Search

Background: Abnormal sarcoplasmic reticulum calcium (Ca) cycling is increasingly recognized as an important mechanism for increased ventricular automaticity that leads to lethal ventricular arrhythmias. Previous studies have linked lethal familial arrhythmogenic disorders to mutations in the ryanodine receptor and calsequestrin genes, which interact with junctin and triadin to form a macromolecular Ca-signaling complex. The essential physiological effects of junctin and its potential regulatory roles in sarcoplasmic reticulum Ca cycling and Ca-dependent cardiac functions, such as myocyte contractility and automaticity, are unknown., Methods and Results: The junctin gene was targeted in embryonic stem cells, and a junctin-deficient mouse was generated. Ablation of junctin was associated with enhanced cardiac function in vivo, and junctin-deficient cardiomyocytes exhibited increased contractile and Ca-cycling parameters. Short-term isoproterenol stimulation elicited arrhythmias, including premature ventricular contractions, atrioventricular heart block, and ventricular tachycardia. Long-term isoproterenol infusion also induced premature ventricular contractions and atrioventricular heart block in junctin-null mice. Further examination of the electrical activity revealed a significant increase in the occurrence of delayed afterdepolarizations. Consistently, 25% of the junctin-null mice died by 3 months of age with structurally normal hearts., Conclusions: Junctin is an essential regulator of sarcoplasmic reticulum Ca release and contractility in normal hearts. Ablation of junctin is associated with aberrant Ca homeostasis, which leads to fatal arrhythmias. Thus, normal intracellular Ca cycling relies on maintenance of junctin levels and an intricate balance among the components in the sarcoplasmic reticulum quaternary Ca-signaling complex.

Junctin is one of the components of the ryanodine receptor Ca release channel complex in sarcoplasmic reticulum. To determine the role of acute alteration of junctin protein levels on cardiomyocyte contractility, we used adenoviral-mediated gene transfer techniques in adult rat cardiomyocytes. Acute downregulation of junctin by 40% resulted in significant increases in cell shortening, rate of contraction (+dL/dt), and rate of relaxation (-dL/dt). The alteration of contractile parameters was associated with increased Ca transient peak and accelerated Ca decay. However, all these contractile and Ca kinetic parameters were depressed significantly when junctin levels were upregulated by 60%. Importantly, there were no alterations in other Ca-cycling protein levels when junctin levels were either decreased or increased. These findings suggest that junctin plays a prominent role in cardiomyocyte Ca-cycling and contractility.

Depressed sarcoplasmic reticulum (SR) Ca-cycling is a hallmark of human and experimental heart failure. Strategies to improve this impairment by either increasing SERCA2a levels or decreasing phospholamban (PLN) activity have been suggested as promising therapeutic targets. Indeed, ablation of PLN gene in mice was associated with greatly enhanced cardiac Ca-cycling and performance. Intriguingly, this hyperdynamic cardiac function was maintained throughout the lifetime of the mouse without observable pathological consequences. To determine the cellular alterations in the expression or modification of myocardial proteins, which are associated with the enhanced cardiac contractility, we performed a proteomics-based analysis of PLN knockout (PLN-KO) hearts in comparison to isogenic wild-types. By use of 2-dimensional gel electrophoresis (2-DE), approximately 3300 distinct protein spots were detected in either wild-type or PLN-KO ventricles. Protein spots observed to be altered between PLN-KO and wild-type hearts were subjected to tryptic peptide mass fingerprinting for identification by MALDI-TOF mass spectrometry in combination with LC/MS/MS analysis. In addition, two-dimensional 32P-autoradiography was performed to analyze the phosphorylation profiles of PLN-KO cardiomyocytes. We identified alterations in the expression level of more than 100 ventricular proteins, along with changes in phosphorylation status of important regulatory proteins in the PLN-KO. These protein changes were observed mainly in two subcellular compartments: the cardiac contractile apparatus, and metabolism/energetics. Our findings suggest that numerous alterations in protein expression and phosphorylation state occurred upon ablation of PLN and that a complex functional relationship among proteins involved in calcium handling, myofibrils, and energy production may exist to coordinately maintain the hyperdynamic cardiac contractile performance of the PLN-KO mouse in the long term.

