Arterial Hypertension May Involve Sarcoplasmic Reticulum Calcium Atpase (Serca) Proteolysis By Matrix Metalloproteinase (Mmp)-2

Objective: Aortic SERCA protein level and activity are reduced in hypertension. Increased MMP-2 activity may cleave SERCA in acute myocardial ischemia and reperfusion injury. Our objective was to verify whether increased vascular MMP-2 activity in hypertension proteolyzes SERCA to alter cytosolic calcium, thus causing enhanced vasoconstriction or maladaptive remodeling. Design and method: Male Sprague-Dawley rats were submitted to two kidney-one clip (2K-1C) or sham surgery and treated with doxycycline (Doxy, 30 mg/kg/day) by gavage from the third to seventh day post-surgery. Systolic blood pressure (SBP; tail-cuff plethysmography) was daily measured, and after seven days aortas were collected and processed for gelatin and in situ zymography, SERCA protein levels by Western blot and calcium immunofluorescence using Rhod-2AM. Aortas were isolated from normal rats were incubated at 37°C for 6 or 12 hours in DMEM with angiotensin (Ang) II (0.1 μM) +/- MMP inhibitors, ONO-4817 (10 μM) or Doxycycline (30 μM). Aorta segments were processed as above and used for co-immunoprecipitation of SERCA and MMP-2. Statistical analysis was done by two-way ANOVA followed by Tukey post-hoc test. The Ethics Committee for Animal Research of the Ribeirao Preto Medical School approved all protocols (122/2019). Results: MMP-2 activity in 2K-1C aorta was elevated 1.5-fold vs. controls and Doxy decreased it by 1.3-fold (n = 5–8; p < 0.05). SERCA proteolysis was 3-fold increase in 2K-1C aorta (n = 4–8; p < 0.05 vs. controls). Treatment with Doxy seemed to prevent this effect (0.49 ± 0.07 2K1C+Doxy vs 1.42 ± 0.42 2K1C). These effects were not associated with changes in SBP as Doxy did not alter it. Cytosolic calcium concentrations trended to increase in 2K1C aorta from 13 to 19 a.u. (n = 3; p = 0.08) and Doxy decreased it (n = 3; p < 0.05). In AngII treated aorta, MMP-2 activity also increased by 1.5-fold (n = 4-5; p < 0.05 vs. controls) and both ONO-4817 and Doxy reduced its activity by the half (n = 4-5; p < 0.05). MMP-2 was co-immunoprecipitated with SERCA in the incubated aortas, and AngII seemed to increase it. Conclusions: SERCA levels may be decreased by MMP-2 activity in the aortas of hypertensive rats which may contribute to calcium-induced vascular alterations in hypertension. These effects are currently being investigated.