Alterations of tension-dependent ATP utilization in a transgenic rat model of hypertrophic cardiomyopathy

Although it is established that familial hypertrophic cardiomyopathy (FHC) is caused by mutations in several sarcomeric proteins, including cardiac troponin T (TnT), its pathogenesis is still not completely understood. Previously, we established a transgenic rat model of FHC expressing a human TnT molecule with a truncation mutation (DEL-TnT). This study investigated whether contractile dysfunction and electrical vulnerability observed in DEL-TnT rats might be due to alterations of intracellular Ca(2+) homeostasis, myofibrillar Ca(2+) sensitivity, and/or myofibrillar ATP utilization. Simultaneous measurements of the force of contraction and intracellular Ca(2+) transients were performed in right ventricular trabeculae of DEL-TnT hearts at 0.25 and 1.0 Hz. Rats expressing wild-type human TnT as well as nontransgenic rats served as controls. In addition, calcium-dependent ATPase activity and tension development were investigated in skinned cardiac muscle fibers. Force of contraction was significantly decreased in DEL-TnT compared with nontransgenic rats and TnT. Time parameters of Ca(2+) transients were unchanged at 0.25 Hz but prolonged at 1.0 Hz in DEL-TnT. The amplitude of the fura-2 transient was similar in all groups investigated, whereas diastolic and systolic fura-2 ratios were found elevated in rats expressing nontruncated human troponin T. In DEL-TnT rats, myofibrillar Ca(2+)-dependent tension development as well as Ca(2+) sensitivity of tension were significantly decreased, whereas tension-dependent ATP consumption ("tension cost") was markedly increased. Thus, a C-terminal truncation of the cardiac TnT molecule impairs the force-generating capacity of the cycling cross-bridges resulting in increased tension-dependent ATP utilization. Taken together, our data support the hypothesis of energy compromise as a contributing factor in the pathogenesis of FHC.     

Abnormal contractile function in transgenic mice expressing a familial hypertrophic cardiomyopathy-linked troponin T (I79N) mutation

This study characterizes a transgenic animal model for the troponin T (TnT) mutation (I79N) associated with familial hypertrophic cardiomyopathy. To study the functional consequences of this mutation, we examined a wild type and two I79N-transgenic mouse lines of human cardiac TnT driven by a murine alpha-myosin heavy chain promoter. Extensive characterization of the transgenic I79N lines compared with wild type and/or nontransgenic mice demonstrated: 1) normal survival and no cardiac hypertrophy even with chronic exercise; 2) large increases in Ca(2+) sensitivity of ATPase activity and force in skinned fibers; 3) a substantial increase in the rate of force activation and an increase in the rate of force relaxation; 4) lower maximal force/cross-sectional area and ATPase activity; 5) loss of sensitivity to pH-induced shifts in the Ca(2+) dependence of force; and 6) computer simulations that reproduced experimental observations and suggested that the I79N mutation decreases the apparent off rate of Ca(2+) from troponin C and increases cross-bridge detachment rate g. Simulations for intact living fibers predict a higher basal contractility, a faster rate of force development, slower relaxation, and increased resting tension in transgenic I79N myocardium compared with transgenic wild type. These mechanisms may contribute to mortality in humans, especially in stimulated contractile states.     

R-92L and R-92W mutations in cardiac troponin T lead to distinct energetic phenotypes in intact mouse hearts

It is now known that the flexibility of the troponin T (TnT) tail determines thin filament conformation and hence cross-bridge cycling properties, expanding the classic structural role of TnT to a dynamic role regulating sarcomere function. Here, using transgenic mice bearing R-92W and R-92L missense mutations in cardiac TnT known to alter the flexibility of the TnT tropomyosin-binding domain, we found mutation-specific differences in the cost of contraction at the whole heart level. Compared to age- and gender-matched sibling hearts, mutant hearts demonstrate greater ATP utilization measured using (31)P NMR spectroscopy as decreases in [ATP] and [PCr] and |DeltaG(~ATP)| at all workloads and profound systolic and diastolic dysfunction at all energetic states. R-92W hearts showed more severe energetic abnormalities and greater contractile dysfunction than R-92L hearts. The cost of increasing contraction was abnormally high when [Ca(2+)] was used to increase work in mutant hearts but was normalized with supply of the beta-adrenergic agonist dobutamine. These results show that R-92L and R-92W mutations in the TM-binding domain of cardiac TnT alter thin filament structure and flexibility sufficiently to cause severe defects in both whole heart energetics and contractile performance, and that the magnitude of these changes is mutation specific.     

