The E22K mutation of myosin RLC that causes familial hypertrophic cardiomyopathy increases calcium sensitivity of force and ATPase in transgenic mice.
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Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease caused by mutations in all of the major sarcomeric proteins, including the ventricular myosin regulatory light-chain (RLC). The E22K-RLC mutation has been associated with a rare variant of cardiac hypertrophy defined by mid-left ventricular obstruction due to papillary muscle hypertrophy. This mutation was later found to cause ventricular and septal hypertrophy. We have generated transgenic (Tg) mouse lines of myc-WT (wild type) and myc-E22K mutant of human ventricular RLC and have examined the functional consequences of this FHC mutation in skinned cardiac-muscle preparations. In longitudinal sections of whole mouse hearts stained with hematoxylin and eosin, the E22K-mutant hearts of 13-month-old animals showed signs of inter-ventricular septal hypertrophy and enlarged papillary muscles with no filament disarray. Echo examination did not reveal evidence of cardiac hypertrophy in Tg-E22K mice compared to Tg-WT or Non-Tg hearts. Physiological studies utilizing skinned cardiac-muscle preparations showed an increase by DeltapCa50>or=0.1 in Ca(2+) sensitivity of myofibrillar ATPase activity and force development in Tg-E22K mice compared with Tg-WT or Non-Tg littermates. Our results suggest that E22K-linked FHC is mediated through Ca(2+)-dependent events. The FHC-mediated structural perturbations in RLC that affect Ca(2+) binding properties of the mutated myocardium are responsible for triggering the abnormal function of the heart that in turn might initiate a hypertrophic process and lead to heart failure. (+info)
F110I and R278C troponin T mutations that cause familial hypertrophic cardiomyopathy affect muscle contraction in transgenic mice and reconstituted human cardiac fibers.
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We have studied the physiological effects of the troponin T (TnT) F110I and R278C mutations associated with familial hypertrophic cardiomyopathy (FHC) in humans. Three to four-month-old transgenic (Tg) mice expressing F110I-TnT and R278C-TnT did not develop significant hypertrophy or ventricular fibrosis even after chronic exercise challenge. The F110I mutation impaired acute exercise tolerance, whereas R278C did not. Skinned papillary muscle fibers from transgenic mice expressing F110I-TnT demonstrated increased Ca(2+) sensitivity of force and ATPase activity, and likewise an increased Ca(2+) sensitivity of force was observed in F110I-TnT-reconstituted human cardiac muscle preparations. In contrast, no changes in force or the ATPase-pCa dependencies were observed in transgenic R278C fibers or in human fibers reconstituted with the R278C-TnT mutant. The maximal level of force development was dramatically decreased in both transgenic mice. However, the maximal ATPase was not different (R278C-TnT) or only slightly less (F110I-TnT) than that of non-Tg and WT-Tg littermates. Consequently, their ratios of ATPase/force (energy cost) at all Ca(2+) concentrations were dramatically higher compared with non-Tg and WT-Tg fibers. This increase in energy cost most likely results from a decrease in force per myosin cross-bridge, because forcing all cross-bridges into the force generating state by substitution of MgADP for MgATP in maximum contracting solutions resulted in the same increase in maximal force (15%) in all transgenic and non-transgenic preparations. The combination of increased Ca(2+) sensitivity and energy cost in the F110I hearts may be responsible for the greater severity of this phenotype compared with the R278C mutation. (+info)
Reversal of inappropriate peripheral vascular responses in hypertrophic cardiomyopathy.