Multiple studies have shown that phospholamban (PLN) plays a key role in regulation of frequency-dependent increase of cardiac contraction, a hallmark of the contractile reserve in myocardium. However, the mechanisms underlying this relationship remain elusive. Phosphorylation of PLN occurs on residues: serine-16 (Ser(16)) and threonine-17 (Thr(17)) in vivo. In isolated wild-type cardiomyocytes, we found that increases of stimulation frequency from 0.5 to 5 Hz were associated with increased Thr(17) phosphorylation of PLN and cardiac contractility. To further delineate the role of PLN phosphorylation in the frequency-dependent increases of cardiac function, three transgenic mouse models, expressing wild-type, Ser16Ala (S16A), or Thr17Ala (T17A) mutant PLN in the null background were generated. Transgenic lines expressing similar levels of wild-type or mutant PLN were selected and isolated cardiomyocytes were paced from 0.5 to 5 Hz. Upon increases in pacing frequency, the fractional shortening (FS) and rates of contraction (+dL/dt) and relaxation (-dL/dt) increased in wild-type and S16A mutant PLN cardiomyocytes. In contrast, in myocytes expressing the T17A mutant PLN, there were no increases in FS and +/-dL/dt upon increasing the frequency of stimulation. The time to 50% peak shortening (TTP(50)) and to 50% relaxation (TTR(50)) were also abbreviated to a much higher extent (two-fold) in wild-type and S16A mutant compared to T17A mutant PLN cardiomyocytes. These results indicate that Thr(17) phosphorylation of PLN is the major contributor to frequency-dependent increases of contractile and relaxation parameters in mouse cardiomyocytes, although some increases in these parameters occur even in the absence of PLN phosphorylation. Thus, the positive force-frequency relationship in cardiomyocytes is mechanistically and mainly related to PLN phosphorylation.

Objective: Phospholamban ablation or ectopic expression of SERCA1a in the heart results in significant increases in cardiac contractile parameters. The aim of the present study was to determine whether a combination of these two genetic manipulations may lead to further augmentation of cardiac function., Methods: Transgenic mice with cardiac specific overexpression of SERCA1a were mated with phospholamban deficient mice to generate a model with SERCA1a overexpression in the phospholamban null background (SERCA1(OE)/PLB(KO)). The cardiac phenotype was characterized using quantitative immunoblotting, sarcoplasmic reticulum calcium uptake and single myocyte mechanics and calcium kinetics., Results: Quantitative immunoblotting revealed an increase of 1.8-fold in total SERCA level, while SERCA2 was decreased to 50% of wild types. Isolated myocytes indicated increases in the maximal rates of contraction by 195 and 125%, the maximal rates of relaxation by 200 and 124%, while the time for 80% decay of the Ca(2+)-transient was decreased to 43 and 75%, in SERCA1(OE)/PLB(KO) hearts, compared to SERCA1a overexpressors and phospholamban knockouts, respectively. These mechanical alterations reflected parallel alterations in V(max) and EC(50) for Ca(2+) of the sarcoplasmic reticulum Ca(2+) transport system. Furthermore, there were no significant cardiac histological or pathological alterations, and the myocyte contractile parameters remained enhanced, up to 12 months of age., Conclusions: These findings suggest that a combination of SERCA1a overexpression and phospholamban ablation results in further enhancement of myocyte contractility over each individual alteration.

