Reduced myocardial sarcoplasmic reticulum Ca(2+)-ATPase protein expression in compensated primary and secondary human cardiac hypertrophy.

Pathological intracellular calcium handling has been proposed to underlie the alterations of contractile behavior in hypertrophied myocardium. However, the myocardial protein expression of intracellular calcium transport proteins in compensated human left ventricular hypertrophy has not yet been studied. We investigated septal myocardial specimens of patients suffering from hypertrophic obstructive cardiomyopathy (n=14) or from acquired aortic valve stenosis (n=11) undergoing myectomy or aortic valve replacement, respectively. For comparison, we studied non-hypertrophied myocardium of six non-failing hearts which could not be transplanted for technical reasons. The myocardial density of the calcium release channel of the sarcoplasmic reticulum (SR) was determined by(3)H-ryanodine binding. Myocardial contents of SR Ca(2+)-ATPase, phospholamban, calsequestrin and Na(+)/Ca(2+)-exchanger were analysed by Western blot analysis. The myocardial SR calcium release channel density was not significantly different in hypertrophied and non-failing human myocardium. In both hypertrophic obstructive cardiomyopathy and in aortic valve stenosis, SR Ca(2+)-ATPase expression was reduced by about 30% compared to non-failing myocardium (P<0.05), whereas the expression of phospholamban, calsequestrin, and the Na(+)/Ca(2+)-exchanger was unchanged. The decrease of SR Ca(2+)-ATPase expression was still observable when related to its regulatory protein phospholamban or to the myosin content of the homogenates (P<0.05). Furthermore, the SR Ca(2+)-ATPase expression was inversely correlated to the septum thickness assessed by echocardiography, but not to age, cardiac index or outflow tract gradient. In primary as well as in secondary hypertrophied human myocardium, the expression of SR Ca(2+)-ATPase is reduced and inversely related to the degree of the hypertrophy. The diminished SR Ca(2+)-ATPase expression might result in reduced Ca(2+)reuptake into the SR and might contribute to altered contractile behavior in hypertrophied human myocardium.     

Heterogeneous transmural gene expression of calcium-handling proteins and natriuretic peptides in the failing human heart.

OBJECTIVE: Human heart failure is associated with a disturbed intracellular calcium (Ca2+) homeostasis. In this regard, ventricular wall stress is considered to be a determinant for expression of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a). In the present study, we analyzed the transmural protein and/or mRNA levels of SERCA2a, other Ca(2+)-handling proteins, and of atrial and brain natriuretic peptides (ANP and BNP) in the human heart. METHODS: Subepicardial (epi), midmyocardial (mid), and subendocardial (endo) sections of the left ventricular free wall from end-stage failing (n = 17) and nonfailing (n = 5) human hearts were analyzed by Western blot for immunoreactive protein levels of SERCA2a, phospholamban (PLN), and calsequestrin (CS). Subepi- and subendocardial sections were analyzed by Northern blot for steady-state mRNA levels of SERCA2a, Na(+)-Ca2+ exchanger (NCX1), ANP, and BNP. RESULTS: SERCA2a protein and mRNA levels were reduced by 40 +/- 5% (P < 0.01) and 25 +/- 7% (P < 0.05) in endo compared to epi in the failing heart and by 27 +/- 14% and 16 +/- 12% (non-significant) in the nonfailing heart, respectively. PLN protein levels were reduced by 23 +/- 6% (P < 0.05) in endo compared to epi in the failing heart and by 17 +/- 25% (non-significant) in the nonfailing heart, whereas CS protein levels and NCX1 mRNA levels were similar across the left ventricular wall. Strikingly, in the failing heart, both BNP and ANP mRNA levels were upregulated predominantly in endo. CONCLUSIONS: In the failing human heart, SERCA2a and PLN, as well as natriuretic peptides but not CS and NCX1 are differentially expressed across the left ventricular wall, implicating (1) different susceptibility of subendocardium and subepicardium to factors affecting expression of these proteins and (2) differences in regulation of the distinct calcium-cycling proteins.     

