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Conventional blood pressure (BP) measurement devices based on an inflatable cuff only provide a narrow view of the continuous BP profile. Cuffless BP measuring technologies could permit numerous BP readings throughout daily life and thereby considerably improve the assessment and management of hypertension. Several wearable cuffless BP devices based on pulse wave analysis (applied to a photoplethysmography or tonometry waveform) with or without use of pulse arrival time are now available on the market. The key question is: Can these devices provide accurate measurement of BP? Microsoft Research recently published a complex article describing perhaps the most important and highest resource project to date (Aurora Project) on assessing the accuracy of several pulse wave analysis and pulse wave analysis-pulse arrival time devices. The overall results from 1125 participants were clear-cut negative. The present article motivates and describes emerging cuffless BP devices and then summarizes the Aurora Project. The study methodology and findings are next discussed in the context of regulatory-cleared devices, physiology, and related studies, and the study strengths and limitations are pinpointed thereafter. Finally, the implications of the Aurora Project are briefly stated and recommendations for future work are offered to finally realize the considerable potential of cuffless BP measurement in health care.

Introduction: Atrial fibrillation (AF) is the most common arrhythmia that leads to thrombus formation, most commonly in the left atrial appendage (LAA), and a resultant five-fold increase in the risk of ischemic stroke. The current standard of stratifying stroke risk, based on the CHA 2 DS 2 -VASc score, does not consider subject-specific LAA hemodynamics and blood stasis risk. We have developed a methodology for computing mean residence time of blood-borne particles in the LAA ( t m ), an index of LAA blood stasis, using 3D left atrial (LA) and LAA geometries and pulmonary venous (PV) inlet blood flow data. While subject-specific 3D geometry can be obtained readily, it is not easy to measure all LA inlet blood flow waveforms in vivo . Therefore, a sensitivity study of LAA t m to the inlet blood flow waveform characteristics (shape and magnitude) is needed. Hypothesis: PV flow pulsatility does not affect LAA t m . Methods: Cardiac computed tomography images of 10 AF subjects were obtained and LA and LAA geometries were reconstructed in 3D and subsequently meshed for computational fluid dynamics (CFD) analysis. We employed CFD analysis, modeling blood cells as neutrally buoyant tracers transported in the blood and used a systems-based approach, to quantify LAA t m . Each subject was simulated using four PV blood flow characteristics: (1) Normal-S , steady PV flow with cardiac output (CO) of 5.5 L/min; (2) AF-S , steady PV flow with reduced CO of 4.4 L/min; (3) Normal-P , normal pulsatile PV flow with normal CO = 5.5 L/min; and (4) AF-P , a typical AF pulsatile waveform where systolic phase and the reversal phase are diminished with reduced CO of 4.4 L/min. Results: Comparing the two steady flow groups, LAA t m significantly increased as CO was decreased ( Normal-S: 2.6±0.3 s, AF-S: 3.1±0.3 s, P =0.02). However, the presence of pulsatility did not affect LAA t m at both levels of CO: Normal-P: 2.4±0.2 s; Normal-S: 2.6±0.3 s, P =0.2 and AF-P: 2.9±0.3 s; AF-S: 3.1±0.3 s, P =0.3. Conclusions: LAA blood stasis risk, as quantified by LAA t m , is significantly affected by the steady PV flow magnitude (i.e., cardiac output) and not by PV flow pulsatility. Therefore, the subject-specific LAA blood stasis risk can be reliably estimated using subject-specific LA and LAA 3D geometries and subject-specific CO.

De novo synthesis of cytoskeleton-regulatory proteins triggered by the megakaryoblastic leukemia (MKL)/serum response factor (SRF) transcriptional system in response to pro-angiogenic growth factors lies at the heart of endothelial cell (EC) migration (a critical element of angiogenesis) and neovascularization. This study explores whether pharmacological intervention of MKL/SRF signaling axis by CCG-1423 is able to suppress angiogenesis. Our studies show that CCG-1423 inhibits migration and cord morphogenesis of EC in vitro and sprouting angiogenesis ex vivo and in vivo, suggesting CCG-1423 could be a novel anti-angiogenic agent. Kymography analyses of membrane dynamics of EC revealed that CCG-1423 treatment causes a major defect in membrane protrusion. CCG-1423 treatment led to attenuated expression of several actin-binding proteins that are important for driving membrane protrusion including ArpC2, VASP, and profilin1 (Pfn1) with the most drastic effect seen on the expression of Pfn1. Finally, depletion of Pfn1 alone is also sufficient for a dramatic decrease in sprouting angiogenesis of EC in vitro and ex vivo, further suggesting that Pfn1 depletion may be one of the mechanisms of the anti-angiogenic action of CCG-1423.

