Renal Denervation Improves Cardiac Diastolic Dysfunction by Restoring Serca2a Transcription in Uninephrectomized Rats

Hirohama D1,2, Kawakami-Mori F2, Ogura S2,3, Mu S4, Jimbo R5, Uetake U6, Yatomi Y7, Nangaku M1, Fujita T2 and Shimosawa T7 1Department of Nephrology and Endocrinology, The University of Tokyo Graduate School of Medicine Tokyo, Japan 2Reserch Center for Advanced Science and Technology, Division of Clinical Epigenetics, The University of Tokyo, Tokyo, Japan 3Department of Pathology and Microbiology, Division of Laboratory Medicine, Nihon University School of Medicine, Tokyo, Japan 4Department of Pharmacology & Toxicology, University of Arkansas for Medical Science, Fayetteville, Arkansas 5Department of Internal Medicine, Tohto Bunkyo Hospital, Tokyo, Japan 6Office for Research Ethics Support, The University of Tokyo Graduate School of Medicine and Faculty of Medicine, Tokyo, Japan 7Department of Clinical Laboratory, The University of Tokyo Graduate School of Medicine, Tokyo, Japan


Introduction
The mortality and morbidity of heart failure (HF) has increased due to the increased prevalence of hypertension and the aging of the population. The proportion of patients with HF with preserved ejection fraction (HF-PEF) accounts for more than 50% of the total HF population [1]. Left ventricular (LV) diastolic dysfunction is considered the major underlying pathology in HF-PEF [2]. However, clinical trials to date have failed to show improvements in diastolic dysfunction or cardiovascular outcome [3,4].
Sarcoplasmic reticulum Ca 2+ -ATPase type 2a (SERCA2a) plays an essential role in Ca 2+ homeostasis and regulates cardiac functions. Reductions in SERCA2a expression have been widely documented in LV systolic [5] as well as diastolic dysfunction [6,7]. By contrast, SERCA2a overexpression has been shown to improve LV systolic [8] and diastolic [7] dysfunction. Furthermore, clinical trials have demonstrated the beneficial effects of transferring the SERCA2a gene to the heart of systolic HF patients [9], but several obstacles remain to be overcome. The transcriptional regulation of SERCA2a may be a new therapeutic target; however, knowledge of SERCA2a transcription is limited [10][11][12][13][14].
below. The procedures were carried out under anesthesia with sodium pentobarbital (20 mg/kg body weight, intraperitoneally). Thereafter, the rats were fed either a normal-salt diet (NS; 0.3% NaCl) or a high-salt diet (HS; 8% NaCl) for 6 weeks. The rats were randomly divided into four groups as follows: 1) NS and sham operation of denervation (NS); 2) NS renal nerve denervated (NS-RDx); 3) HS and sham operation of denervation (HS); and 4) HS and renal nerve denervated (HS-RDx).

Renal denervation
Renal denervation was performed as described previously [17]. The left renal sympathetic nerve was isolated through a retroperitoneal incision, and total renal denervation was achieved by cutting all of the visible renal nerves from the renal artery and vein. These vessels were then stained with a solution of 10% phenol in ethanol. In the sham operation, the renal nerves were isolated but preserved. After the rats were killed, renal tissue norepinephrine (NE) content was measured to confirm total renal denervation [17]. The supernatants were analyzed for endogenous NE by using a high-performance liquid chromatography assay with electrochemical detection. The NE content of the renal tissue in the NS-RDx group was significantly smaller than that in the NS group (2.99 ± 0.52 vs. 100.0 ± 9.11 ng/g tissue; P < 0.01) and nearly undetectable, which indicated that renal afferent and efferent denervation was complete [17].

RNA extraction and quantitative real-time reverse transcription-polymerase chain reaction (PCR)
RNA was prepared from rat LV tissues with an RNeasy fibrous kit (Qiagen, Venlo, Netherlands). Total RNA was reverse-transcribed with Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA). Gene expression was quantitatively analyzed with real-time reverse transcription-PCR as previously described [18]. We used TaqMan Gene Expression Assays with a 7300 Real-Time PCR System (Invitrogen). The ID numbers for the assays were as follows: Rn00667869_m1 for β-actin, RN01499544_m1 for SERCA2a, RN01488777_g1 for β-myosin heavy chain, RN01463848_m1 for collagen 1a, and RN01437681_m1 for collagen 3a.

Physiological studies
Systolic BP was measured with the tail-cuff method (P-98A, Softron, Tokyo, Japan) in conscious rats in the NS (n = 9), NS-RDx (n = 8), HS (n = 22), and HS-RDx (n = 13) groups and recorded at 4 and 6 weeks. We measured systolic BP five times at each time point for each rat and calculated the average.
Twenty-four-hour urine samples were collected via metabolic cages at 6 weeks from the NS (n=6), NS-RDx (n=8), HS (n=19), and HS-RDx (n=12) groups, 18 and urinary protein levels were measured. At the completion of invasive LV-pressure measurements, the animals were killed. Blood samples were obtained from the vena cava, and then the heart tissues were harvested and snap-frozen for RNA and protein analyses.

