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Abstract

Background:

Cardiac shock wave therapy (CSWT) can improve myocardial ischemia and cardiac function in patients with coronary artery disease and refractory angina. The aim of the study was to test its potential role to relieve symptoms in patients with ischemic heart failure (HF) and to identify CSWT-affected genes.

Methods:

Cardiac shock wave therapy was performed on 23 patients (mean age: 67 ± 6 years) with ischemic HF 3 times per week for 3 weeks. Clinical assessment parameters were measured for all patients, and peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of all patients 3 days before CSWT and 1 week after the 3-week CSWT schedule. RNA sequencing of PBMCs collected from 3 patients before and after CSWT was performed on the Illumina Genome Analyzer. Gene expression was determined by quantitative reverse transcription–polymerase chain reaction.

Results:

Cardiac shock wave therapy significantly attenuated myocardial ischemia and severity of angina, health-related quality of life, and myocardial blood flow as estimated by New York Heart Association class, Canadian Cardiovascular Society classification, Seattle Angina Questionnaire, and single photon emission computed tomography images, respectively. We then tried to investigate how CSWT improved myocardial ischemia by RNA sequencing on PBMCs. Gene set enrichment analysis on the sequencing data revealed that CSWT treatment was positively correlated with cytokine and cytokine receptor interaction and chemokine signaling pathway. Furthermore, we demonstrated that CSWT resulted in a significant increase in the expression of promoters of neovascularization (vascular endothelial growth factor A [VEGF-A], VEGF-B, chemokine (C-X-C motif) ligand 1 [CXCL1], CXCL2, CXCL3 and TNFRSF12A) and a notable decrease in the expression of a mediator of cell apoptosis (mitogen-activated protein kinase 9).

Conclusions:

Cardiac shock wave therapy can improve myocardial ischemia and represents as a treatment option for patients with ischemic HF through promoting neovascularization and inhibiting cell apoptosis.

Keywords

cardiac shock wave therapy ischemic heart failure angiogenesis

Introduction

Ischemic heart failure (HF) is a sequela of coronary artery disease (CAD) and caused by reduced myocardial oxygen supply. Ischemic HF has become one of the major global health care issues.^1,2 Different therapeutic options, including medical treatment, percutaneous coronary intervention (PCI), coronary artery bypass grafting (CABG), and cardiac transplantation, are being applied for treating ischemic HF. However, limitations of these therapies persist owing to the variable efficacy for ischemic HF. A growing number of patients with ischemic HF receiving optimal medical treatment still experience severe symptoms, and mortality remains unacceptably high in these patients.³ Therefore, there is an urgent need to develop new and more effective therapies for ischemic HF.

Cardiac shock wave therapy (CSWT) is a newly developed therapy that can improve myocardial ischemia and ameliorate cardiac function in patients with CAD and refractory angina.^4

–7 Evidence indicates that low-energy pulse waves produced by CSWT could induce “cavitation effect,” which utilize mechanical shear force on vascular and myocardial endothelial cells. Cardiac shock wave therapy may reduce ischemia and alleviate angina by stimulating angiogenesis and revascularization in ischemic myocardium.^4,8,9 Furthermore, in vivo animal studies demonstrated that CSWT could increase capillary density and improve regional myocardial blood flow.^8,9 Shock wave treatment upregulates vascular endothelial growth factor (VEGF),⁸ endothelial nitric oxide synthase,^10,11 and basic fibroblast growth factor.¹²

The present study was aimed to investigate the potential benefits of CSWT on ischemic HF. Cardiac shock wave therapy was performed on 23 patients with ischemic HF 3 times per week for 3 weeks. Our study confirmed the efficacy of CSWT in the treatment of ischemic HF. Moreover, we tried to investigate how CSWT improved myocardial ischemia by identifying differentially expressed transcripts in peripheral blood mononuclear cells (PBMCs) before and after CSWT treatment.