A mutation in the human phospholamban gene, deleting arginine 14, results in lethal, hereditary cardiomyopathy

The sarcoplasmic reticulum Ca(2+)-cycling proteins are key regulators of cardiac contractility, and alterations in sarcoplasmic reticulum Ca(2+)-cycling properties have been shown to be causal of familial cardiomyopathies. Through genetic screening of dilated cardiomyopathy patients, we identified a previously uncharacterized deletion of arginine 14 (PLN-R14Del) in the coding region of the phospholamban (PLN) gene in a large family with hereditary heart failure. No homozygous individuals were identified. By middle age, heterozygous individuals developed left ventricular dilation, contractile dysfunction, and episodic ventricular arrhythmias, with overt heart failure in some cases. Transgenic mice overexpressing the mutant PLN-R14Del recapitulated human cardiomyopathy exhibiting similar histopathologic abnormalities and premature death. Coexpression of the normal and mutant-PLN in HEK-293 cells resulted in sarcoplasmic reticulum Ca(2+)-ATPase superinhibition. The dominant effect of the PLN-R14Del mutation could not be fully removed, even upon phosphorylation by protein kinase A. Thus, by chronic suppression of sarcoplasmic reticulum Ca(2+)-ATPase activity, the nonreversible superinhibitory function of mutant PLN-R14Del may lead to inherited dilated cardiomyopathy and premature death in both humans and mice.     

Gender influences on sarcoplasmic reticulum Ca2+-handling in failing human myocardium.

Gender has recently been implicated as an important modulator of cardiovascular disease. However, it is not known how gender may specifically influence the Ca2+-handling deficits that characterize the depressed cardiac contractility of human heart failure. To elucidate the contributory role of gender to sarcoplasmic reticulum (SR) Ca2+ cycling alterations, the protein levels of SR Ca2+-ATPase (SERCA), phospholamban, and calsequestrin, as well as the site-specific phospholamban phosphorylation status, were quantified in a mixed gender population of failing (n=14) and donor (n=15) myocardia. The apparent affinity (EC50) and the maximal velocity (Vmax) of SR Ca2+-uptake were also determined to lend functional significance to any observed protein alterations. Phospholamban and calsequestrin levels were not altered; however, SERCA protein levels were significantly reduced in failing hearts. Additionally, phospholamban phosphorylation (serine-16 and threonine-17 sites) and myocardial cAMP content were both attenuated. The alterations in SR protein levels were also accompanied by a decreased V(max)and an increased EC50 (diminished apparent affinity) of SR Ca2+-uptake for Ca2+ in failing myocardia. Myocardial protein levels and Ca2+ uptake parameters were then analyzed with respect to gender, which revealed that the decreases in phosphorylated serine-16 were specific to male failing hearts, reflecting increases in the EC50 values of SR Ca2+-uptake for Ca2+, compared to donor males. These findings suggest that although decreased SERCA protein and phospholamban phosphorylation levels contribute to depressed SR Ca2+-uptake and left ventricular function in heart failure, the specific subcellular alterations which underlie these effects may not be uniform with respect to gender.     

Hypertrophy and functional alterations in hyperdynamic phospholamban-knockout mouse hearts under chronic aortic stenosis

OBJECTIVE: To determine whether the hyperdynamic phospholamban-knockout hearts are capable of withstanding a chronic aortic stenosis. METHODS: The transverse section of the aorta was banded in phospholamban-knockout and their isogenic wild-type mice, which were followed with echocardiography in parallel, along with sham-operated mice, before and at 2.5, 5 and 10 weeks after surgery. RESULTS: Cardiac decompensation was evidenced by the presence of lung congestion in some banded knockouts and wild-types, giving rise to a subset of non-failing and failing hearts within each group. The incidence of heart failure was not genotype-dependent but rather associated with higher heart rates before surgery. The development of left ventricular hypertrophy was similar between knockouts and wild-types and longitudinal assessment of end-diastolic dimension indicated progressive increases after banding, with a greater dilation in failing mice. Fractional shortening was reduced in failing knockouts and wild-types to a similar degree, with an earlier onset in the knockouts. In addition, fractional shortening was decreased in non-failing knockouts but not wild-types. Ejection times shortened after aortic banding particularly for failing hearts. Assessment of the SR Ca(2+)-ATPase protein levels indicated similar downregulation for failing knockouts and wild-types, while the phospholamban levels were not significantly altered in wild-types. CONCLUSION: The hyperdynamic phospholamban-knockout hearts are able to compensate against a sustained aortic stenosis similar to wild-types.     

