Antisense Strategies in Animal Myocytes SERCA2a Overexpression Compared to Phospholamban Downregulation in Rat and Rabbit Myocytes

Adenoviral gene transfer into isolated myocytes has allowed the investigation of changes in phospholamban in species other than mouse, as well as direct comparison of phospholamban depletion with SERCA2a overexpression in the same preparation. Rat is similar to mouse in the predominance of SERCA2a (92%) compared to other mechanisms of calcium removal, while in rabbit heart (like human) SERCA2a controls approx 70%, and there is a much larger role for the Na+/Ca2+ exchanger to remove excess calcium from the cell (15). Initial results with an antisense strategy for phospholamban in adult myocytes were disappointing (14), but later work showed that even partial (approx 50%) reduction of phospholamban could have effects almost equivalent to SERCA2a overexpression (approx fivefold) on contraction amplitude (Fig. 1) and only slightly less on relaxation (16,17). This correlates well with results from transgenic animals, in which phospholamban knockout can be even more effective than SERCA2a. In mice overexpressing SERCA2a, the maxi-

Fig. 1. Frequency-dependent increase in contraction amplitude of rat or rabbit myocytes cultured for 48 h without infection. Rabbit: uninfected controls (Con) (n = 14 cells), infected with Ad.SERCA2a.GFP (SERCA) (n = 17) or Ad.PlbAs.GFP (Plb-As) (n = 17). Both SERCA and Plb-As curves were significantly different from Con (p < 0.001 in each case) but were not significantly different from each other. Rat: Con (n = 9 cells), SERCA (n = 13) or Plb-As (n = 11). Both SERCA and Plb-As curves were significantly different from Con (p < 0.01 for SERCA; p < 0.02 for Plb-As) but were not significantly different from each other. (Adapted from ref. 13.)

Fig. 1. Frequency-dependent increase in contraction amplitude of rat or rabbit myocytes cultured for 48 h without infection. Rabbit: uninfected controls (Con) (n = 14 cells), infected with Ad.SERCA2a.GFP (SERCA) (n = 17) or Ad.PlbAs.GFP (Plb-As) (n = 17). Both SERCA and Plb-As curves were significantly different from Con (p < 0.001 in each case) but were not significantly different from each other. Rat: Con (n = 9 cells), SERCA (n = 13) or Plb-As (n = 11). Both SERCA and Plb-As curves were significantly different from Con (p < 0.01 for SERCA; p < 0.02 for Plb-As) but were not significantly different from each other. (Adapted from ref. 13.)

mum velocity of myocyte relengthening was accelerated by a modest 40% (18) compared with 340% in cells from PLB-KO mice (19). In that and another study (20), robust overexpression of SERCA2a mRNA translated into increases in protein of only 20-50%. An upper limit on SERCA2a incorporation has been suggested previously in a quantitative study using adenoviral vectors in chick embryo myocytes (21). This may be particularly relevant to the mouse heart, where the activity of SERCA2a (relative to other Ca2+ removal systems) is already high, possibly leaving little scope for increased gain via overexpression of SERCA2a.

Increases in contraction amplitude were similar in either rat or rabbit ventricular myocytes and, like human (see Subheading 3.), showed a frequency dependence (Fig. 1). As might be predicted from the mouse models, adenovirally mediated acceleration of relaxation was less evident in rat than in rabbit, but this was true for both phospholamban-depleted and SERCA2a-overexpressing cells (17). In both rat and rabbit myocytes, the maximum effect of phospholamban antisense/SERCA2a was similar to that of catecholamines, which is consistent with phospholamban phosphorylation as the main mechanism for p-adrenoceptor-mediated lusitropy. Interestingly, however, frequency-dependent acceleration of relaxation, which is not related to phospholamban, was additive with that by SERCA2a overexpression or phospholamban depletion (17).

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