Hypertrophic cardiomyopathy caused by a novel alpha-tropomyosin mutation (V95A) is associated with mild cardiac phenotype, abnormal calcium binding to troponin, abnormal myosin cycling, and poor prognosis.

BACKGROUND: We report hypertrophic cardiomyopathy (HCM) in a Spanish-American family caused by a novel alpha-tropomyosin (TPM1) mutation and examine the pathogenesis of the clinical disease by characterizing functional defects in the purified mutant protein. METHODS AND RESULTS: HCM was linked to the TPM1 gene (logarithm of the odds [LOD] score 3.17). Sequencing and restriction digestion analysis demonstrated a TPM1 mutation V95A that cosegregated with HCM. The mutation has been associated with 13 deaths in 26 affected members (11 sudden deaths and 2 related to heart failure), with a cumulative survival rate of 73+/-10% at the age of 40 years. Left ventricular wall thickness (mean 16+/-6 mm) and disease penetrance (53%) were similar to those for the ss-myosin mutations L908V and G256E previously associated with a benign prognosis. Left ventricular hypertrophy was milder than with the ss-myosin mutation R403Q, but the prognosis was similarly poor. With the use of recombinant tropomyosins, we identified several functional alterations at the protein level. The mutation caused a 40% to 50% increase in calcium affinity in regulated thin filament-myosin subfragment-1 (S1) MgATPase assays, a 20% decrease in MgATPase rates in the presence of saturating calcium, a 5% decrease in unloaded shortening velocity in in vitro motility assays, and no change in cooperative myosin S1 binding to regulated thin filaments. CONCLUSIONS: In contrast to other reported TPM1 mutations, V95A-associated HCM exhibits unusual features of mild phenotype but poor prognosis. Both myosin cycling and calcium binding to troponin are abnormal in the presence of the mutant tropomyosin. The genetic diagnosis afforded by this mutation will be valuable in the management of HCM.

A new model of cooperative myosin-thin filament binding.

Cooperative myosin binding to the thin filament is critical to regulation of cardiac and skeletal muscle contraction. This report delineates and fits to experimental data a new model of this process, in which specific tropomyosin-actin interactions are important, the tropomyosin-tropomyosin polymer is continuous rather than disjointed, and tropomyosin affects myosin-actin binding by shifting among three positions as in recent structural studies. A myosin- and tropomyosin-induced conformational change in actin is proposed, rationalizing the approximately 10,000-fold strengthening effect of myosin on tropomyosin-actin binding. Also, myosin S1 binding to regulated filaments containing mutant tropomyosins with internal deletions exhibited exaggerated cooperativity, implying an allosteric effect of tropomyosin on actin and allowing the effect's measurement. Comparisons among the mutants suggest the change in actin is promoted much more strongly by the middle of tropomyosin than by its ends. Regardless of calcium binding to troponin, this change in actin facilitates the shift in tropomyosin position to the actin inner domain, which is required for tight myosin-actin association. It also increases myosin-actin affinity 7-fold compared with the absence of troponin-tropomyosin. Finally, initiation of a shift in tropomyosin position is 100-fold more difficult than is its extension from one actin to the next, producing the myosin binding cooperativity that underlies cooperative activation of muscle contraction.

Three-dimensional reconstruction of thin filaments containing mutant tropomyosin.

Interactions of the components of reconstituted thin filaments were investigated using a tropomyosin internal deletion mutant, D234, in which actin-binding pseudo-repeats 2, 3, and 4 are missing. D234 retains regions of tropomyosin that bind troponin and form end-to-end tropomyosin bonds, but has a length to span only four instead of seven actin monomers. It inhibits acto-myosin subfragment 1 ATPase (acto-S-1 ATPase) and filament sliding in vitro in both the presence and absence of Ca(2+) (, J. Biol. Chem. 272:14051-14056) and lowers the affinity of S-1.ADP for actin while increasing its cooperative binding. Electron microscopy and three-dimensional reconstruction of reconstituted thin filaments containing actin, troponin, and wild-type or D234 tropomyosin were carried out to determine if Ca(2+)-induced movement of D234 occurred in the filaments. In the presence and absence of Ca(2+), the D234 position was indistinguishable from that of the wild-type tropomyosin, demonstrating that the mutation did not affect normal tropomyosin movement induced by Ca(2+) and troponin. These results suggested that, in the presence of Ca(2+) and troponin, D234 tropomyosin was trapped on filaments in the Ca(2+)-induced position and was unable to undergo a transition to a completely activated position. By adding small amounts of rigor-bonded N-ethyl-maleimide-treated S-1 to mutant thin filaments, thus mimicking the myosin-induced "open" state, inhibition could be overcome and full activation restored. This myosin requirement for full activation provides support for the existence of three functionally distinct thin filament states (off, Ca(2+)-induced, myosin-induced; cf.;, J. Mol. Biol. 266:8-14). We propose a further refinement of the three-state model in which the binding of myosin to actin causes allosteric changes in actin that promote the binding of tropomyosin in an otherwise energetically unfavorable "open" state.

