Cardiac-selective gene delivery of medium-chain acyl-CoA dehydrogenase (MCAD) does not protect against diabetes-induced cardiac dysfunction

doi:10.21203/rs.3.rs-3140983/v1

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Cardiac-selective gene delivery of medium-chain acyl-CoA dehydrogenase (MCAD) does not protect against diabetes-induced cardiac dysfunction

https://doi.org/10.21203/rs.3.rs-3140983/v1

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published 20 Nov, 2023

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People with diabetes are at significantly higher risk of developing heart failure. Diabetic cardiomyopathy describes heart disease in patients with diabetes who have no other cardiac conditions. Specific therapies to treat the diabetic heart are limited. A key mechanism involved in the progression of diabetic cardiomyopathy is dysregulation of cardiac energy metabolism. The aim of this study was to determine if increasing the expression of medium chain acyl-coenzyme A dehydrogenase (MCAD; encoded by Acadm), a key regulator of fatty acid oxidation, could improve function of the diabetic heart. Male mice were administered streptozotocin to induce diabetes, which led to diastolic dysfunction 8 weeks post-injection. Mice then received cardiac-selective adeno-associated viral vectors encoding MCAD (rAAV6:MCAD) or control AAV and were followed for 8 weeks. rAAV6:MCAD did not improve diabetes-induced diastolic dysfunction or alter the expression of key metabolic genes (Ppargc1a, Ppara, Cpt1b, Slc2a4) or proteins (OXPHOS complexes). An inverse correlation between MCAD and perilipin 5 was observed, suggesting that altered MCAD expression may have an impact on lipid droplet accumulation in the diabetic heart.

Cardiac & Cardiovascular Systems

diabetes

diabetic cardiomyopathy

gene therapy

metabolism

dehydrogenase

The effects of increasing MCAD in the diabetic heart were unknown
Increasing MCAD via AAV did not improve diastolic function in the diabetic heart
Perilipin 5, a lipid droplet protein, was lower in diabetic hearts with higher MCAD
AAV transduction efficiency was reduced in the diabetic heart, which has clinical implications

Cardiovascular disease (CVD) is a leading cause of morbidity and mortality for patients with diabetes [1, 2]. In many cases, diabetic patients develop cardiac structural and functional abnormalities even in the absence of common risk factors such as hypertension or coronary heart disease [3, 4]. The term ‘diabetic cardiomyopathy’ describes the pathology exhibited by these patients. Effective treatment options specific to the diabetic heart are limited [5]. The ability of sodium–glucose co-transporter 2 inhibitors to reduce cardiovascular deaths and hospitalization due to heart failure has revolutionized heart failure management in both type 2 diabetic and non-diabetic populations [6, 7]. However, heart failure remains an important issue for patients with T2D, and SGLT-2 inhibitors are not registered for use in patients with type 1 diabetes (T1D)[8]. Thus, there is a need to explore new treatment options for diabetic cardiomyopathy.

The diabetic heart is over-reliant on fatty acid oxidation as a means for producing ATP. Higher fatty acid oxidation rates and lower glucose oxidation rates decrease ATP production and cardiac efficiency, which are hallmarks of diabetic cardiomyopathy [9, 10]. Therefore, inhibiting fatty acid oxidation or stimulating glucose oxidation directly have been proposed as potential therapeutic strategies for diabetic heart disease [10]. While some studies have demonstrated an ability to improve cardiac function and pathology in experimental heart failure models by inhibiting fatty acid oxidation, [11–15], other studies support a role for enhancing fatty acid oxidation as a therapeutic strategy. For example, increasing cardiac fatty acid oxidation via deletion of acetyl-CoA carboxylase 2 prevented high-fat diet-induced cardiac dysfunction and remodeling in mice [16]. Further, in mouse models of heart disease with elevated cardiac fatty acid oxidation, heart function was preserved [17, 18]. A mismatch between cellular fatty acid uptake and mitochondrial β-oxidation can lead to the accumulation of triacylglycerols and other lipids in the cytosol, which impairs contractility [19] and alters signaling, leading to lipotoxicity [20]. Significant lipid accumulation has been observed in failing hearts from T2D patients [21], and lipid accumulation plays a key role in the development of cardiomyopathy in T1D [22]. Strategies to prevent lipotoxicity in the diabetic heart (e.g. inhibiting fatty acid uptake [23] or enhancing triacylglycerol hydrolysis and fatty acid metabolism to reduce cardiomyocyte lipid burden) may be of therapeutic benefit.

Acyl-CoA dehydrogenases are mitochondrial enzymes that catalyze the first step in each cycle of fatty acid β-oxidation. Fatty acids with medium length hydrocarbon chains (C_6 − 10) are oxidized by medium chain acyl-CoA dehydrogenase (MCAD; encoded by Acadm). Reduced expression of MCAD has been observed in failing human and canine myocardial tissue [24–26], and cardiac abnormalities have been documented in infants with MCAD deficiency [27–29]. Several lines of evidence suggest that increasing MCAD expression in the heart may be beneficial. We identified MCAD as a gene that was upregulated in a mouse model of cardiac protection (due to increased phosphoinositide 3-kinase [PI3K] signaling) and downregulated in a mouse model with decreased PI3K signaling [30], which is more susceptible to diabetic cardiomyopathy [31]. Further, MCAD expression was positively correlated with cardiac function in PI3K transgenic mice subjected to myocardial infarction (i.e. mice with higher fractional shortening had higher Acadm expression, while mice with poor systolic function had lower levels of Acadm expression [30]). Supplementation with medium chain fatty acids increased MCAD expression and ameliorated cardiac remodeling in a rat model of hypertension [32] and prevented hypertrophy induced by arginine vasopressin treatment in H9c2 cells [33]. Furthermore, gene delivery of Acadm using cardiac-selective adeno-associated viral vectors (AAV) promoted physiological cardiac hypertrophy in mice and had favorable effects in a mouse model of pressure overload with established cardiac remodeling and dysfunction (less cardiac fibrosis, higher sarco(endo)plasmic reticulum calcium-ATPase 2 expression) [34].

