Cytostatic gene therapy for vascular proliferative disorders with a constitutively active form of the retinoblastoma gene product.

Vascular smooth muscle cell (SMC) proliferation in response to injury is an important etiologic factor in vascular proliferative disorders such as atherosclerosis and restenosis after balloon angioplasty. The retinoblastoma gene product (Rb) is present in the unphosphorylated and active form in quiescent primary arterial SMCs, but is rapidly inactivated by phosphorylation in response to growth factor stimulation in vitro. A replication-defective adenovirus encoding a nonphosphorylatable, constitutively active form of Rb was constructed. Infection of cultured primary rat aortic SMCs with this virus inhibited growth factor-stimulated cell proliferation in vitro. Localized arterial infection with the virus at the time of balloon angioplasty significantly reduced SMC proliferation and neointima formation in both the rat carotid and porcine femoral artery models of restenosis. These results demonstrate the role of Rb in regulating vascular SMC proliferation and suggest a gene therapy approach for vascular proliferative disorders associated with arterial injury.

Transcriptional targeting of replication-defective adenovirus transgene expression to smooth muscle cells in vivo.

Gene transfer using replication-defective adenoviruses (RDAd) holds promise for the treatment of vascular proliferative disorders, but is potentially limited by the capacity of these viruses to infect multiple cell lineages. We have generated an RDAd vector, designated AdSM22-lacZ, which encodes the bacterial lacZ reporter gene under the transcriptional control of the smooth muscle cell (SMC)-specific SM22alpha promoter. Here, we show that in vitro AdSM22-lacZ programs expression of the lacZ reporter gene in primary rat aortic SMCs and immortalized A7r5 SMCs, but not in primary human umbilical vein endothelial cells (HUVECs) or NIH 3T3 cells. Consistent with these results, after intraarterial administration of AdSM22-lacZ to control and balloon-injured rat carotid arteries, beta-galactosidase activity was detected within SMCs of the tunica media and neointima, but not within endothelial or adventitial cells. Moreover, intravenous administration of AdSM22-lacZ did not result in lacZ gene expression in the liver or lungs. Finally, we have shown that direct injection of AdSM22-lacZ into SMC-containing tissues such as the ureter and bladder results in high-level transgene expression in visceral SMCs. Taken together, these results demonstrate that transgene expression after infection with an RDAd vector can be regulated in an SMC lineage-restricted fashion by using a transcriptional cassette containing the SMC-specific SM22alpha promoter. The demonstration of an efficient gene delivery system targeted specifically to SMCs provides a novel means to restrict expression of recombinant gene products to vascular or visceral SMCs in vivo.

The myeloid-cell-specific c-fes promoter is regulated by Sp1, PU.1, and a novel transcription factor.

The protein product of the c-fps/fes (c-fes) proto-oncogene has been implicated in the normal development of myeloid cells (macrophages and neutrophils). mRNA for c-fes has been detected exclusively in myeloid cells and vascular endothelial cells in adult mammals. Although a 13-kilobase-pair (kb) human c-fes transgene exhibits high levels of expression in mice, the sequences that confer myeloid-cell-specific expression of the human c-fes gene have not been defined. Transient-transfection experiments demonstrated that plasmids containing 446 bp of c-fes 5'-flanking sequences linked to a luciferase reporter gene were active exclusively in myeloid cells. No other DNA element within the 13-kb human c-fes locus contained positive cis-acting elements, with the exception of a weakly active region within the 3'-flanking sequences. DNase I footprinting assays revealed four distinct sites that bind myeloid nuclear proteins (-408 to -386, -293 to -254, -76 to -65, and -34 to +3). However, the first two footprints resided in sequences that were largely dispensable for transient activity. Plasmids containing 151 bp of 5'-flanking sequences confer myeloid-cell-specific gene expression. Electrophoretic mobility shift analyses demonstrated that the 151-bp region contains nuclear protein binding sites for Sp1, PU.1, and/or Elf-1, and a novel factor. This unidentified factor binds immediately 3' of the PU.1/Elf-1 sites and appears to be myeloid cell specific. Mutation of the PU.1/Elf-1 site or the 3' site (FP4-3') within the context of the c-fes promoter resulted in substantially reduced activity in transient transfections. Furthermore, transient-cotransfection assay demonstrated that PU.1 (and not Elf-1) can transactivate the c-fes promoter in nonmyeloid cell lines. We conclude that the human c-fes gene contains a strong myeloid-cell-specific promoter that is regulated by Sp1, PU.1, and a novel transcription factor.

