There is little data in the literature regarding the preparation methods of the intra-articular portion of quadruple hamstring-tendon grafts for anterior cruciate ligament (ACL) reconstruction. The aim of this study was to compare the biomechanical properties of a sutured transplant to that of a non-sutured transplant. The hypothesis was that adding stitches to the intra-articular portion of the graft increased its resistance.p < 0.05.A comparative cadaveric study was carried out on five pairs of knees. The average age of the cadavers was 68 years. The exclusion criterion was past knee surgery. In the Sutured Group (SG) two stitches were made on the grafts. No stitches were made on the grafts of the Non-sutured Group (NSG). A tensile failure test was carried out using an Instron® loading machine. The maximal load to failure and stiffness were recorded and we observed the mode of failure for each graft. Statistical analysis was performed using the Wilcoxon rank sum test. Level of significance was set at p = 0.188). The stiffness of the grafts for the NSG was 23.5 N/mm ± 5.3 (17.8–29 N/mm) and 19.7 N/mm ± 5.5 (13.2–24.7 N/mm) for the SG grafts. Overall stiffness values for the NSG were 19% higher than those of the SG; however, the results were not statistically significant (p = 0.438). The failure mode was a rupture at the fixation point except for one sample from the SG which failed at an intra-articular stitch.The hypothesis proposed was not confirmed; adding stitches to the intra-articular portion of the four-strand hamstring-tendon graft does not increase its biomechanical properties. The maximal load to failure was 233.5 N ± 40.6 (186.7–274.5 N) for the NSG, 19.6% higher than for the SG which was 195.2 N ± 42.9 (139.0–238.2 N). Nevertheless, the difference observed was not statistically significant (Whilst the initial hypothesis was not verified, nevertheless, the maximal loads to failure and stiffness were approximately 20% higher when there were no intra-articular stitches compared to the situation where stitches were added to the intra-articular portion of the graft. This was a cadaveric pilot study and, therefore, whilst we are not able to extend our results to clinical practice, the outcomes would indicate the need for further development of this and related protocols deriving from the question of whether there is weakening the graft when adding stitches to its mid-substance. These results remain to be confirmed by further research. Anterior cruciate ligament (ACL) reconstructions performed increase year on year and closer attention has been paid to revision cases in order to understand the reasons for re-rupture . SeveralDespite the existence of extensive literature on ACL reconstruction using hamstring-tendon grafts , there iSome surgeons do not add any stitches, whilst others join the four strands together with several stitches, or perform homogeneous suturing along the entire length of the transplant. There is no consensus and few publications regarding the preparation of the intra-articular portion of the four-strand hamstring transplant. To our knowledge, three articles have been published , 7 to daThe aim of this study was to evaluate whether complementary stitches passing through the four-strands of the ‘central’ portion of the transplant, would increase its tolerable load to failure and stiffness. A cadaveric study was carried out to compare the biomechanical characteristics of a ‘sutured’ transplant to that of a ‘non-sutured’ transplant. The assumed cadaveric study hypothesis was that complementary stitches would increase the resistance of the hamstring autograft. The main outcome was the maximum load at failure of the graft and the secondary outcomes were stiffness and the failure mode.This cadaveric study was performed at the GEBOAS-ICube Biomechanics Laboratory of the Institute of Anatomy of Strasbourg, France. Five pairs of fresh-frozen cadaver legs (10 knees) were used. The donors were three men and two women with a mean age of 68 years 61–79 years). Knees with evidence of prior surgery were excluded from the study. None of the knees had visible incisions, ligament damage nor osteoarthritis [9 years. ° to preserve the mechanical properties of the tissues [The specimens were stored at − 20 tissues , 5. Prio tissues . The cadThe preparation of the grafts was identical for every subject according to the surgical reference technique . The semTM, Ethicon®, Somerville, NJ, USA). The area between the two attachments of the graft ends was measured and divided into three parts. At the junction of each third, an additional stitch was applied which passed through each of the four strands  Fig. . The secnds Fig. .Fig. 2NTo prevent desiccation, the tendons were moistened and wrapped in saline-soaked gauze sponges. The sponges were then wrapped in aluminium foil, placed in an airtight bag, and stored at − 20 °C until further use .The stiffness, ultimate load to failure and the mode of failure were recorded in testing to failure. The specimens were thawed for 12 h at room temperature (18 °C) before the mechanical testing. Each end of the tendon was then fixed in a grasping clamp over a length of 2.5 cm. Additional sandpaper was used to reinforce the grip of the clamp and avoid slippage during the test  Fig. 4)4). The tThe load to failure test was initiated by applying a 10-N preload followed by a tensile test at 10 mm/min constant speed until failure, using a standard and validated test protocol . The loaThe failure mode was defined by the level where the graft rupture occurred. Two types of rupture were distinguished. Failure of the graft in zone 1 meant a rupture at the grip interface of the graft on the clamp and failure in zone 2 was mid-substance failure between the two fixation points  and XLSTAT 2018 .p < 0.05.Statistical analysis of the failure load and stiffness was performed using the Wilcoxon rank sum test with a 5% alpha risk. The level of significance was set at All the tests were performed without incident. No slippage of tendons in the grips during testing was detected. The values collected for each group are summarized in Table p = 0.188). Pull-out strengths for the non-sutured tendons were higher than for the respective paired, sutured tendons with one exception. The stiffness of the grafts for the NSG was 23.5 N/mm ± 5.3 (17.8–29 N/mm) and 19.7 N/mm ± 5.5 (13.2–24.7 N/mm) for the SG. Overall stiffness values for the NSG were 19% higher than those of the SG; however, the results were not statistically significant   for the NSG, 19.6% higher than for the SG which was 195.2 N ± 42.9 (139.0–238.2 N). The difference observed was not statistically significant (Regarding the failure mode, the location of the rupture was closely analysed for all the grafts whilst performing the tests. In the SG group, the transplant rupture occurred in zone 1 in four cases and in zone 2 in one case. Concerning the latter, the rupture occurred by a dilaceration of the tendon fibres from one of the intra-articular stitches Fig. . In the The working hypothesis was that additional stitches would increase the resistance of the transplants and, therefore, reduce their risk of breakage. The results of this study do not support that hypothesis. There was no significant difference between the two groups, even though the maximum loads to failure and stiffness were 20% greater in the NSG compared to those in the SG. Adding stitches to the intra-articular portion of the transplant does not seem to reinforce its properties. The biomechanical test outcomes even suggest that such added stitches could constitute points of weakness as observed in SG subject 4. This assumption is supported by all other transplant ruptures occurring at the fixation point and not on the graft mid-substance.. have sought to determine the causes of these failures [Considering all the different techniques, the iterative rupture rate of a reconstructed ACL is 6% according to the results of a meta-analysis by Jenny et al. . Van Eckfailures . In 58% The comparison with other study results was difficult because of the scarcity of similar publications. Furthermore, this study differs from previous ones because we investigated the use of isolated stitches instead of braided tendons or uniform sutures.Nicklin et al. and Millet et al. studied the effects of continuous braiding of the four strands of a hamstring graft on the tensile strength and rigidity of the graft , 12. ThePailhé et al. found that a uniform suture of the patellar tendon over its entire length increased the graft strength . They coThe results are also tempered by this study’s limitations. The maximum load values in the current study were much lower than those of published values and lower than the tension needed for walking or running. One step produces a tension of about 150 N on the ACL whilst walking and about 450 N whilst running. In order to meet the specifications of an ACL reconstruction, a transplant is expected to be able to support a load of 500 N. In this study, the tensile-strength breaking values are all well below 500 N. These values remain much lower than in other cadaveric study publications , with vaThe experimentation on cadavers, and especially on elderly subjects, makes it difficult to extrapolate to clinical practice. However, all the data available in the literature is also experimental. Due to the cadaveric nature of this study, we could not take into account the biological phenomenon of transplant integration during the post-operative phase .. who have shown that freezing has no influence on elongation during loading, but that the maximum breaking load and rigidity are reduced [The samples were stored frozen and underwent two freeze-thaw steps. These steps do not significantly alter the samples’ biomechanical properties , 5. This reduced . The exp reduced , 15.We acknowledge some limitations to the technique used to fix the samples . The chaThe sample used was small, a limitation inherent to the availability of cadaver parts. This restricted sample may explain the lack of significant difference between the two groups even though the results in the NSG were superior.We believe, however, that this work has its own strengths as no study of this type has yet been published to our knowledge. Furthermore, the knees were paired and so were their own comparators. The weakness of this pairing is that we did not know the dominant character of the knees. Nevertheless, there were no significant differences in terms of length and thickness between the two groups.Whilst the initial hypothesis was not verified, nevertheless, the maximal load to failure and stiffness was approximately 20% higher when there were no intra-articular stitches compared to the situation where stitches were added to the intra-articular portion of the graft. This study remains important due to the absence of consensus on how to prepare the intra-articular portion of hamstring transplants whilst approximately 17% of iterative ACL ruptures are secondary to an intra-articular rupture of the transplant. This was a cadaveric pilot study and, therefore, whilst we are not able to extend our results to clinical practice, the outcomes would indicate the need for further development for this and related protocols deriving from the question of weakening the graft when adding stitches on its mid-substance. These results remain to be confirmed by further research.


