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• W1529573891 abstract "Familial hypertrophic cardiomyopathy (FHC)-associated missense mutations in the tail portion of cardiac troponin T (cTnT) lead to a phenotype characterized by an increased cost of cardiac contraction, contractile dysfunction and impaired energetics. Arginine 92 (R92) in cTnT is a ‘hotspot’ with many FHC-associated missense mutations; each mutation leads to a clinically distinct cardiomyopathy. Both R92Q and R92L cTnT mutant mouse hearts exhibit the FHC phenotype. Here we found that genetically remodelling the sarcomere by substituting αα-myosin heavy chain (αα-MyHC) with ββ-MyHC normalizes the increased cost of cardiac contraction, rescuing both contractile dysfunction and energetic abnormalities, in R92Q cTnT mutant hearts, while wild-type R92 and R92L cTnT mutant hearts were unaffected. Our results demonstrate that the conformation of the tropomyosin-binding domain of cTnT induced by each unique amino acid substitution at R92 is a major determinant of sarcomere function. Abstract The thin filament protein troponin T (TnT) is a regulator of sarcomere function. Whole heart energetics and contractile reserve are compromised in transgenic mice bearing missense mutations at R92 within the tropomyosin-binding domain of cTnT, despite being distal to the ATP hydrolysis domain of myosin. These mutations are associated with familial hypertrophic cardiomyopathy (FHC). Here we test the hypothesis that genetically replacing murine αα-MyHC with murine ββ-MyHC in hearts bearing the R92Q cTnT mutation, a particularly lethal FHC-associated mutation, leads to sufficiently large perturbations in sarcomere function to rescue whole heart energetics and decrease the cost of contraction. By comparing R92Q cTnT and R92L cTnT mutant hearts, we also test whether any rescue is mutation-specific. We defined the energetic state of the isolated perfused heart using 31P-NMR spectroscopy while simultaneously measuring contractile performance at four work states. We found that the cost of increasing contraction in intact mouse hearts with R92Q cTnT depends on the type of myosin present in the thick filament. We also found that the salutary effect of this manoeuvre is mutation-specific, demonstrating the major regulatory role of cTnT on sarcomere function at the whole heart level. Muscle contraction occurs when the thick filament protein myosin interacts with specific domains of the thin filament protein actin, coupling the energy released from ATP hydrolysis (ΔG∼ATP) by myosin to force generation. Availability of the myosin-binding domains of actin for cross-bridge formation depends on the conformation of the thin filament regulatory proteins tropomyosin (TM) and troponin (Tn). Each of the subunits of troponin, TnT, TnC and TnI, plays a regulatory role in cross-bridge dynamics. Cross-bridge cycling depends on both Ca2+-dependent activation of TnC and Ca2+-independent conformational changes in TnT and TnI. The tail portion of TnT binds to TM where the two TM polypeptide chains overlap, changing the flexibility of the thin filament. This determines the affinity of the TnT–TM complex for actin, which in turn determines the availability of myosin-binding domains on actin for cross-bridge formation. Experiments using model systems have shown that changing the TnT–TM interaction by deleting the TnT tail domain alters actomyosin ATPase activity, in vitro motility of actin-TM filaments on myosin heads and cooperativity of myosin subfragment-1 binding to thin filaments (Maytum et al. 2002; Tobacman et al. 2002). Thus, in addition to playing the classical structural role in the thin filament, TnT also plays a crucial dynamic role in the regulation of the contractile cycle and ATP utilization by the sarcomere. Missense mutations in the tail portion of cardiac TnT (cTnT) discovered by association with the cardiac disease familial hypertrophic cardiomyopathy (FHC) have been shown to lead to cardiac phenotypes characterized by contractile dysfunction with modest or no left ventricular (LV) hypertrophy but early sudden death (reviewed in Tardiff, 2011). Arginine 92 (R92) in cTnT is a ‘hotspot’ with many FHC-associated missense mutations; each mutation leads to a clinically distinct cardiomyopathy with a unique progression of disease, making treatment challenging. R92 