Where Will I Go? Relative Location of Physical Medicine & Rehabilitation Residencies Compared to Medical Schools Among Recent Residents.

ABSTRACT: Physical Medicine and Rehabilitation (PM&R) has rapidly been garnering interest as healthcare increases the emphasis on rehabilitation and management for acute and chronic diseases. This study analyzes recent geographical trends of PM&R residents via PM&R residents from 2019 to 2023 which were identified from publicly available data. The relative distribution from medical school to residency, medical school to preliminary program, and preliminary program to residency were analyzed. These locations were categorized as within 100 miles, same state, same region, or different region. Odds ratio (OR) were calculated for the aforementioned relative locations with respect to the presence of a home residency program. A total of 1836 residents were included. The majority of residents (51%) stayed within the same region as their medical school. Residents from medical schools with a home program were more likely to stay within 100 miles (OR: 3.64), the same state (OR: 3.19), and same region (OR: 2.56). Overall, PM&R residents are likely to stay within the same region as their medical school and preliminary year. Additionally, the presence of a home program significantly increases the odds of matching within 100 miles, same state, and same region.

The effectiveness of vaccination on the COVID-19 epidemic in California.

BACKGROUND: The COVID-19 pandemic has caused overwhelming morbidity, mortality, and hospitalization worldwide, including in the state of California. Vaccination efforts have been an important measure in curtailing the adverse outcomes of COVID-19. METHODS: To quantify the effectiveness of COVID-19 vaccinations in California, we conducted a retrospective cohort study investigating how vaccination has impacted the extent of COVID-19 contraction, hospitalizations, and death totals. We compared outcomes of the Delta Wave, Omicron Wave, and Pre-Delta Period. RESULTS: Vaccinated individuals have far-lower incidence risk ratio (IRR) of and odds of contracting a COVID-19 case (Delta IRR: 0.197) being hospitalized from COVID-19 (Delta IRR: 0.105), and dying from COVID-19 compared with an unvaccinated individual (Delta IRR: 0.0941). The preventive fraction of the unexposed and population-preventive fractions for cases, deaths, and hospitalizations also showed significant proportions. All tests showed P < .001. DISCUSSION: Vaccination was most effective in the Delta Wave, then in the Omicron Wave, and least effective in the Pre-Delta Period. Deaths were the most prevented outcome, followed by hospitalizations, then cases. CONCLUSIONS: This study exposes the massive impact of vaccinations in California in reducing COVID-19 outcomes and the potential for fewer adverse outcomes had there been greater vaccination compliance, demonstrating the need to increase vaccination efforts.

COVID-19 Vaccine Acceptance in Pregnant Women in the United States: A Systematic Review and Meta-Analysis.

Purpose: Pregnant women are vulnerable to Coronavirus Disease 2019 (COVID-19) complications, yet may hesitate to get vaccinated. It is important to identify racial/ethnic and other individual characteristics associated with COVID-19 vaccine acceptance in the United States during pregnancy. Methods: We searched PubMed, Embase, and Web of Science for articles published through January 2023 for keywords/terms related to immunization, COVID-19, and pregnancy, and performed a systematic review and meta-analysis to examine characteristics associated with vaccine acceptance. Results: Of 1,592 articles, 23 met inclusion criteria (focused on pregnant women in the United States, and their willingness or hesitation to vaccinate). Twenty-two of the studies examined receipt of ≥1 COVID-19 vaccine dose and/or intention to vaccinate, while one examined vaccine hesitancy. Vaccine acceptance rates ranged from 7% to 78.3%. Meta-analyses demonstrated that compared with Whites, Hispanics (odds ratios [OR] 0.72; 95% confidence interval [CI] 0.58-0.91) and Blacks (OR 0.44; 95% CI 0.30-0.63) had less COVID-19 vaccine acceptance, while Asians (OR 1.78; 95% CI 1.10-2.88) had greater vaccine acceptance. College graduation or more (OR 3.25; 95% CI 2.53-4.17), receipt or intention to receive the influenza vaccine (OR 3.46; 95% CI 2.22-5.41), and at least part-time employment (OR 2.12; 95% CI 1.66-2.72) were significantly associated with vaccine acceptance. Conclusions: COVID-19 vaccine nonacceptance in pregnant women is associated with Hispanic ethnicity and Black race, while acceptance is associated with Asian race, college education or more, at least part-time employment, and acceptance of the influenza vaccine. Future COVID-19 vaccination campaigns can target identified subgroups of pregnant women who are less likely to accept vaccination.

