Augmented protein kinase C-alpha-induced myofilament protein phosphorylation contributes to myofilament dysfunction in experimental congestive heart failure

It is becoming clear that upregulated protein kinase C (PKC) signaling plays a role in reduced ventricular myofilament contractility observed in congestive heart failure. However, data are scant regarding which PKC isozymes are involved. There is evidence that PKC-alpha may be of particular importance. Here, we examined PKC-alpha quantity, activity, and signaling to myofilaments in chronically remodeled myocytes obtained from rats in either early heart failure or end-stage congestive heart failure. Immunoblotting revealed that PKC-alpha expression and activation was unaltered in early heart failure but increased in end-stage congestive heart failure. Left ventricular myocytes were isolated by mechanical homogenization, Triton-skinned, and attached to micropipettes that projected from a force transducer and motor. Myofilament function was characterized by an active force-[Ca(2+)] relation to obtain Ca(2+)-saturated maximal force (F(max)) and myofilament Ca(2+) sensitivity (indexed by EC(50)) before and after incubation with PKC-alpha, protein phosphatase type 1 (PP1), or PP2a. PKC-alpha treatment induced a 30% decline in F(max) and 55% increase in the EC(50) in control cells but had no impact on myofilament function in failing cells. PP1-mediated dephosphorylation increased F(max) (15%) and decreased EC(50) ( approximately 20%) in failing myofilaments but had no effect in control cells. PP2a-dependent dephosphorylation had no effect on myofilament function in either group. Lastly, PP1 dephosphorylation restored myofilament function in control cells hyperphosphorylated with PKC-alpha. Collectively, our results suggest that in end-stage congestive heart failure, the myofilament proteins exist in a hyperphosphorylated state attributable, in part, to increased activity and signaling of PKC-alpha.     

Vascular alterations during the development and progression of experimental heart failure

BACKGROUND: Congestive heart failure (HF) is a multifactorial and progressive condition associated with multiple systemic and vascular alterations. The onset and progression of these alterations and the cause of the condition remain undefined. The main purpose of the present study was to help understand the temporal evolution of vascular alterations and their contribution to the pathogenesis of HF. Vascular reactivity to angiotensin II (Ang II) and norepinephrine (NE), as well as circulating and local angiotensin-converting enzyme (ACE) activity, were assessed in the Syrian cardiomyopathic hamster (SCH) model. METHODS AND RESULTS: We have shown previously that in 2-month-old SCH animals that had not yet developed the clinical manifestations of HF, the contractile response of aortic rings to Ang II was markedly enhanced compared with normal animals. In addition, SCHs showed increased ACE activity in aortic tissue. To assess the relevance of these findings to the development and progression of HF, the temporal evolution of the contractile response of aortic rings to Ang II and NE was evaluated in hamsters at 2, 6, and 11 months of age. Age-matched normal hamsters were used as controls. Within the SCH group, the maximal contraction induced to 10 mumol/L of NE in 2- and 11-month-old animals was similar, but significantly greater than in the age-matched controls (for 2-month-old animals; 1.43 +/- 0.21 g in SCHs v 1.04 +/- 0.15 g in controls; P < .05 and for 11-month-old animals; 1.41 +/- 0.14 g in SCHs v 1.06 +/- 0.07 g in controls; P < .05). The drug concentrations necessary to obtain 50% of the maximal response from the NE concentration-response curves were similar for SCHs and controls at all ages tested. In contrast, the contractility induced by 0.1 mumol/L of Ang II increased progressively in cardiomyopathic animals from 2 to 11 months of age (from 1.3 +/- 0.1 to 1.8 +/- 0.2 g; n = 9; P < .05). In age-matched normal hamsters, the contractile response to Ang II (0.9 +/- 0.1 g) did not vary with age. These findings were observed concomitantly with an increased ACE activity in plasma (18.65 +/- 1.77 nmol/mg x min in controls v 26.5 +/- 1.79 nmol/mg x min in SCHs; P < .05; n = 7) and in heart tissue (0.244 +/- 0.016 nmol/mg x min in controls v 0.563 +/- 0.027 nmol/mg x min in SCHs; P < .05; n = 20) of 11-month-old SCHs. CONCLUSIONS: These results suggest that, in young animals, increased vascular response to elevated levels of NE and hyperreactivity to Ang II could be critical factors in the development and progression of HF. Indeed, Ang II-induced contractility, as well as plasma and heart ACE activity, are good predictors of the progression and severity of HF.     

