Alterations of sarcoplasmic reticulum-mediated Ca2+ uptake in a model of premature ventricular contraction (PVC)-induced cardiomyopathy

Premature ventricular contractions (PVCs) are the most frequent ventricular arrhythmias in the overall population. PVCs are known to acutely enhance contractility by the post-extrasystolic potentiation phenomenon, but over time persistent PVCs promote PVC-induced cardiomyopathy (PVC-CM), characterized by a reduction of the left ventricular (LV) ejection fraction. Ca2+ cycling in myocytes commands muscle contraction and in this process, SERCA2 leads the Ca2+ reuptake into the sarcoplasmic reticulum (SR) shaping cytosolic Ca2+ signal decay and muscle relaxation. Altered Ca2+ reuptake can contribute to the contractile dysfunction observed in PVC-CM. To better understand Ca2+ handling using our PVC-CM model (canines with 50% PVC burden for 12 weeks), SR-Ca2+ reuptake was investigated by measuring Ca2+ dynamics and analyzing protein expression. Kinetic analysis of Ca2+ reuptake in electrically paced myocytes showed a ~ 21 ms delay in PVC-CM compared to Sham in intact isolated myocytes, along with a ~ 13% reduction in SERCA2 activity assessed in permeabilized myocytes. Although these trends were not statistically significant between groups using hierarchical statistics, relaxation of myocytes following contraction was significantly slower in PVC-CM vs Sham myocytes. Western blot analyses indicate a 22% reduction in SERCA2 expression, a 23% increase in phospholamban (PLN) expression, and a 50% reduction in PLN phosphorylation in PVC-CM samples vs Sham. Computational analysis simulating a 20% decrease in SR-Ca2+ reuptake resulted in a ~ 22 ms delay in Ca2+ signal decay, consistent with the experimental result described above. In conclusion, SERCA2 and PLB alterations described above have a modest contribution to functional adaptations observed in PVC-CM.


Introduction
Premature ventricular contractions (PVC) are prevalent in adults, and chronic frequent PVCs are associated with nonischemic cardiomyopathy and cardiac sudden death [1][2][3]. PVC-induced cardiomyopathy (PVC-CM) is potentially reversible with the suppression of PVCs [4][5][6][7]. Large animal models of chronic frequent PVCs are exceptionally suited to study molecular and pathophysiological adaptations of the heart underlying this cardiomyopathy [8][9][10]. The imposition of chronic PVCs on healthy experimental animals leads to LV structural remodeling, resulting in eccentric hypertrophy and cardiac dysfunction characterized by altered inotropy and lusitropy [11].
Ca 2+ transients command cardiac muscle contraction, and alterations in proteins involved in Ca 2+ handling are likely contributors to the cardiac dysfunction observed in PVC-CM. Previous studies showed modifications in key proteins related to dyad organization and Ca 2+ cycling in chronic PVC-CM models [8,9,12], including a reduced expression of the Ca 2+ pump (SERCA2) of the sarcoplasmic reticulum (SR) [9]. SERCA2 pumps Ca 2+ back into the SR lumen at the expense of ATP hydrolysis, influencing both the 1 3 relaxation rate and the size of the intracellular Ca 2+ pool in myocytes [13][14][15]. Moreover, SERCA2 together with other Ca 2+ -binding proteins highly expressed in myocytes (e.g., troponin C, calmodulin, etc.) contribute to the Ca 2+ buffering capacity observed in the myocyte's cytosol [16,17]. Thus, changes in SERCA2 function and/or expression can alter Ca 2+ handling by myocytes. Although thus far unsuccessful, gene therapy to overexpress SERCA2 has been studied to recover cardiac function in other cardiomyopathies in which the expression of this Ca 2+ pump is reduced [18]. In this study, we performed functional and biochemical studies to characterize Ca 2+ uptake components of the SR in the PVC-CM model.

Animal model
Mongrel canines (> 10-month-old, ~ 21 kg) underwent left thoracotomy to implant a wireless telemetry device (Data Scientific International, USA) and a modified pacemaker (Abbott, USA) to reproduce frequent PVCs (50% of PVC burden, 200-220 ms coupling interval) for the PVC-CM group or without PVCs (Sham group) during 12 weeks as previously described [8]. All animals underwent echocardiography and ambulatory ECG telemetry at baseline and 12 weeks. All procedures were approved by the Guide for the Care and Use of Laboratory Animals approved by the Institution Animal Care and Use Committee (IACUC) at McGuire Research Institute. Note: when this report was written, new regulations are in place and canine research stopped, thus further research is no longer available using the presented animal model at our institution.