The diabetic heart undergoes profound changes in lipid metabolism [35–38], but to date, the effect of manipulating MCAD abundance in the diabetic heart has not been established. A recent study showed that increasing fatty acid oxidation in the heart protected against cardiac dysfunction induced by obesity in adult mice [16]. Thus, we hypothesized that increasing MCAD expression in the diabetic heart using AAV mediated gene delivery would improve cardiac function and reduce pathology. Administration of cardiac-selective adeno-associated viral vectors encoding MCAD (rAAV6:MCAD) in an established model of type 1 diabetic cardiomyopathy did not improve diabetes-induced diastolic dysfunction or alter the expression of key metabolic genes (Ppargc1a, Ppara, Cpt1b, Slc2a4) or proteins (OXPHOS complexes). We observed an inverse correlation between MCAD expression and perilipin 5 levels, suggesting that altered MCAD expression may have an impact on lipid accumulation in the diabetic heart. Another important observation from this study was that of reduced AAV transduction efficiency in the diabetic heart. This finding may have important implications for the translation of AAV gene therapies into the clinic.

Experimental animals

All experiments using animals were conducted in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes (National Health & Medical Research Council of Australia, 8th Edition, 2013). All animal procedures and care were approved by the Alfred Research Alliance Animal Ethics Committee (Animal Ethics Committee Approved Project Number E/1867/2018/B). Wildtype mice on a FVB/NJ background (The Jackson Laboratory, Bar Habor, MA, U.S.A) were bred and housed onsite, in a macroenvironmental temperature and humidity-controlled environment (18–23°C, 40–70% humidity) with a 12:12 hr light-dark cycle. All of the mice had access to water and a standard chow diet ad libitum (Specialty Feeds, Western Australia, Australia). Mouse models of diabetes typically urinate more, thus, to reduce excessive soiling of bedding, lower housing densities were used for diabetic mice (2–3 mice per cage). Additionally, all mice were supplied with a cellulose based bedding (ALPHA-Dri®) for superior absorbency, cages were changed more frequently (3–5 times per week), and water supply checked daily. To minimize distress and promote normal behaviors, enrichment was provided to the cages, such as plastic mouse houses or igloos and soft nesting material (e.g. facial tissues, shredded paper, soft cardboard rolls).

Induction of diabetes and study design

At six weeks of age, aged matched male mice were randomly allocated into non-diabetic and diabetic groups. Each day prior to administration of streptozotocin (STZ) (AdipoGen Life Sciences, San Diego, CA, U.S.A, batch number LOTA00514) or citrate buffer vehicle, mice were fasted for 4–6 hrs. Following fasting, mice received five consecutive daily intraperitoneal (i.p.) injections of citrate buffer vehicle (non-diabetic) or STZ (55 mg/kg in 0.02 mol/L citrate buffer, pH 4.5) to induce diabetes and were monitored for 16 weeks (Figure S1). Confirmation of diabetes was performed by measuring blood glucose in conscious mice every 2 to 4 weeks using a glucometer (mice were considered diabetic if blood glucose concentrations exceeded 28 mmol/L) (Figure S1). After eight weeks of untreated diabetes, mice were randomized into AAV treatment groups by an independent researcher using a computer-generated random number program (RAND function in Microsoft Excel, [39]). Mice were subsequently administered with either a recombinant AAV vector (serotype 6) encoding a FLAG-tagged MCAD (rAAV6:MCAD) [34] or no translated product (empty vector control, referred to as rAAV6:CON). A dose of 5×10¹¹ vector genomes (vg) in a total volume of 150 µL saline was injected intravenously via the tail vein using a 29 G syringe as previously described [40] (Figure S1). After AAV administration, mice were monitored for a further eight weeks. Diastolic function was assessed by echocardiography prior to AAV administration (8 weeks after the induction of diabetes) and at endpoint (8 weeks after AAV administration, Figure S1). Consolidated Standards of Animal Experiment ReporTing (CONSAERT) flow charts for reporting animal numbers and exclusions are provided in Figure S2.

Assessment of hyperglycemia

Hyperglycemia (> 28 mmol/L) was confirmed from blood collected via a tail vein prick from conscious non-diabetic and diabetic mice. Glucose was measured using a glucometer (Accu-Chek® Performa, Roche, Basel, Switzerland). The upper detection limit of the glucometer was 33.3 mmol/L. Readings at or above this value (displayed as “HI” on the glucometer) were recorded as 33.3 mmol/L. At endpoint, glycated hemoglobin (HbA1c) was assessed on a Cobas b 101 machine (Roche) from blood collected by cardiac puncture. The normal range for HbA1c is 4-5.6% and < 42 mmol/mol. Mice administered STZ were excluded from further study if they did not develop diabetes (i.e. blood glucose did not exceed 28 mmol/L and HbA1c readings did not exceed 5.6% and 42 mmol/mol) (see CONSAERT flow charts, Figure S2).

Adeno-associated viral vector generation

The rAAV6:MCAD and rAAV6:CON vectors were produced as previously described [34]. The titre of purified vector stocks was determined by RT-qPCR using primers for the CMV promoter: forward, 5-gcggtaggcgtgtacggtgg-3’; reverse, 5’-cgtggatggcgtctccaggc-3’.

