Aref Najafi

Perturbed Length-Dependent Activation in Human Hypertrophic Cardiomyopathy With Missense Sarcomeric Gene Mutations

Rationale: High-myofilament Ca2+ sensitivity has been proposed as a trigger of disease pathogenesis in familial hypertrophic cardiomyopathy (HCM) on the basis of in vitro and transgenic mice studies. However, myofilament Ca2+ sensitivity depends on protein phosphorylation and muscle length, and at present, data in humans are scarce. Objective: To investigate whether high myofilament Ca2+ sensitivity and perturbed length-dependent activation are characteristics for human HCM with mutations in thick and thin filament proteins. Methods and Results: Cardiac samples from patients with HCM harboring mutations in genes encoding thick (MYH7, MYBPC3) and thin (TNNT2, TNNI3, TPM1) filament proteins were compared with sarcomere mutation-negative HCM and nonfailing donors. Cardiomyocyte force measurements showed higher myofilament Ca2+ sensitivity in all HCM samples and low phosphorylation of protein kinase A (PKA) targets compared with donors. After exogenous PKA treatment, myofilament Ca2+ sensitivity was similar (MYBPC3mut, TPM1mut, sarcomere mutation-negative HCM), higher (MYH7mut, TNNT2mut), or even significantly lower (TNNI3mut) compared with donors. Length-dependent activation was significantly smaller in all HCM than in donor samples. PKA treatment increased phosphorylation of PKA-targets in HCM myocardium and normalized length-dependent activation to donor values in sarcomere mutation-negative HCM and HCM with truncating MYBPC3 mutations but not in HCM with missense mutations. Replacement of mutant by wild-type troponin in TNNT2mut and TNNI3mut corrected length-dependent activation to donor values. Conclusions: High-myofilament Ca2+ sensitivity is a common characteristic of human HCM and partly reflects hypophosphorylation of PKA targets compared with donors. Length-dependent sarcomere activation is perturbed by missense mutations, possibly via posttranslational modifications other than PKA hypophosphorylation or altered protein–protein interactions, and represents a common pathomechanism in HCM.

Contractile Dysfunction Irrespective of the Mutant Protein in Human Hypertrophic Cardiomyopathy With Normal Systolic Function

Background— Hypertrophic cardiomyopathy (HCM), typically characterized by asymmetrical left ventricular hypertrophy, frequently is caused by mutations in sarcomeric proteins. We studied if changes in sarcomeric properties in HCM depend on the underlying protein mutation. Methods and Results— Comparisons were made between cardiac samples from patients carrying a MYBPC3 mutation (MYBPC3mut; n=17), mutation negative HCM patients without an identified sarcomere mutation (HCMmn; n=11), and nonfailing donors (n=12). All patients had normal systolic function, but impaired diastolic function. Protein expression of myosin binding protein C (cMyBP-C) was significantly lower in MYBPC3mut by 33±5%, and similar in HCMmn compared with donor. cMyBP-C phosphorylation in MYBPC3mut was similar to donor, whereas it was significantly lower in HCMmn. Troponin I phosphorylation was lower in both patient groups compared with donor. Force measurements in single permeabilized cardiomyocytes demonstrated comparable sarcomeric dysfunction in both patient groups characterized by lower maximal force generating capacity in MYBPC3mut and HCMmn, compared with donor (26.4±2.9, 28.0±3.7, and 37.2±2.3 kN/m2, respectively), and higher myofilament Ca2+-sensitivity (EC50=2.5±0.2, 2.4±0.2, and 3.0±0.2 &mgr;mol/L, respectively). The sarcomere length-dependent increase in Ca2+-sensitivity was significantly smaller in both patient groups compared with donor (&Dgr;EC50: 0.46±0.04, 0.37±0.05, and 0.75±0.07 &mgr;mol/L, respectively). Protein kinase A treatment restored myofilament Ca2+-sensitivity and length-dependent activation in both patient groups to donor values. Conclusions— Changes in sarcomere function reflect the clinical HCM phenotype rather than the specific MYBPC3 mutation. Hypocontractile sarcomeres are a common deficit in human HCM with normal systolic left ventricular function and may contribute to HCM disease progression.

