A stacking sodium dodecyl sulfate polyacrylamide gel electrophoresis system has been used to resolve and quantify all the major myofibrillar protein components (actin, myosin, tropomyosin, and troponin C, T, and I). Quantification was achieved by densitometry of the fast green-stained gels calibrated with the use of purified proteins. The approximate molar ratios of these proteins in rabbit muscle are: actin : myosin: tropomyosin: troponin T: troponin I: troponin C = 7:1:1:1:1:1. On the basis of these results and available structural information one obtains an estimate of 254 myosin molecules per thick filament. (c) 2022 Published by Elsevier Inc.

This article was withdrawn by the authors before final publication on April 22, 2008.

The potential alterations to structure and associations with thin filament proteins caused by the dilated cardiomyopathy (DCM) associated tropomyosin (Tm) mutants E40K and E54K, and the hypertrophic cardiomyopathy (HCM) associated Tm mutants E62Q and L185R, were investigated. In order to ascertain what the cause of the known functional effects may be, structural and protein-protein interaction studies were conducted utilizing actomyosin ATPase activity measurements and spectroscopy. In actomyosin ATPase measurements, both HCM mutants and the DCM mutant E54K caused increases in Ca2+-induced maximal ATPase activities, while E40K caused a decrease. Investigation of Tm's ability to inhibit actomyosin ATPase in the absence of troponin showed that HCM-associated mutant Tms did not inhibit as well as wildtype, whereas the DCM associated mutant E40K inhibited better. E54K did not inhibit the actomyosin ATPase activity at any concentration of Tm tested. Thermal denaturation studies by circular dichroism and molecular modeling of the mutations in Tm showed that in general, the DCM mutants caused localized destabilization of the Tm dimers, while the HCM mutants resulted in increased stability. These findings demonstrate that the structural alterations in Tm observed here may affect the regulatory function of Tm on actin, thereby directly altering the ATPase rates of myosin.

Little information exists concerning the functional roles of the human slow skeletal troponin T isoforms (HSSTnT isoforms) in slow skeletal muscle. Three HSSTnT isoforms have been found in human slow skeletal muscle: HSSTnT1 (+ exons 5 and 12), HSSTnT2 (+5, ‐12), and HSSTnT3 (‐5, ‐12). Another potential isoform HSSTnT‐Hyp (‐5, +12) was recently found at the mRNA level. The objective of this study was to determine the physiological role of these SSTnT isoforms in slow skeletal muscle. To investigate these SSTnT isoforms several methods including skinned fiber mechanics, peptide spot blot, and mammalian two‐hybrid assays were utilized. Skinned rabbit slow soleus muscle fibers were displaced with HSSTnT1, 2, 3 or Hyp and reconstituted with the human slow skeletal troponin I (HSSTnI)/human cardiac troponin C (HCTnC) complex. The calcium sensitivity increased between SSTnT isoforms: isoform 1 (pCa50 = 5.74) < Hyp isoform (pCa50 = 5.80) < isoform 2 (pCa50 = 5.81) < isoform 3 (pCa50 = 5.84). In a reconstituted skeletal muscle system, the HSSTnT 1‐3 isoforms had similar actomyosin ATPase activation or inhibition in the presence or absence of calcium. Peptide spot blot analysis revealed novel interactions between HSSTnT peptides and HCTnC, RSTm and HCTnI. Mammalian two‐hybrid studies showed that the alternatively spliced regions affect the interaction of HSSTnT with other thin filament components. These results suggest that the functional differences that occur between HSSTnT isoforms may be partially due to alternative splicing of exon 12. Overall, HSSTnT isoforms were found to have distinct functional properties in slow skeletal muscle regulation and are likely to be relevant for the understanding of skeletal muscle diseases.Grant Funding Source: UC Davis research funds and NIH

Dr. John Gergely passed away on July 26, 2013 after a long and distinguished career. His publications spanned 67 years. He founded the Department of Muscle Research in the Retina Foundation (which later became the Boston Biomedical Research Institute) and served as director for 34 years. Dr. Gergely served on the editorial boards of ten scientific journals. He was elected as a Fellow of both the Biophysical Society and the American Association for the Advancement of Science. Dr. Gergely made major contributions concerning muscle protein structure and function. He was best known for his work on the troponin complex. The insights of John and his associates have provided the foundation for our understanding of calcium regulation in skeletal and cardiac muscle.