is located in the elongated tail domain of cTnT adjacent to the critical site of overlap of TM monomers. Using molecular dynamics simulations, our group has found that substituting the FHC-associated amino acids tryptophan (W), leucine (L) or glutamine (Q) for R at residue 92 of cTnT alters the flexibility and dynamics of the cTnT domain that binds to TM (Guinto et al. 2007). Moreover, each mutation yields a unique average conformation. A recent molecular dynamics analysis of the thin filament protein complex shows how seemingly small changes in the conformation of one component, in this case TnC, propagates throughout the entire thin filament (Manning et al. 2011). The conformational changes induced by FHC-associated amino acid substitutions at R92 in cTnT produce distinct phenotypes. Murine hearts genetically manipulated to replace R92 cTnT with R92Q, R92W or R92L demonstrate contractile dysfunction, and the changes for R92Q and R92W hearts are more severe than for R92L hearts (Ertz-Berger et al. 2005; He et al. 2007; Guinto et al. 2009; Rice et al. 2010). Thus each mutation produces a unique peptide conformation and dynamics sufficient to alter thin filament flexibility and cross-bridge dynamics, which lead to mutation-specific phenotypes in murine cardiac fibres, cardiomyocytes and intact hearts. Using these well-characterized transgenic mouse hearts, we have also found that these R92 cTnT mutations lead to an increased cost of contraction assessed as decreased free energy available from ATP hydrolysis, |ΔG∼ATP|, and diminished ability of the heart to increase contractile performance (Javadpour et al. 2003; He et al. 2007). This is the case despite the fact that R92 cTnT is physically distant from the ATP hydrolysis pocket in myosin. Importantly, we have also found that the increase in the cost of contraction is mutation-specific (He et al. 2007). Increases in the cost of contraction have also been observed in mouse hearts bearing the FHC-associated R403Q mutation in the actin-binding loop of myosin (Spindler et al. 1998), suggesting that a common consequence of the different malignant FHC-associated mutations in sarcomeric proteins is an increased cost of cardiac contraction. These findings would explain clinical observations that the phosphocreatine (PCr) to ATP ratio is lower in hearts of FHC patients with diverse sarcomeric mutations (Watkins et al. 1995a,b) and thereby provide a structural-energetic basis for their cardiac dysfunction. These observations also suggest that strategies designed to increase the efficiency of contraction would increase contractile performance of those hearts bearing FHC-associated mutations that affect the interaction of the actin-binding loop of myosin in the thick filament and actin in the thin filament. We recently tested the novel hypothesis that genetically remodelling the sarcomere by substituting the endogeneous murine myosin heavy chain, αα-MyHC, with ββ-MyHC in mouse hearts bearing the FHC-associated mutations at R92 in cTnT would rescue abnormalities observed during baseline contractile performance (Rice et al. 2010). The rationale for this experiment was that, despite the fact that the myosin isozymes are highly homologous (93% amino acid identity; McNally et al. 1989), their physiological and biochemical properties differ markedly. In in vitro motility studies, for example, αα-MyHC has twice the actin-activated and Ca2+-stimulated ATPase activity and three times the actin filament sliding velocity whereas it produces only half of the average cross-bridge force as ββ-MyHC (Alpert et al. 2002). The actin- and Ca2+-activated ATPase activities of the different myosin isozymes determine the velocity of sarcomere contraction (Barany, 1967). Furthermore, recent thermodynamic analyses of the dynamics of the nucleotide-binding pocket of myosin provide a mechanistic basis explaining why slow myosins are more efficient at force generation (Purcell et al. 2011). Our experiments using R92Q cTnT mutant mouse hearts showed that genetically replacing αα-MyHC (95%αα–5%ββ) with ββ-MyHC (80%ββ–20%αα) rescued baseline systolic but not diastolic dysfunction (Rice et al. 2010). Neither wild-type R92 cTnT nor R92L cTnT mutant hearts, which demonstrated less contractile dysfunction, were affected. Using R92Q and R92L cTnT mouse hearts, here we test whether