Sprr1a is a key downstream effector of mir-150 during both maladaptive cardiac remodeling in mice and human cardiac fibroblast activation

ABSTRACT MicroRNA-150 (miR-150) is conserved between rodents and humans, is significantly downregulated during heart failure (HF), and correlates with patient outcomes. We previously

reported that miR-150 is protective during myocardial infarction (MI) in part by decreasing cardiomyocyte (CM) apoptosis and that proapoptotic small proline-rich protein 1a (_Sprr1a_) is a

direct CM target of miR-150. We also showed that _Sprr1a_ knockdown in mice improves cardiac dysfunction and fibrosis post-MI and that _Sprr1a_ is upregulated in pathological mouse cardiac

fibroblasts (CFs) from ischemic myocardium. However, the direct functional relationship between miR-150 and SPRR1A during both post-MI remodeling in mice and human CF (HCF) activation was

not established. Here, using a novel miR-150 knockout;_Sprr1a_-hypomorphic (_Sprr1a__hypo/hypo_) mouse model, we demonstrate that _Sprr1a_ knockdown blunts adverse post-MI effects caused by

miR-150 loss. Moreover, HCF studies reveal that _SPRR1A_ is upregulated in hypoxia/reoxygenation-treated HCFs and is downregulated in HCFs exposed to the cardioprotective β-blocker

carvedilol, which is inversely associated with miR-150 expression. Significantly, we show that the protective roles of miR-150 in HCFs are directly mediated by functional repression of

profibrotic _SPRR1A_. These findings delineate a pivotal functional interaction between miR-150 and SPRR1A as a novel regulatory mechanism pertinent to CF activation and ischemic HF. SIMILAR

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FIBROBLASTS-DERIVED MIR-320 PROTECTED AGAINST HEART FAILURE INDUCED BY TRANSVERSE AORTIC CONSTRICTION Article Open access 18 February 2021 INTRODUCTION Controlling microRNA (miRNA or miR)

biogenesis in the heart is an important underlying mechanism of heart failure (HF) [1,2,3,4,5]. Intriguingly, novel miR therapies are being investigated in clinical trials for other diseases

[6,7,8,9] and more recently for HF [10]. We reported that miR-150 is upregulated by the β-blocker carvedilol (Carv), which acts through β-arrestin1-mediated β1-adrenergic receptor (β1AR)

protective signaling [11]. Significantly, using a systemic miR-150 knockout (KO) mouse model, we also showed that miR-150 plays a vital Carv/β1AR/β-arrestin1-mediated protective role in

myocardial infarction (MI) in part by decreasing cardiomyocyte (CM) apoptosis [12]. More recently, we demonstrated that cardiac-specific miR-150 conditional KO (cKO) mice exhibit enhanced

apoptosis and maladaptive post-MI remodeling [13]. Notably, cardiac-specific overexpression of miR-150 attenuated transverse aortic constriction (TAC)-induced cardiac dysfunction [14]

whereas miR-150 loss caused a higher degree of cardiac fibrosis after TAC. MiR-150 was also downregulated in cardiac fibroblasts (CFs), not CMs isolated from TAC mice [15]. Moreover, miR-150

inhibited mouse CF activation in vitro [15]. Interestingly, circulating or cardiac miR-150 is downregulated in patients with multiple cardiovascular diseases (CVDs) [16,17,18,19] and mouse

models of HF [12, 14, 20]. MiR-150 is conserved between rodents and humans and is significantly associated with HF severity and outcome in humans [21]. Collectively, previous studies support

the clinical relevance and potential therapeutic application of miR-150 in HF; however, the detailed mechanisms by which miR-150 modulates HF remain elusive. Small proline-rich protein 1a

(SPRR1A) is induced by stress and is highly conserved. SPRR1A is a substrate of transglutaminase (TGase) I/II-catalyzed crosslinking reactions in forming the keratinocyte envelope [22]. We

showed that CMs with _Sprr1a_ knockdown are protected against apoptosis in the simulated ischemia/reperfusion (sI/R: hypoxia/reoxygenation [H/R]) condition [13]. Adenovirus-mediated ectopic

overexpression of _Sprr1a_ promoted cardiac fibrosis in vivo after TAC whereas protecting CMs and isolated hearts against 2-deoxyglucose and ex vivo I/R [23]. Moreover, _Sprr1a_

overexpression did not affect CM survival after reactive oxygen species treatment or serum deprivation [23]. This prior study indicated both the potential stress-dependent effects of

_Sprr1a_ and the requirement of in vivo genetic loss-of-function approaches to define the role of _Sprr1a_ in the heart. Interestingly, we recently demonstrated that _Sprr1a_-hypomorphic

