Fibroblasts produce the majority of collagen in the heart and are thought to regulate extracellular matrix (ECM) turnover. Although fibrosis accompanies many cardiac pathologies and is generally deleterious, the role of fibroblasts in maintaining the basal ECM network and in fibrosis in vivo is poorly understood. We genetically ablated fibroblasts in mice to evaluate the impact on homeostasis of adult ECM and cardiac function after injury. Fibroblast-ablated mice demonstrated a substantive reduction in cardiac fibroblasts, but fibrillar collagen and the ECM proteome were not overtly altered when evaluated by quantitative mass spectrometry and N-terminomics. However, the distribution and quantity of collagen VI, microfibrillar collagen that forms an open network with the basement membrane, was reduced. In fibroblast-ablated mice, cardiac function was better preserved following angiotensin II/phenylephrine (AngII/PE)-induced fibrosis and myocardial infarction (MI). Analysis of cardiomyocyte function demonstrated altered sarcomere shortening and slowed calcium decline in both uninjured and AngII/PE-infused fibroblast-ablated mice. After MI, the residual resident fibroblasts responded to injury, albeit with reduced proliferation and numbers immediately after injury. These results indicate that the adult mouse heart tolerates a significant degree of fibroblast loss with a potentially beneficial impact on cardiac function after injury. The cardioprotective effect of controlled fibroblast reduction may have therapeutic value in heart disease. Fibroblasts are the primary source of extracellular matrix (ECM) in the heart . The maiIn response to cardiac injury, fibroblasts rapidly adopt an activated phenotype resulting in increased proliferation and deposition of a collagen-rich ECM . An initGrk2, a downstream effector of G protein-coupled receptors known to be elevated in patients with heart failure  and angiotensin II/phenylephrine (AngII/PE) infusion, suggesting that an underlying reduction in fibroblast numbers or cardiac adaptation to fewer fibroblasts may be protective. Our findings reveal that fibroblast reduction before injury does not negatively affect the heart and may provide beneficial effects when cardiac remodeling or repair are provoked by injury.CreERT2/+Pdgfra mouse line to deplete resident fibroblasts (Col1a1-GFP). This reporter expresses GFP under the control of the Col1a1 promoter  and myeloid cells (CD11b+) were unaffected -proteomics and cardiac function analysis, also supports a relatively mild impact of fibroblast ablation overall on the heart, although significant changes in several contractile proteins and metabolic enzymes were identified. Perhaps, one potent contributor to unaltered ECM turnover is the unexpected reduction of proteolytic activity detected by gelatin zymography. Indeed, in addition to gelatin, MMP2 and MMP9, the enzymes principally detected by gelatin zymography are known to cleave numerous ECM components.In support of previous findings , there wCol6a1 knockout mice had improved heart function after MI 26Sor (DTAGt(ROSA)26Sor 26SortdT/DTAPdgfra. All control mice used in these experiments were tamoxifen-induced and age-matched. Control genotypes were CreERT2/+; Gt(ROSA)26SortdT/+Pdgfra or +/+; Gt(ROSA)26SortdT/DTAPdgfra (littermate controls). A mix of male and female littermate (Cre-) and non-littermate (Cre+) controls were used. All experiments were performed on adult mice older than 8 weeks.All animal protocols and experiments were approved by the University of Hawaii at Manoa Institutional Animal Care and Use Committee. Both male and female with a mixed C57Bl/6 background were used for these studies. The  Brenner . The PdgCol1a1-GFP transgene expression, or Pdgfra transcript levels in the heart or kidney. Kidneys were collected, stored in RNAlater Stabilization Solution , and processed for RNA extraction as described below. Mice with less than 45% reduction of either fibroblasts or Pdgfra expression were excluded from studies , and saturated with 3 M KCl to arrest the heart in diastole. Hearts were bisected coronally and fixed with freshly prepared 4% paraformaldehyde (PFA) for 2 hr at room temperature or 10% neutral buffered formalin (NBF) for 24 hr at 4°C. Tissue fixed with 4% PFA was cryoprotected and frozen embedded. Immunostaining was performed on 10 μm tissue cryosections as previously described . PrimaryMice were administered with EdU  by intraperitoneal (IP) injection (50 µg/g) 24 hr before isolation. Hearts were fixed and cryosectioned. The EdU was detected using Click-iT EdU cell proliferation kit .g for 20 min at 4°C and the supernatant was collected. Blots were probed with primary antibody overnight at 4°C, and then incubated with the corresponding Li-Cor IRDye secondary antibody for 1 hr at room temperature. Primary antibodies used for western blot are listed in Atria and valves were removed from isolated hearts. The whole ventricle was homogenized in Radioimmunoprecipitation assay buffer with a protease inhibitor cocktail  using a Dounce homogenizer. Samples were centrifuged at 16,000 × g for 20 min at 4°C, and the supernatant was collected. About 50 µg of protein were loaded per lane. Novex 10% Zymogram Plus gels  were used, and gelatin zymography was performed according to the manufacturer’s instructions.Left ventricle tissue was trimmed to 50 mg, followed by homogenization in Triton X-100 lysis buffer  with protease inhibitor cocktail  using a Dounce homogenizer. Samples were centrifuged at 16,000 × 18s or Gapdh expression. The 2-ΔΔCt method was used for determining relative gene expression levels. Primer sequences used for qRT-PCR are listed in RNA isolation was performed on whole ventricles using IBI Isolate DNA/RNA reagent  and PureLink RNA mini kit  according to the manufacturer’s instructions. RNA quality and concentration were determined by spectrophotometry using a NanoDrop 2000 instrument (Thermo Fisher). The cDNA was synthesized using M-MLV Reverse Transcriptase  and random hexamer primer . The qPCR analysis was performed using PowerUp SYBR Green Master Mix  and a LightCycler 480 instrument (Roche). Samples were run in triplicate and normalized to 2+, and atria and valves were removed. Single-cell suspensions were obtained as previously described  or LIVE/DEAD Fixable Violet Dead Cell Stain kit  was used for live/dead discrimination. Cells were then incubated in Fc block  followed by indicated antibodies and fixed with 1% PFA in flow cytometry buffer. Primary antibodies used for flow cytometry are listed in Adult hearts were transcardially perfused with PBS containing 0.9 mM Caescribed . Briefly2O and hydrolyzed in 6 N HCl for 20 hr at 95°C. Hydrolyzed contents were processed according to the manufacturer’s instructions. The absorbance of the supernatant was read using Molecular Devices SpectraMax M3 microplate reader at 540 nm wavelength.Hydroxyproline assay  was performed on whole ventricles isolated from adult hearts. Samples were homogenized in distilled H2O and decellularized in 10% NaOH until the tissue was clear. Decellularized tissue was washed in distilled H2O twice for 30 min, and then washed overnight at room temperature. Tissue was fixed with 4% tannic acid for 4 hr, post-fixed with 1% osmium tetroxide in 0.1 M sodium cacodylate, dehydrated through an ethanol series, and dried in a Tousimis Samdri-795 critical point dryer. Samples were mounted on aluminum stubs with double-stick carbon tape and coated with gold/palladium in a Hummer 6.2 sputter coater. Samples were viewed and digital images were acquired with a Hitachi S-4800 field emission scanning electron microscope at an accelerating voltage of 5 kV.The LV tissue was cut into a 3 × 3 × 3 mm cube and fixed in 10% buffered formalin for 24 hr at room temperature. Fixed tissue was washed with distilled H2O with protease inhibitor for 30 min, and decellularized in 1% sodium dodecyl sulfate in DPBS with protease inhibitor until the tissue was clear. The timing of decellularization was determined by DNA quantification using DAPI staining to confirm the lack of cells in tissue samples. Decellularized cardiac tissue was washed in distilled H2O with protease inhibitor three times for 5 min at room temperature with light agitation, and then washed overnight to completely remove detergent. Decellularized tissue was placed in 200 μL of Protein Extraction Reagent type 4 with protease inhibitor and sonicated 10 times for 10 s. At least 100 μg of protein was sent to the University of Mississippi Medical Center for mass spectrometry analysis. Mass spectrometry experiments were performed blinded.Whole ventricles from adult mice were cut into 8–10 uniform pieces, washed in distilled HLysates from decellularized heart tissue were obtained as described above and reduced, alkylated, and trypsin-digested into peptides. The peptides were cleaned using a Sep-Pak Vac C18 cartridge (Waters Corporation) and analyzed label-free by LC-MS/MS using a Q Exactive mass spectrometer (Thermo Fisher). A 15 cm Å ~75 μm C18 column (5 μm particles with 100 Å pore size) was used and the nano-ultra-performance liquid chromatography (UPLC )run at 300 nL/min with a 150 min linear acetonitrile gradient . The MS/MS was set up with the exclusion of 25s, and the samples were run with high-energy collisional dissociation fragmentation at a normalized collision energy of 30% and an isolation width of 2m/z. The resolution setting was 70,000 for target values of the MS at 1e6 ions and in MS2 at a resolution setting of 17,500 for 1e5 ions. Mass spectrometry analyses were performed at the University of Mississippi Medical Center.The RAW files were analyzed using Proteome Discoverer 2.2 (Thermo Fisher). Precursor mass tolerance was set at 10 ppm and fragment mass tolerance was set at 0.6 Da. Dynamic modification was set to oxidation (+15.995 Da [M]) and static modification was set to carbamidomethyl (+57.021 Da [C]). Samples were searched against the reviewed mouse database downloaded from Uniprot . A strict false discovery rate of 1% was applied. Label-free quantification was done based on precursor ion intensity and normalization was done using the total peptide amount . Proteins were included only if they were identified by at least two high-confidence peptides.For further visualizations such as multi-scatter plots, PCA, volcano plot, and heat map, normalized abundances from all the samples were imported in Perseus 1.6.5.0. Values were log 2(x) transformed and data were filtered by excluding proteins that were not identified in at least 50% of the samples. The missing values for proteins present in >50%, but not all samples were imputed from the normal distribution feature. Multi-scatter plots and PCA analysis were performed using all proteins, whereas only ECM proteins were used for generating the volcano plot and unsupervised hierarchical clustering. Perseus software was used to perform a t-test with inbuilt multiple hypothesis testing corrections using a permutation-based false discovery rate.A two-step protein extraction method was performed. The first used a commercially available mild detergent T-Per tissue protein extraction reagent (Thermo Fisher) with protease inhibitor cocktail (Roche), and the second used 4 M GuHCl to solubilize the remaining pellet. Samples were prepared for TAILS analysis as previously described . BrieflyAdult mice >22.0 g were subjected to MI as previously described . BrieflyAdult mice >20.0 g were infused with AngII/PE to induce cardiac hypertrophy and fibrosis. Mice were anesthetized with 1–2% isoflurane and mini-osmotic pumps  were implanted subcutaneously. A combination of 1.5 μg/g/day angiotensin II  and 50 μg/g/day phenylephrine hydrochloride  or saline, was infused for 7, 14, or 28 days.Echocardiography was performed using a Vevo 2100 system  to analyze cardiac function after MI and AngII/PE infusion. Systolic function was analyzed in conscious mice. Briefly, hair removal cream was applied to the chest to remove all fur. A layer of ultrasound gel was applied to the animal’s chest, and the probe was lowered at the parasternal line, 90° between the probe and the heart. B- and M-modes were performed to record 2-dimensional and 1-dimensional transverse cardiac measurements and used to analyze LV function. Independent echocardiography on mice 7 months post-induction at The Ohio State University also found no differences in cardiac measurements between control and fibroblast-ablated hearts.Diastolic function was measured in mice anesthetized with 4% isoflurane in 100% oxygen. Anesthesia and body temperature were maintained at 1% isoflurane and 37°C, respectively. M-mode in the short axis at the level of papillary muscles was used to measure systolic function. The 4-chamber apical view with color and tissue Doppler mode was used to measure E/A and E/E’ ratios. All measurements were performed by an experienced operator blinded to the mouse genotypes.