Altered regulation of cardiac muscle contraction by troponin T mutations that cause familial hypertrophic cardiomyopathy

To study the effect of troponin (Tn) T mutations that cause familial hypertrophic cardiomyopathy (FHC) on cardiac muscle contraction, wild-type, and the following recombinant human cardiac TnT mutants were cloned and expressed: I79N, R92Q, F110I, E163K, R278C, and intron 16(G(1) --> A) (In16). These TnT FHC mutants were reconstituted into skinned cardiac muscle preparations and characterized for their effect on maximal steady state force activation, inhibition, and the Ca(2+) sensitivity of force development. Troponin complexes containing these mutants were tested for their ability to regulate actin-tropomyosin(Tm)-activated myosin-ATPase activity. TnT(R278C) and TnT(F110I) reconstituted preparations demonstrated dramatically increased Ca(2+) sensitivity of force development, while those with TnT(R92Q) and TnT(I79N) showed a moderate increase. The deletion mutant, TnT(In16), significantly decreased both the activation and the inhibition of force, and substantially decreased the activation and the inhibition of actin-Tm-activated myosin-ATPase activity. ATPase activation was also impaired by TnT(F110I), while its inhibition was reduced by TnT(R278C). The TnT(E163K) mutation had the smallest effect on the Ca(2+) sensitivity of force; however, it produced an elevated activation of the ATPase activity in reconstituted thin filaments. These observed changes in the Ca(2+) regulation of force development caused by these mutations would likely cause altered contractility and contribute to the development of FHC.     

Mutual Rescues between Two Dominant Negative Mutations in Cardiac Troponin I and Cardiac Troponin T

Troponin T (TnT) and troponin I (TnI) are two evolutionarily and functionally linked subunits of the troponin complex that regulates striated muscle contraction. We previously reported a single amino acid substitution in the highly conserved TnT-binding helix of cardiac TnI (cTnI) in wild turkey hearts in concurrence with an abnormally spliced myopathic cardiac TnT (cTnT) (Biesiadecki, B. J., Schneider, K. L., Yu, Z. B., Chong, S. M., and Jin, J. P. (2004) J. Biol. Chem. 279, 13825–13832). To investigate the functional effect of this cTnI mutation and its potential value in compensating for the cTnT abnormality, we developed transgenic mice expressing the mutant cTnI (K118C) in the heart with or without the deletion of the endogenous cTnI gene to mimic the homozygote and heterozygote of wild turkeys. Double and triple transgenic mice were created by crossing the cTnI-K118C lines with transgenic mice overexpressing the myopathic cTnT (exon 7 deletion). Functional studies of ex vivo working hearts found that cTnI-K118C alone had a dominantly negative effect on diastolic function and blunted the inotropic responses of cardiac muscle to β-adrenergic stimuli without abolishing the protein kinase A-dependent phosphorylation of cTnI. When co-expressed with the cTnT mutation, cTnI-K118C corrected the significant depression of systolic function caused by cTnT exon 7 deletion, and the co-existence of exon 7-deleted cTnT minimized the diastolic abnormality of cTnI-K118C. Characterization of this naturally selected pair of mutually rescuing mutations demonstrated that TnI-TnT interaction is a critical link in the Ca²⁺ signaling and β-adrenergic regulation in cardiac muscle, suggesting a potential target for the treatment of troponin cardiomyopathies and heart failure.     