To define the differential effect of site-specific ventricular counterpacing efficacy during cardiac resynchronization therapy (CRT) to identify the most informative imaging views to quantify it. Cross-sectional and long-axis views commonly are used to assess left ventricular (LV) contractility.The effects of LV apical (LVa) and free-wall (LVfw) pacing during CRT on long- and short-axis contraction, cardiac output, and stroke work were assessed in an open-chested acute canine model to determine whether LVa and LVfw would induce earlier apical than basilar LV radial contraction and earlier free-wall than septal contraction, respectively. Apical (CRTa) and free-wall (CRTfw) using right ventricular (RV) pacing-induced dyssynchrony also were examined.University large animal research laboratory.Ten acutely anesthetized and instrumented open-chested purpose-bred dogs.RV pacing served as the model of cardiac dyssynchrony. Selective LVfw and LVa pacing alone or with RV (CRTfw and CRTa, respectively) were studied relative to right atrial pacing (RA) as the control.Two pairs of 3 ultrasonic crystals were place along the LV longitudinal axis-apex and mid-to-base pairs along septal and free wall lines. Conductance catheter-defined longitudinal LV segmental volumes and pressure-volume data were collected. RV decreased cardiac output and stroke work compared with RA (2.0±0.3 v 1.4±0.1 L/min; 137±22 v 60±14 mJ; p0.05, respectively). LVfw but not LVa decreased stroke work (130±35 mJ), and CRTa but not CRTfw improved both (2.1±0.2 L/min; 113±13 mJ; p0.01 v RV pacing). No difference in time to minimal length free wall-to-septal crystal was seen with pacing. Both LVa and CRTa displayed increased apical-to-basilar shortening delay compared with RA, RV, and LVfw (42±47, 9±105, and 1±46 msec, respectively; p0.05). No matching regional LV volume changes were seen during LVa.LV functional analysis from only a cross-sectional plane may be insufficient to characterize improved LV contraction synchrony during multisite CRT.

Dynamic regulation of the actin cytoskeleton is an essential feature of cell motility. Action of Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP), a family of conserved actin-elongating proteins, is an important aspect of regulation of the actin cytoskeletal architecture at the leading edge that controls membrane protrusion and cell motility. In this study, we performed mutagenesis experiments in overexpression and knockdown–rescue settings to provide, for the first time, direct evidence of the role of the actin-binding protein profilin1 (Pfn1) in VASP-mediated regulation of cell motility. We found that VASP's interaction with Pfn1 is promoted by cell–substrate adhesion and requires down-regulation of PKA activity. Our experimental data further suggest that PKA-mediated Ser(137) phosphorylation of Pfn1 potentially negatively regulates the Pfn1–VASP interaction. Finally, Pfn1's ability to be phosphorylated on Ser(137) was partly responsible for the anti-migratory action elicited by exposing cells to a cAMP/PKA agonist. On the basis of these findings, we propose a mechanism of adhesion–protrusion coupling in cell motility that involves dynamic regulation of Pfn1 by PKA activity.

Rationale: Atrial fibrillation (AF) contributes significantly to morbidity and mortality in elderly and hypertensive patients and has been correlated to enhanced atrial fibrosis. Despite a lack of direct evidence that fibrosis causes AF, reversal of fibrosis is considered a plausible therapy. Objective: To evaluate the efficacy of the antifibrotic hormone relaxin (RLX) in suppressing AF in spontaneously hypertensive rats (SHR). Methods and Results: Normotensive Wistar-Kyoto (WKY) and SHR were treated for 2 weeks with vehicle (WKY+V and SHR+V) or RLX (0.4 mg/kg per day, SHR+RLX) using implantable mini-pumps. Hearts were perfused, mapped optically to analyze action potential durations, intracellular Ca 2+ transients, and restitution kinetics, and tested for AF vulnerability. SHR hearts had slower conduction velocity (CV; P P P P P + current density, I Na (≈2-fold in 48 hours) in human cardiomyocytes derived from inducible pluripotent stem cells (n=18/18; P Conclusions: RLX treatment suppressed AF in SHR hearts by increasing CV from a combination of reversal of fibrosis and hypertrophy and by increasing I Na . The study provides compelling evidence that RLX may provide a novel therapy to manage AF in humans by reversing fibrosis and hypertrophy and by modulating cardiac ionic currents.

AimsDiabetes may promote myocardial extracellular matrix (ECM) expansion that increases vulnerability. We hypothesized that: (i) type 2 diabetes would be associated with quantitative cardiovascular magnetic resonance (CMR) measures of myocardial ECM expansion, i.e. extracellular volume fraction (ECV); (ii) medications blocking the renin-angiotensin-aldosterone system (RAAS) would be associated with lower ECV; and (iii) ECV in diabetic individuals would be associated with mortality and/or incident hospitalization for heart failure.Methods and resultsWe enrolled 1176 consecutive patients referred for CMR without amyloidosis and computed ECV from measures of the haematocrit and myocardial and blood T1 pre- and post-contrast. Linear regression modelled ECV; Cox regression modelled mortality and/or hospitalization for heart failure. Diabetic individuals (n = 231) had higher median ECV than those without diabetes (n = 945): 30.2% (IQR: 26.9-32.7) vs. 28.1% (IQR: 25.9-31.0), respectively, P < 0.001). Diabetes remained associated with higher ECV in models adjusting for demographics, comorbidities, and medications (P < 0.001). Renin-angiotensin-aldosterone system blockade was associated with lower ECV (P = 0.028) in multivariable linear models. Over a median of 1.3 years (IQR: 0.8-1.9), 38 diabetic individuals had events (21 incident hospitalizations for heart failure; 24 deaths), and ECV was associated with these events (HR: 1.52, 95% CI: 1.21-1.89 per 3% ECV increase) in multivariable Cox regression models.ConclusionDiabetes is associated with increased ECV. Extracellular volume fraction detects amelioration of ECM expansion associated with RAAS blockade, and is associated with mortality and/or incident hospitalization for heart failure in diabetic individuals. Extracellular matrix expansion may be an important intermediate phenotype in diabetic individuals that is detectable and treatable.