GSK3β phosphorylates newly identified site in the proline-alanine-rich region of cardiac myosin-binding protein C and alters cross-bridge cycling kinetics in human: short communication.

Rationale: Cardiac myosin–binding protein C (cMyBP-C) regulates cross-bridge cycling kinetics and, thereby, fine-tunes the rate of cardiac muscle contraction and relaxation. Its effects on cardiac kinetics are modified by phosphorylation. Three phosphorylation sites (Ser275, Ser284, and Ser304) have been identified in vivo, all located in the cardiac-specific M-domain of cMyBP-C. However, recent work has shown that up to 4 phosphate groups are present in human cMyBP-C. Objective: To identify and characterize additional phosphorylation sites in human cMyBP-C. Methods and Results: Cardiac MyBP-C was semipurified from human heart tissue. Tandem mass spectrometry analysis identified a novel phosphorylation site on serine 133 in the proline-alanine–rich linker sequence between the C0 and C1 domains of cMyBP-C. Unlike the known sites, Ser133 was not a target of protein kinase A. In silico kinase prediction revealed glycogen synthase kinase 3&bgr; (GSK3&bgr;) as the most likely kinase to phosphorylate Ser133. In vitro incubation of the C0C2 fragment of cMyBP-C with GSK3&bgr; showed phosphorylation on Ser133. In addition, GSK3&bgr; phosphorylated Ser304, although the degree of phosphorylation was less compared with protein kinase A–induced phosphorylation at Ser304. GSK3&bgr; treatment of single membrane–permeabilized human cardiomyocytes significantly enhanced the maximal rate of tension redevelopment. Conclusions: GSK3&bgr; phosphorylates cMyBP-C on a novel site, which is positioned in the proline-alanine–rich region and increases kinetics of force development, suggesting a noncanonical role for GSK3&bgr; at the sarcomere level. Phosphorylation of Ser133 in the linker domain of cMyBP-C may be a novel mechanism to regulate sarcomere kinetics.

GSK3β Phosphorylates Newly Identified Site in the Pro-Ala Rich Region of Cardiac Myosin Binding Protein C and Alters Cross-Bridge Cycling Kinetics in Human

Rationale: Cardiac myosin–binding protein C (cMyBP-C) regulates cross-bridge cycling kinetics and, thereby, fine-tunes the rate of cardiac muscle contraction and relaxation. Its effects on cardiac kinetics are modified by phosphorylation. Three phosphorylation sites (Ser275, Ser284, and Ser304) have been identified in vivo, all located in the cardiac-specific M-domain of cMyBP-C. However, recent work has shown that up to 4 phosphate groups are present in human cMyBP-C. Objective: To identify and characterize additional phosphorylation sites in human cMyBP-C. Methods and Results: Cardiac MyBP-C was semipurified from human heart tissue. Tandem mass spectrometry analysis identified a novel phosphorylation site on serine 133 in the proline-alanine–rich linker sequence between the C0 and C1 domains of cMyBP-C. Unlike the known sites, Ser133 was not a target of protein kinase A. In silico kinase prediction revealed glycogen synthase kinase 3&bgr; (GSK3&bgr;) as the most likely kinase to phosphorylate Ser133. In vitro incubation of the C0C2 fragment of cMyBP-C with GSK3&bgr; showed phosphorylation on Ser133. In addition, GSK3&bgr; phosphorylated Ser304, although the degree of phosphorylation was less compared with protein kinase A–induced phosphorylation at Ser304. GSK3&bgr; treatment of single membrane–permeabilized human cardiomyocytes significantly enhanced the maximal rate of tension redevelopment. Conclusions: GSK3&bgr; phosphorylates cMyBP-C on a novel site, which is positioned in the proline-alanine–rich region and increases kinetics of force development, suggesting a noncanonical role for GSK3&bgr; at the sarcomere level. Phosphorylation of Ser133 in the linker domain of cMyBP-C may be a novel mechanism to regulate sarcomere kinetics.

