Co-expression of skeletal and cardiac troponin T decreases mouse cardiac function.

In contrast to skeletal muscles that simultaneously express multiple troponin T (TnT) isoforms, normal adult human cardiac muscle contains a single isoform of cardiac TnT. To understand the significance of myocardial TnT homogeneity, we examined the effect of TnT heterogeneity on heart function. Transgenic mouse hearts overexpressing a fast skeletal muscle TnT together with the endogenous cardiac TnT was investigated in vivo and ex vivo as an experimental system of concurrent presence of two classes of TnT in the adult cardiac muscle. This model of myocardial TnT heterogeneity produced pathogenic phenotypes: echocardiograph imaging detected age-progressive reductions of cardiac function; in vivo left ventricular pressure analysis showed decreased myocardial contractility; ex vivo analysis of isolated working heart preparations confirmed an intrinsic decrease of cardiac function in the absence of neurohumoral influence. The transgenic mice also showed chronic myocardial hypertrophy and degeneration. The dominantly negative effects of introducing a fast TnT into the cardiac thin filaments to produce two classes of Ca(2+) regulatory units in the adult myocardium suggest that TnT heterogeneity decreases contractile function by disrupting the synchronized action during ventricular contraction that is normally activated as an electrophysiological syncytium.

Metabolic gene expression in fetal and failing human heart.

BACKGROUND: Previous studies suggest that the failing heart reactivates fetal genes and reverts to a fetal pattern of energy substrate metabolism. We tested this hypothesis by examining metabolic gene expression profiles in the fetal, nonfailing, and failing human heart. METHODS AND RESULTS: Human left ventricular tissue (apex) was obtained from 9 fetal, 10 nonfailing, and 10 failing adult hearts. Using quantitative reverse transcription-polymerase chain reaction, we measured transcript levels of atrial natriuretic factor, myosin heavy chain-alpha and -beta, and 13 key regulators of energy substrate metabolism, of which 3 are considered "adult" isoforms (GLUT4, mGS, mCPT-I) and 3 are considered "fetal" isoforms (GLUT1, lGS, and lCPT-I), primarily through previous studies in rodent models. Compared with the nonfailing adult heart, steady-state mRNA levels of atrial natriuretic factor were increased in both the fetal and the failing heart. The 2 myosin heavy chain isoforms showed the highest expression level in the nonfailing heart. Transcript levels of most of the metabolic genes were higher in the nonfailing heart than the fetal heart. Adult isogenes predominated in all groups and always showed a greater induction than the fetal isogenes in the nonfailing heart compared with the fetal heart. In the failing heart, the expression of metabolic genes decreased to the same levels as in the fetal heart. CONCLUSIONS: In the human heart, metabolic genes exist as constitutive and inducible forms. The failing adult heart reverts to a fetal metabolic gene profile by downregulating adult gene transcripts rather than by upregulating fetal genes.

Downregulation of matrix metalloproteinases and reduction in collagen damage in the failing human heart after support with left ventricular assist devices.

BACKGROUND: Left ventricular assist device (LVAD) support of the failing heart induces salutary changes in myocardial structure and function. Matrix metalloproteinases (MMPs) are increased in the failing heart and are induced by stretch in cardiac cells in vitro. We hypothesized that mechanical unloading may affect LV plasticity by regulating MMPs and their substrates. METHODS AND RESULTS: LV samples were collected from patients with dilated cardiomyopathy (DCM, n=14) or ischemic cardiomyopathy (ICM, n=16) at the time of implantation of the LVAD and again during cardiac transplantation. MMP-1, -3, and -9 were measured by ELISA, MMP-2 and -9 gelatinolytic activity by gelatin zymography, and tissue inhibitors of metalloproteinases (TIMPs) by Western blot. Total soluble and insoluble collagens were separated by pepsin solubilization, and the contents were determined by quantification of hydroxyproline. The undenatured soluble collagen was measured by Sircol collagen assay. The results showed that MMP-1 and -9 were decreased, whereas TIMP-1 and -3 were increased, but there was no change in MMP-2 and -3 and TIMP-2 and -4 after LVAD support. The undenatured collagen was increased, with the ratio of undenatured to total soluble collagens increased in ICM and that of insoluble to total soluble collagens increased in DCM after LVAD support. CONCLUSIONS: The reduced MMPs and increased TIMPs and ratios of undenatured to total soluble collagens and insoluble to total soluble collagens after LVAD support suggest that reduced MMP activity diminished damage to the matrix. These changes may contribute to the functional recovery and LV plasticity after LVAD support.