Background Myocardial fibrosis ( MF ) in noninfarcted myocardium may be an interstitial disease pathway that confers vulnerability to hospitalization for heart failure, death, or both across the spectrum of heart failure and ejection fraction. Hospitalization for heart failure is an epidemic that is difficult to predict and prevent and requires potential therapeutic targets associated with outcomes. Method and Results We quantified MF with cardiovascular magnetic resonance extracellular volume fraction ( ECV ) measures in 1172 consecutive patients without amyloidosis or hypertrophic or stress cardiomyopathy and assessed associations with outcomes using Cox regression. ECV ranged from 16.6% to 47.8%. Over a median of 1.7 years, 111 patients experienced events after cardiovascular magnetic resonance, 55 had hospitalization for heart failure events, and there were 74 deaths. ECV was more strongly associated with outcomes than “nonischemic” MF observed with late gadolinium enhancement, thus ECV quantified MF in multivariable models. Adjusting for age, sex, renal function, myocardial infarction size, ejection fraction, hospitalization status, and heart failure stage, higher ECV was associated with hospitalization for heart failure (hazard ratio 1.77; 95% CI 1.32 to 2.36 for every 5% increase in ECV ), death (hazard ratio 1.87 95% CI 1.45 to 2.40) or both (hazard ratio 1.85, 95% CI 1.50 to 2.27). ECV improved classification of persons at risk and improved model discrimination for outcomes (eg, hospitalization for heart failure: continuous net reclassification improvement 0.33, 95% CI 0.05 to 0.66; P =0.02; 0.16, 95% CI 0.01 to 0.33; P =0.02; integrated discrimination improvement 0.037, 95% CI 0.008 to 0.073; P Conclusion MF measured by ECV is associated with hospitalization for heart failure, death, or both. MF may represent a principal phenotype of cardiac vulnerability that improves risk stratification. Because MF can be reversible, cells and enzymes regulating collagen could be potential therapeutic targets.

Background Most current indices of synchrony quantify left ventricular (LV) contraction pattern in terms of a single, global (integrated) measure. We report the development and physiological relevance of a novel method to quantify LV segmental contraction synchrony.

Administering relaxin to conscious rats and humans elicits systemic and renal vasodilation. The molecular mechanisms vary according to the duration of relaxin exposure-so-called "rapid" (within minutes) or "sustained" (hours to days) vasodilatory responses-both being endothelium-dependent. Rapid responses are mediated by G(αi/o) protein coupling to phosphoinositol-3 kinase/Akt (protein kinase B)-dependent phosphorylation and activation of nitric oxide synthase. Sustained responses are mediated by vascular endothelial and placental growth factors, as well as increases in arterial gelatinase activity. Thus, after hours or days of relaxin treatment, respectively, arterial MMP-9 or MMP-2 hydrolyze "big" endothelin (ET) at a gly-leu bond to form ET(1-32), which in turn activates the endothelial ET(B) receptor/nitric oxide vasodilatory pathway. Administration of relaxin to conscious rats also increases global systemic arterial compliance and passive compliance of select isolated blood vessels such as small renal arteries (SRA). The increase in SRA passive compliance is mediated by both geometric remodeling (outward) and compositional remodeling (decreased collagen). Relaxin-induced geometric remodeling has also been observed in brain parenchymal arteries, and this remodeling appears to be via the activation of peroxisome proliferator-activated receptor-γ. Given the vasodilatory and arterial remodeling properties of relaxin, the hormone may have therapeutic potential in the settings of abnormal pregnancies, heart failure, and pathologies associated with stiffening of arteries.

Reversible lysine acetylation is a widespread post-translational modification controlling the activity of proteins in different subcellular compartments. We previously demonstrated that a class II histone deacetylase (HDAC), HDAC4, and a histone acetyltransferase, PCAF, associate with cardiac sarcomeres, and a class I and II HDAC inhibitor, trichostatin A, enhances contractile activity of myofilaments. In this study, we show that a class I HDAC, HDAC3, is also present at cardiac sarcomeres. By immunohistochemical and electron microscopic analyses, we found that HDAC3 was localized to the A band of sarcomeres and was capable of deacetylating myosin heavy chain (MHC) isoforms. The motor domains of both cardiac α- and β-MHC isoforms were found to be reversibly acetylated. Biomechanical studies revealed that lysine acetylation significantly decreased the K(m) for the actin-activated ATPase activity of both α- and β-MHC isoforms. By an in vitro motility assay, we found that lysine acetylation increased the actin sliding velocity of α-myosin by 20% and β-myosin by 36%, compared to their respective non-acetylated isoforms. Moreover, myosin acetylation was found to be sensitive to cardiac stress. During induction of hypertrophy, myosin isoform acetylation increased progressively with duration of stress stimuli, independent of isoform shift, suggesting that lysine acetylation of myosin could be an early response of myofilaments to increase contractile performance of the heart. These studies provide the first evidence for localization of HDAC3 at myofilaments and uncover a novel mechanism modulating the motor activity of cardiac MHC isoforms.