Protein kinase Cepsilon interacts with and inhibits the permeability transition pore in cardiac mitochondria

Although functional coupling between protein kinase Cepsilon (PKCepsilon) and mitochondria has been implicated in the genesis of cardioprotection, the signal transduction mechanisms that enable this link and the identities of the mitochondrial proteins modulated by PKCepsilon remain unknown. Based on recent evidence that the mitochondrial permeability transition pore may be involved in ischemia/reperfusion injury, we hypothesized that protein-protein interactions between PKCepsilon and mitochondrial pore components may serve as a signaling mechanism to modulate pore function and thus engender cardioprotection. Coimmunoprecipitation and GST-based affinity pull-down from mouse cardiac mitochondria revealed interaction of PKCepsilon with components of the pore, namely voltage-dependent anion channel (VDAC), adenine nucleotide translocase (ANT), and hexokinase II (HKII). VDAC1, ANT1, and HKII were present in the PKCepsilon complex at approximately 2%, approximately 0.2%, and approximately 1% of their total expression, respectively. Moreover, in vitro studies demonstrated that PKCepsilon can directly bind and phosphorylate VDAC1. Incubation of isolated cardiac mitochondria with recombinant PKCepsilon resulted in a significant inhibition of Ca2+-induced mitochondrial swelling, an index of pore opening. Furthermore, cardiac-specific expression of active PKCepsilon in mice, which is cardioprotective, greatly increased interaction of PKCepsilon with the pore components and inhibited Ca2+-induced pore opening. In contrast, cardiac expression of kinase-inactive PKCepsilon did not affect pore opening. Finally, administration of the pore opener atractyloside significantly attenuated the infarct-sparing effect of PKCepsilon transgenesis. Collectively, these data demonstrate that PKCepsilon forms physical interactions with components of the cardiac mitochondrial pore. This in turn inhibits the pathological function of the pore and contributes to PKCepsilon-induced cardioprotection.     

Extracellular matrix remodeling during the progression of volume overload-induced heart failure

Volume overload-induced heart failure results in progressive left ventricular remodeling characterized by chamber dilation, eccentric cardiac myocyte hypertrophy and changes in extracellular matrix (ECM) remodeling changes. The ECM matrix scaffold is an important determinant of the structural integrity of the myocardium and actively participates in force transmission across the LV wall. In response to this hemodynamic overload, the ECM undergoes a distinct pattern of remodeling that differs from pressure overload. Once thought to be a static entity, the ECM is now regarded to be a highly adaptive structure that is dynamically regulated by mechanical stress, neurohormonal activation, inflammation and oxidative stress, that result in alterations in collagen and other matrix components and a net change in matrix metalloproteinase (MMP) expression and activation. These changes dictate overall ECM turnover during volume overload hear failure progression. This review will discuss the cellular and molecular mechanisms that dictate the temporal patterns of ECM remodeling during heart disease progression.     