Materials and Methods

Patients

Twenty-three patients admitted to the Department of Cardiology, Shanghai Chest Hospital Affiliated to Shanghai Jiao Tong University (Shanghai, China) from September 2013 to January 2014 were enrolled in this study. All patients received regular medical treatment and given written informed consent prior to enrollment. This study received ethical approval from the Ethics Committee, Shanghai Jiao Tong University (Shanghai, China) and conformed to the Declaration of Helsinki guidelines. Inclusion criteria were based on the guideline developed by the First Affiliated Hospital of Kunming Medical University (Kunming, China) and listed as follows: age of at least 18 years; with a clear history of acute myocardial infarction (over 3 months) or moderate to severe coronary stenosis diagnosed by coronary angiography; receiving regular medical treatment but hospitalized 2 or more times due to exertional dyspnea and other performance of chronic HF; left ventricular ejection fraction (LVEF) between 20% and 45%; still having HF symptoms after surgical revascularization, such as PCI and CABG; and unwilling to accept surgical revascularization. The exclusion criteria were recent acute myocardial infarction (within 3 months) or receiving PCI or CABG within 1 month; LVEF less than 20% or greater than 45%; combined with other heart disease, including valvulopathy; intracardiac thrombus; arrhythmia (lower than 40 bpm or greater than 120 bpm) that cannot be controlled by drugs; patients who cannot be placed in a supine position with cardiogenic shock and patients with severe HF (New York Heart Association [NYHA] functional class IV); patients with severe obstructive pulmonary disease, uncontrolled diabetes retinopathy, pulmonary infarction, aortic dissection, thoracic aortic aneurysm, or cancer; pregnancy women; ever with breast plastic surgery; with damage or infection on the treatment area; receiving thrombolysis, interventional, or surgical treatment during this study; and without completed follow-up or participating in other clinical studies.

Cardiac Shock Wave Therapy Procedure and Treatment Program

Cardiac shock wave therapy was delivered by an electromagnetic shockwave device (Storz Modulith SLC, Switzerland). Patients were placed in the supine position. After target myocardial regions were detected by the ultrasound probe, CSWT was performed using a standardized protocol of 9 sessions (200 shocks per session). Considering that CSWT causes a tingle on the treated location, wave energy was progressively increased (from 0.0024 to 0.09 mJ/mm²) to help patients adapt to the treatment. During the procedure, all patients were closely monitored for vital signs (pulse rate, blood pressure, respiratory rate, and temperature) and cardiac rate.

The CSWT regimen was performed 3 times per week for 3 weeks. Three days before CSWT schedule (baseline) and 1 week after CSWT schedule, clinical assessments of cardiac function were performed in all patients, and blood samples were obtained for PBMCs isolation.

Clinical Assessment Parameters

The efficacy of CSWT was assessed using the NYHA class, Canadian Cardiovascular Society (CCS) angina scale, and Seattle Angina Questionnaire (SAQ) scale. Transthoracic echocardiography was performed to detect LVEF, left ventricular end diastolic volume (LVEDV), and left ventricular end systolic volume (LVESV) on all patients before and after CSWT treatment. To evaluate myocardial perfusion, single photon emission computed tomography (SPECT) with ¹⁸F-fluorodeoxyglucose imaging was carried out at baseline and 1 week after the last CSWT treatment. Summed stress score (SSS) and summed rest score (SRS) were calculated in the stress and rest images by a blinded observer, respectively.

RNA Extraction, Sequencing, and Bioinformatics Analysis

Peripheral blood mononuclear cells were isolated from patients’ whole blood collected before and after the 3-week CSWT schedule by centrifugation at 300g for 30 minutes on mononuclear/polynuclear cell-resolving medium (Flow Laboratories, Rockville, Maryland). Total RNA was then extracted from PBMCs using the TRIzol reagent (Invitrogen, Carlsbad, California) according to the manufacturer’s instructions. RNA concentrations were determined by ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, Delaware). RNA-Seq libraries were prepared from the PBMC RNA (1 μg) using Illumina’s messenger RNA (mRNA) TruSeq protocol, and next-generation sequencing was performed on the Illumina Genome Analyzer (Illumina, San Diego, California). All of our original sequence data have been deposited in NCBI (http://www.ncbi.nlm.nih.gov/sra,AC:SRR2059921).