EF-hand proteins are ubiquitous in cell signaling. Parvalbumin (Parv), the archetypal EF-hand protein, is a high-affinity Ca2+ buffer in many biological systems. Given the centrality of Ca2+ signaling in health and disease, EF-hand motifs designed to have new biological activities may have widespread utility. Here, an EF-hand motif substitution that had been presumed to destroy EF-hand function, that of glutamine for glutamate at position 12 of the second cation binding loop domain of Parv (ParvE101Q), markedly inverted relative cation affinities: Mg2+ affinity increased, whereas Ca2+ affinity decreased, forming a new ultra-delayed Ca2+ buffer with favorable properties for promoting cardiac relaxation. In therapeutic testing, expression of ParvE101Q fully reversed the severe myocyte intrinsic contractile defect inherent to expression of native Parv and corrected abnormal myocardial relaxation in diastolic dysfunction disease models in vitro and in vivo. Strategic design of new EF-hand motif domains to modulate intracellular Ca2+ signaling could benefit many biological systems with abnormal Ca2+ handling, including the diseased heart.

Two troponin T mutations (I79N and ΔE96), which cause hypertrophic cardiomyopathy and restrictive cardiomyopathy respectively, were examined using the thin-filament extraction and reconstitution technique. Effects of Ca2+, ATP, phosphate and ADP concentrations on force and its transients were studied at 25°C and compared to WT. I79N and ΔE96 showed no significant change in maximum tension generated compared to WT, but both of them showed significantly increased Ca2+ sensitivity (pCa50: 5.65±0.04 and 5.62±0.04, respectively) than WT (5.44±0.04). The cooperativity increased in I79N (2.53±0.18) but remained the same in ΔE96 (1.75±0.19) compared to WT (1.71±0.18). Both I79N and ΔE96 showed lowered low-Ca (pCa 8.0) stiffness (YLC, 14.52±2.67 and 23.93±2.73) than WT (30.21±4.36). High-Ca (pCa 4.66) stiffness remained similar among all three Tns. However, The Ca2+ activatable stiffness of I79N and ΔE96 (Yact=YHC-YLC, 55.43±4.92 and 48.49±6.91) was significantly higher than WT (Yact=33.54±4.22) (±sem, N=9-20 for all studies). The All tensions were normalized to Ta, the tension of actin-filament reconstituted myocardium at the standard activating condition. Five equilibrium constants were deduced using sinusoidal analysis. I79N and ΔE96 showed significantly decreased K0 (ADP dissociation constant, 30.20±2.43 mM−1 and 34.61±3.89 mM−1, respectively) than WT (46.8±1.24 mM−1). I79N showed significantly lower K2 (cross-bridge detachment step: 0.80±0.11) than ΔE96 (1.74±0.24) and WT (1.41±0.19). I79N and ΔE96 also showed significantly higher K4 (force generation step: 0.70±0.06 and 0.28±0.01, respectively) than WT (0.21±0.03). The cross-bridge distribution of the mutants and WT was calculated from the equilibrium constants. I79N showed ∼33% more force generating cross-bridges than WT, but ΔE96 remained similar to WT. These results indicate that, under pCa4.66, force/cross-bridge is ∼25% less in I79N than WT, but remains similar in ΔE96.

