Phosphoinositide 3-kinase (p110α) gene delivery limits diabetes-induced cardiac NADPH oxidase and cardiomyopathy in a mouse model with established diastolic dysfunction

Abstract

Phosphoinositide 3-kinase [PI3K (p110α)] is able to negatively regulate the diabetes-induced increase in NADPH oxidase in the heart. Patients affected by diabetes exhibit significant cardiovascular morbidity and mortality, at least in part due to a cardiomyopathy characterized by oxidative stress and left ventricular (LV) dysfunction. Thus, PI3K (p110α) may represent a novel approach to protect the heart from diabetes-induced cardiac oxidative stress and dysfunction. In the present study, we investigated the therapeutic potential of a delayed intervention with cardiac-targeted PI3K gene therapy, administered to mice with established diabetes-induced LV diastolic dysfunction. Diabetes was induced in 6-week-old male mice by streptozotocin (STZ). After 8 weeks of untreated diabetes, LV diastolic dysfunction was confirmed by a reduction in echocardiography-derived transmitral E/A ratio. Diabetic and non-diabetic mice were randomly allocated to receive either recombinant adeno-associated viral vector-6 carrying a constitutively-active PI3K construct (recombinant adeno-associated-virus 6-constitutively active PI3K (p110α) (caPI3K) (rAAV6-caPI3K), single i.v. injection, 2 × 10 vector genomes) or null vector, and were followed for a further 6 or 8 weeks. At study endpoint, diabetes-induced LV dysfunction was significantly attenuated by a single administration of rAAV6-caPI3K, administered 8 weeks after the induction of diabetes. Diabetes-induced impairments in each of LV NADPH oxidase, endoplasmic reticulum (ER) stress, apoptosis, cardiac fibrosis and cardiomyocyte hypertrophy, in addition to LV systolic dysfunction, were attenuated by delayed intervention with rAAV6-caPI3K. Hence, our demonstration that cardiac-targeted PI3K (p110α) gene therapy limits diabetes-induced up-regulation of NADPH oxidase and cardiac remodelling suggests new insights into promising approaches for the treatment of diabetic cardiomyopathy, at a clinically relevant time point (after diastolic dysfunction is manifested).

Defining the Progression of Diabetic Cardiomyopathy in a Mouse Model of Type 1 Diabetes

“…Since the first study to characterize diabetic cardiomyopathy in diabetic patients in 1972, most human and animal studies have demonstrated that this relationship is primarily associated with the onset of diastolic dysfunction (Rubler et al, 1972;Wold et al, 2005). Previous studies in our laboratory in this same mouse model of T1D demonstrated diastolic dysfunction, in terms of reduced E/A and increased peak A wave velocity, deceleration time, and IVRT, is evident at 8 weeks of diabetes; however, now we reveal that some of these key markers of diastolic function are impaired earlier than we had previously reported (Huynh et al, 2010;De Blasio et al, 2015;Prakoso et al, 2017). We can now confirm that a reduction in LV Monocytes (%WBC) 4.3 ± 0.5 3.6 ± 0.6 3.6 ± 0.7 4.2 ± 0.6 4.0 ± 0.5 5.4 ± 0.5 6.0 ± 1.1* 5.6 ± 0.7* 4.0 ± 0.5 7.0 ± 1.2* Neutrophils (%WBC) 11.6 ± 1.7 16.9 ± 2.0 17.4 ± 1.5 18.5 ± 2.1 11.6 ± 2.3 10.6 ± 2.1 15.1 ± 1.5 17.4 ± 3.5 17.5 ± 2.4 10.4 ± 1.6…”

“…This then leads to structural and functional alterations, confirming hyperglycemia as a major mediator of diabetic cardiomyopathy (Fiordaliso et al, 2001;Tate et al, 2017). These observations have been confirmed in rodent models of both type 1 diabetes (T1D) and type 2 diabetes (T2D) (Evans et al, 2003;Huynh et al, 2012Huynh et al, , 2013De Blasio et al, 2015;Prakoso et al, 2017;Tate et al, 2017). Diastolic dysfunction, the most common functional deficit seen in the diabetic heart, is regarded as a consequence of this increased stiffening of the heart, attributed to morphological changes such as myocardial fibrosis as well as cardiomyocyte hypertrophy (van Heerebeek et al, 2008).…”

“…Positively stained apoptotic cells were stained blue while negatively stained cells were counterstained with Nuclear Fast Red. Apoptotic cells were quantified as a percentage of non-apoptotic cells and expressed as fold change from age-matched citrate control mice (20X magnification, 10 fields per image) Prakoso et al, 2017).…”

“…Cardiac gene expression of pro-hypertrophic markers β-myosin heavy chain (β-mhc), Nppa (atrial natriuretic peptide), the pro-fibrotic markers Ctgf (connective tissue growth factor), periostin (Postn) and Tgf-β, Nox2, Cd36, the macrophage marker Cd68, the rate-limiting enzymes for hexosamine biosynthesis, glutamine:fructose-6-phosphate amidotransferase (Gfat) 1 and 2, and O-GlcNAc-transferase (Ogt) and O-GlcNAc-ase (Oga), and the housekeeper 18S, were determined via real-time PCR using SYBR R Green (Applied Biosystems, Scoresby, VIC, Australia) using primers generated from murine sequences. Quantitative real-time analysis was performed as previously described using the C t method to detect relative fold differences of diabetic compared with non-diabetic mice (De Blasio et al, 2015Prakoso et al, 2017).…”

“…For many years, oxidative stress, the result of an imbalance of ROS production and antioxidant status, has been implicated in the development and progression of diabetic cardiomyopathy (Wold et al, 2005;Huynh et al, 2013;Prakoso et al, 2017). The increase in ROS generation observed in the diabetic heart results FIGURE 4 | Measures of inflammation.…”