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OBJECTIVES: We assessed the frequency of abnormal forearm vasodilator responses during lower body negative pressure (LBNP) in 21 non-obstructive hypertrophic cardiomyopathy (HCM) patients (31 +/- 8 [20 to 43] years) with abnormal blood pressure response (ABPR) to exercise and the effects of three drugs used to treat vasovagal syncope (propranolol, clonidine, and paroxetine) in a double-blind crossover study. BACKGROUND: Some HCM patients have an ABPR to exercise, which may be due to paradoxical peripheral vasodilatation. A similar proportion has paradoxical forearm vasodilatation during central volume unloading using LBNP. These abnormal reflexes may be caused by left ventricular mechanoreceptor activation. Similar mechanisms may also contribute to some cases of vasovagal syncope. METHODS: Blood pressure changes were assessed during exercise, and forearm vascular responses and baroreceptor sensitivity were assessed during LBNP using plethysmography. RESULTS: Nine (43%) patients (group A) had paradoxical vasodilator responses (forearm vascular resistance [FVR] fell by 7.5 +/- 4.6 U), and 12 (57%) patients (group B) had normal vasoconstrictor responses during LBNP (FVR increased by 7.7 +/- 4.9 U). Paroxetine augmented systolic blood pressure (SBP) during exercise in group A (21 +/- 6 mm Hg vs. 14 +/- 11 mm Hg at baseline, p = 0.02); no effect was detected in group B. Paroxetine reversed paradoxical vascular responses during LBNP in seven (78%) patients from group A. Propranolol and clonidine had no significant effect on SBP during exercise but reversed paradoxical vascular responses in some patients from group A (n = 5 and n = 3). CONCLUSIONS: Paradoxical vasodilatation during LBNP occurs in 40% of patients with ABPR during exercise and is reversed by propranolol, clonidine, and paroxetine. Paroxetine also improved SBP response to exercise. (+info)
Functional consequences of a mutation in an expressed human alpha-cardiac actin at a site implicated in familial hypertrophic cardiomyopathy.
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Point mutations in human alpha-cardiac actin cause familial hypertrophic cardiomyopathy. Functional characterization of these actin mutants has been limited by the lack of a high level expression system for human cardiac actin. Here, wild-type (WT) human alpha-cardiac actin and a mutant E99K actin have been expressed and purified from the baculovirus/insect cell expression system. Glu-99 in subdomain 1 of actin is thought to interact with a positively charged cluster located in the lower 50-kDa domain of the myosin motor domain. Actin-activated ATPase measurements using the expressed actins and beta-cardiac myosin showed that the mutation increased the K(m) for actin 4-fold (4.7 +/- 0.7 mum for WT versus 19.1 +/- 3.0 mum for the mutant), whereas the V(max) values were similar. The mutation slightly decreased the affinity of actin for S1 in the absence of nucleotide, which can partly be accounted for by a slower rate of association. The in vitro motility for the E99K mutant was consistently lower than WT over a range of ionic strengths, which is likely related to the lower average force supported by the mutant actin. The thermal stability of the E99K was comparable to that of WT-actin, implying no folding defects. The lower density of negative charge in subdomain 1 of actin therefore weakens the actomyosin interaction sufficiently to decrease the force and motion generating capacity of E99K actin, thus providing the primary insult that ultimately leads to the disease phenotype. (+info)
E22K mutation of RLC that causes familial hypertrophic cardiomyopathy in heterozygous mouse myocardium: effect on cross-bridge kinetics.
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Familial hypertrophic cardiomyopathy is a disease characterized by left ventricular and/or septal hypertrophy and myofibrillar disarray. It is caused by mutations in sarcomeric proteins, including the ventricular isoform of myosin regulatory light chain (RLC). The E22K mutation is located in the RLC Ca(2+)-binding site. We have studied transgenic (Tg) mouse cardiac myofibrils during single-turnover contraction to examine the influence of E22K mutation on 1) dissociation time (tau(1)) of myosin heads from thin filaments, 2) rebinding time (tau(2)) of the cross bridges to actin, and 3) dissociation time (tau(3)) of ADP from the active site of myosin. tau(1) was determined from the increase in the rate of rotation of actin monomer to which a cross bridge was bound. tau(2) was determined from the rate of anisotropy change of the recombinant essential light chain of myosin labeled with rhodamine exchanged for native light chain (LC1) in the cardiac myofibrils. tau(3) was determined from anisotropy of muscle preloaded with a stoichiometric amount of fluorescent ADP. Cross bridges were induced to undergo a single detachment-attachment cycle by a precise delivery of stoichiometric ATP from a caged precursor. The times were measured in Tg-mutated (Tg-m) heart myofibrils overexpressing the E22K mutation of human cardiac RLC. Tg wild-type (Tg-wt) and non-Tg muscles acted as controls. tau(1) was statistically greater in Tg-m than in controls. tau(2) was shorter in Tg-m than in non-Tg, but the same as in Tg-wt. tau(3) was the same in Tg-m and controls. To determine whether the difference in tau(1) was due to intrinsic difference in myosin, we estimated binding of Tg-m and Tg-wt myosin to fluorescently labeled actin by measuring fluorescent lifetime and time-resolved anisotropy. No difference in binding was observed. These results suggest that the E22K mutation has no effect on mechanical properties of cross bridges. The slight increase in tau(1) was probably caused by myofibrillar disarray. The decrease in tau(2) of Tg hearts was probably caused by replacement of the mouse RLC for the human isoform in the Tg mice. (+info)
Differential effect of troponin T mutations on the inotropic responsiveness of mouse hearts--role of myofilament Ca2+ sensitivity increase.