Determination of the human cytochrome P450 isoforms involved in the metabolism of zolmitriptan.

1. Zolmitriptan was extensively metabolized by freshly isolated human hepatocytes to a number of components including the three main metabolites observed in vivo (N-desmethyl-zolmitriptan, zolmitriptan N-oxide and the indole acetic acid derivative). In contrast, metabolism of zolmitriptan by human hepatic microsomes was extremely limited with only small amounts of the N-desmethyl and indole ethyl alcohol metabolites being produced. 2. Furafylline, a selective inhibitor of CYP1A2, almost completely abolished the hepatocellular metabolism of zolmitriptan and markedly inhibited formation of the N-desmethyl metabolite in microsomes. Chemical inhibitors, selective against other major human cytochrome P450 (CYP2C9, 2C19, 2D6 and 3A4), had no obvious effects. In addition, expressed human CYP1A2 was the only cytochrome P450 to form the N-desmethyl metabolite. 3. N-desmethyl-zolmitriptan was extensively metabolized by both human hepatocytes and microsomes. The indole acetic acid and ethyl alcohol derivatives were the major metabolites formed by hepatocytes, whereas only the indole ethyl alcohol derivative was produced by microsomes. Metabolism of N-desmethyl-zolmitriptan was not inhibited by cytochrome P450-selective chemical inhibitors nor was it observed following incubation with expressed human cytochrome P450. Clorgyline, a selective inhibitor of monoamine oxidase A (MAO-A), markedly inhibited the microsomal formation of the indole ethyl alcohol derivative. 4. Primary metabolism of zolmitriptan is dependent mainly on CYP1A2, whereas MAO-A is responsible for further metabolism of N-desmethyl-zolmitriptan, the active metabolite. Since the in vivo clearance of zolmitriptan is primarily dependent on metabolism, interactions with drugs that induce or inhibit CYP1A2 or MAO-A may be anticipated.

Functional consequences of troponin T mutations found in hypertrophic cardiomyopathy.

Missense mutations in the cardiac thin filament protein troponin T (TnT) are a cause of familial hypertrophic cardiomyopathy (FHC). To understand how these mutations produce dysfunction, five TnTs were produced and purified containing FHC mutations found in several regions of TnT. Functional defects were diverse. Mutations F110I, E244D, and COOH-terminal truncation weakened the affinity of troponin for the thin filament. Mutation DeltaE160 resulted in thin filaments with increased calcium affinity at the regulatory site of troponin C. Mutations R92Q and F110I resulted in impaired troponin solubility, suggesting abnormal protein folding. Depending upon the mutation, the in vitro unloaded actin-myosin sliding speed showed small increases, showed small decreases, or was unchanged. COOH-terminal truncation mutation resulted in a decreased thin filament-myosin subfragment 1 MgATPase rate. The results indicate that the mutations cause diverse immediate effects, despite similarities in disease manifestations. Separable but repeatedly observed abnormalities resulting from FHC TnT mutations include increased unloaded sliding speed, increased or decreased Ca(2+) affinity, impairment of folding or sarcomeric integrity, and decreased force. Enhancement as well as impairment of contractile protein function is observed, suggesting that TnT, including the troponin tail region, modulates the regulation of cardiac contraction.

Roles for the troponin tail domain in thin filament assembly and regulation. A deletional study of cardiac troponin T.

