Cardiac t-tubules are critical for efficient excitation-contraction coupling but become significantly remodeled during various stress conditions. However, the mechanisms by which t-tubule remodeling occur are poorly understood. Recently, we demonstrated that recovery of mouse ventricular myocytes after hyposmotic shock is associated with t-tubule sealing. In this study, we found that the application of Small Membrane Permeable Molecules (SMPM) such as DMSO, formamide and acetamide upon washout of hyposmotic solution significantly reduced the amount of extracellular dextran trapped within sealed t-tubules. The SMPM protection displayed sharp biphasic concentration dependence that peaks at ∼140 mM leading to >3- to 4-fold reduction in dextran trapping. Consistent with these data, detailed analysis of the effects of DMSO showed that the magnitude of normalized inward rectifier tail current (IK1,tail), an electrophysiological marker of t-tubular integrity, was increased ∼2-fold when hyposmotic stress was removed in the presence of 1% DMSO (∼140 mM). Analysis of dynamics of cardiomyocytes shrinking during resolution of hyposmotic stress revealed only minor increase in shrinking rate in the presence of 1% DMSO, and cell dimensions returned fully to prestress values in both control and DMSO groups. Application and withdrawal of 10% DMSO in the absence of preceding hyposmotic shock induced classical t-tubule sealing. This suggests that the biphasic concentration dependence originated from an increase in secondary t-tubule sealing when high SMPM concentrations are removed. Overall, the data suggest that SMPM protect against sealing of t-tubules following hyposmotic stress, likely through membrane modification and essentially independent of their osmotic effects.

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Cardiac t-tubules undergo significant remodeling in various pathological and experimental conditions, which can be associated with mechanical or osmotic stress. In particular, it has been shown that removal of hyposmotic stress can lead to sealing of t-tubules. However, the mechanisms underlying the sealing process remain essentially unknown. In this study we use dextran trapping assay to demonstrate that in adult mouse cardiomyocytes t-tubular sealing can also be induced by hyperosmotic challenge, and that both hypo- and hyperosmotic sealing display a clear threshold behavior requiring ≈100 mOsm minimal stress. Importantly, during both hypo- and hyperosmotic challenges the sealing of t-tubules occurs only during the shrinking phase. Analysis of the time course of t-tubular remodeling following removal of hyposmotic stress shows that t-tubules become sealed essentially instantly, well before any significant reduction in cell size can be observed. Overall, the data support the hypothesis that the critical event in the process of t-tubular sealing during osmotic challenges is detachment (peeling) of the membrane from the underlying cytoskeleton due to suprathreshold stress.

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Background Although universal Hepatitis C Virus (HCV) Screening in the US was recommended in 2020, the optimal implementation method is unknown. We characterized the efficacy of an automated letter HCV screening program at the Veteran’s Affairs (VA) Greater Los Angeles Healthcare System (VAGLAHS) and evaluated associations with linkage to care. Methods From January 2017 to May 2020, 14,804 Veterans born between 1945-1965 who did not have an HCV antibody (Ab) test result within the last 10 years and who were within the VAGLAHS catchment area were identified. Veterans were mailed a letter recommending HCV screening via a centralized process. Veterans then used the letter to present to a VA laboratory for HCV Ab testing, which included reflex HCV viral load. Those who were HCV viremic were referred to Hepatology/Infectious Diseases clinics for initiation of HCV treatment. Baseline characteristics of those with subsequent HCV viremia were collected. To determine associations with the first HCV visit (linkage to care), we performed independent chi-squared tests. Flowchart of Automated Letter HCV Screening for Veterans Results A total of 12,875 Veterans were identified, 4,011 (31%) Veterans presented for HCV Ab testing, 167/4011 were HCV Ab + (4.2%), and 69/167 (41.3%) had HCV viremia. Of those viremic, 94 % were male, 26% were African-American, 62% had stable housing, 24% lived >90 miles from the nearest VA clinic, and 17% had cirrhosis. Fifty-five Veterans (80%) were evaluated in a viral hepatitis clinic and 84 % (46/55) initiated HCV treatment (Figure 1). Patients’ housing status (p = 0.02), cirrhosis (p< 0.0001), and distance to clinic (p=0.063) were associated with non-linkage to an initial HCV appointment. Conclusion One third of Veterans approached via mail participated in HCV Ab testing. Overall HCV Ab positivity rates were 4% and nearly half had HCV viremia. The majority of Veterans were linked to care but housing status, cirrhosis, and distance to clinic were associated with non-linkage to care. Automated letter screening is a promising approach to HCV screening, including universal screening. Future research should include investigations of telehealth and e-consults for linkage to care, especially for those who have marginalized housing status and live far from clinic. Disclosures Debika Bhattacharya, MD, MSc, Gilead: Grant/Research Support|Regeneron: Grant/Research Support.