Activity of cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase in failing and nonfailing human hearts.

OBJECTIVES: A hallmark of human heart failure is prolonged myocardial relaxation. Although the intrinsic mechanism of phospholamban coupling to the Ca(2+)-ATPase is unaltered in normal and failed human hearts, it remains possible that regulation of phospholamban phosphorylation by cAMP-dependent mechanisms or other second messenger pathways could be perturbed, which may account partially for the observed dysfunctions of the sarcoplasmic reticulum (SR) associated with this disease. METHODS: cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaM kinase) were characterized initially by DEAE-Sepharose chromatography in hearts from patients with end-stage dilated cardiomyopathy. We measured the activity of PKA and CaM kinase in left ventricular tissue of failing (idiopathic dilated cardiomyopathy; ischemic heart disease) and nonfailing human hearts. RESULTS: Basal PKA activity was not changed between failing and nonfailing hearts. One major peak of CaM kinase activity was detected by DEAE-Sepharose chromatography. CaM kinase activity was increased almost 3-fold in idiopathic dilated cardiomyopathy. In addition, hemodynamical data (left ventricular ejection fraction, cardiac index) from patients suffering from IDC positively correlate with CaM kinase activity. CONCLUSIONS: Increased CaM kinase activity in hearts from patients with dilated cardiomyopathy could play a role in the abnormal Ca2+ handling of the SR and heart muscle cell.     

Reduced sarcoplasmic reticulum Ca(2+) load mediates impaired contractile reserve in right ventricular pressure overload

Myocardial contractile reserve is significantly attenuated in patients with advanced heart failure. The aim of this study was to identify mechanisms of impaired contractile reserve in a large animal model that closely mimics human myocardial failure. Progressive right ventricular hypertrophy and failure were induced by banding the pulmonary artery in kittens. Isometric contractile force was measured in right ventricular trabeculae (n=115) from age-matched Control and Banded feline hearts. Rapid cooling contractures (RCC) were used to determine sarcoplasmic reticulum (SR) Ca(2+) load while assessing the ability of changes in rate, adrenergic stimulation and bath Ca(2+) to augment contractility. The positive force-frequency relationship and robust pre- and post-receptor adrenergic responses observed in Control trabeculae were closely paralleled by increases in RCC amplitude and the RCC2/RCC1 ratio. Conversely, the severely blunted force-frequency and adrenergic responses in Banded trabeculae were paralleled by an unchanged RCC amplitude and RCC2/RCC1 ratio. Likewise, supraphysiologic levels of bath Ca(2+) were associated with severely reduced contractility and RCC amplitude in Banded trabeculae compared to Controls. There were no differences in myofilament Ca(2+) sensitivity or length-dependent increases in contractility between Control and Banded trabeculae. There was a significant decrease in SR Ca(2+)-ATPase pump abundance and phosphorylation of phospholamban and ryanodine receptor in Banded trabeculae compared with Controls. A reduced ability to increase SR Ca(2+) load is the primary mechanism of reduced contractile reserve in failing feline myocardium. The similarity of impaired contractile reserve phenomenology in this feline model and transplanted hearts suggests mechanistic relevance to human myocardial failure.     

Evidence for calcineurin-mediated regulation of SERCA 2a activity in human myocardium

