Omecamtiv mecarbil augments cardiomyocyte contractile activity both at resting and systolic Ca2+ levels.

AIMS: Heart failure with reduced ejection fraction (HFrEF) is a disease with high mortality and morbidity. Recent positive inotropic drug developments focused on cardiac myofilaments, that is, direct activators of the myosin molecule and Ca2+ sensitizers for patients with advanced HFrEF. Omecamtiv mecarbil (OM) is the first direct myosin activator with promising results in clinical studies. Here, we aimed to elucidate the cellular mechanisms of the positive inotropic effect of OM in a comparative in vitro investigation where Ca2+ -sensitizing positive inotropic agents with distinct mechanisms of action [EMD 53998 (EMD), which also docks on the myosin molecule, and levosimendan (Levo), which binds to troponin C] were included. METHODS: Enzymatically isolated canine cardiomyocytes with intact cell membranes were loaded with Fura-2AM, a Ca2+ -sensitive, ratiometric, fluorescent dye. Changes in sarcomere length (SL) and intracellular Ca2+ concentration were recorded in parallel at room temperature, whereas cardiomyocyte contractions were evoked by field stimulation at 0.1 Hz in the presence of different OM, EMD, or Levo concentrations. RESULTS: SL was reduced by about 23% or 9% in the presence of 1 µM OM or 1 µM EMD in the absence of electrical stimulation, whereas 1 µM Levo had no effect on resting SL. Fractional sarcomere shortening was increased by 1 µM EMD or 1 µM Levo to about 152%, but only to about 128% in the presence of 0.03 µM OM. At higher OM concentrations, no significant increase in fractional sarcomere shortening could be recorded. Contraction durations largely increased, whereas the kinetics of contractions and relaxations decreased with increasing OM concentrations. One-micromole EMD or 1 µM Levo had no effects on contraction durations. One-micromole Levo, but not 1 µM EMD, accelerated the kinetics of cardiomyocyte contractions and relaxations. Ca2+ transient amplitudes were unaffected by all treatments. CONCLUSIONS: Our data revealed major distinctions between the cellular effects of myofilament targeted agents (OM, EMD, or Levo) depending on their target proteins and binding sites, although they were compatible with the involvement of Ca2+ -sensitizing mechanisms for all three drugs. Significant part of the cardiotonic effect of OM relates to the prolongation of systolic contraction in combination with its Ca2+ -sensitizing effect.

Role of 3D echocardiography-determined atrial volumes in distinguishing between pre-capillary and post-capillary pulmonary hypertension.