Assessment of Left ventricular (LV) structure and function

All ultrasound images were acquired from anaesthetized mice (ketamine/xylazine/atropine, KXA: 80/8/0.96 mg/kg, i.p.) by a single trained individual from the Preclinical Cardiology Microsurgery and Imaging Platform (Baker Heart and Diabetes Institute, Melbourne, Australia), who was blinded to disease and AAV treatment. Images were acquired using a MS550D transducer and a Vevo 2100 High Frequency Ultrasound System (Visual Sonics, Toronto, Canada) as previously described [41]. Echocardiographic evaluation of diastolic function was performed by measurement of transmitral flow parameters including early (E) and late (A) diastolic filling velocities, E/A ratio, deceleration time (DT), and isovolumic relaxation time (IVRT) from an apical four chamber view with pulsed wave Doppler. Tissue Doppler imaging was also performed to obtain early (e’) and late (a’) diastolic mitral annular velocity (e’/a’ ratio) and E/e' ratio to estimate LV filling pressures. All data were analyzed blinded offline using the Vevo Lab Software (Version 3.2.6, Visual Sonics) and independently validated (blinded) [42].

Tissue collection

At study endpoint, mice were culled by exsanguination under anesthesia (KXA: 100:10:1.2 mg/kg, i.p.). Tissues were excised whole, rinsed in 1x PBS, patted dry, weighed and snap frozen in liquid nitrogen for storage at -80°C. For the heart, the atria were removed, weighed and snap frozen. The ventricles were cut along the short axis. The base was fixed in cold 4% PFA for processing and embedding in paraffin, and the apex portions snap frozen for protein and RNA analysis. To determine tibia bone length, hind legs were removed, submerged in 1M NaOH and incubated at 37°C overnight. Tibias were cleaned, rinsed in dH₂O, air dried and measured using a Vernier caliper.

Protein isolation

For protein lysates, frozen mouse ventricles were homogenized in a lysis buffer (10% glycerol, 137 mM NaCl, 20 mM Tris/HCl (pH 7.4), 20 mM NaF, 10 mM EDTA, 1 mM EGTA, 1 mM sodium pyrophosphate, 1 mM vanadate, 1 mM PMSF, 4 µg/mL pepstatin, 4 µg/mL aprotinin, 4 µg/mL leupeptin) using a homogenizer (PRO Scientific, Model Pro 200) and total protein concentration was determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, U.S.A).

Western blotting

Protein lysates (60 µg) were separated by SDS/PAGE, blotted on to PVDF membrane (Merck, Frankfurt, Germany), incubated with primary antibody at 4°C overnight. The next day, blots were washed in 1x TBST and incubated with HRP-conjugated secondary antibodies for 1.5 hrs at room temperature. Blots were incubated in chemiluminescent substrate (ThermoFisher Scientific #34580) and chemiluminescence was detected on a ChemiDoc™ MP Imaging System with Image Lab™ Software (Bio-Rad Laboratories). Signals were quantified using ImageJ 1.44p pixel analysis (NIH Image Software, Bethesda, MD, U.S.A) and data expressed as fold change relative to control. Blots were probed with antibodies for FLAG (Cell Signaling Technologies, Danvers, MA, U.S.A, #2368, 1:1500,), α-tubulin (Cell Signaling Technologies, #2144, 1:2500), MCAD (Santa Cruz Biotechnology, Dallas, TX, U.S.A, sc-49046, 1:1000), perilipin (PROGEN Biotechnik GmbH, Heidelberg, Germany, GP31, 1:5000), OXPHOS cocktail (Mitosciences, Eugene, OR, U.S.A; MS604, 1:5000) or vinculin (Sigma-Aldrich, St. Louis, MO, U.S.A, V9131, 1:2500). The Precision Plus Protein™ All Blue Prestained Protein Standards (Bio-Rad, #1610373) was used as a molecular weight marker. Full-unedited blots are included in the Supplementary Information.

RNA isolation

Total RNA was isolated from frozen ventricles using TRI reagent (Sigma–Aldrich, St. Louis, MO, U.S.A). Briefly, ventricles were simultaneously disrupted in 500 µL of TRI reagent using the TissueLyser II Bead Mill (Qiagen, Düsseldorf, Germany) with stainless steel beads. Following centrifugation, 100 µL of chloroform was added to cause phase separation and the aqueous phase containing RNA was collected. RNA was recovered by precipitation with 2-propanol overnight at -20°C. RNA was quantified on a Nanodrop™ Spectrometer (ThermoFisher Scientific, Waltham, MA, U.S.A).

Quantitative real-time PCR (qPCR)

For gene expression analysis, 2 µg of total RNA was reverse transcribed using the Applied Biosystems™ High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) according to manufacturer’s recommendations. The reverse transcription protocol consisted of 10 min at 25°C, 120 min at 37°C, and 5 min at 85°C. Following cDNA synthesis, heart cDNA was diluted to a final concentration of 25 ng/uL and stored at -20°C. qPCR was performed on an Applied Biosystems Quant Studio 7 Flex real-time PCR instrument using TaqMan® Fast Advanced Master Mix and TaqMan® Gene Expression Assays (20X primer/probe mix, see Table S1 for a list of assays) according to manufacturer’s recommendations. Hypoxanthine phosphoribosyltransferase 1 (Hprt1) was used to standardize for cDNA concentration. The relative changes in gene expression from real-time qPCR experiments was analyzed using the 2^−ΔΔCt method of quantification.