Thin myofilament proteins in norm and heart failure I. Polymerizability of myocardial Straub actin in acute and chronic heart failure

Summary The reduced and intrinsic viscosities of myocardial Straub F-actin from the left ventricle of a practically healthy man were equal to 3.05±0.2 and 2.4±0.32 and from the right ventricle were 2.37±0.2 and 2.1±0.3 dl/g, respectively (the difference between ventricles was not significant). The average length of filaments measured by flow birefringence technique was equal to 1.3±0.04 m, the number-average length (Ln), determined by the electron microscopy was 1.4 m, the weight-average length (Lw), was 2 m and the maximal one was 5.5 m. The histograms showed that the most characteristic length was that of 0.8–1.2 m.According to the flow birefringence data canine myocardial F-actin had a length similar to that of myocardial F-actin from a practically healthy man, though its reduced and intrinsic viscosities were higher.In acute and especially chronic congestive heart failure the actin polymerizability was sharply reduced. In consequence, in acute heart failure the number-average length of F-actin filaments was decreased by 43% and in congestive heart failure by 65.7%.The characteristic length in acute heart failure shifts to the range of 0.2–0.6 m, while in congestive heart failure the range is 0.2–0.4 m. This fact can possibly explain why during preparation of actin from the pathologically changed myocardium according to the methods including purification by the cycles of polymerization-sedimentation-depolymerization, the pathologically changed actin is discarded and the normal actin remains.A definite parallel was observed between the reduction of actin polymerizability and the ability of myocardial glycerinated fiber bundles (MBGF) to generate force.We conclude that the changes of actin properties in heart failure may cause a decrease in contractibility of the myocardial contractile protein system.     

The effects of endothelin-A receptor blockade during the progression of pacing-induced congestive heart failure

Objectives. We sought to identify the effects of endothelin (ET) subtype-A (ET_A)) receptor blockade during the development of congestive heart failure (CHF) on left ventricle (LV) function and contractility.Background. Congested heart failure causes increased plasma levels of ET and ET_Areceptor activation.Methods. Yorkshire pigs were assigned to four groups: 1) CHF: 240 beats/min for 3 weeks; n = 7; 2) CHF/ET_A-High Dose: paced for 2 weeks then ET_Areceptor blockade (BMS 193884, 50 mg/kg, b.i.d.) for the last week of pacing; n = 6; 3) CHF/ET_A-Low Dose: pacing for 2 weeks then ET_Areceptor blockade (BMS 193884, 12.5 mg/kg, b.i.d.) for the last week, n = 6; and 4) Control: n = 8.Results. Left ventricle fractional shortening decreased with CHF compared with control (12 ± 1 vs. 39 ± 1%, p < 0.05) and increased in the CHF/ET_AHigh and Low Dose groups (23 ± 3 and 25 ± 1%, p < 0.05). The LV peak wall stress and wall force increased approximately twofold with CHF and remained increased with ET_Areceptor blockade. With CHF, systemic vascular resistance increased by 120%, was normalized in the CHF/ET_AHigh Dose group, and fell by 43% from CHF values in the Low Dose group (p < 0.05). Plasma catecholamines increased fourfold in the CHF group and were reduced by 48% in both CHF/ET_Ablockade groups. The LV myocyte velocity of shortening was reduced with CHF (32 ± 3 vs. 54 ± 3 μm/s, p < 0.05), was higher in the CHF/ET_AHigh Dose group (39 ± 1 μm/s, p < 0.05), and was similar to CHF values in the Low Dose group.Conclusions. ET_Areceptor activation may contribute to the progression of LV dysfunction with CHF.     