Ouabain decreases sarco(endo)plasmic reticulum calcium ATPase activity in rat hearts by a process involving protein oxidation

The effect of cardiac glycosides to increase cardiac inotropy by altering Ca(2+) cycling is well known but still poorly understood. The studies described in this report focus on defining the effects of ouabain signaling on sarcoplasmic reticulum Ca(2+)-ATPase function. Rat cardiac myocytes treated with 50 microM ouabain demonstrated substantial increases in systolic and diastolic Ca(2+) concentrations. The recovery time constant for the Ca(2+) transient, tau(Ca(2+)), was significantly prolonged by ouabain. Exposure to 10 microM H(2)O(2), which causes an increase in intracellular reactive oxygen species similar to that of 50 microM ouabain, caused a similar increase in tau(Ca(2+)). Concurrent exposure to 10 mM N-acetylcysteine or an aqueous extract from green tea (50 mg/ml) both prevented the increases in tau(Ca(2+)) as well as the changes in systolic or diastolic Ca(2+) concentrations. We also observed that 50 microM ouabain induced increases in developed pressure in addition to diastolic dysfunction in the isolated perfused rat heart. Coadministration of ouabain with N-acetylcysteine prevented these increases. Analysis of sarcoplasmic reticulum Ca(2+)-ATPase protein revealed increases in both the oxidation and nitrotyrosine content in the ouabain-treated hearts. Liquid chromatography-mass spectrometric analysis confirmed that the sarcoplasmic reticulum Ca(2+)-ATPase protein from ouabain-treated hearts had modifications consistent with oxidative and nitrosative stress. These data suggest that ouabain induces oxidative changes of the sarcoplasmic reticulum Ca(2+)-ATPase structure and function that may, in turn, produce some of the associated changes in Ca(2+) cycling and physiological function.     

Atrial contractile dysfunction, fibrosis, and arrhythmias in a mouse model of cardiomyopathy secondary to cardiac-specific overexpression of tumor necrosis factor-{alpha}

Transgenic mice overexpressing the inflammatory cytokine TNF-alpha in the heart develop a progressive heart failure syndrome characterized by biventricular dilatation, decreased ejection fraction, decreased survival compared with non-transgenic littermates, and earlier pathology in males. TNF-alpha mice (TNF1.6) develop atrial arrhythmias on ambulatory telemetry monitoring that worsen with age and are more severe in males. We performed in vivo electrophysiological testing in transgenic and control mice, ex vivo optical mapping of voltage in the atria of isolated perfused TNF1.6 hearts, and in vitro studies on isolated atrial muscle and cells to study the mechanisms that lead to the spontaneous arrhythmias. Programmed stimulation induces atrial arrhythmias (n = 8/32) in TNF1.6 but not in control mice (n = 0/37), with a higher inducibility in males. In the isolated perfused hearts, programmed stimulation with single extra beats elicits reentrant atrial arrhythmias (n = 6/6) in TNF1.6 but not control hearts due to slow heterogeneous conduction of the premature beats. Lowering extracellular Ca(2+) normalizes conduction and prevents the arrhythmias. Atrial muscle and cells from TNF1.6 compared with control mice exhibit increased collagen deposition, decreased contractile function, and abnormal systolic and diastolic Ca(2+) handling. Thus abnormalities in action potential propagation and Ca(2+) handling contribute to the initiation of atrial arrhythmias in this mouse model of heart failure.     

Mechanisms of impaired calcium handling underlying subclinical diastolic dysfunction in diabetes

