Timed pregnant Sprague-Dawley dams (Envigo, Indianapolis, IN) were allowed to deliver naturally at term in house. Irrespective of sex, the newborn pups were divided into two groups within 12 h of birth: (1) room air (normoxic), and (2) 14-day hyperoxia (hyperoxic). Both groups were housed in standard cages within a 30″ × 20″ × 20″ polypropylene chamber with a clear acrylic door. Oxygen concentration within the hyperoxia chamber was maintained at a fraction of inspired oxygen of 0.85 ± 0.03 using a continuous oxygen sensor, while the normoxia chamber was maintained at 0.21. Dams were rotated between room air and hyperoxia every 24 h to prevent oxygen-induced maternal toxicity. After 14 days, hyperoxic pups were returned to room air. Pups were weaned at 24 days. The UW School of Medicine and Public Health Animal Care and Use Committee approved all procedures involving animal care and handling.

RV pressure measurements were performed at the University of Wisconsin Cardiovascular Physiology Core, as previously described (Hacker et al., 2006; Tabima et al., 2010). Briefly, 21 and 35-day-old rats were anesthetized with urethane (1.2 g/kg via intraperitoneal injection), orally intubated, and mechanically ventilated (Harvard Apparatus). The chest cavity was entered through the sternum and the chest wall and lungs were gently retracted to expose the RV. A 1.9F variable segment length admittance pressure catheter (Scisense, London, Ontario, Canada) was introduced into the RV using a 24-gauge needle. The magnitude and phase of the electrical E3 admittance and RV pressure were continuously recorded and analyzed using commercial software (Notocord Systems, Croissy Sur Seine, France).

Right ventricular trabeculae were isolated as described previously (Olsson et al., 2004; Patel et al., 2012). Briefly, the hearts were dissected from 21- and 35-day old normoxic and hyperoxic rats anesthetized with inhaled isoflurane. The hearts were then pinned down to the base of a dissecting dish filled with modified Ringer's solution (in mmol/L: NaCl, 120; NaHCO₃, 19; Na₂HPO₄, 1.2; MgSO₄, 1.2; KCl, 5; CaCl₂, 1; glucose, 10; pH 7.4; ~22°C) pre-equilibrated with 95% O₂/5% CO₂. After the right ventricles were cut open, the hearts were exposed to fresh Ringer's solution containing 20 mM 2,3-butanedione monoxime (BDM) for 30 min (2x solution change). BDM was used to protect the trabeculae from cell contracture and muscle damage during dissection (Mulieri et al., 1989). Right ventricular trabeculae were then dissected free, tied to wooden applicator sticks to hold muscle length fixed, and transferred to relaxing solution (in mmol/L: 100 KCl, 20 imidazole, 7 MgCl₂, 2 EGTA, and 4 ATP; pH 7.0; 4°C) containing 1% Triton X-100. Following overnight skinning, the trabeculae were washed in fresh relaxing solution (~1 h) and then stored at −20°C in relaxing solution containing glycerol (50:50 v/v). The skinned trabeculae were used in experiments within 1 week.

Solutions composition used for mechanical measurements were calculated using the computer program of Fabiato (1988) and the stability constants (corrected to pH 7.0 and 22°C) listed by Godt and Lindley (1982). Both pCa 9.0 and pre-activating solution contained (in mmol/L) 100 BES, 15 creatine phosphate, 4.66 ATP and 5 DTT. In addition, pCa 9.0 solution contained (in mmol/L) 7 EGTA, 0.02 CaCl₂, 5.49 MgCl₂, and 63.14 potassium propionate, whereas pre-activating solution contained (in mmol/L) 0.07 EGTA, 5.29 MgCl₂, and 84.01 potassium propionate. pCa 4.5 solution contained (in mmol/L) 100 BES, 15 creatine phosphate, 4.72 ATP, 7 EGTA, 7.01 CaCl₂, 5.29 MgCl₂, 5 DTT, and 49.61 potassium propionate. pH of all solutions was adjusted to 7.0 with KOH. A range of Ca²⁺ activating solutions (pCa 6.2 to 5.4) were prepared by mixing solutions of pCa 9.0 and pCa 4.5.

