Ablation of the murine alpha myosin heavy chain gene leads to dosage effects and functional deficits in the heart.

The alpha-myosin heavy chain (alpha-MyHC) is the major contractile protein expressed in the myocardium of adult mice. We have produced mice carrying a null mutation of alpha-MyHC by homologous recombination in murine ES cells. Homozygous null animals die between 11 and 12 d in utero of gross heart defects, while alpha-MyHC+/- heterozygotes survive and appear externally normal. The presence of a single functional alpha-MyHC+ allele in heterozygous animals results in reduced levels of the transcript and protein as well as fibrosis and alterations in sarcomeric structure. Examination of heart function using a working heart preparation revealed severe impairment of both contractility and relaxation in a subset of the alpha-MyHC+/- animals. Thus, two alpha-MyHC+ alleles are necessary for normal cardiac development, and hemizygosity for the normal allele can result in altered cardiac function.

Mouse model of a familial hypertrophic cardiomyopathy mutation in alpha-tropomyosin manifests cardiac dysfunction.

To investigate the functional consequences of a tropomyosin (TM) mutation associated with familial hypertrophic cardiomyopathy (FHC), we generated transgenic mice that express mutant alpha-TM in the adult heart. The missense mutation, which results in the substitution of asparagine for aspartic acid at amino acid position 175, occurs in a troponin T binding region of TM. S1 nuclease mapping and Western blot analyses demonstrate that increased expression of the alpha-TM 175 transgene in different lines causes a concomitant decrease in levels of endogenous alpha-TM mRNA and protein expression. In vivo physiological analyses show a severe impairment of both contractility and relaxation in hearts of the FHC mice, with a significant change in left ventricular fractional shortening. Myofilaments that contain alpha-TM 175 demonstrate an increased activation of the thin filament through enhanced Ca2+ sensitivity of steady-state force. Histological analyses show patchy areas of mild ventricular myocyte disorganization and hypertrophy, with occasional thrombi formation in the left atria. Thus, the FHC alpha-TM transgenic mouse can serve as a model system for the examination of pathological and physiological alterations imparted through aberrant TM isoforms.

Characterisation of Na/K-ATPase, its isoforms, and the inotropic response to ouabain in isolated failing human hearts.

OBJECTIVE: The aim was to determine whether failing human hearts have increased sensitivity to the inotropic and toxic effects of ouabain, and to examine alterations in Na/K-ATPase that might explain the observed higher ouabain sensitivity. METHODS: For contractility studies, a total of 57 trabeculae were isolated from two non-failing (death from head injury) and 10 terminally failing, explanted human hearts. After the experiment, each trabecula was inspected under the light microscope for morphological alterations consistent with heart failure. Samples for biochemical and molecular studies were obtained from five non-failing and 13 failing hearts. Total Na/K-ATPase was measured in desoxycholate treated homogenates and expressed per unit of tissue wet or dry weight, DNA, protein, or myosin. Interference from residual bound digoxin due to previous therapy was excluded. The expression of the three alpha isoforms was studied at both the mRNA level using northern blots and the protein level by analysis of dissociation kinetics of the [3H]ouabain-enzyme complex. RESULTS: Trabeculae showing morphological alterations and decreased contractility were sensitive to lower concentrations of ouabain (3-100 nM) than control trabeculae (100-1000 nM); the inotropic EC50 and the minimum toxic concentration were both reduced. [3H]Ouabain binding was significantly lower (p << 0.001) in failing than in non-failing hearts, at 293(SD 74) v 507(48) pmol.g-1 wet weight. No significant change was observed in maximum ATPase turnover rate, or in sensitivities to Na+, K+, vanadate, and dihydro-ouabain. All three alpha isoforms were expressed at the mRNA level in both normal and failing hearts. CONCLUSIONS: This study shows conclusively, for the first time, that failing human hearts are more sensitive to ouabain. This may be at least partly due to a mean reduction of 42% (95% confidence interval, 26 to 56%) in the concentration of Na/K-ATPase (decrease in Na,K pump reserve), but not to an alteration in its catalytic properties or in its isoform composition.

Differential cardiovascular effects of calcium channel blocking agents: potential mechanisms.