Striated muscle contraction is regulated by Ca2+ binding to troponin, which has a globular domain and an elongated tail attributable to the NH2-terminal portion of the bovine cardiac troponin T (TnT) subunit. Truncation of the bovine cardiac troponin tail was investigated using recombinant TnT fragments and subunits TnI and TnC. Progressive truncation of the troponin tail caused progressively weaker binding of troponin-tropomyosin to actin and of troponin to actin-tropomyosin. A sharp drop-off in affinity occurred with NH2-terminal deletion of 119 rather than 94 residues. Deletion of 94 residues had no effect on Ca2+-activation of the myosin subfragment 1-thin filament MgATPase rate and did not eliminate cooperative effects of Ca2+ binding. Troponin tail peptide TnT1-153 strongly promoted tropomyosin binding to actin in the absence of TnI or TnC. The results show that the anchoring function of the troponin tail involves interactions with actin as well as with tropomyosin and has comparable importance in the presence or absence of Ca2+. Residues 95-153 are particularly important for anchoring, and residues 95-119 are crucial for function or local folding. Because striated muscle regulation involves switching among the conformational states of the thin filament, regulatory significance for the troponin tail may arise from its prominent contribution to the protein-protein interactions within these conformations.

Effects of propofol on human hepatic microsomal cytochrome P450 activities.

1. The potential of propofol to inhibit the activity of major human cytochrome P450 enzymes has been examined in vitro using human liver microsomes. Propofol produced inhibition of CYP1A2 (phenacetin O-deethylation), CYP2C9 (tolbutamide 4'-hydroxylation), CYP2D6 (dextromethorphan O-demethylation) and CYP3A4 (testosterone 6beta-hydroxylation) activities with IC50 = 40, 49, 213 and 32 microM respectively. Ki for propofol against all of these enzymes with the exception of CYP2D6, where propofol showed little inhibitory activity, was 30, 30 and 19 microM respectively for CYPs 1A2, 2C9 and 3A4. 2. Furafylline, sulphaphenazole, quinidine and ketoconazole, known selective inhibitors of CYPs 1A2, 2C9, 2D6 and 3A4 respectively, were much more potent than propofol having IC50 = 0.8, 0.5, 0.2 and 0.1 microM; furafylline and sulphaphenazole yielded Ki = 0.6 and 0.7 microM respectively. 3. The therapeutic blood concentration of propofol (20 microM; 3-4 microg/ml) together with the in vitro Ki estimates for each of the major human P450 enzymes have been used to estimate the extent of cytochrome P450 inhibition, which may be produced in vivo by propofol. This in vitro-in vivo extrapolation indicates that the degree of inhibition of CYP1A2, 2C9 and 3A4 activity which could theoretically be produced in vivo by propofol is relatively low (40-51%); this is considered unlikely to have any pronounced clinical significance. 4. Although propofol has now been used in > 190 million people since its launch in 1986, there are only single reports of possible drug interactions between propofol and either alfentanil or warfarin. Consequently, it is difficult to conclude from either the published literature or the ZENECA safety database whether there is any evidence to indicate that propofol produces clinically significant drug interactions through inhibition of cytochrome P450-related drug metabolism.

Enzyme-inducing effects of bicalutamide in mouse, rat and dog.

1. Bicalutamide, a non-steroidal antiandrogen, produced dose-related increases in total cytochrome P450 (P450) and aldrin epoxidase, but had no effect on ethoxyresorufin O-deethylase, when administered for 10 weeks at 0, 25, 75 and 150 mg/kg/day to the male dog. 2. In the male and female mouse, bicalutamide, administered orally at 75 mg/kg/day for 3 months, produced marked induction of total P450, ethoxycoumarin O-deethylase, pentoxyresorufin O-dealkylase and aldrin epoxidase. Immunoblotting showed that bicalutamide produced substantial induction of CYP2B isoforms, with lower increases in CYP3A. Immunohistochemistry of mouse liver sections also showed marked increases in the level of CYP2B isoforms, with an increase in the extent of distribution from centrilobular to panlobular; CYP3A isoforms were also increased, but to a lesser degree. 3. Bicalutamide, administered as 14 daily oral doses (250 mg/kg) to groups of male rats, produced increases primarily in ethoxycoumarin O-deethylase and erythromycin N-demethylase, together with smaller increases in ethoxyresorufin O-deethylase and pentoxyresorufin O-dealkylase; these changes were reversible within 7 days. Immunoblotting of microsomes and immunocytochemistry of liver sections showed that bicalutamide markedly induced CYP3A1, but had little effect on CYP2B1 in rat. Compared with dexamethasone, bicalutamide is a more selective inducer of CYP3A1 in rat. 4. Bicalutamide, administered to rats as 14 daily oral doses of 10 mg/kg, induced its own metabolism by stimulating both aromatic hydroxylation and direct glucuronidation. This effect was apparently offset by a concomitant decrease in hydrolysis of bicalutamide, resulting in no marked change in total amounts of dose eliminated over 2 days. 5. Although the secondary effects of enzyme induction result in thyroid hypertrophy and adenoma in rat and hepatocellular carcinoma in mouse following chronic administration of bicalutamide, these changes are considered to have little clinical relevance. In any case, bicalutamide does not produce enzyme induction in man at clinically relevant dose levels.