大鼠压力负荷性心力衰竭进展过程中三磷酸腺苷敏感性钾通道功能观察

目的 建立压力负荷性心力衰竭(心衰)大鼠模型,在模拟缺血的条件下,研究心衰发展的不同阶段大鼠左室心肌细胞膜三磷酸腺苷敏感性钾通道(K_(APT)通道)的功能变化.方法 雄性Wistar大鼠被随机分配到4周假手术组(F4)11只、12周假手术组(F12)10只、4周手术组(T4)13只和12周手术组(T12)10只.使用腹主动脉缩窄法建立大鼠心衰模型.通过右侧颈总动脉插管记录血流动力学指标.使用改良的Langendorff法分离大鼠左室心肌细胞.应用全细胞膜片钳技术,在电压钳模式下,记录F4、T4、F12、T12各组大鼠左室心肌细胞在正常状态及模拟缺血液灌流条件下K_(ATP)通道电流情况,比较0 mV电压下各组电流密度大小.结果 动脉收缩压、舒张压及平均动脉压从术后4周开始明显升高,但是到12周的时候有所降低.左心室舒张末压和左心室内压上升/下降最大速率在T4组没有明显变化,在T12组左心室舒张末压明显升高而左心室内压上升/下降最大速率明显降低.膜片钳实验结果显示,基础状态下,全细胞膜电流密度在各组之间差异均无统计学意义,但是在模拟缺血液灌流25 min时,T12组大鼠K_(ATP)通道的开放幅度比F12组明显增加,电流密度明显增大[(28.11±3.91)pA/pF比(11.55±1.17)pA/pF,P<0.01],而T4组与F4组之间差异无统计学意义[(14.09±5.74)pA/pF比(11.74±3.68)pA/pF,P>0.05].无论是T12组还是T4组,大鼠心肌中K(ATP)通道的基因表达并没有增多.结论 成功构建压力负荷性大鼠心衰模型,T4组心肌肥厚,T12组呈慢性心衰.心肌细胞膜K_(ATP)通道在慢性心衰期对缺血的敏感性增强,全细胞膜电流明显增大,而这种反应发生在心肌细胞K_(ATP)通道表达增多之前.     

Reorganization of myofilament proteins and decreased cGMP-dependent protein kinase in the human uterus during pregnancy

Excessive or premature contractions of uterine smooth muscle may contribute to preterm labor. Contractile stimuli induce myosin and actin filament interactions through calcium-dependent myosin phosphorylation. The mechanisms that maintain myometrial quiescence until term are not well established, but may include control of calcium levels by nitric oxide and cGMP signaling and thin filament (caldesmon and calponin) regulation. Previously, we reported that myometrial tissues from pregnant rats are not responsive to cGMP due to decreases in cGMP-dependent protein kinase. Considering the well documented differences in the endocrinology of parturition among species, this study was conducted to test the hypothesis that the levels and subcellular distribution of caldesmon, calponin, and cGMP-dependent protein kinase are regulated with the hormonal milieu of human pregnancy. Whereas cGMP-dependent protein kinase was significantly reduced in the human uterus during pregnancy, caldesmon expression was significantly increased, and both caldesmon and calponin were redistributed to a readily extractable subcellular pool. These data suggest that cGMP-dependent protein kinase does not mediate gestational quiescence. Redistribution of thin filament-associated proteins, however, may alter uterine smooth muscle tone or the cytoskeletal framework of myocytes to maintain gestation despite the substantial distention that accompanies all intrauterine pregnancies.     

Role of inflammation in the progression of heart failure

Chronic heart failure (HF) is a disorder characterized in part by immune activation and inflammation. Thus, patients with HF have elevated levels of a number of inflammatory cytokines, both in the circulation and in the failing heart itself. Several mechanisms for this immune activation, which are not mutually exclusive, have been suggested, including neurohormonal activation, hemodynamic overload, and activation of the innate immune system secondary to cardiac stress. Importantly, experimental studies have shown that inflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1b, and monocyte chemoattractant peptide-1 may contribute to the development and progression of HF by promoting myocardial hypertrophy, activating matrix metalloproteinases, provoking contractile dysfunction, and inducing apoptosis. However, inflammatory cytokines may also have adaptive and cardioprotective effects. This important aspect of cytokine biology must be kept in mind when designing new immunomodulatory treatment modalities in HF.     