increasing the efficiency of the sarcomere by substituting the slow murine ββ-MyHC for the fast murine αα-MyHC would rescue the dysfunctional R92Q and R92L cTnT energetic phenotypes and thereby alter the cost of increasing contraction. We chose these two mutations because they represent widely differing clinical phenotypes that are reproduced in transgenic mouse hearts. While both R92Q and R92L cTnT mouse hearts have impaired energetics, demonstrate diastolic contractile dysfunction even at low work states and have severely impaired contractile reserve, R92Q cTnT hearts also have impaired systolic contractile function even at low work states (Javadpour et al. 2000; He et al. 2007; Rice et al. 2010). Rescuing the R92Q cTnT mutant heart and improving contractile and energetic reserve would therefore pose the greater challenge. We chose to test this hypothesis under conditions that we have previously demonstrated come the closest to normalizing the cost of increasing contraction in R92 cTnT mutant mouse hearts, namely perfusion with the inotrope dobutamine (He et al. 2007). Imposing a high work state with this inotrope for αR92L hearts improved contractile performance and normalized the cost of increasing contraction compared to other methods of increasing work but did not rescue its abnormal energetic phenotype. Studying R92L cTnT hearts therefore tests whether remodelling the thick filament would rescue the energetic dysfunction caused by this mutant thin filament. We defined the energetic state of the isolated perfused mouse hearts using 31P-NMR spectroscopy while simultaneously measuring contractile performance at four work states. We found that the cost of increasing contraction in intact beating mouse hearts bearing the FHC-associated mutation R92Q cTnT depends on the type of myosin present in the thick filament, and that the salutary effect of this manoeuvre is mutation-specific. These results demonstrate the crucial role the conformation of the thin filament protein Troponin T plays in the regulation of cardiac contraction. C57BL/6 mice, 19–23 weeks old, bearing c-myc tagged murine cTnT with R92Q and R92L mutations were generated as described (all animals at F4 or above) (Tardiff et al. 1999; Ertz-Berger et al. 2005). The R92Q and R92L lines express 67% and 60% of total cTnT as the mutant form, respectively, and were driven by −2996 bp of a 5′ upstream sequence derived from the rat α-MyHC promoter. β-MyHC expression was increased in the cardiac ventricles of these animals genetically by crossing them with a transgenic line expressing 80%ββ-MyHC in the LV (Krenz et al. 2003). This line expressed the full-length β-MyHC mouse cDNA driven by the mouse α-MyHC promoter. All relevant genotypes were produced and were viable: wild-type cTnT/wild-type α-MyHC (αR92, n = 29), wild-type cTnT/β-MyHC (βR92, n = 26), R92 mutations/α-MyHC (αR92Q, n = 16 and αR92L, n = 11) and R92 mutations/β-MyHC (βR92Q, n = 16 and βR92L, n = 11). Cardiomyocytes rigidly control the stoichiometry of the contractile proteins within the sarcomere, and the total amounts of myosin and cTnT in the six types of hearts were the same (Rice et al. 2010). The genotype of each animal was identified via PCR. The experimental protocols were approved by the Institute of Animal Studies at the Albert Einstein College of Medicine and the Standing Committee on Animals of Harvard Medical Area, and followed the recommendations of current National Institutes of Health and American Physiological Society guidelines. Mice were heparinized (100 units, i.p.) 15 min before experiments and were humanely killed via cervical dislocation. Hearts were quickly isolated and perfused in the Langendorff mode as described (Saupe et al. 1998). Briefly, the chest was opened and the heart was excised, arrested in ice-cold buffer and connected via the aorta to the perfusion cannula. Retrograde perfusion was carried out at a constant coronary perfusion pressure of 75 mmHg at 37°C with phosphate-free Krebs–Henseleit (KH) buffer containing (in mm): NaCl 118, KCl 5.3, CaCl2 2.5, MgSO4 1.2, EDTA 0.5, NaHCO3 25, glucose 10 and pyruvate 0.5 equilibrated with 95% O2–5% CO2, yielding a pH of 7.4. Right ventricular drainage was accomplished by incision of the pulmonary artery. A thin polyethylene tube (PE-10) placed through the apex of the LV was used to drain the effluent