On the day of an experiment, skinned trabeculae were incubated in relaxing solution for 30 min before cutting them free from the sticks and trimming their ends. The trimmed trabeculae were then transferred to a stainless steel experimental chamber containing pCa 9.0 solution (Moss et al., 1983). The ends of each trabecula were tied to the arms glued to a motor (model 312B, Aurora Scientific) and a force transducer (model 403; Aurora Scientific), as previously described (Moss et al., 1983). The chamber assembly was then placed on the stage of an inverted microscope (Olympus) fitted with a 40 × objective and a CCTV camera (model WV-BL600, Panasonic). The light from a halogen lamp was used to illuminate the skinned preparations. Bitmap images of the preparations were acquired using an AGP 4X/2X graphics card and associated software (ATI Technologies) and were used to assess mean sarcomere length (SL) during the course of each experiment. Changes in force and motor position were sampled (16-bit resolution, DAP5216a, Microstar Laboratories) at 2.0 kHz using SLControl software developed in this laboratory (http://www.slcontrol.com). Data were saved to computer files for later analysis.

Passive force, active force-pCa, and k_tr-pCa/force relationships were established at a mean SL of ~2.2 μm as described previously (Olsson et al., 2004; Patel et al., 2012). Briefly, the skinned trabeculae were stretched to a mean SL ~2.2 μm and after measuring length and width, the preparations were transferred first to pre-activating solution, then to Ca²⁺ activating solution, and finally back to pCa 9.0 solution. Once in Ca²⁺ activated solution, steady-state force and the apparent rate constant of force redevelopment (k_tr) was measured simultaneously using the modified multi-step protocol developed by Brenner and Eisenberg (1986) as described in detail previously (Patel et al., 2001) and illustrated in Figure 1. Briefly, after force reached a steady level in activating solution (pCa 6.2–4.5), the length of the preparation was rapidly reduced by ~20%, held for ~20 ms, and then re-stretched back to its original length. As a result, there was an initial transient increase, followed by a decrease in force (seen as a spike in the force trace) and subsequent slower recovery of force to near the initial steady-state level. k_tr reported in the present study is the rate constant of force redevelopment after the spike. The drop in force recorded in solution of pCa 9.0 was considered to be passive force and was therefore subtracted from the drop in total force at each pCa to yield Ca²⁺ activated force (P). The protocol was repeated to establish active force-pCa and k_tr-pCa/relative active force relationships. After completing mechanical measurements, the trabeculae were detached from the points of attachment, placed in sodium dodecyl sulfate (SDS) sample buffer (8M Urea, 2 M thiourea, 0.05 M Tris pH6.8, 75 mM DTT, 3% SDS, and 0.01% bromophenol blue) and stored at −80°C until subsequent protein analysis.