The three major calcium channel blocking agents, diltiazem, nifedipine and verapamil, inhibit calcium entry into excitable cells. Despite this apparent common action at the cell membrane, these drugs produce quantitative and frequently qualitative differences in cardiovascular variables (for example, heart rate, atrioventricular [A-V] conduction and myocardial inotropic state) when evaluated at equieffective vasodilator doses. All three drugs increase coronary blood flow in a dose-dependent fashion (nifedipine greater than diltiazem = verapamil), and produce a negative inotropic effect in vitro in isolated atria and ventricles, also in a dose-dependent manner (verapamil greater than nifedipine greater than diltiazem). However, in conscious dogs nifedipine increases, verapamil decreases and diltiazem has little effect on the inotropic state. A-V conduction is slowed by diltiazem and verapamil but not by nifedipine in anesthetized dogs and in conscious dogs as judged from the P-R interval in the electrocardiogram. Heart rate is slowed in pentobarbital-anesthetized animals but is accelerated in conscious dogs (nifedipine greater than verapamil greater than diltiazem). Nifedipine also appears to interfere significantly with the arterial baroreceptor reflex by an apparent vagolytic action that is less evident with diltiazem and verapamil. Diltiazem, and possibly verapamil and nifedipine as well, appears to retard myocardial damage that accompanies ischemia. The mechanisms and sites of action of these drugs are presumed to be at the cell membrane; however, intracellular sites may also be involved.

Effects of RMI 12330A, a new inhibitor of adenylate cyclase on myocardial function and subcellular activity.

1 RMI 12330A, a lactam-imine, at concentrations of 10(-4) M and higher, inhibited basal as well as isoprenaline and NaF-stimulated adenylate cyclase activity of guinea-pig heart homogenates. However, RMI 12330A was a more potent inhibitor of histamine-stimulated adenylate cyclase (IC50 of 1.5 X 10(-5) M). 2 In the isolated work-performing heart of the guinea-pig, RMI 12330A (IC50 of 1.1 X 10(-6) M) depressed all cardiac functions: pressures developed, dP/dt, contractile force, dF/dt, work performance and stroke work. Left atrial pressure rose and the positive inotropic response to increasing heart rate (staircase) became negative. Histamine, isoprenaline and ouabain no longer caused positive inotropic effects. 3 Increasing the perfusate calcium concentration from 2.5 mM to 4.5 and 6.5 mM completely restored cardiac function after its depression by RMI 12330A. 4 RMI 12330A uncoupled mitochondrial oxidative phosphorylation; the classical uncoupler, dinitrophenol, had the same effects on cardiac dynamics as RMI 12330A. 5 RMI in high doses inhibited hydrolytic activity of Na+, K+-ATPase of crude and purified heart preparations (IC50 of 1.7 X 10(-4) M) and inhibited ouabain binding to the same enzymes (IC50 of 1.5 X 10(-4) M). 6 A lactam-imine analogue of RMI 12330A that had no effect on adenylate cyclase, was also without effect on any of the systems examined.

Benzodiazepine Ro 5-4864 increases coronary flow.

Ro 5-4864 (chlorodiazepam) increased coronary flow in isolated retrograde perfused Langendorff rat heart preparations without affecting heart rate and left ventricular contractility (dP/dt). On the other hand Ro 5-4023 (clonazepam) produced very little effect. PK 11195 which has been shown to inhibit the binding of Ro 5-4864 to cardiac muscle did not antagonize this vasodilatory effect of Ro 5-4864 but increased coronary flow by itself. The data indicate a specific vasodilatory effect of certain benzodiazepines. The mechanism of action remains unknown.

The relative phospholamban and SERCA2 ratio: a critical determinant of myocardial contractility.