Pathological studies
Left ventricles (four hearts each from the NS, HS, and HS-RDx groups) were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into sections of 3-µm thickness. Azan staining was performed to evaluate perivascular and myocardial interstitial fibrosis.

Hemodynamic measurements
At 6 weeks, rats in the NS (n=9), NS-RDx (n = 8), HS (n=22), and HS-RDx (n=13) groups were anesthetized with sodium pentobarbital (40 mg/kg body weight, intraperitoneally). LV pressures were assessed with a Millar Tip catheter (SPR-320NR, 2 Fr, Millar Instruments, Houston, TX), which was introduced from the right carotid artery and advanced into the LV cavity. After the catheters were inserted, the animals were stabilized hemodynamically for 5 minutes. Thereafter, the heart rate, mean arterial pressure (carotid artery), maximal positive LV pressure development (+dP/dt max ), and time constant at the isovolumic relaxation phase (Tau) were measured.

Statistical analysis
The data are presented as the means ± standard error of the mean. Comparison among groups was performed with one-way analysis of variance followed by the Tukey-Kramer post hoc test. A P value of less than 0.05 was considered statistically significant.

Changes in SERCA2a messenger RNA (mRNA) and protein expression
SERCA2a mRNA expression in the HS group was significantly lower than that in the NS group (Figure 1a), and the abundance of SERCA2a protein followed the same trend (Figure 1b). The mRNA expression and protein abundance of SERCA2a were restored by renal denervation (Figure 1a and 1b).
Total PLB protein and PLB phosphorylation at serine 16 and threonine 17 were comparable among the groups (Figure 1c). The protein abundance of nitro tyrosine and 4HNE were not altered by the HS (Figure 1d).

Cardiac function and structural remodeling
BP was elevated in the HS group and was not lowered by renal denervation (Figure 1e and Supplementary Figure S1a and 1b).
In accordance with changes in SERCA2a transcription, rats in the HS group showed deteriorated diastolic function, which was evaluated with Tau and restored by renal denervation (Figure 1f).
LV systolic function, measured with +dp/dt max , in the HS group was higher than that in the NS group which indicated that LV systolic      Table S1). Compared with the LV weight-to-body weight ratio in the NS group, that in the HS group was significantly higher and was not reversed by renal denervation (Figure 1g).

Renal damage
Urinary protein levels in the HS group were significantly higher than those in the NS group. Renal denervation did not affect proteinuria (Supplementary Table S1).
To confirm that low levels of SERCA2a protein impair cardiac function, we evaluated LV diastolic function by measuring Tau. In concordance with SERCA2a level, LV diastolic function was reduced by uninephrectomy and the HS, and this deterioration was reversed by renal denervation. PLB phosphorylation at serine 16 and threonine 17 functionally enhances SERCA2a activity and Ca 2+ uptake in the sarcoplasmic reticulum [19]. In the present study, however, PLB and its phosphorylation were not significantly different among the experimental groups. This result suggests that the restoration of cardiac diastolic function occurs independently of PLB and its phosphorylation.
As shown in Figure 1f-1h and Supplementary Figures S1a, 1b and 1d neither BP nor cardiac hypertrophy was lowered by renal denervation throughout the experiment. Moreover, a regression curve showing the relationship between cardiac weight and Tau showed significantly different relationships between the renal nerve intact (the NS and HS) group and the denervated group (the NS-RDx and HS-RDx) (Supplementary Figure S1c). These data suggest that renal denervation reversed SERCA2a transcription and diastolic function independently of BP changes or cardiac hypertrophy. Furthermore, we observed no fibrotic changes in the heart (Figure 1h and Supplementary Figures S1e, 1f), which suggests that in vivo SERCA2a transcription can be altered independently of profibrotic stimuli.
Owing to our experimental design, we could not discern whether the restoration of SERCA2a transcription was due to the direct or indirect effects of renal denervation on the heart. In the uninephrectomy and HS group, we observed prominent proteinuria that may induce cardiorenal syndrome and affect SERCA2a transcription. However, renal denervation did not restore renal function; therefore, it is doubtful that renal denervation indirectly restored SERCA2a transcription via the preservation of kidney function. A previous study showed that oxidative stress impairs SERCA2a transcription [11]. In the present study, uninephrectomy and the HS did not induce higher oxidative stress, which was evaluated by measuring nitro tyrosine and 4HNE (Figure 1d). Thus, oxidative stress appears unlikely to have played a role in the regulation of SERCA2a transcription in our animal model.
A limitation of our study was the lack of 24-hour continuous BP monitoring. The minor effect of renal denervation on BP reduction is comparable with the result of recent randomized trial [20]. Indeed, 24hour continuous BP monitoring would provide additional information with which to understand the cardioprotective effect of renal denervation. Another limitation of the present study was the inability to clarify the differential role of the afferent and efferent nerves, also a consequence of our experimental design. Further in vivo studies are required to clarify the factors that alter SERCA2a transcription in this model of cardiac failure and via renal denervation.
Taken together, our data may provide new therapeutic insights into LV diastolic dysfunction, and these findings warrant further study.

Financial Disclosure
This study was supported by Grants-in-Aid for Scientific Research (c) (No. 26461249).