To examine the expression profile of MHC isoforms, samples were prepared using RV free wall isolated from normoxic and hyperoxic treated rats and gels were prepared as described previously (Warren and Greaser, 2003). Briefly, RV free wall was homogenized in relaxing solution and homogenate washed with fresh relaxing solution. Next, the homogenate was incubated for 30 min in relaxing solution containing 1% Triton-100. After the incubation period, the homogenate was washed three times with relaxing solution and 2.4 mg (wet weight) of homogenate was suspended in 100 μL SDS sample buffer and stored at −80°C until subsequent protein analysis. A 17 mL solution of resolving gel (6%T and 2% C) was prepared by mixing 8.27 mL water, 1.7 ml 50% glycerol (v/v), 2.55 mL 40% acrylamide (37.5:1 cross-linked with DATD), 4.24 mL 1.5 M Tris, pH 8.8, 170 μL 10% SDS (w/v), 50 μL 10% ammonium persulfate (w/v), and 20 μL TEMED. The resolving gel was poured into an empty BioRad Criterion cassette and water was added to the top of the resolving gel to form a flat surface. After an hour of polymerization, the gel was stored in the cold room overnight. Next day, water was drained out and stacking gel (3%T and 1.5%C; 1.15 mL water, 1 ml 50% glycerol (v/v), 1.5 mL 10% acrylamide (5.6:1 cross-linked with DATD), 1.3 mL 0.5 M Tris, pH 6.8, 50 μL 10% SDS (w/v), 30 μL 10% ammonium persulfate (w/v), and 25 μL TEMED) was poured over the resolving gel. A 12 well comb was inserted and stacking gel was allowed to polymerize for an hour. The gel cassette was then inserted into a BioRad Criterion gel box pre-filled with ice cold lower running buffer (25.09 mM Tris-base, 19.98 mM glycine, 3.47 mM SDS, and 2 mM 2-mecaptoethanol). The comb was removed, wells were washed with water and the chamber was filled with ice cold upper running buffer (50.18 mM Tris-base, 39.96 mM glycine, 6.94 mM SDS, and 10 mM 2-mecaptoethanol). The samples were defrosted and 4 μL of sample was added to 36 μL of sample buffer. The diluted sample was heated (95°C) for 3 min and allowed to cool down before loading 10 μL on to the gel. Electrophoresis was done at 16 mA constant current for 4 h in the cold room. At the end of electrophoresis, the gel was removed from the cassette and silver stained using method described previously (Shevchenko et al., 1996) with following modification (Stelzer et al., 2006). The gels were (a) incubated overnight in fixing solution containing 50% methanol and 10% acetic acid, (b) washed for 20 min (4x ddH₂O changes) with ddH₂O, (c) incubated for 1.5 min in 0.01% sodium thiosulfate solution and then rinsed 4x with ddH₂O, (d) incubated for 20 min in 0.09% silver nitrate solution and then rinsed 4x with ddH₂O, (e) incubated in developing solution containing 0.0004% sodium thiosulfate, 2% potassium carbonate, and 0.0068% formaldehyde until protein bands were visible and then rinsed 4x with ddH₂O, (f) incubated for 20 min in destaining solution containing 10% methanol and 10% acetic acid, and (g) finally washed for 30 min (6x ddH₂O changes) with ddH₂O.

To examine expression profile of myofibrillar proteins, the normoxic and hyperoxic trabeculae stored in SDS sample buffer were electrophoresed using 12% Tris-HCl Precast Criterion gels (BioRad, Hercules, CA). The gel cassette was inserted into BioRad Criterion gel box pre-filled with running buffer (25.09 mM Tris-base, 19.98 mM glycine, 3.47 mM SDS). The comb was removed, wells were washed with water and the chamber was filled with running buffer. The samples were defrosted, sonicated for 15 min and 8 μL of sample was loaded on to the gel. Electrophoresis was done at constant volts (150 V) for 1.5 h at room temperature. At the end of the run, the gels were removed from the cassette and silver stained as described above. Both gels were imaged and protein bands quantified using BioRad Chemi Doc MP Imaging System (BioRad, Hercules, CA).

Cross-sectional areas of skinned trabeculae were calculated by assuming that the trabeculae were cylindrical and by equating the width, measured from video images of the mounted preparations, to diameter. Each Ca²⁺ activated force (P) at pCa between 6.2 and 5.4 was expressed as a fraction of the maximum Ca²⁺ activated force (P_o) developed by the same preparations at pCa 4.5, i.e., P/P_o. To determine the Ca²⁺ sensitivity of isometric force (pCa₅₀), force-pCa data were fitted with the Hill equation: P/P_o = [Ca²⁺]ⁿ/(kⁿ + [Ca²⁺]ⁿ)], where n is the slope (Hill coefficient) and k is the Ca²⁺ concentration for half-maximal activation (pCa₅₀). k_tr was determined by linear transformation of the half-time of force recovery (k_tr = −ln 0.5 × (t_1/2)⁻¹), as described previously (Chase et al., 1994; Patel et al., 2001). All data are presented as means ± standard error (SE). Statistical analysis of the RV pressure data was performed using two-way ANOVA (age and group main effects; GraphPad Prism 6;GraphPad Software Inc., San Diego, CA), all other analysis with unpaired t-tests (Sigma Plot 11.Ink; Systat Software Inc., San Jose, CA), p-values <0.05 were taken as indicating significant differences.