Hemorrhage due to a ruptured splanchnic pseudoaneurysm followed by the formation of a postoperative pancreatic fistula is the most severe complication of a pancreatectomy, sometimes leading to a fatal outcome. Stent graft placement to control the hemorrhage due to the pseudoaneurysm is a validated treatment option, but once the stent graft is infected, infection control is complicated. We report a case of a ruptured pseudoaneurysm of the splanchnic artery after pancreaticoduodenectomy to evaluate the stent graft treatment.A 77-year-old man underwent pylorus-preserving pancreaticoduodenectomy for suspected distal bile duct cancer. Hemorrhage from a pseudoaneurysm of the common hepatic artery due to the formation of the pancreatic fistula was detected on postoperative day 9, and a stent graft was successfully placed with the preservation of hepatic arterial blood flow. On postoperative day 12, new-onset hemorrhage from a pseudoaneurysm of the right hepatic artery developed, and a stent graft was similarly placed, but immediately occluded. Refractory pancreatic and biliary fistulas developed and required continuous drainage. On postoperative day 85, computed tomography revealed the presence of air within the latter stent graft, which indicated infection of the stent graft. The patient died due to sepsis caused by the graft infection.Stent graft placement for the treatment of hemorrhage of a pseudoaneurysm secondary to a postoperative pancreatic fistula, following pancreaticoduodenectomy, is an effective treatment option as it achieves immediate hemostasis and maintains end-organ perfusion. However, stent graft infection is the most detrimental complication. Pancreaticoduodenectomy (PD) is a standard surgical procedure for the treatment of pancreatic head and distal bile duct disease. The mortality rate due to a PD has decreased to less than 5%, but the morbidity rate remains high, ranging between 30 and 50% . HemorrhEscherichia coli was confirmed by blood culture, and the patient died on POD 91 due to sepsis caused by the graft infection. This study was approved by the ethics committee of our institution, and informed consent was obtained from the patient’s next-of-kin.A 77-year-old man, with a background of hypertension and diabetes mellitus, underwent pylorus-preserving PD for suspected distal bile duct cancer. He developed a pancreatic anastomotic leakage on postoperative day (POD) 6, which was draining from a drain placed at the pancreaticojejunal anastomosis during surgery. Sentinel bleeding from the drain was noted on POD 8, and an immediate contrast-enhanced computed tomography (CT) scan revealed no obvious bleeding points. However, a massive hemorrhage from the drain suddenly developed on POD 9, and emergency angiography detected a pseudoaneurysm at the CHA, which had caused the bleeding Fig. a. The riPseudoaneurysms are believed to be caused by anatomical leakages, mainly due to pancreatic fistulas or injury of the arterial wall during operative dissection and lymphadenectomy . It is cDespite the identification of the sentinel bleeding and having performed the CT scan in our case, the pseudoaneurysms were not clearly detected, and angiography was canceled. Sentinel bleeding is considered a sign of pseudoaneurysm bleeding prior to massive hemorrhage and is often observed in 45–78% of patients who have undergone PD .Surgical re-exploration is the conventional management of hemorrhage due to a pseudoaneurysm. However, the surgery is difficult to perform due to tissue fragility and postoperative adhesions, which results in a significant risk of mortality and morbidity. The operative mortality rate has been reported as 43–50%. Therefore, surgical re-exploration is recommended when an endovascular approach is difficult . EndovasTo avoid these complications, stent graft placement is preferred over TAE. Stent graft placement provides excellent flexibility, enables precise deployment of the stent, and maintains end-organ arterial blood flow. Several reports on stent graft placement for the treatment of pseudoaneurysms after PD have recently been published , 10. AsaStent graft placement for the treatment of hemorrhage of a pseudoaneurysm secondary to a POPF after PD is an effective treatment option. It immediately achieves hemostasis and maintains end-organ perfusion better than that in TAE and surgical re-exploration. If local infection around the pseudoaneurysm is uncontrolled, and the stent graft is occluded, a lack of immediate infection control may lead to a fatal outcome.


Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) show tremendous promise for cardiac regeneration, but the successful development of hESC-CM-based therapies requires improved tools to investigate their electrical behavior in recipient hearts. While optical voltage mapping is a powerful technique for studying myocardial electrical activity ex vivo, we have previously shown that intra-cardiac hESC-CM grafts are not labeled by conventional voltage-sensitive fluorescent dyes. We hypothesized that the water-soluble voltage-sensitive dye di-2-ANEPEQ would label engrafted hESC-CMs and thereby facilitate characterization of graft electrical function and integration.We developed and validated a novel optical voltage mapping strategy based on the simultaneous imaging of the calcium-sensitive fluorescent protein GCaMP3, a graft-autonomous reporter of graft activation, and optical action potentials (oAPs) derived from di-2-ANEPEQ, which labels both graft and host myocardium. Cardiomyocytes from three different GCaMP3+ hESC lines  were transplanted into guinea pig models of subacute and chronic infarction, followed by optical mapping at 2 weeks post-transplantation.Use of a water-soluble voltage-sensitive dye revealed pro-arrhythmic properties of GCaMP3+ hESC-CM grafts from all three lines including slow conduction velocity, incomplete host-graft coupling, and spatially heterogeneous patterns of activation that varied beat-to-beat. GCaMP3+ hESC-CMs from the RUES2 and ESI-17 lines both showed prolonged oAP durations both in vitro and in vivo. Although hESC-CMs partially remuscularize the injured hearts, histological evaluation revealed immature graft structure and impaired gap junction expression at this early timepoint.Simultaneous imaging of GCaMP3 and di-2-ANEPEQ allowed us to acquire the first unambiguously graft-derived oAPs from hESC-CM-engrafted hearts and yielded critical insights into their arrhythmogenic potential and line-to-line variation. Human embryonic stem cells (hESCs) have a number of attractive properties for the repair of injured hearts, including tremendous capacity for in vitro expansion and the ability to differentiate into phenotypically unambiguous cardiomyocytes –8. The tOur group has previously reported one approach to obtain useful insights into the electrical integration of hESC-CM grafts in normal and injured hearts . For thiWhile this approach based on GCaMP3 imaging alone has provided crucial insights into the integration of hESC-CM grafts, it does have a number of important limitations. First, GCaMP3 imaging yields no information as to electrical activation of host myocardium, precluding study of the complex spatiotemporal electrical interactions between graft and host tissue. Second, GCaMP3 senses the rise in intracellular calcium, which is obviously delayed relative to membrane depolarization. Moreover, because GCaMP3 is a particularly slow calcium sensor , GCaMP3 We report here our efforts to overcome the above limitations using a novel approach that involves the simultaneous imaging of GCaMP3, the aforementioned genetically encoded calcium-sensitive fluorescent protein that functions as a graft-autonomous reporter of graft activation, and di-2-ANEPEQ, a water-soluble voltage dye that labels both host and graft tissue. This approach allows one to reliably distinguish between host- and graft-derived oAPs and to obtain previously unavailable electrophysiological measurements of hESC-CM graft tissue in injured hearts.We created transgenic H7, ESI-17, and RUES2 hESC lines that stably expressed GCaMP3  via the All hESC experiments were conducted with the approval of either the University of Washington ESC Research Oversight Committee or the Canadian Institutes of Health Research (CIHR) Stem Cell Oversight Committee (SCOC). Note that, while experiments were initiated with the RUES2 hESC line when the laboratory was at the University of Washington, all work with this line had to be discontinued upon relocation to our present institution (RUES2 hESCs are not included in the CIHR SCOC registry of lines approved for use in Canada).6 hESC-CMs per well. Absorbance was measured from 350 to 700 nm in 5 nm steps, while emission was determined from 500 to 800 nm in 5 nm steps following excitation at 480 nm (with a 495 nm cutoff filter). Confocal experiments were performed using a Zeiss LSM510-Meta confocal microscope  operated in lambda-scanning mode, with excitation set to 488 nm and emission detected from 502 to 748 nm in 11 nm steps. We also simultaneously recorded GCaMP3 and di-2-ANEPEQ fluorescence transients by confocal microscopy in time-series mode using 505–530 nm and 650–754 nm bandpass filters, respectively.Spectrofluorimetry and spectral confocal microscopy were used to define the excitation and emission spectra of GCaMP3 and di-2-ANEPEQ  in intact cardiomyocytes. WT and GCaMP3+ hESC-CMs were stained by incubation with di-2-ANEPEQ (5 μM) at 37 °C for 10 min, spun down, switched to dye-free buffer, and used immediately. For spectrofluorimetry experiments, we employed a Spectra Max M2 microplate reader  and 96-well plates loaded with 2 × 108 GCaMP3+ hESC-CMs. Cells were delivered in a pro-survival cocktail of factors that we have previously shown enhances graft retention and survival [All animal procedures were conducted with the approval of the local institutional animal care committees of either the University of Washington or University Health Network (Toronto) in compliance with corresponding national guidelines. We have previously described in detail our methods for the cardiac cryoinjury procedure, intra-cardiac cell injection, and subsequent harvesting of the heart for ex vivo imaging , 14, 36.survival . To prev3, 1.2 MgSO4, 4.7 KCl, 118.0 NaCl, 1.2 KH2PO4, 11.0 glucose, 1.0 Na-pyruvate and 1.8 CaCl2, bubbled with 95% O2/5% CO2, pH adjusted to 7.35, and warmed to 37 °C. To arrest motion during ex vivo imaging, the perfusate was supplemented with blebbistatin . To label hearts with the conventional lipophilic voltage dye RH237, hearts were either perfused with RH237-containing buffer (40 μM) for 30 min or bolus-loaded with RH237 (80 μM in 5 ml volume) over 5 min. Identical results were obtained with either method. To label hearts with the water-soluble voltage dye di-2-ANEPEQ, the latter was added to the perfusion buffer (20 μM concentration) and applied via continuous recirculation loop. Supplementary Fig. SCryoinjured hearts with GCaMP3+ hESC-CM grafts were harvested at 2 weeks post-transplantation and immediately mounted on a modified Langendorff apparatus perfused with the following buffer (in mM): 25.0 NaHC0Supplementary Fig. S6 ohm) electrodes coated with parylene-C insulation . The resultant signals were amplified using a high-impedance intracellular electrometer with variable-capacity neutralization  and then fed into the PowerLab system.During these ex vivo imaging experiments, a pseudo-ECG was acquired using a PowerLab 430 Data Acquisition System (Model ML866) outfitted with a bioamplifier , and the resultant signals were aligned with camera outputs via Labchart software. For recordings obtained under paced conditions, we used the PowerLab 