(_Sprr1a__hypo/hypo_) mice are protected against MI [13]. We also reported that _Sprr1a_ is upregulated in CMs isolated from mouse hearts post-MI [13], which is consistent with a report on

mouse hearts after TAC [23]. Moreover, we showed that left ventricular (LV) _SPRR1A_ is upregulated in patients with HF [13] in agreement with mouse studies showing _Sprr1a_ upregulation in

myocardial injury [24] and renal I/R injury [25]. Notably, we identified proapoptotic _Sprr1a_ as a novel direct and functional target of miR-150 in CMs [13]. We also showed that _Sprr1a_ is

downregulated by Carv in hearts and CMs [13] concurrent with miR-150 upregulation [11]. Given that rodent and human genes of SPRR1A have almost identical genomic organization and

exon/intron sizes [26] and have at least one miR-150 binding site, the regulation of SPRR1A by miR-150 and their roles might be conserved. Importantly, we also showed that _Sprr1a_ knockdown

in mice improves fibrosis post-MI and that _Sprr1a_ is upregulated in mouse CFs isolated from ischemic myocardium [13]. A recent proteomic study in CFs also noted that SPRR1A levels are

significantly higher in infarct CFs than remote CFs during MI [27]. These previous findings indicate a possible role of _Sprr1a_ in CFs; however, whether _Sprr1a_ is functionally regulated

by miR-150 in HF and human CF (HCF) activation remains unknown. Using a novel miR-150 KO;_Sprr1a_-hypomorphic (_Sprr1a__hypo/hypo_) mouse model and primary HCFs, we demonstrate here that (i)

_Sprr1a_ knockdown alleviates cardiac dysfunction, damage, apoptosis, and fibrosis after MI mediated by miR-150 deletion; (ii) _Sprr1a_ is upregulated in HCFs subjected to H/R whereas its

expression is downregulated in HCFs by Carv, which is inversely associated with the expression of miR-150; and (iii) the protective actions of miR-150 in HCFs are mediated by the functional

repression of profibrotic _SPRR1A_. These data directly establish the functional relationship between miR-150 and _Sprr1a_ during both post-MI fibrotic remodeling in mice and HCF activation.

Our novel findings suggest that profibrotic SPRR1A is a crucial downstream effector of miR-150 in repressing CF activation and maladaptive cardiac remodeling. The miR-150/SPRR1A axis,

therefore, may be considered a novel therapeutic target for ameliorating ischemic heart disease and pathological fibrosis. MATERIALS AND METHODS MIR-150 KNOCKOUT AND HYPOMORPHIC _SPRR1A_

MUTANT MICE (_SPRR1A_ _HYPO/HYPO_) AS WELL AS THE GENERATION OF MIR-150 KO;_SPRR1A_ _HYPO/HYPO_ MICE Systemic miR-150 KO mice were purchased from the Jackson Laboratory (007750), and their

cardiac phenotypes post-MI were reported in our previous study [12]. _Sprr1a__hypo/+_ were obtained from the Mutant Mouse Resource & Research Centers (RRID: MMRRC_049856-UCD). We

previously described the detailed methods regarding this mouse line, including the targeting strategy, generation of _Sprr1a__hypo/hypo_ mice, and genotyping strategy [13]. We also reported

the cardiac phenotypes of _Sprr1a__hypo/hypo_ mice after MI in a previous study [13]. In the current study, _Sprr1a__hypo/hypo_ mice were bred with miR-150 KO mice to generate the novel

miR-150 KO;_Sprr1a__hypo/hypo_ mouse line. All mice were maintained on a C57BL/6J background, and wild-type (WT) littermates were used as controls. ETHICS COMMITTEE APPROVAL The animal

experiments conducted as a part of this study complied with the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Mice were euthanized

by thoracotomy under 1–4% inhaled isoflurane. All experiments with mice were performed according to the protocols approved by the Institutional Animal Care and Use Committee at the Indiana

University School of Medicine (approval reference #21189). 8–16 week-old C57BL/6J mice of both sexes were used for this study. Genotype- and sex-matched mice were randomly assigned to

experimental groups to mitigate the cage effect. The genotypes of the animals were masked for researchers until the end of the analysis. STATISTICS Data are presented as the mean ± SEM

(except Fig. 1 using SD because no clear variation bars are shown otherwise) from independent experiments with different biological samples per group. Triplicate experiments were performed

for all biochemical and cell biology studies. The number of in vitro biological samples per group was 3–6. The number of mouse samples per group was 3–18. The exact sample size for each

experimental group/condition is given as a number in the figure/table legend. To ensure the robustness of the data and to allow the direct evaluation of the distribution of the data, we

present graphical data as scatter/dot plots. Normality was assessed with the Kolmogorov-Smirnov test. The following statistical tests were used: unpaired two-tailed t-test for comparisons

between 2 groups, one-way ANOVA with Tukey’s multiple comparison test for multiple groups, two-way ANOVA with Tukey’s multiple comparison test for comparisons between 2 groups with different

treatments, and two-way repeated-measures ANOVA with Bonferroni’s post hoc test for 2 groups over time. The unpaired 2-tailed t-test was based on assumed normal distributions. A _P_ value 