A serum response factor-dependent transcriptional regulatory program identifies distinct smooth muscle cell sublineages.

The SM22alpha promoter has been used as a model system to define the molecular mechanisms that regulate smooth muscle cell (SMC) specific gene expression during mammalian development. The SM22alpha gene is expressed exclusively in vascular and visceral SMCs during postnatal development and is transiently expressed in the heart and somites during embryogenesis. Analysis of the SM22alpha promoter in transgenic mice revealed that 280 bp of 5' flanking sequence is sufficient to restrict expression of the lacZ reporter gene to arterial SMCs and the myotomal component of the somites. DNase I footprint and electrophoretic mobility shift analyses revealed that the SM22alpha promoter contains six nuclear protein binding sites (designated smooth muscle elements [SMEs] -1 to -6, respectively), two of which bind serum response factor (SRF) (SME-1 and SME-4). Mutational analyses demonstrated that a two-nucleotide substitution that selectively eliminates SRF binding to SME-4 decreases SM22alpha promoter activity in arterial SMCs by approximately 90%. Moreover, mutations that abolish binding of SRF to SME-1 and SME-4 or mutations that eliminate each SME-3 binding activity totally abolished SM22alpha promoter activity in the arterial SMCs and somites of transgenic mice. Finally, we have shown that a multimerized copy of SME-4 (bp -190 to -110) when linked to the minimal SM22alpha promoter (bp -90 to +41) is necessary and sufficient to direct high-level transcription in an SMC lineage-restricted fashion. Taken together, these data demonstrate that distinct transcriptional regulatory programs control SM22alpha gene expression in arterial versus visceral SMCs. Moreover, these data are consistent with a model in which combinatorial interactions between SRF and other transcription factors that bind to SME-4 (and that bind directly to SRF) activate transcription of the SM22alpha gene in arterial SMCs.

Identification and characterization of a cardiac-specific transcriptional regulatory element in the slow/cardiac troponin C gene.

The slow/cardiac troponin C (cTnC) gene has been used as a model system for defining the molecular mechanisms that regulate cardiac and skeletal muscle-specific gene expression during mammalian development. cTnC is expressed continuously in both embryonic and adult cardiac myocytes but is expressed only transiently in embryonic fast skeletal myotubes. We have reported previously that cTnC gene expression in skeletal myotubes is controlled by a developmentally regulated, skeletal muscle-specific transcriptional enhancer located within the first intron of the gene (bp 997 to 1141). In this report, we show that cTnC gene expression in cardiac myocytes both in vitro and in vivo is regulated by a distinct and independent transcriptional promoter and enhancer located within the immediate 5' flanking region of the gene (bp -124 to +32). DNase I footprint and electrophoretic mobility shift assay analyses demonstrated that this cardiac-specific promoter/enhancer contains five nuclear protein binding sites (designated CEF1, CEF-2, and CPF1-3), four of which bind novel cardiac-specific nuclear protein complexes. Functional analysis of the cardiac-specific cTnC enhancer revealed that mutation of either the CEF-1 or CEF-2 nuclear protein binding site abolished the activity of the cTnC enhancer in cardiac myocytes. Taken together, these results define a novel mechanism for developmentally regulating a single gene in multiple muscle cell lineages. In addition, they identify previously undefined cardiac-specific transcriptional regulatory motifs and trans-acting factors. Finally, they demonstrate distinct transcriptional regulatory pathways in cardiac and skeletal muscle.

Analysis of SM22alpha-deficient mice reveals unanticipated insights into smooth muscle cell differentiation and function.