The cardiac TnT deletion (HcTnT-ΔE96) linked to Restrictive Cardiomyopathy (RCM) dramatically increases the Ca2+ sensitivity of force development in skinned fibers (J. Biol. Chem. 283(4):2156–66). This study explores whether HcTnT-ΔE96 interferes with convergent regulation by Protein Kinase A (PKA) which decreases myofilament Ca2+ sensitivity. Native cTnT of porcine cardiac skinned fibers was displaced by HcTnT-WT or HcTnT-ΔE96 and reconstituted with binary complex containing pseudo non-phosphorylatable cTnI (cTnI-SS/AA.cTnC) or pseudo-phosphorylated (cTnI-SS/DD.cTnC). For comparison, skinned fibers were also reconstituted with binary cTnI.cTnC phosphorylated by PKA in vitro (cTnI-2P.cTnC) to assess effects of HcTnT-ΔE96 on Ca2+ sensitivity of force development. As expected, fibers displaced with HcTnT-WT reconstituted with cTnI-SS/DD.cTnC decreased Ca2+ sensitivity of force compared to control reconstituted with cTnI-SS/AA.cTnC. In contrast, fibers displaced with HCTnT-ΔE96 and reconstituted with cTnI-SS/DD.cTnC or cTnI-SS/AA.cTnC had similar Ca2+ sensitivities. When fibers were displaced with HcTnT3-WT or HcTnT3-ΔE96 and reconstituted with cTnI-2P.cTnC, the expected decrease in Ca2+ sensitivity was observed for both. These results suggest that phosphorylation mimetic cTnI-SS/DD may not always mimic phosphorylation by PKA. Furthermore, the mutant HcTnT-ΔE96 protein revealed reduced α-helical content by CD spectroscopy when compared to WT. Thermo-denaturation studies indicated that the mutant protein unfolded earlier than WT and was partially unfolded at physiological temperatures. Fluorescence studies containing CTnC-IAANS demonstrated that a high order of complexity (thin filament + myosin S1) is needed to better approach changes in Ca2+ affinity produced by the RCM cTnT mutation in skinned fibers. The effects of the HcTnT mutation on Ca2+ sensitivity properties were likely due to altered interactions of TnT with other thin filament proteins. Supported AHA 09POST2300030 (MSP), NIH R01-HL42325 (JDP) and J&E King 1KN13-34001 (JRP).

Human slow skeletal troponin T (HSSTnT) shares a high degree of homology with cardiac TnT (CTnT). Although the presence of HSSTnT has not been confirmed in the heart at the protein level, detectable levels of HSSTnT mRNA have been found. Whether HSSTnT isoforms are expressed transiently remains unknown. Because transient re-expression of HSSTnT may be a potential mechanism of regulating function, we explored the effect of HSSTnT on the regulation of cardiac muscle. At least three HSSTnT isoforms have been found to exist in slow skeletal muscle: HSSTnT1 (+exons 5 and 12), HSSTnT2 (+exon 5, −exon 12), and HSSTnT3 (−exons 5 and 12). Another isoform, HSSTnT hypothetical (Hyp) (−exon 5, +exon 12), has only been found at the mRNA level. Compared with HCTnT3 (adult isoform), Tn complexes containing HSSTnT1, -2, and -3 did not alter the actomyosin ATPase activation and inhibition in the presence and absence of Ca2+, respectively. HSSTnTHyp was not evaluated as it did not form a Tn complex under a variety of conditions. Porcine papillary skinned fibers displaced with HSSTnT1, -2, or -3 and reconstituted with human cardiac troponin I and troponin C (HCTnI·TnC) complex showed a decrease in the Ca2+ sensitivity of force development and an increase in maximal recovered force (HSSTnT1 and -3) compared with HCTnT3. In contrast, HSSTnTHyp showed an increase in the Ca2+ sensitivity of force development. This suggests that re- or overexpression of specific SSTnT isoforms might have therapeutic potential in the failing heart because they increase the maximal force of contraction. In addition, circular dichroism and proteolytic digestion experiments revealed structural differences between HSSTnT isoforms and HCTnT3 and that HSSTnT1 is more susceptible to calpain and trypsin proteolysis than the other HSSTnTs. Overall, HSSTnT isoforms despite being homologues of CTnT may display distinct functional properties in muscle regulation.