A total of 49 rats were exposed to postnatal normoxia, and 40 rats were exposed to postnatal hyperoxia. Sixteen normoxia and hyperoxia exposed rats were used for the determination of RV pressures at 21 and 35 days of age (n = 8 in each group), respectively. There was a significant difference (p < 0.05) in body weight at day 21 (51.4 ± 1.4 vs. 42.4 ± 1.6 g) but no difference in body weight at day 35 (127.0 ± 4.7 vs. 117.1 ± 3.2 g) in normoxia treated vs. hyperoxia treated rats, respectively. The systolic and diastolic RV pressure (Figure 2) was significantly higher in the hyperoxic group at day 21 compared to the normoxic group at the same time point. This difference between the normoxic and hyperoxic group resolved by day 35 with RV pressure in the hyperoxic group being significantly lower compared to day 21. This suggests a recovery of the hyperoxia-induced PH seen at day 21.

Steady-state mechanical properties were assessed using right ventricular trabeculae isolated from a second cohort of 21-day normoxic (n = 18 trabeculae/15 rats) and hyperoxic (n = 16 trabeculae/12 rats) and 35-day normoxic (n = 15 trabeculae/8 rats) and hyperoxic (n = 9 trabeculae/8 rats) (Table 1). At pCa 9.0, passive force generated by 21-day hyperoxic trabeculae was almost twice (p < 0.001) than age matched normoxic trabeculae, and by 35-day hyperoxic trabeculae generated similar amount of passive force as age matched normoxic trabeculae (Table 1). At pCa 4.5, maximum Ca²⁺ activated force generated by 21-day hyperoxic trabeculae was ~70% greater (p = 0.002) than age matched normoxic trabeculae, and by 35-day hyperoxic trabeculae were similar to age matched normoxic trabeculae (Table 1). At sub-maximal Ca²⁺ (pCa 6.2–5.4), 21-day hyperoxic trabeculae also generated more force than age matched normoxic trabeculae, resulting in a left shift of hyperoxic trabeculae sigmodal force-pCa relationships compared to age matched normoxic trabeculae (Figure 3A). Fitting the force-pCa relationships with the Hill equation yielded significantly higher pCa₅₀ values (ΔpCa₅₀ = 0.23; p < 0.001), implying elevated Ca²⁺ sensitivity of force, and lower n_H values (Δ n_H = 0.3; p = 0.005), which implies depressed apparent cooperativity in activation of force, for hyperoxic compared to normoxic trabeculae. On the other hand, sub-maximal forces generated by 35-day hyperoxic trabeculae were similar to those measured in age matched normoxic trabeculae and as a result there was no discernable difference between the sigmodal force-pCa relationships established in hyperoxic and age matched normoxic trabeculae (Figure 3B). Fitting the force-pCa relationships with the Hill equation yielded similar pCa₅₀ values and n_H values for hyperoxic and age matched normoxic trabeculae. These results suggest that the neonatal exposure to hyperoxia has profound early effects on steady-state force production and that these effects wane as the PH resolves.