Impact of California Statute on Naloxone Availability and Opioid Overdose Rates.

Introduction: Ensuring that people at risk of overdosing on opioids have easy access to naloxone is an essential part of the fight against the opioid crisis. This study evaluates the impact of the 2016 California law (CA AB1535) permitting pharmacies to dispense this life-saving medication without a physician's prescription. Methods: California counties were categorized on the basis of population density (rural, suburban, urban), rate of opioid-related deaths by population density (high, medium, low), and rate of opioid prescriptions by population density (high, medium, low). Ten diverse pharmacies from each category were selected for inclusion. In a brief 1-minute interview conducted between July and August 2021, pharmacists from 146 California pharmacies were surveyed regarding their knowledge of CA AB1535, their practice of dispensing naloxone without a physician's prescription, and whether they normally stock naloxone. Chi-square tests were used to compare responses. Results: Although almost all pharmacies interviewed (94%) were aware of the law and most of them (64%) dispensed naloxone without a physician's prescription, few statistically significant differences were found between surveyed categories. There were no significant relationships between naloxone availability at pharmacies and overdose death rates. Conclusions: Our results suggest that the number of California pharmacies dispensing naloxone without a physician's prescription has continued to increase since the implementation of CA AB1535. However, despite increased access to naloxone at pharmacies, opioid overdose rates have continued to rise since 2016, indicating the need for a multifaceted harm reduction approach.

Bacterial toxins and heart function: heat-labile Escherichia coli enterotoxin B promotes changes in cardiac function with possible relevance for sudden cardiac death.

Bacterial toxins can cause cardiomyopathy, though it is not its most common cause. Some bacterial toxins can form pores in the membrane of cardiomyocytes, while others can bind to membrane receptors. Enterotoxigenic E. coli can secrete enterotoxins, including heat-resistant (ST) or labile (LT) enterotoxins. LT is an AB5-type toxin that can bind to specific cell receptors and disrupt essential host functions, causing several common conditions, such as certain diarrhea. The pentameric B subunit of LT, without A subunit (LTB), binds specifically to certain plasma membrane ganglioside receptors, found in lipid rafts of cardiomyocytes. Isolated guinea pig hearts and cardiomyocytes were exposed to different concentrations of purified LTB. In isolated hearts, mechanical and electrical alternans and an increment of heart rate variability, with an IC50 of ~0.2 µg/ml LTB, were observed. In isolated cardiomyocytes, LTB promoted significant decreases in the amplitude and the duration of action potentials. Na+ currents were inhibited whereas L-type Ca2+ currents were augmented at their peak and their fast inactivation was promoted. Delayed rectifier K+ currents decreased. Measurements of basal Ca2+ or Ca2+ release events in cells exposed to LTB suggest that LTB impairs Ca2+ homeostasis. Impaired calcium homeostasis is linked to sudden cardiac death. The results are consistent with the recent view that the B subunit is not merely a carrier of the A subunit, having a role explaining sudden cardiac death in children (SIDS) infected with enterotoxigenic E. coli, explaining several epidemiological findings that establish a strong relationship between SIDS and ETEC E. coli. Supplementary Information: The online version contains supplementary material available at 10.1007/s12551-023-01100-6.

Improved Workflow for Analysis of Vascular Myocyte Time-Series and Line-Scan Ca2+ Imaging Datasets.