430 system connected to a pencil-point concentric electrode  placed into the LV apex. For a subset of experiments, we obtained simultaneous intracellular voltage recordings via sharp electrodes, using methods modified from Omichi et al. . For thiz-score of each pixel through time played to screen to facilitate identification of active graft regions. Next, we manually selected regions of interest (ROIs) and plotted their mean fluorescence intensity over time . Activation maps of tissue were created by determining for each pixel the timepoint associated with the maximal rate of change in fluorescence activity between the initiation and peak of a single activation [Custom Matlab (MathWorks) scripts were developed to analyze imaging data. In brief, time-synchronized fluorescent images and ECG recordings  were imported into the Matlab environment, and the former were subjected to the following sequence of initial processing steps. First, the images were masked to exclude signals outside the heart, then spatial, low pass frequency, and temporal filters were applied to improve signal quality. The results of filtering were then assessed at the single pixel level before removing signal drift. Next, the data was inverted for voltage dye-signals (which exhibit a decrease in fluorescence intensity upon tissue depolarization through the emission filters described), and a pseudocolored movie of activation based on the tivation . To aid tivation , whereby6 hESC-CMs were aliquoted per well into the commercially available AggreWell 800 system  as per the manufacturer’s recommendations. Eight days later, the formed aggregates were loaded with di-2-ANEPEQ (20 μM) for 5 min at 37 °C, then dispersed and transferred into 23-mm fluorodishes for imaging at 37 °C. hESC-CM aggregates were imaged using the modified epifluorescence dissecting microscope and dual EM-CCD system described above for ex vivo epicardial imaging. Both the single-cell and aggregate preparations were field-stimulated at 1 Hz using custom-made parallel 0.25 mm diameter silver wire electrodes and the PowerLab system (with pulse duration set at 5 ms and voltage at 10 V). Analysis of in vitro optical recordings was performed using custom Matlab scripts as described above. The oAPD from each single cell or aggregate was defined as the mean of > 4 measured oAPDs, and reported results reflect at least > 12 recordings obtained from at least 3 differentiation runs per cell line.oAPs were recorded from WT and GCaMP3+ hESC-CMs in vitro as both single cells and ~ 300 μm diameter aggregates. For the single-cell recordings, hESC-CMs were plated onto gelatin-coated 23-mm glass-bottom fluorodishes . After 4–5 days, the cells were loaded with di-2-ANEPEQ (40 μM) for 5 min at 37 °C, then were transferred to dye-free buffer at 37 °C. Images were acquired using a × 20 objective and an Olympus IX-7 inverted microscope outfitted with an external mercury lamp , filtered to 450–490 nm, and the same emission light path and EM-CCD cameras as described above. To form the aggregates, 3.6 × 10Histological endpoints were obtained using methods previously detailed by our group , 13, 40.Host-graft contact was determined using histological sections immunostained for GFP and counterstained with picrosirius red. Graft expression of Cx43 and cadherin expression was assessed by confocal immunofluorescence using a Zeiss LSM 780 NLO confocal microscope . All measurements were performed by an observer blinded to hESC line and experimental condition.t test, paired or unpaired as appropriate with Welch’s correction for unequal variances as necessary. Multiple comparisons were made using one-way ANOVA with Tukey’s multiple comparison test correction. A p value less than 0.05 was considered significant, and asterisks (*) used in figures indicate a significant difference between experimental groups.GraphPad Prism  was used to perform all statistical analyses. All data groups were first checked for normality by submission to the D’Agostino-Pearson omnibus test, and parametric data is presented as mean ± standard error of the mean. Two-sample comparisons were made using a two-tailed Student’s n = 3). For these imaging experiments, we used a custom-built, dual-channel, EM-CCD-based system to simultaneously acquire the RH237 (“red” channel) and GCaMP3 (“green” channel) signals. As in our previous work [Our group has previously reported that when hESC-CM-engrafted hearts are stained with conventional lipophilic voltage-sensitive fluorescent dyes , these dyes label host but not graft tissue . To confous work , we consous work .Fig. 1Rn = 3). In contrast to our prior experience with RH237, when these hearts were transversely sectioned and examined, we observed strong, uniform staining of both host and GCaMP3+ graft tissue by di-2-ANEPEQ  and injured (n = 3) guinea pig hearts without grafts and consistently detected robust di-2-ANEPEQ-derived oAPs. We next correlated host di-2-ANEPEQ-derived oAPs to simultaneously acquired direct electrode recordings [n = 2) , while APD90 measurements based on RH237 and direct electrode recordings were 146.2 ± 1.5 vs 144.7 ± 1.3 ms (p = 0.3). Note that the amplitude of di-2-ANEPEQ fluorescence transients was larger than those obtained with RH237 in the same heart (4.7% vs 2.4% ΔF/F), an encouraging finding given our goal of detecting oAPs in graft tissue.Because there has been very limited published experience with the use of di-2-ANEPEQ in cardiac optical mapping, we next performed experiments to verify its suitability as a myocardial voltage reporter. For this, we first imaged uninjured  would report electrical activity in both graft and host tissue, while GCaMP3 fluorescence (“green” channel) would function as a critical graft-autonomous reporter of graft activation. If successful, this approach would provide us a reliable means of distinguishing between host- and graft-derived electrical signals with high spatial and temporal resolution.8 GCaMP3+ RUES2 hESC-CMs into cryoinjured guinea pig hearts at 28 days post-injury (n = 5). We then harvested engrafted hearts at 2 weeks post-transplantation and imaged them ex vivo using the dual-channel CCD-based system and the experimental protocol depicted in Supplementary Fig. STo test this approach, we transplanted 1 × 10graft-derived oAPs that were superimposed on the continuing host-derived oAPs . In this case, we observed excellent temporal agreement between the di-2-ANEPEQ and directly recorded APs, with graft APDn = 3–5 per condition). Due to restrictions that arose during experiments, the RUES2 line was not tested at the 10-day timepoint. Qualitatively similar di-2-ANEPEQ dye wash-in and wash-out kinetics was noted across all conditions, and grafts from all lines showed similar oAP amplitudes.Given this capacity to reliably detect and distinguish between host- and graft-derived electrical activity, we advanced to experiments to investigate how parameters including cell line, the timing of cell transplantation, and spontaneous versus paced conditions might affect the electrical behavior of engrafted hearts. To examine this, we transplanted GCaMP3+ hESC-CMs from each of the three cell lines  into cryoinjured guinea pig hearts at either 10 or 28 days post-injury  that were not observed in grafts formed with their H7 counterparts   Fig. a. These Although grafts formed using hESC-CMs from the three different parental lines showed differences in their oAPDs and host-graft coupling outcomes, we did not find significant differences in other parameters including visible graft area Fig. d or grafAdditional file 7: Supplementary Movie File 2. GCaMP3+ hESC-CM graft acting as an ectopic pacemaker. A cryoinjured heart with GCaMP3+ H7 hESC-CM graft was harvested at 14 days post-transplantation, loaded with di-2-ANEPEQ, and imaged ex vivo during spontaneous beating . This video shows the dynamic epicardial di-2-ANEPEQ fluorescent signal, which reports electrical activation in both host and graft tissue, as well as the time-synchronized host ECG, both displayed at one-twentieth actual speed. The heart is oriented with the apex on the right and the base on the left. Host and graft voltage activation have been pseudo-colored with black indicating rest and white depolarization. Note that during the first, second, and fourth beats shown, electrical activation actually begins first in hESC-CM graft tissue located near the center of the heart. Only during the third beat, in what could otherwise be interpreted as a premature ventricular contraction, does electrical activation initiate normally in host tissue near the left ventricular apex.Our group has previously examined in detail the composition of hESC-CM grafts as well as their structural effects on recipient hearts , 16, 42,To rule out a difference in intercalated disc structure between grafts formed with cardiomyocytes from different hESC-CM lines, we immunostained recipient hearts with antibodies against the major gap junction protein connexin-43 (Cx43) and the adherens junction protein N-cadherin. Consistent with our prior work , 13, 17,In prior work, our group has used patch-clamp techniques to obtain measurements of APD in WT hESC-CMs on the order of ~ 200 ms , 40. OurhESC-CMs are a promising cell source for potential novel regenerative therapies, but concerns about the risk of graft-related arrhythmias have highlighted the need for new tools to investigate their electrophysiological properties in vivo. Optical mapping is an attractive experimental modality for this application, and our efforts to apply this technology here led to several observations of interest. First, we confirmed our earlier findings that it can be very challenging to distinguish between hESC-CM graft- and host-derived optical voltage signals; in fact, in our hands, conventional lipophilic voltage dyes do not label hESC-CM graft tissue at all. To overcome this problem, we tested a novel strategy based on the simultaneous imaging of a graft-autonomous reporter of activation (hESC-CM grafts expressing GCaMP3) and the water-soluble voltage dye di-2-ANEPEQ (which stains both graft and host). In brief, after a sufficient duration of perfusion with di-2-ANEPEQ, graft regions showed the superposition of two signals: graft-derived oAPs  and host-derived oAPs . However, during washout, labeling was much more rapidly lost from host myocardium, resulting in the transient isolation of graft-derived oAPs . Hence, while problematic for the longer-term goal of remuscularizing the injured heart with electrically integrated new myocardium, the phenomenon of uncoupled hESC-CM grafts  is that GCaMP3 expression itself appears capable of significantly altering the electrophysiological properties of hESC-CMs. Indeed, in our hands, GCaMP3 exerted significant AP-prolonging effects on cardiomyocytes derived from two different hESC lines (RUES2 and ESI-17), both in vitro and after transplantation into injured hearts. AP prolongation was observed in recordings from both individual transgenic cardiomyocytes and submillimeter multicellular aggregates. This phenomenon was unexpected, as to our knowledge, no one has previously reported such off-target effects by GCaMP3 expression. Indeed, transgenic mice created with systemic and cardiomyocyte-restricted overexpression of the closely related GCaMP molecule GCaMP2 had seemingly normal cardiac function . While bdirect demonstration of graft ectopy , but it is entirely consistent with recent observations from our group based on electroanatomic mapping following hESC-CM transplantation in a swine infarct model [Notwithstanding these limitations, the imaging approach developed in the present study allowed us to obtain important new insights into