Intraoperative Wound Irrigation for the Prevention of Surgical Site Infection After Laparotomy: A Randomized Clinical Trial by CHIR-Net.

Importance: Surgical site infections frequently occur after open abdominal surgery. Intraoperative wound irrigation as a preventive measure is a common practice worldwide, although evidence supporting this practice is lacking. Objective: To evaluate the preventive effect of intraoperative wound irrigation with polyhexanide solution. Design, Setting, and Participants: The Intraoperative Wound Irrigation to Prevent Surgical Site Infection After Laparotomy (IOWISI) trial was a multicenter, 3-armed, randomized clinical trial. Patients and outcome assessors were blinded to the intervention. The clinical trial was conducted in 12 university and general hospitals in Germany from September 2017 to December 2021 with 30-day follow-up. Adult patients undergoing laparotomy were eligible for inclusion. The main exclusion criteria were clean laparoscopic procedures and the inability to provide consent. Of 11 700 screened, 689 were included and 557 completed the trial; 689 were included in the intention-to-treat and safety analysis. Interventions: Randomization was performed online (3:3:1 allocation) to polyhexanide 0.04%, saline, or no irrigation (control) of the operative wound before closure. Main Outcome and Measures: The primary end point was surgical site infection within 30 postoperative days according to the US Centers for Disease Control and Prevention definition. Results: Among the 689 patients included, 402 were male and 287 were female. The median (range) age was 65.9 (18.5-94.9) years. Participants were randomized to either wound irrigation with polyhexanide (n = 292), saline (n = 295), or no irrigation (n = 102). The procedures were classified as clean contaminated in 92 cases (8%). The surgical site infection incidence was 11.8% overall (81 of 689), 10.6% in the polyhexanide arm (31 of 292), 12.5% in the saline arm (37 of 295), and 12.8% in the no irrigation arm (13 of 102). Irrigation with polyhexanide was not statistically superior to no irrigation or saline irrigation (hazard ratio [HR], 1.23; 95% CI, 0.64-2.36 vs HR, 1.19; 95% CI, 0.74-1.94; P = .47). The incidence of serious adverse events did not differ among the 3 groups. Conclusions and Relevance: In this study, intraoperative wound irrigation with polyhexanide solution did not reduce surgical site infection incidence in clean-contaminated open abdominal surgical procedures compared to saline or no irrigation. More clinical trials are warranted to evaluate the potential benefit in contaminated and septic procedures, including the emergency setting. Trial Registration: drks.de Identifier: DRKS00012251.

Microbiome Dysbiosis with Enterococcus Presence in the Upper Gastrointestinal Tract is A Risk Factor for Mortality in Patients Undergoing Surgery for Pancreatic Cancer.

BACKGROUND: Recent retrospective studies suggest a role for distinct microbiota in the perioperative morbidity and mortality of pancreatic head resections. OBJECTIVE: We aimed to prospectively investigate the microbial colonization of critical operative sites of pancreatic head resections to identify microbial stratification factors for surgical and long-term oncologic outcomes. METHODS: Prospective biomarker study applying 16S rRNA sequencing and microbial culturing to samples collected from various sites of the GI tract and surgical sites of patients during pancreatic head resections at a German single high-volume pancreatic center. RESULTS: A total of 101 patients were included (38 non-cancer, 63 cancer patients [50 PDAC patients]) in the study. In a first data analysis series, 16S rRNA sequencing data were utilized from 96 patients to assess associations of microbiome profiles with clinical parameters and outcomes. In general, microbiome composition varied according to sampling site, cancer, age or preoperative ERCP intervention, notably for the bile microbiome. In the PDAC subcohort, compositional variance of the bile or periampullary microbiome was significantly associated with postoperative complications such as ICU admission; on a taxonomic level we observed Enterococcus spp. to be significantly more abundant in patients developing deep or organ-space surgical site infections (SSI). Elevated Enterococcus relative abundances in the upper GI tract, in turn, were associated with 6-months mortality rates. In a second step, we focused on microbiological cultures collected from bile aspirates during surgery and investigated associations with perioperative complications and long-term survival. Notably, Enterococcus spp. were among the most prevalent pathobiont isolates observed in cancer patient bile specimens that were associated with severe SSIs, and thereby elevated mortality rates up to 24 months. Clinically relevant postoperative pancreatic fistulas or severe SSI were found as other major variables determining short-term mortality in this cancer patient cohort. In the context of adverse microbiological factors, a preoperative ERCP was also observed to segregate long-term survival, and it appeared to interact with the presence of Enterococcus spp. as highest mortality rates were observed in PDAC patients with both preoperative ERCP and presence of E. faecalis in bile aspirates. CONCLUSIONS: The presence of Enterococcus spp. in bile ducts of PDAC patients undergoing pancreatic surgery represents a significant risk factor for perioperative infections and, thereby, elevated postoperative and long-term mortality. This finding supports previous data on the use of the antibiotic drug piperacillin-tazobactam as appropriate perioperative antibiotic prophylaxis for preventing adverse outcomes after pancreatoduodenectomy.

