Compromised diabetic heart function is not affected by miR-378a upregulation upon hyperglycemia

Background Cardiac-abundant microRNA-378a (miR-378a) is associated with postnatal repression of insulin–like growth factor 1 receptor (IGF-1R) controlling physiological hypertrophy and survival pathways. IGF-1/IGF-1R axis has been proposed as a therapeutic candidate against the pathophysiological progress of diabetic cardiomyopathy (DCM). We ask whether hyperglycemia-driven changes in miR-378a expression could mediate DCM progression. Methods Diabetes mellitus was induced by streptozotocin (STZ) (55 mg/kg i.p. for 5 days) in male C57BL/6 wild type (miR-378a+/+) and miR-378a knockout (miR-378a−/−) mice. As a parallel human model, we harnessed human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM miR378a+/+ vs. hiPSC-CM miR378a−/−) subjected to high glucose (HG) treatment. Results We reported miR-378a upregulation in cardiac diabetic milieu arising upon STZ administration to wild-type mice and in HG-treated hiPSC-CMs. Pro-hypertrophic IGF-1R/ERK1/2 pathway and hypertrophic marker expression were activated in miR-378a deficiency and upon STZ/HG treatment of miR-378a+/+ specimens in vivo and in vitro suggesting miR-378a-independent hyperglycemia-promoted hypertrophy. A synergistic upregulation of IGF-1R signaling in diabetic conditions was detected in miR-378a−/− hiPSC-CMs, but not in miR-378a−/− hearts that showed attenuation of this pathway, pointing to the involvement of compensatory mechanisms in the absence of miR-378a. Although STZ administration did not cause pro-inflammatory or pro-fibrotic effects that were detected in miR-378a−/− mice, the compromised diabetic heart function observed in vivo by high-resolution ultrasound imaging upon STZ treatment was not affected by miR-378a presence. Conclusions Overall, data underline the role of miR-378a in maintaining basal cardiac structural integrity while pointing to miR-378a-independent hyperglycemia-driven cardiac hypertrophy and associated dysfunction. Supplementary Information The online version contains supplementary material available at 10.1007/s43440-023-00535-8.


Introduction
Rising from the long subclinical period, diabetic cardiomyopathy (DCM) proceeds in the systolic dysfunction stage, becoming a chronic cardiac complication eventually leading to heart failure [1].Sustained cardiomyocyte hypertrophy and excessive interstitial fibrosis lay at the pathophysiological roots of DCM [2,3].The causal molecular background is associated with metabolic disturbances (hyperglycemia) [1,4], however, in long-term randomized trials normalization of blood glucose levels in diabetic patients did not halt DCM progression [5].
Whether diabetes-related alterations of cardiac miRNAs (miRs) landscape may contribute to the pathogenesis of DCM is of growing interest [1,5].Being one of the cardiac-enriched miRs, miRNA-378a exhibits cardiomyocyte-selective expression while is not detected in non-muscle cells [6][7][8].It is located in the first intron of the Ppargc1b gene, encoding PGC-1β, a key regulator of energy metabolism, and gives rise to two functional mature strands, miR-378a-3p and miR-378a-5p [9].Proangiogenic and anti-inflammatory activity of miR-378a was reported by us recently in murine skeletal muscles [6].
Anti-hypertrophic action of miR-378a was demonstrated in cardiac in vitro model of phenylephrine-induced cardiomyocyte hypertrophy and in an in vivo model of cardiac hypertrophy (pressure overload by thoracic aortic constriction) [8,10] but has not examined so far in the context of diabetes.In vitro, in rat primary cardiomyocytes miR-378a inhibited phenylephrine-induced hypertrophy and repressed pro-hypertrophic mitogen-activated protein kinase (MAPK) signaling at four levels, targeting insulinlike growth factor-1 (IGF-1) receptor (IGF-1R) [7] and downstream mediators: MAPK1, GRB2 and KSR1 [10].Our recent study indicates also miR-378a-dependent regulation of hypertrophic growth of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) [11].In addition, previous data point to anti-fibrotic effects of miR-378a in the murine model of pressure overload [8,10].
MiR-378a expression is detected at a relatively low level in the fetal heart, progressively increasing after birth and continuing in aged mice, which is associated with postnatal repression of cardiac IGF-1R [7].Closing a miR-378a/ IGF-1R/IGF-1 feedback loop, IGF-1 may act as an inhibitor of miR-378a triggering cell survival signals in the heart [7].The level of IGF-1 is decreased in the serum of animals and patients with diabetes [12].Accordingly, in the diabetic environment a strategy of IGF-1/IGF-1R overexpression ameliorated the pathophysiological progress of DCM through antioxidative and anti-inflammatory processes [12][13][14].Here, we consider the involvement of miR-378a in the pathogenesis of DCM with particular emphasis on the regulation of IGF-1R signaling.