Evolution of matrix metalloprotease and tissue inhibitor expression during heart failure progression in the infarcted rat

OBJECTIVE: Characterize the timecourse of matrix metalloproteinase (MMP-1, -2, -3, -7, -9, -11, -12, -13, and -14) and endogenous tissue inhibitors of MMPs (TIMP-1, -2, -3, and -4) upregulation during left ventricular (LV) remodeling following myocardial infarction (MI) in rats. METHODS: The descending left coronary artery of male rats (Rattus norvegicus) was ligated to produce a MI. LV function and dilation were assessed from 1 day to 16 weeks post-MI. Protein and mRNA extraction was done on LV samples containing scar and myocardium together. Gelatinase activity was measured by zymography. Westerns were run on the MMPs known to cleave fibrillar collagen in the rat (MMP-8, -13, and -14) as well as TIMP-1, -2, and -4. RESULTS: Average infarct size was 38.6+/-1.1%, and produced LV dysfunction and progressive LV dilation. Thoracic ascites, a marker of congestive heart failure (HF), was not present until 12 weeks post-MI. Upregulation of MMP-2, -8, -9, -13, and -14 and TIMP-1 and TIMP-2 was detected at different timepoints during HF progression. Increased MMP protein levels occurred sometimes without a corresponding elevation in mRNA levels, and increased TIMP mRNA levels without increased protein levels. MMP-13 active form was elevated during the first 2 weeks post-MI while TIMP-1 and TIMP-2 protein levels were not significantly elevated until 2 weeks post-MI. MMP-8 and MMP-14 protein levels increased later during heart failure progression. CONCLUSION: MMP/TIMP upregulation evolves over time following infarction in the rat LV. Some MMPs were significantly elevated during the first week post-MI (MMP-13, -2, and -9) and another was not until 16 weeks post-MI (MMP-14). The dissociation between LV MMP/TIMP mRNA and protein levels shows that post-translation processing occurs in the rat heart.     

Protein kinase D is a novel mediator of cardiac troponin I phosphorylation and regulates myofilament function

Protein kinase D (PKD) is a serine kinase whose myocardial substrates are unknown. Yeast 2-hybrid screening of a human cardiac library, using the PKD catalytic domain as bait, identified cardiac troponin I (cTnI), myosin-binding protein C (cMyBP-C), and telethonin as PKD-interacting proteins. In vitro phosphorylation assays revealed PKD-mediated phosphorylation of cTnI, cMyBP-C, and telethonin, as well as myomesin. Peptide mass fingerprint analysis of cTnI by liquid chromatography-coupled mass spectrometry indicated PKD-mediated phosphorylation of a peptide containing Ser22 and Ser23, the protein kinase A (PKA) targets. Ser22 and Ser23 were replaced by Ala, either singly (Ser22Ala or Ser23Ala) or jointly (Ser22/23Ala), and the troponin complex reconstituted in vitro, using wild-type or mutated cTnI together with wild-type cardiac troponin C and troponin T. PKD-mediated cTnI phosphorylation was reduced in complexes containing Ser22Ala or Ser23Ala cTnI and completely abolished in the complex containing Ser22/23Ala cTnI, indicating that Ser22 and Ser23 are both targeted by PKD. Furthermore, troponin complex containing wild-type cTnI was phosphorylated with similar kinetics and stoichiometry (approximately 2 mol phosphate/mol cTnI) by both PKD and PKA. To determine the functional impact of PKD-mediated phosphorylation, Ca2+ sensitivity of tension development was studied in a rat skinned ventricular myocyte preparation. PKD-mediated phosphorylation did not affect maximal tension but produced a significant rightward shift of the tension-pCa relationship, indicating reduced myofilament Ca2+ sensitivity. At submaximal Ca2+ activation, PKD-mediated phosphorylation also accelerated isometric crossbridge cycling kinetics. Our data suggest that PKD is a novel mediator of cTnI phosphorylation at the PKA sites and may contribute to the regulation of myofilament function.     