Intracellular Ca2+ signals are key for the regulation of cellular processes ranging from myocyte contraction, hormonal secretion, neural transmission, cellular metabolism, transcriptional regulation, and cell proliferation. Measurement of cellular Ca2+ is routinely performed using fluorescence microscopy with biological indicators. Analysis of deterministic signals is reasonably straightforward as relevant data can be discriminated based on the timing of cellular responses. However, analysis of stochastic, slower oscillatory events, as well as rapid subcellular Ca2+ responses, takes considerable time and effort which often includes visual analysis by trained investigators, especially when studying signals arising from cells embedded in complex tissues. The purpose of the current study was to determine if full-frame time-series and line-scan image analysis workflow of Fluo-4 generated Ca2+ fluorescence data from vascular myocytes could be automated without introducing errors. This evaluation was addressed by re-analyzing a published "gold standard" full-frame time-series dataset through visual analysis of Ca2+ signals from recordings made in pulmonary arterial myocytes of en face arterial preparations. We applied a combination of data driven and statistical approaches with comparisons to our published data to assess the fidelity of the various approaches. Regions of interest with Ca2+ oscillations were detected automatically post hoc using the LCPro plug-in for ImageJ. Oscillatory signals were separated based on event durations between 4 and 40 s. These data were filtered based on cutoffs obtained from multiple methods and compared to the published manually curated "gold standard" dataset. Subcellular focal and rapid Ca2+ "spark" events from line-scan recordings were examined using SparkLab 5.8, which is a custom automated detection and analysis program. After filtering, the number of true positives, false positives, and false negatives were calculated through comparisons to visually derived "gold standard" datasets. Positive predictive value, sensitivity, and false discovery rates were calculated. There were very few significant differences between the automated and manually curated results with respect to quality of the oscillatory and Ca2+ spark events, and there were no systematic biases in the data curation or filtering techniques. The lack of statistical difference in event quality between manual data curation and statistically derived critical cutoff techniques leads us to believe that automated analysis techniques can be reliably used to analyze spatial and temporal aspects to Ca2+ imaging data, which will improve experiment workflow.

Adenylyl cyclase isoform 1 contributes to sinoatrial node automaticity via functional microdomains.

Sinoatrial node (SAN) cells are the heart's primary pacemaker. Their activity is tightly regulated by ß-adrenergic receptor (ß-AR) signaling. Adenylyl cyclase (AC) is a key enzyme in the ß-AR pathway that catalyzes the production of cAMP. There are current gaps in our knowledge regarding the dominant AC isoforms and the specific roles of Ca2+-activated ACs in the SAN. The current study tests the hypothesis that distinct AC isoforms are preferentially expressed in the SAN and compartmentalize within microdomains to orchestrate heart rate regulation during ß-AR signaling. In contrast to atrial and ventricular myocytes, SAN cells express a diverse repertoire of ACs, with ACI as the predominant Ca2+-activated isoform. Although ACI-KO (ACI-/-) mice exhibit normal cardiac systolic or diastolic function, they experience SAN dysfunction. Similarly, SAN-specific CRISPR/Cas9-mediated gene silencing of ACI results in sinus node dysfunction. Mechanistically, hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) channels form functional microdomains almost exclusively with ACI, while ryanodine receptor and L-type Ca2+ channels likely compartmentalize with ACI and other AC isoforms. In contrast, there were no significant differences in T-type Ca2+ and Na+ currents at baseline or after ß-AR stimulation between WT and ACI-/- SAN cells. Due to its central characteristic feature as a Ca2+-activated isoform, ACI plays a unique role in sustaining the rise of local cAMP and heart rates during ß-AR stimulation. The findings provide insights into the critical roles of the Ca2+-activated isoform of AC in sustaining SAN automaticity that is distinct from contractile cardiomyocytes.

Use of Team-Based Learning Pedagogy to Prepare for a Pharmacy School Accreditation Self-Study.

Ensuring adequate engagement and preparation of all stakeholders in an accreditation self-study can be challenging for many reasons, including lack of motivation and inadequate understanding of expectations and procedures. The goal of this exploratory study was to determine whether using team-based learning (TBL) pedagogy to deliver an accreditation preparation workshop could effectively prepare and engage participants. A Likert-scale questionnaire was administered to workshop attendees (n = 52) to determine whether they found TBL-based training helpful and whether it promoted engagement. Twenty-four attendees completed the survey (46%). More than 80% of participants strongly agreed or agreed with 12 statements relating to perceptions of self and participant engagement within team activities and the usefulness of team activities. More than 65% of participants strongly agreed or agreed with statements relating to the helpfulness of the TBL approach in preparing for the self-study (five questions). Subgroup analysis showed no significant difference in responses based on whether on not participants had previously been involved in an accreditation self study. Our data indicate that a TBL approach can be an effective way to engage and prepare stakeholders for an accreditation self-study, and that TBL pedagogy has utility outside of the classroom setting.