Mice
miR-378-/-mice at 129SvEv/C57BL/6 background were the gift of Eric Olson (Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA) [15].Mice were backcrossed with C57Bl/6J mice for ten generations to obtain animals on a clear C57BL/6 background.Animal experiments were performed on male mice after approval (No. 108/2021) by the second Institutional Animal Care and Use Committee (IACUC) in Krakow, Poland, according to Polish and European legislation.Mice were housed under specific pathogen-free (SPF) conditions in individually ventilated cages with a 14 h/10 h light/dark cycle and were kept on a normal, chow diet with water and food available ad libitum.For fasting mice were transferred for 4 h to new cages without food, with water ad libitum.

STZ injections and material collection
Type 1 diabetes was induced in 10-13-week-old miR-378a−/− mice (C57Bl6 background) and age-matched wildtype counterparts (miR-378a+/+) by STZ treatment.STZ (55 mg/kg b.w., Sigma-Aldrich, cat.no.S0130) dissolved in 0.1 mol/l citrate buffer (pH 4.5) or citrate buffer alone was administered to mice (5.5 µL/ 1 g) via intraperitoneal injections (i.p.) for five consecutive days [14].Blood glucose level was measured upon 4 h-starvation using AccuCheck glucometer (Roche Diagnostics) 4 and 8 weeks after the last STZ injection.Mice were considered hyperglycemic when their blood glucose level was a minimum 240 mg/dL.Upon 8 weeks of STZ treatment, the assessment of cardiac parameters in vivo by high-resolution ultrasound imaging was performed and the mice were sacrificed thereafter.Peripheral blood was immediately collected, and hearts were perfused with heparinized saline (0.5 IU/ml) and potassium chloride (30 mM) to arrest heart contraction in diastole.

Visualization and quantification of heart function by ultrasonography
For the assessment of cardiac parameters in vivo highresolution ultrasound imaging system for the examination of small animals (Vevo 2100, Visual Sonics) with an MS-400: 38 MHz MicroScan transducer was used.Hair from the chest was removed the day before scanning.Anesthesia was induced in 3% isoflurane (Aerrane, Baxter)/ O2 filled induction chamber.Once anesthetized, the mouse was placed dorsally on heated platform and anesthesia was redirected to the nose cone (maintained at 1.5-2.0%isoflurane, 2.5 L/min).Each paw pad was taped down to platform on ultrasonic gel dots for ECG readings.Prewarmed ultrasonic gel was applied to the shaved spot.The heart was observed using parasternal long axis (PSLAX) view at first and upon 90 clockwise rotation of the probe short axis (SAX) view was captured with M mode line on the center of the LV.Heart rate was kept between 300-600 BPM.For quantification of cardiac function anterior and posterior wall edge were traced through 3-4 beats on 2-4 independent tracings.

RNA-based analysis
Dissected cardiac tissues were collected into RNAlater tissue storage reagent (Sigma-Aldrich, cat.no.R0901), frozen in liquid nitrogen, and stored at -80 °C.Samples were homogenized in phenol/guanidine-based QIAzol Lysis Reagent (Qiagen, cat.no.79306).Total RNA was isolated by organic extraction upon the addition of chloroform.
Total RNA isolation from hiPSC-CMs was done using Fenozol reagent (A&A Biotechnology, cat.no.203-100) with chloroform.Upon isopropanol-based precipitation of RNA from the aqueous phase and ethanol washing, the RNA pellet was resuspended in RNase-free water.RNA concentration and quality were determined by NanoDrop Spectrophotometer (ThermoFisher Scientific).
Total RNA was used for reverse transcription (RT) of either mRNA (500 ng) or miRNA (10 ng) performed with RevertAid reverse transcriptase (200 U/μL, Thermo Fisher Scientific, cat.no.EP0442) and miRCURY LNA RT kit (Qiagen, cat.no.339340), respectively, carried out in Pro-Flex PCR System (ThermoFisher Scientific).Gene expression analysis and miRNA detection were done using quantitative PCR (qPCR) as described previously [6] based on Table 1 The sequences of primers used for the determination of gene expression at mRNA level by qPCR