Compromised SERCA 2a activity is a key malfunction leading to the Ca(2+) cycling alterations in failing human myocardium. SERCA 2a activity is regulated by the Ca(2+)/calmodulin-dependent protein kinase (CaM-kinase) but alterations of the CaM-kinase pathway regarding SERCA 2a in heart failure are unresolved. Therefore we investigated the CaM-kinase and phosphatase calcineurin mediated regulation of SERCA 2a in failing and non-failing human myocardium. We studied human myocardial preparations from explanted hearts from non-failing organ donors (NF, n=8) and from patients with terminal heart failure undergoing cardiac transplantation (dilated cardiomyopathy, DCM, n=8). SERCA 2a activity was determined using a NADH-coupled enzyme assay [expressed in nmol ATP/(mg protein x min)] and by(45)Ca(2+) uptake. Protein expression of SERCA 2a, phospholamban, calsequestrin and calcineurin was assessed by Western blotting (expressed as densitometric units/microg protein); phosphorylation of cardiac proteins was detected with specific phospho-antibodies for phospholamban at threonine-17 (PT17) or by incorporation of [gamma -(32)P] (expressed as pmol(32)P/mg). Maximal(45)Ca(2+) uptake (in pmol/mg/min) (NF: 3402+/-174; DCM: 2488+/-189) and maximal SERCA 2a activity were reduced in DCM compared to NF (V(max): NF: 125+/-9; DCM: 98+/-5). The V(max) reduction could be mimicked by calcineurin in vitro in NF (NF(control): 72.1+/-3.7; NF(+calcineurin): 49.8+/-2.9) and restored in DCM by CaM-kinase in vitro (DCM(control): 98+/-5; DCM(+CaM-kinase): 120+/-6). Protein expression of SERCA 2a, phospholamban and calsequestrin remained similar, but calcineurin expression was significantly increased in failing human hearts (NF: 11.6+/-1.5 v DCM: 17.1+/-1.6). Although the capacity of endogenous CaM-kinase to phosphorylate PT17 was significantly higher in DCM (DCM(control): 128+/-36; DCM(+endogenous CaM-kinase): 205+/-20) compared to NF myocardium (NF(control): 273+/-37; NF(+endogenous CaM-kinase): 254+/-31), net phosphorylation at threonine-17 phospholamban was significantly lower in DCM (DCM 130+/-11 v NF 170+/-11). A calcineurin-dependent dephosphorylation of phospholamban could be mimicked in vitro by incubation of NF preparations with calcineurin (NF(control) 80.7+/-4.4 v NF(+calcineurin) 30.7+/-4.1, P<0.05). In human myocardium, the V(max) of SERCA 2a and the phosphorylation of phospholamban is modulated by CaM-kinase and calcineurin, at least in vitro. In failing human myocardium, despite increased CaM-kinase activity, calcineurin dephosphorylation leads to decreased net phosphorylation of threonine-17 phospholamban in vivo. Increased calcineurin activity contributes to the impaired V(max) of SERCA 2a in failing human myocardium and the disorder in Ca(2+)-handling in heart failure.     

Regulation of myocardial Ca2+-ATPase and phospholamban mRNA expression in response to pressure overload and thyroid hormone.

The sarcoplasmic reticulum (SR) and the contractile protein myosin play an important role in myocardial performance. Both of these systems exhibit plasticity--i.e., quantitative and/or qualitative reorganization during development and in response to stress. Recent studies indicate that SR Ca2+ uptake function is altered in adaptive cardiac hypertrophy and failure. The molecular basis (genetic and phenotypic) for these changes is not understood. In an effort to determine the underlying causes of these changes, we characterized the rabbit cardiac Ca2+-ATPase phenotype by molecular cloning and ribonuclease A mapping analysis. Our results show that the heart muscle expresses only the slow-twitch SR Ca2+-ATPase isoform. Second, we quantitated the steady-state mRNA levels of two major SR Ca2+ regulatory proteins, the Ca2+-ATPase and phospholamban, to see whether changes in mRNA content might provide insight into the basis for functional modification in the SR of hypertrophied hearts. In response to pressure overload hypertrophy, the relative level of the slow-twitch/cardiac SR Ca2+-ATPase mRNA was decreased to 34% of control at 1 week. The relative Ca2+-ATPase mRNA level increased to 167% of control after 3 days of treatment with thyroid hormone. In contrast, in hypothyroid animals, the relative Ca2+-ATPase mRNA level decreased to 51% of control at 2 weeks. The relative level of phospholamban mRNA was decreased to 36% in 1-week pressure overload. Hyperthyroidism induced a decrease to 61% in the phospholamban mRNA level after 3 days of treatment, while hypothyroidism had virtually no effect on phospholamban mRNA levels. These data indicate that the expression of SR Ca2+-ATPase and phospholamban mRNA may not be coordinately regulated during myocardial adaptation to different physiological conditions.     

