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Abstract Background Heart failure (HF) is classified by ejection fraction: reduced EF (<40%) is HFrEF and preserved EF (>50%) is HFpEF. Unlike HFrEF, no therapeutic agent improves mortality in HFpEF. The molecular mechanism that produces HFpEF is not completely understood, but the cascade of pathology that produces HFpEF is thought to begin with changes in vascular reactivity, including a decrease in NO mediated vasodilatation, which coupled with subsequent changes in contractility, energetics and coronary blood flow produce HFpEF. If abnormal vascular reactivity is the initial step in the pathological cascade that produces HFpEF, restoring and/or improving vascular reactivity could represent a novel treatment strategy. Vascular reactivity is primarily regulated by myosin light chain phosphatase, which has catalytic, myosin targeting (MYPT1) and 20kDa subunits. Alternative mRNA splicing of exon24 (E24) of the MYPT1 transcript produces MYPT1 isoforms that differ by the presence or absence of a COOH-terminal leucine zipper (LZ+/LZ-); E24 exclusion produces an NO responsive LZ+ MYPT1, while E24 inclusion produces an NO unresponsive LZ- MYPT. Methods We used the mouse two-hit model of HFpEF (high fat diet and L-NAME) and treated mice with an anti-sense octo-guanidine targeting the 5’ splice site of E24 (ASO-E24) to increase the expression of the NO responsive, LZ+ MYPT1 isoform in vascular smooth muscle. Invasive and noninvasive hemodynamics were used to determine LV function. Results Compared to mice with HFpEF, ASO-E24 treatment maintains LZ+ MYPT1 expression (4.7±0.7au v 1.0±0.4au v 2.0±0.4au, control v HFpEF v ASO-E24 Rx, p<0.05), improves diastolic function; LVEDP (10±1mmHg v 20±4mmHg v 14±3mmHg, p<0.05), dP/dtmin (−8000±300mmHg/s v 6000±500mmHg/s v 8500±700mmHg/s, p<0.05), both early (E; 0.60±0.05m/s v 0.42±0.06m/s v 0.64±0.06m/s, p<0.05) and late diastolic filling (A; 0.38±0.03m/s v 0.24±0.02m/s v 0.47±0.04m/s, p<0.050 and also prevents the increase in lung weight (167±5g v 175±7g v 166±5g, p<0.05). Further, mice treated with ASO-E24 maintained normal relaxation to 8Br-cGMP (65±5% v 44±9% v 72±9%, p=0.05). Conclusion These data demonstrate that maintaining normal LZ+ MYPT1 expression and vascular reactivity prevent the development of HFpEF. These results are consistent with the hypothesis that abnormal vascular reactivity is the initial and primary step in the pathological cascade that produces HFpEF and ASO-E24, which is designed to preserve normal LZ+ MYPT1 expression and vascular reactivity, could represent a novel and effective treatment strategy for HFpEF. Clinical Prospective What is New? In HFpEF, an anti-sense octo-guanidine targeting the 5’ splice site of exon 24 (ASO-E24) of the MYPT1 transcript increases the expression of the NO responsive, LZ+ MYPT1 isoform in vascular smooth muscle. Preserving LZ+ MYPT1 expression and normal vascular reactivity prevents the development of HFpEF. A decrease in LZ+ MYPT1 expression contributes to the development of HFpEF. What are the Clinical Implications? The results of the study demonstrate that abnormal vascular reactivity is an important step in the pathological cascade that produces HFpEF Increasing LZ+ MYPT1 expression could represent a novel treatment strategy for HFpEF.

AbstractAging is associated with cardiac contractile abnormalities, but the etiology of these contractile deficits is unclear. We hypothesized that cardiac contractile and regulatory protein expression is altered during aging. To investigate this possibility, left ventricular (LV) lysates were prepared from young (6 months) and old (24 months) Fischer344 rats. There are no age‐related changes in SERCA2 expression or phospholamban phosphorylation. Additionally, neither titin isoform expression nor phosphorylation differed. However, there is a significant increase in β‐isoform of the myosin heavy chain (MyHC) expression and phosphorylation of TnI and MyBP‐C during aging. In permeabilized strips of papillary muscle, force and Ca2+ sensitivity are reduced during aging, consistent with the increase in β‐MyHC expression and TnI phosphorylation. However, the increase in MyBP‐C phosphorylation during aging may represent a mechanism to compensate for age‐related contractile deficits. In isolated cardiomyocytes loaded with Fura‐2, the peak of the Ca2+ transient is reduced, but the kinetics of the Ca2+ transient are not altered. Furthermore, the extent of shortening and the rates of both sarcomere shortening and re‐lengthening are reduced. These results demonstrate that aging is associated with changes in contractile and regulatory protein expression and phosphorylation, which affect the mechanical properties of cardiac muscle.

