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Heart failure due to dilated cardiomyopathy is frequently caused by myocarditis. However, the pathogenesis of myocarditis remains incompletely understood. Here, we report the presence of neutrophil extracellular traps (NETs) in cardiac tissue of patients and mice with myocarditis. Inhibition of NET formation in experimental autoimmune myocarditis (EAM) of mice substantially reduces inflammation in the acute phase of the disease. Targeting the cytokine midkine (MK), which mediates NET formation in vitro, not only attenuates NET formation in vivo and the infiltration of polymorphonuclear neutrophils (PMNs) but also reduces fibrosis and preserves systolic function during EAM. Low-density lipoprotein receptor-related protein 1 (LRP1) acts as the functionally relevant receptor for MK-induced PMN recruitment as well as NET formation. In summary, NETosis substantially contributes to the pathogenesis of myocarditis and drives cardiac inflammation, probably via MK, which promotes PMN trafficking and NETosis. Thus, MK as well as NETs may represent novel therapeutic targets for the treatment of cardiac inflammation., (© 2019 Weckbach et al.)

Objectives: The aim of this study was to investigate the disparity between the extent of myocardial injury as assessed by thallium and the severity of left ventricular (LV) dysfunction in chronic ischemic heart disease., Background: Although it is believed that thallium differentiates between viable and nonviable myocardium, in some patients with chronic ischemic heart disease, viable regions by thallium may fail to improve function after revascularization., Methods: Thirteen transplant candidates with chronic ischemic heart disease (LV ejection fraction = 14 +/- 6% at rest) were studied prospectively with stress-redistribution-reinjection thallium single-photon emission computed tomography. We examined pretransplantation quantitative thallium uptake and post-transplantation extent and the histological distribution of collagen replacement in infarcted and noninfarcted myocardium and in 13 age-matched control hearts., Results: The volume fraction of collagen varied inversely with wall thickness (r = -0.70, p < 0.001) and was higher in irreversible (30.9 +/- 15.8%) compared with reversible (20.2 +/- 12.6%, p < 0.001) or normal thallium segments (15.0 +/- 8.7%, p < 0.001). The irreversible thallium segments had lower wall thickness and more severe coronary artery narrowing (9.7 +/- 2.8 mm and 95 +/- 8%) compared with reversible (11.7 +/- 2.7 mm and 87 +/- 13%, p < 0.001) and normal thallium segments (12.8 +/- 2.6 mm and 80 +/- 14%, p < 0.001). Mean volume fraction of collagen was significantly lower in noninfarcted than it was in infarcted segments (13 +/- 6% vs. 36 +/- 13%, p < 0.001) but exceeded that in the control hearts (4 +/- 2%, p < 0.001). Noninfarcted segments had predominantly interstitial fibrosis with either microscopic or patchy areas of replacement fibrosis., Conclusions: In chronic ischemic heart disease with severe LV dysfunction, patterns of normal, reversible and irreversible thallium uptake correlated with the magnitude of collagen replacement, segmental wall thickness and severity of coronary artery narrowing. The finding of scattered areas of replacement fibrosis in noninfarcted myocardium may explain the observed disparity between LV contractile dysfunction and the extent of myocardial injury assessed by thallium.

