Sanjeev G. Shroff

Collagen remodeling of the pressure-overloaded, hypertrophied nonhuman primate myocardium.

Cardiac muscle is tethered within a fibrillar collagen matrix that serves to maximize force generation. In the human pressure-overloaded, hypertrophied left ventricle, collagen concentration is known to be increased; however, the structural and biochemical remodeling of collagen and its relation to cell necrosis and myocardial mechanics is less clear. Accordingly, this study was undertaken in a nonhuman primate model of left ventricular hypertrophy caused by gradual onset experimental hypertension. The amount of collagen, its light microscopic features, and proportions of collagen types I, III, and V were determined together with diastolic and systolic mechanics of the intact ventricle during the evolutionary, early, and late phases of established left ventricular hypertrophy (4, 35, and 88 weeks, respectively). In comparison to controls, we found 1) increased collagen at 4 weeks, as well as a greater proportion of type III, in the absence of myocyte necrosis; 2) collagen septae were thick and dense at 35 weeks, while the proportion of types I and III had converted to control; 3) necrosis was evident at 88 weeks, and the structural remodeling and proportion of collagen types I and III reflected the extent of scar formation; and 4) unlike diastolic myocardial stiffness, which was unchanged at 4, 35, or 88 weeks, the systolic stress-strain relation of the myocardium was altered in either a beneficial or detrimental manner in accordance with structural remodeling of collagen and scar formation. Thus, early in left ventricular hypertrophy, reactive fibrosis and collagen remodeling occur in the absence of necrosis while, later on, reparative fibrosis is present. In this study, the remodeled collagen matrix appeared responsible for variations in force generation observed during various phases of left ventricular hypertrophy.

Fibrillar collagen and myocardial stiffness in the intact hypertrophied rat left ventricle.

This study tested the hypothesis that with hypertrophy, the proportion, distribution, and structural alignment of fibrillar collagen are important determinants of myocardial stiffness. Toward this end, the collagen volume fraction (morphometry), the transmural or subendocardial distribution of collagen, and the structural arrangement of fibrillar collagens (picrosirius red) were examined in the hypertrophied ventricle secondary to pressure overload (abdominal aorta banding or perinephritis), isoproterenol, and pressure overload plus isoproterenol. In the same hearts, the slopes of the systolic and diastolic stress-strain relations of the left ventricle, representing its active and passive stiffness, respectively, were obtained. In comparison with controls, we found 1) for a moderate rise in transmural collagen, active and passive stiffness increased with pressure-overload hypertrophy; 2) following isoproterenol alone there was a marked increase in subendocardial collagen, and active and passive stiffness increased; 3) in pressure-overload hypertrophy plus isoproterenol, active stiffness declined. Passive stiffness was increased except when fibrosis and thinning of the interventricular septum occurred, in which case it decreased; and 4) fibrillar collagens involved in remodeling included the formation of either collagen strands and fibers in a greater number of previously collagen-free intermuscular spaces in pressure-overload hypertrophy, or a dense crisscrossing latticework of fibers that encircled muscle fibers after isoproterenol. Thus, an increase in fibrillar collagen in pressure- overload hypertrophy is partially adaptive in that it enhances the tensile strength and three- dimensional delivery of force by the myocardium, but at the expense of reducing distensibility. The appearance of a dense collagen meshwork within the subendocardium after isoproterenol can be considered pathological in that it entraps muscle fibers causing active stiffness to fall while impairing distensibility. Finally, fibrosis may paradoxically reduce passive stiffness if it leads to a thinning of the interventricular septum.

Association Between Extracellular Matrix Expansion Quantified by Cardiovascular Magnetic Resonance and Short-Term Mortality