Phospholamban is a regulatory phosphoprotein which modulates the active transport of Ca2+ by the cardiac sarcoplasmic reticular Ca(2+)-ATPase enzyme (SERCA2) into the lumen of the sarcoplasmic reticulum. Phospholamban, which is a reversible inhibitor of SERCA2, represses the enzyme's activity, and this inhibition is relieved upon phosphorylation of phospholamban in response to beta-adrenergic stimulation. In this way, phospholamban is an important regulator of SERCA2-mediated myocardial relaxation during diastole. This report centers on the hypothesis that the relative levels of phospholamban: SERCA2 in cardiac muscle plays an important role in the muscle's overall contractility status. This hypothesis was tested by comparing the contractile parameters of: a) murine atrial and ventricular muscles, which differentially express phospholamban, and b) murine wild-type and phospholamban knock-out hearts. These comparisons revealed that atrial muscles, which have a 4.2-fold lower phospholamban: SERCA2 ratio than ventricular muscles, exhibited rates of force development and relaxation of tension, which were three-fold faster that these parameters for ventricular muscles. Similar comparisons were made via analyses of left-ventricular pressure development recorded for isolated, work-performing hearts from wild-type and phospholamban knock-out mice. In these studies, hearts from phospholamban knock-out mice, which were devoid of phospholamban, exhibited enhanced parameters of left-ventricular contractility in comparison to wild-type hearts. These results suggest that the relative phospholamban: SERCA2 ratio is critical in the regulation of myocardial contractility and alterations in this ratio may contribute to the functional deterioration observed during heart failure.

Mitochondrial dysfunction accompanies diastolic dysfunction in diabetic rat heart.

The objective of this study was to determine whether a defect in mitochondrial respiratory function accompanies the development of diabetic cardiomyopathy. The hypothesis tested in this study is that a decrease in Ca2+ uptake into mitochondria may prevent the stimulation of Ca(2+)-sensitive matrix dehydrogenases and the rate of ATP synthesis. Streptozotocin (55 mg/kg)-induced diabetic rats were used as a model of insulin-dependent diabetes mellitus. Hearts from 4-wk diabetic rats had basal heart rates and rates of contraction and relaxation similar to control. Isoproterenol caused a similar increase in the rate of contraction in diabetic and control hearts, whereas the peak rate of relaxation was reduced in diabetic hearts. Mitochondrial Ca2+ uptake was reduced in mitochondria from diabetic hearts after 2 wk of diabetes. Na(+)-induced Ca2+ release was unchanged. State 3 respiration rate was depressed in mitochondria from diabetic rats only when the respiration was supported by the substrate of a Ca(2+)-regulated matrix enzyme. The pyruvate dehydrogenase activity was reduced in diabetic mitochondria compared with that of control. It was concluded that mitochondria from diabetic hearts had a decreased capacity to upregulate ATP synthesis via stimulation of Ca(2+)-sensitive matrix dehydrogenases. The impairment in the augmentation of ATP synthesis rate accompanies a decreased rate of relaxation during increased work load.

Diltiazem potentiates the negative inotropic action of nimodipine in heart.

In Langendorff perfused rat hearts, nimodipine enhances coronary flow and inhibits contractility. The binding of [3H]nimodipine (160 Ci/mmol) to sarcolemma isolated from dog heart revealed a KD of 0.2 nM. d-cis-Diltiazem, but not 1-cis-diltiazem, a less active stereoisomer, stimulated [3H]nimodipine (0.17 nM) binding to sarcolemmal membranes (ED50 for diltiazem = 1.1 microM). In the presence of 10 microM d-cis-diltiazem, [3H]nimodipine binding sites were doubled, but there was no change in the apparent affinity. Perfused rat hearts were treated with 250 nM d-cis-diltiazem. The negative inotropic response to nimodipine was dramatically potentiated (I50, from 1.1 to 0.033 microM). The pharmacological and binding effects were observed only at 37 degrees C. It is possible that diltiazem in some way converts low affinity to high affinity sites.

Analysis of the inotropic action of ouabain in rat ventricles: two apparent ouabain inotropic responses.

Two distinct inotropic effects of ouabain were observed in 68 of 82 right ventricular strips of rats with ED50s of 0.5 and 20 microM referred to as "low-dose" and "high-dose" effects, respectively. The other 14 strips showed monophasic dose-response curves, with an apparent ED50 of 0.3 microM; the inotropic response to ouabain or isoproterenol developed by these atypical strips did not exceed 50% over control. In the strips showing the biphasic inotropic response curves, the proportion of the "low-dose" effect varied from 6 to 89% of the maximum response. After treatment with 160 microM ouabain, followed by 60 min of washout, the maximum developed tension in response to ouabain was unchanged, but the "low-dose" effect was abolished or dramatically reduced. In those cases with a remaining "low-dose" response, the response was only 12% or less of the maximum and the ED50 was shifted from 0.3 to about 3 microM ouabain by the washout. The atypical strips, which showed only the "low-dose" effects during the first ouabain exposure, revealed after washout a consistent "high-dose" effect with an ED50 of about 12 microM ouabain. The data show that the two inotropic responses exist in all ventricular strips and that in some strips, after ouabain washout, a residue of the "low-dose" effect remains.

