Calcium sensitivity of force in human ventricular cardiomyocytes from donor and failing hearts

In failing human myocardium changes occur, in particular, in isoform composition and phosphorylation level of the troponin T (TnT) and troponin I (TnI) subunits of the actin filament and the myosin light chains (MLC-1 and -2), but it is unclear to what extent they influence cardiac performance. This overview concentrates on the relation between contractile function, contractile protein composition and phosphorylation levels in small biopsies from control (donor) hearts, from biopsies obtained during open heart surgery (NYHA Class I – IV) and from end-stage failing (explanted, NYHA class IV) hearts. Furthermore, attention is paid to the effect of the catalytic subunit of protein kinase A on isometric force development in single Triton-skinned human cardiomyocytes isolated from donor and end-stage failing left ventricular myocardium at different resting sarcomere lengths. A reduction in sarcomere length from 2.2 to 1.8 µm caused reductions in maximum isometric force by approximately 35 % both in donor and in failing cardiomyocytes. The midpoints of the calcium sensitivity curves (pCa ₅₀) of donor and end-stage failing hearts differed markedly at all sarcomere lengths (mean ?pCa ₅₀ = 0.22). Our findings indicate that 1) TnI phosphorylation contributes to the differences in calcium sensitivity between donor and end-stage failing hearts, 2) human ventricular myocardium is heterogeneous with respect of the phosphorylation of TnT, MLC-2 and the isoform distribution of MLC-1 and MLC-2, and 3) the Frank-Starling mechanism is preserved in end-stage failing myocardium.     

Impaired diastolic function after exchange of endogenous troponin I with C-terminal truncated troponin I in human cardiac muscle

The specific and selective proteolysis of cardiac troponin I (cTnI) has been proposed to play a key role in human ischemic myocardial disease, including stunning and acute pressure overload. In this study, the functional implications of cTnI proteolysis were investigated in human cardiac tissue for the first time. The predominant human cTnI degradation product (cTnI(1-192)) and full-length cTnI were expressed in Escherichia coli, purified, reconstituted with the other cardiac troponin subunits, troponin T and C, and subsequently exchanged into human cardiac myofibrils and permeabilized cardiomyocytes isolated from healthy donor hearts. Maximal isometric force and kinetic parameters were measured in myofibrils, using rapid solution switching, whereas force development was measured in single cardiomyocytes at various calcium concentrations, at sarcomere lengths of 1.9 and 2.2 mum, and after treatment with the catalytic subunit of protein kinase A (PKA) to mimic beta-adrenergic stimulation. One-dimensional gel electrophoresis, Western immunoblotting, and 3D imaging revealed that approximately 50% of endogenous cTnI had been homogeneously replaced by cTnI(1-192) in both myofibrils and cardiomyocytes. Maximal tension was not affected, whereas the rates of force activation and redevelopment as well as relaxation kinetics were slowed down. Ca(2+) sensitivity of the contractile apparatus was increased in preparations containing cTnI(1-192) (pCa(50): 5.73+/-0.03 versus 5.52+/-0.03 for cTnI(1-192) and full-length cTnI, respectively). The sarcomere length dependency of force development and the desensitizing effect of PKA were preserved in cTnI(1-192)-exchanged cardiomyocytes. These results indicate that degradation of cTnI in human myocardium may impair diastolic function, whereas systolic function is largely preserved.     

Functional effects of protein kinase C-mediated myofilament phosphorylation in human myocardium

OBJECTIVE: In human heart failure beta-adrenergic-mediated protein kinase A (PKA) activity is down-regulated, while protein kinase C (PKC) activity is up-regulated. PKC-mediated myofilament protein phosphorylation might be detrimental for contractile function in cardiomyopathy. This study was designed to reveal the effects of PKC on myofilament function in human myocardium under basal conditions and upon modulation of protein phosphorylation by PKA and phosphatases. METHODS: Isometric force was measured at different [Ca(2+)] in single permeabilized cardiomyocytes from non-failing and failing human left ventricular tissue. Basal phosphorylation of myofilament proteins and the influence of PKC, PKA, and phosphatase treatments were analyzed by one- and two-dimensional gel electrophoresis, Western immunoblotting, and ELISA. RESULTS: Troponin I (TnI) phosphorylation at the PKA sites was decreased in failing compared to non-failing hearts and correlated well with myofilament Ca(2+) sensitivity (pCa(50)). Incubation with the catalytic domain of PKC slightly decreased maximal force under basal conditions, but not following PKA and phosphatase pretreatments. PKC reduced Ca(2+) sensitivity to a larger extent in failing (DeltapCa(50)=0.19+/-0.03) than in non-failing (DeltapCa(50)=0.08+/-0.01) cardiomyocytes. This shift was reduced, though still significant, when PKC was preceded by PKA, while PKA following PKC did not further decrease pCa(50). Protein analysis indicated that PKC phosphorylated PKA sites in human TnI and increased phosphorylation of troponin T, while myosin light chain phosphorylation remained unaltered. CONCLUSION: In human myocardium PKC-mediated myofilament protein phosphorylation only has a minor effect on maximal force development. The PKC-mediated decrease in Ca(2+) sensitivity may serve to improve diastolic function in failing human myocardium in which PKA-mediated TnI phosphorylation is decreased.     

Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins

OBJECTIVE: The alterations in contractile proteins underlying enhanced Ca(2+)-sensitivity of the contractile apparatus in end-stage failing human myocardium are still not resolved. In the present study an attempt was made to reveal to what extent protein alterations contribute to the increased Ca(2+)-responsiveness in human heart failure. METHODS: Isometric force and its Ca(2+)-sensitivity were studied in single left ventricular myocytes from non-failing donor (n=6) and end-stage failing (n=10) hearts. To elucidate which protein alterations contribute to the increased Ca(2+)-responsiveness isoform composition and phosphorylation status of contractile proteins were analysed by one- and two-dimensional gel electrophoresis and Western immunoblotting. RESULTS: Maximal tension did not differ between myocytes obtained from donor and failing hearts, while Ca(2+)-sensitivity of the contractile apparatus (pCa(50)) was significantly higher in failing myocardium (deltapCa(50)=0.17). Protein analysis indicated that neither re-expression of atrial light chain 1 and fetal troponin T (TnT) nor degradation of myosin light chains and troponin I (TnI) are responsible for the observed increase in Ca(2+)-responsiveness. An inverse correlation was found between pCa(50) and percentage of phosphorylated myosin light chain 2 (MLC-2), while phosphorylation of MLC-1 and TnT did not differ between donor and failing hearts. Incubation of myocytes with protein kinase A decreased Ca(2+)-sensitivity to a larger extent in failing (deltapCa(50)=0.20) than in donor (deltapCa(50)=0.03) myocytes, abolishing the difference in Ca(2+)-responsiveness. An increased percentage of dephosphorylated TnI was found in failing hearts, which significantly correlated with the enhanced Ca(2+)-responsiveness. CONCLUSIONS: The increased Ca(2+)-responsiveness of the contractile apparatus in end-stage failing human hearts cannot be explained by a shift in contractile protein isoforms, but results from the complex interplay between changes in the phosphorylation status of MLC-2 and TnI.     

The effect of myosin light chain 2 dephosphorylation on Ca2+ -sensitivity of force is enhanced in failing human hearts

OBJECTIVE: Phosphorylation of the myosin light chain 2 (MLC-2) isoform expressed as a percentage of total MLC-2 was decreased in failing (21.1+/-2.0%) compared to donor (31.9+/-4.8%) hearts. To assess the functional implications of this change, we compared the effects of MLC-2 dephosphorylation on force development in failing and non-failing (donor) human hearts. METHODS: Cooperative effects in isometric force and rate of force redevelopment (K(tr)) were studied in single Triton-skinned human cardiomyocytes at various [Ca(2+)] before and after protein phosphatase-1 (PP-1) incubation. RESULTS: Maximum force and K(tr) values did not differ between failing and donor hearts, but Ca(2+)-sensitivity of force (pCa(50)) was significantly higher in failing myocardium (Deltap Ca(50)=0.17). K(tr) decreased with decreasing [Ca(2+)], although this decrease was less in failing than in donor hearts. Incubation of the myocytes with PP-1 (0.5 U/ml; 60 min) decreased pCa(50) to a larger extent in failing (0.20 pCa units) than in donor cardiomyocytes (0.10 pCa units). A decrease in absolute K(tr) values was found after PP-1 in failing and donor myocytes, while the shape of the K(tr)-Ca(2+) relationships remained unaltered. CONCLUSIONS: Surprisingly, the contractile response to MLC-2 dephosphorylation is enhanced in failing hearts, despite the reduced level of basal MLC-2 phosphorylation. The enhanced response to MLC-2 dephosphorylation in failing myocytes might result from differences in basal phosphorylation of other thin and thick filament proteins between donor and failing hearts. Regulation of Ca(2+)-sensitivity via MLC-2 phosphorylation may be a potential compensatory mechanism to reverse the detrimental effects of increased Ca(2+)-sensitivity and impaired Ca(2+)-handling on diastolic function in human heart failure.     