Baseline Interleukin-6 as a Preoperative Biomarker for Liver Fibrosis.

Introduction: Liver (hepatic) fibrosis (LF) is characterized by impaired function and regenerative capacity of the liver and can lead to significantly increased morbidity and mortality in the context of surgical liver resection (LR). For this reason, it is crucial to identify the extent of LF preoperatively. Interleukin-6 (IL-6) is known to play a key role in the pathogenesis of LF, but its exact value as a preoperative marker is unknown. This study aimed to investigate the correlation between preoperatively determined IL-6 and the presence of LF. Methods: In this prospective study, IL-6 was determined in 134 consecutive patients undergoing LR. Patients with liver cirrhosis (LC) and patients with clinical or laboratory signs of inflammation were excluded. LF was graded by a blinded pathologist with regard to the degree of LF according to the Desmet classification (0-4). Baseline IL-6 and degree of LF were correlated. Results: A total of 134 patients were prospectively included prior to LR. For 104 patients, LF was graded and inflammatory parameters were available. Thirty-five of these patients showed LC (Desmet 4), and another 33 patients showed preoperatively elevated inflammatory markers. Two of the remaining patients were liver transplant patients. These patients were excluded from the final analysis. According to Desmet, the remaining 34 patients had LF grade 0 or 1 (none or minimal LF) in 26 cases and LF grade 2 or 3 (moderate-to-severe LF) in 8 cases. Correlation of LF with preoperatively determined IL-6 yielded significantly higher IL-6 levels in the group of patients with moderate-to-severe LF (Desmet 2 or 3) compared to the group with none or minimal LF (Desmet 0 or 1; p = 0.0495). Conclusion: In the context of LR, our results showed a correlation of preoperatively determined IL-6 with the extent of LF present. Higher serum baseline IL-6 concentrations were associated with a higher degree of LF, whereas no other blood parameter or score was that predictive for LF. Our results suggest that baseline IL-6 might serve as a valuable parameter to assess LF prior to LR. More patients need to be analyzed to further evaluate and confirm the predictive accuracy of IL-6 for LF.

Effect of Surgery on Postoperative Levels of the Gut Homeostasis-Regulating Enzyme Intestinal Alkaline Phosphatase.