SM22alpha is a 22-kDa smooth muscle cell (SMC) lineage-restricted protein that physically associates with cytoskeletal actin filament bundles in contractile SMCs. To examine the function of SM22alpha, gene targeting was used to generate SM22alpha-deficient (SM22(-/-LacZ)) mice. The gene targeting strategy employed resulted in insertion of the bacterial lacZ reporter gene at the SM22alpha initiation codon, permitting precise analysis of the temporal and spatial pattern of SM22alpha transcriptional activation in the developing mouse. Northern and Western blot analyses confirmed that the gene targeting strategy resulted in a null mutation. Histological analysis of SM22(+/-LacZ) embryos revealed detectable beta-galactosidase activity in the unturned embryonic day 8.0 embryo in the layer of cells surrounding the paired dorsal aortae concomitant with its expression in the primitive heart tube, cephalic mesenchyme, and yolk sac vasculature. Subsequently, during postnatal development, beta-galactosidase activity was observed exclusively in arterial, venous, and visceral SMCs. SM22alpha-deficient mice are viable and fertile. Their blood pressure and heart rate do not differ significantly from their control SM22alpha(+/-) and SM22alpha(+/+) littermates. The vasculature and SMC-containing tissues of SM22alpha-deficient mice develop normally and appear to be histologically and ultrastructurally similar to those of their control littermates. Taken together, these data demonstrate that SM22alpha is not required for basal homeostatic functions mediated by vascular and visceral SMCs in the developing mouse. These data also suggest that signaling pathways that regulate SMC specification and differentiation from local mesenchyme are activated earlier in the angiogenic program than previously recognized.

The GATA-4 transcription factor transactivates the cardiac muscle-specific troponin C promoter-enhancer in nonmuscle cells.

The unique contractile phenotype of cardiac myocytes is determined by the expression of a set of cardiac muscle-specific genes. By analogy to other mammalian developmental systems, it is likely that the coordinate expression of cardiac genes is controlled by lineage-specific transcription factors that interact with promoter and enhancer elements in the transcriptional regulatory regions of these genes. Although previous reports have identified several cardiac muscle-specific transcriptional elements, relatively little is known about the lineage-specific transcription factors that regulate these elements. In this report, we demonstrate that the slow/cardiac muscle-specific troponin C (cTnC) enhancer contains a specific binding site for the lineage-restricted zinc finger transcription factor GATA-4. This GATA-4-binding site is required for enhancer activity in primary cardiac myocytes. Moreover, the cTnC enhancer can be transactivated by overexpression of GATA-4 in non-cardiac muscle cells such as NIH 3T3 cells. In situ hybridization studies demonstrate that GATA-4 and cTnC have overlapping patterns of expression in the hearts of postimplantation mouse embryos and that GATA-4 gene expression precedes cTnC expression. Indirect immunofluorescence reveals GATA-4 expression in cultured cardiac myocytes from neonatal rats. Taken together, these results are consistent with a model in which GATA-4 functions to direct tissue-specific gene expression during mammalian cardiac development.

A novel myogenic regulatory circuit controls slow/cardiac troponin C gene transcription in skeletal muscle.