from the Thebesian veins. A water-filled balloon, custom-made of polyvinylchloride film, connected to a pressure transducer (Statham P23Db, Gould Statham Inc., Medical Provision Department, Oxnard, CA, USA) was used for continuous recording of LV contractile performance. The balloon matched the size of the ventricular cavity and was inflated to set LV end-diastolic pressure (EDP) to ∼10 mmHg; the balloon volume was then held constant. Systolic and diastolic isovolumic contractile performance data were collected online at a sampling rate of 200 Hz using a commercially available data acquisition system (MacLab; ADInstruments Pty, Milford, MA, USA). Coronary flow rate was measured by collecting coronary sinus effluent through the suction tube. Isolated perfused hearts were placed in a 10 mm glass NMR sample tube and inserted into a custom-made 1H/31P double-tuned probe situated in an 89 mm bore 9.4 T superconducting magnet. To improve homogeneity of the NMR-sensitive volume, the perfusate level was adjusted so that the heart was submerged in buffer. 31P-NMR spectra were obtained without proton decoupling by using a 60°C flip angle, 15 μs pulse width, 2.4 s recycle time, 6000 Hz sweep width and 2 K data points at 161.94 MHz on a GE-400 wide-bore Omega spectrometer (General Electric, Fremont, CA, USA). Spectra were collected during either 16 or 40 min periods and consisted of data averaged from 312 or 1040 free induction decays, respectively. Spectra were analysed using 20 Hz exponential multiplication and zero and first-order phase corrections. The resonance areas corresponding to ATP, PCr and inorganic phosphate (Pi) were quantified using Bayesian Analysis software (G. L. Bretthorst, Washington University, St. Louis, MO, USA). Bayesian Analysis software uses a direct statistical analysis of the free induction decay amplitudes, which corresponds to the resonance areas. By comparing the amplitude under the peaks from fully relaxed (recycle time 15 s) and those of partially saturated (recycle time 2.4 s) spectra, the correction factors for saturation were calculated for ATP (1.0), PCr (1.2), and Pi (1.15). To determine the cytosolic concentration of ATP, the absolute resonance amplitude corresponding to [γ-P]ATP in the 31P-NMR spectra during baseline perfusion was normalized by heart weight. Since the Lowry protein content (which minimizes detection of extracellular matrix protein and thereby approximates myocyte protein content) (Lowry et al. 1951) was indistinguishable among the six groups (not shown), we make the assumption that the ratio of intracellular volume to cardiac mass of 0.48 μl (mg wet weight)−1 was the same for all hearts. In this case, amplitude units (g wet weight)−1 is directly proportional to the absolute intracellular concentrations. The value of 10 mm for [ATP] for αR92 mouse myocardium was used to calibrate the mean [γ-P]ATP peak amplitude of the 31P-NMR spectra obtained during baseline perfusion period. Changes in ATP, PCr and Pi concentrations during the protocols were calculated by multiplying the ratio of their resonance peak amplitude to the mean amplitude of the [γ-P]ATP peaks of the initial baseline spectrum for wild-type hearts by 10 mm. Intracellular pH was determined by comparing the differences in the chemical shifts of Pi and PCr resonances in each spectrum to values from a standard curve; the chemical shift of Pi but not PCr varies with pH. Cytosolic free [ADP] was calculated using the equilibrium expression for the creatine kinase reaction and values for ATP, PCr, creatine (Kammermeier, 1973), and H+ concentrations obtained by NMR spectroscopy and biochemical assay: [ADP] = ([ATP][free Cr])/([PCr][H+]Keq), where Keq is 1.66 × 109 m−1 for a [Mg2+] of 1.0 mm. Cytosolic free [AMP] was calculated using the equilibrium expression for the adenylate kinase reaction and values for ATP and ADP: [AMP] = [ADP]2/[ATP]Keq, where Keq is 1.05. The free energy released by ATP hydrolysis (ΔG∼ATP) is used to drive the ATPase reactions in the cell. Although ΔG∼ATP is a negative value, the change in free energy due to ATP hydrolysis is a positive value. Here we describe changes in ΔG∼ATP in absolute values, denoted as |ΔG∼ATP|, and calculated as |ΔG∼ATP| (kJ mol−1) = |ΔG°+RTln([ADP][Pi]/[ATP])|, where ΔG° (−30.5 kJ mol−1) is the value of ΔG∼ATP under standard conditions of