Alterations in the inotropic responses to forskolin and Ca2+ and reduced gene expressions of Ca2+-signaling proteins induced by chronic volume overload in rabbits

Volume overload results in eccentric cardiac hypertrophy, but it is still unknown how this mechanical overload modulates the inotropic response to exogenous Ca2+ or adenylyl cyclase stimulation. Inotropic responsiveness in vivo and the levels of gene expression of Ca2+ signaling proteins were studied in rabbit hearts hypertrophied as a result of volume overload at 4 and 12 weeks after arteriovenous shunt formation. In sham-operated control rabbits, left ventricular (LV)+dP/dt was augmented in response to graded doses of CaCl2. Dose-related changes of LV+dP/dt to CaCl2 were attenuated significantly in shunt rabbits with volume overload. Forskolin dose-dependently augmented LV+dP/dt in sham rabbits, which was also attenuated significantly in rabbits with volume overload. The mRNA levels of dihydropyridine receptor, Na+/Ca2+ exchanger, sarcoplasmic reticulum Ca2+-ATPase, and ryanodine receptor decreased significantly at 4 and 12 weeks in the volume-overload rabbits compared with the sham rabbits, but the mRNA levels of phospholamban and calsequestrin remained unchanged. Chronic volume overload alters contractile responsiveness to Ca2+ or adenylyl cyclase stimulation, and downregulation of steady state mRNA levels of Ca2+ signaling proteins might be, at least in part, related to this pathologic process.     

Regional expression and functional characterization of the L-type Ca2+-channel in myocardium from patients with end-stage heart failure and in non-failing human hearts

The purpose of the present study was to investigate the expression and functional relevance of sarcolemmal L-type Ca2+-channels in failing and non-failing human myocardium. The protein expression of sarcolemmal L-type Ca2+-channels was determined with 3H-(+)-PN 200-110-binding experiments and Western blot analysis using a specific antibody against the alpha1-subunit in membrane preparations of ventricular and atrial myocardium from both failing (n = 15) and non-failing hearts (n = 8). The gene expression of the ion conducting pore of the L-type Ca2+-channel was examined with Northern blot technique in human failing and non-failing RNA. For normalization the RNA expression of calsequestrin was used. In electrically driven ventricular papillary muscle strips and auricular trabeculae, the responses to nifedipine and Ca2+ as parameters of myocardial function were studied. The protein expression as measured by 3H-(+)-PN 200-110-binding (Bmax) and Western Blot analysis with calsequestrin as reference was similar in left ventricular failing and non-failing myocardium. However, both were reduced in atrial compared to ventricular tissue in failing and non-failing hearts. The KD remained unchanged. Calsequestrin levels were unaltered in failing and non-failing hearts. The gene expression of the alpha1-subunit was similar in human failing and non-failing hearts. The L-type Ca2+-channel antagonist nifedipine reduced force of contraction with the same potency and efficiency in ventricular failing and non-failing myocardium. In contrast, the potency of nifedipine was higher in atrial than in ventricular tissue. Consistently, atrial myocardium from patients with dilated cardiomyopathy was more sensitive towards Ca2+ than those of the control group. In conclusion, the altered Ca2+-homeostasis in failing human myocardium may be less due to changes in sarcolemmal L-type Ca2+-channel expression or function than due to an altered intracellular Ca2+-handling.     

Sarcoplasmic reticulum Ca2+-ATPase modulates cardiac contraction and relaxation

The cardiac SR Ca(2+)-ATPase (SERCA2a) regulates intracellular Ca(2+)-handling and thus, plays a crucial role in initiating cardiac contraction and relaxation. SERCA2a may be modulated through its accessory phosphoprotein phospholamban or by direct phosphorylation through Ca(2+)/calmodulin dependent protein kinase II (CaMK II). As an inhibitory component phospholamban, in its dephosphorylated form, inhibits the Ca(2+)-dependent SERCA2a function, while protein kinase A dependent phosphorylation of the phospho-residues serine-16 or Ca(2+)/calmodulin-dependent phosphorylation of threonine-17 relieves this inhibition. Recent evidence suggests that direct phosphorylation at residue serine-38 in SERCA2a activates enzyme function and enhances Ca(2+)-reuptake into the sarcoplasmic reticulum (SR). These effects that are mediated through phosphorylation result in an overall increased SR Ca(2+)-load and enhanced contractility. In human heart failure patients, as well as animal models with induced heart failure, these modulations are altered and may result in an attenuated SR Ca(2+)-storage and modulated contractility. It is also believed that abnormalities in Ca(2+)-cycling are responsible for blunting the frequency potentiation of contractile force in the failing human heart. Advanced gene expression and modulatory approaches have focused on enhancing SERCA2a function via overexpressing SERCA2a under physiological and pathophysiological conditions to restore cardiac function, cardiac energetics and survival rate.     