–1) plus xylazine (15 mg kg–1) in saline solution and placed in the supine position on a heat pad. Following a midline neck incision, the underlying muscles were pulled to expose the carotid artery. Using a 4–0 suture, the artery was tied and the pressure-volume catheter was advanced through the artery into the left ventricle of the heart. After 5–10 min of stabilization, values at baseline and stimulation at varying frequencies (4–10 Hz) were recorded. To measure the beta-adrenergic response, 5 mg kg–1 dobutamine was injected IP. All the measurements and analysis were performed on LabChart7 (ADInstruments).Cardiac hemodynamic measurements were assessed via a closed chest approach using a 1.4 rodent pressure-volume catheter (Transonic) advanced into the left ventricle through the right carotid artery . In brieCardiomyocyte boundaries were identified by WGA labeling in 10 μm tissue sections that were processed as described above. Cardiomyocytes were defined by having a circular to oval shape in cross-section, surrounded by circular capillaries. A total of 100 cardiomyocytes in a section were outlined and CSA was calculated using ImageJ (NIH).2, 1.2 mM CaCl2, pH 7.4) for Ca2+ transients and contraction measurements.Cardiomyocytes from whole ventricle tissue were isolated from adult mice using a modified Langendorff-free collagenase digestion protocol . Briefly2+ transient was calculated as the difference between peak and diastolic Ca2+ levels according to the equation (F–F0)/F0 after subtraction of background fluorescence. The kinetics of Ca2+ transient time constants were determined using exponential curve fitting. Measurements were made in more than 45 cardiomyocytes for each group from n=5–6 mice.Cardiomyocytes were loaded with 2 μM Fura-2  in Tyrode’s solution for 15 min. Cells were electrically paced at 1 Hz using an IonOptix Myopacer. Measurements were performed using IonWizard version 6.1 software (IonOptix). Images were obtained by a Nikon Eclipse TE2000-U camera. The amplitude of intracellular Ca2 (fold change) ≥2 or ≤–2 and p≤0.05 were analyzed by the functional annotation tool in the database for annotation, visualization and integrated discovery (DAVID) version 6.8 program to search for enriched GO terms.Total RNA was isolated as described above. The quality of RNA was assessed by an Agilent 2100 Bioanalyzer and samples with an RNA integrity number value ≥8.0 were used for microarray analysis. The mRNA transcription profile was determined by Clariom S assay for a mouse . Analysis was performed using Transcriptome Analysis Console software version 4.0.1. The differentially expressed genes with a logBlood was obtained from the submandibular vein and evaluated using an iStat Chem8+ cartridge (Abbot) using an i-STAT 1 handheld analyzer (Zoetis) according to the manufacturer’s recommendations.Band intensity of western blots was measured using ImageJ (version 2.3.0/1.53q). Subtracted background intensity was determined for the same gel image. Fluorescent images were processed and measured using ImageJ. Split channel images were generated, followed by binarization. Fused cells were divided by the watershed function. The number of cells was counted with the analyze particle function or Cell Counter plugin. The identical setting of thresholds was used for each session through automated macros.All statistical analyses were conducted using Prism 8 (Graphpad Software). A Shapiro-Wilk test was performed to determine the data distribution. If the distribution was normal, an unpaired t-test was used for two group comparison (parametric), otherwise, a Mann-Whitney U test was used as a non-parametric analysis. For multiple comparisons with normal distribution, a one- or two-way ANOVA with the Tukey’s test were performed. Statistical variability is expressed as mean ± SD; ns: not significant, p>0.05; *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001. N-values for experiments are indicated in the figure or figure legends.All mouse experiments were performed according to the animal experimental guidelines issued and approved by the Institutional Animal Care and Use Committees of the University of Hawaii at Manoa (APN12-1421 and APN12-1469) and The Ohio State University Wexner Medical Center (#2021 A00000070). This study is directed at cardiac fibrotic change, which is widespread both under conditions of both normal development and pathological responses, and it is associated with both pump failure and arrhythmic change. A new genetic platform is introduced, in which such processes are reduced. What emerges is a reduction in ventricular, atrial and septal fibroblast density. This study is extremely well done, rigorous, and offers valuable insight for investigators interested in fibrosis, cardiac fibroblast biology, and mechanisms of extracellular matrix remodeling. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Christopher L-H Huang as Reviewing Editor and Reviewer #1. and the evaluation has been overseen by a Reviewing Editor and a Senior Editor.Thank you for submitting your article "Consequences of PDGFRalpha+ fibroblast reduction in adult murine hearts" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this letter to help you prepare a revised submission.Essential revisions:The reviewers are positive about the work, but between them, generate a large number of significant revisions, likely because of the unexpected findings in this study. This is reflected in one of the evaluation studies which suggest that "This study is extremely well done, rigorous, and offers valuable insight for investigators interested in fibrosis, cardiac fibroblast biology, and mechanisms of extracellular matrix remodeling", but however, "….the finding is completely unexpected and the paper…. does not extend to the mechanistic depth is needed to understand the basis for the finding. For this reason this review includes all the detailed comments made by the reviewers, in addition to drawing attention to each of the major points indicated particularly from reviewers 2-3.Reviewer 2:1) In some cases, the results are expressed a "stained area/tissue area", when clearly it would have been more appropriate to provide cell counts. At least, they should provide FACS plots showing the gating used for quantification to reassure the readers that PDGFRa was expressed at comparable levels in mice of different ages. Given that it is now widely accepted that cardiac fibroblasts are heterogenous in many ways, it would be important to understand whether this ablation affects this heterogeneity.2) The hearts contained somewhere between half and one third of their normal number of fibroblasts, suggesting that one or two cell divisions would be sufficient to bring these cells back to normal levels. Following an MI, these cells enter rapid proliferation, and their numbers increase significantly. What happens in the ablated heart to fibroblast numbers?3) A time resolved analysis of PDGFRa cells in the context of the two damage models would be very informative and possibly hint at mechanism.4) Does the increase in NG2 indicate an upregulation of the protein in mural cells or increase (maybe compensatory) in the numbers of mural cells? Adding mural cells to the FACS-based quantification of inflammatory, endothelial and fibroblastic cells would help in answering this.5) The authors did not attempt to measure diastolic function in the fibroblast-ablated hearts following damage in either the MI or the Ang II/phenylephrine models. The parameters they report are more relevant to systolic function (ejection fraction) and chamber dimension (LVID). However, I would have expected some measurement of transmitral flow such as E/A ratio or E wave deceleration time from a 4- chamber apical view, or even IVRT. This is important as ECM/fibrotic changes are expected to affect LV compliance/stiffness (i.e. diastolic function) usually before systolic involvement.6) How was the collagen area measured in the 2 damaged models? By antibody staining or using Trichrome staining?7) An important pathway that regulates calcium sensitivity and the actin cytoskeleton at the same time is the RhoA/Rho kinase pathway. Did the authors find any changes in expression of the proteins in this pathway?Reviewer 3:8) It is fascinating that no rupture is observed and that the relative collagen fraction between the ablated mice and controls is largely similar by 10 days. (A) Have the non-ablated fibroblasts proliferated and normalized the fibroblast numbers back to wild type levels? (B) Is it possible that collagen area as a percentage of tissue area may not be the most informative metric of fibrosis due to the significantly altered cardiac dimensions in mice with ablated fibroblasts? (C) It is puzzling that fibroblast ablation has so little impact on the ECM opening up questions regarding the cell state heterogeneity of the non ablated fibroblasts populations- are they all behaving like activated fibroblasts or matrifibrocytes to maintain the matrix?9) This study paid a lot of attention to mechanisms of ECM deposition and remodeling but very little attention to degradation and turnover of the matrix. Are these mechanisms inhibited such that the matrix is no longer turning over once fibroblasts are ablated?10) What is the basis for the reduction in collagen deposition observed for the Ang/PE model but not for the infarction model?11) If the TAILS proteomics data suggests that cardiomyocyte remodeling may be occurring, is this finding reflected in the geometry of isolated myocytes at baseline measured for their contraction characteristics in figure 7?Reviewer #1 (Recommendations for the authors):(a) In the summary, there are a few ambiguities that slightly obscure the meaning: the term 'uninjured' is unclear; the following sentence ["Analysis of cardiomyocyte function demonstrated weaker 42 contractions and slowed calcium decline in both uninjured and AngII/PE infused 43 fibroblast-ablated mice."] apparently differs from the comment that ", cardiac function was better 40 preserved following angiotensin II/phenylephrine (AngII/PE)-induced fibrosis and 41 myocardial infarction".