Nuclear translocation of the cardiac L-type calcium channel C-terminus is regulated by sex and 17ß-estradiol.

The cardiac voltage gated l-type Ca(2+) channel (Cav1.2) constitutes the main entrance gate for Ca(2+) that triggers cardiac contraction. Several studies showed that the distal C-terminus fragment of Cav1.2 α1C subunit (α1C-dCT) is proteolytically cleaved and shuttles between the plasma membrane and the nucleus, which is regulated both developmentally and by Ca(2+). However, the effects of sex and sex hormone 17ß-estradiol (E2, estrogen) on α1C-dCT nuclear translocation are still unexplored. To investigate the sexual disparity in the α1C-dCT nuclear translocation, we first generated an antibody directed against a synthetic peptide (GRRASFHLE) located in α1C-dCT, and used it to probe ventricular myocytes from adult female and male mice. Immunocytochemistry of isolated mouse primary adult ventricular myocytes revealed both nuclear staining and cytosolic punctuate staining around the T-tubules. The ratio of nuclear to cytosolic intensity (Inuc/Icyt) was significantly higher in isolated female cardiomyocytes (1.42±0.05) compared to male cardiomyocytes (1.05±0.02). Western blot analysis of nuclear fraction confirmed these data. Furthermore, we found a significant decrease in nuclear staining intensity of α1C-dCT in both female and male cardiomyocytes upon serum withdrawal for 18h (Inuc/Icyt 1.05±0.02 and 0.89±0.02, respectively). Interestingly, subsequent E2 treatment (10(-8)M) for 8h normalized the intracellular distribution of α1C-dCT in male cardiomyocytes (Inuc/Icyt 1.04±0.02), but not in female cardiomyocytes. Acute treatment of male cardiomyocytes with E2 for 45min revealed a similar effect. This effect of E2 was revised by ICI indicating the involvement of ER in this signaling pathway. Taken together, our results showed that the shuttling of α1C-CT in cardiomyocytes is regulated in a sex-dependent manner, and E2-activated ER may play a role in the nuclear shuttling of α1C-dCT in male cardiomyocytes. This may explain, at least partly, the observed sex differences in the regulation of cardiac Cav1.2 channel activity.

Interaction of epoxyeicosatrienoic acids and adipocyte fatty acid-binding protein in the modulation of cardiomyocyte contractility.

BACKGROUND: Adipocyte fatty acid-binding protein (FABP4) is a member of a highly conserved family of cytosolic proteins that bind with high affinity to hydrophobic ligands, such as saturated and unsaturated long-chain fatty acids and eicosanoids. Recent evidence has supported a novel role for FABP4 in linking obesity with metabolic and cardiovascular disorders. In this context, we identified FABP4 as a main bioactive factor released from human adipose tissue that directly suppresses heart contraction in vitro. As FABP4 is known to be a transport protein, it cannot be excluded that lipid ligands are involved in the cardiodepressant effect as well, acting in an additional and/or synergistic way. OBJECTIVE: We investigated a possible involvement of lipid ligands in the negative inotropic effect of adipocyte factors in vitro. RESULTS: We verified that blocking the CYP epoxygenase pathway in adipocytes attenuates the inhibitory effect of adipocyte-conditioned medium (AM) on isolated adult rat cardiomyocytes, thus suggesting the participation of epoxyeicosatrienoic acids (EETs) in the cardiodepressant activity. Analysis of AM for EETs revealed the presence of 5,6-, 8,9-, 11,12- and 14,15-EET, whereas 5,6-EET represented about 45% of the total EET concentration in AM. Incubation of isolated cardiomyocytes with EETs in similar concentrations as found in AM showed that 5,6-EET directly suppresses cardiomyocyte contractility. Furthermore, after addition of 5,6-EET to FABP4, the negative inotropic effect of FABP4 was strongly potentiated in a concentration-dependent manner. CONCLUSIONS: These data suggest that adipocytes release 5,6-EET and FABP4 into the extracellular medium and that the interaction of these factors modulates cardiac function. Therefore elevated levels of FABP4 and 5,6-EET in obese patients may contribute to the development of heart dysfunction in these subjects.

Adipocyte-derived factors suppress heart contraction.