the electrophysiological properties of hESC-CM grafts in injured hearts. In addition to acquiring previously unknown parameters such as graft CV and APD, we were able to directly observe complex host-graft electrical interactions and extrapolate to outcomes at the organ level. For example, in the case of the heart depicted in Supplementary Movie File 2 and Fig. ct model . The facct model .functional block to integration. Such a block might be due to low levels of Cx43 gap junction expression in graft tissue and/or delays in recovery and depolarization after a preceding long AP (as was frequently observed in these experiments). Low expression and/or isotropic distribution of Cx43 gap junctions may have also contributed to the very slow graft CVs found in this study . There are multiple factors that could have contributed to this outcome. One obvious explanation for the lack of host-graft coupling would be an absence of physical contact between host and graft, but this parameter did not obviously vary in histological sections between grafts from different lines. We also speculate that failure to couple could result from a ocardium .)Finally, we anticipate that the optical approaches developed in this report will be very useful for testing new approaches to improve graft electrical integration and function , as well as for exploring how recipient parameters may affect these same outcomes. For example, while here we used male and relatively young (~ 8-week-old) animals in the present experiments, we intend future work to address how graft electrophysiological function may differ in male versus female or young versus aged recipients. Next, while we have compared outcomes in both subacute and chronic cardiac injury models, it will be important to investigate how co-morbidities or pharmacological treatments that might be encountered in human patients with ischemic cardiomyopathy  may affect graft electrophysiology. Finally, we are particularly interested to understand the molecular basis of the line-to-line differences in electrophysiological behavior noted in vivo  with details of the filter set used for excitation (C). In brief, excitation light was collimated and bandpass filtered to 450–490 nm before being reflected onto the Langendorff-mounted heart. Emitted light was collected and split into the “green” and “red” channels first by a 565 nm longpass dichroic mirror inside the DC2 dual-channel splitter. The “green” channel  signal was then further bandpass filtered to 500–530 nm before being imaged by an EM-CCD camera, while the “red” channel  signal was longpass filtered at 650+ nm for di-2-ANEPEQ imaging or 716+ nm for RH237 imaging before being imaged on a separate EM-CCD camera operated simultaneously.Additional file 2: Supplementary Fig. S2. Spectra of GCaMP3 and di-2-ANEPEQ in hESC-CMs in vitro. A: Absorbance (ABS) and emission (EM) spectra of di-2-ANEPEQ by spectrofluorimetry after loading into hESC-CMs. Note di-2-ANEPEQ-loaded hESC-CMs absorb with a peak at ~ 488 nm and emit with a peak at ~ 600 nm. B: Emission spectra of WT hESC-CMs before and after loading with di-2-ANEPEQ, as determined by confocal lambda scanning. C: Emission spectra of GCaMP3+ hESC-CMs during systole and diastole . D: Emission of GCaMP3+ hESC-CMs after loading with di-2-ANEPEQ as recorded in both systole and diastole. GCaMP3 mediates a large increase in emission signal during systole, while di-2-ANEPEQ emission exhibits a small spectral shift to the left . The shaded areas denote filter sets selected to separate these fluorophores in all subsequent experiments. E: Mean fluorescence activity over time in a single GCaMP3+ hESC-CM after loading with di-2-ANEPEQ, using the filter sets depicted in panel D. Note that while GCaMP3 fluorescence increases upon depolarization, di-2-ANEPEQ fluorescence decreases at the emission wavelengths selected for detection (> 650 nm).Additional file 3: Supplementary Fig. S3. di-2-ANEPEQ reliably reports myocardial electrical activity. Still images and fluorescence traces on the “red” channel from a naïve heart (uninjured and not transplanted) acquired after di-2-ANEPEQ loading (A&B), after di-2-ANEPEQ washout (C&D), and then after loading with RH237 (E&F). These fluorescent oAPs (red traces) were recorded simultaneously and showed good temporal agreement with direct voltage recordings via sharp electrode (black traces). G. Still image (left) and voltage activation map (right) from a naïve heart.Additional file 4: Supplementary Fig. S4. Simultaneous di-2-ANEPEQ imaging and direct electrode recording of hESC-CM graft electrical activity. A cryoinjured heart with hESC-CM graft (H7 line) was impaled with a sharp electrode within the graft tissue to correlate the di-2-ANEPEQ-derived voltage signal with direct intracellular voltage recordings. A: Epicardial still image taken on the di-2-ANEPEQ channel, showing the uncoupled hESC-CM graft footprint (yellow dotted line) inside the cryoinjury region (white dotted line) and the recording electrode positioned in graft tissue. Two ROIs are indicated: one in host myocardium outside the cryoinjury zone and one in hESC-CM graft tissue overlying the tip of the recording electrode. B: Simultaneously acquired oAPs for these two ROIs, as well as the simultaneously acquired direct voltage and ECG recordings. Note the excellent temporal correlation between the direct electrode recordings and di-2-ANEPEQ-derived oAPs from this hESC-CM graft, which was uncoupled from the host.Additional file 5: Supplementary Fig. S5. In vitro optical action potentials from cardiomyocytes from multiple hESC lines. A, B: Representative fluorescence traces from WT or GCaMP3 cardiomyocyte aggregates generated from either the H7 (A) or RUES2 (B) hESC lines. Aggregates were simultaneously imaged on the di-2-ANEPEQ  and GCaMP3  channels during pacing at 1 Hz. While oAPD was similar between WT and GCaMP3+ H7 hESC-CM aggregates, GCaMP3+ aggregates from the RUES2 line typically showed oAPD prolongation relative to their WT counterparts. C: To rule out the possibility that the apparent AP-prolonging effect of GCaMP3 was just reflective of idiosyncratic hESC clones, oAPs were acquired from hESC-CM aggregates formed from four different GCaMP3+ ESI-17 hESC lines. Cardiomyocytes from all four clones showed similar increases in oAPD relative to WT controls.Additional file 6: Supplementary Movie File 1. GCaMP3+ hESC-CM grafts commonly activate along vectors distinct from host tissue and with variable spatial propagation patterns. Representative cryoinjured and GCaMP3+ hESC-CM engrafted heart with the graft exhibiting beat-to-beat changes in spatial activation that are all distinct from the direction of activation in host tissue (host tissue activation not shown). In this movie, graft activation from the GCaMP3 channel is shown at one tenth speed over a still image of the injured heart oriented with the apex at the right, base at the left. Graft activation is pseudocolored black to indicate resting tissue, and white to indicate depolarized tissue. Note this GCaMP3+ H7 hESC-CM graft was uncoupled from the host heart.


V) values are often associated with σ- and π-holes, regions of lower electron density which can interact with electron-rich sites to form noncovalent interactions. Factors impacting σ- and π-holes may thus be monitored in terms of the shape and values of the resulting V. Further precious insights into such factors are obtained through a rigorous decomposition of the V values in atomic or atomic group contributions, a task here achieved by extending the Bader–Gatti source function (SF) for the electron density to V. In this article, this general methodology is applied to a series of 4,4′-bipyridine derivatives containing atoms from Groups VI  and VII , and the pentafluorophenyl group acting as a π-hole. As these molecules are characterized by a certain degree of conformational freedom due to the possibility of rotation around the two C–Ch bonds, from two to four conformational motifs could be identified for each structure through conformational search. On this basis, the impact of chemical and conformational features on σ- and π-hole regions could be systematically evaluated by computing the V values on electron density isosurfaces (VS) and by comparing and dissecting in atomic/atomic group contributions the VS maxima  values calculated for different molecular patterns. The results of this study confirm that both chemical and conformational features may seriously impact σ- and π-hole regions and provide a clear analysis and a rationale of why and how this influence is realized. Hence, the proposed methodology might offer precious clues for designing changes in the σ- and π-hole regions, aimed at affecting their potential involvement in noncovalent interactions in a desired way.Positive electrostatic potential ( VS) is beneficially used to assess electronic properties and interaction capability of specific atoms and sites  × 100.Each VS,max values are largely dominated by the SF(Ch) positive contribution. In particular, the one due to Se is about twice as large as that due to the S atom, for a given R and conformation. The SF(Ch) contribution is in most cases even larger than the VS,max value and ranges, in percentage, from 76% to 134% for Ch = S, and from 169% to 234% for Ch = Se, depending on the conformation. This roughly doubled effect  of the Se is not surprising, due to the lower electronegativity and thus the larger positive charge of Se relative to S (see infra). Conversely, this same reason increases the electronic charge on the R group. It so lowers the SF(R) contribution, changing it from moderately positive  to moderately negative  or from negligibly negative  or moderately negative  to largely negative  on passing from Ch = S to Ch = Se. The trend SF(Me) > SF(Ph) > SF(C6F5) holds true for both Ch = S and Ch= Se and mirrors the trends of the Bader’s net charges q of the R group or, though understandably reversed, the trend of the positive Bader’s net charge on the Ch atom. For instance, for the A1 conformers, the q(R) charges decrease from 0.055 (Me), through −0.001 (Ph), to −0.149 (C6F5) for Ch = S and from −0.038 (Me), through −0.088 (Ph), to −0.251 (C6F5), for Ch = Se, while the q(Ch) charges increase from 0.091 (1-A1), through 0.122 (2-A1), to 0.209 (3-A1) for Ch = S and from 0.278 (4), through 0.315 (5), to 0.410 (6) for Ch = Se.With reference to the decomposition of such hole potentials  into Ch,6F5 group, shifts SF%(Bipy) to slightly positive values (4% to 9%) or to less negative values (about −26%) for Ch = S and Ch= Se, respectively. Although the positive SF(Ch) contributions always dominate the other two SF contributions to the VS,max values, the relative magnitude of the generally negative (or seldom negligibly positive) SF(Bipy) and SF(R) contributions obviously depends on the R group and on the Ch type.Analogously to the SF(R) contribution, also the SF contribution of the Bipy moiety decreases with the decrease of Ch electronegativity. For Ch = Se and for a given R, SF(Bipy) is always negative and greater in magnitude relative to Ch = S. For a given chalcogen, the SF%(Bipy) values are similar for R = Me or Ph (ranging from −14% to −20% for Ch = S and from −46% to –54% for Ch = Se), while the much larger electron-withdrawing power of the R = C6F5) value is from 2 to 10 times larger in magnitude than the SF(Bipy) value, while for R = Ph both contributions are small. However, SF(Bipy) is larger in magnitude than SF(Ph), which is negligibly negative or negligibly positive, depending on the conformer. Conversely, for Ch = Se, the SF(R) and SF(Bipy) contributions are more alike to each other in magnitude and always