(b) The Introduction is an appropriate review of the role of fibroblasts in cellular matrix formation and matrix formation, but should be slightly revised to provide a brief paragraph on the relationships between fibroblasts and the cardiomyocytes themselves, particularly in relation to influencing gap junction cohesion as well as, through their fusion, effects on capacitative properties of the cardiomyocyte syncytium with a potential for arrhythmic substrate.(c) When the discussing the Results, the following merit further discussion.(i) This appeared to be a system that did not show pathological hypertrophy: it exhibited downregulated cell adhesion, collagen binding, collagen fibril organization, and ECM organization, and upregulated membrane, mitochondrial and Z-disc genes, but an absence of uupregulation in Nppa, Nppb, and Myh7 and Myh6 genes related to pathological hypertrophy.2+ transients in control and ablated cardiomyocytes both before and following AngII/PE infusion, with greater transients in the latter, in contrast to:(ii) There was a contrast between: Normal indicators of excitation contraction coupling with similar ca(iii) Mechanical indicators: Cardiomyocytes from ablated hearts showing reduced baseline sarcomeric shortening, speed of contraction with and slowed relaxation.Reviewer #2 (Recommendations for the authors):The authors use an induction protocol that seems to lead to an efficiency of CRE activation somewhat below what has been reported for similar strains by others. They use a threshold of at least 45% reduction to include individual animals in the analysis. This does not seem a major improvement over their recent work in which they report the effects of a 50% reduction is minimal. Also, if their threshold is 45%, why do they keep referring to a 60 to 80% ablation in the text?One important and surprising finding in this paper is that the cells surviving the ablation did not expand to make up for the lost numbers. However, I was surprised to see that most of the evaluation of the extent of depletion over time relied on the expression of PDGFRa itself, a gene whose expression has been reported to be modulated, that is expressed at different levels in different subsets (based on published scRNAseq data) and that is expressed by a single allele in this experimental setup. In some cases, the results are expressed a "stained area/tissue area", when clearly it would have been more appropriate to provide cell counts. At least, they should provide FACS plots showing the gating used for quantification to reassure the readers that PDGFRa was expressed at comparable levels in mice of different ages. Given that it is now widely accepted that cardiac fibroblasts are heterogenous in many ways, it would be important to understand whether this ablation affects this heterogeneity. In other words, does ablation preferentially remove subpopulations of fibroblasts (such as more proliferative cells that express higher levels of Pdgfra) more than others? Knowledge of this may explain possible reasons for the lack of replenishment of fibroblasts. I think it would be worth using scRNAseq in this context.The CRE strain used is active in a number of other organs and anatomical locations, ranging from lung fibroblast to adipogenic cells in fat depots to oligodendrocyte progenitors in the CNS. Did the authors look into these organs, or notice any systemic effects of their depletion protocol?Based on the data presented, the hearts contained somewhere between half and one third of their normal number of fibroblasts, suggesting that one or two cell divisions would be sufficient to bring these cells back to normal levels. Following an MI, these cells enter rapid proliferation, and their numbers increase significantly. What happens in the ablated heart to fibroblast numbers? Do they increase as expected, do they plateau at lower or similar levels to the ones observed in controls? Does the scar still contain the same number of collagen-expressing cells?A time resolved analysis of PDGFRa cells in the context of the two damage models would be very informative and possibly hint at mechanism, and I was surprised to see the analysis limited to one time point and performed by quantifying genes rather than cells themselves.The proteomic analysis of the de-cellularized heart is a strong point of the paper. However, some of the changes observed may not be a direct effect of fibroblast ablation. For example, one of the proteins the authors reported to be upregulated in the fibroblast-ablated hearts is NG2, which is not expressed to any significant extent in fibroblasts. Rather, it is expressed at high levels in mural cells such as pericytes and vascular smooth muscle cells. Does the increase in NG2 indicate an upregulation of the protein in mural cells or increase (maybe compensatory) in the numbers of mural cells? Adding mural cells to the FACS-based quantification of inflammatory, endothelial and fibroblastic cells would help in answering this.I am a bit confused by the fact that the authors, although reported decreased LV internal diameter, found that cardiomyocytes did not increase in size. Usually, the reason for decreased chamber size would be hypertrophy, especially if it is observed in systole and diastole. How do the authors explain this?I am surprised that the authors did not attempt to measure diastolic function in the fibroblast-ablated hearts following damage in either the MI or the Ang II/phenylephrine models. The parameters they report are more relevant to systolic function (ejection fraction) and chamber dimension (LVID). However, I would have expected some measurement of transmitral flow such as E/A ratio or E wave deceleration time from a 4- chamber apical view, or even IVRT. This is important as ECM/fibrotic changes are expected to affect LV compliance/stiffness (i.e. diastolic function) usually before systolic involvement.How was the collagen area measured in the 2 damaged models? By antibody staining or using Trichrome staining? Why in the authors' opinion did fibroblast ablation decrease collagen area in the Ang II/phenylephrine model but not the MI model? Again these questions may be answered by analyzing the proliferative response to these insults.A decrease in shortening without a decrease in calcium transient amplitude, as seen in the ablated hearts, indicates calcium desensitization. An important pathway that regulates calcium sensitivity and the actin cytoskeleton at the same time is the RhoA/Rho kinase pathway. Did the authors find any changes in expression of the proteins in this pathway?Reviewer #3 (Recommendations for the authors):In this manuscript, Kuwabara and colleagues use genetic ablation to reduce the number of fibroblasts resident to the heart. At baseline, the authors observe that fibroblast numbers stay proportionally low after ablation, but with very minimal effects to the structure or composition of the extracellular matrix. Fibroblast ablation prior to myocardial infarction is shown to be beneficial to cardiac function without affecting relative abundance of scar tissue, whereas in an Ang/PE model of fibrosis collagen deposition is impaired and systolic function is preserved.Major Concerns1. For the myocardial infarction model presented in Figure 4, it is fascinating that no rupture is observed and that the relative collagen fraction between the ablated mice and controls is largely similar by 10 days. (A) Have the non-ablated fibroblasts proliferated and normalized the fibroblast numbers back to wildtype levels? (B) Is it possible that collagen area as a percentage of tissue area may not be the most informative metric of fibrosis due to the significantly altered cardiac dimensions in mice with ablated fibroblasts? (C) It is puzzling that fibroblast ablation has so little impact on the ECM opening up questions regarding the cell state heterogeneity of the non ablated fibroblasts populations- are they all behaving like activated fibroblasts or matrifibrocytes to maintain the matrix?2. This study paid a lot of attention to mechanisms of ECM deposition and remodeling but very little attention to degradation and turnover of the matrix. Are these mechanisms inhibited such that the matrix is no longer turning over once fibroblasts are ablated?3. What is the basis for the reduction in collagen deposition observed for the Ang/PE model but not for the infarction model?4. If the TAILS proteomics data suggests that cardiomyocyte remodeling may be occurring, is this finding reflected in the geometry of isolated myocytes at baseline measured for their contraction characteristics in figure 7? Essential revisions:The reviewers are positive about the work, but between them, generate a large number of significant revisions, likely because of the unexpected findings in this study. This is reflected in one of the evaluation studies which suggest that "This study is extremely well done, rigorous, and offers valuable insight for investigators interested in fibrosis, cardiac fibroblast biology, and mechanisms of extracellular matrix remodeling", but however, "….the finding is completely unexpected and the paper…. does not extend to the mechanistic depth is needed to understand the basis for the finding. For this reason this review includes all the detailed comments made by the reviewers, in addition to drawing attention to each of the major points indicated particularly from reviewers 2-3.Regarding a better understanding of the fibroblast responses after injury we have extensively updated the myocardial section including data in the first two weeks after injury. These data include proliferation results, investigation of PDGFRb expressing cells, measurement of diastolic function, quantification of cells by flow, and examination of collagen expressing cells in infarcts. The specific revisions related to reviewers’ queries are described below:Reviewer 2:1) In some cases, the results are expressed a "stained area/tissue area", when clearly it would have been more appropriate to provide cell counts. At least, they should provide FACS plots showing the gating used for quantification to reassure the readers that PDGFRa was expressed at comparable levels in mice of different ages. Given that it is now widely accepted that cardiac fibroblasts are heterogenous in many ways, it would be important to understand whether this ablation affects this heterogeneity.We have now included the gating strategy for PDGFRa expressing cells in Figure 1 supplement 1. The flow cytometry demonstrates a relatively constant level of PDGFRa expression, independent of age, and the technique is sensitive to surface expression as we observed a decreased mean fluorescence intensity when animals were heterozygous for PDGFRa (as in the PDGFRa CreERT2/+ mice). As the reviewer was concerned about fibroblast heterogeneity, for updated analyses we have used Col1a1-GFP as an unbiased tag of collagen producing cells. The collagen GFP quantification identifies cells with type I collagen production and provides a non-PDGFRa focused view of the hearts at baseline, with aging, and after injury Figures 1A-E.In regard to cell counts, in new data we have included cell counts/nuclei . We agree with the reviewer that it is difficult to normalize cell counts. After injury, when quantifying fibroblasts, distinguishing individual fibroblasts, which have significant cellular extensions and increase in volume and can amass in one location is difficult. In addition, immune cell infiltration can vary greatly, altering the relative cell counts. In areas with cardiomyocytes, nuclear numbers can be skewed due to cardiomyocyte area. As these technical issues are most prevalent during MI and can confound quantification by IHC, we have included flow cytometry data to provide an additional perspective on cell