BACKGROUND: Intestinal homeostasis is a crucial factor for complication-free short- and long-term postoperative recovery. The brush border enzyme intestinal alkaline phosphatase (IAP) is an important regulator of gut barrier function and intestinal homeostasis and prevents endotoxemia by detoxifying lipopolysaccharides (LPSs). As IAP is predominantly secreted by enterocytes in the duodenum, we hypothesized that pancreaticoduodenectomy (PD) leads to a significantly stronger decrease in IAP than other major abdominal surgery. STUDY DESIGN: Pre- and postoperative blood, stool, and intestinal samples were collected from patients undergoing PD, as well as other major surgical procedures without duodenectomy. The samples were analyzed using enzyme histochemistry, the para -nitrophenyl phosphate method for IAP, and the limulus amebocyte lysate assay for LPS. RESULTS: Overall, 88 patients were prospectively enrolled in the study. Fecal IAP activity negatively correlated with serum LPS (r = -0.3603, p = 0.0006). PD led to a significant decline in IAP compared to preoperative baseline levels (p < 0.0001). The decline in IAP correlated with the length of proximal small intestinal resection (r = 0.4271, p = 0.0034). Compared to controls, PD was associated with a much more pronounced reduction in IAP-also after adjusting for surgical trauma (operative time, blood loss; r = 0.4598, p = 0.0086). Simultaneously, PD triggered a clearly more prominent increase in serum LPS compared to controls (p = 0.0001). Increased postoperative LPS was associated with an elongated hospitalization (r = 0.7534, p = 0.0062) and more prominent in pancreatic cancer (p = 0.0009). CONCLUSIONS: Based upon the functional roles for IAP, supplementation with exogenous IAP might be a new treatment option to improve short- and long-term outcome after PD.

Preoperative ultrasound elastography for postoperative pancreatic fistula prediction after pancreatoduodenectomy: A prospective study.

BACKGROUND: Postoperative pancreatic fistulas are the most frequent major complications after pancreatoduodenectomy. The soft pancreatic texture is a critical, independent risk factor for postoperative pancreatic fistulas after pancreatoduodenectomy. The current gold standard for postoperative pancreatic fistula risk evaluation consists of the surgeon's intraoperative palpation of the pancreatic texture and, thus, lacks objectivity. In this prospective study, we used ultrasound-based shear-wave elastography, image data analysis, and a fistula risk score calculator to correlate the stiffness of pancreatic tissue with the occurrence of clinically relevant postoperative pancreatic fistulas. METHODS: We included 100 patients with pancreatic pathologies (71% pancreatic ductal adenocarcinoma) and 100 healthy individuals who were preoperatively assessed via real-time tissue ultrasound-based shear-wave elastography on a Philips EPIQ 7 ultrasound device and had pancreatic parenchyma histologically evaluated with manually stained images. RESULTS: We found a significant difference in the mean elasticity between the soft (1.22 m/s) and the hard pancreas group (2.10 m/s; P < .0001). The mean elasticity significantly correlated with the pancreatic fibrosis rate and the appearance of a postoperative pancreatic fistula after pancreatoduodenectomy. Low elasticity (≤1.2 m/s, mean) correlated with soft and high elasticity (>2.0 m/s, mean) with hard pancreatic parenchyma, as assessed by pathologic evaluation. Multivariate analysis revealed a mean elasticity of <1.3 m/s as a significant cut-off predictor for clinically relevant postoperative pancreatic fistulas (P = .003; Youden-Index = 0.6945). CONCLUSION: Preoperative ultrasound-based shear-wave elastography is a feasible and objective clinical diagnostic modality in evaluating pancreatic tissue stiffness. A mean pancreatic elasticity of <1.3 m/s was a significant independent risk predictor of clinically relevant postoperative pancreatic fistulas after pancreatoduodenectomy.

Diagnostic and Therapeutic Management of Early Colorectal Cancer.

Background: Early colorectal cancer (eCRC) is defined as cancer that does not cross the submucosal layer of the colon or rectum, including carcinoma in situ (pTis), pT1a, and pT1b. Early carcinomas differ in their prognosis depending on the risk profile. The differentiation between low and high risk is essential. The low-risk group includes R0-resected, well (G1) or moderately (G2) differentiated tumors without lymphatic vessel invasion (L0), without blood vessel invasion (V0) and a tumor size ≤3 cm. In this constellation, the estimated risk of lymph node metastasis is around 1% or below. The high-risk group includes tumors with incomplete resection (Rx), poor (G3) or undifferentiated (G4) carcinomas, and/or lymphatic and blood vessel invasion (L1) and size ≥3 cm. In a "high-risk" situation, there is a risk for lymph node metastasis of up to 23%. Summary: The incidence of eCRC is rising with a rate of 10% in all endoscopically removed lesions during colonoscopy. For a correct histological evaluation, all suspected lesions should be completely resected. In case of a pT1 lesion in the rectum, pelvic magnetic resonance imaging should be performed to evaluate for suspicious lymph nodes. The therapeutic approach for eCRC is based on histological assessment and ranges from endoscopic resection to radical oncological surgery. The advantages, disadvantages, and associated risks of the individual treatment strategy need to be carefully discussed on a tumor board and with the patient. Key Messages: Treatment options for early colorectal cancer depend on the histological assessment. Poorly differentiated carcinomas, a Kudo ≥ SM2 classified lesion, and a Haggitt level 4 always represent a "high-risk" situation. It should also be mentioned that in rectal cancer, local surgical tumor excision (full-wall excision) is also sufficient for pT1 carcinomas with a "low-risk" constellation (G1/G2; L0, size <3 cm) and an R0 resection.