Previously, we reported the generation and characterization of an R21C cardiac troponin I (cTnI) knock-in (KI) mouse model linked to hypertrophic cardiomyopathy in humans (Biophys J Vol 98, Issue 3, Suppl 1, 148a). The R21C+/+ (homozygote) mice displayed decreased cTnI phosphorylation at serines23/24, decreased Ca2+ sensitivity response (rightward shift) of the myofilament upon PKA incubation and marked late stage heart hypertrophy (≥ 12 months of age). Here, we further investigated effects of the cTnI R21C mutation on intracellular Ca2+ ([Ca2+]i) and contractility in R21C+/+ cardiomyocytes before and after 100 nmol/L isoproterenol (ISO) treatment. Incubation with ISO slightly reduced diastolic [Ca2+]i in R21C+/+ cardiomyocytes, consistent with increased resting sarcomere length (SL) (relaxation), however diastolic [Ca2+]i increased in WT cardiomyocytes without altering resting SL. Despite similar SL shortening between both strains at baseline or after ISO, WT had a strong response to ISO with increased ΔCa2+ amplitude (systolic [Ca2+]i - diastolic [Ca2+]i) but no significant increase in R21C+/+ throughout a range of frequencies was observed. Ca2+ decay was similar between strains before or after ISO, however SL relaxation in R21C+/+ was slower but with stronger response to ISO compared to WT. Furthermore, the rate of relaxation of R21C+/+ skinned fibers was measured by flashphotolysis prior to and after PKA incubation. Data was fit with a double exponential and major changes were identified in the first rate constant (k1). After PKA incubation, k1 in WT skinned fibers significantly increased (19.02/sec to 28.49/sec). In comparison, k1 in R21C+/+ skinned fibers increased from (22.66/sec to 26.40/sec) before and after PKA treatment. These data taken together demonstrates that the HCM R21C-cTnI mutation increases intracellular calcium buffering and delays sarcomere relaxation without changing Ca2+ decay. Supported by NIH R01-HL432325 (JDP).

Defined as clinically unexplained hypertrophy of the left ventricle, hypertrophic cardiomyopathy (HCM) is traditionally understood as a disease of the cardiac sarcomere. Mutations in TNNC1-encoded cardiac troponin C (cTnC) are a relatively rare cause of HCM. Here, we report clinical and functional characterization of a novel TNNC1 mutation, A31S, identified in a pediatric HCM proband with multiple episodes of ventricular fibrillation and aborted sudden cardiac death. Diagnosed at age 5, the proband is family history-negative for HCM or sudden cardiac death, suggesting a de novo mutation. TnC-extracted cardiac skinned fibers were reconstituted with the cTnC-A31S mutant, which increased Ca(2+) sensitivity with no effect on the maximal contractile force generation. Reconstituted actomyosin ATPase assays with 50% cTnC-A31S:50% cTnC-WT demonstrated Ca(2+) sensitivity that was intermediate between 100% cTnC-A31S and 100% cTnC-WT, whereas the mutant increased the activation of the actomyosin ATPase without affecting the inhibitory qualities of the ATPase. The secondary structure of the cTnC mutant was evaluated by circular dichroism, which did not indicate global changes in structure. Fluorescence studies demonstrated increased Ca(2+) affinity in isolated cTnC, the troponin complex, thin filament, and to a lesser degree, thin filament with myosin subfragment 1. These results suggest that this mutation has a direct effect on the Ca(2+) sensitivity of the myofilament, which may alter Ca(2+) handling and contribute to the arrhythmogenesis observed in the proband. In summary, we report a novel mutation in the TNNC1 gene that is associated with HCM pathogenesis and may predispose to the pathogenesis of a fatal arrhythmogenic subtype of HCM.

The cardiac troponin C (cTnC) subunit of the troponin complex is expressed in both slow skeletal and cardiac muscle. Therefore, investigating the effects of cTnC mutations associated with hypertrophic cardiomyopathy (HCM) in slow skeletal muscle may reveal new insights into the mechanisms underlying cardiomyopathies. Glycerol SDS-PAGE analysis was performed on rabbit soleus muscle in order to determine the amount of slow myosin heavy chain type I (MHC I) present and its ratio to other myosin isotypes. The results show that the entire rabbit soleus muscle consists of MHC-1 slow type fibers; thereby, making it possible to use rabbit soleus muscle as a model system for slow skeletal muscle experiments. In order to determine the effects of TnC mutations on the Ca2+ sensitivity of contraction in skinned slow muscle fibers, native TnC was extracted and reconstituted with WT or mutant TnCs. The A8V, E134D, D145E and C84Y cTnC mutants were all tested and C84Y-cTnC was the only mutant that increased the Ca2+ sensitivity of force development in both the skinned cardiac and slow skeletal fibers. Additionally, none of the mutants affected the restored maximal force in the soleus fibers. The absence of a phenotype arising from most of the mutants in the soleus muscle suggests that the aberrant effects of cardiomyopathic cTnCs may be tissue specific, such that other proteins present in slow skeletal muscle may potentially rescue the deleterious effects of the mutations. In the next set of experiments we will determine if these mutations alter the energetics of crossbridges by evaluating the Actomyosin ATPase activities of soleus myofibrils that have their native TnC extracted and reconstituted with recombinant mutant cTnCs. Supported by J&E King 1KD03-33923 (DD), NIH R01-HL42325 (JDP) and NIH 1K99HL103840-01 (JRP).