BACKGROUND: Obesity is strongly associated with cardiovascular diseases including systemic hypertension, coronary artery disease and heart failure. Despite several investigations the pathophysiological mechanisms involved remain unclear. We have previously shown that adipose tissue exerts a highly potent activity with an acute depressant effect on cardiomyocytes, thus suggesting direct involvement of adipose tissue in the development of heart dysfunction. OBJECTIVE AND DESIGN: This study investigates the effects of adipocyte factors obtained from subcutaneous adipose tissue on the whole cardiac function by using isolated perfused rat hearts in a Langendorff mode. We recorded changes in coronary flow, developed isovolumetric left ventricular pressure, contraction rate and relaxation rate. RESULTS: We observed a significant decrease in heart contractility parameters as well as in coronary flow within a few seconds of incubation with adipocyte factors. The cardiodepressant effects could not be blocked by the nonselective cyclooxygenase-inhibitor indomethacin. Human adipocytes release tumor necrosis factor-α, interleukin-6 (IL-6) and IL-1ß into extracellular medium. These cytokines were tested for their potential effect but were, however, not responsible for the cardiodepressant effect observed. CONCLUSION: These data indicate that human adipocytes secrete factors with a strong acute depressant effect on cardiac force generation and coronary flow due to contraction of the coronary vessels, thus suggesting a direct role of adipose tissue in the pathogenesis of cardiac dysfunction.

Regulation of naturally occurring antisense RNA of myosin heavy chain (MyHC) in neonatal cardiomyocytes.

Naturally occurring antisense RNA has been detected for a range of eukaryotic genes. Its abundance compared to levels of its complementary sense mRNA appears to be a factor indicating its possible regulatory function. In previous studies, we detected appreciable levels of antisense RNA against the two isoforms (alpha and beta) of the heavy myosin-chain (MyHC) in the myocardium of rats. If this is to play a significant role in gene expression antisense levels should vary in response to external and internal cellular influences. Recently, a bidirectional promoter located in the alpha/beta MyHC intergenic region was described, which was proposed to regulate coordinated transcription of alpha-MyHC sense and beta-MyHC antisense. To study MyHC antisense regulation in neonatal heart, we investigated cultivated myocytes stimulated with either trijodthyronin (T3) as an inductor of alpha-MyHC or phenylephrine with stimulation of beta-MyHC. RNA-quantification of sense and antisense transcripts of both isoforms was performed by real-time RT-PCR. Stimulation by T3 led to an induction of both sense and antisense of alpha-MyHC and to a decrease of beta-MyHC sense and antisense. Phenylephrine increased sense and antisense beta-MyHC but reduced antisense alpha-MyHC. The sense/antisense of alpha- and beta-MyHC ratio was unchanged compared to control. Results indicate a coregulation of sense and antisense MyHC RNA under stimulation of T3 and phenylephrine in neonatal cardiomyocytes.

Increased expression of non-muscle myosin heavy chain-B in connective tissue cells of hypertrophic rat urinary bladder.

Expression of the non-muscle myosin heavy chain-B (NM-MHC-B, also denoted as the embryonic smooth muscle myosin heavy chain, SMemb) was examined in rat urinary bladder during growth in response to a partial urinary outflow obstruction. Following obstruction, the weight of the urinary bladder increased more than five-fold within 10 days. Immunohistochemistry with a polyclonal antiserum against the C-terminal sequence of NM-MHC-B revealed very few NM-MHC-B immunoreactive cells in the control urinary bladders. In hypertrophic bladders, the number of NM-MHC-B immunoreactive cells markedly increased. The majority of such cells were found in the interstitium surrounding smooth muscle bundles and also in the subserosal and submucosal layers. Western blot analysis showed that the NM-MHC-B expression was transient; the content of NM-MHC-B immunoreactive material had doubled 10 days after obstruction and then declined towards the control level after 6 weeks. Immunohistochemistry revealed co-localization of NM-MHC-B and vimentin within the same cells. NM-MHC-B did not co-localize with smooth muscle actin, suggesting that the source of NM-MHC-B is not a de-differentiated smooth muscle cell or myofibroblast but a non-muscle cell possibly reacting to tissue distension or stress. The NM-MHC-B-positive cells could have a role in the production of extracellular matrix and growth factors or be involved in modulation of spontaneous contractile activity.

The human beta-myosin heavy chain gene: sequence diversity and functional characteristics of the protein.