Retrospective cross sectional analysis of demographic disparities in outcomes of CPR performed by EMS providers in the United States.

OBJECTIVE: To investigate demographic disparities in prehospital cardiopulmonary resuscitation (CPR) initiation and successful outcomes of patients with out-of-hospital cardiac arrest (OHCA) treated by emergency medical services (EMS) providers. METHODS: We analyzed the National Emergency Medical Service Information Systems (NEMSIS) 2017 database, analyzing patient gender, age and race against CPR initiation and Return of Spontaneous Circulation (ROSC). The analysis was performed for a subset of patients who received bystander interventions (n = 3,362), then repeated for the whole cohort of patients (n = 5,833). RESULTS: Within the subgroup of patients that received CPR or AED application prior to the arrival of the paramedics, a logistic regression for CPR initiation rates as a function of race, gender and age reported the following adjusted odds ratios: African American (AA) to White 0.570 (95%CI [0.419, 0.775]), Hispanic to White 0.735 (95%CI [0.470, 1.150]); female to male 0.768 (95%CI [0.598, 0.986]); senior to adult 0.708 (95%CI [0.545, 0.920). Similarly, a logistic regression of ROSC as a function of race, gender and age reported the following adjusted odds ratios: AA to White 0.652 (95%CI [0.533, 0.797]) Hispanic to White 1.018 (95%CI [0.783, 1.323]); female to male 0.887 (95%CI [0.767, 1.025]); senior to adult 0.817 (95%CI [0.709, 0.940]). Similar trends existed in the entire cohort of patients. CONCLUSIONS: These results suggest that there are discrepancies in patient care during cardiopulmonary arrest performed by EMS for OHCA, inviting further exploration of healthcare differences in the prehospital EMS approach to OHCA.

Long-Term Hypoxia Negatively Influences Ca2+ Signaling in Basilar Arterial Myocytes of Fetal and Adult Sheep.

Cerebral arterial vasoreactivity is vital to the regulation of cerebral blood flow. Depolarization of arterial myocytes elicits whole-cell Ca2+ oscillations as well as subcellular Ca2+ sparks due to activation of ryanodine receptors on the sarcoplasmic reticulum. Previous evidence illustrates that contraction of cerebral arteries from sheep and underlying Ca2+ signaling pathways are modified by age and that long-term hypoxia (LTH) causes aberrations in Ca2+ signaling pathways and downstream effectors impacting vasoregulation. We hypothesize that age and LTH affect the influence of membrane depolarization on whole-cell intracellular Ca2+ oscillations and sub-cellular Ca2+ spark activity in cerebral arteries. To test this hypothesis, we examined Ca2+ oscillatory and spark activities using confocal fluorescence imaging techniques of Fluo-4 loaded basilar arterial myocytes of low- and high-altitude term fetal (∼145 days of gestation) and adult sheep, where high-altitude pregnant and non-pregnant sheep were placed at 3,801 m for >100 days. Ca2+ oscillations and sparks were recorded using an in situ preparation evaluated in the absence or presence of 30 mM K+ (30K) to depolarize myocytes. Myocytes from adult animals tended to have a lower basal rate of whole-cell Ca2+ oscillatory activity and 30K increased the activity within cells. LTH decreased the ability of myocytes to respond to depolarization independent of age. These observations illustrate that both altitude and age play a role in affecting whole-cell and localized Ca2+ signaling, which are important to arterial vasoreactivity and cerebral blood flow.

GRK5 Controls SAP97-Dependent Cardiotoxic ß1 Adrenergic Receptor-CaMKII Signaling in Heart Failure.