Background— Extracellular matrix expansion may be a fundamental feature of adverse myocardial remodeling, it appears to be treatable, and its measurement may improve risk stratification. Yet, the relationship between mortality and extracellular matrix is not clear because of difficulties with its measurement. To assess its relationship with outcomes, we used novel, validated cardiovascular magnetic resonance techniques to quantify the full spectrum of extracellular matrix expansion not readily detectable by conventional cardiovascular magnetic resonance. Methods and Results— We recruited 793 consecutive patients at the time of cardiovascular magnetic resonance without amyloidosis or hypertrophic cardiomyopathy as well as 9 healthy volunteers (ages 20–50 years). We measured the extracellular volume fraction (ECV) to quantify the extracellular matrix expansion in myocardium without myocardial infarction. ECV uses gadolinium contrast as an extracellular space marker based on T1 measures of blood and myocardium pre— and post–gadolinium contrast and hematocrit measurement. In volunteers, ECV ranged from 21.7% to 26.2%, but in patients it ranged from 21.0% to 45.8%, indicating considerable burden. There were 39 deaths over a median follow-up of 0.8 years (interquartile range 0.5–1.2 years), and 43 individuals who experienced the composite end point of death/cardiac transplant/left ventricular assist device implantation. In Cox regression models, ECV related to all-cause mortality and the composite end point (hazard ratio, 1.55; 95% confidence interval, 1.27–1.88 and hazard ratio, 1.48; 95% confidence interval, 1.23–1.78, respectively, for every 3% increase in ECV), adjusting for age, left ventricular ejection fraction, and myocardial infarction size. Conclusions— ECV measures of extracellular matrix expansion may predict mortality as well as other composite end points (death/cardiac transplant/left ventricular assist device implantation).

Serial Assessment of the Cardiovascular System in Normal Pregnancy Role of Arterial Compliance and Pulsatile Arterial Load

BACKGROUND Temporal changes in systemic arterial compliance and wave propagation properties (pulsatile arterial load) and their role in ventricular-systemic arterial coupling during gestation have not been explored. Noninvasive methods combined with recently developed mathematical modeling techniques were used to characterize vascular and left ventricular (LV) mechanical adaptations during normal gestation. METHODS AND RESULTS Fourteen healthy women were studied at each trimester of pregnancy and again postpartum. Experimental measurements included instantaneous aortic pressure (subclavian pulse tracings) and flow (aortic Doppler velocities) and echocardiographic imaging of the LV. A small increase in LV muscle mass and end-diastolic chamber dimension occurred by late gestation, with no significant alterations in myocardial contractility. Cardiac output increased and the steady component of arterial load (total vascular resistance) decreased during pregnancy. Several changes in pulsatile arterial load were noted: Global arterial compliance increased (approximately 30%) during the first trimester and remained elevated thereafter. The magnitude of peripheral wave reflections at the aorta was reduced. The mathematical model-based analysis revealed that peripheral wave reflections at the aorta were delayed and that both conduit and peripheral vessels contributed to the increased arterial compliance. Finally, coordinated changes in the pulsatile arterial load and LV properties were responsible for maintaining the efficiency of LV-to-arterial system energy transfer. CONCLUSIONS The rapid time course of compliance changes and the involvement of both conduit and peripheral vessels are consistent with reduced vascular tone as being the main underlying mechanism. The pulsatile arterial load alterations during normal pregnancy are adaptive in that they help to accommodate the increased intravascular volume while maintaining the efficiency of ventricular-arterial coupling and diastolic perfusion pressure.

Determination of pulse wave velocities with computerized algorithms

Careful determination of pulse wave velocity is important in the study of arterial viscoelastic properties, wave reflections, and ventricular-arterial interactions. In spite of its increasingly widespread use, there is as yet no standardized method for its determination. Most studies have manually identified the transit time of the pressure wave front as it travels over a known distance in the arterial system, but the issues of accuracy and reproducibility have not been addressed. This study was designed to investigate the efficacy of four computerized algorithms in the determination of pulse wave velocities in invasive as well as in noninvasive pressure determinations. The four methods were the identification of: (1) the point of minimum diastolic pressure, (2) the point at which the first derivative of pressure is maximum, (3) the point at which the second derivative of pressure is maximum, and (4) the point yielded by the intersection of a line tangent to the initial systolic upstroke of the pressure tracing and a horizontal line through the minimum point. High-fidelity aortic pressure recordings were obtained in 26 patients with a multi-sensor micromanometer catheter. Noninvasive brachial and radial pressure waveforms were recorded in 11 volunteers with external piezoelectric transducers. The results show that the first derivative method consistently provided results that were different from the other methods for both the invasive and noninvasive methods because of changes in the structure of the upstroke as the arterial pulse propagates distally. Although the minimum method worked well for the invasive determinations, it was erratic with the noninvasive determinations, probably because of the higher amount of noise and reflection in the latter. Among the four algorithms, the second derivative and the intersecting tangents methods worked well with both invasive and noninvasive determinations with mean variation coefficients of less than 7% and correlation coefficients between the methods of greater than 0.90 for all data. In conclusion, computerized algorithms allow accurate determination of pulse wave velocity in invasively and noninvasively measured arterial pressure waveforms.