To quantify the amount of mouse and human Acadm within the same sample, primers spanning a highly homologous region between the mouse and human Acadm sequence were designed using the Primer3 database (National Library of Medicine, Bethesda, MD, U.S.A; see Table S2 for primer sequences). Primers were synthesized by Integrated DNA technologies (Coralville, IA, U.S.A). qPCR was performed on a real-time PCR instrument (Applied Biosystems Quant Studio 7 Flex) using SYBR™ Green PCR Master Mix according to manufacturer’s recommendations. Hypoxanthine phosphoribosyltransferase 1 (Hprt1) was used to standardize for cDNA concentration. Standard curve and melt curve analysis was performed, confirming comparable primer efficiencies and a single peak in the melt curve. Relative changes in Acadm gene expression was analyzed using the 2^−ΔΔCt method of quantification.

Assessment of heart histology and immunofluorescence

The ventricles of mouse hearts were fixed in 4% paraformaldehyde at 4°C overnight, placed into a pathology cassette and dehydrated through a series of graded ethanol prior to embedding in paraffin (Monash Histology Platform, Monash University, Australia). Cardiac collagen deposition/interstitial fibrosis was assessed by staining 6 µM sections with Masson’s trichrome (Monash Histology Platform, Melbourne, Australia). To determine LV fibrosis, a custom script was written in the FIJI distribution of ImageJ to quantify the number of blue pixels and total pixels in LV sections captured using a 10x objective lens on an Olympus BX61 light microscope coupled to an Olympus DP80 camera.

Statistical analyses

Statistical analyses were performed using GraphPad Prism (Version 9.3.0, GraphPad Software, San Diego, CA, U.S.A). Results are presented as means ± SEM (unless stated otherwise in the figure legend). Differences between groups were identified using 2-way analysis of variance (ANOVA), followed by Fisher’s post-hoc test as appropriate. A 2-way repeated ANOVA was used when one factor was repeated. Unpaired t-tests or Mann-Whitney tests were used when comparing two groups for a single measure. Exact P-values are reported (to 2 decimal places for P-values larger than 0.01 and 3 decimal places for P-values between 0.01 and 0.001, as per New England Journal of Medicine guidelines). All relative units are expressed as a fold change with the relevant control group normalized to 1.

Administration of rAAV6:MCAD increases cardiac MCAD expression in diabetic and non-diabetic mice

To induce diabetes, 6-week old FVB/NJ male mice were administered 5 consecutive daily injections of STZ (Supp Figure 1). This protocol induces diastolic dysfunction in mice [41,43], which was evident by pulse wave and tissue Doppler echocardiography 8 weeks post-STZ injection in the current study (Figure 1a, Table 1). Diabetic mice had lower E/A and e’/a’ ratios and longer deceleration and isovolumic relaxation times (Figure 1a, Table 1) of impaired relaxation. E/e’ ratio, a marker of diastolic stiffness, was also higher in diabetic mice (Figure 1a, Table 1).

Blood glucose was >28 mmol/L 8 weeks post-STZ in all mice included in the diabetic cohort (Figure 1b), when mice were randomized to receive an intravenous injection of rAAV6:CON or rAAV6:MCAD. Blood glucose levels remained elevated until study endpoint and were unaffected by rAAV6:MCAD treatment (Figure 1b). HbA1c levels were also elevated at study endpoint (16 weeks post-STZ injection) and did not differ between rAAV6:CON and rAAV6:MCAD groups (Figure 1c). Consistent with a diabetic phenotype and prior studies [41], organ weights (wet weight and normalized to body weight or tibia length) of kidney, liver and spleen were increased in diabetic mice. These organ weights were not different between mice administered rAAV6:CON or rAAV6:MCAD (Table 2).

To confirm that injection of rAAV6:MCAD led to expression of the FLAG-MCAD construct in the heart, we performed qPCR on RNA extracted from ventricles of mice dissected at study endpoint. We used Taqman assays to detect endogenous Acadm transcripts (mouse Acadm; Figure 1d) or the exogenous construct(human Acadm; Figure 1e), and a SYBR Green assay to amplify both mouse and human transcripts within the same sample (total Acadm; Figure 1f). Diabetes was associated with a small (~10-20%) increase in Acadm expression (Figure 1d). Human Acadm transcripts, corresponding to the FLAG-MCAD construct, were readily detectable in ventricles from both non-diabetic and diabetic mice administered rAAV6:MCAD (Figure 1e). Interestingly, expression of the AAV-delivered construct was significantly lower in diabetic compared with non-diabetic hearts (Figure 1e). This may reflect reduced uptake in the diabetic myocardium due to compromised vasculature [44], or enhanced clearance of AAV genomes in the diabetic myocardium [45]. Nevertheless, in both the non-diabetic and diabetic cohorts, there was a significant increase in total Acadm expression with rAAV6:MCAD treatment (~13-fold increase in non-diabetic rAAV6:MCAD vs rAAV6:CON, P<0.001; ~2.5-fold increase in diabetic rAAV6:MCAD vs rAAV6:CON, P<0.04; Figure 1f).

FLAG-MCAD was readily detectable in ventricular lysates of rAAV6:MCAD-treated hearts by Western blot analysis (Figure 1g). As with the mRNA expression data, protein expression of FLAG-MCAD was higher in non-diabetic compared with diabetic animals (Figure 1h). There was a strong correlation between mRNA and protein expression of FLAG-MCAD (Figure 1i), demonstrating that the difference in expression between non-diabetic and diabetic groups is most likely due to a difference in uptake or post-transduction transcription, rather than translation.