Mechanical unloading restores beta-adrenergic responsiveness and reverses receptor downregulation in the failing human heart.

BACKGROUND: Mechanical unloading of the failing human heart with a left ventricular assist device (LVAD) results in clinically documented reversal of chamber dilation and improvement of cardiac function. We tested the hypothesis that LVAD support normalizes the ability of cardiac muscle to respond to sympathetic nervous system stimulation by reversing the downregulation of beta-adrenergic receptors. METHODS AND RESULTS: Human LV tissue was obtained from nonfailing hearts of unmatched organ donors and failing hearts at the time of transplantation, with or without LVAD. Baseline contractile parameters and inotropic response to a beta-adrenergic agonist were measured in isolated trabecular muscles. beta-Adrenergic receptor density was quantified by radioligand binding. Results showed a significant increase in the response to beta-adrenergic stimulation after LVAD (developed tension increased by 0.76+/-0.09 g/mm(2) in nonfailing, 0.38+/-0.07 in failing, and 0.68+/-0.10 in failing+LVAD; P<0.01), accompanied by an increased density of beta-adrenergic receptors (58.7+/-9.6 fmol/mg protein in nonfailing, 26.2+/-3.8 in failing, and 63.0+/-8.3 in failing+LVAD; P<0.05). These changes were unrelated to the duration of support. CONCLUSIONS: Data demonstrate that mechanically supporting the failing human heart with an LVAD can reverse the downregulation of beta-adrenergic receptors and restore the ability of cardiac muscle to respond to inotropic stimulation by the sympathetic nervous system. This indicates that functional impairment of cardiac muscle in human heart failure is reversible.

The effects of propofol on the contractility of failing and nonfailing human heart muscles.

We determined the direct effects of propofol on the contractility of human nonfailing atrial and failing atrial and ventricular muscles. Atrial and ventricular trabecular muscles were obtained from the failing human hearts of transplant patients or from nonfailing hearts of patients undergoing coronary artery bypass surgery. Isometric contraction variables were recorded before and after propofol was added to the bath in concentrations between 0.056 and 560 microM. The effects of propofol were compared with its commercial vehicle intralipid. To test beta-adrenergic effects in the presence of propofol, 1 microM isoproterenol was added at the end of each experiment. To determine the cellular mechanisms responsible for the actions of propofol, we examined its effects on actomyosin ATPase activity and sarcoplasmic reticulum (SR) Ca(2+) uptake in nonfailing atrial tissues. Propofol caused a concentration-dependent decrease in maximal developed tension in all muscles, which became significant (P < 0.05) at concentrations exceeding the clinical range (> or =56 microM). Isoproterenol restored contractility to the level achieved before exposure to propofol (P > 0.05 compared with baseline). Failing ventricular muscle exposed to propofol exhibited somewhat diminished ability to recover contractility in response to isoproterenol (P < 0.05 versus failing muscle exposed to intralipid only). Propofol induced a concentration-dependent decrease in the uptake of Ca(2+) into SR vesicles. At the same time, in the presence of 56 microM propofol, the Ca(2+)-activated actomyosin ATPase activity was shifted leftward, demonstrating an increase in myofilament sensitivity to Ca(2+). We conclude that propofol exerts a direct negative inotropic effect in nonfailing and failing human myocardium, but only at concentrations larger than typical clinical concentrations. Negative inotropic effects are reversible with beta-adrenergic stimulation. The negative inotropic effect of propofol is at least partially mediated by decreased Ca(2+) uptake into the SR; however, the net effect of propofol on contractility is insignificant at clinical concentrations because of a simultaneous increase in the sensitivity of the myofilaments to activator Ca(2+).

Gender influences on sarcoplasmic reticulum Ca2+-handling in failing human myocardium.