This spectroscopic study examined the steady-state and kinetic parameters governing the cross-bridge effect on the increased Ca2+ affinity of hypertrophic cardiomyopathy-cardiac troponin C (HCM-cTnC) mutants. Previously, we found that incorporation of the A8V and D145E HCM-cTnC mutants, but not E134D into thin filaments (TFs), increased the apparent Ca2+ affinity relative to TFs containing the WT protein. Here, we show that the addition of myosin subfragment 1 (S1) to TFs reconstituted with these mutants in the absence of MgATP(2-), the condition conducive to rigor cross-bridge formation, further increased the apparent Ca2+ affinity. Stopped-flow fluorescence techniques were used to determine the kinetics of Ca2+ dissociation (k(off)) from the cTnC mutants in the presence of TFs and S1. At a high level of complexity (i.e. TF + S1), an increase in the Ca2+ affinity and decrease in k(off) was achieved for the A8V and D145E mutants when compared with WT. Therefore, it appears that the cTnC Ca2+ off-rate is most likely to be affected rather than the Ca2+ on rate. At all levels of TF complexity, the results obtained with the E134D mutant reproduced those seen with the WT protein. We conclude that strong cross-bridges potentiate the Ca2+-sensitizing effect of HCM-cTnC mutants on the myofilament. Finally, the slower k(off) from the A8V and D145E mutants can be directly correlated with the diastolic dysfunction seen in these patients.

TNNC1, which encodes cardiac troponin C (cTnC), remains elusive as a dilated cardiomyopathy (DCM) gene. Here, we report the clinical, genetic, and functional characterization of four TNNC1 rare variants (Y5H, M103I, D145E, and I148V), all previously reported by us in association with DCM (Hershberger, R. E., Norton, N., Morales, A., Li, D., Siegfried, J. D., and Gonzalez-Quintana, J. (2010) Circ. Cardiovasc. Genet. 3, 155–161); in the previous study, two variants (Y5H and D145E) were identified in subjects who also carried MYH7 and MYBPC3 rare variants, respectively. Functional studies using the recombinant human mutant cTnC proteins reconstituted into porcine papillary skinned fibers showed decreased Ca2+ sensitivity of force development (Y5H and M103I). Furthermore, the cTnC mutants diminished (Y5H and I148V) or abolished (M103I) the effects of PKA phosphorylation on Ca2+ sensitivity. Only M103I decreased the troponin activation properties of the actomyosin ATPase when Ca2+ was present. CD spectroscopic studies of apo (absence of divalent cations)-, Mg2+-, and Ca2+/Mg2+-bound states indicated that all of the cTnC mutants (except I148V in the Ca2+/Mg2+ condition) decreased the α-helical content. These results suggest that each mutation alters the function/ability of the myofilament to bind Ca2+ as a result of modifications in cTnC structure. One variant (D145E) that was previously reported in association with hypertrophic cardiomyopathy and that produced results in vivo in this study consistent with prior hypertrophic cardiomyopathy functional studies was found associated with the MYBPC3 P910T rare variant, likely contributing to the observed DCM phenotype. We conclude that these rare variants alter the regulation of contraction in some way, and the combined clinical, molecular, genetic, and functional data reinforce the importance of TNNC1 rare variants in the pathogenesis of DCM.