Myocardial extracellular volume fraction quantified by cardiovascular magnetic resonance is increased in diabetes and associated with mortality and incident heart failure admission

AIMS Diabetes 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 RESULTS We 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. CONCLUSION Diabetes 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.

Myocardial extravascular extracellular volume fraction measurement by gadolinium cardiovascular magnetic resonance in humans: slow infusion versus bolus

BackgroundMyocardial extravascular extracellular volume fraction (Ve) measures quantify diffuse fibrosis not readily detectable by conventional late gadolinium (Gd) enhancement (LGE). Ve measurement requires steady state equilibrium between plasma and interstitial Gd contrast. While a constant infusion produces steady state, it is unclear whether a simple bolus can do the same. Given the relatively slow clearance of Gd, we hypothesized that a bolus technique accurately measures Ve, thus facilitating integration of myocardial fibrosis quantification into cardiovascular magnetic resonance (CMR) workflow routines. Assuming equivalence between techniques, we further hypothesized that Ve measures would be reproducible across scans.MethodsIn 10 volunteers (ages 20-81, median 33 yr, 3 females), we compared serial Ve measures from a single short axis slice from two scans: first, during a constant infusion, and second, 12-50 min after a bolus (0.2 mmol/kg gadoteridol) on another day. Steady state during infusion was defined when serial blood and myocardial T1 data varied <5%. We measured T1 on a 1.5 T Siemens scanner using a single-shot modified Look Locker inversion recovery sequence (MOLLI) with balanced SSFP. To shorten breath hold times, T1 values were measured with a shorter sampling scheme that was validated with spin echo relaxometry (TR = 15 sec) in CuSO4-Agar phantoms. Serial infusion vs. bolus Ve measures (n = 205) from the 10 subjects were compared with generalized estimating equations (GEE) with exchangeable correlation matrices. LGE images were also acquired 12-30 minutes after the bolus.ResultsNo subject exhibited LGE near the short axis slices where Ve was measured. The Ve range was 19.3-29.2% and 18.4-29.1% by constant infusion and bolus, respectively. In GEE models, serial Ve measures by constant infusion and bolus did not differ significantly (difference = 0.1%, p = 0.38). For both techniques, Ve was strongly related to age (p < 0.01 for both) in GEE models, even after adjusting for heart rate. Both techniques identically sorted older individuals with higher mean Ve values.ConclusionMyocardial Ve can be measured reliably and accurately 12-50 minutes after a simple bolus. Ve measures are also reproducible across CMR scans. Ve estimation can be integrated into CMR workflow easily, which may simplify research applications involving the quantification of myocardial fibrosis.

HDAC4 and PCAF Bind to Cardiac Sarcomeres and Play a Role in Regulating Myofilament Contractile Activity

Reversible acetylation of lysine residues within a protein is considered a biologically relevant modification that rivals phosphorylation ( Kouzarides, T. (2000) EMBO J. 19, 1176-1179 ). The enzymes responsible for such protein modification are called histone acetyltransferases (HATs) and deacetylases (HDACs). A role of protein phosphorylation in regulating muscle contraction is well established ( Solaro, R. J., Moir, A. J., and Perry, S. V. (1976) Nature 262, 615-617 ). Here we show that reversible protein acetylation carried out by HATs and HDACs also plays a role in regulating the myofilament contractile activity. We found that a Class II HDAC, HDAC4, and an HAT, PCAF, associate with cardiac myofilaments. Primary cultures of cardiomyocytes as well as mouse heart sections examined by immunohistochemical and electron microscopic analyses revealed that both HDAC4 and PCAF associate with the Z-disc and I- and A-bands of cardiac sacromeres. Increased acetylation of sarcomeric proteins by HDAC inhibition (using class I and II HDAC inhibitors or anti-HDAC4 antibody) enhanced the myofilament calcium sensitivity. We identified the Z-disc-associated protein, MLP, a sensor of cardiac mechanical stretch, as an acetylated target of PCAF and HDAC4. We also show that trichostatin-A, a class I and II HDAC inhibitor, increases myofilament calcium sensitivity of wild-type, but not of MLP knock-out mice, thus demonstrating a role of MLP in acetylation-dependent increased contractile activity of myofilaments. These studies provide the first evidence that HATs and HDACs play a role in regulation of muscle contraction.

