Diabetes Mellitus and Tuberculosis

Scientists Discover New Treatment Approach To Curb Severe Myocarditis

Feb 22 2018

The outcome of viral myocarditis is closely associated with the immune response of the affected individual. An inhibitor of the immunoproteasome, a protein degradation complex in immunocompetent cells, reduces the extent of the inflammation and thus also the damage to the heart during myocarditis. Scientists of the German Centre for Cardiovascular Research (DZHK) and Charité - Universitätsmedizin Berlin have recently discovered this new treatment approach, as published in the scientific journal EMBO Molecular Medicine.

Viral infections of the myocardium can cause a devastating reaction of the immune system, which can lead to a severe inflammation with ensuing heart failure or even sudden cardiac death, especially in children and young adults. Activation of the immune response curbs the viral disease on the one hand, but causes pathological reactions in the myocardium on the other. The researchers are therefore attempting to identify new targets to weaken the immune response and simultaneously enable secure control of the virus. This is because the course of the disease appears to mainly depend on the interaction between the virus-mediated cell damage and the individual reaction of the body's own defense system. Thus, viral myocarditis is almost asymptomatic in most people.

Stable cardiac function

The DZHK scientist Professor Dr. Antje Beling of the Institute of Biochemistry at Charité-Universitätsmedizin Berlin and her team utilized an immunoproteasome-specific inhibitor, ONX 0914, with the aim of moderating severe courses of viral myocarditis. The immunoproteasome is a protease complex with various enzymatic activities that exists particularly in human immune cells where it degrades proteins. In an animal model with high susceptibility to severe viral myocarditis, this inhibitor could curb the destructive inflammatory reaction. Mice were infected with coxsackievirus B3, which also affects human cardiomyocytes, and were treated with the inhibitor ONX 0914 from the onset of the infection. Although the viral burden only sank fractionally with the inhibitor, less heart tissue was damaged and the cardiac function remained stable. "No severe inflammations that impair the filling of the myocardium and damage the myocardial tissue occur with the inhibitor. The heart can therefore pump unabated", explains Beling.

Inhibitor curbs cytokines

Monocytes and macrophages are those immunocompetent cells that substantially contribute to myocardial damage during a viral infection. For this reason, the Berlin-based researchers investigated how these cells in infected animals reacted to the administration of the inhibitor. They observed that ONX 0914 indeed led to an increased mobilization of monocytes from the bone marrow. However, these cells produced substantially fewer proinflammatory cytokines during the infection. Especially because of the low production of molecules that attract monocytes in the myocardium, no severe tissue-damaging inflammation could develop. "The inhibition of the production of proinflammatory cytokines is the main effect of the inhibitor ONX 0914, which leads to a better course of the myocarditis", says Beling.

In addition, the effect of ONX 0914 on other cells of the immune system, such as lymphocytes and neutrophils, was analyzed. During a viral infection, the scientists could observe in untreated mice that there was a sharp decline in the number of lymphocytes in the body, which was completely prevented by the treatment with ONX 0914. Similarly, they found more antibodies directed against the virus after administration of the inhibitor, which indicates that the immune system's antibody response remained intact or was even improved with ONX 0914. Neutrophil defense cells reacted clearly to the treatment with ONX 0914, yet it did not affect the course of the disease.

Great therapeutic potential

The researchers are currently trying to discover on a molecular level how the immunoproteasome-specific inhibition curbs the production of proinflammatory cytokines. They have already been able to ascertain that a particular cellular signal pathway, the so-called MAP kinase pathway, is involved in the effect's transmission. Moreover, Beling and her colleagues are examining in another myocarditis model whether ONX 0914 can develop the same protective effect there. "The active agent has great potential, not just for myocarditis, but also for other diseases which essentially arise from an exaggerated immune response", says Beling. An analogous inhibitor to ONX 0914 is already being tested in phase I/II clinical studies in patients with inflammatory autoimmune diseases.

Neutralizing Anti-4-1BBL Treatment Improves Cardiac Function In Viral ...