Fibrosis in the heart may result from loss of myocytes, which are replaced by collagens. Apoptosis is now known to contribute to myocyte loss in the failing human heart. The mechanisms underlying the induction of cardiomyocyte apoptosis, and thus the expansion of fibrotic foci in the failing heart, are poorly understood. We hypothesized that viable heart cells adjacent to fibrotic foci might become "predisposed" to apoptosis by expression of the receptor FAS (APO1, CD95). We therefore studied the spatial relationship of FAS expression and fibrosis in patients with heart failure. Left ventricular biopsies were obtained from seven patients undergoing coronary artery bypass grafting. All patients had reduced ejection fraction but varied in New York Heart Association class score at the time of surgery. Heart cell apoptosis, fibrosis, and FAS expression were studied by propidium iodide and in situ end labeling (ISEL) of DNA, Picrosirius red staining, and immunohistochemistry. All patient samples exhibited, albeit to varying degrees, apoptosis detected by ISEL, chromatin condensation, and nuclear fragmentation. In all samples, fibrosis (collagen) was evident both perivascular and in isolated regions of scarring. Regardless of the extent of fibrosis or detectable apoptosis, FAS expression was observed in regions immediately adjacent to the fibrosis, but not in regions distal to fibrosis, nor in fibrotic areas devoid of nuclei. Expression of FAS was found adjacent to both perivascular and diffuse fibrosis, and ISEL-positive nuclei were found within cells reacting positively with anti-FAS antibodies. However, ISEL-positive nuclei were no more abundant in FAS-positive regions (67.6 +/- 5.8% of total nuclei) than in FAS-negative areas (69.5 +/- 9.8%). We conclude that expression of FAS occurs in remaining heart cells adjacent to fibrosis of either perivascular or presumed reparative origin. Although this phenomenon could contribute to the expansion of fibrotic foci, FAS-induced apoptosis in the failing heart may not be more prevalent than apoptosis initiated by other signaling mechanisms.

Although the role of angiotensin II (Ang II) in the pathogenesis and progression of the failing heart is uncertain, previous reports have suggested that myocyte injury may be a component in this process. In this study, we investigated this possibility in more detail. Cardiotoxic effects of nonacutely hypertensive doses of Ang II were examined in 90 rats, including those receiving an angiotensin infusion (200 ng/min i.p.) and those with renovascular hypertension, where endogenous stimulation of Ang II occurred. Myocyte injury and wound healing resulting from these treatments were evaluated by 1) immunofluorescence after in vivo monoclonal antibody labeling of myosin to detect abnormal sarcolemmal permeability, 2) [3H]thymidine incorporation into DNA, to detect fibroblast proliferation, and 3) light microscopic evidence of myocytolysis and subsequent scar formation. We found that exogenous Ang II produced multifocal antimyosin labeling of cardiac myocytes and myocytolysis, which were maximal on days 1-2 of the infusion. Subsequently, DNA synthesis rates were increased, with fibroblast proliferation reaching peak levels on day 2 (Ang II-treated rats, 90.0 +/- 18.6 cpm/micrograms DNA; control rats, 11.4 +/- 2.3 cpm/micrograms DNA; p less than 0.05); microscopic scarring was found on day 14 and represented 0.12 +/- 0.02% of the myocardium. Concurrent treatment with both propranolol (30 mg/kg/day s.c.) and phenoxybenzamine (5 mg/kg/day i.m.) did not attenuate Ang II-induced antimyosin labeling. Increased endogenous Ang II, resulting from renal ischemia after abdominal aortic constriction, produced both antimyosin labeling and increased rates of DNA synthesis like that observed with Ang II infusion. Both myocyte injury and fibroplasia were prevented with captopril (65 mg/day p.o.), but this protective effect was not seen with reserpine pretreatment. Infrarenal aortic banding without renal ischemia, on the other hand, produced hypertension without necrosis. We conclude that pathophysiological levels of endogenous as well as low-dose exogenous Ang II were associated with altered sarcolemmal permeability and myocytolysis with subsequent fibroblast proliferation and scar formation. Myocyte injury was unrelated to the hypertensive or enhanced adrenergic effects of Ang II or to hypertension per se. Captopril was effective in preventing myocyte injury in renovascular hypertension. The mechanism(s) responsible for Ang II-induced necrosis will require further study.

