SERCA2a stimulation by istaroxime improves intracellular Ca2+ handling and diastolic dysfunction in a model of diabetic cardiomyopathy

Abstract

Aims Diabetic cardiomyopathy is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD) that precedes the development of systolic impairment. Mechanisms that can restore cardiac relaxation improving intracellular Ca2+ dynamics represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime has the dual properties to accelerate Ca2+ uptake into sarcoplasmic reticulum (SR) through the SR Ca2+ pump (SERCA2a) stimulation and to inhibit Na+/K+ ATPase (NKA). This project aims to characterize istaroxime effects at a concentration (100 nmol/L) marginally affecting NKA, in order to highlight its effects dependent on the stimulation of SERCA2a in an animal model of mild diabetes. Methods and Results Streptozotocin (STZ) treated diabetic rats were studied at 9 weeks after STZ injection in comparison to controls (CTR). Istaroxime effects were evaluated in vivo and in left ventricular (LV) preparations. STZ animals showed 1) marked DD not associated to cardiac fibrosis, 2) LV mass reduction associated to reduced LV cell dimension and T-tubules loss, 3) reduced LV SERCA2 protein level and activity and 4) slower SR Ca2+ uptake rate, 5) LV action potential (AP) prolongation and increased short-term variability (STV) of AP duration, 6) increased diastolic Ca2+, and 7) unaltered SR Ca2+ content and stability in intact cells. Acute istaroxime infusion (0.11 mg/kg/min for 15 min) reduced DD in STZ rats. Accordingly, in STZ myocytes istaroxime (100 nmol/L) stimulated SERCA2a activity and blunted STZ-induced abnormalities in LV Ca2+ dynamics. In CTR myocytes, istaroxime increased diastolic Ca2+ level due to NKA blockade albeit minimal, while its effects on SERCA2a were almost absent. Conclusions SERCA2a stimulation by istaroxime improved STZ-induced DD and intracellular Ca2+ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment. Translational perspective Deficient sarcoplasmic reticulum (SR) Ca2+ uptake has been identified in cardiomyocytes from failing human hearts with impaired diastolic relaxation (e.g. diabetic hearts) and has been associated with a decreased SERCA2a expression and activity and/or with a higher SERCA2a inhibition by phospholamban. Thus, SERCA2a may represent a pharmacological target for interventions aimed at improving cytosolic Ca2+ compartmentalization into the SR to limit diastolic dysfunction pathologies. In this context, istaroxime is the first-in-class luso-inotropic agent targeting SERCA2a that has already demonstrated its efficacy in clinical trials and may be useful to clarify the relevance of SERCA2a stimulation in controlling cytosolic Ca2+ level.

Istaroxime metabolite PST3093 selectively stimulates SERCA2a and reverses disease-induced changes in cardiac function

“…In STZ rat preparations, baseline SERCA2a Vmax was lower (by -27%) than in healthy ones, with no difference in KdCa (Table S1), accordingly to our recent paper 13 . As also reported previously 19 , the analysis of compounds effects suggests a species-specific response of enzyme kinetics to modulation: whereas in rat preparations both PST3093 (Figure 2, Table S2) and istaroxime (Figure S2, Table S3) increased Vmax, in guinea-pig ones the compounds decreased KdCa instead (Table S3).…”

“…uptake under V-clamp conditions (Figure 3C-D), it failed to clearly affect CaT decay kinetic and CaSR content in field-stimulated myocytes (Fig 3A-B). This apparent discrepancy may result from failure to control the AP profile during field-stimulation measurements as previously described 13 . Overall, results in isolated myocytes are consistent with SERCA2a…”

“…INaK was recorded in isolated rat left ventricular (LV) myocytes as the holding current at -40 mV under conditions enhancing INaK and minimizing contamination by other conductances 13,16 .…”

“…A sample INaK recording is shown in Figure 1C; INaK inhibition curves for PST3093 and istaroxime are shown in Figure 1D. The estimated IC50 for INaK inhibition by istaroxime was 32 ± 4 µM (modified from 13 ); for PST3093 a 9.2 ± 1.1% reduction was observed at the limit concentration for solubility (100 µM), an effect that can be considered insignificant.…”

Hypoglycemia Induced Mitochondrial Connexin-43 Accumulation Aggravates Diabetic Cardiomyopathy

“…Compared to coronary artery disease induced dilated cardiomyopathy, DCM has worse prognosis [4,5]. Compared to other cardiomyopathies, DCM exhibit metabolic dysfunction, electrophysiology, and insulin resistance [6,7] -making DCM management much more challenging. Moreover, our understanding of the molecular underpinnings of DCM remains limited.…”