Aging is a significant risk factor for cardiovascular disease and is known to be associated with an increase in vascular stiffness and decrease in sensitivity to NO mediated vasodilatation. However, the underlying mechanism for these changes in vascular function is unknown. Smooth muscle tone is known to be regulated by the expression and phosphorylation of both nonmuscle (NM) and smooth muscle (SM) myosin. NM and SM phosphorylation is regulated by the activity of myosin light chain kinase (MLCK) and MLC phosphatase. MLC phosphatase is a trimeric enzyme consisting of enzymatic, myosin targeting (MYPT1) and a 20kDa subunit of unknown function. Two isoforms of MYPT1 are produced by alternative mRNA splicing, which differ by the presence or absence of a COOH-terminal leucine zipper (LZ+/-), and the relative expression of LZ+/LZ- MYPT1 determines the sensitivity to NO mediated vasodilatation. We defined NM myosin expression using two-dimensional SDS PAGE and LZ+/LZ- MYPT1 expression using immunoblotting in aortic and tertiary mesenteric smooth muscle from 6mo and 24mo old Fischer344 rats. The data demonstrate that aging is associated with a significant decrease in the expression of both LZ+ MYPT1 and NM myosin. The reduction in LZ+ MYPT1 expression would both decrease the sensitivity to NO mediated smooth muscle dilatation and increase vascular tone, which is a hallmark of the vasculature in aging. On the other hand, the reduction in NM myosin would result in a decrease in vascular tone and could represent a compensatory mechanism to normalize blood pressure during aging. These data demonstrate that there are changes in the expression of smooth muscle regulatory and contractile proteins during aging and these changes in protein expression contribute to molecular mechanism for the decrease in sensitivity to NO mediated vasodilatation as well as the increase in vascular tone or the vascular dysfunction that occurs with aging. Supported by a Mayo CV Prospective Grant (FVB). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Aging is associated with an increased incidence of cardiac dysfunction. We hypothesized that aging associated changes in cardiac regulatory and contractile proteins produce contractile dysfunction. We assessed regulatory and contractile protein expression as well as contractility in left ventricular cardiac muscle from Fischer F-344 rats at 6-, 18-, and 24-month of age. Age-related changes in the expression of β-MHC as well as the expression and phosphorylation of troponin I (TnI) and cardiac myosin binding C protein (MyBP-C) were assessed by Western blot. Aging was associated with an increase in the expression of β-MHC and the phosphorylation of both TnI at Ser23/24 and MyBP-C. Mechanical properties were assessed in Triton-X permeabilized anterior papillary muscle from the left ventricle from both 6- and 24-month old rats. In papillary muscle of 24-month old rats, there was a decrease in maximum Ca2+ activated force, Ca2+ sensitivity of force and the rate constant for force recovery after quick release (ktr). Additionally, cardiomyocytes were isolated from the left ventricle at 6-, 18-, and 24-month of age using a modified Langendorff system. Cardiomyocytes were then loaded with fura-2 AM to measure cytosolic calcium ([Ca2+]cyt) responses to electrical field stimulation (0.5 Hz). [Ca2+]cyt and contractile (sarcomere length shortening) responses to stimulation were simultaneously measured using an IonOptix system. In older cardiomyocytes, the amplitude of evoked [Ca2+]cyt responses was reduced while there was no difference in the kinetics of the [Ca2+]cyt transient. The extent of sarcomere length shortening as well as the rates of both shortening and relaxation were reduced. These results demonstrate that aging is associated with changes in the expression and phosphorylation of contractile and regulatory proteins, which produce changes in cardiac contractility. Supported by a Mayo CV Prospective Grant (FVB). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

