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

AbstractPhospholamban is the regulator of the Ca2+-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 δ-CaM kinase isoforms, but not by an anti-α-CaM kinase. The two polypeptides were selectively immunoprecipitated from solubilized SR vesicles with the anti-δ-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 (Ca2+-dependent) produced a partially Ca2+-independent enzyme while further autophosphorylation (Ca2+-independent) made the enzyme completely Ca2+-independent. Based on these results we suggest that the SRCaM kinase is a distinct δ-CaM kinase isozyme

Distinct contractile systems for electromechanical and pharmacomechanical coupling in smooth muscle

Electromechanical coupling by KCl depolarization of bladder preparations elicits an initial phasic and subsequent tonic contraction. Using a smooth-muscle myosin heavy chain (SM-MyHC) knock-out mouse model we could previously demonstrate, that phasic and tonic contraction of intact neonatal bladder preparations could be elicited through the recruitment of SM-MyHC and non-muscle myosin heavy chains (NM-MyHC), respectively. Inhibition of myosin light chain kinase (MLCK) by ML-7 eliminated the phasic contraction of wild-type (+/+), rather than tonic contraction of neonatal bladder strips prepared from both +/+ and homozygous SM-MyHC knock-out (-/-) mice. Pharmacomechanical coupling upon PDBu-induced activation of protein kinase C of neonatal bladder preparations elicited tonic contraction of both +/+ and -/- murine. We suggest that: i) electromechanical coupling activates both SM-MyHC and NM-MyHC systems via a ML-7 sensitive and insensitive pathway, respectively. ii) Pharmacomechanical coupling recruits part of the NM-MyHC system rather than SM-MyHC

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

AbstractWe investigated the effects of β-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 β-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

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

Analysis of the energetic state of heart cells after adenovirus-mediated expression of hALC-1

Expression of the human atrial myosin light chain 1 (hALC-1) in the cardiac ventricle in vivo as well as in primary cultivated adult cardiomyocytes caused a pronounced positive inotropic effect. Therefore, it is one of the most promising candidate gene to treat congestive heart failure (CHF). In this work, we investigated, whether hALC-1 expression also modifies the energetic state of cardiomyocytes. Primary cultivated neonatal rat hearts cells (NRHC) were infected with adenoviral vectors (Ad vectors) containing a hALC-1 cDNA (AdCMV.hALC-1) or a control Ad vector. Infection efficiency of NRHC reached 100% at 50 multiplicity of infection (MOI). Interestingly and in contrast to primary cultures of liver cells, there were no cytotoxic side effects or induction of apoptosis up to MOI 50 in Ad vector infected NRHC. NRHC expressed large amounts of hALC-1 upon infection with AdCMV.hALC-1 which could easily been detected by protein staining and Western blot analysis. Analysis of intracellular hALC-1 localization by double-labeling immunofluorescence of AdCMV.hALC-1 infected cardiomyocytes revealed the typical myofibrillar striation pattern, as well as co-localization of hALC-1 with myosin heavy chains. There was no difference in the oxygen consumption between controls and AdCMV.hALC-1 infected NRHC. These data suggest that first: adenoviral vectors could be used as a safe and effective tool for gene transfer to cardiomyocytes, and second: that a positive inotropic effect of hALC-1 is not associated with enhanced oxygen consumption