Gender has recently been implicated as an important modulator of cardiovascular disease. However, it is not known how gender may specifically influence the Ca2+-handling deficits that characterize the depressed cardiac contractility of human heart failure. To elucidate the contributory role of gender to sarcoplasmic reticulum (SR) Ca2+ cycling alterations, the protein levels of SR Ca2+-ATPase (SERCA), phospholamban, and calsequestrin, as well as the site-specific phospholamban phosphorylation status, were quantified in a mixed gender population of failing (n=14) and donor (n=15) myocardia. The apparent affinity (EC50) and the maximal velocity (Vmax) of SR Ca2+-uptake were also determined to lend functional significance to any observed protein alterations. Phospholamban and calsequestrin levels were not altered; however, SERCA protein levels were significantly reduced in failing hearts. Additionally, phospholamban phosphorylation (serine-16 and threonine-17 sites) and myocardial cAMP content were both attenuated. The alterations in SR protein levels were also accompanied by a decreased V(max)and an increased EC50 (diminished apparent affinity) of SR Ca2+-uptake for Ca2+ in failing myocardia. Myocardial protein levels and Ca2+ uptake parameters were then analyzed with respect to gender, which revealed that the decreases in phosphorylated serine-16 were specific to male failing hearts, reflecting increases in the EC50 values of SR Ca2+-uptake for Ca2+, compared to donor males. These findings suggest that although decreased SERCA protein and phospholamban phosphorylation levels contribute to depressed SR Ca2+-uptake and left ventricular function in heart failure, the specific subcellular alterations which underlie these effects may not be uniform with respect to gender.

Myocardial tumor necrosis factor-alpha expression does not correlate with clinical indices of heart failure in patients on left ventricular assist device support.

BACKGROUND: Mechanical unloading with a left ventricular assist device (LVAD) can improve clinical indices of heart failure and alter myocardial tumor necrosis factor-alpha (TNFalpha) expression, but a correlation between clinical and molecular indices has not been established. METHODS: We enrolled 14 patients with end-stage heart failure treated with drugs and mechanical unloading in a protocol including the collection of myocardial tissue samples at LVAD implantation and explantation. Ten nonfailing donor hearts served as controls. TNFalpha expression was measured by quantitative reverse transcription polymerase chain reaction. Clinical indices of heart failure were retrospectively analyzed and correlated with myocardial TNFalpha expression. RESULTS: Left ventricular end-diastolic dimension decreased (p < 0.01) and cardiac index (p < 0.001) increased with unloading. Abnormal values of serum sodium, creatinine, blood urea nitrogen, glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, and albumin showed a trend toward normalization with mechanical unloading. TNFalpha expression was increased in 5 of 14 patients and decreased with mechanical unloading in 4 of them. Surprisingly, there was no correlation between mRNA levels of TNFalpha and any of the clinical indices studied. CONCLUSIONS: Although clinical indices of heart failure improve and elevated levels of myocardial TNFalpha expression decrease with mechanical unloading, there is no correlation between the two. Thus, clinical and molecular indices of heart failure in LVAD-supported patients do not always correlate.

Alterations in cardiac function and gene expression during autoimmune myocarditis in mice.

Although myocarditis has been implicated in the pathogenesis of heart failure, a definitive relationship between myocardial inflammation, cardiac dysfunction, and changes in myocyte gene expression has not been established. In this study, we examined the hypothesis that myocardial inflammation and replacement fibrosis following an autoimmune response can progress to cardiac dysfunction and may result in progression to the heart failure phenotype. SWXJ mice were immunized with cardiac myosin on day 0 and day 7, in order to induce an autoimmune response to the myosin protein. Cardiac catheterization via the right carotid artery was performed on days 14, 21, 28, 35, and 42, using a 1.4F Millar transducer-tipped catheter. Hearts were weighed, and cross-sections were cut and stained with either haematoxylin and eosin or Masson's trichrome, in order to identify areas of inflammation and/or fibrosis. Myocardial gene expression was determined by Northern blot analysis. In mice with histological evidence of myocarditis, the heart weight/body weight ratio increased beginning on day 14, and cardiac function decreased beginning on day 21. Myocardial inflammation was accompanied by significant fibrosis beginning on day 21. Quantitation of mRNA showed expression of ventricular atrial naturietic factor, as well as a decrease in myosin heavy chain alpha, beginning on day 21. These data demonstrate that autoimmune inflammation of the heart results in significant cardiac dysfunction, leading to phenotypic alterations similar to those demonstrated in human heart failure and animal models of heart failure.