number before and after MI Figures 1F and 8H,I.2) The hearts contained somewhere between half and one third of their normal number of fibroblasts, suggesting that one or two cell divisions would be sufficient to bring these cells back to normal levels. Following an MI, these cells enter rapid proliferation, and their numbers increase significantly. What happens in the ablated heart to fibroblast numbers?We have now included information documenting fibroblast numbers from multiple perspectives with several time points after LAD ligation. The new data is included in new figure 8. These data demonstrate that the remaining fibroblasts expand, but do not reach the levels observed in the control hearts.3) A time resolved analysis of PDGFRa cells in the context of the two damage models would be very informative and possibly hint at mechanism.We have now provided additional time points addressing cell numbers and fibroblast proliferation in the first two weeks after injury including both PDGFRa and collagen expressing cells after injury in Figure 8 and Figure 4 supplemental figure 1.4) Does the increase in NG2 indicate an upregulation of the protein in mural cells or increase (maybe compensatory) in the numbers of mural cells? Adding mural cells to the FACS-based quantification of inflammatory, endothelial and fibroblastic cells would help in answering this.To address this reviewer’s query regarding mural cells, we have included stains for NG-2 and/or PDGFRb at baseline and after MI to investigate mural cell expansion. There appears to be limited mural cell expansion in the collagen type I producing population. Table 1, Figure 1 supplemental figure 3 and Figure 8 supplemental figure 1.5) The authors did not attempt to measure diastolic function in the fibroblast-ablated hearts following damage in either the MI or the Ang II/phenylephrine models. The parameters they report are more relevant to systolic function (ejection fraction) and chamber dimension (LVID). However, I would have expected some measurement of transmitral flow such as E/A ratio or E wave deceleration time from a 4- chamber apical view, or even IVRT. This is important as ECM/fibrotic changes are expected to affect LV compliance/stiffness (i.e. diastolic function) usually before systolic involvement.We have now performed E/E’ and E/A measurements after permanent ligation of LAD as well as in old control and ablated mice. We observe a significant difference between control and ablated hearts after MI suggesting that reducing the number of fibroblasts helps retain heart diastolic function, but even after a year of deletion ablated mice have similar heart function to controls in the absence of injury Figure 7 and Figure 1 supplement figure 2.6) How was the collagen area measured in the 2 damaged models? By antibody staining or using Trichrome staining?We have now indicated that collagen area was measured using Masson’s or Gomori trichrome stain in the Materials and methods.7) An important pathway that regulates calcium sensitivity and the actin cytoskeleton at the same time is the RhoA/Rho kinase pathway. Did the authors find any changes in expression of the proteins in this pathway?Our microarray analysis documented no change in expression of RhoA/Rho kinase either at baseline or after angiotensin/PE. We performed Reactome pathway analysis with the microarray data of baseline and AngII cohorts and observed no significant terms associated with Rho, RhoA, or Rho-GTPase.Reviewer 3.8) It is fascinating that no rupture is observed and that the relative collagen fraction between the ablated mice and controls is largely similar by 10 days. (A) Have the non-ablated fibroblasts proliferated and normalized the fibroblast numbers back to wild type levels?We performed EdU labeling at day 5 and day 10 after MI and surprisingly observed lower rates of proliferation, in line with an overall reduced number of fibroblasts after MI Figure 8 J,K.(B) Is it possible that collagen area as a percentage of tissue area may not be the most informative metric of fibrosis due to the significantly altered cardiac dimensions in mice with ablated fibroblasts?This is entirely possible, but for the purpose of this manuscript we simply wanted to demonstrate that remaining fibroblasts can compensate in an injury scenario.(C) It is puzzling that fibroblast ablation has so little impact on the ECM opening up questions regarding the cell state heterogeneity of the non ablated fibroblasts populations- are they all behaving like activated fibroblasts or matrifibrocytes to maintain the matrix?To address this question, we performed qPCR on the adherent cells from ablated hearts and compared their gene expression to controls and at baseline and we observed no obvious compensatory gene expression from the adherent cells. Postn, Chad, and Cthrp1 were not detected in these cultures. The data even demonstrates a decrease in many fibroblast genes, suggesting that the adherent cells may not have a definitive fibroblast gene profile. Possibly smooth muscle cells and pericytes could bias the gene expression in these cultures Figure 1J.9) This study paid a lot of attention to mechanisms of ECM deposition and remodeling but very little attention to degradation and turnover of the matrix. Are these mechanisms inhibited such that the matrix is no longer turning over once fibroblasts are ablated?MMP2 and MMP9 presence supporting the notion that matrix is no longer turned over Figure 3D.To address this question we included zymography from total heart and observed a consistent reduction in 10) What is the basis for the reduction in collagen deposition observed for the Ang/PE model but not for the infarction model?We suspect the difference is because of the type of insult. With MI there is a rapid inflammatory response with an exponential burst of fibroblasts in a matter of days, while the angII/PE is more gradual with less fibroblast expansion, relatively. We add this speculation to the discussion page 24.11) If the TAILS proteomics data suggests that cardiomyocyte remodeling may be occurring, is this finding reflected in the geometry of isolated myocytes at baseline measured for their contraction characteristics in figure 7?We did not note any overt shape changes in the isolated cardiomyocytes.Reviewer #1 (Recommendations for the authors):(a) In the summary, there are a few ambiguities that slightly obscure the meaning: the term 'uninjured' is unclear; the following sentence ["Analysis of cardiomyocyte function demonstrated weaker 42 contractions and slowed calcium decline in both uninjured and AngII/PE infused 43 fibroblast-ablated mice."] apparently differs from the comment that ", cardiac function was better 40 preserved following angiotensin II/phenylephrine (AngII/PE)-induced fibrosis and 41 myocardial infarction".The term uninjured was removed from the text of the manuscript. Also, to address reviewer’s two concerns regarding terminology, we have now changed our statements to reflect the cardiomyocyte measurement being performed. Thus, we have removed terms such as weaker contractions.(b) The Introduction is an appropriate review of the role of fibroblasts in cellular matrix formation and matrix formation, but should be slightly revised to provide a brief paragraph on the relationships between fibroblasts and the cardiomyocytes themselves, particularly in relation to influencing gap junction cohesion as well as, through their fusion, effects on capacitative properties of the cardiomyocyte syncytium with a potential for arrhythmic substrate.We have included a brief statement regarding known fibroblast-cardiomyocyte interactions in the introduction.(c) When the discussing the Results, the following merit further discussion.(i) This appeared to be a system that did not show pathological hypertrophy: it exhibited downregulated cell adhesion, collagen binding, collagen fibril organization, and ECM organization, and upregulated membrane, mitochondrial and Z-disc genes, but an absence of uupregulation in Nppa, Nppb, and Myh7 and Myh6 genes related to pathological hypertrophy.2+ transients in control and ablated cardiomyocytes both before and following AngII/PE infusion, with greater transients in the latter, in contrast to:(ii) There was a contrast between: Normal indicators of excitation contraction coupling with similar ca(iii) Mechanical indicators: Cardiomyocytes from ablated hearts showing reduced baseline sarcomeric shortening, speed of contraction with and slowed relaxation.We observed that the ablated and control mice had increased LV chamber sizes in response to MI. However, ablated mice resisted further dilation of the chamber and remained constant till the end of the study. We have made changes in the text to avoid this confusion. Other queries were addressed above.Reviewer #2 (Recommendations for the authors):The authors use an induction protocol that seems to lead to an efficiency of CRE activation somewhat below what has been reported for similar strains by others. They use a threshold of at least 45% reduction to include individual animals in the analysis. This does not seem a major improvement over their recent work in which they report the effects of a 50% reduction is minimal. Also, if their threshold is 45%, why do they keep referring to a 60 to 80% ablation in the text?To our knowledge, no previous protocols have removed resting fibroblasts. Instead, they have focused on removal of activated fibroblasts and only examine deletion efficiency within this subset. While the cutoff we used was 45%, the average deletion was ~70% (69.9% +/- 13.9). In the previous manuscript, we also did not perform any injury. Nonetheless, we can appreciate the reviewer’s concerns and have removed the range of deletion statements throughout the manuscript.One important and surprising finding in this paper is that the cells surviving the ablation did not expand to make up for the lost numbers. However, I was surprised to see that most of the evaluation of the extent of depletion over time relied on the expression of PDGFRa itself, a gene whose expression has been reported to be modulated, that is expressed at different levels in different subsets (based on published scRNAseq data) and that is expressed by a single allele in this experimental setup. In some cases, the results are expressed a "stained area/tissue area", when clearly it would have been more appropriate to provide cell counts. At least, they should provide FACS plots showing the gating used for quantification to reassure the readers that PDGFRa was expressed at comparable levels in mice of different ages. Given that it is now widely accepted that cardiac fibroblasts are heterogenous in many ways, it would be important to understand whether this ablation affects this heterogeneity. In other words, does ablation preferentially remove subpopulations of fibroblasts (such as more proliferative cells that express higher levels of Pdgfra) more than others? Knowledge of this may explain possible reasons for the lack of replenishment of fibroblasts. I think it would be worth using scRNAseq in this context.We have addressed most of these concerns above with the exception of performing single cell sequencing. We hope that the primary cell isolation and use of Col1a1GFP help to alleviate the heterogeneity concerns raised by this reviewer.The CRE strain used is active in a number of other organs and anatomical locations, ranging from lung fibroblast to adipogenic cells in fat depots to oligodendrocyte progenitors in the CNS. Did the authors look into these organs, or notice any systemic effects of their depletion protocol?We