Loss of SM-B myosin affects muscle shortening velocity and maximal force development.

We used an exon-specific gene-targeting strategy to generate a mouse model deficient only in the SM-B myosin isoform. Here we show that deletion of exon-5B (specific for SM-B) in the gene for the heavy chain of smooth muscle myosin results in a complete loss of SM-B myosin and switching of splicing to the SM-A isoform, without affecting SM1 and SM2 myosin content. Loss of SM-B myosin does not affect survival or cause any overt smooth muscle pathology. Physiological analysis reveals that absence of SM-B myosin results in a significant decrease in maximal force generation and velocity of shortening in smooth muscle tissues. This is the first in vivo study to demonstrate a functional role for the SM-B myosin isoform. We conclude that the extra seven-residue insert in the surface loop 1 of SM-B myosin is a critical determinant of crossbridge cycling and velocity of shortening.

The mechanism of action of cantharidin in smooth muscle.

1. The aim of this study was to investigate the mechanism(s) of the vasoconstrictor effect of cantharidin in bovine preparations. 2. Catalytic subunits of protein phosphatase type 1 (PP 1) and type 2A (PP 2A) were immunologically identified in coronary arteries, isolated smooth muscle cells and ventricular myocardium. 3. The mRNAs coding for catalytic subunits of PP 1alpha, PP 1beta and PP 2Aalpha were identified by hybridization with specific cDNA-probes in total RNA from coronary arteries, isolated smooth muscle cells and ventricles. 4. The activities of catalytic subunits of PP 1 and PP 2A separated by column chromatography from coronary arteries, isolated smooth muscle cells and ventricles were inhibited by cantharidin in a concentration-dependent manner. 5. Cantharidin increased the phosphorylation state of smooth muscle proteins including the regulatory light chains of myosin in 32P-labelled intact smooth muscle cells in a concentration-dependent manner. 6. Cantharidin did not affect cytosolic calcium concentrations in aortic smooth muscle cells. 7. It is suggested that cantharidin contracts smooth muscle preparations by increasing the phosphorylation state of regulatory proteins due to inhibition of phosphatase activities. Thus, cantharidin might be a useful tool to study the function of phosphatases in smooth muscle.

On the contractile function of protein phosphatases in isolated human coronary arteries.

It is unknown whether protein phosphatases types 1 and 2A are present in and can regulate the tone of human vascular tissue. The expression and possible function of serine/threonine protein phosphatases (PP) type 1 (PP1) and type 2A (PP2A) were studied in isolated human coronary arteries. Catalytic subunits of PPI and PP2A were identified by means of phosphatase activity measurement in tissue homogenates, by separation of enriched extracts through affinity column chromatography, by immunoblotting with specific antibodies, by hybridization of mRNA with specific DNA probes and PCR of reverse transcribed mRNA. Based on these methods, the catalytic subunits of PP1(alpha,beta,gamma) and PP2A(alpha,beta) were identified. Appropriately, cantharidin, an inhibitor of PP1 and PP2A, increased basal tone of human isolated coronary artery rings with an EC50 of about 16 micromol/l by increasing the phosphorylation state of the regulatory light chains of myosin. In summary, PP1 and PP2A are expressed in human coronary arteries and they can alter vascular tone.

SERCA pump level is a critical determinant of Ca(2+)homeostasis and cardiac contractility.

The control of intracellular calcium is central to regulation of cardiac contractility. A defect in SR Ca(2+)transport and SR Ca(2+)ATPase pump activity and expression level has been implicated as a major player in cardiac dysfunction. However, a precise cause-effect relationship between alterations in SERCA pump level and cardiac contractility could not be established from these studies. Progress in transgenic mouse technology and adenoviral gene transfer has provided new tools to investigate the role of SERCA pump level in the heart. This review focuses on how alterations in SERCA level affect Ca(2+)homeostasis and cardiac contractility. It discusses the consequences of altered SERCA pump levels for the expression and activity of other Ca(2+)handling proteins. Furthermore, the use of SERCA pump as a therapeutic target for gene therapy of heart failure is evaluated.

Role of protein phosphatases in regulation of cardiac inotropy and relaxation.