AIMS: The current guidelines on pulmonary hypertension (PH) recommend the use of invasive examination for differentiating between left-sided heart disease-related (post-capillary) and pre-capillary PH. However, atrial sizes are considered markers of ventricular filling pressures. Therefore, we aimed to test the clinical applicability of atrial volumes measured by transthoracic three-dimensional echocardiography (3DE) in differentiating between pre-capillary and post-capillary PH. METHODS AND RESULTS: Seventy-five consecutive patients with PH were prospectively examined with transthoracic 3DE. After less than 24 h, the patients underwent right heart catheterization and 3DE and were classified as pre-capillary or post-capillary PH according to the recommendations of the ESC guidelines. The atrial volumes were measured offline with dedicated commercial software. Thirty-eight patients (13 men, age 65 ± 18 year) had pre-capillary PH, and 37 (23 men, age 62 ± year) had post-capillary PH. The mean pulmonary artery pressures were similar in patients with pre-capillary and post-capillary PH (38 [IQR 26, 54] mmHg vs. 41 [IQR 33, 48] mmHg, respectively, P = 0.49). The left atrial indexed maximum (LAVi max) and minimum (LAVi min) volumes were significantly larger in the post-capillary PH patient group than in the pre-capillary PH patient group (LAVi max: 64 ± 32 mL/m2 vs. 41 ± 25 mL/m2 , P = 0.001; LAVi min: 50 ± 22 mL/m2 vs. 26 ± 24 mL/m2 , P < 0.0001). The indexed right atrial minimum volume (RAVi min) was also higher in patients with post-capillary PH (51 ± 27 mL/m2 vs. 38 ± 26 mL/m2 ; P = 0.02). Both the left atrial (LA) and right atrial (RA) volumes, especially the LA minimum volume, correlated with the pulmonary artery wedge pressure (PAWP) (r = 0.62 (P < 0.0001) for LAV min vs. r = 0.49 (P < 0.0001) for LAV max; r = 0.32 (P = 0.005) for RAV min vs. r = 0.24 (P = 0.04) for RAV max). Multivariate logistic regression analysis showed that LAVi min was an independent predictor of post-capillary PH. In the receiver operating characteristic (ROC) curves of parameters predicting the post-capillary PH, the areas under the curve (AUC) for LAVi min, LAVi max, and RAVi min were 0.86 (95% CI, 0.76-0.95), 0.78 (95% CI, 0.67-0.89), and 0.66 (0.53-0.78), respectively. Concerning the performance of the atrial volume ratio for differentiating post-capillary PH, the AUC of the atrial volume ratio was significantly lower [AUC: 0.66 (95% CI, 0.53-0.78)]. The ROC analysis indicated a possible cutoff value of 27.7 mL/m2 for LAVi min to predict post-capillary PH (AUC = 0.86; sensitivity = 86%, specificity = 76%). CONCLUSIONS: The BSA-indexed left atrial minimum volume measured by transthoracic 3DE is a useful parameter for differentiating pre-capillary from post-capillary pulmonary hypertension.

Smoking impairs and circulating stem cells favour the protective effect of the T allele of the connexin37 gene in ischemic heart disease--A multinational study.

BACKGROUND: The connexin 37 (Cx37) gene is considered to be a candidate gene for ischemic heart disease (IHD). We analyzed the association between the C1019 > T (Pro319 > Ser) variant of the Cx37 gene and IHD in patients in the Czech Republic, Croatia, Hungary and Romania with regard to the presence/absence of selected cardiovascular risk factors (RF). In a complementary study, we analyzed the association between the Cx37 gene and circulating stem and endothelial progenitor cells in healthy women. METHODS: The study population comprised 2396 patients (663 women) with IHD. The control population comprised 2476 subjects (1, 337 women). Additionally, in 662 healthy women, the association between the Cx37 gene and circulating stem and endothelial progenitor cells was analyzed. RESULTS: The strongest protective effect of the Cx37 T allele was detected in non-smoking patients without diabetes mellitus and hypertension (OR 0.610, 95% CI 0.377-0.990); a similar effect was found in non-smoking men (OR 0.781, 95% CI 0.628-0.971); weaker effect was found in non-smoking women (OR 0.768, 95% CI 0.560-1.050). In non-smoking healthy women, stem cells were significantly higher in TT than in CT and CC carriers (p for trend 0.011). Additionally, non-smoking TT carriers had significantly higher number of stem cells than past and current smoking TT carriers (p for trend = 0.006); no such trend was found in CT and CC carriers. CONCLUSIONS: The protective effect of the T allele of the Cx37 gene might be strongly modified by smoking; in women, this effect could be mediated through stem cells.

Sex-specific chronic stress response at the level of adrenal gland modified sexual hormone and leptin receptors.

