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.

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.

The endogenous cardiac sarcoplasmic reticulum Ca2+/calmodulin-dependent kinase is activated in response to beta-adrenergic stimulation and becomes Ca2+-independent in intact beating hearts.

We investigated the effects of beta-adrenergic stimulation on the activity of the endogenous cardiac sarcoplasmic reticulum Ca2+/calmodulin-dependent protein kinase (SRCaM kinase) in Langendorff-perfused rat hearts. We found that isoproterenol induced generation of autonomous (Ca2+-independent) SRCaM kinase activity to 28 +/- 4.4% of the total activity. Moreover, dephosphorylation of the autonomous SRCaM kinase with protein phosphatase 2A resulted in an enzyme that was again dependent on Ca2+ and calmodulin for its activity. Activation of SRCaM kinase was coupled to phospholamban phosphorylation and activation of the cAMP-signaling system. Our results suggest that the cardiac SRCaM kinase is activated in response to beta-adrenoceptor stimulation. This activation stimulates autophosphorylation at its regulatory domain and converts it to an active Ca2+-independent species that may be the basis for potentiation of Ca2+ transients in the heart.

Effects of zinc on phospholamban phosphorylation.

The effects of zinc on the phosphorylation of phospholamban (PLB) were studied in sarcoplasmic reticulum (SR) membranes prepared from swine ventricular muscle. Zinc produced a dose dependent inhibition of PLB phosphorylation. With the use of phosphorylation site specific antibodies, it was shown that this inhibition was specific for the PLB phosphorylation at Thr-17. Since phosphorylation of this site is known to be mediated by the Ca2+/calmodulin-dependent protein kinase endogenous to the cardiac SR (SRCaM kinase), the action of zinc on SRCaM kinase was investigated. It was found that (i) zinc inhibited the activity of SRCaM kinase (IC50: 15 microM) and (ii) zinc concentrations, at the millimolar range, stimulated Ca(2+)-independent SRCaM kinase autophosphorylation. This ability of zinc to differentiate between autophosphorylation and substrate phosphorylation activities of SRCaM kinase raises the possibility that zinc mediated independent regulation of these processes can occur in the intact heart.

Alternative splicing and cycling kinetics of myosin change during hypertrophy of human smooth muscle cells.

We investigated in vivo expression of myosin heavy chain (MHC) isoforms, 17 kDa myosin light chain (MLC17), and phosphorylation of the 20 kDa MLC (MLC20) as well as mechanical performance of chemically skinned fibers of normal and hypertrophied smooth muscle (SM) of human myometrium. According to their immunological reactivity, we identified three MHC isoenzymes in the human myometrium: two SM-MHC (SM1 with 204 kDa and SM2 with 200 kDa), and one non-muscle specific MHC (NM with 196 kDa). No cross-reactivity was detected with an antibody raised against a peptide corresponding to a seven amino acid insert at the 25K/50K junction of the myosin head (a-25K/50K) in both normal and hypertrophied myometrium. In contrast, SM-MHC of human myomatous tissue strongly reacted with a-25K/50K. Expression of SM1/SM2/NM (%) in normal myometrium was 31.7/34.7/33.6 and 35.1/40.9/24 in hypertrophied myometrium. The increased SM2 and decreased NM expression in the hypertrophied state was statistically significant (P < 0.05). MHC isoform distribution in myomatous tissue was similar to normal myometrium (36.3/35.3/29.4). In vivo expression of MLC17a increased from 25.5% in normal to 44.2% in hypertrophied (P < 0.001) myometrium. Phosphorylation levels of MLC20 upon maximal Ca(2+)-calmodulin activation of skinned myometrial fibers were the same in normal and hypertrophied myometrial fibers. Maximal force of isometric contraction of skinned fibers (pCa 4.5, slack-length) was 2.85 mN/mm2 and 5.6 mN/mm2 in the normal and hypertrophied state, respectively (P < 0.001). Apparent maximal shortening velocity (Vmax(appt), extrapolated from the force-velocity relation) of myometrium rose from 0.13 muscle length s-1 (ML/s) in normal to 0.24 ML/s in hypertrophied fibers (P < 0.001).

Site-specific phosphorylation of a phospholamban peptide by cyclic nucleotide- and Ca2+/calmodulin-dependent protein kinases of cardiac sarcoplasmic reticulum.