Gene set enrichment analysis (GSEA) was conducted using GSEA version 2.0 from the Broad Institute at MIT to identify the pathways that were significantly enriched in samples before and after CSWT treatment. All genes considered for GSEA were 2.5-fold differentially expressed, with P value less than .05. The “c2.KEGG.v4.0.symbols.gmt” gene sets including 165 pathways from the Molecular Signatures Database (http://www.broad.mit.edu/gsea/msigdb/index.jsp) were used for enrichment analysis.

Quantitative Reverse Transcription–Polymerase Chain Reaction

Total RNA (1 μg) was subjected to the synthesis of the first-strand complementary DNA with random primers and Moloney-murine leukemia virus (M-MLV) reverse transcriptase (Fermentas Hanover, Maryland). The quantitative real-time polymerase chain reaction (PCR) was conducted using SYBR-green PCR Master Mix on an ABI 7300 real-time PCR machine (Applied Biosystems, Foster City, California). Glyceraldehyde-3-phosphate dehydrogenase was amplified as the internal control. The gene-specific primers were listed in Table 1. The PCR reactions were run at 95°C for 10 minutes and 40 cycles of 95°C for 15 seconds and 60°C for 45 seconds. Fold change of interested genes was calculated by the equation 2^−ΔΔC.

Table 1.

Primers Sequences for Quantitative PCR.

Primer	Primer Sequence	Size (bp)
VEGF-A (NM_001171627)	F: 5′-AGGGCAGAATCATCACGAAGT-3′; R: 5′-AGGGTCTCGATTGGATGGCA-3′	75
VEGF-B (NM_001243733.1)	F: 5′-GGATAGCCCAGTCAATACAG-3′; R: 5′-CAAGCAAGGTCACTCAGTAG-3′	127
CXCL1 (NM_001511.3)	F: 5′-ATGCTGAACAGTGACAAATC-3′; R: 5′-AAACACATTAGGCACAATCC-3′	147
CXCL2 (NM_002089.3)	F: 5′- CCCAAACCGAAGTCATAGC-3′; R: 5′-GGAACAGCCACCAATAAGC-3′	152
CXCL3 (NM_002090.2)	F: 5′-AGAACAGCAGCTTTCTAGGG-3′; R: 5′-AGGTGGCTGACACATTATGG-3′	248
TNFRSF12A (NM_016639.2)	F: 5′-CTCGCCCACTCATCATTC-3′; R: 5′-GAGGCTCCCTTTCTGTTC-3′	195
MAPK9 (NM_001135044.1)	F: 5′-CAGCAGAAAGGAGAATAAC-3′; R: 5′-AAGGGAAGATGTGAAGAAC-3′	112
P21 (NM_000389.4)	F: 5′-TAGCAGCGGAACAAGGAG-3′; R: 5′-AAACGGGAACCAGGACAC-3′	249
E-cadherin (NM_004360.3)	F: 5′-GAGAACGCATTGCCACATACAC-3′; R: 5′-AAGAGCACCTTCCATGACAGAC-3′	164
ICAM1 (NM_000201.2)	F: 5′-GTTGTTGGGCATAGAGAC-3′; R: 5′-CAGGGCAGTTTGAATAGC-3′	125
GADPH NM_001256799.1)	F: 5′-CACCCACTCCTCCACCTTTG-3′; R: 5′-CCACCACCCTGTTGCTGTAG-3′	110

Abbreviations: CXCL1, chemokine (C-X-C motif) ligand 1; ICAM-1, intercellular adhesion molecule 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAPK9, mitogen-activated protein kinase 9; PCR, polymerase chain reaction; VEGF, vascular endothelial growth factor.

Statistical Analysis

Data were presented as the mean ± standard deviation (SD). Values before and after CSWT schedule were compared using paired Student t test. Statistical analyses were performed using SPSS 17.0 statistics software (SPSS Inc, Chicago, Illinois).