AIM: To compare cardiometabolic risk-related biochemical markers and sexual hormone and leptin receptors in the adrenal gland of rat males, non-ovariectomized females (NON-OVX), and ovariectomized females (OVX) under chronic stress. METHODS: Forty six 16-week-old Sprague-Dawley rats were divided into male, NON-OVX, and OVX group and exposed to chronic stress or kept as controls. Weight, glucose tolerance test (GTT), serum concentration of glucose, and cholesterol were measured. Adrenal glands were collected at the age of 28 weeks and immunohistochemical staining against estrogen beta (ERß), progesterone (PR), testosterone (AR), and leptin (Ob-R) receptors was performed. RESULTS: Body weight, GTT, serum cholesterol, and glucose changed in response to stress as expected and validated the applied stress protocol. Stressed males had significantly higher number of ERß receptors in comparison to control group (P = 0.028). Stressed NON-OVX group had significantly decreased AR in comparison to control group (P = 0.007). The levels of PR did not change in any consistent pattern. The levels of Ob-R increased upon stress in all groups, but the significant difference was reached only in the case of stressed OVX group compared to control (P = 0.033). CONCLUSION: Chronic stress response was sex specific. OVX females had similar biochemical parameters as males. Changes upon chronic stress in adrenal gland were related to an increase in testosterone receptor in females and decrease in estrogen receptor in males.

Single acute stress-induced progesterone and ovariectomy alter cardiomyocyte contractile function in female rats.

AIM: To assess how ovarian-derived sex hormones (in particular progesterone) modify the effects of single acute stress on the mechanical and biochemical properties of left ventricular cardiomyocytes in the rat. METHODS: Non-ovariectomized (control, n=8) and ovariectomized (OVX, n=8) female rats were kept under normal conditions or were exposed to stress (control-S, n=8 and OVX-S, n=8). Serum progesterone levels were measured using a chemiluminescent immunoassay. Left ventricular myocardial samples were used for isometric force measurements and protein analysis. Ca(2+)-dependent active force (Factive), Ca(2+)-independent passive force (Fpassive), and Ca(2+)-sensitivity of force production were determined in single, mechanically isolated, permeabilized cardiomyocytes. Stress- and ovariectomy-induced alterations in myofilament proteins (myosin-binding protein C [MyBP-C], troponin I [TnI], and titin) were analyzed by sodium dodecyl sulfate gel electrophoresis using protein and phosphoprotein stainings. RESULTS: Serum progesterone levels were significantly increased in stressed rats (control-S, 35.6±4.8 ng/mL and OVX-S, 21.9±4.0 ng/mL) compared to control (10±2.9 ng/mL) and OVX (2.8±0.5 ng/mL) groups. Factive was higher in the OVX groups (OVX, 25.9±3.4 kN/m(2) and OVX-S, 26.3±3.0 kN/m(2)) than in control groups (control, 16.4±1.2 kN/m(2) and control-S, 14.4±0.9 kN/m(2)). Regarding the potential molecular mechanisms, Factive correlated with MyBP-C phosphorylation, while myofilament Ca(2+)-sensitivity inversely correlated with serum progesterone levels when the mean values were plotted for all animal groups. Fpassive was unaffected by any treatment. CONCLUSION: Stress increases ovary-independent synthesis and release of progesterone, which may regulate Ca(2+)-sensitivity of force production in left ventricular cardiomyocytes. Stress and female hormones differently alter Ca(2+)-dependent cardiomyocyte contractile force production, which may have pathophysiological importance during stress conditions affecting postmenopausal women.

Cellular mechanisms for diastolic dysfunction in the human heart.

Left ventricular (LV) diastolic dysfunction is an important contributor to many different cardiovascular diseases. LV diastolic dysfunction can manifest itself as slow LV relaxation, slow LV filling or high diastolic LV stiffness. Diastolic abnormalities have been described in the senescent heart, in heart failure with preserved ejection fraction (HFPEF), in diabetic cardiomyopathy, in aortic valve stenosis (AVS), in hypertrophic cardiomyopathy (HCM), as well as in Fabry disease (FD), however, exact cellular and molecular alterations behind the diastolic deterioration in these diseases are not yet completely characterized. Several studies thoroughly investigated altered cardiomyocyte function, changes of contractile myofilaments, extracellular collagen deposition and advanced glycation end products (AGEs) cross-linking in the background of diastolic dysfunction. These clinical and experimental data suggest that underlying mechanisms of LV diastolic dysfunction are divergent in different cardiac pathologies, therefore the present review aims to summarize mechanisms at the cellular level of diastolic abnormalities in various cardiovascular diseases.