The beta-myosin heavy chain gene (MYH7) encodes the motor protein that drives myocardial contraction. It has been proven to be a disease gene for hypertrophic cardiomyopathy (HCM). We analyzed the DNA sequence variation of MYH7 (about 16 kb) of eight individuals: six patients with HCM and two healthy controls. The overall DNA sequence identity was up to 97.2% compared to Jaenicke and coworkers (Jaenicke et al. [1990] Genomics 8:194-206), while the corresponding amino acid sequences revealed 100% identity. In HCM patients, eleven nucleotide substitutions were identified but no causative disease mutation was found: six were detected in coding, four in intronic, and one in 5' regulatory regions. The average nucleotide diversity across this locus was 0.015% with an average of 0.02% in the coding and 0.012% in the noncoding sequence. Analysis of the kinetic behaviour of beta-MHC in the intact contractile structure of normal individuals and HCM patients revealed apparent rate constants of tension development ranging between 1.58 s(-1) and 1.48 s(-1).

Smooth-muscle contraction without smooth-muscle myosin.

Here we have used gene-targeting to eliminate expression of smooth-muscle myosin heavy chain. Elimination of this gene does not affect expression of non-muscle myosin heavy chain, and knockout individuals typically survive for three days. Prolonged activation, by KCl depolarisation, of intact bladder preparations from wild-type neonatal mice produces an initial transient state (phase 1) of high force generation and maximal shortening velocity, which is followed by a sustained state (phase 2) characterized by low force generation and maximal shortening velocity. Similar preparations from knockout neonatal mice do not undergo phase 1, but exhibit a normal phase 2. We propose that, in neonatal smooth muscle phase 1 is generated by recruitment of smooth-muscle myosin heavy chain, whereas phase 2 can be generated by activation of non-muscle myosin heavy chain. We conclude that phase 1 becomes indispensable for survival and normal growth soon after birth, particularly for functions such as homeostasis and circulation.

Modulation of the smooth-muscle L-type Ca2+ channel alpha1 subunit (alpha1C-b) by the beta2a subunit: a peptide which inhibits binding of beta to the I-II linker of alpha1 induces functional uncoupling.

Modulation of the smooth-muscle Ca(2+) channel alpha1C-b subunit by the auxiliary beta2a subunit was studied in the HEK 293 (cell line from human embryonic kidney cells) expression system. In addition, we tested whether the alpha1-beta interaction in functional channels is sensitive to an 18-amino-acid synthetic peptide that corresponds to the sequence of the defined major interaction domain in the cytoplasmic I-II linker of alpha1C (AID-peptide). Ca(2+) channels derived by co-expression of alpha1C-b and beta2a subunits exhibited an about 3-fold higher open probability (P(o)) than alpha1C-b channels. High-P(o) gating of alpha1C-b.beta2a channels was associated with the occurrence of long-lasting channel openings [mean open time (tau)>10 ms] which were rarely observed in alpha1C-b channels. Modulation of fast gating by the beta2a subunit persisted in the cell-free, inside-out recording configuration. Biochemical experiments showed that the AID-peptide binds with appreciable affinity to beta2 subunits of native Ca(2+) channels. Binding of the beta2 protein to immobilized AID-peptide was specifically inhibited (K(i) of 100 nM) by preincubation with free (uncoupled) AID-peptide, but not by a corresponding scrambled peptide. Administration of the AID-peptide (10 microM) to the cytoplasmic side of inside-out patches induced a substantial reduction of P(o) of alpha1C-b.beta2a channels. The scrambled control peptide failed to affect alpha1C-b. beta2a channels, and the AID-peptide (10 microM) did not modify alpha1C-b channel function in the absence of expressed beta2a subunit. Our results demonstrate that the beta2a subunit controls fast gating of alpha1C-b channels, and suggest the alpha1-beta interaction domain in the cytoplasmic I-II linker of alpha1C (AID) as a possible target of modulation of the channel. Moreover, our data are consistent with a model of alpha1-beta interaction that is based on multiple interaction sites, including AID as a determinant of the affinity of the alpha1-beta interaction.

Expression of Ca(2+) channel subunits during cardiac ontogeny in mice and rats: identification of fetal alpha(1C) and beta subunit isoforms.

