M. Louis Handoko

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.

Right Ventricular Diastolic Impairment in Patients With Pulmonary Arterial Hypertension

Background— The role of right ventricular (RV) diastolic stiffness in pulmonary arterial hypertension (PAH) is not well established. Therefore, we investigated the presence and possible underlying mechanisms of RV diastolic stiffness in PAH patients. Methods and Results— Single-beat RV pressure-volume analyses were performed in 21 PAH patients and 7 control subjects to study RV diastolic stiffness. Data are presented as mean±SEM. RV diastolic stiffness (&bgr;) was significantly increased in PAH patients (PAH, 0.050±0.005 versus control, 0.029±0.003; P<0.05) and was closely associated with disease severity. Subsequently, we searched for possible underlying mechanisms using RV tissue of PAH patients undergoing heart/lung transplantation and nonfailing donors. Histological analyses revealed increased cardiomyocyte cross-sectional areas (PAH, 453±31 &mgr;m2 versus control, 218±21 &mgr;m2; P<0.001), indicating RV hypertrophy. In addition, the amount of RV fibrosis was enhanced in PAH tissue (PAH, 9.6±0.7% versus control, 7.2±0.6%; P<0.01). To investigate the contribution of stiffening of the sarcomere (the contractile apparatus of RV cardiomyocytes) to RV diastolic stiffness, we isolated and membrane-permeabilized single RV cardiomyocytes. Passive tension at different sarcomere lengths was significantly higher in PAH patients compared with control subjects (>200%; Pinteraction<0.001), indicating stiffening of RV sarcomeres. An important regulator of sarcomeric stiffening is the sarcomeric protein titin. Therefore, we investigated titin isoform composition and phosphorylation. No alterations were observed in titin isoform composition (N2BA/N2B ratio: PAH, 0.78±0.07 versus control, 0.91±0.08), but titin phosphorylation in RV tissue of PAH patients was significantly reduced (PAH, 0.16±0.01 arbitrary units versus control, 0.20±0.01 arbitrary units; P<0.05). Conclusions— RV diastolic stiffness is significantly increased in PAH patients, with important contributions from increased collagen and intrinsic stiffening of the RV cardiomyocyte sarcomeres.

Bisoprolol Delays Progression Towards Right Heart Failure in Experimental Pulmonary Hypertension

Background— In pulmonary arterial hypertension (PH), sympathetic adrenergic activity is highly elevated. Sympathetic overactivity is a compensatory mechanism at first, but might be detrimental for cardiac function in the long run. We therefore investigated whether chronic low-dose treatment with bisoprolol (a cardioselective &bgr;-blocker) has beneficial effects on cardiac function in experimental PH. Methods and Results— PH was induced in rats by a single injection of monocrotaline (60 mg/kg). Pressure telemetry in PH rats revealed that 10 mg/kg bisoprolol was the lowest dose that blunted heart rate response during daily activity. Ten days after monocrotaline injection, echocardiography was performed and PH rats were randomized for bisoprolol treatment (oral gavage) or vehicle (n=7/group). At end of study (body mass loss >5%), echocardiography was repeated, with additional pressure-volume measurements and histomolecular analyses. Compared with control, right ventricular (RV) systolic pressure and arterial elastance (measure of vascular resistance) more than tripled in PH. Bisoprolol delayed time to right heart failure (P<0.05). RV afterload was unaffected, however, bisoprolol treatment increased RV contractility and filling (both P<0.01), and partially restored right ventriculo-arterial coupling and cardiac output (both P<0.05). Bisoprolol restored RV &bgr;-adrenergic receptor signaling. Histology revealed significantly less RV fibrosis and myocardial inflammation in bisoprolol treated PH rats. Conclusions— In experimental PH, treatment with bisoprolol delays progression toward right heart failure, and partially preserves RV systolic and diastolic function. These promising results suggest a therapeutic role for &bgr;-blockers in PH that warrants further clinical investigation.

