Various combinations of cardiogenic transcription factors, including Gata4 (G), Hand2 (H), Mef2c (M) and Tbx5 (T), can reprogram fibroblasts into induced cardiac-like myocytes (iCLMs) in vitro and in vivo. Given that optimal cardiac function relies on distinct yet functionally interconnected atrial, ventricular and pacemaker (PM) cardiomyocytes (CMs), it remains to be seen which subtypes are generated by direct reprogramming and whether this process can be harnessed to produce a specific CM of interest. Here, we employ a PM-specific Hcn4-GFP reporter mouse and a spectrum of CM subtypespecific markers to investigate the range of cellular phenotypes generated by reprogramming of primary fibroblasts. Unexpectedly, we find that a combination of four transcription factors (4F) optimized for Hcn4-GFP expression does not generate beating PM cells due to inadequate sarcomeric protein expression and organization. However, applying strict single-cell criteria to GHMT-reprogrammed cells, we observe induction of diverse cellular phenotypes, including those resembling immature forms of all three major cardiac subtypes (i.e. atrial, ventricular and pacemaker). In addition, we demonstrate that cells induced by GHMT are directly reprogrammed and do not arise from an Nxk2.5 + progenitor cell intermediate. Taken together, our results suggest a remarkable degree of plasticity inherent to GHMT reprogramming and provide a starting point for optimization of CM subtype-specific reprogramming protocols.

A prospective cohort study was designed to evaluate the long-term outcome and health-related quality of life (HRQoL) in patients with posterior canal benign paroxysmal positional vertigo (PC-BPPV) treated by the particle repositioning maneuver (PRM) in the outpatient clinic of a general community hospital. Fifty individuals with PC-BPPV were included, and 45 (90%) completed the study. The diagnosis was based on the history of short episodes of vertigo and a positional nystagmus during the Dix-Hallpike test (DHT). All patients were treated by a single PRM, and relapses were evaluated by DHT at 30, 180 and 360 days post-treatment; a new PRM was performed if the DHT was positive. The main outcome measures were: percentage of patients with a negative DHT after treatment, scores obtained on the Medical Outcomes Study 36-Item Short Form Health Survey (SF-36) and the Dizziness Handicap Inventory Short Form (DHI-S) before and 30, 180 and 360 days post-treatment. The DHT was found negative in 80% (40/50) of individuals at 30 days. Ten, seven and five patients presented a positive DHT at 30, 180 and 360 days, respectively. Persistent BPPV was observed in 5% (2/50) of patients at 360 days, despite repeated PRM. Relapses (DH+ after successful PRM) were observed in 7.5% (3/50) at 180 days and 360 days. Both questionnaires showed a reliability Cronbach's alpha >0.7. The average standardized score for each SF-36 scale was compared with the reference population normative data, showing differences with norms for all scales except for vitality. After PRM, patients improved their scores with both instruments, indicating a restoration of HRQoL at 30 days. Physical dimension scores of the SF-36 improved from day 30 to 360. DHI-S scores were statistically better after PRM (P < 0.001). Our results show that the effectiveness of PRM is 88% after 1 year of follow-up. Patients with BPPV experienced a decrease in HRQoL, which was restored after PRM. Although relapses were observed in 7.5% of individuals, they did not affect HRQoL.

Twenty-nine individuals (41.43%) presented an affected unilateral posterior canal. Fifteen patients (21.43%) presented a pure horizontal direction-changing positional nystagmus consistent with a diagnosis of horizontal canal BPPV. Twelve individuals (17.14%) presented a unilateral down-beating nystagmus, suggesting possible anterior canal BPPV. In addition, 14 patients (20%) showed multiple positional nystagmus during the examination corresponding to simultaneous multi-canal BPPV, 5 had bilateral posterior canal BPPV and 2 presented a positional down-beating nystagmus in both left and right Dix-Hallpike manoeuvres and the head-hanging manoeuvre, which is highly suggestive of anterior canal BPPV. However, seven individuals showed positional horizontal and vertical side-changing nystagmus that could not be explained by single-canal BPPV. These patients with multiple positional nystagmus showed changing patterns of positional nystagmus at follow-up.

Posterior canal benign paroxysmal positional vertigo has a significant impact on health-related quality of life, and patients experienced a decrease in quality of life as compared with norms. The particle repositioning maneuver improves health-related quality of life in posterior canal benign paroxysmal positional vertigo.

Direct reprogramming of fibroblasts into cardiomyocytes is a promising approach for cardiac regeneration but still faces challenges in efficiently generating mature cardiomyocytes. Systematic optimization of reprogramming protocols requires scalable, objective methods to assess cellular phenotype beyond what is captured by transcriptional signatures alone. To address this question, we automatically segmented reprogrammed cardiomyocytes from immunofluorescence images and analyzed cell morphology. We also introduce a method to quantify sarcomere structure using Haralick texture features, called SarcOmere Texture Analysis (SOTA). We show that induced cardiac-like myocytes (iCLMs) are highly variable in expression of cardiomyocyte markers, producing subtypes that are not typically seen in vivo. Compared to neonatal mouse cardiomyocytes, iCLMs have more variable cell size and shape, have less organized sarcomere structure, and demonstrate reduced sarcomere length. Taken together, these results indicate that traditional methods of assessing cardiomyocyte reprogramming by quantifying induction of cardiomyocyte marker proteins may not be sufficient to predict functionality. The automated image analysis methods described in this study may enable more systematic approaches for improving reprogramming techniques above and beyond existing algorithms that rely heavily on transcriptome profiling.