Effect of thyroid hormone on the expression of mRNA encoding sarcoplasmic reticulum proteins.

The purpose of this study was to determine the expression of genes encoding various sarcoplasmic reticulum components that are functionally coupled with calcium release, uptake, and storage function during cardiac hypertrophy induced by thyroid hormone. Hyperthyroidism was induced in two groups of rabbits by the injection of 200 micrograms/kg L-thyroxine (T4) daily for 4 days (T4-4-day group) and 8 days (T4-8-day group). Hypothyroidism was induced in another group of rabbits by adding 0.8 mg/ml propylthiouracil to the drinking water for 4 weeks. The relative expression level of mRNA encoding different sarcoplasmic reticulum proteins was determined by RNA slot blot and Northern blot analysis. In hyperthyroid hearts, the steady-state level of cardiac ryanodine receptor mRNA and sarcoplasmic reticulum cardiac/slow-twitch Ca(2+)-ATPase mRNA were both increased to 147% (T4-4-day group) and 186% (T4-8-day group) of control, respectively, but decreased to 71% and 75%, respectively, in hypothyroid ventricles. The mRNA level for phospholamban was decreased in both hyperthyroidism (T4-8-day group, 72%) and hypothyroidism (77%) in these hearts. On the other hand, calsequestrin mRNA levels did not change in hyperthyroid and hypothyroid ventricles. In accord with the changes in Ca(2+)-ATPase mRNA levels, the Ca(2+)-ATPase protein was increased to 199% (T4-8-day group) in hyperthyroid ventricles and decreased to 86% of control in hypothyroid ventricles. The expression levels of ryanodine receptor, Ca(2+)-ATPase, phospholamban, and calsequestrin mRNAs were similarly altered in skeletal muscle tissues from hyperthyroid and hypothyroid rabbits. These results indicate that the mRNA levels of sarcoplasmic reticulum proteins responsible for calcium release and calcium uptake are coordinately regulated in response to changes in thyroid hormone level in both heart and skeletal muscle. These changes in mRNA level should lead to changes in protein levels and thus to altered calcium release and uptake in the chronic stages of hyperthyroidism and hypothyroidism.     

Altered myocardial Ca2+ cycling after left ventricular assist device support in the failing human heart

OBJECTIVES: The objective of the present study was to determine whether improved contractility after left ventricular assist device (LVAD) support reflects altered myocyte calcium cycling and changes in calcium-handling proteins. BACKGROUND: Previous reports demonstrate that LVAD support induces sustained unloading of the heart with regression of pathologic hypertrophy and improvements in contractile performance. METHODS: In the human myocardium of subjects with heart failure (HF), with non-failing hearts (NF), and with LVAD-supported failing hearts (HF-LVAD), intracellular calcium ([Ca(2+)](i)) transients were measured in isolated myocytes at 0.5 Hz, and frequency-dependent force generation was measured in multicellular preparations (trabeculae). Abundance of sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (SERCA), Na(+)/Ca(2+) exchanger (NCX), and phospholamban was assessed by Western analysis. RESULTS: Compared with NF myocytes, HF myocytes exhibited a slowed terminal decay of the Ca(2+) transient (DT(terminal), 376 +/- 18 ms vs. 270 +/- 21 ms, HF vs. NF, p < 0.0008), and HF-LVAD myocytes exhibited a DT(terminal) that was much shorter than that observed in HF myocytes (278 +/- 10 ms, HF vs. HF-LVAD, p < 0.0001). Trabeculae from HF showed a negative force-frequency relationship, compared with a positive relationship in NF, whereas a neutral relationship was observed in HF-LVAD. Although decreased SERCA abundance in HF was not altered by LVAD support, improvements in [Ca(2+)](i) transients and frequency-dependent contractile function were associated with a significant decrease in NCX abundance and activity from HF to HF-LVAD. CONCLUSIONS: Improvement in rate-dependent contractility in LVAD-supported failing human hearts is associated with a faster decay of the myocyte calcium transient. These improvements reflect decreases in NCX abundance and transport capacity without significant changes in SERCA after LVAD support. Our results suggest that reverse remodeling may involve selective, rather than global, normalization of the pathologic patterns associated with the failing heart.     