Increased expression of MCAD did not ameliorate diastolic dysfunction in diabetic mice

Having confirmed diastolic dysfunction in the diabetic cohort prior to AAV administration (i.e. 8 weeks post-STZ injection; Figure 1a), we compared diastolic function between the four experimental groups at study endpoint. Representative mitral flow patterns from pulse wave and tissue Doppler imaging are shown in Figure 2a. By 2-way ANOVA, there was a significant effect of diabetes, but not of AAV treatment, for each of the parameters measured (E/A ratio, Figure 2b; e’/a’ ratio, Figure 2c; deceleration time, Figure 2d; IVRT, Figure 2e; and E/e’ ratio, Figure 2f; also see Table 1).

Increased expression of MCAD did not attenuate diabetes-induced cardiac pathology

Gross heart weight (normalized to tibia length, Figure 3a, Table 2) was not altered by diabetes (P=0.07 by 2-way ANOVA) or rAAV6:MCAD administration (P=0.56). Both atrial weight and lung weight (normalized to tibia length, Figure 3a, Table 2) were increased in diabetic mice compared to non-diabetic mice, but did not differ between the rAAV6:CON and rAAV6:MCAD groups. We did not detect an increase in collagen deposition in LV cross-sections from diabetic animals by Masson’s Trichrome staining (Figure 3b), suggesting that increased stiffness in diabetic hearts (increased E/e’, Figure 1a & 2f) may be due to differences in the intrinsic contractile properties of the cardiomyocytes, rather than interstitial fibrosis. Finally, in hearts of diabetic mice we observed increases in the expression of genes that are typically altered in settings of pathological cardiac remodeling, including atrial natriuretic peptide (ANP, encoded by Nppa), b-type natriuretic peptide (BNP, encoded by Nppb) and β-myosin heavy chain (βMHC, encoded by Myh7). Treatment with rAAV6:MCAD did not attenuate the expression of these cardiac stress markers (Figure 3c).

Diabetes alters the cardiac metabolic landscape

In our model, expression of Ppargc1a and Ppara were both unaffected by diabetes or rAAV6:MCAD treatment (Figure 4a). Consistent with other studies [36,46-48], diabetes was associated with an increase in the expression of the mitochondrial membrane fatty acyl-CoA transporter Cpt1b, and a decrease in the expression of the plasma membrane glucose transporter, Slc2a4 (encoding GLUT4). However, expression of both of these genes was unaffected by rAAV6:MCAD treatment (Figure 4a).

Decreased activity of OXPHOS complexes has been reported in the cardiac mitochondria in a rat model of T1D [49] and MCAD has been shown to physically associate with the oxidative phosphorylation (OXPHOS) supercomplex [50]. Therefore, we examined OXPHOS protein expression in the hearts of control and rAAV6:MCAD non-diabetic and diabetic mice. Diabetes led to a significant decrease in the expression of complex I (NDUFB8) and complex IV (MTCO1) also tended to be lower in diabetic compared with non-diabetic animals (Figure 4b). However, rAAV6:MCAD treatment had no effect on the expression of complexes I-V.

MCAD expression correlates with perilipin expression

Finally, we investigated the impact of diabetes and rAAV6:MCAD treatment on lipid accumulation by quantifying perilipin 5 (PLIN5) expression by Western blot analysis. PLIN5 associates with lipid droplets [51,52] and is increased in the diabetic heart [46,53]. In the current study, diabetes induced a robust increase in cardiac PLIN5 expression (Figure 4c). PLIN5 in diabetic mice treated with rAAV6:MCAD tended to be lower than in diabetic mice treated with rAAV6:CON, but this was not statistically significant (P=0.16 by unpaired t-test). PLIN5 expression was more variable in the diabetic rAAV6:MCAD group (standard deviation 41% of mean) compared with the diabetic rAAV6:CON group (standard deviation 25% of mean), prompting us to determine whether PLIN5 levels correlated with FLAG-MCAD expression. Correlation analysis revealed that there was a strong inverse correlation between FLAG-MCAD expression and PLIN5 expression in the diabetic rAAV6:MCAD hearts (r=0.73, P=0.003; Figure 4c), suggesting that rAAV6:MCAD treatment may reduce lipid droplet burden in the diabetic heart.

Increasing MCAD levels as a therapeutic strategy for the diabetic heart

The aim of this study was to determine if increasing expression of MCAD, a key regulator of fatty acid oxidation, could improve diastolic function and reduce pathology in mice with diabetic cardiomyopathy induced by STZ injection. Administration of cardiac-selective adeno-associated viral vectors encoding MCAD (rAAV6:MCAD) led to an increase in MCAD expression, but did not improve diabetes-induced diastolic dysfunction or markers of pathological cardiac remodeling (Nppa, Nppb, Myh7). Although our experimental approach was clinically relevant, as we initiated rAAV6:MCAD treatment after the onset of diastolic dysfunction, it is possible that we may have seen a beneficial effect had we adopted a preventative approach, similar to other studies [54,55]. The age of mice at treatment with rAAV6:MCAD was 14-weeks old, representing a human in their early twenties [56]. Given that children with T1D can display signs of reduced diastolic function as early as 8 years of age (irrespective of the duration of diabetes) [57], treatment at an earlier time-point may also be warranted.