Immunohistochemical Staining for 4-1BB and 4-1BBL

Seko et al22 reported an induction of 4-1BBL expression in cardiomyocytes following CVB3 infection. Our previous observations have shown that 4-1BB and 4-1BBL can be induced in cardiomyocytes after various stimuli, such as doxorubicin treatment and MI in mice (unpublished data). Therefore, we examined the presence and amount of these two proteins in sham and CVB3-infected hearts at 9 days pi. Figure 1a demonstrates minimal expression of the 4-1BB receptor (green, top small panel) in sham mouse hearts and strong expression of 4-1BBL (red, lower small panel), whereas at 9 days pi (Figure 1b), there was increased expression of 4-1BB (39±14 vs 0%, P<0.01), which colocalized with cardiomyocytes (long arrow), in infected hearts as compared to sham. The expression of 4-1BBL remained constant throughout the infection. The receptor and ligand colocalized predominantly in cardiomyocytes (Figure 1b, yellow, long arrow) but were also expressed by small round cells, possibly immune cells (small arrow). Figure 1f shows the graphical representation of the densitometric analyses.

Figure 1

Expression of 4-1BB and 4-1BBL in mouse hearts. Representative myocardial sections from (a) sham (N=4), (b) CVB3-infected and untreated mouse at day 9 (N=4), (c) infected and M522-treated (N=9), and (d) infected and M525 control animals (N=10) showing staining for 4-1BB (green, top small panels) and 4-1BBL (red, lower small panels). The large panels represent overlay of nuclear staining (blue) and colocalization of the two antigens (yellow). (e) Negative controls for 4-1BB and 4-1BBL using isotype-matched IgG. (f) Morphometric quantitation of 4-1BB and 4-1BBL protein expression. Scale bars=50 μm. Data are mean±s.D. **P<0.01 sham vs infected.

For the chronic study, to confirm target coverage of the anti-4-1BBL antibodies (M522 and control Abs) into the mouse hearts, immunostaining for rat IgG2a was performed and immunopositivity was observed in all samples (data not shown). Immunofluorescent staining for 4-1BB and 4-1BBL at 10 weeks pi (Figure 1c and d) showed myocardial colocalization. At this time point, 4-1BBL was still strongly expressed in all tissue and no difference was found between the M522-treated (Figure 1c, red stain, lower small panel) and control hearts (Figure 1d, P=0.7). In contrast, there was faint, diffuse expression of 4-1BB (green stain, top small panels) and for the most part it was coexpressed with its ligand (orange, large panels). Both cardiomyocytes and small globular cells, possibly infiltrating cells expressed 4-1BB, whereas mostly endogenous cells expressed 4-1BBL. In M522-treated hearts, a trend towards increased 4-1BB immunopositivity as compared to the control group was detected although the difference did not reach statistical significance (43±16 vs 23±21%, p=0.1). Figure 1f shows the graphical representation of the densitometric analyses.

Echocardiography

Serial cardiac function measurements were taken by echocardiography. Figure 2a displays the histological cardiac images at various time points following infection (top four panels) and the mid-section and lower panels show the tracings for systolic and diastolic short axis ventricular measurements, respectively. At day 3 post-CVB3 infection, histological myocyte vacuolization was associated with a nonsignificant trend in reduced LV wall thickening but normal wall thickness, LVEF, and cardiac volumes in virus-infected mouse hearts as compared to shams (Figure 2b–e). By day 9 pi, the degree of wall thickening was reduced (30±17 vs 70±19%, P<0.01) and wall thickness was increased (0.15±0.04 vs 0.09±0.01 cm in diastole and 0.19±0.04 vs 0.15±0.02 cm in systole, both P<0.05) in CVB3-infected myocardium as compared to sham hearts, suggestive of wall edema and inflammation. Due to the increased wall thickness in the infected hearts, end-diastolic volume (EDV) was reduced at day 9 pi in myocarditic hearts as compared to sham (0.013±0.003 vs 0.024±0.003 ml, P<0.002), whereas a decrease in end-systolic volume (ESV) was also observed in CVB3-infected hearts but did not reach statistical significance (0.004±0.002 vs 0.007±0.001, P=0.07). On day 30 pi, wall contractility remained depressed (33±15 vs 70±19%, P<0.05), left ventricular posterior wall (LVPW) thickness was still increased although this did not reach statistical significance (0.12±0.01 vs 0.09±0.01 cm in diastole (P=0.08) and 0.16±0.01 vs 0.15±0.02 cm in systole (NS)), the EF was reduced (62±8 vs 72±3%, P<0.005), and ESV was increased by 33% to 0.011±0.003 ml (P<0.01) in myocarditic animals as compared to sham, indicative of intrinsic contractile deficits and systolic dysfunction.