Prostanoids suppress the coronary vasoconstrictor influence of endothelin after myocardial infarction

Myocardial infarction (MI) is associated with endothelial dysfunction resulting in an imbalance in endothelium-derived vasodilators and vasoconstrictors. We have previously shown that despite increased endothelin (ET) plasma levels, the coronary vasoconstrictor effect of endogenous ET is abolished after MI. In normal swine, nitric oxide (NO) and prostanoids modulate the vasoconstrictor effect of ET. In light of the interaction among NO, prostanoids, and ET combined with endothelial dysfunction present after MI, we investigated this interaction in control of coronary vasomotor tone in the remote noninfarcted myocardium after MI. Studies were performed in chronically instrumented swine (18 normal swine; 13 swine with MI) at rest and during treadmill exercise. Furthermore, endothelial nitric oxide synthase (eNOS) and cyclooxygenase protein levels were measured in the anterior (noninfarcted) wall of six normal and six swine with MI. eNOS inhibition with N(ω)-nitro-L-arginine (L-NNA) and cyclooxygenase inhibition with indomethacin each resulted in coronary vasoconstriction at rest and during exercise, as evidenced by a decrease in coronary venous oxygen levels. The effect of l-NNA was slightly decreased in swine with MI, although eNOS expression was not altered. Conversely, in accordance with the unaltered expression of cyclooxygenase-1 after MI, the effect of indomethacin was similar in normal and MI swine. L-NNA enhanced the vasodilator effect of the ET(A/B) receptor blocker tezosentan but exclusively during exercise in both normal and MI swine. Interestingly, this effect of L-NNA was blunted in MI compared with normal swine. In contrast, whereas indomethacin increased the vasodilator effect of tezosentan only during exercise in normal swine, indomethacin unmasked a coronary vasodilator effect of tezosentan in MI swine both at rest and during exercise. In conclusion, the present study shows that endothelial control of the coronary vasculature is altered in post-MI remodeled myocardium. Thus the overall vasodilator influences of NO as well as its inhibition of the vasoconstrictor influence of ET on the coronary resistance vessels were reduced after MI. In contrast, while the overall prostanoid vasodilator influence was maintained, its inhibition of ET vasoconstrictor influences was enhanced in post-MI remote myocardium.

Synergistic role of ADP and Ca2+ in diastolic myocardial stiffness

Diastolic dysfunction in heart failure patients is evident from stiffening of the passive properties of the ventricular wall. Increased actomyosin interactions may significantly limit diastolic capacity, however, direct evidence is absent. From experiments at the cellular and whole organ level, in humans and rats, we show that actomyosin‐related force development contributes significantly to high diastolic stiffness in environments where high ADP and increased diastolic [Ca2+] are present, such as the failing myocardium. Our basal study provides a mechanical mechanism which may partly underlie diastolic dysfunction.

ADP-stimulated contraction: A predictor of thin-filament activation in cardiac disease

Significance Diastolic dysfunction is characteristic of patients with cardiomyopathy. Evidence indicates that diseased hearts show basal sarcomeric activation capable of impairing diastolic performance. By activating human cardiomyopathy muscle in ADP-containing solutions without Ca2+, we showed that actin–myosin blockade is disrupted. This may be caused by the presence of mutations and/or the reduced phosphorylation of myofilament proteins. Our mechanistic study supports the novel idea that protein kinase A-target phosphorylation and myosin-binding protein C regulate the OFF–ON transition of the thin filaments. ADP increased myofilament force and stiffness in the presence of Ca2+ in cardiomyopathy samples, suggesting this condition limits muscle relaxation through increased actin–myosin interactions. We conclude that ADP-stimulated contraction can be used to reveal conformational changes in the three-state model of thin-filament activation. Diastolic dysfunction is general to all idiopathic dilated (IDCM) and hypertrophic cardiomyopathy (HCM) patients. Relaxation deficits may result from increased actin–myosin formation during diastole due to altered tropomyosin position, which blocks myosin binding to actin in the absence of Ca2+. We investigated whether ADP-stimulated force development (without Ca2+) can be used to reveal changes in actin–myosin blockade in human cardiomyopathy cardiomyocytes. Cardiac samples from HCM patients, harboring thick-filament (MYH7mut, MYBPC3mut) and thin-filament (TNNT2mut, TNNI3mut) mutations, and IDCM were compared with sarcomere mutation-negative HCM (HCMsmn) and nonfailing donors. Myofilament ADP sensitivity was higher in IDCM and HCM compared with donors, whereas it was lower for MYBPC3. Increased ADP sensitivity in IDCM, HCMsmn, and MYH7mut was caused by low phosphorylation of myofilament proteins, as it was normalized to donors by protein kinase A (PKA) treatment. Troponin exchange experiments in a TNNT2mut sample corrected the abnormal actin–myosin blockade. In MYBPC3trunc samples, ADP sensitivity highly correlated with cardiac myosin-binding protein-C (cMyBP-C) protein level. Incubation of cardiomyocytes with cMyBP-C antibody against the actin-binding N-terminal region reduced ADP sensitivity, indicative of cMyBP-C’s role in actin–myosin regulation. In the presence of Ca2+, ADP increased myofilament force development and sarcomere stiffness. Enhanced sarcomere stiffness in sarcomere mutation-positive HCM samples was irrespective of the phosphorylation background. In conclusion, ADP-stimulated contraction can be used as a tool to study how protein phosphorylation and mutant proteins alter accessibility of myosin binding on actin. In the presence of Ca2+, pathologic [ADP] and low PKA-phosphorylation, high actin–myosin formation could contribute to the impaired myocardial relaxation observed in cardiomyopathies.

