The effects of acute muscle stretch from 92 to 100% of maximum length (Lmax) on contractile function of rabbit papillary muscles were assessed under basal conditions, in the absence (control group, n=8) or the presence of a cell-permeable PKG activator (8-bromo-cGMP, 10−6 M; PKGa group, n=7) and a PKG inhibitor (Rp-8-Br-PET-cGMPS, 10−6 M; PKGi group, n=7). These substances were dissolved in Krebs-Ringer solution and myocardial stretch was elicited 20 min later.

In a second set of protocols, the contractile response to stretch was studied under ischemic conditions. After stabilization, the bathing solution was replaced by another without glucose and without O2 supply. After 5 min of ischemia, muscles were stretched from 92 to 100% of Lmax (Isch group, n=8). Furthermore, to explore the influence of modulation of the PKG pathway on the systolic response to stretch during ischemia, the latter protocol was also performed in the presence of a PKG activator (8-bromo-cGMP, 10−6 M); Isch-PKGa group, n=7; (H) an NO donor (S-nitroso-N-acetyl-penicillamine (SNAP), 10−5 M; Isch-NO group, n=9) and (I) a PDE5 inhibitor (sildenafil, 10−6 M; Isch-PDE5i group, n=7). These drugs interfere with the NO/PKG pathway at different targets and allow better characterization of SFR modulation. Ischemia was maintained for 15 min. To confirm myocardial viability after the ischemic period, the muscles were reperfused with a glucose-containing Krebs-Ringer solution and O2 supply.

A schematic representation of the protocols is shown in Figure 1.

Figure 1.

Representative bands of the contractile response to acute stretch (upper images) in the control group (A) and Isch group (B) and schematic illustration of experimental protocols (bottom diagrams). Gray bar: stabilization period; white bar: presence of O2 and glucose; dark bar: absence of O2 and glucose (ischemia). AT: active tension.

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The doses were selected according to the literature data and our preliminary studies. All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Values are means±standard error. Within the same group, the effects of acute muscle stretch on the various experimental parameters at specific time points were analyzed with a paired Student's t test. The time-dependent effects of muscle stretch within the same group were analyzed by repeated measures one-way ANOVA, while repeated measures two-way ANOVA was used to compare the time-dependent effects of muscle stretch between several groups. When significant differences were detected with any of the ANOVA tests, the Holm-Sidak method was used to perform multiple comparisons. A value of p<0.05 was accepted as significant.

The muscles stabilized in the presence of the drug for 20 min, during which no significant variations in active tension were observed.

In the control group, active tension (AT) at Lmax, following stretch, was 23.4±3.8 mN/mm2. In the following 15 min there was a progressive increase in AT to 29.1±4.3 mN/mm2 (p<0.001), corresponding to an SFR of 27.8±3.9%. A representative band of the contractile response of a muscle in the control group is shown in Figure 1A.

In the PKGa group no differences were found in SFR after 15 min (20.9±8.13% vs. 27.8±3.9%, p=0.324) (Figure 2). On the other hand, myocardial stretch in the presence of the PKG inhibitor was associated with a significant attenuation of SFR (SFR at 15 min: 11.5±5.9% vs. 27.8±3.9% in controls, p=0.034) (Figure 2).

SFR by PKG under normoxia. The SFR was impaired in the PKGi group. *p<0.05 vs. control. AT: active tension.'> SFR by PKG under normoxia. The SFR was impaired in the PKGi group. *p<0.05 vs. control. AT: active tension.' title='Modulation of the SFR by PKG under normoxia. The SFR was impaired in the PKGi group. *p<0.05 vs. control. AT: active tension.'/>

Figure 2.

Modulation of the SFR by PKG under normoxia. The SFR was impaired in the PKGi group. *p<0.05 vs. control. AT: active tension.

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In the first 5 min of ischemia a significant decrease in myocardial contractility of 51.0±4.0% was seen (AT decreased from 20.1±3.1 mN/mm2 to 10.2±2.2 mN/mm2, p<0.001). After stretch, muscles did not display SFR; instead, they exhibited a significant and progressive decrease in contractility (AT decreased from 12.4±2.3 mN/mm2 to 5.7±1.9 mN/mm2, p<0.001) (Figure 3). A representative band of a muscle of the Isch group is shown in the Figure 1B.

SFR by ischemia. In the Isch group, muscles did not display SFR; instead, they exhibited a significant and progressive decrease in contractility during the 15 min after stretch. *p<0.05 vs. control. AT: active tension.'> SFR by ischemia. In the Isch group, muscles did not display SFR; instead, they exhibited a significant and progressive decrease in contractility during the 15 min after stretch. *p<0.05 vs. control. AT: active tension.' title='Modulation of the SFR by ischemia. In the Isch group, muscles did not display SFR; instead, they exhibited a significant and progressive decrease in contractility during the 15 min after stretch. *p<0.05 vs. control. AT: active tension.'/>

Figure 3.