Diabetes mellitus worsens diastolic left ventricular dysfunction in aortic stenosis through altered myocardial structure and cardiomyocyte stiffness.

BACKGROUND: Aortic stenosis (AS) and diabetes mellitus (DM) are frequent comorbidities in aging populations. In heart failure, DM worsens diastolic left ventricular (LV) dysfunction, thereby adversely affecting symptoms and prognosis. Effects of DM on diastolic LV function were therefore assessed in aortic stenosis, and underlying myocardial mechanisms were identified. METHODS AND RESULTS: Patients referred for aortic valve replacement were subdivided into patients with AS and no DM (AS; n=46) and patients with AS and DM (AS-DM; n=16). Preoperative Doppler echocardiography and hemodynamics were implemented with perioperative LV biopsies. Histomorphometry and immunohistochemistry quantified myocardial collagen volume fraction and myocardial advanced glycation end product deposition. Isolated cardiomyocytes were stretched to 2.2-µm sarcomere length to measure resting tension (F(passive)). Expression and phosphorylation of titin isoforms were analyzed with gel electrophoresis with ProQ Diamond and SYPRO Ruby stains. Reduced LV end-diastolic distensibility in AS-DM was evident from higher LV end-diastolic pressure (21±1 mm Hg for AS versus 28±4 mm Hg for AS-DM; P=0.04) at comparable LV end-diastolic volume index and attributed to higher myocardial collagen volume fraction (AS, 12.9±1.1% versus AS-DM, 18.2±2.6%; P<0.001), more advanced glycation end product deposition in arterioles, venules, and capillaries (AS, 14.4±2.1 score per 1 mm(2) versus AS-DM, 31.4±6.1 score per 1 mm2; P=0.03), and higher F(passive) (AS, 3.5±1.7 kN/m2 versus AS-DM, 5.1±0.7 kN/m2; P=0.04). Significant hypophosphorylation of the stiff N2B titin isoform in AS-DM explained the higher F(passive) and normalization of F(passive) after in vitro treatment with protein kinase A. CONCLUSIONS: Worse diastolic LV dysfunction in AS-DM predisposes to heart failure and results from more myocardial fibrosis, more intramyocardial vascular advanced glycation end product deposition, and higher cardiomyocyte F(passive), which was related to hypophosphorylation of the N2B titin isoform.

More severe cellular phenotype in human idiopathic dilated cardiomyopathy compared to ischemic heart disease.

Activation of the ß-adrenergic receptor (ßAR) pathway is the main mechanism of the heart to increase cardiac output via protein kinase A (PKA)-mediated phosphorylation of cellular target proteins, and perturbations therein may contribute to cardiac dysfunction in heart failure. In the present study a comprehensive analysis was made of mediators of the ßAR pathway, myofilament properties and cardiac structure in patients with idiopathic (IDCM; n = 13) and ischemic (ISHD; n = 10) cardiomyopathy in comparison to non-failing hearts (donor; n = 10) for the following parameters: ßAR density, G-coupled receptor kinases 2 and 5, stimulatory and inhibitory G-proteins, phosphorylation of myofilament targets of PKA, protein phosphatase 1, phospholamban, SERCA2a and single myocyte contractility. All parameters exhibited the expected alterations of heart failure, but for most of them the extent of alteration was greater in IDCM than in ISHD. Histological analysis also revealed higher collagen in IDCM compared to ISHD. Alterations in the ßAR pathway are more pronounced in IDCM than in ISHD and may reflect sequential changes in cellular protein composition and function. Our data indicate that cellular dysfunction is more severe in IDCM than in ISHD.

Molecular determinants of heart failure with normal left ventricular ejection fraction.