To test the hypothesis that in the failing volume-overloaded ventricle, the extracellular matrix and fibrillar collagen in particular are major determinants of the architectural remodeling of the myocardium, this histopathological study of the dilated, postmortem canine left ventricle secondary to rapid ventricular pacing or aortocaval fistula was undertaken. Using the picrosirius-polarization technique to enhance collagen birefringence, we sought to examine the structural integrity of the collagen matrix and interstitium. In the dilated failing ventricle secondary to rapid pacing, we found 1) interstitial edema and a disruption or disappearance of collagen fibers that were apparent within 6 hours of pacing, persisted for weeks, and subsequently were associated with muscle fiber disorganization within the endomyocardium, 2) interstitial fibrosis that was present in the midwall and epimyocardium with chronic pacing, and 3) an early remodeling of intramyocardial coronary arteries that included medial swelling with smooth muscle degeneration followed by proliferative lesions involving fibroblasts and a subsequent perivascular and medial fibrosis. Many of these findings were still evident 48 hours after pacing had been discontinued. In contrast, the collagen matrix and interstitium seen with ventricular dilatation secondary to the circulatory overload that accompanies an aortocaval fistula were indistinguishable from that in sham-operated controls. Thus, we conclude that unlike the chamber enlargement and preserved ventricular function that accompany an aortocaval fistula, ventricular dilatation and failure caused by rapid pacing are based on an architectural remodeling of the myocardium. This structural dilatation involves the extracellular matrix and interstitium and appears to be related to altered permeability of intramyocardial coronary arteries. The mechanism or mechanisms involved in the pathogenesis of myocardial remodeling with rapid ventricular pacing require further investigation.

Study Objective: The aim was to compare the temporal sequence and structural relationship between perivascular and interstitial fibrosis and microscopic scarring seen in the left ventricle in response to either a transient or sustained stimulus to fibrosis., Design: In 72 male Wistar rats (250-350 g) the transient stimulus model was based on the administration of isoprenaline (500 micrograms.kg-1) while the sustained stimulus model was produced by abdominal aortic banding with right renal artery constriction. Serial sections of myocardium were examined and compared at 4 and 12 weeks in each model and to corresponding controls., Experimental Material: The collagen specific stain, Sirius Red F3BA, was used to determine collagen volume fraction and the fibrillar nature of the fibrous tissue response seen by light microscopy., Measurements and Main Results: Following isoprenaline a stable reparative fibrosis of the endomyocardium and increase in collagen volume fraction was seen without an interstitial or perivascular fibrosis of the non-involved myocardium. In unilateral renal ischemia, on the other hand, a progressive perivascular fibrosis was evident throughout the myocardium and from which fibrillar collagen extended into the extracellular space between muscle bundles creating an interstitial fibrosis; microscopic scarring of the endomyocardium became evident at 12 weeks., Conclusions: The reactive perivascular fibrosis of intramyocardial coronary arteries seen in renovascular hypertension is a progressive process that leads to an interstitial fibrosis and eventual microscopic scarring. In contrast, the endomyocardial scarring that follows isoprenaline induced myocyte necrosis is stable and intramural vessels in remote regions are not involved.

Characteristics of pressure overload hypertrophy are known to include an accumulation of collagen (or fibrosis) and a biochemical remodeling of fibrillar type I and III collagens. The corresponding structural nature of myocardial fibrosis is less clear. This light morphologic study was undertaken to address this issue in the hypertrophied left ventricle of the nonhuman primate with experimental hypertension. For this purpose, the picrosirius red technique and polarization microscopy were used to examine the myocardium during the evolutionary, early, and late phases of established hypertrophy corresponding to 4, 35, and 88 weeks of experimental hypertension. Evidence of increased thin perimysial fiber formation, together with collagen fiber disruption and edema at 4 weeks of hypertrophy, was found when the chamber volume to left ventricular mass ratio was reduced. After 35 weeks, when this ratio was again normal, a greater number of intermuscular spaces contained both thick and thin perimysial fibers. In addition to this interstitial fibrosis, a reactive fibrosis consisting of a meshwork of thick and thin perimysial fibers was seen extending over muscle fibers. Finally, at 88 weeks, this fibrous meshwork had encircled muscle and there now was evidence of cell necrosis. The accompanying replacement fibrosis consisted of yet another distinctive orthogonal grid of thick and thin collagen fibers. Thus, a continuum of fibrillar collagen remodeling was seen in pressure overload hypertrophy in the nonhuman primate myocardium. Structurally distinct patterns of myocardial fibrosis were recognized based on the alignment of perimysial fibers with muscle that may explain the cellular remodeling and altered mechanical behavior of the concentrically hypertrophied myocardium.