Favorable effects of rAAV6:MCAD treatment on lipid accumulation in the diabetic heart

Similar to previous reports [36,46-48], we found that the expression of key metabolic genes and proteins was altered in the diabetic heart. We observed an increase in the expression of Cpt1b (encoding CPT1B) and a decrease in the expression of Slc2a4 (encoding GLUT4) with diabetes, reflecting increased reliance of the diabetic heart on fatty acids as a fuel source. Interestingly, these gene expression changes were not accompanied by an increase in the expression of Ppara (PPARa), a master regulator of cardiac fatty acid uptake and oxidation pathways. Consistent with our findings, Ppara mRNA expression was also not different between T1D diabetic and non-diabetic rat hearts [36]. PPARa has been postulated to play a key role in the development of diabetic cardiomyopathy, as cardiac-specific overexpression of PPARa in mice induced a metabolic phenotype reminiscent of the diabetic heart [58] and exacerbated lipid accumulation and cardiac dysfunction induced by STZ injection [59], while cardiac-specific knockout of PPARa prevented fasting-induced cardiac lipid accumulation [60]. In our study, it is possible that Ppara mRNA expression increased to induce changes in Cpt1b and Slc2a4 expression but returned to control levels by study endpoint. In addition, post-translational modifications may play an important role in regulating PPARa activity in the diabetic heart [61].

We also observed lower protein levels of OXPHOS complexes I and IV in hearts of diabetic vs non-diabetic mice, suggesting impaired function of the mitochondrial respiratory chain. Reduced expression of OXPHOS genes has been observed in human T2D skeletal muscle [62,63], and protein levels of complexes IV and V were significantly reduced in T1D rat hearts [49]. We hypothesized that increasing MCAD levels might have favorable effects on OXPHOS expression, as acyl-CoA dehydrogenases, including MCAD, physically associate with OXPHOS supercomplexes to facilitate substrate channeling and electron transfer [50]. However, we did not observe a significant effect of rAAV6:MCAD treatment on protein expression of any of the OXPHOS complexes.

Lipid accumulation is a key feature of the diabetic heart that is thought to contribute to cardiac dysfunction [22]. As hearts were embedded in paraffin at the time of tissue harvesting, we were unable to assess lipid accumulation directly, e.g. by oil red O staining. However, Western blot analysis of PLIN5, a protein that associates with lipid droplets, revealed a trend for reduced PLIN5 in diabetic rAAV6:MCAD versus diabetic rAAV6:CON hearts, and there was a strong negative correlation between FLAG-MCAD and PLIN5 protein expression. Perilipins localize to the phospholipid surface of lipid droplets and play key roles in triacylglycerol storage and lipolysis [64]. PLIN5 and PLIN2 are the predominant isoforms that associate lipid droplets in cardiomyocytes [60,65-69]. Our finding of reduced PLIN5 levels in hearts with greater FLAG-MCAD expression suggest that rAAV6:MCAD treatment can reduce overall lipid accumulation in the diabetic heart, although the effect was mild and was not sufficient to improve diastolic function. An 8-week exercise training program initiated 2 weeks after the induction of diabetes in rats prevented cardiac lipid accumulation, and this was associated with preserved diastolic and systolic function [70]. Thus, therapeutic strategies that have a more profound effect on reducing cardiac steatosis are likely to be more successful than rAAV6:MCAD gene therapy at improving outcomes in settings of diabetes.

Reduced AAV transduction efficiency in the diabetic myocardium

An important observation from the current study was that expression of the AAV-delivered MCAD construct was significantly lower in diabetic hearts compared with non-diabetic hearts, at both the mRNA and protein level. This was not due to a batch effect (the same AAV stock was used for non-diabetic and diabetic animals within each experimental cohort) or the order in which animals were injected with the AAV. In reviewing the literature to determine if reduced transduction had been observed previously in diabetic rodent models, we found that: although studies could show effective and robust expression of AAV-delivered constructs in the diabetic heart, either i) studies did not include the necessary controls to compare the transduction efficiency between diabetic and non-diabetic animals [55,71,72]; or ii) expression of the AAV-delivered construct was unable to be detected at the protein level due to the unavailability of suitable antibodies [73]; or iii) quantitation of the AAV-delivered construct in non-diabetic and diabetic animals was not directly compared or performed [74,75]. Consistent with our findings, Prakoso and colleagues reported significantly less cardiac mRNA and protein expression of their target gene in type 2 diabetic hearts compared with non-diabetic control hearts following AAV delivery [45]. The authors speculated that this may be due to a loss of genomes in the diabetic heart over time and suggested using a higher dose of AAV. In the current study, we adopted a higher dose of AAV (5 x 10¹¹vg vs. 2 x 10¹¹vg of Prakoso et al [45]) but still observed significantly lower expression of our AAV-delivered construct in diabetic hearts compared with non-diabetic control hearts. This may reflect reduced uptake of AAVs in the diabetic myocardium due to compromised vasculature (e.g. vascular rarefaction [44]) or increased uptake in non-cardiac tissues, a loss of AAV genomes over time (as suggested by Prakoso and colleagues [45]), or dysregulation of transcriptional/translational processes in the diabetic heart that reduce its capacity to express AAV genomes [76]. Further research is needed to elucidate the mechanisms of AAV uptake and gene expression in the diabetic heart, as this may have significant clinical implications for the use of AAVs as gene therapy tools in patients with diabetes.