< 0.05 was considered statistically significant. _P_ values are indicated as follows: *,#,or§_P_ < 0.05, **,##,or§§_P_ < 0.01, and ***,###,or§§§_P_ < 0.001. RESULTS SPRR1A

KNOCKDOWN IN MIR-150 KO MICE LARGELY CORRECTS CARDIAC DYSFUNCTION MEDIATED BY MIR-150 DELETION _Sprr1a_ is a direct target of miR-150 in vitro, miR-150 acts as a gatekeeper of CM survival in

part by inhibiting proapoptotic _Sprr1a_ [13], and their correlative cardiac actions are shown [12, 13]; but an in vivo functional relationship between miR-150 and _Sprr1a_ in the heart has

not been established. To directly investigate their in vivo functional interaction in the heart, we generated a novel miR-150 KO;_Sprr1a__hypo/hypo_ mouse line by breeding miR-150 KO mice

with _Sprr1a__hypo/hypo_ mice. We first conducted permanent ligation of the left anterior descending (LAD) artery in mice to induce MI. Consistent with a previous report [12], we observe

that miR-150 KO mice exhibit normal cardiac function at baseline (Supplementary Table 1 and Fig. 1) but respond differently to MI. Cardiac function is significantly compromised in

miR-150-null mice following MI. First, MI significantly worsens the cardiac function of miR-150 KO mice at 3 days as indicated by a decreased ejection fraction (EF), fractional shortening

(FS), diastolic left ventricular anterior wall thickness (LVAW), and systolic left ventricular posterior wall thickness (LVPW) as well as an increase in end-systolic volume (ESV) and

systolic left ventricular internal diameter (LVID) compared to those of WT controls (Supplementary Table 2 and Fig. 1). MiR-150 KO mice also display impaired cardiac function at 4 weeks

post-MI, shown by a significant decrease in EF, FS, diastolic LVPW, and systolic LVPW as well as a significant increase in end-diastolic volume (EDV), ESV, diastolic LVID, and systolic LVID

(Supplementary Table 3 and Fig. 1). MI also causes augmented cardiac dysfunction in miR-150 KO mice at 8 weeks as evidenced by a significant decrease in EF, FS, diastolic LVAW, diastolic

LVPW, and systolic LVPW as well as a significant increase in EDV, ESV, diastolic LVID, and systolic LVID (Supplementary Table 4 and Fig. 1). In contrast, WT controls show less functional

impairment at 4 weeks (Supplementary Table 3 and Fig. 1) and 8 weeks following MI (Supplementary Table 4 and Fig. 1). We next show that miR-150 KO;_Sprr1a__hypo/hypo_ mouse hearts are

functionally normal at baseline (Supplementary Table 1 and Fig. 1). However, a significant improvement in cardiac function at 3 days after MI is observed in miR-150 KO;_Sprr1a__hypo/hypo_

mice compared to miR-150 KO mice, indicated by an increase in cardiac output (CO), EF, FS, and diastolic LVAW as well as a decrease in EDV, ESV, diastolic LVID, and systolic LVID

(Supplementary Table 2 and Fig. 1). MiR-150 KO;_Sprr1a__hypo/hypo_ mice also display enhanced cardiac function at 4 weeks post-MI as evidenced by a significant increase in EF, FS, diastolic

LVAW, systolic LVAW, diastolic LVPW, and systolic LVPW as well as a significant decrease in EDV, ESV, diastolic LVID, and systolic LVID (Supplementary Table 3 and Fig. 1) compared to those

of miR-150 KO mice. Last, we show improved cardiac function in miR-150 KO;_Sprr1a__hypo/hypo_ mice at 8 weeks post-MI compared to miR-150 KO mice as shown by a significant increase in CO,

EF, FS, heart rate (HR), diastolic LVAW, systolic LVAW, and systolic LVPW as well as a significant decrease in EDV, ESV, diastolic LVID, and systolic LVID (Supplementary Table 4 and Fig. 1).