now include a blood metabolic panel. While we appreciate that this does not reflect other organ function such as the liver, spleen, skin etc. We hope that it provides an idea of the general health of the animals. Supplemental table 2. For the reviewer’s information, we do observe reduced fibroblasts in the lung, spleen, kidney, and liver. The lung is complicated by the fact that there are other fibroblasts that never express PDGFRa. We have not examined skin or cornea . Surprisingly, oligodendrocytes do not seem to be perturbed by PDGFRa ablation. Potentially because they either express too low of a level to induce DTA or they are repopulated by some progenitor cell. We have not explored the “other” fibroblast population numbers in these organs.Based on the data presented, the hearts contained somewhere between half and one third of their normal number of fibroblasts, suggesting that one or two cell divisions would be sufficient to bring these cells back to normal levels. Following an MI, these cells enter rapid proliferation, and their numbers increase significantly. What happens in the ablated heart to fibroblast numbers? Do they increase as expected, do they plateau at lower or similar levels to the ones observed in controls? Does the scar still contain the same number of collagen-expressing cells?A time resolved analysis of PDGFRa cells in the context of the two damage models would be very informative and possibly hint at mechanism, and I was surprised to see the analysis limited to one time point and performed by quantifying genes rather than cells themselves.These concerns are addressed above.The proteomic analysis of the de-cellularized heart is a strong point of the paper. However, some of the changes observed may not be a direct effect of fibroblast ablation. For example, one of the proteins the authors reported to be upregulated in the fibroblast-ablated hearts is NG2, which is not expressed to any significant extent in fibroblasts. Rather, it is expressed at high levels in mural cells such as pericytes and vascular smooth muscle cells. Does the increase in NG2 indicate an upregulation of the protein in mural cells or increase (maybe compensatory) in the numbers of mural cells? Adding mural cells to the FACS-based quantification of inflammatory, endothelial and fibroblastic cells would help in answering this.These concerns are addressed above.I am a bit confused by the fact that the authors, although reported decreased LV internal diameter, found that cardiomyocytes did not increase in size. Usually, the reason for decreased chamber size would be hypertrophy, especially if it is observed in systole and diastole. How do the authors explain this?Our data indicate that shortening and Ca kinetics are slowed, while shortening amplitude is decreased. Thus, the slowed kinetics for both shortening and Ca match, with the slowed Ca decline most likely leading to slowed relaxation. While Ca transient amplitude is normal, shortening is decreased. These data would suggest that there is a decrease in Ca sensitivity. In our ablated hearts, the basement membrane is disrupted that could decrease myofilament calcium sensitivity (PMID: 28018228) through various mechanisms such as changes in myofilament phosphorylation (PMID: 34641704).I am surprised that the authors did not attempt to measure diastolic function in the fibroblast-ablated hearts following damage in either the MI or the Ang II/phenylephrine models. The parameters they report are more relevant to systolic function (ejection fraction) and chamber dimension (LVID). However, I would have expected some measurement of transmitral flow such as E/A ratio or E wave deceleration time from a 4- chamber apical view, or even IVRT. This is important as ECM/fibrotic changes are expected to affect LV compliance/stiffness (i.e. diastolic function) usually before systolic involvement.Addressed above.How was the collagen area measured in the 2 damaged models? By antibody staining or using Trichrome staining? Why in the authors' opinion did fibroblast ablation decrease collagen area in the Ang II/phenylephrine model but not the MI model? Again these questions may be answered by analyzing the proliferative response to these insults.Addressed above.A decrease in shortening without a decrease in calcium transient amplitude, as seen in the ablated hearts, indicates calcium desensitization. An important pathway that regulates calcium sensitivity and the actin cytoskeleton at the same time is the RhoA/Rho kinase pathway. Did the authors find any changes in expression of the proteins in this pathway?Addressed above.Reviewer #3 (Recommendations for the authors):In this manuscript, Kuwabara and colleagues use genetic ablation to reduce the number of fibroblasts resident to the heart. At baseline, the authors observe that fibroblast numbers stay proportionally low after ablation, but with very minimal effects to the structure or composition of the extracellular matrix. Fibroblast ablation prior to myocardial infarction is shown to be beneficial to cardiac function without affecting relative abundance of scar tissue, whereas in an Ang/PE model of fibrosis collagen deposition is impaired and systolic function is preserved.Major Concerns1. For the myocardial infarction model presented in Figure 4, it is fascinating that no rupture is observed and that the relative collagen fraction between the ablated mice and controls is largely similar by 10 days. (A) Have the non-ablated fibroblasts proliferated and normalized the fibroblast numbers back to wildtype levels? (B) Is it possible that collagen area as a percentage of tissue area may not be the most informative metric of fibrosis due to the significantly altered cardiac dimensions in mice with ablated fibroblasts? (C) It is puzzling that fibroblast ablation has so little impact on the ECM opening up questions regarding the cell state heterogeneity of the non ablated fibroblasts populations- are they all behaving like activated fibroblasts or matrifibrocytes to maintain the matrix?Addressed above.2. This study paid a lot of attention to mechanisms of ECM deposition and remodeling but very little attention to degradation and turnover of the matrix. Are these mechanisms inhibited such that the matrix is no longer turning over once fibroblasts are ablated?Addressed above.3. What is the basis for the reduction in collagen deposition observed for the Ang/PE model but not for the infarction model?Addressed above.4. If the TAILS proteomics data suggests that cardiomyocyte remodeling may be occurring, is this finding reflected in the geometry of isolated myocytes at baseline measured for their contraction characteristics in figure 7?Addressed above.


Background and Objectives: The constantly increasing prevalence of type 2 diabetes mellitus (T2DM) and the advent of new treatment options have made management of T2DM patients more demanding. We aimed to (a) estimate the familiarity of general practitioners with novel T2DM treatment options, (b) determine whether a digital tool can aid in their treatment decisions and (c) demonstrate that an evidence-based digital clinical support tool can be made using an existing digital platform. Materials and methods: This proof-of-concept study consisted of two parts: We first conducted a simple online survey among general practitioners of three European countries to estimate their familiarity with novel T2DM treatment options and to determine whether they believe that a digital tool can aid in their T2DM treatment decisions. We then proceeded to develop a new digital tool that provides quick, evidence-based support for treatment of patients with T2DM using an existing digital platform. Results: The online survey was completed by 129/5278 physicians . Only 30.7% of all general practitioners reported to be either very or extremely familiar with novel T2DM treatments; the vast majority of participating general practitioners (82.8%) reported that they would find a digital clinical decision support tool for treating T2DM patients either very or extremely useful. A digital tool which features the characteristics deemed most important by the polled physicians was subsequently developed. Conclusions: The results of the online survey showed that familiarity of general practitioners with novel T2DM treatment options is relatively low and that there is a need for digital clinical decision support tools intended to facilitate treatment decisions in T2DM patients. We demonstrated that such a tool can easily be developed using an existing digital platform. Type 2 diabetes mellitus (T2DM) is an expanding global health problem, characterized by dysregulation of carbohydrate, lipid, and protein metabolism. Insulin resistance, one of the hallmarks of T2DM, is closely associated with obesity. T2DM also carries a highly elevated risk of both microvascular complications  and macrovascular complications  .The rapid evolution of our understanding of T2DM has expedited the development of novel medications and several new antidiabetic medications have gained widespread use in the last decade, including dipeptidyl peptidase-4 (DPP-4) inhibitors, glucagon-like peptide-1 (GLP-1) receptor agonists, and sodium/glucose cotransporter 2 (SGLT2) inhibitors, which have been also incorporated in the latest international scientific guidelines for the management and treatment of T2DM ,4,5. SinThe constantly increasing prevalence of T2DM worldwide has made management of T2DM more demanding ; in ordeFortunately, these challenges are paralleled by the digital evolution. Recent decades have seen an increased adoption of mobile devices by the general public as well as the healthcare professionals ,12. MobiThe reasons behind the gap between the guidelines and the real-world management of T2DM patients are not yet fully elucidated . They caThis proof-of-concept study consisted of two parts: first, a simple online survey was conducted among general practitioners of three European countries; then a new digital tool that provides quick, evidence-based support for treatment of patients with T2DM was developed using an existing digital platform.The anonymous online survey consisted of seven multiple-choice questions  that werThe survey included licensed general practitioners of three European countries , who were registered users of the Mediately application  and who had previously provided consent to be contacted for research purposes. In order to achieve a homogenous survey population, physicians of other specialties as well as trainees and residents were not included in the survey. The three countries with different healthcare systems were deemed to be representative of the region where Mediately currently operates . The study was approved by the Slovenian National Medical Ethics Committee .In December 2021, an email with an explanation of the purpose of the questionnaire and a hyperlink to a web-based survey platform was sent to 5278 licensed general practitioners, users of the Mediately application. Based on Mediately’s previous experience with online surveys , the survey was active for 7 days, from 21 December 2021 to 27 December 2021. The results were collected using a dedicated survey platform and analyzed using one of the commonly used spreadsheet programs.Clinical decision support tools are systems that can link individual patient’s characteristics and physician’s findings with evidence-based data and thus assist in the healthcare decision-making process. The T2DM tool was conceptualized as an interactive digital step-by-step algorithm based on the treatment algorithm proposed by the 2019 ADA/EASD update