in combination with captopril in rat and rabbit postinfarction models of heart failure, cardiac output was increased and left ventricular end-diastolic pressure (LV EDP) was decreased without increasing heart rate. A pilot clinical study was undertaken to evaluate the safety and efficacy of DITPA. In a dose-ranging study in 7 normal volunteers the drug was well tolerated. A double-blind comparison then was made of DITPA versus placebo in a group of 19 patients with moderately severe heart failure. Patients were randomly assigned to receive either 1.875 mg/kg of DITPA or placebo daily. After 2 weeks the drug was increased to 3.75 mg/kg daily for an additional 2 weeks. In heart failure patients receiving the drug for 4 weeks, cardiac index was increased (p = 0.04) and systemic vascular resistance index was decreased (p = 0.02). Total serum cholesterol (p = 0.013) and triglycerides (p = 0.005) also were decreased significantly. These results indicate that DITPA is well tolerated and could represent a useful new agent for treatment of congestive heart failure.

Expression of α and β integrins during terminal differentiation of cardiomyocytes

Background: In the myocardium, myocyte cell division is irreversibly blocked shortly after birth. The signal that initiates cell cycle withdrawal is unknown. The purpose of this study was to relate changes in expression of β1 integrin and its associated α subunits to cardiomyocyte cell cycle progression during the fetal-to-neonatal developmental transition in rat. Methods and results: The developmental expression pattern and function of β1 integrin and several of its associated α subunits were examined using reverse transcription (RT) polymerase chain reaction (PCR) and β1 blocking antibodies. During the fetal to neonatal transition, a dramatic shift occurred in the levels of β1 and α isoforms. At the 17-day fetal stage only β1A was present, which remained relatively constant until immediately after birth then decreased by 30% at the adult stage. By contrast, β1D appeared at fetal day 18, increased at neonatal day 2, and afterwards remained constant. This resulted in a ratio of β1A to β1D of about 1:1 in the adult heart. The integrin β1-associated subunits, α3, α6, and α7, were expressed at extremely low levels in 17-day fetal cardiomyocytes. After birth α3 and α6 transiently increased at the 2-day neonatal stage, while α7 isoforms B, C, and X2 progressively increased to the adult stage. Unlike skeletal muscle cells, fluorescence-activated cell sorting analysis (FACS) showed no down regulation of the α5β1 fibronectin receptor during cell cycle withdrawal. Treatment of cultured cardiomyocytes with β1 blocking antibody inhibited the cell cycle in fetal but not in neonatal cells. Conclusion: These results suggest that progression through the cardiomyocyte cell cycle may be dependent upon cell attachment via integrin β1 and correlate with changes that occur in β1 spliced variants and their respective α isoforms.

Pilot studies on the use of 3,5-diiodothyropropionic acid, a thyroid hormone analog, in the treatment of congestive heart failure.

After an initial safety study in 7 normal volunteers, a randomized double-blind comparison was made between 3,5-diiodothyropropionic acid (DITPA) and placebo in 19 patients with moderately severe congestive failure. In heart failure patients receiving the drug for 4 weeks, cardiac index was increased (p = 0.04) and systemic vascular resistance index was decreased (p = 0.02). Systolic cardiac function was unchanged but isovolumetric relaxation time was decreased significantly, suggesting improvement in diastolic function. Total serum cholesterol (p = 0.005) and triglycerides (p = 0.01) also were decreased significantly. DITPA could represent a useful new agent for treatment of congestive heart failure.

Effects of the Thyromimetic Agent Diiodothyropropionic Acid on Body Weight, Body Mass Index, and Serum Lipoproteins: A Pilot Prospective, Randomized, Controlled Study

CONTEXT Widespread thyroid hormone actions offer the possibility of developing selective thyromimetic analogs with salutary metabolic properties. Consequently, effects of diiodothyropropionic acid (DITPA) on body weight, serum lipoproteins, and bone metabolism markers were studied in a prospective, controlled, double-blind 24-wk trial, which was primarily designed to assess treatment of stable chronic heart failure. DESIGN Eighty-six patients (aged 66 +/- 11 yr, mean +/- sd) were randomized (1:2) to placebo or an escalating DITPA dose (90 to 180, 270, and 360 mg/d) over 8 wk until serum TSH was less than 0.02 mU/liter. Patients were studied at 2, 4, 6, 8, 16, and 24 wk and after 4 wk off study drug. Only 21 DITPA-treated and 27 placebo patients completed the full 24 wk of therapy. RESULTS DITPA therapy lowered serum TSH levels and, to a lesser extent, serum T(3) and T(4), but there were no differences in clinical manifestations of thyrotoxicosis or hypothyroidism. Serum total and low-density lipoprotein cholesterol levels both decreased on DITPA; there was a transient decrease in triglycerides and no change in high-density lipoprotein cholesterol. DITPA therapy was associated with significant reduction in body weight, 12.5 lb at 24 wk. Increases in serum osteocalcin, N-telopeptide, and deoxypyridinoline levels were consistent with increased bone turnover on DITPA. CONCLUSION This investigation of DITPA actions demonstrated its efficacy in reducing body weight and lowering total and low-density lipoprotein cholesterol levels. However, DITPAs adverse effects at doses used resulted in a high dropout rate and potentially dangerous skeletal actions were observed.

Treatment of verapamil toxicity in intact dogs.

The treatment of verapamil toxicity was examined in lightly sedated dogs. Verapamil, administered as a bolus (0.72 mg/kg) followed by a continuous infusion (0.11 mg/kg per min), decreased cardiac output (CO) from 3.1 +/- 0.1 to 1.7 +/- 0.1 liter/min (P less than 0.001), heart rate (HR) from 85 +/- 4 to 57 +/- 3 beats/min (P less than 0.001), left ventricular derivative of pressure with respect to time (LV dP/dt) from 2,085 +/- 828 to 783 +/- 78 mm Hg/s (P less than 0.001), mean aortic pressure (AO) from 77 +/- 4 to 38 +/- 2 mm Hg (P less than 0.001) and stroke volume from 39 +/- 3 to 28 +/- 2 ml/beat (P less than 0.01). In verapamil-toxic animals isoproterenol increased HR, CO, LV dP/dt, and AO; calcium chloride increased LV dP/dt and AO; norepinephrine, epinephrine, and dopamine increased CO, AO, and LV dP/dt, atropine increased HR, CO, and AO. Phenylephrine (13-55 micrograms/kg per min) produced no changes except a small increase in AO while very high dose phenylephrine (300 micrograms/kg per min) increased AO, CO, and LV dP/dt. 4-Aminopyridine (4-AP) increased HR, CO, LV dP/dt, and AO. When administered prior to verapamil, 4-AP prevented the development of verapamil toxicity as shown by the significantly higher AO (P less than 0.001), CO (P less than 0.01), and LV dP/dt (P less than 0.01) when 4-AP followed by verapamil was compared to verapamil alone. In conclusion, there does not appear to be a single specific therapy for verapamil toxicity, however it can be partially corrected by presently available pharmacologic therapy and 4-AP.

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.