Limitations and clinical challenges

Males and females respond to drugs and disease differently [77-80], and diabetic cardiomyopathy is no exception [81,82]. The Framingham Heart study [83], and subsequent meta-analysis [84], revealed that women with diabetes are more likely to develop heart failure or suffer a fatal cardiovascular event compared to diabetic men. Further, diastolic dysfunction and significant adverse changes in cardiac structure were reported in children and adolescents with type 1 diabetes, with girls more affected than boys [85]. Despite this, many preclinical studies [54,73,75], including the current study, only include male mice. Female mice are less sensitive to STZ-induced b-cell toxicity (see [86] for review), although a higher dose can be used to induce diabetes and increase blood glucose and HbA1c levels to the same levels as male mice [87]. Interestingly, female diabetic mice were more susceptible to diastolic dysfunction compared with male mice, despite lower hyperglycaemia [88]. It is critical that female animals are included in preclinical studies for successful translation of new therapies into the clinic [89]. A sex-dependent response to a metabolic intervention was recently reported in a mouse model of chronic pressure overload-induced heart failure [90], which has implications for future therapies targeting metabolic pathways.

AAVs are promising gene therapy tools due to their safety profile, versatility, low immunogenicity, ease of manufacturing, and ability to achieve sustainable and stable exogenous gene expression. The number of AAV gene therapies approved for clinical use is growing [91], with the most recent being approved in November 2022 [92]. Despite this, there has been limited clinical success using AAVs to treat cardiovascular disease (see reviews [5,93]). Further, AAV-based therapies are currently very expensive to manufacture (Hemgenix to treat patients with hemophilia B costs US $3.5 million per patient [92]) and would be unaffordable, especially in low- and middle-income countries. Though, with methodological advancements, it is hoped costs will dramatically fall [93]. Our study revealed that expression of AAV-delivered constructs was significantly lower in diabetic hearts compared with non-diabetic hearts. Thus, very high doses of AAV may be required in settings of diabetic heart failure, making therapy even more expensive or scale-up production more difficult [91,94,95]. Enhanced interventions that may reduce costs and improve transduction of the myocardium may come through the development of physical targeting strategies that maximize delivery of vectors to the tissue of interest (e.g. cardiac perfusion strategies [96]), the development of stronger cell-specific promoters, and the development of modified AAV capsids (e.g. directed evolution strategies) that have been optimized for transduction of diseased cardiomyocytes [97-100].

Increasing cardiac MCAD expression using AAV gene therapy at a dose of 5 x 10¹¹ vg for 8 weeks did not improve diabetes-induced diastolic dysfunction nor alter the metabolic profile of key metabolic genes or OXPHOS proteins in a mouse model of established diabetic cardiomyopathy induced by STZ. MCAD expression was inversely correlated with PLIN5 protein levels, suggesting that rAAV6:MCAD treatment reduced lipid accumulation in the diabetic heart. However, the effect size was small, and other interventions (such as exercise training, or gene therapies combined with exercise training) are likely to confer greater therapeutic benefit for patients with diabetic cardiomyopathy [101-103]. This study also revealed that expression of AAV-delivered constructs is significantly lower in the diabetic heart compared with the non-diabetic heart. This may be due to loss of AAV genomes in the diabetic heart over time, reduced transduction efficiency of AAVs in diabetic vs non-diabetic myocardium, or dysregulation of AAV genome transcription/translation in the diabetic heart. For an AAV gene therapy for diabetic heart disease to progress to the clinic, additional studies are required to investigate the mechanisms of AAV uptake and expression in the diabetic myocardium, and to determine the most efficient AAV serotype, optimal dose and route of delivery.

Acknowledgements

We wish to thank Ms Stephanie Jansen (Alfred Research Alliance Animal Facility) for performing the AAV tail vein injections, Ms Amy Hsu (Baker Heart and Diabetes Institute) for technical assistance, and Dr Hongwei Qian (University of Melbourne) for producing the AAV vectors.

Funding

This work was supported by a grant to B.C.B and K.L.W from the Diabetes Australia Research Program (Y19G-BERB), Women in Science Grants from the Baker Heart and Diabetes Institute (to B.C.B), and the Victorian Government’s Operational Infrastructure Support Program. B.C.B is supported by a Baker Fellowship (Baker Heart and Diabetes Institute, Australia). K.L.W is supported by a Future Leader Fellowship from the National Heart Foundation of Australia (award ID 102539). N.M.S. is supported by a Research Training Program scholarship from Monash University. P.G, L.M.D.D. and J.R.M are supported by funding from the National Health and Medical Research Council of Australia.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

Conceptualization: B.C.B, K.L.W; Methodology: B.C.B, K.L.W; Investigation: B.C.B, K.L.W, H.K, N.M.S, A.J.A.R, C.A.H, G.S.Y; Formal Analysis: B.C.B, K.L.W, H.K, N.M.S, A.J.A.R; Writing - original draft preparation: K.L.W, B.C.B; Writing - review and editing: K.L.W, H.K, P.G, L.M.D.D, J.R.M, B.C.B; Funding acquisition: B.C.B, K.L.W; Resources: P.G, L.M.D.D, J.R.M; Supervision: B.C.B, K.L.W, J.R.M. Project Administration: B.C.B. All authors read and approved the final manuscript.

Data Availability Statement

Data generated or analyzed during this study are included in this published article (and its supplementary information files).

Ethics approval

This study does not involve human subjects. All experiments using animals were conducted in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes (National Health & Medical Research Council of Australia, 8th Edition, 2013). All animal procedures and care were approved by the Alfred Research Alliance Animal Ethics Committee (Animal Ethics Committee Approved Project Number E/1867/2018/B).

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Table 1: Transmitral Doppler echocardiography data from non-diabetic and diabetic mice.