Results

Cardiac Shock Wave Therapy Improved Myocardial Ischemia in Patients With Ischemic HF

Twenty-three patients (17 males and 6 females) with ischemic HF were enrolled in this study. The age was ranged from 57 to 78 years (67 ± 6 years). Cardiac shock wave therapy was performed 3 times per week for 3 weeks. In order to evaluate the efficacy of CSWT on myocardial ischemia, severity of angina was estimated by NYHA class, CCS scale, and SAQ 3 days before CSWT schedule (baseline) and 1 week after CSWT schedule. Significant reductions in NYHA class (from 3.0 to 2.0, P < .0001) were observed after CSWT treatment as compared with baseline. Patients before therapy had CCS class III angina symptoms with marked limitation of ordinary physical activity. After CSWT treatment, patients had CCS class II angina with slight limitation of ordinary physical activity (Figure 1B). Cardiac shock wave therapy increased the SAQ score of patients by 83.0% (Figure 1C), which indicated the quality of life of patients was improved by CSWT treatment. Left ventricular ejection fraction, LVEDV, and LVESV showed no significant differences between patients before and after CSWT (Figures 1D-F).

Figure 1.

Clinical parameters before and after cardiac shock wave therapy (CSWT; n = 23). A, New York Heart Association (NYHA) class. B, Canadian Cardiovascular Society (CCS) angina scale. C, Seattle Angina Questionnaire (SAQ). D, Left ventricular ejection fraction (LVEF). E, Left ventricular end diastolic volume (LVEDV). F, Left ventricular end systolic volume (LVESV). Before indicates 3 days before CSWT; after, 1 week after the 3-week CSWT schedule.

To evaluate the effect of CSWT on myocardial blood flow, SPECT images of all patients were also taken 3 days before CSWT schedule (baseline) and 1 week after CSWT schedule. Although there was no significant difference in SRS before and after CSWT, significant improvement in SSS by the 3-week schedule CSWT was observed (from 22.5 ± 3.9 to 18.8 ± 2.9; P < .001, Figure 2B). Representative images depicted in Figure 2A showed that CSWT improved myocardial blood flow in the anterior wall near the apex and the inferior wall of the left ventricle.

Figure 2.

Typical single-photon emission computed tomography (SPECT) of the patient. Marked improvement in summed stress score (SSS) and summed rest score (SRS) was observed after cardiac shock wave therapy (CSWT) treatment. A, Representative images of SPECT from 1 patient before and after CSWT treatment. 0 indicates normal; 1, equivocal; 2, moderate reduction; 3, severe reduction; 4, absent. B, The CSWT treatment significantly decreased SSS, whereas it did not affect SRS (n = 23). Before indicates 3 days before CSWT; after, 1 week after the 3-week CSWT schedule.

RNA Sequencing and Quantitative Reverse Transcription–PCR

In order to explore how CSWT improved myocardial ischemia, gene expression profiling was carried out on PBMCs from 3 patients before and after CSWT using Illumina Genome Analyzer. Comparing with samples before CSWT, 227 genes were significantly upregulated and 183 genes were notably downregulated in samples after CSWT (Supplemental Figure 1). To further characterize the effects of CSWT on ischemic HF, we performed GSEA using the sequencing data. Briefly, a ranked gene list was generated by comparing the RNA sequencing data of patients before and after CSWT. Then, enrichment of different pathway gene sets in this ranked gene list was evaluated using GSEA. Among 165 Kyoto Encyclopedia of Genes and Genomes pathways, 12 pathways were upregulated (Supplemental Table 1) and 5 pathways (Supplemental Table 2) were downregulated after CSWT treatment (nominal P < .05). It is noteworthy that CSWT treatment was positively correlated with multiple genes in cytokine and cytokine receptor interaction (P = .0; Figure 3A) and chemokine signaling pathway (P = .0396, Figure 3B), indicating that CSWT may exert its effects by regulating both pathways. Further, mRNA levels of several genes in the above 2 pathways were then measured in 23 pairs of PBMC samples by quantitative PCR. As shown in Figure 4, VEGF-A, VEGF-B, chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL2, CXCL3, and tumor necrosis factor receptor superfamily member 12A (TNFRSF12A) were upregulated by CSWT treatment. One downregulated gene, mitogen-activated protein kinase 9 (MAPK9), was also confirmed by quantitative reverse transcription–PCR (Figure 4G).

Figure 3.

The signaling pathways bioinformatically predicted to be regulated by CSWT treatment. Using gene set enrichment analysis (GSEA) algorithm, cytokine and cytokine receptor interaction (A) and chemokine signaling pathway (B) were significantly enriched based on CSWT treatment were predicted. Enrichment score (ES) and P value are shown as indicated.