RATIONALE: Cardiotoxic ß1 adrenergic receptor (ß1AR)-CaMKII (calmodulin-dependent kinase II) signaling is a major and critical feature associated with development of heart failure. SAP97 (synapse-associated protein 97) is a multifunctional scaffold protein that binds directly to the C-terminus of ß1AR and organizes a receptor signalosome. OBJECTIVE: We aim to elucidate the dynamics of ß1AR-SAP97 signalosome and its potential role in chronic cardiotoxic ß1AR-CaMKII signaling that contributes to development of heart failure. METHODS AND RESULTS: The integrity of cardiac ß1AR-SAP97 complex was examined in heart failure. Cardiac-specific deletion of SAP97 was developed to examine ß1AR signaling in aging mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the ß1AR-SAP97 signaling complex is reduced in heart failure. Cardiac-specific deletion of SAP97 yields an aging-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA (protein kinase A)-dependent association of ß1AR with arrestin2 and CaMKII and turns on an Epac (exchange protein directly activated by cAMP)-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 (G-protein receptor kinase-5) is necessary to promote agonist-induced dissociation of SAP97 from ß1AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of ß1AR-SAP97 complex and increases in CaMKII activity in hearts. CONCLUSIONS: These data reveal a critical role of SAP97 in maintaining the integrity of cardiac ß1AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy. Graphical Abstract: A graphical abstract is available for this article.

Caveolae Link CaV3.2 Channels to BKCa-Mediated Feedback in Vascular Smooth Muscle.

Objective- This study examined whether caveolae position CaV3.2 (T-type Ca2+ channel encoded by the α-3.2 subunit) sufficiently close to RyR (ryanodine receptors) for extracellular Ca2+ influx to trigger Ca2+ sparks and large-conductance Ca2+-activated K+ channel feedback. Approach and Results- Using smooth muscle cells from mouse mesenteric arteries, the proximity ligation assay confirmed that CaV3.2 reside within 40 nm of caveolin 1, a key caveolae protein. Methyl-ß-cyclodextrin, a cholesterol depleting agent that disrupts caveolae, suppressed CaV3.2 activity along with large-conductance Ca2+-activated K+-mediated spontaneous transient outward currents in cells from C57BL/6 but not CaV3.2-/- mice. Genetic deletion of caveolin 1, a perturbation that prevents caveolae formation, also impaired spontaneous transient outward current production but did so without impairing Ca2+ channel activity, including CaV3.2. These observations indicate a mistargeting of CaV3.2 in caveolin 1-/- mice, a view supported by a loss of Ni2+-sensitive Ca2+ spark generation and colocalization signal (CaV3.2-RyR) from the proximity ligation assay. Vasomotor and membrane potential measurements confirmed that cellular disruption of the CaV3.2-RyR axis functionally impaired the ability of large-conductance Ca2+-activated K+ to set tone in pressurized caveolin 1-/- arteries. Conclusions- Caveolae play a critical role in protein targeting and preserving the close structural relationship between CaV3.2 and RyR needed to drive negative feedback control in resistance arteries.

An economical strategy for early medical education in ultrasound.

BACKGROUND: A movement to include ultrasound training in undergraduate medical education is slowly taking place. However, many educational institutions are hesitant to include formal ultrasound training as a part of their curricula due to curricular time constraints, high cost of ultrasound equipment, and a lack of sufficient faculty skilled with ultrasound. We suggest that an economical ultrasound training strategy is needed to resolve these obstacles and enable hesitant medical programs to include ultrasound training. METHODS: Twenty-eight first year medical students volunteered to attend extra-curricular ultrasound training sessions covering topics related to 11 commonly used sonographical imaging categories. Study assessments included subjective pre/post-training skill evaluation surveys, and objective numerical scores awarded by the session instructor during real-time evaluation of each participant's performance in obtaining each target ultrasound view. RESULTS: A Wilcoxon matched-pairs signed rank test was performed to evaluate the difference between pre-training and post-training survey questions. P values < 0.05 were considered significant. Moreover, following analysis the p value for all test was found to be < 0.0001. Of the 308 total ultrasound-related tasks attempted collectively by all 28 participants, only 7 (2.3%) tasks were deemed unsuccessful by an instructor. CONCLUSIONS: The training program presented in this study requires one faculty member, a single ultrasound machine, and time to conduct six 30-min training sessions with small groups of students over 4 weeks. Many medical schools are concerned that they don't have adequate time or resources to include ultrasound training in their curricula. Our intention is to negate these concerns by providing a simple and practical training method that is both temporally and fiscally economical.

Long-term high-altitude hypoxia influences pulmonary arterial L-type calcium channel-mediated Ca2+ signals and contraction in fetal and adult sheep.