Isolated diastolic dysfunction is found in almost half of asymptomatic patients with well-controlled diabetes and may precede diastolic heart failure. However, mechanisms that underlie diastolic dysfunction during diabetes are not well understood. We tested the hypothesis that isolated diastolic dysfunction is associated with impaired myocardial Ca(2+) handling during type 1 diabetes. Streptozotocin-induced diabetic rats were compared with age-matched placebo-treated rats. Global left ventricular myocardial performance and systolic function were preserved in diabetic animals. Diabetes-induced diastolic dysfunction was evident on Doppler flow imaging, based on the altered patterns of mitral inflow and pulmonary venous flows. In isolated ventricular myocytes, diabetes resulted in significant prolongation of action potential duration compared with controls, with afterdepolarizations occurring in diabetic myocytes (P < 0.05). Sustained outward K(+) current and peak outward component of the inward rectifier were reduced in diabetic myocytes, while transient outward current was increased. There was no significant change in L-type Ca(2+) current; however, Ca(2+) transient amplitude was reduced and transient decay was prolonged by 38% in diabetic compared with control myocytes (P < 0.05). Sarcoplasmic reticulum Ca(2+) load (estimated by measuring the integral of caffeine-evoked Na(+)-Ca(2+) exchanger current and Ca(2+) transient amplitudes) was reduced by approximately 50% in diabetic myocytes (P < 0.05). In permeabilized myocytes, Ca(2+) spark amplitude and frequency were reduced by 34 and 20%, respectively, in diabetic compared with control myocytes (P < 0.05). Sarco(endo)plasmic reticulum Ca(2+)-ATPase-2a protein levels were decreased during diabetes. These data suggest that in vitro impairment of Ca(2+) reuptake during myocyte relaxation contributes to in vivo diastolic dysfunction, with preserved global systolic function, during diabetes.     

Proteolytic N-terminal truncation of cardiac troponin I enhances ventricular diastolic function

Besides the core structure conserved in all troponin I isoforms, cardiac troponin I (cTnI) has an N-terminal extension that contains phosphorylation sites for protein kinase A under beta-adrenergic regulation. A restricted cleavage of this N-terminal regulatory domain occurs in normal cardiac muscle and is up-regulated during hemodynamic adaptation (Z.-B. Yu, L.-F. Zhang, and J.-P. Jin (2001) J. Biol. Chem. 276, 15753-15760). In the present study, we developed transgenic mice overexpressing the N-terminal truncated cTnI (cTnI-ND) in the heart to examine its biochemical and physiological significance. Ca(2+)-activated actomyosin ATPase activity showed that cTnI-ND myofibrils had lower affinity for Ca(2+) than controls, similar to the effect of isoproterenol treatment. In vivo and isolated working heart experiments revealed that cTnI-ND hearts had a significantly faster rate of relaxation and lower left ventricular end diastolic pressure compared with controls. The higher baseline relaxation rate of cTnI-ND hearts was at a level similar to that of wild type mouse hearts under beta-adrenergic stimulation. The decrease in cardiac output due to lowered preload was significantly smaller for cTnI-ND hearts compared with controls. These findings indicate that removal of the N-terminal extension of cTnI via restricted proteolysis enhances cardiac function by increasing the rate of myocardial relaxation and lowering left ventricular end diastolic pressure to facilitate ventricular filling, thus resulting in better utilization of the Frank-Starling mechanism.     

Chronic coexistence of two troponin T isoforms in adult transgenic mouse cardiomyocytes decreased contractile kinetics and caused dilatative remodeling

Our previous in vivo and ex vivo studies suggested that coexistence of two or more troponin T (TnT) isoforms in adult cardiac muscle decreased cardiac function and efficiency (Huang QQ, Feng HZ, Liu J, Du J, Stull LB, Moravec CS, Huang X, Jin JP, Am J Physiol Cell Physiol 294: C213-C22, 2008; Feng HZ, Jin JP, Am J Physiol Heart Circ Physiol 299: H97-H105, 2010). Here we characterized Ca(2+)-regulated contractility of isolated adult cardiomyocytes from transgenic mice coexpressing a fast skeletal muscle TnT together with the endogenous cardiac TnT. Without the influence of extracellular matrix, coexistence of the two TnT isoforms resulted in lower shortening amplitude, slower shortening and relengthening velocities, and longer relengthening time. The level of resting cytosolic Ca(2+) was unchanged, but the peak Ca(2+) transient was lowered and the durations of Ca(2+) rising and decaying were longer in the transgenic mouse cardiomyocytes vs. the wild-type controls. Isoproterenol treatment diminished the differences in shortening amplitude and shortening and relengthening velocities, whereas the prolonged durations of relengthening and Ca(2+) transient in the transgenic cardiomyocytes remained. At rigor state, a result from depletion of Ca(2+), resting sarcomere length of the transgenic cardiomyocytes became shorter than that in wild-type cells. Inhibition of myosin motor diminished this effect of TnT function on cross bridges. The length but not width of transgenic cardiomyocytes was significantly increased compared with the wild-type controls, corresponding to longitudinal addition of sarcomeres and dilatative remodeling at the cellular level. These dominantly negative effects of normal fast TnT demonstrated that chronic coexistence of functionally distinct variants of TnT in adult cardiomyocytes reduces contractile performance with pathological consequences.     