Figure 2

Depressed cardiac function in mice following CVB3 infection. (a) Representative H&E sections of myocardium at 3, 9, and 30 days pi. Below are representative short axis ultrasound images taken at systole (middle panels) and diastole (lower panels) at the level of the papillary muscle, showing tracings of the myocardial wall and chamber. Echocardiography was performed on days 3, 9, and 30 pi and measurements for (b) LVPW thickening, (c) LVPW thickness, (d) LVEF, and (e) end-diastolic and systolic volumes were recorded. Scale bars denote 50 μm. Data are mean±s.D. *P<0.05, **P<0.01, ***P<0.005, baseline vs infected, N=4 at each time point.

To examine chronic cardiac dysfunction and to determine the role of the 4-1BB pathway, we conducted a therapeutic treatment study and extended our observations to 10 weeks pi. Mice were infected with CVB3 and randomly assigned into two groups. On day 15 pi, one group was administered a 4-1BBL neutralizing Ab (M522) and the other group was given a nonblocking Ab, M525 or control. Sham mice administered neutralizing or nonblocking antibodies experienced no functional changes as compared to sham mice with vehicle (data not shown). M522 therapy improved the rate of wall thickening, reduced wall thickness, and reduced EDV at 10 weeks pi as compared to the control group. Comparison of the rate of wall thickening at 10 weeks pi showed that the percentage change from baseline, defined as measurements taken from infected mice prior to infection, following M522 administration was −9±13%, whereas it was −49±16% (P≪0.0001) in control animals (Figure 3a). This indicates that there was improved contractility in the M522-treated group, whereas the control animals still suffered severe contractile impairment.

Figure 3

Effects of M522 treatment on cardiac function in mice. (a) Rate of LV wall thickening, (b) LVPW thickness, and (c) end ventricular volumes were measured at 10 weeks pi and we observed a significant difference in LV wall thickening rate between M522 and controls as well as moderate decreases in LVPW thickness and end-diastolic cardiac volume in M522-treated mice. Data are mean±s.D., *P<0.05, ***P<0.005. M522 (N=9) and controls (N=10).

M522 treatment also appeared to normalize LVPW thickness towards baseline values (Figure 3b). Diastolic LVPW thickness, expressed as percentage change from baseline, was significantly lower in the M522 group as compared to controls (17±10 vs 57±47%, P=0.03), indicating that during chronic myocarditis, the LVPW were still profoundly thickened in the untreated animals. However, systolic wall thickness was not statistically different between the M522-treated and control animals (8±19 vs 13±29%, P=0.66).

Figure 3c demonstrates that at 10 weeks pi as cardiac expansion and remodeling progressed, M522 administration significantly improved EDV as compared to control (16±14 vs 42±25%, P=0.03), as determined by percentage change from baseline. No difference was observed in ESV between the M522 group and controls (67±27 vs 85±49%, P=0.37). Systolic function, as measured by EF was reduced to 67±5% in both groups at 10 weeks pi as compared to 75±5% at baseline (data not shown).

Histological Examination

There was no significant difference in mortality rate between M522-treated and control mice (data not shown). As shown in Figure 4a, M522-treated animals exhibited significantly less myocardial injury than control animals (0 vs 1.3±1.5, P<0.05), as assessed by scoring of H&E-stained cardiac sections. The M522 group showed little or no cardiac injury while controls experienced a great amount of damage, as evidenced by small diffuse lesions and scars (arrows) in their hearts. Some control animals experienced considerable histological injuries, such as multiple large scars and calcific lesions.

Figure 4

Improvement of pathological findings in chronic myocarditis after anti-4-1BBL neutralizing Ab treatment of mice following CVB3 infection. (a) Representative myocardial sections showed markedly decreased inflammation and tissue damage in M522-treated hearts (N=9) as compared to control hearts (N=10). Semiquantitative histological grades for necrosis and tissue granulation as well as for inflammation and edema were significantly lower in M522-treated hearts as compared to the control group (0 vs 1.3±1.5). Sham tissues exhibited no measurable pathological changes (data not shown). (b) Picrosirius staining for total collagen distribution demonstrated decreased collagen accumulation and scars or lesions (arrows) in the M522 group (N=9) as compared to control hearts (N=10). Quantitative assessment for collagen volume fraction as measured using the Image-Pro Plus® program was significantly lower in M522-treated hearts as compared to control (3.2±0.6 vs 4.9±2.2%). Sham tissues exhibited no measurable pathological changes (data not shown). Scale bars=50 μm. Data are mean±s.D., *P<0.05, M522 vs control.