Length modulation of active force in rat cardiac myocytes: is titin the sensor?

The intrinsic cellular mechanisms by which length regulates myocardial contraction, the basis of the Frank-Starling relation, are uncertain. The aim of this work was to test the hypothesis that passive force, possibly via titin, participates in the modulation of Ca2+ sensitivity of cardiac contractile proteins induced by stretch. Titin degradation by a mild trypsin digestion modulated passive force induced by increasing from 1.9 to 2.3 microm sarcomere length in skinned rat cardiac cells. Force-pCa curves were established at these two sarcomere lengths after various durations of trypsin application that induced different passive force levels. They allowed us to evaluate myofilament Ca2+ sensitivity by the pCa of half-maximal activation (pCa50). In control conditions, stretching cells from 1.9 to 2.3 microm induced a leftward shift of pCa50 (DeltapCa50) of 0.39+/-0.03 pCa units (mean+/-SEM, n=8 cells), reflecting an increase in Ca2+ sensitivity of the contractile machinery. Passive force measured every 2 min decreased exponentially after the beginning of the trypsin application (t1/2 approximately 12 min). The first 30% decrease of passive force did not affect the stretch-induced variation in Ca2+ sensitivity. Then, with further decrease in passive force, DeltapCa50 decreased. At the lowest passive force investigated 20% of initial passive force, DeltapCa50 decreased by approximately 55%. These effects were not accompanied by a significant modification of either maximal activated force at pCa 4.5 solution or pCa50 at 1.9 microm sarcomere length. This indicates that there was no major functional alteration of the contractile machinery during the protocol as also suggested by contractile and regulatory protein electrophoresis on 2.5-12% gradient and 15% SDS-PAGE gels. Thus, besides modulation induced by the reduced lattice spacing during enhanced heart refilling, Ca2+ sensitivity of the cardiac contractile machinery may be controlled at least partially by internal passive load, which is known to be largely attributable to titin.     

Protein kinase C α and ε phosphorylation of troponin and myosin binding protein C reduce Ca2+ sensitivity in human myocardium

Previous studies indicated that the increase in protein kinase C (PKC)-mediated myofilament protein phosphorylation observed in failing myocardium might be detrimental for contractile function. This study was designed to reveal and compare the effects of PKCα- and PKCε-mediated phosphorylation on myofilament function in human myocardium. Isometric force was measured at different [Ca²⁺] in single permeabilized cardiomyocytes from failing human left ventricular tissue. Activated PKCα and PKCε equally reduced Ca²⁺ sensitivity in failing cardiomyocytes (ΔpCa₅₀ = 0.08 ± 0.01). Both PKC isoforms increased phosphorylation of troponin I- (cTnI) and myosin binding protein C (cMyBP-C) in failing cardiomyocytes. Subsequent incubation of failing cardiomyocytes with the catalytic subunit of protein kinase A (PKA) resulted in a further reduction in Ca²⁺ sensitivity, indicating that the effects of both PKC isoforms were not caused by cross-phosphorylation of PKA sites. Both isozymes showed no effects on maximal force and only PKCα resulted in a modest significant reduction in passive force. Effects of PKCα were only minor in donor cardiomyocytes, presumably because of already saturated cTnI and cMyBP-C phosphorylation levels. Donor tissue could therefore be used as a tool to reveal the functional effects of troponin T (cTnT) phosphorylation by PKCα. Massive dephosphorylation of cTnT with alkaline phosphatase increased Ca²⁺ sensitivity. Subsequently, PKCα treatment of donor cardiomyocytes reduced Ca²⁺ sensitivity (ΔpCa₅₀ = 0.08 ± 0.02) and solely increased phosphorylation of cTnT, but did not affect maximal and passive force. PKCα- and PKCε-mediated phosphorylation of cMyBP-C and cTnI as well as cTnT decrease myofilament Ca²⁺ sensitivity and may thereby reduce contractility and enhance relaxation of human myocardium.     

p21-activated kinase increases the calcium sensitivity of rat triton-skinned cardiac muscle fiber bundles via a mechanism potentially involving novel phosphorylation of troponin I