Gut Barrier Dysfunction and Bacterial Lipopolysaccharides in Colorectal Cancer.

BACKGROUND: Inflammation is known to be an essential driver of various types of cancer. An increasing number of studies have suggested that the occurrence and development of colorectal cancer (CRC) are linked to the inflammatory microenvironment of the intestine. This assumption is further supported by the fact that patients with inflammatory bowel disease (IBD) are more likely to develop CRC. Multiple studies in mice and humans have shown that preoperative systemic inflammatory response is predictive of cancer recurrence after potentially curative resection. Lipopolysaccharides (LPS) are membrane surface markers of gram-negative bacteria, which induce gut barrier dysfunction and inflammation and might be significantly involved in the occurrence and development of CRC. METHODS: A selective literature search was conducted in Medline and PubMed, using the terms "Colorectal Cancer", "Gut Barrier", "Lipopolysaccharides", and "Inflammation". RESULTS: Disruption of intestinal homeostasis, including gut barrier dysfunction, is linked to increased LPS levels and is a critical factor for chronic inflammation. LPS can activate the diverse nuclear factor-κB (NF-κB) pathway via Toll-like receptors 4 (TLR4) to promote the inflammatory response, which aggravates gut barrier dysfunction and encourages CRC development. An intact gut barrier prevents antigens and bacteria from crossing the intestinal endothelial layer and entering circulation. In contrast, a damaged gut barrier triggers inflammatory responses and increases susceptibility to CRC. Thus, targeting LPS and the gut barrier might be a promising novel therapeutic approach for additional treatment of CRC. CONCLUSION: Gut barrier dysfuction and bacterial LPS seem to play an important role in the pathogenesis and disease progression of colorectal cancer and therefore require further investigation.

Prophylactic effect of retromuscular mesh placement during loop ileostomy closure on incisional hernia incidence-a multicentre randomised patient- and observer-blind trial (P.E.L.I.O.N trial).

BACKGROUND: Incisional hernia is a frequent complication following loop ileostomy reversal. Incisional hernias are associated with morbidity, loss of health-related quality of life and costs and warrant the investigation of prophylactic measures. Prophylactic mesh implantation at the time of surgical stoma reversal has shown to be a promising and safe method to prevent incisional hernias in this setting. However, the efficacy of this method has not yet been investigated in a large multicentre randomised-controlled trial (RCT) with adequate external validity. The P.E.L.I.O.N. trial will evaluate the efficacy of prophylactic mesh reinforcement after loop ileostomy closure in decreasing the rate of incisional hernia versus standard closure alone. METHODS: P.E.L.I.O.N. is a multicentre, patient- and observer-blind RCT. Patients undergoing loop ileostomy closure will undergo intraoperative 1:1 randomisation into either abdominal wall closure with a continuous slowly absorbable suture in small-stitch technique without mesh reinforcement (control group) or abdominal wall closure with an additional reinforcement with a retromuscular non-absorbable, macro-pore (pore size ≥ 1000 µm or effective porosity >0%) light-weight monofilament or mixed structure mesh. A total of 304 patients (152 per group) will need to be randomised in the study. Based on inclusion and exclusion criteria, 1,014 patients are expected to be screened for eligibility in order to recruit the necessary number of patients. The primary endpoint will be the frequency of incision hernias within 24 months according to the European Hernia Society definition. Secondary endpoints will be the frequency of surgical site occurrences (including surgical site infections, wound seromas and hematomas, and enterocutaneous fistulas), postoperative pain, the number of revision surgeries and health-related quality of life. Safety will be assessed by measuring postoperative complications ≥ grade 3 according to the Dindo-Clavien classification. DISCUSSION: Depending on the results of the P.E.L.I.O.N. trial, prophylactic mesh implantation could become the new standard for loop ileostomy reversal. TRIAL REGISTRATION: DRKS00027921, U1111-1273-4657.