Aims Right ventricular (RV) pacing is an iatrogenic cause of heart failure (HF) that has not been well studied. We assessed whether HF patients paced from the right ventricle (RVp) adversely remodel and respond to cardiac resynchronization therapy (CRT) in a similar way to HF patients without right ventricular pacing (nRVp). Methods and results Echocardiograms were performed before and ~5 months after CRT in 31 RVp and 49 nRVp HF patients. Longitudinal intraventricular dyssynchrony using tissue Doppler imaging (TDI) was calculated as the standard deviation of time to peak systolic displacement by tissue tracking (SD-TT) of 12 segments. Longitudinal dyssynchrony within a wall (intramural dyssynchrony) was assessed by two methods: quantifying the number of segments with initial abnormal apical displacement (IMD score) and using a cross-correlation synchrony index (CCSI). Despite similar ejection fractions (EFs) of 28% prior to CRT, left ventricular end-diastolic volume was significantly smaller (143 ± 54 vs. 183 ± 62, P = 0.004) in RVp. The standard deviation of time to peak systolic displacement by tissue tracking (83.4 ± 34.9 vs. 67.9 ± 26.6, P = 0.03) and IMD score (3.1 ± 1.8 vs. 1.3 ± 1.7, P < 0.001) were greater in RVp. Cardiac resynchronization therapy significantly improved EF and volumes in both groups. Ejection fraction increased more in RVp (12.8 ± 9.2% vs. 7.4 ± 7.6%, P = 0.007). Intraventricular dyssynchrony and both measures of intramural septal dyssynchrony improved to a greater extent post-CRT in RVp. Conclusion Right ventricular pacing patients differ from nRVp HF patients in that they have smaller ventricles and greater intraventricular and intramural septal dyssynchrony. Right ventricular pacing HF patients respond better to CRT with greater improvements in EF, and intraventricular and intramural septal dyssynchrony.

Background Some studies suggest that right ventricular (RV) pacing has an adverse impact on left ventricular ejection fraction (LVEF), particularly in subjects with preexisting left ventricular (LV) dysfunction, and that direct LV pacing may be relatively protective. Interactions between pacing site and LVEF remain unclear. Objective The purpose of this study was to examine the relative impact of RV and LV pacing on LVEF by serial study during a period in which LV dysfunction, induced by tachypacing, was introduced and then resolved. Methods In each of five dogs, RV, LV, and simultaneous RV and LV (BiV) pacing modes were compared to native ventricular activation (1) prior to tachypacing (baseline), (2) weekly during a 5-week continuous tachypacing period, and (3) weekly during a 3-week post-tachypacing recovery period. At each evaluation, LVEF and LV contraction synchrony were assessed during each pacing mode. Results The decrease in LVEF during the tachypacing period was more pronounced during RV pacing than during native activation or LV or BiV pacing. The magnitude of this effect correlated with a diminishment in LV contraction synchrony that was not observed during native activation or LV or BiV pacing. During the post-tachypacing period, gradual reversal of these changes toward baseline was observed. Conclusion Compared to native activation, RV pacing worsens LVEF in a manner proportional to the severity of preexisting LV dysfunction, attributable to reduced LV contraction synchrony. In comparison, both LV and BiV pacing preserve LVEF and contraction synchrony.

Right ventricular (RV) failure is associated with poor outcomes in pulmonary hypertension (PH). We sought to phenotype the RV in PH patients with compensated and decompensated RV function by quantifying regional and global RV structural and functional changes. Twenty-two patients (age 51 +/- 11, 14 females, mean pulmonary artery (PA) pressure range 13-79 mmHg) underwent right heart catheterization, echocardiography, and ECG-gated multislice computed tomography of the chest. Patients were divided into three groups: Normal, PH with hemodynamically compensated, and decompensated RV function (PH-C and PH-D, respectively). RV wall thickness (WT) was measured at end-diastole (ED) and end-systole (ES) in three regions: infundibulum, lateral free wall, and inferior free wall. Globally, RV volumes progressively increased from Normal to PH-C to PH-D and RV ejection fraction decreased. Regionally, WT increased and fractional wall thickening (FWT) decreased in a spatially heterogeneous manner. Infundibular wall stress was elevated and FWT was lower regardless of the status of global RV function. In PH, there are significant phenotypic abnormalities in the RV even in the absence of overt hemodynamic RV decompensation. Regional changes in RV structure and function may be early markers of patients at risk for developing RV failure.

