Intracoronary gene transfer of fibroblast growth factor-5 increases blood flow and contractile function in an ischemic region of the heart.

Increased coronary blood vessel development could potentially benefit patients with ischemic heart disease. In a model of stress-induced myocardial ischemia, intracoronary injection of a recombinant adenovirus expressing human fibroblast growth factor-5 (FGF-5) resulted in messenger RNA and protein expression of the transferred gene. Two weeks after gene transfer, regional abnormalities in stress-induced function and blood flow were improved, effects that persisted for 12 weeks. Improved blood flow and function were associated with evidence of angiogenesis. This report documents, for the first time, successful amelioration of abnormalities in myocardial blood flow and function following in vivo gene transfer.

Mitochondrial fragmentation and superoxide anion production in coronary endothelial cells from a mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: Mitochondria frequently change their shapes by fusion and fission and these morphological dynamics play important roles in mitochondrial function and development as well as programmed cell death. The goal of this study is to investigate whether: (1) mitochondria in mouse coronary endothelial cells (MCECs) isolated from diabetic mice exhibit increased fragmentation; and (2) chronic treatment with a superoxide anion (O(2)(-)) scavenger has a beneficial effect on mitochondrial fragmentation in MCECs. METHODS: MCECs were freshly isolated and lysed for protein measurement, or cultured to determine mitochondrial morphology and O(2)(-) production. For the ex vivo hyperglycaemia experiments, human coronary endothelial cells were used. RESULTS: Elongated mitochondrial tubules were observed in MCECs isolated from control mice, whereas mitochondria in MCECs from diabetic mice exhibited augmented fragmentation. The level of optic atrophy 1 (OPA1) protein, which leads to mitochondrial fusion, was significantly decreased, while dynamin-related protein 1 (DRP1), which leads to mitochondrial fission, was significantly increased in MCECs from diabetic mice. Diabetic MCECs exhibited significantly higher O(2)(-) concentrations in cytosol and mitochondria than control MCECs. Administration of the O(2)(-) scavenger TEMPOL to diabetic mice for 4 weeks led to a significant decrease in mitochondrial fragmentation without altering the levels of OPA1 and DRP1 proteins in MCECs. High-glucose treatment for 24 h significantly induced mitochondrial fragmentation, which was restored by TEMPOL treatment. In addition, excess O(2)(-) production, either in cytosol or in mitochondria, significantly increased mitochondrial fragmentation. CONCLUSIONS/INTERPRETATION: These data suggest that lowering the O(2)(-) concentration can restore the morphological change in mitochondria and may help improve mitochondrial function in diabetic MCECs.

Expression of inducible stress protein 70 in rat heart myogenic cells confers protection against simulated ischemia-induced injury.

Myocardial ischemia markedly increases the expression of several members of the stress/heat shock protein (HSP) family, especially the inducible HSP70 isoforms. Increased expression of HSP70 has been shown to exert a protective effect against a lethal heat shock. We have examined the possibility of using this resistance to a lethal heat shock as a protective effect against an ischemic-like stress in vitro using a rat embryonic heart-derived cell line H9c2 (2-1). Myogenic cells in which the heat shock proteins have been induced by a previous heat shock are found to become resistant to a subsequent simulated ischemic stress. In addition, to address the question of how much does the presence of the HSP70 contribute to this protective effect, we have generated stably transfected cell lines overexpressing the human-inducible HSP70. Embryonal rat heart-derived H9c2(2-1) cells were used for this purpose. This stably transfected cell line was found to be significantly more resistant to an ischemic-like stress than control myogenic cells only expressing the selectable marker (neomycin) or the parental cell line H9c2(2-1). This finding implicates the inducible HSP70 protein as playing a major role in protecting cardiac cells against ischemic injury.

Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury.

Myocardial protection and changes in gene expression follow whole body heat stress. Circumstantial evidence suggests that an inducible 70-kD heat shock protein (hsp70i), increased markedly by whole body heat stress, contributes to the protection. Transgenic mouse lines were constructed with a cytomegalovirus enhancer and beta-actin promoter driving rat hsp70i expression in heterozygote animals. Unstressed, transgene positive mice expressed higher levels of myocardial hsp70i than transgene negative mice after whole body heat stress. This high level of expression occurred without apparent detrimental effect. The hearts harvested from transgene positive mice and transgene negative littermates were Langendorff perfused and subjected to 20 min of warm (37 degrees C) zero-flow ischemia and up to 120 min of reflow while contractile recovery and creatine kinase efflux were measured. Myocardial infarction was demarcated by triphenyltetrazolium. In transgene positive compared with transgene negative hearts, the zone of infarction was reduced by 40%, contractile function at 30 min of reflow was doubled, and efflux of creatine kinase was reduced by approximately 50%. Our findings suggest for the first time that increased myocardial hsp70i expression results in protection of the heart against ischemic injury and that the antiischemic properties of hsp70i have possible therapeutic relevance.