Effects of a new positive inotropic agent, 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)- quinolinone (OPC-8212) and its solvent sulfolane on isolated heart preparations of the rat, guinea pig, and dog.

3,4-Dihydro-6-[4-(3,4- dimethoxybenzoyl )-1-piperazinyl]-2(1H)- qu inolinone ( OPC -8212) has positive inotropic effects in guinea pig and dog isolated heart preparations. The rat heart ventricle does not respond to OPC -8212 but the atria do. OPC -8212 produces positive inotropic effects in the rat heart ventricle made hypodynamic by pretreatment with calcium entry blockers. Sulfolane , the solvent of OPC -8212, produced negative inotropic effects in most preparations. In isolated heart preparations of the rat and guinea pig, OPC -8212 produced increased coronary flow; sulfolane had only minor effects.

ATP depletion and mitochondrial functional loss during ischemia in slow and fast heart-rate hearts.

In the present study, isolated dog and rat hearts were perfused in the Langendorff mode with Krebs bicarbonate buffer in the absence and presence of 10(-5) M oligomycin. The perfusion protocols employed allowed tissue pH to drop during subsequent ischemic incubations essentially as it would in blood-perfused hearts. Tissue pH, ATP, lactate, and mitochondrial respiratory function were measured during the course of subsequent zero-flow ischemic incubations. The adenosinetriphosphatase (ATPase) activities attributable to both mitochondrial and nonmitochondrial ATPases in sonicated heart homogenates and the actomyosin ATPase in isolated cardiac myofibrils were measured in both species. Consistent with earlier results with a different model in which tissue pH was buffered during the ischemic incubations [W. Rouslin, J. L. Erickson, and R. J. Solaro. Am. J. Physiol. 250 (Heart Circ. Physiol. 19): H503-H508, 1986], the inhibition of the mitochondrial ATPase in situ by oligomycin markedly slowed both tissue ATP depletion and the loss of mitochondrial function during ischemia in the dog. However, oligomycin had only a very small and transient effect on ATP depletion and mitochondrial function in the rat. This was apparently so because of the fivefold higher rate of glycolytic ATP production as well as the nearly threefold higher total nonmitochondrial ATPase activity of ischemic rat compared with ischemic dog heart. These results suggest that although the inhibition of the mitochondrial ATPase makes a major contribution to ATP conservation in ischemic dog heart, it makes only a very small contribution in rat.

Reversibility of inotropic effects of ouabain on canine atrial and ventricular tissues.

The experiments reported here were designed to quantitate reversibility of ouabain-induced inotropy in atrial and ventricular trabeculae of the dog heart. At 30 degrees C, 1 Hz, 2 mM Ca2+ and 5.9 mM K+, the positive inotropic effects, measured after a 1 h exposure to a single priming concentration of 0.5 microM ouabain, decreased after the removal of the drug with a half time (t1/2) of washout of 9.52 +/- 2.72 h in atrial and 7.64 +/- 1.58 h in ventricular trabeculae. When toxicity occurred it was of three types: transient toxicity, toxicity resembling a 'fast washout,' and severe toxicity characterized by profound and persistent negative inotropy and contracture. Use of the classical glycoside bioassay (Hatcher's digitalis titration) during and after washout, provided a semiquantitative estimation of ouabain remaining in the trabeculae; after a 4 h washout of the drug, the trabeculae were challenged by the addition of one-half of the initial concentration of ouabain (0.25 microM). Trabeculae, which had responded to ouabain with only positive inotropic effects and no toxicity, still contained substantial concentrations of ouabain. Trabeculae which reacted clearly with toxicity to the priming dose of ouabain, showed a significant loss of contractile force and development of contracture. The fastest ouabain washout we observed was 3.1 h.

Alterations in sarcoplasmic reticulum calcium uptake, relaxation parameters and their responses to beta-adrenergic agonists in the developing rabbit heart.