BACKGROUND The effects of dyssynchrony on global left ventricular (LV) mechanics have been well documented; however, its impact on LV energetics has received less attention. OBJECTIVE To assess the effects of LV contraction dyssynchrony on global LV mechano-energetic function in a pacing-induced acute model of dyssynchrony. METHODS Using blood-perfused isolated rabbit heart preparations (n = 11), LV pressure, coronary flow, and arteriovenous oxygen content difference were recorded for isovolumic contractions under right atrial (RA) pacing (control) and simultaneous RA and right ventricular outflow tract (RVOT) pacing (dyssynchrony). LV mechanical function was quantified by the end-systolic pressure-volume relationship (ESPVR). Myocardial oxygen consumption-pressure-volume area (MVO(2)-PVA) relationship quantified LV energetic function. Internal PVA for MVO(2 RVOT) was calculated based on the MVO(2)-PVA relationship for RA pacing. Thus, lost PVA (internal PVA-PVA(RVOT)) represents the mechanical energy not observable at the global level. RESULTS Compared to RA pacing, RVOT pacing depressed LV mechanics as indicated by a rightward shift of ESPVR (i.e., increase in V(d) from 0.58 +/- 0.10 to 0.67 +/- 0.10 mL, P < 0.05). Despite depressed mechanics, RVOT pacing was associated with greater MVO(2) such that the MVO(2)-PVA relationship intercept was markedly increased from 0.025 +/- 0.003 to 0.029 +/- 0.003 mL*O(2)/beat/100gLV (P < 0.05). Excess MVO(2) (i.e., MVO(2 RVOT)- MVO(2 RA)) significantly correlated with lost PVA (R(2)= 0.54, P < 0.001). CONCLUSION A potential mechanism explaining the observed increase in MVO(2) with dyssynchrony may be that the measured PVA at the global level underestimates the internal PVA at the cellular level, which is likely to be the true determinant of MVO(2).

Atrial fibrillation (AF) contributes significantly to morbidity and mortality in elderly patients and has been correlated with enhanced age-dependent atrial fibrosis. Reversal of atrial fibrosis has been proposed as therapeutic strategy to suppress AF.To test the ability of relaxin to reverse age-dependent atrial fibrosis and suppress AF.Aged F-344 rats (24 months old) were treated with subcutaneous infusion of vehicle or relaxin (0.4 mg/kg/day) for 2 weeks. Rat hearts were excised, perfused on a Langendorff apparatus, and stained with voltage and Ca(2+) indicator dyes. Optical mapping and programmed electrical stimulation was used to test arrhythmia vulnerability and changes in electrophysiological characteristics. Changes in protein expression and Na(+) current density (INa) were measured by tissue immunofluorescence and whole-cell patch clamp technique.In aged rats, sustained AF was readily induced with a premature pulse (n = 7/8) and relaxin treatment suppressed sustained AF by a premature impulse or burst pacing (n = 1/6) (P.01). Relaxin significantly increased atrial action potential conduction velocity and decreased atrial fibrosis. Relaxin treatment increased Nav1.5 expression (n = 6; 36% ± 10%) and decreased total collagen and collagen I (n = 5-6; 55%-66% ± 15%) in aged atria (P.05) and decreased collagen I and III and TGF-β1 mRNA (P.05). Voltage-clamp experiments demonstrated that relaxin treatment (100 nM for 2 days) increased atrial INa by 46% ± 4% (n = 12-13/group, P.02).Relaxin suppresses AF through an increase in atrial conduction velocity by decreasing atrial fibrosis and increasing INa. These data provide compelling evidence that relaxin may serve as an effective therapy to manage AF in geriatric patients by reversing fibrosis and modulating cardiac ionic currents.

Most studies of the mechanisms leading to hereditary dilated cardiomyopathy (DCM) have been performed in reconstituted in vitro systems. Genetically engineered murine models offer the opportunity to dissect these mechanisms in vivo. We generated a gene-targeted knock-in murine model of the autosomal dominant Arg141Trp (R141W) mutation in Tnnt2, which was first described in a human family with DCM. Mice heterozygous for the mutation (Tnnt2R141W/+) recapitulated the human phenotype, developing left ventricular dilation and reduced contractility. There was a gene dosage effect, so that the phenotype in Tnnt2R141W/+mice was attenuated by transgenic overexpression of wildtype Tnnt2 mRNA transcript. Male mice exhibited poorer survival than females. Biomechanical studies on skinned fibers from Tnnt2R141W/+ hearts showed a significant decrease in pCa50 (-log[Ca2+] required for generation of 50% of maximal force) relative to wildtype hearts, indicating Ca2+ desensitization. Optical mapping studies of Langendorff-perfused Tnnt2R141W/+ hearts showed marked increases in diastolic and peak systolic intracellular Ca2+ ([Ca2+]i), and prolonged systolic rise and diastolic fall of [Ca2+]i. Perfused Tnnt2R141W/+ hearts had slower intrinsic rates in sinus rhythm and reduced peak heart rates in response to isoproterenol. Tnnt2R141W/+ hearts exhibited a reduction in phosphorylated phospholamban relative to wildtype mice. However, crossing Tnnt2R141W/+ mice with phospholamban knockout (Pln-/-) mice, which exhibit increased Ca2+ transients and contractility, had no effect on the DCM phenotype. We conclude that the Tnnt2 R141W mutation causes a Ca2+ desensitization and mice adapt by increasing Ca2+-transient amplitudes, which impairs Ca2+ handling dynamics, metabolism and responses to β-adrenergic activation.