Phosphorylation of myofilament proteins by kinases such as cAMP-dependent protein kinase and protein kinase C has been shown to lead to altered thin-filament protein-protein interactions and modulation of cardiac function in vitro. In the present study, we report that a small GTPase-dependent kinase, p21-activated kinase (PAK), increases the calcium sensitivity of Triton-skinned cardiac muscle fiber bundles. Constitutively active PAK3 caused an average 1.25-fold (25.0+/-6.0%, n=6) increase in force at pCa 5.75, 1.44-fold (44.0+/-7.78%, n=6) at pCa 6.25, and 2.41-fold (141.2+/-23.7%, n=4) at pCa 6.5, representing a change in pCa50 value of approximately 0.25. Constitutively active PAK3 produced no change in force under conditions of relaxation (pCa 8.0) or maximal contraction (pCa 4.5). Furthermore, an inactive, kinase-dead form of PAK3 failed to produce any change in force development at any pCa value. The myofilament proteins phosphorylated by PAK3, at pCa 6.5, are desmin, troponin T, troponin I, and an unidentified 70-kDa protein. Importantly, cardiac troponin I was found to be phosphorylated at serine 149 of human cardiac troponin I, representing a novel phosphorylation site. These findings suggest a novel mechanism of modulating the calcium sensitivity of cardiac muscle contraction.     

Myocardial length-force relationship in end stage dilated cardiomyopathy and normal human myocardium: analysis of intact and skinned left ventricular trabeculae obtained during 11 heart transplantations

The Frank-Starling-mechanism (FSM) was analyzed in isolated intact and skinned human left ventricular myocardium obtained from 11 heart transplantations (normal donor hearts (NDH), n=8; dilated cardiomyopathy (DCM), n=11). The new technique to utilize muscle strips from normal donor hearts which were actually implanted is described in detail.Methods I) In electrically stimulated left ventricular trabeculae (37°C, oxygenated Krebs-Henseleit solution, supramaximal, electrical stimulation, frequency 1 Hz) force development was analyzed as a function of muscle length (NDH=8; DCM=11). II) In an additional series left ventricular myocardium was demembranized (skinned) by Triton-X-100. At different sarcomere lengths and calcium concentrations corresponding to pCa values of 4.3, 5.5, and 8.0 force development was measured (DCM=11; NDH=9).Results I) Developed force increased up to an optimum as a function of muscle length in intact NDH- and DCM-myocardium. However, the relative increment of developed force after any length step was smaller in DCM than in NDH. Near Lmax (muscle length associated with maximum developed force) passive resting tension was considerably elevated in DCM, indicating significantly incresed diastolic stiffness II) In skinned left ventricular DCM- and NDH-myocardium developed force depended on sarcomere length with an optimum near 2.2 m. However, a reduction of activator calcium concentration from pCa 4.3 to pCa 5.5 produces a smaller percent decline in force at short sarcomere lengths in DCM than it does in NDH.Conclusion the present study shows that except for diastolic stiffness and a smaller relative force increment after any, length step in DCM the Frank Starling mechanism is still present in isolated human left ventricular DCM-as in NDH-myocardium. The current study does not allow to decide whether in skinned myocardium the smaller percent decline in force after reduction of activator calcium concentrations in DCM is caused by an increased calcium sensitivity at short sarcomere lengths or decreased sensitivity at long sarcomere lengths.     

Calcium sensitivity and the Frank-Starling mechanism of the heart are increased in titin N2B region-deficient mice

Previous work suggests that titin-based passive tension is a factor in the Frank-Starling mechanism of the heart, by increasing length-dependent activation (LDA) through an increase in calcium sensitivity at long sarcomere length. We tested this hypothesis in a mouse model (N2B KO model) in which titin-based passive tension is elevated as a result of the excision of the N2B element, one of cardiac titin's spring elements. LDA was assessed by measuring the active tension-pCa (− log[Ca²⁺]) relationship at sarcomere length (SLs) of 1.95, 2.10, and 2.30 µm in WT and N2B KO skinned myocardium. LDA was positively correlated with titin-based passive tension due to an increase in calcium sensitivity at the longer SLs in the KO. For example, at pCa 6.0, the KO:WT tension ratio was 1.28 ± 0.07 and 1.42 ± 0.04 at SLs of 2.1 and 2.3 µm, respectively. There was no difference in protein expression or total phosphorylation of sarcomeric proteins. We also measured the calcium sensitivity after PKA treating the skinned muscle and found that titin-based passive tension was also now correlated with LDA, with a slope that was significantly increased compared to no PKA treatment. Finally, we performed isolated heart experiments and measured the Frank-Starling relation (slope of developed wall stress-LV volume relation) as well as diastolic stiffness (slope of diastolic wall stress-volume relation). The FSM was more pronounced in the N2B KO hearts and the slope of the FSM correlated with diastolic stiffness. These findings support that titin-based passive tension triggers an increase in calcium sensitivity at long sarcomere length, thereby playing an important role in the Frank-Starling mechanism of the heart.     