The structural nature of fibrillar collagen involved in the replacement fibrosis which accompanies discrete areas of cell necrosis remains uncertain, as does its influence on the diastolic and systolic stiffness of the intact myocardium. This study, using 15 micron diameter microsphere embolisation of the rat myocardium, was undertaken to address these issues. Collagen volume fraction (trichrome), fibrillar collagens (picrosirius-polarisation technique), and the stress-strain relations of the intact myocardium (isolated hearts) were determined 30 d after the infusion of microspheres into the left ventricle. Significant differences from controls included: (a) the presence of hypertension secondary to renovascular embolisation; (b) a greater volume fraction of collagen that included not only a meshwork of short, taut appearing, thick and thin collagen fibres, interposed between muscle in areas of cell loss, but also a perivascular fibrosis involving intramyocardial coronary arteries; (c) elevated active stiffness, and (d) a more exponential diastolic stress-strain relation with increased stiffness at strains of 5% or more. These findings suggest that the replacement fibrosis accompanying myocyte necrosis has distinguishing morphological features involving fibrillar collagen and which because of its structure, alignment, and location relative to muscle leads to enhanced myocardial stiffness, including a more exponential rise in the diastolic stress-strain relation. The perivascular accumulation of collagen suggests that additional factors other than microsphere induced necrosis were responsible for this reactive fibrosis.

This study had two objectives: (a) to determine the accumulation of collagen and its structural remodelling in the hypertrophied rat left ventricle after 4 and 8 weeks of abdominal aorta banding; and (b) to correlate these findings with the diastolic stress-strain relation of the intact myocardium. In comparison to age and sex matched controls, the collagen volume fraction of the hypertrophied myocardium after 4 and 8 weeks of aortic banding increased significantly from 3.5(SD1.0)% to 7.8(4.2)% and 6.2(2.0)% respectively. This accumulation of collagen, or fibrosis, occurred in the absence of myocyte necrosis. Scanning electron microscopy showed increased density and thickness of the collagen weave and tendons. At 4 weeks, light microscopy showed interstitial oedema and disrupted collagen fibrils. Left ventricular diastolic stress-strain relations of both pressure overload groups were significantly steeper than that of the control group. Thus the response of the interstitium to the hypertrophic process that accompanies abdominal aorta banding is a complex process that includes a structural remodelling of the fibrillar collagen matrix and the early appearance of interstitial oedema, each of which may contribute to a rise in the passive stiffness of the intact myocardium.

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.

Isoproterenol treatment leads to endomyocardial fibrosis with muscle fibers encircled by fibrillar collagen. This study was undertaken in the rat to determine if muscle encased in collagen would subsequently become either necrotic or atrophic. For this purpose, we monitored the fibrillar nature of myocardial collagen, its alignment with muscle, and the morphology of the endomyocardium, together with the response in diastolic and systolic myocardial stiffness, immediately on completion (10 days) and 30 days after a course of subcutaneous isoproterenol (500 micrograms/kg/day). We found 1) left ventricular hypertrophy at 10 and 30 days with an increase in collagen volume fraction (p less than 0.01) that consisted of a meshwork of thick and thin collagen fibers that encircled endomyocardial muscle, 2) a variable reduction in endocardial muscle fiber diameter at 30 days with the greatest thinning seen in muscle encircled by fibrous tissue, and 3) an elevation (p less than 0.01) in the slope of the diastolic stress-strain relation at 10 and 30 days. The developed systolic stress-strain relation, which was elevated at 10 days (p less than 0.01), declined (p less than 0.05) with the reduction in endomyocardial muscle mass. Thus, endomyocardial muscle, encircled by fibrillar collagen, will atrophy over time, and this leads to a reduction in active stiffness. These findings may, in part, explain why progressive ventricular dysfunction accompanies chronic myocardial disease with endomyocardial fibrosis.