molarity, temperature, pH and [Mg2+], R is the gas constant (8.3 J mol−1 K−1), and T is the temperature in kelvins. For each protocol, experiments were performed using littermate mouse hearts from one of the following cohorts. Cohort 1 consisted of 14 αR92, 12 βR92, 11 αR92Q and 12 βR92Q hearts; cohort 2 consisted of 6 αR92, 10 βR92, 11 αR92L and 11 βR92L; cohort 3 consisted of 6 αR92, 4 βR92, 4 αR92Q and 4 βR92Q hearts; the results are presented as four-way matches. The total number of hearts studied was 105. Two protocols were used. In the first protocol, hearts were perfused with KH buffer containing 2 mm free Ca2+ (total 2.5 mm); hearts were not paced. After a 30 min stabilization period, isovolumic contractile performance and 31P-NMR spectroscopy were measured simultaneously for 16 min. Hearts were then paced at 420 beats min−1 using monophasic square-wave pulses delivered from a stimulator (model S88; Grass Instrument Co., Quincy, MA, USA) through salt-bridge pacing wires consisting of PE-90 tubing filled with 4 m KCl in 2% agarose. Parameters of isovolumic contractile performance and energy metabolites detected by 31P-NMR spectroscopy were measured simultaneously for 16 min. The pacing was then turned off and, after 16 min, hearts were perfused with KH buffer containing 300 mm dobutamine (final concentration). Based on preliminary experiments, this dose of dobutamine produced >90% of the maximum contractile response. After steady state was reached (<3 min), cardiac function and 31P-NMR spectroscopy were again measured simultaneously for another 16 min. This protocol was used for cohorts 1 and 2. In the second protocol, hearts from cohort 3 were perfused with normal KH buffer, unpaced, and then subjected to KCl-arrest. Preliminary experiments showed that increasing the KCl concentration in the buffer to 20 mm was sufficient to arrest the mouse heart; higher concentrations injured the hearts. Isovolumic contractile performance (rate pressure product, RPP = 0 during KCl-arrest) and 31P-NMR spectroscopy (40 min at baseline and during arrest) were measured simultaneously throughout the protocol. At the end of each experiment, hearts were removed from the perfusion apparatus, blotted and weighed. Atria and ventricular weights, body weights and tibia lengths were determined. All results are expressed as means ± SEM. For each protocol, differences among groups were compared by one- or two-way factorial ANOVA followed by post hoc Bonferoni tests as appropriate, and changes between baseline and high workload were compared by repeated-measures. Linear relationships between RPP and |ΔG∼ATP| were fitted using the least squares method. Statistical analyses were performed with GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA), and differences were declared statistically significant at P < 0.05. Physical characteristics of the mice and their hearts are presented in Table 1. Briefly, body weights, heart weights and right ventricular weights were similar for the six groups; atrial weights were ∼2-fold higher in all transgenic mouse hearts while LV weights were ∼25% smaller for βR92Q and βR92L mice. For each protocol, we used littermate mouse hearts perfused in the Langendorff mode under identical conditions of perfusion pressure, temperature, buffer composition, pacing rate, initial end diastolic pressure and coronary flow rate, and the same stimuli to either increase or decrease workload. To increase workload abruptly and thereby assess contractile reserve, we used two stimuli: pacing to increase heart rate (HR) (from 360 to 420 beats min−1) without increasing developed pressure (DevP) and supplying the inotropic agent dobutamine, which increases both HR and DevP. To decrease workload, we increased the concentration of KCl in the buffer to 20 mm to arrest the mouse heart. By combining the non-invasive tool of 31P-NMR spectroscopy and the well-controlled isolated Langendorff-perfused mouse heart (Saupe et al. 1998), we made simultaneous measurements of systolic and diastolic isovolumic contractile performance and energetics at different levels of work for each heart. Indices of systolic contractile performance measured were left ventricle (LV) systolic pressure (LVSP), DevP (the difference between LVSP and EDP), the product of HR and DevP (RPP) and the rate of tension development (+dP/dt); indices