Functional cardiac L-type calcium channels are composed of the pore-forming alpha(1C) subunit and the regulatory beta(2) and alpha(2)/delta subunits. To investigate possible developmental changes in calcium channel composition, we examined the temporal expression pattern of alpha(1C) and beta(2) subunits during cardiac ontogeny in mice and rats, using sequence-specific antibodies. Fetal and neonatal hearts showed two size forms of alpha(1C) with 250 and 220 kDa. Quantitative immunoblotting revealed that the rat cardiac 250-kDa alpha(1C) subunit increased about 10-fold from fetal days 12-20 and declined during postnatal maturation, while the 220-kDa alpha(1C) decreased to undetectable levels. The expression profile of the 85-kDa beta(2) subunit was completely different: beta(2) was not detected at fetal day 12, rose in the neonatal stage, and persisted during maturation. Additional beta(2)-stained bands of 100 and 90 kDa were detected in fetal and newborn hearts, suggesting the transient expression of beta(2) subunit variants. Furthermore, two fetal proteins with beta(4) immunoreactivity were identified in rat hearts that declined during prenatal development. In the fetal rat heart, beta(4) gene expression was confirmed by RT-PCR. Cardiac and brain beta(4) mRNA shared the 3 prime region, predicting identical primary sequences between amino acid residues 62-519, diverging however, at the 5 prime portion. The data indicate differential developmental changes in the expression of Ca(2+) channel subunits and suggest a role of fetal alpha(1C) and beta isoforms in the assembly of Ca(2+) channels in immature cardiomyocytes.

Signaling from beta-adrenoceptor to L-type calcium channel: identification of a novel cardiac protein kinase A target possessing similarities to AHNAK.

A novel calcium channel-associated protein of approximately 700 kDa has been identified in mammalian cardiomyocytes that undergoes substantial cAMP-dependent protein kinase (PKA) phosphorylation. It was therefore designated as phosphoprotein 700 (pp700). The pp700 interacts specifically with the beta(2) subunit of cardiac L-type calcium channels as revealed by coprecipitation experiments using affinity-purified antibodies against different calcium channel subunits. It is surprising that amino acid sequence analysis of pig pp700 revealed homology to AHNAK-encoded protein, which was originally identified in human cell lines of neural crest origin as 700-kDa phosphoprotein. Cardiac AHNAK expression was assessed on mRNA level by reverse transcriptase-polymerase chain reaction. Sequence-directed antibodies raised against human AHNAK recognized pp700 in immunoblotting and immunoprecipitation experiments, confirming the homology between both proteins. Anti-AHNAK antibodies labeled preferentially the plasma membrane of cardiomyocytes in cryosections of rat cardiac tissue and isolated cardiomyocytes. Sarcolemmal pp700/AHNAK localization was not influenced by stimulation of either the PKA or the protein kinase C pathway. In back-phosphorylation studies with cardiac biopsies, we identified distinct pp700 pools. The membrane-associated fraction of pp700 underwent substantial in vivo phosphorylation on beta-adrenergic receptor stimulation by isoproterenol, whereas the cytoplasmic fraction of pp700 was not accessible to endogenous PKA. It is important that in vivo phosphorylation occurred in that pp700 fraction which coprecipitated with the calcium channel beta subunit. We hypothesize that both phosphorylation of pp700 and its coupling to the beta subunit play a physiological role in cardiac beta-adrenergic signal transduction. Haase, H., Podzuweit, T., Lutsch, G., Hohaus, A., Kostka, S., Lindschau, C., Kott, M., Kraft, R., Morano, I. Signaling from beta-adrenoceptor to L-type calcium channel: identification of a novel cardiac protein kinase A target that has similarities to AHNAK.

Expression of atrial myosin light chains but not alpha-myosin heavy chains is correlated in vivo with increased ventricular function in patients with hypertrophic obstructive cardiomyopathy.

The adult rodent heart adapts to increased work load by reexpression of its fetal genes, for example, beta-myosin heavy chain (MHC), in order to improve contractile function. However, the human ventricle regulates contractility by expression of atrial essential myosin light chain (ALC-1) rather than beta-MHC. We evaluated the impact of both mechanisms in patients with hypertrophic cardiomyopathy. MHC isoform expression was quantified at the mRNA and protein levels by reverse transcriptase polymerase chain reaction and immunoblotting, respectively. Although alpha-MHC mRNA was detected in control and hypertrophied human ventricular tissue, alpha-MHC protein was not observed. Similarly, we investigated the expression of ALC-1 by two-dimensional polyacrylamide gel electrophoresis and the clinical and hemodynamic parameters of the patients with hypertrophic cardiomyopathy. We found a significant positive correlation between ALC-1 protein expression and dP/dtmax in the hypertrophied human ventricle in vivo. Correlations between dP/dtmax and expression of protein for the ryanodine receptor and L-type Ca2+ channel were excluded. Our data suggest that reexpression of ALC-1 improves the contractile state of the adult human heart. We propose that two evolutionarily divergent compensatory mechanisms for increased work demand exist in the mammalian heart: MHC regulation in rodents and essential MLC regulation, of cardiac contractility, in humans.

