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

Figure 1. Istaroxime affinity for rat NKA. Changes in SERCA2, PLN levels, and TT expression in STZ vs. CTR rats. (A) Top: recordings of NKA current (INKA) and Ion NaTRIUM Green-2 fluorescence (Hp −40 mV) during exposure to increasing concentrations of istaroxime and, finally, to 1 mmol/L ouabain (OUA). Bottom: concentration-dependent INKA inhibition by istaroxime in isolated CTR LV myocytes (the best logistic fit and confidence intervals are shown, N = 5, n = 6–27). Concentration-dependent NKA activity inhibition by istaroxime and OUA in cardiac preparations is shown in the inset (N = 5). (B) Left: western blot for SERCA2, monomeric (m) and pentameric (p) PLN, pSer16-PLN and pThr17-PLN in STZ (N = 6,7) and CTR (N = 5,6) cardiac homogenates. Right: densitometric analysis; values are expressed as optical density in arbitrary units. *P<0.05 vs. CTR (unpaired t-test). (C) Left: Ca2+ activation curves of SERCA2a activity measured as cyclopiazonic acid sensitive component in cardiac SR homogenates from CTR (N = 8) and STZ (N = 10) rats with or w/o 500 nmol/L istaroxime. Right: statistics of the maximum velocity (Vmax) of the Ca2+ activation curves estimated by sigmoidal fitting. *P<0.05 vs. CTR…

Figure 2. STZ-induced changes in Cai2+ dynamics in field stimulated myocytes. Analysis of istaroxime effects. (A) Left: post-rest potentiation protocol in Fluo4 field stimulated (2 Hz) myocytes; steady state Ca2+ transients (ssCaT) and superimposed first Ca2+ transients (1st CaT) following increasing resting pauses (1–5–10–20 s) are reported in CTR and STZ myocytes, with or w/o 100 nmol/L istaroxime. Traces were normalized to own diastolic Ca2+ (CaD) level (dotted lines). Right: analysis of the 1st CaT amplitude normalized to the amplitude of the pre-pause ssCaT and its pause-dependency. CTR N = 5 (w/o istaroxime n = 44, with istaroxime n = 34), STZ N = 3 (w/o istaroxime n = 35, with istaroxime n = 23). *P<0.05 vs. CTR w/o istaroxime; #P<0.05 vs. STZ w/o istaroxime (two-way ANOVA plus post hoc Sidak’s multiple comparisons). (B) ssCaT and caffeine-induced CaT evocated in field stimulated CTR and STZ myocytes with or w/o 100 nmol/L istaroxime (the dotted line indicates the CaD in CTR w/o istaroxime). (C) Statistics for ssCaD, ssCaT amplitude, caffeine-induced CaT (named CaSR), and ssCaT half decay time (t0.5). CTR N = 5 (n = 36 w/o istaroxime, n = 31 with istaroxime), STZ N = 7 (n = 52 w/o…

Figure 3. STZ-induced changes in electrical activity. Analysis of istaroxime effects. (A) Top: representative AP recorded at 1 Hz in CTR and STZ myocytes with or w/o 100 nmol/L istaroxime. Bottom: rate-dependency of AP parameters (APD50, APD90, Ediast) in CTR and STZ myocytes with or w/o 100 nM istaroxime. CTR N = 4 (n = 29 w/o istaroxime, n = 25 with istaroxime), STZ N = 3 (n = 24 w/o istaroxime, n = 19 with istaroxime). *P<0.05 vs. CTR w/o istaroxime, #P<0.05 vs. STZ w/o istaroxime (two-way ANOVA plus post hoc Sidak’s multiple comparisons). (B) Effects of 100 nmol/L istaroxime superfusion on Ediast (top, AP y axis zoomed to highlight changes) and Nai+ (bottom) in comparison to the effect of 1 mmol/L OUA in CTR myocytes loaded with Ion Natrium Green-2 and stimulated at 7 Hz. (C) Top: rate-dependency of APD90 STV in each experimental group. CTR N = 4 (n = 27 w/o istaroxime, n = 21 with istaroxime), STZ N = 3 (n = 24 w/o istaroxime, n = 20 with istaroxime). *P<0.05 vs. CTR w/o istaroxime (two-way ANOVA plus post hoc Sidak’s multiple comparisons). Bottom: linear correlation between STV of APD90 and APD90 values in CTR and STZ groups; data from all stimulation rates were pooled.

Figure 4. STZ-induced Cai2+ handling changes under control of membrane potential. Analysis of istaroxime effects. (A) Top: APs waveforms (CTR and STZ APs named short and long APs, respectively) and corresponding CaT evocated in V-clamped STZ myocyte through AP-clamp experiments (2 Hz). Caffeine-induced CaT (caff-CaT) and the corresponding NCX current (INCX) were recorded at −80 mV following steady state stimulation with short and long AP to estimate changes in SR Ca2+ content (CaSR). Bottom: statistics of CaT amplitude (N = 5, n = 30) and CaSR (integral of inward INCX, marked as striped area) (N = 5, n = 22) under short and long AP stimulation. Fluorescence signals were converted to free Ca2+ estimating Fmax in each cell. *P<0.05 vs. short AP (paired t-test). (B) Top: transmembrane currents and CaT simultaneously recorded in voltage-clamped cells (Hp −35 mV) from CTR and STZ myocytes with or w/o 100 nmol/L istaroxime. Bottom: statistics of CaT amplitude, CaSR, Ca2+ influx through L-type Ca2+ channel (CaL influx), and ER gain. CTR N = 3 (n = 22–24), STZ N = 5 (w/o istaroxime n = 26–33, with istaroxime n = 28). Fluorescence signals were converted to free Ca2+ estimating Fmax in each cell.…

Figure 5. STZ-induced changes in Ca2+ sparks rate and characteristics. Analysis of istaroxime effects. (A) Representative xt images showing Ca2+ sparks at resting in CTR and STZ myocytes with or w/o 100 nmol/L istaroxime. (B) Statistics of Ca2+ sparks characteristics and rate for each group. *P<0.05 vs. CTR; #P<0.05 vs. STZ w/o istarpoxime (one-way ANOVA plus Tukey’s multiple comparison); CTR N = 7 (n = 62, sparks # = 2789), STZ N = 5 (w/o istaroxime n = 53, sparks # = 2019, with istaroxime n = 47, sparks # = 1940). FWHM, full width at half maximum; FDHM, full duration at half maximum. Spark mass (spark amplitude*1.206* FWHM3).