Western blot
Whole protein lysates were obtained from dissected cardiac tissues (pre-frozen in liquid nitrogen and stored at − 80 °C) upon homogenization in 500 μL of ice-cold 1% Triton X100 in PBS and proteinase inhibitors (Roche Diagnostic) using TissueLyser (Qiagen).Proteins from hiPSC-CMs were isolated using Pierce RIPA Buffer (Thermo Scientific, cat.no.89900) with proteinase inhibitors.Protein samples (30 μg) were subjected to SDS-PAGE and Western Blot under conditions specified previously [11] using primary antibodies against IGF-1R (
Cell area was calculated by the blinded investigator using ImageJ software.

Histological procedures and IF of paraffin cardiac sections
Hearts were fixed for 48 h in 10% formalin and paraffinembedded.Paraffin sections cut with the automatic microtome (ThermoFisher Scientific) (8 μm-thick) were deparaffinized and subjected to hematoxylin and eosin (H&E) staining to visualize tissue morphology, inflammatory infiltration, and necrosis, Masson's Trichrome staining to assess fibrosis, according to the vendor's instructions (Sigma-Aldrich), and IF stainings.For IF, a previously established protocol was used with modifications [16] described in detail in Supplementary material.
Pictures were taken with Carl-Zeiss LSR-510 meta laser scanning confocal microscope or Leica DMi8 microscope with CMOS Leica MC170 HD camera with Leica Application Suite X (Las X) Life Science microscope software platform.
Semi-quantitative analysis of leukocyte infiltration and necrotic events (H&E) was performed within each scan of the whole heart cross-section (1 scan per mouse) from all fields of view (~ 50 images per scan) and expressed as the average arbitrary unit per field of view.Quantitative analysis of collagen area (Masson's Trichrome) was performed from each scan of the whole heart cross-section using QuPath-0.2.3 software and expressed as the % of collagen area per total heart area.α-SMA-positive vessels were counted from all fields of view (35-50 images per scan) within each scan of the whole heart section and expressed as the average number of arterioles per field of view.

Blood cell count
The peripheral blood from the eye was collected directly into EDTA-coated tubes and was analyzed by the ABC Vet device (Horiba ABX).
Both, in miR-378a+/+ and miR-378a−/− cardiac tissue sections, IGF-1R expression colocalized with ⍺-smooth muscle actin (⍺-SMA)-positive blood vessels and connexin 43 (Cx43)-positive CMs (Fig. 2J).Considering that miR-378a expression is selective to CMs [7], the presence and action of IGF-1R in other cardiac cell types or the involvement of other rescue mechanisms in the absence of miR-378a should also be considered.

High glucose increases miR-378a level and promotes pro-hypertrophic IGF-1R pathway in hiPSC-CMs
Due to difficulties with obtaining cardiac muscle material from humans, we harnessed hiPSC-CMs to examine the effect of high glucose (HG) on human CMs.hiPSC-CMs genetically engineered with CRISPR/Cas9 system (hiPSC-CM miR378a+/+ vs. hiPSC-CM miR378a−/−) as reported by us formerly [11], were used to examine the effect of miR378a deprivation.
The activation of the pro-hypertrophic pathway in miR-378a−/− hiPSC-CMs was reflected by the hypertrophic phenotype of those cells.The effect of miR-378a knockout was detected by two-way ANOVA for the genotype × cell area interaction (F 1,89 = 14.85, p = 0.0002, Fig. 3G, Suppl.Figure 1D).Specifically, cell area increased in the absence of miR-378a in control conditions (p = 0.0132) and under HG stimulation (t 49 = 2.067, p = 0.0440) (Fig. 3G) with no apparent glucose effect in any of the genotypes (p > 0.1).The cardiac identity was confirmed by the presence of ⍺-actinin in both miR-378a+/+ and miR-378a−/− cells (Fig. 3G).