Our morphometric data also show that miR-150 KO;_Sprr1a__hypo/hypo_ mice have a significant decrease in the ratio of heart weight/body weight (HW/BW) and the ratio of left ventricle

weight/body weight (LVW/BW) at 8 weeks after MI compared to miR-150 KO controls (Supplementary Table 4). Notably, we do not observe any difference in post-MI mortality between groups

(Supplementary Tables 1, 3, and 4: see n for animal numbers per each group at week 0, week 4, and week 8 after MI). SUSTAINED SPRR1A KNOCKDOWN ALLEVIATES CARDIAC DAMAGE, INFLAMMATION, AND

APOPTOSIS POST-MI MEDIATED BY MIR-150 LOSS We previously reported that miR-150 KO mice display excessive maladaptive post-MI remodeling, such as cardiac damage, inflammation, and apoptosis

[12]. To determine whether repression of _Sprr1a_ mediates the major functions of miR-150 in vivo, we employed miR-150 KO;_Sprr1a__hypo/hypo_ mice and assessed post-MI remodeling compared to

that of miR-150 KO controls. We find that miR-150 KO;_Sprr1a__hypo/hypo_ hearts exhibit a decrease in the loss of normal architecture and cellular integrity (Fig. 2A) as well as decreased

mRNA levels of fetal _Nppa_ (Fig. 2B) after 8 weeks of MI compared to miR-150 KO hearts. We next examined whether an improved cardiac inflammatory cell (CI) response contributes to the

decreased disorganized structure in miR-150 KO;_Sprr1a__hypo/hypo_ hearts post-MI. Notably, inflammatory _Il-6_, _Tnf-α_, and _Ptprc_ are also downregulated in miR-150 KO;_Sprr1a__hypo/hypo_

hearts (Fig. 2C, D and Supplementary Fig. 1) compared to miR-150 KO hearts post-MI. Finally, we find that miR-150 KO;_Sprr1a__hypo/hypo_ hearts contain significantly lower numbers of

cleaved caspase-3-positive cells (Fig. 3A, B), indicating decreased apoptosis in miR-150 KO;_Sprr1a__hypo/hypo_ hearts. Our data further show that miR-150 KO;_Sprr1a__hypo/hypo_ hearts have

decreased mRNA levels of apoptotic _P53_, _Bak1_, and _Bax_ (Fig. 3C–E) compared to levels in miR-150 KO hearts. Altogether, our data suggest that sustained _Sprr1a_ downregulation

ameliorates adverse post-MI remodeling caused by miR-150 deletion and that miR-150 is a functionally important upstream negative regulator of _Sprr1a_ in the heart. KNOCKDOWN OF SPRR1A IN

MIR-150 KO MICE BLUNTS CARDIAC FIBROSIS POST-MI OBSERVED FOLLOWING MIR-150 DEFICIENCY To further determine the response of miR-150 KO;_Sprr1a__hypo/hypo_ mice to MI, we assessed the degree

of fibrosis using Masson’s trichrome staining and picrosirius red staining of the hearts at 8 weeks post-MI. We find larger regions of fibrosis in miR-150 KO hearts than in WT MI controls,

as reported previously [12]. We next observe reduced fibrosis post-MI in miR-150 KO;_Sprr1a__hypo/hypo_ hearts compared to miR-150 KO hearts (Figs. 4, 5A, B, and Supplementary Fig. 2).

MiR-150 KO MI hearts also exhibit increased expression of fibrotic _Col5a1_, _Col6a1_, _Col1a1_, _Col3a1_, and _Ctgf_ (Figs. 5C, D, and 6A–C) compared to expression in WT controls, but

miR-150 KO;_Sprr1a__hypo/hypo_ MI hearts exhibit decreased expression of these profibrotic genes (Figs. 5C, D, and 6A–C) compared to miR-150 KO controls. Next, our in vivo protein analysis

reveals significantly elevated levels of VIMENTIN and α-SMA in miR-150 KO MI mouse hearts compared to WT controls and significantly decreased levels of VIMENTIN and α-SMA in miR-150

KO;_Sprr1a__hypo/hypo_ hearts at 8 weeks post-MI compared to miR-150 KO controls (Fig. 6D, E, and Supplementary Fig. 3); this is consistent with the mRNA data for the profibrotic genes

(Figs. 5C, D, and 6A–C). Collectively, these results demonstrate for the first time that genetic knockdown of _Sprr1a_ significantly attenuates adverse postinfarct remodeling mediated by

miR-150 deletion. MIR-150 IN HUMAN CFS ELICITS PROTECTIVE EFFECTS IN PART THROUGH DIRECT FUNCTIONAL REPRESSION OF PROFIBROTIC SPRR1A Because of the cardiac upregulation of miR-150 by Carv

[11] concurrent with the downregulation of _Sprr1a_ [13], and the downregulation of miR-150 in CFs isolated from TAC mice [15] concurrent with the upregulation of _Sprr1a_ in CFs during MI

[13], we next studied primary adult human CFs (HCFs) to test whether miR-150 and _SPRR1A_ are inversely regulated in HCFs treated with Carv as well as HCFs subjected to H/R conditions.

