Inhibition of Carbonic Anhydrase Prevents the Na+/H+ Exchanger 1-dependent Slow Force Response to Rat Myocardial Stretch.

DÍAZ, RG; VARGAS, LA; SWENSON, ER; PÉREZ, NG ; ALVAREZ, BV

San Diego, California

Congreso; International Society for Heart Research XXI World Congress; 2013

Background* Myocardial stretch is an established signal that leads to hypertrophy. Myocardial stretch induces a first immediate force increase followed by a slow force response (SFR), which is a consequence of increased Ca²+ transient that follows the NHE1 Na+/H+ exchanger activation. Carbonic anhydrase II (CAII) binds to the extreme C-terminus of NHE1 and regulates its transport activity. We aimed to test the role of CAII bound to NHE1 in the SFR. Methods* The SFR and changes in intracellular pH (pHi) were evaluated in rat papillary muscle bathed with CO2/HCO3- buffer and stretched from 92% to 98% of the muscle maximal force development length for 10 min, in the presence of the CA inhibitor 6-ethoxzolamide (ETZ, 100 uM). pHi changes in rat papillary muscles were monitored by fluorescent measurements of BCECF-AM. NHE1/CAII physical association was examined in the SFR by coimmunoprecipitation using rat papillary lysates. Methods* The SFR and changes in intracellular pH (pHi) were evaluated in rat papillary muscle bathed with CO2/HCO3- buffer and stretched from 92% to 98% of the muscle maximal force development length for 10 min, in the presence of the CA inhibitor 6-ethoxzolamide (ETZ, 100 uM). pHi changes in rat papillary muscles were monitored by fluorescent measurements of BCECF-AM. NHE1/CAII physical association was examined in the SFR by coimmunoprecipitation using rat papillary lysates. * Myocardial stretch is an established signal that leads to hypertrophy. Myocardial stretch induces a first immediate force increase followed by a slow force response (SFR), which is a consequence of increased Ca²+ transient that follows the NHE1 Na+/H+ exchanger activation. Carbonic anhydrase II (CAII) binds to the extreme C-terminus of NHE1 and regulates its transport activity. We aimed to test the role of CAII bound to NHE1 in the SFR. Methods* The SFR and changes in intracellular pH (pHi) were evaluated in rat papillary muscle bathed with CO2/HCO3- buffer and stretched from 92% to 98% of the muscle maximal force development length for 10 min, in the presence of the CA inhibitor 6-ethoxzolamide (ETZ, 100 uM). pHi changes in rat papillary muscles were monitored by fluorescent measurements of BCECF-AM. NHE1/CAII physical association was examined in the SFR by coimmunoprecipitation using rat papillary lysates.Methods* The SFR and changes in intracellular pH (pHi) were evaluated in rat papillary muscle bathed with CO2/HCO3- buffer and stretched from 92% to 98% of the muscle maximal force development length for 10 min, in the presence of the CA inhibitor 6-ethoxzolamide (ETZ, 100 uM). pHi changes in rat papillary muscles were monitored by fluorescent measurements of BCECF-AM. NHE1/CAII physical association was examined in the SFR by coimmunoprecipitation using rat papillary lysates. Results* SFR control was120±3% (n=8) of the rapid initial phase and was fully blocked by ETZ, 99±4% (n=6). The SFR corresponded with a maximal increase in pHi of 0.18±0.02 pH units (n=4), and pHi changes were blocked by ETZ (0.04±0.04, n=6), monitored by epifluorescence. NHE1/CAII physical association was examined in the SFR by coimmunoprecipitation, using muscle lysates. CAII immunoprecipitated with anti-NHE1 antibody and the CAII immunoprecipitated protein levels increased 58±9% (n=6) upon stretch of muscles, assessed by immunoblots. The p90RSK kinase inhibitor SL0101-1 (10 uM) blocked the SFR of heart muscles after stretch 102±2% (n=4), and reduced the binding of CAII to NHE1, suggesting that the stretch-induced phosphorylation of NHE1 increases its binding to CAII.* SFR control was120±3% (n=8) of the rapid initial phase and was fully blocked by ETZ, 99±4% (n=6). The SFR corresponded with a maximal increase in pHi of 0.18±0.02 pH units (n=4), and pHi changes were blocked by ETZ (0.04±0.04, n=6), monitored by epifluorescence. NHE1/CAII physical association was examined in the SFR by coimmunoprecipitation, using muscle lysates. CAII immunoprecipitated with anti-NHE1 antibody and the CAII immunoprecipitated protein levels increased 58±9% (n=6) upon stretch of muscles, assessed by immunoblots. The p90RSK kinase inhibitor SL0101-1 (10 uM) blocked the SFR of heart muscles after stretch 102±2% (n=4), and reduced the binding of CAII to NHE1, suggesting that the stretch-induced phosphorylation of NHE1 increases its binding to CAII. Conclusion* CAII/NHE1 interaction constitutes a component of the SFR to heart muscle stretch which potentiates NHE1-mediated H+ transport in the myocardium.* CAII/NHE1 interaction constitutes a component of the SFR to heart muscle stretch which potentiates NHE1-mediated H+ transport in the myocardium.