	8 WEEKS POST DIABETES						ENDPOINT
							Non-diabetic						Diabetic
	Non-diabetic			Diabetic			rAAV6:CON			rAAV6:MCAD			rAAV6:CON			rAAV6:MCAD
No. of animals	12			22			6			6			9			13
Body weight (g)	29.5	±	0.9	31.6	±	0.3*	29.1	±	1.1	29.8	±	1.5	31.5	±	0.5	31.6	±	0.4

*Pulsed Wave Doppler*
Heart rate (bpm)	372	±	9	377	±	6	348	±	10	343	±	9	372	±	8*	367	±	5*
E (mm/s)	800.4	±	23.5	750.7	±	15.1	784.6	±	31.1	802.8	±	31.1	747.7	±	19.4	770.4	±	23.1
A (mm/s)	318.6	±	14.4	407.8	±	16.6*	271.6	±	15.6	323.0	±	14.2	411.5	±	19.1*	402.9	±	12.9*

*Tissue Doppler*
Heart rate (bpm)	373	±	9	376	±	7	343	±	10	345	±	8	378	±	10*	367	±	5*
e' (mm/s)	32.3	±	0.9	24.6	±	0.8*	34.0	±	1.6	30.9	±	2.2	27.6	±	1.9*	24.6	±	1.0*
a' (mm/s)	13.5	±	0.5	18.2	±	0.8*	12.6	±	0.6	11.8	±	0.7	19.1	±	1.6*	15.7	±	0.8*

Abbreviations: A wave, peak late mitral inflow wave velocity; a’, peak late mitral annular tissue velocity; E wave, peak early mitral inflow wave velocity; e’, peak early mitral annular tissue velocity. Data shown as mean ± SEM. Data at 8 weeks post diabetes analyzed by unpaired t-test. Data at endpoint analyzed by 2-way ANOVA. *P<0.05 vs. Non-diabetic. There was no significant effect of AAV treatment by 2-way ANOVA.

Table 2: Morphological and systemic characteristics of non-diabetic and diabetic mice at endpoint

	Non-diabetic						Diabetic
	rAAV6:CON			rAAV6:MCAD			rAAV6:CON			rAAV6:MCAD
No. of animals	6			6			11			13
Age (weeks)	23.2	±	0.6	23.1	±	0.5	23.2	±	0.5	23.3	±	0.5
HbA1c (IFCC mmol/mol)	20.3	±	0.3	20.0	±	0.0	82.9	±	4.1*	91.5	±	3.2*
Body weight (g)	28.6	±	1.1	28.7	±	1.1	30.9	±	0.5*	31.7	±	0.4*
Tibial length (mm)	16.4	±	0.1	16.4	±	0.1	16.5	±	0.1	16.6	±	0.1
Heart weight (mg)	123.7	±	2.8	120.4	±	3.8	127.3	±	2.5	128.8	±	2.5
Atrial weight (mg)	9.2	±	0.5	8.6	±	0.7	10.1	±	0.5*	10.4	±	0.3*
Lung weight (mg)	149.3	±	5.7	150.5	±	3.5	162.5	±	3.6*	166.2	±	2.5*
Liver weight (mg)	1182.0	±	38.9	1211.3	±	38.7	2011.5	±	48.1*	2062.8	±	49.4*
Kidney weight (mg)	463.9	±	13.2	438.2	±	16.7	691.7	±	26.4*	712.7	±	26.0*
Spleen weight (mg)	80.7	±	2.9	87.1	±	4.0	95.5	±	2.3*	101.2	±	2.9*
HW/BW (mg/g)	4.3	±	0.1	4.2	±	0.1	4.1	±	0.1	4.1	±	0.1
AW/BW (mg/g)	0.32	±	0.02	0.30	±	0.02	0.33	±	0.02	0.33	±	0.01
LW/BW (mg/g)	5.2	±	0.1	5.3	±	0.16	5.3	±	0.1	5.2	±	0.07
LivW/BW (mg/g)	41.4	±	0.9	42.3	±	1.28	65.3	±	1.8*	65.0	±	1.1*
KW/BW (mg/g)	16.3	±	0.4	15.3	±	0.32	22.4	±	0.7*	22.4	±	0.7*
SW/BW (mg/g)	2.8	±	0.1	3.0	±	0.14	3.1	±	0.1	3.2	±	0.09
LivW/TL (mg/mm)	72.2	±	2.0	73.9	±	2.0	122.1	±	2.7*	124.3	±	2.7*
KW/TL (mg/mm)	28.3	±	0.6	26.7	±	0.8	42.0	±	1.5*	42.9	±	1.5*
SW/TL (mg/mm)	4.9	±	0.1	5.3	±	0.2	5.8	±	0.1*	6.1	±	0.2*

Abbreviations: AW, atria weight; BW, body weight; HbA1c, hemoglobin A1c; HW, heart weight; IFCC, International Federation of Clinical Chemistry and Laboratory Medicine; KW, kidney weight; LW, lung weight; LivW, liver weight; SW, spleen weight; TL, tibial length.

Data are shown as mean ± SEM. Data analyzed by 2-way ANOVA. *P<0.05 vs. non-diabetic. There was no significant effect of AAV treatment by 2-way ANOVA.

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Cardiac-selective gene delivery of medium-chain acyl-CoA dehydrogenase (MCAD) does not protect against diabetes-induced cardiac dysfunction

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Abstract

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KEY MESSAGES

INTRODUCTION

MATERIALS AND METHODS

Experimental animals

Induction of diabetes and study design

Assessment of hyperglycemia

Adeno-associated viral vector generation

Assessment of Left ventricular (LV) structure and function

Tissue collection

Protein isolation

Western blotting

RNA isolation

Quantitative real-time PCR (qPCR)

Assessment of heart histology and immunofluorescence

Statistical analyses

RESULTS

DISCUSSION

CONCLUSION

Declarations

References

Tables

Supplementary Files

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