Objective: Impaired myocyte Ca2+ handling is a common characteristic of failing hearts and increases in calcineurin activity, a Ca2+-sensitive phosphatase, have been implicated in heart failure phenotype. Transgenic mice with cardiac-specific expression of an active form of calcineurin display depressed function, hypertrophy and heart failure. We examined whether defects in cardiomyocyte Ca2+ handling properties contribute to the impaired cardiac function in calcineurin transgenic mice., Methods: The levels of SR Ca2+ handling proteins, SR Ca2+ transport function and cardiomyocyte mechanics, as well as Ca2+ kinetics were examined in mice overexpressing a constitutively active form of calcineurin., Results: Transgenic expression of activated calcineurin catalytic subunit resulted in significant protein increases (66%) in SERCA2 and decreases (35%) in phospholamban, as well as enhanced (approximately 80%) phospholamban phosphorylation. These alterations in the SR Ca2+-transport proteins resulted in increased V(max) and Ca2+-affinity of SERCA2. The myofibrillar Mg-ATPase activity was also significantly increased at pCa>6.0. The enhanced SR Ca2+ handling and Mg-ATPase activity reflected significant elevation in myocyte contractile parameters (3-fold), Ca2+ transient amplitude (1.5-fold) and the rate of Ca2+ signal decay (2-fold). In contrast, in vivo cardiac function assessed by echocardiography, indicated severely depressed contractility in calcineurin hearts. The apparent disparity in contractile properties between the cellular and multicellular preparations may be partially due to tissue remodeling, including interstitial fibrosis and a marked reduction (45%), dephosphorylation (81%) and redistribution of the gap junctional protein connexin-43, which could compromise intercellular communication., Conclusion: Despite enhanced SR Ca2+ handling and contractility in myocytes, pathological remodeling and defects in intercellular coupling may underlie contractile dysfunction of the calcineurin hearts.

Activation of cAMP-dependent protein kinase A (PKA) in ventricular myocytes by isoproterenol (Iso) causes phosphorylation of both phospholamban (PLB) and troponin I (TnI) and accelerates relaxation by up to twofold. Because PLB phosphorylation increases sarcoplasmic reticulum (SR) Ca pumping and TnI phosphorylation increases the rate of Ca dissociation from the myofilaments, both factors could contribute to the acceleration of relaxation seen with PKA activation. To compare quantitatively the role of TnI versus PLB phosphorylation, we measured relaxation rates before and after maximal Iso treatment for twitches of matched amplitudes in ventricular myocytes and muscle from wild-type (WT) mice and from mice in which the PLB gene was knocked out (PLB-KO). Because Iso increases contractions, even in the PLB-KO mouse, extracellular [Ca] or sarcomere length was adjusted to obtain matching twitch amplitudes (in the presence and absence of Iso). In PLB-KO myocytes and muscles (which were allowed to shorten), Iso did not alter the time constant (tau) of relaxation ( approximately 29 ms). However, with increasing isometric force development in the PLB-KO muscles, Iso progressively but modestly accelerated relaxation (by 17%). These results contrast with WT myocytes and muscles where Iso greatly reduced tau of cell relaxation and intracellular Ca concentration decline (by 30-50%), independent of mechanical load. The Iso treatment used produced comparable increases in phosphorylation of TnI and PLB in WT. We conclude that the effect of beta-adrenergic activation on relaxation is mediated entirely by PLB phosphorylation in the absence of external load. However, TnI phosphorylation could contribute up to 14-18% of this lusitropic effect in the WT mouse during maximal isometric contractions.

Phospholamban is a low molecular weight phosphoprotein in cardiac sarcoplasmic reticulum. The regulatory role of phospholamban in vivo has recently been elucidated by targeting the gene of this protein in embryonic stem cells and generating phospholamban-deficient mice. The phospholamban knockout hearts exhibited significantly enhanced contractile parameters and attenuated responses to beta-agonists. The hyperdynamic cardiac function of the phospholamban knockout mice was not accompanied by any cytoarchitectural abnormalities or alterations in the expression levels of the cardiac sarcoplasmic reticulum Ca(2+)-ATPase, calsequestrin, Na(+)-Ca2+ exchanger, or the contractile proteins. Furthermore, the attenuation of the cardiac responses to beta-agonists was not due to alterations in the phosphorylation levels of the other key cardiac phosphoproteins in the phospholamban knockout hearts. However, ablation of phospholamban was associated with down-regulation of the ryanodine receptor, which suggests that a cross-talk between cardiac sarcoplasmic reticulum Ca2+ uptake and Ca2+ release occurred in an attempt to maintain Ca2+ homeostasis in these hyperdynamic phospholamban knockout hearts.