Evaluation of Ca 2+ transients and sarcomere length
Cell isolation was performed as previously described [12]. Isolated cardiomyocytes were plated on coverslips, incubated with Fura-2 AM (3 µM, Invitrogen), and visualized on an inverted microscope connected to an IonOptix Ca 2+ and contractibility system. Electric field stimulation at 1 Hz, 5 V at 0.5 ms duration was applied to the cells for at least 1 min until reaching steady state. The cells were perfused with normal Tyrode extracellular solution (135 mM NaCl, 4 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM Hepes, 10 mM Glucose, pH 7.3) at 37 °C. Canine isolated cardiomyocytes showed weak responses to electrical field stimulation (likely because of the Fura-2 Ca 2+ buffering properties); therefore 0.8 µM isoproterenol was added to the perfusing solution to enhance Ca 2+ cycling allowing a clear simultaneous visualization of the Ca 2+ transient and contractility. Only healthy cells that maintained a consistent response to the electrical field stimulation without signs of spontaneous waves or arrhythmic responses were used in the analysis. A total of 15 contractions per cell were averaged and analyzed. Ca 2+ transients and sarcomere length were recorded simultaneously. The peak Ca 2+ signal is not reported because some microscope settings were optimized along the extension of the project. Nevertheless, in all cases the signal detection was optimized within the dynamic range of the detector, and kinetic parameters of the signal reported here are not affected by such changes. Therefore, the Ca 2+ transients presented in Fig. 1A are normalized to their maximal response. The changes in cytoplasmic Ca 2+ during the reuptake phase of Ca 2+ transients were reported as the exponential decay time constant of the function (Exp decay τ) that indicates the speed of Ca 2+ reuptake, as analyzed using the IonWizard Software (IonOptix, Westwood, MA, USA). Average sarcomere spacing was determined by a Fast Fourier Transform algorithm using the IonWizard Software at a selected area of isolated myocytes using an acquisition frequency of 0.5 kHz. These measurements were carried out in an area containing a pattern of striation where many sarcomeres were clearly observed. Each electrical pulse defines the time zero and kinetic parameters of the contraction (maximal shortening, time to peak contraction, and the single exponential τ of the relaxation phase) were determined using the IonWizard Analysis Software (IonOptix, Westwood, MA, USA).

SR-Ca 2+ uptake estimated in permeabilized myocytes
SR-Ca 2+ signal was studied in permeabilized myocytes as previously described [19]. Isolated myocytes were plated in Matrigel-precoated coverslips and loaded with 5.5 µM Fluo-5N AM dissolved in the normal Tyrode solution (see composition above). Ca 2+ uptake experiments were performed using an internal solution containing 120 mM potassium aspartate, 20 mM KCl, 3 mM MgATP, 10 mM phosphocreatine, 5 U ml −1 creatine phosphokinase, 0.5 mM EGTA, 20 mM Hepes, and 0.1% saponin (pH 7.2 with KOH). Images were acquired using an EMCDD camera (Andor) at 10 Hz and a monochromatorbased Polychrome V illumination system (Till Photonics) both coupled to an Olympus IX70 microscope. The excitation wavelength used for epifluorescence imaging was 490/10 nm (set by the monochromator), the dichroic mirror cutoff was 505 nm, and the bandpass emission filter 535/50 nm. Fast perfusion was achieved using a pressurized system (Automate Scientific), and images were background-subtracted before further analysis [20]. Ca 2+ concentration in the SR was estimated using the following equation: [Ca 2+ ] SR = Kd × (F-F min )/(F max -F), where F min is determined by exposing the cells to the internal solution modified with 2.5 mM EGTA and 10 mM caffeine. F max was determined by exposing the cells to the internal solution modified with 4Br-A23187 (5 µM), high Ca 2+ (10 mM), and BDM (10 mM) at the end of the experiment. The SR-Ca 2+ loading measurement was performed after caffeine depletion using the internal solution supplemented with ruthenium red (10 µM) to block the RyR2. The estimated free [Ca 2+ ] during the Ca 2+ uptake phase of the experiment was in the diastolic range ~ 150 nM [21]. This was fine-adjusted empirically by using the least concentration of EGTA in the internal solution that produced no contraction of the permeabilized myocytes. Experiments were performed under constant perfusion following the timeline described in Fig. 2. The Kd of Fluo-5N used for calculations was 400 µM as previously described [22,23]. For each cell, the maximal rate of Ca 2+ uptake was determined by computing the maximum of the first derivative of the curve and the maximal Ca 2+ load was determined at the steady-state phase after loading; both determinations were carried out using Origin software (see Fig. 2).