Cardiac-specific overexpression of a superinhibitory pentameric phospholamban mutant enhances inhibition of cardiac function in vivo

Phospholamban is a regulator of the Ca(2+) affinity of the cardiac sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) and of cardiac contractility. In vitro expression studies have shown that several mutant phospholamban monomers are superinhibitory, suggesting that monomeric phospholamban is the active species. However, a phospholamban Asn(27) --> Ala (N27A) mutant, which maintained a normal pentamer to monomer ratio, was shown to act as a superinhibitor of SERCA2a Ca(2+) affinity. To determine whether the pentameric N27A mutant is superinhibitory in vivo, transgenic mice with cardiac-specific overexpression of mutant phospholamban were generated. Quantitative immunoblotting revealed a 61 +/- 6% increase in total phospholamban in mutant hearts, with 90% of the overexpressed protein being pentameric. The EC(50) value for Ca(2+) dependence of Ca(2+) uptake was 0.69 +/- 0.07 microM in mutant hearts, compared with 0.29 +/- 0.02 microM in wild-type hearts or 0. 43 +/- 0.03 microM in hearts overexpressing wild-type PLB by 2-fold. Myocytes from phospholamban N27A mutant hearts also exhibited more depressed contractile parameters than wild-type phospholamban overexpressing cells. The shortening fraction was 52%, rates of shortening and relengthening were 46% and 38% respectively, and time for 80% decay of the Ca(2+) signal was 146%, compared with wild-types (100%). Langendorff-perfused mutant hearts also demonstrated depressed contractile parameters. Furthermore, in vivo echocardiography showed a depression in the ratio of early to late diastolic transmitral velocity and a 79% prolongation of the isovolumic relaxation time. Isoproterenol stimulation did not fully relieve the depressed contractile parameters at the cellular, organ, and intact animal levels. Thus, pentameric phospholamban N27A mutant can act as a superinhibitor of the affinity of SERCA2a for Ca(2+) and of cardiac contractility in vivo.     

A troponin T mutation that causes infantile restrictive cardiomyopathy increases Ca2+ sensitivity of force development and impairs the inhibitory properties of troponin

Restrictive cardiomyopathy (RCM) is a rare disorder characterized by impaired ventricular filling with decreased diastolic volume. We are reporting the functional effects of the first cardiac troponin T (CTnT) mutation linked to infantile RCM resulting from a de novo deletion mutation of glutamic acid 96. The mutation was introduced into adult and fetal isoforms of human cardiac TnT (HCTnT3-DeltaE96 and HCTnT1-DeltaE106, respectively) and studied with either cardiac troponin I (CTnI) or slow skeletal troponin I (SSTnI). Skinned cardiac fiber measurements showed a large leftward shift in the Ca(2+) sensitivity of force development with no differences in the maximal force. HCTnT1-DeltaE106 showed a significant increase in the activation of actomyosin ATPase with either CTnI or SSTnI, whereas HCTnT3-DeltaE96 was only able to increase the ATPase activity with CTnI. Both mutants showed an impaired ability to inhibit the ATPase activity. The capacity of the CTnI.CTnC and SSTnI.CTnC complexes to fully relax the fibers after TnT displacement was also compromised. Experiments performed using fetal troponin isoforms showed a less severe impact compared with the adult isoforms, which is consistent with the cardioprotective role of SSTnI and the rapid onset of RCM after birth following the isoform switch. These data indicate that troponin mutations related to RCM may have specific functional phenotypes, including large leftward shifts in the Ca(2+) sensitivity and impaired abilities to inhibit ATPase and to relax skinned fibers. All of this would account for and contribute to the severe diastolic dysfunction seen in RCM.     