The R21C substitution in cardiac troponin I (cTnI) is the only identified mutation within its unique N-terminal extension that is associated with hypertrophic cardiomyopathy (HCM) in man. Particularly, this mutation is located in the consensus sequence for beta-adrenergic-activated protein kinase A (PKA)-mediated phosphorylation. The mechanisms by which this mutation leads to heart disease are still unclear. Therefore, we generated cTnI knock-in mouse models carrying an R21C mutation to evaluate the resultant functional consequences. Measuring the in vivo levels of incorporated mutant and WT cTnI, and their basal phosphorylation levels by top-down mass spectrometry demonstrated: 1) a dominant-negative effect such that, the R21C+/- hearts incorporated 24.9% of the mutant cTnI within the myofilament; and 2) the R21C mutation abolished the in vivo phosphorylation of Ser(23)/Ser(24) in the mutant cTnI. Adult heterozygous (R21C+/-) and homozygous (R21C+/-) mutant mice activated the fetal gene program and developed a remarkable degree of cardiac hypertrophy and fibrosis. Investigation of cardiac skinned fibers isolated from WT and heterozygous mice revealed that the WT cTnI was completely phosphorylated at Ser(23)/Ser(24) unless the mice were pre-treated with propranolol. After propranolol treatment (-PKA), the pCa-tension relationships of all three mice (i. e. WT, R21C+/-, and R21C+/-) were essentially the same. However, after treatment with propranolol and PKA, the R21C cTnI mutation reduced (R21C+/-) or abolished (R21C+/-) the well known decrease in the Ca2+ sensitivity of tension that accompanies Ser(23)/Ser(24) cTnI phosphorylation. Altogether, the combined effects of the R21C mutation appear to contribute toward the development of HCM and suggest that another physiological role for the phosphorylation of Ser(23)/Ser(24) in cTnI is to prevent cardiac hypertrophy.

A novel double deletion in cardiac troponin T (cTnT) of two highly conserved amino acids (Asn-100 and Glu-101) was found in a restrictive cardiomyopathic (RCM) pediatric patient. Clinical evaluation revealed the presence of left atrial enlargement and marked left ventricle diastolic dysfunction. The explanted heart examined by electron microscopy revealed myofibrillar disarray and mild fibrosis. Pedigree analysis established that this mutation arose de novo. The patient tested negative for six other sarcomeric genes. The single and double recombinant cTnT mutants were generated, and their functional consequences were analyzed in porcine skinned cardiac muscle. In the adult Tn environment (cTnT3 + cardiac troponin I), the single cTnT3-ΔN100 and cTnT3-ΔE101 mutations had opposing effects on the Ca(2+) sensitivity of force development compared with WT, whereas the double deletion cTnT3-ΔN100/ΔE101 increased the Ca(2+) sensitivity + 0.19 pCa units. In addition, cTnT3-ΔN100/ΔE101 decreased the cooperativity of force development, suggesting alterations in intrafilament protein-protein interactions. In the fetal Tn environment, (cTnT1 + slow skeletal troponin I), the single (cTnT1-ΔN110) and double (cTnT1-ΔN110/ΔE111) deletions did not change the Ca(2+) sensitivity compared with control. To recreate the patient's heterozygous genotype, we performed a reconstituted ATPase activity assay. Thin filaments containing 50:50 cTnT3-ΔN100/ΔE101:cTnT3-WT also increased the myofilament Ca(2+) sensitivity compared with WT. Co-sedimentation of thin filament proteins indicated that no significant changes occurred in the binding of Tn containing the RCM cTnT mutation to actin-Tm. This report reveals the protective role of Tn fetal isoforms as they rescue the increased Ca(2+) sensitivity produced by a cTnT-RCM mutation and may account for the lack of lethality during gestation.

Two DCM mutations (E40K and E54K) of Tropomyosin (Tm) were examined using the thin-filament extraction and reconstitution technique. Effects of Ca2+, ATP, phosphate and ADP concentrations on force and its transients were studied at 25°C and compared to WT. Both E40K and E54K showed significantly lower high-Ca (pCa 4.66) tension (THC, 1.21±0.06 and 1.24±0.07, respectively), low-Ca (pCa 7.0) tension (TLC, 0.07±0.02 and 0.06±0.02 at pCa 7.0), and Ca2+ activatable tension (Tact=THC-TLC, 1.15±0.08 and 1.18±0.06) compared to WT (THC=1.53±0.07, TLC=0.12±0.01, Tact=1.40±0.07) (±sem, N=8-21 for all studies).