Immunological identification of five troponin T isoforms reveals an elaborate maturational troponin T profile in rabbit myocardium

Myocardium is generally thought to express no more than two isoforms of troponin T (TnT). We have recently reported that TnT purified from rabbit myocardium is resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into five proteins (TnT1, TnT2, TnT3, TnT4, and TnT5). In this study, these proteins are characterized immunologically and a novel elaborate maturational profile is described. Myocardium was obtained from 23 days of gestation fetal rabbits and 2-day, 6-week, 3-month, and 6-month postnatal rabbits. The major species in the adult myocardium, TnT4, was identified on sodium dodecyl sulfate-polyacrylamide gels and excised. The protein was electroeluted and purified. An amino acid microsequence of a cleaved fragment of this protein was found to be virtually identical to residues 86-99 from adult rabbit cardiac TnT. The protein, TnT4, was used to raise a polyclonal antibody. This antibody recognized all five isoforms from purified cardiac TnT, but none of the TnT isoforms from fast skeletal muscle. A monoclonal antibody, Mab JLT-12, raised against a highly conserved epitope of rabbit fast skeletal muscle, recognized all five cardiac as well as five skeletal muscle isoforms. Western blots performed on intact myocardial preparations demonstrated that TnT1, the cardiac isoform with the slowest electrophoretic mobility, was expressed prominently in the immature hearts, in addition to TnT2, TnT3, and TnT4, but TnT1 was not evident in the 3-month and 6-month postnatal hearts. The expression of TnT2 also decreased with maturation. Thus, the number of TnT isoforms expressed in the rabbit decreases with maturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Maximal ATPase activity and calcium sensitivity of reconstituted myofilaments are unaltered by the fetal troponin T re-expressed during human heart failure

Re-expression of a fetal isoform of troponin T (TnT(4)) has been demonstrated in failing human ventricular myocardium and associated with a decrease in myofibrillar ATPase activity. In order to elucidate the regulatory role of the re-expressed TnT(4) in the failing human heart, we measured ATPase activity in reconstituted cardiac myofilaments prepared with recombinant human TnT(4) or the adult human isoform of troponin T (TnT(3)). Neither the maximal calcium-activated ATPase activity nor the calcium sensitivity of this biochemical assay was significantly different between reconstituted myofilaments containing adult TnT(3) or fetal TnT(4). Our results suggest that the re-expressed fetal TnT(4) is not responsible for the depressed ATPase activity of failing ventricular myofibrils. The increased expression of the fetal isoform of this thin filament regulatory protein in the failing ventricle may be a consequence of a programmed change in gene expression occurring in response to hemodynamic stress, but probably does not contribute to depressed ventricular function characteristic of dilated cardiomyopathies.     

Functional analysis of human cardiac troponin by the in vitro motility assay: comparison of adult, foetal and failing hearts

OBJECTIVE: Human cardiac development and heart failure are associated with altered troponin isoform expression and phosphorylation. As the functional effects of these changes in troponin are unknown, we isolated troponin from human foetal, normal adult and failing adult hearts and investigated their regulatory function. METHODS: Human cardiac troponin was assayed for regulatory function by in vitro motility assay and for protein content by SDS PAGE and immunoblotting. RESULTS: Human cardiac troponin regulated movement of actin-tropomyosin filaments over a bed of immobilised heavy meromyosin. At pCa 9, troponin from foetal and adult hearts reduced the fraction of filaments moving from 90% to less than 15% with a modest (25-30%) decrease in velocity. At pCa 5, troponin from normal adult hearts increased filament velocity by up to 47 +/- 3% with no change in the fraction of filaments moving. Foetal troponin increased velocity by only 4 +/- 6% and the effect of troponin from failing hearts was between these values at 31 +/- 5%. Foetal hearts showed different troponin I and T isoform expression compared with adult hearts. No differences in troponin isoform expression were demonstrated between normal and failing adult hearts. CONCLUSIONS: Functioning troponin and tropomyosin may be isolated from human heart and their properties investigated by in vitro motility assay. Both functional and isoform expression differences exist between foetal and adult cardiac troponin. The regulatory function of troponin from adults with end stage heart failure is different from normal adult troponin. These data suggest a role for altered troponin function in human cardiac development and heart failure.     