Recently, it has been reported that the protein kinase C (PKC) beta isoform plays a critical role in the development of hypertrophy and heart failure. The purpose of the present study was to clarify the mechanism by which activation of PKCbeta led to depressed cardiac function. Thus, we used a PKCbeta2 overexpressing mouse, an animal model of heart failure, to examine mechanical properties and Ca2+ signals of isolated left ventricular cardiomyocytes. The percentage of shortening, rate of shortening, and rate of relengthening of cardiomyocytes were markedly reduced in PKCbeta2 overexpression mice compared to wild-type control mice, although the baseline level and amplitude of Ca2+ signals were similar. These findings suggested a decreased myofilament responsiveness to Ca2+ in transgenic hearts. Therefore, the incorporation of [32P] inorganic phosphate into cardiac myofibrillar proteins was studied in Langendorff-perfused hearts. There was a significant increase in the degree of phosphorylation of troponin I in PKCbeta2-overexpressing transgenic mice. The depressed cardiomyocyte function improved after the superfusion of a PKCbeta selective inhibitor. These findings indicate that in vivo PKCbeta2-mediated phosphorylation of troponin I may decrease myofilament Ca2+ responsiveness, and thus causes cardiomyocyte dysfunction. Since chronic and excess activation of PKCbeta2 plays a direct and contributory role in the progression of cardiac dysfunction, the PKCbeta selective inhibitor may provide a new therapeutic modality in the setting of heart failure.

Increases in heart rate are accompanied by acceleration of relaxation. This effect is apparent at the single myocyte level and depends on sarcoplasmic reticulum (SR) Ca transport and Ca/calmodulin dependent protein kinase [CaMKII; see R. A. Bassani, A. Mattiazzi, and D. M. Bers. Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H703-H712, 1995]. Because phosphorylation of phospholamban (PLB) by CaMKII can stimulate SR Ca transport, it is a plausible candidate mechanism. We examined this issue using ventricular myocytes isolated from wild-type (WT) mice and those in which the PLB gene was ablated by gene targeting (PLB-KO). During steady-state (SS) stimulation, twitch relaxation and intracellular Ca concentration ([Ca]i) decline were significantly faster than after a rest in both WT and PLB-KO myocytes. Furthermore, the CaMKII inhibitor KN-93 (1 microM) abolished the stimulation-dependent acceleration of twitch [Ca]i decline in PLB-KO. This indicates that neither PLB nor its phosphorylation are required for the CaMKII-dependent acceleration of the SS twitch [Ca]i decline and relaxation. Other quantitative aspects of Ca transport in WT and PLB-KO myocytes were also examined. As expected, the time constant (tau) of [Ca]i decline during the SS twitch is much faster in PLB-KO than in WT myocytes (112 +/- 6 vs. 188 +/- 14 ms, P < 0.0001). There was also an increase in SS SR Ca load, based on the change of [Ca]i during rapid caffeine-induced contractures (CafC) with Na/Ca exchange blocked (565 +/- 74 nM for WT, 1118 +/- 133 nM for PLB-KO, P < 0.01). Accounting for cytosolic Ca buffering, this implies a 37% increase in SR Ca content. The tau for [Ca]i decline of the cafC with Na present indicated slower extrusion by Na/Ca exchange in the PLB-KO mouse (2.2 +/- 0.2 s in WT vs. 3.2 +/- 0.2 in PLB-KO, P < 0.01), although exchanger protein expression was unchanged. Integrated Ca flux analysis in WT and PLB-KO myocytes, respectively, shows that 90 and 96% of Ca during twitch relaxation is removed by the SR Ca-ATPase, 9 and 3.4% by Na/Ca exchange, and 0.5 and 0.1% by slow mechanisms (mitochondria Ca uniporter and sarcolemmal Ca-ATPase). We conclude that the PLB-KO myocytes retain a CaMKII-dependent acceleration of SS twitch [Ca]i decline. The PLB-KO (vs. WT) myocytes also have higher SR Ca pump activity, higher SR Ca load, and reduced Na/Ca exchange activity.