Left ventricular pressure overload will result in the hypertrophic growth of the myocardium and in the rat may include a remodeling of both the structural and contractile proteins. As a result, an adaptive rise in active stiffness, or the force generating capacity of the myocardium, may occur. The relative importance of structural vs. contractile protein remodeling to the hypertrophic response and active stiffness is unclear. Accordingly, we monitored the ratio of V1/V3 isomyosin and the fibrillar nature and volume fraction of myocardial collagen, together with the developed systolic stress-strain relation of the intact myocardium in the adult male Wistar rat after 8 weeks of abdominal aorta banding. In comparison to controls and for the 20% increase in left ventricular mass obtained with banding we found: (a) collagen volume fraction had increased significantly (6.2 +/- 2.0 vs. 3.6 +/- 1.0%) while the V1/V3 ratio did not change; (b) interstitial compartment remodeling included a perivascular accumulation of collagen around small intramyocardial coronary arteries and the appearance of more extensive fibrillar collagens; and (c) active stiffness increased significantly. Thus, the increase in active stiffness of the hypertrophied adult rat myocardium, seen with abdominal aorta banding, appears to be related to interstitial fibrosis and not a conversion of myosin isoforms.

The accumulation of collagen within the myocardium is termed fibrosis. In left ventricular pressure overload a reactive interstitial fibrosis, having distinctive biochemical and structural features, is seen. This reactive fibrosis occurs in the absence of myocyte necrosis, is progressive in nature, and initially is an adaptive response that preserves the force generating capacity, or active (systolic) stiffness, of the hypertrophied myocardium. Later in hypertrophy a reparative (or replacement) fibrosis occurs in response to cell loss, the pathogenesis of which is not clear. Nevertheless, independently of cell loss, interstitial fibrosis can have a detrimental influence on the diastolic and systolic stiffness of the myocardium and can result in pathologic hypertrophy with heart failure. In established hypertrophy with disproportionate collagen matrix remodeling (ie, interstitial heart disease), it would be desirable to retard the continued formation of collagen and, if necessary, degrade collagen fibers that are responsible for impeding the stretching and shortening of muscle fibers. Prevention of interstitial fibrosis in pressure overload hypertrophy with pharmacologic agents with both antihypertensive and antifibrotic properties must also be considered. Future research should address these issues with a view toward developing corrective and preventative forms of therapy. Such advances will require a better understanding of cardiac fibroblast growth, collagen synthesis and the regulation of collagen gene expression in the heart.

To determine if a remodeling of the collagen matrix would occur in the absence of hypertrophy and cell necrosis and if such a remodeling could alter active and passive stiffness of the intact myocardium, five rats with genetic hypertension (SHR) were treated (SHRT) with hydralazine for 32 weeks, beginning at four weeks of age, and compared to six age- and sex-matched SHR and seven Wistar-Kyoto genetic control rats (WKY). Left ventricular (LV) weight of SHRT was 17% lower (P less than .001) than that of SHR and 19% higher (P less than .01) than that of WKY. Collagen volume fraction of SHR (13.7 +/- 3.2%) and SHRT (9.9 +/- 1.8%) were greater (P less than .01) than WKY (5.0 +/- 1.9%). Diastolic and systolic stress-strain relations were determined in the isolated heart. A comparison of these relations revealed: 1) a 24% increase in passive stiffness for SHR and SHRT; and 2) a reduced zero-strain intercept (41% to 54%) and slope (36% to 48%) of the developed stress-strain relation for the SHRT. Thus, in SHR, collagen remodeling occurred in the absence of hypertrophy which suggests that the muscular and collagenous compartments of the myocardium are under separate controls. The excess accumulation of collagen in SHR and SHRT leads to abnormal passive stiffness, and the prevention of hypertrophy with hydralazine reduces active stiffness.