negative, hence they are always opposing the SF(Se) contribution. For Ch = Se, SF(Bipy) is twice as large in magnitude than SF(Me), while it is 2/3  or 1/3  smaller in magnitude than SF(R) for R = Ph or C6F5, respectively. Dissection of the Bipy source in the contribution from the pyr and pyr’ ring moieties provides further insight. Regardless of the R group, of the system conformation and of the nature of Ch, the source from the pyr ring carrying the chalcogen atom is always negative, while that of pyr’ ring is invariably positive. The magnitude of the source from the pyr ring significantly increases on passing from S to Se, becoming largely negative and bounded to a limited range of values (−0.020/−0.028 au), while that from the pyr’ ring remains relatively small and in the range of 0.0035/0.0064 au for both Ch = S and Ch = Se. The different sign of the two ring sources, along with the large impact that the chalcogen nature has on the negative source of the pyr ring is a clear indication of the electron-donor effect of the chalcogen substituent on such ring. The SF contributions to the Cpyridyl–Ch σ-holes from the 3′-H and 5′-Br atoms are almost constant for all 1–6 conformers, confirming that for such holes, due to their spatial location and at variance with the case of the CR–Ch σ-holes, the molecular conformation does not play any role. We conclude the discussion of VS,max values, is by far less accurate (Err%: 3–13%).In particular, for Ch = S, the SF %, 95–203; SF(Se)%, 175–317), compared to the case of the Cpyridyl–Ch σ-holes (SF(S)%, 80–134; SF(Se)%, 169–235), the magnitudes of SF contributions do follow similar qualitative trends . Given these similarities between the SF(Ch) values and trends of the CR–Ch and Cpyridyl–Ch σ-holes, analogous interpretations to those put forward for R–Ch holes in 3-B2) where it slightly (7%) concurs to the overall VS,max value of the CR–Ch σ-hole. The way they oppose is very much dependent on the system and on its conformation. It may be just a small opposition as for systems 2-A2 and 3-A2 (−11/−20%) or definitely a far larger one as for the two 6-B1 holes (−171/−216%) or the 4-A1 and 5-A1 holes (−198/−162%). The Bipy and the R groups may either cooperate or oppose each other in their action as the ratio of their contributing sources ranges from −6.3 to 2.5, depending on systems and their conformers. Despite such a variety of behaviors, there are some general trends worth to be mentioned. In the case of Ch = Se, the SF contribution for both Bipy and R moieties is invariably negative (as it is for the Cpyridyl–Ch σ-holes), with that of the Bipy generally and significantly prevailing in magnitude over the Me and Ph for systems 4 and 5. Instead, when R = C6F5, it is the negative source of this group which is larger in magnitude in three out of the five reported cases. Analogously to the case of the Cpyridyl–Ch σ-holes, the SF(R) values follow the trend SF(Me) > SF (Ph) > SF(C6F5) for both Ch = Se and S, due to the corresponding increase of the net negative charge of the R group along the series. For Ch = S, SF(R) is positive (R = Me) or still positive but smaller (R = Ph), while it is negative (−19/−59%) for R= C6F5. Regarding the distinct contributions from the two rings of the Bipy moiety, some of the considerations reported for the Cpyridyl–Ch σ-holes apply. In particular, i) the observation about the constantly negative source from the pyr ring and the always positive contribution from the pyr’ ring, and ii) the large increase of the magnitude of the SF(pyr) on passing from Ch = S to Ch = Se, yet to values (−0.039/−0.532) which are almost twice as big as for the Cpyridyl–Ch σ-holes. It is thus once more evident that the contribution of the Bipy moiety is the result of quite distinct sources from its two composing ring moieties. As anticipated, sources from the 3′-H and the 5′-Br atoms to the CR–Ch σ-holes potential maxima are significantly affected in their magnitudes by molecular conformation.Likewise the C σ-holes  are largR–Ch σ-holes maxima), while it is marginally negative or even positive for other conformers (A2 and B2). To be facing (A1 and B1) or not facing (A2 and B2) the σ-hole, drastically changes the role the 5′-Br atom plays in determining the corresponding VS,max value. The source from 3′-H is invariably positive and large (0.010/0.026 au), but particularly large for those conformers where SF(5′-Br) is almost negligible. For instance, SF(3′-H) is as large as 0.025/0.026 au for the B2 conformers.In particular, SF(5′-Br) is quite high in magnitude and negative for A1 (−0.009/−0.017 au) and B1 conformers , the negative SF(R) contribution is roughly of the same order of magnitude as the positive SF(Ch) contribution and the two sources almost cancel each other. As a result, VS,max turns out to be mostly determined by the negative and small SF(Bipy) contribution. Conversely, when R = C6F5 (compounds 3 and 6), SF(R) becomes much smaller in magnitude than SF(Ch). In fact, the former source drastically decreases its magnitude relative to that observed for compounds 2 and 5, while the latter source slightly increases its positive value relative to these compounds because C6F5 withdraws more electrons from the Ch atom than the Ph group does. Combined with the fact that also SF(Bipy) of 3 and 6 becomes smaller in magnitude than for compounds 2 and 5 and definitely smaller than SF(Ch), the largely positive SF(Ch) source provides about 90% of the VS,max value for conformers 3 or even overdetermines largely (140–190%) this value for conformers 6. The fact that for conformers 6 SF(Bipy) is (much) less negative than for compounds 2 and 5 or that it becomes close to zero, or even positive for conformers 3, is easily explained. Indeed, the strong electron-withdrawing power of C6F5 dampens the electron flow from the Ch atom to the Bipy moiety making this moiety less negatively charged and with a correspondingly (much) less negative source, relative to the case of R = Ph . Dissection of SF(Bipy) in its two ring components SF(pyr) and SF(pyr’) shows that the former is negative, as its six-membered-ring (6MR) carries the Ch substitution, and, except for 3-A1, always prevails in magnitude, while the latter is always positive. The SF(Ph) for the π-hole potentials is always negative, because the negative contribution from the 6MR C atoms overweighs the positive SF contribution from their linked H atoms. On the other hand, for R = C6F5 the 6MR C atoms significantly increase their positive charge because of the H→F substitution and so provide an overall positive source contribution at the π-holes. When Ch = S, the negative SF contribution from the fluorine atoms prevails over the positive contribution from the 6MR, while just the opposite occurs when Se replaces S, because the C atoms become even more positively charged and their positive SF contribution significantly increases.Decomposing π-hole potentials into Ch, R and Bipy SF contributions  neatly h2-A2 and 5-A2, whose internal π-holes exhibit significantly more negative VS,max values (−0.0138 and −0.0160 au) than those of their external π-holes (−0.0090 and −0.0081 au). Please note that the potential values for the internal π-holes are the more negative ones for the whole set of investigated systems. The apparent anomaly (illustrated in 5-A2 π-holes) finds an easy explanation in terms of SF(5′-Br) values and considering that in the 2-A2 and 5-A2 conformers the 5′-Br atom points its π-cloud towards the phenyl ring internal π-hole. Indeed, the SF(5′-Br) value is always negative for these conformers but as small as −0.0023 and −0.0025 au for the external and about 6 times as large (−0.0153 and −0.0173 au) for the internal 2-A2 and 5-A2 π-holes. Such an increase in magnitude qualitatively explains the potential differences between the external and internal π-holes for these two conformers.External and internal π-hole potentials exhibit qualitatively the same SF contribution trends as a function of the R and Ch nature. There is, however, an important exception for conformers VS,max values and their atomic (group) sources may be gained by decomposing the changes in their values upon chemical substitution into chemically meaningful contributions. VS,max variations  and corresponding SF contributions changes (ΔSF) upon substitution of either Ch or R atom/group or of both of them for a given conformer of systems 1–6. ΔVS,max values and their composing ΔSF contributions for a system change X→Y are evaluated as ΔZ = Z(Y)-Z(X) where Z = VS,max or SF. Conformers were kept fixed for any considered X→Y change avoiding to introduce a further (and possibly second-order) variable in the analysis. The ΔVS,max values may be decomposed asVS,max value. In substitution term includes all the sources of groups undergoing a change of chemical composition (shown in bold in rearrangement term is the sum of ΔSF (Bipy) and of those ΔSF(Ch) or ΔSF(R) contributions arising from the Ch and R moieties that are not substituted in the X→Y process  larger in magnitude than the ΔVS,max value they reconstruct . Thus, a chemical moiety substitution quite often induces a substantial charge rearrangement also in those moieties whose chemical formula remains fixed. Such a moiety rearrangement is mirrored in its SF contribution change following the X→Y process. Said in other words, each ΔVS,max value for a system change X→Y results from both the substituted and the only apparently untouched moieties. For the sake of conciseness, the analysis worked out in pyridyl–Ch and the CR–Ch σ-holes.Both ΔSFonstruct . When lapyridyl–Ch σ-holes remains positive and almost constant (0.0552/0.0582 au) for all S→Se changes, while a larger spread (0.0324/0.0744 au) is observed for the CR–Ch holes. A similar behavior is found for ΔSF(R) and ΔSF(Bipy) which are however negative and thus opposing rather than contributing to the positive ΔVS,max values. In particular, ΔSF(R) and ΔSF(Bipy) amount to −0.0281/−0.0325 au and −0.0163/−0.0186 au, respectively, for the Cpyridyl–Ch σ-holes and to −0.0164/−0.0177 au and −0.0124/−0.0462 au, respectively, for the CR–Ch holes. Since ΔSF(Ch) values are all largely positive and fairly exceeding the corresponding ΔVS,max values, ΔSFrearrangement turns out to be also largely negative, almost constant for Cpyridyl–Ch holes (−0.0453/−0.0514 au) and significantly more spread out (−0.0291/−0.0629 au) for the CR–Ch holes.Looking at →Se substitution, keeping the Ch fixed and changing R to R’ leads to more complex variations. On passing from R = Me to R’ = Ph or R’ = C6F5 or from R = Ph to R’ = C6F5, ΔSF(R→R’) is always negative. It is also largely spread out (−0.0069/−0.0371 au) and in all cases, save two from R = Me to R’ = Ph , significantly opposing the potential change ΔVS,max. Conversely, ΔSF(Bipy) is always positive and largely varying (0.0033/0.0266 au). ΔSF(Ch) is also usually positive and quite large (0.0104/0.0315 au), despite being only due to charge density rearrangements within the S or Se atomic basins (plus changes due to variations in the σ-holes maxima locations). In three specific cases, however, ΔSF(Ch) is negligibly small  or even negative . ΔSFrearrangement is always positive, generally quite large (0.0144−0.0430 au) and significantly overdetermining the potential change, except for the two cases where ΔVS,max is negative rather than positive. In such an event  ΔSFrearrangement is quite small (about 0.0050 au) and counteracts the potential change.At variance with the case of fixed R and Srearrangement by definition, is always negative and large or moderately large in magnitude. It also opposes the potential change except in one single case  where ΔVS,max is also negative. Secondly, ΔSF(Ch) is