Protection against myocardial dysfunction after a brief ischemic period in transgenic mice expressing inducible heat shock protein 70.

Brief ischemic periods lead to myocardial dysfunction without myocardial infarction. It has been shown that expression of inducible HSP70 in hearts of transgenic mice leads to decreased infarct size, but it remains unclear if HSP70 can also protect against myocardial dysfunction after brief ischemia. To investigate this question, we developed a mouse model in which regional myocardial function can be measured before and after a temporary ischemic event in vivo. In addition, myocardial function was determined after brief episodes of global ischemia in an isolated Langendorff heart. HSP70-positive mice and transgene negative littermates underwent 8 min of regional myocardial ischemia created by occlusion of the left descending coronary artery, followed by 60 min of reperfusion. This procedure did not result in a myocardial infarction. Regional epicardial strain was used as a sensitive indicator for changes in myocardial function after cardiac ischemia. Maximum principal strain was significantly greater in HSP70-positive mice with 88+/-6% of preischemic values vs. 58+/-6% in transgene-negative mice (P < 0.05). Similarly, in isolated Langendorff perfused hearts of HSP70-positive and transgene-negative littermates exposed to 10 min of global ischemia and 90 min of reperfusion, HSP70 transgenic hearts showed a better-preserved ventricular peak systolic pressure. Thus, we conclude that expression of HSP70 protects against postischemic myocardial dysfunction as shown by better preserved myocardial function.

Overexpression of the rat sarcoplasmic reticulum Ca2+ ATPase gene in the heart of transgenic mice accelerates calcium transients and cardiac relaxation.

The Ca2+ ATPase of the sarcoplasmic reticulum (SERCA2) plays a dominant role in lowering cytoplasmic calcium levels during cardiac relaxation and reduction of its activity has been linked to delayed diastolic relaxation in hypothyroid and failing hearts. To determine the contractile alterations resulting from increased SERCA2 expression, we generated transgenic mice overexpressing a rat SERCA2 transgene. Characterization of a heterozygous transgenic mouse line (CJ5) showed that the amount of SERCA2 mRNA and protein increased 2. 6-fold and 1.2-fold, respectively, relative to control mice. Determination of the relative synthesis rate of SERCA2 protein showed an 82% increase. The mRNA levels of some of the other genes involved in calcium handling, such as the ryanodine receptor and calsequestrin, remained unchanged, but the mRNA levels of phospholamban and Na+/Ca2+ exchanger increased 1.4-fold and 1.8-fold, respectively. The increase in phospholamban or Na+/Ca2+ exchanger mRNAs did not, however, result in changes in protein levels. Functional analysis of calcium handling and contractile parameters in isolated cardiac myocytes indicated that the intracellular calcium decline (t1/2) and myocyte relengthening (t1/2) were accelerated by 23 and 22%, respectively. In addition, the rate of myocyte shortening was also significantly faster. In isolated papillary muscle from SERCA2 transgenic mice, the time to half maximum postrest potentiation was significantly shorter than in negative littermates. Furthermore, cardiac function measured in vivo, demonstrated significantly accelerated contraction and relaxation in SERCA2 transgenic mice that were further augmented in both groups with isoproterenol administration. Similar results were obtained for the contractile performance of myocytes isolated from a separate line (CJ2) of homozygous SERCA2 transgenic mice. Our findings suggest, for the first time, that increased SERCA2 expression is feasible in vivo and results in enhanced calcium transients, myocardial contractility, and relaxation that may have further therapeutic implications.

Transgenic expression of sarcoplasmic reticulum Ca(2+) atpase modifies the transition from hypertrophy to early heart failure.