Identification and functional significance of troponin I isoforms in neonatal rat heart myofibrils

We investigated the mechanism(s) responsible for differences in the effects of acidic pH on Ca2+ activation of the activity of adult and neonatal rat heart myofilaments. Studies on preparations of myofilaments reconstituted with adult troponin-tropomyosin (Tn-Tm) and either adult or neonatal thick filaments indicated that the difference in effect of acidic pH is related to differences in Tn-Tm and not other myofilament proteins. Immunoblotting analysis showed that development of the rat heart myofibrils is associated with isoform switching from slow skeletal TnI to cardiac TnI and from a slow mobility isoform of TnT (TnT1) to a faster Mr isoform (TnT2. Expression of slow skeletal TnI was associated with a relative insensitivity of myofilament Ca2+ activation to deactivation by acidic pH. Moreover, the effect of acidic pH on Ca2+ activation of ATPase activity of soleus myofibrils, which contain cardiac TnC and slow skeletal TnI, was essentially the same as the effect of acidic pH on rat cardiac myofibrils in the early neonatal period. Neonatal myofilaments also contained a relative abundance of a set of polypeptides copurifying with the thin filaments. We have identified these proteins as histones. The relative amount of histones among a variety of preparations from different species was not correlated with the pH sensitivity of myofibrillar Ca2+ activation. Shifts in TnT isoforms among these species were also not correlated with an altered response to acidic pH. Our data provide evidence in support of the hypothesis that the relative insensitivity of neonatal myofilament activity to acidic pH is due to the presence of slow skeletal TnI in the thin-filament regulatory complex.     

AMPK isoform expression in the normal and failing hearts

AMP-activated protein kinase (AMPK) is a master metabolic switch that plays an important role in energy homeostasis at the cellular and whole body level, hence a promising drug target. AMPK is a heterotrimeric complex composed of catalytic α-subunit and regulatory β- and γ-subunits with multiple isoforms for each subunit. It has been shown that AMPK activity is increased in cardiac hypertrophy and failure but it is unknown whether changes in subunit composition of AMPK contribute to the altered AMPK activity. In this study, we determined the protein expression pattern of AMPK subunit isoforms during cardiac development as well as during cardiac hypertrophy and heart failure in mouse heart. We also compared the findings in failing mouse heart to that of the human failing hearts in order to determine whether the mouse heart is a good model of AMPK in human diseases. In mouse developmental hearts, AMPK was highly expressed in the fetal stages and fell back to the adult level after birth. In the failing mouse heart, there was a significant increase in α2, β2, and γ2 subunits both at the mRNA and protein levels. In contrary, we found significant increases in the protein level of α1, β1 and γ2c subunits in human failing hearts with no change in the mRNA level. We also compared isoform-specific AMPK activity in the mouse and human failing hearts. Consistent with the literature, in the failing mouse heart, the α2 complexes accounted for ~2/3 of total AMPK activity while the α1 complexes accounted for the remaining 30-35%. In the human hearts, however, the contribution of α1-AMPK activity was significantly higher (>40%) in the non-failing hearts, and it further increased to 50% in the failing hearts. Thus, the human hearts have a greater amount of α1-AMPK activity compared to the rodent hearts. In summary, the protein level and the isoform distribution of AMPK in the heart change significantly during normal development as well as in heart failure. These observations provide a basis for future development of therapeutic strategies for targeting AMPK.     

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.     

An embryonic-like myosin heavy chain is transiently expressed in nodal conduction tissue of the rat heart

In the bovine nodal conduction tissue we have described the existence of a novel cardiac myosin isoform, immunologically related to the myosin types expressed during skeletal muscle development. Using different monoclonal antibodies specific for the embryonic and the neonatal skeletal myosin heavy chain types we investigated the myosin composition of the rat sino-atrial and atrio-ventricular nodes. We find that nodal conduction tissue fibers of the rat heart contain a distinct cardiac myosin isoform antigenically similar to the skeletal embryonic myosin heavy chain. The expression of this myosin isoform in nodal tissue appears to be developmentally regulated and partially controlled by thyroid hormone. Reactive cardiac fibers were detected in the nodal regions only during fetal development and a few days after birth, whereas very rare labelled fibers could be observed in the adult nodes. This myosin type does not represent a primordial cardiac myosin isoform since it was not detected in the embryonic heart before 13.5 days of gestation. When congenital hypothyroidism was induced in rats, the post-natal disappearance of reactive fibers in the nodal regions was delayed. On the other hand, hypothyroidism induced in the adult rats did not change the number of the reactive nodal fibers with respect to the euthyroid hearts.     

Apoptotic/mytogenic pathways during human heart development

OBJECTIVE: The aim of our study was to assess myocytes apoptosis/mitosis and associated intracellular signalling pathways during heart development. SETTING AND PATIENTS: Eight human fetal hearts (at different gestation ages) and seven human adult hearts were chosen as controls (five normal and two pathological) and studied from both a histological and a molecular point of view. RESULTS: Our results are as follows: (i) all Shc isoforms are expressed and activated in the human fetal heart; (ii) a progressive fading of Shc and ERK expression are evident during gestation; (iii) JNK is present but it is not activated in the human fetal heart; (iv) CD95 is present in the first week of gestation and fades progressively; (v) apoptotic/proliferative processes are present in the early gestation phase and fades progressively; (vi) in the human heart, Shc isoform with medium weight is 55 kD and not 52 kD and it is upregulated in adult myocardial ischaemia. CONCLUSIONS: Myocyte underwent apoptosis/mitosis during gestation. Shc isoforms, together with ERK maintain the homeostasis of the heart.     