Irrespective of age, both normoxic and hyperoxic trabeculae exhibited [Ca²⁺]_free (and force)-dependent changes in the rate of force redevelopment (k_tr), confirming earlier results from rat (Wolff et al., 1995; Palmer and Kentish, 1998; Patel et al., 2012) myocardium. That is, increasing the [Ca²⁺]_free from pCa 6.2 to pCa 4.5 elevated the values of k_tr from 2.62 ± 0.14 to 10.48 ± 1.08 s⁻¹ and 1.93 ± 0.21 to 8.28 ± 1.02 s⁻¹ in 21-day normoxic and hyperoxic trabeculae and from 2.61 ± 0.12 to 11.74 ± 0.58 s⁻¹ and 2.79 ± 0.17 to 10.43 ± 0.78 s⁻¹ in 35-day normoxic and hyperoxic trabeculae. To illustrate this, records of force redevelopment at various levels of [Ca²⁺]_free are shown in Figure 4 for 21-day normoxic (Figure 4A) and hyperoxic (Figure 4B) and 35-day normoxic (Figure 4C) and hyperoxic (Figure 4D) trabecula, where steady state forces at each pCa were normalized to 1.0 to provide better visualization of variations in kinetics of force redevelopment. Figure 5 shows the curvilinear k_tr-relative force relationships observed in 21-day (Figure 5A) and 35-day (Figure 5B) normoxic and hyperoxic trabeculae. At pCa 4.5, both 21 and 35-day hyperoxic trabeculae redeveloped maximum Ca²⁺ activated force at similar rates as age matched normoxic trabeculae. At sub-maximal free [Ca²⁺] (pCa 6.2–5.4), 21-day hyperoxic trabeculae redeveloped sub-maximal forces at significantly slower rates than age matched normoxic trabeculae and as a result the curvilinear k_tr-relative force relationships established in hyperoxic trabeculae were to the right of those established in normoxic trabeculae (Figure 5A). On the other hand, 35-day hyperoxic trabeculae redeveloped sub-maximal forces at rates similar to age matched normoxic trabeculae and as a result there was no discernable difference between curvilinear k_tr-relative force relationships established in hyperoxic and age matched normoxic trabeculae (Figure 5B). These results indicate that the neonatal exposure to hyperoxia has profound effects on cross-bridge cycling kinetics and that the effects are reversible in rat myocardium. These reversible effects are coincident with the normalization of RV pressure at 35 days.

Figure 6 shows a typical SDS-PAGE analysis of MHC isoforms (Figures 6A,B) and myofibrillar protein expression (Figures 6C,D) in 21- and 35-day normoxic and hyperoxic myocardium. Figures 6A,B shows that 21-day hyperoxic RV expressed 14% less α-MHC and more β-MHC than aged matched normoxic RV. Whereas, 35-day hyperoxic RV expressed similar levels of both α-MHC and β-MHC as aged matched normoxic RV. It is also apparent from Figure 6C that both 21- and 35-day hyperoxic trabeculae expressed similar isoforms of key myofibrillar proteins as normoxic trabeculae (cardiac myosin binding protein C (cMyBP-C), actin, troponin T (TnT), tropomyosin (Tm), ventricular myosin light chain 1, and 2 (vMLC1 and vMLC2), respectively. While both 21 and 35-day normoxic trabeculae were found to express 100% cTnI, an observation consistent with a previous study which reported complete conversion of ssTnI to cTnI by 15 days after birth in rat (Warren et al., 2004), 21-day hyperoxic trabeculae expressed both cTnI (33 ± 2%) and ssTnI (67 ± 2%) (Figure 6D). With the exception of one hyperoxic trabeculae expressing both cTnI (31%) and ssTnI (69%), 35-day normoxic and hyperoxic rat trabeculae expressed predominately cTnI. In addition, 21-day, but not 35-day, hyperoxic trabeculae expressed aMLC1 (15 ± 3%) and vMLC1 (85 ± 3%) (Figure 6D), an observation consistent with PH-induced expression of aMLC1in neonatal porcine right ventricle (Morano et al., 1998). These results suggest that neonatal exposure to hyperoxia elevates expression of β-MHC, disrupts transition of ssTnI to cTnI, stimulates expression of aMLC1, and indicates that most of these effects are reversible in rat myocardium.