To examine the contribution of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) to early heart failure, we subjected transgenic (TG) mice expressing SERCA2a gene and wild-type (WT) mice to aortic stenosis (AS) for 7 weeks. At an early stage of hypertrophy (4-week AS), in vivo hemodynamic and echocardiographic indices were similar in TG and WT mice. By 7 weeks of AS, which is the stage of early failure in this model, TG mice with AS had lower mortality than WT mice with AS (6.7% versus 29%). The magnitude of left ventricular (LV) hypertrophy was similar in WT and TG 7-week AS mice. In vivo LV systolic function was higher in TG than in WT 7-week AS mice. In LV myocytes loaded with fluo-3, fractional cell shortening and the amplitude of the [Ca(2+)](i) transients were higher in TG than in WT 7-week AS mice under baseline conditions (0.5 Hz, 1.5 mmol/L [Ca(2+)](o), 25 degrees C). The rates of relengthening and decay in [Ca(2+)](i) were faster in TG than in WT 7-week AS myocytes. In myocytes from WT 7-week AS compared with sham-operated WT mice, contractile reserve in response to rapid pacing was depressed with impaired augmentation of both peak-systolic [Ca(2+)](i) and the SR Ca(2+) load. In contrast, contractile reserve and the capacity to augment SR Ca(2+) load were maintained in TG 7-week AS mice. SERCA2a protein levels were depressed in WT 7-week AS mice, but were preserved in TG 7-week AS mice. These data suggest that defective SR Ca(2+) loading contributes to the onset of contractile failure in animals with chronic pressure overload.

A role for HSP27 in sensory neuron survival.

Peripheral nerve injury in neonatal rats results in the death of the majority of the axotomized sensory neurons by 7 d after injury. In adult animals, however, all sensory neurons survive for at least 4 months after axotomy. How sensory neurons acquire the capacity to survive axonal injury is not known. Here we describe how the expression of the small heat shock protein 27 (HSP27) is correlated with neuronal survival after axotomy in vivo and after NGF withdrawal in vitro. The number of HSP27-immunoreactive neurons in the L4 DRG is low at birth and does not change significantly for 21 d after postnatal day 0 (P0) sciatic nerve axotomy. In contrast, in the adult all axotomized neurons begin to express HSP27. One week after P0 sciatic nerve section the total number of neurons in the L4 DRG is dramatically reduced, but all surviving axotomized neurons, as identified by c-jun immunoreactivity, are immunoreactive for HSP27. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling reveals that very few HSP27-expressing neurons are dying 48 hr after neonatal axotomy. In vitro, a similar correlation exists between HSP27 expression and survival; in P0 DRG cultures, neurons that express HSP27 preferentially survive NGF withdrawal. Finally, overexpression of human HSP27 in neonatal rat sensory and sympathetic neurons significantly increases survival after NGF withdrawal, with nearly twice as many neurons surviving at 48 hr. Together these results suggest that HSP27 in sensory neurons plays a role in promoting survival after axotomy or neurotrophin withdrawal.

Combined and individual mitochondrial HSP60 and HSP10 expression in cardiac myocytes protects mitochondrial function and prevents apoptotic cell deaths induced by simulated ischemia-reoxygenation.

BACKGROUND: The mitochondrial heat-shock proteins HSP60 and HSP10 form a mitochondrial chaperonin complex, and previous studies have shown that their increased expression exerts a protective effect against ischemic injury when cardiac myocytes are submitted to simulated ischemia. The more detailed mechanisms by which such a protective effect occurs are currently unclear. We wanted to determine whether HSP60 and HSP10 could exert a protection against simulated ischemia and reoxygenation (SI/RO)-induced apoptotic cell death and whether such protection results from decreased mitochondrial cytochrome c release and caspase-3 activation and from the preservation of ATP levels by preservation of the electron transport chain complexes. In addition, we explored whether increased expression of HSP60 or HSP10 by itself exerts a protective effect. METHODS AND RESULTS: We overexpressed HSP60 and HSP10 together or separately in rat neonatal cardiac myocytes using an adenoviral vector and then subjected the myocytes to SI/RO. Cell death and apoptosis in myocytes were quantified by parameters such as enzyme release, DNA fragmentation, and caspase-3 activation. Overexpression of the combination of HSP60 and HSP10 and of HSP60 or HSP10 individually protected myocytes against apoptosis. This protection is accompanied by decreases in mitochondrial cytochrome c release and in caspase-3 activity and increases in ATP recovery and activities of complex III and IV in mitochondria after SI/RO. CONCLUSIONS: These results suggest that mitochondrial chaperonins HSP60 and HSP10 in combination or individually play an important role in maintaining mitochondrial integrity and capacity for ATP generation, which are the crucial factors in determining survival of cardiac myocytes undergoing ischemia/reperfusion injury.