Indeed, _SPRR1A_ is downregulated in HCFs subjected to H/R conditions after Carv treatment (Supplementary Fig. 4) concurrent with the upregulation of miR-150 [28]. We also observe that

_SPRR1A_ is increased in HCFs after H/R (Supplementary Fig. 4), consistent with our in vivo results in post-MI hearts and isolated CFs from ischemic myocardium [13]. Notably, we previously

reported that miR-150 is downregulated in HCFs after H/R [28]. Together with other previous reports on miR-150 downregulation in H/R and MI [12] as well as I/R [29, 30], our results indicate

that _Sprr1a_ is a critical functional target of miR-150 in CFs. Because _Sprr1a_ expression is upregulated in CFs isolated from ischemic mouse hearts [13] concurrent with the

downregulation of miR-150 in CFs isolated from TAC mice [15], and miR-150 negatively regulates mouse CF activation in vitro [15], we first confirmed whether a direct target of miR-150,

_SPRR1A_ is repressed by miR-150 in HCFs. Our loss-of-function studies indeed show that _SPRR1A_ is increased after miR-150 inhibition in HCFs (Fig. 7A, B). We next investigated whether

_SPRR1A_ regulates HCF activation. We first observe that _SPRR1A_ knockdown in HCFs decreases the expression of profibrotic _ACTA2_ and _CTGF_ (Fig. 7C and Supplementary Fig. 5), and miR-150

knockdown increases the expression of _ACTA2, CTGF_, and _POSTN_ (Supplementary Fig. 6). To further assess the effects of _SPRR1A_ knockdown, we examined HCF proliferation using

bromodeoxyuridine assay. We find that compared to controls, _SPRR1A_ knockdown decreased HCF proliferation (Fig. 7D–F) under both normoxic and H/R conditions. This is consistent with our

gene expression data, showing that HCFs with _SPRR1A_ knockdown have decreased mRNA levels of S-phase marker _PCNA_, mitosis (M) marker _AURKB_, and G2/M-phase marker CCNB1 compared with

controls (Supplementary Fig. 7). Moreover, our wound migration studies reveal that compared to controls, _SPRR1A_ knockdown decreased HCF migration (Fig. 8A–C) under both normoxic and H/R

conditions. This is consistent with our gene expression data, showing that _SPRR1A_ knockdown in HCFs subjected to H/R decreases mRNA levels of cell migration markers, _CTHRC1_ and _TNC_

compared with controls (Supplementary Fig. 8). _SPRR1A_ knockdown in HCFs also suppresses mRNA levels of CF differentiation markers, _COL4A1_, _COL8A1_, and _SRF_ (Supplementary Fig. 9), as

well as the protein levels of profibrotic α-SMA and FIBRONECTION (Supplementary Fig. 10). Because TGF-β1/SMAD signaling pathway plays a key role in CF activation, we next investigated the

role of SPRR1A in the regulation of TGF-β1 and SMADs. We observe that _SPRR1A_ knockdown in HCFs subjected to H/R decreases mRNA levels of _TGFB1_, _SMAD2_, and _SMAD3_ compared with

controls (Supplementary Fig. 11). This is consistent with our in vivo data, showing that _Sprr1a_ knockdown in mice decreases _Smad3_ expression as well as mRNA and protein levels of TGF-β1

compared with controls (Supplementary Figs. 12, 13). Our data thus suggest that SPRR1A is sufficient to increase HCF activation in part by activating TGF-β1/SMAD signaling pathway. Finally,

to establish the functional relationship between miR-150 and _SPRR1A_ in HCF activation, we applied an antimiR/siRNA-based rescue strategy to validate the functional relevance of the direct

miR-150 target _SPRR1A_. MiR-150 knockdown increases HCF proliferation (Fig. 7D–F and Supplementary Fig. 7) and migration (Fig. 8A–C and Supplementary Fig. 8), which are attenuated by siRNA

against _SPRR1A_ (Figs. 7D–F, 8A–C, Supplementary Figs. 7, 8). We also show that miR-150 knockdown increases the expression of profibrotic _TGFB1_, _SMAD2, SMAD3, COL1A1, COL3A1_, _COL4A1_,

_COL8A1_, and _SRF_ under normoxic and/or H/R conditions, which are attenuated by _SPRR1A_ knockdown (Supplementary Figs. 9, 11, 14). Taken together, our data indicate that profibrotic

SPRR1A is a key direct and functional target of miR-150 in HCFs and whole mouse hearts. DISCUSSION In this study, we identify the direct functional interaction between miR-150 and SPRR1A as

a new regulatory mechanism pertinent to both MI in mice and HCF activation. Mice deficient in miR-150 are sensitized to MI as indicated by the increased cardiac fibrosis, apoptosis,

inflammation, and damage as well as impairment of left ventricular function. Using a novel miR-150 KO;_Sprr1a__hypo/hypo_ mouse model, we demonstrate that _Sprr1a_ knockdown attenuates

excessive adverse postinfarct remodeling mediated by miR-150 loss. Of note, miR-150 KO;_Sprr1a__hypo/hypo_ rescues in part the phenotype exhibited in miR-150 KO, which reaches similar levels

shown in WT MI or higher levels than WT MI (Figs. 2–6 and Supplementary Figs. 1, 3). Using primary adult HCFs, we also discover for the first time that miR-150 functionally inhibits

profibrotic _SPRR1A_ such that the increased expression of _SPRR1A_ in HCFs lacking miR-150 results in a higher degree of sustained CF activation. Thus, the current study suggests that