The effects of etomidate on the contractility of failing and nonfailing human heart muscle.

UNLABELLED: We measured the effects of etomidate on contractility of human cardiac muscle. Muscles were obtained from the left ventricle and right atrium of 12 patients undergoing cardiac transplantation, and from the right atrium of 12 patients undergoing coronary artery bypass surgery. Muscles were studied at 37 degrees C and 1.0 Hz. Variables of isometric contraction were recorded before and after etomidate (0.04-80 microM) or its solvent, propylene glycol. The ability of beta-adrenergic stimulation to cause an inotropic effect after etomidate was also assessed. Etomidate caused a dose-dependent decrease in developed tension, which was statistically significant only at concentrations exceeding clinical doses (> or =20 microM; P < 0. 05). Decreases in maximum rates of contraction and relaxation paralleled changes in developed tension. beta-Adrenergic stimulation reversed the etomidate-induced decreases in developed tension and rates of contraction and relaxation to baseline (P > 0.05 compared with baseline). Thus, in human myocardium, etomidate exerts a dose-dependent negative inotropic effect, which is reversible with beta-adrenergic stimulation. Concentrations required to produce these negative inotropic effects are, however, in excess of those reached during clinical use. Therefore, etomidate-induced negative inotropy is unlikely to be a problem clinically, even in patients with cardiac dysfunction. IMPLICATIONS: Etomidate produced a similar dose-dependent negative inotropic effect in both failing and nonfailing human myocardium. This effect was present only at concentrations exceeding those attained clinically and was reversible with beta-adrenergic stimulation.

Regulation of PKA binding to AKAPs in the heart: alterations in human heart failure.

BACKGROUND: cAMP-dependent protein kinase (PKA) regulates a broad range of cellular responses in the cardiac myocyte. Downstream regulation of the PKA pathway is mediated by a class of scaffolding proteins called A-kinase anchoring proteins (AKAPs), which sequester PKA to specific subcellular locations through binding to its regulatory subunit (R). However, the effect of RII autophosphorylation on AKAP binding and the degree of RII autophosphorylation in failing and nonfailing human hearts remains unknown. METHODS AND RESULTS: We investigated AKAP-RII binding by overlay analysis and surface plasmon resonance spectroscopy and measured RII autophosphorylation in human hearts by backphosphorylation. Binding of Ht31 peptide (representing the RII-binding region of AKAPs) to cardiac RII was increased approximately 145% (P<0.01) for autophosphorylated RII relative to unphosphorylated control. By surface plasmon resonance, RII autophosphorylation significantly increased binding affinity to Ht31 by approximately 200% (P<0.01). Baseline PKA-dependent phosphorylation of RII was significantly decreased approximately 30% (P<0.05) in human hearts with dilated cardiomyopathy compared with nonfailing controls. CONCLUSIONS: These results suggest that AKAP binding of PKA in the heart is regulated by RII autophosphorylation. Therefore AKAP targeting of PKA may be reduced in patients with end-stage heart failure. This mechanism may be responsible for the decreased cAMP-dependent phosphorylation of proteins in dilated cardiomyopathy that we and others have previously observed.

Immunolocalization of annexins IV, V and VI in the failing and non-failing human heart.

UNLABELLED: The failing human heart is characterized by changes in the expression and function of proteins involved in intracellular Ca2+ cycling, resulting in altered Ca2+ transients and impaired contractile properties of cardiac muscle. The role of the cardiac annexins in this process remains unclear. Annexins may play a role in the regulation of Ca2+ pumps and exchangers on the sarcolemma, and have been shown to be altered in some cardiac disease states. OBJECTIVE: The goal of this study was to compare the immunolocalization and expression of annexins IV, V and VI in failing and non-failing human hearts. METHODS: We used immunostaining to identify the subcellular location of annexins IV, V and VI proteins within the myocardial cell, and Western blot analysis to quantify the proteins in the same hearts. RESULTS: Annexin IV showed a cytoplasmic distribution in both failing and non-failing human heart cells. Annexin V was localized at the z-line, around lipofuscin granules, and in the cytosol in the non-failing heart cells. Annexin VI was localized at the sarcolemma and intercalated disc. Protein levels of annexins IV and V were up-regulated in failing human hearts, while the expression of annexin VI was unchanged. CONCLUSIONS: Alterations in the intracellular localization of annexins, along with up-regulation of annexins IV and V in the failing human heart cells, suggests differential regulation of these Ca2+ regulatory proteins during heart failure.