Reversible lysine acetylation is a widespread post-translational modification controlling the activity of proteins in different subcellular compartments. We previously demonstrated that a class II histone deacetylase (HDAC), HDAC4, and a histone acetyltransferase, p300/CREB-binding protein-associated factor, associate with cardiac sarcomeres and that a class I and II HDAC inhibitor, trichostatin A, enhances contractile activity of myofilaments. In this study we show that a class I HDAC, HDAC3, is also present at cardiac sarcomeres. By immunohistochemical and electron microscopic analyses, we found that HDAC3 was localized to A-band of sarcomeres and capable of deacetylating myosin heavy chain (MHC) isoforms. The motor domains of both cardiac α- and β-MHC isoforms were found to be reversibly acetylated. Biomechanical studies revealed that lysine acetylation significantly decreased the Km for the actin-activated ATPase activity of MHC isoforms. By in vitro motility assay, we found that lysine acetylation increased the actin-sliding velocity of α-myosin by 20% and β-myosin by 36% compared with their respective non-acetylated isoforms. Moreover, myosin acetylation was found to be sensitive to cardiac stress. During induction of hypertrophy, myosin isoform acetylation increased progressively with duration of stress stimuli independently of isoform shift, suggesting that lysine acetylation of myosin could be an early response of myofilaments to increase contractile performance of the heart. These studies provide the first evidence for localization of HDAC3 at myofilaments and uncover a novel mechanism modulating the motor activity of cardiac MHC isoforms.

Embryonic myocardium has a high rate of cell proliferation and regulates cellular proliferation, contractile function, and myocardial architecture in response to changes in external mechanical loads. However, the small and complex three-dimensional (3D) structure of the embryonic myocardium limits our ability to directly investigate detailed relationships between mechanical load, contractile function, and cardiomyocyte proliferation. We developed a novel 3D engineered early embryonic cardiac tissue (EEECT) from early embryonic ventricular cells to test the hypothesis that EEECT retains the proliferative and contractile properties of embryonic myocardium. We combined freshly isolated White Leghorn chicken embryonic ventricular cells at Hamburger-Hamilton (HH) stage 31 ( day 7 of a 46-stage, 21-day incubation period), collagen type I, and matrix factors to construct cylindrical-shaped EEECTs. We studied tissue architecture, cell proliferation patterns, and contractile function. We then generated engineered fetal cardiac tissue (EFCT) from HH stage 40 ( day 14) fetal ventricular cells for direct comparison with EEECT. Tissue architecture was similar in EEECT and EFCT. EEECT maintained high cell proliferation patterns by culture day 12, whereas EFCT decreased cell proliferation rate by culture day 9 ( P < 0.05). EEECT increased active contractile force from culture day 7 to day 12. The culture day 12 EEECT contractile response to the β-adrenergic stimulation was less than culture day 9 EFCT ( P < 0.05). Cyclic mechanical stretch stimulation induced myocardial hyperplasia in EEECT. Results indicate that EEECT retains the proliferative and contractile properties of developing embryonic myocardium and shows potential as a robust in vitro model of developing embryonic myocardium.

We explored the relationship between left ventricular (LV) pressure and intracellular free calcium concentration ([Ca]i) in the isolated perfused mouse heart. [Ca]i (rhod-2) and LV pressure were recorded simultaneously. In response to increases in LV volume (Frank-Starling, FS, protocol), there were increases in developed pressure (up to 250%), with no changes in pressure morphology (rise or relaxation time) or [Ca]i (magnitude and morphology) for up to 10 min. During transient increases in the stimulus interval at a fixed LV volume (mechanical restitution, MR, protocol), developed pressure increased significantly (31.3 ± 1.2%), with relatively small changes in peak systolic [Ca]i (7.4 ± 1.4%). The relaxation of [Ca]i, however, was prolonged (30.0 ± 5.5%), resulting in prolonged pressure relaxation (21.2 ± 1.9%) and increased area under the calcium transient that paralleled the increase in developed pressure (1:1 ratio). A model-based analysis showed that changes in LV pressure during the MR protocol could be completely explained by altered [Ca]i; it was not necessary to invoke any changes in model parameters (i.e., dynamic processes that link calcium to pressure). For the FS data, the model predicted only a change in the gain parameter; however, this change alone cannot reproduce well-established length-dependent changes in the steady-state force-pCa relationship. In summary, the mouse myocardium appears to be unique in that significant changes in peak developed pressure can occur with little or no change in the peak [Ca]i. Additionally, unlike other mammalian species, load-dependent prolongation of pressure relaxation is absent in the mouse heart, and pressure relaxation is primarily governed by intracellular free calcium relaxation.

The mechanisms by which α-adrenergic stimulation of the heart in vivo can cause contractile dysfunction are not well understood. We hypothesized that α-adrenergic-mediated contractile dysfunction is mediated through protein kinase C phosphorylation of troponin I, which in in vitro experiments has been shown to reduce actomyosin Mg-ATPase activity. We studied pressure-volume loops in transgenic mice expressing mutant troponin I lacking protein kinase C phosphorylation sites and hypothesized altered responses to phenylephrine. As anesthesia agents can produce markedly different effects on contractility, we studied two agents: avertin and α-chloralose-urethane. With α-chloralose-urethane, at baseline, there were no contractile abnormalities in the troponin I mutants. Phenylephrine produced a 50% reduction in end-systolic elastance in wild-type controls, although a 9% increase in troponin I mutants ( P < 0.05). Avertin was associated with reduced contractility compared with α-chloralose-urethane. Avertin anesthesia, at baseline, produced a reduction in end-systolic elastance by 31% in the troponin I mutants compared with wild-type ( P < 0.05), and this resulted in further marked systolic and diastolic dysfunction with phenylephrine in the troponin I mutants. Dobutamine produced no significant difference in the contractile phenotype of the transgenic mice with either anesthetic regimen. In conclusion, these data (α-chloralose-urethane) demonstrate that α-adrenergic-mediated force reduction is mediated through troponin I protein kinase C phosphorylation. β-Adrenergic responses are not mediated through this pathway. Altering the myofilament force-calcium relationship may result in in vivo increased sensitivity to negative inotropy. Thus choice of a negative inotropic anesthetic agent (avertin) with phenylephrine can lead to profound contractile dysfunction.