invariably positive, quite large (0.0324−0.0886 au) and significantly overdetermining ΔVS,max, except the single case where the latter is negative. ΔSF(R) is instead always negative and smaller in magnitude than ΔSF(Ch).The double substitution case is also complex to analyze, being the result of the rearrangement of the electron distribution of the Bipy moiety and of the Ch and R group replacements. However, two clear indications may be deduced from VS,max analysis. Differently from substitution equal to zero, ΔVS,max (X→Y) ≅ ΔSFrearrangement (Equation (3)). Thus, also the ΔSFrearrangement values should be small, since in this case they represent a response to only a conformational change, rather than to a substitutional change in another moiety of the molecule. For the sake of simplicity, VS,max values for the Cpyridyl–Ch σ-holes are indeed small and arising, in general, from somewhat higher in magnitude ΔSF values since the Ch, R, and Bipy source differences do not have all the same sign. In practice, they may individually concur to or oppose the observed ΔVS,max value. The potential difference values for the CR–Ch σ-holes are instead quite large in many cases. The composing source differences ΔSF(W) show different behaviors among themselves and an exemplar case is shown in pyridyl–Ch holes. Thus, the ΔSF(R) values do not appreciably contribute to the σ-hole potential changes due to the conformational changes. Conversely, ΔSF(Ch) and ΔSF(Bipy) are in most cases quite large and may both concur or may, either one or the other, oppose to the observed ΔVS,max values, according to the investigated conformational change. In summary, the quite large ΔVS,max values for CR–Ch σ-holes are in many cases the result of even larger changes in the source contributions of the Ch and the Bipy moieties. These results show the noteworthy importance of the ΔSFrearrangement contributions in determining hole potential changes, even when arising from the sole molecular conformational change. Indeed, the ΔSFrearrangement (W) value include two contributing factors, one due to the change of the electron distribution within the W moiety and the other due to the changes in the 1/|r − r’| electron density weighting function. According to the location of the potential hole and the nature of the conformational change, the second geometrical factor may acquire a great relevance, while the former factor is generally much less important for a conformational change only. This is indeed the case for the ΔSF  values of CR–Ch σ-holes, where large displacements (>0.2 au) of the holes from the Ch nucleus are observed upon many of the conformational changes listed in M isomers of compounds 1–6 were prepared using the build function, and model kits and tools provided by Spartan’ 10 Version 1.1.0  [VS values, given in au (electrons/bohr), was performed by using Gaussian 09  and at DFT/B3LYP/6–311G* level [VS,max was made through the Multiwfn code [VS on molecular electron density isosurfaces  [VS representations of all conformers of 1–6 depicted in the text  [VS may be simultaneously reconstructed at the same set of reference points (rps) [VS SF reconstruction is quantitatively analogous to evaluating VS as a sum of the electrostatic potential contribution of all QTAIM atomic basins of a molecule [VS field), was written to extract VS atomic SF data calculated by the generalized SF code and to suitably combine them to get VS SF contributions from all atomic groups defined in input [The 3D structures of the configurational CA, USA)  for builG* level . Search wfn code  and thro.002 au) . VS reprthe text  were gra VEXTLOC , was wri SF code  SF_ESI,  (QTAIM)  basin cots (rps) . Such VSmolecule . The SF_molecule . Anotherin input .VS,max values of eight holes carried by the heteroaromatic scaffold were calculated and compared in order to evaluate the impact of chemical and conformational variations on the hole depth. With the aim to gain further insights on the contribution of atoms or groups of the molecule to generate certain VS,max values, the Bader–Gatti SF for the electron density was extended to the electrostatic potential. Naively, a hole VS,max is thought to increase as the remainder of the molecule becomes more electron-attracting. However, VS,max analysis and reconstruction showed that the VS,max values associated with σ- and π-holes located in complex molecular systems depend on the subtle balance of several effects which contribute to increase or decrease the potential value associated with the electron charge density depletion. In particular, negative or positive V contribution from neighboring portions of the molecule were found to significantly concur in determining VS,max along with electronegativity and polarizability properties of bound atoms and groups. The results of this study serve the molecular design of XB, ChB, and π-hole bond donors for applicative purposes, clarifying how chemical and conformational features impact hole regions and affect their potential involvement in noncovalent interactions.This study focused on the interplay between halogen and chalcogen σ-holes, and aromatic π-holes located in the same class of molecules as chemical and conformational variations occur. For this purpose, conformers of six 5,5′-dibromo-2,2′-dichloro-3-chalcogen-4,4′-bipyridines were used as test probes, and


Finally, we will highlight recent studies that described modulations of cardiac stiffness and thus myocardial performance in vivo. Mechanobiology research is just at the cusp of systematic investigations related to mechanical changes in the diseased heart but what is known already makes way for new therapeutic approaches in regenerative biology.Cardiomyocytes are permanently exposed to mechanical stimulation due to cardiac contractility. Passive myocardial stiffness is a crucial factor, which defines the physiological ventricular compliance and volume of diastolic filling with blood. Heart diseases often present with increased myocardial stiffness, for instance when fibrotic changes modify the composition of the cardiac extracellular matrix (ECM). Consequently, the ventricle loses its compliance, and the diastolic blood volume is reduced. Recent advances in the field of cardiac mechanobiology revealed that disease-related environmental stiffness changes cause severe alterations in cardiomyocyte cellular behavior and function. Here, we review the molecular mechanotransduction pathways that enable cardiomyocytes to sense stiffness changes and translate those into an altered gene expression. We will also summarize current knowledge about when myocardial stiffness increases in the diseased heart. Sophisticated  The heart is both an electrical and mechanical organ. A heartbeat is initiated when pacemaker cells at the sinus venosus produce action potentials. This triggers the contraction of cardiomyocytes, which generates the mechanical force required to move blood throughout the body. During each contractile cycle of the heart, cardiomyocytes experience various types of forces, including the hemodynamic pressure by blood, contraction-induced elongation/shortening, and passive elasticity/stiffness from the surrounding extracellular matrix (ECM). Sensitivity of cardiomyocytes to changes in these physical parameters is critical and the mechanical work of the heart has to be adjusted to the levels needed for the physiological blood-based transport of nutrients, gases, and metabolic waste products. Myocardial infarction and cardiomyopathies are characterized by cardiac remodeling, a process that includes increases of cardiomyocyte cell size (hypertrophy), fibroblast proliferation, and the deposition of ECM proteins. These changes alter tissue stiffness and extracellular mechanical stimulation of cardiomyocytes, which affects their biomechanical signaling and heart function. Although cardiomyocytes possess the machinery to sense those mechanical alterations in the diseased heart, ultimately, this does not prevent a reduced cardiac function. However, we are just beginning to understand how biomechanical signaling changes in the diseased heart affect cardiomyocytes. This knowledge promises the development of regenerative cardiac therapies. Many of the proteins covered in this review have been linked to various types of cardiomyopathies. This clearly demonstrates the relevance of cardiac mechanobiology for both normal physiology and for understanding the etiology of cardiomyopathies. Due to the brevity of this review article, we refer to other review articles that cover the disease aspects of these proteins .Here, we review the current state and rapid advances in the field of cardiomyocyte mechanobiology. These have also contributed to a better understanding of myocardial diseases and led to the discovery that cardiac regenerative processes can be modulated by changing mechanical properties of the heart. First, we focus on cytoskeletal and extracellular factors that determine physiological myocardial stiffness. Understanding how changes of tissue stiffness affect the diseased heart requires a clear understanding of which cellular components sense alterations of extracellular mechanical stimuli and transduce these into biochemical signaling and gene expression. For instance, mechanosensitive YAP signaling is highly responsive to matrix stiffness changes in cardiomyocytes and potently regulates cardiomyocyte proliferation . We willE, which describes the relation between tensile stress and axial strain of a material (unit in Pa). During the cardiac contractile cycle, the passive stiffness of the myocardium determines its maximal diastolic capacity/volume.Cardiac conduction imposes a constant mechanical deformation on the myocardium. Intra- and extracellular factors contribute to tissue stiffness, a cardiac mechanical property that allows cardiomyocytes to withstand constant mechanical impact. Stiffness is defined as the resistance of an object to elongation or shortening. The measure of stiffness is the elastic modulus or Young’s modulus Cardiac stiffness changes dramatically during early life. In a comparative study, the murine heart was found to stiffen progressively between embryonic development day E2 with values of < 1 kPa until E14 with a 10-fold increase in stiffness, which is a value that was measured also in neonate and adult myocardium . ConcomiOther studies revealed that Young’s modulus values vary in different vertebrate hearts . Berry eThe maintenance of physiological levels of cardiac stiffness not only determines overall ventricular diastolic function but also ensures proper cardiomyocyte functionality. When neonatal rat myocardium was cultured on substrates with a stiffness value of an adult heart, cardiomyocytes acquired cell morphologies and functions that were characteristic of a differentiated myocardium . SimilarExtra- and intracellular components contribute to myocardial stiffness. On its extracellular side, cardiomyocytes are embedded within an ECM with a characteristic composition of glycosaminoglycans, proteoglycans, collagens, laminins, and fibronectins. These ECM proteins determine cell adhesion, cell motility, and contribute to outside-in signaling into cardiomyocytes . StrikinIntracellular properties of cardiomyocytes also contribute to cardiac stiffness. The main regulator of intracellular stiffness of cardiomyocytes is titin, a giant protein that spans half of the sarcomere  and thatTitin comes in two isoforms within the mammalian heart . The morWhile the overall amount of titin in cardiomyocytes is similar, the ratio of both isoforms varies in the myocardium of different mammalian species . Wu et aMicrotubule networks and intermediate filaments also