The slow/cardiac troponin C (cTnC) gene is expressed in three distinct striated muscle lineages: cardiac myocytes, embryonic fast skeletal myotubes, and adult slow skeletal myocytes. We have reported previously that cTnC gene expression in cardiac muscle is regulated by a cardiac-specific promoter/enhancer located in the 5' flanking region of the gene (bp -124 to +1). In this report, we demonstrate that the cTnC gene contains a second distinct and independent transcriptional enhancer which is located in the first intron. This second enhancer is skeletal myotube specific and is developmentally up-regulated during the differentiation of myoblasts to myotubes. This enhancer contains three functionally important nuclear protein binding sites: a CACCC box, a MEF-2 binding site, and a previously undescribed nuclear protein binding site, designated MEF-3, which is also present in a large number of skeletal muscle-specific transcriptional enhancers. Unlike most skeletal muscle-specific transcriptional regulatory elements, the cTnC enhancer does not contain a consensus binding site (CANNTG) for the basic helix-loop-helix (bHLH) family of transcription factors and does not directly bind MyoD-E12 protein complexes. Despite these findings, the cTnC enhancer can be transactivated by overexpression of the myogenic bHLH proteins, MyoD and myogenin, in C3H10T1/2 (10T1/2) cells. Electrophoretic mobility shift assays demonstrated changes in the patterns of MEF-2, CACCC, and MEF-3 DNA binding activities following the conversion of 10T1/2 cells into myoblasts and myotubes by stable transfection with a MyoD expression vector. In particular, MEF-2 binding activity was up-regulated in 10T1/2 cells stably transfected with a MyoD expression vector only after these cells fused and differentiated into skeletal myotubes. Taken together, these results demonstrated that distinct lineage-specific transcriptional regulatory elements control the expression of a single myofibrillar protein gene in fast skeletal and cardiac muscle. In addition, they show that bHLH transcription factors can indirectly transactivate the expression of some muscle-specific genes.

The GATA-4 transcription factor transactivates the cardiac-specific troponin C promoter-enhancer in non-muscle cells.

The unique contractile phenotype of cardiac myocytes is determined by the expression of a set of cardiac-specific genes. By analogy to other mammalian developmental systems, it is likely that the coordinate expression of cardiac genes is controlled by lineage-specific transcription factors that interact with promoter and enhancer elements in the transcriptional regulatory regions of these genes. Here, we demonstrate that the slow/cardiac-specific troponin C (cTnC) enhancer contains a specific binding site for the lineage-restricted, zinc finger transcription factor, GATA-4 and that GATA-4 mRNA and protein is expressed in cardiac myocytes. In addition, GATA-4 binding sites were identified in several previously characterized cardiac-specific transcriptional regulatory elements. The cTnC GATA-4 binding site is required for transcriptional enhancer activity in primary cardiac myocytes. Moreover, the cTnC enhancer can be transactivated by over-expression of GATA-4 in non-cardiac muscle cells such as NIH 3T3 cells. Taken together, these results are consistent with a model in which GATA-4 functions to direct tissue-specific gene expression during mammalian cardiac development.

Structure and expression of the murine slow/cardiac troponin C gene.

Cardiac troponin C (cTnC) is the calcium-binding subunit of the myofibrillar thin filament that regulates excitation-contraction coupling in cardiac muscle. We have utilized a novel polymerase chain reaction cloning procedure to isolate cDNA clones encoding murine cTnC. Murine cTnC is a 161-amino acid polypeptide that has been highly conserved during evolution. Southern blot analyses demonstrated that the cTnC gene is a member of a multigene family. Northern blot analyses revealed that the cTnC gene is expressed in murine cardiac tissue and slow skeletal muscle (soleus), but is not expressed in fast skeletal muscle (extensor digitorum longus and anterior tibialis) or in neonatal or adult brain, kidney, lung, liver, or testis. In addition, while the cTnC gene is not expressed in murine C2C12 myoblasts, differentiation of these cells into myotubes was shown to result in a dramatic induction of cTnC gene expression. A full length cTnC genomic clone was isolated from a murine genomic library by hybridization with a cTnC cDNA probe and structurally characterized by DNA sequence, primer extension, and S1 nuclease protection analyses. The cTnC gene is 3.4 kilobase pairs long and is composed of six exons. The introns do not appear to divide the gene into functional domains. Analysis of the 5'-flanking region of the gene revealed the presence of a consensus TATA box 24 base pairs 5' of the transcription start site. Despite the finding that the gene is expressed only in cardiac and slow skeletal muscle, it lacks the previously described CArG and M-CAT transcriptional regulatory sequence motifs that are involved in regulating the expression of a number of other myofibrillar genes.

Effect of aging on brain respiration and carbohydrate metabolism of Syrian hamsters.

Syrian hamsters were used to study the effect of aging on brain slice respiration and metabolism. Young animals (average age 8 months) and old animals (average age 18 months) were incubated under standard conditions with the following parameters being measured: oxygen uptake, 14CO2 production, glucose utilization, lactate and pyruvate formation. No differences were found in the two groups. It is still very likely that subtle differences exist but can only be documented under conditions of metabolic stress.