Aim: To investigate the effects of endocardial endothelium (EE) in myocardial mechanics of hypertrophied myocardium of rats., Methods: Hypertrophied myocardium has been developed through long-term renovascular hypertension in rats. Selective stripping of EE was achieved chemically and the effects of EE on the activation and relaxation of hypertrophied myocardium were investigated and compared to those in the presence and absence of intact EE., Results: Resting tension (RT) and peak developed tension (DT) of isometric contraction of the left ventricular papillary muscles of renovascular hypertensive rats (RHR) remained similar, but maximal rate of tension development and fall (+dT/dtmax and -dT/dtmax) were lowered, time to +dT/dtmax (TPP) and time to -dT/dtmax (TPN) were increased, and time to peak developed tension (TPT) and half relaxation time (RT1/2) were prolonged, vs sham-operated rats (Sham). After EE denudation, TPT, and RT1/2 were significantly abbreviated in RHR. The -dT/dtmax was significantly augmented while the +dT/dtmax was unaltered, resulting in a prominent decrease in the ratio of +dT/dtmax to -dT/dtmax., Conclusion: EE predominantly influences the relaxation in isolated myocardium and early diastolic filling events in hearts, and is involved in the cardiac compensatory mechanism in hypertrophied myocardium.

The selective removal of endocardial endothelium of rat left ventricular papillary muscles by 1-second immersion in 0.5% Triton X-100 showed little influence on resting tension and only a small decrease in peak isometric tension (8.3 +/- 1.4 vs 9.6 +/- 2.4 mN/mm2 at Lmax, p > 0.05) with no reduction in maximal rate of tension development (+dT/dtmax; 136 +/- 21 vs 137 +/- 18 mN/mm2/s, p > 0.05). In contrast, there was a marked increase in maximal rate of tension decline (-dT/dtmax) from 71 +/- 14 to 92 +/- 15 mN/mm2/s (p < 0.05), so that the ratio between +dT/dtmax and -dT/dtmax fell from 1.98 +/- 0.27 to 1.51 +/- 0.13 (p < 0.01). Removal of endocardial endothelium led to a significant shortening of isometric twitch contractions. Time to peak tension was abbreviated from 111 +/- 20 to 84 +/- 8 ms (p < 0.05) and the half relaxation time from 92 +/- 9 to 68 +/- 8 ms (p < 0.01). Time to +dT/dtmax was also shortened from 31 +/- 6 to 44 +/- 9 ms (p < 0.05) and time to -dT/dtmax from 90 +/- 12 to 62 +/- 10 ms (p < 0.01). These effects were not influenced by alterations in stimulation frequency or muscle length. The early onset of relaxation and abbreviated duration of relaxation together with an increased rate of decline in tension led to a shorter total twitch which may explain the slightly lower peak tension once the endocardial endothelium was removed. Our findings confirm that endocardial endothelium modulates myocardial contraction, with a predominant influence on relaxation.

Selective removal of endocardium by 1-s immersion of the muscle into 0.5% Triton X-100 resulted in a significant reduction of PT (peak isometric twitch tension) at 1.25 mmol.L-1 [Ca2+]o over the stimulation frequency from 0.2 to 2.0 Hz (3.2 +/- 0.2 vs 4.4 +/- 0.4 mN/mm2 at 1 Hz, P < 0.01), while +dT/dtmax was unaltered. Tension-[Ca2+]o relation was shifted in accordance to [Ca2+]o, but with no significant change on PT at high [Ca2+]o compared with endocardium-intact muscles. TPT (time to peak isometric tension) and RT1/2 (half isometric relaxation time) were typically shortened at all [Ca2+]o or various stimulation frequencies (TPT: 203 +/- 18 vs 265 +/- 37 ms; RT1/2: 77 +/- 10 vs 108 +/- 26 ms, at 1.25 mmol.L-1 and 1 Hz, P < 0.01). Stimulation duration-threshold curve was slightly shifted to the left, yet no change in ERP (effective refractory period) was found. The data demonstrated that endocardium was an important modulator of myocardial contractile performance.