Protein extraction
At the end of the animal protocol, hearts were dissected and washed with ice-cold PBS and the left ventricular tissue was snap frozen in liquid nitrogen and stored at − 80 °C. The tissues were pulverized cautiously in liquid nitrogen using a mortar. Tissues were then solubilized in RIPA buffer with following composition: 25 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, 1 mM PMSF, 1 mM Orthovanadate, 50 mM NaF, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SD, and supplemented with protein inhibitor cocktail (complete TM , Roche). The homogenates were centrifuged at 12,000 rpm at 4 °C, supernatants were recovered, and protein concentration was determined using the Bradford method. Then samples were stored at − 80 °C until further use.

Western blot experiments
Lysates (30-50 µg of protein) were subjected to electrophoresis using an SDS-polyacrylamide gel at 100 V at room temperature for 2-2.5 h. Proteins were electrically transferred onto polyvinylidene difluoride (PVDF) membranes (Invitrogen) at 100 V at 4 °C for 1 h. Membranes were blocked in 0.1% TBS-T containing 5% fat-free milk (Biorad) for 1 h and incubated overnight with antibodies directed against SERCA2 (cat: MA3-919, Invitrogen), phospholamban (PLN, cat: MA3-922, Invitrogen), phospho-PLN (Ser16/ Thr17, cat: 8496, Cell Signaling Technology), cardiac troponin I (cTnI, cat: MA1-20112, Invitrogen), or phospho-cTnI (Ser23/24, cat: 4004, Cell Signaling Technology), supplemented with 5% BSA, all at 4 °C. Then the blots were washed with 0.1% TBS-T and incubated with their respective secondary antibodies conjugated to horseradish peroxidase, and the chemiluminescent signal was acquired using the system FluorChem gel imager (ProteinSimple, CA), and the signals were analyzed using the software AlphaView (ProteinSimple, CA). The protein GAPDH (cat: PA1-988, Invitrogen) was used as a loading control for all experiments. Each lane of the western blot corresponds to a sample of a different animal and each western blot experiment was performed in triplicate. Three Sham and three PVC samples were run per western blot, and each PVC data point was normalized to the average of the Sham samples.

Computer simulation of the Ca 2+ transient
Simulation of the Ca 2+ transient was carried out using Lab-HEART v5.3 (computer simulation of a rabbit ventricular myocyte), by Donald M. Bers and José Puglisi [24]; https:// www. labhe art. org/.

Statistical analyses
Data obtained from cells were segregated by animal (nested data sets) and analyzed using hierarchical statistics [25,26]. Comparison between experimental groups (Sham and PVC) was performed using Nested t test considering p < 0.05 as a significant difference. Additional descriptive statistical parameters such as mean, median, and interquartile range were calculated for each animal. Data are presented using Ca 2+ uptake in permeabilized ventricular myocytes. A Representative trace of the permeabilization method used to estimate the SR-Ca 2+ uptake using the low-affinity Ca 2+ sensor Fluo-5N,, the inset represents the first derivative of the curve to obtain the maximal rate of Ca 2+ uptake, and the asterisk indicates the luminal-free Ca 2+ concentration at steady state after loading (Ext. Sol. = extracellular solution, Caff = caffeine, RR = ruthenium red, EGTA concentration in mM). B Box and whisker plots (including individual values) for isolated myocytes segregated by animal for the maximal rate of SR-Ca 2+ uptake and C Free Ca 2+ in the SR at steady state 1 3 box & whiskers plot including individual values. Western blot data are represented as mean ± SD and individual values are included in the plot. These data were compared using unpaired t test considering p < 0.05 as a significant difference. Echocardiographic data were compared using two-way repeated measurement analysis and Šídák's multiple comparisons post-test considering p < 0.05 as a significant difference. All statistical analyses were done using GraphPad Prism 9.3.1 software.