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Troponin T (TnT) mutations that cause familial hypertrophic cardiomyopathy (FHC) and sudden cardiac death frequently increase myofilament Ca2+ sensitivity, suggesting that their Ca2+-sensitizing effect contributes importantly to the FHC pathogenesis. To test this hypothesis, we compared transgenic mice expressing the Ca2+-sensitizing TnT-I79N mutant (I79N), which causes a high rate of sudden cardiac death in patients, with mice expressing the more benign TnT-R278C mutant (R278C) that does not affect myofilament Ca2+ sensitivity. Acutely increasing myofilament Ca2+ sensitivity with EMD57033 served as a positive control. Isovolumically contracting hearts were compared over a range of loading conditions (Frank-Starling curve). Consistent with their increased myofilament Ca2+ sensitivity, I79N-Tg hearts demonstrated significantly higher systolic performance at low perfusate [Ca2+] compared with R278C-Tg hearts, which were not statistically different from control hearts expressing either human wild-type TnT or no transgene (CON). Diastolic function was impaired in both FHC mutants (time to 90% relaxation: I79N 48 +/- 1.0 ms, n = 10 or R278C 47 +/- 0.4 ms, n = 7, versus CON 44 +/- 1.0 ms, n = 20, P < 0.05). In the presence of isoproterenol, almost all contractile parameters of R278C hearts became indistinguishable from control hearts, whereas both systolic and diastolic function of I79N hearts significantly worsened (end-diastolic pressure: I79N 20 +/- 4 mmHg versus CON 13 +/- 2 mmHg or R278C 11 +/- 2 mmHg, P < 0.05). The Ca2+ sensitizer EMD57033 produced an even greater contractile dysfunction than the I79N mutation at fast pacing rates. In vivo, maximal exercise tolerance was significantly impaired only in I79N mice. Pretreatment with beta-adrenergic receptor antagonists abolished differences in exercise tolerance. In conclusion, the Ca2+-sensitizing effects of TnT mutations may reduce the responsiveness of mouse hearts to inotropic stimuli. (+info)
Alterations of tension-dependent ATP utilization in a transgenic rat model of hypertrophic cardiomyopathy.
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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. (+info)
Microarray analysis of gene expression during early stages of mild and severe cardiac hypertrophy.
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Familial hypertrophic cardiomyopathy (FHC) is a disease characterized by ventricular hypertrophy, fibrosis, and aberrant systolic and/or diastolic function. We previously developed two transgenic mouse models that carry FHC-associated mutations in alpha-tropomyosin (TM): FHC alpha-TM175 mice show patchy areas of mild ventricular disorganization and limited hypertrophy, whereas FHC alpha-TM180 mice exhibit severe hypertrophy and fibrosis and die within 6 mo. To obtain a better understanding of the molecular mechanisms associated with the early onset of cardiac hypertrophy, we conducted a detailed comparative analysis of gene expression in 2.5-mo-old control, FHC alpha-TM175, and alpha-TM180 ventricular tissue. Results show that 754 genes (from a total of 22,600) were differentially expressed between the nontransgenic (NTG) and the FHC hearts. There are 178 differentially regulated genes between NTG and the FHC alpha-TM175 hearts, 388 genes are differentially expressed between NTG and FHC alpha-TM180 hearts, and 266 genes are differentially expressed between FHC alpha-TM175 and FHC alpha-TM180 hearts. Genes that exhibit the largest increase in expression belong to the "secreted/extracellular matrix" category, and those with the most significant decrease in expression are associated with "metabolic enzymes." Confirmation of the microarray analysis was conducted by quantitative real-time PCR on gene transcripts commonly associated with cardiac hypertrophy. (+info)