Cardiac sections were also subjected to picrosirius red staining for visualization of collagen amount and architecture, which was quantitated using Image-Pro Plus®. Figure 4b shows that control hearts stained with picrosirius red revealed a marked increase in collagen deposition as well as soft tissue distortion as compared to M522-treated myocardium (4.9±2.2 vs 3.2±0.6%, P<0.05) via assessment of collagen volume fraction. Increased perivascular, replacement, and reactive fibrosis were also observed in control hearts, whereas the M522 group demonstrated little fibrotic alterations. Such findings correlate with the histological grades, which indicate that control hearts had more damage and lesions. During this late phase, majority of the lesions were likely not active but had evolved into collagenous replacement and reactive fibrosis.

Protease Expression

To determine if proteases were involved in the differential remodeling between M522-treated and control animal hearts, we used RT-PCR to compare the transcriptional levels of MMP-2, MMP-9, and MMP-12. These enzymes have previously been shown to be involved in acute myocarditis and may play a role in long-term remodeling.26 We found that neither expression of MMP-2 nor MMP-9 transcripts were significantly altered between the two groups, as shown in Figure 5. MMP-2 transcript was present abundantly in all samples, whereas MMP-9 was produced minimally in both groups. Interestingly, MMP-12 or metalloelastase was also expressed abundantly by both groups but the control group had increased expression of this transcript as compared to M522-treated animals, P<0.05. Densitometric quantitation of the PCR bands is presented in Figure 5b.

Figure 5

Effects of 4-1BB inhibition on MMP-2, MMP-9, and MMP-12 expression. (a) Representative images showing semiquantitative RT-PCR for MMP-2, MMP-9, and MMP-12 transcription. Strong expression of MMP-2 and MMP-12, but minimal expression of MMP-9 was observed. (b) Densitometric quantitation of the PCR bands showed that while the gelatinases expression did not differ between the two groups, MMP-12 was significantly lower in the M522-treated animals. Data are mean±s.D., *P<0.05, M522 (N=9) vs control (N=10).

Immunohistochemical Staining for Immune Cells

To compare the degree of immune cell infiltration in M522-treated and control animals after CVB3 infection, we stained for CD45, a pan-leukocyte marker, and CD3, a pan-T cell marker. At 10 weeks pi, active myocarditis had largely subsided but various stimuli, such as persistent viral infection or autoimmunity, may cause ongoing low-grade inflammation. Consistent with the histological results, we found that control animals exhibited increased immunopositivity for CD45 (Figure 6a, arrows), indicating more leukocyte infiltration than M522-treated animals (8.5±4.1 vs 5.9±1.3% of total cell fraction, P=0.08), although this differential CD45 infiltration did not reach statistical significance (Figure 6b). Of these immunopositive cells, the majority were T cells as shown by staining for CD3 (Figure 6), and the increase in this subset of lymphocytes in control hearts is statistically significant as compared to M522-treated animals (4.3±3.8 vs 1.3±0.9%, P<0.05). Owing to the scarcity of lesions and scars within the M522-treated animals, most of the leukocytes were diffuse and dispersed among cardiomyocytes. In control animals, the number and size of lesions were increased and we detected extensive immunopositivity for both CD45 and CD3 within and around these lesions (Figure 6a, arrows).

Figure 6

Inflammatory cell infiltration in M522-treated and control mouse hearts. (a) Representative myocardial sections showed markedly decreased inflammation as assessed by immunostaining for CD45 and CD3 in the M522-treated group as compared to control. In control hearts, immune cells, stained in brown, can be detected within lesions as well as diffusely among cardiomyocytes (arrows). (b) Quantitation of the immunoreactivity for CD45 and CD3 staining showed fewer immune infiltrates in M522-treated hearts as compared to control. Scale bars=50 μm. Data are mean±s.D. *P<0.05, M522 (N=9) vs control (N=10).