We previously showed that chronic administration of recombinant human relaxin (rhRLX; 4 microg/h) to conscious female, nonpregnant rats to reach serum levels corresponding to early to midgestation (approximately 20 ng/ml) increases cardiac output (CO) and global arterial compliance (AC) and decreases systemic vascular resistance (SVR), comparable to changes observed in midterm pregnancy. The goals of this study were to test whether chronic administration of rhRLX (4 microg/h) to conscious male rats will yield similar changes in CO and systemic arterial load and to determine whether higher infusion rates of rhRLX (50 microg/h) administered to nonpregnant female rats yielding serum concentrations corresponding to late pregnancy ( approximately 80 ng/ml) will further modify CO and SVR and global AC comparable to late gestation. CO and systemic arterial load, as quantified by SVR and AC, were obtained by using the same methods as in our previous studies. With respect to baseline, chronic rhRLX administration to male rats over 10 days at 4 mug/h increased both CO (20.5 +/- 4.2%) and AC (19.4 +/- 6.9%) and reduced SVR (12.7 +/- 3.9%). These results were comparable to those elicited by the hormone in nonpregnant female rats. In contrast, neither acute (over 4 h) nor chronic (over 6 days) infusion of the higher dose of rhRLX administered to conscious female rats resulted in significant changes in CO, AC, or SVR from baseline. We conclude that 1) rhRLX increases CO and AC and reduces SVR irrespective of sex, and 2) the rhRLX dose response is biphasic insofar as significant alterations in CO and systemic arterial load fail to occur at high serum concentrations.

Relaxin emanates from the corpus luteum of the ovary and circulates during pregnancy. Because the hormone is a potent renal vasodilator and mediates the renal vasodilation and hyperfiltration of pregnancy in conscious rats, we reasoned that it might also contribute to the broader cardiovascular changes of pregnancy. We began investigating this concept by testing whether relaxin can modify systemic arterial hemodynamics and load when chronically administered to nonpregnant rats. The major objectives of the present work were to determine whether relaxin administration to nonpregnant rats 1) modifies cardiac output (CO), systemic vascular resistance, and global arterial compliance (AC), and 2) regulates the passive mechanics of isolated arteries. To accomplish the first objective, we developed a conscious rat model for assessment of global AC. Passive mechanics of small renal arteries were assessed using a pressure arteriograph. Chronic administration of recombinant human relaxin by sc osmotic minipump to conscious, female, nonpregnant rats reduced the steady arterial load by decreasing systemic vascular resistance, increased CO, and reduced the pulsatile arterial load by increasing global AC as quantified by two indices—AC estimated from the diastolic decay of aortic pressure and CO and AC estimated by the ratio of stroke volume-to-pulse pressure. In another group of rats, relaxin administration also regulated the passive mechanics of small renal arteries, indicating that, in addition to reduction in vascular smooth muscle tone, modification of the vascular structure (e.g. extracellular matrix) contributes to the increase in global AC. These findings suggest a role for relaxin in the systemic hemodynamic changes of pregnancy, as well as novel therapeutic potential for relaxin in modifying arterial stiffness and cardiac afterload.

The renin-overproducing transgenic rat (mREN2) has recently been proposed as a model of left ventricular (LV) hypertension and fibrosis. Comparing mREN2 (n=7) to sex- and age-matched Wistar-Han control rats (WHAN, n=11), we have confirmed hypertension (mean blood pressure: 174±9 vs. 107±3 mmHg, P

Objective: CREB A133 transgenic mice that express a dominant negative CREB transcription factor in cardiomyocytes develop a dilated cardiomyopathy that is anatomically, physiologically, and clinically similar to human idiopathic dilated cardiomyopathy. The goals of this study were to quantitate left ventricular (LV) contractility and measure cardiac reserve in CREB A133 mice by using the relation of end-systolic wall stress to the velocity of fiber shortening. Methods: A total of 37 adult CD-1 mice (including both nontransgenic and CREB A133 transgenic mice) were studied with simultaneously acquired high-fidelity instantaneous aortic pressures and 2-dimensionally targeted M-mode echocardiograms. Results: CREB A133 mice displayed significantly lower values of LV fiber shortening velocities over a wide range of afterloads, and they displayed smaller dobutamine-induced shifts from baseline contractility relations. Counterbalancing effects of differences in LV geometry and aortic pressures resulted in comparable levels of LV wall stress during ejection in both groups. Conclusion: These results demonstrate directly that CREB A133 mice display reduced LV contractility at baseline and decreased cardiac reserve. (J Am Soc Echocardiogr 2001;14:209-18.)