In vitro motility analysis of thin filaments from failing and non-failing human heart: troponin from failing human hearts induces slower filament sliding and higher Ca(2+) sensitivity

Contractility of the myocardium is altered in end-stage heart failure. We investigated whether this was related to functional changes in troponin. We isolated troponin from 1 g samples of end-stage failing, non-failing and foetal human heart and studied its regulation of actin-tropomyosin movement over immobilised HMM by in vitro motility assay. At pCa5.4 the sliding velocity of thin filaments reconstituted with non-failing heart troponin was 52+/-4% more than actin-tropomyosin, with failing heart troponin velocity increased by 35+/-2% and with foetal heart troponin velocity increased by 11+/-4%. Thin filaments containing troponin from failing hearts were more Ca(2+)-sensitive than non-failing heart troponin. EC(50) for the fraction of filaments motile and filament velocity decreased 1.76+/-0.20 and 1.89+/-0.62-fold respectively relative to non-failing heart troponin. With foetal heart troponin the EC(50) decreased 2.16+/-0.23 and 3.50+/-1.73-fold for fraction and velocity respectively. Western blots revealed no difference in troponin T or troponin I isoform expression in troponin from failing and non-failing adult hearts but foetal isoforms of troponin I and T were observed in troponin from foetal heart. The level of PKA phosphorylation of troponin from failing and non-failing heart was not significantly different, however, complete non-specific dephosphorylation of troponin abolished most of the difference between failing and non-failing heart troponin. These findings show functional alterations in troponin in failing hearts which could account for the reduced contractile function but there is no change in troponin isoform expression or PKA phosphorylation. Differential phosphorylation by other kinases may account for altered troponin function.     

Further studies on the effects of myosin P-light chain phosphorylation on contractile properties of skinned cardiac fibres

Summary We investigated the influence of myosin P-light chain phosphorylation by Ca²⁺-calmodulin dependent myosin light chain kinase (MLCK) on the sensitivity of the tension-pCa relation and maximum unloaded shortening velocity (v _max) of chemically skinned heart fibres of the pig.Submaximum Ca²⁺ stimulation (pCa 5.5) induced 20±5% of the isometric tension achieved at maximum Ca²⁺ activation (pCa 4.3).MLCK-induced myosin P-light chain phosphorylation increased the isometric force development at pCa 5.5 by 40% whereas maximum tension at pCa 4.3 was not affected.Unloaded shortening velocity (v _max) was not altered by myosin P-light chain phosphorylation either at maximum or at submaximum Ca²⁺ concentration, being c. 1.2 muscle length/s at pCa 5.5 and 2.2 muscle length/s at pCa 4.3.The MLCK-induced increase of the myosin P-light chain phosphorylation level was evaluated by determination of³²P-incorporation. Two phosphorylatable myosin P-light chains could be demonstrated.     

Cell-to-cell variability in troponin I phosphorylation in a porcine model of pacing-induced heart failure

We tested the hypothesis that myocardial contractile protein phosphorylation and the Ca²⁺ sensitivity of force production are dysregulated in a porcine model of pacing-induced heart failure (HF). The level of protein kinase A (PKA)-dependent cardiac troponin I (TnI) phosphorylation was lower in the myocardium surrounding the pacing electrode (pacing site) of the failing left ventricle (LV) than in the controls. Immunohistochemical assays of the LV pacing site pointed to isolated clusters of cardiomyocytes exhibiting a reduced level of phosphorylated TnI. Flow cytometry on isolated and permeabilized cardiomyocytes revealed a significantly larger cell-to-cell variation in the level of TnI phosphorylation of the LV pacing site than in the opposite region in HF or in either region in the controls: the interquartile range (IQR) on the distribution histogram of relative TnI phosphorylation was wider at the pacing site (IQR = 0.53) than that at the remote site of HF (IQR = 0.42; P = 0.0047) or that of the free wall of the control animals (IQR = 0.36; P = 0.0093). Additionally, the Ca²⁺ sensitivities of isometric force production were higher and appeared to be more variable in single permeabilized cardiomyocytes from the HF pacing site than in the healthy myocardium. In conclusion, the level of PKA-dependent TnI phosphorylation and the Ca²⁺ sensitivity of force production exhibited a high cell-to-cell variability at the LV pacing site, possibly explaining the abnormalities of the regional myocardial contractile function in a porcine model of pacing-induced HF.     