In population-based studies, heart failure with normal left ventricular (LV) ejection fraction (HFNEF) is now increasingly recognized and referred to as diastolic heart failure. However, the pathogenic mechanisms underlying HFNEF are incompletely understood, mainly because of limited availability of human myocardial biopsy material. Nevertheless, recent studies have examined in vivo hemodynamics, in vitro cardiomyocyte function, myofilamentary protein composition, collagen content and deposition of advanced glycation end products from LV endomyocardial biopsies. These measures were compared between HFNEF patients, subjects without symptoms of heart failure (controls), patients with heart failure and reduced ejection function (HFREF), and patients with HFNEF and HFREF with diabetes mellitus. This article summarizes the various findings of these studies and focuses on the possible correlations among altered LV myocardial structure, cardiomyocyte function, myofilamentary proteins, and extracellular matrices. These findings revealed novel mechanisms responsible for diastolic LV dysfunction, and they have important therapeutic implications, particularly HFNEF, for which a specific heart failure treatment strategy is largely lacking.

Hypophosphorylation of the Stiff N2B titin isoform raises cardiomyocyte resting tension in failing human myocardium.

High diastolic stiffness of failing myocardium results from interstitial fibrosis and elevated resting tension (F(passive)) of cardiomyocytes. A shift in titin isoform expression from N2BA to N2B isoform, lower overall phosphorylation of titin, and a shift in titin phosphorylation from N2B to N2BA isoform can raise F(passive) of cardiomyocytes. In left ventricular biopsies of heart failure (HF) patients, aortic stenosis (AS) patients, and controls (CON), we therefore related F(passive) of isolated cardiomyocytes to expression of titin isoforms and to phosphorylation of titin and titin isoforms. Biopsies were procured by transvascular technique (44 HF, 3 CON), perioperatively (25 AS, 4 CON), or from explanted hearts (4 HF, 8 CON). None had coronary artery disease. Isolated, permeabilized cardiomyocytes were stretched to 2.2-microm sarcomere length to measure F(passive). Expression and phosphorylation of titin isoforms were analyzed using gel electrophoresis with ProQ Diamond and SYPRO Ruby stains and reported as ratio of titin (N2BA/N2B) or of phosphorylated titin (P-N2BA/P-N2B) isoforms. F(passive) was higher in HF (6.1+/-0.4 kN/m(2)) than in CON (2.3+/-0.3 kN/m(2); P<0.01) or in AS (2.2+/-0.2 kN/m(2); P<0.001). Titin isoform expression differed between HF (N2BA/N2B=0.73+/-0.06) and CON (N2BA/N2B=0.39+/-0.05; P<0.001) and was comparable in HF and AS (N2BA/N2B=0.59+/-0.06). Overall titin phosphorylation was also comparable in HF and AS, but relative phosphorylation of the stiff N2B titin isoform was significantly lower in HF (P-N2BA/P-N2B=0.77+/-0.05) than in AS (P-N2BA/P-N2B=0.54+/-0.05; P<0.01). Relative hypophosphorylation of the stiff N2B titin isoform is a novel mechanism responsible for raised F(passive) of human HF cardiomyocytes.

The peroxynitrite evoked contractile depression can be partially reversed by antioxidants in human cardiomyocytes.

In this study, we aimed to determine the contribution of peroxynitrite-dependent sulfhydryl group (SH) oxidation to the contractile dysfunction in permeabilized left ventricular human cardiomyocytes using a comparative approach with the SH-oxidant 2,2'-dithiodipyridine (DTDP). Additionally, different antioxidants: dithiothreitol (DTT), reduced glutathione (GSH) or N-acetyl-L-cysteine (NAC) were employed to test reversibility. Maximal isometric active force production (F(o)) and the maximal turnover rate of the cross-bridge cycle (k(tr,max)) illustrated cardiomyocyte mechanics. SH oxidation was monitored by a semi-quantitative Ellman's assay and by SH-specific protein biotinylation. Both peroxynitrite and DTDP diminished F(o) in a concentration-dependent manner (EC(50,peroxynitrite) = 49 microM; EC(50,DTDP) = 2.75 mM). However, k(tr,max) was decreased only by 2.5-mM DTDP, but not by 50 microM peroxynitrite. The diminution of F(o) to zero by DTDP was paralleled by the complete elimination of the free SH groups, while the peroxynitrite-induced maximal reduction in free SH groups was only to 58 +/- 6% of the control (100%). The diminutions in F(o) and free SH groups evoked by 2.5-mM DTDP were completely reverted by DTT. In contrast, DTT induced only a partial restoration in F(o) (DeltaF(o,): approximately 13%; P < 0.05) despite full reversion in protein SH content after 50 microM peroxynitrite. Although, NAC or DTT were equally effective on F(o) after peroxynitrite exposures, NAC or GSH did not restore F(o) or k(tr,max) after DTDP treatments. Our results revealed that the peroxynitrite-evoked cardiomyocyte dysfunction has a small, but significant component resulting from reversible SH oxidation, and thereby illustrated the potential benefit of antioxidants during cardiac pathologies with excess peroxynitrite production.