In excitation‐contraction coupling, a transient elevation of cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) triggers a force response. The Ca 2+ sensitivity of muscle reflects the relationship between [Ca 2+ ] cyt and force, and in skeletal and cardiac muscle is often defined as the [Ca 2+ ] cyt at which 50% force is generated. In smooth muscle, Ca 2+ sensitivity has been assessed as the ratio of peak force to peak cytosolic [Ca 2+ ] cyt . However, this measurement overlooks the dynamic nature of Ca 2+ regulation of force generation. In the present study, in order to provide a dynamic index of Ca 2+ sensitivity in airway smooth muscle (ASM), we simultaneously measured [Ca 2+ ] cyt and force and determined their temporal dependence using a phase loop plot. We hypothesized that [Ca 2+ ] cyt /force phase loop plot in ASM would be altered by conditions in which the extent of regulatory myosin light chain (rMLC) phosphorylation was affected. Intact ASM strips were isolated from porcine tracheas obtained from local abattoir. ASM strips, were loaded with Fura‐2 AM (Ca 2+ fluorescent indicator) and stimulated using 1 μM ACh. Isometric force and [Ca 2+ ] cyt responses were simultaneously measured using a Guth Muscle Research System. In the first set of experiments we examined the effect of Y27623, a Rho‐kinase inhibitor, and calyculin A, a protein phosphatase inhibitor that affected Ca 2+ ‐induced rMLC phosphorylation. In a second set of experiments, we examined the effect of cytochalasin D (Cyto‐D), an inhibitor of actin polymerization, which reduces force independent of Ca 2+ ‐induced rMLC phosphorylation. As expected, Y27623 decreased ACh‐induced isometric force but had no effect on evoked [Ca 2+ ] cyt transients. Cyto‐D also decreased force with no effects on evoked [Ca 2+ ] cyt . By comparison, calyculin A increased force with no effect on [Ca 2+ ] cyt . Although these results suggest changes in Ca 2+ sensitivity, the phase loop plots showed no significant shift indicative of changes in dynamic Ca 2+ sensitivity. These results do not support our hypothesis that dynamic Ca 2+ sensitivity in ASM is modulated by the extent of rMLC phosphorylation. Under these experimental conditions, it appears that isometric force generation in ASM is not entirely dependent on [Ca 2+ ] cyt . Support or Funding Information Supported by NIH grant HL126451 (GCS)

In previous studies, we found that prolonged hypothermia (2 h or greater) followed by rewarming induces cardiac contractile dysfunction (rewarming shock) mimicking the effects of accidental hypothermia followed by rapid rewarming in trauma patients. We found that rewarming shock was also presented in isolated cardiomyocytes in which reduced contractility was attributed to PKA‐mediated TnI phosphorylation and reduced Ca 2+ sensitivity. During hypothermia rewarming in cardiomyocytes there is increased production of reactive oxidant species (ROS ‐ superoxide anion), which may trigger the intracellular mechanisms underlying reduced contractility. We hypothesized that in cardiomyocytes, hypothermia/rewarming‐induced ROS production is due to altered mitochondrial respiratory function. To test this hypothesis, isolated cardiomyocytes from 6 rats underwent the following hypothermia/rewarming protocol in which they were electrically stimulated every 2 s: experimental temperature was cooled from 35°C to 15°C during the initial 30 min, then maintained at 15°C for 2 h, followed by rewarming to 35°C in the final 30 min. Time‐matched control cardiomyocytes were also electrically stimulated every 2 s and maintained at 35°C for 3 h. After the 3‐h protocol, cardiomyocytes were placed into separate chambers of an Oroboros Oxygraph (high‐resolution respirometry) to measure O 2 consumption rate (OCR) under varying conditions. Initially, basal OCR was measured for 5 min followed by serial exposure to oligomycin (1 μM for 5 min) to block ATP synthase activity, FCCP (0.5 μM repeated 3 times for 5 min each) an H + ionophore to dissipate mitochondrial membrane potential; and antimycin‐A (0.5 μM for 5 min) to block the electron transport chain. This procedure allowed assessment of the key parameters of mitochondrial respiratory function: basal and maximal respiration (after FCCP exposure), ATP production and proton leak (after oligomycin), and reserve respiratory capacity (calculated after antimycin‐A). We found that compared to control cardiomyocytes, hypothermia/rewarming decreased key parameters of mitochondrial function, which suggests that altered mitochondrial function may have contributed to increased ROS production. In addition, decreased OCR following rewarming may contribute to contractile dysfunction by limiting ATP supply in response to metabolic demand, as well as by contributing to a decline in cell viability (e.g., triggering apoptosis). Support or Funding Information 5T32HL105355 (NSS); Norwegian Air Ambulance Foundation (YSH)