Cooperative effect of calcium binding to adjacent troponin molecules on the thin filament-myosin subfragment 1 MgATPase rate.

The myosin subfragment 1 (S1) MgATPase rate was measured using thin filaments with known extents of Ca2+ binding controlled by varying the ratio of native cardiac troponin versus an inhibitory troponin with a mutation in the sole regulatory Ca2+ binding site of troponin C. Fractional MgATPase activation was less than the fraction of troponins that bound Ca2+, implying a cooperative effect of bound Ca2+ on cross-bridge cycling. Addition of phalloidin did not alter cooperative effects between bound Ca2+ molecules in the presence or absence of myosin S1. When the myosin S1 concentration was raised sufficiently to introduce cooperative myosin-myosin effects, lower Ca2+ concentrations were needed to activate the MgATPase rate. MgATPase activation remained less than Ca2+ binding, implying a true, not just an apparent, increase in Ca2+ affinity. MgATPase activation by Ca2+ was more cooperative than could be explained by cooperativeness of overall Ca2+ binding, the discrepancy between Ca2+ binding and MgATPase activation, or interactions between myosins. The results suggest the thin filament-myosin S1 MgATPase cycle requires calcium binding to adjacent troponin molecules and that this binding is cooperatively promoted by a single cycling cross-bridge. This mechanism is a potential explanation for Ca2+-mediated regulation of cross-bridge kinetics in muscle fibers.

Effects of cardiac thin filament Ca2+: statistical mechanical analysis of a troponin C site II mutant.

Cardiac thin filaments contain many troponin C (TnC) molecules, each with one regulatory Ca2+ binding site. A statistical mechanical model for the effects of these sites is presented and investigated. The ternary troponin complex was reconstituted with either TnC or the TnC mutant CBMII, in which the regulatory site in cardiac TnC (site II) is inactivated. Regardless of whether Ca2+ was present, CBMII-troponin was inhibitory in a thin filament-myosin subfragment 1 MgATPase assay. The competitive binding of [3H]troponin and [14C]CBMII-troponin to actin.tropomyosin was measured. In the presence of Mg2+ and low free Ca2+ they had equal affinities for the thin filament. When Ca274+ was added, however, troponin's affinity for the thin filament was 2.2-fold larger for the mutant than for the wild type troponin. This quantitatively describes the effect of regulatory site Ca2+ on troponin's affinity for actin.tropomyosin; the decrease in troponin-thin filament binding energy is small. Application of the theoretical model to the competitive binding data indicated that troponin molecules bind to interdependent rather than independent sites on the thin filament. Ca2+ binding to the regulatory site of TnC has a long-range rather than a merely local effect. However, these indirect TnC-TnC interactions are weak, indicating that the cooperativity of muscle activation by Ca2+ requires other sources of cooperativity.

Compensated coronary microvascular growth in senescent rats with thyroxine-induced cardiac hypertrophy.

