Abstract

Section 1

Section 2

Section 3

Section 4

Section 5

Discussion
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Phospholamban (PLB) is a low molecular weight phosphoprotein associated with cardiac sarcoplasmic reticulum (SR). The dephosphorylated form of PLB is an inhibitor of the SR Ca²⁺-ATPase affinity for Ca²⁺. Phosphorylation of PLB relieves its inhibitory effects on the SR Ca²⁺ pump, with subsequent acceleration of Ca²⁺ transport into the SR lumen, which is thought to underlie the positive inotropic and lusitropic actions of beta-adrenergic agonists in the mammalian heart. The role of PLB in the regulation of basal myocardial contractility has been recently elucidated through the development of a PLB knockout (KO) mouse via gene-targeting methodology in embryonic stem cells. The PLB knockout mouse hearts exhibited significant increases in the SR Ca²⁺-pump affinity for Ca²⁺ and in the basal contractile parameters compared with wild-type littermates. The cardiac phenotype was analyzed utilizing an integrative approach and combining studies at the subcellular, organ and whole animal levels. Furthermore, the inotropic and lusitropic effects of beta-agonists on cardiac function were significantly attenuated in PLB knockout mice compared with wild-type mice. These studies indicated that PLB is a critical inhibitor of basal cardiac function and a major regulator of the heart's responses to beta-adrenergic agonists.

Phospholamban was originally proposed to form homopentamers through interactions between its transmembrane domain in the SR, based on its migration pattern in SDS-polyacrylamide gels. Recently, alanine-scanning mutagenesis studies in expression systems showed that a monomeric form of PLB, in which Leu-37 was changed to Ala, suppressed SR Ca2+-ATPase Ca²⁺ affinity more effectively than wild-type or pentameric PLB. It was thus postulated that there might be an equilibrium between pentameric and monomeric PLB in SR membranes. Pentameric PLB may represent a less active reservoir, which dissociates to provide active monomeric PLB subunits. However, co-expression of other monomeric forms of PLB, associated with mutation of Cys-41 to Phe or Ser, with SR Ca²⁺-ATPase in HEK-293 cells failed to demonstrate super-inhibition (or gain of function) by PLB monomers. Furthermore, recent studies indicated that a pentameric form of PLB, resulting from mutation of Asn-27 to Ala was more potent than a monomeric mutant (L37A) PLB in inhibiting the SR Ca²⁺-ATPase Ca²⁺ affinity, and suggested that the oligomeric structure of PLB may be important in SR Ca²⁺-ATPase regulation.

The availability of the PLB knockout mouse in conjunction with site-specific mutagenesis technology has provided us with an excellent opportunity to examine structure-function relations of PLB in a background free of the endogenous protein. In this article, we will describe the feasibility of reinsertion of the PLB gene in the PLB knockout heart and present evidence for the functional unit (pentamers vs. monomers) of PLB in vivo.

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Fig. 1: The wild type and PLB knockout hearts were perfused in the absence (-) and presence (+) of isoproterenol (ISO). * P < 0.05 vs. wild type without ISO; # P < 0.05 vs. PLB knockout with ISO.

The time to peak pressure (TTP) and half-relaxation time (RT1/2) were also significantly shortened by PLB deficiency. Maximal isoproterenol perfusion was associated with significant stimulation in the rates of contraction (+dP/dt: 208%) and relaxation (-dP/dt: 260%) in the wild-type hearts, whereas it only slightly stimulated the relaxation rate in the PLB knockout hearts (Fig. 1).

To determine whether ablation of PLB and the accompanied enhancement of cardiac contractile parameters were associated with any alterations of the SR Ca²⁺-ATPase, the activity of this enzyme was examined in Ca²⁺-uptake experiments. The initial rates of the SR Ca²⁺ uptake in cardiac homogenates were assessed as a function of [Ca²⁺]. There was no alteration in the V_maxof the SR Ca²⁺ transport system, indicating that the expression levels and the activity of the SR Ca²⁺-ATPase were not altered in the PLB knockout hearts. However, the affinity of the SR Ca²⁺-ATPase for Ca²⁺, a property modulated by PLB, was increased upon ablation of PLB. Furthermore, dot blot analysis and quantitative immunoblotting revealed no differences in either the transcript or the protein levels of the SR Ca²⁺-ATPase between wild-type and PLB knockout hearts (Fig. 2A).

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Fig. 2: Quantification of the major calcium cycling proteins. SERCAS: SR Ca²⁺-ATPase; CSQ: calsequestrin; RyR: ryanodine receptor; NEC: Na⁺-Ca²⁺ exchanger.

Examination of the transcript or the protein levels of calsequestrin and the sarcolemmal Na⁺-Ca²⁺ exchanger indicated no significant differences between PLB knockout and wild-type hearts. However, phospholamban deficiency was associated with a significant reduction (26%) in the levels of the ryanodine receptor protein (Fig. 2B). The reduction in protein levels was consistent with a 24% decrease in ryanodine binding in PLB knockout hearts compared with wild-type hearts (Fig. 2C), while the ryanodine binding affinity was similar between PLB knockout and wild-type hearts.