Right ventricular pacing improves right heart function in experimental pulmonary arterial hypertension: a study in the isolated heart

Right heart failure in pulmonary arterial hypertension (PH) is associated with mechanical ventricular dyssynchrony, which leads to impaired right ventricular (RV) function and, by adverse diastolic interaction, to impaired left ventricular (LV) function as well. However, therapies aiming to restore synchrony by pacing are currently not available. In this proof-of-principle study, we determined the acute effects of RV pacing on ventricular dyssynchrony in PH. Chronic PH with right heart failure was induced in rats by injection of monocrotaline (80 mg/kg). To validate for PH-related ventricular dyssynchrony, rats (6 PH, 6 controls) were examined by cardiac magnetic resonance imaging (9.4 T), 23 days after monocrotaline or sham injection. In a second group (10 PH, 4 controls), the effects of RV pacing were studied in detail, using Langendorff-perfused heart preparations. In PH, septum bulging was observed, coinciding with a reversal of the transseptal pressure gradient, as observed in clinical PH. RV pacing improved RV systolic function, compared with unpaced condition (maximal first derivative of RV pressure: +8.5 + or - 1.3%, P < 0.001). In addition, RV pacing markedly decreased the pressure-time integral of the transseptal pressure gradient when RV pressure exceeds LV pressure, an index of adverse diastolic interaction (-24 + or - 9%, P < 0.01), and RV pacing was able to resynchronize time of RV and LV peak pressure (unpaced: 9.8 + or - 1.2 ms vs. paced: 1.7 + or - 2.0 ms, P < 0.001). Finally, RV pacing had no detrimental effects on LV function or coronary perfusion, and no LV preexcitation occurred. Taken together, we demonstrate that, in experimental PH, RV pacing improves RV function and diminishes adverse diastolic interaction. These findings provide a strong rationale for further in vivo explorations.

Neurohormonal axis in patients with pulmonary arterial hypertension: friend or foe?

Despite its description some 25 years ago, neurohormonal activation has long been neglected as an important factor in the pathophysiology of pulmonary arterial hypertension (PAH). Neurohormonal activation was interpreted as a necessary compensatory response to maintain cardiac contractility and systemic blood pressure. Therefore, inhibitors of neurohormonal activity (like β-blockers or angiotensin-converting enzyme inhibitors) are considered contraindicated in current PAH management guidelines. However, recent data revealed that sympathetic overstimulation is strongly related to mortality, and blockade of neurohormonal activity in experimental PAH improved survival and cardiac function. These novel insights shed new light on the role of neurohormonal activity in PAH.

Contractile Dysfunction of Left Ventricular Cardiomyocytes in Patients With Pulmonary Arterial Hypertension

BACKGROUND After lung transplantation, increased left ventricular (LV) filling can lead to LV failure, increasing the risk of post-operative complications and mortality. LV dysfunction in pulmonary arterial hypertension (PAH) is characterized by a reduced LV ejection fraction and impaired diastolic function. OBJECTIVES The pathophysiology of LV dysfunction in PAH is incompletely understood. This study sought to assess the contribution of atrophy and contractility of cardiomyocytes to LV dysfunction in PAH patients. METHODS LV function was assessed by cardiac magnetic resonance imaging. In addition, LV biopsies were obtained in 9 PAH patients and 10 donors. The cross-sectional area (CSA) and force-generating capacity of isolated single cardiomyocytes was investigated. RESULTS Magnetic resonance imaging analysis revealed a significant reduction in LV ejection fraction in PAH patients, indicating a reduction in LV contractility. The CSA of LV cardiomyocytes of PAH patients was significantly reduced (~30%), indicating LV cardiomyocyte atrophy. The maximal force-generating capacity, normalized to cardiomyocyte CSA, was significantly reduced (~25%). Also, a reduction in the number of available myosin-based cross-bridges was found to cause the contractile weakness of cardiomyocytes. This finding was supported by protein analyses, which showed an ~30% reduction in the myosin/actin ratio in cardiomyocytes from PAH patients. Finally, the phosphorylation level of sarcomeric proteins was reduced in PAH patients, which was accompanied by increased calcium sensitivity of force generation. CONCLUSIONS The contractile function and the CSA of LV cardiomyocytes is substantially reduced in PAH patients. We propose that these changes contribute to the reduced in vivo contractility of the LV in PAH patients.