We tested the hypothesis that coronary angiogenesis in response to chronic thyroxine (T4) treatment is not limited by age. Male Fischer 344 rats aged either 8 (young adult) or 24 (senescent) mo were studied after receiving either L-thyroxine (0.2 mg/kg sc) or vehicle for 2 mo. Heart weight-to-body weight ratio, compared with age-matched controls, increased by 47 and 44% in 8- and 24-mo T4 groups, respectively. Maximal myocardial perfusion per unit mass, measured in diastole-arrested, maximally dilated, isolated hearts, was similar in T4 rats and their age group controls; however, flow tended to be lower in senescent than in young adult rats. Thus the cross-sectional area of the coronary vessels grew in proportion to the increase in cardiac mass. Morphometric analyses, based on image analysis, showed that capillary length density was slightly lower in the midmyocardium but not the epimyocardium of the 24-mo T4 group compared with their age group controls. However, volume density, surface density, and intercapillary distance were not influenced by T4 treatment and the presence of cardiac hypertrophy. We conclude that in this model of cardiac hypertrophy 1) coronary vessel growth parallels the increase in ventricular mass, 2) capillaries grow by proliferation and an increase in diameter, and 3) vascular growth is not notably compromised during senescence.

Equilibrium linkage analysis of cardiac thin filament assembly. Implications for the regulation of muscle contraction.

A major focus in studies of muscle contraction has been the effect of Ca2+ on the interactions among the thin filament's five constituent polypeptides: actin, tropomyosin, troponin C (TnC), troponin T (TnT), and troponin I (TnI). We have investigated these interactions by analyzing thin filament assembly as a linear lattice binding problem with linkage relationships in the associations of tropomyosin, actin, and troponin. Binding of TnT, the binary TnT.TnI complex, or the ternary troponin complex (+/- Ca2+) to tropomyosin was measured spectrofluorimetrically after labeling cardiac tropomyosin with N-(1-pyrene)iodoacetamide. The affinity constants ranged between 0.2 and 0.6 microM-1 in the presence of 300 mM KCl. Also, the affinities of tropomyosin, tropomyosin-troponin, tropomyosin.TnT, and tropomyosin.TnT.TnI for an isolated site on F-actin were determined. The actin association constants were 0.0006 microM-1 for tropomyosin, 1 microM-1 for tropomyosin.TnT, 2 microM-1 for tropomyosin.TnT.TnI, 0.5 microM-1 for tropomyosin.troponin, and 0.5 microM-1 for tropomyosin-troponin.Ca2+. Linked equilibrium analysis permitted calculation of the affinities for actin.tropomyosin of TnT (400 microM-1), TnT.TnI (1600 microM-1), troponin (500 microM-1), and troponin.Ca2+ (300 microM-1). Therefore, both troponin and tropomyosin.troponin retain high actin-affinity even when Ca2+ is present or when TnI is removed, and even in the absence of cooperative contributions. The results are discussed in consideration of increasing evidence for a Ca(2+)-regulated azimuthal movement of tropomyosin on F-actin (Lehman, W., Craig, R., and Vibert, P. (1994) Nature 368, 65-67). It is proposed that tropomyosin movement may be due to switching between TnI-mediated and TnT-mediated binding of troponin-tropomyosin to distinct sites on F-actin.

Cooperative interactions between adjacent troponin-tropomyosin complexes may be transmitted through the actin filament.

Recent analyses of the assembly of thin filaments containing altered forms of troponin (or no troponin) suggested that the strongly cooperative nature of troponin-tropomyosin binding to actin might be primarily caused by indirect interactions involving the actin lattice, rather than by direct contacts between neighboring troponin-tropomyosin molecules. To test this hypothesis, thin filament assembly was examined using either cardiac tropomyosin digested with carboxypeptidase A (cbpTm) or a tropomyosin with defective function at both amino and carboxyl termini (unacetylated cbpTm). Compared to intact troponin-tropomyosin, both troponin-cbpTm and troponin-unacetylated cbpTm had much weaker binding to actin; however, cooperative interactions were only slightly reduced. These data support the implication that the primary source of the cooperativity involves troponin-tropomyosin-promoted conformational changes within the actin polymer. Surprisingly, the effects of tropomyosin amino- and carboxyl-terminal structural defects on troponin-tropomyosin binding to actin were not additive. In the presence of troponin, tropomyosin molecules with either defect had the same diminution in actin affinity as molecules with both defects. Finally, the Ca2+ sensitivity of troponin-tropomyosin binding to actin was increased by alteration of either end of tropomyosin.