Analysis of Circulating Immune Subsets in Primary Colorectal Cancer.

The development and progression of colorectal cancer (CRC) are known to be affected by the interplay between tumor and immune cells. However, the impact of CRC cells on the systemic immunity has yet to be elucidated. We aimed to comprehensively evaluate the circulating immune subsets and transcriptional profiles of CRC patients. In contrast to healthy controls (HCs), CRC patients had a lower percentage of B and T lymphocytes, T helper (Th) cells, non-classical monocytes, dendritic cells, and a higher proportion of polymorphonuclear myeloid-derived suppressor cells, as well as a reduced expression of CD69 on NK cells. Therefore, CRC patients exhibit a more evident systemic immune suppression than HCs. A diagnostic model integrating seven immune subsets was constructed to distinguish CRC patients from HCs with an AUC of 1.000. Moreover, NR3C2, CAMK4, and TRAT1 were identified as candidate genes regulating the number of Th cells in CRC patients. The altered composition of circulating immune cells in CRC could complement the regional immune status of the tumor microenvironment and contribute to the discovery of immune-related biomarkers for the diagnosis of CRC.

Endoscopic Treatment Options for Gastrointestinal Leaks.

Background: Spontaneous or postoperative gastrointestinal defects are still life-threatening complications with elevated morbidity and mortality. Recently, endoscopic treatment options - up and foremost endoscopic vacuum therapy (EVT) - have become increasingly popular and have shown promising results in these patients. Methods: We performed an electronic systematic search of the MEDLINE databases (PubMed, EMBASE, and Cochrane) and searched for studies evaluating endoscopic options for the treatment of esophageal and colorectal leakages and/or perforations until March 2022. Results: The closure rate of both esophageal and colorectal defects by EVT is high and even exceeds the results of surgical revision in parts. Out of all endoscopic treatment options, EVT shows most evidence and appears to have the highest therapeutic success rates. Furthermore, EVT for both indications had a low rate of serious complications without relevant in-hospital mortality. In selected patients, EVT can be applied without fecal diversion and transferred to an outpatient setting. Conclusion: Despite multiple endoscopic treatment options, EVT is increasingly becoming the new gold standard in endoscopic treatment of extraperitoneal defects of the upper and lower GI tract with localized peritonitis or mediastinitis and without close proximity to major blood vessels. However, further prospective, comparative studies are needed to strengthen the current evidence.

Laparoscopic intraarterial catheterization with selective ICG fluorescence imaging in colorectal surgery.

The quality of mesorectal resection is crucial for resection in rectal cancer, which should be performed by laparoscopy for better outcome. The use of indocyanine green (ICG) fluorescence is now routinely used in some centers to evaluate bowel perfusion. Previous studies have demonstrated in animal models that selective intra-arterial ICG staining can be used to define and visualize resection margins in rectal cancer. In this animal study, we investigate if laparoscopic intra-arterial catheterization is feasible and the staining of resection margins when performing total mesorectal excision with a laparoscopic medial to lateral approach is possible. In 4 pigs, laparoscopic catheterization of the inferior mesenteric artery (IMA) is performed using a seldinger technique. After a bolus injection of 10 ml ICG with a concentration of 0.25 mg/ml, a continuous intra-arterial perfusion was established at a rate of 2 ml/min. The quality of the staining was evaluated qualitatively. Laparoscopic catheterization was possible in all cases, and the average time for this was 30.25 ± 3.54 min. We observed a significant fluorescent signal in all areas of the IMA supplied, but not in other parts of the abdominal cavity or organs. In addition, the mesorectum showed a sharp border between stained and unstained tissue. Intraoperative isolated fluorescence augmentation of the rectum, including the mesorectum by laparoscopic catheterization, is feasible. Inferior mesenteric artery catheterization and ICG perfusion can provide a fluorescence-guided roadmap to identify the correct plane in total mesorectal excision, which should be investigated in further studies.