In isovolumically beating hearts, the speed of left ventricular (LV) relaxation is uniquely determined by peak active stress (ςmax). In contrast, such a succinct description of relaxation is lacking for the ejection beats, although ejection is generally thought to hasten relaxation. We set out to determine how ejection modifies the relaxation-ςmax relationship obtained in the isovolumically beating hearts. Experiments were performed on five isolated rabbit hearts subjected to various loading conditions. Instantaneous LV pressure and volume were recorded and converted to active stress, from which isovolumic relaxation time ( T r) was defined as the time for stress to fall from 75 to 25% of ςmax (isovolumic beats) or its end-ejection value (ejection beats). Steady-state and transient isovolumic beat and steady-state ejection beat data were used to develop a multiple regression model. This model identified stress, current beat ejection, and previous beat ejection history as independent predictor variables of T r and fit the data well in all hearts ( r 2 > 0.98). Furthermore, this model could predict relaxation in transient ejection beats ( r 2 = 0.80 for all hearts). Whereas the coefficient for the current beat ejection was negative (i.e., negative effect or hastening relaxation), the ejection history coefficient was positive (i.e., positive effect or slowing relaxation). The sum of these two coefficients was negative, corresponding to the commonly observed net negative effect of ejection on relaxation. The expected positive inotropic effect of ejection was also observed. The dissipations of both positive inotropic and relaxation effects were slow, suggesting a nonmechanical underlying mechanism(s). We postulate that these two effects are linked and caused by ejection-mediated changes in myofilament Ca2+ sensitivity.

Cardiac ultrasound is a noninvasive technique that is commonly used to serially evaluate cardiac structure and function. Recent advances in Doppler-Echocardiography enable the ultrasonographer to perform a sophisticated noninvasive assessment of cardiovascular physiology. The Rhesus monkey is a frequently used non-human primate animal model of human cardiovascular disease because this species closely models human anatomy and physiology. However, while this species is frequently used in cardiovascular research, standardized echocardiographic values generated from large numbers of normal Rhesus are not available. In the present study, we performed cardiac ultrasound imaging on 28 healthy Rhesus monkeys to obtain normal reference values of cardiovascular structure and function in this species. Nomograms were generated from these data by plotting parameters of cardiovascular geometry and function with body weight. These normal reference data were compared to previously reported values obtained from prior studies that used noninvasive, invasive, and morphometric techniques.

1 To probe the cross-bridge cycle and to learn more about the cardioplegic agent BDM (2,3-butanedione monoxime), its effects on the force–velocity properties and tension transients of skinned rabbit muscle fibres were studied at 1–2 °C and pH 7.0. 2 Three millimolar BDM decreased isometric force by 50%, velocity by 29%, maximum power by 73%, and stiffness by 25%, so that the relative stiffness (stiffness/force ratio) increased by 50% compared with reference conditions in the absence of BDM. 3 Tension transients obtained under the reference condition (0 BDM) could be represented by three components whose instantaneous stiffness accounted for the initial (Phase 1) force deviation and whose exponential recoveries caused the rapid, partial (Phase 2) force recovery following the step. The fastest component had non-linear extension–force properties that accounted for about half the isometric stiffness and it recovered fully. The two slower components had linear extension-force properties that together accounted for the other half of the sarcomere stiffness. These components recovered only partially following the step, producing the intermediate (T2) level which the force approached during Phase 2. 4 Matching the force transients obtained under test conditions (3 mm BDM) required three alterations: (1) reducing the amplitude of the two slower components by 50%, in proportion to isometric force, (2) adding a non-relaxing component and (3) decreasing the amplitude of the rapidly recovering component by 12.5% so that its relative amplitude (amplitude/isometric force) was increased by 75%. The non-recovering component and the increase in relative amplitude of the rapid component were responsible for the increase in relative stiffness of the fibres produced by BDM. The rapidly recovering component had the same time constant and step-size-dependent recovery rates as the fastest of the three mono-exponential components isolated from the tension transient response under the reference condition. BDM therefore appeared to augment the fastest component of the tension transient under the reference condition. 5 The results suggest that BDM detains cross-bridges in low-force, attached states. Since these bridges are attached, they contribute to sarcomere stiffness. Since they are detained, relaxation or reversal of their immediate responses is probably due to bridge detachment rather than to their undergoing the power stroke. The observation that a portion of the test response matched the fastest component of the reference response when the amplitude of the fastest component was increased suggests that a part of the normal rapid, transient tension recovery following a release step is due to detachment of low-force bridges moved to negative-force positions by the step.

Model parameters, estimated from experimentally measured data, can provide insight into biological processes that are not experimentally measurable. Whether this optimized parameter set is a physiologically relevant complement to the experimentally measured data, however, depends on the optimized parameter set being unique, a model property known as a priori global identifiability. However, a priori identifiability analysis is not common practice in the biological world, due to the lack of easy-to-use tools. Here we present a program, Differential Algebra for Identifiability of Systems (DAISY), that facilitates identifiability analysis. We applied DAISY to several cardiovascular models: systemic arterial circulation (Windkessel, T-Tube) and cardiac muscle contraction (complex stiffness, crossbridge cycling-based). All models were globally identifiable except the T-Tube model. In this instance, DAISY was able to provide insight into making the model identifiable. We applied numerical parameter optimization techniques to estimate unknown parameters in a model DAISY found globally identifiable. While all the parameters could be accurately estimated, a sensitivity analysis was first necessary to identify the required experimental data. Global identifiability is a prerequisite for numerical parameter optimization, and in a variety of cardiovascular models, DAISY provided a reliable, fast, and simple platform to provide this identifiability analysis.