Effects of mutant and antisense RNA of phospholamban on SR Ca(2+)-ATPase activity and cardiac myocyte contractility.

BACKGROUND: The delayed cardiac relaxation in failing hearts has been attributed to a reduced activity of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2). Phospholamban (PLB) inhibits SERCA2 activity and is therefore a potential target to improve the cardiac performance in heart failure. METHODS AND RESULTS: Mutants of PLB (Adv/mPLB) or antisense RNA of PLB (Adv/asPLB) was expressed in cardiac myocytes by recombinant adenovirus, and their effects on SERCA2 activity and myocyte contractility were studied. One mPLB, K3E/R14E, pentamerized with endogenous PLB in neonatal myocytes and resulted in a 45% increase in the affinity of SERCA2 for Ca(2+) and 27% faster diastolic Ca(2+) decline as determined by SR (45)Ca uptake assays and by indo 1-facilitated Ca(2+) transient measurement, respectively. Edge-detection analysis of adult myocyte contractility showed a 74% increase in fractional shortening, accompanied by 115% increase in velocity of relengthening and 25% decrease in time to half-maximal relengthening. In parallel, infection of neonatal cardiac myocytes by Adv/asPLB decreased the endogenous PLB level by 54%, which was associated with a 35% increase in Ca(2+) affinity of SERCA2 and 21% faster diastolic Ca(2+) decline. However, in adult cardiac myocytes, Adv/asPLB failed to significantly alter the endogenous PLB level, the SERCA2 activity, or most of the contractile parameters. CONCLUSIONS: K3E/R14E is a dominant negative mutant of PLB that disrupts the structural integrity and function of the endogenous PLB and consequently enhances SERCA2 activity and myocyte contractility. In neonatal myocytes, the decrease in steady-state abundance of PLB by asPLB also leads to increased SERCA2 activity.

Heat shock and adaptive response to ischemia.

A mild heat treatment is known to confer a transient protection to cells against a subsequent lethal heat shock. This phenomenon is better known as thermotolerance and seems to be mediated by the expression of a group of proteins, the heat-shock proteins. Recent studies have shown that a heat-shock pretreatment improves postischemic recovery in an isolated perfused rat heart model. In the present brief review, we cover the more recent findings related to heat shock and the adaptive response that it may produce in the cardiac cell against ischemic stress.

Sarcoplasmic reticulum Ca(2+)-ATPase overexpression by adenovirus mediated gene transfer and in transgenic mice.

The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) is a major determinant of cardiac relaxation. It has been demonstrated that the steady state levels of the mRNA coding for this pump are reduced in human heart failure due to dilated cardiomyopathy. Although results regarding the protein level are controversial, most functional studies indicate decreased SERCA2a activity in heart failure. The extent to which a potential decrease in the calcium sequestering function of this protein could contribute to the contractile dysfunction in heart failure, and whether a reconstitution of SERCA2a could alleviate heart failure, are yet unknown. To further investigate these questions two methodological approaches were chosen. Adenovirus mediated gene transfer provides an approach to study functional consequences of SERCA2a overexpression in cardiac myocytes in vitro [1], and a transgenic mouse model allows the effects of cardiac overexpression of SERCA2a to be examined in vivo [2].

Overexpression of sarcoplasmic reticulum Ca(2+)-ATPase improves cardiac contractile function in hypothyroid mice.

OBJECTIVE: Prolonged cardiac contraction and relaxation in hypothyroidism are in part related to diminished expression of the gene coding for the calcium pump of the sarcoplasmic reticulum (SERCA2a). Therefore, we examined whether or not transgenic SERCA2a gene expression in mice may compensate for the cardiac effects of hypothyroidism. METHODS: SERCA2a mRNA and protein were analyzed from hearts of euthyroid and hypothyroid mice of wild-type or SERCA2a transgene status. Contractile function was studied in isolated left ventricular papillary muscles. RESULTS: We found significant decreases of SERCA2a mRNA and protein levels in hearts of hypothyroid wild-type mice in comparison with euthyroid wild-type mice (controls). Papillary muscles from hypothyroid wild-type mice showed significant increases in time to peak contraction and relaxation times compared with controls. In contrast, SERCA2a mRNA and protein levels were significantly higher in hypothyroid SERCA2a transgenic mice than in hypothyroid wild-type mice. The transgene led to a functional improvement by compensating for the prolonged contraction and relaxation of papillary muscles. CONCLUSIONS: Our murine model of hypothyroidism revealed decreases in SERCA2a gene expression accompanied by prolonged contraction and relaxation of papillary muscles, and an improvement of the contractile phenotype due to compensated SERCA2a gene expression in SERCA2a transgenic mice.