LVEF, LVEDV, and LVESV at baseline and after 12 weeks of PVCs in animal subjects
Left ventricular ejection fraction (LVEF), left ventricular end-diastolic volumes, and left ventricular end-systolic volumes (LVEDV and LVESV, respectively) were measured in nineteen canines implanted with modified pacemakers (see "Materials and methods" section). Nine animals were subjected to bigeminal PVCs for 12 weeks (PVC-CM group), whereas the remaining ten subjects had no exposure to PVCs (Sham group). After 12 weeks, LVEF decreased significantly from 62.4 ± 6.1 to 44.4 ± 4.4% (mean ± SD) in the PVC-CM group (two-way repeated measures ANOVA, Šídák's multiple comparisons test p < 0.0001, Table 1) while no changes in LVEF were seen in the Sham group (61.8 ± 5.4% vs 62.5 ± 4.3%, Table 1). In contrast to Sham (52.7 ± 7.3 ml baseline vs 51.7 ± 6.6 ml after 12 weeks), the PVC-CM group underwent significant increase in LVEDV from a baseline of 49.4 ± 4.5 ml to 63.7 ± 8.9 ml after 12 weeks of PVCs (two-way repeated measures ANOVA, Šídák's multiple comparisons test p < 0.0001, Table 1). In the PVC-CM group, LVESV had a significant increase (baseline 18.6 ± 2.9 ml vs 35.5 ± 5.7 ml after 12 weeks; twoway repeated measures ANOVA, Šídák's multiple comparisons test p < 0.0001, Table 1) without change in the Sham group (baseline 20.2 ± 4.2 ml vs 12 weeks 19.4 ± 4.1 ml). These data show that the cohort of animals used in this study effectively developed a cardiomyopathy when exposed to PVCs for 12 weeks, with significantly reduced ejection fraction.

Ca 2+ reuptake and contraction in isolated cardiomyocytes
To study Ca 2+ reuptake in intact cells, intracellular Ca 2+ signal and cell shortening were determined simultaneously in ventricular adult myocytes that were isolated from the animals at the end of the 12-week period in both PVC and sham groups. Experiments were carried out in the presence of isoproterenol only in myocytes that maintained stable response to electrical pacing (see "Materials and methods" section). Examples of Ca 2+ transient (normalized by the peak intensity, see "Materials and methods" section) and shortening measurements are shown in Fig. 1A and B, respectively. The reuptake phase of the Ca 2+ transient was fitted to a single exponential function, and the time constant reflects the rate at which Ca 2+ concentration is reduced in the cytosol. The difference in time constants was 21 ± 21 ms (18%) slower in PVC-CM vs Sham group (135 ms vs 114 ms, respectively). This difference did not reach statistical significance (p = 0.3360, Nested t test). Both the mean median (119 ms vs 115 ms) and mean interquartile range (57 ms vs 42 ms) were 3 and 35% slower in PVC-CM vs Sham grouped animals, respectively (Fig. 1C). Although the overall dispersion of the data limits a definitive conclusion, the PVC-CM group shows a tendency to have slower and more disperse Ca 2+ decay than the Sham group.
The maximal shortening induced by electrical pacing in myocytes was equivalent in both experimental groups (Fig. 1D). The difference in the maximal shortening was 2.2 ± 1.3% larger in PVC-CM compared to the Sham group (12.8% vs 10.6%, respectively). This difference was not statistically significant (p = 0.1199, Nested t test). However, the time to reach maximal contraction (time to peak) was significantly slower by 19 ± 8 ms in PVC-CM vs Sham (136 ms vs 117 ms, respectively; 95% CI 37.5 ms to 0.4 ms, p = 0.047 Nested t test, Fig. 1E). The relaxation phase of the shortening traces was fitted to a single exponential function and the  Fig. 1F). Although the relaxation time of the shortening is complex to interpret because of the multiple factors involved, the trend toward slower Ca 2+ reuptake in PVC-CM vs Sham may contribute to the slower shortening relaxation velocity in the former group.