Cardiac troponin I threonine 144: role in myofilament length dependent activation

Myofilament length-dependent activation is the main cellular mechanism responsible for the Frank-Starling law of the heart. All striated muscle display length-dependent activation properties, but it is most pronounced in cardiac muscle and least in slow skeletal muscle. Cardiac muscle expressing slow skeletal troponin (ssTn)I instead of cardiac troponin (cTn)I displays reduced myofilament length-dependent activation. The inhibitory region of troponin (Tn)I differs by a single residue, proline at position 112 in ssTnI versus threonine at position 144 in cTnI. Here we tested whether this substitution was important for myofilament length-dependent activation; using recombinant techniques, we prepared wild-type cTnI, ssTnI, and 2 mutants: cTnI(Thr>Pro) and ssTnI(Pro>Thr). Purified proteins were complexed with recombinant cardiac TnT/TnC and exchanged into skinned rat cardiac trabeculae. Force-Ca2+ relationships were determined to derive myofilament Ca2+ sensitivity (EC50) at 2 sarcomere lengths: 2.0 and 2.2 microm (n=7). Myofilament length-dependent activation was indexed as deltaEC50, the difference in EC50 between sarcomere lengths of 2.0 and 2.2 microm. Incorporation of ssTnI compared with cTnI into the cardiac sarcomere reduced deltaEC50 from 1.26+/-0.30 to 0.19+/-0.04 micromol/L. A similar reduction also could be observed when Tn contained cTnI(Thr>Pro) (deltaEC50=0.24+/-0.04 micromol/L), whereas the presence of ssTnI(Pro>Thr) increased deltaEC50 to 0.94+/-0.12 micromol/L. These results suggest that Thr144 in cardiac TnI modulates cardiac myofilament length-dependent activation.     

Force-pCa relation and troponin T isoforms of rabbit myocardium

We have previously reported the existence of at least four troponin T isoforms in rabbit ventricular muscle and described the changes in their distribution with development. In this report we test whether the proportions of the troponin T isoforms are related to the sensitivity of the myofilaments to calcium. We measured the force-pCa relations in 12 detergent-skinned ventricular strands of cardiac muscle from newborn (2-5-day-old) rabbits. We determined from each strand the amount of each troponin T isoform relative to the total amount of troponin T by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometric scans of Western blots probed with a cardiac-specific troponin T monoclonal antibody, MAb 13-11. To assess the presence of different relative amounts of cardiac and slow skeletal troponin I among the strands, we determined the amount of cardiac troponin I relative to tropomyosin. We determined the Hill coefficient and the pCa for half-maximal force, pCa50, for each strand. pCa50 was related directly to the relative amount of troponin T2 (pslope = 0.037). Our results do not indicate a relation between the Hill coefficient and troponin T2. We also did not find a relation between pCa50 and the cardiac troponin I/tropomyosin ratio, which suggests that the correlation between pCa50 and troponin T2 was not a result of changes in the relative amounts of cardiac and slow skeletal muscle troponin I. Our findings indicate that a relation exists between the force-pCa characteristics of rabbit myocardium and the troponin T isoforms that it expresses, suggesting a role for troponin T in modulating the sensitivity of cardiac myofilaments to calcium.     

Effects of calcium on shortening velocity in frog chemically skinned atrial myocytes and in mechanically disrupted ventricular myocardium from rat.