Author Correction: Risk factors for serious morbidity, prolonged length of stay and hospital readmission after laparoscopic appendectomy - results from Pol-LA (Polish Laparoscopic Appendectomy) multicenter large cohort study.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Risk factors for serious morbidity, prolonged length of stay and hospital readmission after laparoscopic appendectomy - results from Pol-LA (Polish Laparoscopic Appendectomy) multicenter large cohort study.

Laparoscopic appendectomy (LA) for treatment of acute appendicitis has gained acceptance with its considerable benefits over open appendectomy. LA, however, can involve some adverse outcomes: morbidity, prolonged length of hospital stay (LOS) and hospital readmission. Identification of predictive factors may help to identify and tailor treatment for patients with higher risk of these adverse events. Our aim was to identify risk factors for serious morbidity, prolonged LOS and hospital readmission after LA. A database compiled information of patients admitted for acute appendicitis from eighteen Polish and German surgical centers. It included factors related to the patient characteristics, peri- and postoperative period. Univariate and multivariate logistic regression models were used to identify risk factors for serious perioperative complications, prolonged LOS, and hospital readmissions in acute appendicitis cases. 4618 laparoscopic appendectomy patients were included. First, although several risk factors for serious perioperative complications (C-D III-V) were found in the univariate analysis, in the multivariate model only the presence of intraoperative adverse events (OR 4.09, 95% CI 1.32-12.65, p = 0.014) and complicated appendicitis (OR 3.63, 95% CI 1.74-7.61, p = 0.001) was statistically significant. Second, prolonged LOS was associated with the presence of complicated appendicitis (OR 2.8, 95% CI: 1.53-5.12, p = 0.001), postoperative morbidity (OR 5.01, 95% CI: 2.33-10.75, p < 0.001), conversions (OR 6.48, 95% CI: 3.48-12.08, p < 0.001) and reinterventions after primary procedure (OR 8.79, 95% CI: 3.2-24.14, p < 0.001) in the multivariate model. Third, although several risk factors for hospital readmissions were found in univariate analysis, in the multivariate model only the presence of postoperative complications (OR 10.33, 95% CI: 4.27-25.00), reintervention after primary procedure (OR 5.62, 95% CI: 2.17-14.54), and LA performed by resident (OR 1.96, 95% CI: 1.03-3.70) remained significant. Laparoscopic appendectomy is a safe procedure associated with low rates of complications, prolonged LOS, and readmissions. Risk factors for these adverse events include complicated appendicitis, postoperative morbidity, conversion, and re-intervention after the primary procedure. Any occurrence of these factors during treatment should alert the healthcare team to identify the patients that require more customized treatment to minimize the risk for adverse outcomes.

Intraarterial indocyanine green (ICG) fluorescence augmentation by marking embryonal resection areas in colorectal surgery: a feasibility study in a porcine model.

Aim: In this pilot animal study we examined whether it is possible to visualize the embryonal resection layers by using intraarterial indocyanine green (ICG) staining when performing total mesorectal excision (TME) for carcinoma of the rectum. Material and methods: We injected ICG into the inferior mesenteric artery (AMI) of four swines to see whether the watershed area of the arterial supply zone can be sufficiently visualized by fluorescence imaging in order to mark the right dissection area along the fascia parietalis before and during resection. Results: We observed a fluorescence signal in all the supplied areas of AMI but not in other parts of the abdominal cavity or other organs. Additionally, the mesorectum also showed a sharp border between colored and non-colored tissue. Conclusion: In this study we present that resection borders may be determined before resection based on ICG-perfusion and we showed that intraoperative exclusive coloring of the rectum including the mesorectum is possible. Visualizing resection borders based on ICG-perfusion before settling the first cut may be a new approach in oncological surgery.