SPRR1A is a crucial ischemic injury-responsive target of miR-150 in whole mouse hearts and HCFs and that miR-150 confers protective actions on both HF in mice and HCF activation by

repressing profibrotic SPRR1A. β1AR is predominantly expressed in the heart, and β-arrestin-mediated β1AR signaling elicits protective effects after Isoproterenol (ISO)-induced injury [31].

In our previous study, miR-150 was activated by the β-blocker Carv acting through β-arrestin-mediated β1AR protective signaling [11]. We recently reported that miR-150 is a critical

downstream mechanism by which β1AR-mediated β-arrestin signaling pathways confer protection [32]. Together with the results presented here, we posit that β-arrestin-mediated β1AR regulatory

mechanisms of miR-150 activation elicit beneficial remodeling in failing hearts by repressing CF activation through inhibiting profibrotic genes, including _Sprr1a_. Interestingly, we

previously showed that miR-150 deletion significantly compromised cardiac function and remodeling following MI by increasing cell death without affecting neovascularization [12], whereas Liu

Z et al. reported that miR-150 overexpression mediated by AgomiR injection protected the mouse heart against acute MI (AMI) by inhibiting monocyte migration [20]. More recently, using a

novel mouse model, we demonstrated that cardiac-specific miR-150 cKO mice had enhanced maladaptive post-MI remodeling. Mechanistically, miR-150 represses a proapoptotic and direct CM target,

_Sprr1a_ [13]. We also showed that _Sprr1a__hypo/hypo_ mice are protected against MI [13]. Although these previous studies showed the correlative relationship between miR-150 and _Sprr1a_

in HF, our overall knowledge of their functional actions remains elusive in part because of (i) the lack of mechanistic insight by which the miR-150/_Sprr1a_ dyad regulates HF and (ii) the

absence of rigorous studies to establish their direct in vivo functional relationship in HF. Our previous fractionation studies of different cardiac cell types showed that the expression of

miR-150 was significantly higher in normal CMs than CFs at baseline and its expression was not altered in stressed CMs, CFs, CIs, and cardiac endothelial cells (CEs) isolated from ischemic

myocardium at 1 week post-MI [13]. Other studies reported that cardiac-specific overexpression of miR-150 alleviates TAC-induced cardiac hypertrophy and HF [14] and that miR-150 loss results

in a higher degree of cardiac fibrosis after TAC and miR-150 is downregulated in CFs (not CMs) isolated from TAC mice [15]. MiR-150 also negatively regulates mouse CF activation in vitro

[15]. It is known that TGF-β1 or TGF-β/Smad signaling inhibits miR-150 expression [33, 34], coincident with SPRR1A upregulation [35]. MiR-150 can also suppress TGF-β in rat hearts and

primary CFs [36]. These previous studies indicate that miR-150 or SPRR1A plays an antifibrotic or profibrotic role, respectively. Interestingly, prior studies showed that SRF and c-Myb are

possible downstream mechanisms that explain antifibrotic effects of miR-150 in the heart [14, 15]. Liu W et al. reported that SRF is repressed by miR-150 in mouse hearts without presenting

any functional rescue experiments [14]. Deng P et al. showed that c-Myb is a key functional target of miR-150 in mouse CFs, but they did not establish their in vivo functional link [15].

Thus, the significance of SRF- and c-Myb-dependent mechanisms of miR-150 actions in vivo has not been defined. Here, we make novel discoveries to establish the functional miR-150/SPRR1A axis

in whole mouse hearts and HCFs as well as to define the role of SPRR1A in CF activation. SPRR1A is a known substrate of TGase II, and prior studies have linked TGase II to HF [37, 38] and

apoptosis of noncardiac cells [39, 40], suggesting a potential role of SPRR1A in HF and apoptosis. In agreement with this idea, we previously showed that CMs with knockdown of _Sprr1a_ are

protected against apoptosis in H/R and that _Sprr1a__hypo/hypo_ mice are protected against MI [13]. Interestingly, _Sprr1a_ expression is increased in post-MI hearts [13, 23] concurrent with

miR-150 downregulation [12]. Our previous cardiac cell fractionation study showed that _Sprr1a_ was ubiquitously expressed in CMs, CFs, CIs, and CEs at baseline. Despite this ubiquitous

expression pattern at baseline, _Sprr1a_ is upregulated in CMs isolated from mouse hearts post-MI [13], consistent with a report of SPRR1A upregulation in CMs from TAC-induced myocardium