Diastolic stiffness of the failing diabetic heart: importance of fibrosis, advanced glycation end products, and myocyte resting tension.

BACKGROUND: Excessive diastolic left ventricular stiffness is an important contributor to heart failure in patients with diabetes mellitus. Diabetes is presumed to increase stiffness through myocardial deposition of collagen and advanced glycation end products (AGEs). Cardiomyocyte resting tension also elevates stiffness, especially in heart failure with normal left ventricular ejection fraction (LVEF). The contribution to diastolic stiffness of fibrosis, AGEs, and cardiomyocyte resting tension was assessed in diabetic heart failure patients with normal or reduced LVEF. METHODS AND RESULTS: Left ventricular endomyocardial biopsy samples were procured in 28 patients with normal LVEF and 36 patients with reduced LVEF, all without coronary artery disease. Sixteen patients with normal LVEF and 10 with reduced LVEF had diabetes mellitus. Biopsy samples were used for quantification of collagen and AGEs and for isolation of cardiomyocytes to measure resting tension. Diabetic heart failure patients had higher diastolic left ventricular stiffness irrespective of LVEF. Diabetes mellitus increased the myocardial collagen volume fraction only in patients with reduced LVEF (from 14.6+/-1.0% to 22.4+/-2.2%, P<0.001) and increased cardiomyocyte resting tension only in patients with normal LVEF (from 5.1+/-0.7 to 8.5+/-0.9 kN/m2, P=0.006). Diabetes increased myocardial AGE deposition in patients with reduced LVEF (from 8.8+/-2.5 to 24.1+/-3.8 score/mm2; P=0.005) and less so in patients with normal LVEF (from 8.2+/-2.5 to 15.7+/-2.7 score/mm2, P=NS). CONCLUSIONS: Mechanisms responsible for the increased diastolic stiffness of the diabetic heart differ in heart failure with reduced and normal LVEF: Fibrosis and AGEs are more important when LVEF is reduced, whereas cardiomyocyte resting tension is more important when LVEF is normal.

Myofilament dysfunction in cardiac disease from mice to men.

In healthy human myocardium a tight balance exists between receptor-mediated kinases and phosphatases coordinating phosphorylation of regulatory proteins involved in cardiomyocyte contractility. During heart failure, when neurohumoral stimulation increases to compensate for reduced cardiac pump function, this balance is perturbed. The imbalance between kinases and phosphatases upon chronic neurohumoral stimulation is detrimental and initiates cardiac remodelling, and phosphorylation changes of regulatory proteins, which impair cardiomyocyte function. The main signalling pathway involved in enhanced cardiomyocyte contractility during increased cardiac load is the beta-adrenergic signalling route, which becomes desensitized upon chronic stimulation. At the myofilament level, activation of protein kinase A (PKA), the down-stream kinase of the beta-adrenergic receptors (beta-AR), phosphorylates troponin I, myosin binding protein C and titin, which all exert differential effects on myofilament function. As a consequence of beta-AR down-regulation and desensitization, phosphorylation of the PKA-target proteins within the cardiomyocyte may be decreased and alter myofilament function. Here we discuss involvement of altered PKA-mediated myofilament protein phosphorylation in different animal and human studies, and discuss the roles of troponin I, myosin binding protein C and titin in regulating myofilament dysfunction in cardiac disease. Data from the different animal and human studies emphasize the importance of careful biopsy procurement, and the need to investigate localization of kinases and phosphatases within the cardiomyocyte, in particular their co-localization with cardiac myofilaments upon receptor stimulation.