contribute to myocardial elasticity. Microtubules are supramolecular assemblies of α/β-tubulin heterodimers that are organized as highly dynamic filaments with multiple roles in mitosis, cell motility, or intracellular molecular transport. Within cardiomyocytes, microtubules generate resistance to the compressive load of the heart, which does not only depend on microtubule density but also on specific de-tyrosinations, which allow crosslinking with intermediate filaments. This affects myocardial stiffness and viscoelasticity . In cardThe etiology of many cardiac pathologies has remained unresolved. However, increasing evidence points at a contribution of defective biomechanics in these diseases. These findings raise the question whether changes in mechanical properties of cardiomyocytes are causative to cardiac pathologies or whether pathologies affect biomechanical properties of cardiomyocytes. While it has been difficult to resolve the causality of events in many cardiac pathologies, increasing evidence suggests that physiological levels of cardiac stiffness are disturbed in many instances of cardiac diseases. Both, the cardiac ECM and intracellular components (titin) contribute to physiological levels of cardiac stiffness, which is disturbed in many cardiac diseases . The rolThe giant molecule titin is the main intracellular regulator of passive tension in cardiomyocytes. Beside the ratio of N2B/N2BA isoforms, passive resistance of cardiomyocytes also depends on posttranslational titin modifications. Most studies reported important phosphosites within the “spring-like” I-band domain .. IncreaThe intracellular stiffness factors desmin and α-actinin are additional players in cardiac diseases. In a mouse model of diastolic dysfunction, levels of desmin and α-actinin increased at Z-disks. The authors suggested this to be a response to the increased strain to cardiomyocytes in this pathologic condition , which pcaveolin-1 mutant hearts. However, overall regeneration was not affected. This was in contrast with previous observations, based on a different caveolin-1 mutant, in which cardiomyocyte proliferation and cardiac regeneration were disturbed. Yet, mechanical properties and cardiac function were not examined in those animals , one of the main causes of heart failure, presented with increased myocardial passive stiffness together with ventricular remodeling and abnormalities in left ventricular diastolic function , 2015. TTo date, we are lacking treatment options to avoid the detrimental changes in cardiac stiffness However, studies in mouse demonstrated that cardiac stiffness can be limited by exercise. In a mouse model with HFpEF, the authors examined the effects of free wheel running exercise on diastolic stiffness. This revealed a beneficial effect that was caused by beneficial phosphorylation changes in the PEVK and N2B spring elements of titin, which control stiffness properties of this important mechanical sarcomeric protein. In comparison, exercise did not decrease stiffness of the ECM. In pharmacological experiments, the authors used the drug ivabradine to lower the heart rate, which mimics one of the physiological effects of effective exercise. However, this treatment did not change the passive stiffness of the heart in the HFpEF mouse model. Taken together, this study demonstrated a beneficial effect of exercise-induced regulatory phosphorylations in titin that softens cardiac stiffness .In conclusion, several reports showed that cardiac stiffness increases and cardiac functionality is severely impaired within the diseased heart . These fUnlike most cardiac diseases characterized by increased tissue stiffness, patients suffering from dilated cardiomyopathy (DCM) present with reduced myocardial passive tension causing ventricular enlargement and impaired systolic function. DCM frequently results from genetic defects in the titin gene  after myocardial infarction. Unfortunately, this study lacked data from later timepoints . These sThe heartbeat constantly exposes cardiomyocytes to physical forces by stress and strain. In the previous chapter, we discussed changes of ECM composition, titin phosphorylation, and cardiac tissue elasticity that occur within the diseased heart. These tissue stiffness changes are detected by cardiomyocytes and converted into gene expression changes. Here, we will focus on the machinery that is involved in these mechanosensitive signal transduction processes.The mechanotransduction processes which translate mechanical stimuli into cellular signals are sensitive to changes in shear stress, cell adhesion forces, substrate rigidity, membrane or cytoskeletal stretching, and compression due to pressure. Focal adhesion integrin-based multi-protein complexes are crucial for these processes and mediate inside-out and outside-in signaling in response to mechanical stimuli or signals from the ECM or neighboring cells. Focal adhesions also couple the mechanical tension between ECM and the cytoskeleton . Within In vitro studies on neonatal rat ventricular myocytes revealed the importance of FAK complex signaling for the response of cardiomyocytes to mechanical stretching. FAK became more strongly phosphorylated and changed its subcellular localization when cardiomyocytes were stretched , Rho-associated coiled-coil containing protein kinases (ROCKs), and mitogen-activated protein kinases (MAPKs)  Figure . In vitrtretched . Signalitretched . Mef2 antretched .Mrtfa and Mrtfb impaired the development of the coronary microvasculature . Cellular and biochemical studies showed that the inactive form of MRTF-A is bound to cytoplasmatic globular actin (G-actin). It is released and translocated to the nucleus in a Rho-dependent manner when G-actin polymerizes to form actomyosin  Miralle. There iculature .The Hippo signaling pathway controls organ size and cell proliferation in response to mechanical tension . Two keyWhile YAP is an important regulator and inducer of cardiomyocyte proliferation in the embryonic and postnatal heart, it does not induce hypertrophic growth of the myocardium. Strikingly, nuclear YAP/TAZ localizes to the nucleus of the infarcted murine myocardium at 3, 5, 14, and 36 days post myocardial infarction but not in remote, non-injured regions . In humaThe mode by which YAP/TAZ becomes activated by mechanical stiffness has raised much interest. This led to the discovery of the ECM protein agrin as a stiffness sensor involved in YAP pathway activation. Its function is directly dependent on matrix stiffness and mediates YAP signaling in cells surrounded by hard but not soft matrices. While this has only been shown in mouse hepatocarcinoma cells , it is cThe nuclear envelope has received much attention for its role in mediating mechanical stress into the nucleus. The structural nuclear envelope protein lamin A is stiffness-dependent and regulates chromatin organization, gene expression, and DNA replication . It has These findings highlight the complex regulation involved in cardiac mechanotransduction. The sensation of physical changes and mechanotransduction within cardiomyocytes involves the costamere, a specialized complex of focal adhesion proteins, which mediates inside-out and outside-in signaling. Also, several downstream mechanosensitive signaling pathways become activated in cardiomyocytes in response to changes of matrix stiffness. These include the nuclear sensors YAP/TAZ, lamin A, and MRTF. These studies showed that general mechanisms of mechanotransduction signaling also function in cardiomyocytes and are required for adequate responses to physical changes and during differentiation.Changes of the cardiac ECM affect the compliance of the cardiac ventricle, which impacts the diastolic volume . What arHeras-Bautista et al. used polyacrylamide (PAA) hydrogel matrices with ranges of stiffness corresponding with embryonic (12 kPA), adult (30 kPA), or fibrotic (123 kPA) cardiac tissue as substrates for murine induced pluripotent stem cell-derived cardiomyocytes. Cardiomyocytes that were grown on a stiff matrix had an impaired contractile function with disarranged sarcomeres whereas cardiomyocytes grown on soft and medium matrices had highly organized sarcomeres. Soft matrix conditions also resulted in comparative transcriptomes in contrast to cardiomyocytes exposed to a stiff hydrogel. Gene expression analyses of cardiomyocytes grown on stiff matrices revealed many deregulated genes, especially of genes related to developmental programs . In addiCulturing embryonic rat cardiomyocytes on substrates representing physiological or fibrotic stiffness values revealed that most cells were angular in shape on stiff substrates and roundish on soft substrates. However, neither myofibril organization and function, nor costamere numbers were affected by substrate stiffness . TractioRibeiro et al. examined human pluripotent stem cell-derived cardiomyocyte behaviors when cultured on substrates with a stiffness ranging from 4 to 100 kPA. Contractile forces increased on very stiff matrices (90 kPA), which was in agreement with a previously mentioned study . In contThose studies revealed the importance of a cardiac matrix with physiological stiffness values. Aberrations can dramatically change cardiomyocyte behavior and performance by influencing differentiation, contractile function, gene expression, cell morphology, and force generation . How mecIn a related study, Pandey et al. sought to address how cardiomyocytes probe and sense their environment. For this, they cultured neonatal rat cardiomyocytes on nanopillar arrays with different levels of stiffness. This revealed that the induction of cardiomyocyte hypertrophy on a stiff matrix was dependent on protein kinase C (PKC). PKC regulated the non-receptor tyrosine kinase Src, which activated non-muscle myosin contractions at cell edges that are involved in sensing of cell rigidity. By using a tension sensor for the costamere adaptor protein talin, the authors revealed that cyclic stretching of talin is induced downstream of PKC and Src under conditions of physiological stiffness but is continuously induced on matrices representing fibrotic stiffness. In infarcted mouse hearts, a model for DCM, PKCδ and PKCα localized to integrin adhesion sites at costameres together with non-muscle myosin light chain. This suggested an involvement of PKC- and Src-induced non-muscle myosin contractility for rigidity sensing in the diseased myocardium .Another recent study provided further insights into the complex relationship, upon cardiac infarction, between ventricular stiffness, ECM protein availability, YAP signaling activation, and induction of cardiomyocyte proliferation . The autin vitro studies. These enabled researchers to use well-defined experimental modulations. Several recent studies revealed direct cellular effects due to matrix stiffness changes. Yet, we are far from understanding the detailed interplay between cardiac stiffness and mechanotransduction in the context of the diseased or regenerating heart.Much of our knowledge related to the cellular changes in cardiomyocytes in response to increased tissue stiffness has come from Cardiac diseases often result in fibrotic tissue deposition or cardiomyocyte hypertrophy, two irreversible physiological changes that have a major impact on cardiac function. In this review, we focused on changes of cardiac stiffness that occur in diseased hearts. Myocardial stiffness, which is the potential of cardiomyocytes to resist contraction-induced elongation or shortening, relies on intra- and extracellular components. Stiffness increases in diseased hearts often result from alterations of ECM components. For instance, ECM becomes deposited