Collagen‐Specific HSP47 ⁺ Myofibroblasts and CD163 ⁺ Macrophages Identify Profibrotic Phenotypes in Deceased Hearts With SARS‐CoV‐2 Infections

Abstract

Background

Cardiac fibrosis complicates SARS‐CoV‐2 infections and has been linked to arrhythmic complications in survivors. Accordingly, we sought evidence of increased HSP47 (heat shock protein 47), a stress‐inducible chaperone protein that regulates biosynthesis and secretion of procollagen in heart tissue, with the goal of elucidating molecular mechanisms underlying cardiac fibrosis in subjects with this viral infection.

Methods and Results

Using human autopsy tissue, immunofluorescence, and immunohistochemistry, we quantified Hsp47⁺ cells and collagen α 1(l) in hearts from people with SARS‐CoV‐2 infections. Because macrophages are also linked to inflammation, we measured CD163⁺ cells in the same tissues. We observed irregular groups of spindle‐shaped HSP47⁺ and CD163⁺ cells as well as increased collagen α 1(I) deposition, each proximate to one another in “hot spots” of ≈40% of hearts after SARS‐CoV‐2 infection (HSP47⁺ P<0.05 versus nonfibrotics and P<0.001 versus controls). Because HSP47⁺ cells are consistent with myofibroblasts, subjects with hot spots are termed “profibrotic.” The remaining 60% of subjects dying with COVID‐19 without hot spots are referred to as “nonfibrotic.” No control subject exhibited hot spots.

Conclusions

Colocalization of myofibroblasts, M2(CD163⁺) macrophages, and collagen α 1(l) may be the first evidence of a COVID‐19–related “profibrotic phenotype” in human hearts in situ. The potential public health and diagnostic implications of these observations require follow‐up to further define mechanisms of viral‐mediated cardiac fibrosis.

Fibrosis is an essential adaptive response after tissue injury. However, excess production causes organ failure, accounting for one‐third of deaths worldwide. ¹ Proteostatic networks composed of stress‐inducible molecular chaperones (eg, HSPs [heat shock proteins] and glucose regulated proteins) facilitate protein homeostasis (ie, proteostasis) and enhance (patho) physiological repair and recovery. ² Chaperones, including HSP47, maintain proteostasis in the endoplasmic reticulum and regulate the biosynthesis, folding, and secretion of procollagen. GRP78, an endoplasmic reticulum–resident chaperone and a SARS‐CoV‐2 coreceptor, is elevated in pneumocytes and alveolar macrophages (CD68⁺ cells) in autopsies of patients with COVID‐19. ³ Because cardiac fibrosis predicts arrythmias and sudden cardiac death in adjudicated human autopsies, efforts to understand mechanisms that lead to cardiac fibrosis among COVID‐19 survivors are urgently needed. ⁴ , ⁵ , ⁶

Herein, we identified COVID‐19–associated increases in 2 cardiac cell types, myofibroblasts and macrophages, which have been associated with tissue repair and fibrosis and have also been linked to enhanced fibrosis experimentally and theoretically. ⁷ To circumvent challenges in accuracy of tissue sampling by invasive endomyocardial biopsy, we used human autopsy tissue, which uniquely enables the simultaneous assessment of clinicomorphological, molecular, and immunohistochemical events at single‐cell resolution in situ.

Methods

The data that support the findings of this study are available from the corresponding author on reasonable request.

Archival autopsy tissues for the investigations described in this work were made available from the Department of Pathology and the Medical College of Wisconsin Tissue Bank under institutional review board–approved protocols. Deidentified demographic and clinical data were obtained from electronic health records under institutional review board–approved protocols.

Immunofluorescent staining was visualized using TSA Plus Cyanine 3 System (Perkin Elmer, Waltham, MA), and Agilent/DAKO with magenta chromogen was used for visualization. Digital immunohistochemical slides were evaluated in a blinded manner for groups of cells positive for HSP47 (termed “hot spots”). Numbers of either HSP47⁺ or CD163⁺ cells in 10, ×400 fields centered on an HSP47⁺ hot spot in each sample were summed (diameter, ≈4 mm²). In nonfibrotic and control samples without hot spots, areas of the same size of those in profibrotic samples were selected and scored. The highest values within any cardiac section (left or right ventricle or septum) were used for comparison.