The 21-day hyperoxic trabeculae generated twice as much force as normoxic trabeculae (Table 1), an effect similar to the PH-induced increase in maximum Ca²⁺ activated force observed in the adult human (Rain et al., 2013), rat (Kogler et al., 2003), and mice (unpublished observation). While these results are consistent with the idea that higher density of thick and thin filaments allows 21-day hyperoxic trabeculae to generate more force, the finding of similar amount of maximum Ca²⁺ activated forces in 35-day hyperoxic and normoxic trabeculae is inconsistent with this mechanism. Previous studies reported that myocardium expressing ssTnI generates a maximum Ca²⁺ activated force similar to myocardium expressing cTnI (Fentzke et al., 1999; Arteaga et al., 2000; Konhilas et al., 2003; Ford and Chandra, 2012) whereas expression of aMLC1 results in a higher maximum Ca²⁺ activated force than expression of vMLC1 (Morano et al., 1998). Interestingly, our findings of increased maximum Ca²⁺activated force and aMLC1 in 21-day old hyperoxia exposed rats was associated with PH, which is similar to a report in a porcine model of PH (Morano et al., 1998). Taken together, the earlier observation of increased maximum Ca²⁺activated force in 21-day hyperoxic trabeculae and the similar amount of maximum Ca²⁺activated force generated by 35-day hyperoxic trabeculae compared to normoxic trabeculae suggests that expression of aMLC1 is likely to play a prominent role in increasing maximum Ca²⁺activated force in 21-day hyperoxic trabeculae.

At sub-maximal Ca²⁺, 21-day hyperoxic trabeculae generated more force than age matched normoxic trabeculae and as a result, the force-pCa relationships established in hyperoxic trabeculae were left-shifted by ~0.23 pCa units compared to those in normoxic trabeculae (Figure 3A), which corresponds to similar changes found in monocrotaline-induced PH (Kogler et al., 2003). In cardiac muscle, expression of either ssTnI (Fentzke et al., 1999; Arteaga et al., 2000; Konhilas et al., 2003; Ford and Chandra, 2012) or aMLC1 (Morano et al., 1997; Diffee and Nagle, 2003; Diffee, 2004) are known to shift force-pCa relationships to the left. Since 21-day hyperoxic trabeculae expressed both ssTnI and aMLC1, it is difficult to say with certainty that the increased Ca²⁺ sensitivity of force in 21-day hyperoxic trabeculae was due to the presence of ssTnI or aMLC1, or both. However, the finding of similar Ca²⁺ sensitivities of force in 35-day hyperoxic trabeculae and age-matched normoxic trabeculae (Figure 3B), in which there is no aMLC1 present and predominant expression of cTnI (Figure 6C), suggest that the hyperoxia-induced changes in expression of MLC1/TnI isoforms at 21 days is reversible and may be responsible for the increase in Ca²⁺ sensitivity in hyperoxic myocardium at this age. Taken together, our findings of increased maximum Ca²⁺activated force, Ca²⁺ sensitivity of force and changes in expression of MLC1/TnI isoforms at 21 days of age in our hyperoxia exposed rats are likely an adaptive response to PH. This is highlighted by the findings at 35 days of age where there were no differences in RV pressure, RV myofibrillar isoform expression and contractile properties.