Initiation of cardiac hypertrophy in response to thyroxine is not limited by age.

We examined the role of age in the initiation of thyroxine-induced (T4) cardiac hypertrophy. T4 (0.4 mg/kg sc) was administered to prepubescent (2 mo), young adult (6 mo), and senescent (24 mo) Fischer 344 rats for 4 days. While significant increases in left ventricular (LV) mass and RNA/LV were evident at 4 days in all T4-treated groups, the elevation in RNA/LV occurred earlier (2 days) in the senescent group. A 0.2-mg/kg dose of T4 elevated RNA values within 24 h in senescent, but not in prepubescent, rats. LV norepinephrine levels were measured to determine whether it plays a role in this model of cardiac hypertrophy. When synthesis of this catecholamine was blocked with alpha-methyl-p-tyrosine, tissue levels fell significantly in all groups, and the decrement was similar in T4-treated and control rats in the two younger groups. We conclude that: 1) the initiation of T4-induced cardiac hypertrophy is not compromised in senescent rats, 2) hearts of senescent rats respond earlier and to a lower dose of T4 than young rats, and 3) the cardiac hypertrophy that occurs in hyperthyroidism is not due to enhanced levels of available cardiac norepinephrine.

Effects of the amino-terminal regions of tropomyosin and troponin T on thin filament assembly.

Bacterially expressed alpha-tropomyosin lacks the amino-terminal acetylation present in muscle tropomyosin and binds poorly to actin (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). Using a linear lattice model, we determined the affinity (Ko) of unacetylated tropomyosin or troponin-unacetylated tropomyosin for an isolated site on the actin filament and the fold increase in affinity (y) when binding is to an adjacent site. The absence of tropomyosin acetylation decreased Ko 2 orders of magnitude in the absence of troponin. Tropomyosin acetylation also enhanced troponin-tropomyosin binding to actin, not by increasing cooperativity (y), but rather by increasing Ko. These results suggest that the amino-terminal region of tropomyosin is a crucial actin binding site. Troponin promoted unacetylated tropomyosin binding to actin, increasing Ko more than 1,000-fold. Troponin70-259, which lacks the troponin T peptide (1-69) spanning the overlap between adjacent tropomyosins, behaved similarly to intact troponin. Cooperative interactions between adjacent troponin-tropomyosin complexes remained strong despite the use of a nonpolymerizable tropomyosin and a troponin unable to bridge neighboring tropomyosins physically. The Ko for troponin70-259-unacetylated tropomyosin was 500-fold greater than for troponin159-259-unacetylated tropomyosin, indicating that troponin T residues 70-158 are critical for anchoring troponin-tropomyosin to F-actin. The mechanism of cooperative thin filament assembly is discussed.

Analysis of troponin-tropomyosin binding to actin. Troponin does not promote interactions between tropomyosin molecules.

The binding of tropomyosin to actin and troponin-tropomyosin to actin was analyzed according to a linear lattice model which quantifies two parameters: Ko, the affinity of the ligand for an isolated site on the actin filament, and gamma, the fold increase in affinity when binding is contiguous to an occupied site (cooperativity). Tropomyosin-actin binding is very cooperative (gamma = 90-137). Troponin strengthens tropomyosin-actin binding greatly but, surprisingly, does so solely by an 80-130-fold increase in Ko, while cooperativity actually decreases. Additionally, troponin complexes containing TnT subunits with deletions of either amino acids 1-69 (troponin70-259) or 1-158 (troponin159-259) were examined. Deletion of amino acids 1-69 had only small effects on Ko and y, despite this peptide's location spanning the joint between adjacent tropomyosins. Ca2+ reduced Ko by half for both troponin and troponin70-159 and had no detectable effect on cooperativity. Troponin159-259 had much weaker effects on tropomyosin-actin binding than did troponin70-259 and had no effect at all in the presence of Ca2+. This suggests the importance of Ca(2+)-insensitive interactions between tropomyosin and troponin T residues 70-159. Cooperativity was slightly lower for troponin159-259 than tropomyosin alone, suggesting that the globular head region of troponin affects tropomyosin-tropomyosin interactions along the thin filament.

Late-onset renal hypertension in old rats alters myocardial microvessels.