AHA: Time Plays Critical Role In Diagnosis, Treatment Of ... - Healio

January 06, 2020

3 min read

Add topic to email alerts

Receive an email when new articles are posted on

Please provide your email address to receive an email when new articles are posted on . Subscribe We were unable to process your request. Please try again later. If you continue to have this issue please contact customerservice@slackinc.Com.

Back to Healio

Leslie T. Cooper Jr.

Robb D. Kociol

The timely diagnosis and treatment of patients with fulminant myocarditis is critical for favorable outcomes, according to a scientific statement by the American Heart Association published in Circulation.

The scientific statement has also been endorsed by the Heart Failure Society of America and the Myocarditis Foundation.

"The point is to have an awareness about the signs and symptoms of cardiogenic shock, which can present in young and previously healthy people," Leslie T. Cooper Jr., MD, FAHA, professor of medicine and chair of the cardiovascular department at Mayo Clinic in Jacksonville, Florida, and vice chair of the writing group, told Healio. "Because young previously healthy people don't usually become critically ill, it's often possible to dismiss or not be thinking about advanced heart failure as you would in an older, sicker population with comorbidities. In this young population, fulminant myocarditis, which is often due to a viral infection or, sometimes, cancer therapy like a checkpoint inhibitor, can develop suddenly and be acutely life threatening with over an 80% mortality. It is, however, in the setting of myocarditis, completely treatable. The majority of people — in our case, 75% — can be bridged to recovery with no transplant and no long-term cardiac pump support, but you do have to think about it and get people on support early."

Signs and symptoms

Robb D. Kociol, MD, MS, medical director of the heart failure program at Beth Israel Deaconess Hospital-Plymouth in Massachusetts and chair of the writing group, and colleagues detailed some of the signs and symptoms of fulminant myocarditis, which can vary with or without manifestations of an inflammatory disorder or infection. Some symptoms may include cardiogenic shock, dyspnea followed by arrhythmias and chest pain or even sudden death.

Multimodality imaging, particularly echocardiography, and endomyocardial biopsy can play a role in diagnosing and managing patients with fulminant myocarditis. The first test should be echocardiography, which has the ability to rapidly process a diagnosis and to assess cardiac and valvular function and morphology, according to the statement. Endomyocardial biopsy during catheterization should be decided upon based on operator experience, clinical suspicion for myocarditis and pretreatment with lytic therapy, antiplatelet agents and anticoagulants.

"Both the American Heart Association and the European Society of Cardiology recommend heart biopsy be performed in those patients with otherwise unexplained cardiogenic shock looking like suspected myocarditis," Cooper said in an interview. "That is the reason for that, is because there are specific forms of myocarditis diagnosed by biopsy only, which would have specific management therapies that affect outcomes."

PAGE BREAK

Cooper added that some health care providers may ask about the role of cardiac MRI in these patients.

"Generally, what our statement says is that it's really very secondary. Most of the people who are going to be in this setting are far too sick to have an MRI. That's where a biopsy is more important than an MRI because it can be done in that sick population."

Some conditions that may result in fulminant myocarditis are lymphocytic myocarditis, giant cell myocarditis and acute necrotizing eosinophilic myocarditis. Novel cancer therapies may also cause myocarditis.

Treatment options

Timely treatment is important. Initial management of patients with cardiac arrest or pulseless arrhythmia should follow the AHA guidelines for advanced cardiac life support, which is geared toward resuscitation and stabilizing. Transferring a patient to a tertiary referral hospital may be warranted for institutions without necessary management capabilities.

"There's a reluctance in some institutions to transfer patients to another institution," Cooper told Healio. "It tends to happen very late when they're very, very sick and sometimes too sick to be saved. Early transfer in cardiogenic shock when indicated is the best thing to do." – by Darlene Dobkowski

For more information:

Leslie T. Cooper Jr., MD, FAHA, can be reached at Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224; email: cooper.Leslie@mayo.Edu.

Disclosures: Cooper reports he received a research grant from the NHLBI and other research support from the Myocarditis Foundation as a volunteer board member. Kociol reports no relevant financial disclosures. Please see the statement for all other authors' relevant financial disclosures.

Add topic to email alerts

Receive an email when new articles are posted on

Please provide your email address to receive an email when new articles are posted on . Subscribe We were unable to process your request. Please try again later. If you continue to have this issue please contact customerservice@slackinc.Com.

Back to Healio

---