Tuning the human heart molecular motors by myosin light chains.

Cardiac contraction is triggered by the cyclic interaction of the "molecular motor" protein myosin with the actin filament, consuming ATP as the energy source to produce tension or shortening. The myosin heavy chain (MHC) contains the actin- and ATP-binding sites and represents the molecular motor of muscle contraction. This review describes the various subunits of human heart myosin in health and disease and discusses their functions. Two different MHC genes (alpha and beta) with distinct biochemical features are expressed in the human heart. Alpha-MHC confers a higher ATPase activity and higher shortening velocity to the heart than beta-MHC. Motor function is regulated by myosin light chain (MLC) isoforms. Expression of the atrial MLC-1 isoform in the hypertrophied human ventricle increases cross-bridge cycling and contractility. It is suggested that MLC-1 acts as a MHC/actin tether. Weakening of this tether increases myosin function. MLC-2 slows the rate of tension development of myosin. This relative inhibition is relieved upon phosphorylation of the MLC-2 perhaps caused by "swing-out" of cross-bridges from the myosin filament. Mutations in all ventricular myosin subunits have been found in patients with hypertrophic cardiomyopathy.

Remodeling of the hypertrophied human myocardium by cardiac bHLH transcription factors.

The basic helix-loop-helix transcription factors eHAND and dHAND are involved in developmental cardiac growth and differentiation. We investigated HAND gene expression in the normal and in the hypertrophied right and left ventricle of patients with tetralogy of Fallot (ToF) and hypertrophic obstructive cardiomyopathy (HOCM). HAND mRNA was constitutively expressed in the hypertrophied heart and increased in the hypertrophic tissue of both patient groups. HAND genes had a complementary left-right cardiac asymmetry of expression with dHAND predominantly in the right and eHAND in the left ventricle. The two cardiac bHLH factors have the ability to form heterodimers with the ubiquitous bHLH protein E12, subsequently recognizing E-boxes in the promoter region of target genes like ALC-1. We found a highly significant positive correlation between HAND and ALC-1 mRNA. The total ALC-1 protein level in ToF was smaller than in HOCM, although ALC-1 mRNA as well as HAND mRNA levels were significantly higher. ToF patients expressed around four times more ALC-1 mRNA for similar amounts of ALC-1 than HOCM patients. Suggesting disturbed ALC-1 translation in ToF, we found ALC-1 antisense mRNA expression in the hypertrophied, but not in the normal, ventricles. The higher the antisense/sense ALC-1 mRNA ratio, the lower ALC-1 protein was expressed.

Inhibition of crossbridge function in the normal human heart by hypoxic endothelial superfusate.

Endothelial cells release diffusible substances which modulate myocardial function. Oxygen pressure is one important factor for stimulation and modulation of endothelial function. Here we investigated the effects of a superfusate obtained from hypoxic (pO(2) 40-50 mmHg) porcine endothelial cell culture on human myocardial crossbridge cycling rate. Isometric force development and the rate constant for tension development of demembranated multicellular fibers from the left myocardium of a normal human heart were determined from the low-tension rigor by photolytic release of ATP from caged-ATP. Incubation with hypoxic or normoxic superfusates did not change maximal isometric force development. However, rate constant of tension development of the normal human heart fibers significantly decreased to 43.3% upon incubation with the hypoxic but not normoxic endothelial cell superfusate.

Regulation of Limulus skeletal muscle contraction.