GATA-6: a zinc finger transcription factor that is expressed in multiple cell lineages derived from lateral mesoderm.

Members of the GATA family of zinc finger transcription factors play important roles in the development of several mesodermally derived cell lineages. In the studies described in this report, we have isolated and functionally characterized the murine GATA-6 cDNA and protein and defined the temporal and spatial patterns of GATA-6 gene expression during mammalian development. The GATA-6 and -4 proteins share high-level amino acid sequence identity over a proline-rich region at the amino terminus of the protein that is not conserved in other GATA family members. GATA-6 binds to a functionally important nuclear protein binding site within the cardiac-specific cardiac troponin C (cTnC) transcriptional enhancer. Moreover, the cTnC promoter enhancer can be transactivated by overexpression of GATA-6 in noncardiac muscle cells. During early murine embryonic development, the patterns of GATA-6 and -4 gene expression are similar, with expression of GATA-6 restricted to the precardiac mesoderm, the embryonic heart tube, and the primitive gut. However, coincident with the onset of vasculogenesis and development of the respiratory and urogenital tracts, only the GATA-6 gene is expressed in arterial smooth muscle cells, the developing bronchi, and the urogenital ridge and bladder. These data are consistent with a model in which GATA-6 functions in concert with GATA-4 to direct tissue-specific gene expression during formation of the mammalian heart and gastrointestinal tract, but performs a unique function in programming lineage-restricted gene expression in the arterial system, the bladder, and the embryonic lung.

Protein synthesis and degradation during starvation-induced cardiac atrophy in rabbits.

To determine the relative importance of protein degradation in the development of starvation-induced cardiac atrophy, in vivo fractional synthetic rates of total cardiac protein, myosin heavy chain, actin, light chain 1, and light chain 2 were measured in fed and fasted rabbits by continuous infusion of [3H] leucine. In addition, the rate of left ventricular protein accumulation and loss were assessed in weight-matched control and fasted rabbits. Rates of total cardiac protein degradation were then estimated as the difference between rates of synthesis and growth. Fasting produced left ventricular atrophy by decreasing the rate of left ventricular protein synthesis (34.8 +/- 1.4, 27.3 +/- 3.0, and 19.3 +/- 1.2 mg/day of left ventricular protein synthesized for 0-, 3-, and 7-day fasted rabbits, respectively). Inhibition of contractile protein synthesis was evident by significant reductions in the fractional synthetic rates of all myofibrillar protein subunits. Although fractional rates of protein degradation increased significantly within 7 days of fasting, actual amounts of left ventricular protein degraded per day were unaffected. Thus, prolonged fasting profoundly inhibits the synthesis of new cardiac protein, including the major protein constituents of the myofibril. Both this inhibition in new protein synthesis as well as a smaller but significant reduction in the average half-lives of cardiac proteins are responsible for atrophy of the heart in response to fasting.

Expression of recombinant genes in myocardium in vivo after direct injection of DNA.

The ability to program recombinant gene expression in cardiac myocytes in vivo holds promise for the treatment of many inherited and acquired cardiovascular diseases. In this report, we demonstrate that a recombinant beta-galactosidase gene under the control of the Rous sarcoma virus promoter can be introduced into and expressed in adult rat cardiac myocytes in vivo by the injection of purified plasmid DNA directly into the left ventricular wall. Cardiac myocytes expressing recombinant beta-galactosidase were detected histochemically in rat hearts for at least 4 weeks after injection of the beta-galactosidase gene. These results demonstrate the potential of this method of somatic gene therapy for the treatment of cardiovascular disease.

The structure and regulation of expression of the murine fast skeletal troponin C gene. Identification of a developmentally regulated, muscle-specific transcriptional enhancer.