Cardiac remodeling and subcellular defects in heart failure due to myocardial infarction and aging

Although several risk factors including hypertension, cardiac hypertrophy, coronary artery disease, and diabetes are known to result in heart failure, elderly subjects are more susceptible to myocardial infarction and more likely to develop heart failure. This article is intended to discuss that cardiac dysfunction in hearts failing due to myocardial infarction and aging is associated with cardiac remodeling and defects in the subcellular organelles such as sarcolemma (SL), sarcoplasmic reticulum (SR), and myofibrils. Despite some differences in the pattern of heart failure due to myocardial infarction and aging with respect to their etiology and sequence of events, evidence has been presented to show that subcellular remodeling plays a critical role in the occurrence of intracellular Ca(2+)-overload and development of cardiac dysfunction in both types of failing heart. In particular, alterations in gene expression for SL and SR proteins induce Ca(2+)-handling abnormalities in cardiomyocytes, whereas those for myofibrillar proteins impair the interaction of Ca(2+) with myofibrils in hearts failing due to myocardial infarction and aging. In addition, different phosphorylation mechanisms, which regulate the activities of Ca(2+)-cycling proteins in SL and SR membranes as well as Ca(2+)-binding proteins in myofibrils, become defective in the failing heart. Accordingly, it is suggested that subcellular remodeling involving defects in Ca(2+)-handling and Ca(2+)-binding proteins as well as their regulatory mechanisms is intimately associated with cardiac remodeling and heart failure due to myocardial infarction and aging.     

Quantitation and distribution of beta-tubulin in human cardiac myocytes

Increasing evidence suggests that derangements of cytoskeletal proteins contribute to alterations in intracellular signaling, myocyte function, and the coupling of myocytes to the extracellular matrix during cardiac hypertrophy and failure. Data from animal studies have shown an increased density of beta-tubulin protein in the right or left ventricle subjected to pressure overload, and have demonstrated that interfering with excess polymerization of beta-tubulin improves contractility. We tested the hypothesis that beta-tubulin is increased in human left ventricular hypertrophy and end-stage heart failure. Confocal microscopy of fluorescently labeled beta-tubulin protein revealed an increased density of the beta-tubulin network in cardiomyocytes from both hypertrophied and failing human hearts as compared to cells from nonfailing hearts. Western blot analysis on total heart homogenate showed no change in beta-tubulin when data were normalized to either actin or calsequestrin, although there was a significant increase in failing human hearts when data were normalized only for a constant amount of protein per heart. The mRNA for beta-tubulin was not changed in hypertrophied hearts, but was significantly decreased in failing human hearts. Thus, similar to animal models, we have shown that the density of the microtubular network within the cardiomyocyte is increased in end-stage failing human hearts. We have also shown for the first time that beta-tubulin density is increased in cells from hypertrophied human hearts. Although the functional implications of this finding in the human heart remain to be explored, data from animal studies suggest that increased beta-tubulin protein contributes to cardiac dysfunction.     