Skeletal muscle contraction of Limulus polyphemus, the horseshoe crab, seemed to be regulated in a dual manner, namely Ca2+ binding to the troponin complex as well phosphorylation of the myosin light chains (MLC) by a Ca2+/calmodulin-dependent myosin light chain kinase. We investigated muscle contraction in Limulus skinned fibers in the presence of Ca2+ and of Ca2+/calmodulin to find out which of the two mechanisms prevails in Limulus skeletal muscle contraction. Although skinned fibers revealed high basal MLC mono- and biphosphorylation levels (0.48 mol phosphate/mol 31 kDa MLC; 0.52 mol phosphate/mol 21 kDa MLC), the muscle fibers were fully relaxed at pCa 8. Upon C2+ or Ca2+/calmodulin activation, the fibers developed force (357+/-78.7 mN/mm2; 338+/-69.7 mN/mm2, respectively) while the MLC phosphorylation remained essentially unchanged. We conclude that Ca2+ activation is the dominant regulatory mechanism in Limulus skeletal muscle contraction.

Plakoglobin is essential for myocardial compliance but dispensable for myofibril insertion into adherens junctions.

Plakoglobin (gamma-catenin), a member of the armadillo family of proteins, is a constituent of the cytoplasmic plaque of cardiac junctions and is involved in anchorage of cytoskeletal filaments to specific cadherins. Its genetic inactivation leads to an embryonic lethal phenotype due to heart dysfunction related to an impairment in the architecture of intercalated discs and in the stability of the heart tissue. To elucidate the functional consequences of the loss of plakoglobin for myofibrillar function, we monitored passive stress-strain relationship and contractility parameters of demembranated embryonic fibers. Heart fibers obtained from plakoglobin-deficient embryonic mice were significantly less compliant than were fibers from wild-type embryos. This difference was especially pronounced at lower fiber extension levels: at 120% of slack length, compliance was 2.5-fold lower in plakoglobin-deficient mice than in the corresponding wild-type group. Contractile paramenters (force per cross-section; Ca2+ sensitivity of isometric force and shortening velocity at near-zero load) were comparable in all experimental groups. Therefore, we suggest that plakoglobin is important for cardiac compliance but not necessary for the attachment of the myofibrillar apparatus to adherens junctions. Thus, we conclude that the loss of function of desmosomes and the profound disarrangement of junctional components in plakoglobin null embryos is associated with a decreased passive compliance, which may explain the ventricular rupture and consequent pericardial tamponade in embryos lacking plakoglobin.

Expression of smooth muscle myosin heavy chain B in cardiac vessels of normotensive and hypertensive rats.

We investigated expression of the 5'-spliced isoform of smooth muscle myosin heavy chain (SM-MHC-B) in smooth muscle cells of cardiac vessels of the left ventricle of normotensive (Wistar-Kyoto) and spontaneously hypertensive rats of the stroke-prone strain by immunofluorescence microscopy. In parallel, liver and bladder were studied for characterization of the nature of vessels expressing SM-MHC-B and for semiquantitative evaluation of its abundance. Smooth muscle cells were detected by staining with a monoclonal antibody specific for alpha-smooth muscle actin. Abundance of the SM-MHC-B isoform in these cells was evaluated by using an antibody raised against the seven-amino acid insert at the 25K/50K junction of the myosin head (a25K/50K) that specifically recognized SM-MHC-B. In the ventricle, a25K/50K immunoreactivity was observed in smooth muscle cells of precapillary arterioles but not in larger vessels or aorta. The a25K/50K immunoresponse of those vessels with the highest expression level of SM-MHC-B closely resembled the signal observed in the smooth muscle layer of urinary bladder known to preferentially express SM-MHC-B. Interestingly, in left ventricles of stroke-prone spontaneously hypertensive rats, there was a significantly reduced fraction of a25K/50K-positive precapillary arterioles compared with normotensive control rats.

Phosphorylation of phospholamban correlates with relaxation of coronary artery induced by nitric oxide, adenosine, and prostacyclin in the pig.