Effects of [Ca2+] on isometric tension and unloaded shortening velocity were characterized in single chemically skinned myocytes from frog atrium and in mechanically disrupted myocardium from rat ventricle. The preparations were attached to a force transducer and piezoelectric translator and were viewed with an inverted microscope to allow continuous monitoring of sarcomere length during mechanical measurements. Unloaded shortening velocity was determined by measuring the time required to take up various amounts of slack imposed at one end of each preparation. Ca2+ sensitivity of isometric tension was assessed as pCa50, i.e., the Ca2+ concentration at which tension was 50% maximal, and was greater for frog atrial myocytes (pCa50 6.17) than for rat ventricular myocytes (pCa50 6.06). This difference in Ca2+ sensitivity may be due to variations in myofibrillar protein isoform composition in the two preparations. Inclusion of caffeine in the activating solutions substantially increased the Ca2+ sensitivity of tension, which may be a manifestation of a direct effect of caffeine on the myofibrillar proteins. Unloaded shortening velocity during maximal activation averaged 4.32 muscle lengths per second in frog atrial myocytes and 4.46 muscle lengths per second in rat ventricular myocytes. When [Ca2+] was reduced, unloaded shortening velocity decreased substantially in both preparations. Possible mechanisms for the effect of Ca2+ on shortening velocity in myocardium include Ca2+ dependence of the rate of ADP dissociation from actomyosin complexes or a shortening-dependent internal load involving structures such as C protein or long-lived myosin cross-bridges.     

Calcium-activated force in a turkey model of spontaneous dilated cardiomyopathy: adaptive changes in thin myofilament Ca2+ regulation with resultant implications on contractile performance.

Using saponin skinned fibers, we investigated whether decreased myofilament calcium responsiveness and contractile activation may in part contribute to heart failure in an animal model of idiopathic spontaneous cardiomyopathy (SCM). We addressed the question as to whether there are adaptive changes at the level of the thin myofilaments in turkey poults with SCM. The calcium concentration ([Ca2+]) required for 50% activation ([Ca2+]50%) was 0.80 +/- 0.12 microM (n = 12) vs. 0.76 +/- 0.08 microM (n = 12) and the Hill coefficient was 1.98 +/- 0.20 (n = 12) vs. 2.14 +/- 0.38 (n = 12) for control and SCM muscles respectively. Maximal Ca(2+)-activated force was not different between control fibers and fibers from failing hearts (3.83 +/- 0.88 g/mm2 vs. 3.65 +/- 0.39 g/mm2). These data indicate there are no differences in calcium-activation between fibers from control and failing myocardium. The effects of caffeine, an agent that increases myofilament Ca2+ sensitivity, were also studied. Addition of 10 mM caffeine resulted in a 0.06 pCa unit leftward shift of the force-pCa relationship in control hearts and 0.14 pCa units in SCM hearts. Caffeine (30 mM) increased force by 26 +/- 2.1% (n = 7) in control fibers and 44.5 +/- 8.7% (n = 8) in myopathic fibers at a pCa of 6.0. The increased responsiveness of muscles from failing hearts to caffeine indicates adaptive changes at the level of the thin myofilaments. Addition of dibutyryl-3',5'-cyclic-Adenosine Monophosphate (D-cAMP) resulted in a 0.21 pCa rightward shift on the calcium axis to higher intracellular calcium concentrations in control myocardium and 0.38 pCa units in SCM failing myocardium. The muscles were also sinusoidally oscillated at frequencies ranging between 0.01 and 100 Hz. In this analysis the frequency at which dynamic stiffness is minimum is taken as a measure of cross-bridge cycling rate. In control muscles, the frequency of minimum stiffness (fmin) was 1.20 +/- 0.11 (n = 4) whereas it was 0.71 +/- 0.08 Hz (n = 4) in myopathic muscles. The addition of 10 microM D-cAMP shifted fmin from 1.20 +/- 0.11 Hz to 1.68 +/- 0.09 Hz (delta = 0.48 +/- 0.06) in control fibers whereas in SCM fibers it caused greater shift of fmin from 0.71 +/- 0.08 Hz to 1.73 +/- 0.08 Hz (delta = 1.02 +/- 0.07). This differential effect of D-cAMP indicates adaptive changes at the level of the myofilaments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Length dependence of cardiac myofilament Ca(2+) sensitivity in the presence of substitute nucleoside triphosphates

Although ATP is the immediate source of energy for muscle contraction other nucleoside triphosphates (NTP) can substitute for ATP as substrates for myosin and as sources of energy for contraction of skinned muscle fibers. However, experiments with skinned skeletal muscle fibers in the presence of substitute NTP indicate significant differences with respect to cross-bridge kinetics, force generation, and Ca(2+) regulation. In this study the length dependence of Ca(2+) sensitivity of skinned bovine cardiac muscle was analyzed in the presence of MgATP, MgCTP, MgUTP, and MgITP. Ca(2+) regulation in the presence of MgCTP and MgUTP was essentially the same as in the presence of MgATP, although the maximum force generated (at sarcomere length 2.4 microm) was about 25% less. However, the length dependence of Ca(2+) sensitivity was eliminated in the presence of MgUTP. With MgITP the maximum force generated (at sarcomere length 2.4 microm) was about the same as in the presence of MgATP, but there was an impairment of relaxation such that at pCa 8 the force developed was about 50-60% of that developed at pCa 5. Moreover, the Ca(2+)-dependent component showed no length-dependent sensitivity. Thus length modulation of Ca(2+) sensitivity is a function of the myosin substrate. Taken in conjunction with other data, the results are consistent with the hypothesis that length-dependence of Ca(2+) sensitivity is modulated at a step upstream from the force-generating reaction.     