Modulation of the SFR by ischemia. In the Isch group, muscles did not display SFR; instead, they exhibited a significant and progressive decrease in contractility during the 15 min after stretch. *p<0.05 vs. control. AT: active tension.

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The use of an NO donor did not significantly influence the myocardial response to acute stretch under ischemic conditions (AT variation after stretch: −27.7±15.2% in Isch-NO vs. −52.4±7.9% in Isch, p=0.229) (Figure 4).

Figure 4.

Contractile response to acute myocardial stretch under ischemic conditions. Isch-PDE5i demonstrated a significantly attenuated AT decline after stretch. *p<0.05 vs. Isch. AT: active tension.

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The decrease in contractility over the 15 min after stretch was not significantly different in the presence of the PKG activator compared to the Isch group (−34.9±18.2% vs. −52.4±7.9% in Isch, p=0.373) (Figure 4).

In contrast to Isch-PKGa, PDE5 inhibition significantly prevented the decrease in contractility after stretch (−8.6±17.6% vs. −52.4±7.9% in Isch, p=0.018) (Figure 4).

Acute myocardial ischemia induces immediate contractile dysfunction, resulting in sudden hemodynamic overload. Hence, it is interesting to characterize the myocardial response to stretch under ischemic conditions. Despite its importance, there is limited knowledge about the effect of ischemia on the myocardial adaptation to stretch. We reported a reversed SFR in ischemic myocardium. This observation may be related to the profound changes in calcium sensitivity observed during ischemia.14,15 Other mechanisms contributing to this dysfunction may include the limited availability of adenosine triphosphate under ischemic conditions, which is known to cause an immediate increase in contractility16 and to promote the development of myocardial rigor due to impairment of sarcomere cross-bridge cycling.17,18

We describe some interesting results regarding the role of PKG in the myocardial response to stretch. Our work demonstrates that inhibition of PKG leads to reduced SFR, but activation does not affect this response. These results suggest a permissive role of the PKG signaling pathway in the normal SFR. These results can be explained by a dynamic interaction between PKG and PDE (Figure 5). Under normal conditions, the basal concentration of cGMP in the human myocardium inhibits PDE3, thus preventing the hydrolysis of cAMP,19,20 the presence of which contributes to the normal SFR.21 We therefore hypothesize that removal of the basal PKG pathway increases the action of PDE3, reducing cAMP levels and thereby the SFR. Another possible mechanism linking PKG activity to the SFR is the troponin I (TnI) phosphorylation level. PKG is known to phosphorylate TnI at serine 23/24, increasing the cross-bridge cycling rate,22 which probably contributes to a more efficient SFR.23

PKG signaling pathway in the normal SFR. The pathway appears to have a permissive role in the SFR, probably through phosphorylation of TnI at serine 23/24 and through inhibition of PDE3.'> PKG signaling pathway in the normal SFR. The pathway appears to have a permissive role in the SFR, probably through phosphorylation of TnI at serine 23/24 and through inhibition of PDE3.' title='Role of the PKG signaling pathway in the normal SFR. The pathway appears to have a permissive role in the SFR, probably through phosphorylation of TnI at serine 23/24 and through inhibition of PDE3.'/>

Figure 5.

Role of the PKG signaling pathway in the normal SFR. The pathway appears to have a permissive role in the SFR, probably through phosphorylation of TnI at serine 23/24 and through inhibition of PDE3.

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One of the most intriguing results of our work is the observation that sildenafil, but neither 8-bromo-cGMP nor an NO donor significantly prevented the gradual decrease in contractility after acute stretch in ischemia. As the best-characterized action of sildenafil is PDE5 inhibition, preventing cGMP degradation, its effects would be expected to be similar to those of a cGMP analog. Interestingly, data published by Elrod et al.24 suggest a cardioprotective role for sildenafil, independent of its action on the NO/cGMP pathway. It is possible that the contractile preservation in ischemia induced by sildenafil is dependent on a still obscure effect of this molecule, independent of its action on PDE5.

Our findings in the rabbit myocardium should be extrapolated to other species with caution. The known intracellular mechanisms of the SFR depend on the animal species under consideration.10,25,26 However, the increase in calcium transients due to activation of the Na+/H+ exchanger and reverse operating mode of the Na+/Ca2+ exchanger seems to be angiotensin-II independent in both rabbit and human myocardium, depending on acute mechanical stretch alone.25,26 In this way, it is probably easier to extrapolate the findings of this work to the human myocardium than to the other commonly used animal models such as cat, rat, and mouse.

The authors declare that the procedures followed were in accordance with the regulations of the relevant clinical research ethics committee and with those of the Code of Ethics of the World Medical Association (Declaration of Helsinki).

The authors declare that no patient data appear in this article.

The authors declare that no patient data appear in this article.