Measurement of Ca2+-sensitivity and maximum Ca2+-activated force
Suramin has long been used in the treatment of various human diseases. Intravenous infusions of Suramin are commonly administered to patients over extended periods of time but there are a number of significant contraindications with peripheral muscle weakness being one of the most frequently reported. Previous work has shown that even after a single infusion (300 mg kg1 ) Suramin remains in skeletal muscle in effective concentrations
(11.6 lg mL1 ; 84 days) for prolonged periods. These observations provide a strong rationale for investigation of the specific effects of Suramin on skeletal muscle function. Single mechanically skinned fibers were directly exposed to Suramin (10, 100 or 500 lmol L1 ) for defined durations (2–10 min) in controlled physiological solutions that mimic the intracellular ionic environment of a fiber.
Suramin is a broad acting polyanionic compound that has been used over many decades, initially for the treatment of African sleeping sickness (African trypanosomiasis) (Reincke et al. 1994; Joshi et al. 2005) and subsequently for river blindness due to onchocerciasis (Schulz-Key et al. 1985). More recently it has also become a treatment modality for adults with recurrent high-grade gliomas (Takano et al. 1994; Grossman et al. 2001). Suramin is a polysulfonated naphthyl urea that is typically administered i.v. (reported dosages vary), and less frequently by i.m. injection, on a weekly basis over a prolonged period of time, often several months.
Investigations that focused on optimal in vivo concentrations for anticancer treatments found that 10% solution Suramin, with a t1/2 of 45–55 days, did not exceed 150 lg mL1 (104 lmol L1 ) on a weekly administration program (Van Rijswijk et al. 1992). This study reported an optimal serum concentration in the anticancer treatment of 319 lg mL1 (223 lmol L1 ). Due to global administration protocols and the broad acting nature of Suramin, a number of contraindications have been reported. Among the most significant side effects are severe fatigue, malaise and lethargy, which all may be manifest as a broad spectrum neuromuscular peripheral weakness of the hands,arms, legs and feet of patients (Eisenberger et al. 1995;Grossman et al. 2001).
Addition of exogenous Troponins to Suramin-treated fibers
The skeletal muscle itself represents a large volume target for Suramin action, with the relative skeletal muscle mass of the body making up 30.6% 5.5 of women and 38.4% 5.1 in men (Janssen et al. 2000). Following a single dose of Suramin, persistently high intracellular concentrations have been observed in various tissues, notably the cortex of the kidney, spleen red pulp, bone marrow, and basement membranes surrounding bone, muscle groups and other organs (McNally et al. 2000).
Therefore, the reduced muscular contractile ability or weakness described in patients treated with Suramin may be a consequence of Suramin directly affecting the Ca2+-handling properties of the sarcoplasmic reticulum (SR). Suramin has been shown to also affect SERCA (Emmick et al. 1994) in addition to RyR. In addition, there is a distinct possibility that Suramin also affects the ability of the contractile apparatus to produce force, which has not previously been investigated. In this study, we use the mechanically-skinned skeletal muscle fiber preparation (Lamb and Stephenson 1990b) that retains a structurally intact SR and contractile apparatus to probe the effect of Suramin on the contractile apparatus and Ca2+-release and uptake by SR over a wide, clinically relevant, concentration range (10– 500 lmol L1 ).
We show that Suramin treatment greatly affects both the Ca2+-sensitivity and maximum Ca2+-activated force and SR Ca2+ release through the RyR’s. We hypothesize that as a result of Suramin treatment there will be changes in Ca2+-sensitivity and maximum Ca2+- activated force associated with direct effects on the contractile apparatus, as well as effects on the SR Ca2+ release through the RyR’s. The combination of these actions helps to explain the Suramin-induced peripheral muscle weakness noted in human pharmacological treatments.
This resulted in the production of increasing force responses that plateaued once maximum Ca2+-activated force was achieved at saturating free [Ca2+] resulting in the identification of the force-Ca2+ relationship for the fiber (forcepCa; where pCa is the ve log10 of the free [Ca2+]). Once the maximum Ca2+-activated force of fibers was confirmed, fibers were relaxed by re-immersion in the relaxing solution. This activation sequence was repeated three times and fibers were only used if the maximum Ca2+-activated force response in a sequence did not decrease by more than 5% between repetitions.
Fibers were then incubated in a relaxing solution containing Suramin (100 lmol L1 for 2 or 10 min or 500 lmol L1 for 2 min). Fibers were then washed in fresh relaxing solution for 10 min to remove residual Suramin and the activation protocol was repeated three more times. The average of the force responses from the three activations was then used to construct a composite force-pCa curve for each experimental group. For each of the three repetitions (both before and after Suramin treatment), the steady-state submaximal force responses were normalized to its own maximum Ca2+-activated force back to the 30 mmol L1 caffeine solution triggering complete Ca2+ emptying of the SR. The ensuing area of the caffeine-induced force transient was subsequently recorded.