Statistical Analysis

Statistical analyses appear in each figure legend, with P<0.05 considered as significant. Analyses were performed in SAS 9.4 (SAS Institute, Cary, NC) and R 4.2.1. ⁸

Results

Demographics of selected patients who died from SARS‐CoV‐2 pneumonia (n=12) between June 2020 and December 2021 are shown in Table; Black patients (67%) outnumbered Hispanic patients (17%) and White patients (17%). Among 14 non–COVID‐19 control subjects who died of nonhematopoietic solid tumors during the same time frame, there were more White people (57%) than Black people (43%). Comorbidities, including dyslipidemia, hypertension, type 2 diabetes, coronary artery disease/prior myocardial infarction, heart failure, or end‐stage renal disease, were not different among the groups (data are not presented).

Table  .

Patient Demographic and Clinical Data

	Patients with COVID‐19			Controls
Characteristic	Overall (n=12)	Profibrotic hot spot (n=5)	Nonfibrotic (n=7)	Overall (n=14)	P values*	P values†
Age, y	65 (28–89)	69 (28–89)	63 (35–71)	63 (31–79)	0.66‡	0.57‡
Sex, female	7 (58)	2 (29)	5 (71)	7 (50)	0.67§	0.56§
Black race	8 (67)	3 (60)	5 (71)	6 (43)
Hispanic ethnicity	2 (17)	0 (0)	2 (29)	0 (0)	0.037§	0.36§
White race	2 (17)	2 (40)	0 (0)	8 (57)
First‐dose vaccine	2 (17)	2 (40)	0 (0)	0 (0)
Fully vaccinated	1 (8)	0 (0)	1 (14)	0 (0)	0.12§	0.15§
Vaccinated	0 (0)	…	…	2 (14)
Not vaccinated	9 (75)	3 (60)	6 (86)	12 (86)
Symptom onset to time of death, d	21 (8–52)	21 (15–43)	17 (8–52)	…	…	0.63‡

Data are presented as median (range) or number (percentage); percentages may not add to 100 because of rounding. The χ² or Fisher exact test compared categorical variables, whereas Mann‐Whitney‐Wilcoxon test compared continuous variables between patients with COVID‐19 and controls or between profibrotic and nonfibrotic hot spots.

^*Overall patients with COVID‐19 vs controls.

^† Profibrotic hot spot vs nonfibrotic patients with COVID‐19.

^‡ Mann‐Whitney‐Wilcoxon test.

^§ The χ² or Fisher exact test.

Although HSP47 expression is found in multiple cardiac cells (ie, fibroblasts, cardiomyocytes, and endothelial cells), cell‐specific targeted deletion studies have definitively established the essentiality of myofibroblast‐specific HSP47 for deposition of type I and V collagen, postinjury scar formation, and survival in mice. ⁹ We observed HSP47⁺ immunostaining was strikingly increased in clusters (1–3 mm diameter) of spindle‐shaped activated fibroblasts (ie, myofibroblasts) in 5 of 12 (42%) hearts but not in other interstitial myocardial cells of COVID‐19 samples or controls (Figure 1). Many HSP47⁺ cells were validated immunohistochemically as periostin positive (Figure 1K), consistent with their identity as myofibroblasts. We used quantitative scoring of HSP47⁺ clusters to identify 2 COVID‐19 subgroups, termed “profibrotic” versus “nonfibrotic.” Profibrotic subjects exhibited HSP47⁺ hot spots in left ventricular, right ventricular, and septal regions, with median (interquartile range) of HSP47 of 482 cells/10 high power fields (369–538); by contrast, HSP47⁺ in nonfibrotic subjects or controls scored median (interquartile range) of HSP47 of 28 ‐ (19–37) or 16 cells/10 high power fields (0–26) (Figure 2A), with adjusted P=0.012 and P<0.01, respectively.

Figure 1. Multiplexed immunostaining of representative control (A, D, and G), nonfibrotic (B, E, and H), and profibrotic COVID‐19 hearts (C, F, I, and K), showing HSP47 (heat shock protein 47), CD163, and collagen α 1(l) (Colα1[I]) expression in myofibroblasts and macrophages.

Cardiomyocytes are marked by gray (cardiac muscle troponin T [cTnT]; Abcam, number ab8295), and nuclei are visualized by royal blue 4′,6‐diamidino‐2‐phenylindole (DAPI) (fluorescent DNA stain) (Akoya Biosciences, number FP1490) staining. C, We observed increased HSP47⁺ (cyan blue; Santa Cruz, number SC‐5293) and CD163⁺ (white; Leica Biosystems, number NCL‐L‐CD163) in COVID‐19 profibrotic samples. F, Extracellular matrix deposition of Colα1(I) (red; Abcam, number ab138492) is increased in profibrotic COVID‐19 samples. I, HSP47, DAPI, cTnT, and CD163 merged with Colα1(I). J, Secondary antibody (ab) alone showed no fluorescence. K, Myofibroblasts were identified by a second protein, periostin (Sigma, number HPA012306).