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.     

Return to the fetal gene program protects the stressed heart: a strong hypothesis

A common feature of the hemodynamically or metabolically stressed heart is the return to a pattern of fetal metabolism. A hallmark of fetal metabolism is the predominance of carbohydrates as substrates for energy provision in a relatively hypoxic environment. When the normal heart is exposed to an oxygen rich environment after birth, energy substrate metabolism is rapidly switched to oxidation of fatty acids. This switch goes along with the expression of “adult” isoforms of metabolic enzymes and other proteins. However, the heart retains the ability to return to the “fetal” gene program. Specifically, the fetal gene program is predominant in a variety of pathophysiologic conditions including hypoxia, ischemia, hypertrophy, and atrophy. A common feature of all of these conditions is extensive remodeling, a decrease in the rate of aerobic metabolism in the cardiomyocyte, and an increase in cardiac efficiency. The adaptation is associated with a whole program of cell survival under stress. The adaptive mechanisms are prominently developed in hibernating myocardium, but they are also a feature of the failing heart muscle. We propose that in failing heart muscle at a certain point the fetal gene program is no longer sufficient to support cardiac structure and function. The exact mechanisms underlying the transition from adaptation to cardiomyocyte dysfunction are still not completely understood.     

L-type Ca2+ channel alpha 1c subunit isoform switching in failing human ventricular myocardium

The objectives of this study were to determine the relative abundance of the L-type Ca channel alpha 1c IVS3 isoforms that result from alternative splicing in normal human ventricular myocytes and to measure the changes in isoform expression in end stage heart failure. METHODS: mRNA was isolated from left ventricular tissue and myocytes from non-failing and failing human hearts. RT-PCR with isoform-specific primers was used to obtain cDNAs that were then mutated for use in competitive PCR reactions. An RNase protection assay was also used to confirm the presence of one of the novel isoforms. RESULTS: Four different alpha 1c IVS3 isoforms were found in non-failing human ventricular myocytes using RT-PCR. Two isoforms contained exon 31 (termed IVS3A isoforms) and two isoforms contained exon 32 (termed IVS3B isoforms). One of these isoforms has not been observed previously and contains exon 31 and all but the last six base pairs of exon 32. In non-failing human ventricular myocytes the IVS3A isoform is 2.5 times more abundant than the IVS3B isoform. There were significant changes in the relative abundance of these isoforms in failing hearts, with the IVS3B isoform being twice as abundant as the IVS3A isoform. All isoforms were confirmed by RNase protection analysis. CONCLUSIONS: These experiments show that there are at least four L-type Ca channel mRNA isoforms in the normal human heart and that the relative abundance of these isoforms changes significantly in heart failure. These alpha 1c isoform changes in heart failure are associated with dysfunctional electromechanical disturbances, but the specific physiological role of each L-type Ca channel isoform in normal and failing hearts needs to be defined.     

Alterations in protein kinase A and protein kinase C levels in heart failure due to genetic cardiomyopathy.

BACKGROUND: It is becoming evident that both cardiac and skeletal muscles are affected in congestive heart failure. Although protein kinases are known to regulate cardiac function, very little is known about their status in cardiac and skeletal muscles during the development of congestive heart failure. OBJECTIVE: To determine changes in the activities and protein levels of protein kinase A (PKA) and protein kinase C (PKC) in cardiac and skeletal muscles in congestive heart failure due to genetic cardiomyopathy on the basis that PKA and PKC are crucial for protein phosphorylation. ANIMALS AND METHODS: Genetically cardiomyopathic UM-X7.1 hamsters (250 to 300 days old) and age-matched Syrian hamsters were used in this study. PKA and PKC activities were assayed by measuring 32P from [gamma-32P]ATP incorporated into synthetic substrates. Relative protein contents of these protein kinases were obtained by using immunoblot analysis in control and failing hamster hearts and skeletal muscles. RESULTS: PKC activity was significantly increased in the failing hearts compared with control preparations. The relative protein contents of cytosolic PKC-alpha and -epsilon , and of particulate PKC-epsilon isozymes were significantly increased in failing hearts. PKC activity was also markedly increased in cardiomyopathic skeletal muscle. Furthermore, PKA activity and protein level in both cardiac and skeletal muscles were significantly increased in the failing heart group compared with control values. CONCLUSIONS: Increased PKC activity in heart failure may be due to changes in PKC-alpha and -epsilon isozymes in cardiomyopathic hearts. Alterations of PKA and PKC in congestive heart failure were not limited to the heart because similar changes in enzyme activities were evident in skeletal muscle.     