Long-term hypoxia (LTH) has a profound effect on pulmonary arterial vasoconstriction in the fetus and adult. Dysregulation in Ca2+ signaling is important during the development of LTH-induced pulmonary hypertension. In the present study, we tested the hypothesis that L-type Ca2+ channels (CaL), which are voltage dependent and found in smooth, skeletal, and cardiac muscle, are important in the adaptation of pulmonary arterial contractions in postnatal maturation and in response to LTH. Pulmonary arteries were isolated from fetal or adult sheep maintained at low or high altitude (3,801 m) for >100 days. The effects were measured using an L-type Ca2+ channel opener FPL 64176 (FPL) in the presence or absence of an inhibitor, Nifedipine (NIF) on arterial contractions, intracellular Ca2+ oscillations, and ryanodine receptor-driven Ca2+ sparks. FPL induced pulmonary arterial contractions in all groups were sensitive to NIF. However, when compared with 125 mM K+, FPL contractions were greater in fetuses than in adults. FPL reduced Ca2+ oscillations in myocytes of adult but not fetal arteries, independently of altitude. The FPL effects on Ca2+ oscillations were reversed by NIF in myocytes of hypoxic but not normoxic adults. FPL failed to enhance Ca2+ spark frequency and had little impact on spatiotemporal firing characteristics. These data suggest that CaL-dependent contractions are largely uncoupled from intracellular Ca2+ oscillations and the development of Ca2+ sparks. This raises questions regarding the coupling of pulmonary arterial contractility to membrane depolarization, attendant CaL facilitation, and the related associations with the activation of Ca2+ oscillations and Ca2+ sparks.

MarkoLAB: A simulator to study ionic channel's stochastic behavior.

Mathematical models of the cardiac cell have started to include markovian representations of the ionic channels instead of the traditional Hodgkin & Huxley formulations. There are many reasons for this: Markov models are not restricted to the idea of independent gates defining the channel, they allow more complex description with specific transitions between open, closed or inactivated states, and more importantly those states can be closely related to the underlying channel structure and conformational changes. METHODS: We used the LabVIEW® and MATLAB® programs to implement the simulator MarkoLAB that allow a dynamical 3D representation of the markovian model of the channel. The Monte Carlo simulation was used to implement the stochastic transitions among states. The user can specify the voltage protocol by setting the holding potential, the step-to voltage and the duration of the stimuli. RESULTS: The most studied feature of a channel is the current flowing through it. This happens when the channel stays in the open state, but most of the time, as revealed by the low open probability values, the channel remains on the inactive or closed states. By focusing only when the channel enters or leaves the open state we are missing most of its activity. MarkoLAB proved to be quite useful to visualize the whole behavior of the channel and not only when the channel produces a current. Such dynamic representation provides more complete information about channel kinetics and will be a powerful tool to demonstrate the effect of gene mutations or drugs on the channel function. CONCLUSIONS: MarkoLAB provides an original way of visualizing the stochastic behavior of a channel. It clarifies concepts, such as recovery from inactivation, calcium- versus voltage-dependent inactivation, and tail currents. It is not restricted to ionic channels only but it can be extended to other transporters, such as exchangers and pumps. This program is intended as a didactical tool to illustrate the dynamical behavior of a channel. It has been implemented in two platforms MATLAB® and LabVIEW® to enhance the target users of this new didactical tool. The computational cost of implementing a stochastic simulation is within the range of a personal computer performance; making MarkoLAB suitable to be run during a lecture or presentation.

Interplay among distinct Ca2+ conductances drives Ca2+ sparks/spontaneous transient outward currents in rat cerebral arteries.