on management of T2DM  and the European Association for the Study of Diabetes (EASD)) [The Mediately application is based on a uniform digital framework, which provides content to mobile and web-based platforms. The developers set up a certified quality management system for medical devices (ISO 13485:2016); the content, function and ergonomics of the tools are reviewed and tested by professionals of different fields, including medical doctors working in the company and several external experts. Most tools are also translated into users’ local languages by professional translators. The Mediately Clinical Tools collection is registered as a medical device and is available free of charge on mobile and web platforms in several European countries.The questionnaire designed to estimate the familiarity of general practitioners with novel T2DM treatment options and to appraise some of their digital needs pertaining to management of T2DM patients was completed by 129/5278 physicians .In general, only 30.7% (39/127) of general practitioners reported to be either very or extremely familiar with novel T2DM treatments. However, there were considerable variations among different countries: the Czech physicians were most familiar with the whole group of novel medications (63.6% either very or extremely familiar) while the Italian physicians more vaguely (only 22.3% were either very or extremely familiar). Slovenian and Italian physicians reported to have the best knowledge of SGLT2 inhibitors , whereas the Czech physicians were the ones most well-informed about the DPP-4 inhibitors and the GLP-1 receptor agonists .Regarding the digital needs, the vast majority of physicians  reported that they would find very or extremely useful a digital tool to support their clinical decisions for treating T2DM patients, a kind of digital tool that, to our knowledge, did not exist prior to the development of our digital T2DM algorithm. The features that were considered to be of utmost importance were the compliance of the tool content with local guidelines, the ease of use, and the cost-free availability. Not all participants provided answers to all the questions of the survey; the question with the lowest number of valid responses was question No. 3 (familiarity with GLP-1 agonists) with 126 valid responses.The algorithm provides a digitalized version of main recommendations covered by the 2019 ADA/EASD update on management of T2DM  and the European Association for the Study of Diabetes (EASD)) [More than two-thirds of the polled physicians were at best moderately familiar with the new generation of T2DM medications that have now been available for about a decade. These groundbreaking medications provide some previously unseen and somewhat unexpected benefits; they address several important aspects of T2DM, the metabolic syndrome and a significant spectrum of the most substantial comorbidities . In addiReports dating back to 2014 have posited that digital tools can be used to address the challenges presented by modern management of chronic conditions . A more We recognize that this study has certain limitations, including the relatively small number of participants to the survey, the lack of data on demographic characteristics of the participants, and the low response rate. Even though almost every European physician probably owns a mobile phone, the digital-only distribution of the questionnaire might have exposed the results to selection bias. Since the process of developing, updating, and validating a digital tool takes some time, the tool content is inevitably updated with some delay after the publication of new guidelines.While response rates of <10% are not unusual for online surveys, we acknowledge that additional factors may have existed that resulted in an even lower response rate: the holiday season, lack of reminders and incentives for completion of the survey, the well-known time-constraints of general practitioners and the relatively short timeframe in which the online survey was active. However, in our experience, incentives and reminders do not consistently increase response rates and can appear disagreeable or annoying, especially to overburdened physicians. We also doubt that increasing the duration of the survey would majorly impact the response rate, potential participants usually become involved with online surveys in the first few days after receiving them or not at all. While it is possible that the low response rate might have impacted the validity of the survey to some extent, we believe that the results at the very least point to the existence of some unmet digital needs (that can demonstrably be fulfilled).We wish to highlight that reliable digital tools have an even greater importance during the current coronavirus disease (COVID-19) pandemic. Indeed, different solutions of telemedicine have been implemented, and it is likely that daily usage of digital tools will be maintained during post-COVID-19 time for patient management . This haBearing in mind the limitations of the current proof-of-concept study, we are convinced that novel digital tools can have a significant role in relieving some of the burden imposed on physicians by the constantly changing evidence-based medicine, and in optimizing the worldwide use of modern diagnostic and therapeutic procedures. More importantly, the use of reliable and effective novel digital tools can potentially improve the care of patients suffering from chronic diseases, including diabetes. We believe potential areas for future research include determining the reasons behind the low familiarity of general practitioners with novel T2DM medications and finding new innovative ways to improve digital clinical decision support systems.The results of our online survey show that (a) the familiarity of general practitioners from Italy, Czechia, and Slovenia with novel T2DM treatment options is relatively low; (b) there is a need for a digital clinical decision support tool intended to facilitate treatment decisions in T2DM patients.We demonstrated that an easy-to-use, evidence-based T2DM treatment clinical decision support tool can easily be developed using an existing digital platform, potentially contributing to improved patient care.


Globally, countries are still battling health challenges and the negative economic stress on the citizenry caused by COVID-19. This study explored the perspectives of market women in Ghana and South Africa on COVID-19. Data collection was executed in both Ghana and South Africa between March 2021 and December 2021. Employing semi-structured questionnaires, face-to-face interviews were conducted. Most of the market women in Ghana described COVID-19 as a global pandemic, while market women in South Africa described the disease as the deadly flu. There were similarities in the perceived signs and symptoms of COVID-19. Market women in both countries specifically observed that not adhering to the safety protocols was the major mode of transmission. Lemon, garlic and ginger were the most common foodstuffs used by the market women to fight COVID-19. To prevent COVID-19 at their places of work, market women stressed the importance of observing the safety protocols. An overwhelming majority of market women in both countries bemoaned the negative impact of COVID-19 on their businesses and suggested the need for financial assistance from their respective governments. The findings are intended to assist policymakers in both Ghana and South Africa to implement interventional projects to assist women whom the literature suggests are the most vulnerable during pandemics such as COVID-19. The novel coronavirus disease was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on 11 February 2020, by experts of the international committee on the taxonomy of viruses. SARS is a zoonotic coronavirus and falls under the β-coronavirus cluster . COVID-1In our quest to fight COVID-19 in our societies, we cannot ignore the contributions of market women. They are considered essential workers who ensure that foodstuffs and other commodities are readily available for the general population amid the COVID-19 pandemic. A study conducted to determine the community drivers affecting adherence to WHO guidelines against COVID-19 amongst rural traders in Uganda revealed that most of the participants  had sufficient information regarding COVID-19 [However, the perspectives of market women on COVID-19 in different countries are not well documented. Comparison of the perspectives of market women in different jurisdictions of Africa could be important drivers for governmental policy decisions on the continent. A classic example is the formulation of policies to reduce the negative impact of COVID-19 on the health and business of market women. The choice of the two countries under investigation was due to the structure of the respective economies. Ghana has a relatively informal economy compared to South Africa. Considering the structure of the two economies, it is inherent to access the views of market women in these two countries due to the differences therein. This study therefore aimed to explore the perspectives of market women on COVID-19 in both Ghana and South Africa.A cross-sectional mixed method (embedded approach) was employed for this study. Analysing both qualitative and quantitative data assists the researcher in deeply understanding the research problem which leads to the drawing of salient conclusions .The study took place in the new Agogo Market, Ashanti region of Ghana, and Ladysmith market, KwaZulu-Natal Province, South Africa. The new Agogo Market is located in the Asokwa Municipal Assembly about 12 km from Kumasi, the Ashanti regional capital. The Asokwa municipal assembly has Asokwa as its capital and a population of about 125,642 . The AshIt is a category B municipality within the uThukela District Municipality with a population of about 237,437. Category B refers to the municipality that shares both municipal executive and legislative authority in its area with a category C within whose area it plunges. Category C denotes a municipality that has both municipal executive and legislative authority that consist of more than one municipality . The KwaMarket women aged 18 years and above in both Ghana (new Agogo Community Market) and South Africa (Ladysmith Market) were random/purposively approached at their places of work to participate in the face-to-face interviews using researcher-administered questionnaires. Market women below the age of 18 years as well as those exhibiting signs and symptoms related to COVID-19 during the face-to-face interviews were excluded.The population of market women at the new Agogo Community Market (Ghana) and Ladysmith (South Africa) Market are about 350 and 200, respectively. Assuming an error of 5%, a confidence level of 95% and a response distribution of 50%, the sample sizes for both markets were 198 and 130, respectively . The 50%Market women were sampled using systematic random sampling  by makinEthical approval was obtained from the Committee on Human Research, Publications and Ethics (CHRPE) of the Kwame Nkrumah University of Science and Technology (KNUST) in Kumasi, Ghana (reference number: CHRPE/AP/060/21) and Sefako Makgatho University Research Ethics Committee (SMUREC), South Africa (reference number: SMUREC/P/120/2021:IR).The study took place in both Ghana and South Africa concurrently between March 2021 and December 2021. Market women in both Ghana and South Africa were visited at their places of work for data collection. All COVID-19 protocols were observed during data collection