We studied the effects of the protein phosphatase (PP) inhibitor cantharidin (Cant) on time parameters and force of contraction (FOC) in isometrically contracting electrically driven guinea pig papillary muscles. We correlated the mechanical parameters of contractility with phosphorylation of the inhibitory subunit of troponin (TnI-P) and with the site-specific phosphorylation of phospholamban (PLB) at serine-16 (PLB-Ser-16) and threonine-17 (PLB-Thr-17). Cant (after 30 min) started to increase FOC (112 +/- 4% of control, n = 10) and TnI-P and PLB-Thr-17 (120 +/- 5 and 128 +/- 7% of control) without any alteration of relaxation time. Cant (10 microM) started to increase PLB-Ser-16, but the relaxation was shortened at only 100 microM (from 140 +/- 9 to 116 +/- 12 ms, n = 9). Moreover, 100 microM Cant, 3 min after application, started to increase PLB-Thr-17, TnI-P, and FOC. Cant (100 microM) began to increase PLB-Ser-16 after 20 min. This was accompanied by shortening of relaxation time. Differences in protein kinase activation or different substrate specificities of PP may explain the difference in Cant-induced site-specific phosphorylation of PLB in isometrically contracting papillary muscles. Moreover, PLB-Thr-17 may be important for inotropy, whereas PLB-Ser-16 could be a major determinant of relaxation time.

On the cardiac contractile, electrophysiological and biochemical effects of endothall, a protein phosphatase inhibitor.

Protein phosphatase inhibitors, e.g. cantharidin, exert positive inotropic effects in mammalian heart preparations. Endothall, a synthetic herbicide which is chemically related to cantharidin, inhibits protein phosphatase activities in mouse liver preparations. However, the cardiac effects of endothall have hitherto not been studied. In guinea pig papillary muscles, endothall (1-100 micromol/l) failed to affect force of contraction, whereas cantharidin (1-100 micromol/l) increased force of contraction maximally to 313.4 +/- 32% of control at 10 micromol/l. In isolated guinea pig ventricular cardiomyocytes, endothall did neither change the free intracellular calcium concentration nor the amplitude of calcium current nor the phosphorylation state of regulatory phosphoproteins like phospholamban. In contrast, cantharidin (30 micromol/l) increased the free intracellular calcium concentration and the L-type calcium current to 149.6 +/- 9% and to 157.6 +/- 12% of control, respectively. Furthermore, cantharidin (1-100 micromol/l) augmented the phosphorylation of phospholamban maximally to 140.8 +/- 7% of control. Nevertheless, in guinea pig ventricular homogenates, both endothall and cantharidin inhibited phosphatase activity with EC(50) values of 1.92 and 0.32 micromol/l, respectively. Thus, in contrast to cantharidin, endothall failed to increase force of contraction, though it inhibited protein phosphatase activity. Clearly, endothall is not an appropriate tool to study the function of protein phosphatases in the mammalian heart.

Contractility and inhibition of protein phosphatases by cantharidin.

1. Cantharidin is a natural defensive toxicant produced by blister beetles. 2. Cantharidin shares structural similarity with highly toxic commercial herbicides (e.g., endothall, endothall anhydride and endothall thioanhydride). 3. Cantharidin inhibits the activity of purified catalytic subunits of serine/threonine protein phosphatases (PP) type 1 and type 2A. 4. Cantharidin increases force of contraction in isolated myocardial and vascular preparations. 5. Cantharidin enhances the phosphorylation state of myocardial and vascular regulatory proteins. 6. Cantharidin is a valuable tool for studying the function of PP in regulatory phosphorylation-dephosphorylation events.

Functional properties of transgenic mouse hearts overexpressing both calsequestrin and the Na(+)-Ca(2+) exchanger.