Ca 2+ uptake by the SR estimated in permeabilized myocytes
To assess the SR-Ca 2+ uptake without the interference of other Ca 2+ fluxes (e.g., NCX, RyR2, and L-type Ca 2+ channel), experiments were carried out in permeabilized cells.
The rate of SR-Ca 2+ uptake and the free SR-Ca 2+ concentration in steady state at the end of the loading phase of the experiment were estimated in isolated myocytes loaded with the low-affinity Ca 2+ dye Fluo-5N. First, the Ca 2+ stored in the SR was depleted using caffeine, and then fast-switched to a caffeine-free buffer supplemented with the RyR2 inhibitor ruthenium red, in the presence ATP and its regeneration system (phosphocreatine and creatine kinase; Fig. 2A). Traces were calibrated to Ca 2+ concentration (see "Materials and methods" section). The maximal rate of Ca 2+ uptake was obtained by calculating the peak of the first derivative of the uptake phase of the curve (inset in Fig. 2A), and SR-free Ca 2+ concentration (in steady state) was estimated by the amplitude of the signal at the end of the Ca 2+ loading phase (asterisk in Fig. 2A). The results showed a 28 ± 31 μM/s difference, equivalent to 13% reduction in the maximum Ca 2+ uptake rate in PVC-CM vs Sham group (193 μM/s vs 221 μM/s, respectively). This difference was not statistically significant in the Nested t test (p = 0.3939). Descriptive statistical analysis shows a mean median (181 μM/s vs 203 μM/s) and a mean interquartile range (97 μM/s vs 128 μM/s), 10 and 24% slower in PVC-CM vs Sham group, respectively (Fig. 2B). The SR-free Ca 2+ at equilibrium was 22.5 µM smaller in the PVC-CM group, equivalent to 2.6% smaller in PVC-CM than in the Sham group (845 vs 867 µM, respectively) without reaching statistical significance (p = 0.8367, Nested t test). Descriptive statistics analysis shows a mean median (788 vs 821 µM) and mean interquartile range (353 vs 421 µM) 3.9 and 16% smaller in PVC-CM vs Sham animals, respectively (Fig. 2C). Similar to the Ca 2+ determinations in intact (non-permeabilized) cells, a tendency toward slower SR-Ca 2+ uptake was found in PVC-CM vs Sham group without statistical significance.

Expression and phosphorylation of proteins involved in SR-Ca 2+ uptake in PVC-CM
To further characterize potential differences in SR-Ca 2+ uptake mechanism between the Sham and PVC-CM groups, western blot (WB) analysis was performed to study the expression of proteins directly involved in SR-Ca 2+ uptake. Cardiac protein extracts were prepared from LV free wall tissue of six animals per group and subjected to WB using GAPDH as loading control (Fig. 3A). While the total amount of SERCA2 was reduced by 22% in PVC-CM vs Sham (p = 0.0364, t test, Fig. 3B), the total phospholamban (PLN) increased by 23% in the PVC-CM group compared to Sham (p = 0.001, t test, Fig. 3B). In addition, PLN phosphorylation decreased by 50% in PVC-CM vs Sham (p = 0.0006, t test, Fig. 3B). In contrast, cardiac Troponin I (cTnI, also regulated by phosphorylation without direct involvement in Ca 2+ uptake) was unchanged between groups (t test, Fig. 3B), whereas its phosphorylation level was reduced by 23% in PVC-CM vs Sham, almost reaching significance between groups (p = 0.0569, t test, Fig. 3B).
To model the hypothetical effect of ~ 20% reduction of SERCA2 (as described above) on cardiomyocyte function, electrically evoked Ca 2+ signals in the presence of isoproterenol were simulated using LabHEART v5.3 software [24]. Reducing SR-Ca 2+ uptake by 20%, the mathematical model produced a ~ 22 ms delay in the reuptake phase of the Ca 2+ transient (at 1 Hz stimulation frequency, Fig. 4).

Discussion
The molecular adaptations induced by persistent PVCs leading to cardiomyopathy are elusive and likely involve a combination of factors. In this study, SERCA2 activity and its regulation were studied in the context of PVC-CM. An adequate Ca 2+ reuptake activity in the SR is critical for muscle relaxation and the maintenance of intracellular Ca 2+ stores in the beating heart [27,28]. One clear result obtained in this work was a ~ 20% decrease in SERCA expression. When this reduction in SERCA activity was modeled in silico, it showed a ~ 22 ms delay in Ca 2+ reuptake (Fig. 4) that was similar to a non-statistically significant delay of ~ 21 ms (p = 0.336) in Ca 2+ reuptake empirically observed in paced PVC-CM isolated myocytes. Moreover, the reduction in SERCA2 expression can contribute to the comparable significant delay in the shortening relaxation time observed (34 ms). However, the insufficient cTnI phosphorylation observed in the WB analysis (p = 0.0569, t test) can also contribute to the delayed contraction and relaxation kinetics observed in electrically paced PVC-CM myocytes [29,30]. A more dramatic effect of SERCA2 reduction (assessed by WB) could be compensated by an upregulation of NCX.