Myocardial structure and function differ in systolic and diastolic heart failure.

BACKGROUND: To support the clinical distinction between systolic heart failure (SHF) and diastolic heart failure (DHF), left ventricular (LV) myocardial structure and function were compared in LV endomyocardial biopsy samples of patients with systolic and diastolic heart failure. METHODS AND RESULTS: Patients hospitalized for worsening heart failure were classified as having SHF (n=22; LV ejection fraction (EF) 34+/-2%) or DHF (n=22; LVEF 62+/-2%). No patient had coronary artery disease or biopsy evidence of infiltrative or inflammatory myocardial disease. More DHF patients had a history of arterial hypertension and were obese. Biopsy samples were analyzed with histomorphometry and electron microscopy. Single cardiomyocytes were isolated from the samples, stretched to a sarcomere length of 2.2 microm to measure passive force (Fpassive), and activated with calcium-containing solutions to measure total force. Cardiomyocyte diameter was higher in DHF (20.3+/-0.6 versus 15.1+/-0.4 microm, P<0.001), but collagen volume fraction was equally elevated. Myofibrillar density was lower in SHF (36+/-2% versus 46+/-2%, P<0.001). Cardiomyocytes of DHF patients had higher Fpassive (7.1+/-0.6 versus 5.3+/-0.3 kN/m2; P<0.01), but their total force was comparable. After administration of protein kinase A to the cardiomyocytes, the drop in Fpassive was larger (P<0.01) in DHF than in SHF. CONCLUSIONS: LV myocardial structure and function differ in SHF and DHF because of distinct cardiomyocyte abnormalities. These findings support the clinical separation of heart failure patients into SHF and DHF phenotypes.

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

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

Cardiomyocyte stiffness in diastolic heart failure.

BACKGROUND: Heart failure with preserved left ventricular (LV) ejection fraction (EF) is increasingly recognized and usually referred to as diastolic heart failure (DHF). Its pathogenetic mechanism remains unclear, partly because of a lack of myocardial biopsy material. Endomyocardial biopsy samples obtained from DHF patients were therefore analyzed for collagen volume fraction (CVF) and sarcomeric protein composition and compared with control samples. Single cardiomyocytes were isolated from these biopsy samples to assess cellular contractile performance. METHODS AND RESULTS: DHF patients (n=12) had an LVEF of 71+/-11%, an LV end-diastolic pressure (LVEDP) of 28+/-4 mm Hg, and no significant coronary artery stenoses. DHF patients had higher CVFs (7.5+/-4.0%, P<0.05) than did controls (n=8, 3.8+/-2.0%), and no conspicuous changes in sarcomeric protein composition were detected. Cardiomyocytes, mechanically isolated and treated with Triton X-100 to remove all membranes, were stretched to a sarcomere length of 2.2 microm and activated with solutions containing varying [Ca2+]. Compared with cardiomyocytes of controls, cardiomyocytes of DHF patients developed a similar total isometric force at maximal [Ca2+], but their resting tension (F(passive)) in the absence of Ca2+ was almost twice as high (6.6+/-3.0 versus 3.5+/-1.7 kN/m2, P<0.001). F(passive) and CVF combined yielded stronger correlations with LVEDP than did either alone. Administration of protein kinase A (PKA) to DHF cardiomyocytes lowered F(passive) to control values. CONCLUSIONS: DHF patients had stiffer cardiomyocytes, as evident from a higher F(passive) at the same sarcomere length. Together with CVF, F(passive) determined in vivo diastolic LV dysfunction. Correction of this high F(passive) by PKA suggests that reduced phosphorylation of sarcomeric proteins is involved in DHF.