Phospholamban (PLB), the regulator of the cardiac sarcoplasmic reticulum (SR) Ca2+ pump is specifically phosphorylated at Ser16 and Thr17 by cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase (CaMK), respectively. The regulation of this dual-site phosphorylation of amino acid residues in direct proximity is only poorly understood. In order to study the site-specific phosphorylation of PLB, we used a synthetic peptide (PLB-24) corresponding to the cytosolic part of the PLB monomer with the phosphorylation sites as a model substrate. PLB-24 possesses substrate properties as the native PLB as demonstrated by phosphorylation with exogenous, purified PKA, cGMP-dependent protein kinase (PKG) and a type II CaMK (CaMKII). In isolated vesicles of cardiac SR there was a rapid phosphorylation of the peptide by the endogenous PKA (SR-PKA) and CaMK (SR-CaMK), but not under conditions that activate PKG. Both SR-PKA and SR-CaMK incorporated the same amount of 32P into PLB-24, 0.60 +/- 0.01 nmol 32P/mg SR protein and 0.61 +/- 0.03 nmol 32P/mg SR protein, respectively. Phosphorylation by SR-PKA was abolished by the specific PKA inhibitor (IC50 = 0.2 microM), whereas SR-CaMK phosphorylation was inhibited by calmidazolium (IC50 = 1.6 microM) and a CaMKII-specific inhibitor peptide (IC50 = 2.5 microM). Phosphorylation by SR-PKA was exclusively at Ser, whereas SR-CaMK phosphorylated only Thr. After simultaneous activation of both SR-kinases 32P incorporation into PLB-24 was additive and occurred at Ser as well as at Thr. Sequential activation of SR-PKA and SR-CaMK also caused the additive phosphorylation of PLB-24 independently of which kinase was activated first. Thus, at the monomeric level of PLB the respective phosphorylation site appears to be accessible to its related SR protein kinase in vitro even when the adjacent site is phosphorylated.

The cardiac sarcoplasmic reticulum phospholamban kinase is a distinct delta-CaM kinase isozyme.

Phospholamban is the regulator of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum (SR). It is phosphorylated by a Ca2+/calmodulin-dependent protein kinase (SRCaM kinase) which is closely associated with cardiac SR membrane preparations. We found that, upon renaturation of pig cardiac SR proteins, blotted onto PVDF membrane, two polypeptides of 54 and 52 kDa showed Ca2+/calmodulin-dependent autophosphorylation. In Western blots of SR proteins, the 54/52 kDa polypeptides were recognized by an antibody specific for the delta-CaM kinase isoforms, but not by an anti-alpha-CaM kinase. The two polypeptides were selectively immunoprecipitated from solubilized SR vesicles with the anti-delta-CaM kinase. The CaM kinase inhibitors KN-62 and peptide CaMK-(281-302) inhibited the activity of the SRCaM kinase with IC50 values in the same range with those obtained for the brain isozyme. In addition, initial autophosphorylation (Ca(2+)-dependent) produced a partially Ca(2+)-independent enzyme while further autophosphorylation (Ca(2+)-independent) made the enzyme completely Ca(2+)-independent. Based on these results we suggest that the SRCaM kinase is a distinct delta-CaM kinase isozyme.

Modulation of phosphorylase kinase activity by sphingolipids.

Psychosine (galactosyl sphingosine) potently inhibits the activity of both nonactivated and activated by covalent modification (autophosphorylation and limited proteolysis) rabbit skeletal muscle phosphorylase kinase. Half-maximal inhibition was observed at 44 microM or 66 microM when the kinase activity was assayed at pH 6.8 or 8.2 respectively. Sphingosine was also inhibitory, but only at pH 6.8 (half-maximal inhibition was observed at 130 microM). In this respect, sphingomyelin, cerebroside and cerebroside sulfate were ineffective. On the other hand, a number of gangliosides stimulated the activity of nonactivated phosphorylase kinase at neutral pH. Among the individual gangliosides tested the activation potency was GD1a greater than GT1b greater than GM1, while GM3 was without effect. Most important, GD1a dramatically increases the activity of the kinase at low Ca2+ concentrations. Both psychosine and GD1a increased the rate of kinase autophosphorylation on alpha- subunit only, but although ganglioside-induced stimulation of autophosphorylation was accompanied with an enhancement of the rate of autoactivation at pH 6.8, psychosine completely blocked autoactivation.