Functional properties of skeletal muscle from transgenic animals with upregulated heat shock protein 70.

The influence of inducible heat stress proteins on protecting contracting skeletal muscle against fatigue-induced injury was investigated. A line of transgenic mice overexpressing the inducible form of the 72-kDa heat shock protein (HSP72) in skeletal muscles was used. We examined the relationship between muscle contractility and levels of the constitutive (HSC73) and inducible (HSP72) forms of the 72-kDa heat shock protein in intact, mouse extensor digitorum longus (EDL), soleus (SOL), and the diaphragm (DPH). In all transgenic muscles, HSP72 was expressed at higher levels compared with transgene-negative controls, where HSP72 was below the level of detection. At the same time, HSC73 levels were downregulated in all transgenic muscle types. Shipment-related stress caused an elevation in the levels of HSP72 in all muscles for 1 wk after arrival of the animals. We also found that, although no statistical differences in response to intermittent fatiguing stimulation in the contractile properties of intact transgene-positive muscles compared with their transgene-negative counterparts were observed, the response of intact transgene-positive EDL muscles to caffeine was enhanced. These findings demonstrate that elevated HSP72 does not protect EDL, SOL, or DPH muscles from the effects of intermittent fatiguing stimulation. However, HSP72 may influence the excitation-contraction coupling (ECC) process, either directly or indirectly, in EDL muscle. If the effects on ECC were indirect, then these results would suggest that manipulation of a specific gene might cause functional effects that seem independent of the manipulated gene/protein.

Myosin isoenzyme distribution and Ca+2-activated myosin ATPase activity in the rat heart is influenced by fructose feeding and triiodothyronine.

Studies were conducted to determine if the level of cardiac Ca+2-activated myosin ATPase activity and ventricular myosin isoenzyme distribution are influenced by both T3 administration and fructose feeding. Previous studies have shown that in the cardiac ventricle of hypothyroid rats, only myosin V3 is present, and the Ca+2-activated myosin ATPase activity is markedly decreased. Hypothyroid [thyroidectomized (Tx)] rats were fed a diet containing 60% fructose or a regular diet (47% complex carbohydrates) for 4 weeks. Fructose feeding of hypothyroid rats led to a significant increase in Ca+2-activated myosin ATPase activity (Tx regular diet, 0.33 +/- 0.02 mumol Pi/mg protein X min; Tx fructose diet, 0.54 +/- 0.04 mumol Pi/mg protein X min). In addition, myosin V1 was detectable in the heart of fructose-fed Tx rats, but was absent in Tx rats on the regular diet. To determine if fructose had an effect of similar magnitude in animals of different thyroid states, Tx rats were injected with 0.075, 0.150, 0.225, and 0.300 micrograms T3/100 g BW daily and placed on fructose or regular diets. The fructose-induced increase in Ca+2-myosin ATPase activity was between 24-27% in Tx rats receiving 0-0.15 micrograms T3/100 g BW daily. In animals receiving 0.225 and 0.300 micrograms T3/100 g BW daily, fructose feeding did not induce a significant increase in myosin ATPase activity. This is due to the fact that the Ca+2-activated myosin ATPase activities of euthyroid and hyperthyroid animals are not significantly different from each other. In hypothyroid rats receiving a 60% glucose diet, Ca+2-myosin ATPase activity showed a significant 20% increase above the value in regular diet-fed Tx rats. Fructose- and glucose-induced changes in Ca+2-myosin ATPase activity occurred in the absence of changes in thyroid hormone or insulin levels. Our findings may indicate that cardiac carbohydrate consumption influences the predominance of ventricular myosin isoenzymes in the rat heart.

Fructose feeding increases Ca++-activated myosin ATPase activity and changes myosin isoenzyme distribution in the diabetic rat heart.