of diastolic contractile performance measured were EDP and the rate of relaxation (−dP/dt). Indices of energetics determined were [ATP], [ADP], [AMP], [PCr], [Pi], pH and |ΔG∼ATP|. We used these results to make comparisons for three pairs of work states: (1) unpaced at baseline conditions and during perfusion with dobutamine, (2) paced during baseline conditions followed by perfusion (unpaced) with dobutamine and (3) unpaced at baseline followed by arrest. This approach allowed us to calculate the change in the free energy available from ATP hydrolysis caused by increasing or decreasing workload, and hence the energetic cost of tension development. It also allowed us to define indices of contractile performance and energetics at each workload for six unique genotypes. For the R92Q cohort, contractile performance results are shown in Figs 1 and 2; representative 31P-NMR spectra are shown in Fig. 3 and average metabolite results are shown in Figs 4 and 5. For the R92L cohort, contractile performance data are shown in Fig. 6; results from the 31P-NMR measurements are shown in Fig. 7. Indices of contractile performance averaged over 1 min intervals before, during (20 s intervals) and after supplying the inotrope dobutamine to a subset of isolated perfused αR92, βR92, αR92Q and βR92Q mouse hearts showing that substituting β-myosin heavy chain (MyHC) for α-MyHC substantially rescues the dysfunctional R92Q phenotype A, left ventricular systolic pressure (LVSP); B, LV end diastolic pressure (EDP); C, rate of LV tension development; (+dP/dt); D, rate of LV relaxation (−dP/dt); E, LV developed pressure (DevP); F, rate pressure product (RPP). Average heart rate was 410 beats min−1 at baseline and 560 beats min−1 with dobutamine. Data shown are means ± SEM, n = 4–5 per group. Select indices of contractile performance for isolated perfused αR92, βR92, αR92Q and βR92Q mouse hearts at baseline (paced, left) and high workload (right) in response to 300 nm dobutamine Increasing β-MyHC expression substantially reversed impaired contractile performance characteristics of intact hearts bearing the R92Q cTnT mutation. Data shown are individual values for n = 11–14 per group, and the horizontal lines are mean values. There were no sex differences in these parameters. A and B, left ventricular pressure, LVSP; C and D, the rate of LV tension development, +dP/dt; E and F, the rate of LV relaxation, −dP/dt. Representative 31P-NMR spectra from 5-month-old αR92, βR92, αR92Q and βR92Q mouse hearts, showing that increased β-MyHC expression rescued the energetic phenotype of αR92Q mouse hearts both at baseline (left) and high workload (right) in response to 300 nm dobutamine Resonance areas from left to right correspond to inorganic phosphate (Pi), phosphocreatine (PCr), and [γ-P]-, [α-P]- and [β-P]ATP. Resonance areas are scaled to the ATP resonance area at baseline for αR92 mouse hearts. See Methods for the parameters for data acquisition. Note the high Pi and the low PCr and ATP resonance areas for αR92Q hearts and the normal resonance areas for βR92Q. Select indices of whole heart energetics for αR92, βR92, αR92Q and βR92Q hearts at baseline (paced, left) and high workload (right) in response to 300 n m dobutamine; note the different scales for the y-axes for some parameters Increasing β-MyHC expression substantially reversed the impaired energetic phenotype of intact hearts bearing the R92Q cTnT mutation. Data shown are individual values for n = 11–14 per group, and the horizontal lines are mean values. There are no sex differences in these parameters. A and B, inorganic phosphate, Pi; C and D, phosphocreatine, PCr; E and F, ATP; G and H, ADP; I and J, AMP; K and L, free energy of ATP hydrolysis, |ΔG∼ATP|. *P < 0.05. Select indices of whole heart energetics for αR92, βR92, αR92Q and βR92Q hearts at baseline (left) and during KCl-arrest (right) Note the different scales for y-axes of most comparisons. Increasing β-MyHC expression substantially rescued the impaired energetic phenotype of intact hearts bearing the R92Q cTnT mutation. Data shown are individual values for n = 4–6 per group, and the horizontal lines are mean values. *P < 0.05. A and B, inorganic phosphate, Pi; C and D, phosphocreatine, PCr; E and F, ATP; G and H, ADP; I and J, AMP; K and L, free energy of ATP hydrolysis, |ΔG∼ATP|. Select indices of