Alterations in myofilament function contribute to left ventricular dysfunction in pigs early after myocardial infarction

Myocardial infarction (MI) initiates cardiac remodeling, depresses pump function, and predisposes to heart failure. This study was designed to identify early alterations in Ca2+ handling and myofilament proteins, which may contribute to contractile dysfunction and reduced beta-adrenergic responsiveness in postinfarct remodeled myocardium. Protein composition and contractile function of skinned cardiomyocytes were studied in remote, noninfarcted left ventricular (LV) subendocardium from pigs 3 weeks after MI caused by permanent left circumflex artery (LCx) ligation and in sham-operated pigs. LCx ligation induced a 19% increase in LV weight, a 69% increase in LV end-diastolic area, and a decrease in ejection fraction from 54+/-5% to 35+/-4% (all P<0.05), whereas cardiac responsiveness to exercise-induced increases in circulating noradrenaline levels was blunted. Endogenous protein kinase A (PKA) was significantly reduced in remote myocardium of MI animals, and a negative correlation (R=0.62; P<0.05) was found between cAMP levels and LV weight-to-body weight ratio. Furthermore, SERCA2a expression was 23% lower after MI compared with sham. Maximal isometric force generated by isolated skinned myocytes was significantly lower after MI than in sham (15.4+/-1.5 versus 19.2+/-0.9 kN/m2; P<0.05), which might be attributable to a small degree of troponin I (TnI) degradation observed in remodeled postinfarct myocardium. An increase in Ca2+ sensitivity of force (pCa50) was observed after MI compared with sham (DeltapCa50=0.17), which was abolished by incubating myocytes with exogenous PKA, indicating that the increased Ca2+ sensitivity resulted from reduced TnI phosphorylation. In conclusion, remodeling of noninfarcted pig myocardium is associated with decreased SERCA2a and myofilament function, which may contribute to depressed LV function. The full text of this article is available online at http://circres.ahajournals.org.     

Calpain-I induced alterations in the cytoskeletal structure and impaired mechanical properties of single myocytes of rat heart

OBJECTIVE: The involvement of Calpain-I mediated proteolysis has been implicated in myofibrillar dysfunction of reperfused myocardium following ischemia (stunning). This study addresses the question whether ultrastructural alterations might be responsible for the depressed contractility. METHODS: Mechanical properties and protein composition of isolated myocytes after Calpain-I exposure (1.25 U/ml; 10 min; 15 degrees C; pCa 5.0) and of ischemic rat hearts following reperfusion were characterized. RESULTS: Maximal isometric force (44 +/- 5 kN/m2) at pCa 4.5 (pCa = -log[Ca2+]) decreased by 42.5% in Triton permeabilized myocytes (n = 11) after Calpain-I treatment. Force (and consequent myofilament disarrangement) during Calpain-I treatment was prevented by 40 mM BDM. The contractile force of Calpain-I exposed myocytes was significantly higher at submaximal levels of activation (pCa 5.5, 5.4 and 5.3) before maximal force development (pCa 4.5) than after maximal force development. The pCa50 value (5.40 +/- 0.02) determined from these initial test contractures did not differ significantly from that of untreated controls (5.44 +/- 0.03). However, after full activation Ca(2+)-sensitivity of force production in Calpain-I treated myocytes was significantly reduced (pCa50 5.34 +/- 0.02). This change in pCa50 was positively correlated with the reduction in maximal isometric force and was accompanied by sarcomere disorder. These findings imply that at least part of the Calpain-I induced mechanical alterations are dependent on force history. Measurements of the rate of force redevelopment after unloaded shortening suggested that Calpain-I did not affect cross-bridge kinetics. SDS gel electrophoresis and Western immunoblotting of Calpain-I treated myocytes revealed desmin degradation. The desmin content of postischemic myocardium was also reduced. CONCLUSION: Our results indicate that ultrastructural alterations may play an important role in the Calpain-I mediated cardiac dysfunction.