Diaphragm muscle fiber weakness in pulmonary hypertension.

RATIONALE Recently it was suggested that patients with pulmonary hypertension (PH) suffer from inspiratory muscle dysfunction. However, the nature of inspiratory muscle weakness in PH remains unclear. OBJECTIVES To assess whether alterations in contractile performance and in morphology of the diaphragm underlie inspiratory muscle weakness in PH. METHODS PH was induced in Wistar rats by a single injection of monocrotaline (60 mg/kg). Diaphragm (PH n = 8; controls n = 7) and extensor digitorum longus (PH n = 5; controls n = 7) muscles were excised for determination of in vitro contractile properties and cross-sectional area (CSA) of the muscle fibers. In addition, important determinants of protein synthesis and degradation were determined. Finally, muscle fiber CSA was determined in diaphragm and quadriceps of patients with PH, and the contractile performance of single fibers of the diaphragm. MEASUREMENTS AND MAIN RESULTS In rats with PH, twitch and maximal tetanic force generation of diaphragm strips were significantly lower, and the force-frequency relation was shifted to the right (i.e., impaired relative force generation) compared with control subjects. Diaphragm fiber CSA was significantly smaller in rats with PH compared with controls, and was associated with increased expression of E3-ligases MAFbx and MuRF-1. No significant differences in contractility and morphology of extensor digitorum longus muscle fibers were found between rats with PH and controls. In line with the rat data, studies on patients with PH revealed significantly reduced CSA and impaired contractility of diaphragm muscle fibers compared with control subjects, with no changes in quadriceps muscle. CONCLUSIONS PH induces selective diaphragm muscle fiber weakness and atrophy.

Right ventricular oxygen supply parameters are decreased in human and experimental pulmonary hypertension

BACKGROUND In pulmonary arterial hypertension (PAH), high right ventricular (RV) power output requires increased myocardial oxygen consumption. Oxygen supply, however, does not increase in proportion. It is unknown what cellular mechanisms underlie this lack of adaptation. We therefore determined oxygen supply parameters in RV tissue slices of deceased PAH patients and compared them with RV tissue of patients who died from left ventricular myocardial infarction (MI). Because autopsy tissue only reflects end-stage disease, rat models with stable and progressive pulmonary hypertension (PH) were studied as well. METHODS Myocardial tissue of 10 PAH and 10 MI patients was collected at autopsy. In rats, stable PH (n = 6) and progressive PH (n = 6) was induced by 40 or 60 mg/kg monocrotaline, respectively. Six rats were used as controls. RESULTS RV cardiomyocyte cross-sectional area was strongly increased in PAH compared with MI patients (p < 0.001), whereas capillary density decreased (p < 0.01). Rat data showed similar RV hypertrophy in stable and progressive PH, and RV capillary density was decreased in both (p < 0.01 and p < 0.0001 vs control rats, respectively). RV myoglobin protein content and functional concentration were reduced in both human and rat PH RVs. In rats, this results from a lack of increase in myoglobin mRNA transcription per cardiomyocyte nucleus. CONCLUSIONS All measured cellular oxygen supply parameters are decreased in the failing human and rat pulmonary hypertensive RV. In contrast to stable PH rats, compensatory adaptations do not occur in end-stage PAH, despite higher myocardial oxygen consumption.