3
Although a possibility, it was previously shown that expression of NCX was unaltered in the canine PVC-CM model [31].
Additional studies performed using permeabilized myocytes showed maximal SR-Ca 2+ uptake and steady-state Ca 2+ SR loading levels at a similar range than in a previous study using a genetically encoded Ca 2+ sensor targeted to the SR in isolated myocytes [28]. Here, we consistently showed a 13% reduction in the maximal SR-Ca 2+ uptake rate and ~ 3% reduction in the SR-free Ca 2+ at steady state in PVC-CM myocytes, also consistent with SERCA reduction, but these results were not statistically significant. Similarly, a volumeoverload model has shown a reduction in SERCA2 after the first month that persisted for 3 months without changes in the expression levels or phosphorylation of PLN [32]. Although SERCA2 expression was permanently reduced, its activity decreased after the first month but recovered after 3 months of starting the volume-overload protocol [32]. This discrepancy between SERCA2 expression and its activity has been previously reported [33]. In the PVC-CM model, we observed a similar result: after 3 months, the activity of SERCA2 in myocytes is marginally compromised despite its reduced level and a significant increase in PLN mass and reduced phosphorylation. These results suggest that another undetermined SERCA2 modulator should be altered to compensate for its decreased level (and changes in the PLN state). For instance, the volume-overload model showed an increase in expression and activity of an acylphosphatase after 3 months that could affect SERCA2 activity [32]. It has been proposed that this acylphosphatase may interfere with the inhibitory action of PLN over SERCA2 [34]. These observations suggest that PVC-CM may have a large "physiological reserve" for SERCA2 activity that can protect SR-Ca 2+ uptake when other adaptations tend to reduce Ca 2+ cycling. Altogether our results suggest that the reduction in SERCA2 expression observed in PVC-CM translates only into mild functional consequences in myocytes. The maximal contraction of the myocytes showed a non-significant trend to be larger in PVC-CM myocytes (Fig. 1D). This result is important since, despite a reduction in LVEF, isolated PVC-CM myocytes have a potential to fully contract in the presence of isoproterenol. Conversely, isoproterenol strongly potentiates the Ca 2+ signal and cell shortening, and it also filters out myocytes that cannot keep the pace under constant stimulation. All these effects of isoproterenol could diminish differences between different experimental conditions [15], therefore suggesting that some results can be different in the absence of isoproterenol. Indeed, the maximal Ca 2+ amplitudes appear reduced (as we previously reported) in the absence of this β-adrenergic agonist in PVC-CM [12].
As part of the sympathetic response of the heart, adrenergic stimulation phosphorylates PLN disrupting its inhibitory action at SERCA2 [35]. Conversely, dephosphorylated PLN interacts with SERCA2 decreasing its affinity to Ca 2+ [33]. Our WB experiment showed an increase in PLN expression and a decreased phosphorylation of PLN in PVC-CM samples compared to Sham, resembling results in a previously reported swine PVC model where PLN expression was increased and SERCA2 reduced [9]. Other studies have also shown that chronic PVCs augmented the sympathetic tone of the heart [36] concomitant with a reduction of the parasympathetic efferent signaling [37]. Thus, chronic sympathetic activity may produce a long-term adjustment of the β-adrenergic signaling pathway (i.e., down-regulation and/ or desensitization) resulting in a PLN dephosphorylation in the PVC-CM chronic state. This is further supported by a 23% reduction in cTnI phosphorylation (p = 0.0569, t test) in PVC-CM, a regulatory protein of the contractile machinery that participates in the inotropic and lusitropic effect of the β-adrenergic stimulation [29,30].
In summary, long-term PVCs result in a decrease in LVEF, and in this context, a reduction in SERCA2 activity appears to promote to some extent a slowing down of the SR-Ca 2+ reuptake, and delayed relaxation of myocytes. Although further experimentation will be necessary to confirm some of the findings, our data suggest that SERCA2 alterations contribute modestly to the cardiac functional abnormalities observed in PVC-CM. Fig. 4 Model simulating the effect of decreasing 20% SERCA activity in myocytes. Simulation of the Ca 2+ transient using LabHEART v5.3 (computer simulation of a rabbit ventricular myocyte) [24]. The simulation was performed first using the default parameters in the presence of 100% isoproterenol (Default + ISO) and then decreasing in 20% the SR uptake parameter from 0.005 to 0.004 mM (− 20% SR uptake + ISO)