Synergistic role of ADP and Ca(2+) in diastolic myocardial stiffness.

Diastolic dysfunction in heart failure patients is evident from stiffening of the passive properties of the ventricular wall. Increased actomyosin interactions may significantly limit diastolic capacity, however, direct evidence is absent. From experiments at the cellular and whole organ level, in humans and rats, we show that actomyosin‐related force development contributes significantly to high diastolic stiffness in environments where high ADP and increased diastolic [Ca2+] are present, such as the failing myocardium. Our basal study provides a mechanical mechanism which may partly underlie diastolic dysfunction.

GSK3β Phosphorylates Newly Identified Site in the Proline-Alanine–Rich Region of Cardiac Myosin–Binding Protein C and Alters Cross-Bridge Cycling Kinetics in Human

Rationale: Cardiac myosin–binding protein C (cMyBP-C) regulates cross-bridge cycling kinetics and, thereby, fine-tunes the rate of cardiac muscle contraction and relaxation. Its effects on cardiac kinetics are modified by phosphorylation. Three phosphorylation sites (Ser275, Ser284, and Ser304) have been identified in vivo, all located in the cardiac-specific M-domain of cMyBP-C. However, recent work has shown that up to 4 phosphate groups are present in human cMyBP-C. Objective: To identify and characterize additional phosphorylation sites in human cMyBP-C. Methods and Results: Cardiac MyBP-C was semipurified from human heart tissue. Tandem mass spectrometry analysis identified a novel phosphorylation site on serine 133 in the proline-alanine–rich linker sequence between the C0 and C1 domains of cMyBP-C. Unlike the known sites, Ser133 was not a target of protein kinase A. In silico kinase prediction revealed glycogen synthase kinase 3&bgr; (GSK3&bgr;) as the most likely kinase to phosphorylate Ser133. In vitro incubation of the C0C2 fragment of cMyBP-C with GSK3&bgr; showed phosphorylation on Ser133. In addition, GSK3&bgr; phosphorylated Ser304, although the degree of phosphorylation was less compared with protein kinase A–induced phosphorylation at Ser304. GSK3&bgr; treatment of single membrane–permeabilized human cardiomyocytes significantly enhanced the maximal rate of tension redevelopment. Conclusions: GSK3&bgr; phosphorylates cMyBP-C on a novel site, which is positioned in the proline-alanine–rich region and increases kinetics of force development, suggesting a noncanonical role for GSK3&bgr; at the sarcomere level. Phosphorylation of Ser133 in the linker domain of cMyBP-C may be a novel mechanism to regulate sarcomere kinetics.

Synergistic role of ADP and Ca2+in diastolic myocardial stiffness: Cross-bridging the gap between energetics and Ca2+

Diastolic dysfunction in heart failure patients is evident from stiffening of the passive properties of the ventricular wall. Increased actomyosin interactions may significantly limit diastolic capacity, however, direct evidence is absent. From experiments at the cellular and whole organ level, in humans and rats, we show that actomyosin‐related force development contributes significantly to high diastolic stiffness in environments where high ADP and increased diastolic [Ca2+] are present, such as the failing myocardium. Our basal study provides a mechanical mechanism which may partly underlie diastolic dysfunction.