Characterization of troponin T dilated cardiomyopathy mutations in the fetal troponin isoform

The major goal of this study was to elucidate how troponin T (TnT) dilated cardiomyopathy (DCM) mutations in fetal TnT and fetal troponin affect the functional properties of the fetal heart that lead to infantile cardiomyopathy. The DCM mutations R141W and DeltaK210 were created in the TnT1 isoform, the primary isoform of cardiac TnT in the embryonic heart. In addition to a different TnT isoform, a different troponin I (TnI) isoform, slow skeletal TnI (ssTnI), is the dominant isoform in the embryonic heart. In skinned fiber studies, TnT1-wild-type (WT)-treated fibers reconstituted with cardiac TnI.troponin C (TnC) or ssTnI.TnC significantly increased Ca(2+) sensitivity of force development when compared with TnT3-WT-treated fibers at both pH 7.0 and pH 6.5. Porcine cardiac fibers treated with TnT1 that contained the DCM mutations (R141W and DeltaK210), when reconstituted with either cardiac TnI.TnC or ssTnI.TnC, significantly decreased Ca(2+) sensitivity of force development compared with TnT1-WT at both pH values. The R141W mutation, which showed no significant change in the Ca(2+) sensitivity of force development in the TnT3 isoform, caused a significant decrease in the TnT1 isoform. The DeltaK210 mutation caused a greater decrease in Ca(2+) sensitivity and maximal isometric force development compared with the R141W mutation in both the fetal and adult TnT isoforms. When complexed with cardiac TnI.TnC or ssTnI.TnC, both TnT1 DCM mutations strongly decreased maximal actomyosin ATPase activity as compared with TnT1-WT. Our results suggest that a decrease in maximal actomyosin ATPase activity in conjunction with decreased Ca(2+) sensitivity of force development may cause a severe DCM phenotype in infants with the mutations.     

Enhanced gene expression of Na(+)/Ca(2+) exchanger attenuates ischemic and hypoxic contractile dysfunction

Enhanced gene expression of the Na(+)/Ca(2+) exchanger in failing hearts may be a compensatory mechanism to promote influx and efflux of Ca(2+), despite impairment of the sarcoplasmic reticulum (SR). To explore this, we monitored intracellular calcium (Ca(i)(2+)) and cardiac function in mouse hearts engineered to overexpress the Na(+)/Ca(2+) exchanger and subjected to ischemia and hypoxia, conditions known to impair SR Ca(i)(2+) transport and contractility. Although baseline Ca(i)(2+) and function were similar between transgenic and wild-type hearts, significant differences were observed during ischemia and hypoxia. During early ischemia, Ca(i)(2+) was preserved in transgenic hearts but significantly altered in wild-type hearts. Transgenic hearts maintained 40% of pressure-generating capacity during early ischemia, whereas wild-type hearts maintained only 25% (P < 0.01). During hypoxia, neither peak nor diastolic Ca(i)(2+) decreased in transgenic hearts. In contrast, both peak and diastolic Ca(i)(2+) decreased significantly in wild-type hearts. The decline of Ca(i)(2+) was abbreviated in hypoxic transgenic hearts but prolonged in wild-type hearts. Peak systolic pressure decreased by nearly 10% in hypoxic transgenic hearts and >25% in wild-type hearts (P < 0.001). These data demonstrate that enhanced gene expression of the Na(+)/Ca(2+) exchanger preserves Ca(i)(2+) homeostasis during ischemia and hypoxia, thereby preserving cardiac function in the acutely failing heart.     