The intracellular mechanisms underlying the action of the endogenous vasodilators such as NO/EDRF, adenosine, and prostacyclin acting through cGMP and cAMP, respectively, are not well understood. One important action of cyclic nucleotides in smooth muscle relaxation is to lower the cytosolic Ca2+ concentration by enhanced sequestration into the sarcoplasmic reticulum. The present study was undertaken to elucidate the potential role of phosphorylation of phospholamban, the regulator of sarcoplasmic reticulum Ca2+ pump, for the control of coronary vascular tone by NO/EDRF, adenosine, and prostacyclin. Phospholamban was identified in pig coronary artery preparations by immunofluorescence microscopy, Western blotting and in vitro phosphorylation. Segments of pig coronary artery, with either intact or denuded endothelium, were precontracted with prostaglandin F2alpha (PGF2alpha). In endothelium-denuded preparations 3-morpholinosydnonimine (SIN-1), 5'-N-ethylcarboxiamidoadenosine (NECA), and iloprost (ILO) caused both relaxation and phospholamban phosphorylation with the potency: SIN-1 > NECA > ILO. The regulatory myosin light chain was significantly dephosphorylated only by SIN-1. In endothelium-intact pig coronary artery, L-NAME caused additional vasoconstriction and a decrease in phospholamban phosphorylation, while phosphorylation of myosin light chain remained unchanged. An inverse relationship between phospholamban phosphorylation and vessel tone was obtained. Our findings demonstrate significant phospholamban phosphorylation during coronary artery relaxation evoked by NO, prostacyclin, and adenosine receptor activation. Because of the close correlation between phosphorylation of phospholamban and vessel relaxation, we propose that phospholamban phosphorylation is an important mechanism by which endogenous vasodilators, especially endothelial NO/EDRF, control coronary vascular smooth muscle tone.

Characterization of naturally occurring myosin heavy chain antisense mRNA in rat heart.

Analysis of mRNA by Northern blot and reverse transcription-polymerase chain reaction demonstrated the expression of sense and considerable amounts of naturally occurring antisense mRNA for beta-myosin heavy chain (MHC) and alpha-MHC in the neonatal rat heart: antisense MHC mRNA expression of alpha-MHC and beta-MHC was approximately half of the corresponding sense MHC mRNA expression. Using a computational approach, we could identify a reverse Pol II promoter in the beta-MHC gene. Both sense and antisense MHC mRNA demonstrated similar sizes of approximately 6,000 bp in the Northern blot. Alpha-MHC antisense mRNA consisted of approximately 3,700 bp of complementary exon sequences and beta-MHC consisted of approximately 2,700 bp, suggesting a higher probability of alpha-MHC mRNA dimerization. Hence, sense mRNA transcripts and protein of alpha-MHC should exist at different relative levels in the neonatal state. In fact, the relative proportion of alpha-MHC was 52.0 +/- 2.6% on the sense mRNA but only 36.3 +/- 1.8% on the protein level. Because of its high abundance in the heart, we suggest that in the neonatal heart naturally occurring antisense mRNA may play a role in the regulation of MHC expression and, therefore, in the control of the energetical and contractile behaviour of the heart.

Modulation of contractility in human cardiac hypertrophy by myosin essential light chain isoforms.

Cardiac hypertrophy is an adaptive response that normalizes wall stress and compensates for increased workload. It is accompanied by distinct qualitative and quantitative changes in the expression of protein isoforms concerning contractility, intracellular Ca(2+)-homeostasis and metabolism. Changes in the myosin subunit isoform expression improves contractility by an increase in force generation at a given Ca(2+)-concentration (increased Ca(2+)-sensitivity) and by improving the economy of the chemo-mechanical transduction process per amount of utilised ATP (increased duty ratio). In the human atrium this is achieved by partial replacement of the endogenous fast myosin by the ventricular slow-type heavy and light chains. In the hypertrophic human ventricle the slow-type beta-myosin heavy chains remain unchanged, but the ectopic expression of the atrial myosin essential light chain (ALC1) partially replaces the endogenous ventricular isoform (VLC1). The ventricular contractile apparatus with myosin containing ALC1 is characterised by faster cross-bridge kinetics, a higher Ca(2+)-sensitivity of force generation and an increased duty ratio. The mechanism for cross-bridge modulation relies on the extended Ala-Pro-rich N-terminus of the essential light chains of which the first eleven residues interact with the C-terminus of actin. A change in charge in this region between ALC1 and VLC1 explains their functional difference. The intracellular Ca(2+)-handling may be impaired in heart failure, resulting in either higher or lower cytosolic Ca(2+)-levels. Thus the state of the cardiomyocyte determines whether this hypertrophic adaptation remains beneficial or becomes detrimental during failure. Also discussed are the effects on contractility of long-term changes in isoform expression of other sarcomeric proteins. Positive and negative modulation of contractility by short-term phosphorylation reactions at multiple sites in the myosin regulatory light chain, troponin-I, troponin-T, alpha-tropomyosin and myosin binding protein-C are considered in detail.