to minimize the risk of infection between the research assistants and the market women. Face-to-face interviews with the help of semi-structured questionnaires were used to collect data from the research participants after informed consent was obtained. The researchers administered a semi-structured questionnaire developed in an English language but translated into Twi (Ghana) and isiZulu languages (South Africa) languages. The developed semi-structured questionnaire was pilot tested with market women to determine its validity. The pilot study included about 20 market women in both Ghana and South Africa to determine the viability of the intended study to be carried out. The responses received from the market women in both Ghana and South Africa helped us modify the semi-structured questionnaire which was used in this study. The first part of the semi-structured questionnaire collected information on the socio-demographics . The second part of the questionnaire obtained information on their general understanding of COVID-19, signs and symptoms of the disease, natural therapies employed by them to fight the disease, measures put in place to fight the disease at the workplace and their general perspectives about the impact of COVID-19 on their businesses and the economies of their respective countries .In Ghana, the face-to-face interviews were conducted by a research assistant who was fluent in both Twi and English languages. In South Africa, the research assistant who conducted the interviews was fluent in both isiZulu and English languages. Each interview lasted for about forty minutes.The interviews conducted in both Ghana and South Africa with the market women were conducted in Twi and isiZulu languages, respectively. The interviews were transcribed verbatim. Experts in both Twi and isiZulu were hired to translate the transcripts to the English language. Back translations were conducted by independent language consultants to determine the correctness of the transcripts . To ensure the credibility of the study, market women in both Ghana and South Africa were invited by the researchers to confirm the contents of the transcripts .Based on Tesch’s method , the texThe average age of market women in Ghana was 39.30 ± 11.76 while in SA it was 43.72 ± 9.24. The majority of market women in both Ghana  and South Africa  had completed secondary education. The mean age of years of working experience stood at 9.22 ± 7.38 and 9.93 ± 6.31 for Ghana and South Africa, respectively. The average monthly profit recorded for market women in Ghana was 590.37 ± 752.38 Ghana Cedis which is approximately 90 USD, while South African market women registered an average monthly profit of 5127.30 ± 2297.94 Rand approximately 338 USD. Averagely, Ghanaian market women were making a profit of 3 USD which is higher than the daily minimum daily wage (2.07) announced by the Government of Ghana in 2021 [The women expressed their understanding of what COVID-19 was, their statements varied from it being a “global pandemic” (44.72%) to a killer disease (0.50%) in Ghana (GH) and “deadly flu affecting lungs” (67.77%) in South Africa (SA), as summarized in The majority of the market and women in Ghana described it as a global pandemic . Some described it as an airborne disease  with a few describing it as a viral disease . An overwhelming majority of market women in South Africa described COVID-19 as a deadly flu .The following statements were made by market women in both Ghana and South Africa regarding their understanding of COVID-19.“…COVID-19 is a global pandemic.”GH 5.“This is a foreign disease, it originated from China”GH17.“COVID-19 is a viral disease.”GH 48.“It is an airborne disease.”GH 30.“Deadly flu affecting the lungs.”SA 10.“COVID-19 is a pandemic flu.”SA 81.The perceived signs and signs of symptoms of COVID-19 mentioned by the market women are exhibited in “To me, cough and fever are the signs that you have COVID-19.”GH 4.“Catarrh and headache are some of the signs and symptoms of COVID-19.”GH 59.“Some of the signs and symptoms of COVID-19 are cough, fever, sweating and difficulty to breathe.”SA 8.“Cough and body pains are the symptoms associated with COVID-19.”SA 30.“Not wearing masks and not washing of hands”GH 17.“Not wearing nose masks and not washing of hands”GH 36.“Not wearing masks, not washing hands and not practising social distancing”SA 2.“Not wearing nose masks and not practising social distancing”SA 14.The natural therapies employed by the participants to mitigate the effects of COVID-19 are represented in Azadirachta indica (neem) tree and Hibiscus sabdariffa L. “sobolo” as those they rely on to fight COVID-19. Similarly, an overwhelming majority of market women in South Africa  mentioned these foodstuffs  and herbs such as Artemisia afra as those they lean on to fight the pandemic. In addition, market women in South Africa emphasized the need to drink hot water regularly to help fight COVID-19.Most of the market women in Ghana  mentioned at least one of these foodstuffs  and medicinal plants such as According to the market women, the therapies and foodstuffs employed by them to fight COVID-19 were as follows:“I used to drink “sobolo” tea and boiled neem tree leaves to fight the virus.”GH 182.“I leaned on these foodstuffs (garlic and ginger) to boost my immune system.”GH 82.“These foodstuffs (lemon and herbs) I ate every day help me to boost my immune system.”SA 87.“I depend on lemon, garlic, ginger and hot water to fight COVID-19.”SA 82.To access their basic knowledge about the measures put in place at their workplace to limit the spread of the virus, market women in both Ghana and South Africa stated that they rely on the safety protocols such as the wearing of nose masks, hand washing, social distancing and the use of sanitisers.An overwhelming majority of the market women in both Ghana  and South Africa  indicated a reduction in income as the main consequence of COVID-19. Some market women in South Africa  mentioned the collapse of businesses as a consequence of the disease.The participants’ observations about the impact of COVID-19 on their businesses are represented in The participants’ general views about the impact of COVID-19 on their respective country’s economies are represented in Market women thought that the respective governments must reduce taxes and assist them with loans to support their businesses.“Our government must help us by reducing taxes”GH 198.“Loans must be readily accessible to cushion our businesses”GH 181.“The government must provide loans to support our businesses”SA 29.“The government must reduce taxes……”SA 9.Our data analysis has shown that the market women in both Ghana and South Africa have a basic understanding about the COVID-19 pandemic and its negative impact on their businesses and the economy of their respective countries. This is evident in the responses they gave. Regarding the participants’ general understanding of COVID-19, market women’s descriptions of COVID-19 in both Ghana and SA is congruent with scientific literature which described the disease as a viral disease and associated with flu-like symptoms .Market women in both Ghana and South Africa had a basic knowledge of the perceived signs and symptoms of COVID-19. An overwhelming majority of market women in Ghana  and South Africa  mentioned that a cough, fever, body pains, catarrh, sore throat, cold, sweating difficulty breathing were signs and symptoms of the disease. The aforementioned perceived signs and symptoms of COVID-19 by the participants are in agreement with scientific literature . This maArtemisia afra has been proven to be effective against SARS-CoV-2 and feline coronavirus (FCoV) [Hibiscus sabdariffa L.“sobolo” employed by market women in Ghana to manage COVID-19 has been proven scientifically to possess inhibitory activity against human A virus (IAV) [Azadirachta indica (neem) tree. The study found that seeds and leaves of Azadirachta indica (neem) showed antiviral activity against herpes simplex virus type 1 [Allium sativum (garlic) has been proven to be effective against the influenza virus [Zingiber officinale) was effective against the human respiratory syncytial virus in human respiratory tracts [The majority of market women in both Ghana and SA mentioned the same foodstuff and therapies, which they relied upon to fight COVID-19. Interestingly, Nie et al., 2021, found that s (FCoV) , Similarus (IAV) . Workerss type 1 . Allium za virus . A studyy tracts . The recy tracts . A studyy tracts .The market women in both Ghana and South Africa were able to mention the basic protocols  that must be observed to fight the COVID-19 pandemic. This interesting observation seems to suggest that the respective governments’ health and communication teams’ message about what must be carried out (protocols) to fight the pandemic has reached the grassroots in both countries. This is a major boost towards the two respective countries’ quest to become COVID-19-free nations in the distant future.The market women in both Ghana and South Africa bemoaned the negative impact of COVID-19 on their business due to the loss of income as a result of the lockdown restrictions imposed by the respective governments in these two countries. This is in agreement with a study conducted in Zimbabwe where women entrepreneurs had no option but to close their businesses due to the loss of revenues as a result of COVID-19 lockdown restrictions imposed by the Zimbabwean government . SimilarMarket women in both Ghana and South Africa spoke about the negative impact of COVID-19 on their respective country’s economies. Available data suggest that Ghana had a decrease in economic growth from 5.8% to 1.5% , while tStudies have shown that compared to men, women entrepreneurs are generally affected during a pandemic because they are more susceptible to difficult situations ,42. The This study was carried out in two countries, Ghana and South Africa. To understand the problem from a wider perspective, the study should be replicated in other countries. The fear of being infected with COVID-19 by getting into contact with different people among the studied population should have contributed to the low sample size recorded in South Africa. The study did not consider vaccination status of the participants, which would have added to the body of knowledge on vaccine uptake in this population.This study explored market women’s perspectives about COVID-19 on the African continent. The data suggest that market women in both Ghana and South Africa had a good general understanding of COVID-19 and its effects on their livelihoods and their respective country’s economies. Market women’s description of COVID-19 as a disease associated with flu-like symptoms is consistent with scientific literature. Market women’s perception of the mode of transmission of COVID-19 (being in contact with an infected person) is congruent with the scientific literature. Natural remedies such as lemon, garlic and ginger employed by the market women to mitigate the effects of COVID-19 have been scientifically proven to possess antiviral activities. The strategies (wearing of masks and handwashing) adopted by market women to limit the transmission of the virus at their workplaces conform to the safety protocols adopted by the World Health Organization (WHO) to fight the pandemic. The market women believed that COVID-19 had harmed their businesses and the economies of their respective countries. Market women in both Ghana and South Africa pleaded with their respective governments to make loans readily accessible to cushion their businesses.