Abnormalities of calcium cycling in the hypertrophied and failing heart

Progressive deterioration of cardiac contractility is a central feature of congestive heart failure (CHF) in humans. In this report we review those studies that have addressed the idea that alterations of intracellular calcium (Ca(2+)) regulation is primarily responsible for the depressed contractility of the failing heart. The review points out that Ca(2+)transients and contraction are similar in non-failing and failing myocytes at very slow frequencies of stimulation (and other low stress environments). Faster pacing rates, high Ca(2+)and beta-adrenergic stimulation reveal large reductions in contractile reserve in failing myocytes. The underlying cellular basis of these defects is then considered. Studies showing changes in the abundance of L-type Ca(2+)channels, Ca(2+)transport proteins [sarcoplasmic reticulum Ca(2+)ATPase (SERCA2), phospholamban (PLB), Na(+)/Ca(2+) exchanger (NCX)] and Ca(2+) release channels (RYR) in excitation-contraction coupling and Ca(2+)release and uptake by the sarcoplasmic reticulum (SR) are reviewed. These observations support our hypotheses that (i) defective Ca(2+)regulation involves multiple molecules and processes, not one molecule, (ii) the initiation and progression of CHF inolves defective Ca(2+)regulation, and (iii) prevention or correction of Ca(2+)regulatory defects in the early stages of cardiac diseases can delay or prevent the onset of CHF.     

A SERCA2 pump with an increased Ca2+ affinity can lead to severe cardiac hypertrophy, stress intolerance and reduced life span

Abnormal Ca(2+) cycling in the failing heart might be corrected by enhancing the activity of the cardiac Ca(2+) pump, the sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) isoform. This can be obtained by increasing the pump's affinity for Ca(2+) by suppressing phospholamban (PLB) activity, the in vivo inhibitor of SERCA2a. In SKO mice, gene-targeted replacement of SERCA2a by SERCA2b, a pump with a higher Ca(2+) affinity, results in cardiac hypertrophy and dysfunction. The stronger PLB inhibition on cardiac morphology and performance observed in SKO was investigated here in DKO mice, which were obtained by crossing SKO with PLB(-/-) mice. The affinity for Ca(2+) of SERCA2 was found to be further increased in these DKO mice. Relative to wild-type and SKO mice, DKO mice were much less spontaneously active and showed a reduced life span. The DKO mice also displayed a severe cardiac phenotype characterized by a more pronounced concentric hypertrophy, diastolic dysfunction and increased ventricular stiffness. Strikingly, beta-adrenergic or forced exercise stress induced acute heart failure and death in DKO mice. Therefore, the increased PLB inhibition represents a compensation for the imposed high Ca(2+)-affinity of SERCA2b in the SKO heart. Limiting SERCA2's affinity for Ca(2+) is physiologically important for normal cardiac function. An improved Ca(2+) transport in the sarcoplasmic reticulum may correct Ca(2+) mishandling in heart failure, but a SERCA pump with a much higher Ca(2+) affinity may be detrimental.     

Prevention of abnormal sarcoplasmic reticulum calcium transport and protein expression in post-infarction heart failure using 3, 5-diiodothyropropionic acid (DITPA)

Heart failure of diverse causes is associated with abnormalities of sarcoplasmic reticulum (SR) Ca(2+)transport. The purpose of this study was to determine whether the thyroid hormone analogue, 3,5-diiodothyropropionic acid (DITPA), prevents abnormal Ca(2+)transport and expression of SR proteins associated with post-infarction heart failure. New Zealand White rabbits were randomly assigned to circumflex artery ligation or sham operation, and to DITPA administration (3.75 mg/kg/day) or no treatment in a two-by-two factorial design. After 3 weeks, echo-Doppler and LV hemodynamic measurements were performed. From ventricular tissue, single myocyte shortening and relaxation were determined, and Ca(2+)transport was measured in homogenates and SR-enriched microsomes. Levels of mRNA and protein content were determined for the SR Ca(2+)-ATPase (SERCA2a), phospholamban (PLB), cardiac ryanodine receptor (RyR-2) and calsequestrin. The administration of DITPA improved LV contraction and relaxation and improved myocyte shortening in infarcted animals. The improvements in LV and myocyte function were associated with increases in V(max)for SR Ca(2+)transport in both homogenates and microsomes. Also, DITPA prevented the decrease in LV protein density for SERCA2a, PLB and RyR-2 post-infarction, without measurable changes in mRNA levels. The thyroid hormone analogue, DITPA, improves LV, myocyte and SR function in infarcted hearts and prevents the downregulation of SR proteins associated with post-infarction heart failure. The specific effects of DITPA on post-infarction SR Ca(2+)transport and the expression of SR proteins make this compound a potentially useful therapeutic agent for LV systolic and/or diastolic dysfunction.