A study of isoproterenol-induced (1 mg/kg) myocardial fibrosis in the rat was undertaken, taking advantage of the differential colorization provided by thick and thin collagen fibers to picrosirius red and polarization microscopy. The objective was to monitor the sequence (day 1, 2, 3, 4, and 8), fibrillar composition, and nature of isoproterenol-induced collagen remodeling, which was found previously to adversely influence myocardial stiffness. The following were found: 1) a distortion and widening of intermuscular spaces on day 1 that was accompanied by the disruption of collagen fibers; 2) by day 2, these spaces were closing and a new fibrillar collagen network had appeared consisting primarily of thinner collagen fibers that crossed over muscle fibers; 3) the new fibrillar network took on a clear crisscrossing pattern on day 3 and 4 as an ever-increasing number of thicker fibers became entwined perpendicular to the thinner fibers; and 4) by day 8, a dense mesh of thick and thin collagen fibers had formed to encircle muscle while a greater number of intermuscular spaces, previously devoid of collagen, were now filled with thicker and thinner collagen fibers. Thus, isoproterenol-induced myocardial fibrosis is initiated by the appearance of interstitial edema and fibrillar collagen disruption and is followed soon thereafter by the formation of thinner collagen fibers that extend across muscle fibers and into which thicker collagen fibers become entwined in a crisscrossing pattern. Once formed, this mesh of collagen fibers encircles cardiac muscle. This pattern of fibrous tissue formation may entrap and isolate myocytes so that the mechanical behavior of muscle and the intact myocardium becomes abnormal.

The interstitium of the myocardium is composed of predominantly type I collagen; type III collagen is present to a lesser extent. The fibrillar collagens serve as tethers between muscle cells, muscle fibers, and blood vessels while also providing a scaffolding that supports the muscular and vascular compartments. In pressure overload hypertrophy, a continuous structural remodeling of the fibrillar collagen matrix is seen. What is initially an adaptive process that enhances tensile strength can eventuate in pathologic hypertrophy with muscle fiber entrapment, cell loss, and abnormal diastolic and systolic stiffness of the myocardium. Morphologically distinct patterns of myocardial collagen accumulation, or fibrosis, have been identified based on the alignment of thick and thin collagen fibers to one another and to cardiac muscle. Each pattern, representing either a reactive (without necrosis) or reparative process, can alter stiffness in a unique manner. The manner in which the interstitium regulates the nature and proportion of fibrillar collagen formation is unknown and deserving of further study. Such information may lead to the development of antifibrotic agents that counteract, prevent or modify disproportionate collagen remodeling in pressure overload hypertrophy. These agents may thereby ultimately represent corrective forms of therapy for the management of heart failure.

The extracellular structural protein, collagen, is responsible for the functional integrity of the myocardium permitting reversible interdigitation and transmission of force between contracting myocytes. In the pressure-overloaded, hypertrophied myocardium, clinical and experimental evidence indicates that the proportion of collagen relative to muscle is increased. Factors that appear to influence collagen growth during the hypertrophic process include age, species, the rapidity with which the overload occurs, the nature of the lesion leading to the pressure-overload, and the severity and duration of the overload. Morphologically, the heart's collagen matrix consists of a complex weave with tendinous insertions that surrounds myocytes grouping them into myofibers, strands of collagen that connect adjoining myofibers, and collagenous struts that join myocytes to other myocytes and capillaries. In a primate preparation of perinephritis with systemic hypertension, it was observed that the tendinous elements of the weave and the strands of collagen lying between myofibers were increased in number and physical dimension. The functional consequences of a remodeling of the collagen matrix that accompanied myocardial hypertrophy remain to be elucidated. A better understanding of the dynamic behavior of the collagen matrix may offer new insights into the pathogenesis of ventricular dysfunction that accompanies the chronic pressure-overloaded state.