According to the present data, treatment with telmisartan prevents damage to contractility orchiectomy-dependent, in papillary muscles isolated from SHR. Orchiectomized rats showed a reduction in the inotropic response to calcium and beta-adrenergic activation, without impairment of calcium influx and preserving arterial blood pressure.
Telmisartan treatment, on the other hand, seems to increase the transsarcolemal influx of calcium, which may be the main mechanism by which the drop in response to calcium and an adrenergic activation was prevented. This suggestive increase in calcium influx was accompanied by a reduction in protein expression of NCX and total phospholambam, which suggests greater intracellular calcium handling.
Several studies have already demonstrated that testosterone deprivation is able to decrease the left ventricular contractility [27–32]. Mechanisms that include increased relaxation time, reduced maximum myofilament response to calcium and the activity of ATPase Myosin, reduced maximum SERCA response  and decreased calcium content in SR  have already been described.
In the present study, the orchiectomized animals showed a reduction in isometric strength as a result of reduced response to calcium and beta-adrenergic activation. However, the speed of contraction and relaxation were not altered, as well as the function of RS, which suggests that there was preservation of the function of the calcium extrusion mechanisms, even in the condition of orchiectomy.
Nevertheless, SERCA 2A protein expression was increased in the orchiectomized group (OCT) compared to the other groups, without changing the expression of NCX and PBL, which suggested that the calcium uptake by SR was not impaired. This is reflected in the potentiation result after pause and relaxation time, which are similar between the groups and indicate, albeit indirectly, that the calcium transit was not altered in this model of orchiectomy. Previous study demonstrated that orchiectomy in normotensive rats increases the sensitivity of SERCA 2A to calcium, but its maximum activity is reduced, without altering its protein expression, which reflected in the increase in the relaxation time (TC50%) of hearts of orchiectomized rats . In our orchiectomy model, we believe that the increase in SERCA 2A protein expression found may be a response to this change in the sensitivity and maximum activity of SERCA 2A, which may have culminated in the normalization of the relaxation time of the isolated papillary muscles.
Because our results suggest that apparently the SR function and the transarcolemal calcium influx have not been altered, we speculated that the orchidectomy induced a reduction in the sensitivity of contractile proteins. In fact, a previous study show that testosterone deficiency can alter the calcium interaction with contractile proteins, besides reducing the activity of myosinic ATPase and sensitivity of contractile proteins, which, in turn, could reducing cardiac contraction force in normotensive animals .
In addition, testosterone modulates cardiac function by affecting the renin-angiotensin system. As already shown, testosterone induces an increase in the production of angiotensinogen and increased renin activity resulting in an enhancing in angiotensin II production [5, 33]. Indeed, we observed that the inhibition of the renin-angiotensin system might improves the contractility of hypertensive-orchiectomized rats. Moreover, the treatment with telmisartan prevented the decline of inotropic responses induced by calcium and isoproterenol observed in OCT rats, probably by increasing of transarcolemal calcium influx. In addition, telmisartan treatment prevented the increased SERCA 2A protein expression observed in OCT group besides induces a reduction NCX and the total PBL protein expression in OCT rats.
These data suggest that the treatment with telmisartam, in addition to improving the calcium influx, may be increasing the calcium content in SR, due to the greater activity of SERCA. The reduction in NCX protein expression translates into less calcium extrusion by the sarcolemma and the reduction in PBL makes up a scenario where there will be a greater availability of myoplasmic calcium compared to a more efficient SERCA. Together, our results suggest an important relationship between testosterone deficiency and RAS, resulting in impairment of cardiac function. Furthermore, it also shows that the use of an RAS antagonist can bring benefits to the contractile dysfunction of the heart when an occurring association of hormonal deprivation and hypertension.