Hypothermia/Rewarming (H/R) is poorly tolerated by the myocardium; however, the underlying intracellular basis of H/R‐induced cardiac injury remains elusive. We hypothesized that in cardiomyocytes, H/R leads to contractile dysfunction primarily through reduced Ca 2+ sensitivity of the myofilaments. To test this hypothesis, isolated rat cardiomyocytes (13–15 cells from 6 rats per group) were electrically stimulated to evoke both cytosolic Ca 2+ ([Ca 2+ ] cyto ) and contractile (sarcomere shortening) responses that were simultaneously measured using an IonOptix system. These measurements were subjected to the following temperature protocols: 1) hypothermia (15°C) followed by rewarming (35°C) or 2) time‐matched control (35°C). Contractile dysfunction after H/R was indicated by reduced shortening velocity and reduced extent of shortening of sarcomere length (SL) compared to time‐matched controls. Throughout hypothermia, basal [Ca 2+ ] cyto increased and the duration of the evoked [Ca 2+ ] cyto transients was prolonged. Phase‐loop plots of [Ca 2+ ] cyto versus contraction in cells exposed to H/R indicate that Ca 2+ sensitivity of contraction is reduced. Consistent with decreased Ca 2+ sensitivity, we found that cardiac troponin I (cTnI) phosphorylation (measured by Western blot) increased after H/R. Our results support our overall hypothesis and suggest that reduced Ca 2+ sensitivity and increased cTnI phosphorylation contributes to the altered excitation‐contraction coupling of cardiomyocytes following H/R. The clinical importance of our results brings focus to Ca 2+ sensitivity as a plausible therapeutic target in treating patients exposed to accidental hypothermia. Support or Funding Information NIH Grant 5T32HL10535 (NSS)

Hypothermia/Rewarming (H/R) induces myocardial dysfunction although the underlying intracellular mechanisms remain elusive. We hypothesized that in cardiomyocytes, H/R disrupts intracellular Ca 2+ ([Ca 2+ ] cyt ) homeostasis via an effect on both release and clearance of cytosolic Ca 2+ leading to a prolongation of evoked [Ca 2+ ] cyt transients associated with activation with downstream impact on contractility. To test this hypothesis, isolated rat cardiomyocytes (13–15 cells from 6 rats per group) were loaded with the Ca 2+ indicator, Fura‐2AM (0.5 μM), and exposed to H/R with or without electrical pacing at 0.5 Hz. In both groups, evoked [Ca 2+ ] cyt and contractile (sarcomere length (SL) shortening) responses were simultaneously measured at 35°C before and after hypothermia using an IonOptix system. H/R experiments were conducted as follows: experimental temperature was cooled from 35°C to 15°C in 30 min, maintained at 15°C for 2 h, and then rewarmed back to 35°C in 30 min. Time‐matched controls were implemented for 3 h at 35°C. With electrical pacing throughout hypothermia, basal [Ca 2+ ] cyt increased and the kinetics of the evoked [Ca 2+ ] cyt responses were prolonged. In addition, the extent and velocity of SL shortening was reduced in cardiomyocytes that were paced during hypothermia. In contrast, in cardiomyocytes that were not electrically paced during hypothermia, basal [Ca 2+ ] cyt was maintained and the kinetics of evoked [Ca 2+ ] cyt transients were unaffected. In addition, in the absence of pacing during hypothermia, contractility of cardiomyocytes was unaffected. These results suggest that H/R affects the release and clearance of evoked [Ca 2+ ] cyt transients (e.g., RyR mediated Ca 2+ release and SERCA mediated clearance) such that basal [Ca 2+ ] cyt increases. This effect of H/R on [Ca 2+ ] cyt homeostasis is associated with reduced cardiac contractility.