Overexpression of calsequestrin (CSQ) induces severe cardiac hypertrophy, whereas overexpression of Na(+)-Ca(2+) exchanger (NCX) does not affect cardiac weight. To investigate a possible beneficial effect of NCX in hypertrophy, we produced transgenic mice overexpressing both NCX and CSQ (NCX/CSQ). Surprisingly, these mice developed severe heart failure. The heart/body weight ratio was enhanced and the mRNA expression of ANF, as a marker of hypertrophy, was highest in double transgenic mice. In isolated muscle strips, the basal relaxation time was prolonged in CSQ and NCX/CSQ mice. Moreover, in the presence of caffeine, force of contraction was increased only in CSQ and NCX/CSQ and was accompanied by elevated diastolic tension. In some respects, however, additional overexpression of NCX altered the CSQ phenotype into the wild-type phenotype. The expression of sarcoplasmic reticulum (SR)-Ca(2+)-ATPase and phospholamban, proteins involved in the Ca(2+) uptake of the SR, were only increased in CSQ, indicating a possible influence of NCX in the regulation of SR-Ca(2+) uptake proteins. The Ca(2+) transients and the L-type Ca(2+) currents in the presence of caffeine were very large in CSQ, but smaller increases were noted in double transgenic mice. Therefore, the successful co-overexpression of CSQ and NCX in these mice provides a novel model in which to investigate the interaction of proteins tightly linked to maintain Ca(2+) homeostasis.

The protein phosphatase inhibitor cantharidin alters vascular endothelial cell permeability.

In this study, we characterized the effects of the protein phosphatases type 1 (PP 1) and type 2A (PP 2A) inhibitor cantharidin in endothelial cells. We identified catalytic subunits of PP 1alpha, PP 2Aalpha, and PP 2Abeta immunologically in bovine aortic endothelial cells. Moreover, we detected mRNAs coding for catalytic subunits of PP 1alpha, PP 1beta, and PP 2Aalpha by hybridization with specific DNA probes in total RNA from these cells. Okadaic acid and cantharidin inhibited the activities of catalytic subunits of PP 1 (okadaic acid, 0.01-1 microM; cantharidin, 1-100 microM) and PP 2A (okadaic acid, 0.1 nM to 1 microM; cantharidin, 0.1-100 microM) separated by column chromatography in a concentration-dependent manner. Moreover, cantharidin (1 microM to 1 mM) increased the phosphorylation state of endothelial proteins including the regulatory light chains of myosin without affecting cytosolic calcium concentrations. Cantharidin (5-100 microM) increased the permeability of cultured endothelial cells in a time- and concentration-dependent manner. We suggest that inhibition of PP 1 and PP 2A activities by cantharidin increases endothelial permeability by enhancing the phosphorylation state of endothelial regulatory proteins. Thus, cantharidin might be a useful tool to study the function of protein phosphatases in endothelial barrier function.

Inotropic effects of diadenosine tetraphosphate (AP4A) in human and animal cardiac preparations.

Diadenosine tetraphosphate (AP4A) is an endogenous compound and exerts diverse physiological effects in animal systems. However, the effects of AP4A on inotropy in ventricular cardiac preparations have not yet been studied. The effects of AP4A on force of contraction (FOC) were studied in isolated electrically driven guinea pig and human cardiac preparations. Furthermore, the effects of AP4A on L-type calcium current and [Ca]i were studied in isolated guinea pig ventricular myocytes. In guinea pig left atria, AP4A (0.1-100 microM) reduced FOC maximally by 36.5 +/- 4.3%. In guinea pig papillary muscles, AP4A (100 microM) alone was ineffective, but reduced isoproterenol-stimulated FOC maximally by 29.3 +/- 3.4%. The negative inotropic effects of AP4A in atria and papillary muscles were abolished by the A1-adenosine receptor antagonist 1, 3-dipropyl-cyclopentylxanthine. In guinea pig ventricular myocytes, AP4A (100 microM) attenuated isoproterenol-stimulated L-type calcium current and [Ca]i. In human atrial and ventricular preparations, AP4A (100 microM) alone increased FOC to 158.3 +/- 12.4% and 167.5 +/- 25.1%, respectively. These positive inotropic effects were abolished by the P2-purinoceptor antagonist suramin. On the other hand, AP4A (100 microM) reduced FOC by 27.2 +/- 7.4% in isoproterenol-stimulated human ventricular trabeculae. The latter effect was abolished by 1,3-dipropyl-cyclopentylxanthine. In summary, after beta adrenergic stimulation AP4A exerts negative inotropic effects in animal and human ventricular preparations via stimulation of A1-adenosine receptors. In contrast, AP4A alone can exert positive inotropic effects via P2-purinoceptors in human ventricular myocardium. Thus, P2-purinoceptor stimulation might be a new positive inotropic principle in the human myocardium.