Figure 4.

The messenger RNA (mRNA) levels of (A) vascular endothelial growth factor A (VEGF-A), (B) VEGF-B, (C) CXCL1, (D) CXCL2, (E) CXCL3, (F) TNFRSF12A, and (G) mitogen-activated protein kinase 9 (MAPK9) in PBMC from 23 patients were detected by quantitative reverse transcription–polymerase chain reaction (qRT-PCR). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was served as an internal control. Before indicates 3 days before CSWT; after, 1 week after the 3-week CSWT schedule.

Discussion

Previous studies have demonstrated the treatment effects of CSWT on patients with severe CAD and refractory angina.^4
–6 Here, we investigated the effects of CSWT on 23 patients with ischemic HF. We reported that CSWT significantly relieved angina as evaluated by NYHA class and CCS Scale and improved life quality as assessed by SAQ (Figure 1). The SPECT imaging studies revealed the improvement in myocardial blood flow in ischemic myocardium following CSWT treatment (Figure 3), which might ascribe to the promotion of angiogenesis by CSWT. However, this study is limited by lacking of a control group who received sham CSWT. Medical devices are thought to have placebo effects, which can only be identified with a sham-based randomized clinical trial. Further research with sham CSWT group will better evaluate the efficacy of CSWT.

Previous studies suggested a role of PBMCs on the prognosis of chronic HF and might be a target for the treatment of cardiovascular disease. The adhesive ability of PBMCs from patients with severe chronic HF to cultured human aortic endothelial cells was greater than cells from healthy controls and patients with mild chronic HF,¹³ which can supply prognostic information of these patients. Expression of IL-1 superfamily in PBMCs from patients with CAD was significantly higher than that from the healthy controls. The mRNA levels of IL-1α and IL-1β decreased significantly by atorvastatin or simvastatin therapy.¹⁴ Here, in order to explore how CSWT improved myocardial ischemia, we performed RNA sequencing on PBMCs collected from 3 patients before and after CSWT. The GSEA revealed that CSWT treatment was positively correlated with multiple genes in cytokine and cytokine receptor interaction (Figure 3A) and chemokine signaling pathway (Figure 3B). A previous study has demonstrated the role of VEGF-B in the revascularization of the ischemic myocardium.¹⁵ Cardiac shock wave therapy markedly increased the levels of VEGF-A in an in vivo animal model.⁸ Chemokines, including CXCL1, CXCL2 and CXCL3,¹⁶ and TNFRSF12A¹⁷ are recognized as potential promoters of neovascularization. Here, the mRNA level of VEGF-A, VEGF-B, CXCL1, CXCL2, CXCL3, and TNFRSF12A was found increased following CSWT treatment (Figure 4), which suggested that CSWT may exert its effects by upregulating VEGF, chemokines, and TNFRSF12A. Moreover, Kaiser et al reported that less injury and cellular apoptosis were found in MAPK9-knockout mice following cardiac ischemia–reperfusion injury.¹⁸ Thus, our finding that CSWT decreased the expression of MAPK9 (Figure 4) indicated that CSWT may improve myocardial ischemia by suppressing MAPK9 expression. Further investigations are needed to clarify the detailed mechanisms how CSWT affected angiogenesis and apoptosis through regulating VEGF, TNFRSF12A, chemokines, and MAPK9.

Conclusion

Our results confirm the efficacy of CSWT in the treatment of patients with ischemic HF and identify gene expression affected by CSWT treatment. Collectively, CSWT is an effective repeatable therapy for myocardial ischemia in patients with ischemic HF.

Footnotes

Author Contributions

Wenxia Wang contributed to design, acquisition and interpretation, and critically revised the manuscript. Hua Liu contributed to acquisition and critically revised the manuscript. Mengxian Song contributed to analysis. Weiyi Fang contributed to acquisition. Fang Yuan contributed to conception and design, critically revised the manuscript, and gave final approval. All authors agrees to be accountable for all aspects of work ensuring integrity and accuracy.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by Science and Technology Development Program of Shanghai Chest Hospital (2014 YZDH20101).

Supplemental Material

The online figures and tables are available at

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