The contractile proteins, myosin, actin and troponins are known to play a major role in controlling the muscle contractile machinery. Thus, the transcript levels of these proteins were evaluated by dot blot analysis. There were no significant alterations in the mRNA levels of alpha-myosin heavy chain, alpha-cardiac actin, alpha-skeletal actin, or troponin I between PLB knockout and wild-type hearts. Furthermore, there were no alterations in the protein levels of myosin, actin, troponin T and troponin I. Examination of beta-myosin heavy chain expression indicated that these transcript levels were undetectable in PLB knockout or wild-type hearts, suggesting that PLB ablation did not trigger any transition between alpha- and beta-myosin heavy chain proteins in an effort to accommodate the enhanced SR Ca²⁺-ATPase activity and hyperdynamic cardiac function in the intact mouse heart.

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Fig. 3: Langendorff-perfused heart preparations. WT: wild type control; KO: PLB knockout hearts; KO+WT: reintroduction of PLB in the knockout mouse hearts.

Reinsertion of PLB in the knockout background was associated with significant depression of the contractile parameters (Fig. 3). Please note that reversal of the enhanced cardiac contractile parameters was not complete, or to the levels observed in control hearts, since the levels of reintroduced PLB were 0.7-fold of those present in control hearts. Interestingly, when the relative levels of PLB or PLB/SR Ca²⁺-pump in the three animal models were plotted against the maximal basal rates of contraction (+dP/dt) and relaxation (-dP/dt), a close linear correlation was observed (Fig. 4), consistent with our previous observations in PLB knockout, PLB-heterozygous and control hearts.

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Fig. 4: The relative ratios of PLB to the SR Ca²⁺-ATPase (SERCA) were plotted against the basal maximal rates of contraction (+dP/dt) and relaxation (-dP/dt) in the perfused hearts from various mouse models: wild type control (control), PLB knockout (KO), and transgenic mice expressing the wild type PLB in the null background (KO+WT).

These findings indicate that the hyperdynamic PLB knockout phenotype can be reversed by reintroduction of PLB in the null background, and demonstrate the potential power of this technology in performing structure-function studies of PLB in vivo.

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Fig. 5: Quantitative immunoblotting of PLB in wild type control (WT) and transgenic mice expressing C41F mutant PLB in the PLB knockout background (KO+C41F).

One of the lines expressing 0.7-fold PLB, which was similar to the PLB levels expressed in KO+WT hearts, was selected for breeding and further studies. Analysis of the SERCA levels showed that there was no alteration upon introduction of mutant PLB in the mouse heart.

As described above, the rates of cardiac contraction and relaxation were significantly increased upon ablation of PLB compared to wild-type hearts. Conversely, reintroduction of wild-type PLB in the knockout hearts reversed their hyperdynamic function. To determine whether reintroduction of monomeric PLB had similar regulatory effects as wild-type or pentameric PLB, the hearts from transgenic mice with wild-type or mutant PLB reintroduced into the knockout background were perfused in parallel in a retrograde mode. Langendorff-perfusion indicated that there was no difference in +dP/dt values between the two transgenic groups, but the -dP/dt values were depressed to a greater extent by reintroduction of wild-type PLB than mutant PLB in the knockout hearts (Fig. 6).

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Fig. 6: Effects of the C41F mutant PLB on contractile parameters in Langendorff-perfused hearts. +dP/dt and -dP/dt: the maximal rates of contraction and relaxation; TTP: time to peak pressure; RT1/2: time to half relaxation.

This observation was also consistent with the time parameters in the same cardiac preparations. There was no difference in the time to peak pressure (TTP), while the time to half-relaxation (RT1/2) was prolonged significantly more in wild-type PLB than in mutant PLB hearts (Fig. 6). These findings indicate that the mutant monomeric PLB was not capable of depressing the enhanced relaxation parameters of the PLB knockout hearts to the same extent as wild-type PLB.

The hypothesis that monomeric PLB is less effective in inhibiting the cardiac contractility was further tested at cellular levels, using isolated cardiomyocytes. Left ventricular myocytes were isolated from KO+C41F and KO+WT transgenic mice, paced at 0.5Hz and used for measurements of contractile parameters and Ca²⁺ transients. Reintroduction of wild-type PLB in the knockout background was associated with significant depression of the cell shortening fraction and prolongation of the time constant of relaxation, compared to PLB knockout cardiomyocytes. Figure 7 shows representative traces of steady-state cell shortening of transgenic myocytes with wild-type PLB or mutant PLB reintroduced in the knockout background.

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Fig. 7: Myocyte twitch contractions. Left ventricular myocytes were isolated and paced at 0.5 Hz. All measurements were obtained during steady-state (SS) twitches at 1 mM Ca²⁺ and room temperature. KO+WT and KO+C41F: transgenic mice with wild type or C41F mutant PLB reintroduced in the knockout background.

Myocytes expressing wild-type PLB relaxed slower than those expressing mutant PLB. The time constant for cell relaxation during the steady-state twitch was significantly faster in the mutant PLB myocytes. The amplitude of cell shortening was slightly but not significantly higher in mutant PLB myocytes. Consistent with cell mechanics, the decline of the Ca²⁺ transient was faster in myocytes with mutant PLB than wild-type PLB. Peak [Ca²⁺]i tended to be higher with the mutant PLB, but again this effect was not significant. Thus, the time constants of relaxation and Ca²⁺ transient decline in isolated cardiomyocytes were diminished to a greater extent by wild-type than mutant phospholamban, even without significant differences in the amplitudes of myocyte contraction and Ca²⁺ transient between the two groups.

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