Decreased SLIM1 expression and increased gelsolin expression in failing human hearts measured by high-density oligonucleotide arrays.

BACKGROUND: Failing human hearts are characterized by altered cytoskeletal and myofibrillar organization, impaired signal transduction, abnormal protein turnover, and impaired energy metabolism. Thus, expression of multiple classes of genes is likely to be altered in human heart failure. METHODS AND RESULTS: We used high-density oligonucleotide arrays to explore changes in expression of approximately 7000 genes in 2 nonfailing and 2 failing human hearts with diagnoses of end-stage ischemic and dilated cardiomyopathy, respectively. We report altered expression of (1) cytoskeletal and myofibrillar genes (striated muscle LIM protein-1 [SLIM1], myomesin, nonsarcomeric myosin regulatory light chain-2 [MLC(2)], and ss-actin); (2) genes responsible for degradation and disassembly of myocardial proteins (alpha(1)-antichymotrypsin, ubiquitin, and gelsolin); (3) genes involved in metabolism (ATP synthase alpha-subunit, succinate dehydrogenase flavoprotein [SDH Fp] subunit, aldose reductase, and TIM17 preprotein translocase); (4) genes responsible for protein synthesis (elongation factor-2 [EF-2], eukaryotic initiation factor-4AII, and transcription factor homologue-HBZ17); and (5) genes encoding stress proteins (alphaB-crystallin and mu-crystallin). In 5 additional failing hearts and 4 additional nonfailing controls, we then compared expression of proteins encoded by the differentially expressed genes, alphaB-crystallin, SLIM1, gelsolin, alpha(1)-antichymotrypsin, and ubiquitin. In each case, changes in protein expression were consistent with changes in transcript measured by microarray analysis. Gelsolin protein expression was also increased in cardiomyopathic hearts from tropomodulin-overexpressing (TOT) mice and rac1-expressing (racET) mice. CONCLUSIONS: Altered expression of the genes identified in this study may contribute to development of the heart failure phenotype and/or represent compensatory mechanisms to sustain cardiac function in failing human hearts.

The human phospholamban gene: structure and expression.

Phospholamban, through modulation of sarcoplasmic reticulum calcium-ATPase activity, is a key regulator of cardiac diastolic function. Alterations in phospholamban expression may define parameters of muscle relaxation. In experimental animals, phospholamban is differentially expressed in various striated and smooth muscles, and within the four chambers of the heart. Decreased phospholamban expression within the heart during heart failure has also been observed. Furthermore, regulatory elements of mammalian phospholamban genes remain poorly defined. To extend these studies to humans, we (1) characterized phospholamban expression in various human organs, (2) isolated genomic clones encoding the human phospholamban gene, and (3) prepared human phospholamban promoter/luciferase reporter constructs and performed transient transfection assays to begin identification of regulatory elements. We observed that human ventricle and quadriceps displayed high levels of phospholamban transcripts and proteins, with markedly lower expression observed in smooth muscles, while the right atria also expressed low levels of phospholamban. The human phospholamban gene structure closely resembles that reported for chicken, rabbit, rat, and mouse. Comparison of the human to other mammalian phospholamban genes indicates a marked conservation of sequence for at least 217 bp upstream of the transcription start site, which contains conserved motifs for GATA, CP1/NFY, M-CAT-like, and E-box elements. Transient transfection assays with a series of plasmids containing deleted 5' flanking regions (between -2530 and -66 through +85) showed that sequences between -169 and the CP1-box at -93 were required for maximal promoter activity in neonatal rat cardiomyocytes. Activity of these reporters in HeLa cells was markedly lower than that observed in rat cardiomyocytes, suggesting at least a partial tissue selectivity of these reporter constructs.