Altered SR protein expression associated with contractile dysfunction in diabetic rat hearts

The goal of this study was to examine whether alteration of sarcoplasmic reticulum (SR) protein levels is associated with early-onset diastolic and late-onset systolic dysfunction in streptozotocin (STZ)-induced diabetic rat hearts. Four-week diabetic rat hearts exhibited slow relaxation, whereas 6-wk diabetic rat hearts exhibited slow and depressed contraction. Total phospholamban level was increased, and phosphorylated level was decreased in 4- and 6-wk diabetic rat hearts. Sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) protein level was unchanged in 4-wk but decreased in 6-wk diabetic rat hearts. Only the apparent affinity of SR Ca2+ uptake for Ca2+ was decreased in 4-wk diabetic rat hearts, but the apparent affinity and the maximum rate was decreased in 6-wk diabetic rat hearts. Insulin treatment of the diabetic rats normalized SR protein expression and function. It was concluded that an increase in nonphosphorylated phospholamban and a decrease in the apparent affinity of SR Ca2+ pump for Ca2+ are associated with early-onset diastolic dysfunction and decreases in SERCA2 protein level and apparent affinity and maximum velocity of SR Ca2+ pump are associated with late-onset systolic dysfunction in diabetic rats.     

Expression of cardiac troponin T with COOH-terminal truncation accelerates cross-bridge interaction kinetics in mouse myocardium

Transgenic mice expressing an allele of cardiac troponin T (cTnT) with a COOH-terminal truncation (cTnT(trunc)) exhibit severe diastolic and mild systolic dysfunction. We tested the hypothesis that contractile dysfunction in myocardium expressing low levels of cTnT(trunc) (i.e., <5%) is due to slowed cross-bridge kinetics and reduced thin filament activation as a consequence of reduced cross-bridge binding. We measured the Ca(2+) sensitivity of force development [pCa for half-maximal tension generation (pCa(50))] and the rate constant of force redevelopment (k(tr)) in cTnT(trunc) and wild-type (WT) skinned myocardium both in the absence and in the presence of a strong-binding, non-force-generating derivative of myosin subfragment-1 (NEM-S1). Compared with WT mice, cTnT(trunc) mice exhibited greater pCa(50), reduced steepness of the force-pCa relationship [Hill coefficient (n(H))], and faster k(tr) at submaximal Ca(2+) concentration ([Ca(2+)]), i.e., reduced activation dependence of k(tr). Treatment with NEM-S1 elicited similar increases in pCa(50) and similar reductions in n(H) in WT and cTnT(trunc) myocardium but elicited greater increases in k(tr) at submaximal activation in cTnT(trunc) myocardium. Contrary to our initial hypothesis, cTnT(trunc) appears to enhance thin filament activation in myocardium, which is manifested as significant increases in Ca(2+)-activated force and the rate of cross-bridge attachment at submaximal [Ca(2+)]. Although these mechanisms would not be expected to depress systolic function per se in cTnT(trunc) hearts, they would account for slowed rates of myocardial relaxation during early diastole.     

Functional consequences of troponin T mutations found in hypertrophic cardiomyopathy.

Missense mutations in the cardiac thin filament protein troponin T (TnT) are a cause of familial hypertrophic cardiomyopathy (FHC). To understand how these mutations produce dysfunction, five TnTs were produced and purified containing FHC mutations found in several regions of TnT. Functional defects were diverse. Mutations F110I, E244D, and COOH-terminal truncation weakened the affinity of troponin for the thin filament. Mutation DeltaE160 resulted in thin filaments with increased calcium affinity at the regulatory site of troponin C. Mutations R92Q and F110I resulted in impaired troponin solubility, suggesting abnormal protein folding. Depending upon the mutation, the in vitro unloaded actin-myosin sliding speed showed small increases, showed small decreases, or was unchanged. COOH-terminal truncation mutation resulted in a decreased thin filament-myosin subfragment 1 MgATPase rate. The results indicate that the mutations cause diverse immediate effects, despite similarities in disease manifestations. Separable but repeatedly observed abnormalities resulting from FHC TnT mutations include increased unloaded sliding speed, increased or decreased Ca(2+) affinity, impairment of folding or sarcomeric integrity, and decreased force. Enhancement as well as impairment of contractile protein function is observed, suggesting that TnT, including the troponin tail region, modulates the regulation of cardiac contraction.     