Discussion
Here, we report the upregulation of miR-378a in cardiac hyperglycemic milieu and HG-treated human hiPSC-CMs.Although we underlined the involvement of miR-378a in maintaining basal cardiac structural integrity and vascularization, we questioned the importance of endogenous miR-378a in the development of DCM.Results point to miR-378a-independent hyperglycemia-driven cardiac hypertrophy and associated dysfunction.

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CM hypertrophy is initiated in response to increased cardiac workload (exercise) or injury acting as a compensatory response to maintain cardiac output [3].Pathological hypertrophy is associated with fibrosis and myofiber disarray [23] eventually increasing cardiac stiffness and compromising heart function [19].IGF-1 as a critical mediator of physiological hypertrophy acts against cardiac dysfunction [24,25].In a diabetic environment, IGF-1/IGF-1R axis was shown to ameliorate the pathophysiological progress of DCM through antioxidative and anti-inflammatory processes [12][13][14].However, any link to miR-378a has not been reported so far.
In the current study, STZ-induced diabetes did not change significantly cardiac IGF-1R expression.It is in agreement with previous studies that also showed comparable [14,26] or even decreased [27] IGF-1R protein levels in STZtreated rodents.It could be explained by the upregulation of miR-378a observed by us in murine diabetic hearts, and in human model, HG-treated hiPSC-CMs.Our data expand and verify previous studies that showed an insignificant increase of miR-378a in diabetic whole heart tissue upon multiple low-dose STZ injections [28] and are in line with reported upregulation of miR-378a in LV specimens of mice given a single high-dose injection of STZ [5].
We previously reported that miR-378a-deficient hiPSC-CMs exhibit hypertrophic growth with the involvement of NFATc3 (nuclear factor of activated T cells c3) and the activation of ERK and AKT kinases [11].In the current study, we verified the hypertrophic phenotype of miR-378a−/− hiPSC-CMs both in control conditions and under HG stimulation with associated increase of IGF-1R signaling, and p-ERK1/2 and p-AKT proteins.The level of biomarkers of cardiac hypertrophy, β-MHC and ANP, increased in miR-378a deficiency (in non-diabetic mice) and STZ-treated miR-378a+/+ mice.This correlated with the pattern of pro-hypertrophic IGF-1R pathway activation in respective murine cardiac specimens as well as miR-378−/− hiPSC-CMs and HG-cultured miR-378a+/+ hiPSC-CMs that also showed increased size, and suggests miR-378a independent hyperglycemia-promoted hypertrophy.Interestingly, STZ-dependent increase in hypertrophic markers was not reflected by cardiac weight.It could be explained by the early stage of cardiac hypertrophy detected by markers expression in cardiac lysates but not yet reflected by enhanced heart weight.
Moreover, we detected a synergistic upregulation of the IGF-1R pathway and cell growth (although not significant) in miR-378a-deficient HG-treated hiPSC-CMs.Thus, miR-378a upregulation in HG conditions could be considered as a compensatory mechanism to decrease pro-hypertrophic pathways.Nonetheless, at the tissue level, mir-378a does not appear to be a key regulator of the IGF-1R pathway in the diabetic heart, as even in its absence this pathway is blocked.The involvement of other rescue mechanisms in the absence of miR-378a in the hyperglycemic milieu needs to be further investigated.The presence and action of IGF-1R in other cardiac cell types should also be considered.
Myocardial fibrosis associated with the accumulation of extracellular matrix proteins constitutes a critical component of DCM pathology [2] and was reported in STZ-diabetic animal models [12,14,22].However, in our experimental setting STZ-driven hyperglycemia although promoted myocardial hypertrophy, did not affect the extent of fibrotic lesions and collagen expression which requires further analysis and constitutes the limitation of the current study.
The impact of miR-378a on fibrotic structural changes seems to be tissue-dependent and affected by disease states [17,29,30].Previous data point to anti-fibrotic effects of miR-378a in the murine model of pressure overload [8,10].Compensation for miR-378 downregulation by AAV9-miR378 administration diminished cardiac fibrosis and increased cardiac contractility [10].In the current work we showed that collagen deposition increased in miR-378a−/− hearts of both non-diabetic controls and upon STZ treatment.Pro-fibrotic effects of miR-378a deficiency were not mediated by TGF-β, but an STZ-dependent drop of TGF-β was detected in wild-type animals where miR-378a was increased.Nonetheless, bioinformatic screening did not find Tgfb as a direct target of miR-378a-3p or miR-378a-5p, hence the effect requires further mechanistical investigation.