Pulmonary hypertension in heart failure with preserved ejection fraction: a plea for proper phenotyping and further research†

Heart failure (HF) with preserved ejection fraction (HFpEF) is a common disease affecting the elderly in particular. Up to 80% of these patients develop pulmonary hypertension (PH), which is associated with worse symptoms and increased mortality.1 It is a matter of concern that drugs approved for pulmonary arterial hypertension (PAH) are sometimes used in such patients despite insufficient data for their safety and efficacy. On the other hand, the impact of PH and right ventricular (RV) dysfunction on morbidity and mortality in HFpEF call for proper attention both at the clinical and scientific level. Here we discuss the clinical problem, pathophysiology, diagnostic shortfalls, gaps in evidence, and future strategies for PH-HFpEF. HFpEF is currently the dominant form of HF in aging societies globally. Epidemiologic trends over the past two decades showed that HFpEF increased relative to HF with reduced ejection fraction (HFrEF).2 Overall mortality did not improve over time, with more than 50% dead in 5 years from diagnosis.2 Differences between epidemiologic and trial populations of HFpEF reflect potential selection bias and lack of uniformity of diagnostic criteria. Epidemiologic studies utilize the most widely applicable definition of HFpEF: (i) clinically diagnosed HF (e.g. by Framingham criteria) and (ii) preserved EF (e.g. ≥50%).2 While such definitions capture the broad unselected population with the syndrome of HFpEF, they are rarely specific enough for clinical trials since the accurate diagnosis relies on symptoms and signs of HFpEF, both non-discriminating particularly in elderly patients with multiple comorbidities. The ESC guidelines included additional criteria, i.e. elevated levels of natriuretic peptides or objective evidence of left ventricular (LV) hypertrophy, left atrial enlargement, and/or LV diastolic dysfunction.3 Yet, the diagnosis of HFpEF remains difficult as many presumably healthy elderly patients fulfil at least some of these echocardiographic criteria. Invasive demonstration of increased pulmonary …

Protein changes contributing to right ventricular cardiomyocyte diastolic dysfunction in pulmonary arterial hypertension.

Background Right ventricular (RV) diastolic function is impaired in patients with pulmonary arterial hypertension (PAH). Our previous study showed that elevated cardiomyocyte stiffness and myofilament Ca2+ sensitivity underlie diastolic dysfunction in PAH. This study investigates protein modifications contributing to cellular diastolic dysfunction in PAH. Methods and Results RV samples from PAH patients undergoing heart‐lung transplantation were compared to non‐failing donors (Don). Titin stiffness contribution to RV diastolic dysfunction was determined by Western‐blot analyses using antibodies to protein‐kinase‐A (PKA), Cα (PKCα) and Ca2+/calmoduling‐dependent‐kinase (CamKIIδ) titin and phospholamban (PLN) phosphorylation sites: N2B (Ser469), PEVK (Ser170 and Ser26), and PLN (Thr17), respectively. PKA and PKCα sites were significantly less phosphorylated in PAH compared with donors (P<0.0001). To test the functional relevance of PKA‐, PKCα‐, and CamKIIδ‐mediated titin phosphorylation, we measured the stiffness of single RV cardiomyocytes before and after kinase incubation. PKA significantly decreased PAH RV cardiomyocyte diastolic stiffness, PKCα further increased stiffness while CamKIIδ had no major effect. CamKIIδ activation was determined indirectly by measuring PLN Thr17phosphorylation level. No significant changes were found between the groups. Myofilament Ca2+ sensitivity is mediated by sarcomeric troponin I (cTnI) phosphorylation. We observed increased unphosphorylated cTnI in PAH compared with donors (P<0.05) and reduced PKA‐mediated cTnI phosphorylation (Ser22/23) (P<0.001). Finally, alterations in Ca2+‐handling proteins contribute to RV diastolic dysfunction due to insufficient diastolic Ca2+ clearance. PAH SERCA2a levels and PLN phosphorylation were significantly reduced compared with donors (P<0.05). Conclusions Increased titin stiffness, reduced cTnI phosphorylation, and altered levels of phosphorylation of Ca2+ handling proteins contribute to RV diastolic dysfunction in PAH.