KEY POINTS: Distinct Ca2+ channels work in a coordinated manner to grade Ca2+ spark/spontaneous transient outward currents (STOCs) in rat cerebral arteries. The relative contribution of each Ca2+ channel to Ca2+ spark/STOC production depends upon their biophysical properties and the resting membrane potential of smooth muscle. Na+ /Ca2+ exchanger, but not TRP channels, can also facilitate STOC production. ABSTRACT: Ca2+ sparks are generated in a voltage-dependent manner to initiate spontaneous transient outward currents (STOCs), events that moderate arterial constriction. In this study, we defined the mechanisms by which membrane depolarization increases Ca2+ sparks and subsequent STOC production. Using perforated patch clamp electrophysiology and rat cerebral arterial myocytes, we monitored STOCs in the presence and absence of agents that modulate Ca2+ entry. Beginning with CaV 3.2 channel inhibition, Ni2+ was shown to decrease STOC frequency in cells held at hyperpolarized (-40 mV) but not depolarized (-20 mV) voltages. In contrast, nifedipine, a CaV 1.2 inhibitor, markedly suppressed STOC frequency at -20 mV but not -40 mV. These findings aligned with the voltage-dependent profiles of L- and T-type Ca2+ channels. Furthermore, computational and experimental observations illustrated that Ca2+ spark production is intimately tied to the activity of both conductances. Intriguingly, this study observed residual STOC production at depolarized voltages that was independent of CaV 1.2 and CaV 3.2. This residual component was insensitive to TRPV4 channel modulation and was abolished by Na+ /Ca2+ exchanger blockade. In summary, our work highlights that the voltage-dependent triggering of Ca2+ sparks/STOCs is not tied to a single conductance but rather reflects an interplay among multiple Ca2+ permeable pores with distinct electrophysiological properties. This integrated orchestration enables smooth muscle to grade Ca2+ spark/STOC production and thus precisely tune negative electrical feedback.

CaMKII-dependent phosphorylation of RyR2 promotes targetable pathological RyR2 conformational shift.

Diastolic calcium (Ca) leak via cardiac ryanodine receptors (RyR2) can cause arrhythmias and heart failure (HF). Ca/calmodulin (CaM)-dependent kinase II (CaMKII) is upregulated and more active in HF, promoting RyR2-mediated Ca leak by RyR2-Ser2814 phosphorylation. Here, we tested a mechanistic hypothesis that RyR2 phosphorylation by CaMKII increases Ca leak by promoting a pathological RyR2 conformation with reduced CaM affinity. Acute CaMKII activation in wild-type RyR2, and phosphomimetic RyR2-S2814D (vs. non-phosphorylatable RyR2-S2814A) knock-in mouse myocytes increased SR Ca leak, reduced CaM-RyR2 affinity, and caused a pathological shift in RyR2 conformation (detected via increased access of the RyR2 structural peptide DPc10). This same trio of effects was seen in myocytes from rabbits with pressure/volume-overload induced HF. Excess CaM quieted leak and restored control conformation, consistent with negative allosteric coupling between CaM affinity and DPc10 accessible conformation. Dantrolene (DAN) also restored CaM affinity, reduced DPc10 access, and suppressed RyR2-mediated Ca leak and ventricular tachycardia in RyR2-S2814D mice. We propose that a common pathological RyR2 conformational state (low CaM affinity, high DPc10 access, and elevated leak) may be caused by CaMKII-dependent phosphorylation, oxidation, and HF. Moreover, DAN (or excess CaM) can shift this pathological gating state back to the normal physiological conformation, a potentially important therapeutic approach.

Genetic ablation of CaV3.2 channels enhances the arterial myogenic response by modulating the RyR-BKCa axis.

OBJECTIVE: In resistance arteries, there is an emerging view that smooth muscle CaV3.2 channels restrain arterial constriction through a feedback response involving the large-conductance Ca(2+)-activated K(+) channel (BKCa). Here, we used wild-type and CaV3.2 knockout (CaV3.2(-/-)) mice to definitively test whether CaV3.2 moderates myogenic tone in mesenteric arteries via the CaV3.2-ryanodine receptor-BKCa axis and whether this regulatory mechanism influences blood pressure regulation. APPROACH AND RESULTS: Using pressurized vessel myography, CaV3.2(-/-) mesenteric arteries displayed enhanced myogenic constriction to pressure but similar K(+)-induced vasoconstriction compared with wild-type C57BL/6 arteries. Electrophysiological and myography experiments subsequently confirmed the inability of micromolar Ni(2+), a CaV3.2 blocker, to either constrict arteries or suppress T-type currents in CaV3.2(-/-) smooth muscle cells. The frequency of BKCa-induced spontaneous transient outward K(+) currents dropped in wild-type but not in knockout arterial smooth muscle cells upon the pharmacological suppression of CaV3.2 channel. Line scan analysis performed on en face arteries loaded with Fluo-4 revealed the presence of Ca(2+) sparks in all arteries, with the subsequent application of Ni(2+) only affecting wild-type arteries. Although CaV3.2 channel moderated myogenic constriction of resistance arteries, the blood pressure measurements of CaV3.2(-/-) and wild-type animals were similar. CONCLUSIONS: Overall, our findings establish a negative feedback mechanism of the myogenic response in which CaV3.2 channel modulates downstream ryanodine receptor-BKCa to hyperpolarize and relax arteries.