We tested the hypothesis that late-onset hypertension in middle-aged (15 mo) and senescent (24 mo) rats would adversely affect the coronary microvasculature. Morphometric analyses were performed on coronary capillaries and arterioles from rats with one-kidney, figure-8 renal wrap hypertension of 3-mo duration. Compared with control rats, wall-to-lumen ratios of arterioles with lumen diameters less than 25 microns were higher in the two hypertensive groups by approximately 30%; larger arterioles did not show consistent intergroup differences. A comparison of the two control groups revealed that wall-to-lumen ratio of arterioles with lumen diameters less than 50 microns tended to be greater in the senescent rats. Capillary numerical density was markedly reduced in the hypertensive animals of both age groups and caused an increase in the mean Krough cylinder radius and in the mean capillary domain. The latter increased by 28-63%; the largest increment occurred in the endomyocardium of the senescent group. A trend toward increased heterogeneity of capillary spacing was also noted in the hypertensive rats. The observed microvascular alterations occurred in the absence of an absolute increase in left ventricular mass but in the presence of cardiocyte hypertrophy. Thus the decrements in capillary numerical density are not only due to inadequate growth but reflect an absolute reduction in the number of these vessels associated with cardiocyte loss. It is concluded that late-onset hypertension in middle-aged and senescent rats is characterized by left ventricular wall remodeling that includes microvascular alterations that would be expected to limit maximal myocardial flow and O2 supply to the cardiocyte.

Characterization of Ca2+ uptake in plasma membrane vesicles isolated from guinea pig ileum smooth muscle.

We have characterized ATP-dependent Ca2+ transport into highly purified plasma membrane fraction isolated from guinea pig ileum smooth muscle. The membrane fraction contained inside-out sealed vesicles and was enriched 30-40-fold in 5'-nucleotidase and phosphodiesterase I activity as compared to post nuclear supernatant. Plasma membrane vesicles showed high rate (76 nmol/mg/min) and high capacity for ATP dependent Ca2+ transport which was inhibited by addition of Ca2+ ionophore A23187. The inhibitors of mitochondrial Ca2+ transport, i.e., sodium azide, oligomycin and ruthenium red did not inhibit ATP-dependent Ca2+ uptake into plasma membrane vesicles. The energy dependent Ca2+ uptake into plasma membranes showed very high specificity for ATP as energy source and other nucleotide triphosphates were ineffective in supporting Ca2+ transport. Phosphate was significantly better as Ca2+ trapping anion to potentiate ATP-dependent Ca2+ uptake into plasma membrane fraction as compared to oxalate. Orthovanadate, an inhibitor of cell membrane (Ca2+-Mg2+)-ATPase activity, completely inhibited ATP-dependent Ca2+ transport and the Ki was approximately 0.6 microM. ATP-dependent Ca2+ transport and formation of alkali labile phosphorylated intermediate of (Ca2+-Mg2+)-ATPase increased with increasing concentrations of free Ca2+ in the incubation mixture and the Km value for Ca2+ was approximately 0.6-0.7 microM for both the reactions.

Alterations in the plasma membrane properties of the myocardium of spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were used to investigate the adaptive biochemical changes in the myocardium in response to chronic afterload. Ouabain-inhibited Na+,K+-adenosine triphosphatase (ATPase) activity was decreased by 40% in myocardium of SHR compared with that from WKY, which may lead to increased intracellular Ca2+ through Na+-Ca2+ exchange. Similarly, alpha 1-adrenergic receptor density, estimated by [3H]prazosin binding, was decreased by 42% in myocardial membranes of SHR, while the affinity for the agonist and the antagonist was not altered. In contrast, the number of Ca2+ channels estimated by [3H]nitrendipine binding was increased by 45% in myocardial membranes of SHR, while the affinity was comparable between SHR and WKY. These differences between WKY and SHR in the membrane properties were not due to differential contamination of plasma membranes because the activities of other putative plasma membrane marker enzymes were comparable between WKY and SHR. There were no differences between WKY and SHR in the myosin ATPase activity estimated using myofibrils, actomyosin, and myosin. These results suggest that specific alterations have occurred in the plasma membrane properties of myocardium of SHR that result in altered intracellular Ca2+ metabolism. These alterations may have an important bearing on excitation-contraction coupling in myocardium of SHR.