The 8-week study period upon STZ treatment was shown to be sufficient to induce cardiac dysfunction in C57BL6J mice [14,22].The pattern of diabetes-related decrease of end-diastolic dimension and end-systolic dimension with increased LVPW thickness was demonstrated previously by Huynh et al. [14] although statistically insignificant.In the current study, we confirmed genotype-independent STZ-driven decrease of end-systolic and end-diastolic LV diameter and LV volume while showing a drop in stroke volume (SV) and cardiac output (CO) in diabetic conditions.It is in agreement with diastolic dysfunction shown by Huynh et al. using Doppler echocardiography and LV catheterization that was consistent with increased fibrosis [14].Despite that, in our model in both miR-378a+/+ and miR-378a−/− mice, we detected increased contractility assessed by LV EF and FS upon STZ treatment.Although diabetes-related increase of FS was also detected by others [14] it requires further investigation.
Here, we examined for the first time the involvement of miR-378a in diabetic heart function.Neither STZ-driven miR-378a upregulation in wild-type mice nor miR-378a deficiency changed the pattern of decreased SV and CO observed in vivo by high-resolution ultrasound imaging.
MiR-378a-independent compromised diabetic heart function may be related to ERK signaling and enhanced hypertrophic markers in STZ-treated animals.On the other hand decreased AKT activity in cardiac tissue shown in diabetic hearts of mice that were given a single injection of STZ [31] and also detected in the current study, could be involved.Despite the involvement of AKT in cardiac hypertrophy, the correlation of improvement of cardiac In vivo high-resolution ultrasound imaging (n = 4-8).Data are presented as mean ± SEM. # p < 0.05, ## p < 0.001, ### p < 0.0001-two-way ANOVA variation; *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA with Tukey's post hoc test; $ p < 0.05, $$ p < 0.01 by unpaired twotailed Student's t test function and structure with elevated AKT activity was reported in diabetic IGF-1R transgenic mice as compared to nontransgenic diabetic mice, suggesting the protective effects of IGF-1R signaling in diabetes [14].In parallel, in the same study elevated ventricular AKT phosphorylation was also detected in nontransgenic diabetic animals compared to non-diabetic controls [14].Thus, one may consider disturbed balance between physiological and pathological cardiac hypertrophy and underlying mechanisms that could determine the diabetic phenotype.
Having in mind a progressive increase of cardiac miR-378a expression from birth to aged mice [7] the effect of miR-378a deficiency might not be severe enough to affect overall cardiac function in used in our study of 10-13-week-old mice.In line with this explanation, in 25-week-old Db/Db mice, a model of type 2 diabetes, miR-378a loss was shown to improve cardiac function [32] which further enhances the hypothesis of the involvement of other rescue mechanisms in the absence of miR-378a in hyperglycemic milieu still to be investigated.
In summary, this work proves hyperglycemia/ HG-driven upregulation of miR-378a expression in the murine heart and novel human CM model.MiR-378a deficiency impaired basal cardiac structural integrity, but changes in mir-378a levels seem to be compensated in the diabetic milieu by other mechanisms regulating the IGF-1R pathway, which questions the importance of endogenous miR-378a in this disease model.

Fig. 6
Fig. 6 miR-378a does not affect STZ-induced changes in cardiac parameters.A Heart weight and B ratio of heart weight to body weight (HW/BW) 8 weeks upon STZ treatment.C Heart rate (HR).Left ventricular (LV) D, E diameter and F, G volume at systole (sys) and diastole (dias), H LV posterior wall, I LV interior diameter, J LV anterior wall, K LV ejection fraction (EF), L LV fractional shorten-

Table 2
The sequences of primers used for the determination of miRNA level using miRCURY LNA PCR kit number indicating the number of animals per group or in vitro experimental repetitions as well as the specific statistical test used with associated markings are underlined in the figure captions.
The values of statistics (main effects and interactions) were provided where applicable (for ANOVA: F, df, and p values; for Student's t test: t, df, and p values).The n