Protein kinase A (PKA)-dependent troponin-I phosphorylation and PKA regulatory subunits are decreased in human dilated cardiomyopathy.

BACKGROUND: Most studies indicate that failing human hearts have greater baseline myofibrillar Ca2+ sensitivity of tension development than nonfailing hearts. Phosphorylation of cardiac troponin I (TnI) by cAMP-dependent protein kinase (PKA) decreases the affinity of the troponin complex for Ca2+, thus altering the Ca2+ sensitivity of force production. We tested the hypothesis that PKA-dependent TnI phosphorylation is altered in the failing human heart and investigated changes in PKA regulatory subunits as a potential mechanism. METHODS AND RESULTS: Using in vitro back-phosphorylation with [gamma-32P]ATP, we demonstrated a significant (P<0.05) approximately 25% reduction in baseline PKA-dependent TnI phosphorylation in human hearts with dilated cardiomyopathy (DCM) compared with nonfailing (NF) human hearts. There was no significant difference in cAMP content or maximal PKA activity between DCM and NF hearts, but expression of the regulatory subunits of PKA-I (RI) and PKA-II (RII) was significantly decreased in DCM versus NF hearts (RI by approximately 40%, P<0.05; RII by approximately 30%, P<0.01). CONCLUSIONS: PKA activity is regulated at the substrate level through interactions of PKA regulatory subunits with A-kinase anchoring proteins. The reduced baseline PKA-dependent phosphorylation of TnI in DCM may be due to decreased expression of RI and RII and consequently reduced anchoring of PKA holoenzyme. These findings provide new evidence of deficiencies in downstream regulation of the beta-adrenergic pathway in the failing human heart and may account for increased baseline myofibrillar Ca2+ sensitivity.

Effects of ketamine on the contractility of failing and nonfailing human heart muscles in vitro.

BACKGROUND: Induction of anesthesia with ketamine may decrease cardiac output in critically ill patients. The direct effects of ketamine on the failing human myocardium are unknown. This study examined the effects of ketamine on contractility of human failing and nonfailing myocardium in vitro. METHODS: Trabecular muscles were obtained from the left ventricles and right atria of 10 patients with heart failure undergoing transplantation and from the right atria of 14 patients undergoing coronary artery bypass surgery. Muscles were dissected and mounted in a 37 degrees C bath and stimulated at 1 Hz. Isometric contraction variables were recorded before and after addition of ketamine (concentrations between 0.44 and 440.0 microM) to the bath. The effects of ketamine were compared with those of buffer. To test muscle contractility, at the end of each experiment, 1 microM isoproterenol was added. RESULTS: Ketamine caused a significant dose-dependent decrease in developed tension in nonfailing atrial and failing atrial and ventricular muscles (P < 0.01 for all). In vehicle-treated muscles, developed tension remained stable, and isoproterenol increased developed tension 136% (nonfailing atrial muscles) and 178% (failing atrial and ventricular muscles; P < 0.01). In nonfailing atrial muscle, isoproterenol restored the ketamine-induced decrease in developed tension toward the baseline value. In failing atrial and ventricular muscles exposed to ketamine, isoproterenol did not counteract the ketamine. CONCLUSIONS: Ketamine exerts a direct dose-dependent negative inotropic effect in human heart muscles. The failing myocardium exposed to ketamine has reduced ability to increase contractility even in the presence of increased beta-adrenergic stimulation.

Troponin I phosphorylation and myofilament calcium sensitivity during decompensated cardiac hypertrophy.