Kinin B1 receptor participates in the control of cardiac function in mice

The kinins have an important role in control of the cardiovascular system. They have been associated with protective effects in the heart tissue. Kinins act through stimulation of two 7-transmembrane G protein-coupled receptors, denoted B(1) and B(2) receptors. However, the physiological relevance of B(1) receptor in the heart has not been clearly established. Using B(1) kinin receptor gene knock-out mice we tested the hypothesis that the B(1) receptor plays an important role in the control of baseline cardiac function. We examined the functional aspects of the intact heart and also in the isolated cardiomyocytes to study intracellular Ca(2+) cycling by using confocal microscopy and whole-cell voltage clamp techniques. We measured heart rate, diastolic and systolic tension, contraction and relaxation rates and, coronary perfusion pressure. Whole-cell voltage clamp was performed to measure L-type Ca(2+) current (I(Ca,L)). The hearts from B(1)(-/-) mice showed smaller systolic tension. The average values for WT and B(1)(-/-) mice were 2.6+/-0.04 g vs. 1.6+/-0.08 g, respectively. This result can be explained, at least in part, by the decrease in the Ca(2+) transient (3.1+/-0.06 vs. 3.4+/-0.09 for B(1)(-/-) and WT, respectively). There was an increase in I(Ca,L) at depolarized membrane potentials. Interestingly, the inactivation kinetics of I(Ca,L) was statistically different between the groups. The coronary perfusion pressure was higher in the hearts from B(1)(-/-) mice indicating an increase in coronary resistance. This result can be explained by the significant reduction of eNOS (NOS-3) expression in the aorta of B(1)(-/-) mice. Collectively, our results demonstrate that B(1) receptor exerts a fundamental role in the mammalian cardiac function.     

Co-expression of skeletal and cardiac troponin T decreases mouse cardiac function

In contrast to skeletal muscles that simultaneously express multiple troponin T (TnT) isoforms, normal adult human cardiac muscle contains a single isoform of cardiac TnT. To understand the significance of myocardial TnT homogeneity, we examined the effect of TnT heterogeneity on heart function. Transgenic mouse hearts overexpressing a fast skeletal muscle TnT together with the endogenous cardiac TnT was investigated in vivo and ex vivo as an experimental system of concurrent presence of two classes of TnT in the adult cardiac muscle. This model of myocardial TnT heterogeneity produced pathogenic phenotypes: echocardiograph imaging detected age-progressive reductions of cardiac function; in vivo left ventricular pressure analysis showed decreased myocardial contractility; ex vivo analysis of isolated working heart preparations confirmed an intrinsic decrease of cardiac function in the absence of neurohumoral influence. The transgenic mice also showed chronic myocardial hypertrophy and degeneration. The dominantly negative effects of introducing a fast TnT into the cardiac thin filaments to produce two classes of Ca(2+) regulatory units in the adult myocardium suggest that TnT heterogeneity decreases contractile function by disrupting the synchronized action during ventricular contraction that is normally activated as an electrophysiological syncytium.     

Cardiac disease in mucopolysaccharidosis type I attributed to catecholaminergic and hemodynamic deficiencies

Cardiac dysfunction is a common cause of death among pediatric patients with mutations in the lysosomal hydrolase α-l-iduronidase (IDUA) gene, which causes mucopolysaccharidosis type I (MPS-I). The purpose of this study was to analyze adrenergic regulation of cardiac hemodynamic function in MPS-I. An analysis of murine heart function was performed using conductance micromanometry to assess in vivo cardiac hemodynamics. Although MPS-I (IDUA(-/-)) mice were able to maintain normal cardiac output and ejection fraction at baseline, this cohort had significantly compromised systolic and diastolic function compared with IDUA(+/-) control mice. During dobutamine infusion MPS-I mice did not significantly increase cardiac output from baseline, indicative of blunted cardiac reserve. Autonomic tone, measured functionally by β-blockade, indicated that MPS-I mice required catecholaminergic stimulation to maintain baseline hemodynamics. Survival analysis showed mortality only among MPS-I mice. Linear regression analysis revealed that heightened end-systolic volume in the resting heart is significantly correlated with susceptibility to mortality in MPS-I hearts. This study reveals that cardiac remodeling in the pathology of MPS-I involves heightened adrenergic tone at the expense of cardiac reserve with cardiac decompensation predicted on the basis of increased baseline systolic volumes.