Inhibition of glycogen synthase kinase 3beta during heart failure is protective

Glycogen synthase kinase (GSK)-3, a negative regulator of cardiac hypertrophy, is inactivated in failing hearts. To examine the histopathological and functional consequence of the persistent inhibition of GSK-3beta in the heart in vivo, we generated transgenic mice with cardiac-specific overexpression of dominant negative GSK-3beta (Tg-GSK-3beta-DN) and tetracycline-regulatable wild-type GSK-3beta. GSK-3beta-DN significantly reduced the kinase activity of endogenous GSK-3beta, inhibited phosphorylation of eukaryotic translation initiation factor 2B epsilon, and induced accumulation of beta-catenin and myeloid cell leukemia-1, confirming that GSK-3beta-DN acts as a dominant negative in vivo. Tg-GSK-3beta-DN exhibited concentric hypertrophy at baseline, accompanied by upregulation of the alpha-myosin heavy chain gene and increases in cardiac function, as evidenced by a significantly greater Emax after dobutamine infusion and percentage of contraction in isolated cardiac myocytes, indicating that inhibition of GSK-3beta induces well-compensated hypertrophy. Although transverse aortic constriction induced a similar increase in hypertrophy in both Tg-GSK-3beta-DN and nontransgenic mice, Tg-GSK-3beta-DN exhibited better left ventricular function and less fibrosis and apoptosis than nontransgenic mice. Induction of the GSK-3beta transgene in tetracycline-regulatable wild-type GSK-3beta mice induced left ventricular dysfunction and premature death, accompanied by increases in apoptosis and fibrosis. Overexpression of GSK-3beta-DN in cardiac myocytes inhibited tumor necrosis factor-alpha-induced apoptosis, and the antiapoptotic effect of GSK-3beta-DN was abrogated in the absence of myeloid cell leukemia-1. These results suggest that persistent inhibition of GSK-3beta induces compensatory hypertrophy, inhibits apoptosis and fibrosis, and increases cardiac contractility and that the antiapoptotic effect of GSK-3beta inhibition is mediated by myeloid cell leukemia-1. Thus, downregulation of GSK-3beta during heart failure could be compensatory.     

Myosin heavy chain synthesis during the progression of chronic tachycardia induced heart failure in rabbits

Chronic tachycardia causes LV dilatation and dysfunction, with no hypertrophy. However, the contributing mechanisms responsible for the left ventricular (LV) remodeling in the absence of myocardial growth in this model of heart failure remain unclear. Therefore, the goal of the present study was to serially examine changes in LV function, steady state myosin heavy chain (MHC) mRNA levels, in vivo synthesis rates, and abundance with the progression of chronic tachycardia induced heart failure. Adult rabbits (3.5–4.5 kg) were studied after one, two, or three weeks of pacing ventricular tachycardia (VT; 400 bpm) and in controls (n=6 for all groups). LV fractional shortening was reduced by 30 % at week one and by over 50 % at week three of chronic VT. End‐diastolic dimension (EDD) increased at week two compared to controls (1.66 ± 0.10 vs 1.35 ± 0.11 cm, p < 0.05) and increased further at week three of VT (1.70 ± 0.06 cm, p < 0.05). The progressive changes in LV geometry and function with chronic VT were not associated with concomitant time dependent changes in LV mass or MHC mRNA levels. In contrast, MHC fractional synthesis rates increased and reached statistical significance at week three of VT compared to controls (8.3 ± 0.8 vs 5.5 ± 0.5 %/day, p < 0.05). Despite the stable or increased MHC protein synthesis rates, there was no change in MHC protein abundance at any point during the progression of VT induced heart failure, implicating enhanced MHC protein degradation. Thus, this study demonstrated that a contributory mechanism for the LV remodeling and lack of myocardial growth, which occurs with VT induced heart failure, is enhanced contractile protein degradative processes. Received: 17 July 1997 , Returned for revision: 20 August 1997, Revision received: 8 September 1997, Accepted: 7 October 1997