Fast skeletal muscle troponin C (sTnC) is the calcium-binding subunit of the myofibrillar thin filament that regulates excitation-contraction coupling. Utilizing a polymerase chain reaction cloning strategy, we have isolated cDNA clones encoding murine sTnC. The 160-amino acid sTnC protein shares 70% amino acid sequence identity with the slow/cardiac isoform of troponin C (cTnC). However, three areas of significant sequence divergence were identified. Southern blot analyses demonstrated that murine sTnC is encoded by a single copy gene that is distinct from that which encodes cTnC. Northern blot analyses showed that the sTnC gene is expressed exclusively in skeletal muscle (extensor digitorum and anterior tibialis) and not in neonatal or adult heart, brain, kidney, liver, lung, or testes. Studies of the murine C2C12 myoblast cell line demonstrated that sTnC gene expression is developmentally regulated during the differentiation of these myoblasts into myotubes. A full-length murine sTnC genomic clone was isolated and characterized by DNA sequence, primer extension, and S1 nuclease protection analyses. The sTnC gene is composed of six exons spanning 2.6 kilobase pairs of genomic DNA. Although the introns do not divide the gene into functional domains, the intron-exon borders are nearly identical to those of the other members of the troponin C multigene family. Transient transfection assays using chloramphenicol acetyltransferase reporter plasmids demonstrated that the sTnC promoter alone is relatively inactive in muscle cells and that high level sTnC gene expression in these cells is controlled by a potent transcriptional enhancer element located within the first intron of the gene. In additional transfection experiments, the sTnC enhancer was shown to display three important biological activities. (i) It was required for high level transcription from the sTnC promoter in muscle cells; (ii) its activity was muscle cell specific; and (iii) its activity was developmentally regulated during the differentiation of C2C12 myoblasts to myotubes. Taken together, these data define the sTnC gene as an excellent model system for studies of developmentally regulated gene expression in skeletal muscle.

GATA-4 activates transcription via two novel domains that are conserved within the GATA-4/5/6 subfamily.

GATA-4 is one of the earliest developmental markers of the precardiac mesoderm, heart, and gut and has been shown to activate regulatory elements controlling transcription of genes encoding cardiac-specific proteins. To elucidate the molecular mechanisms underlying the transcriptional activity of the GATA-4 protein, structure-function analyses were performed. These analyses revealed that the C-terminal zinc finger and adjacent basic domain of GATA-4 is bifunctional, modulating both DNA-binding and nuclear localization activities. The N terminus of the protein encodes two independent transcriptional Activation Domains (amino acids 1-74 and amino acids 130-177). Amino acid residues were identified within each domain that are required for transcriptional activation. Finally, we have shown that regions of Xenopus GATA-5 and -6 corresponding to Activation Domains I and II, respectively, function as potent transcriptional activators. The identification and functional characterization of two evolutionarily conserved transcriptional Activation Domains within the GATA-4/5/6 subfamily suggests that each of these domains modulates critical functions in the transcriptional regulatory program(s) encoded by GATA-4, -5, and -6 during vertebrate development. As such these data provide novel insights into the molecular mechanisms that control development of the heart.

GATA-5: a transcriptional activator expressed in a novel temporally and spatially-restricted pattern during embryonic development.

Members of the GATA family of zinc finger transcription factors regulate critical steps of cellular differentiation during vertebrate development. In the studies described in this report, we have isolated and functionally characterized the murine GATA-5 cDNA and protein and defined the temporal and spatial pattern of GATA-5 gene expression during mammalian development. The amino terminus of the mouse GATA-5 protein shares high level amino acid sequence identity with the murine GATA-4 and -6 proteins, but not with other members of the GATA family. GATA-5 binds to the functionally important CEF-1 nuclear protein binding site in the cardiac-specific slow/cardiac troponin C (cTnC) transcriptional enhancer and overexpression of GATA-5 transactivates the cTnC enhancer in noncardiac muscle cell lines. During embryonic and postnatal development, the pattern of GATA-5 gene expression differs significantly from that of other GATA family members. In the primitive streak embryo, GATA-5 mRNA is detectable in the precardiac mesoderm. Within the embryonic heart, the GATA-5 gene is expressed within the atrial and ventricular chambers (ED 9.5), becomes restricted to the atrial endocardium (ED 12.5), and is subsequently not expressed in the heart during late fetal and postnatal development. Moreover, coincident with the earliest steps in lung development, only the GATA-5 gene is expressed within the pulmonary mesenchyme. Finally, the GATA-5 gene is expressed in tissue-restricted subsets of smooth muscle cells (SMCs), including bronchial SMCs and SMCs in the bladder wall. These data are consistent with a model in which GATA-5 performs a unique temporally and spatially restricted function in the embryonic heart and lung. Moreover, these data suggest that GATA-5 may play an important role in the transcriptional program(s) that underlies smooth muscle cell diversity.