Succinate dehydrogenase (SDH) is a key enzyme in the tricarboxylic acid (TCA) cycle, as well as Complex II of the electron transport chain (ETC). In the SDH reaction, succinate is oxidized to fumarate in the TCA cycle, producing FADH2 contributing to the proton gradient that drives the reduction of ubiquinone to ubiquinol in the ETC. Thus, measurement of the maximum velocity of the SDH reaction (SDHmax) provides insight into both TCA cycle and ETC contributions to the maximum respiratory capacity of mitochondria. The goal of the present study was to use a quantitative histochemical technique, which we previously developed in skeletal muscle fibers, to quantify the SDHmax in permeabilized (45 μg/mL saponin) cardiomyocytes isolated from male Sprague-Dawley rats. In the SDH reaction, the oxidation of succinate (80 mM succinate; determined to be the maximum substrate concentration) to fumarate is coupled to the reduction of nitroblue tetrazolium (NBT; 1.5 mM) to its diformazan (NBTdfz) using 1-methoxyphenazine methosulphate (mPMS; 1 mM) as an exogenous electron carrier and azide (0.75 mM) to inhibit Complex IV. The progressive accumulation of NBTdfz as the reaction proceeds is measured by repeated densitometric (at 570 nm, the peak absorbance wavelength for NBTdfz) measurements. The optical density (OD) measurements are converted to mM fumarate using the Beer-Lambert-Bouger equation based on pathlength for light absorbance and the molar extinction coefficient for NBTdfz. Importantly, the pathlength for light absorbance must be controlled. In cross-sections of skeletal muscle fibers, pathlength was controlled by cryosectioning at 6 μm. In the present study, pathlength was controlled by optical sectioning using a confocal microscope. The isolated cardiomyocytes were imaged in 3D using a Nikon Eclipse A1 laser scanning confocal system with a ×40x/1.3 NA oil-immersion lens with an optical slice set at 0.5 μm. Changes in OD within a series of 10 optical slices were measured every 15 s over a 5 min period and expressed as mM fumarate/min using the Beer-Lambert-Bouger equation. We found that the SDH reaction was linear across 5 min, and that the change in OD was consistent throughout all 10 optical slices within a cardiomyocyte, suggesting that NBTdfz accumulates in the cytoplasm. Our results confirm that this assay of SDHmax is reproducible in cardiomyocytes and offers a single cell alternative to high-resolution respirometry. The ability to measure SDHmax in single cells is in contrast to respirometry, which only measures the average O2 consumption of aggregates of cells (isolated or in tissue). Thus, a direct measurement of SDHmax can provide valuable physiological insight into variation in mitochondrial function across a population of cardiomyocytes, e.g., as might occur with ischemic injury. Supported by the Mayo Clinic This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

AbstractNitric oxide (NO) is thought to increase cardiac contractility by increasing cytosolic Ca2+ concentration ([Ca2+]cyt) during excitation. Alternatively, NO could increase the sensitivity of the contractile response to [Ca2+]cyt (Ca2+ sensitivity). Arginase regulates NO production by competing with NO synthase (NOS), and thus, arginase inhibition should increase cardiac contractility by increasing NO production. We hypothesized that arginase inhibition increases cardiac contractility by increasing both [Ca2+]cyt and Ca2+ sensitivity. [Ca2+]cyt and contractile (sarcomere length [SL] shortening) responses to electrical stimulation were measured simultaneously in isolated rat cardiomyocytes using an IonOptix system. In the same cardiomyocytes, measurements were obtained at baseline, following 3‐min exposure to an arginase inhibitor (S‐[2‐boronoethyl]‐l‐cysteine; BEC) and following 3‐min exposure to BEC plus a NOS inhibitor (NG‐nitro‐l‐arginine‐methyl ester; l‐NAME). These responses were compared to time‐matched control cardiomyocytes that were untreated. Compared to baseline, BEC increased the amplitude and the total amount of evoked [Ca2+]cyt, and the extent and velocity of SL shortening in cardiomyocytes, whereas addition of l‐NAME mitigated these effects. The [Ca2+]cyt at 50% contraction and relaxation were not different across treatment groups indicating no effect of BEC on Ca2+ sensitivity. The [Ca2+]cyt and SL shortening responses in time‐matched controls did not vary with time. Arginase inhibition by BEC significantly increased the amplitude and the total amount of evoked [Ca2+]cyt, and the extent and velocity of SL shortening in cardiomyocytes, but did not affect Ca2+ sensitivity. These effects of BEC were mitigated by l‐NAME. Together, these results indicate an effect of NO on [Ca2+]cyt responses that then increase the contractile response of cardiomyocytes.