We observed no changes in AT1 protein expression in the OCT group. However, an increase of AT1 protein expression was observed after telmisartan treatment, probably due to positive feedback on prolonged inhibition of these receptors. It is possible to note that the AT1 protein expression was not altered in orchiectomized rats treated with telmisartan plus BADGE. This result suggests that the positive feedback observed in the OCT + Tel group probably depends on the activation of PPAR-γ.
Telmisartan is an AT1 receptor antagonist with pharmacological properties defined by a long duration of action and high lipophilicity . It also has partial agonistic properties of PPAR-γ (a nuclear transcription factor receptor superfamily) that may confer beneficial properties in hypertensive patients . It has been used in some studies to demonstrated the multiple changes caused by diseases such as hypertension, diabetes, myocardial infarction and coronary artery disease [35–38]. However, few studies show the effect of this drug on myocardial contractility, which further highlights the importance of our study. Telmisartan is used mainly due to its effect on RAS, but the effects on activate PPAR-γ has been explored in some studies. According to these studies, PPAR-γ activation appears to be an innovative mechanism to mediate the beneficial effects of inhibitors angiotensin receptors [20, 21, 34, 36].
To evaluate the participation of PPARγ in preventing the fall of contractility observed in the telmisartan group, the OCT + Tel + BADGE group was added. Thus, we observed that the prevention of decline in contractility after treatment with telmisartan was maintained after treatment with telmisartan plus BADGE.
However, the inotropic responses induced by calcium and isoproterenol were increased in telmisartan plus BADGE group when compared to the group that received only telmisartan. This result suggests that the effect of telmisartan in preventing the impairment of contractility on OCT group is greater when occur select blocking AT1 receptor (Fig. 3).
Interestingly, when we indirectly assessed the calcium uptake by SR, using the PPP protocol, the telmisartan plus BADGE group showed a reduction compared to the other groups. For right, an increase of NCX protein expression and a reduction of total PLB without changing in SERCA protein expression was observed in the telmisartan plus BADGE group. Together, these results suggest that calcium is probably being extruded mainly by sarcolemma instead of being recaptured by SR, which in turn could reduce the PPP in telmisartan plus BADGE group.
Calcium is one of the most abundant ions in the extracellular environment and its presence in large quantities in the intracellular environment can trigger several other deleterious mechanisms for the heart, such as activation of apoptotic pathways and SR saturation with the possibility of arrhythmia due to spontaneous emissions of SR calcium . Thus, the upregulation of NCX observed in the telmisartan plus BADGE group may have been a compensatory mechanism for this intracellular calcium increase.
In this context, we suggest that a possible increase in calcium influx with the use of the PPAR y inhibitor. In addition to the increase in PRC, there was an increase in the responsiveness to calcium and beta-adrenergic activation in telmisartan plus BADGE group when compared to the group treated only with telmisartan. Since the responsiveness to calcium involves the entry of calcium through the sarcolemma and the saturation of the calcium-binding sites in the sarcomere, the results obtained suggest that the effect of PPAR y inhibition occurs mainly by increasing the calcium inflow without changing the sensitivity of contractile proteins.
It is known that sex hormones and RAS have the ability to induce changes in cardiac morphology. Cardiac hypertrophy is a mechanism in which cardiac mass is increased in response to functional overload that can influence myocardial contractile function. In our findings, orchiectomy did not change the heart's weight either the heart collagen content. However, the perimeter and the nuclear area were reduced, suggesting that testosterone deficiency seems to reduce cardiac trophism, without altering collagen deposition. This result also suggests that the impairment of cardiac force in OCT group does not involve morphological alteration.
In conclusion, it has been shown that orchiectomy causes impairment in the contractility of SHR papillary muscles. Likewise, it has been shown that this damage was prevented by treatment with telmisartan, probably by increasing the transarcolemal flow of calcium, with a consequent increase in the inotropic responses of the heart. In addition, this study created evidence that activation of PPAR-γ may contribute to the beneficial effects of RAS inhibition in some diseases, such as hypertension.