Developmental changes in cardiac sarcoplasmic reticulum function, which may reflect alterations in the myocardial rate of relaxation and its responses to beta-adrenergic stimulation, were assessed using fetal, 4-day-old, 21-day-old and adult rabbit hearts. The fetal hearts exhibited the slowest rate of relaxation (-dP/dt) and the lowest Vmax and EC50 of the sarcoplasmic reticulum Ca(2+)-pump for Ca2+ compared to the other age groups. These parameters were similar among the 4-day-old, 21-day-old and adult hearts. The low physiological and biochemical parameters in the fetal hearts reflected reduced levels of expression of the sarcoplasmic reticulum Ca(2+)-pump and its inhibitor, phospholamban, assessed by quantitative immunoblotting. Isoproterenol perfusion of fetal hearts had no significant effect on their relaxation parameters or on the EC50 of the Ca(2+)-pump for Ca2+, consistent with the low relative levels of phospholamban expressed in these hearts. However, perfusion of the 4-day-old, 21-day-old and adult hearts with isoproterenol resulted in significant increases in the rates of relaxation of each group. The increases in relaxation parameters were associated with decreases in the EC50 of the cardiac sarcoplasmic reticulum Ca(2+)-pump for Ca2+, suggesting a phosphorylation-mediated relief of the phospholamban inhibitory effects. These findings indicate that developmental regulation of the levels of the activity of the cardiac sarcoplasmic reticulum Ca(2+)-pump may reflect alterations in cardiac relaxation parameters and their modulation by beta-adrenergic agonists.

Cardiac myosin heavy chain mRNA expression and myocardial function in the mouse heart.

The vertebrate heart contains two myosin heavy chain isoforms, alpha and beta, which are differentially expressed. To establish a murine model for gene-targeting experiments, we defined the precise temporal expression of the myosin isoforms during cardiogenesis and obtained quantitative measurements of cardiac performance. The relative levels of the alpha- and beta-cardiac transcripts were determined by isolating the RNA from the hearts of CD-1 mice during development and hybridizing the preparations to probes that detect specifically the alpha- or beta-cardiac myosin heavy chain mRNAs. The data indicate that, although both isoforms are present from the onset of cardiogenesis, the beta-isoform predominates during embryogenesis and fetal development. This relation is reversed after the first day of life with a significant drop in the absolute transcript levels during the switch; and alpha/beta ratio of 16:1 is maintained in the neonate, and the relatively high levels of the alpha-transcript remain throughout the adult stages. To be able to make functional comparisons between normal and transgenic mice, we obtained indexes of myocardial function in isolated retrogradely perfused and in work-performing heart preparations in normal and hypodynamic mouse hearts. We found that the physiology of the mouse heart is similar to the rat heart in that we observed a positive staircase in the force-frequency relation of the mouse Langendorff preparation. We also saw contractile responses of more than twice control induced by paired stimulation and persistent postextrasystolic potentiation. As is the case for the rat, in the work-performing mouse heart, afterload (Starling resistance, pressure) changes produced a steeper Starling function curve than did changes in preload (volume, venous return).

Functional evidence in diseased human heart fibers for multiple sensitivities of the inotropic ouabain receptor Na+,K+-ATPase (NKA).

Through contributions of many investigators from our group at the University of Cincinnati Medical Center, several facts emerged. It is clear that the rat ventricle has 2 inotropic sensitivities to ouabain (Grupp et al., 1981), related to high and low affinity ouabain binding sites (Adams et al., 1982) and the presence of an appropriate abundance of alpha and alpha + catalytic subunits of NKA (Young and Lingrel, 1987). The rat atria which demonstrate only the low affinity inotropic response (Grupp et al., 1981) also show the alpha catalytic subunit in vast predominance, with little or no alpha + (Young and Lingrel, 1987). This scheme has now also been confirmed in the ferret (Ng and Akera, 1987). By analogy, we suggest that the possibility exists that in diseased hearts part of the normally prevailing alpha catalytic subunit (sensitivity in normal human fibers about 140 nM ouabain) is converted to a more sensitive alpha + catalytic subunit leading to an increase of the ouabain sensitivity to about 50 nM. There is also an earlier occurrence of toxic effects and a reduction of the number of low affinity (alpha) ouabain binding sites, resulting in a decrease of the maximally achievable contractile force effect of ouabain. This hypothesis is presently being tested in the genetics laboratory of the University of Cincinnati Medical Center where the relative abundance of the alpha, alpha + and perhaps other isoforms of the NKA are being determined in the tissues of the same hearts from which the trabeculae used in this study were obtained.