After the last loading period in the sequence described above (120 sec load), fibers were then washed for 12 min in the wash solution to account for the Suramin treatment time (see below) and then the SR was depleted again with 30 mmol L1 caffeine solution to empty the SR of all Ca2+ before the load protocol (as above) was repeated for each loading period (10–120 sec) once more. This was done to test for reproducibility of the SR Ca2+- loading protocol over time. In separate fibers, this same procedure was repeated with the exception that after the initial control load curve was established, fibers were treated with 100 lmol L1 Suramin (added to the wash solution) for 2 min followed by a further 10 min wash in a different wash solution to remove Suramin. A second load protocol was then repeated.
Conversion of force transients to free calcium estimates
To distinguish between the potential direct effects of Suramin on the pharmacological release of SR Ca2+ by caffeine and any concomitant Suramin-induced alterations in Ca2+ sensitivity and/or maximum Ca2+-activated force (see below), it was necessary to estimate the profile of the Ca2+- change underlying the caffeine-induced force response. The graphical relationship between force and the activating Ca2+ concentration represents a force-pCa relationship as shown in Figure 1A. This relationship can be used to mathematically derive a relationship (Ca2+- force graph; Fig. 1B) that allows for determination of the Ca2+ concentration, this will account for the possibility that Suramin will affect the calcium sensitivity of each fiber and is required for each of the force transients generated by the pharmacological release (e.g., caffeine) of SR Ca2+ of the same fiber using the following equation
Figure 4 shows the conversion of recorded force responses into Ca2+ transients as indicated in the Methods (Equation 1 and Fig. 1B). Panels A and C show force transients for caffeine-induced Ca2+ release after 120 sec of Ca2+ loading before and after exposure to 100 lmol L1 Suramin for 2 min, respectively and Panels B and D illustrate the free Ca2+ estimates. Figure 5 compares collated data for caffeine-induced Ca2+-release force responses converted into free Ca2+ transients. Panels A & B show the peak (% of 120 sec control) of the free Ca2+ transients for control and Suramin treated fibers, respectively, C & D show the rate of Ca2+ rise (% nmol ms1 ) called “rate of release” measured between the period 20 through to 80% of peak, and E & F show the area under the free Ca2+ transient (a reflection of the total amount of Ca2+ released from the SR (Endo and Iino 1980; Fink and
Stephenson 1987; Launikonis and Stephenson 1997).
The mechanically-skinned fiber retains an intact and functional SR (Lamb and Stephenson 1990b). The properties of the SR Ca2+ uptake via the SR Ca2+-ATPase and Ca2+ release from the SR via the RyRs were investigated using the contractile apparatus as an indicator of the cytoplasmic free Ca2+ movements. Treatment of skinned fibers with 100 lmol L1 Suramin for 10 min produced a marked increase in the rate of caffeine-induced Ca2+ release at all levels of SR Ca2+ loading (see Fig. 5D) indicating the Suramin treatment clearly affected the RyRs in our preparation. It is worth noting that in this study Suramin was washed from the fibers following the 10 min treatment period and therefore, the observed effects of Suramin described in this study must result from some irreversible action of Suramin on the RyRs (in addition to the contractile myofibrillar components described earlier).
Suramin is thought to compete with CaM on the RyR displacing CaM from this site (Klinger et al. 1999; Papineni et al. 2002; Sigalas et al. 2009). If this were the case, then our results are most likely explained by an increase in the open probability when activated following the removal of CaM from the RyRs as reported in other studies (Hill et al. 2004). In our study calcium, loading by the SR was not affected after exposure and washout of Suramin suggesting that the SR Ca2+-ATPase was not affected. A previous study by Emmick et al. (1994) reported the SR Ca2+- ATPase was reduced in the presence of Suramin. Taken together, the results indicate that the Suramin effect on the SR Ca2+-ATPase is fully reversible. Indeed, this was also observed by Sigalas et al. (2009) who showed that the SR Ca2+-ATPase in permeablized cardiac cells was not affected by Suramin treatment for a similar time.
Unlike the peak and the rate of rise of the Ca2+-transient described above, which are affected directly by any change in RyR channel activity, the relative area under the caffeine-induced Ca2+-transient provides (see Methods) information about both the amount of Ca2+ loaded into the SR and thus an indication of the activity of the SR Ca2+-ATPase (SERCA) and the total amount of Ca2+ subsequently released from the SR. Using the equation Y ¼ Ymaxð1 AðKXÞ Þ to plot a line of best fit through all loading times, we could assess the amount of Ca2+ loaded into the SR in control and Suramin treated fibers (Fig. 5E and F). It is important to note that loading concentration used in this investigation (10 lmol L1 for 2 min) the maximum Ca2+-activated force elicited at pCa 4.5 (see Table 1) was reduced by 14% and the sensitivity to Ca2+ decreased by 38% (dpCa50 = 0.14).
Author: W. Williams, Dimitrie George Stephenson & Giuseppe S. Posterino