We have measured myocyte cell shortening, troponin-I (Tn-I) phosphorylation, Ca2+ dependence of actomyosin adenosinetriphosphatase (ATPase) activity, adenosine 3',5'-cyclic monophosphate (cAMP) levels, and myofibrillar isoform expression in the spontaneously hypertensive rat (SHR) during decompensated cardiac hypertrophy (76 wk old) and in age-matched Wistar-Kyoto rat (WKY) controls. The decreased inotropic response to beta-adrenergic stimulation previously observed in myocytes from 26-wk-old SHR was further reduced at 76 wk of age. In response to beta-adrenergic stimulation, Tn-I phosphorylation was greater in the 76-wk-old SHR than in the WKY, although cAMP-dependent protein kinase A (PKA)-dependent Tn-I phosphorylation in the SHR did not increase with progression from compensated (26 wk) to decompensated (76 wk) hypertrophy. We also observed a dissociation between the increased PKA-dependent Tn-I phosphorylation and decreased cAMP levels in the 76-wk-old SHR versus WKY during beta-adrenergic stimulation. Baseline Tn-I phosphorylation was significantly reduced in 76-wk-old SHR versus WKY and was associated with decreased basal cAMP levels and increased Ca2+ sensitivity of actomyosin ATPase activity. The change in myofilament Ca2+ sensitivity during beta-adrenergic stimulation in the 76-wk-old SHR (0.65 pCa units) was over twofold greater than in the 76-wk-old WKY (0.30 pCa units). We also determined whether embryonic troponin T isoforms were reexpressed in decompensated hypertrophy and observed significant reexpression of the embryonic cardiac troponin T isoforms in the 76-wk-old SHR. The significant decrease in Ca2+ sensitivity with beta-adrenergic stimulation in 76-wk-old SHR may contribute to the severely impaired inotropic response during decompensated hypertrophy in the SHR.

Mitochondrial calcium content in isolated perfused heart: effects of inotropic stimulation.

We tested the hypothesis that in the intact heart, mitochondrial metabolism is activated by mitochondrial Ca2+ uptake during increased work. We measured left ventricular pressure (LVP), pyruvate dehydrogenase (PDH) activity, and mitochondrial and A band elemental content by electron probe microanalysis (EPMA) in Langendorff-perfused hamster hearts under control conditions, after isoproterenol (10(-6) M) stimulation, and after increasing perfusion pressure from 60 to 100 mmHg. Hearts were rapidly frozen, then EPMA was performed on cryosections cut from the surface of the frozen hearts; PDH activity was measured from the same area. Isoproterenol and elevated perfusion pressure increased LVP by 185 +/- 21 and 58 +/- 14%, respectively, versus controls. PDH activity increased from 10.4 +/- 1.5 (mean +/- SE) nmol.min-1. mg protein-1 (controls) to 21.6 +/- 3.5 (isoproterenol) and 18.5 +/- 3.2 nmol.min-1.mg protein-1 (increased perfusion pressure). There was no significant change in mitochondrial Ca1 in response to isoproterenol [1.2 +/- 0.1 (mean +/- SE) mmol/kg dry wt] or increased perfusion pressure (1.1 +/- 0.1) versus controls (1.0 +/- 0.1). These results suggest that, in the intact heart, mechanisms other than mitochondrial Ca2+ uptake may contribute to PDH activation and increased cardiac work.

Troponin I phosphorylation in spontaneously hypertensive rat heart: effect of beta-adrenergic stimulation.

We compared baseline and protein kinase A (PKA)-dependent troponin I (TnI) phosphorylation in 32Pi-labeled left ventricular myocytes from hearts of 26-wk spontaneously hypertensive rats (SHR) and Wistar-Kyoto controls (WKY). TnI phosphorylation was normalized to myosin light chain 2 phosphorylation, which was invariant. There was no difference in baseline TnI phosphorylation in SHR and WKY, but stimulation with isoproterenol, norepinephrine plus prazosin, forskolin, chloroadenosine 3',5'-cyclic monophosphate, or 3-isobutyl-1-methylxanthine caused a greater increase in TnI phosphorylation in the SHR than in the WKY. This was observed both in the presence and absence of the phosphatase inhibitor calyculin A; thus the differences in TnI phosphorylation between SHR and WKY are not due to decreased phosphatase activity in the SHR. After stimulation of the beta-adrenergic pathway, phospholamban phosphorylation was not different in SHR and WKY, indicating that the observed differences may be specific for PKA phosphorylation of TnI. The increased PKA-dependent TnI phosphorylation in the SHR resulted in decreased Ca2+ sensitivity of actomyosin adenosinetriphosphatase activity as compared with the WKY. We conclude that increased PKA-dependent TnI phosphorylation in the SHR may contribute to the impaired response to sympathetic stimulation.