contractile performance for isolated perfused αR92, βR92, αR92L and βR92L mouse hearts at baseline (left) and high workload (right) in response to 300 nm dobutamine Increasing β-MyHC expression had no effect on impaired contractile performance characteristics of intact hearts bearing the R92L cTnT mutation. Data shown are individual values for n = 6–11 per group, and the horizontal lines are mean values. A and B, left ventricular pressure, LVSP; C and D, the rate of LV tension development, +dP/dt; E and F, the rate of LV relaxation, −dP/dt. Select indices of whole heart energetics for αR92, βR92, αR92L and βR92L hearts at baseline (paced, left) and high workload in response to 300 nm dobutamine Note the different scales for the y-axes for most comparisons. Increasing β-MyHC expression failed to rescue the impaired energetic phenotype of intact hearts bearing the R92L cTnT mutation. Data shown are individual values for n = 6–11 per group, and the horizontal lines are mean values. *P < 0.05. A and B, Pi; C and D, PCr; E and F, ATP; G and H, ADP; I and J, AMP; K and L, free energy of ATP hydrolysis, |ΔG∼ATP|. Previous studies by our group have found that switching the MyHC composition in wild-type C57BL/6 mouse hearts from 95/5%αα/ββ-MyHC to 20/80%αα/ββ-MyHC had no significant effect on baseline isovolumic contractile performance at either the whole heart or myocyte levels (Rice et al. 2010). Here we test whether replacing the endogenous fast murine MyHC with the slow murine MyHC in hearts with normal thin filament composition altered any energetic or contractile performance parameter at four work states: baseline without pacing, baseline with pacing, high work elicited by the inotrope dobutamine and during arrest. As each litter contained αR92 and βR92 cTnT mice, every litter yielded one or more of these comparisons. We describe in detail the expected values for indices of systolic and diastolic contraction and energy-metabolites at different work states for hearts with normal thick and thin filament proteins, αR92 hearts, and whether they differ from hearts with β-MyHC (βR92 hearts). These indices were then used to calculate two key parameters: RPP, the index of contractile performance that takes into account changes in HR, LVSP and EDP, and ΔG∼ATP, the free energy available from ATP hydrolysis. In subsequent sections describing the other genotypes studied, we follow this same approach. Baseline indices of cardiac performance for both αR92 and βR92 hearts were typical of isolated perfused mouse hearts: LVSP was >100 mmHg, the ratio of +dP/dt to −dP/dt was >1 and RPP was ∼41 × 103 mmHg min−1 (Figs 1, 2 and 6). Increasing HR by 17% (from 360 to 420 beats min−1) did not change DevP. αR92 and βR92 hearts had equally robust responses to inotropic challenge with dobutamine, increasing HR to >520 beats min−1 and DevP from ∼100 to ∼180 mmHg; EDP and the rates of tension development and relaxation doubled (Figs 1, 2 and 6). RPP, the index of contractile performance that takes into account changes in HR, LVSP and EDP, increased from ∼41 × 103 to ∼85 × 103 mmHg min−1. The minute-to-minute values for the systolic parameters LVSP, +dP/dt, DevP and RPP and for the diastolic parameters EDP and −dP/dt observed before, during and after dobutamine infusion shown for a subset of these hearts in Fig. 1 are essentially superimposable. αR92 and βR92 hearts also arrested at the same perfusate [KCl]. At each work state, αR92 and βR92 hearts had comparable values for [PCr], [ATP], [Pi] and pH measured by 31P-NMR spectroscopy and for [ADP] and [AMP] calculated using the creatine kinase and adenylate kinase equilibrium expressions (Figs 3, 4 and 7). αR92 and βR92 hearts at baseline had the expected ratio for PCr/ATP of ∼1.7 and normal [ATP] (∼10 mm), [ADP] (∼0.040 mm), [AMP] (∼0.15 μm), [Pi]" @default.
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• W1529573891 date "2012-10-04" @default.
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• W1529573891 title "Myosin-driven rescue of contractile reserve and energetics in mouse hearts bearing familial hypertrophic cardiomyopathy-associated mutant troponin T is mutation-specific" @default.
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• W1529573891 doi "https://doi.org/10.1113/jphysiol.2012.234252" @default.
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