Effect of aging on brain respiration and carbohydrate metabolism of CBF1 mice.

Brain slices of mice (strain CBF1) were used to study the effect of aging on cerebral cortex respiration and metabolism. Young animals (average age 6 months) were compared with old animals (average age 34 months). Metabolism was measured at a normal temperature (37 degrees C) and under hyperthermic stress (40 degrees C). The brain slices were incubated with 14C-glucose under standard conditions with the following parameters being measured: oxygen uptake, 14CO2 production, glucose utilization, and lactate and pyruvate formation. At the normal temperature, there were significant age-associated decreases in oxygen uptake and 14CO2 production but the other parameters were unchanged. At hyperthermic conditions there were significant age-associated decreases in oxygen uptake, 14CO2 production, lactate production, and glucose utilization. Also, in the hyperthermia study, all values were raised from control study values (37 degrees C) with old animals showing smaller increases in glucose utilization and lactate formation. These findings indicate the dysfunction of a number of metabolic pathways in the aged animal.

Cardiac protein synthesis and degradation during thyroxine-induced left ventricular hypertrophy.

Assessment of cardiac protein metabolism in thyroxine-induced left ventricular hypertrophy requires measurements of both protein synthesis and degradation. In vivo protein degradative rates can best be measured as the difference between rates of protein synthesis and growth. Accordingly, rates of left ventricular protein accumulation were determined in growing rabbits, and in animals administered intravenous L-thyroxine (200 micrograms X kg-1 X day-1) for up to 15 days. Left ventricular protein fractional synthetic rates in euthyroid and thyroxine-treated rabbits were measured by continuous infusion of [3H]leucine (200 mu Ci/h X 6 h), and results converted to milligrams protein synthesized and degraded per day. Thyroxine administration produced left ventricular hypertrophy by increasing the rate of total protein synthesis (35.7 +/- 2.0, 71.0 +/- 7.0, and 62.6 +/- 4.0 mg of left ventricular protein synthesized per day for 0-, 3-, and 9-day, thyroxine-treated rabbits, respectively). However, the increased rate of total protein synthesis was greater than the measured rate of total protein accumulation (8.1 vs. 15.9 mg protein/day for euthyroid and thyroxine-treated animals), indicating that left ventricular protein degradative rates were increased as well. These studies indicate that accelerated proteolysis may be important in the molecular and architectural remodeling of the rapidly hypertrophying heart during thyrotoxicosis.

Lysosomal changes during thyroxine-induced left ventricular hypertrophy in rabbits.

By use of a combined morphologic, immunocytochemical, and biochemical approach, this study demonstrates the changes in the lysosomal vacuolar apparatus that accompany thyroxine-induced cardiac hypertrophy. During the 1st wk of thyroxine administration, immunocytochemical studies revealed a decline in cathepsin D within many myocytes, but an increase within interstitial cells. These events transpired with only a modest rise in cathepsin D activity. During the 2nd wk, cathepsin D reappeared within most myocytes and continued to increase within the interstitial population and was associated with a general rise in lysosomal enzyme activity. These data demonstrate the importance of employing both immunocytochemical and biochemical approaches to evaluate the status of the lysosomal vacuolar apparatus, and suggests that the lysosomal vacuolar apparatus may be involved in the apparent remodeling that attends rapid cardiac growth, which may represent one component of the enhanced rates of proteolysis documented in this model of cardiac hypertrophy.