Coronavirus disease 2019 (COVID-19) is associated with an increased prevalence and mortality from diabetic ketoacidosis (DKA) globally. With limited access to specialised care, most patients with DKA in South Africa are managed at district hospital level. This study describes the profile of patients admitted to a district hospital in South Africa with DKA and COVID-19 and examines associated risk factors encountered.This was a case series of all patients presenting to a district hospital with DKA and COVID-19 infection between July 2020 and July 2021. Data extracted included patients’ demographic profiles, biochemical results, comorbidities and clinical outcomes.The median age of the 10 patients admitted during the study period was 39 years old (±12), six of whom were male. The hemoglobin A1c (HbA1c) values on admission ranged from 9.7 to 13.8. Five of the patients had pre-existing type 2 diabetes mellitus (DM). Four of the known DM patients were on metformin only, and one was on biphasic insulin. Three patients had other pre-existing comorbidities, two patients with hypertension and one with human immunodeficiency virus (HIV). Three patients demised, two of whom were hypoxic on admission.Diabetic ketoacidosis appears more commonly in COVID-19 infected patients with type 2 DM and at a young age. Suboptimal glycaemic control was associated with DKA, and hypoxia was a strong predictor for mortality. Treatment inertia was evident in the known DM group, who were on monotherapy despite persistent hyperglycaemia. Greater vigilance is required to detect ketosis in type 2 DM and intensify therapy to improve glycaemic control. Once the viral complex has entered the cell, ACE2 is downregulated; this leads to unopposed angiotensin II, contributing to impaired insulin secretion and therefore hyperglycaemia.15There are different speculations regarding COVID-19’s role in triggering DKA. Studies suggest that COVID-19 aggravates beta cell function to precipitate DKA in both pre-existing and newly diagnosed diabetic patients.9 Some studies in North America indicate that this cohort of patients are young (median age of 47 years) with long-standing hyperglycaemia (median hemoglobin A1c [HbA1c] of 13.8%) on presentation.16 A recent case series in South Africa reported significantly elevated HbA1c levels (10.9% – 13.4%) among four patients at presentation, of which only one was known to have pre-existing DM.9 It is unclear whether COVID-19 unmasks DM or triggers stress hyperglycaemia, a transient state of hyperglycaemia that occurs during serious medical illnesses.17Even though there are case reports of known and previously undiagnosed diabetic patients with COVID-19 presenting with DKA, there is a paucity of data from sub-Saharan Africa on the associated risk factors for DKA in COVID-19 patients.Given the increased incidence and mortality of DKA in COVID-19 patients, it is imperative to investigate the risk factors associated with admission for DKA in order to facilitate early diagnosis and improve clinical outcomes. In this study, a series of 10 patients are discussed, all of whom were admitted to a district hospital in South Africa with DKA and COVID-19. The aim of the case series report is to describe the clinical characteristics of the patients and explore possible risk factors and potential interventions to improve outcomes.This study was a retrospective single-centre case series at an urban district hospital in South Africa. All patients admitted with a diagnosis of DKA between July 2020 and July 2021 were identified from the hospital admissions register. During the study period, all admissions had nasopharyngeal and oropharyngeal swabs sent for identification of the novel coronavirus responsible for COVID-19 disease. Patients with a positive result on the real-time reverse transcription polymerase chain reaction (RT-PCR) assay were then included in the study.18 The hospital has a high care ward but not an intensive care unit (ICU). Patients admitted with DKA are usually managed by the Family Medicine Department in the high care ward.The setting was an urban district hospital in South Africa. This busy facility serves a population of approximately 980 000 people and provides general medical, surgical and obstetric services. Approximately 100 patients with DKA are admitted annually. The high incidence of DKA has been attributed to the high prevalence of DM in South Africa.19 The criteria for diagnosing DKA were as follows:This case series describes all adult patients (> 18 years old) admitted to the facility with DKA who were also infected with the novel coronavirus. The 12-month study period included the tail end of the first COVID-19 wave, the second wave and the early part of the third COVID-19 wave as experienced in South Africa.random blood glucose > 13.9blood pH < 7.3ketonuria.20 Patients with DKA who were managed as outpatients were excluded from this study. All eligible patients were included in this study.Evidence of coronavirus infection was confirmed by laboratory result (RT-PCR). This test confirms presence of the viral genetic material in the body.A data collection tool was used to extract data from selected patient files. Medical records were reviewed, and data were extracted regarding patients’ demographic details, clinical progress and laboratory results.Descriptive analysis was conducted on the data. Descriptive statistics were calculated for all variables, with mean ± standard deviation (s.d.) or median and interquartile range reported for continuous variables, and number and percentage of total were reported for categorical variables.Consent was obtained from the hospital management and the University of KwaZulu-Natal Biomedical Research Ethics Committee (ref. no. BREC/00000907/2019).A total of 106 patients were admitted with DKA at the district hospital during the study period (01 July 2020 – 31 July 2021). Ten of the identified patients with DKA also tested positive for the novel coronavirus (9.4%). As indicated in n = 5) were reported to have pre-existing type 2 diabetes mellitus (T2DM), for which they were on treatment. One of the known T2DM patients was on biphasic insulin only, and the other four were on metformin. None of the known DM patients were on two or more antidiabetic agents. Two of the patients with DM had pre-existing hypertension, and one DM patient was also human immunodeficiency virus (HIV)-positive. All of the patients with comorbid conditions were on treatment prior to admission.Half of the patients . The patients with the two lowest pH values on presentation (pH of 6.88 and 7.00) both demised. The third patient who demised had an initial pH of 7.23.The clinical presentation of patients: six of the patients presented with symptoms suggestive of COVID-19 infection . Hypoxia, as denoted by oxygen saturation of less than 90% on room air, was only present in two of the patients, both of whom demised.21 Although it is a recommendation for patients with severe DKA to be managed in an ICU setting, only three of the non-COVID-19 DKA patients were transferred to ICU during the study period.22 Under normal circumstances, patients with DKA are managed in the high care ward at the district hospital. Only 1 of the 10 patients in this case series was managed in the high care ward; the rest were managed in the COVID-19 isolation wards. Three of the patients recovered in the district hospital ward and were discharged. Three of the patients were transferred to the field hospital from the district hospital, where management was continued. Three patients demised at the district hospital.Despite seven patients requiring step-up care in an ICU, as indicated by national guidelines, only one was successfully transferred to ICU, where he recovered in seven days and was discharged.Two of the three patients who demised had severe DKA; the third patient who demised had features of combined DKA/hyperosmolar hyperglycaemic state (HHS).The average length of stay in hospital was 3.3 days, ranging from 1 to 10 days. The short duration of hospital stay was highly influenced by the COVID-19 pandemic. The district hospital provided acute care and thereafter patients with COVID-19 were stabilised and transferred to one of the COVID-19 field hospitals.Of note, all of the patients (both with known and previously undiagnosed DM) were noted to have abnormal HbA1c values on admission ranging from 9.7 to 13.8. Also, the known diabetic group had an average HbA1c of 11% and unknown diabetics HbA1c 12.5%, yet the previously undiagnosed diabetic patients had better outcomes  compared to the group of known diabetic patients . This could possibly be because of the relatively short duration of hyperglycaemia in the previously undiagnosed patients, and possible presence of comorbidities and diabetic complications in the known diabetic patients may have adversely affected their prognosis.13 Furthermore, patients with COVID-19 and DKA have shown more severe complications and have had higher mortality than those without COVID-19.24 However, there was little representation of the African region in those reviews.13 The high mortality in our cohort (30%) also suggests that COVID-19 related DKA has a poor prognosis. However, other factors such as the absence of ICU facilities and suboptimal glycaemic control during the pandemic could have also contributed to the poor outcomes.This case series provides data on patients admitted to an urban district hospital in South Africa with DKA and COVID-19. Recent systemic reviews concluded that the mortality rate among patients with DKA and COVID-19 was as high as 50%.12 Misra et al. noted an increased incidence of DKA admissions in England during the COVID-19 pandemic, with a higher occurrence among type 2 diabetes and newly diagnosed diabetes than in type 1 diabetes.25 However, because of the small sample size in this case series, it is unclear if people with type 2 DM patients infected with COVID-19 in South Africa are more prone to DKA than those with type 1 DM. There is an evident need for improved surveillance and data in the African region, especially at primary care level.An unusual finding in this case series is that all the patients had T2DM, half of whom were diagnosed on admission. The majority of patients admitted with DKA elsewhere in South Africa are reported to be type 1 DM patients.23 Of note, the five patients with known DM had suboptimal glycaemic control, yet 60% were still on oral antidiabetic agents and monotherapy. Further investigation is required into possible physician inertia and lack of adherence to treatment guidelines. It is also possible that other factors during the pandemic contributed to the poor glycaemic control, such as reduced access to chronic care management and more sedentary lifestyles because of social restrictions and lockdown regulations.23As reported elsewhere, a high HbA1c was associated with risk for hospital admission with DKA and poor outcomes.27 Those patients who presented with significant hypoxia requiring more than simple facemask oxygen therapy demised.In this case series, patients who demised were hypoxic on presentation, or became hypoxic shortly after admission, and those who survived were not oxygen dependent. Studies have shown that the level of hypoxia on presentation to a facility is the most reliable biomarker of critical illness, ICU admission and poor outcomes.28 Further contributors to severe COVID-19 disease are age, obesity and other comorbidities, with obesity and hyperglycaemia seemingly being independent risk factors for a worsened course of illness.23 In this study, comorbidities included hypertension, obesity and HIV. However, the majority of patients were under 45 years old and had no chronic conditions other than DM. Improved glycaemic control alone could potentially result in significantly reduced risk for admission for DKA. There is an evident need for improved attention to community education and health provider adherence to diabetes guidelines.It has become evident that type 2 diabetes is one of the leading contributors to poorer outcome in patients with COVID-19. Despite this, severe illness can occur, even in previously healthy individuals, at any age.The sample size in this case series is small, and therefore the characteristics of this cohort of patients is not statistically significant. Furthermore, the patients selected were a convenience sample from one facility and may not be representative of all patients with DKA and COVID-19. The period of sampling occurred during the height of the COVID-19 pandemic and therefore other factors such as staff shortages and changes in facility resources may have influenced the inpatient management and clinical outcomes of the patients.This study provides useful insight into the risk factors for admission among patients with both DKA and COVID-19 infection in a sub-Saharan African setting. As there were no type 1 DM patients in this case series, it is unclear how COVID-19 influences DKA in type 1 DM in our setting. A significant finding was the suboptimal glycaemic control in all the known diabetic patients who were on monotherapy despite markedly elevated HbA1c levels. Greater attention is required to intensify treatment for poorly controlled type 2 DM patients at primary care level.