[23]. Of importance, we also showed that _Sprr1a_ is upregulated in CFs isolated from post-MI hearts [13], suggesting a potential role of _Sprr1a_ in CFs and cardiac fibrosis. This notion is

supported by previous studies showing that adenovirus-mediated ectopic overexpression of _Sprr1a_ in vivo promotes cardiac fibrosis after TAC [23] and that SPRR1A levels are more highly

expressed in CFs from the infarct zone than in those from the remote region following MI [27]. Our current study also demonstrates for the first time that _SPRR1A_ knockdown suppresses HCF

proliferation and migration (Figs. 7, 8 and Supplementary Figs. 7, 8) and that _Sprr1a_ knockdown attenuates cardiac fibrosis post-MI observed following miR-150 deletion (Figs. 4–6 and

Supplementary Figs. 2,3). In our previous study, we also reported that LV _SPRR1A_ is upregulated in patients with HFrEF [13], consistent with other studies in mice with ISO-induced

myocardial injury [24] and renal I/R injury [25]. Notably, _SPRR1A_ expression was inversely associated with the survival of cancer patients [41,42,43]. Moreover, our recent study reported

that _Sprr1a_ is downregulated in hearts and CMs by Carv [13], concurrent with the upregulation of miR-150 [11]. Here, we also show that _SPRR1A_ is downregulated in HCFs by Carv

(Supplementary Fig. 4), concurrent with the upregulation of miR-150 [28]. Consistent with our findings, the cardiac upregulation of _Sprr1a_ after injury is suppressed by treatment with

cardioprotective Danshen [24]. Thus, the findings of _Sprr1a_ upregulation in CMs and CFs during MI further support that _Sprr1a_ inhibition could be therapeutically beneficial for HF and

cardiac fibrosis. Notably, a regeneration-associated gene, _Sprr1a_ was shown to be regulated by miR-463-3p in tibial nerve tissue [44] as well as by miR-155 in neurons and macrophages [45].

Except for these two other reports, little is known about the regulation of _Sprr1a_ by miRs. Given our findings that profibrotic SPRR1A is an important functional target of miR-150 in

whole mouse hearts (Figs. 1–6 and Supplementary Figs. 1–3) and HCFs (Figs. 7, 8, Supplementary Figs. 7–9, and Supplementary Figs. 11, 14) and that SPRR1A was regulated by miR-150 by direct

interaction [13], future targeted treatment options based on SPRR1A could be considered in MI patients with decreased levels of miR-150. LIMITATIONS Although we demonstrate that systemic

knockdown of _Sprr1a_ in mice alleviates maladaptive post-MI remodeling caused by miR-150 loss and that miR-150 is an important negative regulator of HCF activation in vitro by functionally

repressing profibrotic _SPRR1A_, miR-150 or _Sprr1a_ expression in other myocardial cells may also play a prominent role as supported by our recent study using CM-specific miR-150 cKO mice

[13]. Future studies using conditional cell-specific mouse models are thus warranted to fully understand the possible contribution of miR-150 or _Sprr1a_ expression in other cell types to

postischemic heart remodeling. Especially, whether _Sprr1a_ is a key downstream effector of fibroblast miR-150 during post-MI fibrotic remodeling in mice remains to be determined and is

beyond the scope of the current study. To expand our understanding of the sequence of events and to evaluate fibroblast roles in inflammatory and wound healing responses, additional

immunohistochemical assessments and gene expression studies (e.g., inflammation, acute cardiac cell death, and wound healing) during AMI are also needed. Notably, our functional rescue data

in mouse hearts and HCFs establish a functional link between miR-150 and SPRR1A in HF, supporting that they are in a linear pathway. Nevertheless, miR-150 may also have other targets

mediating distinct functions. We will investigate additional novel functional targets (i.e., SPRR1A-independent mechanisms) in the heart by cross-referencing the gene signature from

cardiac-specific miR-150 cKO mice [13] with miR-150 target prediction analyses in our future mechanistic studies. Importantly, the downstream targets and mechanisms of SPRR1A that regulate

CF activation remain elusive despite the suggestion in our current data (Supplementary Figs. 5 and 7–14) that SPRR1A activates profibrotic markers. Especially, a potential role of SPRR1A as

a regulator of the cytoskeleton remains to be determined because SPRR1A is localized selectively to actin-rich membrane ruffles and is colocalized with F-actin microfilaments [46]. Moreover,

additional methods, such as the Simpson method with multiple B-mode images and pressure-volume loop analysis would be required to measure cardiac function more accurately and to assess

diastolic dysfunction. Atomic force microscopy will be also needed to determine the stiffness of ventricular scar tissue post-MI. In the current study, we use a primary adult HCF model to

bolster the translational significance. Although this human cell model is an appropriate human cell source for HF remodeling in vitro, the mode of action in mice could be different in human