Previous studies have shown that in rats, diabetes mellitus induces a 45% decrease in cardiac Ca++-activated myosin ATPase activity which is accompanied by a decrease in myosin isoenzyme V1 and an increase in myosin isoenzyme V3 levels. Insulin administration reverts Ca++-activated myosin ATPase activity and myosin isoenzyme distribution to normal levels. It is currently unclear whether the effects of insulin on Ca++-myosin ATPase activity and myosin isoenzyme distribution are direct effects of the hormone or are mediated through insulin-induced alterations in cardiac metabolism. To determine if insulin may exert part of its effects by the latter route, diabetic rats were fed a normal, glucose, or fructose diet. Unlike glucose, fructose can enter the initial steps of the glycolytic pathway in the absence of insulin. Placing diabetic rats on different forms of 60% fructose diets for 4 weeks led to a 20-35% increase in Ca++-activated myosin ATPase activity, which was highly significant (normal Ca++-activated myosin ATPase activity, 0.917 mumol Pi/mg protein X min; diabetic, 0.553 mumol Pi/mg protein X min; diabetic + fructose, 0.661 mumol Pi/mg protein X min). The increase in Ca++-activated myosin ATPase activity was accompanied by increased myosin isoenzyme V1 and decreased myosin isoenzyme V3 levels. Feeding animals a 60% glucose diet did not lead to changes in Ca++-activated myosin ATPase activity or myosin isoenzyme distribution. The fructose-induced increase in Ca++-activated myosin ATPase activity and alteration in myosin isoenzyme distribution occurred in the absence of changes in insulin and thyroid hormone levels or improvement in the general metabolic status of fructose-fed diabetic rats.

A physiological dose of triiodothyronine normalizes cardiac myosin adenosine triphosphatase activity and changes myosin isoenzyme distribution in semistarved rats.

The possibility that the lowering of thyroid hormone levels which occurs in the nonthyroidal illness syndrome results in a hypothyroid state at the cardiac tissue level was examined in semistarved rats. Rats were fed 50% of their normal food intake in the form of a regular diet (R. diet) or low carbohydrate diet (L.C. diet) for 8 weeks. Animals semistarved for 8 weeks on the R. diet lost 42% of their body weight, while plasma T3 and T4 levels decreased by 45-50%. Semistarvation on the L.C. diet resulted in a 19% weight loss and a similar 46-49% decrease in plasma T3 and T4 levels. Ca++-activated myosin ATPase activity declined by 28% and 48% with the R. and L.C. diets, respectively [normal rats myosin ATPase, 1.30 +/- 0.18 mumol Pi/(mg protein . min) (mean +/- SD); semistarvation R diet, 0.93 +/- 0.15; semistarvation L.C. diet, 0.67 +/- 0.15]. The administration of physiological amounts of T3 (0.3 micrograms T3/100 g BW daily) restored the cardiac myosin ATPase activity in both groups. To confirm that the T3 effect was due to a normalization of the thyroid status at the tissue level, hypothyroid animals on a normal diet were injected with 0.3 micrograms T3 for 4 weeks, which resulted in normalization of myosin ATPase activity levels. Thyroidectomized rats receiving daily T3 injections, and when placed on a 50% reduction of food intake for 4 weeks still maintained normal myosin ATPase activity even though they lost 36% of their body weight. Distribution of cardiac myosin isoenzymes was determined by pyrophosphate polyacrylamide gel electrophoresis. In normal cardiac ventricles, myosin isoenzyme V1 predominates and represents 68 +/- 7% (+/- SD) of the total myosin. Semistarvation resulted in a redistribution of myosin isoenzymes so that V3 myosin was the predominant species (53 +/- 3% of the total myosin). The administration of 0.3 microgram T3/100 g BW daily for 4 weeks to semistarved rats reverted myosin isoenzyme distribution to V1 predominance (V1 myosin, 54 +/- 3% of the total myosin). These results indicate that the semistarvation-induced lowering plasma T3 and T4 levels is an important determinant of myosin ATPase activity and myosin isoenzyme distribution. Restoration of myosin ATPase activity to its normal level and return to myosin V1 predominance after T3 administration make it likely that these changes are related to the lowering of thyroid hormone levels.

Glucagon administration decreases hepatic nuclear triiodothyronine binding capacity.