Evaluation of pain relief sufficiency using the Cumulative Analgesic Consumption Score (CACS) and its modification (MACS).

Postoperative pain is one of the major complications in general and bariatric surgery, associated with ongoing problems such as ileus, pneumonia and prolonged mobilization. In this study, patients undergoing bariatric surgery were analyzed according to their postoperative pain relief regime. In one group patients were treated with a patient-controlled analgesia (PCA) device, while the other group was treated with oral and intravenous analgesic medication. The aim of this study was to analyze which postoperative pain relief therapy would be more appropriate. We chose the Cumulative Analgesic Consumption Score (CACS) and Numeric Rating Scale (NRS) for pain measurement. For better comparison, we performed a modification of CACS according to PCA treatment. We observed better pain relief in the PCA group. Furthermore, we observed an advantage of treatment with laxatives in patients treated with PCA. In conclusion, PCA devices are appropriate instruments for postoperative pain relief in bariatric patients. CACS is a practical tool for postoperative pain measurement, describing individual pain sensation more objectively, although holding further potential in modification.

TRPC4α and TRPC4ß Similarly Affect Neonatal Cardiomyocyte Survival during Chronic GPCR Stimulation.

The Transient Receptor Potential Channel Subunit 4 (TRPC4) has been considered as a crucial Ca2+ component in cardiomyocytes promoting structural and functional remodeling in the course of pathological cardiac hypertrophy. TRPC4 assembles as homo or hetero-tetramer in the plasma membrane, allowing a non-selective Na+ and Ca2+ influx. Gαq protein-coupled receptor (GPCR) stimulation is known to increase TRPC4 channel activity and a TRPC4-mediated Ca2+ influx which has been regarded as ideal Ca2+ source for calcineurin and subsequent nuclear factor of activated T-cells (NFAT) activation. Functional properties of TRPC4 are also based on the expression of the TRPC4 splice variants TRPC4α and TRPC4ß. Aim of the present study was to analyze cytosolic Ca2+ signals, signaling, hypertrophy and vitality of cardiomyocytes in dependence on the expression level of either TRPC4α or TRPC4ß. The analysis of Ca2+ transients in neonatal rat cardiomyocytes (NRCs) showed that TRPC4α and TRPC4ß affected Ca2+ cycling in beating cardiomyocytes with both splice variants inducing an elevation of the Ca2+ transient amplitude at baseline and TRPC4ß increasing the Ca2+ peak during angiotensin II (Ang II) stimulation. NRCs infected with TRPC4ß (Ad-C4ß) also responded with a sustained Ca2+ influx when treated with Ang II under non-pacing conditions. Consistent with the Ca2+ data, NRCs infected with TRPC4α (Ad-C4α) showed an elevated calcineurin/NFAT activity and a baseline hypertrophic phenotype but did not further develop hypertrophy during chronic Ang II/phenylephrine stimulation. Down-regulation of endogenous TRPC4α reversed these effects, resulting in less hypertrophy of NRCs at baseline but a markedly increased hypertrophic enlargement after chronic agonist stimulation. Ad-C4ß NRCs did not exhibit baseline calcineurin/NFAT activity or hypertrophy but responded with an increased calcineurin/NFAT activity after GPCR stimulation. However, this effect was not translated into an increased propensity towards hypertrophy but rather less hypertrophy during GPCR stimulation. Further analyses revealed that, although hypertrophy was preserved in Ad-C4α NRCs and even attenuated in Ad-C4ß NRCs, cardiomyocytes had an increased apoptosis rate and thus were less viable after chronic GPCR stimulation. These findings suggest that TRPC4α and TRPC4ß differentially affect Ca2+ signals, calcineurin/NFAT signaling and hypertrophy but similarly impair cardiomyocyte viability during GPCR stimulation.