In normoxic trabeculae, the rate of force redevelopment (k_tr) varied with the level of activating [Ca²⁺]_free (or force), increasing as [Ca²⁺]_free (or force) was elevated from sub-maximal to maximal levels (Figures 4, 5). These Ca²⁺- and force-dependent changes in k_tr in normoxic trabeculae are consistent with previous results from rat (Wolff et al., 1995; Palmer and Kentish, 1998; Olsson et al., 2004; Patel et al., 2012), mice (Edes et al., 2007; Colson et al., 2012; Ford and Chandra, 2012), porcine (Edes et al., 2007) and human (Edes et al., 2007) myocardium. Both, 35-day normoxic and hyperoxic trabeculae also exhibited similar Ca²⁺- and force-dependent changes in k_tr, suggesting no remaining significant effects of hyperoxia on these relationships. However, 21-day hyperoxic trabeculae redeveloped sub-maximal forces at a slower rate than age-matched normoxic trabeculae (Figure 4). Thus, when k_tr values were plotted against force normalized to maximum force, k_tr-force relationships in hyperoxic trabeculae were right-shifted compared to those in normoxic trabeculae (Figure 5A), i.e., at equivalent forces, k_tr values were lower in hyperoxic trabeculae. In adult rat myocardium, a decrease in expression of α-MHC, and concomitant increase in expression of β-MHC, is known to slow the rate of force redevelopment (Fitzsimons et al., 1999; Rundell et al., 2005; Locher et al., 2011) and rate of relaxation (Fitzsimons et al., 1998). Thus, the possibility of the depressed cross-bridge cycling kinetics in 21-day hyperoxic trabeculae may be exclusively or in part due to a decrease in expression of α-MHC and concomitant increase in expression of β-MHC (Figure 6B). Previous studies have reported that expression of ssTnI has no significant effects on the rate of force redevelopment in skinned preparations (Ford and Chandra, 2012), whereas expression of aMLC1 increases contraction and relaxation time in whole heart (Morano et al., 1996, 1998; Fewell et al., 1998; Abdelaziz et al., 2004). Since there is expression of aMLC1 and ssTnI in 21-day hyperoxic myocardium but not in normoxic myocardium, both aMLC1and ssTnI appears to be responsible for slower contraction kinetics in hyperoxic myocardium at this age.

Alternatively, the ability of hyperoxic trabeculae to generate more force than normoxic trabeculae can be explained in the context of a two-state kinetic model of cross-bridge interaction proposed by Huxley (1957) and modified by Brady (1991). In this model, multiple states of the cross-bridge kinetic scheme are reduced to just two states, i.e., the transition from non-force-generating to force-generating states is described by f_app, whereas g_app describes the transition from the force-generating state back to the non-force-generating state. Steady isometric force (P) is then equal to N × F × [f_app/(f_app + g_app)], where N is the number of cycling cross-bridges, F is the average force per cross-bridge and k_tr = f _app + g_app. Thus, the hyperoxia-induced increase in Ca²⁺ sensitivity of force in the present study may be due to an increase in N, or F, or the proportion of cross-bridges in the force generating state as a result of an increase in f_app, a decrease g_app, or both. An increase in either the probability of myosin cross-bridge binding to actin (in case of aMLC1; Schaub et al., 1998) or the binding affinity of TnC for Ca²⁺ (in case of ssTnI) would increase N and facilitate cooperative binding of myosin cross-bridges to actin. The latter would be manifested as a decrease in n_H (an index of apparent cooperativity in activation of force; Table 1) of the force-pCa relationship, and a decreased cross-bridge detachment rate (g_app), which would be manifested as decrease in k_tr. Interestingly, g_app derived from natural logarithm of k_tr-relative force relationship (data not shown) was lower in 21-day hyperoxic (1.44 s⁻¹) than normoxic (2.17 s⁻¹) trabeculae. Thus, it appears that the combined effect of β-MHC, ssTnI (increased binding affinity of TnC for Ca²⁺) and aMLC1 (increased probability of myosin cross-bridge binding to actin) are important for the reduced kinetics in the RV at 21 days of age. Although, no previous studies have explored cross-bridge cycling kinetics in a model of BPD, we can infer that reduced cross-bridge cycling kinetics in 21 day hyperoxia exposed rats is likely due to the adaptive response to pulmonary pressure overload and the subsequent myofibrillar isoform expression changes at 21 days of age in this model.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.