The maximal binding capacity (MBC) of nuclear triiodothyronine (T3) receptor sites in rat liver decreases markedly after glucagon administration. Administration of serial doses of glucagon (2.5 microgram/100 g BW) resulted in a 33% decrease in MBC in 3.5 h and MBC was reduced by 45% in 6.25 h. The individual doses used were in the same order of magnitude as those used in the treatment of hypoglycemic human subjects (1.5 microgram/100 g BW). This report presents the first evidence that a peptide hormone can change the number of nuclear T3 receptor sites. The physiological significance of these findings remains to be clarified.

The effects of amiodarone on serum thyroid hormones and hepatic thyroxine 5'-monodeiodination in rats.

Amiodarone (2-n-butyl-3,4'-diethylaminoethoxy-3', 5'-diiodobenzoyl-benzofurane) is an antiarrhythmic drug which increases serum T4 and rT3 levels in patients and lowers serum T3 levels. To investigate its effects on T4 metabolism and its cardiac action, we fed amiodarone to male Fisher rats at doses of 5, 15, and 45 mg/kg BW X day; controls received potassium iodide for 4-7 weeks, and another group received sodium ipodate. At 4 weeks, amiodarone caused a dose-dependent increase in the serum T4 concentration and a slight reduction of serum TSH without a change in the serum T3 concentration. These changes were not present at 7 weeks. Sodium ipodate raised serum T4 concentrations at both times. Rats treated with T4 (150 micrograms/kg BW X day) to suppress thyroidal secretion of hormone and with amiodarone (15 mg/kg) had marked reduction of serum T3 concentrations compared with controls receiving T4 without amiodarone. Liver homogenates from rats treated with amiodarone showed marked reduction on T4 5'-monodeiodinase activity in a dose-related manner. Amiodarone added to liver homogenates in vitro at concentrations of 0.001-1 mM did not inhibit T3 production from T4, whereas ipodate added in vitro (0.01-1 mM) did inhibit T3 production. Rats treated with amiodarone showed a lowering of the resting heart rate and a reduction of the increment in heart rate after iv isoproterenol administration. The cardiac Ca++ myosin ATPase activity was reduced in rats receiving amiodarone (45 mg/kg) compared with that in controls. The data indicate that rats treated with amiodarone have reduced peripheral conversion of T4 to T3 owing to impaired hepatic T4 5'-monodeiodinase activity. In addition, these rats have slowing of heart rate and reduction of cardiac Ca++ myosin ATPase activity. These findings are consistent with the hypothesis that amiodarone blocks some effects of thyroid hormone on the heart, but additional studies are needed to test this hypothesis.

Transcription of the rat sarcoplasmic reticulum Ca2+ adenosine triphosphatase gene is increased by 3,5,3'-triiodothyronine receptor isoform-specific interactions with the myocyte-specific enhancer factor-2a.

Thyroid hormone (T3) increases the transcription of the sarcoplasmic reticulum Ca2+ adenosine triphosphatase (ATPase) gene (SERCA 2) through three thyroid hormone response elements. The existence of repetitive cis elements with different configurations is likely to serve specific functions such as interactions with nuclear transcription factors. In addition, the presence of different T3 receptor isoforms (T3Rs) may contribute to another level of complexity in providing specificity for T3 action. In this study, we investigated T3R alpha 1-vs. T3R beta 1-specific interactions with the myocyte enhancer-specific factor-2 (MEF-2) on the expression of the SERCA 2 gene in transient transfection assays in embryonal heart-derived H9c2 cells. MEF-2a in combination with either T3R alpha 1 or T3R beta 1 isoforms resulted in a 2.5-fold increase in SERCA 2 transgene expression in the absence of T3. Addition of T3 did not induce any further increase in SERCA 2 expression when T3R alpha 1 and MEF-2a expression vectors were cotransfected. In contrast, in the presence of T3R beta 1 and MEF-2, the addition of T3 increased chlorampenicol acetyltransferase activity by an additional 2.2-fold to a total 5.5-fold increase. The interaction between MEF-2a and T3R is transcription factor specific because another factor that binds to MEF-2 consensus sites (heart factor 1b) was not able to interact with T3R. In addition, MEF-2a failed to interact with other nuclear factors (cAMP response element-binding protein and Egr-1) that stimulate SERCA 2 gene transcription. In addition, we found that a single homologous thyroid hormone response element is not able to mediate the interactions between MEF-2a and T3Rs to increase SERCA 2 gene transcription. Our findings point to T3R isoform-specific interactions with a cell type-specific transcription factor (MEF-2) in the regulation of SERCA 2 gene expression.