Physiologic Versus Pathologic Hypertrophy and the Pressure-Overloaded Myocardium

The myocardium consists of myocytes and capillaries embedded in a connective tissue matrix. Myocardial mass, which is predominantly a function of myocyte size, is determined by systolic tension: when systolic pressure is gradually elevated above the normal range, mass will increase. The hypertrophic process is a continuum consisting of subtle transitions that take place within the muscular, collagenous, and vascular compartments; these transitions, however, need not be temporarily concordant. We would identify three phases to the hypertrophic process. First, there is an evolutionary phase, whereby the structural and biochemical remodeling of the various compartments of the myocardium is in transition, with each compartment having its own rate of adjustment. During this evolutionary phase, myocardial contractility, as reflected by stress-length and stress-velocity relations, may or may not be normal, but ventricular pump function and O2 delivery are preserved. Second, there is a physiologic phase during which the structural and biochemical remodeling of the compartments reaches a coordinated balance. The myocardial stress-length relation and ventricular function are each normal, but rate-dependent indices of contractility may be abnormal. During the physiologic phase of hypertrophy, the remodeled myocardium will revert to normal when the abnormal loading condition is removed. Finally, there is a pathologic phase. In this phase, compartment remodeling is no longer balanced (e.g., the ratio of structural versus maintenance proteins), and length and rate-dependent indices of myocardial contractility are depressed. Ventricular pump function is also abnormal in the pathologic phase: consequently, O2 delivery to the tissues is impaired. This imbalance in O2 demand and supply may be apparent at rest in more advanced expressions of disease or may appear during the physiologic stress of exercise in less severe disease. In the latter case, the patients aerobic capacity is reduced to the extent that it can be used to grade the severity of heart failure and to predict the cardiac reserve. During the pathologic phase of hypertrophy, the structural and biochemical remodeling of the myocardium may be irreversible, although this may not be the case for each compartment. Finally, it is important to distinguish cardiac (or myocardial) failure from the clinical syndrome of congestive heart failure. The latter arises from congested organs and hypoperfused tissues; its clinical manifestations are dependent on the activation of the adrenergic nervous and renin-angiotensin-aldosterone systems and the presence of a salt-avid kidney. Congestive heart failure is a late clinical feature of chronic pressure overload and pathologic hypertrophy.

Ultrasonic backscatter system for automated on-line endocardial boundary detection: Evaluation by ultrafast computed tomography

OBJECTIVES The purpose of this study was to evaluate the accuracy of the recently developed echocardiographic on-line endocardial border detection system using ultrafast computed tomography, an independent and proved tomographic imaging modality. BACKGROUND The automated system for on-line endocardial border detection identifies the blood-tissue interface by acoustic quantification of the ultrasonic backscatter signal. METHODS Eighteen subjects were screened by conventional echocardiography and acoustic quantification. Ten of these, with high quality echocardiographic images, were also examined by ultrafast computed tomography. Comparable image planes at the midpapillary level were analyzed. Measurements of left ventricular cavity area were compared at end-diastole and end-systole and time course analyses of cavity area during the cardiac cycle were performed. RESULTS There was good correlation between values for left ventricular end-diastolic area (r = 0.99), end-systolic area (r = 0.93) and fractional area change (r = 0.91) using the two methods. The on-line backscatter system underestimated end-diastolic area (p < 0.001), but the negative bias was small (-1.6 cm2) and the 95% confidence intervals were narrow (-3.6 cm2 to +0.4 cm2). In contrast, the backscatter system overestimated end-systolic area (p < 0.02); the positive bias for this variable was also small (+2.6 cm2) but the confidence intervals were relatively wide (+7.9 to -2.8 cm2). The negative bias of backscatter values for cavity area was fairly constant during diastole and early systole (range -5% to -10%), but during the second half of systole, backscatter values increased progressively relative to computed tomographic values. Real time values for fractional area change measured by the backscatter system were 13% smaller than those determined by ultrafast computed tomography (p < 0.001), with wide confidence intervals (+3% to -30%). Absolute peak rates of area change during systole and diastole were lower by 39% (p < 0.001) and 41% (p < 0.01), respectively, using the on-line ultrasonic backscatter system. Time course analyses revealed the errors to be consistent with cardiac cycle-dependent alterations in gain sensitivity of the ultrasonic backscatter system. CONCLUSIONS The ultrasonic backscatter system is associated with cyclic cavity area measurement errors that need to be addressed if its early promise for on-line assessment of ventricular function is to be fulfilled. Incorporation of an electrocardiographically triggered time-varying gain control may improve accuracy for on-line analysis of ventricular performance.