cells. We would employ CFs isolated from the mice used in this study to confirm the miR-150/SPRR1A axis in CFs. Although we show that modulating the miR-150/SPRR1A axis affects HCF

activation after H/R, most CFs are activated by recruited inflammatory cells after MI, not by ischemia. We would thus use TGF-β in future CF studies. Last, other in vivo injury models as

well as detailed studies on the other roles of the miR-150/SPRR1A dyad in all cell types are warranted before pursuing this axis as a vital therapeutic modality. CONCLUSIONS Our results

using a novel double loss-of-function mouse model and primary adult HCFs suggest that SPRR1A knockdown attenuates adverse post-MI remodeling mediated by miR-150 deletion and that miR-150

plays a vital protective role in part by blunting CF activation through its direct functional repression of profibrotic SPRR1A. Although miR-150 is associated with HF in humans [21] and the

correlative relationship between miR-150 and SPRR1A in the heart has been shown [13], our studies directly establish the functional relationship between miR-150 and SPRR1A during both

post-MI remodeling in mice and HCF activation as well as define an underlying mechanism by which miR-150 affects CF activation. Given that upregulation of SPRR1A [13, 23] or downregulation

of miR-150 [16, 29, 30, 47] also underlies other forms of cardiac disease, the deleterious action of SPRR1A and the protective action of miR-150 in whole mouse hearts and HCFs are likely

applicable to multiple stress settings. Therefore, reducing SPRR1A levels via SPRR1A knockdown and boosting miR-150 levels via Carv or miR-150 overexpression, in part to attenuate CF

activation, could be an attractive adjunctive strategy to provide therapeutic benefits. AVAILABILITY OF DATA AND MATERIALS All data are included in the manuscript and Supplementary

Information. The analytical methods and study materials will be made available to other researchers for the purposes of reproducing the results or replicating the procedures. Other methods

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plasma micoRNAs in atrial fibrillation patients. PLoS ONE. 2012;7:e44906. Article  CAS  PubMed  PubMed Central  Google Scholar  Download references FUNDING This work was supported by the

American Heart Association (AHA) Postdoctoral Fellowship 900453 to S.K., the AHA Career Development Award 931621 to M.N.S., the National Institutes of Health (NIH) R01HL148165 to S.J.C., and

the NIH R01HL146481 to I.K. AUTHOR INFORMATION Author notes * Satoshi Kawaguchi Present address: Department of Emergency Medicine, Asahikawa Medical University, Asahikawa, Hokkaido, Japan *

Bruno Moukette Present address: Internal Medicine Research Unit, Pfizer Inc., Cambridge, MA, USA * Tatsuya Aonuma Present address: Division of Cardiology, Nephrology, Pulmonology, and

Neurology, Department of Internal Medicine, Asahikawa Medical University, Asahikawa, Hokkaido, Japan * These authors contributed equally: Satoshi Kawaguchi, Bruno Moukette. AUTHORS AND

AFFILIATIONS * Department of Anatomy, Cell Biology, and Physiology, Indiana University School of Medicine, Indianapolis, IN, USA Satoshi Kawaguchi, Bruno Moukette, Marisa N. Sepúlveda, Taiki

Hayasaka, Tatsuya Aonuma, Angela K. Haskell, Jessica Mah & Il-man Kim * Division of Gastroenterology and Hepatology, Indiana University School of Medicine, Indianapolis, IN, USA Suthat

Liangpunsakul * Vascular Biology Center, Medical College of Georgia, Augusta University, Augusta, GA, USA Yaoliang Tang * Herman B Wells Center for Pediatric Research, Indiana University

School of Medicine, Indianapolis, IN, USA Simon J. Conway & Il-man Kim * Krannert Cardiovascular Research Center, Indiana University School of Medicine, Indianapolis, IN, USA Il-man Kim

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author inPubMed Google Scholar CONTRIBUTIONS S.K., B.M., M.N.S., T.H., and I.K. designed the research studies, directed the study, and wrote the manuscript. S.K., B.M., M.N.S., and T.H.

conducted the experiments, acquired the data, analyzed the data, and prepared the figures. I.K. supervised the study and provided financial support. T.A., A.K.H., J.M., S.L., Y.T., and

S.J.C. helped to analyze the data and write the manuscript. S.K. and B.M. are listed as co-first authors. The order of co-first authors was determined mainly by the numbers of data presented

in this study, not via any other biases. CORRESPONDING AUTHOR Correspondence to Il-man Kim. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL

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key downstream effector of MiR-150 during both maladaptive cardiac remodeling in mice and human cardiac fibroblast activation. _Cell Death Dis_ 14, 446 (2023).

https://doi.org/10.1038/s41419-023-05982-y Download citation * Received: 02 March 2023 * Revised: 10 July 2023 * Accepted: 11 July 2023 * Published: 19 July 2023 * DOI:

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