ß-adrenergic effects on cardiac myofilaments and contraction in an integrated rabbit ventricular myocyte model.

A five-state model of myofilament contraction was integrated into a well-established rabbit ventricular myocyte model of ion channels, Ca(2+) transporters and kinase signaling to analyze the relative contribution of different phosphorylation targets to the overall mechanical response driven by ß-adrenergic stimulation (ß-AS). ß-AS effect on sarcoplasmic reticulum Ca(2+) handling, Ca(2+), K(+) and Cl(-) currents, and Na(+)/K(+)-ATPase properties was included based on experimental data. The inotropic effect on the myofilaments was represented as reduced myofilament Ca(2+) sensitivity (XBCa) and titin stiffness, and increased cross-bridge (XB) cycling rate (XBcy). Assuming independent roles of XBCa and XBcy, the model reproduced experimental ß-AS responses on action potentials and Ca(2+) transient amplitude and kinetics. It also replicated the behavior of force-Ca(2+), release-restretch, length-step, stiffness-frequency and force-velocity relationships, and increased force and shortening in isometric and isotonic twitch contractions. The ß-AS effect was then switched off from individual targets to analyze their relative impact on contractility. Preventing ß-AS effects on L-type Ca(2+) channels or phospholamban limited Ca(2+) transients and contractile responses in parallel, while blocking phospholemman and K(+) channel (IKs) effects enhanced Ca(2+) and inotropy. Removal of ß-AS effects from XBCa enhanced contractile force while decreasing peak Ca(2+) (due to greater Ca(2+) buffering), but had less effect on shortening. Conversely, preventing ß-AS effects on XBcy preserved Ca(2+) transient effects, but blunted inotropy (both isometric force and especially shortening). Removal of titin effects had little impact on contraction. Finally, exclusion of ß-AS from XBCa and XBcy while preserving effects on other targets resulted in preserved peak isometric force response (with slower kinetics) but nearly abolished enhanced shortening. ß-AS effects on XBCa and XBcy have greater impact on isometric and isotonic contraction, respectively.

Mechanochemotransduction during cardiomyocyte contraction is mediated by localized nitric oxide signaling.

Cardiomyocytes contract against a mechanical load during each heartbeat, and excessive mechanical stress leads to heart diseases. Using a cell-in-gel system that imposes an afterload during cardiomyocyte contraction, we found that nitric oxide synthase (NOS) was involved in transducing mechanical load to alter Ca(2+) dynamics. In mouse ventricular myocytes, afterload increased the systolic Ca(2+) transient, which enhanced contractility to counter mechanical load but also caused spontaneous Ca(2+) sparks during diastole that could be arrhythmogenic. The increases in the Ca(2+) transient and sparks were attributable to increased ryanodine receptor (RyR) sensitivity because the amount of Ca2(+) in the sarcoplasmic reticulum load was unchanged. Either pharmacological inhibition or genetic deletion of nNOS (or NOS1), but not of eNOS (or NOS3), prevented afterload-induced Ca2(+) sparks. This differential effect may arise from localized NO signaling, arising from the proximity of nNOS to RyR, as determined by super-resolution imaging. Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) also contributed to afterload-induced Ca(2+) sparks. Cardiomyocytes from a mouse model of familial hypertrophic cardiomyopathy exhibited enhanced mechanotransduction and frequent arrhythmogenic Ca(2+) sparks. Inhibiting nNOS and CaMKII, but not NOX2, in cardiomyocytes from this model eliminated the Ca2(+) sparks, suggesting mechanotransduction activated nNOS and CaMKII independently from NOX2. Thus, our data identify nNOS, CaMKII, and NOX2 as key mediators in mechanochemotransduction during cardiac contraction, which provides new therapeutic targets for treating mechanical stress-induced Ca(2+) dysregulation, arrhythmias, and cardiomyopathy.