during fibrosis after myocardial infarction . FibroblWhen the diseased heart turns less compliant, stiffness of the ventricular myocardium prevents that the required physiological volume during diastole is reached and blood flow throughout the body is insufficient. However, besides the global impact that an increase of myocardial stiffness has on heart physiology, there are also crucial cellular changes that occur within the affected myocardium. Here, we reviewed the sophisticated molecular machinery that senses stiffness changes in the environment of cardiomyocytes and mediates appropriate responses . MechanoIn vitro studies have potently shown that changing substrate stiffness affects cardiomyocyte function. However, there are only few reports about how stiffness changes may lead to cardiomyocyte de-differentiation and increased proliferation, which is required for cardiac repair. We are just beginning to discover powerful molecules for inducing cardiomyocyte proliferation and to test their potential in vivo in the diseased heart. One pioneering study involved the discovery of the ECM protein agrin, a stiffness sensor in the mechanotransduction cascade regulating cardiomyocyte proliferation (Knowledge about how cardiomyocytes respond to pathological tissue stiffness changes is crucial when it comes to the development of regenerative therapies. External factors that modulate the ECM and tissue stiffness in the diseased heart represent an excellent source for drug and treatment development. They are more easily applicable as part of a therapy than drugs that directly target an intracellular signaling cascade. feration . Its potferation .Another promising approach based on modulating isoforms of the basement membrane protein laminin interfered with myocardial stiffness in HFpEF patients . LamininHowever, besides this amplitude of recent discoveries about the mechanotransduction machinery in cardiomyocytes and the consequences of aberrant tissue stiffness, many questions still remain unanswered. Little is known about the prospects of therapeutic modifications of cardiac ECM and its stiffness, in order to improve cardiomyocyte performance and induce regenerative processes without softening of the ventricular wall. And how exactly can costamere proteins detect substrate stiffness? How does aberrant mechanical signaling due to a stiff matrix interfere with cell migration, intercellular communication, and ligand availability for cardiomyocytes and other cardiac cell types? Several studies have shown the importance of the ECM for cytokine- and growth factor-mediated signaling . It alsoJM established a plan for this manuscript and designed the figures. JM and SA-S wrote the draft and approved the submitted manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Correction to: Pediatric Nephrology10.1007/s00467-019-04344-1The original version of this article unfortunately contained a mistake. In the third paragraph of “Discussion,” two references were missing. The correct wording is “Since several studies have documented that a higher eplet mismatch load between donor and recipient is associated with poor transplant outcomes , 54, it In addition, reference  is missi


We consider how the COVID-19 pandemic has challenged European small- and medium-sized enterprises (SMEs) in the manufacturing sector, and draw suggests policy implications. The sudden onslaught of the pandemic has acted as an economic shock, and we consider how it is likely to affect different types of manufacturing SMEs. We distinguish between immediate effects, a result of the almost-simultaneous lockdowns across Europe and its major trading partners, and longer-term implications for both SMEs and the global value chains where they are inserted. In the shorter run, most SMEs have faced logistical challenges in addition to demand disruptions, although the severity has differed across firms and industries. We argue that in the longer-term, there will be different challenges and opportunities depending on the type of SME. Policy interventions will also need to be sensitive to the different types of SMEs, rather than adopting a one-size-fits-all approach. The policy mix will need to shift from its initial focus on the survival of European SMEs in the short term, towards a more structural and longer-term approach based on promoting their renewal and growth through innovation, internationalization and networking. Small- and medium-sized enterprises (SMEs)The continuing competitiveness of European manufacturing SMEs derives from their capacity to be innovative. However, because of their size and ownership structure, they also generally struggle with profitability and liquidity, thus becoming particularly vulnerable to external shocks  universities and large multinationals.Though the full economic impact of the pandemic is hard to predict, it is already apparent that it represents an unprecedented external shock. On the supply side, SMEs have faced logistical issues due to the disruption of transportation and labor shortages. On the demand side, SMEs have seen their demand decline substantially due to lockdown measures, a drop in consumer confidence and the shutting down of a number of GVCs in affected industries. The severity of these demand and supply shocks is likely to differ according to whether the firm is a stand-alone, a specialized-supplier or a knowledge-based SME. These nuances are important to better understand the medium- and long-term effects, and to set policies accordingly. It is worth distinguishing between the immediate effects of the shock, due to the almost-simultaneous universal lockdowns across major economies between April and June 2020, and its longer term effects.SMEs of all types have experienced immediate effects of the lockdowns on their upstream and downstream activities. Specifically, on the supply side, stand-alone SMEs have faced considerable logistical issues. However, while reduced capacity utilization represents one key challenge, demand-side effects pose a more severe threat. Indeed, most consumer-focused, stand-alone SMEs operate in industries characterized by an elastic demand. With consumers facing employment uncertainties and financial constraints, many of these SMEs have experienced a sudden decline in demand. In contrast, many stand-alone SMEs serving the industrial market may benefit from their niche position, as they face few substitutes and, therefore, customers may be reliant on them. However, with production lines being halted across Europe during the lockdowns, and GVCs disrupted, many of these firms are also likely to face reduced demand or an incapacity to produce.Specialist-supplier SMEs were also severely hit both on their demand-side and their supply-side. With many being engaged in exclusive agreements, orders from key customers which themselves stopped production were likely to be halted. Such effects have occurred in the automotive sector, for example. However, given their specialized focus, these SMEs supply vital components to their customers and, hence, once restrictions are lifted and production restarts, demand is likely to recover. Notably, some SMEs serving the industrial markets, including stand-alone and specialist-supplier SMEs, may have temporarily repurposed their operations towards goods needed during the pandemic, such as face masks and medical equipment; at least making up for some of their lost demand.Finally, demand-side implications for knowledge-based SMEs during the crisis were likely to have been less severe, compared to their supply-side issues. With many of these SMEs requiring laboratories or special equipment, for example, little progress can be made with scientists and engineers having to work from home.There are economic implications associated with the COVID-19 pandemic that go beyond the lockdowns. As outlined in Table In the longer term, however, other challenges (and opportunities) will arise that differ across types of manufacturing SMEs. In particular, the crisis has pointed stand-alone SMEs to the importance of investing in digital technologies. These became essential for SMEs, not only to support crucial downstream activities such as sales and marketing, but also to increase their internal efficiency and productivity PwC . From a Moreover, as outlined in Table It is likely, therefore, that some of the anticipated changes will require GVC ‘lead firms’ to reorganize their supply chains and some specialist-suppliers will see their contracts terminated. In general, SMEs in GVCs are particularly vulnerable to external shocks because ‘lead firms’ often pass their own difficulties on to them OECD . Thus, tDespite these challenges brought by changes in GVCs, there will also be entrepreneurial opportunities, offering new areas of growth and allowing newer SMEs to benefit from creative destruction dynamics  in the territory. All types of SMEs will benefit from strong innovation support schemes, but the focus required might be different. Specifically, product and marketing innovations are most relevant for stand-alone SMEs, including those that differentiate themselves (or operate in niches) and those which—due to the nature of their industry—do not. For the latter group, marketing innovations may be particularly valuable to retain existing and attract new customers. For specialized-suppliers, the main focus will need to be on process and organizational innovations, enabling them to compete on price and quality. Meanwhile, stronger investments in entrepreneurship and start-up support will turn critical to promote knowledge-based SMEs. The most adequate policy mix will also depend on the particularities of each industry and territory, making it necessary to adopt more strategic policy responses, in contrast with the one-size-fits-all approach that characterizes the survival stage.The shift from a focus on survival to the renewal and growth phase is a delicate one that requires a proper sequencing of policy responses. While the survival phase is important, it is very costly and cannot last forever, given its profound effect on fiscal deficits and public debt. Removing the associated initial policy actions should, therefore, be well planned, gradually removing generic financial support measures, especially grants, as the economic situation stabilizes, while introducing new support policies fostering innovation and growth.Policymakers will need to adopt a more proactive role, coordinating new industrial efforts for coupling domestic capacities with the dynamics of GVCs. As we have argued earlier, the COVID-19 crisis can also act as an accelerator of digitalization and sustainability transitions, while spurring a renewed interest in emerging health technologies such as biotechnology and genetics. Policymakers should create new incentives for European firms to benefit from these opportunities, against the context of the ongoing structural reshaping of globalization characterized by the increasing influence by China (Petricevic and Teece This approach -which has been increasingly influencing European, national and subnational policies over recent years- represents a shift towards “transformative” industrial and innovation policies, aimed at setting the direction of change rather than just addressing market failures (Schot and Steinmueller Given the contribution of manufacturing SMEs to the European economy, policymakers need to carefully reassess their policy mix in light of the evolution of the crisis. An ambitious structural policy approach based on promoting the renewal and growth of European manufacturing SMEs—through innovation, internationalization and networking—is of paramount importance to deal with the challenges ahead.