Cardiac contractile proteins in hypertrophied and failing guinea pig heart.

OBJECTIVE: The aim was to study changes in contractile proteins which accompany marked hypertrophy and heart failure in mammalian hearts initially containing predominantly V3 isomyosin. METHODS: Left ventricular myosin and myofibrillar ATPase activity and right ventricular actomyosin ATPase activity were measured in normal guinea pig hearts, in hearts which were hypertrophied as a result of progressive left ventricular systolic overload following ascending aortic banding, and in hypertrophied hearts from animals which showed signs of overt congestive heart failure. Male guinea pigs weighing 225-275 g at the time of aortic banding were used for the studies. RESULTS: Left ventricular myosin and myofibrillar ATPase activity and right ventricular actomyosin ATPase activity were correlated with body weight, left and right ventricular weight, and left ventricular peak systolic pressure during aortic occlusion. Left ventricular myosin ATPase activity and right ventricular actomyosin ATPase activity were markedly depressed in hypertrophied ventricles compared to control ventricles. Cardiac myofibrillar ATPase activity was lower in hypertrophied failing hearts than in control hearts over a wide range of calcium concentrations. In control animals and in those without heart failure, there was a nearly identical inverse relationship between left ventricular mass up to 1600 mg and myosin ATPase activity. Hypertrophied failing hearts were larger but showed little further reduction in cardiac myosin ATPase activity. Representative gel scans of non-dissociating pyrophosphate gels of left ventricular myosin from an 8 d postoperative aortic constricted animal and from its age and weight matched control showed predominantly V3 isomyosin with small amounts of V1 isoenzyme. However, preparations taken from guinea pigs 16 d after aortic constriction showed only the V3 isoform, whereas the V1 isoform was still apparent in control. Hypertrophied failing left ventricles developed less pressure per unit mass during brief aortic occlusion than non-failing left ventricles with comparable myosin ATPase activities. CONCLUSIONS: These observations raise important questions as to the distribution of myosin isoforms in the normal adult guinea pig, and the possibility that myosin ATPase activity might be altered by post-translational modification. Although cardiac myosin ATPase activity correlates with left ventricular performance, it cannot fully explain the depressed performance of failing hearts in this model. Additional immunological studies of cardiac contractile proteins are required as well as studies designed to explore the implications of altered myosin ATPase activity for both contractile function and overall cellular homeostasis.     

Cardiac steroidogenesis in the normal and failing heart.

The present study explores the possibility of local de novo aldosterone production in normal and failing hearts (human and mouse) and the regulation of such putative cardiac steroidogenesis. Total RNA was isolated from human tissue from failing hearts taken at the time of cardiac transplantation, from normal hearts obtained at autopsy, and from normal and pressure-overloaded mouse hearts. Vascular smooth muscle cells from human artery and vein were also analyzed. RNA was reverse transcribed and probed with specific primers for side-chain cleavage enzyme (CYP11A), 3beta-hydroxysteroid dehydrogenase, aldosterone synthase (CYP11B2), 11beta-hydroxylase (CYP11B1), steroidogenic factor-1, and steroid acute regulatory protein. CYP11A, 3beta-hydroxysteroid dehydrogenase-2, and steroid acute regulatory protein were expressed at modest levels in all tissues examined in both mouse and human. In failing human heart, CYP11B1 and CYP11B2 were detected in some samples, in contrast with normal hearts, which expressed neither; in the mouse heart steroidogenic factor-1 was detected, but neither CYP11B1 nor CYP11B2 was found. Steroidogenic factor-1 was detected in no human heart sample tested after 40 cycles of PCR. Although the expression of some steroidogenic genes can be detected in the heart, the likelihood of physiologically relevant levels of aldosterone production by the normal heart is very low. The exact cellular location of steroid synthesis in the failing human heart remains to be established.     

Localisation of atrial natriuretic peptide immunoreactivity in the ventricular myocardium and conduction system of the human fetal and adult heart

Atrial natriuretic peptide immunoreactivity was found in ventricular and atrial tissues with specific antisera raised to the amino and carboxy terminal regions of the precursor molecule. In 13 developing human hearts (7-24 weeks' gestation) the immunoreactivity was concentrated in the atrial myocardium and ventricular conduction system but it was also detected in the early fetal ventricular myocardium. Immunoreactivity in five normal adults was largely confined to the atrial myocardium although it was also found in the ventricular conduction tissues of hearts removed from 10 patients who were undergoing cardiac transplantation. The ventricular conduction system is an extra-atrial site for the synthesis of atrial natriuretic peptide. In the failing heart this synthesis may be further supplemented by expression of the gene in the ventricular myocardium. It is possible that ventricular production of the peptide contributes to the raised circulating concentrations of atrial natriuretic peptide immunoreactivity found in severe congestive heart disease, particularly in patients with dilated cardiomyopathy.