Figure 2. Mann‐Whitney‐Wilcoxon test was used to compare the differences in biomarkers between patients with COVID‐19 and controls, whereas Kruskal‐Wallis test was used to compare profibrotic and nonfibrotic hot spots, and controls.

Dwass, Steel, Critchlow‐Fligner Method ¹³ was used to adjust for multiple comparisons. Box plots with jittered points were shown. Boxes represent median and interquartile range; whiskers indicate minimum and maximum values. A, There is a clear separation of scores for HSP47 (heat shock protein 47) expression. B, The same data for CD163⁺ macrophages. C, Profibrotic product collagen α 1(l) (COLα1[l]) expression in controls, nonfibrotic COVID‐19 samples, and profibrotic COVID‐19 samples. Only profibrotic sections exhibit increased COLα1(l). D, The 5 profibrotic subjects exhibited increases of both HSP47⁺ and COLα1(I) relative to those without HSP47⁺ hot spots. E through G, Unsupervised principal component analysis (PCA) was performed using the following biomarkers: HSP47, CD163, COLA1, and periostin. Missing data were imputed with the R package missMDA, ¹⁴ and unsupervised PCA was performed using R package FactoMine. ¹⁵ Principal component 1 (PC1) (F) and principal component 2 (PC2) (G), which accounted for >95% of the variance, were compared among the 3 groups. There were significant differences in PC1 and PC2 among the 3 groups (P<0.0001 and P<0.01, respectively).

Recent evidence from COVID‐19 acute respiratory distress syndrome and pulmonary fibrosis highlights that CD163⁺ inflammatory macrophages are profibrotic. ¹⁰ , ¹¹ Therefore, we also quantified CD163⁺ cells, which were increased within areas with profibrotic hot spots (median [interquartile range], 421 [322–467]) compared with controls (median [interquartile range], 107 [77–131]), with adjusted P=0.0039 (Figure 1, representative images; Figure 2B, graph). To determine the functional capacity of HSP47⁺ cells, we evaluated collagen α 1(I), a client protein of collagen‐specific HSP47 chaperone that resides in the endoplasmic reticulum–Golgi and facilitates protein maturation before extracellular matrix deposition. Profibrotic patients with COVID‐19 exhibit higher collagen α 1(l) levels (Figure 1D through 1F and Figure 2C and 2D; adjusted P=0.024) than either nonfibrotic or control counterparts. Principal component analysis (Figure 2E through 2G) using HSP47, CD163, periostin, and collagen α 1(l) showed that although subjects with HSP47⁺ profibrotic hot spots were not homogeneous, this group was distinct from either nonfibrotic or control subjects for which principal component 1 and principal component 2 account for >95% of the variance (P<0.0001 and P<0.01, respectively).

Discussion

Our data identify increased HSP47⁺ and CD163⁺ cells and increased collagen I deposition in hot spots within hearts after SARS‐CoV‐2 infection. These changes may be the first evidence of a COVID‐19–related profibrotic phenotype in human hearts in situ. Profibrotic hot spots could represent paracrine‐mediated effector cell (myofibroblast/M‐CSF and macrophage/PDGF) cross talk and acute fibrogenesis (collagen α 1[I]) within the human myocardium, as proposed in a model by Adler et al. ⁷ They might also set the stage for enhanced arrhythmogenesis, even sudden cardiac death. As such, COVID‐19–induced cardiac fibrosis may have critical long‐term public health implications for health care systems and policy makers. ¹² Our work suggests investigations into mechanisms of viral‐induced integrated stress response pathways, myofibroblast lineage transitional states, and paracrine mediators are priorities. One immediate implication is development of noninvasive approaches (ie, plasma biomarkers and noninvasive imaging) to identify antemortem profibrotic phenotypes. These data may also accelerate clinical trials of antifibrotic therapies and could influence policies addressing cardiovascular health disparities among vulnerable populations.

Sources of Funding

The funding for this project to Dr Benjamin was provided by the Bruce and Janine Smith Family, National Institutes of Health (NIH) Director's Pioneer Award (NIH grant 8DP1HL17650), and the Dean's Program Development Funds. Dr Rui was supported by the Department of Pathology, Medical College of Wisconsin. Dr Jacobs was supported by Merit Review BX003833. Dr Puzyrenko was supported by the Michael H. Keelan, Jr, MD, Research Foundation Grant and the Cardiovascular Center at the Medical College of Wisconsin. The project described was also supported by the National Center for Advancing Translational Sciences, National Institutes of Health, Award UL1TR001436. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Disclosures

None.